Macrocyclic lactone (ML) endectocides are used as chemoprophylaxis for heartworm infection (Dirofilaria immitis) in dogs and cats. Claims of loss of efficacy (LOE) of ML heartworm preventives have ...become common in some locations in the USA. We directly tested whether resistance to MLs exists in LOE isolates of D. immitis and identified genetic markers that are correlated with, and therefore can predict ML resistance. ML controlled studies showed that LOE strains of D. immitis established infections in dogs despite chemoprophylaxis with oral ivermectin or injectable moxidectin. A whole genome approach was used to search for loci associated with the resistance phenotype. Many loci showed highly significant differences between pools of susceptible and LOE D. immitis. Based on 186 potential marker loci, Sequenom® SNP frequency analyses were conducted on 663 individual parasites (adult worms and microfilariae) which were phenotypically characterized as susceptible (SUS), confirmed ML treatment survivors/resistant (RES), or suspected resistant/loss of efficacy (LOE) parasites. There was a subset of SNP loci which appears to be promising markers for predicting ML resistance, including SNPs in some genes that have been associated with ML resistance in other parasites. These data provide unequivocal proof of ML resistance in D. immitis and identify genetic markers that could be used to monitor for ML resistance in heartworms.
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 L₃in 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 L₃from 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.
Macrocyclic lactone (ML) molecules have been used for heartworm control for more than 25 years. However, in recent years, there have been reports of loss of efficacy of ML heartworm preventatives ...against
Dirofilaria immitis in some locations in the United States. Macrocyclic lactone resistance is a common problem in nematode parasites of livestock, and more recently, evidence of ivermectin resistance has been reported in the human filarial nematode
Onchocerca volvulus. In this study, four
D. immitis sample groups from the United States with different treatment histories were investigated for evidence of ML-driven genetic selection. DNA from individual adult worms and microfilariae was amplified by polymerase chain reaction to investigate a gene encoding a P-glycoprotein, a protein class known to be involved in ML pharmacology. A significant correlation of a GG–GG genotype with ivermectin response phenotype was found. Moreover, a significant loss of heterozygosity was found in a low responder group; loss of heterozygosity is commonly seen in loci when a population has been under selection. Further studies are required to confirm ML resistance in heartworm populations. However, the genetic changes observed in this study may be useful as a marker to monitor for ML resistance in
D. immitis.
The aim of this work was to evaluate the potential of lufenuron, a benzylphenylurea with ability to interfere with the formation of insect exoskeleton, as a therapeutic drug for larval echinococcosis ...(hydatid disease). For this purpose lufenuron, alone or in combination with albendazole, was administered to CD1 mice bearing Echinococcus granulosus hydatid cysts in the peritoneal cavity. Neither of the drugs alone was able to exert parasiticidal effects. However, in combination with albendazole, lufenuron reduced the growth of cysts (30–40% in cyst diameter respect to control, p<0.05). This effect was associated with ultrastructural alterations of the hydatid cyst wall and a reduction of the content of myo-inositol-hexakisphosphate, the major component of the electron dense granules of the laminated layer. Overall, this work provides evidence that lufenuron could represent a useful compound for the use in chemotherapy against larval echinococcosis, by enhancing albendazole parasiticidal activity.
Oral lufenuron is reportedly an effective treatment for some cats with dermatophytosis. The purpose of this study was to determine if lufenuron, when used as a pre‐treatment prior to challenge ...exposure, would be protective against the development of infection after the direct topical application of fungal macrocondia (Microsporum canis spores). Three groups (n = 6/group) of juvenile cats were treated with either monthly oral lufenuron (30 or 133 mg/kg) or placebo. After 2 months of treatment, kittens were challenged using 105Microsporum canis spores applied to the skin under occlusion. Cats were examined weekly and the following data collected: Wood's lamp examination; scoring for scale/crust, erythema and induration; lesion size; and the development of satellite lesions. Fungal cultures were performed bi‐weekly. All cats became infected; the infections progressed, and then regressed, in a similar fashion in all groups. There were no consistent statistically significant differences in weekly infection scores between treated and untreated cats throughout the study. Treated cats did not recover faster than untreated cats. We conclude that oral lufenuron at the dosing schedule and conditions used in this study did not prevent dermatophytosis or alter the course of infection by direct topical challenge.
Adult cat fleas, Ctenocephalides felis (Bouche), were fed suboptimal in vitro concentrations of lufenuron in blood to allow hatching off flea larvae for cytological study. At concentrations of 0.125, ...0.25, and 0.5 ppm, larval hatch was 64, 15, and 4% respectively. Larvae hatching from eggs laid by adults fed lufenuron at concentrations of 0.025, 0.08, or 0.125 ppm did not differ significantly from the control. However, many larvae from the 0.08-ppm group and higher concentrations died during the 1st instar. Examination of these larvae revealed that they were dying from desiccation caused by bleeding from microscopic lesions in the cuticle or the inability to complete the molt to the next instar. Electron micrographs showed that lufenuron often disrupted formation of the endocuticle resulting in the deposition of an amorphous mass of randomly oriented chitin microfibrils. Other larvae formed normal endocuticle but were unable to digest the old endocuticle or produce new procuticle after apolysis. Failure of larvae to digest old cuticle or form new cuticle was caused by degeneration of the epidermal cells needed for the synthesis of molting fluid and chitin
When cat fleas, Ctenocephalides felis (Bouche), were fed concentrations of lufenuron in cattle blood ranging from 0.5 to 4 ppm, adult mortality increased in a dose-dependent manner to a maximum of ...approximately equal to 24% over a period of 10 d. Fleas treated with 0.5 ppm produced abnormal endocuticle consisting of protein globules embedded in an amorphous chitin matrix. At concentrations of 1.0 ppm or greater, endocuticle formation was inhibited. Ultrastructural studies demonstrated that inhibition of chitin synthesis was associated with degeneration of the epidermal cells. The amount of epidermal cytoplasm decreased and cytoplasmic organelles including mitochondria, ribosomes, and golgi showed lytic changes. At least some mortality of treated fleas was likely the result of a weakened endocuticle and the corresponding decrease in resiliency of the cuticle to expansion during blood-feeding and egg production. An unexpected result of lufenuron treatment was the inhibition of midgut epithelial cell differentiation. At concentrations of 0.5 and 1.0 ppm, partially differentiated epithelial cells were seen in the midgut of bloodfed fleas along with fully differentiated cells.
A new juvenile hormone mimic CCA-255'728, developed by Novartis, was tested for its efficacy on different life stages of the cat flea, Ctenocephalides felis (Bouché). The compound was mixed in bovine ...blood at concentrations of 1, 10, and 100 ppb and fed to adult fleas using an artificial membrane system. Less than 7% of the eggs from fleas fed 10 and 100 ppb hatched, compared with >70% hatch in eggs laid by control fleas or fleas fed 1 ppb. Histological studies demonstrated that approximately 50% of the eggs from fleas fed 10 ppb contained unhatched larvae that died after completing embryological development. At 100 ppb, 24% of the fleas died as fully formed larvae within the egg, but at this concentration nearly 20% of fleas also died before embryogenesis was completed. In addition to embryonic and larval mortality, approximately 28% of eggs from the 100 ppb-treated group were deposited as empty shells. Evidently these eggs were crushed by the muscular action of the oviduct and yolk was expelled into the oviduct during oviposition. None of the test concentrations showed adulticidal effects over a 10-d period. Fecundity was significantly increased with increased concentrations of CGA-255'728 indicating that the compound mimicked the effect of juvenile hormone.
PDF
