Passive and active immunity of broiler chickens against Campylobacter jejuni and ways of disease transmission

The study deals with passive and active immunity of fifty-three broiler chickens after infection with culture of Campylobacter jejuni. Potential transfer of infection by faecal-oral and aerogenic routes was also investigated. Cloacal swabs and ceacal content were analyzed microbiologically. Identification of C. jejuni was carried out by polymerase chain reaction. Observation of passive immunity of broilers from 3 days of age showed that no transfer of C. jejuni infection occurred up to 12 day post-infection (p.i.). Observations of active immunity in fourteen 21 days old chickens infected with C. jejuni showed that 6 chickens were positive on day 3 p.i. and all infected chickens were positive on day 5 p.i. Investigations of the transfer of C. jejuni by faecal-oral route revealed positivity in two broilers on day 3 p.i. and in all tested chickens on day 5 p.i. Aerogenic transfer of infection was not recorded. This was one of the first studies in our country dealing with passive and active immunity of broiler chickens against C. jejuni and spreading of this zoonotic disease. Campylobacteriosis, PCR, microbiological examination, disease transfer The food chain is considered one of the most important ways of transfer of alimentary infections among animals and humans. The spreading of infections in poultry production is related to insufficient hygiene of rearing which is stressful to housed birds and results in increased numbers of heterophils (Bedáňová et al. 2007). Campylobacter jejuni is world-wide considered the principal cause of human diarrhoeal diseases and gastroenteritis transmissible in food. Campylobacteriosis is a zoonosis; many animals serve as reservoirs of this infectious disease. Campylobacter sp. enters the environment also via drinking water contaminated with excrements of animals, birds or infected people (Nogva et al. 2000). The aim of the study was to investigate passive and active immunity of broiler chickens against C. jejuni and potential transfer of this disease by faecal-oral and aerogenic routes. Materials and Methods Experimental animals Fifty-three broiler chickens of the Ross 308 hybrid were obtained from an approved hatchery Častá (No. SK-LH-PK-01-MACH poultry, Budmerice s.r.o., Slovak Republic). The chickens were kept in the experimental facilities of the Department of Microbiology and Immunology, University of Veterinary Medicine and Pharmacy (UVMP) in Košice. Before the experiment, cloacal swabs were taken from all chickens included in the study and all were negative for Campylobacter sp. Then, after 2 days of acclimatization, they were assigned to four experimental groups: Group 1 (n = 13), Group 2 (n = 20), Groups 3 (n = 10) and Group 4 (n = 10). ACTA VET. BRNO 2012, 81: 103–106; doi:10.2754/avb201281020103 Address for correspondence: MVDr. Silvia Ondrašovičová, PhD. Department of Anatomy, Histology and Physiology University of Veterinary Medicine and Pharmacy Komenského 73, 041 81 Košice, Slovak Republic Phone: 532 146 205 Fax: 541 211 214 E-mail: ondrasov1@uvm.sk http://actavet.vfu.cz/ Infection To infect the chickens we administered bacterial culture of reference strain of C. jejuni CCM 6207 (Masaryk University Brno, Czech Republic) per os at a dose of 0.2 ml per experimental chick. The colony-forming unit (CFU) counts at testing of passive and active immunity were 103·ml-1 and 104·ml-1, respectively. Seven chicks from Group 1 were infected with C. jejuni at the age of 3 days, to evaluate their passive immunity. For evaluation of active immunity we infected 14 chickens from Group 2 at the age of 21 days with the same culture of C. jejuni. The remaining six chickens of Groups 1 and 2 were not infected and were housed together with the infected ones to observe potential transfer by the faecal-oral route. Group 3 was kept in a separate cage in the same experimental room to observe the possibility of aerogenic transfer. Group 4 was housed in a separate room and served as control. In the first week of age the chickens were exposed to a temperature of 25–30 °C which was then decreased to 22–27 °C. Standard mixed feed BR1, BR2 and BR3 was supplied to the chickens according to the age. They were fed ad libitum from a feeder and had free access to drinking water. Sampling and microbiological diagnosis Using sterile swabs, two cloacal samples were taken from all broilers on predetermined days (Tables 1 and 2). One swab was transferred to a tube with Bolton liquid multiplication medium and incubated under micro-aerobic conditions for 4 to 6 h at 37 °C and then for 44 h ± 4 h at 41.5 °C. After incubation, we inoculated a loopful of the culture onto two selective modified media, mCCDA and Skirrow agar medium. The second cloacal swab was used to inoculate directly the mCCDA and Skirrow agar media. The inoculated media were incubated at 41.5 °C under micro-aerobic conditions and the colonies were counted after 44 h ± 4 h. The procedures used for confirmation of the genus Campylobacter and for differential diagnostics complied with the Slovak standard STN EN ISO 10272-1. The broilers were killed by cervical dislocation on predetermined days (Tables 1 and 2). Determination of Campylobacter was carried out on 1g of the caecal sac content using decimal dilutions (ISO/TS 10272-2, Slovak standard STN EN ISO 10272-1). PCR diagnosis The DNA was isolated from C. jejuni colonies grown on the above described selective solid media by resuspending them in 500 μl sterile distilled water and incubation at 100 °C for 5–20 min. The sample was then centrifuged at 12 000 g for 5 min (twice) and the supernatant was used for PCR. Concentration of the DNA was determined by a spectrophotometer Spectronic Genesys 5. For PCR we used 100 ng of DNA of the examined sample, 5μl of 10 × concentrated buffer solution according to manufacturer’s recommendation, 0.2 mM dNTPs, 1 μM primers, 3.5 mM MgCl2, 1.0 U AmpliTaq DNA Polymerase (Perkin Elmer, USA). The sample volume was made up to 50 μl with distilled water. The primers for genus Campylobacter were C412F and C1228R, resulting in an amplicon 816 bp and the primers for species C. jejuni were Jej1F and Jej2R, resulting in an amplicon 793 bp (Brown et al. 2004). To identify PCR products we used system GelLogic 100. The results obtained were evaluated statistically using one-way ANOVA, Friedman test, Dunn’s post-test and Mann-Whitney test.


Infection
To infect the chickens we administered bacterial culture of reference strain of C. jejuni CCM 6207 (Masaryk University Brno, Czech Republic) per os at a dose of 0.2 ml per experimental chick.The colony-forming unit (CFU) counts at testing of passive and active immunity were 10³•ml -1 and 10 4 •ml -1 , respectively.
Seven chicks from Group 1 were infected with C. jejuni at the age of 3 days, to evaluate their passive immunity.For evaluation of active immunity we infected 14 chickens from Group 2 at the age of 21 days with the same culture of C. jejuni.The remaining six chickens of Groups 1 and 2 were not infected and were housed together with the infected ones to observe potential transfer by the faecal-oral route.Group 3 was kept in a separate cage in the same experimental room to observe the possibility of aerogenic transfer.Group 4 was housed in a separate room and served as control.
In the first week of age the chickens were exposed to a temperature of 25-30 °C which was then decreased to 22-27 °C.Standard mixed feed BR1, BR2 and BR3 was supplied to the chickens according to the age.They were fed ad libitum from a feeder and had free access to drinking water.

Sampling and microbiological diagnosis
Using sterile swabs, two cloacal samples were taken from all broilers on predetermined days (Tables 1 and 2).One swab was transferred to a tube with Bolton liquid multiplication medium and incubated under micro-aerobic conditions for 4 to 6 h at 37 °C and then for 44 h ± 4 h at 41.5 °C.After incubation, we inoculated a loopful of the culture onto two selective modified media, mCCDA and Skirrow agar medium.
The second cloacal swab was used to inoculate directly the mCCDA and Skirrow agar media.The inoculated media were incubated at 41.5 °C under micro-aerobic conditions and the colonies were counted after 44 h ± 4 h.The procedures used for confirmation of the genus Campylobacter and for differential diagnostics complied with the Slovak standard STN EN ISO 10272-1.
The broilers were killed by cervical dislocation on predetermined days (Tables 1 and 2).Determination of Campylobacter was carried out on 1g of the caecal sac content using decimal dilutions (ISO/TS 10272-2, Slovak standard STN EN ISO 10272-1).

PCR diagnosis
The DNA was isolated from C. jejuni colonies grown on the above described selective solid media by resuspending them in 500 µl sterile distilled water and incubation at 100 °C for 5-20 min.The sample was then centrifuged at 12 000 g for 5 min (twice) and the supernatant was used for PCR.Concentration of the DNA was determined by a spectrophotometer Spectronic Genesys 5.For PCR we used 100 ng of DNA of the examined sample, 5µl of 10 × concentrated buffer solution according to manufacturer's recommendation, 0.2 mM dNTPs, 1 µM primers, 3.5 mM MgCl 2, 1.0 U AmpliTaq DNA Polymerase (Perkin Elmer, USA).The sample volume was made up to 50 µl with distilled water.The primers for genus Campylobacter were C412F and C1228R, resulting in an amplicon 816 bp and the primers for species C. jejuni were Jej1F and Jej2R, resulting in an amplicon 793 bp (Brown et al. 2004).To identify PCR products we used system GelLogic 100.
The results obtained were evaluated statistically using one-way ANOVA, Friedman test, Dunn's post-test and Mann-Whitney test.

Results
The tests of passive immunity of 3-day-old chickens infected with C. jejuni (Group 1) carried out on days 3, 5, 7, 9 and 12 post-infection (p.i.), provided negative results for Campylobacter (Table 1).Throughout the experiment we observed no health changes in the experimental chickens.
The testing of active , carried out on day 3 p.i., showed positivity in 6 infected chickens and 2 chickens were infected by the faecal-oral route.In this period the chickens showed no marked health changes.On day 5, 7, 9 and 12 p.i., the microbiological and PCR methods proved the positivity to C. jejuni in all infected birds but, at the same time, also in all 6 chickens infected by the faecal-oral route (Table 2).
Clinical observations of the chickens from Group 2 showed that on day 7 p.i. two infected chickens suffered from watery diarrhoea.On day 9 p.i. we slaughtered 7 infected chickens and 3 chickens from the group observed for the faecaloral transfer, collected their caecal sacs and carried out pathological-anatomical examination.Two slaughtered chickens infected with C. jejuni showed dilatation of jejunum.On day 12 p.i. additional three chickens from this group, infected with C. jejuni, were affected with diarrhoea.All remaining chickens were slaughtered on this day.Post mortem examination showed dilatation of jejunum in four infected chickens and disseminated haemorrhagic enteritidis in one of them.
The plate counts of C. jejuni in 1g of caecal content of infected chickens (Group 2) ranged from 3.6 × 10 5 to 5.1 × 10 6 CFU and in chickens infected by the faecal-oral route from 7.4 × 10 4 to 4.3 × 10 6 CFU.
Their body mass in Group 2 ranged between 0.9 kg and 1.2 kg (mean 0.98 kg).
Statistical evaluation of the day of colonization of intestinal tract with C. jejuni in Group 2 by means of transformation 0 (negative) and 1 (positive) by Friedman test and Dunn's post-test showed significant difference between days 1 and 5 p.i. at the level of significance α = 0.01.Evaluation of findings by Mann-Whitney test on day 3 p.i. in Group 2 comparing the spreading of infection by direct and faecal-oral route showed no significant differences (α = 0.05) meaning that the infection spread at the same rate.
The cloacal swabs taken from all chickens from Groups 3 and 4 on days 3, 7, 17 and 33 of the experiment were negative for C. jejuni.The health of these chickens was good throughout the experiment.The body mass of chickens from Group 3 ranged between 1.3 and 1.7 kg and that of chickens from Group 4 between 1.3 and 1.9 kg but the mean body mass was the same in both groups (1.5 kg).

105*
sampling of caecal sacs after slaughter

104 Table 1 .
Testing of passive immunity and potential faecal-oral infection transfer in broilers from Group 1.
*collection of samples from caecal sacs after slaughtering the chickens

Table 2 .
Testing of active immunity and potential faecal-oral infection transfer in broilers from Group 2NumberMicrobiological and PCR examination of cloacal swabs and caecal sacs