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.

Prevalence of Dirofilaria repens in dogs living in deltaic coastal plain of the Volturno River (Italy): a geographical risk model of infection.

The prevalence of vector-associated parasitic infections is high in central-southern Italy. The deltaic coastal plain of the Volturno River has been suspected, by veterinary practitioners, to have a high accidental incidence of Dirofilaria repens. Thus, the goal of this study was to evaluate the prevalence of dirofilariasis and other coinfections frequently described in dogs living in the Volturno area. Blood samples of 100 clinical asymptomatic dogs were examined using a Knott's technique and polymerase chain reaction in order to identify microfilariae. Other vector-borne coinfections were also investigated using ELISA kits. The results were analysed using statistical and Geographic Information System (GIS) software. Microfilariae of D. repens were detected in 10% of the dogs surveyed, with a presence of antibodies against Ehrlichia canis (4/10) and Dirofilaria immitis (1/10). Such high incidence should be considered in light of the zoonotic potential for D. repens and the support for more regular use of repellents to prevent the spread of this disease. The GIS analyses indicated that the study area provides suitable conditions to sustain populations of mosquito vectors and D. repens parasites throughout much of the year.

Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infections in donkeys from Southern Italy.

Equine piroplasmosis (EP) has been frequently described in donkeys in subtropical and tropical regions, but published data reflecting large scale surveys are very limited in Europe. The seroprevalence of Babesia caballi and Theileria equi was determined in a donkey population from Campania Region in Southern Italy using a commercial indirect fluorescent antibody test (IFAT), and the risk factors associated with the occurrence of the infection were assessed. Of 203 samples, the overall seroprevalence for EP was 57.1% (116/203), with 35.5% (72/203) for B. caballi and 44.3% (90/203) for T. equi. Co-infection was detected in 46 donkeys (22.6%). The distribution of IFAT antibody titres to B. caballi was: 1:80 (n= 67), 1:160 (n= 2), 1:320 (n= 3); while the distribution of IFAT antibody titres to T. equi was: 1:80 (n= 25), 1:160 (n= 42), 1:320 (n= 12), 1:640 (n= 8), 1:1280 (n= 3). All examined donkeys were asymptomatic, except one adult male (with a titre of 1:640 against T. equi) that showed clinical signs corresponding to the acute stage of EP, reported for the first time in Italy. The unique risk factor associated with a higher B. caballi seroprevalence was the presence of horses in the farms, while risk factors associated with a higher T. equi seroprevalence were poor body condition, presence of ruminants in the farms and milk production. The results indicate a high level of exposure in donkeys living in Southern Italy and suggest that donkeys may be an important reservoir of EP.

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.

Seroprevalence and risk factors of Neospora spp. in donkeys from Southern Italy.

In some European countries there is an increasing interest on donkey. Despite there are few data regarding the donkey's parasitic diseases especially those with a protozoal etiology as neosporosis. Samples used in the study were collected from 238 domestic donkeys during year 2010 in Southern Italy from 207 females and 31 males of five breeds (Martina-Franca, Amiata, Sicilian-Grey, Ragusano, Sardinian) and crossbreeds with the average age 9 years (1 month - 24 year). Sera were tested by a competitive-inhibition enzyme-linked immunosorbent assay for antibodies against Neospora caninum; the sera were marked positive, if more than 30% inhibition was found. Out of a total 238 donkeys, 28 (11.8%) were found positive for Neospora antibodies with 12% in females and 6% in males. Different seroprevalence 15.4%, 16%, 12% and 8.8% were found in age categories <1 year, 1-4 years, 5-9 years and ≥10 years, respectively. The seroprevalence ranged in different breeds from 36% (Sicilian-Grey) to 0% (Sardinian) and in different use from 17% (for breeding) to 0% (for meat production). Logistic regression analysis demonstrated evidence of a significant (P<0.05) association between crossbreed origin of samples and risk of protozoan infection; age of donkeys was also significant risk factor for protozoan infection. No statistical significant difference (P>0.05) was found among genders and use of donkeys and risk of N. caninum infection. This is the first serological survey for Neospora spp. performed in donkeys.

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.

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.