Selection And Immumogenicity Of The Vaccine Strain Of Haemophilus Parasuis

Haemophilus parasuis is a Gram-negative microorganism that colonizes the upper respiratory tract of conventional pigs,but under appropriate conditions can invade and cause severe systemic disease known as Glasser's disease,characterized by fibrinous polyserositis,arthritis and meningitis.With the recent changes in production methods,H.parasuis has become as an important pathogen in all major swine-raising countries,giving rise to important economic losses throughout the world.As the protective antigens and virulence factors are not clear,even now the traditional inactivated vaccine is still used to prevent the disease caused by H.parasuis.In order to study the serotypes of H.parasuis in the West of Fujian Provence and the immunogenicity of the candidate vaccine strain,this reseach focused on the isolation and identification of H.parasuis in the West of Fujian Provence,selection of the vaccine strain and analysis of the immune efficacy.1 Isolation and serovar distribution of Haemophilus parasuis in the West of Fujian ProvenceFrom June 2014 to May 2015,H.parasuis were isolated and identified from clinical pigs,based on morphologic characteristics,biochemical tests,PCR amplification.According to the serotyping,among 41 isolated strains,15 strains(36.6%)were serovar 5,6 strains(14.6%)were serovar 4,followed by sv.1(9.8%),sv.2(4.9%),sv.12(2.4%),sv.13(2.4%),while 7(2.4%)strains were considered as untypeable,5 strains(12.2%)were cross reacted with other serovars.The results suggested that serovars 5(36.6%),4(14.6%)were the most frequently isolated strains in the West of Fujian Provence.2 Selection of vaccine strain of Haemophilus parasuis serotype 57 isolated strains of H.parasuis serotype 5 were used to infect mice intraperitoncally with a median lethal dose(2×109 CFU),among the 7 strains,the mortality of mice was different,the LY02 strain caused a highest death rate(80%),it indicated the virulence of LY02 strain is higher than other strains.Then 5 18-day-old piglets were inoculated intraperitoncally with a lethal dose(7.5 ×109 CFU)per piglet of the LY02 strain and reproduced the typical signs and lesions of Glasser's disease.Piglets challenged showed significantly elevated temperatures,anorexia,dyspnoea,swollen joints,lameness and severe gross and pathological lesions,characterized by arthritis,fibrinosuppurative polyserositis in the pericardial,pleural and peritoneal cavities.The histopathological changes include interstitial pneumonia,focal necrosis of liver,cardiac dilatation,and red pulp dilatation,white pulp atrophy,lymphocyte depletion of spleen.The results indicated the LY02 strain would be a candidate vaccine strain.3 Immunogenicity analysis of Haemophilus parasuis LY02 strainLY02 strain was used for the preparation of H.parasuis bacterin,the suspension containing 5×109 colony-forming units per mL was inactivated 0.4%formalin before the addition of oil emulsion adjuvant.The piglets of vaccinated challenged group and vaccinated control group were vaccinated twice intramuscularly with the water-in-oil H.parasuis inactivated vaccine,three weeks after second vaccination,piglets of vaccinated challenged group and challenged control group were challenged intraperitoncally with a lethal dose(7.5 ×109 CFU)of the LY02 strain,suspended in 3 mL PBS.The immunogenicity was evaluated by clinical and pathological examinations,serologic study and flow cytometry.The results show that:the piglets of challenged control group were all died 3 days post-challenge and showed typical lesions of Glasser's disease,in contrast,the vaccinated challenged group show no appreciable clinical signs or lesions,except for a slight transient rise in rectal temperature;the serum IgG antibody level of vaccinated challenged group showed significant difference compared with challenged control group(P<0.01),black control group(P<0.01)and vaccinated contral group(P<0.05);the concentrations of IL-2,IL-4 in the sera and the CD4 + CD8-numbers in the peripheral blood of piglets of challenged control group decreased significantly,the numbers of CD4-CD8 + in the peripheral blood increased significantly;the concentrations of IL-2 in the sera and the numbers of CD4 + CD8-and CD4-CD8 + in the peripheral blood of piglets in vaccinated challenged group showed no significant difference compared with vaccinated control group and black control group,except for the concentrations of IL-4 in the sera showed significantly higher than that in vaccinated control group(P<0.05).The results indicated that the inactivated vaccine can protect piglets against challenge with a homologous serovar strain.