Molecular detection of Orientia tsutsugamushi in ectoparasites & their small mammal hosts captured from scrub typhus endemic areas in Madurai district, India.

BACKGROUND OBJECTIVES: Scrub typhus, caused by Orientia tsutsugamushi present in small mammals harbouring the ectoparasites. A study was undertaken to detect the pathogen present in small mammals and its ectoparasites in the scrub typhus-reported areas. METHODS: The small mammals (rodents/shrews) and its ectoparasites were screened for O. tsutsugamushi using nested PCR amplification of the groEL gene. Small mammals were collected by trapping and screened for ectoparasites (mites, ticks and fleas) by combing method. RESULTS: All the chigger mites collected were tested negative for O. tsutsugamushi . Interestingly, adult non-trombiculid mites ( Oribatida sp., Dermanyssus gallinae ), fleas ( Xenopsylla astia, X. cheopis, Ctenophalides felis and Ctenophalides sp.) and ticks ( Rhipicephalus sanguineus , R. haemaphysaloides ) screened were found to be positive for O. tsutsugamushi , which the authors believe is the first report on these species globally. Bandicota bengalensis with O. tsutsugamushi infection is reported for the first time in India. The O. tsutsugamushi groEL sequences from the positive samples were similar to the reference strains, Karp and Ikeda and phylogenetically clustered in clade IV with less evolutionary divergence. The blood samples of Rattus rattus , Suncus murinus and B. bengalensis collected from this area were tested positive for O. tsutsugamushi ; interestingly, the sequence similarity was much pronounced with their ectoparasites indicating the transmission of the pathogen to host or vice versa . INTERPRETATION CONCLUSIONS: The outcome of the present investigations widened our scope on the pathogens present in ectoparasites and rodents/shrews from this area. This will help to formulate the required vector control methods to combat zoonotic diseases.

High-level expression of functionally active dengue-2 non-structural antigen 1 production in Escherichia coli.

Detection of nonstructural protein (NS1) is an important diagnostic marker during acute phase of dengue infection. Not only for diagnostic purpose, the protein had important role in vaccine design as well, as a candidate for studying virus assembly and maturation. Various researchers employed different expression systems and strategies for recombinant NS1 protein production. Attempts to express NS1 protein in prokaryotic and yeast expression system result in formation of insoluble protein which needs to undergo refolding to attain native structural and functional forms. Here, we report the production of soluble NS1 protein in E. coli by using appropriate vector and employing suitable culture conditions to maximize protein production. Proteins were purified using metal affinity chromatography. SDS-PAGE and western blot analysis reveal the native structure of NS1 protein. Solid phase ELISA using the recombinantly expressed antigen with positive and negative dengue samples showed that the expressed protein retains its antigenic and immunological properties. To our knowledge, this is the first report on the successful production of functionally active recombinant dengue-2 NS1 protein production without undergoing any in vitro posttranslational modification process.

Molecular characterization of mosquitocidal Bacillus sphaericus isolated from Tamil Nadu, India.

Forty-two Bacillus sphaericus strains were isolated from soil around Tamil Nadu, India. The phylogenetic relationship among the B. sphaericus isolates was analysed by REP-PCR and multiplex PCR was performed for the detection of mosquito larvicidal genes binA, binB, mtx1, mtx2 and mtx3 in B. sphaericus isolates. According to the REP-PCR band pattern, B. sphaericus isolates were divided into group A comprising I-XI clusters and group B comprising cluster XII. Three of the isolates BSTN01, 23 and 24 were gathered under cluster XII showed a high level of larvicidal activity against Culex quinquefasciatus and Anopheles stephensi, the other 39 isolates grouped under I-XI clusters were non-toxic or weak or moderately toxic to mosquito larvae. Even though BSTN23 and 24 were isolated from the same location and both contained all the five mosquito larvicidal genes, their intraspecies difference was clearly elucidated by REP-PCR analysis. Among high toxic isolates, BSTN23 and 24 were observed to contain all the five toxin genes and BSTN01 showed the presence of binary toxin and Mtx1 toxin genes. The isolates BSTN02, 03, 07, 14, 16, 19, 20, 21, 25, 31, 36 and 39 were found to contain mtx1 gene with combination of mtx2 and/or mtx3 showed moderate or low toxicity against mosquito larvae. binA, binB and mtx1 genes were not present in non-toxic isolates. The present study revealed the genetic heterogeneity between both toxic and non-toxic isolates and indicates that there is a good correlation between the presence of toxin genes and toxicity of the strains. These techniques could be developed in screening of novel highly toxic B. sphaericus strains from environment without bioassay on mosquito larvae.

Isolation and characterization of bacteria from the gut of Bombyx mori that degrade cellulose, xylan, pectin and starch and their impact on digestion.

Bombyx mori L. (Lepidoptera: Bombycidae) have been domesticated and widely used for silk production. It feeds on mulberry leaves. Mulberry leaves are mainly composed of pectin, xylan, cellulose and starch. Some of the digestive enzymes that degrade these carbohydrates might be produced by gut bacteria. Eleven isolates were obtained from the digestive tract of B. mori, including the Gram positive Bacillus circulans and Gram negative Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Serratia liquefaciens, Enterobacter sp., Pseudomonas fluorescens, P. aeruginosa, Aeromonas sp., and Erwinia sp.. Three of these isolates, P. vulgaris, K. pneumoniae, C. freundii, were cellulolytic and xylanolytic, P. fluorescens and Erwinia sp., were pectinolytic and K. pneumoniae degraded starch. Aeromonas sp. was able to utilize the CMcellulose and xylan. S. liquefaciens was able to utilize three polysaccharides including CMcellulose, xylan and pectin. B. circulans was able to utilize all four polysaccharides with different efficacy. The gut of B. mori has an alkaline pH and all of the isolated bacterial strains were found to grow and degrade polysaccharides at alkaline pH. The number of cellulolytic bacteria increases with each instar.