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Senecavirus A (SVA) infection in pigs causes vesicular disease and results in a short viremia and transient shedding of the virus, mainly in oral fluids and feces. Here we describe the consistent prolonged shedding of SVA in the semen of 2 boars, and persistence of SVA within the tonsils and testes of 3 adult boars. Two SVA-infected boars that were identified on a Minnesota sow farm in 2017 shed SVA RNA in semen for >3 mo after an outbreak of vesicular disease had occurred on the farm. SVA was isolated from 1 semen sample collected 9 d after clinical disease began on the farm. The third SVA-infected boar was identified on an Indiana sow farm in 2020. All boars had SVA RNA detected in the testes and tonsils by RT-rtPCR, with lower Ct values obtained for the testes than from the tonsils. All boars had multifocal lymphocytic orchitis with segmental degeneration and atrophy of the germinal epithelium within the seminiferous tubules. One boar also had areas of seminiferous tubule collapse and interstitial fibrosis within the testes. In all boars, in situ hybridization demonstrated the presence of SVA mRNA within cells located basally in the seminiferous tubules of the testes, and within the basal surface epithelial cells, crypt epithelial cells, and subepithelial and parafollicular lymphocytes and histiocytes of the tonsil.

Background: There is limited information on the distribution of virulence-associated genes (VAGs) in U.S. Streptococcus suis isolates, resulting in little understanding of the pathogenic potential of these isolates. This lack also reduces our understanding of the epidemiology associated with S. suis in the United States and thus affects the efficiency of control and prevention strategies. In this study we applied whole genome sequencing (WGS)-based approaches for the characterization of S. suis and identification of VAGs., Results: Of 208 S. suis isolates classified as pathogenic, possibly opportunistic, and commensal pathotypes, the genotype based on the classical VAGs (epf, mrp, and sly encoding the extracellular protein factor, muramidase-release protein, and suilysin, respectively) was identified in 9% (epf+/mrp+/sly+) of the pathogenic pathotype. Using the chi-square test and LASSO regression model, the VAGs ofs (encoding the serum opacity factor) and srtF (encoding sortase F) were selected out of 71 published VAGs as having a significant association with pathotype, and both genes were found in 95% of the pathogenic pathotype. The ofs+/srtF+ genotype was also present in 74% of 'pathogenic' isolates from a separate validation set of isolates. Pan-genome clustering resulted in the differentiation of a group of isolates from five swine production companies into clusters corresponding to clonal complex (CC) and virulence-associated (VA) genotypes. The same CC-VA genotype patterns were identified in multiple production companies, suggesting a lack of association between production company, CC, or VA genotype., Conclusions: The proposed ofs and srtF genes were stronger predictors for differentiating pathogenic and commensal S. suis isolates compared to the classical VAGs in two sets of U.S. isolates. Pan-genome analysis in combination with metadata (serotype, ST/CC, VA genotype) was illustrated to be a valuable subtyping tool to describe the genetic diversity of S. suis.

Next generation sequencing (NGS) can be applied to identify and characterize the entire set of microbes within a sample. However, this platform does not provide a morphological context or specific association between the viral or bacterial sequences detected and the histological lesions. This limitation has generated uncertainty whether the sequences identified by NGS are actually contributing or not for the clinical outcome. Although in situ hybridization (ISH) and immunohistochemistry (IHC) can be used to detect pathogens in tissue samples, only ISH has the advantage of being rapidly developed in a context of an emerging disease, especially because it does not require development of specific primary antibodies against the target pathogen. Based on the sequence information provided by NGS, ISH is able to check the presence of a certain pathogen within histological lesions, by targeting its specific messenger RNA, helping to build the relationship between the pathogen and the clinical outcome. In this mini review we have compiled results of the application of NGS-ISH to the investigation of challenging diagnostic cases or emerging pathogens in pigs, that resulted in the detection of porcine circovirus type 3, porcine parvovirus type 2, Senecavirus A, and Mycoplasma hyorhinis ., (Copyright © 2019 Resende, Marshall Lund, Rossow and Vannucci.)

Streptococcus suis is a significant cause of mortality in piglets and growing pigs worldwide. The species contains pathogenic and commensal strains, with pathogenic strains causing meningitis, arthritis, endocarditis, polyserositis, and septicemia. Serotyping and multilocus sequence typing (MLST) are primary methods to differentiate strains, but the information is limited for strains found in the United States. The objective of this study was to characterize the diversity of 208 S. suis isolates collected between 2014 and 2017 across North America (mainly the United States) by serotyping and MLST and to investigate associations between subtype and pathotype classifications (pathogenic, possibly opportunistic, and commensal), based on clinical information and site of isolation. Twenty serotypes were identified, and the predominant serotypes were 1/2 and 7. Fifty-eight sequence types (STs) were identified, and the predominant ST was ST28. Associations among serotypes, STs, and pathotypes were investigated using odds ratio and clustering analyses. Evaluation of serotype and ST with pathotype identified a majority of isolates of serotypes 1, 1/2, 2, 7, 14, and 23 and ST1, ST13, ST25, ST28, ST29, ST94, ST108, ST117, ST225, ST373, ST961, and ST977 as associated with the pathogenic pathotype. Serotypes 21 and 31, ST750, and ST821 were associated with the commensal pathotype, which is composed of isolates from farms with no known history of S. suis -associated disease. Our study demonstrates the use of serotyping and MLST to differentiate pathogenic from commensal isolates and establish links between pathotype and subtype, thus increasing the knowledge about S. suis strains circulating in the United States., (Copyright © 2019 Estrada et al.)

Porcine Pegivirus (PPgV) and Atypical Porcine Pestivirus (APPV) are two recently identified porcine viruses. In this study, the identification of two viruses by metagenomic sequencing, and a duplex semi-quantitative RT-PCR was developed to detect these pathogens simultaneously. The PPgV strain Minnesota-1/2016 had a 95.5%-96.3% nucleotide identity and clustered with the recently identified US PPgV strains, which is a distant clade from the German PPgV strains. The APPV strain Minnesota-1/2016 shared an 87.3%-92.0% nucleotide identity with the other global APPV strains identity but only shared an 82.8%-83.0% nucleotide identity with clade II consisting of strain identified in China. Detection of both PPgV and APPV was 9.0% of the diagnostic cases. Co-infection of PPgV and APPV was identified in 7.5% of the diagnostic cases. The occurrence and genetic characterization of PPgV and APPV further enhance our knowledge regarding these new pathogens in the United States., (© 2019 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH.)

The epidemiology and genetic diversity of transmissible gastroenteritis virus (TGEV) in the United States (US) was investigated by testing clinical cases for TGEV by real time RT-PCR between January 2008 and November 2016. Prevalence of TGEV ranged between 3.8-6.8% and peaked during cold months until March 2013, in which prevalence decreased to < 0.1%. Nineteen complete TGEV genomes and a single strain of porcine respiratory coronavirus (PRCV) from the US were generated and compared to historical strains to investigate the evolution of these endemic coronaviruses. Sixteen of our TGEV strains share 8 unique deletions and 119 distinct amino acid changes, which might greatly affect the biological characteristics of the variant TGEV, and resulted in a "variant" genotype of TGEV. The "variant" genotype shared similar unique deletions and amino acid changes with the recent PRCV strain identified in this study, suggesting a recombination event occurred between the ''variant'' TGEV and PRCV. Moreover, the results indicate the "variant" genotype is the dominant genotype circulating in the US. Therefore, this study provides insight into the occurrence, origin, genetic characteristics, and evolution of TGEV and PRCV circulating in the US.

Background: The study highlights the shedding pattern of Senecavirus A (SVA) during an outbreak of vesicular disease in a sow farm from the South-central Minnesota, USA. In this study, 34 individual, mixed parity sows with clinical signs of vesicular lesions and 30 individual piglets from 15 individual litters from sows with vesicular lesions were conveniently selected for individual, longitudinal sampling. Serum, tonsil, rectal, and vesicular swabs were collected on day1 post outbreak, and then again at 1, 2, 3, 4, 6, and 9 weeks post outbreak. Samples were tested at the University of Minnesota Veterinary Diagnostic Laboratory for SVA via Real Time Polymerase Chain Reaction (RT-PCR) RESULTS: In sows, vesicular lesions had the highest concentration of SVA, but had the shortest duration of detection lasting only 2 weeks. Viremia was detected for 1 week post outbreak, and quickly declined thereafter. SVA was detected at approximately the same frequency for both tonsil and rectal swabs with the highest percentage of SVA positive samples detected in the first 6 weeks post outbreak. In suckling piglets, viremia quickly declined 1 week post outbreak and was prevalent in low levels during the first week after weaning (4 weeks post outbreak) and was also detected in piglets that were co-mingled from a SVA negative sow farm. Similar to sows, SVA detection on rectal and tonsil swabs in piglets lasted approximately 6 weeks post outbreak., Conclusion: The study illustrates the variation of SVA shedding patterns in different sample types over a 9 week period in sows and piglets, and suggests the potential for viral spread between piglets at weaning.

The incursion of highly pathogenic avian influenza (HPAI) into the United States during 2014 resulted in an unprecedented foreign animal disease (FAD) event; 232 outbreaks were reported from 21 states. The disease affected 49.6 million birds and resulted in economic losses of $950 million. Minnesota is the largest turkey-producing state, accounting for 18% of U.S. turkey production. Areas with concentrated numbers of turkeys in Minnesota were the epicenter of the outbreak. The first case was presumptively diagnosed in the last week of February 2015 at the Minnesota Veterinary Diagnostic Laboratory (MVDL) and confirmed as HPAI H5N2 at the National Veterinary Services Laboratories on March 4, 2015. A total of 110 farms were affected in Minnesota, and the MVDL tested >17,000 samples from March to July 2015. Normal service was maintained to other clients of the laboratory during this major FAD event, but challenges were encountered with communications, staff burnout and fatigue, training requirements of volunteer technical staff, test kit validation, and management of specific pathogen-free egg requirements.

Background: Porcine circovirus 2 causes different clinical syndromes resulting in a significant economic loss in the pork industry. Three pigs with unexplained cardiac and multi-organ inflammation that tested negative for PCV2 and other known porcine pathogens were further analyzed., Methods: Histology was used to identify microscopic lesions in multiple tissues. Metagenomics was used to detect viral sequences in tissue homogenates. In situ hybridization was used to detect viral RNA expression in cardiac tissue., Results: In all three cases we characterized the genome of a new circovirus we called PCV3 with a replicase and capsid proteins showing 55 and 35 % identities to the genetically-closest proteins from a bat-feces associated circovirus and were even more distant to those of porcine circovirus 1 and 2. Common microscopic lesions included non-suppurative myocarditis and/or cardiac arteriolitis. Viral mRNA was detected intralesionally in cardiac cells. Deep sequencing in tissues also revealed the presence of porcine astrovirus 4 in all three animals as well as rotavirus A, porcine cytomegalovirus and porcine hemagglutinating encephalomyelitis virus in individual cases., Conclusion: The pathogenicity and molecular epidemiology of this new circovirus, alone or in the context of co-infections, warrants further investigations.

Rotaviruses (RV) are important causes of diarrhea in animals, especially in domestic animals. Of the 9 RV species, rotavirus A, B, and C (RVA, RVB, and RVC, respectively) had been established as important causes of diarrhea in pigs. The Minnesota Veterinary Diagnostic Laboratory receives swine stool samples from North America to determine the etiologic agents of disease. Between November 2009 and October 2011, 7,508 samples from pigs with diarrhea were submitted to determine if enteric pathogens, including RV, were present in the samples. All samples were tested for RVA, RVB, and RVC by real time RT-PCR. The majority of the samples (82%) were positive for RVA, RVB, and/or RVC. To better understand the risk factors associated with RV infections in swine diagnostic samples, three-level mixed-effects logistic regression models (3L-MLMs) were used to estimate associations among RV species, age, and geographical variability within the major swine production regions in North America. The conditional odds ratios (cORs) for RVA and RVB detection were lower for 1-3 day old pigs when compared to any other age group. However, the cOR of RVC detection in 1-3 day old pigs was significantly higher (p < 0.001) than pigs in the 4-20 days old and >55 day old age groups. Furthermore, pigs in the 21-55 day old age group had statistically higher cORs of RV co-detection compared to 1-3 day old pigs (p < 0.001). The 3L-MLMs indicated that RV status was more similar within states than among states or within each region. Our results indicated that 3L-MLMs are a powerful and adaptable tool to handle and analyze large-hierarchical datasets. In addition, our results indicated that, overall, swine RV epidemiology is complex, and RV species are associated with different age groups and vary by regions in North America.

The genomic sequence of a rotavirus group H was identified in the intestine of a diarrheal pig in the United States, designated RVH/Pig-wt/USA/MN9.65/2008/GxP[x]., (Copyright © 2016 Hull et al.)

In early 2014, a Minnesota sow farm with a solid vaccination history suffered a severe porcine reproductive and respiratory syndrome (PRRS) outbreak with unusually high morbidity and mortality in piglets and sows, as well as anorexia and secondary bacterial infections in nursery pigs. Due to the unusual clinical severity in a PRRS-immune herd, genetic characteristics of the virus were examined to determine if a new PRRSV genotype had emerged. Phylogenetic analysis indicated that the virulent strain (PRRSV2/USA/Minnesota414/2014) was related to virulent strains circulating in the mid-western United States in recent years, and that the nonstructural protein 2 (nsp2) gene of MN414 contained an insertion-deletion pattern typical of existing type 2 virulent strains. We conclude that the MN414 isolate is a recently evolved member of the virulent lineage 1 family of type 2 PRRSV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Delinquent reendothelialization (rET) has been shown to have a permissive, if not facilitatory, impact on smooth muscle cell proliferation. This inverse relation has been attributed to certain functions of the endothelium, including barrier regulation of permeability, thrombogenicity, and leukocyte adherence, as well as production of growth-inhibitory molecules. Accordingly, the present investigation was designed to test the hypothesis that an endothelial cell (EC) mitogen could serve as the basis for a novel gene therapy strategy designed to facilitate EC regeneration, reduce neointimal thickening, and promote recovery of EC dysfunction after balloon injury., Methods and Results: New Zealand White rabbits underwent simultaneous balloon injury and gene transfer of one femoral artery with phVEGF165, encoding the 165-amino acid isoform of vascular endothelial growth factor (VEGF), or pGSVLacZ. In each animal transfected with phVEGF165 or pGSVLacZ, the contralateral femoral artery was also subjected to balloon injury but not to gene transfer. For pGSVLacZ, rET remained incomplete at 4 weeks after transfection; in contrast, phVEGF165 produced prompt rET, which was 95% complete by 1 week. Furthermore, rET in the contralateral, balloon-injured, nontransfected limb of the VEGF group was similarly accelerated. Consequently, intimal thickening was diminished, thrombotic occlusion was less frequent, and recovery of EC-dependent vasomotor reactivity was accelerated in VEGF transfectants compared with control animals. A similar benefit was observed for the contralateral, balloon-injured, nontransfected limb., Conclusions: Catheter-mediated, site-specific arterial gene transfer of phVEGF165 can accelerate rET at local and remote sites, leading to inhibition of neointimal thickening, reduction in thrombogenicity, and restoration of endothelium-dependent vasomotor reactivity. These findings support the notion that gene transfer encoding for an EC-specific mitogen may be useful for preventing the complications, including restenosis, of balloon angioplasty.

Background: Striated muscle has been shown to be capable of taking up and expressing foreign genes transferred in the form of naked plasmid DNA, although typically with a low level of gene expression. In the case of genes that encode secreted proteins, however, low transfection efficiency may not preclude bio-activity of the secreted gene product. Accordingly, we investigated the hypothesis that intramuscular (IM) gene therapy with naked plasmid DNA encoding vascular endothelial growth factor (VEGF) could augment collateral development and tissue perfusion in an animal model of hindlimb ischemia., Methods and Results: Ten days after ischemia was induced in one rabbit hindlimb, 500 micrograms of phVEGF165, or the reporter gene LacZ, was injected IM into the ischemic hindlimb muscles. Thirty days later, angiographically recognizable collateral vessels and histologically identifiable capillaries were increased in VEGF transfectants compared with controls. This augmented vascularity improved perfusion to the ischemic limb, documented by a superior calf blood pressure ratio for phVEGF165 (0.85 +/- 0.05) versus controls (0.64 +/- 0.05, P < .01), improved blood flow in the ischemic limb (measured with an intra-arterial Doppler wire) at rest (phVEGF165 = 21.3 +/- 3.9 mL/min, control = 14.6 +/- 1.6 mL/min, P < .01) and after a vasodilator (phVEGF165 = 54.2 +/- 12.0 mL/min, control = 37.3 +/- 8.9 mL/min, P < .01) and increased microspheres in the adductor (phVEGF165 = 4.3 +/- 1.6 mL.min-1.100 g of tissue-1, control = 2.9 +/- 1.2 mL.min-1.100 g of tissue-1, P < .05) and gastrocnemius (phVEGF165 = 3.9 +/- 1.0 mL.min-1.100 g of tissue-1, control = 2.8 +/- 1.4 mL.min-1.100 g of tissue-1, P < .05) muscles of the ischemic limb., Conclusions: Ischemic skeletal muscle represents a promising target for gene therapy with naked plasmid DNA. IM transfection of genes encoding angiogenic cytokines, particularly those that are naturally secreted by intact cells, may constitute an alternative treatment strategy for patients with extensive peripheral vascular disease in whom the use of intravascular catheter-based gene transfer is compromised and/or prohibited.

Proliferation of vascular cells has been previously shown to contribute to spontaneous development of coronary collaterals. Recent studies from several laboratories have established that collateral artery growth in both the heart and limb can be enhanced by administration of angiogenic growth factors, or therapeutic angiogenesis. In this study, we sought (1) to define the extent and time course of endothelial cell (EC) and smooth muscle cell (SMC) proliferation accompanying spontaneous collateral development during limb ischemia and (2) to determine the extent to which proliferative activity of ECs and SMCs is augmented during therapeutic angiogenesis with vascular endothelial growth factor (VEGF), a heparin-binding EC-specific mitogen. Ten days after induction of limb ischemia by surgically excising the femoral artery of rabbits, either VEGF (500 to 1000 micrograms) or saline was administered as a bolus into the iliac artery of the ischemic limb. Cellular proliferation was evaluated by bromodeoxyuridine labeling for 24 hours at day 0 (immediately before VEGF administration) and at days 3, 5, and 7 after VEGF, EC proliferation in the midzone collaterals of VEGF-treated animals increased 2.8-fold at day 5 (P < 0.05 versus control), and returned to baseline levels by day 7. SMC proliferation in midzone collaterals also increased 2.7-fold in response to VEGF (P < 0.05). No significant increase in EC or SMC proliferation was observed in either the stem or re-entry collaterals of VEGF-treated animals compared with untreated ischemic control animals. Reduction of hemodynamic deficit in the ischemic limb measured by lower limb blood pressure was documented at day 7 after VEGF (P < 0.01 versus untreated, ischemic control). These data thus (1) establish the contribution of cellular proliferation to collateral vessel development in limb ischemia and (2) support the concept that augmented cellular proliferation contributes to the enhanced formation of collateral vessels after therapeutic angiogenesis with VEGF.

Background: Most strategies designed to reduce restenosis by the use of pharmacological or biological reagents involve direct inhibition of vascular smooth muscle cell (SMC) proliferation. Alternatively, SMC proliferation might be indirectly inhibited if reendothelialization could be specifically facilitated at sites of balloon-induced arterial injury. Accordingly, we investigated the hypothesis that application of an endothelial cell (EC)-specific mitogen to a freshly denuded intimal surface could accelerate reendothelialization and thereby attenuate intimal hyperplasia., Methods and Results: The left carotid artery of 31 Sprague-Dawley rats was subjected to balloon injury, after which 16 rats were treated with a 30-minute incubation with 100 micrograms of vascular endothelial growth factor (VEGF), an EC-specific mitogen. Control animals (n = 15) received a 30-minute incubation with 0.9% saline. At 2 weeks after balloon injury, carotid artery reendothelialization was markedly superior in the VEGF-treated group compared with the control group (14.59 +/- 1.12 versus 7.96 +/- 0.51 mm2, P < 0.005). The extent of reendothelialization measured at 4 weeks after balloon injury remained superior for arteries treated with VEGF (18.04 +/- 0.90 mm2) versus saline (13.42 +/- 0.84 mm2, P < .005). Neointimal thickening was correspondingly attenuated to a statistically significant degree in arteries treated with VEGF versus the control group at both the 2-week and 4-week time points. Immunostaining for proliferating cell nuclear antigen (PCNA) disclosed a threefold increase in PCNA-positive cells in the neointima of control arteries versus VEGF-treated arteries at 2 weeks after injury., Conclusions: Application of VEGF, an EC-specific growth regulatory molecule, may be effectively used in vivo to promote reendothelialization and thereby indirectly attenuate neointimal thickening due to SMC proliferation.

Background: Little information exists regarding the time course of gene expression after arterial transfection. Accordingly, we sought to determine the time course of gene expression after liposome-mediated arterial gene transfer (lipofectin) using an arterial organ culture model., Experimental Design: Explanted segments of rabbit descending thoracic aorta were maintained in organ culture. Arterial gene transfer, facilitated by cationic liposomes (Lipofectin), was performed with the plasmid pXGH5 encoding the human growth hormone (hGH) under the control of mouse metallothionein-1 promoter., Results: The time course of hGH production after transfection with the plasmid pXGH5 was evaluated. Significant levels (181.0 +/- 33.9 ng/24 hours/gm) of hGH were detected within 24 hours post-transfection, reached a peak on day 7 (238.4 +/- 35.3 ng/24 hours/gm), and declined after day 10. At day 21, hGH could be observed in 50% of the arteries. Immunostaining with a monoclonal antibody for hGH revealed that only a small number of arterial cells (< 1%) were responsible for production of hGH., Conclusions: The organ culture model is a feasible and efficient means for investigating the kinetics of arterial gene transfer. Transfection of pXGH5 results in significant levels of hGH for up to 3 weeks, despite anatomic evidence of only limited gene expression. These data thus support the notion that the magnitude and/or duration of gene expression may be disproportionately high, relative to anatomic assessment of transfection efficiency in the case of a transgene encoding for a secreted protein.

Recombinant bovine interferon-alpha I1 (rBoIFN-alpha) has known antiviral and immunomodulatory effects which have been exploited to reduce clinical disease in a number of clinical situations including bovine respiratory diseases. A slow release rBoIFN-alpha formulation may be of value to reduce bovine respiratory disease under field conditions by extending the period of protection, and hence improving the prophylactic benefits of rBoIFN-alpha. In this report, we describe a formulation of rBoIFN-alpha in sesame oil containing calcium stearate which can successfully sustain the release of rBoIFN-alpha over an 8-day period. Recombinant bovine IFN-alpha could be measured in serum for 8 days following treatment with an initial burst of release 6 h after injection. After a single subcutaneous depot injection of 50 mg and 100 mg of rBoIFN-alpha, initial serum levels reached 12-15 ng/ml and 25 ng/ml respectively. Correlating with this burst of release, there was a decrease in the number of circulating CD4-CD8- gamma delta+ T lymphocytes, and a slight neutropenia. No alterations in other cell phenotypes tested (CD4, CD8, CD2, CD6, B cells, monocytes or MHC class II) were observed, nor were there changes in lymphokine activated killer (LAK), natural killer (NK) cell activity, or oxygen radical formation (assessed by reduction of nitroblue tetrazolium). However, despite the rapid and short-lived burst of rBoIFN-alpha, levels of 2-5 oligoadenylate (2-5 A) synthetase remained elevated for 8 days. The sustained increase of 2-5 A synthetase was not due to the high initial dose released during the burst 6-12 h after injection, since injection of a bioavailable equivalent dose of interferon induced a significant rise in 2-5 A synthetase activity for 4 days only. As 2-5 A synthetase is known to be a correlate of antiviral activity, we propose that this formulation of rBoIFN-alpha may be one approach to increase the window of protection, leading to more effective prevention of bovine respiratory disease.