J Vet Diagn Invest 4: 144-147 (1992)

An indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera In J. Yoon, Han S. Joo, William T. Christianson, Hyun S. Kim, James E. Collins, Robert B. Morrison, Gary D. Dial 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.1-3,8 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 104.5 immunofluorescence infective dose 50 (IFID50)/ml were stored at -70 C. Preparation of test plates. The SAM cells, either freshly prepared or stored at -70 C, were diluted at a concentration of 2 x 106 cells/ml in RPMI-1640 medium supplemented with 10% bovine fetal serum, 0.15% sodium bicarbonate, and antibiotics. The cells were seeded in 96-well microplates and incubated for 3-5 hr at 37 C in an atmosphere of 5% CO,. The supernatant was removed and replaced with 0.2 ml of the medium containing an optimal dilution of virus, except wells in rows 1 and 7, which served as uninfected controls. The plates were incubated again at 37 C for 40-60 hr. The medium was removed when the cell monolayers exhibited an early stage of cytopathic effects (CPE), and 0.2 ml of 100% cold ethanol was added to each well. The plates were then stored at -20 C until use. One test plate from each batch was pretested by IFA with 6 negative and 6 positive reference sera. Only batches producing the same results or a difference of no more than 2-fold in 2 of 12 sera were used for testing samples. The reference sera were collected from pigs before and after experimental infection with SIRS virus. Materials and methods Indirect fluorescent antibody test. After ethanol fixation, Cell culture and virus. Swine alveolar macrophage (SAM) the plates were dried at room temperature for 10-20 min cells were prepared as described previously.8 Stock virus of and washed once with 0.3 ml of phosphate-buffered saline (PBS) (pH 7.2) per well. Test sera were diluted 4-fold serially From the Departments of Clinical and Population Sciences (Yoon, from 1:4 to 1:1,024 in a clean U-bottom 96-well microplate, Joo, Christianson, Kim, Morrison, Dial) and Veterinary Diagnostic and 0.1 ml of each dilution was transferred to the infected Medicine (Collins), College of Veterinary Medicine, University of cell plate. A 1:4 dilution of each serum was added to control wells. The plates were incubated for 45 min at 37 C and then Minnesota, St. Paul, MN 55108. washed 4 times by adding 0.3 ml PBS/well and removing Received for publication January 8, 1992. 144 Downloaded from vdi.sagepub.com at MCGILL UNIVERSITY LIBRARIES on April 6, 2015

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Figure 1. Intensity of indirect fluorescent antibody staining of SIRS virus-infected swine alveolar macrophage monolayers in 96-well microplates. Dilutions of a positive reference serum are 1: 16 (A), 1:64 (B), 1:256 (C), and 1:4 in negative cell control (D).

PBS after l-2 min. After washing, the plates were incubated at 37 C for 45 min with 0.05 ml of an optimal dilution (1: 200-400 in PBS) of rabbit anti-swine IgG conjugated with fluorescein isothiocyanate.a Following incubation, the plates were washed again as above and examined under a fluorescent microscope using a 5 x objective lens. The IFA titers were recorded as the highest serum dilutions with specific cytoplasmic fluorescence. Negative and positive reference sera (IFA titers < 1:4 and 154, respectively) were included in each test. IFA antibody response in experimentally infectedpigs. Six 6-wk-old pigs from a specific-pathogen-free herd were housed in an isolation room. Four pigs were infected intranasally with SIRS virus isolate MN- 1 a (IFID50 103.5/ml), and 2 pigs served as contact controls. Serum samples were collected at 2-7-day intervals from each pig for 4 wk and tested by the IFA method. Serum samples of the pigs collected at time of

infection and from pigs from the source farm during this study were used as negative controls. Preexposure and 1426 days postexposure (PE) sera from 13 pregnant sows infected experimentally in a previous study2 were also used. IFA titers infield samples. A total of 720 serum samples from 25 swine farms collected during the last 10 yr were selected from a serum bank. The selection was based on the previous history of SIRS on each farm. Each sample was tested for IFA to SIRS virus, and the distribution of IFA titers for each farm was analyzed.

Results Various conditions were compared and optimal conditions were determined in the preparation of test plates and the procedure for the IFA test. The optimal conditions for the stock virus (10 IFID 50/ml) used in the 4.5

Table 1. Antibody titer response in 6-week-old pigs following experimental infection with SIRS virus.

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Table 2. Indirect fluorescent antibody (IFA) titers of sows experimentally infected with SIRS virus.

test plate preparations were virus dilution at 1:40 with an incubation period of 48 hours. In a comparison of the incubation times with test sera or fluorescent conjugate for the test, 45 minutes at 37 C was preferred because no difference was found between 45 and 60 minutes but an incubation time of 30 minutes occasionally resulted in lower antibody titers and less intense fluorescence. Typical wells of cell fluorescence in an IFA test are illustrated in Fig. 1. In the experimentally infected pigs, the IFA titers were first detected 7 days PE in 3 of 4 pigs, and the highest titers were reached between 11 and 21 days PE. The first antibody in the 2 contact pigs was detected at 11 days after contact (Table 1). All 45 sera collected from pigs of the source farm (farm 25) during the experiment were < 1:4. All 13 sow sera were < 1:4 at time of infection but were positive (between 1:64 and 21: 1,024) at 14-26 days PE (Table 2). Results of IFA tests for 720 serum samples from 25 different farms are shown in Table 3. Of 344 sera from 15 different swine farms with known clinical histories of SIRS, 257 (74.7%) of the sera had IFA titers of ≥ 1: 4, and 87 (25.3%) of the sera were < 1:4. Of the 257 positive sera, 235 (9 1.4%) had IFA titers of ≥ 1:64. In contrast, 371 of 376 sera (98.7%) from 10 farms with no history of SIRS were < 1:4. Five sera from 2 SIRSnegative farms were positive, with titers of 1: 16- 1:256. The earliest positive serologic results were shown in the samples collected during March 1986. Discussion The IFA is a simple serologic test to detect and quantitate antibody to SIRS virus in swine sera. Initially, 2-fold serum dilutions were used, but the end point

Table 3. Distribution of indirect fluorescent antibody (IFA) titers to SIRS virus in sera collected from swine farms with or without known clinical history of SIRS.

determination was often not clear. This problem was overcome by using 4-fold serum dilutions. Occasionally, end points for a few sow sera were difficult to obtain because of intense background fluorescence. After some experience, specific staining could be differentiated by observing intracytoplasmic fluorescence in the infected cells. Laboratory technicians learned the procedures and performed the test with minimal guidance. Approximately 2 hours were required for the test procedure, but 6-8 plates for 80-120 samples can be tested at the same time by 1 person. Therefore, a trained technician can test at least 100 samples, including serum sorting, in 3-4 hours. In the preparation of test plates, a careful predetermination for optimal virus dilutions and incubation times is necessary. Usually, lower virus dilutions and shorter incubation times resulted in better fluorescence on the monolayers. Virus incubation times varied depending on the batch of SAM cells used. In our lab-

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oratory, 20-30 test plates were routinely prepared in results were highly specific and sensitive. Detection of a batch, and the plates were stored up to 1 month at adult pigs with IFA titers of ≥ 1:64 is an indication of -20 C. The IFA test appeared to be highly specific active infection. and sensitive, although limited numbers of sera from Acknowledgements experimental animals were used to evaluate the test. This work was supported in part by a grant from the MinNone of the 18 experimental pig sera collected at 0, 3 and 5 days PE or the 13 preinoculation sow sera were nesota Agricultural Experimental Station (No. MIN-62-059). positive. Also, none of 45 adult sera collected from the Sources and manufacturers source farm during the experiment were positive. In addition, 98.7% of the sera collected from farms with a. Organon Teknika-Cappel, Malvern, PA. no history of SIRS were negative. These results indicate References that the IFA test is highly specific. Sensitivity of the IFA test with experimentally infected animals was 75% 1. Benfield DA, Nelson E, Collins JE, et al.: 1992, Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate for the sera collected 7 days PE and 100% for the sera ATCC VR-2332). J Vet Diagn Invest 4:127-133. collected 9-28 days PE. 2. Christianson WT, Collins JE, Benfield DA, et al.: 1991, ExperThe IFA titers ranged from 1:64 to 1: 1,024 in piglets imental reproduction of swine infertility and respiratory syndrome in pregnant sows. Am J Vet Res 53:485-488. at and beyond 9 days PE and were 1:64−≥ 1: 1,024 in sows between 14 and 26 days PE. In sera from clinically 3. Collins JE, Benfield DA, Christianson WT, et al.: 1992, Isolation of swine infertility and respiratory syndrome virus (isolate ATCC affected farms, 235 of 257 positive sera (9 1.4%) had VR-2332) in North America and experimental reproduction of IFA titers ranging from 1:64 to ≥ 1: 1,024. These results the disease in gnotobiotic pigs. J Vet Diagn Invest 4: 117-126. suggest that a ≥ 1:64 IFA titer indicates infection and 4. Patron DJ, Brown IH, Edwards S, et al.: 1991, Blue ear disease of pigs. Vet Rec 128:617. could be considered a safe cutoff value. Results of this study, specifically those from farm 1, 5. Terpstra C, Wensvoort G, Pol JMA: 1991, Experimental reproduction of porcine epidemic abortion and respiratory syndrome indicate that SIRS virus infection occurred as early as (mystery swine disease) by infection with Lelystad virus: Koch’s April 1986 in this country. Farm 18 in this study had postulates fulfilled. Vet Q 13: 131-136. a record of severe reproductive problems of unknown 6. Wensvoort G, Terpstra C, Pol JMA, et al.: 1991, Mystery swine disease in The Netherlands: the isolation of Lelystad virus. Vet cause during June 1983, but no serologic evidence for Q 13:121-130. SIRS was found. Two of 37 sera from farm 23 had antibody titers of 1:64 and 1:256, but no clinical signs 7. White M: 1991, Blue ear disease in pigs. Vet Rec 128:574. 8. Yoon IJ, Joo HS, Christianson WT, et al.: 1992, Isolation of a of SIRS were recognized on this farm, which suggests cytopathic virus from weak pigs on farms with a history of swine the existence of subclinical SIRS virus infection. infertility and respiratory syndrome. J Vet Diagn Invest 4:139143. The IFA described was simple to perform, and the

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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 syn...
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