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


METHODS
To 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.


RESULTS
Both 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.


CONCLUSIONS
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. 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.
School of Veterinary Medicine, Louisiana State University and Louisiana Animal Disease Diagnostic Laboratory, Baton Rouge, 70803, LA, USA. cpulaski@vetmail.lsu.edu.
Cassan N
CN
Pulaski
School of Veterinary Medicine, Louisiana State University and Louisiana Animal Disease Diagnostic Laboratory, Baton Rouge, 70803, LA, USA. malone@vetmed.lsu.edu.
John B
JB
Malone
Institute of Parasitology, McGill University, 21 111 Lakeshore Road, Sainte-Anne de Bellevue, H9X3V9, Quebec, Canada. catherine.bourguinat@mcgill.ca.
Catherine
C
Bourguinat
Institute of Parasitology, McGill University, 21 111 Lakeshore Road, Sainte-Anne de Bellevue, H9X3V9, Quebec, Canada. roger.prichard@mcgill.ca.
Roger
R
Prichard
Institute of Parasitology, McGill University, 21 111 Lakeshore Road, Sainte-Anne de Bellevue, H9X3V9, Quebec, Canada. timothy.g.geary@mcgill.ca.
Timothy
T
Geary
School of Veterinary Medicine, Louisiana State University and Louisiana Animal Disease Diagnostic Laboratory, Baton Rouge, 70803, LA, USA. dterry4@lsu.edu.
Danielle
D
Ward
School of Veterinary Medicine, Louisiana State University and Louisiana Animal Disease Diagnostic Laboratory, Baton Rouge, 70803, LA, USA. klei@vetmed.lsu.edu.
Thomas R
TR
Klei
Guidry Animal Hospital, 500 East Gloria Switch Rd, 70507, Lafayette, LA, USA. talguidry@ymail.com.
Tal
T
Guidry
Bocage Animal Hospital, 7353 Jefferson Hwy, 70806, Baton Rouge, LA, USA. bocageanimalhospital@gmail.com.
George 'Bud'
G'
Smith
School of Veterinary Medicine, Louisiana State University and Louisiana Animal Disease Diagnostic Laboratory, Baton Rouge, 70803, LA, USA. bleibe1@tigers.lsu.edu.
Brooke
B
Delcambre
School of Veterinary Medicine, Louisiana State University and Louisiana Animal Disease Diagnostic Laboratory, Baton Rouge, 70803, LA, USA. jbova@vetmail.lsu.edu.
Jonathan
J
Bova
School of Veterinary Medicine, Louisiana State University and Louisiana Animal Disease Diagnostic Laboratory, Baton Rouge, 70803, LA, USA. jpepping@gmail.com.
Jenny
J
Pepping
Novartis Animal Health US, Inc, Greensboro, NC, USA. james.carmichael@novartis.com.
James
J
Carmichael
Novartis Animal Health, Basel, Switzerland. rudolf.schenker@novartis.com.
Rudolf
R
Schenker
School of Veterinary Medicine, Louisiana State University and Louisiana Animal Disease Diagnostic Laboratory, Baton Rouge, 70803, LA, USA. rpariaut@lsu.edu.
Romain
R
Pariaut
D000330:Aedes; D000818:Animals; D000871:Anthelmintics / Q000008:administration & dosage / Q000494:pharmacology*; D018890:Chemoprevention / Q000379:methods; D004183:Dirofilaria immitis / Q000187:drug effects* / Q000302:isolation & purification; D004184:Dirofilariasis / Q000453:epidemiology / Q000469:parasitology*; D004195:Disease Models, Animal; D004283:Dog Diseases / Q000469:parasitology*; D004285:Dogs; D004351:Drug Resistance; D006801:Humans; D007559:Ivermectin / Q000008:administration & dosage / Q000494:pharmacology*; D008145:Louisiana / Q000453:epidemiology; D008297:Male; D008922:Mississippi
D000871:Anthelmintics; D007559:Ivermectin