To describe the midgut microbial diversity and the candidate bacteria for the genetic manipulation for the generation of transgenic mosquitoes refractory to transmission of diseases, the microbiota of wild Culex quinquefasciatus mosquito midgut was studied using a conventional culture technique and analysis of a 16S ribosomal RNA (rRNA) gene sequence library. The culturable microbiota was identified as Acinetobacter junii, Ac. calcoaceticus, Aeromonas culicicola, Bacillus thuringiensis, Microbacterium oxydans, Pantoea agglomerans, Pseudomonas aeruginosa, Staphylococcus epidermidis, Stenotrophomonas maltophila and an unidentified bacterium from the host Drosophila paulistorium. The 16S rRNA gene library was composed of 46% unidentified and uncultured bacteria, 41% Acinetobacter spp., and 13% Lactococcus spp. The coverage calculated for the 150 clones was 83.3%. Thus, the probability of the next cloned sequence falling in a novel operational taxonomic unit (not yet observed) was 16.7%. The majority of the cultured isolates and the 16S rRNA gene library clones belonged to the gamma-proteobacteria class. Most of the bacteria have been previously reported to inhabit the midgut of different mosquito species. Therefore, the results of this study indicate that different mosquito species harbor common representatives of the microbiota that may be the potential candidates for genetic manipulation to control the disease transmission capabilities of the host.
The taxonomic position was examined of three isolates, MTCC 3249T, SH and SLH, from the midgut of female Culex quinquefasciatus and Aedes aegyptii mosquitoes. Numbers of cells of these isolates increased 2000-fold after a blood meal of the mosquitoes. 16S rRNA gene sequence analysis of the novel strains showed that they were highly homologous to strains of Aeromonas. DNA-DNA hybridization studies showed that DNA of strain MTCC 3249T was 96 and 88% similar to that of strains SH and SLH, respectively, and showed 54% relatedness to Aeromonas jandaei and 61% relatedness to Aeromonas sobria, which is below the cut-off value for species differentiation. The biochemical profiles of all three novel strains were identical. On the basis of a polyphasic approach using phenotypic analysis, 16S rRNA gene sequencing and DNA-DNA hybridization studies, a novel species is proposed for these isolates, Aeromonas culicicola sp. nov., with the type strain MTCC 3249T (= NCIM 5147T). Isolation of A. culicicola from the midgut of mosquitoes might help to explain the origin of Aeromonas infections caused without exposure to contaminated water, soil or food.
The taxonomic position was examined of three isolates, MTCC 3249T, SH and SLH, from the midgut of female Culex quinquefasciatus and Aedes aegyptii mosquitoes. Numbers of cells of these isolates increased 2000-fold after a blood meal of the mosquitoes. 16S rRNA gene sequence analysis of the novel strains showed that they were highly homologous to strains of Aeromonas. DNA-DNA hybridization studies showed that DNA of strain MTCC 3249T was 96 and 88% similar to that of strains SH and SLH, respectively, and showed 54% relatedness to Aeromonas jandaei and 61% relatedness to Aeromonas sobria, which is below the cut-off value for species differentiation. The biochemical profiles of all three novel strains were identical. On the basis of a polyphasic approach using phenotypic analysis, 16S rRNA gene sequencing and DNA-DNA hybridization studies, a novel species is proposed for these isolates, Aeromonas culicicola sp. nov., with the type strain MTCC 3249T (= NCIM 5147T). Isolation of A. culicicola from the midgut of mosquitoes might help to explain the origin of Aeromonas infections caused without exposure to contaminated water, soil or food.
Autoantibody response against the small nuclear ribonucleoprotein (snRNP) complex is a characteristic feature of systemic lupus erythematosus. The current investigation was undertaken to determine whether activation of SmD-reactive T cells by synthetic peptides harboring T cell epitopes can initiate a B cell epitope spreading cascade within the snRNP complex. T cell epitopes on SmD were mapped in A/J mice and were localized to three regions on SmD, within aa 26–55, 52–69, and 86–115. Immunization with synthetic peptides SmD31–45, SmD52–66, and SmD91–110 induced T and B cell responses to the peptides, with SmD31–45 inducing the strongest response. However, only SmD52–66 immunization induced T cells capable of reacting with SmD. Analysis of sera by immunoprecipitation assays showed that intermolecular B cell epitope spreading to U1RNA-associated A ribonucleoprotein and SmB was consistently observed only in the SmD52–66-immunized mice. Surprisingly, in these mice, Ab responses to SmD were at low levels and transient. In addition, the sera did not react with other regions on SmD, indicating a lack of intramolecular B cell epitope spreading within SmD. Our study demonstrates that T cell responses to dominant epitope on a protein within a multiantigenic complex are capable of inducing B cell responses to other proteins within the complex. This effect can happen without generating a good Ab response to the protein from which the T epitope was derived. Thus caution must be taken in the identification of Ags responsible for initiating autoimmune responses based solely on serological analysis of patients and animals with systemic autoimmune disorders.
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