Porcine reproductive and respiratory syndrome virus: routes of excretion.

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 (p.i.), 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 p.i. 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 plan an important role in PRRSV transmission. These results support previous reports of persistent infection with PRRSV with prolonged recovery of virus from tonsils of swine.

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.

Antibody-dependent enhancement (ADE) of porcine reproductive and respiratory syndrome virus (PRRSV) infection in pigs.

Infection of porcine alveolar macrophages by the porcine reproductive and respiratory syndrome virus (PRRSV) was significantly enhanced in vitro by antibody raised against the PRRSV isolate ISU-P (p < 0.01). Increased yields and infection rates were highly correlated (r = 0.95) and the ratio of yield to infection rate was greater than 1.4, suggesting that more than one mechanism was responsible for enhanced infection. Antibody-dependent enhancement (ADE) of infection was also demonstrated in vivo using a completely randomized block design (n = 16). The mean level and duration of viremia were greater (p < 0.05) in pigs injected with subneutralizing amounts of PRRSV-specific IgG prior to virus challenge than in control pigs injected with normal IgG. In contrast, virus replication was significantly (p < 0.01) inhibited in pigs with neutralizing antibody titers of 4 log2. The period of time that subneutralizing levels of antibody can persist and contribute to ADE of PRRSV infection was estimated in 4 pigs injected with PRRSV-specific IgG to yield an initial neutralizing antibody titer of 3.8 log2. Neutralizing activity declined to undetectable levels by day 37 postinjection (PI). ADE activity was first detected in undiluted sera on day 20 PI and persisted through day 62 PI. Western immunoblot analysis of sera collected between days 37 and 62 PI detected antibodies specific for the 15-kDa nucleocapsid and 26-kDa glycosylated envelope proteins. These results strongly suggest that ADE has the potential to contribute to the pathogenesis of PRRSV infection and is mediated by antibody specific for the 26-kDa envelope protein.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a devastating disease in swine. The presence and transmission of PRRSV by boar semen has been demonstrated by using a swine bioassay. In this assay, 4- to 8-week-old pigs were inoculated intraperitoneally with semen from PRRSV-infected boars. Seroconversion of these piglets indicated the presence of PRRSV in semen. Seroconversion in gilts has also been demonstrated following artificial insemination with semen from PRRSV-infected boars. These methods of detecting PRRSV in boar semen are time-consuming, laborious, and expensive. The objective of this study was to develop a reliable and sensitive PCR assay to directly detect PRRSV in boar semen. Primers from open reading frames 1b and 7 of the PRRSV genome were used in nested PCRs. Virus was detected at concentrations as low as 10 infectious virions per ml in PRRSV-spiked semen. Specificity was confirmed by using a nested PCR and a 32P-labeled oligonucleotide probe. The primers did not react with related arteriviruses or other swine viruses. The PCR assay showed good correlation with the swine bioassay, and both methods were superior to virus isolation. To consistently identify PRRSV in boar semen, the cell fraction was separated by centrifugation at 600 x g for 20 min, a lysis buffer without a reducing agent (2-mercaptoethanol) was used, and nondiluted and 1:20-diluted cell fractions were evaluated by PCR. PRRSV was not reliably detected in the seminal plasma fraction of boar semen.

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.

Excretion of porcine reproductive and respiratory syndrome virus in semen after experimentally induced infection in boars.

Four boars intranasally inoculated with porcine reproductive and respiratory syndrome (PRRS) virus were monitored for 56 days after exposure for changes in semen characteristics and for the presence of virus in the semen. Clinically, 2 of 4 boars had mild respiratory signs of 1 day's duration after infection. Changes in appetite, behavior, or libido were not detected. All boars seroconverted on the indirect fluorescent antibody and serum virus neutralization tests by day 14 after inoculation. Virus was isolated from serum between days 7 and 14 after inoculation. During the monitoring period, semen volume decreased and pH correspondingly increased; however, this change began 7 to 10 days prior to infection. Differences in sperm morphologic features, concentration, or motility between the preinfection and postinfection samples were not observed. The PRRS virus was detected in semen at the first collection in each of the 4 boars (ie, 3 or 5 days after challenge exposure). Virus was detected in nearly all semen samples collected from the 4 infected boars through days 13, 25, 27, and 43, respectively. Neither gross nor microscopic lesions attributable to PRRS virus were observed in tissues collected at the termination of the experiment (day 56), and virus isolation results from reproductive tissues were negative.

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 10(5)-10(7) 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 > or = 95% of the infected pigs, and 3) the time from initial antibody detection to determination of > or = 95% as positive. By day 10 postchallenge, no statistically significant difference in diagnostic sensitivity was observed among the 8 tests compared in the study.

Influence of dose and route on transmission of encephalomyocarditis virus to swine.

The susceptibility of swine to infection with encephalomyocarditis virus (EMCV) was assessed. Transmission of EMCV in a single exposure by gavage or intranasal routes was highly dose dependent. In a direct comparison, animals were exposed to EMCV by gavage, intranasal, intramuscular, intratracheal, or transdermal routes. A higher proportion of animals exposed by transdermal (5/5), intratracheal (5/5), or intramuscular (5/5) routes than those exposed by intranasal (2/5) or gavage (3/5) routes became infected. The large quantity of virus required to infect animals intranasally or orally suggests that transmission by these routes may not occur routinely in the field. Transmission of EMCV by wound contamination (transdermally) has not been reported previously. Although EMCV was recovered from rectal, external genitalia, and pharyngeal swabs, there was no evidence of pig-to-pig transmission of EMCV from experimentally infected animals to comingled sentinels.

Evaluation of the safety and efficacy of a thymidine kinase, inverted repeat, gI, and gpX gene-deleted pseudorabies vaccine.

A thymidine kinase (TK), inverted repeat, glycoprotein I (gI) and glycoprotein X (gpX) gene-deleted modified live virus pseudorabies vaccine was evaluated for safety in swine and for efficacy in protecting swine against challenge with pseudorabies virus (PRV). Safety was evaluated by inoculating pregnant gilts intravenously and 3-day-old pigs intracerebrally with the vaccine. Efficacy was evaluated by 1) vaccinating 3-day-old pigs with a minimal protective dose intranasally and then challenging with PRV 3 weeks postvaccination or 2) vaccinating weaned pigs with a standard field dose intramuscularly and then challenging with PRV 4 weeks postvaccination. The pigs vaccinated intranasally remained clinically normal following vaccination and challenge with PRV. The pigs vaccinated intramuscularly remained clinically normal following vaccination, but mild respiratory signs were seen in some of the vaccinated pigs following challenge with PRV. Humoral immune response was evaluated with enzyme-linked immunosorbent assays (ELISAs) and a serum virus neutralization test. All of the intramuscularly vaccinated pigs became gI and gpX positive on differential ELISAs following challenge. All of the intranasally vaccinated pigs were seropositive on the indirect gI ELISA following challenge, but not all of the pigs were seropositive on the blocking gI ELISA or the gpX ELISA 3 weeks postchallenge.

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 (gI) 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.

Experimental transmission of an apparent viral pneumonia in conventional and gnotobiotic pigs.

Endemic pneumonia in five- to eight-week-old pigs induced microscopic lesions of proliferative interstitial pneumonia which were compatible with a viral aetiology. The disease was transmitted experimentally to conventional and gnotobiotic pigs by means of a lung homogenate filtered through a 0.22 micron filter. No common viral respiratory pathogens of pigs were isolated. Two types of virus particles were observed in cell culture by electron microscopy; one was about 70 nm in diameter and had an envelope and short surface spicules, the other also had an envelope, was elongated, pleomorphic, measured 80 x 320 nm and was coated by antibodies.

A serosurvey of swine and free-living species on Iowa farms for antibodies against encephalomyocarditis virus.

Swine and free-living nonporcine species from 20 Iowa swine farms were surveyed for antibodies against encephalomyocarditis virus (EMCV). The microtitration serum neutralization test was used, and antibody titers >/= 1:8 were considered positive. The overall prevalence in 267 sows in various groups was 37.8% (range 20-86%). The prevalence in sows maintained in total confinement was significantly lower than in sows not maintained in total confinement (p = 0.01). Prevalence in sows was not associated with other herd characteristics or with abundance estimates of rats and/or mice.Free-living animals tested included 74 domestic cats (Felis domestica), 203 house mice (Mus musculus), 15 mice of the genus Peromyscus, nine Norway rats (Rattus norvegicus), 34 opossums (Didelphis virginiana), 14 raccoons (Procyon lotor), and seven striped skunks (Mephitis mephitis). Of these, the only seropositive animals were two domestic cats.The results of this study failed to implicate the free-living species surveyed as important reservoirs of EMCV for swine, and suggested that swine themselves and/or some species not included in this survey are the main reservoir of EMCV for swine in Iowa.

The epidemiology of toxoplasmosis on Iowa swine farms with an emphasis on the roles of free-living mammals.

Multiparous sows from 19 central Iowa swine farms were tested for antibodies against Toxoplasma gondii by the modified direct agglutination test. Antibody titers of 1:32 or greater were considered positive. Rodents, domestic cats, opossums (Didelphis virginiana), raccoons (Procyon lotor) and striped skunks (Mephitis mephitis) were live-trapped on each farm and similarly tested for antibodies. The overall prevalence of T. gondii antibodies in the species tested was 39/273 (14.3%) swine, 31/74 (41.9%) cats, 2/588 (0.3%) house mice (Mus musculus), 0/21 mice of the genus Peromyscus, 0/9 Norway rats (Rattus norvegicus), 1/34 (2.9%) opossums, 4/14 (28.6%) raccoons and 2/7 (28.6%) striped skunks. The overall prevalence was significantly greater in adult cats versus juvenile cats, adult male cats versus adult female cats, and adult raccoons versus juvenile raccoons. The prevalence of T. gondii antibodies in sows was compared with the prevalence in each non-swine species on a farm basis in order to identify existing associations. The prevalence in sows (and each of the non-swine species) was also analyzed on a farm basis for association with farm characteristics or swine management practices, including the degree of confinement of swine, population size and average parity of breeding female swine, estimated cat population size, and estimated mouse and rat abundance. Average titers of seropositive animals were compared on a species basis. The prevalence in sows which were totally and continuously confined was lower than that in sows which were not totally and continuously confined. The prevalence in sows from farms with an average parity of less than 2.0 was significantly lower than that in sows from farms with an average parity of 2.0 or greater. These results suggested that the prevalence of T. gondii antibodies in swine increased with age and that prevalence in swine could be reduced through total confinement. No associations could be established between prevalence in sows and prevalence in non-swine species or other farm characteristics/swine management practices. However, the high prevalence of T. gondii antibodies in cats suggested that fecal contamination of the environment by cats may be the most significant source of toxoplasmosis for swine. The extremely low prevalence of T. gondii antibodies in house mice suggested that this species was not an important source of T. gondii for swine in Iowa.

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 10(4) PFU 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 (10(2.3) PFU) 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.

Seroprevalence of antibodies against encephalomyocarditis virus in swine of Iowa.

A total of 2,614 swine from 104 herds located throughout Iowa were tested for antibodies against encephalomyocarditis virus (EMCV) by use of the microtitration serum neutralization test. The sample was composed of 587 sows and gilts and 2,027 finishing swine. A statistically significant (P less than 0.002) difference was observed between prevalence in sows and gilts (17.2%) and that in finishing swine (12.2%). Breeding swine maintained in total confinement (20.5%) had significantly (P = 0.04) higher prevalence than did breeders maintained in other types of housing (12.1%), whereas prevalence in finishing swine raised in total confinement (6.4%) was significantly (P = 0.02) lower that in finishers not raised in total confinement (13.6%). Association was not detected between prevalence and herd size or between prevalence and season of the year. Adjusting for test specificity and sensitivity, the true prevalence of EMCV infection in swine in Iowa was estimated to be 13.8% in breeding stock and 8.5% in finishing swine. On a herd basis, 89.4% (93/104) of the herds had one or more EMCV-positive swine.

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.

Reproductive failure associated with Leptospira interrogans serovar bratislava infection of swine.

Specimens from 17 swine herds experiencing reproductive failure were examined for Leptospira interrogans serovar bratislava. Clinical signs observed in these herds included stillborn pigs, weak neonatal pigs, and abortion. Diagnostic tests used to determine L. interrogans serovar bratislava infection were bacteriologic culture, serologic assays to detect antibodies, and immunofluorescence. Examination of fetal serum for antibodies against serovar bratislava and a fluorescent antibody test were the most practical diagnostic procedures.