Development Of Serotype-specific PCR Assays Of Haemophilus Parauis And Streptococcus Suis And Study On The Inactivated Vaccine

Glasser's disease and Streptococcus suis disease are the main bacterial diseases threatening the global swine industry.They are often caused by co-infection or mix-infection of Haemophilus parasuis and S.suis and lead to huge economic losses to the swine industry.At present,the prevention and control of these diseases mainly depend on application of their inactivated or attenuated vaccines.Although these vaccines have good immunity,their cross protections are non-obvious.Therefore,it is imminent to develop a novel and inexpensive new type of bivalent vaccine of H.parasuis and S.suis with high cross-protective immunity.In order to timely understand the latest epidemic dynamics of H.parasuis and S.suis,and to screen candidate vaccine strains,extensive epidemiologica l investigation is needed.Although traditional serotyping methods are commonly used for epidemiological investigation and clinical diagnosis of H.parasuis and S.suis,they are time-consuming and unable to define the serotypes of 10%-40% of strains,bringing great difficulties to clinical diagnosis and epidemiological analysis of the two diseases.To address the deficiencies of existing methods,in this study,the genetic differences in the cluster sites of capsular polysaccharide synthase genes of H.parasuis and S.suis strains with different serotypes were selected as targets to design serotype specific primers for establishing PCR methods for serotyping H.parasuis and S.suis.The newly developed PCR method and traditional agar diffusion method are used to analyze the serotype of 298 H.parasuis strains isolated in 2007-2015 years and survey serotyping rate of S.suis in many farms of Guangdong province,China.The results showed that the serotyping rate of the PCR method was 94.3%(281/298),significantly higher than that of 76.51%(228/298)of the traditional agar diffusion method.In addition,serotyping using the PCR method showed that the serotypes 4,5 and 12 were the most prevalent,accounting for 25.17%,23.15% and 20.47%,respectively,followed by types13(6.04%),2(5.03%)and 7(3.69%).Survey of S.suis using the PCR method showed that pig population at all different stages could carry S.suis with positive rate of 17.11%-100% and S.suis positive rate was 48.35% in farms,44.44% in slaughter house and 85.53% in the diseased pigs.In addition,the healthy pigs were mainly infected with serotypes 29(8.81%),2(8.55%),11(6.22%)and 16(5.70%),while the diseased pigs were mainly infected with serotypes 2(16.41%),3(9.23%),7(7.18%)and 9(6.15%).According to the statistics of 118 farms,the H.parasuis infected farm was 68,accounting for 57.63%,and the S.suis infected farms was 99,accounting for 83.9%,of which the mixed infection of H.parasuis and S.suis was 51 farms,accounting for 43.22%.The above-mentioned epidemiological survey revealed the prevalence and distribution of H.parasuis and S.suis in China in recent years.Moreover,animal experiments screened out two candidate vaccine strains H26 and S3.The former had cross protection against the current epidemic strains of serotypes 4 and 5 while the latter had good cross protection against strains of serotypes 2,7 and 9.Therefore,3 batches of bivalent inactivated H.parasuis and S.suis vaccine were prepared using strains H26 and S3.Animal experiments using 10-days-old piglets by single-dose inoculation,repeated single-dose inoculation and over-dose inoculation of the three batches of vaccines all showed no inflammatory reaction at the injection site,appetite loss,depression and elevated body temperature,indicating that the vaccine had good safety for piglets.Vaccine immunological efficacy test showed that the protection rate of the 3 batches of vaccines was> 4/5 and the titer of the H.parasuis antibody was > 1:16,indicating that the bivalent inactivated vaccine could protect piglets from H.parasuis and S.suis attacks.Further testing showed that the immunization duration and the preservation period of the vaccine was up to 4 months and 18 months,respectively,and the minimum immunization dose was determined to be 2mL.Compared with the commercial similar vaccines,the vaccine had higher immune efficacy.Application of the vaccine effectively solved the problems of commercial vaccines against H.parasuis and S.suis such as requiring many times of single vaccine inoculation,being time consuming and laborious,and having poor immunization effect.Therefore,the research results provide more technical and theoretical basis for the effective prevention and control of S.suis disease and H.parasuis disease.