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Abstract Background Early diagnosis of neosporosis in dogs is challenging. Objectives To evaluate the feasibility of a compound multimodal testing approach for diagnosing in dogs neuromuscular and combined forms of neosporosis. Animals A total of 16 dogs diagnosed with solely neuromuscular neosporosis or with a combination of neuromuscular and central nervous system neosporosis. Methods Retrospective review of clinical signs, laboratory findings, treatment, and outcome with focus on the diagnostic utility of different tests. Development of a chromogenic in situ hybridization (ISH) assay for the identification of Neospora caninum in paraffin-embedded muscle samples. Results 13/16 dogs had only neuromuscular signs of neosporosis, 3/16 had disease signs with concomitant central nervous system (CNS) involvement. Serology was performed in 15/16, with 10/15 showing titers >1 : 160 at admission. PCR on muscle samples detected N. caninum DNA in 11/16. Immunohistochemistry (IHC) detected N. caninum in 9/16 and ISH in 9/16. Histopathology revealed inflammatory myopathy in 10/16, necrotizing myopathy in 5/16, borderline changes in 1/16 and tachyzoites in 9/16. In 4 cases, N. caninum infection was confirmed with all 5 diagnostic methods, 3 cases with 4, 2 with 3, 6 with 2, and 1 animal with 1. Conclusions and Clinical Importance Diagnosis of N. caninum infection should rely on a multimodal diagnostic approach and negativity of 1 single test should not allow for exclusion. Serology in combination with direct parasite identification via histopathology, DNA via PCR, or both modalities, appears a reliable diagnostic approach.

Immune checkpoint inhibition (ICI) is a promising therapeutic strategy for counteracting tumor immune evasion. The therapeutic response largely depends on interactions between cancer cells and the tumor immune microenvironment (TIME). This study aimed to characterize the TIME and its relationship with the immune checkpoint ligand Programmed Death-Ligand 1 (PD-L1) in canine urothelial carcinomas (UCs). UCs were retrospectively selected and tested for PD-L1 using single-antibody immunohistochemistry. Multiplex immunohistochemistry was performed using anti-CD3, -CD20, and -IBA1 antibodies, to co-localize the immune cells (ICs). Both ICs and PD-L1 expression were quantified with computer-assisted image analysis (QuPath software). Based on the spatial distribution and density of ICs, tumors were classified in three distinct immune phenotypes: immune-inflamed, immune-excluded, and immune-desert. Among the 49 UCs analyzed, 11 (22%) were PD-L1+. Forty carcinomas were classified as immune-inflamed (9 PD-L1+; 31 PD-L1-), 7 as immune-excluded (2 PD-L1+; 5 PD-L1-), and 2 as immune-desert (PD-L1-). Macrophages and T-cells were the most numerous ICs, while B-cells were significantly fewer ( p  < 0.0001). PDL1 + tumors exhibited a significantly higher number of macrophages compared to PD-L1- tumors ( p  = 0.003). Immuno-inflamed tumors showed a higher density of T cells ( p  = 0.01) and a lower macrophages-to-T lymphocytes ratio ( p  = 0.02) compared to immune-excluded and immune-desert phenotypes. In summary, most UCs were immune-inflamed and T-cell rich; a subset of tumors was PDL1 + and associated with a higher number of macrophages. Further characterization of T lymphocytes and macrophages polarization is necessary to better stratify the immune response.

The first case of CWD in a Norwegian red deer was detected by a routine ELISA test and confirmed by western blotting and immunohistochemistry in the brain stem of the animal. Two different western blotting tests were conducted independently in two different laboratories, showing that the red deer glycoprofile was different from the Norwegian CWD reindeer and CWD moose and from North American CWD. The isolate showed nevertheless features similar to the classical BSE (BSE-C) strain. Furthermore, BSE-C could not be excluded based on the PrP Sc immunohistochemistry staining in the brainstem and the absence of detectable PrP Sc in the lymphoid tissues. Because of the known ability of BSE-C to cross species barriers as well as its zoonotic potential, the CWD red deer isolate was submitted to the EURL Strain Typing Expert Group (STEG) as a BSE-C suspect for further investigation. In addition, different strain typing in vivo and in vitro strategies aiming at identifying the BSE-C strain in the red deer isolate were performed independently in three research groups and BSE-C was not found in it. These results suggest that the Norwegian CWD red deer case was infected with a previously unknown CWD type and further investigation is needed to determine the characteristics of this potential new CWD strain.

Background Periostin (POSTN) and podoplanin (PDPN) are both proteins playing an important role in humans in the diagnosis and study of many malignant tumours. POSTN affects the regulation of intracellular signalling pathways involving protein kinases PI3-K/Akt and focal adhesion kinase, whereas PDPN increases fibroblast migration and affects the structure of the endothelium; both proteins participate in carcinogenesis, by increasing cell migration and intensifying angiogenesis. In veterinary medicine, their expression has been demonstrated in only a few types of neoplasms, however their presence has not been investigated in canine testicular tumours which are common in dogs and represent a reliable animal model for their human counterparts, due to their structure and biological behaviour. In the present retrospective study, 186 canine testicular tumours namely 61 Leydig cell tumours, 64 Sertoli cell tumours and 61 seminomas and 10 normal canine testicles were immunohistochemically tested for expression of POSTN, PDPN and Ki67 antigens. The polymerase chain reaction (PCR) analysis was also performed on stored frozen samples representative of the three tumour types: 10 Leydig cell tumours, 9 Sertoli cell tumours and 9 seminomas, which confirmed the presence of both proteins. Results Normal testes were negative for both POSTN and PDPN, whereas 27 (44.3%) Leydig cell tumours, 54 (84.4%) Sertoli cell tumours and 56 (91.8%) seminomas were cytoplasmically positive for POSTN and 23 (37.7%), 49 (77.6%) and 44 (72.1%) were cytoplasmically positive for PDPN respectively. The intensity of POSTN and PDPN immunohistochemical reaction was stronger in Sertoli cell tumours and seminomas than in Leydig cell tumours. In addition, Ki67 antigen expression for both POSTN and PDPN correlated significantly with the number of positive cells and the intensity of the reaction in seminomas and Sertoli cell tumours. Conclusions The results showed the absence of expression of POSTN and PDPN in normal canine testes and their expression in neoplastic ones, suggesting a role for these proteins in the carcinogenesis of the testis and encouraging further studies, in particular, on seminomas and Sertoli cell tumours.

Abstract Background Histopathology, immunohistochemistry, and molecular clonality testing are metrics frequently used to diagnose chronic enteropathy (CE) in cats. However, normal values for these metrics have been based mainly on samples from cats that were relatively young, specific pathogen-free, or both. Objectives To describe results of histopathology, immunohistochemistry, and clonality testing of endoscopically-derived biopsy specimens of the upper small intestinal tract from a cohort of clinically healthy client-owned cats. Animals Twenty clinically healthy client-owned cats ≥3 years of age. Methods Tissue specimens were collected from the stomach and duodenum and evaluated single blinded by a board-certified pathologist. In addition, samples were evaluated by routine immunohistochemistry and clonality testing. Cats were followed after the procedure for signs of CE. Results Integrated results from histopathology, immunohistochemistry, and clonality testing were interpreted as consistent with small cell lymphoma (SCL; n = 12), emerging SCL (n = 1), lymphocytic enteritis (n = 6), and pseudoclonality (n = 1). On follow-up, 3 cats eventually developed clinical signs of CE, of which 2 were euthanized 295 and 654 days post-endoscopy. The remaining 17 cats did not show clinical signs of CE after a median of 709 days (range, 219-869 days). Conclusions and Clinical Importance Intestinal biopsy specimens from clinically healthy client-owned cats commonly had abnormal findings on histopathology, immunohistochemistry, clonality testing, or some combination of these without apparent clinical relevance. Current diagnostic metrics for diagnosing CE in cats may need modification to be applicable to the general population of cats.

Mast cell tumours (MCTs) are common malignant neoplasms in dogs, for which prognosis and therapeutic decisions are based on histological features and proliferation markers. Autophagy is a cellular catabolic process responsible for degrading cytoplasmic components to maintain homeostasis, alterations in which are frequently linked to tumour growth and progression. This study was conducted to investigate the occurrence of autophagy in canine MCTs and to verify its value as a prognostic indicator for dogs with the disease. Beclin-1 and LC3B expressions were investigated using immunohistochemistry, and autophagy was ultrastructurally characterised. The autophagic phenomenon was successfully visualised in neoplastic mast cells under transmission electron and immunoelectron microscopy. MCTs from dogs that died due to the disease showed higher positivity for Beclin-1 and dogs with MCTs presenting a LC3B granular immunohistochemical pattern had a significantly shorter post-surgical survival. The occurrence of autophagy is an indicator of poor prognosis. Future studies are needed to elucidate the specific mechanisms and open new opportunities to treatments targeting this cancer cell advantage.

Equine squamous cell carcinomas (eSCCs) are common, and a proportion are likely induced by papillomavirus 2 (EcPV-2). Accurate prediction of clinical outcomes is challenging with no recognized prognostic criteria or consistent histopathological classification scheme for eSCC. The aims of this study were to histopathologically subtype a large case series of eSCCs (genital and ocular) and correlate them with p16 and HER-2 expression, equine papillomavirus infection status, and various clinical and histopathological parameters to predict tumour behavior and prognosis. One hundred and eighty-five samples were examined and subtyped histologically. HER-2 and p16 immunohistochemistry (IHC) and hybridization (ISH) for the EcPV-2 E6/E7 oncogenes were performed on a subset of cases, and follow-up survival data were analyzed. The results were compared and correlated with published guidelines on the categorization of human SCC. Six histopathological subtypes of SCC, according to the WHO, were identified for the first time in horses: usual/invasive (most common), verrucous, pseudoglandular, papillary, warty, and basaloid, with different histological subtypes demonstrating prognostic significance. HER-2 and EcPV-2 statuses were not associated with prognosis in horses with SCC. p16 expression is not associated with EcPV-2 status but could be a potential prognostic factor. ISH demonstrated EcPV-2 genetic material in the majority of eSCCs, except for the papillary subtype, which includes mature, not just pre-cancerous, eSCCs. Widespread HER-2 expression in eSCCs could suggest a role for this cell receptor as a potential therapeutic target.

Telocytes (TCs) are stromal cells characterized by unique long protrusions, known as telopodes (TPs), that establish extensive cellular networks within tissues. They are involved in various functions, including tissue metabolism, mechanical support, and immune regulation. This study aims to investigate the distribution patterns and potential functions of TCs in the myocardium and aortic bulb of young and adult Tibetan sheep by using transmission electron microscopy (TEM), immunohistochemistry, immunofluorescence, and Western blotting techniques. The results demonstrate the existence of TCs in Tibetan sheep hearts and the morphological features changed with age. Compared with the young Tibetan sheep, the TPs were longer and more extensively branched with developed protrusions both in the myocardium and aortic bulb of adult. Immunohistochemical and immunofluorescence staining confirmed that the phenotypic expression of TC markers such as CD34, CD117, α-SMA, and PDGFR-α. Moreover, protein expression analysis revealed that there was no significant differences in CD117 and CD34 between young and adult Tibetan sheep, while α-SMA and PDGFR-α expression was significantly higher in the myocardium of young sheep compared to the adults ( P  < 0.001), but there was no significant difference in the aortic bulb. This study reveals that there are changes in the ultrastructural characterization and immunophenotypic features of TCs in the myocardium and aortic bulb of Tibetan sheep with age, suggesting that TCs play an important role in cardiac development, angiogenesis and functional stability. Therefore, the findings provides clues for further research on characteristics of TCs in plateau hypoxia environment and their potential role in cardiac development.

Background Equine melanocytic neoplasm (EMN) is a skin tumor commonly observed in grey horses. Limited research has yet to investigate proteomic profiles of EMN, particularly in the early stages and their expression patterns. This study, therefore, aimed to identify signature proteins from tissue biopsies to distinguish early EMN, severe EMN, and normal groups. Results Using proteomic analysis of 19 tissue samples (normal: n  = 6, early EMN: n  = 7, severe EMN: n  = 6) through LC–MS/MS, 12,310 proteins were identified. Differentially expressed proteins (DEPs) and functional interaction analysis revealed significant overexpression of Wnt signature proteins, e.g., canonical (Wnt2B) and non-canonical (Wnt5B) Wnt signaling in early EMN stages. Immunohistochemical staining (IHC) towards immunolocalizing Wnt signature protein, particularly the Wnt2B functional signal, further verified its higher expression in early EMN compared to other groups. Conclusions These findings suggest that the Wnt pathway and functional insight are key mediators in signal transduction during early EMN, offering potential markers for initial stage detection. This study enhances the understanding of EMN mechanisms and the role of Wnt proteins, with implications for developing future diagnostic and therapeutic strategies.

Abstract Background Nephrocalcinosis is a common pathological finding in cats with chronic kidney disease and nephrolithiasis. Understanding its pathogenesis may identify future therapeutic targets. Hypothesis Nephrocalcinosis is associated with expression of an osteogenic phenotype. Animals Kidneys with medullary mineralization were obtained from 18 cats (10 with and 8 without nephroliths) undergoing necropsy. Methods Cross-sectional study. Microradiography and histopathology (modified von Kossa stain) were used to confirm parenchymal mineralization. Immunohistochemistry for 5 osteogenic markers was performed to determine their co-localization with nephrocalcinosis. The proportion of kidneys with stronger immunointensity in mineralized versus non-mineralized regions was analyzed using 1-tailed sign tests. The proportion of kidneys with co-localization of nephrocalcinosis and each marker was compared between kidneys with and without nephroliths using Fisher's exact tests. Results Nephrocalcinosis co-localized with osteopontin immunoreactivity in all 18 cats (100%) and with osteocalcin in 12 cats (67%). Both osteogenic markers had stronger immunointensity in mineralized regions compared with non-mineralized regions. Limited co-localization was observed with other markers: bone morphogenic protein-2 in 2 kidneys (both with nephroliths) and tissue non-specific alkaline phosphatase in 1 kidney (without nephroliths); runt-related transcription factor-2 was undetected. No statistically significant differences were found in the co-localization of nephrocalcinosis with osteogenic proteins between kidneys with and without nephroliths. Conclusions and Clinical Importance Expression of osteogenic proteins in areas of nephrocalcinosis indicates that nephrocalcinosis is associated with the development of an osteogenic phenotype. Targeting these processes could offer a novel approach to prevent nephrolithiasis at its origin.