Gene-expression profiling of endomyocardial biopsies from dogs with dilated cardiomyopathy phenotype.

INTRODUCTION: The employment of advanced molecular biology technologies has expanded the diagnostic investigation of cardiomyopathies in dogs; these technologies have predominantly been performed on postmortem samples, although the recent use of endomyocardial biopsy in living dogs has enabled a better premortem diagnostic approach to study the myocardial injury. ANIMALS, MATERIALS, AND METHODS: Endomyocardial biopsies were collected in nine dogs with a dilated cardiomyopathy phenotype (DCM-p) and congestive heart failure and submitted to histologic examination, next-generation sequencing (NGS), and polymerase chain reaction analysis. Data from three healthy dogs (Fastq files) were retrieved from a previously approved study and used as a control group for ribonucleic acid sequencing. RESULTS: Histologic examination revealed endocardial fibrosis in six of nine dogs, whereas lymphocytic interstitial infiltrates were detected in two of nine dogs, and lymphoplasmacytic and macrophage infiltrates were detected in one of nine dogs. On polymerase chain reaction analysis, two dogs tested positive for canine parvovirus two and one dog for canine distemper virus. Gene-expression pathways involved in cellular energy metabolism (especially carbohydrates-insulin) and cardiac structural proteins were different in all DCM-p dogs compared to those in the control group. When dogs with lymphocytic interstitial infiltrates were compared to those in the control group, NGS analysis revealed the predominant role of genes related to inflammation and pathogen infection. CONCLUSIONS: Next-generation sequencing technology performed on in vivo endomyocardial biopsies has identified different molecular and genetic factors that could play a role in the development and/or progression of DCM-p in dogs.

Serum metabolomics assessment of etiological processes predisposing ketosis in water buffalo during early lactation.

Metabolic disorders as ketosis are manifestations of the animal's inability to manage the increase in energy requirement during early lactation. Generally, buffaloes show a different response to higher metabolic demands than other ruminants with a lower incidence of metabolic problems, although ketosis is one of the major diseases that may decrease the productivity in buffaloes. The aim of this study was to characterize the metabolic profile of Mediterranean buffaloes (MB) associated with 2 different levels of ß-hydroxybutyrate (BHB). Sixty-two MB within 50 days in milk (DIM) were enrolled and divided into 2 groups according to serum BHB concentration: healthy group (37 MB; BHB <0.70 mmol/L; body condition score: 5.00; parity: 3.78; and DIM: 30.70) and group at risk of hyperketonemia (25 MB; BHB ≥0.70 mmol/L; body condition score: 4.50; parity: 3.76; and DIM: 33.20). The statistical analysis was conducted by one-way ANOVA and unpaired 2-sample Wilcoxon tests. Fifty-seven metabolites were identified and among them, 12 were significant or tended to be significant. These metabolites were related to different metabolic changes such as mobilization of body resources, ruminal fermentations, urea cycle, thyroid hormone synthesis, inflammation, and oxidative stress status. These findings are suggestive of metabolic changes related to subclinical ketosis status that should be further investigated to better characterize this disease in the MB.

The predictive value of clinical, radiographic, echocardiographic variables and cardiac biomarkers for assessing risk of the onset of heart failure or cardiac death in dogs with preclinical myxomatous mitral valve disease enrolled in the DELAY study.

OBJECTIVES: To identify the predictive value on time to onset of heart failure (HF) or cardiac death of clinical, radiographic, and echocardiographic variables, as well as cardiac biomarkers N-terminal pro brain natriuretic peptide (NT-proBNP) and cardiac troponin I in dogs with preclinical myxomatous mitral valve disease (MMVD). ANIMALS: One hundred sixty-eight dogs with preclinical MMVD and left atrium to aortic root ratio ≥1.6 (LA:Ao) and normalized left ventricular end-diastolic diameter ≥1.7 were included. METHODS: Prospective, randomized, multicenter, single-blinded, placebo-controlled study. Clinical, radiographic, echocardiographic variables and plasma cardiac biomarkers concentrations were compared at different time points. Using receiving operating curves analysis, best cutoff for selected variables was identified and the risk to develop the study endpoint at six-month intervals was calculated. RESULTS: Left atrial to aortic root ratio >2.1 (hazard ratio [HR] 3.2, 95% confidence interval [95% CI] 1.9-5.6), normalized left ventricular end-diastolic diameter > 1.9 (HR: 6.3; 95% CI: 3.3-11.8), early transmitral peak velocity (E peak) > 1 m/sec (HR: 3.9; 95% CI: 2.3-6.7), and NT-proBNP > 1500 ρmol/L (HR: 5.7; 95% CI: 3.3-9.5) were associated with increased risk of HF or cardiac death. The best fit model to predict the risk to reach the endpoint was represented by the plasma NT-proBNP concentrations adjusted for LA:Ao and E peak. CONCLUSIONS: Logistic and survival models including echocardiographic variables and NT-proBNP can be used to identify dogs with preclinical MMVD at higher risk to develop HF or cardiac death.

An integrated approach to control Cystic Echinococcosis in southern Italy.

Cystic echinococcosis (CE) is a severe zoonosis, caused by the larval stage of the tapeworm Echinococcus granulosus. This helminth infection is of increasing public health and socio-economic concern due to the considerable morbidity rates that cause economic losses both in the public health sector and in the livestock industry. Control programmes against E. granulosus are considered long-term actions which require an integrated approach and high expenditure of time and financial resources. Since 2010, an integrated approach to control CE has been implemented in a highly endemic area of continental southern Italy (Campania region). Innovative procedures and tools have been developed and exploited during the control programme based on the following strategies: i) active and passive surveillance in livestock (using geospatial tools for georeferencing), ii) diagnosis in dogs (using the FLOTAC techniques and molecular analysis), iii) targeted treatment of farm dogs (using purpose-built confinement cages), iv) early diagnosis in livestock (by ultrasonography), v) surveillance in humans (through hospital discharge records analysis), vi) monitoring the food chain (analysing raw vegetables), vii) outreach activities to the general public (through dissemination material, e.g. brochures, gadgets, videos, virtual reality). Over eight years, the integrated approach and the new strategies developed have resulted in a noteworthy reduction of the parasite infection rates in livestock (e.g. up to 30 % in sheep). The results obtained so far highlight that using a one health multidisciplinary and multi-institution effort is of pivotal importance in preparing CE control programmes at regional level and could be extended to other endemic Mediterranean areas.

DELay of Appearance of sYmptoms of Canine Degenerative Mitral Valve Disease Treated with Spironolactone and Benazepril: the DELAY Study.

INTRODUCTION: Efficacy of renin-angiotensin-aldosterone system (RAAS) blockade using angiotensin-converting enzyme inhibitors (ACEi) in dogs with preclinical myxomatous mitral valve disease (MMVD) is controversial. HYPOTHESIS: Administration of spironolactone (2-4 mg q 24 h) and benazepril (0.25-0.5 mg q 24 h) in dogs with preclinical MMVD, not receiving any other cardiac medications, delays the onset of heart failure (HF) and cardiac-related death. Moreover, it reduces the progression of the disease as indicated by echocardiographic parameters and level of cardiac biomarkers N-terminal pro brain natriuretic peptide (NT-proBNP) and cardiac troponin I (cTnI). ANIMALS: 184 dogs with pre-clinical MMVD and left atrium-to-aortic root ratio (LA:Ao) ≥1.6 and normalized left ventricular end-diastolic diameter (LVEDDn) ≥1.7. METHODS: This is a prospective, randomized, multicenter, single-blinded, placebo-controlled study. Primary outcome variable was time-to-onset of first occurrence of HF or cardiac death. Secondary end points included effect of treatment on progression of the disease based on echocardiographic and radiographic parameters, as well as variations of NT-proBNP and cTnI concentrations. RESULTS: The median time to primary end point was 902 days (95% confidence interval (CI) 682-not available) for the treatment group and 1139 days (95% CI 732-NA) for the control group (p = 0.45). Vertebral heart score (p = 0.05), LA:Ao (p < 0.001), LVEDDn (p < 0.001), trans-mitral E peak velocity (p = 0.011), and NT-proBNP (p = 0.037) were lower at the end of study in the treatment group. CONCLUSIONS: This study failed in demonstrating that combined administration of spironolactone and benazepril delays onset of HF in dogs with preclinical MMVD. However, such treatment induces beneficial effects on cardiac remodeling and these results could be of clinical relevance.

Short communication: Role of Streptococcus pluranimalium in Mediterranean buffaloes (Bubalus bubalis) with different udder health statuses.

The aims of the current study were to describe presence and clinical role over time of Streptococcus pluranimalium isolated in milk samples of Mediterranean buffalo (MB). Two hundred composite milk samples originating from 40 primiparous MB were collected at 10, 30, 60, 90, and 150d in milk (DIM) and from 20 pluriparous MB at 77 to 120 DIM. Milk samples were used for analysis of somatic cell counts, bacteriological cultures, and identification (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry). Nine of 200 (4.5%) samples of primiparous MB and 3 of 20 (15%) samples of pluriparous MB were positive for Strep. pluranimalium. The prevalence of the bacterium in primipari was 0% (0/40) at 10, 30, and 150 DIM, whereas it was 5 (2/40) and 17.5% (7/40) at 60 and 90 DIM, respectively. Eight primipari were positive only once, whereas 1 was positive at 2 different samplings. Mono-infection was not detected in any of the age categories or udder health status. Infections were transient in primipari. Clinical mastitis was observed in primipari once at 90 DIM, subclinical mastitis detected twice in the same animals at 60 and 90 DIM, and intramammary infections were diagnosed 1 and 5 times at 60 and 90 DIM in primipari, respectively, whereas 3 infections were diagnosed in pluripari. The clinical reflections demonstrate for the first time the presence of Strep. pluranimalium in MB and its association with different udder health status. Nevertheless, it cannot be excluded that the bacterium may simply follow a pattern of commensal or opportunistic behavior, taking advantage of a preexisting bacterial udder infection.

Clinical outcomes and molecular genotyping of Staphylococcus aureus isolated from milk samples of dairy primiparous Mediterranean buffaloes (Bubalus bubalis).

Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows and in Mediterranean buffaloes. Genotype B (GTB) is contagious in dairy cows and may occur in up to 87% of cows of a dairy herd. It was the aim of this study to evaluate genotypes present, clinical outcomes, and prevalence of Staph. aureus in milk samples of primiparous Mediterranean dairy buffaloes. Two hundred composite milk samples originating from 40 primiparous buffaloes were collected from May to June 2012, at d 10, 30, 60, 90, and 150 d in milk (DIM) to perform somatic cell counts and bacteriological cultures. Daily milk yields were recorded. Before parturition until 40 to 50 DIM, all primiparous animals were housed separated from the pluriparous animals. Milking was performed in the same milking parlor, but the primiparous animals were milked first. After 50 DIM, the primiparous were mixed with the pluriparous animals, including the milking procedure. Individual quarter samples were collected from each animal, and aliquots of 1 mL were mixed and used for molecular identification and genotyping of Staph. aureus. The identification of Staph. aureus was performed verifying the presence of nuc gene by nuc gene PCR. All the nuc-positive isolates were subjected to genotype analysis by means of PCR amplification of the 16S-23S rRNA intergenic spacer region and analyzed by a miniaturized electrophoresis system. Of all 200 composite samples, 41 (20.5%) were positive for Staph. aureus, and no genotype other than GTB was identified. The prevalence of samples positive for Staph. aureus was 0% at 10 DIM and increased to a maximum of 22/40 (55%) at 90 DIM. During the period of interest, 14 buffaloes tested positive for Staph. aureus once, 6 were positive twice, and 5 were positive 3 times, whereas 15 animals were negative at every sampling. At 90 and 150 DIM, 7 (17.5%) and 3 buffaloes (7.5%), respectively, showed clinical mastitis (CM), and only 1 (2.5%) showed CM at both samplings. At 60, 90, and 150 DIM, 1 buffalo was found with subclinical mastitis at each sampling. At 30, 60, 90, and 150 DIM, 2.5 (1/40), 22.5 (9/40), 35 (14/40), and 10% (4/40) were considered affected by intramammary infection, respectively. Buffaloes with CM caused by Staph. aureus had statistically significantly higher mean somatic cell count values (6.06 ± 0.29, Log10 cells/mL ± standard deviation) and statistically significantly lower mean daily milk yields (7.15 ± 1.49, liters/animal per day) than healthy animals (4.69 ± 0.23 and 13.87 ± 2.64, respectively), buffaloes with IMI (4.82 ± 0.23 and 11.16 ± 1.80, respectively), or with subclinical mastitis (5.47 ± 0.10 and 10.33 ± 0.68, respectively). Based on our knowledge, this is the first time that Staph. aureus GTB has been identified in milk samples of dairy Mediterranean buffaloes.

Short communication: effects of systemic treatment with penethamate hydriodide on udder health and milk yields in dry primiparous Mediterranean buffaloes (Bubalus bubalis).

The effects of penethamate hydriodide (Mamyzin, Boehringer Ingelheim, Ingelheim, Germany) on udder health and milk yields were evaluated in primiparous Mediterranean buffaloes (Bubalus bubalis). An intramuscular administration of 10 million international units was performed in 20 buffaloes at 7 d precalving (treatment group; TG), and 20 animals were enrolled as the control group (CG). Evening milk samplings were performed at 10, 30, and 60 d in milk (DIM). Somatic cell count (SCC) values were evaluated on composite milk samples, whereas bacteriological culture and California Mastitis Test were performed on quarter milk. Daily milk yields were recorded after all milkings. After 60 DIM, composite milk samples from each animal were collected for monthly SCC and bacteriological culture until drying off. Statistically significant differences were found between the prevalence of mastitic quarters in the 2 groups at 10 and 30 DIM, and between the incidence of mastitic animals during the examined period (TG: 4/20, 20% vs. CG: 10/20, 50%). Even though lower and higher values of SCC and milk yields were found in TG during each sampling, statistically significant differences were only found at 30 (SCC) and 60 DIM (milk yields). In our study, the antibiotic administration precalving showed good bactericidal activity against the most common udder-specific pathogens that cause mastitis in primiparous Mediterranean buffaloes, and greater efficacy was observed at 10 and 30 DIM compared with 60 DIM. Given the significant decrease in SCC and increase in yields achieved, use of this antibiotic could be economically beneficial in buffalo breeding.

Clotting profile in cattle showing chronic enzootic haematuria (CEH) and bladder neoplasms.

Primary haemostasis (bleeding and blood clotting time), activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin III (ATIII), protein C, protein S, fibrinogen and D-dimer were determined in 13 cattle affected by chronic enzootic haematuria (CEH) and bladder neoplasms and 10 healthy cattle (control group). Increases in antithrombin III and protein S activities (P<0.01) and protein C and fibrinogen plasma levels (P<0.05) were observed in sick animals, while activated partial thromboplastin time, prothrombin time, and D-dimer did not show significant differences when compared to healthy animals. The clotting profile observed does not seem responsible for the chronic bleeding typical of CEH. The observed modification of some coagulation markers may derive from multiple interactions among cancer, inflammation and viral infection status typical of this syndrome.

Secondary immune-mediated thrombocytopenia in dogs naturally infected by Leishmania infantum.

Forty-four dogs naturally infected by Leishmania infantum were divided into two groups: 20 thrombocytopenic dogs with fewer than 150 x 10(9) platelets/l, and 24 non-thrombocytopenic dogs with more than 200 x 10(9) platelets/l. Ten clinically healthy dogs were used as controls. A haematological profile was obtained and the dogs' serum was used to assess the presence of platelet-binding IgM and IgG antibodies using a flow cytometry technique. Nineteen of the 20 thrombocytopenic dogs, and 13 of the 24 non-thrombocytopenic dogs had detectable levels of platelet-binding immunoglobulins, but none of the control dogs did so. The differences were significantly different for both IgM and IgG platelet-binding antibodies.

Cutaneous sterile granulomas/pyogranulomas, leishmaniasis and mycobacterial infections.

Cutaneous "sterile" granulomas represent a group of uncommon skin disorders of unknown aetiopathogenesis. Many diseases are included in this group (for example, sterile granuloma/pyogranuloma syndrome and reactive histiocytosis). The definition of sterile is based on the exclusion of other possible aetiological agents (for example, microorganisms or foreign body). Many techniques are used to rule out a microbial aetiology including cytology, histology, immunohistochemistry and culture. However, some organisms are "fastidious" and difficult to culture or to identify with routine methods, and molecular studies are necessary. This is particularly true for mycobacteria (for example, canine leproid granuloma syndrome) and Leishmania. Recently, studies in human and veterinary medicine have proved the presence of microorganisms (mycobacteria and Leishmania) using a polymerase chain reaction technique in specimens previously diagnosed as sterile. Therefore, it is very important, with the development of new technologies, to use a multidisciplinary diagnostic approach to definitively rule out any microorganism before declaring a disease sterile.

Platelet aggregation and haemostatic response in dogs naturally co-infected by Leishmania infantum and Ehrlichia canis.

Haemostatic alterations in dogs naturally infected by ehrlichiosis and/or leishmaniasis were studied. Platelet count, ADP and collagen-induced platelet aggregation, prothrombin time, activated partial thromboplastin time (APTT) and plasma fibrinogen concentration were measured. An evident reduction of platelet aggregation response was shown for Leishmania-Ehrlichia co-infected dogs where platelet aggregation was lower in comparison with control and leishmaniotic dogs (ADP and collagen, P < or = 0.01) and ehrlichiotic dogs (ADP 10 and 7.5 microm, P < or = 0.05). Moreover, a significant increase in APTT as well as a reduction of the albumin/globulin rate (A/G) for leishmaniotic and co-infected dogs versus control and ehrlichiotic dogs was detected. The hypothesis of a synergism between leishmaniosis and ehrlichiosis in altering platelet function by different pathways is discussed.

Presence of anti-platelet IgM and IgG antibodies in dogs naturally infected by Leishmania infantum.

Thirty-three dogs, naturally infected by Leishmania infantum, were enrolled in the study and were classified as oligo-symptomatic (n. 15) and symptomatic or markedly symptomatic (n. 18). A control group was 10 healthy dogs. A haematological profile was obtained and the dogs serum was employed to assess the presence of platelet binding IgM and IgG antibodies (PBIgM, PBIgG) using flow cytometry. FITC labelled goat anti-dog IgM or IgG were used to detect PBIgM and PBIgG. Samples with a mean fluorescence intensity (MFI) that was 100 channels higher on a log scale for more than 30% of the platelets than seen in negative control platelets from a healthy dog were considered positive for the presence of anti-platelet antibodies (PBIg). Twenty-one (63.3%) dogs revealed the presence of PBIg. Six of them were oligo-symptomatic while 15 showed moderate or severe clinical signs of illness. All the dogs with PBIg showed the presence of PBIgM, with nine animals showing both PBIgM and PBIgG. Nine of 18 symptomatic or markedly symptomatic dogs showed thrombocytopenia, while normal platelet counts were observed in all oligo-symptomatic animals. Eight of 9 thrombocytopenic animals showed the presence of PBIgM, while six of them showed PBIgG. One thrombocytopenic dog was negative for PBIg. This study is the first report documenting the presence of PBIg in natural canine leishmaniasis implying a pathogenic association between thrombocytopenia and the presence of antibody against platelet membrane.

Haematological profile on non-lactating Mediterranean buffaloes (Bubalus bubalis) ranging in age from 24 months to 14 years.

Haematological studies were performed on 100 clinically normal non-lactating Mediterranean buffalo species ranging in age from 24 months to 14 years, to determinate the range of normal haematological values for this ruminant species. The animals were divided in 5 groups according to age: Group I, 2-3 years old which had never calved, Group II, 3-4 years old (primipara buffaloes), Group III, 5-7 years old, Group IV 8-10 years old and Group V over 10 years of age. All the haematological values obtained were comparable with the normal values found in adult cattle, and similar to those reported in Indian water buffalo species. The heifer buffalo showed an higher values for packed cell volume (PCV) compared with the older animals, but lower values for mean corpuscular haemoglobin concentration (MCHC) and mean corpuscular haemoglobin (MCH) (P 0.01). In animals above 8 years of age, the white cell count was lower with a significant reduction in absolute values of lymphocytes (P 0.01). Higher absolute values of eosinophils levels was found in the group V (P 0.01).