Dataset Information

Loop-mediated isothermal amplification applied to filarial parasites detection in the mosquito vectors: Dirofilaria immitis as a study model.

ABSTRACT: BACKGROUND: Despite recent advances in our understanding of the basic biology behind transmission of zoonotic infectious diseases harbored by arthropod vectors these diseases remain threatening public health concerns. For effective control of vector and treatment, precise sampling indicating the prevalence of such diseases is essential. With an aim to develop a quick and simple method to survey zoonotic pathogen-transmitting vectors, LAMP (loop-mediated isothermal amplification) was applied to the detection of filarial parasites using a filarial parasite-transmitting experimental model that included one of the mosquito vectors, Aedes aegypti, and the canine heartworm, Dirofilaria immitis. RESULTS: LAMP reactions amplifying the cytochrome oxidase subunit I gene demonstrated high sensitivity when a single purified D. immitis microfilaria was detected. Importantly, the robustness of the LAMP reaction was revealed upon identification of an infected mosquito carrying just a single parasite, a level easily overlooked using conventional microscopic analysis. Furthermore, successful detection of D. immitis in wild-caught mosquitoes demonstrated its applicability to field surveys. CONCLUSION: Due to its simplicity, sensitivity, and reliability, LAMP is suggested as an appropriate diagnostic method for routine diagnosis of mosquito vectors carrying filarial parasites. This method can be applied to the survey of not only canine filariasis but also lymphatic filariasis, another major public health problem. Therefore, this method offers great promise as a useful diagnostic method for filarial parasite detection in endemic filariasis regions.

SUBMITTER: Aonuma H

PROVIDER: S-EPMC2670822 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Loop-mediated isothermal amplification applied to filarial parasites detection in the mosquito vectors: Dirofilaria immitis as a study model.

Aonuma Hiroka H Yoshimura Aya A Perera Namal N Shinzawa Naoaki N Bando Hironori H Oshiro Sugao S Nelson Bryce B Fukumoto Shinya S Kanuka Hirotaka H

Parasites & vectors 20090315 1

<h4>Background</h4>Despite recent advances in our understanding of the basic biology behind transmission of zoonotic infectious diseases harbored by arthropod vectors these diseases remain threatening public health concerns. For effective control of vector and treatment, precise sampling indicating the prevalence of such diseases is essential. With an aim to develop a quick and simple method to survey zoonotic pathogen-transmitting vectors, LAMP (loop-mediated isothermal amplification) was appli ...[more]

PMID: 19284882

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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: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

Loop-mediated isothermal amplification applied to filarial parasites detection in the mosquito vectors: Dirofilaria immitis as a study model.

Publications

Loop-mediated isothermal amplification applied to filarial parasites detection in the mosquito vectors: Dirofilaria immitis as a study model.

Similar Datasets

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