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Expression of Recombinant Leptospiral Surface Lipoprotein-Lsa27 in E. coli and Its Evaluation for Serodiagnosis of Bovine Leptospirosis by Latex Agglutination Test

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Abstract

The expressed recombinant leptospiral surface adhesion lipoprotein (Lsa27) of pathogenic Leptospira in E. coli was evaluated for the detection of Leptospira antibodies in cattle sera by latex agglutination test (LAT). The Lsa27 lacking signal peptide coding gene sequences from L. interrogans serovar Pomona was amplified (~ 660 bp) by PCR and the amplicon was cloned into pETiteN-HisKan vector. The expressed recombinant Lsa27 histidine-tagged fusion protein (rLsa27) was Ni–NTA affinity purified under denaturation followed by renaturation methods. The purified rLsa27 was characterized by SDS-PAGE and immunoblot, which confirmed the leptospiral protein with a MW of ~ 25 kDa. Further, the prepared sensitized latex beads coated with rLsa27 were evaluated as a diagnostic antigen for detection of pathogenic Leptospira antibodies by using known microscopic agglutination test (MAT) positive (n = 74) and negative (n = 62) sera for Leptospira antibodies in LAT, which revealed the relative diagnostic sensitivity of 91.89% and specificity of 87.10% against the gold standard serological test, MAT. Furthermore, on evaluation of developed rLsa27 LAT using serum samples from cattle associated with the history of abortions and reproductive disorder (n = 309), the relative sensitivity of 96.15%, and specificity of 89.11% were observed. Therefore, this rapid field test using the rLsa27 is first of its kind and it could be used as a screening test for the detection of Leptospira antibodies or it can be complemented by other diagnostics for the diagnosis /surveillance of bovine leptospirosis.

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Acknowledgements

The authors wish to thank the Indian Council of Agricultural Research Institute (ICAR), New Delhi, India, for providing facilities, encouragement, and support. This research work is part of the Ph.D. program of First Author and the research project work funded from the ICMR–Adhoc project (F.No. Leptos/7/2013-EID-1 and ECD/ADHOC/27/2016-17). The authors also thank the ICAR-NIVEDI staff for constant support and timely help for filing the patent on “Recombinant leptospiral surface antigen-based immuno-diagnostic test for leptospirosis” (Intellectual Property India-Patent Ref./Application no. 202041022882). We are grateful to all the State Animal Husbandry Department of India, for periodically sending the animal samples to the ICAR-NIVEDI for screening by MAT for the diagnosis of leptospirosis.

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Authors and Affiliations

  1. Indian Council of Agricultural Research -National Institute of Veterinary Epidemiology and Disease Informatics (ICAR-NIVEDI), Yelahanka, Post Box No. 6450, Bengaluru, Karnataka, 560 064, India

    Anusha Alamuri, K. Vinod Kumar, S. SowjanyaKumari, L. Linshamol, R. Sridevi, M. Nagalingam, Parimal Roy & V. Balamurugan

  2. Jain University, Bengaluru, Karnataka, India

    Anusha Alamuri

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Contributions

AA, SS, and LL conducted research work/experiments. RS contributed to the OMP experiment. KVK, MN, and VB analyzed data. PR provided guidance and supported research work. VB conceived, designed research and wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to V. Balamurugan.

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Ethical Approval

The manuscript does not contain animal experimental trials. This work was carried out in the Institutional Biosafety Committee (IBSC) approved ICMR Project “Development of recombinant antigen-based diagnostics for bovine and human Leptospirosis” (F.No. 6-52/NIVEDI/Biosafety/2016/07-19 dated 11.12.2017). The animal sera including human samples available in the Leptospira Laboratory were used in this study. The leptospirosis suspected human samples from the District surveillance unit, Udupi district, Karnataka, India, and the animal sera from various disease investigation units of the state animal husbandry departments or various veterinary colleges and institutes of India were submitted to the ICAR-NIVEDI or collected by NIVEDI teams for screening by MAT for diagnosis.

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Alamuri, A., Kumar, K.V., SowjanyaKumari, S. et al. Expression of Recombinant Leptospiral Surface Lipoprotein-Lsa27 in E. coli and Its Evaluation for Serodiagnosis of Bovine Leptospirosis by Latex Agglutination Test. Mol Biotechnol 62, 598–610 (2020). https://doi.org/10.1007/s12033-020-00278-4

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