Embryonation of Ostertagia ostertagi eggs affects the outcome of real-time quantitative PCR

TY - ABST

T1 - Embryonation of Ostertagia ostertagi eggs affects the outcome of real-time quantitative PCR

AU - Drag, Markus

AU - Höglund, Johan

AU - Nejsum, Peter

AU - Thamsborg, Stig Milan

AU - Enemark, Heidi

PY - 2015

Y1 - 2015

N2 - The aims of this study were to assess how the development of Ostertagia ostertagi eggs into first-stage larva (L1) affects the copy numbers of the Internal Transcribed Spacer region 2 (ITS2) of the ribosomal DNA; and based on these results, to suggest optimal storage conditions for faecal samples prior to detection and quantification by real-time quantitative polymerase chain reaction (qPCR). Fresh O. ostertagi eggs were isolated from cattle faeces and stored at 4°C or 25°C under aerobic or anaerobic conditions. Embryonation was monitored by microscopy and the ITS2 copies were determined by qPCR at predetermined intervals for up to 336 h. Under aerobic conditions, L1 was observed after 24 h at 25°C, while development to L1 took 336 h at 4°C. A corresponding significant increase of the ITS2 copies was also observed (p < 0.0001). However, anaerobic conditions inhibited embryonation at both temperatures and no significant effect of storage on ITS2 copies was noticed (p =0.8984). ITS2 copies were significantly higher in L1 compared with copies in unembryonated eggs (p <0.0001) and with lower coefficients of variation for L1 (33%) compared with unembryonated eggs (266%). In conclusion, storage conditions affect the outcome of qPCR analysis for the quantitative determination of O. ostertagi eggs in cattle faeces. Cold storage at 4°C for up to 3 days or anaerobicvacuum packing at 25°C for up to 336 h will entail no undesirable effects on ITS2 copies.

AB - The aims of this study were to assess how the development of Ostertagia ostertagi eggs into first-stage larva (L1) affects the copy numbers of the Internal Transcribed Spacer region 2 (ITS2) of the ribosomal DNA; and based on these results, to suggest optimal storage conditions for faecal samples prior to detection and quantification by real-time quantitative polymerase chain reaction (qPCR). Fresh O. ostertagi eggs were isolated from cattle faeces and stored at 4°C or 25°C under aerobic or anaerobic conditions. Embryonation was monitored by microscopy and the ITS2 copies were determined by qPCR at predetermined intervals for up to 336 h. Under aerobic conditions, L1 was observed after 24 h at 25°C, while development to L1 took 336 h at 4°C. A corresponding significant increase of the ITS2 copies was also observed (p < 0.0001). However, anaerobic conditions inhibited embryonation at both temperatures and no significant effect of storage on ITS2 copies was noticed (p =0.8984). ITS2 copies were significantly higher in L1 compared with copies in unembryonated eggs (p <0.0001) and with lower coefficients of variation for L1 (33%) compared with unembryonated eggs (266%). In conclusion, storage conditions affect the outcome of qPCR analysis for the quantitative determination of O. ostertagi eggs in cattle faeces. Cold storage at 4°C for up to 3 days or anaerobicvacuum packing at 25°C for up to 336 h will entail no undesirable effects on ITS2 copies.

UR - http://www.waavp2015.com/scientific_programme.php

M3 - Conference abstract for conference

T2 - 25th International Conference of the World Association for the Advancement of Veterinary Parasitology

Y2 - 16 August 2015 through 20 August 2015

ER -