From the 497 samples, 358 sera were from Mato Grosso do Sul state where it represent ∼40% of total beef cattle breed

in Brazil, and the Bos indicus is the main breed, and 139 from Rio Grande do Sul state were ∼15% of the beef cattle is breed, and the main breed is Bos taurus. The samples were collected in different randomly establishments during the period of January to November 2008. The peripheral blood was collected from the jugular vein of Selleck Ku-0059436 adult bovine, using a 19 g needle attached to Vacutainer tubes (Becton-Dickinson, Rutherford, NJ) and stored at −20 °C until use. The antigen for IFAT was prepared as following: the cells infected with N. caninum tachyzoites were diluted in PBS buffer in order to obtain ∼1 × 106 taquizoites/mL, then 20–30 μL (30,000 taquizoites) were add by slide well. http://www.selleckchem.com/Wnt.html Then the slides were dry at 37 °C and stored at −20 °C until use. The sera samples were analyzed at a dilution of 1:50, defined as the cut-off point, using the method previously described ( Pare et al., 1995 and Trees et al., 1993). The sera diluted at 1:50 in PBS buffer were incubated for 45 min at 37 °C. Bound bovine antibodies were detected with fluorescein isothiocyanate-conjugated

anti-bovine IgG (Sigma Chemicals, USA) at a dilution of 1:1125 in PBS buffer for 45 min at 37 °C. Each glass slide included negative and positive control sera. The antigenic domain of NcSRS2, located in the distal ADAMTS5 C-terminal two thirds of the molecule, was amplified by PCR using primers F5′-CAC CAA AGA GTG GGT GAC TGG and R5′-GGT AAG CTT TGC ATC TCC TCT TAA CAC-3′ and cloned into pET100/D-TOPO vectors (Invitrogen Tech, Carlsbad, CA, USA). The recombinant plasmid (pET100/D-TOPO/NcSRS2) was used

for transformation into Escherichia coli BL21 Star (DE3) (Invitrogen Tech, Carlsbad, CA, USA). The E. coli cells in the log phase were treated with 0.75-mM isopropyl α-d-thiogalactoside (IPTG) for 3 h at 37 °C to induce expression of fused fragments of NcSRS2. The recombinant NcSRS2 expression was confirmed by SDS-PAGE and Western blotting using anti-6×His alkaline phosphatase conjugate (1:10,000) (Sigma Chemicals, USA). Antibody-reacting protein bands were revealed using 3,3′-tetrahydrochloride (DAB) and H2O2. The protein was purified using affinity chromatography on a HiTrap chelating column (GE Healthcare, UK) charged with Ni2+ ions. The protein was solubilized in a buffer containing 0.2% N-lauroyl sarcosine or 8-M urea. Subsequently, the concentration and purity of recombinant NcSRS2 were determined using a BCA kit (Pierce, Rockford, IL, USA) and SDS-PAGE, respectively. Purified recombinant NcSRS2 and an unrelated recombinant protein (negative control) were used for Western blotting analysis of positive and negative bovine sera. The samples were mixed with SDS gel-loading buffer (100-mM Tris–HCl at pH 6.8, 100-mM 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) under reducing conditions.