Diagnosis of Human Leptospirosis: Comparison of Microscopic Agglutination Test with Recombinant LigA/B Antigen-Based In-House IgM Dot ELISA Dipstick Test and Latex Agglutination Test Using Bayesian Latent Class Model and MAT as Gold Standard.

Leptospirosis is a spirochaetal infection that possesses a broad host range affecting almost all mammals. In the present study, the microscopic agglutination test (MAT) was compared with recombinant LigA/B antigen-based point-of-care diagnostics such as the in-house IgM dot ELISA dipstick test (IgM- DEDT) and the latex agglutination test (LAT) for the serodiagnosis of human leptospirosis. The comparison of the MAT with these two point-of-care diagnostics was performed using the MAT as the gold standard test and using Bayesian latent class modelling (BLCM), which considers all diagnostic tests as imperfect. The N-terminal conserved region of the LigA/B protein spanning the first to fifth big tandem repeat domains (rLigA/BCon1-5) was employed as a serodiagnostic marker in both of the bedside assays. A total of 340 serum samples collected from humans involved in high risk occupations were screened using the MAT, IgM DEDT and LAT. During the early phase of leptospirosis, BLCM analysis showed that the IgM DEDT and LAT had similar sensitivities (99.6 (96.0-100)) and (99.5 (95.2-100)), respectively, while the single acute phase MAT had the lowest sensitivity (83.3 (72.8-91.3)). Both the IgM DEDT and the LAT may be superior to the single acute phase MAT in terms of sensitivity during the early phase of infection and may be suitable for the early diagnosis of leptospirosis. However, BLCM analysis revealed that the use of both acute and convalescent samples substantially increased the sensitivity of the final MAT (98.2% (93.0-99.8%)) as a test to diagnose human leptospirosis. Both the IgM DEDT and LAT can be employed as bedside spot tests in remote locations where the MAT is not easily accessible.

Functional Diversity of the Excretory/Secretory Proteins of Nematode Parasites.

INTRODUCTION: Parasites release a wide array of protein as excretory and secretory products (ESPs). Irrespective of their mode of propagation, ESPs are found to be secreted or excreted by both naturally occurring and laboratory-cultivated parasites. Mass spectrometry-based approaches have been extensively used to identify and characterize the ESP constituents. ESPs are involved in various cellular activities such as immune modulation, proteolysis, inhibition of proteases and protection of cells against oxidants. Specifically, their role in host immune evasion by down-regulation of pro-inflammatory cytokines and up-regulation of anti-inflammatory cytokines attracts scientific attention. A thorough investigation of functional diversity of ESPs may be helpful in planning control strategies against many parasites. METHODS: This review focuses on diversity of ES proteins, various approaches to identify them and discusses about the biochemical and functional aspects of such proteins. RESULTS: The diverse array of proteins secreted or excreted (a, GST-1, acetylcholinesterase, GAPDH) by the parasites are also described emphasizing their role in cellular physiology. CONCLUSION: Finally, it concludes by citing some of these proteins as potential therapeutic agents against helminth challenge.

Immunoinformatic Study of Recombinant LigA/BCon1-5 Antigen and Evaluation of Its Diagnostic Potential in Primary and Secondary Binding Tests for Serodiagnosis of Porcine Leptospirosis.

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.

Enhancing the Stability of Haemonchus contortus Glyceraldehyde-3-Phosphate Dehydrogenase and Binding of Host Albumin to the Parasite Enzyme.

INTRODUCTION: Haemonchus contortus is an economically important parasite of domestic animals. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) excreted in the ES product of H. contortus can be a promising vaccine candidate for controlling the parasite infection. Unfortunately, the parasite enzyme breaks down rapidly. The current study focusses on stabilizing the recombinant GAPDH (rGAPDH) of H. contortus. METHODS: The rGAPDH was purified and stored in two different buffers (sodium phosphate + EDTA and bicarbonate-sodium chloride) to check the stability. The affinity of the parasite enzyme towards host serum (Goat) components was evaluated by affinity chromatography. The interacting component was identified by mass spectrometry. RESULTS: Here, we report that the enzyme can be stabilized for at least 3 months if stored in bicarbonate-sodium chloride. This should facilitate testing of the enzyme in challenge trials. Additionally, we show that the parasite enzyme has affinity for host albumin; this interaction may have significance in host-parasite relationship. CONCLUSION: The present study reports a combination of sodium bicarbonate (0.1 M) with 0.5 M sodium chloride as a suitable buffer to enhance the stability of H. contortus GAPDH.

Salmonella Typhimurium methionine sulfoxide reductase A (MsrA) prefers TrxA in repairing methionine sulfoxide.

Intraphagocytic survival of Salmonella Typhimurium (ST) depends (at least in part) upon its ability to repair oxidant-damaged macromolecules. Met residues either free or in protein bound form are highly susceptible to phagocyte-generated oxidants. Oxidation of Mets leads to Met-SO formation, consequently loss of protein functions that results in cell death. Methionine sulfoxide reductase (Msr) reductively repairs Met-SO to Met in the presence of thioredoxin (trx) and thioredoxin reductase (trxR). Earlier we reported that methionine sulfoxide reductase A (msrA) gene deletion strain of ST suffered oxidative stress.[1] Thioredoxin system of ST comprises of two thioredoxins (trxA and trxC) and one thioredoxin reductase (trxB). Preferred trx utilized in MsrA-mediated repair of Met-SO is not known. In current study, we cloned, expressed, and purified ST TrxA, TrxB, TrxC, and MsrA in recombinant forms. The migration of TrxA, TrxB, TrxC, and MsrA proteins was approximately 10, 36, 16, and 26 kDa on SDS-gels. The nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-linked reductase assays interpreted that MsrA utilized two times more NADPH for the reduction of S-methyl p-tolyl sulfoxide when TrxA was included in the assays as compared to TrxC.