Selection Of Attenuated Haemophilus Parasuis Strains Using Guinea Pigs Model

Haemophilus parasuisis(HPS), the causative agent of Gl?sser’s disease, belongs to Pasteurellaceae family. So far, at least 15 kinds of serovars were idenified. After broke through the body immune defense, HPS would resulted in severe systemic bacterial infection characterized by fibrinous, polyserositis, polyarthritis, meningitis. In recent years, with the rapid development of the large scale pig farms, the disease was becoming prevalent in the world. Conventional control measures mainly relied on antibiotics and inactivated vaccines. However, the commercial inactivated vaccines could only produce short-term and low level of immune cross protection along with high cost and severe side reaction. Thus, the inactivated vaccines were not effective enough to confront the challenges from multiple serovars. On the contrary, the attenuated vaccines could induce long-term immune protection and produce relatively high level of cross immune protection. So, the screening of HPS attenuated candidate strains for vaccine may be one of the best ways to the development of new vaccines.In our laboratory, HPS strain was continuously subcultured to 250 th generations respectively in 37℃ and 40℃as the vaccine candidate strains. The paper attempted to establish guinea pig infection model for HPS and apply the animal model to obtain the attenuated strains. The main research results are as follows:1. Research of guinea pig model for HPS infection: in the experiments, male guinea pigs weighing 250 to 300 g were used. Nasal cavity infection and intraperitoneal infection were both carried out. In the nasal cavity infection, three and three guinea pigs were inoculated in the nasal cavity with 0.1ml of saline suspension containing6.69×109CFU, 2.23×109CFU and 5.58×108CFU organisms respectively. Three guinea pigs were injected with 0.1ml saline alone. In the intraperitoneal infection, guinea pigs were randomly divided into four groups. Three groups were infected intraperitoneally with 1.0ml of saline suspension containing 4.83×109CFU, 1.93×109CFU and4.83×108CFU organisms respectively. The control group was inoculated with 1.0ml saline alone.The experimental results indicated that the infected guinea pigs showed depression,dullness and anorexia within 4 hours after inoculation in the nasal cavity infection experiment. No clinical signs were observed in control guinea pigs. However, 3 days later,all guinea pigs recuperated and none of guinea pigs in the 4 groups died in the end. 7days later, the pathological lesions of infected guinea pigs were unapparent except thegroup inoculated with 6.69×109CFU organisms. In this group, the guinea pigs showed systemic hemorrhagic symptom. In the intraperitoneal infection experiment, infected guinea pigs appeared serious depression, dyspnea, dullness, anorexia, lie prone within 2hours after infection and subsequently died. In the end, five of six guinea pigs inoculated with 4.83×109CFU organisms died, two died in the 1.93×109CFU infection group and none of guinea pig died in the 4.83×108CFU infection group. The died guinea pigs showed obvious septicemia, fibrinous serositis and ascites. The anatomical lesions differences of the survival guinea pigs in each group were also distinct. Besides,Haemophilus parasuis was recovered successfully from the major organs in these died guinea pigs. In the follow-up tests, the results of guinea pig blood routine, biochemical examination detection and histological section provided further confirms that HPS caused real damage to guinea pigs. So, the results of study proved that guinea pig could act as an laboratory animal for Haemophilus parasuis. At the same time, we found that the intraperitoneal infection route was better than nasal cavity infection route both in operation and the experimental results. Based on the guinea pigs infection model,established the evaluation index and methods to assess the virulence of batches strains.2. Preliminary screening of HPS attenuated strains: Haemophilus parasuis SH0165 was respectively continuously subcultured to 200 generations under 37℃and40 ℃ in BHI medium. Intraperitoneally inoculated guinea pigs with the 100 th, 150 th,200th generations respectively subcultured in 37℃and 40 ℃ with 2×LD50 organisms.Then estimated the results of guinea pigs infected by different passages of SH0165 depended on the evaluation standards. The evaluation scores showed that the virulence of strains in 37℃ decreased as a whole. However, the virulence of strains in 40℃ decreased inconspicuously. In the end, the 200 th generation cultivated in 37℃ could act as the candidate strain for attenuated vaccine.