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Israel Journal of Veterinary Medicine  Vol. 69 (4)  December 2014 Kshirsagar, D.P. 228
INTRODUCTION
Food borne diseases caused by nontyphoid salmonella
represent an important public health problem worldwide.
Underdeveloped and technologically developed countries are
struggling with foodborne outbreaks which result in illness,
death and large economic losses. In underdeveloped coun-
tries there are more than one billion cases of gastroenteritis
and up to 5 million deaths annually (1). In the United States
alone, an estimated 1.4 million cases of salmonellosis is
thought to occur annually, of which about 200,000 cases are
reported to the CDC. Salmonellosis is more common in the
warmer months of the year and accounts for 30% (about 400
yearly) of deaths resulting from foodborne illnesses in USA
and the most commonly isolated serovar are Typhimurium
and Enteritidis (2, 3). A variety of foods have been implicated
as vehicles transmitting salmonellosis to humans, including
poultry, beef, pork, eggs, milk, cheese, fsh, shellfsh, fresh
fruit and juice, and vegetables (4).
Young children, the elderly and patients with chronic ill-
nesses or immunocompromised are particularly susceptible
to salmonellosis. Infective dose of Salmonella bacterium re-
quired overcoming host defenses and cause disease varies,
usually about 10
6
to 10
8
CFU. It has been reported that lower
numbers of S. enterica may be capable of causing outbreaks,
especially in cases involving foods with a high fat content
(5, 6).
Tere are three syndromes observed following the con-
sumption of Salmonella as salmonellosis, typhoid fever and
paratyphoid fever. Although diferent Salmonella serovars
may vary in their degree of virulence, it is presumed that all
are pathogenic S. enterica serovar Typhimurium and S. en-
terica serovar Enteritidis have been implicated, in particular,
Isolation and Molecular Characterization of Virulence-Associated Genes
of Salmonella from Bufalo Meat Samples in Western Region of India
Kshirsagar, D.P.*, Singh, S., Brahmbhatt, M.N. and Nayak, J.B.
Dept. of Veterinary Public Health & Epidemiology, College of Veterinary & A.H, Anand Agricultural University,
Anand-388001 (Gujarat), India.
*
Corresponding Author: Dr. D.P. Kshirsagar, Tel: 919510658407, Email: drdpk04v@gmail.com
ABSTRACT
Salmonella is found worldwide in cattle and is considered among the most important animal related zoonotic
disease. Salmonella is a leading cause of foodborne illness viz. enteric illness. In the present investigation 16
(10.66%) of isolates of Salmonella spp. were found from 150 raw bufalo meat and ofals viz. liver, lung,
muscle, intestine and ground beef (30 each), collected from the retail meat market of Anand, (Gujarat)
India. All the 16 isolates of Salmonella spp. were screened for the presence or absence of virulence associated
genes by using the polymerase chain reaction (PCR). All the Salmonella isolates were subjected to serotyping
and all the isolates of Salmonella spp. revealed the presence of invA, stn and fmA genes. Fourteen (87.5%)
of the isolates showed the presence of spvR gene and 8 (50%) had the spvC gene. Serotyping of Salmonella
isolates revealed that Salmonella enterica serovar Typhimurium was the only detected serovar. Te presence of
invasiveness and enterotoxicity of salmonella isolates in bufalo meat and ofals showed their ability to cause
systemic infections and which may appear to be threat to the public and a health concern.
Keywords: Salmonella; Bufalo Meat; Zoonotic Disease; Serotyping; PCR.
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Israel Journal of Veterinary Medicine  Vol. 69 (4)  December 2014 229 Salmonella from Bufalo Meat and Ofals in India
as causes of human salmonellosis. However, in South-East
Asia, S. enterica serovar Weltevreden has been reported as a
frequent and increasing cause of human infection (7).
Diferent virulence genes such as inv, stn, fm and spv
have been identifed as major genes responsible for virulence
factors in Salmonella. Te invasion (invA) gene found to be
present in Salmonella pathogenicity islands (SPI) and re-
sponsible for invasion in the gut epithelial tissue of human
and animals, whereas, stn gene causes enterotoxic efect to
epithelial cells, leading to enteric disorder (8, 9).
Tere are only few reports of incidence of Salmonella in
retail raw bufalo meat and ofals so the present study was
undertaken with the aim to isolate and identify Salmonella
from raw bufalo meat and ofals sold in retail market from
Anand, (Gujarat) India. Te recovered Salmonella isolates
were subjected to biochemical characterization and detection
of virulence genes by PCR.
MATERIAL AND METHOD
Samples
Altogether 150 raw bufalo meat and ofals comprising of
ground beef (keema), muscle, intestine, liver and lung (30
samples each) were collected into sterilized polyethylene
bags in the morning hours as they are ofered for public sale
from diferent retail bufalo meat shops located in Anand,
(Gujarat) India and transported to the P.G. research labora-
tory of the Veterinary Public Health & Epidemiology de-
partment in an icebox for further processing and microbio-
logical analysis.
Isolation and Identifcation of Salmonella
Culture media and antibiotic supplements used in the study
were procured from Hi-Media Laboratories, Mumbai. Te
standard protocol described in Bacteriological Analytical
Manual (BAM), U.S. Food and Drug Administration
(USFDA) method was adopted for the isolation of
Salmonella spp. from bufalo meat and ofals (10). Briefy,
25 g of each type of sample was thoroughly triturated in
a sterile mortar and pestle and transferred to 225 ml pre-
enrichment in lactose broth. Subsequently 0.1 and 1ml
of pre-enriched sample was transferred to enrichment in
Rappaport-Vassiliadis Soybean Meal (RVSM) broth and
Tetrathionate Broth (TTB), respectively, followed by 24h
of incubation at 42°C and 37°C, respectively. Te enrich-
ments were streaked on Brilliant Green agar (BGA) and
Xylose Lysine Deoxycholate (XLD) agar and incubated for
24 hour at 37°C. Typical colonies on XLD (pink colonies
with or without black centers) and BGA (colourless or pink
or opaque-white colonies often surrounded by pink or red
zone) were picked and streaked further on Bismuth Sulphite
Agar (BSA) for purifcation. Te pure cultures were streaked
on Triple Sugar Iron (TSI) agar and incubated at 37°C for
18 hours. Tose producing typical reaction on TSI (red slant
and yellow butt with H
2
S production-blackening of agar)
were further characterized by biochemical tests viz., cata-
lase, oxidase, decarboxylation of lysine using lysine iron agar,
production of indole, methyl red test, Voges Proskauer test,
utilization of citrate and urease test. Te colonies identifed
as Salmonella were preserved in 20 per cent glycerol broth at
-20°C for further characterization.
Table 1: Primer pairs used for virulence characterization of Salmonella isolates
Primer pair target Primer sequence (5’→3’)*
Annealing
a
Length
b
Reference
invA
F:GTG AAA TTA TCG CCA CGT TCG GGCAA
R:TCA TCG CAC CGT CAA AGG AAC C
64°C 284 bp 11
stn
F:CTT TGG TCG TAA AAT AAG GCG
R:TGC CCA AAG CAG AGA GAT TC
55°C 260 bp 12
fmA
F:CCT TTC TCC ATC GTC CTG AA
R:TGG TGT TAT CTG CCT GAC CA
56°C 85 bp 13
spvR
F:CAG GTT CCT TCA GTA TCG CA
R:TTT GGC CGG AAA TGG TCA GT
57°C 310 bp 14
spvC
F: ACT CCT TGC ACA ACC AAA TGC GGA
R: TGT CTT CTG CAT TTC GCC ACC ATC A
63°C 571 bp 15
* Te Forward primer (F) listed frst followed by the Reverse primer (R).
a
Annealing temperature in °C.
b
Length of amplifcation product in base pairs
Research Articles
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Israel Journal of Veterinary Medicine  Vol. 69 (4)  December 2014 Kshirsagar, D.P. 230
Serotyping of Salmonella isolates
Cultures identifed as Salmonella were serotyped at National
Salmonella and Escherichia Centre (NSEC), Central
Research Institute (CRI), Kasauli (Himachal Pradesh, India).
DNA extraction and Polymerase Chain Reaction
Te DNA of isolates of Salmonella was prepared by bacterial
lysis method. A loopful of culture was taken in a micro-
centrifuge tube in 100µl of sterilized DNAse and RNAse-
free Milli-Q water (Millipore, USA). After being vortexed
the samples were heated at 95°C for 10 min; cell debris was
removed by centrifugation and 3µl of the supernatant was
used as a DNA template in PCR reaction mixture. All the
Salmonella isolates were frst screened for the presence or
absence of virulence associated genes by using the PCR pro-
tocols separately standardized for the detection of diferent
genes. Te PCR was standardized for the detection of fve
genes viz. invA, stn, fmA, spvR and spvC as per methodology
with suitable modifcations given in Table 1. Standardization
of PCR was done by using standard strain of S. Typhimurium
obtained from NICED, Kolkata.
All the PCR primers and molecular reagents were pro-
cured from Bangalore Genei, Bangalore, India. Te reaction
was carried out in thin walled PCR tubes in 25 µl reaction
volume with diferent concentration of reactants under dif-
ferent annealing temperatures and cycling conditions.
Briefy, the reaction mixture was optimized to contain
12.5µl 2X PCR master mixes (MBI Fermentas), 10 ρmol of
each forward and reverse primer, 7.5µl nuclease free distilled
water and 3µl of DNA template. Te reaction was performed
in the thermal cycler with pre-heated lid (Lid temperature
105°C). Reaction conditions employed were: initial denatur-
ation at 94°C for 5 min, followed by 35 cycles of 94°C for 1
min, 58°C for 1.5 min, and 72°C for 1.5 min. A fnal exten-
sion of 7 min at 72°C was employed. Reaction condition
was the same as earlier described except for the annealing
temperature of respective primer used as shown in Table 1.
On completion of the reaction the amplifed products were
analysed on agarose gel electrophoresis through 2% agarose
gel stained with 5 µg/ml of ethidium bromide with a 100
bp DNA ladder as molecular weight markerand visualized
under UV light.
RESULT AND DISCUSSION
In the present study out of 150 raw bufalo meat and of-
fal samples, 16 (10.66%) were found positive for Salmonella
spp. All the sixteen isolates revealed characteristic features
of Salmonella producing pink colonies with or without black
centers from Xylose Lysine Deoxycholate (XLD) and co-
lourless or pink or opaque-white colonies often surrounded
by pink or red zone from Brilliant Green (BG) agar. On
preliminary biochemical characterization they revealed char-
acteristic IMViC pattern as shown in Table 2.
Organ wise, a higher prevalence of (26.66%) was ob-
served among the samples of ground beef tested followed
by (20%) in intestine and (6.66%) in muscle, whereas no
samples of lung and liver was found positive for Salmonella
spp. Earlier studies indicated variable prevalence ranging
from 0% (16) to 28.3% (17) in beef. In addition to this re-
ported prevalence rates of 3.4%, 5.8%, 12%, 14.4%, 8.5%,
Figure 1: Agarose gel showing PCR Amplifed product (284 bp) for
invA gene in Salmonella isolates
Figure 2: Agarose gel showing PCR Amplifed product (260 bp) for
Stn gene in Salmonella isolates
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Israel Journal of Veterinary Medicine  Vol. 69 (4)  December 2014 231 Salmonella from Bufalo Meat and Ofals in India
4.2%, 16.9% and 8.5%, respectively from beef samples (18,
19, 20, 21, 22, 23, 24, 25).
In the present investigation all the 16 isolates of Salmonella
isolated from 150 retail market bufalo meat and ofals were
subjected to PCR assays for the detection of virulence-asso-
ciated genes. All the16 isolates of Salmonella yielded desired
amplifed product of approximately 284 bp, 260 bp and 85 bp
similar to that of reference strain of Salmonella using the primer
pairs for invA, stn and fmA respectively (Figure 1, 2 and 3).
Te virulence profle of Salmonella isolates is shown in Table 3.
Te fndings in this study are in agreement with reports
on detection of these genes in Salmonella enteritidis (26, 27,
28, 29 and 30). Detection of these genes may indicate the
virulence potential of the isolates.
Moreover, among 16 isolates of Salmonella 14 (87.5%)
were positive for spvR gene yielding the desired amplifed
product of approximately 310 bp (Figure 4) and 8 (50%)
were positive for spvC gene yielding the desired amplifed
product of approximately 571 bp (Figure 5). In contrast to
our results higher prevalences of spvR gene and spvC gene
was reported (31, 32) while (33, 34 and 35) reported a lower
prevalence of these genes.
In the present study all the sixteen isolates belonged to
serovar Typhimurium. Te results of present investigation
are in agreement with reports of (19, 20, 21, 25 and 36) for
Figure 3: Agarose gel showing PCR Amplifed product (85 bp) for
flmA gene in Salmonella isolates
Figure 4: Agarose gel showing PCR Amplifed product (310 bp) for
spvR gene in Salmonella isolates
Table 2: Specifc identifcation of Salmonella isolates-
Test Typical Salmonella reaction
Catalase +
Oxidase -
Indole test -
Methyl red (MR) test +
Voges Proskauer (VP) -
Citrate test +
Urease test -
Lysine decarboxylation +
Triple Sugar Iron agar slant
Red/Yellow/H
2
S+
Table 3: Te Organwise prevalence and virulence profle of
Salmonella isolates
Source of
Sample
No. of
sample
analyzed
Total No.
of positive
samples
invA stn fmA spvR spvC
Ground Beef 30 8 (26.66%) 8 8 8 7 4
Muscle 30 6 (20%) 6 6 6 5 2
Intestine 30 2 (6.66%) 2 2 2 2 2
Liver 30 0 0 0 0 0 0
Lung 30 0 0 0 0 0 0
Total 150 16 (10.66%) 16 16 16 14 8
Figure 5: Agarose gel showing PCR Amplifed product (571 bp) for
spvC gene in Salmonella isolates
Research Articles
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Israel Journal of Veterinary Medicine  Vol. 69 (4)  December 2014 Kshirsagar, D.P. 232
presence of S. Typhimurium from bufalo meat samples. In
conclusion, our study highlights the microbiological hazard
of Salmonella contamination of raw bufalo meat and ofal
products during storage and improper handling or cooking
of bufalo meat which can lead to human food-borne illness.
Hence, epidemiological data are needed to inform public
health authorities about the nature and magnitude of the
problem and to monitor trends over time.
ACKNOWLEDGEMENTS
Te authors are grateful to the Dean, College of Veterinary Science
and Animal Husbandry, Anand (Gujarat) for providing the fa-
cilities to pursue this work. Authors are also thankful to National
Institute of Cholera and Enteric Diseases (NICED) Kolkata for
procuring the reference strains and to National Salmonella &
Escherichia Centre, Central Research Institute, Kasauli, India, for
Serotyping of the Salmonella isolates.
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