Herein we review broad issues that affect test performance for agents that produce persistent infections. Using PRRSV as an example, the relationship between “disease transition stages” and “diagnostic transition stages” is discussed using meta-analyses of diagnostic data (n = 4307 results) from the refereed literature to highlight the key issues. Although diagnostic technology will continue to improve, it may be concluded from the analysis that there can be no single best diagnostic approach; rather, the choice of specimen and test must be tailored to the specific testing objective. In most cases, meeting the testing objective(s) will require the use of more than one assay and/or specimen type.

The emergence and spread of highly pathogenic avian influenza virus A subtype H5N1 (HP H5N1-IAV), particularly clade H5N1 2.3.4.4b, pose a severe global health threat, affecting various species, including mammals. Historically, cattle have been considered less susceptible to IAV, but recent outbreaks of H5N1-IAV 2.3.4.4b in dairy farms suggest a shift in host tropism, underscoring the urgency of expanded surveillance and the need for adaptable diagnostic tools in outbreak management. This study investigated the presence of anti-nucleoprotein (NP) antibodies in serum and milk and viral RNA in milk on dairy farms affected by outbreaks in Texas, Kansas, and Michigan using a multi-species IAV ELISA and RT-qPCR. The analysis of ELISA results from a Michigan dairy farm outbreak demonstrated a positive correlation between paired serum and milk sample results, confirming the reliability of both specimen types. Our findings also revealed high diagnostic performance during the convalescent phase (up to 96%), further improving sensitivity through serial sampling. Additionally, the evaluation of diagnostic specificity using serum and milk samples from IAV-free farms showed an excellent performance (99.6%). This study underscores the efficacy of the IAV NP-blocking ELISA for detecting and monitoring H5N1-IAV 2.3.4.4b circulation in dairy farms, whose recent emergence raises significant animal welfare and zoonotic concerns, necessitating expanded surveillance efforts.

This study characterized the susceptibility and dynamic of porcine deltacoronavirus infection in grower pigs under experimental conditions using a combination of syndromic and laboratory assessments. Seven-week-old conventional pigs (n = 24) were randomly distributed into PDCoV- (n = 12) and mock-inoculated (n = 12) groups. Serum was collected at −7, 0, 3, 7, 10, 14, 17, 21, 28, 35, and 42 days post-inoculation (DPI) to evaluate viremia (RT-qPCR) and antibody response (S1-based ELISA). Viral shedding and potential infectivity were determined using pen-based oral fluids and feces collected every other day between DPI 0 and 42. Pigs showed no clinical signs or viremia throughout the study. Active virus shedding was detected in feces (6-22 DPI) and oral fluids (2-30 DPI), peaking at DPI 10. IgG was first detected at DPI 10, being statistically significant after DPI 14 and increasing thereafter, coinciding with the progressive resolution of the infection. Likewise, a significant increase in proinflammatory IL-12 was detected between DPI 10 and 21 in PDCoV-inoculated pigs, which could enhance innate resistance to PDCoV infection. This study demonstrated that active surveillance based on systematic sampling and laboratory testing combining molecular and serological tools is critical for the accurate detection of subclinical circulation of PDCoV in pigs after weaning.

Lactogenic immunity is important for the protection of piglets against many pathogens including porcine epidemic diarrhea virus. Circulating neutralizing antibodies levels in sow sera may help determine if a detectable immune response could confer protection to piglets. Neutralizing antibodies can be detected through various diagnostic assays. This study evaluated the diagnostic characteristics of two neutralizing antibody assays for porcine epidemic diarrhea virus neutralizing antibodies in serum of challenged gilts. Four treatment groups, control, non-vaccinated, vaccinated prior to challenge, and vaccinated following challenge, were comprised of 20 gilts. Serum sample were collected from each gilt prior to and following challenge with porcine epidemic diarrhea virus. Samples were evaluated for the presence of neutralizing antibodies via a fluorescent focus neutralization assay and a high-throughput neutralization assay. Diagnostic sensitivity and specificity for the fluorescent focus neutralization and high-throughput neutralization assays for this study were optimized at a cutoff of a dilution of 80 and 80% fluorescent reduction respectively and demonstrated moderate agreement based off the kappa statistic. The focus fluorescent neutralization and high-throughput neutralization assays can be used to monitor the status of neutralizing antibodies within animals or a population of animals. The high-throughput assay has advantages over the focus fluorescent assay in that it has a higher specificity at the indicated cut-off and the nature of the results allows for more discrimination between individual results.

Endogenous reference genes are used in gene-expression studies to “normalize” the results and, increasingly, as internal sample controls (ISC) in diagnostic quantitative polymerase chain reaction (qPCR). Three studies were conducted to evaluate the performance of a porcine-specific ISC in a commercial porcine reproductive and respiratory syndrome virus (PRRSV) reverse transcription-qPCR. Study 1 evaluated the species specificity of the ISC by testing serum from seven non-porcine domestic species (n = 34). In Study 2, the constancy of ISC detection over time (≥42 days) was assessed in oral fluid (n = 130), serum (n = 215), and feces (n = 132) collected from individual pigs of known PRRSV status. In Study 3, serum (n = 150), oral fluid (n = 150), and fecal samples (n = 75 feces, 75 fecal swabs) from commercial herds were used to establish ISC reference limits. Study 1 showed that the ISC was porcine-specific, i.e., all samples from non-porcine species were ISC negative (n = 34). In Study 2, the ISC was detected in all oral fluid, serum, and fecal samples, but differed in concentration between specimens (p < 0.05; mixed-effects regression model). The results of Study 3 were used to establish ISC reference limits for the 5th, 2.5th and 1.25th percentiles. Overall, the ISC response was consistent to the point that failure in detection is sufficient justification for re-testing and/or re-sampling.

Modern commercial pig production is a complex process that requires successful producers to understand and resolve factors associated with perturbations in production. One important perturbation is inventory loss due to mortality. In this study, data on 60 lots of approximately 2000 weaned pigs (n = 115,213) from one commercial production system were collected through the wean-to-finish (WTF) cycle with the objective of establishing patterns of mortality, estimating differences in profit/loss among patterns of mortality, and identifying production practices associated with mortality patterns. Information provided by the production system included the number of pigs in each lot at the time of placement (beginning inventory), weaning weight, barn dimensions, number of dead pigs (NDP) daily, capacity placed (proportion pigs actually placed versus what had been planned to be placed) and average weight sold. Analysis of NDP revealed three mortality patterns (clusters I, II, III) composed of 6, 40, and 14 lots, respectively, that differed in the temporal onset and/or level of mortality. Average daily gain (ADG) and feed conversion ratio (FCR) were calculated by growth phase for each cluster. An economic model showed profit differences among clusters due to poor biological performance by clusters I and III in the late finishing phase. Cluster II (n = 40) had fewer dead pigs and the highest profit compared to clusters I (n = 6) and III (n = 14). Area per pig (stocking density) was the only factor associated with the differences in mortality patterns. Routine monitoring and the analysis of mortality patterns for associations with production and management factors can help swine producers improve biological performance and improve profit.