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Title: IMPROVED METHODS FOR ISOLATING DNA FROM OSTERTAGIA OSTERTAGI EGGS IN CATTLE FECES

Author
item HARMON, AARON - SOUTH DAKOTA STATE UNIV
item Zarlenga, Dante
item HILDRETH, MICHAEL - SOUTH DAKOTA STATE UNIV

Submitted to: Veterinary Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/21/2005
Publication Date: 2/18/2006
Citation: Harmon, A.F., Zarlenga, D.S., Hildreth, M.B. 2006. Improved methods for isolating dna from ostertagia ostertagi eggs in cattle. Veterinary Parasitology. 135:297-302.

Interpretive Summary: 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. Accurate and rapid identification of species infecting the host is essential to achieving these goals. We previously developed a rapid and sensitive DNA test to diagnose gastrointestinal (GI) nematode eggs from cattle feces; however, methods for increasing the yield, purity and speed of DNA isolation from eggs must be improved to optimize genetic identification. Herein we evaluate a matrix of isolation and purification methods to ultimately enhance the sensitivity of the identification test, and develop a set of recommendations to follow based upon the needs of the user. These recommendations along with the PCR test will allow more selective drug treatment of animals, reduce the potential of drug resistance developing in parasites, reduce the potential for harmful drug residues to collect in animals and reduce costs to the producer groups by identifying and treating only those animals harboring pathogenic parasites. Application of these methods extends well beyond use with GI nematodes of cattle.

Technical Abstract: A multiplex PCR assay for differentiating strongyle eggs from cattle has recently been described; however, the egg disruption and DNA extraction procedures, though effective, are inadequate for large studies or clinical application. The purpose of this research was to evaluate methods for disrupting trichostrongyle eggs, and then assess commercial kits for extracting egg DNA using Ostertagia ostertagi as a model species. Egg disruption procedures tested included probe sonication, bath sonication, bead beating, boiling, microwaving, proteinase K/SDS digestion, freezing, and various combinations of the above with the incorporation of sodium dodecyl sulfate. These procedures were evaluated in conjunction with four commercial DNA extraction kits: DNA Stool mini kit and DNeasy Plant kit (Qiagen), Fast DNA kit (QBiogene), and the MAP extraction kit (Tetracore). Results showed that egg disruption was best accomplished with the bead beater and ceramic beads, resulting in 100% disruption within 1 min. When DNA extraction was preceded by the isolation of eggs from feces, all procedures except the Fast DNA kit produced PCR-ready DNA from at least two eggs. The DNeasy Plant kit allowed consistent detection of DNA released from one egg. Due to the morphological similarities among trichostrongyle eggs in ruminants, strongyle eggs in equids, and hookworm eggs, the methods described herein may have broad application to other nematodes.