Development and Evaluation of ELISA and PCR
Sergey C. Artiushin, Ph.D.
Development of diagnostic and preventive methods for control of leptospirosis in horses is a long term goal of our research program. Our laboratory was the first to create a gene library of Leptospira interrogans serovar Pomona type kennewicki, the most prevalent leptospira in horses in North America.
Using sequential screening with sera from vaccinated and convalescent animals we isolated and investigated several novel genes encoding proteins that may play an important role in host protective immunoresponse. Although these proteins are potential vaccine components, they may also be valuable as antigens in ELISA for detection of leptospira infection.
Our laboratory has pioneered the use of PCR for detection of leptospiras in horse urine. The recent discovery of several novel genes specific for pathogenic leptospiras provides an opportunity for improving the PCR for detection and identification of leptospira.
Specific aims of this project include:
Our research has shown that recombinant proteins, Lk73 and LigA were superior in detection of antibodies in sera from a local farm which had experienced an outbreak. These proteins have been selected for further evaluation as antigen for ELISA.
Previously described 16S rRNA specific primers L16-F/R and L16N-F/R were redesigned following comparative analysis of 16S rRNA of pathogenic and saprophytic leptospira. These primers have been tested in PCR reactions with DNA isolated from 15 strains of pathogenic and 9 strains of saprophytic leptospira belonging to different serovars and genospecies. Positive results were detected only for pathogenic leptospira.
Two sets of primers were designed to differentiate L. kennewicki from L. bratislava and other pathogenic leptospira. The first, for nested PCR includes four primers LG1F/R and LG1NF/R flanking the 5’-end of lk73.5 and its upstream sequence. Tests of these primers in PCR with DNA of pathogenic and saprophytic leptospira have shown positive results only for pathogenic leptospira. Although sizes of amplicons vary slightly for different leptospira, a distinguishably large amplicon was produced from L. kennewicki.
The second set consists of primers (RepF/R) flanking a short insertion element initially discovered upstream of lk73.5 of L. kennewicki. Amplification of leptospira DNA using these primers showed that this IS element was present only in pathogenic leptospira. All pathogenic leptospira, except L. kennewicki and L. pomona, demonstrated distinctive PCR band profiles. Evaluation of these primers for identification of L. kennewicki and other leptospira in urine and other fluids from horses is now in progress.
Maxwell H.Gluck Equine Research Center
Department of Veterinary Science, University of Kentucky
Lexington, Kentucky 40546-0099
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