Dataset Information

Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

ABSTRACT: A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2?, 79.0±0.3?, 76.8±0.1?, and 79.9±0.1?, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

SUBMITTER: Thanchomnang T

PROVIDER: S-EPMC3916452 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

Thanchomnang Tongjit T Intapan Pewpan M PM Tantrawatpan Chairat C Lulitanond Viraphong V Chungpivat Sudchit S Taweethavonsawat Piyanan P Kaewkong Worasak W Sanpool Oranuch O Janwan Penchom P Choochote Wej W Maleewong Wanchai W

The Korean journal of parasitology 20131231 6

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancro ...[more]

PMID: 24516268

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Project description:BackgroundCanine dirofilariasis due to Dirofilaria immitis is known to be endemic in continental Portugal. However, information about the transmitting mosquito species is still scarce, with only Culex theileri identified to date, albeit with L1-2, through dissection. This study was carried out to investigate the potential vectors of Dirofilaria spp. in continental Portugal.MethodsMosquitoes were collected in three distinct seasons (Summer, Autumn and Spring), 2011-2013, in three districts. CDC traps and indoor resting collections were carried out in the vicinity of kennels. Mosquitoes were kept under controlled conditions for 7 days to allow the development of larval stages of Dirofilaria spp.. DNA extraction was performed separately for both head+thorax and abdomen in order to differentiate infective and infected specimens, respectively, in pools, grouped according to the species and collection site (1-40 specimen parts/pool), and examined by PCR using pan-filarial specific primers. Mosquito densities were compared using non-parametric tests. Dirofilaria development units (DDU) were estimated.ResultsIn total, 9156 female mosquitoes, from 11 different species, were captured. Mosquito densities varied among the 3 districts, according to capture method, and were generally higher in the second year of collections. From 5866 specimens screened by PCR, 23 head+thorax and 41 abdomens pools, corresponding to 54 mosquitoes were found positive for D. immitis DNA. These belonged to 5 species: Culex (Cux) theileri (estimated rate of infection (ERI)=0.71%), Cx. (Cux) pipiens f. pipiens and f. molestus (ERI=0.5%), Anopheles (Ano) maculipennis s.l. (ERI=3.12%), including An. (Ano) atroparvus, Aedes (Och) caspius (ERI=3.73%) and Ae. (Och) detritus s.l. (ERI=4.39%). All but Cx. pipiens, had at least one infective specimen. No D. repens infected specimens were found. Infection rates were: 3.21% in Coimbra, 1.22% in Setúbal and 0.54% in Santarém. DDU were at least 117/year in the study period.ConclusionsCulex theileri, Cx. pipiens, An. maculipennis s.l. An. atroparvus, Ae.caspius and Ae. detritus s.l. were identified as potential vectors of D. immitis in three districts of Portugal, from Spring to Autumn, in 5 of the 6 collection dates in 2011-2013. Implications for transmission, in the context of climate changes, and need for prophylactic measures, are discussed.

Project description:BackgroundMosquitoes transmit filarial nematodes to both human and animal hosts, with worldwide health and economic consequences. Transmission to a vertebrate host requires that ingested microfilariae develop into infective third-stage larvae capable of emerging from the mosquito proboscis onto the skin of the host during blood-feeding. Determining the number of microfilariae that successfully develop to infective third-stage larvae in the mosquito host is key to understanding parasite transmission potential and to developing new strategies to block these worms in their vector.MethodsWe developed a novel method to efficiently assess the number of infective third-stage filarial larvae that emerge from experimentally infected mosquitoes. Following infection, individual mosquitoes were placed in wells of a multi-well culture plate and warmed to 37 °C to stimulate parasite emergence. Aedes aegypti infected with Dirofilaria immitis were used to determine infection conditions and assay timing. The assay was also tested with Brugia malayi-infected Ae. aegypti.ResultsApproximately 30% of Ae. aegypti infected with D. immitis and 50% of those infected with B. malayi produced emerging third-stage larvae. Once D. immitis third-stage larvae emerged at 13 days post infection, the proportion of mosquitoes producing them and the number produced per mosquito remained stable until at least day 21. The prevalence and intensity of emerging third-stage B. malayi were similar on days 12-14 post infection. Increased uptake of D. immitis microfilariae increased the fitness cost to the mosquito but did not increase the number of emerging third-stage larvae.ConclusionsWe provide a new assay with an associated set of infection conditions that will facilitate assessment of the filarial transmission potential of mosquito vectors and promote preparation of uniformly infectious third-stage larvae for functional assays. The ability to quantify infection outcome will facilitate analyses of molecular interactions between vectors and filariae, ultimately allowing for the establishment of novel methods to block disease transmission.

Project description:BackgroundIn the past, immune responses to several Brugia malayi immunodominant antigens have been characterized in filaria-infected populations; however, little is known regarding Wolbachia proteins. We earlier cloned and characterized few B. malayi (trehalose-6-phosphate phosphatase, Bm-TPP and heavy chain myosin, BmAF-Myo) and Wolbachia (translation initiation factor-1, Wol Tl IF-1 and NAD+-dependent DNA ligase, wBm-LigA) proteins and investigated the immune responses, which they triggered in animal models. The current study emphasizes on immunological characteristics of these proteins in three major categories of filarial endemic zones: endemic normal (EN, asymptomatic, amicrofilaraemic; putatively immune), microfilariae carriers (MF, asymptomatic but microfilaraemic), and chronic filarial patients (CP, symptomatic and mostly amicrofilaraemic).MethodsImmunoblotting and ELISA were carried out to measure IgG and isotype antibodies against these recombinant proteins in various clinical categories. Involvement of serum antibodies in infective larvae killing was assessed by antibody-dependent cellular adhesion and cytotoxicity assay. Cellular immune response was investigated by in vitro proliferation of peripheral blood mononuclear cells (PBMCs) and reactive oxygen species (ROS) generation in these cells after stimulation.ResultsImmune responses of EN and CP displayed almost similar level of IgG to Wol Tl IF-1 while other three proteins had higher serum IgG in EN individuals only. Specific IgA, IgG1, IgG3 and IgM to Bm-TPP were high in EN subjects, while BmAF-Myo additionally showed elevated IgG2. Enhanced IgA and IgG3 were detected in both EN and CP individuals in response to Wol Tl IF-1 antigen, but IgG1 and IgM were high only in EN individuals. wBm-LigA and BmAF-Myo exhibited almost similar pattern of antibody responses. PBMC isolated from EN subjects exhibited higher proliferation and ROS generation when stimulated with all three proteins except for Wol Tl IF-1.ConclusionsOverall, these findings display high immunogenicity of all four proteins in human subjects and revealed that the EN population was exposed to both B. malayi and Wolbachia proteins simultaneously. In addition, immune responses to Wol Tl IF-1 suggest possible role of this factor in Wolbachia-induced pathological responses while immune responses to other three proteins suggest that these can be explored further as vaccine candidates.

Project description:BackgroundHeartworm (Dirofilaria immitis) in dogs is considered endemic in Australia, but the clinical heartworm disease caused by the heartworm is rare and prevalence is low. The mainstream prevention of the heartworm is based on macrocyclic lactone (ML) administration. The aim of this study was to confirm endemism of the heartworm under current Australian conditions using a cohort of recent microfilaria-positive dogs which were on variable heartworm prevention.MethodsA hotspot of canine heartworm antigen-positive and microfilaria-positive dogs has been detected recently in Queensland, Australia. Blood samples from 39 dogs from Queensland and two dogs from New South Wales were investigated for canine filarioids. Rapid antigen diagnostic tests capable of detection of D. immitis and real-time PCR for quantification and differentiation between D. immitis from Acanthocheilonema reconditum with quantification of microfilariae in canine blood samples, together with D. immitis specific real-time PCR assay, were applied to microfilaria-positive dogs. The P-glycoprotein genotype was determined to test whether Australian-sourced heartworm shared the same genetic markers as those suspected of ML-resistance in North America.ResultsOnly D. immitis was detected in the samples from Queensland and New South Wales, Australia. Using high resolution melt real-time PCR and D. immitis specific real-time PCR, the calculated microfilaria concentration ranged from 1 to 44,957 microfilariae/ml and from 7 to 60,526 microfilariae/ml, respectively. DNA sequencing of the PCR products confirmed D. immitis. Fifteen of the examined dogs were on putative, rigorous ML prevention. For the remaining dogs, compliance with heartworm prevention was unknown or reported as inconsistent. Wild-type genotype AA-GG of the P-glycoprotein locus of D. immitis sequence has been obtained for three blood samples. Due to the incomplete history, any suggestion of a loss of efficacy of MLs must be treated as 'remotely possible'. In the immediate future, records of preventative administration and annual antigen testing would be required to determine any problems with the efficacy of preventatives.ConclusionsThe prevalence of canine heartworm in Australia remains poorly understood. It is generally assumed to be low by veterinary practitioners. The localised increase in the study area confirms endemism of canine heartworm and a requirement for ongoing vigilance through annual heartworm testing to better understand the changing distribution of canine heartworm, client compliance, as well as to detect any change in ML-susceptibility.

Dataset Information

Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

Publications

Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

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