Analysis of developmentally regulated genes of the parasite Haemonchus contortus☆
Introduction
Haemonchus contortus is an abomasal parasite of significant agricultural importance, primarily affecting sheep, as well as other ruminants world-wide. With the clinical effects ranging from mild anaemia to death in young animals, the control of H. contortus and other gastrointestinal nematodes costs the livestock industry an estimated £1000 million annually (Newton and Munn, 1999). Current means of control depend on repeated treatment with anthelmintic chemicals. However, due to increasing drug resistance and concern over residual chemicals in the environment and food chain, an alternate or supplementary means of control must be pursued. Other means of control include the development of molecular vaccines against these parasites (Newton and Munn, 1999).
The life cycle of H. contortus consists of free-living stages on the pasture, as well as parasitic stages within its hosts. However, little is known about the molecular biology and regulation of gene expression of H. contortus. The ability to characterise the expression of developmentally regulated molecules during the different stages of its life cycle may provide a better understanding of mechanisms involved in host invasion, attachment, feeding and evasion of the host immune response. With more comprehensive information, new targets for parasite control may be identified.
Very recently, 370 expressed sequence tags (ESTs) from the free-living and parasitic stages of H. contortus have been published (Hoekstra et al., 2000). These results showed a shift in gene expression during transition to parasitism. However, because this approach does not eliminate house-keeping genes, many sequences which would be expected to be common between the stages, such as ribosomal proteins, were identified. Advances in the field of molecular biology have led to the development of techniques, such as ‘differential display’ (Liang et al., 1992, Liang and Pardee, 1992) and ‘RNA arbitrarily-primed PCR’ (RAP-PCR; Welsh et al., 1992), for the comparison of differential gene expression in different cell types, or under altered conditions. Such techniques provide the ability to rapidly identify changes occurring at the transcriptional level between various stages of development. For example, a study using differential display has been conducted to identify stage-specific genes in the parasitic nematode Angiostrongylus cantonensis (Joshua and Hsieh, 1995). More recently, a splice-leader differential display approach has been used to study differential gene expression in male and female Brugia malayi (Michalski and Weil, 1999). Similarly, RAP-PCR has been used to successfully identify gender-specific transcripts from the porcine nodule worm, Oesphagostomum dentatum (Boag et al., 2000). This study reports the isolation and characterisation of stage-associated H. contortus molecules using the RAP-PCR technique.
Section snippets
Parasite preparation
A benzimidazole-resistant strain of H. contortus (strain VSRG originally obtained from the McMaster Lab, CSIRO, Sydney, Australia) was maintained by serial passage in Merino sheep, reared helminth-free and 3–6 months of age. Infective third-stage larvae (L3) were cultured from the faeces of weaned sheep with patent mono-specific infections (Ministry of Agriculture, Fisheries and Food, 1986); L3 were harvested from faecal cultures after 6–7 days of incubation at 27°C. Adult nematodes were
Identification and recovery of differentially amplified cDNA molecules
To determine the usefulness of RAP-PCR as a tool for the identification of stage-associated molecules of the nematode H. contortus, a comparison was performed between the mRNA populations of the L3 and adult stages using arbitrary primers. In total, 234 cDNA products were displayed, with an average of 33/primer used. Of those products visualised, 32% were unique to L3 and 30% were unique to adult H. contortus (data not shown).
cDNA products (146) that appeared to be differentially amplified in
Discussion
It was expected that the development of H. contortus from the L3 to the adult worm would be accompanied by the differential regulation of many genes, due to profound biochemical changes in response to changes in the environment and development. For example, those genes required for attachment and feeding mechanisms would be expected to be upregulated in the L4 and adult stages. This was reflected in the pattern of differential gene expression seen with RAP-PCR between L3 and adult stages, and
Acknowledgements
This work was supported by Novartis Animal Health Australia, and the Department of Natural Resources and Environment (DNRE) Victoria, Australia. Many thanks to the staff of the Molecular Vaccines Department (VIAS, Attwood) for their technical assistance. The supply of Haemonchus contortus L3 by Novartis Animal Health Australia is greatly appreciated.
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Note: Nucleotide sequence data reported in this paper are available in GenBank™, EMBL, and DDBJ databases under the accession numbers: AF305957–AF305967.