William T. Christianson

Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs

A recent epizootic of swine infertility and respiratory syndrome (SIRS) in a Minnesota swine herd was investigated. Examination of a sow, neonatal piglets, and stillborn fetuses obtained during the epizootic from the affected herd revealed interstitial pneumonitis, lymphomononuclear encephalitis, and lymphomononuclear myocarditis in the piglets and focal vasculitis in the brain of the sow. Fetuses did not have microscopic lesions. No cause for the infertility and respiratory syndrome was determined. Therefore, attempts were made to experimentally reproduce the disease. Eleven 3-day-old gnotobiotic piglets exposed intranasally to tissue homogenates of piglets from the epizootic became inappetent and febrile by 2–4 days postexposure and had interstitial pneumonitis and encephalitis similar to that seen in the field outbreak. After 2 blind passages in gnotobiotic piglets, tissue homogenates were cultured on continuous cell line CL2621, and a cytopathic virus (ATCC VR-2332), provisionally named SIRS virus, was isolated. Gnotobiotic piglets exposed intranasally to the SIRS virus developed clinical signs and microscopic lesions that were the same as those in piglets exposed to the tissue homogenates, and the virus was reisolated from their lungs. This is the first isolate of SIRS virus in the United States that fulfills Kochs postulates in producing the respiratory form of the disease in gnotobiotic piglets and the first report of isolation and propagation of the virus on a continuous cell line (CL2621). The virus is designated as American Type Culture Collection VR-2332.

Characterization of Swine Infertility and Respiratory Syndrome (SIRS) Virus (Isolate ATCC VR-2332)

The characterization of an isolate of swine infertility and respiratory syndrome (SIRS) virus (ATCC VR-2332) is reported. A commercial cell line (CL262 1) was used for the propagation of the virus for all assays. Laboratory studies indicate that this isolate is a fastidious, nonhemagglutinating, enveloped RNA virus. Cesium chloride-purified virions visualized by electron microscopy were spherical particles with an average diameter of 62 nm (range: 48–83 nm) and a 25–30 nm core surrounded by an envelope. Virus replication was restricted to the cytoplasm, as demonstrated by immunofluorescence. The virus did not react serologically with antisera to several common porcine viruses or with antisera to known viruses in the alphavirus, rubivirus, pestivirus, and ungrouped lactic dehydrogenase virus genera of the Togaviridae. However, convalescent sow sera and rabbit hyperimmune sera neutralized the SIRS virus at titers of 1:256 and 1:512, respectively. The virus was stable at 4 and −70 C, but was labile at 37 and 56 C. The properties of this isolate of SIRS virus resemble those of the family Togaviridae but do not match the described genera.

Antigenic Comparison of Lelystad Virus and Swine Infertility and Respiratory Syndrome (SIRS) Virus

This study reports the antigenic relatedness of isolates of Lelystad virus collected in the Netherlands, Germany, and the United States. The binding of antibodies directed against these isolates was tested in a set of field sera collected during outbreaks of porcine epidemic abortion and respiratory syndrome in Europe and outbreaks of swine infertility and respiratory syndrome (SIRS) in North America. Two sets of sera from pigs experimentally infected with Lelystad virus or SIRS virus were also tested. Although all 7 isolates reacted with anti-Lelystad virus sera, antigenic variation was considerable. The 4 European isolates resembled each other closely, but differed from the American isolates, and the 3 American isolates differed antigenically from each other. To reliably diagnose Lelystad virus infection, a common antigen must first be identified.

An indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera

An indirect fluorescent antibody (IFA) test was developed and standardized to detect and quantitate antibody for swine infertility and respiratory syndrome (SIRS) virus in swine sera. Test results were evaluated using sera of pigs infected both experimentally and naturally with SIRS virus. The IFA test used swine alveolar macrophage (SAM) monolayers prepared in 96-well microplates and infected with SIRS virus. The monolayers were incubated with test sera, washed, and stained with fluorescein isothiocyanate-labeled rabbit anti-swine IgG. After another wash step, the monolayers were examined under a fluorescent microscope. A noninfected SAM control well was included for each sample. The antibody titers for each serum sample were recorded as the highest serum dilutions with specific cytoplasmic fluorescence but no fluorescence in the control wells. To evaluate the test, sera of 4 6-week-old pigs that had been infected with SIRS virus, 2 contact pigs, and 13 experimentally infected sows were used. In the experimentally infected pigs, antibody was first detected at 7 days postexposure (PE) and peaked (1:256–1,024) between 11 and 21 days PE. All 13 sow sera were negative at time of infection but were positive (1:64-> 1: 1,024) at 14–26 days PE. Seven hundred twenty sera collected from 25 different swine farms with or without a history of SIRS were also tested. Of 344 sera from 15 swine farms with a clinical history of SIRS, 257 (74.7%) sera had IFA titers ≥ 1:4, whereas 371 (98.7%) of 376 sera from herds with no history of SIRS were negative. The present results indicate that the IFA is a useful test for the detection and quantitation of SIRS virus antibody in swine sera.

Stillbirths, mummies, abortions, and early embryonic death.

Stillbirths, mummies, abortions, and early embryonic death have a substantial impact on the profitability of a farm in both endemic and epidemic conditions. Fetal death is highly dependent on stage of gestation. Implantation occurs around day 14 postmating in sows, and fetal death of an entire litter at this time usually results in a regular return to service. If more than four embryos remain alive, the sow may go on to farrow normally. If fetal death occurs after implantation but before calcification (around 35 days gestation), the sow will either return to estrus at an irregular interval or will farrow a normal litter of reduced size. Although fetuses are normally resorbed prior to calcification, fetal death after that stage of development leads to mummification. Abortions are more directly related to maternal control of pregnancy than fetal failure. Stillbirths are those pigs that appear normal at birth but have lungs that do not float in water. Causes of fetal death can be divided into infectious and noninfectious categories. Infectious causes perhaps are overemphasized but are certainly important in epidemic situations. Some infectious causes of fetal death are primarily systemic maternal pathogens, whereas others may attack the fetus and/or placenta, directly such as PPV, PEV, PRV, SIRS virus, and Leptospira sp. Several other infectious agents have been associated with fetal death. Noninfectious causes of stillborns, mummies, abortions, and early embryonic death are most common in endemic situations. Most stillbirths are due to difficulty at or around parturition, primarily extended duration causing fetal anoxia. Environmental factors such as increased ambient temperature and seasonal infertility affect death rates, as do specific individual sow characteristics, nutritional factors, and toxicities. The causes of stillborns, mummies, abortions, and early embryonic death are often difficult to ascertain, but the potential rewards make investigation efforts worthwhile.

Serologic evidence incriminating a recently isolated virus (ATCC VR-2332) as the cause of swine infertility and respiratory syndrome (SIRS).

and swine infertility and respiratory syndrome (SIRS). tality ranged from 37% to 83% and was accompanied by late term (>day 100) abortion in all but 1 herd. The absence of anorexia in 2 herds and absence of dyspnea in 1 herd may reflect a failure of detection rather than absence of these clinical signs (Table 2). Each case herd was matched with a herd located within 10 miles, of similar size and type of production, and having no history of undiagnosed reproductive failure or respiratory disease in the last 3 years. Sera were collected from approximately 30 representative sows at each of these 16 herds.

Pathogenic properties of encephalomyocarditis virus isolates in swine fetuses.

SummaryThe pathogenicity of 5 different encephalomyocarditis (EMC) virus isolates was investigated in swine fetuses following injection of each virus in utero. Laparotomies were performed on 3 pregnant sows in early mid-third (39–40 days) of gestation and on 5 sows in the late mid-third (70–72 days) of gestation, and groups of fetuses were inoculated with different viruses into the amniotic sacs. The uninoculated fetuses served as controls. Thirty-five (71.4%) of 49 infected and 1 of 26 control fetuses were grossly abnormal. Virus was recovered from 18 of 28 infected fetuses and 1 of 16 control fetuses examined. Antibody to EMC virus was detected in all of 14 fetuses infected at 70–72 days of gestation and examined 11–26 days post-infection. The fetal pathogenicity was different depending on the virus strains and the fetal age at the time of virus infection. The EMC ATCC-VR 129 virus was not pathogenic but NVSL-PR, MN-25 and MN-30 were highly pathogenic to the fetuses in both early and late mid-thirds of gestation, while NVSL-MDV was pathogenic to the fetus in early but not in late mid-third of gestation. Possible mechanisms for differences in the pathogenicity between the virus strains are discussed.

Evaluation of serologic methods for the detection of antibodies to encephalomyocarditis virus in swine fetal thoracic fluids

Hemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) tests were compared to the serum neutralization (SN) test to evaluate their ability to detect antibodies to encephalomyocarditis virus (EMCV). Swine fetal thoracic fluids of known EMCV SN antibody titers (200 samples 2 1:2, 100 samples < 1:2) were selected from a collection of field cases. The thoracic fluids were tested for EMCV antibodies by HI and AGID, and the results were compared to those of the SN test. Of 200 SN antibody-positive samples, 183 (9 1.5%) and 173 (86.5%) were positive in HI and AGID tests, respectively. Of 100 SN-negative samples, 81 (8 1%) and 94 (94%) were negative in HI and AGID tests, respectively. Agreement between the tests was analyzed by calculating Kappa values. The values were 0.73 between SN and HI tests and 0.77 between SN and AGID tests, indicating very good to excellent agreement for HI and AGID tests with the SN test. Of 200 SN-positive samples, 19 samples with low SN titers (1:2–1:16) were further tested by Western immunoblotting, and all were confirmed as positive. Interpretation of the present results suggests that both HI and AGID tests can be used as alternatives to the SN test.