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.

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.

Serological detection of Epstein-Barr virus infection in dogs and cats.

Epstein-Barr virus (EBV) is widespread in humans world-wide. The virus, which is linked to Burkitt's and Hodgkin lymphoma in humans, has recently been detected in pet dogs in Taiwan. The aim of this study was to determine if EBV is present in other canine populations, whether infection is associated with lymphoma in dogs, and whether infection is present in cats. Canine and feline serum samples were analysed by immunofluorescence assay. Antibodies to EBV or an EBV-like virus were detected in 38.4% of UK dog sera, 64% of US dog sera and 38% of cat sera. Canine blood samples, tissue samples from the palatine tonsil and formalin-fixed, paraffin-embedded biopsy samples from canine lymphoma cases were analysed by PCR. Using EBV-specific primers, EBV was detected in one tonsil sample, whereas all other samples tested were negative. PCR using consensus herpesvirus primers identified canine herpesvirus in twelve tonsil samples and one biopsy but no gammaherpesviruses were detected. Canine blood samples from EBV antibody-positive dogs were analysed by RT-PCR to determine if transcripts associated with lytic EBV infection (BcLF1) or latency (LMP2) were present, however all samples tested were negative. In conclusion, there is serological evidence of exposure to EBV or an EBV-like virus in dogs and cats but persistent infection in the canine palatine tonsil is rare and no evidence was found of EBV in canine peripheral blood mononuclear cells. The effect of EBV on canine and feline cells and the possible outcome of the infection for the host require further investigation.

Characterization of pneumonia due to Streptococcus equi subsp. zooepidemicus in dogs.

Streptococcus equi subsp. zooepidemicus has been linked to cases of acute fatal pneumonia in dogs in several countries. Outbreaks can occur in kenneled dog populations and result in significant levels of morbidity and mortality. This highly contagious disease is characterized by the sudden onset of clinical signs, including pyrexia, dyspnea, and hemorrhagic nasal discharge. The pathogenesis of S. equi subsp. zooepidemicus infection in dogs is poorly understood. This study systematically characterized the histopathological changes in the lungs of 39 dogs from a large rehoming shelter in London, United Kingdom; the dogs were infected with S. equi subsp. zooepidemicus. An objective scoring system demonstrated that S. equi subsp. zooepidemicus caused pneumonia in 26/39 (66.7%) dogs, and most of these dogs (17/26 [65.4%]) were classified as severe fibrino-suppurative, necrotizing, and hemorrhagic. Three recently described superantigen genes (szeF, szeN, and szeP) were detected by PCR in 17/47 (36.2%) of the S. equi subsp. zooepidemicus isolates; however, there was no association between the presence of these genes and the histopathological score. The lungs of S. equi subsp. zooepidemicus-infected dogs with severe respiratory signs and lung pathology did however have significantly higher mRNA levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 8 (IL-8) than in uninfected controls, suggesting a role for an exuberant host immune response in the pathogenesis of this disease.

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.

Expression of beta-defensins in the canine respiratory tract and antimicrobial activity against Bordetella bronchiseptica.

Beta-defensins are cationic peptides which form part of the innate immune response of the respiratory epithelium. Due to their antimicrobial properties and immunostimulatory activity, beta-defensins are potential tools for the treatment and prevention of respiratory disease. In dogs, infectious respiratory disease is a common problem, particularly in housed animals. This study aimed to assess the presence of four beta-defensins in the canine respiratory tract and to use quantitative real-time PCR to determine mRNA levels following microbial challenge. Three beta-defensins, CBD1, CBD103 and CBD108, were detected in respiratory cells. All three defensins were also readily expressed in skin samples, while their expression in lymphoid tissues and the kidney was low and inconsistent. Treatment of primary tracheal epithelial cells with lipopolysaccharide (LPS) or infection with canine respiratory coronavirus led to decreased expression of CBD103 and CBD108, while cells infected with canine parainfluenza virus had lower levels of CBD1 and CBD108. Furthermore CBD103 was demonstrated to have antimicrobial activity against the respiratory pathogen Bordetella bronchiseptica.

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.

Longitudinal study of viruses associated with canine infectious respiratory disease.

In this investigation a population of dogs at a rehoming center was monitored over a period of 2 years. Despite regular vaccination of incoming dogs against distemper, canine adenovirus type 2 (CAV-2), and canine parainfluenza virus (CPIV), respiratory disease was endemic. Tissue samples from the respiratory tract as well as paired serum samples were collected for analysis. The development of PCR assays for the detection of CPIV, canine adenovirus types 1 and 2, and canine herpesvirus (CHV) is described. Surprisingly, canine adenovirus was not detected in samples from this population, whereas 19.4% of tracheal and 10.4% of lung samples were positive for CPIV and 12.8% of tracheal and 9.6% of lung samples were positive for CHV. As reported previously, a novel canine respiratory coronavirus (CRCoV) was detected in this population (K. Erles, C. Toomey, H. W. Brooks, and J. Brownlie, Virology 310:216-223, 2003). Infections with CRCoV occurred mostly during the first week of a dog's stay at the kennel, whereas CPIV and CHV were detected at later time points. Furthermore, the evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to CPIV and an immunofluorescence assay for detection of antibodies to CHV is described. This study shows that CPIV is present at kennels despite vaccination. In addition, other agents such as CHV and CRCoV may play a role in the pathogenesis of canine respiratory disease, whereas CAV-2 and canine distemper virus were not present in this population, indicating that their prevalence in the United Kingdom is low due to widespread vaccination of dogs.

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.

Detection of a group 2 coronavirus in dogs with canine infectious respiratory disease.

An investigation into the causes of canine infectious respiratory disease was carried out in a large rehoming kennel. Tissue samples taken from the respiratory tract of diseased dogs were tested for the presence of coronaviruses using RT-PCR with conserved primers for the polymerase gene. Sequence analysis of four positive samples showed the presence of a coronavirus with high similarity to both bovine and human coronavirus (strain OC43) in their polymerase and spike genes, whereas there was a low similarity to comparable genes in the enteric canine coronavirus. This canine respiratory coronavirus (CRCV) was detected by RT-PCR in 32/119 tracheal and 20/119 lung samples, with the highest prevalence being detected in dogs with mild clinical symptoms. Serological analysis showed that the presence of antibodies against CRCV on the day of entry into the kennel decreased the risk of developing respiratory disease.