Isolation And Characterization Of Haemophilus Parasuis And Propolis Vaccine Immune Protection Effect Research

Haemophilus parasuis is a member of the family Pasteurellaceae commensal organism of the upper respiratory tract of conventional pigs.Under appropriate conditions,it can invade and cause severe systemic bacterial disease,characterized by fibrinous polyserositis, meningitis and arthritis.at the present time, the disease has become one of the most serious impact of global pig industry one of the bacterial transmitted diseases, the incidence and mortality also showed a significant upward trend,cause serious economic losses to China’s pig industry.This study was conducted to bacteria isolation and identification as well as indirect ELISA for detection of antibodies to the establishment of method and propolis vaccine immunogenicity research.The main results are as follows:1.In this study, suffering polyserositis and arthritis pigs isolated from Haemophilus parasuis from a few large-scale pig farms in Sichuan Province identified their cultural characteristics, morphology, and biochemical characteristics, the results show that the bacteria did not grow on the basic agar plate or the MacConkey medium, did not grow well on the fresh blood agar plate and the chocolate plate, and grew well on the TSA plate and NAD fresh blood agar plate. Results indicate that the strain is NAD dependent bacteria,with microscopic examination of Gram-stained sample the bacteria was determined to be Gram negative. Morphology of the bacteria was multiple, and exhibited filament, short rod and even ball in shape.It is consistent with the basic characteristics of Haernophilus parasuis;According to the Haemophilus parasuis strains isolated from16SrRNA sequence design primer pairs PCR amplification, further confirmation of the isolated strains of Haemophilus parasuis.The results show that prevalence of HPS in some areas in Sichuan province. 2.On the isolated strains of antibiotic susceptibility tests, and the serotype identification.The results revealed that the isolated strain was tolerable to only gentamycin, enrofloxacin, compound sulfamethoxazole and penicilin, and highly susceptible to cephalosporin Ⅳ, cefotaxime, sulbactam, furaxone, chloromycetin.The isolates strain were made of saline bacterial suspension,detection was carried out according to the method described in the directions of standard serum kit of Haemophilus parasuis.The result showed that isolated strain has stronger coagulation reaction with the positive serum of Hps serovar5, in which white laminar coagulate was formed, but no coagulation reaction with other standard positive serum samples and normal saline.Show that the isolates of HPS type5and named SCHPS5.3.Study on grouth curve of SCHPS5,show that the bacteria in Typtic Soy Broth liquid medium around4h began a slow growth,16～18h growth is best, as the extension of time, OD600began to gradually decline, about24h OD600has risen again, after falling.This test provides a basis for the best time of preparation of inactivated vaccine for Haemophilus parasuis.4.In this study,indirect ELISA method of Haemophilus parasuis antibody for detection was studied, by utilizing the isolated strain bacteria of treated with ultrasonication as the antigen, with preparation of positive serum, optimization of concentration of the coating antigen, concentration of the secondary antibody and reaction time, and susceptibility test, specificity test and repeatability test,established by detection of HPS antibodies by indirect ELISA method.The test determines of the HPS whole bacterial coating concentration was diluted in a ratio of1:100and the serum was diluted in a ratio of1:160,and indirect ELISA optimization of reaction conditions of HPS.The detection sensitivity of the target antibody in indirect ELISA method based on this isolate can reach over1:10000, and this detection method has high specificity and high repeatability.5.In this study, the preparation of Haemophilus parasuis propolis vaccine,and testing its security,after testing safety to pregnant sows prenatal2weeks muscle vaccinated to observe the piglets of maternal antibody levels.And the newborn piglets were vaccinated, and in1,7,14,21,30,35,40,45days after immunization, blood serum separation, detection in newborn piglets immunized serum antibody level.The results show that, through to the pregnant sow prenatal immune for newborn piglets with a higher level of maternal antibody, but piglet at lactation stage did not receive immunity, its in vivo in maternal antibody after weaning will soon disappear, and in newborn piglets immunity can make the piglets at weaning still had higher serum antibody level.