广东省1株猪2型链球菌的分离鉴定及部分生物学特性研究

为确诊引起广东省某规模化猪场保育仔猪发生死亡的病因，本试验采集病猪肝脏、脾脏、心脏、脑、关节液和淋巴结等病料进行细菌分离，并对分离菌株进行纯化培养、菌落形态观察、革兰氏染色、形态学观察、生化试验，用血清型特异性引物进行PCR扩增，并进行毒力基因检测、药敏试验、生长曲线测定及动物试验。结果显示，分离菌株在成牛血清琼脂平板上呈圆形、灰白色、表面光滑、边缘整齐的菌落，镜检为革兰氏阳性球菌，呈链状排列。该菌株的生化试验结果符合链球菌的特征，PCR扩增结果显示猪链球菌保守基因片段（gdh和gapdh）和猪2型链球菌特异的cps2J基因片段均为阳性，表明该分离菌株为猪2型链球菌。毒力基因检测结果表明，该菌株毒力因子基因型为gdh⁺/gapdh⁺/epf⁺/mrp⁺/sly⁺/orf2⁺/fbps⁺。分离菌株用营养肉汤培养6 h达到对数生长期，培养10 h的菌液D_{600 nm}值为0.310。药敏试验结果表明，该菌株对青霉素、阿莫西林、阿莫西林-克拉维酸钾等药物敏感，但对强力霉素耐药。动物试验结果表明，感染分离株的BALB/c小鼠未出现死亡且无明显临床症状，毒力较低。本研究为该猪场链球菌病的防控提供了重要参考信息。

Isolation,Identification and Biological Characteristics of Streptococcus suis Serotype 2 Strain from Guangdong Province

In order to determine the cause of death of piglets in a large-scale pig farm in Guangdong province,the disease materials such as liver,spleen,heart,brain,joint fluid and lymph nodes were collected for bacterial isolation,and the isolated strains were purified and cultured.And colony morphology observation,Gram staining,morphology observation,biochemical test,PCR amplification with serotype specific primers,virulence gene test,drug sensitivity test,growth curve test and animal test were carried out.The results showed that there were round,gray white,smooth surface and neat edge colonies on the agar plate of adult bovine serum.Microscopic examination showed Gram-positive cocci which were arranged in chains.The biochemical test results of this strain were in line with the characteristics of Streptococcus suis,and the PCR amplification results showed that both the conserved gene fragment(gdh and gapdh)of Streptococcus suis and the specific cps 2 J gene fragment of Streptococcus suis serotype 2 were positive,indicating that the isolated strain was Streptococcus suis serotype 2.The results of virulence gene detection showed that the virulence factor genotype of the strain was gdh⁺/gapdh⁺/epf⁺/mrp⁺/sly⁺/orf 2⁺/fbps⁺.The isolated strain reached the logarithmic growth stage after cultured in nutrient broth for 6 h,and the D 600 nm value of the bacterial solution cultured to 10 h was 0.310.The results of drug sensitivity test showed that the strain was sensitive to penicillin,amoxicillin,amoxicillin-clavulanate potassium,but resistant to doxycycline.The animal test results showed that BALB/c mice infected with the isolated strain did not die and had no obvious clinical symptoms,and its virulence was low.This study provided important reference information for the prevention and control of Streptococcus in this pig farm.