Dataset Information

Evaluation of heat-treating heartworm-positive canine serum samples during treatment with Advantage Multi® for Dogs and doxycycline.

ABSTRACT: BACKGROUND:The use of heat-treatment in canine and feline serum has been hypothesized to break the formation of antigen-antibody complexes, thereby freeing the heartworm antigen allowing for detection by commercially available heartworm antigen kits. While studies have analyzed the effect of heat-treating serum and plasma samples in the detection of heartworm antigen, these studies have not utilized necropsy verified results for validation. This study evaluated the use of heat-treating serum samples in experimentally infected dogs during adulticidal treatment in comparison with necropsy adult heartworm recovery. METHODS:As part of a primary study, a total of 16 dogs were experimentally infected with 16 sexually mature adult heartworms using surgical transplantation, allocating 8 dogs in both the control and treated group. Treated dogs received 10 months of topical administration of Advantage Multi® for Dogs (10% Imidacloprid +?2.5% Moxidectin) every 4 weeks and 30 days of 10 mg/kg doxycycline BID. Blood samples were collected from all study animals prior to surgical transplantation of adult heartworms, on study days 0, 1, 3, 7, 14, 21, 28, and every 4 weeks thereafter for the duration of this study. Concentration of heartworm antigen was tested using the DiroCHEK® heartworm antigen test kit using serum samples both pre- and post-heat-treatment. Serum samples were heat-treated at 103 °C in a dry heat block for 10 min and centrifuging at 1818× g for 20 min. RESULTS:There were a total of 4 instances (days 56, 140, 224 and 252) in 3 treated dogs in which a serum sample converted from negative for the detection of heartworm antigen prior to heat-treatment to positive for the detection of heartworm antigen post-heat-treatment. At necropsy, these dogs had no adult heartworms recovered and were all negative on antigen testing prior to and after heat treatment. There was 100% accuracy in the detection of either no infection, or 1-2 adult heartworm infections using the DiroCHEK in serum samples with and without heat-treatment at the time of necropsy. CONCLUSIONS:The DiroCHEK accurately diagnosed all dogs with live adults recovered at necropsy as heartworm antigen positive and all those dogs with no live adults recovered at necropsy as heartworm antigen negative without the use of heat-treatment for samples taken on the day of necropsy. Therefore, these results indicate that the use of heat-treating serum samples did not provide data of any additional value in the diagnosis of heartworm-positive dogs receiving treatment in this study. Additionally, these results may indicate that the conversion of serum samples from negative to positive for the presence of heartworm antigen with heat-treatment may not always accurately diagnose live adult heartworm infections since no adult heartworms were recovered at necropsy for those dogs in which a conversion event occurred. These conversion events may be detecting residual antigen leftover after all adult worms have died or may even be detecting off- target antigens, which have been denatured during heat-treatment. While a necropsy was not performed at the time of the conversion events, no live adult worms were recovered from any of the dogs in which a conversion event occurred earlier in treatment.

SUBMITTER: Savadelis MD

PROVIDER: S-EPMC5819156 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Evaluation of heat-treating heartworm-positive canine serum samples during treatment with Advantage Multi<sup>®</sup> for Dogs and doxycycline.

Savadelis Molly D MD Roveto Jennifer L JL Ohmes Cameon M CM Hostetler Joe A JA Settje Terry L TL Dzimianski Michael T MT Moorhead Andrew R AR

Parasites & vectors 20180220 1

<h4>Background</h4>The use of heat-treatment in canine and feline serum has been hypothesized to break the formation of antigen-antibody complexes, thereby freeing the heartworm antigen allowing for detection by commercially available heartworm antigen kits. While studies have analyzed the effect of heat-treating serum and plasma samples in the detection of heartworm antigen, these studies have not utilized necropsy verified results for validation. This study evaluated the use of heat-treating s ...[more]

PMID: 29458396

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Project description:BACKGROUND:The American Heartworm Society currently recommends the use of a monthly macrocyclic lactone, a 28-day course of 10 mg/kg doxycycline BID, and the 3-dose protocol of melarsomine dihydrochloride for the treatment of canine heartworm disease. Doxycycline is necessary for the reduction of the bacterium Wolbachia, found in all heartworm life-stages. Previous price increases and decreasing availability prompted us to evaluate alternative tetracycline antibiotics, i.e. minocycline, for the reduction of Wolbachia during canine heartworm treatment. METHODS:Thirty-two heartworm-positive dogs were randomized to receive 10 mg/kg or 5 mg/kg of either doxycycline or minocycline for 28 days BID, for a total of 8 dogs per experimental group. All dogs received 6 months of Heartgard Plus® (ivermectin/pyrantel) and the 3-dose protocol of 2.5 mg/kg melarsomine dihydrochloride. Blood samples were collected prior to the initiation of treatment, every 7 days throughout tetracycline treatment, and then monthly thereafter until the dog tested negative for the presence of heartworm antigen. DNA was isolated from circulating microfilarial samples and qPCR was performed on each sample. RESULTS:A greater number of dogs in the 10 mg/kg doxycycline and minocycline treated groups experienced gastrointestinal side effects as compared to the 5 mg/kg doxycycline and minocycline treated groups. All eight dogs in the 10 mg/kg doxycycline-treated group tested negative for the presence of Wolbachia DNA by 28 days post-tetracycline treatment. A total of two dogs in both the 5 mg/kg doxycycline- and 10 mg/kg minocycline-treated groups and three dogs in the 5 mg/kg minocycline-treated group remained positive for the presence of Wolbachia DNA by the end of tetracycline treatment. CONCLUSIONS:No lung pathology was assessed in this clinical trial, therefore the clinical effect of the remaining Wolbachia DNA in the 10 mg/kg minocycline-, 5 mg/kg doxycycline- and 5 mg/kg minocycline-treated groups cannot be determined. Owner compliance in the proper administration of these tetracyclines may be impacted by the increased severe gastrointestinal side effects reported for the 10 mg/kg doxycycline- and minocycline-treated groups. We recommend that veterinarians prescribe the recommended 10 mg/kg doxycycline for canine heartworm treatment and reduce the dosage to 5 mg/kg in cases of severe gastrointestinal side effects in order to improve owner compliance in administration of medications.

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: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: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.

Dataset Information

Evaluation of heat-treating heartworm-positive canine serum samples during treatment with Advantage Multi® for Dogs and doxycycline.

Publications

Evaluation of heat-treating heartworm-positive canine serum samples during treatment with Advantage Multi<sup>®</sup> for Dogs and doxycycline.

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