Abstract
Leptospirosis is responsible for hampering the productivity of swine husbandry worldwide. The aim of this study was to assess the efficacy of bioinformatics tools in predicting the three-dimensional structure and immunogenicity of recombinant LigBCon1-5 (rLigBCon1-5) antigen. A battery of bioinformatics tools such as I-TASSER, ProSA and SAVES v6.0 were used for the prediction and assessment of the predicted structure of rLigBCon1-5 antigen. Bepipred-2.0, DiscoTope v2.0 and ElliPro servers were used to predict linear and conformational epitopes while T-cell epitopes were predicted using NetMHCpan 4.1 and IEDB recommended 2.22 method for MHC Class I and II peptides respectively. The results obtained using various in silico methods were then compared with wet lab experiments comprising of both primary (IgG Dot ELISA Dipstick test) and secondary-binding assays (Latex Agglutination Test [LAT]) to screen 1153 porcine serum samples. The three-dimensional structure of rLigA/BCon1-5 protein as predicted by I-TASSER was found to be reliable by Ramachandran Plot and ProSA. The ElliPro server suggested 10 and three potential linear and conformational B-cell-epitopes, respectively, on the peptide backbone of the rLigA/BCon1-5 protein. The DiscoTope prediction server suggested 47 amino acid residues to be part of B-cell antigen. Ten of the most efficient peptides for MHC-I and II grooves were predicted by NetMHCpan 4.1 and IEDB recommended 2.22 method, respectively. Of these, three peptides can serve dual functions as it can fit both MHC I and II grooves, thereby eliciting both humoral-and cell-mediated immune responses. The prediction of these computational approaches proved to be reliable since rLigBCon1-5 antigen-based IgG Dot ELISA Dipstick test and LAT gave results in concordance to gold standard test, the Microscopic Agglutination Test (MAT), for serodiagnosis of leptospirosis. Both the IgG Dot ELISA Dipstick test and LAT were serodiagnostic assays ideally suited for peripheral level of animal health care system as “point of care” tests for the detection of porcine leptospirosis.
Highlights
Leptospirosis causes huge economic burden to swine husbandry, primarily due to reproductive losses ranging from abortion, decreased number of piglets per litter, birth of runt piglets, increased weaning-to-oestrus interval, agalactia and so-called SMEDI syndrome (Stillbirth; Mummification/Maceration; Embryo Death; Infertility) [1,2]
The Microscopic Agglutination Test (MAT) results revealed a seropositivity of 23.85% (275/1153) for porcine leptospirosis with maximum agglutinins detected for serovar Icterohaemorrhagiae (n = 143) followed by Grippotyphosa (n = 97), Pomona (n = 50), Tarassovi (n = 37), Javanica (n = 18), Australis (n = 06) and Pyrogenes (n = 06) as shown in Figure 1.The cumulative figure of sera positive for various serovars (N = 357) exceeded thetotal MAT positive sera (n = 275) as several sera reacted with multiple leptospiral serovars
Western Blot Analysis Western blot analysis showed that rLigA/BCon1-5 protein was highly immunogenic against anti-Leptospira antibodies present in swine sera since clearly visible bands of either brown or purple colour were obtained when either 3,3 -Di amino benzidine (DAB) or 4– choloro-1– naphthol were used as substrate for HRP conjugated secondary antibodies (Figure 6B).Further, we have found that rLigBCon1-5 antigen reacted in western blot to field sera seropositive for L
Summary
Leptospirosis causes huge economic burden to swine husbandry, primarily due to reproductive losses ranging from abortion, decreased number of piglets per litter, birth of runt piglets, increased weaning-to-oestrus interval, agalactia and so-called SMEDI syndrome (Stillbirth; Mummification/Maceration; Embryo Death; Infertility) [1,2]. A noteworthy example for field-oriented spot test is Latex Agglutination Test (LAT) which is a highly economical, rapid screening penside test ideally suited for large-scale screening of sera samples in endemic areas without using any sophisticated equipment [10]. Another field-oriented test is Dot-ELISA which is a simple, inexpensive and user-friendly test that can be used either in a single test format or for screening a large number of sera specimens [11,12]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
