Mycoplasma hyopneumoniae Surveillance in Pig Populations: Establishing Sampling Guidelines for Detection in Growing Pigs.

Antemortem detection of Mycoplasma hyopneumoniae infection in swine production systems has relied on antibody testing, but the availability of tests based on DNA detection and novel diagnostic specimens, e.g., tracheal swabs and oral fluids, has the potential to improve M. hyopneumoniae surveillance. A field study was performed over a 14-week period during which 10 pigs in one pen at the center of a room with 1,250 6-week-old pigs housed in 46 pens were intratracheally inoculated with M. hyopneumoniae Thereafter, one tracheal sample, four serum samples, and one oral fluid sample were collected from every pen at 2-week intervals. Tracheal and oral fluid samples were tested for M. hyopneumoniae DNA and serum samples for M. hyopneumoniae antibody. Test results were modeled using a hierarchical Bayesian model, based on a latent spatial piecewise exponential survival model, to estimate the probability of detection by within-pen prevalence, number of positive pens in the barn, sample allocation, sample size, and sample type over time. Analysis showed that tracheal samples provided the earliest detection, especially at large sample sizes. While serum samples are more commonly collected and are less expensive to test, high probability of detection estimates were only obtained 30 days postexposure at large sample sizes. In all scenarios, probability of detection estimates for oral fluids within 30 days were significantly lower than those for tracheal and serum samples. Ultimately, the choice of specimen type, sample number, and assay will depend on testing objectives and economics, but the estimates provided here will assist in the design of M. hyopneumoniae surveillance and monitoring programs for different situations.

Normalizing real-time PCR results in routine testing.

Normalization, the process of controlling for normal variation in sampling and testing, can be achieved in real-time PCR assays by converting sample quantification cycles (Cqs) to "efficiency standardized Cqs" (ECqs). We calculated ECqs as E-ΔCq, where E is amplification efficiency and ΔCq is the difference between sample and reference standard Cqs. To apply this approach to a commercial porcine reproductive and respiratory syndrome virus (PRRSV) RT-qPCR assay, we created reference standards by rehydrating and then diluting (1 × 10-4) a PRRSV modified-live vaccine (PRRS MLV; Ingelvac) with serum or oral fluid (OF) to match the sample matrix to be tested. Sample ECqs were calculated using the mean E and reference standard Cq calculated from the 4 reference standards on each plate. Serum (n = 132) and OF (n = 130) samples were collected from each of 12 pigs vaccinated with a PRRSV MLV from -7 to 42 d post-vaccination, tested, and sample Cqs converted to ECqs. Mean plate Es were 1.75-2.6 for serum and 1.7-2.3 for OF. Mean plate reference standard Cqs were 29.1-31.3 for serum and 29.2-31.5 for OFs. Receiver operating characteristic analysis calculated the area under the curve for serum and OF sample ECqs as 0.999 (95% CI: 0.997, 1.000) and 0.947 (0.890, 1.000), respectively. For serum, diagnostic sensitivity and specificity of the commercial PRRSV RT-qPCR assay were estimated as 97.9% and 100% at an ECq cutoff ≥ 0.20, and for OF, 82.6% and 100%, respectively, at an ECq cutoff ≥ 0.45.

Effect of extrinsic factors on the detection of PRRSV and a porcine-specific internal sample control in serum, oral fluid, and fecal specimens tested by RT-rtPCR.

We characterized the effect of 1) temperature × time, 2) freeze-thaw cycles, and 3) high porcine reproductive and respiratory syndrome virus (PRRSV) RNA concentrations on the detection of PRRSV and a porcine-specific internal sample control (ISC) in serum, oral fluid, and fecal samples using a commercial PRRSV RT-rtPCR assay (Idexx). In study 1, the effect of temperature × time on PRRSV and ISC detection was shown to be specimen dependent. In serum stored at 4, 10, or 20°C, PRRSV detection was consistent for up to 168 h, but storage at 30°C reduced detectable PRRSV RNA. ISC RNA was stable in serum held at 4 and 10°C, but not at 20 and 30°C. In contrast, PRRSV and ISC RNAs in oral fluid and fecal samples continuously decreased at all temperature × time treatments. Based on these data, serum samples should be stored at ≤ 20°C to optimize PRRSV RNA detection. Oral fluid and fecal samples should be frozen in a non-self-defrosting freezer until tested. In study 2, freeze-thaw cycles had little impact on PRRSV and ISC detection, but more so in oral fluids than serum or fecal samples. Thus, freeze-thaw cycles in oral fluids should be minimized before RT-rtPCR testing. In study 3, the ISC was not affected by high concentrations of PRRSV RNA in serum, oral fluid, or fecal samples. It should not be assumed that data from our PRRSV study are applicable to other pathogens; additional pathogen-specific studies are required.

Evaluation of a Porcine Endogenous Reference Gene (Internal Sample Control) in a Porcine Reproductive and Respiratory Syndrome Virus RT-qPCR.

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.

Emergent Molecular Techniques Applied to the Detection of Porcine Viruses.

Molecular diagnostic tests have evolved very rapidly in the field of human health, especially with the arrival of the recent pandemic caused by the SARS-CoV-2 virus. However, the animal sector is constantly neglected, even though accurate detection by molecular tools could represent economic advantages by preventing the spread of viruses. In this regard, the swine industry is of great interest. The main viruses that affect the swine industry are described in this review, including African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and porcine circovirus (PCV), which have been effectively detected by different molecular tools in recent times. Here, we describe the rationale of molecular techniques such as multiplex PCR, isothermal methods (LAMP, NASBA, RPA, and PSR) and novel methods such as CRISPR-Cas and microfluidics platforms. Successful molecular diagnostic developments are presented by highlighting their most important findings. Finally, we describe the barriers that hinder the large-scale development of affordable, accessible, rapid, and easy-to-use molecular diagnostic tests. The evolution of diagnostic techniques is critical to prevent the spread of viruses and the development of viral reservoirs in the swine industry that impact the possible development of future pandemics and the world economy.

Detection of pseudorabies virus antibody in swine serum and oral fluid specimens using a recombinant gE glycoprotein dual-matrix indirect ELISA.

Pseudorabies (Aujeszky disease) virus (PRV) was eliminated from domestic swine in many countries using glycoprotein E (gE)-deleted vaccines and serum antibody gE ELISAs, but PRV continues to circulate in some regions and in most feral swine populations in the world. We created a dual-matrix (serum and oral fluid) indirect IgG gE ELISA (iELISA) and evaluated its performance using samples from 4 groups of 10 pigs each: negative control (NC), vaccination (MLV), PRV inoculation (PRV), and vaccination followed by challenge (MLV-PRV). All serum and oral fluid samples collected before PRV challenge and all NC samples throughout the study were negative for gE antibodies by commercial blocking ELISA (bELISA) and our iELISA. Nasal swab samples from 9 of 10 animals in the PRV group were gB quantitative PRC (qPCR) positive at 2 days post-inoculation (dpi). The oral fluid iELISA detected a significant S/P response in the PRV (p = 0.03) and MLV-PRV (p = 0.01) groups by 6 dpi. ROC analyses of serum bELISA (n = 428), serum iELISA (n = 426), and oral fluid iELISA (n = 247) showed no significant differences in performance (p > 0.05). Our data support the concept of PRV surveillance based on oral fluid samples tested by an indirect gE ELISA.

Adapting a porcine reproductive and respiratory syndrome virus (PRRSV) oral fluid antibody ELISA to routine surveillance.

Distinct from tests used in diagnostics, tests used in surveillance must provide for detection while avoiding false alarms, i.e., acceptable diagnostic sensitivity but high diagnostic specificity. In the case of the reproductive and respiratory syndrome virus (PRRSV), RNA detection meets these requirements during the period of viremia, but antibody detection better meets these requirements in the post-viremic stage of the infection. Using the manufacturer's recommended cut-off (S/P ≥ 0.4), the diagnostic specificity of a PRRSV oral fluid antibody ELISA (IDEXX Laboratories, Inc., Westbrook, ME, USA) evaluated in this study was previously reported as ≥ 97 %. The aim of this study was to improve its use in surveillance by identifying a cut-off that would increase diagnostic specificity yet minimally impact its diagnostic sensitivity. Three sample sets were used to achieve this goal: oral fluids (n = 596) from pigs vaccinated with a modified live PRRSV vaccine under experimental conditions, field oral fluids (n = 1574) from 94 production sites of known negative status, and field oral fluids (n = 1380) from 211 sites of unknown PRRSV status. Based on the analysis of samples of known status (experimental samples and field samples from negative sites), a cut-off of S/P ≥ 1.0 resulted in a diagnostic specificity of 99.2 (95 % CI: 98.8, 99.7) and a diagnostic sensitivity of 96.5 (95 % CI: 85.2, 99.2). Among 211 sites of unknown status, 81 sites were classified as antibody positive using the manufacturer's cut-off; 20 of which were reclassified as negative using a cut-off of S/P ≥ 1.0. Further analysis showed that these 20 sites had a small proportion of samples (18.0 %) with S/P values just exceeding the manufacturer's cut-off (x̄ = 0.5). Whereas the remainder of positive sites (n = 61) had a high proportion of samples (76.3 %) with high S/P values (x̄ = 6.6). Thus, the manufacturer's cut-off (S/P ≥ 0.4) is appropriate for diagnostic applications, but a cut-off of S/P ≥ 1.0 provided the higher specificity required for surveillance. A previously unreported finding in this study was a statistically significant association between unexpected reactors and specific production sites and animal ages or stages. While beyond the scope of this study, these data suggested that certain animal husbandry or production practices may be associated with non-specific reactions.

Pseudorabies (Aujeszky's disease) virus DNA detection in swine nasal swab and oral fluid specimens using a gB-based real-time quantitative PCR.

In this study, the detection of PRV DNA in nasal swab (n = 440) and oral fluid (n = 1,545) samples collected over time from experimentally PRV vaccinated and/or PRV inoculated pigs (n = 40) was comparatively evaluated by real-time PCR. Serum samples (n = 440) were tested by PRV gB/gE blocking ELISAs (Pseudorabies Virus gB Antibody Test Kit and Pseudorabies Virus gpI Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME) to monitor PRV status over time. Following exposure to a gE-deleted modified live vaccine (Ingelvac® Aujeszky MLV, Boehringer Ingelheim, Ridgefield, CT) and/or a wild-type virus (3 CR Ossabaw), PRV gB DNA was detected in oral fluid specimens in a pattern similar to that of nasal swabs. For quantitative analyses, PRV PCR quantification cycle (Cq) results were re-expressed as "efficiency standardized Cqs (ECqs)" as a function of PCR efficiency using plate-specific positive amplification controls. ROC analyses of the PRV gB PCR ECqs results showed a similar performance of the PRV gB PCR for nasal swab and oral fluid specimens (area under the ROC curve = 85 % vs 83 %) and, based on an ECq cutoff of 0.01 a diagnostic specificity of 100 % and diagnostic sensitivities for oral fluid and nasal swab specimens of 53 % (95 % CI: 43 %, 62 %) and 70 % (95 % CI: 55 %, 83 %), respectively. Thus, the results described herein demonstrated the detection of PRV gB DNA in swine oral fluid and supported the use of this specimen in PRV diagnosis and surveillance.

Guidelines for oral fluid-based surveillance of viral pathogens in swine.

Recent decades have seen both rapid growth and extensive consolidation in swine production. As a collateral effect, these changes have exacerbated the circulation of viruses and challenged our ability to prevent, control, and/or eliminate impactful swine diseases. Recent pandemic events in human and animal health, e.g., SARS-CoV-2 and African swine fever virus, highlight the fact that clinical observations are too slow and inaccurate to form the basis for effective health management decisions: systematic processes that provide timely, reliable data are required. Oral fluid-based surveillance reflects the adaptation of conventional testing methods to an alternative diagnostic specimen. The routine use of oral fluids in commercial farms for PRRSV and PCV2 surveillance was first proposed in 2008 as an efficient and practical improvement on individual pig sampling. Subsequent research expanded on this initial report to include the detection of ≥23 swine viral pathogens and the implementation of oral fluid-based surveillance in large swine populations (> 12,000 pigs). Herein we compile the current information regarding oral fluid collection methods, testing, and surveillance applications in swine production.

Understanding and interpreting PRRSV diagnostics in the context of "disease transition stages".

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.

Detection of pseudorabies virus antibody in swine oral fluid using a serum whole-virus indirect ELISA.

We evaluated the detection of pseudorabies virus (PRV) antibodies in swine oral fluid. Oral fluid and serum samples were obtained from 40 pigs allocated to 4 treatment groups (10 pigs/group): negative control (NC); wild-type PRV inoculation (PRV 3CR Ossabaw; hereafter PRV); PRV vaccination (Ingelvac Aujeszky MLV; Boehringer Ingelheim; hereafter MLV); and PRV vaccination followed by PRV inoculation at 21 d post-vaccination (MLV-PRV). Using a serum PRV whole-virus indirect IgG ELISA (Idexx Laboratories) adapted to the oral fluid matrix, PRV antibody was detected in oral fluid samples from treatment groups PRV, MLV, and MLV-PRV in a pattern similar to serum. Vaccination alone produced a low oral fluid antibody response (groups MLV and MLV-PRV), but a strong anamnestic response was observed following challenge with wild-type virus (group PRV). Analyses of the oral fluid PRV indirect IgG ELISA results showed good binary diagnostic performance (area under ROC curve = 93%) and excellent assay repeatability (intra-class correlation coefficient = 99.3%). The demonstrable presence of PRV antibodies in swine oral fluids suggests the possible use of oral fluids in pseudorabies surveillance.

Evaluation of three commercial porcine reproductive and respiratory syndrome virus (PRRSV) oral fluid antibody ELISAs using samples of known status.

Oral fluid (n = 564) samples collected longitudinally from twelve 14-week-old pigs vaccinated with a porcine reproductive and respiratory syndrome virus (PRRSV) modified live vaccine were used to evaluate and compare the diagnostic performance of three commercial PRRSV oral fluid (OF) ELISAs (ELISAs 1, 2, 3). Serum samples (n = 132) tested by a PRRSV serum ELISA (ELISA 'S') provided an antibody response baseline for comparison. The initial analysis comparing the rate of positivity between each OF ELISA versus ELISA 'S' and then pairwise among the three OF ELISAs determined that ELISA 2 (143 false negative results) was significantly different from ELISAs 1 and 3, and from ELISA 'S' (Cochran's Q test, p < 0.05). Receiver operating characteristic (ROC) analyses based on the manufacturers' recommended cutoff were used to estimate the diagnostic sensitivities and specificities of ELISA 1 (100%, 100%), ELISA 2 (62%, 97%), and ELISA 3 (94%, 100%). As an additional aid for interpreting results, the diagnostic sensitivities and specificities of each OF ELISA were also estimated over a range of cutoffs. Area under the curve comparisons found no significant difference between ELISAs 1 and 3, but ELISA 2 differed from both ELISA 1 and 3 (ROC Chi-square, p < 0.05). Based on these analyses, the overall diagnostic performance of the three OF ELISAs ranked ELISA 1 ≥ ELISA 3 > ELISA 2.

Mortality Patterns in a Commercial Wean-To Finish Swine Production System.

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.