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The combined treatment of electrochemotherapy (ECT) and interleukin-12 (IL-12) gene electrotransfer (GET) has already been used in clinical studies in dogs to treat various histological types of spontaneous tumors. The results of these studies show that the treatment is safe and effective. However, in these clinical studies, the routes of administration of IL-12 GET were either intratumoral (i.t.) or peritumoral (peri.t.). Therefore, the objective of this clinical trial was to compare the two IL-12 GET routes of administration in combination with ECT and their contribution to the enhanced ECT response. Seventy-seven dogs with spontaneous mast cell tumors (MCTs) were divided into three groups: one treated with a combination of ECT + GET peri. t. (29 dogs), the second with the combination of ECT + GET i.t. (30 dogs), and the third with ECT alone (18 dogs). In addition, immunohistochemical studies of tumor samples before treatment and flow cytometry of peripheral blood mononuclear cells (PBMCs) before and after treatment were performed to determine any immunological aspects of the treatment. The results showed that local tumor control was significantly better in the ECT + GET i.t. group (p < 0.050) than in the ECT + GET peri.t. or ECT groups. In addition, disease-free interval (DFI) and progression-free survival (PFS) were significantly longer in the ECT + GET i.t. group than in the other two groups (p < 0.050). The data on local tumor response, DFI, and PFS were consistent with immunological tests, as we detected an increased percentage of antitumor immune cells in the blood after treatment in the ECT + GET i.t. group, which also indicated the induction of a systemic immune response. In addition, we did not observe any unwanted severe or long-lasting side effects. Finally, due to the more pronounced local response after ECT + GET i.t., we suggest that treatment response assessment should be performed at least two months after treatment, which meets the iRECIST criteria., 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 © 2023 Elsevier B.V. All rights reserved.)

Extracellular vesicles (EVs) are membrane-enclosed fragments shed from all cell types, including tumour cells. EVs contain a wide range of proteins, biolipids and genetic material derived from mother cells and therefore may be potential biomarkers for tumour diagnosis, disease progression and treatment success. We studied the effect of canine mast cell tumours (MCTs) on EV concentrations in blood isolates in association with MCT's histological grade, Ki-67 proliferative index, KIT-staining pattern and number of PLT. The average EV concentration in blood isolates from nine dogs with MCTs was considerably higher than that in blood from eight healthy dogs. But there were no statistically significant differences in EVs concentration in the population of dogs with MCT according to a different histological grade of malignancy (Patnaik, Kiupel), KIT-staining pattern and Ki-67 proliferation index. The results show that these variables statistically do not significantly predicted EV concentrations in blood isolates (P > .05), except the KIT-staining pattern I which added statistically significantly to the prediction (P < .05). The results confirmed the impact of neoplasms on the morphological changes to cell membranes, which result in greater vesiculability and higher EV concentrations., (© 2019 John Wiley & Sons Ltd.)

The aim of this study was to evaluate the safety and efficacy of the combination of electrochemotherapy (ECT) with bleomycin and gene electrotransfer (GET) of plasmid encoding canine interleukin 12 (IL-12) for the treatment of canine oral malignant melanoma (OMM). Our focus was to determine the effect of the treatment on achieving local tumor control and stimulation of an antitumor immune response. Nine dogs with histologically confirmed OMM stage I to III were included in a prospective, non-randomized study. The dogs were treated with a combination of cytoreductive surgery, ECT and IL-12 GET, which was repeated up to five times, depending on the clinical response to the treatment, evaluated according to the follow-up protocol (7, 14 and 28 days after, the last treatment). One month after treatment, the objective response (OR) rate was 67% (6/9). Median survival time (MST) was 6 months and, even though the disease progressed in 8/9 patients at the end of the observation period (2 to 22 months), four animals were euthanized due to tumor-unrelated reasons. In addition, we observed a decline in the percentage of regulatory T cells (T reg ) in the peripheral blood in the course of the treatment, which could be attributed to a systemic antitumor response to IL-12 GET. The results of this study suggest that a combination of ECT and IL-12 GET may be beneficial for dogs with OMM, especially when other treatment approaches are not acceptable due to their invasiveness or cost., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Electrochemotherapy combined with peritumoral interleukin-12 (IL-12) gene electrotransfer was used for treatment of mast cell tumours in 18 client-owned dogs. Local tumour control, recurrence rate, as well as safety of combined therapy were evaluated. One month after the therapy, no side effects were recorded and good local tumour control was observed with high complete responses rate which even increased during the observation period to 72%. IL-12 gene electrotransfer resulted in 78% of patients with detectable serum IFN-γ and/or IL-12 levels. In the treated tumours vascular changes as well as minimal T-lymphocytes infiltration was observed. After 1 week, the plasmid DNA was not detected intra- or peritumorally and no horizontal gene transfer was observed. In summary, our study demonstrates high antitumour efficacy of electrochemotherapy combined with IL-12 electrotransfer, which also prevented recurrences or distant metastases, as well as its safety and feasibility in treatment of canine mast cell tumours., (© 2016 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Mycoplasma (M.) cynos is a proven pathogen of dogs causing respiratory infections including pneumonia. We examined 19 M. cynos strains isolated from different organs of dogs in Austria, Denmark and Israel. All strains agglutinated mammalian and chicken erythrocytes. Using erythrocytes of chickens or dogs as specific ligands we isolated an approximately 65 kDa protein from cell-free supernatants of 3 M. cynos strains, which showed an apparent capacity for haemagglutination. The N-terminal sequence of a 25 kDa fragment of this protein was identified as NNEMTPKVTVEAKSMELLLSVEK. The identical amino acid sequence is encoded by the gene MCYN&#95;0308 in the genome of M. cynos C142. This gene belongs to a family of some 20 genes which encode putative lipoproteins with proline-rich regions (PRR) in the first third of their molecules. We termed the 65 kDa haemagglutinin HapA and sequenced hapA gene homologues of 16 M. cynos strains. Analyses of hapA gene homologues revealed similar but not identical sequences, some having insertions and/or deletions in the PRR. We produced a recombinant HapA protein (rHapA) and also mouse monoclonal antibodies (mAbs) recognizing HapA. However, enzyme immunoassays using native M. cynos colonies and mAbs 5G2 or 3B7 showed variable expression of HapA in all M. cynos strains. This was further confirmed by Western blot analyses which showed different HapA quantities and also size-variation of HapA among strains. Analyses of cDNA of the expressed hapA genes showed that besides the hapA gene cultures of M. cynos (strains 105, 2002, 2297) can also express other forms of hap genes. In addition, in cloned cultures of strain 2297 altered HapA epitopes for mAbs 5G2 and 3B7 with distinct hapA gene mutations that resulted in altered HapA amino acid sequence were found. Most of the dogs examined had serum antibodies to rHapA. In conclusion, we characterized the M. cynos haemagglutinin HapA protein and encoding gene hapA, a factor involved in cytadherence to host cells and therefore important for M. cynos infection, and showed that expression of HapA is varied in M. cynos by two distinct mechanisms; differential gene expression and nucleic acid substitution within hapA homologues., (Copyright © 2014. Published by Elsevier B.V.)

Alterations in serum protein profile, presence of circulating immune complexes (CIC) and proteinuria were investigated in a large group of dogs naturally infected with the Anaplasma phagocytophilum bacterium. Our aim was to evaluate the presence of hypergammaglobulinaemia, CIC and proteinuria as a possible result of an immune-mediated disease following infection by or exposure to A. phagocytophilum. Dogs were divided into three groups - IFA positive (188 dogs with confirmed exposure to A. phagocytophilum), PCR positive (31 dogs with confirmed infection), and control (IFA and PCR negative) (19 dogs). Serum and urine protein patterns were determined by electrophoresis and CIC concentrations by absorbance nephelometry. No significant differences in hypergammaglobulinaemia were observed between the different groups, as shown by the presence of acute phase proteins α2 and β1-2 globulins. CIC concentrations in the IFA and PCR positive groups were, on average, higher than in controls by 151.3μg/ml, though the differences were not significant. The proportion of dogs with proteinuria did not differ significantly between groups. Our results confirm the assumption that anaplasmosis in dogs is most probably a disease with an acute course, with a good prognosis under the right treatment., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Laboratory and clinical parameters of 149 dogs, exposed to Anaplasma phagocytophilum (A. phagocytophilum), and 19 control dogs were evaluated and compared retrospectively. The aim of our study was to determine statistically significant differences of selected parameters between groups of patients, divided according to the immunofluorescence (IFA) titres, in attempt to improve current diagnostic and treatment criteria. Exposure to A. phagocytophilum was confirmed by IFA and infection by PCR. Based on the results, the dogs were divided into 8 groups (6 groups of seropositive dogs according to the antibody titre, 1 group of PCR positive dogs, and a control group). Selected parameters were compared between groups. Thrombocytopenia was confirmed to be the most prominent haematological change in IFA and/or PCR positive dogs. There were no statistically significant differences in clinical and haematological observations between groups of different IFA titre but clear overall differences between each IFA and PCR positive groups compared to the control group. Our results showed the necessity of introducing additional diagnostic procedures in clinical practice, since antibody titre and haematological parameters are not sufficient to confirm the clinical relevance of exposure to A. phagocytophilum in a particular patient., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Unlabelled: The aim of this study was to evaluate the effectiveness of local treatment electrochemotherapy (ECT) with cisplatin and to compare it with effectiveness of surgery for treatment of mast cell tumours (MCT) in dogs., Materials and Methods: In the present retrospective study, 25 dogs of different breeds with MCT were divided into two treatment groups: surgery (16 dogs with 16 tumours) and those whose owners refused surgery being included into the ECT group (9 dogs with 12 tumours). Response rate and duration of response to the treatment were evaluated and comparison between groups was made., Results: The clinical stages of the tumours were stage I in 4 (45%) and stage III in 5 (55%) dogs treated by ECT; 12 (75%) dogs treated by surgery were stage I and 4 (25%) dogs were in clinical stage III. The median size of the tumours was 5.2 cm3 and 2.9 cm3 of tumours treated by surgery and ECT, respectively. ECT resulted in as comparable antitumor effectiveness as surgical treatment. However, the estimated median duration of response in dogs treated with complete surgical excision was 31.5 months, while it was not reached for the ECT group at the time of writing., Clinical Significance: ECT is an easy, effective and safe local treatment of MCT. It can be an alternative treatment to surgery, specifically for smaller nodules in which a complete response with long duration can be obtained after only one treatment session, or when the nodule is unresectable because of the location.

Background: Electrochemotherapy is an antitumor therapy that utilizes locally-delivered, short intense direct current electric pulse to the tumor nodule plus chemotherapy. The aim of the present study was to evaluate the electrochemotherapy treatment of perianal tumors of different sizes in dogs., Materials and Methods: In 12 dogs, 26 tumor nodules of perianal tumors of different size, and clinically expected to be of different histological type, were treated with electrochemotherapy. Electrochemotherapy consisted of intratumoral injection of cisplatin (1 mg/cm3) or bleomycin (3 mg/cm3), followed by application of electric pulses (8 electric pulses; amplitude, 910 V, duration, 100 micros, frequency, 1 Hz) to the tumor nodule., Results: Responses to treatment were assessed 4 weeks after the therapy; 82% of all tumors treated with electrochemotherapy responded with objective response (OR) (complete response (CR)=41%, partial response (PR)=41%), 16% responded with no change (NC) and 1 tumor (2%) went to progressive disease (PD). At the end of the observation period for each tumor, ranging from 1 to 34 months, 92% OR (CR=65%, PR=27%), 8% NC and no PD were obtained. No major local or general side-effects were noted., Conclusion: Electrochemotherapy with cisplatin or bleomycin is an effective treatment of perianal tumors in dogs. The advantages of this therapy are its simplicity, short duration of treatment sessions, low chemotherapeutic doses and insignificant side-effects, as well as the fact that the subject does not have to stay in hospital.

Canine babesiosis, caused by intraerythrocytic Babesia spp., is a tick-borne disease of worldwide importance. No information on canine babesiosis has been documented in Slovenia. Therefore, 238 dogs admitted to the Small animal clinic in Ljubljana from the years 2000 to 2002 were tested for the presence of babesial parasites in the blood. Based on clinical, microscopic and molecular investigations, 14 dogs (5.9%) were determined as being infected with babesiae. Clinical signs relating to acute haemolysis, fever, anorexia, depression and haematological abnormalities such as anaemia and thrombocytopenia were noticed in most of the 14 infected dogs. The morphology of the parasites was indicative of Babesia canis infection. Two subspecies were detected, namely B. canis canis (11 dogs, 4.6%) and B. canis vogeli (3 dogs, 1.3%) using PCR and subsequent sequence analysis of portions of nns rRNA gene. In addition, based on nucleotide sequence analysis, the 11 isolates of B. c. canis could be subdivided into three groups, whereas the three B. c. vogeli isolates were genetically identical. The results of this study demonstrate the presence of canine babesiosis due to B. c. canis and B. c. vogeli in Slovenia., (Copyright 2004 INRA, EDP Sciences)

The aim of this study was to introduce electrochemotherapy with cisplatin into veterinary medicine, where there is a need for inexpensive and effective treatment of cutaneous and subcutaneous tumours of various histological types. The response to treatment was assessed on tumour nodules in 3 cats with mammary adenocarcinoma and fibrosarcoma, and in 7 dogs with mammary adenocarcinoma, cutaneous mast cell tumour, hemangioma, hemangiosarcoma, adenocarcinoma glandulae paranalis and neurofibroma. Twenty-four tumour nodules of different size were treated; 5 with cisplatin injected intratumourally and 19 with electrochemotherapy, i.e. intratumoural administration of cisplatin followed by delivery of electric pulses to the tumour nodule. Electrochemotherapy with cisplatin had a good antitumour effect on all tumours treated. Their average size 4 weeks after treatment was also greatly reduced (0.01 cm3) compared to those treated by intratumoural cisplatin injection alone (3.0 cm3). Altogether, electrochemotherapy- treated tumours responded with 84% objective responses, whereas only one tumourpartially responded to cisplatin treatment alone. Evaluated by contingency table, the response to treatment with electrochemotherapy was significantly better than that of the cisplatin treated group (p=0.014). Furthermore, there was a significant prolongation of the duration of response in electrochemotherapy treated tumours (p = 0.046). This study showed that electrochemotherapy with cisplatin is an effective, safe and simple local treatment of different histological types of cutaneous and subcutaneous tumours in cats and dogs.