CHAPTER-1: CLASSIFICATION OF PATHOGENIC BACTERIA

Cell shape Characteristics Genus Family

Gram negative bacteria
Cocci Aerobic Neisseria Neisseriaceae
Veilonella
Coccobacilli - Brucella Brucellaceae
Bordetella
Pasteurella,
Mannheimia
Haemophilu s
Bacilli Facultative Escherichia, Shigella, Enterobacteriaceae
anaerobic, motile Salmonella, Proteus,
with peritrichous Erwinia, Yersinia,
flagella or non motile Enterobacter, Serratia
Aerobic, motile with Azotobacter Rhizobium Azotobacteraceae
peritrichous flagella
or non motile
Aerobic, motile with Nitrosomonas, Rhizobiaceae
polar flagella Nitrobacter,
Thiobacillus,
Pseudomonas,
Acetobacter, Legionella
Facultative anaerobic Campylobacter, Nitrobacteraceae
with polar flagella Zymomonas, Pseudomona-daceae
Aeromonas
Curved rods with Vibrio, Spirillum, Spirillaceae
polar flagella Desulfovibrio
Gram positive bacteria
Cocci Cells in irregular Staphylococcus, Micrococcaceae
clusters Micrococcus, Sarcina
Cells in chains Streptococcus, Streptococcaceae
Leuconostoc
Bacilli Aerobic Spore Bacillus Bacillaeceae
forming Clostridium
Anaerobic Spore
forming
Lactic fermentation Lactobacillus Lactobacillacaeae
Propionic Propionibacterium Propioni-
fermentation bacteriaceae
Oxidative, weakly Corynebacterium -
fermentative Listeria
 Erysipelothrix
Other major groups
Acid-fast - Mycobacterium Actinomycetales
rods Actinomyces, Nocardia,
Ray-forming Streptomyces
rods
Spiral motile Treponema Spirochetales
organisms Borrellia
Leptospira
Spirocheta
Small lack rigid wall Mycoplasma Mollicutes
pleomorphic
Small - Rickettsiae Rickettsiaceae
intracellular Coxiella Chlamydiaceae
parasites Chlamydia
Intracellular borderline with Bartonella Bartonellaceae
parasites protozoa
CHAPTER-2: STREPTOCOCCI

Learning objectives

To know in detail about

 Morphology, cultural and biochemical characteristics of streptococci

 Classification of streptococci
 Toxins and virulence factors of streptococci
 Diseases caused by streptococci in domestic animals
 Distinguish between streptococci and staphylococci
 General approaches uesed to isolation and identification streptococci
 Dick test, scarlet fever and CAMP test
 Strangles in horses

 SYSTEMATICS

Domain Bacteria
Phylum Firmicutes
Class Bacilli
Order Lactobacillales
Family Streptococcaceae
Genus Streptococcus
Species S. agalactiae, S.dysgalactiae, S. equi subsp zooepidemicus, S.uberis,
S. equi subsp equi, S. canis, S.suis, S. pyogenes(human)
 HISTORY

 Rivolta (1873) described chain forming organisms in pus from a case of strangles in
horses.
 In 1878-79, Pasteur recognized this organism as a pus-forming agent.
 In 1903, Hugo Schottmuller introduced blood to differentiate various types of
hemolysis.
 In 1928, Rebecca Lancefield reported a serological method of grouping streptococci.

HABITAT

 Streptococci are world wide in distribution.

 Most of the streptococci of Veterinary interest live as commensals in the mucosa of
the upper respiratory and lower urogenital tracts.
 They do not survive for long away from the animal hosts.

MORPHOLOGY

 Streptococci are Gram positive, spherical or ovoid cells, arranged in chains or pairs.
 Chain formation is due to the cocci dividing in one plane only and the daughter cells
failing to separate completely. Each coccus is about 1 mm in diameter.
 They are facultative anaerobes, catalase negative, oxidase-negative, and non-spore
forming and non-motile with exception of some of the enterococci.
 Capsulation is not a regular feature of streptococci but some strains
of S.pyogenes and some group C strains have capsules composed of hyaluronic acid
while polysaccharide capsules are encountered in members of group B and D.
 Protoplasts (L-forms) may be induced by penicillin or phage associated lysine and
may be propagated on hyper tonic media.

CLASSIFICATION

 Several systems of classification have been employed. Based on growth

characteristics, type of haemolysis and biochemical activities they can be divided into
6 principal categories.

Group Examples
Pyogenic Streptococci S. pneumoniae, S.pyogenes, S.equi, S. dysgalactiae
Oral Streptococci S.salivarius
Enterococci S.faecalis, S.avium, S. gallinarum
Lactic acid Streptococci S.lactis
Anaerobic Streptococci S. monbillorum
Other Streptococci S.bovis, S.uberis, S.equi subsp zooepidemicus

 The aerobic and facultative anaerobic streptococci are classified, based on their
haemolytic properties.
 Brown (1919) established this method by employing meat infusion peptone agar with
5% horse blood. He recognized three types of reactions.
 o alpha - haemolytic streptococci: They produce a greenish discolouration with
partial haemolysis around the colonies.
o The zone of lysis is small (1or2 mm wide), within which the unlysed
erythrocytes are seen.
o These ά - streptococci are generally commensals in the throat. Because of the
distinctive green color, they produce; they are called as greening or viridans
streptococci. Eg. S.pneumoniae
o Beta haemolytic streptococci produces a sharply defined, clear, colourless
zone of haemolysis, 2 or 4mm wide, within which the red cells are completely
lysed.
o Most of the pathogenic streptococci fall into the beta group and are called as
the haemolytic streptococci.
o Gamma or non-haemolytic streptococci produces no change in the medium.
o The gamma streptococci includes the faecal streptococci ( S. faecalis) and
related species. They are called the enterococcus or indifferent streptococci.
 Anotherimportant way in which the beta haemolytic streptococci were classified by
Rebecca lancefield (1933) was based on the nature of a carbohydrate (C) antigen on
the cell wall. They are known as Lancefield groups, 19 of which have been identified
so far and named by the capital letters A-U (without I and J)

Lancefield Species Host Disease

group
A Streptococcus Humans Scarlet fever , Septic sore
pyogenes throat, erysipelas, abscesses
and rheumatic fever
B S. agalactiae Cattle, sheep Chronic mastitis
and goats
Human and Neonatal septicaemia
dogs
Cats Kidney and uterine infections
C S. dysgalactiae Cattle Acute mastitis
Lambs Polyarthritis
S. dysgalactiae Horse Abscesses, endometritis and
subsp. equismilis mastitis
S. equi. subsp. Horse Strangles , genital and
equi suppurative conditions,
Mastitis and purpura
haemorrhagica
S. equi subsp. Horse Mastitis, abortion, secondary
zooepidemicus Pneumonia and navel
infections
Cattle Metritis & Mastitis
Pigs Septicaemia & arthritis in 1-3
wk old piglets
 Poultry Septicaemia & Vegetative
Endocarditis
Lambs Pericarditis and Pneumonia
D Enterococcus Many species Opportunistic infections
faecalis
S. equines and S. Many species Opportunistic infections
bovis
E (P,U,V) S. Porcinus Pigs Jowl abscesses and
lymphadenitis
G S. canis Carnivores Neonatal septicaemia, genital,
skin and wound infections
Cattle Occasional mastitis
N Lactococcus lactis Cattle unknown
Q Enterococcus Many species unknown
avium
R S.suis type 2 Pigs (4 to 6 Meningitis and arthritis
months)
S S.suis type 1 Pigs (2 to 4wks Meningitis and arthritis
old)
Ungroupable S.uberis Cattle Mastitis
S.pneumoniae Guinea pigs, Pneumonia
rats and
primates

 Haemolytic streptococci of group A are known as S.pyogenes . These may be further

subdivided into types based on the protein (M,T and R) antigens present on the cell
wall.
 The M protein is acid and heat labile and the T protein is acid labile and trypsin
resistant.
 Some of the Lancefield groups may be further subdivided by means of the
agglutination test and designated by Arabic numbers-Griffith typing

CULTURAL CHARACTERISTICS

 It is an aerobe and facultative anaerobe, growing best at a temperature of 37°C. They

grow best in media enriched with blood, serum and fermentable carbohydrates.
 On blood agar after incubation for 24hrs small, circular, semitransparent colonies
with an area of clear haemolysis are produced.
 Virulent strains from fresh isolates produce matt (finely granular) colony and
avirulent strains form glossy colonies. Strains producing capsules form mucoid
colonies.
 In glucose or serum broth, growth occurs as a granular turbidity with a powdery
deposit. No pellicle is formed.
 In Edwards‘s medium (selective media) it produces dewdrop like black colonies.
  Edwards medium containing blood agar, crystal violet and aesculin (differentiate
among different species of streptococci which do or do not hydrolyse aesculin).
 S. pneumoniae is alpha haemolytic, produces mucoid or flat colonies with smooth
borders and a central concavity after 48-72hrs on blood agar (draughts man
colonies).
 Ability to grow in 0.1 % tellurite broth is characteristic of S. faecalis. Majority of
streptococcal species do not grow on Mac Conkey agar except Enterococcus faecalis.

BIOCHEMICAL PROPERTIES

 Streptococci are catalase and oxidase negative . (Click here for visual - catalse test)
 Ferment several sugars producing acid but no gas.
 They ferment sorbitol, trehalose, lactose, maltose, dextrin, and mannitol.
 Gelatin not liquified.
 Nitrates not reduced.
 Indole is negative.
 They are not soluble in 10% bile unlike pneumococci

RESISTANCE

 It is a delicate organism easily destroyed by heat (54° C for 30 minutes).

 It can survive in dust for several weeks, if protected from sunlight.
 It is rapidly inactivated by antiseptics.
 It is more resistant to crystal violet and susceptible to sulphonamides and other
antibiotics.
 Sensitivity to bacitracin is employed as a convenient method for differentiating S.
pyogenes from other hemolytic streptococci.

ANTIGENECITY

 The hyaluronic acid capsule of S. pyogenes inhibits phagocyotosis.

 The cell wall is composed of an outer layer of fimbria containing proteins and
lipoteichoic acid, a middle layer of group specific carbohydrate and an inner layer of
peptidoglycan.
 The Cell wall polysaccharide has been shown to have a toxic effect on connective
tissue in experimental animals.
 The peptidoglycan is responsible for cell wall rigidity. Several protein antigens have
been identified in the cell wall (M, T and R).
 They are responsible for type specificity in S.pyogenes. Among these the M protein
acts as a virulence factor by inhibiting phagocytosis.
 Hair-like pili project through the capsule of group A streptococci.
 The pili consist partly of M protein and are covered with lipoteichoic acid which
isimportant in the attachment of streptococci to epithelial cells.

TOXINS AND VIRULENCE FACTORS

 Streptococci form several exotoxins and enzymes, which contribute to its virulence.

Extra cellular toxins

Hemolysins
  Streptolysins O and S are produced by groups A, C and G.

Streptolysin O

 It is so called because it is oxygen labile. It is an antigenic protein and is active in the

reduced form.
 On blood agar, its activity is seen only in pour plates and not in surface cultures. It is
also heat labile.
 It is lethal on I/V inj. into animals and has a specific cardiotoxic activity. It is a
general cytotoxin.
 Red cells of all animal species except mouse are lysed. Streptolysin O is antigenic and
antistreptolysin regularly appears in sera following Streptococcal infection.
 Estimation of this antibody (ASO) titre is a standard serological procedure for the
retrospective diagnosis of infection with S.pyogenes.

Streptolysin S

 It is an oxygen stable haemolysin and so is responsible for the beta haemolysis seen
around streptococcal colonies on the surface of blood agar plates.
 It is called Streptolysin S since it is soluble in serum. Addition of serum to broth
increased the yield of haemolysin.
 It is protein but not antigenic. It has been shown experimentally to be nephrotoxic.
Erythrogenic toxin (Streptococcal pyogenic exotoxins/ Dick toxin)
 Four erythrogenic toxins are known and most strains of S. pyogenes produce one or
more. They are pyogenic andenhance susceptibility to lethal shock by endotoxin.
 The toxin is thermostable and antigenic. The intradermal injection in rats leads to
development of erythema.
 This reaction is called as ―Dick test‖ or Schultz-charlton reaction and it is useful for
diagnosis of scarlet fever.

Enzymes

 Streptokinase (Fibrinolysin): Filtrates of streptococci gp, A, E & G produces

fibrinolysin. This toxinpromotes the lysis of fibrin clots by activating a plasminogen.
 Fibrinolysin plays a biological role in streptococcal infections by breaking down the
fibrin barrier around the lesions and facilitating the spread of infection.
 Deoxyribonucleases (Streptodornase): These cause depolymerisation of DNA,
pyogenic exudates contains large amount of DNA, derived from the nuclei of necrotic
cells.
 Streptodornase helps to liquefy the thick pus and may be responsible for the thin
serous character of streptococcal exudates.
 Four antigenically distinct streptodornase, A, B, C & D have been recognized.
 Hyaluronidase: This enzyme breaks down the hyaluronic acid of the tissues. This
might favour the spread of infection along intercellular spaces.
 Streptococci possess a hyaluronic acid capsule and also elaborate a hyaluronidase- a
seemingly self-destructive process. This is produced by group A, C, G and B
Streptococci.
 Proteinase: This is another instance of an apparently self-destructive enzyme, since it
is capable of breaking down the M protein, streptokinase and hyaluroindase.
 The enzyme is, however, produced only underspecial conditions such as an acidic
pH (5.5 –6.5). Such conditions may be produced by tissue destruction, as in
abscesses.
 Most strains of S. pyogenes form proteinase.
 Neuraminidase: This activity is detected in streptococci groups A, B, C, G and L. This
enzyme is a virulence factor for pathogens surviving on mucosal surface.
 In addition to this M types of S.pyogenes produce NADase and other many strains
also produce esterase, amylase and N-acetyl glucosaminidase.

Serum opacity factor

 When group A. streptococci is grown in horse serum it produces an opalescence of

the serum. This opacity factor is a protein.
 This is a lipoproteinase and opalescence is a result of an agglomeration of the
bacterial antigen.

PATHOGENESIS

 The natural habitat of the species of streptococci are skin, nose, throat, digestive and
urogenital tracts of man and animals.
 S.pyogenes are present in human nose and throat without causing any disease,
while S.agalactiae and S.uberis can exist in bovine udder without causing mastitis.

PATHOGENECITY

Cattle : Bovine mastitis

 It is caused by S. agalactiae (group B), S. dysgalactiae (grp C), S.

equi subsp. zooepidemicus (grp. C), S. uberis (grp C, D, E, P, V).
 Mastitis arises from the multiplication of streptococci in the teat sinus and
extends into the ducts.
 It causes parenchymatous mastitis, which is characterized by progressively
chronic condition resulting in fibrosis.
 In acute stages milk is composed of purulent exudate, dead tissue cells,
coagulated milk protein and bacteria.
 Peptostreptococcus indolicus is an anaerobic streptococcus, which is responsible
 for summer mastitis in cattle in association with Arcanobacterium pyogenes.

Horse

 S. equi and S. equisimilis are the main causes of strangles in young horses.
 It is characterized by a catarrhal discharge, with inflamation of the nasal mucous
membranes, followed by swelling of pharyngeal LN‘s in which abscesses
develop.
 The infection spreads through lymph channels. It also causes metritis and
cervicitis in horses.
 Purpura haemorrhagica, considered to be an immune mediated disease, occur in
horses 1 to 3 weeks after illness.
 Bastard strangles – in which abscesses developed in many organs. It is a very
serious complication.

Chicken

 S. gallinarum causes typical acute septicemia with peritonitis in chicken.

Dogs

 S. canis is considered to be the cause of acid milk in puppies and canine

tonsillitis. It is also associated with neonatal septicaemia and toxic shock
syndrome.

Pigs

 S. suis causes porcine cervical lymphadenitis and also isolated from pneumonia,
septicaemia, arthritis, endocarditis, meningitis and reproductive tract infections.
 It also causes erosive arthritis in young pigs.

DIAGNOSIS

 It involves clinical, microscopical and bacteriological examination.

Clinical examination

 Palpation of the udder and supramammary lymphnodes will be helpful in

distinguishing the chronic and insidious form of mastitis produced
by S.agalactiae and S.uberis.
 In contrast S.dysgalactiae and S. zooepidemicus causes sudden onset of acute
inflammation of one quarter only with an acute systemic disturbance followed by
joint infections and lameness.

Microscopical examination

 When long chains of organisms are detected in milk samples from chronic mastitis, it
is caused by S. agalactiae.

Based on type of haemolysis on blood agar

Bacteriological examination

 When 0.1 ml of secretions inoculated on Edward‘s medium (blood agar, crystal violet
and aesculin) S.agalactiae produces bluish-grey colonies and S.uberis produces dark
color colonies.
 CAMP test (Christie, Atkins, Munch and Peterson, 1944)
o This test is based on the observation that ruminant red blood cells lysed by
the beta toxin of staphylococci at 370C are completely lysed in the presence
of S.agalactiae (group B).
o Differentiation between the pneumococcus and S.viridans organisms can be
achieved by bile solubility and the optochin test.
 Bile solubility test
o Autolysis of pneumococcal cultures takes place within 15 minutes at 370C in
the presence of 10 per cent sodium deoxycholate.
o These substances have no effect on S. viridans organisms.
 The optochin test
o The majority of pneumococcal strains are sensitive to optochin (Ethyl
hydrocuprein hydrochloride). Whereas S.viridans organisms are not.
o This test consists of placing a small circular piece of filter paper, impregnated
with 1:4000 aqueous solution of optochin, in the center of a blood agar plate
after inoculating the test cultures in streaks across the full width of the
medium.
o The growth of pneumococcal strains will be inhibited to a distance of some 5
mm from the circumference of the filter paper.

CONTROL AND PREVENTION

 In streptococcal infections in animals, including mastitis, the most satisfactory

method of control is antibiotic therapy using penicillin preparations to other
substances because streptococci develop resistance to penicillin comparatively
infrequently.
 Vaccines are of very limited value for the immunization of animals against
streptococcal infections, with the possible exception of equine strangles.
 S.pyogenes (group. A) is the most usual cause of septic sore throat and scarlet fever
in humans.

CHAPTER-3: STAPHYLOCOCCI

Learning objectives

To know in detail about,

 Morphology, cultural and biochemical characteristics of Staphlyococci

 Disease caused by Staphlyococci species in domestic animals
 Clinical symptoms and laborator findings to a possible diagnosis of a gangrenous
mastitis, bumble foot and greasy pig disease
 The role of Mannitol salt agar in isolation of staphylococcus
 Double or hot-cold haemolysis, Coagulase test and Hotis test

SYSTEMATICS
  First discovered by Scottish surgeon Sir Alexander ogston (1880) in infected
tissues. He named it as staphylococcus (Greek staphyle, bunch of grapes; KOKKAS,
berry).

Domain Bacteria
Phylum Firmicutes
Class Bacilli
Order Bacillales
Family Staphylococcaceae
Genus Staphylococcus
Species S. intermedius.
S. hyicus.
HISTORY

 First discovered by Scottish surgeon sir Alexander ogston (1880) in infected tissues.
He named it as staphylococcus‖ (Greek staphyle, bunch of grapes; KOKKAS, berry).

HABITAT

 Staphylococcus occurs worldwide in mammals although the spread of staphylococcal

strains between different animal species is limited.
 They colonize the nasal cavity, skin and mucous membranes and can be transient in
the intestinal tract. It is an opportunist type organism

MORPHOLOGY

 Spherical cells, 0.8 to 1 micrometer in diameter, on agar media the cocci are arranged
in grape like clusters.
 In broth they occur as small groups, pairs, short chains of not more than four
members.
 They are Gram positive, non-motile, non-acid fast, non-spore forming and have no
flagella.

CULTURAL CHARACTERISTICS

 They are aerobic and facultatively anaerobic.

 They grow at an optimum temperature of 35-37°C.
 Staphylococcus grows in the presence of 7-10% Sodium chloride. Hence media-
containing salts is selective for this organism (eg; Mannitol Salt agar) and it is highly
inhibitory to many other bacteria particularly Gram-negative bacteria. (Click here for
visual)

 In Nutrient agar plate the colonies are round, smooth, glistening, opaque, low,
convex, edge, entire end of a golden yellow or white colour.
 In nutrient broth an uniform turbidity is present with powdery sediment.
  Phenolpthaline diphosphate or tellurite agar selectively inhibits non-pathogenic
strains.
 Haemolysis on blood agar. Capable of liberating ‗V‘ factor into the medium, which
favours the growth of Haemophilus organism.
 Purple agar, containing bromocresol purple as a pH indicator and 1% maltose, is used
to differentiate S. aureus and S. intermedius.
 Most strains form pigments, hence previously classified as per the colour of the
pigment produced.
o S. aureus – yellowish or golden orange pigment.
o S. albus - white colonies.
o S. citreus - lemon yellow colour pigment
 Later on it was found that pigment formation was variable. Hence such classification
was no longer followed.
 In sheep or rabbit blood agar plate ―double haemolysis‖ around colonies are formed
with incubation at 37°C it produces an incomplete haemolysis, which develops into a
complete haemolysis when held at 4°C. This is called as hot cold lysis phenomenon.

BIO-CHEMICAL PROPERTIES

 Staphylococcus aureus produces acid from glucose, maltose, mannitol, lactose, and
sucrose and not from salicin, raffinose & inulin.
 The organism is indole negative, positive for NH3, methyl red and Voges - Proskauer
and catalase.
 Hydrolyses gelatin and coagulates serum. Negative for oxidase and H2S production.

RESISTANCE

 Staphylococci are the most resistant of the cocci.

 Usually a temperature of 60°C for half an hour destroys all the cells.
 Death is accomplished in 1% phenol in 35 minutes or 10% formaldehyde in 10
minutes.

ANTIGENICITY

It consists of

Group antigen

 Carbohydrates
o The cell wall of S. aureus contains ribitol teichoic acid.
o S. intermedius contains glycerol teichoic acid.
 Protein A - Present only in S. aureus .

Type antigen

 Proteins other than protein A.

TOXINS AND VIRULENCE FACTORS

  It consists of capsule, Teichoic acid, which have antiphagocytic ability.

Exotoxins

o Hemolysin
 Four antigenically distinct hemolysins causes hemolysis of
erythrocytes. They are Alpha, Beta, delta and Epsilon toxin, which
produce partial hemolysis (hot – cold hemolysis). The alpha toxin is
the major toxin in gangrenous mastitis. It causes spasm of smooth
muscle and is necrotizing and potentially lethal.
o Leukotoxin
 It has the leukocidal activity and includes a and d toxins. By doing so,
the organisms may spread more easily to other parts where they
develop secondary lesions.
o Enterotoxin
 It is seldom produced by animal strains. There are several
antigenically distinct types of heat stable enterotoxins. They are not
destroyed at 100°C for 80 minutes. They are responsible for food
poisoning in man.
o Exfoliative or Dermonecrotoxin
 This toxin causes necrosis of skin by exfoliation and intraepidermal
seperation.
o Toxic shock syndrome toxin (TSST)
 Induce excessive lymphokine production and results in tissue damage.
Bovine and human strains of Sta. aureus produce TSST.

Extracellular enzymes

o Coagulase
 On their ability to coagulate plasma they are classified as coagulase
positive staph. (CPS) and coagulase negative staph. CPS are
considered to be significant pathogens. They are resistant to heat. The
coagulation of plasma produces a fibrin film on the surface of the
organisms, which allows multiplying.
o Hyaluronidase
 It hydrolyses hyaluronic acid, the mucoid ground substance of
connective tissue.
o Nucleases
 Deoxyribonucleases (DNAase) hydrolyze DNA and Ribonucleases
hydrolyze RNA.
o Fibrinolysin
 Fibrinolysin is commonly referred to as staphylokinase, is an activator
of the plasma system leading to the breakdown of fibrin.
o Lipases and esterases - They hydrolyze lipids.
o Lysozyme - Hydrolyses the peptidoglycan in the cell wall of many bacteria

PATHOGENESIS
 PATHOGENICITY

 Horse: Botryomycosis: Infrequent chronic granulomatous lesions involving the udder

of the mare, cow and sow and the spermatic cord of horses.
 Cattle: Mastitis: Staphylococcal bovine mastitis may be chronic, acute and peracute.
Gangrenous mastitis due to a toxin is seen in postparturient cows.
 Sheep: Tick pyemia in lambs occurs in 2-5 week old lambs, which is heavily infected
with Ixodes ricinus.
 Periorbital eczema is an infection due to abrasions, Staphylococcal dermatitis due to
scratches from vegetation.
 Poultry
o Bumble foot: A pyogranulomatous process of subcutaneous tissue of foot that
can involve the joints.
o Staphylococcal arthritis and septicemia in turkeys, omphalitis – yolk sac
infection, wing rot or gangrenous dermatitis infection in poultry
 Pig: Exudative epidermitis (greasy pig disease) is an acute generalized infection of
suckling and weaned pigs caused by S. hyicus. This disease is characterized by excess
sebaceous secretion, exfoliation and exudation.
 Dogs and Cats: Pyoderma is one of the most common skin diseases of dogs.
 In addition to this, Otittis externa and other suppurative conditions are caused by S.
intermedius.
 Staphylococcal antigens produce intense inflammatory reaction andpromote
persistence of the bacteria.
 Other Staphylococcal organisms
o S. aureus sub sp.anaerobius causes caseous lymphadenitis. They are
anaerobic and catalase negative.
o S. caprae in goat‘s milk.
o S. gallinarum and S. arlettae - skin of chickens
o S. lentus in skin of sheep and goats.
o S. equorum in skin of horses
o S. simulans and S. felis - clinical specimens in cats
o S. delphini in skin of dolphins
o S. aureus in Staphylococcal scalded skin syndrome (SSSS) and Toxic shock
syndrome (TSS) in humans.
o MRSA in Methicillin resistant Staphylococcus aureus
 DIAGNOSIS

 Cultural characters in selective media.

 In case of food poisoning identification of the enterotoxin is of diagnostic use.
 CPS produces typical reaction in the Hotis test of milk samples.
 These organisms produced green or greenish brown colonies with white centres after
the milk samples are incubated for 16-20 hours.
 DNA test and protein A tests are also employed.

Pathogenicity test

 Coagulase test: When culture added to Rabbit plasma, fibrinogen isconverted to

fibrin by coagulase enzymes.
 In this test, a suspension of staphylococci is mixed with rabbit plasma either on a
slide or in a small tube.
 The slide test detects the presence of a bound coagulase or clumping factor on the
bacterial surface.
 A positive reaction is indicated by clumping of bacteria within 1 to2 min.
 The tube test detects the free coagulase or staphylocoagulase, which is secreted by
bacteria into the plasma.
 It is the definitive test for coagulase production and positive reaction is indicated by
clot formation in the tube following incubation at 37ºC for 24 hrs. (Click here for
visual)

Phage typing

 Phage typing is carried out for epidemiological purposes, particularly for S.

aureus strains of bovine mastitis.

TREATMENT

 Penicillin is the drug of choice if strains are susceptible.

 New synthetic penicillins such as methicillin, oxacillins are effective.
 Tetracyclines, bacitracin, nitrofurans, Trimethoprims, Cephalosporins, enrofloxacins
are effectiv

CHAPTER-4: BACILLUS

Learning objectives

To know in detail about,

 The diseases caused by Bacillus species in domestic animals and human

 Morphology, cultural and biochemical characteristics of B.anthracis
 Toxins of B.anthracis
 Pathogenesis, control and prevention of anthrax
 Different diagnostic methods of B.anthracis
 Explain the role of PLET agar in isolation of B.anthracis
 Mcfadyen's reaction, Ascoli's preciptatioon and string of pearl's test
 Difference between B.anthracis and anthracoids

SYSTEMATICS
Domain Bacteria
Phylum Firmicutes
Class Bacilli
Order Bacilliales
Family Bacilliaceae
Genus Bacillus
Species B.anthracis, B.cereus, B.subtilis, B.mycoides, B.megaterium,
B.mesentricus

 B.anthracis causes Anthrax in animals and Wool sorter‘s disease, hide porter‘s
disease, Malignant Pustule in humans.
 B.cereus causes food poisoning in humans.
 Other bacilli in this group are non-pathogenic and they are called as anthracoids.
 B.licheniformis is an emerging pathogen and it is implicated in sporadic abortions in
cattle and sheep.

HISTORY

 Discovery of the anthrax bacillus is credited to Davaine and Rayer (1863 –1868).
 Considerable historic interest is attached to anthrax bacilli.
 Pollender 1849 – Anthrax bacillus was the first pathogenic bacterium observed under
the Microscope
 Davaine 1850 – first communicable disease shown to be transmitted by inoculation
of infected blood
 Koch 1863 – first bacillus to be isolated in pure culture
 Pasteur 1881 – used for the preparation of attenuated vaccine.

HABITAT

 Bacillus anthracis spores remain viable for many years in soil, water and animal
hides and products.
 Spores have been isolated from naturally infected soil as long as 60 years.

MORPHOLOGY

 Members of the family are Gram +ve large rods, aerobic (facultative anaerobic),
endospore forming, capsulated, mostly catalase positive and fermentative organisms.
They are motile by peritrichous flagella.
 The anthrax bacillus is one of the largest pathogenic bacteria. They are Gram +ve,
straight, rod shaped, non-motile organisms measuring 4-8 μm x 1-1.5 μm.
 In cultures the bacilli are arranged end to end in long chains. The ends of the bacilli
are truncated or often concave and somewhat swollen.
 Chain of bacilli presents a bamboo stick appearance (and also called as Box car
bacillus - looks like linked rail carriages).
 In tissues or in blood smear, it is found singly, in pairs or in short chains, the entire
bacilli being surrounded by capsule.
 The capsule is polypeptide in nature, being composed of a polymer of d-glutamic
acid.
 Capsules are not formed under ordinary conditions of culture, but only if the media
contains serum, albumen, charcoal, starch or bicarbonates with reduced partial
pressure of carbon dioxide.
 When blood films containing bacilli are stained with polychrome methylene blue for a
few seconds and examined under the microscope, an amorphous purplish material is
noticed around the bacilli. This represents the capsular material and is characteristic
of the anthrax bacilli. This is called asreaction. This reaction depends on the degree of
heat employed for fixation of a blood film.

 Sporulation occurs readily outside the body in the presence of oxygen. Spores are
formed in culture or in the soil, but never in the animal body during life.
 Sporulation occurs under unfavourable conditions for growth and is encouraged by
distilled water, 2% NaCl or growth in oxalated agar. Sporulation takes place at an
optimum temperature of 25-30°C and in atmosphere containing low partial pressure
of oxygen. Sporulation is inhibited by anaerobic conditions and by CaCl2
 Spores are central, elliptical or oval in shape and are of the same width as the
bacillary body. So that they do not cause bulging of the vegetative cell. The spores do
not stain by ordinary methods. But can be stained with Sudan black B.

CULTURAL CHARACTERISTICS

 Grows readily under aerobic and as facultative anaerobic at an optimum temperature

of 35-37°C.
 On agar plates, irregular, round, raised, dull, opaque, grayish white, 2-3mm in
diameter frostedglass appearance colonies are produced.
 Under the low power microscope, the slightly serrated edge of the colony is composed
of long, interlacing chains of bacilli, resembling locks of matted hair. This is referred
to as medusa head or judge's wig or woman‘s curling hair type of growth.
 B. licheniformis produces characteristic hair-like outgrowths from streaks of the
organisms on agar media. Colonies become brown with age.
 The name of this species derives from the similarity of its colonies to lichen.
 In medium containing iron salts, virulent B.anthracis produces pink or purple
coloured pigmented colonies.
 Virulent capsulated strains form rough colonies, while avirulent attenuated strains
form smooth colonies.
 In gelatin stab, fine filaments of growth develop laterally along the line of inoculum.
 The growth nearer to the surface of the medium is the longest and then progressively
shorter where there is less oxygen, resembling inverted fir tree appearance.
 On haemolysis, the B.anthracis produces slight haemolysis with capsule production,
compared with anthracoid organisms.
 B. cereus produces a wide zone of complete haemolysis around the colonies.
 A selective medium (PLET) consisting of polymyxin, lysozyme, EDTA and thallous
acetate added to heart infusion agar is useful for isolating B.anthracis from mixtures
containing other spore-bearing bacilli.

BIOCHEMICAL PROPERTIES

 Bacillus anthracis organisms are catalase +ve .

 They ferment glucose, maltose, saccharose, trehalose and dextrin, produce acid but
no gas.
 Nitrates reduced to nitrites. Indole is negative.

RESISTANCE
  The vegetative bacilli are destroyed at 60°C in 30mts.
 In the carcases of animals, the bacilli remain viable in the bone marrow for a week
and in the skin for two weeks.
 Normal heat fixation of smears may not kill the bacilli in blood film.
 The spores are highly resistant to drying, heat, cold and disinfectants. Spores remain
viable for many years in soil, water and animal hides and products.
 Spores have been isolated from naturally infected soil as long as 60years.
 They resists dry heat at 140°C for 2-3hrs and boiling for 10mts.
 They survive in 5% phenol for weeks. Spores can be killed at 120°C for 10min and 4%
KMnO4 treatment for 15mts.
 Destruction of the spores in animal products is achieved by HCHO.
 Treat 2% solution of HCHO at 39-40°C for 20mts for disinfection of wool and as
0.25% at 60°C for 6 hrs for animal hair and bristles. This process is called as
duckering.
 The anthrax bacillus is susceptible to sulphonamides, penicillin, erythromycin,
streptomycin, tetracycline and chloramphenicol.

ANTIGENICITY

 The complex antigenic structure includes a capsular polypeptide, a somatic protein

and a somatic polysaccharide antigen.
 The capsule contains poly-d-glutamic acid, which is only detectable in virulent
strains, having antiphagocytic property.
 The somatic protein is protective antigen present in the edema fluid.

TOXINS AND VIRULENCE FACTORS

 B. anthracis produces an extracellular toxin which is composed of three components,

Factor I, II & III namely oedema factor (EF), protective antigen (PA) and lethal factor
(LF) respectively.
 Factor I consists of chelating compound containing phosphorus with protein and
carbohydrate moieties.
 Factor II & III consists of proteins.
 All three factors are essential to exhibit their toxic properties.
 They are not toxic individually but the whole complex produces local odema and
generalized shock.

PATHOGENESIS OF BACILLUS

 Anthrax can occur in virtually all mammalian species but birds are highly resistant.
 The main routes of entry of endospores are by ingestion, from soil when grazing or in
contaminated food, and by infection of wounds.
 Inhalation of spores occurs in man, but to a lesser extent in animals.
 Transmission by biting insects may beimportant , especially during an outbreak of
anthrax.
 Cattle, sheep and goats are most susceptible to infection, while horses and humans
occupy an intermediate position and pigs and carnivores are comparatively resistant,
but can succumb if the infective dose is high.

PATHOGENICITY

CLASSIFICATION
Symptoms

Horses

 Acute form is very common and death may take place one day after edematous
swelling of the throat and neck region.
 There may be symptoms of colic. In less acute, oedmatous swelling become
generalized and death occurs after 2-3 days.

Cattle

 Bulls are more susceptible than cows. They have a mortality rate of 90%.
 There are three clinical causes of bovine anthrax.
 In peracute sepeticemia death occurs within 2 hours after animal collapsing with
convulsions, sudden death in animals that appeared normal is common.
 In acute septicemia death occurs within 48 to 96 hours clinical signs include fever,
anorexia, ruminal stasis, hematuria and blood tinged diarrhea.
 Pregnant animals may abort and milk production often abruptly decreases.
 Terminal signs include severe depression, respiratory distress and convulsions.
 In chronic cases, clinical signs are manifested for more than 6 days and are rare.
 B.licheniformis infection is associated with the feeding of contaminated silage and is
responsible for abortion in cattle and sheep.

Sheep

 Disease is acute and death occurs rapidly after convulsions.

Pigs

 They have a greater natural resistance than herbivores.

 The usual signs are oedematous swellings in the region of throat and neck interfering
normal respiration, enteritis and rise in temperature. The disease is not always fatal .

Dogs

 The throat will be inflamed and swollen, gastroenteritis

Lesions

 The carcass of animals will putrify rapidly and develop incomplete rigor mortis.
 The blood is dark,(tarry colored), clots poorly & exudes from the natural orifices.
 The spleen is greatly enlarged, dark and friable. The spleen reveals black cherry jam
consistency.
 The LN (lymph node) at the region of initial infection site is hemorrhagic and
edematous.
 Ecchymotic hemorrhages on the serosal surface of the abdomen, thorax, epicardium
and endocardium are common.
 Subcutaneous edematous swellings are present on the ventral aspect of the neck

Note

 When suspected for anthrax care should be taken not to open the carcass. Muzzle
piece or ear piece is usually sent for examination.
 DIAGNOSIS

Clinical symptoms

 Blood films from dead animals made by puncturing the superficial vein of the ear or
in the region of the foot.
 Care should be taken to seal the injection site by placing cotton soaked in alcohol and
ignited.
 The smears are heat fixed and stained by Wright's or Giemsa‘s stain to reveal B.
anthracis as large blue rods with characteristic dark pink or purple coloured
capsules.
 In case of horses and pigs since peripheral blood contains fewer organisms, smears
should be made from the edematous fluid or LN‘s.

Cultural examination

 Swabs from blood are inoculated on to blood agar plates and incubated at 37°C for 24
hrs and examined for their typical growth.
 Swabs are inoculated in agar enriched with blood or serum and incubated for 6 hrs at
37°C and examined by stained smears.

Bacteriological examination of hair, wool, hide, bone, bone meal & others

 Samples are added to cold saline and shaken intermittently for 3 hours.
 The supernatant fluid is then heated to 70°C for 10 minutes.
 Then they are filtered through two layers of muslin cloth, added to melted agar, and
poured into petridishes, allowed to set and incubated at 37°C.
 After 12 hours incubation, plates are to be examined for characteristic colony
morphology.

Ascoli’s Precipitation test: (Ascoli 1911)

 Grind up the organ or blood of suspected animal and suspend in 5-10 parts of saline
and boil for 15 minutes.
 Filter through filter paper and allow it to cool. Place 0.5 ml of anti-anthrax serum
(1:50) in a small test tube and overlay with 0.5 ml of clear filtrate.
 Stand at room temperature for 15 minutes. A white ring of precipitation indicates a
positive reaction.

String of Pearl’s test: (Charlton,1980)

 B.anthracis produces swollen round cells in chains (string of pearls) when incubated
for 3-6 hrs on tryptose agar containing 0.05 –0.5 I.U of penicillin/ml.
 To 100ml of molten nutrient agar, add required Sodium beznyl penicillin and mix
carefully.
 Pour into petridishes and allow to set. With a scalpel cut a block about 1.6 cm 2 from
the penicillin agar plate and place it on a microscopic slide in a petridish containing a
small piece of moistened absorbent cotton wool to prevent the agar drying out.
 Use a young colony to streak the center of the agar block.
 Place a clean cover slip on the agar block and incubate the petridish at 370C.
 After 2 hrs, remove the slide and examine the inoculum microscopically by oil
immersion for the string of pearls growth.
 Differentiation from non-pathogenic Bacillus sp. It is considered reliable but needs
experience.
  Cherry gamma phage is used. It can be propagated on B. anthracis strain 14, which
makes a suitable control.
 On one half of blood agar plate, streak B. anthracis strain 14 culture as control and
on the other half streak the culture to be identified.
 Add drops of phage preparation (1:10 dilution) on both halves. Incubate the plates in
the upright position for 8-12 hrs.
 Zones of clearing will be seen on control culture and on the suspected half if it is B.
anthracis.

Animal Inoculation

 Guinea pigs & mice are highly susceptible. Materials are inoculated by dermal
scarification, subcutaneous or intramuscular.
 Death occurs in 2-3 days and the organisms will be readily identified in the blood &
tissues.
 Apart from this laser pyrolysis, gas-liquid chromatography and mass spectrometry
are used for detection of anthrax toxin productions.

 DIFFERENTIAL DIAGNOSIS

Difference between B. anthracis & Anthracoid organisms in culture

B. anthracis Anthracoid organisms

Non motile generally motile
Capsulated non capsulated
Grows in long chains Grows in short chains
No turbidity in broth Turbidity in broth
Inverted fir tree in gelatin Atypical or absent
Methylene blue reduced weakly Reduced strongly
Haemolysis weak Strong
Liquefaction of gelatin is slow Rapid
Lecithinase reaction is weak Rapid
Ferments salicin slowly Rapid
Produces toxin, neutralised by B.anthracis antitoxin not neutralized
Pathogenic to g.pigs & mice non pathogenic
Susceptible to gamma phage not susceptible

Difference between B. anthracis & Anthracoid organisms in blood smear

stained with Polychrome methylene blue stain

B. anthracis Anthracoid
Dark pink or Purple coloured capsules No capsule
Organism rod stained blue Organism rod stained blue
Ends truncated Ends bulged and rounded
Single, pair or short chain Usually long chain
Absence of spores Spores may be present
TREATMENT, IMMUNITY AND PUBLIC HEALTH
ASPECTS

Treatment

 The organism is susceptible to penicillin-G, tetracyclines, erythromycin and

chloramphenicol.

Immunity

 Prevention of anthrax in animals is aided by active immunization.

 Initially Bail demonstrated that oedema fluid and tissues obtained from anthrax
lesion, which had been freed from viable organism, had protective properties.
 He termed the active substances as "aggresins".
 Pasteur‘s vaccine was anthrax bacillus attenuated by growth at 42 –430 C .
 As the spore is the common infective form in nature, vaccines consisting of spores of
attenuated strains were developed.
 The strene vaccine contained spores of noncapsulated avirulent mutant strain.
 It should be given 1 month before anticipated outbreaks.
 The Mazucchi vaccine contained spores of stable attenuated carbazzoo strain in 2%
saponin.
 It gives good protection when given subcutaneously.
 Protective humoral antibodies develop in 7-10 days against Factor II of the exotoxin
complex and lasts about one year.

Public Health aspects

 There is need for great care in performing necropsy on animals.

 Infections most often result from spores entering through injuries to the skin,
causing cutaneous anthrax.
 Spores are present in soil, hair, hides, wool (wool sorter’s disease-Pulmonary
anthrax), faeces, milk, meat and blood products.
 The skin lesion (cutaneous anthrax) is usually solitary, painless, seropurulent,
necrotizing, hemorrhagic and ulcerous.
 It leaves a black scar (anthrax=coal), which accounts for the name malignant
pustule.

CHAPTER-5: CLOSTRIDIUM

Learning objectives

To know in detail about,

 Diseases caused by neurotoxic clostridia in domestic animals

  Morphology, cultural and biochemical characteristics of Cl.tetani and Cl.botulinum
 Classification of Cl.botulinum'
 Toxins and pathogenesis of Cl.tetani and Cl.botulinum
 Locked jaw, spinal typhus, Lamsiekte and Western duck sickness
 Distinguish toxins of Cl.tetani and Cl.botulinum
 General approaches used to demonstrate and identify neuro toxins of clostridium

 SYSTEMATICS

Domain Bacteria
Phylum Firmicutes
Class Clostridia
Order Clostridiales
Family Clostridiaceae
Genus Clostridium
CLASSIFIACTION

 The clostridia can be divided into four major groups according to the kind of disease
they produce. They are as follows.
 The Histotoxic clostridia causes a variety of tissue (often muscle) infections
frequently following wounds or other trauma (eg).

C. chauvoei Cattle, sheep (pigs) Black quarter (Black leg)

C. septicum Cattle Malignant edema
Sheep B raxy
Chicken Necrotic dermatitis
C. novyi type A Sheep Big head of rams
Cattle and Sheep Gas gangrene
Type B Sheep (Cattle) Black disease (necrotic hepatitis)
Type C Water buffaloe Osteomyelitis

 Hepatotoxic clostridia produces their toxins in the liver, thus resulting in the disease
Bacillary haemoglobinuria and Black disease (Eg.).

C. haemolyticum ( C. Cattle, (sheep) Bacilliary haemoglobinuria

novyi type D)
C.sordellii Cattle, Sheep, Horses Gas gangrene
C. colinum Birds Quail disease,Ulcerative
enteritis
C. piliforme foals, laboratory foals, laboratory animals
animals Calves, dogs and cats
Calves, dogs and cats
  The Enterotoxigenic clostridium produces mainly enterotoxaemia and food poisoning
although they are occasionally histotoxic (Eg).

C. perfringens ( Humans Food poisoning, gas gangrene

C.welchii ) Type A Lambs Enterotoxaemic Jaundice
Broiler chickens (Yellow lambs disease) Necrotic
enteritis
C. perfringens Lambs (Under 3 Lamb dysentery
(C.welchii ) Type B weeks old)
C. perfringens ( Piglets, lambs, Haemorrhagic
C.welchii ) Type C calves and foals enterotoxaemia(Clostridial enteritis)
Broiler chickens Necrotic enteritis Struck
Adult sheep and
goat
C. perfringens Sheep(except Pulpy kidney disease
(C.welchii ) Type D neonates)
C. perfringens Calves and lambs Enterotoxaemia
(C.welchii ) Type E

 The Neurotoxic clostridia cause the disease by the production of the potent exotoxins
(Neurotoxins) (eg.)

C. tetani Tetanus
C. botulinum Botulism

FAMILY CHARACTERS

Classification

 They are pleomorphic, rod shaped; long filaments and involution forms are common.
 Spore formation occurs with varying frequency in different species.
 The shape and position of the spores vary in different species.
 The clostridia are motile with peritrichous flagella
except C.welchii and C.tetani typeVI. C.welchii is capsulated, while others are not.
 Clostridia are anaerobic. C.odematiens are strict anaerobes and die on exposure to
oxygen.
 C.histolyticum and C.welchii are aerotolerent and may even grow aerobically.
 The clostridia are fermentative, oxidase negative and catalase negative organisms.
 A very useful media for isolation of clostridia is Robertson‘s cooked meat broth.
 Clostridia grow in the medium, rendering the broth turbid most species produces gas.
 Saccharolytic species turn the meat pink – C.odematiens, C.septicum,
C.chauvoei and C.welchii.
  Proteolytic species turn the meat black and produce foul and pervasive odour -
C.tetani, C. botulinum, C.haemolyticum.
In litmus milk medium, the production of acid, clot and gas can be detected

HISTORY

 Tetanus has been known from very early times, having been described by
Hippocrates.
 But the knowledge of the disease was achieved only in 1884.
 Rosenbach –1886 - demonstrated a slender bacillus with round terminal spores in a
case of tetanus.
 Kitasato –1889 – isolated C.tetani in pure culture and reproduced the disease in
animals by inoculation of pure culture.
 The Greek term ―tetanus‖ which means ‗contracture‘ has been taken from the Latin
medicine ―rigor‖.

HABITAT

 Soil, especially that contaminated by animal faeces, is the natural habitat

as C.tetani is often transient in the intestines of horses and other animals.
 It is ubiquitous and has been recovered from a wide variety of other sources,
including street and hospital dust, cotton wool, bandages, catgut, plaster of paris,
clothing etc. It may occur as an apparently harmless contaminant in wounds.

MORPHOLOGY

 C.tetani is a straight, slender, Gram positive rod that characteristically produces a

terminal, spherical endospores that bulges the cell giving the characteristic drumstick
appearance. (The young spore may be oval rather than spherical).
 It occurs singly and occasionally in chains.
 It is non-capsulated and motile by peritrichous flagella.

CULTURAL AND BIOCHEMICAL CHARACTERISTICS

Cultural characters

 Very strict anaerobe, grows at an opt.temp. of 370C and pH7.4.

 It grows on ordinary media and the growth is improved by blood and serum and not
by glucose.
 Surface colonies are very difficult to obtain as the growth has a marked tendency to
swarm over the surface of the agar especially if the medium is moist.
 The swarming nature /spreading can be inhibited by increasing the concentration of
agar upto 3% (stiff agar).
 In this stiff agar individual rhizoid colonies are formed.
 On blood agar, it develops partially translucent, grayish colonies with filamentous
edges giving a fuzzy appearance.
 On horse blood agar, alpha haemolysis is produced, which later develops into beta
haemolysis due to the production of haemolysin (tetanolysin ).
 C.tetani grows well in Robertson‘s cooked meat broth, with turbidity.
 The meat is not digested, but is turned black on prolonged incubation.
 In gelatin stab cultures a fir tree type of growth occurs, with slow liquefaction.
 A greenish fluorescence is produced on media containing neutral red.
Biochemical properties

 C.tetani has feeble proteolytic property, so it does not ferment any sugars.
 It forms indole. It is MR and VP negative, nitrates not reduced.

RESISTANCE

 The endospores are highly resistant and while boiling kills the spores of most strains
in 15mts.
 Autoclaving at 121ºC for 15mts and dry heat temp of 150ºC for more than one hour is
completely sporicidal.
 Spores are able to survive in soil for years and they are resistant to most antiseptics.
 They are not destroyed by 5% phenol or 0.1% mercuric chloride solution in two weeks
or more.
 Iodine (1% aqueous solution) and H2O2 kill the spores within a few hours.

ANTIGENICITY

 Ten serological types have been recognized based on the flagellar antigen
(types I to X). Type VI contains non flagellated strains.
 All types produce the same neurotoxin- tetanospasmin. Which can be
neutralized by one common antitoxin.
 They have a common heat stable somatic antigen shared by all types.
 A second somatic antigen is shared by type II, IV, V and X.

TOXINS

 C.tetani produces atleast two distinct toxins.

o Tetanospasmin
o Tetanolysin
 They are antigenically and pharmacologically distinct and their production is
mutually independent.
 A third toxin – a nonspasmogenic peripherally active neurotoxin has been identified
recently.
 It is not known whether this plays any role in the pathogenesis of tetanus.

Tetanolysin

 It is a heamolysin or cytotoxin causing lysis of rabbit and horse RBC‘S.

 It is heat and oxygen labile, similar to those of streptolysin O, d toxin. (C.
oedematiens) and j toxin (C. welchii).

Tetanospamin

 It is a very potent neurotoxin responsible for the clinical manifestations of tetanus.

 It is oxygen stable, but relatively heat labile, being inactivated at 650C in 5mts.
 The highly purified form gets toxoided spontaneously.
 It is a good antigen and is specifically neutralized by the antitoxin. The toxin exists in
two forms.
 A monomer of MW 68000, which is toxic and a dimer is which is non toxic but
antigenic.
 The purified toxin is active in extremely small amounts and it contains about
3x107 minimum mouse lethal dose per mg protein.
  There is considerable variation in the susceptibility of different species of animals to
the toxin.
 The horses and human are the most susceptible.
 Guinea pigs, mice, goats and rabbits are susceptible in that descendingorder . Birds
and reptiles are highly resistant.

PATHOGENESIS

 C.tetani has little invasive power. The endospores enter traumatized tissue or surgical
wounds, especially after castration or docking, via the umbilicus or into the uterus,
following dystocia in cattle and sheep.
 The spore implanted in a wound can germinate and multiply only if the conditions
are favourable.
 Destruction and necrosis of tissue, lack of drainage in the area, presence of
extraneous matter especially of soil, all create anaerobic conditions and favour
germination of C.tetani spores.
 The resultant vegetative cells multiply at the site and produce the potent
tetanospasmin.
 This travels via peripheral nerves or blood stream to ganglioside receptors of the
motor nerve terminals and eventually to cells of the ventral horn of the spinal cord,
thus affecting many groups of muscles at various levels.
 The toxin acts presynaptically on motor neurons, blocking synaptic inhibition and
causing a spastic paralysis and the characteristic tetanic spasms.
 Tetanospasmin binds specifically to gangliosides in nerve tissue and once bound
cannot be neutralized by antitoxin.
 When toxin travels up to a regional motor nerve in a limb, tetanus first develops in
the muscles of that limb, then spreads to the opposite limb and moves upwards.
 This is known as ascending tetanus and is usually seen only in the less susceptible
animals such as dogs and cats.
 Descending (generalized) tetanus is the common form in susceptible species such as
human and horses.
 In this form toxin circulating in the blood stream affects the susceptible motor nerve
centers that serve the head and neck first and later the limbs. Once established, signs
of tetanus are similar in all animals.

PATHOGENICITY

Symptoms

 It is influenced by several factors, such as the site and nature of the wound, the dose
and toxigenicity of the contaminating organism.
 The incubation period is variable from 2 days to several weeks but is commonly 6-12
days.
 Initial symptoms include mild stiffness and unwillingness to move. This may proceed
to with head, neck and tail becoming rigid.
 Mild twitching of muscles develops into obvious spasms of muscles, which can occur
in response to sudden noises, animal fall over to one side and unable to rise.
 In the terminal stages the rigidity of muscles extend from the limbs to the trunk,
nostrils get dilated, earserect , nictitating membrane protruded and mastication
becomes impossible because the mouth cannot be opened – hence called Locked Jaw.
 Respiration becomes shallow and rapid before final respiratory failure.

Lesions::No characteristic lesion for this disease but there may be a superficial wound
which has developed from accidental injury or from surgery.
 DIAGNOSIS

Direct microscopy

 Demonstration of characteristic drumstick spores of C.tetani by Gram stained smears

of material from a wound (but it is not confirmative,
because C.tetanomorphum and C.tetanoides also produce drumstick spores).

Isolation

 Necrotic tissue from a wound or wound exudates can be heated to 800C for 20mts
and used to inoculate a blood agar plate and another blood agar plate containing stiff
agar.
 A tube of thioglycollate medium or cooked meat broth could also be inoculated and
sub cultured into blood agar.
 The plates are to be incubated anaerobically for 2-3 days. Growth is noticed using a
hand lens as a filamentous growth spreading through out the medium.
 The edges of this growth give pure culture on sub cultivation.
 Confirmation is done by identification of toxin.
 The toxin present in animal‘s serum or in filtrate from cooked meat broth or
thioglycolate medium can be inoculated in to mice S/C or I/M-ly and identified by
neutralization or protection tests using specific antitoxin.

CONTROL AND PREVENTION

 The disease is due to the action of the toxin, and hence, the obvious and most
dependable method of prevention is to build up antitoxic immunity by active
immunization.
 Theavailable methods of prophylaxis are
o Surgical attention
o Antibiotics
o Immunization – passive, active or combined.
 Surgical attention aims at removal of foreign bodies, necrotic tissue and blood clots,
inorder that an anerobic environment favourable for the tetanus bacillus is not
provided.
 Flushing with hydrogen peroxide in the wound area is produces aerobic conditions.
 Tetanus can be prevented by antibiotics (Large doses of penicillin) when
administered 4hrs after infection but not after eight hrs. Hence, prompt
administration is essential.
 Bacitracin or neomycin may be applied locally.
 Penicillin can be given as both injections and orally till healing is established.
Antibiotics have no action on the toxin.
 Antitoxin should be administered promptly, either i/vly or into the sub arachnoid
space, on three consecutive days to neutralize unbound toxin.
 Toxoid may be given subcutaneously topromote an active immune response even on
those animals, which have received antitoxin.
 For prevention, the farm animals should be vaccinated routinely with tetanus toxoid.

HISTORY

 The name botulism is derived from sausage (botulus, latin for sausage), an article of
food that used to be associated with the type of food poisoning.
 C.botulinum was first isolated by Van Ermengam (1896) from a piece of ham that
caused an outbreak of botulism.
 C. botulinum denotes a group of bacteria that produce extremely potent neurotoxins.
 These toxins cause botulism , a disease characterized by flaccid paralysis in many
animals and humans.
 Botulism is most common in water birds, ruminants, horses, mink and poultry.
 Botulism in animals has been called by a variety of names,
o Horses: Spinal typhus / Shaker foal syndrome
o Cattle: Lamsiekte, loin disease and contagious bulbar paralysis
o Water fowl: Limber neck, alkali poisoning and western duck sickness
 Botulism is rare in domestic cats. Pigs and dogs are relatively resistant.

HABITAT

 The endospores are widely distributed in soils and aquatic environment through out
the world.
 The disease botulism is mainly due to the ingestion of preformed toxin.
 Germination of the endospores, with growth of vegetative cells and production of
toxin, occurs in anaerobic situations such as contaminated cans of meat, fish or
vegetables, carcases of invertebrate and vertebrate animals, rotting vegetation and
baled silage.

MORPHOLOGY

 Gram +ve, straight rods, occur in single, pairs or occasionally in chains.

 Spores are oval, wider than the bacilli, situated centrally, terminally or subterminally.
 Non-capsulated, motile by peritrichous flagella.

CULTURAL AND BIOCHEMICAL CHARACTERISTICS

 It is a strict anaerobe, opt.temp is 30-370 C and pH is 7-7.6. Good growth occurs on

ordinary media.
 Surface colonies are large, irregular, and semitransparent with fimbriate border.
 Spores are produced consistently when grown in alkaline glucose gelatin media at 20
to 25 0C.
 In horse blood agar, large transparent colonies with irregular edges are developed
with a narrow zone of haemolysis.
 On sheep blood agar C.botulinum produce beta-haemolysis, the colonies are slightly
domed with a ragged edge.
 In cooked meat medium, the proteolytic strains type A, B and F produces blackening
of meat and non-proteolytic strains C, D and E do not blacken meat.
 Gelatin liquefied rapidly by type A&B and slowly or not at all by type C, D and E.

RESISTANCE

 Clostridium botulinum spores are highly resistant, surviving several hours at 1000C
and for upto 10mts at 1200C.
 But spores can be killed at 1210C for 15mts, while the toxins are destroyed at 1000C
for 20mts.
 ANTIGENS AND TOXINS

C.botulinum possesses a number of H, O and spore antigens

 Eight types of C.botulinum have been identified (Types A to G) based on the

immunological difference in the toxins produced by them.
 These neurotoxins are identical in pharmacological action but differ in potency,
distribution and antigenicity.
 They are neutralized only by the homologus antiserum.

Type Toxin Most Sources of toxin Disease

produced susceptible
animals

A A Humans, Vegetables, fruits, meat and Food borne

chickens, pigs fish botulism

Cα C1 Waterfowl Invertebrate carcases, rotting Limberneck in

vegetation and material on long necked birds
refuse dumps

Cβ C2 Cattle, horses, Carcases, baled silage, Forage poisoning

mink, dogs chicken manure as feed
supplement

D D Cattle, sheep Eating contaminated bones Lamsiekte

and carcases of small
mammals
(Phosphorus deficiency-Pica)

 The toxins differ from other exotoxins in that it is not released during the life of the
organism.
 It is appears in the medium only on the death and autolysis of the cell.
 It is believed to be synthesized initially as a nontoxic protoxin or progenitor toxin.
 Trypsin and other enzymes activate progenitor toxin to active toxin.
 The toxin is heat labile and its mol.wt. is 70,000.
 One mg of neurotoxins contains more than 120 million mouse lethal doses.
 The lethal dose for human is 1-2μg. This toxin acts slowly taking several hours to kill.

Comparison of the toxins of C.tetani and C.botulinum

C.tetani C.botulinum

Site of toxin Wounds Carcases, decaying vegetation and

production occasionally wounds and intestine
Mode of action Centrally by blocking Peripherally by blocking
synaptic inhibition neuromusculartransmission

Type of Spastic paralysis Flaccid paralysis

paralysis

Antigenic types Tetanospasmin (one Eight different toxins produced by types A-G
of toxin antigenic type)

PATHOGENESIS

 C.botulinum is non invasive and virtually non infectious. Botulism is of three types.

Food borne botulism

 It is due to the ingestion of preformed toxin in foodstuffs.

 The toxin is adsorbed from the intestinal tract and is transported via the blood
stream to peripheral nerve cells, where it binds to susceptible cells and suppresses
the release of acetylcholine at the myoneural junctions.
 This result in flaccid paralysis , death being caused by circulatory failure and
respiratory paralysis.

Wound botulism

 The spores are introduced into wounds where they germinate.

 Toxin is formed at this localized site and spreads through the body.
 The shaker foal syndrome in horse is thought to be caused in this way.

Infant botulism

 It occurs when spores are ingested in food and get germinate in the intestines when
the normal flora has not been fully established.
 This form is seen in human infants (Floppy baby syndrome) and as rare epidemics of
type C in broiler chickens and turkey poults.

PATHOGENICITY

Symptoms

 In cattle, the incubation period varies between 2-10 days depending upon the dose of
toxin ingested.
 Initially there will be excitation, followed by incoordination and paralysis of the hind
limbs.
 There will be paralysis of muscles in the mouth, pharynx and neck, resulting in the
animal being unable to swallow and the tongue protruding from the mouth. This is
followed by death.
 In South Africa this condition is termed as lamsiekte in cattle caused by type D,
especially in the phosphorus deficient animals.
  In poultry it results with the ingestion of type C toxin and the disease is known as
duck sickness or western duck disease and Limberneck in chicken.
 The symptoms include paralysis of the wings, legs and neck, protrusion of nictitating
membrane, diarrhoea and comatase before recovering in 5-6 days time.

Lesions

 Pathological changes are noticed in the CNS, especially the brain stem and
3rd ventricle, catarrhal gastroenteritis, hepatitis and nephrosis

DIAGNOSIS

 The diagnosis of botulism is based on history, clinical signs and demonstration and
identification of toxin in serum of moribund or recently dead animals as well as the
detection of toxin and /or C.botulinum in the suspected foodstuff.

Toxin demonstration

 Serum or centrifiuged serum exudates from animals can be directly inoculated i/v ly
(0.3ml) or i/p ly (0.5ml) into mice.
 If toxin is present the characteristic wasp waist appearance in the mice will be seen in
a few hrs or upto 5 days.
 The appearance is due to abdominal breathing because of paralysis of respiratory
muscles.
 Extraction of toxin in foodstuffs is accomplished by grinding the material in saline.
 The suspension is centrifuged and the supernatant is filtered through a 0.45μm filter.
 As the toxin can be in a protoxin form 9 parts of filtrate are treated with one part of
1% trypsin solution and incubated at 370C for 45mts.
 Mice or guinea pigs are inoculated intra-peritoneally.

Toxin identification

 Mouse (or guinea pig) neutralization tests using a polyvalent antitoxin initially,
followed by monovalent antitoxin.

Isolation of C.botulinum from foodstuffs

 Several samples of the foodstuffs are macerated in a small amount of physiological

saline.
 The suspension is heated at 65-800C for 30mts to kill most of the contaminating
organism and to induce the C.botulinum spores to germinate.
 Blood agar plates are inoculated with the suspension and incubated under Co2 at
350C for up to 5 days.
 To determine whether the isolate is a toxin producing strain, a cooked meat broth is
inoculated and incubated at 300C for 5-10 days.
 Filtrates are prepared and lab. animals can be used for demonstration and
identification of the toxin.

CONTROL AND PREVENTION

 Neutralization by polyvalent antiserum is effective.

  Therapeutic agents such as tetraethylamide and guanidine hydrochloride,
whichenhance transmitter release at neuromuscular junctions, may be of value
when given intravenously.
 Immunization is not followed. In South Africa attempts were made by giving two
injections of types C and D toxoid at an interval of several weeks.
 Bivalent & Trivalent antitoxins areavailable .

CHAPTER-6: ENTEROTOXIGENIC CLOSTRIDIA

Learning objectives

To know in detail about,

 Classificaiton of Cl.perfringens
 Diseases caused by Clostridium perfringens in domestic animals and poultry
 Morphology cultural and biochemical characteristics of Clostridium perfringens
 Stormy fermentation and Nagler's reaction
 Enterotoxaemias, struck, pulpy kidney disease and necrotic enteritis
 General approaches used to demonstrate and identify toxins of enterotoxigenic
clostridia

HISTORY

 Clostridial enterotoxaemias are acute, highly fatal intoxications that affect sheep,
lambs, calves, piglets and occasionally foals.
 The diseases are caused by the major exotoxins (enterotoxins) of Clostridium
perfringens types B, C, D and occasionally types A and E.
 The bacillus was originally cultivated by Achalme (1891), but it was first described in
detail by Welch and Nuttal (1892)-who isolated it from the blood and organs of
cadaver.

HABITAT

 Based on toxin production Clostridium perfringens organisms are classified

into 5 types (A to E).
 Clostridium perfringens Type A occurs in the intestinal tract of human and
animals and in most soils.
 Type B to E are more adapted to survival in the intestines but in outbreaks of
disease they survive long enough in soil to infect other animals

MORPHOLOGY

 Gram +ve, bacillus, straight, parallel sides, rounded or truncated ends, occur either
singly or in chains or small bundles.

 It is highly pleomorphic, filamentous and involution forms are common. It is

capsulated and non-motile .
 The spores are oval, sub-terminal and bulge the mother cell.They are rarely produced
and their absence is one of the characteristic morphological features of C.welchii.

CULTURAL AND BIOCHEMICAL CHARACTERISTICS

 It is an anaerobe, but can also grow under micro aerophilic conditions.

 Oxygen is not actively toxic to the bacillus and cultures do not die on exposure to air.
 Though, this bacillus is grown at 370C, pH 5.5-8.0, the temperature of 450C is optimal
for many strains.
 The generation time at this temperature is ten minutes only.
 This property can be utilized for obtaining pure cultures of C.welchii.
 Robertson's cooked meat broth inoculated with mixtures of C.welchii and other
bacteria and incubated at 450C for 4-6hrs serves as enrichment.
 Subcultures from this onto blood agar plates yield pure or predominant growth
of C.welchii.Good growth occurs in Robertson‘s cooked meat medium.

 The meat is turned pink but it is not digested.

 In litmus milk, fermentation of lactose leads to formation of acid, which is indicated
by the change in the color of litmus from blue to red.
 The acid coagulates the casein (acid clot) and the clotted milk is disturbed due to the
vigorous gas production.
 The paraffin plug is pushed up and shreds of clot are seen sticking to the sides of the
tube. This is known as stormy fermentation. (Click here for visual)
 After overnight incubation on rabbit or sheep blood agar, colonies of most strains
show a target haemolysis resulting from a narrow zone of complete haemolysis due to
theta toxin and a much wider zone of incomplete haemolysis due to alpha toxin. This
double zone haemolysis pattern is characteristic for C.welchii.

 All the five types of C.welchii (A –E) produce alpha toxin.

 This is a lecithinase C, which, in the presence of calcium and magnesium ions splits
lecithin into phosphotidyl choline and diglyceride.
 This specific lecithinase effect can be demonstrated by Nagler‘s reaction.
 Type A antitoxin is spread over half of an egg yolk agar plate and allowed to dry.
 The suspect C.perfringens is streaked across both sides of the plate. Click here for
visual

 All the types of C.perfringens produce the alpha toxin, that is a lecithinase.
 On the half of the plate without the antitoxin, the lecithin in the medium is attacked
causing opalescence around the streak.
 The lecithinase reaction is neutralized on the other half of the plate with the antitoxin
but the growth of C.perfringens is unaffected.
 C.welchii ferments several sugars (glucose, maltose, lactose and sucrose) and
produces acid and gas. Indole –ve, MR +ve, VP-ve, H2S +ve.

RESISTANCE
  Spores are usually destroyed within 5 minutes by boiling, but those of the food
poisoning strains of type A and type C resist boiling for 1-3 hrs.
 Autoclaving temp is lethal.

TOXINS

Classification

 C.welchii is one of the most prolific of toxin producing bacteria forming at

least 12 different toxins , besides many other enzymes and biological active
substances.

C. perfringens Major toxins

Type Alpha Beta Epsilon Iota
A + - - -
B + + + -
C + + - -
D + - + -
E + - - +

 Alpha toxin is produced by all types. Mostly by type A strains. It is lethal,

dermonecrotic and haemolytic.
 This is a lecithinase C (phospholipase) that attacks cell membranes causing
cell death and destruction and also responsible for Nagler‘s reaction .
 It is haemolytic for the red cells of most species except horse and goat. This
toxin gives a zone of partial haemolysis on blood agar.
 The haemolysis is of hot-cold variety being best seen after incubation at 370C
followed by chilling at 40C.
 Beta toxin is lethal and necrotising. It is sensitive to trypsin and this explains
the predilection of types B and C for neonates as colostrum has anti trypsin
activity.
 It is a labile toxin and may be destroyed if there is a delay in small intestinal
contents, containing the toxin, reaching the laboratory.
 Epsilon toxin is secreted as a protoxin (proto toxin) and is activated in the
intestines by proteases such as trypsin.
 Pulpy kidney disease is not usually seen in neonatal lambs as colostrum
contains an antitrypsin factor that can prevent the epsilon toxin being
activated.
 The toxin itself increases gut permeability, assuring absorption of the toxin
into the blood stream.
 It damages vascular endothelium (including blood vessels in the brain)
leading to fluid loss and edema.
 This epsilon toxin can be regarded as an enterotoxin and neurotoxin.
 Iota toxin is also produced as a protoxin and is not unique
to C.perfringens type E as it is also formed by C.spiroforme and C.difficile
 Besides several minor toxins are produced such as the theta (haemolysin),
 Kappa (collagenase), lambda (Proteinase), Mu(hyaluronidase) and
Nu(DNase) – all these may contribute to tissue damage.
 Based on the type of toxin productions, Clostridium perfringens are classified
into 5 types

C.perfringens types Major Host Disease

toxins
A Enterotoxins Human Food poisoning
Alpha Lambs Enterotoxaemic jaundice
Broiler chickens Necrotic enteritis
B Beta and Lambs under 3 Lamb dysentry
alpha weeks old
C Beta and Piglets 1-3days Haemorrhagic enteritis
alpha old (Clostridial enteritis)
Broiler chickens Necrotic enteritis
(2 weeks old)
Adult sheep and Struck
goats
D Epsilon Sheep all ages Pulpy kidney disease
(except (over eating disease)
neonates)
E Iota and Calves and lambs Enterotoxaemia
alpha (Haemorrhagic enteritis)
PATHOGENESIS

 The enterotoxaemias are often precipitated by certain husbandry and environmental

factors such as abrupt changes in feeding, usually to a richer diet and overeating and
voracity on high protein and energy rich feeds.
 This leads to slowing of peristalsis with retention of bacteria in the intestines,
absorption of toxins, inadequately digested carbohydrate and the provision of a rich
medium for the proliferation of C.perfringens
 The bacterium inhabits the large intestine in normal animals, but if overgrowth
occurs C.perfringens can spill over into the small intestine with the production of a
large amount of toxin and enterotoxaemia.

 PATHOGENECITY

Disease Clinical and postmortem signs

Food poisoning Sudden onset, diarrhoea, abdominal pain and nausea. But vomiting is
uncommon. Short course and rarely fatal

Enterotoxaemic jaundice Depression, anaemia, icterus, haemoglobinuria and lambs die within 6-12hrs
 of first signs known as the yellows or yellow lamb disease

Lamb dysentry A haemorrhagic and rapidly fatal enterotoxaemia. Lambs are often found
dead

Haemorrhagic enterotoxaemia Dysentery, collapse and death. Small intestine is dark red and has gas
(Clostridial enteritis) bubbles in mucosa

Necrotic enteritis in broilers Depression, diarrhoea, death in a few hrs. Mortality 2-50%. Mucosa of small
intestine has a brown psuedomembrane. Most common in deep litter units.

Struck Sudden death due to an enterotoxaemia

Pulpy kidney disease (Over Odema of brain, glycosuria, sudden death. Excess fluid in body cavity, focal
eating disease) symmetrical encephalomalacia occurs in well-grown lambs.

DIAGNOSIS

 Gram stained smears can be made from the mucosa of the small intestine of a
recently dead animal.
 Large numbers of Gram-positive rods are suggestive of C.welchii.
 Saccharolytic in Robertson's cooked meat media, opalescence in egg yolk agar,
haemolysis on blood agar, stormy clot fermentation and sugar fermentation tests are
helpful for identification.
 Histopathology on brain sections helps to demonstrate focal symmetrical
encephalomalacia in pulpy kidney disease.
 Rapid kidney autolysis, pulpy cortical softening and Glucosuria are suggestive of
pulpy kidney disease.

Demonstration of toxin in the small intestine

 Collect 20-30ml of ileal contents from a recently dead animal and send it to the
laboratory as soon as possible.
 The ileal contents are centrifuged and the clear supernatant is tested for toxin.
 In ileal contents, the epsilon and iota toxins are usually in the active form.
 To demonstrate the toxin 0.4ml of the clarified ileal content can be inoculated i/v ly
into mice.
 If mouse dies within 5 mts this is probably due to shock. Death from toxin usually
occurs within 10hrs.
 Identification of toxin in the clarified ileal content is carried out by a neutralization
test by using suitable antitoxin.

CONTROL AND PREVENTION

 Before the lambing season the ewes are vaccinated with formalised whole culture or
alum precipitated vaccine.
  The resulting passive immunity, with unweaned lambs, derived from colostrum
protect lambs for first 3 weeks of life.
 Similarly alum precipitated trypsin –treated toxoid is also satisfactory.
 Lambs can also be vaccinated by giving the first dose within 72 hrs of birth and
repeated at 4 weeks of age.
 Immunity may not last more than 6-12 months unless booster dose is given.

CHAPTER-7: HISTOTOXIC CLOSTRIDIA

Learning objectives

To know in detail about,

 Diseases caused by histotoxic clostridia in domestic animals

 Morphology, cultural and biochemical characteristics of Cl.novyi, Clostridium
septicum and Clostridium chauvoei.
 Pathogenesis of Histotoxic Clostridia
 Describe antigens and toxins of Cl.novyi.
 B raxy, black quarter and gas gangrene
 Difference diagnostic methods for histo toxic clostridia

HABITAT

 World wide in distribution. The principal habitat of C.novyi (also called

as Clostridium oedematiens) is soil and the intestine of animals.
 Based on toxin production Clostridium novyi is classified into four types (A-D).
 The type A is commonly found in soil. Type B is rarely found in soil, and it is common
in the normal intestinal tract of herbivores.
 Strains of type A and B are recovered from the livers of normal animal. Type D (C.
haemolyticum)- is found in the ruminant digestive tract, liver and in the soi

MORPHOLOGY, CULTURAL AND BIOCHEMICAL

CHARACTERS

 Large, pleomorphic, Gram+ve rods with oval to cylindrical subterminal spores are
characteristic.
 There is little or no swelling of the mother cell, non-capsulated, motile by
peritrichous flagella.
 Clostridium novyi type B and C.haemolyticum are very demanding in both their
anaerobic and nutritional requirements.
 Very strict anaerobic procedures are necessary and media containing cysteine should
be used. These clostridia can die within 15mts of being exposed to atmospheric O2 .
 These organisms are difficult to grow on primary culture and the growth isenhanced
by agar enriched with glucose or freshly prepared blood or fresh brain infusions.
 On blood agar C.novyi produces characteristic large, irregular colonies with a rhizoid
edge and a large zone of clear haemolysis.
 On moist surface of solid media after 3-4 days of incubation colonial motility
develops which is characterized by the movement of daughter colonies moving away
from parent colonies in spirals or arc and few return and fuse to the parent colony.
 In horse blood agar, the colonies are haemolytic, small and usually rhizoidal in
nature.
 Areas of hemolysis develop beneath the colonies and develop into wider zone after
48-72hrs incubation.
 In Sheep blood agar very slight haemolysis develop. In Robertson‘s cooked meat
medium C.novyi type D is very strongly proteolytic.
 Type A, B and C are saccharolytic. The lecithinase activity of beta toxin of type B and
D, and Gamma toxin of type A produces quite distinct opacity changes on egg-yolk
agar.
 C.novyi type A exhibits lipase activity on egg-yolk agar.
 It will produce characteristic iridescent pearly layer on the surface of the colonies,
extending on to the surface of the medium immediately surrounding them.
 C.novyi type A is the only species among clostridia that produces both a lecithinase
and a lipase.
 Saccharolytic type ferment glucose and maltose but not lactose.

RESISTANCE

 Spores of most strains survive heating to 950C for 15mts. But are killed at
autoclaving.
 Spores are resistant to 5% phenol, 10% formalin or 0.1% merthiolate.
 They are killed rapidly by exposure to hypochlorite.
 Spores remain viable for years in soil.

TOXINS

 C.novyi possess several somatic and flagellar antigens, which are not of
muchimportance .
 Based on toxin production C.novyi is classified into 4 types.
 C.novyi synthesizes five major toxins, α, β, γ, δ and ε.

Alpha Beta

C. novyi type A + -

Type B + +

Type C - -

Type D C. haemolyticum - +++

 Type A produces all toxins except beta. Type C isolates are non toxigenic.
 In addition to this type B also produce zeta, eta and theta toxin.
o Alpha toxin produced by type A and B is lethal, necrotizing, causes increased
capillary permeability, and is toxic to several tissues including muscle, heart
and liver.
o Beta toxin is a lecithinase and produced by type B and by C.haemolyticum in
greater amounts.
o This may account for the haemolytic crisis and death in bacillary
haemoglobinuria.
o Gamma toxin is a necrotising phospholipase D.
 o Delta toxin is an oxygen labile haemolysin.
o Epsilon toxin is a lipolytic enzyme.
o Zeta toxin is partly haemolytic.
o Theta toxin is a lipase.
o Eta toxin is a tropomyosinase, which degrades tropomyosin and myosin and
may play a role in destruction of infected muscles.

PATHOGENESIS

 In black disease and bacillary haemoglobinuria, the spores, normally present in the
intestine, may reach the liver and remain dormant in the kupffer cells.
 Any destruction of liver tissue could be the initiating factor.
 The tissue damage is usually due to migration of immature liver fluke
(Fasciola hepatica), and anaerobic conditions permits germination of spores, growth
of vegetative cells and subsequent production of toxin.
 Alpha toxin produced in the local area of necrosis and in the liver is adsorbed into the
circulation and results in systemic effects.
 In case of bacillary haemoglobinuria the dominant toxin is beta toxin.
 Big head in rams develop when sub cutaneous tissues traumatized during fights are
subsequently invaded by C.novyi type A.
 The oedema is the result of vascular damage inflicted by the alpha toxin.

PATHOGENECITY

Symptoms

 Big head
o Odematous swelling occurs in head, face and neck.
o It will be followed by collapse and death of animals.
o The mortality rate may be more than 90%.
 Black disease
o Acute toxaemia leads to sudden death.
o The signs include rapidly decreasing ability to move, unsteady gait and
collapse.
 Bacillary haemoglobinuria
o Common in summer months, affected animals suffer from fever, abdominal
pain, port-wine coloured urine , diarrhoea and haemoglobinuria. The
mortality rate is 90%.

Lesions

 Black disease
o Number of clearly defined gray-yellow foci (necrotic areas) in the liver.
o The lesion consists of a central core of necrosis surrounded by a zone of
leucocytes in which there will be masses of C.novyi. Excess fluid in body
cavities.
o Straw-coloured exudates will be present in pericardial and peritoneal cavities.
o Extensive subcutaneous and bloodstained odema can be noticed in the
carcass.
o Venous congestion occurs that darkens the skin(Black disease).
 Bacillary haemoglobinuria:
o Number of typical anaemic infarcts in the liver.
o Pale and raised areas surrounded by a blue-red zone.
 o There will be blood stained intestinal contents, dark colored urine in the
bladder, marked icterus of the carcass, widespread odema and haemorrhages
in the myocardium

DIAGNOSIS, CONTROL AND PREVENTION

 Based on history
 Direct Gram stained smears
o Presence of characteristic liver lesions together with large number of Gram
+ve rods in liver impression smears from a recently dead animal is suggestive
of the disease.
 Flourescence Antibody Test is useful for the identification of C.novyi type B
and C.haemolyticum in acetone fixed liver impression smears.
 Isolation of organism from affected tissue (as like other clostridial infections) and by
characteristic cultural characters.
 Animal inoculation
o Toxin in the liver can be demonstrated by intra muscular injection of
homogenates into guinea pigs.
o The pathogenicity isenhanced if the homogenate is added to an equal
amount of 5% CaCl2 solution before inoculation.
o The guinea pigs die in 1-2 days with very extensive subcutaneous edema.
Specific antitoxin is not readilyavailable for neutralization tests.

Control and prevention

 Elimination of liver flukes through destruction of snail.

 Aluminum hydroxide adsorbed formalized whole culture vaccines are available.
 Outbreaks of the disease may be controlled by the prompt injection of hyper immune
sera.

HISTORY AND HABITAT

 Clostridum septicum is very closely related with Clostridum chauvoei, hence it is

called as Clostridum chauvoei type A.
 The bacillus was first described by Pasteur and Jourbert (1887) and named it as
Vibrion septique .
 Clostridum septicum causes
o Malignant odema in Cattle, sheep and pigs
o Braxy (Bradsot) in Sheep
o Necrotic dermatitis in Chickens
 It is found in soil and the intestine of animals.

MORPHOLOGY, CULTURAL AND BIOCHEMICAL

CHARACTERS

 Gram +ve, highly pleomorphic, chracteristic long filamentous forms are seen in
stained smears of affected muscle.
 Cigar shaped rods and citron forms are more common. Spores are oval, central or
subterminal. Non-capsulated, motile by peritrichous flagella.
 Strict anaerobe, growth at an opt.temp of 370C, growth ispromoted by glucose.
 On ordinary media, the colonies are irregular and transparent initially, turning
opaque (large, grayish white on continued incubation).
 The colonies are swarming and spreading over the entire surface.
  On stiff agar, the colonies are irregular with a rhizoid edge. Some strains produce
smooth, round colonies.
 In cooked meat medium meat turns pink with rancid odour, and produces abundant
gas (because, it is saccharolytic).
 Like, C.perfringens, the C.septicum inoculated into litmus milk produces the classical
stormy clot or stormy fermentation reaction.
 Ferment glucose, lactose, maltose and salicin but not sucrose. Acid and gas are not
produced.

ANTIGENS AND TOXINS

 Six groups have been recognized, based on somatic and flagellar antigens.
 Clostridum septicum produces at least four distinct toxins and fibrinolysin.
o The α toxin is oxygen stable haemolysin, dermonecrotic and lethal.
o The β toxin is leucotoxic and DNAse.
o The γ toxin is a hyaluronidase.
o The δ toxin is an oxygen labile haemolysin.
 α toxin has direct effect on cardiac muscle and is capable of causing capillary damage.
 Iron is required both for growth of bacteria and for production of α toxin.

PATHOGENESIS

Braxy in sheep

 The disease is more common in winter months.

 When animals ingest large volume of frozen grass, the spores that are present in the
soil are ingested with feed.
 The mucosa of the abomasum is damaged due to cold conditions from an adjacent
rumen.
 Any C. septicum spores present can germinate and replication of the bacterium leads
to toxin production, toxaemia and rapid death.

Malignant odema (Anaerobic cellulites)

 In this exogenous gas gangrene infection, spores are introduced into wounds where
they may germinate in the anaerobic necrotic material and toxin is produced by the
vegetative cells.

PATHOGENECITY

Symptoms

 Braxy usually occurs in well-nourished one-year-old sheep, ailing animals show signs
of abdominal pain and diarrhoea. Death occurs within few hours.
 In malignat odema, infected wound, become gas gangrenous.
 Fever, soft swelling around wound spreading to muscles.
 Swelling odematous and wet with much exudates and gas. Muscles appear dark red to
black color.

Lesions
  In braxy, the lesion may be confined to the abomasum; there will be the characteristic
area of haemorrhagic inflammation in the wall of the abomasum.
 There will be an extensive quantity of blood stained fluid in the peritoneal cavity.

DIAGNOSIS, CONTROL AND PREVENTION

 Based on history, symptoms and characteristic lesions strongly suggestive of this

disease.
 FAT is to be employed for differentiate it from C.chauvoei .
 Identification of specific clostridial toxin by mixing 1.2ml of culture fluid with 0.3ml
of specific antitoxin, allowing the mixture to stand for 30 minutes at 370C and
inoculate two guinea pigs intra peritoneally. If specific toxin present it will be
neutralized.
 Penicillin alone or with hyper immune serum can be used to treat infections.
 Sheep can be effectively immunized against the disease and multi component vaccine
is used.

MORPHOLOGY, CULTURAL AND BIOCHEMICAL

CHARACTERS

 Worldwide distribution in soil and pastures. C. chauvoei (also called as

C. chauvoei type B, C. feseri) causes black quarter or black leg in Cattle & Sheep.
 Gram positive, rod shaped with rounded ends 3-8mm in length & 5mm in width.
 Sometimes pleomorphic, large cigar shaped rods or citron forms occur.
 Non-capsulated and motile by peritrichous flagella. Non-motile variants do occur.
 Spores are oval and located centrally or subterminally. Old cultures stain Gram
negative.
 Strict anaerobe. Growth occurs at an optimum temperature of 370C.
Growthenhanced by the addition of liver extract or glucose.
 In blood agar whitish grey colonies with irregular edges develop, surrounded by a
zone of haemolysis.
 In cooked meat medium growth is slow and meat is turned pink with sour odour
 C. chauvoei ferments glucose, lactose, sucrose, maltose with acid & gas, but not
salicin.

ANTIGENS AND TOXINS

Classification

 C. chauvoei has somatic and flagellar antigens and produces 4 toxins

o Alpha toxin - oxygen stable haemolysin, necrotoxin that causes
dermonecrosis and fibrinolysis
o Beta toxin – DNA ase
o Gamma toxin – hyaluronidase
o Delta toxin – Oxygen – labile haemolysin.
o This toxin is lethal for mice & guinea pigs when given I/v.

PATHOGENESIS
 PATHOGENECITY

Symptoms

 The disease usually occurs in young cattle of 6 months to about 2-3 years of age.
 The most obvious sign is crepitating swelling particularly in the hind or fore quarter
which rackels when rubbed with the fingers as a result of gas production.

 The affected animal will become lame and the affected muscles shows trembling with
violent twitching. Death usually occurs within 24 hours.
 In sheep an acute febrile condition develops within 1-2 days following aninjury and
a typical black quarter lesion can be observed at the site. Death occurs suddenly

Lesion

 In the central part of the lesion there is usually a well-defined area of muscle, which is
dark red in colour, dry, necrotic and filled with small gas bubbles, which give a
swollen appearance to the muscles.
 The lesion has a characteristic rancid odour. Surrounded this area of muscle there
will be yellowish or blood stained oedematous fluid.
 In ewes there will be necrosis of the vaginal mucosa and skin with extensive oedema
involving the hind limbs and thigh muscles.
 DIAGNOSIS, CONTROL AND PREVENTION

 Based on History
 Based on Symptoms - The most obvious sign is crepitate swelling particularly
in the hind or fore quarter which rackels when rubbed with the fingers as a
result of gas production
 Smears prepared from the lesions and oedmatous fluid reveal Gram positve
rod.
 Isolation can be done from the center of the lesion, oedematous fluid and from
heart blood & spleen.
 FAT used to differentiate from C. septicum
 Broth cultures or oedematous fluid from the lesions can be tested for toxicity
and specific neutralization by antitoxin in mice or guinea pigs.
o The most reliable results are obtained from using a formalized alum
precipitated whole culture that confers immunity against the bacteria
as well as the toxin.
o For economic reasons a multi-component vaccine containing C.
chauvoei, C. septicum, C.welchii type D and C. tetani is used.
o A stronger immunity is stimulated by two doses of vaccine at a time
interval of at least 2-3 weeks

Control and prevention

 Hyper immune serum (HIS) is used to control explosive outbreaks. Penicillin

along with HIS is used to treat the disease.
 Oxytetracycline & Chlortetracycline can also be employed effectively in early
stages.

CHAPTER-8: LISTERIA
Learning objectives
To know in detail about,

 Disease syndromes of pathogenic Listeria sp in animals

 Morphology and cultural characteristics of Listeria sp
 Pahogenesis of circling disease in cattle
 Different forms of listeriosis in cattle
 Tumbling motility, Anton's test and cold enrichment

SYSTEMATICS
Domain Bacteria

Phylum Firmicutes

Class Bacilli
 Order Bacillales

Family Listeriaceae

Genus Listeria

 Listeria has been divided into seven species with two distinct groups.
 Among which the Listeria monocytogenes and Listeria ivanovii are haemolytic and
pathogenic for animals.
 The Listeria murrayi and Listeria grayi are nonhaemolytic, rarely isolated and
considered to be non pathogenic.
 Among which the genus L. monocytogenes, is the cause of septicaemia, abortion and
CNS infections in a wide range of animal species including humans.

HISTORY

 L.monocytogenes first described by Murray (1926) who named it as bacterium

monocytogenes because of characteristic monocytosis infection in laboratory
animals.
 It was renamed Listerella hepatolytica by Pirie (1927) and the present name given by
him in 1940.
 The Listeria monocytogenes was first isolated by Gill (1929) from sheep.

HABITAT

 Listeria species are widely distributed in the environment and can be isolated from
soil, faeces, plants, decaying vegetation and silage (pH 5.5) in which the bacteria can
multiply.
 Silage is commonly implicated in outbreaks of listeriosis in cattle and sheep.
 In poor quality sailage the listerial numbers may reach 107 cfu/kg of silage.
 Asymptomatic faecal carriers occur in man and many animal species.
 L.monocytogenes can be excreted in bovine milk.
 Human foods associated with listeriosis in man include soft cheeses, milk and poultry
meat.

MORPHOLOGY AND CULTURAL CHARACTERISTICS

Morphology

 L.monocytogenes are medium sized, Gram+ve rods, non-spore forming and non-acid
fast.
 Old cultures stain Gram –ve. From rapidly growing cultures or animal tissues the
cells can appear coccal.
 They are motile by few (1-5) peritrichous flagella. They are motile at room
temperature, but not at 37°C.

Cultural characteristics

 L.monocytogenes is able to grow at temperature ranges from 4 to 45°C and grow at

pH range of 5.5 to 9.6.
  It is relatively resistant to high salt (10%) concentrations. They are facultative
anaerobes.
 The growth isenhanced by agar enriched with glucose, blood, liver extract and by
10% Co2.
 They grow on nutrient agar, blood agar but not on MacConkey agar.
 Small transparent colonies with smooth borders appear on blood agar in 24hrs,
becoming grayish white in 48hrs.
 L.monocytogenes and other non-pathogenic listeria produce narrow zones of beta
haemolysis, often only under the colony itself.
 L.ivanovii produces a comparatively wide zone of haemolysis and is very similar in
appearance to beta haemolytic streptococci.
 L.monocytogenes produces a CAMP reaction with the haemolysis of S.aureus.
 In contrast, Listeria ivanovii is negative in the CAMP reaction with S.aureus.
 On TSA or BHI agar, these colonies have a characteristic blue-green sheen when light
is reflected obliquely at a 45° angle off their surface.
 In fluid medium, slimy tenacious precipitate forms after incubation for several days.
 L.monocytogenes, particularly shows the characteristic tumbling motility when a 2-4
hr broth culture, incubated at 25°C, is examined by the hanging drop method.
 This motility is an end-over-end tumbling of individual cells with periods of
quiescence.
 When grown in semisolid motility media the Listeria spp. give an unusual umbrella
shaped growth in the subsurface.

BIOCHEMICAL PROPERTIES, RESISTANCE, ANTIGENS AND

TOXINS

Biochemical properties

 All the Listeria species hydrolyse aesculin, Catalase +ve, Oxidase –ve, Indole –ve.
 They produced acid from glucose and rhamnose, but not from xylose and mannitol.
Nitrates not reduced.

Resistance

 It is killed by moist heat at 55°C for 40 minutes and is readily susceptible to the lethal
effects of disinfectants.
 Under natural conditions, in summer they survive for 1 month and in winter for 3-4
months.

Antigens and toxins

 Based on somatic and flagellar antigens, so far 16 serovars have been identified.
 Of these 16 serovars, all cases of animal and human infections are caused by 3
serotypes. ½ a, ½ b and 4 b.
 Numbers indicate O antigen and alphabet indicate H antigens.

PATHOGENESIS

 In both cattle and sheep, listeriosis can manifest itself in four ways
o as a CNS infection (meningo encephalitis in adults and meningitis in the
young)
o as abortion
o as a generalized septicaemia with involvement of the liver and other organs
 o as mastitis in dairy cattle.

Flow Chart

 Silage is commonly implicated in outbreaks of Listeriosis in cattle and sheep. Most

pathogenic bacteria require theavailability of iron in the host for metabolic
activities.
 High iron levels in silage that lead to elevated tissue concentrations of iron may
predispose cattle and sheep fed on silage to Listeriosis.
 The pathogenic listeria can penetrate the epithelial barrier in the intestine and
multiply in hepatic and spleenic macrophages aided by the haemolysin named
listeriolysin O. It leads to septicaemia.
 If the pathogen penetrates through damaged mucosal surfaces to the CNS, via the
trigeminal nerve or an alternate route it may penetrate through the dental pulp
(when sheep are cutting or losing teeth) to the CNS, resulting in neural form.

PATHOGENECITY

Symptoms

 Four syndromes
o Subclinical
 Infections are the most common form of infection.
 Usually outbreak occurs when fed with poor quality, high pH silage,
particularly during cold weather.
o Neonatal infections
 Characterised as visceral infections with a septicemia.
 Often gastroenteritis and bilateral meningitis.
 Deaths are frequent in neonatal animals.
o Listerial abortion
 It is a sporadic condition in cattle and sheep.
 It occurs after 4-8 months pregnancy. Retained placenta is common.
o Neural Listeriosis (circling disease):
 The incubation period ranges from 14 to 40 days.
 The disease is more common in winter or early spring.
 The clinical presentation of meningoencephalitis in adult ruminants
may begin with signs of depression and confusion.
 The ears droop; animal holds its head to one side.
 Protrusion of the tongue and salivation are common and twitching or
paralysis of the facial and throat muscles may occur.
 When the animal moves, it tends to be in a single direction, giving rise
to the common name of circling disease.
 In the terminal stages, the animal may fall and will be unable to rise.
o In poultry, there are signs of
 Torticolis
 Weakness
 Incoordination of legs and
 Sudden death in young birds
 The disease is usually fatal in sheep, pigs and horses.

Lesions

 Micro-abscess in the brain stem, usually unilateral, together with perivascular cuffing
is very characteristic of listeriosis.
  The lesions are most common in the mid brain, pons and medulla oblongata.
 In addition to this there will be generalized septicaemia, focal necrosis of the liver
and spleen will be seen.

DIAGNOSIS

 Stained smears from lesions may reveal Gram +ve rods (often coccobacillary)
 Histopathological examination of brain tissue can often give a presumptive diagnosis
of neural listeriosis
 Isolation and Identification
o Inoculation of specimens on selective media include blood agar with an
antibiotic supplement or blood agar containing 0.05% potassium tellurite
(inhibitory to Gram –ve).
o Specimens from the visceral form of the disease or from abortion cases are
inoculated directly onto the laboratory media.
o A cold-enrichment procedure is necessary for brain tissue from neural
listeriosis.
o Small pieces of spinal cord and medulla are homogenized and a 10%
suspension is made in a nutrient broth.
o The broth suspension is placed in the refrigerator at 40C and sub cultured on
to blood agar once weekly for upto 12 weeks.
 Inoculation in developing chicken embryos causes development of focal necrotic
lesions on the chorio allantoic membrane.(CAM)
 Anton‘s test
o Inoculation of live bacterial suspension into the conjunctiva of a rabbit or
guinea pig only L.monocytogenes causes a purulent keratoconjunctivitis
within 24-36hrs of inoculation.
 Intra peritoneal inoculation of mice with a 24hr broth culture.
o Both L.monocytogenes and L.ivanovii are pathogenic for mice.
o They die within in 5 days with necrotic lesions present in the liver.

CONTROL, PREVENTION AND PUBLIC HEALTH

SIGNIFICANCE

Specimens to be collected

 Visceral form
o Material from lesions in liver, kidneys or spleen
 Neural form
o Spinal fluid, brain stem, and tissue from several sites in the medulla
oblongata
 Abortion
o Placenta (cotyledon), foetal abomasal contents and/or uterine discharges.
 Ruminants in early stages of septicaemic listeriosis respond to systemic therapy with
ampicillin or amoxicillin.
 Response to antibiotic therapy may be poor in neural listeriosis although prolonged
higher doses of ampicillin or amoxicillin combined with an aminoglycoside may be
effective.
 Ocular listeriosis requires treatment with antibiotics and corticosteroids injected
subconjuctivily.
 Poor quality silage should be avoided. Vaccination with killed vaccines, which do not
induce effective cell-mediated immune response, is not protective
because L.monocytogenes is an intracellular pathogen. Live, attenuated vaccines,
 which contain serovars 1/2a, 1/2b, and 4b are reported to reduce the prevalence of
listeriosis in sheep.

Public health significance

 L.monocytogenes causes meningoencephalitis, meningitis, encephalitis and uterine

infection with abortion, stillbirths, granulomatosis and valvular endocarditis in
humans.
 The source could be soil, contaminated milk, cheese, meat, vegetables and human
carriers.
 Infection is frequently associated with immuno-compromised persons.

CHAPTER-9: ERYSIPELOTHRIX

Learning objectives

To know in detail about,

 Principal host and diseases caused by E.rhusiopathiae

 Morphology, cultural and biochemical characteristics of E.rhusiopathiae
 Different forms of diseases cauesd by E.rhusiopathiae in swine
 Clinical symtoms and laboratory findings to a possible diagnosis of diamond skin
disease in pigs
 Explain bottle brush type growth of E.rhusiopathiae in gelatin stab culture

 SYSTEMATICS

Domain Bacteria

Phylum Firmicutes

Class Mollicutes

Order Incertae sedis

Family Erysipelotrichaceae

Genus Erysipelothrix

Species Erysipelothrix
rhusiopathiae

HABITAT
 The bactrerium is widespread in nature and has been recovered from a wide variety
of wild and domestic animals including mammals, fish (both fresh and salt water),
birds, reptiles and amphibians.
 It is present in the soil and can survive for 20 days or longer in alkaline soil.
 The major source of infection for swine and turkeys is carrier animals of the same
species.
 It is reported that 30-50% of pigs carry the bacterium in their tonsils, other lymphoid
tissues.
 It is present in slurry of piglets and can be recovered from the faeces of carrier pigs.

MORPHOLOGY

 Erysipelothrix rhusiopathiae(previously named Erysipelothrix insidiosa form S

(Smooth) - form colonies and usually from acute syndromes is a Gram-positive rod,
the R (rough) form colonies usually from chronic disease is a Gram-positive filament.
 The organism is non-motile, non-spore forming, non-acid fast, occur either in singly,
in groups or in chains.

CULTURAL CHARACTERISTICS

 It is a facultative anaerobe, but growth isenhanced by 10% C02.

 It is able to grow in a temperature range of 5ºC to 42ºC, within a pH range 6.7 to 9.2
and 8% NaCl2.
 Growth occurs on nutrient agar but is improved by the addition of serum or blood. It
will not grow on Mac Conkey agar.
 Media contain either sodium azide (0.1%) or crystal violet (0.001%) may be used as
selective media.
 On blood agar, non-haemolytic pinpoint colonies (0.5 mm) appear at 24 hrs
incubation.
 Colonial variation becomes obvious at 48 hrs incubation when a zone of greenish
haemolysis often develops under and just around the colonies.
 The smooth form colonies are convex, circular with an entire edge.
 The large rough form colonies are flatter, more opaque and have an irregular edge.
 A characteristic reaction is produced when triple sugar iron agar is stab inoculated.
 When incubated at 37ºC for 24 hrs H2S is produced as a thin, black line just along the
inoculation stab.
 The R forms gives a bottle-brush type of growth in stab cultures of gelatin incubated
at 21ºC for 5 days.

BIOCHEMICAL PROPERTIES

 The bacterium is coagulase positive, catalase negative and oxidase negative.

 It does not hydrolyse aesculin or produce urease.
 Erysipelothrix rhusiopathiae usually ferments lactose, glucose, levulose and dextrin.
But the acid production is poor.
 To obtain good result, carbohydrate tests can be carried out in peptone water with
added sterile horse serum (5-10%) with phenol red as the indicator.
 Indole, Methyl red and Voges proskauer tests are negative.

RESISTANCE, ANTIGENS AND TOXINS

 Erysipelothrix rhusiopathiae is resistant to several chemicals including sodium

azide, and to drying, pickling, salting and smoking.
  It is capable of surviving for nearly a year in putrefying meat. But they are susceptible
to caustic soda and hypochlorites.
 They are readily killed in moist heat at a temperature of 55ºC for 10 mts.

Antigens and toxins

 Based on heat labile and heat stable antigens so far 23 serotypes have been identified.
 Strains of serotype 1 are subdivided into 1a and 1b. Serotypes 1a, 1b and 2 are most
frequently involved in disease in swine.
 Hyaluronidase and neuraminidase are produced by some strains.

PATHOGENESIS

 The carrier animals are animportant source of the organism.

 Entry of the organism may be by the oral, cutaneous or respiratory route.
 Ingestion of contaminated feed or water or contamination of abraded skin are the
most common means of infection in swine.
o Contaminate environment
o Carrier animals
o Organism ingested
o Enter small intestine
o Adhere to epithelium
o Penetrate intestine
o Blood stream
o Localization Vascular damage
o Immune complex Thrombosis
o Fever
o Vascular damage
o Arthritis
o Endocarditis
o Skin lesions
 Erysipelothrix rhusiopathiae is able to adhere to epithelial cells, and that they invade
the blood stream and cause localization.
 The more virulent strains produce high levels of neuraminidase that can cause
vascular damage and thrombus formation.
 Congestion of dermal capillaries results in diamond skin disease.
 Arthritis is associated with initial infection of joints and prolonged retention of
bacterial antigen in the joints.
 The diseases caused by Erysipelothrix rhusiopathiae are as follows

Main host (s) Disease syndrome

Pigs  Swine erysipelas

 Acute septicaemic form (Pregnant sows may abort)
 Urticarial form (Diamond skin disease)
 Vegetative endocarditis and Polyarthritis (Chronic
form)

Sheep  Poly arthritis in lambs

 Post-dipping lameness
  Valvular endocarditis and pneumonia

Turkeys , Geese and other  Acute septicaemia (Turkey erysipelas)

birds  Vegetative endocarditis and arthritis (Chronic form)

PATHOGENECITY

Symptoms

 Erysipelas occurs in pigs of all ages, but pigs from 2 months to one year age are highly
susceptible.
 Four forms of clinical disease in swine have been described.
o Acute septicaemia
o Urticarial or diamond skin lesions
o Vegetative endocarditis
o Arthritis
 These may occur alone or in combination. Swine erysipelas manifest in three forms.

Acute

 The acute disease is characterized by high fever, inappetance, depression, a rapid

course of illness, and death within 2-3 days in untreated animals.
 Some animals may show a stiff gait and reluctance to stand or move, and urticarial
cutaneous lesions may develop.
 The diamond shaped raised skin lesions are pathognomonic. Pregnant sows may
abort.

sub acute

 Sub acute disease is similar to the acute except that it is less severe and animals are
likely to recover within 5-7 days.

chronic course

 In the chronic form arthritis is more common.

 The hock, stifle, elbow and carpal joints are most likely to be affected resulting in
severe lameness.
 The mitral valves are involved in valvular endocarditis.

DIAGNOSIS, TREATMENT, PUBLIC HEALTH SIGNIFICANCE

 Diamond shaped skin lesions are pathognomonic.

Specimens to be collected

 It includes liver, spleen, heart valves or synovial tissues.

 Organisms are rarely recovered from skin lesions or chronically affected joints.

Diagnosis can be achieved by

 Gram-positive rods in acute cases and Gram-positive filaments in chronic cases.

  Based on cultural characters and biochemical tests.
* Serological tests are not applicable for diagnosis.

Treatment

 In addition to hyper immune serum, treatment with antibiotics such as penicillin and
tetracyclines are effective.

Public health significance

 Workers engaged in the fish and poultry industries are highly susceptible.
 The organism enters through minor skin abrasions causing localized cellulitis
referred as erysipeloid.

CHAPTER-10: MYCOBACTERIA

Learning objectives

To know in detail about,

 Classification of Mycobacteria
 Morphology, cultural and biochemical characteristics of Mycobacteria
 Cultivation methods of Mycobacteria
 Pathogenesis of tubercle formation
 Isolation and identification of Mycobacteria
 Explain the Acid fast staining and tuberculin tests

SYSTEMATICS
Domain Bacteria

Phylum Actinobacteria

Class Actinobacteria

Subclass Actinobacteridae

Order Actinomycetales

Suborder Corynebacterineae

Family Mycobacteriaceae

Genus Mycobacterium

 The genus includes animal and human pathogens as well as saprophytic members
often referred to as atypical, anonymous, opportunistic, tuberculoid and MOTT
(Mycobacteria other than typical tubercle) bacilli.
 CLASSIFICATION

Classification of Mycobacteria (Tubercle Bacilli)

I. Slowly growing mycobacteria

 Mycobacterium tuberculosis causes human tuberculosis in human and dogs.

 Mycobacterium bovis causes bovine tuberculosis in many animal species and also
cause tuberculosis in human
 Mycobacterium africanum causes human tuberculosis.
 The human type (Mycobacterium tuberculosis) is primarily a pathogen for man.
 But can cause disease in cattle, pigs, dogs, monkeys, parrots and other species.
 The bovine type (Mycobacterium bovis) is a common cause of disease in domestic
animals particularly cattle, pigs, cat, dogs and horse.
 The avian type (Mycobacterium avium) is primarily a pathogen for birds. But can
cause disease in cattle, sheep, goat and pigs.

II. Atypical mycobacteria

 Runyon (1959) grouped the atypical mycobacteria on the basis of pigmentation,

colonial morphology and growth rate.
 The photochromogens will produce pigment only if exposed to light. The
scotochromogens are those that produce yellowish orange pigments in the dark.
 The slow growing mycobacteria are those that require over 7 days incubation and
rapid growers are those requiring less than 7 days.
o Slowly growing photochromogens
 Mycobacterium kansasi, Mycobacterium marinum, Mycobacterium
simiae
o Slowly growing scotochromogens
 Mycobacterium gordonae (tap water scotochromogens)
o Slowly growing non-chromogens
 Mycobacterium avium (Avian tuberculosis)
 Mycobacterium intracellulare
 Mycobacterium paratuberculosis (Johne‘s disease – chronic
hypertrophic enteritis in cattle)
 Mycobacterium lepraemurium (Feline leprosy)
o Rapid growing mycobacteria
 Mycobacterium phlei (timothy grass bacillus)
 Mycobacterium smegmatis

III. Non-cultivable mycobacteriae

 Mycobacterium leprae
o Addition to this the unspecified acid-fast bacilli such as Mycobacterium
senegalense and Mycobacterium farcinogens were isolated from Bovine
farcy.

HISTORY

 The generic name Mycobacterium (fungus bacterium) was proposed by Lehmann

and Neumann (1896).
 The first member of this genus to be identified was the lepra bacillus discovered by
Hansen (1868) – Hansen bacillus.
  Koch (1882) isolated the mammalian tubercle bacillus and proved its causative role
in tuberculosis by satisfying Koch‘s postulates.
 The acid-fast property of Mycobacterium was discovered by Ehrlich (1882).
 Johne (1895) described Johne‘s bacillus - Mycobacterium paratuberculosis.

HABITAT

 It has a worldwide distribution. The usual habitats of the great majority of the
cultivable mycobacteria are water and watery habitats, marshes, wet soil, streams,
lakes, rivers.
 The source of the pathogenic mycobacteria is usually infected animals.
 Mycobacterium bovis is excreted in respiratory discharges, faeces, milk, urine and
semen.
 Mycobacterium avium and Mycobacterium paratuberculosis are shed in faeces
and Mycobacterium tuberculosis mainly in respiratory discharges.
 The atypical mycobacteria are widespread in soil, pastures, grass and water.
 A few are commensals in animals and may infect them.

MORPHOLOGY

 Mycobacteria are slender rods of varying lengths that sometimes show branching
filamentous form resembling ‗fungal mycelium‘.
 Hence, the name mycobacteria, meaning fungus like bacteria.
 Although cytochemically Gram positive, the Mycobacteria do not take up the dyes of
the Gram stain because the cell walls are rich in lipids – Mycolic acid.
 Once a dye has been taken up by the cells they are not easily decolourised, even by
acid-alcohol. Mycobacteria are therefore called as acid-fast bacilli.
 They are usually straight or slightly curved rod occurring singly, pairs or in small
groups. The morphology varies from cells of species to species.
 Mycobacterium tuberculosis is often arranged in serpentine cords.
 Mycobacterium kansasi is distinct banded or beaded appearnce, while
 Mycobacterium avium is often almost coccoid.
 In clinical materials they may appear as bundle of faggots. They are non-motile, non-
sporing and non-capsulated.

CULTURAL AND BIOCHEMICAL CHARACTERISTICS

Cultural characteristics

 A comparatively slow growth rate is characteristic of the mycobacteria, with

generation time ranging from 14-20 hours.
 Colonies appear only in about two weeks and sometimes may be delayed upto 6-8
weeks. Optimum temperature is 37°C and pH is 6.4 –7.0.
 Mycobacterium tuberculosis is an obligate aerobe while Mycobacterium bovis is
microaerophilic. Growth is stimulated by 5-10% CO2.
 Tubercle bacilli do not have exact growth requirements. But they are highly
susceptible to even traces of toxic substances like fatty acids in culture media.
 The toxicity is neutralized by addition of serum, albumin or charcoal.
 Several media, both solid and liquid, areavailable . The egg based Lowenstein
Jensen medium and Stone Brink's medium are most commonly used.
 Malachite green dye (0.025 g/100ml) is commonly used as the selective agent.
  Mycobacterium tuberculosis, Mycobacterium avium and many of the atypical
mycobacteria require glycerol for growth. However glycerol is inhibitory
to Mycobacterium bovis, while sodium pyruvate enhances its growth.
 On Lowenstein Jensen medium (i.e. glycerol containing media), Mycobacterium
tuberculosis giving the characteristic rough, tough and buff colonies – is known as
eugonic.
 The growth of Mycobacterium avium in this medium is also described as
eugonic. Mycobacterium bovis has sparse, thin growth on glycerol containing media
that is called dysgonic.
 Mycobacterium bovis however grows well on pyruvate containing media without
glycerol (i.e. Stone brink's medium).
 Pigment formation is tested with young, well-developed colonies on Lowenstein
Jensen medium.
 The cultures are exposed to a 100-watt, clear electric bulb, at a distance of 50 cm, for
atleast an hour and then incubated again in darkness for a further 1-3 days.
 After this treatment the photochromogens will develop pigement.
 Many of the mycobacteria produce yellow/orange pigments while Mycobacterium
tuberculosis, Mycobacterium bovis and Mycobacterium avium are non-
chromogenic.
 In liquid media, the growth begins at the bottom, creeps up the sides and forms a
prominent surface pellicle (mould like pellicle) that may extend along the sides above
the medium.
 Virulent stains tend to form long serpentine cords on liquid media, while avirulent
strains grow in a more dispersed fashion.
 Supplementation of media with mycobactin (extracted from non-mycobactin
dependant isolates of M.avium subsp. paratuberculosis) is required
for M.avium subsp. paratuberculosis.

Biochemical Properties

 They are oxidative. Atypical mycobacteria are catalse positive, while tubercle bacilli
are peroxidase positive.
 Niacin production and nitrate reduction is only by Mycobacterium tuberculosis.
 Urease is reduced by Mycobacterium tuberculosis and Mycobacterium bovis, but not
by avian strain.

RESISTANCE

 The Mycobacteria are resistance to physical influences and will retain their viability
in soil and particles of dried faeces for many months.
 They are not specifically heat resistant; being killed at 60ºC in 15-20 mts .
 Cultures may be killed by exposure to direct sunlight for two hours.
 Bacilli in sputum may remain alive for 20-30 hrs and in droplet nuclei for 8-10 days .
 They are relatively resistant to disinfectants i.e. exposure to 5% phenol, 15% H2SO4,
3% nitric acid, 5% oxalic acid and 4% NaOH.
 It is destroyed by tincture of iodine in 5mts and by 80% ethanol in 2-10 mts.

ANTIGENS AND TOXINS

 Many antigens have been identified in mycobacteria. Group specificity is due to

polysaccharide and type specificity is due to protein antigen.
 They do not produce any exotoxins. The cell wall of the mycobacterium is composed
of peptidoglycan, arabinogalactan and mycolic acid.
  In addition to this it contains wide range of lipids. The outer layer of the cell wall is
composed of mycosides. (Peptidoglycolipids or Phenolic glycolipids).
 Mycosides are responsible for the control of cellular permeability, resistance to action
of water-soluble enzymes, antibiotics and disinfectants.
 Cord factor (Trehalose – 6,6‘ dimycolate) and Wax D - inhibits chemotaxis,
leukotaxis, responsible for delayed hypersensitivity
 Sulfatides- sulfur containing glycolipids –promote the survival of virulent tubercle
bacilli within macrophages by inhibiting phagolysosome formation and avoiding
exposure to hydrolytic enzymes present in the lysosomes.
 Virulence appears to reside in the lipids of the cell wall. Mycosides, phospholipids
and sulpholipids are protecting the tubercle bacilli against phagocytosis.

PATHOGENESIS

Lesions

 Infection is usually by inhalation and ingestion. The mucociliaryclearance by mucus

and epithelial cilia in the upper respiratory passages provides defense against
infection.
 However, microorganisms on small particles (1-4 μm in size), such as, dust and water
droplets reach alveolar spaces.
 In previously unexposed animals, local multiplication of the mycobacteria occurs and
the resistance to phagocytic killing allows continued intra cellular and extra cellular
replication.
 Infected host cells with mycobacteria can reach local lymphnodes and from there may
pass to the thoracic duct with general dissemination.
 After 10-14 days, CMI responses develop and activated macrophages are able to kill
some mycobacteria.
 The aggregation of macrophages contributes to the formation of a tubercle, and a
fibrous layer may encompass the lesion.
 Caseous necrosis due to the cell death and tissue destruction occurs at the center of
the lesion and this may proceed to calcification or liquefaction.
 Once CMI is established, the lymphatic spread is retarded but occurs via the erosion
of bronchi or blood vessels to new area.
 Haematogenous spread may produce miliary tuberculosis (in deer). This involves
multifocal tubercle formation in an organ.

PATHOGENECITY

Cattle

 Tuberculosis consists of a characteristic lesion – the tubercle. This is an avascular

granuloma composed of a caseous necrosis in a central area encircled by a zone of
epitheloid cells, and a peripheral zone of lymphocytes, granulocytes and fibroblasts.
 Calcification may be present in the necrotic centers.
 An outer boundary of fibrous tissue is usually present between the lesions and
normal tissue.
 Tubercle lesions are more commonly present in the lymphnode, lungs and pleura.
 Military tuberculosis resembling millet seeds with similar lesions in various organs
can be formed by the haematogenous route.

Sheep and Goats

 Similar lesions as that of cattle are seen.

Horses

 Common sites are liver, spleen and lungs. In pigs, the skeleton, especially vertebrate
and long bones, are common sites.

Birds

 The grayish white granulomatous lesions are found in the liver and they are also
present in the intestines, spleen and bone marrow.

DIAGNOSIS, CONTROL AND PREVENTION

Specimens to be collected

 Specimens from live animals include aspirates from cavities, lymph nodes, biopsies,
tracheobronchial lavages and centrifuged deposit from about 50 ml of milk in the
case of suspected tuberculous mastitis. With dead animals, collect fresh and fixed
(10% formalin) samples of lesions.

Diagnosis

 Based on history, signs and post mortem lesions

 Direct microscopy
o The Ziehl-Neelsen (acid-fast) stain is used to stain smears from lesions and
other specimens.
o Organisms are appearing as slender, often beaded, red staining rods against a
blue background.
o Smears stained by fluorescent dyes (auramine, acridine orange or
fluorochrome) allow the mycobacteria to be seen more easily if relatively
small numbers are present.
 Isolation
o Several preliminary procedures are necessary inorder to recover the
comparatively slow growing mycobacteria
o Selective decontamination to reduce significantly the number of fast growing
contaminating bacteria.
o Digestion or liquefaction of mucus is necessary. Mucin-trapped mycobacteria
in specimens, such as broncho-tracheal exudates, may not beavailable for
growth in cultures.
o If mycobacteria are present, they must be concentrated by centrifugation.
o For decontamination, the ground-up specimens must be treated with
decontaminating agents, such as 5% oxalic acid, 4% sodium hydroxide
followed by neutralization of the acid or alkali or 20% antiformin can be used
and subsequently cultured in stone brink or Lowenstein Jensen media.
 By animal inoculation

Inoculated Mycobacterium Mycobacterium Mycobacterium

suspected tuberculosis bovis avium
material

Rabbits (I/V) + ++ ++
Guinea pig (S/C) ++ ++ -

Chicken (I/V) - - ++

 Bacteriophage typing: A, B, C, I
 Tuberculin test (Intra dermal, double intra dermal, ophthalmic tests in cattle and
wattle test in case of poultry)
 Gamma interferon assay, Gas liquid chromatography, PCR, ELISA for detecting
circulating antibodies, FAT , LTT assays can also be used in the diagnosis

Control and prevention

 Treatment and vaccination are inappropriate in control programmes for cattle.

 In many countries, tuberculin testing followed by isolation and slaughter of reactors
has been implemented as the basis of national eradication schemes.

CHAPTER-11: PARA TUBERCLOSIS AND NOCARDIA

Learning objectives

To know in detail about,

 Johne's disease and bovine farcy

 Pathogenesis and pathogenicity of Johne's disease
 Cultivation and diagnostic methods for Johne's disease
 Johnin test
 Diseases caused by Nocardia in domestic animals
 General approaches used to isolate and identify Nocardia

MORPHOLOGY

 Johne‘s disease or paratuberculosis is caused by Mycobacterium avium subsp.

paratuberculosis (also referred as Mycobacterium johnei).
 The disease causes chronic, contagious fatal enteritis, which can affect cattle, sheep,
goats, camels and wild ruminants.
 Note: Mycobacterium avium subsp. paratuberculosis infection in humans is called
as Crohn‘s disease (chronic enteritis in human).
 Mycobacterium avium subsp. paratuberculosis are acid-fast organisms.
 They are short rods measuring 1-2μm in width with rounded edges. They are motile
and does not form spores.
 On artificial media the organism tends to be in shorter club form.

CULTURAL CHARACTERISTICS

 Mycobacterium avium subsp paratuberculosis requires mycobactin - Killed extract

of M.phelei or other killed acid-fast organism- enriched media for growth.
 Slants of Herrold‘s egg yolk medium with mycobactin are highly suitable for isolation
of organism from specimens.
 The slants are incubated at 370C for upto 16 weeks and examined weekly for evidence
of growth.
  They produce minute grayish white, friable, irregular colonies, less than 1 mm in
d.m., in 5-16 weeks. Isolates from sheep may be pigmented.

PATHOGENESIS

 The organism is shed in the faeces, milk and semen of infected animals.
 They remain viable in the environment for up to one year under suitable
conditions.
 Calves under one month of age are highly susceptible and they develop clinical
disease than animals infected later in life.
 Infection is acquired mainly through ingestion.
 The organism is an intra cellular pathogen and cell mediated reactions are mainly
responsible for the enteric lesion.
 Ingested mycobacteria, engulfed by macrophages in which they survive and
replicate, are found initially in Peyer‘s patches.
 As the disease progresses, an immune mediated granulomatous reaction
develops, with marked lymphocyte and macrophage accumulation in the lamina
propria and submucosa.
 The resulting enteropathy leads to loss of plasma proteins and malabsorption of
nutrients and water.

PATHOGENICITY

Symptoms

 Clinical signs develop after prolonged subclinical phase of infection. Affected cattle
are usually more than 2 years of age when signs are first observed.
 In cattle, the disease is characterized by diarrhoea, initially intermittent, dark and
semisolid, but becoming persistent and profuse.
 Progressive weight loss results without loss of appetite, leading to emaciation and
eventually death.
 The mortality rate may approach 100%. Asymptomatic carrier cattle have an
increased incidence of mastitis and infertility.
 In sheep and goats, the disease is clinically evident only in mature animals. The
diarrhoea is less marked and may be absent.

Lesions

 Chronic catarrhal inflammation of the intestine is characteristic.

 In cattle, the mucosa of affected areas of the terminal small intestine and the large
intestine is usually thickened and folded into transverse corrugations.
 The mesenteric and ileocaecal lymphnodes are enlarged and oedematous.
 Thickening of the intestinal mucosa is less marked in sheep, and necrosis and
caseation may be present in the regional lymphnodes.

DIAGNOSIS

Specimens to be collected

 Specimens for direct microscopy from live animals include scrapings /pinch biopsies
from the rectum. Faeces may be submitted for culture.
  In case of dead animals, tissues from affected region of the intestines and from
regional lymphnodes are useful for histopathology.

Diagnosis by

 Microscopical examination by staining the faecal smears with acid-fast stain.

 Bacteriological examination
o Materials decontaminated with 0.3% benzalkonium chloride and
concentrated by centrifugation and subsequently cultured in Herrold's egg
yolk medium and are incubated at 370C for up to 16 weeks.
 Based on PM lesions
 Serological tests
o Complement fixation test can be used. But CFT is laborious and relatively
insensitive.
o Agar gel precipitation test has been used for confirming clinical infection.
ELISA, using serum absorbed with M.phlei may detect subclinically infected
animals.
 Johnin test
o Intra dermal Johnin test
 Inoculate Johnin PPD into the skin of the neck region.
 The delayed hypersensitivity reaction is measured after 48 hours.
 Peak response usually develops a month or so after infection.
o Intra venous Johnin test
 The intravenous Johnin test reaction is measured by increase in body
temperature following intra venous Johnin PPD injection.
 DNA probes, which are highly sensitive, are being used to detect organisms in faeces.

PREVENTION AND CONTROL

 Animals with clinical signs suggestive of paratuberculosis should be isolated, because

they shed large number of bacteria, which can contaminate buildings and pasture.
 Detection and elimination of subclinically infected animals is effective. Inactivated
adjuvanated vaccines areavailable .
 A live vaccine consisting of nonpathogenic strain of Mycobacterium
avium subsp. paratuberculosis is inoculated subcutaneously into calves soon after
birth and before 4 weeks of age.
 It reduces the incidence of Johne‘s disease in the herd.

Phylum Actinobacteria

Class Actinobacteria

Subclass Actinobacteridae

Order Actinomycetales

Suborder Corynebacterineae

Family Nocardiaceae
Genus Nocardia

HISTORY AND HABITAT

History

 Nocard described this organism in 1888, following its isolation from a case of bovine
farcy , hence the name of the type species is Nocardia farcinica.

Habitat

 Nocardia species are soil borne saprophytes.

MORPHOLOGY

 Nocardia has the ability to form Gram-positive, branching filaments of less than 1um
in d.m.
 It is closely related to Corynebacterium, Mycobacterium and Rhodococcus species.
 They are obligate aerobes. Some produce true mycelia and some strains are acid-fast.
All species are non-motile.
 Gram stained smears from lesions revealed Gram-positive branching filaments that
often showed fragmentation into coccobacillary elements.
 The modified ZN stained smears exhibit a similar morphology but most of the
filaments retain the carbol-fuchsin dye and stain red.

CULTURAL AND BIOCHEMICAL PROPERTIES

 The Nocardia species grow very well in blood agar incubated aerobically at 370C for
up to 7 days.The colonies on blood agar are often vivid white and powdery if aerial
filaments and spores are formed. Occasionally the colonies are smooth, heaped and
variably pigmented.
 Inoculate the suspected colonies from blood agar into Sabouraud dextrose agar
(SDA) and incubate at 370C for up to 10 days.
 Both types of colonies firmly adherents to the agar surface. The colonies on SDA are
dry, wrinkled and yellow, becoming deep orange color with age.
 Gram-stained smears from colonies show Gram-positive branching filaments that
characteristically break up into rods or coccobacillary elements with age.
 An MZN –stained smear from young culture reveals red staining, branching
filaments.

There are three morphological forms

 Group I strains have limited mycelia development due to early fragmentation of

hyphae into coccoid forms within 2 to 14 hours of incubation.
 Group II strains produce mycelia, which fragment in about 18 to 20 hours after
incubation, though these mycelia break up into mycelial fragments within two days of
growth.
 The pathogenic Nocardia species belong to Group III. The colonies are usually
leathery in appearance and pigmented . Extensive mycelium produced because
fragmentation does not begin until after 5 days incubation.

Biochemical Properties
  To differentiate Nocardia species tests such as decomposition of casein,
hypoxanthine, tyrosine, urea and xanthine are useful.
 They are oxidase and Catalase positive. Reduce nitrates to nitrites. Gelatin not
hydrolyzed.

PATHOGENESIS

 Nocardia are aerobic and essentially saprophytic.

 They cause suppurative and pyogranulomatous reactions in
immunocompromised hosts or animals that have been exposed to large doses
of the bacterium.
 The pathogenic Nocardia survive within phagocytic vacuoles by preventing
phagolysosome formation.
 This is probably due to the surface lipids as Nocardia species have a cell wall
similar to the mycobacteria.
 Other cell wall lipids may provoke the characteristic granulomatous reaction.
 Exudates are sanguinopurulent and can sometimes contain soft granules
consisting of bacteria, neutrophils and debris.
 They lack the microstructure of the sulphur granules produced by some of the
Actinomyces species.
 Diseases caused by the pathogenic Nocardia are

Species Host (s) Disease

Nocardia Dogs/ Localised cutaneous granulomatous abscesses
asteroides Cats nocardiosis
Nocardia Cattle Pyothorax and granulomas in the thoracic cavity
farcinica Chronic granulomatous mastitis
Bovine farcy in tropical regions
PATHOGENICITY

 Nocardia species have been isolated from a variety of clinical situations, though the
genus is in general an opportunistic pathogen.
 In primary nocardiosis, severe, suppurative or cavitary pulmonary infection
simulating tuberculosis is often observed and in some cases, show cutaneous and
subcutaneous abscesses which are diagnostic of N. asteroids.
 Blood stream invasion with secondary, often fatal, involvement of the internal organs
and the central nervous system are seen in N. asteroids.
 In the bovine species the mostimportant disease condition is mastitis whose
presentation is in the form of extensive fibrosis. N. asteroids is the most often
isolated species.

DIAGNOSIS

 Specimens to be collected
o Specimens should include exudates, aspirates, mastitic milk samples, tissue
from granulomas and thin sections from granulomas in 10% formalin for
histopathology.
 Based on direct microscopy
 o Soft granules are not common in exudates from N. asteroides infections.
Smears made from exudates, aspirates, granulomatous tissue and from
centrifuged deposits of bovine mastitic milk are stained by Gram's and MZN
stain.
o Gram-stained smears reveal Gram-positive branching filaments that often
show some fragmentation into coccobacillary elements.
o The MZN –stained smears exhibit a similar morphology but most of the
filaments retain the carbol fuchsin dye and stain red.
 Based on isolation and identification
o Characteristic colonial morphology on blood and SDA agar and microscopic
appearance.
 Based on biochemical reactions
 Differential diagnosis with Actinomyces
 Actinomyces infections respond well to penicillin and other commonly used
antibiotics, nocardial infections are often refractory to treatment and N. asteroides is
susceptible only to limited range of antimicrobial agents such as Trimethoprim-
sulphamethoxazole or erythromycin.

Characters Nocardiosis Actinomycosis

Granules in exudates Not common Usually present
Filaments MZN positive + -
Fragmentation of filaments + -
Growth on SDA + -
Powdery, white colonies ( aerial hyphae) + -
Susceptibility to penicillin - +

Note

 Infection with Nocardia is by inhalation from the environment, while Actinomyces

infection begins in the host as a normal flora invading damaged tissues.
 Disseminated disease caused by Nocardia is more common in the dogs, while
granuloma formation is the rule with Actinomyces infection and spreads by local
extension.
 When there is a doubt as to whether an animal has actinomycosis or nocardiosis,
precautionary measures to preserve the anaerobic Actinomyces should be instituted,
including prompt delivery to the laboratory under anaerobic condition, culturing on
brain heart infusion agar, blood plates and incubating under anaerobic,
microaerophilic, and aerobic conditions.
 While Actinomyces fails to grow on Sabouraud agar, Nocardia grows uninhibited.
 Acid-fast staining procedure of the sample exudate before culturing will also be
helpful in the presumptive identification of the infecting agent.

CHAPTER-12: ACTINOMYCETES

Learning objectives

To know in detail about,

 Principal host and diseases caused by Actinomyces

 Ray fungus and summer mastitis
 Morphology and cultural characteristics of Actinomyces
 Pathogenesis of lympy jaw
 Specimens to be collected and the general approaches used to diagnose the lumpy jaw
in cattle

SYSTEMATICS

 The actinomycetes comprise a heterologous group of prokaryotes that have the ability
to form Gram positive, branching filaments of less than 1μm in diameter.
 The main animal pathogens in the actinomycetes are in the
genera Actinomyces, Arcanobacterium, Actinobaculum, Nocardia and Dermatophil
us.
 Non-pathogenic, prolific producers of antimicrobial substances – streptomyces are
also included in Actinomycetes

Domain Bacteria

Phylum Actinobacteria

Class Actinobacteria

Subclass Actinobacteridae

Order Actinomycetales

Suborder Actinomyceneae

Family Actinomycetacea

Genus Actinomyces, Arcanobacterium

HABITAT AND HISTORY

 The Actinomyces species are present on mucous membrane of the host animal, often
in the oral cavity, tonsils, and nasopharynx.
 The soil is the natural habitat of many Actinomyces species.
 The generic name Actinomyces was first used by Harz (1879). Boestrom (1891)
isolated Actinomyces bovis.
 Cummins (1962) clearly demonstrated Actinomyces were bacteria and they were
distinct from other branching genera.

MORPHOLOGY

 The organisms show considerable pleomorphism. Actinomyces species are usually

long and filamentous although short V, Y, and T configuration also occur.
 In lesions of actinomycosis, the pus contains small pale yellow granules referred as
sulfur granules .
  The sulphur granule is composed of bacterial filaments and mineralized calcium
phosphate of host origin.
 When the granules are crushed and Gram stained, a mass of Gram-positive
branching filaments about 1μm in width, short rods, and cocci are evident.
 Around this mass, a circle of club shaped bodies with their narrow ends pointing
towards the centre-staining Gram negative. Hence, called ray fungus .
 They are non-acid fast, non-spore forming, nonmotile, non-capsulated and do not
form endospores or conidia.
 In case of Arcanobacterium pyogenes infections the pus or mastitic milk does not
contain any granules.
 Gram stained smears reveal large numbers of small, highly pleomorphic, Gram-
positive rods, cocci and pear shaped cells.
 Occasionally short branching typical Chinese letter appearances are also seen.

CULTURAL CHARACTERISTICS

 They cannot grow on Sabouraud dextrose agar. Actinomyces require enriched media
for growth. They grow well on sheep or ox blood agar.
 Actinomyces bovis is capnophilic (i.e. required 5-10% CO2 for its growth).
 Arcanobacterium pyogenes and Actinomyces viscous will grow aerobically but 5-
10% CO2will enhance their growth.
 Actinomyces bovis and Actinomyces viscous usually require 2-4 days but the growth
of Arcanobacterium pyogenes can usually be seen in 24 hrs.
 Actinomyces bovis colonies are non-haemolytic, very small (< 1nm), white, rough or
smooth and adhere tenaciously to solid medium.
 Gram stained smears show Gram positive, slightly branched filaments or short forms.
On subculture, the bacterium may become diphtheroidal or coccobacillary.
 Actinomyces bovis grows well in thioglycollate medium, giving a characteristic
diffuse growth in about 7-10 days.
 In broth cultures, it grows in coarse aggregates, which in some cases may result in a
granular deposit with a completely clear supernatant.
 Arcanobacterium pyogenes produce a hazy- haemolysis after 24hrs incubation along
the streak lines.
 At 48 hrs incubation, the colonies are surrounded by a narrow zone of complete
haemolysis.
 Arcanobacterium pyogenes has the ability to pit a loeffler serum slope in 24-48 hrs.
(i.e. A loopful of pure culture of the medium is taken and a heavy inoculum is made in
a small area in the center of the slope, taking care not to break the surface of the
medium. The medium is incubated at 370C for 24 –48 hrs).
 Arcanobacterium pyogenes will give positive CAMP test with Staphylococcus
aureus (i.e.enhancement of staphylococcal haemolysis).
 In litmus milk, the organism produces acid and clot after 3 days of growth.
 Actinomyces viscous commonly produces two colonial forms, one being smooth,
entire, convex and glistening and the other is smaller, rough dry and irregular.
 Neither is haemolytic. The larger colonial type yields Gram-positive diphtheroid
forms and the smaller colony has short branching filaments.

BIOCHEMICAL PROPERTIES,RESISTANCE,ANTIGENS AND

TOXINS

Biochemical tests

 Both Arcanobacterium pyogenes and Actinomyces bovis are catalase negative,

ferments several sugars and produce acid.
  Reduction of nitrate is negative. Actinomyces viscous is catalse positive.

Resistance

 Actinomyces are killed at a moist heat temperature of 600C for 20 mts and they are
susceptible to various disinfectants.

Antigens and toxins

 With the exception of Arcanobacterium pyogenes, Actinomyces species have not

been shown to produce any toxin.
 Arcanobacterium pyogenes produces a haemolytic exotoxin, which is dermonecrotic
and lethal and it also produces a protease and an extracellular neuraminidase.

PATHOGENESIS

 Actinomyces bovis, present as part of the normal flora of the mouth.

 Trauma to the tissues is the initiating event in disease and may occur as a result of
shedding of teeth or as a result of coarse feed.
 Whenever there is a trauma, the organism invades a variety of tissues and often
produces lesions in bone.
 Growth of the organism may involve maxillary bone, tongue, pharynx, lungs,
lymphnodes and S/c tissues of the head and neck.
 It initiates rarefying osteomyelitis and soft tissue reaction, the condition being
referred to as lumpy jaw.
 Granulation, mononuclear infiltration and fibrosis occur in the lesions with sinus
tracts leading to the outside.
 Exudate from the tracts contains pus with sulphur granules.
 Arcanobacterium pyogenes is a commensal on the exposed mucosal surfaces of
cattle, sheep and swine
 Arcanobacterium pyogenes infection is often a sequel to earlier tissueinjury , or to
infection with other bacteria. (i.e. Fusobacterium necrophorus , Peptostreptococcus
indolicus ). It produces toxins and established mastitis with abscess formation. The
acute bovine mastitis is refered as summer mastitis.
 Actinomyces viscous serotype 1 appears to be responsible for disease in dogs.
 Two syndromes can occur, either separately or together.
 One is a localized granulomatous lesion involving skin and subcutis; the other is a
pyothorax, with granulomas in the thoracic cavity and often a large accumulation of
sanguinopurulent pleural fluid containing soft white granules.
 Diseases caused by the pathogenic actinomycetes

Actinomycete Host (s) Disease

Actinomyces bovis Cattle Bovine actinomycosis (Lumpy jaw)

(Syn: Ray fungus)

Horses Poll evil/Fistuous withers (occur as a mixed infection
with Brucella species)

Arcanobacterium Cattle, Chronic or acute suppurative mastitis, suppurative

pyogenes Sheep pneumonia, septic arthritis, vegetative endocarditis
(Actinomyces and Pigs (Cattle), endometritis, umbilical infections, wound
pyogenes ) mainly infections and Seminal vesiculitis (Bulls and Boars).
Summer mastitis – a mixed infection
with Peptostreptococcus indolicus

Actinomyces viscous Dogs Canine actinomycosis

 Localised cutaneous granulamatous abscess

and/or
 Pyothorax and granulomas in the thoracic cavity.

Actinomyces isralii Human Human actinomycosis

Actinobaculum suis Pigs Pyogranulamatous mastitis, ascending pyelonephritis,

(Actinomyces suis) cystitis.

PATHOGENICITY

Symptoms

 In case of lumpy jaw in cattle there is marked swelling associated with

suppurative and proliferative osteomyelitis in the region.
 Lumpy jaw produces ill health by interfering with mastication.
 Arcanobacterium pyogenes infection occurs most frequently in heifer and dry
cows during summer months.
 Hence, it is named as summer mastitis.
 The affected quarter become enlarged and firm.
 Animals have fever with general toxaemia.
 The mortality and morbidity rate may be high.

Lesions

 In lumpy jaw, area of suppuration accompanied by the granulation tissues,

erosion of old bones and formation of new bones.
 The pus characteristically contains small sulphur granules.
 In summer mastitis, abscess develops at any site containing greenish yellow foul
smelling pus.

DIAGNOSIS, CONTROL AND PREVENTION

Diagnosis

 Specimens to be collected
 It includes pus, exudates, aspirates, tissue and scrapings from the wall
of abscesses.
 If they have been incised. A volume of fluid or pus should be collected
and submitted, if possible, rather than just a small amount on a swab.
 Thin sections of granulomas in 10% formalin are useful for
histopathology.
 Direct microscopy
 o The pus or exudate is placed in a Petridish and washed carefully with a little
distilled water to expose the yellowish sulphur granules of Actinomyces
bovis or the softer greyish white granules of Actinomyces viscous.
o A granule is placed on a microscopic slide in a drop of 10% KOH and gently
crushed by applying pressure on the cover slip.
o The characteristic clubs can be examined under the low power microscope.
o If it is stained with Gram‘s, the ray fungus can be demonstrated.
 Isolation and Identification of organism
 Fat
 Pitting of loeffler serum slope and CAMP test in case of Arcanobacterium pyogenes

Control and Prevention

 Actinomycetes are highly sensitive to tetracycline, chloramphenicol and penicillin

including benzyl penicillin and ampicillin.

CHAPTER-13: CORYNEBACTERIUM AND RHODOCOCCUS

Learning objectives

To know in detail about,

 Ulcerative lymphagitis in horses

 Diseases caused by Corynebacterium and Rhodococcus species in domestic animals
 Metachromatic granules, cuneiform arrangement, Diphtheroids and Pizzle rot
 Morphology, cultural and biochemical characteristics of Corynebacterium
Rhodococcus species

MORPHOLOGY

 Corynebacteria are Gram positive, non-acid fast, non-motile, non-capsulated, small

pleomorphic rods.
 They frequently occur in rods, coccoid, cub and fiamentous shape.
 The major pathogen is Corynebacterium diphtheriae, which causes diptheria in
children.
 Corynebacteria associated with animals are called diphtheroids.

Domain Bacteria

Phylum Actinobacteria

Class Actinobacteria

Subclass Actinobacteridae

Order Actinomycetales

Suborder Corynebacterineae
 Family Corynebacteriaceae

Genus Corynebactrerium

Family Nocardiaceae

Genus Rhodococcus

MORPHOLOGY

 They are Gram-positive slender rods with a tendency to clubbing at one or both ends;
they are non-sporing, non-motile, non-capsulated and non-acid fast.
 They have granules composed of (high energy phosphate stores) –
polymetaphosphate.
 The granules are more strongly Gram positive than the rest of the bacterial cell.
 Stained with Loeffler‘s methylene blue, the granules take up a reddish purple color
and hence they are called metachromatic granules. They are called as volutin or
Babes Ernst Granules.
 They are often situated at the poles of the bacilli and are called polar bodies.
 Special stains, such as Albert‘s, Neisser‘s and Ponder‘s have been devised for
demonstrating the granules clearly.
 Stained smears from animal tissues often reveal groups of cells in parallel (Palisades)
or cells at sharp angles to each other (Chinese letter or Cuneiform arrangement).
 This is due to the incomplete separation of the daughter cells after binary fission.
 Rhodococcus equi can appear as a Gram-positive coccus or a rod or club shaped form
arranged in clusters. It is capsulated and sometimes weakly acid fast.

CUTURAL CHARACTERISTICS

 Growth is scanty on ordinary media. Enrichment with blood, serum or egg is

necessary for good growth. The optimum temperature for growth is 37ºC and
optimum pH is 7.2. It is an aerobe and facultative anaerobe. Sheep or ox blood agar is
used routinely along with MacConkey agar to detect any Gram-negative
contaminants that may be present.

 On blood agar, Corynebacterium ovis colonies are small, white, dry and non-
haemolytic at 24hr incubation.
 A narrow zone of haemolysis occurs at 72 hrs incubation. After several days
incubation the colonies can reach 3mm in d.m and appear dry, crumbly and cream in
color.
Corynebacterium bovis colonies are small, white, dry and non-haemolytic.
 As it is a lipophilic corynebacterium, they grow very well on media enriched with 0.5
–1% tween 80.
 Rhodococcus equi colonies are small, smooth, shiny and non-haemolytic after 24 hrs
incubation. But on 4-day culture, the colonies become larger, mucoid and salmon-
pink in color.
 The salmon-pink pigmentation is not easily seen against a red background.
 So, the mucoid colonies and salmon-pink pigmentation can easily be demonstrated
on nutrient agar (4-day culture).
  Nutrient agar enriched with yeast extract and glucose is useful forenhancing the
salmon pink pigmentation.
 The renale groups are non-haemolytic. On nutrient agar, after 48 hrs
incubation, Corynebacterium renale produces dull yellow colonies.
 The Corynebacterium pilosum produces distinct yellow and Corynebacterium
cystitidis exhibit white colonies.
 On milk agar, Corynebacterium renale showing casein digestion.
While Corynebacterium pilosum and Corynebacterium cystitidis do not give this
reaction.
 On CAMP tests, the Corynebacterium ovis, Rhodococcus equi and Corynebacterium
renale interacting with beta haemolytic strains of Staphylococcus aureus gives the
following results.

Staphylococcal beta haemolysis

Corynebacterium ovis Inhibition

Rhodococcus equi Enhancement

Corynebacterium renale Enhancement

BIOCHEMICAL CHARACTERS, RESISTANCE AND ANTIGENS

AND TOXINS

Biochemical characters

 They are catalase positive, oxidase negative. Except Corynebacterium bovis others are
urease positive.
 The renale group is very strong urease positive (less than one hour).
 All diptheroids ferments sugar except Rhodococcus equi. Corynebacterium bovis and
Corynebacterium renale ferments both glucose and maltose.
 Two biotypes of Corynebacterium ovis are recognized.
 The ovine/caprine strains lack nitrate-reducing capacity, while the equine/bovine
strains usually reduce nitrate.

Resistance

 Diptheroids are readily destroyed by heat, 60ºC for one hour.

 They are highly susceptible to disinfectants.
 It is more resistant to the action of light, dessication and freezing.
 Rhodococcus equi is resistant to 2.5% oxalic acid for one hour.

Antigens and toxins

 The diphtheroid antigen and toxins are not well documented.

 Corynebacterium ovis produces a filterable toxin similar to that produced by
Corynebacterium diphtheriae.
 It is a haemolytic toxin, which has phospholipase activity to the cell wall lipids.
 In Corynebacterium renale, the pili is antigenic, Renalin- a Corynebacterium renale
extracellular protein may play a role in lysis of cell membranes.
  Rhodococcus equi produces diffusible Rhodococcus equi factors (Phospholipase C
and Cholesterol oxidase) and these as well as the capsule and cell wall constituents
probably play a major role.

PATHOGENESIS

 The diphtheroids infections are characterized by the development of suppurative

lesions and clinical manifestations do not develop in the absence of predisposing
factors.

Corynebacterium ovis

 The prevalence of caseous lymphadenitis may be as high as 50% in adult sheep.

 Some clinically normal sheep may carry the organism in the digestive tract, excrete in
the faeces and contaminate the environment.
 When bacteria enter the host via skin wounds (or tick bite), multiply and are
phagocytosed.
 Phagosome-lysosome fusion takes place. But Corynebacterium ovis multiplies in the
phagolysosome and phagocytic cells die.
 Permeability of local blood vessels increases, encouraging the spread of infection
from the initial site to other locations, often-regional lymphnodes.
 Produces toxins – Phopholipase-D. Abscesses may develop at either primary or
secondary sites, eventually rupturing and discharging a thick, caseous pus containing
large numbers of viable bacteria. In some instances, lesions become metastatic and,
as they increase in number, the thin ewe syndrome develops, resulting in progressive
debilitation and death.

Corynebacterium renale

 Corynebacterium renale is a normal flora in the lower urogenital tract. This group
possess fimbriae which allow attachment to the urogenital mucosa.
 The major predisposing factors that put a cattle at risk are the shortness of the female
urethra and the effects of pregnancy and parturition, thus, disease occurs most
frequently in mature cows.
 The vulva may be animportant portal entry for Corynebacterium renale into the
urinary tract.
 Bacteria grow readily in urine and ascend (through vesiculo uretharal reflex) to the
kidney.
 Corynebacterium renale has high urease activity. The urease is nephrotoxic and
produces pyelonephritis.

Rhodococcus equi

 Rhodococcus equi may be a commensal in the intestine of horses and it is largely a

soil organism.
 The soil enriched with equine faeces and summer temperatures are favours the rapid
multiplication of this bacterium.
 The disease is usually seen in 2-4 month old foals, possibly due to the decline in
maternal antibody at about 6 weeks of age.
 The main route of infection is by inhalation. Rhodococcus equi is a facultative
intracellular pathogen.
 Its ability to survive, persist in, and eventually to destroy alveolar macrophages is the
basis of its pathogencity.
 It causes granulomatous inflammation and abscesses in the lung tissue.
  Heavily infected sputum may be swallowed by the affected foal leading to ulcerative
colitis and mesentric lymphadenitis.
 Corynebacteria are pyogenic bacteria causing a variety of suppurative conditions in
animals.
 The main diseases, hosts and natural habitats of the Corynebacteria are

Species Main Diseases Natural habitat

host (s)

C. Sheep and Caseous Skin, mucous

pseudotuberculosis ( Corynebacterium Goats lymphadenitis membrane and
ovis or Preisz Nocard Bacillus ) (Non- G.I. tract
nitrate
reducing
biotype)

Horses Ulcerative
and Cattle lymphangitis
(Nitrate
reducing
biotype)

Corynebacterium renale Cattle Pyelonephritis Prepuce and

and cystitis semen of
asymptomatic
Pigs Kidney abscess bulls, vaginal
mucous
membrane of
Male Balanoposthitis
heathy cows
sheep (Pizzle rot)

Corynebacterium cystitidis Cattle Severe cystitis, Male genital tract

rarely
Pyelonephritis

Corynebacterium pilosum Cattle Pyelonephritis Male genital tract

and urine

Corynebacterium bovis Cattle Subclinical Udder and teat

mastitis canal of cows

Rhodococcus equi (Corynebacterium Foals (2-4 Suppurative Soil and Faeces of

equi ) months) bronchonephritis foals and other
herbivores

Pigs Cervical Soil

 lymphadenitis

PATHOGENICITY

Symptoms

Corynebacterium ovis

 Caseous lymphadenitis in sheep is characterized by skin wounds, enlarged lymph

glands and abscesses distributed throughout the body.
 But the disease can be a mild infection and it is unnoticed until postmortem is done.
 Ulcerative lymphangitis in horses are similar to glanders.
 Initially there is a pain and swelling of the hind limbs and enlargement of lymphatic
vessels.
 Ulcers develop, discharging purulent material. In severe cases it spreads to abdomen,
forelegs and neck, leading to death.

Corynebacterium renale

 In pyelonephritis, characteristically frequent passage of turbid or blood stained urine

by animals, which are pregnant or recently calved.
 Restlessness and kicking at the abdomen may indicate renal pain. The urine contains
red blood cells, pus cells and albumin.
 Ulcerative balanoposthitis (Pizzle rot), particularly common in Merino sheep and
Angora goats, is characterized by ulceration around the preputial orifice, with a
brownish crust developing over the lesion

Rhodococcus equi

 In suppurative bronchopneumonia, the foals develop cough, fever and increased

respiratory rate.
 In severe cases purulent discharge from nose just before death.

Lesions

Corynebacterium ovis

 In caseous lymphadenitis the superficial lymphnode contains a mass of greenish

yellow caseation in concentric layers, which have an onion ring appearnce in, cross
section (hence, named it as Corynebacterium pseudotuberculosis). In advanced
cases, similar lesions are seen in lungs, kidney, liver and spleen.

Corynebacterium renale

 The kidneys are enlarged, necrosis and suppuration in the medulla. Wedge shaped
suppurative foci in the cortex.
 The kidney pelvis contains blood and pus
 The bladder contains blood and pus with petechial haemorrhages and ulceration of
the tract.

Rhodococcus equi
  Characterized by pyogranulamatous lesions with abscesses in the lung, associated
lymphnodes and pus in the bronchi.

DIAGNOSIS, CONTROL AND PREVENTION

Diagnosis

 Specimens
o Pus or exudates are collected from suppurative conditions and mid stream
urine for isolation of the Corynebacterium renale.
o A tracheal wash technique with infusion of saline, can be used for the recovery
of Rhodococcus equi from affected foals.
 Diagnosis is mainly based on history, symptoms and lesions
 Isolation and identification of the organism
o Based on microscopical appearance, colonial morphology on blood agar,
CAMP tests and Biochemical tests.

Control and prevention

 Diphtheroids are susceptible to penicillin, tetracycline, erythromycin, lincomycin,

neomycin and gentamicin.

CHAPTER-14: SYSTEMATICS
Domain Bacteria

Phyum Proteobacteria

Class Gamma proteobacteria

Order Enterobacteriales

Family Enterobacteriaceae

Genus Escherichia

Genus Salmonella

Genus Shigella

Genus Citrobacter

Genus Enterobacter

Genus Edwardsiella
 Genus Klebsiella

Genus Morganella

Genus Proteus

Genus Providencia

Genus Serratia

Genus Yersinia

 Most members of the family Enterobacteriaceae share the following characters

 Gram negative, non sporing rods, often motile, usually by means of peritrichate
flagellae, capsulate or non capsulate.
 Easily cultivable on ordinary laboratory media. Aerobic and facultative anaerobic.
 All species ferment glucose with the formation of acid or of acid and gas. Reduce
nitrate to nitrite with exception of some strains of Erwinia and Yersinia.
 Oxidase negative, catalase positive except Shigella dysenteriae
 They are at present 28 genera and more than 80 well defined species in
the Enterobacteriaceae (not including the large number
of Salmonella serotypes/species) have been recognized.
 Note: The term Coliform or Coliform bacteria is used to refer to those members
of Enterobacteriaceae that usually ferment lactose, such
as E.coli, Klebsiella and Enterobacterspecies.
 Based on the pathogenicity the Enterobacteriaceae can be divided into 3 groups
o Major pathogens of animals such as E.coli, Salmonella and Yersinia
o Opportunistic pathogens that are known occasionally to cause infection in
animals.
 These include species within the
genera Klebsiella, Enterobacter. Proteus,
Serratia, Edwardsiella, Citrobacter. Morganella and Shigella.
o Uncertain significance for animals. These include species from 17 genera of
this family. Eg. Erwinia, Hafnia, Providencia.

LEARNING OBJECTIVE

To know in detail about,

 Coliforms, white scours, watery mouth, mushy-yolk disease and Hjarre's disease
 Diseases caused by E.coli in domestic animals
 Antigens, toxins and virulence factors of E.coli
 IMViC test
 Classification of pathogenic E.coli
 Role of Mac Conkey agar in diagnosis of Enterobacteriaceae
 General approaches used to isolate, identify and serotyping of E.coli

HABITAT
  E. coli is a natural inhabitant of the large intestine and lower small intestine of all
mammals.
 It is usually present in large numbers in carnivores, omnivores than in herbivores.
 E .coli is voided in faeces and can survive in faecal particles, dust and water for weeks
or months.
 The presence of E .coli in water samples, being tested for potability, is taken as
evidence of faecal pollution.
 This genus is named after Theobald Escherich, who was first to describe the Colon
bacillus under the name Bacterium coli commune (1885).

MORPHOLOGY AND CULTURAL CHARACTERISTICS

Morphology

 Gram negative, rods, non-spores, non-acid fast, occurs either in singly or in pairs.
 Usually motile with peritrichous flagella. Some strains possess a polysaccharide
capsule and fimbriae.

Cultural characteristics

 It is an aerobe and facultative anaerobe, optimum temp. is 37oC, good growth occurs
on ordinary media.
 Colonies are large, thick, grayish white, moist, smooth and opaque.
 The 'S' forms are seen on fresh isolation.
 The 'R' forms seen on repeated subculture.
 On blood agar, many strains, especially those isolated from pathological conditions,
are Beta haemolytic.
 As they are very strong lactose fermenters, the colonies on MacConkey agar are bright
pink. Because of its ability to ferment sugars, it gives yellow - green colonies on
Brilliant green agar (BGA) and yellow colonies on XLD agar.

 On Eosin methylene blue agar (EMB), E.coli colonies have a unique and
characteristic metallic sheen. Click here for visual

 In nutrient broth, growth occurs as general turbidity and a heavy deposit, which
disperses completely on shaking.

BIOCHEMICAL PROPERTIES AND RESISTANCE

Biochemical properties

 The four-biochemical tests- IMViC- (Indole, Methyl Red, Voges- Proskauer, Citrate
utilization) are widely employed to identify E.coli.

 It gives IMViC, +,+, -, -.

 Click here for visual -Indole test
 Click here for visual - Methyl red test
 Click here for visual - Voges-Proskauer test
 Click here for visual -citrate test
 They ferment several sugars and produce acid and gas.
 H2S negative, Urease negative, Oxidase negative and Catalase positive.

Resistance
  E.coli remains in the environment (water, feed, dust) for weeks.
 Some strains are more heat resistant and they will survive at 60°C for 15 min or 55oC
for 60 min.
 They are more sensitive to lethal action of phenol and cresol.

ANTIGENS AND TOXINS

 The capsular antigens (K) are polysaccharides and the cell wall or somatic (O)
antigens are determined by the sugar side chains on the LPS of the outer membrane.
 The Flagellar (H) and Fimbrial (F) antigens are proteins.
 Three kinds of K antigens L, A, B have been described based on the effect of heat on
the agglutinability, antigenicity and antibody binding power.
 The O, H and K antigens can be used to serotype strains of E.coli. So far, 164 types of
O antigens, 103 K antigens and 75 H antigens have been recognized.
 The antigenic pattern of a strain is recorded as the number of particular antigen it
bears for eg: 0157: K85: H19.

Toxins and virulence factors of E.coli

 Capsule
o The capsular polysaccharide is antiphagocytic and also protects the cell wall
from the damaging effects of complement.
 Cell wall
o The endotoxin (lipid A) is the toxin associated with colisepticaemias and the
toxaemia in coliform mastitis.
o Lipid A also interferes with the complement components that are responsible
for the attack on the outer membrane of E.coli.
o Endotoxin is released when bacteria die and lyse.
o The effects of endotoxin in the animal body include fever, leukopenia followed
by leukocytosis and hyperglycemia with a subsequent fall in blood sugar and
lethal shock after a latent period.
 Fimbriae/ Pili
o Fimbrial antigens are adhesins (i.e. they confer the adhesive properties on the
organism).
o Several major specific adhesins Type I (mannose sensitive), mannose
resistant K88 (F4) and K99 (F5), F6 and F 41 allow the attachment of E.coli to
RBC's or glycoproteins on the surface of the epithelial cells of jejunum and
ileum with consequent colonization.
 Enterotoxins
o Both heat labile (LT) and heat stable (ST) enterotoxins are produced by the
ETEC strains that also have the K88, K99 or other colonizing agents.
o The LT enterotoxin is a large molecular weight protein and is antigenically
related to cholera toxin, which causes stimulation of asenylate cyclase activity.
o The ST enterotoxin is also a protein and there are two types, STa and STb. STa
toxin increases the guanylate cyclase system.
 Verotoxins or shiga like toxins or Odema disease toxin
o It inhibits protein synthesis in host cells following interaction with 60S
ribosomal subunit.
o There are two types,SLT I / VT-1- that are neutralized by shiga antitoxin, SLT
2 / VT2e - which causes odema disease in pigs and haemorrhagic colitis in
man and it is not neutralized by shiga antitoxin.
 o Shiga like toxin can cause destruction of intestinal epithelial cells where there
is dense adherence of the pathogenic E.coli.
 Haemolysins
o E.coli strains produce both alpha and beta haemolysin.
o The alpha haemolysin is a protein that damages host cell membranes and also
increases the availbility of iron, which is required for invading organisms in
the host.
 Siderophores
o Siderophores – iron-binding molecules are particularlyimportant for the
invasive strains.
o In response to low level of iron, the siderophores – aerobactin and
enterobactin are synthesized and released which allows the bacteria to
multiply in an environment of limited concentration of free iron.
 Colicins
o Colicins are distinctive class of proteinaceous antibiotic substances produced
by strains of E.coli, which was described, by Gratia and Frederiq in 1946.
o They are 40-60 Kda, plasmid encoded proteins that are released
extracellularly.
o Characteristically, they become attached to specific receptors on the surface of
susceptible bacteria, causing the death of these organisms.
o Colicin V (COLv) is found primarily among virulent bacteria.
o It is plasmid encoded, small molecule and is released by an export
mechanism.
o The Col V plasmid also encode for virulence, increased serum survival, altered
motility, changes in hydrophobicity and facilitates attachment to appropriate
host cells.

PATHOGENESIS

Predisposing factors

 They are paramountimportance for establishing the disease. Young neonates,

particularly one week of age, are particularly susceptible because,
o Normal flora of the intestine is not fully established
o Have a naive immune system
o Receptors for the adhesions of E.coli (i.e. ETEC strains) are present for the
first week of life only in calves and for the first 6 weeks of life in piglets.
 In addition to this, poor husbandry practices, insufficient passive immunity, change
of environment and healthy grain diets in weaned pigs leads to massive colonization
of the intestine by E.coli.
S.No Animals Disease

1  Cattle  Colibacillosis (White scours) or

 Calves less than one week old Colisepticaemia
 Calves surviving colisepticaemia  Joint ill
 Cows  Coliform mastitis

2  Sheep  Colibacillosis and Colisepticaemia

 Neonatal lambs  Watery mouth
 Ewes  Coliform mastitis

3  Pigs  Colibacillosis and colisepticaemia, Piglet

 Piglets less than one week old meningitis
 Pigs about 2 weeks Weanling enteritis
 Weaned pigs  Odema disease
 Sow  Coliform mastitis, MMA(Mastitis, Metritis,
Agalactia syndrome)

4  Dogs  Colisepticaemia (Fading puppy syndrome)

 Neonatal pups  Pyometra
 Bitches  Urinary tract infection
 Adult dogs

5  Poultry  Omphalitis (Mushy -yolk disease)

 Young chicks  Airsacculitis, Colibacillosis, Hjarre's disease
 All ages or Coligranuloma
 Swollen head syndrome in broilers

PATHOGENICITY
Classification of pathogenic E.coli

 Classification of enteric E.coli pathogens

o ETEC: Enterotoxigenic E.coli, which has the firmbrial adhesins K88, K99 etc.
the production of these colonization factors, correlates with enterotoxin
production. These strains cause the majority of cases of neonatal
Colibacillosis. An adhesin, termed intimin is necessary for the binding of
EPEC to enterocytes.
o EPEC: Enteropathogenic E.coli -Do not produce any toxins but they can cause
destruction and stunting of microvilli, atrophy and shedding of enterocytes
which leads to enteritis and diarrhoea.
o EIEC: Enteroinvasive E.coli- strains adhere to cells of the distal small
intestine; invade the erythrocytes and deeper layers of the intestinal mucosa.
They reach the lymphatic system where there is multiplication. Several
virulent factors areimportant as survival for these invasive strains, which are
responsible for colisepticaemia.
o AEEC: Attaching and Effacing E.coli strains colonise the small intestine,
attach to target cells and kill them. The verotoxins destroy the microvilli.
These strains have been isolated from calves and rabbits.
o EHEC: Enterohaemorrhagic E.coli: Two types are recognized. VTEC-
Verotoxigenic E.coli. Eg. O157:H7-which is responsible for the haemorrhagic
colitis-haemolytic ureamic syndrome in humans and SLTEC- Shiga like toxin
producing E.coli.
 Classification based on Haemolysis
o E.coli may produce atleast 4 types of haemolysins- alpha , beta , gamma and
enterohaemolysin.
 Classification based on plasmid
 Colicin typing
 Phage typing

Symptoms

Horse

 Newborn foals suffer from a disease known as Joint ill, navel ill or sleepy foal disease.
There will be a rise in temperature, general weakness, diarrhoea, lameness and death.

Cattle (K99)

 It causes white scour or colisepticaemia in calves. In adult cases the symptoms are
scouring, weakness, prostration and death within hours after initial symptoms.
 In less acute cases the calves become listless, fail to suck and develop diarrhoea.
 The faeces are grayish white in colour (hence the term white scour) with fetid odour.
 Other signs include swollen joints and pneumonia. Faeces with blood stained mucus,
subnormal temperature, animal becomes comatose and dies.
 E.coli is also associated with acute bovine mastitis.
 The udder becomes swollen, hot and painful. Milk production falls rapidly and cease
with systemic disturbances.

Pigs (Serotypes O8, O138, O141 and O147) (K88.K99)

 E.coli infection causes piglet diarrhoea and edema disease. Piglets 4 days old to 3
weeks of age are susceptible.
 There will be diarrhoea, listlessness. They may collapse and die.
  There may be odematous lesions in various parts of the body.
 They will show twitching of the ears and trembling staggering gait.

Poultry (O2,O78,O1)

 E.coli infection causes colibacillosis, airsacculitis, Hjarre‘s disease or coligranuloma

and yolk sac infection.
 Death of some birds occurs rapidly while some birds show loss of appetite,
respiratory distress, diarrhoea and weakness.
 Coligranuloma is a chronic condition characterized by granulomatous lesions in the
epithelium of the intestine and other organs. In broilers it causes swollen head
syndrome.

Dogs (O42)

 It causes fading puppy syndrome in newborn puppies.

 The only symptoms before death are weakness and lack of appetite.
 In older dogs it is associated with acute enteritis and pyometra in bitches.

Sheep and goats (K99)

 Similar lo that of calves. The watery mouth is characterized by severe depression, loss
of appetite, profuse salivation and abdominal distension.

Lesions

Cattle

 In cases of pneumonia the lungs may show areas of congestion and necrosis.
 The spleen and mesenteric lymph nodes are enlarged and congested. Joint infections
develop as synovitis.

Sheep and Goats

 Similar to that of calves.

Pigs

 The intestine shows area of congestion and the stomach is filled with clotted milk.
 In oedema disease the carcass shows edematous areas of eyelids, ears and face.
 The abdominal cavity, pleural and pericardial sac may contain clear fluid containing
fibrin.

Poultry

 Characteristically there is pericarditis, perihepatitis containing a quantity of purulent

exudate.
 Other lesions include inflammation of air sacs, which may contain caseous material,
congested liver with gelatinous exudate and an enlarged spleen.

Dogs
  The lesions include petechial haemorrhages throughout the carcass, congested lungs
and haemorrhagic gastroenteritis.

DIAGNOSIS

 Clinical signs and pathology

 Specimens to be collected
o It includes faecal samples in case of enteric disease, tissue specimens from
septicaemic cases, mastitis milk, mid-stream urine and cervical swabs from
suspected cases of pyometra or metritis.
 Isolation, Identification and serotyping
o Isolation can be done in blood agar, MacConkey agar and EMB agar.
o The organisms can be isolated in pure culture from the small intestine.
o In septicemic conditions E.coli can be recovered from the liver, spleen,
kidneys and lungs.
 Lab. techniques for detection of enterotoxins.
 Detection of heat labile enterotoxin by using mouse adrenal cells,
Chinese hamster ovary cells and vero monkey kidney cells.
 Ligated ileal loops of rabbits are injected with bacterial culture
supernatants.
 A positive test is indicated by the accumulation of fluid in the ligated
loop.
 Detection of heat stable enterotoxins (ST) is done by injecting culture
supernatants into milk filled stomach of infant mice.
 After 4 hours the mice are sacrificed & examined for dilatation of the
intestine due to fluid accumulation.
 The enterotoxins or fimbrial antigens can also be confirmed by
immunological methods or by PCR.
 For expression of fimbrial antigens, isolates should be subcultured on
minca medium.
 Slide agglutination tests for O and H antigens are employed for
serotype identification.

CONTROL AND PREVENTION

Note

 Colostral antibodies can prevent colonization of the intestine by pathogenic E.coli.

 Absorption of gammaglobulin from the intestine declines progressively after birth
and is negligible by 36 hours.

Passive immunization

 Passive immunization can be achieved by immunizing sows with E.coli K88 antigen
during gestation.
 This results in the production of anti K-88 antibodies in the colostrum and milk.
 This when ingested by piglets prevent the adhesive capacity of K88 antigen on the
bacteria.
 In cattle the mother should not be moved to new environment shortly before calving.

Active immunization
  Three types of vaccines areavailable . They are live E.coli K88 antigen (oral vaccine),
killed E.coli bacteria with K88 antigen (bacterin) and bacteria free K88 antigen
(subunit vaccine).
 Vaccination of pregnant cows with purified E.coli K99 fimbrial or whole cell
preparations, often combined with rotavirus antigen, can be used toenhance
colostral protection.
 Antibiotics have been used in the prevention and treatment of colibacillosis,
particularly oxytetracycline, chlortetracycline, streptomycin, ciprofloxacin, and
enrofloxacin. But this has resulted in the development of resistant strains of E.coli.

CHAPTER-15: SALMONELLA

Learning objectives

To know in detail about,

 Definition for pullorum disease and fowl typhoid

 Cultural and biohmeical characters of Salmonella sp
 Role of enrichment broth in isolationm of salmonella sp
 Antigens, toxins and antigenic variations of Salmonella
 Different classification scheme of Salmonella sp

HISTORY

 Salmonella has long been recognized asimportant zoonotic pathogen of worldwide

economic significance in humans and animals.
 Infections of animals with various species of Salmonella sometimes result in serious
disease.
 The interplay of Salmonella with its host takes a variety of forms, including
remarkable host specificity, inapparent infection, recovered carriers, enteritis,
septicaemia, abortion and combination of disease syndromes.
 The typhoid bacillus Salmonella typhi was first observed by Eberth (1880) in the
mesenteric nodes and spleen of fatal cases of typhoid fever and was isolated by Gaffky
(1884).
 It came to be known as Eberth – Gaffky bacillus or Eberthella typhi.
 Salmon and Smith (1885) described a bacillus, which was believed to cause hog
cholera in swine.
 This bacillus, now called Salmonella cholera suis was the first of a series of similar
organisms to be isolated from animal and man- the genus Salmonella
 Salmonellae currently comprise about 2400 serotypes, of which 50 of them are
potentially pathogenic.

HABITAT

 The reservoir for salmonellae is the intestinal tract of warm blooded and cold-
blooded animals.
 The majority of infected animals become sub clinical excretors resulting in
contamination of water, food and the environment.
 In Poultry some serotypes, such as Salmonella enteritidis infect the ovaries and be
transmitted through eggs.
 The undercooked egg dishes may result in human food poisoning.
 The salmonellae can survive for 9 months or more in moist soil, water, faecal
particles and animal feeds, especially in blood, bone and fish meal.
 MORPHOLOGY

 Salmonella are Gram-ve, non-capsulated, non-spore forming, short rods.

 They are motile by peritrichous flagella, except S.gallinarum and S.pullorum.
 Most salmonella strains possess type –1 fimbriae associated with mannose sensitive
adhesive properties.
 Strains of S.gallinarum and S.pullorum form type 2 fimbriae, which are
morphologically and antigenically like type 1 fimbriae but non-adhesive.

CULTURAL CHARACTERISTICS, BIOCHEMICAL PROPERTIES

AND RESISTANCE

Cultural characteristics

 They are aerobic and facultative anaerobic, growing readily on simple media over a
pH range of 6.8. Optimum temperature for growth is 370C.
 On nutrient agar the colonies are large, circular, low convex and smooth.
 The selective enriched media for salmonellae are tetrathionate broth, selenite broth
and rappaport vasiliadis medium.
 The host adapted serotypes from pigs and poultry are more fastidious than others.
 They do not tolerate selenite broth and tetrathionate broth. In this case, Rappaport is
highly suitable.
 After 24-48 hr incubation on selective broth the subculture will be made on
MacConkey agar, Brilliant green agar, XLD and Salmonella Shigella (SS) agar.
 The majority of salmonellae, except some strains of S.arizonae, are non-lactose
fermenters and produce pale or colorless colonies on MacConkey agar.
 Most salmonellae give an alkaline reaction in brilliant green agar and have red
colonies

 On XLD medium, they produce H2S and have red colonies with a black center (Black
center with red skirt).
 On Salmonella and Shigella agar they produce colorless colonies with black center.
 The typical reaction for salmonellae in TSI (triple sugar iron) agar is an alkaline slant
(red), acid butt (yellow) and superimposed H2S (black) production
(R/Y/H2S+). Click here for visual
 The test for lysine decarboxylation is positive.

Biochemical properties

 Salmonella gives IMViC test -, +,-,+. They ferment maltose, mannitol, mannose and
glucose and produce acid and gas. But do not ferment lactose, sucrose and salicin.
 Urease negative. Most salmonellae produce H2S except S.cholera
suis and S.paratyphi A.
 Salmonella pullorum ferments glucose and rhamnose while S.gallinarum ferments
dulcitiol and maltose.

Resistance

 The bacilli are destroyed at 55 oC in one hour or at 60 oC in 15mts.

 Boiling or chlorination of water and pasteurization of milk destroys the bacilli.
 Cultures may be viable for years if prevented from drying.
 They are killed within 5 minutes by mercuric chloride (1:500) or 5% phenol.
 ANTIGENS AND ITS VARIATIONS

Antigens

Flagellar antigen

 It is heat labile protein when mixed with H antisera, agglutinates rapidly producing
large, loose fluffy clumps.
 The H antigen is strongly immunogenic. They have the unique character of diphasic
variation.

Somatic antigen

 It is a heat stable phospholipid protein polysachharide complex, which forms an

integral part of the cell wall.
 It is identical with endotoxin.
 Boivin is extracted from the bacterial cell by treating with trichlor acetic acid hence, it
is called as Boivin antigen.
 When mixed with O antiserum, it forms compact chalky granular clumps. The O
antigen is less immunogenic than H antigen.

Vi antigen

 Many strains of S.typhi, S.paratyphi and S.dublin fail to agglutinate with O

antiserum due to the presence of Vi antigen enveloping the O antigen.
 The Vi antigen is related to virulence and may be lost by serial subculture. The Vi
antigen is poorly immunogenic.
 In addition to endotoxin, enterotoxins similar to heat labile entero toxin of E.coli and
cholera toxin are produced. The cytotoxin similar to shigella cytotoxin is also
produced.

Antigenic variations

 The antigens of salmonellae frequently undergo phenotypic and genotypic variations

H-O variation

 This variation is associated with loss of flagella. When Salmonella are grown in agar
containing phenol (1 in 800), flagella are inhibited. This change is phenotypic and
temporary.
 To attain a population of Salmonella rich in H antigen Craigie‘s tube or U tube on
soft agar can be employed.

Phase variation

 Phase 1 antigen is either specific for a species or shared by a few species only. Hence
termed as specific phase.
 Phase 2 antigens are widely shared and hence termed as nonspecific or group phase.
 Phase 1 antigens are designated as a, b, c…Z and after Z as Z1, Z2, Z3…etc. Phase 2 is
designated as 1,2…etc.
 The flagellar antigens of salmonellae occur in 1 or 2 phases.
 Strains that posses both phases are called diphasic and strains having 1 phase are
called as monophasic.
V-W variation

 Fresh isolates of S. typhi carry a surface Vi antigen that completely masks the O
antigen.
 Such bacillus does not agglutinate with O antiserum but agglutinable with Vi
antiserum. This is called as V form.
 After repeated sub culturing the Vi antigen is completely lost and does not
agglutinate with Vi antiserum but agglutinate with O antiserum.
 This is called as W form. Intermediate stages which agglutinate with both Vi and O
antisera is called VW form.

S-R variation

 The smooth to rough variation is associated with the change in colony morphology,
loss of O antigen and virulence.
 The colonies become large, rough and irregular.
 R forms occur by conversion into mutation.
 It can be prevented by maintaining the cultures by lyophilisation.

Variation in O antigens

 Changes in the structure of O antigens may be induced by lysogenisation with certain

phages resulting in alteration of serotypes.

CLASSIFICATION OF SALMONELLA

O, H and Vi antigens

 Based on biochemical reactions,(Reeves, et.al., 1989), the genus Salmonella is

divided into two species, S.enterica and S.bongori.
 S.enterica is subdivided into 6 subspecies. Enterica (I), Salamae (II), Arizonae
(III a), Diarizonae (III b), Indica (IV) and Houtenae (V).
 Sub genus enterica is the largest and most important, containing all the species
that commonly cause human and animal infections.
 Members of this sub genus are given a name like S.enterica sub sp. enterica
serovar typhimurium.
 Serological classification is done by Kauffmann – white scheme. It depends on
the identification by O and H antigens of the strain and agglutination
 Example

O serogroup * Serotype Antigens

O H
Phase 1 Phase II
2 A Paratyphi A 1,2,12 a -
4 B Paratyphi B 1,4,5,12 b 1,2
Typhimurium 1,4,5,12 c 1,2
7 C Cholera suis 6,7 c 1,5
Paratyphi C 6,7 (W) c 1,5
 9 D Typhi 9 ,12 (W) d -
Enteritidis 1,9,12 g, m 1,7
Pullorum and gallinarum 1,9,12
Dublin - -
1,9,12 (Vi)
g, p -

 Primary subdivision is into O serogroups, each of which shares a common

somatic antigen.
 Where more than one O antigen is present, one of them is the major O Ag and is
regarded as determining the group to which the strain shall be allocated.
 O serogroups were formerly designated by letters of the alphabet (A-Z).
 But now are indicated by numbers 1,2,3… The series of O antigens numbered O1
–67 is not continuous because some O antigens were originally assigned to
bacteria that proved subsequently not to be salmonellae. Thus, only 46 O
serogroups are defined by the 67 O antigens described.

PATHOGENESIS

Salmonella

 Ingestion
 Gastroenteritis form Enteric fever form
 Colonize intestine Colonize intestine
 Penetrate epithelial cells of intestinal villi Penetrate epithelial cells of intestinal villi
 Produce enterotoxins Enter blood stream
 Gastroenteritis Endotoxaemia

Abortion

 Following adherence with fimbriae to the surface of the intestinal mucousal cells, the
bacteria induce ruffling of cell membranes.
 The ruffles facilitate uptake of the bacteria in membrane bound-vesicles, which often
coalesce.
 The org. replicate in these cells and eventually released from the cells.
 The virulence of salmonellae relates to their ability to invade host cells and replicate
in them.
 The O-antigen chains of LPS resist phagocytosis and long chains of LPS prevent
complement action, which will facilitate the spread of organisms.
 The LPS is also responsible for endotoxin activity.
 Salmonellae often localize in the mucosae of the ileum, caecum and colon, and in the
mesenteric lymphnodes of infected animals. Subclinical infection may persist with
small numbers of salmonellae in the faeces. Latent infections, in which salmonellae
are present in the gall bladder but are not excreted, also occur.
 Stress factors such as inter-current infections, transportation, overcrowding,
pregnancy, extreme ambient temperature, sudden changes in rations etc may activate
latent or sub clinical salmonellosis.

Diseases caused by Salmonella

Host Salmonella serotypes Disease

Cattle  S.dublin  Enteritis, septicaemia, meningitis
 S.typhimurium in calves, abortion, osteomyelitis,
joint ill, terminal dry gangrene in
calves.
 Enteritis or septicaemia

Pigs  S.cholera suis  Outbreaks clinically similar to

 S. typhisuis swine fever
 S.typhimurium  Chronic enteritis in young pigs
 Enteritis or septicaemia

Sheep  S.abortus ovis  Abortion

 S.typhimurium  Enteritis or septicaemia
 S. brandenburg  Abortion

Horse  S.abortus equi  Abortion in mares

 S.typhimurium  Enteritis or septicaemia

Poultry  S.pullorum  Pullorum disease (bacillary white

 S.gallinarum diarrhoea) in chicks
 S.arizonae  Fowl typhoid in all ages. Mainly
 S.enteritidis, adults.
S.typhimurium and many  Severe enteritis and septicaemia in
other serotypes chicks, turkey poults ( Arizona ).
 Fowl paratyphoid

Human  S.typhi  Typhoid fever

 S.paratyphi  Paratyphoid fever
 (S.paratyphi A)  Food poisoning
 S.schottmuelleri
 (S.paratyphi B)
 S.hirschfeldri
 (S.paratyphi C)
 S.enteritidis
 S.typhimurium

PATHOGENICITY

Pathogenesis

Cattle

 S. typhimurium and S. dublin infections in adult cattle gives rise to a rise in

temperature, inappetance, a sudden drop in milk yield and diarrhoea. This is
followed by dysentery.
 Pregnant animals may abort and death occurs in 2 to 3 days.
  Terminal dry gangrene and bone lesions are common in chronic infections
with S. dublin in calves.

Horses

 The serotypes commonly affecting horses are S. abortus equi and S. typhimurium.
 Abortion occurs during the last 2 months of pregnancy or at full term.
 In newborn foals, infection results in septicaemia, weakness, diarrhoea, pyrexia,
laboured breathing and death in 2-3 days.
 In some cases infections occur in the naval and joints.

Sheep and Goat

 The common serotypes isolated are S.abortus ovis and S. typhimurium.

 Abortion occurs during the last month of pregnancy.
 Death in lambs occurs during the first day after birth with diarrhoea and dysentery.

Pigs

 The serotypes commonly found are S. cholera suis and S. typhimurium.

 Pig‘s paratyphoid can occur as an acute, subacute and chronic disease.
 In acute form there will be rise in temperature, inappetance and discoloration of the
skin.
 Bluish discoloration of the ears and snout is characteristic.
 Death occurs in 1-4 days after the onset of symptoms.
 In less acute cases there will be profuse yellowish diarrhoea, poor appetite, weakness
and death in animals.

Poultry

 Diseases in poultry include infections with S. pullorum (Pullorum disease/Bacillary

white diarrhoea) , S. gallinarum (Fowl typhoid) and S. typhimurium (Avian
paratyphoid).
 In Pullorum disease the newly hatched chicks may die without any marked
symptoms.
 Other symptoms include huddling near the heat source, loss of appetite, drooping of
wings and head, whitish diarrhoea with pasty vent and respiratory distress.
 The majority of infected adults have lowered egg production.
 In fowl typhoid disease occurs among growers and adults with a mortality rate of 50
%.
 The symptoms include listlessness, diarrhoea with greenish coloured faeces and
paleness of wattle and comb. This is followed by death.
 In avian paratyphoid newly hatched birds are susceptible and symptoms are similar
to that of pullorum disease.

Lesions

Cattle

 There are areas of haemorrhagic inflammation and necrosis of the large intestine.
 The mesenteric lymph nodes are oedematous and haemorrhagic.
 Areas of necrosis occur in the liver and spleen.

Horse
  Petechial haemorrhages occur in the heart, spleen and lungs of the aborted fetuses.
 Fetal membranes are oedematous, haemorrhagic with areas of necrosis.

Sheep and Goat

 The abomasum and small intestine have areas of congestion and haemorrhage.
 Haemorrhages occur in the myocardium and kidney cortex with oema of the
mesenteric lymph nodes.

Pigs

 In acute cases the mucosa of stomach, intestine, kidney cortex and myocardium
reveals haemorrhagic spots.
 Mesenteric lymph nodes and spleen are enlarged and hyperaemic. There may be
discoloration of the skin.

Poultry

 In Pullorum disease, the liver is yellowish in color with haemorrhagic streaks.

 In chronic cases the ovary consists of pedunculate and misshapen ovules, which is
characteristic.
 In fowl typhoid the most obvious lesion includes enlarged and congested liver, which
becomes dark red or brown (bile stained liver) after exposure to the atmosphere.
 There may be multiple necrotic areas through out the liver.
 In Avian paratyphoid there will be congestion and necrosis of the liver and spleen
with catarrhal enteritis.

DIAGNOSIS

Specimens to be collected

 It includes faeces and blood from live animals, intestinal contents and tissue lesions
from dead animals, abomasal contents from aborted fetuses and parenchymatous
organs from septicaemic conditions.

Diagnosis based on

 Symptoms and lesions

 Isolation and identification of the organism - If the organism is less, initially it is
enriched in peptone water and then inoculated in selective enrichment broth.
 After incubation for 48 hours they are streaked onto selective enriched media.
 The samples used are aborted fetus, feces, blood, milk, egg and visceral organs.
 Agglutination test: In horses the O agglutinin titre of 1/1000 or more is considered
positive.
 Presence of agglutinin titre against H antigen indicates active infection.
 In cattle a titre of 1 in 40 against O antigen and l in 320 against H antigen is
considered positive.
 In poultry a titre of l in 50 & l in 25 is indicative of active infection.
 (Note: The agglutinin titre will vary with different areas for positive cases) (Click here
for visual)

 Whole blood agglutination test: 1 drop of colored or plain pullorum antigen is mixed
with 1 drop of blood and allowed for 2 minutes. In positive cases there will be clumps.
  Tube agglutination test: In this test the sample serum is diluted serially to which 0.5
ml of constant amount of antigen is added and kept at 37°C in a water bath overnight.
 Agglutinations in dilutions at a titre of 1 in 40 indicates a positive test.

CONTROL, PREVENTION AND PUBLIC HEALTH ASPECTS

Control and prevention

 In horses, cattle, sheep and goat live vaccines give rise to solid immunity but the
animal may remain as carrier throughout its life.
 Killed vaccines are safe but can stimulate only a temporary resistance.
 For cattle attenuated live culture of S. dublin vaccine has been developed.
 In poultry either killed or live attenuated vaccine stimulates good immune response.
 Various chemotherapeutic agents have been used to treat salmonellosis including
Chloromycetin, Terramycin, neomycin, furazolidone and sulfasuxidine.

Public Health aspects

 The majority of serotypes are potential pathogens for both man and animals.
 Theimportant route of transmission is through milk, meat and egg.
 This will result in Salmonella food poisoning with symptoms of severe
gastroenteritis.

CHAPTER-16: YERSINIA - INTRODUCTION

 Yersinia species are non-lactose fermenters and, with the exception of Y. pestis are
motile.
 Although there are more than 10 Yersinia species, only Yersinia pestis , Yersinia
enterocolitica , Yersinia pesudoteuberculosis are pathogenic for animals and man.
 They characteristically demonstrate bipolar staining in Giemsa-stained smears from
animal tissues.
 Yersinia pesudotuberculosis and Yersinia enterocolitica are found in the intestinal
tracts of a wide range of wild mammals, birds and domestic animals.
 All these animals may be reservoirs of infection. Many avian species may act as
amplifier hosts and may also transfer the organisms mechanically.
 They able to grow in a wide temperature range (5 to 420C) and survive for long
periods in cool wet conditions.
 In endemic areas, wild rodents areimportant reservoirs of Yersinia pestis.
 Fleas, especially Xenopsylla cheopis, the oriental rat flea, transmit the infection to
man and other animals.
 Diseases caused by Yersinia

Species Host Disease

Yersinia enterocolitica Pigs, other domestic Enteritis (sub clinical infections)

animals

Ewes Sporadic abortion

Yersinia Guinea-pigs and other Septicemia (Pseudotuberculosis) and

pseudotuberculosis laboratory animals Focal hepatic necrosis

Sheep, goats, cattle, Enteritis, mesenteric lymphadenitis

buffalo and pigs

Cattle, sheep, goats Sporadic abortion

Yersinia pestis Humans Bubonic and pneumonic plague

Rodents Sylvatic plague

Cats Feline plague

YERSINIA PSEUDOTUBERCULOSIS

Synonym: Pasteurella pseudotuberculosis

Distribution

 The organisms has a world wide distribution and is particularly associated with a
disease known as pseudotuberculosis in guinea pigs and to a less extent in turkeys,
pigeons, rats, rabbits and horses.
 It has occasionally been isolated from sheep, goats, pigs, cattle, cats & man.

Morphology

 Y. pseudotuberculosis occur as Gram negative short ovoid rods.

 They are motile at 22°C and show bipolar appearance with leishman's staining.

Cultural characters

 It can grow on MacConkey agar, which is a point of differentiation

between Pasturella multocida and Yersinia.
 Indole is not produced & nitrates are reduced to nitrates.

Antigens and Toxins

 Of the ten serotypes of Yersinia pseudotuberculosis, serotypes I,II, III contain the
majority of pathogens.
 Yersinia posses a common flagellar antigen and a common somatic antigen.

Symptoms

 The acute form of the disease develops as a sudden and acute septicaemia.
 The septicaemic form is called as pseudotuberculosis.
 Deaths may be sudden and mortality may be high.
  In chronic cases there will be intermittent diarrhoea with emaciation before death.

Lesions

 The spleen and mesenteric lymph nodes are enlarged and congested.
 In chronic cases necrotic and caseous lesions in the spleen, liver and mesenteric
Lymph nodes will be noticed, resembling pseudotuberculosis.

Diagnosis

 By isolation & identification of the organism from the liver, spleen, heart blood and
bone marrow.
 A cold enrichment procedure may facilitate recovery of yersiniae from faeces,
especially if they are present in very low numbers.
 A 5% suspension of faeces in PBS, held at 40C for 3 weeks, is subcultured weekly on
MacConkey agar.

Control and Prevention

 The organisms are susceptible to chloramphenicol and streptomycin.

Public health aspects

 Y. pseudotuberculosis in man produce chronic infection of the mesenteric lymph

nodes and produce symptoms suggestive of an appendicitis.

YERSINIA PESTIS

 This organism causes bubonic plague or pneumonic plague in man. It is carried by

rats & other rodents.
 They are Gram-negative short coccobacilli showing bipolar staining with Leishmans
stain.
 It can grow aerobically and anaerobically in the presence of serum and blood.
 It grows on MacConkey agar and capsule formation is noticed at
37°C. Y.pestis produces a capsular antigen and a somatic antigen.
 It causes pneumonic or bubonic plague and the lesions include swollen and
haemorrhagic lymphatic glands and spleen with multiple necrotic areas in the spleen
and liver.
 Diagnosis is done by isolation & identification of the organism.
 Live and killed vaccines are used for prevention and plague cases are treated with
oxytetracycline & other antibiotics.
 Three clinical forms of feline plague are recognized; bubonic, septicaemic and
pneumonic.
 The most common form of the disease is characterized by enlarged lymphnodes
(Buboes) associated with lymphatic drainage (serosanguineous fluid or pus) from the
site of infection.
 Septicaemia may occur without lymphadenopathy and is potentially fatal.
 Pneumonic lesions may result from haematogenous spread.
 Cats with pneumonic lesions are potential source of human infection through aerosol
generation.

YERSINIA ENTEROCOLITICA
 Y. enterocolitica has been identified from pigs, horse, ox, sheep and goat, dogs, cats,
rodents etc. 57 O-factors, 6 K -antigens and 19 H - antigens have been identified.
 Based on this there are five biotypes and more than 50 serotypes have been
identified.
 Somatic antigens 2,3,5,8 and 9 are present in isolates from clinical infections caused
by this species.
 Serotype O:9 is of particularimportance because it shares common antigens
with Brucella species and it may induce false-positive reactions in brucella
agglutination tests.
 They produce either generalized infections or enteritis.
 It can grow on blood agar and the optimum temperature for growth is 28°C.
 A preceding cold enrichment improves the isolation rate.
 It produces gastroenteritis in human infants and terminal ileitis, mesenteric
lymphadenitis and pseudo appendicitis in adolescents.
 The pig is the natural reservoir for Yersinia enterocolitica serotype O3 biotype 4,
which is animportant pathogen in humans

SHIGELLA

 Four species (S. boydii, S. dysenteriae, S. flexneri and S. sonnei) are etiological
agents of diarrhoea and dysentery in man and subhuman primates.
 They are Gram –ve, non-motile, rod shaped organisms found in the intestinal flora of
animals.
 Shigella are facultatively anaerobic and grows on blood agar and MacConkey agar at
37oC without haemolysis.
 Shigella produces an enterotoxin which acts on the epithelial lining of the terminal
illeum and large intestine, producing bloody mucus stained stool.

CITROBACTER

 Citrobacter freundi forms part of the normal intestinal flora of domestic animals
and are also found in soil and water.
 They are Gram negative, facultatively anaerobic, motile, rod shaped organisms,
which grows on blood agar and MacConkey agar at 37°C producing no
haemolysis.
 In domestic animals, Citrobacter are opportunistic pathogens in a variety of
extra intestinal infections.

EDWARDSIELLA

 Edwardsiella tarda appears to have a worldwide distribution and has been isolated
from all classes of vertebrates, fish, amphibia, reptiles, birds and mammals.
 They are Gram negative, motile rods. They are facultatively anaerobic and grow on
blood agar and MacConkey agar at 37°C.
 They are non-haemolytic and lactose negative organisms.
 E. tarda has been implicated as the cause of diarrhoea,wound infections and sepsis in
animals and humans.

ENTEROBACTER

 E. cloacae and E.agglomerans are the most commonly isolated species.

  They inhabit mainly the environment and they are also found in the digestive tract of
animals.
 They are Gram negative, motile rod shaped organisms.
 They are facultatively anaerobic and grow on blood agar and MacConkey agar at
37°C.
 In domestic animals, Enterobacter species are opportunistic pathogens in a variety of
extra intestinal infections.
 Enterobacter species is attributed to abortion and infection of genital tract in horses,
abortion and mastitis in cattle and agalactia syndrome in pigs.

KLEBSIELLA

Klebsiella (Friedlanders bacillus)

 Kelbsiella species occur naturally in soil and water and are normal intestinal flora of
domestic animals.
 They are non-motile, encapsulated rods arranged singly, in pairs or short chains.
 The Klebsiella are non haemolytic on blood agar and lactose positive on MacConkey
agar.
 Klebsiella pneumoniae has O antigen (11 types) and K antigen (70 types).
 In horses Klebsiella causes inflammation of the genital mucosa and abortion and
generalized infections of foals.
 In cattle it causes mastitis, generalized infections and enteritis of calves.
 In pigs it causes atrophic rhinitis and in poultry it causes air sac and yolk sac
infections.
 K. pneumoniae and K. oxytoca are the most commonly isolated species from
domestic animals.

MORGANELLA

 M. morganii is the normal intestinal flora of domestic animals.

 They are Gram negative, motile rod shaped organisms.
 They are facultatively anaerobic and grow on blood agar and MacConkey agar at 37°
C.
 In domestic animals, M. morganii is an opportunistic pathogen and is most
frequently isolated from wound and urinary tract infections.

PROTEUS

 Proteus mirabilis and P.vulgaris are normal intestinal flora of domestic animals and
also are found in soil and polluted water.
 Proteus is Gram-negative rod shaped organism but often show pleomorphism.
 All strains are normally motile with peritrichous flagella and majority produce
fimbriae. Spores and capsules are not produced.
 These organisms are aerobic and facultatively anaerobic and growth takes place at
37°C growth on solid media shows the feature of swarming.
 All species are positive to the phenylpyruvic acid. They are responsible for
endometritis, gastroenteritis and arthritis.
 In pigs it causes diarrhoea and urinary tract infections.
 In dogs and cats it causes urinary tract infections, cystitis, otits externa and
sepsis. Click here for visual

PROVIDENCIA
 Domain Bacteria

Phylum Proteobacteria
 Providencia rettgeri and P.stuartii are
found in the normal Class Gamma intestinal flora of domestic
animals. proteobacteria
 They are Gram- negative rods and motile by
peritrichous flagella.
 They are facultatively anaerobic and growth on
blood agar and MacConkey agar at 37°C
produces no haemolysis and is lactose
negative.
 P.rettgeri is frequently isolated in pure culture from
urinary infections.

SERRATIA

 S. liquefaciens and S. marcescens form part of normal intestinal flora of domestic

animals and also occur in soil and water.
 They are Gram-negative rods and are motile by peritrichous flagella.
 They are facultatively anaerobic. On blood agar the colonies are white, pink or red in
color.
 S. marcescens produces a deep red pigment and on MacConkey agar at 37°C produce
lactose positive colonies.
 S. marcescens are isolated from conjunctivitis, pneumonia, septicaemia and
endometritis in horses and in cases of abortion and mastitis in cattle.

CHAPTER-17: PASTEURELLA

Learning objectives

To know in detail about,

 Fowl cholera, Haemorrhagic septicaemia and shipping fever

 Morphology, cultural and biochemical characters of Pasteurella sp
 Difference between P.multocida and P.haemolytica (Mannheimia haemolytica)
 Antigns, toxins and pathogenesis of Pasteurella sp
 General approaches used to isolate and identify Pasterurella sp
 Leishman staining technique

 SYSTEMATICS
 Order Pasteurellales

Family Pasteurellaceae

Genus Pasteurella

Genus Mannheimia

Genus Haemophilus

Genus Actinobacillus

Genus Lonepinella

HISTORY AND HABITAT

History

 The first significant report of the P.multocida was made by Bollinger (1878) and
Perroncito (1878) and it was extensively investigated in 1880 by Pasteur as the cause
of Fowl cholera.
 The P.haemolytica was isolated from disease of calves and sheep by Jones (1921).
 The name Pasteurellacea was proposed by Mannheim (1981).

Habitat

 Pasteurella organisms are world wide in distribution with a wide spectrum of hosts.
 They infect practically all domesticated and many wild animals and birds.
 They occur as commensals in the mucous membrane of the Upper respiratory and
intestinal tract of animals.

MORPHOLOGY

 They are of small Gram –ve rods or coccobacilli that showbipolar

staining particularly when fresh isolates stained with Leishman‘s or Wrights or
Giemsa. They are non motile and non spore forming. ,

Cultural characteristics

 Growth takes place at an optimum temperature of 37oC. Growth isenhanced by the

addition of blood or serum. On serum agar Pasteurella exhibit 3 different colonial
forms
o Smooth form: Virulent for rabbits, growing diffusely in broth. Forming
smooth, moderately opaque iridescent colonies on serum agar. It contains a
type specific polysaccharide capsular antigen.
o Rough form: Completely avirulent for rabbits, giving a granular deposit in
broth and forming translucent bluish colonies. It has neither a capsular nor a
mucoid antigen.
o Mucoid form: Intermediate virulence. It is rich in hyaluronic acid.
  The routine medium for isolation of Pasteurella species is Brain heart Infusion agar
and Ox or Sheep blood agar.
 P. multocida has a characteristic sweetish odour, is non-haemolytic, and does not
grow on MacConkey agar
 Mannheimia haemolytica and P. trehalosi are beta haemolytic. On sheep blood agar,
colonies are surrounded by a single narrow zone of hamolysis, but in lamb blood agar
a double zone haemolysis may be seen. Mannheimia haemolytica and P.
trehalosi usually tolerate the bile salts in MacConkey agar to grow as pin point pink
red colonies. On TSI produce Y/Y/H2S-ve .
 P. anatipestifer produces dewdrop colonies within 48 hrs.

BIOCHEMICAL PROPERTIES, ANTIGENS, TOXINS AND

RESISTANCE

Biocehmical properties

 They are oxidase +ve, catalase +ve and fermentative (except P.

trehalosi and R.anatipestifer). P.multocida is indole positive.
 Mannheimia haemolytica is indole negative. They ferment several sugars (glucose,
sucrose, galactose and sorbitol) with acid and no gas.
 P.anatipestifer is non fementative, non haemolytic, does not grow on MacConkey
agar, indole –ve, urease –ve.

Antigens and toxins

 Based on capsular polysaccharide antigens, using PHA, Carter

classified P.multocida into 5 serotypes (A-F). A type C is not a valid capsular
serotype.
 The capsular types may be subdivided further into 16 somatic types based on heat
stable somatic antigen, using AGPT (i.e. serologic differences in LPS). The capsular
type followed by the number representing the somatic type designates a serotype
 For e.g. Type A:5, A:8, A:9 produce fowl cholera
 Type B:6 cause haemorrhagic septicaemia in Asia
 Type E:6 cause haemorrhagic septicaemia in Africa
 The toxin produced by P.multocida (mostly type A and D strain) is mainly cell
associated, polypeptide, heat stable, dermonecrotic, lethal and immunogenic.
 In case of Mannheimia haemolytica/ P. trehalosi, on the basis of soluble surface
antigens so far 17 serotypes have been recognized.
 There are two distinct biotypes, A and T, based on fermentation of arabinose and
trehalose respectively.
 Serotypes 3,4,10 and 15 are P. haemolytica biotype T - P. trehalosi, all others are A -
Mannheimia haemolytica.
 Biotype A strains are associated with ruminant pneumonias, septicaemia in young
lambs and ovine mastitis,while biotype T strains are usually associated with
septicaemic infection in older lambs and sheep.
 All strains of Mannheimia haemolytica elaborate a soluble cytotoxin (Leukotoxin).
 It is a protein of relatively large mol.wt, thermolabile, resembles the alpha
haemolysin in E.coli and is immunogenic.

Resistance

 They are readily killed at 550C for 15mts or if exposed to sunlight for 3-4 hrs.
 They are also killed by 0.5% phenol in 15 mts.
 They are susceptible to penicillin but resistant to sulphonamides.
 PATHOGENESIS

 The mechanism of disease production is not fully understood.

 Pasteurella infections result from the invasion of commensal organism during period
of stress such as overcrowding, chilling, transportation or intercurrent infection. But
exogenoustransmission may occur by aerosal or contact exposure.
 In birds, the organisms are transmitted from nasal, oral or conjuctival secretions of
infected birds, often-convalescent carriers, and by aerosal or contamination of
drinking water and feed. Clinical disease is precipitated by stress such as
overcrowding, laying, moulting and severe climatic change.
 Passage of infecting agent from animal to animal results inenhancement of
virulence.
 P.multocida is a primary cause of fowl cholera and Haemorrhagic septicaemia.
However, P.multocida is a frequent secondary invader in pneumonic disease. When it
is primary, septicaemia frequently occurs.
 Septicaemia results from penetration of the pharyngeal mucous membrane by highly
pathogenic strains. They resist phagocyotsis and elaborate toxins.
 Pneumonias of cattle and sheep are the most important Mannheimia
haemolytica infection.
 Disease usually follows within 1-2 weeks of a stressing experience such as
transportation, hence the name shipping fever in cattle.
 Serotype 1 predominates in bovine pneumonia, serotype 2 in ovine disease.
 Pathogenesis is related to the adherence and colonization of the lower respiratory
epithelium.
 All serotypes produce soluble leucotoxin. The toxin impairs phagocytosis and causes
of lysis of cells, which allows the movement of K, Na and calcium through trans
membrane gradients. This leads to lung damage.
 Lysis of platelets results in pulmonary vascular thrombosis and fibrin exudation
typically associated with shipping fever pneumonia.

Diseases caused by Pasteurella

Speices Disease

P.multocida (Based on Capsular polysaccharide typing)  Fowl cholera

 Haemorrhagic septicaemia / Barbone ( Asia
and Australia )
 Type A  Atrophic rhinits of pigs
 Type B  Haemorrhagic septicaemia ( Africa )
 Type D  Disease in turkey
 Type E
 Type F

 Mannheimia haemolytica (P.haemolytica Biotype  Pneumonia (Shipping fever) in cattle and sheep
A)  Septicaemia in lambs
 Pasteurella trehalosi (P.haemolytica Biotype T)

 P.anatipestifer (Rimerella anatipestifer)  New duck disease ( 1-8 wk old ducklings)

PATHOGENECITY

Symptoms

Cattle

 In acute cases of haemorrhagic septicemia the symptoms include a rise in

temperature, a sudden drop in milk yield, signs of abdominal pain, severe diarrhoea
and dysentery.
 Respiration becomes rapid and shortly before death the mucous membranes appear
cyanotic.
 In less acute cases there will be odema development in the region of the head, neck
and brisket.
 The nasal discharge may be blood stained or purulent. Death occurs within 2-4 days.
 P.haemolytica give rise to symptoms of bronchopneumonia with pleuritis. This
condition is referred as transit or shipping fever.

Sheep

 Pasteurella infection results in pneumonia and mastitis.

Pigs

 Pasteurella type D infection causes atrophic rhinitis in pigs and is a secondary

invader in respiratory infections.

Poultry

 The acute form of fowl cholera can affect many species of wild and domesticated
birds including chicken, turkey, ducks and geese causing high mortality.
 Death occurs suddenly without any predominant symptoms.
 In ailing birds, breathing is rapid through the open beak, the feathers are ruffled and
the comb and wattles become cyanotic.
 In chronic cases swollen wattles and comb, hot and painful joints are characteristic.

Lesions
Cattle

 In acute form, the lesions include multiple haemorrhages on the serous membranes
and organs together with blood stained exudates in the thorax and abdomen.
 There will be severe gastroenteritis with enlargement of mesenteric lymphnodes.
 In edematous form the striking lesion is odema occurring in the subcutaneous tissues
in the region of throat, and brisket region.
 In pectoral form, the lesions are confined to the lungs and pleural cavity with
enlargement of bronchial and mediastinal lymph glands.

Sheep and goat

 In acute cases there may be exudates in the pleural cavity and pericardial sac.

Poultry

 The liver and spleen are enlarged, hyperaemic with necrotic foci.
 There will be peritonitis, fluid in the pericardial sac and petechial haemorrhages on
the serous surfaces.
 In chronic infections there will be swelling of the comb and wattles and accumulation
of serofibrinous fluid in the joints.

DIAGNOSIS, CONTROL AND PREVENTION

Diagnosis

 By symptoms and lesions

 Leishman‘s stain from smears of heart blood, liver, spleen, lungs or exudates reveal
bipolar stained organisms.
 Isolation and Identification of the organisms from heart blood and fluid exudates in
blood agar and brain heart infusion agar will show dewdrop like colonies after 18hrs
incubation.
 In putrified material bone marrow of long bones are useful to get pure cultures.
 Animal inoculation is done by inoculating the suspected material or bacterial culture
in mice and rabbits by scarification or subcutaneous route.
 The animal dies in 24-72 hrs with haemorrhagic tracheitis.

Control and prevention

 Immunity is predominantly humoral. Pasteurella multocida P52 strain have proved

useful. It gives protection for 6 months to 1 year.
 Vaccines for fowl cholera are not successful. So vaccines should be prepared from
types prevalent in that area for protection.
 Pasteurella is amenable to Penicillin-G, streptomycin, chloramphenicol,
chlortetracycline, sulpha and tripmethoprim, enrofloxacin and oxytetracycline.
 It is one of the few Gram-negative bacteria sensitive to penicillin.

CHAPTER-18: HEMOPHILUS AND ACTINOBACILLUS

Learning objectives

To know in detail about,

  Morphology, cultural and biochemial characters of Haemophilus species
 Collection and transport of speciments for diagnosis of Haemophilus infection
 Wooden tongue and morse biochemical characters of A. ligneresi
 Antigens, toxins and pathogenesis of A.ligneresi in cttle
 Difference betweeen Actinobacillosis and Actinomycosis
 General approaches used to isolate and identify A.ligneresi

HISTORY AND HABIT

Haemophilus

 Species of the genus Haemophilus are small, non-motile, non-sporing, Gram-

negative rods and filaments.
 They are characterized by their requirement of one or both of twoaccessory growth
factors (X and V) present in blood. (Haemophilus, means blood loving) .

History

 In 1883, Koch had described a bacillus causing conjunctivitis in Egypt , eventually

named H.aegypticus.
 In 1892, Pfeiffer isolated H.influenza from sputum of patients. H.parasuis was first
described by Glasser in 1910. H.paragallinarum was identified by DeBlieck (1932).

Habitat

 Haemophilus species are commensals or parasites of the mucous membranes of

human and animals, most commonly of the upper respiratory and lower genital
tracts.
 H.somnus is part of the bacterial flora of the male and female bovine genital
tract. H.paragallinarum is more closely associated with the upper respiratory tract
and sinuses of sick or recovered birds.

MORPHOLOGY AND CULTURAL CHARACTERISTICS

Morphology

 Gram negative, small, medium sized coccobacilli or rods, often markedly

pleomorphic, sometimes filamentous, non-motile, non-spore forming and non-acid
fast.
 Capsules can be produced. H.inflenzae, H.parasuis and H.paragallinarum require
one or both of twoaccessory growth factors X and V.

Cultural characteristics

 They are aerobic and facultatively anaerobic. The opt.temp.is 370C.

 They are nutritionally fastidious, will not grow on nutrient agar and MacConkey agar.
 The X and V factors must be supplied for all the Haemophilus species
except H.somnus.
 The X factor is a heat labile iron-porphyrin-haematin or other haemins.
 It is necessary for the synthesis of catalase and other enzymes involved in aerobic
respiration.
 The V factor is a heat labile factor, present in RBC‘s and in many other animal and
plant cells.
  It is synthesized by fungi and some bacteria ( eg: Staphylococcus aureus).
 The V factor can be supplied as co-enzyme I, NAD or NADP+.
 It appears to act as a hydrogen acceptor in the metabolism of the cell.
 X and V factors requirement of Haemophilus species

Species X factor V factor

H. influenzae + +

H. aegyptius + +

H.parasuis - +

H.paragallinarum - +

H.parinfluenzae - +

H.somnus - -

 In addition to X and V factors, the growth of the many of the Haemophilus species
isenhanced by 10% C02.
 Haemophilus grows on blood agar, but growth is scanty, as the V factor is present
mainly intra cellularly in red cells.
 The chocolate agar is the most suitable medium for isolation of Haemophilus.
 In chocolate agar, the V factor is released from the red cell, and the heat stable X
factor is still present.

 On chocolate agar, H.paragallinarum produces typical dewdrop like colonies

and H.somnus shows characteristic yellow tinge colonies.

Sattelitism

 When Staphylococcus aureus is streaked across a plate of blood agar on which a

specimen containing Haemophilus has been inoculated, after 18-24hrs incubation at
370C under 5-10% Co2, the colonies of the Haemophilus will be large, well developed
alongside the streak of Staphylococcus, and smaller farther away.
 This phenonmenon is called sattelitism and demonstrate the dependence
of Haemophilus on V factor, which isavailable in high concentration near the
staphylococcal growth and only in smaller quantities away from it. Click here for
visual

 Media supplemented with yeast extract, Levinthals medium (clear transparent media
may be prepared by boiling and filtering a mixture of blood and nutrient broth) or
Filde‘s agar (by adding a peptic digest of blood to nutrient agar) are also suitable for
the primary isolation of Haemophilus.

MORPHOLOGY

Biochemical properties
  Biochemical reactions are not helpful in identification.
 The fermentation reactions are irregular. Nitrates are reduced to nitrites.
 H.parasuis and H.paragallinarum are oxidase negative but H.somnus are oxidase
positive and catalase negative.

Resistance

 Haemophilus species are fragile. They are readily destroyed by heating (550C for
30mts), refrigeration (00C to 40C), drying and disinfectants.
 In cultures the cells die within 2 to 3 days due to autolysis. For long-term
preservation the cultures may be lyophilized.

Antigens

 Non-capsulated strains are antigenically heterogenous. Some somatic antigens have

been identified.
 The capsular antigens are polysaccharide in nature and they resemble pneumococcal
capsular antigens.
 It is immunochemically similar to the K antigens of E.coli.
 Based on immunodiffusion test, using heat stable antigens 15 serovars of H.parasuis,
9 serotypes of H.paragallinarum and 15 serotypes of H.somnus have been
recognized.

PATHOGENESIS

 Young or previously unexposed animals are highly susceptible. Stress factors

contribute to the development of disease.
 The capsule and cytotoxic factor are thought to be virulence factors and endotoxin
may play a role in the disease process.
 During stress, the bacteria may invade the mucosal barrier. The invasive mechanism
is not known.
 In respiratory tract initially nasopharyngitis. If this infection is not checked, it may
lead to sinusitis, otitis media and pneumonia.
 If a bacteraemia develops, joint infections and meningitis may follow.
 Diseases caused by the Haemophilus species

Species Host Disease

H. somnus Cattle Thrombo embolic meningo encephalitis (TEME)
(Sleepers)
Sheep Pneumonia and pleurisy
Arthritis
Endometritis and abortion
Epididymitis and orchitis in rams
H.parasuis Pigs Polyserositis and meningitis in young pigs (Glasser‘s
disease )
H.paragallinarum Poultry Infectious coryza
H.influenzae Human Variety of diseases ranging from respiratory infections to
meningitis
PATHOGENICITY
Symptoms

 Haemophilus infection of cattle is manifested by 4 principal syndromes.

In sub clinical form animal won't exhibit any clinical signs.
 In acute form if there is respiratory involvement with pneumonia and septicaemia,
the symptoms are fever, dry cough and dyspnoea.
 If the bacteria localized in the CNS with TEME, (i.e. sleepers) the signs are lameness
and CNS disturbance with high mortality.
 In chronic form, there will be joint infections accompanied by arthritis and
reproductive failure.
 In H.parasuis infection mostly very young, weaned pigs are highly susceptible.
 The infections occur concurrently with virus infections. In chronic form, the affected
pigs exhibit lameness, pyrexia, depression and anorectic.
 In poultry, H.paragallinarum causes acute rhinitis, sinusitis with odema and
conjunctivitis.
 The disease is characterized by nasal discharge, sneezing and odema of the face.
Reduction in growth and egg production also occur.

Lesions

 In Glasser's disease during acute death conditions, the PM reveals large deposition of
fibrin in joints and on any or all of the serosal surfaces (poly serositis) of the body.
 In addition there will be fibrinous pericarditis, pleuritis and peritonitis. Catarrhal
inflammation of the infra orbital sinus is characteristic of infectious coryza.

DIAGNOSIS AND TREATMENT

Diagnosis

Specimens

 In H.somnus infections, the organism can be demonstrated in brain lesions, they can
be recovered from semen samples and preputial washings of healthy bulls.
 Haemophilus species are highly delicate and do not survive long when removed from
the host.
 Clinical material is best frozen (dry ice preferred) and delivered to the laboratory
within 24 hrs.
 Refrigeration and transport media may not assure viability.
 A presumptive identification of Haemophilus can be made based on the host species,
clinical signs and lesions, colony characters, X and V factor requirements, oxidase
and catalase reactions and whether or not C02enhances growth.
 Serological tests including agglutination, AGPT, ELISA are used to
detect H.paragallinarum and H.somnus infection.

Treatment

 Haemophilus species are susceptible to gentamicin, tetracycline, sulfonamides,

chloramphenicol, neomycin and erythromycin.
 Vaccines prepared from H.paragallinarum grown in egg yolk, if inoculated
intramuscularly reduces the incidence of infectious coryza.

HISTORY AND HABITAT

  The Actinobacillus species are Gram negative, very small, non-motile, non-sporing
and non acid-fast bacilli.
 Small coccal elements are often lying at the pole of a larger form, giving a
characteristic ‗Morse-code‘ appearance

History

 Actinobacillus lignieresii was isolated by Ligniers and Spitz (1902).

 The generic name actinobacillus was first used by Brumpt (1910).

Habitat

 Actinobacillus species is worldwide in distribution. They are commensals of the

respiratory, alimentary or genital mucosa.
 A.lignieresii is a commensal in the oral and rumen of cattle and sheep.
 A.equuli occurs as a commensal in the equine intestinal tract and in the mouth.
 A.suis is present in the tonsil and upper respiratory tract of healthy pigs.
 Actinobacilli cannot survive in the environment, carrier animals play a major role
intransmission .

MORPHOLOGY AND CULTURAL CHARACTERISTICS

Morphology

 Gram –ve, small, rod shaped organism. They are non-motile, non-sporing and non-
acid fast.
 They are non capsulated (except A.pleuropneumoniae) but extracellular slime is
present in three major species (A.lignieresii, A.equuli and A.suis)
 In media containing fermentable carbohydrates, the occurrence of rather long,
almost filamentous forms is seen.
 Small granules are found scattered along the bacilli, often lying at the pole of a
bacillary or filamentous form, giving a characteristic ‗Morse code‘ form.
 In lesion in the animal body small grayish white granules are present.
 If these granules are crushed on a slide and stained, club colonies are seen consisting
of club-like processes of calcium phosphate, with Gram-negative rods of A.
lignieresii in the center.
 Both bacilli and club forms are Gram negative.
 They can be distinguished with ZN stain in which the club appears red and the bacilli
blue.

Cultural characteristics

 They are aerobic, or micro aerophilic, and facultative anaerobe.

 The optimum temperature is 370C on blood agar, A.lignieresii develops small,
glistening, non-haemolytic colonies within 24 hrs.
 They are usually slightly sticky (cohesive properties) on primary isolation, but lose
this character on subculture.
 The organism grows well on MacConkey agar and it is a late lactose fermenter. The
colonies are first pale but become pinkish.
 A. equuli strains are haemolytic. The colonies are sticky with this feature remaining
on subculture. It is lactose fermenter on MacConkey agar.
 A.suis are also haemolytic. Colonies are stickier. It grows well on MacConkey agar.
 In CAMP test A.pleuropneumoniaeenhances beta haemolysis of Staphylococcus
aureus. (i.e. Positive).
BIOCHEMICAL PROPERTIES, RESISTANCE, PATHOGENS AND
TOXINS

Biochemical properties

 They are catalase positive (except A.pleuropneumoniae). Oxidase and urease

positive.
 Ferment several sugars, produce acid and gas. IMViC negative, H2S positive.

Resistance

 They are rapidly killed by heating at 620C for 10 mints and by drying.
 Culture lose their viability rapidly and should be subcultured every 5-7 days.

Antigens and Toxins

 In A.lignieresii, heat stable somatic and heat labile surface antigens are described.
 Six antigenic types (1-6) and two subtypes (1a and 1c) have been demonstrated.
 In A.equuli, as in A.lignieresii, both heat labile and heat stable antigens can be
demonstrated.
 In A.suis, the antigens have not been studied in any detail.
 In A.pleuropneumoniae, 12 serotypes and 2 biotypes have been described,.
 Biotype 1 requires NAD for its growth while biotype 2 is NAD independent.
 In A.pleuropneumoniae, capsule, LPS, outer membrane proteins and toxins
(haemolytic and cytotoxic) play a major role in pathogenicity.
 Exotoxin is not formed in A.lignieresii.

PATHOGENESIS

 A.lignieresii , is a commensal of oral cavity and the intestinal tract.

 It can survive for upto five days in hay or straw.
 The actual mechanisms of actinobacilli are unknown.
  Bovine actinobacillosis is spread by the lymphatics. The specific disease is
wooden (timber) tongue, but granulomatous lesions can also involve the head,
neck and limbs.
 Less commonly, the lungs and other internal organs are also affected.
 A.suis : Infection occurs via the aerosal route by close contact or through skin.
 Once the organism has entered the blood stream it spreads rapidly throughout
the body.
 Several factors, LPS, cytotoxin etc are responsible for gross lesions and they are
usually seen in the lungs, kidney, heart, spleen, intestines and skin.
 The lungs may also be filled with serous or serofibrinous exudates with
pleuropneumonia.
 A.pleuropneumoniae : The organism enters the lungs, multiplies rapidly.
 During growth the organism releases a large quantity of OMP, LPS, cytokines
and other factors which causes destruction of neutrophils that is likely to be
responsible for the massive and tissue damage.
 Diseases caused by the pathogenic actinobacilli

Species Host Disease

A. lignieresii Cattle Bovine actinobacillosis (Wooden
(timber) tongue )
Polygranulomatous lesions
around head, neck and limb
A.equuli Neonatal foals Sleepy foal disease or Joint ill
Mares Abortion./septicaemia
A.pleuropneumoniae Pigs Arthritis, nephritis and
(Haemophilus endocardtitis
pleuropneumoniae)
A.suis Pigs under 3 Acute fatal septicaemia
months of age Arthritis, pneumonia and
Older pigs pericarditis
A.seminis Rams Epididymitis
PATHOGENICITY

Symptoms

 Cattle
o Chronic pyogranulomatous lesions occur on tongue and other soft tissues.
o Enlargement and protrusion of the tongue that interferes feeding. The pus
contains soft grayish white granules.
 Pigs
o A.suis can infect pigs of all ages. But infection is most serious in very young
animals.
o In neonates and suckling pigs, A.suis can cause an acute and rapidly fatal
septicaemia.
o Death occurs within 15hrs. Affected animal may show signs of cyanosis,
petechial haemorrhages, fever, respiratory distress, neurologic disturbances
and arthritis.
o In older animals, the disease is less severe and may be characterized by fever,
anorexia and persistent cough. The mortality is also much lower.
o A.pleuropnemoniae can cause acute and rapidly fatal pleuropneumonia.
o The acute form is characterized by extensive haemorrhage and fibrin deposit
in the lungs.
o Affected animals show signs of severe respiratory distress, cyanosis, fever and
vomiting.
o In chronically infected animals the organism may be sequestrated in the lungs
in the necrotic lesions, tonsils and URT and may responsible for spread of
infection.
 Sheep
o Actinobacillus seminis is a common cause of epididymitis in young rams.
o The organism is found in the prepuce. The infection occurs probably following
an ascending opportunistic infection.
o Abscesses and purulent discharge through fistulae on the scrotal skin are
most commonly seen.
 Horse
o Sleepy foal disease is an acute, potentially fatal septicaemia of newborn foals
caused by Actinobacillus equuli.
o Occasionally it causes abortion and peritonitis in adult horses.
o The organism is found in the reproductive and intestinal tracts of mares.
o Foals can be infected in utero and after birth via the umbilicus.
o Affected foals are febrile and recumbent. Death usually ocuurs in 1 to 2 days.

DIAGNOSIS, TREATMENT, PREVENTION AND CONTROL

Diagnosis

 Specimens to be collected
o Pus, biopsy material and tissues in case of wooden tongue.
o Tracheal washings or affected portions of lung in pleuropneumonia cases.
 Based on history and symptoms
  Isolation and identification of organism
o Club colonies in tissue sections, growth pattern on blood and MacConkey agar
and biochemical test are highly useful.

Treatment

 In wooden tongue, sodium iodide parentrally or potassium orally is effective.

 Sulphonamides or combination of penicillin and streptomycin are usually effective.
 Oral isoniazid for 30 days has been used in animals with refractory lesions.
 Ampicillin, carbenicillin, potentiated sulphonamides and tetracyclines are effective
against A.suis infection.

Control and prevention

 Polyvalent bacterins may induce protective immunity but fail to preventtransmission

or the development of a carrier state.

CHAPTER-19: PSEUDOMONAS

Learning objectives

After reading this chapter, the learner will understand the following,

 Diseases produced by the pseudomonas in domestic animals

 Define white more's bacillus, Glanders, Farcy and fleece rot
 Morphology, cultural and biochemical characters of pseudomonas species
 Pigments produced by the strains of Pseudomonas aeruginosa
 Antigens, toxins and pathogenesis of Pseudomonas
 Strauss reaction and Mallein test
 General approaches used to isolate and identify Pseudomonas species

SYSTEMATICS

 The Pseudomonas is Gram –ve, aerobic, medium-sized rods, motile by one or several
polar flagella (except P.mallei).
 Catalase and oxidase positive and some species produce water-soluble pigments and
most will grow on MacConkey agar.

Domain Bacteria

Phylum Proteobacteria

Class Gamma proteobacteria

Order Pseudomonadales

Family Pseudomonadaceae
Genus Pseudomonas

HISTORY AND HABITAT

History

 P. aeruginosa was first isolated by Gessary 1882

 P. mallei was discovered by Loeffler and Schutz –1882
 P.pseudomallei was first described by Whitemore and Krishnaswamy 1912

Habitat

 Most of the species in this genus are saprophytes,

including P.aeruginosa and P.pseudomallei.
 The P.aeruginosa is ubiquitous in the environment and is found in water, soil and on
plants, as well as on skin and mucous membrane of healthy animals.
 The P.pseudomallei mainly is present in the tropics and it is considered to be a soil
organism.
 Wild rodents act asimportant reservoir of this organism. P.mallei is an obligate
parasite.

MORPHOLOGY

 Gram negative, rod shaped bacteria. They have parallel sides and rounded ends.
 They are arranged singly, in small bundles, in short chains or in filaments.
 All except P.mallei are motile by means of polar flagella at one or both ends.
 Some strains are lopotrichate and some are monotrichate. P.pseudomallei is
peritrichous.
 Non spore forming and non acid fast.
 It is non capsulated but when grown in the absence of sucrose an extracellular
polysaccharide slime layer may be formed.
 Most of the strains possess fimbriae.

CULTURAL CHARACTERISTICS

 Very strict aerobes, growth occurs at wide range of temperature 5-420C.

 Pseudomonas species are non fastidious and will grown on TSA, blood agar and on
less complex media.
 Agar that contains cetrimide is the selective medium for P.aeruginosa.
 On nutrient agar, the colonies are grayish blue with a characteristic fruity, grape like
odour (amino acetophenone) and gives different types of colonies (Smooth form,
Mucoid form and rough form). Some strains have colonies with distinct metallic
sheen.
 The strains of P.aeruginosa produce diffusible pigments
o Pyocyanin – bluish green in color, which dissolves in Chloroform
o Pyoverdin – greenish yellow in color which is soluble in water but not in
chloroform (Fluorescin)
o Pyorubin – red
o Pyomelanin – dark brown to black pigment
 Some strains produce all 4 pigments, pyorubin and pyomelanin are less commonly
produced.
 The pigment production is best seen by growing the strains on nutrient agar slants at
room temperature for upto two weeks.
  Pyocyanin-, which is unique to P.aeruginosa , gives the blue color associated with
many cultures.
 Most strains give a clear zone of haemolysis on blood agar.
 On MacConkey agar, P.aeruginosa produce (non-lactose fermenter) pale colonies
with greenish blue pigment superimposed.
 They also grow on Brilliant green agar and XLD medium.
 P.pseudomallei colonial growth varies from smooth and mucoid, to rough form with
a dull, wrinkled, corrugated, honeycomb surface.
 In smooth form the colonies are shiny and grayish yellow. After several days the
colonies become yellowish brown and umbonate.
 The growth has a characteristic earthy or musty odour due to the production of
ammonia.
 Partial and later complete haemolysis is observed on sheep blood agar.
 It is a lactose fermenter on MacConkey agar but there is no growth on deoxycholate
or salmonella shigella (SS) agar.
 P.mallei growth is very slow; the colonies are smooth, and white to
cream. P.mallei cannot be grown on MacConkey agar.

BIOCHEMICAL PROPERTIES, ANTIGENS AND TOXINS

Biochemical properties

 In addition to pigment production (P.aeruginosa only) and characteristic odour the

pseudomonads are strongly oxidase positive, reduce nitrate to nitrite, liquefy gelatin,
IMViC -,-,-,+.
 All ferment glucose and produce less acid. P.pseudomallei ferments lactose. OF test is
positive for P.aeruginosa.

Antigens and toxins

 The Pseudomonas is highly heterogenous. Serotyping, immunotyping, pyocin typing

and phage typing are all used for characterization of isolates.
 Based on somatic antigens 17 serotypes have been described in P.aeruginosa.
 P.aeruginosa produces numerous extracellular toxins and enzymes. Those that may
play role in pathogenesis are fibrinolysin, elastase, lecithinase, lipase, protease,
haemolysin (heat stable and heat labile), leucocidin, alginate, phospholipase C,
esterase and exotoxin A.
 In addition to this, the pigment produced by P.aeruginosa exhibits antimicrobial
activities against a wide range of bacteria and some fungi. P.pseudomallei produces
very potent exotoxin (similar to exotoxin A), protease, lipase and a lecithinase.
 No exotoxin has been described in Pseudomonas mallei.

RESISTANCE

 They are more resistant to high dilutions of quaternary ammonium compounds and
phenolic compounds.
 P.aeruginosa can survive for long periods on water faucets, utensils, floors,
instruments, baths, humidifiers and respiratory equipments.
 They grow very well in antiseptic lotions kept for use in hospitals. Pseudomonas is
susceptible to ethylene oxide and heat (550C for one hour).
 Pseudomonads are resistant to wide range of antimicrobial drugs.
 Much of this intrinsic resistance is attributed to the outer membrane porins, which
restrict passage of many antimicrobial agents into the periplasm.
  Resistance to penicillin, Ampicillin, tetracycline, first and second-generation
cephalosporin, sulfonamides, neomycin, streptomycin, kanamycin, chloramphenicol,
nitrofuran and trimethoprim-sulfonamide.
 Susceptible to gentamicin, amikacin, colistin, Polymyxin B, carbenicillin, cefoxamine,
third generation cephalosporins and Ciprofloxacin

PATHOGENESIS

 In P.aeruginosa the source of infection may be either endogenous or exogenous.

 Some defect in local defense or generalized defense is necessary for this opportunistic
pathogen to cause disease.
o P.aeruginosa + Breach in host defense
o (Ubiquitous in environ ment)
o Adhere
o (Pili, exoenzymes)
o Multiply
o Antiphagocyitc LPS and slime
o Iron scavenging system (Pyocyanin and Pyoverdin)
o Resistance to killing by serum
o Produce toxins
o (Exotoxin A, Elastase and Protease)
o Tissue damage due to toxins Tissue damage due to immune complex
mechanism
o (Acute disease) (Chronic disease)
o Elimination of bacteria Persistence of bacteria
 In P.pseudomallei, the animal gets infections mainly occurring as a result of
ingestion, inhalation or skin contamination. The pathogenesis of melidiosis is not
known.
 In P.mallei (Cattle, swine, birds and rats etc are resistant to infection) equidae gets
infections most commonly by ingestion and also by inhalation and skin
contamination.
 After ingestion, there is invasion of the gut wall and a subsequent septicaemia.
 The endotoxin plays a major role. Inhalation usually leads to a bronchopneumonia
and lesions in the nasal mucosa. Infections of abraded skin leads to Farcy.
 Glanders is a contagious, usually chronic disease of horse, characterized by the
formation of tubercle like nodules (granulomas) that frequently break down to form
ulcers.

Diseases produced by the Pseudomonads

Species Host Disease

P. aeruginosa  All species  Wound infections
 Dogs and cats  Otitis externa, keratitis,
 Cattle dermatitis and cystitis
 Horses  Mastitis, enterits and
 Pigs arthritis
 Sheep  Ulcerative keratitis,
metritis and abortion
 Enteritis and
respiratory infection
  Mastitis, pneumonia,
green wool (Fleece
rot/Skin infection)

P.pseudomallei Horses, cattle, sheep, Melioidosis

(Whitemore‘s bacillus ) goats, pigs and dogs (Pseudoglanders)
P. mallei Horses, mules and Glanders
(Malleomyces donkeys Farcy (Cutaneous glanders)
mallei or Loefflerella
mallei or Pfeiferella mallei )
PATHOGENECITY

Symptoms and lesions

 Depends upon the degree and site, the P.aeruginosa produces wide variety of
suppurative infections.
 In localized lesions there will be yellowish green pus.
 The acute form of P.pseudomallei occurs more often in young animlas.
 The chronic form is characterized by nodules in the lungs, liver, spleen, lymphnodes
and subcutis, the lesions resembling those of caseous lymphadenitis and numerous
visceral abscesses.
 Three forms of glanders are seen in horse. They are classified as

Pulmonary form: rise in temperature, coughing with blood stained mucosa.

Nasal form

 Nasal form initially there is reddening of the nasal mucosa and mucoid discharge
from nostrils.
 It will become purulent, blood stained and adherent as brown crust. Ulcers may form
on the nasal mucosa.

Cutaneous form: (Farcy):

 Development of small cutaneous or subcutaneous nodules in the limbs and flanks.

 These nodules develop into hollow ulcers exuding yellow or oily pus.
 The local lymphatic vessels become corded and tubercle like nodules are seen in
lungs and other visceral organs.

DIAGNOSIS

Specimens

 Urine, pus, affected tissues and swab from infected tissue surfaces are suitable
for P.aeruginosa.
 In case of P.mallei, collect tissue containing early nodules or pus from ulcers.

Diagnosis

 Based on symptoms and lesions

  Based on pigment production
 Based on cultural and Biochemical characters
 Based on animal inoculation
o Straus reaction
 The Straus reaction is seen in male guinea pigs inoculated intra
peritoneally with infective material containing
either P.pseudomallei or P.mallei.
 The reactions of swelling of the testes, inflammation of the tunica
vaginalis and ulceration of the scrotal skin develops in 2-3 days. (Not
confirmatory one.
 Because Straus reaction is also produced by Brucella, Preisz-nocard
bacillus, and Actinobacillus ligniersii)
 Mallein test
o This is used to demonstrate the hypersensitivity developed after infection
with P.mallei.
o Mallein is a glycoprotein extracted from the bacterium.
 Subcutaneous test: swelling at the injection site and fever.
 Opthalmic test: Instilled on conjuctival sac. Inflammatory and
purulent reaction occurs within 6-12 hrs.
 Intrapalpebral: Inoculate at skin of the lower eyelid. Localized,
odematous swelling and purulent conjunctivitis
 Serological tests
o CFT, Agglutination, IHA, and CIE are used in the diagnosis of glanders.
o CFT and IHA are useful in melioidosis.

CHAPTER-20: BRUCELLA

Learning objectives

To know in detail about,

 Diseases produced by the pathogenic brucella in domestic animals

 Morphology, cultural and biochemical characters of Brucella sp
 Selective media for Brucella
 Antigens and biotyping of Brucella
 Pathogenesis of brucellosis in domestic animals
 Different diagnostic methods of brucellosis
 ABRT, Coombs test, RBPT, Card test and Whey agglutination test
 General approaches used to control brucellosis

SYSTEMATICS
Domain Bacteria

Phylum Proteobacteria

Class Alpha proteobacteria

Order Rhizobiales

Family Brucellaceae
 Genus Brucella

 Brucella are small Gram negative, non-motile, non-spore forming rods.

 They are aerbic and carboxyphilic. Catalse, oxidase and urease positive.
 They are partially acid fast, in that they are not decolourised by 0.5% acetic acid in
the Modified Ziehl –Neelsen acid stain.
 All the six recognised
species, B.abortus, B.melitensis, B.suis, B.ovis, B.canis and B.neotomae are
pathogenic, facultative, intra cellular parsites with a prediliction for the
reticuloendothelial system and the reproductve tract.

HISTROY AND HABITAT

History

 B.melitensis was identified by Bruce in Malta in 1887. B.abortus was first recognised
by Bang in 1897. B.suis was discoverd by Traum in 1914.
 Alice Evans (1918) identified the first Brucella of human origin in the USA. Buck
(1930) developed live attenuated strain 19 vaccine.

Habitat

 Brucella species are obligate parasites and each species has a preferred natural host
that serves as a reservoir of infection.
 Brucellae have a predilection for ungulate placentas, foetal fluids and testes of bulls,
rams, boars and dogs.

MORPHOLOGY AND CULTURAL CHARACTERISTICS

Morphology

 Brucellae are coccobacilli or short rods, arranged singly or in short chains. They are
non-motile, non-capsulated, non-sporing and partial acid fast.
 They are stained by Grams, MZN and Koster‘s stain.
 Koster‘s stain is mostly useful in demonstrating Brucella in smears from the
cotyledons in bovine abortion (Cells of chorion are packed with organism).
 MZN isspecially useful to stain smears from foetal membranes, uterine swabs,
smear or stomach contents of aborted foetus.

Cultural characteristics

 Brucella is strict aerobe.

 B.abortus is carboxyphilic, requiring 5-10% CO2.
 The optimum temp. is 37ºC. The growth very slow on simple media, the plates should
be incubated for as long as 30 days.
 Growth is improved by addition of serum or liver extract.
 Growth is also improved by addition of several aminoacids, thiamine, biotin,
magnesium, isoerythritol and manganese.
 Selective media for Brucella are Albimi medium and Columbia agar.
 Serum Dextrose Agar, Potato infusion agar, Tryptose Soya Agar, TryptoseThiamine
Agar are useful for primary isolation.
  The SDA enriched with bacitracin, cycloheximide, nalidixic acid, nystatin and
polymyxin B is used as selective medium.
 After 3-5 days incubation on selective serum agar, pinpoint smooth, glistening,
bluish, translucent colonies will appear.
 Strains of B.ovis and B.canis are always in the rough form. Others are initially
smooth form and become rough on subsequent cultivation.
 Brucella is non haemolytic on blood agar. In liquid media growth is uniform and in
old cultures a powdery or viscous deposit is formed.

BIOCHEMICAL PROPERTIES AND RESISTANCE

Biochemical properties

 Brucellae are catalse positive, oxidase positive (except B.ovis and B.neotomae), and
urease +ve (except B.ovis and B.melitensis), reduce nitrate, IMViC, -,-,-,-. (indole,
methyl red, voges proskauer and citrate utilization negative).

Resistance

 Readily killed by commonly used disinfectants and pasteurization.

 Brucellae are destroyed by heat at 60ºC in 10mts and by 1% phenol in 15mts. They
survive in soil and manure for several weeks.
 Remain viable for 10 days in refrigerated milk, one month in ice cream, 4 months in
butter and many weeks in meat.
 B.abortus will survive for 41/2 hrs when exposed to direct sunlight, 4 days in urine, 5
days in cloth at room temperature and 75 days in an aborted fetus.

ANTIGENS AND BRUCELLA

Antigens

 The somatic anigens of brucellae contain two main antigenic determinants

designated as A and M, which are present on the LPS proten compelx.
 Brucella abortus contains more A antigen than M (20:1).
 B.melitensis has more M than A (20:1). B.suis has an intermediate pattern.
 B.canis and B.ovis are antigenically rough and do not possess the A and M antigens.
Both possess a surface antigen R.
 Like many other Gram-negative bacteria, the change from S to R is associated with
loss of virulence, a tendency toauto agglutination and loss of antigen.
 The vaccine strain, B.abortus 19 is an intermediate mutant that, although low in
virulence, is antigenic.
 Antigenic cross-reactions exist between brucellae with several bacteria like Yersinia
enterocolitica, Vibrio cholerae and some strains
of E.coli and Salmonella (Heterophile antigens).
 No exotoxins have been described. In addition to endotoxin, the surface cell wall
carbohydrate is responsible for binding to B-lymphocytes and play a major role in
pathogenesis.

Brucella biotyping

 Based on CO2 requirement, H2S production (B.abortus biotype 1,2,3,4

and B.neotomae), urease activity, growth in the presence of dyes (thionin, basic
fuchsin), agglutination with monospecific sera (B.canis and B.ovis – agglutinate only
 with anti R sera) and Phage typing, three biotypes have been recognized
in B.melitensis, eight in B.abortus and four in B.suis.
 B.abortus biotype 3 is commonly present in India.

PATHOGENESIS

 The common routes of infection in animals and humans are via the mucous
membrane of the digestive tract, genital tract and skin.
 Veneral transmission is main route for B.ovis. Less commonly infection may occur
through inhalation or by conjuctiva.
 Organisms may penetrate the mucosa of nasal or oral cavities. Soon after entry into
the host the brucellae are engulfed by phagocytic cells, in which they survive, multiply
and are transported to the regional lymphnodes.
 After multiplication, the organism pass to the thoracic duct and then via blood stream
to parenchymatous organs and other tissues such as joints, granulomatous foci
develop in lymphatic tissues, liver, spleen, bone marrow and other locations. On
occasion it will become abscess.
 Brucellosis is essentially disease of the sexually mature animal, the predilection sites
being the reproductive tracts of males and females especially the pregnant uterus.
 Allantoic factors such as erythritol (Polyhydric OH),steroid hormones and other
substances favour the growth of most brucellae.
 Erythritol is present in the placenta and male genital tract of cattle, sheep, goats and
pigs but not humans.
 Erythritol does not stimulate the growth of B.ovis and inhibits B.abortus strain 19,
the attenuated vaccine strain.
 A pyogranulomatous reaction occurs in affected placentae and abortion occurs from 6
month onwards.
 Note: Females usually abort only once, after which a degree of immunity develops,
and the animals remains infected and large numbers of brucellae can be excreted in
foetal fluids at subsequent parturitions. Permanent infertily may occur in male dogs
infected with B.canis.
 Diseases caused by Brucella species

Species Host Disease

B. abortus  Cattle  Abortion and orchitis ( Bang's disease / Abortion storm)

 Sheep, Goat and Pig  Sporadic abortion
 Horses  Poll evil and Fistulous withers
 Human  Undulent fever

B.melitensis  Goat and Sheep  Abortion

 Cattle  Occasional abortion
 Human  Malta fever

B.suis  Pig  Abortion, orchitis, arthritis, spondylitis and infertility

 Human  Undulent fever

B.ovis  Sheep  Epididymitis in rams and abortion in ewes

B.canis  Dogs  Abortion, epididymitis and permanent inferlity in milk
 Human  Undulent fever

B.neotomae  Desertwood rat  Non pathogenic

PATHOGENECITY

Symptoms

Cattle

 The disease in cattle is almost always caused by B.abortus.

 The incubation period is usually from 30 to 60 days.
 After bacteraemia the infection localizes in the placentae, if the animal is not
pregnant, the infection localizes in udder (interstitial mastitis).
 In the bull, it localize in the testicle and cause orchitis and epididymitis.
 Abortion at 6 months and retained placentae are the cardinal signs.

Sheep

 B.melitensis infections results in abortion at about 3rd or 4th month of pregnancy.

Other signs are lameness and mastitis.

Swine

 Swine brucellosis is characterized by abortion, sterility, and birth of stillborn or weak

pigs, focal abscesses in various organs, spondylitis and lameness.
 Abortion may occur at any time during gestation.

Dogs

 Expelled tissues and vaginal discharges of aborted bitches and the urine of infected
males are primary sources of the infectious agent.
 The incubation period is 6 to 21 days. Infected bitches usually aborted in the last
trimester.
 Following abortion there is yellow brown to dark brown discharges that persist for 1
to 6 weeks.
 Epididymitis and testicular atrophy with decreased spermatogenesis are common in
the male and may result in irreversible sterility.

Lesions

 The gravid bovine uterus infected with B.abortus develops a necrotic placentitis.
 The cotyledons become swollen, hyperaemic and surrounded by brownish exudates.
 The inter cotyledonary spaces are thickened and have a characteristic leathery
appearance.
 Orchitis in bull result in abscess formation or areas of necrosis in the testicles,
surrouned by fibrous tissues.
  Brucella suis causes placentitis, metritis in ewes and epididymitis and orchitis in
boars.
 In horses, B.abortus infection associated with fistulous withers (a chronic
inflammatory condition of the supraspinous bursa) and poll evil.

DIAGNOSIS

Specimens to be collected

 In abortion cases a full range of specimens should be collected and submitted for a
differential diagnosis.
 A whole foetus should be sent, if feasible.
 Alternatively foetal stomach contents, any foetal lesions, cotyledons, uterine
discharges, urine, coloustrum, paired serum samples and sections of cotyledons and
foetal lesions in 10% formalin for histopathology. Semen and tissue from epididymis
or testes from males could be examined.

Based on symptoms and lesions

Direct examination

 Modified ZN and Koster‘s stain are useful in demonstrating brucellae in smear from
the placenta (cotyledons) in bovine abortion.
 Cells of the chorion are packed with organism, which stain red against a blue
background.
 Organism can also be demonstrated directly in smears from vaginal mucous, semen
and various tissues. Direct and indirect FAT is also used.

Based on isolation and identification

Based on biotyping

Animal inoculation

 Straus reaction: Orchitis in male guinea pigs.

 It is the most sensitive test for isolating pathogenic brucellae.
 It is highly useful if the material is badly contaminated or the numbers of organisms
are very small.
 Guinea pigs are inoculated intra muscularly with 0.5 – 1 ml of tissue homogenate.
 Euthanise 6 wks after inoculation. Serum is taken for serology and the spleen,
together with any abnormal tissue, is collected for bacteriological examination.

Immunological tests

 A wide range of immunological tests have been developed

o Serum agglutination test/ Tube agglutination test (titre > 1:40 is +ve, titre
<1:20 is doubtful). Click here for visual

o ABRT/Milk ring test - Click here for visual

o Coombs test: to detect incomplete antibodies
o RBPT: Antigen consists of suspension of brucellae organisms stained with
rose Bengal and adjust to pH 3.6
 o Card test: Which uses only one serum dilution and stained antigen, is rapid,
sensitive and used as a field screening test.
o Whey agglutination test
o CFT, AGID and ELISA
o Rivanol precipitation and Mercaptoethanol agglutination: to detect primary
IgM antibodies
o PCR

CONTROL AND PREVENTION

 The immunity acquired from natural infection is not always sufficient to prevent
reinfection.
 The general basis for elimination of brucellosis is testing and removal of reactors
from the herd.
 Because the cattle will be less susceptible to reinfection, calf hood vaccination is
recommended.
 The attenuated live vaccine (Strain 19 B.abortus biotype 1) is used in female calves 4
to 12 months of age.
 Because strain 19 cause infertility in some male calves, its use is restricted to females.
 The adjuvant bacterins eg: vaccine 45/20 is used as booster
vaccine. B.melitensis Rev.1 live attenuated vaccine has been used with success to
immunize rams against B.ovisinfection.

CHAPTER-21: CAMPYLOBACTER

Learning objectives

To know in detail about,

 Principal host and diseases caused by Campylobacter species

 Morphology, cultural and biochemical characters of Campylobacter species
 Selective media for Campylobacter
 Pathogenesis and pathogenicity of Campylobacteriosis in domestic animals
 Different diagnostic methods fo Campylobacteriosis
 Mating test or virgin heifer test for Campylobacteriosis

SYSTEMATICS
Domain Bacteria

Phylum Proteobacteria

Class Epsiloproteobacteria

Order Campylobacterales

Family Campylobacteraceae

Genus Campylobacter
  The mostimportant animal pathogens are C.
fetus subsp. fetus, C. fetus subsp. venerealis, C. jejuni and possibly C.
mucosalis and C. hyointestinalis.
 The Campylobacter species were once placed in the genus Vibrio and some of the
diseases are still occasionally referred to as 'vibriosis'.

HABITAT

 The Campylobacter species are worldwide in distribution.

 Many of the animal species are commensals on the mucosa of the oral cavity and
intestinal tract.
 C. fetus subsp. venerealis occurs in the prepuce of bulls and in the genital tract of
cows in herds where bovine genital campylobacteriosis is, or has been, present.
 There are also some nonpathogenic species that are saprophytes in the environment.

MORPHOLOGY AND CULTURAL CHARACTERISTICS

Morphology

 The Campylobacter species are thin, curved, Gram-negative, microaerophilic, motile

rods.
 The cells are 0.2-0.5 u.m in width and when daughter cells remain joined, S-shaped,
seagull-shaped and sometimes long spiral forms may be seen.
 They are motile by means of polar flagella. Small, curved or seagull-shaped rods can
be demonstrated by using DCF-stained (dilute carbol fuchsin for 4 minutes) smears
prepared from colonies. Wet mounts under phase contrast or darkfield microscopy
reveal the characteristic curved forms with darting motility.

Cultural characterstics

 The organisms grow at 25 °C or 45 °C. They grow best on nutritious basal media
supplemented with 5-10 per cent blood under reduced oxygen tension.
 The Campylobacter fetus (both subspecies) grows very well on nutritious base
(Brucella, Columbia or brain heart infusion agars) supplemented with 5-10 per cent
blood.
 The medium can be made selective by the addition of polymyxin B sulphate (2
units/ml), novobiocin (2 µg/ml) and cycloheximide (20 µg/ml).
 Incubate the plates at 37°C for 4-6 days in a microaerophilic atmosphere containing 6
per cent O2, 10 per cent CO2 and 84 per cent N2.
 These atmospheric conditions must be adhered to strictly. Both subspecies of C.
fetus have small (1 mm), round, slightly raised, smooth, translucent colonies- said to
have a 'dewdrop' appearance.
 C. jejuni grows very well on charcoal-cefopera-zole-deoxycholate agar or Blaser's
Campy-BAP medium.
 This latter medium is Brucella agar with 5 per cent sheep blood and vancomycin (10
ug/ml), polymyxin B sulphate (2-5 units/ml), trimethoprim lactate (5 ug/ml)
cephalothin (15 ug/ml), and amphotericin B (2 ug/ml).
 The plates should be incubated under the atmosphere described above for 2-3 days at
42° C.
 The colonies of C. jejuni are usually flat, grey, and larger than those of C. fetus and
can be spreading and watery on moist plates. C. mucosalis / C. coli grows on
Columbia blood agar with and without 1:60,000 brilliant green.
 Incubate the plates anaerobically at 37°C for 2-5 days. After primary isolation the
bacterium will grow well microaerophilically.
  C. coli produces a pink-tan pigment and the growth of C. mucosalis may appear as a
dirty-yellow colour.

BIOCHEMICAL PROPERTIES, RESISTANCE AND

ANTIGENECITY

Biochemical properties

 Campylobacter species are non-fermentative, non-hemolytic, oxidase-positive,

produced hydrogen sulphide, reduced nitrate and catalase-variable.
 C. jejuni is the only species that hydrolyses sodium hippurate.

Resistance

 C. fetus is killed at a temperature of 600C for 5 minutes.

 It will survive for 10-20 days in soil, hay and manure, depending upon humidity and
temperature. They are readily killed by disinfectants.

Antigencity

 C. fetus subsp. venerealis has "0" and "W' cell wall antigens. "0" antigen is stable,
whereas "W" antigen is altered once specific (sIgA) antibodies are produced.
 Now "W" antigen is called Surface Array protein. As sIgA immunologic memory is
poor, the bacterium can shift between antigens.
 C. fetus subsp. fetus has"0" A-2 and B. But the protein antigens areimportant

PATHOGENESIS

 Little is known of the pathogenic mechanisms of most Campylobacter species. C.

jejuni produces an adhesin, a cytotoxin and a heat-labile toxin similar to that
of Escherichia coli.
 Transmission of many of the Campylobacter species, including C. fetus subsp. fetus,
is by the faecal-oral route. C. fetus subsp. venerealis is transmitted by coitus and
infection of the female genital tract may lead to metritis with resulting death and
resorption of the embryo (infertility), or occasionally to abortion.
 C. fetus subsp. venerealis - is an obligate parasite of bovine genitalia.
 Encounter is usually through coitus or artificial insemination - in bulls the infection
is usually unapparent and involves the eptihelium of the penis and the fornix of the
prepuce.
 The organism localizes in the anterior vagina and cervix during the ovulatory phase
but does not invade the uterus and oviducts until progesterone release - and then
causes endometritis and salpingitis for several weeks to a few months during which
the animal is infertile - animals usually regain fertility within 5 months.
 C. fetus subsp. fetus - Ovine abortions, however organism enters via ingestion,
(venereal transmission does not occur) causes bacteraemia and finally localizes in the
placenta causing placentitis and abortion near the end of gestation.
 Infection of ewes in the first half of the gestation period does not result in abortion.
 Occasionally causes abortion during latter half of gestation in cattle - organism enters
through ingestion.
 C. sputorum subsp. mucosalis - found in mouth of piglets for about 2 weeks following
weaning - if conditions favour (stress, overcrowding, underlying gastroenteritis), the
organisms can invade intestinal mucosa and multiply intracellularly.
  The result is excessive proliferation of functionally (absorptive capacity) immature
epithelial cells which carry the organism.
 Affected pigs do not thrive; lesions are in the lower ileum and cecum (proliferating
epithelia) with loss of villi and development of polypoid (polyp-like) masses.
 Animals usually recover in 6 weeks. The diseases caused by Campylobacter
species are

Species Host Disease

C. fetus subsp. venerealis  Cattle  Epizootic bovine infertility (Bovine genital

campylobacteriosis) , early embryonic death and
occasional abortion

C. fetus subsp. fetus  Sheep  Abortion (Ovine genital campylobacteriosis)

 Cattle  Occasional abortion
Syn: C. fetus intestinalis

C. jejuni  Cattle  Winter dysentery & Mastitis

 Sheep  Abortion
 Poultry  Avian vibrionic hepatitis
 Dogs and cats  Enteritis with diarrhoea

C. mucosalis  Pigs Swine proliferative enteritis (Porcine intestinal

adenomatosis complex)

C. hyointestinalis  Pigs Swine proliferative enteritis (Porcine intestinal

adenomatosis complex)
( Serpulina
hyointestinalis)

C.coli  Pigs, Man  Mild diarrhoea in pigs and entertis in man.

 Swine dysentery (along with Serpulina
hyodysenteriae)

PATHOGENECITY

Symtpoms

Cattle

 Affected cattle usually show signs of acute endometritis with in 2 weeks after
exposure.
 Failure to implant or early abortions, Abortions at 5 -7 months, retained placenta and
increase number of services per conception resulting in repeat breeders are most
commonly seen.

Sheep
  Pregnant ewes that become infected may lose weight and appear unthrifty. Diarrhea
may be present. They usually abort late in their pregnancy.
 They may also deliver stillborn or weak lambs. In unvaccinated flocks, the abortion
rate may reach 70% of the ewes.
 The aborted fetus is autolyzed (already showing signs of decomposition).
 This is in contrast to a fresh fetus in Chlamydial/Enzootic Abortion of Ewes (EAE),
which is another common cause of ovine abortion.
 The placenta is often hemorrhagic (bloody), necrotic (decomposition), and
edematous (swollen, leathery).
 Blood tinged fetal fluids. Characteristic necrotic spots in the fetal liver.
 Following the abortion, the ewe may develop an infection of the uterus (metritis) and
require additional medical attention.
 Mortality in the ewes may exceed 5%. Surviving ewes may become carriers.

Swine

 Proliferative enteritis in swine with adenomatous changes due to excessive

proliferation of immature epithelial cells. 6 to 20 week old pigs are affected mostly.

DIAGNOSIS

Based on Direct microscopy

 Both subspecies of Campylobacter fetus can be demonstrated in foetal abomasal

contents using the DCF stain.
 Fluorescent antibody staining of smears from foetal abomasal contents, cervical
mucus and preputial washings is most reliable, especially when small numbers of the
bacterium are present.
 C. jejuni can be seen in wet mounts of faces by phase contrast or darkfield
microscopy.
 The typical darting motility of corkscrew-like organism is suggestive
of Campylobacter species.
 Characteristic slender, curved rods can be demonstrated in DCF-stained smears, or
by phase contrast of ovine foetal abomasal contents and in bile from chickens with
hepatitis.
 C. mucosalis can be visualised using the modified Ziehl-Neelsen (MZN) stain on
heat-fixed smears from mucosal scrapings.
 Large numbers of pink-staining, slender, curved rods, located intracellularly are seen.
Inoculate the above said specimens on to any one of the selective agar and
identification of the organism is based on colonial morphology as described earlier.

Based on Isolation and identification

 Campylobacter fetus (both subspecies): cervical mucus and preputial washings can
be passed through a 0.65 µm membrane filter to reduce contamination.
 Foetal abomasal contents and filtrates are useful for isolation and identification.
 C. jejuni: rectal swabs or faeces are the most sutiable specimens.
 C. mucosalis: Homogenates of mucosal scrapings from the affected intestine are
useful.

Based on Biochemical and other tests

 Susceptibility or resistance to nalidixic acid or cephalothin, hydrogen sulphide

production, nitrate reduction growth at 25º C or 45º C and the catalase reaction, are
 some of the criteria on which a definitive identification of the Campylobacter
species is made.

Hippurate test

 C. jejuni is the only species that hydrolyses sodium hippurate.

 For this test a large loopful of a 24-48 hour culture of C. jejuni is emulsified in 0.4 ml
of a 1 per cent aqueous solution of sodium hippurate and incubated at 37°C for 2
hours.
 Then 0.2 ml of a ninhydrin solution is added to the tubes at 37°C.
 A positive reaction is given by a deep purple colour developing after 10 minutes.

By Serology

 The cervical mucus agglutination test for C. fetus subsp. venerealis is accurate if
carried out 2-7 months post-infection.
 A vaginal mucus agglutination test has been found useful but the serum agglutination
test is unreliable.
 As C. jejuni is present in the intestines of many normal animals, its isolation from
faeces may not necessarily be significant.
 A four-fold increase in an agglutinating antibody titre to the bacterium would suggest
involvement of the organism in the diarrhoea.
 Fluorescent antibody-stained smears can be used to identify C. fetus, but the
technique does not distinguish between the subtypes.

By mating test or vigin heifer test

 To detect infected bull, virgin heifers are inseminated with semen and preputial
washings from suspected animals and examined the heifer‘s vaginal mucous during
the following 3-4 weeks.
 This test is effective but expensive and time consuming.

DNA probes areavailable for diagnosis

CONTROL, PREVENTION AND PUBLIC HEALTH

SIGNIFICANCE

 This disease is very contagious and spreads rapidly among the remaining animals
unless very strict hygiene is practiced.
 The fetus, placenta, birth fluids, vaginal discharge, and feces from the affected
animals are all sources of infection.
 If the water or feeding areas become contaminated with these materials, the abortion
rate can be very high.
 Isolate the aborting animals immediately, along with proper disposal of the aborted
fetus/placenta, and disinfection procedures.
 Prevent the disease from spreading by limiting access to the aborted materials by
wild birds and wild or domestic mammals, which can carry the bacteria to other lots
or ranches. Cleanliness is absolutely essential.
 Many Campylobacter species produce beta-lactamase, thus accounting for their
resistance to penicillin and ampicillin.
 Killed bacterins are effective both prophylactically and therapeutically. Heifers and
cows should be vaccinated 1 to 4 months before breeding.
 Segregate affected bulls and test new additions. If infected, suspend breeding for 90
days and use antibiotics and artificial insemination.
  In Sheep, vaccinate all incoming and unvaccinated ewes thirty days prior to breeding
season and again sixty to ninety days later.
 Follow up with a booster every year at the onset of breeding season.
 While some immunity is obtained following an outbreak, immunity against one strain
of Campylobacter is not cross-protective against the other strain.
 This false sense of security combined with the presence of carrier animals can result
in further abortion storms.

Public health significance

 Campylobacter has potential public health significance. C. jejuni - a veryimportant

zoonotic pathogen - Milk, minced meat, dogs, cats, and poultry are important
reservoirs of infection for humans.
 It is widely distributed in the intestine of domestic and wild animals but is rarely
isolated from the feces of normal humans.
 In human even 500 c.f.u./ml in milk are infective, the signs are fever, abdominal
pain, nausea, vomiting, and bloody stools. Some may become persistently infected.

CHAPTER-22: MYCOPLASMA

Learning objectives

To know in detail about,

 Mostimportant species of Mycoplasma and the mycoplasmal diseases of domestic

animals and poultry
 Morphology, cultural and biochemical characters of Mycoplasma
 Diene's staining method
 Difference betweeen L forms and Mycoplasma
 Diagnostic methods used to identify Mycoplasma

MORPHOLOGY

 Class: Mollicutes (Soft skin)

Domain Bacteria

Phylum Firmicutes

Class Mollicutes

Order Mycoplasmatales

Family Mycoplasmataeceae

Genus Mycoplasma

Order Acholeplasmatales
 Family Acholeplasmataeceae (not required cholestrol)

Genus Acholeplasma

Order Entomoplasmatales

Family Spiroplasmataeceae

Genus Spiroplasma

 Family: Mycoplasmataceae -Sterol or Cholestrol requirement for their growth

 Genus
o Mycoplasma
o Ureaplasma (hydrolyses urea)
 Division: Tenericutes
o The term mycoplasma that refers to the fungus-like forms of the branching
filaments.
o They are the smallest and simplest prokaryotic cells capable of self-
replication.
o The genome of mycoplasmas and ureaplasmas is the smallest for any self-
replicating prokaryotic cells.
o They are 5x108 daltons compared to the average bacterial genome of 2.5
x109 daltons.
o These organisms lack the genetic ability to form a cell wall and are enclosed in
triple layerd plasma membrane composed of protein, glycoprotein, glycolipid
and phospholipid.
o As there is no rigid cell wall, the mollicutes are plastic, filterable and
pleomorphic.
o Mycoplasmas are parasitic and pathogenic for animals. More than 60 species
of Mycoplasma are known to cause disease in variety of animals.

HISTROY AND HABITAT

History

 The first mycoplasmal species, the causative agent of bovine pleuropneumonia, was
first isolated by Nocard and Roux (1898).
 The species discovered later were called PPLO, because of their resemblance to the
organisms causing pleuropneumonia.

Habitat

 The Mycoplasmas occur worldwide as free-living saprophytes or as parasites of

animals.
 Both pathogenic and non-pathogenic species are found as commensals on the
mucous membrane of the URT, intestinal and genital tracts, on articular surfaces and
in the bovine mammary gland.
 Outside the host, the pathogenic species can survive in microenvironments, protected
from sunlight, for several days.
 MORPHOLOGY

 Mycoplasmas are the smallest and most pleomorphic microorganism.

 They occur as cocci, spirals, flaments and rings. The characteristic feature is the
complete absence of cell wall or cell wall precursors such as muramic acid and
diaminopimelic acid.
 They stain poorly with Gram's stain (gives Gram –ve). Gives better resuts with
Giemsa and other Romanowsky stain.
 The method of reproduction is not fully understood. But some divide by binary
fission, or by budding.
 Some have a reproductive cycle by the development within the filaments of
elementary bodies and their subsequent release by fragmentation and disintegration
of the filaments.
 The minimal reproductive unit is about 0.3 µm in d.m. but because the cells are
pliable they are able to pass through a 0.22 µm membrane filter.
 Dark field and Phase contrast microscopy are recommended for studying the
morphology of Mycoplasma.
 M. mycoides has a galactan capsule. They are generally non- motile and non-spore
forming.

CULTURAL, BIOCHEMICAL CHARACTERISTICS AND

RESISTANCE

 Mycoplasmas are highly fastidious organisms and most require specific growth
factors, an isotonic medium and the absence of inhibitory substances for growth.
 Mycoplasma and Ureaplasma require reduced sterol or cholestrol for their growth.
 Because the Mycoplasma are unable to synthesise purines and pyrimidines, they
require complex media.
 The agar which contains bovine heart infusion, 20% horse serum, (Pooled serum
from several animals), 10% yeast extract, 20ml of adenine dinucleotide, 50 units of
penicillin and 0.25 mg of thallous acetate (inhibitory to Gram –ve and fungi) and the
optimal pH is 7.5.
 Most grow aerobically but some require N2 with 5% to 10% Co2 after 2 to 6 days of
aerobic incubation at 370C, colonies on solid media are 0.1 to 0.6 mm in d.m.
 under low power magnification, the colonies appear transparent, flat and often
resemble a fried egg.
 Colonies grow into the medium and are difficult to remove from the agar surface.

 The colonies are best studied by stained with Diene's method

 A block of agar containing microcolonies is placed, colony side upwards, on a
microscopic slide.
 A light film of Diene‘s stain (alcoholic solution of methylelne blue and azure) is
placed on a coverslip and allowed to dry.

 This is then put, stain-slide downwards on the microcolonies on the agar block then
examined under low power of microscope.
 The dense center of the microcolonies, which grow down into the agar, stain dark
blue, the less dense peripheral zone, resembling surface growth, stains light blue.
 Most mycoplasmas are haemolytic in swine blood agar. Mycoplasmas may grow in
chicken embryos (yolk sac route) and cell cultures.
 Ureaplasma produce tiny colonies (T-mycoplasma) than the
conventional mycoplasma
 Biochemical charactersMycoplasmas are chemoorganotrophs, the metabolism being
mainly fermentative.

 Most species utilize glucose or arginine as the major source of energy.

 Urea is not hydrolysed except by ureaplasmas.
 They are generally not proteolytic.

Resistance

 Mycoplasmas are more fragile because of the absence of cell wall.

 Drying, sunlight and the usual disinfectants readily kill them.
 They are resistant to penicillin and sulfonamides.

PATHOGENESIS

 Transmissions are usually veneral, vertical or by aerosols and manyimportant avian

mycoplasmas are egg transmitted.
 Various stress factors predispose the mycoplasmal infection. They parasitic
mycoplasma tends to adhere firmly to the host‘s mucous membranes with specific
attachment structures.
The organisms are extra cellular and produce haemolysins, proteases, nucleases and
other toxic factors like capsular carbohydrates, ammonia that can lead to death of
host cells or to a chronic infection.
 Some pathogenic species have a predilection for mesenchymal cells lining joints and
serous cavities.
 The respiratory tract and lungs are most frequent sites of infection.
 Mycoplasmas are capable of destroying the cilia of cells in the respiratory tract, thus
predisposing secondary bacterial invasion.
 The fibrinous exudates frequently present in infections protects them from antibody
and antimicrobial drugs.

Host Species Disease

Poultry  M.gallisepticum  Chronic Respiratory Disease

(CRD) and air sac disease in chicken
 Infectious sinusitis in chicken

Turkeys  M.synoviae  Infectious synovitis

 M.meleagridis  Airsacculitis and bursitis

Cattle  M.mycoides subsp mycoides (Small  Contagious Bovine Pleuro Pneumonia

colony type) (CBPP)
 M.bovis  Mastitis, arthritis, pneumonia,
 M.bovigenitalium abortion and genital infection
 Vaginitis, arthritis, seminal vesiculitis
and mastitis

Goats  M.mycoides subsp Capri  Contagious Caprine Pleuro

 M.mycoides subsp mycoides (Large Pneumonia (CCPP)
 Pneumonia, septicaemia, polyarthritis
 colony type) and mastits

Sheep  M.agalactiae  Contagious agalactia

Pigs  M.hyorhinis  Polyserositis and chronic arthritis in

 M.hyosynoviae young pigs
 M.hyopneumoniae  Polyarthritis in adult
 Enzootic pneumoniae of pigs

DIAGNOSIS

Specimens

o Mycoplasmas are fragile and specimens must be kept refrigerated and

delivered to laboratory within 24-48 hrs of collection.
o The samples may include mucousal scrapings, tracheal exudates, aspirates,
and pneumonic tissue from the edge of the lesion, cavity or joint fluids and
mastitic milk.
o Swabs should be submitted in transport medium.

Diagnosis

 Based on direct microscopy

o FAT is most reliable than simple staining.
 By isolation and identification
o The inoculation technique will vary according to the nature of the specimen.
 Fluid materials such as foetal fluids and exudates can be inoculated
directly into broth and spread over the surface of the agar medium.
 Specimens such as semen, joint fluids and tissues may contain
inhibitors for mycoplasmas.
 Make ten fold dilutions in mycoplasma broth and then culture.
 Tissues, such as pieces of lung can be moved across the surface of an
agar plate for inoculation.
 Alternatively the tissue can be homogenized in broth, made ten fold
dilutions and inoculated.
o Differentiation from bacterial L forms
 Bacteria that have temporarily failed to form cell walls (L-form) can
produce microcolonies similar to those of the mycoplasmas (the
failure to form cell wall is often due to the bacteria being exposed to
penicillin or other antimicrobial agents that affect cell wall formation).
 The L-forms may also produce fried-egg colonies like mycoplasmas,
but they differ in a number of respects.
 L-forms resemble the parent bacteria biochemically and antigenically,
they are not filterable, the minimum reproductive unit being about
600nm, sterols are not required for their growth, they are non
pathogenic and they show nucleic acid homology with parent bacteria.
o Differentiation between these two can be carried out by
  Subculturing the suspected bacterial L-form on media without
antibacterial substances should cause reversion of the L-forms with
the formation of normal sized bacterial colonies.
 Staining microcolonies with Diene's stain. Mycoplasmal colonies
retain the stain indefinitely,whereas bacterial L-form microcolonies
tend to decolourise in about 15 mts.
 Growth inhibition test
o This test is based on the ability of antisera to specifically inhibit the growth of
homologus species on solid media.
 Sensitivity to digitonin
o This test reflects the requirement of cholestrol for growth.
o Mycoplasma and Ureaplasma species are sensitive to digitonin
whereas Acholeplasma are not.
o Digitonin filter paper discs are commonly used.
 Serological test
o Rapid plate agglutination test, HI test and AGID: for screening of avian
mycoplasmas.

 CFT for CCPP and enzootic pneumonia. FAT, ELISA, LAT, species specific DNA
probes are highly useful in the identification of mycoplasmas.

TREATMENT AND CONTROL

 Tetracycline, gentamycin, kanamycin, tylosin, erythromysin and quinolones are

effective in some infections.
 Dipping hatching eggs in an antibiotic solution has been effective in producing chicks
free of M.gallisepticum.
 Cattle are vaccinated with live attenuated M.mycoides subsp mycoides strain are
useful to prevent CBPP.
 Live, attenuated and inactivated vaccines give partial protection against losses in egg
production and infections due to M.gallisepticum.

CHAPTER-23: LEPTOSPIRA

Learning objectives

To know in detail about,

 Spirocheates, Weil's disease, sugarcane fever and stuttgart disease

 Disease syndrome caused by leptospira in domestic animals
 Media for leptospira
 Morphology and cultural characters of leptospira
 Dark field examination
 Microscopic agglutination test
 Diagnostic methods used to identify leptospira

SYSTEMATICS
Domain Bacteria
 Phylum Spirochaetes

Class Spirochaetes(Spira- coil, Chaite- hair)

Order Spirochaetales

Family Spirochaetaceae

Genus Borrelia

Family Serpulinaceae

Genus Serpulina

Family Leptospiraceae

Genus Leptospira(Leptos-fine or thin) Leptonema

 The spirochaetes are slender, motile, unicellular, helically coiled, bacteria ranging
from 0.1 -3µm in width.
 Based on pathogenicity, DNA composition, and inability to grow at 13ºC the
genus Leptospira has traditionally been divided into two groups
o The pathogenic species - L.interrogans
o Free-living non-pathogenic strains - L.biflexa
 The common Leptospira interrogans serovars are L.interrogans serovar
Pomona, L.interrogans serovar Hardojobovis, L.interrogans serovar
Icterohaemorrhagiae, L.interrogans serovar Canicola, L.interrogans serovar
Grippotyphosa, L.interrogans serovar Bratislava, and L.interrogans serovar
Tarassovi.

HISTORY AND HABITAT

History

 Weil described the first case of leptospirosis in man in 1886.

 The causative agent of Weil‘s disease was isolated by India in 1915 and named L.
icterohaemorrhagiae.
 Subsequently a very large number of leptospires have been isolated from human
patients and animals from different parts of the world.

Synonyms of leptospirosis

 Weil‘s disease, Seven-day fever, Japanese autumnal fever, Rice field fever, Sugar cane
fever and Stuttgart disease (dogs).

Natural habitat
 Leptospirosis occurs worldwide. Leptospires are present in the proximal convoluted
tubules of mammalian kidneys (Sometimes genital tract) and are excreted in urine,
often for several months. Characteristically, a reservoir host shows minimal or no
clinical signs.
 Several animals act as carrier. Rats are particularlyimportant and they carry most
pathogenic serotypes.
 Animal carriers often excrete upto 100 million leptospirae per ml of urine.
 A warm moist environment with the presence of ground water with a neutral or
slightly basic pH favours the survival of the Leptospira outside the host.
 In India the disease has been more frequently reported from southern states than rest
of the country.

MORPHOLOGY

 Leptospires are actively motile, helically shaped, slender spirochaetes possessing

large number of light and fine spirals.
 They are 0.2 –0.3 µm in d.m. and 6-20 µm in length.
Characteristically, Leptospira have hooked ends and two periplasmic flagella (PF),
also known as axial filament and endoflagella.
 Rotation of the PF results in the distinct spinning mobility of Leptospira.
 They stain weakly or not at all with both aniline and Romanowsky stains. They may
be stained with giemsa stain.
 But they can be bestviewed under dark field illumination microscope and can be
stained by silver impregnation technique of Fontana. Leptospira divide by binary
fissio

CULTURAL CHARACTERISTICS

 Leptospira are obligate aerobes that use long chain fatty acids or fatty alcohols rather
than CHO and aminoacid as their energy and carbon sources.
 In addition to this it also requires vitamins B1, B12 and purines. Optimum
temperature for growth is 25ºC to 35ºC. They are highly fastidious.
 They grow very well in media enriched with rabbit plasma (rabbit plasma contain
high concentration of bound vitamin B12).
 Several liquid and semisolid media areavailable . Korthof‘s modified medium,
Stuart‘s liquid medium, Fletcher‘s semi solid medium, EMJH (Ellinghausen,
McCullough (1965), Johnson and Harris (1967), protein free medium (commomly
used for preparation of vaccine) and Ellis medium (mainly for isolation of leptospires
from the genital tract of cows).
 Among these, the EMJH medium contains bovine albumin (fraction V), Tween 80
and rabbit plasma and is most commonly used for isolation of Leptospira.
 The generation time in laboratory media is 12 –16 hrs. The cultures are incubated at
30ºC for upto 8 weeks.
 In semisolid media, growth occurs characteristically a few millimeters below the
surface.
 Addition of 5-flurouracil (100 mgm/ml) in medium is inhibitory for most of the
microorganism but not for leptospires.
 The colonies of Leptospira strains appear colorless and below the surface of the agar.
They may not be visible until held against opaque light.
 The leptospires are identified on the basis of their typical morphology and motility
under dark field microscopy.
 In fluid medium, the leptospires appear to rotate alternatively along their axis,
moving backward and forward with no apparent polar differentiation.
 In semisolid media, flexing, boring and serpentine movements are seen.
 RESISTANCE, ANTIGENS AND TOXINS

Resistance

 Leptospira are highly susceptible to heat, being killed in 10 mts at 50ºC and in 10 sec
at 60ºC.
 They are also sensitive to acid and are destroyed by gastric juice in 30 mts. Bile
destroys them rapidly.
 They are also readily destroyed by most antiseptics and disinfectants.
 They can survive for several days in alkaline water and only 12 –14 wks in sewage.
 Dogs may shed leptospires in their urine for 2 to 6 months, cattle for 3 months and
rats for longer periods.
 Leptospira have remained viable for atleast 6 days in coagulated blood and also they
remain viable in unfrozen kidneys for several days after the death of the animal.

Antigens and toxins

 A lipopolysaccharide antigen appears to be present in all members of the genus.

 Based on surface antigens, probably composed of protein-polysachharide
complexes L. interrogans is divided into more than 190 different serovars, arranged
into 25 major antigenically related serogroups. Animals and humans can be infected
with wide variety of serovars.
 The serovars causing disease in animals vary between countries and sometimes
between regions in the same country.
 Pathogenic leptospires produce haemolytic, lipolytic and cytotoxic substances.

PATHOGENESIS

 The accidental (incidental) hosts are infected by directtransmission through

infected urine, placental or fetal tissues or indirectly through contact with a
contaminated environment.
 Venereal transmission plays a major role in pigs. Vertical transmission from the
mother to the foetus may also occur in cattle.
 Leptospires gain entry through mucous membrane (nasal, genital, ocular, intestinal)
or through abraded or water-softened skin.
 After epithelial penetration there is haematogenous spread (Leptospires able to
invade the blood stream more rapidly than other bacteria), with localization and
proliferation in parenchymatous organs, particularly the liver, kidneys, spleen and
sometimes meninges for upto 16 days.
 It causes damage to endothelium of small blood vessels, leading to extravasion of
blood and secondary ischaemia result in damage to liver, kidney and adrenals.
 In the kidneys, the organism reaches and localize in the lumen of proximal
convoluted tubules.
 Penetration and multiplication in the fetus leads to fetal death and resorption,
abortion or weak-off spring. (In foetus, if infection occur at 3rd trimester, can
produce specific antibodies and may overcome the infection).
 The leptospires tend to persist in sites such as renal tubules, eyes and uterus where
antibody activity is minimal.
 Leptospira causes following disease syndromes in domestic animals.

Host Disease syndrome

Cattle  Subclinical with leptospiruria (Hardjo)
 Milk drop syndrome (Hardjo)
 Abortion and neonatal mortality (Pomona and Hardjo)
 Infertlity (Hardjo)
 Haemoglobinuria, jaundice and fever in calves (Pomona, Grippotyphosa and
Icterohaemorrhagiae)

Pigs  Subclinical with leptospiruria (Pomona)

 Fever and non-suppurative mastitis (Pomona)
 Infertility, abortion and still birth (Canicola, Pomona, Icterohaemorrhagiae)
 Haemoglobinuria, jaundice and fever (Icterohaemorrhagiae)

Dogs  Subclinical with leptospiruria (Pomona)

 Acute haemorrhagic type characterized with fever, vomiting, prostration and
death (Icterohaemorrhagiae)
 Less acute icterus type (Canicola and Icterohaemorrhagiae)
 Ureamic type (Canicola)

Horse  Recurrent iridocyclitis/periodic opthalmia / moon blindness (Pomona)

 Abortion at 6 month of gestation

Sheep  Subclinical infection with leptospiruria (Hardjo)

PATHOGENECITY

Symptoms and lesions

 Leptospirosis can occur as

o Acute severe disease (Septicaemia with endotoxaemia such as haemorrhages,
hepatitis, nephritis and meningitis)
o Subacute moderately severe disease (nephritis, agalactiae, hepatitis and
meningitis)
o Chronic disease (iridocyclitis, abortion, still birth, infertility)
o Subclinical form (recognize only by the development of antibody)
o Symptoms include fever, severe muscle pain, haemoglobinuria, jaundice,
anemia, anuria, meningeal signs, gastro intestinal signs, skin rashes and
photophobia.
o Cattle: Acute form characterized with marked drop in milk production
associated with pyrexia in chronic form- abortion, still birth or premature
calves.
o Pigs: Haemoglobinuria and jaundice in piglets, abortion in the last 3rd of
pregnancy and stillbirth.
o Dogs: The acute form of leptospirosis is known as Stuttgart disease.
o It is characterized by fever, haemorrhage, anemia and jaundice.

DIAGNOSIS
Specimens

 PBS containing 1%BSA is used as transport medium

o Heparinised blood for culture
o Whole blood/serum for serological tests (blood must be collected during early
febrile stage)
o Mid-stream urine for dark filed examination
 Leptospires can disintegrate quite quickly in a urine sample, especially if it is acidic.
 So, if the urine cannot be examined within 20 mts, it should be neutralized with N/10
Hcl, or N/10 NaOH.
 To preserve the morphology of the leptospires for several days, 20 ml of midstream
urine should be added immediately to 1.5ml of 10% formalin.
 By adding formalin, the leptospires will be killed but will retain their morphology for
several days and can be examined by dark field microscopy.
 Kidneys for both darkfield microscopy and culture. Kidneys/or liver sections in 10%
formalin for histopathology.
 Foetal kidney smears (cryostat sections or smears) for FAT. Foetal abomasal
contents, cotyledons and uterine discharge for differential diagnosis.
 CSF and semen from bull can also be useful for demonstration of leptospires.

Direct microscopy

 Leptospires can be demonstrated in urine, other body fluids, and tissues by darkfield
microscopy (DFM) and by FAT.
 To detect under DFM, one probably needs 10,000 to 20,000 leptospires/ml in the
sample toview atleast one Leptospira in the high power field.
 Urine is centrifuged to concentrate the leptospires. Unclotted blood is centrifuged at
low speed to sediment the RBC after which the plasma can be removed and
centrifuged at high speed.

Isolation in culture media

 After inoculating the suitable media incubate at 30ºC for upto 8 weeks. A drop of the
culture is examined by DFM.

Animal inoculation

 Guineapigs, hamsters and weaning gerbils can be inoculated i/p with 0.5 to 1 ml of
neutralized urine, unclotted blood or a 10% tissue suspension in EMJH or 1% BSA.
 Cardiac blood is taken aseptically when a temperature rise is detected or at 5,8,10 and
14 days post infection.
 Media are inoculated with 2-3 drops of the freshly collected blood.

By serology

 Macroscopic agglutination test: it is a screening test and uses dead antigen (lack of
specificity).
 Microscopic agglutination test (MAT): it uses live leptospires as antigen and is highly
sensitive and serovar specific.
 CFTand ELISA are also useful for detection of leptospiral antibodies in serum.
 To identify the serovar, MAT, restriction endonucleae, DNA analysis and monoclonal
antibodies are useful.

TREATMENT AND CONTROL

  Immunity appears to be mainly humoral in that the organisms are not intracellular
and bacterins give protection for short duration.
o Dogs: Bacterins usually contain serovars, Canicola and Icterohaemorrhagiae.
o Cattle: Bacterins usually contain serovars Hardjo, Grippotyphosa, Canicola
and Icterohaemorrhagiae.
 Combined penicillin, streptomycin are highly effective. The tetracycline and
macrolide antibiotics are also effective.

CHAPTER-24: BACTERIOIDES AND FUSOBACTERIUM

Learning objectives

To know in detail about,

 Bacterioides and Fusobacterium species

 Media and cultivation methods for Bacterioides and Fusobacterium species
 Colonial morphology and microscopic appearance of Bacterioides species
 Different types of foot rot in sheep
 Choice of specimens and collection procedures, isolation and identification methods
of Bacterioides infections in animals

SYSTEMATICS
Domain Bacteria Bacteria Bacteria

Phylum Proteobacteria Bacteroidetes Fusobacteria

Class Gammaproteobacteria Bacteroidetes Fusobacteriales

Order Cardiobacteriale Bacteroidales Fusobacteriales

Family Cardiobacteriaceae Bacteroidaceae Fusobacteriaceae

Genus Dichelobacter Bacteroides -

 Bacteroides, Fusobacterium, Campylobacter mucosalis, Campylobacter

hyointestinalis and Serpulina hyodysenteriae are non spore forming, Gram-negative
anaerobic bacteria.
 They are commonly implicated in necrotic and suppurative conditions, often as
mixed infections with facultative anaerobic bacteria.

HABITAT AND MORPHOLOGY

Habitat

 B. nodosus are obligate anaerobic bacteria of the digital epidermis of sheep under
normal climatic conditions.
 They will not survive on pastures for more than a week.
  Other Bacteroides species can be normal inhabitants of the skin, mucous membrane
and G.I. tract of domestic animals.
 Fusobacterium species occurs as a commensal in the alimentary tract and mucous
membranes of a variety of animals.

Morphology

 Bacteroides nodosus appear as Gram-negative, fairly large (1.7 x 3-6 µm), slightly
curved and non-motile rods.
 Often swollen at one or both ends. They occur singly or occasionally in pairs
 Fusobacterium necrophorum is Gram-negative, long and filamentous but does not
branch.
 Filaments can be up to 100 µm in length and 0.5-0.7 µm in diameter.
 May have tapered or rounded ends. Irregular staining is characteristic

CULTURAL CHARACTERISTICS

 Anaerobic jars with a catalyst, an anaerobic indicator and an atmosphere free of

oxygen (10% Hydrogen, 5% Co2 and 85% Nitrogen) is essential for the culture of
these strict anaerobes.
 Enriched blood agar is highly suitable for these fastidious anaerobes.
 Eugon, Columbia, trypticase soy or brain-heart infusion agar enriched with 0.5 per
cent yeast extract, vitamin K (10 µg/ml), haemin (5 µg/ml), kanamycin (100 µg/ml)
and vancomycin (7.5 µg/ml) are most commoly used.
 Bacteroides spp. (except B. ureolyticus) are resistant to kanamycin but
the Fusobacterium spp. is sensitive to this antibiotic.
 A 'Fastidious Anaerobe agar' isavailable commercially with various antibiotic
supplements, depending on the anaerobe that is being sought.
 Eugon agar base with 0.2 per cent (w/v) yeast extract, 10 per cent defibrinated horse
blood and one µg/ml lincomycin is the selective medium for Bacteroides nodosus.
 Lemco agar containing pulverized hoof powder can also be used. Members of the B.
fragilis group will grow on bile aesculin medium with 5 per cent sheep blood.
 Agar media should be pre-reduced in an anaerobic jar for 6-24 hours before use. The
plates are incubated at 35-37°C immediately after streaking for 4-8 days.
 Liquid media such as Cooked meat broth with 0.4 per cent glucose or thioglycollate
medium is also suitable with the addition of the vitamin K-haemin supplement.

Colonial morphology and microscopic appearance

 The cellular morphology, and sometimes the colonial morphology, can be very
variable depending on the strain, medium and cultural conditions.

Bacteroides nodosus

 B. nodosus in a Gram-stained smear from enriched blood agar, appears as straight or

slightly curved rods with the characteristic terminal knobs on one or both ends of the
cells
 Three basic colonial types are described
o B-type: papillate or beaded (most pathogenic) from ovine foot rot.
o M-type: mucoid (less pathogenic) from non-invasive infections of sheep and
cattle.
o C-type: circular (non-pathogenic) and resulting from repeated passage in
media.
 o The colonies, generally, are greyish-white and 0.5-3.0 mm, diameter, in 3-7
days.

Bacteroides melaninogenicus

 Circular, entire, convex and shiny colonies, 0.5- 2.0 mm in diameter.

 Colonies become darker after 5-14 days, being black in the centre with a grey-brown
periphery.
 Haematin (black or brown) pigment is seen best on media containing blood.
 A few strains are haemolytic on rabbit blood agar. The colonies fluoresce under ultra
-violet light.

Bacteriodes asaccharolyticus

 Colonies are 0.5-1.0 mm in diameter, round, convex, opaque and light grey after 48
hours' incubation.
 In 6-14 days the colonies may become black. Some strains are haemolytic on rabbit
blood agar

Bacteroides fragilis

 Colonies are circular, entire, low convex, translucent to semi-opaque.

 They tend to have concentric rings of growth. Less than 1 % of strains are haemolytic.

Fusobacterium necrophorum

 F. necrophorum produces grey to yellowish shiny colonies on blood agar, that are
about 2-3 mm in diameter after 48 hours' incubation.
 Haemolysis is variable.
 A Gram-stained smear from the colonies shows long Gram-negative filaments that
are less characteristic than those from direct microscopic examination of specimens.
 Lipase, but not lecithinase, activity is exhibited by many strains
of F. necrophorum on egg yolk agar.

BIOCHEMICAL CHARACTERISTICS AND RESISTANCE

Biochemical characters

 All pathogenic Bacteroides species are catalase negative except B. fragilis. B.

nodosus does not ferment carbohydrates.
 F. necrophorum is indole and H2S positive. Gelatin is not liquefied.
 They ferment glucose and maltose with production of acid and gas but not lactose.

Resistance

 Both the species are highly susceptible to atmospheric oxygen.

 They are readily killed at 50 –60º C for 15 minutes.

TOXINS AND PATHOGENESIS

Toxins

 B. nodosus causing foot rot produces keratinolytic enzymes in greater amounts.

  F. necrophorum produces an exotoxin (leukotoxin) and necrotizing endotoxin.
 The endotoxin when inoculated intradermally into rabbit causes necrosis.
 The exotoxin when inoculated in skin of rabbits causes mild erythema but when
administerd intravenously causes maciation or death of rabbits in a few hours.

Pathogenesis

 The infections are often endogenous, arising from normal flora at the site or by
wounds contaminated by nearby flora.
 For these strict anaerobes multiply at a focus in animal tissue if the redox potential of
the area is lowered.
 This can occur through trauma and necrosis, ischaemia, parasitic invasion or
concomitant multiplication of facultative anaerobes.
 B. nodosus produces keratinolytic enzymes and F. necrophorum produces leukotoxin
– which protects Corynebacterium pyogenes from phagocytosis. C.
pyogenes produces a diffusible factor that stimulates the proliferation
of Fusobacterium in tissue.
 The conditions caused by these non-sporing anaerobes include soft tissue abscesses
and cellulitis, post-operative wound infections, periodontal abscesses, aspiration
pneumonia, lung and liver abscesses, peritonitis, pleuritis, myometritis,
osteomyelitis, mastitis and foot rot. The excessive odour is due to production of
volatile fatty acids.
 Diseases caused by Bacteroides and Fusobacterium species

Species Host(s) Disease

Dichelobacter (Bacteroides) Sheep Contagious (virulent) foot rot

nodosus Cattle, Goats and Contagious interdigital dermatitis
Pigs

B. melaninogenicus Cattle Foot rot

B. asaccharolyticus Dogs, cats, Osteomyelitis

horses, cattle

B. fragilis Calves, lambs, Diarrhoeal disease

foals, piglets

Fusobacterium necrophorum Sheep Foot abscess , Ovine interdigital

(associated dermatitis (Scald)
with Corynebacterium Cattle
pyognes ) Calf diphtheria
Horses
Thrush - infections of front of hoof.
Pigs
Ulcerative stomatitis
Chickens ("sore mouth" or "Bull nose" - viainjury
from fitting nose rings)
 Necrotic enteritis

Avian diptheria (Secondary to fowl pox)

PATHOGENICITY

Symptoms

 Infections in animals generally occur when animals are kept in filthy, manure-laden
surroundings.
 Among the Fusobacterium only necrophorum regularly causes disease in animals.
 It is frequently a secondary invader (e.g., liver abscesses in cattle often found
with Corynebacterium pyogenes and is characterized by a necrotic process,
commonly causing diseases collectively referred to as necrobacilloses and present as
necrosis, abscess formation, and putrid odour (most common fermentation product
is butyric acid).

Symptoms will vary according to the sites of the lesions.

Cattle

 In foot rot – acute painful swelling of a hoof which leads to lameness.

 Necrosis in digits spreads to tendons, ligaments and joints.
 Calf diphtheria is more common in 3 to 18 months old.
 Excessive salivation, purulent discharge from nose, coughing, temperature and loss
of appetite and is found in necrotic foci in the mouth, larynx and trachea.

Sheep

 Foot rot (interdigital dermatitis, infective bulbar necrosis and heel abscess), mouth
lesions and abortions (rare).

Swine

 Principal cause of bull nose or Ulcerative stomatitis viainjury from fitting nose
rings.

Cats

 Opportunistic. Highly suppurative. Involves nasal passages, oral cavity and bone.
 Secondary invaders to tissue damage.
 Dental tartar leads to gingivitis and periodontal disease.

DIAGNOSIS

Choice of specimens

 As the non-sporing anaerobes constitute a major portion of the normal flora, the
specimens must be collected with care to avoid contamination from the normal
anaerobic flora, situated mainly on mucous membranes and in the intestinal tract.
The following samples are suitable for culture of the non-spore-forming anaerobes.
o Pus from abscesses
 o Discharges from wounds (surgical and traumatic)
o Direct pleural aspirates
o Peritoneal aspirates
o Joint fluids
o Urine if taken by suprapubic puncture
o Tissue specimens (biopsy, necropsy and post-operative)

Collection of specimens

 Specimens for the isolation of these strict anaerobes should be placed immediately in
an oxygen-free container, especially small pieces of tissue or material taken on swabs.
 Larger pieces of tissue (over 2 cm3) usually maintain an anaerobic microenvironment
deep in the tissue and can be placed in an air-tight jar for transportation.
 Fluid specimens can be collected in a sterile syringe, the air expelled and the needle
bent over or plugged. However, if the specimen cannot be processed within an hour, a
fluid specimen should be placed in an oxygen-free tube or vial.
 All specimens for anaerobic culture should be processed within a few hours of collec-
tion.
 It is best to keep the specimens at ambient temperature rather than in the
refrigerator, as oxygen absorption is greater at lower temperatures.
o Direct examination
 Gram-stained smears of the specimens are useful as a screening
process, although many of these anaerobes are not morphologically
distinctive.
 Dilute carbol fuchsin (4-8 minutes) stained smears are more useful
for Bacteroides and Fusobacterium species as they tend to stain
faintly with the Gram-stain.
 Fusobacterium necrophorum in clinical specimens is long and
filamentous (about 1µm in diameter) and characteristically stains in
an irregular manner.
 Bacteroides nodosus is a large rod characterised by the presence of
terminal enlargements at one or both ends (barbell or club shaped).
o FAT is reported as being specific and sensitive
o By Isolation and identfication in anerobic media
o By animal inoculation
 Mice, rats and guinea pigs inoculated with cultures of B. nodosus will
produce abscess and sepsis.
 Inoculate rabbit subcutaneously with material suspected
for F. necrophorum - produces lesions throughout the body.
 Differential diagnosis should be made with strawberry foot rot caused
by Dermatophilus species, FMD and pyogenic infections associated
with Corynebacterium pyogenes.

TREATMENT AND CONTROL

 In B. nodosus infection the affected hooves should be trimmed and treated with
10% formalin or chloramphenicol or tetracycline.
 Parental treatment with penicillin and streptomycin may be of value.
 In Fusobacterium infections, 5-10% CuSO4 is recommended for the treatment
of foot lesions.
 For early treatment sulphonamides, tetracycline and erythromycin are useful.
 CHAPTER-25: DERMATOPHILUS

Learning objectives

To know in detail about,

 Disease caused by Dermatophilus species

 Development cycle of Dermatophiuls congolensis
 Tram-track appearance and strawberry foot rot
 Pathogenesis of mycotic dermatitis in sheep
 Haalstra's method for the isolation of Dermatophilus congolensis
 Different diagnostic methods of dermatophilosis

SYSTEMATICS
Domain Bacteria

Phylum Actinobacteria

Class Actinobacteria

Subclass Actinobacteridae

Order Actinomycetales

Suborder Micrococcineae

Family Dermatophilaceae

Genus Dermatophilus

 Dermatophylaceae is a group of bacteria with mycelial filaments which divide

transversely and in at least two longitudinal planes to form masses of coccoid or
cuboidal cells, which characteristically become motile.
 They are Gram positive, non acid fast, aerobic and produce aerial mycelium when
their growths are stimulated by 10% CO2.
 Dermatophilus congolensis, Dermatophilus dermatonomus and Dermatophilus
pedis are the pathogens causing variety of skin lesions in mammals, including man

HISTORY AND HABITAT

History

 Bovine disease was first described by Van Saceghem in 1915 in the Congo now known
as Zaire in Africa.

Habitat
  Dermatophilus congolensis is the only species in the genus thought to maintain itself
in small foci of infection on a carrier animal or within scab particles in dust.
 It can survive in scab material for periods upto 3 years.

MORPHOLOGY

 D. congolensis is Gram positive, non acid fast and aerobic to facultatively anaerobic.
 It is filamentous and branching. Mature filaments are composed of motile, coccal
zoospores, in parallel lines, at least two abreast, resulting in a 'tram-track'-like
appearance.
 The zoospores are about 1 um in diameter. If the flakes of scab (collected from
infections) are treated too roughly, when the smears are made, the filaments will
disintegrate and only Gram-positive cocci (zoospores) will be seen.

Life cycle

 Transverse, horizontal and vertical septa form in the immature filaments dividing it
into coccal zoospores.
 When mature, these zoospores are motile by polar flagella and are infective.

CULTURAL AND BIOCHEMICAL CHARACTERISTICS

Cultural characteristics

 The bacterium is comparatively easy to culture and grows well on sheep or ox blood
agar.
 An atmosphere of 5-10 per cent CO2enhances the growth of the organism, espe-
cially on primary isolation.
 The inoculated plates are incubated at 37°C (Optimum temp. 37oC.) for up to 5 days,
although colonies may be seen after 24-48 hours incubation.

Colonial morphology

 Small (about 1 mm) greyish-yellow, distinctly haemolytic colonies can be seen after
24-48 hours incubation.
 They are firmly adherent to the medium and are embedded in the agar.
  After 3-4 days, isolated colonies can be 3 mm in diameter and are rough, wrinkled
with a golden-yellow colour. Older colonies can become mucoid.
 No growth occurs on Sabouraud dextrose agar.

Microscopic appearance

 Gram-stained smears from colonies do not show the characteristic 'tram-track'

appearance seen on direct microscopy.
 Usually the smears reveal uniformly staining, Gram-positive, branching filaments but
sometimes coccal forms predominate.

Biochemical characters

 D. congolensis is catalase-positive, urease-positive, gelatin-positive and produces

acid from glucose, fructose and maltose.
 It is indole-negative, does not reduce nitrate and non-fermentative although acid is
produced from certain carbohydrates.

PATHOGENESIS

 Dermatophilus congolensis causes very severe clinical disease and its infection is
most common in tropical and subtropical regions.

Diseases caused by Dermatophilus species

 Dermatophilus congolensis mainly affects Cattle, horses, sheep and goats, but many
animal species and man can be infected.
 The disease has many names
o Cattle : Rain scald, Streptothricosis, Dermatophilosis
o Sheep: Mycotic dermatitis (general infection), lumpy wool (wool- covered
skin) and strawberry foot rot (skin of lower leg and coronet)
 The bacterium produces disease in many animal species. It is also a zoonosis.
 The common name for the disease is dermatophilosis or streptothricosis.
 As far as is known the bacterium is considered an obligate parasite, living only on
animals.
 Infection is spread by contact, biting insects, fomites and by other unknown means.
 Moist conditions and high relative humidity are known topromote the prevalence of
the disease.
 D. congolensis causes skin infections most commonly seen in cattle, sheep, goats,
horses and polar bears in zoological collections.
 The infection is characterised by the formation of thick crusts which come away easily
with a tuft of hair, leaving a moist, depressed area with bleeding points from capil-
laries.
 Infections can be localised but have a tendency to spread over large areas of the body
and the morbidity and mortality can be high, especially in tropical regions.

 The position of the lesions varies with the predisposing conditions.

 In periods of high rainfall the lesions tend to occur along the backs of animals.
 Where there is a heavy infestation with Amblyomma ticks the lesions are present in
the predilection sites of the ticks: dewlap, axillae, udder and scrotum.
 In the dry season, in tropical regions, when feed is scarce the lesions are on the
muzzle, head and lower limbs due to the animals foraging in thorn-covered scrub.
 Though the disease does not lead to death in the adult cattle, deaths in young goats
and cattle have been reported.
  In sheep, the disease is called mycotic dermatitis and is seen in three forms

Dermatitis of the wool-covered areas of the body or lumpy wool

o Dermatitis of the face and scrotum; and

o Dermatitis of the lower leg and foot, which may result in severe ulcerative
dermatitis referred to as "strawberry foot rot".
 Infections have been reported in dogs and cats. In dogs, spontaneous
dermatophilosis has been confined to the intugementary structures, whereas deeper
tissue lesions in the form of abscesses have been reported in the felines.
 Lymph node enlargements with draining fistulas of the subcutaneous and
submucosal tissues have also been reported in cats.

DIAGNOSIS AND IMMUNITY

Diagnosis

Based on Direct microscopy

 A tuft of hair that is plucked from the lesion usually detaches with scab material
adhering to it.
 Grind up a small amount of scab material and place a little in 2 ml distilled water in a
container and incubated for 3.5 hours at room temperature.
 Place the container, with lid removed, in a candle jar at room temperature for 15
minutes.
 The motile zoospores are chemotactically attracted to the carbon dioxide-enhanced
atmosphere in the candle jar and move to the surface of the distilled water.
 Remove a loopful of fluid from the surface and inoculate a blood agar plate.
 Incubate the inoculated plate at 37°C for 72 hours under 5-10 per cent CO2.
 Identification of this bacterium does not seem to pose any problems. Lesion and
typical morphology are quite diagnostic.

Haalstra's Method for the Primary Isolation of Dermatophilus congolensis

 The organism grows well on blood agar plates within 24 to 48 hours as small grayish
white colonies turning yellow to orange upon further incubation.
 Although the isolation of D. congolensis may not be necessary for a diagnosis of
streptothricosis, Scab material contains many contaminants and Haalstra's method
was developed to overcome this problem.

Based on Isolation and Identification

 Small pieces of material are shaved from the scab with a scalpel and the flakes of scab
are softened in a few drops of distilled water on a microscope slide.
 A smear is made, taking care to leave a few flakes of scab material intact. The smear
can be stained by either Giemsa or Gram stains.
 Giemsa is the better stain to show the characteristic morphology of the bacterium.
 Segmenting filaments and coccoid spores stain deep purple. The spores are seen in
packets.

Immunity
  There is no known vaccine for immunization against the disease produced
by Dermatophilus.
 Recovery from the disease seems to confer permanent immunity to reinfection.

CHAPTER-26: RICKETTSIALES

Learning objectives

To know in detail about,

 Characters of rickettsiales
 Principal diseases, host and mode oftransmission of rickettsial pathogens
 Q-fever and Heart water in cattle
 Weil-felix reaction
 Stains used to identify the rickettsiales
 Cultivation methods of rickettsiales
 Antigens and toxins of rickettsiales
 Pathogenesis of rickettsiales
 General approaches used to diagnose Q-fever canine ehrlichiosis, Salmon poisoning
and Heart water

SYSTEMATICS

o Domain : Bacteria
o Phylum : Proteobacteria
o Class : 1. Alphaproteobacteria
o Order : 1. Rickettsiales
o Family : 1. Rickettsiaceae
o Family : 2. Ehrichiaceae
o Genus : Ehrlichia
o Genus : Anaplasma
o Genus : Cowdria
o Genus : Neorickettsia
o Genus : Aegyptianella
o Order : 2. Rhizobiales
o Family : Bartonellaceae
o Genus : Bartonella
o Class : 2. Gammaproteobacteria
o Order : Legionellales
o Family : Coxiellaceae
o Genus : Coxiella
o Phylum : Firmicutes
o Class : Mollicutes
o Order : Mycoplasmatales
o Family : Mycoplasmataceae
o Genus : Haemobartonella
o Genus : Eprythrozoon
 They are minute obligate intra cellular parasites requiring living cells for
multiplication.
 They were formerely considered closely related to virus.
 But based on their characters like
o cell walls similar to those of other Gram negative bacteria.
o divide by binary fission
o possessing cell wall containing muramic acid
 o metabolic enzymes independanat of the host cell
o posess both DNA and RNA
o large enough to be seen under the light microscope
o held back by bacterial filters
o susceptible to antibiotics
o They are considered true bacteria,specially adapted to obligate intra cellular
parasitism.

CLASSIFICATION

 Depending on the diseases they produce, the vectors that transmit them, antigenic
relationships, growth properties and resistance to physical and chemical agents.

S.No Genus Cell parasitised in host

1 Rickettsia Macrophages, leucocytes and endothelial cells

2 Coxiella Circulating leucocytes

3 Ehrlichia Vascular endothelial cells

4 Cowdria Vascular endothelial cells

5 Neorickettsiae Reticular cells of lymphoid tissue

 Rickettsiales are usually parasites of alimentary tract of arthropods such as fleas, lice,
ticks and mites.
 Transmission is from artropod to animal.
 The principal diseases, hosts, mode of transmission of pathogens in
the Rickettsiales are:

Species Disease Main host Transmission

R.Prowazekii Epidemic typhus Human Louse

R.Mooseri Endemic typhus Human Rat flea

R.Rickettsii Rocky mountain Human, dogs, Ticks (Dermacentor spp)

spotted fever rabbit

R.tsutsugamushi Scrub typhus Small Mite

rodents,Birds

Rochalimae Trench fever Human Louse

quintana

Coxiella burnetti Q-fever Human, Cattle Human

and small
ruminants Contaminated dust mainly by
inhalation and ingestion of
contaminated milk

Contact with birth fluid of

ruminants

Ixodes tick

Animals

Ehrlichia bovis Bovine Cattle Ixodes tick

ehrlichiosis

Ehrlichia canis Canine Dog Rhipicephalus sanguineus

ehrlichiosis
(Brown dog tick)
(Tropical canine
pancytopenia)

E.phagocytophilia Grazing fever in Cattle &Sheep Ixodes tick

cattle

E.risticii Potomac horse Horse Vector not known

fever

(equine
monocytic
ehrlichiosis)

Cowdria Heart water Cattle, Sheep, Ambylomma ticks

ruminantium Goat and wild
ruminants

Neorickettsia Salmon Dogs, foxes, bears, Ingestion of salmon fish contain

helminthoeca poisoning ferrets the infected helminth
fluke(Nanophytes salmincola)

HISTORY AND HABITAT

History
  The name Rickettsiales has been given in honor of American Pathologist Howard
Taylor Rickets (1909) who first observed these microorganisms in Rocky Mountain
spotted fever.
 Derrick (1935) investigated cases of fever occurring in abattoir workers in Brisbane,
Australia.
 As the etiology of the disease was unknown, it was referred to as Querry or Q fever.
 The causative agent was identified by Burnert (Australian) and Cox (American)- so, it
named as Coxiella burnetti.

Habitat

 The Rickettsiales are essentially parasites of arthropods, replicating in the cells of

gut.
 Some can be passed transovarially, in ticks and mites but others such
as Cowdria and Ehrlichia, are passed transtadially.
 They do not survive the outside the living cells (host or vector) with the exception
of Coxiella burnetii, which produce endospore like forms, that can survive in dust
particles for 50 days or more.
 Several Rickettsiales may persist in the host in a latent form.

MORPHOLOGY AND CULTIVATION

Morphology

 Rickettsiae are small, non-motile, non-capsulated pleomorphic, coccobacillary (0.3 –

0.6 x 0.8-2µm in size) forms existing as obligate intra cellular parasite.
 Under the EM, the rickettsiae are seen to have a 3 layered cell wall and trilaminar
plasma membrane, thus resembling Gram –ve bacteria.
 They stain reasonably well with Giemsa, Castaneda, (bluish purple), Gimenez,
Machiavello (deep red), and Leishman stains, but poorly with Gram‘s stain.

Cultivation

 Rickettsiae multiply by simple binary fission. They have cytochromes and their
metabolic reactions are aerobic.
 They possess many of the metabolic functions of bacteria but require exogenous co-
factors from animal cells.
 Rickettsiae can genrate their own energy, but they also depend on their host for some
energy.
 Rickettsiales require living cells for replication. They are readily cultivated in the yolk
sac of developing chicken embryo (first shown by Cox), or in cell lines like mouse
fibroblast, HeLa and HEp2.
 Growth generally occurs in the cytoplasm of the infected cells or in some cases
(spotted fever) in the nucleus.
 E.canis can be propagated very well in dog monocytes culture.
 Rochalimae quintana –the only rickettsiae which have the ability to grow on blood
agar.
 Guinea pig and Mice are useful for primary isolation.

RESISTANCE, ANTIGENS AND TOXINS

Resistance
  Rickettsiae are readily inactivated by physical and chemical agents.
 Rickettsiae can lose their viability in storage due to loss of their intercellular ATP pool
and several coenzymes.
 They can be preserved in skimmed milk or a suspending medium containing sucrose,
K, Po4 and glutamate (SPG) medium.
 Coxiella burnetti is relatively resistant to physical and chemical agents.
 In dried tick faeces and in wool, it survives for a year or more at 40C and in meat for
atleast a month.
 Holding method of pasteurisaton is not effective, but the flash method is effetive.
 Rickettsiae are susceptible to tetracycline and chloramphenicol.
 Penicillin and sulphonamides are ineffective. Sulphonamides may actuallyenhance
the growth of rickettsiae.

Antigens and toxins

 Atleat 3 types of antigens have been demonstrated

o Group specific soluble antigen
o Species specific antigen
o Alkaline stable polysaccharide- found in some rickettsiae and in some strains
of Proteus organism.
 The sharing of antigens between rickettsiae and Proteus is the basis for the Weil-Felix
reaction used for the diagnosis of rickettsial infections by the demonstration of
agglutinins to Proteus strains OX19, Ox2 and OX k.
 Coxiella burnetti is the only rickettsiae to exhibit phase variation. Fresh isolates are
in Phase I.
 They become Phase II on repeated passage in yolk sac but reversion to phase I take
place by passaging in guinea pigs.
 Phase II cells are autoagglutinable. Phase I activity is attributed to a surfcace CHO
antigen.
 Phae I immunogen is more powerful than Phase II and elicits high titre antibodies.
 Q fever sera react with other rickettsial antigens or with Proteus.
 The toxins have not been isolated and identified. Haemolysins are produced by some
typhus rickettsiae.
 Rickettsiae contains (endotoxin like) LPS. They are different from true endotoxins of
Gram-negative bacteria.

PATHOGENESIS

o Adherence-, which is facilitated by the surface receptors of the host cell

o Endocytosis
o Phagosome destruction- Rickettsiae destroy the phagosomal mambrane by
phospholipase
o Multiply within the cytoplasm or in certain cases (spotted fever) nucleus.
 Infection begins in the vascular system, organism proliferate in the endothelial and
phagocytic cells and are disseminated via blood stream.
 There is obstruction of small blood vessels because of hyperplasia of infected
endothelial cells and resulting thrombi.
 If capillary endothelium is affected, producing thrombi that result in haemorrhagic
skin rashes.
 Q fever organism has prediliction for mammary gland and placentae in cattle and
sheep. Occasionally asymptomatic infections occur.
 Q fever infection causes abortion in sheep, goats and cattle and bronchopneumonia
in sheep.
  Fever, hamorrhagic rash, stupor, shock and patchy gangrene of subcutis and skin are
the common signs and lesions noticed in rickettsial infections
 In case of Ehrlichiosis, the affected animals show symptoms of congested mucous
membrane, purulent discharge from eyes and nose, gastritis, oedema of the hind legs
and enlarged lymphnodes.
 The mortality rate is 100% in acute cases. On P.M., the lesions are pulmonary
oedema, haemorrhages of lung, hydrothorax, splenomegaly and hyperplasia of
lymphnodes.
 In Salmon poisoning, the mortality reaches 90%, affected dogs become weak, with
vomition, depression and diarrhoea.
 Theimportant lesion is hyperplasia of lymphnodes with necrosis.

Steps involved in parasitization include

 In mammals, by direct penetration of skin as a result of feeding by an infected

arthropod (tick, louse, flea or mite)
 In arthropods as the result of ingestion of blood of infected animals
 From arthropod to progeny by infected ova.
 In Q fever, wild animals such as bandicoot may constitute the primary reservoir, the
infection being transmitted among them by Ixodes tick.
 Transovarial transmission has been demonstrated in Ixodes tick. The rickettsiae are
abundant in tick faeces and survive in them for long periods in the dry state.
 Ticks transmit the disease to cattle, sheep and poultry. The rickettsiae are shed large
numbers in the milk of infected cattle, uterine discharges, after-birth and other
secretions.
 The infected material gets dried up in the atmosphere and becomes suspended in the
air and transported to long distances.
 In human, the principal route of infection is mainly by inhalation of contaminated
dust particle, ingestion of infected milk, and contact with contaminated material.
 In Salmon poisoning in dogs, the disease is transmitted by eating raw salmon fish,
which contained the infected fluke (Nanophytes salminicola).
 Infected metacercariae encysted in the muscles of fish are ingested by dogs; flukes
will get mature and release invasive rickettsiae.
 The fluke eggs are passed in the dog intestine. These eggs develop into meracids,
which infect the snail.
 The cercaria develops within the snail and passes from snail and infects susceptible
species of fish.

DIAGNOSIS

 It vary with the disease, but usually include unclotted blood for blood smears,
affected tissues (such as brain in heart water), paired serum samples for serology are
appropriate.
 In Q fever, besides blood, the sputum and less often the urine, may yield the causative
agent.
 By Direct microscopy
Both blood and tissue smears, stains such as Giemsa, Gimenez, Machiavello and
Leishman as well as FAT are useful.
 By Isolation and cultivation
This is often difficult and is not usually necessary for a laboratory diagnosis.
 Guineapigs and mice are useful for primary isolation. Suspected materials are
inoculated i/peritoneally and the animals have to be observed for 3-4 weeks for
raising their temperature. (Rochalimae quintana will not grow in guinea pigs and
mice).
 Disease Diagnosis
Q-fever  Detection of organisms in
o Giemsa or FAT stained smears from ruminant placentas
o Inoculation of chicken embryos, guinea pigs and hamsters
o Paired sera samples for agglutination, CFT and ELISA
o Allergic test
 The cattle and horses are inoculated in the lower eyelids with the antigen. After
3-4 days, there is acute swelling of the eyelid in the positive case with rise in
temperature.

Canine  Giemsa stained blood smears (best at 13th day post infection) - characteristic
ehrlichiosis inclusion in monocytes and neutrophils
 Serology – indirect FAT

Salmon  Observation of characteristic fluke eggs in faeces

poisoning  Giemsa, Gimenez or machiavello stained smears of fluid aspirated from
lymphnode

Heart water  Giemsa or FAT stained smears from brain tissue

 Inoculation of susceptible cattle or mice

Weil-felix reaction

 This test was developed by Weil and Felix (1916).

 The weil-felix reaction is a simple and specific agglutination test for the diagnosis of
some rickettsial disease.
 The basis of the test is the sharing of an alkali stable carbohydrate antigen by some
rickettsiae and by certain strains of Proteus, P.vulgaris OX19 and OX2 and Proteus
mirablisOX k. This test is usually done as either tube or rapid slide agglutination test.
 The test is of no value in diagnosis of trench fever and Q fever.

Weil- felix reaction in rickettsial disease

Disease Agglutination with

OX 19 OX 2 OX k
Epidemic typhus +++ ± -
Endemic typhus +++ ± -
Rocky Mountain spotted fever ++ ++ -
Scrub typhus - - +++
Trench fever - - -
Q-fever - - -
TREATMENT AND CONTROL
  Immunity is both cellular and humoral. Vaccines are notavailable for the prevention
of the rickettsial diseases of animals.
 Tetracyclins and chloramphenicol are effective in control of Q-fever Requires
adequate pasteurisation of milk and care in the handling of animals an their
products.
 Eradication of ticks is very helpful in control of rickettsial infection.

CHAPTER-27: CHLAMYDIAE

Learning objectives

To know in detail about,

 Basophilic virus, energy parasites, psittacosis, omithosis and Bedsonian

agents
 Difference between elementary bodies and reticulate bodies
 Development cycle of chlamydiae
 Stain used to identify chlamydiae
 Different cultivation methods of chlamydiae
 Antigens, toxin and pathogenesis of chlamydiae organisms
 General approaches used to diagnose chlamydiae

SYSTEMATICS
Domain Bacteria
Phylum Chlamydiae
Class Chlamydiae
Order Chlamydiales
Family Chlamydiaceae
Genus Chlamydia
Chlamydophila

 Chlamydia are derived from the characteristic appearance of the inclusion bodies
produced by these agents, which are seen close to the nuclei of infected cells as a
cloak or mantle (chlamys meaning mantle).
 Chlamydiae are obligate intra cellular parasites, filterable, requiring living system for
multiplication.
 They differ from viruses in
o They possess cell wall, resembling Gram-negative bacteria but lacking
muramic acid
o Do not have an eclipse phase following an infection.
o Produce basophilic intracytoplasmic inclusion bodies in infected cells (Hence,
referred as basophilic virus).
o Pocesses both DNA and RNA
o Multiply by binary fission.
o Sensitive to antibiotics.
 Therefore, they occupy a position intermediate between rickettsia and viruses.
o Chlamydiae are more dependant on host cell for high energy compounds such
as ATP. Hence, they are called as energy parsites.
 o C.psittaci causes variety of infections in animals such as gastritis, diarrhoea,
pneumonia, enzootic abortion in ewes, abortion, orchitis, epididymitis,
seminal vesiculitis, feline pneumonitis, sporadic bovine encephalomyelitis
(BUSS disease), conjuctivitis, rhinitis, hepatitis, and polyserositis and poly
arthritis.
o Chlamydia infection in psittacine birds such as parrots called as psittacosis
and this infection in non-psittacine birds such as pigeons, sparrows, turkey
and domestic poultry called as ornithosis.

HISTROY AND HABITAT

History

 The preliminary study in psittacosis was carried out by Sir Samuel Bedson.
 Hence, the psittacosis agents are also termed as Bedsoniae.

Natutal habitat

 In both birds and mammals the G.I.tract appears to be natural inhabitant

of C.psittaci.
 The infection with prolonged faecal shedding is characteristic.
 C.psittaci is commonly carried in the spleen and kidney of normal appearing birds.

MORPHOLOGY

 Chlamydiae occur in two forms, elementary bodies and reticulate bodies.

Developmental cycle of Chlamydiae

o They have a unique developmental cycle with alternating, morphologically

distinct, infectious and reproductive forms.
o The elementary bodies (EB) are small, spherical 200-300nm in d.m.,
infectious and represents the extracellular form of the organism.
o The elementary bodies enters a cell by endocytosis and differentiate into the
large (500 –1000nm size), non infectious, but metabolically active reticulate
body (RB) inside an expanding vacuole.
o The RB multiplies by binary fission producing further RB‘s.
o At about 20hrs following infection, some of the RB‘s start to condense and
mature within the inclusion to form EB‘s.
o In general, release of infectious EB‘s begins at about 40hrs post infection due
to lysis of the cell.
 Chlamydiae are Gram negative; the EBs can be demonstrated by the use of either
chemical stains or FAT and IPT.
 In modified ZN stain the EBs tend to occur in clumps and stain red against a blue
background.
 When the modified ZN stained smears examined under darkfield microscopy, the
EBs appears as bright green, coccal structures.
 In methylene blue stain under dark field illumination the EBs show autofluorescence,
they are revealed as refractile, yellow green bodies surrounded by a halo.
 In machiavello stain, the EBs stain red against a blue background. In castanedas
stain, the EBs are stained blue with a reddish background.
 Giemsa stain: Particularly useful in smears from conjuctival swab of feline
pneumonitis cases.
  Infected conjuctival epithelial cells contain basophilic intracytoplasmic aggregates of
C.psittaci.

CULTIVATION

 They require living systems for multiplication.

 They do not have cytochromes and their metabolic reactions are essentially
anaerobic.
 They are highly dependant on the host cells for energy.
 Mouse inoculation
o Suspected material is inoculated into mice intranasally, intra peritonelly.
o The mice die within in ten days and smears of the lung, peritoneal exudates,
spleen or brain will show the elementary bodies. (Levinthal cole Lillie or LCL
bodies).
 Inoculation on 6-7 days old embryonated eggs via the yolk sac route.
o The LCL bodies can be demonstrated in the smears of yolk sac from infected
eggs.
 A variety of continuous cell lines are useful for cultivation of Chlamydiae, such as
McCoy, BHk-21, L929 and vero.
o Chemical treatment of the cells with cycloheximide (1-2mg/ml) and 5-iodo-2-
deoxy uridine (80mg/ml) will greately enhance the cultivation.

RESISTANCE,ANTIGENSAND TOXINS

Resistance

 Chlamydiae are heat labile, being inactivated within minutes at 560C.

 They are susceptible to ethanol, ether and low concentration of phenol and formalin.
 Infectivity is maintained for several days at 40C.
 They can be preserved frozen at –700C or lyophilized.
 The elementary bodies are relatively resistant and remain viable for several days
under sutiable environmental condition.
 They are highly sensitive to oxytetracycline, erythromycin and tylosin.

Antigens and toxins

 All Chlamydiae share a group (genus) specific antigen.

 This antigen, also known as complement fixation antigen, is LPS in nature.
 In addition Chlamydiae possess species specific and serovar (serotype) specific
antigen.
 Based on OM protein antigens, more than 10 serovars of C.psittaci have been
described.
 Chlamydiae produce a toxin, probably protein in nature, which is lethal to mice on
intra venous inoculation.
 The toxin is specifically neutralized by the antitoxin.

PATHOGENESIS

 Chlamydial organisms may be shed in faeces of carrier animals. Chlamydial

elementary bodies are shed in the semen.
 Young one gets infection mainly through milk of the dam.
 Animals and humans are infected by the inhalation of infectious dust and droplets.
  In case of enzootic abortion and enteritis in ewes, the infection may take place by
ingestion.
 The severity of the disesase depends on
o Strain and virulence of the agent
 Ovine and bovine type 1 isolates are more frequently associated with
abortion, genital infections and enteritis.
 Type II isolates are associated with conjunctivitis, pneumonia,
encephalomyelitis and polyarthritis.
o Age, sex, physiological state and species of host.
o Route of infection and degree of exposure to Chlamydiae
o Environment and management practices.
 The Chlamydiae have a predilection for epithelial cells of the mucous membrane.
 After inhalation of infectious dust and droplets, pneumonia may develop.
 In enzootic abortion of ewes the organism localize in cells of the placenta, abortion
will occur in the last 2-3 weeks of pregnancy and this is associated with a diffuse,
necrotic placentitis.
 In BUSS disease, inflammation of vascular endothelium and nervous signs are
predominant.
 In avian psittacosis, as a result of certain stress conditions, the organism multiply in
the spleen and kidney of normal appearing birds and are shed in the faeces in large
numbers.
 The faeces dry, producing a dust that is infectious to susceptible avian and human. In
birds, respiratory, intestinal and systemic signs are seen.

DIAGNOSIS

 Chalmydiae can be isolated from the blood during the early stage and from the
sputum on later stage.

Specimens

 In case of abortion, smears from affected cotyledons or chorion, prepared from

vaginal swabs and from the wet surface of aborted fetuses.
 Uterine discharges, fetal membranes, fetal tissues are also useful.
 Aspirated synovial fluid in polyarthritis, conjuctival swab, samples of lung, liver,
spleen and paired serum samples are mostly helpful.
 Demonstration of Chlamydophila psittaci elementary bodies on
smears by using either chemical stains or Immunofluorescence
staining.
In stained smears, Brucella species may look very similar
to Chlamydophila psittaci. But can be differentiated by serology,
isolation and immunological staining methods.
 Isolation and cultivation
By inculation into mouse, yolk sac route of embryonated eggs, cell
culture and demonstration of LCL bodies.
 Serological tests like ELISA, CFT, IFAT, LAT
Cross reactivity between Chlamydiae species and other Gram negative
bacteria will complicate the interpretation of serological test.
 PAGE, Restriction endonuclease analysis and monoclonal antibody
typing will help in diagnosis.

TREATMENT AND CONTROL

 Immunity is both cell mediated and humoral.

  The serotype specific antigen stimulates production of protective antibody.
 Vaccines consisting of suspension of killed organism are available for the prevention
of feline pneumonitis and enzootic abortion of ewes.
 Tetracycline and chloramphenicol are effective. Penicillin and sulphonamides are not
useful.

CHAPTER-28: VETERINARY MYCOLOGY

Introduction

 Fungi, unlike bacteria, are eukaryotic organisms that lack chlorophyll and absorb all
nutrients from the environment. Fungi are ubiquitous and the majority are
saprophytic; only a few of the thousands of recognized species are pathogenic for
animals or humans.
 Most species that do infect animals are limited, by nutritional requirements and host
defenses, to the superficial skin or subcutaneous tissues. However, fungal infections
in the immunocompromised host can be very serious.
 Systemic fungal infections are the most serious and some are limited to particular
geographical regions. Because fungi are eukaryotic, they are not susceptible to most
of the antibiotics used to treat bacterial infections. There are only a few drugs used in
the treatment of fungal infections and most of them have some toxicity for mammals.
 The fungi that are most commonly isolated as agents of disease in animals are yeasts,
dermatophytes, and opportunistic fungi (e.g. Aspergillus).

GENERAL CHARACTERS OF FUNGI

 Fungi are eucaryotic, chemoorganotrophic and spore bearing organisms.

 They are non motile.
 They absorb nutrients and have no photosynthetic activity.
 They reproduce both sexually and asexually.
 The body or vegetative structure of fungus is called as thallus (P: Thalli).
 It varies in size and complexity, ranging from the single cell microscopic yeast to
multicellular molds, puff balls and mushrooms.
 Fungi are usually aerobic (Particularly Molds), some yeast, however, are facultatively
anaerobic and can obtain energy by fermentation.
 Obligate or strict anaerobic fungi are found in the rumen of the cattle.
 Fungi grow in wide range of temperature between 10°C and 40 °C. Some fungi can
grow at temperature of 50°C or even in freezing temperature.
 Optimum temperature is 22 to 30°C (Saprophytic fungi) and for parasitic fungi 30 -
37°C.
 Mostly fungi prefer an acidic pH for their growth. The optimum pH range is between
3.8 to 5.6.
 However, some fungi can grow even in salt water and some can grow in high
concentration of solute.
 Some fungi that exist in the mycelial form in nature at room temperature will convert
to a yeast form at 37°C in the tissues of animals.
 These fungi are called as dimorphic and the shift is called as YM (Yeast to Mold) shift.

CLASSIFICATION OF PATHOGENIC FUNGI

 Pathogenic fungi can be classified according to

o Macroscopic Morphology
o Microscopic Morphology
 o Type of Reproduction
o Taxonomy and
o Mycosis

Macroscopic Morphology

 Certain fungus species of veterinary importance are known to multiply through the
budding of blastospores (Imperfect yeast); resembling the budding of perfect yeast.
 The budding type of multiplication produces a pasty to mucoid appearance on culture
media, resembling appearance of a bacterial culture, these forms are termed as
Monomorphic yeasts.
 E. g. Candida albicans and other Candida spp. Cryptococcus neoformans,
Geotrichum candidurn, Trichosporon cutaneum.
 Some species are known to multiply only by sending out germ tubes from the spores.
 These germ tubes develop into long filaments, called hyphae, which may become
septate, branched or both, depending upon the species.
 Hyphae develop into a mat of filamentous growth known as mycelium.
 Mycelial growth can be observed macroscopically as a flilamentous (mold) growth
shortly after its development commences.
 The hyphae of the mycelium usually give rise to spores in which case the process of
development repeats under proper conditions for growth; these type of fungi are
termed as Monomorphic molds
e.g. Microsporum, Trichophyton, Aspergillus and Penicillium species.
 A few species of pathogenic importance are known to multiply at 25°C in the manner
of the monomorphic molds and to multiply at 37°C in the manner of monomorphic
yeasts.
 Such type of species which are known to multiply as either a yeast form or a mold
form, depending upon the temperature of Incubation, are termed as dimorphic fungi
eg. Histoplasma capsulatum. Blastomyces dermatitidis, Sporothrix schenckii.

Microscopic Morphology

 Basic microscopic morphological features pertinent to veterinary importance include:

o Type of hyphae
o Type of spores
o Type of spore bearing structure
 Type of hyphae
o Septate ; Presence of cross walls in a hyphal filament.
o Aseptate : Absence of cross walls in a hyphal filament, also known as
coenocytic hypha.
o Pseudohypha: Chain of elongated budding cells that have failed to detach.
 Type of spores
o Two types of spores are produced by fungi i. e. asexual and sexual
o t. Asexual spores are of the following types
 Macroconidium: Term referring to large, often multi cellular
conidium.
 Microconidium: Small, single celled conidium.
 Chlamydospores: These are thick walled, resistant spores formed by
the direct differentiation of the mycelium (i. e. concentration of
protoplasm and nutrients) e.g. Candida albicans.
 Arthrospore: This is an asexual spore formed by the disarticulation of
mycelium e.g. Geotrichum candidum.
 Blastospore: A spore produced as a result of budding process the
mycelium or from a single spore e. g. Saccharomyces spp .
  Sporangiospore: An asexual spore produced by closed, often spherical
structure called sporangium e.g. Rhizopus.
 The specialised hypha bearing sporangium is known as
sporongiophore and the persisting dome shaped upper portion of the
sporongiophore is called columella.
o Sexual spores are as follows
 Ascospores: A sexual spore characteristic of the true yeasts
and Ascomycetes.
 They are produced in a sac like structure called ascus. This ascospore
results from the fusion of two nuclei e. g. Saccharomyces spp.
 Basidiospore: Sexual spore, characteristic of the class Basidiomycetes,
produced on a specialised club like structure called basidium.
 Zygospore: A thick walled sexual spore produced through fusion of
two similar gametangia found in the class Phycomycetes.
 Gametangium is a structure in which gametes are produced. Gamete :
A sexual cell, especially a cell formed in a gametangium.
 Type of spore bearing structure
o Conidiophore: is a stalk like branch of the mycelium on which conidia develop
either singly or in numbers e.g. Penicillium. These vary from extremely short,
near obsolete hyphae to those that are long intricately branched.
o Sporangiophore: is specialised hypha bearing sporangium and the persisting
dome shaped upper portion of the sporangiophore is called columella.

Type of Reproduction

 Fungi reproduce asexually or sexually or both depending upon the species and
environmental conditions.
o If a fungus species demonstrate sexual reproduction alone or sexual and
asexual reproduction, the fungus is called perfect fungus eg. Saccharomyces
cerevisiae.
o If a fungus species demonstrates only asexual reproduction, this fungus is
called an imperfect fungus, also known as fungi-imperfecti and these belong
to the class Deuteromycetes.

Taxonomy

 Class Phycomycetes
o The Phycomycetes are the most primitive class of fungi.
o They produce broad, aseptate hyphae and reproduce asexually by forming
sporangia that contain sporangiospores.
o Sexual reproduction occurs by means of thick walled resting spores, which
can be zygospores or oospores.
 Class Ascomycetes
o Ascomycetes are represented by two morphologically distinct types.
o The first type has unicellular, round or oval forms reproducing asexually by
budding of blastospores.
o The perfect yeast, genus Saccharomyces, represents this type. If conditions
are favourable, sexual ascospores are formed.
o Four or eight ascospores develop within each sac-like enclosure called an
ascus. The asci break open to release the ascospores.
o The second type of Ascomycetes have septate hyphae, producing filamentous
forms, which reproduce asexually by spores called conidia and sexually by
ascopores.
o In this type, the asci are usually enclosed within a tightly meshed network of
hyphae (mycelia) called perithecium.
  Class Basidiomycetes
o Basidiomycetes develop sexual basidiospores from specialized club shaped
structures called basidia.
o Each basidium usually bears four exogenous basidiospores resembling toes on
a foot.
 Class Deuteromycetes
o The majority of pathogenic fungi belong to this class. Deuteromycetes (fungi
imperfecti) are composed of those fungi that lack a demonstrable means of
sexual reproduction, therefore, are considered imperfect.
o The Deuteromycetes are represented by two morphologically distinct types: a
filamentous (mold) form and an imperfect yeast form resembling the perfect
yeast, Saccharomyces.
o Asexual spores of two major types are produced in this class. These are the
thallospores and conidia.
o The thallospores are formed by a change in portions of the thallus or body of
the fungus and include the arthrospores, blastospores and chlamydospores.
o The conidia are produced by abstrictions from specialized hyphae called
conidiophores.
o Large conidia mav be called macroconidia and small conidia as microconidia.

Mycosis

 Mycoses can be divided into four categories according to the tissues usually invaded.
 Superficial
o The etiological agents of the superficial mycoses are confined to the outermost
layers of skin and hair.
o The superficial mycoses are less serious than the other mycoses
e.g. Trichosporon cutaneum.
 Cutaneous
o Most of the etiological agents of the cutaneous mycoses possess the special
ability to invade and destroy keratin in skin, hair and nails, e.g. Candida
albicans, Trichophyton, Microsporum, & Epidermophyton spp.
 Subcutaneous
o The agents responsible for subcutaneous mycoses are more serious than the
cutaneous mycoses and they invade primarily the muscle tissue,
e.g. Rhinosporidium seebri, Sporothrix schenckii.
 Systemic
o The agents of systemic mycoses invade deep tissue and create symptoms
resembling other diseases of the particular tissues or organ invaded.
o Systemic mycoses may also have cutaneous manifestations.
o The systemic mycoses are the most serious of the mycoses.
o E.g. Candidiasis, cryptococcosis, histoplasmosis, geotrichosis, blastomycosis
etc.,

PREDISPOSING FACTORS FOR FUNGAL INFECTIONS

 Prolonged antibiotic therapy. Antibiotics may lower host resistance in some ways, but
the best known effect is through alteration of the normal bacterial flora.
 The most common fungal infection resulting from antibiotic treatment is intestinal
candidiasis.
 Immunosuppressive treatment and drugs. Drugs that interfere with inflammation
and humoral and cellular immunity, make animals particularly more susceptible to
opportunistic fungal infections.
 Radiation therapy is particularly toxic for antibody-producing cells.
  Infectious and noninfectious diseases (e.g. cancer), and pregnancy can reduce host
resistance and make animals more susceptible to fungal infections.
 Genetic immune deficiencies.
 Environmental factors, such as trauma, general lowered resistance due to stress, a
moist environment and exposure to a large number of organisms.

CHARACTERISTIC OF FUNGUS INFECTIONS

 Most fungal diseases are not contagious or zoonotic; an exception is

dermatophytosis.
 Most fungi are opportunistic and demonstrate relatively low invasiveness and low
virulence for healthy hosts.
 Host predisposed on exposure to a large number of spores.
 Fungal infections often result in a chronic, granulomatous, inflammatory process
similar to that seen in mycobacterial disease.
 Due to the chronic nature of fungal infections, cell-mediated immunity is usually
more important than humoral immunity in regard to protection.
 Animals exposed or infected by some fungi may develop a hypersensitivity reaction.
 Most fungal infections are asymptomatic or self-limited.
 Most fungi that cause disease in animals have no recognized sexual state.

REFERENCE BOOKS

 Essentials of Veterinary Microbiology - Fifth edn.,

 G.R. Carter, M.M. Chengappa and A.W. Roberts
 Clinical Veterinary Microbiology -P.J. Quinn and M.E. Carter
 Veterinary Microbiology and Microbial Diseases
 J. Quinn, B.K.Marky, M.E Carter, W.J. Donnetty and F.C. Leonard
 Veterinary Mycology - S.K. Fand and K.B. Singh
 Medically important fungi - a guide to identification- Davise Howig Larone
 Laboratory Medical Mycology- Yousef-AI – Doory
 Veterinary Medical Mycology - Paul F. Jungerman and Robert M. Schwartzman

CHAPTER-29: DERMATOPHYTOSIS

Learning objectives

To know in detail about,

 Diseases caused by Dermatophilus species

 Developmental cycle of Dermatophiuls congolensis
 Tram-track appearance and strawberry foot rot
 Pathogenesis of mycotic dermatitis n sheep
 Haalstra's method for the isolation of Dermatophilus congolensis
 Different diagnostic methods of dermatophilosis

INTRODUCTION

 Dermatophytosis is an infection produced by molds capable of parasitizing only

keratinized epidermal structures: superficial skin, hair, feathers, horn, hooves, claws
and nails.
  Those that have a sexual reproductive phase belong to the Ascomycetes.
Dermatophyte infections are called ringworm (tinea).
 The dermatophytes, more than any other group of fungi, have developed a close host-
parasite relationship with animals.
 Many dermatophytes have evolved into obligate parasites.
 Dermatophyte infections are contagious and a zoonotic source of infections for
humans.
 Infections from animal to human are referred to as zoophilic, while those from soil to
animal or human are referred to as geophilic.
 The most common animal dermatophytes are
o Zoophilic pathogens

Pathogens Animals affected

Trichophyton All domestic animals
mentagrophytes
Trichophyton verrucosum Cattle and sheep
Trichophyton gallinae Fowl
Trichophyton equinum Horses
Microsporum canis predominately dogs and cats primates and
horses also
Microsporum nanum Swine and humans

o Geophilic Pathogen: Microsporum gypseum – mainly affects dogs, cats,

horses, and humans.
 Rare causes of animal dermatophytes are the anthropophilic, globally
prevalent Trichophyton rubrum, Microsporum audouinii, Trichophyton
schoenleinii, Microsporum cookei, Microsporum distortum, Trichophyton megnini.

HISTORY AND HABIT

History

 Ringworm, the common name for dermatophyte infections, has been described from
the earliest historical times.
 The name came about due to the fact that the fungus grows equally in all directions
and forms lesions with circular or ring forms.
 The Romans associated the disease with insects and referred to it as tinea, meaning
small insect.
 Tinea is still used to refer to different clinical settings of the disease (e.g., tinea pedis -
ringworm of the feet).
 In 1910 Sabouraud published a detailed work on systematic and scientific studies of
the dermatophytes. There are currently 37 species of dermatophytes.
 In 1959 the sexual state of some of the dermatophytes was identified, and they are
now classified in the class Ascomycetes.
 In 1958 Gentles published the first report on the oral administration of griseofulvin,
which cured experimental dermatophytosis in a guinea pig.

Natural habitat
 The dermatophytes are closely related in appearance, physiology, and antigenicity.
 Although the soil is rich with dermatophytes, most of the agents that cause animal
disease are obligate parasites of animals.
 In general, the more chronic the infection and the more adapted the parasite is to the
host, the less severe the inflammatory response will be.
 M. gypseum is a natural soil inhabitant that is a common cause of dermatophytosis,
while most of the other common animal pathogens are normally found only on
animals.
MORPHOLOGY

 In their nonparasitic phase, including culture, dermatophytes produce septate,

branching hyphae collectively called mycelium.
 The asexual reproductive units (conidia) are found in the aerial mycelium. These
units may be either macroconidia or microconidia.
 Shape, size, structure, arrangement and abundance of conidia are diagnostic criteria.
 A general rule that can be applied (but not always) is that most species in the
genus Microsporum produce predominately macroconidia, and most Trichophyton
speciesproduce predominately microconidia with few or no macroconidia.
 Hyphal peculiarities — spirals, nodules, rackets, chandeliers and chlamydoconidia —
are more common in some species than others, but they are rarely diagnostic.
 Pigmentation is useful in dermatophyte differentiation.
 In tissue sections, arthroconidia can often be identified. This parasitic phase
arthrospore can remain infectious for years.
 Except in size ranges, which overlap among dermatophyte species, arthroconidia are
indistinguishable from species to species.
 Chlamydoconidia are also commonly seen in some dermatophytes in vitro and their
presence may be of use diagnostically in the absence of other conidia.
 Sexual spores (ascospores) are absent in the parasitic phase.

CULTURAL CHARACTERISTICS

 The most common media for propagating dermatophytes are dermatophyte test
medium (DTM) or Sabouraud's dextrose agar, a 2% agar containing 1% peptone and
4% glucose.
 Its acidity (pH 5.6) renders it mildly bacteriostatic and selective.
 The selectivity is enhanced by addition of cycloheximide (500µg/ml), which inhibits
other fungi, and gentamicin and tetracycline (100 µg/ml of each), or chloramphenicol
(50 µg/ml).
 Dermatophytes are aerobes-and nonfermenters. Some attack proteins and deaminate
amino acids.
 They grow optimally at 25°C to 30°C and require several days to weeks of incubation.
 Some dermatophytes in skin and hair (but not in culture) produce a green
fluorescence due to a tryptophan metabolite that is visible under a Wood's light. Of
animal dermatophytes, only Microsporum canis produces this reaction.

PATHOGENESIS

 Proteolytic enzymes (elastase, collagenase, keratinase) may determine virulence,

particularly in severe inflammatory disease.
 The dermatophytes are highly specialized for utilizing keratin as food source.
Localization in keratinized epidermis has been attributed to the lack of sufficient
available iron elsewhere.
 Keratinase is therefore a clearly recognized virulence factor for dermatophytes.
  It is thought that those dermatophytes that are highly species-specific have a
keratinase that can only hydrolyze keratin from a particular animal species, whereas
those with more broad acting keratinases can invade the skin of many different
species (e.g. T. mentagrophytes).
 The infectious unit conidia enters the skin through an abrasion, germinate and
hyphae begin to grow in the stratum corneum.
 Portions of mycelium differentiate into arthroconidia. This growth pattern in the
hairless skin predominates with some dermatophytes (M. nanum, T. rubrum).
 Hair invasion, which is prominent in most animal ringworm, begins with
germination of a spore near a follicular orifice.
 The hyphae invade the hair follicle and enter the cortex of the hair by dissolving the
keratin.
 The hyphae and conidia are carried to the surface by the growing hair, which often
breaks off.
 Hair invasion may be endothrix (arthroconidia develop within the hair shaft only and
the cuticle remains intact) or ectothrix (arthroconidia develop outside the hair shaft
and hyphae are within the hair shaft; the cuticle is destroyed).

PATHOGENICITY

Symptoms

 Although not fatal, dermatophytosis can be a cause of significant economic loss and a
source of infection for man.
 One of the first clinical signs is loss of hair, followed by an inflammatory reaction of
the skin due to the host response.
 Dermatophytosis occurs more commonly in very young, old, or sick animals and
most often in stabled rather than pastured animals.
 The peak incidence occurs in the winter. The characteristic lesion is a hyperkeratosis
with septate hyphae and arthroconidia in the stratum corneum.
 Invasion of the hair causes the shaft to become weak and break, resulting in circular,
scaly areas of alopecia with or without crust formation.
 Arthroconidia within or outside the hairshaft are also referred to as arthrospores.
 Manifestations range from erythema to vesiculopustular reactions and suppuration.
 Mild forms are seen in T. verrucosum infection of calves. Severe reactions are typical
in T. mentagrophytes infection of dogs and M. gypseum infection of horses.
 Local plaques (kerion) may resemble certain skin tumors, especially in dogs.
 The inflammatory reaction may arrest the mycotic infection but become the primary
problem through secondary suppurative bacteria! infection.
 The roughly circular pattern of the lesions and their inflamed margins suggested the
terms ringworm and tinea (Latin for worm). Different tineas are
o Tinea barabe – beard
o Tinea capitis – scalp
o Tinea corporis – body
o Tinea cruris – groin
o Tinea favosa – favus
o Tinea imbricata and Tinea manum – hands
o Tinea pedis – feet
o Tinea unguium – nails

Lesions

 Lesions are very similar in different animal species.

 The most common areas of infection on dogs and cats are the head and extremities.
  In horses and sheep the neck and girth are most often infected.
 In cattle, the most common sites are the head and neck.
 In fowl, the disease is referred to as fowl favus or white comb, due to the white, moldy
crusts that develop on the comb and wattle.
 Hyphae that invade the stratum corneum induce a hyperkeratosis.
 The resulting inflammatory response by the host is most intense at the area of recent
invasion.
 The infection spreads in all directions, giving a ring-like appearance.
 The most active site of infection is at the periphery, while the central area begins to
heal.
 Therefore, specimens for culture and examination should always be collected from
the edge of the lesion.
 The living skin is not infected. The optimal growth temperature of dermatophytes is
30° C; most grow poorly, if at all at 37° C and, therefore cannot infect deeper tissues.
 The host may develop a hypersensitivity to the invading fungus that may result in
vesicular lesions developing in various parts of the body.
 These "id" reactions are thought to be due to fungi or their products disseminating in
the body and to an immune reaction.
 The resulting hypersensitivity reaction to the fungus may be delayed-type, or
immediate.
 Infections due to M. canis are often associated with kerions, which are vesicles in and
around the lesion.

DIAGNOSIS

Diagnosis

 Based on Direct Examination

o Fluorescence of hairs is useful for identification of hairs that may be infected
with dermatophytes.
o In 50% to 70% of cases, hairs and skin scales infected with M. canis or M.
audouinii may emit a bright greenish fluorescence under ultraviolet light
 Based on Microscopic examination
o Skin scrapings and hair are examined microscopically for the presence of
hyphae and arthroconidia.
o The scraping should include material from the margins of any lesion and the
full thickness of the keratinized epidermis.
o The hair is plucked, so as to include the intrafollicular portion.
o The sample is placed on a slide, flooded with 10% to 20% KOH or NaOH ,
with a cover slip, and heated gently.
o Microscopic examination should begin under low power and subdued light.
o Infected hairs are encased in an irregular sheath of arthrospores that may
double their normal thickness.
o At higher magnification of such hairs, individual, spherical arthroconidia are
recognizable.
o In hairless skin, branching hyphae and chains of arthroconidia occur.
 Based on culture
o Hair and skin scrapings should be inoculated to dermatophyte test medium
(DTM) and Sabouraud dextrose agar medium, with and without
cycloheximide and chloramphenicol, and incubated at 25-30º C for up to 4
weeks. Samples suspected of containing T. verrucosum are incubated at 37ºC.
o The DTM medium will turn red as the dermatophyte is growing, and the
fungus itself will usually be hyaline and fluffy.
o Although selective, other molds may grow on DTM and therefore idenification
should be confirmed by microscopy.
 o Very long, narrow hyphae with distinctive shapes and micro or macroconidia
are indicative of dermatophyte infection.

Differential diagnosis

 Differentiate from Insect bites, Urticaria, bacterial infections, seborrheic dermatitis

and interdigital dermatitis.

TREATMENT AND PREVENTION

Treatment

 Ringworm generally regresses spontaneously within a few weeks or months, unless

complicated by secondary infections or constitutional factors.
 The agents may persist after clinical cure.
 Combined topical and systemic treatment is often preferable. Of two systemic agents
available, griseofulvin and ketoconazole, the latter is more costly and less proven.
 Both drugs are given orally and are relatively well tolerated.
 For small animals, griseofulvin given orally is most beneficial for severe infections.
 The drug is incorporated into the keratin of the tissue and renders the skin resistant
to infection.
 The fungus is shed with the dead skin, and therefore requires prolonged therapy
(given for at least a month, or 2 weeks), particularly if the infection is in the nail.
Some toxicity may occur.
 Ketoconazole and fluconazole may also be used, but would be very expensive.
 Topical treatments can be used for large animals and for skin infections, but are not
useful for nail infections.
 These treatments include salicylic and benzoic acids, iodine, natamycin-s, and
imidazole derivatives. Infections can recur.

Prevention

 Wood light screening of scalps and suspected animal reservoirs. (Cats and dogs), hair
brush technique for culture of none fluorescing scalps, improve hygiene and
discourage sharing of clothing and accessories.
 In cattle, vaccines for T. verrucosum have been reported to be successful for 3-5
years. However, this has not been shown with vaccines to other dermatophytes.

CHAPTER-30: CANDIDIASIS

Learning objectives

To know in detail about,

 Diseases and main host of Candida albicans

 Candidiasis, moniliasis, thrush and crop mycosis
 Pathogenesis and pathogenicity of candidiasis
 Characterizaton of different candida species based on biochemical tests
 Different diagnostic techniques used to diagnose candidiasis
 Importance of biggy agar in isolation of C.albicans
 Germ tube test and Dalmu's technique

INTRODUCTION
  Candidiasis is a general term covering diseases caused by yeasts of the
genus Candida, especially C.albicans.
 Candidiasis can occur either as a superficial or as a systemic infection.
 There are more than 150 species of Candida, but only C.albicans is commonly
associated with disease in animals.
 Diseases and main hosts of Candida albicans

Host (s) Disease (s)

Chicken, turkey, pigeon, ducklings Crop mycosis or Avian moniliasis
Swine Stomach ulcers and cutaneous candidiasis
Puppies, kittens, calves and foals Mycotic stomatitis and enteritis
Mares and bitches Genital tract infection

 Candidiasis of the alimentary tract often referred as thrush.

 In addition to C.albicans bovine mastitis is caused
by C.tropicalis, C.pseudotropicalis, C.parapsilosis, C.krusei and C.rugosa.
 C.parapsilosis cause bovine abortion and C.rugosa has been implicated in pyometra
in a mare.
 Candida albicans infections are also called as moniliasis, candidiasis and candidosis.

HABITAT

 C.albicans is worldwide in distribution. All Candida occur saprophytically.

 The C.albicans is a commensal of the oral, gastro-intestinal and genital tract of many
species of animals and humans.
 Candida species are commonly present in the crop of birds.
 Faecal contamination of feed also responsible for disease transmission.

PATHOGENESIS

 Most infections are endogenous in origin and predisposing causes such as

immunosuppression, prolonged antibiotic therapy and malnutrition.
 Disseminated candidiasis (or) systemic candidiasis is more common in
immunosuppressed animals.
 Candida possesses adhesions consisting of fibrillar peptide, Mannans, which have an
affinity for the fibronectin on the surface of cells.
 The yeast forms are responsible for tissue damage.
 Inhibition of yeast cell division results in hyphal elements that invade tissues.
 Possible virulence factors are cell wall glycoprotein, proteases, neuraminidase, chitin,
mannoprotein and lipids.
 The cell-wall glycoproteins have an endotoxin like activity.
 Infection caused by C.albicans frequently involves mucous membranes.
Granulomatous lesions are rare.

PATHOGENECITY

Lesions
  In acute cases the lesions appear as tiny, discrete yellowish white (or) grayish white
pustules which loosely adhere to the mucous membranes and rather resemble a small
quantity of curdled milk.
 In acute cases, the wall of the crop is thickened and covered by a corrugated
pseudomembrane of yellowish grey necrotic material giving the characteristic ‗turky-
towelling‘ appearance.
 In avian moniliasis (or) thrush lesion are confined to the crop, less frequently invade
the mouth, oesophagous, proventriculus, gizzard and intestine.

Symptoms

 Vomiting, diarrhoea and emaciation occur in pigs.

 In Cattle, candidiasis occurs following prolonged antibiotic therapy. Pneumonic and
enteric symptoms are seen.
 The mastitis may be mild and self-limiting, spontaneous recovery may occur within a
week.
 The disease has no diagnostic symptoms except that the affected birds shows
unsatisfactory growth, stunted appearance, roughness of feathers, listlessness, loss of
appetite, shrunken appearance of chest and tendency to stand around with head
drowned on shoulder.
 High mortality occurs in young birds in acute cases.
 The mouth and oesophagous may show ulcer like patches.

DIAGNOSIS

Specimens

 Scrapings from lesions, centrifuged milk samples, biopsy or tissue samples in 10%
formalin for histopathology.

 Based on morphology
o C.albicans grows as oval, budding yeast cell on agar cultures & in animal
tissues.
o Pseudohyphae are also produced in animal tissue by elongation of yeast cells
that fail to separate.
o In Gram stained smears C.albicans appear as purple-blue yeast cell.
o It can also be demonstrated in specimens by 10% KOH (or) by lacto phenol
cotton blue.
o The tissue sections stained by PAS-haematoxylin (or) methaneamine silver
stains, the C.albicans appear as thin walled oval, budding yeast cells and/or in
the form of pseudohyphae.
 Based on isolation and identification
o C.albicans grows well on blood agar or SDA without inhibitors (Candida spp
may be inhibited by cycloheximide).
o The plates are streaked with a small inoculum as for bacteria. The cultures are
incubated at 37ºC, aerobically, for upto 5 days.
o Colonies of C.albicans are white to cream, shiny, high convex and have a
pleasant beery smell.
o Smears from the colonies stained with Gram's or lactophenol cotton blue or
methylene blue stain reveal thin walled budding yeast cell and pseudohyphae.
o BiGGy agar (Bismuth-sulphite-glucose- glycine- yeast agar) can also be used
for the isolation and identification of C.albicans.
 o Most bacterial contaminants are inhibited by the Bismuth
sulphite. C.albicans and C.tropicalis strongly reduce the Bismuth sulphite to
Bismuth sulphide.
o C.albicans gives smooth, circular, brownish colonies and no color diffusion
into the surrounding medium.
o The colonies of C.tropicalis are similar but there is diffuse blackening of the
medium after 72 hrs.
 Germ tube or serum tube test
o A small inoculum from an isolated colony is suspended in 0.5 ml of sheep,
bovine, rabbit or human serum and incubated at 370C for 2-3 hrs.
o A drop of the preparation is examined under phase contrast or high objective
of the light microscope.
o Small, thin walled tubes will be seen projecting from some of the yeast cells.
This is charcteristic of C.albicans.
 Demonstration of Chlamydospore (Dalmu's technique)
o Subsurface inoculation is made on corn meal- tween 80 or chlamydospore
agar and the plates are incubated at 30ºC for 2-4 days.
o A thin coverslip is placed on the surface of the agar and examined under high
power microscope to demonstrate thick walled chlamydospores borne on the
tips of pseudohyphae.
o Clusters of smaller blastospores may also be present.
 Based on biochemical test

Ability to utilize C.albicans C. C. C.

tropicalis pseudotropicalis parapsilosis
Glucose Acid (A) and A&G A&G A&G
Gas (G)
Sucrose A A&G A&G -
Maltose A&G A&G - -
Lactose - - A&G -
Chlamydospore + - - -
on corn meal agar

 Animal inoculation
o Rabbits and mice are susceptible to intra venous and intra peritoneal
inoculation respectively. Abscesses develop in the kidney.

PREVENTION AND CONTROL

 The majority of the candidiasis cases are associated with predisposing diseases,
unsanitary conditions or medication with antibiotics.
 Correction of this condition is the first step in therapy.
 Nystatin (Mycostatin) administered in the feed to treat candidiasis in chickens,
turkeys, swine, dogs and cats.
 It has been administered in the mammary gland to treat mastitis in cattle.
 Amphotericin B is the most effective drug for the treatment of systemic candidiasis.
 Ketoconazole and clotrimazole have been effective in the treatment of
mucocutaneous candidiasis.
 HISTORY AND HABITAT

 Rhinosporidiosis is mycosis of cattle, horses, mules, dogs and humans and is

characterized by large polyps, tumors or wart like lesions on the nasal and ocular
mucous membrane.
 The causative agent is Rhinosporidium seeberi.

History

 Seeberi described the Rhinosporidiosis in Man during 1910.

Natural habitat and distribution

 The natural habitat of the organism is thought to be associated with stagnant water.
 The disease has worldwide distribution but its occurrence is most common in India
and Srilanka.
 The disease has been reported in India more frequently in human beings.
 It is of interest that 90% infections involve the nose of male animals.
 In south India, particularly humid climatic areas, the disease is more in animals and
in dry areas the disease mostly occur in man.

PATHOGENESIS

 The mode of transmission and mechanism of infection is not known.

 No evidence of animal-to-animal, animal to man and man-to-man transmission.
 It is however probable that injury plays a part in determining infection and that
infection is sometimes from airborne soil particles.
 Inhalation of contaminated dust may also be a mode of transmission.

PATHOGENICITY

 Rhinosporidium lives in soil and it is believed that water is a necessary medium of

transmission.
 Infection usually results from a local traumatic inoculation and is associated with
water activities e.g. swimming in stagnant water.
 The infection is typically limited to the mucosal epithelium.
 Its life cycle begins with a round endospore(6-10 μm in diameter), which grows to
become a thick-walled sporangium (100-450 μm in diameter) that contains up to
several thousand endospores.
 Mature sporangiospores are approximately 7-9 um in size and escape through a pore
that develops in the sporangial wall.
 The disease progresses with the local replication of R. seeberi and associated
hyperplastic growth of host tissue and a localized immune response.
 Infection of the nose and nasopharynx is common; other parts include palpebral
conjunctivae, skin, ear,genitals, and rectum.
 These polyps are pink to deep red, are sessile or pedunculated, and are often
described as strawberrylike in appearance.
 Because the polyps of rhinosporidiosis are vascular and friable, they bleed easily
upon manipulation.
 The polyps are chronic but are not painful.
 They can cause obstruction of the respiratory tract resulting in asphyxia.
 The rhinosporidial mass has been classically described as a strawberry like mulberry
mass.
  This mass may extend from the nasal cavity into the nasopharynx and present itself
in the oral cavity. These lesions commonly cause bleeding from the nasal cavity.
 Rhinosporidium seeberi can also affect the lacrimal gland and also rarely the skin
and genitalia.

Rhinosporidiosis in dogs

 Rhinosporidiosis is a very rare chronic (long-term) infection that typically occurs in

the mucous membranes of dogs.
 It most commonly occurs in the nose and nostrils, but can also take hold in the nose
and eyes. Rhinosporidiosis belongs to the zoonotic class of fungal infections, meaning
that it can be transmitted to humans.
 Signs and symptoms of rhinosporidiosis include the following: sneezing, bleeding,
wheezing, or labored breathing; an infection of the nostrils with a cauliflower-like
growth; a polyp or other growth located near or on the nostril - this growth may be
white or yellowish in color and may appear speckled or spotted because of the fungus
associated with the growth

DIAGNOSIS AND TREATMENT

 Rhinosporidium seeberi has not been grown in culture and no laboratory animals are
available for cultivation.
 Only method of diagnosis is demonstration of spores and sporangia in wetmount
preparations of nasal discharge and section of polyps.
 Spores are 6 - 7 µm in d.m. Spores increase in size and attaining a size of
approximately 100 µm become transformed into sporangia by the deposition of a
layer of cellulose within the chitinous wall. Numerous nucleoid division occur and it
attains 200 -300µm in d.m.
 A sporangium contains approximately 16,000 to 20,000 spores.
 At one point, the sporangium thins to form a pore and the spores are escaped.
 In the tissue sections stained by H &E stain, various forms of sporangia are seen
 Young trophic forms: 10 to 100 µm in d.m. with single central basophilic karyosomes
and amorphous cytoplasm.
 Mature forms 100 -300µm in d.m. containing sporangiospore.
 Empty and collapse form of sporangia.

Treatment

 Surgical excision of polyps. But there is a chance of recurrence.

CHAPTER-31: CRYPTOCOCCOSIS

Learning objectives

To know in detail abot,

 Diseases caused by Cryptococcus neoformans

 Cryptococcosis or Torulosis
 Morphology and cultural characters of C.neoformans
 Media for cryptococcosis
 General approaches used to isolate and identify C.neoformans

INTRODUCTION
  Cryptococcosis also known as European blastomycosis or Torulosis is a subacute or
chronic mycotic infection of man and various species of animals involving the CNS,
the respiratory system and eye.
 It is caused by encapsulated yeast – Cryptococcus neoformans. Of the 19 species
of Cryptococcus, only C.neoformans is pathogenic for animals and humans.
 Diseases and main hosts of Cryptococcus neoformans

Host (s) Disease (s)

Dogs and cats Subcutaneous and nasal granulomas, Meningitis, Blindness
Horses Nasal granuloma
Cattle Cryptococcal mastitis
Human Cryptococcal meningitis
MORPHOLOGY

 There are two varieties Cryptococcus neoformans var neoformans (serotypes A and
D) and Cryptococcus neoformans var gattii (Serotypes B and C).
 C. neoformans is a member of the fungi imperfecti. But Filobasidiella neoformans is
the teleopmorphic (sexual stage) state of serotypes A and D, Filobasidiella
bacillispora is the teleomorphic state of serotypes B and C.
 C. neoformans is a spherical to oval, thin walled, budding yeast that varies greatly in
d.m.
 The cells are surrounded by a mucoid polysaccharide capsule that varies in thickness,
but in animal tissues it is usually very large, the width of the capsule exceeding the
d.m. of the parent cell.
 Daughter cells are usually single and bud from the parent cell by a thin neck.
 Yeast cells are Gram positive. Lactophenol cotton blue or nigrosin stains are
commonly used to demonstrate the spherical budding yeast surrounded by a capsule.

HABITAT

 C. neoformans is present in dust and has been isolated from the skin, mucous
membrane and intestinal tract of normal animals and birds.
 The reservoir of types A and D is the faeces of birds, particularly pigeons and soil
contaminated by avian excreta.
 The pigeon is not infected, the organism colonise the faeces after they have been
passed.
 The organisms are concentrated in pigeon faeces due to their high content of
creatinine.
 The creatinine inhibits many other micro organism but can be utilized by C.
neoformans.
 It can survive in pigeon droppings for more than a year. C. neoformans has a
worldwide distribution.

CULTURAL AND BIOCHEMICAL CHARACTERS

Cultural characters

 C. neoformans will grow well on blood agar or on SDA (without cycloheximide).

  The plates are streaked out as for bacteria and incubated aerobically at 370C for upto
two weeks.
 Capsular growth can be enhanced by culture on chocolate agar under 5% Co2 at 370C.
 The majority of the Cryptococcus species are unable to grow at 370C, where as C.
neoformans can grow at temperature upto 400C.
 Colonial growth is often not apparent until after nearly two weeks incubation0.
 The colonies are smooth, moist, shiny, white and become wrinkled, cream to
brownish granular colonies on further incubation.
 Bird seed agar (which contain Guizotia abyssinica seeds) is the selective medium
for C. neoformans.
 It contains di and polyphenolic compounds. Cryptococcus species use creatinine and
produce melanin-pigmented (brown) colonies.
 The dark brown pigment colonies occur after the plates are incubated at 370C for
atleast a week.

Biochemical characters

 C. neoformans is highly urease positive.

 It will produce urease on heavily inoculated Christensen‘s urea agar slope.

PATHOGENESIS

 Infections are exogenous and are usually acquired by inhalation. Animal to

animal transmission is not known to occur.
 The route of infection is usually respiratory, often with localization in the
nasal cavity or paranasal sinuses and later extension to the brain and
meninges.
 The virulence of C. neoformans is largely associated with the antiphagocytic
and immunosuppressive capsule and a unique enzyme diphenol oxidase.
 The cryptococcal lesions, on gross examination, resemble myxomatous
neoplasm; infection of the meninges can resemble tubercular meningitis.
 Infections extend to the optic nerve resulting in blindness. Subcutaneous
granuloma is often occurring in cervical or pedal regions.

PATHOGENECITY

 In cryptococcal mastitis cows with mild infections often show no clinical signs except
swelling of the affected glands.
 In severe infections, animal exhibits typical clinical signs of bacterial mastitis (fever,
swelling and firmness of the udder).
 Milk secretion gradually diminishes. Milk will appear grey, white, highly viscid and
mucoid.
 In dogs, it affects the CNS, causing incoordination, hyperaesthesia and nasal
discharge.
 Subcutaneous granuloma around the ear, face and feet. In cats, chronic nasal and
ocular discharge, granulomas and blindness are common.

DIAGNOSIS

Diagnosis
  Specimens: CSF, lesions or exudates, mastitic milk, biopsies and tissues should be
collected.
 Direct microscopy
o Demonstration of budding yeast with a large capsule by India ink, Nigrosin
and LPCB staining methods.
 Histological sections on biopsies of tissue from lesions can be stained by the PAS-
haemotoxylin stain. This will stain or outline, the yeast cell but not the capsule, which
appear as a clear area surrounding the cell.
 Based on isolation and identification: production of brown pigment on birdseed agar.
 Based on urease production
 By Animal inoculation
o Mice are susceptible to pathogenic strains.
o When intra cerebral or intra peritoneal inoculation of suspected material, the
mice will die within 4 days to two weeks.
o PM reveals gelatinous lesions in the abdominal cavity and lungs.
o The budding encapsulated yeast can be demonstrated from the lesions.
 By serology
o Slide LAT, tube agglutination, CFT, ELISA, IFAT are useful to detect
antibodies.

Treatment

 Amphotericin B is the drug of choice. Imidazole derivatives such as ketoconazole and

fluconazole are also useful.

CHAPTER-32: CLASSICAL HISTOPLASMOSIS

Learning objectives

To know in detail about,

 Synonyms of classical histoplasmosis

 Natural habitat and morphology of Histoplasma capsulatum
 Cultural characteristics of Histoplasma capsulatum
 Pathogenesis and pathogenicity of classical histoplasmosis
 General approaches used to diagnose classical histoplasmosis

INTRODUCTION

 Synonyms of this disease are small form histoplasmosis, Darling‘s disease and
reticuloendothelial cytomycosis.
 Histoplasmosis is a chronic, granulomatous disease caused by Histoplamsa
capsulatum var capsulatum (Teleomorph : Ajellomyces capsulatus).

NATURAL HABITAT AND MORPHOLOGY

 The organisms become heavily concentrated in the feces of birds (particularly black
birds, sea gulls, starlings and pigeons); bats are also important vectors of this disease.
Thus, the fungus has been isolated from soil in bat caves, bird roosts, chicken houses,
and silos inhabited by pigeons.
 The organisms are facultative, intracellular parasites of macrophages.
  The tissue form cells of H. capsulatum var.capsulatum appear as small, oval yeast
cells with or without buds.
 Daughter cells are attached to mother cells by a narrow attachment point.
 The yeast cells are relatively small (2-4 um). A clear halo is seen around darker-
staining central material.

CULTURAL CHARACTERISTICS

 H. capsulatum may be cultured on the Sabouraud agar with chloramphenicol but

without cycloheximide at 25° C, or on brain heart infusion agar with blood at 37° C.
At 25° C, the mycelial phase has two types of conidia: small, round microconidia and
large (7-18 µm) thick-walled, macroconidia with knob-like projections. The yeast
phase grows at 37° C on blood agar.

EPIDEMIOLOGY

 Prevalent in America, parts of Africa and Asia. Rare in Australia and Europe.
 In the United States, histoplasmosis occurs throughout the midwest and much of the
eastern half of the United States.
 Infection usually occurs through inhalation of spores of the dimorphic
fungus Histoplasma capsulatum.
 Infections may also occur through ingestion. Most infections are subclinical or
benign.
 Histoplasmosis may occur commonly in animals in endemic areas.
 Dogs are particularly susceptible, but the disease has also been reported in cattle,
cats, swine, horses, sheep and wild animals.
 The disease has not been reported in birds. Histoplasma capsulatum is not
contagious.

PATHOGENESIS

 The disease may vary from small granulomatous nodules to an acute, disseminating,
rapidly fatal form.
 Usually, the disease manifests itself either as a pulmonary or intestinal infection, and
may be inapparent or subclinical, mild, acute, chronic, or disseminated.
 Ulcerations and tuberculosis-type lesions may occur in many of the organ systems.
 Mice are highly susceptible to infection, whereas other laboratory animals vary in
susceptibility.
 Following inhalation of spores, macrophages phagocytize the organisms and an
inflammatory response ensues.
 The fungus is either killed, or local granulomas form with calcification.
 Host immunity and the number of spores inhaled determine which form the disease
manifests itself as.
 The macrophages may carry the organisms to various body sites and actually help to
disseminate it.
 Thus, histoplasmosis has been referred to as a disease of the reticuloendothelial
system.
 Enlargement of the liver and spleen, and nodules on the tongue, ocular involvement,
and abortion have also been reported.

PATHOGENECITY

Clinical signs
  Basically pulmonary. Most infections are asymptomatic or benign and self
limiting.
 Symptomatic forms with fever, night sweats, weight loss and hemoptysis.
 In disseminated cases hepatomegaly and splenomegaly develop, with anemia and
leucopenia.
 Chronic pulmonary infections associated with cough, dyspnea, chest pain,
hemoptysis and weight loss.
 Cavities may develop in the apex or subapical regions of the lungs.

Lesions

 Epithelioid and giant cell granulomas of the lung are charactersitic.

 Invasion of cells of the reticuloendothelial system in the adrenal glands, bone
marrow, gastro intestinal tract, liver, lymphnode and spleen. Lesions tend to
become calcified.

DIAGNOSIS

Diagnosis

 The organisms are rarely seen extracellularly. Specimens (CSF, biopsies, bone
marrow, lymph nodes, or buffy coat) stained with a variety of histological stains
(Gomori methanamie or Periodic acid-schiff stains) reveal the organisms within
macrophages. The yeast cells are relatively small (2-4 um).
 A clear halo is seen around darker-staining central material.
 The skin test becomes positive after exposure to the fungus and remains so for the life
of the animal.
 Thus, the skin test is of little usefulness in detecting active disease.
 Furthermore, skin testing may induce antibody formation, thereby, interfering with
more useful serological tests.
 The complement fixation test for detection of specific antibodies is useful for
diagnosis.
 Animals develop a rapid rise in titer following infection; the titer falls off gradually
and disappears by nine months.
 However, cross-reactivity to antigens of the other systemic fungal pathogens may
occur.
 The immunodiffusion test is a useful adjunct to the CF test; the development of 2
bands may indicate active or past infection, respectively.
 A latex agglutination test and fluorescent antibody test are useful screening tests, but
complement fixation is considered the confirmatory test.

Treatment

 Amphotericin B is the drug of choice, but ketoconazole, sulfonamides, and ethyl

vallinate may also be effective.

CHAPTER-33: EPIZOOTIC LYMPHANGITIS

Learning objectives

To know in detail about,

 Synonyms of Histoplasma farciminosum

  Causative agents of epizootic lymphangitis
 Morphology, cultural and biochemical characteristics of H.farciminosum
 Different forms of epizootic lymphangitis
 Different diagnostic methods used for epizootic lymphangitis
 Treatment, control and prevention of epizootic lymphangitis

HISTORY AND MORPHOLOGY

 Histoplosma capsulalum and H. farciminosum cause clinical disease in horses.

 Epizootic lymphangitis is characterised by a cord-like appearance of the
subcutaneous lymphatic vessels, especially of the limbs, neck and chest and the
development of a series of pyogranulomas, the discharge from which contains yeast-
like cells of the pathogen. Rarely, infection may lead to pneumonia and conjunctivitis.
 Histoplasmosis caused by H. capsulatum has been recognised in horses in certain
areas of the world.
 Histoplosma farciminosum (Synonyms: Cryptococcus farciminosum, Zymonema
farciminosum, Histoplasma capsulatum var. farciminosum) is the cause of epizootic
lymphangitis.
 Histoplosma farciminosum will be used to name the agent, although according to
Ajello the organism does not belong to the genus Hisloplosma.
 Epizootic lymphangitis is a disease which is distributed world-wide, with endemic
foci in North Africa and Asia.
 The organism was first demonstrated in pus by Rivolta in 1873 but was not
successfully cultivated until 1896 when the first pure cultures were obtained by
Tokishiga in Japan.
 The yeast form of the organism appears in pus as a double-contoured oval or ovoid
body, measuring 2.5-3.5 µm by 3-4 um. The saprophytic stage is mycelial form.

CULTURAL CHARACTERISTICS

 Both yeast and mycelial forms can be cultivated if suitable media, temperature of
incubation and carbon dioxide tenson are provided.
 The organism grows slowly when the yeast phase is grown on media rich in protein
and in an atmosphere enriched with CO2.
 Several culture media have been used, but the most satisfactory were Sabouraud's
dextrose agar enriched with 2.5% glycerol; brain heart infusion agar enriched with
10% horse blood; nutrient agar supplemented with 2% dextrose; mycobiotic agar and
mycoplasma-like organism medium.
 Growth on all media is very slow and appears after four to eight weeks of incubation
at 25°C.
 Colonies of the mycelial form are a yellowish/light brown to deep brown, convoluted,
waxy and cauliflower-like.
 In body tissues, the ability of H. farciminosum to convert from the mycelial form to
the yeast form appears to be dependent on temperature and nutrition as well as the
strain, However, in vitro, conversion of the mycelial form to the yeast form of H.
farciminosum can be achieved by incubating at 35°C to 37°C.
 Complete conversion to the yeast form is achieved only after four to five repeated
serial transfers onto fresh media every eight days.

BIOCHEMICAL CHARACTERISTICS AND RESISTANCE

Biochemical characteristics
  The biochemical characteristics of the mycelial form include positive reactions to
catalase and urease tests as well as the assimilation of ammonium sulphate as the
sole source of nitrogen.
 No Fermentation of carbohydrate sugars, liquefaction of gelatine or reduction of
nitrate occurs.

Resistance

 The organism is highly resistant to the effects of physical and chemical agents and
can survive for at least a month in the dust of stables or kraals.
 This pathogen is also known to be viable and virulent after desiccation in the
laboratory for 25 months.
 H. farciminosum may survive for up to ten weeks in non-sterile water at 26°C.

EPIDEMIOLOGY

 Epizootic lymphangitis is a contagious disease which can infect humans.

 The disease mainly affects horses, mules and donkeys, although infection may occur
in camels and cattle.
 Mice and rabbits may be infected experimentally.
 Horses under six years of age are most susceptible.
 The mode of transmission of the disease is not well established.
 Direct contact with infective materials through injured skin or through cutaneous
abrasions is the most common mode of infection.
 Spread of infection can also occur indirectly through contaminated objects such as
grooming tools, feeding and watering utensils and harnesses and wound dressings.
 Flies that feed on open wounds may act as possible vectors.
 The organism has been isolated from the alimentary tract of biting flies that had
alighted in open lesions and the disease has developed in horses 4.8 km from the
nearest case.
 Experimentally, flies (Musca spp. and Stomoxys spp.) have been shown to be capable
of transmitting the infection.
 Transmission also possible via stallions to mares by copulation.

PATHOGENESIS

 The incubation period ranges from several weeks to six months.

 Following the initial invasion of the skin, the organism spreads through the
lymphatic vessels to the regional lymph nodes, and in more advanced cases involves
the internal organs.
 Nodular and chronic suppurating lesions are evident in the skin overlying lymph
vessels and nodes.
 When mucosal lesions occur, most are confined to the upper respiratory tract and
eyes.
 The nasal infection is usually accompanied by mucopurulent discharge containing
large numbers of the fungus.

PATHOGENECITY

Clinical signs

 The cases of epizootic lymphangitis can be grouped into four different forms, namely:
cutaneous, respiratory, ocular and asymptomatic carriers.
  The cutaneous form of the disease, after which the disease was named, is the most
common.
 The initial lesion is an open granulomatous wound along the course of a lymphatic
vessel, which has a tendency to ulcerate, or to undergo alternating periods of
discharge and closure for some weeks before healing with residual scar formation.
 Lesions are most common in the forelimbs, the chest wall and the neck. In severe
cases, skin over the entire body may be affected.
 The lesions begin as indolent, chancre-like papules, becoming larger over the course
of weeks and eventually form irregular pyogranulomatous nodules, which frequently
ulcerate.
 Mortality does not usually exceed 10% to 15% and the main loss results from the
inability of animals to work for several weeks because of extremely painful lesions.
 The ophthalmic form of the disease is less frequent. Infection may occur as
conjunctivitis or a naso-lachrymal infection.
 The infection rarely becomes generalised. Initial infection is characterised by a
watery discharge from one or both eyes and some swelling of the eyelids, followed by
the development of papules and ulcerating button-like growths on the conjunctiva
and/or on the nictitating membrane .
 The respiratory form of the disease is characterised by lesions which are mostly
confined to the upper respiratory tract.
 This form usually occurs as a late development in the cutaneous form of the disease.
 On the nasal mucosa, the lesions begin as yellowish papules or nodules and these
soon form crater-like granulating ulcers that bleed easily.
 The lesions are usually found near the external nares. These lesions may also occur in
the lungs.
 Asymptomatic carriers can be identified clinically by the identification of fibrocalcific
skin lesions at previous sites of infection. Such horses will give a positive result to an
intradermal sensitivity test and positive reactions to serological tests.

Lesions

 Gross lesions are manifested by pyogranulomas, purulent discharge of thickened

superficial lymphatic vessels and enlargement of regional lymph nodes.
 Histopathologically, a typical granulomatous tissue reaction occurs with a
predominance of the large macrophages, many of which contain oval organisms in
the cytoplasm.
 Affected tissues stained by Gram stain revealed the presence of ovoid double-
contoured yeast-like cells.
 Periodic-acid Schiff or Gomori's methanamine silver stains are very useful to
demonstrate the presence of the organisms.
 Typical nodules of liquefied foci have also been recorded in the pleura, spleen, liver
and bone marrow.

DIAGNOSIS

Diagnosis

 Laboratory tests used in the diagnosis of epizootic lymphangitis include isolation of

the causative agent by culture and tests for the presence of antibodies in the blood.
 Haemalological picture showed leucocytosis, neutrophilia and an increase in the
erythrocyte sedimentation rates.
 Direct smear examination and culture technique
 o Diagnosis is usually based upon demonstration of the typical yeast-like,
double-contoured cells in pus collected aseptically from the lesion and
confirmed by culturing the pathogen.
o H. farciminosum is a Gram-positive organism and is successfully cultivated
on a vanety of media.
o Growth is relatively slow; most isolates require from four to eight weeks for
development of characteristic colonies.
 Serological tests
o In the absence of positive culture of H. farciminosum, a presumptive
diagnosis is usually made, based on the presence of antibodies in the serum.
o Although several serological tests have been used for the diagnosis of
epizootic lymphangitis, none of the tests are sufficiently sensitive or specific to
confirm diagnosis.
o The four serological tests such as FAT, AGID, ELISA and serum agglutination
are relevant.
 Electron microscopic examination
o Tissues taken from cutaneous lesions revealed the presence of oval bodies.
o Most of the details of the fine internal structures could be observed.
 Animal inoculation
o Experimental transmission of H. farciminosum has been attempted in mice,
guinea-pigs and rabbits.
o Imnunosuppressed mice were highly susceptible to experimental infection
and can be used for diagnostic purposes.
o Intra dermal test
o An accurate and reliable method of skin testing is the intradermal test.
o This consists of intradermal injection of 0.1 ml of soluble antigen prepared
from H. farciminosum.
o An increase in the thickness of the skin of 8 mm to 20 mm, 24 h after
injection of the antigen can be regarded as a positive result.

Differential diagnosis

 A number of diseases may be confused with epizootic lymphangitis (e.g. glanders,

strangles, ulcerative lymphangitis and sporotrichosis), especially when these diseases
occur under the same environmental conditions.

TREATMENT

 Epizootic lymphangitis is a chronic disease, although some cases may heal

spontaneously a few weeks after the development of clinical signs.
 The intravenous injection of 100 ml of sodium iodide of a 10% solution, repeated
weekly for four weeks, gives good results.
 The infected horses were treated with an intravenous injection of amphotencin B at a
dose of 0.2 mg/kg body weight three times on alternate days.
 The scabs were removed and the areas cleaned daily with an iodine solution for seven
days.
 Administration of griseofulvin, repeated if necessary, has given good results when
combined with iodides and local surgical treatment.
 The surgical treatment usually consists of opening the nodules and packing with
gauze soaked in 7% tincture of iodine.

CONTROL AND PREVENTION

  Outbreaks in non-endemic areas are probably best controlled by the slaughter of
affected animals.
 The long incubation period of the disease, the high resistance of the causative agent
and the presence of clinically healthy carriers make control of the disease difficult in
endemic areas.
 Control of the disease depends upon elimination of the infection by culling infected
horses and preventing spread by hygieneic precautions.
 Cleaning and disinfection will help to prevent the disease from spreading.
 This method of control is the most satisfactory and proven to be mandatory for large
breeding companies in endemic areas.
 A killed formalised vaccine prepared from the yeast form of the fungus, administered
subcutaneously in a dose of 5 ml once a year, has given good results.
 An attenuated vaccine was developed by exposure of the causative agent to high
temperature.
 Horses inoculated subcutaneously with 3 ml in a single dose have given a higher
protection rate.

CHAPTER-34: COCCIDIOIDOMYCOSIS

Learning objectives

To know in detail about,

 Morphology and cultural characteristics of Coccidioides immitis

 Coccidioidomycosis
 Diagnostic methods for coccidioidomycosis
 Sporotrichosis
 Blastomycosis

INTRODUCTION

 Coccidioidomycosis is usually a benign, inapparent, or mildly severe upper

respiratory infection that resolves naturally.
 On occasion, the disease may become an acute or chronic, disseminating, fatal
mycosis.
 The etiologic agent, Coccidioides immitis, is the most virulent of the fungal
pathogens.
 The disease is not uncommon among laboratory workers who isolate the agent.
 It is endemic in the soils of the Southwest United States and Central South America.
 Dust storms increase the incidence of disease.
 Of domestic animals, dogs are most frequently affected, although horses are
occasionally affected as well.
 Infections also occur in cats, swine, sheep, cattle, human and nonhuman primates,
and some 30 species of non-domestic mammals.

MORPHOLOGY

 In the soil, C. immitis is a mould made up of slender septate hyphae that give rise, on
thicker secondary branches, to chains of infectious arthroconidia (arthrospores,
arthroaleuriospores, arthroaleurioconidia).
 These are bulging, thick-walled cells, separated by empty cells, through which breaks
occur when arthroconidia are dispersed.
  In tissue, arthroconidia grow into spherical sporangia with birefringent walls,
"spherules", which by internal cleavage produce several hundred "endospores".
 The walls disintegrate, allowing dissemination of endospores, each of which may
repeat the cycle or, on a nonliving substrate, give rise to mycelial growth.
 Though only arthroconidia are naturally infectious, endospores can experimentally
initiate disease. Sexual spores are not known.
 "Coccidioidin" in supernatants of mycelial C. immitis broth cultures is largely
polysaccharide, but contains some amino acid nitrogen.
 It is used in cutaneous hypersensitivity and serologic tests.
 "Spherulin," a lysate of cultured spherules, is also used in skin tests. Both are
leukotactic.

CUTURAL CHARACTERISTICS AND RESISTANCE

Cultural characteristics

 This dimorphic fungus grows more quickly than the other dimorphic pathogens (1-2
weeks), but the same cultivation media are used.
 On Sabouraud's or blood agar, at 25° C or 37° C, a moist white colony develops that
later is covered with a fluffy mycelium. Bovine blood agar is hemolyzed.
 Arthroconidia are produced in 5 to 7 days. Mycelial growth should be evident within a
week and is examined for presence of arthroconidia in a lactophenol cotton blue wet
mount.
 Thick-walled, barrel-shaped arthroconidia alternating with empty disjunctor cells is
characteristic.
 The isolate can be reconverted to the sporangial phase by animal inoculation or
cultivation in a spherule medium.
 The sporangial phase is produced at 40°C in media containing casein hydrolysate,
glucose, biotin, glutathione and a salt mixture.

Resistance

 Arthroconidia resist drying and tolerate heat and salinity better than do competing
soil organisms.
 In summer heat, C. immitis survives in soil layers nearer the surface than its
competitors.
 When conditions favor growth again after rains, C. immitis repopulates the
superficial soil layers first, ensuring its widespread dispersal.

PATHOGENESIS

 Infection is mainly by inhalation of dust. Primary cutaneous infections are rare.

Infection is initiated by inhalation of the arthrospores.
 Relevant cell products include proteases, T suppressor cell activator, and leukotactic
agents. Leukocytes in vitro encourage arthroconidial metamorphosis to spherules.
 The agent triggers an inflammatory response in the lung, is engulfed but not killed by
phagocytes and is conveyed to the lymph node, where another inflammatory focus
develops.
 Inflammation is stimulated in part by a potent serine protease, which is liberated
during the growth of the fungus in vivo (digests elastin, collagen, and
immunoglobulins).
 Normally, cell-mediated immune responses arrest the process at this stage following
stimulation of TH -1, lymphocytes that activate macrophages.
  With inadequate cell-mediated immunity, dissemination can occur to bones, skin,
abdominal viscera, heart, genital tract, and eye (and rarely in animals to brain and
meninges).

CLINICAL SIGNS AND LESIONS

Clinical Signs

 In all species, overt disease is the exception. Highest prevalence of canine systemic
coccidioidomycosis is observed in male dogs, 4 to 7 years of age.
 Young Boxer dogs and Doberman pinschers are highly susceptible.
 Pulmonary disease may be asymptomatic, symptomatic of variable degree, benign
and chronic, or progressive.
 Dissemination may occur, but only in the dog and human, and depends on host
resistance and the level of exposure.
 There may be respiratory signs (including cough), fever, lameness due to bone
involvement, or discharging sinuses from deep lesions.
 The disease is most common in Boxers and Doberman Pinschers.
 The disease has not been reported in cats.
 In cattle, sheep, and swine, the disease is usually asymptomatic, limited to lungs and
regional lymph nodes and undiagnosed until slaughter.

Lesions

 Gross lesions are white granulomas varying from miliary nodules to irregular masses.
Peritoneal, pleural, and pericardial effusions occur.
 The initial lesions are found in the lungs. Systemic disease may involve the meninges,
bones, joints, and subcutaneous and cutaneous tissues.
 Lesions may also occur in the lung, brain, liver, spleen, and kidney.
 In acute cases, burrowing abscesses are common.
 In chronic and slowly advancing cases, focal and suppurative granulomatous lesions
are common without caseation or calcification.
 Severe, disseminated disease is usually seen only in dogs and humans.
 Cattle and swine are often infected, but the disease is restricted to a few tuberculous-
like lesions in the lymph nodes and sometimes the lung.

DIAGNOSIS, TREATMENT AND CONTROL

Diagnosis

 Based on Direct Examination of Specimens

o Animal fluids and tissues are examined for spherules by wet mount in saline
containing 10% KOH.
o Spherules are 10 to 80 μm in diameter, have a thick wall, and contain
endospores.
o When the spherule bursts, it releases endospores (2-5 um) and leaves a ghost
spherule and the endospores are stained by a fungal stains such as
hematoxylin and eosin and Gomori methanamine silver.
 Based on Culture
o Blood agar and Sabouraud's agar with antibiotics are inoculated, tape-sealed,
and incubated at 37°C and 25°C, respectively.
o Mycelial growth should be evident within a week and is examined for
presence of arthroconidia in a lactophenol cotton blue wet mount, the isolate
 can be reconverted to the sporangia! phase by animal inoculation or
cultivation in a spherule medium.
 Based on serological test
o Serologic tests include the coccidioidin skin test (which is useful for
prognostic purposes), immunodiffusion test, complement fixation test, latex
agglutination test, and tube precipitin test.
o Serological tests may be more useful than culture. A negative skin test is a
poor prognostic sign.
o For the immunodiffusion test, multiple lines are associated with progressive
infection, whereas a single band usually indicates a stable, chronic infection.

Treatment and control

 As for the other fungal pathogens, amphotericin B is the drug of choice.

 Nystatin is also effective, but is more toxic. Ketoconazole may be effective. Vaccines
are not available.

SPOROTRICHOSIS

 Sporotrichosis is caused by the dimorphic fungus Sporothrix schenckii.

 The disease is characterized by nodular lesions that suppurate, ulcerate, drain, and
involve the cutaneous and subcutaneous tissues and the adjacent lymphatics.
 The fungus is widespread in nature, found in soil, on wood, and on other vegetation.
 The organism gains entrance to the skin through wounds or by traumatic
implantation.
 Occasionally (especially in dogs), the infection may spread to involve bone, muscle,
the central nervous system, lungs, or the genitourinary tract.
 Infections are common, particularly in horses and dogs. In dogs, the disease is more
likely to disseminate and result in a fatal infection.
 In horses, the disease must be differentiated from epizootic lymphangitis (caused
by Histoplasma farciminosum).
 The lymphocutaneous form may be nonsuppurative, or may result in ulcerations and
pus that discharge at several sites along the lymphatic channel, which serves as a
means of transmission for the organism within the animal.
 Laboratory rodents are highly susceptible to experimental infection, indicating the
virulence of this organism is greater than that of the opportunistic fungi.
 The disease is infectious, but not contagious, and is chronic.
 This dimorphic fungus can be isolated from lesions on brain heart infusion agar,
blood agar, or Sabouraud agar with cycloheximide and chloramphenicol.
 The mold is white when young, then turns brown to black.
 The hyphae are septate and fine, and the microconidia form in clusters at the hyphal
tips or as sessile forms at the sides of the hyphae.
 Differentiation from similar looking fungi is by conversion of the mold to the yeast
phase.
 Single-celled, cigar-shaped yeasts may or may not be seen in pus from lesions.
 Fluorescent antibody enhances visualization and confirmation of the disease.
 Serological diagnosis can be made by demonstration of a rise in complement-fixing
antibody.
 Potassium iodide, Amphotericin B, ketoconazole and micoconazole are effective for
treatment.

BLASTOMYCOSIS

 Etiologic agent: Blastomyces dermatitidis

  Source: soil enriched with decaying organic materils
 Epidemiology: Primarily North America with sporadic cases reported from India,
Africa and the Middle East.
 Clinical Signs
o Cutaneous lesions (Pustules, verrucous or ulcerated lesions) or subcutaneous
abscesses.
o Acute pulmonary disease with cough, pleuritic pain, chills, and low-grade
fever.
o Chronic pulmonary disease with productive cough, fever, weight loss,
dyspnoea, and fatigue.
o Dissemination to skin, bone and genitourinary tract may also occur.
 Lesions: Tissue responses suppurative and granulamatous
 Treatment: Amphotericin B or Ketoconazole

CHAPTER-35: ASPERGILLOSIS

Learning objectives

To know in detail about,

 Diseases caused by Aspergillus species

 Morphology and colony characters of Aspergillus species
 Pathogenesis of Aspergillosis in cattle
 General approaches used to diagnose Aspergillosis

INTRODUCTION

 It is primarily a disease of the respiratory system characterized by inflammatory,

granulomatous, necrotising lesions.
 Haematogenous spread of the organism leads to lesions in eye, skin, meninges and
respiratory tract.
 The disease is mainly caused by A.fumigatus, A.flavus and A.niger.

Host (s) Disease (s)

Avian species (Chicken, turkey, Acute Aspergillosis
ducks, pigeon, quails) (Brooder‘s pneumonia) Chronic Aspergillosis
Bovine Abortion, pneumonia and mastitis
Ovine Pneumonia, and abortion
Horse Abortion and diarrhoea
Dog Ear and nasal infection
Cat Fatal pulmonary aspergillosis

 When compared to infection in avian species, the intensity of infection in other

species is less.
 Aspergillosis is an economically important disease because of its high mortality and
morbidity in brooder chicks.

CULTIVATION
Cultivation of the organism

 The Aspergillus grows very well in ordinary Sabouraud's Dextrose Agar with
chloramphenicol.
 Cycloheximide should never been incorporated in the media.

Characters A.fumigatus A.niger A.flavus

Macroscopic  Velvetty or  Wooly. At first  Velvetty,
morphology of powdery, at white to yellow yellow to
the colony first white and then green or
then turning turning to dark brown
to dark bluish brown to black.  Reverse: Red
green.  Reverse: white brown
 Reverse of the to yellow
colony will be
white to tan.

Morphology of Single, usually one on Double, cover entire Single and double,
conidiospore upper half of the vesicle, form radiate cover entire vesicle,
and vesicle, parellel to head point out in all
sterigmata axis of stalk. directions
PATHOGENECITY

 In avian species aspergillosis encountered in two main forms

Acute Aspergillosis

 In which there is high morbidity and mortality in very young chicks.

 This form of the disease is commonly known as brooder pneumonia.
 Typical symptoms are loss of appetite, high temperature, listlessness, foetid
diarrhoea, convulsion and affected chicks die with in 24-48hrs of the onset of
symptoms.

Chronic Aspergillosis

 It is seen in individual adult birds or few birds in a flock.

 The affected birds may survive for longer periods in a gradually declining state.
 Symptoms are very mild and it is associated with anaemia, yellowing of faeces and
the presence of respiratory rattle.

Cattle

 Conditions of high humidity and temperature encourage the growth of molds when
hay and straw is stored and this constitutes the source of infection for cattle.
 Aspergillus fumigatus is considered as the primary cause of mycotic abortion,
however many other Aspergillus species, A.flavus, A.nidulans, A.niger,
A.terreus and A.versicolor are also found to be associated with abortion.
 Infections mainly occur by inhalation into lungs or by ingestion, and then carried to
the placenta in the blood stream from lesions in the respiratory tract or ulcers,
mycotic ruminits or other lesions of the digestive tract.
  This results in a slowly developing fungal placentitis (one to two months) and intefere
with the nutrition of the foetus, resulting in foetal death and abortion.
 Chronic form may lead to purulent vaginitis, cervicitis and endometritis, resulting in
infertility.
 Abortion most commonly occurs in 6-7 months of gestation.
 The aborted foetus shows discrete, raised ringworm type lesions on the skin of head
and neck.
 The placenta is found thickened, haemorrhagic, odematous and necrotic.
 The cotyledons will be grey in color, inter cotyledonary area will be leathery, grey and
tan in color.
 On necropsy grayish or yellowish gaseous exudates with mycelia are commonly seen
in the lung and airsac.
 Sometimes the organism colonise the bronchi, forming a compact spherical colony,
which is called fungus ball.
 The fungal balls are produced most frequently by A.niger than A.fumigatus.
 Characteristic nodular lesions are also seen in alimentary canal, kidneys and ovaries.

DIAGNOSIS

 Diagnosis required continuous effort because it is one of the common contaminant of

laboratory and it can be cultured routinely from the skin and URT of healthy animals.
 For confirmatory diagnosis, consider the following points:
o Repeated isolation
o Absence of any other pathogen
o Recovery from unexposed tissue and demonstration of hyphae.

Method of diagnosis

 Direct microscopic examination

o Wet mount prepartion of sputum, nasal discharge, milk, uterine discharge,
fetal stomach contents.
 Diagnosis is confirmed by isolation of pathogens from the stomach contents of the
aborted foetuses and placenta.
o Demonstration of fungal hyphae in the foetal tissue (By using 10% KOH or
LPCB).
 Demonstration of pattern of condiospore and sterigmata by slide culture method.
 Animal pathogenicity test
 Allergic test.

PREVENTION AND TREATMENT

Prevention

 Aspergillosis can be prevented by

o Reducing spore exposure to the animals
o Removal of potential source of spore contamination
o Maintain stress free environment
o Prophylactic use of 5-fluorocytosine inhibits inhaled spore germination.

Vaccines

 Several types of vaccines are used involving different parts of the fungal elements. i.e.
whole cell filterate, spores, mycelial fragments, germinating cells and they are
 inactivated with the use of heat, phenol, formalin etc. some vaccines of live cell origin
are also available.

Treatment

 Amphotericin-B, 5-fluorocytosine :- fungistaic, administerd orally, it will inhibit

spore germination.
 Ketoconazole effective against cutaneous and gastro intestinal aspergillosis.

CHAPTER-36: MYCOTOXIN AND MYCOTOXICOSES

Learning objectives

To know in detail about,

 Mycotoxins and mycotoxicoses

 General features of mycotoxin formation
 Toxigenic fungi and their toxins of vet.importance
 Toxins and their target organs/tissues
 Characteristics of mycotoxins
 Three forms of mycotoxicosis
 Important features of mycotoxicoses

HISTORY

 Fungal toxins are referred to as mycotoxins and the diseases they produced are
termed as mycotoxicosis.
 The epidemics of ergot poisoning caused by eating cereal grains infected with the
parasitic fungus Claviceps purpura have been recorded since the middle ages, the
toxin compounds responsible for this ergotism were identified in 1875. During 1942 –
1947, the disease alimentary toxic aleukia (ATA) outbreak occurred due to the
consumption of mouldy cereal grains.
 One of the first, well-documented outbreaks of animal mycotoxicosis (aflatoxicosis)
occurred in East Anglia, England in 1960 when more than 1,00,000 turkey poults
died of an unknown disease (Turkey X disease). Subsequently, the examination of the
incriminated grounnut meal revealed the presence of A.flavus mycelia and the toxic
metabolites revealed by thin layer chromatography (TLC) were called aflatoxins.

GENERAL FEATURES OF MYCOTOXIN FORMATION

 Many of the toxigenic fungi, over 100 known species, are capable of elaborating
mycotoxins.
 The same mycotoxin can be produced by different fungi and the same fungus can
produce different mycotoxins.
 Toxin production occurs only under specific conditions of moisture, temperature,
suitability of substrate and appropriate oxygen tension.
 The optimum conditions for toxin production are relatively specific for each fungus.
 For e.g. Fusarium elaborates its toxin at freezing temperature, while A.
flavus requires a temperture of 250C.
 The susceptibility of different crops to mould infection is goverened by the presence
of suitable substrates.
 Damage to the seed coat by insects, mechanical harvesting, severe frost or other
factors may predispose crops to fungal attack.
 Insects may also serve as carriers of fungal spores.
 The fungi associated with cereal grains have been divided mainly into two types.
o Field fungi which invade the grains before harvest and required greater water
activity for growth
 e.g. Fusarium, Helminthosporium and Cladosporium
o Storage fungi which invade the grains after harvest during drying and in
storage
 e.g. Aspergillus, Penicillium
 The main types of toxigenic fungi, which produce mycotoxins of animal/ poultry
importance, are

Species Toxins
A.flavus and A.parasiticus Aflatoxins
A. ocheraceus Ochratoxin
Fusraium roseum Trichothecane (t-2) toxin
Penicillium citrinum Citrinin
A.nidulans and A.versicolor Sterigmatocyosin
CHARACTERISTICS OF MYCOTOXINS

 The term mycotoxin is derived from the Greek word – ‗mykes‘ meaning ‗fungus‘ and
the Latin word – ‗toxicum‘ meaning ‗poison‘.
 Mycotoxins are group of compounds produced by some strains of certain fungi that
cause illness or death when ingested by man or animals.
 They are low molecular weight, non-antigenic, heat stable secondary fungal
metabolites.
 They can activate at low concentrations. They have a wide spectrum of toxic effects,
like carcinogenic, mutagenic, teratogenic and immunosuppressive.
 Acquired immunity does not occur following exposure.
 Each toxin affects specific target organs or tissues.

Target organs/ tissues Toxins

Vascular system Aflatoxins
Digestive system Aflatoxins
Mucous membrane Trichothecane (t-2) toxin
Urinary system Ochratoxin
Reproductive system Zearalenone (Fusarium toxin)
Cutaneous system Sporidesmin
MYCOTOXICOSIS

 Mycotoxicosis is disease syndrome that result from the ingestion of mycotoxins.

 They are neither infectious nor contagious, but they cause heavy economic losses to
the poultry and cattle farmers by affecting growth and prodcution performance.
 The severity of mycotoxicosis in animals is determined by several factors.
o Species of toxigenic fungus
 o Concentration of mycotoxin in the food
o Age, sex and health status of the exposed animal
o Target organs or tissue affected
o Duration of exposure to contaminated feed.
 Mycotoxins can enter the system of birds and animals by ingestion, inhalation or
direct skin contact. Mycotoxicosis occurs in three forms.
o Acute primary mycotoxicosis
 These are produced when high to moderate amounts of mycotoxins
are consumed.
 Acute mycotoxicosis often causes marked signs of disease or death.
o Chronic primary mycotoxicosis
 These types of syndromes will occur from moderate to low levels of
mycotoxin intake.
 They often cause reduced productivity in the form of slower growth
rate, reduced reproductivity and inferior market quality.
o Mycotoxicosis of domestic animals and poultry

Disease Fungus Crop or Mycotoxin Animals

substrate affected
Aflatoxicosis Aspergillus Ground nut, Afaltoxins B1, Cattle, pig,
flavus Aspergillus maize and B2, G1,G2 poultry
parasiticus nut crops and dogs
Ergotism Claviceps purpura Seed heads Ergotamine Cattle,
of many and Sheep, Pig,
grasses and ergometrine Horse and
grains Poultry
Facial Eczema Pithomyces charatarum Pasture, Sporidesmin Sheep and
litter Cattle
Oestrogenism Fusarium graminareum Maize, Zearalenone Pigs
Barley and
cereals
Leukoencephalo Fusarium moniliforme Maize Fumonisins Horses
malacia B1 (A1, A2, and
B2) Donkey
Trichothecane Many Fusarium species Cereals T-2 toxin, Many
toxicosis diacetoxy - species
seripenol
Ocharatoxicosis A. ochraceus Barley, Ochratoxin - Pigs and
P. viridicatum wheat and A Poultry
Maize
CLINICAL FEATURES OF MYCOTOXICOSIS

 Some important clinical features of mycotoxicoses are

o The disesase produced are not transmissible to incontact animals.
o Outbreaks are often seasonal and sporadic, and may be associated with
certain batches of stored food or particular types of pasture.
o Intially, the signs of illness are decreased growth rate or immunosuppression.
 o Treatments such as antibodies are usually ineffective.
o Recovery generally depends on the type and amount of mycotoxin ingested
and the duration of the exposure to contaminated feed.
o The only acceptable evidence for the presence of mycotocoses in animals is
the laboratory demonstration of mycotoxins in suspected food, or in the
tissues, secretions or excretions of affected animals.
o Characteristic lesions in target organs of affected animals are important
supporting diagnostic evidence

AFLATOXICOSIS

 The name aflatoxin derives from Aspergillus flavus toxin. Afalatoxins are a group of
approx.
 20 related toxic compounds produced by some strains of A.
flavus and A.parasiticus during growth on a variety of cereal grains and food stuffs
such as maize, cotton seed and groundnuts.
 High humidity and high temperature during pre-harvesting, harvesting,
transporatation and storage, as well as damage to feed crops by insects, drought and
mechanical injury during harvesting, favours the growth and toxin production
of Aspergillus flavus.
 Mould growth and toxin formation require a moisture content of the substrate
greater than 15%, temp.250C and adequate aeration.
 Aflatoxins are a group of related bisfuranocumasin compounds with toxic,
carcinogenic, teratogenic and mutagenic activity.
 The four major aflatoxins are B1, B2, G1 and G2. These mycotoxins are named
according to their position and fluorescent colour on thin layer chromatography
(TLC).
 B1and B2 produce blue colour and G1, G2 gives green fluorescence.
 Aflatoxins M1, M2 are hydroxylated metabolites of B1 and B2 that are excreted in the
milk of lactating animals such as dairy cows.

BIOLOGICAL EFFECTS OF AFLATOXIN

Acute toxicity

 Hepatic injury and nervous signs such as ataxia and convulsions. Death may occur
suddenly.

Chronic toxicity

 There is reduction in efficiency of food conversion, depressed daily weight gain,

decreased milk production in dairy cattle and enhanced susceptibility to intercurrent
infections due to immunosuppression.

PATHOGENECITY

Symptoms

 Young animals, particularly young pigs, calves, turkey poults and ducklings are highly
susceptible.
 Aflatoxin B1 produce the most hepatogenic, carcinogenic, teratogenic and
embryotoxic effects.
  Prominent signs in calves include blindness, circling, grinding of teeth, diarrhoea,
tenesmus and convulsions.
 Aflatoxicosis has ben described in goats. But sheep are highly resistant.
 In dairy cattle, afalatoxin M1 and M2 are excreted in the milk.
 In pigs, signs include drowsiness, inappetance, jaundice, weight loss and yellow
urine.
 Ducklings are considered to be the most susceptible avain species to aflatoxins.
 Signs include anorexia, poor growth rate, ataxia and opisthotonus, followed by death.
 In birds over three weeks of age, subcutaneous haemorrhages of legs and feet.

Lesions

 Principle target organ is liver. Depending on the severity of intoxication,

hepatomegaly with necrosis and marked bile duct hyperplasia will occur.
 Acute hepatic failure and massive haemorrhage due to impaired blood clotting,
increased capillary fragility leading to death may occur with higher doses.
 In chronic toxicity, in additon to liver damage, degenerative changes in the kidney,
thymus cortical aplasia leading to decreased cell mediated immune response will
occur.

DIAGNOSIS

 Chemical identification of mycotixins in food samples and biological assays for

toxicity are important confirmatory steps.
 Demonstration of toxigenic strains of Aspergillus flavus and Aspergillus
fumigatus and of potentially toxic levels of mycotoxins in the food, tissues, secretions
are helpful for diagnosis.
 Concentration of aflatoxin B1 in excess of 100μg /kg of feed are considered toxic for
cattle.
 Thinlayer chromatography and HPLC are more sensitive analytical methods for
determing afaltoxins levels in the food.
 Radio immuno assay and ELISA methods are also available.

Biological assays

 Ducklings are mostly susceptible. Bile duct proliferation in one-day-old ducklings

and chick embryo bioassay are highly useful.

CONTROL AND PREVENTION

 Prevention of contamination at all stages of food production, storage and use is the
preferred method of preventing aflatoxicosis.
 Decontamination procedures like physical removal and chemical treatment of
aflatoxin contaminated feeds such as with acids, alkalies, aldehydes, oxidizing agents
of selected gases (ammonia) have been used for degrading aflatoxins.
 High affitnity inorganic compounds such as benzoic and propionic acid have been
widely used as preservatives for stored agricultural products.