HAEMOFLAGELLATES

Live in blood or tissues of man or animals which serve as definitive hosts. They pass through a
cycle in gut of insect vectors which serve as intermediate hosts.Family Trypanosomatidae
(Haemoflagellates of medical importance). Has 2 genera. Trypanosoma and Leishmania.

Blood or tissue flagellates

BLOOD TISSUE

Trypanasoma Leishmania
1. T. brucei 1. L. donovani
Sub Species 2. L. tropica
T. brucei rhodesiense 3. L. braziliensis
T. brucei gambiense
T. beucei brucei
2. T. cruzi

Morphology:
Body, nucleus, kinetoplast, Flagellum and undulating membrane.
Body: Elongated & curved in falgellate stage. Ovoid in non-flagellate stage.
Nucleus: Round/oval centrally located.
Kinetoplast: Round/rod shaped infront or behind nucleus. Has parabasal body & blepharoplast.
Flagellum: Arises from blepharoplast. The portion lying inside the body is called axoneme or
axial filament.
Undulating membrane: Ribbon of protoplasm formed by flagellum.

Developmental stages:
Haemoflagellates occur in different forms in vertebrates & invertebrates. 4 forms seen
Amastigote, promastigote, epimastigote, trypomastigote.
Morphological variations: based on the position of flagellum and kinetoplast
Amastigote form:
Rounded, no external flagellum. Seen intracellularly in vertebrate hosts. Eg: T.cruzi &
Leishmania.
Promastigote form:
Lanceolate shaped. Flagellum seen. Kinetoplast anterior to nucleus. Found in insect vector of
Leishmania.
Epimastigote form:
Elongated cell, undulating membrane seen. Kinetoplast anterior and close to nucleus. Seen in
T.cruzi within insect vectors.
Trypomastigote form:
Spindle shaped. Infective stage of trypanosomes. Kinetoplast posterior to nucleus flagellum form
long undulating membrane.Absent in Leishmania. Found in vector arthropods & blood of
infected vertebrates.

Trypanosoma
 Protista: (unicellular eukaryotes)
 Sarcomastigophora: (with pseudopodia and/or flagella)
 Mastigophora: (flagellates)
 Kinetoplastida: (presence of extranuclear DNA, kinetoplast)
 Order : trypanosomatidae
 Genus: trypanosoma
Exist in the blood & connective tissues as trypomastigote forms passes life cycle in two hosts
Vertebrate and insect. Transmission from vertebrate host to another by blood sucking insects.
Trypanasoma brucei group: causes African trypanasomiasis or sleeping sickness.
Consist of
T.brucei rhodesience
T.brucei gambience
T.brucei brucei – animal strain.
2. T. cruzi: Causes chagas disease (South American trypanasomiasis)
African trypanasomiasis or sleeping sickness
 Trypanasoma brucei group
Morphology: It exist as Trypomastigote in vertebrate host. Trypanasomes are pleomorphic
Single flagellum arising from kinetoplast. Small dot like kinetoplast stains darkly. Centrally
placed nucleus.
Habitat
Connective tissue of man regional lymph nodes through lymphatics blood stream causing
parasitaemia.
Epidemiology:
Distribution:
• T.b.gambiense – W.Africa.
• T.b.rhodesiense – E.Africa.
• Transmission: By insect bite of vector Tsetse fly.
• Host Range: Man definitive & reservoir host.
• Tsetse fly- Intermediate host.
• Infective form: Metacyclic trypomastigotes present in salivary glands of Tsetse fly.
Life cycle: It possess its life cycle in two different hosts.Definitive host: man
Intermediate host: many species of tsetse fly.
Transmission
1. Trypomatigotes injected through skin when tsetse fly takes a blood meal .
Human host
2. Trypomastigote multiply in blood, lymph and in later stages in CNS
Tsetse fly
3. Trypomastigote ingested by tsetse fly
4. Parasite multiply in midgut. Migrate to salivary gland.
5. Become epimastigote and multiply. Develop into infective metacyclic trypomastigotes.
Lifecycle continue.
Clinical features
At the site of inoculation painful chancre,Invade the blood stream,Lymphadenopathy,CNS-
fever, head ache, loss of nocturnal sleep.Erythematous rashes on chest and
sholders.,Meningoencephalopathy.Patient thin, weak, sign of malnutrition.Death earlier-
Rhodesian.
W.African sleeping sickness:
IP: One- several weeks.
Nodule or “Chancre” at site of bite. Resolves in 2 weeks.Parasitemia for months (stage1). May
self limit.Lymph node invasion after few months. Intermittent fever, chills, headache.
A classic sign of T. b. gambiense infection is the enlargement of the cervical lymph glands at the
back of the neck (known as Winterbottom’s sign).
E.African sleeping sickness:
IP: About 2 weeks to year.
Chancre 10 days later. Parasitemia after 2-3 weeks. Cervical lymphadenopathy , recurrent fever.
Spleen & kidney involved. Later stages meningoencepahilitis and ends fatally.
Lab diagnosis
Samples:Peripheral blood,Bone marrow, CSF
Methods:
Microscopy
Giemsa stained smear for demonstration of Trypomastigote form.
Cultivation
 Animal inoculation
 Serology: IFA, ELISA, IHA
 CATT (card agglutination trypanasomiasis test).

Prophylaxis
Destruction of vectors.Isolation of human population from infected area
Chemoprophylaxis
Treatment: Suramine,Pentamidine. Melarsoprol, Eflornithine for CNS involvement.

South American trypanosomiasis (Chaga’s disease)

Caused by T.cruzi. A zoonotic disease.
HABITAT
Muscular, nervous system & RE system.
Epidemiology:
Seen in central & S.America.
Reservoirs: Humans, Dogs, Cats, armadillos, rodents etc.
Transmission: Reduviid bugs (Triatoma species) act as vectors. Spread through their faeces.
By blood transfusion (5%), congenital & sexual transmission.
Morphology:
2 forms seen:
A) Trypomastigote form: Found in blood as thin flagellates.
B) Amsatigote form: Ovoid non-flagellate form found in tissues.
Life cycle:
hosts. Vertebrate hosts (man & animals) & invertebrate hosts (reduviid bugs).
In Humans:
• Reduviid Bugs (Triatomine) while feeding on blood, defecates on the skin which contains
metacyclic trypomastigote.
• The trypomastigotes enters body through bite wound when the person rubs or scratches
or into mucosal surface through fingers
• Trypomastigotes invade tissues & get converted into amastigotes in RE cells, heart
muscles (cardiomyopathy), oesophagus, colon (megacolon).
• Undergo binary fission, transform into trypomastigotes & burst out to bloodstream,
which infects fresh tissues.
In reduviid bugs
• Trypomastigotes enter midgut after blood meal.
• Transforms to epimastigote form & Multiply.
• Reaches hindgut & transform to metacyclic trypomastigote.
Clinical disease:
Acute stage:
IP:1-2 wks. Firm swelling at entry site “Chagoma”. Swelling of eye lids “Romana’s sign”.
Common in infants. Fever, generalized lymphadenopathy etc. may be seen.
Chronic stage:
Damage to Auerbach’s plexus leads to dilatation of alimentary canal. Chaga’s cardiomyopathy
(cardiac dilatation).
Lab diagnosis:
Microscopy: Thick & thin blood smears reveal trypomastigotes. Geimsa staining done.
Xenodiagnosis: Infection free, lab bred reduviid bugs fed on patient’s blood & faeces examined
after 2-3 weeks for parasites.
Blood culture: Novy Nicolle-McNeal (NNN) medium or warren medium.
Serology: Chronic stages detected by CFT, direct agglutination & FAT.
Prevention of trypanosomes: use of DDT. Use of concrete roof houses.

LEISHMANIA
Blood and tissue flagellates
3 species are parasitic to man
1. Leishmania donovani: causing
- Visceral leishmaniasis or Kala-azar (Black sickness)
- Post Kala-azar dermal leishmaniasis
2.Leishmania tropica
 Causing : Oriental sore (Delhi boil). (Old world cutaneous leishmaniasis)
3. Leishmania braziliansis: Causing: Espundia (mucocutaneous leishmaniasis). (New world
cutaneous leishmaniasis)
Clinical classification
1. Visceral leishmaniasis – Kala azar
2. Cutaneous leishmaniasis – Oriental sore
- Espundia
- Post kala azar lesion
Leishmania donovani
Reported simultaneously by Leishman from London and Donovan from Madras in 1903
Geographical distribution: China, Africa, India ,S.America, and Russia
Habitat
It inhabits the reticuloendothelial system of vertebrate host (man). Found intracellularely as
amastigote form.
Morphology
It appear in two forms
1) Amastigote form (in definitive host: Man, dog, hamster etc)
Round or oval in shape (2-4 m).It is also known as L.D bodies (Leishman-Donovan body) It
occur in infected lesion of man in reticuloendothelial cells.
2) Promastigote stage
Long slender spindle shaped 15-20 m long. It occur in the digestive tract of insect vector
(sand fly) or laboratory culture. It has nucleus, kinetoplast, free flagellum projecting from
the anterior end.

Life cycle
Occur in two stages
- Amastgote form in definitive host (man)
- Promastigote form in intermediate host (Sand fly)
Sand fly of the genus Phlebotomus
Transmission
1.Promastigote injected through skin when sand fly takes a blood meal
Human Host
2. Promastigote taken up by the macrophages and become amastigotes. Multiply in
Reticuloendothelial cells (Visceral leishmaniasis) or Skin macrophages (cutaneous
leishmaniasis)
Sand fly
3. Amastigote ingested by sand fly.
4. Amastigote become promastigotes (midgut)
5. Promastigote multiply. Migrate to head and mouth part of the fly.
Life cycle continue
Other mode of transmission
• congenital
• Blood transfusion
• Accidental transmission to lab workers
Pathogenesis
L. donovani causes Kala -azar or visceral leishmaniasis Incubation period varies from 3-6
months to 1-2 years.
Clinical features
Pyrexia – continuous or remittent, Spleenic enlargement,Hepatomegaly, lymph adenopathy,
Epistaxis, anaemia .Skin become rough, dry, and dark pigmented so named Kala –azar (black
sickness). Hair tend to be brittle and fall out. untreated case death occur upto 15-95%.Cell
mediated immunity suppressed.
Lab diagnosis
Specimen
peripheral blood, aspirated materials of bone marrow, spleen or enlarged lymphnode.
A. Direct evidence
Amastigote form of Leishmania donovani can be detected in Leishman or Giemsa stained
smears of blood and aspirates.
Positive result of examination of aspirate
Spleen - 98 %
Bone marrow - 50 - 85 %
Enlarged lymph node - 65 %
Amastigote of Leishmania:
Small round or oval bodie. Usually found in groups inside mononuclear phagocytic cells or
lying free between cells. Nucleus and rod shaped kinetoplast in each amastigote stain dark red
color. Cytoplasm takes pale staining.
Isolation (culture)
Culturing of aspirates in visceral Leishmaniasis is useful in detecting light infection.
NNN medium.(Novy-Nicolle-McNeal medium) Consist of :
Difco blood agar – 8gm
Glass distilled water – 200ml
Defibrinated rabbit blood(10%) – 20ml
Sterilise the medium and dispense 5 ml of medium into 20ml sterile screw cap bottle.Sample
inoculated into the media and culture incubated at 240c for 1-4 weeks. The parasite grow as
promastigote and can be demonstrated by examining a drop of fluid under microscope(high
power objective).
other media used
Shneiders insect tissue culture medium.
Preparation of medium:
Schneider’s Drosophila tissue culture medium - 80ml
Foetal calf serum - 20ml
Antibiotic- antimycotic solution – 1.2ml
3. Animal inoculation: in Hamster
4. Demonstration of antigen by PCR

Indirect evidence
1. Blood examination
- Leucopenia
- Erithrocyte count decrease
- Thrombocytopenia
- Serum globulin level increase
2. Serological tests
- Aldehyde test
- Indirect Immuno Fluorescent test
- ELISA, CIE
Aldehyde tests:
The test depends on nonspecific serum globulin in the patient’s serum. This become positive
only when the disease is more then 3 month.
Procedure:
 To 1-2 ml of serum in a test tube, add 2 drops of concentrated formalin and mix well.
Allow to stand upto 20 min.
 Positive reaction : jellification of milk white opacity (like hard boiled egg)
Leishmanin Skin test
Prophylaxis
- Control of parasite
- Control of reservoirs
- avoid the bite of sand fly
Treatment
- Antimonials
- Pentamidine
- Cytokine therapy.
Post kala- azar dermal leishmaniasis (PKDL)
It is a cutaneous form of leishmaniasis that occur s in about 2-10 % kala azar patients generally 1
or 2 years after completion of antimonial treatment for original disease.Visceral infection
disappears but skin infection persist.
Clinical manifestation: hypopigmented patches, erythematous patches,yellowish pink nodules.