ADRIAN WOLSTENHOLME
Associate Professor

CURRENT RESEARCH
Anthelmintic Resistance in Parasitic Nematods
Parasitic nematodes (roundworms) are serious pathogens of humans and their domesticated animals. These infections can cause serious disease and economic losses, but for many years have been effectively controlled in livestock and companion animals by the use of anthelmintic drugs. Recently, however resistance to these drugs in veterinary parasites has reached very serious levels in many parts of the world, including the South Eastern USA. There is also the suggestion that resistance may be emerging in human parasites, which cause debilitating diseases, especially of the poor. We are interested in discovering more about how anthelmintic drugs act, how parasites become resistant to them and in the development of new techniques to detect and reverse drug resistance. There are three major classes of anthelmintic in current use, plus another two that have recently become available. The three major classes of anthelmintic are the benzimidazoles, the macrocyclic lactones and the ; the two recent classes are the cyclodepsipeptides, such as emodepside, and the amino-acetonitrile derivatives (AADs), such as monepantel. The mechanism of action of the benzimidazoles is to prevent tubulin repolymerisation and resistance has been widely reported to be due to a Single Nucleotide Polymorphism (SNP) that causes a single amino-acid change in b-tubulin, F200Y. We are investigating the most reliable and quantitative method for detecting this SNP in Haemonchus contortus, a parasite of small ruminants. To date, we have produced a real-time PCR assay using locked nucleic acid (LNA) probes that can reliably detect resistance alleles at a frequency of 5-10% within a few hours. The results correlate well with in vivo tests, such as the egg-hatch assay. We will continue to develop and improve this assay, comparing it with other molecular techniques, such as Pyrosequencing, in collaboration with Prog. Georg von Samson-Himmelstjerna of the Hanover School of Veterinary Medicine.
Nicotinic acetylcholine receptors in the parasitic nematode, Ascaris suum.
Sally Williamson and Hayley Bennett are studying the nicotinic receptors of the large parasite of pigs, A. suum, supported in part by Pfizer Animal Health. This worm is closely related to an important human parasite, A. lumbricoides, and the physiology of its nicotinic receptors has been widely studied because of the size and convenience of the preparation. However, we know little about the molecular biology of the receptor, and of any mutations that may be associated with resistance to nicotinic drugs, which include not only the imidazothiazoles, such as levamisole, but also the AADs. Sally has successfully cloned several subunit cDNAs of the Ascaris nicotinic receptor and has been able to show that the subunits are expressed on Ascaris muscle, and can be expressed in the Xenopus oocyte to form functional nicotinic receptors susceptible to levamisole, pyrantel and oxantel, as well as nicotine. Hayley is also studying nicotinic acetylcholine receptors, in this case a novel subunit, designated ACR-26, which seems to be only found in parasitic nematodes. She has found that expression of this subunit is restricted to head muscles and is actively investigating its other properties.
Transgenic C. elegans as a method for studying drug resistance genes of parasitic nematodes.
This is a new project, in which we will be trying to find out whether the model organism, C. elegans, is a useful way of studying parasite genes. C. elegans is easy to grow in the lab, and there are lots of useful mutant strains that are readily available, plus many fairly simple phenotypic assays. None of these things are available for parasitic nematodes, so can we express parasite genes in the model worm? Yes, we can, but the question is how specific are the results that we obtain: many genes exist as families and we want to know how specific the effects are. We are using our favourite gene family, which encodes the glutamate-gated chloride channels, which are the targets of the macrocyclic lactones, as a way of answering these questions.
The molecular basis of ivermectin resistance in parasitic nematodes.
Despite some intensive research, we still do not understand how parasitic nematodes become resistant to the macrocyclic lactones. We are taking several approaches to solving this problem; one of the most promising is by making a detailed comparison of the genes that are expressed in a very closely related pair of H. contortus isolates, one of which is susceptible to the drug; it was used to rapidly select a resistant population in only three populations. This close relationship should minimise the problems of genetic heterogeneity that have bedevilled work in this area.

RECENT PUBLICATIONS

Search PubMed for "Wolstenholme, A.J"

Cook, A., Aptel, N., Portillo, V., Siney, E., Sihota, R., Holden-Dye, L., Wolstenholme, A. (2006) Caenorhabditis elegans ivermectin receptors regulate locomotor behaviour and are functional orthologues of Haemonchus contortus receptors. Mol. Biochem. Parasitol. 147, 118-125. doi:10.1016/j.molbiopara.2006.02.003

Walsh, T.K. Donnan, A., Jackson, F., Skuce, P., Wolstenholme, A.J. (2007). Sensitive detection and measurement of benzimidazole resistance alleles in Haemonchus contortus. Vet. Parasitol. 144, 304-312. doi:10.1016/j.vetpar.2006.10.014

McCavera, S., Walsh, T.K., Wolstenholme, A.J. (2007) Nematode ligand-gated chloride channels: an appraisal of their involvement in macrocyclic lactone resistance and prospects for developing molecular markers. Parasitology
134, 1111-1121. doi:10.1017/S0031182007000042

Williamson, S.M., Walsh, T.K., Wolstenholme, A.J. (2007). The cys-loop ligand-gated ion channel gene family of Brugia malayi and Trichinella spiralis: a comparison with Caenorhabditis elegans. Inv. Neurosci. 7, 219-226. doi: 10.1007/s10158-007-0056-0

Lavender, V., Chong, S., Ralphs, K., Wolstenholme, A.J., Reaves, B.J. (2008). Increasing expression of calcium permeable TRPC3 and TRPC7 channels enhances constitutive secretion. Biochem J. 413, 437-446. doi:10.1042/BJ20071488

von Samson-Himmelstjerna, G., Walsh, T.K., Donnan, A.A., Carrière, S., Jackson, F., Skuce, P.J., Rohn, K., Wolstenholme, A.J. (2009) Molecular detection of benzimidazole resistance in Haemonchus contortus as a tool for routine field diagnosis. Parasitology, 136, 349-358. doi:10.1017/S003118200800543X

von Samson-Himmelstjerna, G., Prichard, R.K., Wolstenholme, A.J. (2009) Anthelmintic resistance as a guide to the discovery of new drugs? In, ‘Drug Discovery in Infectious Diseases’, Selzer, P.M. (ed), pp17-32. Wiley-VCH, Weinheim.

McCavera, S., Rogers, A.T., Yates, D.M., Woods, D., Wolstenholme, A.J. (2009) An ivermectin-sensitive glutamate-gated chloride channel from the parasitic nematode, Haemonchus contortus. Mol Pharmacol, 75, 1347-1355. doi: 10.1124/mol.108.053363

Williamson, S.M., Robertson, A.P., Brown, L., Williams, T., Woods, D.J., Martin, R.J., Sattelle, D.B., Wolstenholme, A.J. (2009). The nicotinic acetylcholine receptors of Ascaris suum: formation of two distinct receptors by varying the relative expression levels of two subunits. PLoS Pathogens, 5: e1000517

Email
adrianw@uga.edu

Office
G9, Wildlife Building
706-542-2404

Degrees