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A significant proportion of canine urothelial carcinomas carry the driver valine to glutamic acid variation (V595E) in BRAF kinase. The detection of V595E may prove suitable to guide molecularly targeted therapies and support non-invasive diagnosis of the urogenital system by means of a liquid biopsy approach using urine. Three cohorts and a control group were included in this multi-step validation study which included setting up a digital PCR assay. This was followed by investigation of preanalytical factors and two alternative PCR techniques on a liquid biopsy protocol. Finally, a blind study using urine as diagnostic sample has been carried out to verify its suitability as diagnostic test to complement cytology. The digital PCR (dPCR) assay proved consistently specific, sensitive, and linear. Using the dPCR assay, the prevalence of V595E in 22 urothelial carcinomas was 90.9%. When compared with histopathology as gold standard in the blind-label cases, the diagnostic accuracy of using the canine BRAF (cBRAF) variation as a surrogate assay against the histologic diagnosis was 85.7% with 92.3% positive predictive value and 80.0% negative predictive value. In all the cases, in which both biopsy tissue and the associated urine were assayed, the findings matched completely. Finally, when combined with urine sediment cytology examination in blind-label cases with clinical suspicion of malignancy, the dPCR assay significantly improved the overall diagnostic accuracy. A liquid biopsy approach on urine using the digital PCR may be a valuable breakthrough in the diagnostic of urothelial carcinomas in dogs.

Dwarfism phenotypes occur in many species and may be caused by genetic or environmental factors. In this study, we investigated a family of nine Dogo Argentino dogs, in which two dogs were affected by disproportionate dwarfism. Radiographs of an affected dog revealed a decreased level of endochondral ossification in its growth plates, and a premature closure of the distal ulnar physes. The pedigree of the dogs presented evidence of monogenic autosomal recessive inheritance, combined linkage and homozygosity mapping assigned the most likely position of a potential genetic defect to 34 genome segments, totaling 125 Mb. The genome of an affected dog was sequenced and compared to 795 control genomes. The prioritization of private variants revealed a clear top candidate variant for the observed dwarfism. This variant, PRKG2:XM_022413533.1:c.1634+1G&gt, T, affects the splice donor site and is therefore predicted to disrupt the function of the PKRG2 gene encoding protein, kinase cGMP-dependent type 2, a known regulator of chondrocyte differentiation. The genotypes of the PRKG2 variant were perfectly associated with the phenotype in the studied family of dogs. PRKG2 loss-of-function variants were previously reported to cause disproportionate dwarfism in humans, cattle, mice, and rats. Together with the comparative data from other species, our data strongly suggest PRKG2:c.1634+1G&gt, T to be a candidate causative variant for the observed dwarfism phenotype in Dogo Argentino dogs.

‘Candidatus Neoehrlichia mikurensis’ is an emerging tick-borne zoonotic agent that primarily affects immunocompromised human patients. Dogs and foxes are frequently exposed to ticks,and both species are in close proximity to humans. This is the first study to systematically investigate the occurrence of ‘Candidatus Neoehrlichia mikurensis’ in Canidae in Europa. We analyzed 1’739 blood samples from dogs in Switzerland,Italy,Spain and Portugal and 162 blood samples from free-ranging red foxes (Vulpes vulpes) in Switzerland. All samples were tested using a previously described multiplex real-time PCR for the Anaplasmataceae family,the ‘Candidatus Neoehrlichia’ genus and the ‘Candidatus Neoehrlichia mikurensis’ species. All Anaplasmataceae positive samples were subsequently tested using specific real-time PCRs for Anaplasma phagocytophilum,Anaplasma platys,Ehrlichia canis and Rickettsia helvetica. Among the tested animals,one dog from Zurich tested positive for ‘Candidatus Neoehrlichia mikurensis’. The 12-year old West Highland white terrier had been splenectomized 3 months prior to the blood collection and presented with polyuria/polydipsia. Fanconi syndrome was diagnosed based on glucosuria with normoglycemia and hyperaminoaciduria. A. platys and E. canis were detected in 14/249 dogs from Sicily and Portugal; two of the dogs were coinfected with both agents. Four Swiss foxes tested positive for A. phagocytophilium. R. helvetica was detected for the first time in a red fox. In conclusion,‘Candidatus Neoehrlichia mikurensis’ infection should be considered in sick dogs,particularly when immunocompromised. The pathogen seems not to be widespread in Canidae in the investigated countries. Conversely,other Anaplasmataceae were more readily detected in dogs and foxes.

The conventional polymerase chain reaction (PCR)/sequencing methods may be poorly suited for the detection of somatic mutations in canine mast cell tumour (MCT) samples owing to limited sensitivity. This study was aimed at establishing novel and more sensitive methods, assessing their limit of detection and comparing their sensitivity with conventional methods.Two different 'driver' somatic mutations of c-KIT, together with the wild-type counterparts, were cloned in plasmids to prepare standard samples with known concentrations of mutated alleles in a background of wild-type alleles; the plasmids standards were assayed using either conventional or novel, highly sensitive technique. Conventional PCR/sequencing showed a sensitivity of 50-20%. Conversely, all the novel methods obtained higher sensitivities allowed reaching as low as 2.5-1.2% of the mutated DNA.The study demonstrates that early conventional methods could likely have underestimated the prevalence of KIT mutations of MCTs, therefore affecting the assessment of their relevance in prognosis and tyrosine kinase inhibitor (TKI) treatment effectiveness.

Introduction Feline cutaneous mast cell tumours (cMCTs) are characterized by a variable biologic behaviour. Development of multiple cutaneous nodules and concurrent visceral involvement, along with inconsistency of conventional prognostic aids, justify present uncertainty in differentiating benign from malignant forms. c-kit proto-oncogene activating mutations have been reported in 68% of feline MCTs, however their prognostic relevance has not been determined. This is a prospective study performed on feline cMCTs with variable clinical presentation to assess whether KIT immunohistochemical overexpression can be regarded as indicative of c-kit mutations, and evaluate their relationship with disease progression. Materials and Methods Cats with a histological diagnosis of cMCT in the absence of visceral involvement were enrolled. Patients underwent surgery and were followed for at least 6 months. KIT/CD117 immunohistochemistry and c-kit mutation analysis (exons 8,9,11) were performed on tumour samples and correlated with DFI and OS. Results Twenty-one cats with 11 solitary, 4 multiple, and 6 disseminated cMCTs were included. Determinations were performed on 28 tumour samples. KIT cytoplasmic expression was observed in 20 tumours, showing a shorter DFI (P=0.013) and OS (P=0.04). c-kit encoding mutations were detected in 58% of cases (exon 8, 12%; exon 9, 46%; exon 11, 4%), but they were not directly correlated with clinical behaviour. Conclusion c-kit mutations occur frequently in feline cMCT, and their incidence is higher among tumours with a CD117 overexpression, but their prognostic relevance needs to be assessed in a larger series. Multiple nodules from any one cat did not harbour the same mutations, suggesting that they may represent separate events.

Canine Leishmaniasis (CanL) is associated with a high prevalence of glomerular damage and Chronic Kidney Disease. Quantitative albuminuria in humans is considered an early marker of renal damage, however its measurement in dogs is still limited in the clinical setting. The aim of the study was to describe clinicopatological findings in a population of dogs affected by CanL with particular regard to proteinuria, albuminuria and renal damage.The medical records of 135 dogs affected by CanL, admitted to the Veterinary Teaching Hospital (2004-2011), were available. CanL diagnosis was based on clinical signs and serology or cytology. Clinicopathological evaluation upon admission included CBC, a full biochemistry profile with Total Iron Binding Capacity (TIBC) and Antithrombin, and urinalysis comprehensive of Urine Total Protein and Urine Albumin to Creatinine ratio (UPC and UAC), and Urine Albumin % (UA%). UAC and Serum Immunoglobulins (IgG and IgM) were measured in 114/135 and 20/135 dogs, respectively, and were quantified with immunoturbidimetric methods. These assays were previously validated in our lab and reference intervals were obtained using 65 healthy dogs. Data were analyzed with descriptive and non parametric statistics. A difference was considered significant for p0.5) was found in 80/135 (59%; Median 1.42; Range 0.01- 29.9). Albuminuria (UAC >0.024) was found in 75/114 (65%; Median 0.13; Range 0.001-14.846). Based on sCrea >1.4 mg/dl and USG < 1030 or UPC > 0.5, 85/135 dogs (63%) were considered Kidney Diseased (KD). KD dogs had significantly higher concentrations of serum globulins, IgG and IgM with a significant reduction of Hematocrit value and serum Albumin, A/G, Antithrombin, Calcium, TIBC concentrations. Among non proteinuric patients who had UAC performed (50/114), 13/50 (28%) were albuminuric. UA% on total urinary proteins ranged from 0.15 to 90% and was positively correlated with UPC (r=0.54) and UAC (r=0.80).Our results suggest that during CanL, KD dogs have a greater humoral response with more severe clinicopathological alterations; furthermore UAC could be a biomarker for early detection of renal damage.

In dogs, persistent renal proteinuria with Urinary Protein to Creatinine ratio (UPC) ≥2.0 usually is due to glomerular renal disease. In humans, Nephrotic-Range Proteinuria (NRP) is defined as UPC ≥ 3.5, while Overt Albuminuria (OA) is defined as Urinary Albumin to Creatinine ratio (UAC) > 0.3. Analogous cut-off values are lacking for dogs. The aim of this study was to characterize clinical and clinicopathological features associated with 'glomerular proteinuria' in dogs, with particular regard to UAC.Retrospective analysis included a total of 338 dogs admitted to our Teaching Hospital, between January 2002 and September 2011, with glomerular proteinuria defined by UPC ≥2.0. Data collected were signalment, clinical and clinicopathological findings including Blood Pressure, CBC, serum Albumin (Alb), Total Protein, Cholesterol, Creatinine (Crea), Urea, Electrolytes, CRP, Total Iron, Total Iron Binding Capacity (TIBC), Antithrombin%, D-Dimers, Fibrinogen, and urinalysis comprehensive of UPC and UAC (immunoturbidimetric method). Data were analyzed with descriptive and non parametric statistics. A difference was considered significant for p 0.3). Hypoalbuminemia (Alb ≤2.5 g/dl) was detected in 57.2% of cases (Median 2.35; range 0.74-4.29). A significant negative correlation between UPC, UAC and Alb (r=-0.43 and r=-0.25, respectively), was detected. Crea concentration was above the reference interval in 53% of dogs (Median 1.85; range 0.47-22.07; RI 0.65- 1.35). Median Fibrinogen concentration was 5.28 g/l (range 0.64-10.63; RI 1.45-3.85), Median D-Dimers concentration was 0.26 μg/ml (range 0.01-8.84; RI

Introduction: Feline mast cell tumours (FeMCTs), overall Accounting for 1e9% of feline neoplasms, are characterized by a highly variable biological behaviour. Frequent post- surgical recurrence, de-novo development of multiple tumours and concurrent visceral and cutaneous involvement justify uncertainty in differentiating benign from malignant forms, with tendency to systemic spread. Materials and Methods: A series of FeMCTs with variable clinical presentation were examined by histology (cell morphology, differentiation, growth pattern, mitotic activity), CD117 immunohistochemistry and c-Kit mutation analysis (exons 8, 9 and 11). Data were correlated with clinical records (clinical signs, TNM stage, haematological abnormalities and 2-year follow-up) to assess their prognostic significance. Results: Twenty cats with 10 solitary cutaneous, five multiple cutaneous and five systemic MCTs were included; nine cats were still alive at the end of the follow-up period. Overall, 29 tumour samples were examined. There were 21 well-differentiated, three pleomorphic and five atypical FeMCTs; mean mitotic activity was 9/10 high power fields. Low to high CD117 expression was observed in 18 cases. Further c-Kit mutations, beside those previously described in FeMCT, were found. Conclusions: This study investigated the effects of c-Kit dysregulation on FeMCT biological behaviour and also potentially allowed the identification of those patients that may benefit from molecular targeted therapies.