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Objectives: This multicentre, retrospective observational study aimed to describe the clinical presentation, diagnostic methods, treatment and outcomes of cats with tracheal masses., Methods: Eighteen cats from five academic or secondary/tertiary animal hospitals were included., Results: The median age at diagnosis was 10.7 years (mean 9.5; range 1-17). There were nine castrated males, seven spayed females, one intact male and one intact female. Fourteen (78%) were domestic shorthairs, one (6%) was an Abyssinian, one (6%) was an American Shorthair, one (6%) was a Bengal and one (6%) was a Scottish Fold. The most common presenting complaints included chronic respiratory distress or dyspnoea (n = 14), followed by wheezing/gagging (n = 12), coughing (n = 5) and voice changes (n = 5). There was cervical tracheal involvement in 16/18, and two showed involvement of the intrathoracic trachea. The following methods were used for diagnosis: ultrasound-guided fine-needle biopsy (UG-FNB) and cytology (n = 8), bronchoscopic forceps biopsy and histopathology (n = 5), surgical resection and histopathology (n = 3), forceps biopsy via an endotracheal tube (n = 1) and histology of tissue sputtered from a cough (n = 1). Lymphoma was most often diagnosed (n = 15), followed by adenocarcinoma (n = 2) and squamous cell carcinoma (n = 1). Most lymphoma cases received chemotherapy with or without radiation according to various protocols, and partial (n = 5) or complete responses (n = 8) were noted. Kaplan-Meier survival data for cats with lymphoma revealed a median survival time of 214 days (95% confidence interval >149 days), which was significantly longer than that of other types of tumours (21 days)., Conclusions and Relevance: Lymphoma was the most prevalent diagnosis, and showed a good response to chemotherapy with or without radiation therapy. Various diagnostic procedures were performed, and UG-FNB and cytology are good diagnostic procedures for cervical tracheal lesions. Owing to the variety of treatment protocols at different centres, it was impossible to compare outcomes.

Severe adverse reactions in cats after vaccination were examined from 316 cases reported to the Ministry of Agriculture, Forestry and Fisheries (MAFF) in Japan during 15-year period from April 2004 to March 2019. We found that 130 (41%) showed anaphylaxis, and 99 (76%) of the 130 cases of anaphylaxis resulted in death. Veterinarians should be well prepared to deal with vaccine-associated anaphylaxis in cats. Bovine serum albumin (BSA) as indicator of purification was detected at high levels in commercially available feline vaccines. BSA might derive from fetal calf serum in culture media. This study provides useful information about anaphylaxis including critical details of the potential clinical signs associated with adverse events to feline vaccination.

Corticosteroid administration prior to the application of chemotherapy in small animal lymphoma patients is a concern, as it is discussed to negatively influence the therapeutic outcome due to corticosteroid-induced drug resistance. Using feline lymphoma cell lines FT-1 and MS4 we have shown, that prednisolone pre-treatment alters the susceptibility of these cells towards doxorubicin or vincristine treatment in vitro. The observed effect was negative as for the killing potential and it was cell line and drug (doxorubicin or vincristine) dependent. Furthermore, increase in mRNA expression of selected proteins with multidrug resistance potential (MDR1, BCRP, LRP, MT) was observed after prednisolone pre-treatment. Administration of chemical inhibitors of these proteins did not lead to reversal in sensitivity of tested cell lines to doxorubicin or vincristine., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nasal lymphoma (NL) is the most common nasal tumor in cats, and radiotherapy, chemotherapy, or a combination of these treatments have been described as the treatment for this disease. However, the previous studies included various machines and protocols of radiotherapy. Therefore, we aimed to retrospectively compare the prognosis among cases treated with palliative hypofractionated radiotherapy, chemotherapy, and a combination of them with united machine and protocol of radiotherapy. When compared overall survival and progression free survival, there was no significant difference among these three groups. The data of this study suggested that similar efficacy could be achieved by palliative hypofractionated radiotherapy, chemotherapy, or a combination of them.

Objectives: Primary copper-associated hepatopathy (PCH) has been reported in young cats. Although our group recently reported a young cat with PCH harbouring single-nucleotide variations in ATP7B , limited information is available regarding its association with the pathogenesis of feline PCH. The objective of this study was to investigate the prevalence of ATP7B variations in cats with PCH., Methods: Rhodanine staining was performed to detect hepatic copper accumulation (HCA) in intraoperative liver tissue specimens from 54 cats. In cats with HCA, variations in ATP7B and COMMD1 and serum ceruloplasmin activity were analysed., Results: Based on age, liver histopathological findings and hepatic distribution of accumulated copper, PCH was suspected in 4/54 cats. Sequence analysis of ATP7B and COMMD1 revealed single-nucleotide variations in ATP7B in 3/4 cats with PCH. Among the cats with PCH, one showed remarkably low serum ceruloplasmin activity, while the other three did not., Conclusions and Relevance: The results of this study suggest that some cats with PCH harbour single-nucleotide variations in ATP7B , suggesting that feline PCH is an equivalent disorder to human Wilson's disease. This study provides basic evidence facilitating further studies of the pathophysiology and treatment of feline PCH.

Objectives: To investigate the clinical characteristics of feline acute lymphoblastic leukaemia patients diagnosed according to the recent diagnostic criteria for the equivalent canine condition., Materials and Methods: The medical records of six cats diagnosed with acute lymphoblastic leukaemia were retrospectively reviewed to extract data on clinicopathological characteristics and outcomes. The lymphoid origin of the tumour cells was confirmed by polymerase chain reaction for antigen receptor gene rearrangement, flow cytometry or immunohistochemistry., Results: Non-specific clinical signs such as lethargy and anorexia were common, and anaemia and thrombocytopenia were also commonly identified. Leucocytosis was observed in four cats and leucopenia was observed in two; the number of lymphoblasts in the peripheral blood samples varied among the cases. Lymphoblasts originated from B-cell lineage in four cats and T-cell lineage in one, and those of another cat were positive for both B-cell marker CD21 and T-cell marker CD8. Five of the six cats were treated with cytotoxic chemotherapy, and a partial response was obtained in two. The median overall survival was 55 days (range: 1 to 115)., Clinical Significance: Acute lymphoblastic leukaemia should be considered if lymphoblasts are observed in peripheral blood, even if their number is small. The prognosis for cats that have acute lymphoblastic leukaemia is as poor as that for dogs, and further studies are needed to develop effective treatment., (© 2018 British Small Animal Veterinary Association.)

A 6-year 5-month-old spayed female Scottish Fold cat presented with a one-month history of gait abnormalities, increased salivation, and decreased activity. A blood test showed hyperammonemia and increased serum bile acids. Imaging tests revealed multiple shunt vessels indicating acquired portosystemic shunt. Histopathologic analysis of liver biopsy showed features consistent with liver hypoperfusion, such as a barely recognizable portal vein, increased numbers of small arterioles, and diffuse vacuolar degeneration of hepatocytes. These findings supported the diagnosis of primary hypoplasia of the portal vein/microvascular dysplasia, (PHPV/MVD). To our knowledge, this is the first case of feline PHPV/MVD that developed multiple acquired portosystemic shunts and presented with hepatic encephalopathy.

Gammaherpesviruses (GHVs) are members of an emerging subfamily of the family Herpesviridae. A recent study identified a novel GHV in domestic cats (Felis catus GHV1, FcaGHV1), and epidemiological surveys have found that FcaGHV1 is distributed worldwide. In this study, we investigated the prevalence of GHVs in domestic cats in Japan with a molecular epidemiological survey. Blood samples were collected from 1,738 domestic cats and GHV-derived DNA was detected with PCR in 1.3% (23/1,738) of the Japanese domestic cats. The FcaGHV1 detected in this study was very similar to FcaGHV1 detected in a domestic cat in North America. Older age (>5 years old) and Feline immunodeficiency virus infection were identified as risk factors for GHV infection.

A 4-y-old cat exhibited neurologic signs such as wobbling, right head tilt, and intention tremor, and MRI revealed a mass in the cerebellum. The cat died 5 mo after initial presentation, and no neoplastic lesions, other than the cerebellar mass, were observed at autopsy. Histologically, large atypical cells resembling Hodgkin cells, with single large inclusion-like nucleoli, and those resembling Reed-Sternberg cells, with symmetrically arranged nuclei, had infiltrated the left side of the cerebellum and were admixed with small lymphocytes. These atypical cells were positive for feline leukemia virus (FeLV), CD20, BLA36, vimentin, p16, p53, and Pax5, and negative for CD3, CD79a, and Iba1 by immunohistochemistry. Multiplex PCR for immunoglobulin heavy-chain gene rearrangement revealed monoclonal proliferation of B-lymphocytes. We describe this feline primary cerebellar B-cell lymphoma that displayed Hodgkin lymphoma-like tumor cells with FeLV protein expression.

A retrospective study of intrahepatic cholelithiasis (IC) in 9 dogs and 2 cats was conducted. Only 1 dog showed clinical signs related to hepatobiliary disease before referral and during the follow-up period. Intrahepatic cholelithiasis might be a subclinical finding in both dogs and cats.

Epidemiological and pathological studies on Helicobacter spp. in feline stomachs in Japan were conducted using genus- and species-specific (H. felis, H. bizzozeronii, H. heilmannii sensu stricto [s.s.] and H. pylori) polymerase chain reactions (PCRs), ureAB gene sequencing and histopathology. PCR results showed that 28 of 56 cats were infected with Helicobacter spp., and H. heilmannii s.s. was the most prevalent species by both PCR (28/28) and ureAB gene sequencing (26/28). Some of the sequences showed high similarities with those from human patients with gastric diseases (99%). There were no significant differences between Helicobacter spp.-positive and -negative cats in the severity of chronic gastritis (P=0.69). This is the first extensive epidemiological study on feline gastric Helicobacter spp. in Japan.

Serum amyloid A (SAA) concentration and plasma matrix metalloproteinase-9 (MMP-9) levels are increased in cats with lymphoma. In the present study, the association between SAA and MMP-9 production was evaluated using recombinant feline SAA (rfSAA) and three feline lymphoma-derived cell lines: 3201, MS4, and MCC. MMP-9 mRNA expression was significantly increased by rfSAA stimulation only in MCC cells. Secreted MMP-9 protein in culture media was confirmed by gelatin zymography, with clear bands of MMP-9 detected in MCC cells following rfSAA stimulation. A significant increase in semi-quantified MMP-9 levels was observed with 5 and 25μg/ml of rfSAA stimulation. The infiltrative activities of feline lymphoma cells, assessed by the matrigel transwell assay, showed that rfSAA stimulated cell infiltration in MCC cells, in addition to MMP-9 expression. Although the response to rfSAA stimulation varied between cell lines, the results showed that rfSAA can stimulate MMP-9 production and infiltration of feline lymphoma-derived cells. The findings of this study have identified a novel role for SAA in the progression of some forms of feline lymphoma., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study, plasma MMP-9 activity was evaluated in cats with lymphoma. Plasma samples were obtained from 26 cats with lymphoma before treatment. From 13 of the included 26 cats, plasma samples were obtained 4 weeks after the initiation of treatment. Plasma samples were also obtained from 10 healthy cats as a control. Plasma MMP-9 activity was examined by gelatin zymography and semi-quantitative value (arbitrary unit; a.u.) for each sample was calculated. Relatively high levels of MMP-9 were observed in cats with lymphoma compared with those in healthy control cats. MMP-9 quantification through zymography showed significantly higher activity in cats with lymphoma (median, 0.63 a.u.; range, 0.23-3.24 a.u.) than in healthy controls (0.22 a.u.; 0.12-0.46 a.u.; P < 0.01). MMP-9 activities were significantly different before (0.73 a.u.; 0.30-3.24 a.u.) and after treatment (0.50 a.u.; 0.14-1.32 a.u.; P = 0.017). Measuring plasma MMP-9 activity in cats with lymphoma may become an appropriate monitoring tool for feline lymphoma., (© 2014 John Wiley & Sons Ltd.)

The p16 gene acts as a tumor suppressor by regulating the cell cycle and is frequently inactivated in human and canine cancers. The aim of this study was to characterize genetic and epigenetic alterations of the p16 in feline lymphoid and non-lymphoid malignancies, using 74 primary tumors and 11 tumor cell lines. Cloning of feline p16 and subsequent sequence analysis revealed 11 germline sequence polymorphisms in control cats. Bisulfite sequencing analysis of the p16 promoter region in a feline lymphoma cell line revealed that promoter methylation was associated with decreased mRNA expression. Treatment with a demethylating agent restored mRNA expression of the silenced p16. PCR amplification and sequencing analysis detected homozygous loss (five tumors, 6.7%) and a missense mutation (one tumor, 1.4%) in the 74 primary tumors analyzed. Methylation-specific PCR analysis revealed promoter methylation in 10 primary tumors (14%). Promoter methylation was frequent in B cell lymphoid tumors (7/21 tumors, 33%). These genetic and epigenetic alterations were also observed in lymphoma and mammary gland carcinoma cell lines, but not detected in non-neoplastic control specimens. These data indicate that molecular alterations of the p16 locus may be involved in the development of specific types of feline cancer, and warrant further studies to evaluate the clinical value of this evolutionarily-conserved molecular alteration in feline cancers., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pancreatic lipase immunoreactivity (Spec fPL) is currently considered to be the most accurate blood test for the diagnosis of feline pancreatitis. In this study, we measured lipase activity in cats using a newer catalytic lipase assay of dry-chemistry system (FDC-v-LIP) to determine the reference range and compared the results with those for Spec fPL. Based on the results of healthy cats, the reference range of FDC-v-LIP was determined to be less than 30 U/l. FDC-v-lip did not show a strong correlation with Spec fPL in cats with various diseases, which resulted in the low sensitivity and positive predictive value. However, the relatively high (>90%) specificity and negative predictive value indicated that FDC-v-LIP could be a useful patient-side screening test for the exclusion of feline pancreatitis.

In 2012 and 2013, the World Small Animal Veterinary Association (WSAVA) Vaccination Guidelines Group (VGG) undertook fact-finding visits to several Asian countries, with a view to developing advice for small companion animal practitioners in Asia related to the administration of vaccines to dogs and cats. The VGG met with numerous first opinion practitioners, small animal association leaders, academic veterinarians, government regulators and industry representatives and gathered further information from a survey of almost 700 veterinarians in India, China, Japan and Thailand. Although there were substantial differences in the nature and magnitude of the challenges faced by veterinarians in each country, and also differences in the resources available to meet those challenges, overall, the VGG identified insufficient undergraduate and postgraduate training in small companion animal microbiology, immunology and vaccinology. In most of the countries, there has been little academic research into small animal infectious diseases. This, coupled with insufficient laboratory diagnostic support, has limited the growth of knowledge concerning the prevalence and circulating strains of key infectious agents in most of the countries visited. Asian practitioners continue to recognise clinical infections that are now considered uncommon or rare in western countries. In particular, canine rabies virus infection poses a continuing threat to animal and human health in this region. Both nationally manufactured and international dog and cat vaccines are variably available in the Asian countries, but the product ranges are small and dominated by multi-component vaccines with a licensed duration of immunity (DOI) of only 1 year, or no description of DOI. Asian practitioners are largely unaware of current global trends in small animal vaccinology or of the WSAVA vaccination guidelines. Consequently, most practitioners continue to deliver annual revaccination with both core and non-core vaccines to adult animals, with little understanding that "herd immunity" is more important than frequent revaccination of individual animals within the population. In this paper, the VGG presents the findings of this project and makes key recommendations for the Asian countries. The VGG recommends that (1) Asian veterinary schools review and increase as needed the amount of instruction in small animal vaccinology within their undergraduate curriculum and increase the availability of pertinent postgraduate education for practitioners; (2) national small animal veterinary associations, industry veterinarians and academic experts work together to improve the scientific evidence base concerning small animal infectious diseases and vaccination in their countries; (3) national small animal veterinary associations take leadership in providing advice to practitioners based on improved local knowledge and global vaccination guidelines; (4) licensing authorities use this enhanced evidence base to inform and support the registration of improved vaccine product ranges for use in their countries, ideally with DOI for core vaccines similar or equal to those of equivalent products available in western countries (i.e. 3 or 4 years). The VGG also endorses the efforts made by Asian governments, non-governmental organisations and veterinary practitioners in working towards the goal of global elimination of canine rabies virus infection. In this paper, the VGG offers both a current pragmatic and future aspirational approach to small animal vaccination in Asia. As part of this project, the VGG delivered continuing education to over 800 Asian practitioners at seven events in four countries. Accompanying this document is a list of 80 frequently asked questions (with answers) that arose during these discussions. The VGG believes that this information will be of particular value to Asian veterinarians as they move towards implementing global trends in small companion animal vaccinology., (© 2014 WSAVA.)

The present study was carried out to analyze the prognosis of 163 cats with lymphoma classified anatomically and cytomorphologically. Anatomically, alimentary lymphoma was the most common form and showed significantly shorter survival than mediastinal and nasal lymphomas in cats. Cytomorphologically, there was no predominant subtype in feline lymphomas. Immunoblastic type (18%), centroblastic type (16%), globule leukocyte type (15%), lymphocytic type (12%), lymphoblastic type (12%), pleomorphic medium and large cell type (10%) and anaplastic large cell type (7%) were relatively common subtypes. Most of the cats with globule leukocyte lymphoma had the alimentary form. Comparing median survival time among classifications, cats with globule leukocyte lymphoma showed significantly shorter survival than those with high-grade and other low-grade lymphomas. Furthermore, cats with high-grade lymphomas showed significantly shorter survival than cats with other low-grade lymphomas. The present study indicated the clinical significance of anatomical and cytomorphological evaluation in feline lymphomas.

Chronic myelogenous leukaemia was diagnosed in a 7-year-old male neutered domestic shorthair cat. Leukocytosis (74,900/µl)--mature neutrophilia, eosinophilia and basophilia--was observed. Bone marrow aspiration revealed hypercellularity with proliferation of cells of myeloid lineage. An underlying condition leading to leukocytosis was not identified. The severe leukocytosis did not respond to antibiotic therapy. Based on these findings, chronic myelogenous leukaemia was diagnosed. Because of the absence of clinical signs, the cat was monitored without treatment until 7 months after diagnosis, when it developed pruritic skin lesions. Pruritus was controlled with oral prednisolone. Forty-two months after diagnosis, the cat developed nasal lymphoma, which was treated with radiation therapy, resulting in complete remission. The cat was still in good physical condition 63 months after diagnosis, despite the persistence of marked neutrophilia, eosinophilia and basophilia., (© ISFM and AAFP 2013.)

Feline leukemia virus (FeLV) induces neoplastic and nonneoplastic diseases in cats. The transduction of cellular genes by FeLV is sometimes observed and associated with neoplastic diseases including lymphoma and sarcoma. Here, we report the first natural case of feline Notch2 transduction by FeLV in an infected cat with multicentric lymphoma and hypercalcemia. We cloned recombinant FeLVs harboring Notch2 in the env gene. Notch2 was able to activate expression of a reporter gene, similar to what was previously reported in cats with experimental FeLV-induced thymic lymphoma. Our findings suggest that the transduction of Notch2 strongly correlates with FeLV-induced lymphoma.

Serum amyloid A (SAA) is reported not only as a marker for the presence of inflammation but also as a prognostic indicator in human beings. In cats, however, there is no report on the association between SAA concentration and prognosis. The objective of the current study was to evaluate SAA concentration as a prognostic marker in diseased cats. A total of 175 cats with neoplastic diseases, inflammatory diseases, and other diseases were retrospectively recruited, and the medical records of these cats, including follow-up data on mortality, were reviewed. Cats were divided into 2 groups according to SAA concentration, and differences in survival between each group were assessed. Median survival time of cats in the elevated SAA (>0.82 mg/l) group was significantly shorter than that in the nonelevated SAA (≤0.82 mg/l) group (P < 0.001). Furthermore, by multivariate analysis, SAA concentration was shown as a significant and independent prognostic marker in cats with various diseases (P = 0.015). Serum amyloid A concentration in diseased cats is a useful predictive indicator of prognosis regardless of diagnosis.

Aspergillus udagawae and A. viridinutans are members of the section Fumigati; both cause invasive aspergillosis in humans. These two Aspergillus species are discriminated from A. fumigatus by molecular methods. Herein, we report two cases of feline orbital aspergillosis, one caused by A. udagawae and the other by A. viridinutans. To our knowledge, Case 1 represents the first reported case of treatment of A. udagawae with a high dosage of itraconazole, and Case 2 represents the first reported case of A. viridinutans infection associated with sarcoma. Identification of the etiologic agents of these cases was confirmed by comparative analyses of the sequences of β-tubulin-encoding genes. With the spread of non-fumigatus aspergillosis, increasing emphasis should be placed on molecular identification of the infecting Aspergillus species and the use of in vitro drug susceptibility tests to ensure the selection of appropriate antibiotics.

Of the various classification systems for non-Hodgkin's lymphoma, the updated Kiel classification is valuable for veterinary practice, because of its utility to classify the subtypes not only by histopathology but also by cytomorphology. However, there are only a few reports of small number of feline lymphomas to apply the updated Kiel classification. In this study, immunogical subtypes and morphology of 76 feline lymphomas were evaluated and classified into subtypes of the updated Kiel classification. Of the cases, 49% were T-cell lymphoma, 25% were B-cell lymphoma and 26% were undetermined immunological subtype. Based on the updated Kiel classification, most subtypes were identified also in feline lymphomas as in dogs and humans. Globule leukocyte lymphoma was specific for cats and relatively common in feline alimentary lymphomas. Of the present cases, 64% were high-grade subtypes, whereas 36% were low-grade subtypes. The present study indicated that feline lymphomas could be morphologically classified by the modified updated Kiel classification.

X-chromosome inactivation pattern (XCIP) analysis has been widely used to assess cell clonality in various types of neoplasms in humans. In the present study, a polymerase chain reaction-based feline XCIP analysis using the feline androgen receptor gene was developed. To construct the system of the analysis, polymorphism in CAG tandem repeats within the feline androgen receptor gene was explored using somatic DNAs from 50 male and 103 female cats. CAG tandem repeats in exon 1 of the feline androgen receptor gene were found to be polymorphic, containing 15 to 22 CAG repeats. Of the 103 female cats, 70 (68%) were heterozygous for the number of CAG repeats, indicating the possible usefulness of XCIP analysis in cats. Application of the feline XCIP analysis to 3 feline mammary gland adenocarcinoma cell lines revealed distinctly skewed XCIPs in these cell lines, indicating their clonal origins. Twelve (80%) of the 15 primary tissue/cell samples obtained from cats with various neoplastic diseases showed skewed XCIPs. Moreover, bone marrow samples from 3 cats with myelodysplastic syndrome were also found to have skewed XCIPs. The polymerase chain reaction-based XCIP analysis developed in this study can provide information on cell clonality in female cats, potentially facilitating the differential diagnosis of various disorders in cats.

A Japanese domestic long-hair cat of about 8 years of age was presented with vomiting and hematochezia and was found to have significant hypereosinophilia. Bone marrow aspiration revealed moderate increases of eosinophilic lineages. Histopathological examination revealed mild eosinophilic and epitheliotropic T-lymphocytic infiltrations in the duodenum. Although the cat remained asymptomatic with only prednisolone administration, the cat presented with hematemesis, weight loss, and severe anorexia 512 days after the initial presentation. Subsequently, gastrointestinal perforation developed, and the cat died on Day 536. Histopathological examination of autopsy specimens revealed mixed cellular infiltration including eosinophils and neoplastic lymphocytes in the intestinal lymph nodes, intestine, liver, spleen, and pancreas. Immunohistochemical examination supports a diagnosis of intestinal T-cell lymphoma with severe hypereosinophilic syndrome.

The P53 tumor suppressor protein is a multifunctional transcription factor that prevents the malignant transformation of normal cells. In human malignancies, p53 is the most frequently altered gene and is mutated in approximately 50% of all malignancies. In contrast, p53 gene mutation has been rarely detected in feline malignancies, and most feline malignancies conceivably retain the wild-type p53 (wt-p53) gene. MDM2 negatively regulates the P53 protein by inhibiting its transcriptional activity and nuclear transport and by inducing its degradation. Inhibition of P53-MDM2 interaction stabilizes P53 protein and activates P53 pathway. Nutlin-3, a small molecule that inhibits P53-MDM2 interaction, was shown to have an antitumor effect in several human cancer cells retaining the wt-p53 gene. In the present study, we evaluated and compared the antitumor effect of nutlin-3 in 5 different feline lymphoma cell lines, of which 3 harbored wt-p53, and 2, mutated p53 (mt-p53). Treatment with nutlin-3 resulted in increased amounts of P53 protein in conjunction with augmented expression of P53-target genes in 3 feline lymphoma cell lines with the wt-p53 gene, but not in 2 feline lymphoma cell lines with the mt-p53 gene. Nutlin-3 treatment also induced G1-S and/or G2-M cell cycle arrest and apoptosis in lymphoma cell lines with wt-p53. Nutlin-3 treatment induced cell cycle arrest but not apoptosis in the cell lines with mt-p53. From these results, we concluded that nutlin-3 has an antitumor effect on feline lymphoma cell lines harboring the wt-p53 gene through accumulation and activation of P53 leading to cell cycle arrest and apoptosis. The present study suggests that inhibition of P53-MDM2 interaction using nutlin-3 may be a new therapeutic strategy for treating feline lymphoma retaining the wt-p53 gene., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. Polymerase chain reaction (PCR) amplification of the complementarity-determining region (CDR) 3 of the T-cell receptor (TCR) γ gene can be used to assess clonality of T-cell populations as a supportive diagnostic tool for T-cell neoplasms. Because the length variation in the TCRγ CDR3 is relatively small, false positive results may occur in non-neoplastic T-cell populations in the absence of high-resolution analytical methods for PCR products. In the present study, a PCR assay system was developed to detect clonal TCRγ gene rearrangement in feline lymphoid cells using GeneScan analysis. Thirty T-cell neoplasms, 27 B-cell neoplasms, and 34 non-neoplastic tissues were subjected to the newly developed TCRγ gene rearrangement analysis. Clonal TCRγ gene rearrangement was detected in 26 of 30 (87%) T-cell neoplasms, 2 of 27 (7%) B-cell neoplasms, and 1 of 34 (3%) non-neoplastic tissues. To compare GeneScan analysis with conventional PAGE and heteroduplex analysis, 20 clonal and 20 polyclonal samples were subjected to both analyses. Most of the results were concordant between the 2 analyses; however, several clonal peaks (bands) appeared as a single band when analyzed via conventional PAGE with heteroduplex analysis in 4 of the 20 (20%) clonal samples as a result of the difference in resolution. The PCR assay system to detect clonal TCRγ gene rearrangement in feline lymphoid cells, using GeneScan analysis, would be a useful molecular diagnostic tool for feline T-cell neoplasms, with high fidelity., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. PCR amplification of complementarity-determining region 3 (CDR3) of the immunoglobulin heavy-chain variable region (IGHV) gene can be used to assess clonality of B-cell populations as a supportive diagnostic tool for B-cell neoplasms. Because of the sequence variation and possible somatic hypermutation of the IGHV gene, sensitivity of the PCR-based assay to detect clonal IGHV gene rearrangement largely depends on the sequences and numbers of primer sets. Prior to the development of an efficient assay, we cloned and sequenced 97 IGHV complementary DNAs (48 IGHV-1 and 49 IGHV-3 clones) from normal cat spleens. On the basis of these sequences, we designed 6 forward primers at the variable region and 5 reverse primers at the joining region. Using each of 6 forward primers and a mixture of 5 reverse primers, we amplified CDR3 of IGHV genes and analyzed the PCR products by conventional PAGE and Genescan analyses using fluorescence-labeled primers. Twenty-six feline B-cell neoplasms diagnosed by histopathological and immunohistochemical examinations were subjected to the newly developed analysis of IGHV gene rearrangement. Clonal IGHV gene rearrangement was detected in 22 of 26 (84%) samples by both PAGE and Genescan analyses. To reduce the number of PCR reactions, we constructed a multiplex PCR analysis system using a mixture of IGHV-1- and IGHV-3-specific primers as forward primers and a mixture of 5 joining region reverse primers. Results of the multiplex PCR were 100% concordant with those obtained by each of the singleplex PCRs. The multiplex PCR-based assay and Genescan analysis developed in the present study would be useful and practical tools to detect clonal IGHV gene rearrangement in feline B-cell neoplasms., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Clinical application of genetic clonality analysis by PCR has been available in feline lymphoma. However, it is in under dispute whether the analysis can be applied for cellular lineage determination. To evaluate utility of the analysis for lineage determination, the results between immunohistochemistry and the genetic analysis were compared. Based on the result with immunohistochemistry, sensitivity of the primer for IgH was 89% and specificity was 75%. Sensitivity of the primer for TCRγ was 25% and specificity was 100%. Cross-lineage IgH rearrangement was found in 25% of T-cell lymphomas. The present study suggests that the genetic analysis had better be applied as not a definite but an adjunctive tool for lineage determination in feline lymphomas.

We established a novel feline B-cell line, MS4, from the neoplastic pleural effusion of a cat with cutaneous B-cell lymphoma. Immunophenotype staining of the MS4 cells was positive for CD20, CD79α, and IgA and negative for CD3, CD4, CD5, CD8α, CD18, CD21, CD22, IgM, IgG, Ig light chain, and MHC class II. PCR analysis for immunoglobulin heavy chain gene rearrangements revealed a monoclonal rearrangement, whereas no clonal rearrangement of the T-cell receptor γ gene was detected. Southern blotting with an exogenous feline leukemia virus (FeLV) U3 probe revealed no integration of exogenous FeLV provirus. The MS4 cell line is the first FeLV-negative feline B-cell lymphoma cell line, and may be used to investigate the pathogenesis of spontaneously occurring feline lymphoma and the development of new therapies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A 10-year-old American Shorthair cat with nasal discharge, anorexia, and weight loss was found to have pancytopenia and hyperproteinaemia. Bone marrow aspiration revealed atypical plasma cells that totalled 50% of the nucleated bone marrow cells. The number of atypical plasma cells progressively increased in the peripheral blood during the observation period of 64 days. The cat did not respond to treatments with melphalan, chlorambucil, and prednisolone, and died 71 days after the initial presentation. Clinical, cytological, histopathological, and immunohistochemical findings in this case supported the diagnosis of myeloma-related disorder (MRD) with leukaemic progression., (Copyright © 2010 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.)

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 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.

A 10-year-old Japanese domestic cat was presented with anorexia and weight loss. Severe anemia and thrombocytopenia were detected. Abdominal radiography and ultrasonography revealed the presence of multiple masses in the spleen. Cytological analyses of the masses revealed several atypical histiocytic cells and considerable hemophagocytosis. A splenectomy was performed, and the mass was diagnosed as histiocytic sarcoma on the basis of histopathological, cytochemical and immunohistochemical analyses. Further, abnormal hemophagocytosis was observed in the bone marrow. The cat was administered prednisolone and lomustine, and it survived for 107 days after admission. An autopsy revealed the presence of neoplastic histiocytic cells in the bone marrow, liver, pancreatic lymph node and glomeruli. This is the first case of histiocytic sarcoma in a cat to be reported in Japan.

Time-course changes in the concentration of serum amyloid A (SAA), a major acute phase protein, were measured in a cat with pancreatitis over an 831-day period and compared with changes in WBC count and feline trypsin-like immunoreactivity (fTLI). SAA concentration was increased at the onset of the disease and gradually decreased over 5 days of treatment with an improvement in the clinical condition. In contrast, fTLI concentration and WBC count were not increased at the onset of the disease but increased gradually during the 5 days of treatment. Long-term monitoring from days 68 to 831 revealed a good correlation between SAA concentration and the reoccurrence of clinical signs in the cat; however, WBC count did not increase even with the exacerbation of disease. These findings suggest that the SAA concentration may be a useful marker for evaluating response to treatment and disease exacerbation in feline pancreatitis.

CXC-chemokine receptor 4 (CXCR4) functions as a receptor for feline immunodeficiency virus (FIV). Although we previously found that a CXCR4 antagonist, T140, inhibited the FIV replication in vitro, it was not effective in cats infected with FIV because of its low stability in feline serum. To resolve this problem, several T140 derivatives have been developed. Here, we examined the efficacy of T140 analogs, TF14016 and TF14013, on the inhibition of FIV infection. These compounds were shown to significantly inhibit the syncytia formation in CXCR4-expressing cells after co-cultivation with FIV-infected cells and the replication of FIV in a feline lymphoid cultured cell line. These results indicated that TF14016 and TF14013 could be useful as antiviral drugs for cats infected with FIV.

Two cats showing chronic vomiting, diarrhea and weight loss were found to have leukocytosis with marked eosinophilia. Both cats were diagnosed with hypereosinophilic syndrome by the findings of increased eosinophils and their precursors in the bone marrow, eosinophilic infiltration into multiple organs, and exclusion of other causes for eosinophilia. Although cytoreductive chemotherapy with hydroxycarbamide and prednisolone was performed, these two cats died 48 days and 91 days after the initial presentation.

Feline leukemia virus (FeLV), which is subclassified into three subgroups of A, B and C, is a pathogenic retrovirus in cats. FeLV-A is minimally pathogenic, FeLV-C can cause pure red cell aplasia, and FeLV-B is associated with a variety of pathogenic properties such as lymphoma, leukemia and anemia. FeLV-induced neoplasms are caused, at least in part, by somatically acquired insertional mutagenesis in which the integrated provirus may activate a proto-oncogene or disrupt a tumor suppressor gene. The common integration sites for FeLV have been identified in six loci with feline lymphomas: c-myc, flvi-1, flvi-2 (contains bmi-1), fit-1, pim-1 and flit-1. Oncogenic association of the loci includes that c-myc is known as a proto-oncogene, bmi-1 and pim-1 have been recognized as myc-collaborators, fit-1 appears to be closely linked to myb, and flit-1 insertion is shown to be associated with over-expression of a cellular gene, e.g. ACVRL1. Thus, identification of common integration sites for FeLV is a tenable model to clarify oncogenesis. Recent advances in molecular biology and cytogenetics have developed to rapidly detect numbers of retroviral integration sites by genome-wide large-scale analyses. Especially, polymerase chain reaction (PCR)-based strategies and chromosome analyses with fluorescence in situ hybridization (FISH) will be applicable for studies on FeLV.

RNA interference (RNAi) is a process in which double-stranded RNA induces the post-transcriptional sequence-specific degradation of homologous messenger RNA. The present study was carried out to apply the RNAi technology to inhibit the replication of feline immunodeficiency virus (FIV). Four small interfering RNAs (siRNAs) homologous to the FIV gag gene were synthesized and transfected into a feline fibroblastic cell line chronically infected with FIV (CRFK/FIV). These synthetic siRNAs efficiently inhibited the replication of FIV. Next, we examined the effect of retroviral vector-mediated transfer of FIV-specific short hairpin RNA (shRNA) on the replication of FIV in a feline T-cell line chronically infected with FIV (FL4). The retroviral vector-mediated transfer of FIV-specific shRNA was shown to markedly inhibit the replication of FIV in the FL4 cells. These results provide useful information for the development of RNAi-based gene therapy strategy to control FIV infection.

Transforming growth factor-beta1 (TGF-beta1), an inflammatory cytokine, plays a role in tissue fibrosis, such as glomerular sclerosis and tubulointerstitial fibrosis of the kidneys. In the present study, the urinary TGF-beta1 level of cats diagnosed with chronic renal failure (CRF) was measured to investigate its relationship to the pathogenesis of feline CRF. Urinary TGF-beta1 levels (TGF-beta1/creatinine ratio) were significantly increased compared with healthy controls, whereas serum levels of TGF-beta1 were not. These results indicate that TGF-beta1 is expressed in the kidneys of CRF cats, and that it was reflected in the urinary TGF-beta1 level. Therefore, TGF-beta1 may play a role in feline CRF, and urinary TGF-beta1 could be used as a clinical marker for renal fibrosis.

Eight new feline mammary adenocarcinoma cell lines derived from either primary or metastatic lesions were established. The morphology of all the cell lines was epithelioid and round to spindle in shape, with cell growth occurring in a monolayer fashion. On immunohistochemistry, these cells reacted with anti-keratin and anti-vimentin antisera. The doubling time of these cells was between 19 and 54 hr. Tumor masses were developed in nude mice by subcutaneous inoculation of the cells that were histologically identical to their original mammary tumor lesions. Telomerase activities measured using the telomeric repeat amplification protocol assay revealed high telemetric activity in all of the cells.

Tumors at the cranial base in 2 cats (a 9 1/2-year-old, castrated male Chinchilla and a 7-year-old, castrated male American Shorthair) were diagnosed as malignant craniopharyngioma. The tumor lesion was histopathologically divided into four parts: 1) a small acinus part, in which relatively large cells with a pale cytoplasm composed small acini; 2) a duct part, in which small cuboidal cells composed ducts; 3) a cyst part, in which there were large cysts lined with flat cells; and 4) a pavement part, in which large multi-angular-shaped cells proliferated in a pavement pattern. The epithelial cells of some parts were positive for keratin by immunohistochemistry. Histopathologic findings of the present feline cases were identical to those of malignant craniopharyngioma in other animal species.

A feline lymphoblastoid cell line (KO-1) was established from a 5-year-old neutered female cat with naturally occurring thymic lymphoma. KO-1 cells had a rearrangement of T-cell receptor beta-chain gene and a germ-line configuration of immunoglobulin heavy chain gene, however, they were devoid of T-cell-specific surface phenotype. Cytogenetically, KO-1 cells showed a hyperploidy (2n = 41) due to the trisomy of B2, F2 and X chromosomes. Although KO-1 cells were shown to be clonally expanded cells integrated with feline leukemia virus (FeLV) proviruses and expressed its structural proteins in their cytoplasm, they did not produce virus particles as shown by transmission electron microscopy and the absence of the viral protein and reverse transcriptase activity in the culture supernatant. The present study showed that the KO-1 cell line established here was a feline T-cell lymphoma cell line having a unique characteristic as an FeLV nonproducer.

One of the mechanisms of eosinophil infiltration is its induction by chemoattractants such as regulated upon activation, normal T-expressed and secreted (RANTES) which is a cysteine-cysteine chemokine that mediates chemotaxis and activation of eosinophils in humans and mice. Skin lesions of feline eosinophilic plaque are characterized by a predominant infiltration of eosinophils. The mechanism(s) of eosinophilic infiltration in the skin and/or mucosa of cats is unknown. It is possible that RANTES is involved. To investigate the presence of RANTES in the skin of cats with eosinophilic plaques and nonaffected skin, we cloned and sequenced the full-length feline RANTES cDNA gene, in order to determine whether it is present in the skin of cats with eosinophilic plaques and/or if it is present in normal adjacent skin. We were able to document the the expression of RANTES mRNAs in skin with feline eosinophilic plaque as well as in normal cat skin. The full-length cDNA sequence of the RANTES gene (742 bp) contained a single open reading frame of 276 bp encoding a protein of 92 amino acids. The amino acid sequence of feline RANTES shared 67 and 74% sequence identity with that of bovine and mouse RANTES genes, respectively. RT-PCR analysis on RANTES mRNA in the skin of cats with eosinophilic plaque revealed that its expression was higher in the eosinophilic plaque skin lesions than in the normal skin. The result suggested that RANTES might play a role to induce eosinophil infiltration in feline eosinophilic plaque lesions.

Thymus and activation-regulated chemokine (TARC) is a member of CC chemokine and plays an essential role in recruitment of CC chemokine receptor 4 positive Th2 cells to allergic lesion. To investigate the association of TARC in allergic inflammation of cats, a TARC cDNA was cloned from feline thymus by RT-PCR with 3' rapid amplification of cDNA ends (RACE) method. The feline TARC clone contained a full length open reading frame encoding 99 amino acids which shared 80.8%, 72.5%, 65.6% and 67.8% homology with dog, human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lung, lymph node, kidney, small intestine, colon and skin of the normal cat tissues examined. Furthermore, it was found that TARC mRNA was strongly expressed in lesional skin of cats with eosinophilic plaque. The present results demonstrated that TARC might be involved in the pathogenesis of eosinophilic plaque in cats.

A polymerase chain reaction assay was developed to detect Cryptococcus neoformans in biopsy samples. The assay detects the CAP59 gene of Cryptococcus neoformans and was used to substantiate cutaneous cryptococcosis in a 5-year-old cat submitted to the Veterinary Medicine Center at the University of Tokyo.

We investigated the hematologic abnormalities and prognoses in 16 cats with myelodysplastic syndromes (MDS). Nonregenerative anemia, thrombocytopenia, and neutropenia were observed in 15, 13, and 4, respectively, of the 16 cats with MDS. Morphologic abnormalities characteristic of MDS included megaloblastoid rubricytes (9 cats), hyposegmentation of neutrophils (7 cats), nuclear abnormality of rubricytes (10 cats) and neutrophils (13 cats), and micromegakaryocytes (10 cats). Disease in these 16 cats was subclassified into refractory anemia (RA; 8 cats), RA with excess of blasts (RAEB; 5 cats), RAEB in transformation (RAEB in T; 1 cat), and chronic myelomonocytic leukemia (CMMoL; 2 cats), according to the human French-American-British (FAB) classification. In the cats in which the clinical outcome was known, 3 of 6 cats with high blast cell count MDS, including RAEB, RAEB in T, and CMMoL, developed acute myeloid leukemia, but only 1 of 8 cats with low blast cell count MDS (RA) developed acute myeloid leukemia. Based on the Dusseldorf scoring system for the prognosis of human MDS, the survival times of the cats showing high scores (> or =3 points) were significantly shorter than those of the cats with low scores (<3 points). The FAB classification and Dusseldorf scoring system were considered to be useful for predicting the prognosis of feline MDS. Furthermore, 15 of the 16 cats with MDS in this study were infected with feline leukemia virus, indicating its possible etiologic role in the pathogenesis of feline MDS.

Feline myelodysplastic syndromes (MDS) has been diagnosed in many cats infected with feline leukemia virus, although the pathogenesis of this hematopoietic deficiency has been unclear. In this study, we assayed the bone marrow erythroid colony-forming units (CFU-E) and granulocyte-machrophage CFUs (CFU-GM) to investigate the pathogenesis of feline MDS. The number of CFU-E colonies was decreased in 4 of 7 cats with MDS, and the number of CFU-GM colonies was also decreased in 4 cats. Furthermore, small colonies of CFU-GM were found in all 7 cases. These findings indicated that refractory cytopenia of feline MDS could be caused by abnormal maturation and differentiation of hematopoietic stem cells in bone marrow, as it is in human MDS. The pathogenesis of feline MDS might be similar to that of human MDS.

A cat showing seasonal allergic symptoms of rhinitis was examined for reactivities to Japanese cedar (Cryptomeria japonica, CJ) pollen allergen by intradermal skin test (IDST), Prausnitz-Kustner (P-K) test, and lymphocyte blastogenic response. In IDST for 26 common allergens. the cat showed a positive reaction to CJ pollen allergen. P-K test using CJ pollen allergen also showed a positive reaction, indicating the presence of serum IgE specific to CJ pollen. In the lymphocyte blastogenic response, the stimulation index in the presence of CJ pollen allergen was 2.4. These data suggested that the seasonal rhinitis observed in the cat was caused by the sensitization to CJ pollen allergen.