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First analysis of the secretome of the canine heartworm, Dirofilaria immitis.

ABSTRACT: BACKGROUND: The characterization of proteins released from filariae is an important step in addressing many of the needs in the diagnosis and treatment of these clinically important parasites, as well as contributing to a clearer understanding of their biology. This report describes findings on the proteins released during in vitro cultivation of adult Dirofilaria immitis , the causative agent of canine and feline heartworm disease. Differences in protein secretion among nematodes in vivo may relate to the ecological niche of each parasite and the pathological changes that they induce. METHODS: The proteins in the secretions of cultured adult worms were run on Tris-Glycine gels, bands separated and peptides from each band analysed by ultra mass spectrometry and compared with a FastA dataset of predicted tryptic peptides derived from a genome sequence of D. immitis. RESULTS: This study identified 110 proteins. Of these proteins, 52 were unique to D. immitis. A total of 23 (44%) were recognized as proteins likely to be secreted. Although these proteins were unique, the motifs were conserved compared with proteins secreted by other nematodes. CONCLUSION: The present data indicate that D. immitis secretes proteins that are unique to this species, when compared with Brugia malayi. The two major functional groups of molecules represented were those representing cellular and of metabolic processes. Unique proteins might be important for maintaining an infection in the host environment, intimately involved in the pathogenesis of disease and may also provide new tools for the diagnosis of heartworm infection.

SUBMITTER: Geary J

PROVIDER: S-EPMC3439246 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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First analysis of the secretome of the canine heartworm, Dirofilaria immitis.

Geary James J Satti Mohamed M Moreno Yovany Y Madrill Nicole N Whitten Doug D Headley Selwyn A SA Agnew Dalen D Geary Timothy T Mackenzie Charles C

Parasites & vectors 20120710

<h4>Background</h4>The characterization of proteins released from filariae is an important step in addressing many of the needs in the diagnosis and treatment of these clinically important parasites, as well as contributing to a clearer understanding of their biology. This report describes findings on the proteins released during in vitro cultivation of adult Dirofilaria immitis , the causative agent of canine and feline heartworm disease. Differences in protein secretion among nematodes in vivo ...[more]

PMID: 22781075

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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.

Project description:BACKGROUND:Canine heartworm disease, caused by Dirofilaria immitis, has global veterinary importance. In Australia, the prevalence of canine heartworm infection decreased markedly following the introduction of over-the-counter macrocyclic lactones. We aimed to estimate the prevalence of canine heartworm infection in at-risk populations of dogs in eastern Australia and analyse published prevalence data from Australia. METHODS:In total, 566 dogs from eastern Australia were tested for the presence of D. immitis antigen. Four cohorts were studied: pig-hunting dogs from Queensland (Cohort 1, n = 104), dogs from remote New South Wales (NSW) (Cohort 2, n = 332), urban pets from rural NSW (Cohort 3, n = 45) and ex-racing Greyhounds from Sydney, NSW (Cohort 4, n = 85). Serum samples were screened for D. immitis antigen using a reference laboratory microwell-based assay (DiroChek®) or a point-of-care immunochromatography test kit (Anigen Rapid®). Risk factors associated with the odds of D. immitis antigen seropositivity were identified using binary logistic regression models. Seropositive blood samples were tested for the presence and quantity of D. immitis DNA using a species specific real-time (q)PCR assay. A metanalysis of the Australian canine heartworm literature was conducted. RESULTS:The prevalence of dirofilariasis in pig-hunting dogs from Queensland (Cohort 1) was 12.5% (95% CI: 6.5-18.9%), with a subpopulation of dogs from Central Queensland having a prevalence of 21% (95% CI: 12.3-33.4%). Age was significantly associated with D. immitis antigen seropositivity (increased risk with increased age). The odds of being > 5 years versus ≤ 5 years was 3.7-times (95% CI: 1.1-12.5) greater in antigen positive versus antigen negative dogs. No D. immitis antigen positive dogs were detected in dogs from NSW (Cohorts 2-4). The Australian canine heartworm disease literature includes 98 peer-reviewed publications (1901-2019) with 30 studies reporting on D. immitis prevalence in dogs. Throughout the publication peak period (1980s), the primary antemortem diagnostic test was detection of microfilariae. CONCLUSIONS:Canine heartworm infection in dogs used for pig hunting is a previously unexplored topic in Australia. Pig-hunting dogs are infected with canine heartworm in Queensland, Australia, placing pet dogs and cats at increased risk of infection.

Project description:Canine heartworm disease is a potentially deadly cardiopulmonary disease caused by the mosquito-borne filarial nematode Dirofilaria immitis. In Australia, the administration of macrocyclic lactone (ML) drugs has successfully reduced the prevalence of D. immitis infection. However, the recent re-emergence of D. immitis in dogs in Queensland, Australia and the identification of ML-resistant isolates in the USA poses an important question of whether ML-resistance has emerged in this parasite in Australia. The aim of this study was to utilise phenotypic and genotypic approaches to examine the sensitivity to ML drugs in D. immitis in Australia. To do this, we surveyed 45 dogs from Queensland and New South Wales across 3 years (2019-2022) for the presence of D. immitis infection using an antigen test, quantitative Modified Knott's test, and qPCR targeting both D. immitis and the D. immitis symbiont Wolbachia. A phenotype observed by utilising sequential quantification of microfilariae for 23/45 dogs was coupled with genetic testing of filtered microfilariae for SNPs previously associated with ML-resistance in isolates from the USA. Sixteen (16/45) dogs tested positive for D. immitis infection despite reportedly receiving 'rigorous' heartworm prevention for 12 months prior to the study, according to the owners' assessment. The phenotype and genotypic assays in this study did not unequivocally demonstrate the presence of ML-resistant D. immitis in Australia. Although the failure of 16 dogs to reduce microfilaremia by >90% after ML treatment was considered a suspect phenotype of ML-resistance, no genotypic evidence was discovered using the genetic SNP analysis. The traditional quantitative Modified Knott's test can be substituted by qPCR targeting D. immitis or associated Wolbachia endosymbiont DNA for a more rapid measurement of microfilariae levels. More definitive phenotypic evidence of resistance is critically needed before the usefulness of SNPs for the detection of ML-resistance in Australia can be properly assessed.

Project description:BackgroundThe canine heartworm Dirofilaria immitis, a filarioid nematode of dogs and other carnivores, is widespread in the USA and the world. Over 20 different mosquito species serve as intermediate hosts of D. immitis, but their contribution to transmission varies according to factors like host feeding patterns, geographic locations and climatic conditions. The yellow fever mosquito, Aedes aegypti, is a competent vector of D. immitis but is often dismissed as a vector of veterinary relevance given its anthropophilic feeding behavior. We evaluated the prevalence of D. immitis in pet dogs along the USA-Mexico border and assessed whether Ae. aegypti in the area are naturally infected with heartworm and are potentially acting as a vector.MethodsA total of 200 whole blood samples collected from pet dogs in the Lower Rio Grande Valley in south Texas from 2016 to 2019 were included in this study. Canine serum samples for D. immitis were tested using the DiroCHEK® Canine Heartworm Antigen Test Kit pre- and post-immune complex dissociations (ICD) and blood samples were tested using high-resolution melt (HRM) quantitative PCR (qPCR) and a probe-based qPCR. Additionally, mosquito specimens were collected and identified, and Ae. aegypti heads, abdomens and pools were tested using conventional PCR (cPCR) and HRM qPCR.ResultsOverall, heartworm prevalence in dogs aged > 6 months was 40.8% (64/157) when the results from all testing modalities were considered. Heartworm antigen was detected in 33.5% and 40.7% of the dogs using DiroCHEK® pre- and post-ICD, respectively. By molecular screening, 20.1% of dogs tested positive with probe-based qPCR, while only one tested positive with HRM qPCR. Of the Ae. aegypti abdomens from blood-fed Ae. aeygpti tested, 20 (21.7%) from mosquitoes that fed on dogs and four (7%) from those that fed on humans tested positive for heartworm. Among Ae. aegypti heads from blood-fed Ae. aeygpti, two (1.1%) were positive based on cPCR and four (2.5%) were positive based on HRM qPCR. No D. immitis DNA was detected in the 208 pools of whole bodies (358 individuals) of Ae. aegypti gravid females.ConclusionsOur study highlights a high prevalence of heartworm in dogs in south Texas and provides evidence that Ae. aegypti could be contributing to heartworm transmission in canine populations in this region.

Project description:BACKGROUND:For dogs and cats, chemoprophylaxis with macrocyclic lactone (ML) preventives for heartworm disease is widely used in the United States and other countries. Since 2005, cases of loss of efficacy (LOE) of heartworm preventives have been reported in the U.S. More recently, ML-resistant D. immitis isolates were confirmed. Previous work identified 42 genetic markers that could predict ML response in individual samples. For field surveillance, it would be more appropriate to work on microfilarial pools from individual dogs with a smaller subset of genetic markers. METHODS:MiSeq technology was used to identify allele frequencies with the 42 genetic markers previously reported. Microfilaria from ten well-characterized new isolates called ZoeKY, ZoeMI, ZoeGCFL, ZoeAL, ZoeMP3, ZoeMO, ZoeAMAL, ZoeLA, ZoeJYD-34, and Metairie were extracted from fresh blood from dogs. DNA were extracted and sequenced with MiSeq technology. Allele frequencies were calculated and compared with the previously reported susceptible, LOE, and resistant D. immitis populations. RESULTS:The allele frequencies identified in the current resistant and susceptible isolates were in accordance with the allele frequencies previously reported in related phenotypes. The ZoeMO population, a subset of the ZoeJYD-34 population, showed a genetic profile that was consistent with some reversion towards susceptibility compared with the parental ZoeJYD-34 population. The Random Forest algorithm was used to create a predictive model using different SNPs. The model with a combination of three SNPs (NODE_42411_RC, NODE_21554_RC, and NODE_45689) appears to be suitable for future monitoring. CONCLUSIONS:MiSeq technology provided a suitable methodology to work with the microfilarial samples. The list of SNPs that showed good predictability for ML resistance was narrowed. Additional phenotypically well characterized D. immitis isolates are required to finalize the best set of SNPs to be used for large scale ML resistance screening.

Dataset Information

First analysis of the secretome of the canine heartworm, Dirofilaria immitis.

Publications

First analysis of the secretome of the canine heartworm, Dirofilaria immitis.

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