, 1988). Rabbit anti-Leptospira Androgen Receptor antagonist antisera to serogroup Icterohaemorrhagiae serovars: Copenhageni, Icterohaemorrhagiae (strain RGA), Icterohaemorrhagiae (strain Kantorowics), and Lai; serogroup Canicola serovars: Canicola (strain Hond Utrecht IV), Galtoni (strain LT1014), Jonsis (strain Jones); serogroup Sarmin serovars: Sarmin (strain Sarmin), Machiguenga (strain MMD3), Waeveri (strain CZ 390); serogroup Grippotyphosa serovars: Grippotyphosa (strain Moskva

V), Valbuzzi (strain Valbuzzi), Liangguang (strain 1880); serogroup Sejroe serovars: Hardjo (strain Hardjoprajitno), Sejroe (strain M84), Wolffi (strain 3705); serogroup Pomona serovar Pomona; and serogroup Australis serovars: Bratislava (strain Jez Bratislava), Australis (strain Ballico), Fugis

(strain Fudge) were generated as described previously (Stallman, 1984). The medium used for the selection of escape mutants was EMJH containing 10% v/v mouse ascites fluid containing the mAb F70C7. Initially, a stationary-phase culture of Lai wild type (LaiWT) grown in EMJH medium was supplemented with mouse ascites fluid (mAb: F70C7) to 10% v/v and incubated at 30 °C for a week. One millilitre of the culture was then inoculated into 10 mL of EMJH, 10% mouse ascites (mAb: F70C7) selection medium. This procedure was repeated five times until no agglutination with the F70C7 mAb was observed by MAT, indicating the loss of the reactive epitope next (Zuerner & Trueba, 2005). The mutant

strain RG7422 cost was then cloned by performing 10 serial 10-fold dilutions in EMJH liquid medium and selecting the tube showing growth obtained from the highest dilution. The genetic relatedness of LaiMut and LaiWT was investigated by restriction fragment length polymorphism (RFLP) using EcoRI and BamHI and by sequencing regions of the lipopolysaccharide locus using a series of primers designed to provide double-stranded coverage of the targeted region of the genome (Victoria et al., 2008; Ahmed et al., 2009). For sequencing, DNA extraction was performed using the QIAamp DNA extraction kit according to the manufacturer’s instructions (Qiagen GmbH, D-40724 Hilden, Germany). DNA sequencing was carried out using the BigDye Ready Reaction Dye Deoxy terminator cycle sequencing kit. Sequence products were resolved on an Applied Biosystems 3730 capillary sequencer. The MAT was performed with 16 mAbs reacting with serogroup Icterohaemorrhagiae antigens (Table 1) and three polyclonal antisera reacting with other serogroups using a standard protocol (Cole et al., 1973). The use of a 1/10 starting dilution was intended to increase the sensitivity of the assay.