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

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.


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).
Initially, 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). But later on, at least 21 more species have been identified under Leptospira with more than 200 specific serovars.
Studies have meant that leptospirosis is prevalent in Brazilian herds, showing 6.76%; 11.8%; in the south, northeast regions of the country, respectively (Dewes et al., 2020b;Pimenta et al., 2019;Sanches et al., (2018) analyzed blood samples of 11 herds (representing 400 animals of São Paulo, Brazil.) and 66% of animals were positive to definitive serological test against at least one pathogen serogroup of Leptospira spp. definitive serological test. Magalhães et al. (2020), determined the prevalence of bovine leptospirosis in the Triângulo Mineiro region, Minas Gerais, Brazil, identified 48% of the cows were reagents for the Wolffi, Hardjo and Hebdomadis serovars in a large number of rural properties.
Several serological tests are used to diagnose bovine leptospirosis, such as ELISA, complement fixation, and immunofluorescence microscopy, but the definitive serological test is the microscopic agglutination test (MAT), according to the International Organization for Animal Health (OIE) (Who, 2003;Levett, 2004). However, it is a laborious and potentially Research, Society andDevelopment, v. 10, n. 6, e22710615091, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i6.15091 3 risky test since several serovar cultures must be kept alive in a liquid medium (Levett, 2004;Quinn et al., 2005). Also, the risk of cross-contamination between antigens makes necessary to check periodically for contamination of the culture medium, which can affect the titration results of the test (Sykes et al., 2011).
Current diagnostic techniques, such as MAT and even the polymerase chain reaction (PCR), are not entirely satisfactory. Although the leptospirosis diagnosis is confirmed, the tools required to perform the tests are restricted to laboratories with high infrastructure costs. For this reason, there is a need to develop rapid, sensitive, and specific tests that can facilitate the diagnosis and require fewer resources to be implemented (Picardeau, 2013).
The Dot Blot test, also known as dot-blot ELISA, dot immunobinding assay, is a technique based on the sensitization of the nitrocellulose membranes (NM) with specific antigens, subjecting them to the reaction with the primary (serum) and secondary antibody, marked with peroxidase to develop coloring. The principle is similar to ELISA, with the additional advantages of high sensitivity, convenience, and low cost, since it does not require sophisticated equipment (Pinheiro, 2001;Pinheiro, Olortegui, Gouveira, Araujo, & Andrioli, 2006). The Dot Blot test is a potential tool for diagnostic purposes; however, there are few reports of its use to detect leptospirosis.
The aim of this study was to standardize and validation the dot-blot technique for the serological diagnosis of bovine leptospirosis, using as an antigen the outer membrane proteins (OMP) of the Hardjo and Wolffi.

Sampling
The 96 serum samples analyzed in this study were collected from cattle in the region of Uberlândia, MG. The samples were analyzed in the infectious diseases laboratory at the School of Veterinary Medicine (FAMEV) of the Universidade Federal de Uberlândia (UFU).
A volume of 250 ml of the Hardjo serovar culture was centrifuged at 8,000 x g (15 minutes at 4°C) and the supernatant discarded. The pellet was resuspended three consecutive times in 2 mL PBS (pH 7.2) containing 5 mM MgCl2, and then three more times in 2 mL TE buffer (10 mM Tris hydrochloride -2 mM EDTA, pH 7.4). Subsequently, 1% Triton X114 (Sigma-Aldrich) (v/v) was added and incubated at 4°C for 30 minutes. Then, centrifuged at 8,500 x g (10 minutes at 4°C). The supernatant was collected, and 2% Triton X114 (v/v) was added. The mixture was heated at 37°C for 10 minutes in a water bath, centrifuged at 2,000 x g (10 minutes at 25°C) and the aqueous and detergent phases were separated using a micropipette.
After separation, the two phases were placed in an ice bath for 10 minutes, followed by the addition of 60μL Triton X114 to the aqueous phase and 2 mL T.E. (pH 7.4) to the detergent phase, which were then centrifuged at 3,000 x g (10 minutes at 4°C). This procedure was repeated three times. The proteins were precipitated with 10 volumes of acetone (Vetec, PA) in an ice bath for 45 minutes and thereafter, the two fractions were centrifuged at 12,600 x g at 4°C for 30 minutes. The acetone present in the supernatant was discarded, and the pellet suspended in 1 mL of ultrapure water. This solution was divided into aliquots and stored at -20°C until analysis. The procedure was repeated with 250 ml of Wolffi serovar culture.
The extracted solutions were subjected to AMICON system, diluted in PBS, in an ultrafiltration Ultracel -10k Millipore® cell (Pinheiro, Gouveia, Yorinori, & Andrioli, 2005). Finally, the extracted proteins were measured following the Bradford (1976) method, set at 595 nm in a spectrophotometer using bovine serum albumin (BSA) as standard.

Standardization of the dot blot test
The test was developed in three steps as follows (Jacobson, 1998): (I) checking test feasibility; (II) test development and optimization; (III) performance assessment.

Feasibility
To verify the test feasibility, ten non-hemolyzed serum samples (Jacobson, 1998), of which six positive and four negative according to MAT results, were used. The antibody titers ranged from low to high and were positive for both Hardjo and/or Wolffi serovars, acknowledging, however, the simultaneous occurrence of Hebdomadis serovar.

Development
The development of the Dot Blot technique was based on the methodology described by Fenoll, Jado, Vicioso, & Casal et al. (1997), following the OIE recommendations for the standardization of equipment and apparatus. The NM (nitrocellulose membrane, Santa Cruz Biotechnology, 0.22 µM pore -Ultra Cruz SC -3718) was cut into thin strips of approximate dimensions 0.6 x 6 cm, and sensitized with pipette-applied purified bovine immunoglobulin G (IgG) obtained from bovine with leptospirosis; outer membrane protein of the Hardjo serovar (OMPH); outer membrane protein of the Wolffi serovar (OMPW); and BSA, disposed separately according to the scheme shown in section 7 of Figure 1. The IgG and BSA were used as positive and negative control, respectively, in all the strips. Three different concentrations of the antigenic portion, 0.5μL, 1μL and 2μL of OMPH and OMPW solution, were used to assess the optimal concentration of antigen in the NM sensitization.
The strips were washed three times with 1 ml PBS containing 0.05% Tween 20 (PBST) for 2 minutes, under stirring.
Three different washing times were used (1, 2 and 5 minutes). Subsequently, the peroxidase conjugated anti-rabbit IgG antibody was diluted in 1 mL PBSTM solution for 60 minutes, stirring constantly. The strips were washed three more times with PBST for 2 minutes in the shaker. The dilutions 1:5000 and 1:10000 were tested to determine the optimal dilution of conjugate antibody.
The color was developed by adding 1 mL of the developing solution (15 mL TrisNaCl, 0.01g DAB [3,3'-Diaminobenzidine (DAB) -. Sigma Cat. nº D5637] and 12μL peroxide hydrogen) to each channel in the dark while stirring for 5 minutes. The samples were considered positive for bovine leptospirosis when the color changed for one or both OMPs.

Assessment
A total of 96 bovine serum samples previously tested by MAT were used to evaluate and standardize the proposed methodology. The NMs were coated with bovine IgG, OMPH, OMPW and BSA, and prepared as shown in Fig.1b. The 96 samples were tested to evaluate the test performance compared to the gold standard. The test efficiency, sensitivity and specificity relative values, positive and negative predictive values and the degree of agreement using the Kappa index, were obtained using the Bioestat 5.0 software, always referring to the results achieved in the microscopic agglutination test in dark field (Armitage, Berry, & Matthews, 2008;Jacobson, 1998;Ferreira & Ávila, 2001;Ayres, Ayres Júnior, Ayres, & Santos, 2007). Figure 1 shows the results for the different antigen concentrations (PMEH and PMEW) used in the nitrocellulose membrane during the standardization process. The antigen production resulted in a total volume of 600μL for each OMP serovar, with concentrations of 0.11 and 0.08 μg/μL for OMPH and OMPH, respectively. The best results for the washing with PBST buffer were observed in 2 minutes since the negative control samples were still lightly colored in 1 minute. On the other hand, the positive control samples lost color after 5 minutes.   Research, Society andDevelopment, v. 10, n. 6, e22710615091, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i6.15091 7 The best results were obtained using 1μL antigen solution (OMP) to sensitize the NM with 1 mL primary antibody (test serum) diluted to 1:500 and 1 mL conjugate antibody diluted to 1:5000. This combination best distinguished the positive and negative samples, especially negative samples and those positive but with low titers such as 1:100.

Results and Discussion
The efficiency of the Dot Blot test was determined by comparing the test results with a total of 96 samples previously analyzed by MAT. The MAT results showed that 37 (38%) samples were positive and 59 (62%) negative. The titers were calculated as the highest dilution of serum that had at least 50% agglutination in each evaluated serogroup. These same samples were subjected to Dot Blot, and the results showed that 58 (60%) were positive, and 38 (40%) negative.
The analysis of test performance showed 71.87% efficiency, 91.89% sensitivity and 59.32% relative specificity. The predictive values were 58.62% and 59.32% for positive and negative samples, respectively ( Table 1).
The comparison of the results from the MAT and Dot Blot tests shows 71.9% agreement and 0.46 kappa index (p<0.0001), according to the Bioestat 5.0 software (Table 1).

Discussion
All antigen concentrations used to sensitize the MN caused the color to change in all serum dilutions (Figure 1), regardless of titrable levels of antibodies ( Figure 2). However, 1μL OMP resulted in the best color definition compared to others. Therefore, 1µL antigen (0.11 and 0.08 μg/μL for OMPH and OMPH) was used to perform one test and, in this case, the protein extract had good yield, the initial 250 ml culture produced antigen to perform 600 tests. Dewes et al. (2020) standardized the dot blot test for bovine leptospirosis with the use of inactivated antigen suspended in PBS buffer and obtained satisfactory results with 10uL.
The best results for (PBST) buffer washing after adding the tested serum samples was 2 minutes. At 1 minute, samples with low titers could not be distinguished from the negative samples and/or nonspecific reactions. The Tween 20 buffer, the surfactant component, removes nonspecific antibodies, especially when present in small amounts or weakly bound to hydrophobic substrates such as nitrocellulose (Juhl, Nørgaard, & Bjerrum, 1984). The 1-minute wash did not allow the Tween 20 to remove satisfactorily non-specific antibodies bound to the substrate.
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. Souza et al. (2012) standardized and validated the indirect ELISA test using the OMP of the Hardjo serovar as antigen (ELISA-OMP/Hardjo), which evidenced the potential of the Hardjo membrane proteins to be applied in diagnostic screening of bovine leptospirosis with 100% sensitivity and 73% specificity.
The MAT technique is considered to have low sensitivity, especially in the acute phase, and its interpretation is hampered by the high degree of cross-reactivity between different serogroups (Levett, 2004). It is, therefore, better classified as serogroup-specific test rather than serovar-specific since it does not guarantee specificity when the serovars belong to the same sorogroup (WHO, 2003).
The test showed high sensitivity (91.89%) and a potential to detect highly infected animals. It is appropriate to assist the diagnosis of bovine leptospirosis, especially if used as a screening test followed by another test with greater specificity. The positive and negative predictive values were 58.62% and 59.32%, respectively. Dewes et al. (2020) observed similar results, with 94% sensitivity, and 53% specificity.
Agreement of 71.9% was observed between MAT and Dot Blot tests and kappa index of 0.46 (p <0.0001), with good reproducibility. The kappa index suggests that the observed difference between the results did not occur randomly, which indicates moderate but significant (Fenoll et al., 1997) test reproducibility.
Compared to MAT, in this study was possible to observe lower cost, less time and dispenses with the need for laboratory equipment. Similar results were shown by Dewes et al. (2020), However, they used 10uL of antigen while in this study only 1uL (0.11 and 0.08 μg/μL for OMPH and OMPH) was used.
The good yield, since the antigen produced from 250 mL of Leptospira spp. culture allowed to perform 600 tests, the antigen's ability to sensitize the NM and induce satisfactory answer, indicate that the Dot Blot test is feasible. Similar results were found by Dewes et al. (2020a) As reported in recent research this technique had proving suitable for the serological diagnosis other diseases, with the bovine brucellosis, the antigen adheres to the center of the nitrocellulose membrane, which ensures the use of an established antigen concentration, and its simplicity, precision and speed demonstrated that the assay can be used in the field for large scale diagnosis (Bastos et al., 2018).
The test is also convenient, low cost and does not require sophisticated equipment (Pinheiro et al., 2006). It is possible to analyze large numbers of samples under unfavorable conditions (Bamdad, Rodstai, Solimandjahi, & Malekaneh, 2001) since nitrocellulose technical characteristics allow its successful use in field studies and minimally equipped laboratories (Rossi, Tsang, & Pilcher, 1991).
The hydrophobic plastic support prevents leakage of substances (antigens, antibody, buffers) from one well to another, which would cause cross-reactions (Lin & Halbert, 1986). The stability of the NM adhered antigen enables to use this test in the field (Hebeling & Kalter, 1986).
The development of diagnostic kits with NM sensitized with OMPs of several serovars would allow veterinarians to conduct field screening tests to detect cattle serum reagent to bovine leptospirosis, conveniently and with no need for sophisticated equipment.

Conclusion
The Hardjo and Wolffi OMPs were effective antigens in enzyme immunoassays to detect antibodies to bovine leptospirosis regardless of the infecting serovar.
The Dot Blot test standardized in this study proved to be feasible for the serological diagnosis of bovine leptospirosis, using the MAT as a reference, particularly if used as a screening test followed by a test with higher specificity. The Dot Blot test sensitivity is promising as an alternative for the diagnosis of bovine leptospirosis. Further studies are necessary to determine the feasibility of developing a diagnostic kit containing nitrocellulose membranes coated with OMPs of various serovars.