Pigs are highly susceptible to but do not transmit mink-derived highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b.

ABSTRACTRapid evolution of highly pathogenic avian influenza viruses (HPAIVs) is driven by antigenic drift but also by reassortment, which might result in robust replication in and transmission to mammals. Recently, spillover of clade 2.3.4.4b HPAIV to mammals including humans, and their transmission between mammalian species has been reported. This study aimed to evaluate the pathogenicity and transmissibility of a mink-derived clade 2.3.4.4b H5N1 HPAIV isolate from Spain in pigs. Experimental infection caused interstitial pneumonia with necrotizing bronchiolitis with high titers of virus present in the lower respiratory tract and 100% seroconversion. Infected pigs shed limited amount of virus, and importantly, there was no transmission to contact pigs. Notably, critical mammalian-like adaptations such as PB2-E627 K and HA-Q222L emerged at low frequencies in principal-infected pigs. It is concluded that pigs are highly susceptible to infection with the mink-derived clade 2.3.4.4b H5N1 HPAIV and provide a favorable environment for HPAIV to acquire mammalian-like adaptations.

Development and Evaluation of Antigen-Specific Dual Matrix Pestivirus K ELISAs Using Longitudinal Known Infectious Status Samples.

Pestivirus K, commonly known as atypical porcine pestivirus (APPV), is the most common cause of congenital tremor (CT) in pigs. Currently, there is limited information on the infection dynamics of and immune response against APPV and no robust serologic assay to assess the effectiveness of preventative measures. To that end, known infection status samples were generated using experimental inoculation of cesarean-derived, colostrum-deprived pigs. Pigs (2 per pen) were inoculated with minimum essential medium (n = 6; negative control) or APPV (n = 16). Serum, pen-based oral fluid samples, and nasal swabs were collected through 70 days postinoculation (dpi). The immune response to recombinant APPV Erns, E2, or NS3 antigens was evaluated using both serum and oral fluids via indirect enzyme-linked immunosorbent assays (ELISAs). APPV was detected by real-time reverse transcription-PCR (RT-qPCR) in all oral fluid and serum samples from APPV-inoculated animals by 24 and 35 dpi, respectively. All samples remained genome positive until 70 dpi. Detection of nasal shedding was less consistent, with APPV being detected by RT-qPCR in all inoculated animals at 42, 49, and 56 dpi. Antibodies were first detected in oral fluids at 14 dpi, 10 days before serum detection, and concurrently with the first oral fluids RT-qPCR detection. Across sample types and time points, the Erns ELISA outperformed the other targets. In conclusion, both oral fluid and serum APPV Erns ELISAs can be used to economically evaluate the individual and herd status prior to and following intervention strategies.

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.

Polioencephalomyelitis in Domestic Swine Associated With Porcine Astrovirus Type 3.

In the past decade, different members of the genus Mamastrovirus have been associated with outbreaks of neurologic disease in humans, cattle, sheep, mink, and, most recently, porcine astrovirus 3 (PoAstV3) in swine. We performed a retrospective analysis of 50 cases of porcine neurologic disease of undetermined cause but with microscopic lesions compatible with a viral encephalomyelitis to better understand the role and pathogenesis of PoAstV3 infection. Nucleic acid was extracted from formalin-fixed paraffin-embedded (FFPE) tissue for reverse transcription quantitative polymerase chain reaction (RT-qPCR) testing for PoAstV3. In addition, 3 cases with confirmed PoAstV3-associated disease were assayed by RT-qPCR to investigate PoAstV3 tissue distribution. PoAstV3 was detected in central nervous system (CNS) tissue via RT-qPCR and in situ hybridization in 13 of 50 (26%) FFPE cases assayed. PoAstV3 was rarely detected in any tissues outside the CNS. Positive cases from the retrospective study included pigs in various production categories beginning in 2010, the earliest year samples were available. Based on these results, PoAstV3 appears to be a recurring putative cause of viral encephalomyelitis in swine that is rarely detected outside of the CNS at the time of clinical neurologic disease, unlike other common viral causes of neurologic disease in swine.

Early detection and differential serodiagnosis of Mycoplasma hyorhinis and Mycoplasma hyosynoviae infections under experimental conditions.

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.