Evaluation of the presence of porcine reproductive and respiratory syndrome virus in pig meat and experimental transmission following oral exposure

A study was performed to evaluate the presence of porcine reproductive and respiratory syndrome virus (PRRSV) in pig meat collected at slaughterhouses and its potential transmission to pigs via pig meat. A total of 1039 blood samples were collected from pigs upon their arrival at the abattoir. The following day, meat samples (n = 1027) were collected from the carcasses of these same pigs. Samples originated from 2 Canadian slaughterhouses, 1 situated in the province of Quebec and the other situated in the province of Manitoba. Serum samples were tested for antibodies to PRRSV and both serum and meat samples were also tested for PRRSV nucleic acid by polymerase chain reaction (PCR). Seropositivity to PRRSV for all serum samples was 74.3%. Furthermore 45 (4.3%) of the total serum samples and 19 (1.9%) of the 1027 meat samples were positive for PRRSV by PCR. Sequence analysis of open reading frame (ORF) 5 performed on 15 of the 19 PRRSV strains identified in pig meat indicated that 9 were field strains and 6 were vaccine-like (98% to 99.7% nucleotide homology with the Ingelvac RespPRRS/Repro vaccine). One of these 6 strains presented an intermediate 2-6-2 restriction fragment length polymorphism (RFLP) cut pattern and the others showed the characteristic 2-5-2 RFLP pattern of the vaccine strain. All strains sequenced were determined to be North American strains. In only 1 of the 19 PRRSV-positive meat samples could PRRSV be isolated. To test the potential infectivity of meat samples containing residual PRRSV, 11 of the PCR-positive meat samples (weighing 1.05 to 1.8 kg) were each used in feeding experiments of 2 PRRSV antibody-negative specific pathogen-free pigs of 9 wk of age. Samples were cut into several pieces and fed to each pair of pigs on 2 consecutive days. Each pig pair was housed in a separate cubicle and serum samples were collected at -7, 0, 7, 14, and 20 to 21 days post exposure. Seven pig pairs were found to be infected by PRRSV following ingestion of meat samples, including meat samples containing vaccine-like virus, as judged by the demonstration of PRRSV antibodies and/or PRRSV nucleic acid in the serum. In summary, the present study indicated that low residual quantities of PRRSV may be found in a small percentage of pig meat collected at slaugtherhouses. Furthermore, when this meat was fed raw to pigs in the experimental setting designed, pigs could be infected by PRRSV.     

Monitoring porcine reproductive and respiratory syndrome virus infection status in swine herds based on analysis of antibodies in meat juice samples

An indirect ELISA test was developed as a novel tool aimed at monitoring the herd infection status of swine herds. Meat juice samples from pig carcasses were analysed for the presence of antibodies against porcine reproductive and respiratory syndrome virus (PRRSV). A study of samples from herds with known PRRS status was undertaken. The PRRS status of the herds was evaluated based on the analysis of blood samples by another serological test (blocking ELISA) capable of differentiating between infection with PRRSV of the American type and European type. The specificity of the indirect ELISA test on meat juice samples was 0.98. The sensitivity of the test depended on the type of the PRRSV strain involved. The apparent prevalence in herds infected with the American type of PRRSV was 0.44. The apparent prevalence in herds infected with the European type of PRRSV was 0.64. Herd level sampling and herd level criteria for assessing the PRRS status of herds by the new test were developed. Herds were classified as PRRS negative or PRRS seropositive based on 10 meat juice samples collected randomly at slaughter throughout a 3-month-period. Herd PRRS status classification by the indirect ELISA was validated in 47 herds by collection of blood samples from the herds. Eighteen herds were classified as PRRS negative by both test systems. Twenty-nine herds were classified as PRRS seropositive by both test systems. Acceptable herd classification was achieved using this test.     

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。     

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。     

High risk of false positive results in a widely used diagnostic test for detection of the porcine reproductive and respiratory syndrome virus (PRRSV)

During 2003 and 2004, increasing numbers of positive PRRSV RT-PCR results were reported from herds negative for PRRSV infection. Interestingly, three herds represent nucleus herds with no animal contacts from outside and without clinical symptoms of PRRS until now. Since these positive results that were obtained using a PCR protocol adapted to routine laboratory conditions could not be reproduced with other PRRSV specific RT-PCRs, controlled negative and positive samples were used to examine this phenomenon. A RT-PCR assay for detection and differential diagnosis of the European and North American genotypes of the porcine reproductive and respiratory syndrome virus (PRRSV) according to the method previously published by Oleksiewicz et al. [Oleksiewicz, M.B., Botner, A., Madsen, K.G., Storgaard, T., 1998. Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes. Vet. Microbiol. 64, 7-22] was investigated in parallel to another recently published method [Pesch, S., 2003. Etablierung einer Nachweismethode für die zwei Genotypen von dem porcine reproductive and respiratory syndrome virus (PRRSV) und ein Beitrag zu seiner molekularen Epidemiologie. Thesis, Institute of Virology, Faculty of Veterinary Medicine, University of Leipzig]. A panel of 228 clinical samples sent in for PRRSV routine diagnostics served as test panel. It was found that both methods have similar analytical sensitivity. However, the primers published by Oleksiewicz were shown to yield a very high proportion of false positive results under routine diagnostic laboratory conditions, i.e. they resulted in RT-PCR products with non-PRRSV sequences, that were indistinguishable from truly positive reagents in standard gel electrophoresis settings. The reason for and possible implications of this finding as well as the risk of modifying published methods without control are discussed.     

Effect of formalin fixation on the immunohistochemical detection of PRRS virus antigen in experimentally and naturally infected pigs.

The purpose of this study was to determine the effect of formalin fixation on the immunohistochemical detection of porcine reproductive and respiratory syndrome (PRRS) viral antigen in lungs of experimentally and naturally infected pigs. In separate trials, five 24-day-old pigs and six 10-day-old pigs were housed as separate groups in isolation and inoculated intranasally with 10(5.5) TCID50 of an isolate of PRRS virus (PRRSV; P129). The older and younger pigs were euthanatized at 7 and 10 days post inoculation (dpi), respectively. At necropsy, all pigs had gross and microscopic lung lesions typical of PRRS, and PRRSV was isolated from all pigs. To insure uniform fixation, lungs from each pig were cut into 1-cm-thick slices and immersed into 10% neutral-buffered formalin. After fixation in formalin for 8 hours or 1, 2, 3, 5, 6, 8, 10, and 15 days, 3 lung sections from some or all pigs were processed for histological examination using routine methods. Immunohistochemical staining for PRRSV antigen was positive at the following times (days unless otherwise stated) after fixation (percentage of pigs staining positive for PRRSV in parentheses): 8 hours (100); 1 (100); 2 (100); 3 (80); 5 (33); and 6, 8, 10, and 15 (0-all negative). To further evaluate the effects of formalin fixation on PRRSV immunodetection, 31 field cases of PRRS were selected for immunohistochemistry (IHC). Over a 3-month period, submitted cases were selected from the Purdue University Animal Disease Diagnostic Laboratory, W. Lafayette, Indiana, for IHC if 1) the clinical history included respiratory disease, 2) PRRSV was isolated from lung and/or serum from the submitted pigs or tissues, 3) at least 1 section of lung fixed in 10% neutral-buffered formalin was submitted for IHC, and 4) the duration of fixation could be accurately determined from the case history. Of the 31 PRRSV-infected pig cases meeting the selection criteria, 23 were fixed in formalin for 4 days or less. Twenty-one of these 23 (91%) were positive by IHC. Two of 8 cases fixed for greater than 4 days (25%) were positive by IHC. In practical terms, 1-day shipping of fixed samples to a laboratory followed by routine tissue processing within a laboratory should not adversely affect immunohistochemical detection of PRRS viral antigen. But a delay in shipping or processing of more than 2 days could reduce or prevent the detection of PRRS viral antigen by IHC.     

Oral transmission of porcine reproductive and respiratory syndrome virus by muscle of experimentally infected pigs

The current study was performed to determine if porcine reproductive and respiratory syndrome virus (PRRSV) could be transmitted to pigs by feeding muscle tissue obtained from recently infected pigs. Muscle obtained from pigs infected with either a European strain (EU donor pigs) or American strain (US donor pigs) of PRRSV was fed to PRRSV-free receiver pigs. The donor pigs were slaughtered 11 days post-infection (dpi). PRRSV was detected by conventional virus isolation in muscle at 11 dpi from 7 of 12 EU donor pigs and 5 of 12 US donor pigs. In contrast to conventional virus isolation, all muscle samples from infected pigs were positive for viral nucleic acid by PCR, except for muscle from one animal infected with the American strain of PRRSV. Five hundred grams of raw semimembranosus muscle from each of the donor pigs was fed over a 2 days period (250 g per day) to each of two receiver pigs (48 receiver pigs). The receiver pigs were housed separately in five groups. One of the five groups was fed muscle obtained from US donor pigs that was also spiked with the American strain of PRRSV. Sentinel pigs were placed in-contact with the group of receiver pigs fed spiked muscle. All receiver pigs became viraemic by 6 days post-feeding (dpf). There was evidence of horizontal transmission with sentinel pigs, in-contact with receiver pigs, becoming viraemic. The study demonstrates that PRRSV could be infectious through the oral route via the feeding of meat obtained from recently infected pigs.     

Immunohistochemical detection of porcine reproductive and respiratory syndrome virus using colloidal gold.

Two cytopathic agents were isolated on porcine alveolar macrophages following inoculation with homogenates of lung tissues from pigs showing respiratory problems. These isolates were identified as porcine reproductive and respiratory syndrome (PRRS) virus isolates by indirect immunofluorescence using a PRRS virus (PRRSV) specific monoclonal antibody (MAb) and were designated as LHVA-92-1 and LHVA-92-2. Immunogold electron microscopy using a porcine PRRS positive serum pool and protein A-gold resulted in an intense labelling of aggregates of viral particles. Dark specific cytoplasmic staining of porcine alveolar macrophages infected with both virus isolates could be observed by immunogold silver staining (IGSS) using the specific MAb. This method proved effective in detecting PRRSV antigens in several ethanol-fixed tissues of piglets intranasally inoculated with the supernatants of macrophages infected with each isolate. Immunogold silver staining was also successfully used for the detection of PRRSV antigens on sections of formalin-fixed paraffin-embedded lung tissues and on frozen sections of lungs. The present results indicate that colloidal gold may be useful for the identification and immunohistochemical detection of PRRSV in tissues.     

Studies on the carriage and transmission of porcine reproductive and respiratory syndrome virus by individual houseflies (Musca domestica)

The objectives of the study were to determine the site of porcine reproductive and respiratory syndrome virus (PRRSV) in individual houseflies, to assess whether an individual housefly could transmit PRRSV to a susceptible pig, and to compare the ability of PCR, virus isolation and a pig bioassay to detect PRRSV in houseflies. In the first experiment 26 houseflies were fed on a pig infected experimentally with PRRSV; 13 were processed as a whole fly homogenate, while an exterior surface wash and a gut homogenate were collected from the other 13. Infectious PRRSV was recovered from nine of the whole fly homogenates, 12 of the gut homogenates and one of the exterior surface washes. In the second experiment, two of 10 individual houseflies, which had fed on an infected pig, transmitted PRRSV to a susceptible pig in a controlled manual transmission protocol. In the third experiment, single flies or pools of 30 flies were immersed in different concentrations of a PRRSV inoculum, then tested by PCR, virus isolation and bioassay. The virus was detected at a concentration of 10(1) TCID50/ml by PCR, 10(2) TCID50/ml by the bioassay and 10(3) TCID50/ml by virus isolation.     

Increase of CD163 but not sialoadhesin on cultured peripheral blood monocytes is coordinated with enhanced susceptibility to porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted tropism mainly for porcine alveolar macrophages (PAMs), but not for peripheral blood monocytes (BMo) in vivo. Previous research showed that only a few BMo became susceptible to PRRSV infection after 1 day culture. Porcine sialoadhesin (PoSn) and CD163 are identified to be the two main PRRSV receptors for binding and internalization. Both receptors are not expressed on BMo, or only expressed at low levels, which may explain why PRRSV cannot infect them. The relationship of BMo differentiation/aging, PRRSV receptor level, and susceptibility to PRRS virus infection has not been thoroughly investigated. In this study, BMo were successfully cultured with pig serum plus L929 cell culture supernatant. Our results showed that both the mRNA and protein expression levels of PoSn were significantly increased after 5-day culture. The mRNA level of CD163 was enhanced more than 20-fold after 1-day culture; CD163-positive BMo increased dramatically from about 2% after 2h- culture to about 50% after 96-h culture. Furthermore, cultured BMo became much more permissive to PRRSV infection, and the percentage of PRRSV-infected BMo was at least the same as PAMs, if not higher, when infected with CH-1a, the first PRRSV strain isolated in China, or HV, a highly virulent strain. Three other PRRSV strains including VR2332, and two classical Chinese isolates could also infect cultured BMo as well. Most importantly, PRRS virus was successfully isolated from 14 of 15 antibody-positive serum samples using cultured BMo. These results suggest that the enhanced susceptibility of cultured BMo to PRRS virus is coordinated with increased CD163 expression, but less related to the delayed (day 5) increased expression of PoSn. Thus, cultured BMo could be an alternative choice for PRRS virus isolation and identification.     

猪CD163游离受体的体内表达与抗猪繁殖与呼吸综合征病毒感染效果的研究

为了验证构建的重组腺病毒rAd-SRCR59-Fc能否在猪体内有效表达游离受体及其能否抑制PRRSV感染,将重组腺病毒注射断奶仔猪,用夹心ELISA检测SRCR59-Fc表达情况;选择PRRSV易感仔猪肌肉注射重组腺病毒rAd-SRCR59-Fc(4×109 TCID50/头),5 d后与JXA1株PRRSV人工感染猪同居饲养,定期测定体温、观察临床症状,采集血样和粪样进行PRRSV定量检测,对病死猪进行病理剖检和主要器官病毒载量测定。结果显示,重组腺病毒接种猪能表达SRCR59-Fc游离受体蛋白,并持续15 d左右;与感染对照组相比,rAd-SRCR59-Fc注射组首次高热时间和临床症状均推迟3 d,第5天~第13天临床症状评分显著低于感染组(P<0.01),至试验结束(25 d)仅有1头猪死亡;肺脏无明显肉眼病变,仅见少量坏死灶,病理切片显示肺泡壁轻微增厚;第3天~第13天病毒血症水平显著低于感染组(P<0.01),第5~第13天粪排毒量低于感染组(P<0.05),器官病毒载量均显著低于感染组(P<0.01)。研究表明CD163游离受体的重组腺病毒能有效阻断PRRSV毒株感染,可以作为PRRS预防或治疗性疫苗进一步研究。     

华东某猪场猪繁殖与呼吸综合征的诊断与防制

华东区某猪场暴发以妊娠母猪繁殖障碍为特征的传染病。在该场采集可疑病例血样 62份进行检测 ,其中 53份血清为猪繁殖与呼吸综合征(PRRS)阳性 ,阳性率高达 85 4 %。结合病毒分离鉴定确诊为PRRS病毒 (PRRSV)感染 ,RT PCR试验证实分离毒属美洲型PRRSV ,且与ATCC VR2 332株属同一基因亚型。确诊后采取隔离、消毒及采用当地病猪肺组织灭活苗紧急接种等措施 ,取得了较满意的效果。跟踪血清学调查显示 ,1年后该场抽检血样PRRS阳性率仍达 80 %     

猪繁殖与呼吸综合征病毒和牛病毒性腹泻病毒双重RT-PCR检测方法的建立

根据牛病毒性腹泻病毒(BVDV)5'端非编码区基因序列,设计合成了1对特异性引物,参考本实验室针对猪繁殖与呼吸综合征病毒(PRRSV)N蛋白设计的引物,经过PCR反应条件的优化,建立了BVDV和PRRSV双重RT-PCR的检测方法。对于PRRSV和BVDV的cDNA最低检测量分别为3.8×10-4 ng和7×10-4 ng,对于猪瘟病毒(CFSV)、脑心肌炎病毒(EMCV)和猪圆环病毒2型(PCV-2)的PCR扩增结果均为阴性;用该方法对江苏省不同地区采集的75份仔猪的肺脏、脾脏和淋巴结等病料进行了检测,结果PRRSV有55份阳性,BVDV有14份阳性,PRRSV和BVDV混合感染的有12份,与PRRSV和BVDV单一RT-PCR的检测结果符合率分别为89.3%和92%。证明建立的双重RT-PCR检测方法可用于临床样品中BVDV和PRRSV的检测。     

应用RT-PCR检测猪粪便中PRRSV

通过逆转录-聚合酶链反应(RT-PCR),设计特异性引物检测猪粪便中猪繁殖与呼吸综合征病毒ORF7基因片段.结果发现,所检测的8份猪粪便样本中有6份扩增出了目的条带,判定为阳性.