Thesis - Open Access
Master of Science (MS)
Biology and Microbiology
Michael B. Hildreth
Six primer pairs, each with a pair of genus-specific and universal second internal transcribed spacer (ITS2) primers were evaluated to amplify and sequence the ITS2 region of nuclear ribosomal DNA to identify the species and genotypes of Haemonchus from livestock fecal samples containing mixtures of trichostrongyle nematode eggs. Based upon the use of universal ITS2 primers on Haemonchus adults, the ITS2 region has been shown to contain three fixed single-nucleotide differences between H. placei and H. contortus. It is clinically useful to identify the species and genotypes of Haemonchus infecting livestock, but it has not been possible in fecal samples containing trichostrongyle eggs from multiple species. Three primers came from publications showing genus-level specificity for Haemonchus. Two primers were published universal ITS2 primers. A genus specific and a universal primer was developed based upon analyses of sequences available on the National Center for Biotechnology Information website. PCR products from these primers amplifying individual worm DNA were sent to GenScript USA Inc. for Sanger sequencing, and then compared with egg DNA recovered from mixed infections. Comparisons included sequence quality values (QV) coupled with analyses of their ability to differentiate H. placei from H. contortus. A set composed of a forward universal primer in the 5.8S subunit gene coupled with a genus-specific reverse primer (at the ITS2 3' end, extending into the 28S gene) provided excellent QV values over most of the ITS2 region except for the 3' end. Two other primer sets provide good QV values in the 3' half of ITS2. These whole-worm DNA results remained consistent, and so did egg DNA from mixed infections for most samples except one fecal egg sample from one ewe. Two egg DNA samples from a cattle herd and two egg DNA and one adult worm DNA from a sheep flock were identified as H. placei and H. contortus, respectively. Three distinct genotypes were identified based on nucleotide diversity. Two of them are already reported and one is unique but needs further study to confirm the finding.
Includes bibliographical references
Number of Pages
South Dakota State University
Khanal, Pratiksha, "A PCR/Sequencing Technique to Identify Species and Genotypes of Haemonchus from Mixed Trichostrongyle Egg DNA Recovered from Livestock Fecal Samples" (2018). Electronic Theses and Dissertations. 2636.