Cairo University Faculty of Veterinary Medicine Department of Microbiology. Thesis Presented By

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1 Cairo University Faculty of Veterinary Medicine Department of Microbiology STUDIES ON ESCHERICHIA COLI IN CALVES Thesis Presented By Rehab Fathy El-Shafey El-Sayed (B.V.SC., Cairo University, 2000) For Master Degree In Veterinary Medical Sciences - Microbiology (Bacteriology, Immunology and Mycology) Under the Supervision of 2009

2 Under the Supervision of Prof. Dr. Mohamed Kamal Refai Professor of Microbiology Faculty of Veterinary Medicine Cairo University Prof. Dr. Soheir Shoukry Elias Ex.Chief Researchers of Bacteriology Animal Health Research Institute Dokki - Giza

3 1. INTRODUCTION Escherichia coli was first isolated from intestine of human and animals at the dawn of bacteriology was first described by Escherich as Bacterium coli commune (Escherich, 1885) Late 1950 and early 1960 witnessed a resurgence of interest in E. coli as an animal pathogen. The pioneering studies of Williams Smith and Walter. Sojka s group, Weybridge, led to important discoveries about the role of E. coli in enteric and septicaemic disease in animals.

4 1. INTRODUCTION Serotyping emerged as a major tool in understanding the association of specific groups of E. coli with disease in various animal species and its use by Sojka and the Фrskovs resulted in the discovery of the first fimbrial E. coli antigen (K88) shown to be responsible for intestinal colonization. Since 1965, there have been impressive progress in our understanding of virulence factors of E. coli and their contributions to the increasingly wide variety of disease recognized to be cuased by E. coli, other fimbriae that could confer intestinal colonizing ability and to the discovery by Sojka and Williams Smith of K99 fimbriae on E. coli from calf diarrhoea.

5 1. INTRODUCTION Enterotoxigenic E. coli (ETEC) infection, the most common type of diarrhoea in calves, has been associated with E. coli, which has two virulence factors namely adhesins and enterotoxins, which are mostly regulated on plasmids. Fimbrial antigens enable them to attach to and colonize on the villi of the small intestine. Strains in calves most commonly possess k99 (F5) or F41 fimbrial antigens or both. These antigens are the focus of immunological protection. Enterotoxigenic E. coli also elaborate non-antigenic, protein, thermostable enterotoxin (STa) which reduces absorption and increases fluid and elecetrolyte secretion of small intestinal epithelial cells (Nagy and Fekete, 2005).

6 2. The aim of this study was to: 1. Determine the incidence of pathogenic E. coli in diarrhoeic and apparently healthy cow calves. 2. Detect E. coli K99 antigen 3. Clarify the possible correlation between pathogenic E. coli and calf age. 4. Determine some of virulence factors of E. coli 5. Investigate the correlation between virulence factors and antigenic structure of E. coli isolated from calves. 6. Test sensitivity of E. coli isolated from claves to antimicrobial agents.

7 3. MATERIAL AND METHODS 1. Material: 1.1. Samples: 233 faecal samples were collected from cow-calves, out 193 were suffering from diarrhoea 40 from apparently healthy normal calves All calves aged from one day to six months. The samples were collected from calves before any trial of treatment has been applied.

8 3. MATERIAL AND METHODS 1.2. Diagnostic antisera The isolates were serologically identified by "Seiken" Escherichia coli diagnostic antisera for pathogenic types in this study. Sera 51 vials (polyvalent 8 vials and 43 monovalent vials. Product code , Japan. Polyvalent 1: O1, O26, O86A, O111, O119, O127A, O128. Polyvalent 2: O44, O55, O125, O126 and O146, O166. Polyvalent 3: O18, O114, O142, O151, O157, O158. Polyvalent 4: 6, O27, O78. O148, O159, O168. Polyvalent 5: O20, O25, O63, O513, O167. Polyvalent 6: O8, O15, O115, O169. Polyvalent 7: O28ac, O112AC, O124, O136, O144. Polyvalent 8: O29, O143, O152, O164.

9 3. MATERIAL AND METHODS 1.3. E. coli K99 antisera Standard type cultures strain of E. coli (E. coli 431 (O101: K30-99: F41: N:M.) was used in the preparations of K99 antisera. This strain was kindly supplied from National Animal Disease. Center, Agriculture Research Service, U.S. Department of Agriculture, Ames, Iowa

10 3. MATERIAL AND METHODS 1.4. Experimental laboratory animals: o Bouscat rabbits, kg body weight were used for the production of K99 diagnostic antisera. o These animal were brought from clean colony and they were apparently healthy. o They were kept separately for 4.5 weeks in clean boxes and a preventive course of broad septum antibiotic (tetracycline) was given to them, two weeks before use.

11 3. MATERIAL AND METHODS 1.5. Antibiotic sensitivity discs: Cephaloxin CL 30 Mg Penicillin P 10 units Ampicillin G ABP 10 µg Erythromycin E 15 µg Trimethoprim SXT 25µg Streptomycin S 10 µg Neomycin N 30 µg Amikacillin AK 30 µg Danox DNF 5 µg Gentamycin CN 120 µg Amoxicillin AML 10 µg

12 3. MATERIAL AND METHODS 2. Methods 1. Samples collection. 2. Bacteriological examination. 3. Determination of E. coli k99 antigen. 4. Bacterial culturing of faecal samples.

13 3. MATERIAL AND METHODS 2. Methods 5. Identification of the isolates. Morphological identification. Serotyping of isolates. Slide agglutination test for k99 pilus antigen. Preparation of immune sera (k99 antisera).

14 Table (1): The biochemical reaction of E. coli isolates (Quinn et al., 2002) V= Variable Test Result Motility + Catalase + Oxidase - Indole + Methyl red + Voges proskauer - Simmon's citrate - TSI Acid slant/acid butt Urease - Glucose AG Lactose AG Maltose AG Mannitol AG Sucrose V AG = Acid and gas - = Negative + = Positive

15 3. MATERIAL AND METHODS 2. Methods 5. Virulence tests: 1. Congo red binding activity (CR) (Berkhoff and Vinal, 1986) 2. Haemolytic activity 3. Haemagglutination test (HA) (Evans et al., 1979) 4. Detection of heat stable enterotoxin (STa) of E. coli 5. Infant mouse assay 6. Antibiogram assay of E. coli isolates

16 Table (2): Interpretation of zones of inhibition in agar diffusion method for antimicrobial susceptibility Antimicrobial agent Diameter of inhibition Control disc Resistant Intermediate Sensitive Amikacillin 30 mg 14 or less or more Ampicillin 10 µg 11 or less or more Cephaloxin 30 µg 14 or less or more Erythromycin 15 µg 13 or less or more Gentamicine 10 µg 12 or less or more Streptomycin 10 µg 11 or less or more

17 4. RESULTS Table (3): Incidence of E. coli in diarrhoeic and apparently healthy calves. No. of Positive cases Animal status examined animals No. % Diarrhoeic calves App. healthy calves Total

18 Table (4): Incidence of E. coli in relation to the age of calves. Age Diarrhoeic calves App.healthy calves Examined +ve % Examined +ve % First month month month Total

19 Table (5): Incidence of K99+ve E. coli in diarrhoeic and apparently healthy calves in the first month of age. Animal status No. of cases +ve E. coli K99+ve K99-ve No. % No. % Diarrhoeic App healthy Total

20 K99+ve K99-ve Diarrhoeic App. Healthy Total Fig. (1): Incidence of K99+ve E. coli in diarrhoeic and apparently healthy calves in the first month of age

21 Table (6): Prevalence of piliated E. coli in relation to the age of diarrhoeic calves. No. of K99 +ve Age/ day examined +ve E. coli animals No. % Total

22 Table (7): Serogrouping of K99 ve E. coli from diarrhoeic calves (1-15 days old). Serogroup No. % O O O O O Untypable Total 68

23 Table(8): Enterotoxins production in K99+ve E. coli isolated from diarrhoiec calves ( aged 1-15 days old). Age/day K99 +ve ST +ve ST ve No. % No. % Total

24 K99+ve K99-ve Total Figure (2): Percentage of enterotoxins production in K99+ve E. coli isolated from diarrhoiec calves (aged 1-15 days old).

25 Table (9): Enterotoxins production in K99 ve E. coli strains with regard to serogroups (aged 1-15 days old. Heat stable toxin +ve (ST Heat stable toxin ve (ST O No. of +ve) ve) serogroup strains No. % No. % O O O O O Untypable Total

26 K99 +ve K99 -ve O78 O119 O125 O55 O26 Untypable Total Figure (3): Percentage of enterotoxins production in K99 ve E. coli isolated from diarrhoiec calves regard to serogroups (aged 1-15 days old).

27 Table (10): Haemagglutination activity of K99 +ve E. coli isolated from diarrhoeic calves (aged 1-15 days old). RBCs Examined No. HA ve HA +ve MRHA MSHA No. No. % No. % No. % No. % Cow Sheep Camel Rabbit

28 HA-ve HA+ve MRHA MSHA Cow Sheep Camel Rabbit Figure (4): Percentage of haemagglutination activity of K99 +ve E. coli isolated from diarrhoeic calves (aged 1-15 days old).

29 Table(11): Haemagglutination activity of K99 -ve E. coli isolated from diarrhoeic calves (aged 1-15 days old). RBCs HA ve HA +ve MRHA MSHA No. % No. % No. % No. % Cow Sheep Camel Rabbit

30 HA-ve HA+ve MRHA MSHA Cow Sheep Camel Rabbit Figure (5): Percentage of Haemagglutination activity of K99 -ve E. coli isolated from diarrhoeic calves (aged 1-15 days old).

31 Table(12): Haemagglutiantion activity of K99 ve E. coli isolated from diarrhoeic calves with regard to serogroups (aged 1-15 days old). O sero No. of Cow RBCs Sheep RBCs Camel RBCs Rabbit RBCs group isolates No % No % No % No % O O O O O Untypable Total

32 Cow RBCs % Sheep RBCs % Camel RBCs % Rabbit RBCs % O78 O119 O125 O55 O26 Untypable Total Figure (6): Percentage of haemagglutination activity of k99-vee. coli isolated from diarrhoeic calves with regard to serogroups (aged 1-15 days old).

33 Table (13): Haemolytic activity of K99 +ve E. coli isolated from diarrhoiec calves (aged 1-15 days old ). Age/day K99 +ve Haemolytic strains Non haemolytic strain No. % No. % Total

34 Haemolytic Non haemolytic Total Figure (7): Percentage of haemolytic activity of K99 +ve E. coli isolated from diarrhoiec calves (aged 1-15 days old).

35 Table (14): Haemolytic activity of K99 ve E. coli isolates from diarrhoeic calves with regard to serogroups. (aged 1-15 days old). O No. of Haemolytic Non-haemolytic serogroup strains No. % No. % O O O O O Untypable Total

36 Haemolytic % Non-haemolytic % O-78 O-119 O-125 O-55 O-26 Untypable Total Figure (8): Percentage of haemolytic activity of K99 ve E. coli isolated f rom diarrhoeic calves with regard to serogroups (aged 1-15 days old).

37 Table (15): Congored binding ability of K99 +ve E. coli isolated from diarrhoiec calves (aged 1-15 days old). Age/day K99+ve CR +ve CR ve No. % No. % Total

38 CR +ve % CR -ve % to 5 6 to to 15 Total Figure (9): Percentage of Congored binding ability of K99 +ve E. coli isolated from diarrhoeic calves (aged 1-15 days old).

39 Table (16): Congored binding ability of K99 ve E. coli isolated from diarrhoeic calves with regard to serogroups. (aged 1-15 days old). O No. of CR +ve CR ve serogroup isolates No. % No. % O O O O O Untypable Total

40 CR +ve % CR -ve % O-78 O-119 O-125 O-55 O-26 Untypable Total Figure (10): Percentage of Congored binding ability of K99 ve E. coli isolated from diarrhoeic calves with regard to serogroups. (aged 1-15 days old)

41 Table (17): Correlation between K99 antigen and enterotoxins production of E. coli recovered from diarrhoiec calves aged 1-15 days old. No. of ST +ve ST ve K99 E. coli isolates No. % No. % K99 +ve E. coli K99 ve E. coli

42 Table (18): Correlation between K99 antigen and haemagglutination activity of E. coli strains isolated from diarrhoeic calves aged 1-15 days old. K99 E. coli No. of HA +ve HA ve isolates No. % No. % K99 +ve E. coli K99 ve E. coli

43 Table (19): Correlation between K99 antigen and haemolytic activity of E. coli recovered from diarrhoiec calves aged 1-15 days old. K99 E. coli No. of Haemolytic Non-Haemolytic isolates No. % No. % K99 +ve E. coli K99 ve E. coli

44 Table (20): Correlation between K99 antigen and Congored binding ability of E. coli strains isolated from diarrhoeic calves aged 1-15 days old. K99 E. coli No. of CR+ve CR-ve isolates No. % No. % K99 +ve E. coli K99 ve E. coli

45 K99 +ve E. coli K99 ve E. coli ST+ve% Haemolytic+ve% CR+ve% HA+ve% Figure (11): Percentage of virulence factors of E. coli isolated from diarrhoeic calves (aged 1-15days old).

46 Table (21): Antibiogram for K99 +ve E. coli isolated from diarrhoeic and apparently healthy calves. Chemotherapeutic Sensitive Resistant agent No. % No. % Danox (Danofloxacin) Amikacillin Gentamycin Ampicillin Neomycin SXT Streptomycin Amoxicillin Erythromycin Cephaloxin Penicillin

47 Sensitive % Resistant % Amikacillin Danox (Danofloxacin) Gentamycin Ampicillin Neomycin SXT Streptomycin Amoxicillin Erythromycin Cephaloxin Penicillin Figure (12): Percentage of antimicrobial susceptibility of k99+ve E. coli isolated from diarrhoeic and apparently healthy calves.

48 Conclusion 1- E. coli is one of the bacterial pathogens that plays an essential role in inducing diarrhoea in newborn calves. 2- Synergestic role was found between adhesion and toxin producing strains. This was indicated by the high percentage of E. coli K99+ve which produced enterotoxin. 3- HA activity showed high incidence when using the RBCs of the same species of diarrhoiec calves. 4- CR test may be used as a phenotypic marker or virulence factor of E. coli isolates. Cultures that CR+ve and negative in other virulence factors need further studies to clarify this point. 5- It is tempting to suspect that E. coli virulence may play a significant but not a whole part in the pathogenesis of diarrhoeal diseases. 6. It should be emphasized that there are multiple factors related to the host such as age, gastric ph, intestinal flora complicating viral infection,in addition to, feeding, management and hygiene measures.