Steven R. Kopp

Anthelmintic activity of cyclotides: in vitro studies with canine and human hookworms.

Hookworm infection is a leading cause of maternal and child morbidity in countries of the tropics and subtropics, as well as being an important parasite in companion-animal medicine. The cyclotides are a novel family of cyclic cystine knot containing peptides from plants that have been shown to possess anthelmintic activity against Haemonchus contortus and Trichostrongylus colubriformis, two important gastrointestinal nematodes of sheep. In the current study we demonstrated the in vitro effects of three representative cyclotides, kalata B1, kalata B6 and cycloviolacin O14, on the viability of larval and adult life stages of the dog hookworm Ancylostoma caninum, and larvae of the human hookworm Necator americanus. The cyclotides showed significant anthelmintic activity towards both hookworm species. The different cyclotides showed similar patterns of relative activity as that seen previously with the livestock nematode species. This study demonstrates that cyclotides have promising activity in vitro against important parasites of companion animals and humans.

Acetylcholine receptor subunit genes from Ancylostoma caninum: Altered transcription patterns associated with pyrantel resistance ☆

The molecular mechanism of resistance to nicotinic agonist anthelmintics such as pyrantel and levamisole in nematodes of medical and veterinary significance is poorly understood. The identification of pyrantel-resistant isolates of the canine hookworm, Ancylostoma caninum, provides an opportunity to explore, at a molecular level, the mechanism of cholinergic resistance in a species that is a model for the human hookworms. Here we describe the cloning of three A. caninum genes orthologous to components of the pyrantel-sensitive nicotinic acetylcholine receptor in Caenorhabditis elegans (UNC-29, -38, -63). Analysis of mRNA levels by quantitative PCR was also performed on these genes, plus an additional three nicotinic acetylcholine receptor subunit genes thought not to be constituents of the pyrantel-sensitive receptor, for which a partial sequence was obtained. Gene sequences and mRNA levels were compared between two isolates of A. caninum showing either high- or low-level resistance to pyrantel (as shown previously by in vivo efficacy and in vitro comparative studies). While no polymorphisms of likely significance between the two A. caninum isolates were observed, quantitative analysis of transcription revealed significantly lower levels for the three putative pyrantel receptor subunits (AAR-29, -38 and -63) in the highly pyrantel-resistant isolate compared with the isolate with low-level resistance. In contrast, transcription of the three subunits thought not to constitute the pyrantel receptor (AAR-8, -15 and -19) was either not significantly different between the two isolates, or slightly higher in the highly-resistant isolate. This data suggests that reduced transcription of the mRNA coding for nicotinic acetylcholine receptor subunits that form the pyrantel-sensitive receptors may be a component of the pyrantel resistance mechanism in A. caninum.

Molecular Evidence Supports the Role of Dogs as Potential Reservoirs for Rickettsia felis

Rickettsia felis causes flea-borne spotted fever in humans worldwide. The cat flea, Ctenocephalides felis, serves as vector and reservoir host for this disease agent. To determine the role of dogs as potential reservoir hosts for spotted fever group rickettsiae, we screened blood from 100 pound dogs in Southeast Queensland by using a highly sensitive genus-specific PCR. Nine of the pound dogs were positive for rickettsial DNA and subsequent molecular sequencing confirmed amplification of R. felis. A high prevalence of R. felis in dogs in our study suggests that dogs may act as an important reservoir host for R. felis and as a potential source of human rickettsial infection.

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.

The potential impact of density dependent fecundity on the use of the faecal egg count reduction test for detecting drug resistance in human hookworms

Current efforts to control human soil-transmitted helminth (STH) infections involve the periodic mass treatment of people, particularly children, in all endemic areas, using benzimidazole and imidothiazole drugs. Given the fact that high levels of resistance have developed to these same drugs in roundworms of livestock, there is a need to monitor drug efficacy in human STHs. The faecal egg count reduction test (FECRT), in which faecal egg output is measured pre- and post-drug treatment, is presently under examination by WHO as a means of detecting the emergence of resistance. We have examined the potential impact of density dependent fecundity on FECRT data. Recent evidence with the canine hookworm indicates that the density dependent egg production phenomenon shows dynamic properties in response to drug treatment. This will impact on measurements of drug efficacy, and hence drug resistance. It is likely that the female worms that survive a FECRT drug treatment in some human cases will respond to the relaxation of density dependent constraints on egg production by increasing their egg output significantly compared to their pre-treatment levels. These cases will therefore underestimate drug efficacy in the FECRT. The degree of underestimation will depend on the ability of the worms within particular hosts to increase their egg output, which will in turn depend on the extent to which their egg output is constrained prior to the drug treatment. As worms within different human cases will likely be present at quite different densities prior to a proposed FECRT, there is potential for the effects of this phenomenon on drug efficacy measurements to vary considerably within any group of potential FECRT candidates. Measurement of relative drug efficacy may be improved by attempting to ensure a consistent degree of underestimation in groups of people involved in separate FECRTs. This may be partly achieved by omission of cases with the heaviest infections from a FECRT, as these cases may have the greatest potential to increase their egg output upon removal of density dependent constraints. The potential impact of worm reproductive biology on the utility of the FECRT as a resistance detection tool highlights the need to develop new drug resistance monitoring methods which examine either direct drug effects on isolated worms with in vitro phenotypic assays, or changes in worm genotypes.

Molecular evidence of Rickettsia felis infection in dogs from northern territory, Australia

The prevalence of spotted fever group rickettsial infection in dogs from a remote indigenous community in the Northern Territory (NT) was determined using molecular tools. Blood samples collected from 130 dogs in the community of Maningrida were subjected to a spotted fever group (SFG)-specific PCR targeting the omp B gene followed by a Rickettsia felis-specific PCR targeting the glt A gene of R. felis. Rickettsia felis omp B and glt A genes were amplified from the blood of 3 dogs. This study is the first report of R. felis infection in indigenous community dogs in NT.

Seroprevalence and risk factors for Rickettsia felis exposure in dogs from Southeast Queensland and the Northern Territory, Australia

BackgroundThe recent detection of Rickettsia felis DNA in dogs in Australia suggests that dogs are potential mammalian reservoir hosts for this emerging rickettsia. To date, there is no published report addressing the seroprevalence of R. felis in dogs in Australia.MethodsAntigens for R. felis were produced by inoculating confluent XTC-2 monolayer cell cultures with three pools of cat flea (Ctenocephalides felis) homogenates. Infection was confirmed by real-time (qPCR), conventional or nested PCRs targeting the omp B, glt A, 17 kDa and omp A genes. Two hundred and ninety-two dogs from Southeast Queensland and the Northern Territory were tested for the presence of R. felis antibodies using a microimmunofluorescence (IF) test and the seroprevalence and associated risk factors for exposure were determined using both uni- and multi-variate analyses.ResultsRickettsia felis was successfully isolated in cell culture from all three cat-flea pools. One hundred and forty-eight dogs (50.7%) showed seropositivity with titres ≥64 and 54 (18.5%) with titres ≥128. At antibody titres ≥64, dogs with active ectoparasite control were less likely to be seropositive to R. felis (OR: 2.60; 95% CI: 1.20 - 5.56).ConclusionsThis first reported isolation of R. felis in cell culture in Australia allowed for the production of antigen for serological testing of dogs. Results of this serological testing reflects the ubiquitous exposure of dogs to R. felis and advocate for owner vigilance with regards to ectoparasite control on domestic pets.

Large-scale monitoring of imidacloprid susceptibility in the cat flea, Ctenocephalides felis

Although on‐animal topical treatment with compounds such as imidacloprid has revolutionized the control of the cat flea, Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae), the development of insecticide resistance is a continuing threat. As part of a highly co‐ordinated and unprecedented resistance monitoring programme for C. felis, 1437 flea isolates were collected by veterinary clinics in Australia, Germany, France, the U.K. and 29 states in the U.S.A. from 2002 to 2009. About 65% of the collections were made from June to October each year and 71% of the collections were from cats. Collections of flea eggs were sent to one of five different laboratories, where they were tested with a diagnostic dose of imidacloprid (3 p.p.m.) applied to larval flea‐rearing medium. Of the 1437 collections received, 1064 contained adequate numbers of eggs for testing. Of these isolates, untreated eggs failed to hatch in 22.7% and were not considered valid bioassays. Survival rates >5% and development of adult fleas (a threshold for further testing) occurred in only 22 isolates. They were re‐tested with the same diagnostic dose and none produced >5% adult emergence. Complete dose–response bioassays were performed on three of the isolates that had triggered a second test and produced slopes, intercepts and LC50 values similar to those for existing susceptible laboratory strains. Results confirmed sustained susceptibility of C. felis to imidacloprid, despite its widespread use for over a decade.

Canine vector‐borne disease pathogens in dogs from south‐east Queensland and north‐east Northern Territory

OBJECTIVES To determine the prevalence of canine vector-borne diseases (CVBD: Babesia spp., Anaplasma spp., Ehrlichia spp., haemotropic mycoplasmas and Hepatozoon) in Australian dogs; namely, dogs from pounds in south-east Queensland and an indigenous Aboriginal community in the north-east of the Northern Territory. DESIGN AND PROCEDURE Blood samples were collected from 100 pound dogs and 130 Aboriginal community dogs and screened for the CVBD pathogens using polymerase chain reaction (PCR). All positive PCR products were sequenced for species confirmation. RESULTS In total, 3 pound dogs and 64 Aboriginal community dogs were infected with at least one CVBD pathogen. Overall, B. vogeli was detected in 13 dogs, A. platys in 49, M. haemocanis in 23, Candidatus Mycoplasma haematoparvum in 3 and C. M. haemobos in 1 dog. Co-infections were detected in 22 Aboriginal community dogs. CONCLUSIONS This study found B. vogeli, A. platys and haemotropic mycoplasma infections to be common in dogs in subtropical and tropical areas of Australia. This study also reports for the first time the prevalence and genetic characterisation of haemotropic mycoplasmas in dogs in Australia.

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

Graphical abstract