Case summary A 3-year 10-month-old spayed female domestic shorthair cat was presented for subacute progressive hyporexia, vomiting and lethargy. On presentation, the cat was dyspnoeic, and venous blood gas analysis revealed metabolic acidosis, hypercalcaemia (both total and ionised), hyperlactaemia and hyperglycaemia. Physical examination identified a 2 × 3 cm crusted cutaneous lesion on the cranium, reduced mentation, mild tachycardia, harsh bronchovesicular sounds and approximately 5% dehydration. Owing to welfare concerns, the owners elected euthanasia. Post-mortem examination revealed moderate autolytic changes. The organs most affected by vascular lesions included the heart, brain, kidneys, liver and pancreas. Histopathology revealed mild to severe multifocal intraluminal and mural proliferations of atypical endothelial cells, accompanied by multifocal thrombosis and mild perivascular oedema. Immunohistochemistry showed that the proliferating cells were negative for alpha-smooth muscle actin, and quantitative PCR for Bartonella species was also negative. However, 50% of the proliferating cells were positive for factor VIII. These findings supported a diagnosis of feline systemic reactive angioendotheliomatosis. Relevance and novel information In the authors’ opinion, this case contributes to the growing body of literature on this rare condition and raises the possibility of an association with hypercalcaemia.

Mesotheliomas are relatively rare and affect many animal species but have not been reported in donkeys (Equus asinus). Here we report a case of bicavitary epithelioid mesothelioma in a 33-year-old female donkey presented with weight loss, lethargy and anorexia. Physical examination revealed tachycardia, ileus and delayed capillary refill time. The animal was humanely euthanized and submitted for necropsy. Macroscopic examination revealed cachexia, 24.5 L of serosanguineous peritoneal effusion, 500 ml of serosanguineous pleural effusion and multifocal to coalescing exophytic, white, occasionally haemorrhagic, pinpoint to 5 × 5 × 2 cm nodules on the pleural and peritoneal surfaces and occasionally infiltrating into viscera. Histopathology of the omentum revealed an infiltrative, unencapsulated, poorly demarcated and moderately cellular neoplasm composed of cuboidal to polygonal cells arranged in acini, tubules and nests and, rarely, micropapillary projections, on a moderate fibrovascular stroma. Neoplastic cells had variably distinct cell borders, abundant eosinophilic granular to indistinctly vacuolated cytoplasm, a pleomorphic, central nucleus with finely stippled to marginated chromatin and up to three occasionally very large and distinct magenta nucleoli. There was marked anisocytosis and anisokaryosis and frequent mitoses. Neoplastic cells had diffuse and strong cytoplasmic vimentin immunolabelling and most also had moderate cytoplasmic immunolabelling of cytokeratin. Macroscopic, histopathological and immunohistochemical examinations were consistent with a bicavitary epithelioid mesothelioma, a rare neoplasm in domestic animals that has not been reported in the donkey. Dual vimentin and cytokeratin immunopositivity has been reported in other neoplasms in domestic animals, including ovarian, pulmonary and hepatocellular carcinomas, but no evidence of a primary carcinoma or of any other primary neoplasm was identified in this animal.

Pathology is a discipline that relies on the description and interpretation of changes occurring in organs and tissues, and it is largely a “hands-on” experience, both during training and professional practice. Instigated by the need to provide a solution for online learning and teaching, a plethora of different approaches have been tested during the Covid-19 pandemic. The enforced inability to meet in person created the necessity to quickly replace the hands-on experience of practical classes, routinely considered the “gold standard” in undergraduate pathology teaching, with alternative and innovative digital solutions that could allow the students to appreciate most, if not all, features of the specimen to describe and interpret. Here we present a successful deployment of photogrammetry for the purpose of teaching gross veterinary pathology to undergraduate students. Fresh specimens obtained during routine diagnostic post-mortem activity have been photographed using Digital Single-Lens Reflex cameras and rendered into high quality 3D models, preserving almost unaltered morphology, color, and texture, when compared to the original specimen. Once processed using photogrammetry software, exported and uploaded into an online repository, 3D models become readily available via our digital learning platform (CANVAS) to all undergraduate students for self-study and consolidation, as well as to teaching staff for use during online lectures, traditional face-to-face classes, small group teaching and seminars. Preliminary data collected from students’ feedback highlighted the positive reception from users, and the enriched learning experience, while prolonging indefinitely the availability of rare and perishable teaching material.

Snakebite envenoming is a neglected tropical disease that causes >100,000 deaths and >400,000 cases of morbidity annually. Despite the use of mouse models, severe local envenoming, defined by morbidity-causing local tissue necrosis, remains poorly understood, and human-tissue responses are ill-defined. Here, for the first time, an ex vivo, non-perfused human skin model was used to investigate temporal histopathological and immunological changes following subcutaneous injections of venoms from medically important African vipers (Echis ocellatus and Bitis arietans) and cobras (Naja nigricollis and N. haje). Histological analysis of venom-injected ex vivo human skin biopsies revealed morphological changes in the epidermis (ballooning degeneration, erosion, and ulceration) comparable to clinical signs of local envenoming. Immunostaining of these biopsies confirmed cell apoptosis consistent with the onset of necrosis. RNA sequencing, multiplex bead arrays, and ELISAs demonstrated that venom-injected human skin biopsies exhibited higher rates of transcription and expression of chemokines (CXCL5, MIP1-ALPHA, RANTES, MCP-1, and MIG), cytokines (IL-1β, IL-1RA, G-CSF/CSF-3, and GM-CSF), and growth factors (VEGF-A, FGF, and HGF) in comparison to non-injected biopsies. To investigate the efficacy of antivenom, SAIMR Echis monovalent or SAIMR polyvalent antivenom was injected one hour following E. ocellatus or N. nigricollis venom treatment, respectively, and although antivenom did not prevent venom-induced dermal tissue damage, it did reduce all pro-inflammatory chemokines, cytokines, and growth factors to normal levels after 48 h. This ex vivo skin model could be useful for studies evaluating the progression of local envenoming and the efficacy of snakebite treatments.

Astrocytomas are relatively common primary brain tumours of humans and companion animals. In dogs, they represent approximately 17-28% of primary central nervous system tumours. However, extracranial metastasis is extremely rare. This case report describes a grade IV astrocytoma (glioblastoma) in the cerebrum of a young Cane Corso dog with pulmonary metastases. The diagnosis was obtained via histopathological morphology and immunophenotyping, which showed strong positivity for glial fibrillary acidic protein, vimentin and connexin-43. The glioblastoma in this Cane Corso had epithelioid morphology with histological features of malignancy including high mitotic count, microvascular proliferation, serpentine necrosis and subventricular zone involvement. Epithelioid glioblastoma is a rare subtype that has only relatively recently been formally acknowledged in human medicine and it can also pose a diagnostic challenge in veterinary medicine.

A 10-year-old crossbreed (labradoodle) was presented with an acute history of vomiting and diarrhoea, with supraventricular tachycardia and ventricular premature complexes. Physical examination revealed mild tachycardia, but no significant abnormalities otherwise. Investigations with echocardiogram and computed tomography identified a right atrial mass, nodular interstitial lung pattern, multiple nodules throughout the hepatic parenchyma and peritoneal effusion. Abdominocentesis confirmed a haemoabdomen. Treatment was declined and the dog was euthanased the same day. Histopathology of the cardiac mass confirmed a cardiac myxoma, which stained Alcian blue-positive, demonstrating the mucin content of the tumour. The hepatic lesions were factor VIII-positive, consistent with a visceral haemangiosarcoma.

A 6-year old, male, neutered German shepherd dog, with a bodyweight of 58 kg, was presented for investigation of relapsing dysuria following 2 years of adequate medical control of suspected detrusor urethral dyssynergia. During retrograde contrast urethrocystography, contrast media was visible in the prostatic vessels and caudal vena cava, suggesting rapid vascular uptake. Postmortem histopathological evaluation of the prostate was consistent with pyogranulomatous prostatitis.

The induction of antiviral effector proteins as part of a homeostatically controlled innate immune response to infection plays a critical role in limiting the propagation and transmission of respiratory pathogens. However, the prolonged induction of this immune response can lead to lung hyperinflammation, tissue damage, and respiratory failure. We hypothesized that tissues exposed to the constant threat of infection may constitutively express higher levels of antiviral effector proteins to reduce the need to activate potentially harmful innate immune defences. By analysing transcriptomic data derived from a range of human tissues, we identify lung tissue to express constitutively higher levels of antiviral effector genes relative to that of other mucosal and non-mucosal tissues. By using primary cell lines and the airways of rhesus macaques, we show the interferon-stimulated antiviral effector protein TRIM22 (TRIpartite Motif 22) to be constitutively expressed in the lung independently of viral infection or innate immune stimulation. These findings contrast with previous reports that have shown TRIM22 expression in laboratory-adapted cell lines to require interferon stimulation. We demonstrate that constitutive levels of TRIM22 are sufficient to inhibit the onset of human and avian influenza A virus (IAV) infection by restricting the onset of viral transcription independently of interferon-mediated innate immune defences. Thus, we identify TRIM22 to confer a pre-existing (intrinsic) intracellular defence against IAV infection in cells derived from the respiratory tract. Our data highlight the importance of tissue-specific and cell-type dependent patterns of pre-existing immune gene expression in the intracellular restriction of IAV from the outset of infection.

The intestinal microbiota plays an essential role in the metabolism and immune competence of chickens from the first day after hatching. In modern production systems, chicks are isolated from adult chickens, instead hatching in a clean environment. As a result, chicks are colonized by environmental bacteria, including potential pathogens. There is a need to investigate methods by which chicks can be exposed to a more appropriate microbial community at hatching. Such methods must be easy to apply in a hatchery and produce consistent results. The development of the intestinal microbiota of chicks hatched from eggs sprayed with dilute adult cecal content during incubation was observed at 0, 3, 7, and 14 days post-hatching (dph) across two experiments. High-throughput Illumina sequencing was performed for the V4 hypervariable region of the 16S rRNA gene. A topical treatment of dilute adult cecal content was sufficient to transplant spore-forming bacteria such as Lachnospiraceae and Ruminococcaceae. However, this treatment was not able to transplant other taxa that are considered to be core elements of the chicken cecal microbiota, such as Bacteroidaceae, Lactobacillaceae, Bifidobacteriaceae, and Burkholderiaceae. The topical treatment significantly altered the microbiota of chicks immediately posthatching and accelerated the normal development of the microbiota with earlier colonization by Ruminococcaceae in the cecum and "Candidatus Arthromitus" in the ileum. The effect of the treatment on the cecal microbiota was maximal at 3 dph but diminished over time. IMPORTANCE Over the last 60 years poultry production has intensified in response to increased demand for meat. In modern systems, chicks hatch without contacting chickens and their gut bacteria. Consequently, they are colonized by environmental bacteria that may cause disease. The normal bacteria that live in the gut, or intestinal microbiota, play an important role in the development of the immune system. Therefore, it is essential to find easy ways to expose chicks to the more appropriate bacteria at hatching. This experiment investigated whether spraying eggs with adult cecal contents was sufficient to transfer an adult microbiota to chicks. Our findings show that spore-forming bacteria were transplanted, but other members of the microbiota were not. In this respect, the spray application was partially successful, but the timing of the spray needs to be modified to ensure that more bacteria are transferred.

We hypothesized that increased expression of antiviral host factors at portals of viral entry may protect exposed tissues from the constant threat of invading pathogens. Comparative transcriptomic analysis identified the broad-acting restriction factor TRIM22 (TRIpartite Motif 22) to be among the most abundantly expressed antiviral host factors in the lung, a major portal of entry for many respiratory pathogens. This was surprising, as TRIM22 is currently considered to be an interferon stimulated gene (ISG) product that confers protection following the activation of pathogen-induced cytokine-mediated innate immune defences. Using human respiratory cell lines and the airways of rhesus macaques, we experimentally confirmed high levels of constitutive TRIM22 expression in the lung. In contrast, TRIM22 expression in many widely used transformed cell lines could only be observed following immune stimulation. Endogenous levels of TRIM22 in non-transformed cells were sufficient to restrict human and avian influenza A virus (IAV) infection by inhibiting the onset of viral transcription independently of cytokine-mediated innate immune defences. Thus, TRIM22 confers a pre-existing (intrinsic) tissue-specific immune barrier to IAV infection in the respiratory tract. We investigated whether the constitutive expression of TRIM22 was a characteristic shared by other ISGs in human lung tissue. Transcriptomic analysis identified a large group of ISGs and IAV immuno-regulatory host factors that were similarly enriched in the lung relative to other mucosal tissues, but whose expression was downregulated in transformed cell-lines. We identify common networks of immune gene downregulation which correlated with enhanced permissivity of transformed cells to initiate IAV replication. Our data highlight the importance of tissue-specific and cell-type dependent patterns of pre-existing immune gene expression in the intrinsic intracellular restriction of IAV; findings highly relevant to the immune regulation of many clinically important respiratory pathogens. Author Summary The respiratory tract is a major portal of virus entry for many clinically important viruses, including seasonal and pandemic influenza A virus (IAV). We reasoned that cells within the respiratory tract might differentially express antiviral host factors to protect against the constant challenge of viral infection. We found the broad-acting antiviral protein TRIM22, conventionally regarded as an interferon stimulated gene (ISG) product upregulated in response to virus infection, to be constitutively expressed to high levels in the lung. We found that constitutive expression of TRIM22 restricted the initiation of human and avian IAV infection independently of cytokine-mediated innate immune defences. We identified pre-existing tissue-specific and cell-type dependent patterns of constitutive immune gene expression that strongly correlated with enhanced resistance to IAV replication from the outset of infection. Importantly, we show that these constitutive patterns of immune gene expression are lost or downregulated in many transformed cell lines widely used for respiratory virus research. Our data highlight the importance of pre-existing tissue-specific and cell-type dependent patterns of constitutive antiviral gene expression in the intracellular restriction of respiratory viral pathogens not captured in conventional cell culture model systems of infection.

Cases of arthropod-infested, abandoned or abused animals are sometimes brought to the attention of veterinarians by animal welfare authorities, with the requirement for a full postmortem examination towards criminal or civil proceedings. In these situations, entomology is an important support tool for the pathologists' investigation since the presence of arthropod life cycle stages serve as reliable forensic markers, especially for blowflies which form the first waves of activity following death. In the present study, 70 cadavers from a total of 544 referred to the Institute of Veterinary Science, University of Liverpool, between 2009 and 2014 displayed evidence of infestation. Here, the authors introduce principles of applied entomology and simplified approaches for estimating the minimum time since death, relevant in the context of routine submissions and the broad remit of individual cases. Despite often limited availability of scene of the crime and local thermal data, the interpretation of the minimum postmortem interval has nonetheless proved valuable as an adjunct to the expert pathology report. However, future developments and enhanced accuracy in this area of animal welfare require resource and training in expertise, and agreed standardisation of both laboratory and field procedures.

Feline herpesvirus type 1 (FeHV-1) is one of the etiological agents of feline respiratory disease. FeHV-1 is an epitheliotropic and cytopathic virus that mainly causes rhinitis and conjunctivitis, although pneumonia is also occasionally seen. In this study, the authors investigated the pathogenesis of FeHV-1-associated pneumonia, comparing natural cases with viral infection of tracheal ring and cell cultures in vitro, using histology, immunohistology, double immunofluorescence, and transmission electron microscopy as investigative tools. The results confirm that FeHV-1 targets both respiratory epithelial cells and pneumocytes and indicate that FeHV-1 pneumonia is the consequence of continuous cell-to-cell viral spread from the upper airways via the trachea into the lungs. They provide strong evidence that FeHV-1-infected cells die primarily via apoptosis, following loss of cell-to-cell contact, rounding, and detachment. However, virus-induced lesions in vivo are dominated by marked neutrophil infiltration and extensive necrosis with less prominent apoptosis; in the airways, the tissue necrosis can extend into the submucosa. The necrosis appears to result from virus-induced neutrophil influx and release of proteolytic enzymes, such as matrix metalloproteinase-9, from the neutrophils.

Feline herpesvirus type 1 (FeHV-1) is one of the etiological agents of feline respiratory disease. FeHV-1 is an epitheliotropic and cytopathic virus that mainly causes rhinitis and conjunctivitis, although pneumonia is also occasionally seen. In this study, the authors investigated the pathogenesis of FeHV-1-associated pneumonia, comparing natural cases with viral infection of tracheal ring and cell cultures in vitro, using histology, immunohistology, double immunofluorescence, and transmission electron microscopy as investigative tools. The results confirm that FeHV-1 targets both respiratory epithelial cells and pneumocytes and indicate that FeHV-1 pneumonia is the consequence of continuous cell-to-cell viral spread from the upper airways via the trachea into the lungs. They provide strong evidence that FeHV-1–infected cells die primarily via apoptosis, following loss of cell-to-cell contact, rounding, and detachment. However, virus-induced lesions in vivo are dominated by marked neutrophil infiltration and extensive necrosis with less prominent apoptosis; in the airways, the tissue necrosis can extend into the submucosa. The necrosis appears to result from virus-induced neutrophil influx and release of proteolytic enzymes, such as matrix metalloproteinase-9, from the neutrophils.

Non-human primates are the animals closest to humans for use in influenza A virus challenge studies, in terms of their phylogenetic relatedness, physiology and immune systems. Previous studies have shown that cynomolgus macaques (Macaca fascicularis) are permissive for infection with H1N1pdm influenza virus. These studies have typically used combined challenge routes, with the majority being intra-tracheal delivery, and high doses of virus (> 107 infectious units). This paper describes the outcome of novel challenge routes (inhaled aerosol, intra-nasal instillation) and low to moderate doses (103 to 106 plaque forming units) of H1N1pdm virus in cynomolgus macaques. Evidence of virus replication and sero-conversion were detected in all four challenge groups, although the disease was sub-clinical. Intra-nasal challenge led to an infection confined to the nasal cavity. A low dose (103 plaque forming units) did not lead to detectable infectious virus shedding, but a 1000-fold higher dose led to virus shedding in all intra-nasal challenged animals. In contrast, aerosol and intra-tracheal challenge routes led to infections throughout the respiratory tract, although shedding from the nasal cavity was less reproducible between animals compared to the high-dose intra-nasal challenge group. Intra-tracheal and aerosol challenges induced a transient lymphopaenia, similar to that observed in influenza-infected humans, and greater virus-specific cellular immune responses in the blood were observed in these groups in comparison to the intra-nasal challenge groups. Activation of lung macrophages and innate immune response genes was detected at days 5 to 7 post-challenge. The kinetics of infection, both virological and immunological, were broadly in line with human influenza A virus infections. These more authentic infection models will be valuable in the determination of anti-influenza efficacy of novel entities against less severe (and thus more common) influenza infections.

Defence proteins produced by epithelial cells are a critical component of the host response to respiratory infection. BPIFA1/SPLUNC1 is a member of the bactericidal/permeability-increasing (BPI) fold-containing (BPIF) protein family and is secreted in the mammalian respiratory tract. While it has been implicated in airway host defence its function is yet unresolved. The objective of this study was to use system biology tools to assess SPLUNC1 function in the respiratory tract in response to Influenza A virus (IAV) infection. We have established an in vivo model for studying SPLUNC1 function using knockout mice. As early as day 1 post infection, there is a 1 log increase in IAV titre and a more rapid spread of virus. Analyses of both label-free quantitative proteomics and transcriptomics data sets show an increased infiltration of inflammatory cells in the lungs of infected SPLUNC1 KO compared with wild-type controls. This has been complemented by histopathology as well as flow cytometric analysis, which shows a 3-fold increase in neutrophils in the lungs and BAL. Pathway analyses also reveals a modulation in cell movement and humoral immune responses in the KO mice. Gene set enrichment analysis of RNAseq output shows a significantly reduced enrichment in the expression of B and T lymphocyte surface markers in KO mice compared to wild-type mice, hence identifying molecular signatures associated with SPLUNC1 action. Our data indicate that SPLUNC1 is a crucial component of the mucosal host defence against Influenza A virus and provide further support for its function in influencing inflammation and adaptive immunity.

BPIFA1 is a member the bactericidal/permeability-increasing (BPI) protein family and is structurally similar to other BPIF family members with demonstrated innate immune roles. It is produced mainly in the mammalian respiratory tract and has been implicated in airway host defense against respiratory pathogens. However, the function of this protein is yet unresolved in many species and by elucidating this will aid to clarify its physiological role within the lungs. The objective of this study was to assess BPIFA1 expression and function in the respiratory tract in response to Influenza A virus. Using BPIFA1 knockout mice, we have shown increased virus load and inflammation in the lungs of these mice infected with the X31 strain (H3N2) as compared with wild-type controls. Interestingly, there were large peri-vascular aggregations of B cells in the lungs of BPIFA1-/- mice suggesting a role of BPIFA1 in modulating antibody responses. Lung tissues from mice infected with different strains of varying pathogenicities of Influenza A virus were analysed by immunohistochemical methods at specific time points during the infection period. Imaging software was then used to quantify the level of protein expression within the respiratory epithelium. It was found that the level of BPIFA1 decreased in response to highly pathogenic H5N1 and H7N9 infections but not to low pathogenicity H5N1 or H1N1 viruses. This alteration in expression was associated with an inflammatory response within the respiratory tissue surrounding the epithelium. Our data indicate that BPIFA1 influences inflammation and possibly signals to immune cells during Influenza A virus infection.