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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 – [email protected]

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

2

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

8

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 .

9

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. 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



stool samples

Chicken meat Stool sample

Prevalence(%)

in %



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%


Chhattisgarh


Chhattisgarh

Durg

Rajnandgaon

Raipur

Dhamtari

Bilaspur

12.50%

7.50%10%

5%

Durg

Rajnandgaon

Raipur

Dhamtari

Bilaspur

5%

12.50%

7.50%


Chhattisgarh


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



% Resistant

% Intermediate

% Sensitive



% 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


1000900800700600500400

300

200

100

Lane 1 2 3 4 5 6 7 8 9 10 11 12

bp

284 bp


284 bp

617 bp


1000900800700600500400

300

200

100

Lane 1 2 3 4 5 6 7 8 9 10 11 12

bp

284 bp


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



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


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



Yeast extract 5.000


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


Bile salts 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


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


14) Nutrient broth




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




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




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


Dissolve the potassium hydroxide in the water.

19) Simmons Citrate Agar


Magnesium sulphate 0.200

Ammonium dihydrogen phosphate 1.000

Dipotassium phosphate 1.000

Sodium citrate 2.000


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




Yeast extract 3.000

Beef extrac t 3.000

Lactose 10.000

Sucrose 10.000

Dextrose 1.000


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


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

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