An assessment of sanitation protocols for commercial transport vehicles contaminated with porcine reproductive and respiratory syndrome virus

The objective of this study was to develop and test a rapid (< 2 h) sanitation protocol designed for porcine reproductive and respiratory syndrome virus (PRRSV) positive commercial transport vehicles involving cold water washing and disinfection via fumigation using scale models of weaned pig trailers. The study consisted of 2 phases. Following experimental contamination of model trailers with PRRSV MN 30-100 (5 x 10(5)TCID50), phase 1 evaluated the presence or absence of PRRSV RNA by polymerase chain reaction (PCR) on swabs collected from the trailer interiors 0, 60, and 90 min after treatment. Phase 2 consisted of evaluating the infectivity of trailers 90 min posttreatment by monitoring changes in the PRRSV-status of naive sentinel pigs housed for 2 h. Treatments included washing only (treatment 1), washing plus formaldehyde fumigation (treatment 2), washing plus fumigation with glutaraldehyde-quaternary ammonium chloride (treatment 3), and washing plus overnight drying (treatment 4). Porcine reproductive and respiratory syndrome virus RNA was detected in all trailers (20 out of 20 replicates) at 60 and 90 min following the application of treatments 1 and 2. These trailers also contained infectious PRRSV, as determined by the infection of naive pigs housed in treated trailers and the testing of organic debris collected from the interior of trailers by swine bioassay. At 90 min posttreatment, all trailers treated with glutaraldehyde-quaternary ammonium chloride were PCR-negative, non-infectious to sentinel pigs, and swine bioassay negative. Similar results were observed in trailers allowed to dry for 8 h. Under the conditions of this study, it appears certain disinfectants may possess different levels of efficacy against PRRSV and PRRSV-positive models may be effectively sanitized in the absence of overnight drying.     

An evaluation of disinfectants for the sanitation of porcine reproductive and respiratory syndrome virus-contaminated transport vehicles at cold temperatures

The objective of this study was to evaluate the efficacy of commercially available disinfectants to sanitize porcine reproductive and respiratory syndrome virus (PRRSV) contaminated trailer models in cold climates (-20 degrees C and 4 degrees C). Disinfectants evaluated included Synergize, Aseptol 2000, Biophene, Sentramax, Virkon, Tek Trol, and DC&R. All products were applied to trailers via fumigation at 4 degrees C. Following experimental contamination of model trailers with PRRSV MN 30-100 (5 x 10(5) TCID50), models were tested for the presence or absence of PRRSV-RNA by polymerase chain reaction (PCR) on swabs collected 0, 30, and 60 min after treatment. Treatments included washing only, washing plus disinfectant fumigation, washing plus fumigation, and washing plus overnight drying. The PRRSV-RNA detected across trailers ranged from 0/12 replicates in trailers treated with Synergize or allowed to dry for 8 h. These trailers were also negative for the presence of infectious PRRSV, based on the lack of sentinel pig infection (0/4 replicates). In contrast, the detection of PRRSV-positive swabs by PCR ranged from 3/12 (Aseptol) to 10/12 (Biophene). Based on these results, the efficacy of Synergize was evaluated at -20 degrees C. In an attempt to reduce the impact of freezing on disinfectant activity, 30 mL of disinfectant was added to a 3840 mL of a 40% methanol solution, a 10% propylene glycol (PG) solution, or water alone. The PRRSV-contaminated trailers were treated with 1 of 3 disinfectant mixtures via fumigation, stored for 8 h at -20 degrees C, allowed to thaw, and sampled as described. Trailers treated with 40% methanol or 10% PG did not freeze and were negative for PRRSV-RNA and infectious virus following thawing. In contrast, trailers treated with disinfectant and water were frozen within 60 min at -20 degrees C, and decontamination was not successful.     

An evaluation of test and removal for the elimination of porcine reproductive and respiratory syndrome virus from 5 swine farms.

The objective of this field study was to evaluate the protocol of test and removal (T&R) for the elimination of porcine reproductive and respiratory syndrome virus (PRRSV) from 5 chronically infected breeding herds. The T&R protocol involved sampling the entire breeding herd in one day, testing sera by polymerase chain reaction and ELISA to detect previously exposed and/or infected animals, and subsequently removing them from the herd. Following completion of T&R, breeding herds were monitored for 12 consecutive months, using ELISA, for the presence of antibodies to PRRSV. In order to be classified as a PRRSV-negative herd, all samples collected over the 12-month monitoring period were required to be negative by ELISA (s/p ratio < 0.4). At the conclusion of the monitoring period, all 5 farms were PRRSV-negative, according to the defined testing criteria. Approximately 2.2% (74/3408) ELISA false positive samples were detected across all 5 farms during the monitoring period. The diagnostic cost required during the T&R protocol was approximately US $10.66 per animal tested. Limitations of the study were a lack of herds with large (> 2000 sows) breeding herd inventories, and herds with a history of PRRSV vaccination.     

Further assessment of fomites and personnel as vehicles for the mechanical transport and transmission of porcine reproductive and respiratory syndrome virus

This study re-evaluated the role of fomites and personnel in the mechanical transport and transmission of porcine reproductive and respiratory syndrome virus (PRRSV) between pig populations. Swabs were collected from hands, boots, coveralls, and other fomites following contact with infected pigs and compared with identical samples collected in the absence of PRRSV exposure. Naïve pigs were provided contact with contaminated fomites/personnel and blood tested periodically post-exposure [positive exposure population (PEP)] and compared with populations that did not gain exposure via these routes [negative exposure population (NEP)]. The majority of swab samples from hands, coveralls, and boots from personnel and fomite samples (cable snare and bleeding equipment) following contact with the PRRSV-infected Source Population. Transmission of PRRSV to the PEP was observed (7/7) cases but not in the NEP. In conclusion, under the proper conditions, transport and transmission of PRRSV by fomites and personnel may occur between swine populations in the absence of intervention.     

An experimental model to evaluate the role of transport vehicles as a source of transmission of porcine reproductive and respiratory syndrome virus to susceptible pigs

The objectives of this study were to determine the concentration of porcine reproductive and respiratory syndrome virus (PRRSV) in a scale-model trailer that was required to infect susceptible pigs, evaluate the potential of PRRSV-contaminated transport vehicles to infect na?ve pigs and assess 4 sanitation programs for the prevention of virus spread. To maximize study power, scale models (1:150) of weaned-pig trailers were constructed that provided an animal density equal to that of an actual weaned-pig trailer capable of transporting 300 pigs. The 1st aim involved contaminating the interior of the model trailers with various concentrations (10(1) to 10(4) TCID50/mL) of PRRSV MN 30-100, then housing sentinel pigs in the trailers for 2 h. Pigs exposed to trailers contaminated with > or = 10(3) TCID50/mL became infected. The 2nd aim involved housing experimentally infected seeder pigs in trailers for 2 h, then directly introducing sentinel pigs for 2 h. Infection of sentinels was demonstrated in 3 of 4 replicates. The 3rd aim involved applying 1 of 4 sanitation procedures (treatments) to contaminated trailers. Treatment 1 consisted of manual scraping of the interior to remove soiled bedding (wood chips). Treatment 2 consisted of bedding removal, washing (80 degrees C, 20,500 kPa), and disinfecting (with 1:256 phenol; 10-min contact time). Treatment 3 consisted of treatment 2, followed by freezing and thawing. Treatment 4 consisted of bedding removal, washing, disinfecting, and drying. Ten replicates were conducted per treatment. Pretreatment swabs from all trailers tested positive by polymerase chain reaction (PCR). Post-treatment swabs were PCR-positive for all trailers except those that were washed, disinfected, and dried. Infection of sentinel pigs by PRRSV was also detected by PCR after all treatments except washing, disinfecting, and drying. Under the conditions of this study, drying appeared to be an important component of a sanitation program for ensuring PRRSV biosecurity of transport vehicles.     

猪繁殖与呼吸综合征病毒的研究进展

猪繁殖与呼吸综合征 (Porcine reproductiveand respiratory syndrome,PRRS)是 1 987年发现的一种新的猪病 ,它主要是引起母猪的繁殖障碍 ,如流产、早产、死胎、木乃伊胎和呼吸道症状的一种由猪繁殖与呼吸综合征病毒 (PRRSV)引起的接触性传染病。此病最初在美国首先被发现 ,随后相继在世界各国报道 ,我国台湾省也有疫情的报道。 1 995年我国首次从进口猪中检出 PRRS阳性猪 ,并从中分离到 PRRSV。 1 996年郭宝清等首次从国内 PRRS血清阳性猪群中分离到 PRRSV,从而证实了我国也有此病的流行[1] 。由于 PRRS主要是引起母猪的繁殖…     

猪繁殖与呼吸综合征病毒的分离与鉴定

从广东地区发病猪场的病料中,分离到1株致Marc-145细胞病变的病毒ShB6。扩增猪繁殖与呼吸综合征病毒(PRRSV)主要结构蛋白基因ORF2-ORF7并进行序列分析,结果表明,该分离株与国内PRRSV分离株HB-1(sh)/2002的同源性为96.9%;与ATCC VR-2332株的同源性为91%;而与Lelystad株的同源性仅为59.8%;用美洲型PRRSV单抗进行免疫组化染色,结果显示在细胞病变处呈现明显的阳性着色(为棕黄色)。综合可见,所分离的病毒为美洲型PRRSV。     

猪繁殖与呼吸综合征病毒分子生物学研究进展

猪繁殖与呼吸综合征病毒(PRRSV)是引起猪繁殖与呼吸综合征的病原体,本文对PRRSV的分子生物学研究进展进行了综述,主要包括PRRSV的基因组结构、病毒蛋白及其功能、抗原变异等,旨在为诊断技术、免疫机理研究、疫苗设计与疫病防制提供借鉴.     

高致病性猪繁殖与呼吸综合征病毒(PRRSV)气溶胶的发生与传播机制

为深入认识猪繁殖与呼吸综合征病毒(PRRSV)气溶胶的发生与传播机制,将35日龄无PRRSV抗体仔猪预饲1周后分为4组(PRSSV攻毒组、直接接触组、间接接触组和阴性对照组),饲养于正负压隔离器中。2个隔离器置于不同室内并通过管道相连,采用AGI-30收集器收集其空气样品。然后,将其接种Marc-145细胞,再用RT-PCR检测病毒;并检测血常规和抗体变化。结果发现,攻毒组于攻毒后4d开始形成气溶胶,并持续到试验结束。气溶胶高峰出现在攻毒后15d。气溶胶传播的间接接触组从临床症状、病理变化、提取病理组织核酸和抗体检测等多个方面都证明其感染了PRRSV,并经鼻腔向外排毒;直接接触组和间接接触组几乎同时感染PRRSV。试验表明,PRRSV不仅能形成气溶胶并且能够通过其迅速感染临近猪群。     

Survival of porcine reproductive and respiratory syndrome virus in houseflies

The objectives of the study were to determine the duration of porcine reproductive and respiratory syndrome virus (PRRSV) survival in houseflies (Musca domestica Linnaeus) following feeding on an infected pig, and to determine whether the virus was present on the exterior surface or within the internal viscera of the fly. A total of 210 laboratory-colonized houseflies were allowed to feed to repletion on a pig, experimentally infected with PRRSV on day 7 postinoculation, and then maintained alive under laboratory conditions (27°C). Two subsets (A and B) of 30 flies were collected at each of the following sampling points; 0, 6, and 12 hours post feeding (pf). Subset A contained an extra group of 30 flies collected at 24 hours pf due to the availability of extra flies. Flies in subset A were processed as whole fly homogenates, while the exterior surface washes and digestive organs were collected from flies in subset B. Whole fly homogenates, collected at 0, 6, and 12 hours pf, were positive by both polymerase chain reaction (PCR) and swine bioassay. Digestive organs, collected at 0 and 12 hours pf, were positive by PCR and swine bioassay. The PRRSV RNA was detected by PCR from the exterior surface wash of subset B flies collected at 0, 6, and 12 hours pf; however, only the subset collected at 0 hour pf was swine bioassay-positive. This study indicates that infectious PRRSV can survive within the intestinal tract of houseflies for up to 12 hours following feeding on an infected pig, but only for a short period on the exterior surface of the flies.     

Molecular evolution of porcine reproductive and respiratory syndrome virus isolates from central China

To investigate the genetic diversity of prevailing porcine reproductive and respiratory syndrome virus (PRRSV) in Henan Province of China, 61 ORF5 gene sequences, originating from Henan Province during 2003–2010, were subjected to amino acid variation and phylogenetic analysis. The analyzed PRRSV ORF5 sequences carried evidence of one unique recombination event. Phylogenetic analysis revealed that all Henan isolates belonged to type 2 genotype and were divided into two subgroups. The dominant isolates had shifted from subgroup 1 to subgroup 2 during 2003–2010. Amino acid variation analysis of the glycoprotein 5 revealed that Henan PRRSV strains tended to accumulate more substitutions within the N-terminus and hypervariable region. Selective pressure analysis revealed evidence that some ORF5 sites have likely evolved in response to immune pressure.     

Construction and evaluation of genetically engineered replication-defective porcine reproductive and respiratory syndrome virus vaccine candidates

Porcine reproductive and respiratory syndrome virus (PRRSV) is an emerging pathogen causing significant economic losses in the swine industry worldwide. Two novel gene-deleted viruses were constructed and evaluated as vaccine candidates. Using the full-length infectious cDNA clone of North American PRRS isolate P129, the ORF2 and ORF4 genes (which encoded minor structural glycoproteins GP2a/2b and GP4, respectively) were individually deleted from the viral genome. Both deletion mutants were non-viable in MARC-145 cells and porcine alveolar macrophages, indicating that both genes are essential for virus replication. To rescue the replication-defective PRRSV, two complementing cell lines, MARC-2000 and MARC-400, were established to stably express the PRRSV GP2 and GP4 proteins, respectively. These cells were able to complement the deleted gene function of PRRSV in trans and supported production of the replication-defective DeltaORF2-PRRSV and DeltaORF4-PRRSV viruses. Both DeltaORF2-PRRSV and DeltaORF4-PRRSV viruses were propagated for 40-50 generations in the corresponding complementing cells and remained replication-defective in MARC-145 cells. To examine the immunogenic potential of the replication-defective PRRSV as vaccine candidates, four groups of pigs, 20 pigs per group, were immunized twice with DeltaORF2-PRRSV or DeltaORF4-PRRSV and challenged with the homologous virulent virus at 3 weeks post-immunization. In spite of the fact one group showed significant reduction in virus load, we could not demonstrate improvement from clinical diseases in this vaccination/challenge study. However, we did show that the cDNA clone of PRRSV can be a useful tool to genetically engineer PRRSV vaccine candidates and to study pathogenesis and viral gene functions.     

猪繁殖与呼吸道综合征病毒RT-PCR检测方法研究

为建立一种能够快速、确切诊断猪繁殖与呼吸综合征(PRRS)的方法,根据GenBank上已发表的猪繁殖与呼吸综合征病毒(PRRSV)核苷酸序列设计并合成了一对能特异性扩增PRRSV基因片段的引物,经过条件优化后,建立了检测PRRSV的RT-PCR方法,扩增病毒核酸片段长432bp.试验证明,该方法具有良好的特异性、敏感性、重复性和稳定性,简便、快速、高效,为PRRSV的快速准确诊断及进一步的PRRSV的流行病学研究提供了重要的技术手段.     

乳猪"高热症"猪繁殖与呼吸综合征病毒的分离与致病性分析

本研究针对疑似高热症疫情的猪场,从分离病原的致病特性及基因特征,对高热症的病因进行了探索。以RT—PCR／PCR从临床病料中检测出猪繁殖与呼吸综合征病毒（PRRSV）、猪圆环病毒2型（PCV2）、猪瘟病毒（CSFV）核酸,利用Marc-145细胞从病料中分离到PRRSV（XS070425毒株）。测序结果显示,XS070425毒株Nsp2基因无高致病性PRRSV Nsp2基因533～561位连续29个氨基酸“RPVTPLSEPIPVPAPRRKFQQVKRLSSAA”的缺失;但遗传进化分析显示,XS070425毒株Nsp2、ORF5基因与近期我国报道的高致病性PRRSV-HuN4和JXA1有较高的同源性,达95％～96％。致病性试验显示,PRRSV-XS070425原始病料悬液和Marc-145细胞分离物接种健康猪后,病毒血症可持续26d,并出现典型的PRRS临床症状和体温升高,但不致死感染猪。这些结果说明,无NSP2蛋白29个氨基酸残基缺失的PRRSV也是猪高热症的重要病原。     

Laboratory model to evaluate the role of aerosols in the transport of porcine reproductive and respiratory syndrome virus

The aim of this study was to develop a model to evaluate the aerosol transmission of porcine reproductive and respiratory disease virus (PRRSV). PRRSV (MN 30-100 strain, total dose 3 x 10(6) virus particles) was aerosolised and transported up to 150 m and a portable air sampler was used to collect air samples at 1, 30, 60, 90, 120 and 150 m (five replicates at each distance) and the air samples were tested by TaqMan PCR and virus isolation. The infectivity of the aerosolised PRRSV was tested by exposing six PRRSV-naive pigs for three hours to aerosolised virus that had been transported 150 m. PRRSV RNA was detected in all five replicate air samples collected at 1, 30, 60 and 90 m, in four of the five collected at 120 m, and in three of the five collected at 150 m. Infectious PRRSV was detected by virus isolation at 1 and 30 m (all five replicates), 60, 90 and 120 m (three of the five) and 150 m (two of the five). There was a 50 per cent reduction in the log concentration of PRRSV RNA every 33 m. Three of the six pigs exposed to PRRSV-positive aerosols became infected, and PRRSV RNA was detected in air samples and on swab samples collected from the interior of the chambers that housed the infected pigs while they were being exposed.     

高致病性猪蓝耳病病毒吉林株的分离鉴定及生物学特性

从吉林某猪场采集曾接种过猪繁殖与呼吸综合征疫苗的发病猪病料,经RT-PCR初步鉴定为猪繁殖与呼吸综合征病毒后,利用反转录合成cDNA,用针对PRRSV M、N基因片段设计的特异性引物进行扩增及电泳,在紫外凝胶成像系统下可见约为660bp特异性扩增条带。解剖发病仔猪,发现其有HP-PRRSV发病的特征,两耳及鼻端淤血,呈蓝紫色,肺部等内脏器官淤血呈暗红色。病理组织切片显示,病猪有典型的间质性肺炎的特征性病理变化。将其接种于Marc-145细胞后,在培养至第4代时出现典型的细胞病变（CPE）,表现为细胞聚集成丛、随后固缩、变圆、脱落;经Reed-Muench法计算得出两PRRSV分离株的病毒滴度分别为10-5.30 TCID50/0.1mL和10-5.53TCID50/0.1mL。用间接免疫荧光试验观察到在接种病料的Marc-145细胞胞浆内出现特异性荧光,而未接种PRRSV的细胞对照则未见到荧光反应。     

Retention of ingested porcine reproductive and respiratory syndrome virus in houseflies

OBJECTIVE: To evaluate retention of porcine reproductive and respiratory syndrome virus (PRRSV) in houseflies for various time frames and temperatures. SAMPLE POPULATION: Fifteen 2-week-old pigs, two 10-week-old pigs, and laboratory-cultivated houseflies. PROCEDURE: In an initial experiment, houseflies were exposed to PRRSV; housed at 15 degrees, 20 degrees, 25 degrees, and 30 degrees C; and tested at various time points. In a second experiment to determine dynamics of virus retention, houseflies were exposed to PRRSV and housed under controlled field conditions for 48 hours. Changes in the percentage of PRRSV-positive flies and virus load per fly were assessed over time, and detection of infective virus at 48 hours after exposure was measured. Finally, in a third experiment, virus loads were measured in houseflies allowed to feed on blood, oropharyngeal washings, and nasal washings obtained from experimentally infected pigs. RESULTS: In experiment 1, PRRSV retention in houseflies was proportional to temperature. In the second experiment, the percentage of PRRSV-positive houseflies and virus load per fly decreased over time; however, infective PRRSV was found in houseflies 48 hours after exposure. In experiment 3, PRRSV was detected in houseflies allowed to feed on all 3 porcine body fluids. CONCLUSIONS AND CLINICAL RELEVANCE: For the conditions of this study, houseflies did not support PRRSV replication. Therefore, retention of PRRSV in houseflies appears to be a function of initial virus load after ingestion and environmental temperature. These factors may impact the risk of insect-borne spread of PRRSV among farms.