Analysis of developmentally regulated genes of the parasite Haemonchus contortus

Abstract

Differential regulation of gene expression in the development of Haemonchus contortus was analysed using RNA arbitrarily-primed PCR. A study of third-stage larval and adult H. contortus revealed large differences between the two stages; 32 and 30% unique third-stage larval and adult RNA arbitrarily-primed PCR products, respectively. This finding is consistent with a high degree of differential gene expression between these developmental stages. A number of adult products were sequenced, revealing 11 molecules to be similar to deposits within sequence databases. Four other molecules that did not have significant similarity to sequences in the databases may represent developmentally regulated genes specific to H. contortus. Northern analysis of the putative adult-expressed molecules with homologues in the databases confirmed that four were expressed only in adults, while four were expressed in both stages, but had different sized transcripts. This may reflect differential splicing, or expression of closely related but different molecules at different life cycle stages. Two molecules were present in mRNA populations from both stages, suggesting these were false stage-associated molecules. No transcript was detected for one molecule by Northern analysis, probably due to low level of expression. In situ hybridisation analysis was used to localise expression of transcripts in the adult parasite, in particular, to gain some insight into the nature of those molecules with no known predicted function.

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.