Drug resistance is one of the greatest challenges facing parasitologists worldwide at this time.While the livestock and equine industries have been most affected to date, the fields of human andcompanion animal medicine are also potentially vulnerable. The study of drug resistance incompanion animal parasites has been a neglected field, with little published information on theresistance status of important companion animal parasites. Following anecdotal reports of pyranteltreatment failures in south east Queensland dogs, a placebo-controlled trial of the efficacy ofpyrantel in dogs experimentally infected with local isolates of A. caninum was undertaken. Whileaverage adult A. caninum burdens in pyrantel-treated dogs were significantly lower than incontrols (178.0 ± 24.4 vs 239.6 ± 13.9; p<0.01), parasitologic efficacy of pyrantel, calculated bycomparing the average hookworm burden in treated and untreated groups, was just 25.7%. This isthe first confirmed report of drug resistance in a helminth parasite of dogs. Analysis of the faecalegg count trends among dogs revealed that egg counts of pyrantel-treated dogs rose by an average17.3 ± 7.6% in the six days following treatment, a significantly higher increase than the 11.6 ±8.3% rise in control dogs (p<0.05). This observation highlights the dubious relevance of thestandard FECRT in testing for anthelmintic resistance in hookworm infections, and reinforces theneed for alternative monitoring methods.One such alternative is the use of in-vitro bioassays to measure worm sensitivity to pyrantel. Theisolate established in the aforementioned placebo-controlled trial (PR isolate) was designated ashighly-resistant, while a second isolate sourced from a remote Aboriginal community,subsequently demonstrated to be 75% susceptible to pyrantel in-vivo, was designated to be of lowlevelresistance. These isolates were used to assess the discriminatory ability of a larval motilityassay (LMA) and larval feeding inhibition assay (LFIA). In addition, a further bioassay, the larvalarrested morphology assay (LAMA) was developed and tested Observations suggest that theLAMA is the most effective of these assays, showing an IC50 value of 0.043 μg/ml for the PRisolate, compared to an IC50 value of 0.0025 μg/ml for the NT isolate, representing a resistanceratio of 17.2 for this assay. While biphasic response curves were observed with the LMA,complicating mathematical description, this method also shows promise, particularly as asecondary assay coupled with the LAMA.The PR and NT isolates were also examined in a series of phenotypic studies designed to bettercharacterize pyrantel resistance at this level. The LAMA and whole-worm cannulation with forcetransduction were used to characterize the L3 and adult lifestages of A. caninum, respectively.Both larval and adult life-stages of the pyrantel resistant isolate (PR) were less sensitive to bothpyrantel and levamisole than the isolate with low-level pyrantel resistance (NT). In contrast, aninverse response was observed for larval responses to bephenium, with the NT isolate two-foldless sensitive to this drug compared to the PR isolate. However, this difference was not observedin the adult life-stage. While no difference in response to nicotine was observed at the larval stage,PR adults were two-fold less sensitive to nicotine than their NT counterparts, paralleling theirrelative sensitivity to pyrantel and levamisole. These observations highlight the complexity ofnicotinic pharmacology, and suggest that the resistance phenotype differs between the L3 andadult lifestages.Elucidation of the molecular basis of resistance to nicotinic agonist drugs such as pyrantel isdesirable, since this anthelmintic class continues to be widely utilised in both veterinary andhuman medicine. To this end, a comparative molecular study was undertaken utilising the PR andNT isolates. Three key orthologues of the pyrantel-sensitive nicotinic acetylcholine receptor in C.elegans were cloned from A. caninum. Analysis of mRNA levels by quantitative PCR was alsoperformed on these three genes plus three additional nicotinic acetylcholine receptor subunitgenes, thought not to be constituents of the pyrantel-sensitive receptor, for which partial sequencewas obtained. No significant polymorphisms were observed between the two A. caninum isolates,however quantitative analysis of transcription revealed significantly lower expression of the threeputative pyrantel receptor subunits in the highly pyrantel resistant A. caninum isolate compared tothe A. caninum isolate with low-level resistance. In contrast, expression of the three subunitsthought not to constitute the pyrantel receptor was never lower in the highly-resistant isolate. Thisdata suggests that alterations to nicotinic acetylcholine receptor subunit expression may be acomponent of the pyrantel resistance mechanism in A. caninum.