) قالوا سبحانك لا علم لنا الا ما علمتنا انك انت العليم الحكيم ( البقرة 32 البقرة 32

SPOTLIGHT ON CONTROL OF CHICKEN CAMPYLOBACTERIOSIS Cairo University Faculty of Veterinary Medicine Department of Poultry Diseases

Wafaa Abdel-Ghani Abdel-Ghani B.V.Sc., Cairo University, 1996 M.V.Sc., Cairo University, 2000 Thesis Presented By For The Degree of Doctor Philosophy in (Birds and Rabbits Diseases) 2003

Under the supervision of Prof. Dr. Mohamed H. H. Awaad Professor of Poultry Diseases Faculty of Veterinary Medicine Cairo University

Prof. Dr. Mamdouh Abdel-Ghani Professor of Microbiology Faculty of Veterinary Medicine Cairo University

Dr. Nagwa Saad Rabie Senior Researcher of poultry diseases National Research Center

Prof. Dr. AHMED ALI SAMY Professor of Poultry Diseases Faculty of Veterinary Medicine, Alex. University. Prof. Dr. FATHY SAAD Professor of Poultry Diseases Faculty of Veterinary Medicine, Cairo University.

Campylobacter jejuni (C.jejuni) Campylobacter jejuni (C.jejuni) infection in chickens has been implicated as contagious disease charcterized by chronic course, high morbidity, low mortality and reduction in egg production (Peckham,1984). C.jejuni C.jejuni is the primary cause of human gatrointestinal infection among campylobacter species. Consumption of chickens and retail poultry products is incriminated as the main vehicle for transmission of infection to human (Nachamkin et al.,1993).

campylobacter Hence, campylobacter enteritis constitutes a zoonosis of major concern in public health and it has been shown to be greater problem than salmonellosis in several countries, so, it is important to address intervention strategies by which these bacteria can be reduced or removed from the food animals. Such strategies could include vaccines or probiotic cultures. C.jejuni (Burr et al.,1988; Pavlovskis et al.,1991 and Rollwagen et al.,1993). Various types of C.jejuni bacterins were tested in laboratory animals like mice and rabbits with successful results (Burr et al.,1988; Pavlovskis et al.,1991 and Rollwagen et al.,1993).

C.jejuni (Stern et al.,1990;Cowthrow et al.,1994;Rice et al.,1997;Lee et al.,1999 and Ziprin et al.,2002). In chickens, different types of living and killed vaccines against C.jejuni have been used with variable results (Stern et al.,1990;Cowthrow et al.,1994;Rice et al.,1997;Lee et al.,1999 and Ziprin et al.,2002). The wide misuse of chemotherpeutics leads to development of antibiotic resistant strains and destruction of GIT microflora, so using of what is called competitive exclusion (CE) products or probiotics has been considered as a safe and an alternative approach to the use of chemotherapeutics. (Mulder,1996). Probiotics are composed of pure culture of one or more of identified and non-pathogenic living intestinal microorganisms that are able to proliferate in the bird’s intestinal tract, resulting in balanced microflora (Mulder,1996).

Increase feed intake and digestion: Intestinal bacterial flora takes part in the metabolism of nutrients and synthesis of vitamins. Neutralizes enterotoxins enterotoxins produced by pathogenic bacteria Altering metabolism by: * Increase digestive enzyme activity(amylase, protease and lipase). * Decrease bacterial enzyme activity(Reduction in b-glucuronidase ). * Reduce ammonia in the excreta and litter of broilers. Stimulation of IgA production Maintaining beneficial microbial population in GIT (Antagonistic activity and competition for attachment sites)

The aim of the study C.jejuni 1. Preparation of safe and effective C.jejuni bacterins from the local field strains following these steps: C.jejuni a. Determination of whether the locally isolated C.jejuni strains were having the same protein make-up or having heterogenous structure through electrophoretic characterization of their outer membrane proteins. campylobacter b. Serodiagnosis to evaluate the production of specific IgG humoral campylobacter antibodies in the sera of immunized meat-type and egg-type chickens.

c. Evaluation of the protective potential of the locally prepared bacterins in the challenged chickens. d. Histopathological examination of organs collected from immunized and challenged birds. C.jejuni 2. Study the efficacy of certain acidifiers and probiotic preparations in reducing C.jejuni colonization in intestine of chicken broilers.

Strains used: Three local strains of C.jejuni representing biotype I and II were used in bacterins preparation and used as challenge bacteria in all experiments. Preparation of sarcosyl insoluble OMPs using SDS- PAGE according to Laemmeli, 1970, Newell et al., 1984 and Jin and Penner, Preparation of whole cell bivalent C.jejuni bacterins (AHAB & IFAB) as described by Williams et al., 1976 and Bryner et al., 1978 and Experiment (1) Experiment (2)

Quality control tests of the prepared bacterins (British veterinary codes, 1970) Purity test C.jejuni Completion of C.jejuni inactivation Sterility test Safety test

Experiment (3) Immunization of chicken broilers with the prepared bivalent C.jejuni (AHAB & IFAB) at 1 & 3 weeks of age

Experiment (4a) Immunization of egg-type chickens with the prepared bivalent C.jejuni (AHAB & IFAB) at 1 & 3 weeks of age

Experiment (4b) Immunization of egg-type chickens with the prepared bivalent C.jejuni (AHAB & IFAB) at 9 weeks of age

Serum samples were collected from immunized birds at 0 hr (just before immunization) and weekly after each immunization dose. These serum samples were subjected to ELISA (Cawthraw et al., 1994) to detect humoral IgG antibodies. All immunized challenged birds were kept under close observation for further three weeks for clinical signs and mortalities. Faecal swabs were collected from challenged birds at 3,7,11,15,18 and 21 days post challenge to determine the frequency of C.jejuni shedding. At the end of observation period survived birds were sacrificed and subjected to post mortem examination for lesion scoring (Rabie, 1998).

Specimens were collected from liver and intestine for reisolation and identification using: Motility test (Simbert, 1974). Morphological identification (Holt et al., 1994). Biochemical identification (Koneman et al., 1995). Liver and intestine specimens were subjected to histopatological examination (Carlton, 1967).

Experiment (5) Acidifier and probiotics were added to the ration from day-old till the end of the experiment (5 weeks). C.jejuni All the groups were orally challenged with 0.5 ml containing 5x10 8 CFU/ml of C.jejuni at 2 weeks of age. The birds were kept under observation for 3 weeks post challenge for signs and mortalities. The effect of acidifiers and probiotics on campylobacteriosis in chicken broilers

C.jejuni Faecal swabs were collected from all groups at 3,7,11,15,18 and 21 days post infection for exploring the frequency of C.jejuni shedding. C.jejuni Five chickens were sacrificed weekly at 7,14 and 21 days post infection and were subjected to C.jejuni reisolation from the liver and intestine. Sacrificed chickens at 7 and 14 days and all survived birds at the end of observation period were examined for post mortem lesion scoring.

Bands No.Marker (Mol.W)KDa Strain No (1) (Mol.W)KDa Strain No (2) (Mol.W)KDa Strain No (3) (Mol.W)KDa Table (1): Electrophoretic characterization of outer membrane Proteins (OMPS) of C.jejuni strains using SDS-PAGE KDa=Kilo Dalton

Fig. (1): Electrophoretic characterization of outer membrane proteins (OMPS) of C.jejuni strains using SDS-PAGE. proteins (OMPS) of C.jejuni strains using SDS-PAGE.

Results of quality control tests of bivalent C.jejuni bacterins C.jejuni Microscopical and biochemical identification of the grown seed culture onto Skirrow’s media revealed presence of pure culture of C.jejuni cells. C.jejuni Inactivated C.jejuni cells showed no growth on Skirrow’s agar plates after formalin inactivation. 1. Purity test 2. Completion of C.jejuni inactivation

3. Sterility test C.jejuni The formalin killed C.jejuni cells gave no bacterial growth after cultivation onto PPLO agar and broth and also no fungal growth was obtained after cultivation onto sabouraud dextrose agar plates. 4. Safety test The prepared bivalent bacterins were found to be safe for day-old-chicks, producing neither clinical signs nor local reactions and deaths during seven successive days observation period.

Fig. (2): Comparison between the results of ELISA in immunized chicken broilers with (AHAB) and (IFAB) at 1 & 3 wks of age

Fig. (3): Post mortem lesion scoring of C.jejuni in immunized and unimmunized chicken broilers at the end of 21 days observation period.

broiler Fig. (4): Shedding of C.jejuni in immunized and unimmunized broiler chickens during 21 days observation period

Fig. (5): Reisolation of C.jejuni from immunized and unimmunized chicken broilers at the end of 21 days observation period

liver Table (2): The main histopapatological lesions in liver and degree of broilers severity in chicken broilers immunized with two types of C.jejuni bacterins LesionsControl (AHAB) Broilers immunized with (AHAB) (IFAB) Broilers immunized with (IFAB) 1. Congested blood vessels Present(++)Absent 2. Inflammatory cells in blood vessels Present(++)Present(+) 3. Multifocal necrosis Present(+++)Present(++) specially biotype I Present(++) 4. Cellular infiltration Present(++++)Present(+++)Present(++) 5. Bile duct hyperplasia and newly formed bile ductules Present(+++)Absent 6. Hepatocytes vaculation Present(++++)Present(++) specially biotype II Absent 7. Hepatocytes granulation AbsentPresent(++) specially biotype II Absent 8. Activation of Kupffer cells Present(+++)Absent ++++=Severe+++=Moderate++ & +=Mild

intestine Table (3): The main histopapatological lesions in intestine and degree broilers of severity in chicken broilers immunized with both bacterins LesionsControl (AHAB) Broilers Immunized with (AHAB) (IFAB) Broilers Immunized with (IFAB) 1.Necrosis of enterocytes Present(++++)Present(+++)Present(++) 2.Hyperplasia of enterocytes Present(+++)AbsentPresent(+) 3.Goblet cells transformation Absent Present(+) specially biotype II 4.Hyperplasia of Crypt Present(+++)Present(++) specially biotype II Present(+) specially biotype II 5.Cellular infiltration in lamina propria Present(++++)Present(+++)Present(+) ++++=Severe+++=Moderate++ & +=Mild

(IFAB) Fig. (6): Liver of broilers immunized with (IFAB) and infected with C.jejuni showing few numbers of inflammatory cells in blood vessels. (H & E X33). Fig. (7): Liver of broilers immunized with (AHAB) and infected with C.jejuni showing focal area of necrosis (arrow). (H & E X33). Fig. (8): Liver of broilers immunized with (IFAB) and infected with C.jejuni showing mild vacculation of hepatocytes. (H & E X66). Fig. (9): Liver of control (unimmunized and infected) broilers showing severe hyperplasia of the epithelial lining the bile duct. (H & E X66).

Fig. (10): Intestine of control (unimmunized and infected) broilers showing severe hyperplasia of enterocytes (arrow). Notice severe inflammatory cells aggregation in lamina propria. (H & E X66). Fig. (11): Intestine of broilers immunized with (IFAB) and infected with C.jejuni showing mild hyperplasia of enterocytes, goblet cells transformation (arrow) and mild inflammatory cells aggregation. (H & E X33). Fig. (12): Intestine of broilers immunized with (AHAB) and infected with C.jejuni showing hyperplasia of crypt of Luberkuhn with moderate numbers of mitotic figures (arrow).(H & E X66).

Fig. (13): Comparison between the results of ELISA in immunized egg-type chickens with AHAB and IFAB at 1 & 3 wks of age

Figure (14): Post mortem lesion scoring of C.jejuni in immunized and unimmunized egg-type chickens at the end of 21 days observation period

egg-type Fig. (15): Shedding of C.jejuni in immunized and unimmunized egg-type chickens during 21 days observation period

Fig. (16): Reisolation of C.jejuni from immunized and unimmunized egg-type chickens at the end of 21 days observation period

LesionsControl (AHAB) Egg-type chickens immunized with (AHAB) (IFAB) Egg-type chickens immunized with (IFAB) 1. Congested blood vessels Present(++)Absent 2. Inflammatory cells in blood vessels Present(++++)Present(+++)Absent 3. Multifocal necrosis Present(+++) Present(++) specially biotype II Absent 4. Cellular infiltration Present(++++)Present(+++)Present(++) 5. Bile duct hyperplasia and newly formed bile ductules Present(+++)Present(++)Absent 6. Hepatocytes vaculation Present(++++)AbsentPresent(+++) 7. Hepatocytes granulation Present(+++) Absent 8. Activation of Kupffer cells Present(+++) Absent ++++=Severe+++=Moderate++ & +=Mild liver Table (4): The main histopathological lesions in liver and degree of egg-type severity in immunized egg-type chickens at 3 rd or 9 th week of age with two types of bacterins

LesionsControl (AHAB) Broilers Immunized with (AHAB) (IFAB) Broilers Immunized with (IFAB) 1.Necrosis of enterocytes Present(++++) specially biotype II Present(+) 2.Hyperplasia of enterocytes Present(++) specially biotype II AbsentPresent(+) 3.Goblet cells transformation Present(++)Present(+) specially biotype II Present(+) 4.Hyperplasia of Crypt Present(++++)AbsentPresent(+) 5.Cellular infiltration in lamina propria Present(++++)Present(++)Present(+) intestine Table (5): The main histopathological lesions in intestine and degree of egg-type severity in immunized egg-type chickens at 3 rd or 9 th week of age with two types of bacterins ++++=Severe+++=Moderate++ & +=Mild

control Fig. (17): Liver of control (unimmunized and infected) egg-type chickens showing destruction of the cell wall of the blood vessels (arrow). (H & E X33) (AHAB) Fig. (18): Liver of egg-type chickens immunized with (AHAB) at 3 weeks of age and infected with C.jejuni showing focal aggregation of mononuclear cells around the portal area and inside the blood vessel (arrow). (H & E X66). control Fig. (19): Liver of control (unimmunized and infected) egg-type chickens showing multifocal necrotic area (arrow).(H & E X33). control Fig. (20): Liver of control (unimmunized and infected) egg-type chickens showing multifocal nuclear cells aggregation in between hepatic parenchyma. (H & E X33).

Fig. (21): Liver of egg-type chickens immunized with (AHAB) at 9 weeks of age and infected with C.jejuni showing moderate mononuclear cells aggregation. (H & E X33). Fig. (22): Liver of egg-type chickens immunized with (IFAB) at 9 weeks of age and infected with C.jejuni showing slight mononuclear cells aggregation (arrow). (H & E X33). Fig. (23): Liver of egg-type chickens immunized with (AHAB) at 9 weeks of age and infected with C.jejuni showing mild activation of Kuppfer cells (arrow). (H & E X132). Fig. (24): Liver of egg-type chickens immunized with (AHAB) at 9 weeks of age and infected with C.jejuni showing newly formed bile ductule (arrow). (H & E X132).

control Fig. (25): Intestine of control (nuimmunized and infected) egg-type chickens showing severe necrosis of enterocytes leaving denuded surface of villi. Notice severe inflammatory cells infiltration in lamina propria. (H & E X33). (AHAB) Fig. (26): Intestine of egg-type chickens immunized with (AHAB) at 3 weeks of age and infected with C.jejuni showing mild necrosis of enterocytes at tip of villi (arrow). (H & E X33).

Fig. (27): Comparison between the results of ELISA in immunized egg-type chickens with AHAB and IFAB at 9 weeks of age

Fig. (28): Post mortem lesion scoring of C.jejuni in immunized and unimmunized egg-type chickens at the end of 21 days observation period

Fig. (29): Shedding of C.jejuni in immunized and umimmunized egg- type chickens during 21 days observation period

Fig. (30): Reisolation of C.jejuni from immunized and unimmunized egg-type chickens at the end of 21 days observation period

Fig.(31): Post mortem lesion scoring of C.jejuni in acidifier and probiotics treated and untreated chicken broilers

Fig.(32): Shedding of C.jejuni in acidifier and probiotics treated and untreated chicken broilers

Fig.(33): Reisolation of C.jejuni from acidifier and probiotics treated and untreated chicken broilers

CONCLUSION

C.jejuni The most important point of view in this task was how to control C.jejuni infection in poultry. The results proved that application of water-type or oil-type bacterin was effective, but the later was more effective than the former. These bacterins gave short period of immunity so, they need to be boostered to prolong the period of immunity. (lactic and formic acid) (P. acidilactici and S. boulardii) S.boulardii C.jejuni Addition of acidifier (lactic and formic acid) and probiotics (P. acidilactici and S. boulardii) to poultry feed was very successful. However, S.boulardii was the superior in complete elimination of C.jejuni infection.