| Campylobacter jejuni (Cj) is an important zoonotic foodborne pathogen. It can cause human bacterial gastroenteritis and acute diarrhea if being infected. Immunocompromised persons can lead to endocarditis, arthritis, osteomyelitis, encephalitis, sepsis and other systemic diseases. But so far, Cj has no appropriate animal models yet and its pathogenesis is not fully understood. This study attempted to establish infant rabbits model for Cj infection induced diarrhea and to study the dynamic distribution of bacteria in rabbit model, the regular pattern of the immune response and possible infection mechanisms. Using selective capture of transcribed sequences method to identify Cj genes expressed during the process of infection of infant rabbit cecum. The results of this study achieved would be meaningful for in-depth study and control of Cj infection.1、Establishment of infant rabbit model for C.jejuni induced diarrheaThe infant rabbits were selected as the experimental animals because they were phylogenetically more closely related to primates than rodents were and had an anatomy and physiology that more closely resembles that of primates. Also they’re small in body type, convenient to feed, and easy to manage. Based on the optimal modeling condition from experiments, orogastric inoculation of109CFU C.jejuni into2-day-old,40±2g rabbits pretreated with cimetidine(50mg/kg), and then evaluate the model by collecting clinical symptoms, pathological changes, pathogen isolation and pathogenicity information. The results indicated that C.jejuni can artificially infect the infant rabbit and induce self-limiting, watery diarrhea disease. The gross pathological changes showed the fluid-filled distended cecum and the enlarged colon with a thicked wall. Histological analyses of sections from the intestines of infected rabbits revealed mild abnormalities including edema, congestion of capillaries and lamina propria. C.jejuni was isolated and identified from infected organs. Dose-response experiments showed that the degree of diarrheal diseases was positive correlation between the challenge dose and the amount of bacterial colonization. Diarrhea occurs when as little as106CFU of C.jejuni was orally inoculated into rabbits. And the cecum and colon colonization occurs when the dose was as little as104CFU. Different isolates of C.jejuni showed significantly different in pathogenicity. This study successfully established infant rabbit model of diarrhea caused by Cj, and the model can effectively evaluate Cj strain’s virulence.2、The dynamic study of C.jejuni in infant rabbit modelBased on the model above-mentioned, the rabbits were euthanized at fixed times after infection (12,24,36,48or60hr PI). At necropsy, the regularity and distribution of Cj in vivo organism was detected by bacteria isolation and PCR. The Cj invasive effect and morphological changes of intestinal epithelial cells were observed by scanning and transmission electron microscopy. Common cytokines, chemoattractants, antimicrobials and enzymes of the rabbit immune response were detected by real-time RT-PCR. The experiment results indicated that Cj could quickly invade into blood, showed no significant difference in adhesiveness to liver, kidney, and other organon parenchymatosums and eliminated at48hr PI. Cj could colonize longer than60hr and as high as108CFU/g in the cecum and colon. Scanning and transmission electronmicrographs showed Cj can adherent on the villi of rabbits, partly brush border of the epithelium is destroyed at24-36hr PI. At48-60hr, overall architecture of the epithelium is disorganized with necrotic epithelial cells with severe inflammatory infiltrate IEL. IL-10and antimicrobial peptide CAP-18were upregulated firstly at12hr PI then IL-18and CCL20upregulated at36hr PI. At the late time point of infection (60hr PI), IFN-y、IL-10、IL-8and iNOS played as key innate responders to bacterial challenge in the intestine.3、Identification of C.jejuni genes expressed within rabbit cecum by selective capture of transcribed sequencesThe selective capture of transcribed sequences (SCOTS) was used to identify Cj NCTC11168genes expressed during the process of infection of infant rabbit cecum. Total RNA was isolated from infected tissues (cecum)24hr PI and converted to cDNAs. By using the cDNA selection method of SCOTS, cDNA clones derived from transcripts in the cecum were selected and sequenced. A total of9genes were identified, of which encoded enzymes for metabolism, cell surface biosynthesis, replication and general microbial stress response. Five genes were chosen randomly and verified by real-time RT-PCR analysis. All5genes (pnp、katA、cj0609c、 cj1481c and acs) were upregulated by Cj in infected rabbit cecum, with changes ranging from4.49-fold to116-fold when compared with in vitro cultures. The genes will provide a molecular basis for further study of the Cj pathogenesis. |
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