David H. Baum

Serological and Molecular Detection of Senecavirus A Associated with an Outbreak of Swine Idiopathic Vesicular Disease and Neonatal Mortality

ABSTRACT We performed a longitudinal field study in a swine breeding herd that presented with an outbreak of vesicular disease (VD) that was associated with an increase in neonatal mortality. Initially, a USDA Foreign Animal Disease (FAD) investigation confirmed the presence of Senecavirus A (SVA) and ruled out the presence of exotic agents that produce vesicular lesions, e.g., foot-and-mouth disease virus and others. Subsequently, serum samples, tonsil swabs, and feces were collected from sows (n = 22) and their piglets (n = 33) beginning 1 week after the onset of the clinical outbreak and weekly for 6 weeks. The presence of SVA RNA was evaluated in all specimens collected by reverse transcriptase quantitative PCR (RT-qPCR) targeting a conserved region of the 5′ untranslated region (5′-UTR). The serological response (IgG) to SVA was evaluated by the weekly testing of sow and piglet serum samples on a SVA VP1 recombinant protein (rVP1) indirect enzyme-linked immunosorbent assay (ELISA). The rVP1 ELISA detected seroconversion against SVA in clinically affected and non-clinically affected sows at early stages of the outbreak as well as maternal SVA antibodies in offspring. Overall, the absence of vesicles (gross lesions) in SVA-infected animals and the variability of RT-qPCR results among specimen type demonstrated that a diagnostic algorithm based on the combination of clinical observations, RT-qPCR in multiple diagnostic specimens, and serology are essential to ensure an accurate diagnosis of SVA.

Novel Senecavirus A in Swine with Vesicular Disease, United States, July 2015.

To the Editor: Senecavirus A (SVA; formerly known as Seneca Valley virus [SVV] belongs to the genus Senecavirus, family Picornaviridae (1,2). SVA was first isolated in 2001 as a contaminant of the PER.C6 cell line and designated as SVV-001 (1,3). Since its discovery, SVA has been infrequently detected in swine with idiopathic vesicular disease (IVD) (4–6), which clinically resembles foot-and-mouth disease, swine vesicular disease, vesicular exanthema of swine, and vesicular stomatitis. The virus has also been retrospectively detected in previous cases with various clinical conditions in the United States during 1988–2001 (7). However, the clinical significance of SVA in swine could not be determined (7,8).

Sampling guidelines for oral fluid-based surveys of group-housed animals

Formulas and software for calculating sample size for surveys based on individual animal samples are readily available. However, sample size formulas are not available for oral fluids and other aggregate samples that are increasingly used in production settings. Therefore, the objective of this study was to develop sampling guidelines for oral fluid-based porcine reproductive and respiratory syndrome virus (PRRSV) surveys in commercial swine farms. Oral fluid samples were collected in 9 weekly samplings from all pens in 3 barns on one production site beginning shortly after placement of weaned pigs. Samples (n=972) were tested by real-time reverse-transcription PCR (RT-rtPCR) and the binary results analyzed using a piecewise exponential survival model for interval-censored, time-to-event data with misclassification. Thereafter, simulation studies were used to study the barn-level probability of PRRSV detection as a function of sample size, sample allocation (simple random sampling vs fixed spatial sampling), assay diagnostic sensitivity and specificity, and pen-level prevalence. These studies provided estimates of the probability of detection by sample size and within-barn prevalence. Detection using fixed spatial sampling was as good as, or better than, simple random sampling. Sampling multiple barns on a site increased the probability of detection with the number of barns sampled. These results are relevant to PRRSV control or elimination projects at the herd, regional, or national levels, but the results are also broadly applicable to contagious pathogens of swine for which oral fluid tests of equivalent performance are available.

Reactivity of Porcine Epidemic Diarrhea Virus Structural Proteins to Antibodies against Porcine Enteric Coronaviruses: Diagnostic Implications

ABSTRACT The development of porcine epidemic diarrhea virus (PEDV) antibody-based assays is important for detecting infected animals, confirming previous virus exposure, and monitoring sow herd immunity. However, the potential cross-reactivity among porcine coronaviruses is a major concern for the development of pathogen-specific assays. In this study, we used serum samples (n = 792) from pigs of precisely known infection status and a multiplex fluorescent microbead-based immunoassay and/or enzyme-linked immunoassay platform to characterize the antibody response to PEDV whole-virus (WV) particles and recombinant polypeptides derived from the four PEDV structural proteins, i.e., spike (S), nucleocapsid (N), membrane (M), and envelope (E). Antibody assay cutoff values were selected to provide 100% diagnostic specificity for each target. The earliest IgG antibody response, mainly directed against S1 polypeptides, was observed at days 7 to 10 postinfection. With the exception of nonreactive protein E, we observed similar antibody ontogenies and patterns of seroconversion for S1, N, M, and WV antigens. Recombinant S1 provided the best diagnostic sensitivity, regardless of the PEDV strain, with no cross-reactivity detected against transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), or porcine deltacoronavirus (PDCoV) pig antisera. The WV particles showed some cross-reactivity to TGEV Miller and TGEV Purdue antisera, while N protein presented some cross-reactivity to TGEV Miller. The M protein was highly cross-reactive to TGEV and PRCV antisera. Differences in the antibody responses to specific PEDV structural proteins have important implications in the development and performance of antibody assays for the diagnosis of PEDV enteric disease.

SEROLOGIC SURVEY AND RESULTS OF URINARY PCR TESTING FOR LEPTOSPIROSIS IN CAPTIVE BLACK-TAILED PRAIRIE DOGS (CYNOMYS LUDOVICIANUS)

Abstract:  Leptospirosis is an important zoonotic disease occurring clinically and subclinically in humans and a wide variety of mammal species worldwide. Often, rodents and wild animals are identified as important reservoirs for the disease. Twenty-two captive black-tailed prairie dogs (Cynomys ludovicianus) housed within a zoo were examined as part of a routine census and preventive medicine program. During examinations, blood and urine were collected to screen for exposure to, or infection with, leptospirosis. All animals were apparently healthy at the time of examination. Leptospira microscopic agglutination test identified 12 of 22 (54.5%) prairie dogs with antibody titers ≥1 : 100 against Leptospira interrogans serovar bratislava on initial serologic examination. All prairie dogs within this collection were serologically negative for L. interrogans serovars canicola, hardjo, icterohaemorrhagiae, and pomona and Leptospira kirschneri serovar grippotyphosa. Leptospira polymerase chain reaction (PCR) testing of urine was negative in all animals tested. This report describes evidence that captive prairie dogs may be exposed to leptospirosis, most likely from wild rodent reservoirs; however, serum titers are low, and lack of leptospiral DNA detected by PCR indicates that these captive animals are unlikely to be important reservoirs for the disease.

Detection of porcine reproductive and respiratory syndrome virus (PRRSV)-specific IgM-IgA in oral fluid samples reveals PRRSV infection in the presence of maternal antibody

The ontogeny of PRRSV antibody in oral fluids has been described using isotype-specific ELISAs. Mirroring the serum response, IgM appears in oral fluid by 7days post inoculation (DPI), IgA after 7 DPI, and IgG by 9 to 10 DPI. Commercial PRRSV ELISAs target the detection of IgG because the higher concentration of IgG relative to other isotypes provides the best diagnostic discrimination. Oral fluids are increasingly used for PRRSV surveillance in commercial herds, but in younger pigs, a positive ELISA result may be due either to maternal antibody or to antibody produced by the pigs in response to infection. To address this issue, a combined IgM-IgA PRRSV oral fluid ELISA was developed and evaluated for its capacity to detect pig-derived PRRSV antibody in the presence of maternal antibody. Two longitudinal studies were conducted. In Study 1 (modified-live PRRS vaccinated pigs), testing of individual pig oral fluid samples by isotype-specific ELISAs demonstrated that the combined IgM-IgA PRRSV ELISA provided better discrimination than individual IgM or IgA ELISAs. In Study 2 (field data), testing of pen-based oral fluid samples confirmed the findings in Study 1 and established that the IgM-IgA ELISA was able to detect antibody produced by pigs in response to wild-type PRRSV infection, despite the presence of maternal IgG. Overall, the combined PRRSV IgM-IgA oral fluid ELISA described in this study is a potential tool for PRRSV surveillance, particularly in populations of growing pigs originating from PRRSV-positive or vaccinated breeding herds.

The effect of chemical clarification of oral fluids on porcine epidemic diarrhea virus antibody responses

Routine testing of breeding herd oral fluid (OF) samples for porcine epidemic diarrhea virus (PEDV) IgG and/or IgA is used to track levels of PEDV immunity over time. However, OFs contain particles of feed, feces, and inorganic material that detract from the quality of the sample. We clarified swine OF samples using lyophilized chitosan-based formulas (A–C) tested by PEDV IgG and IgA ELISAs. To evaluate both the immediate and residual effects of treatment on antibody detection, samples were tested immediately post-treatment, then stored at 4°C and retested at 2, 4, and 6 days post-treatment (DPT). Formulations were shown to effectively clarify samples. Statistical analysis comparing treated to untreated OF samples at 0 DPT found that neither chitosan nor Tween 20 affected the OF ELISA IgA and IgG sample-to-positive (S/P) ratio results (p > 0.05). Furthermore, pairwise comparisons of 0 DPT to 2, 4, and 6 DPT results detected no significant differences (p > 0.05) in IgA and IgG S/P ratios (i.e., treated OF samples were stable over time). Therefore, chitosan efficiently clarified OF specimens without affecting the results of the PEDV IgG and IgA antibody ELISAs.

Effect of chemical clarification of oral fluids on the detection of porcine reproductive and respiratory syndrome virus IgG

Routine collection and testing of oral fluid (OF) samples facilitates porcine reproductive and respiratory syndrome virus (PRRSV) surveillance in commercial swine herds in a cost-effective, welfare-friendly fashion. However, OFs often contain environmental contaminants that may affect liquid handling and test performance. Traditional processing methods (e.g., filtration or centrifugation) are not compatible with high-throughput testing because of the burden of additional processing costs and time. OF “clarification” using chemical flocculants is an alternative approach not widely explored. Therefore, we evaluated the effect of chitosan-based clarification treatment on a commercial PRRSV OF ELISA. Serum and individual OFs were collected from vaccinated pigs (n = 17) at −7 to 42 d post-vaccination and subdivided into 4 aliquots. Each aliquot was clarified (treatment A, B, C), with the 4th aliquot serving as untreated control. All samples were tested by PRRSV OF ELISA immediately after treatment and then were held at 4°C to be re-tested at 2, 4, 6, and 14 d post-treatment. Quantitative and qualitative treatment effects were evaluated. A Kruskal–Wallis test found no significant difference in ELISA S/P responses among treatments by days post-treatment. No difference was detected in the proportion of positive PRRSV antibody samples among treatments (Cochran Q, p > 0.05). Treatment of swine OFs using chitosan-based formulations did not affect the performance of a commercial PRRSV OF ELISA. Chitosan (or other flocculants) could improve OF characteristics and could be adapted for use in the field or in high-throughput laboratories.