| In this research, the identification of pathogens from samples originating from 16 farms that were suspected to be affected with Streptococcus suis (S. suis ) revealed: 14 infections caused by S. suis, one caused by Toxoplasma.gondii, and for one case the pathogen was not identified. Of the S. suis samples that were isolated, three were S. suis serotype 2, five were S. suis serotype 7 and six were not identified. Serological detection of S. suis serotype 2 from the sera collected from different regions from Gansu, Qinghai, and Ningxia provinces revealed the positive rates for S. suis serotype 2 ranged from 0 to 16.7%. In the 2007-2011 period, S. suis strains were isolated from tonsils of pigs from Wuwei: 272 S. suis isolates were collected and the positive sample rate was 27.2%, of which, 5.5% were S. suis serotype 2, 13.2 % were serotype 9, 11.4% were serotype 9, and 3.7% were serotype 1. The drug resistance ability test revealed that 84% of the isolated S. suis strains had multidrug resistance.To obtain the best possible phylogenetic analysis among genus Streptococcus, the Multilocus sequence typing (MLST) method was used and the concatenated sequence of partial sequences from dnaK-EFTu-potA-eno-uvrA-guaB genes was found to be most suitable for analysis. The bootstrap values in every node for the 14 Streptococcus species described in this research were≥86. From the phylogenetic analysis, it was found that the species used in this research could be divided into three major clusters. Cluster one includes the mitis group-related S. suis and S. thermophilus which belonged to the Salivarious group. The second cluster includes the pyogenic group and the third cluster includes S. mutans and S. gallolyticus which belong to the equines group. Based on this research, a partial 638 bp sequence from the gene coding for ABC-type spermidine/putrescine transport system was found to be usable for genotyping among genus Streptococcus at the species level. To make a safe and highly efficient vaccine, we produced, using the pSET4s vector, twenty four D-alanine authophic S. suis serotype 2 strain 06TS08 by gene knockout of the alanine racemase gene that is responsible for the supply of D-alanine. One D-alanine authophic S. suis serotype 2 strain that was named 11△alr 2/24 was selected for S. suis serotype 2 live vaccine research. To assess the immune dosage of the authophic S. suis serotype 2 strain as a vaccine, 30 piglets that were S. suis serotype 2 negative were distributed into 6 groups of 5 piglets, each of which were administrated by intramuscular injection with 5×10~8, 1×10~9,2×10~9,3×10~9,4×10~9,5×10~9 CFU of 11△alr 2/24. Simultaneously, five piglets were used as control which were injected with PBS. Four weeks later, all the piglets were challenged with 1.7×10~7 CFU S. suis serotype 2 by auricular vein injection. The non-immunized piglets all died within one week. For immunized pigs: in the 5×10~8 group, three survived;in the 1×10~9 group three survived; 2×109 group four survived; in the 3×10~9 group four survived; in the 4×10~9 group five survived and in the 5×10~9 group five survived. Based on these results, 4×10~9 CFU was considered to be a suitable dosage. Two batches of vaccines were prepared based on this dosage and the safety test of the vaccine revealed that S. suis serotype 2 11△alr 2/24 was not found in liver, heart, spleen, lung and tonsils of immunized piglets 28 days post injection. At the same time, for every vaccine batch, 10 piglets were distributed into two groups: 5 piglets were administrated by intramuscular injection with 1ml of the prepared vaccine and 5 piglets were used as the control group which injected PBS. Four weeks later, all the piglets were challenged with 2.1×10~7 CFU Serotype 2 by auricular vein injection. The non-immunized piglets were all dead within one week while all the immunized piglets survived. These results revealed that the mutant S. suis serotype 2 strain 11△alr 2/24was safe and effective to make vaccines. |
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