ADDIS ABABA UNIVERSITY

FACULTY OF VETERINARY MEDICINE

PREVALENCE AND SEROTYPE DISTRIBUTION OF SALMONELLA

IN SLAUGHTERED SHEEP AND GOATS AND ABATTOIR ENVIRONMENT
IN AN EXPORT ABATTOIR, MODJO, ETHIOPIA

BY
AKAFETE TEKLU FITE

JUNE 2008
DEBRE ZEIT, ETHIOPIA
 ADDIS ABABA UNIVERSITY
FACULTY OF VETERINARY MEDICINE

PREVALENCE AND SEROTYPE DISTRIBUTION OF SALMONELLA

IN SLAUGHTERED SHEEP AND GOATS AND ABATTOIR ENVIRONMENT
IN AN EXPORT ABATTOIR, MODJO, ETHIOPIA

A thesis submitted to the School of Graduate Studies of Addis Ababa University

in partial fulfillment of the requirements for the Degree of Master of Veterinary Science in
Tropical Veterinary Public Health

BY
AKAFETE TEKLU FITE

JUNE 2008
DEBRE ZEIT, ETHIOPIA

2
 PREVALENCE AND SEROTYPE DISTRIBUTION OF SALMONELLA
IN SLAUGHTERED SHEEP AND GOATS AND ABATTOIR ENVIRONMENT
IN AN EXPORT ABATTOIR, MODJO, ETHIOPIA

BY
AKAFETE TEKLU FITE

Board of External Examiners: Signature

Dr. Mohammed Abdella ______________________

Dr. Karim Tunkara ______________________

Dr. Berhe Gebre-Egziabher ______________________

Dr. Jeffery Mosser ______________________

Academic Advisor:

Dr. Moses Kyule (BVM, MSc, MPVM, PhD, Associate Professor) __________________
II
ACKNOWLEDGMENTS

I would like to express my deepest gratitude to my academic advisor Dr. Moses Kyule (Associate
Professor) for his overall guidance and taking his time to correct the manuscript.

I am deeply grateful to the USAID Ethiopian Sanitary and Phytosanitary Standards and Livestock
and Meat Marketing (USAID SPS-LMM) program especially, Drs. Wondosson Asfaw and
Leakemariam Yigezu for the financial support. I heartily appreciate all the staff at the study
abattoir, especially, Dr. Reta Nigatu and Mr. Habtamu Mammo for allowing me to work in the
abattoir and for their cooperation during sample collection. I wish also to express my sincere
thanks to Dr. Rene Hendrikson and his colleagues for serotyping of our Salmonella isolates.

Dr. Kelay Belihu, Associate Dean, also deserves special thanks for establishing collaborative
relationship with USAID SPS-LMM program, which enabled me to complete the research work,
and for his moral and overall administrative support. I would also like to extend my deep sense of
indebtedness to Dr. Bayleyegn Molla for his scholarly guidance, unreserved reference material
provision and encouragement.

A lot of thanks are due to Drs. Gashaw, Yenehiwot, Gebreyohans and Desalegn who made the
difficult situation in the lab pleasant through humors and understanding. I am also grateful to all
staff of the microbiology laboratory, especially Hirut Tesfaye for her friendly approach and
support.

My final gratitude is reserved to my Dad Teklu Fite, Mom Muluwork Regassa, brothers
Misganaw and Abdisa, sister Hawine and the rest of all my beloved families for their invaluable
material and moral support in all facets of life and their constant encouragement to prepare this
paper.

I
TABLE OF CONTENTS
PAGES

LIST OF ABBREVIATIONS ...................................................................................................... V

LIST OF TABLES ..................................................................................................................... VII
LIST OF FIGURES ..................................................................................................................VIII
LIST OF APPENDICES ............................................................................................................. IX
ABSTRACT................................................................................................................................... X
1. INTRODUCTION ..................................................................................................................... 1
2. LITRATURE REVIEW ............................................................................................................ 4
2.1. Historical perspectives ........................................................................................................ 4
2.2. Taxonomy and nomenclature ............................................................................................ 5
2.3. Characteristics of Salmonella ............................................................................................ 6
2.3.1. Biochemical characteristics ........................................................................................... 7
2.3.2. Growth and destruction .................................................................................................. 7
2.4. Antigenic composition ........................................................................................................ 8
2. 5. Virulence factors .............................................................................................................. 10
2.6. Epidemiology ..................................................................................................................... 11
2.6.1. Distribution .................................................................................................................. 12
2.6.2. Host range .................................................................................................................... 13
2.6.3. Source of infection and transmission ........................................................................... 14
2.6.4. Pathogenesis................................................................................................................. 15
2.6.5. Carrier states ................................................................................................................ 16
2.7. Clinical feature .................................................................................................................. 16
2.7.1. Salmonella infections in humans ................................................................................. 16
2.7.2. Salmonella infections in animals ................................................................................. 18
2.7.3. Ovine and caprine salmonellosis ................................................................................. 18
2.8. Detection ............................................................................................................................ 19
2.8.1. Pre-enrichment in nonselective liquid medium ........................................................... 20
2.8.2. Enrichment in selective liquid media........................................................................... 20
2.8.3. Plating out and identification ....................................................................................... 21

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 2.8.4. Confirmation ................................................................................................................ 22
2.8.5. Typing .......................................................................................................................... 23
2.9. Treatment .......................................................................................................................... 25
2.10. Prevention and control ................................................................................................... 25
2.10.1.Vaccination ................................................................................................................. 27
2.10.2. Competitive exclusion ............................................................................................... 29
2.11. Antimicrobial resistance................................................................................................. 29
2.12. Zoonotic implication ....................................................................................................... 30
2.13. Economic importance ..................................................................................................... 31
2.14. Salmonella in Ethiopia .................................................................................................... 31
3. MATERIALS AND METHODS ............................................................................................ 37
3.1. Description of the study site and study population ........................................................ 37
3.1.1. Study site...................................................................................................................... 37
3.1.2. Study population .......................................................................................................... 37
3.2. Study design ...................................................................................................................... 38
3.3. Sampling ............................................................................................................................ 38
3.3.1. Sampling procedure ..................................................................................................... 39
3.3.2. Sample processing ....................................................................................................... 41
3.4. Isolation and identification of Salmonella ...................................................................... 41
3.4.1. Pre-enrichment in non selective liquid medium .......................................................... 42
3.4.2. Enrichment in selective liquid media........................................................................... 42
3.4.3. Plating out and identification ....................................................................................... 42
3.4.4. Confirmation ................................................................................................................ 43
3.5. Data management and analysis ....................................................................................... 46
4. RESULTS ................................................................................................................................. 47
4.1. Prevalence of Salmonella .................................................................................................. 47
4.2. Distribution of serotypes .................................................................................................. 50
5. DISCUSSION ........................................................................................................................... 54
5.1. Salmonella prevalence ...................................................................................................... 54
5.2. Serotypes ............................................................................................................................ 60

III
6. CONCLUSIONS AND RECOMMENDATIONS ................................................................ 64
7. REFERENCES......................................................................................................................... 66
8. APPENDICES .......................................................................................................................... 77
9. CURRICULUM VITAE.......................................................................................................... 88
10. SIGNED DECLARATION SHEET ..................................................................................... 91

IV
LIST OF ABBREVIATIONS

AAU Addis Ababa University

AM Abdominal muscles
Aw Water activity
BGA Brilliant green agar
BGS Brilliant green sulfa
BPW Buffered peptone water
BSA Bismuth sulfite agar
CC Cecal content
CDC Centers for Disease Control and Prevention
CI Confidence interval
CS Carcass swab
DIASALM Diagnostic Semi-Solid Salmonella medium
DM Diaphragmatic muscles
DNA Deoxyribonucleic acid
EHS Eviscerator’s hand swab
FAO Food and Agriculture Organization
FDAM Final draft amendment
FHS Flayer’s hand swab
FVM Faculty of Veterinary Medicine
H Flagellar antigen
HACCP Hazard analysis and critical control point
Hek Hektoen enteric
HS Eviserator’s hand swab
ISO International Organization for Standardization
KS Eviscerating knife swab
LIA Lysine iron agar
LPS Lipopolysaccharide
MAP Modified atmosphere packaging
MKTTn Muller-Kauffmann tetrathionate with novobiocin

V
MLN Mesenteric lymph node
MSRV Modified-semisolid Rappaport-Vassiliadis
O Somatic antigen
OIE Office International des Epizooties
OR Odds ratio
RC Rumen content
RV Rappaport-Vassiliadis
RVS Rappaport-Vassiliadis with Soya
SC Selenite cysteine
SkS Skin swab
SPI Salmonella Pathogenicity Islands
Spp Species
SS Salmonella – Shigella
TBG Tetrathionate brilliant green
TSI Triple sugar iron
Vi Capsular antigen
VP Voges-Proskauer
WHO World Health Organization
WS Water sample
XLD Xylose lysine desoxycholate

VI
LIST OF TABLES
PAGES

Table 1: Antigenic structure of some Salmonella serotypes............................................................ 9

Table 2: Salmonella serotypes isolated from samples of food animals in Ethiopia ...................... 32
Table 3: Salmonella serotypes isolated from animal products in Ethiopia.................................... 35
Table 4: Salmonella serotypes/serogroups isolated from humans in Ethiopia .............................. 36
Table 5: Distribution of type and number of samples collected .................................................... 39
Table 6: Comparative results by Pearson’s X2 test of species-specific Salmonella prevalence in
MLN and CC samples at Modjo Export abattoir, Ethiopia, October 2007 to April 2008 ..... 47
Table 7: Prevalence of Salmonella by sample types and species of animals examined ................ 48
Table 8: Summary results of multiple stepwise logistic regression of the associations of carcass
contamination with Salmonella with the risk factors............................................................. 49
Table 9: Distribution of Salmonella serotypes isolated from apparently healthy slaughtered sheep
and goats, abattoir personnel and abattoir environment at Modjo Export abattoir, Ethiopia,
October 2007 to April 2008 ................................................................................................... 50
Table 10: Distribution of serotypes on samples of Salmonella positive animals at Modjo Export
abattoir, Ethiopia, October 2007 to April 2008 ..................................................................... 51
Table 11: Distribution of serotypes by samples of carcass positive animals at Modjo Export
abattoir, Ethiopia, October 2007 to April 2008 ..................................................................... 53

VII
LIST OF FIGURES
PAGES

Figure 1: Flow diagram showing ISO method for the detection of Salmonella ............................ 44
Figure 2: Proportion of Salmonella serotypes isolated from sheep and goat carcasses ................ 52

VIII
LIST OF APPENDICES
PAGES

Appendix I: Map showing the areas where the slaughtered sheep and goats are originated…….77
Appendix II: Media used and preparations for the isolation and identification of Salmonella…..78
Appendix III: Work sheet for recording colony morphology and biochemical reactions of
presumptive Salmonella isolates…………………………………………………...83
Appendix IV: Pictures……………………………………..……………………………………..84

IX
ABSTRACT

A survey study was conducted on 142 and 60 apparently healthy slaughtered sheep and goats,
respectively at an export abattoir, Modjo, Ethiopia from October 2007 to April 2008. The
objectives were to determine prevalence and serotype distribution of Salmonella in slaughtered
sheep and goats and abattoir environment, investigate the associations between some potential
risk factors and Salmonella contamination rates of carcasses and forward strategies to minimize
them. A total of 1240 samples consisting of skin swabs, eviscerator’s hand swabs, eviscerating
knife swabs, mesenteric lymph nodes, cecal contents, carcass swabs and water samples were
collected. The samples were examined for the presence of Salmonella following standard
techniques and procedures outlined by the International Organization for Standardization. In a
total of 202 animals examined for Salmonella, 18 (8.9%) were positive of which 11 (7.7%) were
sheep and 7 (11.7%) were goats. In a total of 1240 different samples, Salmonella was isolated in
89 (7.2%) samples of which 25 (12.4%) were carcass swabs, 11 (5.5%) mesenteric lymph nodes,
8 (4.0%) cecal contents, 10 (5.0%) skin swabs, 18 (8.9%) eviscerator’s hand swabs, 15 (7.4%)
eviscerating knife swabs and 2 (7.1%) water samples. Salmonellae were detected in all test
samples obtained from sheep and goats. Although the associations of carcass contamination with
potential risk factors were assessed, no statistically significant results were observed except with
eviscerating knife swab, which was found to be significantly associated with carcass
contamination. Sheep and goat carcasses that were eviscerated using Salmonella positive knives
were 4.175 times more likely to be contaminated with Salmonella compared to those that were
eviscerated with Salmonella negative knives. The 89 isolates of Salmonella recovered were
composed of 12 different serotypes. Salmonella Infantis (16.9%), S. Typhimurium (15.7%) and S.
Heidelberg (13.5%) were prevalent in test samples. Other serotypes isolated included S. Reading
(10.1%), S. Braenderup and S. Enteritidis (9.0% each), S. Butantan (6.7%), S. Anatum (5.6%), S.
Newport and S. Poona (4.5% each), S. Give (3.4%) and S. Hadar (1.1%). Results of the present
study showed that Salmonella are widespread in slaughtered sheep and goats, eviscerator’s hands
and the abattoir environment. Furthermore, eviscerating knife was the main source of carcass
contamination. Therefore, proper sterilization of knives at 82oC should be implemented at
abattoirs in order to reduce contamination of the meat and other edible offals during slaughtering

X
operations. Moreover, the current findings create momentum for further research to determine
other sources of carcass contamination in abattoirs.

Key words: Slaughtered sheep and goats, Salmonella, Prevalence, Serotypes, Modjo, Ethiopia

XI
1. INTRODUCTION

Food safety has been a concern of mankind since the dawn of history. Despite advances in food
science and technology, foodborne diseases remain one of the major public health problems all
over the world; they are also an important cause of reduced economic productivity (WHO, 1995;
Cullor, 1997; Unnevehr and Jensen, 1998; Legnani et al., 2004; Busani et al., 2005). The world
Declaration on Nutrition, adopted by the FAO/WHO International Conference on Nutrition,
emphasizes that hundreds of millions of people suffer from communicable and non-
communicable diseases caused by contaminated food and water (WHO, 1995; Unnevehr and
Jensen, 1998; FAO/WHO, 2004; Busani et al., 2005).

Wide spectrums of pathogens play major role in causing foodborne diseases. Most of them are
zoonotic and have reservoirs in healthy food animals from which they spread to a variety of
foods. Therefore, foods of animal origin are considered as major vehicles of foodborne infections
(Busani et al., 2005). In many registers, non-typhoid Salmonella species are documented as one
of the leading causes of foodborne bacterial diseases. Foodborne Salmonella typically causes
acute gastroenteritis and may cause a more serious septicaemic disease, usually in the very
young, the elderly or immunocompromised subjects (David et al., 2001; Giovannacci et al.,
2001; Hjartardóttir et al., 2002; Molla et al., 2003; Mølbak et al., 2006).

Salmonella species (spp.) occur widely in the natural environment and in different sectors of the
global food chain. The ability of these microorganisms to survive under adverse conditions and to
grow in the presence of low levels of nutrients and at suboptimal temperatures and pH values
presents a formidable challenge to the agricultural and food processing industries in marketing
safe products. The continued prominence of raw meats, eggs, dairy products, vegetable sprouts,
fresh fruits, and fruit juices as the principal vehicles of human foodborne salmonellosis arises
from major difficulties to coordinate sectorial control efforts within each industry. The problem
of salmonellosis is further compounded by the massive and unrestricted movement of foods in
international trade, the national disparities in the hygienic agricultural and aquacultural
production of foods and the non-uniform government and industry food safety controls during the
processing, distribution and marketing of fresh and processed food products (Molla et al., 2003;

1
Forshell and Wierup, 2006). Moreover, the emergence and persistence of highly virulent and
antibiotic-resistant Salmonella strains in recent years are major public health concerns (D’Aoust,
2001).

Food animals harbor a wide range of Salmonella serotypes and so act as sources of
contamination, which is of paramount epidemiological importance in non-typhoid human
salmonellosis. The process of removing the gastrointestinal tract during slaughtering of food
animals is regarded as one of the most important sources of carcass and organ contamination with
Salmonella at abattoirs. Moreover, contamination of meat by Salmonella may occur at abattoirs
from the excretion of symptomless animals, contaminated abattoir equipment, floors and
personnel and the pathogen can gain access to meat at any stage during butchering. Cross
contamination of carcasses and meat products could continue during subsequent handling,
processing, preparation and distribution. Therefore, periodic surveillance of levels of Salmonella
contamination in the different food animals, food products and environment is necessary to
prevent the spread of this pathogen and infection of humans (Hjartardóttir et al., 2002; Molla et
al., 2003; Vieira-Pinto et al., 2005; Woldemariam et al., 2005).

A number of studies conducted on poultry, pig, cattle, poultry meat, minced beef, and humans in
Ethiopia showed that salmonellae are prevalent in various food animals and meat products
(Pegram et al., 1981; Nyeleti et al., 2000; Molla et al., 2003; Molla and Mesfin, 2003; Ejeta et
al., 2004; Woldemariam et al., 2005; Aragaw et al., 2007) and humans (Mache et al., 1997;
Mache, 2002). Although little study has so far been undertaken to determine the prevalence of
Salmonella in sheep and goats in Ethiopia (Wassie, 2004; Woldemariam et al., 2005), studies
carried out elsewhere indicated that salmonellae are widespread in small ruminants (Sojka et al.,
1977; Nabbut and Al-Nakhli, 1982; Sharma et al., 1996; Chandra et al., 2007). Moreover, none
of the previous studies in Ethiopia on small ruminant determined the occurrence, magnitude and
distribution of Salmonella in the environment where sheep and goats are slaughtered. There is
paucity of information on the occurrence, distribution and prevalence of Salmonella in abattoir
environments including those export abattoirs located in central Ethiopia. It has not yet been
determined to what extent these environments serve as sources of Salmonella particularly to red
meat contamination. A study of such types would provide valuable information as to the major

2
sites of contamination in abattoir environments and help in the implementation of strategies to
minimize contamination levels. Therefore, this study was undertaken at a modern export abattoir
in Modjo with the following objectives:

 Establish the occurrence and prevalence of Salmonella in slaughtered sheep and

goats
 Determine serotype diversity in slaughtered sheep and goats and abattoir
environment
 Investigate the major sources of carcass contamination in abattoirs and
 Forward strategies to minimize the contamination.

3
2. LITRATURE REVIEW

2.1. Historical perspectives

In 1885, the bacteriologist Theobald Smith (1859-1934), who worked in the US Department of
Agriculture, isolated S. Choleraesuis from porcine intestine, and the genus Salmonella was
named after D.E. Salmon, his laboratory chief (D’Aoust, 1997; Mølbak et al., 2006).

The first report of a laboratory-confirmed outbreak of foodborne salmonellosis described an

episode in which 58 persons in 25 different families who had eaten beef developed acute
gastroenteritis; one died. Gartner isolated the ‘Gartner-bacillus’ from infected cow from which
the meat came, and from organs of the fatal case. Kauffmann determined that the ‘Gartner-
bacillus’ from this outbreak was serotype Enteritidis, but other outbreaks of ‘Gartner-bacillus’
were of serotype Dublin and possibly other serotypes. Mice, rabbits, guinea pigs and goats were
affected when inoculated with the bacillus. In the following years, several outbreaks of
salmonellosis affecting man or animals were reported, and the old concept of ‘meat poisoning’
was linked with the etiologic agent Salmonella. Subsequently, human salmonellosis occurred
primarily among individuals who ate meat from ill animals, mainly cattle, but also pigs or goats
(Mølbak et al., 2006).

The development of serotyping was fundamental for the understanding of the epidemiology of
Salmonella infections. While S. Typhi was easy to recognize by biochemical tests, it was for
example, impossible to distinguish between S. Typhimurium and S. Paratyphi B on the basis of
fermentation of sugars or other biochemical properties. Many bacteriologists considered S.
Typhimurium and S. Paratyphi B to be identical. However, it was an enigma why infections with
seemingly identical bacteria often did not share pathological, clinical and epidemiological
features. This and several other questions were resolved at the end of the 1920s when White and
Kauffmann, through the use of improved serological methods, succeeded in designing a
classification system for Salmonella. The foundation for this serotyping scheme was the
discovery of the flagellar H antigen and the thermostable somatic O antigen by Weil and Felix
and the phase-shift in the H antigen (D’Aoust, 1997; Mølbak et al., 2006).

4
2.2. Taxonomy and nomenclature

The classification and nomenclature of salmonellae has been controversial for many years.
According to the latest nomenclature, which reflects recent advances in taxonomy, the genus
Salmonella consists of three spp: S. enterica, the type species, S. bongori, the former subspecies
V and S. subterranea (Shelobolina et al., 2004; Heyndrickx et al., 2005; Euzéby and Barrett,
2007). Salmonella enterica in turn is further divided in to six subspecies, which are referred to by
a Roman numeral and a name (I, S. enterica subsp. enterica; II, S. enterica subsp. salamae; IIIa,
S. enterica subsp. arizonae; IIIb, S. enterica subsp. diarizonae; IV, S. enterica subsp. houtenae;
VI, S. enterica subsp. indica). S. enterica subspecies are differentiated biochemically and by
genomic relatedness (Brenner et al., 2000; Jay, 2000; Heyndrickx et al., 2005; OIE, 2005;
Forshell and Wierup, 2006; Euzéby and Barrett, 2007).

Salmonella species are further classified into serotypes (serovars) using the Kauffmann-White
scheme, which is defined and maintained by the WHO Collaborating Centre for Reference and
Research on Salmonella at the Pasteur Institute, Paris, France. The classification is on the basis of
extensive diversity of lipopolysaccharide (LPS) antigens (O), flagellar protein antigens (H) and
sometimes the capsular (Vi) antigens (OIE, 2005; Euzéby and Barrett, 2007). Currently there are
about 2,541 serotypes of Salmonella and new serotypes are listed in annual updates of the
Kauffmann-White scheme (Euzéby and Barrett, 2007).

The most common serotypes that cause infections in humans and food animals belong to S.
enterica subspecies enterica. The serotypes of the other subspecies are common in
poikilothermic animals and in the environment, although some serotypes of S. arizonae and S.
diarizonae have been associated with disease in turkeys and sheep (Brenner et al., 2000; OIE,
2005).

Center for Disease Control and Prevention (CDC) of the U.S. uses names for serotypes in
subspecies I (for example, serotypes Enteritidis, Typhimurium, Typhi, and Choleraesuis) whereas
antigenic formulas for unnamed serotypes described after 1996 in subspecies II, IV, and VI and
in S. bongori. For named serotypes, to emphasize that they are not separate species, the serotype
name is not italicized and the first letter is capitalized (Brenner et al., 2000, OIE, 2005). At the

5
first citation of a serotype the genus name is given followed by the word ‘’serotype’’ or the
abbreviation ‘’ser.’’and then the serotype name. Subsequently, the name may be written with the
genus followed directly by the serotype name (for example, Salmonella Typhimurium or S.
Typhimurium) (Brenner et al., 2000; Jay, 2000; Chiu et al., 2004; OIE, 2005).

Serotype names designated by antigenic formulae include: (i) subspecies designation (subspecies
I through VI), (ii) O antigens followed by a colon, (iii) H antigens (phase 1) followed by a colon,
and (iv) H antigens (phase 2, if present) (for example, Salmonella serotype IV 45:g, z51:-). For
formulae of serotypes in S. bongori, V is still used for uniformity (for example, S. V 61:z35:-).
Before 1996 all serotypes in all subspecies except subspecies IIIa and IIIb were given names. In
1996 the WHO collaborating center began naming serotypes only in subspecies I and dropped all
existing serotype names in subspecies II, IV and VI and S. bongori from the Kauffmann-White
scheme. For surveillance purposes, i.e., for compatibility with old data, CDC continues to use
pre-1996 names for serotypes in subspecies II, IV and VI and S. bongori (Brenner et al., 2000,
OIE, 2005).

Salmonella serotypes were named according to the disease they caused (S. Enteritidis, S. Typhi,
S. Paratyphi, S. Abortus equi, and S. Bovismorbificans) or the animals from which they were
isolated. For example, S. Gallinarum and S. Pullorum were important pathogens in poultry, S.
Choleraesuis an important swine pathogen, and S. Typhimurium got its name because it was
originally isolated from ill laboratory mice. A limited number of serotypes were named after the
person who isolated (e.g. S. Virchow). Currently each new antigenically distinguishable type is
typically named after the geographic place at which it was first isolated (S. London, S. Miami, S.
Richmond and so on) (Jay, 2000; Mølbak et al., 2006).

2.3. Characteristics of Salmonella

The family Enterobacteriaceae consists of Gram-negative, facultatively anaerobic non-spore-

forming rods. Salmonella conforms to the general definition of the family (D’Aoust, 1997; Quinn
et al., 1999; Intorre et al., 2005; Forshell and Wierup, 2006). Members of the genus are motile by
peritrichous flagellation except S. Pullorum and S. Gallinarum, which lack flagella. Nonmotile

6
variants can also arise as a result of a faulty assembly of flagellar subunits or deficiencies in the
motor functions of these appendages (D’Aoust, 1997; 2000). Salmonellae are
chemoorganotrophic, with an ability to metabolize nutrients by the respiratory and fermentative
pathways (D’Aoust, 1997).

2.3.1. Biochemical characteristics

Salmonellae are generally unable to ferment lactose, sucrose or salicin, although glucose and
certain other monosaccharides are fermented with the production of gas. They are usually
catalase positive, oxidase-negative and reduce nitrates to nitrites. The microorganisms use citrate
as the sole carbon source, decarboxylate lysine, arginine and ornithine and produce hydrogen
sulphide. The methyl-red reaction is positive, the Voges-Proskauer test is negative and indole is
negative. Phenylalanine is not delaminated, urea is not hydrolysed, gelatin is not liquified rapidly
in nutrient media and neither DNAase nor lipase are produced. Salmonellae may harbor
temperature phages or plasmids that code for metabolic characters used in identification (e.g.
H2S, lactose or sucrose fermentations) (Coetzer et al., 1994; D’Aoust, 1997; 2000; Jay, 2000;
Mølbak et al., 2006).

2.3.2. Growth and destruction

Salmonellae are able to grow on a large number of culture media and produce visible colonies
well within 24 h at about 37oC. The parameters of pH, water activity (aw), nutrient content and
temperature are all interrelated for salmonellae, as they are for most other bacteria. The pH for
optimum growth is around neutrality (between 6.6 and 8.2), with values above 9.0 and below 4.0
being bactericidal. A minimum growth pH of 4.05 has been recorded for some (with hydrochloric
and citric acids), but depending on the acid used to lower the pH, the minimum may be as high as
5.5. Aeration was found to favor growth at lower pH values (Jay, 2000; D’Aoust, 2001).

Salmonella grows in the temperature range of 2 - 47oC with rapid growth between 25 and 43oC
(D’Aoust, 2000; 2001). The lowest temperatures at which growth has been reported are 5.3oC for
S. Heidelberg and 6.2oC for S. Typhimurium. Temperatures of around 45oC have been reported

7
by several investigators to be the upper limit for growth. Regarding available moisture, growth
inhibition has been reported for aw values below 0.94 in media with neutral pH, with higher aw
values being required as the pH is decreased toward growth minima (Coetzer et al., 1994; Jay,
2000; D’Aoust, 2001).

Salmonellae are unable to tolerate high salt concentration. Brine above 9% is reported to be
bactericidal. Nitrite is effective, with the effect being greatest at the lower pH values. This
suggests that the inhibitory effect of this compound is referable to the undissociated HNO2
molecule (Jay, 2000).

With respect to heat destruction, all salmonellae are readily destroyed at milk pasteurization
temperatures. Some investigators also found that cells grown at 44oC were more heat resistant
than those grown at either 15oC or 35oC (Jay, 2000).

Some food products are packaged under vacuum or gaseous mixtures of carbondioxide, nitrogen
and oxygen to increase product quality and extend shelf life. The benefits of modified
atmosphere packaging (MAP) stem are from the inhibition of endogenous spoilage micro flora
and enhanced organoleptic stability of the product. The high concentrations of carbondioxide
(>50%v/v) commonly used in MAP inhibit the growth of Salmonella spp. but exert little or no
effect on survival (D’Aoust, 2001).

2.4. Antigenic composition

The antigenic classification or serotyping of Salmonella used today is a result of extensive studies
of antibody interactions with bacterial surface antigens by Kauffman and White. Three kinds of
surface antigens, somatic (O, Ger. ohne), flagellar (H, Ger. hauch) and capsular (K, Ger. kapsel)
determine the reactions of the organisms to specific antisera (Jay, 2000; Chiu et al., 2004).

Species and serotypes are placed in groups designated A, B, C and so on, according to
similarities in content of one or more O antigens (Table 1) (Jay, 2000; Chiu et al., 2004). The O
antigens are part of the lipopolysaccharide component of the cell wall that also contains lipid A

8
and a core portion. The O antigens or O-specific side chains consist of repetitive oligosaccharide
units of which the type, order and repetition of sugar moieties differ between serotypes (Coetzer
et al., 1994). The O antigens are quite stable to heat whereas the K and H antigens are heat labile.
Because flagellar proteins are less heterogeneous than the carbohydrate side chains, considerably
fewer H antigenic type exist (Jay, 2000).

The H antigens are further classified in to two types: specific phase or phase 1 and group phase or
phase 2. Phase 1 antigens are shared with only a few other species or varieties of Salmonella;
phase 2 may be more widely distributed among several species. Any given culture of Salmonella
may consist of organism in only one phase or of organisms in both flagellar phases. The H
antigens of phase 1 are designated with small letters and those of phase 2 are designated by
Arabic numerals (Jay, 2000).

Table 1: Antigenic structure of some Salmonella serotypes (adapted from Jay, 2000)

Serogroups Serotypes O-antigens* H-antigens

Phase 1 Phase 2
A S. Paratyphi 1,2,12 a (1,5)
B S. Heidelberg (1), 4, (5), 12 r 1,2
S. Typhimurium 1,4, (5), 12 i 1,2
C1 S. Virchow 6,7, (14) r 1,2
S. Choleraesuis 6,7 (c) 1,5
S. Oranienburg 6,7 m, t -
S. Montevideo 6,7 g, m, s, (p) (1,2,7)
C2 S. Newport 6,8 e, h 1,2
D S. Typhi 9,12, (vi) d -
S. Enteritidis 1,9,12 g, m (1,7)
S. Gallinarum 1,9,12 - -
E S. Anatum 3, 10 e, h 1, 6
* The italicized antigens are associated with phage conversion. ( ) = May be absent.

9
2. 5. Virulence factors

The biological effects of the virulence factors of Salmonella are interrelated and are responsible
for enteric and systemic clinical signs and lesions of salmonellosis (Coetzer et al., 1994;
Radostits et al., 1994; Forshell and Wierup, 2006).

Lipopolysaccharide (LPS): the lipid component anchors the complex to the outer membrane of
the bacteria and is the base to which the sugar chain is attached. The polysaccharide component
of LPS is based around the core antigen, which is a short chain of sugars. To this core antigen is
attached a chain of up to 40 repeating oligosaccharide units, the O antigen. It is this part of the
LPS which interacts with the immune system and which is involved in host–bacterium interaction
and bacterial virulence (Cogan and Humphrey, 2003). Apart from the fact that the O-specific side
chain of the LPS is immunogenic and is recognized by the host’s immune system, it has also been
associated with invasiveness of Salmonella and enterotoxin production (Coetzer et al., 1994;
Radostits et al., 1994; D’Aoust, 2000).

Enterotoxin: several serotypes of Salmonella produce enterotoxin that is closely related, both
functionally and immunologically, to cholera toxin produced by Vibrio cholerae and the
thermolabile toxin of Escherichia coli (LT). Diarrheagenic enterotoxin is a major bacterial
virulence factor in human salmonellosis. The toxin is released into the intestinal lumen and in the
cytoplasm of host cells during the epithelial translocation of endosomal salmonellae and invasion
of the underlying mucosa. Then the enterotoxin binds to intestinal epithelial cells and stimulates
increased intracellular cyclic adenosine monophosphate, which leads to the net secretion of Cl-,
HCO-3, Na+ and water into the intestinal lumen, resulting in diarrhoea (Coetzer et al., 1994;
D’Aoust, 2000; Jay, 2000; Mølbak et al., 2006; Forshell and Wierup, 2006).

Cytotoxin: some serotypes of Salmonella produce a cytotoxic factor that is related to the shiga
neurotoxin. Salmonella cytotoxin is a thermolabile protein, which bound to the outer membrane
of the bacterial cell wall and causes increased permeability of intestinal epithelial cell membranes
in the host tissue. The cytotoxin appears to chelate cations such as Ca++ and Mg++, which appears

10
to cause structural changes in the cells and selective leakage of molecules (D’Aoust, 1991;
Coetzer et al., 1994; Radostits et al., 1994; Jay, 2000; Forshell and Wierup, 2006).

Adhesion pili (fimbriae): fimbriae facilitate the adhesion of bacteria to epithelial cells (Coetzer et
al., 1994; Radostits et al., 1994; Cogan and Humphrey, 2003; Mølbak et al., 2006; Forshell and
Wierup, 2006).

Plasmids: the virulence of the most common serotypes (e.g. S. Dublin, S. Typhimurium and S.
Choleraesuis) that cause disease in livestock is enhanced by serotype-specific plasmids, which
provide these serotypes with the ability to survive in macrophages. Salmonella may carry R-
plasmids that provide resistance to some antimicrobial drugs (Coetzer et al., 1994; Jay, 2000;
D’Aoust, 2000; Chiu et al., 2004; Forshell and Wierup, 2006; Mølbak et al., 2006).

In general, many of the virulence genes of Salmonella enterica are located on pathogenicity
islands of the chromosomes, referred to as ‘Salmonella Pathogenicity Islands’ (SPI). These genes
are believed to be acquired by Salmonella from other bacterial species through horizontal gene
transfer. They include functions such as host cell invasion and intracellular pathogenesis
(Forshell and Wierup, 2006).

2.6. Epidemiology

Salmonella is one of the leading causes of bacterial foodborne disease in industrialized as well as
developing countries even though the incidence seems to vary between countries (Radostits et al.,
1994; D’Aoust, 1997; 2000; Giovannacci et al., 2001; Molla et al., 2003; Chiu et al., 2004;
Vieira-Pinto et al., 2005). The wide variations in the national prevalence of salmonellosis likely
arise from limited scope of studies and lack of coordinated epidemiological surveillance systems,
under-reporting of cases and the presence of other diseases considered to be of high priority
(Radostits et al., 1994; D’Aoust, 2000; 2001; Molla et al., 2003).

The last few decades have witnessed marked increases in the incidence of human Salmonella
infections. Several factors, including tourist travel, international movement of foods and food

11
ingredients, animal feed trade and the importation of infected animal replacement stocks
contribute to the national succession of Salmonella serotypes in human populations and in the
food chain. The prominence of agricultural products as major reservoirs of Salmonella spp. arises
from the ubiquity of the microorganism in the natural environment, the intense on-farm
husbandry practices that favor the spread of salmonellae among reared animals, the use of
untreated sludge to fertilize agricultural land and rendering of animal offals into frequently
contaminated feed proteins (D’Aoust, 1997; 2001; Mølbak et al., 2006).

According to the WHO Global Salm-Surv, during 2000-2002, S. Enteritidis was by far the most
common serotype reported from humans globally. In 2002, it accounted for 65% of all isolates,
followed by S. Typhimurium at 12% and S. Newport at 4%. Among nonhuman isolates, S.
Typhimurium was the most commonly reported serotype in all the 3 years, accounting for 17% of
isolates in 2002 followed by S. Heidelberg (11%) and S. Enteritidis (9%). Salmonella Enteritidis,
S. Typhimurium and S. Typhi were ranked among the 15 most common human serotypes in all
regions of the world throughout the 3-year study period. Salmonella Agona, S. Infantis, S.
Montevideo, S. Saintpaul, S. Hadar, S. Mbandaka, S. Newport, S. Thompson, S. Heidelberg, and
S. Virchow were also widespread. In Africa in 2002, S. Enteritidis and S. Typhimurium were
each reported from approximately one fourth of isolates from humans. Among nonhuman
sources, S. Anatum and S. Enteritidis constituted the largest proportion of isolates (Galanis et al.,
2006; Swaminathan et al., 2006).

2.6.1. Distribution

The primary habitat of Salmonella is the intestinal tract of farm animals, humans, birds, reptiles,
and occasionally insects. Although their primary habitat is the intestinal tract, they have also been
found in spleen, liver, bile, mesenteric and portal lymph nodes, diaphragm, and pillar as well. As
intestinal forms, the organisms are excreted in feces from which they may be transmitted by
insects and other living creatures to a large number of places. Moreover, they may also be found
in polluted water (Radostits et al., 1994; Quinn et al., 1999; Jay, 2000; Vieira-Pinto et al., 2005).

12
Salmonellae can survive for 9 months or more in the environment in sites such as moist soil,
water, fecal particles and animal feeds, especially in blood-and-bone and fish meals (Quinn et al.,
1999; D’Aoust, 2001; Mølbak et al., 2006). The ubiquity of salmonellae in the natural
environment, coupled with the intensive husbandry practices used in the meat, fish and shellfish
industries and the recycling of offal and inedible raw materials into animal feeds, has favored the
continued prominence of this human bacterial pathogen in the global food chain (D’Aoust, 1997).

European Union scientific committee concluded that the food categories that possibly pose the
greatest hazard to public health include: raw meat and some meat products intended to be eaten
raw, raw or undercooked poultry meat products, eggs and products containing raw eggs and
unpasteurised milk and some milk products. Sprouted seeds, and unpasteurised fruit juices are
also of concern (D’Aoust, 1997; Jay, 2000; Chiu et al., 2004; Forshell and Wierup, 2006).
Contamination of milk usually occurs after the milk leaves the cow, even though the organism
can be excreted into the milk during the acute phase of the disease and occasionally by carrier
animals (Radostits et al., 1994). Moreover, salmonellae have been found in commercially
prepared and packaged foods. Among the contaminated foods were cake mixes, cookie dough,
cornbread mixes, coconut meal, salad dressing, mayonnaise, milk and other foods (Jay, 2000;
D’Aoust, 2001; Mølbak et al., 2006).

2.6.2. Host range

Salmonellae have a wide variety of domestic and wild animal hosts. All members of the genus
are considered to be potentially pathogenic, although serotypes may differ widely in their host
range and the pathogenic syndromes that they produce. From the epidemiological point of view,
salmonellae can be classified in to three main groups. The first group (human-adapted) comprises
S. Typhi, S. Paratyphi A and S. Paratyphi C, which infect humans only and are spread directly or
indirectly from person to person. The second group includes serotypes that are host-adapted for
particular species of vertebrates. Included are S. Gallinarum (poultry), S. Dublin (cattle), S.
Abortus-equi (horses), S. Abortus-ovis (sheep) and S. Choleraesuis (swine). Some of these are
also pathogenic for human (especially S. Dublin and S. Choleraesuis). The third group (nonhost-
adapted) contains the majority of the other Salmonella serotypes with no particular host

13
preference that infect both humans and other animals (WHO, 1988; Jay, 2000; Forshell and
Wierup, 2006).

2.6.3. Source of infection and transmission

Salmonellae are mainly transmitted by the fecal-oral route. They are carried asymptomatically in
the intestines or gall bladder of many animals, and are continuously or intermittently shed in the
feces (Radostits et al., 1994; OIE, 2005; Forshell and Wierup, 2006). They can also be carried
latently in the mesenteric lymph nodes or tonsils; these bacteria are not shed, but can become
reactivated after stress or immunosuppression. Fomites and mechanical vectors (insects) can
spread Salmonella (OIE, 2005).

Vertical transmission occurs in birds, with contamination of the vitelline membrane, albumen and
possibly the yolk of eggs. Salmonella spp. can also be transmitted in utero in mammals (OIE,
2005).

Animals can become infected from contaminated feed (including pastures), drinking water or
close contact with an infected animal (including humans). Birds and rodents can spread
Salmonella to livestock. Carnivores are also infected through meat, eggs and other animal
products that are not thoroughly cooked. Cats sometimes acquire Salmonella Typhimurium after
feeding on infected birds or spending time near bird feeders (OIE, 2005; Forshell and Wierup,
2006).

People are often infected when they eat contaminated foods of animal origin such as meat or eggs
(OIE, 2005). Ingesting organisms in animal feces can also infect them, either directly or in
contaminated food or water. Directly transmitted human infections are most often acquired from
the feces of reptiles, chicks and ducklings. Livestock, dogs, cats, adult poultry and cage birds can
also be involved (OIE, 2005).

The trade of live animals within and between countries spreads Salmonella. Moreover, trade in
contaminated animal feed products has also significantly contributed to the spread of Salmonella

14
and several large outbreaks in humans have been traced back to contaminated animal feed.
Salmonella is additionally spread between countries by humans as a result of foodborne
infections acquired abroad. The overall importance of these routes of transmission may reflect the
prevalence of Salmonella contamination of food (including food of animal origin) in a particular
country (Forshell and Wierup, 2006).

2.6.4. Pathogenesis

After entering the small bowel, salmonellae must traverse the intestinal mucus layer before
encountering and adhering to cells of the intestinal epithelium. Salmonellae express several
fimbriae that contribute to their ability to adhere to intestinal epithelial cells (Radostits et al.,
1994; Quinn et al., 1999; Ohl and Miller, 2001). Once across the intestinal epithelium,
salmonellae encounter another obstacle of innate immunity, the submucosal macrophage.
Salmonella serotypes that cause systemic infection enter macrophages, again apparently by
induced macropinocytosis, and subsequently activate virulence mechanisms that allow evasion of
the microbiocidal functions of the phagocyte, permitting survival and replication in the
intracellular environment. Migration of infected phagocytes to other organs of the
reticuloendothelial system probably facilitates dissemination of bacteria in the host (Ohl and
Miller, 2001).

The invasive strains that produce septicemia are able to escape destruction by the host and to
multiply within the macrophages of the liver and spleen as well as intravascularly. The invasive
abilities of some strains of S. Typhimurium are increased by the presence of genes carried on a
plasmid. O-repeat units of the lipopolysaccharide prevent destruction within the blood stream. It
is thought that they may mask determinants on the bacterial cell surface that would normally bind
complement and activate it by means of the alternate pathway. This would reduce chances of
chemotaxis, opsonization and phagocytosis. As a result there will be multiplication of the
organisms in the body that subsequently leads to a severe endotoxaemia. Furthermore, these
invasive salmonellae secrete siderophores, which remove iron from iron-binding proteins of the
host (Radostits et al., 1994; Quinn et al., 1999).

15
2.6.5. Carrier states

Because salmonellae are facultatively intracellular organisms that survive in the phagolysome of
macrophages, they can evade the bactericidal effects of antibody and complement. Thus,
persistence of infection in animals and in the environment is an important epidemiological
feature of salmonellosis. When an animal is infected with S. Dublin it may become a clinical case
or an active carrier, passing organisms constantly or intermittently in the feces. It may also
become a latent carrier with infection persisting in lymph nodes or tonsils but no salmonellae in
the feces, or even a passive carrier, which is constantly picking up infection from pasture, but is
not invaded so that when is removed from the environment the infection disappears. The
importance of the latent carriers is that they become active carriers or even clinical cases under
stressful conditions. Although all infected adults become carriers it is rarely for any length of
time, and calves rarely become carriers. In sheep and cattle the carrier state may persist for as
long as 10 weeks and in horses up to 4 months (Radostits et al., 1994).

The intermittent fecal shedding that may follow the acute phase of human salmonellosis may be
of short duration (convalescent carrier) or may persist for one or more years (chronic carrier) if
the condition is not effectively treated. Carrier states are of concern to the food manufacturing
and food service industries because of the perceived risk of cross contamination of foods by
infected food handlers and potentiation of foodborne disease outbreaks. Although temporary
exclusion of food handlers with non-typhoid diarrheal illness from work in sensitive areas is
common practice, there is lack of unanimity on the need to exclude asymptomatic workers from
their food-related duties and need to subject them to follow-up stool testing (D’Aoust, 1991).

2.7. Clinical feature

2.7.1. Salmonella infections in humans

Infections with non-typhoid Salmonella serotypes most often result in self-limited acute
gastroenteritis that does not require antimicrobial therapy (D’Aoust, 1991; Chiu et al., 2004).
Nevertheless, approximately 5% of individuals with gastrointestinal illness caused by non-

16
typhoid Salmonella serotypes develop bacteremia (Chiu et al., 2004). Non-typhoid salmonellosis
in humans is usually manifested as a localized enterocolitis. The incubation period ranges from
5h to seven days, but clinical signs usually begin 12 h to 36 h after ingestion of a contaminated
food. Shorter incubation periods are generally associated with either higher doses of the pathogen
or highly susceptible people (Forshell and Wierup, 2006).

Clinical signs include abdominal pain, nausea, watery diarrhoea with occasional mucus and
traces of blood, mild fever and chills. The diarrhoea varies from a few thin vegetable-soups like
stools to massive evacuations with accompanying dehydration. Vomiting, prostration, anorexia,
headache and malaise may also occur. The syndrome usually lasts for two to seven days.
Susceptibility to infection is highest in infants, elderly people and in immuno-compromised
hosts. A fatal outcome is rare. With the exception of S. Choleraesuis and S. Dublin, which show a
definite predilection for systemic spread, non-typhoid salmonellosis is generally restricted to the
intestinal tract. The excreta of infected patients contain large numbers of Salmonella spp. at the
onset of illness. Those numbers decrease with the passing of time. Some patients become
carriers, and some are still excreting Salmonella spp. after three months (D’Aoust, 1991; Chiu et
al., 2004; Forshell and Wierup, 2006).

In contrast to non-typhoid salmonellosis, human infections of S. Typhi (enteric fever) and

paratyphoid strains are more severe and characteristically involve a systemic spread of the
pathogen and invasion of extra intestinal tissues. Following a period of incubation ranging from 8
to 28 days, non-specific symptoms of fever, chills and abdominal pain with underlying
bacteremia gradually build up in intensity within the first few week of illness. Appearance of
watery diarrhea (or constipation) with persistent abdominal pain, prostration and cutaneous rose
spots that rarely affect the extremities are commonly encountered in the second week of disease.
The course of the disease in the next 2 weeks may involve gradual resolution of symptoms with
possible relapses 7 to14 days after completion of antibiotic therapy or more serious conditions
such as intestinal perforation, osteomyelities and meningitis. The fatality case ratio of S. Typhi
infection ranges from 1% in developed countries to more than 10% in third world countries
(D’Aoust, 1991).

17
2.7.2. Salmonella infections in animals

Salmonellae are often carried asymptomatically (OIE, 2005; Forshell and Wierup, 2006). Clinical
disease usually appears when animals are stressed by factors such as transportation, crowding,
food deprivation, weaning, parturition, a concurrent viral or parasitic disease, sudden change of
feed, or overfeeding following a fast. Salmonellosis is common in horses after major surgery. In
some cases, oral antibiotics may also precipitate disease. Although salmonellosis can be seen in
all domestic animals, pregnant, lactating or young mammals and birds are the most susceptible
(OIE, 2005).

The clinical signs vary with the infecting dose, health of the host, Salmonella serotypes and other
factors (OIE, 2005). Host adapted serotypes primarily cause abortions or severe gastroenteritis in
their animal hosts. In food animals, a group of more frequently isolated serotypes, such as S.
Typhimurium, S. Enteritidis, S. Hadar and S. Infantis, manifest themselves clinically through per-
acute septicemia, acute enteritis or chronic enteritis. In the subclinical form of the disease, the
animal may either have a latent infection or become a temporary or persistent carrier. The
remaining, less frequently isolated serotypes can colonize animals, usually without significant
clinical signs, but they are all considered capable of causing gastrointestinal infection of varying
severity in humans (Forshell and Wierup, 2006).

2.7.3. Ovine and caprine salmonellosis

The only recognized form of the disease in sheep is acute enteritis on a flock scale. However, in
the early stages of the outbreak there may be some cases of the septicemic form. After
experimental infection of sheep with S. Dublin, fever and diarrhea are followed in pregnant ewes
by abortion. Abortion is also common in the naturally occurring disease and has come to exceed
S. Abortusovis as a cause of abortion in sheep in the United Kingdom. Some ewes die after
abortion and many of lambs born alive die subsequently. Fever and diarrhea followed by abortion
have also been produced experimentally in sheep by administration of S. Dublin (Radostits et al.,
1994).

18
In goats, naturally occurring cases are not reported often. Salmonella Dublin is the usual
pathogen in those countries where it is a resident, but S. Typhimurium is also recorded as a cause
(Radostits et al., 1994).

Experimental S. Typhimurium infection in 10 Indian goats revealed that all the animals except
one showed dyspnoea, anorexia, pyrexia and progressive weakness. A rise in body temperature of
almost 20C was observed at 3 days post inoculation, which gradually decreased. Diarrhoea was
observed in one animal at 2 days post inoculation and in two others at 3 days post inoculation.
The remaining animals developed diarrhoea from 4 days post inoculation onwards. Diarrhoea did
not continue beyond 7 days post inoculation in any of the animals. The animals lost body weight
with the progression of infection. A general improvement in the condition of animals coincided
with the decrease in temperature and diarrhoea. One animal did not suffer clinically, though it
lost a little weight (Sharma et al., 2001).

2.8. Detection

Salmonella can be isolated either from tissues collected aseptically at necropsy or from feces,
rectal swabs, environmental samples, food products and feedstuffs. When infection of the
reproductive organs, abortion or conceptus occurs, it is necessary to culture fetal stomach
contents, placenta and vaginal swabs and, in the case of poultry, embryonated eggs. Individual
samples for bacteriological tests should be collected as aseptically as possible by following the
respective standards. Moreover, precautions should be taken to avoid cross contamination of
samples during transit and at the laboratory. Packages should also be kept cool and accompanied
by adequate information (OIE, 2005).

Conventional cultural methods for the detection of foodborne Salmonella spp. generally consist
of five distinct and successive steps: pre-enrichment in nonselective media, selective enrichment
in broth media, plating on differential agar, biochemical screening and serological confirmation
(D’Aoust, 2000; 2001).

19
2.8.1. Pre-enrichment in nonselective liquid medium

The number of Salmonella in feces from asymptomatic animals, environmental samples, animal
feed and food is usually low, and are often accompanied by considerably larger numbers of other
Enterobacteriaceae or other families. Therefore, it is necessary to use pre-enrichment media to
assist the isolation. Furthermore, pre-enrichment is necessary to permit the detection of
sublethally injured Salmonella (ISO 6579, 2002).

Pre-enrichment in a nonselective broth medium for 18-24h at 35-37oC allows the small numbers
of salmonellae, which may otherwise be killed by the toxic effect of enrichment media, to
multiply, or it may help to resuscitate salmonellae that have been sublethally damaged, e.g. by
freezing, heating, exposure to biocides or desiccation. Pre-enrichment may not be the best
method for isolating less vigorous Salmonella strains, such as the host-adapted strains, from feces
because of overgrowth by competing organisms during nonselective pre-enrichment (D’Aoust,
2000; 2001). The use of short (6-8h) pre-enrichment for greater method brevity is not
recommended because it fails to provide salmonellae with sufficient time to adapt to its new
environment, repair cellular damage and actively grow to high numbers (D’Aoust, 2001).

The traditional pre-enrichment media include nonfat dry milk with added brilliant green dye for
the pre-enrichment of cocoa and chocolate products, brilliant green water for milk powder,
tripticase soy broth supplemented with potassium sulfite to neutralize spice-dependent
bacteriostasis and buffered peptone water, nutrient or lactose broths for other foods and
agricultural products, however, buffered peptone water is the most commonly used pre-
enrichment medium in the recovery of Salmonella in foods (D’Aoust, 2001).

2.8.2. Enrichment in selective liquid media

Enrichment media are liquid or semi-solid agar media that contain additives that selectively
permit salmonellae to grow while inhibiting the growth of other bacteria (D’Aoust, 2000; 2001).
Some, however, are also relatively toxic to certain serotypes of Salmonella, e.g. selenite inhibits
S. Choleraesuis, and brilliant green is toxic to many strains of S. Dublin. Elevated temperatures

20
have also been used to increase the selectivity of enrichment medium, and a temperature of 43°C
is used in some laboratories, although this may be inhibitory with some media, e.g. tetrathionate
and Rappaport-Vassiliadis at 43°C inhibit temperature-sensitive strains, especially S. Dublin and
41.5°C is now recommended for incubation of Rappaport-Vassiliadis broth. Selective motility
enrichment may also be used to increase the sensitivity of Salmonella isolation and semi-solid
enrichment media, e.g. modified semi-solid Rappaport-Vassiliadis or Diagnostic Semi-Solid
Salmonella medium (DIASALM), may provide greater sensitivity. The formulation of the
medium, temperature and duration of incubation, and the volume of the samples used to inoculate
the medium, may all serve to improve the isolation rate, and these variables should always be
taken into account. Examples of selective enrichment medium are sodium tetrathionate, as in
Muller-Kauffman broth, selenite cysteine (SC), tetrathionate brilliant green (TBG) broth and
Rappaport-Vassiliadis (RV) broths, or semi-solid Rappaport-Vassiliadis medium. Additions such
as Ferrioxamine E may be added to selective media to enhance isolation of Salmonella from iron
or nutrient-limited samples such as eggs, water or soil (D’Aoust, 2001; ISO 6579, 2002).

The concurrent use of two enrichment media where one medium is incubated at 41-43oC and the
other at a more permissive temperature (35-37oC) maximizes detection of the target
microorganism. These conditions accommodate fastidious Salmonella spp. that grow poorly or
are inhibited at elevated temperatures and whose growth is hampered in highly selective
enrichment media such as TBG and RV. Numerous studies on the reliability of enrichment
conditions support the following decreasing order of effectiveness: TBG43 ≥ RV43 > TBG35 >
SC35 (D’Aoust, 2000; 2001).

2.8.3. Plating out and identification

Enrichment cultures are plated onto selective agar media for the presumptive identification of
Salmonella colonies on the basis of discriminating biochemical reactions. Standard plating media
include brilliant green agar (BGA), brilliant green sulfa (BGS), Bismuth sulfite agar (BSA),
xylose-lysine-desoxycholate (XLD) and Hektoen enteric (Hek) agars that report on acid
production from lactose and/or sucrose utilization through determinant colour changes in the
media. Salmonella spp that typically produce hydrogen sulfide appear as charcoal black colonies

21
with or without a black halo that produce a metallic sheen under reflected light. The Rambach
and SM-ID plating media produce discriminating colour reaction in the presence of isolates that
are -galactosidase positive and that produce acid from propylene glycol (Rambach) and
glucuronate (SM-ID). Plating media generally yield suspect colonies within 18-24 h incubation at
35-37oC, except BSA, which may require 48 h incubation for the development of presumptive
Salmonella colonies. Comparative studies support the following ranking in decreasing order of
effectiveness: BSA > BGS > BGA  Rambach = SM-ID > XLD > Hek (D’Aoust, 2000; 2001;
ISO 6579, 2002).

2.8.4. Confirmation

For confirmation, at least five presumptive (typical or suspect) Salmonella colonies will be
selected from every selective plating media. If the suspected colonies on each plate are fewer
than five, all the colonies will be selected. The selected colonies will be streaked onto the surface
of pre-dried nutrient agar plates, in a manner that will allow well-isolated colonies to develop.
Then the inoculated plates will be incubated at 37 oC  1oC for 24h  3h. The pure cultures on
nutrient agar will be used for biochemical and serological confirmation (ISO 6579, 2002).

Biochemical confirmation

The use of lactose and/or sucrose, the production of H2S and presence of lysine decarboxylase
and urease are key determinants in the biochemical screening of presumptive Salmonella isolates
(D’Aoust, 2000; 2001; ISO 6579, 2002).

Salmonellae are facultatively anaerobic and, with few exceptions, all produce gas from
fermentable sugars. In culture, nitrates are reduced to nitrites, and hydrogen sulfide is produced.
Glucose and maltose are fermented, dulcitol and inositol are variably used, and sucrose is not
fermented (Coetzer et al., 1994).

22
Serological confirmation

The three kinds of surface antigens, O antigens, H antigens, and Vi antigens, determine the
reactions of the organisms to specific antisera. The detection of the presence of Salmonella O, Vi
and H-antigens is done by the slide agglutination with the appropriate sera, from pure colonies
after auto-agglutinable stains have been eliminated. This method relies on the antibody/antigen
reaction between a test culture and commercially prepared antiserum (Popoff and Le Minor,
2001; ISO 6579, 2002).

2.8.5. Typing

Because of the importance of Salmonella in foodborne disease, numerous typing methodologies

have been developed and have been used to trace salmonellosis outbreaks to the contaminated
source and to delineate the epidemiology of Salmonella infections (Kotetishvili et al., 2002;
Botteldoorn et al., 2004). Some of the typing techniques include serotyping and phage typing
(Kotetishvili et al., 2002; Demczuk et al., 2003; Botteldoorn et al., 2004). These techniques are
useful for defining relationships between strains (Botteldoorn et al., 2004).

Serotyping

Serotyping is based on the O and H antigens using a slide agglutination test (Coetzer et al., 1994;
Quinn et al., 1999; Mortimer et al., 2004). Most serotypes exhibit diphasic flagellar antigen
expression by alternately expressing two genes, fliC (phase 1) and fljB (phase 2) which encode
flagellins of different antigenicity. Salmonella serotyping methods recognise 63 distinct phase 1
flagellar antigenic factors and 37 phase 2 flagellar antigenic factors although the latter are not
always present (Mortimer et al., 2004).

Bacterial growth for serotyping should be taken from a triple sugar iron (TSI) agar slant or from
nutrient agar as culture from selective media is often unsuitable for typing. Then a loopfull of
culture of the Salmonella to be serotyped should be suspended in a drop of saline on a
microscope slide and examined for autoagglutination. This can occur with rough strains and will

23
invalidate the serotyping. Smooth-rough dissociation occurs after subculture and most frequently
from media containing carbohydrates (Coetzer et al., 1994; Quinn et al., 1999). Smooth
Salmonella to be serotyped is emulsified in a drop of 0.85% saline on a clean microscope slide. A
drop of antiserum is added to and mixed well with the Salmonella suspension. The slide is rocked
gently for about 30 seconds and the antigen-antibody mixture examined for agglutination. The
Salmonella is first tested against antisera to the O antigens and then the H antigen (Quinn et al.,
1999).

Phage typing

Phage typing is based on the specificity of a given phage for its host bacterium, and this
relationship allows one to use known phages to identify their specific hosts (Jay, 2000).
Therefore, phage typing of Salmonella isolates is based on the sensitivity of a particular isolates
to a series of bacteriophages at appropriate dilutions. This can be useful to determine whether
isolates, which come from different places at different times, are similar or different in their
reactions with specific sets of phages used for typing (Quinn et al., 1999).

In more detail, phage typing is based on specific bacteriophage reacting with specific receptors
on bacterial cell walls or pili so that they can then invade and lyse the bacteria. The test isolate is
exposed to a series of different phage and the pattern of lysis is observed and compared with
other patterns recorded previously. Banks of phage have been developed for a number of
serotypes including S. Typhimurium and S. Enteritidis. Very dry agar plates containing a rich
nutrient source are flooded with a liquid culture of the bacterial isolate. The liquid culture is
removed; the culture film allowed to dry and a set of phage suspensions are inoculated onto the
surface of the plate by means of a multipoint inoculator. Phage spots are left to dry, and plates are
incubated inverted overnight at 37oC. The next day, phage lysis reactions are recorded, and
compared to a set of standards developed by the Public Health Laboratory Service, Colindale,
England guidelines (Schmieger, 1999).

24
2.9. Treatment

Antimicrobial agents should not be used routinely to treat uncomplicated non-typhoid Salmonella
gastroenteritis. However, antimicrobial therapy is essential in the treatment of serotype
Choleraesuis infection, in view of the high rate of extra intestinal infections caused by this
organism. Because of the increasing prevalence of resistance to conventional antimicrobial agents
such as ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole, empirical therapy for
life-threatening bacteremia or focal infection suspected to be caused by non-typhoid Salmonella
should include a broad-spectrum cephalosporin or a fluoroquinolone until susceptibility patterns
are known. It is also important to search for endovascular abnormalities by using imaging
techniques in older patients with or without evidence of atherosclerosis. Although there is no
consensus on the optimal duration of postoperative antibiotic therapy for endovascular infections
caused by Salmonella, most investigators still recommend a minimum of 6 weeks. The duration
of therapy for other extra intestinal infections should be considered based on the site of infection.
In general, 10 to 14 days for bacteremia, 4 to 6 weeks for osteomyelitis, and 4 weeks for
meningitis are suggested. Prolonged therapy may be needed in immunocompromised patients.
Many consultants would prescribe some months of suppressive therapy, following parenteral
treatment, especially for human immunodeficiency virus-infected patients. For patients with a
focal suppurative process, surgical drainage should be undertaken as soon as possible in addition
to antibiotic treatment for the best chance of achieving a cure (Chiu et al., 2004).

2.10. Prevention and control

WHO had formulated three lines of defense against Salmonella, which still comprise valid
strategic approaches to risk mitigation (WHO, 1988; Forshell and Wierup, 2006).

The first approach focuses on controlling Salmonella in the food-producing animal (pre-harvest
control). Pre-harvest control of Salmonella at the farm level has long been considered an
important part of pathogen reduction schemes, because traditional meat inspection cannot control
Salmonella-contaminated carcasses. Therefore, it is fundamental that monitoring programmes
should be established to identify Salmonella-infected herds and animals and that efforts are made

25
to find and control the sources of infection and prevent further spread. The ultimate objective is
to produce Salmonella-free animals. According to several studies, improving hygiene and
husbandry management programmes, including feed control are of major importance in aiding
animals to withstand exposure to Salmonella, and to minimize the possible subsequent spread of
the agent on the farm (WHO, 1988; Forshell and Wierup, 2006).

The second approach involves improving hygiene during the slaughter and further processing of
the meat (harvest control). Appropriate slaughter and dressing procedures need to be developed
and strictly followed to achieve any reduction in Salmonella contamination. Such contamination
can be limited using the hazard analysis critical control point (HACCP) concept (WHO, 1988;
Forshell and Wierup, 2006).

In general, all animals intended for slaughter should pass ante mortem veterinary inspection.
Clinically healthy carriers of salmonellae cannot be detected by visual inspection, but they could
play an important role in the spread of Salmonella contamination during slaughtering and
processing. These animals may harbour salmonellae in intestinal and other lymph nodes, on the
skin and in other parts as a result the microorganisms may spread to other animals during
transport to the abattoir, in lairages and during slaughter. The hooves, hides, fleece and hair of
slaughtered animals can be contaminated with salmonellae, so that cross contamination of the
carcasses and the meat could be resulted during the removal of hooves and skin. Therefore,
proper hygiene during skinning process can eliminate this contamination (WHO, 1988).

One of the most important precautions to be taken in the evisceration area is to prevent
contamination of the carcass by intestinal contents. Hands and knives could become
contaminated with the gut contents, as well as from cutting lymph nodes as part of veterinary
inspection. Knives should be decontaminated in hot water (80oC for at least 2 minutes) and hands
should be washed regularly. If the carcasses are not intended for immediate human consumption,
they should be chilled and inspected. For the chilling process to suppress the proliferation of
bacteria, including salmonellae, the temperature should be below 4oC. To achieve quick and
effective chilling, whole carcasses or parts of carcasses must not touch one another or the walls

26
of the chill rooms. In addition, carcasses emerging from the slaughter line and carcasses already
chilled should not be put in the same chill room (WHO, 1988).

The third approach targets the final preparation of food by educating the food industry and
consumers about good hygiene practices (post-harvest control) (WHO, 1988; Forshell and
Wierup, 2006). During processing, raw food of animal origin undergoes different treatments
intended to inhibit the multiplication of microorganisms or even to eliminate them fully. The
number of salmonellae in raw foods can be reduced by treatments with heat, irradiation or other
methods known to decrease the microbial load. It has been stressed that different food treatments
during processing should not be used to cover up deficient manufacturing practices and the lack
of basic food hygiene. Factors such as the cross contamination of final products by raw materials,
failure during heat treatments or other decontamination processing, inadequate refrigeration and
poor personal hygiene of food handlers are well documented and will contribute to outbreaks of
foodborne salmonellosis (WHO, 1988).

Generally the successful prevention of foodborne salmonellosis originating from animal

production must involve all the three lines of defense (WHO, 1988; Forshell and Wierup, 2006).

2.10.1. Vaccination

Different investigations and practical experiences have provided evidence that vaccination play a
role in the prevention and control of animal salmonellosis. Nowadays, three major types of
Salmonella vaccines are widely used: parenterally administered killed vaccine, parenterally
administered, rough avirulent live vaccines and orally or parenterally administered, genetically
altered, auxotrophic live vaccines (WHO, 1988; OIE, 2005)

Parenterally administered killed vaccines (heat or formalin inactivation, possibly with the
addition of potent adjuvants such as alhydrogel) are commercially produced against S. Dublin, S.
Typhimurium, S. Abortusovis and S. Abortusequi. In addition, autogenous killed vaccines are
frequently prepared for outbreaks of salmonellosis involving these or other serotypes.
Parenterally applied live vaccines have also been used in a number of countries; these include the

27
semi-rough strains, such as 9R for fowl typhoid and HWS51 for S. Dublin infections. Live
vaccines have also been prepared from genetically defective (auxotrophic) mutants, selected for
their requirement for metabolites that are absent or present in insufficient quantities in the
immunized animal. For example vaccines of purine-requiring S. Dublin, S. Choleraesuis and S.
Typhimurium and aromatic amino acid-requiring S. Typhimurium and S. Dublin are available
commercially (WHO, 1988; OIE, 2005).

In general, live vaccines stimulate a much greater cell-mediated immune response than do
inactivated vaccines, which produce significant humoral antibody titres when administered
parenterally. Oral immunization with live or inactivated vaccines stimulates local intestinal
immunity and has been shown to be effective in decreasing the excretion of salmonellae by
infected animals. Cross-protection against other Salmonella serotypes is not observed following
immunization with inactivated vaccines. However, an orally administered, live vaccine against S.
Typhimurium has been shown to protect calves against challenge by S. Dublin (WHO, 1988).

The development of human parenteral heat-inactivated, phenol-preserved whole cell typhoid and
paratyphoid vaccines at the close of the nineteenth century contributed significantly to the
reduction of enteric fever in endemic areas. These vaccines confer 80% to 90% protection for 3 –
7 years and are associated with low to moderate levels of adverse reactions. An oral typhoid
vaccine manufactured by the Swiss Serum and Vaccine Institute (Berne) under the trade name
Vivotif Berna provides 60% to 80% protection for up to 7 years with booster doses recommended
at 3-year intervals. The vaccine S. Typhi Ty21a strain was originally derived from random
mutagenesis with nitrosoguanidine and ultraviolet light irradiation. The mutant lacks the Vi
surface antigen and contains a mutation in the galactose epimerase locus, which prevent the
synthesis of complete LPS. The latter condition results in a rough variant that is highly
susceptible to lysis by the host complement system. Another marketed typhoid vaccine uses the
Vi capsular polysaccharide as immunogen. This injectable preparation is distributed by the
Pasteur Merieux Serums et Vaccines (Lyon, France) under the trade name Typhim Vi. The
clinical success of the oral Ty21a and the injectable Vi vaccine has not dampened research
interests in the development of novel typhoid and paratyphoid vaccines providing high levels of

28
immunogenicity, biological safety and storage stability and engendering few side effects
(D’Aoust, 2000).

2.10.2. Competitive exclusion

The use of competitive exclusion, in which the normal intestinal flora protects the host against
invading pathogens, is a valuable part of Salmonella control in poultry farming (Forshell and
Wierup, 2006). Susceptibility to Salmonella infection in poultry can be substantially reduced by
spray or oral treatment prior to exposure (ideally in the hatchery) with an anaerobic culture of
adult bird cecal microflora that inhibits colonization by Salmonella. This application is known by
the name of the scientist who first described it – the Nurmi concept (WHO, 1988). Competitive
exclusion cultures have been used and tested in various countries and positive results from the
use of competitive exclusion have also been reported in pigs (Forshell and Wierup, 2006).

2.11. Antimicrobial resistance

Salmonella display high natural susceptibility levels to the most commonly used antibacterial
agents (Cabrera et al., 2004). Conventional antimicrobial agents, such as ampicillin,
chloramphenicol, and trimethoprim-sulfamethoxazole, had been the drugs of choice in the
treatment of salmonellosis before the 1980s (Chiu et al., 2004). However, the occurrences of
isolated Salmonella strains showing resistance to one or more antibacterial agent have steadily
increased world wide, probably due to continuous antibiotic pressure (Cabrera et al., 2004; Chiu
et al., 2004; Schroeter et al., 2004; Agustı´n et al., 2005). This is an important public health
problem that may be related to therapeutic failure. Moreover, the problem is especially relevant
in developing areas, where the lack of economic resources does not allow a wide antibacterial
armamentarium (Cabrera et al., 2004). Extended-spectrum cephalosporins and fluoroquinolones
have been suggested as appropriate alternative agents in the treatment of infections caused by
such multidrug-resistant Salmonella serotypes; however, since 1991, outbreaks or cases of
infections caused by Salmonella serotypes resistant to extended-spectrum cephalosporins or
fluoroquinolones have been increasingly reported (Chiu et al., 2004).

29
The emergence of antimicrobial resistance in Salmonella is complicated because the use of
antibiotics for therapeutic purposes in veterinary medicine and as growth promoters in animal
feed, thus presenting a potential risk to public health from zoonotic infections (Chiu et al., 2004;
Schroeter et al., 2004; Agustı´n et al., 2005). In addition, pet animals such as frogs and turtles
and their water environment were shown to carry multidrug-resistant Salmonella strains, which
could subsequently cause infections in humans. Therefore, to curb the resistance problem in
Salmonella, it has been suggested that inappropriate use of antimicrobial agents in food animals
should be prohibited (Chiu et al., 2004).

2.12. Zoonotic implication

National and global epidemiological registries continue to highlight the importance of Salmonella
spp. as one of the leading cause of foodborne bacterial zoonotic disease in humans (WHO, 1988;
D’Aoust, 2000; David et al., 2001; Giovannacci et al., 2001; Hjartardóttir et al., 2002; Molla et
al., 2003; Mølbak et al., 2006). It is noteworthy that the problem of human salmonellosis from
the consumption of contaminated foods generally remains on the increase worldwide (D’Aoust,
2000). The cumulative losses of salmonellosis are due to productivity losses and absenteeism,
pain and suffering, lost leisure time and medical costs. The costs of food safety regulatory
programs and costs to the food industry for product recalls and plant closures due to foodborne
salmonellosis outbreaks would increase the size of the estimated lose. Moreover, the most serious
risk is that the transmitted bacteria will have acquired resistance to specific antibiotics because
the animals from which they originate have been treated with the particular antibiotics repeatedly
over long period (Radostits et al., 1994).

Various clinical forms of Salmonella such as gastroenteritis, bacteremia and other systemic
abnormalities can occur in veterinarians working with Salmonella-infected animals (Radostits et
al., 1994). Moreover, cutaneous salmonellosis has been reported in veterinarians attending to
infected cattle at the time of parturition. The disease was characterized by pustular dermatitis
from which S. Virchow and S. Dublin were isolated (Visser, 1991). Similarly, pustular dermatitis
caused by S. Stanley developed on the arm of a veterinary surgeon after the delivery of a dead

30
bovine calf. Unlike the previous reports, the veterinarian did not develop any systemic symptoms
and made a full recovery (Lazarus et al., 2007).

2.13. Economic importance

Salmonellosis is a significant cause of economic loss in farm animals because of the costs of
clinical disease, which include deaths, diagnosis and treatment of clinical cases, diagnostic
laboratory costs, the cost of cleaning and disinfections, and the cost of control and prevention. In
addition, when the disease is diagnosed in a herd it can create considerable apprehension in the
producer because of its difficulty in identifying infected animals. An estimation of the economic
impact of an outbreak of S. Dublin infection in a calf-rearing unit indicated that the cost of the
disease represented a substantial proportion of the gross margin of rearing calves. The losses
incurred by livestock producers include reduced feed efficiency and reduced weight gains or
deaths because of salmonellosis (Radostits et al., 1994).

2.14. Salmonella in Ethiopia

A number of studies conducted by different individuals on various slaughtered food animals such
as avian, swine, bovine, camel, ovine and caprine species showed the presence of a number of
Salmonella serotypes (Table 2) indicating the potential of carcass contamination during
slaughtering operations.

31
Table 2: Salmonella serotypes isolated from samples of food animals in Ethiopia
Species Samples No of Animals Serotypes isolated References

Examined Positive
Bovine  280 6 S. Dublin, S. Bredeny Pegram et al.
MLN
(1981)
Porcine MLN 160 1 S. Saintpaul "
(pooled)
Avian Organs - 77 S. Galinarum "
Bovine Feces 235 5 S. Dublin, S. Muenchen Nyeleti et al.
(2000)
" MLN 235 9 S. Anatum, S. Dublin "
(pooled)
" AM 235 23 S. Anatum, S. Dublin "
" DM 235 28 S. Anatum, S. Dublin "
Bovine Feces 323 2 S. Mishmarhaemek Alemayehu et al.
(pooled) (2003)
" MLN 323 3 S. Typhimurium, S. Enteritidis "
" AM 323 9 S. Mishmarhaemek, S. Dublin, "
S. Typhimurium, S. Guildford
" DM 323 10 S. Dublin, S. Mishmarhaemek, "
S. Typhimurium, S. Guildford,
S. Enteritidis
Camel Feces 119 18 S. Saintpaul, S. Muenchen, Molla et al.
S. Kottbus, S. Havana, (2004)
S. Heidelberg, S. Derby,
S. Enteritidis, S. Anatum
" MLN 119 19 S. Saintpaul, S. Braenderup, "
S. Muenchen, S. Typhimurium var.
Copenhagen, S. Kottbus,
S. Hadar, S. Bovismorbificanse,
S. Butantan, S. Infantis
" Liver 119 14 S. Saintpaul, S. Braenderup, "
S. Typhimurium var. Copenhagen, S.
Hadar, S. Kottbu
" Spleen 119 17 S. Saintpaul, S. Typhimurium var. "
Copenhagen, S. Heidelberg,
S. Braenderup, S.Infantis, S.Kottbus
S. Anatum, S. Butantan, S. Havana
" AM 119 25 S. Saintpaul, S. Typhimurium, "
S.Braenderup, S. Muenchen,
S. Kottbus, S. Hadar, S. Havana
" DM 119 23 S. Saintpaul, S. Braenderup, "
S. Havana, S. Infantis,
S. Muenchen, S. Kottbus

32
Species Samples No of Animals Serotypes isolated References
Examined Positive
Ovine Feces 104 5 S. Typhimurium, S. Enteritidis, Wassie (2004)
S. Reading, S. Heidelberg
" MLN 104 8 S. Typhimurium var. Copenhagen, "
S.Typhimurium, S.Reading, S.Give,
S. Heidelberg, S. Niederoderwitz
" Liver 104 2 S. Typhimurium, S. Give "
" AM 104 2 S. Typhimurium, S. Heidelberg "
" Spleen 104 1 S. Typhimurium, "
Caprine Feces 100 2 S. Typhimurium, S. Poona "
" MLN 100 2 S. Poona "
Ovine Feces 47 1 S. Kottbus Woldemariam et
al. (2005)
" Liver 47 2 S. Infantis "
" AM 47 5 S. Infantis, S. Braenderup "
" DM 47 2 S. Kingbawa, S. Infantis "
Caprine Feces 60 2 S. Infantis, S. Kottbus "
" MLN 60 7 S. Zanzibar, S. Infantis, "
S. Anatum, S. Butantan,
S. Typhimurium, S. Kingbawa
" Spleen 60 2 S. Infantis, S. Butantan "
" Liver 60 3 S. Butantan, S. Braenderup "
" AM 60 2 S. Butantan, S. Infantis "
" DM 60 7 S. Hadar, S. Infantis, S. Butantan "
Bovine HS 100 31 S. Anatum, S. Newport, Berhanu (2006)
S. Bredeney, S. Eastbourne,
" RC 100 19 S. Uganda, S. Anatum, S. Newport "
" CC 100 6 S. Typhymurium, S. Anatum "
S. Newport
" MLN 100 8 S. Reading, S. Anatum "
S. Newport
" CS 100 2 S. Eastbourne, S. Urbana "
Porcine MLN 101 42 S. Hadar, S. Kentuckey, Molla et al.
S. Anatum, S. Havana, S. Leoben, (2006)
S. Enteritidis, S. Blockley,
S. Kiambu, S. Livingstone
" Tongue 101 22 S. Hadar, S. Kentuckey, S. Anatum, "
S. Blockley, S. Leoben, S. Havana, S.
Gaminara, S. Eastbourne
" CC 101 17 S. Hadar, S. Kentuckey, "
S. Blockley, S. Leoben,
S. Gaminara, S. Anatum

33
Species Samples No of Animals Serotypes isolated References
Examined Positive
" Liver 99 11 S. Hadar, S. Uganda, S. Anatum, "
S. Kentuckey, S. Havana,
S. Blockley,
" Muscle 99 2 S. Newport, S. Kentuckey "
" MLN 278 99 S. Typhimurium var. Copenhagen, S. Aragaw et al.
Eastbourne, S. Saintpaul, (2007)
S. Newport, S. Kentuckey
" CC 278 63 S. Anatum, S. Kentuckey, "
S. Hadar, S. Typhimurium
var. Copenhagen
S. Saintpaul, S. Eastbourne
" Carcass 278 11 S. Hadar, S. Eastbourne, "
S. Havana, S. Anatum,
S. Kentuckey

MLN = Mesenteric lymph nodes, AM = Abdominal muscles, DM = Diaphragmatic muscle, HS = Hide
swab, RC = Rumen content, CC = Cecal content, CS = Carcass swab.

European Union scientific committee concluded that the food categories that possibly pose the
greatest hazard to public health include: raw meat and some meat products intended to be eaten
raw, raw or undercooked poultry meat products, eggs and products containing raw eggs and
unpasteurised milk and some milk products. Sprouted seeds, and unpasteurised fruit juices are
also of concern (D’Aoust, 1997; Jay, 2000; Chiu et al., 2004; Forshell and Wierup, 2006). In
Ethiopia, various serotypes of Salmonella were isolated from samples of meat (Molla and
Mesfin, 2003; Tibaijuka et al., 2003; Endrias, 2004), ready to eat foods (Tegegne and Ashenafi,
1998; Endrias, 2004) and other food items in Ethiopia (Endrias, 2004) (Table 3) indicating the
potential of human infections through consumption of raw or undercooked food of animal origin.

34
Table 3: Salmonella serotypes isolated from animal products in Ethiopia

Sample type No. of samples Serotypes isolated Reference

Examined Positive
Ayib 84 1 S. Braenderup Becker et al.
(1996)
Raw milk 159 1 S. Hall "
Kitfo 73 1 S. Hall "
Raw ‘Kitfo’ 50 21 - Tegegne and
Ashenafi (1998)
Minced beef 330 26 S. Anatum, S. Dublin, S. Saintpaul Nyeleti et al.
(2000)
Chicken meat 301 54 S. Braenderup, S. Anatum, S. Saintpaul, Tibaijuka et al.
and giblets S. Uganda (2003)
Chicken meat 378 80 S. Braenderup, S. Typhimurium var. Molla and Mesfin
and giblets Copenhagen, S. Anatum, S. Kottbus, (2003)
S. Typhimurium

Minced beef 160 23 S. Infantis, S. Braenderup, S. Anatum, Ejeta et al. (2004)

S. Bovismorbificans, S. Vejle,
S. Dublin, S. Saintpaul

Mutton 85 12 S. Infantis, S. Braenderup, S. Anatum, "

S. Bovismorbificans
Pork 55 9 S. Infantis, S. Braenderup, S. Vejle "

Chicken meat 208 29 S. Newport, S. Braenderup, S. Hadar, Endrias (2004)

S. Typhimurium, S. Kentucky,
S. Anatum

Pork 194 22 S. Newport, S. Infantis, S. Kottbus "

Mutton 212 23 S. Newport, S. Hadar, S. Typhimurium, "

S. Kentucky, S. Anatum

Minced beef 142 12 S. Newport, S. Typhimurium, "

S. Infantis, S. Kentucky, S. Anatum,
S. Saintpaul

Fish 128 3 S. Newport "

Cheese 190 4 S. Newport "

35
Non-typhoid salmonellosis is one of the most important foodborne diseases throughout the world.
Different workers in Ethiopia investigated prevalence and serotype distribution of Salmonella in
humans (Table 4).

Table 4: Salmonella serotypes/serogroups isolated from humans in Ethiopia

Sample No of Samples Serotypes/serogroups Reference

type Examined Positive isolated

Blood, - - Serogroup A, B, C, E, Gedebou and Tassew

stool S. Typhi (1981)

Stool 1000 45 Serogroup A, C, B, D, E, Ashenafi and Gedebou

S. Typhi (1985)

Stool 700 45 Serogroup A, B, C, D, E, Mache et al. (1997)

S. Typhi

Stool 300 18 S. Anatum, S. Dublin, Nyeleti et al. (2000)

S. Meleagridis

Stool 384 59 Serogroups A, B, C, D, E, Mache (2002)

S. Typhi

Stool 68 5 S. Newport Endrias (2004)

 100 7 S. Anatum, S. Newport Berhanu (2006)
FHS

EHS 100 2 S. Anatum, S. Newport "


FHS = Flayer’s hand swab, EHS = Eviscerator’s hand swab

36
3. MATERIALS AND METHODS

3.1. Description of the study site and study population

3.1.1. Study site

The study was conducted in an export abattoir at Modjo, Ethiopia. Modjo town is the center of
Lume District in Eastern Showa administrative zone of Oromia Regional State. It is located 73
kms southeast of Addis Ababa at an altitude of 1777 meters above sea level. It experiences a
bimodal pattern of rainfall with the main rainy season extending from June to September and a
short rainy season that extends from March to May with an average annual rainfall of 800mm.
The average maximum and minimum temperatures are 28°C and 18°C respectively (ILRI, 2005).

In the abattoir 200-600 sheep are slaughtered every Thursday and Sunday and 500 to 1500 goats
every day based on the demand from importers. Unscheduled slaughters, which include small
group of camels, cattle and calves, are also conducted up on the demand from their customers.
The abattoir has one main slaughter hall with one overhead rail. The rail is higher and serves for
both small ruminant and cattle slaughter operations. In addition, the abattoir has one emergency
slaughter hall, one detaining room, and four chilling rooms. Furthermore, head, skin and
gastrointestinal tract content collection rooms are available separately.

Sheep and goats are slaughtered separately one after the other and the same personnel are
involved in slaughtering both groups of animals. The slaughtering process involves bleeding
using “Halal” method followed by hanging and washing the skin with tap water, flaying,
evisceration, carcass washing with pressurized water and wiping with cloths. Personnel hands
and carcass washing was done using tap water.

3.1.2. Study population

The study was carried out on apparently healthy slaughtered sheep and goats, apparently healthy
abattoir personnel and the abattoir environment. The sheep and goats slaughtered at the abattoir

37
were male and adult animals originated from different parts of the country mainly from Borana,
Awash-Metehara, Bati Wolo, Babile and Ginir (Appendix I). The animals were transported on
double-decked trucks. After arriving at the abattoir, the animals were rested for 24 to 72hrs in
concrete floored and roofed shades till they were slaughtered.

3.2. Study design

A survey study was undertaken on apparently healthy slaughtered sheep and goats, apparently
healthy abattoir personnel and the abattoir environment at an export abattoir from October 2007
to April 2008. The variable of interest considered as an output variable at the slaughterhouse was
carcass Salmonella status. The explanatory variables considered were Salmonella status of sheep
and goats skin, eviscerating knives, eviscerator's hands, mesenteric lymph nodes, caecal contents
and the water used to wash the carcass.

3.3. Sampling

The sample size required for this study was determined depending on the expected prevalence of
Salmonella and the desired absolute precision according to Thrusfield (2005) as follows:

n = 1.962 Pexp (1- Pexp)

2
d
Where:
n = required sample size
P = expected prevalence
exp

d = desired absolute precision

Previous study on Salmonella in sheep and goats in Modjo Export Abattoir recorded a prevalence
of 10.3 and 3.9% respectively (Wassie, 2004). Therefore, 95% confidence interval, 5% precision
and respective 10.3 and 3.9% expected prevalence of sheep and goats were used to estimate the
sample size. Using this information, the number of slaughter sheep and goats needed to
demonstrate the prevalence of Salmonella was estimated at 142 and 60 respectively (Table 5).

38
Table 5: Distribution of type and number of samples collected

Type of sample Number of samples

Sheep Goats
Skin swab 142 60
Mesenteric lymph node 142 60
Cecal content 142 60
Carcass swab 142 60
Eviscerator’s hand swab 142 60

Eviscerating knife swab  142 60

Water sample 28

Total 852 360

*
Sample types collected during sampling of the respective species of animals.

3.3.1. Sampling procedure

Individual animals were systematically sampled depending on the number of animals slaughtered
on each day. Samples were collected weekly and on each visit 7 animals and equal numbers of
environmental samples were collected.

From each selected slaughtered sheep and goats, skin swabs, mesenteric lymph nodes, cecal
contents and carcass swabs were collected in separate sterile containers. Samples were also
collected from eviscerating knives, eviscerator’s hand and water used to wash the carcass.
Selected animals were identified using two similar numbers attached by safety pin on the hind
leg. One of these numbers was transferred to the eviscerated abdominal organs to match them
with the carcasses.

Skin swabs were taken before bleeding was done. A swab moistened with 10ml buffered peptone
water (BPW) (AES loboratoire, Cedex, France) was used to rub the external skin where
slaughtering incision was made and over the skin where incisions were made for flaying.

39
Knives used for evisceration were sampled before each animal was eviscerated. The knife blade
was twice swabbed from the tip to the base using sterile cotton wool moistened with 10ml BPW
(Botteldoorn et al., 2003).

Abdominal contents were removed to the gut room and sampled. About 25 gm of mesenteric
lymph node samples were collected in sterile universal bottles (Vieira-Pinto et al., 2005). After
disinfection with an alcohol-impregnated wipe, the cecal wall was incised using a sterile
disposable scalpel and approximately 50g of the content were also collected aseptically
(McDowell et al., 2007).

Carcass swabs were collected at the end of slaughtering process according to Botteldoorn et al.
(2003). The swab sampling was performed according to the protocol described by Bhandare et al.
(2007) and ISO 17604 (2003). Briefly, carcass swabs were collected from the brisket and rib
medial, brisket and rib lateral, loin and flank region by the swab technique for which sterile
cotton wool swabs (3 cm long and 1 cm in diameter) held by wooden sticks moistened with 10ml
BPW were used. An area of about 100 cm2 was marked with a sterile template of 10 cm · 10 cm
on each site on the carcass. Then the swabs were rubbed over the whole area with pressure
continuously for 30seconds, moving first horizontally and turning the swab so that all sides were
used, and the swabs were transferred to a screw-capped test tubes containing 10 ml of BPW,
breaking off the wooden shaft against the inside of the tube. Subsequently, the same procedure
was repeated to sample the same area with dry swab that was placed into the same container
having 10ml BPW.

Swabs from eviscerator’s hands were taken immediately before the animal identified for
sampling was eviscerated. Both hands were swabbed using a swab moistened with 10ml BPW. In
addition, water sample of 25ml was also collected aseptically on every visit to the abattoir.

During sample collection, each sample was legibly and clearly labeled with identifying
information, which included date of sampling, type of sample and species of the animal from
which the sample was obtained. The samples were then transported on ice to the Addis Ababa

40
University, Faculty of Veterinary Medicine microbiology laboratory at Debre Zeit for processing
and analysis upon arrival.

3.3.2. Sample processing

The mesenteric lymph node samples were aseptically freed from the surrounding tissue, and
25gm was weighed, immersed briefly in boiling water approximately for 10 seconds separately to
decontaminate the surface following the method by Vieira-Pinto et al. (2005). Each lymph node
was then cut into smaller pieces on sterile cutting board by using sterile scalpel blade. The
minced lymph nodes were then put into sterile stomacher bags and 225 ml of BPW was added
and homogenized for two minutes with stomacher (Seward Stomacher 400, London, UK) at high
speed. Whenever samples were less than 25 gm, BPW was added to the samples in 1: 9 ratio.

Ten grams of cecal contents were weighed on sterile aluminum foil and were put in sterile flasks.
About 90 ml BPW was added and the resulting mixture was agitated to disperse the content as
described in McDowell et al. (2007).

Test tubes containing swab samples from the skin, carcasses, eviscerator’s hands and knife blades
were shaken on a vortex mixer for 30seconds for uniform distribution of microorganisms and
then the samples were homogenized in 90 ml BPW and pre-enriched (Swanenburg et al., 2001;
Botteldoorn et al., 2003; Bhandare et al., 2007).

Twenty-five ml of the water sample was transferred into a sterile flask, and 225 ml BPW was
added. Then the sample was stirred gently (Swanenburg et al., 2001).

3.4. Isolation and identification of Salmonella

Salmonella was isolated and identified according to the techniques outlined in the International
Organization for Standardization (ISO 6579, 2002; ISO 6579:2002/FDAM 1, 2007). The
bacteriological media used in different stages of the isolation and identification of Salmonella
were prepared according to the manufacturer’s recommendations (Appendix II). All samples

41
were processed separately. In general, the detection of Salmonella necessitates four successive
stages: pre-enrichment in nonselective liquid medium, enrichment in selective liquid media,
plating out and identification and confirmation of identity (Figure 1).

3.4.1. Pre-enrichment in non selective liquid medium

All the samples were processed separately as in section 3.3.2. Then, the processed samples in
appropriate amount of BPW (1:9) were incubated for 18h  2h at 37 oC 1 oC.

3.4.2. Enrichment in selective liquid media

Rappaport-Vassiliadis with Soya (RVS) broth (Titan Biotech Ltd., Bhiwadi, India) and Muller-
Kauffmann tetrathionate with novobiocin (MKTTn) broth (Oxoid Ltd., Basingstoke Hampshire,
England) were used for selective enrichment of all the samples except the cecal contents (ISO
6579, 2002). In the case of cecal content samples modified semi-solid Rappaport-Vassiliadis

(MSRV) medium (Bacto , Difco Laboratories, USA) and MKTTn broths were used (ISO
6579:2002/FDAM 1, 2007).

A 0.1 ml pre-enriched sample was transferred aseptically into a tube containing 10 ml of MSRV
medium for cecal content samples or 10 ml of RVS broth for the remaining sample types and
incubated at 41.5 oC 1 oC for 24h  3h. Another 1 ml of the culture obtained in pre-enrichment
broth was transferred aseptically into a tube containing 10 ml of MKTTn broth and incubated at
37 oC 1 oC for 24h  3h.

3.4.3. Plating out and identification

Xylose lysine desoxycholate (XLD) agar (Titan Biotech Ltd., Bhiwadi, India) and Salmonella -
Shigella (SS) agar (Titan Biotech Ltd., Bhiwadi, India) plates were used for plating out and
identification purpose.

42
A loopfull of inoculum from each RVS, MSRV and MKTTn broth cultures was streaked onto
XLD and SS agar plates and the inoculated plates were incubated at 37 oC for 24  3h. After
proper incubation, the plates were examined for the presence of typical Salmonella colonies.
Typical colonies of Salmonella grown on XLD medium produce hydrogen sulphide and have a
black (H2S) center and a lightly transparent zone of reddish colour due to the colour change of
the indicator (ISO 6579, 2002) while on SS medium they become colorless colonies with black
center. Salmonella H2S negative variants (e.g. S. Paratyphi A) grown on XLD agar are pink with
a darker pink center whereas lactose-positive salmonellae are yellow with or without blackening.

3.4.4. Confirmation

For confirmation, at least five presumptive Salmonella colonies were selected from every
selective plating media. Whenever the suspected colonies on each plate were fewer than five, all
the colonies were selected. The selected colonies were streaked onto the surface of pre-dried
nutrient agar (Oxoid Ltd., Basingstoke Hampshire, England) plates, in a manner that allow well-
isolated colonies to develop and incubated at 37oC  1 oC for 24h  3h. Then, the pure cultures on
nutrient agar were used for biochemical and serological confirmation (ISO 6579, 2002).

43
 Test sample +Buffered

ENRICHEMENT
peptone water
PRE- temperature

Incubation for
18h  2h at 37 oC  1 oC
ENRICHEMENT
SELECTIVE

0.1ml 0f culture 1ml of culture

+ +
10ml of RVS or 10ml of MKTTn broth
MSRV incubation for
incubation for 24h  3h at 37 oC  1 oC
24h  3h at 41.5 oC 1 oC
PLATING-OUT

XLD and SS agar

incubation for
24h  3h at 37 oC  1 oC

From each plate, select at least five

presumptive Salmonella colonies
CONFIRMATION

Nutrient agar, incubation for

24h  3h at 37 oC  1 oC

Biochemical Serological
Confirmation Confirmation

Expression of results

Figure 1: Flow diagram showing ISO method for the detection of Salmonella (ISO 6579, 2002;
ISO 6579:2002/FDAM 1, 2007)

44
Biochemical confirmation

Colonies suspected to contain Salmonella were tested biochemically according to the

recommendations of ISO 6579 (2002). The biochemical tests included glucose, lactose and
sucrose fermentation and gas and H2S production in TSI agar, growth in L-lysine decarboxylation
medium, urease, Voges-Proskauer (VP) and Indole production tests.

TM
TSI (Oxoid Ltd., Basingstoke Hampshire, England) and lysine iron agar (LIA) (Difco , Becton
Dickinson, Claix, France) slants were inoculated by stabbing the butt and streaking the slant. In
addition, pure colonies were inoculated onto urea (Oxoid Ltd., Basingstoke Hampshire, England)
broth, methyl red - Voges-Proskauer (Titan Biotech Ltd., Bhiwadi, India) medium and SIM
(BBL, Becton Dickinson and Company Cockeysville, USA) medium for further
characterization. For comparison purposes and ease of identification on plates, presumptive
TM
Salmonella colonies were also inoculated onto Rambach agar (CHROMagar, Paris France)

plates. The inoculated media were then incubated at 37°C for 24 ± 2 h according to the
recommendations of ISO 6579 (2002). Isolates presumptive of Salmonella on the biochemical
tests were cultured on tryptic soy agar (AES laboratoire, Cedex, France) and sent to National
Food Institute (Copenhagen, Denmark) for serotyping.

Serological confirmation and serotyping

Serotyping of the Salmonella isolates was carried out by identification of O and H-antigens by
slide agglutination using Salmonella O- and H-antisera obtained from National Salmonella
Centre – Statens Seruminstitut, Copenhagen, Denmark. The definition of the serotypes was based
on the antigen combination present and serotypes were named according to the Kauffmann-White
scheme (Popoff and Le Minor, 2001).

For O-typing a loop full of saline was placed on a slide and a second loop full next to the first. A
loop full of growth from the inoculated nutrient agar plate was taken and mixed into the first
saline drop on the slide. The same procedure was repeated for the second drop (negative control
test) ensuring a smooth, opaque suspension in both drops. Then a drop of poly O antisera was

45
added to the first drop. Antisera and culture (antigen) were mixed with a loop for up to 1 minute.
Appearance of drop 1 was compared with that of drop 2 because if drop 2 has lumps in it, the
culture is autoagglutinating and no further typing is possible. Once the strains were tested in the
O-sera-pools, they were then tested in the individual O-sera represented in the positive O-pool.
The procedure for H-typing was the same as that of O-typing except in H-typing, which was first,
sub-cultured from nutrient agar to Swarm agar and incubated overnight at 37°C. Here also the
strains were first tested in the H-antisera-pools. Afterwards, the strains were tested in the
individual H-antisera represented in the positive H-pool (Global Salm-Surv, 2004).

3.5. Data management and analysis

All data were entered into a Microsoft Excel spreadsheet and checked for accuracy. After
validation, data were transferred to SPSS release 11.5.0 (SPSS, 2002) for analyses.

Species-and sample-specific prevalences of Salmonella were expressed as percentages. The

prevalence was defined as the number of Salmonella positives per the number of samples
examined. An animal was considered positive when a mesenteric lymph node and/or cecal
content sample was culture positive for Salmonella. The agreement of the mesenteric lymph node
and cecal content results was measured using the Kappa statistic. The data were analysed by
comparing proportions using Pearson’s chi-square or Fisher's exact test based on the number of
observations per contingency table cells.

For association of risk factors considered in the abattoir with carcass contamination, multiple
stepwise logistic regression analysis was used. The explanatory variables considered (skin swab,
eviscerating knife swab, eviscerator’s hand swab, cecal content, mesenteric lymph node and
water sample Salmonella status and total slaughter volume) were separately analysed to see their
associations with the outcome of the bacteriological status of the carcass.

46
4. RESULTS

The present study was conducted on 142 and 60 apparently healthy slaughtered sheep and goats
respectively at an export abattoir, Modjo, Ethiopia from October 2007 to April 2008 with the
objectives of establishing prevalence of Salmonella in slaughtered sheep and goats, determining
serotype diversity in slaughtered sheep, goats and abattoir environment, providing information as
to the major sources of carcass contamination in abattoirs and forward strategies to minimize the
contamination. Bacteriological examination was conducted on skin swab (SkS), eviscerating
knife swab (KS), eviserator’s hand swab (HS), cecal content (CC), mesenteric lymph node
(MLN), carcass swab (CS) (each n = 202) and 28 water (WS) samples.

4.1. Prevalence of Salmonella

Out of the total 202 animals (142 sheep and 60 goats) examined for bacteriological status of
Salmonella, 18 (8.9%) were positive of these, 11 (7.7%) were sheep and 7(11.7%) were goats.
No statistically significant differences (p > 0.05) were found between sheep and goats in being
positive for Salmonella (Table 6). An animal was considered Salmonella positive when it was
bacteriologically positive either for MLN and/or CC. Skin and carcass Salmonella statuses were
considered indicators of contamination and were not used for the calculation of prevalence. Of
the 18 positive animals, only 1 (5.6%) animal was culture positive both for MLN and CC
samples. The rest (94.6%) were culture positive either for MLN or for CC samples and were not
significantly different (P = 0.366). The agreement of the MLN and CC samples was measured
using the Kappa statistics and the result indicated low agreement between the two (Kappa value =
0.062, 95% CI = -0.074 – 0.198).

Table 6: Comparative results by Pearson’s X2 test of species-specific Salmonella prevalence in

MLN and CC samples at Modjo Export abattoir, Ethiopia, October 2007 to April 2008

Sample type Odds Ratio CI for the Odds Ratio P-value

MLN 1.134 0.290 – 4.431 0.579
CC 0.237 0.055 – 1.027 0.052
MLN = Mesenteric lymph node, CC = cecal content, CI = confidence interval

47
Of the total 1240 samples examined from sheep, goats, abattoir personnel and abattoir
environment, Salmonella was isolated in 89 (7.2%) samples of which 25 (12.4%) carcass swab,
11 (5.5%) mesenteric lymph node, 8 (4.0%) cecal content, 10 (5.0%) skin swab, 18 (8.9%)
eviserator’s hand swab, 15 (7.4%) eviscerating knife swab and 2 (7.1%) water samples were
positive for Salmonella (Table 7).

Table 7: Prevalence of Salmonella by sample types and species of animals examined

Number of samples
Sheep Goats Total
Positive (%)

Positive (%)

Positive (%)
Sample
Examined

Examined

Examined
95% CI

95% CI

95% CI
types

SkS 142 7 (4.9) 2.2 – 10.3 60 3 (5.0) 1.3 – 14.8 202 10 (5.0) 2.5 – 9.2
MLN 142 8 (5.6) 2.6 – 11.2 60 3 (5.0) 1.3 – 14.8 202 11 (5.5) 2.9 – 9.8
CC 142 3 (2.1) 0.6 – 6.5 60 5 (8.3) 3.1 – 19.1 202 8 (4.0) 1.9 – 7.9
CS 142 20 (14.1) 9.0 – 21.2 60 5 (8.3) 3.1 – 19.1 202 25 (12.4) 8.3 – 17.9
HS 142 15 (10.6) 6.2 – 17.1 60 3 (5.0) 1.3 – 14.8 202 18 (8.9) 5.5 – 13.9
KS 142 12 (8.5) 4.6 – 14.6 60 3 (5.0) 1.3 – 14.8 202 15 (7.4) 4.4 – 12.2
 20 1 (5.0) 0.3 – 26.9 8 1(12.5) 0.7 – 53.3 28 2 (7.1) 1.3 – 25.0
WS
Overall Positive = 11(7.75%) Positive = 7(11.7%) Positive = 18(8.9%)
SkS = skin swab, MLN = mesenteric lymph node, CC = cecal content, CS = carcass swab, HS =
eviscerator’s hand swab, KS = eviscerating knife swab, WS = water sample and CI = confidence interval
*
Sample types collected during sampling of the respective species of animals.

Salmonellae were detected in all test samples obtained from sheep and goats with different
frequencies of occurrence. There were no statistically significant differences (p > 0.05) in the
proportions of Salmonella between sheep and goat samples.

The level of carcass contamination was considered as an outcome variable taking skin swab,
mesenteric lymph node, cecal content, eviscerator’s hand swab, eviscerating knife swab and
water sample Salmonella status and total slaughter volume as risk factors for carcass
contamination. Therefore, associations of carcass contamination with the risk factors were

48
assessed using logistic regression analysis (Table 8) and no statistically significant associations
could be demonstrated between the carcass contamination and skin swab, mesenteric lymph
node, cecal content, eviscerator’s hand swab, water sample Salmonella status and total slaughter
volume (P > 0.05). However, eviscerating knife swab was found to be significantly associated (P
= 0.017) with carcass contamination and the odds ratio (OR) was 4.175. Therefore, carcasses of
animals that were eviscerated using Salmonella positive knives were 4.175 times (OR = 4.175,
95% CI = 1.297 - 13.444) more likely to be contaminated with Salmonella compared to those that
were eviscerated using Salmonella negative knives. This was also demonstrated by the difference
and similarities in the type of serotypes isolated (Table 9). But, the biological significance of
some potential risks showed high probability of associations for example, MLN with OR = 2.88,
CC with OR = 2.48, HS with OR = 2.22 and WS with OR = 7.67.

Table 8: Summary results of multiple stepwise logistic regression of the associations of carcass
contamination with Salmonella with the risk factors

Risk Coefficient Std. Err. P-value Odds Ratio 95% CI for the
factors Odds Ratio

SkS 0.608 0.821 0.459 1.837 0.367 – 9.186

MLN 1.058 0.714 0.138 2.881 0.711 – 11.673
CC 0.908 0.846 0.283 2.478 0.472 – 13.014
HS 0.796 0.613 0.193 2.218 0.668 – 7.367
KS 1.429 0.597 0.017 4.175 1.297 – 13.444
WS 2.037 1.542 0.186 7.667 0.374 – 157.361
Total slaughter 0.000 0.001 0.576 1.000 0.999 – 1.001
volume
Std. Err. = Standard error, CI = confidence interval, SkS = skin swab, MLN = mesenteric lymph node,
CC = cecal content, HS = eviscerator’s hand swab, KS = eviscerating knife swab and WS = water samples

49
4.2. Distribution of serotypes

The 89 isolates of Salmonella recovered during the study were composed of 12 different
serotypes. The most prevalent serotypes were S. Infantis (16.9%), S. Typhimurium (15.7%) and
S. Heidelberg (13.5%). Other serotypes recovered included S. Reading (10.1%), S. Braenderup
and S. Enteritidis (9.0% each), S. Butantan (6.7%), S. Anatum (5.6%), S. Newport and S. Poona
(4.5% each), S. Give (3.4%) and S. Hadar (1.1%) (Table 9). In the present study, except S. Hadar,
which was recovered only from goat samples, the rest serotypes were isolated from both sheep
and goat samples.

Table 9: Distribution of Salmonella serotypes isolated from apparently healthy slaughtered sheep
and goats, abattoir personnel and abattoir environment at Modjo Export abattoir,
Ethiopia, October 2007 to April 2008

No. of
Salmonella Salmonella
SkS MLN CC CS HS KS WS Overall (%)
serotypes serotypes for
sheep/ goats
S. Infantis - - - 8 5 2 - 12/3 15(16.9)
S.Typhimurium - 5 2 - 5 - 2 11/3 14(15.7)
S. Heidelberg - - - 7 - 5 - 10/2 12(13.5)
S. Reading - - - 3 2 4 - 8/1 9(10.1)
S.Braenderup - - - 6 - 2 - 7/1 8(9.0)
S. Enteritidis - - 3 - 3 2 - 5/3 8(9.0)
S. Butantan - 1 2 - 3 - - 3/3 6(6.7)
S. Anatum 3 2 - - - - - 4/1 5(5.6)
S. Newport 3 1 - - - - - 3/1 4(4.5)
S. Poona 2 1 1 - - - - 2/2 4(4.5)
S. Give 2 1 - - - - - 1/2 3(3.4)
S. Hadar - - - 1 - - - 0/1 1(1.1)
Overall 10 11 8 25 18 15 2 66/23 89
(%) (11.2) (12.4) (9.0) (28.1) (20.2) (16.9) (2.2) (100)
SkS = skin swab, MLN = mesenteric lymph node, CC = cecal content, CS = carcass swab, HS =
eviscerator’s hand swab, KS = eviscerating knife swab and WS = water samples

50
The distribution of Salmonella serotypes from the positive animals showed that S. Typhimurium
(45.5%) was the most prevalent serotype in MLN followed by S. Anatum (18.2%), S. Butantan,
S. Newport, S. Poona and S. Give (9.1% each). On the other hand, S. Enteritidis (37.5%) was the
most prevalent serotype in CC samples followed by S. Poona (12.5%), S. Typhimurium and S.
Butantan (25% each) (Table 10). From the table it can be seen that S. Typhimurium, S. Butantan
and S. Poona were detected both from MLN and CC whereas S. Anatum, S. Newport and S. Give
were detected only from MLN and S. Enteritidis was isolated from a CC samples.

Table 10: Distribution of serotypes on samples of Salmonella positive animals at Modjo Export
abattoir, Ethiopia, October 2007 to April 2008

ID SkS MLN CC CS HS KS WS

G27 - - S. Enteritidis - - - -
G28 - - S. Butantan - - - -
G33 - S. Butantan - - - - -
G34 - S. Give - S. - -
Reading
G36 - - S.Typhimurium - - - -
G37 - S. Poona S. Poona S. Hadar - - -
G54 S. - S. Enteritidis - - - -
Newport
S28 - - S. Enteritidis - - - -
S35 - - S.Typhimurium S.Heidelberg - - -
S37 - - S. Butantan - S. - -
Infantis
S48 - S. Newport - - - - -
S50 - S. Anatum - - - - -
S66 - S.Typhimurium - - - - -
S73 S. S. - S. - - -
Newport Typhimurium Braenderup
S91 - S. - - S. S. -
Typhimurium Infantis Heidelberg
S111 - S. Anatum - - S.Infantis - -
S114 - S. - S. - - -
Typhimurium Heidelberg
S137 - S.Typhimurium - - - - -
ID = identification number, CS = carcass swab, MLN = mesenteric lymph node, CC = cecal content, SkS= skin
swab, HS = eviscerator’s hand swab and KS = eviscerating knife swab

51
Salmonella was recovered from 25 carcass swabs of which 20 were from sheep and 5 were from
goats. Salmonella Infantis (32%), S. Heidelberg (28%) and S. Braenderup (24%) were the most
common serotypes recovered from carcass swab samples while S. Reading (12%) and S. Hadar
(4%) were recovered from carcass swabs with lesser frequencies (Figure 2). Salmonella Hadar
was isolated only from goat carcass, S. Reading was isolated only from sheep carcasses and the
rest 3 serotypes were isolated from both sheep and goat carcasses.

40%

32%
Proportion of the serotypes

30% 28%
24%
20%

12%
10%
4%
0%
ng
is

ar
up
g
er
nt

ad
di
r
elb
a

de

ea

H
nf

en
eid

R
I

S.
ra
S.

S.
H

B
S.

S.

Salmonella serotypes

Figure 2: Proportion of Salmonella serotypes isolated from sheep and goat carcasses

Of the 25 carcasses positive for Salmonella, 5 had the same serotype from knives used for
evisceration. Other 4 carcasses contaminated with Salmonella were slaughtered on the same day
with other animals harboring the same serotypes on their carcasses. The remaining 16 isolates
contaminating carcasses were not isolated either from other samples in the same animal or
animals slaughtered on the same day (Table 11).

52
Table 11: Distribution of serotypes by samples of carcass positive animals at Modjo Export
abattoir, Ethiopia, October 2007 to April 2008

ID Sampling CS MLN CC SkS HS KS WS

Date
G31 S. - - - - - -
1/1/2008 Braenderup
G32 10/1/2008 S. Infantis - - - - S. Infantis -
G37 10/1/2008 S. Hadar S. Poona S. Poona - - - -
G59 24/2/2008 S. Infantis - - - - - -
G60 24/2/2008 S. - - - - S. -
Heidelberg Heidelberg
S22 24/1/2008 S. - - - - - -
Braenderup
S32 27/1/2008 S. Infantis - - - - - -
S35 31/1/2008 S. - S. - - - -
Heidelberg Typhimurium
S39 31/1/2008 S. - - - S. - -
Heidelberg Typhimurium
S56 10/2/2008 S. Infantis - - - S. Reading - -
S67 14/2/2008 S. - - - - - -
Braenderup
S70 21/2/2008 S. Reading - - - S. - -
Typhimurium
S73 21/2/2008 S. S. - S. - - -
Braenderup Typhimurium Newport
S80 28/2/2008 S. - - - - - -
Braenderup
S87 2/3/2008 S. Reading - - - - - -
S90 6/3/2008 S. Infantis - - - - S. Infantis -
S92 6/3/2008 S. - - S. Poona - S. -
Braenderup Braenderup
S96 16/3/2008 S. Reading - - - - - -
S100 16/3/2008 S. - - - - - -
Heidelberg
S103 20/3/2008 S. Infantis - - - - - -
S104 20/3/2008 S. - - - - S. -
Heidelberg Heidelberg
S109 20/3/2008 S. Infantis - - - - - -
S114 23/3/2008 S. S. - - - - -
Heidelberg Typhimurium
S123 27/3/2008 S. - - - S. Enteritidis - -
Heidelberg
S127 30/3/2008 S. Infantis - - - - - 
 had the same serotype in the knives used to eviscerate,  were slaughtered on the same day with other
animals harboring the same serotypes on their carcasses,  = S. Typhimurium

53
5. DISCUSSION

5.1. Salmonella prevalence

In the present study, the prevalence of Salmonella in apparently healthy slaughtered sheep and
goats was 7.7 and 11.7%, respectively. These findings are in agreement with the report of
D’Aoust (1989), which indicated that prevalence of Salmonella ranged between 2 and 51.5% in
sheep and 1–18.8% in goats. Woldemariam et al. (2005) reported respective prevalence of 2.8
and 9.8% in apparently healthy slaughtered sheep and goats in Debre Zeit, Ethiopia. Similarly, a
study undertaken in slaughtered sheep and goats in Hyderabad indicated a higher prevalence (6
%) of salmonellosis in goats as compared to the sheep (1.64 %) (Rajmalliah et al., 1989). In
contrast, Wassie (2004) found a high prevalence of Salmonella in sheep of 11.5% as compared to
goats of 3%. The current study revealed results, which are slightly lower than the respective 14.7
and 18.3% prevalence in slaughtered sheep and goats in Riyadh Public Abattoir, Saudi Arabia
(Nabbut and Al-Nakhli, 1982) and the 17.6% prevalence of Salmonella in goats slaughtered for
chevon in India (Chandra et al., 2006). However, the results are slightly higher than the relatively
lower prevalence in apparently healthy slaughtered sheep (3.1%) and goats (3.8%) in an Indian
abattoir as reported in Kumar et al. (1973) and the respective 1 and 2.3% Salmonella prevalence
in sheep and goats by Sharma et al. (1996).

The difference in the reported prevalences could be associated with the sampling plan and
procedures, sample type, the bacteriological techniques employed in detecting Salmonella or
difference in occurrence and distribution of Salmonella in the study population regardless of test
samples and methods of detection (McEvoy et al., 2003). It is also known that keeping animals to
be slaughtered in crowded waiting pens at abattoirs could facilitate the excretion and
transmission of infection among them. In addition to this, stress could induce higher infection
rates among animals when they are held in the market for long periods before slaughter (Watson,
1975; Radostits et al., 1994).

The respective 2.1 and 8.3% Salmonella prevalence in cecal contents of sheep and goats obtained
in our study compared well with the respective 2.1 and 3.3% prevalences of Salmonella in sheep

54
and goat feces reported by Woldemariam et al. (2005) and the 4.8 and 2% fecal prevalence of
sheep and goats respectively by Wassie (2004). However, a study carried out to estimate the
prevalence of fecal Salmonella in healthy pigs, cattle and sheep at a slaughter in Great Britain
yielded a Salmonella prevalence of 0.1% in cecal contents of sheep (Davies et al., 2004), which
was slightly lower than the findings of the current study. The present result is relatively lower
than 12% fecal prevalence of sheep in Norway (Alvseike and Skjerve, 2002) and 7.8% pelt
prevalence in lambs in UK (Small et al., 2002).

It is well documented that, when animals are starved, salmonellae can survive and multiply in the
rumen. Moreover, healthy carriers intermittently excrete only a few salmonellae, unless they
undergo some kind of stress, for example during transportation or holding in the lairages prior to
slaughter (Moo et al., 1980; Samuel et al., 1981; Nabbut and Al-Nakhli, 1982; Venter et al.,
1994). Therefore, the present high cecal prevalence of Salmonella could be associated with the
exposure of animals to such predisposing factors as starvation, overcrowding, transportation and
longer lairage confinement prior to slaughtering.

The detection of Salmonella of 5.6 and 5.0% in the mesenteric lymph nodes of sheep and goats
respectively, supports earlier observation by Moo et al. (1980) who reported a 4% Salmonella
prevalence in mesenteric lymph nodes in Australian sheep. However, the current study findings
considerably vary from other previous reports. Wassie (2004) reported 7.7 and 2% prevalence of
Salmonella in the mesenteric lymph nodes of sheep and goats respectively. Woldemariam et al.
(2005) also found respective 0 and 11.7% prevalence of Salmonella in sheep and goat mesenteric
lymph nodes. Furthermore, Pegram et al. (1981) failed to identify salmonellae from ovine
mesenteric lymph nodes at the Addis Ababa abattoir. Study undertaken to determine prevalence
of Salmonella in goats slaughtered in India revealed a prevalence of 9.8% in the mesenteric
lymph nodes (Chandra et al., 2006). In addition, Nabbut and Al-Nakhli (1982) recovered 14.7%
salmonellae from the mesenteric lymph nodes of sheep and goats slaughtered in the Riyadh
Public Abattoir. According to Nabbut and Al-Nakhli (1982), the enrichment method revealed
more infected lymph nodes than by direct plating method. Therefore, the differences in the
reported prevalences could be associated with the bacteriological techniques employed for the
detection of Salmonella or differences due to the distribution of the organisms in different study

55
populations in different prevailing conditions. In the present study, there was no statistical
association between mesenteric lymph node Salmonella prevalence and carcass contamination.
This could be due to the fact that lymph nodes are solid enclosed tissues so that they are not
likely to contaminate hands of butchers, environment or carcasses, unless incised during
inspection.

In our study, 7.1% (2/28) Salmonella prevalence was recorded in the water samples used to wash
the carcasses. No comparable data is available for water used to wash sheep and goat carcasses.
However, no Salmonella was recovered from 16 scalding water samples at Addis Ababa abattoir,
Ethiopia (Aragaw et al., 2007) and in 5 Belgian slaughterhouses (Botteldoorn et al., 2003).
Moreover, a study undertaken to determine the level of selected bacteria in the water used for the
rinsing of broiler carcasses at small retail processing operations in Trinidad resulted with 5.1%
(4/78) Salmonella prevalence (Rodrigo et al., 2005). Another study of sensitivity and description
of Salmonella isolated from poultry slaughterhouses and workers in Iraqi revealed 20 and 5%
prevalence of Salmonella from washing and scalding water, respectively (Sultan and Sharif,
2002). The absence of Salmonella in scalding water seems to be due to high scalding water
temperature recorded during slaughter activities. But in this Modjo abattoir survey cold water
was used to wash the carcasses of sheep and goats, which may have contributed to the relative
high prevalence of Salmonella in it.

A 7.4% Salmonella prevalence from the eviscerating knives obtained in this study was in
consistent with the 5% prevalence of evisceration knife-study in Queensland, Australia (Peel and
Simmons, 1978) and the 5% prevalence on the killing knives in poultry slaughterhouses in Iraqi
(Sultan and Sharif, 2002). Another study on knife blades undertaken to indicate the prevalence of
Salmonella reported 26.7 and 10% prevalence at two Botswana abattoirs, A and B, respectively
(Motsoela et al., 2002).

A study was undertaken to examine salmonellae on posts, handrails and hands in a beef abattoir
in Queensland. Salmonellae were isolated from the hands of workers in all stages along the
slaughtering line particularly 30 % on the hands of workers in the evisceration area (Smeltzer et
al., 1980b). Comparatively, that prevalence was higher than the 8.9% recorded in this Modjo

56
abattoir study. Watson (1975) described that washing of the hands with soap and running water
for 15 seconds is needed to remove an inoculum of 100 or less of Salmonella organisms from the
finger tips. While, heavier inocula leave viable salmonellae on the hands even after such
washing. Similarly, Smeltzer et al. (1980b) indicated that washing is an essential part of any
program aimed at reducing cross contamination of carcasses with Salmonella. Therefore, the low
prevalence obtained in this study could be as a result of frequent hand washing which might have
reduced bacterial loads from the hands of those personnel to low levels.

It is clear that the presence of Salmonella excretors in batches of animals in transit and passing
through the lairage could result in contamination of skins. Moreover, Bacon et al. (2002)
indicated that external surfaces of animals serve as a source of contamination for the underlying,
sterile carcass surfaces during the dehiding process. Examination of 100 cattle and 100 sheep
passing through 10 abattoirs in Australia showed a high level of Salmonella contamination of the
hides in cattle (57%) and fleece in sheep (51%) (Watson, 1975). This finding is much higher than
the findings of 4.9 and 5% Salmonella prevalence obtained from the skin of sheep and goats
respectively in this study. However, no statistically significant association was found between
skin swab Salmonella prevalence and carcass contamination. The probable reason for this is that
there was less contact between the skin and the underlying, sterile carcass surfaces as flaying, in
the study abattoir, was carried out automatically. It has been indicated that manual operation of
all the processing steps during slaughtering of the animals in abattoirs, rather than the use of
semi-automatic or automatic systems in operations increases the probabilities of contamination of
edible organs and spreading of salmonellae in abattoir environments (Nabbut and Al-Nakhli,
1982).

This study recorded 14.1% prevalence of Salmonella on sheep carcasses and 8.3% on goats.
These are in consistent with reports of previous works. Woldemariam et al. (2005) reported
respective 7.4 and 7.5% prevalence of Salmonella on carcasses of sheep and goats at Debre Zeit
abattoir, Ethiopia and Sierra et al. (1995) reported 10% prevalence of Salmonella on freshly
dressed lamb carcasses in Spain. On the other hand, microbiological quality survey on sheep
carcasses obtained 1.5 and 0.1% prevalence of Salmonella in USA and Australia respectively,
which are lower than the current findings (Duffy et al., 2001; Phillips et al., 2001). The high level

57
of carcass contamination with Salmonella is of special public health significance for a country
like Ethiopia, where raw and undercooked meat is the favorite meal in most areas.

The surfaces of carcasses are easily contaminated with salmonellae in abattoirs with poor
hygienic control during skinning and evisceration from symptomless animal excretors,
contaminated abattoir equipment and floors. Furthermore, it has been reported that slaughtering
practices such as poor disinfections of knives and other equipment, slaughter floor drains, poor
personal hygiene of slaughterhouse personnel and poor sanitation of slaughterhouse are important
in the contamination of carcasses (Nabbut and Al-Nakhli, 1982; Adesiyun and Oni, 1989; Sierra
et al., 1995). In the present study, the level of carcass contamination was considered as an
outcome variable taking mesenteric lymph node, cecal content, skin swab, eviscerator’s hand
swab, eviscerating knife swab and water sample Salmonella status and total slaughter volume as
risk factors for carcass contamination. Although the associations of carcass contamination with
the potential risk factors was assessed, no statistically significant associations could be
demonstrated between the carcass contamination and the risk factors except with eviscerating
knife swab, which was found to be significantly associated with carcass contamination. However,
there were other risk factors, which showed significant (ORs>1) associations with contamination
of carcasses. Nevertheless, specific attention must be given to the sterilization of knives. As
clearly indicated by different workers (Watson, 1975; Smeltzer et al., 1980a; Motsoela et al.,
2002), it is salutary to note that knives must be immersed in water for 2 minutes at 82oC to reduce
the number of contaminating microorganisms. In line with this, although sterilization of the
knives by immersion in water at 82oC for at least 10seconds may significantly reduce the number
of Salmonella on knives, subsequent contacts with either the steel or scabbard may serve to apply
a fresh inoculum. Therefore, provision should be made for regular sterilization of both steels and
scabbards and the knives.

As indicated by Norval (1961), there is no doubt that the wiping cloths used by slaughter
personnel for cleaning up the carcasses could be an important source of contamination of
carcasses. In this survey it was observed that after washing the carcasses using pressurized water,
slaughter personnel used wiping cloths to clean and dry the surface of the carcasses. Moreover,
the wiping cloths used were not sterile and one wiping cloth was used for a number of continuous

58
carcasses. This situation might considerably contribute to the cross contamination of carcasses
resulting in relatively high prevalence of Salmonella on carcasses. In certain foreign countries,
wiping cloths have been prohibited. For example no cloths are used in piggery abattoirs in
Edinburgh, UK, and only to a very limited extent in the line system for cattle (Norval, 1961).

According to Smeltzer et al. (1980a), contacts between aprons and the carcasses are unavoidable
in many locations and may result in carcass-to-carcass transfer of Salmonella. In addition, results
from different studies showed that equipment that indirectly or accidentally contacts the carcass
such as steels, scabbards, aprons, protective rails, stainless steel sheets or other fixed structures
does contribute to the spread of Salmonella in a meat works. All these factors may also contribute
to the relatively higher prevalence of Salmonella on carcasses of the slaughtered sheep and goats
obtained in our study. Therefore, this role would need to be considered if attempts to reduce
Salmonella contamination of the carcass by sanitizing other equipment, knives, saws, cutting
boards etc. were to be successful. Furthermore, Adesiyun and Oni (1989) emphasized that meat
from the following day’s slaughter could therefore be contaminated with salmonellae from
animals slaughtered the previous day. Similarly, Kumar et al. (1973) indicated that infected
carcasses might contaminate other carcasses during the dressing operations, in transportation or
at butcheries.

In general, the slaughter process inevitably involves some degree of meat contamination, whether
from the animals themselves, the abattoir environment or through contact with operatives and
equipment, as carcasses move through the process. The extent of the contamination can be
reduced by the use of good manufacturing practices and specific measures to safeguard meat
hygiene at all stages of production. A suitable approach to hygiene control is through the
application of the principles of the HACCP system, which is a systematic means of identifying,
evaluating and controlling any hazard in a food production or handling operation that might
affect the consumer. In conclusion, it was found that the knife blades used for evisceration
contributed significantly to the Salmonella found in carcasses. Implementing proper HACCP
procedures and practices to reduce the cross contamination from these sources could decrease the
prevalence of Salmonella at the abattoir.

59
5.2. Serotypes

Out of the total 89 Salmonella isolates, 12 different Salmonella serotypes were identified in our
study. The most prevalent serotypes were S. Infantis, S. Typhimurium, S. Heidelberg, S. Reading,
S. Braenderup and S. Enteritidis. Previously, other workers have reported some of these serotypes
in sheep and goats in Ethiopia (Molla et al., 2003; Wassie, 2004; Woldemariam et al., 2005),
India (Kumar et al., 1973) and Saudi Arabia ( Nabbut and Al-Nakhli, 1982). It should also be
noted that the presence and distribution of Salmonella serotypes could vary geographically from
region to region. While some serotypes remain prevalent over many years, others emerge or
decrease over time. The rapid international trade in agricultural, aquaculture and manufactured
animal food products has also facilitated the introduction of new Salmonella serotypes in many
countries (D’Aoust, 1994).

Among the Salmonella isolates identified, the most frequently isolated serotype was S. Infantis
that accounted for 16.9% of the total isolates. Similarly, S. Infantis was the most frequently
isolated (45.5%) serotype in slaughtered sheep and goats in Debre Zeit, Ethiopia (Woldemariam
et al., 2005). This serotype was also reported previously in Ethiopia from camel (Molla et al.,
2004) and swine (Aragaw et al., 2007). It was also recovered from animal products such as
mutton (Ejeta et al., 2004), minced beef (Ejeta et al., 2004; Endrias, 2004), chicken meat and
giblet (Molla and Mesfin, 2003) and pork (Ejeta et al., 2004; Endrias, 2004). Furthermore, this
serotype was one of the most commonly isolated serotypes in a study undertaken to assess cross
contamination of pig carcasses in selected slaughterhouses in Belgium (Botteldoorn et al., 2003).
Galanis et al. (2006) also reported S. Infantis among the top 10 Salmonella serotypes identified
from human and non-human isolates from all regions of the world. Therefore, the isolation of this
serotype in such a proportion in this study, and its dominance in previous studies in various food
items coupled with the tradition of raw and/or undercooked meat consumption in Ethiopia, is of
significant public health concern, as the organism may reach the consumer along the production
chain from abattoir to table/fork.

The second most frequent serotype (15.7%) recovered in this study was S. Typhimurium. It was
also recovered previously from slaughtered sheep and goats (Wassie, 2004; Woldemariam et al.,

60
2005), cattle (Pegram et al., 1981; Alemayehu et al., 2003; Berhanu, 2006), swine (Aragaw et al.,
2007) and camel in Ethiopia (Molla et al., 2004). It was also isolated from a variety of food items
of animal origin including minced beef (Endrias, 2004), chicken meat and giblets (Mesfin and
Molla, 2003; Endrias, 2004). Moreover, Typhimurium is one of the most common Salmonella
serotype isolated from sheep and goats in Saudi Arabia (Nabbut and Al-Nakhli, 1982) and cattle
in Australia (Smeltzer et al., 1980a; Fegan et al., 2004) and Republic of Ireland (McEvoy et al.,
2003). Salmonella Typhimurium is among the most prevalent serotypes in the world. It has been
for instance one of the 2 most frequent serotypes in human and non-human isolates since 1990 in
Europe and America (Global Salm Surv, 2002). In addition, it is the most common cause of food
poisoning in Europe, America and Australia (Hjartardóttir et al., 2002).

Salmonella Heidelberg was the third most common isolate recovered. It constituted 13.5% of all
the isolates in this current study. This serotype was reported previously in Ethiopia from
slaughtered sheep (Wassie, 2004) and camel (Molla et al., 2004). Salmonella Heidelberg was
also isolated from bone factory in Addis Ababa, Ethiopia (Pegram et al., 1981).

Salmonella Reading constituted 10.1% of all the isolates in this study. The serotype was
previously reported from sheep (Wassie, 2004) and cattle (Berhanu, 2006) in Ethiopia and from
sheep and goats in Riyadh Public Abattoir, Saudi Arabia (Nabbut and Al-Nakhli, 1982).

Salmonella Braenderup and S. Enteritidis were also among the prevalent serotypes (9.0% each)
recovered in the current study. Salmonella Braenderup was one of the most frequently isolated
serotypes, from animals and animal products in Ethiopia. It was reported from sheep and goats
(Woldemariam et al., 2005), camels (Molla et al., 2004), swine (Aragaw et al., 2007), chicken
meat and giblet (Molla and Mesfin, 2003; Endrias, 2004), minced beef and mutton (Ejeta et al.,
2004), pork (Ejeta et al., 2004) and cottage cheese (ayib) (Becker et al., 1996). Likewise, S.
Enteritidis was reported earlier in Ethiopian sheep (Wassie, 2004), cattle (Alemayehu et al.,
2003), camels (Molla et al., 2004) and swine (Aragaw et al., 2007). Moreover, S. Enteritidis is
the most common serotype of Salmonella isolated from humans in different countries (van
Duijkeren et al., 2002; Bangtrakulnonth et al., 2004).

61
In this study S. Butantan accounts for 6.7% of the serotypes identified, which is lower than the
24.2% prevalence obtained by Woldemariam et al. (2005) in apparently healthy slaughtered
sheep and goat in Debre Zeit abattoir, Ethiopia. This serotype was also recovered from camel in
Ethiopia (Molla et al., 2004).

Salmonella Anatum was 5.6% of the serotypes identified in this study. It was reported earlier in
Ethiopian cattle (Nyeleti et al., 2000; Berhanu, 2006), goats (Woldemariam et al., 2005), camel
(Molla et al., 2004), swine (Aragaw et al., 2007), chicken meat (Molla and Mesfin, 2003;
Tibaijuka et al., 2003; Endrias, 2004), minced beef (Nyeleti et al., 2000; Ejeta et al., 2004;
Endrias, 2004), mutton (Ejeta et al., 2004) and abattoir personnel (Nyeleti et al., 2000). Other
investigators also reported S. Anatum from different regions of the world. It is one of the most
commonly isolated serotypes in USA (Dargatz et al., 2003; Wray and Davies, 2003), Australia
(Fegan et al., 2005), Taiwan (Chiu et al., 2004) and Thailand (Bangtrakulnonth et al., 2004).

Salmonella Newport and S. Poona (4.5% each) were also serotypes recovered in this study.
Salmonella Newport was previously reported in slaughtered goats (Woldemariam et al., 2005),
cattle (Berhanu, 2006) and swine (Aragaw et al., 2007). Furthermore, this serotype was the most
common isolate recovered from variety of food samples of animal origin and stool samples from
supermarket personnel in Addis Ababa, Ethiopia (Endrias, 2004). It was the third common
laboratory confirmed Salmonella isolate from humans in USA in 1999 preceded only by S.
Typhimurium and S. Enteritidis, and the seventh in Taiwan during 1991 to 1996 (Chiu et al.,
2004).

Salmonella Poona was previously reported in slaughtered goats in Debre Zeit, Ethiopia (Wassie,
2004). Other workers also reported it from different regions of the world. In Saudi Arabia,
Nabbut and Al-Nakhli (1982) isolated S. Poona in apparently healthy slaughtered sheep and goats
at the Riyadh Public Abattoir. In India, Kumar et al. (1973) reported it in apparently healthy
slaughtered goats and in Nigeria, Falade (1976) isolated S. Poona from feces of healthy goats
slaughtered at Kanu abattoir. Adesiyun and Oni (1989) also isolated S. Poona from slaughtered
goats in Zaria, Nigeria.

62
Salmonella Give (3.4%) was another serotype identified in the present study. It was previously
reported from slaughtered sheep in Ethiopia (Wassie, 2004). It was also reported in Portugal in a
study undertaken to determine the occurrence of Salmonella in the ileum, ileocolic lymph nodes,
tonsils, mandibular lymph nodes and carcasses of slaughtered pigs (Vieira-Pinto et al., 2005).

In this study the least isolated serotype was S. Hadar (1.1%). It was isolated only from goat. This
finding is in agreement with Woldemariam et al. (2005) who detected it only from goats. This
serotype was previously reported in Ethiopia from camels (Molla et al., 2004), poultry (Molla
and Mesfin, 2003), chicken (Endrias, 2004) and mutton (Endrias, 2004) at 2.6, 2.2, 6.1 and 2.0%
serotype prevalence respectively.

Most of the serotypes isolated in this study are incriminated as major causes of foodborne
outbreaks of human salmonellosis in different countries of the world (D’Aoust, 1997; Jay, 2000).
The isolation of serotypes belonging to the serogroups B, C and E reiterates their importance in
the global food chain and their prominent associations with human salmonellosis (D’Aoust,
1989; D’Aoust et al., 1992). Moreover, S. Typhimurium, S. Heidelberg and S. Enteritidis are
listed within the 5 most prevalent Salmonella serotypes reported from different animal food
products (Jay, 2000).

As described previously, no statistically significant associations (P > 0.05) could be demonstrated

between most of the variables considered to be sources of contamination (mesenteric lymph
node, cecal content, skin swab, eviscerator’s hand swab, water sample) and bacterial status of the
carcass. However, eviscerating knife swab was found to be significantly associated (P = 0.017)
with carcass contamination. This was also demonstrated by Salmonella serotype distribution
among the different sample types. Most of the isolates recovered from the twenty five positive
carcasses, S. Infantis, S. Heidelberg, S. Braenderup and S. Reading were among the most
frequently recovered serotypes from eviscerating knife swab. In general, the similarities of
serotypes between the carcass swab and eviscerating knife swabs indicate contamination of
carcasses during the slaughtering process. Of course the possibility of detecting similar serotypes
from different samples due to the distribution of these serotypes in the study area should not be
ignored.

63
6. CONCLUSIONS AND RECOMMENDATIONS

In the present study respective 7.7 and 11.7% prevalence of Salmonella in apparently healthy
slaughtered sheep and goats at an export abattoir in Modjo, Ethiopia was obtained. Salmonellae
were detected in sheep and goats from skin, mesenteric lymph nodes, cecal contents, carcasses
and also on the abattoir personnel hands, eviscerating knives and water samples with different
frequencies of occurrence. There was high contamination of sheep and goats carcasses with
Salmonella indicating the role of slaughter processes followed by the abattoir in carcass
contamination.

Eviscerating knife was found to be the main source of carcass contamination during the
slaughtering process. Carcasses of sheep and goats that were eviscerated using Salmonella
positive knives were more likely to be contaminated with this microorganism compared to those
that were eviscerated using Salmonella negative knives. This was also demonstrated by the
similarity of Salmonella serotypes between those found on carcass swabs and eviscerating knife
swabs.

The most frequently isolated serotype was S. Infantis followed by S. Typhimurium, S.

Heidelberg, S. Reading, S. Braenderup and S. Enteritidis. Other serotypes recovered included, S.
Butantan, S. Anatum, S. Newport, S. Poona, S. Give and S. Hadar.

Based on the above conclusions the following recommendations are forwarded:

 Measures such as operating a two-knife system, where one knife is being sanitized at
820C while the other is in use, should be implemented at the abattoir in order to reduce
contamination of meat and other edible offals during the slaughtering operations.

 Good hygienic practices in the abattoir and applications of the hazard analysis critical
control point (HACCP) concept should be put in place in order to eliminate or reduce
foodborne pathogens to acceptable limits, including Salmonella.

64
 Further, the findings in this study create momentum for more research work to determine
other sources of carcass contaminations in the abattoir and to determine cost-effective
measures that should be taken to reduce contamination from these sources.

65
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8. APPENDICES

Appendix I: Map showing the areas where the slaughtered sheep and goats are originated

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Appendix II: Media used and preparations for the isolation and identification of Salmonella

1. Buffered peptone water (BPW) (AES loboratoire, Cedex, France)

Composition (g/liter): Peptone from casein 10.0; sodium chloride 5.0; di-sodium hydrogen
phosphate 3.5; potassium dihydrogen phosphate1.5. pH  7.2  0.2 at 25 oC.
Preparation: Twenty grams of this medium was dissolved in one liter of distilled water and
o
sterilized by autoclaving at 121 C for 15 minutes.

2. Rappaport-Vassiliadis Soya bean Meal Broth (RVS-Medium) (Titan Biotech Ltd.,

Bhiwadi, India)
Composition (g/liter): Magnesium chloride (anhydrous) 13.58; sodium chloride 7.2; Soya
peptone 4.5; potassium dihydrogen phosphate 1.26; Di-potassium hydrogen phosphate 0.18;
malachite green 0.036. pH  5.2  0.2 at 25 oC.
Preparation: Dissolve 26.75gm in 1 liter of distilled water. Gently heat to dissolve the medium
completely. Dispense 10ml amounts into tubes and sterilize by autoclaving at 115 oC for 15
minutes.


3. Rappaport-Vassiliadis (MSRV) Medium Semisolid Modification (Bacto , Difco
Laboratories, USA)
Composition (g/liter): Bacto tryptose 4.59; casein hydrolysate (acid) 4.59; sodium chloride 7.34;
potassium dihydrogen phosphate 1.47; magnesium chloride anhydrous 10.93; malachite green
oxalate 0.037; bacto agar 2.7.
Preparation: Suspend 31.6gm in 1 liter of distilled or deionized water and boil to dissolve. Do
not autoclave. Cool to 50 oC. Aseptically dispense 10ml amounts into sterile tubes.

4. Muller- Kauffmann Tetrathionate Broth Base (Oxoid Ltd., Basingstoke Hampshire,

England)
Composition (g/liter) (Base medium): Tryptone 7.0; Soya peptone 2.3; sodium chloride 2.3;
calcium carbonate 25.0, sodium thiosulphate 40.7; ox bile 4.75.
Iodine solution: Iodine 20 gm; potassium iodide 25gm; distilled water 100ml.
Brilliant Green solution: Brilliant Green (BDH or Chroma) 0.1gm, distilled water 100ml.

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Novobiocin solution: Novobiocin sodium salt 0.04gm, distilled water 5ml
Preparation: Suspended 82gm of the base medium in 1 liter of distilled water and bring to the
boil. Cool below 45oC and add, just prior to use, 19ml of iodine solution, 9.5ml of a 0.1%
brilliant green solution and 5ml of the Novobiocin solution. Mix well and fill out into sterile
tubes. pH  8.0  0.2 at 25 oC.

5. Xylose lysine desoxycholate agar (XLD-agar) (Titan Biotech Ltd., Bhiwadi, India)
Composition (g/liter): Agar 13.5; lactose 7.5; sucrose 7.5; sodium thiosulphate 6.8; L-Lysine
hydrochloride 5.0; sodium chloride 5.0; D-xylose 3.5; yeast extract 3.0; sodium desoxycholate
2.5; ferric ammonium citrate 0.8; phenol red 0.08. pH  7.5  0.2 at 25 oC.
Preparation: Dissolve 55gm in 1 liter of distilled water. Gently heat to dissolve completely.
Avoid excess heating. Do not autoclave. Cool to 55 oC – 60 oC and dispense as desired.

6. Salmonella-Shigella agar (SS) (Titan Biotech Ltd., Bhiwadi, India)

Composition (g/liter): Agar 15.0; sodium citrate 10.0, lactose 10.0; bile salts 8.5; sodium
thiosulphate 8.5; beef extract 5.0, tryptone 2.5; peptone 2.5; ferric citrate 1.0; neutral red 0.025;
brilliant green 0.00033. pH  7.0  0.2 at 25 oC.
Preparation: Dissolve 63gm in 1 liter of distilled water. Boil with frequent agitation to dissolve
the medium completely. Do not autoclave or overheat. Cool to about 50 oC. Mix and pour into
sterile petri plate.

7. Nutrient agar (Oxoid Ltd., Basingstoke Hampshire, England)

Composition (g/liter): ‘Lab-Lemco’ powder 1.0; yeast extract 2.0; peptone 5.0; sodium chloride
5.0; agar 15.0. pH  7.4  0.2 at 25 oC.
Preparation: Suspend 28 gm in 1 liter of distilled water and bring to the boil to dissolve
o
completely. Then sterilize by autoclaving at 121 C for 15 minutes.

8. Triple sugar iron agar (TSI) (Oxoid Ltd., Basingstoke Hampshire, England)
Composition (g/liter): ‘Lab-Lemco’ powder 3.0; yeast extract 3.0; peptone 20.0; sodium chloride
5.0; lactose 10.0; sucrose 10.0; glucose 1.0; ferric citrate 0.3; sodium thiosulfate 0.3; phenol red
9.5; Agar 12.0. pH  7.4  0.2 at 25 oC.

79
Preparation: Suspend 65gm in 1 liter of distilled water and bring to the boil to dissolve
o
completely. Sterilize by autoclaving at 121 C for 15 minutes. Allow the medium to set in slopped
form with a butt about 1 inch long.

TM
9. Lysine iron agar (LIA) (Difco , Becton Dickinson, Claix, France)
Composition (g/liter): Peptone 5.0; yeast extract 3.0; dextrose 1.0; L-Lysine HCl 10.0; ferric
ammonium citrate 0.5; sodium thiosulphate 0.04; bromcresol purple 0.02; Agar 15.0. pH  6.7 
0.2 at 25 oC.
Preparation: Suspend 34.5gm of the powder in 1 liter of purified water. Mix thoroughly. Heat
with frequent agitation and boil for 1 minute to completely dissolve the powder. Autoclave at
o
121 C for 12 minutes.

10. Urea Broth Base (Oxoid Ltd., Basingstoke Hampshire, England)

Composition (g/liter): Peptone 1.0; glucose 1.0; di-sodium hydrogen phosphate 1.2; potassium
dihydrogen phosphate 0.8; sodium chloride 5.0, phenol red 0.004. pH  6.8  0.2 at 25 oC.
Preparation: Add 0.9gm to 95ml distilled water and sterilize by autoclaving for 20 minutes at
o o
115 C. Cool to 55 C and aseptically add one ampoule of sterile urea solution (SR20). Mix well
and distribute 10ml amounts into sterile containers.

11. MR-VP Medium (Buffered Dextrose Broth) (Titan Biotech Ltd., Bhiwadi, India)
Composition (g/liter): Peptone 7.0; dextrose 5.0; dipotassium phosphate 5.0. pH  6.9  0.2 at 25
o
C.
Preparation: Dissolve 17 gram in 1 liter of distilled water. Gently heat to dissolve the medium
completely. Distribute 10ml amounts in each tube. Sterilize by autoclaving at 121oC for
15minutes.

Reagents required for Voges-Proskauer (VP) reaction

1-Naphihol, ethanolic solution
Composition: 1-Naphihol 6gm; Ethanol, 96% (volume fraction) 100ml
Preparation: Dissolve the1-Naphihol in the ethanol.

80
Potassium hydroxide solution
Composition: Potassium hydroxide 40gm; water 100ml
Preparation: Dissolve the potassium hydroxide in the water.

12. SIM (BBL, Becton Dickinson and Company Cockeysville, USA)

Composition (g/liter): Pancreatic digest of casein 20.0; peptic digest of animal tissue 6.1; ferrous
ammonium sulfate 0.2; sodium thiosulfate 0.2; agar 3.5. pH  7.3  0.2 at 25 oC.
Preparation: Suspend 30 gram of the powder in 1 liter of purified water. Mix thoroughly. Heat
o
with frequent agitation and boil for 1 minute. Autoclave at 121 C for 15 minutes.

Reagent required for Indole reaction

Kovacs reagent
Composition: 4- Dimethylaminobenzaldehyde 5gm; ethanol alcohol 75ml; hydrochloric acid
25ml.
Preparation: Mix the components with constant stirring. The final reagent should be stored in
brown bottle.

TM
13. Rambach agar (CHROMagar, Paris France)

Composition (g/liter): Opaque agar 20.0; propylene glycol 10.4; peptones and yeast extract 8.0;
chromogenic and selective mix 2.7. pH  7.2  0.2 at 25 oC.
Preparation: Suspend the medium in the proportion of 30.7g/l of purified water. Disperse powder
slowly in water by rotating for swelling of the agar. Add the liquid content of the supplement vial
o
in the proportion of 10.4g/l. swirl for mixing. Heat and bring to boiling (100 C) while swirling or
o
stirring regularly. If using an autoclave, do so without pressure. Do not heat to more than 100 C.
the mixture may also be brought to boil in a microwave oven: after initial boiling, remove from
oven, stir gently, then return to oven for short repeated bursts of heating until complete fusion of
the agar grains has taken place (large bubbles replacing foam). Cool in a water bath to 45/50.
Swirl or stir gently to homogenize. Pour in to sterile petri dishes and allow to gel and dry.

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14. Tryptic soy agar (AES laboratoire, Cedex, France)
Composition (g/liter): Pancreatic digest of casein 15.0; soy peptone 5.0; sodium chloride 5.0;
agar 15.0
Preparation: Suspend 40gm in 1 liter of cold distilled water, heat to boiling with frequent
o o
agitation and sterilize by autoclaving at 121 C for 15minutes. Cool to 45 – 50 C. Mix well, pour
into sterile transporting tubes and allow the medium to solidify.

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Appendix III: Work sheet for recording colony morphology and biochemical reactions of presumptive Salmonella isolates

Sample Sample Spp Date of Origin Total Colony TSI LIA

no. type Collection slaughter Character on Lac/ Gluc H2S Gas Butt Slant H2S

Rambach

Remark
Urease
volume XLD SS Suc

Indole
VP
agar agar

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Appendix IV: Pictures

A = Uninoculated RVS broth

B = Uninoculated MKTTn broth

C = MKTTn broth after incubation for 24

h at 37oC

D = RVS broth after incubation for 24 h at

41.5oC

E = Uninoculated MSRV medium

F and G = MSRV medium after

incubation for 24 h at 41.5oC

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 A = Presumptive Salmonella colonies

Salmonella-Shigella agar

A = Uninoculated (control)

B = Lactose and/or sucrose fermentation

negative and H2S production positive

C = Lactose and/or sucrose fermentation

negative, glucose fermentation
positive, gas production positive and
H2S production negative

D = Lactose and/or sucrose and glucose

fermentation positive, gas and H2S
production negative

E = Lactose and/or sucrose and glucose

fermentation positive, gas and H2S
production positive
Triple sugar iron agar

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 A = Uninoculated (control)

B = Lysine decarboxylation and H2S

production positive

C = Lysine decarboxylation and H2S

production negative

Lysine iron agar

A = Uninoculated (control)

B = VP positive

C = VP negative

MR-VP Medium for VP test

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 A = Uninoculated (control)

B = Urease positive

C = Urease negative

Urea broth

A = Uninoculated (control)

B = Indole production positive and H2S

production negative

C = Indole and H2S production positive

D = Indole and H2S production negative

E = Indole production negative and H2S

production positive

SIM medium for Indole production test

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 A = Presumptive Salmonella colonies
(Red)

B = Presumptive E coli colonies

(Blue green)

Rambach agar

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9. CURRICULUM VITAE

A. Biographical Data:

Name: Akafete Teklu Fite

Date of Birth: December 27, 1979
Place of Birth: Addis Ababa, Ethiopia
Sex: Female
Qualification: Doctor of Veterinary Medicine
Nationality: Ethiopian
Languages: Amharic, Afaan Oromo, English
(Speaking, listening, reading and writing)
Address: P.O.Box: 57831, Addis Ababa, Ethiopia
Tele: +251-911-350705 or +251-112-593409
E-mail: ake_tak@yahoo.com

B. Educational Background

Year School attended

1985 – 1997 Yehiwot Birhan Primary and Secondary School
1998 -1999 Addis Ababa University, Faculty of Natural Science
2000 – 2004 Addis Ababa University, Faculty of Veterinary Medicine

C. Professional Experience

2005 - 2007 Instructor at Alage Agricultural Technical Vocational and Educational

Training College), Department of Animal Health (Alagae, Ethiopia)

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D. Research output/ Technical paper

Characterization of Oestrus in Borana and Holstein – Friesian X Borana (F1 crossbred) heifers
treated with Prostaglandin F2. Akafete Teklu and Merga Bekana (2008) Ethiopian
Veterinary Journal, 12: 145 – 159.

E. Training

Computer skill (Application softwares: MS Dose, MS Word, MS Excel, MS Access)

Teaching Methodology (At Kotebe Teacher’s Training College)
Refresher - training entitled Veterinary public health (At the FVM, AAU, Ethiopia)

F. Membership to professional societies

Ethiopian Veterinary Association (EVA)

Ethiopian Animal Welfare Association

G. Scholars

DAAD- In- Country/ In- Region Scholarship

International Association for Food Protection (IAFP) 2008 Student Travel Scholarship

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10. SIGNED DECLARATION SHEET

This thesis is my original work, has not been presented for a degree in any other university and
that all sources of material used for the thesis have been duly acknowledged.

Name _______________________________________
Signature ______________________________________
Date of submission _______________________________

This thesis has been submitted for examination with my approval as University advisor.

Moses N. Kyule (BVM, MSc, MPVM, PhD)___________________________________

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