heartworms Archives - Center for Tropical and Emerging Global Diseases

Evaluation of diagnostic techniques for early detection of heartworm in experimentally infected dogs: identification of Dirofilaria immitis-derived microRNA in the initial 28 weeks post-inoculation

Donna Huber on June 13, 2024

Background: Dirofilaria immitis, commonly known as heartworm (HW), is a parasitic nematode transmitted by various mosquito species, leading to heartworm disease (HWD) in dogs. Diagnosis of HW typically involves antigen or microfilariae detection, or visualization of adult worms through imaging or post mortem examination. Polymerase chain reaction (PCR) and micro RNA (miRNA) detection have been explored for HW diagnosis.

Methods: Three dogs, previously experimentally infected with HW, underwent blood sampling every 4 weeks for 7 months. Samples were assessed for antigen presence after heat treatment, PCR amplification, and microfilaria examination using Giemsa-stained thick smears. Additionally, whole blood aliquots underwent miRNA deep sequencing and bioinformatic analysis.

Results: Heartworm antigen was detectable after heat treatment at 20 weeks post-inoculation and via PCR at 24 weeks, with microfilariae observed in peripheral blood smears at 28 weeks. However, deep miRNA sequencing revealed that the miRNA candidate sequences are not consistently expressed before 28 weeks of infection.

Conclusions: While ancillary molecular methods such as PCR and miRNA sequencing may be less effective than antigen detection for detecting immature larval stages in an early stage of infection, our experimental findings demonstrate that circulating miRNAs can still be detected in 28 weeks post-infection.

Daniel Felipe Barrantes Murillo, Elyssa J Campbell, Andrew R Moorhead, Chengming Wang. Parasit Vectors. 2024 Jun 13;17(1):258. doi: 10.1186/s13071-024-06337-y.

Effects of doxycycline dose rate and pre-adulticide wait period on heartworm-associated pathology and adult worm mass

Donna Huber on July 25, 2023

Background: The American Heartworm Society canine guidelines recommend treatment with doxycycline prior to adulticide administration to reduce levels of Wolbachia and its associated metabolites, which are known to be a leading cause of pulmonary pathology. Studies have determined that doxycycline administered at 10 mg/kg BID for 28 days is an effective dose for eliminating Wolbachia, but what has not been determined is the clinical relevance of this elimination. The current guidelines also recommend a 30-day wait period following administration of doxycycline to allow for clearance of metabolites, such as Wolbachia surface protein, and for further reduction in heartworm biomass before administration of adulticide. Reducing the doxycycline dose and eliminating the wait period may carry practical benefits for the animal, client, and practitioner.

Methods: To investigate these treatment practices, Dirofilaria immitis adults were surgically transplanted into each of 45 dogs, which were divided into nine study groups of five dogs each. Seventy-five days after transplantation, two groups each were administered 5, 7.5, or 10 mg/kg BID doxycycline orally for 28 days and 6 µg/kg ivermectin monthly, with three untreated groups serving as controls. Study animals were necropsied and examined prior to treatment as well as 30 and 60 days post-treatment.

Results: Mean worm weight was unaffected by dosage but exhibited a significant increase at 30 days and significant decrease at 60 days post-treatment, including in control groups. Histopathology lesion scores did not significantly differ among groups, with the exception of the lung composite score for one untreated group. Liver enzymes, the levels of which are a concern in doxycycline treatment, were also examined, with no abnormalities in alanine aminotransferase or alkaline phosphatase observed.

Conclusions: No consistent worsening of tissue lesions was observed with or without the AHS-recommended 30-day wait period, nor did reduced dosages of doxycycline lead to worsening of pathology or any change in efficacy in depleting worm weight. Mean worm weight did significantly increase prior to, and decrease following, the wait period. Future work that also includes adulticide treatment (i.e. melarsomine) will study treatment recommendations that may improve both animal health and owner compliance.

Andrew R Moorhead, Christopher C Evans, Kaori Sakamoto, Michael T Dzimianski, Abdelmoneim Mansour, Utami DiCosty, Crystal Fricks, Scott McCall, Ben Carson, C Thomas Nelson, John W McCall. Parasit Vectors. 2023 Jul 25;16(1):251. doi: 10.1186/s13071-023-05858-2.

Evaluation of renal values during treatment for heartworm disease in 27 client-owned dogs

Donna Huber on June 9, 2023

Background: Canine heartworm disease (CHD) caused by Dirofilaria immitis remains a common preventable disease with increasing incidence in some parts of the USA. The treatment guidelines of the American Heartworm Society (AHS) currently recommend monthly macrocyclic lactone administration, 28 days of doxycycline given orally every 12 h and three injections of melarsomine dihydrochloride (1 injection on day 2 of treatment followed 30 days later by 2 injections 24 h apart). Minocycline has also been utilized when doxycycline is unavailable. The systemic effects of CHD, which particularly impact cardiac and renal function, have been described, with infected dogs often experiencing renal damage characterized by an increase in serum concentrations of renal biomarkers. Although the AHS treatment protocol for CHD has been shown to be safe and effective in most cases, the potential for complications remains. No study as of yet has evaluated changes in symmetric dimethylarginine (SDMA), a sensitive marker of renal function, during treatment for CHD. The purpose of the present study was to evaluate renal function in dogs by measuring serum creatinine and SDMA concentrations during the adulticide treatment period.

Methods: Serum creatinine and SDMA concentrations were measured in 27 client-owned dogs affected by CHD at the following time points: prior to starting doxycycline or minocycline therapy (baseline), during doxycycline or minocycline therapy (interim), at the time of the first dose of melarsomine (first dose), at the time of the second dose of melarsomine (second dose) and at the dog’s follow-up visit after treatment, occurring between 1 and 6 months after completion of therapy (post-treatment). Concentrations of creatinine and SDMA were compared between time points using a mixed effects linear model.

Results: Mean SDMA concentrations following the second dose of melarsomine were significantly lower (-1.80 ug/dL, t-test, df = 99.067, t = -2.694, P-Value = 0.00829) than baseline concentrations. There were no other statistically significant differences in the concentration of either biomarker between the baseline and the other time points in CHD dogs undergoing treatment.

Conclusions: The results suggest that the current AHS protocol may not have a substantial impact on renal function.

C Autumn M Vetter, Alison G Meindl, Bianca N Lourenço, Michael Coyne, Corie Drake, Rachel Murphy, Ira G Roth, Andrew R Moorhead. Parasit Vectors. 2023 Jun 9;16(1):191. doi: 10.1186/s13071-023-05779-0.

Treatment of dogs with Bravecto® (fluralaner) reduces mosquito survival and fecundity

Donna Huber on April 28, 2023

Background: Mosquitoes serve as the vector of canine heartworm (Dirofilaria immitis), which represents a significant and persistent threat to canine health. A reduction in the longevity and/or reproductive success of mosquitoes that take a blood meal from fluralaner-treated dogs may consequently reduce the local transmission of heartworm and prevent new infections. A novel secondary effect of an oral formulation of the ectoparasiticide fluralaner (Bravecto^®) against a laboratory strain of the mosquito Aedes aegypti, a potential major vector of canine heartworm, was investigated in this study.

Methods: Six dogs were administered a single dose of fluralaner orally in the form of Bravecto^® Chews (at the labeled fluralaner dose of 25 mg/kg body weight), while six control dogs received no treatment. Mosquitoes were fed on blood that was collected from each dog prior to treatment and weekly for 15 weeks post-treatment to assess the continued effects of fluralaner as its serum level decreased. Mosquito fitness was assessed by three parameters: rate of successful blood-feeding, survival, and egg laying.

Results: Successful blood-feeding rate was similar between control and treatment groups. In the fluralaner treatment, mosquito survival was significantly reduced within the first 24 h after blood-feeding, for the first 12 weeks post-treatment of the dogs (efficacy range = 33.2-73.3%). Survival of mosquitoes up until a potentially heartworm-infective timepoint (14 days post-blood-feeding) was significantly reduced in the fluralaner-treated group at several timepoints (1, 2, 5, 11, 12, 13, 14, and 15 weeks post-treatment; efficacy range = 49.4-91.4%), but was less consistently reduced at the other timepoints. Egg laying by mosquitoes was almost completely suppressed for the first 13 weeks following treatment of the dogs with fluralaner (treatment efficacy ≥ 99.8%).

Conclusions: Mosquitoes fed blood from fluralaner-treated dogs experienced a significant reduction in survival and fecundity. These findings support the potential for a reduction in heartworm transmission directly by lethal effects on the vector and indirectly through a reduction of the local vector population when mosquitoes are exposed to animals treated with fluralaner.

Christopher Charles Evans, Dorothy Normile, Sheryl Gamble, Frank Guerino, Michael T Dzimianski, Andrew Riddell Moorhead. Parasit Vectors. 2023 Apr 28;16(1):147. doi: 10.1186/s13071-023-05682-8.

Comparative sequences of the Wolbachia genomes of drug-sensitive and resistant isolates of Dirofilaria immitis

Donna Huber on September 15, 2020

The recent identification of isolates of D. immitis with confirmed resistance to the macrocyclic lactone preventatives presents an opportunity for comparative genomic studies using these isolates, and examining the genetic diversity within and between them. We studied the genomes of Wolbachia endosymbionts of five isolates of D. immitis maintained at the University of Georgia. Missouri and Georgia-2 are maintained as drug susceptible isolates, and JYD-27, Yazoo-2013 and Metairie-2014 are resistant to the macrocyclic lactone preventatives. We used whole genome amplification followed by Illumina-based sequencing from 8 to 12 individual microfilariae from each of the five isolates, obtaining a depth of coverage of approximately 40–75 fold for each. The Illumina sequences were used to create new genome assemblies for all the Wolbachia isolates studied. Comparisons of the Wolbachia sequences revealed more than 3000 sequence variations in each isolate. We identified 67 loci specific in resistant isolates but not in susceptible isolates, including 18 genes affected. Phylogenetic analysis suggested that the endosymbionts of the drug-susceptible isolates are more closely related to each other than to those from any of the resistant parasites. This level of variation in the Wolbachia endosymbionts of D. immitis isolates suggests a potential for selection for resistance against drugs targeting them.

Pei-Tsz Shin, Rodrigo de Paula Baptista, Connor M. O’Neill, Connor Wallis, Barbara J.Reaves, Adrian J. Wolstenholme. Veterinary Parasitology, Volume 286, October 2020, 109225. https://doi.org/10.1016/j.vetpar.2020.109225

Using Population Genetics to Examine Relationships of Dirofilaria Immitis Based on Both Macrocyclic Lactone-Resistance Status and Geography

Donna Huber on May 23, 2020

Prevention of infection with canine heartworm (Dirofilaria immitis) is based on the compliant administration of macrocyclic lactone (ML) drugs. Resistance to ML drugs is well documented in D. immitis; however, there remains a paucity of information on the spatial distribution and prevalence of resistant isolates. This project aims to improve understanding of ML-resistance by using a population genetic approach. We developed a large panel of microsatellite loci and identified 12 novel highly polymorphic markers. These 12, and five previously published markers were used to screen pools of microfilariae from 16 confirmed drug-susceptible, 25 confirmed drug-resistant, and from 10 suspected drug-resistant field isolates. In isolates where microfilarial suppression testing indicated resistance, Spatial Principal Component Analysis (sPCoA), Neighbor Joining Trees and Bayesian clustering all revealed high genetic similarity between pre- and post-treatment samples. Somewhat surprisingly, the Neighbor Joining tree and sPCoA generated using pairwise Nei’s distances did not reveal clustering for resistant isolates, nor did it reveal state-level geographic clustering from samples collected in Georgia, Louisiana or Mississippi. In contrast, Discriminant Analysis of Principle Components was able to discriminate between susceptible, suspected-resistant and resistant samples. However, no resistance-associated markers were detected, and this clustering was driven by the combined effects of multiple alleles across multiple loci. Additionally, we measured unexpectedly large genetic distances between different passages of laboratory strains that originated from the same source infection. This finding strongly suggests that the genetic makeup of laboratory isolates can change substantially with each passage, likely due to genetic bottlenecking. Taken together, these data suggest greater than expected genetic variability in the resistant isolates, and in D. immitis overall. Our results also suggest that microsatellite genotyping lacks the sensitivity to detect a specific genetic signature for resistance. Future investigations using genomic analyses will be required to elucidate the genetic relationships of ML-resistant isolates.

Julie Sanchez, Guha Dharmarajan, Melissa M. George, Cassan Pulaski, Adrian J. Wolstenholme, John S. Gilleard, Ray M. Kaplan. 2020 Veterinary Parasitology; 283:109125. doi: 10.1016/j.vetpar.2020.109125.

Efficacy and side effects of doxycycline versus minocycline in the three-dose melarsomine canine adulticidal heartworm treatment protocol

Donna Huber on December 27, 2018

Abstract

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.

Molly D. Savadelis, Katherine M. Day, Jenna L. Bradner, Adrian J. Wolstenholme, Michael T. Dzimianski, and Andrew R. Moorhead. 2018. Parasites & Vectors; 11:671. https://doi.org/10.1186/s13071-018-3264-z

Tag: heartworms