Isolation And Identification Of Riemerella Anatipestifer And Preliminary Study On Artificial Infection Test

Riemerella anatipestifer (RA) is a major cause of disease of ducks across the world and can results in great financial loss. This pathogen mainly infects poultry especially ducklings within 1 month. It can cause an acute or chronic, septic, high contact of infectious diseases with respiratory symptoms, fibrinous pericarditis, perihepatitis, balloon inflammation, meningitis and arthritis as principal symptoms. So researches focusing on the identification and pathogenesis of RA are important for the prevention of anatipestifer disease.In our study, we designed a pair of primers targeting the nucleotide sequences of outer membrane protein gene of RA from GenBank and established a specific PCR for rapid detection of RA. By using the established PCR to test the DNA samples from Avian Pasteurella multocida, poultry Salmonella, Escherichia coli have, we determine specificity of the method. Overall, we collected suspected samples (n=35) of heart, liver and brain from April 2013 to March 2014. After streaking these samples on TSA plates, suspected colonies were picked for preliminary separation and purification. Suspected colonies were identified by PCRs and the results showed that we totally isolated 20 strains of RA. The results of the antibiotics susceptibility tests about the 20 strains of RA against 16 kinds of antibiotics showed that 95%～ 100% were high sensitivity to cephalosporins and piperacillin, while the resistance to streptomycin, tobramycin and kanamycin were up to 90%～100%, indicating that RA in local area was loccephalosporin-sensitive.Type 1 serotype RA strain was selected to determine the LD50 of 14 days old ducklings infected by intraperitoneal injection according to Karber method, and the result turned out to be 2.5 x 107 CFU. We obtained animal models of RA infection using an LD50 dose of infected ducklings and provided further information for the research on ducklings’infected immune organ index at different time points, serum biochemical parameters, dynamic changes of inflammatory factor.By monitor the immune organs, we found that the immune organ index of spleen rose with the development of the course, while those of bursa and thymus decreased. And finally they all became normal after recovery period. The serum biochemical indicators were examined by automatic biochemical analyzer. The followings are overall results:comparing with those in control group, the ALT, AST activity of the infected group appeared with decreasing-increasing trend and the ALT and AST activity were significant decreased 48h～60h and 48h～72h than the control group (P<0.05 or P<0.01). However after 7d, they were significantly increased (P <0.05). TP, GLO activity of the infected group appeared with an increasing-decreasing trend, TP activity were significantly increased after 48h, GLO activity were significantly increased at 48h-84h (P<0.05 or P<0.01); GGT, ALB activity were significantly increased than control groups during recovery period (P<0.05 or P<0.01). And in this study a real-time quantitative PCR based on SYBR Green was established to relatively quantity of the gene expression of inflammatory factor mRNA in spleen and reveal its change rules. The results pointed that the expression of IL-1β、IL-2、IL-6、IL-8、IL-10、IFN-y of the infected group increased(P<0.05 or P<0.01) with the development of the course and reaches the peak after 48h-60h. The gene expression of TNF-a mRNA in infected group increased (P<0.05) during 12 hours to 72 hours after injection. Although gene expression during 12 hours to 72 hours was slightly higher than control group, it has no statistically significance between this two groups (P> 0.05). Compared with control group, gene expression of TGF-β1 mRNA in infected group was significantly higher than control group in recovery period (P<0.05). Gene expression of C3 mRNA in infected group increased at first and decreased slightly then, but was still significantly higher than that in control group(P<0.05 or P<0.01). Gene expression of C5 mRNA in infected group was significantly higher (P> 0.05) at early stage of infection, while in control group it was significantly higher at 72h (P> 0.05), and there was no significant difference at other times. The result suggested that RA infection could cause variation of inflammatory factors which regulate immunoreactions. And all the gene expression of inflammatory factors increased among infected ducklings more or less, accounting for the severe inflammation of ducklings.To conclude, a fluorescence resonance energy transfer (FRET) real-time PCR was established for absolutely quantitative detection of organization pathogens, revealing its dynamic propagation rules in ducklings. Ducklings Infected with RA within 6 hours, organisms could be detected from heart, liver, spleen, lung, brain, bursa of Fabricius and thoracic. Moreover, bacterial number in various tissues and organs of ducklings increased with the development of the course and decreased in the recovery period and was the highest in the heart, lung and liver, followed by immune organ, while that in brain got the lowest.