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.

Helminth infections in Italian donkeys: Strongylus vulgaris more common than Dictyocaulus arnfieldi.

Donkeys have been used as working animals for transport and farm activities worldwide. Recently, in European countries, there has been an increasing interest in donkeys due to their use as pets, onotherapy or milk production. During 2014-2016, a countrywide survey was conducted to determine prevalence and risk factors of principal helminth infections in 1775 donkeys in 77 Italian farms. A questionnaire on management and parasite control practices was filled out for each farm. Faecal samples were examined using a modified McMaster technique, a centrifugation/flotation method and a sedimentation technique. Pooled coprocultures were performed for differentiation of strongylid eggs. Strongyles were the most common parasites detected (84.9%), followed by Dictyocaulus arnfieldi (6.9%), Oxyuris equi (5.8%), Parascaris spp. (3.6%), Anoplocephala spp. (1.0%), Strongyloides westeri (0.3%). Coprocultures revealed an omnipresence of cyathostomins (100%), followed by Strongylus vulgaris (31.0%), Poteriostomum spp. (25.0%), Triodontophorus spp. (9.0%), Strongylus edentatus (7.0%), Strongylus equinus (5.0%). Logistic regression analysis identified breed, co-pasture with horses, living area, herd size and number of treatments as significantly associated with strongyles. Sex, age, living area and herd size were significantly associated with Parascaris spp. Dictyocaulus arnfieldi was significantly associated with sex, grass, co-pasture with horses, living area and herd size. Strongylus vulgaris was significantly associated with living area and herd size. The mean number of anthelmintic treatments/year was 1.4; most of the donkeys (71.8%) were dewormed using an ivermectin drug. It is important to design parasite programs to specifically address both D. arnfieldi and S. vulgaris in donkeys, and this is especially important if donkeys co-graze with horses.

Distribution and risk factors of canine haemotropic mycoplasmas in hunting dogs from southern Italy.

Mycoplasma haemocanis (Mhc) and "Candidatus Mycoplasma haematoparvum" (CMhp) are the main haemoplasma species known to infect dogs. The aim of this study was to determine the prevalence of haemoplasma species infections in hunting dogs from southern Italy and assess related risk factors. 1,433 hunting dogs living in Campania region were tested by qPCR assay. The prevalence was 19.9 %; 13.1 % for Mhc and 11.4 % for CMhp; 4.6 % showed a coinfection with both haemoplasma species. Statistical analysis revealed living in Salerno province (Mhc: OR 3.72; CMhp: OR 2.74), hound (Mhc: OR 5.26; CMhp: OR 8.46) and mixed breed (Mhc: OR 3.38; CMhp: OR 2.80), rural environment (Mhc: OR 12.58; CMhp: OR 10.38), wild mammal hunting (Mhc: OR 8.73; CMhp: OR 8.32), cohabitation with other animals (Mhc: OR 2.82; CMhp: OR 2.78) and large pack size (Mhc: OR 2.96; CMhp: OR 1.61) as risk factors for haemoplasmas. Male gender (OR 1.44) and tick infestation history (OR 1.40) represented risk factors only for Mhc, while adult age (2-7 years - OR 2.01; > 7 years - OR 1.84) and large body size (OR 1.48) were associated only to CMhp. Mhc infection was significantly associated to Babesia vogeli (p < 0.05) and Hepatozoon canis (p < 0.001), while CMhp with H. canis (p < 0.001). This study adds information on haemoplasma species distribution in hunting dogs in southern Italy. Outdoor lifestyle and contact with wild fauna, through greater exposure to tick infestation, or possibly wounds acquired during hunting or fighting, could be factors contributing to haemoplasma infections.

Hepatozoon canis in hunting dogs from Southern Italy: distribution and risk factors.

Hepatozoon canis is a hemoprotozoan organism that infects domestic and wild carnivores throughout much of Europe. The parasite is mainly transmitted through the ingestion of infected ticks containing mature oocysts. The aims of the present survey were to determine the prevalence of H. canis in hunting dogs living in Southern Italy and to assess potential infection risk factors. DNA extracted from whole blood samples, collected from 1433 apparently healthy dogs living in the Napoli, Avellino, and Salerno provinces of Campania region (Southern Italy), was tested by a quantitative real-time polymerase chain reaction (qPCR) assay to amplify H. canis. Furthermore, the investigated dog population was also screened by qPCR for the presence of Ehrlichia canis, a major tick-borne pathogen in Southern Italy, in order to assess possible co-infections. Two hundred dogs were H. canis PCR-positive, resulting in an overall prevalence of 14.0% (CI 12.2-15.9). Breed category (P < 0.0001), hair coat length (P = 0.015), and province of residence (P < 0.0001) represented significant risk factors for H. canis infection. The presence of H. canis DNA was also significantly associated with E. canis PCR positivity (P < 0.0001). Hunting dogs in Campania region (Southern Italy) are frequently exposed to H. canis, and the infection is potentially associated with close contact with wildlife. Further studies are needed to assess the pathogenic potential of H. canis, as well as the epidemiological relationships between hunting dogs and wild animal populations sharing the same habitats in Southern Italy.

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.

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.

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.

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.

Failure of a multi-subunit recombinant leishmanial vaccine (MML) to protect dogs from Leishmania infantum infection and to prevent disease progression in infected animals.

We report results of a Phase III trial of the multi-subunit recombinant Leishmania polyprotein MML for the protection of dogs against infection by Leishmania infantum. The antigen, also known as Leish-111f, is the first antileishmanial human vaccine entered Phase I clinical testing. The study was performed in a leishmaniasis endemic area of southern Italy. Three groups of 15 Leishmania-free beagle dogs each, received 3 monthly injections with vaccines A (MML+MPL-SE adjuvant), B (sterile saline = control) and C (MML+Adjuprime adjuvant), respectively, before transmission season 2002. The surviving dogs received a second three-dose vaccine course 1 year later. The dogs were naturally exposed to sandfly bites for 2.5 months in 2002, and for 5 months in 2003. Every 2 months post vaccination, dogs were examined by clinical and immunological evaluation, and by specific serology, microscopy, culture and PCR. A weak lymphoproliferative response to MML was seen in A and C groups throughout the study period. One year after the first vaccine course, the cumulative incidence of leishmanial infections was 40% in group A, 43% in group B and 36% in group C. Two-year post-vaccination (1 year after the second vaccine course) the cumulative incidence was 87% in group A (with three symptomatic cases), 100% in group B (with no symptomatic cases) and 100% in group C (with two symptomatic cases). The efficacy of the MML vaccine as an immunotherapeutic agent for the prevention of disease progression (subpatent infection-->asymptomatic patent infection-->symptomatic patent infection) was evaluated through follow-up of dogs found infected prior to the second vaccination. Among 15 infected animals, progression to a subsequent stage of infection was found in 5/6 dogs of group A, 3/6 of group B and 2/3 of group C. We conclude that vaccination with MML is not effective to prevent leishmaniasis infection and disease progression in dogs under field conditions.

Biochemical indicators of bone metabolic activity in buffalo (Bubalus bubalis) during late pregnancy and early lactation.

Blood levels of calcium, inorganic phosphorus, magnesium, osteocalcin, intact parathyroid hormone, calcitonin, alkaline phosphatase activity, creatinine and thyroid hormones were estimated in 10 healthy buffalo during late pregnancy (30, 15 days and 7 days before calving), within 12 h after calving and 7-15-30-45 and 60 days after calving. The almost constant serum levels of calcium, phosphorus, intact parathyroid hormone, and the low calcitonin concentration indicate that these buffalo need to utilize only a little of their endogenous mineral resources. Bone-turnover could be demonstrated by variations in the serum levels of osteocalcin and alkaline phosphatase activity. A study of these bone markers could be useful for other research purposes and for future clinical application in pathological conditions.

Decreased lipid fluidity of the erythrocyte membrane in dogs with leishmaniasis-associated anaemia.

In both man and the dog, anaemia resulting from natural leishmaniasis is often severe and mainly associated with a shortened life span of erythrocytes. Lipid fluidity of erythrocyte membranes from 17 dogs with anaemia caused by visceral leishmaniasis was investigated by means of fluorescence polarization. Results were compared with those from three groups of control animals (10 healthy dogs, seven dogs with visceral leishmaniasis but no anaemia, and 10 dogs with anaemia unrelated to leishmaniasis). Fluorescence polarization values recorded for animals with leishmaniasis-associated anaemia were elevated-indicating reduced erythrocyte membrane fluidity-and significantly higher than in the control groups. Mechanical sequestration by the spleen due to increased cell rigidity, or alterations in receptor/ligand erythrocyte cytoadherence mechanisms, or both, may result from decreased membrane fluidity and hence contribute to the anaemia of Leishmania -infected dogs.

Early suppression of lymphoproliferative response in dogs with natural infection by Leishmania infantum.

Dogs are the domestic reservoirs of zoonotic visceral leishmaniasis caused by Leishmania infantum. Early detection of canine infections evolving to clinically patent disease may be important to leishmaniasis control. In this study we firstly investigated the peripheral blood mononuclear cell (PBMC) response to leishmanial antigens and to polyclonal activators concanavalin A, phytohemagglutinin and pokeweed mitogen, of mixed-breed dogs with natural L. infantum infection, either in presymptomatic or in patent disease condition, compared to healthy animals. Leishmania antigens did not induce a clear proliferative response in any of the animals examined. Furthermore, mitogen-induced lymphocyte proliferation was found strongly reduced not only in symptomatic, but also in presymptomatic dogs suggesting that the cell-mediated immunity is suppressed in progressive canine leishmaniasis. To test this finding, naive Beagle dogs were exposed to natural L. infantum infection in a highly endemic area of southern Italy. Two to 10 months after exposure all dogs were found to be infected by Leishmania, and on month 2 of exposure they all showed a significant reduction in PBMC activation by mitogens. Our results indicate that suppression of the lymphoproliferative response is a common occurrence in dogs already at the beginning of an established leishmanial infection.