猪细环病毒的检测及与多种病原混合感染的调查分析

为了解细环病毒(Torque teno virus,TTV)在广西猪群中的感染情况,本研究运用Nest-PCR方法,对2009—2011年采自广西140个规模猪场的156份血液、流产胎儿及肺脏、脾脏、肾脏、肝脏、淋巴结等组织样品进行检测,并对阳性样品的非编码区(UTR)进行克隆测序及遗传进化分析;同时对部分样品进行猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)、典型猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)检测及细菌的分离鉴定。结果发现,广西猪群中TTV总感染率达到93.6%,TTV2的感染率(76.9%)不仅明显高于TTV1(16.7%),且毒株间遗传变异较大。TTV多与PCV2和PRRSV混合感染,且以与PRRSV混合感染率更高(64.29%)。猪群中存在2重、3重,甚至4重TTV与其他病毒的混合感染。临床上TTV与细菌的混合感染(或细菌继发感染)以链球菌和副猪嗜血杆菌多见。本研究证实在广西猪群中存在TTV感染,且存在普遍的TTV与PRRSV、PCV2和CSFV混合感染。     

2010年国内部分省份猪场常见病毒性疾病的病原学调查

随着规模化养猪业的发展,传染性疾病越来越多,混合感染或多重感染十分普遍,给疾病的诊断和预防带来很大困难。本文将我们实验室2010年分别采自福建、广西、河南、上海、内蒙、浙江、江苏和山西等地发病猪场的185份病料,通过RT-PCR和PCR方法对其进行猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus,PRRSV）、猪繁殖障碍的猪瘟病毒（Classical swine fever virus,CSFV）、猪圆环病毒2（Porcine circovirus type 2,PCV2）、猪细小病毒（Porcine parvovirus,PPV）、猪伪狂犬病毒（Pseudorabies virus,PRV）、细环病毒2（Torque teno virus 2,TTV2）等病毒检测。结果表明在所检的病料中PRRSV、PCV2和TTV2的阳性率比较高,分别为49.2%、62.2%和95.1%。有些地方TTV2的阳性率高达100%;同时,还存在很普遍的PRRSV与PCV2、PRRSV与TTV2、PCV2与TTV2等混合感染,混合感染率分别为27.6%、45.4%、58.4%;以及PRRSV、PCV2与TTV2的三重感染率为24.9%。同时对部分PRRSV阳性病料的PRRSV GP5和nsp2基因分别进行测序,分析测序结果表明病料中的PRRSV的GP5和nsp2序列与PRRSV HuN4株的相应序列的亲缘关系分别在98.2%和96.2%以上,且在nsp2区域都存在30个不连续氨基酸的缺失,说明现在临床中所流行的PRRSV可能仍是与高致病性PRRSV基因型相似的病毒。通过本文可以及时了解当前养猪场的病毒性疾病的流行情况,为猪场病毒性疾病的防控提供依据。     

Porcine respiratory disease complex after the introduction of H1N1/2009 influenza virus in Brazil

From 2009 to 2015, 74 lungs from suckling (6.8%), nursing (70.3%), fattening (20.3%) pigs and pregnant sows (2.7%) with respiratory signs from pig farms in Southern Brazil were submitted to a diagnostic laboratory for necropsy and/or histologic examination and screening for respiratory agents by RT‐qPCR, immunohistochemistry (IHC), virus isolation (VI) and subtyping for influenza A virus (IAV), IHC and nested PCR for Mycoplasma hyopneumoniae (Mhyo), PCR for porcine circovirus 2 (PCV2), RT‐qPCR for porcine reproductive and respiratory syndrome virus (PRRSV) and bacterial culture. All lung samples were positive for IAV using RT‐qPCR. Seventy‐two lungs had histologic lesions associated with acute to subacute IAV infection characterized by necrotizing bronchiolitis/bronchitis or bronchointerstitial pneumonia with lymphocytic peribronchiolitis and bronchiolar/bronchial hyperplasia, respectively. Forty‐nine lungs (66.2%) were positive by IHC for IAV nucleoprotein. The H1N1/2009 was the most common subtype and the only IAV detected in 58.1% of lungs, followed by H1N2 (9.5%) and H3N2 (6.8%). Coinfection of IAV and Mhyo was seen in 23 (31%) cases. Although 14.9% of the lungs were positive for PCV2 using PCR, no suggestive lesions of PCV2 disease were observed. Porcine reproductive and respiratory syndrome virus (PRRSV) was not detected, consistent with the PRRS‐free status of Brazil. Secondary bacterial infections (8/38) were associated with suppurative bronchopneumonia and/or pleuritis. Primary IAV infection with Mhyo coinfection was the most common agents found in porcine respiratory disease complex (PRDC) in pigs in Southern Brazil.     

断奶仔猪多系统衰竭综合征相关病原混合感染的流行病学调查

为了解中国江苏省及周边地区猪场断奶仔猪多系统衰竭综合征(PMWS)相关病原的流行情况,本研究采用PCR方法,对2014年1月至2015年5月采自江苏、安徽及浙江等地猪场的125份健康猪样品和261份发病猪样品分别进行猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)、猪细小病毒(PPV)、输血性传播病毒(TTV)和类猪圆环病毒P1的检测。结果显示,所有样品PCV2、PRRSV、PPV、TTV1、TTV2和P1的阳性率分别为39.38%、21.76%、3.11%、15.80%、16.32%和10.10%,其中混合感染主要存在于PMWS的发病猪群,以PCV2与TTV2 (15.32%)和PCV2与PRRSV (11.87%)的混合感染为主。结果表明,江苏省及周边地区猪场普遍存在PMWS相关病原的混合感染现象,加大了PMWS相关病原的防控难度。     

Epidemiological Investigation of Multi-pathogens Co-infection with Post-weaning Multi-systemic Wasting Syndrome

To investigate the epidemiology of multi-pathogens related with post-weaning multi-systemic wasting syndrome (PMWS) in Jiangsu province and surrounding areas,125 healthy samples and 261 diseased samples which obtained in Jiangsu,Anhui and Zhejiang provinces from the January 2014 to May 2015 were screened by PCR for the presence of PCV2,PRRSV,PPV,TTV and P1.These results revealed the following infection rates of PCV2,PRRSV,PPV,TTV1,TTV2 and P1 were 39.38%,21.76%,3.11%,15.80%,16.32% and 10.10%,respectively.In comparison,the co-infection of several viruses occurred in the diseased pigs,and the main co-infections were PCV2+TTV2 (15.32%) and PCV2+PRRSV (11.87%).These observations indicated that the phenomenon of co-infection with multi-pathogens had been widely existing in swine,which increased the difficulty of prevention and controlling of the PMWS.     

2018年广西重要猪源病毒性疫病流行病学调查

为了解2018年广西猪群重要疫病流行情况,试验采集广西各地的病死猪组织样品及病猪腹泻拭子,应用多重实时荧光定量RT-PCR检测猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）,应用多重实时荧光定量PCR检测猪伪狂犬病病毒（PRV）、猪圆环病毒1型（PCV1）、猪圆环病毒2型（PCV2）及猪圆环病毒3型（PCV3）,应用多重RT-PCR检测猪流行性腹泻病毒（PEDV）、猪德尔塔冠状病毒（PDCoV）、猪传染性胃肠炎病毒（TGEV）和猪轮状病毒（PRoV）。结果显示,所检测的694份组织样品中,CSFV、PRRSV、HP-PRRSV、PRV、PCV1、PCV2、PCV3的阳性率分别为11.10%、18.88%、7.20%、5.19%、2.45%、67.00%和5.76%;2种病原混合感染率为41.21%,3种病原混合感染率为4.32%,其中PRRSV和PCV2混合感染率最高。所检测的792份肠内容物及拭子腹泻样品中,PEDV、PDCoV、TGEV、PRoV的阳性率分别为9.72%、5.81%、1.77%和6.31%;2种病原混合感染率为5.30%,其中PEDV和PRoV混合感染率最高。结果表明,当前多种重要病毒性疫病仍在广西猪群发生和流行,并且多重感染普遍存在,应进一步加强监测和防控。     

猪输血传播病毒(TTV)与猪圆环病毒混合感染的检测及TTV遗传变异分析

为了解我国猪群中猪输血传播病毒(TTV)与猪圆环病毒(PCV)混合感染情况,本研究采用PCR方法对采自14个省280份病料样品进行检测,结果显示,TTV1和TTV2阳性检出率分别为51.8%和28.2%,TTV1与TTV2混合感染率为18.2%;PCV1和PCV2阳性率分别为41.1%和37.5%,PCV1与PCV2混合感染率为19.6%;PCV2阳性样品中TTV1和TTV2混合感染率达75.0%。此外,对2株TTV1基因组进行测序,与GenBank中登录的6个TTV1序列比对,相似性结果为67.3%～95.1%;对4株TTV2基因组测序,与GenBank登录的4条TTV2序列比对,相似性为84.7%～90.4%;将TTV1与TTV2进行序列比对,相似性仅为44.0%。TTV1和TTV2基因高变区分别位于520 nt～2 594 nt和720 nt～2 170 nt;TTV1基因保守区位于1 nt～520 nt和2 595 nt～2 800 nt;而TTV2基因保守区位于1 nt～719 nt和2 171 nt～2 800 nt。以上结果表明,我国猪群中存在TTV与PCV2的混合感染,并且两种病毒混合感染引起...     

猪圆环病毒2型与猪TTV混合感染的流行病学调查

根据GenBank_h发表的猪圆环病毒2型（Porcinecircovirustype2,PCV2）基因组序列和TTV（Torquetenovirus）1、2型的UTR序列设计合成引物,建立了分别用于检测PCV2和TTV1、TTV2的PCR及巢式PCR方法。应用建立的PCR方法对送检的广东、福建和江西等7个省份258份血液和组织样品进行了PCV2、TTV1和TTV2的检测,确定猪群中PCV2与TTV1和／或TTV2混合感染情况。结果表明,94份样品表现为PCV2和TTV1的混合感染,占样品总数的36．4％;193份样品表现为PCV2和TTV2的混合感染,占74．8％;另外,还有一些样品为三重感染,占34．5％。由此可以看出,猪群中PCV2和／或TTV1和／或TTV2的混合感染很普遍。     

猪源TTV Taqman实时荧光定量PCR检测方法的建立和应用

根据TTV1和TTV2的非编码区（UTR）的保守序列分别设计并合成两套特异性引物和Taqman探针,建立了鉴别TTV1和TTV2的Taqman实时荧光定量PCR方法。通过常规PCR方法分别克隆TTV1和TTV2的非编码区（UTR）的保守序列并将其连入pMD18-T载体,制备阳性标准品,优化反应条件,以10倍系列稀释的标准品分别绘制标准曲线,TTV1标准曲线的相关系数为0.984,TTV2标准曲线的相关系数为0.994。检测结果显示,两种方法的灵敏度均可达10 copies/μL,除猪源TTV外,对猪繁殖与呼吸综合征病毒、猪瘟病毒、猪2型圆环病毒、猪流感病毒检测结果均为阴性。该方法重复性好,批内和批间变异系数均小于3%。检测采集自广西和内蒙古的44份病料,TTV1的阳性率为47.73%,TTV2阳性率为70.45%,TTV1和TTV2混合感染的阳性率为31.82%。猪源TTV检测方法的建立为该病毒的流行病学调查和定量提供了有效的手段。     

Natural co-infection of torque teno virus and porcine circovirus 2 in the reproductive apparatus of swine

This work aimed to detect and study natural co-infection of Circoviridae torque teno virus (TTV) and porcine circovirus 2 (PCV2) in the swine reproductive apparatus. Semen and organs from 17 boars were tested by nested and real-time PCR. PCV2 was amplified from semen (47%), lymph nodes (84.6%) and testicles (35.3%). TTV2 was amplified from 16/17 testis and 13/13 lymph nodes. TTV1 DNA was detected in fewer testicle samples (2/17), which were also TTV2 positive. Analyzed ovaries, follicular fluid and uteri of 83 culled sows showed TTV2, TTV1 and PCV2 from 49.3%, 30.1% and 6.0% of the sows, respectively. Sperm analysis indicated insignificant differences between PCV2 and TTVs positive and negative boars. The most frequent pathologic lesion in sows was endometritis (28.9%), but this was unassociated with PCV2 or TTVs detection. These findings question the importance of PCV2 and TTV2 natural co-infection in the pathology of porcine reproductive failures.     

广西猪繁殖与呼吸综合征病毒感染状况调查

采用RT-PCR技术,对2004年1月至2005年4月期间,广西13个市104个疑似猪繁殖与呼吸综合征病毒(PRRSV)感染猪场,无菌采取231头病、死猪的组织病料(肺脏、淋巴结、脾脏)进行了病毒检测。同时,对鉴定为PRRSV阳性的组织病料和猪场进行了猪圆环病毒2型(PCV2)、猪瘟病毒(CSFV)和猪伪狂犬病毒(PRV)的检测,以确定猪群中PRRSV与PCV2、CSFV和PRV混合感染情况。结果从12个市的115份组织病料中检出PRRSV,病料的平均阳性率为49.78%(115/231),猪场的平均阳性率为61.54%(64/104),不同地市有一定的差异。PRRSV与PCV2、CSFV和/或PRV二重或多重混和感染的组织病料总数为53份,猪场总数为39个,混合感染的组织病料和猪场的总阳性率分别为22.94%(53/231)和37.50%(39/104)。混合感染的组织病料占PRRSV阳性组织病料的46.09%(53/115),混合感染的猪场占PRRSV阳性猪场的60.94%(39/64)。其中以PCV2和PRRSV混合感染的组织病料和猪场数最多。由此可见,PRRSV感染在广西猪场已普遍存在,与其他病毒混合感染现象逐渐趋向复杂化。     

Commercial spray-dried porcine plasma does not transmit porcine circovirus type 2 in weaned pigs challenged with porcine reproductive and respiratory syndrome virus

The objective of this study was to evaluate if spray dried porcine plasma (SDPP) containing porcine circovirus type 2 (PCV2) genome supplemented in feed could transmit PCV2 to pigs challenged with porcine reproductive and respiratory syndrome virus (PRRSV). Twenty-three PRRSV-free pigs, non-viraemic for PCV2, were housed in bio-safety level 3 facilities and assigned to four groups in a 2×2 factorial design consisting of PRRSV challenge and a negative control. The diet contained 0 or 8kg SDPP per 100kg of feed. PRRSV challenge groups were inoculated intranasally with 2mL of a suspension containing 10(6) TCID(50)/mL PRRSV. The SDPP used in the study contained 7.56×10(5) PCV2 genome copies per gram. Dietary treatments were fed from 4days prior to PRRSV inoculation until 28days post-inoculation (PI). All challenged pigs developed PRRSV viraemia by day 3PI and PRRSV antibodies were detected in sera by day 14PI, with no difference between diet treatments. Neither PRRSV viraemia nor seroconversion was observed in non-challenged pigs. PCV2 was not detected in the serum of any pigs throughout the experimental period. SDPP containing the PCV2 genome supplemented in feed did not result in PCV2 transmission to either healthy or PRRSV-infected pigs under these experimental conditions.     

Detection rates of porcine reproductive and respiratory syndrome virus,porcine circovirus type 2, and swine influenza virus in porcine proliferative and necrotizing pneumonia

A retrospective study on pig lung tissues from 60 cases of proliferative and necrotizing pneumonia (PNP) was performed to determine the presence of porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and porcine circovirus type 2 (PCV2) in these lesions. Cases selected included 30 cases diagnosed between 1988 and 1992 and 30 cases diagnosed between 1997 and 2001. In each group of 30 cases, 10 were from suckling piglets, whereas the other 20 were from postweaned animals representing either nursery or grower-finisher pigs. Immunohistochemistry using a monoclonal antibody to influenza virus type A was used to determine the presence of SIV, and in situ hybridization was used for the detection of PRRSV and PCV2 nucleic acids. PRRSV was detected in 55 of the 60 cases examined (92%), PCV2 in 25 cases (42%), and SIV in only 1 case (2%). In 30 cases (50%), PRRSV was the only virus detected, whereas in 25 other cases (42%), a combination of PRRSV and PCV2 could be detected in the lungs with PNP lesions. PCV2 could not be detected in the lungs of suckling pigs with PNP. All PCV2-positive cases were found in postweaned pigs and were always in combination with PRRSV. In this latter age group, PCV2 was detected in 63% of the cases (25/40). Data from our study indicate that SIV is rarely identified in PNP and that PCV2 infection is not essential for the development of PNP lesions. The results of the present study demonstrate that PRRSV is consistently and predominantly associated with PNP and should be considered the key etiologic agent for the condition.     

PCV2、PRV和PRRSV多重PCR方法的建立及其应用

根据GenBank登录的猪伪狂犬病毒（PRV）、猪圆环病毒2型（PCV2）和猪繁殖与呼吸综合征病毒（PRRSV）的参考基因序列,设计3对引物分别用于扩增PCV2的ORF2基因、PRV的gE基因、PRRSV的N基因的目的片段,通过优化反应中各个影响因素,建立了PRV、PCV2、PRRSV的多重PCR（mPCR）检测方法。敏感性和特异性的结果表明,该方法对这3种病毒的最低核酸检出量分别为32.5（PRV）、25.2（PCV2）、35.9pg（PRRSV）。该方法对猪流感病毒（SIV）、猪圆环病毒1型（PCV1）、大肠杆菌、猪瘟病毒（CSFV）、猪流行性腹泻病毒（TGE）等病毒的检测结果均为阴性。200份临床样品的多重PCR结果表明,PCV2感染率为80%（160/200）,PRV感染率为21%（42/200）,PRRSV的感染率为78%（156/200）。200份临床样品主要为PCV2和PRRSV混合感染,阳性率达56.0%（112/200）。该方法的建立对这3种病毒病的早期快速检测和指导临床实践具有十分重要的意义。     

上海市猪博卡病毒感染情况调查

根据GenBank公布的猪博卡病毒(Porcine Bocavirus,PBoV)序列,在VP1/2基因区域设计引物和TaqMan探针建立实时荧光定量PCR检测方法,对上海市10个区(县)规模场2006～2011年间采集的1800份猪血清、2010年种猪场不同月份采集的45份猪粪便、2010年9个规模场采集的27份猪鼻棉拭以及门诊采集的9份高热病死猪内脏进行检测,阳性率依次为24％、30％、0％、67％.6份PBoV阳性病料进行猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome,PRRSV)、猪圆环病毒2型(Porcine circovirus type 2,PCV2)、猪伪狂犬病毒(Pseudorabies virus,PRV)、猪细小病毒(Porcine parvovirus,PPV)检测,阳性率依次为83％、100％、0％、0％.检测结果表明,PBoV感染在上海市普遍存在,猪内脏中检出率较高,且春秋两季高发,仔猪比较易感,并与PRRSV、PCV2存在混合感染.     

猪五种繁殖障碍性疫病病原体多重PCR的建立及初步应用

为了建立能够同时检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)和猪细小病毒(PPV)的多重PCR,并用于猪场感染情况的动态监控以及临床诊断,根据GenBank中已发表的5种病毒的基因序列,针对CSFV的E2、PRRSV的Nsp2、PRV的gB、PCV2的ORF2和PPV的VP2基因,分别设计了特异性引物。在建立的单项PCR基础上,通过优化反应条件,建立了能同时检测5种病毒的多重PCR,并具有较高的灵敏性和良好的特异性。采用建立的多重PCR对127份疑似病猪的扁桃体活体组织进行检测,检出了5种病毒的存在,其中感染2种及以上病毒的样品比例为38.6%(49/127),表明该方法是一种快速、灵敏、高效的病原学检测手段。     

2014年广西部分地区常见猪群疫病感染情况监测分析

为探明2014年广西猪群主要疫病的感染情况,本研究从发病猪场和屠宰场采集猪组织样品共325份,应用RT-PCR方法检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪流行性腹泻病毒(PEDV),并应用PCR方法检测猪圆环病毒2型(PCV2)和猪伪狂犬病病毒(PRV)。结果发现,发病猪场中,这5种病毒感染率分别为12.00%、28.57%、19.43%、53.71%和9.71%,而屠宰场的感染率分别为5.33%、2.67%、5.33%、59.33%和11.33%。对猪群混合感染情况分析发现,PCV2和其他病原的混合感染率最高。其中,发病猪场二重感染最高的为PRRSV+PCV2,达到11.43%,其次为PEDV+PCV2、CSFV+PCV2和PCV2+PRV,阳性率分别为5.71%、4.00%和4.00%;三重感染率最高的为PRRSV+PEDV+PCV2以及PRRSV+PCV2+PRV,阳性率均为2.29%。屠宰场二重感染最高的是PCV2+PRV,达到3.33%;三重感染最高的是CSFV+PCV2+PRV,阳性率为0.67%。结果表明,在发病猪场和屠宰场中,PCV2的感染率最高,且常与其他病原发生混合感染,PRV感染率呈上升趋势,加强对这2种病毒的监控对控制广西地区猪群发病具有重要意义。     

2016—2019年广西玉林市部分猪场主要病毒性疫病监测

为了解近年来广西玉林市规模猪场主要病毒性疫病的流行动态和免疫保护水平,分析疫情流行趋势和暴发风险,对2016—2019年采集自玉林市规模猪场284个场次的6954份血清样本,以及113个场次的1539份临床健康猪组织样本、249个场次自主送检的536份病死猪组织样本,进行猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(P...     

5种猪病多重PCR检测方法的建立

To establish a method for simultaneous detection of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV), a multiplex PCR was developed with a set of specific primers designed based on the conserved sequences of CSFV, PRRSV, PRV, PCV2 and PPV. Under the optimized conditions of multiplex PCR,five special fragments of 167 (CSFV),433 (PRRSV),305 (PRV), 559 (PCV2) and 882 bp (PPV) were amplified with a detection limit of 220, 1.6, 72, 400 and 370 pg, respectively. But the multiplex PCR amplification results of swine influenza virus （SIV）, Japanese encephalitis virus （JEV）, Streptococcus suis （SS） and porcine epidemic diarrhea virus （PEDV） were negative．The results showed that the multiplex PCR method was capable of CSFV, PRV, PRRSV, PCV2, PPV infection of single or mixed clinical samples for rapid diagnosis.     

猪瘟病毒和不同型猪繁殖与呼吸综合征病毒的多重RT-PCR检测方法的建立

为建立能同时检测猪瘟病毒(CSFV)和猪繁殖与呼吸综合征病毒(PRRSV)的方法,针对CSFV和PRRSV的基因序列设计3对特异性引物,第1对引物扩增CSFV毒株NS2基因508bp片段,第2对引物扩增PRRSV美洲型经典毒株和变异毒株Nsp2基因338bp/248bp片段,第3对引物扩增PRRSV欧洲型毒株ORF5基因614bp片段。经过反应条件的优化,建立了能同时检测并区分CSFV毒株和PRRSV美洲型经典毒株、变异毒株及欧洲型毒株的多重RT-PCR方法。该方法可以特异扩增CSFV和PRRSV,而与猪口蹄疫病毒(FMDV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)、猪圆环病毒2型(PCV-2)均无交叉反应;对CSFV和PRRSV 4种重组质粒标准品的检出下限均为1.67×103拷贝/μL。对采集的106份临床疑似病料进行检测,结果CSFV和PRRSV变异株混合阳性4份,占3.77%(4/106);CSFV阳性7份,占6.60%(7/106);PRRSV变异株阳性17份,占16.04%(17/106)。结果表明,建立的多重RT-PCR检测方法可以用于CSFV和PRRSV的临床快速鉴别诊断和流行病学调查。