《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (6): 1856-1864.doi: 10.16431/j.cnki.1671-7236.2019.06.035

• 临床兽医 • 上一篇    下一篇

猪繁殖与呼吸综合征病毒与副猪嗜血杆菌混合感染的诊断

张海龙, 陈盛楠, 谢博, 李丽, 黄良宗   

  1. 佛山科学技术学院生命科学与工程学院, 佛山 528000
  • 收稿日期:2018-10-08 出版日期:2019-06-20 发布日期:2019-06-19
  • 通讯作者: 黄良宗 E-mail:13925923069@163.com
  • 作者简介:张海龙(1995-),男,广东韶关人,硕士生,研究方向:猪传染病诊断与防控,E-mail:1163615487@qq.com
  • 基金资助:

    广东省教育厅预防兽医学重点实验室项目(2014KTSPT037);广东省自然科学基金项目(2017A030310612)

Diagnosis of Mixed Infection of Porcine Reproductive and Respiratory Syndrome Virus and Haemophilus parasuis

ZHANG Hailong, CHEN Shengnan, XIE Bo, LI Li, HUANG Liangzong   

  1. College of Life Sciences and Engineering, Foshan University, Foshan 528000, China
  • Received:2018-10-08 Online:2019-06-20 Published:2019-06-19

摘要:

为确诊广东省阳江市某规模化猪场(存栏800头母猪)保育猪发病死亡的原因,本试验对从该发病猪场采集的3份肺脏、肝脏、脾脏临床样品进行细菌学检测及药敏试验,采用PCR/RT-PCR检测临床样品中猪伪狂犬病病毒(PRV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)、猪圆环病毒3型(PCV3)和猪肺炎支原体等病原。对特异性扩增的3株PRRSV的ORF5基因产物进行序列测定,与VR2332、HuN4、JXA1、CH-1a等代表毒株进行核苷酸序列同源性分析,并构建系统进化树。结果表明,试验分离鉴定出1株副猪嗜血杆菌(Hps),对7种临床常用药如阿莫西林、头孢拉定等均有较强的敏感性。同源性比对结果表明,3株PRRSV (LJW1、LJW2和LJW3)ORF5基因核苷酸同源性为99.3%~99.8%,与欧洲型代表毒株Lelystad核苷酸同源性为64.0%~64.2%,与HP-PRRSV毒株JXA1、HuN4、CH-1a和TJ核苷酸同源性较高,分别为99.2%~99.5%、99.0%~99.3%、94.5%~94.9%和98.8%~99.2%;与中国河南和广西分离的HP-PRRSV毒株HeNzm1-16和GXLZ05-2015核苷酸同源性较高,分别为99.3%~99.7%和99.2%~99.7%,与美洲型经典疫苗株MLV、美洲型标准株NC、美洲型经典株VR2332核苷酸同源性较低,分别为88.5%~88.8%、85.2%~85.5%和82.3%~82.6%。PRRSV ORF5基因系统进化树分析表明,3株PRRSV均属于美洲型毒株,与国内HP-PRRSV代表毒株JXA1、HuN4和TJ等处于同一分支,亲缘关系较近。本研究揭示了该场保育猪发病病原,并从分子水平上明确了分离的3株PRRSV与不同代表毒株的亲缘关系,为弱毒疫苗的合理选择使用和综合防控PRRSV提供了参考依据。

关键词: 猪繁殖与呼吸综合征病毒(PRRSV); 副猪嗜血杆菌(Hps); ORF5基因; 遗传进化

Abstract:

To ascertain the cause of the death of nursery pigs in Yangjiang city,Guangdong province,we carried out the bacteriological and drug sensitive test of 3 clinical samples (lung,liver and spleen) collected from pigs in this study.PCR/RT-PCR method were used to test pseudorabies virus (PRV),classical swine fever virus (CSFV),porcine reproductive and respiratory syndrome virus (PRRSV),circovirus type 2 (PCV2),circovirus type 3 (PCV3) and Mycoplasma hyopneumoniae in clinical samples.Futhermore,the nucleotide sequence of PRRSV ORF5 gene from three positive samples was determined,which conducted nucleotide sequence similarity analysis and constructed genetic evolution tree with VR2332,HuN4,JXA1,CH-1a and other representative strains.It indicated that a strain of Haemophilias paresis (Hps) was isolated and identified by bacteriological detection.Hps had strong sensitivity to 7 kinds of clinical drugs,such as amoxicilin and cefradine.The gene similarity analysis showed that the PRRSV ORF5 gene nucleotide similarity in three strains (LJW1,LJW2 and LJW3) was 99.3% to 99.8%,the nucleotide similarity to the European-type representative strain Lelystad was 64.0% to 64.2%,the nucleotide similarity with HP-PRRSV strains JXA1,HuN4,CH-1a and TJ were 99.2% to 99.5%,99.0% to 99.3%,94.5% to 94.9%,98.8% to 99.2%,respectively;The higher nucleotides similarity contrast with HP-PRRSV strains HeNzm1-16 and GXL205-2015 isolated from Henan and Guangxi in China were 99.3% to 99.7% and 99.2% to 99.7%,respectively.The nucleotide similarity with the American classic vaccine strains MLV,the American standard strains NC,and the American classic strain VR-2332 was 88.5% to 88.8%,85.2% to 85.5% and 82.3% to 82.6%,respectively.The phylogenetic tree analysis of PRRSV ORF5 showed that all the three strains belonged to the American strain,and they were in the same branch with the domestic HP-PRRSV representative strains JXA1,HuN4,TJ,etc,and the genetic relationship was the closer.This study revealed the pathogens of pigs in this field,and confirmed the genetic relationship between three isolates from PRRSV and different representative strains at the molecular level,which provided a basis of the rational selection and use of attenuated vaccines and comprehensive prevention and control PRRSV.

Key words: porcine reproductive and respiratory syndrome virus(PRRSV); Haemophilus parasuis(Hps); ORF5 gene; genetic evolution

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