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«Degradation of Prion Protein by the Gastrointestinal Microbiota of Cattle CHRISTINA SCHERBEL Vollständiger Abdruck der von der Fakultät ...»

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ABTEILUNG MIKROBIOLOGIE

ZENTRALINSTITUT FÜR ERNÄHRUNGS- UND LEBENSMITTELFORSCHUNG

WEIHENSTEPHAN

TECHNISCHE UNIVERSITÄT MÜNCHEN

angefertigt am

Institut für Mikrobiologie und Toxikologie

Bundesforschungsanstalt für Ernährung und Lebensmittel Standort Kulmbach Degradation of Prion Protein by the Gastrointestinal Microbiota of Cattle

CHRISTINA SCHERBEL

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 (Dr. rer. nat.) genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr.med.vet. Dr.med.vet.habil. Dr.h.c. (Univ.

Kaposvár, Ungarn) Johann Bauer Prüfer der Dissertation: 1. Univ.-Prof. Dr.rer.nat.habil. Siegfried Scherer

2. Univ.-Prof. Dr.med.vet. Dr.med.vet.habil. Erwin Peter Märtlbauer (Ludwig-Maximilians-Universität München)

3. Priv.-Doz. Dr.med.vet. Dr.med.vet.habil. Manfred Gareis (Ludwig-Maximilians-Universität München) Die Dissertation wurde am 25.01.2007 bei der Technischen Universität München eingereicht und durch die Fakultät für Wissenschaftszentrum Weihenstephan am 08.05.2007 angenommen.

Contents I Contents I List of abbreviations IV Summary VI Zusammenfassung VII

1. INTRODUCTION 1 1.1 Transmissible spongiform encephalopathies (TSE) 1 1.1.1 Definition 1 1.1.2 Human TSE diseases 1 1.1.3 Animal TSE diseases 3 1.1.3.1 Bovine spongiform encephalopathy (BSE) 3 1.1.3.2 Scrapie 4 1.1.3.3 Chronic wasting disease (CWD) 4 1.1.3.4 Other animal TSE diseases 4 1.2 Nature of the infectious agent 5 1.2.1 Viral hypothesis 5 1.2.2 Prion hypothesis 6 1.3 Prion protein (PrP) 6 1.3.1 Function of prion protein (PrP) 6

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Summary The influence of complex microflora residing in the gastrointestinal tract of cattle on prion protein plays a crucial role with respect to early TSE pathogenesis and the potential infectivity of faeces resulting in environmental contamination. However, it is unknown whether infectious prion proteins (PrPSc), considered to be very stable, are inactivated by microbial processes in the gastrointestinal tract of animals. Feedstuffs consumed by ruminants are initially exposed to microbial fermentation in the rumen prior to gastric and intestinal digestion. Particularly, the polygastric digestion of ruminants represents an efficient system to degrade food proteins by microbial fermentation processes in rumen and colon.

In this study, rumen and colon contents from healthy cattle, taken immediately after slaughter, were used to assess the ability of these microbial consortia to inactivate PrPSc. For that purpose, the consortia were incubated with brain homogenates of scrapie (strain 263K) infected hamsters and BSE infected cattle, respectively.

Biochemical analyses indicate the ability of complex ruminal and colonic microbiota of cattle to decrease scrapie associated prion protein up to immunochemically undetectable levels in Western blot under physiological conditions. In contrast, incubation with BSE associated prion protein did not result in degradation. This implicates a greater stability of BSE associated prion protein towards microbial degradation processes in the gastrointestinal tract.

In vivo hamster bioassays were performed with degraded samples of scrapie brain homogenates in order to prove the concomitance of the loss of anti-prion antibody 3F4 immunoreactivity and the inactivation of PrPSc. The results demonstrated significant prion infectivity after degradation of infected hamster brain through the gastrointestinal microflora of cattle.

Thus, infectivity is still present, even in the absence of Western blot signals. This might be caused by PrPSc at levels below the threshold of immunochemical detection, or by a subfraction of infectious prion protein not detectable by immunochemical methods. Finally, the possibility of present infectious molecules or structures other than PrPSc must be considered.

Conclusively, these data highlight the deficiency of using Western blot or immunoassay formats in TSE inactivation assessment studies, and raise the possibility that the environment might be contaminated through cattle shedding infected faeces.

Zusammenfassung VII

Zusammenfassung

Ziel der Arbeit war es, die Stabilität von Prion-Proteinen (PrPSc) im Gastrointestinaltrakt von Rindern zu untersuchen, um Aussagen zur Verbreitung und Ausscheidung von TSE-Erregern treffen zu können. Bislang ist nicht bekannt, ob infektiöse Prion-Proteine während der Verdauung durch mikrobielle Prozesse abgebaut und inaktiviert werden. In der Regel werden Proteine aus Futtermitteln im polygastrischen Verdauungssystem der Wiederkäuer nahezu vollständig verdaut. Während 70-90 % der Proteine im Pansen vorwiegend durch Bakterien abgebaut werden, erfolgt ein weiterer Protein-Abbau durch proteolytische Bakterien der Mikroflora im Colon. Um zu überprüfen, ob dies auch auf die Struktur des Prion-Proteins zutrifft, wurde die komplexe Mikroflora des bovinen Gastrointestinaltraktes auf die Fähigkeit des PrPSc-Abbaus getestet.





Hierfür wurden Inkubationsversuche mit den komplexen Pansen- bzw. Coloninhalten von Mastbullen und Scrapie-infizierten Hamsterhirnhomogenaten (Stamm 263K) bzw. BSEinfizierten Rinderhirnhomogenaten durchgeführt.

Dabei konnte Scrapie-assoziiertes PrPSc nach einer Inkubation von bis zu 40 Stunden sowohl mit Pansen- als auch mit Coloninhalt im Western Blot immunochemisch nicht mehr nachgewiesen werden, während BSE-assoziiertes PrPSc unter identischen Bedingungen nicht abgebaut werden konnte. Um Aussagen über eine Inaktivierung von PrPSc durch die bovine Gastrointestinalflora treffen zu können, wurden Tierversuche durchgeführt und die Infektiosität in den Ansätzen nach der Degradation von PrPSc bestimmt. Obwohl PrPSc im Western Blot immunochemisch nicht detektierbar war, konnte im Bioassay dennoch signifikante Prioninfektiosität nachgewiesen werden. Folglich korrelieren die Resultate der biochemischen Analyse nicht mit denen des Bioassays.

Dies wäre durch eine fehlende Sensitivität der immunochemischen Nachweismethode erklärbar, mit der Folge, dass gewisse Mengen an PrPSc unterhalb der Nachweisgrenze des Western Blots bzw. eine nicht detektierbare Subfraktion von PrPSc die Infektiosität verursachen. Darüber hinaus könnten andere infektiöse Moleküle und Strukturen unabhängig von PrPSc vorhanden sein. Letztendlich kann eine Kontamination der Umwelt durch das Ausscheiden von infektiösen Faeces nicht ausgeschlossen werden. Des Weiteren zeigen diese Daten den Bedarf an alternativen Nachweismethoden bezüglich der Diagnose von TSEErkrankungen.

Introduction 1

1. Introduction

The present work was part of the project “Occurrence and Stability of the BSE Agent in Foodstuff (primarily in Milk and Milk Products) and in the Environment” (Grant No. 1205 TG 81 LMU 19a) within the Bavarian Research Cooperation FORPRION. After the occurrence of the first cases of BSE in Bavaria the Bavarian Government decided to fight prion diseases. At the beginning of the year 2001 a research initiative was started, called the "Bavarian Research Cooperation Prions (FORPRION)". This research consortium was granted by the Ministry of Science, Research and Art and the Ministry of Health, Food and Consumer Protection. Through basic and applied research the consortium aims to make progress in the diagnosis and therapy of human and animal prion diseases, as well as in the field of preventive consumer protection.

1.1 Transmissible spongiform encephalopathies (TSE)

1.1.1 Definition Transmissible spongiform encephalopathies (TSE) or prion diseases are a group of fatal neurodegenerative disorders of humans and animals, which always lead to death. Incubation periods prior to clinical symptoms range from months to years. So far, there is no prophylaxis or therapy for any form of TSE. These diseases are experimentally and naturally transmissible to individuals of the same or other species. They are usually characterized by spongiform degeneration of the brain accompanied by the deposition of amyloid plaques consisting of abnormal protease resistant prion protein (PrP) (Soto, 2006 in Prions: The new biology of proteins).

–  –  –

association with a mutant PrP allele or evidence for exposure to TSE agent (Harries-Jones et al., 1988). The transmitted and iatrogenic forms of humans TSE are represented by kuru, iatrogenic CJD and variant CJD (vCJD). Kuru occurred among the aborigines in Papua New Guinea throughout the 1950s and 1960s. Ritual acts of mortuary cannibalism seemed to be responsible for epidemic transmission (Gajdusek, 1977). Iatrogenic CJD has been induced by transplantation of infected tissues, administration of pituitary hormones derived from deceased individuals suffering from unrecognized TSEs, or by neurosurgery using instruments incompletely sterilized following use from TSE patients (Brown et al., 1992).

In 1996, vCJD was described, which is now believed to be a zoonotic disease of bovine spongiform encephalopathy (BSE) agent (Will et al., 1996). The unusual young age range of these patients and their distinctive pathology implies a new clinical form of TSE disease.

Biochemical and histopathological evidence suggests that vCJD represents transmission of BSE prions to humans (Bruce et al., 1997; Hill et al., 1997). Since there is no association of occupational exposure of vCJD patients to cattle on farms or in abattoirs, spread may have occurred through consumption of BSE-contaminated meat products. As of 2006, 164 cases of

vCJD have been reported, mostly from UK (EUROCJD data, 31.10.2006:

http://www.eurocjd.ed.ac.uk/vcjdworldeuro.htm). The incidence of vCJD in humans is low and appears to be stabilizing or even falling since 2003 (Figure 1).

–  –  –

A large proportion of the British population may have been exposed to BSE infection.

Furthermore, animal experiments indicate that the infectious dose (ID50) for oral crossspecies transmission of BSE is as low as 500 mg of brain tissue (Foster et al., 1996).

Considering that only approximately 160 humans have contracted vCJD, it is likely that vCJD susceptibility is controlled by endogenous and/or exogenous factors other than the amount of ingested infectious agent (Bons et al., 1999).

1.1.3 Animal TSE diseases

1.1.3.1 Bovine spongiform encephalopathy (BSE) In 1986, bovine spongiform encephalopathy (BSE) was diagnosed and described in UK for the first time (Wilesmith et al., 1988). Thus far, more than 190 000 cases of BSE have been reported world wide. About 97 % of the BSE cases have been confirmed in UK, while 404 cases have been approved in Germany (OIE, 18.12.2006). The OIE report summarizes all cases of BSE positive tested animals regardless of clinical signs (http://www.oie.int/eng/info/en_esb.htm).

BSE is a massive common-source epidemic caused by contaminated meat and bone meal (MBM) fed primarily to dairy cows (Nathanson et al., 1997). The MBM was prepared from the offal of sheep, cattle, pigs, and chickens as a high-protein nutritional supplement. It is assumed that a change in rendering process in the late 1970s allowed prions to survive rendering and to be passed into cattle (Morgan, 1988). However, it remains unclear whether BSE originated by adaptation from a scrapie strain of sheep or from an unrecognised bovine TSE case. The disease has a long incubation period of 4-5 years and it is fatal for cattle within weeks to months of its onset.

Measures for preventing human exposure have been identified and enforced. They include the ban on meat and bone meal in animal feed in 1994, testing of slaughtered animals since 2001, systemic removal of "high-risk material" from carcasses since 2000 and destruction of suspected and confirmed bovine cases as well as the control of animals potentially exposed at the same time. The peak of the BSE epidemic in cattle occurred in 1992-1993, and the incidence has declined dramatically since regulations have been established preventing feeding of ruminant MBM (Figure 1).

Introduction 4 1.1.3.2 Scrapie Scrapie, a fatal, degenerative disease affecting the central nervous system of sheep and goats, has been concerned since the 19th century (Parry, 1962). Signs of scrapie vary among individual animals and develop very slowly. Due to damage to nerve cells, affected animals usually show behavioural changes, tremor, rubbing, and motoric incoordination that progress to death. A crucial breakthrough was achieved in the 1930s by the experimental transmission of scrapie to goats. Subsequently, the scrapie agent has been transmitted to hamsters, mice, rats, mink, guinea pigs and some species of monkeys by inoculation (Barlow and Rennie, 1976; Crozet et al., 2001; Gibbs, Jr. and Gajdusek, 1972; Hanson et al., 1971). The scrapie agent is thought to be spread most commonly from the ewe to her offspring and to other lambs through contact with the placenta and placental fluids (Detwiler and Baylis, 2003).

Scrapie prions appear to persist for years in the environment. When scrapie infected brain was mixed with soil and buried in the garden, 2-3 log units of infectivity of an initial of 4.8 log units remained after three years (Brown and Gajdusek, 1991).

1.1.3.3 Chronic wasting disease (CWD) Chronic wasting disease (CWD), a transmissible spongiform encephalopathy (TSE) affecting cervids in North America and Canada, is recognized since the late 1970s (Williams and Young, 1980). CWD has been diagnosed in mule deer, white-tailed deer, and Rocky Mountain elk in captive herds and in the wild. Symptoms of CWD include excessive salivation, teeth grinding, lowering of the head, and dropping ears.

CWD is thought to be transmitted horizontally from infected to susceptible animals. Residual infectivity in contaminated environments also appears to be important in sustaining epidemics (Miller and Williams, 2004). Recently, data were published indicating the presence of infectious prions in saliva and blood of deer with CWD (Mathiason et al., 2006). This may explain a facile transmission of the disease via body fluids.



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