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.     

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)'.     

Porcine epidemic abortion and respiratory syndrome (mystery swine disease). Isolation in Spain of the causative agent and experimental reproduction of the disease.

In March of 1991, a disease that affected pregnant sows and caused a high mortality in unweaned piglets was detected in Spain. Based on the clinical signs observed, mystery swine disease, which had been described recently in Germany, Holland and Belgium, was suspected. From the samples obtained from the affected farm, a filtrable agent (0.22 micron) was isolated on cell culture. It produced cytopathic effects, its replication was intracytoplasmic, it was sensitive to chloroform, and cross-reacted with a Lelystad reference serum. When inoculated into pregnant sows, the agent produced inappetence for 2-4 days, without hyperthermia. One of the sows aborted at 100 days of gestation; the two others had delayed parturitions (days 115 and 116). There was a mixture of healthy piglets, mummified fetuses, stillbirths and weak piglets. Microscopic examination of the lungs of healthy piglets killed at 8 and 12 days of life revealed the presence of interstitial pneumonia. The sera from the three sows at 39 days after infection cross-reacted with the Lelystad virus (titres > or = 1/640), whereas pre-inoculation sera did not recognize it (titres < or = 1/10). This is the first report from Spain of the isolation of an agent (antigenically related to the Lelystad virus), capable of reproducing the disease previously designated as 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.     

Outbreaks in Quebec pig farms of respiratory and reproductive problems associated with encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) was isolated from tissues of aborted fetuses and weaned and suckling piglets from 4 different pig farms in Quebec. The farms were experiencing reproductive failure in sows of different parities concomitant to respiratory problems in suckling and postweaning piglets. At necropsy, gross lesions were confined to the lung and consisted of pulmonary congestion and edema of various degrees. Lesions of multifocal interstitial to proliferative pneumonia were found in the lungs of these piglets. Bacteriologic examination of various tissues from necropsied pigs yielded no pathogens in most cases. No significant antibody titers against 3 swine viruses (transmissible gastroenteritis virus, porcine parvovirus, and swine influenza virus) and two bovine viruses (bovine viral diarrhea and infectious bovine rhinotracheitis viruses) were detected in the sera of convalescent pigs. The Quebec EMCV isolates were antigenically related to the reference ATCC-VR129 strain of EMCV, as demonstrated by indirect immunofluorescence, serum neutralization (SN), and Western immunoblotting. However, one of the Quebec isolates could be distinguish by SN. EMCV-specific SN antibody titers up to 1:12,800 were detected in thoracic and ascitis fluids of aborted fetuses and in sera of convalescent pigs. A possible pneumotropic EMCV variant in swine may exist.     

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.     

Actinobacillus suis septicemia in mature swine: two outbreaks resembling erysipelas

In the winter of 1987/88 a previously unrecognized septicemic disease syndrome — actinobacillosis in mature sows and gilts — was diagnosed in two minimal-disease swine herds in southwestern Ontario. In herd 1, 34 sows, 2 boars, 13 feeder pigs, and 30 suckling pigs were affected; 11 sows, 2 feeders, and 18 suckling pigs died. In herd 2, 13 sows and 1 feeder pig were affected; 1 sow and 1 feeder pig died. The disease was manifested by moderate fever (39-40.5°C), round or rhomboid erythematous skin lesions, and inappetence. Sudden deaths without previous clinical signs were frequent. Histologically, coccobacillary thromboemboli in superficial and deep dermal vessels were associated with infarcted dermal and epidermal tissues. The causative organism, Actinobacillus suis, was isolated from the affected pigs. Treatment with commonly used antibacterial drugs was effective.

In many respects, the disease resembled acute swine erysipelas and presented diagnostic problems for this reason.

     

Outbreaks of classical swine influenza in pigs in England in 1986

Serum samples from pig herds in Great Britain have been examined for antibodies to influenza virus since 1968. Antibodies to H3N2 virus strains have been found since 1968 and the serological data presented here suggests that H3N2 virus strains continue to persist in the pig population. An outbreak of acute respiratory disease occurred in a 400-sow unit. The outbreak was characterised by coughing, anorexia, fever, inappetence and loss of condition. The gilts and weaners were affected and the morbidity approached 100 per cent. An influenza A virus designated A/Swine/Weybridge/117316/86 (H1N1) was isolated from the herd and 28 paired serum samples from the affected animals showed increases in the haemagglutination inhibition titres to this isolate. Haemagglutinin and neuraminidase characterisation indicated that the virus is similar to H1N1 viruses isolated recently from pigs in Europe. A total of 91 herds experiencing respiratory disease were investigated, of which 42 gave positive reactions in the haemagglutination inhibition test. Antibodies to A/Port Chalmers/1/73 (H3N2) were also detected in some of the herds but it is not known whether this strain plays any role in the current respiratory disease problems in pigs.     

The Pattern of Enteroviral Infections in a Herd of Swine

Twenty-six pigs from four litters in a healthy herd of swine were examined periodically for the fecal excretion of viruses by the use of porcine kidney cell cultures. Viruses were initially isolated from all pigs between 34 to 64 days of age. The pigs within each litter began shedding virus in their feces approximately at the same time, usually within one week, and the type of virus initially recovered was usually the same. Subsequently, waves of infection with different enteroviruses appeared to occur during the observation period of six months. At least six antigenically different viruses were isolated from this herd over a 26-month period. Most, if not all, of these viruses were considered to belong to the enterovirus group. No disease was associated with these enteroviral infections.

The colostrum and milk of sows contained significant amounts of enteroviral antibodies. Prior to nursing, the serum of new-born pig contained no enteroviral antibodies but, shortly after nursing, high titers of such antibodies were present in the serum. Antibodies were detected in the feces of suckling pigs.

     

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。     

Eradication of some infectious pig diseases by perinatal tiamulin treatment and early weaning

From 10 days before the expected date of farrowing onwards, 97 sows infected by Mycoplasma hyopneumoniae and Treponema hyodysenteriae were given tiamulin daily at a dosage of 20 mg/kg bodyweight via the feed. Three days before farrowing the sows were washed with a disinfectant and transferred to an isolated farrowing house. The sucking piglets remained with their dams for five days, during which time the sows continued to receive the tiamulin-containing feed. The sucking piglets also received tiamulin daily at a dosage of 30 mg/kg bodyweight. At six days old the weaker piglets of the litter were returned to the original herd, together with their dams. A total of 574 piglets of about 1.5 kg bodyweight each were transferred to an isolated and previously disinfected pig farm and reared there. A total of 13.8 per cent of these pigs died by 50 days old. On the isolated farm, 10.9 per cent of the 829 second generation piglets born to the 101 first generation sows, died up to the age of 50 days. On the isolated farm about 2000 pigs were subjected to repeated clinical, pathological and laboratory examinations for M hyopneumoniae, T hyodysenteriae, Aujeszky's disease virus and Leptospira species during the three year period of study. No evidence of infection with any of these agents was found in the 2000 pigs of the isolation herd, although the original sow herd had been latently infected by these pathogens. No maternally derived antibodies against these pathogens were detectable in sera of three-day-old sucking piglets of the second and third generations.     

A novel concept for the control of Aujeszky's disease: experiences in two vaccinated pig herds

A study was conducted to examine the usefulness of a glycoprotein I (gI)-ELISA to monitor Aujeszky's disease virus infection in two vaccinated pig herds; the gI-ELISA can differentiate between pigs infected with Aujeszky's disease virus and pigs vaccinated against Aujeszky's disease with gI-negative vaccines. The two herds had been vaccinated with gI-negative vaccines for several years. The first survey, in September 1986, revealed that approximately 10 per cent of the breeding pigs in a large multiplier herd were seropositive for antibodies to gI of Aujeszky's disease virus, and it was decided to try to eliminate the virus from the herd by gI-ELISA testing and culling of gI-seropositive pigs. A one month quarantine period for incoming stock was established, and only gI-seronegative pigs were admitted to the herd. After two rounds of testing and culling the herd appeared to be free of wild-type Aujeszky's disease virus, and neither Aujeszky's disease virus nor antibodies could be detected either in 21 sentinel pigs placed on the farm or in 347 stillborn piglets or piglets that died shortly after birth. The herd probably remained free of Aujeszky's disease virus until the end of the 27-month period of monitoring except for two of 639 breeding pigs that were unexpectedly found to be positive in the gI-ELISA in November 1987. These sows were culled. A second breeding herd was monitored for antibodies to gI of Aujeszky's disease virus for two years. The gI-seropositive sows constituted approximately 30 per cent of the herd's breeding pigs, but they were not culled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term lack of transmission of Aujeszky's disease virus (ADV) in a chronically infected Swedish weaner pig producing herd

A cohort of 53 swine seronegative to Aujeszky's disease virus (ADV) was monitored in a 1 year study of a chronically infected commercial Swedish weaner pig producing herd. Serum samples were acquired from all 134 adult swine and analyzed by enzyme-linked immmunosorbent assay (ELISA). Animals testing negative, along with introduced replacement gilts, were followed serologically every second month. Movements of animals were recorded for 319 days and exposure to seropositive animals was calculated for each seronegative pig in the cohort. The accumulated daily pig contact between the 53 ADV-non-infected swine and 43 infected swine was 35 660 days and the median number of days in contact for the non-infected swine with infected was 222. Despite the frequent contact with seropositive pigs, no seronegative animals seroconverted during the first 11 months of observation. Forty-six of 53 pigs seroconverted after a clinical outbreak during the twelfth month of observation.     

Serological, pathological and cultural evaluations of swine infected experimentally with Mycoplasma flocculare.

Fourteen caesarean-derived, colostrum-deprived pigs and seven conventional swine were exposed to low passage, cloned, field isolates of Mycoplasma flocculare. Sera were collected at varying intervals postexposure (PE) and tested against M. flocculare and M. hyopneumoniae antigens in a semi-automated ELISA. Swine were killed six to 17 weeks PE and their lungs examined grossly for lesions and culturally for mycoplasmas. Pure cultures of M. flocculare were recovered from the lungs of 11 of 14 swine killed six to 12 weeks PE. Mycoplasmas were not isolated from the swine killed 15 to 17 weeks PE. Only one pig had gross lesions of pneumonia. Immunoassays revealed that swine were slow to seroconvert and titers (expressed in terms of optical density) were low. Three of 21 swine had antibodies to M. flocculare five weeks PE, five of 17 had seroconverted at seven to eight weeks and all surviving swine had antibodies to M. flocculare 76 days PE and beyond. Net optical density of positive sera was in the range of 0.201 to 0.412 (an optical density of 0.2 regarded as the breakpoint between negative and positive reactions in our ELISA). All of the sera were ELISA-negative when tested against M. hyopneumoniae antigen. This is regarded as a very significant finding. There has been concern that field sera might contain antibodies to M. flocculare and that such antibodies could render serodiagnostic tests for mycoplasmal pneumonia of swine nonspecific. Results of the present study suggest that swine infected with M. flocculare do not develop sufficient levels of antibodies to interfere with enzyme immunoassays for M. hyopneumoniae.     

Isolation of a novel viral agent associated with porcine reproductive and neurological syndrome and reproduction of the disease

Disease outbreaks characterized by reproductive failure and/or neurologic disorders, which are commonly referred as "Porcine Reproductive and Neurologic Syndrome (PRNS)", were observed in many swine farms in Iowa and other states. Although an infectious cause was suspected to account for the disease, no conclusive diagnosis had been reached with respect to conventional infectious agents. Extensive laboratory diagnostic investigation on suspect cases repeatedly resulted in the isolation of a cytopathic enveloped virus of 50-60nm in size from nervous and second lymphoid tissues and sera and, to reflect its unknown identity, named "Virus X". The presence of virus particle with morphological characteristics similar to Virus X in tissues from affected animals was also observed on thin-section positive-staining electron microscopy. Isolates of Virus X were not readily recognized by antibodies raised against any known viruses pathogenic to swine but by antisera collected from animals surviving clinical episode, indicating that Virus X is likely a previously unrecognized agent. Pregnant sows experimentally inoculated with Virus X (ISUYP604671) or homogenate (filtrate) of tissues from a clinically affected animal developed clinical signs and pathological changes similar to field observations including the loss of pregnancy. Furthermore, caesarian-derived, colostrum-deprived young pigs developed mild encephalomyelitis lesions in brains after experimental inoculation with the virus or the tissue homogenate although clinical neurologic signs were not observed. More importantly, Virus X was re-isolated from all inoculated animals while control pigs remained negative for the virus during the study. Collectively, Virus X is a novel viral agent responsible for PRNS and remains to be further characterized for taxonomical identity.     

Experimental reproduction of swine infertility and respiratory syndrome in pregnant sows.

The purpose of this study was to experimentally reproduce swine infertility and respiratory syndrome (SIRS). Six multiparous sows were intranasally inoculated at 93 days of gestation with lung homogenates from clinically affected pigs, and 3 additional sows were similarly inoculated with a virus isolated in cell culture from the lung homogenate (SIRS virus, isolate ATCC VR-2332). Inoculated sows developed transient anorexia, farrowed up to 7 days prematurely, and delivered a mean of 5.8 live pigs and 6.0 dead fetuses/litter. Clinical signs of disease were not observed in 3 sham-inoculated control sows that delivered a mean of 12.7 live pigs and 0.3 stillborn fetuses/litter. The SIRS virus was isolated from 50 of 76 live-born and stillborn fetuses from the 9 infected litters. Virus was not isolated from 26 autolyzed fetuses or 15 control pigs. Six of 9 inoculated sows developed neutralizing antibodies to SIRS virus. The reproductive effects found in these experiments were identical to those found in field cases. On the basis of our findings, virus isolate ATCC VR-2332 causes the reproductive failure associated with SIRS.     

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.     

An outbreak of blue-eared pig disease (porcine reproductive and respiratory syndrome) in four pig herds in Great Britain.

The clinical syndrome of a new disease of pigs in four herds in the Humberside area is described. The first signs of the disease were anorexia, lethargy and pyrexia with up to 60 per cent of the dry sows affected. These signs were followed by an increased incidence of abortions which occurred in up to 3.3 per cent of sows, premature farrowings in up to 20.6 per cent of sows and stillbirths and late mummification which affected up to 26.0 and 18.8 per cent of fetuses, respectively. Mortality in neonatal and pre-weaning pigs reached up to 88 per cent and respiratory disease of high morbidity and low mortality occurred in fattening pigs. There were infertility problems in sows, with an increase in returns to service and a failure to show oestrus after weaning or aborting. The signs of the disease in boars were anorexia and malaise. Cyanosis of the extremities affected up to 2 per cent of the animals. The outbreak lasted 11 weeks in all the herds.     

Effect of virus-specific antibodies on attachment, internalization and infection of porcine reproductive and respiratory syndrome virus in primary macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) induces respiratory distress in young pigs and reproductive failure in sows. In PRRSV infected pigs, virus persists for several weeks to several months. Although IPMA antibodies are detected from 7 days post inoculation (pi), virus neutralizing (VN) antibodies are commonly detected starting from 3 weeks pi with an SN test on Marc-145 cells. Since infection of Marc-145 cells is quite different compared to infection of macrophages, the in vivo target cell, the role of these VN antibodies in in vivo protection is questionable. In our study, we demonstrated that antibodies from pigs early in infection with PRRSV Lelystad virus (14 days pi) showed no neutralization in the SN test on Marc-145 cells, but partially reduced Lelystad virus infection of porcine alveolar macrophages. At 72 days pi, VN antibodies were detected by the SN test on Marc-145 cells, and these protected macrophages completely against Lelystad virus infection. In contrast, these VN antibodies only partially reduced porcine alveolar macrophage infection of a Belgian PRRSV isolate (homologous virus), and had no effect on infection of porcine alveolar macrophages with the American type VR-2332 strain (heterologous virus). Confocal analysis of Lelystad virus attachment and internalization in macrophages showed that antibodies blocked infection through both a reduction in virus attachment, and a reduction of PRRSV internalization. Western immunoblotting analysis revealed that sera from 14 days pi, which showed no neutralization in the SN test on Marc-145 cells but partially reduced Lelystad virus infection of macrophages, predominantly recognized the Lelystad virus N protein, and reacted faintly with the M envelope protein. Sera from 72 days pi, with VN antibodies that blocked infection of Marc-145 cells and PAM, reacted with the N protein and the two major envelope proteins M and GP5. Using the Belgian PRRSV isolate 94V360 an identical but less intense reactivity profile was obtained. VN sera also recognized the VR-2332 N and M protein, but not the GP5 protein.