Evidence for divergence of restriction fragment length polymorphism patterns following in vivo replication of porcine reproductive and respiratory syndrome virus

OBJECTIVE: To determine stability of the restriction fragment length polymorphism (RFLP) pattern of a porcine reproductive and respiratory syndrome vaccine virus and patterns of other viral strains as they replicate in pigs. SAMPLE POPULATION: Field samples of porcine reproductive and respiratory syndrome virus (PRRSV) and samples from 2 weaned pigs, 2 nursery-age pigs, and 5 gilts experimentally infected with PRRSV. PROCEDURE: PRRSV was isolated from field samples, experimentally infected pigs, or pigs that were in contact with experimentally infected pigs. For each virus, RNA was isolated from infected cells, and RFLP patterns were determined. RESULTS: 61% of field samples had 2-5-2 RFLP patterns characteristic of the vaccine virus, 32% had field virus RFLP patterns, and 7% had intermediate RFLP patterns that indicated a virus with a close relationship to the vaccine virus. Viruses isolated from experimentally infected pigs had no change in RFLP patterns after up to 13 weeks of in vivo replication and transmission to contact pigs. CONCLUSIONS AND CLINICAL RELEVANCE: RFLP patterns distinguish the vaccine and field strains of PRRSV; however, as the vaccine virus spreads among a swine population, the RFLP pattern can change to a related intermediate pattern. A glycine at residue 151 of open reading frame 5 is another marker for the vaccine virus; this glycine is rapidly lost and eventually replaced with arginine as the vaccine virus replicates in pigs.     

湖南高致病性猪蓝耳病隐性感染情况调查

用ELISA和RT-PCR的方法对采集自湖南省内20个规模场1007份血清和3个市级定点屠宰场50份猪肺门淋巴结进行蓝耳病血清抗体检测和高致病性猪蓝耳病病毒的检测。结果1007份血清中,蓝耳病抗体阳性率为72.9%(734/1007),高致病性猪蓝耳病病毒携毒率为3.2%(32/1007),50份肺门淋巴结病毒阳性率为16%(8/50),其中,蓝耳病免疫与非免疫猪血清其抗体阳性率相差不显著,种猪的抗体阳性率明显高于商品猪,而其病毒携毒率为0%。部分规模猪场和眼观健康的育肥猪存在高致病性猪蓝耳病病毒的隐性感染。     

华南地区猪圆环病毒和猪繁殖与呼吸综合征病毒检测分析

为了解华南地区猪圆环病毒及猪繁殖与呼吸综合征病毒的最新流行情况,采集了华南地区11个规模猪场各饲养阶段猪血清807份,用套式PCR(nPCR)检测猪圆环病毒1型(PCV-1)和猪圆环病毒2型(PCV-2);采集了若干猪场2010年1月至2011年8月期间有咳嗽、喘气、消瘦及疑似PDNS等临床症状的猪血清312份,以及无临床症状猪血清104份,以nPCR检测PCV-2,以一步法反转录PCR(RT-PCR)检测猪繁殖与呼吸综合征病毒。结果发现,所调查的11个规模猪场中只有5个检出PCV-1,所有猪场均检出PCV-2。PCV-2阳性率为30.61%,而PCV-1阳性率仅为4.21%;经产母猪和7周龄以上保育猪PCV阳性率最高;有临床症状的猪血清PCV-2阳性率为58.65%,PRRSV阳性率为37.82%,PCV-2阳性猪群中有39.89%的猪同时感染PRRSV;有症状猪群7月～9月的PCV-2感染率最高,而1月～3月最低;无临床症状猪血清PCV-2阳性率为27.9%,PRRSV阳性率为0.96%,PCV-2与PRRSV无混合感染。证明PCV尤其是PCV-2在华南地区仍广泛传播并流行,而且PCV-2与PRRSV混合感染致病情况较多。PCV-2的感染率与季节有一定的相关性,种猪带毒情况严重。     

抗独特型抗体对猪繁殖与呼吸综合征病毒感染的免疫作用

用PRRSV感染SPF猪，血清检测结果显示，机体不仅产生抗PRRSV抗原的各种抗体(Ab1)，而且产生针对这些抗体的抗独特型抗体(Ab2)。根据各种蛋白质的等电点不同，应用IEF技术分离纯化出PRRSV感染猪血清中的不同IgG。分别以纯化的抗PRRSV—GP5蛋白、抗PRRSV-M蛋白的Ab2免疫SPF猪各5头，7d后经鼻腔感染PRRSV，定期采集血样进行病毒分离或鉴定试验。抗PRRSV-GP5蛋白的Ab2免疫的猪，其血样自感染后3～7d均检出PRRSV；3头猪在感染后14～63d未检出PRRSV；2头猪在感染后14～35d检出PRRSV，从42～56d转为阴性，其中1头猪在63d时检出PRRSV。抗PRRSV—M蛋白的Ab2免疫的猪，其血样自感染后3～7d均检出PRRSV；2头猪在感染后14～63d未检出PRRSV；3头猪在感染后14～35d检出PRRSV，从42～56d转为阴性，其中1头猪在63d时检出PRRSV。抗PRRSV—GP5和抗PRRSV—M蛋白的Ab2免疫作用显著，可作为PRRSV-GP5和PRRSV—M蛋白的替代抗原产生具有中和效应的抗体，保护机体免受PRRSV的感染。     

Persistence of porcine reproductive and respiratory syndrome virus in intensive farrow-to-finish pig herds.

An epidemiological study of porcine reproductive and respiratory syndrome (PRRS) within pig herds was conducted in 8 intensive farrow-to-finish pig farms. Persistence of PRRS virus (PRRSV) in pig herds was demonstrated by regular postmortem examination on 2 farms for a period of 2 y. Virus isolation and serum neutralization (SN) tests were performed on the sera collected from 9 groups of pigs (10 pigs/group) of various ages on 8 pig farms. Except for 1 farm, isolation rates of PRRSV reached the highest level of 70 to 100% of pigs 6 to 8 wk of age, which coincided with the lowest levels of maternal immunity. In 1 pig herd, sows (39 in total) with SN titers of < or = 1:2, 1:4-1:8, and > or = 1:16 were designated as groups 1, 2, and 3, respectively. Sera were obtained from their progeny (3 pigs randomly selected from each litter) at various ages from 0 to 22 weeks. A positive correlation (r = 0.377, P < 0.001) between the SN titers of sows and those of their progeny (1-week-old piglets) was observed. Pigs at the age of 6 wk, only 7.9% of group 1 pigs compared to 72.4% of group 3 pigs were seropositive. A significant difference (P < 0.01) in the percentage of pigs with PRRSV viremia among the 3 groups was observed, with the lowest level found in group 3 pigs. The isolation rates of PRRSV from serum reached the maximum at the age of 9 wk for all 3 groups. The results indicated that passively acquired serum antibodies conferred a protective effect for piglets; however, loss of passive immunity at various ages of pigs produced susceptible pigs that resulted in PRRSV persistence in the pig herds. Pigs 6 to 9 weeks old were the major reservoir for PRRSV in farrow-to-finish pig herds.     

我国五省区部分猪场高致病性猪蓝耳病毒感染情况检测与分析

对2008年-2009年我国五省区发病猪场235份、屠宰场的218份样品进行了高致病性猪蓝耳病病毒(HP-PRRSV)RT-PCR检测,挑选具有代表性的HP-PRRSV阳性样品进行HP-PRRSV ORF5基因扩增、测序及分析.结果表明,这五个省区发病场蓝耳病的检出率平均74.6%.屠宰场的检出率平均44%,混合感染主要以二重感染为主,且发病场较屠宰场严重.对获得的13株HP-PRRSV ORF5进行测序分析,发现序列长度均为603 bp,未见缺失或插入,仅在9 aa～29 aa存在点突变;序列比较发现,与普通株标准序列VR-2332株核苷酸同源性达85.9%～87.2%;与高致病性毒株的同源性JXA1-06核苷酸同源性达96.0%～99.0%.而其推导氨基酸序列与普通型、高致病性毒株的同源性分别为87.1%～88.1%和97.5%～98.0%.从遗传进化以及变异情况看,获得的PRRSV ORF5序列均为与JXA1类似的高致病性PRRSV序列,与JXA1和HB-1同属美洲型中的一个亚群.     

2006-2011年唐山地区猪蓝耳病病毒隐性感染情况调查

为了了解唐山地区猪蓝耳病的带毒情况,采用RT-PCR方法对2006-2011年采自64个县级屠宰场276份商品猪的肺脏或肺门淋巴结进行猪繁殖与呼吸综合征病毒(PRRSV)病原学检测,结果屠宰场阳性率为51.56%,样品总阳性率为20.29%,其中高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)样品总阳性率为15.94%,经典猪繁殖与呼吸综合征病毒(C-PRRSV)样品总阳性率为4.35%。总之,猪繁殖与呼吸综合征病毒,尤其是高致病性猪繁殖与呼吸综合征病毒在唐山地区猪群中污染面积较大,应值得高度重视。     

豫南地区规模化猪场猪繁殖与呼吸综合征病毒和猪圆环病毒2型抗体检测与分析

为掌握豫南地区规模化猪场中猪繁殖与呼吸综合征病毒（PRRSV）和猪圆环病毒2型（PCV2）疫苗免疫场猪群抗体水平和非疫苗免疫场猪群病原感染情况,试验利用ELISA方法对该地区猪群进行了PRRSV和PCV2抗体水平检测。结果表明,在疫苗免疫猪群中,PRRSV和PCV2抗体合格率分别为68.8%和58.7%,规模越大的猪场合格率越高,200~500头母猪规模场抗体阳性率最高（分别为76.8%和77.4%）,50头母猪以下场最低（分别为48.3%和44.2%）,种猪的合格率均最高,而育肥猪群合格率均最低。在非疫苗免疫猪群中,PRRSV和PCV2抗体阳性率分别为55.6%和65.3%,PRRSV抗体阳性率最低的是100~200头母猪规模场（46.5%）,最高的是50头母猪以下规模场（68.8%）,断奶仔猪和育肥猪群抗体阳性率均在71%以上;随猪场规模越大,PCV2抗体阳性率越低,200~500头母猪规模场抗体阳性率为41.4%,50~100头规模场和50头以下场的阳性率分别为78.9%和78.6%,种公、母猪的PCV2抗体阳性率最低（分别为37.5%和40.4%）,断奶仔猪和育肥猪抗体阳性率较高（分别为82.2%和79.5%）。本研究反映了豫南地区猪繁殖与呼吸综合征（porcine reproductive and respiratory syndrome,PRRS）和猪圆环病毒病（porcine circovirus disease,PCVD）疫苗免疫猪场的疫苗免疫效果和非疫苗免疫猪场病原感染的实际情况和规律,为该地区PRRS和PCVD防制工作提供了理论依据和指导。     

Polymerase chain reaction-based restriction fragment length polymorphism pattern of porcine reproductive and respiratory syndrome virus directly from lung tissues without virus isolation in Korea.

Polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis was developed for directly typing porcine reproductive and respiratory syndrome virus (PRRSV) from lung specimens without virus isolation. Twenty nine lung specimens collected from postweaning pigs were isolated for PRRSV. When the PCR products from the 29 lung specimens were digested by the restriction enzymes MluI, HincII, SacII and HaeIII, the RFLP patterns from the 29 lung specimens matched with those from the corresponding PRRSV isolates from each pig. The results suggest that the PCR-based RFLP analysis method may be useful to distinguish PRRSV isolates directly from lung specimens without virus isolation.     

高致病性猪繁殖与呼吸综合征病毒GD07b株的分离鉴定及Nsp2基因同源性分析

在某集约化猪场采集疑似高致病性猪繁殖与呼吸综合征病死猪的肺、淋巴结等病料,进行检测,将RT-PCR检测结果为阳性的病料处理后接种Marc-145细胞,培养72～96 h后细胞单层出现明显的CPE。收获病毒经RT-PCR方法检测,并将此PCR产物经纯化后连同引物送相关公司进行测序,结果显示,该病毒为繁殖与呼吸综合征病毒Nsp2基因缺失株,将该病毒命名为HPPRRSV-GD07b株。将GD07b株序列与国内外19株PRRSV Nsp2基因进行比较分析,结果表明,该毒株Nsp2基因与CH-1a株等4株PRRSV经典株同源性较低,为60.5%～81.6%;而与14株变异株同源性较高,为90.6%～98.9%。     

河北屠宰场高致病性猪蓝耳病带毒情况调查

为了了解河北省屠宰场猪只高致病性猪蓝耳病的带毒情况,采用RT-PCR方法对2006～2009年采自全省11个地市228个县级屠宰场1565份商品猪的肺脏或肺门淋巴结进行高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)病原学检测,结果4年间猪场的阳性比率分别为55.56％、72.50％、44.62％和28.74％;4年样品阳性检出率分别为28.45％、40.74％、12.01％和7.31％.HP-PRRSV同伪狂犬病病毒(PRV)、圆环病毒2型(PCV-2)和猪瘟病毒(CSFV)混合感染所占的比率为61.66％,多以PCV-2和PRV的二重或三重混合感染为主.总之,HP-PRRSV在河北省猪群中污染面积较大,值得高度重视.     

2016～2018年江苏省规模猪场PRRS血清学调查

为了解2016年1月~2018年12月期间江苏省规模猪场猪繁殖与呼吸障碍综合征(porcine reproductive and respiratory syndrome,PRRS)流行动态,本研究对该省27家规模猪场不同批次送检的2062份未免疫PRRS疫苗血清样本,采用ELISA方法进行了猪繁殖与呼吸障碍综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)抗体检测,并对抗体阳性率≥80%的猪群阶段,依据PRRSV抗体S/P分布频段选取135份血清,采用RT-qPCR方法检测并统计PRRSV阳性率,进行血清学调查。结果显示,27家未免疫PRRS疫苗猪场抗体总阳性率为68.82%。不同地区抗体阳性率较高的为宿迁和南通,分别为72.04%和71.09%,连云港地区最低为58.82%。2016~2018年呈现逐年下降的趋势,从76.41%下降至57.72%。不同猪群阶段中以公猪、后备母猪、12~16周龄育肥猪和18~25周龄育肥猪抗体阳性率较高,分别为80.95%、92.86%、86.84%和98.09%。在上述4个抗体阳性率≥80%的猪群阶段,RT-qPCR检测结果显示,24份PRRSV阳性血清样本C t值与抗体S/P值高低无相关性。血清中PRRSV阳性率以后备母猪最高为20.58%,在PRRSV抗体S/P值<0.4和S/P值≥2.5异常分布频段,血清PRRSV阳性率均≥15.38%,而0.4≤S/P值<2.5正常范围内PRRSV阳性率均≤10%,差异显著,具有统计学意义(P<0.05)。表明江苏省规模化猪场不同地区、不同规模和不同阶段猪群普遍存在 PRRSV 感染,该研究结果为规模猪场PRRS的防控提供了参考。     

河南省部分猪群主要疫病疫苗免疫抗体水平监测与效果分析

2009年1月—2011年12月,从河南省信阳和驻马店地区62个猪场共收集免疫过猪瘟病毒(CSFV)、伪狂犬病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)和猪圆环病毒2型(PCV2)疫苗的猪血液,利用ELISA方法对样品进行抗体水平的检测。结果显示:该地区猪场猪群的疫苗免疫合格率最高是PRV疫苗(87.06%);其次是CSFV疫苗(76.24%);PRRSV和PCV2的疫苗免疫合格率较低,分别为65.20%和50.78%。4种疫苗免疫抗体合格率在不同规模的猪场有较大的差别,规模猪场的猪群某些疫病的抗体未必比散养猪群高。种猪群的4种疫苗的免疫合格率最高,商品猪中各个生长阶段的免疫后抗体合格率比较后发现,断奶仔猪群PRRSV抗体合格率明显高于哺乳仔猪,育肥猪群合格率要比哺乳仔猪群和断奶仔猪群的抗体合格率低。     

High genetic relatedness among Mycobacterium avium strains isolated from pigs and humans revealed by comparative IS1245 RFLP analysis

Members of the Mycobacterium avium complex cause pig mycobacteriosis and opportunistic human infections. Infections due to environmental mycobacteria are increasing in both industrial and developing countries. Mycobacterium-infected pig carcasses can pass for human consumption due to the poor specificity of meat control by visual detection at the slaughter houses. The genetic relatedness of porcine and human MAC isolates in Finland has been unknown. M. avium isolates isolated from pig organs (n=16) and clinical samples (n=13) were compared by IS1245 RFLP analysis to evaluate the similarity of the isolates obtained from human and porcine samples. Nearly identical multicopy M. avium subsp. hominissuis IS1245 RFLP fingerprints were obtained for isolates of porcine and human origin. IS1245 RFLP patterns of 38% of the porcine and human M. a. hominissuis isolates were >90% similar. The RFLP patterns of two porcine and two human isolates showed >95% similarity. The high similarity of the IS1245 RFLP patterns of the human and porcine M. a. hominissuis isolates indicates close genetic relatedness, suggesting that M. a. hominissuis is transmitted between pigs and humans, or that pigs and humans share common environmental sources of infection. Porcine and human isolates with RFLP patterns differing by only one or two bands were found, which shows that the same M. a. hominissuis strains may infect both humans and pigs.     

酒泉市猪圆环病毒2型和3型的流行病学监测和分析

为掌握酒泉市五个农业县猪圆环病毒2型（PCV2）和3型(PCV3)流行情况,在辖区内不同区域、不同养殖规模、不同生长阶段的19个规模化养殖场、38个散养户、4家屠宰场采集血清样品230份、粪便样品250份、猪肺脏、扁桃体、颌下淋巴结等组织样品220份,进行PCV2和PCV3核酸检测,检出阳性样品138份,阳性率为21.23%,PCV2的阳性率为15.54%,PCV3的阳性率为5.69%,表明PCV2的流行比PCV3更普遍、阳性感染率更高,但在不同区域、不同规模猪场的危害程度和流行情况存在一定的差异。规模养殖场的总体阳性感染率明显低于散养户,保育猪的感染率高于其他生长阶段的生猪。本次实验对酒泉市猪场PCV感染综合防控提供技术支撑,为科学养猪产生积极影响。     

Comparative serologic and virologic study of commercial swine herds with and without postweaning multisystemic wasting syndrome

A comparative serologic and virologic study was performed in pigs from 5 herds with postweaning multisystemic wasting syndrome (PMWS) and 2 herds without PMWS in Quebec. In each herd, 60 blood samples were collected at 4-wk intervals from pigs from 3 to 23 wk of age. The serum was evaluated for the presence of antibodies to porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), as well as for the presence of nucleic acid of PCV2, PRRSV, and porcine parvovirus (PPV), by means of the polymerase chain reaction (PCR). Serologic profiles for PCV2 were very similar in 6 of the 7 herds, including the 2 without PMWS, and were characterized by a gradual decrease in antibody titres from 3 until 11 wk of age, followed by seroconversion at 15 wk, and high PCV2 antibody titres thereafter in all pigs. Only starting at 11 to 15 wk of age could PCV2 viremia be detected, except in 1 herd, in which clinical signs were observed at 6 to 7 wk of age. A PCV2 viremia could be detected within the same pigs for a minimum of 8 wk, and the virus could still be detected in 41% of the serum samples obtained at 23 wk of age. The antibody level did not appear to influence the occurrence of disease, since titres were similar in pigs in the herds with or without PMWS. Infection with PRRSV, as demonstrated by PCR and seroconversion, preceded that of PCV2 by at least 1 mo in both types of herd. Both PRRSV and PCV2 were detected in some pigs in 5 of the 7 herds, including 1 herd without PMWS. Porcine parvovirus could be detected in serum by PCR in 2 herds with PMWS after the onset of clinical signs and also in 1 herd without PMWS. Genomic analysis of PCV2 strains identified in the herds without PMWS indicated complete or very high homology (99.4% to 100%) with the PCV2 strains identified in 4 herds with PMWS. In our field study, the triggering of PMWS in the herds could not be linked to coinfection with either PRRSV or PPV or to the use of a specific immunostimulant, such as vaccines, or to particular genomic differences between the PCV2 strains identified.     

Evaluation of the role of mallard ducks as vectors of porcine reproductive and respiratory syndrome virus

To assess the transmission of porcine reproductive and respiratory syndrome virus (PRRSV) from pigs to mallard ducks, 10 adult (one-year-old) female mallard ducks were housed with pigs infected experimentally with PRRSV, and allowed to be in close contact with them for 21 days. To evaluate the transmission of PRRSV from mallard ducks to pigs, two adult ducks were inoculated orally with PRRSV (total dose 10(6.0) TCID50) and allowed to drink PRRsv-infected water; 24 hours later, two four-week-old PRRsv-naive sentinel pigs were housed in pens below the cages housing the ducks for 14 days. In both experiments, cloacal and faecal samples were collected three times a week from the ducks and tested by PCR, virus isolation and a pig bioassay. Blood samples from the pigs were tested by ELISA, PCR and virus isolation. Sera from the ducks were tested by serum neutralisation. The ducks were examined postmortem and selected tissues were tested by PCR, virus isolation, histopathology and pig bioassay. In both experiments all the cloacal swabs, faecal samples, tissues and sera from the ducks were negative by all the tests. The sera from the pigs in the first experiment were PCR positive at three, seven, 14 and 21 days after infection and ELISA positive at 14 and 21 days. Sera from the pigs in the second experiment were negative by all the tests. The virus was isolated from the oral inoculum and the drinking water provided for the ducks in the second experiment. Under the conditions of this study, it was not possible to demonstrate the transmission of PRRSV either from the pigs to the ducks or from the ducks to the pigs.