Daniela Malatesta

Occurrence of fatal canine Angiostrongylus vasorum infection in Italy

Two cases of fatal canine angiostrongylosis, occurred in a short-time between them in Italy, are described. Both animals presented with severe cardio-respiratory signs and died shortly after admission. At necropsy, both dogs showed lung haemorrhagic lesions, thrombosis of the pulmonary arteries and dilatation of the right heart chambers. Adult nematodes, identified as Angiostrongylus vasorum, were recovered from the heart and/or the pulmonary arteries of the dead animals. The present report focuses on the clinical and parasitological aspects of the two cases and underlines that canine angiostrongylosis should be included in the differential diagnosis of dogs with cardio-pulmonary disorders in southern Europe.

PDGFs and PDGFRs in canine osteosarcoma: new targets for innovative therapeutic strategies in comparative oncology.

Platelet derived growth factor receptor (PDGFR)α and PDGFRβ are tyrosine kinase receptors that are overexpressed in 70-80% of human osteosarcomas (OSAs) and may be suitable therapeutic targets for specific kinase inhibitors (TKIs). Canine OSA shows histopathological and clinical features similar to human OSA, and is considered an excellent model in comparative oncology. This study investigated PDGF-A, PDGF-B, PDGFRα and PDGFRβ expression in 33 canine OSA samples by immunohistochemistry and in seven primary canine OSA cell lines by Western blot and quantitative PCR analysis. Immunohistochemical data showed that PDGF-A and PDGF-B are expressed in 42% and 60% of the OSAs analysed, respectively, while PDGFRα and PDGFRβ were expressed in 78% and 81% of cases, respectively. Quantitative PCR data showed that all canine OSA cell lines overexpressed PDGFRα, while 6/7 overexpressed PDGFRβ and PDGF-A relative to a normal osteoblastic cell line. Moreover, in vitro treatment with a specific PDGFR inhibitor, AG1296, caused a dose- and time-dependent decrease in AKT phosphorylation. Collectively, these data show that PDGFRs/PDGFs are co-expressed in canine osteosarcomas, which suggests that an autocrine and/or paracrine loop is involved and that they play an important role in the aetiology of OSA. PDGFRs may be suitable targets for the treatment of canine OSA with a specific TKI.

Gross and histopathological changes associated with Aelurostrongylus abstrusus and Troglostrongylus brevior in a kitten.

Aelurostrongylus abstrusus affects the respiratory system of domestic cats worldwide, causing different clinical signs. Recently, Troglostrongylus brevior, a parasite found only in feral and wild animals until 2010, has been regarded as a cause of potentially severe bronchopneumonia in domestic cats. The life cycle of both nematodes involves the same intermediate (i.e. land mollusks) and paratenic hosts (i.e. lizards, birds and small mammals). Biological and epidemiological drivers may cause the spreading of A. abstrusus along with a possible emergence of T. brevior from marginal areas and limited hosts. Given the merit in increasing knowledge on these potentially emerging nematodes, the present paper describes clinical features and the first post mortem gross and histopathological observations in a case of mixed infection by A. abstrusus and T. brevior in a kitten.

Immunohistochemical investigation of cell cycle and apoptosis regulators (Survivin, β-Catenin, P53, Caspase 3) in canine appendicular osteosarcoma

BackgroundOsteosarcoma (OSA) represents the most common canine primary bone tumour. Despite several pathways have been investigated so far, few molecules have been identified as prognostic tools or potential therapeutic targets, and there is still the need to find out molecular pathways with specific influence over OSA progression to facilitate earlier prognosis and treatment.Aims of the present study were to evaluate the immunohistochemical pattern and levels of expression of a panel of molecules (survivin, β-catenin, caspase 3 -inactive and active forms- and p53) involved in cell cycle and apoptosis regulation in canine OSA samples, known to be of interest in the study also of human OSA, and to detect specific relations among them and with histological tumour grade, disease free interval (DFI) and overall survival (OS).ResultsNuclear β-catenin immunostaining was detected in normal osteoblasts adjacent to the tumour, and in 47% of the cases. Cytoplasmic and/or membranous immunostaining were also observed. Nuclear survivin and p53 positive cells were found in all cases. Moderate/high cytoplasmic β-catenin expression (≥10% positive cells) was significantly associated with the development of metastasis (P = 0.014); moderate/high nuclear p53 expression (≥10% positive cells) was significantly associated with moderate/high histological grade (P = 0.017) and shorter OS (P = 0.049). Moderate/high nuclear survivin expression (≥15% positive cells) showed a tendency toward a longer OS (P = 0,088).ConclusionsThe present results confirmed p53 as negative prognostic marker, while suggested survivin as a potential positive prognostic indicator, rather than indicative of a poor prognosis. The detection of nuclear β-catenin immunostaining in normal osteoblasts and the absent/low expression in most of the OSAs, suggested that this pathway could not play a major role in oncogenic transformation of canine osteoblasts. Further studies are needed to confirm these hypotheses.

Retrospective study on the occurrence of the feline lungworms Aelurostrongylus abstrusus and Troglostrongylus spp. in endemic areas of Italy.

Aelurostrongylus abstrusus is a metastrongyloid nematode infesting the respiratory system of domestic cats worldwide. Troglostrongylus brevior and Troglostrongylus subcrenatus, two lungworms thought to infest wild felids, have been found recently in domestic cats from Spain and Italy. These unexpected findings have raised doubts about the assumed past and present occurrence of Troglostrongylus spp., especially T. brevior, in domestic hosts and suggest that there may have been missed detection or misdiagnosis. The present retrospective study evaluated the presence of lungworms in cats from Italy with a diagnosis of respiratory parasitism or with compatible lung lesions from 2002 to 2013. Sixty-eight samples of DNA and larvae from cats with a diagnosis of aelurostrongylosis, and 53 formalin-fixed paraffin-embedded lung samples from cats confirmed as lungworm infested or with compatible lesions, were investigated using two DNA-based assays specific for A. abstrusus or T. brevior. All DNA and larval samples were positive for A. abstrusus and one was additionally positive for T. brevior. Most paraffin-embedded lung tissues were positive only for A. abstrusus, but two samples tested positive for both lungworms and one for T. brevior only. This study supports the major role of A. abstrusus in causing feline respiratory parasitism in endemic areas of Italy.

β-catenin in canine skin: immunohistochemical pattern of expression in normal skin and cutaneous epithelial tumours.

In normal adult skin, β-catenin is a structural component of the intercellular junction and the Wnt/β-catenin pathway plays a key role in the regulation of cutaneous homeostasis, particularly in the maintenance of hair follicle stem cells. No data are available on the expression pattern of β-catenin in normal canine skin and in canine cutaneous epidermal and follicular tumours. The present study used immunohistochemistry to determine β-catenin expression in four samples of normal canine skin and 62 cutaneous epithelial tumours (14 epidermal, 30 follicular and 18 glandular). β-catenin expression was localized to the nucleus of matrical and dermal papilla cells in anagen hair follicles and was also found in scattered cells of the outer root sheath, suggesting that these follicular epithelial cells may have a high proliferative potential. Nuclear labelling, considered a hallmark of activation of the Wnt/β-catenin signalling pathway, was observed in canine follicular tumours with matrical differentiation (100% of cases of trichoepithelioma and pilomatricoma), suggesting that a possible mutation of the canine CTNBB1 gene may underlie these tumours. In contrast, malignant tumours (squamous cell carcinoma, basal cell carcinoma, sebaceous and apocrine gland carcinoma and epithelioma) were characterized by reduction/loss of β-catenin membrane labelling compared with normal cutaneous epithelial cells and benign tumours, suggesting that reduction/loss of β-catenin expression is important in the acquisition of the malignant phenotype and may have a role in the infiltration and metastasis of these tumours.

Circovirus in domestic and wild carnivores: An important opportunistic agent?

Circoviruses are relatively novel pathogens with increased importance in canids. In this study, we first screened the presence of dog circovirus (DogCV) by molecular methods from a total number of 389 internal organ samples originating from 277 individuals of domestic dogs and wild animals including wolves, foxes and badgers. All the animals originated from Central-Southern Italy, specifically from Abruzzi and Molise regions, areas hosting several natural parks. DogCV was detected in 9/34 wolves (P=26.4%; IC 95%: 14.6-43.1%), 8/209 dogs (P=3.8%; IC 95%: 1.9-7.3%), 0/24 foxes (P=0%; IC 95%: 0-13.8%), 1/10 badgers (P=10%; IC 95%: 1.79-40.4%). However, all DogCV positive animals were shown to be infected at least by an additional key pathogen, including canine distemper virus (CDV) and canine parvovirus type 2. All wolves, but one, presenting DogCV in the internal tissues suffered from CDV infection. The DNA purified from 17 DogCV infected organs was used for whole genome sequencing and phylogenetic analysis.

Expression of heat shock proteins in premalignant and malignant urothelial lesions of bovine urinary bladder.

Abnormal heat shock protein (HSP) levels have been observed in a number of human tumours, where they are involved in all hallmarks of cancer. Since bovine urothelial tumours share striking morphological and biochemical features with their human counterparts, the aim of this study was to evaluate the immunohistochemical levels of Hsp27, Hsp60, Hsp72, Hsp73 and Hsp90 in 28 normal bovine urinary bladders and 30 bovine papillomavirus-positive urothelial tumours (9 in situ carcinomas, 9 low-grade and 12 high-grade carcinomas) and adjacent premalignant lesions obtained from cows suffering from chronic enzootic haematuria, in order to investigate the role of these proteins in the process of urothelial carcinogenesis. A semi-quantitative method was used for the analysis of the results. Western blot analysis was also used to confirm HSP expression in normal controls. All investigated HSPs were expressed in normal bovine urothelium, showing characteristic patterns of immunolabelling throughout urothelial cell layers, which usually appeared to be conserved in urothelial hyperplasia and dysplasia. On the other hand, gradual loss of Hsp27 immunostaining resulted to be significantly associated with increasing histological grade of malignancy (P < 0.01). As well, a significantly reduced immunosignal of Hsp73 and Hsp90 was observed in high-grade and low-/high-grade carcinomas, respectively (P < 0.01). In contrast, Hsp60 (P < 0.01) and Hsp72 (P < 0.05) immunoreactivity appeared to be significantly increased both in premalignant and malignant lesions when compared to that observed in normal urothelium, thus suggesting an early involvement of these proteins in neoplastic transformation of urinary bladder mucosa.

Survivin and Related Proteins in Canine Mammary Tumors Immunohistochemical Expression

Survivin is reexpressed in most human breast cancers, where its expression has been associated with tumor aggressiveness, poor prognosis, and poor response to therapy. Survivin expression was evaluated in 41 malignant canine mammary tumors (CMTs) by immunohistochemistry, in relation to histological grade and stage, and correlated with that of some related molecules (β-catenin, caspase 3, heat shock proteins) to understand their possible role in canine mammary tumorigenesis. An increase in nuclear survivin expression, compared with healthy mammary glands, was observed in CMTs, where nuclear immunolabeling was related to the presence of necrosis. No statistically significant relation was found between the expression of the investigated molecules and the histological grade or stage. The present study may suggest an important involvement of survivin in CMT tumorigenesis. Its overexpression in most of the cases evaluated might suggest that targeting survivin in CMTs may be a valid anticancer therapy.

Nuclear survivin expression as a potentially useful tool for the diagnosis of canine cutaneous sebaceous lesions.

BACKGROUND Sebaceous glands are specialized cutaneous adnexal glands, which work under constant hormonal control to produce sebum. They can give rise to several proliferative lesions, such as hamartoma, hyperplasia and neoplasms (adenoma, epithelioma and carcinoma). Their nomenclature is currently confusing, both in veterinary and in human medicine, owing to the difficulty of differentiating between some of these lesions. METHODS The present study used immunohistochemistry to determine the expression levels and patterns of survivin and Ki67 in five samples of normal canine skin and 44 cases of canine cutaneous lesions with sebaceous differentiation (10 hamartomas, nine hyperplasia, eight adenomas, eight epitheliomas and nine carcinomas). RESULTS In normal glands, survivin, as well as Ki67, was expressed in scattered reserve cells. In hamartomas, survivin was more highly expressed than in normal skin, indicating a possible role of this molecule in the pathogenesis of these congenital lesions. In tumours, a moderate or high level of survivin and Ki67 expression (more than two and four and more than two positive cells, respectively) were significantly correlated with a malignant histotype, infiltrative growth and a moderate or high number of mitoses (more than two). CONCLUSIONS AND CLINICAL IMPORTANCE The level of survivin expression increased with increasing malignancy, designating survivin as a new diagnostic marker in the assessment of malignancy of sebaceous tumours.