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Mycoplasma hyorhinis (MHR) and Mycoplasma hyosynoviae (MHS) are common opportunistic pathogens in the upper respiratory tract and tonsils of swine. The identification of the specific species involved in clinical cases using conventional diagnostic methods is challenging. Therefore, a recombinant chimeric polypeptide based on the seven known variable lipoproteins (A-G) specific of MHR and a cocktail of surface proteins detergent-extracted from MHS cultures were generated and their suitability as antemortem biomarkers for serodiagnosis of MHR- and MHS-infection were evaluated by ELISA. M. hyorhinis and MHS ELISA performance, evaluated using serum samples collected over a 56-day observation period from pigs inoculated with MHR, MHS, M. hyopneumoniae, M. flocculare, or Friis medium, varied by assay, targeted antibody isotype, and cutoffs. The progressions of MHR and MHS clinical diseases were evaluated in relation to the kinetics of the isotype-specific antibody response in serum and bacterial shedding in oral fluids during the observation period. In pigs inoculated with MHR, bacterial DNA was detected in one or more of the 5 pens at all sampling points throughout the study, IgA was first detected at DPI 7, one week before the first clinical signs, with both IgA and IgG detected in all samples collected after DPI 14. The peak of MHS shedding (DPI 8) coincided with the onset of the clinical signs, with both IgA and IgG detected in all serum samples collected ≥ DPI 14. This study demonstrated, under experimental conditions, that both ELISAs were suitable for early detection of specific antibodies against MHR or MHS. The diagnostic performance of the MHR and MHS ELISAs varied depending on the selected cutoff and the antibody isotype evaluated. The high diagnostic and analytical specificity of the ELISAs was particularly remarkable. This study also provides insights into the infection dynamics of MHR-associated disease and MHS-associated arthritis not previously described.

Atypical porcine pestivirus (APPV) is a pestivirus that infects swine and has been associated with congenital tremor (CT) in piglets in the field, as well as those born to experimentally infected sows. However, APPV has also been detected in swine of various ages without clinical signs. Experimental and field studies have demonstrated prolonged detection of viral RNA in serum, secretions, and tissues. For this work, nine gilts from a longitudinal APPV field study were selected based on birth in CT-positive litters and evidence of prolonged detection of virus in the sera. These six-month old animals were transported to a research facility for further sampling, breeding, and necropsy. The gilts were placed in contact with naïve animals for approximately one month at two different timepoints, prior to and during gestation, to assess transmission. After farrowing, litters were monitored for CT and tested for APPV. Following arrival, serum samples were PCR-negative for APPV; however, the gilts consistently tested positive in oral fluids and had detectable APPV in the cerebellum months later at necropsy. The gilts had a delayed humoral immune response, with the majority not seroconverting until approximately ten months of age. There were PCR-positive tissues and evidence of seroconversion in animals from both contact groups; however, none of the litters tested PCR-positive for APPV. These findings improve our understanding of the temporal dynamics and transmission potential of APPV infection and can help guide control measures to reduce viral spread.

Atypical porcine pestivirus (APPV) was found to be associated with pigs demonstrating congenital tremors (CT), and clinical signs in pigs have been reproduced after experimental challenge. Subsequently, APPV has been identified in both symptomatic and asymptomatic swine of all ages globally. The objective of this research was to perform a longitudinal study following two cohorts of pigs, those born in litters with pigs exhibiting CT and those born in litters without CT, to analyze the virus and antibody dynamics of APPV infection in serum from birth to market. There was a wide range in the percentage of affected pigs (8–75%) within CT-positive litters. After co-mingling with CT-positive litters at weaning, pigs from CT-negative litters developed viremia that was cleared after approximately 2 months, with the majority seroconverting by the end of the study. In contrast, a greater percentage of pigs exhibiting CT remained PCR positive throughout the growing phase, with less than one-third of these animals seroconverting. APPV RNA was present in multiple tissues from pigs in both groups at the time of marketing. This study improved our understanding of the infection dynamics of APPV in swine and the impact that the immune status and timing of infection have on the persistence of APPV in serum and tissues.

There is a paucity of information concerning the ecology of porcine hemagglutinating encephalomyelitis virus (PHEV) in commercial swine herds. This study provided evidence that PHEV infection is endemic and highly prevalent in U.S. swine herds. These results raised questions for future studies regarding the impact of endemic PHEV on swine health and the mechanisms by which this virus circulates in endemically infected populations. Regardless, the availability of the validated PHEV S1 enzyme-linked immunosorbent assay (ELISA) provides the means for swine producers to detect and monitor PHEV infections, confirm prior exposure to the virus, and to evaluate the immune status of breeding herds. Members of family Coronaviridae cause a variety of diseases in birds and mammals. Porcine hemagglutinating encephalomyelitis virus (PHEV), a lesser-researched coronavirus, can infect naive pigs of any age, but clinical disease is observed in pigs ≤4 weeks of age. No commercial PHEV vaccines are available, and neonatal protection from PHEV-associated disease is presumably dependent on lactogenic immunity. Although subclinical PHEV infections are thought to be common, PHEV ecology in commercial swine herds is unknown. To begin to address this gap in knowledge, a serum IgG antibody enzyme-linked immunosorbent assay (ELISA) based on the S1 protein was developed and evaluated on known-status samples and then used to estimate PHEV seroprevalence in U.S. sow herds. Assessment of the diagnostic performance of the PHEV S1 ELISA using serum samples ( n  = 924) collected from 7-week-old pigs ( n  = 84; 12 pigs per group) inoculated with PHEV, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, or porcine deltacoronavirus showed that a sample-to-positive cutoff value of ≥0.6 was both sensitive and specific, i.e., all PHEV-inoculated pigs were seropositive from days postinoculation 10 to 42, and no cross-reactivity was observed in samples from other groups. The PHEV S1 ELISA was then used to estimate PHEV seroprevalence in U.S. sow herds (19 states) using 2,756 serum samples from breeding females (>28 weeks old) on commercial farms ( n  = 104) with no history of PHEV-associated disease. The overall seroprevalence was 53.35% (confidence interval [CI], ±1.86%) and herd seroprevalence was 96.15% (CI, ±3.70%). IMPORTANCE There is a paucity of information concerning the ecology of porcine hemagglutinating encephalomyelitis virus (PHEV) in commercial swine herds. This study provided evidence that PHEV infection is endemic and highly prevalent in U.S. swine herds. These results raised questions for future studies regarding the impact of endemic PHEV on swine health and the mechanisms by which this virus circulates in endemically infected populations. Regardless, the availability of the validated PHEV S1 enzyme-linked immunosorbent assay (ELISA) provides the means for swine producers to detect and monitor PHEV infections, confirm prior exposure to the virus, and to evaluate the immune status of breeding herds.

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.

Background Porcine parainfluenza virus 1 (PPIV-1) is a respiratory virus in the family Paramyxoviridae and genus Respirovirus . It is closely related to bovine parainfluenza virus 3, human parainfluenza virus 1, and Sendai virus. Recent reports suggest PPIV-1 is widespread in swine herds in the United States and abroad. However, seroprevalence studies and the ability to evaluate cross neutralization between heterologous strains is not possible without validated antibody assays. This study describes the development of an indirect fluorescence antibody (IFA) assay, a whole virus enzyme-linked immunosorbent assay (wv-ELISA) and a serum virus neutralization (SVN) assay for the detection of PPIV-1 antibodies using 521 serum samples collected from three longitudinal studies and two different challenge strains in swine. Results The area under the curve (AUC) of the wv-ELISA (95% CI, 0.93–0.98) was significantly higher ( p  = 0.03) compared to the IFA (95% CI, 0.90–0.96). However, no significant difference was observed between the IFA and wv-ELISA when compared to the SVN (95% CI, 0.92–0.97). All three assays demonstrated relatively uniform results at a 99% true negative rate, with only 11 disagreements observed between the IFA, wv-ELISA and SVN. Conclusions All three serology assays detected PPIV-1 antibody in swine serum of known status that was collected from experimental studies. The SVN detected seroconversion earlier compared to the IFA and the wv-ELISA. Both the wv-ELISA and the SVN had similar diagnostic performance, while the IFA was not as sensitive as the wv-ELISA. All three assays are considered valid for routine diagnostic use. These assays will be important for future studies to screen seronegative swine for research, determine PPIV-1 seroprevalence, and to evaluate vaccine efficacy against PPIV-1 under experimental and field conditions.

Current measures to prevent TGEV from entering a naive herd include quarantine and testing for TGEV-seronegative animals. However, TGEV serology is complicated due to the cross-reactivity with PRCV, which circulates subclinically in most swine herds worldwide. Conventional serological tests cannot distinguish between TGEV and PRCV antibodies; however, blocking ELISAs using antigen containing a large deletion in the amino terminus of the PRCV S protein permit differentiation of PRCV and TGEV antibodies. Several commercial TGEV/PRCV blocking ELISAs are available, but performance comparisons have not been reported in recent research. This study demonstrates that the serologic cross-reactivity between TGEV and PRCV affects the accuracy of commercial blocking ELISAs. Individual test results must be interpreted with caution, particularly in the event of suspect results. Therefore, commercial TGEV/PRCV blocking ELISAs should only be applied on a herd basis. This study compared the performances of three commercial transmissible gastroenteritis virus/porcine respiratory coronavirus (TGEV/PRCV) blocking enzyme-linked immunosorbent assays (ELISAs) using serum samples ( n  = 528) collected over a 49-day observation period from pigs inoculated with TGEV strain Purdue ( n  = 12), TGEV strain Miller ( n  = 12), PRCV ( n = 12), or with virus-free culture medium ( n  = 12). ELISA results were evaluated both with “suspect” results interpreted as positive and then as negative. All commercial kits showed excellent diagnostic specificity (99 to 100%) when testing samples from pigs inoculated with virus-free culture medium. However, analyses revealed differences between the kits in diagnostic sensitivity (percent TGEV- or PRCV-seropositive pigs), and all kits showed significant ( P <  0.05) cross-reactivity between TGEV and PRCV serum antibodies, particularly during early stages of the infections. Serologic cross-reactivity between TGEV and PRCV seemed to be TGEV strain dependent, with a higher percentage of PRCV-false-positive results for pigs inoculated with TGEV Purdue than for TGEV Miller. Moreover, the overall proportion of false positives was higher when suspect results were interpreted as positive, regardless of the ELISA kit evaluated. IMPORTANCE Current measures to prevent TGEV from entering a naive herd include quarantine and testing for TGEV-seronegative animals. However, TGEV serology is complicated due to the cross-reactivity with PRCV, which circulates subclinically in most swine herds worldwide. Conventional serological tests cannot distinguish between TGEV and PRCV antibodies; however, blocking ELISAs using antigen containing a large deletion in the amino terminus of the PRCV S protein permit differentiation of PRCV and TGEV antibodies. Several commercial TGEV/PRCV blocking ELISAs are available, but performance comparisons have not been reported in recent research. This study demonstrates that the serologic cross-reactivity between TGEV and PRCV affects the accuracy of commercial blocking ELISAs. Individual test results must be interpreted with caution, particularly in the event of suspect results. Therefore, commercial TGEV/PRCV blocking ELISAs should only be applied on a herd basis.

Porcine coronaviruses (PoCoVs) are common etiological viral agents of enteric and respiratory disease in swine, but most epidemiological information derives from intensively managed herds. Data from outdoor systems and wildlife remain scarce, despite the potential role of free-range production and wildlife-livestock interactions in sustaining virus transmission. In southern Spain, the traditional Dehesa agroforestry system supports Iberian pigs that share space and resources with wild boars and other species, creating interfaces where cross-species circulation may occur. To address this gap, we assessed the seroprevalence of five PoCoVs, including porcine hemagglutinating encephalomyelitis virus (PHEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV), in Iberian pigs and wild boars from central-western Spain. A total of 260 Iberian pig sera and 564 wild boar sera collected between 2016 and 2020 were tested using indirect ELISAs for PHEV, PEDV, and PDCoV and a blocking ELISA for PRCV/TGEV. Antibodies to PHEV were highly prevalent in Iberian pigs (68.0%) and detected at lower levels in wild boars (22.6%), a pattern consistent with endemic exposure in domestic pigs and sporadic circulation in wildlife. PEDV antibodies were identified in 8.5% of Iberian pigs and 2.8% of wild boars, with higher prevalence in pigs during 2016 followed by a sharp decline, suggesting past but not ongoing activity. PDCoV antibodies were rare overall (3.1% in pigs, 2.6% in wild boars) but reached 41.6% in pigs from Cáceres in 2017-2018, indicative of a localized event. PRCV antibodies were widespread in Iberian pigs (67.3%), with higher prevalence in Badajoz compared to Cáceres, while wild boars showed rising seropositivity in Ciudad Real/Toledo by 2022 (up to 33.3%). No TGEV antibodies were detected in either host population, supporting the predominance of PRCV in the region. These findings demonstrate that PHEV and PRCV are enzootic in free-range Iberian pigs, while PEDV and PDCoV circulate at low levels, and wild boars are more likely incidental than reservoir hosts. The detection of antibodies in the absence of clinical outbreaks underscores the silent nature of PoCoV circulation in extensive systems and highlights the importance of integrating wildlife-livestock interfaces into surveillance and biosecurity strategies in Mediterranean production landscapes.

The contribution of circulating antibody to the protection of naïve piglets against porcine epidemic diarrhea virus (PEDV) was evaluated using a passive antibody transfer model. Piglets (n = 62) derived from 6 sows were assigned to one of 6 different treatments using a randomized block design which provided for allocation of all treatments to all sows' litters. Each treatment was designed to achieve a different level of circulating anti-PEDV antibody via intraperitoneally administration of concentrated serum antibody. Piglets were orally inoculated with PEDV (USA/IN/2013/19338E, 1 x 103 TCID50 per piglet) 24 hours later and then monitored for 14 days. Piglets remained with their dam throughout the experiment. Sow milk samples, piglet fecal samples, and data on piglet clinical signs, body weight, and body temperature were collected daily. Fecal samples were tested by PEDV real-time reverse transcriptase PCR. Serum, colostrum, and milk were tested for PEDV IgG, IgA, and virus-neutralizing antibody. The data were evaluated for the effects of systemic PEDV antibody levels on growth, body temperature, fecal shedding, survival, and antibody response. The analysis showed that circulating antibody partially ameliorated the effect of PEDV infection. Specifically, antibody-positive groups returned to normal body temperature faster and demonstrated a higher rate of survivability than piglets without PEDV antibody. When combined with previous literature on PEDV, it can be concluded that both systemic antibodies and maternal secretory IgA in milk contribute to the protection of the neonatal pig against PEDV infections. Overall, the results of this experiment suggested that passively administered circulating antibodies contributed to the protection of neonatal piglets against PEDV infection.

Background/Objectives: Environmental enrichment (EE) devices are required in various countries and markets to promote animal welfare, with dual-purpose devices more likely to encourage adoption. We developed an EE device that allows pigs to self-administer liquids, designed to align with natural and play behaviors, and utilized a maternal pheromone (MP) to attract pigs to the device. This study aimed to evaluate the efficacy of this device in delivering vaccines for Erysipelas, Ileitis, Mycoplasma, and Influenza to growing pigs. Methods: Pigs were assigned to three treatments groups: Control (unvaccinated), Hand-Vaccinated (via oral gavage or intramuscular injection), and Self-Vaccinated using the EE device. Baseline samples were collected to determine initial antibody status, and serum and oral fluids’ IgG and IgA levels were measured post-vaccination to assess immune response. Four studies were conducted with 36 pigs (12 per treatment) over a 49-day period. Results: Self-vaccination pigs receiving the avirulent live Erysipelas vaccine developed oral and serum antibodies comparable to Hand-Vaccinated pigs. Pigs self-administering the avirulent live Lawsonia intracelluaris vaccine developed oral fluid antibodies. In contrast, pigs who received Mycoplasma or Influenza vaccines through self-vaccination exhibited significantly lower antibody levels compared to the Hand-Vaccinated group. Conclusions: These findings demonstrated that self-vaccination using EE devices for the oral administration of avirulent live vaccines offers benefits such as reduced labor and improved animal welfare. However, killed vaccines did not elicit sufficient antibody responses, suggesting the need for modified vaccine formulations or administration strategies to improve self-vaccination efficacy.