1株血清2型副猪嗜血杆菌的分离鉴定及致病性研究

doi:10.16431/j.cnki.1671-7236.2025.02.039

摘要/Abstract

摘要： 【目的】明确浙江地区某猪场副猪嗜血杆菌(Haemophilus parasuis,Hps)感染情况，明确分离株的血清型、生物学特性及致病性，为临床控制副猪嗜血杆菌提供理论参考。【方法】从发病猪的肺脏、心脏血液、关节液等病料中分离、纯化菌株，并对其进行革兰染色镜检、16S rRNA基因PCR扩增及测序，明确分离株的血清型，检测分离株携带毒力基因情况、对常见抗菌药物的敏感性及对小鼠的致病性，使用饱和硫酸铵法提取菌株分泌蛋白，并通过电泳分析蛋白主要富集区域。【结果】分离株在TSA培养基上生长出圆形、光滑湿润、颜色灰白、边缘整齐的针尖大小的菌落；接种至TSB培养基后，1～3 h生长缓慢，3～4 h进入对数生长期，4～12 h进入生长稳定期；革兰染色镜检呈红色短杆状、长杆状，长度不等，疑似副猪嗜血杆菌。16S rRNA基因PCR扩增结果显示，出现680 bp的特异性条带，与预期相符。血清型鉴定结果显示，检测到2型血清型副猪嗜血杆菌特异性条带。BLAST比对显示该分离株为血清2型副猪嗜血杆菌。毒力基因检测结果显示，分离株携带cdtA、cdtB、cdtC、vtaA1、rfaD、rfaE、gnd、cpaD、espP2、vacJ、rfaF、vtaA2、htrA和ompP2基因。该菌株以1.0×10⁹ CFU/mL剂量感染BALB/c小鼠，在5～10 h出现死亡，并引起小鼠肝脏细胞核浓缩、坏死,肺泡腔内有大量浆液性渗出物，混有红细胞和菌体，脾脏组织炎性细胞浸润。药敏试验结果显示，分离株对头孢哌酮、头孢呋辛钠、哌拉西林、青霉素等药物耐药，对氯霉素表现中介，对环丙沙星、左氧氟沙星、阿奇霉素等药物敏感。该菌株分泌蛋白大小主要集中在25～180 ku。【结论】本研究分离鉴定获得1株血清2型副猪嗜血杆菌，该分离株编码多种毒力基因，具有致病性，对多种常用抗菌药物有明显的耐药性，并分泌多种蛋白。试验结果提示该猪场可使用喹诺酮类、大环内酯类及氨基糖苷类药物进行防控。

关键词: 副猪嗜血杆菌; 血清型; 分离鉴定; 致病性

Abstract: 【Objective】 The purpose of this experiment was to clarify the infection of Haemophilus parasuis (Hps) in a pig farm in Zhejiang province,and clarify the serotype,biological characteristics and pathogenicity of the isolate,so as to provide theoretical reference for clinical control of Hps. 【Method】 The strain was isolated and purified from lung,heart blood,joint fluid and other disease materials of infected pigs.Gram staining microscopy,16S rRNA gene PCR amplification and sequencing were performed to determine the serotype of the isolate.The virulence genes,susceptibility to common antibiotics and pathogenicity of the isolate were detected.The secreted protein was extracted by saturated ammonium sulfate method,and the main enrichment regions of the protein were analyzed by electrophoresis. 【Result】 The isolate grew round,smooth and moist,pale in color,and pinpoint-sized colonies with neat edges on TSA medium.After inoculation into TSB medium,the isolate grew slowly at 1-3 h,entered the logarithmic phase at 3-4 h,and entered the stable phase at 4-12 h.Gram staining microscopy showed red short rod shape,long rod shape,varying in length,suspected Hps.PCR amplification of 16S rRNA gene showed a specific band of 680 bp,which was consistent with expectations.The results of serotype identification showed that specific bands of Hps serotype 2 were detected.BLAST comparison showed that the isolate was Hps serotype 2.Virulence gene test results showed that the isolate carried cdtA,cdtB,cdtC,vtaA1, rfaD,rfaE, gnd,cpaD,espP2,vacJ,rfaF,vtaA2,htrA and ompP2 genes.When the isolate infected BALB/c mice at a dose of 1.0×10⁹ CFU/mL,the mice died within 5-10 h，and caused nucleus concentration and necrosis in the liver,a large amount of serous exudate in the alveolar cavity,mixed with red blood cells and bacteria,and inflammatory cell infiltration in the spleen.The results of drug sensitivity test showed that the isolate was resistant to cefoperazone,cefuroxime sodium,piperacillin and penicillin,intermediate to chloramphenicol,and sensitive to ciprofloxacin,levofloxacin and azithromycin.The size of secreted protein mainly concentrated in 25-180 ku. 【Conclusion】 In this study,a strain of Hps serotype 2 was isolated and identified.The isolate encoded a variety of virulence genes,was pathogenic,had obvious resistance to a variety of commonly used antibacterial drugs,and secreted a variety of proteins.The results indicated that quinolones,macrolides and aminoglycosides could be used for prevention and control in this farm.

Key words: Haemophilus parasuis; serotype; isolation and identification; pathogenicity

中图分类号:

S855.1

李倩文, 华涛, 常晨, 颜星宇, 李军星, 唐波. 1株血清2型副猪嗜血杆菌的分离鉴定及致病性研究[J]. 中国畜牧兽医, 2025, 52(2): 901-911.

LI Qianwen, HUA Tao, CHANG Chen, YAN Xingyu, LI Junxing, TANG Bo. Isolation,Identification and Pathogenicity Analysis of a Strain of Haemophilus parasuis Serotype 2[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(2): 901-911.

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