T Lymphocytes in Histiocytic Sarcomas of Flat-Coated Retriever Dogs.

Flat-Coated Retriever dogs are predisposed to the development of histiocytic sarcoma (HS), a poorly differentiated, highly malignant neoplasm. The authors have previously documented a significant lymphocytic infiltrate in such tumors. The objective of this study was to examine the presence and expression of regulatory T cells in HS tumor samples. Forty tumors were included in this study. All tumors were immunolabeled for CD3, CD79a, CD25, CD45RA, and FOXP3. The proportion of positive cells was compared between tumors presenting as a localized primary soft tissue mass (soft tissue origin HS) and disseminated HS affecting viscera, especially the spleen (splenic origin HS). By immunohistochemistry, 95% of infiltrating T cells were positive for Foxp3 in all sections, suggesting the presence of regulatory T cells. The proportion of cells positive for FOXP3 was higher in the tumors arising in soft tissues, whereas the proportion of CD45RA-positive cells was higher in the splenic origin HS. Canine HS has an aggressive clinical behavior and is uniformly fatal. The difference in the proportion of tumor-infiltrating lymphocytes positive for these 2 markers in the 2 locations may represent differences in tumor microenvironment between the 2 sites.

An immunohistochemical study of the expression of the hypoxia markers Glut-1 and Ca-IX in canine sarcomas.

Tumor hypoxia has been associated with increased malignancy, likelihood of metastasis, and increased resistance to radiotherapy and chemotherapy in human medicine. Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor that is induced by tumor hypoxia and regulates the pathways involved in cellular response and adaptation to the hostile tumor microenvironment. HIF-1 induces transcription of different proteins, including Ca-IX and Glut-1, which are considered endogenous markers of chronic hypoxia in solid tumors in humans. In this study, sections from 40 canine sarcomas (20 histiocytic sarcomas and 20 low-grade soft-tissue sarcomas) were immunostained for these markers. Expression of Glut-1 was scored based on percentage of positive staining cells (0 = <1%; 1 = 1%-50%; 2 = >50%) and intensity of cellular staining (1 = weak; 2 = strong); Ca-IX was scored based on percentage of positive cells (0 = <1%; 1 = 1%-30%; 2 = >30%). Intratumoral microvessel density was measured using CD31 to assess intratumoral neoangiogenesis. Histiocytic sarcomas showed statistically significant higher Glut-1 immunoreactivity and angiogenesis than did low-grade soft-tissue sarcomas. Intratumoral microvessel density in histiocytic sarcomas was positively associated with Glut-1 immunoreactivity score. These findings suggest a potential role of hypoxia in the biology of these tumors and may provide a base for investigation of the potential prognostic use of these markers in naturally occurring canine tumors.

Transmission of canine transmissible venereal tumour between two dogs in the UK.

Canine transmissible venereal tumour (CTVT) is a contagious cancer spread by transfer of living cancer cells. Occasional cases are observed in the UK in dogs imported from endemic regions. Here, we report a case of imported canine transmissible venereal tumour that was transmitted to a second dog within the UK. Transmission of genital canine transmissible venereal tumour occurred despite neutered status of the second dog. The aggressive course of disease in both cases, which included metastasis, resistance to therapeutic interventions and ultimate euthanasia of both dogs, is described. The diagnosis of canine transmissible venereal tumour was made using a combination of cytology, histology, immunohistochemistry and PCR to detect the LINE-MYC rearrangement. Practitioners unfamiliar with canine transmissible venereal tumour are reminded of this disease of concern, particularly when imported dogs are placed in multi-dog households, irrespective of neuter status.

The clinical presentation and histopathologic-immunohistochemical classification of histiocytic sarcomas in the Flat Coated Retriever.

The Flat Coated Retriever is a breed at risk of development of histiocytic sarcoma (HS), but in contrast to the disseminated form of disease recognized in the Bernese Mountain Dog, most reports of HS in Flat Coated Retrievers describe a localized lesion affecting the musculature or fascia of limbs. The purpose of this study was to review data and material received though an ongoing Flat Coated Retriever tumor survey to better define the presentation of HS in the breed and to determine the utility of subclassification of tumors arising at different sites by histology and immunohistologic phenotyping. Data on 180 dogs bearing HS-like tumors were available for review, which showed that although the majority (101 lesions, 57%) were primary limb lesions, 47 dogs (26%) had visceral, mainly splenic lesions with no peripheral primary tumor. A detailed histologic and immunohistologic review of 20 limb tumors and 20 splenic tumors showed that 2 distinct phenotypic subtypes could be identified: a histiocytic subtype, most prevalent in the splenic tumors, and a histiocytic-spindle-pleomorphic subtype, mainly seen in the limb tumors. Despite their variable morphology, all tumors expressed major histocompatibility complex class II and the leukocyte antigen CD18, but only those tumors in the spleen consistently expressed CD11d. The majority of tumors also contained a mild to moderate infiltrate of T lymphocytes.

Salmonella enterica serovar typhimurium trxA mutants are protective against virulent challenge and induce less inflammation than the live-attenuated vaccine strain SL3261.

In Salmonella enterica serovar Typhimurium, trxA encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of trxA in the mouse-virulent S. Typhimurium strain SL1344 (SL1344 trxA), replacing the gene with a kanamycin resistance gene cassette. SL1344 trxA was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344 trxA induced less inflammation in murine spleens and livers than SL3261, the aroA mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344 trxA was partially attributed to a reduction in the number of both CD4(+) and CD8(+) T cells and B lymphocytes in the spleen and reduced infiltration by CD11b(+) cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a trxA mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting trxA in SL3261. SL3261 trxA was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344 trxA.

Chronic Inflammatory Disease in the Pancreas, Kidney and Salivary Glands of English Cocker Spaniels and Dogs of Other Breeds Shows Similar Histological Features to Human IgG4-related Disease.

Chronic pancreatitis (CP) is a common disease in the English cocker spaniel (ECS) and is characterized histologically by duct destruction, interlobular fibrosis and dense periductular and perivenous lymphocytic aggregates. These features are also found in human autoimmune pancreatitis type 1, part of a glucocorticoid-responsive, multiorgan syndrome, newly recognized as IgG4-related disease (IgG4-RD). Human IgG4-RD affects one or several organs, often showing a predominance of IgG4+ plasma cells histologically, with an IgG4+:total IgG+ plasma cell ratio of >40%. This study investigated whether ECSs with CP and/or inflammatory disease in several organs show an increase in IgG4+ plasma cells within affected tissues. Histological sections of pancreas, liver, kidney, salivary gland and conjunctiva were obtained from ECSs with idiopathic chronic inflammatory disease affecting those tissues. Tissue samples from age-matched dogs of other breeds with similar diseases were also sampled. Control diseased tissue samples, from dogs without a suspected immune-mediated disease, were included. A subset of ECSs and dogs of other breeds presented with disease in more than one organ. Immunohistochemistry was performed with primary reagents detecting total IgG and three of the four canine IgG subclasses (IgG2, IgG3 and IgG4). Normal sections of pancreas and liver showed an absence of labelled plasma cells of any subclass. Normal kidney and salivary gland sections showed the presence of a few labelled plasma cells (<10 plasma cells/high-power field). Fourteen tissue sections from 12 ECSs and seven sections from six dogs of other breeds showed elevated numbers of IgG4+ plasma cells and IgG4+:IgG+ ratios >40%. Individual dogs (ECSs and other breeds) showed marked increases in IgG4+ cells. There were no significant differences in the number of IgG4+ plasma cells between ECSs and dogs of other breeds for affected pancreas, liver, salivary glands and conjunctiva. Kidney sections had more IgG4+ cells, for both ECSs and dogs of other breeds, than did sections from other organs. Dogs of other breeds had significantly more IgG4+ plasma cells in affected kidneys than ECSs. In conclusion, several ECSs and dogs of other breeds fulfilled the histological criteria for the diagnosis of IgG4-RD, supporting the existence of a multiorgan immune-mediated disease in ECSs and some dogs of other breeds.

Studies on the evolution, pathology, and immunity of commercial fattening rabbits affected with epizootic outbreaks of diarrhoeas in Mexico: a case report.

Epizootic outbreaks of diarrhoeas have emerged and disseminated in different rabbit farms in Mexico causing great economical losses, during the past years. Seven, 5-weeks-old New Zealand White (NZW) rabbits chosen at random from 35 ill animals that were remitted for postmortem, histopathology, and ultrastructural examinations were studied. Bacteriological and parasitological studies were carried out in three additional ill rabbits of same age. In a field trail 45, 5-weeks-old apparently healthy NZW rabbits were observed daily for sanitary status for a 5-week period. Some of the rabbits did not response to the preventive drug treatment and were therefore, used to study the development of the disease. Clinical signs, gross lesions, and mortality throughout the fattening period were recorded. Eight, 8-weeks-old NZW rabbits who survived an outbreak were assessed for gamma-globulins in serum of the total protein fraction during a 3-week period. Gamma-globulins were also measured in eight free-disease healthy rabbits of same breed and age. Lesions of the small intestine consisted of mucoid enteropathy, lymphocytic plasmocytic enteritis with atrophy and fusion of villi, and hyperplasia of globet cells. Serosal edema was present. Ultrastructural examinations of jejunum and ileum from 3/7 diseased rabbits, revealed enterocytes in apoptosis, mixed with degenerative and/or necrotic changes together with infiltration of lymphocytes, macrophages, neutrophils, and loss of microvillus. There were electron dense structures suggestive of virus particles inside the nuclei and cytoplasm of some enterocytes. There was lymphoid spleen atrophy and proliferation of reticuloendothelial cells in 7/7 rabbits. Interstitial pneumonia in 4/7 rabbits was found. Encephalitozoon cuniculi was detected in the brain of 1/7 rabbits. Escherichia coli were detected in 3/3 cases and Eimeria spp. in 2/3 cases. Mortality rate in the field study was 51.1% and the spread of the disease occur in 9/9 cages. The proportion of gamma-globulins in rabbits who survive an outbreak was much lower (P=0.0001) than free-disease healthy rabbits (8.1+/-1.0 and 14.0+/-1.0, respectively). The disease was multifactorial and consisted of sub-acute mucoid enteropathy probably induced by viral infection and aggravated by the proliferation of opportunistic pathogens common to rabbits. This may explain the severe degenerative and necrotic changes observed in the small intestine of diarrhoeic rabbits.

Expression of Fibroblast Activating Protein and Correlation with Histological Grade, Mitotic Index and Ki67 Expression in Canine Mast Cell Tumours.

Fibroblast activating protein (FAP) is a membrane serine protease expressed by activated fibroblasts, particularly tumour associated fibroblasts (TAFs). FAP expression has not been reported in canine mast cell tumours (MCTs). The objective of this study was to evaluate the expression of FAP in TAFs and its correlation with histological grade, mitotic index and Ki67 expression in canine MCTs. FAP expression was evaluated by immunohistochemistry (IHC) in 30 canine MCTs. Twenty-eight (90%) of the MCTs expressed FAP in the stroma, 16 cases showed low to intermediate FAP score and 14 cases had a high FAP score. FAP was correlated positively with both Patnaik (P = 0.007) and Kiupel (P = 0.008) grading systems, mitotic index (P = 0.0008) and Ki67 expression (P = 0.009). High stromal FAP expression could be a potential negative prognostic factor in canine MCTs.

Prevalence of pancreatic, hepatic and renal microscopic lesions in post-mortem samples from Cavalier King Charles spaniels.

OBJECTIVES: The objective of this study was to describe the prevalence of microscopic pancreatic, hepatic and renal lesions in post-mortem samples from Cavalier King Charles spaniels. METHODS: The prevalence of microscopic lesions was determined by routine histopathology and compared to ante-mortem clinical signs. RESULTS: There was evidence of chronic pancreatitis in 51·9% of the cases, and age correlated with severity. Renal lesions were diagnosed in 52·2% of cases, most of which were inflammatory. Ante-mortem diagnosis of pancreatic and renal disease was 25 and 16·7%, respectively. Primary hepatic lesions were diagnosed in 11·1% of cases; secondary hepatic lesions were diagnosed in 64·8%. CLINICAL SIGNIFICANCE: Pancreatic and renal lesions are common in Cavalier King Charles spaniels, but they have similar rates of hepatic disease as the general population. The increasing prevalence of pancreatic lesions with age suggests that it might be a progressive condition.

Hepatic carcinoma with spleen metastasis in a California sea lion from the Gulf of California.

A primary hepatic carcinoma with a neuroendocrine pattern was detected in an adult female California sea lion (Zalophus californianus) found dead on Granito Island in the Gulf of California (Mexico) in January 1996. At necropsy, several light yellow nodules of different sizes were observed on the entire surface of the liver and spleen. Microscopic examination of these nodules using routine haematoxylin-eosin stain, revealed cubic, polyhedral and pleomorphic cells with three to four bizarre mitotic figures per field (40X). An immunohistochemistry test revealed a positive reaction of indirect immunoperoxide to cytokeratin (CK2). This is the first known case of a primary hepatic carcinoma in free-ranging California sea lions from Mexican waters.

Histopathology inflammation scoring and classification in 34 dogs with inflammatory nasal disease.

The aims of this study are to suggest a histological classification and a scoring system (0-12) for dogs with inflammatory nasal disease, and to compare and determine statistical associations between histopathology findings and lymphoplasmacytic rhinitis (LPR) or aspergillosis. Twenty-one LPR cases and 13 aspergillosis cases were reviewed and classified at the Queen's Veterinary School Hospital of the University of Cambridge. Statistical analysis was performed using SPSS V.17 and the level of significance accepted was P<0.05. The suggested classification includes the name of the two most common inflammatory cells found, duration of disease, lesion distribution, severity of epithelial and goblet cell hyperplasia and presence of oedema and fungi. The inflammation score was calculated according to the number of inflammatory cells present, and revealed that half the cases had moderate nasal inflammation and that most of the cases (67.6 per cent) were lymphocytic rhinitis. As far as the statistical associations were concerned, only fungal presence was proven to be associated with aspergillosis (P=0.04). The two conditions were found to have similar histological appearance. Implying that the histological diagnosis can sometimes be difficult and that the clinician should always consider the results from other diagnostic tests to reach a final diagnosis.

Loeffler's endocarditis and bicavity eosinophilic effusions in a dog with visceral mast cell tumour and hypereosinophilia.

A 9-year-old crossbred dog was presented with a 2-week history of diarrhoea and tachypnoea. Marked circulating eosinophilia was identified. Pleural and abdominal effusions were detected by radiography and ultrasonography and cytological examination of these fluids revealed a predominance of eosinophils. Splenic and hepatic cytology revealed mast cell neoplasia, which was confirmed as visceral mast cell tumour on post-mortem examination. Histological changes of myocardial inflammation, necrosis and fibrosis were found. These findings are consistent with Loeffler's endocarditis.

Histopathological investigation of syringomyelia in the Cavalier King Charles spaniel.

Syringomyelia (SM) in Cavalier King Charles spaniels (CKCSs) is identified commonly on magnetic resonance images and is sometimes associated with clinical signs of pain and cervical hyperaesthesia. However, the mechanism by which SM develops in this breed has not been fully elucidated and the associated effects on spinal cord structure have not been reported previously. The aims of this study were to describe changes found in the spinal cord of CKCSs, to compare findings between symptomatic and asymptomatic dogs and to determine whether syrinx formation was associated with tissue destruction. Anomalies of the central canal were found in all specimens and many dogs had grossly visible fluid-filled cavities within the spinal cord. Prominent microscopical findings were spongy degenerative changes associated with neuronal necrosis and Wallerian degeneration. The ependyma was discontinuous in many specimens, notably in symptomatic individuals, and there was evidence of angiogenesis and fibrous tissue proliferation around blood vessels adjacent to syrinx cavities. Compared with two different samples of the normal dog population, dogs with syrinxes had significantly less grey matter, although this decrease was associated with generalized loss of spinal cord area. Therefore, SM is associated with degenerative changes in the spinal cord and may develop through primary disruption of ependymal integrity followed by vascular hypertrophy and proliferation. Glial and fibrous proliferation appears to be associated with expression of clinical signs.

Intentional marginal excision of canine limb soft tissue sarcomas followed by radiotherapy.

OBJECTIVES: To evaluate the outcome in a group of dogs treated with postoperative radiotherapy following intentional marginal excision of soft tissue sarcomas from their limbs and to assess parameters for prognostic significance. METHOD: Patients that had had intentional marginal excision of limb soft tissue sarcomas followed by radiotherapy were selected. A coarse fractionated protocol of four once weekly 8 to 9 Gy by 4 MV x-rays was used. The time to local recurrence was determined. Tumour grade, size, site, number of surgeries, surgeon and time from last surgery to radiotherapy were evaluated as potential prognostic indicators. RESULTS: Fifty-six cases were included. Minor surgical complications occurred in four patients (7%). Tumour recurred locally in 10 dogs (18%). Fourteen dogs died from tumour-related causes (25%). From Cox proportional hazard analysis time from surgery to radiotherapy was the only predictor of tumour recurrence (P=0·039); hazard ratio 8·63. Delaying radiotherapy beyond 4 weeks was associated with improved outcomes. Three dogs developed serious but non-life-threatening local complications; wound dehiscence, self-trauma and osteonecrosis of underlying bone. CLINICAL SIGNIFICANCE: Intentional marginal excision followed by hypofractionated radiotherapy is a viable option for canine limb soft tissue sarcomas, providing good long-term clinical outcomes and low morbidity.

Breed, age and gender distribution of dogs with chronic hepatitis in the United Kingdom.

Standardised histological criteria are now available for the diagnosis of canine chronic hepatitis (CH). CH is common in dogs, but no studies have reported breed, age and gender distributions in the United Kingdom (UK). The objective of this study was to determine which breeds had an increased risk for developing CH in the UK and to report the age and gender distribution for those breeds. The databases of six veterinary histopathology laboratories were searched for cases with a histological diagnosis of CH according to standardised criteria. The breed, age and gender of dogs was recorded and compared to a control population to calculate the odds ratio and 95% confidence intervals for developing CH. A total of 551 cases of CH were identified, consisting of 61 breeds. Nineteen breeds were represented by five or more cases. Breeds with an increased risk for developing CH included the American cocker spaniel, Cairn terrier, Dalmatian, Dobermann pinscher, English cocker spaniel, English springer spaniel, Great Dane, Labrador retriever and Samoyed. The median age at diagnosis for all breeds with CH was 8 years (range 7 months to 16 years). Dalmatians, Dobermann pinschers and English springer spaniels with CH were significantly younger than Cairn terriers, English cocker spaniels and Labrador retrievers with CH. Females were over-represented when all cases were examined together. In conclusion, several breeds in the UK have an increased risk of CH, some of which have not been previously reported.

Chronic hepatitis in the English springer spaniel: clinical presentation, histological description and outcome.

Medical records and liver histology of 68 English springer spaniels (ESS) with a histological diagnosis of CH were reviewed retrospectively. PCR was performed on liver tissue for canine adenovirus-1 (CAV-1), canine parvovirus, canine herpesvirus and pathogenic Leptospira species. Follow-up information was obtained to calculate survival times. Median age at presentation was three years seven months (range, seven months to eight years five months) and there were 48 female and 20 male dogs. Clinical signs were non-specific and five dogs were asymptomatic. All dogs had an increase in serum activity of one or more hepatobiliary enzymes. Histopathology demonstrated hepatocyte necrosis and apoptosis with varying amounts of fibrosis. A predominantly lymphoplasmacytic infiltrate throughout the hepatic parenchyma was found in all 68 dogs, but 45 of these dogs also had a neutrophilic component to the inflammatory infiltrate. There was no significant copper accumulation and no aetiological agent was identified by PCR. The median survival time was 189 days (range, 1 to 1211 days), 38 dogs died within three months and 12 dogs survived more than a year following diagnosis.

Salmonella enterica serovar Typhimurium mutants completely lacking the F(0)F(1) ATPase are novel live attenuated vaccine strains.

The F(0)F(1) ATPase plays a central role in both the generation of ATP and the utilisation of ATP for cellular processes such as rotation of bacterial flagella. We have deleted the entire operon encoding the F(0)F(1) ATPase, as well as genes encoding individual F(0) or F(1) subunits, in Salmonella enteric serovar Typhimurium. These mutants were attenuated for virulence, as assessed by bacterial counts in the livers and spleens of intravenously infected mice. The attenuated in vivo growth of the entire atp operon mutant was complemented by the insertion of the atp operon into the malXY pseudogene region. Following clearance of the attenuated mutants from the organs, mice were protected against challenge with the virulent wild type parent strain. We have shown that the F(0)F(1) ATPase is important for bacterial growth in vivo and that atp mutants are effective live attenuated vaccines against Salmonella infection.