YERSINIA ENTEROCOLITICA IN CALVES STUDIES ON THE COMMON ANTIGENS Cairo University Faculty of...

YERSINIA ENTEROCOLITICAYERSINIA ENTEROCOLITICA IN CALVES IN CALVES STUDIES ON THE COMMON ANTIGENSSTUDIES ON THE COMMON ANTIGENS

Cairo University

Faculty of Veterinary Medicine

Department of Microbiology

Eman Ibrahim MahmoudEman Ibrahim Mahmoud

( M. V. Sc. , Cairo University, 1999 )( M. V. Sc. , Cairo University, 1999 )

Eman Ibrahim MahmoudEman Ibrahim Mahmoud

( M. V. Sc. , Cairo University, 1999 )( M. V. Sc. , Cairo University, 1999 )

Thesis Presented

By

For the degree ofDoctor of Philosophy

In Veterinary Medical Science (Bacteriology, Immunology and Mycology )

2006

Under the Under the supervision ofsupervision of

PROFESSOR DR. MOHAMED KAMAL REFAIEmeritus Professor of Microbiology

Department of Microbiology Faculty of Veterinary Medicine

Cairo University

DR. HODA MOHAMED ZAKISenior Researcher

Animal Health Research Institute,Dokki, Cairo

PROFESSOR DR. SALAH EL-DIN ABDEL HAMID YOUSSEF

Professor of PharmacologyDepartment of Pharmacology

Faculty of Veterinary MedicineCairo University

•Yersinia enterocolitica has been implicated in several outbreaks of food and water born illness (Hurvell, 1981 and Bottone, 1997).

•Yersinia enterocolitica has been isolated in different parts of the world from a large number of wild and domesticated animals (Lee et al., 1981), numerous food-stuffs and from domestic water supplies. Although the paths of human infection are largely hypothetical, contact with infected people or animals and intake of contaminated water or food are considered the most likely modes of infection.

Because Yersinia enterocolitica can grow at low temperature, refrigerated foods initially contaminated with a small number of bacteria may serve not only as a vehicle for transmission but as a medium for growth (Stern et al., 1980).

Yersinia enterocolitica can survive for long periods in the environment due to their ability to grow with minimal nutritional requirements and their ability to remain metabolically active at extremes of temperature (Tashiro et al., 1991).

Strains of Yersinia enterocolitica were isolated from animals suffering from acute enteritis (Brewer and Corbel, 1983) and were

considered as true human pathogens manifested by mesenteric lymphadenitis, enterocolitis, abdominal pain, erythema,

septicaemia and artheritic alteration (Cover and Alber, 1989 and Crespo et al., 2002).

The pathogenic potential of this microorganism is partially due to the expression of determinants encoded on a virulence associated

plasmid. Bacterial strains harbouring this plasmid express a series of different temperature dependent phenotypes: Calcium

dependence at 37°C growth (Gemski et al., 1980), autoagglutination activity (Laird and Cavanaugh, 1980),

production of V and W antigens (Perry and Brubaker, 1983), resistance to bactericidal effect of serum (Pai and DeStephano,

1982), ability to adhere HEp-2 cells (Hessemann et al. , 1984) and increased affinity for crystal violet and Congo red (Robins

Browne et al., 1989).

In addition to these plasmid-mediated virulence factors, other chromosomally encoded determinants, including an enterotoxin

production and pyrazinamidase activity are also thought to play a role in virulence, since strains lacking the plasmid have been found

to survive with macrophages (Charnetzly and Shufard, 1985) and cause disease in animal model (Fukushima, 1987).

Lipopolysaccharide (LPS) is a complex outer membrane molecule of Gram negative bacteria. It consists of three moieties:

Lipid A, the core and an oligosaccharide and O side chain (Luderitz et al., 1971 and Bengoechea et al., 2003). The O side chain has been

regarded as a virulence factor in several bacterial species. In Yersinia enterocolitica LPS O side chain plays a role in resistance to

killing by the alternative complement pathway (Wachter and Brade, 1989 and Skurnik and Bengoechea, 2003). Others reported that most

of the antibody response during Yersinia enterocolitica infection is directed against the O side chain (Granfors, 1979).

The major cause of false positivity in the serological diagnosis on brucellosis is infection with Yersinia enterocolitica serovar O:9 (Kittellberger et

al., 1995 and Munoz et al. , 2005).

The aim of the studyThe aim of the study

Therefore, the purpose of the study reported here was initially to estimate the prevalence of Yersinia enterocolitica in diarrhoeic and apparently normal cow and buffalo calves.

The interesting objective goal in this work was to investigate the virulence attributes of Yersinia enterocolitica isolates which include: the capabilities of Congo red dye absorption, Crystal violet binding, haemagglutination, autoagglutination, pyrazinaminidase, calcium dependency, serum sensitivity, invasiveness in HEp-2 cells, enterotoxin production and induction of conjunctivitis in laboratory animals.

Moreover, the study was directed to study and characterize the LPS and OMPs of different Yersinia enterocolitica isolates and to assess the suitability of ELISA using whole cell antigens and LPS or OMPs to provide reliable means for the differential diagnosis of yersiniosis and brucellosis.

3.1.1. Samples:

General health condition

No. of tested calves

Cow calves

Buffalo calves

Apparently normal 160 90 70

Diarrhoeic 198 140 58

Total 358 230 128

3.2.1.1. Faecal samples:

3.2.1.2. Blood samples:

3.2.2. Primary isolation and purification:

3.2.2.1. Direct plating:

CIN agar at 25°C for 48 hours

3.2.2.2. Cold enrichment :

1st week 2nd week 3rd week

Suspected colonies onto the surface of CIN medium were characterized by dark red

center surrounded by an outer zone which was usually translucent “bulls eye”.

The suspected colonies were picked up and subcultured onto nutrient agar plates to

confirm their purity, then transferred (as well as the reference strain) to nutrient agar slopes

for maintenance till they were screened .

3.2.3. Identification of the isolates:3.2.3. Identification of the isolates:The purified colonies were subjected to:The purified colonies were subjected to:

3.2.3.1. Morphological examination:Films from pure suspected colonies were stained with Gram's

stain and examined microscopically.

3.2.3.2. Cultural examination:Motility was assured by stabbing the organism in

two tubes of motility medium. The first one was incubated at 25C for 24 hours and the second at 37C for 24 hours (Pianetti et al., 1990).

3.2.3.3. Primary screening tests:At least three suspected, well separated colonies were

subjected to oxidase and catalase tests .

Oxidase negative and catalase positive colonies were submitted to triple sugar iron (TSI) agar and Christensen's urea agar.

Acid slants and butts or alkaline slants and acid butts with little or no gas and no H2S on TSI tubes and usually were positive for urease test were suspected to be Yersinia enterocolitica and further examined by biochemical tests.

3.2.3.4. Further biochemical tests:•Simmon's citrate.•Phenylalanine deaminase.•Lysine decarboxylase test.•Arginine dihydrolase test.•Ornithine decarobxylase.•Indole production.•Methyl red reaction (at 25°C and 37°C).•Voges- Proskauer test (at 25°C and 37°C).•Nitrate reduction to nitrate.•Lactose (O-F) test.•B- galactosidase (ONPG) activity at 25°C.•Aesculin hydrolysis test.•Acid production from; D-glucose, sucrose, L-sorbose , D-trehalose, L-arabinose, D-cellobiose, D-sorbitol , maltose D-xylose, dulcitol, adonitol, D-raffinose, L-rhamnose and D-melibiose and salicin.

3.2.4. Biotyping of the isolates:Biotyping of the isolates was determined according to the

scheme of Wauters et al. (1987) by the following tests; •Aesculin hydrolysis, •Indole, •Salicin, •lactose (OF), •Xylose, •Nitrate, •Trehalose, •B-galactosidase, •Ornithine decarboxylase, •Voges-proskauer, •Sorbose, •Sorbitol •Sucrose .

3.2.5. Serotyping of the isolates:The isolates were serotyped using the available

monovalent antisera by slide agglutination test (Winblad et al., 1966 and Winblad 1968) .

3.2.6. Virulence and pathogenicity tests : 3.2.6.1. Congo Red (CR) binding test: 3.2.6.2. Crystal Violet (CV) binding assay:3.2.6.3. Haemagglutination (HA) test :3.2.6.4. Autoagglutination :3.2.6.5. Calcium dependency for growth at 37°C:3.2.6.6. Pyrazinamidase activity :3.2.6.7. Serum sensitivity test :3.2.6.8. Invasion of HEp-2 cells:-3.2.6.9. Enterotoxin Production:3.2.6.10. Sereny test:

3.2.7. Preparation of antigens:

3.2.7.1. Preparation Whole bacterial antigen (W.B.) (Granfors et al., 1981):

3.2.7.2. Preparation of outer membrane proteins:

3.2.7.3. Extraction and purification of LPS :

3.2.8. Polyacrylamide gel electrophoresis (SDS-PAGE):

It was performed according to the method described by Laemmli (1970).

3.2.8.1. Casting of the gel:

3.2.8.2. Preparation of the samples:

3.2.8.3. Samples loading:

3.2.8.4. Silver stain: (Tsai and Frasch, 1982).

3.2.9. Production of antisera : (Granfors et al. , 1981)

3.2.9.1. Preparation of whole bacterium antigens used for immunizations:

3.2.9.2. Rabbit immunization : (Granfors et al. , 1981)

3.2.10. Serological examination of the serum: Using enzyme linked immunosorbant assay.

Table ( 4 ) : Prevalence of Yersinia enterocolitica recovered from the examined calves.

Fig. ( 1 ) : Prevalence of Yersinia enterocolitica recovered from the examined calves.

Table ( 5 ) : Comparison of direct plating and cold enrichment techniques in isolation of Yersinia enterocolitica.

TechniqueDirect

plating

Cold enrichment technique

Direct plating 6/35

(17.14 % )13/35

( 37.14 % )

Cold enrichment Technique

13/35 ( 37.14 % )

16/35( 45.71 % )

Total19/35

( 54.29 % )29/35

(82.86 % )

Table ( 7 ) : Wauters biotyping tests of Yersinia enterocolitica isolates (Wauters et al., 1987).

Series of biochemical tests

Biovar*

1A 1B 2

Number of biovars 19* 9 7

Aesculin hydrolysis + - -

Salicin (acid ) + - -

Lipase + + -

Indole + + (+)

Xylose (acid) + + +

Trehalose (acid ) + + +

-galactosidase + - -

Voges Proskauer + + +

Table (8) : Distribution of Yersinia enterocolitica biovarsin examined calves.

Animal

Biovar

Cow calves Buffalo calves

TotalApparently healthy

DiarrhoeicApparently

healthyDiarrhoeic

1A 2 (5.71 %) 8 (22.86 %) 6 (17.14 %) 3 (8.57 %) 19

1B 0 6 (17.14 %) 0 3 (8.57 %) 9

2 1 (2.86 %) 4 (11.43 %) 0 2 (5.71 %) 7

Total 3 (8.57 %)18 (51.42

%)6 (17.14 %) 8 (22.86 %) 35

* Percentage was calculated according to total number of isolates (35 isolates).

Table ( 9 ) : Serovars of Yersinia enterocolitica isolates from examined calves.

Serovar identifiedNo. of positive / No. of

tested

O:3 5/35 ( 14.28 % )

O:5 7/35 ( 20.00 % )

O:8 16/35 ( 45.71 % )

O:9 7/35 ( 20.00 % )

Fig. ( 5 ) : Serovars of Yersinia enterocolitica isolates from examined calves .

5

7

16

7

0

2

4

6

8

10

12

14

16

Nu

mb

er

O:3 O:5 O:8 O:9

Serovar

Table (10) : Distribution of Yersinia enterocolitica serovarsin examined calves.

Animal

Serovar

Cow calves Buffalo calves

Apparently healthy

DiarrhoeicApparently

healthyDiarrhoeic

O:3 1 (2.86 %) 3 (8.57 %) 1 (2.86 %) 0

O:5 1 (2.86 %) 0 3 (8.57 %) 3 (8.57 %)

O:8 0 11 (31.43 %) 2 (5.71 %) 3 (8.57 %)

O:9 1 (2.86 %) 4 (11.43 %) 0 2 (5.71 %)

* Percentage was calculated according to total number of isolates (35 isolates).

Fig. (6) : Yersinia enterocolitica serovars in examined calves.

2.862.86

0

2.86

8.57

0

31.43

11.43

2.86

8.57

5.71

0 0

8.578.57

5.71

0

5

10

15

20

25

30

35

40

%

Apparentlyhealthy calves

Diarrhoeic claves Apparentlyhealthy calves

Diarrhoeic claves

O:3

O:5

O:8

O:9

Cow calves Buffalo calves

Table (11) : Relationship between serovars and biovars of Yersinia enterocolitica isolates .

SerovarsBiovars

Total

1A 1B 2

O:3 5 (14.29 %) 0 0 5 (14.29 %)

O:5 7 (20.00 %) 0 0 7 ( 20.00 %)

O:8 7 (20.00 %) 9 (25.71 %) 0 16 (45.71 %)

O:9 0 0 7 (20.00 %) 7 ( 20.00 )

Total19

(54.29 %)9

(25.71 %)7

(20.00 %)35

( 100 % )

Table (12) : Distribution of serobiovars in examined calves .

Animal

Sero/biovars

Cow calves Buffalo calves

Apparently healthy

DiarrhoeicApparently

healthyDiarrhoeic

No. % No. % No. % No. %

O:3/1A 1 2.86 3 8.57 1 2.86 0 0

O:5/1A 1 2.86 0 0 3 8.57 3 8.57

O:8/1A 0 0 5 14.29 2 5.71 0 0

O:8/1B 0 0 6 17.14 0 0 3 8.57

O:9/2 1 2.86 4 11.43 0 0 2 5.71

Total 3 8.57 18 51.43 6 17.14 8 22.86

Table (13) : Congo red reaction among Yersinia enterocolitica recovered from examined animals.

Fig. (7) : Congo red reaction among Yersinia enterocolitica recovered from examined animals.

33.33

100

16.67

100

0

20

40

60

80

100

%

Cows Buffaloes

Apparently healthy calves Diarrhoeic claves

Photo ( 1 ) : Congo red binding activity of Yersinia enterocolitica isolates A- Congo red positive colonies .B- Congo red negative colonies .

BA

Table (15) : Crystal violet binding assay of Yersinia enterocolitica recovered from examined calves.

Fig. (8) : Crystal violet binding assay of Yersinia enterocolitica recovered from examined calves.

33.33

88.89

16.67

100

0

20

40

60

80

100

%

Cows Buffaloes

Apparently healthy calves Diarrhoeic claves

Photo ( 2 ) : Crystal violet binding activity of Yersinia enterocolitica isolates A- Crystal violet positive colonies.B- Crystal violet negative colonies.

BA

Fig. (9) : Mannose resistant haemagglutinating activities of Yersinia enterocolitica isolates to different RBCs .

33.3333.33

77.78

61.9

33.33 33.33

87.5 87.5

0

10

20

30

40

50

60

70

80

90

100

%

Apparentlyhealthy calves

Diarrhoeicclaves

Apparentlyhealthy calves

Diarrhoeicclaves

Bovine

Guinea pig

Chicken

Human

Cow calves Buffalo calves

Table ( 18 ) : Correlation between the haemagglutination test using different erythrocytes and the isolated Yersinia

enterocolitica serobiovars.

Table (19) : Autoagglutination of Yersinia enterocolitica recovered from examined calves.

Fig. (10) : Autoagglutination of Yersinia enterocolitica recovered from examined calves.

33.33

66.67

16.67

75

0

20

40

60

80

100

%

Cows Buffaloes

Apparently healthy calves Diarrhoeic claves

Fig. (11) : Calcium dependency of Yersinia enterocolitica recovered from examined calves.

33.33

66.67

16.67

75

0

20

40

60

80

100

%

Cows Buffaloes

Apparently healthy calves Diarrhoeic claves

Fig. (12) : Pyrazinamidase activity of Yersinia enterocolitica recovered from examined calves.

100

44.44

100

37.5

0

20

40

60

80

100

%

Cows Buffaloes

Apparently healthy calves Diarrhoeic claves

Fig. (13) : Serum sensitivity of Yersinia enterocolitica recovered from examined calves.

33.33

88.89

33.33

77.78

66.67

87.5

50

75

0

10

20

30

40

50

60

70

80

90

100

%

Survival in serum Growth in serum Survival in serum Growth in serum

Apparently healthy calves Diarrhoeic claves

Cow-calves Buffalo-calves

Fig. (14) : Invasiveness property of Yersinia enterocolitica recovered from examined calves.

66.67

100

50

100

0

20

40

60

80

100

%

Cows Buffaloes

Apparently healthy calves Diarrhoeic claves

A B

Photo (3) : (A): Normal HEp-2 cells.

(B) Invasion of Yersinia enterocolitica in HEp-2 cells (The bacteria seen as dark blue forms within and usually filling the cytoplasm or within cytoplasmic vacuoles).

Fig. (15) : Enterotoxigenicity of Yersinia enterocolitica recovered from examined calves.

0

77.78

33.33

87.51

0

20

40

60

80

100

%

Cows Buffaloes

Apparently healthy calves Diarrhoeic claves

Fig. (16) : Sereny test of Yersinia enterocolitica isolates recovered from examined calves.

0

61.11

0

62.5

0

20

40

60

80

100

%

Cows Buffaloes

Apparently healthy calves Diarrhoeic claves

Photo ( 4 ) : (A) : Normal guinea pig.(B) : Conjunctivitis in guinea pig caused by

of Yersinia enterocolitica isolates (Sereny test).

Photo (5) : SDS-Photo (5) : SDS-PAGE pattern PAGE pattern

of LPS of LPS extracted from extracted from five serobiovars five serobiovars of the examined of the examined

calves.calves.

Photo (6) : Photo (6) : SDS-PAGE SDS-PAGE pattern of pattern of OMPs OMPs extracted extracted from five from five serobiovars serobiovars of the of the examined examined calves.calves.

Fig. (17) : Yersinia enterocolitica antibodies in sera of the examined calves using whole bacterial antigens by ELISA.

1.110

1.110

3.332.86

1.43

4.295

8.57

1.43

4.29

2.86

4.294.293.45

8.62

5.17

8.62

12.07

0

2

4

6

8

10

12

14

16

18

20

%

Apparentlyhealthy calves

Diarrhoeicclaves

Apparentlyhealthy calves

Diarrhoeicclaves

O:3/1A

O:5/1A

O:8/1A

O:8/1B

O:9/2

Cow calves Buffalo calves

Fig. (18) : Yersinia enterocolitica antibodies in sera of the examined calves using LPS antigens by ELISA.

1.110

1.111.110

2.86

1.43

4.295

8.57

1.43

4.29

2.862.86

4.29

1.72

8.62

6.9

12.0712.07

0

2

4

6

8

10

12

14

16

18

20

%

Apparentlyhealthy calves

Diarrhoeicclaves

Apparentlyhealthy calves

Diarrhoeicclaves

O:3/1A

O:5/1A

O:8/1A

O:8/1B

O:9/2

Cow calves Buffalo calves

Fig. (19) : Yersinia enterocolitica antibodies in sera of the examined calves using outer membrane proteins by ELISA.

1.111.110 0

1.112.14

1.43

3.57

5

3.57

1.43

4.29

2.86

1.43

0 0

3.45

5.175.17

6.9

0

2

4

6

8

10

12

14

16

18

20

%

Apparentlyhealthy calves

Diarrhoeicclaves

Apparentlyhealthy calves

Diarrhoeicclaves

O:3/1A

O:5/1A

O:8/1A

O:8/1B

O:9/2

Cow calves Buffalo calves

Table (39) : Whole bacteria (WB) antigen, lipopolysaccharide (LPS) antigen and outer membrane proteins (OMPs) antigen of

Yersinia enterocolitica antibodies in examined calves serum versus to their bacteriological status.

Bacteriological status

No.

ELISA test using

WB LPS OMPs

Positive Negative

35323

3238

3237

3410

Total 358 70 69 44

WB: Whole bacterial antigen.LPS : Lipopolysaccharide.

OMPs : Outer membrane proteins.

Table (40) : Sensitivity and specificity of whole bacterium WB) antigen, lipopolysaccharide (LPS) antigen and outer membrane

proteins (OMPs) antigen in diagnosis of Yersinia enterocolitica .

Bacteriological status

ELISA test using

WB LPS OMPs

Sensitivity* 91.43 % 91.43 % 97.14 %

Specificity** 88.24 % 88.54 % 96.59 %

*Sensitivity =True positive

True positive + False negative

**Specificity =True negative

True negative + False positive

Table (41) : Cross reaction between different whole bacterial (WB) antigens of Yersinia enterocolitica and Brucella abortus in

rabbit antisera.

WB antig

en

Mean optical density of sera positive for

O:3/1A O:5/1A O:8/1A O:8/1B O:9/2 B. abortus

O:3/1A 1.321 0.100 0.167 0.052 0.701 0.021

O:5/1A 0.128 0.606 0.066 0.010 0.077 0.031

O:8/1A 0.082 0.022 0.727 0.741 0.091 0.121

O:8/1B 0.096 0.028 0.351 1.132 0.054 0.023

O:9/2 0.721 0.021 0.044 0.084 0.963 0.921

Results of homologous reactions are printed in boldface type.

Table (42) : Cross reaction between different Lipoploysaccharide (LPS) antigens of Yersinia enterocolitica and

Brucella abortus in rabbit antisera.

LPSantigen

Mean optical density of sera positive for

O:3/1A O:5/1A O:8/1A O:8/1B O:9/2 B. abortus

O:3/1A 1.530 0.022 0.018 0.014 0.022 0.082

O:5/1A 0.011 0.969 0.019 0.021 0.015 0.110

O:8/1A 0.034 0.101 0.464 0.321 0.021 0.082

O:8/1B 0.121 0.024 0.242 0.992 0.051 0.051

O:9/2 0.841 0.050 0.031 0.012 0.891 1.251

Results of homologous reactions are printed in boldface type.

Table (43) : Cross reaction between different outer membrane proteins (OMPs) antigens of Yersinia enterocolitica and Brucella

abortus in rabbit antisera.

OMPsantigen

Mean optical density of sera positive for

O:3/1A O:5/1A O:8/1A O:8/1B O:9/2 B. abortus

O:3/1A 1.530 0.096 0.022 0.019 0.081 0.121

O:5/1A 0.021 0.943 0.021 0.011 0.082 0.180

O:8/1A 0.012 0.091 1.442 0.081 0.033 0.011

O:8/1B 0.011 0.033 0.041 1.502 0.031 0.131

O:9/2 0.130 0.064 0.021 0.081 0.931 0.012

Results of homologous reactions are printed in boldface type.

It was concluded that, Yersinia enterocolitica is incriminated in enteric diseases of calves. Yersinia enterocolitica must be taken in consideration in differential diagnosis as a causative agent of diarrhoeal diseases. Calves are considered as a source of contamination of Yersinia enterocolitica. Care must be taken to avoid transmission of Yersinia enterocolitica to human from calves. The use of OMPs-ELISA as a suitable assay to differentiate clearly between Yersinia enterocolitica O:9 and Brucella abortus infections.

Finally, further studies are needed to verify the cross-reaction between Yersinia enterocolitica O:9 and Brucella abortus and the epidemiological feature of this organism in Egypt.