Enzymatic amplification of latent pseudorabies virus nucleic acid sequences.

To investigate various aspects of the latency of pseudorabies virus in swine (PRV, suid herpesvirus 1) we developed in vitro nucleic acid amplification methods based upon the polymerase chain reaction. Primers flanking a 156-bp region of the pseudorabies virus gp II gene were annealed to purified PRV DNA as well as DNA isolated from the trigeminal ganglia of swine latently infected with PRV and subjected to PCR amplification. Following amplification, 100 fg of PRV DNA was visualizable on stained gels and 1 fg (equivalent to 6 viral genome copies) was detectable when amplification was combined with blot hybridization. PRV-specific DNA sequences which remained undetectable by direct blot hybridization assays were amplified to levels visualizable on ethidium-bromide-stained gels in 5 of 5 experimental latently infected animals. In addition, oligonucleotide primers specific for a 223-bp region of the PRV immediate-early gene (IE 180) were capable of amplifying overlapping latency associated transcripts (LATs), via a cDNA intermediate, in 6 of 6 latently infected swine. These nucleic acid amplification methods should be applicable to the investigation of PRV latency, and gene expression during latency and reactivation, in which few cells harbor latent virus.

DNA hybridization procedure to detect pseudorabies virus DNA in swine tissue.

A DNA hybridization technique was developed to detect the presence of pseudorabies virus (PRV) DNA. P Nick translated probes of high specific activity were prepared from transformed Escherichia coli plasmids into which Bacillus amyloliquefaciens H (Bam H1) restriction fragments of PRV DNA had been inserted. Swine cellular DNA and tissue culture PRV DNA were digested with Bam H1, separated by agarosegel electrophoresis, transferred onto nitrocellulose paper, hybridized to the radioactive probes, and washed under high stringency conditions; autoradiographs were then prepared. Under the optimal hybridization conditions described, the detection limit of these probes was 10(-11)g of PRV DNA. In reconstruction experiments, 3 of the selected probes cross hybridized with digested swine cellular DNA, and 4 probes did not. The addition of polyuridylic acid and polyguanylic acid to the hybridization reactions did not alter the amount of hybridization. The results indicated that this procedure may be useful for studying the latency of pseudorabies viral infection.

Role of the raccoon in the transmission of pseudorabies: a field and laboratory investigation.

Serum samples were collected from 42 raccoons trapped in a pseudorabies enzootic area of Missouri. All samples were negative for neutralizing antibodies to pseudorabies virus (PRV). Raccoons were orally exposed with one of four dose levels of PRV. The raccoons were found to be susceptible to moderately large doses of PRV tissue culture infective dose (10(4) - 10(5) TCID50). All raccoons given 10(3) TCID50 of PRV survived and did not develop clinical signs. The PRV was consistently isolated from tonsillar swabs collected from raccoons that died after exposure. The PRV was also isolated from the brain, tonsils, lungs, and salivary glands of raccoons that died. It was isolated from samples of the brain and tonsils collected from a raccoon 5 days after death. Neutralizing antibodies to PRV could not be detected in the serum of raccoons that survived PRV exposure. Tonsillar swabs and tissue samples collected from these raccoons were free of PRV. The results indicated that the raccoon may serve as a short-term reservoir for PRV, but it is unlikely to have an ipizootiologic role as a long-term, subclinical carrier of the virus.

Confirmation of pseudorabies virus infection, using virus recrudescence by dexamethasone treatment and in vitro lymphocyte stimulation.

A corticosteroid treatment regimen, together with peripheral blood lymphocyte transformation and virus isolation, were used as epizootiologic tools to study pigs seropositive to pseudorabies (PR) virus, but of unknown PR viral infection status. After treatment with 160 mg of dexamethasone daily for 5 days, PR virus was isolated from 2 of 3 adult littermate boars that carried antibodies to PR virus. These pigs also transferred infection to a specific-pathogen-free sentinel penmate . After dexamethasone treatment, increased lymphocyte stimulation indices were detected in all PR virus seropositive pigs. Pseudorabies virus was not isolated from a seronegative littermate gilt of the seropositive boars. The gilt also remained PR virus seronegative and negative for lymphocyte transformation. Before dexamethasone treatment, the 3 boars and 1 gilt were seronegative to the PR viral serum neutralization (SN) test at 11 weeks of age. At 13 weeks of age while still SN negative, these pigs were moved to a building and randomly penned with 76 other SN-negative pigs. At 18 weeks of age, all pigs in the building were PR virus seronegative, but at 25 weeks of age, the 3 boars were seropositive and the gilt was seronegative. There were no other pigs in the building that had antibodies to PR virus before the addition or after the removal of these pigs. Seemingly, the 3 boars were infected with PR virus before 13 weeks of age indicating that PR virus infection of swine may occur in the absence of detectable SN antibodies.

Detection of latent pseudorabies virus in porcine tissue, using a DNA hybridization dot-blot assay.

A DNA-hybridization dot-blot technique was developed to detect the presence of pseudorabies virus (PRV) DNA in porcine tissue. Seven 32P-nick translated probes of high specific activity were prepared from transformed Escherichia coli plasmids into which Bacillus amyloliquefaciens H (Bam HI) restriction fragments of PRV-DNA had been inserted. Samples of DNA that had been extracted from porcine tissue or from PRV grown in tissue culture were transferred to nitrocellulose paper, using a microsample filtration manifold and were hybridized to the probes under high-stringency conditions. Under optimal hybridization conditions, the minimum detection amount of PRV-DNA was 10(-11) g, which is equivalent to 1 copy of the PRV genome/80 host cells. Four probes did not show cross hybridization with DNA extracted from tissues of known PRV-negative swine, and these were subsequently used to detect PRV-DNA in infected porcine tissues. Generally, correlation between virus isolation and hybridization data was good for tissues from swine that had died of acute PRV infection. Furthermore, PRV-DNA was present in specific tissues of all 4 seropositive swine that had recovered from pseudorabies and in which no infective virus or viral products were detected at necropsy. Pseudorabies virus DNA was present in the rostralis cerebral cortex (n = 2) or in the medulla oblongata (n = 1) and trigeminal ganglion (n = 1). This probably indicated the portal of entry of the virus into the CNS. In another seropositive pig, there was evidence of a productive infection in the tonsils, although virus was not isolated in a tissue culture system.(ABSTRACT TRUNCATED AT 250 WORDS)

Pseudorabies virus infection in raccoons: a review.

Pseudorabies is a rarely reported disease of raccoons. Laboratory and field evidence of PRV infection suggests the raccoon is a "dead end" host with little opportunity for raccoon-to-raccoon spread of virus. All reported field cases have been associated closely with infected swine and swine have been considered the source of the raccoon infection. The clinical signs of PRV in raccoons closely resembles those of canine distemper and rabies virus infections. Infection with the latter viruses are considered more prevalent and likely to be mistaken for PRV infection. Both CD and rabies virus may be maintained in raccoon populations with raccoon-to-raccoon transfer while PRV may not. Differentiation of PRV, CD and rabies infections is best achieved by histopathologic analysis of lung and brain tissue, together with virus isolation. It is of utmost public health importance that wildlife authorities recognize the similarities between these diseases, together with the different epidemiologic behavior of the viruses and the means to differentiate clinical cases.

Serologic status of pseudorabies virus in growing-finishing pigs in quarantined herds.

It has been reported that pseudorabies virus (PRV) stops spreading within growing-finishing sections of a large percentage of infected farrow-to-finish herds. This study was designed to follow the PRV status of growing-finishing pigs in a sample of infected herds. Fifteen infected herds were selected, of which 11 had seropositive finishing pigs and 4 had seronegative finishing pigs. These herds were visited quarterly for one year, and a cross section of growing-finishing pigs was tested for the presence of anti-PRV antibodies. The 4 herds that initially were seronegative remained seronegative, whereas of the 11 herds initially seropositive, 4 remained seropositive, 4 became seronegative, and 3 became temporarily seronegative before becoming seropositive again. Three characteristics serologic profiles were observed: one indicating continued viral spread; one indicating no spread for at least the preceding 3 months; and one indicating that PRV spread had recently ceased in this section of the herd. Results of our study indicated that periodic monitoring of a cross section of the growing-finishing pigs for their PRV serologic status was valuable for determining whether PRV was actively spreading in this section of the herd.

Identification of pseudorabies virus-infected swine herds by evaluating the serostatus of boars or finishing pigs.

Data were collected from 39 Minnesota swine farms quarantined for pseudorabies virus (PRV) infection. Each herd was serologically evaluated for antibodies to PRV in the sows, boars, and finishing pigs. To identify PRV-seropositive swine herds, the Kappa statistic was used to estimate the effectiveness of evaluating the PRV serostatus of boars or of finishing pigs. Using the serostatus of all herd boars, the sensitivity (with 95% confidence interval) of identifying PRV-infected herds was 58 +/- 22%, and the specificity was 100 +/- 0%; Kappa statistic was 0.55. Using the serostatus of a representative sample of finishing pigs, the sensitivity of identifying PRV-infected herds (with 95% confidence interval) was 63 +/- 22%, and specificity was 87 +/- 23%; Kappa statistic was 0.40. The PRV serostatus of herd boars or of a representative sample of finishing pigs did not accurately reflect the PRV serostatus of the herd.

Epidemiologic monitoring following an episode of pseudorabies involving swine, sheep, and cattle.

In April 1978, an episode of pseudorabies involved swine, sheep, and cattle on a farm in north-central Missouri. The herd of 48 farrowing sows was vaccinated, then separated from the sheep and cattle by double fencing. Within 4 days, death losses in sheep and cattle ceased, indicating that separation by double fencing is an effective means of controlling interspecies virus spread. Monitoring of pseudorabies in sows and their vaccinated offspring was done by serum neutralization testing and virus culturing of pharyngeal swabs for the following 12 months. Pseudorabies virus carriers were in the sow herd 6 months after the initial episode. The serologic response to vaccination of the offspring from immune sows was poor, but pigs segregated from their dams at weaning remained free of detectable infection. It was concluded that segragation at weaning may be useful when expensive breeding lines must be salvaged from an infected herd.

Atypical staphylococcal mastitis in a dairy herd.

A herd of cattle with a history of increased prevalence of clinical and nonclinical mastitis was investigated. Bacteriologic analysis of milk samples indicated approximately 50% of the herd was producing milk containing coagulase-positive staphylococci. Of these staphylococcal isolates, 55% had characteristics consistent with those of human strains of staphylococci, based on hemolysin production and phage patterns. Human beings in contact with the herd were nasal carriers of these staphylococci, which produced a granulartype coagulase reaction in bovine plasma, rather than the usually expected clot-type reaction. In the herd, the staphylococci caused mainly nonclinical mastitis, which was largely unresponsive to antibiotic therapy.

Spread of pseudorabies virus among breeding swine in quarantined herds.

In theory, pseudorabies virus (PRV) may be eliminated from any size of breeding herd by phased test and removal if replacement gilts are not infected with PRV, culling decisions are partially based on PRV status, and the cull rate is higher than the incidence rate of PRV. Annual cull rates are commonly at least 50%, but little information exists on the incidence of PRV within enzootically infected swine herds. The purpose of this study was to develop a method by which spread of PRV could be detected among breeding swine within enzootically infected herds and to determine the incidence of PRV infection in these herds. Data were collected from 17 herds that were quarantined for PRV and ranged in size from 120 to 1,100 sows. At each herd, within the first 5 days of introduction, a group of approximately 30 replacement gilts was identified, vaccinated with a glycoprotein X-deleted PRV vaccine, and blood sample was collected. The owner of 1 herd had a nonvaccinated breeding herd and elected to leave incoming gilts nonvaccinated. After vaccination, blood samples were collected every 1 to 2 months for an average of 13.6 months. Serum samples from vaccinated gilts were tested for antiglycoprotein X antibodies by a specific differential ELISA. Samples from nonvaccinated gilts were evaluated by serum neutralization test. Product-limit method was used to estimate the probability of not becoming infected with PRV. Spread was detected in 7 of 8 herds that had more than 400 sows and in 2 of 9 herds that had less than 400 sows.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors associated with spread of pseudorabies virus among breeding swine in quarantined herds.

Knowledge of the factors that place susceptible gilts at highest risk of pseudorabies virus (PRV) infection in a quarantined herd is crucial to reduce spread of PRV within the herd. Cohorts of PRV seronegative gilts were monitored in 17 herds that were endemically infected with PRV to determine the location of breeding females at the time of infection with PRV and identify herd characteristics and management and housing factors that may influence spread of PRV in the breeding section of swine herds endemically infected with PRV. Blood samples were collected every 1 to 2 months for an average of 13.6 months. In addition, blood was collected from a representative sample of finishing pigs (greater than or equal to 20 weeks old) 3 times per year to determine their serologic PRV status. Incidence rates and relative risks of PRV infection were estimated for 4 areas of the breeding section: gestation barn, gilt pool, farrowing room, and breeding area. Overall, 28, 11, 8, and 2 females became infected with PRV in each of these areas, respectively. The greater number of females infected in the gestation barns, compared with the number of females infected in other locations, is probably a consequence of being at risk for a longer period rather than of a higher incidence rate. Herd size, common housing for gilts in the gilt pool and sows, and serologic pattern of PRV infection in finishing pigs were associated with the detection of spread of PRV in the breeding section of the 17 herds.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors associated with circulation of pseudorabies virus within swine herds.

Data were collected from 104 Minnesota swine farms quarantined for pseudorabies virus (PRV) infection. Each herd was serologically evaluated for the presence of antibodies to PRV in finishing pigs. Herd management practices, swine housing design, and disease profiles were described for each farm. Multiple logistic regression analysis was used to determine which factors were associated with circulation of PRV in the finishing pigs of farrow-to-finish farms. Sixty-seven (64%) of the herds had no serologic evidence of PRV circulation in the finishing section, whereas 37 herds (36%) contained at least one PRV seropositive finishing pig. The odds of a given finishing herd being seropositive for PRV were 2.85 times higher if the finishing pigs were housed in confinement (P = 0.01), 2 times higher if Actinobacillus (Haemophilus) pleuropneumoniae was a clinical problem in the herd (P = 0.03), 1.36 times less for each year that passed since the herd quarantine was issued (P = 0.01), 1.74 times higher if clinical signs of PRV were reported (P = 0.04), and 1.52 times higher if animal protein was included in at least one of the rations (P = 0.08).

Evaluation of the radial immunodiffusion enzyme assay for the detection of antibodies to pseudorabies virus.

The validity of radial immunodiffusion enzyme assay (RIDEA) as a diagnostic test for antibodies to pseudorabies virus (PRV) in porcine serum was determined. Serum samples from sows and offspring were tested for the presence of antibodies to PRV, using both the RIDEA and the PRV serum-neutralization (SN) test. Overall sensitivity and specificity of the RIDEA done on serums from the sows were 95.7% and 95.2%, respectively. This sensitivity compares with 97.3% sensitivity of the SN test of the same serums. In 658 swine serum samples from routine submissions to the University of Missouri-Columbia Veterinary Diagnostic Laboratory that were tested by the RIDEA, the calculated sensitivity and the specificity were 94.3% and 98.9%. The RIDEA and SN test were equally sensitive (99.0%) to detect antibodies resulting from infection with a field strain of virus. They had reduced sensitivity (RIDEA, 91.7%; SN test, 95.2%) in tests of serums from vaccinated sows. For the detection of passively transferred antibodies in young pigs, sensitivity of the RIDEA was 76.1%, and specificity was 100%. In all instances, RIDEA was 100% sensitive at SN titers of 1:16 or greater. In testing serum samples of swine after field virus infection, sensitivity and specificity of the RIDEA approximated those of the SN test. This reliability, together with its ease of performance, makes the RIDEA an ideal field test in programs to detect PRV-infected herds and in programs designed to free herds of PRV infection.

Factors associated with the seroprevalence of pseudorabies virus in breeding swine from quarantined herds.

Strategies for the elimination of pseudorabies virus (PRV) from swine herds include test and removal, offspring segregation, and depopulation/repopulation. The prevalence of PRV in a herd is a major factor in selection of the most appropriate strategy. The purpose of the study reported here was to describe the prevalence of PRV in adult swine in PRV quarantined herds in Minnesota, and to determine herd factors associated with the seroprevalence. Questionnaires describing the health history of the herd, management practices, and design of the swine facilities were obtained from the owners of 142 quarantined herds. Blood was collected from 29 finishing pigs over the age of 4 months, up to 29 adult females, and all herd boars. Factors considered to be significant in a bivariate analysis were combined in a stepwise multiple logistic regression analysis. The prevalence of PRV-seropositive adults in each herd was bimodally distributed among the 142 herds. In 42 (30%) of the herds, none of the females tested was seropositive, which represented the lower mode. At least 90% of the adults tested were seropositive in 30 (21%) of the herds and represented the higher mode. The odds of the breeding swine of a given herd having a PRV seroprevalence of greater than or equal to 20% as compared with having a seroprevalence of less than 20% was 1.654 times higher per 50 adults in the herd, 13.550 times higher if the finishing pigs were seropositive, 2.378 times higher if sows were housed inside during gestation, and 1.481 times lower per number of years since the imposition of quarantine.(ABSTRACT TRUNCATED AT 250 WORDS)

High- and low-challenge exposure doses used to compare intranasal and intramuscular administration of pseudorabies virus vaccine in passively immune pigs.

We compared 3 modified-live pseudorabies virus (PRV) vaccine strains, administered by the intranasal (IN) or IM routes to 4- to 6-week-old pigs, to determine the effect of high- and low-challenge doses in these vaccinated pigs. At the time of vaccination, all pigs had passively acquired antibodies to PRV. Four experiments were conducted. Four weeks after vaccination, pigs were challenge-exposed IN with virulent virus strain Iowa S62. In experiments 1 and 2, a high challenge exposure dose (10(5.3) TCID50) was used, whereas in experiments 3 and 4, a lower challenge exposure dose (10(2.8) TCID50) was used. This low dose was believed to better simulate field conditions. After challenge exposure, pigs were evaluated for clinical signs of disease, weight gain, serologic response, and viral shedding. When vaccinated pigs were challenge-exposed with a high dose of PRV, the duration of viral shedding was significantly (P less than 0.05) lower, and body weight gain was greater in vaccinated pigs, compared with nonvaccinated challenge-exposed pigs. Pigs vaccinated IN shed PRV for fewer days than pigs vaccinated IM, but this difference was not significant. When vaccinated pigs were challenge-exposed with a low dose, significantly (P less than 0.05) fewer pigs vaccinated IN (51%) shed PRV, compared with pigs vaccinated IM (77%), or nonvaccinated pigs (94%). Additionally, the duration of viral shedding was significantly (P less than 0.05) shorter in pigs vaccinated IN, compared with pigs vaccinated IM or nonvaccinated pigs. The high challenge exposure dose of PRV may have overwhelmed the local immune response and diminished the advantages of the IN route of vaccination.(ABSTRACT TRUNCATED AT 250 WORDS)

Pig herds having a single reactor to serum antibody tests to Aujeszky's disease virus.

The introduction of Aujeszky's disease virus into a herd of pigs usually results in a rapid spread of the virus and a high percentage of pigs become seropositive. However, herd monitoring for the virus occasionally reveals a single seropositive breeding pig, referred to as a single reactor. The seropositive status of single reactors may be due to previous vaccination against Aujeszky's disease, or to exposure to a field strain of the virus, or to a false positive reaction in the serological assay. During a monitoring programme in Minnesota, 30 pig herds with single serological reactors were detected. Twenty-seven of these single reactors from 19 herds were segregated from their herds immunosuppressed with dexamethasone. Aujeszky's disease virus was isolated from four of the 27 pigs. Three of the four herds subsequently had outbreaks of Aujeszky's disease, suggesting that some single reactors were infected with Aujeszky's disease virus and had the potential to spread the virus within and between herds.