MRI of osteosarcoma metastases in the brain of an old English Sheepdog.

A 9-year-old, male neutered old English Sheepdog was presented for further investigation of altered mentation, impaired vision, and hemineglect syndrome of 10 days duration. An MRI study of the brain revealed multifocal, contrast-enhancing intra-axial lesions that had a stippled hypointense appearance in all sequences but lacked evidence of a strong signal void on T2* images. Histological examination of the brain postmortem confirmed the lesions were metastases arising from an osteosarcoma, which was later identified in the right humerus. To the authors' knowledge, these MRI characteristics of osteosarcoma metastases in the canine brain have not been previously reported.

Nonocular Melanocytic Neoplasia in Cats: Characterization and Proposal of a Histologic Classification Scheme to More Accurately Predict Clinical Outcome.

Nonocular melanocytic neoplasia is considered uncommon in cats yet is routinely encountered in diagnostic pathology and recognized to exhibit a wide variation in biological behavior. Accurate prediction of clinical outcomes is challenging with no widely recognized prognostic criteria. Signalment and tumor location were retrospectively evaluated in 324 cats diagnosed with nonocular melanocytic neoplasia. Histologic features were described in 141 neoplasms and outcome data were available in 79 cases. Immunohistochemistry using Melan-A, PNL-2, cyclooxygenase 2 (COX-2), and E-cadherin was performed in a subset (n = 24). Multivariate analysis identified tumor site, mitotic count, and the presence of intratumoral necrosis to be independent predictors of tumor-related death. On the basis of these findings, we propose a novel histologic grading scheme in which nonocular melanocytic neoplasms involving the lips, oral or nasal mucosa, or nasal planum are considered high grade if they fulfill 1 or both of the following criteria: at least 4 mitoses in 10 high-power fields (HPF) or presence of intratumoral necrosis; those arising elsewhere are considered high grade if they fulfill both of the above criteria. Of 79 tumors with outcome data, 43 (54%) were low grade and 36 (46%) were high grade. The grading system had an 80% sensitivity and 92% specificity for predicting tumor-related death in this population of cats. Median survival for cats with low-grade tumors was not reached, and the median survival was 90 days for those with a high-grade tumor. PNL-2 and Melan-A were sensitive markers for feline nonocular melanocytic neoplasia, and although not significantly associated with prognosis, a large proportion expressed COX-2, suggesting a potential therapeutic role for COX-2 inhibitors.

Canine ovarian epithelial tumours: histopathological and immunohistochemical evaluation with proposed histopathological classification system.

Canine ovarian epithelial tumours (OETs) are currently divided into ovarian adenomas and carcinomas, which are further inconsistently subclassified as papillary or cystic, whereas in human medicine, OETs are subdivided into several subtypes. This study aimed to establish clear morphological features enabling more consistent distinction between benign OETs and ovarian carcinomas (OvCas) as well as defining different histopathological patterns of canine OvCas. Analysis revealed a mitotic count threshold of >2 as a potential criterion for differentiating OvCas from benign OETs. Alongside ovarian adenomas, ovarian borderline tumours were introduced as a distinct category among benign OETs. OvCas exhibited five different histopathological patterns, namely papillary, solid with tubular differentiation, micropapillary, cystic and sarcomatous. Since some OvCas can morphologically overlap with other ovarian tumours, the expression of cytokeratin 7, a cytokeratin expressed in ovarian epithelium, was assessed and proved helpful, although it was not expressed in all cases. Furthermore, we investigated the expression of 14-3-3σ and cyclooxygenase 2 (COX-2). Based on the frequent expression of 14-3-3σ, this marker appears to have a role in canine OETs since it is not expressed in normal canine ovaries. The infrequent expression of COX-2 suggests that it is a poor candidate as a potential therapeutic target in canine OvCas.

CD117 expression in canine ovarian tumours.

Canine ovarian cancer poses a significant diagnostic and therapeutic challenge. The heterogeneous nature of ovarian tumours makes accurate histological identification difficult, whilst treatment is limited to surgical excision. The tyrosine kinase receptor CD117 is neo-expressed in many tumours and represents a potential diagnostic and prognostic biomarker and therapeutic target. This study aimed to establish if CD117 is neoexpressed in canine ovarian tumours. Immunohistochemistry was employed to assess expression of CD117 in 29 canine ovarian tumour samples. CD117 labelling was assessed with a semiquantitative immunoreactivity score, and the location of labelling was recorded as membranous, focal cytoplasmic or diffuse cytoplasmic. Histological morphology was assessed and used to assign subgroups based on growth pattern. Cytokeratin 7 labelling was used to indicate the tumour type as epithelial or sex-cord stromal in origin. Mitotic index, percentage of necrosis and vascular invasion were also assessed and evaluated for association with CD117 expression. Overall, 81% of ovarian tumours neoexpressed CD117 and normal ovarian tissue did not express CD117. Positive immunolabelling was seen in a subset of cells in both ovarian carcinomas (n = 20) and ovarian granulosa cell tumours (n = 3). There was no association between CD117 expression and patient age, histological subtype, mitotic index, percentage of necrosis or vascular invasion. This is the largest study to identify the expression of CD117 in canine ovarian tumours, but further research is needed to elucidate its prognostic and therapeutic value.

Genetic diversity of Streptococcus equi subsp. zooepidemicus and doxycycline resistance in kennelled dogs.

The genetic diversity and antibiotic resistance profiles of 38 Streptococcus equi subsp. zooepidemicus isolates were determined from a kennelled canine population during two outbreaks of hemorrhagic pneumonia (1999 to 2002 and 2007 to 2010). Analysis of the szp gene hypervariable region and the 16S-23S rRNA intergenic spacer region and multilocus sequence typing (MLST) indicated a predominant tetO-positive, doxycycline-resistant ST-10 strain during 1999 to 2002 and a predominant tetM-positive doxycycline-resistant ST-62 strain during 2007 to 2010.

Identification of three novel superantigen-encoding genes in Streptococcus equi subsp. zooepidemicus, szeF, szeN, and szeP.

The acquisition of superantigen-encoding genes by Streptococcus pyogenes has been associated with increased morbidity and mortality in humans, and the gain of four superantigens by Streptococcus equi is linked to the evolution of this host-restricted pathogen from an ancestral strain of the opportunistic pathogen Streptococcus equi subsp. zooepidemicus. A recent study determined that the culture supernatants of several S. equi subsp. zooepidemicus strains possessed mitogenic activity but lacked known superantigen-encoding genes. Here, we report the identification and activities of three novel superantigen-encoding genes. The products of szeF, szeN, and szeP share 59%, 49%, and 34% amino acid sequence identity with SPEH, SPEM, and SPEL, respectively. Recombinant SzeF, SzeN, and SzeP stimulated the proliferation of equine peripheral blood mononuclear cells, and tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) production, in vitro. Although none of these superantigen genes were encoded within functional prophage elements, szeN and szeP were located next to a prophage remnant, suggesting that they were acquired by horizontal transfer. Eighty-one of 165 diverse S. equi subsp. zooepidemicus strains screened, including 7 out of 15 isolates from cases of disease in humans, contained at least one of these new superantigen-encoding genes. The presence of szeN or szeP, but not szeF, was significantly associated with mitogenic activity in the S. equi subsp. zooepidemicus population (P < 0.000001, P < 0.000001, and P = 0.104, respectively). We conclude that horizontal transfer of these novel superantigens from and within the diverse S. equi subsp. zooepidemicus population is likely to have implications for veterinary and human disease.

Sequence analysis of divergent canine coronavirus strains present in a UK dog population.

Forty faecal samples were tested by RT-PCR using coronavirus consensus primers to determine faecal shedding of canine coronavirus (CCoV) and canine respiratory coronavirus (CRCoV) in a dog population housed at a rescue centre. Seven samples were positive for CCoV while all samples were negative for CRCoV. Sequence analysis of five CCoV strains showed a high similarity with transmissible gastroenteritis virus (TGEV) at the N-terminus of the spike protein. All strains contained an open reading frame for the nonstructural protein 7b, which is not present in TGEV, indicating that the strains were related to the previously described CCoV strain UCD-1. Two samples contained CCoV strains with 5' spike sequences most similar to type II CCoV while one sample was found to contain type I CCoV. Primers directed to the N gene allowed specific detection of all CCoV strains analysed in this study. This investigation shows that CCoV strains containing spike proteins similar to TGEV are present in the UK dog population. PCR primers directed to conserved regions of the CCoV genome are recommended for detection of CCoV in clinical samples due to high genetic variability.

Development of a quantitative real-time PCR for the detection of canine respiratory coronavirus.

Canine respiratory coronavirus (CRCoV) has been detected recently in dogs with canine infectious respiratory disease and is involved in the clinical disease complex. CRCoV is a group 2 coronavirus most closely related to bovine coronavirus and human coronavirus OC43. A real-time PCR assay was developed for the detection of CRCoV. The assay was validated against cell culture grown virus and shown to have a high level of sensitivity. A range of tissue samples were collected from dogs at a re-homing centre with a history of endemic respiratory disease. The samples were tested using a conventional nested PCR assay and CRCoV was quantitated by real-time PCR. CRCoV was detected most frequently in the nasal mucosa, nasal tonsil and trachea. It was also detected in the lung, and bronchial lymph node. Of the enteric tissues, only one mesenteric lymph node sample was positive. In addition two colon samples were positive for CRCoV by nested PCR only. In conclusion, CRCoV appears to infect the upper respiratory tract preferentially. The CRCoV real-time PCR assay has proved to be a highly specific and sensitive assay that can be applied for diagnostic purposes as well as to investigate further the tissue tropism of CRCoV.

Strain typing of Mycoplasma cynos isolates from dogs with respiratory disease.

The association of Mycoplasma cynos with canine infectious respiratory disease is increasingly being recognised. This study describes the strain typing of 14 M. cynos isolates cultured from trachea and bronchoalveolar lavage samples of six dogs with respiratory disease, from two separate kennels in the United Kingdom. The genetic similarity of the isolates was investigated using pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD). Most of the isolates from four dogs housed at a re-homing kennel were genetically similar and some isolates from different dogs were indistinguishable by both PFGE and RAPD. These isolates were cultured from dogs with non-overlapping stays in the kennel, which may indicate maintenance of some strains within kennels. A small number of isolates showed much greater genetic heterogeneity and were genetically distinct from the main group of M. cynos strains. There was also a high degree of similarity of the M. cynos type strain (isolated from a dog with respiratory disease in Denmark in 1971) to at least one of the United Kingdom isolates using PFGE analysis, which may suggest possible conservation of pathogenic strains of M. cynos.

Quantification of mRNA encoding cytokines and chemokines and assessment of ciliary function in canine tracheal epithelium during infection with canine respiratory coronavirus (CRCoV).

One of the first lines of defence against viral infection is the innate immune response and the induction of antiviral type I interferons (IFNs). However some viruses, including the group 2 coronaviruses, have evolved mechanisms to overcome or circumvent the host antiviral response. Canine respiratory coronavirus (CRCoV) has previously been shown to have a widespread international presence and has been implicated in outbreaks of canine infectious respiratory disease (CIRD). This study aimed to quantify pro-inflammatory cytokine mRNAs following infection of canine air-interface tracheal cultures with CRCoV. Within this system, immunohistochemistry identified ciliated epithelial and goblet cells as positive for CRCoV, identical to naturally infected cases, thus the data obtained would be fully transferable to the situation in vivo. An assay of ciliary function was used to assess potential effects of CRCoV on the mucociliary system. CRCoV was shown to reduce the mRNA levels of the pro-inflammatory cytokines TNF-alpha and IL-6 and the chemokine IL-8 during the 72 h post-inoculation. The mechanism for this is unknown, however the suppression of a key antiviral strategy during a period of physiologic and immunological stress, such as on entry to a kennel, could potentially predispose a dog to further pathogenic challenge and the development of respiratory disease.

Canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex.

Infectious respiratory disease in dogs is a constant challenge because of the involvement of several pathogens and environmental factors. Canine respiratory coronavirus (CRCoV) is a new coronavirus of dogs, which is widespread in North America, Japan, and several European countries. CRCoV has been associated with respiratory disease, particularly in kenneled dog populations. The virus is genetically and antigenically distinct from enteric canine coronavirus; therefore, specific tests are required for diagnosis.

Displacement of an Ununited Medial Humeral Condylar Ossification Centre in the Cat.

A 2-year-old cat was presented with the complaint of acute-onset non-weight-bearing lameness of the right forelimb. When examined, the cat was of short stature and had multiple joint and cartilaginous abnormalities suggestive of chondrodysplasia. The cause of the acute lameness was radiographically identified as a displaced osseous fragment from the medial portion of the right humeral condyle. The features of the osseous fragment were consistent with an ununited medial condylar ossification centre of the distal humerus. Furthermore, a nondisplaced ununited ossified fragment of similar appearance and size was present in the contralateral elbow. Surgical treatment by excision of the displaced fragment resulted in a preinjury level of limb function in the long-term outcome evaluation.

Acquired urea cycle amino acid deficiency and hyperammonaemic encephalopathy in a cat with inflammatory bowel disease and chronic kidney disease.

CASE SUMMARY: A 5-year-old male neutered Persian cat was referred for investigation of a 4 week history of weight loss, inappetence and intermittent vomiting. Chronic kidney disease (CKD) and inflammatory bowel disease were diagnosed, and despite immunosuppressive therapy and assisted enteral nutrition, the cat experienced persistent anorexia, vomiting and severe weight loss. After 2 additional weeks of treatment, the cat developed acute-onset neurological signs associated with severe hyperammonaemia and was euthanased. Plasma amino acid assessment revealed deficiency of several amino acids involved in the urea cycle, including arginine. RELEVANCE AND NOVEL INFORMATION: To our knowledge, this is the first reported case of an acquired urea cycle amino acid deficiency without nutritional deprivation in a cat. Several contributing factors were suspected, including intestinal malabsorption and CKD. This case demonstrates the importance of urea cycle amino acids in feline metabolism and possible necessity for parenteral supplementation, particularly in the context of persistent weight loss despite adequate enteral nutrition.

Isolation and sequence analysis of canine respiratory coronavirus.

Canine respiratory coronavirus (CRCoV) has frequently been detected in respiratory samples from dogs by RT-PCR. In this report the first successful isolation of CRCoV from a dog with respiratory disease is described. The isolate CRCoV-4182 was cultured in HRT-18 cells but failed to replicate in a number of other cell lines. The nucleotide sequence of the 3'-terminal portion of the CRCoV genome was determined including all open reading frames from the NS2 gene to the N gene. Comparison with other coronavirus sequences showed a high similarity to bovine coronavirus (BCoV). The region between the spike and the E gene was found to be the most variable and was used for phylogenetic analysis of several CRCoV strains. CRCoV-4182 showed a mutation within the non-structural protein region downstream of the S gene leading to the translation of an 8.8 kDa putative protein comprising a fusion of the equivalent of the BCoV 4.9 kDa protein to a truncated version of the BCoV 4.8 kDa protein. The culture of CRCoV will enable analysis of the expression and function of this and other CRCoV proteins as well as allowing the study of the role of CRCoV in the aetiology of canine infectious respiratory disease.

Serological prevalence of canine respiratory coronavirus in southern Italy and epidemiological relationship with canine enteric coronavirus.

Canine respiratory coronavirus (CRCoV) has been detected in dogs suffering from respiratory disease and is thought to be involved in canine infectious respiratory disease (CIRD) complex. Canine enteric coronavirus (CECoV) is a widespread pathogen of dogs, responsible for mild to severe diarrhea in pups. The purpose of this study was to establish the seroprevalence of CRCoV in Italy and its relationship to CECoV type II seroprevalence. The age and year of sample collection from seropositive dogs was also assessed. Of adult domestic dogs, 23.3% had antibodies to CRCoV, compared with 86.1% with antibodies to CECoV. Amongst a population of kenneled pups, 4.0% had antibodies to CRCoV, and 97.0% had antibodies to CECoV.

Serological prevalence of canine respiratory coronavirus.

Canine respiratory coronavirus (CRCoV) has recently been detected in dogs; it is a group 2 coronavirus showing similarity to bovine coronavirus (BCoV) but is distinct from canine enteric coronavirus (CECoV). CRCoV may play an important role in canine infectious respiratory disease (CIRD) either by predisposing to further and potentially more serious viral and bacterial infections or possibly as a primary pathogen. The prevalence of serum antibodies to CRCoV, in a population of dogs in the south east of England, has been shown previously to be 30.1% on the first day of entry to a rehoming kennel [Erles, K., Toomey, C., Brooks, H.W., Brownlie, J., 2003. Detection of a group 2 coronavirus in dogs with canine infectious respiratory disease. Virology 310, 216-223]. The purpose of this study was to establish the prevalence of CRCoV in the general canine population within as well as outside the UK. An ELISA, used to test for the presence of antibodies to CRCoV in canine serum samples, identified seropositive dogs in UK, USA, Canada, Republic of Ireland and Greece. The development of an ELISA based on CRCoV antigen and immunofluorescence assay are described here. 54.7% (547/1000) of North American and 36.0% (297/824) of United Kingdom dogs were seropositive for CRCoV. The age and geographical distribution of seropositive dogs was also assessed. The cross-reactivity demonstrated between CRCoV antibodies from different countries and a UK viral isolate suggests immunological similarity. The overall prevalence of this virus in both North America and the UK suggests that CRCoV has international significance and that further epidemiological studies are required.

Prevalence of Mycoplasma agassizii and Chelonian herpesvirus in captive tortoises (Testudo sp.) in the United Kingdom.

During the months of April to August in 1999 and 2002, oral swabs were collected from 146 tortoises (Testudo sp.) in private collections in the United Kingdom and tested by polymerase chain reaction (PCR) for the presence of Mycoplasma agassizii and Chelonian herpesvirus (ChHV). The presence of M. agassizii was confirmed by restriction digestion of the PCR product. A 307-bp fragment of the ChHV UL5 homologue gene was sequenced and found to show most similarity to equine herpesvirus type 1. A prevalence of 15.8 and 8.2% was found for M. agassizii and ChHV, respectively. Comparison of the carriage of both M. agassizii and ChHV in different species of tortoises correlated the presence of M. agassizii with Testudo horsfieldii and ChHV with Testudo marginata and Testudo graeca iberia. An association of ChHV with stomatitis was also found. Mixed infections with both agents were detected. The findings further demonstrate this pathogen-tortoise association and the cross transmission of these infections if different tortoise species are housed together.

Tropism and pathological findings associated with canine respiratory coronavirus (CRCoV).

Canine infectious respiratory disease (CIRD) occurs frequently in densely housed dog populations. One of the common pathogens involved is canine respiratory coronavirus (CRCoV), however little is known regarding its pathogenesis and the role it plays in the development of CIRD. The pathogenesis of five geographically unrelated canine respiratory coronavirus (CRCoV) isolates was investigated. Following experimental infection in dogs, all five CRCoV isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. The presence of CRCoV was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. Viral shedding was readily detected from the oropharynx up to 10 days post infection, but there was little or no evidence of rectal shedding. The successful re-isolation of CRCoV from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of CRCoV for respiratory tissues and fulfils the final requirement for Koch's postulates. By study day 14 dogs had seroconverted to CRCoV and the antibodies raised were neutralising against both homologous and heterologous strains of CRCoV in vitro, thus demonstrating antigenic homogeneity among CRCoV strains from the two continents. Defining the role that CRCoV and other agents play in CIRD is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. Here we have successfully developed a model for studying the pathogenicity and the role of CRCoV in CIRD.

Streptococcus zooepidemicus: an emerging canine pathogen.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) has caused several outbreaks of haemorrhagic pneumonia in dogs in recent years. This highly contagious and often fatal disease is characterised by sudden onset of clinical signs including pyrexia, dyspnoea and haemorrhagic nasal discharge. Post mortem examination typically reveals pulmonary haemorrhage and pleural effusion. Histopathology demonstrates fibrino-suppurative, necrotising and haemorrhagic pneumonia in most cases. The pathogenesis of S. zooepidemicus infection in dogs is incompletely understood. Bacterial virulence factors as well as host factors may contribute to the severe outcome. S. zooepidemicus occasionally causes zoonotic infections with potentially serious consequences. Canine vaccines for S. zooepidemicus are currently not available and prevention of the disease therefore relies on limiting bacterial spread by implementing stringent control measures in kennels. Further research, particularly sequence analysis of canine strains, is required to gain insights into epidemiology and pathogenesis of this emerging disease.