Detection of anti-influenza A nucleoprotein antibodies in pigs using a commercial influenza epitope-blocking enzyme-linked immunosorbent assay developed for avian species.

Influenza virus causes acute respiratory disease in pigs and is of concern for its potential public health significance. Many subtypes of influenza virus have been isolated from pigs, and the virus continues to evolve in swine populations. Current antibody assays have limited antigenic recognition, and accurate, broad-spectrum, high through-put screening tests are needed to detect infections in swine herds and to aid in the implementation of control measures. In the current study, a commercial blocking enzyme-linked immunosorbent assay (ELISA) developed for the detection of Influenza A virus nucleoprotein antibodies in avian species was evaluated for the detection of anti-influenza serum antibodies in swine. Serum samples used to evaluate the test were archived samples from influenza research conducted at the U.S. Department of Agriculture-Agricultural Research Service-National Animal Disease Center and included samples from influenza-inoculated pigs (H1N1, H1N2, H2N3, and H3N2), contact-infected pigs, vaccinated pigs, and negative controls. Based on samples of known status (n = 453), a receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity at 96.6% (95% confidence interval [CI]: 92.3, 98.9) and 99.3% (95% CI: 97.6, 99.9), respectively. By using the cutoff established in the ROC analysis, the assay was evaluated in pigs infected with 2 isolates of the 2009 pandemic H1N1 virus. Overall, the assay showed excellent diagnostic performance against the range of influenza subtypes investigated and could serve as a useful screening assay for swine.

Stability of Porcine reproductive and respiratory syndrome virus at ambient temperatures.

The stability of Porcine reproductive and respiratory syndrome virus (PRRSV) was evaluated for temperatures appropriate to laboratory and field settings. Four North American (type 2) isolates (ATCC VR-2332, JA-142, MN-184, and Ingelvac(R) PRRS ATP vaccine virus) in cell culture medium (pH 7.5) were held at 1 of 4 temperatures (4, 10, 20, and 30 degrees C) and sampled over time. Samples were tested for infectious virus and total PRRSV RNA using median tissue culture infectious dose and quantitative reverse transcription polymerase chain reaction, respectively. The rate of loss of infectious virus was expressed in terms of the time required for virus concentration to decline by one half (i.e., half-life [T(1/2)]). Statistical analysis found that temperature, but not virus isolate, had a significant effect on T(1/2), and a single nonlinear regression model was derived to predict T(1/2) for temperatures between 0 and 50 degrees C: T(1/2) = 243.54 e((-0.109*TEMP)). In contrast to changes over time in the concentration of infectious virus, no change in the concentration of quantitative reverse transcription polymerase chain reaction-detectable PRRSV was detected at any temperature and contact time. This information will be of interest to persons working in laboratory or field situations in which the control of PRRSV is important.

Efficient surveillance of pig populations using oral fluids.

Currently virus surveillance in swine herds is constrained by the cost-effectiveness and efficiency of sampling methods. The objective of this study was to assess the value of using oral fluids collected by barn personnel as a method of surveillance based on PCR testing. Approximately 12,150 pigs in 10 wean-to-finish barns on 10 farms were monitored for the presence of porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus (IAV), and Torque teno virus genogroups 1 (TTV1) and 2 (TTV2) by sampling oral fluid specimens. Oral fluid samples were collected from 6 pens at each site starting at the time of pig placement (∼3 weeks of age) and continuing thereafter at 2-week intervals for a period of 18 weeks. Data were analyzed both on a pen basis and barn basis. Overall, 508 (85%) samples were positive for PCV2, 73 (12%) for PRRSV, 46 (8%) for IAV, 483 (81%) for TTV2, and 155 (26%) for TTV1 during the study period. The estimated arithmetic means of the quantitative PCR-positive oral fluids for PCV2, PRRSV, and IAV were 1×10(4.62), 1×10(4.97), and 1×10(5.49)per ml. With a single exception, all barns were positive for PCV2 and TTV2 at every sampling point in the study. Virus detection varied among barns, particularly for IAV and PRRSV. The pen level, cumulative distribution of agent combinations between all 10 barns were statistically different. The most commonly observed patterns were PCV2+TTV2 (239 pen samples, 40%), PCV2+TTV1+TTV2 (88 pen samples, 15%), and PCV2 alone (66 pen samples, 11%). This "proof-of-concept" project showed that a variety of viruses could be detected either intermittently or continuously in pig populations and demonstrated that barn herd virus status is highly variable, even among barns in the same production system. Oral fluid sampling is a promising approach for increasing the efficiency and cost effectiveness of virus surveillance in swine herds.

Comparison of RNA extraction and real-time reverse transcription polymerase chain reaction methods for the detection of Porcine reproductive and respiratory syndrome virus in porcine oral fluid specimens.

The objective of the current study was to evaluate various RNA extraction and polymerase chain reaction (PCR) protocols for the detection of Porcine reproductive and respiratory syndrome virus (PRRSV) in porcine oral fluids. Extraction protocols were selected based on ease of use and compatibility with high-throughput, automated systems. The results showed marked differences among extraction protocols, PCR protocols, and combinations thereof in detecting PRRSV in the oral fluid matrix. An important finding was that PCR reactions were partially inhibited by unknown factors in the oral fluid matrix and that inhibition was reduced by use of a higher concentration of PCR enzymes. The results suggest that further optimization of PCR assays for porcine oral fluids is needed and that laboratories should not assume that methods optimized for detection of virus in serum will perform equally with porcine oral fluids.

The development of oral fluid-based diagnostics and applications in veterinary medicine.

The purpose of this review was to discuss the history of the development and implementation of oral fluid diagnostics for infectious diseases of humans and domestic animals. The use of oral fluid for the assessment of health and diagnosis of disease in humans and animals has a surprisingly long history. As early as 1909, Pollaci and Ceraulo reported sensitive and specific agglutination of 'Micrococcus melitensis' (Brucella melitensis) by oral fluid from patients diagnosed with Malta Fever. A 1986 report of the detection of antibodies against human immunodeficiency virus (HIV) in oral fluid from patients with acquired immunodeficiency syndrome (AIDS) marked the start of a remarkably rapid series of developments in oral fluid-based assays. Cumulatively, the literature strongly supports implementation of oral fluid-based diagnostics in veterinary diagnostic medicine. Pathogen-specific IgA, IgM and IgG antibodies have all been demonstrated in oral fluid collected from diverse domestic animal species in response to infection. A variety of infectious agents, both local and systemic, are shed in oral fluid, including some of the most economically significant pathogens of production animals (e.g. foot-and-mouth disease virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus) Ultimately, point-of-care rapid assays (i.e. cow-side, sow-side or pen-side tests) and access to real-time infectious disease data will revolutionize our delivery of health management services.

Porcine circovirus type 2 (PCV2)-infection and re-inoculation with homologous or heterologous strains: virological, serological, pathological and clinical effects in growing pigs.

Long-term PCV2 infection and/or concurrent infection with genotypes PCV2a and PCV2b may play a role in the development of clinical porcine circovirus-associated disease (PCVAD). To evaluate this premise, 24 11-week-old specific pathogen-free (SPF) pigs were randomly assigned to 1 of 4 treatments: negative controls, a single inoculation with PCV2a, single inoculation followed by re-inoculation with a homologous PCV2a strain, or repeated inoculations with heterologous strains (PCV2a, PCV2b). Pigs were evaluated for clinical signs daily through 140 days post inoculation (dpi). Serum samples were collected every other day from dpi 0 through 14 and weekly thereafter. PCV2-inoculated pigs were viremic by dpi 2 and 13 of 18 pigs remained viremic at 140 dpi. No statistical differences in the onset, level, or duration of PCV2 viremia were detected among treatment groups. Anti-PCV2 antibodies were detected between 14 and 28 dpi and were present through 140 dpi without statistical differences in antibody response among treatment groups. In the current study, pigs had extended viremia combined with detectable tissue PCV2 antigen levels despite the presence of high levels of anti-PCV2 antibody; however, no clinical disease was observed.