Effect of challenge dose and route on porcine reproductive and respiratory syndrome virus (PRRSV) infection in young swine.

Porcine reproductive and respiratory syndrome virus (PRRSV) is perceived to be highly infectious because of the rapid spread of the virus through populations of domestic swine throughout the world. However, no information has been published on the minimum infectious dose of PRRSV and the effect of challenge dose on clinical response. In this experiment, ten groups of pigs (n = 3 per group) were inoculated with one of five different quantities (10(1)-10(5) fluorescent foci units per millilitre) of PRRSV (isolate ISU-P) by either intramuscular or intranasal routes. Clinical signs and body temperature were monitored for 21 days. Serum was collected periodically throughout the study period to monitor the presence of virus in serum and the early immune response of pigs. A 2-mL inoculum containing 10(1) fluorescent foci units of virus per millilitre was found sufficient to achieve infection by either route. Time to onset of clinical signs was highly associated with challenge dose (P < 0.01), regardless of route of exposure. However, no dose- or route-dependent differences in the severity of clinical manifestation were observed. No significant differences in the time of onset or degree of humoural immune response to PRRSV infection were observed between different treatment groups. However, intramuscular exposure appeared to induce a more uniform antibody response compared to intranasal exposure. These results confirmed that PRRSV is highly infectious; a fact that should be taken into consideration when designing strategies for the prevention and control of PRRSV.

Acute effects of bilateral lung volume reduction surgery on lung and chest wall mechanical properties.

STUDY OBJECTIVES: To characterize acute changes in the dynamic, passive mechanical properties of the lungs and chest wall, elastance (E) and resistance (R), caused by lung volume reduction surgery (LVRS). DESIGN: Prospective data collection. PATIENTS: Nine anesthetized/paralyzed patients with severe emphysema. INTERVENTIONS: Bilateral LVRS. MEASUREMENTS AND RESULTS: From measurements of airway and esophageal pressures and flow during mechanical ventilation throughout the physiologic range of breathing frequency (f) and tidal volume (VT), E and R of the total respiratory system (Ers and Rrs), lungs (EL and RL), and chest wall (Ecw and Rcw) immediately before and after LVRS were calculated. After surgery, Ers, EL, Rrs, and RL were all greatly increased at each combination off and VT (p<0.05). Ecw and Rcw showed no consistent changes (p>0.05). The increases in EL were greatest in those patients with the lowest residual volumes, highest FEV1 values, and highest maximum voluntary ventilations measured 3 months preoperatively (p<0.05); the increases in RL were greatest in those patients with the lowest preoperative residual volumes (p<0.05). The largest increases in RL were in those patients with the largest decreases in residual volume and total lung capacity, measured 3 months postoperatively, caused by LVRS (p<0.05). CONCLUSION: Acute effects of LVRS are large increases in lung elastic tension and resistance; these increases need to be considered in immediate postoperative care, and can be predicted roughly from results of preoperative pulmonary function tests.

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.

Studies of porcine reproductive and respiratory syndrome (PRRS) virus infection in avian species.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently recognized virus of swine. As a newly emerging virus, much of the basic information regarding PRRSV is in the process of discovery. We report three experiments with PRRSV in birds, and a fourth experiment to evaluate the infectivity and transmissibility of avian-derived PRRSV in swine. Experiment 1 compared the susceptibility of Muscovy ducks, Mallard ducks, guinea fowl, and chickens to PRRSV. Birds were exposed to PRRSV (ATCC VR-2402) in drinking water and virus isolation was attempted from feces collected from cages. Based on the duration of fecal shedding of the virus, this experiment showed that Mallard ducks were particularly susceptible to PRRSV. Experiment 2 was done in mallards to corroborate and augment the observations of experiment 1. Virus was isolated from pooled mallard feces up to 25 days post exposure (PE) and from the intestinal contents of 8 of 20 birds euthanized on day 38 PE. No gross or microscopic lesions were observed in ducks collected between 0 and 15 days PE. Experiment 3 evaluated the infectivity and transmissibility of mallard-derived PRRSV in mallards. A cage of mallards orally exposed to PRRSV shed the virus in feces. Exposure of a second cage of mallards to feces from the first cage resulted in fecal shedding of PRRSV by birds in cage two. In turn, exposure to feces from the second cage led to fecal shedding by mallards in a third cage. Experiment 4 assessed the infectivity and transmissibility of mallard-derived virus in swine. Pigs intranasally exposed to PRRSV isolaed from mallard feces in experiment 2 became viremic, seroconverted by ELISA, and transmitted the virus to sentinel swine. Collectively, these studies show that the possibility exists for avian species to be involved in the epidemiology of PRRSV. This is the first report of PRRSV infection in a species other than swine.

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)

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.

Expression of Aleutian mink disease parvovirus capsid proteins in a baculovirus expression system for potential diagnostic use.

A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and VP2 was inserted into the polyhedron gene of Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed by the recombinant virus, AcADV-1, in Spodoptera frugiperda-9 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western immunoblot analysis (WIA) indicated that synthesis of both VP1 and VP2 was being directed by AcADV-1. Fluorescence microscopic examination of AcADV-1-infected S. frugiperda-9 cells indicated that the recombinant protein was present within the nucleus of the cells, and electron microscopic examination of these cells revealed the presence of small particles 23-25 nm in diameter. Structures resembling empty ADV capsids could be purified on CsCl density gradients, thus indicating that the ADV proteins were self-assembling. The antigenicity of recombinant VP1 and VP2 was evaluated by WIA. Sera collected from 16 mink prior to infection with ADV did not react with VP1 and VP2. Ten sera collected from mink with counter current immunoelectrophoresis (CIE) titers greater than 4 (log2) reacted with VP1 and VP2 in WIA. Two of 6 sera with CIE titers of 4 and 1 of 14 sera with CIE titers < 4 reacted with the recombinant proteins. These results suggest that baculovirus recombinant ADV capsid proteins may be useful as diagnostic antigens.

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.

Antibody response of pseudorabies virus subunit-vaccinated pigs to viral nucleocapsid proteins following low-dose virus challenge of immunity.

The antibody response to pseudorabies virus nucleocapsid proteins (NCP) was evaluated by the western immunoblot analysis before and after challenge of immunity by nasal inoculation of 10(2.3) plaque-forming units of virus in 10 pigs that had been vaccinated with pseudorabies virus envelope glycoproteins. Antibody to 5 NCP with molecular mass of 140, 63, 41, 34, and 23 kD was first detected in vaccinated and nonvaccinated pigs on day 14 after challenge of immunity. Antibody to 2 of the 5 NCP continued to be detected through day 113 in 9 of 10 vaccinated pigs. Beyond day 32, antibody to NCP was not detected in 1 vaccinated pig. The 23-, 34-, and 41-kD proteins were the most immunogenic. Antibody to each of these proteins was first detected on day 14 in 10, 10, and 8 pigs, respectively. Seven, 6, and 8 pigs, respectively, were antibody-positive for these proteins on day 113. The 140- and 63-kD proteins were the least immunogenic. Antibody to these proteins was detected in 8 and 9 pigs, respectively, on day 14, and in 4 and 5 pigs, respectively, on day 113. Chi-square analysis for dependency indicated that the antibody response to the 140- and 63-kD proteins was interdependent. These results suggested that combinations of NCP may be useful as nonvaccine diagnostic antigens.

Studies on the ability of a 98-kilodalton pseudorabies virus diagnostic antigen to detect latent infections induced by low-dose exposure to the virus.

The effect of low-dose challenge of immunity with pseudorabies virus (PRV) on subunit-vaccinated pigs was studied in 2 experiments. In the first experiment, we studied the effect of challenge dose on the antibody response to an early excreted 98-kilodalton PRV-glycoprotein that was used as a diagnostic antigen in the ELISA. In the second experiment, we studied the effect of low doses of virus on the establishment of latent infections in subunit-vaccinated pigs. The relationship of virus exposure dose and vaccine dose to the response of pigs to diagnostic antigen was studied in 18 pigs. Two groups of 3 pigs were vaccinated with a total of 200 micrograms of a lectin-derived PRV subunit vaccine over a 5-week period. Two groups of 3 pigs were similarly vaccinated with a total of 100 micrograms. Two groups of 3 pigs served as nonvaccinated controls. One group of pigs from each of the preceding categories was intranasally exposed to 10(6.0) and 10(2.7) plaque-forming units (PFU) of virus. Antibody to diagnostic antigen was detected by the ELISA and radioimmunoprecipitation 3 to 7 days earlier in pigs exposed to 10(6.0) PFU, demonstrating that the size of the virus challenge dose affects the antibody response to diagnostic antigen. The establishment of latent infections by low PRV doses and the ability to detect these infections was studied in 10 subunit-vaccinated pigs. Each pig was intranasally exposed to 10(2.3) PFU of virus (day 0).(ABSTRACT TRUNCATED AT 250 WORDS)