Blood samples were collected from the coccygeal or jugular vein of all cattle in each herd on the respective visit. Each farm was visited over a two-year period, at three-month intervals. It was not possible to collect precolostral samples from calves. Blood samplings of calves younger than four months were not used to avoid false-positive as result of the presence of colostral antibodies against N. caninum ( Cardoso et al., 2008). The samples were collected at Farm I from March 2004 to March 2006, at Farm II from September 2004 to September 2006 and at Farm III from October 2005 to October 2007. After centrifugation at 1000 × g for 20 min, the sera were removed and stored at −20 °C until analysis.

Serum was tested for antibodies

against N. caninum by means of the indirect fluorescent antibody test (IFAT), using whole culture-derived tachyzoites (NC-1 strain) as the antigen, with a cutoff value of 1:100 ( PLX4032 clinical trial Dubey et al., 1988). Fluorescein isothiocyanate conjugated rabbit anti-bovine IgG (Sigma, St. Louis, MO, USA) was used at a dilution of 1:3000. Positive and negative control samples were added to each slide. The prevalence of N. caninum infection in herds was defined as the number CP-868596 purchase of seropositive animals divided by the total number of animals tested within that herd in a given sample collection. Only animals sampled more than once were used in analyzing N. caninum vertical transmission and seroconversion. To minimize the impact of a false-positive or false-negative result, the animals were classified as seropositive if the first two consecutive blood samples were positive, but otherwise, they were isothipendyl considered seronegative if the first two consecutive blood samples were negative ( Hietala and Thurmond, 1999). All dam-daughter serological results were recorded. The efficiency of vertical transmission was estimated as the proportion of

seropositive daughters born to seropositive dams (seropositive calves/seropositive dams × 100). The association between the serological status in dams and their offspring was measured and compared using 2 × 2 tables and the χ2 test. Seroconversion was designated when an animal classified as N. caninum negative or positive had, respectively, two consecutive positive or negative samples thereafter. The seroconversion rate and the proportion culled were expressed per 100 cow-years at risk ( Thrusfield, 2007). Differences in the rates of positive and negative seroconversion and culling and the association between age of seropositive cows and congenital infection rate were assessed by comparison between two independent proportions. Values are expressed as the Mean ± Standard Deviation (S.D.). Differences and association were considered statistically significant when P < 0.05. The statistical analysis was performed using EPIDAT (Pan-American Health Organization, USA). Blood samples from all animals were collected nine times at each of the three farms over the two years of observation.