title: The level of embryonation influences detection of Ostertagia ostertagi eggs by semi-quantitative PCR.
abstract: 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).
  
  
  
  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 %).
  
  
  
  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.
authors.affiliation: Section for Parasitology and Aquatic Diseases, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark. markus.drag@sund.ku.dk.|Section for Bacteriology, Pathology and Parasitology, National Veterinary Institute, Technical University of Denmark, Frederiksberg C, Denmark. markus.drag@sund.ku.dk.
authors.forename: Markus
authors.identifier: 0000-0002-7412-6402
authors.initials: M
authors.lastname: Drag
authors.affiliation: Department of Biomedical Sciences and Veterinary Public Health, Section for Parasitology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Höglund
authors.affiliation: Section for Parasitology and Aquatic Diseases, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Nejsum
authors.affiliation: Section for Parasitology and Aquatic Diseases, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.
authors.forename: Stig M
authors.initials: SM
authors.lastname: Thamsborg
authors.affiliation: Section for Bacteriology, Pathology and Parasitology, National Veterinary Institute, Technical University of Denmark, Frederiksberg C, Denmark.|Norwegian Veterinary Institute, PO Box 750 Sentrum, Oslo, N-0106, Norway.
authors.forename: Heidi L
authors.initials: HL
authors.lastname: Enemark
mesh_terms: D000818:Animals; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D010028:Ostertagia / Q000196:embryology*; D010063:Ovum / Q000502:physiology*; D010100:Oxygen / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D013048:Specimen Handling
chemical_list: D021903:DNA, Ribosomal Spacer; D010100:Oxygen
keywords: Egg development; First-stage larvae; ITS2; Ostertagia ostertagi; Real-time semi-quantitative PCR