Preliminary investigation on feline coronavirus presence in the reproductive tract of the tom cat as a potential route of viral transmission

Objectives: Feline infectious peritonitis (FIP) is an immune-mediated disease initiated by feline coronavirus (FCoV) infection. To date, the only proven route of transmission is the faecal\u2013oral route, but a possible localisation of FCoV in the reproductive tract of tom cats is of concern, owing to the involvement of the male reproductive tract during FIP and to the presence of reproduction disorders in FCoV-endemic feline catteries. The aim of the study was to investigate the presence and localisation of FCoV in semen and/or in the reproductive tract of tom cats, and its possible association with seroconversion and viraemic phase. Methods: Blood, serum, semen and/or testicle samples were obtained from 46 tom cats. Serology was performed on 38 serum samples, nested reverse transcriptase PCR (nRT-PCR) and reverse transcriptase quantitative PCR (RT-qPCR) were performed on 39 blood samples and on 17 semen samples, and histology, immunohistochemistry and nRT-PCR were performed on 39 testicles. Results: Twenty-four of 38 serum samples were positive on serology. Semen samples were negative on RT-PCR and RT-qPCR for FCoV, while all blood samples were negative at both molecular methods, except for one sample positive at RT-qPCR with a very low viral load. All testicles were negative at immunohistochemistry, while six were positive at nRT-PCR for FCoV. Serology and blood PCR results suggest that the virus was present in the environment, stimulating transient seroconversion. FCoV seems not to localise in the semen of tom cats, making the venereal route as a way of transmission unlikely. Although viral RNA was found in some testicles, it could not be correlated with the viraemic phase. Conclusions and relevance: In the light of these preliminary results, artificial insemination appears safer than natural mating as it eliminates the direct contact between animals, thus diminishing the probability of faecal\u2013oral FCoV transmission

Epigenetic silencing of TFPI-2 in canine diffuse large B-cell lymphoma

Epigenetic modifications are important early events during carcinogenesis. In particular, hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a well-known mechanism of gene silencing that contributes to cancer development and progression. Tissue factor pathway inhibitor 2 (TFPI-2) is a tumor suppressor involved in invasiveness inhibition. Although TFPI-2 transcriptional silencing, through promoter hypermethylation, has been widely reported in several human malignancies, it has never been explored in lymphoma. In the present study TFPI-2 methylation and gene expression have been investigated in canine Diffuse Large B-cell lymphomas (cDLBCL). The methylation level of 23 CpGs located within the TFPI-2 promoter was investigated by bisulfite-specific PCR and next generation amplicon deep sequencing (GS Junior 454, Roche) in 22 cDLBCLs and 9 controls. For the same specimens, TFPI-2 gene expression was assessed by means of Real-time RT-PCR. Sequence analysis clearly demonstrated that TFPI2 is frequently hypermethylated in cDLBCL. Hypermethylation of the TFPI-2 promoter was found in 77% of DLBCLs ( 17 out of 22) and in one normal lymph node. Globally, dogs with DLBCL showed a mean methylation level significantly increased compared to controls (p<0.01) and analysis of hypermethylation by site identified 19 loci out of 23 ( 82%) with mean methylation levels from 2- to 120-fold higher in cDLBCL. Gene expression analysis confirmed a significant down-regulation of TFPI-2 ( p<0.05) in DLBCLs compared with normal lymph nodes, suggesting that TFPI-2 hypermethylation negatively regulates its transcription. In addition, a significant positive correlation ( p<0.01) was found between TFPI-2 methylation levels and age providing the first indication of age-associated epigenetic modifications in canine DLBCL. To conclude, our findings demonstrated that epigenetic dysregulation of TFPI-2, leading to its reduced expression, is frequently detected in canine DLBCL. In the next future, the aberrant TFPI-2 promoter hypermethylation may be considered in association with prognosis and therapy

Immunophenotype predicts survival time in dogs with chronic lymphocytic leukemia

Background: Chronic lymphocytic leukemia (CLL) is a hematologic disorder in dogs, but studies on prognostic factors and clinical outcome are lacking. In people, several prognostic factors have been identified and currently are used to manage patients and determine therapy.Objectives: The aim of the study was to determine if the immunophenotype of neoplastic cells predicts survival in canine CLL.Design: Retrospective study.Animals: Forty-three dogs with CLL.Procedures: Records of dogs with a final diagnosis of CLL were reviewed. For each included dog, a CBC, blood smear for microscopic reevaluation, and immunophenotyping data had to be available. Data on signalment, history, clinical findings, therapy, follow-up, as well as date and cause of death were retrieved.Results: Seventeen dogs had B-CLL (CD21+), 19 had T-CLL (CD3+ CD8+), and 7 had atypical CLL (3 CD3- CD8+, 2 CD3+ CD4- CD8-, 1 CD3+ CD4+ CD8+, and 1 CD3+ CD21+). Among the variables considered, only immunophenotype was associated with survival. Dogs with T-CLL had approximately 3-fold and 19-fold higher probability of surviving than dogs with B-CLL and atypical CLL, respectively. Old dogs with B-CLL survived significantly longer than did young dogs, and anemic dogs with T-CLL survived a significantly shorter time than dogs without anemia.Conclusions: Although preliminary, results suggested that immunophenotype is useful to predict survival in dogs with CLL. Young age and anemia are associated with shorter survival in dogs with B-CLL and T-CLL, respectively

Splenic marginal zone lymphoma in 5 dogs (2001-2008)

Background: Splenic marginal zone lymphomas (MZL) in dogs arise from the marginal zone of B-cell follicles and can progress slowly.Objectives: To describe clinical features, treatment, and outcome of dogs with splenic MZL.Animals: Five dogs with naturally occurring MZL.Methods: Clinical, laboratory, and follow-up data were retrospectively reviewed. Diagnosis was based on clinical, histopathological, and immunophenotypic features.Results: All dogs had stage IV disease; among them, 2 were symptomatic (substage "b") because of splenic rupture. Four dogs underwent splenectomy and adjuvant doxorubicin, and 1 dog underwent surgery only. Three out of the 4 dogs treated with surgery and chemotherapy died of causes unrelated to lymphoma, after 760, 939, and 1,825 days, whereas the remaining dog was alive and in complete remission after 445 days. The dog not receiving any adjuvant treatment had recurrence of the tumor after 180 days.Conclusions and Clinical Importance: Splenic MZL appears indolent and can benefit from splenectomy, with or without systemic chemotherapy

The dog as a possible animal model for human non-Hodgkin lymphoma: a review

none3noneLaura Marconato;Maria Elena Gelain;Stefano ComazziLaura, Marconato; Gelain, MARIA ELENA; Stefano, Comazz

The dog as a possible animal model for human non-Hodgkin lymphoma : a review

Lymphoma represents the most frequent hematopoietic cancer in dogs, and it shows significant overlap with the human disease. Several environmental factors have been associated with canine lymphoma, suggesting that they may contribute to lymphomagenesis. Canine lymphoma often presents in advanced stage (III-V) at diagnosis and, most commonly, has an aggressive clinical course requiring prompt treatment, which relies on the use of polychemotherapy. In this review, we will summarize the state-of-the-art of canine lymphoma epidemiology, pathobiology, diagnostic work-up and therapy, and will highlight the links to the corresponding human disease, providing evidence for the use of dog as an animal model of spontaneous disease

Use of Sysmex XT-2000iV hematology analyser to identify haematopoietic neoplasms in dogs

Myeloproliferative and lymphoproliferative disorders are common in dogs. In some cases, the differentiation between reactive and neoplastic proliferation can be difficult. Moreover, the correct identification of lineage and maturation stage of neoplastic cells requires experienced hematopathologists and expensive techniques such as flow cytometry. The ability to identify these cells could be enhanced by the introduction of an automated blood cell analyser, such as the Sysmex XT-2000iV, that combines an optical laser/fluorescence method with impedance technology. The aim of this work was to assess the possible role of scattergrams generated by the Sysmex XT-2000iV in the diagnostic approach to canine hematopoietic disorders. Peripheral blood samples from healthy dogs without hematological abnormalities were used as a control group in order to determine the best canine gatings and to compare them to pathological samples. Eighteen samples from dogs with lympho/myeloproliferative neoplasms were analysed. In particular, based on morphology of the cells and on flow cytometric analysis, 8 case of lymphoma with and without blood involvement, 6 cases of acute leukemia (myeloid, lymphoid and undifferentiated) and 4 of chronic lymphocytic leukemia were included in the study. Specific gates were developed in order to identify a typical pattern in acute and chronic leukemia and in lymphoma with blood involvement. In the majority of samples neoplastic cells displayed a typical scattergram that allowed the correct identification of the disease. In particular, in all chronic lymphocytic leukemias, a distinct population of cells characterized by intermediate side scatter properties and fluorescence intensity was easily identified. In contrast, in acute leukemias and stage V lymphoma with circulating malignant lymphocytes, neoplastic cells were characterized by higher levels of fluorescence intensity, presumably due to the presence of blast cells with increased amounts of nucleic acid. In cases of lymphoma in which flow cytometry had excluded peripheral blood involvement, the scattergrams were comparable with those of normal dogs. Only in 2 cases were the scattergrams not consistent with the flow cytometric diagnosis: one case of acute lymphocytic leukemia characterized by extreme leukocytosis was misidentified as a chronic leukemia, and one case of lymphoma without blood involvement displayed a pattern similar to that present in leukemic lymphoma. In conclusion, although a complete and accurate differential was automatically generated by the instrument in only a few cases, the analysis of scattergrams from the Sysmex XT-2000iV can represent a first and rapid step capable of identifying neoplastic populations in blood from dogs with lympho/myeloproliferative disorders