TY - JOUR
T1 - The level of embryonation influences detection of Ostertagia ostertagi eggs by semi-quantitative PCR
AU - Drag, Markus
AU - Höglund, Johan
AU - Nejsum, Peter
AU - Thamsborg, Stig Milan
AU - Enemark, Heidi L.
PY - 2016/6/29
Y1 - 2016/6/29
N2 - Background: The Internal Transcribed Spacer 2 (ITS2) is a candidate diagnostic marker of the pathogenic cattle nematode Ostertagia ostertagi. The aims of this study were: (i) to document and quantify how the development of O. 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 of Ostertagia ostertagi were obtained from fresh faeces and stored at 4 degrees C or 25 degrees 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 degrees C, embryonation and a significant increase of ITS2 copies (P < 0.0001) were observed after 12 h. At 4 degrees 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 (33 vs 266 %). Conclusions: Aerobic storage of O. ostertagi eggs at 25 degrees 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 degrees 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 degrees 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.
AB - Background: The Internal Transcribed Spacer 2 (ITS2) is a candidate diagnostic marker of the pathogenic cattle nematode Ostertagia ostertagi. The aims of this study were: (i) to document and quantify how the development of O. 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 of Ostertagia ostertagi were obtained from fresh faeces and stored at 4 degrees C or 25 degrees 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 degrees C, embryonation and a significant increase of ITS2 copies (P < 0.0001) were observed after 12 h. At 4 degrees 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 (33 vs 266 %). Conclusions: Aerobic storage of O. ostertagi eggs at 25 degrees 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 degrees 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 degrees 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.
KW - Egg development
KW - First-stage larvae
KW - ITS2
KW - Ostertagia ostertagi
KW - Real-time semi-quantitative PCR
UR - http://www.scopus.com/inward/record.url?scp=84976412272&partnerID=8YFLogxK
U2 - 10.1186/s13071-016-1657-4
DO - 10.1186/s13071-016-1657-4
M3 - Journal article
C2 - 27357701
SN - 1756-3305
VL - 9
JO - Parasites & Vectors
JF - Parasites & Vectors
IS - 1
M1 - 368
ER -