Paola Scarpa

Development of muscle pathology in canine X-linked muscular dystrophy. I. Delayed postnatal maturation of affected and normal muscle as revealed by myosin isoform analysis and utrophin expression

Abstract Canine X-linked muscular dystrophy (CXMD) is genetically homologous to Duchenne muscular dystrophy and shares the severe myopathy and lethal clinical development of the human disease. We used immunohistochemistry to characterize the time course of postnatal expression of adult fast, adult slow and developmental myosin in the muscle of CXMD dogs, carriers and healthy controls. We also characterized the expression of utrophin and dystrophin. This detailed immunolocalization study confirmed that postnatal muscle maturation is delayed in normal dogs compared to other animals and humans, and is only achieved at around 60 days. In CXMD dogs major derangement of myosin expression became evident from about 15 days; there was a selective loss of fibers expressing fast myosin and persistence of developmental fibers compared to controls. In carriers, the proportion of dystrophin-deficient fibers, which mainly expressed fast myosin, decreased with age. In controls and carriers utrophin was absent from muscle fiber surfaces in 2-day-old animals but present between 15 and 30 days, to mostly disappear by 60 days. In dystrophic animals, sarcolemmal expression of utrophin was more marked and persistent. That immature neonatal muscle from control dogs normally contains sarcolemmal utrophin may have implications for the success of utrophin up-regulation therapy to correct the dystrophic phenotype. The data of this study provide important baseline information for further studies on the development and progression of pathological changes in the muscle of CXMD dogs.

Multiple Endocrine Neoplasia Type-I-like Syndrome in Two Cats

Multiple endocrine neoplasia (MEN) embodies a group of diseases in human patients and domestic animals that are characterized by hyperplasia or neoplasia, or both, of two or more endocrine tissues. The MEN-1 syndrome is associated with menin gene mutations that induce various combinations of parathyroid, pituitary, and pancreatic endocrine tumors in humans. Two male, Domestic Shorthair cats developed symmetric alopecia, insulin-resistant diabetes mellitus, and pituitary-dependent hyperadrenocorticism at 12 and 13 years of age. Examination of skin biopsy specimens revealed atrophic dermatosis associated with hyperadrenocorticism. In one cat, cutaneous lesions consistent with paraneoplastic alopecia associated with pancreatic adenocarcinoma also were evident. Multiple invasive pancreatic beta cell carcinomas, pituitary corticotroph adenomas, and thyroid C-cell and parathyroid chief cell hyperplasia were diagnosed on the basis of results of gross, histologic, and immunohistochemical findings in both cats. Pancreatic exocrine adenocarcinoma was diagnosed in both cats. one cat also had hepatocellular carcinoma. Exons 1-8 of the feline menin gene were sequenced and were found to bear 93% homology with the human gene sequence, and the corresponding amino acid sequences shared 98% homology. Purification of total RNA and amplification of cDNA from lesional tissues to document mutations in the feline menin gene sequence were unsuccessful. The combination of lesions observed was consistent with the diagnosis of MEN-1-like syndrome in both cats.

Echocardiographic values in clinically healthy adult dogue de Bordeaux dogs.

OBJECTIVES To assess the influence of body surface area, age and gender on echocardiographic parameters and to establish echocardiographic reference values for dogue de Bordeaux dogs. METHODS Thirty-nine healthy dogue de Bordeaux dogs of both sexes, older than one year, were recruited and 31 of these were included in the study. The classic linear regression model proved to be the best way to analyse the data. The reference limits of the echocardiographic measurements were calculated using the regression equations. The difference between the mean values of body surface area in both gender groups was evaluated by using one-way ANOVA. RESULTS A significant correlation was seen between several echocardiographic parameters and body surface area or body surface area and age, and high coefficients of determination (R2) were found. No effect of gender was detected on echocardiographic variables, except for the thickness of the left ventricular posterior wall at end diastole. CLINICAL SIGNIFICANCE The echocardiographic parameters related to body surface area, in the absence of correlation with other independent variables (gender and age) should be interpreted with caution because their variation could be significant for the presence of heart disease. The proposed statistical model allows estimation of echocardiographic parameters in dogue de Bordeaux dogs with different body surface areas and ages.

The effect of inter-laboratory variability on the protein:creatinine (UPC) ratio in canine urine

Quantification of proteinuria is a fundamental step in staging dogs with chronic kidney disease and in monitoring the course of disease or the efficacy of anti-proteinuric treatments. Analytical precision and accuracy of the proteinuria assessment could be affected by several factors such as biological variability, different operators and quality control materials. The aim of this study was to assess whether inter-laboratory variability could affect the urinary protein to creatinine (UPC) ratio and whether this variability may affect patient classification according to the International Renal Interest Society (IRIS) sub-staging system. The same urine samples were analysed in three different laboratories using different instruments and different reagent brands. The results of the three laboratories were highly correlated to each other although urinary protein (UP), urinary creatinine (UC) and the UPC ratio of one laboratory were found to be significantly higher than those of the other two. No significant differences between the other two laboratories were recorded. The concordance in classifying dogs according to the IRIS guidelines was good if all three proteinuria categories were analysed separately or if borderline proteinuric (BP) dogs were included in the proteinuric group, and very good if BP dogs were merged into the non-proteinuric group. The inter-laboratory variability in UPC ratio measurement was not so great as to impede the identification of proteinuric dogs, but may influence the estimation of the magnitude of proteinuria.

Measurement of proteinuria in dogs: analytic and diagnostic differences using 2 laboratory methods

BACKGROUND Urinary protein-to-creatinine (UPC) ratio is an early diagnostic and prognostic marker of renal disease in dogs. Pyrogallol red molybdate (PRM) and Coomassie brilliant blue (CBB) are the most popular dye-binding assays for measurement of proteinuria. Published guidelines recommend strict cut-off points to substage patients with chronic renal diseases, irrespective of the assay applied. However, analytic variability and method-dependent differences could affect substaging of patients. OBJECTIVES The aims of this study were to analytically validate the CBB assay to evaluate possible method-dependent differences with PRM in urinary protein (UP) determination, and to assess the influence of such differences in substaging according to the International Renal Interest Society (IRIS). METHODS Urine was collected from healthy and proteinuric dogs. Intra-assay and inter-assay repeatability (imprecision), linearity under dilution (LUD), and spiking recovery (inaccuracy) were determined for the CBB assay. Split samples were measured with PRM and CBB, and agreement between methods and concordance in classification according to IRIS guidelines was determined. RESULTS The CBB assay was precise (< 10%) at all urine protein concentrations after excluding outliers from the intra-assay precision assay of high urine protein concentrations. Acceptable accuracy was demonstrated with both LUD and spiking recovery test. Both UP and UPC determined by CBB were significantly higher (P < .0001) than those obtained with PRM, and both a constant and proportional bias were present. Concordance of IRIS substaging was only moderate. CONCLUSIONS The CBB is precise and accurate, but the higher UPC obtained with CBB vs PRM may affect interpretation of the IRIS guidelines.

Sensitivity and specificity of manual and automated measurements of reticulocyte parameters for classification of anemia in dogs: 174 cases (1993–2013)

OBJECTIVE To assess sensitivity and specificity of manual and automated measurements of reticulocyte percentage, number, and production index for classification of anemia in dogs. DESIGN Retrospective case series SAMPLE 174 blood smears from client-owned dogs with anemia collected between 1993 and 2013 for which reticulocyte parameters were determined manually (nonregenerative anemia, 22; preregenerative anemia, 23; regenerative anemia, 28) or with an automated laser-based counter (nonregenerative anemia, 66; preregenerative anemia, 17; regenerative anemia, 18). PROCEDURES Diagnostic performance was evaluated with receiver operating characteristic (ROC) curves by considering preregenerative anemia as nonregenerative or regenerative. Sensitivity, specificity, and positive likelihood ratio were calculated by use of cutoffs determined from ROC curves or published reference limits. RESULTS Considering preregenerative anemia as non regenerative, areas under the curve (AUCs) for reticulocyte percentage, number, and production index were 97%, 93%, and 91% for manual counting and 93%, 90%, and 93% for automated counting. Sensitivity, specificity, and positive likelihood ratio were 82% to 86%, 82% to 87%, and 4.6 to 6.4, respectively. Considering preregenerative anemia as regenerative, AUCs were 77%, 82%, and 80% for manual counting and 81%, 82%, and 92% for automated counting. Sensitivity, specificity, and positive likelihood ratio were 72% to 74%, 76 to 87%, and 2.7 to 6.2, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Whereas all reticulocyte parameters identified regeneration in anemic dogs, the performance of specific parameters was dependent on the method used. Findings suggested that lower cutoffs than published reference limits are preferred for reticulocyte number and production index and higher cutoffs are preferred for reticulocyte percentage. Reticulocyte production index may be useful when the pretest probability of regeneration is moderate.

Association of amniotic uric acid, glucose, lactate and creatinine concentrations and lactate/creatinine ratio with newborn survival in small-sized dogs – preliminary results

In order to define the normal composition of canine amniotic fluid and to detect differences between surviving and non-surviving newborn puppies, the present study determined the uric acid, glucose, lactate and creatinine concentrations and the lactate to creatinine ratio in amniotic fluids collected during elective Caesarean section from small-sized purebred bitches. The possible relationship between newborn survival and the studied parameters, as well as the effects of maternal parity, fetal gender and Apgar score were assessed. The study enrolled 27 small-sized purebred bitches submitted to elective Caesarean section at term. After opening the fetal membranes, amniotic fluid samples were collected aseptically from the amniotic sac of each fetus. The data obtained from 74 amniotic fluid samples collected from 27 bitches showed that amniotic glucose concentration was lower (P < 0.05) in non-surviving than in surviving puppies. Within the normal, surviving puppies, amniotic glucose concentration was higher (P < 0.05) in male than in female newborns, and the lactate/creatinine ratio was significantly higher in multiparous than in primiparous bitches (P < 0.05). These preliminary results demonstrate the relevance of amniotic glucose, but not of uric acid, lactate, creatinine and the lactate to creatinine ratio for detecting puppies at risk of death immediately after birth.

Serum symmetric dimethylarginine and creatinine in Birman cats compared with cats of other breeds

Objectives The aim of this study was to assess whether, in contrast to serum creatinine, which is higher in Birman cats than in other breeds, the serum concentration of symmetric dimethylarginine (SDMA) is comparable in clinically healthy Birmans and in the general feline population. This could allow, in this breed, to better evaluate chronic kidney disease (CKD). Methods Serum creatinine and SDMA were measured in clinically healthy Birmans (n = 50) and in cats of other breeds (n = 46), and the results were statistically compared. A breed-specific reference interval (RI) was established for Birmans and compared with the RI for the general feline population (0.0–14.0 µg/dl). Results Creatinine (1.58 ± 0.36 mg/dl) and SDMA (12.2 ± 2.8 µg/dl) were higher (P <0.001) in Birmans than in cats of other breeds (1.19 ± 0.17 mg/dl; 10.3 ± 2.5 µg/dl). In 20/50 Birman cats (40.0%) serum creatinine was higher than both the non-breed-specific RI of our laboratory and the threshold recommended to classify cats as IRIS stage 2 (1.6 mg/dl). The concentration of SDMA was higher than the pre-existing RI in 10/50 Birmans (20.0%) and in four cats of other breeds (8.7%). Among Birmans, the proportion of cats with SDMA >14 µg/dl was lower (P <0.017) than the proportion of cats with creatinine >1.60 mg/dl. However, the deviation from the upper limit of the RI was lower than the analytical variability of the method in 7/10 Birmans and in 4/4 cats of other breeds. The breed-specific RI (3.5–18.7 µg/dl) overlapped with the pre-existing one. Conclusions and relevance SDMA may be a better marker of CKD in Birman cats than creatinine when non-breed-specific RIs are utilised. The coupled analysis of creatinine and SDMA could help prevent errors in diagnosing and staging CKD in Birman cats.