猪繁殖与呼吸综合征和猪流感混合感染的诊断

通过流行病学调查、临床症状观察和病理剖检，结合血清学调查和病原学检测，对浙江省部分地区流行的“猪高热症”病因进行了探讨，初步认为这种疫情是由猪繁殖与呼吸综合征（PRRS）和猪流感并发造成的，继发感染则加重了本次疫情所造成的损失。     

猪繁殖-呼吸综合征病毒上海株的分离鉴定

从上海地区５个发病猪场的病料中,分离到３株致Ｍａｒｃ－１４５细胞病变的病毒：ＳＨ１、ＳＨ２和ＳＨ３,理化特性研究表明,３株分离毒均对氯仿、乙醚、甲醛、热（５６℃）、酸（ｐＨ６以下）、碱（ｐＨ８以上）敏感。负染可见病毒以大小不等的具囊膜的球形粒子为主,囊膜上有纤突,直径为６０￣１００ｎｍ;超薄切片可见病毒粒子分散在细胞浆内,直径为４５￣８０ｎｍ。间接免疫荧光试验表明,３株分离毒均与ＰＲＲＳＶ高免血清呈强阳性反应,而与ＨＣＶ、ＰＰＶ、ＰＲＶ、ＴＧＥＶ高免血清的反应均呈阴性。综上可见,所分离的病毒为ＰＲＲＳＶ。     

猪繁殖与呼吸综合征病毒、猪圆环病毒2型与副猪嗜血杆菌混合感染的诊断及病原分析

为确定河南开封某猪场发生猪呼吸系统疾病综合征（PRDC）的病原,本研究无菌采集病死保育猪肺脏、心脏和脾脏等组织样品,进行细菌学检验和药敏试验,通过PCR/RT-PCR检测样品中猪繁殖与呼吸综合征病毒（PRRSV）、猪圆环病毒2型（PCV2）、猪流感病毒（SIV）、猪伪狂犬病病毒（PRV）、猪瘟病毒（CSFV）和猪肺炎支原体（Mhp）等病原,并对核酸阳性病毒性病原的抗原结构基因进行测序和遗传演化分析。结果表明,通过细菌分离培养、形态观察、卫星现象观察和16S rRNA基因鉴定,从病死保育猪体内分离鉴定出1株副猪嗜血杆菌（Hps）,药敏实验表明该菌株对对氨苄西林、阿莫西林克拉维酸、头孢噻呋和四环素几种药物敏感。核酸检测PRRSV和PCV2核酸阳性,分别命名为PRRSV/HN-2019和PCV2/HN11-2019;进一步对PRRSV/HN-2019和PCV2/HN11-2019的结构基因分析发现,PRRSV/HN-2019与与NADC30分支的毒株亲缘关系较近,属于NADC30-like毒株;PCV2/HN11-2019与PCV-2d分支的毒株亲缘关系较近,属于PCV-2d分支。综上所述,本研究确定该猪场存在PRRSV、PCV2和Hps的混合感染,为该猪场下一步的PRDC有效防控提供了参考依据。     

Diagnosis and Pathogen Analysis of Mixed Infection of Porcine Reproductive and Respiratory Syndrome Virus,Porcine Circovirus Type 2 and Haemophilus parasuis

To identify the causative agent of porcine respiratory disease complex (PRDC) occurred at a pig farm in Kaifeng city,Henan province,we identified the potential causative bacteria of the morbid nursing piglets by bacterial test and drug sensitivity test of the clinical samples (lung,liver and spleen),and detected the common potential pathogens causing PRDC diseases by PCR and RT-PCR assays,including porcine reproductive and respiratory syndrome virus (PRRSV),porcine circovirus type 2 (PCV2),swine influenza virus (SIV),pseudorabies virus (PRV),classical swine fever virus (CSFV) and Mycoplasma hyopneumoniae (Mhp),and then sequcing and phylogenetic analysis of the structural gene of positive causative pathogens were carried out.The results showed that a Haemophilus parasuis (Hps) strain were isolated and identified by the observation of bacterial morphology and satellite phenomenon,and the sequence analysis of 16S rRNA gene.The drug sensitivity test showed that the Hps strain was sensitive to ampicillin,amoxicillin-clavulanic acid,ceftiofur and tetracycline.Meanwhile,PCR and RT-PCR assays indicated that all the samples were positive for PRRSV and PCV2,and named the involved strain as PRRSV/HN-2019 and PCV2/HN11-2019,respectively.Sequencing and phylogenetic analysis of the ORF5 gene of the newly identified PRRSV revealed that the PRRSV/HN-2019 strain was closely related to the NADC30-like strains and grouped into NADC30-like genotype clade.And cap gene of the newly identified PCV2 strain the PCV2/HN11-2019 strain was closely related to the PCV-2d strains and grouped into PCV-2d genotype clade.In conclusion,this study demonstrated that the morbid piglets were co-infected with PRRSV,PCV2 and Hps,which provided a basis for the development of effective control strategies in the pig farm.     

猪繁殖与呼吸综合征病毒单克隆抗体的研究进展

单克隆抗体在兽医领域的应用价值越来越受到研究人员的重视。本文概述了近年来猪繁殖与呼吸综合征病毒（PRRSV）结构蛋白单克隆抗体的研究概况，以及它们在该病病毒分子生物学研究中的应用。     

对青岛地区4个猪场的猪细环病毒II型的分子流行病学调查

为了解青岛地区各猪场中猪细环病毒II型（TTV2）的存在和感染情况,本实验对2012年3月至5月期间采自青岛地区4个猪场的59份猪血清样品利用病毒核酸提取试剂盒进行了猪细环病毒DNA提取,在此基础上利用TTv2的特异性引物进行PCR检测。结果显示只有莱西1个猪场的血清样品中检测到阳性,阳性率为13．8％,并且存在TTV1与TTV2混合感染的情况,混合感染率为6．9％。本实验结果证实某些猪场中存在猪细环病毒感染,由于TTV在猪的多系统衰竭综合征的病症发展中可能扮演一定角色,所以了解TTV在猪场中的存在状况具有重要意义。     

安徽省部分规模化猪场猪繁殖与呼吸综合征血清学调查

在安徽部分地区采集了14个猪繁殖与呼吸综合征(PRRS)免疫猪场和13个非PRRS免疫猪场不同猪群血清样品743份,应用ELISA方法检测PRRSV抗体.结果显示,未免疫猪场PRRSV抗体平均阳性率为31.17%,猪场阳性率为76.92%;免疫PRRS猪场抗体平均阳性率为62.99%.通过SPSS3.0软件数据分析表明,PRRS免疫猪场与未免疫猪场PRRS抗体阳性率差异极显著(P<0.01),提示安徽省规模猪场存在PRRSV隐性感染.     

Prevalence of Antibodies to Classical Swine Fever,Aujeszky's Disease,Porcine Reproductive and Respiratory Syndrome,and Bovine Viral Diarrhoea Viruses in Wild Boars in Croatia

During the hunting season in February 1999, a total of 44 blood samples were collected from wild boars shot in the area of MoslavaC?ka gora. These blood samples were examined by enzyme immunoassay for the presence of antibodies to classical swine fever (CSFV), Aujeszky's disease (ADV), bovine viral diarrhoea (BVDV), and porcine reproductive and respiratory syndrome (PRRSV) viruses. Out of 44 serum samples examined, 17 (38.63%) were positive for CSFV, 24 (54.54%) were positive for ADV and two (4.54%) were positive for BVDV. All sera were negative for PRRSV. The results, recorded for the first time in Croatia, supported the hypothesis that wild boar act as a potential reservoir of CSFV, ADV and BVDV, and thus have a role in the epidemiology of these diseases.     

上海地区猪场中家鼠PRRSV和CSFV带毒情况调查

从上海9家猪场内采集的46只家鼠的血清和组织样品,采用IHA、ELISA和RT-PCR方法对猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)的抗体及抗原进行了检测,结果均为阴性。由此说明家鼠对这两种病毒不易感,不可能作为PRRSV和CSFV的传播媒介和贮存宿主。     

陕西省规模化猪场猪繁殖与呼吸综合征流行病学调查

2005年10月至2006年9月．我们对陕西省西安、宝鸡、汉中三市的部分发生繁殖障碍的规模化猪场（存栏母猪300头以上）就PRRS进行了流行病学调查，现报告如下。[第一段]     

贵阳市规模化猪场猪繁殖与呼吸综合征血清学调查研究

试验应用ELISA检测方法对贵阳市3个未免疫猪繁殖与呼吸综合征（PRRS）猪场和9个免疫猪场的后备母猪、生产母猪和5～10周龄断奶仔猪3个猪群进行猪繁殖与呼吸综合征病毒（PRRSV）抗体检测。结果表明：贵阳市3个未免疫猪场中2个有PRRSV感染,猪群抗体阳性率在0％-10％之间,平均阳性率为5％;9个免疫猪场猪群PRRSV抗体阳性率在65％～90％之间,平均阳性率为82．80％,未免疫猪场与免疫猪场猪群抗体平均阳性率相比,差异极显著（P〈0．01）。     

部分猪场H1和H3亚型猪流感的血清学调查

为了解中国部分省市规模化猪场H1和H3亚型猪流感病毒的流行情况,采用血凝抑制试验对采集于广东、湖南、河南省12个市县28个规模化猪场的799份血清进行H1和H3亚型猪流感病毒的抗体检测。结果表明,H1亚型抗体阳性率在0~83.33%之间,猪抗体总阳性率为46.18%(369/799),猪场阳性率为89.29%(25/28)。H3亚型抗体阳性率在0~100.00%之间,猪抗体总阳性率为61.33%(490/799),猪场阳性率为85.71%(24/28)。广东、湖南和河南地区H1亚型抗体阳性率分别为48.91%、40.26%和50.67%,H3亚型抗体阳性率分别为58.55%、70.78%和78.67%。在被调查的上述3个地区的猪群中,H1和H3亚型猪流感病毒的感染较为普遍,其中H3亚型感染率高于H1亚型,且各地区猪流感病毒的流行情况存在地域性差异。     

广东部分地区猪场PRRS的血清学调查

2008年7月至2009年1月,应用酶联免疫吸附反应法对广东省茂名、惠州、东莞、四会、鹤山5个地区的40个中小规模猪场进行了猪繁殖与呼吸综合征(PRRS)的血清学调查。结果显示,未进行疫苗免疫接种的猪场PRRSV的感染率高达100%,平均抗体阳性率为66.77%,其中后备母猪为87.50%,生产母猪为80.77%,仔猪为57.21%;进行过疫苗免疫接种的猪场PRRS平均抗体阳性率82.82%,其中后备母猪为87.06%,生产母猪为82.40%,仔猪为81.89%。由此可见,猪繁殖与呼吸综合征野毒在广东省中小规模猪场普遍存在,而且感染率差异显著(P0.01),这可能与各个猪场的饲养环境、管理水平和防疫措施等有关。     

Clinical Effects of Experimental Dual Infections with Porcine Reproductive and Respiratory Syndrome Virus Followed by Swine Influenza Virus in Conventional and Colostrum‐deprived Pigs

Previous studies demonstrated that experimental dual infections of pigs with porcine reproductive and respiratory syndrome virus (PRRSV) followed by H1N1 influenza virus cause more severe disease and growth retardation than the respective single virus infections. Here three experiments were undertaken to better define the clinical impact of combined PRRSV‐H1N1 infections in conventional and caesarean‐derived colostrum‐deprived (CDCD) pigs. Groups of pigs were inoculated by aerosol with PRRSV followed by H1N1 at 3‐, 7‐ or 14‐day intervals. During the post‐H1N1 period, mean body temperatures, respiratory signs and mean weight gains in the PRRSV‐H1N1 inoculated groups were recorded and compared with those in uninoculated controls (experiments 1 and 2) or in singly virus‐inoculated pigs (experiment 3). In a first experiment with conventional pigs, the PRRSV‐3d‐H1N1 and PRRSV‐7d‐H1N1 infections induced mean body temperatures >40.5°C during 8 days (peaks 41.1 and 41.6°C, respectively) and mean growth reductions of 3.4 and 4.8 kg, respectively, during the 2 weeks after H1N1, along with marked depression and respiratory disease. The PRRSV‐14d‐H1N1 infection, on the contrary, was largely subclinical. In a second experiment with conventional pigs, PRRSV‐3d‐H1N1 and PRRSV‐7d‐H1N1 infections were clinically milder, with smaller increases in mean body temperatures (peak 40.5°C in both groups) and growth reductions (1.4 and 1.6 kg, respectively). In both groups, only one pig showed prominent general and respiratory signs. In a final experiment with CDCD pigs, PRRSV‐7d‐H1N1 infection had minimal effects on mean clinical performances and growth and, except for one pig that was severely affected, differences with the single virus inoculations were negligible. Thus, both the time interval between infections and the sanitary status of pigs can affect the clinical outcome of dual PRRSV‐H1N1 infections. However, factors so far unknown seem to cause large variations in the clinical response between individual pigs.     

猪圆环病毒2型和猪瘟病毒抗体检测与分析

目的检测临汾市某县猪圆环病毒2型(PCV2)和猪瘟病毒(CSFV)的抗体水平.方法分别从该县的企业、散户、大户的猪只中采集血清186份,应用ELISA方法检测抗体水平.结果 PCV2抗体阳性率平均为83.33%,CSFV抗体阳性率平均为54.83%.结论该县猪圆环病毒2型感染严重,猪瘟疫苗免疫抗体水平偏低,与猪瘟疫苗使用不当、猪圆环病毒2型感染存在一定关系.     

猪瘟与猪呼吸障碍综合征病毒混合感染的PCR诊断

猪瘟和猪呼吸障碍综合征病毒病均是养猪生产中的主要病毒性传染病,各种年龄猪均易感,猪感染后死亡率很高。猪瘟的主要症状为发病急、高热、全身出血以及脾梗死;猪呼吸障碍综合征可以引起免疫抑制,猪感染后表现为繁殖障碍和呼吸道传染病,母畜发生流产、产死胎与木乃伊胎。该文对一例疑似猪瘟和猪呼吸障碍综合征病毒混合感染病例进行了PCR诊断,为猪场相关疾病的诊断提供参考。     

上海及周边地区散养猪主要病毒病的流行状况调查

采用PCR方法对上海及周边地区散养临床发病猪进行主要病毒病的病原学检测,主要包括猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒和猪圆环病毒2型。2005年检测病死猪病料97份,4种病的阳性率分别为33.33%、23.81%、35.90%、40.00%;2006年检测357份,4种病的阳性率分别为15.91%、33.33%、10.53%、23.61%;2007年检测1050份,4种病的阳性率分别为13.92%、35.20%、7.78%、20.57%。2005年-2007年的病例中均有不同程度的混合感染,混合感染率分别为3.09%、4.48%、3.62%,表明这些病原在上海及周边地区的散养猪中普遍存在,成为猪病防疫中不可忽视的一个环节。     

PRRS ELISA试剂盒检测猪繁殖与呼吸综合征病毒抗体的应用

利用重组杆状病毒表达的PRRSV核衣壳蛋白作抗原制成ELISA诊断试剂盒，检测人工感染PRRSV猪血清、疫苗免疫抗体和田间血清，并与IDEXX公司生产的试剂盒进行比较。结果表明，该试剂盒在PRRSV感染后8天内就可检出感染性抗体，而用IDEXX公司生产的试剂盒在感染后的18天才检测到感染性抗体，对弱毒疫苗的免疫检测也基本能反映出疫苗的免疫应答状况。对华东地区PRRSV流行病学调查结果表明，PRRSV在国内普遍存在，同时也证明研制的试剂盒，是客观科学调查我国PRRS流行情较理想的检测工具。     

表达猪流感病毒血凝素基因的重组腺病毒的构建及免疫原性分析

通过细菌内同源重组的方法成功构建了表达H3N2亚型猪流感病毒血凝素的重组腺病毒。首先将血凝素基因亚克隆到穿梭载体质粒pAdeno Vator-CMV5-IRES-GFP上，与腺病毒基因组质粒pAdeno Vator△E1／E3共转化大肠杆菌BJ5183菌株，经细菌内同源重组产生重组腺病毒质粒pAd—HA—GFP。将该重组质粒转染293细胞以包装重组腺病毒。根据报告基因GFP的表达及Western-blot分析，证明获得了可正确表达猪流感病毒血凝素的重组腺病毒rAd—HA—GFP。重组腺病毒经一次性腹腔注射接种昆明鼠，2周后可检测到血凝抑制抗体，并且抗体至少可以持续12周，证实重组腺病毒介导表达的血凝素蛋白具有良好的免疫原性，可诱导小鼠产生高滴度的特异性抗体。