Gene expression profiles associated with early relapse during first remission induction in canine multicentric high-grade B-cell lymphoma.

Although chemotherapy using CHOP-based protocol induces remission in most cases of canine multicentric high-grade B-cell lymphoma (mhBCL), some cases develop early relapse during the first induction protocol. In this study, we examined the gene expression profiles of canine mhBCL before chemotherapy and investigated their associations with early relapse during the first whole CHOP-based protocol. Twenty-five cases of mhBCL treated with CHOP-based protocol as first induction chemotherapy were included in this study. Sixteen cases completed the first whole CHOP-based protocol without relapse (S-group), and nine developed relapse during the chemotherapy (R-group). RNA-seq was performed on samples from neoplastic lymph nodes. Differentially expressed genes (DEGs) were extracted by the comparison of gene expression profiles between S- and R-groups, and the differences in the expression levels of these genes were validated by RT-qPCR. Extracted 179 DEGs included the genes related to chemokine CC motif ligand, T-cell receptor signaling pathway, and PD-L1 expression and PD-1 checkpoint pathway. We focused on chemokine CC motif ligand, and CCL4 was confirmed to be significantly downregulated in the R-group (P=0.039). We also focused on the genes related to T-cell signaling pathway, and CD3E (P=0.039), ITK (P=0.023), and LAT (P=0.023) genes were confirmed to be significantly upregulated in the R-group. The current results suggest that both changes in tumor cells and the interactions between tumor cells and immune cells are associated with the efficacy of the chemotherapy for first remission induction.

Abnormal movement of the pulmonary valve in dogs diagnosed with patent ductus arteriosus.

In the typical left-to-right patent ductus arteriosus (PDA), the shunt flows from the ductus arteriosus towards the pulmonary valve. Although hemodynamic changes have been carefully studied in dogs with PDA, there is very little information on the outcomes of the pulmonary valve after surgical correction of PDA. This study aimed to visualize the pulmonary valve by transthoracic echocardiography in dogs with PDA before and after surgical ligation. Prior to surgery, the movement of the anterior semilunar cusp of the pulmonary valve was obstructed by the shunted blood flow during systole in all nine dogs with PDA in this study. M-mode echocardiography revealed a continuous trajectory of the cusp, because the cusp was pushed towards the right ventricle during the whole cardiac cycle by the shunted flow. Epicardial echocardiography performed in one dog during surgical ligation of the ductus arteriosus revealed that the movement of the anterior semilunar cusp normalized immediately after ligation. B- and M-mode echocardiography may be used to support the diagnosis of PDA through observation of the pulmonary valve when color Doppler echography is not available. The findings in this study may be of importance in distinguishing PDA from PDA-mimicking diseases worth considering before the treatment process (e.g. aorticopulmonary fistulas or aberrant arteriovenous shunts).

Effects of toceranib phosphate (Palladia) monotherapy on multidrug resistant lymphoma in dogs.

We examined whether multidrug resistant (MDR) canine lymphoma increases gene expression for platelet-derived growth factor receptor α (PDGFRα), vascular endothelial growth factor receptor 2 (VEGFR2), and c-KIT, and whether toceranib phosphate (TOC) has potential as a treatment for MDR canine lymphoma. The clinical data showed that PDGFRα expression was higher in canine subjects with MDR T-cell lymphoma than in those with untreated T-cell lymphoma, and that c-KIT expression was greater in subjects with T-cell lymphoma than in those with B-cell lymphoma. TOC monotherapy was well tolerated without serious adverse effects, and two of the five subjects that received TOC exhibited partial responses. The data presented here could contribute to the assessment of TOC-based therapy for dogs with MDR or T-cell lymphoma.

Hypoxia-activated prodrug TH-302 decreased survival rate of canine lymphoma cells under hypoxic condition.

We tested the hypotheses that hypoxic stimulation enhances growth potentials of canine lymphoma cells by activating hypoxia-inducible factor 1α (HIF-1α), and that the hypoxia-activated prodrug (TH-302) inhibits growth potentials in the cells. We investigated how hypoxic culture affects the growth rate, chemoresistance, and invasiveness of canine lymphoma cells and doxorubicin (DOX)-resistant lymphoma cells, and influences of TH-302 on survival rate of the cells under hypoxic conditions. Our results demonstrated that hypoxic culture upregulated the expression of HIF-1α and its target genes, including ATP-binding cassette transporter B1 (ABCB1), ATP-binding cassette transporter G2 (ABCG2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and survivin, and enhanced the growth rate, DOX resistance, and invasiveness of the cells. Additionally, TH-302 decreased the survival rate of the cells under hypoxic condition. Our studies suggest that hypoxic stimulation may advance the tumorigenicity of canine lymphoma cells, favoring malignant transformation. Therefore, the data presented may contribute to the development of TH-302-based hypoxia-targeting therapies for canine lymphoma.

Rapid immunocytochemistry for the detection of cytokeratin and vimentin: assessment of its diagnostic value in neoplastic diseases of dogs.

BACKGROUND: Immunocytochemistry (ICC) is an advanced diagnostic technique used in the field of veterinary cytology. We recently developed a rapid ICC method for the detection of cytokeratin and vimentin in dogs, which helps to determine whether tumor cells are of epithelial or nonepithelial origin. However, the diagnostic value of this rapid ICC method in neoplastic diseases of dogs has not been assessed yet. OBJECTIVES: The aim of the present study was to assess the diagnostic accuracy of rapid ICC compared to standard immunohistochemistry (IHC). METHODS: Air-dried smear samples and formalin-fixed paraffin sections were prepared from tumors excised from dogs (n = 30). Immunosignals for cytokeratin and vimentin were detected in smear samples by rapid ICC, and in paraffin sections by standard IHC. Signals in smear samples detected by rapid ICC were compared with positive staining in paraffin sections detected by standard IHC and analyzed for statistical significance (kappa statistic). RESULTS: Rapid ICC detected specific immunosignals in 25/30 cases (83.3%), and nonspecific signals were detected in 5/30 cases. Statistical analysis revealed fair agreement in epithelial tumors (n = 16) with cytokeratin (κ = 0.236) and vimentin (κ = 0.294). In nonepithelial tumors (n = 14), almost perfect agreement was demonstrated with cytokeratin (κ = 0.857) and vimentin (κ = 0.857). CONCLUSIONS: The rapid ICC method can be a useful tool for the diagnostic cytology of neoplastic tissues in dogs.

Minimal residual disease in canine lymphoma: An objective marker to assess tumour cell burden in remission.

Lymphoma is the most common haematopoietic malignancy in dogs. Since a high proportion of dogs with lymphoma achieve remission soon after initiation of chemotherapy, an objective marker assessing treatment efficacy is required. Following clinical remission, the residual population of tumour cells can be referred to as the minimal residual disease (MRD). MRD traditionally has been detected by cytology and flow cytometry; however, if the burden of malignant cells is low, these methods might not be sufficiently sensitive to detect MRD. As an extension of the development of PCR for antigen receptor gene rearrangements (PARR) in dogs, there has been recent progress in the application of real-time quantitative PCR (RT-qPCR) to canine lymphoma. With the RT-qPCR system, a very high sensitivity (1 cell per 10,000 cells) has been achieved by preparing allele-specific oligonucleotide primers and probes designed from neoplastic clones of each dog. A series of MRD diagnostics studies employing the RT-qPCR system has revealed its usefulness as a prognostic indicator, an objective marker of treatment efficacy and a predictor of relapse for dogs with lymphoma receiving chemotherapy. Introduction of the MRD monitoring system will provide an innovative scientific tool in the development of superior treatments and monitoring strategies for canine lymphoma.

Development and application of multiple immunofluorescence staining for diagnostic cytology of canine and feline lymphoma.

BACKGROUND: Immunophenotyping of canine and feline lymphoma to determine B-cell or T-cell origin is important for predicting prognosis and for development of treatment protocols. For advanced diagnostic cytology tests that can be performed on smears are required to predict the immunophenotype of lymphomas. OBJECTIVES: The aim of this study was to develop a multiple immunofluorescence (MIF) staining method for the determination of lymphocyte immunophenotype in cytologic specimens, and to evaluate its clinical utility. METHODS: B cells and T cells were detected using anti-CD79α and anti-CD3 antibodies, respectively, followed by specific fluorescence-labeled secondary antibodies. The MIF staining method was first developed using fresh-frozen sections of normal canine lymph nodes. The optimal fixative, the necessity of antigen retrieval (AR), and the optimal concentration of the antibodies were determined. The MIF method was then applied to smears of normal lymph nodes, and to clinical samples from dogs and cats with lymphoma. The MIF results were compared to genetic clonality results. RESULTS: B and T cells were detected based on specific fluorescence in frozen sections, using formalin fixation without AR. Specific fluorescence was also detected in smears from normal lymph nodes and lymphomas, and the immunophenotypes predicted from this MIF staining method completely corresponded to those from genetic clonality analysis. CONCLUSIONS: The MIF staining method that we developed in this study effectively distinguished lymphocyte immunophenotypes with high specificity and sensitivity using a single smear sample, and was useful as a diagnostic tool for canine and feline lymphoma.

Molecular survey of arthropod-borne pathogens in ticks obtained from Japanese wildcats.

The Iriomote cat (IC), Prionailurus bengalensis iriomotensis, and the Tsushima leopard cat (TLC), Prionailurus bengalensis euptilurus, are endangered subspecies of leopard cats in Japan. In addition to habitat destruction and road kills, infectious diseases may threaten their populations, and infection with arthropod-borne pathogens has been reported in both subspecies. Infestations with ectoparasites, especially ticks, have frequently been observed in ICs and TLCs. In the present study, ticks collected from captured ICs and TLCs between November 2011 and January 2012 were morphologically identified and the prevalence of the pathogens Bartonella sp., Babesia sp., Ehrlichia sp., Anaplasma sp., Hepatozoon sp., and hemoplasmas in the ticks was molecularly evaluated. The ticks Haemaphysalis longicornis, H. hystricis, and Amblyomma testudinarium were obtained from ICs, and H. megaspinosa, Ixodes tanuki, H. campanulata, and A. testudinarium were collected from TLCs. The pathogens Hepatozoon felis, Babesia sp., and Anaplasma bovis were detected in ticks obtained from ICs, while H. felis. Babesia sp., Ehrlichia sp., E. muris, 'Candidatus Mycoplasma haemominutum', and Bartonella henselae were found in ticks from TLCs. To protect and conserve these endangered animals, continuous monitoring and additional surveys will be necessary to understand the role of ticks as disease vectors in Japanese wildcats.

The clinical utility of two human portable blood glucose meters in canine and feline practice.

BACKGROUND: Portable blood glucose meters (PBGMs) are useful for serial measurements of blood glucose and creation of blood glucose curves in veterinary practice. However, it is necessary to validate PBGMs designed for people for veterinary use. OBJECTIVES: Our objective was to evaluate the accuracy of 2 PBGMs designed for people for use in dogs and cats. METHODS: The blood glucose levels were determined in blood samples collected from 69 dogs and 26 cats admitted to the Kagoshima University Veterinary Teaching Hospital, using a MEDISAFE [PBGM-T] and an Antsense III [PBGM-H], and a FUJI DRI-CHEM 7000V as reference method. The correlations and agreements among the results were statistically analyzed. RESULTS: Simple regression analyses revealed a high correlation between values from both PBGMs and the reference method in both dogs and cats. However, Passing-Bablok regression and Bland-Altman analyses revealed that the data from both PBGMs did not show statistical agreement with the reference values. Concordance correlated coefficients were moderate for the PBGM-T and almost perfect for the PBGM-H for canine samples, and were poor for the PBGM-T and substantial for the PBGM-H for feline samples. Hematocrit values significantly affected the results of the PBGM-T, but not the PBGM-H. Error grid analyses revealed that all measurements from both PBGMs would lead to acceptable treatment decisions. CONCLUSIONS: Our findings suggest that both PBGMs, especially the PBGM-H, would be clinically useful in small animal practice, although there was a bias between each PBGM and the reference method.

Molecular epidemiologic survey of Bartonella, Ehrlichia, and Anaplasma infections in Japanese Iriomote and Tsushima leopard cats.

The Iriomote cat (IC; Prionailurus iriomotensis) and the Tsushima leopard cat (TLC; Prionailurus bengalensis euptilura) are endangered wild felids in Japan. As a part of ongoing conservation activities, we conducted a molecular, epidemiologic survey of Bartonella, Ehrlichia, and Anaplasma infections in wild IC and TLC populations. Blood samples (47 from 33 individual IC; 22 from 13 TLC) were collected between August 2002 and January 2011. Using PCR analysis, we confirmed the presence of Bartonella henselae in ICs and Bartonella clarridgeiae in TLCs, with prevalences of 6% and 8%, respectively. Using PCR and basic local alignment search tool analyses, we identified Ehrlichia canis in both cats and Anaplasma bovis in TLCs. The prevalence of E. canis was 12% in ICs and 8% in TLCs, and the prevalence of A. bovis was 15% in TLCs. This is the first report, to our knowledge, of B. henselae, B. clarridgeiae, E. canis, and A. bovis infections in these two endangered species. Continuous monitoring of these pathogens is needed for their conservation.

Epidemiological survey of tick-borne protozoal infection in iriomote cats and tsushima leopard cats in Japan.

This epidemiological survey was conducted to determine the prevalence of Hepatozoon, Babesia and Theileria infection in the Iriomote cat (IC) and the Tsushima leopard cat (TLC). Blood samples from 43 ICs and 14 TLCs were collected between November 2002 and January 2012. Polymerase chain reaction and DNA sequencing analyses detected a Hepatozoon felis infection prevalence of 72.0% (31/43 cats) and 100% (14/14 cats) in ICs and TLCs, respectively. The degree of Hepatozoon parasitemia observed on blood smears ranged from 0.1 to 4.7%. However, no cases had obvious clinical signs of hepatozoonosis. Neither Babesia- nor Theileria-infected wildcats were detected in this study.

An epidemiological survey of hemoplasma infection in Iriomote cats (Prionailurus bengalensis iriomotensis).

An epidemiological survey of Iriomote cats (Prionailurus bengalensis iriomotensis) was conducted to understand the prevalence and molecular characteristics of hemotropic mycoplasma (hemoplasma). A series of ecological surveys of Iriomote cats were performed between November 2003 and September 2010. During this period, 31 Iriomote cats were captured or found, and 39 blood samples were collected. Polymerase chain reaction screening for hemoplasmas and BLAST searches revealed that 4 of the 31 cats were positive for hemoplasma infection (n=3, Mycoplasma haemofelis [Mhf]; n=1, 'Candidatus M. turicensis' [CMt]). The 4 infected cats were captured or found in the northern area of the island of Iriomote. Phylogenetic analyses revealed close relationships between Mhf and CMt isolated from Iriomote cats compared with those from domestic cats and other wild felids. In our study, we identified two species of hemoplasma in Iriomote cats. The number and location of the hemoplasma-positive cats appeared to be limited; however, continuous surveillance of hemoplasma infection in Iriomote cats is necessary.

Molecular epidemiological survey of the Babesia gibsoni cytochrome b gene in western Japan.

In this study, we conducted a survey of the cytochrome b (cytb) gene of Babesia gibsoni (B. gibsoni) isolated from clinical cases to determine the prevalence of potential atovaquone (ATV)-resistant variants. Ninety-two blood samples were collected from naturally B. gibsoni infected dogs. The cytb nucleotide sequence was determined by direct sequencing. Twelve non-synonymous amino acid substitutions were identified in cytb. The principal ATV-resistant substitution, M121I, was detected in three cases. This survey determined that potentially ATV-resistant B. gibsoni strains are present in dogs in Japan.

A case of Iriomote cat (Prionailurus bengalensis iriomotensis) with Hepatozoon felis parasitemia.

We herein present clinical findings of an Iriomote cat with Hepatozoon felis parasitemia. A male Iriomote cat was captured for ecological analyses three times from January 2010 to January 2011. Although this cat did not show any hematological abnormalities at the time of the first capture, H. felis parasitemia and increased serum creatine kinase levels were detected at the second and third captures. H. felis infection was confirmed by polymerase chain reaction, and amplified 18S ribosomal RNA gene fragments were 100% identical to those of H. felis in leopard cats in Korea. Although the virulence of H. felis in this cat was suggested to be low, this is the first report of an H. felis-infected Iriomote cat with parasitemia.

An epidemiological survey of feline hemoplasma infection in Japan.

Hemoplasma (hemotropic mycoplasma) often causes hemolytic anemia in infected cats, especially those with immune suppression. An updated nationwide epidemiological survey of feline hemoplasmosis was conducted in Japan. Blood samples were collected from 1,770 outdoor-accessing cats from March to October 2008. The infections were molecularly detected by PCR analyses, which are able to distinguish Mycoplasma haemofelis (Mhf), `Candidatus M. haemominutum' (CMhm), and `Candidatus M. turicensis' (CMt) infections. Of the 1,770 cats, 468 cases (26.4%) revealed a single- or co-infection of feline hemoplasmas [Mhf alone, 42 cases (2.4%); CMhm alone, 280 cases (15.8%); CMt alone, 48 cases (2.7%); Mhf+CMhm, 28 cases (1.6%); Mhf+CMt, 6 cases (0.3%); CMhm+CMt, 50 cases (2.8%); Mhf+CMhm+CMt, 14 cases (0.8%)]. In addition, male gender, middle to old age, history of fight wounds, and feline immunodeficiency virus infection were shown to be risk factors for hemoplasma infection. Close attention must be paid to the acute onset of disease in feline practice because a prevalence of hemoplasma infection was detected even in clinically healthy cats.

An updated nation-wide epidemiological survey of feline immunodeficiency virus (FIV) infection in Japan.

An updated nation-wide epidemiological survey of feline immunodeficiency virus (FIV) infection was conducted in Japan. Blood samples were collected from 1,770 outdoor accessing cats from March to October 2008. Serologically, 410 cats (23.2%) were positive for anti-FIV antibody. Proviral DNA of the FIV env V3-V5 region isolated from 348 cases could be phylogenetically analyzed. The present study disclosed a geographic distribution of four subtypes (A, B, C and D) of FIV in Japan. Even though an FIV vaccine was introduced in Japan, we do not currently know whether this vaccine is effective against all strains of FIV in Japan or not. Therefore, close attention still has to be paid to epidemic and genotypic trends of FIV.

Evaluation of prognostic factors and establishment of a prognostic scoring system for canine primary immune-mediated hemolytic anemia.

Clinical courses of primary immune-mediated hemolytic anemia (pIMHA) in dogs are highly variable, however, limited information is available to predict their accurate prognoses. To evaluate the prognostic significance of clinical factors and to propose a scoring system to predict prognoses, the medical records of seventy-one dogs with pIMHA were reviewed. Overall mortality rate of dogs with pIMHA was 39% and most of the dogs died within 3 months from diagnosis. Sex, body weight, seasonality, packed corpuscular volume (PCV), platelet count (PLT), total plasma protein (TP), blood urea nitrogen, albumin, total bilirubin, sodium ion, prothrombin time, and fibrin/fibrinogen degradation products before immunosuppressive treatment can influence on survival time in dogs with pIMHA. A prognostic scoring system using a combination of sex, seasonality, PCV, PLT and TP can be statistically significant for raising the accuracy of prognostic prediction. Using the scoring system for prognostication in dogs with pIMHA may enable veterinarians to predict a prognosis easily and accurately.

Cutaneous nocardiosis in a cat.

A 4-year-old spayed female domestic shorthair cat was presented to us for swelling of all foot pads. The skin lesions were histopathologically diagnosed as suppurative pyogranulomatous panniculitis. The lesions did not respond to cephalexin, prednisolone or itraconazole. However, complete resolution of the skin lesions was obtained with doxycycline. A littermate living in the same household developed similar skin lesions that were also successfully treated with doxycycline. Polymerase chain reaction analysis detected the 16S ribosomal RNA gene of Nocardia spp. in DNA extracted from lesion pus, and direct nucleotide sequencing analysis revealed 100% homology with Nocardia elegans. We diagnosed this case as nocardiosis.

A case report: a dog with acute onset of Hepatozoon canis infection.

We present a clinical overview of a dog with acute onset of Hepatozoon canis infection. A stray female beagle dog of unknown age was referred to Kagoshima University showing anemia. Blood tests revealed the presence of anemia, thrombocytopenia, hyperproteinemia, polyclonal gammopathy, hypoalbuminemia, and elevated creatine kinase and alkaline phosphatase activities. In addition, capsule-like organisms were detected in the cytoplasm of approximately 50% of neutrophils in blood smears. H. canis infection was confirmed by polymerase chain reaction and DNA sequencing analyses. Amplified DNA fragments revealed 100% identity to the 18S ribosomal RNA gene of H. canis. The clinical symptoms improved after the administration of antibiotics. Hepatozoonosis in dogs is rare, but veterinarians should be alert to its possible acute onset.

Antiviral activity of membrane fusion inhibitors that target gp40 of the feline immunodeficiency virus envelope protein.

For the entry of lentivirus into target cells, fusion between its viral membrane and cellular membrane is essential. The present study was conducted to examine the inhibitory effect of modified peptides corresponding to heptad repeats (HR) 1 and 2 of feline immunodeficiency virus (FIV) envelope gp40 on the fusion between the viral and cellular membranes. FIV-N36 and FIV-C35 were synthesized as authentic peptides of the N-terminal HR1 domain and C-terminal HR2 domain of FIV gp40, respectively. FIV-C35EK1, FIV-C35EK2, and FIV-C35EK3 were peptides synthesized by modifying FIV-C35 as the X-EE-XX-KK concept to increase their solubility in water and the stability of their alpha-helicity. FIV-C35 and FIV-C35EK1 inhibited the cell membrane fusion mediated by FIV-infected cells and the replication of FIV. FIV-N36, FIV-C35EK2, and FIV-C35EK3 did not show any apparent inhibitory effect. These results indicated that the newly developed membrane fusion inhibitors could facilitate the development of novel anti-lentiviral chemotherapies.