Plant cysteine proteinases (CPs) from papaya, pineapple, and some fig species are capable of killing parasitic nematode worms in vitro and also possess anthelmintic effects in vivo by a mechanism different to all commercially available synthetic anthelmintics. There exists, nonetheless, a vast repository of as yet untested plant resources with potential for development into novel anthelmintics. For screening purposes, parasitic nematodes are not an ideal animal model mainly because of the need to maintain complex life-cycles in the laboratory, involving passage through infected animals as well as ensuing costs and ethical considerations. A cheap and effective rapid Caenorhabditis elegans-based assay for screening plant CP extracts for nematicidal activity has been developed based on neutral red retention colorimetric assay. More sensitive ways of identifying nematicidal activity of plant products were explored using wild type and different mutant and transgenic strains. These methods involved assessments of behavioural end points (e.g. motility), lethality and cuticular damage. The effect of CPs on wild type (Bristol N2) and cystatin null mutant (cpi-l-t- and cpi-2-t-) C. e/egans was concentration-, temperature- and time-dependent. (Ps were able to induce elevated cpi-l and cpi- 2 cystatin expression thereby suggesting that c. elegans deploy cystatins CPI-l and CPI-2 to resist CP attack. When taken together, the results of lethality, motility assays and cuticle damage in wild type, bacterially unswollen (bus) strains and cpi-null mutants point towards a cheap and effective rapid through-put C. e/lgans-based assay for screening the nematicidal activities of plant-derived CPs.

Parasitic nematodes continue to be the causative agents of some of the most prevalent diseases of man and cause significant economic losses to the worldwide agri-food industry. With anthelmintic resistance on the rise and environmental concerns over the use of many nematicides the effectiveness of current control options is diminishing. The only viable alternative is the creation of new, environmentally safe drugs/control options. The reverse genetic technique of RNA interference (RNAi) facilitates the examination of gene function by the application of double stranded (ds)RNA complementary to a gene of interest which triggers endogenous cellular mechanisms that destroy the target gene transcript. Of particular interest here is the application of RNAi to nematode species and how this technology could be harnessed to improve our understanding of nematode biology and, ultimately, exploited to inform drug target selection. This study demonstrates that RNAi remains a potentially useful tool for the validation of gene function despite the pressing need to improve experimental rigor and statistical analysis when validating RNAi induced gene knockdown. Although RNAi failed to silence gene expression in the free-living nematode Panagrellus redivivus and was limited in its efficacy in l3 stage worms of the pig parasite Ascaris suum, it did trigger consistent knockdown of selected genes in A. suum adults. A bioinformatic analysis of the RNAi pathway components in the genomes/transcriptomes of selected nematodes indicated the occurrence of all functional groupings within diverse nematodes, suggesting that RNAi mechanisms are in place broadly in the phylum. There were no RNAi effector deficiencies that specifically mapped to RNAi incompetency. As such there remains hope that with further optimisation, and as culture and delivery techniques continue to improve, RNAi could be an important tool for screening gene function and validating drug targets in nematode parasites.

No description available.

Gastrointestinal (GI) nematodes are important helminth pathogens responsible for severe losses to livestock industries and human health throughout the world. Control of these infections relies primarily on chemotherapy; however there is rapid development of resistance to all available classes of anthelmintic drugs, and therefore new alternative treatments are urgently required. Plant cysteine proteinases (CPs) from papaya latex, pineapple fruit and stem extracts have been demonstrated to be effective against GI nematodes of rodents, chickens, pigs and sheep. The current study extended evaluation of different plant extracts and the factors affecting the efficacy of papaya latex supernatant (PLS) as an anthelmintic against GI nematodes in a mouse model system and formulation and delivery for use in ruminants. The study started with purification and concentration of CPs in PLS using different methods to determine which of them would provide high yield of CPs. It was found that concentration by dialysis provided a high yield of active enzyme in PLS. Storage of PLS at -200C and -800C retained more active enzymes for prolonged period of time than at ambient temperature and 4oC. Motility assay conditions showed to have no influence on enzyme activity. While the in vitro experiment results showed significant detrimental effect of pineapple fruit extract, stem bromelain and little effect of kiwi fruit extract against Heligmosomoides bakeri motility. In vivo experiments showed less efficacy of these enzymes than expected when compared with PLS. The first factor to be assessed in this study was the effect of fasting on the anthelmintic efficacy of PLS. The results showed that PLS was equally effective in reducing worm burdens whether mice were fasted before treatment or not, and by avoiding fasting the side effects of treatment were minimized. Comparison of efficacy in a range of mouse strains indicated that efficacy varied between mice of different genotype. At the dose used, the treatment was most effective in C3H mice ranging from 90.5% to 99.3% in reducing worm burdens and less effective in NIHS, CD1 and BALB/c strains (7.9%, 36.0% and 40.5% reduction respectively). However, host sex and body size were shown not to have any influence on the anthelmintic efficacy of PLS. Since CPs are particularly sensitive to pH, variation between mouse strains in gut pH was investigated but no significant differences in pH were found along the GI tract of the poor (BALB/c) and high responder mice (C3H) to PLS treatment and concurrent administration of the antacid cimetidine also did not improve efficacy. The study also explored the potential of formulation and delivery of PLS as an anthelmintic drug for ruminants. In vitro studies involving both immediate and slow release dosage formulations simulating the physiological conditions (pH, temperature and peristaltic movement) in the GI tract of the animal were conducted. In the slow release experiments, two hydrophilic matrices were tested, the xanthan gum and hydroxypropyl methylcellulose (HPMC) (both Methocel-LVCR and Methocel-CR). Methocel-CR provided better slow release results compared to the others. In the immediate release experiments 3 disintegrants (Primojel, L-HPC and Ac-Di-sol) were investigated and Ac-Di-Sol® was found to produce the faster immediate drug release rate. Preliminary in vitro studies also showed that PLS was highly effective against equine GI nematodes. Finally the empirical findings in this study provide useful information for improvement of formulation and delivery of these naturally occurring plant-derived enzymes for treatment of intestinal worm infections in humans and livestock, while achieving maximum efficacy and minimal side-effects.

Parasitic nematodes of grazing livestock represent an increasing economic and welfare problem for British agriculture. By investigating specific life-cycle stages of these parasites, it may be possible to identify key molecules or pathways that are required for the survival of the worms, and thus exploit these for future control strategies. It has been shown previously that the third larval stages (L3) of the ovine parasitic nematode Teladorsagia circumcincta produce high levels of transcript for the enzyme GTP-Cyclohydrolase relative to later developmental stages. As the ratelimiting factor in the production of tetrahydrobiopterin, GTP-Cyclohydrolase is required for a number of different biochemical pathways, including those involved in the production of serotonin and melanin. As the L3 do not feed, it can be hypothesised that, if finite resources are being used in the production of transcript encoding this enzyme, then it may be important for survival. In this thesis, a number of approaches were taken to explore the function of GTPCyclohydrolase in the life-cycle development of T. circumcincta. The closely related parasite, Dictyocaulus viviparus, was used as a model organism to explore the role of GTP-Cyclohydrolase and serotonin production with regards to larval arrest, or hypobiosis. This process occurs readily under experimental conditions in D. viviparus, which is not possible with T. circumcincta. Quantitative PCR was used to examine GTP-Cyclohydrolase transcript levels in two different strains of D. viviparus, one that enters larval arrest when exposed to cold conditions and one that does not. No differences were observed between the two strains suggesting that GTP-Cyclohydrolase was unlikely to be involved in hypobiosis. The model nematode, Caenorhabditis elegans, was used to perform functional complementation experiments to assess the role of GTP-Cyclohydrolase in the cuticle, as it has been shown previously that C. elegans GTP-Cyclohydrolase mutants have a ‘leaky cuticle’ and are killed by lower doses of anthelmintics and bleach than the wild-type worms. The T. circumcincta gene for GTP-Cyclohydrolase was able to restore cuticular integrity of C. elegans GTP-Cyclohydrolase-deletion mutants, suggesting that the role played by the protein in both species is similar. In vitro inhibition experiments using a chemical inhibitor of GTP-Cyclohydrolase showed that T. circumcincta larval development was disrupted in the presence of the inhibitor. It was also shown that T. circumcincta L3 that were exposed to sunlight produced melanin, suggesting that the levels of GTP-Cyclohydrolase observed in the preparasitic stages of T. circumcincta may be required for the synthesis of melanin. Together, these data suggest that GTP-Cyclohydrolase is required by the preparasitic stages to survive on pasture. Ultraviolet radiation has been shown previously to be harmful to T. circumcincta L3, so if the melanin production provides protection from this, then it would be crucial for the survival of the pre-parasitic stages.

Potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are economically important pests of potato (Solanum tuberosum) crops in potato growing regions worldwide. Integrated management is under threat, with effective nematicides increasingly being withdrawn on environmental and health grounds. Alternative strategies are urgently needed and trap cropping could be one of them. The non-tuber-bearing Solanum sisymbriifolium is regarded as an effective trap crop for PCN with strong hatching ability and immunity to PCN infection and has been used in the UK and The Netherlands. However, its mode of action is unknown. In order to shed light on the mode of action so that a novel control strategy could be identified, the interactions between G. pallida and S. sisymbriifolium were investigated using in vitro bioassays. In choice assays, G. pallida J2s were equally attracted to the roots of S. sisymbriifolium and to those of S. tuberosum. However, potato root diffusate (PRD), which is routinely used to induce PCN hatch, failed to attract G. pallida J2s in chemotaxis bioassays, indicating hatching factors (HFs) and soluble compounds present in PRD are not involved in attraction of G. pallida J2s to potato roots. The J2s invaded the roots of S. sisymbriifolium in large numbers but failed to develop further. To facilitate continuous observation of nematode development, a novel in vitro method was devised with the use of Pluronic F-127, which requires no sterilisation, and the life cycle of G. pallida was successfully observed in S. tuberosum roots. Quantitative real-time polymerase chain reaction analyses of defence related genes of S. tuberosum and S. sisymbriifolium infected with G. pallida revealed up-regulation of the chitinase gene (ChtC 2.1) at 3 days post inoculation in S. sisymbriifolium but not in S. tuberosum. Electrospray ionisation-mass spectrometry analyses of root exudate extracts of the two Solanum species and subsequent bioassay-guided fractionation showed that the HF of S. sisymbriifolium differs from that of S. tuberosum. Previously, attention had been solely paid to the hatching ability of the root exudate of S. sisymbriifolium, but this study revealed for the first time that the aerial part extract possesses a significant hatching ability.