Correlation between real-time qPCR and development of strongyle eggs from cattle

TY - ABST

T1 - Correlation between real-time qPCR and development of strongyle eggs from cattle

AU - Drag, Markus

AU - Nejsum, Peter

AU - Höglund, Johan

AU - Thamsborg, Stig Milan

AU - Enemark, Heidi

PY - 2014

Y1 - 2014

N2 - Differentiation of veterinary important parasitic strongyle eggs is time-consuming, because morphologically distinct third-stage larvae (L3) must be cultured for species/genus identification. A recently published qPCR technique provides a non-labour intensive method for detection and quantification of the two most important nematode eggs in cattle faeces. However, as quantification correlates with DNA content, quantification of copy numbers of the second internal transcribed spacer (ITS2) region is problematic as DNA content increases during egg development. The aim of this study was to assess the impact of oxygen availability and temperature on the multiplication of ITS2 copy numbers in O. ostertagi eggs. Fresh eggs were recovered from cattle faeces by sieving, flotation and entrapment in nylon-mesh filters and subsequently deposited in aliquots (n=18) of 5 ml distilled water with air circulation. To test the effect of oxygen deprivation, fresh faecal samples (n=18) were vacuum packed. A total of 36 aliquotswere stored at temperatures of 4°C or 25°C for up to 336 hours. Morphological changes were observed, and DNA content was measured at nine time points throughout the study period. Preliminary morphological analysis demonstrated developmental progression. In waterdeposited eggs, first-stage larvae (L1) were observed after 336 hours at 4°C and after 24 hours at 25°C. An additional 24 hour study showed formation of L1 already after 12 hours incubation at 25°C. In oxygendeprived eggs, no development was observed neither at 4°C nor at 25°C throughout the 336 hours study. Thus, the importance of oxygen and temperature as regulators of egg development was verified. Ongoing studies will quantify ITS2 copy numbers by qPCR. The results will allow us to correlate observed developmental progression with ITS2 copy numbers, and thereby provide knowledge on optimal storage conditions and sources of errors for data interpretation of this diagnostic molecular technique.

AB - Differentiation of veterinary important parasitic strongyle eggs is time-consuming, because morphologically distinct third-stage larvae (L3) must be cultured for species/genus identification. A recently published qPCR technique provides a non-labour intensive method for detection and quantification of the two most important nematode eggs in cattle faeces. However, as quantification correlates with DNA content, quantification of copy numbers of the second internal transcribed spacer (ITS2) region is problematic as DNA content increases during egg development. The aim of this study was to assess the impact of oxygen availability and temperature on the multiplication of ITS2 copy numbers in O. ostertagi eggs. Fresh eggs were recovered from cattle faeces by sieving, flotation and entrapment in nylon-mesh filters and subsequently deposited in aliquots (n=18) of 5 ml distilled water with air circulation. To test the effect of oxygen deprivation, fresh faecal samples (n=18) were vacuum packed. A total of 36 aliquotswere stored at temperatures of 4°C or 25°C for up to 336 hours. Morphological changes were observed, and DNA content was measured at nine time points throughout the study period. Preliminary morphological analysis demonstrated developmental progression. In waterdeposited eggs, first-stage larvae (L1) were observed after 336 hours at 4°C and after 24 hours at 25°C. An additional 24 hour study showed formation of L1 already after 12 hours incubation at 25°C. In oxygendeprived eggs, no development was observed neither at 4°C nor at 25°C throughout the 336 hours study. Thus, the importance of oxygen and temperature as regulators of egg development was verified. Ongoing studies will quantify ITS2 copy numbers by qPCR. The results will allow us to correlate observed developmental progression with ITS2 copy numbers, and thereby provide knowledge on optimal storage conditions and sources of errors for data interpretation of this diagnostic molecular technique.

KW - Cattle

KW - Parasitology

KW - Molecular diagnostics

KW - Diagnostics

KW - Nematodes

M3 - Conference abstract in proceedings

SP - 32

BT - Parasites in an ever changing world

T2 - DSP Spring Symposium 2014

Y2 - 28 March 2014

ER -