Dataset Information

Molecular detection of Dirofilaria immitis and its Wolbachia endosymbionts in dogs from Myanmar.

ABSTRACT: Heartworm disease in dogs and cats caused by Dirofilaria immitis continues to be a major clinical issue globally. This study focused on dogs suspicious of having tick-borne diseases (TBD) brought to a clinic and a veterinary teaching hospital in Myanmar. Blood samples were collected and initially screened using SNAP® 4Dx® Plus test kit. All dog blood samples were subjected to conventional PCR to detect both Dirofilaria spp. (cox1 gene) and Wolbachia spp. (16S rDNA) infections. Infection with D. immitis was detected in 14 (28.0%) of 50 examined samples, while the detection rate of TBD causative agents, including Anaplasma phagocytophilum and Ehrlichia canis, was 26.0% (13/50) and 26.0% (13/50), respectively, as determined by ELISA rapid test. In this study, D. immitis infection was moderately but significantly correlated with TBD infections (Pearson's r = 0.397, P = 0.008). Comparative sequence and phylogenetic analyses provided molecular identification of D. immitis in Myanmar and confirmed the identity of its Wolbachia endosymbiont with Wolbachia endosymbionts isolated from D. immitis, Rhipicephalus sanguineus and Aedes aegypti. The present study contributes to our understanding of the coexistence of D. immitis and Wolbachia endosymbiosis in dogs, and the findings may benefit the future prevention and control of dirofilariasis in dogs.

SUBMITTER: Bawm S

PROVIDER: S-EPMC10665652 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Molecular detection of Dirofilaria immitis and its Wolbachia endosymbionts in dogs from Myanmar.

Bawm Saw S Khaing Yadanar Y Chel Hla Myet HM Hmoon Myint Myint MM Win Shwe Yee SY Bo Min M Naing Tint T Htun Lat Lat LL

Current research in parasitology & vector-borne diseases 20231004

Heartworm disease in dogs and cats caused by Dirofilaria immitis continues to be a major clinical issue globally. This study focused on dogs suspicious of having tick-borne diseases (TBD) brought to a clinic and a veterinary teaching hospital in Myanmar. Blood samples were collected and initially screened using SNAP® 4Dx® Plus test kit. All dog blood samples were subjected to conventional PCR to detect both Dirofilaria spp. (cox1 gene) and Wolbachia spp. (16S rDNA) in ...[more]

PMID: 38021190

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Project description:BackgroundWolbachia is present in all life stages of Dirofilaria immitis. Wolbachia surface protein (WSP) can be highly immunogenic and induce acute inflammatory reactions in the host upon worm death. To avoid the abrupt release of Wolbachia and its antigens from deceased parasites, the American Heartworm Society (AHS) has recommended using doxycycline (DOXY) and having a 1-month wait period between the DOXY treatment and the adulticidal process for Wolbachia elimination. Studies have shown that the 28 day, 10 mg/kg twice daily (BID) administration of DOXY can effectively clear Wolbachia in the bloodstream of the host. The 1-month wait period is hypothesized to allow for further reduction of Wolbachia. However, the levels of Wolbachia in adult parasites after the DOXY treatment remain unknown.MethodsForty-five purposely bred dogs were intravenously transplanted with 20 Dirofilaria immitis adults, consisting of 12 females and 8 males. The dogs were divided into nine groups of five dogs each. Two groups each received 5, 7.5, or 10 mg/kg DOXY BID orally for 28 days, and ivermectin (IVM) monthly (6 µg/kg.) Three groups remained untreated as controls. Study animals were necropsied on day 0, day 30, and day 60, following the start of treatment. Adult worms were collected at necropsy and preserved for analysis. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry for WSP were performed on worms collected at each time point. The data were analyzed using a linear mixed model (LMM). Multiple comparisons were adjusted using Tukey's test.ResultsThe qPCR results showed that all treatment doses significantly reduced Wolbachia levels compared with the control groups at 30 and 60 days. The intradose comparison indicated a significant decrease on day 60 compared with day 30. No significant differences were found between different doses on the two examination dates. Immunohistochemistry indicated the markedly reduced presence of Wolbachia in treatment groups.ConclusionsAll DOXY dosages can be considered effective in reducing Wolbachia on both tested dates (30 and 60 days). On the basis of the further reduction of Wolbachia levels in adult D. immitis, the 1-month rest period in the AHS heartworm treatment guidelines is beneficial. Wolbachia can still be detected on day 60 in all dosage groups.

Project description:BackgroundThe diagnosis and management of canine heartworm disease is a growing concern for shelter veterinarians. Although the accuracy of commercial antigen test kits has been widely studied, recent reports have renewed interest in antigen blocking as a causative factor for false "no antigen detected" results. The objectives of this study were to determine the prevalence of false "no antigen detected" results in adult dogs entering shelters in northern, southern, and western regions of the country and to identify historical and clinical risk factors for such results.MethodsSerum samples were evaluated for Dirofilaria immitis antigen using a commercially available point-of-care ELISA; samples in which no antigen was detected underwent a heat treatment protocol and repeat antigen testing. Whole blood samples underwent Knott testing to identify the presence of microfilariae. Historical and clinical findings were analyzed using exact logistic regression.ResultsA total of 616 samples were analyzed. Overall prevalence of positive antigen test results (prior to heat treatment) was 7.3% and frequency of false "no antigen detected" results due to antigen blocking (ie, samples with no antigen detected prior to heat treatment and positive after heat treatment) was 5.2%. Among dogs that had no detectable antigen on the initial tests, dogs that had microfilariae detected via modified Knott testing (OR = 32.30, p-value = 0.013) and dogs that previously received a heartworm preventive (OR = 3.81, p-value = 0.016) had greater odds of antigen blocking than dogs without these factors. Among dogs that were heartworm positive, those without microfilariae detected had greater odds of antigen blocking than dogs with this factor (OR = 11.84, p-value = 0.0005). Geographic region of origin was significantly associated with occurrence of antigen blocking (p = 0.0036); however, blocking occurred in all regions sizably contributing to heartworm diagnoses. Of the 74 dogs found to be infected with heartworms in this study, 39.2% (29) had no detectable antigen prior to heat treatment.ConclusionsHeat treatment of serum samples should be considered to improve diagnostic test accuracy, particularly in dogs that reportedly received a heartworm preventive prior to antigen testing regardless of region of origin.

Project description:BackgroundAs a result of globalization and climate change, Dirofilaria immitis and Dirofilaria repens, the causative agents of dirofilariosis in Europe, continue to spread from endemic areas in the Mediterranean to northern and northeastern regions of Europe where autochthonous cases of dirofilarial infections have increasingly been observed in dogs and humans. Whilst D. repens was recently reported from mosquitoes in putatively non-endemic areas, D. immitis has never been demonstrated in mosquitoes from Europe outside the Mediterranean.MethodsFrom 2011 to 2013, mosquitoes collected within the framework of a German national mosquito monitoring programme were screened for filarial nematodes using a newly designed filarioid-specific real-time PCR assay. Positive samples were further processed by conventional PCR amplification of the cytochrome c oxidase subunit I (COI) gene, amplicons were sequenced and sequences blasted against GenBank.ResultsApproximately 17,000 female mosquitoes were subjected to filarial screening. Out of 955 pools examined, nine tested positive for filariae. Two of the COI sequences indicated D. immitis, one D. repens and four Setaria tundra. Two sequences could not be assigned to a known species due to a lack of similar GenBank entries. Whilst D. immitis and the unknown parasites were detected in Culex pipiens/torrentium, D. repens was found in a single Anopheles daciae and all S. tundra were demonstrated in Aedes vexans. All positive mosquitoes were collected between mid-June and early September.ConclusionThe finding of dirofilariae in German mosquitoes implies the possibility of a local natural transmission cycle. While the routes of introduction to Germany and the origin of the filariae cannot be determined retrospectively, potential culicid vectors and reservoir hosts must prospectively be identified and awareness among physicians, veterinarians and public health personnel be created. The health impact of S. tundra on the indigenous cervid fauna needs further investigation.

Project description:BackgroundHeartworms, Dirofilaria immitis, are known to be widespread in dogs and cats in the USA, but there have been no country-wide prevalence studies performed to date. There have also been no large-scale studies to determine whether the closely related species, Dirofilaria repens, occurs in the USA.MethodsTo provide this large-scale data, we examined whole blood samples (n = 2334) submitted from around the USA to the Molecular Diagnostic Laboratory at Auburn University between 2016 and 2022. Quantitative PCRs for D. immitis (targeting 16S rRNA) and D. repens (targeting cytochrome c oxidase subunit 1 gene) were performed to determine the presence of Dirofilaria DNA. DNA sequencing was performed to confirm the results.ResultsDirofilaria immitis DNA was found in 6.3% (68/1080) of the dogs from 17/39 states, and 0.3% (4/1254) of the cats from 4/42 states. None of the dogs or cats were positive for D. repens. The average 16S rRNA copy number of D. immitis in the dogs was 1,809,604 in 200 µl whole blood, while only a single copy was found in each of the four D. immitis-positive cats. The prevalence of D. immitis in dogs of different ages, sexes, and breeds did not differ significantly, but the prevalence in Southern states (7.5%, 60/803) was significantly higher than in the Western (1.7%, 1/58), Midwest (3.3%, 4/120), and Northeastern states (3.1%, 3/98) (P < 0.05). Dogs positive for D. immitis were identified in each study year (2016: 4.2%, 2/48; 2017: 9.8%, 4/41; 2018: 5.1%, 8/156; 2019: 4.9%, 15/306; 2020: 9.8%, 26/265; 2021: 4.9%, 13/264). Interestingly, dogs infected with Hepatozoon spp. (11.8%, 37/313) were significantly more likely to also be positive for D. immitis than dogs without evidence of Hepatozoon infection (3.9%, 30/760) (P < 0.0001).ConclusionsTo our knowledge, this is the first nationwide molecular survey of Dirofilaria spp. in dogs and cats in the USA, and the largest molecular survey of canine and feline dirofilariosis worldwide. Further studies are warranted to combine PCR with standard heartworm diagnostics to better understand the prevalence of Dirofilaria spp. and aid in determining the risks posed to dogs and cats in the USA.

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:BackgroundInfection by the canine heartworm, Dirofilaria immitis, causes significant cardiopulmonary disease, with progression impacted by increasing parasite numbers and duration of infection. The renin-angiotensin-aldosterone system (RAAS) is an important mediator of cardiac and pulmonary disease. Angiotensin-converting enzyme 2 (ACE2) mitigates the maladaptive effects of angiotensin II by converting it to angiotensin (1-7). We hypothesized that circulating ACE2 activity would be altered in dogs with high heartworm infection intensities relative to dogs without heartworms.MethodsFrozen serum samples (-80 °C) from 30 dogs euthanized at Florida shelters were analyzed for ACE2 activity using liquid chromatography-mass spectrometry/mass spectroscopy and a kinetics approach with and without an ACE2 inhibitor. A convenience sample of 15 dogs without heartworms (HW0) and 15 dogs with > 50 heartworms (HW>50) was included. Heartworm number and microfilariae presence were determined at necropsy. The effects of heartworm status, body weight, and sex on ACE2 were evaluated using regression analysis. Values of P < 0.05 were considered significant.ResultsAll HW0 dogs were D. immitis microfilariae-negative and all HW>50 dogs were D. immitis microfilariae-positive with a median adult worm count of 74 (minimum = 63, maximum = 137). The ACE2 activity of HW>50 dogs (median = 28.2 ng/ml; minimum = 13.6, maximum = 76.2) was not different from HW0 dogs (median 31.9 ng/ml; minimum = 14.1, maximum = 139.1; P = 0.53). The ACE2 activity was higher in dogs with high body weight (median 34.2 ng/ml minimum = 14.1, maximum = 76.2) than in dogs with low weight (median 27.5 ng/ml; minimum = 16.4, maximum = 139.1; P = .044).ConclusionsHeartworm infection did not impact ACE2 activity in shelter dogs with or without heartworms, but heavier dogs had higher ACE2 activity compared to lighter dogs. Comprehensive RAAS evaluation and additional clinical information would aid in understanding how ACE2 activity relates to the entire cascade and clinical status in dogs with heartworm disease.

Dataset Information

Molecular detection of Dirofilaria immitis and its Wolbachia endosymbionts in dogs from Myanmar.

Publications

Molecular detection of <i>Dirofilaria immitis</i> and its <i>Wolbachia</i> endosymbionts in dogs from Myanmar.

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