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Astroviruses infect mammals and birds resulting in either gastroenteritis, neurologic disease, or asymptomatic infection. Porcine astrovirus 4 (PoAstV4) has previously been detected in the upper respiratory tract of pigs with clinical respiratory disease; however, proof of respiratory tract infection and association of the virus with respiratory pathology have not been shown. In this retrospective study of young pigs with clinical respiratory disease of unknown etiology, PoAstV4 was detected with RNA in situ hybridization in lesions consistent with epitheliotropic viral infection in 85 of 117 pigs. This is the first report associating an astrovirus with respiratory pathology.

Purpose. To describe the pathology and imaging findings in two neonatal piglets with congenital limb deformities. Methods. The litter from a second parity crossbred sow presented with four mummified fetuses, three stillborn piglets, and two live piglets with notable limb deformities that were unable to effectively ambulate. The piglets were euthanized and submitted for gross and histological evaluation. Results. Both pigs had bilateral secondary cleft palates, with hypoplasia of the nasal turbinates, and external rotation of the forelimbs. One pig displayed bilateral cryptorchidism, markedly thin and shortened hindlimbs, and syndactyly of both hind feet. Radiographs and gross dissection confirmed the presence of single ossified proximal to distal phalanges of both feet, bilaterally shortened tibias with fibular aplasia, and delayed ossification of tarsal as well as carpal bones. Conclusions. To the author’s knowledge, this is the first reported case of hindlimb meromelia with syndactyly in a pig.

Bovine coronavirus (BCoV) is a known cause of enteric disease in cattle; however, its role in bovine respiratory disease (BRD) is poorly understood, with a dearth of evidence of the detection of the virus in respiratory tract lesions. We coupled histologic evaluation of tracheal and lower airway tissues from 104 calves with BRD in which BCoV was detected in the lungs via PCR followed by direct detection of BCoV by immunohistochemistry and an RNA in situ hybridization assay (ISH; RNAscope technology). RNAscope ISH detected BCoV in respiratory epithelium in more cases than did IHC. Using both methods of direct detection, tracheal epithelial attenuation and identification of the virus within lesions were observed commonly. Our results confirm a role of BCoV in respiratory tract infection and pathology, and show that the virus likely plays a role in the development of BRD.

Accurate and timely results of diagnostic investigations and laboratory testing guide clinical interventions for the continuous improvement of animal health and welfare. Infectious diseases can severely limit the health, welfare, and productivity of populations of animals. Livestock veterinarians submit thousands of samples daily to veterinary diagnostic laboratories (VDLs) for disease diagnosis, pathogen monitoring, and surveillance. Individual diagnostic laboratory reports are immediately useful; however, aggregated historical laboratory data are increasingly valued by clinicians and decision-makers to identify changes in the health status of various animal populations over time and geographical space. The value of this historical information is enhanced by visualization of trends of agent detection, disease diagnosis, or both, which helps focus time and resources on the most significant pathogens and fosters more effective communication between livestock producers, veterinarians, and VDL professionals. Advances in data visualization tools allow quick, efficient, and often real-time scanning and analysis of databases to inform, guide, and modify animal health intervention algorithms. Value is derived at the farm, production system, or regional level. Visualization tools allow client-specific analyses, benchmarking, formulation of research questions, and monitoring the effects of disease management and precision farming practices. We present here the approach taken to visualize trends of disease occurrence using porcine disease diagnostic code data for the period 2010 to 2019. Our semi-automatic standardized creation of a visualization platform allowed the transformation of diagnostic report data into aggregated information to visualize and monitor disease diagnosis.

Technologic advances in information management have rapidly changed laboratory testing and the practice of veterinary medicine. Timely and strategic sampling, same-day assays, and 24-h access to laboratory results allow for rapid implementation of intervention and treatment protocols. Although agent detection and monitoring systems have progressed, and wider tracking of diseases across veterinary diagnostic laboratories exists, such as by the National Animal Health Laboratory Network (NAHLN), the distinction between detection of agent and manifestation of disease is critical to improved disease management. The implementation of a consistent, intuitive, and useful disease diagnosis coding system, specific for veterinary medicine and applicable to multiple animal species within and between veterinary diagnostic laboratories, is the first phase of disease data aggregation. Feedback loops for continuous improvement that could aggregate existing clinical and laboratory databases to improve the value and applications of diagnostic processes and clinical interventions, with interactive capabilities between clinicians and diagnosticians, and that differentiate disease causation from mere agent detection, remain incomplete. Creating an interface that allows aggregation of existing data from clinicians, including final diagnosis, interventions, or treatments applied, and measures of outcomes, is the second phase. Prototypes for stakeholder cooperation, collaboration, and beta testing of this vision are in development and becoming a reality. We focus here on how such a system is being developed and utilized at the Iowa State University Veterinary Diagnostic Laboratory to facilitate evidence-based medicine and utilize diagnostic coding for continuous improvement of animal health and welfare.

Chondrosarcomas are common tumors of the canine appendicular and axial skeleton; however, extraskeletal chondrosarcomas are very rare. Herein we report a case of extraskeletal chondrosarcoma in the tongue of a dog. Histologically, glossal skeletal muscle was infiltrated and effaced by islands of cartilage and streams of spindle-shaped cells. Retrospective analysis of 236 tongue masses submitted to the Iowa State University surgical biopsy service between 2011 and 2019 showed that the majority of submitted tongue masses are either non-neoplastic or benign, with granular cell tumors identified as the most prevalent benign neoplasms. Malignant tumors accounted for nearly 30% of all submitted masses, with malignant melanoma diagnosed most frequently.

Vaccine control and prevention of porcine reproductive and respiratory syndrome (PRRS), the most important disease of swine, is difficult to achieve. However, the discovery of broadly neutralizing antibody activity against porcine reproductive and respiratory syndrome virus (PRRSV) under typical field conditions opens the door to new immunologic approaches for robust protection. We show here that passive administration of purified immunoglobulins with neutralizing antibodies reduced PRRSV2 infection by up to 96%, and PRRSV1 infection by up to 87%, whereas immune immunoglobulins lacking neutralizing activity had no effect on viral infection. Hence, immune competence of passive immunoglobulin transfer was associated specifically with antibody neutralizing activity. Current models of PRRSV infection implicate a minor envelope glycoprotein (GP) complex including GP2, GP3, and GP4, as critical to permissive cell infection. However, conserved peptides comprising the putative cell attachment structure did not attenuate neutralization or viral infection. The results show that immunological approaches aimed at induction of broadly neutralizing antibodies may substantially enhance immune protection against PRRSV. The findings further show that naturally occurring viral isolates are able to induce protective humoral immunity against unrelated PRRSV challenge, thus removing a major conceptual barrier to vaccine development.

The lack of available, well characterized, established, domestic porcine cell lines hinders the advancement of porcine cellular immunology. A case of multicentric lymphoma was diagnosed in a market weight pig at the time of slaughter. Affected lymph nodes and spleen were collected and used for single cell isolation and analysis. Cell lines were established by 3 rounds of limiting dilution from splenic and subiliac lymph node lymphomas. Surface marker staining identified the cells as CD21+, CD79a+, CD20+, PAX5+, and CD3- and cells were grown and easily passaged in cell culture. Transcriptome analysis was carried out to further characterize these rapidly proliferating cells validating the initial cytometric findings, confirming their identity as B cell lymphomas, and suggesting that they arose from germinal center centroblasts with aberrant control of BCL6 expression. Functional analysis identified the cells as being involved in cancer, cell movement, cell survival, and apoptosis. These new porcine B cell lymphoma cell lines will be a valuable resource for more in-depth cellular investigations into the porcine immune system and cancer, as well as providing a potential tool for the growth of lymphotropic viruses of pigs and humans.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine health and well-being worldwide largely due to an insufficient understanding of the adaptive immune response to infection leading to ineffective PRRSV control. The memory and anamnestic response to infection are critical gaps in knowledge in PRRSV immunity. The lack of effective tools for the evaluation of the memory response previously hindered the ability to effectively characterize the porcine memory response to infection. However, the creation and validation of a PRRSV nsp7-specific B cell tetramer now facilitates the ability to detect very rare memory B cells and thus define the memory response of the pig. Here, we describe the PRRSV nsp7-specific B cell response following vaccination and challenge in six key secondary lymphoid organs including the identification of PBMCs as the tissue of interest for the memory immune response in pigs. Following live virus challenge of immune animals, an anamnestic response of nsp7-specific memory B cells and neutralizing antibodies was observed. This characterization of the functional humoral immune response to PRRSV answers key questions involved in regional specialization of the immune response following intramuscular inoculation of PRRSV MLV.

Immunological memory is elicited after either vaccination or natural exposure to a pathogen and is essential for protection against re-exposure. Despite its critical importance, the ability to interrogate the veterinary animal memory immune response has long been hindered by a paucity of tools to assess immunological memory. As a result, the evaluation and analysis of protective immune responses that predict immune protection in food and fiber animals and facilitate vaccine development are obstructed. To fill this gap in knowledge in swine, we created a B cell tetramer to porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 7 (nsp7) to efficiently and effectively investigate the memory B cell response, a hallmark of anti-viral immunity. This novel reagent was validated by using a modified capture ELISA, tetramer pulldowns, and flow cytometry, and it was shown to detect rare, antigen-specific B cells that were present at a frequency of about 0.001% of total B lymphocytes in immune animals. The nsp7-B cell tetramer will help to characterize the PRRSV-specific memory B cell response, which is fundamentally important for understanding immunological competence and animal variation in resistance to PRRSV infection. We expect that the method will be widely applicable to the exploration of immunity to veterinary pathogens.

The adaptive immune response is necessary for the development of protective immunity against infectious diseases. Porcine reproductive and respiratory syndrome virus (PRRSV), a genetically heterogeneous and rapidly evolving RNA virus, is the most burdensome pathogen of swine health and wellbeing worldwide. Viral infection induces antigen-specific immunity that ultimately clears the infection. However, the resulting immune memory, induced by virulent or attenuated vaccine viruses, is inconsistently protective against diverse viral strains. The immunological mechanisms by which primary and memory protection are generated and used are not well understood. Here, we summarize current knowledge regarding cellular and humoral components of the adaptive immune response to PRRSV infection that mediate primary and memory immune protection against viruses.

In the pig, the memory B cell is largely uncharacterized and as a result, our ability to determine long-term vaccine efficacy has been hindered. To address this unmet need, CD21+ porcine B cells were activated in vitro with CD40L and stimulated with various cytokines to test for cellular activation and expansion into antibody secreting cells (ASC). IL-21 was a potent inducer of proliferation and differentiation, inducing a 3-fold expansion in total cell numbers with about 15% of all B cells differentiating to IgM or IgG ASCs. But even with robust proliferation the viability of CD21+ B cells deteriorated rapidly following initial expansion. Thus, APRIL and BAFF were tested as cell survival and maintenance factors. BAFF enhanced mature B cell and ASC viability, but APRIL was only effective for ASCs. They induced a 2-fold increase in secreted IgM and IgG. CD21+ B cells from porcine reproductive and respiratory virus (PRRSV) immune and naïve pigs were expanded and tested for reactivity to nonstructural protein 7 (nsp7), a highly immunogenic PRRSV protein. Immune, but not naïve, pigs displayed positive reactivity on both ELISPOT and ELISA. These results highlight the expansion and differentiation potential of memory B cells in vitro without the stimulation of surface Ig, as well as the establishment of a culture system to interrogate the porcine memory B cell. A PRRSV nsp7-B-cell tetramer was produced for identification and FACS sorting of antigen-specific B cells. Combining B cell tetramers with the in vitro model of memory B cell expansion is expected to facilitate the evaluation of vaccination efficacy through analysis of memory B cell response quality and magnitude without requiring live animal challenge.

Immunological prevention of infectious disease, especially viral, is based on antigen-specific long-lived memory B cells. To test for cellular proliferation and differentiation factors in swine, an outbred model for humans, CD21+ B cells were activated in vitro with CD40L and stimulated with purported stimulatory cytokines to characterize functional responses. IL-21 induced a 3-fold expansion in total cell numbers with roughly 15% of all B cells differentiating to IgM or IgG antibody secreting cells (ASCs.) However, even with robust proliferation, cellular viability rapidly deteriorated. Therefore, a proliferation inducing ligand (APRIL) and B cell activating factor (BAFF) were evaluated as survival and maintenance factors. BAFF was effective at enhancing the viability of mature B cells as well as ASCs, while APRIL was only effective for ASCs. Both cytokines increased approximately two-fold the amount of IgM and IgG which was secreted by IL-21 differentiated ASCs. Mature B cells from porcine reproductive and respiratory virus (PRRSV) immune and naïve age-matched pigs were activated and treated with IL-21 and then tested for memory cell differentiation using a PRRSV non-structural protein 7 ELISPOT and ELISA. PRRSV immune pigs were positive on both ELISPOT and ELISA while naïve animals were negative on both assays. These results highlight the IL-21-driven expansion and differentiation of memory B cells in vitro without stimulation of the surface immunoglobulin receptor complex, as well as the establishment of a defined memory B cell culture system for characterization of vaccine responses in outbred animals.

The lack of well characterized, established domestic porcine cell lines hinders advancement of porcine cellular immunology understanding in disease resistance and anti-viral immunity. Numerous cases of multicentric lymphoma were diagnosed in pigs at the time of slaughter. Affected organs were harvested and submitted for single cell isolation and analysis. Culture of disaggregated single cell suspensions in RPMI 1640 with weekly passage resulted in clusters of dividing cells in about 2% of attempted isolations. In one pig, cells grew in approximately 50% of wells. Cell lines were established by limiting dilution repeated 3 times from splenic and subiliac lymph node lymphomas. Initial flow cytometry analysis showed a population of CD3+, CD79a+, CD21+, CD4−, and CD8− cells which have grown and been maintained in culture for more than 7 months and more than 10 subcultures. To further characterize the nature of this paradoxical homogenous population of CD3+ and CD79a+ cells, transcriptome analysis is being carried out to validate initial cytometric findings and identify key gene pathways. This new porcine lymphoma cell line will be a valuable resource for more in-depth cellular investigations into the porcine immune system, as well as providing a potential tool for the growth of lymphotropic viruses of pigs and humans.

Autophagy is an important component of the innate immune response, directly destroying many intracellular pathogens. However, some pathogens, including several RNA viruses, subvert the autophagy pathway, or components of the pathway, to facilitate their replication. In the present study, the effect of inhibiting autophagy on the growth of dengue virus was tested using a novel inhibitor, spautin-1 ( s pecific and p otent aut ophagy in hibitor 1). Inhibition of autophagy by spautin-1 generated heat-sensitive, noninfectious dengue virus particles, revealing a large effect of components of the autophagy pathway on viral maturation. A smaller effect on viral RNA accumulation was also observed. Conversely, stimulation of autophagy resulted in increased viral titers and pathogenicity in the mouse. We conclude that the presence of functional autophagy components facilitates viral RNA replication and, more importantly, is required for infectious dengue virus production. Pharmacological inhibition of host processes is an attractive antiviral strategy to avoid selection of treatment-resistant variants, and inhibitors of autophagy may prove to be valuable therapeutics against dengue virus infection and pathogenesis.