Susceptibility of Adult Cat Fleas (Siphonaptera: Pulicidae) to Insecticides and Status of Insecticide Resistance Mutations at the Rdl and Knockdown Resistance Loci.

The susceptibility of 12 field-collected isolates and 4 laboratory strains of cat fleas, Ctenocephalides felis was determined by topical application of some of the insecticides used as on-animal therapies to control them. In the tested field-collected flea isolates the LD50 values for fipronil and imidacloprid ranged from 0.09 to 0.35 ng/flea and 0.02 to 0.19 ng/flea, respectively, and were consistent with baseline figures published previously. The extent of variation in response to four pyrethroid insecticides differed between compounds with the LD50 values for deltamethrin ranging from 2.3 to 28.2 ng/flea, etofenprox ranging from 26.7 to 86.7 ng/flea, permethrin ranging from 17.5 to 85.6 ng/flea, and d-phenothrin ranging from 14.5 to 130 ng/flea. A comparison with earlier data for permethrin and deltamethrin implied a level of pyrethroid resistance in all isolates and strains. LD50 values for tetrachlorvinphos ranged from 20.0 to 420.0 ng/flea. The rdl mutation (conferring target-site resistance to cyclodiene insecticides) was present in most field-collected and laboratory strains, but had no discernible effect on responses to fipronil, which acts on the same receptor protein as cyclodienes. The kdr and skdr mutations conferring target-site resistance to pyrethroids but segregated in opposition to one another, precluding the formation of genotypes homozygous for both mutations.

Client Perspectives on Desirable Attributes and Skills of Veterinary Technologists in Australia: Considerations for Curriculum Design.

Client or service user perspectives are important when designing curricula for professional programs. In the case of veterinary technology, an emerging profession in the veterinary field in Australasia, client views on desirable graduate attributes, skills, and knowledge have not yet been explored. This study reports on a survey of 441 veterinary clients (with 104 responses) from four veterinary practices in Brisbane, Queensland, conducted between October 2008 and February 2009. The included veterinary practices provided clinical placements for veterinary technology undergraduates and employment for veterinary technology graduates (2003-2007). Client socio-demographic data along with ratings of the importance of a range of technical (veterinary nursing) skills, emotional intelligence, and professional attributes for veterinary technology graduates were collected and analyzed. Overall, the majority of clients viewed technical skills, emotional intelligence, and professional attributes as important in the clinical practice of veterinary technology graduates with whom they interacted in the veterinary practice. Client interviews (n=3) contextualized the survey data and also showed that clients attached importance to graduates demonstrating professional competence. Agglomerative hierarchical cluster analysis revealed four distinct groupings of clients within the data based on their differing perceptions. Using a multivariable proportional-odds regression model, it was also found that some client differences were influenced by demographic factors such as gender, age, and number of visits annually. For example, the odds of female clients valuing emotionality and sociability were greater than males. These findings provide useful data for the design of a professionalizing and market-driven veterinary technology curriculum.

Monitoring field susceptibility to imidacloprid in the cat flea: a world-first initiative twelve years on.

In 2001, an international surveillance initiative was established, utilising a validated larval development inhibition assay to track the susceptibility of cat flea isolates to imidacloprid. In 2009, an Australian node was incorporated into the programme, joining laboratories in the United States and Europe. Field isolates of Ctenocephalides felis eggs were submitted to participating laboratories and, where egg quantity and quality was sufficient, were placed in the imidacloprid discriminating dose bioassay for evaluation. Between 2002 and 2012, a total of 2,307 cat flea isolates were received across all sites; 1,685 submissions (73 %) were suitable for placement into the bioassay. In the Northern Hemisphere, isolate submission rate was influenced by season, with highest numbers submitted between June and October. In Australia, pets with flea infestations could be sourced year-round, and submission rate was largely influenced by programme factors and not climate. A total of 1,367 valid assays were performed between 2002 and 2012 (assay validity data was not recorded in 2001); adult flea emergence 5 % or greater at 3 ppm imidacloprid was observed in 38 of these assays (2.8 %). For these isolates that reached the threshold for further investigation, re-conduct of the assay using either a repeat challenge dose of 3 ppm of imidacloprid or a dose response probit analysis confirmed their susceptibility to imidacloprid. From 2009 to 2012, the Australian node performed valid assays on 97 field isolates from a total of 136 submissions, with no adult emergence observed at the 3-ppm imidacloprid discriminating dose. In addition to reviewing the data generated by this twelve-year initiative, this paper discusses lessons learned from the coordination and evolution of a complex project across geographically dispersed laboratories on three continents.

Prevalence of canine heartworm infection in Queensland, Australia: comparison of diagnostic methods and investigation of factors associated with reduction in antigen detection.

BACKGROUND: The prevalence of Dirofilaria immitis infection in dogs is increasing globally and spreading into new areas. Prevalence of dirofilariosis in the state of Queensland, Australia, was as high as 90% before the introduction of macrocyclic lactones. Limited research on prevalence of D. immitis infection in dogs in Queensland has been reported in the last 30 years. Antigen testing is the most common method for detection of dirofilariosis but its accuracy is reduced by antigen getting trapped (blocked antigen) in immune complexes (ICs). The objectives of this research were to determine the prevalence of D. immitis infection in dogs from two geographical areas (Brisbane and Townsville) in Queensland, to determine the extent to which blocked antigen affects the validity of antigen testing, and to explore whether this was associated with microfilaraemia, location, age or sex. METHODS: Blood samples from Brisbane (sub-tropical climate) and Townsville (tropical climate) shelter dogs were evaluated for the presence of D. immitis antigen before (conventional antigen testing, CAT) and after dissociation of ICs by heat treatment (antigen testing after heat treatment, ATHT), using a commercially available test. Microfilariae were detected using modified Knott's test (MKT). Test proportions were compared with McNemar's test and the association between antigen test-discordant results (positive for antigen after dissociation of ICs) and microfilaraemia, location, sex and age was modelled using logistic regression. RESULTS: Dirofilaria immitis prevalence in dogs from Townsville (22% by CAT, 32.1% by ATHT and 16.7% by MKT) was significantly higher than in dogs from Brisbane (1.1% by CAT and MKT and 1.7% by ATHT) [Formula: see text]. Dissociation of ICs allowed detection of significantly more D. immitis infected dogs than either conventional antigen testing or microfilariae detection, or the combined antigen and microfilariae detection [Formula: see text]. The odds of dogs being positive for antigen after dissociation of ICs were significantly higher for microfilaraemic, 3-4-year-old female dogs from Townsville. CONCLUSIONS: The high prevalence of infection with D. immitis in dogs from Townsville poses a health risk for local susceptible host species, including humans. Dissociation of ICs increases antigen detection and should be considered in dogs suspected of D. immitis infection but negative on routine testing.

Attributes of clinical role models as described by senior veterinary students in Australia.

Role models incite admiration and provide inspiration, contributing to learning as students aspire to emulate their example. The attributes of physician role models for medical trainees are well documented, but they remain largely unexplored in the context of veterinary medical training. The aim of the current study was to describe the attributes that final-year veterinary students (N=213) at the University of Queensland identified when reflecting on their clinical role models. Clinical role model descriptions provided by students were analyzed using concept-mapping software (Leximancer v. 2.25). The most frequent and highly connected concepts used by students when describing their role model(s) included clients, vet, and animal. Role models were described as good communicators who were skilled at managing relationships with clients, patients, and staff. They had exemplary knowledge, skills, and abilities, and they were methodical and conducted well-structured consultations. They were well respected and, in turn, demonstrated respect for clients, colleagues, staff, and students alike. They were also good teachers and able to tailor explanations to suit both clients and students. Findings from this study may serve to assist with faculty development and as a basis for further research in this area.

Veterinary students' perceptions of their day-one abilities before and after final-year clinical practice-based training.

The present study evaluated the impact of final-year clinical practice-based training on veterinary students' perceptions of competence in "Day One" abilities by administering a pre- and post-training self-assessment checklist. This study also investigated the influence of student demographics on their perceptions of satisfaction about their own knowledge and skills and preparedness for practice. Perceptions regarding the usefulness of the checklist as a self-audit tool were also sought. Final year students (N=85) were surveyed on commencement and upon completion of the training using a checklist that had been adapted from the list of essential new-graduate abilities that was developed by the Royal College of Veterinary Surgeons and adopted by the Australasian Veterinary Boards Council. Significant improvements in student perceptions of competence were observed for 38 of the 41 abilities. Students' satisfaction with their knowledge and skill base and their perceptions of preparedness for practice were only weakly correlated with overall perceptions of competence for individual ability items and did not vary significantly with student age, gender, background, intended field and location of work, or with their work experience as veterinary nurses-if any-while studying. Two thirds of students believed that access to the self-assessment checklist on commencement of the training helped them identify areas for improvement before graduation. This article concludes that clinical practice-based training results in considerable improvement in senior veterinary students' perceptions of competence in Day One abilities and that a self-assessment checklist may help students guide their learning. Results from the present study may be useful for veterinary schools as they develop or enhance strategies used for outcomes assessment.

Demographics and employment destinations of a new group of veterinary technologists in Australia.

This article provides a descriptive analysis of the demographics and employment destinations of the first three cohorts (2003-2005) of graduates (N=69) from a program that is unique in Australia: the Bachelor of Applied Science (Veterinary Technology) at the University of Queensland. Data for this study were collected in February 2006 via e-mail, telephone, or personal communication with graduates, and from university records. Ninety-three percent (64/69) of the graduates were female. The mean age was 23 years, and 58% (40/69) had entered university directly from high school. Employment destinations were determined for 96% of the graduates (N=66). Of those, 52% (34/66) were employed in veterinary practices. Government agencies and allied animal industries accounted for 15% (10/66). Another 14% (9/66) had enrolled in further undergraduate study. Three percent (2/66) had enrolled in a research honors year or a doctor of philosophy (PhD) degree program at the School of Veterinary Science. Eight percent (5/66) were employed in wildlife parks, zoos, or universities, and the remaining 9% (6/66) were traveling overseas, seeking employment, or employed outside the field. The study revealed that graduates were employed in diverse veterinary and allied animal health occupations. There appears to be a niche for Australian veterinary technology graduates educated in a university environment that complements the role of the veterinary profession in the twenty-first century. This reflects trends emerging in other countries, most notably the United States and the United Kingdom.

Phenotypic characterization of two Ancylostoma caninum isolates with different susceptibilities to the anthelmintic pyrantel.

The anthelmintic pyrantel plays an important role in the control of gastrointestinal helminths of humans and domestic animals. Despite the demonstration of pyrantel resistance in several helminth species over the last 20 years, the resistance mechanism remains unclear. It has been hypothesized that resistance may arise as a consequence of changes to the relative proportions of subpopulations of nicotinic acetylcholine receptors (nAchRs). To test this hypothesis, we examined the responses of two isolates of the canine hookworm Ancylostoma caninum with low-level resistance (isolate NT) and high-level resistance (isolate PR) to pyrantel to nicotinic agonist drugs reported to be selective for three nAchR subtypes. We used larval motility and conformation assays and force transduction experiments with adult worms. Pyrantel and levamisole were less potent against larvae of isolate PR than larvae of isolate NT (up to an 18-fold increase in the 50% inhibitory concentration); on the other hand, bephenium was more potent against larvae of isolate PR than larvae of isolate NT (twofold) and nicotine had the same potency against larvae of both isolates. In adults, pyrantel, levamisole, and nicotine were less potent against isolate PR than isolate NT (two- to threefold), but the potency of bephenium against the two isolates was equivalent. Our data indicate a complex pattern of nAchRs in this species and suggest that the two isolates differ in their relative sensitivities to agonists targeting different nAchRs.

Application of in vitro anthelmintic sensitivity assays to canine parasitology: detecting resistance to pyrantel in Ancylostoma caninum.

Resistance of the canine hookworm Ancylostoma caninum to anthelmintic therapy with pyrantel is an emerging problem in canine veterinary practice. Detecting anthelmintic resistance in parasites of pets is problematic because traditional resistance-monitoring techniques used with livestock parasites, such as the faecal egg count reduction test, are often impractical for use in small animals. We used two field-collected isolates of A. caninum in an abbreviated critical trial to test their pyrantel resistance status. The strains showed high-level and low-level resistance, with in vivo pyrantel efficacies of 28% and 71%, respectively. We noted a distinct worm density dependence effect on faecal egg count during the critical trial; egg counts in the dogs containing the low-level resistant isolate were 41% higher 6 days after drug treatment, despite the removal of 71% of the adult worms by the drug treatment. We then assessed four candidate in vitro assays for their ability to detect pyrantel resistance in A. caninum larvae, using these two isolates. The assays included a new format termed the larval arrested morphology assay (LAMA), based on observation of the effects of pyrantel on the body shape adopted by infective stage A. caninum larvae in vitro. Our data suggests that three of these assays, the LAMA, the larval motility assay (LMA), and larval feeding inhibition assay (LFIA) show promise with regards to detection of pyrantel resistance in A. caninum, but the complexity of the LFIA would likely limit its suitability for field studies. In vivo pyrantel efficacies of 28% and 71% in the two A. caninum isolates were associated with a 17-fold shift in the in vitro IC(50) values measured using the LAMA. Further testing with isolates of varying degrees of resistance is required to determine which of these assays is suitable as a rapid in vitro laboratory test for pyrantel resistance in A. caninum. The present study also indicates that potential exists for the novel LAMA or the LMA to be of use in detecting pyrantel resistance in the human hookworms, Necator americanus and Ancylostoma duodenale.

Pyrantel in small animal medicine: 30 years on.

Pyrantel, a tetrahydropyrimidine nicotinic agonist anthelmintic, has been used in companion animal medicine since the 1970s to control two important nematode groups, the hookworms and the roundworms. Given the zoonotic potential of these parasites, pyrantel has served a dual role in helping to protect the health of both companion animals and the public for more than 30 years. This review describes the history and mechanism of action of this drug, and collates evidence that resistance to pyrantel has developed in at least one canine nematode, the hookworm Ancylostoma caninum. The role of in vitro diagnosis tests in managing anthelmintic resistance in companion animal parasites is discussed, as are management practices that may reduce the rate at which resistance develops.

Hematology and serum biochemistry of the brush-tailed rock-wallaby (Petrogale penicillata).

In Australia the brush-tailed rock-wallaby (Petrogale penicillata) is the subject of a national recovery plan, and several sites have been selected for reintroductions. Condition of wild populations and individual animals can be monitored using hematologic and serum biochemistry analytes, and hematologic variables have been correlated with postrelease survival in other species. Prior to such monitoring, reference values for blood variables are required, but these data have not been available for the brush-tailed rock-wallaby. During four trapping periods from November 2004 to August 2005, 116 blood samples were collected from 44 brush-tailed rock-wallabies in a wild colony in southeast Queensland. Some variables varied with sex, age, method of restraint, lactation demands, and trapping period. After partitioning, when required, reference ranges for hematology and serum biochemistry variables were established. This study provides the most comprehensive serum biochemistry reference range for any macropodid marsupial yet published.

Development and evaluation of immunoblot-based serodiagnostic tests for hydatid infection in macropodids.

This study examined the suitability of immunoblot assays using antigen B from hydatid cyst fluid (HCF) and the recombinant protein EpC1 to estimate the prevalence of hydatid infection in threatened small macropodid populations. When HCF antigen B was used on sera from 29 wild eastern grey kangaroos (Macropus giganteus) examined for the presence of hydatid cysts at necropsy (17 positive and 12 negative), and from five captive-bred macropodids, the sensitivity and specificity were 88% and 41%, respectively. Following optimization, the use of rEpC1 on sera from 51 wild eastern grey kangaroos (48 positive and 3 negative at necropsy), and from 9 captive bred macropodids, resulted in sensitivity and specificity of 48% and 92% respectively.

Clustering of hydatid infection in macropodids.

The introduced parasite, Echinococcus granulosus, has been reported in numerous macropodid species in various areas of Australia, but no extensive studies investigating the prevalence and risk factors for infection in wild macropodids have been reported. In this study, 2998 macropodid carcasses were examined following commercial culling on 21 properties in southern Queensland. Of the 71 infected animals, all had cysts in the lung tissue, while two also had cysts in the pleural cavity and one animal had a liver cyst. The number of cysts in infected animals ranged from 1 to 17, with the majority of animals (n=64) having one to three cysts. Estimated total cyst volume varied from 0.2 to 1075 cm3. Some animals had a total lung cyst volume likely to have impacted significantly upon respiratory function and cyst degeneration was only seen in approximately one-third of infected animals. Multilevel models were used to investigate putative risk factors at both kangaroo and property levels. At the kangaroo level, females were twice as likely to be infected as males. After adjusting for sex, no property-level risk factors were significantly associated with the presence of hydatid infection. Prevalence varied substantially between properties (range 0-12%) and this high degree of clustering of infection was reflected in a high intra-class correlation co-efficient in the final model (0.333). These results have important implications for both public health and conservation strategies, and suggest that there are important unidentified risk factors for hydatid infection associated with properties. They also demonstrate that spatial clustering should be considered when analysing hydatid infection data in macropodids, particularly when assessing area-level risk factors.

Precocious development of hydatid cysts in a macropodid host.

This study describes the pathological changes associated with an experimental infection of captive wallabies with Echinococcus granulosus. Adult and juvenile tammar wallabies (Macropus eugenii) were infected orally with 0, 1,000, 2,500 or 8,000 E. granulosus eggs. Lung cyst progression was monitored by chest radiography every 4 months until 16 months p.i. Animals were necropsied from 9 to 16 months after infection. Cysts were detected radiographically from 4 months onwards. The number of cysts per animal varied from one to 10 and the majority (36/40) of cysts established in the lungs. Infection rate was low (35.5%), but cyst development was more rapid and onset of fertility much earlier than has been recorded in sheep. Cyst growth resulted in loss of functional lung capacity, up to an estimated 28% within 14 months of infection. Degenerative changes in cysts were less common in tammars than has been reported in sheep, with gross degeneration of cysts identified in only two animals. Complications associated with lung cyst development included fatal anaphylaxis, pneumothorax and atelectasis. Seven of the 11 infected tammars died or were euthanased as a result of infection during the experiment. From the parasite's perspective, infection of this host allows a shortened life cycle and correspondingly greater biotic potential. We believe this is the first published study that demonstrates the susceptibility of tammar wallabies to hydatid disease and confirms their suitability as a laboratory model for studying the disease in macropodids.

High-level pyrantel resistance in the hookworm Ancylostoma caninum.

While anthelmintic resistance is now a widely recognized issue in the livestock industries, its existence within companion animal medicine has been rarely established conclusively. We undertook a placebo-controlled in vivo trial to measure the efficacy of pyrantel embonate against pooled isolates of the hookworm Ancylostoma caninum from Brisbane, Australia. A statistically significant fall in adult worm burden was observed among dogs in the pyrantel treatment group compared to the control dogs (178.0+/-24.5 versus 239.7+/-14.0; p=0.02), equating to an efficacy of just 25.7% (95% CI, 15.0-35.1%), as based upon reduction in mean worm burden. Analysis of faecal egg count trends through the course of the study revealed that egg counts rose in both control and pyrantel-treated dogs, with a greater rise observed in the latter group (11.6+/-8.3% versus 17.3+/-7.6%; p=0.04), despite the decrease in adult worm numbers in this group. Our results indicate that high-level anthelmintic resistance does occur in companion animal medicine, and highlight the need for greater vigilance and more judicious use of anthelmintics in small animal practice. They further indicate that the faecal egg count reduction test needs to be used with caution with this parasite.

Animal handling as an integrated component of animal and veterinary science programs at the University of Queensland.

Students in animal science and veterinary science at the University of Queensland (UQ) have similar introductory courses in animal handling in year 1 of their degree programs. Veterinary students take animal-handling instruction in farm and companion animals, whereas animal science students are instructed in handling farm animals, horses, and rodents. Veterinary students are introduced to rodents, and animal science students to dogs and cats, in subsequent years of the curriculum. Both cohorts receive additional training, with clinical emphasis for veterinary students in years 3, 4, and 5 of their five-year curriculum. The introductory course is well received by students; both student cohorts appreciate the opportunity provided and the effort that goes into the animal-handling classes. Undergraduates realize that acquiring animal-handling skills will increase their proficiency in their subsequent careers; veterinary graduates recognize that their handling prowess will give their clients confidence in their abilities. Most clients cannot judge the competence of a veterinarian's diagnosis or treatment but will judge their ability based on their handling skills. Ongoing practice allows students to become competent in animal handling.

Use of big data in the surveillance of veterinary diseases: early detection of tick paralysis in companion animals.

BACKGROUND: Tick paralysis, resultant from envenomation by the scrub-tick Ixodes holocyclus, is a serious threat for small companion animals in the eastern coast of Australia. We hypothesise that surveillance systems that are built on Internet search queries may provide a more timely indication of high-risk periods more effectively than current approaches. METHODS: Monthly tick paralysis notifications in dogs and cats across Australia and the states of Queensland (QLD) and New South Wales (NSW) were retrieved from Disease WatchDog surveillance system for the period 2011-2013. Internet search terms related to tick paralysis in small companion animals were identified using Google Correlate, and corresponding search frequency metrics were downloaded from Google Trends. Spearman's rank correlations and time series cross correlations were performed to assess which Google search terms lead or are synchronous with tick paralysis notifications. RESULTS: Metrics data were available for 24 relevant search terms at national level, 16 for QLD and 18 for NSW, and they were all significantly correlated with tick paralysis notifications (P < 0.05). Among those terms, 70.8, 56.3 and 50 % showed strong Spearman's correlations, at national level, for QLD, and for NSW respectively, and cross correlation analyses identified searches which lead notifications at national or state levels. CONCLUSION: This study demonstrates that Internet search metrics can be used to monitor the occurrence of tick paralysis in companion animals, which would facilitate early detection of high-risk periods for tick paralysis cases. This study constitutes the first application of the rapidly emerging field of Internet-based surveillance to veterinary science.

In vitro levamisole selection pressure on larval stages of Haemonchus contortus over nine generations gives rise to drug resistance and target site gene expression changes specific to the early larval stages only.

There is some evidence that resistance to levamisole and pyrantel in trichostrongylid nematodes is due to changes in the composition of nicotinic acetylcholine receptors (nAChRs) which represent the drug target site. Altered expression patterns of genes coding for nAChR subunits, as well as the presence of truncated versions of several subunits, have been implicated in observed resistances. The studies have mostly compared target sites in worm isolates of very different genetic background, and hence the ability to associate the molecular changes with drug sensitivity alone have been clouded to some extent. The present study aimed to circumvent this issue by following target site gene expression pattern changes as resistance developed in Haemonchus contortus worms under laboratory selection pressure with levamisole. We applied drug selection pressure to early stage larvae in vitro over nine generations, and monitored changes in larval and adult drug sensitivities and target site gene expression patterns. High level resistance developed in larvae, with resistance factors of 94-fold and 1350-fold at the IC50 and IC95, respectively, in larval development assays after nine generations of selection. There was some cross-resistance to bephenium (70-fold increase in IC95). The expression of all the putative subunit components of levamisole-sensitive nAChRs, as well as a number of ancillary protein genes, particularly Hco-unc-29.1 and -ric-3, were significantly decreased (up to 5.5-fold) in the resistant larvae at generation nine compared to the starting population. However, adult worms did not show any resistance to levamisole, and showed an inverse pattern of gene expression changes, with many target site genes showing increased expression compared to the starting population. A comparison of the larval/adult drug sensitivity data with the known relationships for field-derived isolates indicated that the adults of our selected population should have been highly resistant to the drug if the larval/adult sensitivity relationships were in accordance with previous field isolates. Hence, our selected worms showed a life-stage drug sensitivity pattern quite different to that seen in the field. The present study has highlighted an association between drug target site changes and resistance to levamisole in H. contortus larvae. However, it has also highlighted the artificial nature of the larval selection method with levamisole, as the resistance phenotype and the associated molecular changes were only observed in the drug-pressured life stage. The study therefore reinforces the need for caution in extrapolating larval-based laboratory selection outcomes to field resistances.

Drug-efflux and target-site gene expression patterns in Haemonchus contortus larvae able to survive increasing concentrations of levamisole in vitro.

While there is some evidence that changes in nicotinic acetylcholine receptor (nAChR) subunits confer resistance to levamisole in gastrointestinal helminth parasites, the exact nature of the resistance mechanism(s) is unclear. We utilised the presence of a resistant fraction within the Wallangra 2003 isolate of Haemonchus contortus larvae in order to subdivide the population into three subpopulations of larvae able to survive increasing concentrations of the drug. We then measured gene expression levels in the subpopulations and the larval population as a whole, focusing on genes encoding the subunit components of levamisole-sensitive receptors, genes encoding ancillary proteins involved in receptor assembly, and P-glycoprotein (P-gp) genes. The subpopulation surviving the lowest levamisole concentration showed increases of 1.5- to 3-fold in a number of P-gp genes (Hco-pgp-3, -4, -10, and -14) alongside unchanged receptor genes, compared to the whole Wallangra larval population. On the other hand, the subpopulation surviving the intermediate levamisole concentration showed an increase in only a single P-gp (Hco-pgp-14), alongside decreases in some receptor subunit (Hco-unc-63a) and ancillary protein genes (Hco-unc-50, Hco-ric-3.1 and 3.1). The subpopulation surviving the highest levamisole concentration showed further decreases in receptor subunit genes (Hco-unc-63a and Hco-unc-29 paralogs) as well as genes involved in receptor assembly (Hco-unc-74, Hco-unc-50, Hco-ric-3.1 and 3.1), alongside no increased P-gp gene levels. This suggests a biphasic pattern of drug resistance in the larvae of this worm isolate, in which a non-specific P-gp-mediated mechanism confers low levels of resistance, while higher level resistance is due to altered receptor subunit composition as a result of changes in both subunit composition and in the levels of proteins involved in receptor assembly.

Acetylcholine receptor subunit and P-glycoprotein transcription patterns in levamisole-susceptible and -resistant Haemonchus contortus.

The mechanism of resistance to the anthelmintic levamisole in parasitic nematodes is poorly understood, although there is some evidence implicating changes in expression of nicotinic acetylcholine receptor (nAChR) subunit genes. Hence, in order to define levamisole resistance mechanisms in some Australian field-derived isolates of Haemonchus contortus we examined gene expression patterns and SNPs in nAChR subunit genes, as well as expression levels for P-glycoprotein (P-gp) and receptor ancillary protein genes, in various life stages of one levamisole-sensitive and three levamisole-resistant isolates of this species. Larvae of two isolates showed high-level resistance to levamisole (resistance ratios at the IC50 > 600) while the third isolate showed a degree of heterogeneity, with a resistance factor of only 1.1-fold at the IC50 alongside the presence of a resistant subpopulation. Transcription patterns for nAChR subunit genes showed a great degree of variability across the different life stages and isolates. The most consistent observation was the down-regulation of Hco-unc-63a in adults of all resistant isolates. Transcription of this gene was also reduced in the L3 stage of the two most resistant isolates, highlighting its potential as a resistance marker in the readily accessible free-living stages. There was down regulation of all four Hco-unc-29 paralogs in adults of one resistant isolate. There were no consistent changes in expression of P-gps or ancillary protein genes across the resistant isolates. The present study has demonstrated a complex pattern of nAChR subunit gene expression in H. contortus, and has highlighted several instances where reduced expression of subunit genes (Hco-unc-63a, Hco-unc-29) may be associated with the observed levamisole resistance. The data also suggests that it will be difficult to detect resistance using gene transcription-based methods on pooled larval samples from isolates containing only a resistant subpopulation due to the averaging of gene expression data across the whole population.