Amplifications were
done in a total volume of 25 μl containing Tris–HCl, KCl, (NH4)2SO4, 8.0 mM of MgCl2, and 1.25 U HotStar Taq Polymerase (Qiagen, Hilden), as well as 200 μM of each dNTP, 1 μM of each primer, 0.2 μM of each probe and 250 ng of DNA. For each PCR test, positive controls of genomic babesial DNA from each subspecies and negative controls containing DNA from uninfected dogs were included in every run. Amplifications of target genes were done using an iCycler (Biorad, Munich) with an initial Taq Polymerase activation step of 13 min at MS-275 concentration 95 °C, followed by 50 cycles at 95 °C for 30 s, annealing at 50 °C for 30 s and elongation at 72 °C for 30 s. Fluorescence was measured at each annealing step. Reactions were evaluated using the software version 3.1 (iCycler iQ Real Time PCR detection system, Biorad) and were regarded as positive if the amount of fluorescence exceeded a threshold value (basal emission
plus the 10 fold of its standard deviation) and followed a curve of a sigmoid shape. Blood samples were collected from dogs living in farms in three regions within the State of Minas Gerais, Brazil: Lavras (latitude – S 21°20′; longitude – W 45°00′), Belo Horizonte (latitude – S 19°55′; longitude – W 43°56′) and Nanuque (latitude – S 17°49′; longitude – W 40°20′). In these areas two seasons are well defined during the year: a dry season (from April to September) and a rainy season (from October to March). The climatic data, as referred in http://www.agritempo.gov.br/agroclima/pesquisaWeb?uf=MG, were obtained throughout the experimental HIF inhibitor period for each region. Blood samples were collected during a dry season of 2004 from 252 dogs living in the following locations: Lavras (n = 100), found Belo Horizonte (n = 50) and Nanuque (n = 102). In the subsequent rainy season, a total of 166 dogs were re-sampled, as followed: Lavras (n = 71), Belo Horizonte (n = 29) and Nanuque
(n = 66). From each sample, DNA was extracted using the Wizard Genomic DNA Purification (Promega, Madison, USA) and Giemsa stained smears were microscopically examined for direct detection of Babesia parasites. DNA concentration was determined using the spectrophotometer NanoDrop ND-1000 (NanoDrop, Wilmington, USA) and DNA samples were diluted using ultrapure water to reach a concentration of 50 ng/μl. The Chi-square test was used to evaluate associations between prevalence and incidence among municipalities and seasons. The Kappa test was performed to compare detection in blood smears and by the Real Time PCR. Although the Real Time PCR developed in this study had been designed to detect all subspecies of B. canis ( Fig. 1), only B. canis vogeli was found in all three regions analyzed. Prevalence rates by region and season are shown in Table 2. Direct examination of blood smears detected few positive animals (0.8% during dry season, and 0.0% during the rainy season), while the Real Time PCR detected 9.9% of positive animals during the dry season and 10.8% during the rainy season.