Development of enzyme-linked immunosorbent assay for human leptospirosis serodiagnosis using leptospira secretome antigen

Secretome (extracellular proteins) has been considered as a potential diagnostic biomarker, vaccine and therapeutic candidates for bacterial infections. In this research, secretomes of two reference Leptospira spp, namely, pathogenic L. interrogans serovar Autumnalis strain Akiyami and saprophytic L. biflexa serogroup Semaranga serovar Patoc strain P136 were evaluated for their immunogenicity to microscopic agglutination test (MAT)-positive leptospirosis patients’ sera in IgM- and IgG-ELISAs in comparison to a whole Leptospira homo-genate antigen. At a single serum dilution of 1:1,000, sensitivity of the pathogenic Leptospira secretome antigen-based IgM- and IgG-ELISAs was 90% (18/20) and 75% (15/20), respectively, when compared with that of the MAT assay. Thus, Leptospira secretome provides a potential antigen source in serodiagnosis of leptospirosis

Development of enzyme-linked immunosorbent assay for human leptospirosis serodiagnosis using leptospira secretome antigen

Secretome (extracellular proteins) has been considered as a potential diagnostic biomarker, vaccine and therapeutic candidates for bacterial infections. In this research, secretomes of two reference Leptospira spp, namely, pathogenic L. interrogans serovar Autumnalis strain Akiyami and saprophytic L. biflexa serogroup Semaranga serovar Patoc strain P136 were evaluated for their immunogenicity to microscopic agglutination test (MAT)-positive leptospirosis patients’ sera in IgM- and IgG-ELISAs in comparison to a whole Leptospira homo-genate antigen. At a single serum dilution of 1:1,000, sensitivity of the pathogenic Leptospira secretome antigen-based IgM- and IgG-ELISAs was 90% (18/20) and 75% (15/20), respectively, when compared with that of the MAT assay. Thus, Leptospira secretome provides a potential antigen source in serodiagnosis of leptospirosis

Genetic typing of the 56-kDa type-specific antigen gene of contemporary Orientia tsutsugamushi isolates causing human scrub typhus at two sites in north-eastern and western Thailand.

Orientia tsutsugamushi is the causative agent of scrub typhus, a major cause of febrile illness in the rural areas of Southeast Asia. Twenty-three strains of O. tsutsugamushi were isolated from patients with scrub typhus in north-east (Udorn Thani province) and western Thailand (Tak province) between 2003 and 2005. The isolates were characterized by sequencing the entire ORF of the 56-kDa-type-specific antigen gene, followed by phylogenetic analysis. The majority (15/23) of isolates clustered with the Karp-type strain, six with a Gilliam-type strain and one each with the TA716- and TA763-type strains. Overall, there was considerable diversity in sequence, comparable to that seen in strains from across the rest of the scrub typhus-endemic world. There was no significant difference in the distributions of strains between the two provinces (P=0.08, Fisher's exact) nor a temporal change in distribution with year of isolation (P=0.80, Fisher's exact). Within this diversity there were also examples of isolates with identical 56-kDa genotypes that were cultured from patients from the same geographical areas

Mimotope identification from monoclonal antibodies of Burkholderia pseudomallei using random peptide phage libraries.

This study used random peptide libraries, displayed by bacteriophage T7 and M13, to identify mimotopes from four monoclonal antibodies (mAbs) specific to Burkholderia pseudomallei. Bound phages, selected from fourth-round panning with each mAb, were tested for binding specificity with each mAb using ELISA, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. Overall, 75 of 90 phages (83.3%) were ELISA-positive with each mAb. Mimotopes from all 75 phages (100%) were found to match protein sequences of Burkholderia spp. from GenBank. The predominant mimotopes were TP-GRTRVT found in 13.3%, LTPCGRTxD (8%), AREVTLL (6.7%), NxVxKVVSR (5.3%), PCAPRSS (4%), LGRVLAN (4%), RNPKKA (2.7%) and CPYPR (2.7%). The following GenBank-matched proteins (i.e. the hypothetical proteins) were located at the outer membrane of Burkholderia spp.: BPSL2046 of B. pseudomallei K96243 (matched with mimotope CGRTxD), BpseP_02000035 (matched with LGRVLAN), BPSS0784 of B. pseudomallei K96243 (matched with CPYPR), BURPS1710b_1104 of B. pseudomallei 1710b (matched with CARQY) and TonB-dependent siderophore receptor of B. cenocepacia H12424 (matched with CVRxxLTPC and TPCRxRT). These phage mimotopes and matched proteins may have the potential for further use as diagnostic reagent and immunogen against melioidosis. These results demonstrate that phage-display technique has the potential for rapidly identifying phage mimotopes that interact with B. pseudomallei mAbs

Mimotope identification from monoclonal antibodies of Burkholderia pseudomallei using random peptide phage libraries.

This study used random peptide libraries, displayed by bacteriophage T7 and M13, to identify mimotopes from four monoclonal antibodies (mAbs) specific to Burkholderia pseudomallei. Bound phages, selected from fourth-round panning with each mAb, were tested for binding specificity with each mAb using ELISA, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. Overall, 75 of 90 phages (83.3%) were ELISA-positive with each mAb. Mimotopes from all 75 phages (100%) were found to match protein sequences of Burkholderia spp. from GenBank. The predominant mimotopes were TP-GRTRVT found in 13.3%, LTPCGRTxD (8%), AREVTLL (6.7%), NxVxKVVSR (5.3%), PCAPRSS (4%), LGRVLAN (4%), RNPKKA (2.7%) and CPYPR (2.7%). The following GenBank-matched proteins (i.e. the hypothetical proteins) were located at the outer membrane of Burkholderia spp.: BPSL2046 of B. pseudomallei K96243 (matched with mimotope CGRTxD), BpseP_02000035 (matched with LGRVLAN), BPSS0784 of B. pseudomallei K96243 (matched with CPYPR), BURPS1710b_1104 of B. pseudomallei 1710b (matched with CARQY) and TonB-dependent siderophore receptor of B. cenocepacia H12424 (matched with CVRxxLTPC and TPCRxRT). These phage mimotopes and matched proteins may have the potential for further use as diagnostic reagent and immunogen against melioidosis. These results demonstrate that phage-display technique has the potential for rapidly identifying phage mimotopes that interact with B. pseudomallei mAbs

Immune responses of selected phagotopes from monoclonal antibodies of Burkholderia pseudomallei.

Random peptide libraries displayed by bacteriophage T7 and M13 were employed to identify mimotopes from 4 monoclonal antibodies (MAbs) specific to Burkholderia pseudomallei. Insert DNA sequences of bound phages selected from four rounds of panning with each MAb revealed peptide sequences corresponding to B. pseudomallei K96243 hypothetical protein BPSL2046, hypothetical protein BpseP_02000035, B. pseudomallei K96243 hypothetical protein BPSS0784, B. pseudomallei 1710b hypothetical protein BURPS1710b_1104, and B. cenocepacia H12424 TonB-dependent siderophore receptor, all located at the outer membrane. The immune responses from all selected phagotopes were significantly higher than that of lipopolysaccharide. The study demonstrates the feasibility of identifying mimotopes through screening of phage-displayed random peptide libraries with B. pseudomallei MAbs

Patient and sample-related factors that effect the success of in vitro isolation of Orientia tsutsugamushi.

Orientia tsutsugamushi is the causative agent of scrub typhus infection, a major cause of human disease in rural areas of Southeast Asia. Twenty-six blood samples collected from patients with serologically proven scrub typhus during a six month period were sent to Bangkok (535 km from the clinical site) by road at ambient temperature (average daily temperature range: 27.1-29.1 degrees C) for attempted in vitro isolation in Vero cells. O. tsutsugamushi was isolated from 12 samples (sensitivity 46.7%) with the time to isolation ranging from 16 to 37 days [median 27 days, inter-quartile range (IQR) 22.5-33.5 days]. Patient factors such as days of fever and O. tsutsugamushi IgM antibody titer, transport factors such as transit time, and isolate genotype (Karp and Gilliam/Kawasaki) were assessed to determine their influence on the outcome of in vitro isolation. None of the factors significantly influenced the isolation outcome. This study demonstrates that O. tsutsugamushi can often be isolated in vitro from the blood of scrub typhus patients when transported at ambient tropical temperatures for many days

Sensitivity and uncertainty of analytical footprint models according to a combined natural tracer and ensemble approach

Evaluations of analytical footprint models using data from several stations located in different land use types are still scarce, but valuable for defining the spatial context of the measurements. Therefore, we evaluated two analytical footprint models by applying a ‘forward’ and an ‘inversion’ method. We used eddy covariance measurements from a flat agricultural landscape in western Germany in the summer of 2009, with seven eddy covariance systems over three different land use types with contrasting sensible heat fluxes. We found that the model of Hsieh et al. (2000. Adv. Water Resour. 23, 765–772) and of Kormann and Meixner (2001. Boundary Layer Meteorol. 99, 207–224) are both overestimating the distance of the peak contribution of the footprint. In our evaluation, the former model performs slightly better, independent of whether the crosswind dispersion was used from the latter model, or from the proposed model by Detto et al. (2006. Water Resour. Res. 42, 1–16)