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Miranda D Vieson,* Pablo Piñeyro,* Tanya LeRoith Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech University, Blacksburg, VA, USA *These authors contributed equally to this workAbstract: Numerous infectious agents are responsible for causing primary or secondary respiratory disease in dogs. These agents can cause upper or lower respiratory infections commonly observed in veterinary practices. Clinical signs might vary from mild dyspnea, sneezing, and coughing to severe pneumonia with systemic manifestations. Depending on the etiologic agent, the gross and microscopic changes observed during these infections can be rather unspecific or have highly characteristic patterns. While histopathology and cytology are not always required for diagnosis of respiratory infections, they are often useful for establishing a definitive diagnosis and identifying specific etiologic agents. Research regarding epidemiology, pathogenesis, diagnostics, and clinical manifestations related to these infectious pathogens provides valuable information that has improved treatments and management of the diseases they cause. This review discusses the epidemiology, general clinical characteristics, and pathologic lesions for some of the important viral, bacterial, fungal, and parasitic etiologies of canine respiratory disease.Keywords: bacterial, fungal, parasitic, pneumonia, rhinitis, tracheobronchitis, viral

Abstract: Porcine circovirus associated disease (PCVAD) is currently one of the most economically important diseases in the global swine industry. Porcine circovirus type 2 (PCV2) is the primary causative agent, however co-infection with other swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV) is often required to induce the full spectrum of clinical PCVAD. While the specific mechanisms of viral co-infection that lead to clinical disease are not fully understood, immune modulation by the co-infecting viruses likely plays a critical role. We evaluated the ability of dendritic cells (DC) infected with PRRSV, PCV2, or both to induce regulatory T cells (Tregs) in vitro. DCs infected with PCV2 significantly increased CD4+CD25+FoxP3+ Tregs (p <0.05) and DCs co-infected with PRRSV and PCV2 induced significantly higher numbers of Tregs than with PCV2 alone (p <0.05). Cytokine analysis indicated that the induction of Tregs by co-infected DCs may be dependent on TGF-β and not IL-10. Our data support the immunomodulatory role of PCV2/PRRSV co-infection in the pathogenesis of PCVAD, specifically via Treg-mediated immunosuppression. [Copyright &y& Elsevier]

Background: Multiple studies have demonstrated associations between the early-life gut microbiome and incidence of inflammatory and autoimmune disease in childhood. Although microbial colonization is necessary for proper immune education, it is not well understood at a mechanistic level how specific communities of bacteria promote immune maturation or drive immune dysfunction in infancy., Objectives: In this study, we aimed to assess whether infant microbial communities with different overall structures differentially influence immune and gastrointestinal development in healthy mice., Methods: Germ-free mice were inoculated with fecal slurries from Bifidobacterium longum subspecies infantis positive (BIP) or B. longum subspecies infantis negative (BIN) breastfed infants; half of the mice in each group were also supplemented with a pool of human milk oligosaccharides (HMOs) for 14 d. Cecal microbiome composition and metabolite production, systemic and mucosal immune outcomes, and intestinal morphology were assessed at the end of the study., Results: The results showed that inoculation with a BIP microbiome results in a remarkably distinct microbial community characterized by higher relative abundances of cecal Clostridium senu stricto, Ruminococcus gnavus, Cellulosilyticum sp., and Erysipelatoclostridium sp. The BIP microbiome produced 2-fold higher concentrations of cecal butyrate, promoted branched short-chain fatty acid (SCFA) production, and further modulated serotonin, kynurenine, and indole metabolism relative to BIN mice. Further, the BIP microbiome increased the proportions of innate and adaptive immune cells in spleen, while HMO supplementation increased proliferation of mesenteric lymph node cells to phorbol myristate acetate and lipopolysaccharide and increased serum IgA and IgG concentrations., Conclusions: Different microbiome compositions and HMO supplementation can modulate SCFA and tryptophan metabolism and innate and adaptive immunity in young, healthy mice, with potentially important implications for early childhood health., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Maternal secretor status is one of the determinants of human milk oligosaccharides (HMOs) composition, which, in turn, influences the gut microbiota composition of infants. To understand if this change in gut microbiota impacts immune cell composition, intestinal morphology, and gene expression, 21-day-old germ-free C57BL/6 mice were transplanted with fecal microbiota from infants whose mothers were either secretors (SMM) or non-secretors (NSM) or from infants consuming dairy-based formula (MFM). For each group, one set of mice was supplemented with HMOs. HMO supplementation did not significantly impact the microbiota diversity; however, SMM mice had a higher abundance of genus Bacteroides , Bifidobacterium , and Blautia , whereas, in the NSM group, there was a higher abundance of Akkermansia , Enterocloster , and Klebsiella . In MFM, gut microbiota was represented mainly by Parabacteroides , Ruminococcaceae&#95;unclassified , and Clostrodium&#95;sensu&#95;stricto . In mesenteric lymph node, Foxp3+ T cells and innate lymphoid cells type 2 were increased in MFM mice supplemented with HMOs, while in the spleen, they were increased in SMM + HMOs mice. Similarly, serum immunoglobulin A was also elevated in MFM + HMOs group. Distinct global gene expression of the gut was observed in each microbiota group, which was enhanced with HMOs supplementation. Overall, our data show that distinct infant gut microbiota due to maternal secretor status or consumption of dairy-based formula and HMO supplementation impacts immune cell composition, antibody response, and intestinal gene expression in a mouse model., Importance: Early life factors like neonatal diet modulate gut microbiota, which is important for the optimal gut and immune function. One such factor, human milk oligosaccharides (HMOs), the composition of which is determined by maternal secretor status, has a profound effect on infant gut microbiota. However, how the infant gut microbiota composition determined by maternal secretor status or consumption of infant formula devoid of HMOs impacts infant intestinal ammorphology, gene expression, and immune signature is not well explored. This study provides insights into the differential establishment of infant microbiota derived from infants fed by secretor or non-secretor mothers milk or those consuming infant formula and demonstrates that the secretor status of mothers promotes Bifidobacteria and Bacteroides sps. establishment. This study also shows that supplementation of pooled HMOs in mice changed immune cell composition in the spleen and mesenteric lymph nodes and immunoglobulins in circulation. Hence, this study highlights that maternal secretor status has a role in infant gut microbiota composition, and this, in turn, can impact host gut and immune system., Competing Interests: The authors declare no conflict of interest.

Introduction: Colonocyte oxidation of bacterial-derived butyrate has been reported to maintain synergistic obligate anaerobe populations by reducing colonocyte oxygen levels; however, it is not known whether this process is disrupted during the progression of type 2 diabetes. Our aim was to determine whether diabetes influences colonocyte oxygen levels in the University of California Davis type 2 diabetes mellitus (UCD-T2DM) rat model., Research Design and Methods: Age-matched male UCD-T2DM rats (174±4 days) prior to the onset of diabetes (PD, n=15), within 1 month post-onset (RD, n=12), and 3 months post-onset (D3M, n=12) were included in this study. Rats were administered an intraperitoneal injection of pimonidazole (60 mg/kg body weight) 1 hour prior to euthanasia and tissue collection to estimate colonic oxygen levels. Colon tissue was fixed in 10% formalin, embedded in paraffin, and processed for immunohistochemical detection of pimonidazole. The colonic microbiome was assessed by 16S gene rRNA amplicon sequencing and content of short-chain fatty acids was measured by liquid chromatography-mass spectrometry., Results: HbA1c % increased linearly across the PD (5.9±0.1), RD (7.6±0.4), and D3M (11.5±0.6) groups, confirming the progression of diabetes in this cohort. D3M rats had a 2.5% increase in known facultative anaerobes, Escherichia-Shigella , and Streptococcus (false discovery rate <0.05) genera in colon contents. The intensity of pimonidazole staining of colonic epithelia did not differ across groups (p=0.37). Colon content concentrations of acetate and propionate also did not differ across UCD-T2DM groups; however, colonic butyric acid levels were higher in D3M rats relative to PD rats (p<0.01)., Conclusions: The advancement of diabetes in UCD-T2DM rats was associated with an increase in facultative anaerobes; however, this was not explained by changes in colonocyte oxygen levels. The mechanisms underlying shifts in gut microbe populations associated with the progression of diabetes in the UCD-T2DM rat model remain to be identified., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Canine soft tissue sarcomas (STS) are common neoplasms and considered immune deserts. Tumour infiltrating lymphocytes are sparse in STS and, when present, tend to organize around blood vessels or at the periphery of the neoplasm. This pattern is associated with an immunosuppressive tumour microenvironment linked to overexpression of molecules of the PD-axis. PD-1, PD-L1 and PD-L2 expression correlates with malignancy and poor prognosis in other neoplasms in humans and dogs, but little is known about their role in canine STS, their relationship to tumour grade, and how different therapies affect expression. The objective of this study was to evaluate the expression of checkpoint molecules across STS tumour grades and after tumour ablation treatment. Gene expression analysis was performed by reverse-transcriptase real-time quantitative PCR in soft tissue sarcomas that underwent histotripsy and from histologic specimens of STS from the Virginia Tech Animal Laboratory Services archives. The expression of PD-1, PD-L1 and PD-L2 was detected in untreated STS tissue representing grades 1, 2, and 3. Numerically decreased expression of all markers was observed in tissue sampled from the treatment interface relative to untreated areas of the tumour. The relatively lower expression of these checkpoint molecules at the periphery of the treated area may be related to liquefactive necrosis induced by the histotripsy treatment, and would potentially allow TILs to infiltrate the tumour. Relative increases of these checkpoint molecules in tumours of a higher grade and alongside immune cell infiltration are consistent with previous reports that associate their expression with malignancy., (© 2023 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Systemic lupus erythematosus (SLE) is an autoimmune disease caused by environmental factors and loss of key proteins, including the endonuclease Dnase1L3. Dnase1L3 absence causes pediatric-onset lupus in humans, while reduced activity occurs in adult-onset SLE. The amount of Dnase1L3 that prevents lupus remains unknown. To genetically reduce Dnase1L3 levels, we developed a mouse model lacking Dnase1L3 in macrophages (conditional knockout [cKO]). Serum Dnase1L3 levels were reduced 67%, though Dnase1 activity remained constant. Homogeneous and peripheral antinuclear antibodies were detected in the sera by immunofluorescence, consistent with anti-double-stranded DNA (anti-dsDNA) antibodies. Total immunoglobulin M, total immunoglobulin G, and anti-dsDNA antibody levels increased in cKO mice with age. The cKO mice developed anti-Dnase1L3 antibodies. In contrast to global Dnase1L3-/- mice, anti-dsDNA antibodies were not elevated early in life. The cKO mice had minimal kidney pathology. Therefore, we conclude that an intermediate reduction in serum Dnase1L3 causes mild lupus phenotypes, and macrophage-derived DnaselL3 helps limit lupus., Competing Interests: Conflicts of interest. P.A.K. and R.B.S. have patents pending on Dnase1L3 modifications to improve serum half-life., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

SARS-CoV-2 is primarily transmitted through droplets and airborne aerosols, and in order to prevent infection and reduce viral spread vaccines should elicit protective immunity in the airways. The neonatal Fc receptor (FcRn) transfers IgG across epithelial barriers and can enhance mucosal delivery of antigens. Here we explore FcRn-mediated respiratory delivery of SARS-CoV-2 spike (S). A monomeric IgG Fc was fused to a stabilized spike; the resulting S-Fc bound to S-specific antibodies and FcRn. Intranasal immunization of mice with S-Fc and CpG significantly induced antibody responses compared to the vaccination with S alone or PBS. Furthermore, we intranasally immunized mice or hamsters with S-Fc. A significant reduction of virus replication in nasal turbinate, lung, and brain was observed following nasal challenges with SARS-CoV-2 and its variants. Intranasal immunization also significantly reduced viral airborne transmission in hamsters. Nasal IgA, neutralizing antibodies, lung-resident memory T cells, and bone-marrow S-specific plasma cells mediated protection. Hence, FcRn delivers an S-Fc antigen effectively into the airway and induces protection against SARS-CoV-2 infection and transmission., (© 2023. The Author(s).)

Competing Interests: The authors declare no conflict of interest.

Hepatitis E virus (HEV) causes adverse clinical outcomes in pregnant women, but the underlying mechanisms remain poorly understood. To delineate the mechanisms of pregnancy-associated adverse effects during HEV infection, we utilized a genotype 3 HEV from rabbit (HEV-3ra) and its cognate host (rabbits) to systematically investigate the clinical consequences, viral replication dynamics, and host immune and hormonal responses of HEV infection during pregnancy. We found a significant fetal loss of 23% in HEV-infected pregnant rabbits, indicating an early-stage miscarriage. HEV infection in pregnant rabbits was characterized by higher viral loads in feces, intestinal contents, liver, and spleen tissues, as well as a longer and earlier onset of viremia than in infected nonpregnant rabbits. HEV infection altered the pattern of cytokine gene expressions in the liver of pregnant rabbits and caused a transient increase of serum interferon gamma (IFN-γ) shortly after a notable increase in viral replication, which may contribute to early fetal loss. Histological lesions in the spleen were more pronounced in infected pregnant rabbits, although moderate liver lesions were seen in both infected pregnant and nonpregnant rabbits. Total bilirubin was elevated in infected pregnant rabbits. The serum levels of estradiol (E2) in HEV-infected pregnant rabbits were significantly higher than those in mock-infected pregnant rabbits at 14 days postinoculation (dpi) and correlated positively with higher viral loads in feces, liver, and spleen tissues at 28 dpi, suggesting that it may play a role in extrahepatic virus dissemination. The results have important implications for understanding the severe diseases associated with HEV infection during pregnancy. IMPORTANCE HEV causes adverse pregnancy outcomes, with a mortality rate of >30% in pregnant women, but the underlying mechanisms are poorly understood. In this study, we utilized HEV-3ra and its cognate host (pregnant rabbit) to delineate the potential underlying mechanisms of pregnancy-associated adverse outcomes during HEV infection. We found that infected pregnant rabbits had a fetal loss of 23%, which coincided with enhanced viral replication and an elevated systemic IFN-γ response, followed by longer viremia duration and extrahepatic viral dissemination. Estradiol levels were increased in infected pregnant rabbits and correlated positively with higher fecal viral shedding and higher viral loads in liver and spleen tissues. Infected pregnant rabbits had more pronounced splenic lesions, higher serum total bilirubin, and an altered cytokine gene expression profile in the liver. The results will contribute to our understanding of the mechanisms of HEV-associated adverse pregnancy outcomes.

Chronic hepatitis E virus (HEV) infection has become a significant clinical problem that requires treatment in immunocompromised individuals. In the absence of an HEV-specific antiviral, ribavirin (RBV) has been used off-label, but treatment failure may occur due to mutations in the viral RNA-dependent RNA polymerase (RdRp), including Y1320H, K1383N, and G1634R. Chronic hepatitis E is mostly caused by zoonotic genotype 3 HEV (HEV-3), and HEV variants from rabbits (HEV-3ra) are closely related to human HEV-3. Here, we explored whether HEV-3ra, along with its cognate host, can serve as a model to study RBV treatment failure-associated mutations observed in human HEV-3-infected patients. By utilizing the HEV-3ra infectious clone and indicator replicon, we generated multiple single mutants (Y1320H, K1383N, K1634G, and K1634R) and a double mutant (Y1320H/K1383N) and assessed the role of mutations on replication and antiviral activity of HEV-3ra in cell culture. Furthermore, we also compared the replication of the Y1320H mutant with the wild-type HEV-3ra in experimentally infected rabbits. Our in vitro analyses revealed that the effects of these mutations on rabbit HEV-3ra are altogether highly consistent with those on human HEV-3. Importantly, we found that the Y1320H enhances virus replication during the acute stage of HEV-3ra infection in rabbits, which corroborated our in vitro results showing an enhanced viral replication of Y1320H. Taken together, our data suggest that HEV-3ra and its cognate host is a useful and relevant naturally occurring homologous animal model to study the clinical relevance of antiviral-resistant mutations observed in human HEV-3 chronically-infected patients. IMPORTANCE HEV-3 causes chronic hepatitis E that requires antiviral therapy in immunosuppressed individuals. RBV is the main therapeutic option for chronic hepatitis E as an off-label use. Several amino acid changes, including Y1320H, K1383N, and G1634R, in the RdRp of human HEV-3 have reportedly been associated with RBV treatment failure in chronic hepatitis E patients. In this study, we utilized an HEV-3ra from rabbit and its cognate host to investigate the effect of these RBV treatment failure-associated HEV-3 RdRp mutations on viral replication efficiency and antiviral susceptibility. The in vitro data using rabbit HEV-3ra was highly comparable to those from human HEV-3. We demonstrated that the Y1320H mutation significantly enhanced HEV-3ra replication in cell culture and enhanced virus replication during the acute stage of HEV-3ra infection in rabbits. The rabbit HEV-3ra infection model should be useful in delineating the role of human HEV-3 RBV treatment failure-associated mutations in antiviral resistance.

Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Ae. aegypti, an urban mosquito vector of many other arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission after escaping the midgut of live Ae. aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N generates reduced viremia and displays less severe tissue pathology in vivo in a mouse model. We found evidence in mouse fibroblasts that MAYV E2-T179N is less dependent on the Mxra8 receptor for replication than WT MAYV. Similarly, exogenous expression of human apolipoprotein receptor 2 and Mxra8 enhanced WT MAYV replication compared to MAYV E2-T179N. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence-but coming at the cost of optimal replication in humans-and may represent a first step towards a future emergence event., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Cereghino et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that has a strong preference for women of child-bearing age. Maternal factors play an essential role in shaping the immune system of the newborn, yet it is unknown whether maternal factors could modulate the development of SLE in the offspring. Activation-induced cytidine deaminase (AID) is an enzyme required for somatic hypermutation and class switch recombination. Given that IgG and IgA isotypes account for the vast majority of passive immunity in rodents, our previously established AID-deficient BXSB mice provide a model in which maternal antibodies that can be transferred to the offspring are greatly diminished and have restricted repertoire. In this study, we compared genotypically identical mice born to either AID-sufficient dams or AID-deficient dams and evaluated the effects of maternal antibodies in disease progression. Offspring from knockout dams developed disease at a faster rate, as shown by more severe nephritis and elevated pathogenic autoantibodies compared to their counterparts born to wild-type dams. When immune competent pups were cross fostered onto AID-deficient dams, these mice exhibited more severe disease characteristics, including exacerbated lupus nephritis, increased levels of circulating antinuclear antibodies, and more activated T cells. These results suggest that a protective antibody effect contributes to the modulation of SLE progression in postnatal period. Overall, these findings highlight the importance of maternal antibodies in programming the immune system and altering SLE development in offspring., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The hepatitis B virus core antigen (HBcAg) tolerates insertion of foreign epitopes and maintains its ability to self-assemble into virus-like particles (VLPs). We constructed a ∆HBcAg-based VLP vaccine expressing three predicted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B and T cell epitopes and determined its immunogenicity and protective efficacy. The recombinant ∆HBcAg-SARS-CoV-2 protein was expressed in Escherichia coli, purified, and shown to form VLPs. K18-hACE2 transgenic C57BL/6 mice were immunized intramuscularly with ∆HBcAg VLP control (n = 15) or ∆HBcAg-SARS-CoV-2 VLP vaccine (n = 15). One week after the 2nd booster and before virus challenge, five ∆HBcAg-SARS-CoV-2 vaccinated mice were euthanized to evaluate epitope-specific immune responses. There is a statistically significant increase in epitope-specific Immunoglobulin G (IgG) response, and statistically higher interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) expression levels in ∆HBcAg-SARS-CoV-2 VLP-vaccinated mice compared to ∆HBcAg VLP controls. While not statistically significant, the ∆HBcAg-SARS-CoV-2 VLP mice had numerically more memory CD8+ T-cells, and 3/5 mice also had numerically higher levels of interferon gamma (IFN-γ) and tumor necrosis factor (TNF). After challenge with SARS-CoV-2, ∆HBcAg-SARS-CoV-2 immunized mice had numerically lower viral RNA loads in the lung, and slightly higher survival, but the differences are not statistically significant. These results indicate that the ∆HBcAg-SARS-CoV-2 VLP vaccine elicits epitope-specific humoral and cell-mediated immune responses but they were insufficient against SARS-CoV-2 infection., (© 2023 Wiley Periodicals LLC.)

A controlled-neonatal piglet trial was conducted to evaluate the impact of a plant-based infant formula containing buckwheat and almonds as the main source of protein compared to a commercially available dairy-based formula on the gut health parameters. Two day old piglets were fed either a plant-based or a dairy-based formula until day 21. Gut microbiome, cytokines, growth and metabolism related outcomes, and intestinal morphology were evaluated to determine the safety of the plant-based infant formula. This study reported that the plant-based formula-fed piglets had a similar intestinal microbiota composition relative to the dairy-based formula-fed group. However, differential abundance of specific microbiota species was detected within each diet group in the small and large intestinal regions and fecal samples. Lactobacillus delbrueckii, Lactobacillus crispatus, and Fusobacterium sp. had higher abundance in the small intestine of plant-based formula-fed piglets compared to the dairy-based group. Bacteroides nordii, Enterococcus sp., Lactobacillus crispatus, Prevotella sp., Ruminococcus lactaris, Bacteroides nordii, Eisenbergiella sp., Lactobacillus crispatus, Prevotella sp., and Akkermansia muciniphila had greater abundance in the large intestine of the plant based diet fed piglets relative to the dairy-based diet group. In the feces, Clostridiales, Bacteroides uniformis, Butyricimonasvirosa, Cloacibacillus porcorum, Clostridium clostridioforme, and Fusobacterium sp. were abundant in dairy-based group relative to the plant-based group. Lachnospiraceae, Clostridium scindens, Lactobacillus coleohominis, and Prevetolla sp. had greater abundance in the feces of the plant-based group in comparison to the dairy-based group. Gut morphology was similar between the plant and the dairy-based formula-fed piglets. Circulatory cytokines, magnesium, triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH), vitamin D, vitamin K, and IgE levels were similar among all piglets independent of dietary group. Overall, the present study demonstrated that a plant-based formula with buckwheat and almonds as the primary source of protein can support similar gut microbiota growth and health outcomes compared to a dairy-based infant formula.

Melanomas in humans and dogs are highly malignant and resistant to therapy. Since the first development of immunotherapies, interest in how the immune system interacts within the tumor microenvironment and plays a role in tumor development, progression, or remission has increased. Of major importance are tumor-infiltrating lymphocytes (TILs) where distribution and cell frequencies correlate with survival and therapeutic outcomes. Additionally, efforts have been made to identify subsets of TILs populations that can contribute to a tumor-promoting or tumor-inhibiting environment, such as the case with T regulatory cells versus CD8 T cells. Furthermore, cancerous cells have the capacity to express certain inhibitory checkpoint molecules, including CTLA-4, PD-L1, PD-L2, that can suppress the immune system, a property associated with poor prognosis, a high rate of recurrence, and metastasis. Comparative oncology brings insights to comprehend the mechanisms of tumorigenesis and immunotolerance in humans and dogs, contributing to the development of new therapeutic agents that can modulate the immune response against the tumor. Therapies that target signaling pathways such as mTOR and MEK/ERK that are upregulated in cancer, or immunotherapies with different approaches such as CAR-T cells engineered for specific tumor-associated antigens, DNA vaccines using human tyrosinase or CGSP-4 antigen, anti-PD-1 or -PD-L1 monoclonal antibodies that intercept their binding inhibiting the suppression of the T cells, and lymphokine-activated killer cells are already in development for treating canine tumors. This review provides concise and recent information about diagnosis, comparative mechanisms of tumor development and progression, and the current status of immunotherapies directed toward canine melanoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Stevenson, Klahn, LeRoith and Huckle.)

A 6 yr old male castrated American Staffordshire terrier was referred for a nonhealing wound at the site of a previously incompletely excised, high-grade soft tissue sarcoma. Physical examination revealed right popliteal lymphadenopathy and a fungating mass of the right pelvic limb at the level of the hock. Thoracic and abdominal computed tomography revealed mild lymphadenopathy of multiple iliac and inguinal lymph nodes. Right pelvic limb amputation and inguinal lymphadenectomy were performed. Histopathology was consistent of a high-grade soft tissue sarcoma with diffuse spread through the lymphatic vessels of the right pelvic limb up to the right inguinal lymph node but not affecting the lymph node itself. Doxorubicin chemotherapy was elected postoperatively as adjuvant therapy. Approximately 4 mo following initiation of chemotherapy, the patient developed a firm, tubular subcutaneous mass starting near the previous amputation site with tracking toward the thorax. Fine needle aspiration of the new mass was consistent with atypical spindle cell proliferation. Palliative care was elected, and the patient was euthanized 3 mo later because of progressive disease. In-transit metastasis is a rare behavior for soft tissue sarcomas across all species, and this is the first report of such a presentation for canine soft tissue sarcoma., (© 2023 by American Animal Hospital Association.)

Usutu virus (USUV, Flaviviridae ) is an emerging mosquito-borne virus that has been implicated in neuroinvasive disease in humans and epizootic deaths in wild birds. USUV is maintained in an enzootic cycle between ornithophilic mosquitoes, primarily Culex spp., and wild birds, predominantly passerine species. However, limited experimental data exist on the species competent for USUV transmission. Here, we demonstrate that house sparrows are susceptible to multiple USUV strains. Our study also revealed that Culex quinquefasciatus mosquitoes are susceptible to USUV, with a significantly higher infection rate for the Netherlands 2016 USUV strain compared to the Uganda 2012 USUV strain at 50% and 19%, respectively. To assess transmission between avian host and mosquito vector, we allowed mosquitoes to feed on either juvenile chickens or house sparrows inoculated with USUV. Both bird models transmitted USUV to C. quinquefasciatus mosquitoes. Linear regression analyses indicated that C. quinquefasciatus infection rates were positively correlated with avian viremia levels, with 3 to 4 log 10 PFU/mL representing the minimum avian viremia threshold for transmission to mosquitoes. Based on the viremia required for transmission, house sparrows were estimated to more readily transmit the Netherlands 2016 strain compared to the Uganda 2012 strain. These studies provide insights on a competent reservoir host of USUV. IMPORTANCE Usutu virus (USUV) is a zoonotic mosquito-borne virus that can cause neuroinvasive disease, including meningitis and encephalitis, in humans and has resulted in hundreds of thousands of deaths in wild birds. The perpetuation of USUV in nature is dependent on transmission between Culex spp. mosquitoes and various avian species. To date, few experimental data exist for determining which bird species are important for the maintenance of USUV. Our studies showed that house sparrows can transmit infectious Usutu virus, indicating their role as a competent host species. By identifying reservoir species of USUV, we can predict areas of USUV emergence and mitigate its impacts on global human and wildlife health.

Human milk harbors complex carbohydrates, including human milk oligosaccharides (HMOs), the third most abundant component after lactose and lipids. HMOs have been shown to impact intestinal microbiota, modulate the intestinal immune response, and prevent pathogenic bacterial binding by serving as decoy receptors. However, the direct effect of HMOs on intestinal function and immunity remains to be elucidated. To address this knowledge gap, 21-day-old germ-free mice (C57BI/6) were orally gavaged with 15 mg/day of pooled HMOs for 7 or 14 days and euthanized at day 28 or 35. A set of mice was maintained until day 50 to determine the persistent effects of HMOs. Control groups were maintained in the isolators for 28, 35, or 50 days of age. At the respective endpoints, intestinal tissues were subjected to histomorphometric and transcriptomic analyses, while the spleen and mesenteric lymph nodes (MLNs) were subjected to flow cytometric analysis. The small intestine (SI) crypt was reduced after HMO treatment relative to control at days 28 and 35, while the SI villus height and large intestine (LI) gland depth were decreased in the HMO-treated mice relative to the control at day 35. We report significant HMO-induced and location-specific gene expression changes in host intestinal tissues. HMO treatment significantly upregulated genes involved in extracellular matrix, protein ubiquitination, nuclear transport, and mononuclear cell differentiation. CD4+ T cells were increased in both MLNs and the spleen, while CD8+ T cells were increased in the spleen at day 50 in the HMO group in comparison to controls. In MLNs, plasma cells were increased in HMO group at days 28 and 35, while in the spleen, only at day 28 relative to controls. Macrophages/monocytes and neutrophils were lower in the spleen of the HMO group at days 28, 35, and 50, while in MLNs, only neutrophils were lower at day 50 in the 14-day HMO group. In addition, diphtheria toxoid and tetanus toxoid antibody-secreting cells were higher in HMO-supplemented group compared to controls. Our data suggest that HMOs have a direct effect on gastrointestinal tract metabolism and the immune system even in the absence of host microbiota., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rosa, Sharma, Gurung, Casero, Matazel, Bode, Simecka, Elolimy, Tripp, Randolph, Hand, Williams, LeRoith and Yeruva.)

Hepatitis E virus (HEV) is an important but understudied zoonotic virus causing both acute and chronic viral hepatitis. A proportion of HEV-infected individuals also developed neurological diseases such as Guillain-Barré syndrome, neuralgic amyotrophy, encephalitis, and myelitis, although the mechanism remains unknown. In this study, by using an in vitro blood-brain barrier (BBB) model, we first investigated whether HEV can cross the BBB and whether the quasi-enveloped HEV virions are more permissible to the BBB than the nonenveloped virions. We found that both quasi-enveloped and nonenveloped HEVs can similarly cross the BBB and that addition of proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has no significant effect on the ability of HEV to cross the BBB in vitro. To explore the possible mechanism of HEV entry across the BBB, we tested the susceptibility of human brain microvascular endothelial cells lining the BBB to HEV infection and showed that brain microvascular endothelial cells support productive HEV infection. To further confirm the in vitro observation, we conducted an experimental HEV infection study in pigs and showed that both quasi-enveloped and nonenveloped HEVs invade the central nervous system (CNS) in pigs, as HEV RNA was detected in the brain and spinal cord of infected pigs. The HEV-infected pigs with detectable viral RNA in CNS tissues had histological lesions in brain and spinal cord and significantly higher levels of proinflammatory cytokines TNF-α and interleukin 18 than the HEV-infected pigs without detectable viral RNA in CNS tissues. The findings suggest a potential mechanism of HEV-associated neuroinvasion.

Equine protozoal myeloencephalitis (EPM) is a debilitating neurologic disease affecting horses across the Americas. Gaps in understanding the inflammatory immune response in EPM-affected horses create difficulties with diagnosis and treatment, subsequently negatively impacting the prognosis of affected horses. The purpose of the current study was to evaluate circulating levels of the inflammatory immune marker soluble CD14 (sCD14), in horses with EPM (n = 7) and determine if they differed from healthy neurologically normal horses (n = 6). Paired sera and cerebrospinal fluid (CSF) samples were analyzed for sCD14. Inclusion criteria for EPM horses consisted of the presence of neurologic signs consistent with EPM, Sarcocystis neurona surface antigens 2, 4/3 (SnSAG 2, 4/3) ELISA serum: CSF antibody ratio ≤ 100, and a postmortem diagnosis of EPM. Control horses were neurologically normal, healthy horses with SnSAG 2, 4/3 ELISA serum: CSF antibody ratios of > 100. Serum anti-Sarcocystis neurona antibodies indicate that healthy control horses were exposed to S. neurona but resistant to developing clinical EPM. EPM cases had significantly greater concentrations of sCD14 in CSF samples compared to control horses and increased serum sCD14 concentrations. A positive correlation between sCD14 serum and CSF concentrations was observed in EPM-affected horses but not healthy horses. Soluble CD14 is an inflammatory marker, and the study results suggest it is elevated in EPM patients. When performed in conjunction with clinical evaluation and standard antibody testing, there may be potential for sCD14 to be utilized as a correlate for EPM., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Usutu virus (USUV; family: Flaviviridae, genus: Flavivirus ), is an emerging zoonotic arbovirus that causes severe neuroinvasive disease in humans and has been implicated in the loss of breeding bird populations in Europe. USUV is maintained in an enzootic cycle between ornithophilic mosquitos and wild birds. As a member of the Japanese encephalitis serocomplex, USUV is closely related to West Nile virus (WNV) and St. Louis encephalitis virus (SLEV), both neuroinvasive arboviruses endemic in wild bird populations in the United States. An avian model for USUV is essential to understanding zoonotic transmission. Here we describe the first avian models of USUV infection with the development of viremia. Juvenile commercial ISA Brown chickens were susceptible to infection by multiple USUV strains with evidence of cardiac lesions. Juvenile chickens from two chicken lines selected for high (HAS) or low (LAS) antibody production against sheep red blood cells showed markedly different responses to USUV infection. Morbidity and mortality were observed in the LAS chickens, but not HAS chickens. LAS chickens had significantly higher viral titers in blood and other tissues, as well as oral secretions, and significantly lower development of neutralizing antibody responses compared to HAS chickens. Mathematical modelling of virus-host interactions showed that the viral clearance rate is a stronger mitigating factor for USUV viremia than neutralizing antibody response in this avian model. These chicken models provide a tool for further understanding USUV pathogenesis in birds and evaluating transmission dynamics between avian hosts and mosquito vectors.

Deterioration in glucose homeostasis has been associated with intestinal dysbiosis, but it is not known how metabolic dysregulation alters the gastrointestinal environment. We investigated how the progression of diabetes alters ileal and colonic epithelial mucosal structure, microbial abundance, and transcript expression in the University of California Davis Type 2 Diabetes Mellitus (UCD-T2DM) rat model. Male UCD-T2DM rats (age ~170 days) were included if <1-month (n = 6, D1M) or 3-month (n = 6, D3M) post-onset of diabetes. Younger nondiabetic UCD-T2DM rats were included as a nondiabetic comparison (n = 6, ND, age ~70 days). Ileum villi height/crypt depths and colon crypt depths were assessed by histology. Microbial abundance of colon content was measured with 16S rRNA sequencing. Ileum and colon transcriptional abundances were analyzed using RNA sequencing. Ileum villi height and crypt depth were greater in D3M rats compared to ND. Colon crypt depth was greatest in D3M rats compared to both ND and D1M rats. Colon abundances of Akkermansia and Muribaculaceae were lower in D3M rats relative to D1M, while Oscillospirales, Phascolarctobacterium, and an unidentified genus of Lachnospiraceae were higher. Only two transcripts were altered by diabetes advancement within the colon; however, 2039 ileal transcripts were altered. Only colonic abundances of Sptlc3, Enpp7, Slc7a15, and Kctd14 had more than twofold changes between D1M and D3M rats. The advancement of diabetes in the UCD-T2DM rat results in a trophic effect on the mucosal epithelia and was associated with regulation of gastrointestinal tract RNA expression, which appears more pronounced in the ileum relative to the colon., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

The COVID-19 pandemic has paralyzed the global economy and resulted in millions of deaths globally. People with co-morbidities like obesity, diabetes and hypertension are at an increased risk for severe COVID-19 illness. This is of overwhelming concern because 42% of Americans are obese, 30% are pre-diabetic and 9.4% have clinical diabetes. Here, we investigated the effect of obesity on disease severity following SARS-CoV-2 infection using a well-established mouse model of diet-induced obesity. Diet-induced obese and lean control C57BL/6 N mice, transduced for ACE2 expression using replication-defective adenovirus, were infected with SARS-CoV-2, and monitored for lung pathology, viral titers, and cytokine expression. No significant differences in tissue pathology or viral replication was observed between AdV transduced lean and obese groups, infected with SARS-CoV-2, but certain cytokines were expressed more significantly in infected obese mice compared to the lean ones. Notably, significant weight loss was observed in obese mice treated with the adenovirus vector, independent of SARS-CoV-2 infection, suggesting an obesity-dependent morbidity induced by the vector. These data indicate that the adenovirus-transduced mouse model of SARS-CoV-2 infection, as described here and elsewhere, may be inappropriate for nutrition studies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Sarcocystis neurona is the predominant etiological agent of the infectious equine neurologic disease, equine protozoal myeloencephalitis (EPM), which is prevalent in the United States. A wealth of knowledge about S. neurona biology and its life cycle has accumulated over the last several decades. However, much remains unknown about the aberrant equine host's immune response to S. neurona and the relatively high prevalence of exposure to the protozoa but relatively infrequent occurrence of clinical neurologic disease. Mouse models simulating EPM are commonly used to study the disease due to numerous challenges associated with studying the disease in horses. The critical role of the cytokine, interferon gamma (IFNγ), in protection against S. neurona encephalitis has been well established as Ifnγ -/- mice are highly susceptible to S. neurona encephalitis. However, there are discrepancies in the literature regarding S. neurona disease susceptibility in lymphocyte deficient mice, lacking T-lymphocytes and their associated Ifnγ production. In the current study, we investigated S. neurona encephalitis susceptibility in 2 genetically different strains of lymphocyte null mice, C57Bl/6 (B6).scid and Balb/c.scid. The B6.scid mouse was determined to be susceptible to S. neurona encephalitis as 100 % of infected mice developed neurologic disease within 60 days post infection (DPI). The Balb/c.scid mouse was nearly disease resistant as only 10 % of mice developed neurologic disease 60 DPI. Encephalitis was histologically demonstrable and S. neurona was identified in cerebellar samples collected from B6.scid but absent in Balb/c.scid mice. To further investigate the importance of T-lymphocyte derived Ifnγ, T- lymphocytes were adoptively transferred into B6.scid mice. The adoptive transfer of Ifnγ competent T- lymphocytes offered complete protection against S. neurona encephalitis but transfer of Ifnγ deficient T- lymphocytes did not with 100 % of these recipient mice succumbing to S. neruona encephalitis. Histological analysis of collected cerebellar samples confirmed the presences of S. neurona and encephalitis in recipient mice that developed neurologic disease. These studies show that the background strain is critical in studying SCID susceptibility to S. neurona disease and suggest a protective role of Ifnγ producing T- lymphocytes in S. neurona encephalitis susceptible mice., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Melanoma in humans and dogs is considered highly immunogenic; however, the function of tumor-infiltrating lymphocytes (TILs) is often suppressed in the tumor microenvironment. In humans, current immunotherapies target checkpoint molecules (such as PD-L1, expressed by tumor cells), inhibiting their suppressive effect over TILs. The role of PD-L2, an alternative PD-1 ligand also overexpressed in malignant tumors and in patients with anti-PD-L1 resistance, remains poorly understood. In the current study, we evaluated the expression of checkpoint molecule mRNAs in canine melanoma and TILs. Analysis of checkpoint molecule gene expression was performed by RT-qPCR (real-time quantitative polymerase chain reaction) using total RNA isolated from formalin-fixed and paraffin-embedded melanomas ( n = 22) and melanocytomas ( n = 9) from the Virginia Tech Animal Laboratory Services archives. Analysis of checkpoint molecule expression revealed significantly higher levels of PDCD1 ( PD-1 ) and CD274 ( PD-L1 ) mRNAs and an upward trend in PDCD1LG2 ( PD-L2 ) mRNA in melanomas relative to melanocytomas. Immunohistochemistry revealed markedly increased numbers of CD3 + T cells in the highest PD-1 -expressing subgroup of melanomas compared to the lowest PD-1 expressors, whereas densities of IBA1 + cells (macrophages) were similar in both groups. CD79a + cell numbers were low for both groups. As in human melanoma, overexpression of the PD-1/PD-L1/PD-L2 axis is a common feature of canine melanoma. High expression of PD-1 and PD-L1 correlates with increased numbers of CD3 + cells. Additionally, the high level of IBA1 + cells in melanomas with low PD-1 expression and low CD3 + cells levels suggest that the expression of checkpoint molecules is modulated by interactions between T cells and cancer cells rather than histiocytes.

As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible gram-negative autotransporter to express vaccine antigens on the surface of genome-reduced bacteria to enhance interaction of vaccine antigen with the immune system. As a proof-of-principle, we utilized genome-reduced Escherichia coli to express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as killed whole-cell vaccines. The FP sequence is highly conserved across coronaviruses; the six FP core amino acid residues, along with the four adjacent residues upstream and the three residues downstream from the core, are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated interferon-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine-appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens., Competing Interests: Competing interest statement: The University of Virginia and Virginia Polytechnic Institute and State University have filed a provisional patent application covering some of the work described., (Copyright © 2021 the Author(s). Published by PNAS.)

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease. Here we report the development of subunit PRRSV-2 vaccines by expressing swine leucocyte antigen (SLA) class I and class II allele-specific epitope antigens in a robust adenovirus vector. SLA I-specific CD8 and SLA II-specific CD4 T cell epitopes of PRRSV-2 NADC20 were predicted in silico . Stable murine leukaemia cell lines (RMA-S), which are TAP-deficient and lacking endogenous class I epitope loading, were established to express different SLA I alleles. The binding stability of PRRSV T cell epitope peptides with SLA I alleles expressed on RMA-S cells was characterized. Two PRRSV poly-T cell epitope peptides were designed. NADC20-PP1 included 39 class I epitopes, consisting of 8 top-ranked epitopes specific to each of 5 SLA I alleles, and fused to 5 class II epitopes specific to SLA II alleles. NADC20-PP2, a subset of PP1, included two top-ranked class I epitopes specific to each of the five SLA I alleles. Two vaccine candidates, Ad-NADC20-PP1 and Ad-NADC20-PP2, were constructed by expressing the polytope peptides in a replication-incompetent human adenovirus 5 vector. A vaccination and challenge study in 30 piglets showed that animals vaccinated with the vaccines had numerically lower gross and histopathology lung lesions, and numerically lower PRRSV RNA loads in lung and serum after challenge compared to the controls, although there was no statistical significance. The results suggested that the Ad-NADC20-PP1 and Ad-NADC20-PP2 vaccines provided little or no protection, further highlighting the tremendous challenges faced in developing an effective subunit PRRSV-2 vaccine.

An 8-y-old Labrador Retriever was presented to a small animal practice in northern Virginia with a history of recent lethargy. Physical examination findings were unremarkable. Ultrasound revealed several large hepatic masses and multiple smaller masses involving the pancreas. Cytologic findings on fine-needle aspirates of the hepatic masses included inflammation and necrosis with eosinophilic, membranous oval structures consistent with cestode infection. Histopathologic findings for biopsies of these masses included extensive necrosis, inflammation, and PAS-positive hyaline-like membranous material interpreted as metacestode cyst wall. A PCR product was generated from aspirate material using primers specific for Echinococcus multilocularis . Subsequent sequence data were 100% homologous to E. multilocularis NADH dehydrogenase subunit I gene sequences. The dog received daily oral albendazole (10 mg/kg) treatment, but its condition deteriorated, and the dog was euthanized. The dog, born in Mississippi, was brought as a puppy to Virginia with no other travel history. To our knowledge, alveolar echinococcosis has not been reported previously in a dog in the United States; E. multilocularis infection was apparently acquired in the mid-Atlantic region of the United States.

Background: Dengue virus (DENV) is estimated to infect 390 million people annually. However, few host factors that alter disease severity are known. Malnutrition, defined as both over- and undernutrition, is a growing problem worldwide and has long been linked to dengue disease severity by epidemiological and anecdotal observations. Accordingly, we sought to establish a mouse model to assess the impact of nutritional status on DENV disease severity., Results: Using transiently immunocompromised mice, we established a model of mild dengue disease with measurable viremia. We then applied it to study the effects of healthy weight, obese, and low-protein diets representing normal, over-, and undernutrition, respectively. Upon infection with DENV serotype 2, obese mice experienced more severe morbidity in the form of weight loss and thrombocytopenia compared to healthy weight groups. Additionally, obesity altered cytokine expression following DENV infection. Although low protein-fed mice did not lose significant weight after DENV2 infection, they also experienced a reduction in platelets as well as increased spleen pathology and viral titers., Conclusions: Our results indicate that obese or undernourished mice incur greater disease severity after DENV infection. These studies establish a role for nutritional status in DENV disease severity.

Porcine epidemic diarrhea virus (PEDV) has had a negative economic impact on the global swine industry for decades since its first emergence in the 1970s in Europe. In 2013, PEDV emerged for the first time in the United States, causing immense economic losses to the swine industry. Efforts to protect U.S. swine herds from PEDV infection and limit PEDV transmission through vaccination had only limited success so far. Following the previous success in our virus-like particle (VLP) based vaccine in mouse model, in this study we determined the immunogenicity and protective efficacy of a VLP-based vaccine containing B-cell epitope 748 YSNIGVCK 755 from the spike protein of PEDV incorporated into the hepatitis B virus core capsid (HBcAg), in a comprehensive pregnant gilt vaccination and piglet challenge model. The results showed that the vaccine was able to induce significantly higher virus neutralization response in gilt milk, and provide alleviation of clinical signs for piglets experimentally infected with PEDV. Piglets from pregnant gilt that was vaccinated with the VLP vaccine had faster recovery from the clinical disease, less small intestinal lesions, and higher survival rate at 10 days post-challenge (DPC)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)