Microscopic agglutination test (MAT) The microscopic agglutination test was performed Luminespib nmr according to [1]. In brief, an array of 22 serovars of Leptospira spp. as antigens were employed: Australis, Autumnalis, Bataviae, Canicola, Castellonis, Celledoni, Copenhageni, Cynopteri, Djasiman, Grippotyphosa, Hardjo, Hebdomadis, Icterohaemorrhagiae, Javanica, Panama, Patoc, Pomona, Pyrogenes, Sejroe, Shermani, Tarassovi and Wolffi. All the strains were maintained in EMJH liquid medium (Difco, USA) this website at 29°C. A laboratory – confirmed case of leptospirosis was defined by the demonstration of a four – fold microagglutination titer rise
between paired serum samples. The probable predominant serovar was considered to be the one with the highest dilution that could cause 50% of agglutination. MAT was considered negative when the titer was below 100. Characterization of the protein in silico Predicted coding sequence (CDSs) LIC11834 and LIC12253 were identified on L. interrogans serovar Copenhageni and selection was based on cellular localization; cellular localization prediction was performed by PSORT, http://psort.nibb.ac.jp[54] and PredictProtein web server, https://www.predictprotein.org/[25]. The SMART [23]http://smart.embl-heidelbergde/ and PFAM [55]http://www.sanger.ac.uk/Software/Pfam/ web servers were used to search for predicted functional and structural domains. The presence
of lipobox putative sequence MK0683 datasheet was evaluated by use of the LipoP program [56]http://www.cbs.dtu.dk/services/LipoP/. selleck screening library The predicted sequence of the lipobox was also assessed by use of the SpLip program, as described by Setubal
et al. [57]. Secondary structure, solvent accessibility and cellular localization predictions were also performed by using PredictProtein web server, https://www.predictprotein.org/[25]. DNA isolation and PCR analysis Leptospira cultures were harvested by centrifugation at 11,500 g for 30 min and gently washed in sterile PBS twice. Genomic DNA was isolated from the pellets by guanidine – detergent lysing method using DNAzol® Reagent (Invitrogen), according to the manufacturer’s instructions. Primers were designed according to L. interrogans serovar Copenhageni genome sequences (GenBank accession AE016823) and are listed in Table 1. PCR was performed in a reaction volume of 25 μl containing 100 ng of genomic DNA, 1 × PCR buffer (20 mM Tris – HCl, pH 8.4, 50 mM KCl), 2 mM MgCl2, 20 pmol of each specific primer, 200 μM of each dNTP, and 2.5 U Taq DNA Polymerase (Invitrogen). Cycling conditions were: 94 ° C – 4 min, followed by 40 cycles at 94°C – 50 sec, 57°C (LIC11834) or 56°C (LIC12253) – 50 sec, 72°C – 90 sec, and a final extension cycle of 7 min at 72°C. PCR amplified products were loaded on a 1% agarose gel for electrophoresis and visualization with ethidium bromide.