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

3. A Trial for Prevention of Campylobacter jejuni Infection in Broiler Chickens using Autogenus Bacterin M. H. Awaad 1, Nagwa, S. R. 2 and Wafaa A. Abd El-Ghany 1 1 Faculty of Veterinary Medicine, Cairo University 2 National Research Centre, Egypt

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

5. 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. 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).

6. The aim of the study C.jejuni Preparation of two types of C.jejuni bacterins from the local field strains and evaluation of their protective potential in broiler chickens through immuno-Assay [mean Enzyme Linked Immuno- sorbent Assay (ELISA) titres] and bio-Assay (clinical signs, mean lesion score, rate of shedding, reisolation rate and histopathological examination)

7. Materials & Methods Three local strains of C.jejuni representing biotype I and II were used in bacterins preparation and as a challenge bacteria. Preparation of whole cell bivalent C.jejuni bacterins aluminium hydroxide (AHAB) and incomplete Freund’s oil adjuvant bivalent bacterins (IFAB) )10 9 CFU/ml C.jejuni) as described by Williams et al., 1976 and Bryner et al., 1978 and 1988. Quality control tests of the prepared bacterins (purity, sterility and safety) tests (British veterinary codes, 1970)

8. Experimental design

9. Serum samples were collected from immunized birds at 0 hr (just before immunization) and weekly after each immunization dose. Sera were subjected to ELISA (Cawthraw et al., 1994) to detect humoral IgG antibodies. All immunized challenged birds (0.5ml C.jejuni 10 8 CFU biotype I&II at 4 wks old) were kept under close observation for 3 weeks for signs, mortalities & lesions Cloacal 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 C.jejuni re-isolation, post mortem examination for lesion scoring (Rabie, 1998) & histopatholgy.

10. 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

11. 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.

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

13. Results of Bio-Assay C.jejuni Signs of depression, sleepy appearance and mucoid greenish diarrhea were seen only in control C.jejuni challenged group. C.jejuni No deaths were recorded in all C.jejuni challenged groups. Clinical signs Mortalities

14. Shedding of C.jejuni in immunized and unimmunized broiler chickens during 21 days observation period

15. Reisolation of C.jejuni from immunized and unimmunized chicken broilers at the end of 21 days observation period

16. Gross lesion scoring of C.jejuni in immunized and un- immunized sacrificed chicken broilers at the end of 21 days observation period.

17. liver The main histopapatological lesions in liver and degree of severity in chicken broilers immunized with two types of C.jejuni bacterins Lesions Control (AHAB) Broilers immunized with (AHAB) (IFAB) Broilers immunized with (IFAB) Congested blood vessels ++Absent Inflammatory cells in blood vessels ++++ Multifocal necrosis +++++ specially biotype I++ Cellular infiltration +++++++++ Bile duct hyperplasia and newly formed bile ductules +++Absent Hepatocytes vaculation ++++++ specially biotype IIAbsent Hepatocytes granulation Absent++ specially biotype IIAbsent Activation of Kupffer cells +++Absent ++++=Severe+++=Moderate++ & +=Mild

18. intestine The main histopapatological lesions in intestine and degree of severity in chicken broilers immunized with both bacterins LesionsControl (AHAB) Broilers Immunized with (AHAB) (IFAB) Broilers Immunized with (IFAB) Necrosis of enterocytes+++++++++ Hyperplasia ofenterocytes+++Absent+ Goblet cells transformationAbsent + especially biotype II Hyperplasia of Crypt+++++ especially biotype II + especially biotype II Cellular infiltration in lamina propria ++++++++ ++++=Severe+++=Moderate++ & +=Mild

19. (IFAB) Fig. (1): Liver of broilers immunized with (IFAB) and infected with C.jejuni showing few numbers of inflammatory cells in blood vessels. (H & E X33). Fig. (2): Liver of broilers immunized with (AHAB) and infected with C.jejuni showing focal area of necrosis (arrow). (H & E X33). Fig. (3): Liver of broilers immunized with (IFAB) and infected with C.jejuni showing mild vacculation of hepatocytes. (H & E X66). Fig. (4): Liver of control (unimmunized and infected) broilers showing severe hyperplasia of the epithelial lining the bile duct. (H & E X66). 12 3 4

20. Fig. (5): 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. (6): 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. (7): 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). 56 7

21. C.jejuni Application of C.jejuni water-type or oil-type bacterin was effective considering stimulation of immune response, reduction of signs, lesions and the organism shedding. Oil type bacterin was more effective than water type one. CONCLUSION