An Indirect Fluorescent Antibody Test for the Detection of Antibody to Swine Infertility and Respiratory Syndrome Virus in Swine Sera

Abstract

Abstract. 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.Swine infertility and respiratory syndrome (SIRS) virus has recently been recognized as an important pathogen in the United States because the virus causes severe reproductive failure in pregnant sows and high mortality due to respiratory disease in young pigs. [1][2][3]8 A similar syndrome has been reported in several European countries, 4-7 and the causative agent, Lelystad virtus, 6 was identified. Antibody to the Lelystad virus has been detected by an immunoperoxidase monolayer assay (IPMA), 6 whereas a serum neutralization (SN) test was described for the detection of antibody to SIRS virus.2 The purpose of the present study was to develop a simple indirect fluorescent antibody (IFA) method for detection and quantitation of SIRS virus antibody in swine sera. The method was evaluated using sera of pigs experimentally infected with SIRS virus. Test results of field serum samples collected from 25 different farms with or without a clinical history of SIRS are also described.SIRS MN-lb isolate at the fifth passage level was prepared in SAM cells, and virus aliquots of 10 4.5 immunofluorescence infective dose 50 (IFID 50 )/ml were stored at -70 C. Materials and methodsPreparation of test plates. The SAM cells, either freshly prepared or stored at -70 C, were diluted at a concentration of 2 x 10 6 cells/ml in RPMI-1640 medium supplemented w...

Diagnostic Performance of Assays for the Detection of Anti-Porcine Reproductive and Respiratory Syndrome Virus Antibodies in Serum and Muscle Transudate (“Meat Juice”) Based on Samples Collected under Experimental Conditions

“…The IFAT was performed as previously described. 18,23 In brief, MARC-145 cells at a concentration of 4 3 10 5 cells per milliliter (150 ml per well) suspended in MEM h supplemented with 10% heat-inactivated FBS, n were grown in 96-well plates. i Diluted PRRSV (ATCC VR-2332) was added to alternative rows of cells.…”

Characterization of Antibody Response to Porcine Reproductive and Respiratory Syndrome Virus ORF5 Product following Infection and Evaluation of its Diagnostic Use in Pigs

“…These sera were collected from farms known to have a history of PRRSV-free status. Their status had also been confirmed by an IFA test 25 and the commercial ELISA. a These sera were used as negative controls.…”

“…On this basis, the International Committee on Taxonomy of Viruses recently established a new order, Nidovirales, to contain the 2 families. 6,11 In infected cells, the approximately 15-kb From the USDA/ARS, Meat Animal Research Center, Clay Center, NE 68933 (Kwang, Yang) 15,22,25,26 All of these tests employ native viral antigens, which are produced in a tissue culture system. Among these tests, the ELISA is used routinely for PRRSV antibody detection.…”

Evaluation of Enzyme-Linked Immunosorbent Assay (ELISA)and Immunofluorescent Antibody (IFA) Test for the Detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Antibody in Pigs from Conventional Farms.

“…At the positive farms, some animals had the antibodies by IFA (titer ≥64). This tentative cut-off point of IFA (titer of 64) was referred to Yoon et al [27]. They speculated that IFA cut-off titer might be set at 64, when conventional pigs were tested.…”

“…Therefore a highly sensitive and specific assay is needed. The IFA, the immunoperoxidase monolyer assay (IPMA), the virus neutralization test (VNT), and the haemaglutination inhibition test (HI) have been reported as antibody assays of PRRS virus (PRRSV) [1,7,8,13,23,27,29]. IFA has been most frequently used for PRRS in Japan.…”