Establishment of macrocyclic lactone resistant Dirofilaria immitis isolates in experimentally infected laboratory dogs.

Cassan N Pulaski,John B Malone,Roger Prichard,Jonathan Bova,Thomas R Klei,Timothy Geary,Tal Guidry,Catherine Bourguinat,James Carmichael,Danielle Ward,Rudolf Schenker,George `Bud’ Smith,Romain Pariaut,Brooke Delcambre,Jenny Pepping

doi:10.1186/s13071-014-0494-6

Cassan N Pulaski, John B Malone + Show 13 more

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https://doi.org/10.1186/s13071-014-0494-6

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Abstract

BackgroundStrains of Dirofilaria immitis suspected of lack of efficacy (LOE) to macrocyclic lactone (ML) preventive drugs have been increasingly reported in dogs by practicing veterinarians since 2005 in the Lower Mississippi Delta region. If proven, and not controlled in the early stages, the emergence of ML drug resistance threatens to become a widespread problem in the US that may limit the effectiveness of current preventive drug treatment methods.MethodsTo validate practice reports, a statewide survey of Louisiana veterinarians was done to define the extent of the problem and identify focal ‘hotspots’ of reported ML LOEs using Geographic Information Systems (GIS) methods. The present study then utilized microfilariae (Mf) from two canine field cases from different state locations that fit criteria for a high index of suspicion of LOE against heartworms by ML drugs. Blood containing Mf from the canine field cases was used to infect and produce L3 in Aedes aegypti for experimental infection of two groups of dogs, each of which contained two laboratory dogs, one treated with prophylactic ivermectin (12 μg/kg) monthly for 6 months at twice the label dose (6 μg/kg), and one untreated control.ResultsBoth treated and untreated dogs from Group I and Group II developed patent D. immitis infections by 218 DPI and 189 DPI, respectively, as evidenced by a positive occult heartworm antigen test and microfilaremia by the Knott’s test. Mf counts gradually increased post-patency in test and control dogs. Infective larvae raised from microfilariae from the treated Group I dog were used to successfully establish a second generation isolate, confirming heritability of resistance in the face of a monthly ivermectin challenge dose of 24 μg/kg, given monthly for 3 months.ConclusionsThese experimental infection studies provide in vivo evidence of the existence of ML drug resistance in dogs infected by D. immitis L3 from suspect field LOE cases in the Lower Mississippi Delta. Results encourage further work on mechanisms underlying the emergence of ML resistance in D. immitis and development of evidence-based resistance management strategies for heartworm preventives in order to extend the useful life of current drugs.

Highlights

Strains of Dirofilaria immitis suspected of lack of efficacy (LOE) to macrocyclic lactone (ML) preventive drugs have been increasingly reported in dogs by practicing veterinarians since 2005 in the Lower Mississippi Delta region
LSU 10a ivermectin (12 μg/kg, monthly for 6 months) LSU 10b untreated control LSU 10-II ivermectin (24 μg/kg, monthly for 3 months) LSU 13a ivermectin (12 μg/kg, monthly for 6 months) LSU 13b untreated control We propose that we have successfully isolated two strains from proven cases of resistance in client-owned dogs in Louisiana that can be used to further study and characterize the emergence of ML resistance by D. immitis
ML drugs for livestock, protected against heartworms, intestinal helminthes and other internal parasites with very striking benefits for dogs [26,27]. This was soon followed by controlled pharmaceutical company experimentation leading to Food and Drug Administration (FDA) approval and marketing in the late 1980’s of current ML drug formulations at the very low dose levels used for prevention of canine heartworms [26,27]. These experimental infection studies provide in vivo evidence of the existence of ML drug resistance in dogs infected by D. immitis L3 from suspect field LOE cases in the Lower Mississippi Delta

Summary

Introduction

Strains of Dirofilaria immitis suspected of lack of efficacy (LOE) to macrocyclic lactone (ML) preventive drugs have been increasingly reported in dogs by practicing veterinarians since 2005 in the Lower Mississippi Delta region. Anecdotal reports of increasing LOE cases from the veterinary community in heartworm endemic areas, pharmaceutical firms initiated reimbursement programs in 2004 to cover costs of treatment of LOE cases if owners and their veterinarians could document full compliance with recommended preventive treatment practices. The objectives of studies reported here are to record results of: 1) a 2009 survey to document the experience of veterinarians in Louisiana on potential emerging ML resistance by D. immitis, and 2) establishment of ML resistant isolates of D. immitis in experimentally infected laboratory dogs using L3 raised in Aedes aegypti fed on microfilaremic blood from 2 dogs with a ‘high index of suspicion of resistance’ Several research efforts were initiated, investigating the spatio-temporal scale and dynamics of LOE reports [2], potential in vitro indicators of resistance [3,4], possible genetic markers of resistance via genomic studies [5,6,7,8,9,10] and in vivo experiments, the gold standard, to establish confirmed resistant strains from canine field cases in the laboratory [11,12].

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