Multiple congenital ocular anomalies in three related litters of Jack Russell Terrier puppies.

OBJECTIVE: To describe multiple congenital ocular anomalies in three litters of Jack Russell Terrier puppies. ANIMALS STUDIED: Seven purebred Jack Russell Terrier puppies from three related litters and their four parents. PROCEDURES: Medical records of the puppies and their parents were evaluated. All dogs underwent a complete ophthalmic examination, followed by bilateral ocular ultrasonography in two of the puppies with complete corneal opacity. One eye from an affected puppy was subjected to histopathology. A complete database of pedigrees was built, and individual inbreeding was evaluated. RESULTS: The most commonly diagnosed ocular anomalies in the puppies were: various anomalies of the fundus (12/14 eyes); microphthalmia (10/14 eyes); sclerocornea (8/14 eyes); and persistent pupillary membranes (7/14 eyes). Six out of seven puppies had at least two ocular abnormalities, and only one puppy was normal. Four out of seven puppies had sclerocornea, a particular corneal opacity to date described only in Spanish Podenco dogs. No ocular abnormalities were found in the parents examined (4/4). Analysis of the pedigrees showed that all the puppies and two parents were inbred, and the individual values of the inbreeding puppies were greater than 6.25% in two litters. CONCLUSIONS: Inbreeding with closely related Jack Russell Terriers may result in severe congenital eye abnormalities in puppies.

Combined immunohistochemical protocols to differentiate macrophages within the mononuclear-phagocyte system.

BACKGROUND: The "mononuclear phagocyte system" (MPS) refers to dispersed mononuclear monocytes and macrophages and is used to distinguish them from polymorphonuclear cells. The term "histiocyte" indicates large cells with voluminous granulated cytoplasm, sometimes containing engulfed particles, recognized as fully differentiated end cells of the MPS. Dendritic cells (DC) represent another diversified population whose inclusion in the MPS is still debated. The diverse cells of the MPS cannot all be characterized by single antigen markers or unique functions expressed at all stages of cell differentiation or activation. Nevertheless, in a diagnostic setting, their reliable identification plays a major role when a specific therapy must be established. Understanding the heterogeneity among MPS cell populations is indeed relevant to define different therapeutic approaches that can range from the use of antibiotics to immunomodulatory agents. For this reason, we attempted to establish a protocol to reliably identify the proportion of macrophages within the mononuclear phagocyte system in a tissue and/or in a given inflammatory population. METHODS: the Tafuri method was used in different double immunofluorescence protocols using an anti-Iba-1, anti-MAC387, and anti-CD11b-CD68-CD163-CD14-CD16 antibody. RESULTS AND DISCUSSION: in normal canine skin the anti-Iba-1 antibody stained an epidermal cell population (i.e. Langerhans cells) and scattered cells within the dermal compartment. MAC387 was unable to stain cells containing Leishmania amastigotes in leishmaniasis-diagnosed samples as the anti-CD11b-CD68-CD163-CD14-CD16 antibody did. By using a combination of staining protocols to differentiate macrophages within the whole histiocytic infiltrate we validated the use of a cocktail of rabbit monoclonal antibodies raised against CD11b, CD68, CD163, CD14, CD16 to stain skin macrophages.

Osteopontin expression in healing wounds of horses and in human keloids.

REASONS FOR PERFORMING STUDY: Convincing evidence shows that persistent or excessive expression of osteopontin (OPN) is linked to fibroproliferation of various organs in laboratory animals and in man, such that its downregulation is a logical therapeutic objective. OBJECTIVES: To investigate OPN expression in an equine model of wound healing and in clinical specimens of equine exuberant granulation tissue and human keloids in an effort to better understand the contribution of this protein to inflammation-associated skin fibrosis. STUDY DESIGN: Description of gene and protein expression in an experimental equine model of wound healing and clinical specimens in horse and man. METHODS: Osteopontin gene expression was evaluated by quantitative PCR, while protein expression was investigated by means of immunohistochemical staining. RESULTS: Quantitative PCR showed that the OPN gene is expressed in normal intact skin of horses and continues to be expressed during the wound-healing process. An increase in gene expression was observed throughout the phases of wound healing, with a final decrease at wound closure. The protein was not detected in normal skin. Keratinocytes in wound-edge samples did not express the protein, whereas dermal immunoreactivity was confined to inflammatory cells. Healed wounds were devoid of staining. Equine exuberant granulation tissue showed immunoreactivity of the surrounding epidermis, infiltrating neutrophils, mononuclear cells, endothelial cells and fibroblasts. Human keloids showed OPN immunoreactivity throughout the epidermis as well as in mononuclear cells and scattered fibroblasts. CONCLUSIONS: Immunohistochemical data show a different pattern of expression between normally healing and fibrotic wounds (exuberant granulation tissue and keloids), thus suggesting a role in fibroproliferation in horses and man.

Histology as a valid and reliable tool to differentiate fresh from frozen-thawed fish.

Selling fish products as fresh when they have actually been frozen and thawed is a common fraudulent practice in seafood retailing. Unlike fish products frozen to protect them against degenerative changes during transportation and to extend the product's storage life, fish intended for raw consumption in European countries must be previously frozen at -20° C for at least 24 h to kill parasites. The aim of this study was to use histological analysis to distinguish between fresh and frozen-thawed fish and to evaluate this method for use as a routine screening technique in compliance with the requirements of European Commission Regulation No. 882/2004 on official food and feed controls. Method performance (i.e., accuracy and precision) was evaluated on tissue samples from three common Mediterranean fish species; the evaluation was subsequently extended to include samples from 35 fish species in a second experiment to test for method robustness. Method accuracy was tested by comparing histological results against a "gold standard" obtained from the analysis of frozen and unfrozen fish samples prepared for the study. Method precision was evaluated according to interrater agreement (i.e., three laboratories with expertise in histopathology in the first experiment and three expert analysts in the second experiment) by estimating Cohen's kappa (and corresponding 95 % confidence intervals) for each pair of laboratories and experts and the combined Cohen's kappa for all three experts and laboratories. The observed interrater agreement among the three laboratories and the three experts indicated high levels of method accuracy and precision (high sensitivity and specificity) and method reproducibility. Our results suggest that histology is a rapid, simple, and highly accurate method for distinguishing between fresh and frozen-thawed fish, regardless of the fish species analyzed.

Association between waste management and cancer in companion animals.

BACKGROUND: Increased cancer rates have been documented in people residing in areas around Naples characterized by illegal dumping and incineration of waste. HYPOTHESIS: Risk of cancer in dogs and cats is associated with waste management. ANIMALS: Four hundred and fifty-three dogs and cats with cancer and 1,554 cancer-free animals. METHODS: Hospital-based case-control study in Naples (low danger) and nearby cities having a history of illegal waste dumping (high danger). Odds ratio (OR) between high- and low-danger areas was calculated for all tumors and various malignancies in dogs and cats. RESULTS: An increased risk for cancer development was identified in dogs but not in cats residing in high-danger areas (OR: 1.55; 95% confidence interval: 1.18-2.03; P < .01). A 2.39-fold increased risk of lymphoma (P < .01) accounted for the greater tumor frequency in dogs residing in high-danger areas. The risk of mast cell tumor and mammary cancer did not differ in dogs residing in high- or low-danger areas. CONCLUSIONS AND CLINICAL IMPORTANCE: Waste emission from illegal dumping sites increases cancer risk in dogs residing in high-danger areas. An increased prevalence of lymphoma has been previously recognized in humans living close to illegal waste dumps. Thus, epidemiological studies of spontaneous tumors in dogs might suggest a role for environmental factors in canine and human carcinogenesis and can predict health hazards for humans.

Anaplastic and aggressive subcutaneous sarcoma in a seven-month-old dog.

A seven-month-old female, Italian hound which was presented with anorexia, dyspnoea and weight loss showed two subcutaneous masses at physical examination. Radiographs showed multiple opacities in the lungs, thorax, liver and spleen, and cytology showed discrete pleomorphic cells, with variable nuclear : cytoplasmic ratio, foamy cytoplasm and round to oval nuclei. Incisional biopsy histology showed the infiltrative proliferation of highly pleomorphic spindle to roundish large cells, discrete or arranged in a storiform pattern, with scattered multinucleated giant cells. Immunohistochemistry exhibited strong positivity for vimentin and mild labelling for alpha-smooth muscle actin and lysozyme; other mesenchymal or histiocytic lineage markers stained negatively. Because of the poor prognosis, the owner elected euthanasia. Post-mortem examination confirmed massive metastatic spread. Cytology, histology and immunohistochemistry suggested the diagnosis of anaplastic sarcoma with giant cells, although disseminated histiocytic sarcoma was a reliable differential diagnosis. The authors underline the difficulties in definitively categorising poorly differentiated sarcomas, even if a large panel of markers is applied.

Effect of a hydrocolloid dressing on first intention healing surgical wounds in the dog: a pilot study.

OBJECTIVES: To evaluate the efficacy of a hydrocolloid dressing for the treatment of surgical wounds in dogs. METHODS: Six healthy young female dogs of medium size and different breed underwent ovariohysterectomy. Histological evaluation was performed on biopsies taken from the edges of the wounds at day 7. The dressing was applied on one half of the wound according to manufacturer's instructions; the second half served as control. Biopsy specimens were fixed in a 10% formalin buffered solution pH 7.4, paraffin embedded and stained with haematoxylin and eosin. For clinical assessment, the presence and quality of exudate, erythema of the surrounding area, swelling and correct apposition of the wound margins were evaluated. RESULTS: The hydrocolloid dressing was easy to use. The clinical quality of the treated skin wounds was superior to the non-treated ones. Comparison of histological features between treated and untreated wounds showed a more regular organisation of the granulation tissue in the treated wounds, with fibroblasts being aligned parallel to the overlying epidermis. The number of inflammatory cells and the extension of granulation tissue were less prominent and less widespread in treated compared to untreated wounds. CONCLUSION: The dressing performed very well in terms of adhesiveness and flexibility. It was useful in the management of surgical wounds to avoid contamination and ameliorate the epithelialisation rate and granulation tissue morphology of the surgical scar.

Adjuvant gemcitabine after surgical removal of aggressive malignant mammary tumours in dogs.

Canine mammary tumours are generally treated with surgery alone, despite the fact that 50% of them are malignant and many will eventually lead to recurrence or metastases. A prospective clinical trial in which dogs with aggressive mammary carcinoma of clinical stages IV and V were treated with surgical excision (n = 9) or with surgery and adjuvant weekly gemcitabine (n = 10) for at least four cycles was conducted. Gemcitabine was given as an intravenous infusion at the dose of 800 mg m(-2). Aim of the study was to explore potential beneficial effects of gemcitabine on time to local recurrence (TTR), time to distant metastases (TTM) and overall survival (OS) in canine patients with operated mammary tumours bearing high risk for locoregional failure and distant metastases. Also, factors associated with OS, including neutering status, body weight, age, clinical stage at presentation, tumour size, histological grade and, in dogs receiving chemotherapy, the number of gemcitabine treatments, were investigated. Finally, acute toxicities related to chemotherapy and quality of life were assessed in dogs receiving gemcitabine. Dogs treated with surgery alone or surgery followed by gemcitabine had no difference in TTR, TTM or OS (P > 0.05). In the group of dogs receiving adjuvant chemotherapy, the number of gemcitabine treatments was positively correlated with OS (P = 0.017). Gemcitabine treatment was well tolerated, with no dogs experiencing clinically relevant haematological or gastrointestinal toxicity. Despite being safe at the present dose, gemcitabine chemotherapy as an adjunct treatment to surgical excision may not be recommended in dogs with aggressive mammary carcinoma.

Morphometrical approach for predicting regional lymph node micrometastatic load in canine mast cell tumours: preliminary results.

Canine cutaneous mast cell tumours (MCTs) have a variable biologic behaviour, and accurate staging is necessary to dictate therapy and predict outcome. Regional lymph node (RLN) involvement is a relevant prognostic factor. While obvious lymph node (LN) metastases are relatively easy to be diagnosed, micrometastatic disease recognition is challenging. The main aim of the study was to evaluate the number of mast cells (MCs) in the LNs of clinically healthy dogs (n = 4, group 1), dogs with inflammatory diseases (n = 31, group 2) and dogs with cutaneous MCT (n = 27, group 3), including animals with no RLN metastases (subgroup 3.1), those with occasional MCs in RLNs (3.2) and those with obvious RLN metastasis (3.3). MCs also were morphometrically evaluated for the following nuclear parameters: mean nuclear area (MNA), mean nuclear perimeter (MNP), largest to smallest diameter length (LS ratio), mean nuclear form factor and coefficient of variation of nuclear area. The average percentages of MCs were 0.0 and 0.01 in groups 1 and 2, respectively, and 0.07, 2.4 and 47.1 in subgroup 3.1, 3.2 and 3.3. MNA and MNP were significantly higher in subgroup 3.3 than in group 2 (P < 0.05). MNA and MNP in subgroup 3.2 suggested the presence of neoplastic MCs; this prediction of micrometastatic load correlated with outcome. Analysis of preliminary results shows that nuclear morphometry is useful to detect micrometastatic disease in RLN of dogs bearing cutaneous MCTs.

Postsurgical intra-incisional 5-fluorouracil in dogs with incompletely resected, extremity malignant spindle cell tumours: a pilot study.

The safety and efficacy of intra-incisional 5-fluorouracil (5-FU) in the management of incompletely resected malignant spindle cell tumours of extremities was evaluated in six dogs. After marginal surgery, the dogs underwent weekly intra-incisional 5-FU for a minimum of six cycles. Treatment was well tolerated by all dogs, with no systemic adverse effects and only one episode of local cutaneous hyperpigmentation, which completely and spontaneously resolved. Median follow-up for all the dogs was 546 days (mean 619; range 297-1207). At the date of analysis, four dogs were still alive with no evidence of local recurrence, and two dogs had died as a result of their disease. The cause of death was development of distant metastases in one dog and tumour regrowth in the other. Despite the small sample size, this study documents that intra-incisional 5-FU chemotherapy is a safe and efficacious adjuvant treatment in the case of incompletely resected malignant spindle cell tumours in dogs and that long disease control can be achieved.

Quantitative analysis of tryptase- and chymase-containing mast cells in eosinophilic conditions of cats.

The presence and density of tryptase-positive/chymase-positive mast cells (MCs) (MC(TC)), chymase-positive/tryptase-negative MCs (MCC), and tryptase-positive/chymase-negative MCs (MC(T)) in lesional skin from cats with eosinophilic conditions were investigated. Skin biopsy specimens from eight cats with eosinophilic plaque (three cats), eosinophilic granuloma (two cats), and eosinophilic dermatitis (three cats) were studied. Toluidine blue staining and a double-enzyme-immunohistochemical staining technique were performed to determine MC density and MC subtypes, respectively. MC density varied from 170.3 to 503 cells/mm2 (mean value of 314.9 cells/mm2). In the superficial dermis, 5.9% of the MC belonged to the MC(T), 12.8% to the MC(C), and 81.2% to the MC(TC) subtype. In the deep dermis, 12.8% belonged to the MC(T), 12.8% to the MC(C), and 73.8% to the MC(TC) subtype. It is the first time that MC(C) have been identified. The double-labeling procedure proved to be a reliable tool for identifying simultaneously the presence of MC subtypes in feline skin.

Feline cutaneous phaeohyphomycosis due to Cladophyalophora bantiana.

A case of feline cutaneous phaeohyphomycosis due to Cladophyalophora bantiana is described. The cat was presented with breathing difficulty and a swollen, ulcerated nodule on the dorsal nose and left nostril. Histological examination of the nodule revealed a cystic granulomatous dermatitis characterised by neutrophils, macrophages and giant cells. Pigmented, yeast-like fungus cells and hyphal elements were easily identified in haematoxylin-eosin stained tissue sections. Cladophyalophora bantiana was isolated from a tissue specimen. This organism, primarily known to cause cerebral infection in humans and cats, only rarely causes cutaneous infection. Despite anti-fungal chemotherapy two relapses occurred. The cat was feline immunodeficiency virus- and feline leukemia virus-negative and even if the owner was unaware of trauma, the hypothesis of wound contamination is the most likely.

Two cases of dermatophytic pseudomycetoma in the dog: an immunohistochemical study.

Two cases of canine dermatophytic pseudomycetoma resulting in subcutaneous nodules resembling those previously reported in Persian cats are described. Culture performed from one nodule yielded dark yellow colonies consistent with Microsporum canis. Immunohistochemistry, using rabbit anti-M. canis, demonstrated specific binding to fungal elements in paraffin sections. The specificity of the antiserum was further tested by an agar gel immunodiffusion assay using a soluble extract from a feline isolate of M. canis as antigen. The antiserum did not cross-react with an Aspergillus fumigatus antigen. These are the first two reported cases of canine dermatophytic pseudomycetoma and immunohistochemical staining supported the diagnosis.

Clinical and histological evaluation of an analogue of palmitoylethanolamide, PLR 120 (comicronized Palmidrol INN) in cats with eosinophilic granuloma and eosinophilic plaque: a pilot study.

Fifteen cats with eosinophilic granuloma or eosinophilic plaque were given PLR 120 at the dosage of 10 mg kg-1 twice daily for one month. PLR-120 down-modulates mast cell degranulation via a receptor-mediated mechanism. No other drugs were permitted and cats were kept free of parasites throughout the study. A clinical evaluation and skin biopsies were performed before and after the treatment. Clinical improvement was assessed at 15 and 30 days. Mast cell numbers were counted and their granular content was assessed by densitometric analysis on toluidine blue-stained sections before and after the treatment. Ten of 15 (67%) cats showed clinical improvement of signs and lesions. There was no significant difference between mast cell numbers in skin biopsies taken before and after the trial, whereas the number of granules was significantly increased (P < 0.009). This pilot study suggests that PLR-120 might be a useful drug for the treatment of eosinophilic granuloma and eosinophilic plaque.

Survey of canine and feline follicular tumours and tumour-like lesions in central Italy.

Between January 1985 and June 1997, a total of 4005 skin biopsies were received from dogs and 898 from cats. Follicular tumours and tumour-like lesions together represented 10.4 per cent and 8.1 per cent of all skin tumours in the dog and cat, respectively. The prevalence of tumour-like lesions, such as follicular and dermoid cysts, dilated pore and focal adnexal dysplasia, was highest, representing 41.2 per cent and 68 per cent of all follicular lesions in dogs and cats, respectively. In the dog, follicular tumours were distributed as follows: trichoblastoma (25.6 per cent of all tumours and tumour-like lesions), infundibular keratinising acanthoma, (14 per cent), pilomatricoma (13 per cent), trichoepithelioma (3.9 per cent) and tricholemmoma (2.3 per cent). In the cat, the distribution was 26 per cent for trichoblastoma, 4 per cent for trichoepithelioma and 2 per cent for pilomatricoma. Tumour-like lesions and infundibular keratinising acanthoma in the dog were mostly located on the trunk, trichoblastoma and tricholemmoma on the head, and pilomatricoma on the neck. In the cat, both tumour-like lesions and trichoblastoma were frequently present on the neck and head.

Tumor necrosis factor-alpha and virus expression in the central nervous system of cats infected with feline immunodeficiency virus.

Cytokine disregulation has been implicated in the pathogenesis of lentivirus-induced diseases. In the present study, 18 specific pathogen free (SPF) cats were inoculated with feline immunodeficiency virus (FIV) Petaluma strain and sacrificed at different times post-infection. Five additional SPF cats were used as controls. The cell localization of the cytokine tumor necrosis factor alpha (TNF-alpha) in the central nervous system (CNS) was determined by immunohistochemical and morphometric analyses with a polyclonal rabbit anti-human TNF-alpha antibody. TNF-alpha and FIV RNA were measured using competitive reverse transcriptase polymerase chain reaction (PCR) assays and the number of proviral genomes was estimated by competitive PCR. Portions of frontal cortex were collected from each animal and both formalin-fixed and snap-frozen and stored at -80 degrees C until used. The results showed that TNF-alpha is present mainly in astrocytes and microglial cells. Morphometric analysis showed that areas of TNF-alpha production increased in the early phases of infection. Molecular analyses demonstrated that the kinetics of proviral loads in the CNS were comparable to what observed in lymph nodes and peripheral blood mononuclear cells, with the peaks in the early and late stages of infection. A positive correlation was found between viral parameters and TNF-alpha transcription, the strongest relationship was found between the transcription of the cytokine and viral RNA load. These results confirm that invasion of CNS by FIV occurs soon after virus exposure and that during this phase there is an increase of local viral loads with concomitant up-regulation of TNF-alpha expression. During the asymptomatic phase viral replication remains low in spite of the progression of CNS alterations. The dissociation between the viral load and the lesions observed suggests the importance of an indirect mechanism for the progression of these lesions, even if TNF-alpha seems to play a role particularly in the early phase of infection.

Neuropathology in cats experimentally infected with feline immunodeficiency virus: a morphological, immunocytochemical and morphometric study.

Neuropathological changes have been described associated with feline immunodeficiency virus (FIV) infection. The objective of our study was to characterize the lesions found in the brain and spinal cord of experimentally FIV-infected cats and to quantify, by morphometric analysis, the intensity of gliosis found in these subjects at different time post infection (pi). The brains and spinal cords appeared grossly normal. Gray matter of cortical and subcortical structures showed a moderate to pronounced gliosis particularly in all cerebral cortex and hippocampus. Morphometric analysis demonstrated that GFAP immunoreactivity was markedly higher in infected animals. Gliosis was present 15 days pi and did not appear to progress during the infection, whereas neuronal changes when present were observed only in long-term infected animals (15-23 months pi). In a large proportion of infected cats a diffuse gliosis of white matter and vacuolar myelinopathy was also present. Despite some discrepancies observed between neuropathological changes in FIV-infected animals and HIV-infected individuals, the presence in the cerebral cortex of cats with FIV infection of alterations similar to those observed in AIDS patients demonstrates that FIV is an interesting animal model particularly that may be useful for clarifying the pathogenesis of neuropathological changes associated with HIV infection.

Regional distribution of lesions in the central nervous system of cats infected with feline immunodeficiency virus.

Neuropathological examination of the central nervous system of 13 naturally and 13 experimentally feline immunodeficiency virus (FIV)-infected cats revealed diffuse gliosis of gray and white matter and vacuolar myelinopathy in a large proportion of infected animals, sometimes associated with lymphocytic meningitis. Multinucleated giant cell formation, the hallmark of multifocal giant cell encephalitis in HIV infection, was never observed. Morphometric analysis confirmed a marked increase of GFAP reactivity in infected cats. Gliosis was mainly present in cortical structures of frontal, parietal, and occipital lobes. Only one naturally infected animal evidenced clinical symptoms of neurological damage. This study confirms that FIV provides an interesting model for studying HIV-induced cortical and subcortical brain pathology believed to be the cause of the neurological manifestations frequently observed in AIDS patients.