Experimental inoculation of pigs with monkeypox virus results in productive infection and transmission to sentinels.

Monkeypox virus (MPXV) is a re-emerging zoonotic poxvirus responsible for producing skin lesions in humans. Endemic in sub-Saharan Africa, the 2022 outbreak with a clade IIb strain has resulted in ongoing sustained transmission of the virus worldwide. MPXV has a relatively wide host range, with infections reported in rodent and non-human primate species. However, the susceptibility of many domestic livestock species remains unknown. Here, we report on a susceptibility/transmission study in domestic pigs that were experimentally inoculated with a 2022 MPXV clade IIb isolate or served as sentinel contact control animals. Several principal-infected and sentinel contact control pigs developed minor lesions near the lips and nose starting at 12 through 18 days post-challenge (DPC). No virus was isolated and no viral DNA was detected from the lesions; however, MPXV antigen was detected by IHC in tissue from a pustule of a principal infected pig. Viral DNA and infectious virus were detected in nasal and oral swabs up to 14 DPC, with peak titers observed at 7 DPC. Viral DNA was also detected in nasal tissues or skin collected from two principal-infected animals at 7 DPC post-mortem. Furthermore, all principal-infected and sentinel control animals enrolled in the study seroconverted. In conclusion, we provide the first evidence that domestic pigs are susceptible to experimental MPXV infection and can transmit the virus to contact animals.

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.

Experimental co-infection of calves with SARS-CoV-2 Delta and Omicron variants of concern.

Since emerging in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has repeatedly crossed the species barrier with natural infections reported in various domestic and wild animal species. The emergence and global spread of SARS-CoV-2 variants of concern (VOCs) has expanded the range of susceptible host species. Previous experimental infection studies in cattle using Wuhan-like SARS-CoV-2 isolates suggested that cattle were not likely amplifying hosts for SARS-CoV-2. However, SARS-CoV-2 sero- and RNA-positive cattle have since been identified in Europe, India, and Africa. Here, we investigated the susceptibility and transmission of the Delta and Omicron SARS-CoV-2 VOCs in cattle. Eight Holstein calves were co-infected orally and intranasally with a mixed inoculum of SARS-CoV-2 VOCs Delta and Omicron BA.2. Twenty-four hours post-challenge, two sentinel calves were introduced to evaluate virus transmission. The co-infection resulted in a high proportion of calves shedding SARS-CoV-2 RNA at 1- and 2-days post-challenge (DPC). Extensive tissue distribution of SARS-CoV-2 RNA was observed at 3 and 7 DPC and infectious virus was recovered from two calves at 3 DPC. Next-generation sequencing revealed that only the SARS-CoV-2 Delta variant was detected in clinical samples and tissues. Similar to previous experimental infection studies in cattle, we observed only limited seroconversion and no clear evidence of transmission to sentinel calves. Together, our findings suggest that cattle are more permissive to infection with SARS-CoV-2 Delta than Omicron BA.2 and Wuhan-like isolates but, in the absence of horizontal transmission, are not likely to be reservoir hosts for currently circulating SARS-CoV-2 variants.

Case report: Malignant chemodectoma with hepatic metastasis in a cat.

A 10-year-old, male-neutered, domestic short-hair cat was examined at the Veterinary Health Center Emergency Service at Kansas State University for a one-day history of dyspnea. Prior to thoracocentesis, sedation was provided. The cat stopped breathing after sedation and went into cardiac arrest. Cardiopulmonary resuscitation (CPR) was unsuccessful. At necropsy, there was severe pleural effusion and bilateral pulmonary atelectasis. The myocardium of the atria and ventricles, and tunica adventitia of coronary vessels, pulmonary artery, and aorta, had pale, firm, multinodular masses ranging from 0.3 to 0.5 cm in diameter. Multiple nodules were also present in the liver. Multifocally expanding the epicardial fat and compressing the underlying epicardium, infiltrating, and expanding the myocardium, and expanding the walls of major vessels, there was a multinodular, unencapsulated, densely cellular neoplasm composed of polygonal epithelial cells arranged in nests and packets and supported by a fine fibrovascular stroma. The nodules in the liver had similar histologic features. In this case, neoplastic cells at the primary and metastatic sites were intensely immunoreactive to synaptophysin, variably reactive to chromogranin A, and negative for neuron specific enolase, cytokeratin, vimentin, thyroglobulin, and smooth muscle actin. The gross, histologic, and immunohistochemical findings support the diagnosis of chemodectoma, with metastases to the liver. Synaptophysin and chromogranin A were the most useful immunohistochemical markers to diagnose malignant chemodectoma in this cat.

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.

Bovine Respiratory Syncytial Virus Genome Sequences from Cattle with Clinical Respiratory Disease in Kansas, 2021.

We report two near-complete bovine respiratory syncytial virus genome sequences collected from 10-month-old cattle with respiratory disease in Kansas in December 2021. No other respiratory pathogens were confirmed in the samples. These genome sequences update the currently circulating BRSV field strains in the United States.

Comparative Analysis of Novel Strains of Porcine Astrovirus Type 3 in the USA.

Porcine astrovirus type 3 (PoAstV3) has been previously identified as a cause of polioencephalomyelitis in swine and continues to cause disease in the US swine industry. Herein, we describe the characterization of both untranslated regions, frameshifting signal, putative genome-linked virus protein (VPg) and conserved antigenic epitopes of several novel PoAstV3 genomes. Twenty complete coding sequences (CDS) were obtained from 32 diagnostic cases originating from 11 individual farms/systems sharing a nucleotide (amino acid) percent identity of 89.74-100% (94.79-100%), 91.9-100% (96.3-100%) and 90.71-100% (93.51-100%) for ORF1a, ORF1ab and ORF2, respectively. Our results indicate that the 5'UTR of PoAstV3 is highly conserved highlighting the importance of this region in translation initiation while their 3'UTR is moderately conserved among strains, presenting alternative configurations including multiple putative protein binding sites and pseudoknots. Moreover, two predicted conserved antigenic epitopes were identified matching the 3' termini of VP27 of PoAstV3 USA strains. These epitopes may aid in the design and development of vaccine components and diagnostic assays useful to control outbreaks of PoAstV3-associated CNS disease. In conclusion, this is the first analysis predicting the structure of important regulatory motifs of neurotropic mamastroviruses, which differ from those previously described in human astroviruses.

Environmental Sampling for Avian Influenza Virus Detection in Commercial Layer Facilities.

The present study was designed to evaluate the utility of environmental samples for convenient but accurate detection of avian influenza virus (AIV) in commercial poultry houses. First, environmental samples from AIV-negative commercial layer facilities were spiked with an H5N2 low pathogenic AIV and were evaluated for their effect on the detection of viral RNA immediately or after incubation at -20 C, 4 C, 22 C, or 37 C for 24, 48, or 72 hr. Second, Swiffer pads, drag swabs, and boot cover swabs were evaluated for their efficiency in collecting feces and water spiked with the H5N2 LPAIV under a condition simulated for a poultry facility floor. Third, environmental samples collected from commercial layer facilities that experienced an H5N2 highly pathogenic AIV outbreak in 2014-15 were evaluated for the effect of sampling locations on AIV detection. The half-life of AIV was comparable across all environmental samples but decreased with increasing temperatures. Additionally, sampling devices did not differ significantly in their ability to collect AIV-spiked environmental samples from a concrete floor for viral RNA detection. Some locations within a poultry house, such as cages, egg belts, house floor, manure belts, and manure pits, were better choices for sampling than other locations (feed trough, ventilation fan, and water trays) to detect AIV RNA after cleaning and disinfection. Samples representing cages, floor, and manure belts yielded significantly more PCR positives than the other environmental samples. In conclusion, environmental samples can be routinely collected from a poultry barn as noninvasive samples for monitoring AIV.

Muestreo ambiental para la detección del virus de la influenza aviar en instalaciones de aves de postura comerciales. El presente estudio fue diseñado para evaluar la utilidad de las muestras ambientales para la detección rápida pero precisa del virus de la influenza aviar (AIV) en casetas avícolas comerciales. Primero, muestras ambientales de las instalaciones comerciales de aves de postura negativas para influenza aviar se inocularon con un virus de la influenza de baja patogenicidad (LPAIV) H5N2 y se evaluaron para determinar su efecto en la detección de ARN viral inmediatamente o después de la incubación a -20 C, 4 C, 22 C o 37 C durante 24 hr, 48 hr o 72 horas. En segundo lugar, se evaluaron las esponjas marca Swiffer, los hisopos de arrastre y los cubre botas para muestreo ambiental para determinar su eficiencia en la recolección de heces y agua inoculada con el virus de influenza aviar de baja patogenicidad H5N2 en una condición simulada de piso de una instalación avícola. En tercer lugar, muestras ambientales recolectadas de instalaciones comerciales de ponedoras que experimentaron un brote de influenza aviar altamente patógena H5N2 en 2014-15, se evaluaron para determinar el efecto de la ubicación de muestreo en la detección de influenza aviar. La vida media del virus de la influenza aviar fue comparable en todas las muestras ambientales, pero disminuyó con el aumento de la temperatura. Además, los dispositivos de muestreo no difirieron significativamente en su capacidad para recolectar muestras ambientales inoculadas con influenza aviar de un piso de concreto para la detección de ARN viral. Algunas ubicaciones dentro de la caseta aviar, como jaulas, bandas transportadoras de huevo, piso de la caseta, bandas transportadoras de gallinasa y fosas de gallinasa, fueron las mejores opciones para el muestreo en comparación con otras ubicaciones (comederos, ventiladores y bandejas de agua) para detectar el ARN del virus de influenza después de la limpieza y desinfección. Las muestras que representan jaulas, piso y bandas transportadoras de gallinasa arrojaron significativamente más resultados positivos de PCR que las otras muestras ambientales. En conclusión, las muestras ambientales se pueden recolectar rutinariamente de uns granja avícola como muestras no invasivas para monitorear al virus de influenza aviar.

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.

Detection and Cellular Tropism of Porcine Astrovirus Type 3 on Breeding Farms.

Astroviruses cause disease in a variety of species. Yet, little is known about the epidemiology of a majority of astroviruses including porcine astrovirus type 3 (PoAstV3), which is a putative cause of polioencephalomyelitis in swine. Accordingly, a cross-sectional study was conducted on sow farms with or without reported PoAstV3-associated neurologic disease in growing pigs weaned from those farms. Additionally, a conveniently selected subset of piglets from one farm was selected for gross and histologic evaluation. The distribution of PoAstV3 in the enteric system was evaluated through in situ hybridization. PoAstV3, as detected by RT-qPCR on fecal samples, was frequently detected across sows and piglets (66-90%) on all farms (65-85%). PoAstV3 was detected subsequently at a similar detection frequency (77% vs 85%) on one farm after three months. Viral shedding, as determined by the cycle quantification value, suggests that piglets shed higher quantities of virus than adult swine. No link between gastrointestinal disease and PoAstV3 was found. However, PoAstV3 was detected by in situ in myenteric plexus neurons of piglets elucidating a possible route of spread of the virus from the gastrointestinal tract to the central nervous system. These data suggest PoAstV3 has endemic potential, is shed in the feces at greater quantities by suckling piglets when compared to sows, and infection is widespread on farms in which it is detected.

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.

Pathogenicity and Competitive Fitness of Salmonella enterica Serovar 4,[5],12:i:- Compared to Salmonella Typhimurium and Salmonella Derby in Swine.

Since 2014, Salmonella 4,[5],12:i:- has emerged as the most common serovar of Salmonella enterica identified from swine samples submitted to veterinary diagnostic laboratories in the United States. To compare the pathogenicity of S. 4,[5],12:i:- in swine to the known pathogenic Salmonella Typhimurium and lesser pathogenic Salmonella Derby, 72 pigs (20 per Salmonella serovar treatment and 12 controls) were inoculated with either S. Typhimurium, S. 4,[5],12:i:-, S. Derby, or sham-inoculated and followed for up to 28 days thereafter via rectal temperature, fecal scoring, and fecal culture. Animals were euthanized on days 2, 4, or 28 to determine the gross and histopathologic signs of disease and tissue colonization. The results clearly demonstrate that for the isolates selected, serovar 4,[5],12:i:- possesses similar ability as serovar Typhimurium to cause clinical disease, colonize the tonsils and ileocecal lymph nodes, and be shed in the feces of infected swine past resolution of clinical disease. To compare the competitive fitness of S. 4,[5],12:i:- to S. Typhimurium in swine when co-infected, 12 pigs were co-inoculated with equal concentrations of both S. Typhimurium and S. 4,[5],12:i and followed for up to 10 days thereafter. When co-inoculated, serovar 4,[5],12:i:- was consistently detected in the feces of a higher percentage of pigs and at higher concentrations than serovar Typhimurium, suggesting an increased competitive fitness of 4,[5],12:i:- relative to serovar Typhimurium when inoculated simultaneously into naïve pigs. Whole genome sequencing analysis of the isolates used in these studies revealed similar virulence factor presence in all S. 4,[5],12:i:- and S. Typhimurium isolates, but not S. Derby, providing additional evidence for similar pathogenicity potential between serovars 4,[5],12:i:- and Typhimurium. Altogether, this data strongly supports the hypothesis that S. 4,[5],12:i:- is a pathogen of swine and suggests a mechanism through increased competitive fitness for the increasing identification of Salmonella 4,[5],12:i:- in swine diagnostic samples over the past several years.

Development of Polioencephalomyelitis in Cesarean-Derived Colostrum-Deprived Pigs Following Experimental Inoculation with Either Teschovirus A Serotype 2 or Serotype 11.

Teschovirus encephalomyelitis is a sporadic disease associated with Teschovirus A (PTV) serotype 1 and, less frequently, other serotypes. In recent years, the number of cases submitted to the Iowa State University Veterinary Diagnostic Laboratory with a history of posterior paresis has increased. Submission histories from various regions of the United States suggest a trend for clinical disease to persist in herds and affect a wider age-range of pigs than historically reported. Polioencephalitis and/or myelitis was consistently present and PTV was detected in affected neural tissue by PCR in a portion of cases. Sequencing from two clinical cases identified PTV-2 and PTV-11. To assess neuropathogenicity of these isolates, 5-week-old cesarean derived and colostrum-deprived pigs were assigned to three groups: negative control (n = 4), PTV-2-inoculated (n = 7), and PTV-11-inoculated (n = 7). Three PTV-2-inoculated pigs developed mild incoordination of the hind limbs, one of which progressed to posterior ataxia. While all PTV-11-inoculated pigs showed severe neurological signs consistent with Teschovirus encephalomyelitis, no evidences of neurological signs were observed in sham-inoculated animals. All PTV-2- and PTV-11-inoculated pigs had microscopic lesions consistent with Teschovirus encephalomyelitis. To our knowledge, this is the first description of PTV-11 and experimental study demonstrating the neuropathogenicity of PTV-11 in the United States.