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1. Auflage 2012
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University of Veterinary Medicine Hannover
Transmission and pathogenesis of the Q fever pathogen Coxiella burnetii in a sheep flock, and the attempt to sanify a Q fever
infected herd
Thesis
Submitted in partial fulfilment of the requirements for the degree -Doctor of Veterinary Medicine-
Doctor medicinae veterinariae ( Dr. med. vet. )
by
Regina Luise Eibach Herrenberg
Hannover 2012
Academic supervision: Univ.-Prof. Dr. Martin Ganter
Clinic for Swine, Small Ruminants, Forensic Medicine and Ambulatory Service
1. Referee: Univ.-Prof. Dr. Martin Ganter
2. Referee: PD Dr. Martin Runge
Date of the oral examination: 05.10.2012
This project was funded by the German Federal Ministry of Education and Research (Q Fever network), Grant 01KI0734.
Meiner Familie
Parts of this work have already been published in the following journals:
Epidemiology and Infection (2012) Volume 140, issue 11, pp. 1939-1949
Berliner und Münchener Tierärztliche Wochenschrift (2013) 126, Heft 1/2, Seite 3-9
Furthermore, the following parts have already been published:
How to deal with an acute Q fever outbreak?
R. Eibach, F. Bothe, M. Runge, M. Ganter Oral presentation
European College of Small Ruminant Health Management, Athens, Greek, 29.-30. October 2011
Q-Fieber Ausbruch in einer Schaf- und Ziegenherde und damit verbundene Humanerkrankungen
R. Eibach, F. Bothe, M. Runge, S. Fischer, W. Philipp, M. Ganter Poster presentation
International DVG Conference on Small Ruminant Health Management, Freiburg 11./12.May 2012
Effect of Vaccination with Coxevac® in a sheep flock with an acute Q fever outbreak R. Eibach, F. Bothe, M. Runge, S. Fischer, W. Philipp, M. Ganter
Poster presentation
National Symposium on Zoonoses Research, Berlin 6./7. October 2011
Management of a Sheep Flock after a Q Fever Outbreak R. Eibach, F. Bothe, M. Runge, S. Fischer, W. Philipp, M. Ganter Poster presentation
AVID Tagung "Bakteriologie" DVG, Kloster Banz 5.-7. October 2011
Human Infections after an acute Q Fever Outbreak in a Sheep and Goat Flock R. Eibach, F. Bothe, M. Runge, M. Ganter
Poster presentation
AVID Tagung "Bakteriologie" DVG, Kloster Banz 5.-7. October 2011
Q-Fieber
F. Bothe, R. Eibach, M. Runge, M. Ganter Oral presentation
Kolloquium der Schafsherdengesundheitsdienste Bayern, Grub 2./3. September 2009
Q fever in Germany
M. Ganter, F. Bothe, R. Eibach, M. Runge Oral presentation
Q-Fieber Tagung in Breda, Netherlands 25./26. February 2010
Q-Fieber in Deutschland
Ganter, M., Bothe, F., Eibach, R., Runge, M.
Oral presentation
Kolloquium der Schafgesundheitsdienste 2010, Rothesütte/Harz 16./17. June 2010
Q-Fieber in Deutschland, der Versuch eines Überblicks Ganter, M., Bothe, F., Eibach, R., Runge, M.
Oral presentation
Tagung der DVG-Fachgruppe Bakteriologie und Mykologie, Jena 22.-24. June 2010
Q-Fieber in Deutschland
Ganter, M., Bothe, F., Eibach, R., Runge, M.
Oral presentation
Vereinigung beamteter Tierärzte, Nienburg 27. October 2010
Q-Fieber
F. Bothe, R. Eibach, M. Runge, M. Ganter Oral presentation
bpt-Kongress Hannover 2010 18.-21. November 2010
Erfahrung mit der Coxevac®-Impfung bei Schafen F. Bothe, R. Eibach, M. Runge, M. Ganter
International DVG Conference on Small Ruminant Health Management, Freiburg 11./12. May 2011
Q fever in small ruminants
Ganter, M., Bothe, F., Eibach, R., Runge, M.
Oral presentation
European College of Small Ruminant Health Management, Athens, Greek 29.-30. October 2011
Q fever: Baseline monitoring of a sheep and a goat flock associated with human infections M. Ganter, R. Eibach, F. Bothe, S. Fischer, W. Philipp, M. Runge
Poster presentation
National Symposium on Zoonoses Research, Berlin 6./7. October 2011
Table of contents
Table of contents
1. Introduction 2. Manuscript I
Q fever: Baseline Monitoring of a Sheep and a Goat Flock associated with Human Infections
2.1. Summary 2.2. Introduction
2.3. Material and Methods 2.4. Results
2.5. Discussion 2.6. Acknowledgements 2.7. References 3. Manuscript II
Long-term monitoring of a Coxiella burnetii-infected sheep flock after vaccination and antibiotic treatment under field conditions
3.1. Zusammenfassung 3.2. Summary
3.3. Material and Methods 3.4. Results
3.5. Discussion 3.6. Acknowledgements 3.7. References 4. General discussion 5. Zusammenfassung 6. Summary 7. References 8. Acknowledgements
List of abbreviations
List of abbreviations
Bp base pair
BW body weight
C. burnetii Coxiella burnetii C. abortus Chlamydia abortus
cf. confer
DNA deoxyribonucleic acid
EDTA edetic acid
e.g. exempli gratia
ELISA Enzyme-linked Immunosorbent Assay
et al. et alii
F female
Fig. Figure
g gravity
IIFA Indirect Immunofluorescence Assay
IgG Immunoglobuline G
IgM Immunoglobuline M
LA long acting
LCV Large cell variants
LPS Lipopolysaccharid layer
M male
min minute
OD optical density
OEA Ovine enzootic abortion PCR Polymerase chain reaction P-value probability value
s second
SCV Small cell variants
SDC Small dense cells
TM Trade mark
V. Vena
vgl. vergleiche
y years
Introduction
1. Introduction
Within the last decade, Q fever has become more and more present in Europe. This maybe due to improved diagnostic possibilities together with an increasing awareness of this disease or even because Q fever has increased absolutely in our latitudes.
Prominent examples are Q fever outbreaks in Soest (2003, North Rhine-Westphalia, Germany) with 299 affected humans (PORTEN et al. 2006), in Jena (2005, Thuringia, Germany) with 331 human Q fever cases (GILSDORF et al. 2008) and the large Q fever outbreak in the Netherlands (2007-2010) with over 2300 human infections in 2009 (HOGERWERF et al. 2011).
Ruminant livestock, particularly goats and sheep, is consistently named in literature as the most common source of human Q fever infections. A synopses by Hellenbrand et al. (2001) already showed the trend of an increase of Q fever cases in Germany between 1947 and 1999 and confirms a transmission by sheep in 24 out of 40 documented outbreaks. They also mention a “north-south divide”, where 33 of the 34 described Q fever outbreaks occurred in Bavaria, Baden-Württemberg. Rhineland-Palatinate, Hesse, North Rhine-Westphalia and Thuringia. Moreover, their data suggests, that urbanization of rural areas may contribute to the increase in Q fever.
In Soest, for example, the human Q fever outbreak occurred after a Q fever positive ewe lambed on a farmers’ market. The human Q fever infections in Jena could also be related to a Coxiella (C.) burnetii infected sheep flock passing by a residential complex during lambing season. In both outbreaks, it can be assumed, that the Q fever infected animals, and therefore the infectious agent C. burnetii, got into contact with a human population not used having contact to farm animals. These circumstances give reason to assume, that in cases in which the bacteria meets an immunological naïve population in urban regions the number of affected people is higher than in rural areas. A possible explanation therefore might be a higher seroprevalence of Q fever in people living in the countryside as it could be shown in a Swiss study by Dupuis et al. (1986).
Introduction
2
The high number of human infections in the above mentioned Q fever outbreaks, some of them including sever clinical symptoms, makes it indispensable to come up with measures to control the Q fever situation in Germany and its surrounding countries.
Due to the fact that Q fever in ruminant livestock and in humans is only notifiable for statistical purposes (meldepflichtig) in Germany no sanctions, based on the animal disease law (Tierseuchengesetz), can be imposed on the owner of an infected flock. Therefore the management of such infected flocks is even more important to reduce or even avoid human infections.
The aim of this study therefore was to describe and understand the pathogenesis and epidemiology of a naturally infected sheep flock which caused human infections.
Additionally, the effect of the vaccination with Coxevac® in combination with an antibiotic treatment was investigated in this Q fever and Chlamydia abortus positive sheep flock, together with its supervision for 18 months.
The work was compiled in the frame of the German Q fever project. The German Q fever network (Q-Fieber-Verbund1) was created to re-evaluate the epidemiological situation in Germany, investigate the aetiopathology of chronic human patients, gain new insides into the pathogenicity of C. burnetii isolates, identify new reservoirs of the agent and improve diagnostic and typing methods. The investigations are funded by the Federal Ministry of Education and Research, Germany and the German Aerospace Centre.
Within this project, the Clinic for Swine and Small Ruminants in cooperation with the Lower Saxony State Office for Consumer Protection and Food Safety, Veterinary Institute Braunschweig-Hannover focuses on transmission and pathogenesis of Q fever in sheep with the attempt of eradication of Q fever in a sheep flock.
1 Within this work the term Q-Fieber-Verbund is used as umbrella term for the following members:
Konsiliarlabor für Coxiella burnetii, Landesgesundheitsamt Baden-Württemberg; National Consulting Laboratory for Coxiella burnetii, Baden-Württemberg State Health Office, Stuttgart, Germany; Institut für Medizinische Mikrobiologie, Universitätsklinikum Jena; Institute of Medical Microbiology, University Hospital Jena; Germany; Institut für Mikrobiologie der Bundeswehr, München; Institute of Microbiology of the Federal Armed Force, Munich, Germany; Friedrich-Loeffler Institut: Institut für bakterielle; Infektionen und Zoonosen, Jena; Institute for Bacterial Infections and Zoonoses, Jena, Germany; Institut für Epidemiologie, Wusterhausen, Institute of Epidemiology, Wusterhausen, Germany; Institut für molekulare Pathogenese Jena, Institute of Molecular Pathogenesis, Jena, Germany
Manuscript I
2. Manuscript I
Epidemiology and Infection
Volume 140, Issue 11, pp. 1939-1949, November 2012
Published online: 05.January 2012: DOI: http://dx.doi.org/10.1017/S0950268811002846
Q fever: Baseline Monitoring of a Sheep and a Goat Flock associated with Human Infections
R. Eibach¹, F. Bothe¹, M. Runge², S. F. Fischer³, W. Philipp4 and M. Ganter¹
¹University of Veterinary Medicine Hannover, Foundation, Germany
²Lower Saxony State Office for Consumer Protection and Food Safety, Veterinary Institute Hannover, Germany
³Baden-Württemberg State Health Office, Stuttgart, Germany
4University of Hohenheim, Germany
Summary
Animal losses due to abortion and weak offspring during a lambing period amounted up to 25% in a goat flock and up to 18% in a sheep flock kept at an experimental station on the Swabian Alb, Germany. Fifteen out of 23 employees and residents on the farm tested positive for Coxiella (C.) burnetii antibodies by ELISA and IIFA. Ninety-four per cent of the goats and 47% of the sheep were seropositive for C. burnetii by ELISA. Blood samples of 8% goats and 3% sheep were PCR-positive. C. burnetii was shed by all tested animals through vaginal mucus, by 97% of the goats and 78% of the sheep through milk, and by all investigated sheep through faeces (PCR testing).
In this outbreak human and animal infection were temporally related suggesting that one was caused by the other
Manuscript II
4 3. Manuscript II
Berliner und Münchener Tierärztliche Wochenschrift
126, Heft 1/2, Seite 3-9 (2013) DOI: 10.2376/0005-9366-126-3
Long-term monitoring of a Coxiella burnetii-infected sheep flock after vaccination and antibiotic treatment under field conditions
Langzeitüberwachung einer Coxiella burnetii-infizierten Schafherde nach Impfung und Antibiose unter Feldbedingungen
Regina Eibach1, Friederike Bothe1, Martin Runge2*, Martin Ganter1*
*beide Senior-Autoren haben zu gleichen Teilen zur Arbeit beigetragen
Klinik für kleine Klauentiere und forensische Medizin und Ambulatorische Klinik der Stiftung Tierärztliche Hochschule Hannover, Hannover, Germany1
Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES) – Lebensmittel- und Veterinärinstitut Braunschweig/Hannover, Hannover, Germany2
Abstract
The purpose of this study was to pursue the development of a sheep flock for a period of 18 months after a Q fever outbreak. In a flock with approximately 250 ewes losses of 18% were caused by abortions and weak offspring. Q fever was diagnosed in April 2009 after detection of Coxiella (C.) burnetii in one placenta and two aborted fetuses by PCR. Shortly afterwards Q fever was diagnosed in humans. Between July 2009 and December 2010 the sheep were sampled regularly. Six weeks before lambing the flock was initially immunised with
Manuscript II
Coxevac®. Moreover, pregnant ewes were treated with oxytetracyclines during late gestation until lambing. Seroprevalence increased from 39% before vaccination up to 98% afterwards.
After these measures bacteraemia and shedding of C. burnetii decreased significantly, indicating that during an acute outbreak the vaccination with Coxevac® can reduce the shedding of C. burnetii effectively, although vaccination is performed without medical approval for sheep and in late pregnancy. Both measures should be taken into consideration in acute Q fever outbreaks to reduce or even avoid transmission to humans.
Keywords: Q fever, coxiellosis, zoonosis, measures
Zusammenfassung
Ziel dieser Studie war es, die Entwicklung einer Schafherde nach einem Q-Fieber-Ausbruch über einen Zeitraum von 18 Monaten zu verfolgen.
In einem Schafbestand (ca. 250 Muttertiere) wurden durch Aborte und lebensschwache Lämmer Tierverluste in Höhe von 18 % verursacht. Die Diagnose Q-Fieber wurde im April 2009 nach PCR-Nachweis von Coxiella (C.) burnetii in einer Nachgeburt und zwei abortierten Feten gestellt und in zeitlichem Zusammenhang Q-Fieber-Infektionen bei Menschen diagnostiziert. Eine regelmäßige Beprobung der Schafe fand im Zeitraum Juli 2009 bis Dezember 2010 statt. Die Herde wurde sechs Wochen vor der Ablammung mit Coxevac® grundimmunisiert.Zudem wurden alle hochtragenden Schafe bis zur Ablammung regelmäßig mit Oxytetracyclin behandelt. Die Seroprävalenz stieg nach erfolgter Grundimmunisierung von 39 % vor der Impfung auf 98 %. Nach diesen Maßnahmen nahm die Bakteriämie und die Ausscheidung von C. burnetii signifikant ab, was darauf hindeutet, dass bei einem akuten Ausbruch Impfungen mit Coxevac® die Ausscheidung von C. burnetii deutlich reduzieren können, ungeachtet fehlender Zulassung beim Schaf und Einsatz in der Hochträchtigkeit.
Daher sollten diese beiden Maßnahmen bei einem Q-Fieber-Ausbruch in Erwägung gezogen werden, nicht zuletzt um den Infektionsdruck auf den Menschen zu reduzieren.
Schlüsselwörter: Q-Fieber, Coxiellose, Zoonose, Kontrollmaßnahmen
General Discussion
6 4. General Discussion
Q fever is a widespread zoonosis and occurs again and again as retail space epidemic with human involvement in Germany (PORTEN et al. 2006; GILSDORF et al. 2008).
The aim of this study was to describe and understand the pathogenesis and epidemiology of a naturally infected sheep flock which caused human infections. Furthermore, we investigated the effect of vaccination with Coxevac® in combination with an antibiotic treatment of this Q fever and Chlamydia abortus positive sheep flock and supervised the development over a period of 18 months.
The first part of this study (manuscript I) describes the initial investigations concerning epidemiology in different animal species and humans, which were made in May 2009, respectively July 2010, on a farm after a Q fever outbreak. The study shows the coherence between the human infections and the affection of a goat and sheep flock on an isolated site.
The second part of this study (manuscript II) traced and investigated the development of the Q fever infection in the sheep flock for 18 months. Within this period a sanitation program was implemented.
In general, farmers and especially veterinarians should be conscious that Q fever outbreaks in small ruminants might be difficult to recognize. In Q fever-positive flocks of small ruminants a variation of the abortion rate from 3% up to 80% is described (PALMER et al. 1983;
ARRICAU-BOUVERY u. RODOLAKIS 2005). Moreover, even if there are not many abortions in a flock but increased losses due to weak offspring, Q fever should always be considered as the etiological agent. The described situation was also found on the investigated farm: during the lambing period in 2009, particularly in the sheep flock, losses were dominated by weak offspring.
As described in manuscript I, the rate of losses in the goat flock (25%) was slightly higher in comparison to the one of the sheep flock (18%).The serological responses as well showed a considerably higher seroprevalence in goats (94%) compared to sheep (47%). The same tendency is shown by the results of the EDTA blood samples: DNA of C. burnetii could be detected in 3% of the sheep blood samples and in 8% of the goat blood samples. These results
General Discussion
give reason to assume that goats show more severe clinical signs than sheep in terms of abortions and weak offspring although this should be regarded carefully, as we do not know the precise time of entry of C. burnetii into the farm or the route of transmission. Moreover, it should be considered, that daily milking in a dairy goat flock improves the chances of transmission of C. burnetii from one animal to another in comparison to a sheep flock on pasture. Nevertheless, sheep can carry and shed C. burnetii, even though the animals are clinically inconspicuous (BERRI et al. 2001). The results also demonstrate that goats as well as sheep shed C. burnetii in their vaginal mucus, milk or faeces. This fact reconfirms studies which argue that infected dairy animals shed a high number of C. burnetii with birth products (ABINANTI et al. 1953; HATCHETTE et al. 2001), but also with faeces, urine and milk, even for several months after parturition (ARRICAU-BOUVERY et al. 2003; ASTOBIZA et al. 2010), a fact we could prove in manuscript II for vaginal mucus and faeces. The results of both papers demonstrate that in an acute Q fever outbreak the bacteria are excreted through all possible shedding routes even for some time after parturition, though in manuscript I, a statistical analysis was not possible because of the existing sampling bias.
Due to the different routes and duration of excretion of C. burnetii, but especially due to its high tenacity (SCHLIESSER 1991) and its aerogen distribution, C. burnetii is difficult to fight and can become a serious threat to the neighbouring human population of affected farms. As described in manuscript I it is always difficult to clearly outline the infection route of the bacteria onto animal farms or into flocks retrospectively. Nevertheless, it is very likely, that if human infections occur nearby a Q fever outbreak in farm animals, that these people have either contracted the disease via the inhalation of contaminated particles in the air (VAN DEN BROM u. VELLEMA 2009) or via direct contact with infected animals and their shedding products (TISSOT-DUPONT u. RAOULT 1993; BERRI et al. 2000). In Germany, most of the human infections are associated with sheep, rarely with goats and only sporadically with cattle, even though more than 90% of Q fever notifications in farm animals occur in cattle herds (ANONYMOUS 2009). The reason therefore might be found in the different housing systems of these animals entailing either more or less contact to more or fewer people (BÖTTCHER et al. 2011). Cattle are mostly held indoors and there are usually only a few and stable number of people who get into close contact with them. Furthermore,
General Discussion
8
cattle are calving throughout the whole year and generally inside the stables. This means on the one hand, that massive shedding of C. burnetii is possible throughout the whole year during deliveries, but that on the other hand, the concentration of the bacteria in the environment is much lower as cattle are normally calving one after another, spreading over the whole year. In contrast, in a sheep flock all ewes are lambing within a certain period of time, resulting in high concentrations of the bacteria in the environment in a relatively short time. Another difference is that calves and kits are normally separated from the dams just after parturition, which certainly reduces an infection of the newborns, whereas lambs remain normally with their mothers. Concerning human infections, the most important difference is that sheep are mostly kept outdoors on pastures in most of the time of the year (averagely 300 days per year) and that sheep often deliver outdoors. Due to the seasonality of lambing, a huge number of sheep lamb within a short time. During the lambing season, infected flocks shed a huge amount of C. burnetii into the surroundings, the birth products dry up and the wind disperses the infectious agent into the environment (TISSOT-DUPONT et al. 2004) and any people passing by or local residents may become infected.
Another important fact in this respect may also be the differences of C. burnetii strains isolated form different outbreaks and deriving from different species. This allows correlating molecular typing patterns of the strains with different animal species, infectivity as well as with virulence for humans. These strain differences may also explain why most of the human Q fever infections derive from flocks of small ruminants.
Furthermore, the majority of people are having fewer and fewer contact with farm animals.
This may be due to the fact that people tend to move from rather unpopulated areas into higher urbanized areas where farm animals are usually not to be found. These changes in urbanizations may also play a role concerning human Q fever cases. Residents of rural regions in contrast are still in more or less close contact with livestock. Therefore it can be assumed that a higher percentage of people in rural areas may have antibodies of C. burnetii compared to those living in highly urbanized areas. The study by Dupuis et al. (1986) conducted in Switzerland supports this thesis. The study was able to show that the seroprevalence of the inhabitants of Switzerland differs form 9,5%-12,2% in big cities to 23,6%-31,7% in mountain areas. Presumably, people with antibodies do not get Q fever or
General Discussion
show only mild clinical symptoms, maybe without even noticing them. Therefore, in order to prevent further Q fever outbreaks the contact between humans and sheep should further be reduced, especially in urban areas, as already suggested by Hellenbrand et al. (2001). The importance of such measurements can be illustrated with the Q fever outbreak in Jena in 2005. In this case, a small group of 17 pregnant gimmers lambed on a pasture, situated closely to a residential complex, before the shepherd moved its flock to the next pasture. As this flock was Q fever positive, and therefore, especially during the lambing period a massive shedding of the infectious agent must have taken place, 331 residents of this complex became infected by C. burnetii, some even with severe clinical symptoms (GILSDORF et al. 2008).
As described in manuscript I we can only assume the connection between the Q fever infections of the sheep flock held on the experimental field station and the infections of the residents and employees. Unfortunately, due to rather late reception of the official approval, the sampling of the residents and employees was not possible until 14 months after the acute phase in the flock and we have no control samples of anyone before the Q fever outbreak occurred. Nevertheless, the IIFA results of the serum samples allow us to draw a temporal connection between the human infections and the happenings in the flocks. But even though this connection can be assumed we cannot conclusively prove it. However, the solitude and rural location of the concerned farm described in manuscript I could be reasons for the rather low number of Q fever affected humans in comparison to the outbreaks described earlier. The residents living there are usually used to have contact to farm animals and furthermore, in Baden-Wuerttemberg, Q fever is endemic in cattle and small ruminants (ANONYMOUS 2010) which entails a certain contact between the bacterium and the human population.
The awareness of the disease in the population and the medical fraternity varies depending on the country and, especially in Germany, on each federal state. The announced human (SURVSTAT 2012) and animal Q fever cases clearly reflect this awareness, especially in relation to several epidemic outbreaks (e.g. Soest 2003 and Jena 2005).
A control of Q fever is, because of the highly infectious agent and its high tenacity, very difficult. Additionally, there is a lack of experiences as we could learn from the large outbreak in the Netherlands in 2007-2010. To decrease or even avoid human infections, the shedding
General Discussion
10
and therefore the spread of C. burnetii by small ruminants and cattle must be controlled. Due to the fact, that Q fever is only notifiable for statistic purposes in Germany, only advices can be given but no real sanctions can be imposed on the farmers. Such advices are that infected animals, especially sheep, goats and cattle, should be held indoors for parturition and their birth products should be removed and disinfected immediately and send to the rendering plant. Besides an increased standard of hygiene, only a vaccination and an antibiotic treatment are possible measures to fight Q fever. As the results of manuscript II show, a combined therapy of vaccination with Coxevac® and an antibiotic treatment with oxytetracyclines reduces shedding of C. burnetii. In this case, the antibiotic treatment was primarily implemented because a C. abortus infection was additionally proven in the sheep flock.
However, we are also of the opinion that the treatment with oxytetracyclines has an impact on the Q fever infection, as it reduces the shedding of C. burnetii via birth products, already described by Woernle et al. (1985) for cattle.
Coxevac® was licensed in September 2010 for cows and goats within the EU. For the use in sheep an exceptional approval according to the national epizootic diseases act (Tierseuchengesetz § 17c) was necessary. The vaccine (Coxevac®, CEVA-Phylaxia Veterinary Biologicals Co. Ltd., Budapest, Hungary) consists of the inactivated phase I (strain Nine Mile). Q fever vaccines vary in their composition and preparation process, including the strain, the possible combination of strains and which C. burnetii phase is used. Apparently, the most important difference is the phase (ARRICAU-BOUVERY et al. 2005). A phase I vaccine is meant to be more protective than a phase II vaccine (ORMSBEE et al. 1964).
Further studies have already shown that a phase I vaccine is able to reduce shedding of the infectious agent in bovine and ovine models and protects cows against abortion, infertility and low foetal weight (BEHYMER et al. 1976; BROOKS et al. 1986; SCHMEER et al. 1987).
As we do not have any control groups which are either only vaccinated or only treated with oxytetracyclines or without any sanctions, we cannot conclusively say which of the two measures led to the result. Presumably, a combined therapy always makes sense as soon as C.
abortus, causing the Ovine enzootic abortion (OEA), can be proven as supplemental reason for the reproductive disorders. Although an antibiotic treatment does not necessarily decrease the shedding of C. burnetii, it may reduce abortions, and therefore may reduce uncontrolled
General Discussion
shedding, caused by C. burnetii or C. abortus, respectively (RODOLAKIS et al. 1980).
Nevertheless the shedding via normal parturitions remains and therefore an increased standard of hygiene is required.
The results of manuscript II clearly show that in case of an acute Q fever outbreak the vaccination with Coxevac® in combination with an antibiotic therapy is able to reduce the shedding of C. burnetii effectively, despite the fact that the vaccination is not licensed for sheep within the EU and was also performed during pregnancy.
Particularly, during a Q fever outbreak with human infections the authorities would not accept to simply wait until the possible natural decrease of shedding occurs but demand immediate measures to fight the shedding of the infectious agent and therefore prevent human health risks. In cases of co-infections with C. abortus as found on this farm site a treatment with oxytetracyclines might help to reduce the number of abortions and weak offspring. Therefore both measures should always be considered in an acute Q fever outbreak to reduce or even avoid human infections. On the basis of this paper follow-up study should be performed under high biosafety standards, so that an infection trial with a S3 infectious agent can be carried out with different C. burnetii infected experimental groups: one only vaccinated, one only treated with antibiotics and an untreated control group. Such a follow-up study could conclusively answer which measure decreases shedding of the infections agent and clinical signs.
Investigations on the results of vaccinating with Coxevac® in the Netherlands into acutely infected goat flocks showed that this measure was able to reduce the amount of the infectious agent C. burnetii in milk, vaginal mucus and uterine fluid (HOGERWERF et al. 2011).
Nevertheless, it should furthermore be investigated if the vaccination has any preventive effects and is therefore able to protect small ruminants against a Q fever infection, which in turn would minimize or even prevent the infection of humans by aerosols.
In this context, a compulsory notification of Q fever, limited to infections of small ruminants, seems to be advisable for the future. Only with such a compulsory notification required measures, such as vaccination, treatment and stabling of infected flocks; vaccination of flocks in the immediate vicinity of infected ones or even, if necessary, the culling of chronic carriers
General Discussion
12
could be decreed. On this background farmers would also receive indemnity for treatment and vaccination as well as for losses.
In summary, this study is able to show the danger and high urgency of medical intervention in cases of Q fever outbreaks. Especially authorities, veterinarians and shepherds should be aware of this urgency to act in any way in order to prevent human infections. Regarding the fact that proper handling with an acute Q fever outbreak in flocks of small ruminants is still discussed further investigations, as mentioned earlier in this paper, are indispensable. A further consideration could be the vaccination of entire flocks in Q fever endemic areas, which in turn depends on whether the vaccination induces resilient protection and on the financial feasibility.
Zusammenfassung
5. Zusammenfassung
Regina Eibach
Übertragungswege und Pathogenese des Q-Fieber-Erregers Coxiella burnetii in einer Schafherde sowie der Versuch zur Sanierung dieser Herde
Q-Fieber zählt zu einer der bedeutendsten Zoonosen in unseren Breitengraden. Vor diesem Hintergrund soll im Rahmen des Q-Fieber-Projektes (Q-Fieber Verbund, siehe Fußnote 1, Seite 5) die molekulare Pathogenese des Q-Fiebers und ihre Anwendung in der Diagnostik und Epidemiologie in Deutschland erforscht werden. Die Mehrzahl der Humaninfektionen und insbesondere die in den letzten Jahren gehäuft auftretenden Kleinraumepidemien beim Menschen wurden meist durch infizierte kleine Wiederkäuer hervorgerufen.
Ziel dieser Arbeit war es daher die Epidemiologie eines Q-Fieber-Ausbruchs in einer Schaf- und Ziegenherde mit Humanerkrankungen aufzuarbeiten (vgl. Manuskript I: „Q fever:
Baseline Monitoring of a Sheep and a Goat Flock associated with Human Infections“) um so ein Sanierungsprogramm für eine Q-Fieber-positive Schafherde erstellen und über 18 Monate hinweg verfolgen zu können (vgl. Manuskript II: „Long-term monitoring of a Coxiella burnetii-infected sheep flock after vaccination and antibiotic treatment under field conditions“, „Langzeitüberwachung einer Coxiella burnetii-infizierten Schafherde nach Impfung und Antibiose unter Feldbedingungen“).
Im ersten Teil der Arbeit konnten Erkenntnisse über Ausscheidung, und Antikörperaktivität von Schafen und Ziegen einer akut mit Q-Fieber infizierten Herde gewonnen werden.
Außerdem konnte erstmal bei einem Teil der Tiere eine Bakteriämie nachgewiesen werden.
Darüber hinaus wurden Serumblutproben von 23 Mitarbeitern und Bewohner des betroffenen Hofes ausgewertet.
Die durch vermehrt auftretende Aborte und erhöhte Verluste durch lebensschwache Lämmer auffällig gewordene Schaf- und Ziegenherde einer Versuchsstation auf der schwäbischen Alb wurde nach der Diagnose Q-Fieber im Mai 2009 initial beprobt. Die Auswertung dieser
Zusammenfassung
14
Proben ergab, dass bei 47% der Schafe und 94% der Ziegen eine deutlich erhöhte Antikörperaktivität gegen C. burnetii nachgewiesen werden konnte. Des Weiteren konnten bei 3% der untersuchten Schafe und bei 8% der untersuchten Ziegen C. burnetii spezifische DNA Sequenzen im Vollblut mittels PCR detektiert werden. Alle untersuchten Vaginaltupfer, sowohl von den Schafen als auch von den Ziegen, waren in der PCR positiv. Außerdem konnte C. burnetii DNA bei 97% der untersuchten Ziegenmilchproben und 78% der untersuchten Schafsmilchproben gefunden werden, ebenso wie in 100% der untersuchten Kottupfer von Schafen. Auch wenn die Blutprobenentnahme der Mitarbeiter und Bewohner erst 14 Monate nach dem akuten Q-Fieber Ausbruch in der Herde möglich war, konnte ein Zusammenhang zwischen den Humaninfektionen und den Geschehnissen in der Herde hergestellt werden, da eine erhöhte Antikörperaktivität bei 15 der 23 untersuchten Personen auf eine durchgemachte Q-Fieber Infektion hinwies.
Die Einschleppungspforte des Bakteriums C. burnetii in die Herde ist retrospektiv schwer zu ermitteln. Nahe liegend ist jedoch, dass ein Lehrling sowie sein Hund als Vektor fungierten und das Bakterium vom elterlichen Betrieb, auf dem Q-Fieber ein Jahr zuvor diagnostiziert worden war, mitgebracht hatten.
Im zweiten Teil der Arbeit (vgl. Manuskript II) wurde dieselbe Schafherde 6 Wochen vor Beginn der Ablammung mit Coxevac® grundimmunisiert und alle zwei Wochen bis zur Ablammung mit Oxytetracyclinen behandelt. Die Ausscheidung, Bakteriämie und serologische Entwicklung der Tiere sowie Umgebungsproben wurden über einen Zeitraum von 18 Monaten alle drei bis vier Monate untersucht. Antikörper gegen C. burnetii konnten vor der Grundimmunisierung mit Coxevac® bei 39% bzw. 33% der untersuchten Schafe festgestellt werden. Am ersten Untersuchungstermin nach der Impfung hatten bereits 98% der Schafe serokonvertiert. Der Nachweis von C. burnetii spezifischen DNA Sequenzen im Vollblut sank nach der Impfung und der Antibiotikabehandlung signifikant ab (p=0,001), ebenso der Nachweis von C. burnetii positiven Vaginaltupfern (p=0,001). Bei den Umgebungsproben wurden bei jedem Beprobungstermin mindestens eine C. burnetii positive Probe gefunden.
Somit zeigen diese Untersuchungen, dass die Kombination aus Impfung und antibiotischer
Zusammenfassung
Behandlung zusammen mit verstärkten hygienischen Maßnahmen bei einem akuten Q-Fieber- Ausbruch als Maßnahmen anzuraten sind um humane Q-Fieber-Infektionen zu reduzieren oder sogar gänzlich zu vermeiden. Da die Untersuchungen im Rahmen einer Feldstudie stattgefunden hatten war es leider nicht möglich unbehandelte Kontrollgruppen zu halten (vgl.
Manuskript II). Daher ist es schwierig zu sagen welche der beiden Maßnahmen zu dem Erfolg geführt hatte. Bei einer Co-infektion mit C. abortus, wie sie zudem noch in der Herde vorlag, ist jedoch eine antibiotische Therapie immer anzuraten. Des Weiteren konnte aufgezeigt werden, dass der Impfstoff Coxevac® auch bei einer Anwendung während der Trächtigkeit und in akuten Q-Fieber Fällen bei Schafen sicher und effektiv ist.
Summary
16 6. Summary
Regina Eibach
Transmission and pathogenesis of the Q fever pathogen Coxiella burnetii in a sheep flock, and the attempt to sanify a Q fever infected herd
Q fever is one of the most important zoonotic diseases along our latitudinal lines. The majority of human infections and especially the cumulating retail space epidemics affecting humans over the last years were usually caused by small ruminants. On this background the aim of this study within the Q fever research network (cf. footnote 1, page 5) was to describe and understand the pathogenesis and epidemiology of a naturally infected sheep flock.
Furthermore, we investigated the effect of the vaccination with Coxevac® in combination with an antibiotic treatment of this Q fever positive sheep flock with a Q fever outbreak and a co- infection with C. abortus.
The aim of this paper therefore is twofold. On the one hand we aimed at analyzing and describing the Q fever outbreak in a sheep and goat flock with human infections (cf.
manuscript I), in order to develop and apply a sanitation program for a Q fever positive sheep flock on the other hand, which was additionally monitored for 18 months (cf. manuscript II).
The first part of this study (cf. manuscript I: „Q fever: Baseline Monitoring of a Sheep and a Goat Flock associated with Human Infections“) describes the results of the investigated sheep and goats as well as of the employees and residents at an experimental station in Germany.
The respective animal farm attracted attention because of an increased number of abortions and weak offspring. After Q fever was diagnosed in May 2009, sheep and goats were sampled by vaginal and faecal swabs as well as EDTA blood and serum. Moreover, serum samples of the employees and residents were taken. The evaluation of the samples showed that 47% of the sheep and 94% of the goats were seropositive. Furthermore, C. burnetii-specific DNA sequences could be detected by PCR in blood samples in 3% of the sheep and 8% of the goats. All vaginal swabs taken of sheep and goats as well as all faecal swabs taken of sheep
Summary
were positive in the C. burnetii PCR. C. burnetii DNA could also be detected in 78% of the sheep milk samples and in 97% of the goat milk samples.
Even though the blood samples of the employees and residents were only taken 14 months after the acute infection in the flocks a connection between the human infections and the infection of the goat and sheep flock on this isolated experimental station could be drawn, as 15 of 23 human blood samples showed an increased antibody activity indicating that these persons have already gone through an earlier Q fever infection.
The gateway through which the bacteria C. burnetii infected the flock is retrospectively difficult to determine. One possible assumption, however, is that an apprentice, together with his dog, was the possible gateway through which the bacteria found its way into the flock as the apprentice and his dog came from a farm in which Q fever was diagnosed already one year earlier.
In the second paper (cf. manuscript II: „Long-term monitoring of a Coxiella burnetii-infected sheep flock after vaccination and antibiotic treatment under field conditions), the development of the Q fever infection in the sheep flock was traced and investigated over a period of 18 months. Within this time frame a sanitation program was implemented. From May 2009 to December 2010, EDTA and serum blood samples as well as vaginal and rectal swabs were collected from all sheep of the farm every three to four months. In September 2009, six weeks before the start of the expected lambing period, the sheep were vaccinated with Coxevac®. Moreover, pregnant ewes received oxytetracycline fortnightly until lambing. Prevalence of seropositive animals in the flock increased from 39% and 33%, respectively (results of the two samplings before the vaccination), to 98% afterwards. Bacteraemia and bacterial shedding in vaginal discharge also decreased significantly after vaccination and oxytetracycline administration. The findings indicate that in cases of an acute Q fever outbreak, vaccination with Coxevac® combined with oxytetracycline administration and high standards of hygiene can lead to reduced shedding of C. burnetii and reduced number of abortions and weak offspring, not only in cases of co-infections with C. abortus. As our investigations took place within the framework of a field study, it was not possible to establish untreated and unvaccinated control groups due to legal reasons. Moreover, although Coxevac® is not licenced for pregnant sheep, it was safe and effective. Therefore, the
Summary
18
measures described in this study may be considered as essential enforcement during an acute Q fever outbreak, in order to prevent human Q fever infections.
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Acknowledgement
22 8. Acknowledgement
Mein besonderer Dank gilt meinem Doktorvater Herrn Prof. Dr. Ganter für die Überlassung des Themas, seiner jederzeit gewährten Unterstützung, seiner ständigen Erreichbarkeit und vor allem unermüdlichen Diskussionsbereitschaft ohne die diese Arbeit sicherlich nicht so zustande gekommen wäre.
Außerdem bedanke ich mich ganz herzlich bei Herrn PD Dr. Runge, nicht nur für die Untersuchung meiner ganzen Proben, sondern auch für seine geduldige und hilfreiche Unterstützung während meiner Veröffentlichungsphasen. Dieser Dank gilt auch seinem gesamten Laborteam.
Friederike Bothe danke ich für die gute Zusammenarbeit im Projekt, die stets fachliche und persönliche Unterstützung sowie für viele konstruktive Gespräche.
Bei Frau PD Dr. Silke Fischer, LGA Stuttgart, bedanke ich mich für die Untersuchung und Unterstützung bei der Interpretation der humanmedizinischen Proben dieser Arbeit.
Danke der Universität Hohenheim und besonders dem Leiter der Versuchsstation für Tierhaltung, Tierzüchtung und Kleintierzucht der Universität Hohenheim, Herrn Peitz, für etwaige Unterstützung bei der Durchführung der Versuche. Außerdem danke an alle Mitarbeiter des Oberen Lindenhofs der Universität Hohenheim für Eure Unterstützung während der heißen Probensammelphase, Eure Geduld mit meiner anfänglichen Unbeholfenheit und die zahlreichen lustigen Stunden - vor allem bei nächtlichen Probesammelaktionen.
Ein großes Dankeschön außerdem an alle Mitarbeiter des Labors der Klinik für kleine Klauentiere - vor allem an Malte Diederichsen für Deine unermüdliche Hilfe und gute Laune.
Acknowledgement
Des Weiteren danke ich allen Mitarbeitern der Klinik für kleine Klauentiere für die wunderbare Zeit, besonders Angie, Melanie, Julia, Alex, Dühli, Carina, Doris und Sabine für die Unterstützung in jeglicher Situation.
Danke Hanni für Deinen unermüdlichen, wunderbaren, unübertreffbaren Support!
Danke Kimy und Nora, dafür dass Ihr Euch mein Gejammer Tag und Nacht angehört und mich auch in jeder anstrengenden Stunde ertragen habt.
Außerdem bedanke ich mich ganz besonders bei Knut, der vor allem in der Endphase mit viel Geduld, Verständnis und liebevoller Fürsorge zum Gelingen dieser Arbeit beigetragen hat und mich stets an seinem Vermögen der englischen Sprache hat teilhaben lassen.
Meinen Eltern sei gedankt für eine wunderbare, sorgenfreie Zeit während des Studiums und der Doktorarbeit und dafür dass Ihr immer an mich geglaubt habt und mich bei allen Flusen in meinem Kopf immer unterstütz habt.
Die kleine Schwester dankt außerdem Ihrem großen Bruder für jeglichen Support während der gesamten Studien- und Doktorandenzeit, nicht zuletzt wegen der Lösung von etwaigen Computer-Graphik-Problemen.
