Han S. Joo

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.

Comparison of Porcine Alveolar Macrophages and CL 2621 for the Detection of Porcine Reproductive and Respiratory Syndrome (PRRS) Virus and Anti-PRRS Antibody

The American and European strains of porcine reproductive and respiratory syndrome (PRRS) virus were initially isolated in an established cell line (CL 2621) and porcine alveolar macrophages (PAM), respectively. Subsequent isolation of American strains of this virus in PAM has also been reported. To determine their relative sensitivity for virus isolation, both PAM and CL 2621 cells were inoculated with 98 tissue specimens and 73 serum samples from animals suspected of having PRRS. Four of the 98 tissue samples yielded virus in both cell types, whereas 7 samples were positive only in PAM and 4 samples only in CL 2621. Of the 73 serum samples tested, 18 were positive in PAM of which only 2 were positive in CL 2621. Additionally, 82 isolates obtained initially in CL 2621 were inoculated in PAM cells, and 18 strains isolated originally in PAM were inoculated in CL 2621. Of the 82 CL 2621 isolates, 25 could not be propagated on PAM. Of the 57 that replicated in PAM, as detected by a positive test on indirect fluorescent antibody test, only 28 produced cytopathic effects and 29 did not. Of the 18 PAM isolates, 5 did not grow on CL 2621. Although PAM were relatively more sensitive for virus isolation, their failure to support the growth of certain strains of PRRS virus indicates the existence of variants among PRRS virus strains, and both PAM and CL 2621 should be used for virus isolation from clinical samples. In addition, the sensitivity of these 2 cell types was compared for the detection of fluorescent antibodies to PRRS virus using 179 serum samples from PRRS-infected animals. The results were comparable in both cell systems.

Cloning, Expression, and Sequence Analysis of the ORF4 Gene of the Porcine Reproductive and Respiratory Syndrome Virus MN-1b

Porcine reproductive and respiratory syndrome virus (PRRSV) MN-1b strain open reading frame 4 (ORF4) has been cloned, sequenced, and expressed in Escherichia coli. The homologies of nucleotide and amino acid sequences between MN-1b (US isolate) and LV (European isolate) are 69% and 64%, respectively. The data also showed that ORF4 of MN-1b is 36 bases shorter than that of LV. Western blot analysis of expressed recombinant ORF4 protein reacted with 65% (26/40) of PRRSV-infected pig sera tested. These results demonstrated that ORF4 of PRRSV may not be a well-conserved region.

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.

Serologic, virologic, and histopathologic observations of encephalomyocarditis virus infection in mummified and stillborn pigs.

Stillborn and mummified swine fetuses from swine farms experiencing reproductive problems were investigated for evidence of infection with encephalomyocarditis (EMC) virus by fetal serology, virus isolation, and histopathologic examination. Fetal sera or thoracic fluids of 478 abnormal fetuses collected during January through December 1987 were tested for the presence of antibody specific to EMC virus. Of 478 samples tested, 175 (36.6%) had EMC virus serum neutralizing antibody titers of 1:64 or greater. The samples positive for EMC virus antibody were obtained from 38 swine farms located in 9 states in the United States. In addition to serologic observations, tissue samples of some abnormal fetuses were examined for the presence of virus and histopathologic lesions. The EMC virus was isolated in 1 case from the fetuses of an aborted litter. The isolate was serologically identical to a reference EMC virus. Nonsuppurative encephalitis and myocarditis were observed in the fetal samples collected from 2 different herds. Thoracic fluid of 1 stillborn pig with lesions was positive for EMC virus antibody at a titer of 1:512. We suggest that a widespread reproductive problem recently experienced in several major swine-producing areas of the United States may have been caused by EMC virus infection.

Evaluation of an Indirect Fluorescent IgM Antibody Test for the Detection of Pigs with Recent Infection of Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused major economic losses to swine producers in recent years due to severe reproductive failure in pregnant sows and respiratory disease in young pigs. Antibodies to PRRSV in swine sera can be tested by various serologic methods including immunoperoxidase monolayer assay, 12 indirect fluorescent antibody (IFA) test, 14 enzymelinked immunosorbent assay, and serum neutralization (SN) test 3,15 However, none of the tests can adequately determine the time of infection. Development of a serologic test that could estimate the infection time or identify a recent infection would be valuable to swine farmers when introducing pigs from a PRRSV-infected farm. Demonstration of specific IgM antibodies has been a useful marker for recent infection to different viral infections. Virusspecific IgM antibody has been detected regularly in primary infection with herpes simplex virus, rubella virus, cytomegalovirus, hepatitis A and B viruses and pseudorabies viTUS. For example, IgM antibody to pseudorabies virus in pigs was first detected in serum 6 days postinoculation (PI), reached the highest titers 10 days PI, and was detectable up to 35 days PI. The objectives of this study were to evaluate an IFA test for the detection of IgM antibody specific to PRRSV and to compare the detection of IgM antibody and the presence of virus in sera from pigs with or without known days PI. PRRSV MN-1b was used for the IFA test, and the MN-H and MN-W isolates were used for animal inoculation. A permissive clone (MARC-145) of the African green monkey kidney (MA-104) cell line was used for virus propagation, virus isolation, and serology. The MARC-145 cells were maintained in Eagle’s minimum essential medium supplemented with 3% fetal bovine serum, 0.15% sodium bicarbonate, and antibiotics. Sera used were collected at known intervals from 3-weekold pigs following experimental infection with PRRSV MNH. Sera from sows infected experimentally with PRRSV MN-W or MN-H were also used. Field serum samples were collected from pigs of known age in 2 different farms with endemic PRRSV infection, and the samples were transported on the same day to the laboratory for virus isolation and serology. Virus isolation from sera was performed using 24-well microplates by inoculating 0.05 ml of the undiluted sera to 1 ml of MARC-145 cell suspensions (2 x 10 cell/ml). Infected monolayers were observed daily for cytopathic changes

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.