COST Action 855. Animal Chlamydioses and the Zoonotic Implications PROGRESS REPORT. Period: from to (March 2008)

COST Technical Committee COST Action 855 Animal Chlamydioses and the Zoonotic Implications PROGRESS REPORT Period: from 09-2003 to 11-2007 (March 2008) (Start date of the Action) (last update) This Report is prepared by the Management Committee of the Action and presented to the relevant Technical Committee. The report is a "cumulative" report, i.e. it is updated annually and covers the period beginning from the start date of the Action. 1

CONTENTS 1. OVERVIEW: ACTION IDENTIFICATION DATA COST Action 855 Title Animal Chlamydioses and the Zoonotic Implications TC Recommendation: (day/month/year) First MC meeting: 13/09/2003 CSO Approval: 07/03/2002 Last MC meeting: (day/month/year) Start date: 13/09/2003 (1) Final Report: (day/month/year) (2) Duration: 4 years Evaluation Report: (day/month/year) (2) Extension: 8 months (ending 04/07/2008) TC Evaluation: (day/month/year) End date: 04/11/2007 Number of signatories: 18 Signatories and date of signature: (day/month/year) Country Date Belgium 06/06/2002 Bulgaria 18/06/2002 Croatia 27/05/2004 Former Yugoslav Republic of Macedonia 04/03/2004 France 10/10/2002 Germany 29/05/2002 Greece 02/06/2004 Hungary 29/05/2002 Ireland 29/05/2002 Israel 09/06/2006 Italy 19/06/2002 Netherlands 19/04/2006 Poland 05/06/2002 Slovenia 05/04/2005 Spain 29/05/2002 Sweden 03/06/2003 Switzerland 11/07/2002 United Kingdom 29/05/2002 Institutes of non-cost countries: (list) Area: Agriculture, Biotechnology Action Web site: http://www.vetpathology.uzh.ch/forschung/costaction855.html 2

Chairperson: Title, Name: Dr. Konrad Sachse Tel.: +49-3641-804 334 Affiliation: Friedrich-Loeffler-Institut Fax: +49-3641-804 228 Postal Address: Naumburger Str. 96a, 07743 Jena, Germany E-Mail: konrad.sachse@fli.bund.de TC Rapporteur: Dr. John Williams, France, e-mail john.williams@tours.inra.fr External Evaluator: Prof. Peter Timms, Queensland University of Technology, Brisbane, Australia, e-mail p.timms@qut.edu.au External Evaluator: Title, name, affiliation, country (1) Date of the first MC meeting (2) When the report is received by TC Secretariat 2. OBJECTIVES The main objective of the Action is to better understand the spread and importance of animal chlamydioses in Europe by using epidemiological data. The hazards of animal chlamydioses for human public health arising from contact to reared, companion, synanthropic and wild animals will be assessed. To achieve this goal, effective detection methods based on DNA amplification, monoclonal antibodies or recombinant proteins will be developed and validated by a working group of the Action. Current research in member countries will increase knowledge on the determinants of pathogenicity of chlamydial isolates, an important prerequisite for the assessment of the zoonotic potential and virulence in general. The benefits of the Action include the improvement of general diagnostic standards in Europe in connection with animal chlamydioses. Diagnostic data collected during the Action will be the basis for recommendations on improved management, control and prophylaxis of chlamydial infections, such as the psittacosis/ornithosis complex and OEA. The efficacy of currently used vaccines will be assessed and the prospects of novel vaccines can be evaluated. The development of cheaper, safer alternatives, such as recombinant protein or DNA-based vaccines would be a major advance, benefiting animal welfare, reducing the risk of zoonotic infection and reducing the considerable economic losses resulting from the disease. Generally, the Action will contribute to improvements in animal health and welfare, as well as human health. The negative economic impact on animal husbandry caused by chlamydioses will be reduced. 3. TECHNICAL DESCRIPTION AND IMPLEMENTATION The annual workshops constitute the main scientific discussion forum of the Action. Exchange of scientific information and practical collaboration will be done mainly in the five Working Groups (see below), which will also be responsible for choosing the topics of the workshops. Moreover, Working Groups have organised their own meetings to discuss specialised topics or work out documents of general interest, e.g. the zoonotic risk associated with urban pigeon populations and the harmonisation of diagnostic methods. Short-term missions and other exchange of scientists between laboratories wereinitiated and prepared at the Working Group level before being proposed to the Management Committee. Here is a summary of the main topics addressed by the Working Groups. 3

WG 1: New diagnostic tests Leaders: Andreas Pospischil (CH) and Simone Magnino (IT) The topics of this WG include species-specific serological tests targeting newly identified epitopes, as well as new ELISA and immunohistological tests. Besides conventional PCR, the emphasis is on the exchange of expertise in real-time PCR and the emerging DNA microarray technology. Companies producing equipment and test kits in this area have been invited to take part in this WG. WG 2: Field survey and validation Leaders: Erhard F. Kaleta (DE) and Evangelia Vretou (GR) The activities of this WG are focusing on diagnostic tests for zoonotic chlamydial agents and validation trials. The OIE guidelines for test validation will be applied in these trials. WG 3: Zoonotic aspects of chlamydioses Leaders: Vittorio Sambri (IT) and Yvonne Pannekoek (NL) This group is focusing on increasing the exchange of information and cooperation between human and veterinary medicine in the field of zoonotic chlamydial infections. The main objective is to improve the system of reporting of zoonotic cases throughout Europe. This will lead to more reliable data on the incidence and general importance of these diseases. The data will be used for assessing the risks of transmission of infection to humans and for generating guidelines for risk assessment. COST 855 also encourages scientific collaboration for a comprehensive molecular characterisation of zoonotic chlamydial isolates from humans. WG 4: Research on pathogenesis Leaders: David Longbottom (UK) and Bryan Markey (IE) Many aspects of the pathogenesis of chlamydioses have yet to be elucidated. The WG is encourageing cooperation and scientific exchange on topics like shedding of chlamydiae, vectors of transmission and the incubation period in natural infection. Also the mechanisms of persistence and latency of infection are being addressed. Pathogenesis and cell-mediated immune responses are being investigated by several labs, in both natural and experimental animal model systems. At the molecular level, and following the completion of several Chlamydial Genome Sequencing Projects, surface antigens potentially involved in pathogenesis/virulence such as the polymorphic outer membrane proteins and the type III secretion system structural and effector proteins are investigated, as are processes of attachment, cellular trafficking and apoptosis. WG 5: Development of vaccines Leaders: Daisy Vanrompay (BE) and Annie Rodolakis (FR) Vaccination is thought to be the best way of controlling most animal chlamydioses. New developments will focus on DNA vaccines and subunit vaccines based on recombinant antigens. But also novel vaccine delivery systems and animal models for zoonotic infections are of great interest to this WG. Two important outcomes of any effective vaccine will be to stop shedding of infectious organisms, and also to be able to distinguish vaccinated animals from those with natural infection. Vaccine-producing companies will be invited to take an active part. 4. PARTICIPATION AND COORDINATION 4.1 Management Committee Chairperson: Konrad Sachse, Dr. Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Institut für molekulare Pathogenese Naumburger Str. 96a 4

D-07743 Jena Tel. +49 3641 804 334 Fax: +49 3641 804 228 konrad.sachse@fli.bund.de Vice-chairperson: Andreas Pospischil, Prof. Institute for Veterinary Pathology University of Zurich Winterthurerstrasse 268 CH-8057 Zürich Tel: +41 1 635 85 51 Fax: +41-1-635 89 34 apos@vetpath.unizh.ch Scientific secretary: Bouktje Stol COST Office Av. Louise 149 ( 21/25 ) B-1050 Brussels Tel:+32 2 533 38 25 BStol@cost.esf.org MC Members: Country Name Affiliation Belgium Vanrompay, Daisy University of Ghent, Dept. Molec. Biotechnol., Ghent Bulgaria Martinov, Svetoslav National Res. Vet. Med. Inst., Lab of Chlamydia and Rickettsia, Sofia Croatia Prukner-Radovcic, Estella University of Zagreb, Faculty of Veterinary Medicine, Department of Poultry Diseases, Zagreb France Rodolakis, Annie INRA Tours-Nouzilly, Pathologie Infectieuse et Immunologie, Tours France Laroucau, Karine AFSSA, Unité Zoonoses Bactériennes, Laboratoire d'etude et de Recherche en Pathologie Animale et Zoonoses, Maisons-Alfort Germany Sachse, Konrad Institute of Molecular Pathogenesis at the Friedrich Loeffler Institute, Jena Germany Kaleta, Erhard F. Justus-Liebig-Universität Giessen, Institut für Geflügelkrankheiten, Giessen Greece Vretou, Evangelia Hellenic Pasteur Institute, Athens Hungary Szeredi, Levente Central Veterinary Institute, Budapest Ireland Markey, Bryan University College Dublin, Faculty Vet. Medicine, Dept. Vet. Microbiol. & Parasitol., Dublin Italy Magnino, Simone Istituto Zooprofilattico Sperimentale della Lombardia e dell'emilia Romagna, Pavia Italy Sambri, Vittorio Section of Microbiology, University of Bologna, St Orsola Hospital, Bologna 5

Macedonia (FYRM) Ilieski, Vlatko University of Skopje, Faculty of Veterinary Medicine, Skopje Netherlands Pannekoek, Yvonne University of Amsterdam, Academic Medical Center, Amsterdam Netherlands van Duim, Brigitta University of Amsterdam, Academic Medical Center, Amsterdam Poland Niemczuk, Krzysztof National Veterinary Research Institute in Pulawy, Department of Cattle and Sheep Diseases, Pulawy Poland Kondracki, Marian National Veterinary Research Institute in Pulawy, Pulawy Spain Buendia Marin, Antonio J. Universidad de Murcia, Facultad de Veterinaria, Unidad Anatomía Patológica, Murcia Spain Caro Vergara, Maria Rosa Universidad de Murcia, Facultad de Veterinaria, Unidad Anatomía Patológica, Murcia Sweden Bölske, Göran Dept of Bacteriology, National Veterinary Institute, Uppsala Sweden Alenius, Stefan Dept of Ruminant Medicine and Veterinary Epidemiology, Swedish University of Agricultural Sciences, Uppsala Switzerland Pospischil, Andreas Universität Zürich, Inst. Vet. Pathologie, Zürich Switzerland Borel, Nicole Universität Zürich, Inst. Vet. Pathologie, Zürich United Kingdom Longbottom, David Moredun Research Inst., Intl. Res. Centre, Pentlands Science Park, Midlothian 4.2 Participating Institutions Institution City Country Queensland University of Technology, School of Life Sciences Brisbane Australia Primary Industries Research Victoria Attwood Australia University of Ghent, Faculty of Agriculture and Applied Biological Science, Dept. Mol. Biotechnology Veterinary and Agrochemical Research Centre, Coordination Centre for Veterinary Diagnostics Ghent Brussels Belgium Belgium National Res. Vet. Med. Inst., Lab of Chlamydia and Rickettsia Sofia Bulgaria World Veterinary Poultry Association BirdLife International Zagreb Croatia University of Zagreb, Faculty of Veterinary Medicine Department of Poultry Zagreb Croatia INRA Centre de Tours-Nouzilly, Pathologie Infectieuse et Immunologie Nouzilly France AFSSA Maisons-Alfort France Institut Pourquier Montpellier France Ruhr-University Bochum, BG-Kliniken Bergmannsheil, Institute of Pathology Bochum Germany Landesamt für Verbraucherschutz Sachsen-Anhalt Stendal Germany 6

Institution City Country Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health Jena Germany Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit Oberschleißheim Germany Justus-Liebig-Universität Gießen, Faculty of Veterinary Medicine Gießen Germany Friedrich-Schiller-Universität Jena, Inst. Med. Microbiology Jena Germany Animal Health Service, Landwirtschaftskammer NRW Bonn Germany Hellenic Pasteur Institute Athens Greece CEVA-PHYLAXIA Co. Ltd. Budapest Hungary Central Veterinary Institute Budapest Hungary Veterinary Institute of Debrecen Debrecen Hungary University College Dublin, Faculty of Veterinary Medicine Dublin Ireland Central Veterinary Research Laboratory, Dept. of Agriculture and Food Abbotstown Dublin Ireland University of Bologna, S. Orsola Hospital, Section of Microbiology DMCSS Bologna Italy Università degli Studi di Bari, Facoltà di Medicina Veterinaria Veterinaria Valenzano (Bari) Italy Dipartimento di Sanità e Benessere degli Animali Institut Pourquier Perugia Italy Istituto Zooprofilattico dell Umbria e delle Marche Perugia Italy University of Bologna, Dept. of Veterinary Public Health and Animal Pathology Ozzano Emilia Italy (BO) University of Teramo, Dept. of Biomedical Science Teramo Italy L'IZSLER Sez. di Ferrara Cassana (Ferrara) Italy Università degli Studi di Bari, Facoltà di Medicina Veterinaria Valenzano (Bari) Italy Dipartimento di Sanità e Benessere degli Animali Istituto Zooprofilattico Sperimentale Lazio e Toscana Rome Italy Istituto Zooprofilattico Sperimentale del Mezzogiorno - Sezione Diagnostica Cosenza Italy Provinciale di Cosenza Istituto Zooprofilattico Sperimentale della Lombardia e dell'emilia Romagna Pavia Italy National Reference Laboratory for Animal Chlamydioses Istituto Zooprofilattico Sperimentale Piemonte, Liguria e Valle d Aosta Turin Italy Universita degli Studi di Teramo, Dip.to di Sceinze Biomediche Comparate Teramo Italy Istituto Zooprofilattico Sperimentale Sassari Sassari Italy Istituto Zooprofilattico Sperimentale, Portici Portici (NA) Italy Istituto Zooprofilattico Sperimentale delle Venezie Legnaro Italy Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata - Sede di Foggia Italy Foggia University of Bologna, S. Orsola Hospital, Section of Microbiology, DMCSS Bologna Italy Istituto Zooprofilattico Sperimentale della Sicilia A. Mirri Palermo Italy Instituto Zooprofilattico Sperimentale Delle Venezie Legnaro (PD) Italy University St. Ciryl and Methodius, Faculty of Veterinary Medicine Skopje Macedonia Institute for Animal Disease Control (CIDC) Lelystad Netherlands Academic Medical Centre, Univ. Amsterdam, Med. Microbiol. Amsterdam Netherlands Natl. Veterinary Research Institute, Lab. for Serological Diagnosis and Cattle & Sheep Diseases Pulawy Poland 7

Institution City Country Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica Cátedra de Microbiología Buenos Aires República Argentina World Veterinary Poultry Association, BirdLife International Ljubljana Slovenia Universidad de Murcia, Facultad de Veterinaria, Departamento de Histología y Murcia Spain Anatomía Patológica Universidad de Murcia, Facultad de Veterinaria, Departamento de Sanidad Animal Murcia Spain Swedish University of Agricultural Sciences, SLU, Dept. of Clinical Sciences, Uppsala Sweden Division of Ruminant Medicine and Veterinary Epidemiology National Veterinary Institute (SVA), Dept. of Bacteriology Uppsala Sweden University of Zurich, Vetsuisse Faculty, Institute of Veterinary Pathology Zurich Switzerland University of Lausanne, Faculty of Biology and Medicine Lausanne Switzerland Microbiology Institute IMU 02-219 University of Basel, Institute of Anatomy Basel Switzerland Moredun Research Institute, Pentlands Science Park Edinburgh UK EH26OPZ University of Maryland, Dept. of Biomedical Sciences Baltimore, MD USA Auburn University, College of Veterinary Medicine, Dept. of Pathobiology Auburn, Alabama USA The Institute of Genomic Research Rockville, 20850 USA 4.3 Meetings of the Management Committee Preliminary meeting held on September 13, 2003 at the Faculty of Veterinary Medicine of University College Dublin, Ireland, from 10:00 a.m. till 5:00 p.m. First MC meeting held on November 5, 2003 at the COST Secretariat, Square de Meeus 8, Brussels, from 10:30 a.m. till 4:30 p.m. Second MC meeting held on September 2, 2004 at the Congress Centre of the Faculty of Science of Eötvös Loránd University, Budapest, Hungary, from 4:15 p.m. till 6:00 p.m. Third MC meeting held on September 23, 2005, at Banca di Monteriggioni, Via Cassia, Siena, Italy, from 4:15 till 6:00 p.m. Fourth MC meeting held on September 5, 2006, at Moredun Research Institute, Penicuik, Midlothian, Scotland/UK, from 4:15 till 6:00 p.m. 8

Fifth MC meeting held on September 11, 2006 at National Veterinary Research Institute, Pulawy, Poland from 4:15 till 6:00 p.m. 4.4 Meetings of the Working Groups WG 2+5 Topic: Serological diagnosis in ovine enzootic abortion: Vaccinated versus naturally infected sheep Date: Wednesday, March 23, 2005 Venue: Institute of Veterinary Pathology, University of Zurich, Zurich (CH) Local organiser: Andreas Pospischil Number of attendants: 16 WG 1 Topic: Validation of new diagnostic tests for animal chlamydioses Date: Friday June 10, 2005 Venue: Het Pand, Onderbergen 1, Gent (BE) Local organiser: Daisy Vanrompay Number of attendants: 20 WG 1+3 Topic: Validation of new molecular diagnostic assays and zoonotic aspects of animal chlamydioses Date: Friday, October 14, 2005 Venue: Friedrich-Loeffler-Institut, Jena (DE) Local organiser: Konrad Sachse Number of attendants: 15 (no COST funding) WG 2 Topic: The problem of street pigeons and its implications on public health Date: April 21, 2006 Venue: Pavia (IT), Local organiser: Simone Magnino Number of attendants: 15 WG 4 Topic: Immunopathogenesis of chlamydial infections Date: June 8, 2006 Venue: Dublin (IE) Local organisers: Bryan Markey and Donal Sammin Number of attendants: 15 WG 5 Topic: Vaccine development Date: June 9, 2006, Venue: Dublin (IE) Local organisers: Daisy Vanrompay and Annie Rodolakis 9

Number of attendants: 15 WG 1 Topic: New molecular diagnostic tests for chlamydiae Date: September 21, 2006 Venue: Chur (CH) Local organiser: Andreas Pospischil Number of attendants: 20 (funded by Swiss national COST office) WG 1+2 Topic: New and emerging chlamydia-like organisms Date: October 29-30, 2007 Venue: Lausanne (CH) Local organiser: Gilbert Greub Number of attendants: 24 Expert Meetings (not assigned to a particular WG) May 2004, Budapest, 3 participants May 2005, Bologna, 4 participants November 2006, Jena, 6 participants 2004 4.5 Short-term scientific missions (List: dates, hosts and nationality of scientists and topics) Head of Sending Laboratory Researcher Host Lab Date Scientific Topic Longbottom, David (UK) Karen Kerr (PhD student) M.R. Caro Vergara (ES) 2 weeks (End of May) Detection of immune cells in mouse frozen sections Marsilio, Fulvio (IT) Barbara di Martino A. Rodolakis (FR) Aug 9-13, 2004 PCR-RFLP detection of chlamydiae Pospischil, Andreas (CH) Nicole Borel K. Sachse (DE) Aug 15-20, 2004 Chlamydial cell cultures, different serology methods for chlamydial antibodies (CFT and ELISA), quantitative detection of Chlamydia by real-time PCR, DNA microarray test for chlamydial differentiation Sachse, Konrad (DE) Stefanie Göllner (PhD student) A. Pospischil (CH) May 3-14, 2004 vitro model for induction of persistence of chlamydiae in cell culture Sambri, Vittorio (IT) Elisa Storni (PhD student) K. Sachse (DE) Dec 05-12, 2004 use of DNA microarray technology for identification of chlamydiae Szeredi, Levente (HU) Levente Szeredi A. Pospischil (CH) Nov 21-27, 2004 immunohistochemical and PCR examination of paraffin-embedded samples Vanrompay, Daisy (BE) Tom Geens (PhD student) K. Sachse (DE) Nov 15-26, 2004 DNA-Microarrays 10

2005 Sending Laboratory Researcher Host Lab Date Scientific Topic V. Ilieski Macedonia (fyr) L. Pendovski Estela Prukner Zagreb (CRO) May 16-22 Diagnosis of Animal Chlamydioses using imunohistohemistry and classical histology Bryan Markey (IE) Donal Sammin D. Longbottom (UK) Aug 15-26 Immuopathology of enzootic abortion in ewes D. Longbottom (UK) Morag Livingstone (PhD) E. Vretou (GR) October 17-28 POMPs in 2-D electrophoresis V. Ilieski Macedonia (fyr) D. Mitrov Tomaz Zadnik Ljubljana (SLO) October 17-23 Diagnosis of Animal Chlamydioses using using ELISA and PCR methodology J. Salinas (ES) Nieves Ortega (PhD) K. Sachse (DE) Aug 15 - Sept 03 PCR in chlamydial diagnosis 2006 Macedonia (fyr) V. Ilieski Macedonia (fyr) Sending Laboratory Researcher Host Lab Date Scientific Topic D. Vanrompay (BE) Delphine Beeckman (PhD) K. Sachse (DE) April 24 to May 5 DNA microarray technology and persistence models in chlamydial infection V. Ilieski Macedonia (fyr) Vlatko Ilieski A. Pospischil (CH) 25 June - 2 July Immunohistochem. examination of clinical samples from chlamydioses Bryan Markey (IE) Sheila Worral (PhD) D. Longbottom (GB) Oct 8-13 In situ hybridisation V. Ilieski Macedonia (fyr) Ivanco Naletoski Karine Laroucau (FR) Nov 19-26 PCR-based diagnostic methods V. Ilieski Vlatko Ilieski Karine Laroucau (FR) Nov 19-26 Modern diagnostic methods for chlamydiae Trpe Ristoski A. Pospischil (CH) Dec 4-8 Histopathology 2007 Sending Laboratory Researcher Host Lab Date Scientific Topic S. Martinov (BG) S. Martinov B. Markey (IRL) October 1-7 Diagnostic methods A. Pospischil (CH) Daniela Zweifel K. Sachse (DE) Aug 20-31 DNA microarray technology in diagnosis K. Laroucau (FR) Fabien Vorimore K. Sachse (DE) March 26-30 DNA microarray technology in diagnosis B. Markey (IRL) Erin Williams K. Sachse (DE) June 4-8 Real-time PCR of chlamydiae Fulvio Marsilio (IT) Ilaria Meridiani G. Entrican (UK) March 20-June 20 Development of a vaccine against C. abortus infection 5. RESULTS COST (European Co-Operation in the field of Scientific and Technical research) Action 855 Animal Chlamydioses and the Zoonotic Implications was set up in order to promote and enable cooperation between scientists in veterinary research to address issues relating to chlamydial infections in animals and also their zoonotic potential. To achieve this goal, working groups were tasked to focus on five specific objectives: (1) the development and validation of new and existing diagnostic tests; (2) the 11

field evaluation of diagnostic tests; (3) the assessment of the zoonotic risks associated with animal chlamydioses; (4) to conduct basic research to improve our understanding of chlamydial pathogenesis, virulence factors and associated immune responses; and (5) to develop new vaccines for controlling chlamydial infection. Through the research activities of the member groups of this European network substantial advances have been made in these areas and presented at the annual conferences in Dublin (2003), Budapest (2004), Siena (2005), Edinburgh (2006), Pulawy (2007) and now culminates with this final meeting in Aarhus. Here we review some of the scientific highlights that have been achieved over the course of the 5 years of the Action. New diagnostic methods Recent advances in laboratory diagnosis of chlamydial infections have been reviewed by members of COST 855 (Sachse et al., 2008). Real-time PCR has been recognised as a powerful technique for rapid, specific and sensitive laboratory diagnosis of chlamydial infections. New real-time PCR assays have been developed and evaluated for Chlamydophila (C.) psittaci, the causative agent of psittacosis (Geens et al., 2005; Heddema et al., 2006a; Pantchev et al., 2008), as well as C. pecorum and C. pneumoniae (Markey et al., 2005; Markey et al. 2007), C. abortus (Livingstone et al., 2008; Pantchev et al., 2008) and the family Chlamydiaceae (Ehricht et al., 2006; Godin et al., 2006). DNA microarray technology has been shown to possess a great potential for species identification of chlamydiae (Sachse et al., 2005). In a collaborative study to validate this technology, the microarray findings were compared with real-time PCR, conventional PCR, immunohistochemistry and other tests. The DNA microarray assay was shown to be highly accurate with good performance parameters, demonstrating its suitability for routine diagnosis (Borel et al., 2008). To explore the genotype repertoire and epidemiology of C. psittaci strains in the Netherlands, an ompa gene sequence-based approach was suggested (Heddema et al., 2006b). Geens et al. (2005) developed a new C. psittaci genotype-specific real-time PCR and demonstrated the occurrence of an additional avian genotype, designated E/B. A new DNA microarray assay has been developed for direct genotyping of C. psittaci strains from clinical samples, which was shown to detect all currently accepted genotypes, as well as subgroups and so far untyped strains (Sachse et al., 2007, 2008). With the availability of the genome sequence of C. psittaci 6BC, the typing method termed Multiple Loci Variable Number of Tandem Repeats (VNTR) Analysis (MLVA), which is based on the detection of tandem repeat polymorphisms, has been used successfully to characterise the molecular diversity among C. psittaci isolates and assess its usefulness for future epidemiological investigations (Laroucau et al., 2008a). Similar studies are ongoing for the species of C. pecorum and C. abortus (Laroucau et al., 2008d). Recently developed real-time PCR methods specific for Parachlamydia and Waddlia, respectively, were applied to investigate the presence of these organisms in ruminant abortion (Casson et al., 2008; Ruhl et al., 2008). Species-specific immunohistochemistry protocols using antibodies against Parachlamydia and Waddlia have been established, demonstrating the agent within the placental lesions (Borel et al., 2007). The protocols used are suitable for routine diagnosis and should complete the current examination procedure on bovine abortion cases. The tissue microarray technology was validated in combination with immunohistochemistry by testing antibodies to differentially expressed proteins in an IFN-_-induced model of chlamydial persistence (Borel et al., 2006). A newly developed nested PCR-enzyme immunoassay with an internal inhibition control has proved to be highly sensitive and specific for demonstrating chlamydial DNA in birds (Van Loock et al., 2005b). A recently developed serological test for ovine chlamydioses has been shown to detect infection much earlier in pregnancy than existing tests, which should allow appropriate control measures to be applied earlier, thus limiting the environmental spread of infection to other animals (Livingstone et al., 2005). To improve serological monitoring of flocks free of C. abortus, a new test for simultaneous detection of C. pecorum based on recombinant antigens is being developed (Rodolakis et al., 2006). Several new commercial serological tests have been evaluated using sera from experimentally infected sheep and field sera (Vretou et al., 2007; Rekiki et al., 2006b; Wilson et al., 2006). A recent field study, the first of its kind, compared antibody titres in latently infected, diseased and vaccinated animals over a two-year period (Gerber et al., 2007). In another study, a comparison of 3 commercial serological assays with CFT and 4 in-house ELISAs has been conducted to evaluate their specificity and sensitivity in detecting C. abortus and C. pecorum infected sheep (Wilson et al., 2008). Additionally, a recombinant antibody ELISA suitable for birds, pigs and other ani- 12

mals has been developed and compared to established tests (Vanrompay et al., 2004;Verminnen et al., 2006). Finally, Ortega et al. (2007b) compared several protocols of DNA purification for the diagnosis of OEA from paraffin-embedded samples by PCR. The results showed that PCR from paraffinembedded sections can be a useful tool for analysis of field samples. Research on pathogenesis, immune response and virulence factors A number of peculiarities associated with chlamydial infections make research in pathogenesis particularly difficult, e.g. the absence of classical virulence factors, the wide variety of clinical manifestations (from subclinical or mild to acute), the tendency towards chronicity, as well as poorly understood host immune response. Research of COST 855 member groups has focused on the identification and characterisation of inclusion membrane proteins in C. abortus, which are known as virulence factors involved in the pathogenesis of the disease. Vretou et al. (2006) identified and partially characterised the first inclusion membrane protein of C. abortus, which was designated Inc766 and represents a potential virulence factor. Inc766 has been shown to form dimers and high order oligomers (Vretou et al., 2008a) and to decorate cytoplasmic extra-inclusion vesicles (Vretou et al., 2008b). One of the projects has focused on the chlamydial type III secretion system and its contribution to chlamydial virulence (Geens et al., 2004; Beeckman et al., 2008). Further studies are being conducted to characterise effector proteins and to elucidate their possible role in chlamydial pathogenesis. Examination of the transcriptional response associated with in vitro models of chronic infections has also improved our understanding of the regulation of the chlamydial intra-cellular developmental cycle and has identified new molecular features of the persistent state of C. pneumoniae and C. psittaci (Polkinghorne et al., 2006; Goellner et al., 2006a). Recent work on host cell processes triggered by chlamydial infection revealed that C. psittaci was capable of modulating host cell apoptosis. In particular, inhibition of host cell apoptosis may facilitate permanent colonisation of host tissue by the pathogen and ensure the long-term survival of persistent C. psittaci within the host cell (Goellner et al., 2006b). Studies investigating the persistence and shedding of C. abortus in post abortion sheep at oestrus and subsequent lambing by real-time PCR have revealed low numbers of organisms suggesting that they do not impact significantly on disease epidemiology either through mechanical or venereal transmission by the ram or through the products of lambing (Livingstone et al., 2008). A mathematical/computational epidemiological model for C. abortus infection in sheep has been constructed to simulate disease progression and in order to understand the underlying processes that drive transmission (Milne et al., 2008). A series of experimental infection studies clearly demonstrated the pathogenic interaction between C. psittaci and other respiratory pathogens, such as the avian metapneumovirus (Van Look et al., 2006a), Escherichia coli (Van Look et al., 2006b), and Ornithobacterium rhinotracheale (Van Look et al., 2005a) in the turkey respiratory disease complex. Following the completion of the C. abortus genome (Thomson et al., 2005), an 11k C. abortus microarray for pathogenesis studies has been designed and developed in conjunction with Agilent Technologies, and used to investigate strain variation in the field. Through comparative genomics the genome has revealed significant variation in two families of proteins, the polymorphic membrane proteins or pmps (autotransporter proteins of the type V secretion system) and the TransMembrane Head or TMH proteins (inclusion membrane proteins) and it has been suggested that it is variation in these proteins that are responsible for the niche specificity of this pathogen (Thomson et al., 2005). A C. abortus genome web resource has been developed (Longbottom et al., 2005, 2006) and is now available on the Moredun Research Institute website (http://bioweb-2.mri.sari.ac.uk/cab/). The site can be used to both search and browse the genome using graphical views. Another project is addressing virulence genes of C. abortus identified from the total genome sequence of strain AB7 to compare them with those of vaccine strain 1B and mutant strains (Rodolakis et al., 2006). Vretou et al. (2006) identified and partially characterised the first inclusion membrane protein of C. abortus, which was designated Inc766 and represents a potential virulence factor. Using a murine model, several aspects of the immune response against C. abortus infection were investigated focusing on the role of NK cells (Buendía et al., 2004), or CD4+ and CD8+ T cells (Martínez et al., 2006), respectively. It was concluded that CD8+ T cells may play a role in the regu- 13

latory control of CD4+ T cell response and may have a direct cytotoxic or IFN-_-mediated effect on infected cells. Also, Buendia et al. (2007) have developed a new model of intranasal infection of C. abortus, which is useful for testing the protection offered by different vaccines against C. abortus. Furthermore, knowledge of the pathogenesis of C. abortus experimental infection in the natural host has been considerably extended (Navarro et al., 2004). The development of lesions in placental tissues following experimental infection of sheep with C. abortus have been described early in infection (Maley et al., 2008). Comparison of the foetal and maternal inflammatory responses in the ovine placenta showed that the foetal response is innate in character, while the maternal response represented an acquired, chlamydia-specific response (Sammin et al., 2006) and it is this acquired response of the ewe that prevents the organism reaching the placenta in subsequent pregnancies (Sammin et al., 2005). Innate immune sensory mechanisms (e.g. Toll-like receptors) and defence mechanisms (anti-proteases, defensins) are likely to have an important role in limiting chlamydial infection in the early stages. The expression of ovine Elafin and Secretory Leukocyte Protease (SLPI) Inhibitor, both of which possess anti-microbial and anti-inflammatory properties, has been demonstrated in the female reproductive tract. SLPI has been shown to be expressed in the uterus late in pregnancy and so may form part of the innate defence against C. abortus (Wheelhouse et al., 2006). The pattern of the cytokine interferongamma (IFN-_) mrna expression and its production in foetal and maternal components of the placenta during the advanced stages of chlamydial placentitis were systematically examined to elucidate the host response to enzootic abortion of ewes (Worral et al., 2008). The epidemiological importance of chlamydia-associated abortion in small ruminants was analysed in Italy, Hungary, Ireland and other countries (Masala et al., 2005; Cafiero et al., 2006; Szeredi et al., 2006; Markey, 2006). A serological study was undertaken to estimate the prevalence of C. abortus in samples of sheep and goats originating from Greek organic farms which had outbreaks of abortion. The effect of natural and experimental chlamydial infection on lung function parameters in pigs has been examined (Sachse et al., 2004; Reinhold et al., 2005). A group of subclinically infected calves, i.e. natural carriers of Chlamydophila spp., was shown to display significantly deteriorated health parameters, such as lower body weight, elevated body temperature, altered blood parameters and other signs associated with chronic effects on animal health at a subclinical level, when compared to chlamydia-free calves (Reinhold et al., 2008). Even in the absence of overt of clinical symptoms, respiratory chlamydial infection appeared to be associated with chronic inflammation of lungs and airways, and pulmonary dysfunctions persisted in calves until the age of 7 months (Jaeger et al., 2007). A retrospective survey on the importance of chlamydial infections in cases of bovine abortion was conducted in Switzerland (Borel et al., 2006). Investigations are underway to determine the prevalence of chlamydial infections in cattle and pigs in Great Britain, Sweden, Germany and other European countries and to assess the impact of these infections on infertility and abortion, as well as animal health and performance. The suitability of a horse model for the study of human COPD was addressed by Theegarten et al. (2008). C. psittaci and C. abortus were present in the lung of both clinically healthy horses and those with RAO. Immunohistochemistry revealed acute chlamydial infections with inflammation in RAO horses, whereas mostly persistent chlamydial infection and no inflammatory reactions were seen in clinically healthy animals. In vitro infections of amoebae with C. abortus have been undertaken to study their interaction and to elicit the potential role of amoebae in the epidemiology of chlamydioses (Wirz et al., 2005). Development of new vaccines for improved control of chlamydial infections At present, few commercial vaccines for animal chlamydioses are commercially available, and some of those in use are known to have considerable limitations (Longbottom and Livingstone, 2006). Members of COST 855 have conducted vaccination studies based on DNA vaccines, recombinant MOMP and subunit membrane fractions. Cp. psittaci DNA vaccination significantly protected turkeys against severe clinical disease and significantly reduced chlamydial excretion, as well as the lesions caused by this pathogen, whereas recombinant MOMP vaccination induced lower protective immune responses (Van Loock et al. 2004; Verminnen et al., 2005). The use of adjuvants like vitamin D 3 and CpG motifs was evaluated, but they did not significantly enhance proctective immune responses following DNA vaccination (Verminnen et al., 2005; Loots et al., 2006). A DNA vaccination protein boost protection study in sheep against C. abortus infection has been completed and shown to generate 14

both cellular and humoral immune responses (Frew et al., 2005), although these responses did not appear to be protective. The choice of antigen, adjuvant and delivery method requires further investigation to improve protective efficacy (Longbottom & Livingstone, 2005). The protective efficacies of a purified native soluble MOMP preparation and a purified recombinant MOMP preparation were assessed in a C. abortus challenge mouse model and compared to vaccination with live EBs (Livingstone and Longbottom, 2006b). The protective immunity of a vaccine based on the C. abortus groel gene was tested and evaluated in a murine model (Hechard et al., 2004). Members of COST 855 are currently conducting a study on serological evaluation of titres against C. abortus after vaccination in OEA-affected and OEA-negative sheep flocks to collect and evaluate data for future schemes to control outbreaks of this disease. Using novel adjuvants, new inactivated vaccines against C. abortus have been tested. The data indicate a good performance in sheep (García de la Fuente et al., 2004). Caro et al. (2005) presented a new co-infection model using Nippostrongilus brasiliensis and C. abortus to analyse the protection induced by experimentally inactivated vaccines and new adjuvants, as well as the influence of an established Th2 immune response on the outcome of vaccination. The role of PMNs and NK cells in protection against C. abortus infection conferred by different vaccines was studies by Ortega et al. (2006). The results open up new perspectives to study the innate immune mechanisms underlying effective protection after vaccination. A new model of animal infection has been developed in the hamster: this model has been used to evaluate the immune protection induced by recombinant antigens against a challenge infection by C. pneumoniae (Sambri et al., 2004; Finco et al., 2005). Assessment of zoonotic risks associated with animal chlamydioses The Technical Annex of COST 855 stated that specific data needed to be collected and published to assess the current epidemiological situation concerning zoonotic transmission of chlamydiae in Europe. Laboratories of the participating countries have conducted diagnostic surveys in flocks of poultry (Verminnen et al., 2008) and sheep, herds of cattle, pet animals (Harkinezhad et al., 2007; Vanrompay et al., 2007) and others to evaluate the prevalence of chlamydial agents. Cases of chlamydioses in persons connected with the respective farm, production unit or household are being included in several ongoing studies. A severe outbreak of psittacosis in a poultry flock in Germany led to infection of 24 individuals with one death (Gaede et al., 2008). Molecular diagnostic examinations revealed involvement of two different genotypes of Chlamydophila psittaci, i.e. A and E/B. The high zoonotic potential of genotype A requires particular attention. Although poultry breeding is economically important in France, few data are available concerning the prevalence of C. psittaci and cases of zoonotic transmission. In 2006 and 2007, several outbreaks of human psittacosis linked to ducks or psittacines were investigated by the French National Public Health Surveillance Centre (InVS) and the National Reference Centre (human aspects), and by Veterinary Services and AFSSA (animal aspects). Medical and veterinary epidemiological surveys were carried out, which included diagnostic confirmation by serological and/or PCR testing (Laroucau et al., 2007). Whenever possible, samples were typed by PCR-RFLP and by MLVA, and animal and human samples were compared (Laroucau et al., 2008b; Laroucau et al., 2008c). Considering the potential severity of the human disease and the recurrence of epidemic episodes in various professional contexts, a two-year prospective descriptive study of human psittacosis, which was coordinated by the InVS, was started in January 2008. The aim of this study is to determine the incidence of hospitalised human cases and the frequency of grouped cases, as well as to define the risk of exposures for the patients. Additionally, the analysis of the strains isolated from humans and animals, as well as the description of breeding characteristics and working conditions will improve our knowledge of risk factors for animal-to-human transmission. Ultimately, this study will allow the introduction of improved prevention and control measures (Laroucau et al., 2008c). The potential health hazards caused by urban pigeons as carriers of C. psittaci infection have been addressed by COST 855 (Magnino et al., 2006; Magnino et al., 2008). There is an urgent need for more epidemiological studies, which include cases of zoonotic transmission to contact persons, as well as regular exchange of data and experience among experts and health authorities of European countries. In this context, studies on urban pigeons, wild birds and poultry have been conducted in Croatia, Macedonia, Bulgaria and other countries to assess the zoonotic risk (Prukner-Radovcic et al. 2004, 15

2005; Martinov, 2006). The availability of swabs collected for the French bird flu surveillance offered the opportunity to study the prevalence of avian chlamydiosis among a heterogeneous bird population. In this study based on the molecular detection of Chlamydiaceae by real-time PCR from cloacal and/or tracheal swabs, 7% of the samples were diagnosed positive, which corresponded to 11% of the birds studied. While most of the restriction patterns were genotype B or E-like, in some samples atypical patterns were observed (Laroucau et al., 2007). Furthermore, C. psittaci was demonstrated to be present in aborted fetal membranes from mares (Szeredi et al., 2005). This new finding indicates another possible source of human infection. The significance of chlamydiae in male genital tracts and ejaculates of ruminants and pigs has been studied to assess the potential role of venereal transmission (Kauffold et al., 2006a, 2006b; Teankum et al. 2006, 2007). A recent study has focused on Chlamydiales in guinea pigs and their zoonotic potential (Lutz et al., 2006; Wohlgroth-Lutz et al., 2006). A flock of diseased guinea pigs was found to be infected with C. caviae. The owner of the guinea pig flock was found to be infected (Parachlamydia acanthamoebae was also detected), as were in-contact animals (cat and rabbit). This suggests that zoonotic infection with C. caviae should be considered and appropriate advice given to owners of guinea pigs. The role of chlamydiae as a possible cause of mass deaths of free-ranging amphibians and frogs in Switzerland has been studied (Blumer et al., 2005). Cattle abortion of unknown infectious etiology still remains a major economic problem. Thus, lateterm bovine abortion cases were investigated for possible new abortigenic agents, such as Waddlia and Parachlamydia, resulting in the first description of Parachlamydia in the setting of bovine abortion in Switzerland (Borel et al., 2007). As Parachlamydia may be involved in lower respiratory tract infection in humans, caution should be taken when handling bovine abortion material because of the potential zoonotic risk. The association between infection with C. psittaci and ocular adnexal lymphomas of the MALT (mucosa-associated lymphoid tissue)-type (OAML) in humans has been studied in a patient, owner of a C. psittaci-infected canary, who developed two metachronous C. psittaci associated lymphomas (Ferreri et al., 2007b). The response to C. psittaci-eradicating antibiotic therapy with doxycycline has been assessed in patients with advanced-stage ocular adnexal MALT lymphoma. After therapy, C. psittaci DNA has no longer been detectable by PCR in all six treated patients, and durable complete or partial regression of lesions were achieved in three patients (Ferreri et al., 2007a). The detection of viable and infectious C. psittaci in conjunctival swabs and/or peripheral blood mononuclear cells (PBMC) in a proportion of OAML patients has been also reported (Ferreri et al., 2008). Final Remarks In its 4 years of existence, the Action has attracted the participation of more than 100 chlamydia researchers from 18 COST member states (Belgium, Bulgaria, Croatia, Germany, Former Yugoslav Republic of Macedonia, France, Great Britain, Greece, Hungary, Ireland, Israel, Italy, the Netherlands, Poland, Slovenia, Sweden, Switzerland, Spain) and 6 other countries (Australia, Argentina, Bosnia, China, Japan USA). Continuous exchange and interaction between research groups have led to a measurable increase in quantity and quality of chlamydia research in Europe. The publications of COST 855 scientists have contributed to raising the awareness of chlamydial infections, as well as their economic and zoonotic dimensions in many European countries. The major scientific achievements of this network will be published in a special issue of the journal Veterinary Microbiology later this year. Taking advantage of the financial support of the COST organisation, numerous activities of the participating researchers have created an active and efficient network, which will continue to function way beyond its official conclusion. 6. DISSEMINATION OF RESULTS 6.1 Publications and Reports (Give the total number. Detailed list in an annex) Total number: 345 Details see Annex 1 16

6.2 Conferences and Workshops First workshop on Research in the field of animal chlamydioses: Date: September 13th, 2003 Venue: Faculty of Veterinary Medicine of University College Dublin, Ireland Attendance: 15 participants from 9 countries Second workshop on Zoonotic Aspects of Animal Chlamydioses: Date: September 1st, 2004 Venue: Congress Centre of the Faculty of Science of Eötvös Lorand University, Budapest, Hungary Attendance: 72 participants from 17 countries Third workshop on Diagnosis and Pathogenesis of Animal Chlamydioses: Date: September 22-23, 2005 Venue: Auditorium, Banca di Monteriggioni, Siena, Italy Attendance: 98 participants from 19 countries Fourth workshop on Animal Chlamydioses and Zoonotic Implications: Date: September 3-5, 2006 Venue: Moredun Research Institute, Edinburgh, UK Attendance: 104 participants from 23 countries Fifth workshop on Pathogenesis, Epidemiology and Zoonotic Importance of Animal Chlamydioses Date: September 10-11, 2007 Venue: National Veterinary Research Institute, Pulawy, Poland Attendance: 90 participants from 21 countries 6.3 Web site The COST 855 homepage at http://www.vetpathology.uzh.ch/forschung/costaction855.html is hosted by the University of Zurich. It has basic information on objectives, working organisation, the management committee, progress reports, selected publications and meetings. Additionally, a temporary website for the 4th workshop was set up in 2006 at http://www.moredun.org.uk/cost855/ 6.4 Scientific and Technical Co-operation (List briefly co-operation and contacts established with scientific institutions, with other research programmes, especially in the EU framework programme, and with potential users) Members of COST 855 have attended meetings of COST 854 "Protozoal reproduction losses in farm ruminants", COST 845 on Brucellosis, and vice versa. MC member Dr. B. Markey was invited by the management committee of COST 854 to attend the final meeting of this Action in Liège in September 2006. He presented an overview of chlamydial infections in animals and the activities of COST 855. Discussions were held on possible future links between the two groups of researchers and future COST Action proposals. 17

The chairman of COST 855 participated in the workshop of COST Action B28 "Array technologies for BSL3 and BSL4 pathogens" held on December 10 12, 2007, in Vienna, Austria. There he presented the major achievements of COST 855 in the field of DNA microarray technology. Besides, there have been sporadic contacts to the EU Concerted Action on Wildlife Zoonoses. 6.5 Transfer of results (List briefly co-operation and contacts established with the Commission, with normalisation and standardisation bodies, with industry and operators) In order to disseminate the results of COST 855 members on development of new diagnostic tests, contacts were established with the following companies: Intervet, Boxmeer, Netherlands IDEXX Bern, Switzerland Institut Pourquier, France Veterinaria AG, Zurich, Switzerland Clondiag Chip Technologies, Jena, Germany medac, Hamburg, Germany. In order to disseminate the results of COST 855 members on development of new vaccines, contacts were established with the following companies: CEVA, Budapest, Hungary Pfizer, UK and Germany Impfstoffwerk Dessau, Germany Intervet, Boxmeer, Netherlands 6.6 Contacts in the ERA (List the contacts, if any, with other activities in the European Research Area (ERA), e.g. integrated or targeted projects, NoE, EUROCORES, etc.) none 7. ECONOMIC DIMENSION (List estimate of total manpower expressed in person-year dedicated to the activities of the action for each year and the total duration of the action) (List funds received from the COST budget for each year and for the entire duration of the Action utilised for Secretariat, Publications, Workshops and Seminars, MC meetings, Short-Terms scientific missions, other and Total) Estimate of manpower 2004: 72 participants from 17 countries 2005: 98 participants from 19 countries 2006: 104 participants from 23 countries 2007: 90 participants from 21 countries 18