Leptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulent Leptospira interrogans serovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera (n = 118) from cases of acute leptospirosis along with sera (n = 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable in in vitro cultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation of Leptospira infection.
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Department of Biotechnology, Ministry of Science and Technology (DBT) provided funding to Kalimuthusamy Natarajaseenivasan under grant number BT/PR13502/MED/15/36/2010. Indian Council of Medical Research (ICMR) provided funding to Kalimuthusamy Natarajaseenivasan under grant number SEC/DHR/HRD-FELLOW/1(2)/2011.
Raja, Veerapandian; Shanmughapriya, Santhanam; Kanagavel, Murugesan; Artiushin, Sergey; Velineni, Sridhar; Timoney, John F.; and Natarajaseenivasan, Kalimuthusamy, "In Vivo-Expressed Proteins of Virulent Leptospira interrogans Serovar Autumnalis N2 Elicit Strong IgM Responses of Value in Conclusive Diagnosis" (2016). Gluck Equine Research Center Faculty Publications. 8.
Supplemental File 1 - Table S1. Case definition and grouping of the patients included in the present study. Table S2. Age and sex distribution of the confirmed cases of leptospirosis included in the study. Table S3. Details of the pooled serum samples with reference to difference in days of symptoms. Table S4. Primer sequences used in the present study. Table S5. Median MAT titers of 118 sera selected from the bank of 754 laboratory-confirmed cases of leptospirosis. Table S6. Conservation of the identified proteins among other pathogenic bacteria. Fig. S1. PCR amplification of the identified genes and REA analysis. Fig. S2. SDS-PAGE profile of the purified recombinant proteins. Fig. S3. ArgC and RecA expression is not temperature regulated. Fig. S4. Relationship between MAT titer and ELISA OD for the recombinant proteins. Fig. S5. Evaluation of LigA-C peptides in IgM ELISAs.