Diagnosis of persistent or prolonged porcine reproductive and respiratory syndrome virus infectionsD. Benfielda, J. Nelsona, K. Rossowb, C. Nelsona, M. Steffenc and R. Rowlandc
a Department of Veterinary Science, South Dakota State University, Box 2175, Brookings, SD 57007, USA
b Department of Veterinary Diagnostic Investigation, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, USA
c Department of Biology-Microbiology, South Dakota State University, Box 2140D, Brookings, SD 57007, USA
Abstract - Porcine reproductive and respiratory syndrome (PRRS) emerged as the leading viral disease of swine in this decade. The virus causes abortions, increased numbers of stillborn pigs and pre-weaning mortality in the breeding herd. Respiratory disease with an increased incidence of secondary viral and bacterial infections is common in weaned pigs and most pigs are delayed to market by several weeks. While some herds experience only acute PRRS outbreaks followed by a return to normal production, many continue to have prolonged problems related to the persistence of the PRRS virus (PRRSV) in the herd. The goal of the present study was to use an in utero exposure model to produce persistently infected pigs and then use these pigs to identify tissue(s) and cell type(s) that support PRRSV replication in acute and persistent PRRSV infections. This information in combination with the antibody response of these pigs to PRRSV was used in an attempt to develop a method to identify persistently infected pigs. In this study, sows were intranasally exposed to PRRSV at 90 days gestation and allowed to carry litters to term. The PRRSV was isolated from the serum of 75% of the newborn piglets born to the infected sows. The remaining piglets became virus isolation (VI) positive and seropositive within two weeks after birth. The number of VI-positive serum samples began to decline at 14 days after farrowing, which correlated with peak levels of circulating antibody and appearance of PRRSV neutralizing activity in the serum. The last VI-positive serum sample was obtained at 48 days after farrowing. Virus replication in pigs that died within 21 days after birth is representative of acute PRRS infections. Cells supporting PRRSV replication were observed in all organs and tissues, which is consistent with the macrophage as the principal target of PRRSV replication. Pigs that survived longer than 21 days eventually recovered and showed no clinical signs of PRRS. At 63 days after farrowing, approximately two weeks beyond the last VI-positive serum sample, pigs were randomly removed, euthanazied and tissues examined of PRRSV replication using VI, RT-PCR and in situ hybridization (ISH). All lung samples obtained from pigs at 63 days and beyond were negative for infectious virus, and viral nucleic acid by VI, RT-PCR and ISH, respectively. The absence of virus in lung and serum correlated with the absence of detectable virus in other non-lymphoid organs, including salivary gland, liver, and kidneys. In contrast, PRRSV was isolated from tonsil and lymph nodes up to 132 days after farrowing. RT-PCR confirmed the presence of virus and ISH demonstrated that cells (macrophages) within these tissues supported virus replication. The biological relevance of low-level PRRSV replication in the lymphoid organs was evaluated by determining if PRRSV was transmitted to uninfected pigs. Sentinel pigs were placed in contact with persistently infected pigs at 64 (2 sentinels), 84 (2 sentinels), 98 (one sentinel), 112 (1 sentinel) and 260 (4 sentinels) days after birth. All sentinel pigs were allowed to mingle freely with the persistently infected pigs for one week. Pigs were then removed to an isolation room and serum was monitored weekly for virus and antibody. All sentinel pigs commingled with principals at 64 to 112 days post-farrowing were VI positive within one week after introduction, demonstrating the virus was shed to naïve pigs. No transmission occurred to sentinels placed in contact at 260 days after birth. The results indicate that PRRSV first establishes an acute systemic infection during which virus replication is observed in all organs and tissues. This pattern of virus replication is consistent with the tissue macrophage as the primary host cell for PRRSV. Eventually, virus disappears from the non-lymphoid organs and the blood. Even though neutralizing and non-neutralizing antibodies are present, virus replication is still detected within tonsil and lymph nodes. In the acute phase of PRRS infections, serum and most tissues are excellent samples for detection of the virus by virus isolation and/or PCR. Lymphoid tissues appear to be the best specimens for detection of the virus in prolonged or persistently infected pigs. Tonsils may be the tissue of choice, because virus can be either isolated or detected by PCR longer than in serum. Serum is not a valuable predictor of prolonged infections by PRRSV. However, combinations of declining antibody titers on ELISA with negative serum PCR may indicate elimination of the PRRSV by the host.
Corresponding author: D. Benfield Tel.: (1) 605 688 4317; fax: (1) 605 688 6003;
© INRA, EDP Sciences 2000