Development and standardization of the Dot Blot test for serological diagnosis of bovine leptospirosis

Higor Oliveira Silva,Tatiane Cristina Fernandes Tavares,Anna Monteiro Correia Lima,Pollyanna Mafra Soares,Mariana Assunção De Souza

doi:10.33448/rsd-v10i6.15091

Abstract

Leptospirosis a public health problem and an endemic zoonosis in Brazil, is diagnosed by serological methods. Therefore, low-cost and easy to execute methodologies with good/high sensitivity, such as Dot Blot, are an important diagnostic tool. The aim of this study was to standardize and validation the dot-blot technique for the serological diagnosis of bovine leptospirosis. Several concentrations of antigens applied to nitrocellulose membranes, and different dilutions of conjugated bovine serum were evaluated to develop and standardize the test. The best distinction/contrast between positive and negative samples was observed for 1μL antigen (0.11μg/μL outer membrane protein of the Hardjo serovar (OMPH) and 0.08 μg/μL outer membrane protein of the Wolffi serovar (OMPW)), 1:500 and 1:10000 bovine serum dilution and conjugate, respectively. The Dot Blot test efficiency was 71.87% and kappa index, 0.46 (p<0.0001). The other parameters measured were: sensitivity 91.89%; specificity 59.32%; positive predictive value 58.62%; and, negative predictive value 59.32%. In addition to high sensitivity, other advantages of the Dot Blot technique have been identified, such as practicality, low cost since it does not require sophisticated devices and the fact that the Hardjo and Wolffi OMP also reacted with serovars from other pathogenic serogroups. The results provided positive expectations for the use of Dot Blot as support in the diagnosis of bovine leptospirosis, especially if used as a screening test, stimulating further research for the future development of kits for diagnostic purposes.

Highlights

IntroductionLeptospirosis is a worldwide disseminated zoonosis (Quinn, Markey, Carter, Donnelly, & Leonard et al, 2005)
Leptospirosis is a worldwide disseminated zoonosis (Quinn, Markey, Carter, Donnelly, & Leonard et al, 2005). It is considered a public health problem and more common in developing countries (Bharti et al, 2003). This disease is caused by pathogenic bacteria of the genus Leptospira; it is endemic in Brazil and occurs all year round (Brasil, 2009)
Unlike the LPS, the outer membrane proteins (OMP) were preserved between different L. interrogans serovars (Faine et al, 1999; Levett, 2004), and the OMPs of the Hardjo serovar are similar to the outer membrane protein profile of other serovars (Lafetá et al, 2008). These results show that the Dot Blot test using the OMPH and OMPW can detect anti-Leptospira spp. antibodies in the serum regardless of the serovar involved

Summary

Introduction

Leptospirosis is a worldwide disseminated zoonosis (Quinn, Markey, Carter, Donnelly, & Leonard et al, 2005). It is considered a public health problem and more common in developing countries (Bharti et al, 2003). This disease is caused by pathogenic bacteria of the genus Leptospira; it is endemic in Brazil and occurs all year round (Brasil, 2009). The genus Leptospira, was classified than L. interrogans and L. biflexa ( pathogenic and non-pathogenic species), based on the presence of homologous antigens (nearly 60 serovars under L. biflexa and at least 225 serovars under L. interrogans). Despite the fact that several serovars have been described as causing bovine leptospirosis, serological surveys in Brazil demonstrate the predominance of serovars Hardjo and Wolffi, the first being the most prevalent

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