高致病性猪繁殖与呼吸综合征病毒与副猪嗜血杆菌共感染的综合鉴定

Comprehensive Identification and Typing of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Coinfection with Serum Haemophilus Parasuis Type 5

为了对四川省某肥育猪场疫病原因进行确诊并为其提供临床用药指导,研究从发病猪场采集病变组织样本进行细菌分离纯化;通过PCR分别检测并鉴定猪繁殖与呼吸综合征病毒(PRRSV)和副猪嗜血杆菌(HPS)基因型和血清型,并进行核苷酸变异性分析和遗传进化分析;用K-B纸片法筛选HPS敏感抗生素。根据病猪高热、耳根四肢发绀;全身淋巴结肿大,肺脏、肝脏肿大实变等主要临床症状和病理变化,结合PCR初步确定引发该次疫情的主要病毒为PRRSV。进一步对该毒株Nsp2基因进行克隆测序和比对分析发现其与广西分离株PRRSV HUN4-like GXBH1404株序列相似度高达99.17%。根据本次分离的毒株和参考的毒株建立了基于Nsp2基因的进化树,该毒株属于Lineage8谱系的HP-PRRSV毒株,与谱系8中的代表毒株JXA1和HUN4、GXBH1404及四川流行毒株SCCD16、SC2012等5个毒株相比,SCLZ202201株Nsp2基因共存在9个独有的碱基突变,与四川流行毒株相比共存在21个碱基突变,表明HP-PRRSV仍在四川地区持续流行,其基因多样性也在不断增加。经细菌分离纯化获得一株依赖NAD生长的革兰氏阴性短杆菌,结合PCR和生化鉴定,综合判定该菌为血清5型副猪嗜血杆菌(HPS)菌株。药敏试验结果显示,该细菌对新霉素、恩诺沙星、庆大霉素、头孢克洛、头孢氨苄较敏感;对林可霉素、卡那霉素、多西环素中介;对头孢拉定耐药。由此确定,该次猪场疫情由PRRSV HUN4-like毒株和HPS 5型菌株混合感染所致。研究确定了引发该次疫情的主要原因,为养殖场相关疫病的防控提供了理论依据。

In order to diagnose the cause of the epidemic disease in a finishing pig farm in Sichuan Province and provide clinical medication guidance,the diseased tissue samples were collected from the diseased pig farm for bacterial isolation and purification.The genotypes and serotypes of PRRSV and HPS were detected and identified by PCR,and nucleotide variability and genetic evolution were analyzed.Screening of HPS sensitive antibiotics by K-B disk method.According to the pig fever,ears and limbs cyanosis;the main clinical symptoms and pathological changes such as lymph node swelling in the whole body,swelling and consolidation in the lung and liver were preliminarily determined to be the main virus causing the epidemic situation of PRRSV based on PCR examination.Further cloning,sequencing and comparative analysis of the Nsp2 gene of this strain showed that the sequence similarity with Guangxi isolate PRRSV HUN4-like GXBH1404 was as high as 99.17%.An evolutionary tree based on Nsp2 gene was established according to the isolates and the reference strains.The strain belongs to the HP-PRRSV strain of Lineage 8 lineage,and compared with the five representative strains in lineage 8,namely,JXA1 and HUN4,GXBH1404,and the epidemic strains in Sichuan,namely,SCCD16 and SC2012.There were nine unique base mutations in the Nsp2 gene of SCLZ202201 strain,and a total of 21 base mutations compared with the Sichuan epidemic strain,indicating that HP-PRRSV was still prevalent in Sichuan province,and its gene diversity was also increasing.After bacterial isolation and purification,an NAD-dependent gram-negative Brevibacterium sp.was obtained,which was comprehensively determined to be a serum Haemophilus parasuis type 5(HPS)strain by PCR and biochemical identification.The results of drug sensitivity test showed that the bacteria were more sensitive to neomycin,enrofloxacin,gentamicin,cefaclor and cephalexin.Intermediate for lincomycin,kanamycin and doxycycline;resistant to cefradine and ofloxacin.It was concluded that the combined infection of PRRSV HUN4-like strain and HPS 5 strain caused the outbreak in pig farms.This study identified the main causes of the epidemic and provided a theoretical basis for the prevention and control of diseases related to farms.