Isolation, Identification And Antimicrobial Susceptibility Of Campylobacter In Pig And Chicken And Development Of A Loop-mediated Isothermal Amplification Assay For Detection Of Campylobacter Jejuni

Campylobacter is a Gram-negative, microaerophilic, vibrio or spiral-shapedbacterium. It causes intestinal infections in human and animals worldwide. Poultrycomprises the greatest concentration of Campylobacter and thus the main source of humancampylobacteriosis although no obvious symptoms appeared under infection. In total,19species of the genus Campylobacter have been described thus far. The two species, C.jejuni and C. coli, are the most common bacterial causes of human gastroenteritis.To investigate the prevalence and species of Campylobacter in pig and poultry gut,samples from Beijing suburban livestock and poultry farms and slaughter house werecollected for Campylobacter isolation. After enrichment the samples were simultaneouslyexamined for the genus Campylobacter, the species of C. jejuni and C. coli by triplex PCR.Isolation and identification of Campylobacter strains were performed from the PCR-basedpositive samples. Finally, the Campylobacter isolates were tested antibiotic resistance byantimicrobial susceptibility assay. In this study,132samples from chicken intestinal andanal swabs were identified by PCR. Totally, C. jejuni-positive amplification product wasobserved in6samples. Unfortunately, no isolates of C. jejuni was obtained with bacterialisolation. A total of241samples of pig intestinal contents and anal swabs were screened bytriplex PCR. Totally,178（73.9%） and5（2.1%） samples showed positive for C. coli and C.jejuni respectively. Of all the Campylobacter positive samples,63（35.4%） C. coli isolateswere isolated and identified while no C. jejuni isolate was obtained. Antimicrobialsusceptibility of63C. coli isolates was evaluated with26antibiotics. The results showedthat the C. coli isolates were highly sensitive to chloramphenical （100%）, antimicrobial（93.65%）, doxycycline （91.48%）, gentamicin （82.54%） and amoxycillin （82.54%） whilesignificant resistance to cfeoperazone （100%）, cephalexin （100%）, cephradine （100%）,cefazolin （98.41%）, cefixine （92.06%）, nalidxic Acid （92.06%）, streptomycin （90.48%）,ciprofloxacin （90.32%）, norfloxacin （88.89%） and penicillin （87.30%）. Prevalence ofmultiple antibiotics resistance in the C. coli isolates was observed with a high percentageof14-20antibiotics resistance. This study indicated that C. coli, but not C. jejuni, was themost commonly detected Campylobacter species in pig gut with significant resistance to multiple antibiotics.Loop-mediated isothermal amplifi cation （LAMP） is an established nucleic acidamplification method offering rapid, accurate, and cost-effective diagnosis of infectiousdiseases. In this study, the LAMP assay was developed to detect C. jejuni in animalproducts. Firstly, the specific mapA gene was used to design a set of primers in developinga loop-mediated isothermal amplification （LAMP） assay for detection of C. jejuni. Tooptimize the LAMP assay, some factors involved in the assay were screened to determinethe optimal condition. The final LAMP assay comprised1.6μM each of inner primers FIPand BIP,0.2μM each of outer primers F3and B3,8mM Mg²⁺,1.4mM dNTP,0.8MBetaine. The mixture was incubated at65℃for60min. The specificity test showed thatthe assay correctly identified all C. jejuni strains but not13other bacterial species. Thesensitivity of the assay was100fg for C. jejuni genomic DNA and7.5cfu per test tube forC. jejuni culture. This LAMP assay is a rapid and simple tool for the detection of C. jejuniand will provide an essential role in facilitating the early diagnosis of this organism.