Mystery swine disease in The Netherlands: the isolation of Lelystad virus.

In early 1991, the Dutch pig-industry was struck by the so-called mystery swine disease. Large-scale laboratory investigations were undertaken to search for the etiological agent. We focused on isolating viruses and mycoplasmas, and we tested paired sera of affected sows for antibodies against ten known pig viruses. The mycoplasmas M. hyosynoviae, M. hyopneumoniae, and Acholeplasma laidlawii, and the viruses encephalomyocarditis virus and porcine enterovirus types 2 and 7 were isolated from individual pigs. An unknown agent, however, was isolated from 16 of 20 piglets and from 41 of 63 sows. This agent was characterised as a virus and designated Lelystad virus. No relationship between this virus and other viruses has yet been established. Of 165 sows reportedly afflicted by the disease, 123 (75 per cent) seroconverted to Lelystad virus, whereas less than 10 per cent seroconverted to any of the other virus isolates or to the known viral pathogens. Antibodies directed against Lelystad virus were also found in pigs with mystery swine disease in England, Germany, and in the United States. We conclude that infection with Lelystad virus is the likely cause of mystery swine disease.     

Experimental reproduction of porcine epidemic abortion and respiratory syndrome (mystery swine disease) by infection with Lelystad virus: Koch's postulates fulfilled

Aerosol exposure of eight pregnant sows to cell-culture- propagated Lelystad virus resulted in clinical signs characteristic of so-called mystery swine disease. After an incubation of 4-7 days, all sows were inappetant and listless for 6-9 days. Two sows developed a transient red-blue discolouration of the ears ('abortus blauw' or blue ear disease) accompanied by abdominal respiration, and two had a fever for one day only. One sow aborted at 109 days of gestation. The other seven sows, farrowing between 113 and 117 days of gestation, gave birth to numerous mummified, dead, and weak piglets. Of these seven, the mean number of piglets born dead to each sow was 4.6 and the mean number born alive was 7.7; 3.1 piglets per sow (40%) died within the first week. Lelystad virus was isolated from 31 piglets, which were born dead or died shortly after birth. Antibody was detected in precolostral blood samples or ascitic fluids of 23 piglets, a finding which demonstrated transplacental passage of the virus in six out of eight litters. We conclude that Lelystad virus is the causal agent of mystery swine disease. Since its aetiology is no longer a mystery, we propose the more appropriate name 'porcine epidemic abortion and respiratory syndrome (PEARS)'.     

Highly cited article published in the Veterinary Quarterly in 1991

In early 1991, the Dutch pig industry was struck by the so-called mystery swine disease. Large-scale laboratory investigations were undertaken to search for the aetiological agent. We focused on isolating viruses and mycoplasmas, and we tested paired sera of affected sows for antibodies against ten known pig viruses. The mycoplasmas M. hysonoviae, M. hyopneumoniae, and Acheloplasma laidlawii, and the viruses encephalomyocarditis virus and porcine enterovirus types 2 and 7 were isolated from individual pigs. An unknown agent however, was isolated from 16 of 20 piglets and from 41 of 63 sows. This agent was characterized as a virus and designated Lelystad virus. No relationship between this virus and other viruses has yet been established. Of 165 sows reportedly affected by the disease, 123 (75 per cent) seroconverted to Lelystad virus, whereas less than 10 per cent seroconverted to any of the other virus isolates or to known viral pathogens. Antibodies directed against Lelystad virus were also found in pigs with mystery swine disease in England, Germany, and the United States. We conclude that infection with Lelystad virus is the likely cause of mystery swine disease.     

Highly cited article published in the veterinary quarterly in 1991

Summary

In early 1991, the Dutch pig industry was struck by the so‐called mystery swine disease. Large‐scale laboratory investigations were undertaken to search for the aetiological agent. We focused on isolating viruses and mycoplasmas, and we tested paired sera of affected sows for antibodies against ten known pig viruses. The mycoplasmas M. hysonoviae, M. hyopneumoniae, and Acheloplasma laidlawii, and the viruses encephalomyocarditis virus and porcine enterovirus types 2 and 7 were isolated from individual pigs. An unknown agent however, was isolated from 16 of 20 piglets and from 41 of 63 sows. This agent was characterized as a virus and designated Lelystad virus. No relationship between this virus and other viruses has yet been established. Of 165 sows reportedly affected by the disease, 123 (75 per cent) seroconverted to Lelystad virus, whereas less than 10 per cent seroconverted to any of the other virus isolates or to known viral pathogens. Antibodies directed against Lelystad virus were also found in pigs with mystery swine disease in England, Germany, and the United States. We conclude that infection with Lelystad virus is the likely cause of mystery swine disease.     

Lelystad virus, the cause of porcine epidemic abortion and respiratory syndrome: a review of mystery swine disease research at Lelystad.

This paper reviews the laboratory investigations that led us to isolate the Lelystad virus and demonstrate that this virus causes mystery swine disease. We describe: 1) isolating the virus from the disease; 2) characterizing the virus as a new enveloped RNA virus; 3) reproducing the disease experimentally with the isolated Lelystad virus; 4) isolating the virus from the experimentally induced disease.     

规模化猪场猪圆环病毒2型感染的实验室诊断

为了确诊贵州省某规模化猪场保育仔猪异常死亡原因,从怀孕母猪、产房母猪、后备母猪、种公猪、哺乳仔猪、保育仔猪6个猪群采集90份血清样本采用ELISA方法分别进行血清抗体检测,并对采集的90份血清样本和1份病死猪淋巴结组织采用荧光PCR方法进行病原学检测。结果:6个猪群综合的猪瘟病毒、猪蓝耳病病毒、伪狂犬病病毒g B蛋白、伪狂犬病病毒g E蛋白血清抗体阳性率分别为87.78%、70.00%、88.89%、4.44%;猪瘟病毒、猪蓝耳病病毒、伪狂犬病病毒病原核酸检测显示阴性,猪圆环病毒2型病原核酸检测淋巴结组织样本显示阳性。试验结果表明,引起该猪场保育仔猪死亡的原因为猪圆环病毒2型感染。同时,怀孕母猪群出现了伪狂犬病病毒g E蛋白抗体阳性,提示怀孕母猪群可能存在猪伪狂犬病野毒感染。     

Isolation of a Lelystad virus-like agent from British pigs and scanning electron microscopy of infected macrophages.

Six, one-week-old gnotobiotic piglets were inoculated with tissues or sera collected from field cases of porcine reproductive and respiratory syndrome. The piglets showed little or no illness, and two that were necropsied at 8 and 9 days post infection appeared grossly normal. However, a Lelystad virus-like agent was isolated from most of the inoculated pigs using porcine alveolar macrophage cultures. Seroconversion to the Lelystad virus was observed and some animals developed microscopically detectable interstitial pneumonias. Scanning electron microscopy was used to study the in vitro cytopathic effect of the Lelystad virus on porcine alveolar macrophages.     

Experimental production of congenital persistent swine fever infections: I. Clinical,pathological and virological observations

Twelve sows were inoculated with a low-virulent field strain of swine fever (SF) virus at 40, 65 and 90 days of pregnancy. Transplacental infection occurred in eight sows and these farrowed 88 piglets.Prenatal mortality was highest in litters from sows infected at 40 days after service, and postnatal death was most frequent in litters from sows infected at 65 days. Three sows gave birth to completely infected litters, whereas the five others farrowed litters in which uninfected piglets were found. The later the sows were infected during pregnancy, the more uninfected piglets were born. Twelve piglets recovered from the infection and the percentage of recoveries increased with the stage of pregnancy at which inoculation took place. Twenty-three piglets developed a persistent infection. The earlier infection occurred during pregnancy, the more persistent infections were produced. The persistently infected pigs developed a runting syndrome from about one week after weaning. The clinical signs were more severe in pigs from sows infected at 65 days of pregnancy than in pigs from sows infected at 40 days. A persistent viraemia was present in these pigs with titres ranging between 10^5.7 and 10^7.0 plaque forming units/ml of plasma. At autopsy the most pronounced lesions were an atrophy of the thymus, and swollen and pale lymph nodes. Virus antigen was present in lymphoidal, reticulo-endothelial and epithelial tissues.One persistently infected pig survived superinfection with a virulent strain of SF virus for 45 days.     

Upper respiratory infection of lactating sows with transmissible gastroenteritis virus following contact exposure to infected piglets.

Ten breeding sows were left in direct contact with their newborn piglets that had been experimentally infected with transmissible gastroenteritis (TGE) virus. All sows became infected with the virus. The sows developed fever and showed mild clinical signs of the disease for a few days. The sows excreted virus in the nasal secretion, feces, and milk during the acute febrile phase of illness. Virus was isolated from the nasal secretion of one sow as early as 20 hours after contact exposure to the infected piglets. At necropsy, the virus was more frequently isolated from the tissues of the upper respiratory tract than from small intestines; this finding indicated that the TGE coronavirus replicated in the upper respiratory tract and induced an acute respiratory infection in susceptible adult swine. Neutralizing antibody was present in the sera 8 sows after 12 to 36 days during the convalescent period. From these results, we conclude that susceptible sows in direct contact with ill piglets can become infected and by excreting virus can serve as a source of TGE virus for other susceptible pigs on the premises.     

Long-term studies on maternal immunity for Aujeszky's disease and classical swine fever in wild boar piglets

The aim of the studies was to fathom the duration and the role of maternal immunity for Aujeszky's disease (AD) and classical swine fever (CSF) in wild boar offspring. In one experiment, two wild boar sows were infected with a low pathogenic pseudorabies virus (PRV) in 1999. A total of 51 offspring was born between 1999 and 2002 and was monitored for PRV maternal antibodies. In a second experiment, the maternal immunity for CSF was analysed. Therefore, a sow was orally vaccinated against CSF using vaccine baits containing the live-attenuated C-strain vaccine. The vaccination took place in January 1999. The sow gave birth to four piglets in 2001 and to two piglets in 2002. With respect to maternal immunity for AD, some piglets reacted positive in the ELISA up to 27-week post-partum while in the neutralization test antibodies were detected up to 15-week post-partum. The calculated half-life of neutralizing antibodies was 21 days. Regarding CSF, the neutralization titres of maternal antibodies dropped continuously reaching values of < or =10 ND50 20-week post-partum. After the 12th week post-partum, most of the sera reacted negative in the ELISA. However, after the third month, low levels of neutralization titres were still detectable. The results are discussed with respect to the epidemiology and control of both diseases in wild boar populations.     

Experimental inoculation of late term pregnant sows with a field isolate of porcine reproductive and respiratory syndrome vaccine-derived virus.

The use of a live attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in piglets has been associated with reproductive disorders in non-vaccinated sows. Vaccine-derived virus (VDV) has been isolated from foetuses, stillborn pigs, and dead piglets, indicating that the live vaccine spread from vaccinated piglets to non-vaccinated sows, and that the virus might be implicated in the severe reproductive problems observed. In the present study, one such VDV isolate was used to experimentally infect pregnant sows in the last trimester. The chosen isolate, which had more than 99.6% identity to the attenuated vaccine virus, originated from the lungs of a stillborn pig from a swine herd with a sudden high level of stillborn pigs and increased piglet mortality in the nursing period. Intranasal inoculation of sows with the virus isolate resulted in congenital infection, foetal death, and preweaning pig mortality. As such, the present study showed that vaccine-derived PRRSV can cause disease in swine consistent with PRRS.     

Immunogenicity in Aujeszky's disease virus structural glycoprotein gVI (gp50) in swine.

Aujeszky's disease virus (ADV) envelope glycoprotein gVI (gp50) was purified from virus-infected Vero cells by ion-exchange and immunoaffinity chromatography and its usefulness as a subunit vaccine was evaluated in active and passive immunization studies. Four-week-old piglets were immunized intramuscularly (IM) with purified gVI twice two weeks apart and challenged intranasally (IN) 10 days after the second immunization with 30 LD50 (10(8)PFU) of a virulent strain of ADV. Pigs, vaccinated with 100 micrograms of purified gVI, produced virus neutralizing antibodies and did not develop clinical signs after challenge exposure. The challenge virus was not isolated from nasal swabs and tonsils of gVI-vaccinated pigs, whereas non-vaccinated control pigs developed illness after challenge exposure with the same virulent ADV strain which was later recovered from their nasal swabs and tonsils. Pregnant sows vaccinated twice with purified gVI (IM) at a three week interval produced virus neutralizing antibodies in colostrum. Four-day-old sucking piglets born of vaccinated sows were passively protected by colostral antibodies against intranasal challenge with a lethal dose of virulent ADV. Sera from gVI-vaccinated pigs were distinguished from experimentally infected swine sera by their differential reactivity in enzyme-linked immunosorbent assay (ELISA) using four major viral glycoproteins (excluding gVI) as antigen purified by the use of lentil-lectin.     

Presence of porcine parvovirus in sera from pigs is independent of antibody titers

Porcine parvovirus (PPV) is a widespread DNA virus that causes reproductive failure in swine. The aim of the present study was to investigate the presence of PPV in sera of nursery piglets (healthy n = 191 and wasting n = 132) and regularly vaccinated sows (with different parity rank [PR] n = 129), collected from different herds. Altogether, 452 animals were sampled in 27 herds owned by five companies. All sera were analyzed for the presence of PPV DNA by nested-PCR. The samples from sows were in addition tested for the presence of antibodies by Hemagglutination Inhibition (HI). PPV DNA was detected in healthy piglets (15.7%), wasting piglets (18.2%) and sows (17.8%). 25 herds had at least one positive sample and four companies had positive animals. The serology revealed that 84.7% of the sows had detectable antibodies and the fourth PR sows had the highest mean PPV antibody titers. Thirteen sows (19.1%) were found to be positive for DNA detection in the presence of high levels of antibody titers (> 512). This finding indicates that PPV DNA can be detected in different swine production categories irrespective of antibody titers.     

Swine reproductive and respiratory syndrome in Québec: Isolation of an enveloped virus serologically-related to Lelystad virus

Sera were collected from convalescent sows and sick piglets from six pig farms in southern Quebec that have experienced outbreaks of the so-called porcine reproductive and respiratory syndrome. By indirect immunoperoxidase, a few of these sera (4 of 14) (28.6%) were found to be positive for antibody to the Lelystad virus, whereas by indirect immunofluorescence 30 of 36 (83.3%) were positive for antibody to the antigenically-related American isolate ATCC-VR2332. Pregnant sows inoculated intranasally with filtered homogenates prepared from the lungs of necropsied piglets obtained from a seropositive farm developed fever, inappetence, and reproductive failure characterized by stillbirths and various stages of mummification. Lesions of interstitial pneumonia were induced in experimentally-infected specific pathogen-free piglets. A virus, having morphological and biological characteristics of viruses assigned to the family Togaviridae, was isolated from lung tissues of experimentally-infected animals; it could only be propagated in primary cultures of porcine alveolar macrophages. Identification of the virus was confirmed by indirect immunofluorescence using a monoclonal antibody directed against the nucleocapsid protein of the ATCC-VR2332 isolate and porcine sera that were found positive for antibody to both the Lelystad and ATCC-VR2332 isolates.     

猪伪狂犬病基因缺失活疫苗(SA215)免疫母猪后仔猪母源抗体消长规律及首免日龄

在研究猪伪狂犬病基因缺失活疫苗 (SA 2 15 )免疫接种母猪所产仔猪母源抗体消长规律 ,并绘制其消长曲线的基础上 ,确定了仔猪首免日龄。对免疫母猪所产仔猪于不同日龄进行抗体检测结果表明 ,全部仔猪均获得了高水平母源抗体 ,7日龄高达 2 8.56 ,6 0日龄降至 2 2 .56 ;随着日龄增长 ,抗体水平呈逐渐降低趋势 ,2次大幅下降出现在 14～ 2 1日龄、30～ 6 0日龄。对免疫母猪所产仔猪分别于不同日龄免疫接种 1头份剂量疫苗 (SA2 15 ) ,7d后采血进行抗体检测 ,结果表明 ,7日龄、14日龄、2 1日龄免疫接种仔猪均未引起明显抗体水平升高 ,反而较同期仔猪略有降低 ,30日龄和 6 0日龄免疫接种仔猪则出现了抗体水平的升高 ,其中以 6 0日龄仔猪升高幅度为大。结合母源抗体消长规律 ,确定猪伪狂犬病基因缺失活疫苗 (SA2 15 )免疫母猪所产仔猪的首免日龄为 30日龄     

猪细小病毒N株的生物学和免疫学特性研究

猪细小病毒N株是从广西初产母猪所产死胎脏器分离的自然弱毒株。用这个毒株接种PPV HI抗体阴性的四月龄小猪和怀孕14～23天的后备母猪进行安全性试验,结果无任何异常临床症状、病毒血症和同居感染,母猪分娩正常,初生仔猪在吃初乳前HI抗体阴性。该毒株免疫的小猪、后备母猪和怀孕母猪,完全能抵抗猪细小病毒强毒攻击,攻毒后49天剖杀母猪,结果胎儿正常,胎儿心血HI抗体阴性,取胎儿脏器未分离出病毒,分娩母猪产仔正常,仔猪吃初乳前HI抗体阴性。而对照猪攻毒后产生病毒血症,产下不同组合异常仔,并从死胎儿脏器分离出病毒,健活仔猪吃初乳前能测出HI抗体。从而证明用N株作为弱毒苗能防止由猪细小病毒引起的繁殖障碍性疾病。     

某规模化猪场伪狂犬病的诊断

河南平顶山某猪场母猪出现较严重的流产和产死胎现象,且50日龄~70日龄仔猪出现神经症状,根据临床表现初步诊断为伪狂犬病。为排除猪繁殖与呼吸综合征和猪瘟,进行了实验室诊断。应用ELISA方法检测发病保育猪及母猪血清的伪狂犬病病毒野毒株gE抗体,并对发病仔猪病料进行了伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)的实时荧光定量PCR检测。结果显示,伪狂犬病病毒野毒抗体阳性,实时荧光定量PCR检测确定仔猪病料中PRV核酸阳性,PRRSV和CSFV核酸阴性。结合临床症状及实验室检测,确诊该猪场发生的是猪伪狂犬病。     

Pathogenicity of a bovine viral diarrhoea virus strain in pregnant sows: short communication

The biological properties of bovine viral diarrhoea virus (BVDV) strain Oregon C24V were studied after intranasal and subcutaneous infection of pregnant sows. This virus strain is widely used in Hungary for immunising cattle against bovine viral diarrhoea (BVD). Based upon the results of the clinical, gross pathological, histopathological and virological examinations it can be established that the given strain caused asymptomatic infection and serological conversion in sows that were in the second third of gestation. The virus caused clinically apparent disease in some of the piglets born at term, which indicates that it had crossed the placenta. More than half (57%) of the live-born piglets died within 60 days of birth. The sows and their progeny did not shed the virus. BVDV infection has great differential diagnostic importance in pigs, as classical swine fever (CSF) virus strains of reduced virulence cause similar clinical symptoms and gross and histopathological changes.     

Pathologic consequences of a severe influenza outbreak (swine virus A/H1N1) under natural conditions in the non-immune sow at the beginning of pregnancy

Pathological consequences of a severe outbreak of swine influenza (H1N1 virus) in the non immune sow at the beginning of pregnancy, under natural conditions. A sudden acute outbreak of fever, depression, anorexia and coughing in a group of nulliparous sows from a herd that was currently under epidemiological investigation lead to build a particular disposal of observation. The clinical signs were daily recorded including rectal temperature. Blood was taken from the sows at the beginning of the troubles and 3 weeks later for the detection of Aujesky's disease, coronavirus TGE-like, Influenza viruses A/H1N1 and A/H3N2 and Mycoplasma hyopneumoniae. Viral detection was attempted from nasal swabs and aborted fetuses during the acute phase. The clinical study showed fever reaching near 41 degrees C on most of the pigs and lasting usually from 2 to 5 days. The diagnosis of Influenza (virus swine H1N1) was established both on serology (massive seroconversion) and on the detection of the virus from the nasal swabs and from an aborted fetus. The control of the lungs of sows "not in pig" and culled showed extended lesions of bronchopneumonia and Pasteurella multocida was found. The technical consequences of this severe outbreak of Influenza on reproduction were mainly important at the beginning of pregnancy. Over 13 sows inseminated less than 1 week before the outbreak, only 3 farrowed (respectively 5.5 and 12 piglets); 7 returned to oestrus and 3 "not a pig" at 21 days (echotomography) did not show signs of heat and were culled. Over 8 pregnant sows (1 month of pregnancy), 6 farrowed normal litters and total embryonic resorption occurred in 2 sows. Over 18 pregnant sows (more than 45 days gestation) one aborted.     

Experimental in utero inoculation of late-term swine fetuses with porcine circovirus type 2.

All 37 fetuses of 3 laparotomized pregnant sows at 86, 92, and 93 days of gestation were inoculated intramuscularly through the uterine wall with porcine circovirus type 2 (PCV-2). The sows were allowed to farrow, and blood and tissue samples were collected from their piglets before and after suckling colostrum. Thirteen fetuses from 2 sows at 90 and 103 days of gestation were used as controls. Of the 37 PCV-2 inoculated fetuses, 24 were grossly normal and 13 were mummified, stillborn, or weak-born at farrowing. Infection with PCV-2 was demonstrated in various tissues of grossly normal and abnormal fetuses by virus isolation, polymerase chain reaction, and immunohistochemical methods. Antibodies specific to PCV-2 were also detected from the sera or thoracic fluids of abnormal fetuses and unsuckled normal pigs. No evidence of PCV-2 infection was found in any control fetuses. The present results confirm previous findings that PCV-2 can infect late-term swine fetuses and may cause reproductive abnormalities.