Prevalence and incrimination of Anopheles fluviatilis species S (Diptera: Culicidae) in a malaria endemic forest area of Chhattisgarh state, central India

BACKGROUND: Chhattisgarh state in central India is highly endemic for malaria and contributes about 13% of annually reported malaria cases in the country with predominance of P. falciparum. Entomological investigations were carried out in a tribal forested area of district Bastar located in the southern part of Chhattisgarh state to record the prevalence of sibling species of Anopheles fluviatilis and An. culicifacies complexes. The vector species complexes were investigated at sibling species level for their biology in terms of resting and feeding behavior and malaria transmission potential. METHODS: Indoor resting vector mosquitoes collected during 2010–2011 were identified to sibling species by cytotaxonomy and polymerase chain reaction (PCR) assay. The blood meal source analysis and incrimination studies were done at sibling species level by counter current immunoelectrophoresis and enzyme linked immunosorbent assay (ELISA) respectively. RESULTS: Analysis of sibling species composition revealed predominance of An. fluviatilis species S in the study area, which was found to be highly anthropophagic and rested in human dwellings whereas the sympatric species T was primarily zoophagic. Incrimination studies showed high sporozoite rate in species S, thereby confirming its vectorial efficiency. An. culicifacies was encountered in low numbers and comprised species B and C in almost equal proportion. Both these species were found to be exclusively zoophagic. CONCLUSION: The observations made strongly suggest that species S of Fluviatilis Complex is the principal vector of malaria in certain forest areas of district Bastar, Chhattisgarh state and should be the target species for vector control operation. Vector control strategies based on biological characteristics of Fluviatilis S will lead to substantial decline in malaria incidence in such areas

Knockdown resistance (kdr)-like mutations in the voltage-gated sodium channel of a malaria vector Anopheles stephensi and PCR assays for their detection

<p>Abstract</p> <p>Background</p> <p>Knockdown resistance (<it>kdr</it>) in insects, resulting from mutation(s) in the voltage-gated sodium channel (vgsc) gene is one of the mechanisms of resistance against DDT and pyrethroid-group of insecticides. The most common mutation(s) associated with knockdown resistance in insects, including anophelines, has been reported to be present at residue Leu1014 in the IIS6 transmembrane segment of the vgsc gene. This study reports the presence of two alternative <it>kdr</it>-like mutations, L1014S and L1014F, at this residue in a major malaria vector <it>Anopheles stephensi </it>and describes new PCR assays for their detection.</p> <p>Methods</p> <p>Part of the vgsc (IIS4-S5 linker-to-IIS6 transmembrane segment) of <it>An. stephensi </it>collected from Alwar (Rajasthan, India) was PCR-amplified from genomic DNA, sequenced and analysed for the presence of deduced amino acid substitution(s).</p> <p>Results</p> <p>Analysis of DNA sequences revealed the presence of two alternative non-synonymous point mutations at L1014 residue in the IIS6 transmembrane segment of vgsc, i.e., T>C mutation on the second position and A>T mutation on the third position of the codon, leading to Leu (TTA)-to-Ser (TCA) and -Phe (TTT) amino acid substitutions, respectively. Polymerase chain reaction (PCR) assays were developed for identification of each of these two point mutations. Genotyping of <it>An. stephensi </it>mosquitoes from Alwar by PCR assays revealed the presence of both mutations, with a high frequency of L1014S. The PCR assays developed for detection of the <it>kdr </it>mutations were specific as confirmed by DNA sequencing of PCR-genotyped samples.</p> <p>Conclusions</p> <p>Two alternative <it>kdr-</it>like mutations, L1014S and L1014F, were detected in <it>An. stephensi </it>with a high allelic frequency of L1014S. The occurrence of L1014S is being reported for the first time in <it>An. stephensi</it>. Two specific PCR assays were developed for detection of two <it>kdr</it>-like mutations in <it>An. stephensi</it>.</p

Transcriptional analysis of an immune-responsive serine protease from Indian malarial vector, Anopheles culicifacies

<p>Abstract</p> <p>Background</p> <p>The main vector for transmission of malaria in India is the <it>Anopheles culicifacies </it>mosquito species, a naturally selected subgroup of which is completely refractory (R) to transmission of the malaria parasite, <it>Plasmodium vivax</it>;</p> <p>Results</p> <p>Here, we report the molecular characterization of a serine protease (<it>acsp30</it>)-encoding gene from <it>A. culicifacies</it>, which was expressed in high abundance in the refractory strain compared to the susceptible (S) strain. The transcriptional upregulation of <it>acsp30 </it>upon <it>Plasmodium </it>challenge in the refractory strain coincided with ookinete invasion of mosquito midgut. Gene organization and primary sequence of <it>acsp30 </it>were identical in the R and S strains suggesting a divergent regulatory status of <it>acsp30 </it>in these strains. To examine this further, the upstream regulatory sequences of <it>acsp30 </it>were isolated, cloned and evaluated for the presence of promoter activity. The 702 bp upstream region of <it>acsp30 </it>from the two strains revealed sequence divergence. The promoter activity measured by luciferase-based reporter assay was shown to be 1.5-fold higher in the R strain than in the S. Gel shift experiments demonstrated a differential recruitment of nuclear proteins to upstream sequences of <it>acsp30 </it>as well as a difference in the composition of nuclear proteins in the two strains, both of which might contribute to the relative abundance of <it>acsp30 </it>in the R strain;</p> <p>Conclusion</p> <p>The specific upregulation of <it>acsp30 </it>in the R strain only in response to <it>Plasmodium </it>infection is suggestive of its role in contributing the refractory phenotype to the <it>A. culicifacies </it>mosquito population.</p

Polymorphism and epitope sharing between the alleles of merozoite surface protein-1 of Plasmodium falciparum among Indian isolates

<p>Abstract</p> <p>Background</p> <p>The C-terminal region of merozoite surface protein-1 (MSP-1) is one of the leading candidates for vaccination against the erythrocytic stages of malaria. However, a major concern in the development of MSP-1 based malaria vaccine is the polymorphism observed in different geographical <it>Plasmodium falciparum </it>isolates. To explore whether the sequence heterogeneity of PfMSP-1 leads to variation in naturally acquired anti-MSP-1₁₉antibodies, the present study was undertaken to study PfMSP-1₁₉sequence polymorphism in malaria-endemic villages in eastern India and also carried out a competition enzyme-linked immunosorbent assay using three PfMSP-1₁₉variant forms.</p> <p>Methods</p> <p>The sequence variations in the C-terminal region of PfMSP-1₁₉were determined in a malaria endemic region. Three PfMSP-1₁₉variants were produced in <it>Escherichia coli </it>(PfMSP1₁₉QKNG-L, PfMSP1₁₉EKNG-L and PfMSP1₁₉ETSR-F) and an immunodepletion assay was carried out using the corresponding patients' sera.</p> <p>Results</p> <p>Results revealed predominance of PfMAD20 allele among Indian field isolates. Seven PfMSP-1₁₉variant forms were isolated in a singe geographical location. Three of PfMSP-1₁₉variant forms when expressed in <it>E. coli </it>showed presence of cross-reaction as well as variant specific antibodies in malaria infected patient sera.</p> <p>Conclusion</p> <p>The present study demonstrates the existence of allele specific antibodies in <it>P. falciparum</it>-infected patient sera, however their role in protection requires further investigation. These results thereby, suggest the importance of a multi-allelic PfMSP-1₁₉based vaccine for an effective malaria control.</p

Allelic dimorphism of Plasmodium vivax gam-1 in the Indian subcontinent

BACKGROUND: Genetic polymorphism is an inevitable component of a complex organism especially in multistage infectious organisms such as malaria parasites. Understanding the population genetic structure of the parasites would provide valuable information for effective malaria control strategies. Recently, the development of molecular tools like PCR has made analysis of field samples possible and easier and research on Plasmodium vivax has also been strengthened. Not many reports are available on the genetic polymorphism of P. vivax from the Indian sub-continent. This study evaluates the extent of diversity in field isolates of India with respect to Pvgam-1. METHODS: A study was designed to assess the diversity of Pvgam-1 among field isolates from India, using a nested PCR assay. Field isolates were collected from different regions of the country and the observed variability was confirmed by sequencing data. RESULTS: Both Belem and Chesson type alleles were present either exclusively or in mixed form among isolates of all 10 study sites. The Belem type allele was predominant, occurring in 67% of isolates. The proportion of isolates showing the mixed form (both Belem and Chesson type alleles occurring together in the same isolate) was about 13 overall (up to 38.5% in some isolates). Sequencing of the PCR-amplified Belem and Chesson type alleles confirmed the PCR results. Among the 10 study sequences, 11 polymorphic sites and four singleton variations were observed. All the nucleotide substitutions were non-synonymous. CONCLUSION: Study shows limited diversity of Pvgam-1 marker in Indian isolates with well representation of both Belem and Chesson type alleles

PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

<p>Abstract</p> <p>Background</p> <p><it>Anopheles culicifacies s.l</it>., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (<it>kdr</it>) is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of <it>kdr </it>mutation (L1014F) in a field population of <it>An. culicifacies s.l</it>. and three new PCR-based methods for <it>kdr </it>genotyping.</p> <p>Methods</p> <p>The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant <it>An. culicifacies s.l</it>. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F) in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR), an Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis-PCR (PIRA-PCR) were developed and tested for <it>kdr </it>genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped.</p> <p>Results</p> <p>The genotyping of this <it>An. culicifacies s.l</it>. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the <it>kdr </it>allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium.</p> <p>Conclusion</p> <p>The Leu-Phe mutation, which generates the <it>kdr </it>phenotype in many insects, was detected in a pyrethroid and DDT resistant <it>An. culicifacies s.l</it>. population. Three PCR-based methods were developed for <it>kdr </it>genotyping. All the three assays were specific. The ARMS method was refractory to non-specific amplification in non-stringent amplification conditions. The PIRA-PCR assay is able to detect both the codons for the phenylalanine mutation at <it>kdr </it>locus, i.e., TTT and TTC, in a single assay, although the latter codon was not found in the population genotyped.</p