The level of embryonation influences detection of Ostertagia ostertagi eggs by semi-quantitative PCR. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Drag, Markus; Höglund, Johan; Nejsum, Peter; Thamsborg, Stig; Enemark, Heidi
• Is Part Of: Parasites & vectors. Volume 9:Issue 1(2016)
• Page Start: 1
• Page End: 8
• Publication Date: 2016-12
• Subjects: Ostertagia ostertagi -- Egg development -- First-stage larvae -- Real-time semi-quantitative PCR -- ITS2; Parasitism -- Periodicals; Parasites -- Periodicals; Vector-pathogen relationships -- Periodicals; Animals as carriers of disease -- Periodicals; Insects as carriers of disease -- Periodicals; 616.96
• DOI: 10.1186/s13071-016-1657-4
• Languages: English
• ISSNs: 1756-3305

Abstract

Abstract Background The Internal Transcribed Spacer 2 (ITS2) is a candidate diagnostic marker of the pathogenic cattle nematodeOstertagia ostertagi . The aims of this study were: (i) to document and quantify how the development ofO. ostertagi eggs affects ITS2 copies under different storage conditions, and (ii) to suggest optimal storage conditions for faecal samples in a diagnostic pipeline that involves detection and semi-quantification by real-time semi-quantitative polymerase chain reaction (qPCR). Findings Eggs ofOstertagia ostertagi were obtained from fresh faeces and stored at 4 °C or 25 °C under aerobic or anaerobic (vacuum packing) conditions. Development was monitored by microscopy for up to 336 h, and the ITS2 copies were determined by qPCR from a fixed number of parasites. Under aerobic conditions at 25 °C, embryonation and a significant increase of ITS2 copies (P < 0.0001) were observed after 12 h. At 4 °C, embryonation occurred after 168 h with a trend towards increased ITS2 copies. Anaerobic conditions inhibited egg development at both temperatures and no significant increase in ITS2 copies was noticed (P = 0.90). ITS2 copies were analysed for each parasite stage: first-stage larvae (L1) exhibited significantly higher copy numbers (20, 353 ± 1, 950) than unembryonated eggs (568 ± 168;P < 0.0001) with lower coefficient of variation (33vs 266 %). Conclusions Aerobic storage ofO. ostertagi eggs at 25 °C led to a significant increase in ITS2 copies after 12 h due to embryonation and subsequent hatching. In contrast, anaerobic storage (vacuum packing) at 25 °C completely inhibited egg development and any undesirable semi-quantification bias for up to 336 h. Hence, vacuum packing is an optimal storage strategy prior to molecular diagnostic analyses. Alternatively, aerobic storage at 4 °C for up to 72 h can be used. Due to high copy numbers and lower genetic variation, the L1 stage may be considered for diagnostics and further molecular research.