Characterization of neurologic disease-associated Streptococcus suis strains within the United States swine herd and use of diagnostic tools.

Streptococcus suis negatively impacts swine health, posing diagnostic and preventative challenges. S. suis can induce disease and also quietly reside on mucosal surfaces. The limited use of diagnostic tools to identify disease-associated strains and rule out differential diagnoses, alongside the complex ecology of S. suis, poses significant challenges in comprehending this important pathogen and defining pathotypes. This study evaluated 2,379 S. suis central nervous system (CNS) isolates from diagnostic submissions between 2015 and 2019. Isolates originating from submissions with histologic evidence of CNS infection (n = 1,032) were further characterized by standard and advanced diagnostic techniques. We identified 29 S. suis serotypes and 4 reclassified serotypes as putative causes of CNS disease. Among these, serotypes 1 and 7 emerged as the predominant putative causes of CNS infection (32% of submissions). Furthermore, 51 sequence types (STs), of which 15 were novel, were detected with ST1 predominating. Through whole-genome sequencing of 145 isolates, we observed that five commonly used virulence-associated genes (VAGs; epf, mrp, sly, ofs, and srtF) were not present in most disease-associated isolates, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) yielded false-positive results in 7% of isolates. These data indicate that (i) clinical signs and site of isolation alone are insufficient for defining a pathotype, (ii) S. suis serotypes and STs associated with CNS infection are more diverse than previously reported, (iii) MALDI-TOF MS may need to be supplemented with additional diagnostic tools for precise S. suis identification, and (iv) VAGs remain an unreliable means for identifying isolates associated with CNS disease.IMPORTANCEStreptococcus suis is an important and complex systemic bacterial pathogen of swine. Characterization of S. suis strains originating from pigs with histologic confirmation of neurologic disease is limited. Review of swine diagnostic submissions revealed that fewer than half of cases from which S. suis was isolated from the brain had histologic evidence of neurologic disease. This finding demonstrates that clinical signs and site of isolation alone are not sufficient for identifying a neurologic disease-associated strain. Characterization of strains originating from cases with evidence of disease using classic and advanced diagnostic techniques revealed that neurologic disease-associated strains are diverse and commonly lack genes previously associated with virulence.

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.

Performance of Commercial Mycoplasma hyopneumoniae Serum Enzyme-Linked Immunosorbent Assays under Experimental and Field Conditions.

Mycoplasma hyopneumoniae is an economically significant pathogen of swine. M. hyopneumoniae serum antibody detection via commercial enzyme-linked immunosorbent assays (ELISAs) is widely used for routine surveillance in commercial swine production systems. Samples from two studies were used to evaluate assay performance. In study 1, 6 commercial M. hyopneumoniae ELISAs were compared using serum samples from 8-week-old cesarean-derived, colostrum-deprived (CDCD) pigs allocated to the following 5 inoculation groups of 10 pigs each: (i) negative control, (ii) Mycoplasma flocculare (strain 27399), (iii) Mycoplasma hyorhinis (strain 38983), (iv) Mycoplasma hyosynoviae (strain 34428), and (v) M. hyopneumoniae (strain 232). Weekly serum and daily oral fluid samples were collected through 56 days postinoculation (dpi). The true status of pigs was established by PCR testing on oral fluids samples over the course of the observation period. Analysis of ELISA performance at various cutoffs found that the manufacturers' recommended cutoffs were diagnostically specific, i.e., produced no false positives, with the exceptions of 2 ELISAs. An analysis based on overall misclassification error rates found that 4 ELISAs performed similarly, although one assay produced more false positives. In study 2, the 3 best-performing ELISAs from study 1 were compared using serum samples generated under field conditions. Ten 8-week-old pigs were intratracheally inoculated with M. hyopneumoniae Matched serum and tracheal samples (to establish the true pig M. hyopneumoniae status) were collected at 7- to 14-day intervals through 98 dpi. Analyses of sensitivity and specificity showed similar performance among these 3 ELISAs. Overall, this study provides an assessment of the performance of current M. hyopneumoniae ELISAs and an understanding of their use in surveillance.

Assessment of changes in antibiotic use in grow-finish pigs after the introduction of PRRSV in a naïve farrow-to-finish system.

Responsible antibiotic usage (ABU) is crucial for both animal and human health and requires constant improvement of antimicrobial stewardship (AMS). The presence of porcine reproductive and respiratory syndrome virus (PRRSV), a viral pathogen with immunosuppressive effects on swine, can intensify bacterial co-infections, alter antibiotic pharmacokinetics, and potentially lead to increased ABU. This study aimed to measure ABU changes in the grow-finish population associated with PRRSV infection and describe the antibiotic classes employed to manage clinical signs from a farrow-to-finish genetic multiplier system. Three PRRSV statuses (naïve, positive epidemic, and positive endemic) were established to classify the lots based on PRRSV circulation, with a total of 135,063 animals evaluated. The number of pig treatments per animal days at risk (PTDR) was calculated by administration route to quantify ABU across PRRSV status using negative binomial regression and non-parametric tests (P-value < 0.05). Moreover, to improve ABU comparability in the international scenario, the milligrams per population correction unit (mg/PCU) was calculated according to the European Medicines Agency guidelines. In the nursery phase, there was a statistically significant difference between PRRSV statuses for the overall PTDR (injectable and water routes of administration), with an ABU increase of 3.79 and 2.51 times the naïve PTDR for positive epidemic and endemic status, respectively. For the finishing phase, there was a statistically significant difference between PRRSV statuses in the injectable PTDR, with an ABU increase of 2.74 and 2.28 times the naïve PTDR level for positive epidemic and endemic statuses, respectively. In the nursery phase, the mean mg/PCU was 22.27 mg/PCU for naïve, 86.71 for positive epidemic, and 33.37 for positive endemic statuses; in the finishing phase, 81.31, 76.55, and 67.09 mg/PCU, respectively. The most frequently injected antibiotic in the nursery phase was ampicillin, with 49 % of total injections, followed by lincomycin (31 %) and enrofloxacin (20 %), and in the finishing phase, 72 % of injections were lincomycin, followed by enrofloxacin (28 %). The results highlight that the PRRSV outbreak in the source was associated with a grow-finish ABU increase, revealing the importance of preventing PRRSV infection to potentially decrease ABU and improve AMS within swine production systems.

Comparative assessment of the performance of a commercial fluorescent microsphere immunoassay and three commercial ELISAs for Mycoplasma hyopneumoniae serum antibody detection.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is a significant porcine respiratory disease complex pathogen, prompting many swine farms and production systems to pursue M. hyopneumoniae elimination strategies. Antibody testing is cost-effective in demonstrating sustained freedom from M. hyopneumoniae, often replacing PCR testing on deep tracheal swabs. The process typically involves testing a subpopulation of the herd using an M. hyopneumoniae screening antibody ELISA, with non-negative results further assessed through confirmatory testing, such as PCR. Recently, a commercial (Biovet) fluorescent microsphere immunoassay (FMIA) for detecting M. hyopneumoniae antibodies has been introduced as an alternative to ELISA. Its performance was compared to three commercial ELISAs (Idexx, Hipra, and Biochek) using experimental serum samples from pigs inoculated with M. hyopneumoniae, M. hyorhinis, M. hyosynoviae, M. flocculare, or mock-inoculated with Friis medium. FMIA consistently detected M. hyopneumoniae at earlier time points than the ELISAs, although two false-positive results were encountered using the manufacturer's recommended cutoff. ROC analysis allowed for the evaluation of various cutoffs depending on testing objectives. Poisson regression of misclassification error counts detected no difference in the Biovet FMIA and Hipra ELISA but significantly fewer misclassification errors than Idexx and Biocheck ELISAs. This study showed FMIA as a suitable alternative to traditional ELISAs for screening purposes due to its superior antibody detection rate at early stages. Alternatively, adopting a more stringent cutoff to improve diagnostic specificity could position the FMIA as a viable confirmatory test option. Overall, FMIA is an optimal choice for M. hyopneumoniae antibody surveillance testing, offering versatility in testing strategies (e.g., triplex FMIA M. hyopneumoniae/PRRSV types 1 and 2) and contributing to improved diagnostic capabilities in porcine health management.

A cross-sectional assessment of PRRSV nucleic acid detection by RT-qPCR in serum, ear-vein blood swabs, nasal swabs, and oral swabs from weaning-age pigs under field conditions.

Introduction: The porcine reproductive and respiratory syndrome virus (PRRSV) continues to challenge swine production in the US and most parts of the world. Effective PRRSV surveillance in swine herds can be challenging, especially because the virus can persist and sustain a very low prevalence. Although weaning-age pigs are a strategic subpopulation in the surveillance of PRRSV in breeding herds, very few sample types have been validated and characterized for surveillance of this subpopulation. The objectives of this study, therefore, were to compare PRRSV RNA detection rates in serum, oral swabs (OS), nasal swabs (NS), ear-vein blood swabs (ES), and family oral fluids (FOF) obtained from weaning-age pigs and to assess the effect of litter-level pooling on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection of PRRSV RNA. Methods: Three eligible PRRSV-positive herds in the Midwestern USA were selected for this study. 666 pigs across 55 litters were sampled for serum, NS, ES, OS, and FOF. RT-qPCR tests were done on these samples individually and on the litter-level pools of the swabs. Litter-level pools of each swab sample type were made by combining equal volumes of each swab taken from the pigs within a litter. Results: Ninety-six piglets distributed across 22 litters were positive by PRRSV RT-qPCR on serum, 80 piglets distributed across 15 litters were positive on ES, 80 piglets distributed across 17 litters were positive on OS, and 72 piglets distributed across 14 litters were positive on NS. Cohen's kappa analyses showed near-perfect agreement between all paired ES, OS, NS, and serum comparisons (). The serum RT-qPCR cycle threshold values (Ct) strongly predicted PRRSV detection in swab samples. There was a ≥ 95% probability of PRRSV detection in ES-, OS-, and NS pools when the proportion of positive swab samples was ≥ 23%, ≥ 27%, and ≥ 26%, respectively. Discussion: ES, NS, and OS can be used as surveillance samples for detecting PRRSV RNA by RT-qPCR in weaning-age pigs. The minimum number of piglets to be sampled by serum, ES, OS, and NS to be 95% confident of detecting ≥ 1 infected piglet when PRRSV prevalence is ≥ 10% is 30, 36, 36, and 40, respectively.

Porcine reproductive and respiratory syndrome virus RNA detection in tongue tips from dead animals.

The control of porcine reproductive and respiratory syndrome virus (PRRSV) hinges on monitoring and surveillance. The objective of this study was to assess PRRSV RNA detection by RT-PCR in tongue tips from dead suckling piglets compared to serum samples, processing fluids, and family oral fluids. Tongue tips and serum samples were collected from three PRRSV-positive breeding herd farms (farms A, B, and C) of three different age groups: newborns (<24 h), processing (2 to 7 days of age), and weaning (18 to 22 days of age). Additionally, processing fluids and family oral fluids were collected from 2-7 days of age and weaning age, respectively. In farms A and B, PRRSV RNA was detected in tongue tips from all age groups (100 and 95%, respectively). In addition, PRRSV RNA was detected in pooled serum samples (42 and 27%), processing fluids (100 and 50%), and family oral fluids (11 and 22%). Interestingly, the average Ct value from tongue tips was numerically lower than the average Ct value from serum samples in the newborn age. In farm C, PRRSV RNA was only detected in serum samples (60%) and family oral fluids (43%), both from the weaning age. Further, no PRRSV RNA was detected in tongue tips when pooled serum samples from the same age group tested PRRSV RNA-negative. Taken together, these results demonstrate the potential value of tongue tips for PRRSV monitoring and surveillance.

Comparison of Mycoplasma hyopneumoniae response to infection by route of exposure.

Mycoplasma hyopneumoniae (MHP) is a concern both for pig well-being and producer economic viability. In the absence of fully protective health interventions, producers rely on controlled exposure to induce an immune response in pigs and minimize the clinical outcomes of MHP infection in pig populations. This study compared the effect of route of exposure on MHP infection, antibody response, clinical signs, and pathology. Six-week-old MHP-negative pigs (n = 78) were allocated to negative control (n = 6) or one of three MHP exposure routes: intratracheal (n = 24, feeding catheter), intranasal (n = 24, atomization device), and aerosol (n = 24, fogger). Body weight, cough indices, and samples (serum, oral fluid, tracheal) were collected weekly through 49 days post-exposure (DPE). Intratrachal exposure produced the highest proportion (24/24) of MHP DNA-positive pigs on DPE 7, as well as earlier and higher serum antibody response. Intranasal and aerosol exposures resulted in infection with MHP DNA detected in tracheal samples from 18/24 and 21/24 pigs on DPE 7, respectively. Aerosol exposure had the least impact on weight gain (0.64 kg/day). No difference was observed among treatment groups in coughing and lung lesions at necropsy. While intratracheal inoculation and seeder animals are frequently used in swine production settings, intranasal or aerosol exposure are viable alternatives to achieve MHP infection. Regardless of the route, steps should be taken to verify the purity of the inoculum and, in the case of aerosol exposure, avert the unintended exposure of personnel and animals to other pathogens.