Howard T. Hill

Porcine reproductive and respiratory syndrome virus: a persistent infection.

Persistent infection with porcine reproductive and respiratory syndrome virus (PRRSV) was shown in experimentally infected pigs by isolation of virus from oropharyngeal samples for up to 157 days after challenge. Four 4 week old, conventional, PRRSV antibody-negative pigs were intranasally inoculated with PRRSV (ATCC VR-2402). Serum samples were collected every 2 to 3 days until day 42 post inoculation (PI), then approximately every 14 days until day 213 PI. Fecal samples were collected at the time of serum collection through day 35 PI. Oropharyngeal samples were collected at the time of serum collection from 56 to 213 days PI by scraping the oropharyngeal area with a sterile spoon, especially targeting the palatine tonsil. Turbinate, tonsil, lung, parotid salivary gland, spleen, lymph nodes and serum were collected postmortem on day 220 PI. Virus isolation (VI) on porcine alveolar macrophage cultures was attempted on all serum, fecal and oropharyngeal samples, as well as tissues collected postmortem. Postmortem tonsil tissues and selected fecal samples were also assayed for the presence of PRRSV RNA by the polymerase chain reaction (PCR). Serum antibody titers were determined by IFA, ELISA and SVN. Virus was isolated from all serum samples collected on days 2 to 11 PI and intermittently for up to 23 days in two pigs. No PRRSV was isolated from fecal samples, but 3 of 24 samples were PCR positive, suggesting the presence of inactivated virus. Oropharyngeal samples from each pig were VI positive 1 or more times between 56 and 157 days PI. Oropharyngeal samples from 3 of 4 pigs were VI positive on days 56, 70 and 84 PI. Virus was isolated from one pig on day 157 PI, 134 days after the last isolation of virus from serum from this animal. Virus was isolated from oropharyngeal samples for several weeks after the maximum serum antibody response, as measured by IFA, ELISA and SVN tests. All tissues collected postmortem were VI negative and postmortem tonsil samples were also negative by PCR. An important element in the transmission of PRRSV is the duration of virus shedding. The results of this study provided direct evidence of persistent PRRSV infection and explain field observations of long-term herd infection and transmission via purchase of clinically normal, but PRRSV infected, animals. Effective prevention and control strategies will need to be developed in the context of these results.

Characterization of the humoral immune response to porcine reproductive and respiratory syndrome (PRRS) virus infection

The development of the humoral immune response against porcine reproductive and respiratory syndrome (PRRS) virus was monitored by an indirect fluorescent antibody (IFA) test, immunoperoxidase monolayer assay (IPMA), enzyme-linked immunosorbent assay (ELISA), and serum virus neutralization (SVN) test over a 105-day period in 8 pigs experimentally infected with ATCC strain VR-2402. Specific antibodies against PRRS virus were first detected by the IFA test, IPMA, ELISA, and the SVN test 9-11, 5-9, 9-13, and 9-28 days postinoculation (PI), respectively, and reached their maximum values by 4-5, 5-6, 4-6, and 10-11 weeks PI, respectively, thereafter. After reaching maximum value, all assays showed a decline in antibody levels. Assuming a constant rate of antibody decay, it was estimated by regression analysis that the ELISA, IFA, IPMA, and SVN antibody titers would approach the lower limits of detection by approximately days 137, 158, 324, and 356 PI, respectively. In this study, the immunoperoxidase monolayer assay appeared to offer slightly better performance relative to the IFA test, ELISA, and SVN test in terms of earlier detection and slower rate of decline in antibody titers. Western immunoblot analysis revealed that antibody specific for the 15-kD viral protein was present in all pigs by 7 days PI and persisted throughout the 105-day observation period. Initial detection of antibodies to the 19-, 23-, and 26-kD proteins varied among pigs, ranging from 9 to 35 days PI. Thereafter, the antibody responses to these 3 viral proteins of PRRS virus continued to be detected throughout the 105-day study period. These results clearly indicate that the 15-kD protein is the most immunogenic of the 4 viral proteins identified and may provide the antigenic basis for the development of improved diagnostic tests for the detection of PRRS virus antibodies.

Porcine reproductive and respiratory syndrome virus: routes of excretion.

Abstract This study was conducted to delineate potential sites of exit and duration of shedding of porcine reproductive and respiratory syndrome virus (PRRSV). Two experiments of 6 pigs each were conducted. Pigs were farrowed in isolation, weaned at 7 days of age, and housed in individual HEPA filtered isolation chambers. In each experiment, 3 pigs served as controls and 3 were inoculated intranasally with PRRSV (ATCC VR-2402) at 3 weeks of age. In a first experiment, on days 7, 14, 21, 28, 35, and 42 post inoculation (PI), pigs were anesthetized and intubated. The following samples were collected: serum, saliva, conjunctival swabs, urine by cystocentesis, and feces. Upon recovery from anesthesia, the endotracheal tube was removed, rinsed, and the rinse retained. In the second experiment, the sampling schedule was expanded and serum, saliva, and oropharyngeal samples were collected from day 55 to day 124 PI at 14 day intervals. Virus was isolated in porcine alveolar macrophages up to day 14 from urine, day 21 from serum, day 35 from endotracheal tube rinse, day 42 from saliva, and day 84 from oropharyngeal samples. No virus was recovered from conjunctival swabs, fecal samples, or negative control samples. This is the first report of isolation of PRRSV from saliva. Virus-contaminated saliva, especially when considered in the context of social dominance behavior among pigs, may play an important role in PRRSV transmission. These results support previous reports of persistent infection with PRRSV prolonged recovery of virus from tonsils of swine.

Failure to consider the antigenic diversity of porcine reproductive and respiratory syndrome (PRRS) virus isolates may lead to misdiagnosis.

Porcine reproductive and respiratory syndrome (PRRS) is a recently identified viral disease of swine that is a significant problem in swine producing regions throughout the world. Because of the potential economic impact of PRRS on swine production, extensive resources have been expended on the development of accurate diagnostic tests and efficacious vaccines. One of the potential obstacles to the success of these efforts is antigenic diversity among PRRS virus isolates. Antigenic diversity has been demonstrated between North American and European PRRS virus field isolates by using homologous and heterologous polyclonal swine antiserum in an immunoperoxidase monolayer assay. Antigenic differences between US and European PRRS virus isolates have also been demonstrated using a panel of 3 monoclonal antibodies (MAbs) specific for the 15kD protein of the PRRS virus: SDOW 17, which was generated against the PRRS virus isolate ATCC VR-2332, and EP147 and VO17, which were generated against the PRRS virus isolate SD1. All 3 MAbs reacted, as determined by fluorescence microscopy, with 63 of 63 US isolates originating from the north-central region of the United States. Additionally, MAb SDOW17 reacted with the Lelystad PRRS virus and 56 of 56 European isolates representing 8 different countries (Denmark, France, Germany, Italy, Luxemburg, The Netherlands, Spain, and The United Kingdom). However, neither MAb EP147 nor MAb VO17 reacted with any of these European isolates. Similar observations have been made by Canadian investigators, who reported that the same panel of MAbs reacted with 10 of 10 Canadian PRRS virus isolates. These observations suggest that MAb SDOW17 could be used as a universal diagnostic reagent for the detection of PRRS virus infection in clinical specimens. However, the following report indicates that North American isolates are antigenically more diverse than previously demonstrated and that it may not be possible to rely on a single MAb for diagnostic purposes.

Evaluation of rotavirus infection and diarrhea in Iowa commercial pigs based on an epidemiologic study of a population represented by diagnostic laboratory cases

Group A, B, and C rotaviruses were identified in 9% (96/1,048) of pig fecal specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory during 1987 and 1988. Six of the rotaviruses were group B, 5 were group C, and the remaining 89% were group A. Of the rotavirus cases with more than 1 serotype, 5 were multiple group A serotypes, 1 involved a group A and B serotype, and 1 included 2 group C serotypes. A retrospective epidemiologic evaluation of pig diarrhea in herds of origin was done using data obtained from the accession records of the rotavirus and 88 matched nonrotavirus pig diarrhea control cases. Herds from which rotavirus cases were derived experienced lower morbidity, mortality, and case fatality rates than matched control herds. The incidence of diarrhea decreased rapidly among all pigs from birth to 3 weeks of age. The peak incidence for piglet diarrhea occurred in February, and a moderate rise occurred in August-September. Definitive evidence for transmissible gastroenteritis virus was found in 12% of nonrotavirus cases but none of the rotavirus cases in which it was sought. Other pathogenic microorganisms were identified less frequently and inconsistently.

Evaluation of Serological Pseudorabies Tests for the Detection of Antibodies during Early Infection

Six enzyme-linked immunosorbent assays, a latex agglutination test, and the standard microtitration serum virus neutralization test were compared for their ability to detect antibodies against pseudorabies virus (PRV) during the early stages of infection. Thirty-five pigs were infected intranasally with 105-107 TCID50 of either the Iowa 4892 pneumotropic or the Becker strain of PRV. Blood samples were drawn from experimentally inoculated animals on days 4–10, 14, and 21 postchallenge. Test sensitivity estimates and comparisons among tests were made for each sampling day over the 21-day monitoring period. Results of this study demonstrated differences among tests in 1) the time from inoculation to initial antibody detection, 2) the time to detect ≥ 95% of the infected pigs, and 3) the time from initial antibody detection to determination of ≥ 95% as positive. By day 10 postchallenge, no statistically significant difference in diagnostic sensitivity was observed among the 8 tests compared in the study.

Comparison of in vitro replication and cytopathology caused by strains of canine distemper virus of vaccine and field origin

Three biological properties of canine distemper virus were examined to determine if any would consistently differentiate field from vaccine strains of the virus. The properties were the ability to (1) infect macrophages and epithelial cells, (2) produce distinct cytopathologic effect in alveolar and peritoneal macrophages and Vero cells, and (3) produce pocks on the chorioallantoic membrane of embryonated chicken eggs. Four vaccine strains and 5 field isolates were used in the study. The 5 field isolates were obtained directly from canine tissues. Of the 3 properties studied, only the comparison of the ability of the viruses to infect macrophages and epithelial cells was a consistent marker of virus origin. Virulent field isolates would only infect macrophage cultures, whereas the vaccine strains infected both types of cells. One avirulent field isolate from a case of old dog encephalitis reacted more like a vaccine strain by infecting both cell types.

Diagnostic Compatibility of a Thymidine Kinase, Inverted Repeat, gI, and gpX Modified Live Gene-Deleted PRV Vaccine with Three Differential ELISAs

The differential pseudorabies virus (PRV) vaccines currently in use in the USA have deletions of the genes coding for the glycoprotein I (gl) and/or glycoprotein X (gpX). The absence of gI and/or gpX allow for the serologic differentiation of vaccinated swine from PRV-infected swine using differential enzyme-linked immunosorbent assays (ELISAs). A newly developed pseudorabies vaccine virus has 4 deletions of the viral genome: the genes coding for gI, gpX, and thymidine kinase and a portion of the repeat region to attenuate the virus. The purpose of this work was to evaluate the diagnostic compatibility of the gI/gpX gene-deleted vaccine with 3 differential vaccines and 3 differential ELISAs currently in use. Pigs vaccinated 3 times with the gI/gpX gene-deleted vaccine remained seronegative on the 3 differential ELISAs tested. Pigs previously vaccinated with either a gI or gpX gene-deleted vaccine and then vaccinated with the gI/gpX gene-deleted vaccine remained seronegative on the respective gI or gpX differential assay.

Detection of Pseudorabies Virus Infection in Subunit-Vaccinated and Nonvaccinated Pigs using a Nucleocapsid-Based Enzyme-Linked Immunosorbent Assay

The potential of a pseudorabies virus (PRV) nucleocapsid protein (NC)-based enzyme-linked immunosorbent assay (ELISA) as a screening assay for PRV infection in subunit-vaccinated and nonvaccinated pigs was studied. The NC-ELISA compared favorably to a commercial ELISA for detecting PRV infection in nonvaccinated pigs. Virus-specific antibody was first detected by the NC-ELISA between days 14 and 21 in 5 pigs challenged intranasally with 104PFU of virus. Antibody continued to be detected in these pigs through day 42, when the experiment was terminated. The NC-ELISA also detected antibody in 23 of 24 pigs from PRV-infected herds. In contrast, the commercial ELISA detected antibody 1 week earlier than the NC-ELISA in experimentally infected pigs but failed to detect antibody in 3 naturally exposed pigs that were identified by the NC-ELISA. Infection in these animals was confirmed by radioimmunoprecipitation analysis. The potential usefulness of the NC-ELISA for detecting infection in vaccinated pigs was also evaluated. The nucleocapsid-specific antibody responses of 10 PRV envelope glycoprotein subunit-vaccinated pigs were monitored prior to and following nasal exposure to a low dose (1023PFU) of PRV. Sera were collected periodically for 113 days after infection. Nucleocapsid-specific antibody responses measured by the NC-ELISA remained below the positive threshold before challenge but increased dramatically following virus exposure. Maximum ELISA responses were obtained on day 32 postchallenge (p.c.). Mean ELISA responses decreased thereafter but remained well above the positive threshold on day 113 p.c. PRV nucleocapsid protein can be used effectively as antigen in the ELISA for detecting PRV infection in both nonvaccinated and subunit-vaccinated pigs.