|
Diagnostic Procedures for PRRS By Dr. P.G. Halbur, P.S. Paul, B.H. Janke, K.J.
Schwartz, J.J. Andrews A diagnosis of Porcine Reproductive and Respiratory Syndrome (PRRS) relies on compiling information from the clinical history of the herd, serology, pathology, and ultimately on isolation of the PRRS virus (PRRSV). This paper will briefly review common diagnostic tests that are now in use and give a more in-depth update on the use of the newly developed immunoperoxidase test for detection of PRRSV antigen in tissue sections. Three excellent references that review the diagnosis of PRRSV have been published in the last year.1-3 Clinical signs vary widely between farms and thus are not that useful for a definitive diagnosis except in the case of a severe acute outbreak in naive herds which experience abortion storms, increased numbers of stillborn pigs, and severe neonatal and nursery pig pneumonia. Presently, the most common clinical presentation is pneumonia and miscellaneous bacterial problems in 3-10 week old pigs. Many PRRSV-positive herds have no apparent reproductive or respiratory problems. Gross lesions that are very suggestive of PRRSV infection in growing pigs include lymphadenopathy, and pneumonia. The most consistent experimentally reproducible gross lesion in 3-10 week old pigs inoculated with PRRSV is lymphadenopathy. In particular, iliac and mediastinal lymph nodes are often 3-10 times normal size, tan in color, and sometimes cystic. The lymph nodes are not normally hyperemic as seen in bacterial septicemia. The degree of grossly visible pneumonia is very much strain dependent. In general the lungs fail to collapse and have a patchy distribution of 10-80 percent tan-colored consolidation. Three histologic lesions are consistent with PRRSV infection. Interstitial pneumonia is commonly observed and is characterized by septal infiltration with mononuclear cells, type 2 pneumocyte proliferation, and the presence of necrotic cells in the alveolar spaces. Nonsuppurative perivascular myocarditis and hyperplastic lymph nodes are commonly observed in the subacute stages of disease. Encephalitis is less often observed. Lesions in the fetus and placenta are rarely observed. Most laboratories are routinely using either an indirect-fluorescent antibody (IFA) test or immunoperoxidase monolayer assay (IPMA) for serum antibody detection. With both the IFA and IPMA, one must subjectively determine endpoints and thus the tests are not automated. Serum virus (SVN) neutralization tests have also been developed and ELISA tests are currently used in some research laboratories. Antibodies detected by the IFA test usually appear within 10-14 days of exposure but may be relatively short-lived, sometimes disappearing in 3 months. Antibodies detected by ELISA usually appear within 3 weeks but duration is unknown. SVN antibodies usually are not detected until 4-5 weeks post exposure. The SVN test is considered less sensitive in acute disease, but, improvements have been made in the SVN test using seronegative porcine serum supplementation. SVN titers reportedly are measurable longer and thus may be better suited for detection of positive animals in chronically infected herds. Clinicians are requesting and interpreting antibody titers to determine the appropriate time for vaccination and/or implementation of management or control strategies. It does not appear that serology tests are yet adequate enough to use antibody titer levels to make major management decisions. For now it may be more appropriate to look for a change from seronegative to seropositive status or at least a 4-fold increase in titer. Looking for an increasing percentage of seropositive pigs in a particular age group over time in a herd can be quite useful to determine where the virus is maintained and actively spreading. Keep in mind that sows infected in the early 3rd trimester and aborting near term will likely not show increasing titers. Virus isolation (VI) provides a definitive diagnosis but has some limitations. Virus is rarely isolated from stillborn or autolyzed aborted fetuses. Sows infected early in the last trimester may have transient viremia and not abort until late term. Dead pigs of any age are not the best samples for VI because the virus does not survive well at room temperature. Tissues should be removed from the carcass, packaged separately, and refrigerated as soon as possible. The best tissues for virus isolation are tonsil, lung, lymph nodes, and spleen. Serum is an excellent sample for virus isolation since viremia is often prolonged in growing pigs, the sample is easy to handle, and it can be quickly chilled and processed for shipment to laboratories. Variation between laboratories in the ability to isolate PRRSV is high because tests, reagents, cell lines, and media have not been standardized. The efficacy of isolation varies because not all North American strains will grow on each cell line. Rapid methods for detection of PRRSV antigen in tissues have been developed. Frozen tissue-section IFA tests have been used with limited success. We have recently described the development of an immunoperoxidase test (IPT) for use on formalin-fixed tissue. The IPT appears to be quite useful to confirm the presence of active infection. Examination of one section of each of the following tissues from 26 pigs experimentally inoculated with ATCC VR 2386 PRRSV allowed us to detect the virus in 18/26 lungs, 26/26 tonsils, 15/26 mediastinal lymph nodes, and 14/26 tracheobronchial lymph nodes. The pigs in this study were killed over a 28 day period post inoculation. We were able to detect the virus in at least one tissue in every pig necropsied up to 10 days post inoculation. We have recently described the complete technique for the streptavidin-biotin based immunoperoxidase technique for PRRSV antigen detection in porcine tissues.4 Briefly, after endogenous peroxidase removal by 3 percent hydrogen peroxide and digestion with 0.05 percent protease (Protease XIC, Sigma Chemical Company, St. Louis, MO) primary monoclonal antibody ascites fluid diluted 1:1000 in TRIS/PBS is added for 16 hours at 4C in a humidified chamber. The monoclonal antibody SDOW-17 (Dr. David Benfield, South Dakota State University) has been recently characterized and recognizes a conserved epitope of the PRRSV nucleocapsid protein.5 Biotinylated goat anti-mouse linking antibody (Dako Corporation, Carpintera, CA) is used and followed by treatment with peroxidase-conjugated streptavidin (Zymed Laboratories, South San Francisco, CA) and incubation with 3, 3'-diaminobenzidine tetrahydrochloride (Vector Laboratories Inc., Burlingame, CA) and finally staining with hematoxylin. The use of histopathology, tissue immunoperoxidase antigen detection techniques, virus isolation procedures and serology has allowed us to offer a fairly rapid and reliable diagnosis of PRRSV infection. Continued progress needs to be made in standardizing these techniques and improving their sensitivity and specificity.
Selected References 1. Van Alstine WG, Stevenson GW, Kanitz CL: 1993, Diagnosis of porcine reproductive and respiratory syndrome. Swine Health and Production Volume 1, Number 4, pp24-28.2. Christianson WT, Joo HS: 1994, Porcine reproductive and respiratory syndrome: A review. Swine Health and Production Volume 1, Number 2, pp10-28.3. Goyal SM: 1993 Porcine reproductive and respiratory syndrome. J Vet Diagn Invest 5:656-664.4. Halbur PG, Andrews JJ, Huffman EL, Paul PS, Meng X, Niyo Y: 1994, Development of a streptavidin-biotin immunoperoxidase procedure for the detection of porcine reproductive and respiratory syndrome virus antigen in porcine lung. Accepted for publication. J Vet Diagn Invest.
|