Isolation And Identification Of Riemerella Anatipestifer And Development Of The Bivalent Inactivated Vaccine

Infectious serositis of duck is a contagious disease, caused by Riemerella anatipestifer (RA). The2to8-week-old ducks are most vulnerable to the disease along with acute or chronic septicemia. Sick ducks are characterized by oculonasal discharges, diarrhoea, ataxia and tremor of head and neck, showing pathological changes in fibrinous pericarditis, perihepatitis, airsacculitis and meningitis, and many infected animals show caseous salpingitis, conjunctivitis and arthritis and so forth. The mortality rate for the disease is about10-80%and the ducks slowly grow with the declined feed conversion after recovery from the disease. Since the disease spreads by contaminated feed, water, air and dust, which persist in contaminated duck farms and is difficult to be eliminated, through respiratory and gastrointestinal tracts or skin lesions, it is considered as one of the most serious diseases causing economic loss for duck farming.According to isolation of bacteria from the dead ducks of suspected infectious serositis from Linyi and Qingdao etc., Shandong in2008, a total of14strains of bacteria were isolated, and then through the identification for morphological, biochemical and cultural characteristics, PCR assay and serotype identification, the results showed that all of14isolates were RAs,6and5strains of which were serotype1and2, respectively, and the others were not identified. Using the isolates to prepare fresh broth and regulating its density to1×109CFU/mL, by which the animal regression test was performed for2-week-old ducklings, the result indicated that all of the ducklings, to some extent, fell ill or died during10days post artificial infection. The results of antibiotic sensitivity tests showed that the isolates were sensitive to ceftriaxone, cephalexin, florfenicol, gentamicin, cefotaxime and vanguard ADM Ⅵ; however, most of the isolates, to some extent, resisted to tylosin, Norfloxacin, tetracycline, ofloxacin, ciprofloxacin and cotrimoxazole.The hyperimmune sera against RAs were prepared with the strains of RA-sd3(serotype1) and RA-sd2(serotype2) with good immunogenicity. The microagglutination test (MAT), which detected antibodies from sera against RAs, was developed:in96-wells V-shaped well bottom plate,50μL of serum was diluted following2-fold serial dilutions with50μL0.85%sterile physiological saline and then the50μL of agglutination antigens (OD600=1.452) were added to the diluted sera, and the result could be observed after4hours post reaction of samples in moist box (37℃). The prepared agglutination antigens can be conserved in4℃for5months. The method is characterized by relatively high sensitivity and specificity as well as low cost, and is convenient to employ in clinical diagnosis.The bivalent inactivated vaccine for infectious serositis of duck was prepared complying with the procedure:cultivating the strains of RA-sd3(serotype1) and RA-sd2(serotype2) with good immunogenicity in tryptone soy broth media, respectively, harvesting products, regulating the density of bacteria to1×1010CFU/mL, using0.3%formaldehyde to inactivate the products, equally mixing them together and finally, emulsifying the product with a ratio of water to oil being1:2. The3～7-day-old ducklings were vaccinated through subcutaneous injection in the neck with the bivalent inactivated vaccine (0.3mL/feather) which undergone a safety inspection. The effective rate of protection was over90%after21days post artificial infection and the period of Immune protection could last two months.