Isolation And Identification Of Haemophilus Parasuis And Preliminary Development Of Trivalent Inactivated Bacterin

Haemophilus parasuis belongs to the genus Haemophihis in the family Pasteurella. which is constantly existing in the respiratory tract of pigs, mainly inducing polyserositis and arthritis. Recently, it has caused immense economic losses in the domestic swine industry with increasing prevalence. In present study, Haemophilus parasuis from either the sick or the healthy pigs in Jiangsu Province and Anhui Province were isolated and identified, which could provide evidence for the prevention of Haemophilus parasuis. Meanwhile, trivalent inactivated bacterin of Haemophilus parasuis containing serotype 4,5 and 12 was preliminarily explored and protective immunity was evaluated in BALB/c mice.1 Isolation and identification of Haemophilus parasuisThe samples, collected at the Yangzhou University animal hospital and Yangzhou slaughterhouse, plated onto chocolate medium for culture, and 16 S rRNA of Haemophilus parasuis was detected by the PCR technology. The strain with 16 S rRNA PCR positive were isolated and identified.Total 8 strains of Haemophilus parasuis were isolated from sick pigs. Total 26 strains of Haemophilus parasuis were isolated from healthy pigs. The isolation rate was 7.12%. Three strains of Haemophilus parasuis were gene sequenced, of which the homology was over 99% compared with the isolates of home and abroad deposited in GenBank.All the isolates of Haemophilus parasuiswere serotyped through the glass plate agglutination. The isolates included 3 strains of serotype 5.2 strains of serotype 12,1 strain of serotype 14 and 2 unserotypeable strains in sick pigs. The isolates included 4 strains of serotype 4 (15.38%),1 strains of serotype 5 (3.85%),3 strains of serotype 11 (11.54%),10 strains of serotype 12 (38.46%),1 strains of serotype 13 (3.85%) and 7 unserotypeable strains (26.92%) in healthy pigs.2 Preliminary development of trivalent inactivated bacterinGuinea pigs were challenged intraperitoneally with Haemophilus parasuis (serotype 4, or 5 or 12). The results showed that the guinea pigs all died containing serotype 5 and 12 infection and one died of serotype 4 (2 guinea pigs infected). Peritoneal and pleural effusion and slight fibrin exudation were observed in the died guinea pigs, whose symptoms were similar to the infection pigs of Haemophilus parasuis.BALB/c mice were challenged intraperitoneally with three different bacterial doses of Haemophilus parasuis (serotype 4, or 5 or 12). When the infection dosage reached 5.0×109 CFU, the challenged BALB/c mice were dead. When the infection dosage was 1.0×109 CFU, the BALB/c mice challenged with serotype 4 or 5 isolates exhibited one death, respectively, and those challenged with serotype 12 isolate experienced no deaths.BALB/c mice were immunized subcutaneous used the trivalent inactivated vaccine 1 (containing 3 serotype strains at 1.0×109 CFU), trivalent inactivated vaccine 2 (containing 3 serotype strains at 2.0×109 CFU), trivalent inactivated vaccine 3 (containing 3 serotype strains at 4.0×109 CFU) and commercial inactivated vaccine on day 0. Subsequent booster injections were administered on day 21. Two weeks after the second immunization, all mice were challenged intraperitoneally with Haemophilus parasuis (serotype 4, or 5 or 12, respectively). When considering the serotype 4 and 5, the protection rates of trivalent inactivated vaccine immunized group 1,2,3 and commercial vaccine immunized group were 40%,40%,60%,40%, respectively. When considering the serotype 12, the protection rate of trivalent inactivated vaccine immunized group 1,2,3 and commercial vaccine immunized group were 60%,60%,80%,20%, respectively. In summary, the immune protective effect of trivalent inactivated bacterin of Haemophilus parasuis preliminary developed in this study was better than the commercial in activated vaccine.