Correlation between real-time qPCR and development of strongyle eggs from cattle

Markus Drag, Peter Nejsum, Johan Höglund, Stig M. Thamsborg, Heidi Enemark

    Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsResearchpeer-review

    Abstract

    Differentiation of veterinary important parasitic strongyle eggs is time-consuming, because morphologically distinct third-stage larvae (L3) must be cultured for species/genus identification. A recently published qPCR technique provides a non-labour intensive method for detection and quantification of the two most important nematode eggs in cattle faeces. However, as quantification correlates with DNA content, quantification of copy numbers of the second internal transcribed spacer (ITS2) region is problematic as DNA content increases during egg development. The aim of this study was to assess the impact of oxygen availability and temperature on the multiplication of ITS2 copy numbers in O. ostertagi eggs. Fresh eggs were recovered from cattle faeces by sieving, flotation and entrapment in nylon-mesh filters and subsequently deposited in aliquots (n=18) of 5 ml distilled water with air circulation. To test the effect of oxygen deprivation, fresh faecal samples (n=18) were vacuum packed. A total of 36 aliquots were stored at temperatures of 4°C or 25°C for up to 336 hours. Morphological changes were observed, and DNA content was measured at nine time points throughout the study period. Preliminary morphological analysis demonstrated developmental progression. In waterdeposited eggs, first-stage larvae (L1) were observed after 336 hours at 4°C and after 24 hours at 25°C. An additional 24 hour study showed formation of L1 already after 12 hours incubation at 25°C. In oxygendeprived eggs, no development was observed neither at 4°C nor at 25°C throughout the 336 hours study. Thus, the importance of oxygen and temperature as regulators of egg development was verified. Ongoing studies will quantify ITS2 copy numbers by qPCR. The results will allow us to correlate observed developmental progression with ITS2 copy numbers, and thereby provide knowledge on optimal storage conditions and sources of errors for data interpretation of this diagnostic molecular technique.
    Original languageEnglish
    Title of host publicationDSP - Joint Spring Symposium 2014 : Parasites in an ever changing world
    Number of pages1
    PublisherDanish Society for Parasitology
    Publication date2014
    Pages32
    Publication statusPublished - 2014
    EventJoint Spring Symposium 2014 : Danish Society for Parasitology and Danish Society for Tropical Medicine & International Health - Frederiksberg, Denmark
    Duration: 28 Mar 2014 → …
    http://parasitology.dk/web/pages/dsp-meetings/spring-symposium.php

    Conference

    ConferenceJoint Spring Symposium 2014 : Danish Society for Parasitology and Danish Society for Tropical Medicine & International Health
    Country/TerritoryDenmark
    CityFrederiksberg
    Period28/03/2014 → …
    Internet address

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