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«Quantification of Cryptosporidium parvum and enteroviruses by quantitative Real-Time PCR (qPCR) in environmental samples - Methodological ...»

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TECHNISCHE UNIVERSITÄT MUNCHEN

Lehrstuhl für Bodenökologie

Quantification of Cryptosporidium parvum and enteroviruses by quantitative

Real-Time PCR (qPCR) in environmental samples

- Methodological developments for monitoring anaerobic systems

Gabriela Garcés Sanchez

Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für

Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. Dr. h.c. J. Bauer

Prüfer der Dissertation:

1. Univ.-Prof. Dr. J. Ch. Munch

2. Univ.-Prof. Dr. H. Horn (Karlsruhe Institute of Technology) Die Dissertation wurde am 15.10.2012 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 06.05.2013 angenommen.

Abstract Manure and wastewater are valuable sources of nutrients that are used in agriculture. Besides the nutrient content and their economic value, these matrices can also be carriers of pathogenic microorganisms and viruses that can affect the health of living beings.

Cryptosporidium strains and enteroviruses are human and animal pathogens that can be present. Both show resistance against various environmental stresses and are very infective and of high significance for public health.

Effective hygiene control of manure and wastewater as well as control of the sanitizing efficiency of treatment systems is only possible when suitable detection methods are available. This work aimed at advancing the application of molecular detection tools based on real-time qPCR and RT-qPCR in this field.

A DNA extraction procedure for Cryptosporidium oocysts in manure and for detection of the hsp70 gene by real-time qPCR was developed and optimized. Emphasis was on the oocyst lysis and nucleic acid extraction steps, as these are of high importance for application with complex matrices and oocysts with strong walls. The DNA based qPCR detection method was evaluated with soil from agricultural land and manure and digested manure from anaerobic fermenters to assess the screening potential for oocysts and the treatment efficiencies.

Furthermore, optimized lysis and extraction procedures for RNA and mRNA were developed and evaluated in RT-qPCR based assays. RNA enteroviruses and mRNA production in oocysts were quantified in complex samples within this context. The optimized methods for C. parvum were applied to different environmental matrices and oocysts with different viability in order to assess their viability by targeting hsp70 mRNA. The procedures were used to evaluate the inactivation and degradation of C. parvum in anaerobic digesters treating manure.

This work shows that lysis by cumulative bead beating for 165 s (performed in time intervals) allows rapid and efficient recovery of nucleic acids from oocysts. The optimized DNA extraction and qPCR detection method can be applied to complex substrates like manure and soil, provides specific detection in short time, and allows the quantification of Cryptosporidium pathogens. The DNA based method applied to manure yielded a detection limit of 2.4 x 102 – 8.3 x 102 oocysts / ml sample with a DNA recovery of 83 %. The analyses of manure and digested manure from anaerobic bioreactors show that the DNA measurement Abstract alone underestimates the reduction of pathogen viability and the sanitizing efficiency of the treatment system.

Measuring the hsp70 mRNA content, however, showed a positive relation with viability. This was found for oocysts subjected to thermophilic anaerobic treatment, as well as for oocysts aged over a period of 12 months. An indication of the viability of Cryptosporidium oocysts contained in environmental substrates can be given by measuring the hsp70 mRNA content.

In addition, the measurement of the hsp70 mRNA production response to a short heat shock trigger was shown to provide an indication of the vitality of oocysts present in a sample, even when this response was small. The production of hsp70 mRNA after heat shock for fully viable and infectious oocysts was increased by a mean factor of 1.6 times. For samples containing a reduced fraction of viable oocysts, the factor was lower than 1.2 fold.

The optimized (m)RNA based methods provided reliable and sensitive detection for manure and digested manure in the RT-qPCR assay. The direct extraction and purification of mRNA from these matrices using cumulative bead beating as lysis step and oligo (dT)25-magnetic beads for mRNA extraction in combination with a specific purification procedure yielded a detection limit of hsp70 mRNA from 5.5 x 103 - 8.1 x 103 viable oocysts / ml sample and occasionally even from a lower oocyst concentration. However, qPCR results obtained for manure could not be reproduced for spiked agricultural soil samples with relatively high clay content.

The optimized DNA and RNA based (RT)-qPCR detection systems allow the quantification of C. parvum oocysts and enteroviruses in complex environmental samples. For enteroviruses in manure, the method extraction and detection efficiency was 37 %.

For sanitizing purposes, thermophilic (anaerobic) treatment at 55°C can ensure the inactivation of oocysts in a short time (1 or 4 h), as seen by infectivity test results. At this temperature and retention times, a reduction in hsp70 mRNA content of ≥ 1.5 log units was obtained. The reduction of mRNA content can therefore be associated to the inactivation of oocysts in hygiene tests of treatment systems, e.g. using sentinel chambers.





The developed molecular tools can be useful for the rapid, sensitive and specific screening of Cryptosporidium and enteroviruses in complex environmental samples in order to identify contaminated sources from which these pathogens can disseminate in the environment.

Zusammenfassung Gülle und Abwasser sind wertvolle Reststoffe mit Nährstoffen, die in der Landwirtschaft Verwendung finden. Beide Substrate sind allerdings auch potentielle Träger von pathogenen Mikroorganismen und Viren, die die Gesundheit von Mensch und Tier beeinträchtigen können. Kryptosporidien und Enteroviren gehören zu diesen Pathogenen. Beide zeigen Resistenz gegen verschiedenste Umwelteinflüsse, sind sehr infektiös und haben eine hohe Relevanz für die öffentliche Gesundheit.

Effektive Hygienekontrolle für Gülle und Abwasser sowie die Steuerung der Hygienisierung von Behandlungssystemen ist nur möglich, wenn geeignete Methoden zum Nachweis von Pathogenen zur Verfügung stehen. Ziel dieser Arbeit war die Untersuchung, Entwicklung und Anwendung von auf Real-Time qPCR und RT-qPCR basierenden molekularen Detektionsmethoden für die gennanten Krankheitserreger.

Ein DNA-Extraktionsverfahren für Cryptosporidium-Oozysten in Gülle und die Detektion des hsp70-Gens durch quantitative Real-Time qPCR wurde entwickelt und optimiert. Ein Schwerpunkt lag auf der Oozysten-Lysis und der Nukleinsäureextraktion, da diese von hoher Bedeutung für die Anwendung in komplexen Matrizen und mit dickwandigen Oozysten sind.

Um das Potenzial für den Einsatz im Screening nach Oozysten und die Untersuchung der Effizienz von Behandlungssystemen zu beurteilen, wurde die DNA-basierte qPCRNachweismethode anhand von Bodenproben aus landwirtschaftlichen Flächen, Gülle und Gärresten aus der anaeroben Behandlung evaluiert.

Weiterhin wurden optimierte Lysis- und Extraktionsverfahren für RNA und mRNA entwickelt und mit RT-qPCR-basierten Methoden evaluiert. In diesem Zusammenhang wurden RNA-Enteroviren und mRNA-Produktion in Oozysten in komplexen Proben quantifiziert. Die optimierten Methoden für C. parvum wurden auf verschiedene Umweltproben und Oozysten unterschiedlichen Alters mit dem Ziel angewendet, die Lebensfähigkeit der Pathogene mittels RT-qPCR mit hsp70-mRNA als Zielmolekül zu beurteilen. Die Verfahren wurden verwendet, um die Inaktivierung von C. parvum während der anaeroben Behandlung von Gülle auszuwerten.

Diese Arbeit zeigt, dass die Lysis durch kumulatives Bead-Beating für 165 s (durchgeführt in Zeitintervallen) eine schnelle und effiziente Extraktion von Nukleinsäuren aus Oozysten ermöglicht. Die optimierte DNA-Extraktion und qPCR-Nachweismethode kann auf komplexe Substrate wie Gülle und Boden angewendet werden. Sie bietet einen spezifischen Nachweis in Zusammenfasung kurzer Zeit und ermöglicht die Quantifizierung von Cryptosporidium-Pathogenen. Die Nachweisgrenze der DNA-basierten Methode für Gülle lag bei 2,4 x 102 – 8,3 x102 Oozysten / ml Probe mit einer DNA-Wiederfindungsrate von 83 %. Die Analysen der Proben aus den anaeroben Bioreaktoren haben gezeigt, dass die DNA-Messung keine direkte Beurteilung der Verringerung der Erreger und der Hygienizierungseffizienz des Behandlungsverfahrens erlaubt.

Die gemessene hsp70-mRNA-Produktion zeigte andererseits eine positive Korrelation mit der Lebensfähigkeit. Dies ergab die Untersuchung von Oozysten die einer thermophilen anaeroben Behandlung unterzogen worden sind und von Oozysten die über 12 Monate gelagert wurden. Eine Indikation für die Lebensfähigkeit von Cryptosporidium-Oozysten in Umweltproben kann durch die Messung des hsp70-mRNA-Gehaltes gegeben werden.

Darüber hinaus wurde gezeigt, dass die Messung der hsp70-mRNA-Produktion als Reaktion auf einen induzierten Hitzeschock ebenfalls die Vitalität der Oozysten indiziert, auch wenn diese Reaktion relativ gering war. Die hsp70-mRNA-Produktion für voll lebensfähige und infektiöse Oozystem erhöhte sich nach dem induzierten Hitzeschock um einen mittleren Faktor von 1,6. Für Proben, die einen reduzierten Anteil von lebensfähigen Oozysten enthielten, war der Faktor kleiner als 1,2.

Die optimierten (m)RNA-basierten Methoden erlauben einen zuverlässigen und sensitiven RT-qPCR-basierten Nachweis in Gülle und Gärresten. Für die Methode mit direkter mRNAExtraktion unter Verwendung des kumulativen Bead-Beating für den Lysis-Schritt und mit Oligo(dT)25-magnetischen Partikeln in Verbindung mit den speziellen Reinigungsverfahren für die Extraktion ergab sich für die mRNA eine Detektionsgrenze von 5,5 x 103 bis 8,1 x 103 vitalen Oozysten / ml Probe. Gelegentlich konnten sogar noch niedrigere OozystenKonzentration nachgewiesen werden.

Für mit Oozysten dotierte landwirtschaftliche Bodenproben mit relativ hohem Tongehalt konnten die (m)RNA-basierten Methode jedoch nicht zur quantitativen Bestimmung herangezogen werden, was auf eine verstärkte Inhibition zurückgeführt wurde.

Die optimierten DNA- und RNA-basierten (RT)-qPCR-Nachweismethoden erlauben die Quantifizierung von C. Parvum-Oozysten und Enteroviren in umweltrelevanten Matrizen. Für Enteroviren in Gülle lag die Extraktions- und Detektions-Effizienz bei 37%.

–  –  –

Reduktion der hsp70-mRNA-Gehalte von ≥ 1,5 log-Stufen nachgewiesen werden. Die Messung der mRNA-Reduktion, z.B. unter Verwendung von Diffusions-Keimträgern, kann damit die Inaktivierung der Oozysten bei Hygienetests von Behandlungssystemen nachweisen.

Die entwickelten molekularbiologischen Methoden erlauben ein schnelles, sensitives und spezifisches Screening nach Cryptosporidium-Oozysten und Enteroviren in komplexen Umweltproben. Sie können mithelfen, Kontaminationen zu identifizieren und analysieren, von denen aus sich diese Erreger in der Umwelt verbreiten können.

Acknowledgements This work was initiated in the frame of an inter-institutional project at the Institute of Water Quality Control, Technische Universität München.

I would like to express my profound gratitude to my advisor, Prof. Jean Charles Munch, Director of the Institute of Soil Ecology, Helmholtz Zentrum München, for his continuous support, positiveness and advises. His support has been invaluable during this project, and it would not have been possible to complete it without it. Thank you for the valuable discussions.

It has been an honour to me. I am very thankful for your encouragements, especially in the difficult times.

My special gratitude is also to Prof. Harald Horn from the Karlsruhe Institute of Technology, Germany, for his co-advise and support. I thank him very much for the opportunity he gave me to conduct this research at the Institute of Water Quality Control, while he was chairing the institute. Thank you for your support in completion of this work, the open attitude and the helpful discussions.

I would like to thank deeply my project advisor Dr. Michael Lebuhn, from the Bayerische Landesanstalt fur Landwirtschaft, LfL, Germany, for the very valuable talks about this research, for his suggestions, and for critical and positive comments that helped to shape this work. Many thanks for the strong encouragement at every stage! Working with him and in his team, has been a great experience. Thank you for your friendship.

My gratitude I also would like to express to Prof. Peter A.Wilderer, Institute of Water Quality Control, for his motivating words and the inspiration that he gave me to work on topics related to waste and wastewater management. Thank you for the great opportunity that you gave me to learn and work in this field in Munich.

It is specially acknowledged the scholarships and financial support from: the TUM HWPProgram "Chancengleichheit für Frauen in Forschung und Lehre"; the Institute for Advanced Studies of Sustainability, Munich; and the STIBET-Program from the TUM Graduate School that supported this work.

Ms. Petra Ritter from the International office, TUM: thank you for your support at the times it was needed.

I thank my colleagues and friends Dr. Christoph Bauer and Dr. Mathias Effenberger from the LfL, for their comments and review of parts of this thesis. Besides, it has been always fun the moments we shared. Mathias, it was an interesting and positive time we had working with the model biogas plant in Garching.

I would like to thank Dr. Michael Najdrowski, formerly at the Universität Leipzig, for his technical comments and contribution to this work, and for his friendship and humour. I was always happy to receive the post with the little but hazardous living samples.

My thanks also to Dr. Martin Haslbeck, Institute of Biotechnology, TUM, for providing facilities and samples for experiments at his laboratory.



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