<abstract xmlns="http://www.w3.org/1999/xhtml"><p> Introduction: Chlamydia psittaci is a gram-negative obligate intracellular pathogen of birds. Poultry infections lead to economic losses and can be transmitted to humans. No vaccine is available and the bacterium-host cell interaction is not completely understood. Replicating bacteria cause pneumonia, but C. psittaci can also be non-replicating and persistent inside the cytoplasm of avian cells. RT-qPCR provides insight into the molecular pathogenesis of both active replicating and persistent Chlamydia psittaci in birds, but requires identification of stably expressed reference genes to avoid biases. Material and Methods: We investigated the expression stability of 10 C. psittaci candidate reference genes for gene expression analysis during normal growth and penicillin-induced persistence. C. psittaci Cal10 was cultured in HeLa229 and RNA was extracted. The expression level of each candidate was examined by RT-qPCR and Cq values were analysed using geNorm. Results: The genes tyrS, gidA, radA, and 16S rRNA ranked among the most stably expressed. The final selected reference genes differed according to the bacterial growth status (normal growth versus persistent status), and the time points selected during the duration of the normal chlamydial developmental cycle. Conclusion: The study data show the importance of systematic validation of reference genes to confirm their stability within the strains and under the conditions selected.</p></abstract>

Introduction: Chlamydia psittaci is a gram-negative obligate intracellular pathogen of birds. Poultry infections lead to economic losses and can be transmitted to humans. No vaccine is available and the bacterium-host cell interaction is not completely understood. Replicating bacteria cause pneumonia, but C. psittaci can also be non-replicating and persistent inside the cytoplasm of avian cells. RT-qPCR provides insight into the molecular pathogenesis of both active replicating and persistent Chlamydia psittaci in birds, but requires identification of stably expressed reference genes to avoid biases. Material and Methods: We investigated the expression stability of 10 C. psittaci candidate reference genes for gene expression analysis during normal growth and penicillin-induced persistence. C. psittaci Cal10 was cultured in HeLa229 and RNA was extracted. The expression level of each candidate was examined by RT-qPCR and Cq values were analysed using geNorm. Results: The genes tyrS, gidA, radA, and 16S rRNA ranked among the most stably expressed. The final selected reference genes differed according to the bacterial growth status (normal growth versus persistent status), and the time points selected during the duration of the normal chlamydial developmental cycle. Conclusion: The study data show the importance of systematic validation of reference genes to confirm their stability within the strains and under the conditions selected.

Chlamydia psittaci reference genes for normalisation of expression data differ depending on the culture conditions and selected time points during the chlamydial replication cycle

Department of Animal Production, Faculty of Bioscience Engineering, Ghent University, 9000 Ghent,

Journal of Veterinary Research

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Introduction: Chlamydia psittaci is a gram-negative obligate intracellular pathogen of birds. Poultry infections lead to economic losses and can be transmitted...

Chlamydia psittaci reference genes for normalisation of expression data differ depending on the culture conditions and selected time points during the chlamydial replication cycle

Published Online: Dec 17, 2016

Page range: 403 - 409

Received: Jan 22, 2016

Accepted: Nov 07, 2016

DOI: https://doi.org/10.1515/jvetres-2016-0060

Keywords
birds, Chlamydia psittaci, RT-qPCR, normalisation, references genes

© 2016

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Chlamydia psittaci reference genes for normalisation of expression data differ depending on the culture conditions and selected time points during the chlamydial replication cycle

Published Online: Dec 17, 2016

Page range: 403 - 409

Received: Jan 22, 2016

Accepted: Nov 07, 2016

DOI: https://doi.org/10.1515/jvetres-2016-0060

Keywordsbirds, Chlamydia psittaci, RT-qPCR, normalisation, references genes

© 2016

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Keywords
birds, Chlamydia psittaci, RT-qPCR, normalisation, references genes