Submitted to: American Association of Veterinary Parasitologists
Publication Type: Book / Chapter
Publication Acceptance Date: July 12, 2004
Publication Date: August 10, 2004
Citation: Zarlenga, D.S., Gasbarre, L.C. 2004. Identification and characterization of genes differentially expressed in ostertagia ostertagi fourth stage larvae using subtraction cloning and comparative hybridization [abstract]. American Association of Veterinary Parasitologists. Interpretive Summary: Gastrointestinal (GI) nematode infections cause major losses to the American cattle industry due to decreased productivity of infected animals. The goal of effective nematode control is to protect cattle from production losses by reducing parasite transmission and the rate of parasite establishment in the host. Without increased knowledge of the fundamental interactions between parasites and host immune system components, and those factors which define parasitism within the organism, immunity-based control programs cannot be developed. To this end, a cDNA subtraction library was constructed and differentially screened to identify genes specifically transcribed in the most pathogenic, L4 stage, of Ostertagia. Numerous sequences were identified and partially characterized that were L4 specific. This approach has demonstrated newly developed technology for library construction, and will now allow our research to focus on parasite genes and gene products responsible for the organism escaping the protective responses of the host.
Technical Abstract: The nematode, Ostertagia ostertagi, is the predominant cause of parasite-induced production losses in cattle throughout temperate regions of the world. These losses result primarily from damage caused by fourth stage larvae (L4) present in the gastric glands. Although the parasite is a potent stimulator of the immune system, within the local milieu it has the ability to control, impede and/or evade development of the protective response. Thus, delineating genes specifically expressed in the L4 is tantamount to understanding the mechanism by which O. ostertagi eludes immune intervention by the host upon reinfection. To this end, we developed new methodology to generate a PCR-derived, L4-specific cDNA subtraction library using as little as 50 ng of both tester and driver total RNAs. Upon differentially screening as few as 400 clones with radiolabeled cDNA from L3, L4 or adult parasites, at least 10% of the randomly chosen clones contained gene sequences that were substantially up-regulated during L4 development. Among the sequences examined, a cDNA encoding a cuticle protein was identified that is exclusively and abundantly expressed in L4. Additionally, 1% of the chosen sequences were found to be down-regulated in the L4 relative to the other stages analyzed. Within this group, a putative apyrase (ATP-diphosphohydrolase) sequence was discovered that is predominantly transcribed in L3, and has been documented in other systems for its ability to abrogate platelet aggregation. From preliminary data, we can conclude that the cloning procedure presented here is an effective method for enriching in differentially expressed sequences, and has substantially enhanced our ability to identify and characterize a subset of genes involved in the parasite infection process and/or evasion of host immunity.