ISOLATION AND IDENTIFICATION OF ESCHERICHIA COLI FROM DIARRHOEIC CALF FAECES BY BIOCHEMICAL

TESTS, ANTIBIOGRAM PATTERN AND PCR BASED DETECTION OF TOXIGENIC GENES

A THESIS

SUBMITTED TO THE ANAND AGRICULTURAL UNIVERSITY

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE

OF

MASTER OF VETERINARY SCIENCE

IN

VETERINARY MICROBIOLOGY

BY

GITANJALI ARYA B.V.Sc. & A.H.

(Reg. No. 04 – 05613– 2003)

Department Of Veterinary Microbiology College of Veterinary Science & Animal Husbandry

Anand Agricultural University, Anand 2005

Dr. Ashish Roy B. V. Sc. & A.H., M. V. Sc., Ph. D. Associate Professor Department of Veterinary Microbiology College of Veterinary Science & Animal Husbandry Anand Agricultural University, Anand Gujarat State (India)

CERTIFICATE

This is to certify that the thesis entitled “ISOLATION AND

IDENTIFICATION OF ESCHERICHIA COLI FROM DIARRHOEIC CALF

FAECES BY BIOCHEMICAL TESTS, ANTIBIOGRAM PATTERN AND

PCR BASED DETECTION OF TOXIGENIC GENES” submitted by Gitanjali

Arya (Reg. No. 04-05613-2003) in partial fulfillment of the requirements for the

award of the degree of MASTER OF VETERINARY SCIENCE in the subject of

VETERINARY MICROBIOLOGY of the Anand Agricultural University is a

record of bonafide research work carried out by her under my guidance and

supervision and the thesis has not previously formed the basis for award of any

degree, diploma or other similar title.

Place: Anand

Date: /11 / 2004 (Ashish Roy)

MAJOR ADVISOR

CERTIFICATE

This is to certify that I have no objection for supplying copy of any part of

this thesis at a time through reprographic process, if necessary for rendering

reference services in a library or documentation centre.

Place: Anand

Date: / 11 / 2004 (Gitanjali Arya)

(Ashish Roy)

MAJOR ADVISOR

LIST OF FIGURES

Sr. No.

Figure No.

Title Page No.

1. 4.1 In vitro antimicrobial drug resistance pattern of E. coli isolates

89

2. 4.2 Location wise prevalence of antimicrobial drug resistance among E. coli isolates

92

ISOLATION AND IDENTIFICATION OF ESCHERICHIA COLI FROM DIARRHOEIC CALF FAECES BY BIOCHEMICAL

TESTS, ANTIBIOGRAM PATTERN AND PCR BASED DETECTION OF TOXIGENIC GENES

Student: Gitanjali Arya Major Guide: Dr. Ashish Roy

Department of Veterinary Microbiology College of Veterinary Science and Animal Husbandry

Anand Agricultural University Anand

ABSTRACT The present study was undertaken to investigate biochemical characters, serotypes, biotypes, multiple drug resistance, colicinogeny, haemolytic activity and detection of toxigenic genes of E. coli from diarrhoeic calf faecal samples. In all 91 E. coli isolates obtained from 46 faecal samples revealed typical cultural characters on the MacConkey agar and eosin methylene blue agar. Some of the E. coli isolates showed variation from the standard biochemical pattern viz. H2S production (1.09% isolates), urea hydrolysis (21.97% isolates), citrate utilization (3.29 % isolates) and fermentation of adonitol (2.19% isolates) and raffinose (35.1% isolates). All the 91 isolates were serotyped, of which 82 typable isolates belonged to 36 different ‘O’ serogroups, while 6 isolates were untypable and 3 were rough isolates. In all 17 different serogroups obtained from Livestock Research Station- Anand Agricultural University (LRS-AAU), Anand were in decreasing frequency viz. O86 (7 isolates), four isolates each of O128, O171 and O172, O62 (3 isolates), serogroups O22, O26, O32, O55, O110, O157 having 2 isolates each and one isolate each belonging to serogroups O11, O18, O24, O126, O131 and UT (untypable). Out of six isolates obtained from Veterinary College Clinics (VC), AAU, Anand 4 different serogroups were recorded viz. O2 and O167 two isolate each and one isolate each of O12 and O197. Out of twenty seven isolates obtained from other unorganized farms (OF), in and around Anand twenty different serogroups detected were O5, O9, O11, O22, O66, O78, O86, O100, O101, O108, O126, O157, O161, O162, O171 and O172 one isolate each while two isolates each of serogroup O98 and O131, 3 rough and four untypable isolates.

Eighteen isolates obtained from Livestock Research Station-Junagadh Agricultural University (LRS-JAU), Junagadh comprised of twelve different serogroups viz. O18 (5 isolates), two isolates each of O109, O128, while one isolate each of O3, O15, O22, O68, O76, O131, O168, O171 and UT. The most common serogroups were O22, O131 and O171 followed by O11, O18, O86, O126, O128, O157 and O172 among the E. coli isolates obtained from various locations. Twenty four different biotypes of E. coli were obtained on the basis of fermentation reactions of sugars viz. dulcitol, raffinose, rhamnose, salicin, starch and sucrose.

In vitro antibiotic resistance pattern against 12 antibiotics were detected. Higher percent (72-100%) of E. coli isolates showed resistance against kanamycin, cepahlexin, amikacin, cephaloridine, enrofloxacin and ampicillin. Moderate numbers (28-57%) of isolates were found to be resistant to ciprofloxacin, ceftiofur and tetracycline. Lesser percent (8.79-15.38%) of isolates were resistant to nalidixic acid, colistin and co-trimoxazole.

Among the 91 E. coli isolates studied for colicinogeny 32 (35.16%) were colicin producers.

PCR based detection of toxigenic genes with the primer sets LT1-LT2 and microST1-microST2 gave products of expected size i.e. 132 bp for LT (heat labile) enterotoxin and 171 bp for ST (heat stable) enterotoxin genes. Out of 91 E. coli isolates one (1.09%) possessed ST gene and two isolates (2.19%) harboured LT gene. The primer directed amplification of the verotoxin (VT1 and VT2) genes gave the products of expected size i.e. 130 bp for VT1 gene and 228 bp for VT2 gene. Total 41 (45.05%) isolates were found to be positive for VT genes. Among the isolates 32 (35.16%) isolates harboured both VT1 and VT2 genes while 6 (6.5%) isolates possessed only VT2 gene and 3 (3.25%) had VT1 gene only. Overall 38 (41.75%) isolates were positive for VT2 gene and 35 (38.46%) isolates were positive for VT1 gene.

Dedicated To

My Beloved Parents

ACKNOWLEDGEMENTS With immense pleasure, I express my sincere regards, gratitude and indebtedness to my major advisor, Dr. Ashish Roy, Associate Professor, Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand for his valuable and timely guidance, constructive criticism, constant motivation and encouragement throughout the course of this study and in preparation of this manuscript. I am highly thankful to my minor advisor Dr. P.H. Vataliya, Professor, Department of Animal Genetics and Breeding, and other members of my advisory committee, Dr. M.K. Jhala, Associate Research Scientist, Department of Veterinary Microbiology and Dr. B.P. Joshi, Professor, Department of Veterinary Pathology for their valuable suggestions and inspiration throughout this study. My sincere thanks and obligations are to Dr. J.H. Purohit, Professor and Head, Department of Veterinary Microbiology and Dr. J.V. Solanki, Professor and Head, Department of Animal Genetics and Breeding for their valuable suggestions, advice and providing all the requisite facilities for research work. I take this opportunity to pay my humble thankfulness to Dr. M.C. Desai, Principal and Dean College of Veterinary Science and Animal Husbandry for providing all the necessary facilities and environment for carrying out this post graduate study. It would be unworthy of me, if I miss a chance to express my indebtedness to Dr. C.G. Joshi, Associate Professor and Dr. D.N. Rank, Associate Professor Department of Animal Genetics and Breeding, for their perpetuative encouragement and annexing the intricate problems of my research work. I would like to extend my sincere thanks to the Director Central Research Institute, Kasauli (H.P.) for serotyping of E. coli isolates and to the Director, Institute of Microbial Technology, Chandigarh for supplying the known strain of LT+ST+ E. coli for my research purpose.

I am sincerely thankful to Dr. I.H. Kalyani, Senior Research Assistant, Department of Veterinary Microbiology, for his help during this study. My sincere thanks are also to my seniors, Dr(s): Mahendra Yadav, Trupti Patel, Niraj Makadiya, Anshu Jain, Harshad Goriya, Prakash Koringa, Jayesh Patel and my colleagues Dr(s): Nagaraj, Anitha, Vartika, Mamta, Chandrakant, Vandana, Gayatri, Lata, Shadma, Ranjini Nilen, Tejendra, for their valuable suggestions, kind co-operation and encouragement during the research work and preparation of this thesis.

I am highly thankful to the staff members of Department of Veterinary Microbiology, Shri R.J. Shah, Jayaben, Khodakaka, Mohanbhai, Surabhai and Harishbhai for their co-operation during the entire study period.

I also, owe my thanks to Mr. Kunal Saini, Dr. Manjeet Singh and Dr. Pankaj Khurana for helping me to find and refer relevant literature for this research work.

I would like to express my deepest sense of gratitude and affection to my parents for all the blessings, inspiration and constant help. My humble thanks are to my younger sister and the best friend Anoop, younger brothers Jayprakash and Siddharth for their never failing encouragement shown during this study. My thanks are also to Dr. K.N. Wadhwani for constant encouragement during this study.

In the last, but not the least, I thank all those who helped directly or indirectly in completion of this study. My most sincere thanks to the Almighty God who made everything possible. Place: Anand Date: / /2005 (Gitanjali Arya)

CONTENTS

Chapter No. Chapter Page No.

I INTRODUCTION 1

II REVIEW OF LITERATURE 7

III MATERIALS AND METHODS 46

IV RESULTS 61

V DISCUSSION 108

VI SUMMARY AND CONCLUSIONS 137

REFERENCES i-xxv

APPENDICES I-VI

LIST OF TABLES

Sr. No.

Table No.

Title Page No.

1. 3.1 Sources and number of faecal samples collected

46

2. 3.2 Details of primers used for PCR reaction

56

3. 3.3 Composition of mastermix for PCR reaction

57

4. 3.4 Steps and conditions of thermocycling for PCR

58

5. 4.1 E. coli isolates obtained from diarrhoeic calf faecal samples

63

6. 4.2 Sugar fermentation activity of E. coli isolates

70

7. 4.3 Unusual biochemical characters of E. coli isolates

77

8. 4.4 Biotypes of E. coli isolates on the basis of fermentation reactions of dulcitol, raffinose, rhamnose, salicin, starch and sucrose

82

9. 4.5 Distribution of E. coli serogroups within biotype 84

10. 4.6 Number and per cent of E. coli isolates resistant to antimicrobial drugs

88

11. 4.7 Location wise prevalence of antimicrobial drug resistance among E. coli isolates

91

12. 4.8 Antimicrobial drug resistance pattern and colicinogeny of E. coli isolates

93

13. 4.9 Results of PCR amplification of toxigenic genes in E. coli isolates

103

LIST OF PLATES

Sr. No.

Plate No.

Title Page No.

1. I Lactose fermenting pink colonies of E. coli on MacConkey agar

62

2. II E. coli colonies with characteristic greenish metallic sheen on eosin methylene blue agar

62

3. III Indole, Methyl Red (MR), Voges Proskaeur (VP) and citrate test for E. coli

69

4. IV Reaction on TSI agar by E. coli 76

5. V Urease test for E. coli 76

6. VI In vitro antibacterial drug sensitivity test for E. coli isolates

87

7. VII Detection of colicinogeny among the E. coli isolates 99

8. VIII Agarose gel showing PCR amplified product (132 bp) for LT gene in E. coli isolates

101

9. IX Agarose gel showing PCR amplified product (171 bp) for ST gene in E. coli isolates

101

10. X Agarose gel showing PCR amplified product (130 bp) for VT1 gene in E. coli isolates

102

11. XI Agarose gel showing PCR amplified product (228 bp) for VT2 gene in E. coli isolates

102

ACRONYMS

AEEC Attaching and Effacing E. coli bp base pair BHI Brain Heart Infusion °C Centigrade/ Degree celceius cAMP Cyclic Adenosine Monophosphate cGMP Cyclic Guanosine Monophosphate CNF Cytotoxic Necrotizing Factor

DNA Deoxyribonucleic acid dNTPs Dinucleotide(s) triphosphate E. coli Escherichia coli

EDTA Ethylene diamine tetra acetic acid

et al. et alii/alia ETEC Enterotoxigenic E. coli g gram(s) GPW Glucose-Phosphate Peptone water GIT Gastro intestinal tract HC Haemorrhagic colitis LT Thermolabile M Molar µl Microliter µg Micro gram

mA mili ampere mg Milligram ml Milliliter mM Milimolar Min Minute MTCC Microbial Type Culture Collection and Gene Bank NaCl Sodium Chloride

ng Nanogram OD Optical density PCR Polymerase chain reaction % Per cent

pmole picomole(s)

RLIL Rabbit Ligated Ileal Loop RNA Ribonucleic acid

rRNA Ribosomal RNA

rpm Revolution per minute 28S Svedberg constant/Sedimentation constant sec Seconds SLT Shiga like toxin SLTEC Shiga like toxin producing E. coli ST Thermostable STEC Shiga toxin producing E. coli Stx Shiga toxin TBE Tris borate EDTA buffer TSI Tripple Sugar Iron Agar UV Ultra violet

V Volts viz. Videlicet VT Verotoxin VTEC Verocytotoxigenic/Verotoxigenic E. coli EPEC Enteropathogenic E.coli ETEC Enterotoxigenic E.coli PBA Phosphate buffer agar MH Muller Hinton agar

INTRODUCTION

CHAPTER I INTRODUCTION

Escherichia coli (E. coli) a member of family Enterobacteriaceae is a short Gram- negative, non-spore forming and usually peritrichous and fimbriate bacillus. A capsule or microcapsule is often present and a few strains produce profuse polysaccharide slime. E. coli was first isolated by Theobald Escherich in 1885 from faeces of infants. It serves as a major facultative anaerobe throughout its life as a harmless saprophyte but Larulle (1889) was the first to suggest the possible role of E. coli as a pathogenic organism. E. coli has been shown to be a normal inhabitant of the gastrointestinal tract of animals and man (Smith, 1965). The organism typically colonizes the infant gastrointestinal tract within hours of life and thereafter, both E. coli and the host derive mutual benefit (Drasar and Hill, 1974). In the debilitated or immunosupressed host or when gastrointestinal barriers are violated even normal non pathogenic strains of E. coli can cause infection. Pathogenic E. coli are one of the most important groups of bacteria causing diarrhoea and extra intestinal infections in humans and animals (Levine, 1987). Bovine calf scours or neonatal diarrhoea of calves is a severe form of diarrhoea that causes more financial loss to cow-calf producers than any other disease-related problem. Scours is a clinical sign of disease that may have many causes, although E. coli has been frequently implicated as the primary bacterial cause in calves (Yamamoto and Nakazawa, 1997). About 50% cases of neonatal diarrhoea were ascribed to E. coli (Tripathi and Soni, 1984). Several classes of diarrhoea causing E. coli are now recognized on the basis of production of virulence factors, distinct O:H serogroups, distinct diarrhoeal syndromes and difference in epidemiology (Nataro and Kaper, 1998) viz. Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), Enterohaemorrhagic E. coli (EHEC) or Verocytotoxigenic E. coli (VTEC), Enteroaggregative E. coli (EAEC), Diffusely adherent E. coli (DAEC).

Enterotoxigenic E. coli (ETEC) strains produce plasmid mediated enterotoxins which bind to their specific receptors on the gut epithelium and by a complex interplay of biological mediators cause diarrhoea. Heat Labile (LT) and Heat stable (ST) are the two main classes of enterotoxins identified (Levine, 1987). ETEC strains were first recognized as causes of diarrhoeal disease in piglets, where the disease continues to cause lethal infection in newborn animals (Alexander, 1994). The first descriptions of ETEC in human reported that certain E. coli isolates from the stools of children with diarrhoea elicited fluid secretion in ligated rabbit intestinal loops (Taylor et al., 1961). Enterotoxigenic E. coli (ETEC) possess unique attributes of virulence by virtue of which they can be differentiated from other nonpathogenic micro flora of intestine of healthy animals (Smith and Halls, 1968). Heat labile (LT) enterotoxin activates adenylate cyclase enzyme leading to cAMP accumulation which causes increased secretion of sodium ions and loss of chloride ions and water leading to watery diarrhoea. While ST stimulates guanylate cyclase enzyme leading to increased intracellular cGMP levels. This activity leads ultimately to stimulation of chloride secretion and/or inhibition of sodium chloride absorption, resulting in net intestinal fluid secretion that leads to diarrhoea (Sharma et al., 1992; Sears and Kaper, 1996). ETEC strains from diarrhoeic calves, camels, goats, infants and milk have been isolated in India (Kumar et al., 1982; Kumar, 1990; Chauhan and Kaushik, 1991; Dubey et al., 2000). Verocytotoxigenic/Verotoxigenic E. coli (VTEC) was identified by Konowalchuk and his coworkers (1977) as a distinct group of E. coli named as verotoxic E. coli (VTEC), which had the ability to produce toxins with profound and irreversible effect on vero cells. In 1980s it was recognized that verotoxins (VT) is encoded on a bacteriophage in E. coli (Scotland et al., 1981; O’Brien et al., 1984). Further investigations on VTs led to the discovery of two major toxin types: VT1 and VT2, which were shown to be genetically

(55% DNA homology) and immunologically (not cross reactive) different from each other (Karmali, 1989). VT1 was genetically and immunologically related to Shiga toxin (Stx), which is produced by Shigella dysenteriae type 1 strains (Jackson et al., 1987; Nakao and Takeda, 2000). Despite these differences,VT1, VT2 and Stx genes are similar in function and are genetically organized in an operon structure with two genes encoding the A- (toxin) and the B- (cell receptor binding) subunits. Therefore, SLT (shiga like toxin), shiga toxin (Stx) and VT (verotoxin) nomenclature systems have been used interchangeably. Thus, verotoxin-producing E. coli (VTEC) is also termed as shiga-like toxin producing E. coli (SLTEC) or shiga toxin producing E. coli or STEC (Paton and Paton 1998).

The verocytotoxic activity is due to enzymatic cleavage (N-glycosidase) of an adenine residue in the 28S rRNA leading to inhibition of protein synthesis in the target cell (Melton-Celsa and O’Brien 2003). There are at least 200 serotypes of E. coli that are capable of producing Shiga toxins (Johnson et al., 1996) however, of these serotypes E. coli O157:H7 is the most well known to both microbiologists and the general public. This organism was first recognized in 1982 following an outbreak of haemorrhagic colitis (HC) in the USA (Riley et al., 1983). Since then, STEC O157 has been implicated in sporadic cases and outbreaks of diarrhoea, haemorrhagic colitis and haemolytic syndrome world-wide. Some outbreaks have been large, e.g. Japan where over 9000 children were infected (Michino et al., 1998). Stx-producing E. coli can be found in the faecal flora of a wide variety of animals including cattle, sheep, goats, pigs, cats, dogs, chickens (Beutin et al., 1993; Johnson et al., 1996; Wallace et al., 1997). High rates of colonization of Stx-positive E. coli have been found in bovine herds in many countries (Wells et al., 1991; Clarke et al., 1994; Burnens et al., 1995). These rates are as high as 60% but are more typically in the range of 10 to 25%. In India, there is paucity of information on VTEC. It has not been identified as significant etiologic agent of diarrhoea for humans and various animal species (Wani et al., 2004). However, there is a variety of data showing the involvement of VT in diarrhoea and eneterocolitis, beginning with early demonstrations that purified VT can cause fluid accumulation and histological damage when injected into ligated intestinal loops (O’Brien and Holmes, 1987). One possible mechanism for fluid secretion in response to VT involves the selective killing of absorptive villous tip of intestinal epithelial cells by VT (Keenan et al., 1986; Kandel et al., 1989). VTEC strains from diarrhoeic calves, lambs, infants and beef have been isolated in India (Kumar et al., 1982; Khan et al., 2002; Chattopadhyay et al., 2003; Wani et al., 2004). In recent years, there has been considerable increase in the incidence of drug resistance in bacteria due to extensive and indiscriminate use of antimicrobial agents for therapy, prophylaxis or growth promotion. The emergence of drug resistant strains following continuous feeding of antibiotics to livestock has led to loss of their efficacy (Smith and Hall, 1966; Singh et al., 1992). Resistance to front line antimicrobials is present among E. coli isolates incriminated in bovine calf scours. These isolates have shown multiple drug resistance to β-lactams, including expanded-spectrum cephalosporins, aminoglycosides, sulphonamides, tetracyclines and fluoroquinolones (Bradford et al., 1999). This combination of virulence coupled with multiple drug resistance has posed an increasing threat to successful treatment of E. coli related veterinary diseases. This suggests that the use of antibiotics in animals could lead to a reservoir of antibiotic-resistant bacteria that could potentially infect humans. Thus, keeping in view, the above facts and the magnitude of the problem a more rapid, accurate method to detect diarrhoeagenic E. coli is needed for improved clinical diagnosis, farm biosecurity and epidemiological studies. The proposed study is, therefore, planned and aimed to detect and characterize the field isolates of E. coli from diarrhoeic calves by cultural, biochemical and molecular methods with following objectives:

Objectives: 1. Isolation and characterization of E. coli from diarrhoeic calf feaces. 2. Study on biotyping and serotyping of E. coli isolates. 3. Detection of antibiotic resistance profile of E. coli isolates. 4. Detection of virulence associated factor like colicinogeny among the

isolates. 5. Detection of heat stable (ST) enterotoxin, heat-labile (LT) enterotoxin and

Verotoxin (VT1 and VT2) genes among the isolates by Polymerase Chain Reaction (PCR).

REVIEW OF LITERATURE

CHAPTER II

REVIEW OF LITERATURE

Escherichia coli first described by Theoder Escherich in 1885, is a member of family Enterobacteriaceae which is present as normal flora in the lower intestine of both humans and animals however, some strains can cause gastrointestinal illness ranging from mild to cholera like diarrhoea and may lead to potentially fatal complications such as haemolytic uremic syndrome. De and co-workers (1956), identified enterotoxins secreted by E. coli able to mediate increased fluid secretion in gut leading to diarrhoea. Based on virulence properties of E. coli, their difference in epidemiology and distinct O:H serotypes, Nataro and Kaper (1998) have classified diarrhoea causing E. coli into distinct groups viz: Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), Enterohaemorrhagic E. coli (EHEC) or Verocytotoxigenic E. coli (VTEC), Enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC). Enterotoxigenic E. coli (ETEC) strains produce plasmid mediated enterotoxins which bind to their specific receptors on the gut epithelium and by a complex interplay of biological mediators cause diarrhoea. Two main classes of enterotoxins are identified: Heat Labile (LT) enterotoxin and Heat stable (ST) enterotoxin. There are two major subtypes of LT, LT-I and LT-II which do not cross-react immunologically. LT-I is expressed by E. coli strains that are pathogenic for both humans and animals. LT-II is found primarily in animal E. coli isolates and rarely in human isolates but in neither animals nor humans has it been associated with disease. Thus the term LT refers to the LT-I form (Nataro and Kaper, 1998). Heat stable enterotoxin has two subtypes-STa and STb. STa toxins are produced by ETEC and several other gram-negative bacteria including Yersinia enterocolitica and V. cholerae non-O1, while STb has been found only in ETEC (Levine, 1987). ETEC strains were first recognized as causes of diarrhoeal disease in piglets, where the disease continues to cause lethal infection in newborn animals (Alexander, 1994). The first descriptions of ETEC in humans reported that certain E. coli isolates from the stools of children with diarrhoea elicited fluid secretion in ligated rabbit intestinal loops (Taylor et al., 1961). Konowalchuk and co-workers (1977) identified a distinct group of E. coli named as Verocytotoxigenic/Verotoxic E. coli, which had the ability to produce a toxin with profound and irreversible effect on vero cells. The toxin differed from heat labile (LT) and heat stable (ST) enterotoxins of E. coli and was cytotoxic only for vero cells. Later on O’Brien and La Veck (1983) purified and characterized the cytotoxin produced by one of the Konowalchuk’s isolates (serotype O26:H11) and found that it had similar structure and biological activity to shiga toxin (Stx), produced by Shigella dysenteriae type 1. Moreover, it could be neutralized by anti-Stx. Therefore, the term shiga-like toxin (SLT) was also applied to verotoxin (VT). Therefore, SLT and VT nomenclature systems have been used interchangeably. Thus, verotoxin-producing E. coli is also termed as shiga-like toxin producing E. coli (SLTEC) or shiga-toxin producing E. coli (STEC). The presence of multiple verotoxin type was initially understood from the fact that anti-Stx could not neutralize cytotoxic effect of some VTEC strains (Scotland et al., 1985, Strockbrine et al., 1986). On the other hand crude antisera raised against non-neutralizable strains didin’t neutralize Stx. This demonstrated that some VTEC strains produced only anti-Stx neutralizable toxin (i.e. VT1/SLT1/Stx-1), others produced only the non-neutralizable toxin (VT2/SLT-II/Stx-2) while another group produced both VT1 and VT2. VT1 was essentially identical to shiga-toxin (Stx) and neutralized by anti-Stx but it was not true for VT2 (O’Brien and Holmes, 1987).

Neonatal diarrhoea of calves with high mortality and morbidity causes serious economic losses and assumes greater significance among various infections. About 50 per cent cases of neonatal diarrhoea were ascribed to E. coli (Tripathi and Soni, 1984). Colibacillosis involves initial colonization of the bacteria in the intestine with the help of various adhesins like K99 in case of calf strains and subsequent liberation of enterotoxin which ultimately results in diarrhoea (Sojka, 1971). 2.1 Isolation and Identification of E. coli E. coli is a facultative anaerobe that can be recovered easily from clinical specimens on general or selective media at 37°C under aerobic conditions; growth of these organisms has also been observed at 44°C. E. coli is Gram negative, catalase positive, oxidase negative, non spore forming, and rod shaped bacterium. The motility of the organism occurs due to peritrichous flagella, although some non-motile strains have also been recorded. When growing anaerobically there is an absolute requirement for fermentable carbohydrate. Glucose is fermented to give acid and gas. These organisms are able to utilize carbon and nitrogen sources for all their metabolic and energy needs (Edwards and Ewing, 1972). E. coli in faecal samples are most often recovered on MacConkey or Eosin Methylene Blue (EMB) agar. Merchant and Packer (1967) considered EMB agar as a suitable medium for isolation of E. coli from faeces and foods because of ability to produce distinctive colonies having greenish metallic sheen not produced by other bacteria of Enterobacteriaceae family. For epidemiological or clinical purposes E. coli strains are often selected from MacConkey agar plates after presumptive visual identification of lactose fermenting pink colonies. However, this method should be used only with caution because only 90 per cent of E. coli are lactose positive, some diarrhoeagenic E. coli strains are typically lactose negative. The indole test positive in 99 per cent of E. coli strains is the single best test for differentiation from other members of Enetrobacteriaceae (Nataro and Kaper, 1998). 2.2 Serotypes of E. coli associated with calf diarrhoea Serotyping of E. coli occupies a central place in the history of this pathogen (Lior, 1996). Prior to the identification of specific virulence factors in diarrhoeagenic E. coli strains serotype analysis was the predominant means by which pathogen strains were differentiated. According to modified Kauffman scheme, E. coli is serotyped on the basis of their O (somatic), H (flagellar) and K (capsular) surface antigen profiles (Edwards and Ewing, 1972; Lior, 1996). A total of 170 different O antigens, each defining a serogroup, are recognized currently. E. coli of specific serogroup can be associated reproducibly with certain clinical syndromes but it is not in general the serological antigens themselves that confer virulence. Rather, the serotypes and serogroups serve as readily identifiable chromosomal markers that correlate with specific virulent clones (Whittman et al., 1993). Sivaswamy and Gyles (1976) isolated ETEC strains O9:K- and O101:K from diarrhoeic calves less than two weeks of age having white scours with an isolation rate of 36%. ETEC strains O8:K5, O8:K25, O9:K35, O20: K-, O101:K28, O101: K30 were reported to be isolated from diarrhoeic calves by Braaten and Myers (1977). Srivastava and Arya (1979) reported 56 strains of E. coli from calves. Of these 26 could be serotyped into 13 serogroups viz. O17, O22, O55, O25, O48, O89, O5, O18, O20, O61, O62, O68 and O90. Of these serotypes O5 and O22 were found pathogenic. Ueda et al., (1981) reported various ETEC strains viz. O2, O8, O13, O20, O48, O101, O135, O141 and 8 untypable strains isolated from diarrhoeic calves and piglets.

Scotland and co-workers (1981) isolated ETEC O44, O114 and O128 from diarrhoeic calves in tropical or developing countries including India also. Latif (1982) isolated E. coli from 53 diarrhoeic buffalo calves in Gujarat of which O17, O35, O39, O32, O34, O7 and O43 constituted the dominant serotypes and other strains were O5, O22, O24, O25 and O60. Tripathi and Soni (1984) observed that out of 33 strains of E. coli isolated from cases of neonatal calf diarrhoea, the isolates belonging to serogroups O11, O15, O55, O40, O20, O26, O22 and O60 were enterotoxigenic. Panda and Panda (1987) studied calf diarrhoea in Orissa. Out of 202 diarrhoeic calves (1-30 days of age), 95 yielded E. coli belonging to 23 serotypes viz. O3, O7, O8, O9, O28, O36, O42, O43, O68, O76, O83, O97, O91, O105, O107, O116, O132, O133, O143, O149, O151, O157 and O161. Five ‘O’ types accounted for 66% of isolates viz.O8, O16, O91, O143 and O157. Serotypes O3, O68, O83 and O91 elicited positive reaction in rabbit ileal loop (RLIL) test while O43 and O152 were found negative for RLIL. Beutin et al. (1989) analyzed sixty four verotoxin producing E. coli strains. Strains of human origin were of the following serotypes: O157:H7 or H-, O111:H8 or H-, O26:H11, O114:H4 and rough: H7. Strains of serotypes O157:H7, O113:H21, O116:H21 and rough: H- were from cattle, while those of serotypes O139:K12:H1 were from pigs. Shah (1989) studied E. coli from neonatal diarrhoea in calves in Gujarat. Out of 71 E. coli isolates, 59 (83.10%) isolates could be serotyped which belonged to 36 serogroups while 10 isolates were untypable and two were rough strains. Serotypes frequently isolated were O7, O20, O55, O57, O61, O148, O149 (three isolates) and O2, O4, O5, O12, O32, O91, O114, O120 and O160 (two isolates each). The rest of the serotypes were widely spread over. Kaura et al. (1991) isolated E. coli from diarrhoeic buffalo calves and cow calves. They reported serotypes O4, O7, O9, O11, O17, O20, O24, O25, O40, O55, O74, O86, O101, O110, O123, O129, O131, O142, O149, O153 and O166 from buffalo calves. They reported serotypes O1, O5, O7, O8, O9, O13, O17, O25, O71, O77, O86, O101, O117, O132, O153, and O169 from cow calves. Oswald (1991) studied forty three diarrhoeic bovine isolates of E. coli , out of which 29 strains were typable and belonged to 10 different O serogroups: O78 (eight strains), O123 (four strains) and O4 (four strains). Minakshi et al. (1992) reported E. coli in diarrhoeic buffalo and cow calves. They isolated O4, O9, O25, O7, O8, O11, O26, O101, O109, O110, O129, O123 E. coli serotypes from buffalo calves and serotypes O4, O17,. O18, O8, O19, O20, O22, O25, O55, O170, O101, O103, O113, O132, O153 of E. coli from cow calves. Sutariya (1993) reported out of 15 E. coli isolates from calf diarrhoeal cases, five different serotypes viz. O11, O39, O103, O23 and O54 while ten isolates were untypable. Joon and Kaura (1993) isolated 27 and 1 culture respectively of E. coli from 52 diarrhoeic and 16 non-diarrhoeic cow calves. Diarrhoeic E. coli were O1, O4, O8, O9, O20, O21, O22, O25, O28, O88, O111, O118, O123, O130, O134 and O153 while non-diarrhoeic isolate was identified to be O109. E. coli isolated from diarrhoeic buffalo calves belonged to serogroups O101 (3 strains), O13, O123, O132, O153 (2 strains each) and O8, O49, O68, O102, O111 and O117 (one strain each) and those from non-diarrhoeic calves belonged to serogroups O2, O8, O49 and O123. Mainil (1999) reported serotypes viz.O5:H-, O8:H8, O20:H9, O26:H11, O103:H2, O111:H-, O118:H16, O128:H- and O145:H- associated with diarrhoea in calves. Nishikawa et al. (2002) isolated 7.3% (67/924) diarrhoeagenic E. coli from sporadic cases of human diarrhoeal illness in Japan using PCR technique. In their study several different types of serotypes detected were O111:H-, O26:H11, O157, O6:H16, O25, O8, O55:H7, O86, O126 and O128.

Blanco et al. (2003) isolated VTEC from cattle and sheep. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2 and untypable: H19. Different VTEC serotypes (eg O5: H-, O6:H10, O91: H, O117: H-, O128: H-, O128:H2, O146:H8, O146:H21, O156:H- and untypable :H21) were found more frequently in sheep. Hussain et al. (2003) isolated 101 E. coli strains from diarrhoeic calves. These strains belonged to the serogroups O8, O21 and O116 (two strains each), O7, O15, O18, O22, O101, O111, O123 and O141 (one strain each) and one untypable strain. Sharma et al. (2004) examined a total of 146 faecal samples from diarrhoeic calves. They reported thirty nine serotypes, three strains were untypable and three strains were rough. Serogroups predominantly obtained were O123 (12.12%), O78 (8.33%), O23 (7.5%), O2 (6.06%), O8 (5.30%), O25 (5.30%), O55 (3.03%), O60 (3.03%), O4 (3.03%), O33 (2.27%) and O157 (1.51%). Wani et al. (2004) reported 144 strains of E. coli isolated from a total of 340 faecal samples from calves. Out of 144 strains 5(3.47%) each were untypable and rough. Remaining 134 strains were identified as belonging to different serogroups viz. O2, O3, O6, O7, O8, O9, O10, O12, O15, O16, O20, O22, O25, O26, O33, O45, O46, O50, O54, O55, O60, O75, O77, O78, O85, O86, O88, O89, O91, O96, O100, O101, O102, O105, O107, O109, O111, O114, O115, O123, O125, O131, O132, O146, O148, O150, O153, O156, O157, O1612, O162, O164 and O165. Zweifel et al. (2005) reported a total of 42 STEC strains from slaughtered healthy cattle, the strains belonged to 18 different O serogroups, 12 H types and 26 different O:H serotypes. However 40% of these strains were of three serotypes (O2, O103 an O116) and 62% belonged to three H types (H2, H21 and H-). Thirty eight per cent belonged to 4 O:H serotypes viz. O103:H2 (4 strains), O113:H4 (3 strains), O116:H- ( 5 strains) and ONT:H- (4 strains). In all eight new O:H serotypes that have not previously been reported in bovine strains (O2:H21, O8:H20, O86:H-, O111:H21, O116:H11, O117:H16 and O136:H2) were found. 2.3 Biochemical characterization of E. coli Almost all strains of E. coli are reported to produce acid from glucose, lactose, mannitol and arabinose but not from adonitol and inositol and acid from dulcitol, salicin and sucrose varies from different strains. Gas production has been observed in case of glucose only (Sojka, 1965). Other biochemical tests as proposed by Edwards and Ewing (1972), viz. indole production, positive methyl red and negative Voges Proskauer reaction and inability to grow on Simmon’s citrate medium are considered very useful for preliminary identification of E. coli strains. There are no specific media recommended for isolation of enterotoxigenic E. coli but like other E. coli strains they can grow on nutrient agar, MacConkey Lactose agar, and eosin methylene blue (EMB) agar. Blood agar plates can also be used for isolation of diarrhoeagenic E. coli (Edwards & Ewing, 1972). Sojka (1971) observed that inspite of involvement of many bacteria in neonatal calf diarrhoea about fifty per cent were ascribable to E. coli infection alone. Hussain and Saikia (2000) examined a total of 93 faecal samples from diarrhoeic calves and yielded 101 isolates of E. coli. Single infection only with E. coli in majority of the cases (73.12%) and mixed infection with two or three species was recorded in few cases of calf diarrhoea. 2.4 Unusual Biochemical Characters of E. coli Atypical biochemical behaviour of E. coli strains isolated from diseased conditions has been reported (Sutariya, 1993). Often these characters were found transmissible along

with drug resistance, as reports indicate. Hence it is possible to use this unusual biochemical behaviour as an epidemiological tool in characterizing the isolates from disease outbreaks. 2.4.1 Production of H2S Orskov and Orskov (1973) demonstrated ability to produce hydrogen sulphide (H2S), raffinose fermentation and tetracycline resistance among E. coli isolates from animals. Eleven of the thirty two strains studied were able to transmit the capacity to form H2S; six of the nine strains were able to transmit raffinose fermentation, while 5 of the eleven H2S producing donor strain were capable to transmit tetracycline resistance to the recipient strain. Barbour and co-workers (1985) suggested H2S production ability as a phenotypical marker for epidemiological studies. They found eleven E. coli strains isolated from colisepticaemia in broilers showed a similar antigenic make up (O78: H-) and similar drug resistance pattern (streptomycin, sulfathiazole and tetracycline). Sutariya (1993) found four isolates out of twenty poultry E. coli isolates that produced H2S on TSI. Mishra et al. (2002) isolated few atypical poultry E. coli strains which produced H2S (48 %). 2.4.2 Production of Urease The enzyme urease catalyzes the hydrolysis of urea to give ammonia and carbon dioxide, thereby providing an important nitrogen source for many bacterial species. In addition, urease can contribute to the virulence of several gram-negative bacteria and enhance their acid resistance. However, ureolytic Escherichia coli strains are rarely found among clinical isolates. Urease gene cluster is located within a pathogenicity island called other genetic islands i.e. O- islands (OI): OI 43 and OI 48. Studies have indicated that among various diarrhoeagenic E. coli strains from clinical sources, urease genes are associated with major EHEC groups O26, O111 and O157 but are absent from diarrhoeagenic E. coli strains of other pathogroups, including enetrotoxigenic E. coli, enteroaggregative E. coli, enteroinvasive E. coli and enteropathogenic E. coli (Friedrich et al., 2005). Adetosoye and Ojo (1983) reported urease producing ability along with multiple drug resistance, among isolates obtained from diarrhoeal cases of kids and piglets. Both the characters were found to be transmissible. The urease producing E. coli strains belonged to three serotypes O26:K-:H11; O18ab/ac:K-H-; O68K-H-. Pohl et al. (1989) reported four strains of E. coli out of 2000 strains from calf diarrhoea, produced urease. They belonged to serotypes O5: K-H- Tominaga et al. (1989) reported urease activity of verocytotoxin producing E. coli isolates of serotype O5: K-: NM from cases of calf diarrhoea. These isolates were designated as Attaching and Effacing E. coli (AEEC). Sutariya (1993) observed urease activity in three poultry isolates out of total 20 isolates, which was found to be transferable along with multiple drug resistance and colicinogeny. Dubey et al. (2001) observed that 4.3 % E. coli isolated from diarrhoeic goats produced urease. Mishra et al. (2002) found 16% of ureolytic E. coli isolates from poultry birds. 2.4.3 Ability to utilize citrate Ishiguro et al. (1978) showed twenty seven isolates of citrate positive variants of E. coli from cattle, pigs, horses. These had multiple drug resistance and the ability to transfer multiple drug resistance to recipient strains.

Ishiguro and Sato (1979) reported sixty seven isolates of citrate positive variants of E. coli from man and domestic animals which otherwise were typical E. coli in their biochemical characteristics. The ability to transmit citrate utilization characters was demonstrated in 53 (79%) of the variants. Drug resistance determinants and citrate utilizing characters were co-transmitted into E. coli K12 by conjugation. Lee and Choi (1983) reported citrate utilizing variants of E. coli from cattle, pigs, chickens and pigeons. Of 3,108 E. coli like colonies obtained from faecal samples of 681 different animals, 123 colonies yielded citrate utilizing E. coli. Kim and Tak (1984) showed that 20 of 391 strains of E. coli from chickens with colibacillosis had ability to utilize citrate. In 18 isolates this ability was found transferable. Dubey et al. (2001) observed that E. coli strains isolated from diarrhoeic goats failed to utilize citrate in Simon’s medium but 48.9% were positive for using citrate as a sole carbon source when tested in Christensen’s citrate agar. 2.4.4 Adonitol and Raffinose Fermentation In general the organism E. coli rarely ferments adonitol while variation among strains (upto 50 per cent positive) is being observed for raffinose activity as per Edwards and Ewing (1972). The variation of raffinose activity might be due to its plasmid mediated nature as has been reported by many workers. Braaten and Myers (1977) reported biochemical characterization of enterotoxigenic and non-enetrotoxigenic E. coli isolated from diarrhoeic calves. Nine out of eighteen enterotoxigenic E. coli isolates and one out of fifteen non-enterotoxigenic E. coli isolates showed adonitol fermentation. Six out of 18 ETEC and twelve out of 1% non-ETEC showed raffinose fermentation. Smith and Huggins (1978) reported that K88 pili are determined by genes carried by plasmids which usually carry gene for raffinose fermentation. Cloud et al. (1985) reported biochemical characterization of E. coli isolated from diseased broilers. Thirty out of 197 isolates fermented adonitol while 177 out of 197 fermented raffinose. Mondaca et al. (1985) demonstrated raffinose fermentation and tetracycline resistance in twenty one H2S producing E. coli strains isolated from pigs and man. They suggested that H2S production and tetracycline resistance both are coded by same plasmid. 2.5 Biotypes of E. coli isolates Biochemical reactions have conventionally been used for identification of bacteria to the species level. Extensive studies of biochemical reactions of bacteria have been done to introduce biochemical typing systems in epidemiological studies of bacteria (Barr and Hogg, 1979; Krishnan et al., 1987) and biotyping was also found to be useful tool to distinguish haemolytic strains of E. coli belonging to serogroups O138, O139 and O141 which had been isolated from pigs (Manson, 1962). E. coli are able to ferment a variety of carbohydrate substrates, generally by converting them to glucose or to a substrate on the fermentative chain of the breakdown of glucose. The various fermentable carbohydrates include substances such as compounds of glucose with other sugars. Thus maltose, trehalose and cellobiose are compounds of two glucose molecules linked in different ways. Lactose is a compound of glucose and the related sugar galactose, sucrose is a compound of glucose and the related sugar fructose. Raffinose is a compound of glucose, fructose and galactose. Other carbohydrates like substances which can be fermented include sugars like mannose, sorbose, arabinose and rhamnose and the related sugar alcohols dulcitol, mannitol and sorbitol. Also compounds of sugars with other substances can be fermented particularly if the sugar molecule can be separated from the other part. An example is the substance salicin.

The ability to ferment a given sugar of the types described above by a strain of E. coli is dependent on the strain having the requisite enzymes to convert it to glucose or to a substance on the degradative chain from glucose. It has been found that different strains of E. coli differ in their ability to perform these conversions. Thus while all strains will ferment glucose and over 90% will ferment mannitol and lactose, with many of the other sugars mentioned above the fermentation reactions will vary. This is the basis of biotyping E. coli. These tests are also easy to perform, by determining, whether a strain of E. coli will produce acid following growth in the presence of the carbohydrate (Crichton and Old, 1982). Rao et al. (1975) on the basis of fermentation of 4 sugars viz. sucrose, dulcitol, raffinose and salicin classified 349 E. coli isolates from intestinal tract of 14 adult fowls, into 16 biotypes. Further they noted that it appears difficult to classify the E. coli isolates on biotype basis, since the occurrence of more number of biotypes can not be ruled out. Pandey et al. (1979) classified 29 isolates of E. coli obtained from 106 samples of milk, human urine, calf diarrhoea, poultry enteritis and infantile diarrhoea into 19 biotypes by their fermentation reactions with dulcitol, starch, sucrose, salicin, raffinose and rhamnose. Hinton et al. (1982) examined a total of 2973 E. coli isolated from six different groups of animals for their ability to ferment adonitol, dulcitol, raffinose, rhamnose and sorbose. Twenty fermentation patterns were recorded although 2443 (82%) of the E. coli belonged to 7 of the 32 possible biotypes. Ninety six O-serotypes were identified within the 2973 E. coli. The number of O serotypes represented in the 15 most common biotypes ranged from three to fifteen. Serotypes O8 and O9 were found most commonly in different groups of animals and several biotypes amongst these two O-serotypes were identified in 2 or more groups of animals. Camguilhem and Milon (1989) isolated a total of 575 E. coli strains from weaned diarrhoeic rabbits. More than half of the 575 E. coli strains tested belonged to serogroup O103, less frequent serogroups included O132 (5.7%), O68 (4%), O128 (3.3%) and O2 (3%). A sample of 126 strains was further checked for simplified biotypes by using five carbohydrate fermentation reactions viz. sorbose, dulcitol, raffinose, sucrose and rhamnose. Of the isolates belonging to 72 serogroup O103 seventy strains were shown to belong to biotypes characterized by a rhamnose-negative reaction, 4 of nine serogroup O68 strains showed this type of reaction. Blanco et al. (1996) isolated a total of 305 E. coli strains from diarrhoeic and healthy rabbits. They distinguished a total of 17 different biotypes based on fermentation of sugars viz. sorbose, dulcitol, raffinose, sucrose and rhamnose. The majority of rabbit E. coli strains were assigned to only six biotypes (B6, B13, B14, B27, B28, B30 and B31). Rhamnose negative B6, B13, B14 biotypes were more frequently found among E. coli strains isolated from diarrhoeic rabbits whereas rhamnose positive B27, B30 biotypes were more commonly detected among strains obtained from healthy controls. The most common serobiotypes found among E. coli strains isolated from diarrhoeic rabbits in order of frequency were O103:B14 (72 strains), O103:B6 (16 strains), O26:B13 (12 strains) and O128: B30 (12 strains). Chachra and Katoch (1996) biotyped 48 out of sixty eight E. coli isolates from diarrhoeic poultry into 12 different combinations based on carbohydrate fermentation patterns with six sugars viz. dulcitol, raffinose, rhamnose, salicin, starch and sucrose. Rhamnose and starch combination (17 strains), starch and sucrose combination (7 strains), salicin, starch and sucrose combination (five strains) and dulcitol and rhamnose combination (4 strains) were found more than other sugar combinations. Chattopadhyay et al. (2003) found that thirteen strains out of 20 E. coli samples from diarrhoeic calves found positive for verotoxin gene by PCR test could be isolated and characterized for biotyping on the basis of sugar fermentation reactions in three sugars viz. sorbitol, raffinose, dulcitol and decarboxylase test with lysine, arginine and ornithine and

found six different biotypes. All these strains were untypable for O serogroup against O157:H7 antisera but all of them possessed Stx gene. Murinda et al. (2004) reported diagnostic significance of rhamnose fermentation test. They recorded rhamnose non fermenters belonging to E. coli O26 were hundred per cent STEC producers. 2.5 Multiple antimicrobial drug resistance in E. coli isolates associated with calf

diarrhoea Neonatal calf diarrhoea is one of the stumbling blocks for the dairy farmer in our country. Despite of vaccination programs and management measures, treatment with antibiotics may be required in some cases. Although antimicrobial susceptibility testing is recommended, information on drug resistance trends in a geographic area is helpful to veterinarians in drug selection for empirical therapy. The result of culture and antibiotic sensitivity studies may be valuable as background information for further therapy for effective control of disease. Otherwise indiscriminate use of antimicrobial drugs may lead to serious hazards of drug resistance. Pohl et al. (1969) recorded multiple resistance to antibiotics among E. coli isolated from calves. Majority of the strains showed multiple resistance and were able to transmit resistance to susceptible strains. Saxena (1976) tested 245 haemolytic E. coli strains. Out of which 40.4 percent were found resistant to tetracycline, 2.8 per cent to furazolidone, 1.2 per cent to polymyxin B, 18.8 per cent to neomycin, 54.7 per cent to streptomycin and 10.6 per cent to chloramphenicol. Tripathi and Soni (1982) isolated 33 E. coli strains from fifty newborn calves with diarrhoea. About 25 per cent of isolates were resistant to nalidixic acid, ampicillin and chloramphenicol. Neomaycin and polymyxin B had intermediate inhibitory effects. Forty five per cent strains were resistant to streptomycin, 57.58 per cent to methicillin and 66.0 per cent to kanamycin. Multiple drug resistance was shown by number of strains.

Boro et al. (1983) tested 34 isolates of E. coli recovered from different pathological conditions in animals against 16 antimicrobial agents in vitro. The antibiotics in order of efficacy were gentamicin (92.3%), chloramphenicol (79.4%), colistin sulphate (75%), ampicillin (33.4%) and tetracycline (29.4%).

Sherwood and Snodgrass (1983) isolated 88 of 1529 E. coli isolates from diarrhoeic and clinically normal calves. They studied antibiotic resistance of ETEC (27 isolates) and non-ETEC (33 isolates) and observed that a higher proportion of non ETEC than the ETEC isolates was resistant to five of the antibiotics tested (streptomycin, gentamicin, ampicillin, polymyxin B, chlortetracycline, chloramphenicol, neomycin) but the reverse was the case for streptomycin and neomycin. Mehrotra et al. (1984) conducted antibiotic sensitivity tests on 60 E. coli isolates of cattle, 60 isolates of poultry, 68 isolates of goats and 28 human isolates against 9 antibiotics and furazolidone. Two hundred seventeen of 248 strains were resistant to tetracycline and oxytetracycline, 207 to erythromycin, 204 to doxycycline, 114 to streptomycin. All were sensitive to nalidixic acid. Panda and Panda (1987) tested 50 isolates of E. coli from diarrhoeic calves for antibiotic sensitivity and found that septran, chloramphenicol and nalidixic acid were 100 per cent effective while gentamicin 96 per cent, neomycin 70 per cent, ampicillin 64 per cent and nitrofurazone 60 per cent effective. Shah (1989) reported drug resistance of 71 E. coli isolates from calf diarrhoea. Multiple drug resistance was exhibited by most of the E. coli organisms. One culture was

resistant to seven out of eight antibiotics. Gentamicin was found to be the most effective antibiotic having all the organisms sensitive to it. Resistance of E. coli isolates to various antibiotics was observed in the following ascending order. Neomycin 8.45 per cent, chloramphenicol 23.94 per cent, co-trimoxazole 26.76 per cent, furazolidone 38.04 per cent, streptomycin 46.48 per cent, ampicillin 54.93 per cent and tetracycline 69.02 per cent. Saravanbava et al. (1990) carried out a study on E. coli associated with calf diarrhoea in Tamil Nadu. The antibiotic resistance pattern of 74 E. coli isolates was carried out. All the E. coli isolates showed multiple drug resistance. The percentage of resistant isolates was as follows: co-trimoxazole 6.76 per cent, gentamicin 9.46 per cent, chlopramphenicol 12.16 per cent, nalidixic acid 13.51 per cent, kanamycin 17.57 per cent, streptomycin 41.59 per cent, carbenicillin 67.57 per cent, tetracycline 79.73 per cent, cephaloridine 98.65 per cent, ampicillin 100.00 per cent and sulfamethazole 100.00 per cent. Aalback et al. (1991) studied prevalence of antibiotic resistance of E. coli in Danish pigs and calves. Rectal swabs from 52 piglets and 78 calves were examined. All the animals harboured drug resistant E. coli. Certain resistance patterns (Sulfonamide + Streptomycin; Sulfonamide + Streptomycin + Tetracycline; Sulfonamide + Streptomycin + Tetracycline + Ampicillin) were found to be shared by numerous strains suggesting a genetic linkage of the resistance markers. Sutariya (1993) studied antibiotic resistance pattern of diarrhoeic calf E. coli isolates and found that out of 15 E. coli isolates all of them showed resistance to cloxacillin, erythromycin, tetracycline and sulfamethizole. Fourteen isolates were resistant to neomycin and 10 isolates were resistant to ampicillin and streptomycin. Nine isolates were resistant to chloramphenicol, while 6 were resistant to nitrofurantoin and lesser number of isolates showed resistance to gentamicin (4) and kanamycin(3). None of the isolates were resistant to nalidixic acid.

Cid et al., (1996) determined the in vitro activities of 14 antimicrobial agents against 92 strains of E. coli isolated from lambs and kids affected by neonatal diarrhoea. The overall percentage of resistant strains to tetracycline was very high (above 70%). A high level of resistance (30-50%) to ampicillin and chloramphenicol was also detected. However, the strains were highly susceptible (99 to 100 %) to cephalosporins and nalidixic acid. Most of the strains showed multiple resistances: 77.2% of isolates were resistant to atleast 2 antibiotics, 55.4% were resistant to atleast 4 antibiotics and 33.7% were resistant to at least 6 antibiotics. A total of 34 antibiotypes could be distinguished. Orden et al. (1999) determined the in vitro activities of several cephalosporins and quinolones against 195 strains of E. coli isolated from dairy calves affected by neonatal diarrhoea. One hundred and thirty seven of these strains produced one or more potential factors (F5, F41, F17, CNF, verotoxin and eae gene) but the remaining 58 strains did not produce any of these factors. From 11-18% of E. coli strains were resistant to cephalothin, nalidixic acid enoxacin and enrofloxacin. However cefuroxime, cefotaxime and cefquinome were highly effective against the E. coli isolates tested. Verotoxigenic strains were significantly more sensitive to nalidixic acid, enoxacin and enrofloxacin than non toxigenic strains. Bradford et al. (1999) studied antibiotic resistance among E. coli isolates from diarrhoeal disease in calves. Many of the isolates were multiple resistant to beta lactams, including expanded spectrum cephalosporins, aminogycosides, sulphonamides tetracyclines and fluoroquinolones. All of the isolates tested were resistant to ampicillin and had reduced susceptibility to ticarcillin and piperacillin. Twenty seven of the 32 isolates tested showed increased resistance to the expanded spectrum cepalosporins, aztreonam and cefoxitin. All of the isolates were resistant to kanamycin, streptomycin, sulphisoxazole and tetracycline. In addition, many were resistant to chloramphenicol, gentamicin, trimethoprim-sulphamethoxazole. Four isolates were nalidixic acid resistant but only two strains were

ciprofloxacin resistant. All 32 strains showed varying degrees of resistance to ceftiofur and about 13% of E. coli strains implicated in bovine scours were resistant to ceftiofur. Kobayashi et al. (2001) tested antimicrobial susceptibility among E. coli isolates using 14 antimicrobial agents and found that only eight isolates were resistant to some of the antimicrobial drugs tested. Six of these isolates were resistant to aminoglycosides (kanamycin and dihydrostreptomycin) and /or tetracyclines (chlortetracycline and oxytetracycline) and the other two showed resistance to colistin. Chattopadhyay et al. (2001) studied the antibiotic sensitivity pattern of STEC strains from animal, human and food products and reported that STEC strains were uniformly sensitive to common antibiotics except tetracycline, cephalexin, dicloxacillin, erythromycin and lincomycin. Khan et al. (2002) also examined antimicrobial resistance pattern of 63 shiga toxin producing E. coli isolates from 19 human stool samples, 40 cow stool samples and 4 beef samples in Kolkata using 15 antimicrobials. Resistance was observed most commonly to ampicillin (25.4%), tetracycline (23.8%) and streptomycin (14.3%) and less frequently to cephalothin (1.1%), co-trimoxazole (9.5%), nalidixic acid (6.4%) and neomycin (3.2%). They reported that more than one third of these strains (35%) showed reduced susceptibility to different antimicrobial agents, but were not completely resistant to any of the antibiotics. About 14.3% of the isolates were sensitive to all antimicrobials used. Fourteen E. coli strains showed multiple drug resistance and there was no common resistance pattern among the strains. Chattopadhyay et al. (2003) tested 13 STEC strains for antimicrobial susceptibility against the following antimicrobial agents: amoxicillin, amikacin, cephalexin, cefotaxime, chloramphenicol, ciprofloxacin, co-trimoxazole, furaxone, gentamicin, nalidixic acid and norfloxacin and tetracycline. Antimicrobial susceptibility pattern of STEC showed that 68.2%, 61.5%, 46% and 30.76% were resistant to co-trimoxazole, furaxone, tetracycline/nalidixic acid and cephalexin respectively. Most of the strains were sensitive to norfloxacin, gentamicin and chloramphenicol. Hariharan et al. (2004) evaluated in vitro resistance to eight antimicrobials among enterotoxigenic E. coli from piglets and calves over a period of 13 years. The percentages of resistance of the bovine isolates in ascending order in the first eight years were ceftiofur (4%), gentamicin (6%), spectinomycin (44%), trimethoprim-sulphonamide (TMS) (46%), neomycin (64%) and oxytetracycline (81%). For the last 5 year period least resistance was seen against ceftiofur, followed by florfenicol, TMS and tetracycline, the resistance rates being 8%, 11%, 48% and 75% respectively. Sharma et al. (2004), carried out antibiotic sensitivity test in E. coli strains from diarrhoeic calves and found that E. coli strains were most sensitive to oflaxacin (97.28%) , kanamycin (93.24%) followed by gentamicin (85.62%), co-trimoxazole (71%), nalidixic acid (57.15%), streptomycin (52.50%), doxycycline (50%), oxytetracycline (30.78%) and trimethoprim (26-27%). 2.6 Colicinogeny among E. coli isolates Colicines are substances of high molecular weight, generally proteins that are formed by Enterobacteriaceae family and having strain specific lethal activity on other bacteria. Colicinogeny, the ability to produce colicin, is a stable heritable property of many E. coli strains (Fredericq, 1957). Colicines may help E. coli strains to establish in the gut by inhibiting other resident normal flora although, the role is not very clear (Elwell and Shipley, 1980). Heller and Smith (1973) studied colicinogeny along with other characters among E. coli isolates from dead chickens and they found that colicin production was twice as

common in the pathogenic strains as in a collection of strains isolated from the faeces of healthy chickens; about half of it was transferable. Invasive strains of E. coli are reported more frequently to be the Col-V producer than the faecal strains. Colicin-V production is often determined by virulence associated plasmid. However, Col-V itself is not a virulence factor. Arunachalam et al. (1974) studied serological typing, colicinogeny and colicin sensitivity of E. coli strains associated with colisepticaemia and enteritis in poultry. Thirteen out of fifty nine E. coli strains were colicinogenic. Eleven unknown colicins could be typed. H colicin was produced by all strains. Cooper and James (1984) reported two new E colicines, E8 and E9, produced by strains of E. coli. A strain of E. coli isolated from chicken caeca produced two new E colicines, and colicin M. This strain had seven plasmids, five of which have been transferred to recipient strain of E. coli. Harnett and Gyles (1984) found that colicin production was more common property of porcine ETEC (80.8 %) than of porcine non-ETEC (25 %) and all porcine ETEC of serogroups O101 and O64 were colicinogenic. Equal numbers of bovine ETEC strains were colicinogenic as well as non-colicinogenic. Djonne (1985) studied colicin production in E. coli of piglets in relation to pathogenicity factors, three hundred and fifteen E. coli strains isolated from piglets with neonatal diarrhoea were examined for colicin production. Almost all the E. coli strains positive for enterotoxin production also produced colicin while about 17 per cent of the non ETEC strains did so. Yadav et al. (1986a) studied association of colicinogeny and antibiotic resistance among E. coli strains from human origin. One hundred fifty two strains of E. coli isolated from urinary tract and gastrointestinal tract of man were screened for their colicinogeny, antibiotic resistance and presence of R-factors. Of these 152 E. coli strains 9.2 per cent were detected as colicinogenic and 85.7 per cent of these colicinogenic strains were found resistant to one or more of the antibiotics at a time. Al-Dabbas and Willinger (1986) reported colicin production by E. coli from calves with diarrhoea. Out of 274 E. coli strains, 59 strains (21.53 per cent) were colicinogenic. Corlu (1988) showed that 10 per cent of E. coli isolates from healthy lambs and 13 per cent of those from diarrhoeic lambs were colicinogenic. Ayhan and Aydin (1989) reported that colicin production was detected in 3 (5.1 %) of 59 bovine isolates of E. coli, 2 (12.15%) of 16 fowl isolates and 4 (4.2%) of 95 human isolates of E. coli. Oswald et al. (1991) tested 43 bovine isolates of E. coli for colicin production along with other virulence factors and found that 32% of the strains produced colicin. Sutariya (1993) studied colicinogeny in diarrhoeic calves and poultry E. coli isolates. He found that 6 (40%) of 15 isolates of E. coli from diarrhoeic calves and 11 (55 %) of twenty poultry E. coli isolates were colicin producers. Blanco et al. (1997) studied 625 E. coli strains isolated from visceral organs of chickens with colisepticaemia and from faeces of healthy chickens for production of heat labile (LT) enterotoxin, heat stable (ST) enterotoxin, verotoxins, alpha heamolysin, colicin-V and other types of colicines. They found a clear correlation between the production of colicines and in vitro pathogenicity. Thus, 80% and 66% of strains producing Col-V and other types of colicines respectively were characterized as being of high pathogenicity, whereas only 15% of the non colicinogenic strains were classified as highly pathogenic. Ahmad et al. (2004) screened 194 strains of E. coli of different clinical conditions (diarrhoea, gastroenteritis, urinary tract infection and septicaemia) of human and animals for colicin production. They found incidence of colicin production maximum in E. coli strains of human UTI (26.66%) followed by human GIT, animal GIT and least in poultry septicaemia

(4.16%). Of these colicinogenic strains 19 (57%) were found resistant to one or more antibacterial drug tested. Resistance against tetracycline was observed maximum (48.48%) followed by doxycycline, streptomycin, ampicillin, chloramphenicol, amoxicillin and cotrimoxazole. None of the strains were found resistant to nalidixic acid and norfloxacin. 2.7 Virulence attributes of E. coli 2.7.1 Haemolysin production Bisht et al. (1977) found haemolytic and necrotoxic activity among 156 and 93 isolates respectively out of 560 E. coli isolates from cases of acute gastroenteritis, chronic diarrhoea as well as healthy human population. A positive association between haemolysin and necrotoxin production was observed amongst the isolates from diseased cases but in healthy population they were not common as in pathogenic group. Aditi et al. (1981) studied haemolysin production of E. coli isolates causing urinary tract infection in humans and found that twenty eight strains out of 59 were haemolytic. Boro et al. (1983) isolated 121 E. coli isolates from scouring piglets. Out of them 21 strains were haemolytic. No reaction was observed between haemolytic strains with that of serotypes. Sherwood and Snodgrass (1983) observed a good but not absolute correlation between enterotoxigenicity and haemolysin production in E. coli strains isolated from diarrhoeic and clinically normal calves. Out of the total number of 86 isolates producing haemolysin 81 were ETEC and five were non-ETEC isolates. Whereas of the total 1443 non haemolytic strains seven were ETEC and 1436 were non ETEC Arora and Prabhakar (1984) observed that higher percentage of E. coli isolated from gastroenteritis showed haemolytic activity (36%) than those from healthy patients. Yadav et al. (1986b) found that among the 102 E. coli strains isolated from urinary tract infection of human 36 were ETEC and 66 were non-ETEC. They observed that 33 per cent ETEC isolates were haemolytic while only 9 per cent of non-ETEC isolates showed haemolysis. Shah (1989) studied haemolysin and necrotoxin production in E. coli isolated from diarrhoeic calves. Production of haemolysin and necrotoxin was found in 24 and 15 strains respectively out of 71 strains. Beutin et al. (1989) investigated 66 E. coli strains isolated from humans and animals for production of verotoxins (VT) and haemolysin. They detected 60 of the 64 VT positive strains that produced haemolysin while two VT negative strains failed to do so. They also studied haemolysin production among non-VTEC faecal isolates and observed that out of total 549 strains enterohaemolysin was detected in only four O26 VT negative enteropathogenic E. coli strains and not in any other group. Fifteen per cent of 267 faecal E. coli strains were positive for alpha haemolysin. No haemolysin positive strain was found in a group of 45 ETEC strains obtained from humans (42 strains) animals (2 strains) or food stuffs (1 strain). Kaura et al. (1991) studied a total of 77 strains of E. coli isolated from diarrhoeic buffalo calves and cow calves for their antibiotic resistance, haemolysin production and enterotoxins. Sixteen E. coli isolates from buffalo calves strains and two isolates from cow calves were haemolytic. Oswald et al. (1991) tested haemolysin production in 43 isolates of E. coli obtained from diarrhoeic calves and found that 4 (9%) produced α- haemolysin. Joon and Kaura (1993) isolated E. coli strains from diarrhoeic and non-diarrhoeic cow and buffalo calves and found that 4 each of 28 strains of E. coli from cow-calves and buffalo calves, caused haemolysis of heterologous erythrocytes. However 2 and 4 E. coli strains each from cow and buffalo calves respectively, haemolysed homologous RBCs.

Patil et al. (1999) carried out a study for testing drug resistance and virulence characters viz. haemolysin and enetrotoxin production in E. coli isolated from diarrhoeic calves. Eight (44.4%) of 18 E. coli isolates were found to produce haemolysin and among these 6 (75%) were enterotoxigenic (ETEC). Khan et al. (2002) investigated prevalence of shiga toxin producing E. coli in diarrhoeic patients, healthy domestic cattle and raw beef samples and studied haemolysin production. Fourteen of 30 STEC isolates showed α- haemolytic activity. However three STEC strains didn’t produce haemolysin. Hussain et al. (2003) screened 101 E. coli strains isolated from diarrhoeic calves for virulence characteristics and they found that eight (7.92%) of 101 strains of E. coli belonging to five serogropups (O22, O98, O123 and O164) and one untypable strain produced haemolysin . Salvadori et al. (2003) screened 205 E. coli strains isolated from calves with diarrhoea for the presence of virulence factors associated with bovine colibacillosis, like shigatoxins, α-haemolysin, enterohaemolysin, CNF type 1 and LT and ST enterotoxin production. Of the 205 E. coli strains 20 were α-haemolysin positive. Two (1%) of α-haemolysin strains were CNF producing strains. None of the strains producing haemolysin were associated with enterotoxins. 2.7.2 Detection of toxigenic E. coli isolates

Identification of diarrhoeagenic E. coli strains requires that these organisms be differentiated from nonpathogenic members of the normal flora. Serotypic markers correlate sometimes very closely, with specific categories of diarrhoeagenic E. coli however, these markers are rarely sufficient in and of themselves to reliably identify a strain as diarrhoeagenic. (An exception may be strains of serotype O157:H7, a serotype that serves as a marker for virulent enterohemorrhagic E. coli strains nevertheless, EHEC of serotypes other than O157:H7 are being identified with increasing frequency in sporadic and epidemic cases). Thus, detection of diarrhoeagenic E. coli has focused increasingly on the identification of characteristics which themselves determine the virulence of these organisms. This may include in vitro phenotypic assays which correlate with the presence of specific virulence traits or detection of the genes encoding these traits (Nataro and Kaper, 1998). (a) Phenotypic Assays Based on Virulence Characteristics There are no suitable biochemical markers by which ETEC could be identified and distinguished from other E. coli strains producing diarrhoea. Detection of ETEC has long relied on detection of the enterotoxins LT and/or ST. ST was initially detected in a rabbit ileal loop assay (Evans et al., 1973) but the expenses and lack of standardization caused this test to be replaced by the suckling mouse assay (Gianella, 1976). Several immunoassays like RIA, ELISA have been developed for detection of ST. The traditional bioassay for detection of LT involves the use of cell culture, either the Y1 adrenal cell assay or the Chinese hamster ovary (CHO) cell assay. Immunological assays for LT include Biken test, ELISA, latex agglutination and reversed passive latex agglutination test (Nataro and Kaper, 1998).One of the most useful phenotypic assays for the diagnosis of diarrhoeagenic E. coli is the HEp-2 adherence assay (Donnenberg and Nataro, 1995). This assay provides the best ability to differentiate among all three adherent diarrhoeagenic categories viz.EPEC, EAEC, and DAEC (Vial et al., 1988). Bettelheim and Beutin (2003) have reviewed that VTEC (verocytotoxigenic E. coli) can be detected by detection of verotoxins produced by the bacteria or of the genes associated with VT (verotoxin) production. Vero cell toxicity of verotoxins can be detected by observation of cytopathic effects on vero cells (Konowalchuk et al., 1977). Serological

(ELISA, immunoblot and reversed passive latex agglutination) tests were developed for identification of verotoxins produced by bacteria. (b) Molecular Detection Methods

Diarrhoeagenic E. coli strains were among the first pathogens for which molecular diagnostic methods were developed. Indeed, molecular methods remain the most popular and most reliable techniques for differentiating diarrhoeagenic strains from nonpathogenic members of the stool flora and distinguishing one category from another. Substantial progress has been made both in the development of nucleic acid-based probe technologies as well as PCR methods (Nataro and Kaper, 1998). • Nucleic acid probes: The use of DNA probes for detection of heat-labile (LT) and heat-

stable (ST) enterotoxins in ETEC revolutionized the study of these organisms, replacing cumbersome and costly animal models of toxin detection (Moseley et al., 1982). Since then, gene probes have been introduced for all diarrhoeagenic categories.

• Polymerase Chain Reaction (PCR): is a major advance in molecular diagnostics of pathogenic microorganisms including E. coli. PCR primers have been developed successfully for several of the categories of diarrhoeagenic E. coli.

ETEC strains were among the first pathogenic micro-organism for which molecular diagnostic techniques were developed. Moseley et al. (1982) found DNA probes useful in the detection of LT and ST encoding genes in stool and environmental samples. Since then, several advances in ETEC detection have been made but genetic techniques continue to attract the most attention and use. ETEC produce heat labile (LT) and heat stable (ST) toxins and cause diarrhoea. These enterotoxins attach to specific receptors on luminal surface of intestinal epithelial cells and activate adenylate cyclase which subsequently alter concentration of intracellular cyclic AMP and cyclic GMP and lead to unmask secretory activity of less accessible crypt cells resulting in out pouring of electrolytes and fluid into lumen of small intestine. This overwhelms the absorption capacity of large intestine resulting into typical rice bran diarrhoea (Yadav et al., 1988). For detection of ETEC several PCR assays are developed that are quite sensitive, rapid and specific when used directly on clinical samples or in isolated bacterial colonies (Rich et al., 2001). A useful adaptation of PCR is the multiplex PCR assay (Lang et al., 1994; Stacy-Phipps et al., 1995) in which several PCR primers are combined with the aim of detecting one of several different diarrhoeagenic E. coli pathotypes in a single reaction. Genetic methods (PCR and DNA-hybridization) are used for identification of VT1 and VT2 gene specific DNA sequences. However, PCR techniques have been extensively used to detect VT genes. The PCR test for amplification of VT1 and VT2 gene sequences has demonstrated universality in that both toxin genes can be detected and differentiated across a wide spectrum of verocytotoxigenic E. coli serotypes other than O157:H7, which presently exceed 200 serotypes (Johnson et al., 1996) and have been associated with haemorrhagic colitis and haemolytic uremic syndrome or diarrhoea and for which no biochemical markers are currently available, so the PCR is more rapid, accurate and inexpensive detection of these important genes in clinical material. Shiga toxin or verotoxin induces fluid accumulation by selectively killing the absorptive intestinal villous cells, thereby decreasing fluid absorption and unmasking basal crypt anion secretion. These changes shift the usual balance of intestinal absorption and secretion toward net secretion (Keenan et al., 1986; Kandel et al., 1989). Pollard et al. (1990) used four sets of primers for detection of VT1 and VT2 gene sequences of 130 bp and 346 bp respectively in E. coli strains by PCR method and found forty VT producing E. coli strains.

Lang et al. (1994) developed a triplex PCR method to simultaneously amplify a heat labile toxin sequence (LT) of 258 bp, a shiga like toxin I sequence (SLT-I) of 130 bp and a shiga like toxin II sequence (SLT-II) of 346 bp from toxigenic strains of E. coli. Heuvelink (1998) examined faecal samples from Dutch cattle and sheep collected at slaughterhouses, for the presence of VTEC of serogroup O157 using multiplex PCR. E. coli O157 strains could be isolated from 57 (10.6%) of 540 adult cattle, 2 (0.5%) of 397 veal calves, 2 (3.8%) of 52 ewes and 2 (4.1%) of 49 lambs. With the exception of two isolates from adult cattle which appeared to be negative for VT genes all animal isolates were positive for both VT (VT1 and VT2) genes and therefore were regarded as potential human pathogens. Bradford et al. (1999) examined 32 E. coli isolates obtained from individual bovine calf scours cases for antimicrobial susceptibility and for presence of STX-I and STX-II, LT, ST (STa and STb) along with other virulence factors. Genes for STX-II, LT and STb were not detected among these isolates although eight strains were positive for STa eneterotoxin, indicating that they were ETEC. One isolate was positive for STX-I. Pal et al. (1999) reported the isolation of STEC from non-diarrhoeic animal sources. These workers collected faecal samples from 67 healthy cattle in Kolkata and examined these for STEC by multiplex PCR and eight strains isolated belonged to eight serotypes viz. O146:H1, O149:HNT( not typable), O Untypable:H19. O88: HNT, ONT:H2, O82:H8 and O28ac:H21. Kobayashi et al. (2001) examined the prevalence of STEC, by using stool samples from 87 calves, 88 heifers and 183 cows on 78 farms in Japan. As determined by screening with PCR, the prevalence was 46% in calves, 66 % in heifers and 69% in cows. They found a connection between the serogroups and the types of Stx genes: all of the serogroups O26 strains contained Stx 1, whereas the serogroups O84, O113 and O116 strains contained Stx2. Chattopadhyay et al. (2001) isolated and characterized STEC strains from animal, human and food products. A total of 876 samples (330 animals, 184 human and 362 food samples) were screened for presence of STEC by PCR technique. The isolation rate was higher in diarrhoeic animals (6.02%), followed by diarrhoeic handler (3.12%) and raw beef (1.78%). Khan et al. (2002) studied antibiotic resistance, virulence gene and other molecular profile of STEC strains isolated from human stool samples, cow stool samples and beef samples over a period of two years in Kolkata. The dominant combinations of virulence genes present in the strains studied were Stx1 and Stx2 (44.5% of strains) and Stx2 and hlyA (19% of strains). Resistance to one or more antibiotics was observed in 49.2% of the STEC strains with some strains exhibiting multiple drug resistance. Rahman (2002) studied a total of 37 strains of E. coli isolated from cases of bovine calf diarrhoea and gastrointestinal disorder. He reported prevalence of verotoxin genes among non-EHEC (non O157) strains detected by PCR amplification technique. Of the 37 strains of E. coli tested , five (13.5%) were found to be positive for VT genes: of which two strains harboured both VT1 and VT2 genes, while two strains only VT1 and one strain VT2 gene only. Nishikawa et al. (2002) isolated 7.3% (67/924) diarrhoeagenic E. coli from sporadic cases of diarrhoeal illness in Japan using PCR technique. For detection of LT, ST and VT2 gene sequences of 132 bp, 171 bp and 228 bp respectively in E. coli isolates they used six sets of primers. These 67 strains were composed of 10 (15%) EHEC, 4 (6%) ETEC along with other groups of diarrhoeagenic E. coli. Among the ETEC isolates, all the four strains carried ST gene and three carried LT gene. Chattopadhyay et al. (2003) found a total of 20 STEC strains positive by PCR test out of 415 samples of different categories screened (faecal samples from 77 diarrhoeic calves, 78 healthy calves and 150 diarrhoeic children, 50 animal handlers, 60 food samples).

The isolation rate was highest in diarrhoeic calves where 17 (22.07%) were found positive for STEC out of 77 diarrhoeic calf faecal samples tested. On colony hybridization with Stx1 and Stx 2 gene probes out of 29 samples that were positive by PCR technique, 10 contained both Stx1 and Stx2 genes while 3 contained only Stx2 gene. This study showed PCR to be more sensitive than hybridization technique. Salvadori et al. (2003) isolated 205 E. coli strains from calves with diarrhoea from mid-western Brazil. They screened them for the presence of virulence factors associated with bovine colibacillosis using specific oligonucleotide primers for shiga toxins (1 and 2), LT and ST variants. They detected 9.7% and 6.3% of E. coli strains positive for shiga toxin 1 and 2 respectively; 8.3% and 3.9% E. coli strains positive for LT-II and STa enterotoxins respectively as detected by PCR.

Blanco et al. (2003) isolated 432 non-O157 VTEC strains from cattle and tested them for presence of the virulence gene using PCR. The PCR demonstrated that 99 strains (23%) had VT1 gene, 232 (54%) had the VT2 gene and 101 (23%) had both toxin genes. They found a correlation between the O serogroup and the toxin produced. The majority of VTEC strains of serogroups O26, O64, O103, O128, and O136 had the VT1 gene, whereas the majority of the strains of serogroups O2, O4, O77, O91, O113, O116, O162, O163, O171, and O174 had the VT2 gene. The strains having both VT1 and VT2 genes belonged to the serogroups O20, O22, O82, O105, and O126. They also isolated E. coli O157:H7 from 55 (12%) of 471 calves in a foodlot.

Cobbold and co-workers (2004) studied STEC prevalence among dairy, feedlot and cow-calf herd in Washington and STEC strains were detected in 7.4% of fecal samples and farm environmental samples (of 1440 samples) using PCR technique. The prevalence for Stx gene (6%) in samples from feedlots was significantly lower than those from dairy (20%) and range (Stx: 21%) facilities.

Wani et al. (2003) reported the isolation and characterization of STEC serogroups associated with diarrhoea in calves and lambs in India. They subjected 130 bovine and 15 ovine strains to multiplex PCR for detection of Stx1, Stx2 genes along with other virulence factors. STEC strains belonging to different serogroups were detected in 9.73% of calves and 6% of the lambs studied. One of the most important serogroup O157 known to cause certain life-threatening infections in humans was isolated from both bovine and ovine faecal samples.

Shaw et al. (2004) isolated 170 VTEC strains from 47% of dam samples (40 of 86 samples) and 20% of calf samples (130 of 664 samples). During their study 14 calves showed symptoms of diarrhoea. However, VTEC (serogroup O26) was detected in only one diarrhoeic calf.

Wani et al. (2004) serotyped E. coli isolates obtained from an outbreak of bloody diarrhoea in 1-16 week old crossbred calves in an organized dairy farm in Kashmir. Seven out of 10 calves were affected. Serogroup O116 was recovered from 5 calves with diarrhoea. The virulence gene profile revealed Stx1, eaeA (attaching and effacing gene) and hlyA (α-haemolysin) genes.

Eriksson and co-workers (2005) performed prevalence study of VTEC O157 in 371 randomly selected Swedish dairy herds and analysed by immunomagnetic separation and PCR methods. The isolation rate for VTEC O157 was 8.9%.

Irino et al. (2005) reported a total of 202 individual shiga toxin producing E. coli (STEC) isolates among 1471 E. coli colonies screened for Stx. One hundred and forty (69.3%) of them were typable. The great majority of the isolates carried Stx2 (40.6%) or Stx1Stx2 (56.4%) sequences. Only few isolates harboured Stx1 sequence alone (3%).

In an another study Wani et al. (2005) demonstrated association of STEC O4 serotype with an outbreak of diarrhoea in 4-7 week old calves. Six E. coli O4 strains carried eaeA and EHEC hlyA genes and three possessed Stx1 genes.

Zweifel et al. (2005) reported a total of 42 STEC strains from slaughtered healthy cattle in Switzerland. The PCR analysis showed that 18 (43%) strains carried the Stx1 gene, 20 strains (48%) had the Stx2 gene and four (9.5%) strains had both Stx1 and Stx2 genes.

MATERIALS AND METHODS

CHAPTER III

MATERIALS AND METHODS The present study was carried out to ascertain biochemical characters, multiple drug

resistance, colicinogeny and virulence attributes (toxigenic genes) of E. coli obtained mainly as clinical isolates, from faecal samples of diarrhoeic calves. Faecal samples from diarrhoeic calves from organized farms of Anand Agricultural University and associated agricultural universities, unorganized farms and villages in and around Anand and Veterinary College Clinics, were collected during the period from November 2004 to February 2005 to screen for E. coli with toxigenic potential. The details of collection of samples from diarrhoeic calves (up to two months of age) are presented in Table 3.1.

Table 3.1: Sources and number of faecal samples collected Place No. of faecal

samples (A) Organized farms 1. Livestock Research Station, Anand Agricultural University (LRS-

AAU), Anand 2. Livestock Research Station, Junagadh Agricultural University

(LRS-JAU), Junagadh

20

10

(B) Veterinary College Clinics (VC), AAU, AAU 03 (C) Other Unorganised farms (OF), in and around Anand 13

Total 46 3.1 Glassware and chemicals

During course of the study, glassware of Standard brand and chemicals of Analar or Excellar grade were used. Glassware and other materials of routine use were cleaned and sterilized following the standard procedures. 3.2 Experimental material The samples from field were collected (Table 3.1) for study as follows:- 3.2.1 Test strains

Faecal samples from the rectum of diarrhoeic calves (upto two months) were collected using HiCulture (Himedia) sterile transport swabs. The samples were transported to Microbiology laboratory on ice. 3.2.2 Reference strains of E. coli

• Rowe strain, a universal colicin sensitive strain maintained at Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, AAU, Anand. This strain was used for detection of colicinogeny of test strains.

• MTCC 723 (O78:K80:H11, CFA/I+ LT+ ST+) was obtained from Institute of Microbial Technology, Chandigarh. This strain was used as known strain carrying heat labile (LT) and heat stable (ST) enterotoxin genes.

• A known VT1+ VT2+ gene carrying strain maintained at Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, AAU, Anand.

3.3 Isolation and Characterization of E. coli isolates

3.3.1 Media for isolation and biochemical tests

All media required for isolation and biochemical characterization of E. coli were prepared as per Edwards and Ewing (1972).

3.3.2 Isolation of E. coli

Faecal samples were inoculated on MacConkey agar and incubated at 37° C overnight. From each plate two isolated lactose fermenting colonies were inoculated on Eosin methylene blue (EMB) agar medium for preliminary characterization and colonies showing characteristic metallic sheen on EMB agar were picked up and considered as presumptive E. coli. The purified cultures of E. coli were stored in phosphate buffer agar stabs (Annexure-I) for further identification by biochemical tests and other studies. All the isolates were stained by Gram’s Method to determine the purity of the isolates.

3.3.3 Biochemical characterization of E. coli isolates

E. coli isolates were preliminary identified by biochemical tests viz. Indole, Methyl-red, Voges proskaeur and citrate utilization. The isolates were further characterized for their biochemical activity by the following tests viz. carbohydrate fermentation, urea hydrolysis, production of H2S on TSI as per Edwards and Ewing (1972).

3.3.3.1 Indole test: Few drops of xylene were added into two-day-old growth of the isolate in two ml of tryptone water (Annexure-II) and mixed thoroughly to dissolve indole and about 0.2 ml of Kovac’s reagent (Annexure-II) was added. Pink layer of xylene was considered as positive reaction.

3.3.3.2 Methyl- Red (MR) test: Five to six drops of MR reagent (Annexure-II) were added to a five day growth of the isolate in five ml of glucose-phosphate peptone water (GPW). Development of a pink or bright red colour was considered to be positive.

3.3.3.3 Voges-Proskauer (VP) test (Barritt’s method): Three ml of five percent solution of α- naphthol in absolute ethanol and one ml of 40 per cent KHO were added to a five day growth of the isolate in five ml of GPW (Annexure-II). Development of a pink colour in the mixture was indicative of a positive test.

3.3.3.4 Citrate Utilization: Slant of Simmon’s citrate agar (Annexure-I) was inoculated with the culture and incubated at 37° C for 7d. Growth with a development of blue colour of the medium was considered as a positive reaction.

3.3.3.5 Urease Test:

Urea agar (Annexure-I) slants were inoculated and incubated at 37°C and observed upto 7d. A positive reaction was observed by development of pink colour in the slant (Barrow and Feltham, 1993). 3.3.3.6 Hydrogen Sulphide production on TSI agar: The TSI agar (Annexure-I) slants were inoculated heavily by stabbing the butt and streaking the slope of slant. The tubes were incubated at 37°C for 7d and observed daily. Black discolouration of butt and slant indicated an H2S production while, yellow colour of butt and slant indicated acid production (Collee et al., 1996). 3.3.3.7 Carbohydrate Fermentation Tests: Fermentation reactions of seven sugars viz., adonitol, dulcitol, raffinose, rhamnose, salicin, starch and sucrose were studied. One per cent of each sugar in peptone water (Annexure-II) base with Andrade’s indicator was used. For the test isolates grown in peptone water were inoculated into each

sugar medium. Tubes were incubated at 37°C for seven days and readings were recorded after every 24 hours. Production of pink colour was considered as positive reaction.

Proper controls were kept for each of the biochemical tests performed. 3.3.4 Serotyping of E. coli

All the E. coli isolates were sent to National Salmonella and Escherichia Centre, Central Research Institute, Kasauli (Himachal Pradesh) for serotyping.

3.4 Assay for antimicrobial drug resistance of the E. coli isolates

The bacterial isolates were subjected to in vitro antibiotic sensitivity test as per the method of Bauer et al. (1966). The antibiotic discs were obtained from HiMedia Laboratories Ltd. Mumbai. Isolates were tested against commonly used antibiotics viz. Ampicillin (A, 10µg), Amikacin (Ak, 30µg), Ceftiofur (Fur, 0.2µg), Cephalexin (Cp, 30µg), Cephaloridine (Cr, 10µg), Colistin (Cl, 10µg), Ciprofloxacin (Cf, 05µg), Cotrimoxazole (Co, 25µg), Enrofloxacin (Ex, 10µg), Kanamycin (K, 30µg), Nalidixic acid (Na, 30µg), Tetracycline (T, 30µg).

Isolates were grown in Brain Heart Infusion (BHI) broth (Annexure-II) overnight and plates of Mueller Hinton (MH) agar No.2 (Annexure-I) were seeded with about one ml of inoculum. The inoculum was allowed to dry. Antibiotic discs were placed on inoculated agar surface at about two cm apart. The plates were incubated at 37°C overnight and diameter of the zones of inhibition was measured. The measurements were compared with zone size interpretative chart furnished by the manufacturer and the zones were graded as sensitive and resistant.

3.5 Haemolytic activity

Haemolytic activity of E. coli isolates was observed as per the method of Joon and Kaura (1993). Haemolytic activity was determined by spot inoculation of E. coli isolates on five percent sheep blood agar. After 24 hours of incubation at 37°C, observation was made for haemolytic zone around the colonies.

3.6 Detection of colicinogeny

The test for colicinogeny of the strains was performed as described by Barker and Old (1979) with some modifications. 3.6.1 Reference strain

E. coli Rowe strain, universally colicin sensitive strain, maintained at Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, AAU, Anand. 3.6.2 Media Used (Annexure-I)

(a) Bile salt agar (b) Soft overlay agar

3.6.3 Procedure

Isolated colonies of the test strains grown on MacConkey agar was spot inoculated on bile salt agar media (3.6.2a). Such six isolated colonies were inoculated for each test strain at an approximately distance of 1cm, the inoculated media was incubated for 48 hrs at 37°C. The growth obtained was exposed to chloroform vapour, for 30 minutes at room temperature by placing a disc of Whatman filter paper (of approximately 7 cms diameter) soaked in chloroform on the inner surface of petridish lid. To detect colicin production, eighteen hours broth culture of colicin sensitive strain (3.6.1) was mixed in 0.1 ml amount with 5 ml of molten soft overlay agar (3.6.2b) maintained at 45°C in water bath and mixed thoroughly. The mixture was then poured on to the inoculated test medium as described

above. The petridish was left covered on the working table for half an hour to allow the soft overlay agar to solidify. Then the plates were reincubated at 37°C for 24hrs. Colicin producers were detected by observing formation of a circular zone of no growth of the colicin sensitive strain around the colony of test strain.

3.7 Toxigenic Gene Detection 3.7 .1 DNA Extraction

The genomic DNA of E. coli isolates was isolated according to Wilson (1987) with minor modifications.

3.7.1.1 Preparation of broth culture of E. coli

The culture was prepared by inoculating the isolate in Luria broth (HiMedia, Mumbai) and incubating at 37°C for 24 h in a shaker water bath.

3.7.1.2 Preparation of material for nucleic acid extraction

About 50 ml of broth culture was centrifuged at 10,000 rpm for 10 min at 5°C. The supernatant was discarded and the pellet was used for extraction of nucleic acid extraction.

3.7.1.3 Following solutions were used for extraction

i. Tris-EDTA (pH 8.0) 10 mM Tris-HCl 1 mM EDTA ii. SDS (10% w/v) iii. Proteinase K solution (20mg/ml, w/v) iv. 5 M Sodium chloride

v. CTAB (Hexadecyl trimethyl ammonium bromide, 10% solution in 0.7M NaCl)

vi. Saturated phenol (pH 8.0) vii. Chloroform viii. Isoamyl alcohol ix. 7.5 M Ammonium acetate

x. Chilled absolute ethanol 3.7.1.4 Isolation of genomic DNA by Proteinase-K-SDS method

• Pellet containing bacterial cells was suspended in 2 ml Tris-EDTA (pH-8.0), 250 µl SDS (10% w/v) and10µl of proteinase K solution (20mg/ml, w/v) and incubated for 1 h at 37oC.

• Subsequently, 500 µl of 5M NaCl followed by 100 µl CTAB (10% solution in 0.7 M NaCl) was added and incubated in water bath for 10 min at 65oC.

• The solution was spun at 8,000 rpm for 10 min after mixing with equal volume of chloroform: isoamyl alcohol (24:1) and upper phase was transferred to clean microfuge tube.

• Equal volume of phenol : chloroform : isoamyl alcohol (25:24:1) was added, mixed well by inverting, spun for 10 min at 10,000 rpm and upper aqueous phase was transferred again to a clean microfuge tube.

• In the collected supernant, the DNA was precipitated with one-tenth volumes of ammonium acetate (7.5M) and double the volume with chilled absolute ethanol.

• Tube was centrifuged for 10 min at 11,000 rpm and ethanol was discarded.

• The pellet was washed in 70% ethanol and again spun for 5 min at 11,000 rpm.

• Ethanol was discarded and pellet was dried.

• DNA was resuspended in 200 µl sterile distilled water and kept in water bath at 65oC for one hour and stored at -20oC till use.

3.7.1.5 Quality checking and quantitation of DNA

Quality and purity of DNA were checked by agarose gel electrophoresis. 0.8 percent agarose in 0.5X TBE (PH 8.0) buffer (Sambrook et al., 1989) was used for submarine gel electrophoresis. Ethidium bromide (1 %) was added @ 5µl /100ml. The wells were charged with 5µl of DNA preparations mixed with 1X BPB dye. Electrophoresis was carried out at voltage 5V/cm for 60 min at room temperature. DNA was visualized under UV transilluminator.

Quantity of DNA was calculated by spectrophotometric method. OD at 260 and 280 were taken in UV spectrophotometer with distilled water as reference.

Concentration (µg/ml) = OD at 260 x dilution factor x 50

Where 50 is concentration of dsDNA expressed in 1 µg/ ml at OD of 1.

3.7.2 PCR reaction for LT, ST, VT1 and VT2 genes

The following components were used in PCR mixture

I. 2X PCR Mastermix (Fermentas, Life Sciences):

• 4mM MgCl2

• 0.4mM of each dNTPs (dATP, dCTP, dGTP, dTTP)

• 0.05units/ml of Taq DNA polymerase

• 150 mM tris-HCl PCR buffer

II Primers (Table 3.2)

III DNA samples were diluted to a final concentration of 30ng/µl and 1 µl of this preparation was used as template for PCR.

Table 3.2: Details of primers used for PCR reaction

Primers Sequences

(5’- 3’)

Target Gene

Size of amplified

product(bp)

Reference

LT1(F)

LT2(R)

AGC AGG TTT CCC ACC GGA TCACCA GTG CTC AGA TTC TGG GTC TC

LT

132

Nishikawa et al.,(2002)

microST1

(F)

microST2

(R)

TTT ATT TCT GTA TTG TCT TT

ATT ACA ACA CAG TTC ACA G

ST

171

Nishikawa et al.,(2002)

VT1a(F)

VT1b(R)

GAA GAG TCC GTG GGA TTA CG

AGC GAT GCA GCT ATT AAT AA

VT1

130

Pollard et al.,(1990)

VT1(F)

VT2(R)

TTT ACG ATA GAC TTC TCG AC CAC ATA TAA ATT ATT TCG CTC

VT2

228

Nishikawa et al.,(2002)

3.7.2.1 PCR conditions

PCR was carried out in a final reaction volume of 15 µl using 0.2 ml thin wall PCR tube. A master mix for minimum of 10 samples was prepared and aliquoted in 14 µl quantities in each PCR tube. One µl sample of DNA was added in each tube to make the final volume of 15 µl as in Table 3.3.

PCR tubes containing the mixture were tapped gently and quickly spun at 10,000 rpm for few seconds. The PCR tubes with all the components were transferred to thermal cycler (Eppendorf, Germany).

Table 3.3: Composition of master mix for PCR reaction

Components Quantity Final

concentration 2X PCR Mastermix 7.50 µl 1X

DNase free water 5.50 µl --

0.50 µl 10 pmole/µl Primers Forward

Reverse 0.50 µl 10 pmole/µ

DNA template 1.0 µl --

Total 15.0 µl -- 3.7.2.2 PCR protocol The PCR protocol was as described in Table 3.4 for the primers used.

Table 3.4: Steps and conditions of thermocycling for PCR

Sr. No. Steps

Temp. /Time

(LT1and LT2

primer)

Temp. /Time (microST1 and

microST2primer)

Temp. /Time

(VT1 and VT2

primer)

Temp. /Time(VT1a and

VT1b primer)

Temperature - - - 94 °C 1 Initial denaturation Time - - - 5 min

Temperature 94 °C 94 °C 94 °C 94 °C 2 Denaturation Time 30 sec 30 sec 30 sec 2 min Temperature 45 °C 45 °C 45 °C 55 °C 3 Annealing Time 1 min 1 min 1 min 1 min Temperature 72 °C 72 °C 72 °C 72 °C 4 Extension Time 1.5 min 1.5 min 1.5 min 1 min Temperature - - - 72 °C 5 Final

Extension Time - - - 7 min 6. Cycles 25 cycles

25 cycles

25 cycles 35 cycles

3.7.3 Agarose gel electrophoresis

The following reagents were used for agarose gel electrophoresis i. Agarose

Agarose 2.00 g TBE (0.5X) 100 ml Ethidium bromide (1%) 5 µl

ii. Tris Borate EDTA (TBE) buffer, pH 8.3 (5X)

Tris HCI 0.445 M Boric Acid 0.445 M EDTA 10 mM

iii. Ethidium bromide (1%) Ethidium bromide 10 mg

Distilled water 1.0 ml

To confirm the targeted PCR amplification, 5 µl of PCR product from each tube was mixed with 1µl of 6X gel loading buffer from each tube and electrophoresed on 2.0 per cent agarose gel along with 50bp DNA Ladder (GeneRuler- Fermentas) and stained with ethidium bromide (1 per cent solution at the rate of 5 µl/100 ml) at constant 80 V for 30 minutes in 0.5X TBE buffer. The amplified product was visualized as a single compact band of expected size under UV light and documented by gel documentation system. 3.8 Equipments Some of the important equipments used for the present study were as below. i) Camera CT-1, Super Cosina, Japan ii) Centrifuge Universal 30RF Hettich Zentrifugen, Germany iii) Gel documentation system Syn Gene, Gene Genius Bio

Imaging system, UK iv) Micropipetts Finnpipette, Thermo Electron Corporation, USA

v) Power pack Power pack 1000, BioRad, USA, ATTO, Japan

vi) Spectrophotometer UV/VIS Spectrophotometer Unicam, UK vii) Submarine gel electrophoresis apparatus Bangalore Genie, India viii) Thermal cycler Eppendorf thermal cycler, Germany ix) Ultra centrifuge LE-80 K, Beckman x) Weighing balance BP 210 D, Sartorius, Germany

RESULTS

CHAPTER IV

RESULTS

This study was carried out to isolate and ascertain biochemical characters, multiple drug resistance, colicinogeny and virulence attributes (toxigenic genes) of E. coli from diarrhoeic calf faecal samples from organized farms viz. Livestock Research Station, Anand Agricultural University, Anand (LRS-AAU) and Livestock Research Station, Junagadh Agricultural University (LRS-JAU), other unorganized farms and villages (OF) in and around Anand and Veterinary College Clinics (VC), AAU.

During present study a total 46 faecal samples from the rectum of diarrhoeic calves (up to two months) were collected and processed for isolation of E. coli (Table 4.1). 4.1 Isolation, cultural identification and serotypes of E. coli isolates 4.1.1 Isolation and identification of E. coli isolates

For isolation of E. coli, MacConkey agar (MCA) and eosin methylene blue agar (EMB) were used as differential and selective plating media. Afore mentioned 46 faecal samples were initially screened on MCA. Pink coloured colonies on MCA plates (Plate I) were considered to be lactose fermenting isolates. Two such pink colonies from each MCA plate were picked up and transferred to EMB agar plate. Colonies with greenish metallic sheen (Plate II) on EMB agar plates were tentatively considered to be of E. coli. In all 91 E. coli isolates obtained from the faecal samples were used for further study (Table 4.1). 4.1.2 Serotypes of E. coli isolates

All the 91 E. coli isolates were sent to National Salmonella and Escherichia Centre Kasauli (Himachal Pradesh) for serotyping. All the isolates of E. coli were typed for ‘O’ antigen. Table 4.1 shows serotypes of E. coli isolates.

Out of 91 E. coli isolates 82 isolates belonged to 36 different ‘O’ serogroups while six isolates were untypable and three were rough isolates. Various serogroups were then grouped location wise to study the prevalence of different serogroups in different locations from where samples were collected. 4.1.3 Haemolytic activity of E. coli isolates Out of 91 E. coli isolates tested for haemolytic activity, 4 (4.39 per cent) produced clear zone of haemolysis around the colonies (Table 4.1). Table 4.1 E. coli isolates obtained from diarrhoeic calf faecal samples

Sr. No. Isolate No. ‘O’ Serogroups Haemolysis

LRS, Anand Agricultural University 1. AU-1 O11 - 2. AU-2 O18 - 3. AU-3 O22 - 4. AU-4 O22 - 5. AU-5 O24 - 6. AU-6 O26 - 7. AU-7 O26 - 8. AU-8 O32 - 9. AU-9 O32 -

10. AU-10 O55 - 11. AU-11 O55 -

Sr. No. Isolate No. ‘O’ Serogroups Haemolysis 12. AU-12 O62 - 13. AU-13 O62 - 14. AU-14 O62 - 15. AU-15 O86 - 16. AU-16 O86 - 17. AU-17 O86 - 18. AU-18 O86 - 19. AU-19 O86 - 20. AU-20 O86 - 21. AU-21 O86 - 22. AU-22 O110 - 23. AU-23 O110 - 24. AU-24 O126 - 25. AU-25 O128 - 26. AU-26 O128 - 27. AU-27 O128 - 28. AU-28 O128 - 29. AU-29 O131 - 30. AU-30 O157 - 31. AU-31 O157 - 32. AU-32 O171 - 33. AU-33 O171 - 34. AU-34 O171 - 35. AU-35 O171 - 36. AU-36 O172 - 37. AU-37 O172 - 38. AU-38 O172 - 39. AU-39 O172 - 40. AU-40 UT -

Veterinary College Clinics, AAU 41. VC-1 O2 - 42. VC-2 O2 - 43. VC-3 O12 - 44. VC-4 O97 - 45. VC-5 O167 - 46. VC-6 O167 -

Other Farms 47. OF-1 O5 - 48. OF-2 O9 + 49. OF-3 O11 - 50. OF-4 O22 - 51. OF-552 O66 - 52. OF-6 O78 - 53. OF-7 O86 - 54. OF-8 O98 + 55. OF-9 O98 + 56. OF-10 O100 + 57. OF-11 O101 -

Sr. No. Isolate No. ‘O’ Serogroups Haemolysis 58. OF-12 O108 - 59. OF-13 O126 - 60. OF-14 O131 - 61. OF-15 O131 - 62. OF-16 O157 - 63. OF-17 O161 - 64. OF-18 O162 - 65. OF-19 O171 - 66. OF-20 O172 - 67. OF-21 Rough - 68. OF-22 Rough - 69. OF-23 Rough - 70. OF-24 UT - 71. OF-25 UT - 72. OF-26 UT - 73. OF-27 UT -

LRS, Junagadh Agricultural University 74. JU-1 O3 - 75. JU-2 O15 - 76. JU-3 O18 - 77. JU-4 O18 - 78. JU-5 O18 - 79. JU-6 O18 - 80. JU-7 O18 - 81. JU-8 O22 - 82. JU-9 O68 - 83. JU-10 O76 - 84. JU-11 O109 - 85. JU-12 O109 - 86. JU-13 O128 - 87. JU-14 O128 - 88. JU-15 O131 - 89. JU-16 O168 - 90. JU-17 O171 - 91. JU-18 UT -

4.2 Biochemical characterization of E. coli isolates

The E. coli isolates were stained by Gram’s method to check for the purity and then characterized by biochemical tests viz. IMViC pattern (indole production, Methyl Red (MR) test, Voges-Proskauer (V.P.) test, citrate utilization on Simmon’s citrate medium), urea hydrolysis, production of H2S on TSI agar, carbohydrate fermentation using seven sugars as per the method described by Edwards and Ewing (1972).

On the basis of Gram’s staining the isolates were found to be Gram negative bacilli. The isolates exhibited similar IMViC pattern of + + - - except for three isolates (AU-24, AU-26 and JU-3) which utilized citrate on Simmon’s citrate medium (Plate III).

Ability of the isolates to utilize various sugars were carried out by determining the fermentation activity for the following sugars viz. adonitol (Ad), dulcitol (D), raffinose (Ra), rhamnose (Rh), salicin (Sa), starch (St) and sucrose (Su) (Table 4.2). In decreasing

frequency dulcitol was fermented by thirty five (38.46%) of isolates studied, raffinose by thirty one (34.06%) isolates, rhamnose by thirty (32.96%) isolates, sucrose by twenty one (23.07%) isolates, salicin by fourteen (15.38%) isolates, starch by four (4.39%) isolates and adonitol was fermented by three (3.29%) isolates. Table 4.2 Sugar fermentation activity of E. coli isolates

Sr No. Isolate No. Serogroups Ad D Ra Rh Sa St Su1. AU-1 O11 - - - - - - - 2. AU-2 O18 - - + - - - - 3. AU-3 O22 - - - - - - - 4. AU-4 O22 - - - + - - - 5. AU-5 O24 - - - - - - - 6. AU-6 O26 - - - - - - - 7. AU-7 O26 - - - - - - - 8. AU-8 O32 - - - - - - - 9. AU-9 O32 - - - - - - -

10. AU-10 O55 - + + - - - + 11. AU-11 O55 - - + - + - - 12. AU-12 O62 - - - - - - - 13. AU-13 O62 - - - - - - - 14. AU-14 O62 - - - + - - - 15. AU-15 O86 - + + + - - - 16. AU-16 O86 - + + + - - - 17. AU-17 O86 - + + + - - - 18. AU-18 O86 - + + + - - - 19. AU-19 O86 - - - - - - + 20. AU-20 O86 - - - - - - - 21. AU-21 O86 - - - - - - - 22. AU-22 O110 - - - - - - - 23. AU-23 O110 - - - - - - - 24. AU-24 O126 - - - - - - - 25. AU-25 O128 - + - - - - - 26. AU-26 O128 - - - - - - - 27. AU-27 O128 - - - - - - - 28. AU-28 O128 - - - - - - - 29. AU-29 O131 - + - - - - - 30. AU-30 O157 - + - + - - - 31. AU-31 O157 - + - + - - - 32. AU-32 O171 - + - - - - - 33. AU-33 O171 - + - - - - - 34. AU-34 O171 - - + - + - + 35. AU-35 O171 - - + + - - + 36. AU-36 O172 - - - + - - - 37. AU-37 O172 - - - - - - - 38. AU-38 O172 - - - - - - - 39. AU-39 O172 - - - - - - - 40. AU-40 UT - - - - - - - 41. VC-1 O2 - + + - + - + 42. VC-2 O2 - + - - + - + 43. VC-3 O12 - - - + - - -

Sr No. Isolate No. Serogroups Ad D Ra Rh Sa St Su44. VC-4 O97 - + + + - - - 45. VC-5 O167 - - - - - - - 46. VC-6 O167 - + - - - - + 47. OF-1 O5 - + + + - - - 48. OF-2 O9 - - - - - - - 49. OF-3 O11 - + - - + - - 50. OF-4 O22 - + - + - - - 51. OF-552 O66 - + - + - - - 52. OF-6 O78 - - + - - + + 53. OF-7 O86 - - + + - - - 54. OF-8 O98 - + + - - - - 55. OF-9 O98 - + + - - - - 56. OF-10 O100 - - - + + - - 57. OF-11 O101 - - - + - - + 58. OF-12 O108 - - - + - - - 59. OF-13 O126 - + - + - - - 60. OF-14 O131 - + - + - - - 61. OF-15 O131 - + + + - - - 62. OF-16 O157 - - - + - - - 63. OF-17 O161 - - - + - - + 64. OF-18 O162 + - - - + - - 65. OF-19 O171 - - - + - - - 66. OF-20 O172 - - - - + - - 67. OF-21 Rough - - - + - - - 68. OF-22 Rough - - - + + - - 69. OF-23 Rough - - - + - - - 70. OF-24 UT - - - - + - - 71. OF-25 UT - - - - + - - 72. OF-26 UT - + - + - - - 73. OF-27 UT + - - + - - - 74. JU-1 O3 - - + - - + + 75. JU-2 O15 - - + - - - + 76. JU-3 O18 - + + - + - + 77. JU-4 O18 - + - - + - + 78. JU-5 O18 - + + - + - + 79. JU-6 O18 - + + - - - + 80. JU-7 O18 - + + - - - - 81. JU-8 O22 - + + - - + + 82. JU-9 O68 - + - - - - + 83. JU-10 O76 - + + - - + - 84. JU-11 O109 - - + - - - - 85. JU-12 O109 - - + - - - - 86. JU-13 O128 - - - - - - - 87. JU-14 O128 - - + - - - + 88. JU-15 O131 - + + - - - + 89. JU-16 O168 + + - - - - + 90. JU-17 O171 - - + - - - - 91. JU-18 UT - - + - - - -

4.3 Unusual biochemical characters

Atypical biochemical behaviour (Table 4.3) was observed by three isolates (3.29 per cent) which showed citrate utilization. Only one isolate (1.09 per cent) produced H2S on TSI agar medium while all the isolates produced acidic reaction (Plate IV). Out of 91 E. coli isolates twenty isolates (21.97 per cent) revealed urea hydrolysis (Plate V). Thirty-one (34.06%) E. coli isolates had raffinose fermentation activity while only three isolates (3.29%) showed adonitol fermentation activity.

4.3 Unusual biochemical characters of E. coli isolates Sr No. Isolate No. Serogroup Urease

test H2S production

On TSI Citrate

Test Ad Ra

1. AU-1 O11 - - - - - 2. AU-2 O18 + - - - + 3. AU-3 O22 + - - - - 4. AU-4 O22 - - - - - 5. AU-5 O24 - - - - - 6. AU-6 O26 - - - - - 7. AU-7 O26 - - - - - 8. AU-8 O32 + - - - - 9. AU-9 O32 - - - - -

10. AU-10 O55 - - - - + 11. AU-11 O55 - - - - + 12. AU-12 O62 + - - - - 13. AU-13 O62 - - - - - 14. AU-14 O62 + - - - - 15. AU-15 O86 + - - - + 16. AU-16 O86 - - - - + 17. AU-17 O86 - - - - + 18. AU-18 O86 + - - - + 19. AU-19 O86 - - - - - 20. AU-20 O86 - - - - - 21. AU-21 O86 - - - - - 22. AU-22 O110 - - - - - 23. AU-23 O110 - - - - - 24. AU-24 O126 - - + - - 25. AU-25 O128 - - - - - 26. AU-26 O128 + - + - - 27. AU-27 O128 + - - - - 28. AU-28 O128 - - - - - 29. AU-29 O131 + - - - - 30. AU-30 O157 + - - - - 31. AU-31 O157 - - - - - 32. AU-32 O171 - - - - - 33. AU-33 O171 - - - - - 34. AU-34 O171 - - - - + 35. AU-35 O171 - - - - + 36. AU-36 O172 - - - - - 37. AU-37 O172 - - - - -

Sr No. Isolate No. Serogroup Ureasetest

H2S productionOn TSI

Citrate Test

Ad Ra

38. AU-38 O172 + - - - - 39. AU-39 O172 - - - - - 40. AU-40 UT + - - - - 41. VC-1 O2 - - - - + 42. VC-2 O2 - - - - - 43. VC-3 O12 + - - - - 44. VC-4 O97 - - - - + 45. VC-5 O167 - - - - - 46. VC-6 O167 - - - - - 47. OF-1 O5 + - - - + 48. OF-2 O9 + - - - - 49. OF-3 O11 - - - - - 50. OF-4 O22 - - - - - 51. OF-5 O66 + - - - - 52. OF-6 O78 - - - - + 53. OF-7 O86 - - - - + 54. OF-8 O98 - - - - + 55. OF-9 O98 - - - - + 56. OF-10 O100 - - - - - 57. OF-11 O101 - - - - - 58. OF-12 O108 - - - - - 59. OF-13 O126 - - - - - 60. OF-14 O131 - - - - - 61. OF-15 O131 - - - - + 62. OF-16 O157 - - - - - 63. OF-17 O161 - - - - - 64. OF-18 O162 - - - + - 65. OF-19 O171 - - - - - 66. OF-20 O172 - - - - - 67. OF-21 Rough - - - - - 68. OF-22 Rough - - - - - 69. OF-23 Rough - - - - - 70. OF-24 UT + + - - - 71. OF-25 UT + - - - - 72. OF-26 UT - - - - - 73. OF-27 UT + - - + -

74. JU-1 O3 - - - - + 75. JU-2 O15 - - - - + 76. JU-3 O18 - - + - + 77. JU-4 O18 - - - - - 78. JU-5 O18 - - - - + 79. JU-6 O18 - - - - + 80. JU-7 O18 - - - - + 81. JU-8 O22 - - - - + 82. JU-9 O68 - - - - - 83. JU-10 O76 - - - - +

Sr No. Isolate No. Serogroup Ureasetest

H2S productionOn TSI

Citrate Test

Ad Ra

84. JU-11 O109 - - - - + 85. JU-12 O109 - - - - + 86. JU-13 O128 - - - - - 87. JU-14 O128 - - - - + 88. JU-15 O131 - - - - + 89. JU-16 O168 - - - + - 90. JU-17 O171 + - - - + 91. JU-18 UT - - - - +

4.4 Biotypes of E. coli isolates

All the 91 E. coli isolates were studied for the sugar fermentation activity for the following sugars viz. adonitol, dulcitol, raffinose, rhamnose, salicin, starch and sucrose. Sixty seven isolates showed the ability to utilize one or more sugar while 24 isolates failed to utilize any sugar (Table 4.2).

Thus, 67 E. coli isolates were biotyped based on carbohydrate fermentation patterns with six sugars viz. dulcitol, raffinose, rhamnose, salicin, starch and sucrose into 24 different combinations (Table 4.4). The most commonly occurring biotypes were Biotype III (10 isolates), XV (7 isolates), VII (6), II (5), the rest biotypes were found to have less than five isolates. Table 4.4 Biotypes of E. coli isolates on the basis of fermentation reactions of dulcitol,

raffinose, rhamnose, salicin, starch and sucrose Biotype

No. Isolates Total no.

of isolates

Positive sugars

I AU-25, AU-29, AU-32, AU-33, 4 Dulcitol II AU-2, JU-11, JU-12, JU-17, JU-18 5 Raffinose III AU-4, AU-14, AU-36, VC-3, OF-12,

OF-16, OF-19, OF-21, OF-23, OF-27 10 Rhamnose

IV OF-18, OF-20, OF-24, OF-25 4 Salicin V AU-19 1 Sucrose VI OF-8, OF-9, JU-7 3 Dulcitol and raffinose VII AU-30, OF-4, OF-5, OF-13, OF-14, OF-

26 6 Dulcitol and rhamnose

VIII OF-3 1 Dulcitol and salicin IX VC-6, JU-9, JU-16 3 Dulcitol and sucrose X OF-7 1 Raffinose and rhamnose XI AU-11 1 Raffinose and salicin XII JU-2, JU-14 2 Raffinose and sucrose XIII OF-10, OF-22 2 Rhamnose and salicin XIV OF-11, OF-17 2 Rhamnose and sucrose XV AU-15, AU-16, AU-17, AU-18, VC-4,

OF-1, OF-15 7 Dulcitol,raffinose and

rhamnose XVI JU-10 1 Dulcitol, raffinose and

starch XVII AU-10, JU-6, JU-15 3 Dulcitol, raffinose and

sucrose XVIII AU-35 1 Dulcitol, rhamnose and

sucrose

Biotype No.

Isolates Total no. of

isolates

Positive sugars

XIX VC-2, JU-4 2 Dulcitol, salicin and sucrose

XX AU-35 1 Raffinose, rhamnose and sucrose

XXI OF-6, JU-1 2 Raffinose, starch and sucrose

XXII AU-34 1 Raffinose, salicin and sucrose

XXIII VC-1, JU-3, JU-5 3 Dulcitol, raffinose, salicin and sucrose

XXIV JU-8 1 Dulcitol, raffinose, starch and sucrose

It was observed that there were different serogroups under same biotype as shown in Table 4.5 Table 4.5 Distribution of E. coli serogroups within biotype

Sr. No.

Biotype No. Serogroups detected

1. I (04) O128(01), O131(01), O171 (02) 2. II(05) O18(01), O109 (02), O171 (01), UT (01) 3. III (10) O12 (01), O22 (01), O62 (01), O108 (01), O157 (01), O171 (01),

O172 (01), Rough (02), UT (01) 4. IV (04) O162 (01), O172 (01), UT (02) 5. V (01) O86 (01) 6. VI (03) O98 (02), O18 (01) 7. VII (06) O22 (01), O66 (01), O126 (01), O131 (01), O157 (01), UT (01) 8. VIII (01) O11 (01) 9. IX (03) O167 (01), O68 (01), O168 (01)

10. X (01) O86 (01) 11. XI (01) O55 (01) 12. XII (02) O15 (01), O128 (01) 13. XIII (02) O100 (01), Rough (01) 14. XIV (02) O101 (01), O161 (01) 15. XV (07) O86 (04), O97 (01), O5 (01), O131 (01) 16. XVI (01) O76 (01) 17. XVII (03) O18 (01), O55 (01), O131 (01) 18. XVIII (01) O157 (01) 19. XIX (02) O2 (01), O18 (01) 20. XX (01) O171 (01) 21. XXI (02) O3 (01), O78 (01) 22. XXII (01) O171 (01) 23. XXIII (03) O2 (01), O18 (02) 24. XXIV (01) O22 (01)

Figures in parenthesis indicate number of isolates

4.5 Assay for antimicrobial drug resistance of E. coli isolates In vitro antibiotic resistance pattern of the isolates were determined by disc diffusion

method of Bauer et al. (1966). The E. coli isolates were tested against commonly used antibiotics viz. ampicillin (A), amikacin (Ak), ceftiofur (Fur), cephalexin (Cp), cephaloridine (Cr), colistin (Cl), ciprofloxacin (Cf), co-trimoxazole (Co), enrofloxacin (Ex), kanamycin (K), nalidixic acid (Na), tetracycline (T). 4.5.1 Number and per cent of E. coli isolates resistant to antimicrobial drugs The number and per cent of E. coli isolates resistant to various antimicrobial drugs are as shown in Table 4.6 as observed by disc diffusion method (Plate VIa and VIb). Among the 91 isolates of E. coli tested for resistance against various antibiotics very high per cent of isolates were found to be resistant to the following antibiotics kanamycin (95.6%), cephalexin (92. 3%), amikacin (91.2%), cephaloridine (87.9%) and enrofloxacin (84.61%). High per cent of isolates (72.52%) were resistant to ampicillin while moderately high per cent of isolates were found to be resistant to antibiotics tetracycline (56.04%), ceftiofur (37.3%) and ciprofloxacin (28.57%). However, among the isolates tested lesser per cent of isolates were found to be resistant to the antibiotics co-trimoxazole (15.38%) and colistin (12.08%) while only 8.79% isolates were resistant to nalidixic acid (Figure 4.1). Table 4.6 Number and per cent of E. coli isolates resistant to antimicrobial drugs

Antibiotic No. of isolates resistant Per cent of isolates resistant Ak 83 91.2 A 66 72.52

Fur 34 37.36 Cp 84 92.30 Cr 80 87.91 Cf 26 28.57 Cl 11 12.08 Co 14 15.38 Ex 77 84.61 K 87 95.60 Na 08 8.79 T 51 56.04

• Ak-Amikacin; A- Ampicillin; Fur- Ceftiofur; Cp- Cephalexin; Cr- Cephaloridine;

Cf- Ciprofloxacin; Cl- Colistin; Co- Co-trimoxazole; Ex- Enrofloxacin; K- Kanamycin; Na- Nalidixic acid; T- Tetracycline

4.5.2 Location wise prevalence of antimicrobial drug resistance of the E. coli isolates The E. coli isolates were primarily obtained from 4 locations viz. Livestock Research Station, Anand Agricultural University (LRS-AAU), Livestock Research Station-Junagadh Agricultural University (LRS-JAU), Veterinary College Clinics, Anand (VC) and unorganized farms in and around Anand city (OF). On analysis of the prevalence of antibiotic resistance among the isolates (Table 4.7) collected from various locations, it was found that 80 to 100 % of the E. coli isolates studied revealed resistance against amikacin, cephalexin, cephaloridine and kanamycin except for isolates from JAU which showed 66.66% resistance to cephalorodine. Higher per cent of the E. coli isolates from all the locations showed resistance against antibiotics enrofloxacin (66.6-92.5%) and ampicillin (55.5-80%).

Moderate to very high resistance (51.8-100%) to tetracycline was found among the isolates obtained from AAU, VC, OF while the isolates from JAU showed lower resistance (22.2%) against tetracycline. In general the isolates revealed lower to very low resistance to the following antibiotics viz. nalidixic acid (5.5-16.6%), colistin (5.5- 22.5%) and co-trimoxazole (10 to 25.9%) and ciprofloxacin (22.5-33.3%) obtained from the various locations AAU, VC, OF and JAU, except for the isolates of VC which were more resistant to ciprofloxacin (50%). However, all the isolates from unorganized farms in and around Anand city were sensitive to colistin (Figure 4.2).

4.7 Location wise prevalence of antimicrobial drug resistance among E. coli isolates Per cent of resistant isolates to following antibiotics Place No. of isolatesAk A Fur Cp Cr Cf Cl Co Ex K Na T

LRS, AAU 40 80 80 37.5 100 100 22.5 22.5 10 90 95 7.5 67.5Veterinary College Clinics 06 100 66.6 83.3 100 100 50 16.6 16.6 66.6 100 16.6 100 Other Farms 27 100 74.1 25.9 85.1 81.4 29.6 00 25.9 92.5 96.2 11.1 51.8LRS, JAU 18 100 55.5 38.8 83.3 66.6 33.3 5.5 11.1 66.6 94.4 5.5 22.2

• Ak-Amikacin; A- Ampicillin; Fur- Ceftiofur; Cp- Cephalexin; Cr- Cephaloridine; Cf- Ciprofloxacin; Cl- Colistin; Co- Co-trimoxazole;

Ex- Enrofloxacin; K- Kanamycin; Na- Nalidixic acid; T- Tetracycline

4.5.3 Multiple antimicrobial drug resistance pattern of the E. coli isolates Multiple drug resistance was demonstrated for most of the isolates. All but one strain were resistant to at least three drugs and 83 were resistant to five or more drugs (Table 4.8). Following three patterns of drug resistance were revealed amongst the E. coli isolates.

1. Resistance to cephalexin, cephaloridine and enrofloxacin was observed for 68 (74.72%) isolates.

2. Resistance to cephalexin, cephaloridine, enrofloxacin, amikacin and kanamycin was observed for 67 (73.62%) isolates and

3. Resistance to ampicillin, cephalexin, cephaloridine and kanamycin was shown by 60 (65.93%) isolates.

Table 4.8 Antimicrobial drug resistance pattern and Colicinogeny of E. coli isolates

Sr No. Isolate No. Drug Resistance Pattern Colicinogeny 1. AU-1 AkAFur CpCrExKNa - 2. AU-2 AkACpCrExKT + 3. AU-3 AkACpCrCfClCoExKT - 4. AU-4 AkACpCrCfClCoExKT - 5. AU-5 AkCpCrKT + 6. AU-6 AkCpCrExKT + 7. AU-7 AkCpCrExKT + 8. AU-8 AkFurCpCrEx - 9. AU-9 AkFurCpCrEx -

10. AU-10 AkACpCrKT - 11. AU-11 AkACpCrKT - 12. AU-12 AkACpCrCfExKT + 13. AU-13 AkACpCrCfExKT ++ 14. AU-14 AkACpCrCfExKT ++ 15. AU-15 AkAFurCpCrExKT - 16. AU-16 AkAFurCpCrExKT - 17. AU-17 AkAFurCpCrExKT - 18. AU-18 AkAFurCpCrExKT - 19. AU-19 AkAFurCpCrExKT - 20. AU-20 AkAFurCpCrExKT - 21. AU-21 AkAFurCpCrExKT - 22. AU-22 AkFurCpCrCfExKT + 23. AU-23 AkFurCpCrCfExKT + 24. AU-24 AkAFurCpCrExKT - 25. AU-25 AkACpCrClExKT - 26. AU-26 AkACpCrClExKT + 27. AU-27 AkACpCrClExKT + 28. AU-28 AkACpCrClExKT - 29. AU-29 AkCpCrKT + 30. AU-30 AkAFurCpCrCfClCoExKNa - 31. AU-31 AkAFurCpCrCfClCoExKNa - 32. AU-32 ACpCrExK + 33. AU-33 ACpCrExK + 34. AU-34 ACpCrExK +++

35. AU-35 ACpCrExK -

Sr No. Isolate No. Drug Resistance Pattern Colicinogeny 36. AU-36 ACpCrExK - 37. AU-37 ACpCrExK - 38. AU-38 ACpCrExK + 39. AU-39 ACpCrExK + 40. AU-40 AkACpCrClExKT - 41. VC-1 AkFurCpCrKT - 42. VC-2 AkFurCpCrKT - 43. VC-3 AkAFurCpCrCfClCoExKNaT - 44. VC-4 AkACpCrExKT - 45. VC-5 AkAFurCpCrCfExKT + 46. VC-6 AkAFurCpCrCfExKT + 47. OF-1 AkCpCrExK - 48. OF-2 AkAFurCpCrCfExKT +++ 49. OF-3 AkAFurCpCrKT - 50. OF-4 AkAFurCpCrCfKT - 51. OF-5 AkACrCoExKT - 52. OF-6 AkAFurCpCrCfExKT - 53. OF-7 AkACpCrCfExKT - 54. OF-8 AkACpCrCfExK +++ 55. OF-9 AkACpCrCfExK +++ 56. OF-10 AkACpCrCoExKT ++ 57. OF-11 AkACpCrCoExKT - 58. OF-12 AkFurCpCrCfExKNa - 59. OF-13 AkACrCoExKT - 60. OF-14 AkACpCrExKNa - 61. OF-15 AkACpCrExKNa - 63. OF-17 AkACpCrExKT - 64. OF-18 AkCoExK - 65. OF-19 AkACpExK - 66. OF-20 AkFurExKT - 67. OF-21 AkACpExK - 68. OF-22 AkACpExK - 69. OF-23 AkAFurCpCrCfExKT + 70. OF-24 AkACpCrCoExKT ++ 71. OF-25 AkACpCrCoExKT ++ 72. OF-26 AkCpCrExK - 73. OF-27 AkCpCrExK - 74. JU-1 AkAFurCpCrClCoEx - 75. JU-2 AkACpCrK - 76. JU-3 AkAFurCpCrCfExK - 77. JU-4 AkAFurCpCrCfExK - 78. JU-5 AkAFurCpCrCfExK - 79. JU-6 AkAFurCpCrCfExK - 80. JU-7 AkAFurCpCrCfExK - 81. JU-8 AkCpK ++ 82. JU-9 AkACpCrCoKNaT - 83. JU-10 AkK ++ 84. JU-11 AkExK -

Sr No. Isolate No. Drug Resistance Pattern Colicinogeny 85. JU-12 AkExK - 86. JU-13 AkCpK ++ 87. JU-14 AkCpK - 88. JU-15 AkCpCrExKT - 89. JU-16 AkACpCrExKT + 90. JU-17 AkCpCrExK + 91. JU-18 AkAFurCpCrCfExKT +

• Note: +++ : Highly Positive (25 mm or more), ++ : 18- 25mm, +: less than 18mm

4.6 Assay for Colicinogeny

Colicinogeny is one of the important virulence associated factor and it is exhibited often by multiple drug resistant organism. Colicinogeny was determined as per method described by Barker and Old (1979). The result of positive colicin producing strain is shown in Plate VII.

Out of 91 E. coli isolates tested for colicinogeny, 32 (35.16%) isolates were colicinogenic (Table 4.8). 4.7 Toxigenic Gene Detection

All the E. coli isolates were screened for the presence of the heat stable (ST) enterotoxin, heat labile (LT) enterotoxin and verotoxin (VT1 and VT2) gene sequences by PCR. 4.7.1 Detection of LT and ST genes

In MTCC 723, the primer sets LT1-LT2 and microST1-microST2 gave products of expected sizes, that is, 132 bp for LT (Plate VIII) and 171 bp for ST genes (Plate IX). Only one isolate (1.09%) was found to posses ST gene and two isolates harbored LT gene sequences in PCR test (Table 4.9). 4.7.2 Detection of VT1 and VT2 genes

Results of primer-directed amplification of the VT1 and VT2 genes are represented in Plate X and XI respectively which show the presence and distribution of the two amplified products when DNA from positive control strain (Reference strain E. coli VT1+VT2+ maintained at Department of Veterinary Microbiology) and test isolates were used as template. The sizes of amplified products were as predicted from the design of the primers, that is, 130 bp for the VT1 primers (Plate X) and 228 bp for the VT2 primers (Plate XI). Of 91 E. coli isolates 41 isolates (45.05%) were positive for VT genes: of which 32 (35.16%) harboured both VT1 and VT2 genes, while six E.coli isolates (6.5%) harboured only VT2 and 3 isolates were positive for VT1 gene only. Overall, 38 (41.75%) isolates carried VT2 genes and 35 (38.46%) isolates had VT1 gene. The results of the PCR assay for detection of toxigenic genes in E. coli isolates are presented in Table 4.9.

Table 4.9 Results of PCR amplification of toxigenic genes in E. coli isolates

Sr.No. Isolate No. Serogroup VT1 VT2 VT1 and VT2 LT ST1. AU-1 O11 - - - - - 2. AU-2 O18 - - - - - 3. AU-3 O22 + + + - - 4. AU-4 O22 + + + - - 5. AU-5 O24 + + + - - 6. AU-6 O26 - - - - - 7. AU-7 O26 - - - - - 8. AU-8 O32 - - - - -

Sr.No. Isolate No. Serogroup VT1 VT2 VT1 and VT2 LT ST9. AU-9 O32 - - - - -

10. AU-10 O55 + + + - - 11. AU-11 O55 + + + - - 12. AU-12 O62 - - - - - 13. AU-13 O62 + + + - - 14. AU-14 O62 - - - - - 15. AU-15 O86 + + + - - 16. AU-16 O86 + + + - - 17. AU-17 O86 + + + - - 18. AU-18 O86 + + + - - 19. AU-19 O86 + + + - - 20. AU-20 O86 + + + - - 21. AU-21 O86 - - - - - 22. AU-22 O110 - - - - - 23. AU-23 O110 + + + - - 24. AU-24 O126 - - - - - 25. AU-25 O128 + + + - - 26. AU-26 O128 + + + - - 27. AU-27 O128 + + + - - 28. AU-28 O128 + + + - - 29. AU-29 O131 + + + - - 30. AU-30 O157 + - - - - 31. AU-31 O157 - - - - - 32. AU-32 O171 + + + - - 33. AU-33 O171 - + - - - 34. AU-34 O171 - + - - - 35. AU-35 O171 + + + - - 36. AU-36 O172 + + + - - 37. AU-37 O172 + + + - - 38. AU-38 O172 + + + - - 39. AU-39 O172 + + + - - 40. AU-40 UT + + + - - 41. VC-1 O2 - + - + - 42. VC-2 O2 - - - + - 43. VC-3 O12 - - - - - 44. VC-4 O97 - - - - - 45. VC-5 O167 + + + - - 46. VC-6 O167 + + + - - 47. OF-1 O5 + - - - - 48. OF-2 O9 - - - - - 49. OF-3 O11 - - - - - 50. OF-4 O22 - - - - - 51. OF-552 O66 - - - - - 52. OF-6 O78 - - - - - 53. OF-7 O86 + + + - - 54. OF-8 O98 - - - - - 55. OF-9 O98 - - - - - 56. OF-10 O100 - - - - -

Sr.No. Isolate No. Serogroup VT1 VT2 VT1 and VT2 LT ST57. OF-11 O101 - - - - - 58. OF-12 O108 - - - - - 59. OF-13 O126 - - - - - 60. OF-14 O131 - - - - - 61. OF-15 O131 - - - - - 62. OF-16 O157 + + + - - 63. OF-17 O161 - - - - - 64. OF-18 O162 - - - - - 65. OF-19 O171 - - - - - 66. OF-20 O172 - - - - - 67. OF-21 Rough - + - - - 68. OF-22 Rough - - - - - 69. OF-23 Rough - - - - - 70. OF-24 UT - - - - - 71. OF-25 UT - - - - - 72. OF-26 UT + + + - - 73. OF-27 UT - - - - - 74. JU-1 O3 - - - - - 75. JU-2 O15 - - - - - 76. JU-3 O18 - - - - - 77. JU-4 O18 - - - - - 78. JU-5 O18 - - - - - 79. JU-6 O18 - - - - - 80. JU-7 O18 - - - - - 81. JU-8 O22 - - - - - 82. JU-9 O68 - - - - - 83. JU-10 O76 - - - - - 84. JU-11 O109 - - - - - 85. JU-12 O109 - - - - - 86. JU-13 O128 - + - - - 87. JU-14 O128 - - - - - 88. JU-15 O131 - + - - - 89. JU-16 O168 + + + - + 90. JU-17 O171 + + + - - 91. JU-18 UT + + + - -

DISCUSSION

CHAPTER-V DISCUSSION

Escherichia coli is among the common bacterial enteric pathogens capable of causing intestinal disease. Several classes of diarrhoea causing E. coli are now recognized on the basis of production of virulence factors. These bacteria include strains of Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), Enterohaemorrhagic or Verotoxigenic E. coli (EHEC/VTEC), Enteroaggregative E. coli (EAEC), Diffusely adherent E. coli (DAEC) as reported by Nataro and Kaper (1998). VTEC or shiga like toxin (SLT) producing E. coli have been shown to produce bacteriophage encoded cytotoxins active on vero and Hela cells (Scotland et al., 1981; O’Brien et al., 1984). Among VTEC serotypes, O157:H7 has been shown to be closely associated clinically with sporadic and outbreak cases of haemorrhagic colitis, haemolytic uremic syndrome and thrombocytopenic purpura. Many VTEC representative of non-O157 serogroups have also been associated with well characterized intestinal and extraintestinal illness, and the clinical significance of these cytotoxin producing pathogens should not be neglected (Karmali, 1989). Since colibacillosis/neonatal calf diarrhoea is an important cause of economic loss on farms, detailed studies of the virulence factors produced by E. coli strains in farm animals are needed. Moreover, improved identification of enterotoxigenic and verotoxigenic strains in clinical samples is required in order to further elucidate the association of these toxigenic strains with diseases of farm animals. These days the PCR technology has allowed large scale screening of many colonies from clinical material or contaminated foods. Neonatal calf diarrhea is common in India but few studies reported so far have dealt with PCR based detection of toxigenic genes in E. coli isolates from diarrhoeic calves. In Gujarat, there is paucity of reports regarding PCR based toxigenic gene detection in E. coli from diarrhoeic calf. The present research work was taken up with a view to ascertain characters which might be associated with virulence of E. coli isolated from diarrhoeic calf faecal samples. Multiple antimicrobial drug resistance has often been reported among the E. coli isolated from disease outbreaks. This character is mostly coded by transferable plasmids. These plasmids may also carry certain genes for various activities like colicin production, toxin production etc. these characters might play its role directly in the virulence activity of the organism or can associate for the virulent characters. Some of these characters may simply be associated with these virulence plasmids, thus in turn act as a marker for identification of the virulence plasmid like col V production. A number of E. coli serotypes have been identified for host related diseased state caused by this organism, although a wide variation in serotypes among the isolates have been reported with emerging trend of transmissible nature of the virulent character, the serotype related host specificity becomes a lesser important feature (Sojka, 1971). Hence an attempt is made in the present work to relate to various characters of E. coli as a pathogen which is presented in the following discussion. 5.1 Isolation and Identification of E. coli Isolates During present study, an attempt was made to isolate E. coli from the diarrhoeic calf faecal samples from organized farms viz. Livestock Research Station, Anand Agricultural University (LRS-AAU), Anand and Livestock Research Station (LRS-JAU) Junagadh, other

unorganized farms and villages in and around Anand (OF) and Veterinary college clinics (VC), AAU, Anand. During the present study 91 E. coli isolates were obtained from 46 rectal samples. All the isolates revealed characteristic features of E. coli, which were Gram negative bacilli, produced pink lactose fermenting colonies on MacConkey agar and characteristic greenish metallic sheen on eosin methylene blue agar. On preliminary biochemical characterization they revealed characteristic IMViC pattern that is, + + - - except for three isolates (AU-24, AU-26 and JU-3) which were citrate positive. 5.2 Serotypes of E. coli associated with calf diarrhea E. coli of specific serogroup can be associated with reproducibility of certain clinical syndrome. The distribution of different serotypes of E. coli varies with geographical regions and their prevalence in man and animals in a particular area. A shift in ‘O’ serogroups along with their virulence factors has also been observed (Soderlind et al., 1988). In the present study, out of 91 E. coli isolates different ‘O’ serogroups were recorded in different locations viz. LRS-AAU, Veterinary College Clinics (VC), unorganized farms (OF) in and around Anand city and LRS-JAU, among them 82 belonged to 36 different ‘O’ serogroups while six isolates were untypable and three were rough isolates. Among the 40 isolates obtained from LRS-AAU the 17 different serogroups detected in decreasing frequency were O86 (7 isolates), four isolates each of O128, O171 and O172, O62 (3 isolates), serogroups O22, O26, O32, O55, O110 and O157 having two isolates each and one isolate each belonging to serogroups O11, O18, O24, O126, O131 and UT (untypable). Six isolates obtained from Veterinary College Clinics belonged to four different serogroups O2 and O167 having two isolate each and one isolate each of O12 and O197. The number of isolates from unorganized farms was twenty seven. The twenty different serogroups detected were O5, O9, O11, O22, O66, O78, O86, O100, O101, O108, O126, O157, O161, O162, O171 and O172 one each while two isolates each of serogroup O98 and O131. There were three rough and four untypable isolates. Eighteen isolates obtained from LRS-JAU comprised of twelve different serogroups viz. O18 (5 isolates), two isolates each of O109, O128 while one isolate each of O3, O15, O22, O68, O76, O131, O168, O171 and UT. In present study, among the isolates the serogroups viz. O5, O22, O18, O55, O62 and O68 have been also reported by Srivastava and Arya (1979) from calf diarrhoeal cases. Of these, serogroup O5 have been also reported by various other workers (Latif, 1982; Shah, 1989 and Kaura et al., 1991). The serogroup O22 has been also reported by Latif (1982) from diarrhoeic buffalo calves, Tripathi and Soni (1984), Joon and Kaura (1993), Hussain et al. (2003) and Wani et al. (2004) from cases of neonatal cow calf diarrhoea. In the present study serogroup O2 was isolated carrying LT gene as detected using PCR technique. Ueda et al. (1981) also reported ETEC strain of O2 serogroup. Other workers (Shah, 1989, Sharma et al. 2004 and Wani et al. 2004) also isolated O2 serogroup from calf diarrhoeal cases. In the present investigation two isolates of O55 were recorded. This serogroup was also reported by Tripathi and Soni (1984), Shah (1989), Kaura et al. (1991), Minakshi (1992), Sharma et al. (2004) and Wani et al. (2004) from the cases of neonatal calf diarrhoea. The serogroup O101 isolated in present study have been reported by Ueda et al. (1981), Kaura et al. (1991), Minakshi et al. (1992), Hussain et al. (2003) and Wani et al. (2004) from cases of neonatal calf diarrhea.

The serogroups O32 and O24 isolated in this study from diarrhoeic calf cases were also isolated by Latif (1982) from diarrhoeic buffalo calves, however Shah (1989) isolated serogroup O32 and Kaura et al. (1991) isolated O24 from neonatal diarrhoea in calves. The serogroups O3, O9, O68, O76, O97, O157 and O161 recorded in present study were reported by Panda and Panda (1987) from calf diarrhoea in Orissa. Of these O3 and O9 serogroups have been reported by Wani et al. (2004). The serogroup O9 was reported by Kaura et al. (1991), Joon and Kaura (1993). However, Joon and Kaura reported O68 from diarrhoeic buffalo calves. Serogroups O157 and O161 were also reported by Wani et al. (2004). Serogroups O15 and O18 found in this study were also reported by Hussain et al. (2003). The serogroup O11 recovered in this study has been reported by Tripathi and Soni (1984) and Sutariya (1993) from neonatal calf diarrhoea Kaura et al. (1991) and Minakshi et al. (1992) isolated this serogroup from diarrhoeic buffalo calves. Of the 36 different serogroups obtained in the present study 17 serogroups viz, O2, O3, O9, O12, O15, O22, O26, O55, O78, O86, O100, O101, O109, O131, O157, O161, O162 were also reported by Wani et al. (2004) from diarrhoeic calf faecal samples. Serogroups O78 and O157 were also isolated by Sharma et al. (2004). In the present study it was observed that serogroups O22, O131 and O171 were prevalent in all the four locations except Veterinary College Clinics and the serogroups O11, O18, O86, O126, O128, O157 and O172 were prevalent in at least two locations. In the present study six E. coli isolates were untypable and three were rough isolates. It is not uncommon to isolate strains of E. coli, which could not be typed with the available antisera (Ueda et al., 1981; Beutin et al., 1989; Shah, 1989; Sutariya, 1993; Hussain et al., 2003). Serotypes like O157, O26, O8, O55, O86, O126 and O128 isolated in present study have been found to be associated with infantile diarrhoea among neonates and adult human patients suffering from gastroenteritis as reported by Beutin et al. (1989) and Nishikawa et al. (2002). Thus, these serotypes may be of zoonotic importance. 5.3 Biochemical characterization of E. coli isolates In the present study all the isolates showed the typical biochemical behaviour of E. coli as described by Edwards and Ewing (1972). The biochemical behaviour of the isolates revealed that they all were positive for methyl red and indole production and while negative for Voges-Proskauer test and failed to utilize citrate on Simmon’s citrate agar except for three isolates (AU-24, AU-26 and JU-3) which were citrate positive. The result for citrate utilization was also observed for E. coli isolates by Sutariya (1993). These results are in accordance with the bio-chemical characteristics of E. coli as reported by Edwards and Ewing (1972) and Barrow and Feltham (1993). In this study only one isolate out of a total of 91 E. coli isolates was able to produce H2S on TSI agar this was in accordance to findings of Barrow and Feltham (1993) who reported 0-15 % strains positive for H2S production. In this study a total of 21.97 per cent of E. coli isolates studied showed urea hydrolysis. Out of 41 E. coli isolates positive for VT genes eleven isolates (26.82%) expressed urea hydrolysis. Moreover, in this study out of three O157 E. coli isolates one (33.33 %) showed urease activity. Such high frequency of urease production (between 22 and 40%) has been reported by various workers in verocytotoxin producing E. coli O157 of bovine origin (Friedrich et al., 2005)

In the present investigation ability of all the E. coli isolates to utilize various sugars was carried out by fermentation reaction and it was found that in decreasing frequency dulcitol was fermented by thirty five (38.46%) of isolates studied, raffinose by thirty one

(34.06%) isolates, rhamnose by thirty (32.96%) isolates, sucrose by twenty one (23.07%) isolates, salicin by fourteen (15.38%) isolates, starch by four (4.39%) isolates and adonitol by three (3.29%) isolates. These results were in accordance to that reported by Barrow and Feltham (1993) except for rhamnose for which they found 85-100% strains positive in fermentation reaction. 5.4 Unusual Biochemical Characters of the E. coli isolates Atypical biochemical characters of E. coli strains have been used as an epidemiological tool in characterization of isolates from disease outbreaks (Barbour et al., 1985). In the present study atypical biochemical characters like H2S production, urease production, citrate utilization, and fermentation of adonitol and raffinose were studied. 5.4.1 Production of H2S Occasionally E. coli isolates, which usually do not produce H2S, spontaneously appear to produce H2S (Galton and Hess, 1946). These strains have been recognized as atypical E. coli that have acquired plasmids (Layne et al., 1971). In H2S producing E. coli, these extra chromosomal elements code for an enzyme system that produce H2S from thiosulphate or tetrathionate (Orskov and Orskov, 1973). In the present study one isolate out of 91 E. coli isolates tested produced H2S. Production of H2S by E. coli isolates is also reported by Sutariya (1993) and Mishra et al. (2002). 5.4.2 Production of Urease Urease enzyme can contribute to the virulence of several gram-negative bacteria and enhance their acid resistance but, in general ureolytic Escherichia coli strains are rarely found among clinical isolates. However, among various diarrhoeagenic E. coli strains from clinical sources, urease genes have been reported to be associated with major EHEC groups O26, O111 and O157 but were found to be absent from diarrhoeagenic E. coli strains of other pathogroups, including enterotoxigenic E. coli, enteroaggregative E. coli, enteroinvasive E. coli and enteropathogenic E. coli (Friedrich et al., 2005) In the present study twenty isolates (21.97 per cent) out of 91 E. coli isolates showed urea hydrolysis. Production of urease by E. coli have been reported by earlier workers like, Pohl et al. (1989), Sutariya (1993), Dubey et al.(2001) and Mishra et al. (2002). In this study out of 41 E. coli isolates positive for VT genes eleven isolates (26.82%) expressed urea hydrolysis. Tominaga et al. (1989) also reported urease activity of verocytotoxin producing E. coli isolates from cases of calf diarrhoea. Moreover, in this study out of three O157 E. coli isolates one (33.33 %) showed ureolytic activity. Such high frequency of urease production (between 22 and 40%) has been reported in verocytotoxin producing E. coli O157 of bovine origin isolated by Friedrich et al. (2005). 5.4.3 Ability to utilize citrate Usually E. coli isolates do not utilize citrate but atypical strains of E. coli may show citrate utilization. Barrow and Feltham (1993) reported 0-15% of E. coli strains able to utilize citrate. In the present study three E. coli isolates (3.29%) out of 91 isolates showed citrate utilization. Similar findings have been also reported by Ishiguro et al. (1978), Ishiguro and Sato (1979), Lee and Choi (1983), Kim and Tak (1984) and Dubey et al. (2001). 5.4.4 Fermentation of adonitol and raffinose E. coli rarely ferments adonitol whereas variable results (upto 50% positive) are observed for its raffinose fermentation activity ((Edwards and Ewing, 1972). The variable raffinose fermentation activity might be due to its genetic locus, which is reported to be plasmid coded (Smith and Huggins, 1978).

In the present study, thirty-two (35.1%) E. coli isolates had raffinose fermentation activity while only 2 isolates (2.19%) showed adonitol fermentation activity. However, even higher per cent of E. coli isolates showing fermentation activity of these two sugars have been reported by other workers, Braaten and Myers (1977) reported adonitol and raffinose fermentation in 9 (50%) and 6 (33.33%) isolates respectively out of 18 ETEC strains and Cloud et al. (1985) showed that 30 (15.2%) out of 197 isolates fermented adonitol, while 177 (89.84%) of 197 isolates fermented raffinose. Smith and Huggins (1978) reported that K88 pili are determined by genes carried by plasmids which usually carry gene for raffinose fermentation. In the present study raffinose fermentation and tetracycline resistance were shown simultaneously by 12 E. coli isolates which is in agreement with findings of Mondaca et al. (1985). 5.5 Biotypes of the E. coli isolates Biochemical reactions have conventionally been used for identification of bacteria to the species level. Extensive studies of biochemical reactions of bacteria have been done to introduce biochemical typing systems in epidemiological studies of bacteria (Barr and Hogg, 1979; Krishnan et al., 1987). E. coli are able to ferment variety of carbohydrate substrates, generally by converting them to glucose or to a substrate on the fermentative chain of the breakdown of glucose. The various fermentable carbohydrates include substances such as compounds of glucose with other sugars like maltose, trehalose cellobiose, lactose, sucrose, raffinose, rhamnose, dulcitol, mannitol and sorbitol. Also compounds of sugars with other substances can be fermented particularly if the sugar molecule can be separated from the other part; an example is the substance salicin. The ability to ferment a given sugar of the sugars described above by a strain of E. coli is dependent on the strain having the requisite enzymes to convert it to glucose or to a substance on the degradative chain from glucose. It has been found that different strains of E. coli differ in their ability to perform these conversions. Thus, this is the basis of biotyping E. coli. These tests are easy to perform, by determining, whether a strain of E. coli will produce acid following growth in the presence of carbohydrates (Crichton and Old, 1982). In the present study the fermentation reactions of carbohydrates by all the 91 E. coli isolates were found to be variable. Out of the 91 E. coli isolates obtained 67 isolates were able to utilize one or more sugars while 24 isolates failed to utilize any of the sugars tested. The isolates could be grouped into various biotypes considering the fermentation reactions of six different sugars viz. dulcitol, raffinose, rhamnose, salicin, starch and sucrose. Out of 91 E. coli isolates 67 E. coli isolates were distributed into 24 different biotypes. This is in accordance with findings of Pandey et al. (1979) and Chachra and Katoch (1996) who used the same six sugars for biotyping of E. coli isolates. In the present study, the most commonly occurring biotypes were biotype III (10 rhamnose fermenting isolates), XV (7 dulcitol, raffinose and rhamnose fermenting isolates), VII (6 dulcitol rhamnose fermenting isolates), II (5 raffinose fermenting isolates), the rest of the biotypes were found to be having less than five isolates. In present investigation 33 different serogroups comprising of 59 typable, 5 untypable and 3 rough isolates could be grouped into 24 different biotypes. In many cases it was observed that isolates with same biotype exhibited different serogroups and reverse was also true. Such variability in fermentation reactions biotypes and distribution amongst E. coli isolates have also been reported by Hinton et al. (1982), Camguilhem and Milon (1989) and Blanco et al. (1996). In the present study, 29 (70.73%) out of 41 verotoxigenic E. coli (VTEC) were rhamnose non-fermenters while 33 (66%) out of 50 non-VTEC isolates were rhamnose non-

ferementers. Moreover, it was observed that 12 (29.26%) out of 41 VTEC isolates were rhamnose fermenters. Murinda et al. (2004) also reported similar findings but they observed higher per cent of VTEC O26 (100%) and lower per cent of non-VTEC O26 (40%) rhamnose non-fermenter isolates. Thus, rhamnose fermentation reaction may serve as a diagnostic tool for detection of verotoxigenic E. coli from cases of diarrhoeal illness. 5.6 Multiple antimicrobial drug resistance of the E. coli isolates In the present study in vitro antibiotic resistance patterns of the E. coli isolates were determined by disc diffusion method of Bauer et al. (1966). The E. coli isolates were tested against commonly used antibiotics viz. ampicillin (A), amikacin (Ak), ceftiofur (Fur), cephalexin (Cp), cephaloridine (Cr), colistin (Cl), ciprofloxacin (Cf), co-trimoxazole (Co), enrifloxacin (Ex), kanamycin (K), nalidixic acid (Na) and tetracycline (T). 5.6.1 Prevalence of antimicrobial drug resistance among the E. coli isolates Though antibiotics use has its advantages, the intensive and extensive use of antibiotics has lead to the emergence of antimicrobial resistance. The indiscriminate and uncontrolled use of antimicrobial drugs exerts a selection pressure and encourages the proliferation of drug resistant strains of E. coli in animal population. When this is coupled with poor environmental sanitation and low personal hygiene, the situation may constitute a danger to public health. Among the 91 E. coli isolates studied very high percent of isolates (80-100%) were resistant to kanamycin, cephalexin, amikacin, cephaloridine and enrofloxacin. Ninety-five per cent of E. coli isolates showed resistance to kanamycin and such high resistance among the isolates is agreeable to the findings of Bradford et al. (1999). In the present study 92.3 per cent of isolates showed resistance to cephalexin. In contrast to the findings of Chattopadhyay et al. (2003) who observed 30.7 percent isolates resistant to cephalexin. A high per cent of resistance among the isolates to cephaloridine (87.9 per cent) was detected, even higher resistance (98.65%) to this antibiotic was observed by Saravanbava et al. (1990). In this study higher resistance among isolates was observed against amikacin (91.2%) and enrofloxacin (84.61%), while lower per cent of resistance pattern against amikacin (15.38%) and enrofloxacin (11-18%) was observed by Chattopadhyay et al. (2003) and Orden et al. (1999) respectively. In the present study high per cent of isolates (72.52%) were resistant to ampicillin, similar findings have been reported by Panda and Panda (1987), Saravanbava (1990), Aalback et al. (1991), Sutariya (1993) and Bradford et al.(1999). In this study, moderately high per cent of isolates were found to be resistant to antibiotics tetracycline (56.04%), ceftiofur (37.3%) and ciprofloxacin (28.57%). Higher per cent of (50 per cent or more isolates) resistant to tetracycline has also been reported by Mehrotra et al. (1984), Shah (1989), Saravanbava et al. (1990), Aalback et al. (1991), Sutariya (1993), Bradford (1999) and Hariharan et al. (2004). Moderately high per cent (37.3%) of isolates were resistant to ceftiofur. Ceftiofur, a veterinary expanded spectrum cephalosporin, is active against a variety of animal pathogens associated with bovine and swine respiratory disease. However, it is often used to treat bacterial diarrhoeal diseases when strains are multiple resistant to other veterinary antimicrobials. Survey by Bradford et al. (1999) revealed that about 13% of E. coli strains implicated in bovine scours were resistant to ceftiofur. In this study the ceftiofur resistant E. coli isolates associated with bovine calf scours were also found to be multiple resistant to several other antibiotics. However, Hariharan et al. (2004) observed lower resistance against ceftiofur (8%) among the ETEC isolates tested.

In present study, moderately high per cent of isolates (28.57%) were found to be resistant to ciprofloxacin which is in contrast to the findings of Bradford et al. (1999) and Chattopadhyay et al. (2003) who observed lesser number 6.25% and 15.38% of E. coli isolates respectively resistant to ciprofloxacin. In the present study, among the isolates tested lesser per cent of isolates were found to be resistant to the antibiotics co-trimoxazole (15.38%), colistin (12.08%) and nalidixic acid (8.79%). Lesser number of isolates resistant to co-trimoxazole was also reported by Shah (1989), Saravanbava et al. (1990), and Khan et al. (2002). However, more number of E. coli isolates (25%) resistant to colistin have been reported by Kobayashi et al. (2001). In this study very low resistance (8.79%) among the E. coli isolates was found against nalidixic acid, which is similar to findings of Saravanbava et al.(1990), Orden et al. (1999), Bradford et al. (1999) and Khan et al. (2002). Thus, in the present study highest resistance was observed towards aminoglycosides (93.4%) followed by tetracyclines (56.04%), penicillin and cephalosporins (72.5%), fluoroquinolones (40.6%), sulphonamides (15.38%) and polymyxin antibiotics (12.08%). Aminoglycosides, such as gentamicin, kanamycin and amikacin, are commonly used as antimicrobial agents in the treatment of infections by both Gram negative and Gram positive organisms. In the present study ninety three percent of E. coli isolates showed resistance to this group of antibiotics. Resistance was highest for kanamycin i.e. 95.60 per cent. Bradford et al. (1999) also reported similar resistance pattern for kanamycin, however higher resistance observed for amikacin (91.2 per cent) in this study was contradictory to the findings of Chattopadhyay et al. (2003), who found 15.38% of E. coli isolates resistant to amikacin. Resistance to aminoglycosides is widespread, with more than 50 aminoglycoside-modifying enzymes already described. Most of these genes are associated with Gram negative bacteria. Besides these enzymes, efflux systems and rRNA mutations have been also reported (Schmitz and Fluit, 1999; Shaw et al., 1993). In the present study, 51 (56.04%) out of 91 E. coli isolates were resistant to tetracyclines. Similarly, other workers also (Mehrotra et al., 1984, Shah, 1989, Saravanbava et al., 1990, Aalback et al., 1991, Sutariya, 1993, Bradford et al., 1999 and Hariharan et al., 2004) reported high resistance of E. coli isolates against tetracycline. Although tetracyclines initially were useful for treatment of infections with aerobic Gram negative organisms, many enterobacteriaceae are now becoming relatively resistant to them. Resistance to tetracyclines in E. coli and related species is principally plasmid mediated and an inducible trait. Mechanisms of resistance include decreased accumulation of tetracycline due to either acquisition of an energy-dependant efflux pathway or to decreased influx, or to decreased access of tetracycline to the ribosome (site of action) due to acquisition of ribosome protected proteins and enzymic inactivation (Speer et al., 1992). Micro organisms that have been resistant to one tetracycline frequently exhibit resistance to the others. Tetracyclines were found initially to be highly effective against ETEC, but resistance has been emerging in the recent past and becoming a constraint in the treatment (Kapusnilk-uner et al., 1996). In the present study cephalosporins were placed third in order of resistance (72.5%) along with penicillin. Three drugs of this group were used in present study: cephalexin and cephaloridine – first generation cephalosporins and ceftiofur- a third generation cephalosporins. Ceftiofur was more efficacious (62.64%) than cepahalexin (7.7%) and cephaloridine (12.09%). The present finding is in partial agreement with the reports of Orden et al. (1999) and Hariharan et al. (2004). Higher sensitivity of E. coli isolates to ceftiofur compared to cephalexin and cepahloridine in this present study might be due to the reason that the third generation cephalosporins are more active than the first generation against Gram negative organisms (Donowitz and Mandell, 1988; Karchmer, 1995). Thus, Ceftiofur an expanded spectrum cephalosporin is highly effective against bovine E. coli isolates.

In this study per cent of isolates resistant to, ampicillin (extended spectrum penicillin) were 72.5%. Similar findings have been reported by Panda and Panda (1987), Saravanbava (1990), Aalback et al. (1991), Sutariya (1993) and Bradford et al. (1999). The resistance recorded to penicillin (ampicillin) in the present study might be attributed to the production of β-lactamase group of penicillin destroying enzymes, which have been reported to be of as many as 50 different types. These enzymes are encoded in either chromosomes or plasmids, and they may be constitutive or inducible (Rang et al., 1995). The fluoroquinolones are an exceptionally important and rapidly developing group of antimicrobial drugs and are being introduced into human and veterinary medicine for a wide variety of antimicrobial purposes (Prescott and Baggot, 1993). In the earlier reports about fluoroquinolones, resistance of human (Kojima, et al., 1989 and Visser et al., 1991) and bovine (Bauditz , 1987) E. coli strains to these antibiotics was rarely observed. In the present study three members of quinolone group were tested for their efficacy against isolated strains viz. nalidixic acid, ciprofloxacin and enrofloxacin. The average resistance of the isolates to quinolone group of antibiotics was 40.6%. The frequencies of resistance of enrofloxacin (84.61%) and ciprofloxacin (28.57%) were relatively high. Other investigators have also described increases in the levels of resistance to these antimicrobial agents among E. coli strains isolated from humans (Bauernfeind et al., 1994 and Lehn, et al., 1996) and cattle (Bőttner et al., 1995 and Orden et al., 1999). The increase in the level of resistance of bovine E. coli isolates to fluoroquinolones may indicate a risk to public health because some of these strains principally, VTEC strains may cause diseases in humans (Karmali, 1989) and because resistance to the fluoroquinolones used in veterinary medicine may confer resistance to the fluoroquinolones used in human medicine. In the present study among the total antibiotics used least resistance (8.79%) was observed against nalidixic acid (first generation fluoroquinolone). This high sensitivity of E. coli isolates may be due to the uncommon use of this antibiotic. In the present study lesser number (15.38%) of E. coli isolates were found to be resistant to co-trimoxazole. This finding is in agreement with reports of Shah (1989), Saravanbava et al. (1990) and Khan et al. (2002). There is significant variation in the susceptibility of enterobacteriaceae to sulphonamides group in different geographical locations because of spread of resistance mediated by plasmids and transposons (Goldstein et al., 1986). The resistance to sulphonamides is associated with the acquisition of a plasmid that codes for an altered dihydrofolate reductase, the target enzyme (Houvinen, 1987), or due to alteration in the dihydropteroate synthase enzyme, which utilizes para amino benzoic acid (PABA) in an alternative pathway for synthesis of essential metabolites (Gerald and William, 1996).

In the present study E. coli isolates showed lesser resistance (12.08%) to colistin (polymyxin) antibiotic. Thus, colistin was found to be highly efficacious (87.92%) against E. coli isolates. This present finding is in accordance with the reports of Boro et al. (1983) and Kobayashi et al. (2001). Polymyxin antibiotics have cationic detergent properties and their mechanism of action involves interaction with the phospholipids of the cell membrane and disruption of its structure. They have a selective, rapidly bactericidal action on Gram negative bacilli, especially coliforms (Rang et al., 1995). High sensitivity of colistin sulphate against E. coli isolates might be attributed to its uncommon use in routine work.

5.6.2 Location wise prevalence of antimicrobial drug resistance of the E. coli isolates During this study E. coli isolates numbering 40 from Livestock Research Station, Anand Agricultural University, Anand (LRS-AAU), 6 from Veterinary College Clinics,

AAU (VC), 27 from other unorganized farms (OF) in and around Anand and 18 from Livestock Research Station, Junagadh Agricultural University (LRS-JAU) were analysed for the antimicrobial drug resistance. Prevalence of drug resistance to amikacin, cephalexin, cephaloridine and kanamycin was found among 80-100 % of the isolates from LRS-AAU, VC, OF while percent of resistant isolates for these drugs were less (66.66%) among LRS-JAU isolates. Resistance of the isolates against enrofloxacin and ampicillin ranged from 66.6 to 92.5 per cent and 55.55 to 80 per cent respectively among the isolates from all the locations. Moderate to very high per cent of isolates were resistant to tetracycline (51.8- 100%) among the isolates from all the three locations except for JAU (22.2%). In general the per cent of the isolates resistant to nalidixic acid, co-trimoxazole and ciprofloxacin were lower to very low (33.3 to 5.5 per cent) among the isolates from all the locations. However, the isolates from VC were more resistant to ciprofloxacin (50%) while interestingly all the isolates from OF were sensitive to colistin. Similar work carried out by Sutariya (1993) for the E. coli isolates obtained from Anand calf diarrhoeal cases showed higher resistance for cloxacillin, erythromycin, tetracycline, sulfamethazole and neomycin while lesser number of resistant isolates were reported to ampicillin, streptomycin and chloramphenicol. Least number of resistant isolates were reported against nitrofurantoin, gentamicin and kanamycin while none of the isolates were resistant to nalidixic acid. 5.6.3 Pattern of multiple antimicrobial drug resistance among the E. coli In the present study all the isolates revealed multiple drug resistance to various antibiotics ranging from resistance to two antibiotics up to ten antibiotics. Eighty three isolates were resistant to five or more antibiotics. Predominant resistance pattern was observed for the following antibiotics viz. ampicillin, amikacin, cephalexin, cephaloridine, enrofloxacin and tetracycline. On analysis three en bloc pattern of drug resistance could be identified:

1. Resistance to cephalexin, cephaloridine and enrofloxacin was shown by 68 (74.72%) isolates.

2. Resistance to cephalexin, cephaloridine, enrofloxacin, amikacin and kanamycin was shown by 67 (73.62%) isolates.

3. Resistance to ampicillin, cephalexin, cephaloridine and kanamycin was observed for 60 (65.93%) isolates. Similar multiple drug resistance among the E. coli isolates from calf diarrhoeal cases

have been reported by Pohl et al. (1969), Tripathi and Soni (1982), Shah (1989), Saravanbava et al. (1990), Aalback et al. (1991), Cid et al. (1996), Bradford et al. (1999) and Khan et al. (2002). These workers revealed various combination of multiple antimicrobial drug resistance among the E. coli isolates during their study showing no common en bloc multiple drug resistance pattern prevalence in their report.

In the early 1950s, several enterobacteria were found resistant to many antibiotics and the resistance factors were transferred from resistant bacteria to non-resistant organisms by cell to cell contact (Akiba et al., 1960). Studies on antimicrobial resistance of E. coli from different animal species showed an increase in the incidence of resistance over the years as a result of the wide spread use of antimicrobial drugs in animals (Prescott et al., 1984; Wray et al., 1993; Cid et al., 1996), the problem further aggravated by the transfer of E. coli from livestock to poultry to human (Kapoor et al., 1994). The use of drugs does not induce resistance but rather provides an intense selection pressure which eliminates the susceptible normal flora in the host and spares the resistant ones (Hinton et al., 1986).

In this study, the highest rate of resistance has been detected against the antimicrobial drugs most commonly used either as feed additives or as curative agents in

farm animals or for treatment in human medicine, while the E. coli strains isolated were susceptible to less commonly used antimicrobial agents. This warrants restriction on the use of antibiotics as feed additives and rational use of antimicrobial therapy of infections in man and animals.

5.5 Colicinogeny among E. coli isolates

Colicines may help E. coli strains to establish in gut by inhibiting other normal resident flora (Elwell and Shipley, 1980). Thus it is an important virulence associated factor of E. coli.

In the present study out of the 91 E. coli isolates, 32 (35.16%) isolates were colicin producer. Colicin producing E. coli from calf diarrhoeal cases are also reported by Al-Dabbas and Willinger (1986), Ayhan and Aydin (1989), Oswald et al. (1991), Sutariya (1993) and Ahmad et al. (2004). In this study one of the isolate JU-16 was enetrotoxigenic as well as colicinogenic, this finding was similar to that reported by Sutariya (1993). In present study 17 out of 32 colicinogenic E. coli isolates, were positive for verocytotoxin genes (14 isolates carried both VT1 and VT2 genes while 3 isolates carried only VT2 genes). Moreover, in this study, all the colicinogenic E. coli isolates (32) were found resistant to one or more antibacterial drugs tested. This finding is comparable to the reports of Ahmad et al. (2004) who found 57% of the colicinogenic strains resistant to one or more antibacterial drug tested. 5.8 Virulence attributes of the E. coli isolates 5.8.1 Haemolysin production In the present study out of 91 E. coli isolates 4 (4.39%) revealed haemolytic activity. None of these four haemolytic isolates were associated with any of the toxigenic genes. Similar findings have been reported by Salvadori et al. (2003) who isolated 20 α- haemolysin positive (9.75%) E. coli strains and none of these strains were associated with enterotoxigenic genes. Yadav et al. (1986b) and Beutin et al. (1989) could also find haemolytic E. coli strains among non-ETEC strains. In this study, out of 4 haemolysin producing E. coli isolates, serogroup O98 (2 isolates) was found to be haemolytic. Hussain et al. (2003) also reported haemolysin producing O98 E. coli strain. 5.8.2 Toxigenic gene detection using PCR 5.8.2.1 Detection of heat labile (LT) and heat stable (ST) enterotoxin genes In the present study, with primer sets LT1-LT2 and microST1-microST2 gave products of expected sizes, i.e. 132 bp for LT and 171 bp for ST genes. Nucleic acid from reference enterotoxigenic E. coli (MTCC 723) showed amplification of the requisite fragments for the LT and ST genes in this PCR protocol. This was in accordance with the results obtained for LT and ST gene by using afore mentioned primer sets by Nishikawa et al. (2002). In this study out of the 91 E. coli isolates tested using the above mentioned primers, one isolate (1.09%) revealed to possess ST gene and two isolates (2.19%) harbored LT genes in PCR technique. Such a low frequency may be comparable to French (0%), Spanish (1.3%) and Brazilian (3.9%) data (De Rycke et al. 1986; Blanco et al.1988 and Salvadori et al., 2003). Similarly, Nishikawa et al. (2002) from human diarrhoeal cases and Salvadori et al. (2003) from diarrhoeic calves could detect presence of LT and ST genes using PCR technique. 5.8.2.2. Detection of Verocytotoxin (VT1 and VT2) genes In the present study, primer directed amplification of the VT1 and VT2 genes gave the products as predicted from the design of the primers, that is, 130 bp for VT1 primers

(Pollard et al. 1990) and 228 bp for the VT2 primers (Nishikawa et al.2002). In this study, out of 91 E. coli isolates tested, 41 (45.05%) were found to be positive for VT genes: of which 32 (35.16%) harboured both VT1 and VT2 genes, while six isolates (6.5%) harboured only VT2 gene and 3 isolates were positive for VT1 gene only. Overall, 38 (41.75%) isolates carried VT2 genes and 35 (38.46%) isolates had VT1 gene. Similar results for high per cent (40 per cent or more) of VT gene positive E. coli isolates have been reported by Kobayashi et al. (2001) Khan et al. (2002), Salvadori et al. (2003), Irino et al. (2005) and Zweifel et al. (2005). However, lesser isolation rate for VT positive E. coli isolates have been reported by Rahman (2002) and Chattopadhyay et al., (2003) In the present study 35 (38.4%) and 38 (41.75%) E. coli isolates were found positive for VT1 and VT2 respectively. Similar results for VT1 and VT2 producing strains were reported by Blanco et al. (2003) and Zweifel et al. (2005). The great majority of the E. coli isolates of bovine origin carrying VT2 (40.6%) or VT1VT2 (56.4%) sequences have been reported by Irino et al. (2005). In this study more number of VT gene positive isolates (38.4% VT1 and 41.75% VT2) than LT (2.19%) and ST (1.09%) gene positive E. coli isolates could be found. This finding is comparable to the findings of Salvadori et al., (2003) who found isolation rate of VT genes (9.7% stx1 and 6.3% stx2) higher than compared to LT (8.3%) and ST (3.9%) genes in E. coli isolates from calf diarrhoeal cases. In this study, one of the most important serogroup O157 (3 isolates) known to cause ceratin life-threatening infections in humans and animals was isolated. Isolation of O157 is also reported by Wani et al. (2003) and Sharma et al. (2004). In the present study, prevalence of verocytotoxin genes among non-O157 strains could be detected by PCR amplification technique. Of the 41 verocytotoxin positive E. coli isolates 38 were non-O157 isolates. Similar prevalence of verocytotoxin genes among non-O157 were reported by Rahman (2002) and Blanco et al. (2003). In the present investigation, it was observed that within same serogroups both VT positive and VT negative isolates are prevalent. These serogroups were: O157 (3 isolates: one was positive for both VT1 and VT2 genes, one was positive for only VT1 gene and the third O157 E. coli was negative for both VT1 and VT2 genes), O62 (3 isolates: 1 positive for VT1 and VT2 and 2 negative for both genes), O86 (8 isolates: 7 positive for VT1 and VT2, one isolate negative for both VT1 and VT2 genes), O110 (2 isolates: one isolate positive and one negative for both VT1 and VT2 genes), O128 (6 isolates: 4 positive for both VT1 and VT2 genes, one positive only for VT2 gene and one isolate negative for both VT1 and VT2 genes), O131 (4 isolates: one positive for both VT1 and VT2 genes, one had VT2 gene and two isolates were negative for both VT1 and VT2 genes), O171 (6 isolates: 3 isolates positive for both VT1 and VT2 genes, 2 positive for VT2 genes and one isolate negative for both VT1 and VT2 genes), O172 (5 isolates: 4 isolates positive for VT2 gene only and one negative for both VT1 and VT2 genes), O2 (2 isolates: one positive for VT2 gene only and one negative for both VT1 and VT2 genes). In this study 6 untypable isolates were detected of these, 3 isolates were positive for both VT1 and VT2 genes and 3 were negative for both VT1 and VT2 genes. Of the three rough isolates detected in this study, only one was positive for VT2 gene. This was in accordance with Johnson et al. (1996) who reported that E. coli strains belonging to over 200 serotypes can express stx (shiga like toxin/VT) but with in most serotypes, both stx positive and stx negative strains can be found. During the past decade, VTEC has evolved from a clinical novelty to a global public health concern. VTEC infections have been reported from over 30 countries on six continents, causing a spectrum of human and animal illness ranging from symptom free carriage to severe bloody diarrhoea and even to life-threatening sequelae such as haemolytic uraemic syndrome, haemorrhagic (Khan et al., 2002). However, there is a paucity of reports on VTEC in the developing world including India.

Epidemiological studies to determine the significance of VTEC and ETEC disease have been limited by the lack of simple and rapid assays, since the tissue culture assays are expensive and time consuming. ELISA systems have been developed to detect VT and shiga toxins (Kongmuang et al., 1987), but major drawbacks with these systems are related to specificity and sensitivity. Another test namely receptor-linked-immunosorbent assay for VT1 is very sensitive but detects only VT1 (Basta et al., 1989). The PCR technique used in this study is relatively simple, highly sensitive and specific. Subsequent to extraction of DNA, the PCR protocol yields in 4 hours, data which requires several days by traditional tissue culture assays. Mixed VTEC infections could be easily detected and extensive data could be generated concerning the distribution of VTEC and particularly non-O157 positive E. coli strains in pathogenesis of disease like diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome. Dairy farming in Gujarat is very advanced and rearing of cattle and buffalo is a very common practice, particularly in the rural areas. Again, proper hygienic practices and sanitary measures are lacking while handling of cattle in these areas. Thus it may be presumed that the diarrhoeic cattle, particularly the calves, can be an important source of VTEC causing human infections as high rate of prevalence by VT positive E. coli strains have been found in bovine herds in many countries (Nataro and Kaper, 1998). Therefore, screening of larger number of animals of different bovine races and types of farms (dairy or meat) are required in order to establish precisely the identity and prevalence of virulence factors associated with colibacillosis/neonatal calf diarrhoea. Such studies will enable to reveal the actual magnitude of the problem caused by VTEC and ETEC. This will provide an important epidemiological data about this disease and also give an early warning regarding any outbreaks in future.

SUMMARY AND CONCLUSIONS

CHAPTER-VI

SUMMARY AND CONCLUSIONS The present study was undertaken to investigate biochemical characters, serotypes, biotypes, multiple drug resistance, colicinogeny, haemolytic activity and detection of toxigenic genes of E. coli from diarrhoeic calf faecal samples. During the present study a total of 46 faecal samples from diarrhoeic calves (up to two months) were collected from organized farms of Livestock Research Station, Anand Agricultural University (LRS-AAU), Anand and Livestock Research Station, Junagadh Agricultural University (LRS-JAU), Junagadh, other unorganized farms (OF) and villages in and around Anand city and Veterinary College Clinics (VC), AAU, Anand. All the samples were processed for detection of E. coli. In all 91 E. coli isolates were obtained from the faecal samples. All the isolates revealed characteristic features of E. coli, which were Gram negative bacilli, produced pink lactose fermenting colonies on MacConkey agar and characteristic greenish metallic sheen on eosin methylene blue (EMB) agar. On preliminary biochemical characterization all the isolates revealed characteristic IMViC pattern i.e. + + - - except for three isolates which were citrate positive. Some of the E. coli isolates showed atypical biochemical behaviour viz. H2S production (1.09% isolates), urea hydrolysis (21.97% isolates), citrate utilization (3.29 % isolates) and fermentation of adonitol (2.19% isolates) and raffinose (35.1% isolates). All the 91 E. coli isolates were sent to National Salmonella and Escherichia centre, Central Research Institute, Kasauli. Out of 91 E. coli isolates different ‘O’ serogroups were recorded in different locations viz. LRS-AAU, Veterinary College Clinics, other unorganized farms in and around Anand city and LRS-JAU. Among the 91 isolates 82 belonged to 36 different ‘O’ serogroups while six isolates were untypable and three were rough isolates. Out of 40 isolates obtained from LRS-AAU, 17 different serogroups were recorded in decreasing frequency viz O86 (7 isolates), four isolates each of O128, O171 and O172, O62 (3 isolates), serogroups O22, O26, O32, O55, O110, O157 having 2 isolates each and one isolate each belonging to serogroups O11, O18, O24, O126, O131 and UT (untypable). Out of six isolates obtained from Veterinary College Clinics, 4 different serogroups were recorded viz. O2 and O167 two isolate each and one isolate each of O12 and O197.

Out of twenty seven isolates obtained from unorganized farms, twenty different serogroups detected were O5, O9, O11, O22, O66, O78, O86, O100, O101, O108, O126, O157, O161, O162, O171 and O172 one isolate each while two isolates each of serogroups O98 and O131, 3 rough and four untypable isolates.

Eighteen isolates obtained from LRS-JAU comprised of twelve different serogroups viz. O18 (5 isolates), two isolates each of O109, O128, while one isolate each of O3, O15, O22, O68, O76, O131, O168, O171 and UT.

The most common serogroups were O22, O131 and O171 prevalent in all the four locations except in Veterianry College Clinics, followed by O11, O18, O86, O126, O128, O157 and O172 prevalent in at least two locations.

Out of 91 E. coli isolates 67 were distributed into 24 different biotypes considering the fermentation reactions of sugars viz. dulcitol, raffinose, rhamnose, salicin, starch and sucrose. The most commonly occurring biotypes were biotype III (10 rhamnose fermenting isolates), XV (7 dulcitol, raffinose and rhamnose fermenting isolates), VII (6 dulcitol rhamnose fermenting isolates), II (5 raffinose fermenting isolates) and the rest of the biotypes were found to be having less than five isolates. It was observed that there were different serogroups under the same biotype and vice-versa.

In vitro antibiotic resistance patterns against 12 antibiotics were detected. E. coli isolates showed very high prevalence of resistance against kanamycin (95.6%), cepahlexin (92.3%), amikacin (91.2%), cephaloridine (87.9%) and enrofloxacin (84.61%). High percent of isolates (72.52%) showed resistance against ampicillin. Moderate numbers of isolates were found to be resistant to tetracycline (56.04%), ceftiofur (37.3%) and ciprofloxacin (28.57%). Lesser prevalence of resistant isolates was observed for co-trimoxazole (15.38%), colistin (12.08%) and nalidixic acid (8.79%).

Out of 91 E. coli isolates studied for colicinogeny 32 (35.16%) were colicin producers.

In the present study out of 91 E. coli isolates four (4.39 %) revealed haemolytic activity but none of these four isolates were associated with presence of any of the toxigenic genes tested.

All the 91 E. coli isolates were screened for the presence of toxigenic genes by PCR based detection for heat labile (LT) enterotoxin, heat stable (ST) enterotoxin and verotoxin (VT1 and VT2) genes. In the present study the primer sets LT1-LT2 and micro ST1-micro ST2 gave products of expected sizes i.e. 132 bp for LT and 171 bp for ST genes, using reference enterotoxigenic strain (MTCC 723). Out of 91 E. coli isolates tested using above mentioned primers one isolate (1.09%) revealed to possess ST gene and two isolates (2.19%) harboured LT gene in PCR technique.

In the present study primer directed amplification of the VT1 and VT2 genes gave the products as predicted from the design of primers i.e. 130 bp for VT1 primers and 228 bp for the VT2 primers. Out of 91 E. coli tested, 41 (45.05%) were found to be positive for VT genes. Among the isolates 32 (35.16%) isolates harboured both VT1 and VT2 genes while six E. coli isolates (6.5%) harboured only VT2 gene and 3 (3.25%) isolates had VT1 gene only. Overall 38 (41.75%) isolates revealed presence of VT2 gene while 35 (38.46%) possessed VT1 gene as evidenced by PCR amplification.

In this study, one of the most important serogroup O157 (3 isolates) known to cause certain life-threatening infections in humans and animals was isolated. Out of three O157 isolates two were positive for VT gene. Moreover, prevalence of verotoxin genes among the non-O157 isolates was also detected in PCR amplification technique.

In the present investigation it was also observed that within some serogroups both VT positive and VT negative isolates are prevalent.

The present study led to the following conclusions: 1. A variety of E. coli serotypes might be associated with calf diarrhoea, which needs for

strict monitoring and surveillance for effective measures for controlling neonatal calf diarrhoea.

2. Serotypes like O8, O26, O55, O86, O126, O128 and O157 which were recovered during the present investigation have also been isolated frequently from infantile diarrhoea among neonates, adult human patients suffering from gastroenteritis. These serotypes might be of zoonotic importance.

3. Some VT gene positive untypable isolates were found in this study. They certainly do deserve special attention for further studies and should be considered along with predominant toxin producing E. coli when formulating future control measures.

4. Atypical biochemical characters and colicinogeny of the isolates along with multiple drug resistance may serve as markers or tools for epidemiological surveys to trace the source of infection in disease outbreaks, particularly when it involves a particular area or an organized farm. This is helpful in epidemiological survey whenever the disease occurs among the animals.

5. Distribution of E. coli into different 24 groups of fermentative biotype suggests the biodiversity amongst E. coli population. Further, it is supported by occurrence of different serotypes under same biotype and vice-versa.

6. The pattern of multiple drug resistance prevailed in organized and unorganized farms are also helpful as guideline for clinical approach in forms of antibiotic therapy.

7. Epidemiological studies to determine the significance of toxigenic E. coli viz. verotoxigenic E. coli and LT/ST producing enterotoxigenic E. coli, mediated diseases could be established by PCR technique, which is relatively simple, highly sensitive and specific. PCR technique has also increased the rapidity of detection of toxigenic isolates.

8. High rate of prevalence of VT positive E. coli have been found in bovine herds, thus screening of larger number of cattle population should be carried out for future studies.

9. Future research efforts must target O157 as well as non O157 VTEC isolates for their role in human and animal infections viz. diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome. Studies regarding epidemiological and zoonotic potential of VTEC isolates need special emphasis for improved diagnosis, control and surveillance measures.

REFERENCES

REFERENCES Aalback, B.; Rasmussen, J.; Nielson, B. and Olsen, J.E. (1991). Prevalence of antibiotic

resistant E. coli in Danish pigs and cattle. A.P.M.I.S. 99: 1103-1110. Adetosoye, A.I. and Ojo, M.O. (1983). Transfer of urease production amongst E. coli

isolated from diarrhoeic goats and piglets. Tropical Veterinarian 1: 141-145. Aditi, Prabhu; Mondkar, A.D. and Madkarni, M.S. (1981). Haemolysin and necrotoxin

production of E. coli with special reference to serotypes causing urinary tract infection. Indian J. Pathol. Microbiol. 24:33-38.

Ahmad, I.; Ahmad, S. and Yadava, J.N.S. (2004). Incidence of colicin production among the pathogenic strains of E. coli and transferability to Col. Plasmids to Salmonella typhimurium. Indian Vet. Med. Jour. 28: 340-344.

Akiba, T.; Koyama, K.; Ishiki, Y.; Kimura, S. and Fukushima, T. (1960). Studies on the mechanism of the development of multiple drug-resistant clones of Shigella. Jap. J. Microbiol., 4: 219-227.

Al-Dabbas, A.H.M. and Willinger, H. (1996). Properties of E. coli strains from calves with diarrhoea before weaning. Weiner Tierarztliche Monatssch. 73: 217-222.

Alexander, T.J.L. (1994). Neonatal diarrhea in pigs, p.151-170. In C.L. Gyles (ed.) Escherichia coli in domestic animals and humans. CAB International, Wallingford, United Kingdom.

Arora, N.S. and Prabhakar, H. (1984). A study of pathogenicity of E. coli isolated from different sources. Indian J. Pathol. Microbiol. 27: 57-62.

Arunachalam, T.N.;Jayaraman, M.S. and Balaprakasam, R.A. (1974). Serological typing, colinogenecity and colicin sensitivity of E. coli strains associated with colisepticaemia and enteritis in poultry. Indian Vet. J. 51: 203-209.

Ayhan, H. and Aydin, N. (1989). Studies in the colicinogenic characteristics of E. coli strains isolated from man and animals. Veteriner Mikrobiyoloji Dirgisi. 6: 91-99.

Barbour, E.K.; Nabbut, N.H. and At Nakhi, H.M. (1985). Production of H2S by E. coli isolated from poultry. An unusual character useful for epidemiology of colisepticaemia. Avian Diseases. 29: 341-346.

Barker, R. and Old, D.C. (1979). Biotyping and colicin typing of Salmonella typhimurium strains of phage type 141 isolated in Scotland. J. Med. Microbiol. 12: 265-276.

Barr, J.G. and Hogg, G.M. (1979). Biotypes of Klebsiella pneumoniae and Enterobacter aerogenes characterized by differential substrate metabolism: application of the technique. J. Clin. Pathol. 32: 935-943.

Barrow, G.I. and Feltham, R.K.A. (1993). Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd edn., 140-43, Cambridge University Press, Cambridge.

Basta, M.; Karmali, M. and Lingwood, C. (1989). Sensitive receptor-specified enzyme-linked immunosorbent assay for Escherichia coli verocytotoxin. J. Clin. Microbiol. 27: 1617-1622.

Bauditz, R. (1987). Results of clinical studies with Baytril in calves and pigs. Vet. Med. Rev. 2:122-129.

Bauer, A.W.; Kirly, W.M.M.; Sherris, J.C. and Turck, M.(1966). Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Path. 45: 493-496.

Bauernfeind, A.; Abele-Horn, M.; Emmerling, and Jungwith, R. (1994). Multiclonal emergence of ciprofloxacin-resistant clinical isolates of Escherichia coli and Klebsiella pneumoniae. J. Antimicrob. Chemother.34:1075-1076.

Bettelheim, K.A. and Beutin, L. (2003). Rapid laboratory identification and characterization of verocytoxigenic (Shiga toxin producing) Escherichia coli (VTEC/STEC). J. Appl. Microbiol. 95: 205-217.

Beutin, L.; Geier, D.; Steinrück, H.; Zimmermann, S. and Scheutz, F. (1993). Prevalence and some properties of verotoxin (Shiga-like toxin)-producing Escherichia coli in seven different species of healthy domestic animals. J. Clin. Microbiol. 31:2483-2488.

Beutin, L.; Montenegro, M.A.; Orskov, I.; Orskov, F.; Prada, J.; Zimmermann, S. and Stephan, R. (1989). Close association of verotoxin (shiga-like toxin) production with enterohemolysin production in strains of E. coli. J. Clin. Microbiol. 27: 2559-2564.

Bisht, D.; Pande, R.C.; Gupta, S.C. and Mehrotra, T.N. (1977). Haemolysin and necrotoxin production of E. coli isolated from various sources. Indian J. Pathol. Microbiol. 20: 223-229.

Blanco J.; Blanco M.; Blanco, J.E.; Mora, A.; Gonalez, E.A. and Bernardez, M.I. (1996). O serogroups, biotypes and eae genes in Escherichia coli strains isolated from diarrhoeic and healthy rabbits. J. Clin. Microbiol. 34: 3101-3107.

Blanco J.; Blanco M.; Blanco, J.E.; Mora, A.; Gonalez, E.A.; Bernardez, M.I. and Alonso, M.P. (2003). Verotoxin-producing Escherichia coli in Spain: prevalence, serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants, raw beef products and humans. Exp. Biol. Med. 228:345-351.

Blanco, J.; Gonzalez, E.A.; Garcia,S.; Blanco, M.; Regueiro, B. and Bernardez, I. (1988). Production of toxins by Escherichia coli strains isolated from calves with diarrhea in Galicia (North-western Spain). Vet. Microbiol. 18: 297-311.

Blanco, J.E.; Blanco, M.; Mora, A. and Blanco, J. (1997). Production of toxins (Enterotoxins, verotoxins and necrotoxins) and colicins by Escherichia coli strains isolated from septicaemic and healthy chickens: Realtionship with in vivo pathogenicity. J. Clin. Microbiol. 35:2953-2957.

Boro, B.R.; Sarma, A.K. and Sarma, G. (1983). Studies on the strains of E. coli isolated from various clinical conditions in animals. Indian Vet. J. 60: 245-249.

Bőttner, A.; Schmid, P. and Humke, R. (1995). In vitro efficacy of cefquinome (INN) and other anti-infective drugs against bovine bacterial isolates from Belgium, France, Germany, The Netherlands, and The United Kingdom. J. Vet. Med. 42:377-383.

Braaten, B.A. and L.L., Myers. (1977). Biochemical characteristics of enterotoxigenic and non-enterotoxigenic Escherichia coli isolated from calves with diarrhea. Am. J. Vet Res. 38: 1989-1991.

Bradford, P.A.; Petersen, P.J.; Fingerman, I. M. and White, D.G. (1999). Characterization of expanded-spectrum cephalosporin resistance in E. coli isolates associated with bovine calf diarrheal disease. J. Antimicrobial Chemotherapy. 44: 607-610.

Burnens, A. P.; A. Frey; H. Lior and J. Nicolet. (1995). Prevalence and clinical significance of Vero-toxin-producing Escherichia coli (VTEC) isolated from cattle in herds with and without calf diarrhea. J. Vet. Med. 42:311-318.

Camguilhem, R. and Milon, A. (1989). Biotypes and serogroups of Escherichia coli involved in intestinal infections of weaned rabbits: clues to diagnosis of pathogenic strains. J. of Clin. Microbiol.27: 743-747.

Chachra, D. and Katoch, R.C. (1996). Prevalence of Escherichia coli and Salmonella among domestic poultry in Himachal Pradesh. Indian J. Poult. Sci. 31: 38-44.

Chattopadhyay, U.K.; Dutta, S.; Deb, A. and Pal, D. (2001), Verotoxin producing Escherichia coli- an environment-induced emerging zoonosis in and around Calcutta. Int. J. Environ. Health Res.1: 107-112.

Chattopadhyay, U.K.; Gupta, S. and Dutta, S. (2003). Search for Shiga toxin producing Escherichia coli (STEC) including O157:H7 strains in and around Kolkata. Indian J. Med. Microbiol. 21: 17-20.

Chauhan, R.S. and Kaushik, R.K.(1991). Isolation of enterotoxigenic Escherichia coli from calves with diarrhoea. Vet Microbiol. 29: 195-197.

Cid, O.; Piriz, S.; Riuz-Santa-Quiteria.; J.A., J., Vadillo, S. and de la Fuente, R. (1996). In vitro susceptibility of Escherichia coli strains isolated from diarrhoeic lambs and goat kids to 14 antimicrobial agents. J. Vet. Pharmacol. Therap., 19: 397-401.

Clarke, R. C.; J. B. Wilson.; S. C. Read.; S. Renwick.; K. Rahn.; R. P. Johnson.; D. Alves.; M. A. Karmali.; H. Lior.; S. A. McEwen.; J. Spika, and C. L. Gyles. (1994). Verocytotoxin-producing Escherichia coli (VTEC) in the food chain: preharvest and processing perspectives, p. 17-24. In M. A. Karmali, and A. G. Goglio (ed.), Recent advances in verocytotoxin-producing Escherichia coli infections. Elsevier Science B.V., Amsterdam, the Netherlands.

Cloud, S.S.; Rosenberger, J.K.; Fries, P.A.; Wilson, R.A. and Odor, E.M. (1985). In vitro and in vivo characterization of avian Escherichia coli.I. Serotypes, metabolic activity and antibiotic activity and antibiotic sensitivity. Avian Dis. 29: 1084-1093.

Cobbold, R.N.; Rice, D.H.; Szymanski,M.; Call, D.R,and Hancock, D.D.(2004). Comparison of Shiga- Toxigenic Escherichia coli prevalences among dairy, feedlot and cow calf herds in Washington State. App. Env. Microbiol. 70: 4375-4378.

Collee, J.G.; Fraser, A.G.; Marion, B.P. and Simmons, A. (1996). Mackie and McCartney's Practical Medical Microbiology, 4th edn., Churchill Livingstone, New York.

Cooper, P.C. and James, R. (1984). Two new E colicins, E8 and E9, produced by strains of E. coli. J. Gen. Microbiol. 130: 209-215.

Corlu, M. (1988). Investigation on biochemical, colicinogenic and MRHA characteristics of E. coli strains isolated from healthy and diarrhoeic lambs. Veterina Fakultesi Dergisi 60:4469.

Crichton, P.B. and Old, D.C. (1982). A biotyping scheme for the subspecific discrimination of Escherichia coli. J. Med. Microbiol. 15:233-241.

De Rycke, J.; Bernard, S.; Laport, J.; Naciri, M.; Popoff, M.R. and Rodolakis, A. (1986). Prevalence of various enteropathogens in the faeces of diarrhoeic and healthy calves. Ann. Rech. Vet. 17: 159-168.

De, S.N.; Bhattacharya, K. and Sarkar, J.K. (1956). A study of the pathogenicity of strains of Bacterium coli from acute and chronic eneteritis. J. Path. Bact. 71: 201-209.

Djonne, B.K. (1985). Colicin production in relation to pathogenicity factors in strains of E. coli isolated from the intestinal strains of piglets. Acta Veterinaria Scand 26:145-152.

Donnenberg, M. S.,and Nataro, J. P. (1995). Methods for studying adhesion of diarrheagenic Escherichia coli. Methods Enzymol. 253:324-336.

Donowitz, G. R. and Mandell, G. L. (1988). Beta-Lactam antibiotics, N. Engl. J. Med., 318: 419-426, 490- 500.

Drasar, B.S. and Hill, M.J. (1974). Human intestinal flora.p36-43. Academic Press Ltd. London, United Kingdom.

Dubey, S. and Sharda, R. (2001). Prevalence, serotypes and pathogenecity of E. coli associated with diarrhoea in goats. Indian J. Anim. Sc., 71: 92-94.

Dubey, S.; Sharda, R.; Chhabra, D.; Sharma, V.; Reddy, A.G. and Sharma, R.(2000). Enterotoxigenic Escherichia coli strains isolated from diarrhoeic goats. Ind. J. Comp. Micro. Immuno. Infect. Dis.21:68-69.

Edwards, R. and Ewing, W.N. (1972). Identification of Enterobacteriaceae. 3rd edn., Burgess Publishing Co., Minnesota.

Elwell, L.P and Shipley, P.L. (1980). Plasmid mediated factors associated with virulence of bacteria to animals. Annu. Rev. of Microbiol. 34: 465-496.

Eriksson E.; Aspon A.;Gunnarsson A. and Vagsholm I (2005).Prevalence of verotoxin-producing Escherichia coli (VTEC) O157 in Swedish dairy herds. Epidemiol. Infect. 133: 349-358.

Evans, D.G.; Evans, D.J. and Pierce, N.F. (1973). Differences in the response of rabbit small intestine to heat labile and heat stable enterotoxins of Escherichia coli. Infect. Immun. 7: 873-880.

Friedrich, A.W.; Kőck, R.; Bielaszewska, M.; Zhang, W.; Karch,H. and Mathys, W. (2005). Distribution of the urease gene cluster among and urease activities of enterohemorrhagic Escherichia coli O157 isolates from humans. J. Clin. Microbiol. 43: 546-550.

Fredericq, P. (1957). Colicins. Annu.Rev. Microbiol.11:7-22. Galton, M.M. and Hess, M.E. (1946). Hydrogen sulfide formation by Shigella alkalescens. J.

Bacteriol. 52: 143. Gerald, M. L. and Williams, P. A (Jr.). (1996). Sulfonamide, Trimethoprim-

sulfamethoxazole, Quinolone, and agents for urinary tract infections. Antimicrobial agents. In: The Pharmacological Basis of Therapeutics. Hardman, J. G., Limbrit, L. E., Molinoff, P. B., Ruddon, R. W. and Gilman, A.G. (eds.) 9th edn., pp. 1057-1070., McGraw-Hill, New York.

Giannella, R.A. (1976). Suckling mouse model for detection of heat stable Escherichia coli enterotoxin: characteristics of the model. Infect. Immun. 14:95-99.

Goldstein, F. W.; Papodopoulou, B. and Acar, J. R. (1986). The changing pattern of trimethoprim resistance in Paris, with a view of world wide experience. Rev. Infect. Dis., 8: 725-737.

Hariharan, H.; Mada C.; Poole, D.and Page, R. (2004). Antibiotic resistance among enterotoxigenic Escherichia coli from piglets and calves with diarrhea. Can. Vet. J. 45: 605-606.

Harnett, N.M. and Gyles, C.L. (1984). Resistance to drugs and heavy metals, colicin production and biochemical characterization of selected bovine and porcine E. coli strains. Appl. Environ. Microbiol. 48:930-935.

Heller, E.D. and Smith, H.W. (1973). The incidence of antibiotic resistance and other characteristics amongst Escherichia coli strains causing fatal infection in chickens: the utilization of these characteristics to study the epidemiology of the infection. J. Hyg. 71: 771-781.

Heuvelink, A. E.; Van de Kar, N. C. A. J.; Meis, J. F.; G. M., Monnens, L. A. H. and Melchers, W. J. G. (1998). Characterization of verocytotoxin-producing Escherichia coli O157 isolates from patients with haemolytic uraemic syndrome in Western Europe. Epidemiol. Infect. 115:1-14.

Hinton, M.; Allen, V. and Linton, A.H. (1982). The biotyping of E. coli isolated from healthy farm animals. J. Hyg. (Lond.). 88: 543-555.

Hinton, M.; Kaukas, A.H. and Linton, A.H. (1986). The ecology of drug resistance in enteric bacteria. J. Appl. Bact., (Symposium Suppl.), 577- 592.

Houvinen, P. (1987). Trimethoprim resistance. Antimicrob. Agents Chemother 31: 1451-1456 .

Hussain, I. and Saikia, G.K. (2000). Isolation and characterization of bacteria from diarrhoeic bovine calves. Indian J. Comp. Microbiol. Immunol. Infect. Dis. 21: 125-127.

Hussain, I; Saikia, G.K. and Rahman H.(2003). Virulence factors of Esherichia coli isolated from diarrhoeic calves. Indian. J. Comp. Microbiol. Immunol. Infect. Dis. 24:63-66.

Ishiguro, N.; Oka, C. and Sato, G. (1978). Isolation of citrate positive variants of E. coli from domestic pigeons, pigs, cattle and horses. Appl. Env. Microbiol. 36: 217-222.

Ishiguro, N. and Sato, G. (1979). The distribution of plasmids determining citrate utilization in citrate positive variants of E. coli from humans, domestic animals, feral birds and environments. J. Hyg. 83:331-344.

Irino, K.; Kato, M.A.M.F.; Vaz, T. M. I.; Ramo, I.I.; Souza, M.A.C., Cruz, A.S.; Gomes, T.A.T.; Vieira, M.A.M. and Gath, B.E.C. (2005). Serotypes and virulence markers of shiga toxin producing E. coli (STEC) isolated from dairy cattle in Sãu Paulo State, Brazil. Vet. Microbiol. 105: 29-36.

Jackson, M.P.; Newland, J.W.; Holmes, R.K. and O’Brien, A.D. (1987) Nucleotide sequence analysis of the structural genes for Shiga-like toxin I encoded by bacteriophage 933J from Escherichia coli. Microbial Pathogenesis. 2: 147–153.

Johnson, R. P.; Clarke, R. C.; Wilson, J. B.; Read, S. C.; Rahn, K.; Renwick, S. A.; Sandhu, K. A.; Alves, D.; Karmali, M. A.; Lior, H.; McEwen, S. A.; Spika, J. S. and Gyles, C. L. (1996). Growing concerns and recent outbreaks involving non-O157:H7 serotypes of verotoxigenic Escherichia coli. J. Food Prot. 59:1112-1122.

Joon D.S. and Kaura Y.K.(1993). Isolation and characterization of some of enterotobacteria from diarrhoeic and non-diarrhoeic calves. Indian J. Anim. Sc. 63: 373-383.

Kandel, G.; Donohue-Rolfe, A.; Donowitz, M. and Keusch, G. T. (1989). Pathogenesis of Shigella diarrhea XVI. Selective targetting of shiga toxin to villus cells of rabbit jejunum explains the effect of the toxin on intestinal electrolyte transport. J. Clin. Invest. 84:1509-1517.

Kapoor, K.N. and Kulshreshtha, S.B. (1994). Multiple drug resistance of E. coli isolates from diarrhoel patients in relation to enterotoxin production. Indian Vet. Med. J., 18: 18-22.

Kapusnilk-uner, J. E.; Sande, M. A. and Chambers, H. F. (1996). Antimicrobials agents- Tetracyclines, Chloramphenicol, Erythromycin and miscellaneous antibacterial agents. In: Goodman and Gillman’s, The Pharmacological Basis of Therapeutics. Hardman, J. G., Limbrid, L. E., Molinoff, P. B., Rudden, R. W. and Gillman, A. G. (eds.), 9th edn., pp. 1123-1153, McGraw Hill, New York.

Karchmer, A. W. (1995). Cephalosporin. In: Mandell, Douglas and Bennett’s Principles and Practice of Infectious Disease. Mendell, G. L., Bennett, J. E. and Dolin, R. (eds.), 4th edn., pp. 247-263, Churchill Livingstone, New York.

Karmali, M. A.(1989). Infection by verocytotoxin-producing Escherichia coli. Clin. Microbiol. Rev. 2:15-38.

Kaura, Y.K.; Minakshi; Kumar, T. and Chaturvedi, G.C. (1991). Characteristics of E. coli strains isolated from diarrhoeic buffalo and cow calves. Indian J. Microbiol. 31: 33-47.

Keenan, K. P.; Sharpnack, D. D.; Collins, H.; Formal, S. B. and O'Brien, A. D. (1986). Morphological evaluation of the effects of Shiga toxin and E. coli Shiga-like toxin on the rabbit intestine. Am. J. Pathol. 125:69-80.

Khan, A.; Yamasaki, S.; Sato, T.; Ramamurthy, T.; Pal, A.; Datta, S.; Chowdhury, N.R.; Das, S.C.; Sikdar, A.; Tsukamoto, T.; Bhattacharya, S.K.; Takeda, Y. and Nair, G.B. (2002). Prevalence and genetic profiling of virulence determinants of non-O157 Shiga toxin-producing Escherichia coli isolated from cattle, beef and humans, Calcutta, India. Emerg. Infect. Dis. 8: 54-62.

Kim, K.S. and Tak, R.B. (1984). Studies on pathogenic E. coli isolated from chickens with colibacillosis.II. Antimicrobial drug resistance and transferable R-plasmids in E. coli. III. Biochemical properties and conjugal transfer of citrate utilizing ability in citrate positive strains of E. coli. IV. Isolation and characterization of R-plasmid deoxyribonucleic acid. Korean J. Vet. Pub. Hlth. 8: 1-10.

Kobayashi, H.; Shimada, J.; Nakazawa, M.; Morozumi, T.; Pohjanvirta, T,; Pelkonen, S. and Yamamoto, K. (2001). Prevalence and characteristics of Shiga toxin-producing Escherichia coli from healthy cattle in Japan. Appl. Environ. Microbiol.67: 484-489.

Kojima, T.; Inoue, M. and Mitsuhashi, S. (1989). In vitro activity of AT-4140 against clinical bacterial isolates. Antimicrob. Agents Chemother. 33:1980-1988.

Kongmuang, U., Honda, T. and Miwatani. (1987). Enzyme linked immunosorbent assay to detect Shiga toxin of Shigella dysenteriae and related toxins. J. Clin. Microbiol.25:115-118.

Konowalchuk, J.; Speirs, J.I. and Starvie, S. (1977). Vero response to a cytotoxin of Escherichia coli. Infect. Immun. 18:775-779.

Krishnan, C.; Fitzgerald, V.A.; Dakin, S.J. and Behme, R.J. (1987). Laboratory investigation of outbreak of haemorrhagic colitis caused by Escherichia coli O157:H7. J. Clin. Microbiol. 25: 1043-1047.

Kumar, A.; Sharma, V.D. and Thapliyal, D.C.(1982). Occurrence of enteropathogenic/ enterotoxigenic Escherichia coli in cow calves and infants/children. Indian J. Comp. Micro. Immuno. Inf. Dis. 3:174-177.

Kumar,S. (1990). M.V.Sc. Thesis submitted to CCS Haryana Agriculture University, Hisar. Lang, A. L.; Tsai, Y. L.; Mayer, C. L.; Patton, K. C. and Palmer, C. J. (1994). Multiplex

PCR for detection of the heat-labile toxin gene and Shiga-like toxin I and II genes in Escherichia coli isolated from natural waters. Appl. Environ. Microbiol. 60:3145-3149.

Larulle, L. (1889). Cellule. 5:59. (cited by Sojka, 1965). Latif, M.S.A. (1982). A survey study on the incidence and role of Salmonella and E. coli in

diarrhoeal diseases of buffalo calves. M.V.Sc. thesis, Gujarat Agricultural University. Sardar Krushinagar.

Layne, P.; Hu, A.S.; Balows, A. and Davis, B.R. (1971). Extrachromosomal nature of hydrogen sulfide production in E. coli. J. Bacteriol. 106: 1029-1030.

Lee, H.J. and Choi, W.P. (1983). Isolation of citrate utilizing variants of E. coli from cattle, pigs, chickens,pigeons and wild animals. Korean J. Vet. Res. 23: 173-178.

Lehn, N.; Stőwer-Hoffmann, J.; Kott,T.; Strassner C.; Wagner, H.; Krőnke, M. and Schneider-Brachert W. (1996). Characterization of clinical isolates of Escherichia coli showing high levels of fluoroquinolone resistance. J. Clin. Microbiol. 34:597-602.

Levine, M.M. (1987).Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohaemorrhagic and enteroadherent. J. Infect. Dis. 155: 377-89.

Lior,H.(1996). Classification of Escherichia coli p31-72. In: C.L. Gyles (ed.) Escherichia coli in domestic animals and humans CAB International, Wallingford, United Kingdom.

Mainil. J.A.(1999). Shiga/Verotoxins and Shiga/Verotoxigenic Escherichia coli in animals. Vety. Res. 30:235-257.

Manson, I. (1962). Biochemical properties of haemolytic E. coli strains belonging to O-groups 138, 139 and 141. Acta Veterinaria Scandinavia. 3:79-87.

Mehrotra, P.K.; Lakhotia, R.L. and Mehrotra, P.N. (1984). Occurrence of serotypes and antibiotic resistance in E. coli isolates from different species. Indian J. Anim. Sc. 54: 396-399.

Melton-Celsa, A. and O’Brien, A. (2003). Plant and bacterial toxins as RNA N-glycosidases. In Bacterial Protein Toxins ed. Burns, D.,Barbieri, J.T., Iglewski, B.H. and Rappuoli, R. pp. 245–255.Washington, DC: ASM Press.

Merchant, I.A. and Packer,R.A.(1967).Veterinary Bacteriology and Virology 6th Ed..Iowa State University Press USA.

Michino, H.; Araki, K.; Minami, S.; Nakayama, T.; Ejima, Y and Hiroe, K. et al. (1998). Recent outbreaks of infections caused by Escherichia coli O157:H7 in Japan. In Escherichia coli O157:H7 and Other Shiga-Toxin-Producing E. coli Strains ed. Kaper, J.B. and O'Brien, A.D. pp. 73681.Washington, DC: American Society for Microbiology.

Minakshi; Kaura, Y.K. and Chaturvedi, G.C. (1992). Cell surface hydrobhobic property of E. coli isolated from diarrhoeic buffalo and cow calves. Indian J. Microbiol. 32: 69-73.

Mishra, A.; Sharda, R.; Chhabra, D. and Moghe, M.N. (2002). Escherichia coli isolates from domestic poultry. Indian J. Anim. Sc. 79: 727-729.

Mondaca, M.A.; Jaramilo, S; Henriquez, M.; Montoya, R. and Zemelman, R. (1985). Production of H2S, raffinose ferementation and tetracycline resistance in strains of E. coli isolated from pigs and related sources. Microbios Letters. 29: 75-78.

Moseley, S. L.; Echeverria, P.; Seriwatana, J.; Tirapat, C.; Chaicumpa, W.; Sakuldaipeara, T. and Falkow, S. (1982). Identification of enterotoxigenic Escherichia coli by colony hybridization using three enterotoxin gene probes. J. Infect. Dis. 145:863-869.

Murinda, S.E.; Batson, S.D.; Nguyen, L.T.; Gillespie, B.E. and Oliver, S.P. (2004). Foodborne Pathogens and Disease. 1:125-135.

Nakao, H .and Takeda, T.,( 2000). Escherichia coli Shiga toxin. J. Nat.Toxins. 9: 299– 313. Nataro J.P. and Kaper J.B.(1998). Diarrheagenic Escherichia coli. Clin. Microbiol. Rev.

11:142-201. Nishikawa, Y.; Zhou, Z.; Hase, A.; Ogasawara, J.; Kitase, T.; Abe, N.; Nakamura, H.; Wada,

T.; Ishii, E.; Haruki, K. and the Surveillance Team (2002). Diarrheagenic Escherichia coli isolated from stools of sporadic cases of diarrheal illness in Osaka city, Japan between 1997 and 2000: Prevalence of Enteroaggregative E. coli heat stable enterotoxin 1 gene- possessing E. coli. Jpn. J. Infect. Dis. 55: 182-190.

O’Brein, A.D. and Holmes, R.K. (1987). Shiga and Shiga-like toxin. Microbiol. Rev. 1:206-220.

O’Brien, A.D. and La Veck, G.D. (1983). Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli. Infect. Immun. 40: 675-683.

O'Brien, A. D.; Newland, J. W.; Miller, S. F.; Holmes, R. K.; Smith, H. W. and. Formal, S. B. (1984). Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or infantile diarrhea. Science 226:694-696.

Orden, J.A.; Ruiz-Santa-Quiteria.; J.A.;Garcia S.; Dolores C.I.D. and Fuente R.D.(1999). In vitro activities of cephalosporins and quinolones against Escherichia coli strains isolated from diarrheic calves. Antimicrob. Agents Chemother.43:510-513.

Orskov, I. and Orskov, F. (1973). Plasmid-determined H2S character in Escherichia coli and its relation to plasmid-carried raffinose fermentation and tetracycline characters: examination of 32 H2S- positive strains isolated during the years 1950-1971. J. Gen. Microbiol. 25:467-499.

Oswald, E.; De Rycke, J.; Lintermans, P.; Van Muylem, K.; Mainil, J.; Daube, G. and Pohl, P. (1991). Virulence factors associated with cytotoxic necrotizing factor type 2 in bovine diarrhoeic and septicemic strains of Escherichia coli. J Clin. Microbiol. 29: 2522-2527.

Pal, A.; Ghosh, S.; Ramamurthy, T.; Yamasaki, S.; Tsukamoto, T. and Bhattacharya, S.K. (1999). Shiga-toxin producing Escherichia coli from healthy cattle in a semi-urban community in Calcutta, India. Indian J. Med. Res. 110:83-85.

Panda, N.N. and Panda, S.N. (1987). Studies on calf diarrhea in Orissa with special reference to colibacillosis. Indian J. Anim. Hlth. 26: 109-112.

Pandey, P.N.; Thapliyal, D.C. and Sharma, S.N. (1979). Enterotoxigenicity of some Esherichia coli isolates. Indian J. Anim. Res. 13:1-4.

Patil, R.M.; Karpe, A.G. and Gujar, M.B.(1999). Studies on drug resistance and virulence of Escherichia coli from diarrheic calves. Indian J. Comp. Micro. Immuno. Infect. Dis. 20:29-31.

Paton, J.C. and Paton, A.W. (1998). Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 11, 450–479.

Pohl, P.; Mainil, J.; Lintermans, P. and Kaeckenbeeck, A. (1989). Discovery in Belgium of four toxigenic E. coli strains of type S-102-9. Annales de Medicine Veterinaric. 133: 619-621.

Pohl, P.; Thomas, J. and Pycke, J.M. (1969). Antibiotic resistant Enterobacteriaceae in calf intestinal flora. Annls. Med. Vet. 113: 123-131.

Pollard, D. R.; Johnson, W. M.; Lior, H.; Tyler, S. D. and Rozee, K. R. (1990). Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction. J. Clin. Microbiol. 28:540-545.

Prescott, J.F., and Baggot, J.D. (1993). Fluoroquinolones, p.252-262. in Antimicrobial therapy in veterinary medicine, 2nd ed.Iowa State University Press, Ames.

Prescott, J. F.; Gannon, U. P.; Kittler, G. and Hlywka, G. (1984). Antimicrobial drug susceptibility of bacteria isolated from disease processes in cattle, horses, dogs and cats. Canad. Vet. J., 25: 289-292.

Rahman, H. (2002). Prevalence of verotoxin genes among non-enterohaemorrhagic Escherichia coli. Indian J. Comp. Microbiol. Immunol. Infect. Dis. 23: 189-190.

Rang, H. P.; Dale, M. M. and Ritter, J. M. (1995). Antibacterial agents. In: Pharmacology. 3rd edn., pp. 718-743, Churchill Livingstone, London.

Rao, M.V.S.; Kulshrestha, S.B. and Kumar, S. (1975). Serotyping and biochemical characterization of E. coli isolated from intestinal tract of poultry. Indian J. Anim. Sc. 45:779-783.

Rich, C.; Alfidja, A.; Sirot, J.; Joly, B. and Forestier, C. (2001). Identification of human enterovirulent Escherichia coli strains by multiplex PCR. J. Clin. Lab. Anal. 15: 100-103.

Riley, L.W.; Remis, R.S.; Helgerson, S.D.; Megge, H.B.; Wells, J.G.; Davis, B.R.; Herbent, R.J.; Olcott, E.S.; Johnson, L.M.; Hargrett, N.T.; Blake, P.A.and Cohen, M.L.(1983). Haemorrhagic colitis associated with a rare Escherichia serotype. New Eng. J. Med. 308: 681-685.

Salvadori, M.R.; Valadares, G.F.; Leite, D.S.; Blanco,J. and Yano, T. (2003). Virulence factors of Escherichia coli isolated from calves with diarrhea in Brazil. Brazilian Journal of Microbiology. 34:230-235.

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Analysis and cloning of eukaryotic genomic DNA in Molecular cloning. Cold Spring Harbor Laboratory Press. p 9-19.

Saravanbava, K.; Venugopalan, A.T. and Balaprakassam, R.A. (1990). Enteropathogenic E. coli associated with neonatal calf diarrhea. Cherion 19:29-34.

Saxena, S. (1976). Serologic study of E. coli isolated from animals. Effect of antibiotics and chemotherapeutic agents on E. coli. Indian Vet. J.53: 564-566.

Schmitz, F. J. and Fluit, A. C. (1999). Mechanisms of resistance, pp. 7.2.1-7.2.14. In: Infectious diseases. Armstrong D. and Cohen, S. (eds.), C. V. Mosby, Ltd., London, United Kingdom.

Scotland, S. M.; Smith, H. R. and Rowe, B. (1985). Two distinct toxins active on vero cells from Escherichia coli O157. Lancet. ii. 885-886.

Scotland, S. M.; Smith, H. R.; Willshaw, G. A. and Rowe, B. (1981). Vero cytotoxin production in strain of Escherichia coli is determined by genes carried on bacteriophage. Lancet ii:216.

Sears, C.L. and Kaper, J.B. (1996). Enteric Bacterial toxins: mechanisms of action and linkage to intestinal secretion. Microbiol. Rev. 60: 167-215.

Shah, N.M. (1989). Microbiological investigations of neonatal calf diarrhea with specific reference to detection of rota virus and enterotoxigenic E. coli.Ph. D. Thesis. Gujarat Agaricultural University. Sardar Krushinagar.

Sharma, D.K.; Soni, S.S.; Kashyap, S.K. and Shringi, B.N. (2004). Seroprevalence, antibiotic sensitivity pattern and transfer of plasmid coded characters of E. coli. Indian Vet. J. 81:6-8.

Sharma, V.D.; Thapliyal, D.C.; Singh, S.P. and Malik, P.(1992). Cytotonic and cytotoxic enterotoxin of enterobacteria. Indian J. Microbiol. 32:327-356.

Shaw D.J.; Jenkins C.; Pearce M.C.; Cheasty,T.; Gunno G.J.; Dougan G.; Smith, H.R.; Woolhouse, M.E.J. and Frankel G.(2004). Shedding pattern of Verotoxin producing Escherichia coli strains in a cohort of calves and their dams on a Scottish beef farm. Appl. Environ. Microbiol.70: 7456-7465.

Shaw, K. J.; Rather, P. N.; Hare, R.S. and Miller, G. H. (1993). Molecular genetics of aminoglycoside resistance genes and familial relationship of the aminoglycoside-modifying enzymes. Microbiol. Rev. 57:138-163.

Sherwood, D. and Snodgrass, D.R.(1983). Prevalence of ETEC in calves in Scotland and northern England. Vety. Res. 113:208-212.

Singh, M.; Chaudhari, M.A.; Yadav, J.N.S. and Sanyal, S.C. (1992). The spectrum of antibiotic resistance in human and veterinary isolates of E. coli collected from 1984-1986 in Northern India. J. Antimicrobial Chemotherapy 29:159-168.

Sivaswamy, G. and Gyles, C.L.(1976). The prevalence of enterotoxigenic Escherichia coli in the faeces of calves with diarrhea. Canad. J. Comp. Med. 40:241-246.

Smith H.W. (1965). The development of the flora of the alimentary tract in young animals. J. Path. Bact. 90: 495-513.

Smith, H.W. and Hall, S.(1966). Observations on infective drug resistance in Britain. Vet. Rec. 78:415-424.

Smith, H.W. and Hall, S.(1968). The experimental infection of calves with bacteremia producing strains of Escherichia coli. The influence of colostrums. J. Med. Microbiol.1 : 61-78.

Smith, H.W. and Huggins, M.B. (1978). The influence of plasmid determined and other characteristics of enteropathogenic E. coli on their ability to proliferate in the alimentary tracts of piglets, calves and lambs. J. Med. Microbiol. 11:471-492.

Soderlind, O.; Thafvelin, V. and Moolby, R. (1988). Virulence factors in Escherichia coli strains isolated from Swedish pigs with diarrhoea. J. Clin. Microbiol., 26: 879-884.

Sojka W.J.(1965).Escherichia coli in domestic animals and poultry 1st ed. Commonwealth Agricultural Bureaux, Farnham Royal, Bucks, England.

Sojka, W.J. (1971). Enteric diseases in newborn piglets, calves and lambs due to Escherichia coli infection. Veterinary Bulletin (London) 41: 509-522.

Speer, B. S.; Shoemaker, N. V. and Salyers, A. A. (1992). Bacterial resistance to tetracycline: Mechanisms, transfer, and clinical significance. Clin. Microbiol. Rev., 51: 387-399.

Srivastava, N.C. and Arya, S.C. (1979). E. coli serotypes in calves. Indian Vet. J. 56: 901-903.

Stacy-Phipps, S.; J. J. Mecca. and J. B. Weiss. 1995. Multiplex PCR assay and simple preparation method for stool specimens detect enterotoxigenic Escherichia coli DNA during the course of infection. J. Clin. Microbiol. 33:1054-1059.

Strockbrine, N.A.; Marques, L.R.M.; Newland, J.W.; Smith, H.W.; Holmes, R.k. and O’Brien, A.D. (1986). Two toxin- converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities. Infect. Immun. 53: 135-140.

Sutariya P.H. (1993). Studies on biochemical characters, drug resistance, colicinogeny and virulence associated characters of E. coli isolated from clinical samples. M.V.Sc. Thesis submitted to the Gujarat Agricultural University, Anand Campus, Anand.

Taylor, J.; Wilkins, M. P. and Payne, J. M (1961). Relation of rabbit gut reaction to enteropathogenic Escherichia coli. Br. J. Exp. Pathol. 42:43-52.

Tominaga, K.; Nakazawa, M.; Haritani, M and Hirata, K. (1989). Biochemical characteristics and pathogenicity of attaching and effacing E. coli (AEEC) isolated from calves with diarrhea. J. Japan Vet. Med. Assoc. 42:775-779.

Tripathi, R.D. and Soni, J.L.(1982). Antibiotic sensitivity test with E.coli isolated from cases of neonatal calf diarrhea. Indian Vet. J. 59:413-416.

Tripathi, R.D. and Soni, J.L.(1984). Enteropathogenic E.coli in neonatal calf diarrhea in crossbred calves. Indian Vet. J. 61:4-8.

Ueda, H.; Terakads, N.; Isayama, Y.; Sakurai, K. and Kakazawa, M.(1981). Etiological studies on enterotoxigenic Escherichia coli among isolates from domestic animals in Japan. National Institute of An. Health Quarterly- Japan. 21: 108-109.

Vial, P. A.; Browne, R. R.; Lior, H.; Prado, V.; Kaper, J. B.; Nataro, J. P.; Maneval, D.; Elsayed, A. and Levine. M. M. (1988). Characterization of enteroadherent-aggregative Escherichia coli, a putative agent of diarrheal disease. J. Infect. Dis. 158:70-79.

Visser, M.R.; Rozenberg-Arska, M.; Beumer, M.; Hoepelman, I.M. and Verhoef, J. (1991). Comparative in vitro antibacterial activity of sparfloxacin (AT-4140;RP 64206), a new quinolone. Antimicrob. Agents Chemother.35:858-868.

Wallace, J. S.; T. Cheasty, and K. Jones. (1997). Isolation of vero cytotoxin-producing Escherichia coli O157 from wild birds. J. Appl. Microbiol. 82:399-404.

Wani, S.A.; Bhatt, M.A.; Samanta, I.; Nishikawa, Y. and Buchh, A.S.(2003). Isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and lambs with diarrhoea in India. Lett. Appli. Microbiol. 37: 121-126.

Wani, S.A.; Bhatt, M.A.; Samanta, I.; Nishikawa, Y. and Buchh, A.S. (2004). Escherichia coli O116 associated with an outbreak of calf diarrhoea. Vet Rec. 154: 506-508.

Wani, S.A.; Bhatt, M.A.; Samanta, I.; Nishikawa, Y. and Buchh, A.S. (2005). Escherichia coli O4:NM associated with an outbreak of calf diarrhoea. Vet. J. 169: 300-302.

Wells, J. G., Shipman, L. D., Greene, K. D., Sowers, E. G., Green, J. H., Cameron, D. N., Downes, F. P., Martin, M. L., Griffin, P. M., Ostroff, S. M., Potter, M. E., Tauxe, R. V., and Wachsmuth, I. K. (1991). Isolation of Escherichia coli serotype O157:H7 and other Shiga-like-toxin-producing E. coli from dairy cattle. J. Clin. Microbiol. 29:985-989.

Whittam, T. S.; Wolfe, M. L.; Wachsmuth, I. K.; Orskov, F.; Orskov, I. and Wilson, R. A. (1993). Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea. Infect. Immun. 61:1619-1629.

Wilson, K. (1987). Preparation of genomic DNA from bacteria. In: Current protocols in molecular biology. Vol. 1 (Ausbedal, F.M., Brent, R.E., Kirston, D.D., Moore, J.G.; Seidman, J.A., Smith and Struhl, K. eds). John wiley and Sons. New York. 2.4.1.

Wray, C.; McLaren, I. M. and Carroll, P. J. (1993). E. coli isolated from farm animals in England and Wales between 1986 and 1991. Vet. Rec., 133: 439-442.

Yadava, J.N.S.; Ahmed, C.H. and Singh, M. (1988). Prevalence of enterotoxigenic E. coli in clinical isolates of man and animals. Indian J. Anim. Sc.58: 532-534.

Yadav, J.N.S.; Ansari, M.Q. and Reeta, Goel (1986a). Association of colicinogeny and antibiotic resistance among strains of E. coli of human origin. Indian Vet. J. 63: 261-265.

Yadav, J.N.S.; Saxena, V.K. and Ansari, M.Q. (1986b). Plasmids coding for enterotoxins, haemolysins and haemagglutinins and their role in virulence and pathogenicity of E. coli. Indian J. Path. Micro. 29:303-307.

Yamamoto,T. and Nakazawa M.(1997). Detection and sequences of the enteroaggregative Escherichia coli heat stable enterotoxin 1 gene in ETEC strains isolated from piglets and calves with diarrhea. J. Clin Microbiol. 35:223-227.

Zweifel, C.; Schumacker, S.; Blanco, M.; Blanco, J.E.; Tasara, T.; Blanco, J. and Stephen, R. (2005). Phenotypic and genotypic characteristics of non-O157 Shiga toxin-producing E. coli (STEC) from Swiss Cattle. Vet. Microbiol. 105: 37-45.

ANNEXURE - I Bile salt agar

Tryptone 10.0 g Sodium Chloride 5.0 g Bile salts 0.15 g Agar 13.0 g Distilled water 1000 ml Final pH 7.6 Autoclaved at 15 psi for 15 minutes. Soft overlay agar (3.6.2b) was same in

composition as above (3.5.2a) except agar was used at 0.5 per cent concentration. Blood Agar (BA) Blood agar base (Dehydrated, HiMedia) Ingredients Grams/liter Beef heart, infusion form 500.00 Tryptose 10.00 Sodium chloride 5.00 Agar 15.00 Final pH (at 25oC) 7.3 + 0.2 Suspended 40 gm of dehydrated blood agar base in 1000 ml distilled water and sterilized by autoclaving at 15 psi pressure, 121oC temperature for 20 minutes. The molten medium was cooled to about 50oC temperature and aseptically 5% v/v sterile defibrinated sheep blood was added. The above medium was mixed well and poured into sterile petri plates. Brain Heart Infusion Broth (BHI broth) (Dehydrated, HiMedia) Ingredients Grams/liter Peptic digest of animal tissues 10.00 Calf brain, infusion (solids)Yeast extract 12.50 Beef heart Infusion (solids) 5.00 Dextrose 2.00 Sodium chloride 5.00 Disodium phosphate 2.50 Final pH (at 25oC) 7.4 + 0.2 Suspended 37 gm in 1000 ml distilled water, distributed in test tube and sterilized by autoclaving at 15 psi pressure, 121oC for 20 minutes. Eosin Methylene Blue (EMB) Agar (Dehydrated, HiMedia) Ingredients Grams/litre Peptone 10.00 Lactose 10.00 Dipotassium hydrogen phosphate 2.00 Eosin Yellow 4.00 Methylene blue 0.065 Agar 25.00 Final pH (at 25 oC) 7.2

Suspended 36.00 gm of dehydrated EMB in 1000 ml distilled water and sterilized by autoclaving at 15 psi pressure, 121oC for 20 minutes. The molten medium was cooled to about 50oC temperature and poured into sterile petri plates. MacConkey Agar (MCA) (Dehydrated, HiMedia)

Ingredients Grams/litre Peptic digest of animal tissue 20.00 gm Lactose 10.00 gm Bile salt 5.00 gm Sodium chloride 5.00 gm Neutral red .07 gm Agar 15.00 gm Distilled water 1000.00 ml

Final pH (at 25oC) 7.5 + 0.2 Suspended 55.07 gm of dehydrated MCA in 1000 ml distilled water and sterilized

by autoclaving at 15 psi pressure, 121oC for 20 minutes. The molten medium was cooled to about 50oC temperature and poured into sterile petri plates. Mueller Hinton (MH) Agar No. 2 (Dehydrated, HiMedia) Ingredients Grams/litre Casein acid hydrolysate 17.50 Beef heart infusion 2.00 Starch, soluble 1.5 Agar 17.00 gm Final pH (at 25°C) 7.3 +0.2

Suspended 38 gm in 1000 ml distilled water. Sterilized by autoclaving at 15 psi pressure, 121oC for 20 minutes. The molten medium was cooled to about 50oC temperature and poured into sterile petri plates. Phosphate buffer agar:

Meat extracts 5.0 g Peptone 10.0 g

NaCl 3.0 g Na2HPO4 12 H2O 2.0 g Agar 10.0 g Distilled water 1000 ml Final pH 7.4

Distributed in test tubes and sterilized by autoclaving at 15 psi pressure, 121oC for 20 minutes. Simmons’ Citrate Agar (Dehydrated, HiMedia) Ingredients Grams/liter Magnesium sulphate 0.20 Ammonium dihydrogen phosphate 1.00 Dipotassium phosphate 1.00 Sodium citrate 2.00 Sodium chloride 5.00 Bromo thymol blue 0.08 Final pH (at 25oC) 6.8 + 0.2 Suspended 24.28 gm in 1000 ml distilled water, distributed in test tubes and sterilized by

autoclaving at 15 psi pressure, 121oC for 20 minutes.

2% Urea Agar Solution: 1

Peptone 1.0 gm Nacl 0.5 gm

Glucose 1.0 gm Potassium dihydrogen phosphate 2.0 gm Phenol red 0.012 gm (or 0.2% soln 6ml) Agar 15.0 gm Distilled Water 900.0 ml pH 6.8, Autoclave at, 15 psi pressure for 15 min Solution: 2

Urea 2.0 gm Distilled water 100.0 ml pH 6.8 to 6.9, Sterilize by seitz filter

Total 1000ml

Note: To constitute the medium Solution 2 was added in Solution 1, temperature of Solution 1 was brought down to 50°C in a water bath maintained at 50°C and then distributed in test tubes

ANNEXURE – 2 Indole Test

a) Kovac’s reagent Paradimethylaminobenzaldehyde 50 gm Pure amyl or isoamyl alcohol 75 ml Concentrated pure hydrochloric acid 25 ml Dissolve the aldehyde in the alcohol by gentle warming in a waterbath, cool and add the acid. Protect from light and store at 4°C temperature.

b) Tryptone water

Tryptone 10 gm Sodium chloride 5 gm Distilled water 1000 ml Dissolve the solids by stirring in the waterbath. Leave the reaction unadjusted and sterilize at 15 psi pressure 121oC temperature for 20 minutes.

Methyl Red (MR) Test

a) Glucose Phosphate Peptone Water (GPPW) Glucose 5.00 gm Peptone 5.00 gm Dipotassium hydrogen phosphate 5.00 gm Distilled water 1000.00 ml Sterilize by autoclaving at 10 psi pressure, 115 oC temperature for 30 minutes

b) Methyl Red (MR) reagent

Methyl red 0.1 gm Ethyl alcohol (95 to 96 per cent) 300.0 ml Dissolve the dye in alcohol and then add sufficient distilled water to make 500 ml.

Voges- Proskauer (VP) Test (Barrit’s method)

a) Glucose Phosphate Peptone Water (GPPW) Same as for the MR test

b) VP test reagents I. Five per cent α- naphthol in absolute ethanol

II. 40 per cent KOH containing 0.3 per cent creatine

Citrate Utilization Test Simmons’ Citrate Agar (Dehydrated, HiMedia) Ingredients Grams/liter Magnesium sulphate 0.20 mmonium dihydrogen phosphate 1.00 Dipotassium phosphate 1.00 Sodium citrate 2.00 Sodium chloride 5.00 Bromo thymol blue 0.08 Final pH (at 25oC) 6.8 + 0.2

Suspended 24.28 gm in 1000 ml distilled water, distributed in test tube and sterilized by autoclaving at 15 psi pressure, 121oC for 20 minutes. Carbohydrate fermentation test

a) Peptone water Peptone 10.00 gm Sodium chloride 5.00 gm Distilled water 1000.00 ml Final pH (at 25oC) 7.5

b) Andrade’s indicator 0.5 gm acid fuschin was dissolved in 100 ml distilled water. Then 16 ml of 1M

NaOH was added. 10 ml of indicator was used for each liter of medium. 1 gm of appropriate sugar was dissolved in 100 ml basal medium, distributed in test

tube and sterilized at 10 psi pressure, 115oC temperature for 30 minutes. Gram’s stain 1 Ammonium oxalate crystal violet Solution 1: Crystal violet 2.0 gm Ethyl alcohol (95 per cent) 20.0 ml Solution 2: Ammonium oxalate 0.8 gm Distilled water 80.0 ml Solution 1 and 2 were mixed well and then filtered. 2 Lugol’s (Gram’s) iodine solution Iodine 1.0 gm Potassium iodide 2.0 gm The ingredients were dissolved and then filtered. 3 Acetone or Ethyl alcohol (decolorizer) 4 Safranin (counter stain) Safranin-O (2.5 per cent solution) in 95 per cent alcohol 10 ml Distilled water 100 ml