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In March 2024, the US Department of Agriculture's Animal and Plant Health Inspection Service reported detection of highly pathogenic avian influenza (HPAI) A(H5N1) virus in dairy cattle in the United States for the first time. One factor that determines susceptibility to HPAI H5N1 infection is the presence of specific virus receptors on host cells; however, little is known about the distribution of the sialic acid (SA) receptors in dairy cattle, particularly in mammary glands. We compared the distribution of SA receptors in the respiratory tract and mammary gland of dairy cattle naturally infected with HPAI H5N1. The respiratory and mammary glands of HPAI H5N1-infected dairy cattle are rich in SA, particularly avian influenza virus-specific SA α2,3-gal. Mammary gland tissues co-stained with sialic acids and influenza A virus nucleoprotein showed predominant co-localization with the virus and SA α2,3-gal. HPAI H5N1 exhibited epitheliotropism within the mammary gland, and we observed rare immunolabeling within macrophages.

Prediction and early detection of kidney damage induced by nonsteroidal anti-inflammatories (NSAIDs) would provide the best chances of maximizing the anti-inflammatory effects while minimizing the risk of kidney damage. Unfortunately, biomarkers for detecting NSAID-induced kidney damage in cats remain to be discovered. To identify potential urinary biomarkers for monitoring NSAID-based treatments, we applied an untargeted metabolomics approach to urine collected from cats treated repeatedly with meloxicam or saline for up to 17 days. Applying multivariate analysis, this study identified a panel of seven metabolites that discriminate meloxicam treated from saline treated cats. Combining artificial intelligence machine learning algorithms and an independent testing urinary metabolome data set from cats with meloxicam-induced kidney damage, a panel of metabolites was identified and validated. The panel of metabolites including tryptophan, tyrosine, taurine, threonic acid, pseudouridine, xylitol and lyxitol, successfully distinguish meloxicam-treated and saline-treated cats with up to 75-100% sensitivity and specificity. This panel of urinary metabolites may prove a useful and non-invasive diagnostic tool for monitoring potential NSAID induced kidney injury in feline patients and may act as the framework for identifying urine biomarkers of NSAID induced injury in other species.

The global distribution and constant evolution are challenges for the control of porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting swine worldwide. Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. Here we developed and optimized procedures for real-time genotyping and whole genome sequencing of PRRSV directly from clinical samples based on targeted amplicon- and long amplicon tiling sequencing using the MinION Oxford Nanopore platform. Procedures were developed and tested on 154 clinical samples (including lung, serum, oral fluid and processing fluid) with RT-PCR Ct values ranging from 15 to 35. The targeted amplicon sequencing (TAS) approach was developed to obtain sequences of the complete ORF5 (main target gene for PRRSV genotyping) and partial ORF4 and ORF6 sequences of both PRRSV-1 and PRRSV-2 species. After only 5 min of sequencing, PRRSV consensus sequences with identities to reference sequences above 99% were obtained, enabling rapid identification and genotyping of clinical PRRSV samples into lineages 1, 5 and 8. The long amplicon tiling sequencing (LATS) approach targets type 2 PRRSV, the most prevalent viral species in the U.S. and China. Complete PRRSV genomes were obtained within the first hour of sequencing for samples with Ct values below 24.9. Ninety-two whole genome sequences were obtained using the LATS procedure. Fifty out of 60 sera (83.3%) and 18 out of 20 lung samples (90%) had at least 80% of genome covered at a minimum of 20X sequence depth per position. The procedures developed and optimized in this study here are valuable tools with potential for field application during PRRSV elimination programs.

To evaluate trends in bacterial causes of valvular endocarditis in swine, we retrospectively analyzed 321 cases diagnosed at Iowa State University Veterinary Diagnostic Laboratory (Ames, IA, USA) during May 2015--April 2020. Streptococcus gallolyticus was the causative agent for 7.59% of cases. This emerging infection in swine could aid study of endocarditis in humans.

Mycoplasma hyopneumoniae ( M. hyopneumoniae ), the primary etiological agent of swine enzootic pneumonia, causes significant economic losses in the pork industry. This fastidious pathogen exhibits extremely slow growth in vitro, complicating its quantification. Several quantification methods, including color-changing units (CCU), colony-forming units (CFU), flow cytometry, and ATP luminometry, are documented in the literature, with CCU being the gold standard. However, the correlation among these techniques has not been thoroughly evaluated. Additionally, viability quantitative polymerase chain reaction (v-qPCR) using propidium monoazide (PMA) or ethidium monoazide (EMA) offers a rapid and sensitive alternative for detecting viable bacteria. This study aims to evaluate and compare different methods for quantifying M. hyopneumoniae , validate an accurate real-time quantitative approach, and develop a tailored v-qPCR assay for this challenging pathogen. The in vitro growth kinetics of three M. hyopneumoniae strains (232, J, and 2010) were evaluated using CCU, CFU, flow cytometry, and ATP luminometry. A confocal laser scanning microscopy (CLSM) protocol was established for direct quantification of M. hyopneumoniae in culture media and used to validate flow cytometry-based quantification under controlled conditions. Finally, a rapid v-qPCR assay was developed and optimized for viable M. hyopneumoniae quantification. The comparison of M. hyopneumoniae growth kinetics across CFU, CCU, flow cytometry, and ATP luminometry demonstrated similar growth dynamics and high assay correlation. Flow cytometry and CLSM quantification showed a strong correlation for strain 232 (r = 0.9973) and a moderate correlation for strain J (r = 0.8933). The detection of live M. hyopneumoniae by v-qPCR correlated strongly with viable cell numbers detected by flow cytometry (R² values: 232: 0.9726; J: 0.8628; 2010: 0.9933, p < 0.05). The limit of detection of the v-qPCR assay for the reference strain 232 was 5 × 10⁴ viable M. hyopneumoniae cells/mL. This study presents the first validated PMA-based v-qPCR assay for mycoplasma species, along with established flow cytometry and CLSM protocols for rapid and accurate differentiation of viable and non-viable M. hyopneumoniae cells. These methods significantly reduce quantification time from the four weeks required by CCU or CFU to just a few hours. Furthermore, upon validation in clinical specimens, v-qPCR can potentially serve as a valuable tool in M. hyopneumoniae eradication programs.

Porcine circovirus 2 (PCV2) is an economically significant, ubiquitous pathogen affecting the global swine industry. While vaccines have been highly efficacious in controlling clinical disease, questions have arisen in recent years regarding gaps in clinical protection observed in the field due to heterologous PCV2 infection and coinfections with virulent, contemporary porcine reproductive and respiratory syndrome virus (PRRSV) isolates. Therefore, this study evaluated clinical, pathological, and immunological differences in homologous and heterologous PCV2 vaccinated pigs co-challenged with PCV2d and PRRSV. The study closely mimicked field conditions where 21-day-old commercial pigs were vaccinated with a commercial PRRSV vaccine and either a commercial PCV2a or PCV2d vaccine or left as PCV2 unvaccinated controls. Pigs were co-challenged with PCV2d and PRRSV restriction fragment polymorphism (RFLP) 1–7-4 28 days post-vaccination. In addition to significantly higher mortality, the unvaccinated group had significantly higher tissue viral load and viremia in addition to moderate to severe lymphoid depletion with significantly greater PCV2 antigen detected in the lymph nodes, tonsil, and lung compared to both vaccinated groups. While PCV2 vaccination regardless of subtype prevented the development of severe clinical PCVAD in the majority of vaccinates, the PCV2d vaccinates had reduced tissue viral load, significantly lower viremia, and reduced lymphoid depletion with less PCV2 antigen detected in tissues compared to the PCV2a vaccinates. Additionally, approximately 20 % of the PCV2a vaccinates had moderate to severe lymphoid depletion with moderate to severe antigen detection, which is associated with clinical PCVAD. While total levels of PCV2-specific antibodies measured by ELISA were similar between the PCV2a and PCV2d vaccinates, PCV1–2 chimeric virus neutralization assays revealed differential subtype-specific neutralizing antibody (NA) titers among the PCV2a and PCV2d vaccinates. Prior to challenge on day 28 (28 days post-vaccination), PCV2d vaccinates had significantly higher NA titers against the PCV1–2d vaccine and challenge chimeric viruses, while the PCV2a vaccinates had significantly higher NA titers against the PCV1–2a vaccine chimeric virus. Collectively, homologous vaccination may provide greater protection in virulent co-infection scenarios in the field. This study provides further insight into differences in protection elicited by homologous and heterologous vaccination, resulting in valuable insights to enhance PCV2 control strategies in the current PCV2d/PRRSV co-infection paradigm. •Severe PCVAD and increased mortality were observed in non-vaccinated pigs after PCV2d/PRRSV co-challenge.•PCV2d vaccinates showed reduced viral load, viremia, and minimal lymphoid depletion compared to PCV2a vaccinates.•PCV2d vaccinates had higher neutralizing antibody titers against PCV2d, while PCV2a vaccinates showed delayed antibody responses.•A subset of PCV2a vaccinates developed moderate lymphoid depletion and PCVAD-compatible lesions.•Both vaccines reduced clinical signs but did not prevent PCV2 detection in tissues, highlighting the need for improved vaccine strategies.

Avian reovirus (ARV) has emerged as an important pathogen in turkeys, causing economic losses through tenosynovitis, necrotizing hepatitis, immunosuppression, and enteric disease. Despite its ubiquity, the evolutionary history of ARV cross-species transmission among chickens, turkeys, and wild birds remains poorly understood, hindering effective control and surveillance. This study investigates ARV temporal phylogenetics with an emphasis on interspecies transmission in turkeys. Whole genome sequences (WGSs) from seventy-seven turkey cases and one quail case at the Iowa State University Veterinary Diagnostic Laboratory, along with 74–136 segment sequences per gene from GenBank (1970–2023), were analyzed. Temporal phylogenetic analyses identified chickens as the ancestral host, with spillover into turkeys beginning in the mid-20th century, followed by stable transmission within turkey populations. Migration analyses revealed predominantly unidirectional transmission from chickens to turkeys. WGS analyses showed high variability in the M2 and σC-encoding region of the S1 segment, suggesting selective pressure on outer capsid proteins. M2, S1 σC, and L3 had the highest substitution rates, implicating their role in adaptation and antigenic diversity. These findings highlight the complexity of ARV evolution across hosts and underscore the need for robust genotyping schemes and surveillance strategies to mitigate outbreaks in poultry.

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) infect the small intestine and cause swine enteric coronavirus disease. The mucosal innate immune system is the first line of defense against viral infection. The modulatory effect of PDCoV and PEDV coinfection on antiviral signaling cascades of the intestinal mucosa has not been reported. Here, we investigate the gene expression levels of pattern recognition receptors, downstream inflammatory signaling pathway molecules, and associated cytokines on the intestinal mucosa of neonatal piglets either infected with a single- or co-infected with PDCoV and PEDV using real-time PCR. The results demonstrate that single-PEDV regulates the noncanonical NF-κB signaling pathway through RIG-I regulation. In contrast, single-PDCoV and PDCoV/PEDV coinfection regulate proinflammatory and regulatory cytokines through TRAF6-mediated canonical NF-κB and IRF7 signaling pathways through TLRs. Although PDCoV/PEDV coinfection demonstrated an earlier modulatory effect in these signaling pathways, the regulation of proinflammatory and regulatory cytokines was observed simultaneously during single viral infection. These results suggested that PDCoV/PEDV coinfection may have synergistic effects that lead to enhanced viral evasion of the mucosal innate immune response.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organ’s epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine.

Chronic hepatitis E virus (HEV) infection is a significant clinical problem in immunocompromised individuals such as organ transplant recipients, although the mechanism remains unknown because of the lack of an animal model. We successfully developed a pig model of chronic HEV infection and examined immune correlates leading to chronicity. The conditions of immunocompromised patients were mimicked by treating pigs with an immunosuppressive regimen including cyclosporine, azathioprine, and prednisolone. Immunocompromised pigs infected with HEV progressed to chronicity, because 8/10 drug-treated HEV-infected pigs continued fecal virus shedding beyond the acute phase of infection, whereas the majority (7/10) of mock-treated HEV-infected pigs cleared fecal viral shedding at 8 wk postinfection. During chronic infection, serum levels of the liver enzyme γ-glutamyl transferase and fecal virus shedding were significantly higher in immunocompromised HEV-infected pigs. To identify potential immune correlates of chronic infection, we determined serum levels of cytokines and cell-mediated immune responses in pigs. Results showed that HEV infection of immunocompromised pigs reduced the serum levels of Th1 cytokines IL-2 and IL-12, and Th2 cytokines IL-4 and IL-10, particularly during the acute phase of infection. Furthermore IFN-γ–specific CD4⁺ T-cell responses were reduced in immunocompromised pigs during the acute phase of infection, but TNF-α–specific CD8⁺ T-cell responses increased during the chronic phase of infection. Thus, active suppression of cell-mediated immune responses under immunocompromised conditions may facilitate the establishment of chronic HEV infection. This pig model will aid in delineating the mechanisms of chronic HEV infection and in developing effective therapeutics against chronic hepatitis E.