The level of embryonation influences detection of Ostertagia ostertagi eggs by semi-quantitative PCR

Markus Drag; Johan Höglund; Peter Nejsum; Stig Milan Thamsborg; Heidi L. Enemark

doi:10.1186/s13071-016-1657-4

The level of embryonation influences detection of Ostertagia ostertagi eggs by semi-quantitative PCR

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

6 Citations (Scopus)

74 Downloads (Pure)

Abstract

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 °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 (33 vs 266 %). Conclusions: Aerobic storage of O. 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.

Original language	English
Article number	368
Journal	Parasites & Vectors
Volume	9
Number of pages	8
ISSN	1756-3305
DOIs	https://doi.org/10.1186/s13071-016-1657-4
Publication status	Published - 29 Jun 2016

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The level of embryonation influences detection of Ostertagia ostertagi eggs by semi-quantitative PCRFinal published version, 1.24 MB

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title = "The level of embryonation influences detection of Ostertagia ostertagi eggs by semi-quantitative PCR",

abstract = "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 °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 (33 vs 266 %). Conclusions: Aerobic storage of O. 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.",

author = "Markus Drag and Johan H{\"o}glund and Peter Nejsum and Thamsborg, {Stig Milan} and Enemark, {Heidi L.}",

year = "2016",

month = jun,

day = "29",

doi = "10.1186/s13071-016-1657-4",

language = "English",

volume = "9",

journal = "Parasites & Vectors",

issn = "1756-3305",

publisher = "BioMed Central",

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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 °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 (33 vs 266 %). Conclusions: Aerobic storage of O. 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.

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 °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 (33 vs 266 %). Conclusions: Aerobic storage of O. 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.

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

M1 - 368

ER -

The level of embryonation influences detection of Ostertagia ostertagi eggs by semi-quantitative PCR

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