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Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]2026; 57(1); 52; doi: 10.1007/s42770-025-01854-z

Advances in ELISA-based detection of equine leptospirosis.

Abstract: Leptospirosis is a globally distributed zoonosis that affects both humans and animals, with being the main causative agent. In horses, the disease is associated with considerable economic losses. The Microscopic Agglutination Test (MAT) is the reference test for diagnosis but has limitations, emphasizing the need for effective diagnostic alternatives. In this study, was evaluated the use of a recombinant chimera, composed of ErpY-like and LemA proteins, as an antigen for ELISA-based detection of equine leptospirosis. The chimera was successfully expressed, purified and tested on 915 horse serum samples previously analyzed by MAT. The ELISA correctly identified all positive samples, with no false negative observed. The ROC curve analysis, considering different hypothetical prevalence values, reached up to 0.92. The assay demonstrated sensitivity ranging from 98.8% to 100%, specificity from 77.6% to 84%, positive predictive value between 68.6% and 77.2%, and negative predictive value between 92% and 100%. The Kappa coefficient was 0.7432, indicating good agreement with MAT. When samples were categorized by titers, those with a 1:400 dilution achieved the highest accuracy (99%), while those with a 1:100 dilution showed slightly lower accuracy (94.8%), demonstrating that higher antibody titers resulted in a more effective assay. The major serovars that cause infection in horses, such as Bratislava, Icterohaemorrhagiae, Sejroe, Pomona, and Copenhageni, react with rErpY-lemA. These results suggest that the ErpY-LemA chimera is a promising screening tool for equine leptospirosis, offering a valuable alternative for the rapid and accurate detection of different serovars and titers. The test demonstrated high sensitivity and specificity, with strong agreement with the MAT (Kappa value). No false negatives were identified, further confirming the test’s reliability. This is the first study to use a recombinant chimera for equine leptospirosis diagnosis. The recombinant ErpY-LemA chimera was successfully used as an antigen in an ELISA-based assay for diagnosing equine leptospirosis, proving to be a promising screening tool. [Image: see text]
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Publication Date: 2026-02-09 PubMed ID: 41663676PubMed Central: PMC12886683DOI: 10.1007/s42770-025-01854-zGoogle Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This research evaluated a new ELISA test for detecting leptospirosis in horses, using a recombinant protein chimera as an antigen, showing it to be a reliable alternative to the standard diagnostic method.

Background on Leptospirosis and Diagnosis Challenges

  • Leptospirosis is a widespread zoonotic disease affecting both humans and animals, including horses.
  • The disease in horses causes important economic impacts due to illness.
  • The current reference diagnostic method is the Microscopic Agglutination Test (MAT), which has limitations such as complexity, requirement of live cultures, and potential inaccuracies.
  • There is a need for effective, rapid, and accurate alternative diagnostic methods.

Development of the ELISA-Based Assay Using Recombinant Chimera

  • A recombinant chimera combining two Leptospira proteins, ErpY-like and LemA, was created to be used as the antigen in the ELISA test.
  • This chimera was successfully expressed and purified for use in diagnostics.
  • The purpose was to detect antibodies in horse serum samples, indicating leptospirosis infection.

Testing and Validation of the ELISA Assay

  • 915 horse serum samples previously tested with MAT were used to validate the ELISA test.
  • The ELISA assay correctly identified all positive cases, demonstrating zero false negatives.
  • Sensitivity of the test ranged from 98.8% to 100%, showing it is highly effective at identifying infected horses.
  • Specificity ranged from 77.6% to 84%, indicating a good ability to correctly identify non-infected horses.
  • Positive Predictive Value (PPV) ranged between 68.6% and 77.2%, meaning most positive results are true positives.
  • Negative Predictive Value (NPV) ranged between 92% and 100%, meaning negative results are highly reliable.
  • The ROC curve analysis showed an area under the curve (AUC) of up to 0.92, reflecting very good test accuracy.
  • The Kappa coefficient was 0.7432, indicating good agreement between ELISA and MAT results.

Performance by Antibody Titer Levels

  • Samples were grouped by antibody dilution titers to evaluate test accuracy at different infection levels.
  • Samples with a 1:400 dilution had the highest accuracy of 99%, showing the test works best with higher antibody concentrations.
  • Samples with a 1:100 dilution still had good accuracy at 94.8%, though slightly less than higher titers.
  • This suggests the ELISA test sensitivity improves with higher antibody levels in serum.

Spectrum of Leptospira Serovars Detected

  • The recombinant ErpY-LemA chimera reacted to antibodies against major equine-infecting Leptospira serovars: Bratislava, Icterohaemorrhagiae, Sejroe, Pomona, and Copenhageni.
  • This broad reactivity means the ELISA can effectively detect infections caused by multiple important serovars.

Conclusions and Significance

  • The study represents the first reported use of a recombinant chimera protein in diagnosing equine leptospirosis via ELISA.
  • The test offers a valuable alternative to the MAT, with advantages including rapidity, ease of use, and high sensitivity/specificity.
  • No false negatives occurred, highlighting the test’s reliability for screening purposes.
  • The recombinant ErpY-LemA-based ELISA is a promising screening tool that can improve surveillance and management of equine leptospirosis by enabling accurate and timely detection.

Cite This Article

APA
Cardoso TL, Wozeak DR, Pereira IL, da Silva Ribeiro LD, Rodrigues RO, Hartwig DD. (2026). Advances in ELISA-based detection of equine leptospirosis. Braz J Microbiol, 57(1), 52. https://doi.org/10.1007/s42770-025-01854-z

Publication

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]
ISSN: 1678-4405
NlmUniqueID: 101095924
Country: Brazil
Language: English
Volume: 57
Issue: 1
Pages: 52
PII: 52

Researcher Affiliations

Cardoso, Thayná Laner
  • Bacteriology and Bioassays Laboratory, Department of Microbiology and Parasitology, Institute of Biology, Federal University of Pelotas, Pelotas, RS, Brazil.
  • Biotechnology Center, Biotechnology Development Center, Federal University of Pelotas, Pelotas, RS, CEP 96010-900, Brazil.
Wozeak, Daniela Rodriguero
  • Bacteriology and Bioassays Laboratory, Department of Microbiology and Parasitology, Institute of Biology, Federal University of Pelotas, Pelotas, RS, Brazil.
  • Biotechnology Center, Biotechnology Development Center, Federal University of Pelotas, Pelotas, RS, CEP 96010-900, Brazil.
Pereira, Isabel Ladeira
  • Bacteriology and Bioassays Laboratory, Department of Microbiology and Parasitology, Institute of Biology, Federal University of Pelotas, Pelotas, RS, Brazil.
  • Biotechnology Center, Biotechnology Development Center, Federal University of Pelotas, Pelotas, RS, CEP 96010-900, Brazil.
da Silva Ribeiro, Laura Dias
  • Bacteriology and Bioassays Laboratory, Department of Microbiology and Parasitology, Institute of Biology, Federal University of Pelotas, Pelotas, RS, Brazil.
  • Biotechnology Center, Biotechnology Development Center, Federal University of Pelotas, Pelotas, RS, CEP 96010-900, Brazil.
Rodrigues, Rogério Oliveira
  • Leptospirosis Laboratory, Department of Animal Diagnostics and Research, Secretariat of Agriculture, Livestock and Irrigation, Desidério Finamor Veterinary Research Institute, Eldorado do Sul, RS, Brazil.
Hartwig, Daiane Drawanz
  • Bacteriology and Bioassays Laboratory, Department of Microbiology and Parasitology, Institute of Biology, Federal University of Pelotas, Pelotas, RS, Brazil. daianehartwig@gmail.com.
  • Biotechnology Center, Biotechnology Development Center, Federal University of Pelotas, Pelotas, RS, CEP 96010-900, Brazil. daianehartwig@gmail.com.

Grant Funding

  • 001 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
  • Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul / Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul
  • Conselho Nacional de Desenvolvimento Científico e Tecnológico / Conselho Nacional de Desenvolvimento Científico e Tecnológico

Conflict of Interest Statement

Declarations. Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper

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