Risk factors associated with clinical signs of vesicular stomatitis and seroconversion without clinical disease in Colorado horses during the 2014 outbreak.

Vesicular stomatitis (VS) is caused by a contagious rhabdovirus that affects horses, cattle, and swine. Clinical signs of vesicular stomatitis virus (VSV) infection in pigs and cattle are indistinguishable from foot-and-mouth disease (FMD), a foreign animal disease and reportable disease in the United States (Rodriguez et al., 2000). A VS epidemic occurred in the Rocky Mountain region in 2014-15. A study was conducted in Colorado to evaluate horse- and management-level factors associated with VS. For a horse to be considered a clinical VS horse, there were two requirements. First, clinical VS horses had to have clinical signs consistent with VS, including one or more of the following: vesicles, ulcers, erosions or crusting on the muzzle, nares, lips, oral or nasal mucosa, ears, ventrum, udder or penile sheath, or coronary band lesions. Second, clinical VS horses had to have laboratory confirmation of VSV exposure via virus isolation from lesions or a positive complement fixation test performed on sera. All non-clinical horses residing on VSV-affected premises enrolled in the study were evaluated for exposure (i.e., seroconversion) to VSV. Overall, management and housing data were collected from 334 horses on 48 premises in Colorado. Approximately one-third (31.4%) of enrolled horses were clinical cases and two-thirds (68.6%) were controls. Three premises-matched logistic regression models were constructed in SAS using backward elimination (P-value < 0.05) after univariate screening of a priori-selected variables (P-value < 0.20). Model outcomes included differences in characteristics and management of 1) clinical and nonclinical horses, 2) exposed and unexposed horses, and 3) exposed nonclinical and unexposed nonclinical horses. Overall, factors most strongly associated with risk of being a VS clinical horse were access to pasture (P-value = 0.002), and pregnancy status (P-value = 0.001). Factors most strongly associated with VSV exposure among horses were access to pasture (P-value = 0.003) and lack of any insect control (P-value = 0.001). The only factor associated with VSV-exposed nonclinical horses compared with unexposed VSV horses was contact with clinical horses (P-value = 0.013). There were no associations identified regarding clinical horses compared with exposed nonclinical horses. With regard to severity of lesions (severe vs. moderate or mild), no variables met the criteria for inclusion in the multivariable model. Results of this study provide evidence that pasture access and fly control are important factors associated with VSV exposure.

Active and latent ovine herpesvirus-2 (OvHV-2) infection in a herd of captive white-tailed deer (Odocoileus virginianus).

Malignant catarrhal fever (MCF) is the clinical manifestation of infection of certain ruminant species with one of a group of pathogenic gammaherpesviruses known as MCF viruses. Cattle and numerous exotic ruminant species are susceptible to clinical disease that may be sporadic or occasionally epidemic in nature. The most common MCF virus worldwide is ovine herpesvirus (OvHV)-2. Reservoir hosts such as sheep, carry and excrete OvHV-2, but do not develop clinical signs, while clinically susceptible species develop severe and often fatal disease. The existence of latent infection in clinically susceptible hosts is poorly understood, but is documented in some ruminant species. Twenty-six animals from a captive herd of white-tailed deer (Odocoileus virginianus) died and were examined from October 2006 to December 2010. Fifteen of these animals (58%) showed clinical signs and gross and microscopical lesions consistent with MCF, while 11 (42%) did not. Polymerase chain reaction (PCR) amplification yielded product consistent with OvHV-2 DNA in samples of spleen from all 26 deer. To examine the possibility of latent infection in this herd, peripheral blood mononuclear cells were examined by PCR for OvHV-2 DNA, and the test was positive in 23/32 (72%) clinically normal deer. Archived serum samples were used to examine the history of MCF exposure in the herd using a competitive enzyme-linked immunosorbent assay, which demonstrated that 10/40 (25%) deer tested had MCF viral antibodies, with nine deer being seropositive over multiple years. Combined with previous observations in deer and other species, these results suggest the existence of latent infection of white-tailed deer with OvHV-2.

Identification and analysis of the first 2009 pandemic H1N1 influenza virus from U.S. feral swine.

The first case of pandemic H1N1 influenza (pH1N1) virus in feral swine in the United States was identified in Texas through the United States Department of Agriculture (USDA) Wildlife Services' surveillance program. Two samples were identified as pandemic influenza by reverse transcriptase quantitative PCR (RT-qPCR). Full-genome Sanger sequencing of all eight influenza segments was performed. In addition, Illumina deep sequencing of the original diagnostic samples and their respective virus isolation cultures were performed to assess the feasibility of using an unbiased whole-genome linear target amplification method and multiple sample sequencing in a single Illumina GAIIx lane. Identical sequences were obtained using both techniques. Phylogenetic analysis indicated that all gene segments belonged to the pH1N1 (2009) lineage. In conclusion, we have identified the first pH1N1 isolate in feral swine in the United States and have demonstrated the use of an easy unbiased linear amplification method for deep sequencing of multiple samples.

Simultaneous infection of pigs and people with triple-reassortant swine influenza virus H1N1 at a U.S. county fair.

Influenza-like illness was noted in people and pigs in attendance at an Ohio county fair in August 2007. The morbidity rate in swine approached 100% within 1-2 days of initial clinical signs being recognized, and approximately two dozen people developed influenza-like illness. Triple-reassortant swine H1N1 influenza viruses were identified in both pigs and people at the fair. The identified viruses (A/Sw/OH/511445/2007, A/Ohio/01/2007, and A/Ohio/02/2007) were similar to H1N1 swine influenza viruses currently found in the U.S. swine population. This case illustrates the possibility of transmission of swine influenza in settings where there is close human/swine interaction.

Genetic variation and phylogenetic analyses of the ORF5 gene of acute porcine reproductive and respiratory syndrome virus isolates.

Swine herds in the US have experienced recent outbreaks of a severe form of porcine reproductive and respiratory syndrome (designated acute or atypical PRRS) characterized by abortion and high mortality in pregnant sows. Most of the affected herds had been vaccinated with modified live-vaccines (MLVs) against PRRS. To explore the possible mechanism of the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. The complete ORF5 gene of eight acute PRRSV isolates from herds experiencing acute PRRS outbreaks in Iowa and North Carolina was amplified and sequenced. Sequence analyses revealed that these acute PRRSV isolates shared 88-95% nucleotide and 88-96% amino acid sequence identities to each other, 87-97% nucleotide and 84-96% amino acid sequence identities with other North American PRRSV isolates and the MLVs. Most of the amino acid substitutions locate in the putative signal sequence and two short hypervariable regions at the amino terminus. The ORF5 gene sequence of the acute PRRSV isolate 98-37120-2 from a non-vaccinated swine herd in Iowa is very closely related to that of the RespPRRS MLV, with 97% nucleotide and 96% amino acid sequence identities. Phylogenetic analysis revealed that all eight acute PRRSV isolates are clustered within the North American genotype. Several minor branches that are not associated with geographic origins were also identified within the North American genotype. One acute PRRSV isolate (98-37120-2) is clustered with the RespPRRS MLV and several Danish isolates that were confirmed to be derived from the RespPRRS MLV. The ORF5 gene sequences of other seven acute isolates are more related to those of several earlier PRRSV isolates and the PrimePac MLV than to that of the RespPRRS MLV. Our results showed that the acute PRRSV isolates analyzed in this study differed from each other in ORF5 genes, although they all clustered within the North American genotype. The data from this study do not fully support the hypothesis that the emergence of acute PRRS is due to reversion of MLVs to a pathogenic phenotype, as only one of the eight acute isolates was shown to be very closely related to the RespPRRS MLV.

A comparison of diagnostic assays for the detection of type A swine influenza virus from nasal swabs and lungs.

Nasal swabs and lung samples from pigs experimentally infected with H1N1 swine influenza virus (SIV) were examined for the presence of SIV by the indirect fluorescent antibody assay, immunohistochemistry, cell culture virus isolation, egg inoculation, and 2 human enzyme immunoassays (membrane enzyme immunoassay, microwell enzyme immunoassay). Egg inoculation was considered to be the gold standard for assay evaluation. The 2 human enzyme immunoassays (EIA) and egg inoculation agreed 100% for the prechallenge nasal swabs. Agreement on SIV identification in nasal swabs with egg inoculation following challenge was considered to be good to excellent for membrane EIA (kappa = 0.85) and microwell EIA (kappa = 0.86). Agreement on SIV identification in lung tissue with egg inoculation following challenge was good to excellent for membrane EIA (kappa = 0.75), fair for microwell EIA, fluorescent antibody, and cell culture virus isolation (kappa = 0.48, 0.64, 0.62, respectively), and poor for immunohistochemistry (kappa = 0.36). No assay was 100% accurate, including the "gold standard," egg inoculation. In light of this information, it is important to consider clinical signs of disease and a thorough herd history in conjunction with diagnostic results to make a diagnosis of SIV infection.

Evolution of swine H3N2 influenza viruses in the United States.

During 1998, severe outbreaks of influenza were observed in four swine herds in the United States. This event was unique because the causative agents, H3N2 influenza viruses, are infrequently isolated from swine in North America. Two antigenically distinct reassortant viruses (H3N2) were isolated from infected animals: a double-reassortant virus containing genes similar to those of human and swine viruses, and a triple-reassortant virus containing genes similar to those of human, swine, and avian influenza viruses (N. N. Zhou, D. A. Senne, J. S. Landgraf, S. L. Swenson, G. Erickson, K. Rossow, L. Liu, K.-J. Yoon, S. Krauss, and R. G. Webster, J. Virol. 73:8851-8856, 1999). Because the U.S. pig population was essentially naive in regard to H3N2 viruses, it was important to determine the extent of viral spread. Hemagglutination inhibition (HI) assays of 4, 382 serum samples from swine in 23 states indicated that 28.3% of these animals had been exposed to classical swine-like H1N1 viruses and 20.5% had been exposed to the triple-reassortant-like H3N2 viruses. The HI data suggested that viruses antigenically related to the double-reassortant H3N2 virus have not become widespread in the U.S. swine population. The seroreactivity levels in swine serum samples and the nucleotide sequences of six additional 1999 isolates, all of which were of the triple-reassortant genotype, suggested that H3N2 viruses containing avian PA and PB2 genes had spread throughout much of the country. These avian-like genes cluster with genes from North American avian viruses. The worldwide predominance of swine viruses containing an avian-like internal gene component suggests that these genes may confer a selective advantage in pigs. Analysis of the 1999 swine H3N2 isolates showed that the internal gene complex of the triple-reassortant viruses was associated with three recent phylogenetically distinct human-like hemagglutinin (HA) molecules. Acquisition of HA genes from the human virus reservoir will significantly affect the efficacy of the current swine H3N2 vaccines. This finding supports continued surveillance of U.S. swine populations for influenza virus activity.

Characterization of immune response of young pigs to porcine circovirus type 2 infection.

A longitudinal study was conducted to characterize the immune response of young swine to infection with porcine circovirus type 2 (PCV-2). Five 8-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally and intramuscularly with a field isolate of PCV-2 at a concentration of 10(4) TCID50/mL. Along with monitoring for clinical signs and viremia, serum samples were collected from all pigs at day 0 and thereafter every 7 days postinoculation (PI) until the termination of the study on day 35 PI. No clinical signs were observed in any of the animals during the study period. In all pigs, PCV-2 was detected by polymerase chain reaction (PCR) in serum samples collected on days 7, 14, and 21 PI. Viral DNA and antigens were detected by in situ hybridization and immunohistochemistry in tonsil, spleen, medial iliac lymph nodes, and ileum collected from each pig at the end of the study. Collectively, naïve young swine were shown to be susceptible to PCV-2. Virus-specific antibody was detected by an indirect fluorescent antibody (IFA) assay on day 14 PI, but virus-neutralizing antibody was not detected until day 28 PI. As neutralizing antibodies developed, cross-reactivity with PCV type 1 (PCV-1) also developed on the IFA test. Western immunoblot analysis revealed three PCV-2 proteins with molecular masses of 28 kd, 28.5 kd, and 35 kd. The 35-kd protein was also demonstrated in PCV-1, suggesting that this protein induced the cross-reactivity between PCV types 1 and 2. Antibody to the 28-kd protein was detected on day 14 PI and later, indicating that this protein was the most immunogenic. Because of its immunogenicity and specificity to PCV-2, and 28-kd protein might provide the antigenic basis for the development of diagnostic tests for detection of PCV-2 antibody.

Oral vesicular lesions in horses without evidence of vesicular stomatitis virus infection.

OBJECTIVE: To report clinical and serologic findings in horses with oral vesicular lesions that were consistent with vesicular stomatitis (VS) but apparently were not associated with VS virus (VSV) infection. DESIGN: Serial case study. ANIMALS: 8 horses. PROCEDURE: Horses were quarantined after appearance of oral lesions typical of VS. Severity of clinical signs was scored every 2 to 5 days for 3 months. Serum samples were tested for antibodies by use of competitive ELISA (cELISA), capture ELISA for IgM, serum neutralization, and complement fixation (CF). Virus isolation was attempted from swab specimens of active lesions. RESULTS: 2 horses with oral vesicular lesions on day 1 had antibodies (cELISA and CF) against VSV; however, results of CF were negative by day 19. Five of the 6 remaining horses were seronegative but developed oral lesions by day 23. Virus isolation was unsuccessful for all horses. CONCLUSIONS AND CLINICAL RELEVANCE: Horses were quarantined for 75 days in compliance with state and federal regulations. However, evidence suggests that oral lesions were apparently not associated with VSV infection. The occurrence in livestock of a vesicular disease that is not caused by VSV could confound efforts to improve control of VS in the United States and could impact foreign trade. Vesicular stomatitis is of substantial economic and regulatory concern.

Emergence of H3N2 reassortant influenza A viruses in North American pigs.

In late summer through early winter of 1998, there were several outbreaks of respiratory disease in the swine herds of North Carolina, Texas, Minnesota and Iowa. Four viral isolates from outbreaks in different states were analyzed, both antigenically and genetically. All of the isolates were identified as H3N2 influenza viruses with antigenic profiles similar to those of recent human H3 strains. Genotyping and phylogenetic analysis demonstrated that the four swine viruses had emerged through two different pathways. The North Carolina isolate is the product of genetic reassortment between human and swine influenza viruses, while the others arose from reassortment of human, swine and avian viral genes. The hemagglutinin genes of the four isolates were all derived from the human H3N2 virus circulating in 1995. It remains to be determined if either of these recently emerged viruses will become established in the pigs in North America and whether they will become an economic burden.

Genetic reassortment of avian, swine, and human influenza A viruses in American pigs.

In late summer through early winter of 1998, there were several outbreaks of respiratory disease in the swine herds of North Carolina, Texas, Minnesota, and Iowa. Four viral isolates from outbreaks in different states were analyzed genetically. Genotyping and phylogenetic analyses demonstrated that the four swine viruses had emerged through two different pathways. The North Carolina isolate is the product of genetic reassortment between H3N2 human and classic swine H1N1 influenza viruses, while the others arose from reassortment of human H3N2, classic swine H1N1, and avian viral genes. The hemagglutinin genes of the four isolates were all derived from the human H3N2 virus circulating in 1995. It remains to be determined if either of these recently emerged viruses will become established in the pigs in North America and whether they will become an economic burden.

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.

Evaluation of serological tests for the detection of pseudorabies gE antibodies during early infection.

Two enzyme-linked immunosorbent assays (ELISAs) and a particle concentration fluorescence immunoassay (PCFIA) were compared for their ability to detect antibodies against pseudorabies virus (Aujeszky's disease virus) glycoprotein E (gE) in the early stages of infection in pigs previously vaccinated with gE-deleted pseudorabies vaccines. Seventy pigs were included in the study. Five groups of 6 pigs each were vaccinated with one of 5 different pseudorabies virus (PRV) gE-deleted vaccines, and subsequently infected intranasally with 10(5.6) TCID50 of the Iowa 4892 pneumotropic strain of PRV. This entire procedure was repeated using 10(4.6) TCID50 of the Rice strain of PRV. Five unvaccinated control pigs were also challenged with each virus strain. Three control pigs died before seroconverting, leaving 67 pigs for comparison. Blood samples were drawn from experimentally inoculated pigs on the day of vaccination, the day of challenge, and on 4-10, 14, and 21 days postchallenge (DPC). Serology test sensitivity estimates and comparisons among tests were made for each sampling day. Results of this study demonstrated differences among the tests in the time from inoculation to initial antibody detection, and the time to detect 50% and 75% of the infected pigs. The average time until first detection of pigs as seropositive for gE antibodies by PCFIA was 7.5 DPC. The blocking ELISA detected pigs as seropositive an average of 8.8 DPC, and the indirect ELISA first detected gE antibodies by 9.3 DPC. Fifty percent of the pigs were detected as seropositive by days 7, 8, and 9 for the PCFIA, blocking ELISA, and indirect ELISA, respectively. Similarly, 75% of the pigs were detected as seropositive by days 8, 9, and 10 for the PCFIA, blocking ELISA, and indirect ELISA, respectively. All pigs were detected as seropositive by 14 DPC for all 3 tests.

General overview of PRRSV: a perspective from the United States.

Four years after the report of its discovery, porcine reproductive and respiratory syndrome virus (PRRSV) continues to challenge swine producers, veterinary practitioners, and animal health researchers in the United States. The prevalence of infection is high--60% to 80% of herds is a reasonable estimate--but the clinical effects of infection vary widely among farms. In many herds, infection is unapparent and productivity seemingly unaffected. Some infected herds report occasional respiratory disease outbreaks in young pigs, or periodic outbreaks of reproductive disease, and a few herds experience severe, chronic disease problems, particularly in young pigs. In these herds, secondary infections with viral or bacterial pathogens, particularly Salmonella choleraesuis, Streptococcus suis, or Haemophilus parasuis typically occur concurrently with PRRSV infections. Understanding why some herds undergo devastating episodes of clinical disease and others show no apparent effects is central to solving the problem of clinical PRRS for swine producers. Understanding the ecology and epidemiology of PRRSV is the key to preventing and controlling PRRSV in the future. The objective of this article is to review recent developments in these areas.

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.

Navigating the wards: teaching medical students to use their moral compasses.

The upsurge in formal medical ethics training stems from the desire for more compassionate, less "dehumanized" physicians who can competently face the ethical dilemmas posed by technologic advances and resource constraints. How best to encourage ethical thinking and behavior among medical students remains an open question. However, the authors argue that medical ethics education suffers from an overreliance on strategies that target ethical thinking, with relative inattention to students as ethical actors in specific clinical contexts. In order to produce ethically competent physicians, medical educators must not only teach students to understand and learn from the dilemmas that shape their moral world but also train them to respond to those dilemmas appropriately. The authors discuss current practices in ethics education and how traditional approaches may not equip students with the types of moral "navigating skills" they need to become ethical physicians. They illustrate how medical students can and do learn norms of ethical behavior on the wards and argue why medical education ought to focus more explicitly on this aspect of clinical training. They conclude by recommending ways medical educators can encourage ethical thinking and behavior throughout the entire course of medical training.

Persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars.

Four seronegative adult boars were intranasally inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332. Serum and semen were collected 2-3 times weekly for over 100 days postinoculation (DPI). Serum samples were assayed for PRRSV by virus isolation (VI) and a polymerase chain reaction (PCR) and screened for antibodies to PRRSV using the indirect fluorescent antibody (IFA) and virus neutralization (VN) tests. Semen was assayed for PRRSV RNA by PCR. Virus and viral RNA was detected in the serum of all boars within 1 DPI by Vi and/or PCR. However, VI results indicated that viremia was transient and occurred from 1 to 9 DPI. Viral RNA was detected in serum from 1 to 31 DPI. In the acute stage of the infection, PRRSV RNA was detected in serum by PCR prior to the presence of viral RNA in semen. The PRRSV RNA was detected in semen as early as 3 DPI and persisted for 25 DPI in 2 of the boars and 56 and 92 DPI in the remaining 2 boars. Detection of PRRSV RNA in semen occurred 2-8 and 28-35 days prior to the detection of antibodies by IFA and VN, respectively. PRRSV was isolated from the bulbourethral gland of the boar that shed viral RNA in semen for 92 DPI. These results suggest that PRRSV RNA can be detected by PCR in boar serum and semen, and may persist for variable periods of time. Viremia and the serologic status of the boar are not adequate indicators of when PRRSV or PRRSV RNA is being shed in the semen. Preliminary findings also indicated that neither shipping stress nor reinoculation with homologous PRRSV resulted in viremia or viral RNA shedding in semen.

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.

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.