4株广东地区2型猪链球菌耐药性及致病性研究

doi:10.16431/j.cnki.1671-7236.2023.09.030

摘要/Abstract

摘要： 【目的】了解广东部分地区猪场疑似猪链球菌病的病原血清型、毒力基因特点、耐药性以及致病性。【方法】对4个疑似流行猪链球菌的猪场的送检病料进行细菌分离培养,采用形态学观察、革兰氏染色镜检、16S rRNA测序以及生化试验鉴定分离菌株,通过PCR试验对分离株进行血清型鉴定、毒力基因检测,采用药敏纸片扩散法对分离菌株进行药敏试验,通过活菌计数法绘制生长曲线,通过人工感染昆明小鼠评价各分离菌对小鼠的致病性。【结果】分离株在10%成牛血清的TSA培养基上长出圆形、灰白色、半透明、表面光滑、边缘整齐的小菌落。镜检出革兰氏阳性球菌,短链状排列,生化特性与猪链球菌相符,测序分析后确定为猪链球菌。血清型鉴定结果显示,4株分离菌均为2型猪链球菌,其中有3株的表型为mrp⁺/epf⁺/orf2⁺/gapdh⁺/sly⁺/fbps⁺/gdh⁺,有1株表型为mrp^-/epf^-/orf2^-/gapdh⁺/sly⁺/fbps⁺/gdh⁺,共2种毒力基因表型。药敏结果显示,4株分离菌对红霉素、克林霉素、四环素、复方新诺明耐药,对青霉素、氨苄西林敏感。分离菌在4~6 h期间达到生长对数期,培养6 h到达活菌峰值。试验组小鼠在感染猪链球菌后出现精神沉郁、抱团、呼吸急促、尾部发青等症状,随后出现死亡,剖检有不同程度的病理变化。4株菌株半数致死量分别为4.47×10⁷、3.57×10⁷、5.77×10⁷、1.22×10⁸ CFU。【结论】本研究成功分离鉴定出4株2型猪链球菌,4株分离株具有多重耐药性且具有较强的致病力。本研究结果可为广东地区猪链球菌的防控以及疫苗候选株的筛选提供理论参考。

关键词: 2型猪链球菌; 分离鉴定; 毒力基因表型; 药敏试验; 致病性

Abstract: 【Objective】 The purpose of this study was to understand the pathogenic serotype,virulence gene characteristics,drug resistance and pathogenicity of suspected Streptococcus suis in pig farms in some areas of Guangdong.【Method】 Bacterial isolation and culture were carried out of samples with suspected Streptococcus suis epidemics in four pig farms,and the isolates were identified by morphological observation,Gram staining microscopy,16S rRNA sequencing,and biochemical testing.Serotype identification and virulence gene detection of the isolated strains were performed by PCR assay.Drug sensitivity tests were conducted on the isolated strains using drug sensitive paper diffusion method,and growth curves were drawn using live bacterial count method.The pathogenicity of each isolate was evaluated by artificial infection of Kunming mice.【Result】 The isolated strain grew small colonies on TSA medium containing 10% adult bovine serum,which were round,gray white,semi transparent,with smooth surfaces and neat edges.Gram positive cocci were detected by microscopy,arranged in a short chain,with biochemical characteristics consistent with Streptococcus suis.After sequencing analysis,it was determined to be Streptococcus suis.The serotype identification results showed that all four isolated strains were Streptococcus suis type 2,with three strains having phenotypes of mrp⁺/epf⁺/orf2⁺/gapdh⁺/sly⁺/fbps⁺/gdh⁺ and one strain having phenotypes of mrp^-/epf^-/orf2^-/gapdh⁺/sly⁺/fbps⁺/gdh⁺,totaling two virulence gene phenotypes.The drug sensitivity results showed that the four isolates were resistant to erythromycin,clindamycin,tetracycline and cotrimoxazole,and sensitive to penicillin and ampicillin.The isolates reached the logarithmic growth phase during 4-6 h,and reached the peak viable bacteria at 6 h of incubation.The mice in the test group showed symptoms such as depression,huddling,shortness of breath and blue tail after infection with Streptococcus suis,followed by death and pathological changes of different degrees on autopsy.The LD₅₀of the four strains were 4.47×10⁷,3.57×10⁷,5.77×10⁷and 1.22×10⁸CFU,respectively.【Conclusion】 This study successfully isolated and identified 4 strains of Streptococcus suis type 2,which had multiple drug resistance and strong pathogenicity.The results of this study could provide theoretical reference for the prevention and control of Streptococcus suis in Guangdong and the screening of vaccine candidate strains.

Key words: Streptococcus suis type 2; isolation and identification; virulence gene phenotype; drug sensitivity test; pathogenicity

中图分类号:

S852.61⁺1

字嘉鑫, 连英杰, 陈敏仪, 李秋煦, 张伟泽, 陆梦可, 张阳洋, 区浩炫, 马骏, 白挨泉. 4株广东地区2型猪链球菌耐药性及致病性研究[J]. 中国畜牧兽医, 2023, 50(9): 3740-3751.

ZI Jiaxin, LIAN Yingjie, CHEN Minyi, LI Qiuxu, ZHANG Weize, LU Mengke, ZHANG Yangyang, QU Haoxuan, MA Jun, BAI Aiquan. Drug Resistance and Pathogenicity of 4 Strains of Streptococcus suis Type 2 in Guangdong[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3740-3751.

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