HARAMAYA UNIVERSITY

SCHOOL OF GRATUATE STUDIES

ANTIMICROBIAL RESISTANCE PATTERN AND PUBLIC HEALTH

IMPORTANCE OF Salmonella ISOLATED FROM RAW CHICKEN EGG
IN MIZAN TEFERI, SOUTH WEST ETHIOPIA

MSc Research Proposal

Melkamu Melese

College: Veterinary Medicine

School/Department: Veterinary Public Health
Program: Msc in Veterinary Public Health

Major Advisor: Shimelis Mengistu (DVM, MSc, Asst. Professor)

Co- Advisor: Berhanu Sibhat (DVM, MSc, Assoc. Professor)

November, 2017
Haramaya University, Ethiopia
 ACKNOWLEDGMENT

I would like to express my deepest gratitude to my academic advisors Dr. Shimelis Mengistu and
Dr. Berhanu Sibhat for their overall guidance and advice sharing their time, knowledge and
experience throughout my MSc thesis proposal preparation.

ii
 LIST OF ACRONYMS AND ABBREVIATIONS

ACMSF Advisory Committee on the Microbiological Safety of Food

ACSSuT Ampicillin, Chlorampheniol, Streptomycin, Sulfonamides, and
Tetracycline
AST Antimicrobial Sensitivity Test
ATCC American Type Culture Collection
BPW Buffered Peptone Water
CAFO Concentrated Animal Feeding Operations
CDC Center for Disease Control
CFSAN Center for Food Safety and Applied Nutrition
CFU Colony Forming Unit
CIDRAP Center for Infectious Disease Research and Policy
CLSI Clinical and Laboratory Standards Institute
DSM Diagnostic Services of Manitoba
EC European Commission
EFSA European Food Safety Authority
ERS-USDA Economic Research Service – USDA
ESR Environmental Science and Research Limited
FDA Food and Drug Administration
FRI Food Research International
FSA Food Standards Agency
GPAs Growth Promoting Agents
HPA Health Protection Agency
ICMSF International Committee on the Microbiological Specifications for Foods
ISO International Standard Organization
MAR Multiple Antimicrobial Resistance
m.a.s.l meter above sea level
 TABLE OF CONTENTS

ACKNOWLEDGMENT ii
LIST OF ACRONYMS AND ABBREVIATIONS iii
TABLE OF CONTENTS iv
LIST OF TABLE vii
LIST OF FIGURE viii
SUMMARY ix
1. INTRODUCTION 1
2. REVIEW OF LITERATURE 5
2.1. Evolution of Salmonella 5
2.2. Characteristics of Salmonella 5
2.2.1. Biochemical characteristics 6
2.2.2. Growth and Destruction 6
2.3. Classification and Nomenclature 7
2.4. Epidemiology 10
2.4.1. Distribution 10
2.4.2. Host range 11
2.4.3. Source of infection and transmission 12
2.4.4. Pathogenesis 13
2.4.5. Carrier states 13
2.5. Clinical signs 14
2.6. Diagnosis 15
2.6.1. Pre-enrichment in nonselective liquid medium 15
2.6.2. Enrichment in selective liquid media 16
2.6.3. Plating out and identification 17
2.6.4. Confirmation 18
2.6.4.1. Biochemical confirmation 18
2.6.4.2. Serological confirmation 18
 2.7. Salmonellosis in Man 19
2.8. Salmonellosis in Animals 19
2.8.1 Salmonella in food animals in Ethiopia 20
2.8.2. Salmonella in poultry 20
2.8.3. Salmonella in eggs 21
2.9. Health and Economic Impact of Salmonellosis 21
2.10. Antimicrobial Resistance 22
2.11. Prevention and Control of Salmonellosis 23
3. MATERIALS AND METHODS 25
3.1. Description of the Study Area 25
3.2. Sample Collection and Transportation 25
3.3. Sample Processing 26
3.3.1. Non-selective pre-enrichment 26
3.3.2. Selective enrichment 27
3.3.3. Plating and identification 27
3.3.4. Biochemical tests 27
3.3.4.1. Triple Sugar Iron (TSI) Agar 27
3.3.4.2. Urea agar 28
3.3.4.3. Citrate utilization test 28
3.3.4.4. L-lysine decarboxylation medium 28
3.3.4.5. Indole test 28
3.4. Antimicrobial Susceptibility Testing 30
3.4.1. Disk diffusion testing 30
3.4.1.1. Preparation of Mueller-Hinton Agar 30
3.4.1.2. Turbidity standards (McFarland) for inoculum preparation 30
3.4.1.3. Inoculum preparation 31
3.4.1.4. Inoculation of test plates 31
3.4.1.5. Application of disks to inoculated agar plates 31
3.4.1.6 Reading plates and interpreting results 32
 3.5 Questionnaire Survey 32
3.6 Data Management and Analysis 32
4. WORK PLAN 34
5. LOGISTICS 35
6. REFERENCES 37
7. APPENDICES 44
 LIST OF TABLE

Table 1: Plan of Activities 34

Table 2: Personnel expense 35
Table 3: Transport expense 35
Table 4 : Laboratory services and stationary cost 36
Table 5: Miscellaneous Expense 36
Table 6: Budget summary 36
 LIST OF FIGURE

Figure 1: Classification of the genus Salmonella 9

Figure 2: Procedure of isolation of Salmonella from raw egg chicken 29
 SUMMARY

Cross-sectional study will be conducted on chicken eggs from Mizan Teferi poultry farms and
local markets (directly from the farmers in Mizan) from October, 2017 to April, 2018 with the
objective to isolate and estimate the prevalence and antimicrobial resistance patterns of
Salmonella isolates from chicken eggs. A total of 312 eggs; 156 eggs from Mizan poultry farm
and 156 eggs from Mizan open market directly from the farmers will be collected. Twenty four
eggs from market (one egg from one egg seller) and poultry farm will be collected once per week
using systamatic random sampling technique. Eggs will be collected in sterile bags and aseptic
procedures is strictly adopted during collection. Samples of collected eggs from study area will
be individually placed into a sterile plastic container in an ice box and will be transported to the
Mizan Veterinary regional laboratory. The antimicrobial susceptibility testing will be done by
the agar disk diffusion method as described by the National Committee for Clinical Laboratory
Standards (NCCLS, 2005). The pure Salmonella isolates confirmed by the biochemical testing
procedure as described in ISO 6579: 2002 will be tested for antimicrobial susceptibility. The
antibiotics to be used will be selected among the currently available and commonly used
chemotherapeutic agents for treatment of Salmonella infection in human and animals.
Descriptive statistics such as percentage and proportion will be used to describe samples detected
positive to Salmonella isolation from the total sample analyzed by sources of samples and
sample type. The Pearson’s chi-square (X2) test will be used to determine the significance of
difference or variation of prevalence. P-value of less than 0.05 was considered to determine
statistically significant differences. All statistical analysis was performed using STATA software
package (version 12.1).

Key word: Salmonella, isolation, public health, antimicrobial sensitivity, Mizan, Ethiopia
 1. INTRODUCTION

Poultry, egg and egg products are among the most nutritious foods on earth and they share an
important part of the human diet. However, they are perishable just like meat, fish and other food
items, and consumption of improperly handled poultry and egg products are closely associated
with negative health impacts causing a food-borne illness, such as salmonellosis (FAO/WHO,
2002).

Salmonellosis is an important zoonotic disease caused by the genus Salmonella which constitutes
a major public health burden and represents a significant cost in many countries.The prevalence
of Salmonella in animals is a continuous threat to human health (Murugkar et al., 2005).
Salmonellae are widely distributed in nature and cause a spectrum of diseases in man,animal and
birds. Poultry eggs, meat and their products are the commonest vehicles of Salmonella to humans
(Nagappa et al., 2007). Every year millions of human cases are reported worldwide and the
disease results in thousands of deaths (Herikstad et al., 2002).

Members of the genus Salmonella are Gram-negative, motile, facultatively anaerobic organisms
belonging to the family Enterobacteracae (Ellermeier and Slauch, 2006). The genus Salmonella
contains two species, Salmonella enterica, which consists of six subspecies, and Salmonella
bongori. Currently the genus includes a total of more than 2,500 serotypes (Popoff et al., 2004).
Salmonella nomenclature is complex, and is based on names for serotypes in subspecies I. For
example, Salmonella enterica subsp. enterica serotype Enteritidis, is shortened to S. Enteritidis
(Brenner et al; 2000). Salmonella enterica subspecies enterica (subspecies I) is responsible for
99.5 % of infection in man and animal (Martin et al., 2006). Most of the infections are zoonotic
in origin but some serotypes like S. Typhi and S. Paratyphi infect only humans (Yan et al., 2003).

The infectious dose, incubation period, symptoms and mode of transmission of salmonellosis
caused by different serotypes are similar. Symptoms include diarrhea, fever and abdominal
cramps with incubation periods ranging from 12 to 72 hours. The illness usually lasts from 4 to 7
days and most people recover without treatment. The elderly, infants and those with impaired
immune systems are more likely to have a severe illness (Hans and Dean, 2006).

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Egg contents may be contaminated with salmonellae by two routes: vertical transmission
(transovarian) or horizontal (trans-shell) transmission (FAO/WHO, 2002). In transovarian
transmission, Salmonella are introduced from infected reproductive tissues to eggs prior to shell
formation. Salmonella serotypes associated with poultry reproductive tissues and that are of
public health concern include S. Enteritidis, S. Typhimurium and S. Heidelberg (ICMSF, 1996;
ACMSF, 2001). S. Enteritidis may be better able to achieve invasion (ACMSF, 2001).

Horizontal transmission is usually derived from fecal contamination on the egg shell with
penetration after the egg is laid. It also includes contamination through environmental vectors,
such as farmers, pets and rodents. Many different serotypes of the genus Salmonella may be able
to contaminate egg contents by migration through the egg shell and membranes. Such a route is
facilitated by moist egg shells, storage at ambient temperature and shell damage (ACMSF,
2001).

Food Standards Agency (FSA, 2003) of the United Kingdom has drawn attention to the risk
associated with eating raw and lightly cooked eggs and issued public health advice on the safe
handling and use of eggs. It is estimated that, in the United States, Salmonella transmission
through contaminated shell eggs or egg products results in 700,000 cases of salmonellosis and
costs 1.1 billion United States dollar annually (Rodriguez and Yousef, 2005). In many countries,
Salmonella spp. is controlled in egg production chain. In addition, storing eggs in a cool area
(below 15°C) and keeping eggs separate from other foods is important to avoid possible
Salmonella cross contamination and keep eggs safe (Hans and Dean, 2006). One study in
Ethiopia showed from the total 400 chicken eggs examined for Salmonella prevalence, 46 (11.5
%) were positive, from which 25 (6.3 %) and 27 (6.8 %) were found from egg shell and egg
content, respectively (Minte et al., 2011).

The use of antibiotics in food animals selects bacteria which are resistant to antibiotics used in
humans. These might be spread via the food to humans and cause human infection (Phillips,
2004). Amongst Salmonella spp., antimicrobial resistance is a well confirmed phenomenon
antimicrobial-resistant Salmonella are increasingly associated with the use of antimicrobial
agents. Antimicrobials are substances that have significantly contributed to the prevention and
 3

treatment of infectious diseases in humans, as well as many animal species (CDC, 2008).
However, the excess or overuse of antimicrobials can generate genomic selective pressures to
enable microbes to adapt and acquire resistance (Witte, 2001).

Ultimately, increases in bacterial antimicrobial resistance pose a considerable threat to public

health, especially for vulnerable populations including young children (Shea, 2003), the elderly
and immunocompromised individuals (Hitti and Wolff, 2005). Concentrated animal feeding
operations (CAFOs) in agricultural practices have evolved to accommodate food consumption
rates with increased agricultural output at the risk of introducing antimicrobial resistant
pathogens into the environment. In addition, several studies have suggested that characteristics of
agricultural environmental settings, including animal crowding, CAFO hygiene, temperature,
ventilation control and stress, can influence antimicrobial resistance and pathogen risk
(Silbergeld et al., 2008).

The presence of resistant organisms in the poultry and poultry products for consumption is a
safety concern to the population (Schlundt et al., 2004) and therapeutic concern for the
physicians which might pose prolonged treatment in cases of outbreaks, delayed recovery or
treatment failure (Silbergeld, 2008). There is a scarcity of knowledge concerning poultry farm
development associated with antimicrobial resistance and foodborne bacteria. Information on the
antimicrobial resistance pattern of the Salmonella isolates from chicken table eggs could be
useful for successful treatment, as well as planning strategic use of drugs to minimize resistance
in the future.

In Ethiopia as in other developing countries, it is difficult to evaluate the burden of salmonellosis

because of the limited scope of studies and lack of coordinated epidemiological surveillance
systems. In addition, under-reporting of cases and presence of other diseases considered to be of
high priority may have overshadowed the problem of salmonellosis (Oosterom, 1991).

Salmonellosis is endemic in the country and there is a desire to strengthen the monitoring and
surveillance of salmonellosis using suitable diagnostic tools so as to prevent and control its
occurrence. In addition to this, the extent of Salmonella contamination of eggs sold on local
 4

market and farm, and antimicrobial profile of the Salmonella isolates has not been adequately
studied and very limited information exists in Ethiopia and none in the South West Ethiopia.
Insufficient information at hand in this regard is the main factor that hindered efforts to prevent
and control the disease. Based on the ubiquitous nature of Salmonella and previous studies made
in Ethiopia, it was hypothesized that eggs sold in local markets and poultry farm may serve as a
source of Salmonella species particularly among those raw egg consumers and considerable
proportion of them might have developed resistance to antimicrobials that are commonly used
both in the veterinary and public health sectors.

Therefore, the purpose of the present study will be to isolate and determine the prevalence and
antimicrobial resistance patterns of Salmonella isolates from chicken eggs collected from the
Mizan Teferi local markets and poultry farm.

The specific objectives of the study will be:-

 To estimate the prevalence of Salmonella spp. from egg shell and egg contents of raw
chicken eggs sold at Mizan Teferi local markets and poultry farm.
 To assess farmers and consumers raw chicken egg consumption and handling (storage)
practices
 To determine the antimicrobial susceptibility patterns of the Salmonella isolates to
selected antimicrobial agents using the disk diffusion method
 5

2. REVIEW OF LITERATURE

2.1. Evolution of Salmonella

The genus Salmonella was named after Daniel E. Salmon who first reported the isolation of
Salmonella from a pig in 1885 and named the organism Bacterium choleraesuis (currently
known as Salmonella). A non-human Salmonella spp., S. Choleraesius, was isolated from a
swine’s intestine by Theobald Smith (1859-1934) under the direction of Daniel E. Salmon (1850-
1914) in 1885 (Ellermeier and Slauch, 2006). Dr. Salmon was the administrator of the USDA
research program, and thus the organism was named after him (Rao, 2004; FDA/CFSAN, 2008).

It is conjectured that Salmonella and E. coli, which evolved from a common ancestor emerged
about the time of the emergence of mammals, and emerges as mammalian and avian pathogens
through the acquisition of pathogenicity islands and of a virulence plasmid plus through variation
in lipopolysaccharide antigens (Wray and Wray, 2001). It is hypothesized that accumulation of
single mutations, insertions or deletions with the genome of modern-time Salmonella appears to
have generated many pseudo genes, suggesting its recent evolutionary origin (Papagrigorakis et
al., 2007).

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

2.2.1. 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) (Coetzeret al., 1994; D’Aoust, 2000; Jay, 2000; Mølbaket
al., 2006).

2.2.2. 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 - 47°C with rapid growth between 25 and 43°C
(D’Aoust, 2000; 2001). The lowest temperatures at which growth has been reported are 5.3°C for
S. Heidelberg and 6.2°C for S. Typhimurium. Temperatures of around 45°C have been reported
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 (Coetzeret al., 1994; Jay,
2000; D’Aoust, 2001).
 7

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 preferable 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 44°C 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).

2.3. Classification and Nomenclature

Historically Salmonella had been named based on the original places of isolation such as
Salmonella London and Salmonella Indiana. This nomenclature system was replaced by the
classification based on the susceptibility of isolates to different selected bacteriophages which is
also known as phage typing. Phage typing is generally employed when the origin and
characteristic of an outbreak must be determined by differentiating the isolates of the same
serotype. It is very reproducible when international standard sets of typing phages are used More
than 200 definitive phage types (DT) have been reported so far. For example, S. Typhimurium
DT104 designates a particular phage type for Typhimurium isolates (WHO, 2004, Pui et al.,
2011).

Epidemiologic classification of Salmonella is based on the host preferences. The first group
includes host-restricted serotypes that infect only humans such as S. Typhi. The second group
includes host-adapted serotypes which are associated with one host species but can cause disease
 8

in other hosts serotypes such as S. Pullorum in avian. The third group includes the remaining
serotypes. Typically, Salmonella Enteritidis, Salmonella Typhimurium and Salmonella
Heidelberg are the three most frequent serotypes recovered from humans each year (D’Aoust,
2001; WHO, 2004). The genus consists of two species: the first is S. entericawhich is divided
into six subspecies (Figure 1): S. enterica subsp. enterica, S. enterica subsp. salamae, S.
entericasubsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp. Houtenae and S.
Enterica subsp. indica; and the second is S. bongori (formerly called S. enterica subsp. bongori)
(WHO, 2004). Salmonella enterica subspecies is mainly isolated from warm-blooded animals
and accounts for more than 99% of clinical isolates whereas remaining subspecies and S.
bongoriare mainly isolated from cold-blooded animals and account for less than 1% of clinical
isolates. As an example, the Kauffmann species Salmonella Typhimurium is now designated as
Salmonella entericasubspecies I serotype Typhimurium. Under the modern nomenclature
system, the subspecies information is often omitted and culture is called S. entericaserotype
Typhimurium and in subsequent appearance, it is written as S. Typhimurium. This system of
nomenclature is used nowadays to bring uniformity in reporting (WHO, 2004).

Kauffmann-White scheme classifies Salmonella according to three major antigenic determinants

composed of flagellar H antigens, somatic O antigens and virulence (Vi) capsular K antigens.
This was adopted by the International Association of Microbiologists in 1934. Agglutination by
antibodies specific for the various O antigens is employed to group Salmonellae into the 6
serogroups: A, B, C1, C2, D and E. For instance, S. Paratyphi A, B, C and S. Typhi express O
antigens of serogroups A, B, C1 and D, respectively. More than 99% of Salmonella strains
causing human infections belong to Salmonella enterica. Although not common, cross-reactivity
between O antigens of Salmonella and other genera of Enterobacteriaceae do occur (Puiet al.,
2011).

Therefore, further classification of serotypes is based on the antigenicity of the flagellar H

antigens which are highly specific for Salmonella (D’Aoust, 2001). In brief, O antigens are
lipopolysaccharide (LPS) of the outer bacterial membrane. They are heat stable, resistant to
alcohol and dilute acids. H antigens are heat-labile proteins associated with the peritrichous
flagella and can be expressed in one of two phases. The phase 1 H antigens are specific and
 9

associated with the immunological identity of the particular serovars whereas phase 2 antigens
are non-specific antigens containing different antigenic subunit proteins which can be shared by
many serovars.Therefore, further classification of serotypes is based on the antigenicity of the
flagellar H antigens which are highly specific for Salmonella (Puiet al., 2011). In brief, O
antigens are lipopolysaccharide (LPS) of the outer bacterial membrane. They are heat stable,
resistant to alcohol and dilute acids. H antigens are heat-labile proteins associated with the
peritrichous flagella and can be expressed in one of two phases. The phase 1 H antigens are
specific and associated with the immunological identity of the particular serovars whereas phase
2 antigens are non-specific antigens containing different antigenic subunit proteins which can be
shared by many serovars. K antigens which are heat- sensitive carbohydrates are produced by
Salmonella serovars that express a surface-bound polysaccharide capsular antigen (D’Aoust,
2001).

Figure 1: Classification of the genus Salmonella

Source: Langridge et al., (2008).
 10

Note: Numbers in brackets indicate the total number of serotypes included in each subspecies.
* Common serotypes are listed but other serotypes may cause bacteraemia or focal infection;
subsp = subspecies.

2.4. 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 (Radostitset al.,
1994; D’Aoust, 1997; 2000; Giovannacciet al., 2001; Mollaet 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
(Radostitset al., 1994; D’Aoust, 2000; 2001; Mollaet 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
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 offal into frequently
contaminated feed proteins (D’Aoust, 1997; 2001; Mølbaket al., 2006).

2.4.1. 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
 11

also be found in polluted water (Radostits et al., 1994; Quinn et al., 1999; Jay, 2000; Vieira-
Pinto et al., 2005).

Salmonella 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
unpasteurized milk and some milk products. Sprouted seeds, and unpasteurized 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).

2.4.2. 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
 12

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

2.4.3. 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 (Radostitset 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).

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
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
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the prevalence of Salmonella contamination of food (including food of animal origin) in a

particular country (Forshell and Wierup, 2006).

2.4.4. 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 (Radostitset 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 (Radostitset al., 1994; Quinn et al., 1999).

2.4.5. Carrier states

 14

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, 2001).

2.5. Clinical signs

The disease manifestations depend on the virulence of different Salmonella serovars; the number
of Salmonella ingested and host immunity. Many Salmonella infections are opportunistic
infection in compromised hosts. The majority of Salmonella infections in a herd over time are
subclinical; the clinical infections are only the tip of the iceberg, even during outbreaks of
clinical disease (Gay, 2003). A sudden change in herd resistance caused by concurrent disease,
for example, fascioliasis, bovine virus diarrhea virus, nutritional stress, or severe weather can
result in clinical disease (Wray and Davies, 2003).
 15

Salmonellosis is manifested in animals in three major forms: enteritis, septicemia, and abortion.
Fever, inappetence, and depression are commonly observed in acutely ill animals. Enteritis
caused by Salmonella results in the passage of foul-smelling, watery feces, which may contain
fibrin, mucus, and sometimes blood. When the enteric disease is severe, death may result from
dehydration, electrolyte loss, and acid-base imbalance. Septicaemic form often leads to abortion.
Salmonella Dublin has been associated with outbreaks of abortion in cattle, and several other
serovars adapted to animal hosts have a particular association with abortion. The signs and
lesions are not pathognomonic and in many cases, especially in poultry and pigs, Salmonella
infections may be inapparent (OIÉ, 2000).

2.6. Diagnosis

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).

2.6.1. 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
 16

assist the isolation. Furthermore, pre-enrichment is necessary to permit the detection of sub
lethally 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 sub lethally 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).

2.6.2. 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
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
 17

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 41-43°C and the
other at a more permissive temperature (35-37°C) 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).

2.6.3. 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
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
 18

(Rambach) and glucuronate (SM-ID). Plating media generally yield suspect colonies within 18-
24 h incubation at 35-37°C, 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).

2.6.4. 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 37ºC ± 1ºC for 24h ± 3h. The pure cultures on
nutrient agar will be used for biochemical and serological confirmation (ISO 6579, 2002).

2.6.4.1. 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 (Coetzeret al., 1994).

2.6.4.2. 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
 19

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).

2.7. Salmonellosis in Man

With respect to human disease, Salmonella serotypes can be divided into three groups that cause
distinctive clinical syndromes, typhoid fever, bacteremia and enteritis (Santos et al.,2001).The
non-typhoid Salmonella serotypes can cause protean manifestations in humans, including acute
gastroenteritis, bacteremia and extra intestinal localized infections involving many organs (Chiu
et al., 2004). Within Salmonella enterica subspecies I (Salmonella enterica subspecies enterica),
the most common O-antigen serogroups are A, B, C1, C2, D and E.

Strains within these serogroups cause approximately 99 % of Salmonella infections in humans

and warm-blooded animals. Serotypes in other subspecies are usually isolated from coldblooded
animals and the environment but rarely from humans (Velge et al., 2005).

Following ingestion of contaminated food or water, the pathogenesis of both typhoid and
Salmonella enteritis begins with the intestinal phase, while only typhoid progresses to a systemic
phase (Brown et al., 2005). Transmission of this disease within the human population is
generally a result of poor sanitation of water and food supplies in developing nations. The broad
host-range Salmonella serovars are prevalent within warm-blooded animal populations that make
up the human food supply and bacterial transmission generally results from consumption of raw
or undercooked food products (Jones, 2005).

2.8. Salmonellosis in Animals

Salmonella serotypes have a broad host range (Santos et al., 2003) and prevalent in the warm
blooded animal population including rodents (Jones, 2005). Reptiles kept as pets, such as turtles,
guanas, other lizards and snakes, are often identified as non-food sources of infection (Hans and
Dean, 2006). Some serotypes are highly adapted to animal hosts, such as S. Gallinarum in
 20

poultry and S. Abortus-ovis in sheep. Many nontyphoidal Salmonella strains such as S.

Typhimurium and S. Enteritidis infect a wide range of animal host including poultry, cattle and
pigs (Ohl and Miller, 2001). These serotypes generally cause selflimiting gastrointestinal
infections usually less severe than enteric fever in humans. However, they also have the capacity
to produce typhoid-like infections in mice and in humans or asymptomatic intestinal colonization
in chickens (Velge et al., 2005).

2.8.1 Salmonella in food animals in Ethiopia

Foods of animal origin, especially poultry and poultry products, including eggs, have been
consistently implicated in sporadic cases and outbreaks of human salmonellosis, and chicken
products are widely acknowledged to be a significant reservoir for Salmonella. (Panisello etal.,
2000).

In Ethiopia, there is no Salmonella serotype and antimicrobial resistance surveillance and

monitoring system, therefore the available information are fragmented and made available
through individual publications. One study, conducted on chicken and different chicken products
in Ethiopia indicated the presence of different serotypes of Salmonella (Molla andMesfin, 2003).
In that study out of the total 80 Salmonella isolates, 8 different serotypes were identified of
which S. Braenderup was the most frequent followed by S. Typhimurium var.Copenhagen, S.
Anatum, S. Kottbus and S. Typhimurium. Other serotypes isolated include S.Bovismorbificans,
S. Hadar and S. Infantis. S. Braenderup, S. Anatum and S. Newport appearto be the major
Salmonella serotypes associated with chicken meat and chicken meat products around Addis
Ababa (Tibaijuka et al., 2002; Zewdu, 2008).

2.8.2. Salmonella in poultry

The emergence of Salmonella in poultry and later in egg production has been related to
theindustrialization and the globalization of poultry and egg production (Hans and Dean,
2006).Poultry may acquire Salmonella from various sources, including birds, feedstuffs, rodents
and other vehicles. Clinical disease is uncommon, with the exception of infection caused by the
 21

host-adapted serovar Pullorum/Gallinarum, but most infections are of importance as potential

sources of food poisoning in man. Poultry products may be contaminated at several stages during
the slaughter process, either from feces during evisceration, or by cross-contamination from
contact with contaminated products (Hans and Dean, 2006).

2.8.3. Salmonella in eggs

Salmonella is a leading cause of food-borne illness in many countries with eggs and poultry
being important vehicles of transmission. During the past two decades S. Enteritidis has become
a leading serotype causing human infections, with hen eggs being a principal source of the
pathogen (FSA, 2004).

In United Kingdom, the Department of Health funded a retail survey of UK produced eggs,
sampled in England was undertaken between May 1995 and April 1996. Salmonella spp. were
detected in 0.99 % of the 13,970 samples of 6 eggs (an estimated contamination rate per
individual egg of 1 in every 100 boxes of 6 eggs). There was no significant change in Salmonella
contamination of UK produced eggs since a previous survey in 1991 (FSA, 2004).

Bacterial infections of shell eggs and its content such as Salmonella spp., especially S. Enteritidis
can occur in two different ways: either vertically or horizontally (EFSA, 2005). In vertical
transmission, Salmonella are introduced from infected reproductive tissues to eggs prior to shell
formation. Horizontal transmission is usually derived from fecal contamination on the egg shell.
It also includes contamination through environmental vectors, such as farmers, pets and rodents.
They may be able to contaminate egg contents by migration through the egg shell and
membranes. Such a route is facilitated by moist egg shells, storage at ambient temperature and
shell damage by Salmonella (FAO/WHO, 2002).

2.9. Health and Economic Impact of Salmonellosis

Salmonella is one of the eight microorganisms in the European Union Zoonoses Monitoring
Directive, which shows a disease considered to have a high impact on human health in theUnion
 22

(Jong and Ekdahl, 2006). Poultry products have always topped the incidence of salmonellosis in
many developing countries including India, Egypt, Brazil and Zimbabwe (Henson, 2003) and is
the most seriously perceived food risks in chicken meat, even in the developed countries (Yeung,
2001). S. Enteritidis is transmitted to the human food supply through eggs of hens that appear
healthy (Porwollik et al., 2005). Contamination with Salmonella in poultry products can occur at
multiple steps along the food chain, which includes production, processing, distribution, retail
marketing, handling and preparation (Cui et al., 2005).

The estimated annual costs in 1998 and 2003 of medical care and lost productivity due to
foodborne Salmonella infections in the United States were 2.3 and 2.9 billion dollars
(ERSUSDA, 2003). In the year 2000, 16,983 laboratories confirmed cases of salmonellosis were
reported in the United Kingdom, most commonly associated with the consumption of chicken
and undercooked egg dishes (UK zoonoses report, 2000). In the year 2001, China recorded that
17.9 % of the total food poisoning was caused by Salmonella spp. (FAO/WHO, 2004).

Apart from the impact on the health of the individuals, economic losses on international and
national trade due to Salmonella in the poultry products and product recalls have direct economic
and public perception effects on the processing industries (Kramer et al., 2005).

In Ethiopia as in other developing countries, it is difficult to evaluate the burden of salmonellosis

because of the limited scope of studies and lack of coordinated epidemiological surveillance
systems. In addition, under-reporting of cases and presence of other diseases considered to be of
high priority may have overshadowed the problem of salmonellosis (Oosterom, 1991). Until
1981, comprehensive study on invasive Salmonella was not conducted in Ethiopia (Gedebou and
Tassew, 1981).

2.10. 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
 23

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 worldwide, 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).

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).

2.11. Prevention and Control of Salmonellosis

Prevention of salmonellosis by the implementation of hygiene measures is difficult and use of

antibiotics may give rise to the emergence of resistance problems (Mastroeni and Menager,
2003). Reducing Salmonella prevalence requires a multi-hurdle approach at all stages of
breeding, hatching, grow-out, transportation and processing. Attenuated DNA recombinant live
Salmonella vaccines (Kang et al., 2002), combined with comprehensive control strategy in
animals, feed and animal food stuffs will help to reduce salmonellosis. Additional measures to
 24

control secondary contamination could be prevention of contamination by cleaning and

disinfection, hygiene of personnel and proper processing (Sinell, 1995).

Growth of microorganisms in meat and poultry products can be controlled by maintaining a cold
chain at 10°C, especially for Salmonella during transport and storage (Coleman et al., 2003).

Vaccination does not offer complete protection of the birds against infection. An effective way to
control Salmonella in eggs is by preventing the vertical spread of S. Enteritidis between different
generations of birds. The principle is a top down approach, eliminating Salmonella from the top
of the production pyramid and downwards by test and slaughter (Hans and Dean, 2006).The
post-harvest control level of Salmonella in infected/contaminated shell eggs can be reduced by
decontamination of the surface of the shell by heating, irradiation or other means.

Whole eggs should be pasteurized by the process of heating to 60.0 °C (140 °F) for 3.5 minutes.
Liquid egg white is pasteurized at 56.7 °C (134 °F) for 3.5 minutes or 55.6 °C (132°F) for 6.2
minutes. Liquid egg yolk is pasteurized at 61.1 °C (142 °F) for 3.5 minutes or 60.0°C (140 °F)
for 6.2 minutes. In recent years, pasteurized shell eggs have emerged. The process involves
pasteurization of the shell egg in water baths for extended periods of time (hours) to achieve a
temperature in the center of ~55 °C. Up to a 7-log reduction in Salmonella counts in the interior
has been documented by this method. Special attention should be given to the preparation and
handling of those foodstuffs that have caused a large proportion of outbreaks in recent years,
namely mayonnaise, cold desserts and pre-cooked savouries (Hans and Dean, 2006).
 25

3. MATERIALS AND METHODS

3.1. Description of the Study Area

The study will be conducted in Mizan Teferi which is located in the Bench Maji Zone (BMZ),
Southwestern part of Ethiopia. Mizan, the capital of BMZ is found at a distance of 561 Km from
Addis Ababa and 842 Km from the regional capital Hawassa. BMZ is bordered with keffa Zone
in North, Debub Omo in Northeast, Sheka Zone in southwest, with Gambella and south Sudan
Republic in southern direction. Agroecologically BMZ consists of 52% lowland (500-1500
m.a.s.l), 43% intermediate highland (1500-2300 m.a.s.l) and 5% highland (>2300m.a.s.l). The
mean annual temperature and rainfall varies from 15.1°C-27.5°C and 400-2000mm respectively
(BMZARD, 2014).

3.2. Study Design and Sampling

Cross-sectional study will be conducted on chicken eggs from Mizan Teferi poultry farms and
local markets (directly from the farmers in Mizan) will be conducted from October, 2017 to
April, 2018.

Sample size determination: The sample size required for this study will be determined based on
the prevalence of Salmonella done by Minte et al., (2011) on chicken table eggs at Kombolcha,
Northern Ethiopia. The estimate was desired to be with 5 % sampling error and 95 % confidence.
The sample size is calculated using the following formula for each sampling sites (Thrusfield,
2005).
N = (Zα)2 P (1-P)
d2
Where:
N = number of sample size.
P = prevalence (11.5%)
d = marginal error between the samples and population (0.05)
Zα/2 = the standard normal deviation corresponding 95% of confidence level = 1.96
 26

N = (1.96)2 (0.115) (1-0.115) = 156

(0.05)2

Accordingly, a total of 312 eggs; 156 eggs from poultry farms in Mizan and 156 eggs from
Mizan open market directly from the farmers will be collected. Twenty four eggs from market
(one egg from one egg seller) and poultry farm (12 from each group) will be collected once per
week using systematic random sampling technique. Eggs will be collected in sterile bags and an
aseptic procedure is strictly adopted during collection. Samples of collected eggs from study area
will be individually placed into a sterile plastic container in an ice box and will be transported to
the Mizan Veterinary regional laboratory.

3.3. Sample Processing

The sterile plastic bags containing selected eggs will be open with scissors and the samples
processed immediately. Swab technique will be used to sample the shell surface of the intact
eggs. Sterile cotton swabs dipped into sterile buffered peptone water (BPW) will be used to swab
the entire surface area of the egg shell. The same eggs from which shell sample collected will be
used for interior (egg content) sampling. The egg's surface will be sterilized by immersing in 70
% alcohol for 2 min, air dried in a sterile chamber for 10 min and then cracked with a sterile
scalpel blade. The isolation will be conducted utilizing the conventional methods for the
detection of Salmonella following the standard guidelines from ISO 6579(ISO, 2002).

3.3.1. Non-selective pre-enrichment

The swabs will be directly inoculated into 10 ml BPW in screw capped bottles and incubated at
37 °C for 16-18 hrs. Each 25 ml of the egg’s content will be inoculated into 225 ml of BPW and
homogenized for two minutes with stomacher. After mixing thoroughly, the samples will be
incubated at 37 °C for 16-18 hrs (ISO, 2002).
 27

3.3.2. Selective enrichment

From the pre-enrichment broth after incubation and thoroughly shaking, 0.1 ml of the broth will
be transferred into a tube containing 10 ml of Rappaport-Vassiliadis medium (RV broth).Another
1 ml of the pre-enrichment broth will be transferred into a tube containing 10 ml of Muller-
Kauffmann tetrathionate broth (MKTT broth). The inoculated RV broth will be incubated at 41.5
°C ± 1 °C for 24 ± 3 hours and the inoculated MKTT broth at 37 °C ± 1 °C for 24 ± 3 hours
(ISO, 2002).

3.3.3. Plating and identification

Xylose lysine desoxycholate (XLD) agar plate will be used for plating and identification
purpose. A loop-full of inoculum each from the RV and MKTT broth will be transferred and
streaked separately onto the surface of XLD agar. The plates will be incubated at 37°C ± 1°C for
24 ± 3hours. After proper incubation, the plates will be examined for the presence of suspected
Salmonella colonies, which on XLD agar were pink with a darker center and a lightly transparent
zone of reddish color due to the color change of the indicator whereas lactose positive
salmonellae are yellow with or without blackening. Five Salmonella presumptive colonies were
transferred to nonselective solid media for further confirmatory tests. Confirmation will be done
by using biochemical test according to ISO 6579 (ISO, 2002).

3.3.4. Biochemical tests

3.3.4.1. Triple Sugar Iron (TSI) Agar

The Triple sugar iron agar slants will be prepared with a thick butt. A loopful culture of pure
growth from nutrient agar will stabbed into the butt and streaked on the slant and will be
incubated for 24 hours at 37°C. Typical Salmonella cultures show alkaline (red) slants and acid
(yellow) butts with gas production (bubbles) and formation of hydrogen sulfide (blackening of
the agar).
 28

3.3.4.2. Urea agar

The hydrolysis of urea releases ammonia and production of ammonia increases the pH of the
medium that change color of phenol red (pH indicator) to rose pink, and later to moderate red.
The basal medium will be sterilized by autoclaving at 121°C for 15 minutes. When it has cooled
to about 50°C, 100 ml of a 20 percent solution of pure urea previously sterilized by filtration will
be added and poured into test tubes. The isolates will be inoculated into the urea to determine
urease production. The inoculated tubes will be incubated at 37°C for up to 96 hours. The
observations will be made at an interval of 4, 24, 48 and 96 hours. Urease positive cultures
changed the color of the indicator to red.

3.3.4.3. Citrate utilization test

Simmon’s citrate agar will be sterilized by autoclaving at 121°C for 15 minutes at 15 lb pressure
and cooled for slant formation. The strains will be cultured on the prepared Simmon’s citrate
agar medium, incubated at 37°C for 48 hours and observations will be recorded. Opacity and
change in color of bromothymol from green to blue indicated a positive reaction.

3.3.4.4. L-lysine decarboxylation medium

Lysine decarboxylation broth will be inoculated with the loopful culture of the test organism and
one will kept uninoculated control. Both tubes will be incubated for 24 hours at 37°C. Turbidity
and a purple color after incubation indicate a positive reaction. A yellow color indicates a
negative reaction.

3.3.4.5. Indole test

Indole is a nitrogen-containing compound that can be formed from the degradation of the amino
acid tryptophan by certain bacteria. Tryptone will be used as a substrate because it contains much
tryptophan. The indole reacts with aldehyde compound of Kovac’s reagent and forms red
coloured compound that is more soluble in alcohol. For indole test peptone water will be
 29

prepared and the ingredients will be dissolved in distilled water, dispensed in test tubes and
sterilized by autoclaving at 121°C for 15 minutes. The tubes of the medium will be inoculated
with test isolates using sterile platinum loop and incubated at 37°C aerobically for up to 96
hours. Finally, 0.5 ml of Kovac’s reagent will be added to each of the inoculated and un-
inoculated controls. The tubes will be shaken gently and the results will be recorded. Positive
results will indicated by the development of red colour in the alcoholic layer of the reagent and
no colour in the control tube.

Non-selective pre-enrichment

Swab of egg shell in 10 ml BPW &25ml of egg content in 225ml of 10% buffered pepton water
37°C, 24 h

Selective enrichment

0.1 ml in 10 ml Rappaport-Vassiliadis Soy Broth 37°C, 24 h

1ml of culture in 10ml+ Selenite cystine broth 18-24 hrs, 37oc
Isolation

XLD with an inoculation

BGA or other selective agar plates with an inoculation loop
37°C

Streaking on nutrient agar 37°C, 24 h

Pick five presumptive Salmonella colonies from each agar plate
and inoculate on nutrient agar

Biochemical confirmation 37°C, 24 h

KIA/ TSI and citrate 36+/-10C, 24 h

LDC, Urease, 36+/-10C, 3-24h
VP
Indole 3 minute

Figure 2: Procedure of isolation of Salmonella from raw egg chicken

 30

3.4. Antimicrobial Susceptibility Testing

3.4.1. Disk diffusion testing

The antimicrobial susceptibility testing will be done by the agar disk diffusion method as
described by the National Committee for Clinical Laboratory Standards (NCCLS, 2005), now
known as the Clinical and Laboratory Standards Institute (CLSI). The pure Salmonella isolates
confirmed by the biochemical testing procedure as described in ISO 6579: 2002 will be tested for
antimicrobial susceptibility. The antibiotics to be used will be selected among the currently
available and commonly used chemotherapeutic agents for treatment of Salmonella infection in
human and animals.

3.4.1.1. Preparation of Mueller-Hinton Agar

The Mueller-Hinton agar will be prepared as per the instructions provided by the manufacturer.
After autoclaving the media at 121°C for 15 minutes, it will be cooled to 50°C and
approximately 30 ml to 50 ml will be poured into the 15 x 150 mm Petri dishes. The depth of the
agar in the Petri dishes will be maintained approximately at 4 mm. The freshly prepared plates
will be used on the same day.

3.4.1.2. Turbidity standards (McFarland) for inoculum preparation

McFarland 0.5 turbidity standards will be prepared as per the standard guidelines described by
the Clinical and Laboratory Standards Institute (CLSI). A volume of 0.5 ml of a 1.175 %
(wt/vol) barium chloride dihydrate (BaCl2.2H2O) solution will be added to 99.5 ml of 0.18
mol/L (1 % vol/vol) sulphuric acid with constant stirring to maintain the suspension. The
turbidity standard will be then aliquoted into test tubes of 4 ml each, identical to those used to
prepare the inoculum suspension. McFarland standards then will be stored in the dark at room
temperature (22 °C to 25 °C). Before each use, the standards will be shaken well, mixing the fine
white precipitate of barium sulphate in the tube.
 31

3.4.1.3. Inoculum preparation

At least 3-5 well isolated colonies of the same morphological type will be selected from the agar
plate culture. The top of each colony will be touched with a loop, and the growth will transferred
into a tube containing 4 ml tryptic soy broth. The broth culture will be either directly adjusted to
the McFarland standards or by incubation at 35°C until it achieved or exceeded the turbidity of
the 0.5 McFarland standards (usually 2-6 hours). The turbidity of the actively growing broth
culture will be adjusted with sterile broth to obtain turbidity optically comparable to the point of
the 0.5 McFarland standards.

3.4.1.4. Inoculation of test plates

Optimally within 15 minutes after adjusting the turbidity of the inoculum suspension, a sterile
cotton swab will dipped into the adjusted suspension. Rotate the swab several times and press
firmly on the inside wall of the tube above the fluid level. This removed excess inoculum from
the swab. The dried surface of a Mueller-Hinton agar plate will be inoculated by streaking the
swab over the entire sterile agar surface. This procedure will repeated by streaking two more
times, rotating the plate approximately 60° each time to ensure an even distribution of inoculum.
As a final step the rim of the agar will be swabbed. The procedure will be done under laminar
flow to avoid contamination. The lid will be left ajar for 3-5 minutes but no more than 15
minutes, to allow for any access surface moisture to be absorbed before applying the drug
impregnated disks.

3.4.1.5. Application of disks to inoculated agar plates

The drugs used for disk diffusion testing will be: amoxicillin 25 μg (AMX), ampicillin 10 μg
(AMP), chloramphenicol 30 μg (CHL), ciprofloxacin 5 μg (CIP), clindamycin 2 μg (CLN),
erythromycin 15 μg (ERY), gentamycin 10 μg (GEN), kanamycin 30 μg (KAN), nitrofurantoin
300 μg (NIT), spectinomycin 100 μg (SPC), tetracycline 10 μg (TET) and trimethoprim 5 μg
(TMP). The predetermined battery of antimicrobial disks will be dispensed onto the surface of
 32

the inoculated agar plate. Each disk will be pressed down individually to ensure complete contact
with the agar surface. The disk placed in the agar surface will not closer than 24 mm from center
to center. A total of six disks were placed on one 150 mm plate. The plates will be inverted and
placed in an incubator set to 35°C within 15 minutes after the disks will be applied.

3.4.1.6 Reading plates and interpreting results

After 16-18 hours of incubation, each plate will be examined. The resulting zone of inhibition
will uniformly circular with a confluent lawn of growth. The diameters of the zones of complete
inhibition (judged by the unaided eye) will be measured. Zones will be measured to the nearest
whole millimeter, using sliding callipers, which is held on the back of the inverted Petri plate.
The petri plates will be held a few inches above a black, non-reflecting background. Transmitted
light from the colony counter is used to examine the zones for light growth wherever indicated,
within apparent zones of inhibition. The zone margin will be taken as the area showing no
obvious, visible growth that can be detected with the unaided eye. Faint growth of tiny colonies,
which can be detected only with a magnifying lens at the edge of the zone of inhibited growth,
will be ignored. The sizes of zones of inhibition is interpreted by referring to zone diameter
interpretive standards from NCCLS (2005) and the isolates will be reported as susceptible,
intermediate or resistant to the agents that have been tested.

3.5 Questionnaire Survey

Questionnaire survey will be administered randomly to 150 farmers and consumers to identify
conditions of handling practice (storage), transportation, preparation and utilization patterns of
chicken eggs in the study area. Amharic language will be used in the conditions where the
language is spoken, whereas, in the areas where Amharic is not spoken interpreters will be used.

3.6 Data Management and Analysis

Data management, entry and analysis will be employed using Microsoft Office Excel 2007.
Descriptive statistics such as percentage and proportion will be used to describe samples detected
 33

positive to Salmonella isolation from the total sample analyzed by sources of samples and
sample type. It was generated using the procedure of frequency (FREQ) and expressed in
percent. The Pearson’s chi-square (X2) test will be used to determine the significance of
difference or variation of prevalence. P-value of less than 0.05 was considered to determine
statistically significant differences. All statistical analysis to be performed using STATA
software package (version 12.1).
 34

4. WORK PLAN

The study is anticipated to be completed in definite one year period. The activities within the
study will be out lined as follows.

Table 1: Plan of Activities

No. Major activities to be done Date

1. Proposal development and submission July-September, 2017
2. Proposal defense November, 2017
3. Data collection November - January, 2018
4. Data analysis and Thesis writing February – April ,2018
5. Final preparation and submission May, 2018
6. Thesis defense May, 2018
 35

5. LOGISTICS

Table 2: Personnel expense

No Person Unit No. of days Rate in birr Total

1. Main advisor Days 15 206 3090
2. Co-advisor Days 15 206 3090
3 Researcher Days 7500 7500
4. Lab. technicians Days 20 150 3000
5. Laboratory cleaners Days 20 28 560
6. Supervision and examination Days 3162 3162
Subtotal 20402

Table 3: Transport expense

No. Person Departure Destination Trips Cost per trip in Total

birr
1 Researcher Mizan (study area HU 4 times 700 2800
Mizan AA 4 times 300 1200
Subtotal 4,000
 36

Table 4 : Laboratory services and stationary cost

No. Description Unit Quantity Cost in birr Total cost

1. Chemical purchasing 1750 1750
2. Media purchasing 3000 3000
3 Egg purchasing No. 312 4 1248
4 Stationary cost 700 700
Subtotal 6698

Table 5: Miscellaneous Expense

No. Description Total cost in birr

1. Proposal and Thesis printing and binding 400
2. Questionnaire duplication 500

Subtotal 900

Table 6: Budget summary

No. Description Total Expense in birr

1. Personnel expense 20402
2. Transport expense 4000
3. Laboratory services 6698
4. Miscellaneous Expense 900
Grand total 32, 000
 37

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 44

7. APPENDICES

Appendix I

Nutrient Broth (OXOID)

Bacto-beef extract 3g
Bacto-peptone 5g
Distilled water 1000 ml
pH adjusted to 7.2-7.4
It was sterilized by autoclaving at 121 °C (15 lb pressure) for 15 minutes

Appendix II

Nutrient agar (OXOID)

Peptone 5g
Beef extract 3g
Agar 15 g
Distilled water up to 1 litre
The medium was sterilized in the autoclave at 121 °C for 15 minutes and poured into sterile
Petri dishes in 15 ml quantities

Appendix III

Buffered peptone water (LAB M, UK)

Proteose 10.0 g
Sodium chloride 5.0 g
Disodium phosphate 3.5 g
Monopotassium phosphate 1.5 g
Final pH 7.2 ± 0.2
 45

Prepare by autoclaving 20 gms of media in 1 litre distilled water. Heat to boiling to dissolve the
medium. Dispense in required amount. Sterilize by autoclaving at 121 oC /15 Ibs pressure for 15
minutes.

Appendix IV

Rappaport vassiliadis medium (LAB M, UK, OXOID)

Soy peptone 4.5 g
Sodium chloride 7.2 g
Potassium dihydrogen phosphate 1.26 g
Dipotassium hydrogen phosphate 0.18 g
Magnesium chloride anhydrous 12.4 g
Malachite green 0.036 g
pH 5.2 ± 0.2
Weigh 26 grams of powder and dispense into 1 litre of deionised water. Allow to soak for 10
minutes. Swirl to mix, when dissolved dispense in to 10 ml volume screw capped bottles.
Sterilize by autoclaving at 115 oC for 15 minutes
Appendix V
Muller-Kauffmann tetrathionate broth (LAB M, UK, OXOID)
1. Thiosulphate solution:
Sodium thiosulphate 24.8 g
Sterile water 100 ml
2. Iodine solution:
Potassium iodide 20 g
Iodine 12.7 g
Sterile water 100 ml
Complete medium
Calcium carbonate 2.5 g
Nutrient broth 78 ml
Thiosulphate solution 15 ml
Iodine solution 4 ml
 46

Phenol red 0.02 %

Add calcium carbonate in nutrient broth and sterilize by autoclaving them. Then add iodine,
thiosulphate and then phenol red under aseptic conditions. Distribute in the screw-capped test
tubes

Appendix VI

XLD agar (OXOID)

Yeast extract 3.0 g
Sodium chloride 5.0 g
D (+) xylose 3.5 g
Lactose 7.5 g
Sucrose 7.5 g
L (+) lysine 5.0 g
Sodium deoxcycholate 2.5 g
Sodium thiosulfate 6.8 g
Ammonium iron (III) citrate 0.8 g
Phenol red 2 ml
Agar-agar 13.5
Distilled water 1 litre
The ingredients were steamed to dissolve with final pH 7.0-7.2.

Appendix VII

TSI Agar (OXOID)

Polypeptone peptone 20 g/L
Sodium chloride 5 g/L
Lactose 10 g
Sucrose 10 g
Glucose 1g
Ferrous ammonium sulphate 0.2 g/L
 47

Sodium thiosulphate 0.2 g/L

Phenol red 0.025 g/L
Agar-Agar 13 g
PH 7.3
Suspend 64.50 gms in 1000 ml distilled water. Heat to boiling to dissolve the medium
completely. Mix well and distribute into test tubes. Sterilize by autoclaving at 15 Ibs pressure
(121 oC) for 15 minutes. Allow the medium to set in slopped form with a butt about 1 inch long.

Appendix VIII

Simmon’s Citrate Medium (OXOID)

Sodium chloride 5.0 g
Magnesium sulphate 0.2 g
Ammonium dihydrogen phosphate 1.0 g
Potassium dihydrogen phosphate 1.0 g
Sodium citrate 1.0 g
Bacto agar 20 g
water 1000 ml
Bromothymol blue (0.2 %) 40 ml
pH adjusted to 6.8
Sterilized the media by autoclaving at 121 °C for 15 minutes at 15 lb pressure and cooled for
slope formation.

Appendix IX

Urea extra pure

Melting point 132-133 o C
Insoluble matter 0.003 %
Acidity 0.05 % N
Alkality 0.05 % N
Sulphated ash 0.05 %
 48

Chloride 0.0005 %
Sulphate 0.001 %
Copper 0.0001 %
Iron 0.0001 %
Lead 0.002 %
Biuret 0.05 %

Appendix X

Urea agar base (OXOID)

Peptone 1.0 g
Glucose 1.0 g
Sodium chloride 5.0 g
Disodium phosphate 1.2 g
Potassium dihydrogen phosphate 0.8 g
Phenol red 0.012 g
Suspend 2.4 g in 95 ml of distilled water. Bring to the boil to dissolve completely, sterilize by
autoclaving 115°C for 20 minutes. Cool to 50°C and aseptically add one ampoule of sterile
Urea solution (SR20). Mix well, distribute 10 ml amounts into sterile containers and allow to set
in the slop position

Appendix XI

Peptone Water for Indole Reaction (OXOID)

Peptone 20 g
Sodium chloride 5g
Distilled water 1000 ml
The solids were dissolved by steaming. The reaction at room temperature was adjusted to pH
7.5. The medium was dispensed in 5 ml quantities in test tubes and autoclaved at 121°C for 15
minutes.
 49

Appendix XII

Lysine iron agar (OXOID)

peptone 5.0 g
Yeast extract 3.0 g
Glucose 1.0 g
L-lysine 10.0 g
Ferric ammonium citrate 0.8 g
Sodium thiosulphste 0.04 g
Bromocresol purple 0.02 g

Appendix XIII

Mueller Hinton Agar (OXOID)

Beef 300.0 g
Casein hydrolysate 175.0 g
Starch 1.5 g
Agar 1.7 g
pH 7.3 ± 0.1 at 25 oC
Suspend 38 g in 1 liter of distilled water. Bring to the boil to dissolve the medium completely.
Sterile by autoclaving at 121°C for 15 minutes.

Appendix XIV- Biochemical test result interpretation

Tests Positive Negative

KIA slant butt gas H2S slant butt gas H2S
red yellow crack black yellow red No crack No black
Urease red/purple yellow/orange
LDC blue yellow
Indole red ring on surface yellow ring
Citrate blue green
 50

Appendix XV

Questionnaire

1. Sample collection date _____________________ Site of collection ___________________

2. What is the kind of chickens? _________________________________________________
3. What is the source of the egg used for consumption?

Homemade Purchased from market

4. Do you wash the egg before preparation for consumption? Yes No

5. Do you consume raw egg? Yes No

6. If yes, what is the reason? Justify ______________________________________________
__________________________________________________________________________

7. Does your family consume raw egg? Yes No

If yes to Q7, for what reason do they consume raw egg?
A) For medicinal B) for nutritious purpose C) Other (specify)

If yes to Q7, age group up to 5 yrs 6-10 yrs 11-15yrs above 15 yrs

8. If yes, age group up to 5yrs 6-10yrs 11-15yrs above 15yrs

9. What do you do with cracked egg? Consumed Sold Rejected

10. Do you keep eggs before consumption? Yes No

11. If yes, for how many days do you keep?

1 - 7 days 7 – 15days 15-30 days above 30 days

12. What is your keeping mechanism?

At room/ambient temperature in egg cartons

At room/ambient temperature in box

Together with crops and coffee

13. Would you change after usage or reused it again if you used carton?

Changed Reused
14. Which cooking method do you used for consumption of eggs?
a) poaching b) hard cooking c) scrambling d) frying e) baking
 51

Appendix XV

Laboratory Form
No Sample XLD SS Urea Citra L-lysine
Code agar agar agar te TSI agar Decarbox
test ylationmedium
Slant Butt H2S Gas
production
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17