Proceedings

1

Protozoa are amongst the main source of infectious diseases for humans and animals transmitted

orally. The aim of the workshop is to underline the main key questions for the research concerning

either public and animal health. The symposium excludes the protozoa transmitted by arthropods and

will focus more deeply on the new development on protozoa transmitted by food or water (Giardia,

Toxoplasma, Cryptosporidium…) or orally transmitted (Coccidia, Histomonas…). The symposium will

consider various approaches like the genome sequencing, proteomic development, transgenic

protozoa as a tool particularly to explain the strategy to study virulence factors. The new

developments for the diagnosis and control will also be discussed.







Support and thanks: Société Française de Parasitologie, réseau ZOOPNET (MedVetNet), réseau

ANOFEL Cryptosporidie, Département SA INRA, Centre INRA de Recherche Tours Nouzilly,

ECOS/ANUIES MS06S03, ENVA, AFSSA.







Organisation: INRA Animal Health Department (P Boireau, J Cabaret), Zoopnet (MedVetNet) (SM

Cacciò)





Local organizing committee : P Boireau (INRA Animal Health Department), J Cabaret, D Licois, M

Naciri, F Laurent (INRA UR IASP), Daniel Bout (UMR INRA Univ 483IPV)





Scientific committee: Simone M Cacciò (ISS, Italy), Francis Derouin (ANOFEL cryptosporidia

network, France), Jean Dupouy-Camet (Hôpital Cochin, Service de Parasitologie, France), Alain

Chauvin (UMR INRA ENVN 1034 IPHM, France), Daniel Bout (UMR INRA Univ 483IPV), Lionel

Zenner (UMR INRA ENVL, 958PEV, France), Jacques Cabaret and Dominique Licois (INRA UR IASP

France), Guadalupe Ortega-Pierres (CINVESTAV Mexico)









2

PROGRAMME





International workshop on Protozoa orally

transmitted in public and animal health

14-15th December 2006

INRA Research Center, Tours Nouzilly, France



Thursday December 14th 2006

10:00 - 10:30am: Registration/opening address

 Welcome address from the INRA research Centre of Tours. Catherine Beaumont INRA,

Nouzilly

 Welcome address from the French Society for Parasitology: Jean Dupouy-Camet (French

Society for Parasitology)



 Endemic protistan intestinal parasitoses in Denmark

C. Rune Stensvold and Henrik V. Nielsen. Department for Bacteriology, Mycology and Parasitology,

Artillerivej 5, 2300 Copenhagen S, Statens Serum Institut, Denmark. 15 mn



10:30am - 1:30pm: PROTOZOA AND DRINKING WATER

Chairpersons: Guadalupe Ortega-Pierres, Francis Derouin, Rachel Chalmers

 Information on the II International on Giardia and Cryptosporidium Conference, Morelia,

Mexico, 13-18 May, 2007 (by Guadalupe Ortega-Pierres). 3 mn



EPIDEMIOLOGY

 Cryptosporidium strain collection and genotyping in France: the ANOFEL-Cryptosporidium

National Network. Francis Derouin for the ANOFEL-Cryptosporidium Network. Laboratory of

Parasitology-Mycology, Saint-Louis Hospital, and Faculty Denis Diderot Paris 7, Paris.15 mn

 Environmental transmission of Cryptosporidium and Giardia. Simone M. Cacciò. Department

of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Roma.15 mn

 Giardia and Cryptosporidium in calves in Belgium: importance for animal and human

health. T. Geurden, S. Casaert, J. Vercruysse and E. Claerebout. Laboratory for Parasitology, Faculty

of Veterinary Medicine, Ghent University. 15mn

 Cryptosporidium and Giardia in Danish cattle and pigs: Prevalence of different species and

genotypes. Maddox-Hyttel, Charlotte, Langkjær, Rikke B., Enemark, Heidi L., Vigre, Håkan.

Department of Veterinary Diagnostics and Research, Danish Institute for Food and Veterinary

Research (DFVF), Copenhagen, Denmark. 15mn

 Genotypic analysis of Enterocytozoon bieneusi isolates from Gabon and Cameroon:

reporting a new highly divergent sequence and a wide distribution of genotypes. Breton J.,

Biligui S., Nzamba C., Okome M., Carbone A., Accoceberry I., Kombila M. and Thellier M.

Département de Parasitologie-Mycologie, Faculté de Médecine, Libreville, Gabon/Centre de

Traitement Ambulatoire, Hôpital général de Libreville/Service des Maladies Infectieuses, Hôpital de la

Fondation Jeanne Ebori, Libreville, Génomique Analytique, Université Pierre et Marie Curie-Paris,

Service de Parasitologie-Mycologie, Hôpital Saint-André, Bordeaux, Service de Parasitologie-

Mycologie, Pavillon Laveran, CHU de la Pitié-Salpêtrière, et Unité INSERM 511 Paris. 15 mn

 Molecular genetic characterization of Cryptosporidium in humans in the Netherlands. Peter

R. Wielinga, Ankje de Vries, Laetitia M. Kortbeek, Joke W.B. van der Giessen. National Institute for

Public Health and the Environment (RIVM), Microbiological Laboratory for Health Protection/RIVM,

Laboratory for Infectious Diseases Surveillance and Screening. 15 mn

 Advances in molecular surveillance and investigation of waterborne Cryptosporidium

outbreaks. Rachel Chalmers, Kristin Elwin, Guy Robinson, Stephen Hadfield and the north west

Wales outbreak control team. UK Cryptosporidium Reference Unit (UK CRU), NPHS Microbiology

Swansea, Singleton Hospital. 15 mn







3

MECHANISMS OF INFECTION

 Cryptosporidium parvum induces a specific post-translational regulation of the

oligopeptides transporter in neonatal rats. Perrine Marquet, Laurence Barbot, Aurélia Planté, Jean

François Huneau, Jean Gérard Gobert and Nathalie Kapel. Faculté de Pharmacie, Paris. 15 mn

 Identification of a new surface adhensin protein on sporozoite and oocyst stages of

,

Cryptosporidium parvum by screening of a phage-display cDNA library. Jigang Yin An Guo, Zhe

Hu, Qijun Chen. Research Institute of Zoonosis, Jilin University, Changchun / Key Laboratory of

Zoonosis, Ministry of Education, Changchun. 15 mn

 Giardia duodenalis: role of Calcium and PKC in encystment induction, insights on the

formation of cyst wall. M. Guadalupe Ortega Pierres. Department of Genetics and Molecular

Biology, Centro de Investigación y Estudios Avanzados Mexico. 15 mn



1:30-2:30pm: Lunch

2:30 - 5:00pm: PROTOZOA AND FOOD

Chairpersons: ML Dardé, D. Bout, J. van der Giessen

 Sarcoscystis and Toxoplasma in meat. Astrid Tenter, Germany. 20 mn



DIAGNOSTIC

 Development and evaluation of a new TaqMan real-time PCR assay for quantitative

detection of Toxoplasma gondii DNA. Jean Menotti, Yves J.F. Garin, Marie-Christine Serugue,

Janine Stanislawiak, Patricia Ribaud, Nathalie De Castro, Sandrine Houzé, and Francis Derouin.

Laboratory of Parasitology-Mycology/ Department of Hematopoietic Stem Cell

Transplantation/Department of Infectious Diseases, Saint-Louis Hospital/Laboratory of Parasitology-

Mycology, Bichat Hospital, Paris. 12 mn



EPIDEMIOLOGY

 French organization for Toxoplasma gondii strain collection and genotyping: Biological

Resource Centre (CRB ToxoBS) and National Reference Centre (CNR Toxoplasmosis). Dardé

ML, Ajzenberg D, Aubert D, Derouin F, Demar M, Villena I. CNR de la Toxoplasmose, Faculté de

Médecine / CHU / CRB Toxoplasma, Limoges, Faculté de Médecine / CHU / CRB Toxoplasma,

Reims, Faculté de Médecine, Paris V, CHU Cayenne.12 mn

 Estimation of soil contamination by Toxoplasma gondii in a urban area: preliminary results.

Afonso Eve, Lemoine Mélissa, Romand Stéphane, Aubert Dominique, Ravat Marie-Caroline, Thulliez

.

Philippe, Villena Isabelle, Riche Benjamin, Rabilloud Muriel, Gilot-Fromont Emmanuelle 2C2A-

CERFE, 08240 Boult-aux-Bois, Laboratoire de Biométrie et Biologie Evolutive, UMR CNRS5558,

Université Lyon 1, Institut de Puériculture de Paris, Laboratoire de Parasitologie-Mycologie, Hôpital

Maison Blanche, Reims. 12 mn

 Prevalence of Toxoplasma gondii in Croatian outdoor pigs detected by polymerase chain

reaction. Beck Relja, Marinculić Albert , Department for Parasitology,Veterinary Faculty,Zagreb 12 mn

 Toxoplasma gondii in pigs from different housing systems in The Netherlands.

 Joke van der Giessen, Manoj Fonville, Martijn Bouwknegt, Merel Langelaar, Ant Vollema, National

Institute for Public Health and the Environment (RIVM), Microbiological Laboratory for Health

.

Protection/ Food and Consumer Product Safety Authority, Zutphen. 12 mn

 Seroprevalence and factors associated to Toxoplasma gondii in cattle, sheep and goat

from Champagne-Ardenne, France. Aubert D., Gilot-Fromont E., Amine S., Hermitte, P., Gibout O.,

Geers R., Villena I. 12 mn



MECHANISMS

 Protective mechanisms set following immunization with Toxoplasma gondii extract-pulsed

dendritic cells in a murine chronic toxoplasmosis model. Guiton R., Zagani R., Bout D. and

Dimier-Poisson I. UMR 0483 Immunologie Parasitaire et Vaccinologie, Faculté des Sciences

Pharmaceutiques, Tours. 12 mn





ROUND TABLE TOXOPLASMA IMPACT ON PUBLIC HEALTH (AND

ANIMAL HEALTH) 40 mn





4

Friday December 15th, 2006

9:00am– 1:30pm: PATHOGENIC PROTOZOA AND ANIMAL HEALTH

IN DEARTH FOR CONTROL?

Chairs: J. Cabaret, F. Laurent, M. Shirley



 Uses and abuses of drugs: towards the end of the story

 A drastic reduction in available drugs in Europe: consequences for control. Example with

Histomonas. Lionel Zenner (UMR INRA ENVL Lyon) 15 mn

 Incidence of histomoniasis in turkeys in France since 2003: measures of disease frequency and

first approach to analysing survival data. Callait-Cardinal M.P., Leroux S., Chauve C.M., Le Pottier

G. & Zenner L. (UMR INRA ENVL Lyon) 10 mn

 Phasing out anticoccidial feed additives in poultry: are we ready? J-M Reperant (AFSSA

Ploufragan) 15 mn

 Sporozoan and plants common metabolic pathways and research of new drugs: Isoprenoid

biosynthesis in coccidia. Marc Clastre et al (Tours University) 15 mn

 Plants and microalgae extracts as a source of new drugs: what to expect? Christian Vivares (UMR

CNRS 6023, Clermont-Ferrand University)15 mn



 Vaccines: proposals and achievements?

 The precocious strains from rabbits: a paradigm from nature? Dominique Licois, Alysson

Niepceron and Cedric Neveu (INRA, Nouzilly) 15 mn

 Use of an attenuated Toxoplasma strain in sheep vaccination. Dimier-Poisson I, Moiré N,

Ismael A, Olivier M., Lebrun M, Dubremetz JF, Ducournau C, Bout D, Mévélec MN (UMR-INRA

Pharmacy Faculty, Tours/UMR 5539 CNRS-Université de Montpellier 2, CP 107, Place Eugene

Bataillon, 34090 Montpellier,) 15 mn

 Protective antigens in Eimeria maxima. Martin W Shirley (Compton, UK) 25mn



 Immunostimulation strategies to control neonatal diseases. Fabrice Laurent (INRA Nouzilly)

15 mn



 Open communications:

 Pathogenesis of Histomonas in turkey. Karine Huber (INRA ENVL, Lyon) 10 mn

 Intracellular development of Eimeria tenella: role of parasitic heat shock protein and

modulation of cellular apoptosis. Marylène Péroval and Marie Labbé (INRA Jouy) 10 mn

 Diagnosis of avian coccidioses: do we need molecular tools? Jean-Michel Reperant and

Martine Thomas-Henaff. 10 mn





1:30-2:30pm: Lunch



2:30pm: Poster session: Presentation in front of the poster (1-2mn each). This will be arranged as a

guided visit

3:00pm: European programmes in construction: Presentations

3:30pm: Rules and demands for projects: INRA Cell for EU programmes

4:00pm: End of meeting and return to Tours by bus









5

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006









ABSTRACTS



Oral communications









6

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006





Prevalence of Toxoplasma gondii in Croatian outdoor pigs detected by polymerase chain

reaction



Beck Relja, Marinculić Albert

Department for Parasitology

Veterinary Faculty

Zagreb, Croatia



Consumption of improperly prepared pork meat products has been considered a major risk for

Toxoplasma infection in humans. Outdoor and backyard farming have been associated with the higher

incidence of Trichinella infection while other food borne parasites appear to be neglected. In order to

prove the risk of Toxoplasma we have examined muscle tissue of traditionally slaughtered pigs. Meat

samples were collected during routine inspection for the presence of Trichinella larvae. Totally 68

meat samples were examined by nested polymerase chain reaction. DNA was extracted from one

gram of muscle tissue. Nested polymerase chain reaction analysis, using primers specific for the

Toxoplasma gondii SAG2 locus, revaled the presence of this protozan in 18 meat samples. Meat from

pigs reared in backyards is used to prepare traditional delicacies that are usually cured, smoked,

salted, pickled or air-dried. Usually the sausages are prepared from the meat of several pigs that is

homogenously mixed. This increases the possibility for the spreading of T. gondii cysts in many

different meat products. The prevalence of 26, 47 % infected samples clearly point out that

Toxoplasma gondii is present in high number of husbandries and the real number of infected animals

could be even higher. According to the data of the Croatian Ministry of Agriculture, Forestry and Water

Resources, almost 40 % of pigs are reared in small backyards and outdoor farming condition. High

percentage of infected traditionally reared animals clearly indicate that there is also another very

important food borne parasite beside Trichinella in Croatia.









7

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Genotypic analysis of Enterocytozoon bieneusi isolates from Gabon and Cameroon : reporting

a new highly divergent sequence and a wide distribution of genotypes.

1,6 6 2 3 4 5 1

Breton J.,Biligui S., Nzamba C., Okome M., Carbone A., Accoceberry I., Kombila M. and

6

Thellier M.

1 2

Département de Parasitologie-Mycologie, Faculté de Médecine, Libreville, Gabon; Centre de

3

Traitement Ambulatoire, Hôpital général de Libreville, Gabon; Service des Maladies Infectieuses,

4

Hôpital de la Fondation Jeanne Ebori, Libreville, Gabon; Génomique Analytique, Université Pierre et

5

Marie Curie-Paris 6, France; Service de Parasitologie-Mycologie, Hôpital Saint-André, Bordeaux,

6

France; Service de Parasitologie-Mycologie, Pavillon Laveran, CHU de la Pitié-Salpêtrière, et Unité

INSERM 511 Paris, France.



Intestinal microsporidiosis is a leading cause of chronic diarrhoea and wasting in severely

immunocompromised HIV positive patients. Microsporidia are obligate, intracellular parasitic protozoa

that disseminate through shedding of small, environmentally resistant mature spores. Two species can

cause gastrointestinal disease, Enterocytozoon bieneusi and Encephalitozoon intestinalis. E. bieneusi

has also been isolated from a large number of animals and potential zoonotic transmission is

supported by phylogenetic analysis of the Internal Transcribed Spacer (ITS) region of the rRNA gene.

Since infectious spores are shed in the host‟s faeces, the transmission routes of this pathogen may

involve person-to-person as well as waterborne or foodborne contaminations, which has led to its

classification as NIH category B biodefense pathogens and EPA microbial contaminant candidates.

Intestinal microsporidiosis is a public health problem in Africa due to the magnitude of the HIV

pandemics and poor level of sanitary conditions. However, data on the prevalence of E. bieneusi in

Africa is scarce, and values differ greatly (5% to 50%), depending on the population studied and the

methods used for diagnosis.

We conducted two studies of E. bieneusi prevalence in Central Africa, the first in HIV positive patients

from an urban setting in Gabon and the second in a non selected rural population in Cameroon (HIV

prevalence rate = 0.5%). Stool samples were analysed by IFAT using species specific monoclonal

antibodies and PCR. 25 out of 822 HIV positive patients from Gabon and 22 out of 758 villagers in

Cameroon were found positive for E. bieneusi. The prevalence rates were surprisingly similar in both

studies (2.9% and 3.0%), considering the differences in the populations studied.

Genotypic analysis of the ITS showed a high degree of genetic diversity in samples from both

countries. In Gabon, 15 isolates showed 7 different genotypes: the previously reported genotypes A

(6.6%), D (6.6%), and K (26.6%) as well as 4 new genotypes: CAF1 (19.9%), CAF2 (6.6%), CAF3

(6.6%) and CAF4 (26.6%). Interestingly, the set of genotypes found in the E. bieneusi isolates from

the rural population in Cameroon show some similarities in diversity but not in prevalence. Five

genotypes were found in 20 isolates, the known genotypes A (40%), B (15%), D (15%) and K (5%), as

well as the new genotype CAF4 (25%). Here genotypes A and CAF4 predominate, whereas genotype

K, CAF4 and CAF1 were more prevalent in Gabon. Thus, the risks of infection with different E.

bieneusi genotypes seem to be associated with immune status, occupational and living conditions.

Finally, phylogenetic analysis of the new genotype CAF4 indicates that it is highly divergent. It was

identified in both HIV-negative and positive patients in Gabon and Cameroon and may represent a

new Enterocytozoon species endemic to Central Africa.









8

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Environmental transmission of Cryptosporidium and Giardia



Simone M Cacciò

Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Viale

Regina Elena, 299, 00161 Rome, Italy.



Cryptosporidium and Giardia are genera of protozoan parasites that infect a wide range of vertebrates.

Species within these genera cause human cryptosporidiosis and giardiasis which probably constitute

the most common causes of protozoal diarrhoea, worldwide, and lead to significant morbidity and

mortality in both the developing and developed world. Cryptosporidium and Giardia can be transmitted

to humans via any mechanism by which material contaminated with faeces containing infectious

Cryptosporidium oocysts or Giardia cysts can be swallowed by a susceptible host. Water and food

play an increasingly recognized role in the epidemiology of these diseases. Molecular techniques are

available to determine species and genotypes of Cryptosporidium and Giardia and to distinguish

human from non-human pathogens. Validated methods to determine the species, genotype and

subgenotype present in heterologous mixtures should be applied to environmental samples to permit

monitoring and characterization of infection sources, disease tracking and to establish causative links

to both waterborne and foodborne outbreaks. Meaningful interpretation of population structures and

occurrence / prevalence baselines can only be performed by analysing a well planned set of samples

from all possible sources, taken regularly, over time, rather than focussing on outbreak investigations.

For food, this includes such analyses in its country of origin.









9

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Incidence of histomoniasis in turkeys in France since 2003: measures of disease frequency

and first approach to analysing survival data.

1 2 1 2 1

Callait-Cardinal M.P. , Leroux S. , Chauve C.M. , Le Pottier G. & Zenner L.

1

UMR958 Protozoaires Entéricoles des Volailles, INRA – École Nationale Vétérinaire de Lyon, F-

69280 Marcy l‟Étoile.

2

Comité Interprofessionnel de la Dinde Française (CIDEF), 11 rue de Plaisance, BP24, F-35310

Mordelles



The aim of this study was to describe the temporal dynamics of histomoniasis outbreaks in turkeys in

France since 2003. Two questionnaires were used to collect the data and were combined with several

laboratory samples. The analysis was performed using several measures of disease frequency and

first analyze of survival data.



The results showed a seasonal effect with the majority of cases occurring during the hottest months,

from April to September. A very high number of cases were found among birds from 4 to 8 weeks of

age, but surprisingly some of them arose in younger (3 weeks) and much older birds (up to 17 weeks).

The mortality rate was mostly below 10% but is above 30% in nearly 20% of cases. The study of

survival curves revealed several profiles: a profile with high slope (HSP), with low survival rate before

culling and early mortality peak of high intensity after first clinical signs; a profile with middle slope

(MSP), with middle survival rate and late mortality peak of medium intensity; a profile with low slope

(LSP), with high survival rate and no mortality peak. A preliminary analysis of the survival data showed

that liver lesions were systematically present in the cases with HSP. Moreover, the enteric diseases

(necrotic enteritis, non specific enteritis or coccidiosis) tend to be more frequent in this profile. We will

discuss several hypotheses for a better understanding of these profiles.









10

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Advances in molecular surveillance and investigation of waterborne Cryptosporidium

outbreaks.



Rachel Chalmers, Kristin Elwin, Guy Robinson, Stephen Hadfield and the north west Wales outbreak

control team

UK Cryptosporidium Reference Unit (UK CRU), NPHS Microbiology Swansea, Singleton Hospital,

Swansea SA2 8QA



Since 2000, the UK CRU has been investigating the molecular epidemiology of human

cryptosporidiosis in the United Kingdom. Primary diagnostic laboratories have been sending

Cryptosporidium-positive diagnostic faeces for species/genotype identification, leading to the creation

of an archive of over 14000 isolates (oocysts, DNA and patient data). This work has shown the equal

predominance of C. parvum and C. hominis across the country as a whole, with variation by person,

time and place. Significant species-specific risk factors have been identified. The diversity of infecting

isolates has also been revealed: one of at least 8 other species/genotypes have been found in 4%

human cases.



In the autumn of 2005 an increase in human cases of cryptosporidiosis was identified locally in North

West Wales. Routine testing at the CRU using PCR-RFLP at the Cryptosporidium oocyst wall protein

gene identified C. hominis in nearly all cases. The cases were located predominantly within water

supply zones sourced from an upland reservoir. C. hominis was detected in untreated and treated

drinking water. To investigate whether there was an on-going source of contamination and to locate

this, a programme of environmental sampling was established within the hydrological catchment of the

reservoir. Sampling included septic tanks, sewage treatment works and effluent, and surface waters.

Treated water continued to be sampled by continuous filtration. Samples were analysed by oocysts

capture using immunomagnetic separation, stained using a fluorescent monoclonal antibody and

examined by fluorescence microscopy. Cryptosporidium-positive microscope slides from 59 samples

were processed using an optimised technique for the removal of material and revocery of DNA from

slides containing very low oocyst numbers (e.g.: a single DAPI positive oocyst). Molecular

characterisation was carried out using PCR-RFLP of the SSU rRNA gene, in pentuplicate, followed by

double stranded DNA sequencing and further sequence analysis of the GP60 gene to compare

alleles. C. hominis was prevalent in this catchment, particularly in waters under the influence of

sewage, but other species/genotypes were also identified. The same gp60 C. hominis allele was

identified in human cases and environmental samples.



This work demonstrates that long-term country-wide genotyping is possible, informs the epidemiology

of human cryptosporidiosis, and provides an archive for further investigation of isolates. It also shows

the practical application of techniques during outbreak investigations to provide information in a timely

manner for public health purposes.









11

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Giardia and Cryptosporidium in calves in Belgium: importance for animal and human health



T. Geurden, S. Casaert, J. Vercruysse and E. Claerebout

Laboratory for Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-

9820 Merelbeke, Belgium



A cross-sectional survey on 100 dairy farms in Flanders, Belgium, was conducted to determine the

prevalence of Giardia and Cryptosporidium. Faecal samples were collected from 496 calves between

the age of 1 and 70 days. Because there is no gold standard for the detection of Giardia and

Cryptosporidium, a Bayesian approach was used on a subset of 235 samples to determine the test

characteristics of different diagnostic assays and to obtain prevalence estimates. For the detection of

Giardia, three diagnostic assays were used: microscopical examination (ME), an immunofluorescence

assay (IFA) and an antigen-detection ELISA. Six diagnostic assays were used for Cryptosporidium:

ME, IFA, two ELISAs and two different PCR assays (18S rRNA and COWP). When the results of the

„conventional‟ techniques (ME, IFA, ELISA) were used in a first Bayesian model, ELISA and IFA were

both sensitive and specific diagnostic techniques for Giardia, whereas ME was less sensitive. The

estimated prevalence of Giardia in dairy calves was 19% (42% positive farms). For Cryptosporidium,

the model using the conventional techniques estimated that the calf prevalence was 16% (39%

positive farms) and that the specificity of IFA and ELISA was high, compared to ME. However, when a

six-test Bayesian model was developed, including the two PCR assays, the estimated calf prevalence

increased to 58% and the sensitivity of the conventional techniques decreased. Since clinical

cryptosporidiosis is especially important in calves before the age of one month and oocyst excretion

peaks in calves from this age category, ME, IFA and ELISA are suitable for clinical diagnosis. For

epidemiological studies however, the higher sensitivity of PCR assays might be needed to identify

subclinical reservoir animals. Using the IFA, the prevalence of Giardia and Cryptosporidium was also

estimated in 333 beef calves between the age of 1 and 70 days on 50 beef farms. Giardia was

detected in 34% of the calf samples (64% positive farms) and Cryptosporidium in 6% of the beef

calves (25% positive farms).

Randomly selected samples from dairy (n=49) and beef calves (n=37) as well as samples from calves

with diarrhea (n=26) were sequenced to compare the occurence of different Giardia and

Cryptosporidium genotypes. For the identification of Giardia the ß-giardin gene was used. Both G.

duodenalis assemblage A (34%) and assemblage E or G. bovis (66%) were frequently identified,

indicating that the zoonotic assemblage A might be more prevalent in calves in Europe than previously

assumed. For Cryptosporidium the HSP-70 and the 18SrRNA gene were targeted. C. parvum positive

samples were also sequenced using the 60kDa glycoprotein (gp60) gene for subgenotype analysis. In

the majority of the samples C. parvum was found (86%, subgenotype IIa), next to C. bovis (13%) and

C. suis (1%). In Cryptosporidium positive samples from calves suffering from diarrhea only C. parvum

was identified, confirming that C. parvum is the major species in the ethiology of neonatal

cryptosporidiosis. In the majority of Giardia positive samples from calves with diarrhea assemblage E

was identified (77%), suggesting that clinical symptoms in calves are predominantly caused by the

livestock assemblage.

Because the prevalence of both parasites in calves was high and the majority of positive calves

excreted zoönotic genotypes of Giardia and Cryptosporidium, further work will investigate the possible

role of cattle as a source of Giardia and Cryptosporidium in drinking water in Belgium, and possible

measures to decrease (oo)cyst excretion in calves, based on risk factor analyses.









12

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Isoprenoid biosynthesis in coccidia

a a,b c d d

Clastre Marc , Goubart Armelle , Prel Anne , Mincheva Zoia , Viaud-Massuart Marie-Claude , Bout

b a b c

Daniel , Rideau Marc , Velge-Roussel Florence and Laurent Fabrice

a

EA2106 Biomolécules et Biotechnologies Végétales, UFR Sciences Pharmaceutiques, Université de

Tours, 37200 Tours, France

b

INRA UMR 483 Immunologie Parasitaire et Vaccinologie, UFR Sciences Pharmaceutiques,

Université de Tours, 37200 Tours, France

c

INRA UR1282 IASP, Equipe Contrôle et Immunologie des Maladies Entériques du Nouveau-né,

37380 Nouzilly, France

d

EA3857 Laboratoire de Synthèse et Physicochimie Organique et Thérapeutique, UFR Sciences

Pharmaceutiques, Université de Tours, 37200 Tours, France



The extensive efforts invested in sequencing apicomplexa genomes and especially the wealth of

information now available concerning their metabolic pathways have opened new possibilities for the

development of specific drugs with low toxicity for the mammalian host. The mevalonate-independent

methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis was initially discovered in

eubacteria. Subsequently identified in plastids of higher plants, algae and in many pathogenic

microorganisms including the malaria parasite P. falciparum, this pathway differs from the mevalonate

pathway found in mammals. The existence of the MEP pathway in P. falciparum and the discovery of

the antimalarial activity of the antibiotic fosmidomycin led us to consider the presence of this pathway

in other protozoan parasites collectively referred to as the coccidian like Cryptosporidium parvum and

C. hominis, Toxoplasma gondii and Eimeria tenella.



Extensive nucleotide database searches for genes encoding the isoprenoid enzymes identified

members of the MEP pathway in both T. gondii and E. tenella while Cryptosporidium species lack both

the mevalonate and the MEP pathways.

The operativity of the MEP pathway in E. tenella and T. gondii was confirmed by gene expression

studies. Indeed, RT-PCR analysis showed that the MEP pathway genes are expressed in various

forms of the E. tenella life cycle and in the active infectious form of T. gondii.



The effect of fosmidomycin was investigated in vitro by treating Chicken cells infected with E. tenella

sporozoites and mouse cells infected with T. gondii tachyzoites. The drug was poorly effective even at

high concentrations.



Thus, both fosmidomycin sensitivity and isoprenoid metabolism differs substantially between

apicomplexan species and the results acquired from malaria drug development must therefore only be

extended to other apicomplexa with great precaution.









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International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



French organization for Toxoplasma gondii strain collection and genotyping: Biological

Resource Centre (CRB ToxoBS) and National Reference Centre (CNR Toxoplasmosis)

1,2 1,2 1,3 1,4 1,5 1,3

Dardé ML , Ajzenberg D , Aubert D , Derouin F , Demar M , Villena I

1

. CNR de la Toxoplasmose

2

. Faculté de Médecine / CHU / CRB Toxoplasma, Limoges

3

. Faculté de Médecine / CHU / CRB Toxoplasma, Reims

4

. Faculté de Médecine, Paris V

5

. CHU Cayenne, French Guiana



In December 2002, a grant from the French Ministry of Research allowed the creation of a Biological

Resource Centre (CRB) dedicated to Toxoplasma strains. Isolates collected by a network of 28

French parasitologists are sent to this centre, co-located in Reims and in Limoges. Some

parasitologists from other countries collaborate to this collection (Portugal, Danemark, Belgium, Iran,

Uruguay).

Since 2006, a National Reference Centre for toxoplasmosis is created based on a network of

laboratories; 4 laboratories are recognized as associated laboratories along specific experience

(epidemiology, serology, molecular technics, and characterization of T. gondii strains). Laboratories in

charge of the CRB were recognized as associated laboratories to the National Reference Centre for

toxoplasmosis.

For inclusion in CRB, each isolate is accompanied by clinical and epidemiological data. Isolates are

cryopreserved after multiplication in cell culture or after mouse inoculation. The CRB Toxoplasma also

receive Toxoplasma DNA isolated from pathological products. This bank of strains is available for

scientific projects.

The genotype of each isolate or DNA extract is determined by a microsatellite analysis (Multiplex PCR

with 5 microsatellites) and by PCR-RFLP on 3 genes (SAG1, SAG2, GRA7). In case of atypical

genotypes, a multilocus sequencing typing is performed. Five other more polymorphic microsatellites,

with a high discriminatory power, are also available for fingerprinting (human outbreak,

epidemiological tracking in animals products).

667 isolates or DNA have been sent to the CRB (481 from human cases of toxoplasmosis, 186 from

animal products). Human cases correspond to 340 congenital toxoplasmosis, 42 cases of

toxoplasmosis in immunodeficient patients and 24 cases of acquired toxoplasmosis in

immunocompetent patients. There is an overwhelming predominance of type II strains in cases of

congenital toxoplasmosis in this sample (91%) and the few non-type II isolates are found mainly in

cases where contamination occurred outside the French metropolitan territory. The predominance of

type II is lower in immunodeficient patients (49%). In this group of patients, the influence of the

geographical origin of the contamination can partly explain the different repartition of genotypes

(African patients mainly harboured genotypes with a mixture of type I and III alleles), but it does not

explain the higher proportion of type III. The 24 cases of toxoplasmosis in immunocompetent patients

are severe cases observed mainly in French Guiana (19 cases) associated to atypical genotypes and

cases of ocular toxoplasmosis (4 cases: 3 type II and one genotype I/III).

All the isolates from various species of domestic and wild animals collected in France were type II

isolates.









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International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Cryptosporidium strain collection and genotyping in France: the ANOFEL-Cryptosporidium

National Network

Francis DEROUIN for the ANOFEL-Cryptosporidium Network

Laboratory of Parasitology-Mycology, Saint-Louis Hospital, and Faculty Denis Diderot, university Paris

7, Paris, France.



Background: Cryptosporidium is recognized as a major cause of diarrhea worldwide and in all age

groups. In the past 20 years, this parasite has been responsible for significant outbreaks of

gastrointestinal disease mainly in North America and in United Kingdom but also throughout the world.

In France three outbreaks have been reported since 2000, involving an estimated number of 150 to

900 patients, but the true prevalence and incidence cryptosporidiosis in the French population is not

known. In a report published in 2002, the French Food Safety Agency (AFSSA) pointed out the lack of

information on human cryptosporidiosis in France and strongly suggested to reinforce investigation

means on Cryptosporidium in humans, animals and foods (including water resources)

Aim: Constitute a national network of human cryptosporidiosis in order to provide health public

authorities with information on the incidence and epidemiology of human cryptosporidiosis in France.

Methods: The ANOFEL Cryptosporidium National Network (ACNN) was constituted on a voluntary

basis by the French association of medical parasitologists (ANOFEL), and with the partial support of

AFSSA and the National Institut of Disease Surveillance (Institut National de Veille Sanitaire, INVS).

The network includes 32 hospital parasitology laboratories (mainly university-hospitals) having a

common working field on cryptosporidiosis. Following a proposal launched in April 2004 to the

ANOFEL members, the network was established in October 2004.

Each member is engaged to notify every new case of proven human cryptosporidiosis, collect samples

and related clinical/epidemiological data and participate once a year to an inter-laboratory diagnostic

test on network referenced samples. Cases of Cryptosporidium infection are considered either proven,

on the basis of the demonstration of oocysts by microscopy, or probable when only specific PCR is

positive. Laboratories proceed then to register case-related clinical and epidemiological data by using

data sheet forms and to the shipping of Cryptosporidium isolates or DNA samples together with duly

filled data forms to collecting centers.

Two collecting centers (Lille, Lyon) receive, acknowledge and store Cryptosporidium faecal or DNA

isolates (aliquots), provide aliquots for molecular identification, record isolate-associated data in the

database, provide isolate aliquots to potential requesters.

The ACCN meets at least once a year. A report of activities of the Network is presented at the annual

meeting of the ANOFEL association and to INVS.

rd

From December 2005 to November 23 , 2006, 104 cases have been notified, and 78 isolated were

collected. Forty-four were genotyped.

Conclusion: Within 2 years the ANOFEL Cryptosporidium National Network proved efficient to collect

clinical samples and provide new information on human cryptosporidiosis in France. The collection

and preservation of well-defined samples will markedly support epidemiological research in France on

Cryptosporidium and improvement of diagnostic methods. Network activities will be extended to

giardiasis in 2007 on the same basis of organization.









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International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Estimation of soil contamination by Toxoplasma gondii in a urban area: preliminary results

1, 2, 4 2 3 4

AFONSO Eve , LEMOINE Mélissa , ROMAND Stéphane , AUBERT Dominique , RAVAT Marie-

2 3 4 2 2

Caroline , THULLIEZ Philippe , VILLENA Isabelle , RICHE Benjamin , RABILLOUD Muriel , GILOT-

2

FROMONT Emmanuelle

1

2C2A-CERFE, 08240 Boult-aux-Bois, France.

2

Laboratoire de Biométrie et Biologie Evolutive, UMR CNRS5558, Université Lyon 1, 43 bd du 11

novembre 1918, 69622 Villeurbanne Cedex, France.

3

Institut de Puériculture de Paris, 26 bd Brune, 75014 Paris, France.

4

Laboratoire de Parasitologie-Mycologie, 45 rue Cognacq Jay, Hôpital Maison Blanche, 51092 Reims

cedex., France



In urban areas, feral populations of mammals may reach high local densities and entail serious risk of

zoonosis transmission. Here we aim to estimate the soil contamination by oocysts of Toxoplasma

gondii in the gardens and grounds of the Croix-Rousse hospital in Lyon (France). In this site, a

population of domestic cats lives at high density (9.7 cats/hectare) and seroprevalence of T. gondii is

low (18.6%, Afonso et al. 2006).

We studied the defecating behaviour of cats, in order to search for areas most frequently used and to

analyze which categories of cats used each area. A first series of observations allowed us to identify

16 areas used by cats to defecate. Between February and August 2005, 45 identified cats were

regularly fed individually with food containing plastic marks and the marked feces were searched for in

the defecating areas. Among 260 marks, 66 were recovered. The use of defecating areas depended

on the age and gender of cats, in accordance with previous knowledge on home range in cats. We

predicted that soil contamination should be highly heterogeneous at a local scale.

We sampled soil in order to detect T. gondii, using RT-PCR following the method proposed by

Romand et al. (2003). In a first series of observations in April 2004, the whole site was sampled using

a stratified sampling plan. Three of the 55 samples were positive. In a second series of observations in

November 2005, the 16 areas used by cats were sampled with one to four samples per area. Eight

samples out of 62 were positive. In both experiments, the positive samples were localized only in

areas used by cats for defecation, and specifically in shaded areas, where oocyst survival was

possibly enhanced.

This study shows that oocysts can be detected in soil samples even when the serological prevalence

of T. gondii is low in cats. The distribution of oocysts is expected to be heterogeneous and may

depend on the behaviour of cats and on local characteristics.



Afonso E., Thulliez P., Gilot-Fromont E. 2006. Transmission of Toxoplasma gondii in an urban

population of domestic cats (Felis catus). International Journal for Parasitology 36 : 1373-1382.

Romand S., Fromont E., Pontier D., P. Thulliez P. 2003. Détection par PCR en temps réel de

Toxoplasma gondii dans les prélèvements de sol. Congrès de la Société Française de Parasitologie,

16-18 décembre 2003, AFSSA, Maisons-Alfort.









16

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Protective mechanisms set following immunization with Toxoplasma gondii extract-pulsed

dendritic cells in a murine chronic toxoplasmosis model.



Guiton R., Zagani R., Bout D. and Dimier-Poisson I.

UMR 0483 Immunologie Parasitaire et Vaccinologie, Faculté des Sciences Pharmaceutiques, Tours,

France



Toxoplasma gondii, an obligate intracellular protozoan, is the causative agent of toxoplasmosis. This

disease is usually severe during neurotoxoplasmosis and congenital toxoplasmosis, which is

responsible for numerous abortions or fetal deformations, in humans as well as in animals. No vaccine

is presently available ; so the development of effective vaccinal approches is still a topical question.

In our laboratory, we try to determine the role of dendritic cells at the origin of early and specific

immune responses, in the context of murine toxoplasmosis.

CBA/J mice immunization with a murine splenic dendritic cell line established and characterised in our

lab (SRDC), pulsed with T. gondii extract, induces a specific humoral immune response and a Th1

type cellular immune response (specific lymphoproliferation and IFN-γ secretion) which induce a

significant protection of mice, determined by a 70% decrease in the cerebral cyst load during the

chronic phase of infection.

To define lymphocytic effectors that interact with dendritic cells during the chronic phase of infection,

following a vaccination using pulsed or unpulsed SRDC, we used in vivo depletions of the two major

+ +

lymphocyte populations, LT CD4 or LT CD8 . Until now we were unable to determine the role of LT

+

CD4 in a convincing way. On the other hand, a strong correlation was observed between the

+

increasing number of intracerebral cysts and depletion of LT CD8 , thus showing their decisive role in

+

the protection of mice. Recent results seem to indicate that the LT CD8 act via IFN-γ and IL-10

production as seen following restimulation of mesenteric lymph nodes cells and splenocytes with

parasite extract.









17

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Infection with Histomonas meleagridis in turkeys: dissemination kinetics in tissues related to

pathology

1 2 1 1

Huber Karine , Belli Patrick , Reynaud Marie-Claude , Zenner Lionel

2

UMR958 Protozoaires Entéricoles des Volailles, INRA, Ecole Nationale Vétérinaire de Lyon, Marcy

l‟Etoile, France.

2

Unité d‟histologie, anatomie-pathologique, Ecole Nationale Vétérinaire de Lyon, Marcy l‟Etoile,

France.



Histomonas meleagridis is a flagellated protozoa causing histomoniasis, a disease of gallinaceous

fowl. This disease is characterized by necrotic typhlitis, hepatitis, and high mortality, especially in

turkeys. In an attempt to detect the progression of H. meleagridis in the turkey, birds were infected via

the cloaca. Between day 0 and 19, a group of four turkeys were killed and autopsied every 3 days.

Cecal and hepatic lesion scores were used to measure the severity of infection. For each turkey, 15

tissue samples were taken. Samples were analyzed by PCR and examined for histopathology and

scored. In the caecum the parasite was detected from D2 to D19 both by PCR and by microscopic

observation. First visible cecal lesions were observed at D5. Their intensity increased until D12. After

D12 a progressive recovery was observed. With histopathology, an infiltration by mononuclear cells of

lamina propria and submucosae was observed most of the time. The infiltration can reach the serosa

and lymphocytes can then invade the mesentere. In the liver, parasites were detected between D7

and D12. PCR detection occurred only in some severe lesions, probably due to an irregular

distribution of parasite inside the liver. First macroscopical hepatic lesions appeared at D7. The mean

hepatic lesion score is maximal at D9 and decreases from D12 to D14. Microscopic lesions consist in

areas of necrosis enclosed by mononuclear cells commingled with isolated hepatocytes. For these two

organs a similar evolution for histopatological scores was observed but with a slower lowering of the

scores at the end of the experiment. Parasite DNA was detected sporadically in other organs but the

parasite was never seen by microscopy. With histology, only aspecific inflammatory lesions were

observed. These results allow us to emit and discuss two main hypotheses on the dissemination route

of the parasite: through the bloodstream and by a direct transfer of the parasite along the peritoneum.









18

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Immunostimulation strategies to control neonatal diseases



Laurent, F., Barrier, M., Mancassola, R. and Lacroix-Lamandé, S.

Laboratoire Contrôle et Immunologie des Maladies Entériques du Nouveau-né, UR1282 Infectiologie

Animale et Santé Publique, INRA de Tours, 37380 Nouzilly, France.



During the first weeks of life, animals are particularly susceptible to all kinds of infections due to an

incomplete development of their immune system. Most infectious agents enter the body at mucosal

surfaces and therefore mucosal immune response functions as a first line of defence. In some cases,

maternal immunity (natural or following immunisation of the mother) is not sufficient to avoid the

infection of the newborns. This is the case for cryptosporidiosis which is the first cause of diarrhoeal

enteric diseases in young calves in France. The development of this zoonotic disease which affects

young or immunodeficient animals is dependent of the immune status of the host. Immunostimulation

strategies targeting the innate immune cells that can rapidly participate in the control of the infection in

neonates appear therefore to be a method of choice to reduce the severity of cryptosporidiosis.

Cells of the innate immune compartment express microbial pattern-recognition receptors among which

Toll-like receptors have retained a particular attention these last years. Once stimulated these

receptors transduce the complex signalling responses that are required for inflammation and for the

subsequent development of adaptive immunity. CpG motifs present in bacterial DNA are recognized

by Toll-like receptor 9 and trigger the production of TH1 cytokines and chemokines that are known to

be important in the defence against protozoan infections. We therefore investigated the role of oral or

parenteral administration of synthetic oligodeoxynucleotides (ODN) containing CpG motifs that mimic

those present in bacterial DNA, on the development of Cryptosporidium parvum infection.









19

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



The precocious strains from rabbits: a paradigm from nature?



Licois Dominique, Niepceron Alisson, Neveu Cédric

INRA, UR 1282, IASP-213, 37380 Nouzilly, France



Precocious strains are characterized by a shorter endogenous development than that of the wild

strains from which they derive. These strains have proven to be good candidates to live vaccines as

they were tested as protective in natural conditions. One of the main characteristics of the four

precocious strains of rabbit Eimeria is their very marked morphological difference compared to the

corresponding wild strains, regarding the refractile bodies. Thus precocious strains are easily identified

in rabbit, conversely to what is observed in avian Coccidia. Furthermore, a strong reduction of the

multiplication rate, pathogenicity and the duration of the life-cycle, indicates differences in the host

parasite interactions. Whereas wild strains are highly pathogenic, the precocious lines induce very low

pathogenicity but their immunogenicity is clearly preserved. We infer that those two strains

(precocious vs wild) are an excellent model for study of virulence in Coccidia. We decided to launch a

programme of characterization of the elements of the genome differentiating the precocious lines from

the wild strains, in order to detect genes/expressions implied in virulence. Accordingly, we retained

two techniques: a subtractive differential library, the SSH (Subtractive Suppressive Hybridization) and

the cDNA-AFLP. The parasitic model will be E. intestinalis. Recent methodological developments

enabled us to define the optimum parasitic stage making it possible to obtain the best outputs of

extraction of the RNA, ie the beginning of the sporulation of the oocysts. The methods of extraction of

the total RNA and mRNA or of obtaining cDNA were optimized for the studied parasite. Primers

necessary to control PCR for the SSH were required starting from genes described in Eimeria of

chicken. Those relating to the genes Hsp70 and Hsp90 have been validated for cDNA of E.

intestinalis. Lastly, and this is not one of the least advantages compared to the avian strains, we now

have isogenic lines for the two E. intestinalis strains (wild virulent and attenuated precocious line). The

difference could then be attributed to the status of virulence.









20

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Cryptosporidium and Giardia in Danish cattle and pigs: Prevalence of different species and

genotypes



Maddox-Hyttel Charlotte, Langkjær Rikke B., Enemark Heidi L., Vigre Håkan

Department of Veterinary Diagnostics and Research, Danish Institute for Food and Veterinary

Research (DFVF), Copenhagen, Denmark



An epidemiological survey of Giardia and Cryptosporidium in Danish livestock was conducted

comprising 50 randomly selected dairy and sow herds, respectively. Each herd was visited once for

the collection of faecal samples and registration of management parameters. Faecal samples were

collected from three different age groups of animals, i.e. five sows/cows, ten nursing piglets/calves

less than one month, and ten weaner pigs 8-45 kg / calves 1-12 months and the samples were

analysed for the presence of the two parasites by immunofluorescence microscopy.

The study revealed an age specific herd prevalence of Cryptosporidium of 16, 31 and 100% for sows,

piglets and weaners, respectively, and of 14, 96 and 84% for cows, young calves and older calves,

respectively. For Giardia the age specific herd prevalence was 18, 22 and 84% for the sows, piglets

and weaners, while for cattle herds the prevalence was 60, 82 and 100% for cows, young calves and

older calves, correspondingly.

The genetic diversity was determined by selecting a large proportion of the (oo)cyst containing

samples for molecular characterisation. Sequencing and phylogenetic analysis of the 18S rDNA locus

and/or the HSP70 gene of 183 pig and 154 cattle isolates of Cryptosporidium revealed the presence of

C. suis, pig genotype II, C. parvum (cattle genotype), C. bovis, Cryptosporidium deer-like genotype

and a novel C. suis-like genotype.

For both cattle and pigs, a host age related change in distribution of species/genotypes was observed.

The zoonotic C. parvum (cattle genotype) was most prevalent in young calves. For Giardia, 82 and

145 isolates from pigs and cattle, respectively, were analysed at the 18S rDNA locus and/or the gdh

gene. Giardia isolates belonging to the zoonotic Assemblage A were found in both young and older

calves, as well as in weaners and piglets, whereas cows seemed to be infected purely by isolates of

the livestock group, Assemblage E.









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International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Cryptosporidium parvum induces a specific post-translational regulation of the oligopeptides

transporter in neonatal rats

(1) (1,2) (1) (3)

Perrine Marquet , Laurence Barbot , Aurélia Planté , Jean François Huneau ,

(1) (1,2)

Jean Gérard Gobert and Nathalie Kapel

1. EA209 “Eucaryotes Pathogènes”, Faculté des Sciences Pharmaceutiques et Biologiques,

Université René Descartes, 75006 Paris, France

2. Service de Coprologie Fonctionnelle, Groupe-Hospitalier Pitié-Salpêtrière, 75013 Paris, France

3. UMR INRA/INA-PG Physiologie de la Nutrition et du Comportement Alimentaire, Institut National

Agronomique Paris-Grignon, 75005 Paris, France



Cryptosporidium parvum is a parasitic protozoa increasingly appreciated as a cause of intestinal

malabsorptive syndrome leading to malnutrition and/or growth failure. Since a major mechanism for

apical peptide absorption by small intestine is via the proton-coupled transporter PepT1, we

investigated the expression and functionality of this transporter in our model of acute cryptosporidiosis.

5

Four-day-old Sprague-Dawley rats were inoculated by gavage with 5.10 oocysts of C. parvum and

killed at day 12 (peak of the infection). PepT1 expression (mRNA and protein) and functionality were

quantified both in the distal small intestine, preferential site of C. parvum implantation, and in the

proximal small intestine, free of parasite, using RT-PCR, western blot, immunohistochemistry and

Ussing chambers, respectively. Despite transient mRNA over-expression, no difference in total PepT1

protein expression or in glycyl-sarcosine fluxes was observed in C. parvum-infected rats compared

with controls, both in the proximal and in the distal small intestine. However, a significant decrease of

apical membrane protein expression of PepT1 and a cytoplasmic accumulation were observed in C.

parvum-infected enterocytes compared with controls. These results strongly suggest that C. parvum-

infection leads to a post-translational regulation of PepT1 whereas mucosal aggression is usually

known to induce a regulation of PepT1 at the transcriptional level.









22

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Development and evaluation of a new TaqMan real-time PCR assay for quantitative detection of

Toxoplasma gondii DNA

1 1 1 1

Jean MENOTTI, Yves J.F. GARIN, Marie-Christine SERUGUE, Janine STANISLAWIAK, Patricia

2 3 4 1

RIBAUD, Nathalie DE CASTRO, Sandrine HOUZE, and Francis DEROUIN.

1 2

Laboratory of Parasitology-Mycology, Department of Hematopoietic Stem Cell Transplantation, and

3 4

Department of Infectious Diseases, Saint-Louis Hospital, and Laboratory of Parasitology-Mycology,

Bichat Hospital, AP-HP, Paris, France.



Background: T. gondii can be responsible for fetus infections leading to mild or severe sequelae, and

for life-threatening infections in immunocompromised hosts. As early diagnosis is essential to start

therapy, several PCR assays have been developed, but comparison of their respective sensitivities is

often lacking.

Aim: To develop a new 5‟-nuclease real-time PCR assay that targets the 200- to 300-fold repetitive

AF146527 gene and assess its performances for diagnostic and treatment follow-up.

Materials and methods: Primers and a TaqMan probe were designed to amplify an 83-bp fragment

of T. gondii AF146527 gene. A retrospective analysis was first performed on 144 clinical specimens

previously analyzed for the presence of T. gondii DNA by a PCR-ELISA assay that targets the B1

gene of T. gondii. A prospective analysis was then performed on the 203 clinical specimens received

at the laboratory of Parasitology of Saint-Louis hospital during a 4-month period. Each specimen was

tested by both assays. Additionally, iterative samples from a patient with cerebral and disseminated

toxoplasmosis already tested by a real-time PCR assay that targets B1 gene were tested by the real-

time PCR assay that targets AF146527 gene.

Results: All positive samples by the PCR-ELISA that targets B1 gene were positive by the real-time

PCR that targets AF146527 gene. All 15 samples negative by PCR-ELISA and positive by real-time

PCR belonged to patients with proven toxoplasmosis, presenting other positive samples by both

techniques. Moreover, the real-time PCR assay on AF146527 gene allowed detection with a mean

gain of 7.1 amplification cycles when compared to the real-time PCR assay that targets B1 gene.

Conclusion: This study demonstrating the higher sensitivity of the 5‟-nuclease real-time PCR assay

developed on AF146527 gene confirms the interest of using this highly repeated target to improve the

diagnosis of toxoplasmosis.









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International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Used of an attenuated toxoplasma strain in sheep vaccination

1 1 1 2 3 3 1 1

Dimier-Poisson I, Moiré N, Ismael A, Olivier M., Lebrun M, Dubremetz JF, Ducournau C, Bout

1

D, Mévélec MN

1

UMR Université-INRA d'Immunologie Parasitaire et Vaccinologie Université François-Rabelais de

2

Tours; INRA, UFR des Sciences Pharmaceutiques, 31 Avenue Monge, 37200 Tours, INRA UR 1282

3

Infectiologie Animale et Santé Publique (IASP) 37380 Nouzilly, UMR 5539 CNRS-Université de

Montpellier 2, CP 107, Place Eugene Bataillon, 34090 Montpellier, FRANCE.



We evaluated a new vaccine candidate, Mic1-3KO in mice and sheep. Mic1-3KO is a mutant strain of

Toxoplasma gondii RH that lacks the MIC1 and MIC3 genes. During early invasion, T. gondii secretes

proteins from micronemes that play a central role in the recognition of and adhesion to host cells.

Disruption of either the MIC1 gene or the MIC3 gene slightly reduced virulence in mice, and the

double knockout is markedly impaired in virulence.

We examined the ability of Mic1-3KO to protect OF1 mice against both chronic and congenital

infection and to protect sheep against abortion. We also studied the early migration of Mic1-3KO in

mice and the development of cysts in mice and sheep after injection of the Mic1-3KO.

Swiss OF1 mice were vaccinated with 20 Mic1-3KO tachyzoites and challenged orally with T. gondii

(76K strain). Mic1-3KO induced a strong humoral and cellular Th1 immune response and conferred

highly significant protection against chronic infection in mice (2 years old) and 2-year old cattle. Prevalence

was lower when cattle was introduced recently, suggesting that toxoplasmosis may be acquired

locally. Concerning herd-level factors, the final model retained the effects of presence of cats,

presence of cervids, geographical isolation of the farm and presence of running water as significantly

associated with prevalence. Local characteristics were determinant in herd prevalence.









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International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Microalgae extracts as a source of new drugs: what to expect?

VIVARES C.P., EL ALAOUI H.

UMR CNRS 6023, Laboratoire Biologie des Protistes, Université B. Pascal, 63177 Aubière Cedex,

France



Coccidiosis is recognized as the parasitic disease that has the greatest economic impact on poultry

production. The annual worldwide cost is estimated at about $800 million, and that for the American

broiler industry about $450 million. This includes the costs of prophylactic in-feed medication for

broilers and broiler-breeders, alternative treatments if the medications fail, and losses due to mortality,

morbidity, and poor feed conversions of birds that survive outbreaks. A growing number of natural

products or feedstuffs are tested as anticoccidial dietary additives. The use of and search for drugs

and dietary supplements derived from natural sources (plants and microalgae) have accelerated in

recent years. Ethnopharmacologists, botanists, microbiologists, and natural-products chemists are

combing the biodiversity for phytochemicals and “leads” which could be developed for treatment of

infectious diseases. While 25 to 50% of current pharmaceuticals are derived from plants, few are used

as antimicrobials. Plants and microalgae are rich in a wide variety of secondary metabolites, such as

tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimicrobial

properties.



We tested within the framework of our start-up several extracts of microalgae. Those were tested,

initially, in vitro on Toxoplasma gondii, coccidia model, 7 are active. The most active ones were then

tested in vivo on Eimeria tenella (in chicken). Currently, we have at disposal 2 extracts of microalgae

which reduce or make disappear the parasite in chicken and are considered as coccidiostatic. These

experiments must be optimized to allow an eradication of the parasite.









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International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Molecular genetic characterization of Cryptosporidium in humans in the Netherlands.

1 1 2 1

Peter R. Wielinga , Ankje de Vries , Laetitia M. Kortbeek , Joke W.B. van der Giessen

1

National Institute for Public Health and the Environment (RIVM), Microbiological Laboratory for Health

Protection, Antonie van Leeuwenhoeklaan 9,

2

P.O. Box 1, Bilthoven, the Netherlands. RIVM, Laboratory for Infectious Diseases Surveillance and

Screening. Email: peter.wielinga@rivm.nl



In humans, mainly two species of Cryptosporidium are found: C. hominis, also called C. parvum

genotype H or type 1, and C. parvum (genotype C or type 2). C. parvum is also found in animals. Our

aim was to study the genetic diversity of human Cryptosporidium strains in the Dutch population.

Therefore, we genotyped a cohort of 97 human patients with diarrhea that were Cryptosporidium

positives from different regions in the Netherlands. Stool isolates were typed by DNA sequence

analysis for six loci on the Cryptosporidium genome: 18S rRNA gene (18S), the Cryptosporidium outer

wall protein (COWP), the heat shock protein 70 (HSP70), the two microsatellite markers ML1 and ML2

and the GP15 locus. Results were compared with reference strains and analyzed for to possible

relations between genotypes and amongst other things seasonality, gender, geographic location and

age. Our results show that except for one C. felis isolate, all isolates belonged to the C. parvum or the

C. hominis assemblage. C. hominis was found in approximate three quarter of the cases. The number

of C. hominis cases showed a peak in the period September – November, coinciding with the period

that most cases of cryptosporidiosis were reported. C. parvum was found more constant throughout

the year. Gender difference did not correlate with either of the two assemblages. When age was

considered, the majority (80%) of the cases originated from children between 0 -7 years and

consisting for >70% of C. hominis. The remaining cases were from patients >29 years old and showed

a tendency for more C. parvum.









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International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Identification of a new surface adhensin protein on sporozoite and oocyst stages of

Cryptosporidium parvum by screening of a phage-display cDNA library

1,2 1,2 1,2 1,2

Jigang Yin ,An Guo , Zhe Hu ,Qijun Chen .

1

Research Institute of Zoonosis, Jilin University, Changchun130062,P.R. China

2

Key Laboratory of Zoonosis, Ministry of Education, Changchun130062,P.R. China



Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules

that mediate C.parvum-host interaction and the molecular mechanisms involved in the pathogenesis

are not well known. A novel phage display method were established to identify surface adhensive

protein of C.parvum. A cDNA library of the sporozoite and oocyst stages of C.parvum expressed on

the surface of T7 phage was screened with intestinal epithelial cells(IECs) from the newborn

Cryptosporidium-free Holstein calves. Proteins that selectively and specifically bound to IECs were

then enriched using a multi-step panning procedure. A novel surface adhesive protein (CP12) was

selected. Sequence analysis revealed an ORF of 315bp which was deduced to encode a putative

protein with 104 amino acids. The molecular weight of the putative protein was calculated to be 12KDa

with an isoelectric point of 4.49. ScanProsite analysis showed that CP12 has a N-terminal signal

peptide at aa 1-28, a N-glycosylation site at aa 58-61, a casein kinase II phosphorylation site at aa 83-

86,and two N-myristoylation sites at aa 26-31 and aa 62-67. In order to localize the CP12 protein, the

recombinant protein was successfully expressed in E.coli. The expression were determined by SDS-

PAGE and Western blotting analysis. The Immunofluorescence assay (IFA) using antibody specific for

rCP12 demonstrated that the antibody can specifically recognize the surface of sporozoite and oocyst,

especially apical region of sporozoite. The surface localization of CP12 and its involvement in the

host-parasite interaction suggest that it may serve as an effective target for specific preventive and

therapeutic measures for cryptosporidiosis.

Key words: Cryptosporidium parvum; Adhension protein; Phage-display cDNA library









36

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Consequences of a drastic reduction in available drugs in Europe: the exemple of

histomoniasis.

Zenner Lionel

Unité Mixte de Recherche ENVL/INRA (Ecole Nationale Vétérinaire de Lyon / Institut National de la

Recherche Agronomique) 958 Protozoaires Entéricole des Volailles, École Nationale Vétérinaire de

Lyon, 69280 Marcy l‟Étoile, France.



Histomoniasis is a disease of gallinaceous fowl, caused by a flagellated protozoan, Histomonas

meleagridis, characterized by necrotic thyphlitis with tan-yellow “sulfur” droppings, hepatitis, and high

mortality especially in turkeys. Until 1950, arsenicals were the only compounds which were used to

control of histomoniasis in the field. Then nitroimidazoles, and dimetridazole in particular, became

available. They were used for many years in feed or water for the treatment and prevention of the

disease. In 1995, the European Council (Annex IV of Council Regulation EEC 1798/95) banned the

use of dimetridazole in veterinary medicinal products for food-producing animals because this

substance was identified as potentially carcinogenic for the consumer. So, until the Council of Europe

ban this practice, factory-farmed turkeys systematically received a supplement of Dimetridazole (200

th st

ppm, authorised until the 15 May, 2002) or Nifursol (50 - 75 ppm, authorised until the 31 March,

2003) as a prophylactic against histomoniasis.

With 625,000 tons of meat produced in 2004, the turkey represents 32% of poultry production and

10% of total meat production in France. Of the 25 E.U countries, France is the largest producer of

turkeys, leading Germany (370,000 tons), Italy, the United Kingdom and Poland and accounting for

41% of total production in Europe. The ban imposed on these means of control and treatment is

causing serious problems. The question arose as to the possible consequences of its withdrawal on

the re-emergence of this disease and if histomoniasis is becoming a threat to turkey producers and an

industry which was previously relatively untouched by this disease.

To precisely assess the consequences of the withdrawal of these products on the turkey producers of

France, a procedure to follow up cases of histomoniasis in farms was set up immediately following the

withdrawal of Nifursol and left in place for 2 years (April 2003 – March 2005). While collecting this

data, several epidemiological elements of the disease have been defined.

It is also interesting to look at the consequence of the implementation of this legislation on the

research on histomoniasis. Furthermore, it is necessary to consider others means of control. Great

interest has been focused on the antimicrobial and antiparasitic properties of natural extracts, and

especially volatile oils of natural origin. As an alternative to chemical drugs, they have already

demonstrated some efficacy against protozoa. Another way would be the vaccine. Finally, this

experience could be useful to anticipate other anti-protozooal molecule ban in the future.









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International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006









ABSTRACTS



Poster communications









38

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



TOXOPLASMOSIS IN WILDLIFE IN CHAMPAGNE-ARDENNE, FRANCE: SEROPREVALENCE

AND PARASITE ISOLATION.

1 2 3 3 1 4 1

AUBERT D , TERRIER ME , HERMITTE P , GIBOUT O , GEERS R , DARDE ML , VILLENA I .

1 Laboratory of Parasitology, EA 3800, CHU de Reims, 45 rue Cognacq-Jay, 51092, Reims, France;

2 AFSSA- LERRPAS, Domaine de Pixérécourt BP 9 54220 Malzéville, France.

3 Departmental Veterinary Laboratory, Chemin des Champs de la Loge BP 216, 10006 Troyes,

France.

4 Laboratory of Parasitology, EA 3174, CHU Dupuytren, 2 Avenue Martin Luther King, 87042

Limoges, France.



Toxoplasma gondii, intracellular protozoan parasite, can infect a wide broad of mammals including

birds and man. Geographical distribution of this parasite is large leading to wide prevalence of

toxoplasmosis depending from various regions. Humans can be infected by consumption of cysts

present in various meat or oocysts present in vegetables or water. Since principal source of T. gondii

contamination is attributed to cattle, sheep or domestics pigs (Tenter et al., 2000), consumption of

wildlife meat can lead to Toxoplasma infection. Little is known of genotypes of T.gondii isolates in wild

animals. The purpose of this study was to establish the prevalence of Toxoplasma gondii antibodies

from selected wildlife species in Champagne-Ardenne, to isolate T.gondii and to genetically

characterize them.

Wild pigs (Sus scrofa; n=79), Roe deer (Capreolus capreolus; n=25), red deer (Cervus elaphus;

n=16), fallow deer (Dama dama; n=4), red foxes (Vulpes vulpes; n=13), European brown hares (Lepus

europaeus, n=13), and common mallards (Anas platyrhynchos; n=4) were hunted in Champagne-

Ardennes during the hunting seasons 2003-2005. Sera were tested for antibodies to T.gondii with the

modified agglutination test, and the heart from animals with titers superior or equal to 1:6 were

bioassayed individually in mice. The seroprevalence varies from 6.6% (red deer) to 72% (roe deer).

T.gondii was isolated from the hearts of 13 of 35 seropositive wild boars (Sus scrofa), 6 of 8 red foxes

(Vulpes vulpes), 6 of 16 roe deers (Capreolus capreolus), 1 of 1 deer (Cervus elaphus) and 1 of 2

common mallards (Anas platyrhynchos). Genotyping of the 27 isolates using the SAG2 locus and

microsatellite analysis indicated that all were type II. None were virulent for mice



Toxoplasmosis is a common infection in wild mammals from Champagne-Ardenne and its prevalence

varies considerable according to taxonomic groups. The titer of 1:25 commonly used in the MAT as

cut-off in most of the animal studies must be carefully employed. In this study, T.gondii isolates were

obtained in three wild boar and one red deer with antitoxoplasma titer of 1:6.



Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol.

2000, (12-13): 1217-12158.









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International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Participation of intestinal epithelial cell to dendritic cell recruitment in the intestine of C.

parvum infected neonatal mice.

1 1 1 2 1

Auray , G., Lacroix-Lamandé S., Mancassola , R. Dimier-Poisson , I. and Laurent F.

1

Laboratoire Contrôle et Immunologie des Maladies Entériques du Nouveau-né, UR1282 Infectiologie

Animale et Santé Publique, INRA de Tours, 37380 Nouzilly, France.

2

Laboratoire d'Immunologie Parasitaire et de Vaccinologie, UMR 0483 Université-INRA, Université

des Sciences Pharmaceutiques, 31 Avenue Monge, 37200 Tours, France.



Cryptosporidium parvum is a protozoan parasite which infects mammals and develops in the intestinal

epithelial cells (IEC) of neonates or immunodeficient hosts. During the infection, a wide range of pro-

inflammatory cytokines, and chemokines are produced locally facilitating the recruitment of pro-

inflammatory cells. In the intestine of neonatal mice, only very few immune cells are present in the

+

lamina propria. CD11c dendritic cells (DC) which are potent antigen presenting cells, are among the

first cells to reach the infected mucosa. In this work, the role and recruitment of the different DC

subsets during cryptosporidiosis were investigated. Results: We first studied the DC-attracting

chemokines produced by C. parvum-infected-intestinal epithelial cells. By in vitro studies and real-time

PCR analysis, we observed a strong up-regulation of the chemokines CCL2, CCL3, CCL4, CCL5 and

CCL20. With chemotactic assay, we observed that the response of infected IEC induced the migration

of immature DC of CD11 or CD8 phenotype. Moreover, the supernatants of infected IEC induced

the upregulation of the costimulation molecules CD40 and CD86 and of MHC-II on surface of DC and

the production of the pro-Th1 cytokine IL-12. This in vitro study shows that the infected host cells

produce soluble factors that induce the local activation of DC which may be of great importance for the

development of the protective immune response. In vivo, in a neonatal mouse model the upregulation

of chemokines attracting DC was confirmed in the ileum as soon as 2 days post-infection.

+ +

Immunohistochemical stainings revealed among the CD11c DC that the myeloid (CD11 ) and

- -

double-negative (CD8 CD11 ) subpopulations were the main subsets recruited in the lamina propria

after C. parvum infection. The recruitment of activated CD11+ DC and mPDCA-1+ plasmacytoid DC

was observed in the MLN of infected mice suggesting an initiation of the specific immune response

after infection of enterocytes by C. parvum. This work suggests that IECs participate in the recruitment

and activation of specific subsets of DC after C. parvum infection in a neonatal environment and

highlights the role of myeloid and plasmacytoid in the subsequent protective immune response









40

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Genetic heterogeneity of the triosephosphate isomerase locus on isolates of Giardia

duodenalis in a dog breeding unit.

a b a a

Bahuon C , Bermudez M , Polack B , Boireau P .

a

Joint Research Unit BIPAR, INRA AFSSA ENVA UPVM, 23 avenue du Général De Gaulle 94703

MAISONS-ALFORT cedex, France.

b

CINVESTAV-IPN, Av IPN 2508, Mexico City, 07360, Mexico.



Giardiosis in dog is caused by the intestinal parasite, Giardia duodenalis. Giardia is the main digestive

parasite found in puppies. In a big dog breeding unit, we collected 48 fecal samples from three group

of puppies and 8 sewage samples (3 from washing water of the three same dog housings as for fecal

samples, 4 from sewage pipelines, 1 from the cesspit). Samples were analyzed for Giardia cyst

identification and numeration by direct immunofluorescence. All fecal samples except two were

positive, and the mean parasite load was 8250 cysts per gram, with a maximum of 106 900. All water

samples were positive with a mean of cysts per milliliter load of 98 for washing waters, 28 for sewage

pipeline water, and 6 for the cesspit water sample. Genotypes were determined by PCR-RFLP on the

triosephosphateisomerase locus. Four fecal samples were successfully amplified. Two were identified

as type C, other samples are under current investigation.









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International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Oral and parenteral administration of oligonucleotides to neonate mice increases resistance to

enteric infection by Cryptosporidium parvum.



Barrier, M. Lacroix-Lamandé, S. Mancassola, R., Auray, G., Bernardet, N., Chaussé, A-M. and

Laurent, F.



Laboratoire Contrôle et Immunologie des Maladies Entériques du Nouveau-né, UR1282 Infectiologie

Animale et Santé Publique, INRA de Tours, 37380 Nouzilly, France.



Neonates are particularly vulnerable to infections during the first weeks of life due to an incomplete

development of their immune system. A new strategy based on the stimulation of the mucosal immune

system with orally administered phosphorothioate CpG-ODN have been employed to reduce the

sensitivity to neonatal enteric diseases. Cryptosporidium parvum develops in the intestinal epithelial

cells and causes diarrhea in young or immunodeficient hosts. The development of this zoonotic

disease is strongly related to the immune status and therefore represents a good model to study the

development of the neonatal intestinal immune system. Results: A single oral administration of CpG-

ODN-1668 performed 24h before infection to 1 day-old C57BL/6 neonate mice was able to decrease

by 90% the parasitic load in the intestine. Surprisingly, the non-CpG-ODN-1668 was also as effective

to decrease the infection. Similar data were observed with non-CpG-ODN-1982 suggesting that the

protective effect observed was not due to a particular structure of the non-CpG-ODN-1668.

Subsequent analysis revealed that CpG-ODN administration induced as soon as 4h post-treatment a

strong upregulation of the early activation molecule (CD69), of several proinflammatory cytokines (IL-

1β, IL-6) of Th1 (IFNγ, IL-12) and Th2-type cytokines (IL-10) in the ileum where the parasite grows. On

the other hand, non-CpG-ODN induced only minor increase compared to CpG-ODN administration

excepted for IL-10. These two types of ODN act via two different mechanisms, one dependent of

TLR9 (CpG-ODN) and the other one (non-CpG) independent of the presence of the receptor.

However, both mechanisms require the presence of IFNγ and in a lesser extend of IL-12 to be

effective against parasite development. Recent experiments showed that when the treatments were

inoculated during the course of infection, only CpG-ODN administration reduced dramatically the level

of infection in the next 24h. Current studies are performed to precise the cell populations and

mechanisms involved in the protection process. Overall these data suggest that ODN are potent

immunostimulants of the neonatal intestinal immune system which could be extremely useful for the

treatment of cryptosporidiosis and other enteric diseases.









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International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Survey of Toxoplasmosis in pigs and wildboars in SPAIN AND POLAND

1 1 2 3 1

Perret C , Bahuon C , Serrano F , Cabaj W , Boireau P

1 UMR BIPAR INRA-AFSSA-ENVA, France

2 Unidad de Parasitología y Enfermedades Parasitarias, Departamento de Medicina y Sanidad

Animal, Facultad de Veterinaria, Universidad de Extremadura, Spain

3 Witold Stefanski Institute of Parasitology of the PAS, Poland



Toxoplasmosis is a major public health concern in Europe as an average of 20% of the population

have a positive serology in several European countries (France, Spain and Poland). Toxoplasmosis

was proved to be mainly transmitted by meat consumption. Indeed, a European multicentre case-

control study demonstrated that risk factors most strongly predictive of acute infection in pregnant

women were game meat, beef and lamb (1). Pigs are also one of the animals, which can be

responsible for the transmission of the parasite Toxoplasma.



Sera from pigs and wild boars, originated from Poland and Spain, were tested by an ELISA

(Pourquier) previously validated with pigs experimentally infected with T. gondii. Sera were obtained

from pigs experimentally infected by European species of Trichinella, and showed no cross-reaction

between parasites.



The results showed a high prevalence of Toxoplasma with 2% and 25% of positive pigs in Spain and

Poland respectively, and 24% and 32% of positive wild boars in Spain and Poland respectively. This

high prevalence indicates that pigs and wild boars may play a role in the transmission of the parasite,

and be part of the causative agents within human population.



Serology testing is proved to be useful in the control of toxoplasmosis in pigs and wild boars. A

development of this technology would be to use muscle fluids, which are easier to get from dead

animals at the slaughterhouse.



This work was supported by EU Contract Trichiporse (QLRT-2000-0156).



Key words: Toxoplasmosis, pig, wild boar, ELISA



1. Cook AJ, Gilbert RE, Buffolano W, Zufferey J, Petersen E, Jenum PA, Foulon W, Semprini AE,

Dunn DT (2000) Sources of toxoplasma infection in pregnant women: European multicentre case-

control study. British Medical Journal, 321 (7254) : 142-147









43

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



ETIOPATHOGENESIS AND DIAGNOSIS OF EIMERIOSIS IN LAMBS FROM WESTERN ROMANIA

1 1 2 1

COZMA Vasile , MITSIADI Maria , LEFKADITIS Menelaos , SUTEU Eronim

1

Faculty of Veterinary Medicine, 3 Manastur Street, 400372 Cluj-Napoca, Romania

2

Veterinary Clinic Delfon 111, Z.C. 54644, Thessaloniki, Greece



Etiopathogenesis and diagnosis of diarrhea syndrome were studied in a farm form Western Romania

in 1,850 Transylvania Merino lambs, aged between 4-12 weeks. Incidence of the syndrome was 26%

and outbreaks were recorded in lambs between 4 weeks and 3 months old. Mortality was 16% of sick

lambs. Clinical signs were diarrhea with yellow-brown and mucous feces, apathy, anorexia and

dehydration. Lesions were: catharal enterocolitis with haemorrhagic spots, whitish foci on intestinal

mucosa, hypertrophy of mesenteric lymph nodes. Histopathology revealed atrophy of intestinal villi

and Lieberkuhn glands, desquamative enteritis with epithelial necrosis and eimerian evolutive stages.

TEM revealed fatty degeneration of enterocytes, schizogonic and gametogonic forms of Eimeria.

Etiology of diarrhea in lambs consisted of numerous infective organisms: Eimeria (95% extensivity and

17,500 EPG intensity), Cryptosporidium (17.82%), Giardia (22%), and bacteria (E. coli – 45%; Proteus

– 65%). No rota- or coronaviruses were found. Species structure for Eimeria infection was: E.

ovinoidalis (42%), E. crandalis (28%), E. faurei (10%), E. parva (17%) and E. pallida (3%).









44

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



A molecular epidemiological approach to investigating the sources of Cryptosporidium

parvum in the UK

1 1 2 1

Kristin Elwin , Stephen Hadfield , Paul Hunter , Rachel Chalmers

1

UK Cryptosporidium Reference Unit, NPHS Microbiology Swansea, Singleton Hospital, Swansea,

SA2 8QA, UK

2

University of East Anglia, Norwich



Long term enhanced molecular surveillance has shown that main cause of human cryptosporidiosis is

either C. parvum or C. hominis. While epidemiological investigations have indicated that C. hominis is

acquired from other people or their sewage, C. parvum has a wide range of animal hosts including

humans and the source of infection is often unclear.



Cryptosporidium isolates, identified by polymerase chain reaction– restriction fragment length

polymorphisms in sporadic cases recruited to a case control study in the UK, were further investigated

at three micro-satellite DNA markers: ML1, ML2 and gp15. Subtypes were identified on the basis of

marker fragment size (allele) distribution and compared with detailed exposure history gathered from

the patients.



C. hominis isolates were largely indistinguishable, with 90% of the 106 that were typable at all 3 loci

having the same alleles. C. parvum isolates showed more variation with 31 multilocus genotypes

identified in the 63 typable isolates.



C. parvum genotypes were grouped by hierarchical cluster analysis to further investigate associations

with exposure data. Cluster 1 was significantly associated with a history of animal contact and with

living in a rural area, while clusters 2 and 3 were exclusive to patients who did not report animal

contact. Within cluster 1, one particular ML1 allele was significantly associated with touching farmed

animals while another ML1 allele was mainly found in patients living in urban areas and was not found

in people who reported animal contact.



This study provides further evidence of human–adapted strains of C. parvum in the UK and offers a

method to distinguish these from potentially zoonotic strains. Furthermore, investigation of one marker

may provide a rapid tool for classifying a strain as zoonotic or human.









45

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Prenatal diagnosis of congenital toxoplasmosis by PCR and mice inoculation in Warsaw -

Poland

1 1 2 1

Golab E. , Waloch M. , Nowakowska D. , Dzbenski T.H.

1 2

Department of Medical Parasitology, National Institute of Hygiene, Warsaw, Poland, Department of

Fetal-Maternal Medicine and Gynecology, Research Institute Polish Mother‟s Memorial Hospital, Lodz,

Poland



Although the seropositivity rate in pregnant woman in Poland is high (about 41%), it was evaluated

that the frequency of congenital Toxoplasma infection in our country reaches 1 per 1000 live births.

Identification of pregnant women at high risk for transmitting the infection to the fetus allows for

specific treatment to prevent the development of the most serious sequelae of congenital infection

which include intracranial and ocular damage in children. In the Department of Medical Parasitology of

the National Institute of Hygiene prenatal diagnosis of congenital toxoplasmosis using polymerase

chain reaction (PCR) and/or mice inoculation of amniotic fluid (AF) is conducted, based on the

assumption that the presence of toxoplasmas or their DNA in the fluid testifies fetus infection. Since

the year 2000, twenty cases of fetal infection were recognized for the group of 313 pregnant women

suspected of primary toxoplasmosis on the basis of serological examinations. Using the PCR method

involving detection of the B1 gene of T. gondii, 18 positive cases were recognized, and when primers

for 529 genomic fragments of the parasite were used we managed to qualify as positive two additional

cases. From amongst 17 AF samples which were positive in the PCR method, three negative results

were gained when mice inoculation was used. We found that all these negative results of mice

inoculation were received when AF samples were kept for more than one week since amniopuncture

day, before the inoculation, and were obtained from asymptomatic cases of infection. Thus far, all

examined isolates from cases of congenital toxoplasmosis in Poland were identified as type II strains.









46

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



CCR5-/- mice successfully induce a Th1 response and control Cryptosporidium parvum

infection.



Lacroix-Lamandé, S. Mancassola, R., Auray, G. and Laurent F.

Laboratoire Contrôle et Immunologie des Maladies Entériques du Nouveau-né, UR1282 Infectiologie

Animale et Santé Publique, INRA de Tours, 37380 Nouzilly, France.



Cryptosporidium parvum is a protozoan parasite that infects intestinal epithelial cells and causes

enteric infections and diarrhea in humans and animals. C. parvum infection is characterized by

infiltration of the lamina propria by inflammatory cells. Protection against this parasite has been largely

associated with the cell-mediated immune response and a strong production of IFN. We previously

found that C57BL/6J neonate mice deficient in IFN died from the infection, whereas wild-type

neonate mice were able to clear the infection in 3 weeks. In infected IFN-deficient mice, the intestinal

upregulation of the non-ELR C-X-C chemokines (CXCL9, CXCL10, CXCL11) mRNAs, and of the C-C

chemokines CCL4 and CCL5 mRNAs was dramatically altered suggesting that these molecules could

play an important role in the resolution of cryptosporidiosis. In this study, we further investigate the

role of the chemokine response during cryptosporidiosis, by focusing on the role of the chemokine

receptor 5 (CCR5) that binds CCL3, CCL4 and CCL5 chemokines. Result. We first compared the level

of infection between CCR5-/- and wild-type neonate mice. We observed that 6 days after infection of

the neonates, the number of parasites present in the intestine was higher in CCR5-/- mice than in wild-

type. However, the parasite loads were similar between the two types of mice at day 13 p.i. during the

recovery phase of the infection, suggesting that CCR5 play a significant role in the early stages of the

protective immune response. At day 6 p.i. we observed a similar increase in the total number of cells

within the draining lymph nodes (MLN) in the two types of infected mice when compared to their

respective controls. However, the recruitment of CD8+ cells in the MLNs of infected CCR5-/- mice was

reduced compared to wild-type neonates. Expression of the intestinal IFN, which is a key cytokine for

the control of C. parvum, was similar during the infection in WT and CCR5-/- mice and cannot explain

the difference of sensitivity of these mice. This study shows that CCR5-/- neonatal mice are more

sensitive to C. parvum in the early stage of infection which may be due to a lower recruitment of CD8+

cells but CCR5 is however not essential for the control of cryptosporidiosis most probably due to the

chemokines/chemokine-receptors redundancy of function.









47

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Investigation for human-pathogenic Cryptosporidium sp and Giardia duodenalis in wild

bivalves from coastal Normandy



Le Goff L.*, Gromellon N.*, Girot H*, Ballet J.J.**, Berthe T.***, Favennec L*, Gargala G.*,.

*Laboratoire de Parasitologie, ADEN EA 3234, Université de Rouen, 76183 Rouen cedex 1, France

**Laboratoire d'Immunologie et d'Immunopathologie, CHU de Caen, 14033 Caen Cedex, France

*** LMDF, Groupe Biodiversité et environnement, Upres 2123, Université de Rouen, Rouen, France



Cryptosporidium sp. and Giardia duodenalis are presently identified as the most prevalent causative

agents of human protozoal diarrhea in Normandy. These protists are widely distributed in natural fresh

waters. They also appear resistant for weeks in marine environment. Coastal areas can be

contaminated from estuarian river drainage which is increased by heavy rains and floods which wash

Cryptosporidium sp. oocysts and/or Giardia duodenalis cysts from the land. Thus wild filter-feeder

mollusks may represent significant oocyst/cyst concentrators and vectors in the marine environment.

In this study, the presence of oocysts/cysts was investigated in three wild bivalves intended for human

consumption, i.e. mussels (Mytilus edulis), cockles (Cerastoderma edule) from coastal areas

surrounding the estuary of the Seine. Several sites were investigated in the late Spring and Summer.

Cryptosporidium sp. oocysts were isolated from mussels and cockles, more frequently than Giardia

duodenalis cysts which were only present in mussels. Bivalves harboring parasites were found in

areas nearer the Seine estuary. Data suggest that mussels and/or cockles from sea waters close from

the Seine estuary are a potential source of human Cryptosporidium sp. and/or Giardia duodenalis

contamination.









48

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



Longitudinal study of anti-Toxoplasma antibodies in naturally infected calves



Marieke Opsteegh, Merel Langelaar, Manoj Fonville, Joke van der Giessen

National Institute for Public Health and the Environment (RIVM), Microbiological Laboratory for Health

Protection, Antonie van Leeuwenhoeklaan 9, P.O. Box 1, Bilthoven, the Netherlands



Toxoplasma gondii is an intestinal coccidium of felids with an unusual wide range of intermediate

hosts. Toxoplasmosis in humans can be severe, especially when contracted transplacentally or in

immunocompromised individuals. Also, evidence for deleterious health-effects on immunocompetent

people is accumulating. People contract toxoplasmosis by ingestion of raw or undercooked meat

containing tissue cysts, ingestion of oocysts via contaminated soil, food or water, or congenitally by

transplacental transmission of tachyzoites. There is no critical evidence that natural T. gondii infection

causes clinical disease in cattle, nor is there a well documented natural case of abortion in cattle due

to toxoplasmosis. It has been suggested that cattle are resistant to Toxoplasma infection and are able

to clear infection. This implies that the consumption of beef poses a lower risk for infection than

consumption of other meat. In contrast, Cook et al. (2000) reports eating raw beef as one of the risk

factors that most strongly predicted acute infection in pregnant women. It is our aim to gain further

insight into natural Toxoplasma infections in cattle. Twenty-seven calves kept under natural conditions

were followed during 17 months from birth onward. Serum samples were collected on a regular basis

and tested for anti-Toxoplasma antibodies by ELISA. Twenty-five calves were moved to the pasture

when 12 months old. Twenty-one had increased titers at the first or second sampling after being

moved outside. Two had already seroconverted earlier and only two remained seronegative for the

duration of the experiment. After being moved outside, sampling was continued for five months, and

although most animals (20) were still positive at the last sampling date, a rapid decline in titer was

observed in 10 calves. These results show that calves can contract a Toxoplasma infection, and the

large proportion of calves that do contrasts with suggested resistance. These animals were, however,

all kept on the same, possibly highly contaminated, pasture. The risk of infection appears to be higher

on the pasture than in the stable. Further testing will be performed to evaluate persistence of infection.

In addition, a sample of the Dutch cattle population is tested by ELISA to estimate seroprevalence of

Toxoplasma gondii and preliminary results will be presented.









49

International workshop on Protozoa orally transmitted in public and animal health.

Centre de Recherche INRA Tours Nouzilly, 14-15 December 2006



EVALUATION OF A STRATEGY FOR THE DETECTION OF MULTIPLE WATERNORNE

PARASITES, INCLUDING TOXOPLASMA GONDII OOCYSTS, IN ENVIRONMENTAL WATER

SAMPLES IN CHAMPAGNE-ARDENNE, FRANCE.



Villena I, Marneff F, Pisano E, Aubert D.



Laboratory of Parasitology, Faculty of Medicine, EA 3800, University of Reims, France;



Toxoplasma gondii is a protozoan parasite capable of infecting a variety of birds and mammals,

including humans. Several recent outbreaks of toxoplasmosis were related to drinking water. We

propose a strategy for Toxoplasma oocyst detection, based on a multirisk waterborne parasitic

approach including Giardia and Cryptosporidium recovery by the AFNOR method on the same

sample. Our strategy involves three basic steps: (i) concentration and filtration of the water sample to

recover small numbers of Toxoplasma oocysts, (ii) elution and purification on a density gradient, and

(iii) detection. Water samples are filtered to recover Toxoplasma oocysts, and purified on a sucrose

density gradient. Detection is based on PCR and mouse inoculation (bioassay), to determine the

presence and the infectivity of recovered oocysts. In an experimental seeding assay, a parasite

density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10

oocysts/liter was detected in 100% of cases. Depending on the sample source, the sensitivity of the

PCR assay varuied from less than 10 to more than 1000 oocysts/liter (Villena et al., 2004).

This detection strategy was applied to 241 environmental water samples collected over a 3 year‟s

period. Finally, among the 225 interpretable samples, we detected Toxoplasma DNA in 12 cases

(5.3%). Three cases involved raw surface water, eight cases underground water; these samples were

collected from sites because of frequent pathogen recovery (including Giardia spp. and

Cryptosporidium spp.). More surprising, one case involved public distribution water. None of the

samples were positive by bioassay.

This strategy, which efficiently detects Toxoplasma oocysts in water, may be suitable as a public

health sentinel method.





VILLENA I., AUBERT D., GOMIS P., FERTE H., INGLARD JC., DENIS-BISIAUX H., DONDON JM.,

PISANO E., ORTIS N., PINON JM. Evaluation of a strategy for Toxoplasma gondii oocyst detection in

water. Appl. Environ. Microbiol., 2004, 70, 7. 4035-4039.









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LIST OF PARTICIPANTS





NAME Given Name e-mail

AUBERT Dominique daubert@chu-reims.fr

AURAY Gaël auray@tours.inra.fr

AUTHIÉ Edith edith.authie@tours.inra.fr

AZAS Nadine nadine.azas@pharmacie.univ-mrs.fr

BAHUON Céline celine.bahuon@yahoo.fr

BALLET Jean-Jacques ballet-jj@chu-caen.fr

BECK Relja reljab@vef.hr

BERNARDET Nelly Nelly.Bernardet@tours.inra.fr

BERTHONNEAU Jacques j.berthonneau@chu-poitiers.fr

BESSIERE Marie-Hélène bessieres.mh@chu-toulouse.fr

BLAGA Radu r.blaga@afssa.fr

BOIREAU Pascal pboireau@vet-alfort.fr

BONHOMME Julie juliedak@yahoo.fr

BOUYER Sabrina s.bouyer@chu-poitiers.fr

BRETAGNE Stéphane bretagne@univ-paris12.fr

BRETON Jacques jakbreton@yahoo.fr

CABARET Jacques Jacques.Cabaret@tours.inra.fr

CACCIO Simone simone.caccio@iss.it

CALLAIT-CARDINAL Marie-Pierre mp.callait@vet-lyon.fr

CANDOLFI Ermanno violaine.philippe@medecine.u-strasbg.fr

CHALMERS Rachel Rachel.Chalmers@nphs.wales.nhs.uk

CHAUSSE Anne-Marie Anne-Marie.Chausse@tours.inra.fr

CLAEREBOUT Edwin edwin.claerebout@ugent.be

CLASTRE Marc marc.clastre@univ-tours.fr

COZMA Vasile cozmavasile@yahoo.com

DARDE Marie-Laure marie-laure.darde@unilim.fr

DELMAS Florence Florence.Delmas@Pharmacie.univ-mrs.fr .

DEMAR Magalie mdemar@yahoo.com

DEROUIN Francis paracord@wanadoo.fr

DEVILLE Sébastien sebastien.deville@airliquide.com

DIMIER-POISSON Isabelle dimier@univ-tours.fr

DROUET-VIARD Françoise Francoise.Viard@tours.inra.fr

DUCOURNAU Céline celine.ducournau@univ-tours.fr

DUNOYER Charlotte cdunoyer@chasseurdefrance.fr

DUONG Thanh Hai th.duong@med.uni-tours.fr

DUPOUY-CAMET Jean jean.dupouy-camet@cch.aphp.fr

EL-HMOUZI Jamila Jamila.el-hmouzi@tours.inra.fr

ELWIN Kristin Kristin.elwin@nphs.wales.nhs.uk

EUZEBY Jean jean.euzeby@bacterio.org

FAVENNEC Loïc loic.favennec@chu-rouen.fr

FORT Geneviève Genevieve.Fort@tours.inra.fr







51

GAFA Valérie valerie.gafa@univ-paris5.fr

GARGALA Gilles gilles.garlala@univ-rouen.fr

GASQUET Monique monique.gasquet@pharmacie.univ-mrs.fr

GERMON Stéphanie stephanie.germon@univ-tours.fr

GILES Michaela

GILOT-FROMONT Emmanuelle fromont@biomserv.univ-lyon1.fr

GOLAB Elzbieta egolab@pzh.gov.pl

GUITON Rachel rachelguiton@aol.com

HEDHLI Dorsaf dorsafhedhli@yahoo.fr

HUBER Karine k.huber@vet-lyon.fr

IMBERT Christine chistine.imbert@univ-poitiers.fr

JABBARI Kamel

KAPEL Nathalie nathalie.kapel@univ-paris5.fr

KHALDI Samira samira_khaldi@yahoo.fr

LACROIX-LAMANDE Sonia Sonia.Lacroix@tours.inra.fr

LAURENT Fabrice Fabrice.Laurent@tours.inra.fr

LAWTON Philippe Lawton@univ-lyon1.fr

LE GOFF Laetitia laetitia.le-goff@wanadoo.fr

LICOIS Dominique Dominique.Licois@tours.inra.fr

MACÉ Pauline p.mace@afssa.fr

MADDOX-HYTTEL Charlotte cmh@dfvf@dk

MANCASSOLA Roselyne Roselyne.Mancassola@tours.inra.fr

MARQUET Perinne perrine.marquet@univ-paris5.fr

MENOTTI Jean jean.menotti@sls.ap-hop-paris.fr

MERGEY Thiphaine thiphainemergey@yahoo.fr

MEVELEC Marie-Noelle mevelec@univ-tours.fr

MOIRÉ Nathalie nathalie.moire@inra.tours.fr

NACIRI Muriel Murielle.Naciri@tours.inra.fr

NEVEU Cédric Cedric.Neveu@tours.inra.fr

NIELSEN Henrik Vedel HVN@SSI.dk

NIEPCERON Alisson alisson.niepceron@tours.inra.fr

OPSTEEGH Marieke marieke.opsteegh@rivm.nl

ORTEGA-PIERRES Guadalupe gortega@cinvestav.mx

PARAUD Carine c.paraud@niort.afssa.fr

PEDRAZA-DIAZ Susana spedrazadiaz@phls.nhs.uk

PENARETE Diana diana.penarete@etu.uni-tours.fr

PEROVAL Marylene p.marylene@wanadoo.fr

PERRET Catherine c.perret@afssa.fr

PETERSEN Eskild EPF@sks.aaa.dk

POLACK Bruno bpolack@vet-alfort.fr

RAMIANDRISOA Mahery matanjaka@yahoo.com

REPERANT Jean-Michel jm.reperant@afssa.fr

RICHOMME Céline richomme@corte.inra.fr

RODIER Marie-Hélène m.h.rodier@chu-poitiers.fr

SHIRLEY Martin martin.shirley@bbsrc.ac.uk





52

STENSVOLD Rune Christian HVN@SSI.dk

TENTER Astrid astrid.tenter@tiho-hannover.de

THEBAULT Anne a.thebault@afssa.fr

THELLIER Marc marc.thellier@psl.aphp.fr

THOMAS Myriam m.thomas@afssa.fr

THULLIEZ Phillipe ipp.thulliez@free.fr

TOURATIER Louis louis.touratier@club.francetelecom.fr

TOUROULT Pauline pauline_touroult@yahoo.fr

VALHEIM Mette Mette.Valheim@tours.inra.fr

VALLEE Isabelle ivallee@vet-alfort.fr

Van Der GIESSEN Joke Joke.van.der.Giessen@rivm.nl

Van LANGENDONCK Nathalie langendo@med.univ-tours.fr

VILLENA Isabelle ivillena@chu-reims.fr

VIVARES Christian christian.vivares@univ-bpclermont.fr

WATIER Stéphanie svi.brest@wanadoo.fr

WIELINGA Peter peter.wielinga@rivm.nl

YIN Jigang yjg@jluhp.edu.cn

ZENNER Lionel l.zenner@vet-lyon.fr









53