Full-Genome Sequences of Two Newcastle Disease Virus Strains Isolated in West Java, Indonesia.

The full-genome sequences of strains chicken/Indonesia/Cilebut/010WJ/2015 and chicken/Indonesia/ITA/012WJ/1951, isolated in West Java, Indonesia, in 2015 and 1951, respectively, were examined. Chicken/Indonesia/Cilebut/010WJ/2015 (genotype VII) caused a 2015 disease outbreak in Indonesia, and chicken/Indonesia/ITA/012WJ/1951 (genotype VI) is used as a standard strain for challenge in Newcastle disease virus (NDV) vaccine trials.

A novel quantitative polymerase chain reaction to monitor urinary tract mycoplasma infection in a dog.

The aim of the study was to develop a quantitative real-time PCR assay for diagnosis and monitoring of mycoplasma urinary tract infections (UTI) in a dog. An English Cocker Spaniel dog with the history of urinary tract infection was physically examined and laboratory findings identified chronic renal insufficiency and urinary tract infection. Attempts to culture organisms from pyuric urine failed, and empirical antibiotic therapy did not resolve the pyuria. A mycoplasma species most closely resembling Ureaplasma canigenitalium was identified in urine samples by conventional PCR and sequencing. A quantitative PCR method was developed to monitor and finally verify successful treatment. This novel approach to monitoring mycoplasma urinary tract infections is conceptually simple, and provides rapid results. It may have wider application in monitoring treatment efficacy for infections with other Mycoplasma spp. as well as additional organisms that are difficult to culture. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we highlight two different findings, detection of Ureaplasma canigenitalium in a dog with chronic urinary tract infection and development of a quantitative real-time PCR test to track treatment results in an infected dog. This report is the first report of detection of U. canigenitalium in one dog in Australia. This novel qPCR method for monitoring mycoplasma urinary tract infections is conceptually simple and provides results fast. It will have wider applications in monitoring treatment efficacy for infections with mycoplasmas and mycoplasma-like organisms that are difficult to culture, and provides a sensitive guide to treatment progress.

Prevalence of exon 11 internal tandem duplications in the C-KIT proto-oncogene in Australian canine mast cell tumours.

OBJECTIVE: To measure the prevalence of internal tandem duplications (ITDs) in exon 11 of the proto-oncogene C-KIT in a sample of Australian cutaneous canine mast cell tumours (MCTs) drawn from general practice and to evaluate relationships between tumour mutation status and prognostic factors including signalment, tumour histological grade, tumour anatomical location and tumour size. METHODS: C-KIT exon 11 ITDs were detected by PCR in DNA extracted from formalin-fixed, paraffin-embedded canine MCTs sourced from three veterinary diagnostic laboratories in Adelaide and Melbourne. Tumours were graded according to two different systems (Patnaik and Kiupel systems) by board-certified anatomical pathologists blinded to the PCR results. Relationships between tumour mutation status and prognostic factors were evaluated using a generalised binary logistic regression analysis. RESULTS: ITDs were identified in 13 of 74 cutaneous canine MCT samples, giving an overall prevalence of 17.6% (95% confidence interval: 8.9-26.2%). ITDs were detected in 10 of 18 Patnaik grade III MCTs (55.6%) and 11 of 22 Kiupel high-grade MCTs (50%). Wald chi-square analysis revealed that detection of tumour ITDs was significantly associated with both Patnaik's and Kiupel's histologic grading systems (each: P < 0.001). The presence of the ITDs in MCTs was not associated with signalment, tumour anatomical location or tumour size. CONCLUSION: The prevalence of C-KIT exon 11 ITDs in Australian canine MCTs is similar to the prevalence in overseas canine populations (overall prevalence in Australia approximately 18%). ITDs were more frequently identified in higher grade MCTs.

Genome plasticity in the mouse oocyte and early embryo.

In dissecting the molecules and molecular mechanisms that control mammalian oocyte-to-embryo transition, we found abundant transcripts representing developmentally regulated ERVs (endogenous retroviruses) in mouse oocyte and two-cell stage embryo cDNA libraries. These retrotransposons can act as alternative promoters and first exons for diverse genes, synchronizing their expression. Heritable genetic change due to replication of these retrotransposons probably occurs specifically in oocytes and early embryos. ERVs are usually epigenetically silenced, through DNA methylation and chromatin-based mechanisms. Their activation and silencing indicates a change in the epigenetic state of the genome. The thousands of endogenous retro-elements in the mouse genome provides potential scope for large-scale co-ordinated epigenetic fluctuations and leads to the hypothesis that differential transposable element expression triggers sequential reprogramming of the embryonic genome during the oocyte-to-embryo transition.

Systems biology of the 2-cell mouse embryo.

The transcriptome of the 2-cell mouse embryo was analyzed to provide insight into the molecular networks at play during nuclear reprogramming and embryonic genome activation. Analysis of ESTs from a 2-cell cDNA library identified nearly 4,000 genes, over half of which have not been previously studied. Transcripts of mobile elements, especially those of LTR retrotransposons, are abundantly represented in 2-cell embryos, suggesting their possible role in introducing genomic variation, and epigenetic restructuring of the embryonic genome. Analysis of Gene Ontology of the 2-cell-stage expressed genes outlines the major biological processes that guide the oocyte-to-embryo transition. These results provide a foundation for understanding molecular control at the onset of mammalian development.

MRP1 gene expression level regulates the death and differentiation response of neuroblastoma cells.

We have previously reported a strong correlation between poor prognosis in childhood neuroblastoma (NB) patients and high-level expression of the transmembrane efflux pump, Multidrug Resistance-associated Protein (MRP1), in NB tumour tissue. In this study, we inhibited the endogenous expression of MRP1 in 2 different NB tumour cell lines by stably transfecting an MRP1 antisense expression vector (MRP-AS). Compared with control cells, MRP-AS transfectant cells demonstrated a higher proportion of dead and morphologically apoptotic cells, spontaneous neuritogenesis, and, increased synaptophysin and neurofilament expression. Bcl-2 protein expression was markedly reduced in MRP-AS cells compared to controls. Conversely, we found that the same NB tumour cell line overexpressing the full-length MRP1 cDNA in sense orientation (MRP-S) demonstrated resistance to the neuritogenic effect of the differentiating agent, all-trans-retinoic acid. Taken together, the results suggest that the level of MRP1 expression in NB tumour cells may influence the capacity of NB cells for spontaneous regression in vivo through cell differentiation and death.

Efficacy of doxorubicin as an induction agent for cats with lymphosarcoma.

OBJECTIVE: To determine the efficacy of doxorubicin when used alone in inducing remission in cats with lymphosarcoma. DESIGN: Prospective multi-institutional study of naturally occurring disease. METHODS: Cases were accrued from veterinary institutions in Australia and New Zealand after obtaining consent from informed owners. Cats were treated with doxorubicin every 3 weeks for three treatments. If there was no response to the first dose of doxorubicin or if the cat relapsed during the doxorubicin regimen, the cat was withdrawn from the trial and either euthanased or treated with other agents. Age, breed, gender and anatomic site of the lymphosarcoma (multicentric, alimentary, mediastinal, extranodal) were recorded for each cat. Clinical remission was assessed before each treatment by physical examination, radiography, ultrasonography and computed tomography where appropriate. Complete remission was defined as the disappearance of all clinical signs and clinically detectable tumour. RESULTS: Twenty-one cases were accrued over a 2-year-period but only 19 were available for data analysis. Young Siamese cats were over-represented and all cats with mediastinal tumours were young Siamese. There was a significant difference between the mean ages of cats with mediastinal or multicentric lymphosarcoma (mean +/- SD: 3.5 +/- 3.0 and 4.3 +/- 2.6 years, respectively) and cats with alimentary or extranodal LSA (11.4 +/- 0.9 and 11.0 +/- 0.9 years, respectively). Of 19 cats treated with doxorubicin alone, 6 (32%) had complete remission, 6 (32%) had partial remission and 7 (36%) did not respond. CONCLUSIONS: The results suggest that doxorubicin cannot be recommended as a single agent for treatment of feline lymphosarcoma because of the rather poor remission rate achieved.

Absence of MEN2A- or 2B-type RET mutations in primary neuroblastoma tumour tissue.

Specific germline mutations in the RET proto-oncogene predispose to the familial cancer syndromes: multiple endocrine neoplasia (MEN) types 2A and 2B, and familial medullary thyroid carcinoma. Expression of the RET receptor tyrosine kinase is tightly restricted to tumours of neural crest origin, such as neuroblastoma, and neuroblastoma has been observed in RET transgenic mice. Neuroblastoma tumour cell lines transfected with the MEN2A RET gene exhibit spontaneous neuritic differentiation, whereas MEN2B-type RET transfectants demonstrate altered cell adhesion and enhanced metastatic potential. In this study, the authors examined genomic DNA from 26 primary neuroblastoma tumours for MEN2A and MEN2B RET mutations, using restriction enzyme digestion of polymerase chain reaction products as an alternative to direct sequencing. Examination of RET exons 10 (codons 611, 618, 620), 11 (codons 632, 633, 634) and 16 (codon 918) in all 26 tumours revealed no RET mutations. Taken together these data suggest that abnormalities of the RET signalling pathway, rather than oncogenic, MEN2-type RET activation by mutation, may play a role in neuroblastoma tumorigenesis.

Expression of multiple endocrine neoplasia 2B RET in neuroblastoma cells alters cell adhesion in vitro, enhances metastatic behavior in vivo, and activates Jun kinase.

Point mutations, deletions, and recombinations of the RET proto-oncogene are associated with several inherited human diseases of neural crest-derived cells: Hirschsprung's disease, familial medullary thyroid carcinoma, and the multiple endocrine neoplasia (MEN) syndromes, types 2A and 2B. RET expression is restricted to normal and malignant cells of neural crest origin, such as human neuroblastoma cells. To better understand the role of the activated RET oncogene in neural crest cells, we transfected two adherent human neuroblastoma tumor cell lines with oncogenic MEN2 mutant RET cDNAs. Transfectant clones from both cell lines overexpressing MEN2B RET demonstrated a marked increase in the cell fraction growing in suspension. Both control and MEN2B cells formed tumors at the site of injection in all cases. However, mice injected with MEN2B cells developed lung metastases at a much higher frequency than control mice. Only RET protein derived from MEN2A transfectant cells had increased autokinase activity, whereas MEN2B transfectant cells demonstrated selective activation of the mitogen-activated protein kinase, Jun kinase-1 (Jnk1). These results indicate a biochemical signaling pathway that may link oncogenic RET with the metastatic process.

Retrospective study of 60 cases of feline lymphosarcoma.

OBJECTIVE: To characterise epidemiological and clinical findings, and diagnostic procedures undertaken, in cats with lymphosarcoma at a veterinary teaching hospital. DESIGN: Retrospective case study. PROCEDURE: Hospital records were reviewed for 7159 cats, sick or healthy, examined during a 10-year period (1984 to 1994). Sixty cats with lymphosarcoma were identified and classified by anatomical location of the tumor. Data on breed, age, sex, clinical signs and diagnostic procedures were collated. RESULTS: The prevalence of feline lymphosarcoma in the hospital population was 0.84%. Siamese cats appeared predisposed to lymphosarcoma but other purebreds were not. Males were somewhat overrepresented amongst affected cats. Similar numbers of cases (12 to 18) were seen in each of the four anatomic categories (multicentric, mediastinal, alimentary and extranodal). Cats with mediastinal lymphosarcoma were mostly young and Siamese. Clinical signs in affected cats were varied, usually multiple and often nonspecific. Two of 22 cases tested positive for feline leukaemia virus antigen in blood and 6 of 13 were positive for feline immunodeficiency virus antibody. CONCLUSIONS: Extranodal lymphosarcoma seemed more prevalent in this study than reported elsewhere. Siamese cats in the study population may have had a genetic predisposition to lymphosarcoma. Limited evidence suggested feline leukaemia virus may be less important, and feline immunodeficiency virus more important, in the local population than indicated in overseas reports. Additional studies are needed to investigate breed predisposition and feline leukaemia virus and feline immunodeficiency virus status in Australian cats with lymphosarcoma.

Visceral mast cell tumour with eosinophilia and eosinophilic peritoneal and pleural effusions in a cat.

A cat with weight loss, pyrexia and recurrent lethargy and depression was found to have pleural and peritoneal eosinophilic effusions, peripheral eosinophilia, eosinophilic lymphadenitis and a massively enlarged mesenteric lymph node. Visceral mast cell neoplasia was diagnosed after histopathological examination of a biopsy of the mass. Palliative chemotherapy was attempted unsuccessfully and the cat was euthanased.

Irradiation of nonlymphoproliferative neoplasms of the nasal cavity and paranasal sinuses in 16 cats.

Sixteen cats with malignant tumors (10 carcinomas, 6 sarcomas) of the nasal cavity and paranasal sinuses were treated with curative intent by radiotherapy. Clinical stating was based on radiographic findings, using the tumor, node, metastasis classification system of the World Health Organization. Irradiation was done with a telecobalt-60 unit (13 cats) and an orthovoltage unit (3 cats). Fourteen cats were treated with irradiation alone, and 2 cats had incomplete surgical resections prior to radiotherapy. Treatment dose was 48 Gy (minimum tumor dose), administered by use of 4 Gy per fraction on a Monday/Wednesday/Friday basis over 4 weeks. Survival times after treatment ranged from 1 to 36 months. The 1- and 2-year overall survival rates were 44.3 and 16.6%, respectively. Histologic type and clinical stage did not have prognostic value. Most acute radiation reactions were mild and self-limiting. Chronic ocular complications were seen in 3 cats. These treatment responses compared favorably with those previously described in dogs and cats with intranasal neoplasms treated with teletherapy and provided a perspective for comparison of new treatment methods.

Photodynamic therapy for nasal and aural squamous cell carcinoma in cats.

Eighteen random-bred cats with a total of 19 nasal or aural squamous cell carcinomas were treated with photodynamic therapy, using aluminum phthalocyanine tetrasulfonate as the photosensitizer. Cats were irradiated at power densities of 100 mW/cm2 and energy densities of 100 J/cm2. Successful outcome was obtained in 10 tumors after 1 treatment, and 2 more tumors had complete responses after 1 or 2 additional treatments. Treatments were more effective in tumors of stage T2 or earlier. Five tumors had partial responses, and the response of 2 tumors could not be evaluated. The treatment was safe and well tolerated by most cats, although we found that cats should be kept out of sunlight for 2 weeks after treatment.

Evaluation of commercially available antibodies to cytokeratin intermediate filaments and laminin in normal cat pinna.

The pattern of distribution of cytokeratin (CK) intermediate filaments can be used to characterize subsets of epithelial tissues. The purpose of the study was to examine the CK expression of feline pinna skin. Six normal feline pinnae were routinely processed in formalin. An immunohistochemical method was used to stain the pinnae with 8 commercially available anti-human CK antibodies (Abs) (PKK1, CAM 5.2, UCD 10/11, 35BH11, 34BE12, AE1/AE3, MAK 6, A575) and an anti-human laminin Ab. All the CK Abs selectively localized to epithelium except 35BH11, which did not react with any part of the pinna. Some epithelial subsets were identified by their unique staining pattern with CK Abs. Basal cells but not suprabasal cells of the epidermis stained with PKK1; basal but not lumenal cells of apocrine glands stained with 34BE12. Apocrine glands stained with all CK Abs except 35BH11. All epithelial structures were stained with A575. Basal lamina of epithelial and mesenchymal tissues was clearly identified by the anti-laminin Ab. The results indicate that in cat pinna some commercially available anti-human CK Abs selectively stain subsets of epithelium and adnexa. PKK1, 34BE12, and A575 were the CK Abs with the most consistent staining patterns, the other Abs stained more variably from pinna to pinna. The pattern of epithelial and adnexal staining was similar but not identical to that reported for humans.

Combined chylothorax, chylopericardium, and cranial vena cava syndrome in a dog with thymoma.

A dog was examined because of anorexia and development of submandibular, sternal, and forelimb edema. Physical examination revealed engorged jugular veins and engorged blood vessels of the conjunctivae and nictitating membranes. Thoracic radiography revealed pleural and pericardial effusions, later identified as chyle. Contrast angiography revealed an intravascular mass, later identified as thymoma, in the cranial vena cava.