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STUDIES ON ISOLATION AND MOLECULAR
CHARACTERIZATION OF SALMONELLA spp. OF PUBLIC
HEALTH SIGNIFICANCE IN CHEVON AND CHICKEN
MEAT
M.V. Sc. THESIS
By
VIVEK KUMAR NAIK
DEPARTMENT OF VETERINARY PUBLIC HEALTH AND EPIDEMIOLOGY
COLLEGE OF VETERINARY SCIENCE AND
A. H., ANJORA
CHHATTISGARH KAMDHENU VISHWAVIDYALAYA
DURG (C.G.)
2014
STUDIES ON ISOLATION AND MOLECULAR CHARACTERIZATION
OF SALMONELLA spp. OF PUBLIC HEALTH SIGNIFICANCE IN
CHEVON AND CHICKEN MEAT
Thesis
Submitted to the
Chhattisgarh Kamdhenu Vishwavidyalaya, Durg
By
Vivek Kumar Naik
IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE
DEGREE OF
Master of Veterinary Science
In
Veterinary Public Health
September, 2014
ROLL NO. -4004 I.D. NO. - K130112019
VITA
The author of this thesis, Vivek Kumar Naik was born on June 5th, 1987 at
Ghargoda block, Distt. Raigarh (C.G.). He passed his higher secondary examination in the
year 2005 with first division from Jawahar Navodaya Vidyalaya, Bhupdevpur, Distt.
Raigarh (C.G). In July 2007 he started his career in veterinary profession for his B.V. Sc. And
A.H. from College Of Veterinary Science, Anjora, Durg (C.G.) and completed it in July 2012
with OGPA of 6.98/10.00. Due to his keen interest in research work towards public health,
in the same year he joined Department of Veterinary Public Health and Epidemiology for his
master’s degree in September 2012. He has completed the requisite course works for M.V.Sc.
programme.
Address:
Dr. Vivek Kumar Naik
At and post- CHC, Civil Line
Dharamjaigarh
Distt-Raigarh (C.G.)
Pin- 496116
Email – vetvivek8583@gmail.com
CERTIFICATE-I
This is to certify that the thesis entitled “Studies on isolation and
molecular characterization of Salmonella spp. of public health significance in
chevon and chicken meat’’ submitted in partial fulfillment of the requirements
for the degree of “Master of Veterinary Science” of Chhattisgarh Kamdhenu
Vishwavidyalaya, Durg, is a record of bonafide research work carried out by
Vivek Kumar Naik, under my guidance and supervision. The subject of the
thesis has been approved by Student’s Advisory Committee and the Director of
Instructions.
No part of the thesis has been submitted for any other degree or diploma
(certificate awarded etc.) or has been published/published part has been fully
acknowledged. All the assistance and help received during the course of
investigations have been duly acknowledged by him.
Dr. Sanjay Shakya
Date: Chairman, Advisory Committee
THESIS APPROVED BY STUDENT’S ADVISORY COMMITTEE
Chairman : Dr. Sanjay Shakya ………………...
Member : Dr. Anil Patyal ………………...
Member : Dr. S. D. Hirpurkar ………………...
Member : Dr. S. L. Ali ………………...
Member : Dr. G. K. Dutta ………………...
CERTIFICATE-II
This is to certify that the thesis entitled “Studies on isolation and
molecular characterization of Salmonella spp. of public health significance in
chevon and chicken meat’’ submitted by Vivek Kumar Naik to the
Chhattisgarh Kamdhenu Vishwavidyalaya, Durg, in partial fulfillment of the
requirements for the degree of M.V.Sc. in the Department of Veterinary Public
Health and Epidemiology, College of Veterinary Science and Animal Husbandry,
Anjora, Durg, has been approved by the Student’s Advisory Committee after oral
examination in collaboration with the external examiner.
External Examiner
Major Advisor
Head of the Department
Dean
Director of Instructions
Dated:
ACKNOWLEDGEMENT
My hope is that the quality and significance of this research adequately reflects the
quality of people that have supported me through it.
I am extremely grateful to my major advisor Dr. Sanjay Shakya, Professor,
Department of Veterinary Public Health and Epidemiology and Chairman of Advisory
Committee. His leadership, work ethic, and contagious enthusiasm seem to push the people
beyond the sense of their own limitations. I’m fortunate to have studied under the
mentorship.
In this regard, I wish to express my profound sense of gratitude to Dr. S. P. Tiwari,
Dean, College of Veterinary Science and Animal Husbandry, Anjora, Durg for providing
necessary facility to conduct this study successfully.
I extend my cordial thanks to the members of my advisory committee, Dr. Anil
Patyal, Assistant Professor, Department of Veterinary Public Health and Epidemiology, Dr.
S. D. Hirpurkar, Professor, Department Of Veterinary Microbiology, Dr. S. L. Ali, Professor,
Department of Clinical Medicine and Dr. G. K. Dutta, Professor, Department Of Veterinary
Physiology And Biochemistry for providing their time and knowledge to steer me in the right
direction.
I am highly thankful to Dr. Nitin Gade, Assistant Professor, Department Of
Veterinary Physiology and Biochemistry, Dr. Fateh Singh, Scientist, Central Sheep And Wool
Research Institute, Avikanagar, Dr. Nidhi Rawat, Assistant Professor, Department Of
Veterinary Microbiology, Dr. Neelu Gupta, Associate Professor, Department Of Veterinary
Pathology and Dr. Smita for their valuable suggestion, time to time advice and constructive
help throughout the course of study.
I am most grateful and feel highly esteemed privilege to express my wholehearted
thanks to my friend Dr. Abhinav Verma for his constant inspiration, ever willing co-
operation, moral support and faith which organised me during this endeavour.
I am extremely indebted to Dr. Ashish Wankar, Dr. Bhoomika Sirsant, Dr. Menka,
Dr. Seema Kriplani, Dr. Sourabh Yogi, Dr. Kiran Rout, Dr. Yugesh Choudhary, Dr. Prashant
Dewangan, Dr. Pankaj Sai, Dr. Praveen Kumar, Diamond Sahu, Shiv Sidar, Somesh Joshi
for their moral support, guiding assistance and rewarding co-operation throughout the
research work.
I feel great pleasure in acknowledging my colleague and friends, Dr. Foziya Farzeen
Khan, Dr. Abrar, Dr. Prashant Nalge, Dr. Amol, Dr. Jyoti Sahu, Dr. Prashant Dewangan,
Dr. Dev Kalihari, Dr. Ajay Chaturvedani, Dr. Lakhan Prasad Manhar, Dr. Vikash Jaiswal,
Dr. Ashok Patel, Dr. Sandeep, Dr. Puspraj And Dr. Dilip Painkra for their continuous
support and encouragement during those time when it was most needed.
I profoundly express my gratefulness to my respected seniors Dr. Surendra Naik, Dr.
Tanmay Ottalwar, Dr. Preeti Ekka, Dr. Deepesh Rawte, Dr. Vishwajeet Dilliwar, Dr.
Jitendra Goldie Lall, Dr. Dinesh Kurrey, Dr. Pramod Thakur, Dr. Komal Rai, Dr. Bhuvan
Naik, Dr. Suresh and Dr. Riddhhi Patel for their constant inspiration, moral support and
faith on me.
I am extremely thankful to my dear juniors Dr. Jitendra Naik, Sambhuti Shankar
Sahu, J Suryam Dora, Pranjal Pandey, Shailesh Gupta, Chudamani Chandrakar, Neelkant
Rajwade, Ayush Yadav, Jainendra, Bhuvneshwar Kanwar and Krishna Kushwaha for their
immense co-operation and generous help throughout the course of investigation.
The technical assistance provided by Mr. Lalit deshmukh, Mr. Rajendra Yadav and
and Mr. Sunderlal Dewangan, lab attendant, Department of Veterinary Public Health and
Epidemiology is highly acknowledged.
I am thankful to Sahu bhaiya for preparing this manuscript nicely.
None of this would have been possible if it were not for the personal sacrifices and
unconditional support from my parents. They are the most amazing person I know.
At last, I express my sincere thanks to all those who helped me either directly or
indirectly at various stages during the tenure of this study.
Anjora, Durg Vivek Kumar Naik
September, 2014
CONTENTS
Chapter No. Name of Chapter Page No.
I Introduction 1-4
II Review of literature 5-26
III Materials and methods 27-39
IV Results and discussion 40-59
V Summary, conclusionsand suggestions forfuture research work
60-64
Reference 65-81
Appendix i-ix
Abstract 82-83
LIST OF TABLES
Table No. Title of Table Page No.
01 Details of the primers used for amplification of stn,
invA and pef genes
28
02 Antibiotic discs used in present study 34
03 District wise SPC value (log10cfu/gm) of chevon and
chicken meat samples
41
04 Prevalence of Salmonella spp. in chevon, chicken
meat and stool sample
44
05 District wise prevalence of Salmonella spp. in chevon 44
,chicken meat and stool samples
06 Biochemical profile of suspected Salmonella isolates 46
07 Pattern of antibiogram shown by Salmonella isolates 51
08 Antibiogram assay of Salmonella isolates 54
09 Sample wise antibiogram assay of Salmonella
isolates
55
10 Distribution of Salmonella specific virulent genes
among different isolates
58
LIST OF FIGURES
Figure No. Title of Figure After
Page No.
01 Salmonella isolates showing moderately large, moist,
smooth and colourless colonies with pink background
Brilliant green agar (BGA)
44
02 Salmonella isolates showing black colony surrounded
by brownish-black zone with metallic sheen on
Bismuth sulphite agar (BSA) plate
44
03 Salmonella isolates showing colourless colonies on
MacConkey Lactose Agar (MLA) plate
44
04 Prevalence of Salmonella spp. in chevon, chicken
meat and stool sample
44
05 Prevalence of Salmonella spp. in chevon in different
districts of Chhattisgarh
44
06 Prevalence of Salmonella spp. in chicken meat in
different districts of Chhattisgarh
44
07 Salmonella isolates showing Urease test 48
08 Salmonella spp. showing acid butt, alkaline slant with
H2S production on TSI agar.
48
09 Salmonella isolates showing Citrate utilization test 48
10 Salmonella isolates showing colourless ring on Indole
test
48
11 Antibiotic sensitivity test showing the zone of
inhibition against different antibiotics
56
12 Antibiogram pattern shown by Salmonella isolates 56
13 Agarose gel electrophoresis showing amplified PCR
product of stn gene of Salmonella isolates
59
14 Agarose gel electrophoresis showing amplified PCR
product of invA gene of Salmonella isolates
59
ABBREVIATIONS
Abbreviations Full form
APHA American public health associationα Alpha
BGA Brilliant green agarbp Base pair
BSA Bismuth sulphite agarCDC Centre for disease control and
preventionCfu Colony forming units°C Degree celcious
EFSA European food safety authorityet al. Et alia (and others)FAO Food and Agriculture OrganizationFig. Figure
Gm GramGPPW Glucose phosphate peptone water
Hrs Hoursi.e. id est (that is)
IMViC Indole, Methyl Red, Voges-Proskauer,Citrate
Log1o Logarithm base tenMLA MacConkey’s Lactose Agar
µl Microlitremg Milligramml Millilitremm Milli metremin Minute (s)PBS Phosphate buffer saline
/ Per% Percent
PBS Phosphate buffer salinePCR Polymerase chain reactionrpm Revolution per minuteSPC Standard plate countTBE Tris-Borate EDTATSI Triple sugar ironTT Tetrathionate
USDA United States Department ofAgriculture
V Voltsviz. Vide licet (namely)
WHO World Health Organisation
1
CHAPTER-I
INTRODUCTION
Infectious microbial diseases constitute a major cause of death in many
parts of the world, particularly in developing countries and among them
Salmonella have been identified as a leading cause of food borne illness in
humans and animals resulting in significant morbidity and mortality (Akkina et
al., 1999). In India, salmonellosis is endemic and its importance as potential
zoonosis needs no emphasis. It causes heavy economic losses every year
(Rahman, 2002). Salmonella enterica serovar Typhimurium and Salmonella
enterica serovar Enteritidis are the most frequently isolated serovar from food
borne outbreaks throughout the world (Herikstad et al., 2002). Salmonellae are a
large group of enteric bacteria with a broad range of hosts and can cause
enterocolitis (salmonellosis), enteric fever (typhoid fever), and septicaemia
(Moon, 2011).
The general symptoms of human salmonellosis are fever, diarrhoea,
abdominal cramps, nausea, vomiting, chills, and prostration. Occasionally the
infection can be more serious with loss of fluid and electrolytes and can be fatal
especially to the sick, infants, and the elderly. The most common contaminated
foods resulting in human salmonellosis include beef, chicken, turkey, pork, eggs,
milk and products made from them.
Poultry products are frequent vehicles in the transmission of Salmonella,
dominating other foods of animal origin as potential source of infection (Bryan
and Doyle, 1995; D’Aoust, 1997). In India the prevalence of Salmonella in retail
chicken breast was 13.0% and S. Typhimurium (87.8%) was the most frequently
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associated serotype among non typhoidal Salmonella in chicken meat (NARMS,
2012). It has been suggested that stress associated with transportation,
overcrowding and feed withdrawal in animals before slaughter increases shedding
of Salmonella. Goat meat is the most widely consumed meat in the world and
outbreaks of salmonellosis have also been found to be associated with
consumption of contaminated goat meat. However sufficient information is not
available on occurrence of salmonellosis through contaminated chevon in
Chhattisgarh.
Epidemiology and pathogenic process in salmonellosis are dictated by an
array of factors that act in tandem and ultimately manifest in the typical symptoms
of salmonellosis. Virulence genes encode products that assist the organisms in
expressing its virulence in the host cells. Some genes are known to be involved in
adhesion and invasion viz. sef, pef, spv or inv; others are associated with the
survival in the host system- mgtC or in the actual manifestation of pathogenic
processes viz., sop, stn, pip A, B, D. Nucleic acid based diagnostic techniques are
being employed for the detection of various gene -encoded virulence factors viz.,
Salmonella enterotoxin (stn), Salmonella Enteritidis fimbriae (sef) and plasmid
encoded fimbriae (pef) genes. However, the distribution of these genes among the
various isolates obtained from biological source is yet to be elucidated. The exact
mechanism by which Salmonella induces diarrhoea is not fully understood.
Salmonella harbours an enterotoxin similar to the enterotoxins in E. coli. This
enterotoxin production is mediated by the stn gene. The stn gene is one of the
virulent genes that assist Salmonellae in expressing its virulence in the host cells
3
and manifestation of pathogenic processes, mainly diarrhoea (Chopra et al.,
1987).
The antimicrobial resistance of Salmonella is an increasing problem and
has become a public health issue worldwide (Kaye et al., 2004). Antibiotics with
the greatest percentage of resistant isolates include Amoxicillin, Clavulanic acid,
Ampicillin, Ceftiofur, Cefoxitin, Chloremphenicol, Streptomycin, Sulfonamides,
and Tetracyclines; however, the percentage of isolates resistant to these drugs has
increased since 1997. Contamination of food with antibiotic-resistant bacteria can
be a major threat to public health, causing community outbreaks of infectious
diseases. Moreover the evidences on hazard of therapeutic failure due to the
increasing incidence of antimicrobial resistance among Salmonella species are
increasing now a day (Arslan et al., 2010).
Several methods have been developed for the detection, identification and
molecular characterization of Salmonella species (Sen et al., 2007). Culture can
take from 4 to 7 days in order to isolate and confirm the presence of Salmonella
from the sample (Bennett et al., 1998). Conventional culture methods used for the
isolation of Salmonella include, non-selective pre-enrichment followed by
selective enrichment and plating on selective and differential agars. Suspected
colonies are then confirmed biochemically and serologically. More recently, a
number of alternative methods for the detection of Salmonella in foods have been
developed including, immune-assays, nucleic acid hybridization and polymerase
chain reaction (PCR) techniques (Li et al., 2000).
The Polymerase Chain Reaction (PCR) has become a powerful tool in
microbiological diagnostics during the last decade. PCR based methods combine
4
simplicity with a potential for high specificity and sensitivity in detection of food-
borne pathogens.
In view of the above, the present study has been undertaken with the
following objectives-
1. To determine the microbial load of chevon and chicken meat
2. Isolation and identification of Salmonella of public health significance
from food of animal origin (chevon and chicken meat) and from human
diarrhoeal samples by cultural, morphological and biochemical methods
3. Determination of antibiogram of the Salmonella isolates
4. Molecular characterization of Salmonella isolates by detecting virulence
gene
5
CHAPTER-II
REVIEW OF LITERATURE
The genus Salmonella was named after Dr. Daniel Salmon, a veterinary
bacteriologist at the United States Department of Agriculture (USDA) (Gast,
2003; Salyers and Whitt, 2002). Salmonella belong to the bacterial family
Enterobacteriaceae are short Gram-negative bacilli and nonsporulating. The
genus Salmonella comprises two species, Salmonella bongori and Salmonella
enterica. Within Salmonella enterica there are six subspecies: Salmonella
enterica subspecies enterica (I), Salmonella enterica subspecies salamae (II),
Salmonella enterica subspecies arizonae (IIIa), Salmonella enterica subspecies
diarizonae (IIIb), Salmonella enterica subspecies houtenae (IV) and Salmonella
enterica subspecies indica (VI) (Solari et al., 2003). These subspecies can be
further classified into approximately 50 serogroups based on their
lipopolysaccharide (LPS) O antigen component (Sabbagh et al., 2010).
Salmonella bongori and most subspecies of Salmonella enterica colonize the
environment of cold-blooded animals and in some cases can cause disease in these
animals. However, the most biomedical relevant subspecies is S. enterica
subspecies enterica, whose serovars have special clinical significance in both
veterinary and human diseases (Brenner et al., 2000). Salmonella enterica
subspecies enterica can be further divided into over 2500 serovars based on their
flagellar (H) antigen and lipopolysacharide (LPS) (O) antigen structures (Sabbagh
et al., 2010; Coburn et al., 2007; Tindall et al., 2005; Brenner et al., 2000).
Most of serotypes of Salmonella move using peritrichial flagella, although
serotypes such as S. pullorum and S. gallinarum are nonmotile. They are either
6
aerobic or facultative anaerobic, and grow between 5 and 45°C. Optimum growth
occurs at 37°C. The ideal pH for multiplication is 7, but Salmonella survives in
pH values between 4 and 9. They grow in culture medium for enterobacteria and
on blood agar. Colonies are 2 to 4 mm in diameter, with smooth and round edges.
They are slightly raised in medium containing carbon and nitrogen. Colonies may
remain viable for a long time when stored in peptone broth (Gast, 1997).
Biochemically, Salmonella strains have the ability to catabolize nutrients, and
catabolize D-glucose and other carbohydrates, except lactose and sucrose, with
production of acid and gas. They are catalase positive and oxidase negative, they
do not ferment malonate, do not hydrolyze urea and do not produce indole, they
can use citrate as a sole source of carbon, reduce nitrate to nitrite, and may
produce hydrogen sulphide (Quinn et al., 2002). The bacterium itself is
surrounded by a mucus layer, which contributes to its resistance to phagocyte
digestion, and has a fringe of fimbria located around its outer surface that are used
in cell adhesion (Hirsh et al., 2004; Quinn et al., 2002)., which along with other
major pathogens in this group are often attributed to causing illness within the
small intestine, from which the bacteria can migrate and result in progression to
full systemic body disease (Hirsh et al., 2004).
2.1 Epidemiology
Salmonella species have been reported to cause an estimated 1.4 million
cases of food borne illness and more than 500 deaths per year in the U.S. (CDC,
2005). Each year, approximately 40,000 salmonella infections are culture-
confirmed, serotyped, and reported by the United States Centers for Disease
7
Control and Prevention. Of the total cases, 96% are estimated to be caused by
foods (Mead et al., 1999). In Europe Salmonella was the second most reported
cause of food borne diseases in humans with 160,649 people suffering from
Salmonella infections in 2006, approximately 35 people in every 100,000 (EFSA,
2007).
World Health Organisation (WHO) estimate for annual global incidence of
Salmonella infection are about 20 million cases with greater than six hundred
thousand (>600,000) deaths. It is encountered in tropical countries including
India, South and Central America and Africa, where they constitute serious source
of morbidity and mortality with rapid population growth, increased urbanization,
limited safe water, and infrastructure and health problems (World Health
Organisation, 2006).
Global surveillance data indicates that incidence of gastrointestinal
infections caused by Salmonella enteritidis has increased massively during the
last decade. Salmonella serovars which cause human salmonellosis have been
demonstrated to be transmitted through infected poultry flocks, meat and eggs
(Holt et al., 1994). Salmonella enteritidis was reported to be responsible for 380
salmonellosis out-breaks in USA between 1985 and 1991, involving 13056
illnesses and 50 deaths (Mishu et al., 1994).
Non–typhoidial salmonellosis is a food borne disease of primary concern
in developed, as well as developing countries. The spread of this disease is
favoured by a variety of animal reservoirs and a wide commercial distribution of
both animals and food products. This disease is among one of the major public
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health problems in terms of socio-economic impact (Mushtaq-ul-hassan et al.,
2008; Razzaque et al., 2009).
2.2 Status of salmonellosis in India
Salmonella has shown to be endemic in India in humans and animals
(Verma et al., 2001) and has been associated with foods of animal origin
(Thapliyal, 1999). 209 serovars of Salmonella consisting of important human
pathogens have been documented in India (Rahman, 2002).
Murugkar et al. (2005) carried out a study to report the isolation along
with the serotype, phage types and antibiogram pattern of Salmonella among man,
livestock and poultry in the north-eastern India.
Murugkar et al. (2005) conducted a study in North eastern part of India
where he studied distribution of various serovars of Salmonella among animal
species. Salmonella Typhimurium was the commonest serovars in all species
under investigation. Furthermore, they found prevalence of 14.7% in poultry,
14.2% in piglets and 9.6% in cattle.
Nagappa et al. (2007) conducted a study in 100 samples each of chicken
eggs and meat, collected from various retail outlets of the Tarai region of
Uttaranchal by the presence of S. Typhimurium was reported.
Kumar et al. (2008) reported that the most common serovars from humans
in India are Salmonella Typhi (73%) and Salmonella paratyphi A (24%) among
typhoidal serovars, and S. Worthington (28.2%) and S. Typhimurium (22.5%)
among non-typhoidal serovars .
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Bisht (2009) examined faecal as well as poultry cloacal samples, 6 (1.7%)
of samples yielded Salmonella which comprised of S. Paratyphi B (3), S.
Typhimurium (2) and S. Stanley (1).
Anumolu et al. (2012) employed a set of primers derived from fli C gene
to standardise PCR for detection of salmonella typhimurium from poultry samples
viz. Claoacal swab, egg swabs, poultry faeces, and feed, which gave specific
amplification of a 620 bp fragment. Screening of 112 samples revealed that
samples positive for Salmonella typhimurium by PCR assay.
Das et al. (2012) carried out research work for detection and molecular
characterisation of Salmonella enteric serovar typhi isolated from humans with
typhoidial fever by biochemical, phonotypical and virulence gene based
polymerase chain reaction (PCR) techniques.
Muthu et al. (2014) undertook a study to detect the two genes namely,
salmonella enterotoxin (stn) and plasmid encoded fimbrial (pef) genes among
clinical isolates of three Salmonella species from humans. PCR findings indicated
that the stn gene is widely distributed among Salmonella irrespective of the
serovars and source of isolation.
2.3 Standard Plate Count of chevon and chicken meat:
Nair et al. (1990) reported the total aerobic plate count (APC) in the
dressed birds from the Central Food Technological Research Institute (CFTRI)
processing plant was 6.3717 log10 cfu/g.
10
Singh et al. (1995) examined 67 mutton sample and reported that Standard
Plate Count (SPC) ranged from 8.5×107 to 1.3 × 1010 cfu/g and coliform count
from 2.2×105 to 5.8 ×106 cfu /g.
Zweifel and Stephan (2003) evaluated microbial quality of 580 sheep
carcasses and reported the medium SPC ranging from 2.5 to 3.8log10cfu/cm2.
Janjirkar et al. (2005) estimated the microbiological quality of fresh and
frozen poultry meat. The SPC of fresh meat was found to be 4.82±0.83log10cfu/g.
Kaskhedikar (2007) examined the total viable count of 105 food sample,
the SPC of chicken, chevon, mutton, and buffalo samples ranged between 0.13-
0.29 ×105, 19-29 × 105, 1.7-2.6 × 105 and 17-28 ×105cfu/g respectively.
Eglezos et al. (2008) studied bacteriological profile of 300 raw chicken
nuggets and reported the mean of aerobic plate count to be 5.4 log10cfu/g.
Tompkins et al. (2008) examined the bacteriological quality of 270 poultry
meat samples and reported the SPC range from 7.2787log10cfu/g to 7.3979
log10cfu/g.
Lambey et al. (2009) carried out a cross sectional study of raw goat meat
samples from the local meat markets of Mathura, India to investigate bacterial
load in ready to sale chevon with special emphasis on isolation and identification
of salmonella spp. samples were collected from 40 goat carcass from local retail
meat shops of Mathura. On carcass of goats, the mean of the log10 standard plate
count was 7.03cfug-1.
Nikas (2009) examined bacteriological quality of 100 poultry meat
samples and pork meat samples. The SPC of chicken samples ranged from
6.0414-6.4624log10cfu/g and SPC of pork ranged from 7.1139-7.4472log10cfu/g.
11
Hassan Ali et al. (2010) conducted a study in which raw meat samples
(250) and surface swabs (90) from meat processing equipment and the
surrounding environment were analyzed for microbiological contamination. They
reported the total aerobic counts ranging between 108 –1010 CFU/g or cm2.
Gangil et al. (2011) conducted an experiment to assess the microbiological
quality i.e. total viable count of total 50 raw goat meat samples collected from
hotels and retail meat shops of Jaipur, Rajasthan. The estimation of microbial load
on chevon sample showed log10TVC range from 5.04 to 7.97 (average of
6.67±0.12).
Adu-Gyamfi et al. (2012) screened the microbiological quality of chicken
by analyzing 27 chicken thigh samples collected from the retail outlets. Mean
total viable counts for the supermarkets, local markets and farms were reported as
6.46, 6.91 and 6.57 log10 cfu/g respectively.
Dabassa et al. (2012) analysed the samples composed from cattle , goat
and sheep for microbial load determination using conventional culture method.
The aerobic mesophilic counts varied from 3.0 to 9.0 log10 CFU/g.
Dhanze et al. (2012) evaluated the microbiological quality of 152 food
samples comprising eggs (47), chicken (45), chevon (30) and ready to eat foods of
animal origin (30) collected from retail outlets in and around Palampur (H.P.) by
employing standard plate count (SPC). Among the chevon samples, 60% showed
SPC of <6 log10 CFU/g and all the chicken samples had SPC of <5.77 log10
CFU/g.
Mawia et al. (2012) conducted a study to assess the microbiological
quality of chevon and poultry meat collected from different parts of and around
12
Jammu city. A total of 167 meat samples (85 chevon and 82 poultry) were
collected and processed for standard plate count (SPC). The results were
presented as log10cfu/g. Mean value of SPC for chevon samples was 6.37±0.06
and for poultry meat sample, the count was 6.64 ±0.06.
Patyal et al. (2012) evaluated the bacteriological quality of raw chicken
meat marketed in retail shops of Jaipur city in Rajasthan, India. A total of 50 raw
chicken meat samples were collected aseptically from different retail meat shops
and analysed for the total viable count (TVC). The log10TVC in chicken meat
samples was found between the ranges of 5.52-7.97 with the average (Mean±S.E.)
of 7.14±0.11 log10cfu/g. The results of TVC revealed high bacterial contamination
of chicken meat and only 40% samples were in acceptable category.
Ahmad et al. (2013) conducted a study to assess the microbial load of raw
meat at abattoirs and retail outlets in different areas of Lahore. Beef, mutton
(sheep, goat) and chicken meat samples (n=140) were collected from various
abattoirs (n=60) and retail outlets (n=80). All the samples were subjected to
aerobic plate count (APC). They found that Mean SPCs of beef, sheep, and goat
meat from abattoirs (5.35, 5.42 and 4.84 log10 CFU/cm2 respectively) were
significantly lower as compared to SPC values of retail outlets (7.15, 6.92 and
6.62 log10 CFU/cm2 respectively). Mean SPC of chicken meat from retail outlets
was 7.22 log10 CFU/cm2.
Nnachi et al. (2014) carried out a study and showed that the mean total
aerobic counts (TAC) (expressed as log10cfu/g) for the four meat types were
9.84±0.14, 9.91±0.12, 10.03±0.31 and 14.63±3.82 for Pork, Goat, Donkey and
Beef respectively, projecting beef as the most contaminated and pork as the least.
13
2.4. Prevalence of Salmonella:
2.4.1. Poultry
Panisello et al. (2000) reported that one of the most frequent causes of
infection by Salmonella reported in humans has been through the handling of raw
poultry carcasses and products, together with the consumption of undercooked
poultry meat.
Poppe (1994) Stated that the establishment of S. Enteritidis infection in
chicken breeder flocks could lead to widespread infection in layer and broiler
flocks and subsequently in the human population.
Guard-Petter (2001) stated that S. Enteritidis is the only Salmonella
serovar that contaminates egg routinely, even though chickens are associated with
wide range of serotypes.
Whyte et al. (2002) conducted a experiment to study the prevalence of
Salmonella contamination in raw poultry. A total of 198 neck skin samples were
obtained from within 40 flocks at a commercial broiler slaughtering facility. The
presence of Salmonella was assessed by traditional culture methods and by a
salmonella- specific polymerase chain reaction (PCR) test. Salmonella was
recovered from 32 (16%) of all samples using traditional culture methods.
Salehi et al. (2005) detected the presence of salmonella in 192 samples of
poultry carcasses from poultry farms in Shiraz province (Iran). A total of 30
isolates were found in chicken samples showing prevalence of 15.6%, by
conventional culturing and confirmed by PCR and serology methods.
Dahal et al. (2008) conducted a cross sectional study by analyzing random
samples of broiler carcasses (400) collected directly from the import vessels
14
between November 2006 and April 2007 at national centre for animal health. Of
the 400 samples analyzed, prevalence of Salmonella was 13% with Salmonella
Enteritidis as the most frequently isolated serotype (84.62%), followed by
Salmonella Typhimurium (15.38%).
Akhtar et al. (2010) conducted an experiment to show the prevalence of
Salmonella in chicken meat and human diarrhoeal sample. out of 85 poultry meat
collected 26 was found positive for Salmonella showing a prevalence of 30% and
out of 125 human stool collected 58 shown to be positive for Salmonella showing
a prevalence of 46.40%.
Rousi et al. (2010) conducted a study on 414 faecal samples from flocks
of laying hen and they were found to be positive with S. Enteritidis and S. Cerro
as most prevalent serovar.
Bisht (2010) conducted a study in Pantnagar, where out of 722 faecal and
stool samples 13 samples gave Salmonella isolates, consisting of S. Typhimurium
(5), S. Enteritidis (5) and S. Infantis (3) and showed that serovars were mostly
confined to poultry population and thus, pose a great threat to human population.
Ruban et al. (2010) conducted a study to isolate and identify Salmonella
spp from chicken slaughtered under different processing conditions in modern
processing units in Karnataka, India. In the study breast and thigh samples were
evaluated for presence of Salmonella spp. A total of 450 (225 breast and 225 thigh
muscles) samples were tested. They found that prevalence of Salmonella spp. was
higher in thigh meat (31.99%) compared to breast muscles (24.8%).
Moon et al. (2011) undertook a study in which he analysed the poultry
meat in different markets of Wardha district for the presence of pathogenic
15
Salmonella species. In the study it was revealed that there was a prevalence of
38.33% in poultry meat.
Hue et al. (2011) collected a total of 425 carcases from 58 French poultry
slaughter houses to study the prevalence of Salmonella spp. on broiler chicken
carcases and isolated Salmonella from 32 carcases thus, leading to 7.52%
prevalence. Thirteen different serotypes were identified where S. Indiana was the
most prevalent (33.3%) followed by S. Kottbus (13.9%).
Rabie et al. (2012) carried out a study to report the prevalence of the
serotypes and genetic types of salmonella among broiler chicken and raw chicken
in Toukh, Egypt. Samples collected from (50) diarrheic broiler chicken, (50) raw
frozen chickens meat were bacteriologically and serologically processed for
identification of Salmonella. Isolates were subjected to multiplex PCR using
specific Salmonella primers. The prevalence of Salmonella spp. was 7 (14%) and
2 (4%) in broiler chickens and chicken meat.
Panda et al. (2012) focussed his study to evaluate the bacteriological
quality of meat and meat products from Palam valley over a period of 5 years. A
total of 76 raw chicken meat samples were collected. Out of 76 meat sample, 8
samples were found to be positive for Salmonella showing a prevalence rate of
10.52% in raw chicken meat.
Patyal et al. (2012) conducted an experiment to evaluate the prevalence of
Salmonella spp. in raw chicken meat sample marketed in retail shops of Jaipur
city in Rajasthan, India. A total of 50 raw chicken meat samples were collected
aseptically from different retail meat shops and analyzed for the isolation of
16
Salmonella spp. It was revealed that 6% of chicken meat samples were positive
for Salmonella.
Kumar et al. (2013) screened 200 samples comprising poultry meat (100)
and poultry faeces (100) to report the isolation along with the serotypes, phage
types and antibiogram pattern of Salmonella among poultry meat and
environmental sources in the India. Out of 200 samples only three (one poultry
meat and two poultry faeces) (1.5%) were found positive for Salmonella by
cultural method.
2.4.2 Goats
Molla et al. (2006) conducted a study in Ethiopia and screened a total of
100 goats for the isolation and identification of Salmonella species. Salmonella
was isolated in 3 goats, which was a high prevalence, reported in animals used for
human consumption.
Duffy et al. (2009) reported that goat carcasses contaminated with
Salmonella during slaughter could be a source of infection, if consumed raw or
inadequately cooked, or may also serve as a source of cross- contaminated to
other foods. He reported S. Saintpaul as dominant serovars, followed by S.
Typhimurium whereas in another study made by Molla et al. (2006) the common
serovars isolated were S. Typhimurium, followed by S. Heidelberg, S. Reaiding,
S. Give, and S. Poona.
Lambey et al. (2009) carried out a cross sectional study on raw goat meat
samples collected from the local meat markets of Mathura, India to investigate
bacterial load in ready to sale chevon with special emphasis on isolation and
17
identification of Salmonella spp. A total of 40 goat meat samples were collected
from local retail meat shops of Mathura. Out of which, 1.25% of goat meat
samples were found positive for Salmonella. .
Moon et al. (2011) analyzed the goat meat in different markets of Wardha
district for the presence of pathogenic Salmonella species and revealed that
prevalence rate of Salmonella spp. in goat meat was 38.33%.
Zubair et al. (2012) conducted a study to determine the prevalence of
Salmonella species in slaughtered animals and abattoir sewage from Zakho
Abattoir, Kurdistan Region, Iraq. Result showed that the prevalence of Salmonella
in apparently healthy sheep and goats was 2.5% and 2% respectively.
Dabassa et al. (2012) conducted a study and analysed 60 goat meat
samples from the abattoir of Jimma town, South West, Ethiopia for the presence
of Salmonella spp. over a 5 month period between December, 2009 and May,
2010. After examining he found that the prevalence of Salmonella in chevon was
3.3%.
Eze et al. (2012) evaluated the microbial quality of fresh goat meat sold in
Umuahia market Abia State. A total of 40 samples of fresh goat meat were
collected and analyzed for the presence of Salmonella species. Bacterial genera
isolated showed that among all, Salmonella species had the percentage occurrence
of 8.05%.
Panda et al. (2012) evaluated the bacteriological quality of meat and meat
products from Palam valley over a period of 5 years. A total of 36 raw chevon
meat samples were collected. Out of 36 meat sample, 5 samples were found
positive for Salmonella with a prevalence rate of 13.88% in raw chevon.
18
Ahmad et al. (2013) studied the different meat samples to assess the
microbial load of raw meat at abattoir and retail outlets in different areas of
Lahore. A total of 40 samples (20 from abattoir, 20 from retail outlets) were
collected and subjected to Salmonella detection. The prevalence of Salmonella in
goat meat was found as 10% in both abattoir and retail outlets.
Odey et al. (2013) evaluated the micro-flora of selected meat and ready to
eat meat products in Calabar, Cross Rever State, Nigeria. Samples were collected
randomly and analysed microbiologically. The study showed that Salmonella spp.
was present in 14.20% of goat meat samples analyzed.
2.4.3 Human
Blaser et al. (1982) reported that the prevalence rate of non typhoidal
Salmonellae, isolated from urban and rural area in Bangladesh was 0.29% and
0.26% respectively.
Goldberg and Rubin (1988) conducted a study and stated that in
developing nations frequently salmonellosis is caused by S. Typhi, which is
highly adapted to human host, whereas, the developed nations, such as USA,
suffer from Salmonella not specifically adapted to human or animal hosts.
Herikstad et al. (2002) stated that S. Enteritidis and S. Typhimurium were
the two most frequently isolated serovars among human isolates.
Parry et al. (2002) reported that Salmonella Typhi causing typhoid fever is
also prevalent in many parts of the world, especially in the rural communities.
Between 17 and 33 million cases are reported annually with 600,000 associated
deaths.
19
Clark et al. (2004) conducted a study in New Zealand and reported that
human cases of salmonellosis have occurred through contact with infected animals
and not through consumption of animal products.
Murugkar et al. (2005) carried out a study to report the isolation along
with the serotypes, phage types and antibiogram pattern of Salmonellae among
man, livestock and poultry in northeastern India. He examined 112 human stool
samples and recovered Salmonella from 23 samples showing prevalence of
20.5%.
Kariuki et al. (2006) conducted a study on 332 children in Kenyan hospital
and he grouped them as children suffering from only bacteremia (51.2%), with
gastroenteritis and bacteremia (8.4%) and with gastroenteritis alone (40.4%). The
non typhoidal Salmonella serotypes obtained from all the cases included S.
Typhimurium (59%) and S. Enteritidis (28.3%).
Kumar et al. (2008) reported that the most common serovars from humans
in India are Salmonella Typhi (73%) and Salmonella Paratyphi A (24%) serovars,
and S. Worthington (28.2%) and S. Typhimurium (22.5%) among non serovars.
Akhtar et al. (2010) conducted a study to show the prevalence of
Salmonella in human diarrhoeal samples. Out of 125 human stool samples
collected, 58 shown to be positive for Salmonella showing a prevalence of
46.40%.
CDC (2010) reported that S. Typhimurium was the second most
commonly isolated Salmonella serovar in 2009, after S. Enteriditis and is the most
common serovar responsible for Salmonella-related hospitalization in children
under the age of 4 years in the U.S.
20
EFSA (2011) report revealed that in 2008, salmonellosis accounted for
131,468 and in 2009 confirmed human cases were 108,614 in EU. Thus, the
number of salmonellosis cases in humans decreased by 17.4 %, compared to
2008. In particular, human cases caused by S. Enteritidis decreased markedly.
Nesa et al. (2011) conducted a study to isolate and identify Salmonella
serovars from human stool samples and characterization of the isolated serovars
using biochemical, serological, molecular and antimicrobial sensitivity
techniques. A total of 25 samples were collected of which 16% were found
positive to Salmonella serovars.
Ramyil et al. (2013) compared the diagnostic performance of widal test
and stool culture in the laboratory diagnosis of Salmonella infection in children
(0-14 yrs) and adults (18 yrs and above). The total number of adult found positive
for stool culture was 12 (25%) among which were 10 (31.2%) males, 2 (12.5%)
female, while the total no of children found positive to culture were 9 (20.9%)
among which were 7 (26.9%) males and 2 (11.7%) females, respectively.
2.5 Biochemical screening of samples:
Das et al. (2012) carried out research work in Tamil Nadu in which
detection of Salmonella enterica serovar Typhi isolated from humans with
typhoidial fever was confirmed by biochemical method. In triple sugar iron slants,
the butt and slant turned into yellow and red colour respectively indicating the
fermentation of glucose alone and production of acid in the butt. Isolates showed
positive result for oxidase test, and methyl red test and negative for indole
21
production and urease production. All the isolates were found gram negative,
flagellated and motile.
Odey et al. (2013) conducted a study to evaluate the micro flora of
selected meat and meat products in Nigeria. Suspected Salmonella isolates were
further processed for biochemical testing. Test shown result as indole negative,
methyl red positive, voges proskauer negative and citrate positive. Samples were
catalase positive, oxidase negative, and coagulase negative. Carbohydrate
fermentation test revealed that the samples were lactose negative, sucrose
negative, glucose positive and mannitol positive.
2.6 Antibiotic sensitivity test:
Usage of antimicrobial drugs has played an important role in animal
husbandry, since they are used in prophylaxis, treatment and growth promotion
(Oliveira et al., 2005). Resistance to combinations of several classes of
antimicrobials has led to the emergence of multidrug-resistant (MDR) strains that
may pass from food animals to humans (O’Brien, 2002 and White et al., 2001). In
many countries including India, various studies have been done so far to
investigate resistance pattern of different serovars of Salmonella and different
workers reported different observations.
Murugkar et al. (2005) reported that only 25.26% of strains were resistant
to Cephalexin whereas in another study, Bhatia et al., (1992) had already reported
that 78% of total isolates were resistance to Cephalexin.
Parveen et al. (2007) reported Salmonella isolates for susceptibility to 15
antimicrobial agents of veterinary and human health significance. Results revealed
22
that, 79.8% isolates were resistant to at least one antimicrobial and 53.4% were
resistant to three or more antimicrobials. Overall the most common resistance
phenotypes were those to Tetracycline (73.4%), Ampicilliin (52.9%),
Amoxicillin-clavulanic acid (52%), Cefoxitin (52%) and Ceftiofur (51.7%). Less
resistance was found to Streptomycin (35.2%), Sulfisoxazole (21.8%) and
Kenamicin (6.3%).
Nesa et al. (2011) conducted antibiotic sensitivity testing of isolated
Salmonella isolates against 8 commonly used antibiotics belonging to different
groups. Among the isolates 100% were highly sensitive to ciprofloxacin, 80% and
60% were to Kanamycin and Chloramphenicol respectively while 40% to
Cotrimoxazole and 20% to Cephalexin. 100% were highly resistant to
Erythromycin and 75 % resistant to Amoxicillin while 20 % were resistant to
Nalidixic acid and Chloramphenicol.
Moon et al. (2011) conducted a study to identify antibiotic sensitivity
pattern of Salmonella spp. isolated from chevon and chicken meat. Results
revealed that the Salmonella isolates were sensitive to Ampicillin, Colistin,
Piperacillin (each of three recorded 56.25% sensitivity) & Netillin & Norfloxacin
(both 43.75% sensitive) and resistant to the antibiotics Ciprofloxacin & Ofloxacin
(56.25% resistance observed against both antibiotics).
Jaulkar et al. (2011) processed 11 Salmonella isolates for studying their
antibiogram pattern. All isolates exhibited resistance to penicillin-G, followed by
Ampicillin, Amoxyclav, and Trimethoprim (81.81% each); Erythromycin and
Tetracycline (63.63% each); Doxycyclin hydrochloride (54.54%); Ceftazidime
(45.45%); Streptomycin (18.18%); Azithromicin, Cephotaxim and Nalidixic Acid
23
(9.09% each). Moderate degree of sensitivity was revealed towards Cephotaxim
(45.45%); Neomycin and Nalidixic Acid (27.27% each); Tetracycline (18.18%);
Azithromicin, Ceftazidime, Erythromycin and Trimethoprim (9.09% each).
Highest degree of sensitivity was recorded towards Amikacin and
Gentamicin(100% each); Azithromicin and Streptomycin (81.81% each) and
Neomycin (72.72%).
Jaulkar et al. (2011) estimated MAR (Multiple Antibiotic Resistance)
index of Salmonella isolates in Nagpur. The MAR index of Salmonella ranged
from 0.06 to 0.53. Out of 11 isolates, 10 were found to have MAR index more
than 0.2, thus indicated injudicious use of antibiotics.
Das et al. (2012) conducted a study in which 16 Salmonella isolates were
found. All the isolates (100%) were found resistant to Ampicillin, moderately
sensitive to Nalidixic Acid and Nitrofurantoin and sensitive to Carbenicillin,
Chloramphenicol, Clindamycin, Gentamycin, Kanamycin and Tetracycline.
However, 13 (81.25 %) isolates were also found resistance to Cefuroxime, while
11 (68.75 %) isolates were found resistant to penicillin-G and Cephalothin. The
remaining 3 (18.75 %) were moderately sensitive to Cefuroxime and 5 (31.25 %)
isolates were moderately sensitive to penicillin-G and Cephalothin.
Kumar et al. (2013) isolated Salmonella from chicken meat and subjected
them to antimicrobial susceptibility testing against 16 different antibiotics
employing disc diffusion technique. Results indicated that Ampicillin and
Sulphafurazole showed 100% resistance in comparision to Furazolidone. All
isolates were sensitive to Nalidixic Acid. Fifty percent or more resistance was
observed among these isolates for as many as 5 antimicrobials including
24
Sulphafurazole (100%), Colistin (100%), Ampicillin (100%), Co-trimaxole (50%)
and Furazolidone (50%).
2.7 Polymerase Chain Reaction (PCR) assay for salmonella identification:
Widjojoatmodjo et al. (1991) standardized first PCR, targeting oriC gene
for specific Salmonella detection.
Rahn et al. (1992) published a PCR assay based on invA gene, testing 772
isolates, comprising of 630 Salmonella and 142 non Salmonella strains. Though S.
Senftenberg and Litchfield were found to be negative, yet, invA showed highest
selectivity in a comparison study (Malorny et al., 2003).
Chen and Griffith’s (2001) reported that several workers have used PCR
with varied success for detection of Salmonella from foods using specific gene
sequences for targeting. Of these, invA gene and fliC gene have been the most
frequently targeted genes for primer selection in PCR based Salmonella spp
detection.
Murugkar et al. (2003) conducted a study to observe the distribution of
virulence gene of Salmonella namely Salmonella enterotoxin (stn), Salmonella
enteritidis fimbrial (sef) and plasmid encoded fimbrial (pef) genes, among
different serovars of Salmonella enterica isolated from man and animals. A total
of 95 isolates belonging to S. Typhimurium (51), S.Enteritidis (36), S. Bareilly(3),
and S. Paratyphi B(5) serovars were subjected to polymerase chain reaction
(PCR ) assay for the detection of stn, sef and pef genes using their specific primers
and the PCR products were analyzed by 1% agarose gel electrophoresis for the
presence of the respective genes. Varying distribution pattern of these genes was
25
observed amongst the isolates while stn was found in all the 95 strains, sef was
found only among the S Enteritidis isolates. The pef gene was found to be absent
in 10 isolates including the three S Bareilly isolates.
Salehi et al. (2005) screened 192 sample of poultry carcass for the
presence of Salmonella from poultry farms in Shiraz province, Iran. 30
Salmonella strains were isolated from broiler specimens, by culturing and
selective plating. When subjected to Salmonella specific –PCR using primers
S139 and S141 belong to invA, all isolates including positive control and Arizona
generated a single 284 bp amplified DNA fragment on 1.2% agarose gel.
Nagappa et al. (2007) conducted a study in which 100 chicken meats and
100 chicken egg samples were processed for the presence of Salmonella and
confirmed through PCR targeting invA gene. Presence of Salmonella was
documented by the appearance of an amplified PCR fragment of 284 bp in all four
isolates.
Freitas et al. (2010) conducted mPCR targeting ompC (Salmonella genus
specific gene), Sdf1 (S. Enteritidis specific), ViaB (S. Typhi specific) and Spy (S.
Typhimurium) genes which proved to be highly fruitful producing fragments of
204 bp in all whereas fragments of 304 bp in S. Enteritidis, 738 bp in S. Typhi and
401 bp in S. Typhimurium, respectively.
Shanmugasamy et al. (2011) conducted PCR assay to assess the presence
of Salmonella spp. In collected samples, InvA gene specific primers were
selected. 8.3% of poultry carcass contaminated with Salmonella spp. was found.
Muthu et al. (2014) undertook a study to detect the two genes namely,
Salmonella enterotoxin (stn) and plasmid encoded fimbrial (pef) genes, among
26
clinical isolates of three Salmonella spp from human. A total of 176 isolates
belonging to Salmonella enterica serovar Typhi (133), Salmonella enteric serovar
paratyphi A (41) and Salmonella enterica serovar Typhimurium (2) serovars were
analysed by polymerase chain reaction (PCR) using their specific primers for the
detection of stn and pef genes. Results of study revealed the presence of stn gene
in 140 isolates with overall prevalence of 79.5% and none of the isolates found
positive for pef gene.
27
CHAPTER-IIIMATERIALS AND METHODS
The present study was conducted at the Department of Veterinary Public
Health and Epidemiology, College of Veterinary Science and Animal Husbandry,
Anjora, Durg, Chhattisgarh. In the present investigation, attempts were made to
isolate and identify Salmonella organisms from foods of animal origin (chevon
and chicken meat) and human diarrhoeal samples. The isolates thus recovered
were further subjected to biochemical and molecular characterization.
3.1 Materials
3.1.1 Glass wares and plastic wares
The glass wares used in the present study were procured from Borosil
Glass wares Ltd. India, whereas, plastic wares and other disposables were
procured from Tarson Product Pvt. Ltd. India. The glasswares were washed and
sterilized following the standard procedures and used during the study period.
3.1.2 Media, chemicals and reagents
All the bacteriological media, chemicals and reagents used in the present
study were obtained from Hi-Media, India, Thermo Scientific, USA and
Bangalore Genei, India and prepared according to the instructions provided by
the manufacturing firms and were checked for sterility before use. The details of
culture media, reagents, stains etc. used throughout the study are as per appendix.
28
3.1.3 Equipment and instruments
Autoclave (Obromax), Deep freezer (Remi), Electronic balance
(Sartorius), PCR system mastercycler (Eppendorf), Gel documentation system
(Biorad), Horizontal gel electrophoresis unit (Biometra), Hot air oven (Unitech),
Incubator (Mac), UV spectrophotometer (Biometra TI3), Laminar flow (Klenz
flow), Micropipette (Borosil), Microwave oven (LG), Refrigerator (LG), Ultra
low temperature freezer (Remi), Vortex mixer (Mac) and Water bath (Rivotek)
were used during the course of present study.
3.1.4 Primers
The sequence and length of the primers targeting the gene segments are
given below in table 1.
Table 1: Details of the primers used for amplification of stn, invA and pef
genes
Target gene Sequence of primer
(5’-3’)Length
(bp)
Amplicon
size
References
stn
Forward GTG AAA TTA TCG
CCA CGT TCG GGC
AA
26
617 Murugkar et
al. (2003)Reverse TCA TCG CAC CGT
CAA AGG AAC C
22
invA
Forward TTG TGT CGC TAT
CAC TGG CAA CC
23
284 Rahn et al.
(1992)Reverse ATT CGT AAC CCG
CTC TCG TCC
21
pef
Forward TGT TTC CGG GCT
TGT GCT
18
700 Murugkar et
al. (2003)Reverse CAG GGC ATT TGC
TGA TTC TTC C
22
29
3.1.5 Antibiotic discs:
Antibiotic discs from HiMedia Laboratory Pvt Limited, Mumbai were used.
3.2 Methods
3.2.1 Sample collection
The foods of animal origin i.e. Chevon and chicken meat were collected
for isolation of Salmonella spp. and for estimating microbial load through
standard plate count. A total of 400 samples comprising Chevon (n=200) and
Chicken (n=200 each) were collected from Durg, Rajnandgaon, Dhamtari, Raipur
and Bilaspur districts of Chhattisgarh during the present study. Stool samples
(n=50) were collected from the local pathology laboratory and various hospitals
located in and around Durg, Rajnandgaon, Dhamtari, Raipur and Bilaspur districts
of Chhattisgarh.
The samples were collected following the protocol recommended by
International Commission on Microbiological Specification for Food (1978). All
the samples were collected in poly bags aseptically and transported to the
laboratory under chilled condition for analysis within 4-6 hrs.
Stool samples were collected in small containers and brought to the
laboratory within 4-6 hrs for further processing and analysis.
3.2.2. Standard Plate Count:
In present study Standard Plate Count (SPC) of each sample of chevon and
chicken meat were determined according to the method described by American
Public Health Association (APHA, 1984). For enumeration purpose ten-fold serial
30
dilution of each sample was prepared in sterile NSS. Subsequently 1 ml of 104 and
105 dilutions were aseptically transferred with the help of a sterile pipette on a
liquid media in petri plates. The inoculum with media was mixed thoroughly and
plate was kept at room temperature for 30 minutes to allow the solidification of
media and then incubated at 37 0C for 24 hrs. The test was performed in
duplicates. The bacterial colonies were counted using digital colony counter. For
calculation of bacterial counts, plates with 30 to 300 colonies were selected and
colonies were counted using digital colony counter. Then the number of colonies
was multiplied with the reciprocal of dilution factor. The result was expressed in
cfu/gm of samples.
3.2.3 Isolation of Salmonella spp.
3.2.3.1 Chevon and chicken meat
For isolation of Salmonella spp from fresh chevon and chicken meat,
standard ISO 6579:2002 and CDC manual (2003) was followed with slight
modifications. The following steps were performed.
Pre-enrichment
For pre- enrichment 25g of meat samples was taken, blended and
discharged in 225 ml of buffered peptone water (BPW) and incubated at 370 C for
20-24 hrs.
Selective Enrichment
Selective enrichment was done in Tetrathionate broth (TT). With a sterile
pipette 1.0ml of the pre enrichment culture was transferred into 10 ml of TT
broths and Incubated at 370C for 20-24 hrs.
31
Selective plating
Brilliant Green Agar (BGA) and Bismuth Sulphite Agar (BSA) was
inoculated with loopful of selective enrichment broth culture and incubated at
370C for 20-24 hrs to obtain well isolated colonies.
Purification
Minimum of 2 colonies from each presumptively- positive plates were
streaked onto MacConkey lactose agar in order to differentiate the lactose non-
fermenters from lactose fermenters for purification and then incubated at 370C for
20- 24 hrs.
3.2.3.2 Isolation of Salmonella from human stool sample
Human stool samples were processed for typhoidal as well as non-
typhoidal salmonellae as per the methods described in CDC/WHO manual (2003)
and in standard ISO 6579:2002, respectively. One gram of human stool sample
was transferred into 10 ml TT broth for selective enrichment. Incubation of TT
broth was carried out for 24 hrs at 37°C. A loopful culture from TT Broth was
streaked on BGA and BSA plates. All the plates were incubated at 37°C for 24 hrs
and observed for characteristic colonies i.e. large, moist and colourless colonies
surrounded by pink medium on BGA and black colony surrounded by brownish-
black zone with metallic sheen on BSA. The characteristic colony was picked up
and streaked on McConkey’s lactose agar (MLA) in order to differentiate the
lactose fermenters (pink colonies) with that of non-lactose fermenters (colourless
colonies). The plates were incubated at 37°C for 24 hrs and the putative
Salmonella colonies were picked up and stained. The organisms showing red rods
32
on Gram’s staining were further streaked on nutrient agar and incubated at 37°C
for 24 hrs. On appearance of proper growth these slants were stored in refrigerator
(4°C) till further use.
3.2.4 Biochemical characterization of putative Salmonella isolates
For further identification, Salmonella isolates were subjected to various
biochemical tests as per the protocol described by Ewing (1986).
3.2.4.1 Triple sugar iron agar (TSI)
The suspected cultures from the agar slants were inoculated on TSI with
the help of straight loop and were incubated at 37°C for 18-24 hrs. Thereafter
observed for the appearance of typical reactions such as development of acid butt
and alkaline slant with H2S production. The samples showing pinkish slant and
yellow butt or black slant and yellow butt were considered as positive for
Salmonella spp.
3.2.4.2 Urease test
Salmonella isolates was inoculated in 3 ml sterile urea broth with the help
of loop and incubated at 37°C for 3-12 hrs. Development of pink colour showed
presence of urease. If reaction was negative then the broth was further incubated
for six more days for confirmation as urease negative.
3.2.4.3 Citrate Utilization
Salmonella isolate was streaked on Simmon’s citrate slant surface and
incubated at 37°C for 24 hrs. If organism used citrate, then the growth of bacteria
was indicated by development of bright blue colour.
33
3.2.4.4 Indole production
Five ml of sterile peptone water was inoculated with a loopful of culture
and incubated at 37°C for 48 hrs. After incubating the bacteria, 0.5 ml Kovac’s
reagent was added to the media. The development of a red/pink layer on top of the
media indicated positive result.
3.2.4.5 Methyl Red (MR) test
The test was performed by inoculating the test organism in 5 ml sterile
glucose phosphate peptone water (GPPW) to detect the production of sufficient
acid during the fermentation of glucose. After overnight incubation at 37 °C, a
drop of methyl red solution was added. A positive methyl red test was shown by
the appearance of a bright red colour, indicating acid production. A yellow or
orange colour was treated as negative.
3.2.4.6 Voges –Proskauer test
A loopful of the suspected culture was suspended in a sterile tube
containing 5 ml of sterile GPPW and incubated at 37 °C for 24 hrs. After
incubation, 6 drops of 5% ethanolic solution of α -naphthol and then 4 drops of
40% potassium hydroxide solution were added. The tube was shaken after
addition of each reagent. The formation of pink to bright red colour indicated a
positive reaction.
3.2.4.7 Carbohydrate Utilization
The carbohydrate fermentation test was performed by inoculating test
cultures into tubes containing various carbohydrates. These tubes were incubated
34
at 37°C for 24 hrs and observed for acid production which was indicated when
media turns pink. Gas produced was noted as bubbles in the Durham’s tube in
case of glucose fermentation.
3.2.5 Antibiotic sensitivity test
Antibiotic sensitivity test was performed as per the method of Bauer and
Kirby (1966). Commercially available antibiotic discs (HiMedia laboratories
Limited, Mumbai) were used to test susceptibility of the isolated Salmonella
against different antibiotics.
Table 2: Antibiotic discs used in present study
S.
No.
Antibiotic discs Symbol Concentration
(mcg)
Interpretative criteria
(mm)
R I S
01. Oxytetracyclin O 30 <11 12-14 >15
02. Amoxycillin AX 10 <13 14-16 >17
03. Cephalexin CN 30 <14 15-17 >18
04. Ciprofloxacin CIP 5 <15 16-20 >21
05. Gentamicin GEN 30 <12 13-14 >15
06. Erythromicin E 10 <13 14-22 >23
07. Cefotaxime CTX 10 <22 23-25 >26
08. Nalidixic Acid NA 30 <13 14-18 >19
09. Ampicillin AMP 10 <13 14-16 >17
10. Ceftazidime CAZ 30 <17 18-20 >21
11. Imipenem IPM 10 <19 20-22 >23
12. Amoxyclav AMC 30 <13 14-17 >18
13. Cefixime CFM 5 <15 16-18 >19
14. Meropenem MRP 10 <19 20-22 >23
R=Resistant, I=Intermediate, S=Sensitive
35
3.2.5.1 Inoculation of test plates
Pure culture from the nutrient agar slant was transferred into a tube
containing 5 ml Luria -Bertani broth. The broth culture was incubated at 37 0C.
After incubation, a sterile cotton swab was dipped into the suspension. The swab
was swirled several times and pressed firmly on the clear wall of the tube to
remove excess inoculums. The inoculum in swab was then inoculated on the dried
surface of a Mueller –Hinton agar plates by rubbing the swab over the entire
sterile agar surface. This procedure was repeated two more times, rotating the
plates approximately 600 each time to ensure an even distribution of inoculum.
The plate was left for 3 to 5 min to allow any excess surface moisture to be
absorbed before applying the antibiotic impregnated discs.
3.2.5.2 Application of discs to inoculated agar plates
The predetermined set of antimicrobial discs viz. Oxytetracycline
(30mcg), Amoxycillin (10mcg), Cephalexin (30mcg), Ciprofloxacin (5mcg),
Gentamicin (30mcg), Erythromycin (10mcg), Nalidixic Acid (30mcg),
Cefotaxime (10mcg), Ampicillin (10mcg), Ceftazidime (30mcg), Imipenem
(10mcg), Amoxyclav (30mcg), Cefixime (5mcg), Meropenem (10mcg) were
placed onto the surface of the inoculated agar plates. The discs were distributed
evenly with minimum gap of 24 mm from centre to centre. The plates were placed
in inverted position in an incubator at 370C within 15 min of the placements of
discs. After 16 to 18 hrs of incubation, each plate was examined for the zones of
inhibition. The diameter of the zones of complete inhibition was measured and
compared with the zone size interpretation chart provided by supplier and were
graded as sensitive, intermediate, and resistant.
36
3.2.5.3 Multiple antibiotic resistance (MAR) index
The multiple antibiotic resistance (MAR) index was calculated as per
Krumperman (1985), by applying a/b where “a” is the number of antibiotics to
which an isolate was resistant and “b” is the number of antibiotics to which the
isolates were exposed.
3.2.6 Molecular characterization of Salmonella spp
3.2.6.1 Isolation of Genomic DNA
Template DNA incorporated in PCR reaction was prepared by boiling and
snap chill method. Culture from nutrient agar was inoculated into 5ml of Luria-
Bertani broth (LB) and incubated for 12-16 hrs at 37°C. Cells from 1.5 ml of the
culture were harvested by centrifugation at 8000 rpm (6800 × g) in a
microcentrifuge for 6 min at room temperature. The supernatant was decanted and
pellet in microcentrifuge tube was added with 1.5ml phosphate buffer saline and
mixed by vortexing, the suspension was then centrifuged for 6 min and
supernatant was discarded. The pellet was mixed with 300µl distilled water and
then put in to water bath for 10 min at 100°C followed by immediate chilling on
crushed ice for at least 20 min. Finally tubes were centrifuged at 10000 g for 2
min and clear supernatant was collected and stored at -20°C until further use.
3.2.6.2 Amplification of stn, invA and pef gene by polymerase chain reaction:
For amplification of stn, invA and pef genes; 25 µl volume reaction
mixtures consisting of following component was used.
37
Contents Quantity
10x PCR assay buffer 2.5 µl
dNTP mix 2.5 µl
Primers (forward and reverse) 1 µl each
Taq polymerase 1.25 µl
Genomic DNA 3 µl
Autoclaved milli Q water 13.75 µl
Amplification of stn gene
For amplification of stn gene, method described by Murugkar et al. (2003)
was followed with suitable modification. PCR was performed in a thermal cycler.
The cycling conditions were optimised for PCR reaction mixture which consisted
of an initial denaturation (940C for 5 min) followed by 30 cycles of denaturation
(940C for 1min), primer annealing (590C for 1 min) and extension (720C for 1
min). A final extension at 720C was given for 10 min. The PCR products thus
obtained was stored at 40C until further use.
Amplification of invA gene
PCR targeting invA gene was performed as per the method described by
Rahn et al. (1992). The cycling conditions were optimised for PCR reaction
mixture which consisted of an initial denaturation (940C for 5 min) followed by
30 cycles of denaturation (940C for 1 min), primer annealing (550C for 1 min) and
extension (720C for 1 min). A final extension at 720C was given for 5 min. The
PCR products thus obtained were kept at 40C and used in electrophoresis.
38
Amplification of pef gene
The PCR for amplification of pef gene was performed following the
protocol described by Murugkar et al. (2003). PCR was run for 25 cycles of
denaturation (940C for 1min), primer annealing (550C for 1 min) and extension
(720C for 1 min) followed by incubation at 72cC for 10 min. The PCR products
was analysed by agarose gel electrophoresis.
3.2.6.3 Agarose gel electrophoresis
To analyse the amplified products, the submarine agarose gel
electrophoresis was performed as described by Sambrook and Russell (2001).
Agarose gel (1.6%) was prepared by putting 0.8 gm agarose in 50 ml 1XTris-
Borate EDTA (TBE) buffer and subjected to heat until the agarose was
completely dissolved and appeared as a clear transparent solution. The agarose
solution was allowed to cool to 60°C and then ethidium bromide (0.5µg/ml) dye
was added to it. Thereafter the gel was poured into the gel casting tray held within
the gel holding tray and the comb was placed into the slots on the tray in such a
manner that a gap of 0.5 mm was left between the tips of comb teeth and the floor
of casting tray so that the wells were completely sealed by the agarose. It was
allowed to solidify for 20-30 min and then the comb was gently removed. The
casting gel along with the running tray was submerged into the electrophoresis
tank containing 1X TBE buffer. A total volume of 8 µl DNA samples were taken
on a clean parafilm and mixed evenly with 2 µl of 6X gel loading dye (Thermo
scientific) and loaded carefully into the wells of agarose gel. To determine the size
of the amplified PCR product 100 bp DNA ladder was loaded in one well.
Electrophoresis was performed at 70 V for 45 min and the mobility was
39
monitored by the migration of the dye in the gel. After appropriate migration, the
gel was visualised under UV transilluminator.
3.2.6.4 Visualization of the gel in Gel Doctm XR
The amplified PCR products were visualized under transilluminator and
the gel were documented by Gel Doctm XR.
40
CHAPTER-IV
RESULTS AND DISCUSSION
Salmonella is one of the primary causes of bacterial foodborne infections
in humans. The re-emergence of this food borne pathogen in recent outbreaks has
highlighted this microorganism once again to the frontlines of public health
science. It is now a day a global problem. Approximately 95% of the cases of
human Salmonellosis are associated with the consumption of contaminated food
products such as meat, poultry, eggs, seafood, and dairy products. In the present
study, attempts were made to isolate and identify Salmonella organisms from
foods of animal origin and humans. The putative Salmonella isolates recovered
from these samples were further subjected to biochemical, serological and
molecular characterization.
4.1. Standard Plate Count
The Standard Plate Count (SPC) of 400 samples, comprising of 200
chevon meat and 200 chicken meats, was determined by pour plate technique and
values observed are given in table 3. Highest Mean SPC of chevon was recorded
in Raipur district (4.4099log10cfu/g), followed by Bilaspur (4.4014log10cfu/g),
Dhamtari (4.3692log10cfu/g), Durg (4.3521log10cfu/g), and Rajnandgaon
(4.2671log10cfu/g) districts. Highest Mean SPC of chicken meat was seen in
Raipur district (4.4199log10cfu/g), followed by Dhamtari (4.3944log10cfu/g),
Bilaspur (4.3820log10cfu/g), Durg (4.3692log10cfu/g), and Rajnandgaon
(4.2966log10cfu/g) districts.
The SPC in present study was within the permissible limits of 6log10cfu/g
(BIS, 1995) thus considered chevon acceptable for human consumption. The
41
result of present study are in congruence with findings of Dhanze et al. (2012)
who reported 4.778 log10cfu/g in chevon samples collected from different parts of
Palampur. Similar result was shown by Ahmad et al. (2013) who reported SPCs
of goat meat as 4.84 log10cfu/g. However higher SPC value in chevon were
reported by several researchers (Mawia et al, 2012; Patyal et al., 2012; Lambey et
al., 2009; Gangil et al., 2011; Ahmad et al., 2013).
In case of poultry meat, mean SPCs of 4.3724 was observed. This is
similar to the report shown by Janjirkar et al. (2005) who reported mean SPCs of
fresh poultry meat as 4.82±0.83 log10cfu/g. On the contrary higher mean SPCs of
poultry meat 7.3979, 6.4624 and 6.91 were reported by Tompkins et al. (2008),
Nikas (2009) and Adu-Gyamfi et al. (2012) respectively.
Table 3: District wise SPC values (log10cfu/g) of collected chevon and chicken
samples
S.No.
Districts Chevon Chicken MeatSPC Range Mean SPC
ValueSPC Range Mean
SPCValue
1. Raipur 4.2810-4.5198 4.4099 4.2810-4.6020 4.4199
2. Bilaspur 4.2855-4.4871 4.4014 4.1461-4.6127 4.3820
3. Dhamtari 4.0492-4.6127 4.3692 4.2455-4.5465 4.3944
4. Durg 4.04-4.50 4.3521 4.0211-4.6334 4.3692
5. Rajnandgaon 4.04-4.57 4.2671 4.0453-4.5051 4.2966
Overall Mean SPCValue
4.3645 4.3724
42
4.2. Isolation of Salmonella spp.
In order to get better recovery of salmonellae, the chevon meat (n=200) as
well as chicken meat (n=200) samples were inoculated in buffered peptone water
(BPW) medium for pre-enrichment. Pre-enrichment step is found essential for
meat and milk where organisms are either less in number or are in stressed
condition, as it helps to recover surviving organisms which may have received
sub-lethal damage due to adverse environmental conditions and are unable to
multiply if inoculated directly into enrichment broth (Fricker, 1987). Assuming
that the desired organisms have got conditioned and relieved from the
environmental stress 1 ml BPW broth culture was transferred into 10 ml of TT
broth. Thereafter, a loopful of culture from selective enrichments was streaked
onto BGA and BSA media. At least, five characteristic colonies each from BGA
(moderately large, moist, smooth and colourless colonies with pink background),
BSA (black colony surrounded by brownish–black zone with metallic sheen) and
MLA (colourless colonies) were picked up for further identification (Fig. 1, 2 and
3).
A total of 450 samples comprising chevon (200) and chicken meat (200)
and stool sample (50) were screened for presence of Salmonella spp. Out of 450
samples only 32 (18 chevon and 14 chicken meat) were positive for Salmonella
spp by culture method showed a overall prevalence of 7.11%. The highest
prevalence was observed in chevon (9%) followed by chicken meat (7%) and
human stool sample (0%) (Fig. 4 and Table 4).
In chevon, highest prevalence was observed in Durg district (12.5%),
followed by Raipur and Dhamtari (10% each), Rajnandgaon (7.5%) and Bilaspur
43
(5%) districts as shown in Fig. 5 and Table 5 . In chicken meat highest prevalence
of Salmonella was seen in Rajnandgaon (12.5%) district followed by Raipur and
Bilaspur (10%), Durg (5%) and Dhamtari (2.5%) districts as shown in Fig. 6 and
Table 5.
In the present study prevalence of Salmonella in chevon was found as 9%
which was nearly close to the prevalence (8.05%) reported by Eze et al. (2012) in
Umuahia market Abia State. Similar prevalence of 10%, 13.88% and 14.20%
were reported by Ahmad et al. (2013), Panda et al. (2012) and Odey et al. (2013)
respectively. However lower prevalence rate of 1.25%, 2.5% and 3.3% were
reported by Lambey et al. (2009), Zubair et al. (2012) and Dabassa et al. (2012).
On the contrary higher prevalence rate of 38.33% was reported by Moon et al.
(2011) in Wardha district.
In case of poultry meat 7% prevalence was seen which is similar to the
findings of Patyal et al. (2012) who reported 6% prevalence in chicken meat in
Jaipur city, Rajasthan. Hue et al. (2011) also reported similar prevalence of 7.52%
in France. Similar observation were also reported by Panda et al. (2012), Dahal et
al. (2008), Salehi et al. (2005) and Whyte et al. (2002). However lower
prevalence rate of 1.5% and 4% were reported by Kumar et al. (2013) and Rabie
et al. (2012). On the contrary higher prevalence rate of 30%, 31.99% and 38.33%
were observed by Akhtar et al. (2010), Ruban et al. (2010) and Moon et al.(2011),
respectively.
In the present study no Salmonella spp. was isolated from human stool.
The cause of such a low prevalence rate of Salmonella in human stool could be
44
because of the fact that the samples were collected from non-clinical apparently
healthy humans
Table 4: Prevalence of Salmonella spp. in chevon, chicken meat and stool
samples
S. no Sample No of sample
analysed
No of sample
positive
Prevalence
(%)
1 Chevon meat(m) 200 18 09
2 Chicken meat(c) 200 14 07
3 Stool sample 50 00 00
Table 5: District wise prevalence of Salmonella in chevon, chicken meat and stool
samples
S.
No.
Districts Chevon Chicken meat Stool
No. of
samples
collected
No. of
samples
positive
(%)
No. of
samples
collected
No. of
samples
positive (%)
No. of
samples
collected
No. of
samples
positive
(%)
01. Durg 40 5 (12.5%) 40 2 (5%) 10 0 (0%)
02. Rajnandgaon 40 3 (7.5%) 40 5 (12.5%) 7 0 (0%)
03. Raipur 40 4 (10%) 40 3 (7.5%) 20 0 (0%)
04. Dhamtari 40 4 (10%) 40 1 (2.5%) 7 0 (0%)
05. Bilaspur 40 2 (5%) 40 3 (7.5%) 6 0 (0%)
Fig. 1: Salmonellae isolates showing moderately large, moist, smooth andColourless colonies with pink background on Brilliant green agar(BGA) plate
Fig. 2: Salmonellae isolates showing black colony surrounded bybrownish-black zone with metallic sheen on Bismuth sulphiteagar (BSA)
Fig. 1: Salmonellae isolates showing moderately large, moist, smooth andColourless colonies with pink background on Brilliant green agar(BGA) plate
Fig. 2: Salmonellae isolates showing black colony surrounded bybrownish-black zone with metallic sheen on Bismuth sulphiteagar (BSA)
Fig. 1: Salmonellae isolates showing moderately large, moist, smooth andColourless colonies with pink background on Brilliant green agar(BGA) plate
Fig. 2: Salmonellae isolates showing black colony surrounded bybrownish-black zone with metallic sheen on Bismuth sulphiteagar (BSA)
Fig. 3: Salmonellae isolates showing colourless colonies on MacConkeyLactose Agar (MLA) plate
Fig 4: Prevalence of Salmonella spp. in chevon, chicken meat and human
stool samples
0
1
2
3
4
5
6
7
8
9
Chevon
Fig. 3: Salmonellae isolates showing colourless colonies on MacConkeyLactose Agar (MLA) plate
Fig 4: Prevalence of Salmonella spp. in chevon, chicken meat and human
stool samples
Chicken meat Stool sample
Prevalence(%)
in %
Fig. 3: Salmonellae isolates showing colourless colonies on MacConkeyLactose Agar (MLA) plate
Fig 4: Prevalence of Salmonella spp. in chevon, chicken meat and human
stool samples
Prevalence(%)
in %
Fig 5: Prevalence of Salmonella spp. in chevon in different districts of
Chhattisgarh
Fig 6: Prevalence of Salmonella spp. in chicken meat in different districts of
Chhattisgarh
10%
10%
7.50%
2.50%
Fig 5: Prevalence of Salmonella spp. in chevon in different districts of
Chhattisgarh
Fig 6: Prevalence of Salmonella spp. in chicken meat in different districts of
Chhattisgarh
Durg
Rajnandgaon
Raipur
Dhamtari
Bilaspur
12.50%
7.50%10%
5%
Durg
Rajnandgaon
Raipur
Dhamtari
Bilaspur
5%
12.50%
7.50%
Fig 5: Prevalence of Salmonella spp. in chevon in different districts of
Chhattisgarh
Fig 6: Prevalence of Salmonella spp. in chicken meat in different districts of
Chhattisgarh
Durg
Rajnandgaon
Raipur
Dhamtari
Bilaspur
Durg
Rajnandgaon
Raipur
Dhamtari
Bilaspur
45
4.3 Biochemical characterization of Salmonella isolates
The cultures suspected to be of Salmonella were subjected to various
biochemical tests consisting of TSI, Urease, Methyl red, Voges Proskauer,
Indole, Citrate utilization and Carbohydrate (viz. Glucose, Sucrose, Lactose, and
Mannitol, Sorbitol, Raffinose) fermentation tests. Of all suspected Salmonella
isolates tested, only 32 isolates comprising of: 14 from poultry meat, 18 from
chevon meat showed characteristic biochemical reaction for Salmonella as shown
in Table 6 and figure 7 to 10. The various pattern and reaction shown by
Salmonella isolates in this investigation were similar to the observation seen by
Das et al. (2012) in Tamil nadu and Odey et al. (2013) in Nigeria.
46
Table 6: Biochemical profile of suspected Salmonella isolates
S .No. Isolates
No.
Source Biochemical tests
Urease Indole MR VP Citrate TSI Sorb Raff Suc Lac Mann Glu
1. D-C11 Chicken
meat
- - + - + + + - - - + +
2. D-C20 Chicken
meat
- - + - + + + - - - + +
3. R-C3 Chicken
meat
- - + - + + + - - - + +
4. R-C4 Chicken
meat
- - + - + + + - - + + +
5. R-C18 Chicken
meat
- - + - + + + - - - + +
6. R-C22 Chicken
meat
- - + - + + + - - - + +
7. R-C30 Chicken
meat
- - + - + + + - - - + +
8. RP-
C10
Chicken
meat
- - + - + + + - - - + +
9. RP- Chicken - - + - + + + - - - + +
Cont.
47
C24 meat
10. RP-
C38
Chicken
meat
- - + - + + + - - - + +
11. DH-
C28
Chicken
meat
- - + - + + + - - - + +
12. BP-C7 Chicken
meat
- - + - + + + - - - + +
13. BP-
C21
Chicken
meat
- - + - + + + - - - + +
14. BP-
C33
Chicken
meat
- - + - + + + - - - + +
15. D-M2 Chevon - - + - + + + - - - + +
16. D-M20 Chevon - - + - + + + - - - + +
17. D-M21 Chevon - - + - + + + - - - + +
18. D-M27 Chevon - - + - + + + - - - + +
19. D-M28 Chevon - - + - + + + - - + + +
20. R-M16 Chevon - - + - + + + - - - + +
21. R-M24 Chevon - - + - + + + - - - + +
22. R-M25 Chevon - - + - + + + - - - + +
Cont.
48
23. RP-M1 Chevon - - + - + + + - - - + +
24. RP-
M15
Chevon - - + - + + + - - - + +
25. RP-
M21
Chevon - - + - + + + - - - + +
26. RP-
M27
Chevon - - + - + + + - - - + +
27. DH-M8 Chevon - - + - + + + - - + + +
28. DH-
M11
Chevon - - + - + + + - - - + +
29. DH-
M20
Chevon - - + - + + + - - - + +
30 DH-
M31
Chevon - - + - + + + - - - + +
31. B-M17 Chevon - - + - + + + - - - + +
32. B-M19 Chevon - - + - + + + - - - + +
Sorb=Sorbitol, Raff= Raffinose, Suc= Sucrose, Lac=Lactose, Mann=Mannitol, Glu=GlucoseTSI=Triple Sugar Iron, MR=Methyl Red, VP=Voges-ProskauerM=Chevon,C=Chicken, D=Durg, R=Rajnandgaon, RP=Raipur, DH=Dhamtari, B=Bilaspur
Fig. 7: Salmonella spp. showing negative reaction on urea broth as there is nocolour change (D). A positive reaction was indicated by change incolour to pink (A, B and C).
Fig.8: Salmonella isolates showing typical reactions such as development ofacid butt, alkaline slant with H2S production on TSI Agar slant (A)while, such reactions are absent in negative control (B and C).
B C DA
A B C
Fig.9: Citrate utilization test- A positive reaction for Salmonella spp. isindicated by growth of organism on to the slant as well as changein the colour of the slant from green to blue (B and C) while suchreaction are absent in negative control (A).
Fig. 10: Indole test – Absence of development of bright red colour ringwas suggestive of positive test for Salmonella spp. (A, B and D).
CBA
DCBA
49
4.4 Antibiotic sensitivity test
Antimicrobial resistance has been recognised by World Health
Organization as a major emerging problem of public health. The emergence
and spread of multi-drug resistance against Salmonella species have reinforced
the need for epidemiological studies describing the prevalence and the pattern
of resistance of this strain. Different antibiotic sensitivity pattern was shown in
the present study by different isolates (Fig. 11 and 12). All 32 isolates
obtained from chevon and chicken meat were found to be sensitive to
Ciprofloxacin and 93.75% isolates were resistant to Erythromycin. Majority of
the isolates were sensitive to Gentamicin (96.87%), Imipenem (96.87%) and
Ceftazidime (93.75%) and resistant to Oxytetracyclin (59.37%). Varying
degree of sensitivity was found against Amoxyclav and Cefixime (81.25%
each), Nalidixic Acid, Amoxicillin and Cephalexin (78.12% each), Ampicillin
(75%), Cefotaxime (59.37%) (Table 7 and 8).
Sample wise antibiogram study revealed that all the isolates from
chicken meat were 100% resistant to Erythromycin whereas 88.88% of
Chevon meat isolates were resistant to Erythromycin. In case of
Oxytetracyclin the order of resistance in chevon was (72.22%) followed by
chicken meat (42.85%) (Table 9).
Highest multiple antibiotic resistance (MAR) index was 0.50 (1
isolate) followed by 0.42 (one isolate), 0. 35 (one isolate), 0.28 (three isolates),
0.21 (eight isolates), 0.10 (nine isolates) and 0.07 (eight isolates). The
minimum MAR index 0 was shown by one isolates. Out of 32, 14 isolates
were found to have MAR index equal to or more than 0.2, thus indicated
injudicious use of antibiotics (Table 7). Similar results were noticed by Jaulkar
50
et al. (2011) who reported MAR index ranging from 0.06 to 0.53 and he found
10 out of 11 isolates were having MAR index more than 0.2.
51
Table 7: Pattern of antibiogram shown by the Salmonella isolates
S.No.
Isolates Antibiotic discsMAR index0 AX CN CIP GEN E CTX NA AMP CAZ IPM AMC CFM MRP
1. D-C11 R S S S S R S S S S S S S S 0.10
2. D-C20 S S S S S R I S S S S S S S 0.07
3. R-C3 S S S S S R S I S S S S S S 0.07
4. R-C4 S S S S S R I S S S S S S S 0.07
5. R-C18 R S S S S R S R S S S S S S 0.21
6. R-C22 R S S S S R S S S S S S S S 0.14
7. R-C30 R S S S S R S S S S S S S S 0.14
8. RP-C10 S S S S S R I S S S S S S S 0.07
9. RP-C24 S S S S S R I R S S S S S S 0.14
10. RP-C38 S S S S S R S S S S S S S I 0.07
11. DH-C28 S S S S S R S S S S S S S S 0.07
12. BP-C7 S R R S S R I S R S S R R I 0.42
13. BP-C21 R S S S S R S S R S S S I S 0.21
14. BP-C33 R S S S S R S S S S I S S I 0.14
Cont.
52
15. D-M2 S S S S S R S R S S S S S S 0.14
16. D-M20 R S S S S R S S S S S S S S 0.14
17. D-M21 R I I S S R R S I I S S R S 0.28
18. D-M27 R S S S S R S S S S S S S S 0.14
19. D-M28 R S S S S R S S S S S S R S 0.21
20. R-M16 R S S S S R S R S S S S S I 0.21
21. R-M24 S S S S S R I S S S S S S S 0.71
22. R-M25 R S S S S I S S S S S S S S 0.71
23. RP-M1 R R R S S R R S S S S I S S 0.35
24. RP-M15 R S R S S R S S R S S I I I 0.28
25. RP-M21 S S S S S I I S I I S S S S 0
26. RP-M27 S I S S I R R R S S S S S S 0.21
27. DH-M8 R S S S S R R R S S S S S S 0.28
28. DH-M11 R I R S S R S S S S S I S I 0.21
29. DH-M20 R I R S S R S S I S S I S I 0.21
30. DH-M31 S S S S S R R S R S S S S S 0.21
Cont.
53
31. B-M17 R R R S S R I S R S S R R S 0.50
32. B-M19 R S S S S R S S S S S S S S 0.14
MAR=Multiple antibiotic resistanceD=Durg, R=Rajnandgaon, RP= Raipur, DH=Dhamtari, B=BilaspurM= chevon meat, C= chicken meatR=Resistant, I=Intermediate, S=SensitiveO=Oxytetracyclin, AX= Amoxycillin, CN= Cephalexin, CIP=Ciprofloxacin, GEN=Gentamicin, E= Erythromicin, CTX=Cefotaxime,NA= Nalidixic Acid, AMP=Ampicillin, CAZ=Ceftazidime, IPM=Imipenem, AMC=Amoxyclav, CFM=Cefixime, MRP=Meropenem
54
Similar results were also observed by Nesa et al. (2011) who reported
that 100% isolates were sensitive to ciprofloxacin and 100 % were resistant to
erythromycin. This is in accordance with the present study. In the present
study 75% sensitivity towards Ampicillin was seen. The observation was
corroborative with Moon et al. (2011) who reported 56.25% sensitivity
towards Ampicillin. 78.12% of sensitivity towards Nalidixic Acid was
observed during the present study which is similar to the observation of
Kumar et al. (2013). Jaulkar et al. (2011) recorded highest degree of
sensitivity towards Gentamicin (100%) which is in accordance to the
sensitivity of 96.87% for Gentamicin.
Results showed that the large number of recovered Salmonella isolates
were resistant to multiple antimicrobials. Multidrug-resistant Salmonella
isolates have been reported as the cause of both human and animal
salmonellosis worldwide by numerous investigators and these strains are of
particular clinical concern because they frequently display resistance to key
antimicrobials, notably third generation cephalosporin (Jain et al.,2006; Logue
et al.,2003; White et al.,2001; Zhao et al.,2006).
Table 8: Antibiogram assay of Salmonella isolates
S.
N
o.
Name of
antimicrobial
agent
Pattern of antibiogram
Resistant Intermediate Sensitive
1. Oxytetracyclin 19(59.37%) 0(0%) 13(40.62%)
2. Amoxycillin 3(9.37%) 4(12.5%) 25(78.12%)
3. Cephalexin 6(18.75%) 1(3.12%) 25(78.12%)
4. Ciprofloxacin 0(0%) 0(0%) 32(100%)
5. Gentamicin 0(0%) 1(3.12%) 31(96.87%)
6. Erythromycin 30(93.75%) 2(6.25%) 0(0%)
Cont.
55
7. Cephotaxime 5(15.62%) 8(25%) 19(59.37%)
8. Nalidixic acid 6(18.75%) 1(3.12%) 25(78.12%)
9. Ampicillin 5(15.62%) 3(9.37%) 24(75%)
10. Ceftazidime 0(0%) 2(6.25%) 30(93.75%)
11. Imipenem 0(0%) 1(3.12%) 31(96.87%)
12. Amoxyclav 2(6.25%) 4(12.5%) 26(81.25%)
13. Cefixime 4(12.5%) 2(6.25%) 26(81.25%)
14. Meropenem 0(0%) 7(21.87%) 25(78.12%)
Table 9: Sample wise antibiogram of Salmonella isolates
S.
No.
Name of antimicrobial
agent
Pattern of
antibiogram
Chevon(n=18) Chicken
meat(n=14)
1. Oxytetracyclin (O) R
I
S
13(72.22%)
0(0%)
5(27.77%)
6(42.85%)
0(0%)
8(57.14%)
2. Amoxycillin (AX) R
I
S
2(11.11%)
4(22.22%)
12(66.66%)
1(7.14%)
0(0%)
13(92.85%)
3. Cephalexin (CN) R
I
S
5(27.77%)
1(5.55%)
12(66.66%)
1(7.14%)
0(0%)
13(92.85%)
4. Ciprofloxacin (CIP) R
I
S
0(0%)
0(0%)
18(100%)
0(0%)
0(0%)
14(100%)
5. Gentamicin (GEN) R
I
S
0(0%)
1(5.55%)
17(94.44%)
0(0%)
0(0%)
14(100%)
6. Erythromicin (E) R
I
S
16(88.88%)
2(11.11%)
0(0%)
14(100%)
0(0%)
0(%)
7. Cephotaxime (CTX) R
I
5(27.77%)
3(16.66%)
0(0%)
5(35.71%)
Cont.
56
S 10(55.55%) 9(64.28%)
8. Nalidixic Acid (NA) R
I
S
3(16.66%)
1(5.55%)
14(77.77%)
3(21.42%)
0(0%)
11(78.57%)
9. Ampicillin (AMP) R
I
S
3(16.66%)
3(16.66%)
12(66.66%)
2(14.28%)
0(0%)
12(85.71%)
10. Ceftazidime (CAZ) R
I
S
0(0%)
2(11.11%)
16(88.88%)
0(0%)
0(0%)
14(100%)
11. Imipenem (IPM) R
I
S
0(0%)
0(0%)
18(100%)
0(0%)
1(7.14%)
13(92.85%)
12. Amoxyclav (AMC) R
I
S
1(5.55%)
4(22.22%)
13(72.22%)
1(7.14%)
0(0%)
13(92.85%)
13. Cefixime (CFM) R
I
S
3(16.66%)
1(5.55%)
14(77.77%)
1(7.14%)
1(7.14%)
12(85.71%)
14. Meropenem (MRP) R
I
S
0(0%)
4(22.22%)
14(77.77%)
0(0%)
3(21.42)
11(78.57%)
R=ResistantI=IntermediateS=Sensitive
Fig. 11: Antibiotic sensitivity test showing the zone of inhibition againstdifferent antibiotics
Fig 12: Antibiogram pattern shown by Salmonella isolates
0
20
40
60
80
100
120
Fig. 11: Antibiotic sensitivity test showing the zone of inhibition againstdifferent antibiotics
Fig 12: Antibiogram pattern shown by Salmonella isolates
% Resistant
% Intermediate
% Sensitive
Fig. 11: Antibiotic sensitivity test showing the zone of inhibition againstdifferent antibiotics
Fig 12: Antibiogram pattern shown by Salmonella isolates
% Resistant
% Intermediate
% Sensitive
57
4.5 PCR based molecular characterization
The genomic DNA of conventionly and biochemically confirmed
Salmonella isolates were extracted by hot and cold lysis method (snap chill).
Three genes viz. invA, stn and pef of Salmonella isolates were targeted and
amplified employing PCR with specific reported primers. 8µL of the amplified
PCR product was electrophoresed through 1.6% agarose gel. The
electrophoretic analysis of the PCR products revealed amplification of
fragment targeting invA gene at 284 bp and 617 bp for stn gene, respectively
as shown in Fig 13 and 14 and Table 10.
All Salmonella isolates were found to carry the enterotoxin
determinant stn gene. The results were in agreement with Murugkar et al.
(2003) and Makino et al. (1999). Murugkar et al. (2003) obtained 617 bp in
all 95 Salmonella strains belonging to Salmonella enterica, isolated from man
and animals. The gene stn is reported to be absent in S. bongori strains as well
as other members of Enterobacteraceae or Vibrio families which have
enterotoxigenic potential (Murugkar, 2003). PCR amplicon was obtained in all
32 Salmonella cultures, which were confirmed by conventional and
biochemical methods with no false-negative reactions. However, Muthu et al.
(2014) detected stn gene among clinical isolates of three Salmonella species
from human and found that 79.5% Salmonella isolates were positive for
enterotoxin.
The invA (284 bp) gene was targeted for the confirmation of
Salmonella organisms at the genus level and the pef (700 bp) was targeted for
the virulence of Salmonella. The gene invA is present in all invasive strains of
Salmonella and absent from closely related genera such as Escherichia. The
58
invA gene was amplified from 31 out of 32 Salmonella isolates. These results
are in agreement with Shanmugasamy et al. (2011) who reported 139-141 the
most selective primer set, which targets the invA gene. The specific PCR
assay, which was validated in his project, showed high selectivity on 242
Salmonella strains (sensitivity 99.6%) and 122 non – Salmonella strains
(specificity 100%). The results are consistent with ones observed by Salehi et
al. (2005) who was able to establish 100% reproducibility and specificity of
primer pair. It is speculated that invA is absent in these strains, which are not
invasive, or that they might be using other invasive mechanisms. However,
absence of invA in Salmonella seems to be rare (Malorny et al., 2003).
In the present study none of the isolates were found positive for pef
gene. This is similar to the report of Muthu et al. (2014) who also couldn’t
find pef in any of the Salmonella isolated from human clinical samples.
Table 10: Distribution of Salmonella specific virulent genes among
different isolates
S. No. Isolates Target genes
Stn invA pef
1. D-C11 + + -
2. D-C20 + + -
3. R-C3 + + -
4. R-C4 + + -
5. R-C18 + + -
6. R-C22 + + -
7. R-C30 + + -
8. RP-C10 + + -
9. RP-C24 + + -
10. RP-C38 + + -
59
11. DH-C28 + + -
12. BP-C7 + + -
13. BP-C21 + + -
14. BP-C33 + + -
15. D-M2 + + -
16. D-M20 + + -
17. D-M21 + + -
18. D-M27 + - -
19. D-M28 + + -
20. R-M16 + + -
21. R-M24 + + -
22. R-M25 + + -
23. RP-M1 + + -
24. RP-M15 + + -
25. RP-M21 + + -
26. RP-M27 + + -
27. DH-M8 + + -
28. DH-M11 + + -
29. DH-M20 + + -
30 DH-M31 + + -
31. B-M17 + + -
32. B-M19 + + -
M=Chevon, C=Chicken, D=Durg, R=Rajnandgaon, RP=Raipur,DH=Dhamtari, B=Bilaspurstn= Salmonella enterotoxin, invA= invasive, pef= plasmid encoded fimbrial
Fig. 13: Agarose gel electrophoresis showing amplified PCR product ofstn gene of Salmonella isolatesLane 1- 100 bp ladderLane 2, 3, 4, 5, 6, 7, 8 and 9- Positive samplesLane 10- Negative control
1000900800700600500400
300
200
100
Lane 1 2 3 4 5 6 7 8 9 10 11 12
bp
284 bp
Fig. 14: Agarose gel electrophoresis showing amplified PCR product ofinvA gene of Salmonella isolatesLane 1- 100 bp ladderLane 2, 3, 4, 6, 7, 8, 9, 11 and 12- Positive sampleLane 5- Negative sampleLane 10 – Negative control
284 bp
617 bp
Fig. 13: Agarose gel electrophoresis showing amplified PCR product ofstn gene of Salmonella isolatesLane 1- 100 bp ladderLane 2, 3, 4, 5, 6, 7, 8 and 9- Positive samplesLane 10- Negative control
1000900800700600500400
300
200
100
Lane 1 2 3 4 5 6 7 8 9 10 11 12
bp
284 bp
Fig. 14: Agarose gel electrophoresis showing amplified PCR product ofinvA gene of Salmonella isolatesLane 1- 100 bp ladderLane 2, 3, 4, 6, 7, 8, 9, 11 and 12- Positive sampleLane 5- Negative sampleLane 10 – Negative control
284 bp
617 bp
Fig. 13: Agarose gel electrophoresis showing amplified PCR product ofstn gene of Salmonella isolatesLane 1- 100 bp ladderLane 2, 3, 4, 5, 6, 7, 8 and 9- Positive samplesLane 10- Negative control
1000900800700600500400
300
200
100
Lane 1 2 3 4 5 6 7 8 9 10 11 12
bp
284 bp
Fig. 14: Agarose gel electrophoresis showing amplified PCR product ofinvA gene of Salmonella isolatesLane 1- 100 bp ladderLane 2, 3, 4, 6, 7, 8, 9, 11 and 12- Positive sampleLane 5- Negative sampleLane 10 – Negative control
284 bp
617 bp
60
CHAPTER-V
SUMMARY, CONCLUSIONS AND SUGGESTIONS FOR
FUTURE RESEARCH WORK
Salmonella organisms affect a wide range of animals and cause acute
gastro-intestinal disorder. Two types of syndrome caused by this organism in
humans include enteric fever and other gastroenteritis. Salmonella infections
in humans are frequently associated with faecal contamination of the foods.
The organism may easily spread among animals in a herd or flock and animal
may become intermittent or persistent carriers.
The Standard Plate Count (SPC) of 400 samples consisted of 200
chevon meat and 200 chicken meats were determined by pour plate technique.
Highest mean SPC of chevon was recorded in Raipur district
(4.4099log10cfu/g), followed by Bilaspur (4.4014log10cfu/g), Dhamtari
(4.3692log10cfu/g), Durg (4.3521log10cfu/g), and Rajnandgaon
(4.2671log10cfu/g) districts. Highest Mean SPC of chicken meat was seen in
Raipur district (4.4199log10cfu/g), followed by Dhamtari (4.3944log10cfu/g),
Bilaspur (4.3820log10cfu/g), Durg (4.3692log10cfu/g), and Rajnandgaon
(4.2966log10cfu/g) districts.
A total of 450 samples comprising chevon (n=200) and chicken meat
(n=200) and stool sample (n=50) were screened for the presence of Salmonella
spp. Out of them, 32 samples (18 chevon and 14 chicken meat samples) were
found positive for Salmonella spp by culture method with a overall prevalence
of 7.11%. The highest prevalence was observed in chevon samples (9%)
followed by chicken meat samples (7%) and human stool sample (0%).
61
In chevon, highest prevalence was observed in chevon samples
collected from Durg district (12.5%), followed by Raipur and Dhamtari (10%
each), Rajnandgaon (7.5%) and Bilaspur (5%) districts. Chicken meat samples
collected from Rajnandgaon district showed highest prevalence of Salmonella
(12.5%) followed by samples of Raipur and Bilaspur (10%), Durg (5%) and
Dhamtari (2.5%) districts.
The 32 suspected isolates of Salmonella were further subjected to
various biochemical tests consisting of TSI, Urease, Methyl red, Voges
Proskauer, Indole, Citrate utilization and Carbohydrate (Glucose, Sucrose,
Lactose, and Mannitol, Sorbitol, Raffinose) fermentation tests. Of all
suspected Salmonella isolates tested, 14 from poultry meat, 18 from chevon all
showed characteristic biochemical reaction specific for Salmonella spp.
Antibiotic sensitivity/resistance pattern against Salmonella isolates was
studied in the present study. All 32 isolates obtained from chevon and chicken
meat were found to be sensitive to Ciprofloxacin and 93.75% isolates were
resistant to Erythromycin. Majority of the isolates are sensitive to Gentamicin
(96.87%), Imipenem (96.87%) and Ceftazidime (93.75%) and resistant to
Oxytetracyclin (59.37%). Varying degree of sensitivity found against
Amoxyclav, Cefixime (81.25% each), Nalidixic Acid, Amoxicillin and
Cephalexin (78.12% each), Ampicillin (75%) and Cefotaxime (59.37%).
Sample wise antibiogram study revealed that all the isolates from
chicken meat were resistant to Erythromycin whereas 88.88% of Chevon meat
isolates were resistant to Erythromycin. In case of Oxytetracyclin, isolates
from chevon samples showed higher resistance (72.22%) followed by isolates
from chicken meat samples (42.85%).
62
Highest multiple antibiotic resistance (MAR) index was 0.50 (1
isolate) followed by 0.42 (one isolate), 0. 35 (one isolate), 0.28 (three isolates),
0.21 (eight isolates), 0.10 (nine isolates) and 0.07 (eight isolates). The
minimum MAR index 0 was shown by one isolates. Out of 32 isolates of
Salmonella spp., 14 isolates were found to have MAR index equal to or more
than 0.2, thus indicating injudicious use of antibiotics.
The genomic DNA of conventionly and biochemically confirmed
Salmonella isolates were extracted by hot and cold lysis method (snap chill).
Three genes viz. invA, stn and pef of Salmonella isolates were targeted and
amplified employing PCR with specific reported primers. All Salmonella
isolates were found to carry the enterotoxin determinant stn gene. PCR
amplicon for it was obtained in all 32 Salmonella isolates. The invA (284 bp)
gene was targeted for the confirmation of Salmonella organisms at the genus
level. 31 out of 32 Salmonella isolates were found positive for the invA gene.
In the present study none of the isolates were found positive for the pef gene
(700bp) virulence gene of Salmonella.
63
CONCLUSIONS:
1. The overall prevalence of Salmonella was found 9% in chevon whereas 7% in
chicken meat in this region with little variation in biochemical activities of the
isolate.
2. As all the isolates were from chevon and chicken meat thus it may pose a
major threat for spread of Salmonellosis in humans in and around Durg,
Raipur, Dhamtari, Rajnandgaon and Bilaspur Districts.
3. Salmonella needs special concern because of poor hygienic conditions
prevailing in the areas of sampling which ultimately favour its spread.
Salmonella spp. should be under supervision of public health and veterinary
authorities to ensure the early detection and to implement preventive measures
to control the spread of zoonoses.
4. The result of the study demonstrated the prevalence of Salmonella
contamination and varied spectrum of antimicrobial resistance, including
several MDR phenotypes among Salmonella isolates from chevon and chicken
meat samples.
5. Overall, antimicrobial resistance phenotypes were similar between Salmonella
isolates recovered from chevon and chicken meat samples. This highlighted
the need for continued surveillance of zoonotic foodborne pathogens including
antimicrobial-resistant variants throughout the food production chain.
6. The consumers should be educated, emphasizing the public health importance
of it.
64
SUGGESTIONS FOR FUTURE RESEARCH WORK:
1. A comprehensive study in human and different species of animals with more
number of samples from different districts can be done for ascertaining the
status of Salmonella in Chhattisgarh.
2. Nucleotide sequencing of Salmonella spp. can be carried out to differentiate
the isolates.
3. Phage typing and Pulse Field Gel Electrophoresis (PFGA) of Salmonella
isolates can be carried out.
4. Molecular methods that are commonly used including Multilocus Enzyme
Electrophoresis (MEE), Chromosomal DNA, Restriction Endonuclease
Analysis (REA) as well as Ribotyping of Salmonella spp. can be carried out.
65
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APPENDIX
1) α -naphthol, ethanolic solution for VP test
α -naphthol 6g
Ethanol, 96%(V/V) 100ml
Dissolve the α -naphthol in ethanol.
2) Bismuth sulphite agar
Ingredients Gms / Litre
Pancreatic digest of casein 5.000
Beef extract 5.000
Peptic digest of animal tissue 5.000
Dextrose 5.000
Sodium phosphate 4.000
Ferrous sulphate 0.300
Bismuth sulphite indicator 8.000
Brilliant green 0.025
Agar 20.000
Final pH (at 25°C) 7.6±0.2
Suspend 52.32 grams in 1000 ml purified/ distilled water. Heat to boiling
to dissolve the medium completely. Do not overheat or sterilize in autoclave or by
fractional sterilization since overheating may destroy the selectivity of the
medium. Transfer to a water bath maintained at about 50°C .The sensitivity of the
medium depends largely upon uniform dispersion of precipitated bismuth sulphite
in the final gel, which should be dispersed before pouring into the sterile Petri
plates
3) Brilliant green agar
Ingredients Gms / Litre
Peptic digest of animal tissue 5.000
Pancreatic digest of casein 5.000
Yeast extract 3.000
Lactose 10.000
Sucrose 10.000
Sodium chloride 5.000
Phenol red 0.080
Brilliant green 0.0125
Agar 20.000
pH after sterilization (at 25°C) 6.9±0.2
Suspend 58.09 grams in 1000 ml purified /distilled water. Heat to boiling
to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure
(121°C) for 15 minutes. Avoid overheating. Cool to 50°C. Mix well before
pouring into sterile Petri plates.
4) Buffered peptone water
Ingredients Grams/Litre
Casein enzymic hydrolysate 10.00
Disodium hydrogen phosphate .12H2O 9.00
Sodium chloride 5.00
Monopotassium hydrogen phosphate 1.50
Final pH (at 25°C) 7.0±0.2
5) Ethidium bromide (10mg/ml)
Ethidium bromide 10 mg
Distilled water 1ml
Dissolve ethidium bromide in distilled water.
6) Glucose phosphate peptone water
Peptone 5 g
Di-potassium hydrogen phosphate 5g
Glucose 10 g
Distilled water 1000 ml
Dissolve all the components and sterilize at 1150C for 10 mins.
7) Kovac’s reagent for indole reaction
4-dimethylaminobenzaldehyde 5 g
Hydrochloric acid, p= 1.18 – 1.19 g/ml 25 ml
Amyl alcohol 75 ml
Mix the components. Store at 4°C.
8) Luria bertani broth
Ingredients Gms / Litre
Casein enzymic hydrolysate 10.000
Yeast extract 5.000
Sodium chloride 10.000
Final pH ( at 25°C) 7.5±0.2
Suspend 25 grams in 1000 ml distilled water. Heat if necessary to dissolve
the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15
minutes. Dispense as desired.
9) MacConkey’s Lactose Agar
Ingredients Gms / Litre
Bile salts 5.000
Peptic digest of animal tissue 20.000
Sodium chloride 5.000
Lactose 10.000
Neutral red 0.070
Agar 15.300
Final pH ( at 25°C) 7.4±0.2
Suspend 55.37 grams in 1000 ml distilled water. Heat to boiling with
gentle swirling to dissolve the agar completely. Sterilize by autoclaving at 15 lbs
pressure (121°C) for 15 minutes. Avoid overheating. Cool to 40-50°C and pour
into sterile Petri plates. The surface of the medium should be dry when
inoculated.
10) Media for sugar tests
Peptone water 100 ml
Andrade’s indicator 1 ml
Sugar 1 g
Mix the components and autoclave at 12 lbs for 10 minutes.
11) MR-VP Medium
Composition
Ingredients Grams/Litre
Buffered peptone 7.00
Dextrose 5.00
Dipotassium phosphate 5.00
Final pH (at 25°C) 6.9 ± 0.2
12) Mueller-Hinton agar
Ingredients Gms / Litre
Beef, infusion from 300.000
Casein acid hydrolysate 17.500
Starch 1.500
Agar 17.000
Final pH (at 25°C) 7.3±0.1
Suspend 38 grams in 1000 ml distilled water. Heat to boiling to dissolve
the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15
minutes. Mix well before pouring.
13) 0.85% normal saline
Sodium chloride 0.85 g
Distilled water 100 ml
14) Nutrient broth
Ingredients Gms / Litre
Peptic digest of animal tissue 5.000
Sodium chloride 5.000
Beef extract 1.500
Yeast extract 1.500
Final pH ( at 25°C) 7.4±0.2
Suspend 13 grams in 1000 ml distilled water. Heat, if necessary, to
dissolve the medium completely. Dispense as desired and sterilize by autoclaving
at 15 lbs pressure (121°C) for 15 minutes
15) Nutrient Agar
Ingredients Gms / Litre
Peptic digest of animal tissue 5.000
Sodium chloride 5.000
Beef extract 1.500
Yeast extract 1.500
Agar 15.000
Final pH (at 25°C) 7.4±0.2
Suspend 28 grams in 1000 ml distilled water. Heat to boiling to dissolve
the medium completely. Dispense as desired and sterilize by autoclaving at 15 lbs
pressure (121°C) for 15 minutes. Mix well before pouring.
16) Peptone water
Ingredients Gms / Litre
Peptic digest of animal tissue 10.000
Sodium chloride 5.000
Final pH (at 25°C) 7.2±0.2
Suspend 15.0 grams in 1000 ml distilled water. Heat if necessary to
dissolve the medium completely. Dispense in tubes and sterilize by autoclaving at
15 lbs pressure (121°C) for 15 minutes. Note: If desired add required
carbohydrate for checking fermentation pattern with added 1% phenol red
solution.
17) 1x phosphate buffered saline solution (PBS)
Sodium chloride 8 g
Potassium chloride 1000ml
Di-sodium hydrogen phosphate 1.15 g
Potassium Di-hydrogen phosphate 0.2 g
Distilled water up to 1000ml
Adjust the pH to 7.2
18) Potassium hydroxide solution for VP test
Potassium hydroxide 40 g
Distilled water 100 ml
Dissolve the potassium hydroxide in the water.
19) Simmons Citrate Agar
Ingredients Gms / Litre
Magnesium sulphate 0.200
Ammonium dihydrogen phosphate 1.000
Dipotassium phosphate 1.000
Sodium citrate 2.000
Sodium chloride 5.000
Bromothymol blue 0.080
Agar 15.000
Final pH (at 25°C) 6.8±0.2
Suspend 24.28 grams in 1000 ml distilled water. Heat, to boiling, to
dissolve the medium completely. Mix well and distribute in tubes or flasks.
Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
20) Tris- borate EDTA buffer (TBE) (5xstock solution)
Tris base 54 g
Boric acid 27.5 g
EDTA (0.0 M, Ph 8.0) 20 ml
Distilled water up to 1000 ml
21) TSI agar
Ingredients Gms / Litre
Peptic digest of animal tissue 10.000
Casein enzymic hydrolysate 0.000
Yeast extract 3.000
Beef extrac t 3.000
Lactose 10.000
Sucrose 10.000
Dextrose 1.000
Sodium chloride 5.000
Ferrous sulphate 0.200
Sodium thiosulphate 0.300
Phenol red 0.024
Agar 12.000
Final pH (at 25°C) 7.4±0.2
Suspend 64.52 grams in 1000 ml distilled water. Heat to boiling to
dissolve the medium completely. Mix well and distribute into test tubes. Sterilize
by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Allow the medium to
set in sloped form with a butt about 1 inch long.
22) Urea broth
Ingredients Gms / Litre
Potassium dihydrogen orthophosphate 9.100
Yeast extract 0.100
Anhydrous disodium hydrogen phosphate 9.500
Urea 20.000
Phenol red 0.010
Suspend 38.71 grams in 1000 ml purified/ distilled water. Mix thoroughly
to dissolve the medium completely.Sterilize by filtration. Aseptically dispense in
sterile tubes as desired.
82
THESIS ABSTRACT
Title of thesis: Studies on isolation and molecular characterization ofSalmonella spp. of public health significance in chevon and chicken meat
Name of Student: Vivek Kumar Naik (Roll No. 4004, ID No-K130112019)
Salmonella infections continue to be a major public health problem in
both developed and developing countries. The present study was undertaken to
estimate microbial load, isolate and identify the Salmonella spp. of public
health significance in chevon and chicken meat along with their antibiogram
pattern. A total of 450 samples (200 chevon, 200 chicken meats and 50 human
stool sample) were processed for Salmonella by conventional cultural
technique and further confirmed by biochemical screening and molecular
techniques.
The identification of suspected Salmonella isolates was done using
biochemical methods. The samples were plated on various media like BGA,
BSA and MLA from where the putative isolates were stabbed on TSI and
Simmons citrate agar. They were further tested for Urease activity and the
isolates showing negative reaction were studied for their IMViC pattern as
well as carbohydrate fermentation activity. On biochemical identification all
32 isolates presented the desired reactions.
Further these isolates were subjected to PCR based molecular
characterization. In PCR technique; primers targeting stn, invA and pef genes
were used. All conventionally and biochemically confirmed isolates of
Salmonella amplified 617 bp fragment in PCR reaction targeting enterotoxin
encoding stn gene and 31 among 32 isolates amplified 284 bp fragment in
83
PCR targeting conserved sequence of the invA gene. However in the present
study none of the isolates were found positive for pef gene. A total of 32
isolates 18 from chevon and 14 from chicken meat yielded Salmonella.
However it is interesting that no Salmonella spp. was found in human stool.
All the isolates were subjected to antibiotic susceptibility testing
against 14 antibiotics. Most of the isolates were found to be sensitive to
Ciprofloxacin and Cefotaxim whereas majority of the isolates were resistant to
Erythromicin, Cephalexin and Nalidixic Acid. Highest multiple antibiotic
resistance (MAR) index was 0.50 (1 isolate) followed by 0.42 (one isolate), 0.
35 (one isolate), 0.28 (three isolates), 0.21 (eight isolates), 0.10 (nine isolates)
and 0.07 (eight isolates). The minimum MAR index 0, was shown by one
isolates. Out of 32, 14 isolates were found to have MAR index equal to or
more than 0.2, thus indicated injudicious use of antibiotics.
Dr. Sanjay Shakya
Major Advisor and Chairman
Professor and head
Department of Veterinary Public Health and Epidemiology