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Abstract: Leptospirosis, caused by different Leptospira species, is one of the most widespread zoonotic infections worldwide. Here we expressed three major leptospiral lipoproteins and examined their immunogenicity. All the pathogenic Leptospira strains tested possess the lipL21, lipL32 and lipL41 genes, but the latter two can be further divided into different gene types (lipL32-1, lipL32-2, lipL41-1, lipL41-2). Microscopic agglutination test revealed that rLipLs antisera had extensive cross-immunoagglutination among the 178 leptospiral strains in which rLipL32-1 contributed the highest agglutination titer. The rLipLs-based ELISAs established in this study demonstrated that in the sera of 385 leptospirosis patients infected with different serovars of Leptospira interrogans, rLipL32-1 had the highest positive rates for IgG and IgM (89.4–98.7%), followed by the IgG/IgM positive rates of rLipL21 (87.0–96.1%) and rLipL32-2 (86.5–96.9%), while the two rLipL41s presented the lowest IgG/IgM positive rates (69.9–83.9%). The immunoprotective levels in guinea pigs of rLipL32-1 (58.3% and 66.7%) were the highest, compared to those of the other rLipLs (25.0–58.3%). Multiple different rLipLs would increase immunoprotective levels (from 58.3% and 66.7% to 83.3% and 91.7%). The data suggest that all the rLipLs are the genus-specific superficial antigens of pathogenic Leptospira species and should be considered in designing universal vaccines against leptospirosis. [Copyright &y& Elsevier]