Validation of a human angiopoietin-2 ELISA for measurement of angiopoietin-2 concentrations in canine plasma samples and supernatant of primary canine aortic endothelial cell cultures.

OBJECTIVE To assess 2 human ELISA kits for measurement of angiopoietin-1 and -2 concentrations in canine plasma samples, determine whether plasma angiopoeitin-2 concentration differed between septic and healthy dogs, and determine the effect of tumor necrosis factor-α (TNF-α) stimulation on angiopoeitin-2 release from primary canine aortic endothelial cells (pCAECs) in vitro. ANIMALS 10 healthy dogs and 10 septic dogs. PROCEDURES Human angiopoietin-1 and -2 ELISAs were used to detect recombinant canine angiopoietins-1 and -2 in canine plasma samples. The angiopoietin-2 ELISA was further validated by use of plasma samples from healthy and septic dogs and supernatants of pCAEC cultures. Associations between plasma angiopoeitin-2 and C-reactive protein (CRP) concentrations were examined. RESULTS Angiopoeitin-2 but not angiopoeitin-1 was detected in canine plasma samples by the respective ELISAs. The angiopoeitin-2 ELISA had excellent dilutional linearity, parallelism, accuracy, precision, and reproducibility for measurements in canine plasma samples and pCAEC supernatants. Plasma angiopoeitin-2 concentration was significantly higher in septic dogs (median, 25.5 ng/mL) than in healthy dogs (median, 6.7 ng/mL) and was positively correlated with plasma CRP concentration (R2 = 0.60). Stimulation of pCAECs with TNF-α resulted in a significant increase in supernatant angiopoietin-2 concentration. CONCLUSIONS AND CLINICAL RELEVANCE The tested human angiopoietin-2 ELISA kit was useful for measuring angiopoietin-2 concentrations in canine plasma samples and pCAEC supernatants. Sepsis appeared to increase angiopoietin-2 concentration in dogs in vivo, whereas TNF-α stimulation caused release of angiopoietin-2 from pCAECs in vitro. These findings support the use of angiopoietin-2 as a marker of endothelial cell activation and inflammation in dogs.

Canine leptospirosis in Switzerland-A prospective cross-sectional study examining seroprevalence, risk factors and urinary shedding of pathogenic leptospires.

Leptospirosis is an important worldwide zoonosis. While human leptospirosis remains rare in Switzerland, the incidence of canine leptospirosis is unusually high compared to other European countries. The aims of this cross-sectional study were to determine the exposure of asymtomatic dogs to pathogenic Leptospira in Switzerland, to characterise risk factors associated with seropositivity and to determine the prevalence of urinary shedding. Sampling was stratified to cover the whole of Switzerland. Sera were tested by microscopic agglutination test for antibodies against a panel of 12 serovars. Urine was tested for pathogenic Leptospira using a LipL32 real-time PCR. Of 377 sera, 55.7% (95%CI 51.2-60.7) showed a reciprocal MAT titre of ≥1:40 and 24.9% (95%CI 20.7-29.4) of ≥1:100 to at least one serovar. Seropositivity (MAT ≥1:100) was most common to serovars Australis (14.9%; 95% CI 11.4-18.6) and Bratislava (8.8%; 95%CI 6.1-11.7), followed by Copenhageni (6.1%; 95%CI 3.7-8.5), Canicola (5%; 95%CI 2.9-7.4), Grippotyphosa (4.5%; 95%CI 2.7-6.9), Pomona (4%; 95%CI 2.1-6.1), Autumnalis (2.7%; 95%CI 1.3-4.2) and Icterohaemorrhagiae (1.6%; 95%CI 0.5-2.9). In unvaccinated dogs (n=84) the prevalence of a MAT titre ≥100 was 17.9% (95%CI 10.7-26.2), with a similar distribution of reactive serovars. Variables associated with seropositivity (≥1:40) to any serovar included age (OR 1.29/year; 95%CI: 1.1-1.5) and bioregion with higher risks in the regions Northern Alps (OR 14.5; 95%CI 2.2-292.7), Central Plateau (OR 12.3; 95%CI 2.0-244.1) and Jura (OR 11.2; 95%CI 1.7-226.7) compared to Southern Central Alps. Dogs living with horses were significantly more likely to have antibodies to serovar Bratislava (OR 4.68;95%CI 1.2-17.2). Hunting was a significant risk factor for seropositivtiy to serovar Grippotyphosa (OR 8.03; 95%CI 1.6-30.8). Urine qPCR positivity was uncommon (1/408 dogs; 0.2%; 95% CI0-0.7). These results demonstrate that dogs in Switzerland are commonly exposed to pathogenic Leptospira; however, the risk of dogs contributing to the spread of Leptospira in the environment appears low.