猪源A型和D型多杀性巴氏杆菌强毒菌株的生物学特性比较研究

A comparative study on biological characterization of Pasteurella multocida serogroups A and D isolates from swine in China

为分析猪源A型和D型多杀性巴氏杆菌(PM)的生物学特性差异,本研究对5株A型和5株D型猪源PM强毒菌株的生化特征、生长特性、毒力特性、16SrRNA基因分子特征等生物学特性进行了比较研究。在TSB液体培养基中的生长曲线测定结果表明这些菌株均能够高密度发酵培养;21种生化试验的反应结果表明,10株PM的生化反应均不一致,即使同一血清型的不同菌株之间也具有较大差别。对小鼠毒力试验结果表明,A型PM菌株的毒力(LD_(50)为4 cfu^26 cfu)显著强于D型菌株(LD50为2.1×10~4 cfu^5.2×10~5 cfu)。16SrRNA基因的序列分析结果表明,16SrRNA基因非常保守,这10株PM之间的同源性均在99.8%以上;在1 505 bp序列中,仅存在第57 bp(T/C)、239 bp(T/C)和376 bp(G/C)处3个多态性位点,并且不具有型特异性。本研究为猪源PM灭活疫苗菌株的筛选等提供了实验依据。

Serogroups A and D isolates of the Pasteurella multocida are the dominant causes of swine pasteurellosis in China. The biological characteristics of l0 field isolates of P.multocida, such as the growth rate, carbohydrate utilization, and virulence in mice were tested and the 16S rRNA genes of 5 serogroup A and 5 serogroup D of P.multocida isolates were also determined in this study. Replications of the isolates in tryptic soy broth containing 5% bovine serum showed that the highest colony-forming units （cfu） for serogroups A and D of P.multocida were 2.4x109 to 4.3x109 cfu/mL and 2.8x109 to 5.5x109 cfu/mL, respectively. There were different fermentation patterns between the serogroups A and D isolates for 21 different carbohydrates, and the difference existed even among the isolates in the same serogroup P.multocida. The serogroup A isolates showed more virulent in the BALB/c mice with the LDs0 ranging from 4 to 26 cfu, compared to the serogroup A isolates with the LD~ ranging from 2.1 × 10^4 to 5.2×10^5 cfu, and no significant difference was found among the isolates within the same serogroup. The 16S rRNA gene sequencing and phylogenetic analysis demonstrated that the 16S rRNA genes of the 10 field isolates were relatively conservative.However, three single nucleotide polymorphisms （SNPs） were found in the 16S rRNA genes （1,505 bp） of these 10 P.multocida isolates, which located at 57 bp （T/C）, 239 bp （T/C）, and 376 bp （G/C）, respectively. In conclusion, this study provides a basis for the further study on the pathogenesis and vaccine development of P.multocida.