Cytology represents a useful diagnostic tool in the preliminary clinical approach to canine splenic lesions, and may prevent unnecessary splenectomy. However, few studies have evaluated diagnostic accuracy of cytology in the diagnosis of canine splenic neoplasms. The aim of this study was to determine overall accuracy, sensitivity, specificity, positive and negative predictive values (i.e. diagnostic accuracy indexes) of cytology for canine splenic neoplasms following Standards for the Reporting of Diagnostic Accuracy Studies (STARD) guidelines. A consecutive series of canine splenic cytological samples was retrospectively retrieved from the database of the Diagnostic Pathology Service of the Department of Veterinary Medicine (DIMEVET—University of Milan). Histopathology was set as the diagnostic reference standard. Cytological cases were enrolled when slides were available for review and when the same lesion was submitted for histopathology. Seventy-eight (78) lesions were included in the study. By histopathology, 56 were neoplastic and 22 were non-neoplastic. Cytology had an overall accuracy of 73.08% (95% C.I. 61.84%-82.50%), sensitivity of 64.29% (95% C.I. 50.36%-76.64%), specificity of 95.45% (95% C.I. 77.16%-99.88%), and positive and negative predictive values of 97.3% (95% C.I. 84.01%-99.60%) and 51.22% (95% C.I. 42.21%-60.15%), respectively. Low sensitivity and negative predictive value were balanced by very high specificity and positive predictive value. When positive for neoplasia, cytology represents a useful diagnostic tool to rule in splenic neoplasia, prompting surgery independently from other diagnostic tests. Conversely, a negative cytological result requires additional investigations to confirm the dog to be disease free.

Histiocytic sarcomas represent malignant tumors which require new treatment strategies. Canine distemper virus (CDV) is a promising candidate due to its oncolytic features reported in a canine histiocytic sarcoma cell line (DH82 cells). Interestingly, the underlying mechanism might include a dysregulation of angiogenesis. Based on these findings, the aim of the present study was to investigate the impact of a persistent CDV-infection on oxidative stress mediated changes in the expression of hypoxia-inducible factor (HIF)-1α and its angiogenic downstream pathway in DH82 cells in vitro. Microarray data analysis, immunofluorescence for 8-hydroxyguanosine, superoxide dismutase 2 and catalase, and flow cytometry for oxidative burst displayed an increased oxidative stress in persistently CDV-infected DH82 cells (DH82Ond pi) compared to controls. The HIF-1α expression in DH82Ond pi increased, as demonstrated by Western blot, and showed an unexpected, often sub-membranous distribution, as shown by immunofluorescence and immunoelectron microscopy. Furthermore, microarray data analysis and immunofluorescence confirmed a reduced expression of VEGF-B in DH82Ond pi compared to controls. In summary, these results suggest a reduced activation of the HIF-1α angiogenic downstream pathway in DH82Ond pi cells in vitro, most likely due to an excessive, unusually localized, and non-functional expression of HIF-1α triggered by a CDV-induced increased oxidative stress.

Sarcomas especially of histiocytic origin often possess a poor prognosis and response to conventional therapies. Interestingly, tumours undergoing mesenchymal to epithelial transition (MET) are often associated with a favourable clinical outcome. This process is characterized by an increased expression of epithelial markers leading to a decreased invasion and metastatic rate. Based on the failure of conventional therapies, viral oncolysis might represent a promising alternative with canine distemper virus (CDV) as a possible candidate. This study hypothesizes that a CDV infection of canine histiocytic sarcoma cells (DH82 cells) triggers the MET process leading to a decreased cellular motility. Immunofluorescence and immunoblotting were used to investigate the expression of epithelial and mesenchymal markers followed by scratch assay and an invasion assay as functional confirmation. Furthermore, microarray data were analysed for genes associated with the MET process, invasion and angiogenesis. CDV‐infected cells exhibited an increased expression of epithelial markers such as E‐cadherin and cytokeratin 8 compared to controls, indicating a MET process. This was accompanied by a reduced cell motility and invasiveness. Summarized, these results suggest that CDV infection of DH82 cells triggers the MET process by an increased expression of epithelial markers resulting in a decreased cell motility in vitro.

Histiocytic sarcomas refer to highly aggressive tumors with a poor prognosis that respond poorly to conventional treatment approaches. Oncolytic viruses, which have gained significant traction as a cancer therapy in recent decades, represent a promising option for treating histiocytic sarcomas through their replication and/or by modulating the tumor microenvironment. The live attenuated canine distemper virus (CDV) vaccine strain Onderstepoort represents an attractive candidate for oncolytic viral therapy. In the present study, oncolytic virotherapy with CDV was used to investigate the impact of this virus infection on tumor cell growth through direct oncolytic effects or by virus-mediated modulation of the tumor microenvironment with special emphasis on angiogenesis, expression of selected MMPs and TIMP-1 and tumor-associated macrophages in a murine xenograft model of canine histiocytic sarcoma. Treatment of mice with xenotransplanted canine histiocytic sarcomas using CDV induced overt retardation in tumor progression accompanied by necrosis of neoplastic cells, increased numbers of intratumoral macrophages, reduced angiogenesis and modulation of the expression of MMPs and TIMP-1. The present data suggest that CDV inhibits tumor growth in a multifactorial way, including direct cell lysis and reduction of angiogenesis and modulation of MMPs and their inhibitor TIMP-1, providing further support for the concept of its role in oncolytic therapies.

Cladosporium infections have a poor prognosis in animals, most likely due to a lack of knowledge about diagnosis and treatment. In this study, we described a case of a lethal Cladosporium allicinum infection in a captive bullfrog (Pyxicephalus adspersus) in Europe. One adult male bullfrog was referred with clinical signs of lethargy and a cutaneous nodule. Fungal infection was suspected on cytology and confirmed by histology and cultural isolation. The mold was identified by molecular methods using partial sequencing of the TEF1α gene and the ITS region of rDNA. Climbazole antifungal treatment was started but the frog died after 30 days, and necropsy was done. Pigmented hyphae and structures consistent with muriform bodies were found on a background of diffuse granulomatous inflammation at cytological and histopathological examinations. Fungal culture revealed the presence of pigmented fungi identified as Cladosporium allicinum only by partial sequencing of the TEF1α gene. A focally extensive granuloma with intralesional hyphae and muriform bodies effacing the architecture of head, liver, kidneys, lungs, and large intestine were retrieved after necropsy. This study is the first Italian report of the occurrence of lethal C. allicinum infection in a frog and highlights the role of this Cladosporium sp. in chromoblastomycosis.