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Isolation, Identiﬁcation and Molecular Characterization of Poultry Salmonella
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ISOLATION, IDENTIFICATION AND MOLECULAR
CHARACTERIZATION OF POULTRY SALMONELLA
MS THESIS
MD. SHAFIULLAH PARVEJ
DEPARTMENT OF MICROBIOLOGY AND HYGIENE

BANGLADESH AGRICULTURAL UNIVERSITY
MYMENSINGH
JUNE 2013
A Thesis
Submitted to
Bangladesh Agricultural University, Mymensingh
In partial Fulfillment of the Requirements for the Degree of
Master of Science
In
Microbiology
By

Examination Roll No.: 12 VMicroJJ-09 M
Registration No.: 39400
Semester: January- June, 2013
Session: 2012-2013
DEPARTMENT OF MICROBIOLOGY AND HYGIENE

BANGLADESH AGRICULTURAL UNIVERSITY
MYMENSINGH
JUNE 2013
A Thesis
Submitted to
Bangladesh Agricultural University, Mymensingh
In partial Fulfillment of the Requirements for the Degree of
Master of Science
In
Microbiology
Approved as to style and contents by
……………………………… …………………………………
Dr. Marzia Rahman Dr. Munirul Alam
Supervisor Co-Supervisor
……………………………
Dr. Mst. Minara khatun
Chairman, Defence Committee
&
Head
Department of Microbiology and Hygiene
Bangladesh Agricultural University
Mymensingh
JUNE 2013
ACKNOWLEDGEMENTS
In the name of Allah, the Most Gracious, the Most Merciful.
At the inception, all praises to “The Almighty Allah” the Lord and supreme
ruler of the Universe, the Omnipotent, the Omniscient, the Beneficial and
the Merciful, without whose immeasurable grace and profound kindness
the author would have never be able to pursue the higher studies in this
field of science and to carry out the whole research and to build up this
thesis for the degree of Master of Science (MS) in Microbiology.
The author would like to express his ever indebtedness, deepest sense of
gratitude, sincere appreciation and profound regards to his reverend
teacher and research supervisor, Dr. Marzia Rahman, Associate professor,
Department of Microbiology and Hygiene, Faculty of Veterinary Science
(FVS), Bangladesh Agricultural University, Mymensingh for her scholastic
and dynamic guidance, unflinching cooperation, constant inspiration in
every stage of this study, affectionate feeling, constructive criticism and
encouragement throughout the period of the research and preparation of
the manuscript.
The author also wishes to express his gratefulness and sincere gratitude to
his respected Co-superviser Dr.Munirul Alam, senior scientist, icddr,b,
Mohakhali Dhaka for his scholastic supervision, kind cooperation,
inspiration, valuable advice and nice comments throughout the research
work that made the author successful to complete the thesis.
The author humbly desires to express his profound respect and sincere
appreciation to his respected teacher, Dr. Mst. Minara Khatun, Associate
Professor and Head, Department of Microbiology and Hygiene, FVS, BAU,
Mymensingh, for her constant help, cooperation, inspiration, precious
suggestions and providing laboratory facilities during the research work.
It is a boundless opportunity to express deep sense of gratitude for the

author to express a very special thanks and gratitude to Dr. K.H.M.
Nazmul Hussain Nazir, Associate Professor and Jayedul Hassan,
Assistant Professor, Department of Microbiology and Hygiene, FVS, BAU,
Mymensingh, for their endless supports in conducting research and for
their encouragement to establish a research career.
iii
The author finds a great pleasure in expressing his cordial thanks to
honorable teachers Prof. Dr. Md. Mufizur Rahman, Prof. Dr. Md. Mansurul
Amin, Prof. Dr. Bahanur Rahman, Prof Dr. Md. Alimul Islam, , Prof. Dr. Md.
Shahidur Rahman Khan, Prof. Dr. Sukumar Saha, Dr. Md. Tanvir Rahman,
Dr.Md. Ariful Islam, Dr. S. M Lutful Kabir,Dr. Md. Abdul Kafi, Md.Ferdousur
Rahman Khan, Mahbubul Pratik Siddique Md. Shafiqul Islam and Md.
Golzar Hossain, Department of Microbiology & Hygiene, BAU,
Mymensingh, for their valuable advice and constant inspiration throughout
the entire period of the study.
The author finds it a great pleasure in expressing his cordial thanks to Mst.
Fatema-Tuz-Johura, Senior research officer, icddr,b and Mohammad Saiful
Islam, research officer, CFWD, icddr,b, mohakhali, Dhaka, for their endless
supports in conducting research
The author could not have done it without the help Of all well-wishers,
Murshidul Ahsan,Mueena Jahan,Fahmida Afroj,Ehsanul Hoque,Mahmuda
Akhter, Sharmin akter, Mirza Tasmin nahar, Md. Sayduzzaman
jowel,Tayabur Rahman, Sohel Rana, Shamim Ara Nipa, Ashraful Alam,
Imran, Achinta Kumar Biswas and Tazrin Kamal,thank you for pleasant
times inside and outside the lab, for all the long hours and many laughs!
The author would also like to express his heartiest thanks to all the
laboratory and office staff, Department of Microbiology and Hygiene and
all laboratory researcher and staff in the icddr,b, Mohakhali, Dhaka for
support during study period.
Last, but not the least, the author in ever indebted to his beloved parents,
younger brother, sister and other family members for their heartiest
blessings, sacrifice and encouragement throughout the entire period of
academic life. Finally, the author is also extending his cordial thanks to all
of his other friends, relatives and well-wishers.
The Author
June, 2013
iv
CONTENTS
CHAPTER TOPICS PAGE NO.
ACKNOWLEDGEMENTS iii-iv
CONTENTS v-viii
LIST OF TABLES ix
LIST OF FIGURES x
LIST OF ABBREVIATIONS xi
ABSTRACT xii
1 INTRODUCTION 1-4
2 RIVEW OF LITERATURE 5-21
2.1 Incidence and Prevalence of salmonellosis in chickens 5-10
2.2 Isolation, Identification and cultural characteristics of 10-11

Salmonella spp.
2.3 Biochemical and serotyping of Salmonella serovers 11-13
2.4 Molecular Characterization of Salmonella spp. 13-16
2.5 Antibiotic Susceptibility test 16-19
2.6 Pulsed Field Gel Electrophoresis (PFGE) 20-21
3 MATERIALS AND METHODS 22-35
3.1 Materials 22-24
3.1.1 Study area 22
3.1.2 Glassware and other appliances 22
3.1.3 Solid culture media 22
3.1.4 Liquid media for culture and reagents for biochemical tests 23
3.1.5 Salmonella specific antisera for serotyping 23
v
CONTENTS (Contd.)
3.1.6 23
Primers used for PCR
3.1.7 Agarose gel 24
3.1.8 Antibiotic disc 24
3.1.9 Pulsed-field gel electrophoresis (PFGE). 25
3.2 Methods 25-35
3.2.1 Experimental Design 25-26
3.2.2 Collection and transportation of samples 27
3.2.3 27
Preparation of culture media and reagents
3.2.3.1 Preparation of culture media 27
3.2.3.2 Preparation of reagents 27
3.2.3.2.1 Sugar media 27-28
3.2.3.2.2 Methyl-Red Voges-Proskauer (MR-VP) broth 28
3.2.3.2.3 Methyl-Red solution 28
3.2.3.2.4 Voges-Proskauer solution 28
3.2.3.2.4.1 Alpha- naphthol solution 28
3.2.3.2.4.2 Potassium hydroxide solution 28
3.2.3.2.6 Normal saline solution 28
3.2.4 Cultural characterization of Salmonella Spp 29
3.2.5 Gram’s staining 29-30
3.2.6 Differentiation of isolated salmonella using biochemical 30-31
test
vi
CONTENTS (Contd.)
3.2.6.1 29-30
Sugar (Carbohydrate) fermentation test
3.2.6.2 Methyl-Red (MR) test 30
3.2.6.3 Voges-Proskauer (VP) test 30
3.2.6.4 Indol test 30
3.2.7 Serotyping by slide agglutination test 30
3.2.8 Motility test 31
3.2.9 Molecular Characterization by PCR 32
3.2.9.1 DNA extraction 32
3.2.9.2 32
Preparation of a PCR mixture (25µl)
3.2.10 Antibiotic Sensitivity Test 32
3.2.10.1 Inoculum preparation 32
3.2.10.2 Growth medium 32
3.2.10.3 Inoculation of test plates 33
3.2.10.4 33
Application of antibiotic discs to MH agar plates
3.2.10.5 Reading plates and interpretation of the results 33-34
3.2.10.6 Standard for Antibiotic sensitivity test 34
3.2.11 Pulsed-field gel electrophoresis (PFGE) 34-35
3.2.11.1 Image analysis 35
3.2.12 Maintenance of stock culture 35
4 RESULTS 36-49
4.1 36
Prevalence of Salmonella in Poultry
4.2 Isolation of Salmonella by cultural characteristics 37- 38
4.2.1 Results of culture in different culturing media 37
4.2.1.1 Salmonella-Shigella agar 37
vii
CONTENTS (Contd.)
4.2.1.2 Eosin Methylene Blue (EMB) agar media 37
4.2.1.3 Xylose Lysine Deoxychockolate (XLD) agar media 38
4.3 Results of Gram’s staining technique 38
4.4 Results of biochemical test 39-40
4.4.1 Indole test 39
4.4.2 Methyl Red test 39
4.4.3 Voges Proskauer test 39
4.4.4 Carbohydrate fermentation reaction 39-40
4.5 41
Serotyping of Salmonella
4.6 Results of Motility test 41
4.7 Molecular Characterization by PCR 43-44
4.8 Antibiotic sensitivity test 44-46
4.9 47-48
Pulsed Field Gel Electrophoresis
5 DISCUSSION 49-52
6 53-54
SUMMARY AND CONCLUSION
55-64
REFERENCES
APPENDICES 65-68
viii
LIST OF TABLES
TABLE NO. TOPICS PAGE NO.
1 PCR primers with sequence 23

2 Antimicrobial agents with their disc concentration 24
3 Distinguishing characters of 3 Salmonella serotypes 32
4 Inhibition zone diameter for Salmonella spp 35
5 Results of different biochemical tests 41
6 Results of cultural, staining and morphological 43
characteristics of the Salmonella spp.
7 Antibiotic sensitivity pattern of Salmonella spp 46
ix
LIST OF FIGURES
FIGURE NO. TOPICS PAGE NO.
1 Experimental design for Isolation of Salmonella 26

organism from chicken
2 Prevalence of salmonella in Poultry 37
3 Salmonella colony on SS agar 38
.4 Cultural properties of Salmonella on EMB agar 38

5 Salmonella colony on XLD agar. 39
6 Gram’s staining of salmonella isolates 39

7 Results of biochemical test. 40
8 Sugar test indicating S. gallinarum/S. typhimurium 41
9 Sugar test indicating S. pullorum 41

10 Serotyping of Salmonella spp by group D 42
antiserum.
11 MIU media showing motile and non motile 42

salmonella
12 PCR for Salmonella gallinarum 44
13 PCR for Salmonella typhimurium 45
14 Antibiogram studies against the isolated bacteria 47
15 Antibiotic Sensitivity Test 47
16 PFGE gel image of Salmonella sp. 48

17 Genomic fingerprinting of Salmonella strains 49
x
LIST OF ABBREVIATIONS
AML = Amoxicillin
BAU = Bangladesh Agricultural University
CT = Colistin Sulphate
C = Control
D = Dulcitol
DLS = Department of Livestock Services
Dr./DR. = Doctor
E = Erythromycin
e.g = Example
EMB = Eosin Methylene Blue
et al., = Associated
FAO = Food and Agriculture Organization
Fig. = Figure
i.e. = That is
icddr’b = International Centre for Diarrheal Disease Research,
Bangladesh
MS = Master of Science
NB = Nutrient broth
NaCl = Sodium Chloride
NaOCl = Sodium Hypochloride
% = Percentage
µg = Microgram
µl = Microlitre
/ = Per
+ve = Positive
< = Less than
> = Greater than
°C = Degree Celsius
Lab. = Laboratory
lb = Pound
Min = Minute
Ml = Millilitre
xi
ISOLATION, IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF
POULTRY SALMONELLA
ABSTRACT
Salmonellae are an important group of pathogens responsible for human and animal
diseases. The etiological agents of fowl typhoid and pullorum disease are Salmonella
enterica subsp. enterica serovar Gallinarum, which is divided into two distinct biovars
under the serogroup D1, Gallinarum and Pullorum, which are denoted as S. gallinarum
and S. pullorum, respectively. The present study was undertaken with the aim to
identify (biochemical, serological, and molecular) and characterize Salmonella serovars
isolated from commercial layer (chicken) of Bangladesh. For this purpose a total of 150
cloacal swab samples were collected and were subjected to various cultural,
biochemical, and molecular examinations. Among the 16 positive isolates five
fermented glucose and maltose and produced both acid and gas and did not ferment
dulcitol which is positive for S. pullorum. Six of the isolates fermented glucose, maltose
and dulcitol and produced acid, which are typical for S. gallinarum. Total 11 isolates
were positive in agglutination test with group D antisera, suggesting that among 16
isolates five were Salmonella pullorum, six were Salmonella gallinarum and the remaining
five Salmonella typhimurium. Polymerase chain reaction (PCR) performed with speF
primers revealed two of the isolates to be positive for a 2 kb band, suggesting these two
isolates to be Salmonella gallinarum. PCR performed with fimA primers revealed three of
the isolates to be positive for an 85 bp band, suggesting that these three isolates were
Salmonella typhimurium. Antibiogram using six drugs revealed 5(31.25%) strains to be
resistant and 5(31.25%) intermediate towards Ciprofloxacin. Fourteen (87.5%) isolates
were resistant to Amoxycilin, while fourteen (87.5%) were found intermediate
resistance towards Neomycin. However, none of the isolates were sensitive to
Cloxacilin and Erythromycin, two (12.50%) were intermediate and 14 (87.50%) were
resistant. In case of Colistine sulphate, 8(50%) isolates were resistant while the
remaining 8 (50%) were intermediate. Pulsed-field gel electrophoresis of the XbaI-
digested genomic DNA exhibited banding patterns that were identical (the similarity
coefficient was 100%) for all the tested strains which formed a tight cluster when
dendrogram was constructed using the PFGE patterns, suggesting high level of clonal
relatedness among them. The data in this study suggest the prevalence of Salmonella
enterica, which is multidrug resistant and highly clonal for commercial layers of
Bangladesh.
xii
CHAPTER I
INTRODUCTION
Preliminary Salmonella research is dated back to 1880, when the bacteria were
isolated from a person who died from typhoid fever. Subsequently, in 1886,
Daniel E. Salmon and colleagues isolated from swine the organism currently
known as Salmonella choleraesuis, which was believed to be the causative agent for
hog cholera (Grimont et al., 2000; Le Minor, 1991).
Now a day’s salmonellosis is a common and serious problem in the poultry

industry of the world especially in the developing country including Bangladesh.
Poultry industry is a rising profitable sector in Bangladesh. There has been
tremendous development of this sector over the recent years in this country
(Rahman 2003). The entrepreneurship has, by now, shown its great potential as
income generation activities (IGA) and also a tool for poverty alleviation besides
improving of human dietary nutrition through supply of egg and meat. These two
items are the important sources of animal protein in Bangladesh to meet up the
protein requirement of the people. About 89% of the rural households that rear
livestock also rear poultry in Bangladesh (BBS, 1996). The poultry population in
Bangladesh is estimated to be 221.39 millions of chickens and 41.23 millions ducks
(DLS, 2009). About 50,000 chicken farms and 26,000 duck farms have already been
set up in private sector in addition to the Government farms. At present , there are
more than 130 hatcheries producing 0.476 million day-old-chicks per week and
about one million commercial layer and broiler farms supplying 0.6 million kg of
poultry meat and 9.9 million table eggs per weeks (Kabir, 2005).
However, the advancement of poultry industry is seriously hampered due to the

outbreak of various infectious and non-infectious diseases. Among the bacterial
diseases, salmonellosis caused by the genus Salmonella is of major problems in the
poultry industry in Bangladesh (Haider et al., 2008). It causes a variety of acute
and chronic diseases of poultry in Bangladesh (Bhattacharjee et al., 1996).
1
The disease may occur in chicken, duck, pigeon, quail, insects and rodent and
other animals and birds. More than 2,300 serotypes of all the isolated Salmonella,
10% of these have been isolated from poultry (Gast, 1997). Pullorum and Fowl
Typhoid (FT) are caused by Salmonella enterica serovar Salmonella pullorum and
Salmonella gallinarum respectively. Both are economically important diseases of
chickens but may infect other birds as well. Chickens and turkeys are the natural
hosts for FT (Fowl typhoid) and PD ( Pullorum disease), but it also occurs in other
birds, both wild and domestic. As a naturally occurring disease, salmonellosis
caused by S. pullorum has also been reported in humans (Shiviprasad, 1997).
The genus Salmonella is phylogenically clustered in the family of Enterobacteriaceae

(Bennasar et al., 2000; Grimont et al., 2000). Salmonella is characterized as
ubiquitous, Gram-negative, intracellular, straight rod shaped, nonencapsulated,
facultative, non-spore forming, and generally motile with peritrichous flagella
(Gray and Fedorka-Cray, 2002; Kwang et al., 1996;). The bacterium has a width of
0.7 to 1.5 µm and a length of 2.0 to 5.0 µm (Holt et al., 1994). Salmonella spp. are
typically found in soil, water, food, and the gastro-intestinal tract of humans and
other animals (Anderson and Ziprin, 2001). Most Salmonella are motile, with the
exception of the poultry-specific serotypes of S. gallinarium and S. pullorum
(Grimont et al., 2000). The organism is a facultative anaerobe that grows on food
in the presence or absence of oxygen. Salmonella can grow within a wide range of
temperatures from 8 to 450 C (Hanes, 2003), but the optimum temperature is 37 0
C. Typically, Salmonella pH growth range lies between 4.5 to 9.0 (D'Aoust, 1989);
however, the most favorable pH for growth is between 6.5 to 7.5 (Garcia-Del
Portillo, 1999), Salmonella is tolerant to high moisture and grows best in conditions
with a water activity (aw) of 0.93 (Gray and Fedorka-Cray, 2002;). Salmonella
grows optimally when sodium chloride (NaCl) is between 3 to 4% and 350 mg/L
of sodium nitrite (NaNO2) (Portillo, 2000).
The etiological agents of fowl typhoid and pullorum disease are Salmonella enterica
subsp. enterica serovar Gallinarum, which is divided into two distinct biovars
under the serogroup D1, Gallinarum and Pullorum, which are denoted as
2
S. gallinarum and S. pullorum, respectively (Shivaprashad, 1997 and Le Minor,
1984). The various motile and non-host adapted highly invasive serotypes such as
Salmonella enteritidis and Salmonella typhimurium are commonly referred to as
paratyphoid salmonellae (Gast, 1997).
Clinical signs of fowl typhoid are typical of a septicaemic condition in poultry and
include increased mortality and poor quality in chicks hatched from infected eggs.
Older birds show signs of anaemia, depression, labored breathing and diarrhoea
causing adherence of faeces to the vent. The highest mortality in pullorum disease
occurs in birds of 2–3 weeks of age. In older birds disease may be mild or
inapparent. In breeding and laying flocks susceptibility is increased at the point of
lay (Wigley et al., 2005), but reduced egg production and hatchability may be the
only signs of S.Pullorum. Trans-ovarian infection resulting in infection of the egg
and hatched chicks or poults is one of the most important transmission routes for
both diseases.
Salmonella gallinarum and S. pullorum are host adapted to avian species

(Eswarappa et al., 2009) and are considered to pose a minimal zoonotic risk
(Shivaprasad, 2000), although the genome is continually evolving, which could
theoretically widen the host range in future (Liu et al., 2002).
Pullorum disease and fowl typhoid causes mortality, especially in young chicks,
reduced productivity and hatchability in layer birds. Hossain et al., (2004)
performed a study to determine the seroprevalence and mortality in chickens
caused by pullorum disease and fowl typhoid from 5 different government
poultry farms in Bangladesh. Muktaruzzaman et al., (2010) used several types of
biochemical media and reagents to identify Salmonella isolates. But no work has
been done yet in Bangladesh to characterize the non- motile poultry Salmonella at
molecular level and to detect the clonal relationship, among the Salmonella sp by
PFGE.
3
Considering the above situation the present study was undertaken with the
following objectives:
I. To find out Prevalence of Salmonella in local and commercial chickens.
II. To isolate and identify Salmonella organism from commercial layers and
local chickens by biological methods and molecular characterization
using PCR.
III. To determine antibiotic resistance profile of serovars isolated from

layer.
IV. To find out the clonal relatedness among the poultry Salmonella using
PFGE.
4
CHAPTER II
REVIEW OF LITARATURE
The present research was carried out for the isolation, identification and
molecular characterization of poultry Salmonella. In our knowledge, molecular
characterization of poultry Salmonella by PCR and PFGE had not been done
before in Bangladesh. However, the research works relevant to the present study
in any part of the world have been reviewed and presented in this chapter.
2.1 Incidence and Prevalence of Salmonellosis in chickens
Centre for disease control and prevention (2013) reported that a total of 98
persons infected with the outbreak strains of Salmonella Infantis, Salmonella Lille,
Salmonella Newport, or Salmonella Based on ongoing epidemiologic investigations,
additional ill persons infected with Salmonella serotypes Lille and Newport
reported contact with chicks, ducklings, and other live baby poultry from Mt.
Healthy Hatchery in Ohio, and these strains have been added to the outbreak.
Bae et al., (2013) isolated Salmonella from 10 (66.7%) of the first and 5 (33.3%) of
the last chilling waters and from 32 (42.7%) carcasses originating from 9
slaughterhouses. The major prevalent serotypes of Salmonella originating from 2
duck slaughterhouses and 13 chicken slaughterhouses tested were S. typhimurium
and S. enteritidis, respectively.
Li et al., (2013) identified 165 Salmonella enterica isolates from 1382 samples taken
from conventional farms, abattoirs and retail markets from 2010 to 2011 in
Sichuan, China Among these isolates, S. enterica serotypes Derby (76 isolates, 46%)
and S. typhimurium (16 isolates, 10%) were the most prevalent.
Lapuze et al., (2012) provided a comparison on the prevalence of Salmonella

infection in layer hens from commercial layer farms with high and low rodent
densities was investigated. Out of 280 laying hens sampled from three commercial
5
layer farms with high rodent densities, Salmonella enterica subsp. enterica serovar
Enteritidis (Salmonella Enteritidis) was isolated from 20 (7.14%) hens and
Salmonella enterica subsp. enterica serovar Infantis (Salmonella Infantis) from three
(1.07%) hens.
Centre for disease control and prevention (2012) reported that Salmonella
infections from contact with live poultry (chickens, ducks, turkeys, and geese)
continue to be a public health problem. In summer 2011, two clusters of human
Salmonella infections were identified through Pulse Net, a molecular subtyping
network for foodborne disease surveillance. Standard outbreak and traceback
investigations were conducted. From February 25 to October 10, 2011, a cluster of
68 cases caused by Salmonella serotype Altona and a cluster of 28 cases caused by
Salmonella Johannesburg were identified in 24 states. Among persons infected,
32% of those with Salmonella altona and 75% of those with Salmonella
Johannesburg were aged ≤5 years. Forty-two of 57 (74%) Salmonella altona patients
and 17 of 24 (71%) of Salmonella Johannesburg patients had contact with live
poultry in the week preceding illness. Most patients or their parents reported
purchasing chicks or ducklings from multiple locations of an agricultural feed
store chain that was supplied by a single mail-order hatchery. Live poultry were
purchased for either backyard flocks or as pets.
Lassniq et al., (2012) conducted a survey in Austria an EU-wide baseline on the

prevalence of Salmonella spp. in broilers organized by the EU commission was
conducted from October 2005 to September 2006. In Austria, 363 flocks on farms
consisting of at least 5000 broilers each were tested. 28 flocks (7.7%) showed
infections with Salmonella spp., eight flocks (2.2%) had either S. enteritidis (six
flocks) or S. typhimurium (two flocks). In detail, S. enteritidis (1.7%), S. typhimurium
(0.6%), S. montevideo (4.1%), S. infantis 0.6%, S. senftenberg, S. tennessee and S.
virchow (0.3% each) have been found.
Crespo et al., (2012) found that environmental swabs from pullet houses acts as a
source of Salmonella enteritidis (SE) which was identified by the Rapid Chek SE
immunoassay test. Of the 1162 samples tested in 2011, 20 samples were positive,
6
but only two samples were confirmed positive for SE by culture. Seventeen
positive samples were from pullet houses that had been vaccinated with SE
bacterin 2 to 3 days prior to submission to the lab.
Ellerbroek et al., (2010) isolated Salmonella from 400 imported chicken carcasses
in Bhutan and from 178 pig carcasses in Vietnam for antibiotic resistance analyzed
on a random basis against 14 antimicrobial agents. Among the poultry samples
tested, 13% were positive for Salmonella.
Kwon et al., (2010) reported that Salmonella enterica serovar gallinarum (Salmonella
gallinarum) is the causative agent of fowl typhoid (FT), a severe systemic disease
of chickens that resulted in high mortality since 1992, in Korea. According to the
analysis based on the chicken breeds (n=521 farms), the incidence of FT in
commercial broilers, Baeksemi (a mixed breed of male meat-type breeder and
female commercial layer), commercial layers, native chickens, and broiler
breeders was 47.7, 28.4, 17.2, 5.1, and 1.3%, respectively. Of the affected broilers,
over 90% birds were under 2 wk of age, indicating it was possible that they were
infected with Salmonella gallinarum via vertical transmission.
Ahmed et al., (2009) isolated 10 Salmonella, from retail chicken meat in Hiroshima
prefection, Japan, the samples were assayed for antimicrobial susceptibility, the
presence of integrons and antimicrobial resistance genes.
Oliveira et al., (2006) isolated strains of Salmonella spp. from poultry products in
the State of Ceara, Brazil. A total number of 114 samples were collected from 63
broiler carcasses derived from two processing plants and two supermarkets and
51 extra samples were collected in broiler farms located in the State of Ceara,
which used three live production stages. Each excreta sample considered of a
fresh excreta pool from 100 birds. Samples were submitted to microbiological
analysis and the isolated Salmonella strains were tested for antimicrobial
sensitivity. No Salmonella was isolated from excreta samples, while broiler carcass
samples showed a high contamination rate of 11.8%. Three serotypes were
identified: S. enterica serovar enteritidis 50%, S. enterica serovar panama 33% and S.
enterica serovar newport 17%.
7
Islam et al., (2006) studied on the seroprevalence, isolation and characterization
of Salmonellae from layer chickens during the period from January to May 2006.
The used materials were blood sample, cloacal and liver swabs from live and
dead birds respectively and visceral organs (liver, lungs, spleen and intestine).
The detection methods used were serum plate agglutination (SPA) test; necropsy
and histopathology; cultural, morphological and biochemical test. The overall
seroprevalence was 43.4%. A total of 33 (21.02%) Salmonellae from live and dead
birds were isolated. The isolation rate of Salmonellae was higher in seronegative
(31.6%) group than seropositive (3.2%) group. Out of 33 Salmonella isolates, 25
were S. pullorum, 3 were S. gallinarum and the rest 5 were motile Salmonellae.
Mahendra et al., (2006) conducted a cross-sectional study of raw meat samples

obtained from the local meat market of Kathmandu Metropolitan City, Nepal,
during September 2002-May 2003, with special emphasis on isolation and
identification of Salmonella spp. A total 123 raw meat samples (55 chicken meat, 37
buffalo meat and 31 goat meat) were collected and analysed relative to season.
Salmonella spp. was found in 14 (11.4%) meat samples. Eight (14.5%) samples of
chicken meat, 5 (13.5%) samples buffalo meat, and in one sample (3.3%) of goat
meat were positive for Salmonella. The Salmonella spp. identified were S. pullorum
(3.3%), S. gallinarum (0.8%), S. typhi (1.6%), S. choleraesuis (0.8%) and Salmonella of
subgenus I or II group (4.9%). The seasonal prevalence of Salmonella was highest
in the months of April/May.
Tsai et al., (2005) sampled cloacal swabs from 100 duck farms in Taiwan between
March 2000 and January 2001 for isolation and standard cultivation of Salmonella
spp. and thermophilic Campylobacter spp, Salmonella spp. were isolated from 4.6% of
ducks from 20% of duck farms. Ten serotypes of S. enterica were identified: S.
postsdam (31.9% of isolates, S. dusseldorf (18.7%), S. indiana (14.3%), S. typhimurium
(7.7%), S. hadar (5.5%), S newport (4.4%), S. derby (4.4%), S. montevideo (2.2%).
Hossain et al., (2004) performed a study to determine the seroprevalence and

mortality in chickens caused by pullorum disease and fowl typhoid from 5
different government poultry farms. The chickens were divided according to 3
8
age groups during the period from June 2002 to May 2003. The overall
seroprevalence of salmonellosis, especially pullorum disease and fowl typhoid
was 26.67%. The mean seropositivity values of the 5 farms for the 3 different age
groups from each farm were 18.97±2.27, 33.20±3.53 and 27.84±2.67% on the 10th,
24th and 40th week of age, respectively. The mean seropositivity values of the 3
different age groups from each farm were 36.77±5.40, 24.05±3.9, 26.80±3.90,
25.77±4.49 and 19.93±3.28% in Mirpur, Savar, Bogra, Kishoregonj and Tangail
poultry farms respectively. S. gallinarum, the agent causing fowl typhoid, was the
most predominant organism accounting for 295 isolates. Only 74 isolates were
identified S. pullorum, the agent causing pullorum disease. The highest mean
proportion of mortality due to fowl typhoid among the 5 farms was 43.36±2.39%
and was highly significant (P<0.001) in the 27-39 weeks age group. The proportion
of mortality due to pullorum disease was highly significant (P<0.001) in the 0-13
weeks age group. The highest mean value of pullorum disease was 10.47±1.14
from the 5 farms.
Batabyal et al., (2003) reported that among a total of 298 isolates obtained from
both ailing (n=122) and dead (n=98) quails, only in 10 (3.4%) were Salmonella
gallinarum identified. These strains yielded typical results in biochemical tests, ie.
indole test negative and MR test positive, and were highly pathogenic to adult
quails (inoculated orally), resulting in 100% mortality
Sujatha et al., (2003) isolated Salmonella gallinarum from poultry in and around
Hyderabad and Secunderabad cities in India and was characterized. Six isolates of
S. gallinarum were obtained from 21 clinical samples by employing both pre-
enrichment and selective media. Percentage of positive isolation was 28.67%.
Georgiades and Iordanidis (2002) reported that during the last decade, 618
pigeons, 182 canaries and 71 psittacines, from Thessalonica greater area (Greece)
were examined in the Clinic of Poultry Diseases. Post mortem examination was
performed in all birds and samples were collected from the liver, spleen, heart
and intestine for bacteriological examinations. Blood agar (5% sheep blood),
MacConkey agar and Selenite broth were used for culturing suspect material.
9
Serological and biochemical tests were performed from colonies grown on agar
plates. Salmonella was isolated from 53 out of 618 pigeons (8.6%), 33 out of 182
canaries (18.1%) and 2 out of 71 psittacines (2.8%). S. typhimurium was the most
frequently isolated serotype in pigeons (75.5% of isolates), followed by S.
enteritidis (11.3%). S. gallinarum and S. hadar (3.8%), as well as S. abony (1.9%) were
isolated less frequently. S. typhimurium was also the most frequently isolated
serotype in canaries (90.9% of isolates), followed by S. enteritidis (6.1%). S. infantis
and S. gallinarum were each isolated once from psittacines.
Wieliczko et al., (2001) studied on the prevalence of Salmonella gallinarum and

Salmonella pullorum in commercial layer farms in the southwestern region of
Poland and the characteristics of the isolated strain were studied (1999-2000). The
presence of both microorganisms was detected in 10 farms of brown laying hens
aged 18-55 weeks old. The lesions observed were typical of acute fowl typhoid.
Ten strains (62.5%) of S. gallinarum and 6 strains (37.5%) of S. pullorum were
isolated.
2.2 Isolation, Identification and cultural characteristics of

Salmonella spp.
Hyeon et al., (2012) isolated Salmonella from 118 of the 180 samples (65.5%).
Salmonella were detected in 105 samples (88%) plated on XLD and 111 samples
(94%) plated on SM-ID 2 when RVS broth was used for enrichment, and 43
samples (36.4%) plated on XLD and 67 samples (56.8%) plated on SM-ID 2 when
the MKTTn broth was used. The highest sensitivity was found in the RVS-XLD
combination (0.99), followed by RVS-SM-ID 2 (0.97).
Muktaruzzaman et al., (2010) mentioned that Salmonella organisms showed

different cultural characteristics in different media. These were turbidity in Tetra
Thionate broth, pink white color colonies in Brilliant Green agar, gray white
colony in Nutrient agar, slightly grayish color colonies in Salmonella Shigella
agar, black color colony in Tripple Suger Iron agar, pale color colonies in
MaConkey’s agar, well defined glistening colonies in Blood agar and pinkish
colonies in EMB agar.
10
Sujatha et al., (2003) reported that the liver of chicken was found to be the most
suitable organ for isolation of S. gallinarum. Use of pre-enrichment media was
better than conventional media for the successful isolation of the bacteria. Isolates
revealed moist, pin-sized, circular, non-lactose fermenting colonies on
MacConkey, S-S, BGA, and BHI agar media.
Habrun and Mitak (2003) counducted a study during 2001 and 2002 with two
hundred and sixty-five pooled poultry faeces samples were examined at the
Department of Bacteriology, Croatian Veterinary Institute, using standard culture
methods. Upon enrichment, three different selective broths vz. tetrathionate
broth, selenite cystine broth and Rappaport Vassiliadis broth, inoculated on
xylose-lysine-deoxychockolate (XLD) and Rambach agars after incubation, were
used for isolation. The poorest results were obtained when tetrathionate broth
was used (2 or 12.5% Salmonella spp. isolation on XLD agar and 5 or 31% isolation
on Rambach agar). Selenite cystine broth proved more reliable (4 or 25% isolation
on XLD agar and 12 or 87.5% isolation on Rambach agar). Rappaport Vassiliadis
broth yielded the best results (7 or 44% isolation on XLD agar and 16 or 100%
isolation on Rambach agar). Rambach agar showed a considerably higher
sensitivity than XLD agar. Enrichment, two different selective broths, and two
different selective and differential agars should be used in culture isolation of
Salmonella spp. Tetrathionate broth with 24 hours incubation is not a reliable
medium for Salmonella spp. isolation from the faeces.
2.3 Biochemical and serotyping of Salmonella serovers
Kwon et al., (2010) reported that the phenotypic analysis, Salmonella gallinarum
strains (n=142) isolated during 2001 to 2007 showed the same pattern in the
majority of the biochemical tests such as carbohydrate fermentation and amino
acid decarboxylation. Interestingly, all of the strains could not ferment rhamnose,
but SG 9R could, making rhamnose a potential biomarker to distinguish the
vaccine strain.
Selvam et al., (2010) reported that Cloacal swabs of birds were subjected for
isolation and identification of Salmonella pullorum and Salmonella gallinarum. The
11
biovars Salmonella gallinarum and pullorum were differentiated based on TSI agar
slant inoculation and different sugar fermentation tests. They found that none of
the biovars fermented galactose and dulcitol and this indicated the isolates were
not biovar Gallinarum. All the isolates fermented glucose and were confirmed as
Pullorum.
Muktaruzzaman et al., (2010) conducted several types of biochemical media and

reagents like bacteriological peptone, methyl red, phenol red, liquid paraffin wax,
MR-VP media, potassium hydroxide, V-naphthol, alcohol and dulcitol were used
in this study to identify salmonella isolates. In Methyl red test, Positive reaction
was indicated by the persistence of red color, indication of acidity and the
negative one by the yellow color. In Voges-Proskauer (V-P) test the appearance of
pink color indicated positive test. In case of Indole test A red color in the reagent
layer indicated indole and negative case, there was no development of red colour.
In the carbohydrate fermentation test acid production was indicated by the color
change from red to yellow of the medium and the gas production was noted by
the appearance of gas bubbles in the inverted Durham's tube. Motility test was
performed by the hanging drop slide method. The motile and non-motile
organisms were identified by observing motility in contrasting with to and fro
movement of bacteria.
Brooks et al., (2008) reported that the structure and serological specificities of the
lipopolysaccharides (LPSs) from Salmonella enterica serovar gallinarum biovar
Pullorum provided an improved basis for the distinction between antigenic types
and the development of improved diagnostic tests. Several of the anti-LPS O-PS
Mabs were specific for S. pullorum and other serogroup D1 Salmonella, and are
potentially useful for the development of improved diagnostic tests for these
organisms
Sujatha et al., (2003) reported that all isolates of Salmonella showed positive
reaction to M.R., citrate, nitrate, and H< sub>2</ sub>S. Sugar fermentation tests
revealed acid without gas from glucose, maltose, dulcitol, galactose, trehalose,
12
xylose, and rhamnose. All the isolates were confirmed as S. gallinarum with
antigenic structure 9,12, - -, by N.S.E.C.
Hossain (2002) stated that among five basic sugars the Salmonella ferment
dextrose, maltose and mannitol with production of acid and gas but no
fermentation was observed in lactose and sucrose.
Proux et al., (2002) reported that the biovar Salmonella pullorum and Salmonella
gallinarum were differentiated by the use of sugars such as maltose, dulcitol and
glucose.
Shah et al., (2001) characterized Salmonella enterica subspecies enterica serovar

gallinarum (S. gallinarum) strains (n=92) of avian origin isolated from Gujarat State
by biotyping, antimicrobial drug resistance, bacteriocin production, and serum
resistance. Variability in the phenotypic potentials associated with production of
H2S in TSI, ornithine decarboxylase activity, and fermentation of rhamnose were
observed. Antimicrobial sensitivity test revealed multiple drug resistance and
chloramphenicol was found to be the most effective, whereas sulfamethoxazole
was the least effective drug. Fifteen (16.3%) strains were bacteriocin producers,
whereas 10 strains were resistant to bactericidal activity of guineapig and chicken
sera.
2.4 Molecular Characterization of Salmonella spp.
Pulido-Landínez et al., (2013) described that to assess diversity of Salmonella

enterica serotypes present in poultry and their environment from Southern Brazil,
the Kauffman-White-LeMinor (KWL) scheme was used to serotype a total of 155
isolates. Isolates were then re-examined with nested PCR and sequencing of the
dkgB-linked Intergenic Sequence Ribotyping (ISR) region that assesses single
nucleotide polymorphisms occurring around a 5S ribosomal gene. Serotypes
identified were Heidelberg (40.6%), Enteritidis (34.2%), Hadar (8.4%),
Typhimurium (3.9%), Gallinarum (3.2%), Agona (1.3%), Cerro (1.3%), Livingstone
(1.3%), Infantis (0.6%), Isangi (0.6%), Mbandaka (0.6%), Montevideo (0.6%), and
Senftenberg (0.6%).
13
Temelli et al., (2012) evaluated the capability of the Vitek immunodiagnostic
assay system easy Salmonella (VIDAS ESLM) method and a specific real-time PCR
system (Light Cycler), in detecting Salmonella from a total of 105 naturally
contaminated samples comprised of poultry meat and poultry meat products.
Twelve (33.33%), 11 (30.55%), and 18 (50.00%) out of 36 poultry meat samples
were positive for Salmonella by ISO, VIDAS ESLM, and LCPCR, respectively.
Salmonella detection rates from poultry meat products were 5.80% for ISO and
8.69% for LCPCR, whereas none of these products tested positive by VIDAS
ESLM.
Kang et al., (2011) designed duplex PCR primer to target polymorphic regions of
glgC and speC genes showing multiple mutations in the sequenced S. enterica
subsp. enterica serovar Gallinarum 287/91 genome and were applied to the
specific identification of biovars Gallinarum and Pullorum. Boiled lysates of 131
reference and field strains of Salmonella and other related Gram-negative bacteria
were tested to validate the duplex PCR assay. All strains of biovars Gallinarum
(n=53) and Pullorum (n=21) tested were correctly identified based on this assay
(100% sensitivity) while the other strains (n=57) were PCR negative (100%
specificity). These results demonstrate that a highly accurate biovar-specific
duplex PCR assay can be performed for the rapid identification and
discrimination of biovars Gallinarum and Pullorum from field isolates.
Jawad and Hamadani (2011) described that the PCR technique revealed that 34
isolates of Salmonella spp contained fimA gene ( DNA amplification showed one
distinct band with molecular weight of 85 bp when electrophorised on agarose
gel), while for fimC gene, 32 Salmonella isolates were S. typhimurium appeared to
contain this gene ( DNA amplification showed one distinct band with M. Wt. of
257 bp).The results of this study revealed that the PCR technique had a high
specifity (100%) in the detection of Salmonella spp., Salmonella typhimurium in
comparison to culture and biochemical test, Mini API 20 E and serological tests.
14
Simone et al., (2009) used primer speF for the detection of S. gallinarum. A
forward primer, speF-1 (5’- TTA GCC GTC ATT GCC CGG ATT -3’) and a reverse
primer, speF-4 (5’- ACG AGG TTT AAT GAC GTA GC -3’) were used.
Amplification reaction mixtures contained 30 µL X-mix (916 µL H2O milli-Q, 120
µL 10X buffer, 120 µL dNTP (2 mM), 36 µL MgCl ); 0,5 µL of each primer 1 e 4
(speC or speF) and 0,4 µL taq DNA polymerase (Invitroven 10342-020). The cycling
parameters were 92ºC for 3 min, followed by 24 cycles including denaturation at
92ºC for 20seg, annealing at 50ºC for 1min, extension at 72ºC for 3min, and a final
extension cycle of 72ºC for 5min. The amplification products were observed by
electrophoresis on 1% agarose gel and the size of the products was analyzed in
comparison to a 1Kb ladder M.W. size marker (GIBCO) after ethidium bromide
staining.
Kisiela et al., (2005) stated that digestion of PCR amplicons of the fimH gene from
S. gallinarum biovar gallinarum strains with SacI gave two DNA fragments of 554
and 472 bp and only one fragment of 1026 bp for S. Gallinarum biovar Pullorum.
This allows a clear differentiation between these two biovars.
McClelland et al., (2001) showed that there was unspecific amplification of gene
speF, leading to the production of a fragment greater than was expected.
Hyuk et al., (2000) stated that the variable regions of the flagellin C gene from 41
biotype pullorum and 52 biotype gallinarum were amplified by colony-PCR and
analyzed by single strand conformational polymorphism (SSCP) method.
Differences in SSCP electrophoretic patterns were confirmed by nucleotide
sequencing. In addition, PCR-RFLP with Hinp1I was also successfully applied to
differentiate the two biotypes. These results suggested that the variable regions of
fliC could be used as a genetic marker to differentiate biotype gallinarum from
biotype pullorum
Kwon et al., (2000) differentiated two biotypes based on biochemical

characteristics. However, conventional culture and biochemical assays were time-
consuming, laborious and need sterile laboratory practices. Although the two
biotypes, gallinarum and pullorum were non-motile, they possessed the phase 1
15
flagellin C gene. The variable regions of the flagellin C gene from 41 biotype
pullorum and 52 biotype gallinarum were amplified by colony-PCR and analyzed
by single strand conformational polymorphism (SSCP) method. Differences in
SSCP electrophoretic patterns were confirmed by nucleotide sequencing. In
addition, PCR-RFLP with Hinp1I was also successfully applied to differentiate the
two biotypes. These results suggested that the variable regions of fliC could be
used as a genetic marker to differentiate biotype gallinarum from biotype pullorum.
2.5 Antibiotic Susceptibility test
Seyyedeh et al., (2013) described that the stereotyping results showed that 34 of
44 isolates of Salmonella belonged to Salmonella infantis (79.5 %), one strain (2.3%)
of group C and 8 strain (18.2%) of group D. However, all these strains were
sensitive to Cefotaxime and Ciprofloxacin, and 100% were resistant to Nalidixic
acid, Tetracyclin and Sterptomycin. The most common resistance pattern (34.1%)
was towards six antibiotics, and 6.8% of strains were resistant to at least three
antibiotics.
Ramya et al (2013) described that the sensitivity of S. Enteritidis was 100% for
ciprofloxacin followed by chloramphenicol and amikacin (96%), gentamycin
(90%), amoxicillin (82%), streptomycin (80%), tetracycline (76%), nalidixic
acid (68%), ampicillin (58%) and sulfonamide (10%). The resistance was highest
for sulfonamide (76%) followed by ampicillin (32%), nalidixic acid (30%) and 6-
20% for gentamycin, amoxicillin and tetracycline.
Bae et al., (2013) reported that regarding the characteristics of their antibiotic
resistance, 8 of the 11 ampicillin resistant isolates carried blaTEM only, two carried
blaTEM and blaCTX-M-14 and one carried blaCTX-M-3 and only one AmR isolate with the
blaCTX-M-3 β-lactamase gene Salmonella strain. Twenty seven Salmonella isolates
showed nalidixic acid resistance with a mutation at amino acid codon Asp87 in
gyrA and no mutation in the parC gene.
Gallati et al., (2013) reported that over the years 2007-2011, the reports of
salmonellosis caused by Salmonella enterica serovar 4,[5],12:i:- significantly
increased. A high prevalence of multidrug-resistant isolates, mainly showing an
16
ampicillin-streptomycin-sulfonamide-tetracycline resistance pattern (ASSuT), was
observed. In addition, four extended spectrum beta lactamase (ESBL) (CTX-M-
55)-producing isolates were found.
Li et al., (2013) identified a total of 165 Salmonella enterica isolates from 1382
samples taken from conventional farms, abattoirs and retail markets from 2010 to
2011 in Sichuan, China. Among these isolates, S. enterica serotypes derby (76
isolates, 46%) and typhimurium (16 isolates, 10%) were the most prevalent, and
high antimicrobial resistance observed for tetracycline (77%), nalidixic acid (41%)
and spectinomycin (41%).
Imad et al., (2012) stated that antimicrobial susceptibility test of the 98 isolates of
Salmonella revealed that 32.7% were resistant to one or more of the 24
antimicrobials tested. Generally, resistance for 13 different antimicrobial drugs
was recognized. The most common resistance was to streptomycin (24/32, 75%),
ampicillin (19/32, 59.4%), tetracycline (15/32, 46.9%).
De et al., (2012) stated that the antimicrobial susceptibility of E. coli, Salmonella

spp. in chicken. For E. coli and Salmonella spp. clinical resistance to newer
compounds (Cefepime, cetotaxime and ciprofloxacin) was absent. Colistin
sulphate resistance was absent for E. coli but apparent for Salmonella spp
Iwabuchi et al., (2011) described that among 452 Salmonella isolates, 443 (98.0%)
were resistant to one or more antibiotics, and 221 (48.9%) showed multiple-
antibiotic resistance, thereby implying that multiple-antibiotic resistant
Salmonella organisms are widespread in chicken meat in Japan. Resistance to
oxytetracycline was most common (72.6%), followed by dihydrostreptomycin
(69.2%) and bicozamycin (49.1%).
Hyeon et al., (2011) observed that in salmonella the highest antibiotic resistance
was to erythromycin (100%) followed by streptomycin (22.2%) and tetracycline
and chloramphenicol (16.7%). Of the 18 isolates, 5 (27.8%) were resistant to two or
more antibiotics, and 1 isolate from chicken meat was resistant to eight antibiotics,
including cephalosporins.
17
Lu et al., (2011) evaluated the antimicrobial resistance of Salmonella isolated in
2008 from a chicken hatchery, chicken farms, and chicken slaughterhouses in
China. More than 80% of the S. indiana isolates were highly resistant to ampicillin
(97.7%), amoxicillin/clavulanic acid (87.9%), cephalothin (87.9%), ceftiofur
(85.7%), chloramphenicol (84.9%), florfenicol (90.9%), tetracycline (97.7%),
doxycycline (98.5%), kanamycin (90.2%), and gentamicin (92.5%). About 60% of
the S. indiana isolates were resistant to enrofloxacin (65.4%), norfloxacin (78.9%),
and ciprofloxacin (59.4%). Of the S. indiana isolates, 4.5% were susceptible to
amikacin and 5.3% to colistin. Of the S. enteritidis isolates, 73% were resistant to
ampicillin, 33.1% to amoxicillin/clavulanic acid, 66.3% to tetracycline, and 65.3%
to doxycycline, whereas all of these isolates were susceptible to the other drugs
used in the study.
Petrovic et al., (2011) worked on 480 samples of chicken liver and carcasses swabs
were taken from 7 abattoirs. The presence of Salmonella spp. was found in 69
samples (14.37%). Intermediate susceptibility or resistances to one or more
antimicrobial drugs were found in 28(40.58%) isolates of Salmonella spp. It was
concluded that the common zoonotic pathogens transferred by chicken meat. The
application of prophylactic and sanitary measures in facilities, abattoirs and
during meat processing may considerably reduce the incidence of resistance
zoonotic bacteria.
Kang et al., (2010) determined the antimicrobial susceptibility of Salmonella

enterica serovar gallinarum isolates (n=105) from chickens in South Korea between
2002 and 2007 by minimum inhibitory concentrations of 16 antimicrobials, and
their predominant resistance profiles were genetically characterized. Most isolates
(99/105; 94.3%) were resistant to nalidixic acid and resistant/intermediately
resistant to fluoroquinolones, and 63.8% (67/105) of the isolates were resistant to
three or more antimicrobials.
Wouafo et al., (2010) collected 150 chickens from eight retail markets in Yaounde,
and 90 (60%) tested positive for Salmonella. The isolates were tested for their
susceptibilities to amoxicillin, amoxicillin/clavulanic acid, cefoxitin, cefotaxime,
18
gentamicin, streptomycin, tetracycline, chloramphenicol, sulfonamides, nalidixic
acid, ciprofloxacin, and trimethoprim/sulfamethazole by disk diffusion assay.
Minimum inhibitory concentrations of ampicillin, streptomycin, tetracycline,
sulfonamides, and nalidixic acid were determined for the resistant strains by agar
dilution method. Eleven isolates (10.7%) of the 103 tested were susceptible to all
antimicrobials. Resistance was most observed to tetracycline (84.5%),
streptomycin (44.7%), and nalidixic acid (34%). Forty-one isolates (39.8%) were
multidrug resistant (resistant to three or more antimicrobials from different
classes).
Soomro et al., (2010) described that all salmonella isolates showed sensitivity to
cefotaxime, ceftazidime, gentamicin, tobramycin, ciprofloxacin, ofloxacin, and
chloramphenicol, whereas resistance to streptomycin, tetracycline, and ampicillin
was also detected. A lower proportion of isolates were resistant to kanamycin and
trimethoprim–sulphametoxazole.
Islam et al., (2008) isolated a total of 46 Salmonella spp. from 150 blood cultures.
Salmonella typhi was predominant serotype, followed by S. paratyphi . A Results
of Antimicrobial susceptibility pattern of Salmonella paratyphi against antibiotics
showed that isolates were sensitive to Amoxicillin (75%), Amoxyclave (75%),
Aztreonam (100%), Amikacin (100%), Cefaclor (100%), Cefixime (100%),
Ceftazidime (68.75%), Ceftriaxone (68.75%), Cephalexin (75%), Ciprofloxacin (5
0%), Co-trimoxazole (75%), Gent amycin (100%), Mecillinam (100%), Nalidixic
acid (75%), Netilmicin (100%) and resistant to Amoxicillin (25%), Amoxyclave
(25%), Azithromycin (100%), Ceftazidime (31.25%), Ceftriaxone (31.25%),
Cephalexin (25%), Ciprofloxacin (50%).
19
2.6 Pulsed Field Gel Electrophoresis (PFGE)
Issack et al., (2013) collected forty-nine isolates of salmonella between 2008 and
2011 and were analyzed. Overall characterization of the isolates by PFGE digested
with XbaI and BlnI resulted in eight different patterns. The largest of the clusters
in the composite dataset consisted of 20 isolates, including two raw chicken
isolates, four poultry isolates, and nine human stool isolates from two outbreaks.
A second cluster consisted of 18 isolates, of which 12 originated from human
blood and stool samples from both sporadic and outbreak cases. Six food isolates
were also found in this cluster, including isolates from raw and grilled chicken,
marlin mousse, and cooked pork. One poultry isolate had a closely
related PFGE pattern. The results indicate that one clone
of Salmonella typhimurium found in poultry has been causing outbreaks of
foodborne illness in Mauritius and another clone that has caused many cases of
gastrointestinal illness and bacteremia in humans could also be linked to poultry.
Thus, poultry appears to be a major reservoir for Salmonella typhimurium in
Mauritius.
Bae et al., (2013) reported all the phenotypic and genotypic properties of the 18 S.
enteritidis and 8 S. typhimurium based on PFGE, phage types and antibiotic
resistance pattern, the predominant patterns were XEI/BEI-PT32a-NaR (n=5) and
XTI/BTI-RNDC-no resistant antibiotics (n=6), respectively.
Gallati et al., (2013) stated that the PFGE type STYMXB.0131 dominated among
human as well as food isolates. Multilocus variable-number tandem-repeat
analysis profile 3-12-10-0-0211, which, in many cases, coincided with PFGE type
STYMXB.0131 and phage type DT193 were the most prevalent types found for the
isolates further characterized by these typing methods. Our data provide strong
evidence for a spread of two specific Salmonella serovar 4,[5],12:i:- clones
(PFGE pattern STYMXB.0131, resistance type ASSuT) and (PFGE pattern
STYMXB.0131, resistance type SSuT). In contrast to the human isolates, the
pork/poultry isolates expressed predominantly the SSuT resistance type.
20
Barua et al., (2013) investigated 100 broiler farms and eleven were positive for
motile Salmonella, giving a farm-level prevalence of 11% (95% confidence interval
5-17%). The PFGE genotyping demonstrated that the isolates belonging to the
same serovars were closely related due to variation in only 1-4 bands. All the S.
Virchow and S. Java isolates, irrespective of breeder or broiler farm origin, were
plasmid-free, except for one S. Virchow isolate from a broiler farm that harboured
a 9.7 kb-sized plasmid. The S. Kentucky isolates belonged to three plasmid
profiles having plasmids of four different sizes, ranging from 2.7 to 109 kb.
Li et al., (2013) identified 165 Salmonella enterica isolates from 1382 samples taken
from conventional farms, abattoirs and retail markets from 2010 to 2011 in
Sichuan, China. The PFGE patterns were diverse. The findings indicated that most
isolates from different sampling sites were phenotypically and genetically diverse,
and Salmonella was widespread and may transmit along the food production
chain from farm to market. Isolates with decreased susceptibility to
fluoroquinolones and extended-spectrum cephalosporins, which are used to fight
foodborne Salmonella, pose a serious threat to public health.
Nogrady (2013) isolated 76 strains between 2004 and 2009 from raw meat and
fecal samples of broiler origin in nine European countries - including Hungary -
were examined by phage typing, antimicrobial resistance typing, pulsed-field gel
electrophoresis (PFGE) profile and plasmid analysis. The strains could be divided
into two large PFGE clusters with 92% similarity. Cluster A (n=39) contained 15
German, seven Italian, five British, five Polish isolates, one Austrian and one
Hungarian isolate. Five Hungarian isolates that were isolated prior to the
appearance of the MDR clone also belonged to this cluster. Strains of this cluster
comprised seven pulsotypes and 14 different phage types and were mostly
susceptible to the 12 antimicrobials tested. Cluster B (n=33) contained all but one
of the recent Austrian (n=14) and of Hungarian (n=9), six Polish, one Italian and
one German as well as the single Turkish and the Romanian strains, representing
five pulsotypes.
21
CHAPTER III
MATERIALS AND METHOD
The present research was carried out during the period of January 2013 to June
2013 in the laboratory of the Department of Microbiology and Hygiene, BAU,
Mymensingh. Some experiments were also conducted at Enteric and Food
Microbiology laboratory, iccddr,b, Mohakhali, Dhaka. The detailed outline of the
Materials and Methods are given below.
3.1 Materials
3.1.1 Study area
The samples were collected from commercial layer of BAU poultry farm and
poultry shed Dept. of Microbiology and Hygiene, BAU, Mymensingh and from
local chickens of Fasillamor Village, Mymensingh and transferred to the
laboratory of the Department of Microbiology and Hygiene, BAU, Mymensingh.
The samples were used to isolate and identify and also to characterize poultry
salmonellae by biochemical, serological and molecular methods.
3.1.2 Glassware and other appliances
Glass wares and appliances were used during the course of the experiment are as
follows: Test tubes (with or without Durham’s fermentation tube and stopper),
petridishes, conical flask, pipette, slides and cover slips, incubator, freeze,
thermometer, water bath, jar, microscope, sterilizing instruments, electronic
balance, sterilized cotton, immersion oil, beaker, hand gloves, spirit lamps, match
lighter, bacteriological loop, glass spreader and forceps, scissors and autoclave,
gel electrophoresis apparatus, gel documentation system, thermo cycler.
3.1.3 Solid culture media
Salmonella-Shigella agar (SS), eosin methyline blue agar (EMB) xylose lysine
deoxychockolate agar( XLD), and Muller Hinton (MH) agar were used for this
study.
22
3.1.4 Liquid media for culture and reagents for biochemical tests
The liquid media used in the study were nutrient broth (NB), peptone broth,
methyl-red and Voges-Proskauer broth (MR-VP) and sugar media.
3.1.5 Salmonella specific antisera for serotyping
Salmonella antiserum (S & A Reagent Lab, Bankok, Thailand.) group D was used
[
for the serotyping of Salmonella spp.
3.1.6 Primers used for PCR
Two primer sets were used in this study. SpeF and fimA are the specific primers
to detect f Salmonella gallinarum and Salmonella typhimurium respectively.
Table- 1: PCR primers with sequence
Primer Sequence Size (bp)
FimA(F) 5'-CCTTTCTCCATCGTCCTGAA-3' 85
FimA(R) 5'-TGGTGTTATCTGCCTGACCA-3'
SpeF(F) 5'-TTAGCCGTCATTGCCCGGATT-3' 2000
SpeF(R) 5'-ACGAGGTTTAATGACGTAGC-3'
F = Forward,
R = Reverse
23
3.1.7 Agarose gel
1% and 3% agarose (Sigma) gel were used for electrophoresis of the PCR
products.
3.1.8 Antibiotic disc
The antibiotic sensitivity tests were performed by disc diffusion method.

Commercially available antibiotic discs (Oxoid, UK) were used to determine the
antibiotic sensitivity pattern. This method allowed for the rapid determination of
the efficacy of the drugs by measuring the diameter of the zone of inhibition. The
antibiotics that were used against the isolated organisms with their disc
concentration are mentioned in the Table 1.
Table-2: Antimicrobial agents with their disc concentration
Name of the antibiotics Disc concentration (µg/disc)
Ciprofloxacin (CIP) 5
Cloxacillin (OB) 5
Amoxycillin (AMP) 10
Neomycin (N) 30
Erythromycin (E) 15
Colistin sulphate (CT) 25
Legand: µg= Micro-gram
24
3.1.9. Pulsed-field gel electrophoresis (PFGE).
A. Contour-clamped homogeneous electrical field (CHEF-DRII) apparatus (Bio-

Rad)
B. XbaI restriction enzyme
C. 1% pulsed-field-certified agarose in 0.5X TBE (Tris-borate-EDTA) buffer.
D. UV transilluminator
E. A one-dimensional gel documentation system (Bio-Rad).
F. Bionumeric software (Applied Maths, Belgium).
3.2 Methods
3.2.1 Experimental Design
The entire study was divided in to four steps. The first step consists of collection
of cloacal swab of layer birds followed by isolation, identification and
characterization of Salmonellae on the basis of their colony, staining and
biochemical properties. The third step includes serotyping of the isolated
Salmonellae spp and the last step is the molecular characterization of the isolated
Salmonella spp. by PFGE and amplification of species specific gene. Confirmed
Salmonella organisms were preserved in 50% buffered glycerin.
25
Cloacal swabs were collected by sterile cotton and immediately transferred to Nutrient broth
Incubation at 370 C for 1-2 hrs
Inoculation on to SS agar
Incubation at 37º C for overnight
Bacteria showed pure black colony, subcultured on SS agar. Bacteria showed colorless translucent,pink
colony colony
Gram’s staining
Discarded
Gram (-) ve, small rod shaped bacteria.
Subculture on to XLD agar Subculture on to EMB agar
Incubation at 37º C for overnight
Small, Circular,pink colony with black centre. Small, Circular,Pink colour colony
d l d
Biochemical Characterisation
Agglutination test by Salmonella group D antisera
Motility test
Molecular detection of Salmonella spp by PFGE and PCR
Antibiotic sensitivity test
Fig-1: Experimental design for Isolation of Salmonella organism from chicken
26
3.2.2 Collection and transportation of samples
A total of 150 cloacal swabs were collected using sterile cotton bud of which 100
from of commercial layer 9 and 50 from local chicken. The swabs with bud were
inoculated into nutrient broth and brought to bacteriology laboratory of the
Department of Microbiology and Hygiene, Bangladesh Agricultural University
(BAU), Mymensingh. Aseptic measures were followed during collection and
transportation of samples.
3.2.3Preparation of culture media and reagents
3.2.3.1 Preparation of culture media
Salmonella-Shigella agar (SS), Eosin Methyline Blue agar (EMB), Xylose Lysine
Deoxychockolate agar( XLD), Muller Hinton agar, Motility Indole Urea (MIU)
media were used for this study. All these media were prepared according to the
instruction of manufacturer (Hi- media, India).
3.2.3.2 Preparation of reagents
3.2.3.2.1 Sugar media
Sugar media consisted of peptone water to which fermentable sugar was added to
the proportion of 1 percent. Peptone water was prepared by adding one gram of
peptone and 0.5 grams of sodium chloride in 100 ml distilled water. The medium
was boiled for 5 minutes, adjusted to pH 7.0, cooled and then filtered through
filter paper. Phenol red, an indicator at the strength of 0.2 percent solution was
added to peptone water and then dispensed in 5 ml amount into cotton plugged
test tubes containing a Durham’s fermentation tubes, placed inversely. These
were then sterilized in the autoclave at 1210 C for 15 minutes maintaining a
pressure of 15 lbs per sq. inch (1 kg/ cm2). The sugars used for fermentation were
prepared separately as 10 percent solutions in distilled water (10 grams sugar was
dissolved in 100 ml of distilled water). A gentle heat was necessary to dissolve the
sugar completely and sterilized by stem sterilizer. Before use, the sterility of the
sugar medium was judged by incubating the tubes overnight at 370C. The basic
27
sugars dextrose, maltose, lactose,and dulcitol were used to prepare sugar
medium.
3.2.3.2.2 Methyl-Red Voges-Proskauer (MR-VP) broth
A quantity of 3.4 gm of Bacto MR-VP medium was dissolved in 250 ml of distilled

water dispensed in 2 ml amount in each test tube and then the tubes were
autoclaved at 1210 C for 15 minutes maintaining a pressure of 15 lbs per sq. inch (1
kg/ cm2). After autoclaving, the tubes containing medium were incubated at 370 C
for overnight to check their sterility and then stored in a refrigerator for future
use.
3.2.3.2.3 Methyl-Red solution
The indicator methyl-red (MR) solution was prepared by adding 0.1 gm of Methyl
–red powder in 300 ml of 95% alcohol and diluting this to 500 ml with the adding
of 200 ml of distilled water.
3.2.3.2.4 Voges-Proskauer solution
3.2.3.2.4.1 Alpha- naphthol solution
Alpha- naphthol solution was prepared by dissolving 5 gms of 1-naphthol in 100

ml of 95% ethyl alcohol.
3.2.3.2.4.2 Potassium hydroxide solution
Potassium hydroxide (KOH) solution was prepared by dissolving 40 gms

potassium hydroxide crystals in 100 ml of cold distilled water. All chemical and
reagents were prepared according to instruction of manufacturer. (Hi- media,
India)
3.2.3.2.6. Normal saline solution
Normal saline solution was prepared by adding 0.85 gms of crystalline sodium
chloride in 100 ml of distilled water in a sterilized flask and autoclave at 1210 C for
15 minutes maintaining a pressure of 15 lbs per sq. inch (1 kg/ cm2).
28
3.2.4. Cultural characterization and isolation of Salmonella Spp.
The nutrient broth containing the samples were incubated at 37° C for 1-2 hrs and
then spread onto S-S agar plates followed by further incubation at 37° C for
overnight. Both colorless or translucent and black color colonies were observed on
S-S agar. The colonies showed black in color were subjected to subculture in SS
agar to make pure colony which were further used for Gram’s staining. In Gram’s
staining the organism appeared as gram negative rod under light microscope. The
organisms were sub-cultured into EMBA and XLDA. Thus single pure colony was
obtained. These pure isolates were used for the further study.
3.2.5 Gram’s staining
The Salmonellae colonies were characterized morphologically using Gram’s stain

according to the method described by Merchant and Packer (1967). Briefly, a small
colony was picked up from SS agar with a bacteriological loop, smeared on
separate glass slide with a drop of distilled water and fixed by gentle heating.
Crystal violate (Hi-media, India) was then applied on each smear to stain for two
minutes followed by washing with running water. Few drops of Gram’s Iodine
(Hi-media, India) was then added, which acted as mordant for one minute and
then washed with running water. Acetone alcohol (Hi-media, India) was then
added (acts as decolorizer) for few seconds. After washing with water, safranin
was added as counter stain and allowed to stain for two minutes. The slides were
then washed with water, dried in air and then examined under light microscope
with high power objective (100X) using immersion oil.
3.2.6 Differentiation of isolated salmonella using biochemical test
For this study isolated organisms with supporting growth characteristics of

Salmonella were subjected to sugar (Carbohydrate) fermentation test, MR-VP
reaction, indole reaction.
3.2.6.1 Sugar (Carbohydrate) fermentation test
The carbohydrate fermentation test was performed by inoculating a loopful of

thick test bacterial culture into the individual tubes containing sugars like
29
dextrose, maltose, lactose, dulcitol and incubated at 370 C for 24 hours. Acid
production was indicated by the change of media from pink to yellow color while
gas production was indicated by the appearance of gas bubbles in the inverted
Durham’s fermentation tubes.
3.2.6.2 Methyl-Red (MR) test
The test was performed by inoculating a colony of the test organism in 0.5 ml
sterile glucose phosphate broth. After overnight incubation at 370C, a drop of
methyl red solution was added. A positive methyl red test was shown by the
appearance of bright red colour indicated acidity while a yellow or orange colour
was considered as negative.
3.2.6.3 Voges-Proskauer (VP) test
Two ml of sterile glucose phosphate peptone broth were inoculated with a pure
colony of test organisms and incubated at 370C for 24 hours. A very small amount
(knife point) of creatine was added and mixed and 3 ml of sodium hydroxide
were added and shacked well. The bottle cap was removed and left for an hour at
room temperature. It was observed closely for the slow development of a pink
colour for positive cases.
3.2.6.4 Indol test
Two ml of peptone water was inoculated with a pure colony of bacterial culture
under observation and incubated at 370C for 24 hours after which 0.5 ml Kovac's
reagent was added, shaked well and examined after 1 minute. A red colour in the
reagent indicated positive test.
3.2.7 Serotyping by slide agglutination test
A single isolated colony from SS agar was dissolved with physiological saline
solution. A single drop of thick bacterial suspension was placed on glass slide and
a drop antiserum was added. The slide was gently rotated to mix the fluids
thoroughly. These cultures which agglutinated within one to two minutes were
selected as positive for Salmonella pullorum or Salmonella gallinarum.
30
3.2.8 Motility test
The motility test was performed to differentiate motile Salmonella from non-motile
one. This test was performed in Motility Indole Urea (MIU) medium (Hi-media,
India),where a sterile straight wire used to inoculate 5 ml of sterile MIU medium
taken earlier in a screw caped test tube with a smooth pure colony of the test
organism. When inoculating the MIU medium, a stab was made with a sterile
straight wire and stoppered the tube followed by incubation at 35-37° overnight.
Motility is shown by a spreading turbidity from the stab line or turbidity
throughout the medium (compared with an uninoculated tube)
Table-3: Distinguishing characters of 3 Salmonella serotypes
Tests Salmonella Salmonella Other Salmonella

pullorum gallinarum spp
Lactose - - -
fermentation
Glucose + + +
(Acid and Gas) ( Acid) (Acid and Gas)
Dulcitol - + +
( Acid) ( Acid)
Maltose ± + +
Indole Production - - -
Methyl red test + + +
Voges-Proskauer - - -
test
Motility - - +
Legends
+ = Positive
- = Negativ
31
3.2.9 Molecular Characterization by PCR
3.2.9.1 DNA extraction
A pure bacterial colony was mixed with 100 ul of distilled water which were
boiled for 10 minutes then immediately kept on ice for cold shock followed by
centrifugation at 10000 rpm for 10 minutes. The supernatant were collected and
used as DNA template for PCR.
3.2.9.2 Preparation of a PCR mixture (25µl)
PCR mixture was prepared using nucleus free water (6.5µl), master mixture
(Promega) (12.5µl), primer (F) and Primer (R) (1 µl each) and DNA template (4µl).
The fimA genes were amplified as follows: 20 cycles of denaturation (940C, 1

min), annealing (580C, 30 sec.), and elongation (720C, 1 min). Initial denaturation
(940C, 5min), and final elongation (720C, 5min), final holding temperature was
40C.
The SpeF genes were amplified as follows: 25 cycles of denaturation (920C, 20

sec), annealing (500C, 1 min.), and elongation (720C, 3 min). Initial denaturation
(920C, 3 min), and final elongation (720C, 5min), final holding temperature was
40C.
A 1% and 3% agarose gel were used for electrophoresis of SpeF and fimA gene
respectively.
3.2.10 Antibiotic Sensitivity Test
3.2.10.1 Inoculum preparation
3.2.10.2 Growth medium
Isolated colonies of the same morphological type were selected and the top of the
colonies were touched with a loop which was transferred into a tube containing 4-
5 ml of a suitable broth. The broth cultures were incubated at 37 0C for overnight.
32
3.2.10.3 Inoculation of test plates
1. Sterile cotton swab is dipped into the broth suspension. The swab should be
rotated several times and pressed firmly on the inside wall of the tube above the
fluid level. This will removes excess inoculums from the swab.
2. The dried surface of a Mueller- Hinton agar plate is inoculated by streaking the
swab over the entire sterile agar surface. This procedure is repeated by streaking
two more times, rotating the plate approximately 600 each time to ensure an even
distribution of inoculums. As a final step, the rim of the agar is swabbed.
3. The lid may be ajar for 3 to 5 minutes, but no more than 15 minutes, to allow for
any excess surface moisture to be absorbed before applying the drug impregnated
disks.
3.2.10.4 Application of antibiotic discs (Oxoid, UK) to MH agar plates
The predetermined battery of antimicrobial discs is dispensed onto the surface of

the inoculated agar plate. Each disc must be pressed down to ensure complete
contact with the agar surface. Whether the discs are placed down individually or
with a dispensing apparatus, they must be distributed evenly so that they are no
closer than 24 mm from centre to centre. The plates are inverted and placed in an
incubator set to 37 0C within 15 minutes after the disc are applied.
3.2.10.5 Reading plates and interpretation of the results
1. After 16 to18 hours of incubation each plate was examined. If the plate was
satisfactorily streaked, and the inoculum was correct, the resulting zones of
inhibition will be uniformly circular and there will be a confluent lawn of growth.
If individual colonies are apparent, the inoculum was too light and the test must
be repeated. The diameters of the inhibition zones are measured to the nearest
whole millimeter, using sliding calipers or a ruler, which is held on the back of the
inverted petri plate. The petri plate is held a few inches above a nonreflecting
background and illuminated with reflected light. The results were recorded at 16-
18 hours post incubation. Transmitted light is used to examine the zone of
inhibition.
33
2. The zone margin should 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, is ignored.
3. The sizes of the zones of inhibition are interpreted according to Zone of

Diameter Interpretative Standards of CLSI (2007).
3.2.10.6 Standard for Antibiotic sensitivity test: According to Clinical and

Laboratory Standard Institute (2007), the zone of diameter interpreted the
standard for Salmonella spp., are mentioned in the Table.
Table-4: Inhibition zone diameter for Salmonella spp.

Antibiotic discs Resistance Intermediate Sensitive
Ciprofloxacin (CIP) ≤ 15 mm 16-20 mm ≥ 21 mm
Cloxacillin (OB) 10 mm 10-12 mm 13 mm
Amoxycillin (AMP) ≤ 13 mm 14-17 mm ≥ 18 mm
Neomycin (N) ≤ 12 mm 13-16 mm 17 mm
Erythromycin (E) ≤ 13 mm 14-22 mm ≥ 23 mm
Colistin sulphate 8 mm 9-11 mm 12 mm
(CT)
Source: Clinical and laboratory standard Institute (CLSI), (2007)
3.2.11 Pulsed-field gel electrophoresis (PFGE)
Whole agarose-embedded genomic DNA from the Salmonella isolates were

prepared. PFGE was carried out using a contour-clamped homogeneous electrical
field (CHEF-DRII) apparatus (Bio-Rad), according to procedures described
elsewhere (Ribot et al., 2006). Genomic DNA of the test strains was digested by
XbaI restriction enzyme (Gibco-BRL, Gaithersburg, MD), and Salmonella enterica
serovar Braenderup was digested using XbaI, with fragments employed as
molecular size markers. Restriction fragments were separated in 1% pulsed-field-
certified agarose in 0.5X TBE (Tris-borate-EDTA) buffer. Post-electrophoresis gel-
34
treatment included gel stained and de-stained. The DNA was visualized using a
UV transilluminator, and images were digitized via a one-dimensional gel
documentation system (Bio-Rad).
3.2.11.1 Image analysis
The fingerprint pattern in the gel was analyzed using a computer software
package, Bionumeric (Applied Maths, Belgium). After background subtraction
and gel normalization, the fingerprint patterns were subjected to typing based on
banding similarity and dissimilarity, using the Dice similarity coefficient and
unweighted-pair group method employing average linkage (UPGMA) clustering,
as recommended by the manufacturer. The results were graphically represented
as dendrograms.
3.2.12 Maintenance of stock culture
For further study it was necessary to preserve the isolated Salmonella

organisms. For this purpose pure culture of isolated Salmonella organisms
were preserved in 50% sterile buffered glycerin and stored at -200 C. This
method is more appropriate for preserving bacteria with no deviation of
their original characters for several years.
35
CHAPTER IV
R
RESULTS
S
The present stu

udy was en
nvisaged to determine the prevallence and to
o identify and
a
charracterize different Salm
monella sero
ovars isolateed from com
mmercial laayer.
4.1 Prevalence
P e of salmo
onella in P
Poultry
A to
otal of 150 samples were
w collecteed during the study p
period. Am
mong them 16
sam
mples (10.66
6%) were positive fo
or salmoneella. In casse of comm
mercial lay
yer,
amo
ong 100 sam
mples, 16 samples
s (166%) were positive
p forr Salmonellaa, whereas all
sam
mples (50) co
ollected from
m local chiccken were negative
n fo
or Salmonellaa .
1
16
1
14
Percentage of positive isolates
1
12
1
10
8 number of positive
p
6 isolates
0
Commerrcial layer Local chicken
Types of poultry
Fig-2: Prevalence
P of
o Salmonellla in Poultry
y.
36
4.2. Isolation of Salmonella by cultural characteristics
4.2.1. Results of culture in different culture media

4.2.1.1 Salmonella-Shigella agar
On S-S agar all of the isolates were produced translucent, black, smooth, small
round colonies which are positive for Salmonella.
Single colony of Salmonella spp.
Fig-3 : Salmonella colony on S-S agar
4.2.1.2 Eosin Methylene Blue (EMB) agar media
Salmonella suspected colony were inoculate to EMB agar and produced pink
color colony on EMB agar.
Pink color colony of Salmonella spp.
Fig-4: Cultural properties of Salmonella on EMB agar.
37
4.2.1.3 Xylose Lysine Deoxychockolate (XLD) agar media
All of the suspected Salmonella isolates produced pink color colony with black
centre.
Pink color colony with black centre
Fig-5: Salmonella colony on XLD agar.
4.3 Results of Gram’s staining technique
The microscopic examination of Gram’s stain revealed Gram-negative, pink

colored, short rod shaped bacteria, arranged in single and paired.
Gram-negative, pink colored,

short rod shaped bacteria
Fig-6: Gram’s staining of Salmonella isolates (scale bar 10X100)
38
4.4 Results of biochemical test
4.4.1Indole test: All of the test isolates were indole negative.
4.4.2 Methyl Red test: All of the isolates were positive for Methyl Red test.
4.4.3 Voges Proskauer test : All isolated Salmonellae were negative for V-P test.
-ve -ve +ve C
Fig-7: Results of biochemical test.
4.4.4 Carbohydrate fermentation reaction
Among the 16 positive isolates five were fermented glucose and maltose and
produced both acid and gas and did not ferment dulcitol which is positive for S.
pullorum. Six of the isolates fermented glucose, maltose and dulcitol with
producing acid, which are typical characteristics for S. gallinarum. Acid
production was marked by the color change from reddish to yellow. Gas
production was marked by accumulation of gas in the Durham’s tube. It was
observed after incubation at 370C for 48 hours.
39
AG AG -ve AG C -ve AG -ve -ve C
Fig-8: Sugar test indicating Fig-9: Sugar test indicating S. pullorum.

S. gallinarum/S. typhimurium
Table-5: Results of different biochemical tests.
Tests Salmonella Salmonella Salmonella

pullorum gallinarum typhimurium
Lactose fermentation - - -
Glucose + + +
Dulcitol - + +
( Acid) ( Acid)
Maltose ± + +
Indole Production - - -
Methyl red test + + +
Voges-Proskauer test - - -
Motility - - +
Total positive isolates 5 6 5
40
4.5 Serotypin
S g of Salmo
onella isollated from
m chicken
In slide aggllutination test, the isolates were

w agglutinated wiith group D
antisserum. Agg
glutination was indicaated by clumping with nute. Total 11
hin one min
isolaates were positive
p in agglutinatio
a on test with
h group D aantisera. It indicates th
hat
amo
ong 16 isolaates 11 werre Salmonellla pullorum
m and/or Salmonella gallinarum
g a
and
otheer 5 samples Salmonellaa typhimuriu
um
–Vee with group

p D antiseraa
+Ve with
w group D antisera
+Ve with group D antisera
ontrol
Co
up D antiseerum.
Fig -10: Serrotyping of Salmonella spp by grou
4.6 Results
R off Motility test
t
Amo
ong 16 iso
olates 11 issolates werre found to
o be non motile
m aracterized by
cha
form
ming the sttab line wiithout prod
ducing turb
bidity in th
he Motility
y Indole Urrea
(MIU
U) medium
m and ano
other 5 iso
olates werre found motile
m charracterized by
chan
nging of collour of MIU
U medium.
NM N
NM M M M C
Legends
NM = Noon motile
M = Mottile
C = Conttrol
.
Fig
g-11: MIU media
m show
wing motilee and non m
motile Salm
monella.
41
Table-6: Results of cultural, staining and morphological characteristics of
the Salmonella spp isolated from chicken
Cultural characteristics Staining Motility

No. of the characteristics
+ve SSA XLD EMB
sample Opaque, Pink Pink Color, Gram negative,
translucent, color Circular, short rod
smooth, round colony smooth shaped
colonies with with colonies
black centre black
centre
1 Do Do Do Do Non- motile
5 Do Do Do Do Motile
42
4.7 Molecular Characterization by PCR
4.7.1 PCR by speF primer
Polymerase chain reaction with speF primer, 2 isolates showed positive band at
2000 bp i.e. The 2 isolates were found to be Salmonella gallinarum.
1 2 3 4 5 6
2000 bp
1000 bp
Fig-12: PCR for Salmonella gallinarum (Lane 1 was 1 kb ladder, lane 2, 5 were
negative for speF gene, lane 3, 4 were positive for speF gene, lane 6 was negative
control)
43
4.7.22 PCR by fimA
f prim
mer
Poly
ymerase ch
hain reaction
n with fim
mA primer, 3 isolates sshowed possitive band
d at
85 bp
b i.e. The 3 isolates were
w salmoneella typhimu
urium.
1 2 3 4 5 6 7 8
100
1 bp 85 bp
Fig-13: PCR fo
or Salmonellla typhimuriium. detectting fimA g
gene (85 bp). (Lane1: 1100
bp ladder, lanee 2, 3, 4 werre positive band for fiimA gene.L
Lane5, 6, 7 were
w negative
and lane 8 wass negative control).
4.8 Antibiotic
A c sensitivitty test
Antiibiogram using
u six drugs reveealed 5(31..25%) straiins to be resistant and
a
5(31
1.25%) intermediate towards
t Ciiprofloxacin
n. Fourteen
n (87.5%) isolates were
resisstant to Amoxycilin
A 87.5%) weere found intermediate
n, while ffourteen (8
resisstance towards Neom wever, nonee of the isolates were sensitive to
mycin. How
Clox
xacilin and
d Erythrom
mycin, two (12.50%) were
w interm
mediate and
d 14 (87.500%)
were resistant. In case off Colistine sulphate, 8(50%)
8 isolaates were reesistant wh
hile
the remaining
r 8 (50%) were intermed
diate.
44
Table-7: Antibiotic sensitivity pattern of Salmonella spp.
No. of CIP AML OB N CT E
Positive
isolates
1 S R R I I R
2 S I I I I R
3 S R R S I I
4 I R R I R R
5 R R R I R R
6 R R R I I R
7 I R R I R R
8 I R R I R R
9 R R R I R R
10 S R R I I R
11 S I I I I R
12 S R R S I I
13 I R R I R R
14 R R R I R R
15 R R R I I R
16 I R R I R R
S=Sensitive
I= Intermediate
R=Resistant
45
100
90
Number of isolates 80
70
Sensitive
60
Intermediate
50
Resistant
40
30
20
10
0
CIP AML OB N CS E
Antibiotics
Fig-14: Antibiogram studies against the isolated bacteria. This figure represents
that 31.25% isolates were resistant to Ciprofloxacin while 87.5% isolates were
resistant towards Amoxicillin. However, 87.5% isolates were resistant to
Cloxacilin and Erythromycin. In case of Colistine sulphate, 50% isolates were
resistant while 87.5% were intermediate resistance to Neomycin.
Colistine sulphate
Erythromycine
Cloxaciline
Ciprofloxacin
Neomycin
Amoxyciline
Fig-15: Antibiotic Sensitivity Test
46
4.9 Pulse Field Gel Electrophoresis
The XbaI-digested chromosomal DNA from 11 Salmonella sp. isolated from

Bangladesh Agriculture University in 2013 were subjected to PFGE to determine
their clonal relatedness. The XbaI restriction enzyme digested the genomic DNA
into 16 to 20 fragments (Fig. 16) ranged from 550 to 20 kb of molecular sizes. Only
one pulsotypes was found among the test strains.
Cluster analysis, which was performed with dendrograms (prepared by Dice

similarity coefficient and UPGMA clustering methods) obtained from the PFGE
patterns of XbaI-digested genomic DNA, revealed a single cluster with 100%
similarity coefficient (Fig. 17). The PFGE pattern of all the strains matched with
each other in the number and position of the DNA fragments, suggesting genetic
homogeneity.
Fig: -16: PFGE gel image of Salmonella sp.
47
Figure 17. Genomic fingerprinting of Salmonella isolated from poultry farm of
Bangladesh Agriculture University in 2013. Dendrogram was constructed by Dice
similarity coefficient and UPGMA clustering method by using pulsed-field gel
electrophoresis (PFGE) images of XbaI- restriction digested genomic DNA of the
tested Salmonella strains; the scale bar at the top (left) indicates similarity
coefficient. The banding patterns and the similarity coefficient revealed 100%
similarity for all the tested strains included in the dendrogram, suggesting high
level of clonal relatedness.
48
CHAPTER V
DISCUSSION
To our knowledge this is the first study on molecular characterization of poultry

Salmonella in Bangladesh. The present study was carried out to find the
prevalence of Salmonella in commercial layer and local chickens with isolation,
identification, characterization and differentiation by serotyping. Later the
isolated salmonellae were differentiated by biochemical tests and molecular
techniques such as PCR and PFGE. The samples were collected and immediately
brought to the microbioloy laboratory of the Department of Microbiology and
Hygiene, Bangladesh Agricultural University (BAU), Mymensingh for isolation,
identification and biochemical differentiation of Salmonella serovars.
Among 100 samples of commercial layer 16 samples (16%) were positive for
Salmonella and among 50 samples of local chickens none of the samples (00%)
were positive for Salmonella. Li R. et al., (2013) reported that a total of
165 Salmonella isolates were identified from 1382 samples (11.93%). Lapuze et al.,
(2012), isolated Salmonella from 20 (7.14%) hens out of 280 hens. Petrovic et al.,
(2011) worked on 480 samples of chicken liver and carcasses swabs from 7
abattoirs. Of which Salmonella spp. was found in 69 samples (14.37%). Ellerbroek et
al., (2010) isolated Salmonella from 400 imported chicken carcasses in Bhutan,
among the samples tested, 13% were positive for Salmonella. Kwon et al., (2010)
reported that in Korea, Salmonella organisms found in commercial layers and
native chickens were 17.2% and 5.1% respectively. In our study the prevalence
were found somewhat more than the study of Lapuze et al (2012), Petrovic et al,
(2011), Ellerbroek et al (2010) and less than the study conducted by Kwon et al
(2010).
Since the isolation and correct identification of Salmonella are very crucial for the
characterization purpose, the colonies having typical cultural characteristics were
selected as presumptive for Salmonella serovers. In this study several selective
media such as SS, EMB, XLD were used simultaneously to culture the organism
49
because all of them are not equally suitable for all the serovars of Salmonella. In
the present study, specific enriched media were used for the isolation and
identification of Salmonellae which was also used by a number of researchers such
as Hyeon JY et al., (2012), Muktaruzzaman et al., (2010), Habrun, and Mitak.,
(2003). The colony characteristics of Salmonella spp. found in this study was
translucent, black , smooth, small round colonies on SS agar, Pink color colony on
EMB agar and pink color colony with black centre in XLD agar, were similar to
the findings of other authors (Muktaruzzaman et al., (2010) Sujatha et al., (2003)
Habrun, and Mitak., (2003) ). Of which 16 samples were detected as positive for
salmonella spp in this step.
For serotyping of the isolates commercially available antiserum was used to

differentiate nonmotile Salmonella such as Sulmonella pullorum and Salmonella
gallinarum from other poultry Salmonella. Of 16 isolates 11 isolates were
agglutinated with antiserum which indicated that 11 were belonging to the sero
group D of non-motile Salmonella spp (S. pullorum and S. gallinarum). This result
correlated with the results of Brooks et al. (2008).
Motility test was fundamental basis for the detection of motile and non-motile
Salmonella organisms. In motility test, 11 isolates were non motile and 5 isolates
were motile. That means the non motile 11 isolates were either Salmonella enterica
serotype gallinarum and pullorum or and 5 motile isolates were S. typhimurium,
which was supported by other researcher (Grimont et al., 2000). The isolated
salmonella were differentiated to Salmonella enteric serover pullorum and
Salmonella gallinarun and Salmonella typhumurium on the basis of motility
followed by dulcitol fermentation test and by PCR. Finally we found 5 isolates as
S. pullorum, 6 isolates as S. gallinarum and 5 isolates as S. typhimurium. The
findings of this present study are similar with the result of Blaxland et al., 1956;
Cox and Williams, 1976; Ewing, 1986. They mentioned that differentiation of
Salmonella enterica serotype gallinarum biotype gallinarum and biotype pullorum
known as important bacterial pathogens in chickens has been relied on
biochemical tests. S. gallinarum biovars gallinarum and pullorum represent the
50
same serovar but different biovars, their identification and differentiation is based
mostly on biochemical characteristics. Such methods are the most widely used
and officially recognized, but they are time-consuming, cumbersome, and costly,
especially when many samples have to be analysed in a short time. Therefore,
DNA-based methods, especially those based on PCR, were used for the
differentiation of S. gallinarum biovar gallinarum from S. gallinarum biovar
pullorum. Of these methods, analysis of the SpeF gene by PCR related to ornithine
descarboxylation, are present in SG seems to be the most promising because of its
sensitivity and specificity. In SG, the genes are expressed and the bacterium is
positive for ornithine; in SP, the genes are not expressed and ornithine results are
negative (Christensen et al, 1992).
Molecular characterization was done by PCR, in which fimA gene was amplified
for the detection of isolated Salmonella typhimurium and speF gene for Salmonella
gallinarum. For two isolates speF gene was successfully amplified and 3 isolates
were positive for fimA gene. All conditions and result found in the PCR which
are supported by the findings of the several authors such as Simone et al., (2009)
McClelland et al., (2001) Kisiela et al (2005) Jawad and Hamadani (2011).
We have done antibiotic sensitivity test for all positive isolates. Antibiogram using
six drugs revealed 5(31.25%) strains to be resistant towards Ciprofloxacin.
Fourteen (87.5%) isolates were resistant to Amoxycilin, Cloxacilin and
Erythromycin while fourteen (87.5%) were found intermediate resistance towards
Neomycin. In case of Colistine sulphate, 8(50%) isolates were resistant while the
remaining 8 (50%) were intermediate.
Ramya et al (2013) described that the sensitivity of Salmonella spp. was 100% for
ciprofloxacin followed by amoxicillin (82%). Hyeon et al., (2011) stated that in
salmonella the highest antibiotic resistance observed was to erythromycin (100%)
followed by streptomycin (22.2%) and tetracycline and chloramphenicol (16.7%).
All isolated Salmonella in our study were multidrug resistant because, the farmers
use several types of antibiotics in sick as well as healthy birds also without
maintaining proper dose.
51
Pulsed-field gel electrophoresis of the XbaI-digested genomic DNA exhibited
banding patterns that were identical (the similarity coefficient was 100%) for all
the tested isolates which formed a tight cluster when dendrogram was
constructed using the PFGE patterns, suggesting high level of clonal relatedness
among them. The data in this study suggest the prevalence of Salmonella enterica,
which is multidrug resistant and highly clonal for commercial layers of
Bangladesh.
52
CHAPTER VI
SUMMARY AND CONCLUSION
Salmonella is regarded as the major bacterial food borne pathogen causing human
illnesses worldwide. It cannot be debated that poultry meat and eggs are major
vehicles for Salmonella transmission to human. As such, researchers, veterinarians,
processors and the government are working in tandem to search for new method
of testing and characterization for Salmonella. PCR based methods for identifying
pathogens provide more advantageous options for this purpose than
conventional testing.
The present study was conducted for the isolation and molecular characterization
of the Salmonella from the field infections. For this purpose cloacal swabs were
collected from layer birds and were subjected to different selective culture media.
Then subjected to gram’s staining. After presumptive diagnosis of the Salmonella
spp. biochemical tests were performed for the identification of the Salmonella. Then
all positive isolates were subjected to motility test by MIU media to differentiate
motile and nonmotile Salmonella. All non motile isolates were S. pullorum or S.
gallinarum and motile isolates were S. typhimurium. Then we have done basic
sugar test to differentiate between S. pullorum and S. gallinarum. On the basis of
such tests, one of which fermented glucose and producing both acid and gas but
did not ferment lactose, maltose and dulcitol and negative for VP, Indole
indicating Salmonella pullorumum.
The other group was positive for Methyl Red, fermented glucose, maltose and
dulcitol but did not ferment lactose and negative for VP, Indole indicating
Salmonella gallinarum.
53
Then serotyping was done by group D antisera, which agglutinated all suspected
isolates for S. pullorum and S. gallinarum and did not agglutinate motile Salmonella.
Then PCR was done by fimA primer for S. typhimurium suspected isolates, three
isolates were positive for this gene. Another PCR was performed for S. gallinarum
suspected isolates by speF primer, two isolates were positive for this gene.
Antibiogram using six drugs revealed 5(31.25%) strains to be resistant and 5(31.25%)
intermediate towards Ciprofloxacin. Fourteen (87.5%) isolates were resistant to
Amoxycilin, while fourteen (87.5%) were found intermediate resistance towards
Neomycin. However, none of the isolates were sensitive to Cloxacilin and Erythromycin,
two (12.50%) were intermediate and 14 (87.50%) were resistant. In case of Colistine
sulphate, 8(50%) isolates were resistant while the remaining 8 (50%) were intermediate.
Pulsed-field gel electrophoresis of the XbaI-digested genomic DNA exhibited banding

patterns that were identical (the similarity coefficient was 100%) for all the tested strains
which formed a tight cluster when dendrogram was constructed using the PFGE
patterns, suggesting high level of clonal relatedness among them. The data in this study
suggest the prevalence of Salmonella enterica, which is multidrug resistant and highly
clonal for commercial layers of Bangladesh.
Moreover this study likes to conclude with the following consequences:
i. Another research should be performed to develop vaccine seed for the

most appropriate vaccine of the present pathogenic strain in order to better
protection of both poultry and human.
ii. Detection of efficacy of the present local Salmonella vaccine of Bangladesh.
iii. Genome analysis to have an idea about the genes responsible for
pathogenicity and drug resistance of Salmonella spp. isolated from chicken.
iv. Research should be conducted to find out the incidence and prevalence of
Salmonella spp. among different commercial layer of Bangladesh and to find
out the clonal relationship among poultry Salmonella.
54
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64
APPENDCES
A. Composition of different media

1. Nutrient agar or broth
Lab-Lemco Powder 1.00 gm
Peptone 5.00 gm
Yeast extracts 2.00 gm
Agar 15.0 gm
Distilled water 100 ml
Final p H (at 250C) 7.4 ± 0.2
In case of NB agar is omitted only
2. Salmonella-Shigella agar
Peptic digest of animal tissue 5.00 gm
Beef extract 5.00 gm
Lectose 10.00 gm
Bile salts mixture 8.50 gm
Sodium citrate 10.00 gm
Sodium thisulphate 8.50 gm
Ferric citrate 1.00 gm
Brilliant green 0.00033 gm
Neutral red 0.025 gm
Agar 15 gm
Disstilled water 1000 ml
Final PH (at 25 0C) 7.0 ± 0.2
65
3. Muller- Hington Agar
Beef infusion 300.00 gm
Casein acid hydrolysate 17.50 gm
Starch 1.50 gm
Agar 17.00 gm
Final pH (at 25oC) 7.3 ± 0.1 gm
4. MIU Medium
Casein enzymic hydrolysate 10.000 gm

Dextrose 1.000 gm
Sodium chloride 5.000 gm
Phenol red 0.010 ml
Agar 2.000 gm
Final pH ( at 25°C) 6.8±0.2
B.
a. Peptone water
Bacto-peptone 10.0 gm
0.5% Phenol red 0.1 ml
b. Sugar solutions
Individual sugar 5 gm
c. Sugar media preparation
Peptone water 4.5 gm
Sugar solution 0.5 ml
C. Composition of reagents
a. 0.1% Peptone water
Peptone 1.0 gm
66
b. Kovac’s reagent for indole preparation
P-dimethyl aminobenzal dehyde 5.0 gm
Amylalcohol 75.0 gm
Conc. HCL 25 ml
c. V-P reagent- 1
5% alpha-naphthol in absolute ethyl alcohol
d. V-P reagent- 2
40% potassium hydroxide containing 0.3% creatine. The ingredients were
dissolved by heating gently over a steam bath. When in solution, added 0.05 gm
of cotton blue dye.
e. Phosphate buffered saline solution
Disodium hydrogen Phosphate 2.8 gm
Potassium chloride 0.2 gm
Potassium dihydrogen phosphate 0.2 gm
Distilled water to make 1000 ml
f. Methyl red solution
Methyl red 0.05 gm
Ethanol (absolute) 28 ml
g. Phenol red solution
0.2% aqueous solution of phenol red
h. Gram stain solution
a. Stock Crystal violate
Crystal violate 10.0 gm
Ethyl alcohol (95%) 1000 ml
b. Stock oxalate solution
Ammonium oxalate 1.0 gm
Crystal violet working solution: 20 ml of solution no. (a) mixed with 80 ml of
solution no. (b). Additional dilution was made when desired.
67
c. Lugol’s Iodine solution
Iodine crystal 1.0 gm
Potassium iodide 2.0 gm
Dissolve completely in 10 ml of distilled water, then added to distilled water to
make 300 ml. Stored in amber bottle.
d. Ethyl alcohol 250 ml
e. Acetone 250 ml
f. Counterstain
Safranin 2.5 ml
Ethyl alcohol (95%) 100 ml
68
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Isolation, Identiﬁcation and Molecular Characterization of Poultry Salmonella

Thesis · June 2013

Md. Shafiullah Parvej

The user has requested enhancement of the downloaded file.

MD. SHAFIULLAH PARVEJ

DEPARTMENT OF MICROBIOLOGY AND HYGIENE

MD. SHAFIULLAH PARVEJ

DEPARTMENT OF MICROBIOLOGY AND HYGIENE

Approved as to style and contents by

In the name of Allah, the Most Gracious, the Most Merciful.

It is a boundless opportunity to express deep sense of gratitude for the

2 RIVEW OF LITERATURE 5-21

2.1 Incidence and Prevalence of salmonellosis in chickens 5-10

2.2 Isolation, Identification and cultural characteristics of 10-11

2.3 Biochemical and serotyping of Salmonella serovers 11-13

2.4 Molecular Characterization of Salmonella spp. 13-16

2.5 Antibiotic Susceptibility test 16-19

2.6 Pulsed Field Gel Electrophoresis (PFGE) 20-21

3 MATERIALS AND METHODS 22-35

3.1 Materials 22-24

3.1.1 Study area 22

3.1.2 Glassware and other appliances 22

3.1.3 Solid culture media 22

3.1.5 Salmonella specific antisera for serotyping 23

3.1.8 Antibiotic disc 24

3.1.9 Pulsed-field gel electrophoresis (PFGE). 25

3.2 Methods 25-35

3.2.1 Experimental Design 25-26

3.2.2 Collection and transportation of samples 27

3.2.3.2 Preparation of reagents 27

3.2.3.2.1 Sugar media 27-28

3.2.3.2.2 Methyl-Red Voges-Proskauer (MR-VP) broth 28

3.2.3.2.3 Methyl-Red solution 28

3.2.3.2.4 Voges-Proskauer solution 28

3.2.3.2.4.1 Alpha- naphthol solution 28

3.2.3.2.4.2 Potassium hydroxide solution 28

3.2.3.2.6 Normal saline solution 28

3.2.4 Cultural characterization of Salmonella Spp 29

3.2.5 Gram’s staining 29-30

3.2.6 Differentiation of isolated salmonella using biochemical 30-31

3.2.6.3 Voges-Proskauer (VP) test 30

3.2.6.4 Indol test 30

3.2.7 Serotyping by slide agglutination test 30

3.2.8 Motility test 31

3.2.9 Molecular Characterization by PCR 32

3.2.9.1 DNA extraction 32

3.2.10.1 Inoculum preparation 32

3.2.10.2 Growth medium 32

3.2.10.3 Inoculation of test plates 33

3.2.10.6 Standard for Antibiotic sensitivity test 34

3.2.11 Pulsed-field gel electrophoresis (PFGE) 34-35

3.2.11.1 Image analysis 35

3.2.12 Maintenance of stock culture 35

4.2 Isolation of Salmonella by cultural characteristics 37- 38

4.2.1 Results of culture in different culturing media 37

4.2.1.1 Salmonella-Shigella agar 37

4.2.1.2 Eosin Methylene Blue (EMB) agar media 37

4.2.1.3 Xylose Lysine Deoxychockolate (XLD) agar media 38

4.3 Results of Gram’s staining technique 38

4.4 Results of biochemical test 39-40

4.4.1 Indole test 39

4.4.2 Methyl Red test 39