# Animal-human-environment continuum in the emergence and spread of antibiotic resistance in human pathogens

Axel Cloeckaert

► To cite this version:

Axel Cloeckaert. Animal-human-environment continuum in the emergence and spread of antibiotic resistance in human pathogens. AESA Congress 25 years, Animal epidemiology Past, present and future, Sep 2016, Liège, Belgium., 2016, AESA Congress 25 years, Animal epidemiology Past, present and future. hal-01602872

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Association d'Epidémiologie et de Santé Animale

7th - 9th Sept. 2016 - Liège (Belgium)
Congress 25 years

Animal epidemiology
Past, present and future
# Welcome
to the AESA 25th anniversary International Congress !

It is our pleasure to welcome you in Liege at the first international symposium in Animal Epidemiology of the Association of Epidemiology and Animal Health (in French, Association d'Epidémiologie et de Santé Animale, AESA). The AESA aims to enhance the interest and education and to stimulate research in the field of veterinary epidemiology and animal health, mainly by organizing workshops, conferences and symposia and by providing scientific support in those matters.

Despite increased interest, research and some investment in veterinary epidemiology and animal health, the scale of the task for the future is considerable for epidemiologists and many challenges remain or new challenges will arise. This reason together with the 25th anniversary of our association are explaining the title of our international congress: «25 years of animal epidemiology: past, present and future». With a look at the past and at the present, in order to anticipate the orientation of the future is the main idea of this topic. This big challenge needs sharing of information, exchange, brainstorming, interdisciplinary approach (e.g. social and economic sciences) and connectivity.

Considering these epidemiological challenges, do we need to adapt the epidemiological approach or not? Invited speakers with international appeal will try to find answers for the future in ten different sessions concerning crisis management in animal health, biosecurity in livestock, pets and horses, climate change and diseases, vector borne diseases, one health: antimicrobial resistance, participatory epidemiology, wildlife diseases, epidemiology, surveillance and risk analysis, bee diseases and finally drivers of diseases emergence.

In addition of this variety of interesting subjects, we will give young scientists the opportunity to present their work during different parallel sessions during the two days symposium (oral presentations and posters).

At the end of the symposium, two prizes will be attributed to the best posters by AVIA-GIS and AESA.

We thank the gold sponsors (Ministre de l'Agriculture, de la Nature, de la Ruralité, du Tourisme, des Aéroports, délégué à la Représentation à la Grande Région, Minister of Small Businesses, Self-employment, Small and Medium-sized Enterprises, Agriculture and Social Integration, BioSellal) and silver sponsors (Province of Liège, AWÉ, CODA-CERVA, Fédération Européenne pour la Santé Animale et de Sécurité Sanitaire, Zoetis, AVIA-GIS, Boehringer-Ingelheim, MERIAL, BioX-Diagnostics, ID-Vet, Thermo Fisher Scientific, European Colostrum Industry) for their financial support and the Crowne Plaza Hotel for its hospitality. A special thanks to the board members of the Scientific Committee and the AESA Organization Committee.

We appreciated all the work that had been fulfilled during the organization of this meeting as well during the evaluation of the submitted proceedings, abstracts and posters. We can be proud of the obtained results.

On behalf of the boards of the AESA we wish you a pleasant and stimulating meeting.

Best regards,

E

Prof. Claude Saegerman
AESA President
**Organizing committee**

Juana Bianchini, Géraldine Boseret, Guy Czaplicky, Fabiana Dal Pozzo,
Laurent Delooz, Marc Dispas, Noémie El Agrebi, Jean-Yves Houtain,
Mathieu Hubaux, Marie-France Humblet, Ludovic Martinelle, Quentin Nemery,
Claude Saegerman, Véronique Renault, Damien Thiry, Etienne Thiry

**Scientific committee**

Prof. Claude Saegerman, ULg, Chairman

Prof. Marius Gilbert, ULB, Vice-Chairman

Dr. Marie-France Humblet, PhD, ULg, Member

Dr. Guy Czaplicki, MSc, ARSIA, Member
Our partners

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The Walloon Region
M. René Collin
Minister of Agriculture, Nature and Rurality

biose||al

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The Federal Government of Belgim
M. Willy Borsus
Minister of Small and Medium-sized
Enterprises, Agriculture and Social Integration

<figure><img src="image_5.png" /></figure>

Province
de Liège

Agriculture

<figure><img src="image_6.png" /></figure>

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ID.vet
Innovative Diagnostics

<figure><img src="image_8.png" /></figure>

CODA - CERVA

zoetis™

<figure><img src="image_9.png" /></figure>

Boehringer
Ingelheim

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ThermoFisher
SCIENTIFIC

ave
association wallonne
de l'élevage

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A SANOFI COMPANY

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# Table of content

## WORKSHOPS .................................................................................................................. 10

→ Workshop 1- Estimation of test characteristics and true prevalence using a Bayesian framework .................................................... 1

→ Workshop 2 - Abortion indicators ................................................................................................................................. 2

## CRISIS MANAGEMENT IN ANIMAL HEALTH .................................................................... 3

### KEYNOTE LECTURE .................................................................................................................. 4

→ Crisis management in animal health.................................................................................................................... 4

### SELECTED ORAL PRESENTATIONS ........................................................................................................ 6

→ Coxiella burnetii immunity and shedding dynamics after vaccination of dairy goats in field conditions ................................................ 6

→ Risk categorization in the context of a porcine reproductive and respiratory syndrome (PRRS) regional control program................ 7

→ Implementation of an information system to gather data on dogs' bites and vaccinations against rabies in the Veneto Region ........ 8

→ Epizootic of Contagious Caprine Pleuropneumoniae in sand gazelles (Gazella subgutturosa marica) in the United Arab Emirates ... 9

### POSTERS ........................................................................................................................................ 10

→ Radioscopy laboratory diagnostic of epizootic diseases in Belgium and European countries ................................................ 10

## CLIMATE CHANGES AND DISEASES .................................................................................. 11

### KEYNOTE LECTURE .................................................................................................................. 12

→ Global changes and the emergence of important zoonoses ................................................................................................................ 12

### SELECTED ORAL PRESENTATIONS ........................................................................................................ 14

→ Influence of water temperature on the outcome of a Brucella pinnipedialis hooded seal (Cystophora cristata) strain infection in cod (Gadus morhua) ................................................................................................................ 14

→ Seroprevalence of Borrelia burgdorferi in horses in the southern part of Belgium: a "one health" driven study.................................. 15

→ Effectiveness and cost efficiency of syndromic surveillance compared to conventional surveillance: Bluetongue serotype 8 across countries as case study................................................................................................................ 16

→ Serological monitoring 2008-2015 of Walloon dairy herds exposition to Leptospira hardjo based on BTM Elisa.................................. 17

## BIOSECURITY IN LIVESTOCK, PETS AND HORSES .................................................................... 18

### KEYNOTE LECTURE .................................................................................................................. 19

→ Ensuring continuous biosecurity for high health high performance (HHP) horses: a public-private collaboration ................................ 19

### SELECTED ORAL PRESENTATIONS ........................................................................................................ 21

→ Biosecurity measures applied in the United Arab Emirates: a comparative study between livestock and wildlife sectors ................ 21

→ How to assess the biosecurity level in stud farms? Survey among 70 Belgian and French horse breeders .................................... 22

→ A descriptive study to assess the perception of cattle farmers towards the implementation of biosecurity.................................. 23

→ Assessing motivators and obstacles for implementation of biosecurity measures in animal production in Europe ................................ 24

### POSTERS ........................................................................................................................................ 25

→ Influence of the purchase policy on the risk of PI birth, a retrospective study in Southern Belgium ........................................ 25

→ Analytical Hierarchy Process for the evaluation of biosecurity levels in fattening rabbit farms. ................................................ 26

## VECTOR BORNE DISEASES .................................................................................................... 27

### KEYNOTE LECTURE .................................................................................................................. 28

→ The German Arbovirus Surveillance and Mosquito Monitoring Program, 2009 - 2015 ................................................................ 28

### SELECTED ORAL PRESENTATIONS ........................................................................................................ 30

→ Qualitative spatial assessment of human TBE risk factors in Belgium .................................................................................................... 30

→ Resurgence of Schmallenberg virus in Belgium after 3 years of epidemiological silence ................................................................ 31

→ What are the vector-borne pathogens present in the blood of French cattle? Results of a field study on 130 samples ................ 32

→ Schmallenberg virus infection in sheep: a model of within-herd seroprevalence evolution ................................................................ 34

### POSTERS ........................................................................................................................................ 35

→ Optimisation of an ELISA immunoenzymatic technique for the detection of IgG antibodies against Trypanosomo sp. in cattle population of Ecuador ................................................................................................................ 35

→ Polymerase Chain Reaction in the diagnosis of Anaplasma marginale in cattle populations of Ecuador and its further molecular identification by 16S ribosoma sequencing ................................................................................................................ 36
ONE HEALTH: ANTIMICROBIAL RESISTANCE.................................................................................. 37

KEYNOTE LECTURE .................................................................................................................................................. 38

→ Animal-human-environment continuum in the emergence and spread of antibiotic resistance in human pathogens.................................................................................. 38

SELECTED ORAL PRESENTATIONS .................................................................................................................. 42

→ A quantitative exposure model for the cross-contamination of pig feeding-stuffs as a result of antimicrobial residues.................................................................................. 42

→ The influence of the introduction of antimicrobial use guidelines on prescription habits in first-line, small animal veterinarians.................................................................................. 43

→ Identification of bovine methicillin resistant staphylococci from Europe, Africa and North America by colony hybridization, PCR and antibiotic sensitivity .................................................................................................................................................. 44

→ Identification of bovine and porcine colistin-resistant mcr1-positive Escherichia coli.................................................................................................................................................. 45

POSTERS.................................................................................................................................................................................. 46

→ Isolation of bacteriophages against Klebsiella pneumoniae and in vivo activity.................................................................................................................................................. 46

→ The use of antibiotics in cattle livestock in the north Benin, a real factor of the development of bacterial resistance in the cattle and the food chain .................................................................................................................................................. 47

→ High prevalence of Antimicrobial resistance among Escherichia coli isolated from day old broiler chicks in Mostaganem province, northwestern Algeria .................................................................................................................................................. 48

WILDLIFE DISEASES.................................................................................................................................................. 49

KEYNOTE LECTURE .................................................................................................................................................. 50

→ TB epidemiology in Europe: past, present and future of a multi-host infection .................................................................................................................................................. 50

SELECTED ORAL PRESENTATIONS .................................................................................................................. 53

→ Genotyping analysis of Brucella melitensis at the wildlife-livestock-human interface in the Middle East .................................................................................. 53

→ Occurrence of Cryptosporidium Oocysts in Edible Frogs (Rana Species) Sold for Human Consumption in Hanwa Frog Market Zaria, Kaduna State, Nigeria .................................................................................................................................................. 54

→ Occurrence of the new pathogenic variant of rabbit haemorrhagic disease virus (RHDV2) in wild populations of rabbits in Southern Belgium. .................................................................................................................................................. 55

→ Validation of Bayesian prevalence estimation with an imperfect diagnostic test.................................................................................................................................................. 56

PARTICIPATORY EPIDEMIOLOGY.................................................................................................................. 57

KEYNOTE LECTURE .................................................................................................................................................. 58

→ We need to talk! The added value of participatory epidemiology .................................................................................................................................................. 58

SELECTED ORAL PRESENTATIONS .................................................................................................................. 60

→ Review of Participatory Epidemiology practices in Animal Health (1980-2015) .................................................................................................................................................. 60

→ Applying sociological methods to the study of animal diseases: the case of Bovine Tuberculosis in Spain.................................................................................. 61

→ On the path towards reduced antimicrobial resistance: herd-level quantification of veterinary antimicrobial consumption in Belgian pig herds through the online monitoring tool ‘AB Register’. .................................................................................................................................................. 62

→ Improvement of micro-organisms classification methodology into risk groups.................................................................................................................................................. 63

EPIDEMIOLOGY, SURVEILLANCE AND RISK ANALYSIS.................................................................................. 64

KEYNOTE LECTURE .................................................................................................................................................. 65

→ Animal epidemiology, surveillance and risk analysis: past, present and future .................................................................................................................................................. 65

SELECTED ORAL PRESENTATIONS .................................................................................................................. 69

→ How does pooling of serum or blood samples from young calves permanently infected with BDV-V affect the detectability of the real time PCR test? A field study in the context of the Belgian BVD eradication program. .................................................................................. 69

→ Approach towards benchmarking surveillance: bovine tuberculosis in Belgium as an exemple .................................................................................................................................................. 70

→ Computer and veterinary sciences: an integrated database for disease surveillance.................................................................................................................................................. 71

→ Seroprevalence and risk factor analysis of small ruminant lentiviruses in Belgian sheep and goats .................................................................................................................................................. 72

POSTERS.................................................................................................................................................................................. 73

→ Surveillance to declare freedom and/or detect a case? A case study for bovine brucellosis in Belgium .................................................................................................................................................. 73

→ Characterization of bovine brucellosis in Colombia (2006 – 2012).................................................................................................................................................. 74

→ Identification of virulotypes and serotypes of enteropathogenic (EPEC) and Shigatoxigenic (STEC) Escherichia coli from healthy cattle at slaughterhouses in Wallonia. .................................................................................................................................................. 75

→ Outbreak investigations and molecular characterization of foot-and-mouth disease viruses circulating in Niger. .................................................................................................................................................. 76

BEE DISEASES.................................................................................................................................................. 77

KEYNOTE LECTURE .................................................................................................................................................. 78

→ A sticky mess - understanding the drivers of honey bee losses .................................................................................................................................................. 78
<table><thead><tr><th></th><th></th></tr></thead><tbody><tr><td>SELECTED ORAL PRESENTATIONS</td><td>81</td></tr><tr><td>➢ Development of an original bee health management tool for beekeepers: Bee Best Check</td><td>81</td></tr><tr><td>➢ A multidisciplinary and evidence-based methodology applied for prioritizing bee diseases in France</td><td>82</td></tr><tr><td>➢ Residues from beeswax: a health risk for the consumer?</td><td>83</td></tr><tr><td>➢ Classification of adult cattle diseases in Belgium based on a veterinary and a laboratory survey, years 2013-2015</td><td>84</td></tr><tr><td>DRIVERS OF DISEASES EMERGENCE</td><td>85</td></tr><tr><td>KEYNOTE LECTURE</td><td>86</td></tr><tr><td>➢ Main drivers of emerging and re-emerging infectious diseases: Focus on livestock</td><td>86</td></tr><tr><td>SELECTED ORAL PRESENTATIONS</td><td>91</td></tr><tr><td>➢ Illegal meat trade – a threat to both biodiversity and public health</td><td>91</td></tr><tr><td>➢ Recombination (genetic evolution) as driver of new epidemic norovirus strains</td><td>91</td></tr><tr><td>➢ Bayesian assessment of two competitive ELISAs for the detection of bovine viral diarrhoea virus antibodies in bovine sera</td><td>92</td></tr><tr><td>➢ A Bayesian evaluation of four antibody ELISA tests to certify freedom of bovine viral diarrhea infection in unvaccinated cattle</td><td>92</td></tr><tr><td>POSTERS</td><td>93</td></tr><tr><td>➢ Seasonal variations of micromammal communities and the risk of tick-borne disease in a hedgerow agro-ecosystem</td><td>93</td></tr><tr><td>ZOONOSES AND PUBLIC HEALTH</td><td>94</td></tr><tr><td>POSTERS</td><td>95</td></tr><tr><td>➢ Rabiës: a still present-day deadly thread</td><td>95</td></tr><tr><td>➢ Evaluation of immunofluorescence antibody test used for the diagnosis of canine leishmaniasis in the Mediterranean basin: a systematic review and meta-analysis</td><td>95</td></tr><tr><td>➢ Prevalence of bovine brucellosis and risk factors assessment in cattle herds in Mostaganem, northwestern Algeria</td><td>96</td></tr><tr><td>➢</td><td></td></tr></tbody></table>
# Program

## Wednesday, September 7

<table><thead><tr><th>13:00</th><th>17:30</th><th>Registration</th><th></th><th>Lobby - Reception</th><th></th></tr></thead><tbody><tr><td>13:15</td><td>17:30</td><td>Workshop</td><td>Estimation of test characteristics and true prevalence using a Bayesian framework</td><td>Claude Saegerman</td><td>Salon Gretry</td></tr><tr><td>13:15</td><td>17:30</td><td>Workshop</td><td>Indicators of abortions</td><td>Pascal Hendrikx</td><td>Salon Nagelmackers</td></tr><tr><td>17:45</td><td>18:15</td><td></td><td>Opening ceremony</td><td>Claude Saegerman</td><td>Bar la Cave</td></tr><tr><td>18:15</td><td>21:00</td><td></td><td>Walking dinner</td><td></td><td>Bar la Cave</td></tr></tbody></table>
Thursday, September 8

<table><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>8:30</td><td>12:00</td><td>Registration</td><td></td><td></td><td>Lobby - Reception</td></tr><tr><td>10:00</td><td>10:20</td><td>Introduction</td><td></td><td>Claude Saegerman</td><td>Salle Le Bal</td></tr><tr><td colspan="6"><strong>Session 1</strong> Crisis management in animal health</td></tr><tr><td>10:20</td><td>11:00</td><td>Keynote lecture</td><td>Crisis management in animal health</td><td>Chairman:</td><td>Géraldine Boseret</td></tr><tr><td>11:00</td><td>11:15</td><td>Selected oral presentations</td><td>Coxiella burnetii immunity and shedding dynamics after vaccination of dairy goats in field conditions</td><td></td><td>Géraldine Boseret</td></tr><tr><td>11:15</td><td>11:30</td><td></td><td>Risk categorization in the context of a porcine reproductive and respiratory syndrome (PRRS) regional control program</td><td></td><td>Fabiana Dal Pozzo</td></tr><tr><td>11:30</td><td>11:45</td><td></td><td>Implementation of an information system to gather data on dogs' bites and vaccinations against rabies in the Veneto Region.</td><td></td><td>Alberto Aallepuz</td></tr><tr><td>11:45</td><td>12:00</td><td></td><td>Epizootic of Contagious Caprine Pleuropneumoniae in sand gazelles (Gazella subgutturosa marica) in the United Arab Emirates</td><td></td><td>Donata Trimarchi</td></tr><tr><td>12:00</td><td>13:30</td><td>Lunch Break</td><td>Poster Session</td><td></td><td>Orangerie</td></tr><tr><td colspan="6"><strong>Session 2A</strong> Climate change and diseases</td></tr><tr><td>13:30</td><td>14:00</td><td>Keynote lecture</td><td>Global changes and the emergence of important zoonoses</td><td>Chairman:</td><td>Marius Gilbert</td></tr><tr><td>14:00</td><td>14:15</td><td>Selected oral presentations</td><td>Influence of water temperature on the outcome of a Brucella pinnipedialis hooded seal (Cystophora cristata) strain infection in cod (Gadus morhua)</td><td></td><td>Marius Gilbert</td></tr><tr><td>14:15</td><td>14:30</td><td></td><td>Seroprevalence of Borrelia burgdorferi in horses in the southern part of Belgium: a "one health" driven study</td><td></td><td>Jacques Godfroid</td></tr><tr><td>14:30</td><td>14:45</td><td></td><td>Effectiveness and cost efficiency of syndromic surveillance compared to conventional surveillance: bluetongue serotype 8 across countries as case study</td><td></td><td>Meersschaert Carole</td></tr><tr><td>14:45</td><td>15:00</td><td></td><td>Serological monitoring 2008-2015 of Walloon dairy herds exposition to Leptospira hardjo based on BTM Elisa</td><td></td><td>Sarah Welby</td></tr><tr><td colspan="6"><strong>Session 2B</strong> Biosecurity in livestock, pets and horses</td></tr><tr><td>13:30</td><td>14:00</td><td>Keynote lecture</td><td>Ensuring continuous biosecurity for high health high performance (HHP) horses: a public-private collaboration</td><td>Chairman:</td><td>Morgane Dominguez</td></tr><tr><td>14:00</td><td>14:15</td><td>Selected oral presentations</td><td>Biosecurity measures applied in the United Arab Emirates: a comparative study between livestock and wildlife sectors</td><td></td><td>Morgane Dominguez</td></tr><tr><td>14:15</td><td>14:30</td><td></td><td>How to assess the biosecurity level in stud farms? Survey among 70 Belgian and French horse breeders</td><td></td><td>Anne-Lise Chaber</td></tr><tr><td>14:30</td><td>14:45</td><td></td><td>A descriptive study to assess the perception of cattle farmers towards the implementation of biosecurity</td><td></td><td>Marie-France Humblet</td></tr><tr><td>14:45</td><td>15:00</td><td></td><td>Assessing motivators and obstacles for implementation of biosecurity measures in aimal production in Europe</td><td></td><td>Bert Damiaans</td></tr><tr><td>12:00</td><td>13:30</td><td>Coffee Break</td><td>Posters session</td><td></td><td>Orangerie</td></tr><tr><td colspan="6"><strong>Session 3A</strong> Vector borne diseases</td></tr><tr><td>15:30</td><td>16:00</td><td>Keynote lecture</td><td>The German Arbovirus Surveillance and Mosquito Monitoring Program, 2009 - 2015</td><td>Chairman:</td><td>Jonas Schmidt-Chanasit</td></tr><tr><td>16:00</td><td>16:15</td><td>Selected oral presentations</td><td>Qualitative spatial assessment of human TBE risk factors in Belgium</td><td></td><td>Jonas Schmidt-Chanasit</td></tr><tr><td>16:15</td><td>16:30</td><td></td><td>Resurgence of Schmallenberg virus in Belgium after 3 years of epidemiological silence</td><td></td><td>Sophie Roelandt</td></tr><tr><td>16:30</td><td>16:45</td><td></td><td>What are the vector-borne pathogens present in the blood of French cattle? Results of a field study on 130 samples</td><td></td><td>Laurent Delooz</td></tr><tr><td>16:45</td><td>17:00</td><td></td><td>Schmallenberg virus infection in sheep: a model of within-herd seroprevalence evolution</td><td></td><td>Claire Pelletier</td></tr><tr><td colspan="6"><strong>Session 3B</strong> One health: antimicrobial resistance</td></tr><tr><td>15:30</td><td>16:00</td><td>Keynote lecture</td><td>Animal-human-environment continuum in the emergence and spread of antibiotic resistance in human pathogens</td><td>Chairman:</td><td>Axel Cloeckaert</td></tr><tr><td>16:00</td><td>16:15</td><td>Selected oral presentations</td><td>A quantitative exposure model for the cross-contamination of pig feeding-stuffs as a result of antimicrobial residues</td><td></td><td>Axel Cloeckaert</td></tr><tr><td>16:15</td><td>16:30</td><td></td><td>The influence of the introduction of antimicrobial use guidelines on prescription habits in first-line, small animal veterinarians</td><td></td><td>Marilena Fillipitzi</td></tr><tr><td>16:30</td><td>16:45</td><td></td><td>Identification of bovine methicillin resistant staphylococci from Europe, Africa and North America by colony hybridization, PCR and antibiotic sensitivity</td><td></td><td>Steven Sarrazin</td></tr><tr><td>16:45</td><td>17:00</td><td></td><td>Identification of bovine and porcine colistin-resistant mcr1-positive Escherichia coli</td><td></td><td>Damien Thiry</td></tr><tr><td>17:15</td><td>19:00</td><td></td><td>Visit of Liège</td><td></td><td>Damien Thiry</td></tr><tr><td>20:00</td><td>23:55</td><td></td><td>AESA 25<sup>th</sup> anniversary GALA DINNER</td><td></td><td>Salle Le Bal</td></tr></tbody></table>
<table><tr><td>8:30</td><td>9:00</td><td>Registration</td><td>Lobby - Reception</td></tr></table>

<table><tr><td>Session 4A</td><td>Wildlife diseases</td><td>Chairman:</td><td>Christian Gortázar</td><td>Salle Le Bal</td></tr></table>

<table><tr><td>9:00</td><td>9:30</td><td>Keynote lecture</td><td>TB epidemiology in Europe: past, present and future of a multi-host infection</td><td>Christian Gortázar</td></tr><tr><td>9:30</td><td>9:45</td><td>Selected oral presentations</td><td>Genotyping analysis of Brucella melitensis at the wildlife-livestock-human interface in the Middle East</td><td>Anne-Lise Chaber</td></tr><tr><td>9:45</td><td>10:00</td><td></td><td>Occurrence of Cryptosporidium Oocysts in Edible Frogs (Rana Species) sold for Human Consumption in Hanwa Frog Market Zaria, Kaduna State, Nigeria</td><td>Grace Kia</td></tr><tr><td>10:00</td><td>10:15</td><td></td><td>Occurrence of the new pathogenic variant of rabbit haemorrhagic disease virus (RHDV2) in wild populations of rabbits in Southern Belgium</td><td>Rosario Volpe</td></tr><tr><td>10:15</td><td>10:30</td><td></td><td>Validation of Bayesian prevalence estimation with an imperfect diagnostic test</td><td>Mathias Flor</td></tr></table>

<table><tr><td>Session 4B</td><td>Participatory epidemiology</td><td>Chairman:</td><td>Marisa Peyre</td><td>Salon Gretry-N.</td></tr></table>

<table><tr><td>9:00</td><td>9:30</td><td>Keynote lecture</td><td>We need to talk! The added value of participatory epidemiology</td><td>Marisa Peyre</td></tr><tr><td>9:30</td><td>9:45</td><td>Selected oral presentations</td><td>Review of Participatory Epidemiology practices in Animal Health (1980-2015)</td><td>Alberto Aallepuz</td></tr><tr><td>9:45</td><td>10:00</td><td></td><td>Applying sociological methods to the study of animal diseases: the case of Bovine Tuberculosis in Spain</td><td>Giovanna Ciaravino</td></tr><tr><td>10:00</td><td>10:15</td><td></td><td>On the path towards reduced antimicrobial resistance: herd-level quantification of veterinary antimicrobial consumption in Belgian pig herds through the online monitoring tool 'AB Register'.</td><td>Wannes Vanderhaeghen</td></tr><tr><td>10:15</td><td>10:30</td><td></td><td>Improvement of micro-organisms classification methodology into risk groups</td><td>Aline Baldo</td></tr></table>

<table><tr><td>10:30</td><td>11:00</td><td>Cofee Break</td><td>Posters session</td><td>Orangerie</td></tr></table>

<table><tr><td>Session 5A</td><td>Epidemiology, surveillance and risk analysis</td><td>Chairman:</td><td>Claude Saegerman</td><td>Salle Le Bal</td></tr></table>

<table><tr><td>11:00</td><td>11:30</td><td>Keynote lecture</td><td>Epidemiology, surveillance and risk analysis: past, present and future</td><td>Claude Saegerman</td></tr><tr><td>11:30</td><td>11:45</td><td>Selected oral presentations</td><td>Classification of adult cattle diseases in Belgium based on a veterinary and a laboratory survey, years 2013-2015</td><td>Véronique Renault</td></tr><tr><td>11:45</td><td>12:00</td><td></td><td>How does pooling of serum or blood samples from young calves permanently infected with BDV-V affect the detectability of the real time PCR test? A field study in the context of the Belgian BVD eradication program.</td><td>Jean-Baptiste Hanon</td></tr><tr><td>12:00</td><td>12:15</td><td></td><td>Approach towards benchmarking surveillance: bovine tuberculosis in Belgium as an exemple</td><td>Sarah Welby</td></tr><tr><td>12:15</td><td>12:30</td><td></td><td>Computer and veterinary sciences: an integrated database for disease surveillance</td><td>Simone Rizzo</td></tr></table>

<table><tr><td>Session 5B</td><td>Bee diseases</td><td>Chairman:</td><td>Dennis vanEngelsdorp</td><td>Salon Gretry-N.</td></tr></table>

<table><tr><td>11:00</td><td>11:30</td><td>Keynote lecture</td><td>A sticky mess - understanding the drivers of honey bee losses</td><td>Dennis vanEngelsdorp</td></tr><tr><td>11:30</td><td>11:45</td><td>Selected oral presentations</td><td>Development of an original bee health management tool for beekeepers: Bee Best Check</td><td>Noëmie El Agrebi</td></tr><tr><td>11:45</td><td>12:00</td><td></td><td>A Multidisciplinary and evidence-based methodology applied for prioritizing bee diseases in France</td><td>Suzanne Bastian</td></tr><tr><td>12:00</td><td>12:15</td><td></td><td>Presence of plant protection product and veterinary drug residues in beeswax: risk scenario analysis of the consumer chronic exposure and proposition of action limits</td><td>Olivier Wilmart</td></tr><tr><td>12:15</td><td>12:30</td><td></td><td>Seroprevalence and risk factor analysis of small ruminant lentiviruses in Belgian sheep and goats</td><td>Rodolphe Michiels</td></tr></table>

<table><tr><td>12:30</td><td>14:00</td><td>Lunch Break</td><td>Poster Session</td><td>Orangerie</td></tr></table>

<table><tr><td>Session 6</td><td>Drivers of diseases emergence</td><td>Chairman:</td><td>Juana Bianchini</td><td>Salle Le Bal</td></tr></table>

<table><tr><td>14:00</td><td>14:30</td><td>Keynote lecture</td><td>Main drivers of emerging and re-emerging infectious diseases: Focus on livestock</td><td>Juana Bianchini</td></tr><tr><td>14:30</td><td>14:45</td><td>Selected oral presentations</td><td>Illegal meat trade - a threat to both biodiversity and public health</td><td>Anne-Lise Chaber</td></tr><tr><td>14:45</td><td>15:00</td><td></td><td>Recombination (genetic evolution) as driver of new epidemic norovirus strains</td><td>Louisa Ludwig</td></tr><tr><td>15:00</td><td>15:15</td><td></td><td>Bayesian assessment of two competitive ELISAs for the detection of bovine viral diarrhoea virus antibodies in bovine sera</td><td>Claude Saegerman</td></tr><tr><td>15:15</td><td>15:30</td><td></td><td>A bayesian evaluation of four antibody ELISA tests to certify freedom of bovine viral diarrhea infection in unvaccinated cattle</td><td>Sophie Roelandt</td></tr></table>

<table><tr><td>15:30</td><td>15:45</td><td>AVIA-GIS Poster Award - AESA Poster Award</td><td>Valérie De Waele</td><td>Salle Le Bal</td></tr></table>

<table><tr><td>15:30</td><td>16:00</td><td>Closing Ceremony</td><td>Claude Saegerman</td><td>Salle Le Bal</td></tr></table>
<figure><img src="image_14.png" /></figure>

Association d'Epidémiologie et de Santé Animale

Workshops
# Workshop 1- Estimation of test characteristics and true prevalence using a Bayesian framework

Prof. Claude Saegerman

Research Unit of Epidemiology and Risk Analysis applied to veterinary sciences (UREAR-ULg)

Fundamental and Applied Research for Animal and Health (FARAH) Center,

Faculty of Veterinary Medicine, University of Liege

<figure><img src="image_15.png" /></figure>

claude.saegerman@ulg.ac.be

Bayesian and frequentist philosophies are often used to estimate diagnostic test performance such as sensitivity (Se), specificity (Sp) and the true prevalence (TP) of diseases. However some controversies exist between the two philosophies as the frequentist approach uses fixed parameter values whilst the Bayesian approach uses prior information (Berkvens et al., 2003; Sanogo et al., 2014).

The Bayesian philosophical methods are based on the idea that unknown quantities, such as population means or proportions (Se, Sp and/or TP), have a probability distribution that expresses our prior knowledge or belief about these quantities, before adding the knowledge gained from true observational data. In other words, Bayesian inference considers the data to be fixed and parameters to be random, because they are unknown. In contrast, in frequentist methods prior knowledge is obtained differently and population means or proportions are considered as fixed values (Bland and Altman, 2002). Thus, frequentist inference considers the unknown parameters to be fixed and the data to be random.

By offering the use of prior or expert knowledge on parameters combined with actual field data in the same model, the Bayesian approach is useful to obtain more accurate and reliable estimates in the absence of a gold standard (Berkvens et al., 2006). However, accuracy and validity of Bayesian estimates depend on the availability and the quality of prior information included in the estimation and on the validity of the protocol (e.g. conditional dependence between tests or not, conditions of validation of the procedure). Additionally, original Bayesian approach allows sharing information and results between scientists from different disciplines (e.g. infectology, immunology, epidemiology and Bayesian statistics). Sharing of information and the interdisciplinary approach are useful to ensure the good estimation of test characteristics (Se and Sp) and TP of disease.

Estimation of these parameters is of prime importance to ensure disease surveillance programs (Sanogo et al., 2013), especially when the animal samples are of an unknown status (e.g. absence of gold standard or emerging diseases).

Moreover, the World Assembly of delegates of the OIE added recently the Bayesian approach to the OIE terrestrial manual as a tool to estimate the Se and the Sp of diagnostic tests (OIE, 2014).

The objectives of this workshop are to present some key elements to be considered in the estimation of test characteristics and true prevalence using a Bayesian framework. A practical exercise will also be given to be solved in order to gain confidence and independence but also to learn how to perform a critical appraisal and validity control.

## References

Berkvens, D.L., Speybroeck, N., Lesaffre, E., Saegerman, C., 2003. Comments on "Pouillot, R., Gerbier, G., Gardner, I.A., 2002. 'TAGS', a program for the evaluation of test accuracy in the absence of a gold standard. Preventive Veterinary Medicine 53, 67-81". Preventive Veterinary Medicine 59, 181-183.

Berkvens, D., Speybroeck, N., Praet, N., Adel, A., Lesaffre, E., 2006. Estimating disease prevalence in a Bayesian framework using probabilistic constraints. Epidemiology (Cambridge, Mass.) 17, 145-153.

Bland, J.M., Altman, D.G., 2002. Statistics notes: Validating scales and indexes. British Medical Journal 324, 606-607.

OIE, 2014. Terrestrial Manual 2013, Chapter 1.1.5 Principles and methods of validation of diagnostic assays for infectious diseases. OIE, Paris, France, pp. 17.

Sanogo, M., Abatih, E., Saegerman, C., 2014. Bayesian versus frequentist methods for estimating true prevalence of disease and diagnostic test performance. Vet J. 202(2):204-207.

Sanogo, M., Thys, E., Achi, Y.L., Fretin, D., Michel, P., Abatih, E., Berkvens, D., Saegerman, C., 2013. Bayesian estimation of the true prevalence, sensitivity and specificity of the Rose Bengal and indirect ELISA tests in the diagnosis of bovine brucellosis. Vet J. 195(1):114-120.
# Workshop 2 - Abortion indicators

## Pascal HENDRIKX

Chief of the Surveillance Coordination & Support Unit (unité de coordination et d'appui à la surveillance- UCAS)
Anses - laboratories directorate,
Platform for epidemiological surveillance in animal health (Plateforme ESA)

<figure><img src="image_16.png" /></figure>

Pascal.HENDRIKX@anses.fr

Monitoring of abortions indicators in ruminants may have several objectives. This is primarily an issue for early detection of exotic priority diseases such as brucellosis or Rift Valley fever. This monitoring may also allow early detection of new health events, as was the case for Schmallenberg disease in 2011 or unusual manifestations of known diseases as the "abortion, jaundice and splenomegaly" syndrome detected in Belgium in 2014. In addition, many causes of abortions in ruminants deserve a dedicated follow up to enable farmers and veterinarians to adopt the best prevention practices and treatments to reduce the economic impact on livestock.

Estimation of abortions indicators is carried out by the implementation of various monitoring methods. Direct indicators of abortions are typically measured through data from notifications made by farmers and investigations made by veterinarians to exclude priority threats and sometimes determine the cause abortion. It is commonly accepted and demonstrated that this direct indicator often lack sensitivity and improvement require heavy financial investments disproportionate to the efficiency that can be expected. To overcome the limitations of these direct indicators, several tracks have recently been explored to identify indirect indicators based on the analysis and processing of data already available such as calving intervals, artificial insemination intervals or the number of calves produced by cow. All these direct or indirect indicators show performance differences in terms of reliability, efficiency and cost-effectiveness.

Several types of activities can be implemented to try to improve the performance of abortion indicators. This is primarily to conduct assessment activities such as the global assessment of surveillance systems using qualitative methods or the assessment of the sensitivity of abortions notification systems by quantitative methods such as capture recapture. Quality of indicators also directly depends on the quality of the data collected. Therefore, data quality has to be regularly evaluated and improved.

From an operational perspective, the improvement of classical indicators requires to conduct communication actions based on the knowledge of limits and motivations of the actors of the surveillance system to notify and investigate the abortion events. This knowledge requires performing sociological studies. The operational measure of indirect indicators represents a major challenge in terms of an automated data collection, analysis and processing and production of results.
The objective of this workshop is to share experiences and lead a collective reflection on the objectives, issues and operational modalities of production of abortions indicators in support to effective and coherent health strategies.
<figure><img src="image_17.png" /></figure>

Association d'Epidémiologie et de Santé Animale

# Crisis management
in animal health

7th - 9th Sept. 2016 - Liège (Belgium)
Congress 25 years

Animal epidemiology
Past, present and future
# Keynote lecture

## Crisis management in animal health

Géraldine Boseret¹, DVM, MScV, PhD

¹ FASFC

Géraldine is born in Liège, Belgium. She graduated as Doctor in Veterinary Medicine in 2000 and Doctor in Vet Sciences (PhD) in 2008. After 13 years as assistant in Faculty of Veterinary Medicine of University of Liège (bacteriology, food microbiology and epidemiology, respectively), she was hired as Veterinary Inspector at the Federal Agency for the Safety of the Food Chain (FASFC) in Brussels, and then as Emerging Diseases Expert in Directorate Control Policy of the FASFC, section Animal Health (S2).

Her expertise fields are emerging diseases such as Lumpy Skin Disease, Bluetongue, Foot and Mouth Disease, Swine fevers, Avian Influenza, Rabies, but also endemic diseases in pigs, small ruminants and lagomorphs. She is also conjoint focal point for OIE, regarding wildlife diseases.

The tasks she is committed to are diversified: writing the legislation, giving expert advices, evaluating and submitting European co-financed surveillance programs (e.g. Bluetongue), participating in scientific workshops.
She is now candidate to a residency in the European College of Veterinary Public Health.

<figure><img src="image_18.png" /></figure>

Géraldine.Boseret@afsca.be

<figure><img src="image_19.png" /></figure>

## Background

How could we define a crisis in animal health?

From an epidemiological point of view, a crisis in animal health is defined as the emergence (new disease)/re-emergence (already known disease) of an animal disease in a short time period and in a limited spatial location, which presents a risk of expansion or establishment on the territory (Toma and Thiry, 2003).

From an economic-political point of view, one would consider such a disease as a crisis matter if it presents a major impact in public health (e.g.: zoonosis: avian influenza) and/or significant economic consequences (e.g. foot-and-mouth disease).

In both cases, the threat should be controlled and, at best, finally eradicated.

**Crisis management** include all actions a country will implement to prevent and fight the introduction of a disease on its territory. In Belgium, the Federal Agency for the Security of the Food Chain is responsible for this prominent task, in collaboration with the Ministry of Public Health and all actors involved such as farmers, vets, laboratories, other Ministries, animal health associations and universities.

The Crisis Cell, along with the S2 - Animal Health Section of the Directorate of Control Policy, are the key node of crisis management, as they are at the meeting point between Belgian stakeholders and international authorities, especially the European Union and the World Organization for Animal Health.

But how does crisis management in animal health work?

Three examples will enlighten this difficult and complex process, taking in account not only the specific epidemiological context, but also the drivers which could influence the management of a crisis, such as economic constraints, public opinion and the press.

## European Union: introduction of lumpy skin disease: a true emergence story

The author will present the origin of LSD and its expansion throughout the European Union since August 2015, when the disease was first identified in Greece.

Measures taken by infected countries and the European Union will be discussed, and put in perspective with the 5 T's: Travel, Trade ; Transport, Tourism, Terrorism.

Authorities involved in the decision process will be shortly presented: SCoPAFF, EFSA, CVO meetings, GT-TADs.
<figure><img src="image_20.png" /><figcaption>Fig 1: Number of outbreaks notified per month per country (source: ADNS ©).</figcaption></figure>

### Neighboring countries: the story of bluetongue (re-)emergence

The author will present the spreading process of the bluetongue virus serotype 8 [BTV-8] across France in 2015-2016, and discuss whether it is a real re-emergence or not.

As France shares a border with Belgium, the risk of introduction of BTV-8 has to be pragmatically considered, and therefore, a pre-crisis structure was created by the Belgian Authority along with stakeholders. That would be a good example of *Preparedness*, which will be described based on the Belgian model and compared to the actions taken by other neighbouring countries.

The acute point of vaccination will also be an important aspect of the discussion, as it is forbidden in Belgium since 2012, as well as the economic impact of such prophylactic measures in comparison with the cost of a bluetongue crisis.

### Rabbit haemorrhagic disease virus 2 (RHDV2): a Belgian story

In July 2016, the « new » variant of the rabbit haemorrhagic disease virus (RHDV) was isolated in domestic and wild rabbits in Belgium. This disease, in contrast with bluetongue and lumpy skin disease, is not submitted to a mandatory notification, and will therefore be managed differently compared to both diseases addressed previously.

The author will also discuss what should be considered as “false” emergence and will put forward the omnipresent “media-related” power which could threaten or bias the evaluation of a sanitary crisis.
# Selected oral presentations

## Coxiella burnetii immunity and shedding dynamics after vaccination of dairy goats in field conditions

Fabiana Dal Pozzo<fnref n="1" />, Guy Czaplicki<fnref n="2" />, Fabien Grégoire<fnref n="2" />, Nathalie Kirschvink<fnref n="3" />, Claude Saegerman<fnref n="1" />

<figure><img src="image_21.png" /></figure>

fdalpozzo@ulg.ac.be

### Objective

In Belgium the Ministerial Decree of May 11, 2011 established some surveillance and control measures for Q fever to be applied to ovine and goat dairy farms. As an example, compulsory vaccination of PCR positive farms has been implemented. The aim of this work was to investigate the effects of Q-fever vaccination under field conditions in some Belgian dairy goat farms. Yearly vaccination was performed by the farm's veterinarian under field conditions.

### Methods

At the beginning of the study, farms were classified according to the length of the vaccination protocol (1, 2 and more than 2 years of vaccination). The effect of the vaccination was evaluated based on sampling carried out between 9 to 11 months after the occurrence of the last annual vaccination. Three groups of randomly selected goats were sampled according to their age (1, 2 and older than 2 year-old). Serum, milk, vaginal and rectal swabs were collected, as well as bulk tank milk (BTM), air collected inside the farm and feces on the stable floor by the «overshoes» method. Antibodies against C. burnetii were detected in serum, individual milk and BTM. The bacteria's shedding was evaluated from individual animal samples (milk, vaginal and rectal swabs) and from the BTM, the air samples and the overshoes. The animal ethical commission at the University of Liege approved the study protocol.

### Results

Seven goat farms were visited and included in the study. C. burnetii DNA could be found in the BTM of the some farms as well as in environmental samples (air samples and feces with «overshoes» method). Furthermore, despite vaccination seronegative animals could be found using a commercialized ELISA targeting both phases 1 and 2 antigens. According to their age, it was assumed that 1 year-old goats got one vaccination, 2 year-old goats were vaccinated twice and older goats received at least 3 vaccinations in their life. Vaccinated goats belonging to the 1 year-old category were more frequently seronegative than the oldest category of goats, assuming that the measured antibodies were lasting shorter in young goats. However, this ELISA did not allow to differ between antibodies developed after vaccination and natural infection. Indeed, because environmental contamination was proved (air samples and feces with the «overshoes» method), the natural exposition of the goats to C. burnetii cannot be ruled out. Furthermore, bacterial shedding was detected in vaccinated goats in milk, vaginal and rectal swabs.

### Conclusion

This study allowed to describe the efficacy of Q fever vaccination when applied in field conditions. Further studies will aim to characterize the humoral immunity of goats in function of the occurrence of bacterial shedding and distinguish recent or chronic infections.

**Keywords:** Coxiella burnetii ; Q fever ; dairy goats ; vaccination

<footnote n="1">¹ University of Liege, Fundamental and Applied Research for Animals Health (ULg - FARAH) - ULg FMV Epidemiologie B42 Quartier Vallee 2 Avenue de Cureghem 7A 4000 LIEGE, Belgium</footnote>

<footnote n="2">² Department of Animal Health, Association Régionale de Santé et d'Identification Animales (ARSIA), Ciney, Belgium</footnote>

<footnote n="3">³ Namur Research Institute for Life Sciences (Narilis), UNamur, Belgium</footnote>
# Risk categorization in the context of a porcine reproductive and respiratory syndrome (PRRS) regional control program

Alberto Allepuz<fnref n="1" />, Jordi Casal<fnref n="1" />, Enric Mateu<fnref n="1" />, Gerard Martín<fnref n="2" />

<figure><img src="image_22.png" /></figure>

alberto.allepuz@uab.cat

## Objective

A pilot program of voluntary regional control of PRRS is being initiated in selected areas of Spain. Within this context we are developing a semi-quantitative risk analysis in order to estimate the risk of having an external introduction and a recirculation of PRRS.

## Methods

A 139-question survey has been performed in 79 farms included in the control program in order to obtain data related with the dierent biosecurity measures applied. These measures included those ones related to purchase of animals and semen, location, transport vehicles and visits (i.e. external biosecurity) and to management practices, facilities cleaning and disinfection and employees routines (i.e. internal biosecurity).

In order to estimate the initial risk posed by the purchase of animals and semen, neighborhood, vehicles and visits together with the importance of the dierent biosecurity measures an expert opinion workshop was performed. In this workshop 24 different veterinarians and researchers actively involved in PRRS activities participated and gave their scores based in a ten-points scale as suggested by Dufour et al., (2011). The nal risk estimates of PRRS external introduction and internal recirculation, taking into account the variability associated with the expert opinion procedure, were calculated by Monte Carlo simulations using the mc2d package implemented in R (Pouillot and Delignette-Muller, 2010).

## Results

Preliminary results shows that vehicles presented the higher initial external risk and that despite the reduction derived of the application of external biosecurity measures they continued to have a higher score than the other routes. The percentage of reduction (i.e. measures currently in place in relation to all the possible measures to be applied) indicated that there is high room for improvement in order to minimize the risk of introduction or recirculation.

## Conclusion

By using this methodology we have been able to rank farms in relation to their risk of introduction or recirculation. This information might be useful for PRRS control activities. Nevertheless, the method is very dependent on the initial scores and the importance of the dierent biosecurity measures provided by the panel of experts and therefore it would be desirable to update these values in the light of new knowledge related with PRRS epidemiology. Is also important to consider that these scores might vary depending on the epidemiological context of the disease and therefore the scores should be updated to each particular situation prior to apply the method.

## Reference

Dufour B, Plee L, Moutou F, Boisseleau D, Chartier C, Durand B, Ganiere JP, Guillotin J, Lancelot R, Saegerman C, Thebault A, Hattenberger AM, Toma B. A qualitative risk assessment methodology for scientific expert panels. Rev Sci Tech. 2011, 30, 673-81.

Pouillot, R., Delignette-Muller, M.L., 2010. Evaluating variability and uncertainty inmicrobial quantitative risk assessment using two R packages. Int. J. FoodMicrobiol. 142 (3), 330{340.

**Keywords:** Biosecurity ; risk assessment

<footnote n="1">¹ Departament de Sanitat i Anatomia Animal, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain - Spain</footnote>

<footnote n="2">² Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain</footnote>
# Implementation of an information system to gather data on dogs' bites and vaccinations against rabies in the Veneto Region

Donata Trimarchi¹, Laura Bortolotti¹, Mario Cobianchi¹, Manuela Lanari¹, Simone Rizzo¹, Michele Brichese²

¹ Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe) - Italy

² Sezione Veterinaria e Sicurezza Alimentare. Regione Veneto - Italy

<figure><img src="image_23.png" /></figure>

dtrimarchi@izsvenezie.it

## Objective

Zoonotic infections transmitted by companion animals could be a serious public health problem, as in the case of domestic rabies, which has been eradicated from industrialized countries over the last 60 years by a process of control of the dog population. In autumn 2009, when fox-mediated rabies re-emerged in the Veneto Region (north-eastern Italy), among other

emergency measures, dog vaccination was made compulsory in a specific area at higher risk of rabies transmission and an information system was implemented in order to support the management of the vaccination campaign and to monitor dog's aggressions and bites, due to the importance that bites play in rabies transmission.

## Methods

Since 2003 a Regional Canine Registry Data Bank (BAC) has been operating in the Veneto Region, in which the data (e.g. breed, sex, age, neutering state, etc.) and the movements of dogs (e.g. change of ownership, transfer, death, etc.), together with the personal data of their owners and keepers are stored. The BAC contains also the data on dogs which are captured and identi_ed as stray dogs and are housed in a shelter.

New tools were added to the BAC in order to gather the main data of the vaccinations performed by Veterinary Services (VSs) and Veterinary practitioners (VPs), such as: brand and name of the product, batch number, date and place of vaccination, expiry date of immunization, type of vaccination (privately or within the Public Veterinary Service) and attending veterinarian.

Initially the data was entered only by VSs, then also VPs were allowed to access the system. Other functions were added to the BAC, allowing the reporting of the data on each dog's aggression (i.e. context, date, victims, reporting authority) to obtain an archive of biting dogs and dogs with uncontrolled aggression. Eventually biting dogs are evaluated into a "0-3" scale of dangerousness and speci_c measures are set for safekeeping of the animal depending on the severity of the identi_ed risk: main information about the dog evaluation (e.g. veterinarian, risk category attributed, date) are also recorded in the BAC.

## Results

From 2010 to 2012 these tools were used massively: a total of 343,985 vaccinations and boosters were entered on a population of 287,723 dogs living in the risk area at the end of the mandatory vaccination period and 6,509 bites were recorded, on a population of 778,342 dogs living in the whole territory of the Veneto Region. From 2013 Italy has regained rabies free status, but the above mentioned functions are still in use. From 2013 to 2015 a mean of 24,266 vaccinations, 2,251 bites/aggressions are recorded and 2,182 dogs are evaluated each year.

## Conclusion

The activation of a computerized data collection system in animal health served as working tool for epidemiological surveillance activities and emergencies management. This information system has become an instrument of daily use and has replaced previous paperbased information ows. The centralized database allows VSs and VPs to record and access data directly, detailed reports can be produced in real time, and, in addition, people who handle dogs (e.g. veterinarians, dogcatchers) and usually consult the BAC are immediately informed about the dog's vaccination, its aggressions and its risk category which could be useful for their own safe. Moreover VSs' operating procedures in handling a bite event have been standardized using the same software.

**Keywords:** Information system ; Rabies ; Dog
# Epizootic of Contagious Caprine Pleuropneumoniae in sand gazelles (Gazella subgutturosa marica) in the United Arab Emirates

Anne-Lise Chaber<fnref n="1" />,<fnref n="2" />, Louis Lignereux<fnref n="1" />,<fnref n="3" />, Claude Saegerman<fnref n="1" />, Lucia Manso-Silvan<fnref n="4" />,<fnref n="5" />, Armelle Peyraud<fnref n="4" />,<fnref n="5" />, Andrea Appoloni<fnref n="4" />,<fnref n="5" />, François Thiaucourt<fnref n="4" />,<fnref n="5" />

<figure><img src="image_24.png" /></figure>

alchaber@hotmail.com

Domestic goats were long considered the only species susceptible to Contagious Caprine Pleuropneumonia (CCPP), a devastating bacterial disease transmitted exclusively by direct contact between infected and susceptible animals. However, CCPP has been recently confirmed in an increasing number of wild ruminant species. This work describes the course of a CCPP outbreak within a captive collection of more than 3000 Sand gazelles (Gazella subgutturosa marica) kept in a set of well-separated enclosures located in a desert area.

The study revealed that Sand gazelles are highly susceptible to CCPP, with mortality rates reaching 70%, and that transmission can take place "at a distance" of at least 80 meters. Various antibiotic treatments and immunization with different vaccine preparations were implemented. The commercial vaccines tested showed little effect and did not elicit any measurable serological response by either the reference competitive ELISA test or an in-house indirect ELISA. On the other hand, a single injection of a reference vaccine manufactured according to the OIE guidelines elicited a high specific serological response. The observation of daily deaths allowed the calibration of the parameters of a compartmental model and the first estimation of the basic reproductive ratio (R0) for a CCPP outbreak.

These findings and analyses call for new measures regarding CCPP surveillance and prevention/control: i) endangered wildlife ruminants, kept in captivity for conservation purposes, must be considered at risk of CCPP, which can be transmitted even in the absence of close contact with infected animals; ii) the quality of commercial CCPP vaccines needs to be improved and thoroughly assessed; iii) in silico models may allow establishing more suited intervention strategies combining the use of antibiotics, which reduce Mccp shedding, and vaccines, which decrease animal susceptibility. Biosecurity protocols and vaccination strategies must be aligned with these recommendations in order to control and mitigate the risk that CCPP poses to both the wildlife species conservation and the livestock production sectors.

**Keywords:** Contagious Caprine Pleuropneumonia ; wildlife ; transmission ; vaccines ; UAE

<footnote n="1">¹ Faculty of Veterinary Medicine, University of Liège (UREAR-ULg, FARAH) - Belgium</footnote>

<footnote n="2">² Wildlife Consultant L.L.C, Al Ain.</footnote>

<footnote n="3">³ Environment Agency Abu Dhabi, Abu Dhabi. - United Arab Emirates</footnote>

<footnote n="4">⁴ CIRAD, UMR CMAEE, Montpellier - Centre de coopération internationale en recherche agronomique pour le développement [CIRAD] - France</footnote>

<footnote n="5">⁵ INRA, UMR1309 CMAEE, Montpellier - Institut national de la recherche agronomique (INRA)</footnote>
# Posters

## Radioscopy laboratory diagnostic of epizootic diseases in Belgium and European countries

Mickaël Cargnel

Veterinary and Agrochemical Research Centre (CODA-CERVA) - Groeselenberg 99, 1180 Uccle - Belgium

<figure><img src="image_25.png" /></figure>

micargnel@hotmail.com

### Objective

Belgium plays a key role in international animal trade making it very sensitive to disease introduction via different pathways from around the world or via wildlife. It is therefore crucial to react quickly to these diseases, to establish appropriate contingency plans and to develop reliable diagnostic tests.

### Methods

Based on 29 selected diseases (Bianchini J., personal communication), a mapping of existing techniques was created. An overview of assays available and which would be used in different scenarios (risk importation, early detection, spread and freedom of disease) in national and international laboratories was established. If gaps were detected, international laboratories were contacted in order to create future partnerships. These data were completed by the analysis of the World Animal Health Information System (WAHIS) online tools of the World Organization for Animal diseases (OIE) for the 40 major European countries (possibilities and assays used) encoded for 23 of the 29 diseases included in the OIE list

### Results

These surveys have showed that virus isolation and virus neutralization test are sensitive and specific and are therefore often considered as gold standard confirmation tests. However, they are slow, laborious and require biosafety laboratory facilities. For many diseases, enzyme-linked immunosorbent assays (ELISA) are often used and commercially available. Although its rapidity, often good sensitivity and specificity, ELISA needs to be standardized and fit for purpose which requires training and experience. Molecular methods such as the polymerase chain reaction (PCR or mostly real-time PCR) are very specific and sensitive which give qualitative and/or quantitative result in a few hours but need adequate material (e.g. good extraction protocol). In addition, PCR's are very often sequence-dependent and thus sensitive to mutations and contaminations which can lead to false negative results. Sequencingoffers promising solutions but is currently only used as confirmation for first suspicious samples and is still restrained for research purposes because of the price for the equipment, the samples preparation and the requirement for trained staff. On the other hand, this project suggests the importance of implementing, keeping up-to-dated online data at European level for (re-)emerging diseases, especially for Cache valley virus, Aino disease, Nipah disease, Porcine deltacoronavirus.

### Conclusion

It was showed that Belgium is able to establish a diagnostic for 25 of the 29 of targeted diseases and underlined the importance of collaboration between laboratories permitting Belgium to have the diagnostic capacities to face all situations mentioned in the scenarios.

**Keywords:** emerging diseases ; laboratory assays ; diagnostic
<figure><img src="image_26.png" /></figure>

Association d'Epidémiologie et de Santé Animale

Climate change
and diseases
Keynote lecture

Global changes and the emergence of important zoonoses

Marius Gilbert ¹PhD

Spatial epidemiology lab. (SpELL), Université Libre de Bruxelles, Brussels, Belgium

Marius Gilbert (Brussels, 1972) is Senior FNRS Research Associate at the Université Libre de Bruxelles, where he leads
the Spatial Epidemiology Lab (SpELL, http://lubies.ulb.ac.be/Spatepi.html).

His researches deal with agricultural intensification and changes in agro-ecosystems leading to the emergence and
spread of emerging infectious diseases, such as avian influenza

Marius.Gilbert@ulb.ac.be

<figure><img src="image_27.png" /></figure>

Because of its impact on all components of societies, climate change is seen as the major element of global change. However, when it comes to infectious diseases, climate variability is only one of the many factors that could influence their emergence, spread and persistence and that have changed strongly in the last century. The primary source and genetic pool of an emerging pathogen are most often found in wildlife (Jones et al. 2008) and the presence of livestock has had an important role in the evolution and emergence of infectious diseases in human history (Diamond 2002). When man started domesticating animals, they began to be exposed to their pathogens, and this offered new evolutionary opportunities. During what is called the first epidemiological transition, man moved toward sedentary agricultural societies, establishing larger groups in cities, which provided opportunities for pathogens such as malaria, smallpox, measles or tuberculosis to emerge and spread (Harper & Armelagos 2010). The second epidemiological transition started with the industrial revolution, when improved nutrition, and more effective public health measures resulted in a decline in early mortality due to infectious diseases. As populations aged, they began to experience a concomitant rise in non-communicable diseases such as heart failure, cancer, and diabetes (Barrett et al. 1998). We are now experiencing the third epidemiological transition, which is characterized by the new emergence or re-emergence of pathogens, in a context of fast demographic changes, globalization of production and trade, and changes in land use and climate.

Along with the biggest increase in human population of history, the 20th century has marked a dramatically accelerated rate of urbanization (from 13% living in cities in 1900 to 49% in 2005) providing new habitats for vectors, concentrated populations of human hosts and a substantial increase in demand for livestock products. In parallel, in parallel to the green revolution in crop production, the livestock revolution fundamentally changed the way livestock were raised and traded (Steinfeld et al. 2006). Those changes in human and livestock numbers and distribution have been so significant that people and their livestock represent today more than 95% of the terrestrial vertebrate biomass [estimate based on (Smil 2002; FAO 2009)]. In addition, the epidemiological connectivity of the human and livestock populations has been considerably expanded through the globalization of trade and travel. Whilst epidemiological theory predicts that an increase in the number and connectivity of hosts should result in higher disease persistence and spread, the effects of those changes in human and animal populations have been largely tempered by the rapid parallel developments of vaccination, antibiotics, and increased investments in disease prevention and control. However, as the history of emerging diseases has demonstrated, emerging pathogens are continuously evolving to adapt to these new patterns of human and animal populations (Antia et al. 2003) and we are now facing major challenges in maintain a front-line of efficient antimicrobials to maintain our protection.

Alongside the changes in host abundance and connectivity, emerging and re-emerging diseases affecting people and livestock are strongly influenced by environmental changes in land use (Patz et al. 2004) and climate (Epstein 2001). The World Health Organization (WHO) estimates that one quarter of the global burden of disease in humans, disproportionately felt in the developing world, is due to environmental change (Prüss-Ustün & Corvalán 2007). Similar estimates have not been made for livestock, though given the shared environmental pressures on them, it can be assumed a corresponding statement could be made with animal diseases.

When it comes to climate, several studies have looked at the potential effect of climate change on infectious diseases of animals (de la Rocque 2008). They highlighted that climate change and climate variability may influence virtually all components of disease systems: the pathogen (e.g. influencing the development rate, or survival outside the host or vector), the host (through the immune response, or changes in host distribution), the vectors (arthropod vector development is tightly linked to climatic parameters such as temperature and humidity). In addition, climate change and climate variability may strongly influence disease by indirect effects such as, for example, movements of hosts resulting from floods or heat waves, or climate induced changes in land use or land cover. So, our current capacity to predict the actual impact of climate change on livestock diseases is somewhat limited, but could be improved in the future. There are
many reasons why the direction and magnitude of the influences of climate change and climate variability are difficult to predict and generalize.

Firstly, the relationships between climate change, climate variability and disease are often disease specific. The OIE 2008 review on the impact of climate change on the epidemiology and control of animal diseases demonstrated a contrasting range of potential effects in different diseases systems including Rift Valley Fever (RVF), Bluetongue (BT), avian influenza (AI), tick-borne diseases (TBD), mosquito-borne diseases (MBD), leishmaniasis, and helminthiases.

Secondly, the potential impact of climate on diseases transmitted by arthropod vectors is strongly scale-dependent in space and time. Temperature affects arthropod vector development at embryonic, larval, and pupal stages, influences adult feeding behavior, and affects adult lifespans. Similarly, aquatic or moist environments are often needed for breeding stages, so that high precipitation can for example create more reservoirs and thus amplify the number of breeding sites. All vector species distributions can therefore be defined by their temperature and moisture ecological niche, which are defined by both a lower and an upper bound. The IPCC predictions for future temperature and rainfall are notoriously heterogeneously distributed throughout the globe, and highlight not only changes in the averages but also the extremes. The impact of such changes on different vectors is therefore likely to vary substantially from place to place, with higher vector populations in some parts of the world (regions that have recently become part of the niches) and in lower populations in others (regions that are no longer suitable). In addition, a general rise in annual mean temperature may have very different effects on vector populations according to the season: increased winter temperature may have a positive effect through a lower winter mortality, whereas increased summer temperature may have the opposite effect through an increased mortality in adults. So, both spatial and temporal heterogeneity of the predicted changes, combined to the inherent uncertainties in the projections make predictions extremely difficult, even for a single disease system.

Third, in most disease systems, climate change is taking place alongside other important changes and their respective effects are hence difficult to disentangle. These other changes may involve rapid transformation of the land use, increase in trade and movement of live animals, increase in trade of goods that may harbour breeding vectors, changes in the distribution and abundance of livestock, or changes in the genetic composition of hosts.

However, whilst there are clear difficulties in assessing the overall impact of climate change and variability on livestock diseases, careful assessment and prediction in some regions and disease systems remains possible, as demonstrated by the numerous studies that have used modelling to forecast the future distribution of suitability on a species by species, or disease by disease basis. Those different factors combine when it comes to emerging zoonoses, which are influenced by global changes in human, livestock and wildlife distribution and movements together with other environmental changes.

## References

* Antia, R., Regoes, R.R., Koella, J.C. & Bergstrom, C.T. (2003) The role of evolution in the emergence of infectious diseases.

* Barrett, R., Kuzawa, C.W., McDade, T. & Armelagos, G.J. (1998) Emerging and re-emerging infectious diseases: the third epidemiologic transition. Annual review of anthropology, 247–271.

* Diamond, J. (2002) Evolution, consequences and future of plant and animal domestication. Nature, 418, 700–707.

* Epstein, P.R. (2001) Climate change and emerging infectious diseases. Microbes and Infection, 3, 747–754.

* FAO. (2009) FAO Statistical Database. Available on http://faostat.fao.org/.

* Harper, K. & Armelagos, G. (2010) The Changing Disease-Scape in the Third Epidemiological Transition. International Journal of Environmental Research and Public Health, 7, 675–697.

* Jones, K.E., Patel, N.G., Levy, M.A., Storeygard, A., Balk, D., Gittleman, J.L. & Daszak, P. (2008) Global trends in emerging infectious diseases. Nature, 451, 990–993.

* Patz, J.A., Daszak, P., Tabor, G.M., Aguirre, A.A., Pearl, M., Epstein, J., Wolfe, N.D., Kilpatrick, A.M., Foufopoulos, J., Molyneux, D. & Bradley, D.J. (2004) Unhealthy Landscapes: Policy Recommendations on Land Use Change and Infectious Disease Emergence. Environmental Health Perspectives, 112, 1092–1098.

* Prüss-Ustün, A. & Corvalán, C. (2007) How much disease burden can be prevented by environmental interventions? Epidemiology (Cambridge, Mass.), 18, 167–178.

* de la Rocque, S. (2008) Climate change: impact on the epidemiology and control of animal diseases. Introduction. Revue scientifique et technique (International Office of Epizootics), 27, 303–308.

* Smil, V. (2002) Worldwide transformation of diets, burdens of meat production and opportunities for novel food proteins. Enzyme and Microbial Technology, 30, 305–311.

* Steinfeld, H., Gerber, P., Wassenaar, T.D., Castel, V. & de Haan, C. (2006) Livestock's Long Shadow: Environmental Issues and Options. FAO.
# Selected oral presentations

## Influence of water temperature on the outcome of a Brucella pinnipedialis hooded seal (Cystophora cristata) strain infection in cod (Gadus morhua)

Jacques Godfroid¹, Ingebjørg Nymo¹, Marit Seppola¹, Sascha Al Dahouk¹, Kathrine Bakkemo¹, María Pilar Jiménez De Bagüés¹, Anett Larsen¹

¹ Arctic Infection Biology, Department of Arctic and Marine Biology,UiT - The Arctic University of Norway, Tromsø, Norway.

<figure><img src="image_28.png" /></figure>

jacques.godfroid@uit.no; anett.k.larsen@uit.no

### Objective

In the natural environment, pathology in hooded seal (Cystophora cristata) induced by Brucella pinnipedialis has not been documented. A lack of intracellular survival and multiplication in hooded seal macrophages and epithelial cells indicates that B. pinnipedialis does not induce a chronic infection in this species. This suggests that hooded seals acquire a transient B. pinnipedialis infection from their environment, likely through their diet. We showed that Atlantic cod (Gadus morhua), part of the hooded seal's diet, is able to sustain a B. pinnipedialis infection. In the context of climate change, important questions to understanding infectious disease dynamics and being able to predict changes are: how much is climate change likely to shift the suitability for infectious diseases, and how does this vary among diseases in the Arctic where warming is occurring disproportionately? In this study, we assessed the influence of water temperature on the outcome of a B. pinnipedialis infection in Atlantic cod.

### Methods

Atlantic cod were infected intraperitoneally with 108B. pinnipedialis. Tagged uninfected in-contact cod were kept with infected cod. Uninfected control cod were kept in separate tanks. The experimental infection was run at 6°C and 15°C, mimicking Arctic water and increased water temperatures expected in the Northern Atlantic during the 21st century (International Panel on Climate Change scenarios).

### Results

At 6°C, viable bacteria were found in the blood of all infected cod at all times post infection (pi) (day 1, 7, 14, 21, 35, and 49). Neither gross pathology nor mortality were recorded in fish kept at 6°C. Anti-Brucella antibodies were detected earlier at 15°C than at 6°C. At 15°C, B. pinnipedialis were eliminated more quickly, however mortality was observed between day 7 and 20 pi in 5/60 fish. Importantly, no B. pinnipedialis was cultured from these 5 dead fish, as from any in-contact fish. At 15°C, 1/60 fish in the uninfected in-contact group died at day 9 and 1/60 fish in the uninfected control group died at day 21.

### Conclusion

Our results suggest that B. pinnipedialis is an opportunistic pathogen in Atlantic cod. An increase in water temperature is likely to be an important driver for the enhanced mortality, without observable gross pathology. A possible trade-off between immunocompetence and other vital functions at sub-optimal temperatures may explain the observed mortality. This raises questions on the influence of increased water temperatures in the oceans and the emergence of diseases in aquatic ectotherms.

**Keywords:** Brucella pinnipedialis; Atlantic cod; ectotherms; climate change; water temperature
# Seroprevalence of *Borrelia burgdorferi* in horses in the southern part of Belgium: a "one health" driven study

Carole Meersschaert<fnref n="1" />, Simona Cerri<fnref n="2" />, Rachel Houben<fnref n="2" />, Pierre-Hugues Pitel<fnref n="3" />, Valérie De Waele<fnref n="4" />, Guy Hendrickx<fnref n="4" />, Hélène Amory<fnref n="2" />

<figure><img src="image_29.png" /></figure>

vdewaele@avia-gis.com; helene.amory@ulg.ac.be

## Objective

Lyme borreliosis is the most prevalent tick-borne infection in the northern hemisphere affecting both humans and animals including horses. The complex interactions between the pathogen, *Borrelia burgdorferi* spp. , the vector, mainly *Ixodes ricinus* in Europe and the widespread range of hosts, as well as the impact of several climatic and non-climatic factors affecting these interactions support the necessity of interdisciplinary collaboration for surveillance and prevention. Because humans and horses commonly share the same habitat the epidemiological data collected in horses can be potentially usefull for assessing risk factors for human borreliosis. Unfortunately, the lack of specific symptoms in horses makes diagnosis a challenging task for veterinarians. Antibody testing is for these reasons a valuable tool but must be interpreted with caution.

The objective of our study was to provide accurate data concerning the prevalence of *Borrelia burgdorferi sensu lato* antibodies in horses living in the Southern part of Belgium as well as to assess diagnostic value of the commonly used serological tests (IF and ELISA) in order to promote critical interpretation of positive test results. An invalid diagnosis of Lyme disease will indeed have negative consequences for the horse as well as for public health regarding to the threat this represents for antibiotic resistance. By arguing adapted use of serologic testing we intended to promote rational use of antibiotics.

## Methods

To achieve our objective we conducted three complementary studies. We conducted a prevalence study on a sample of 303 serums collected between April 2014 and April 2016 from horses living in Wallonia who hadn't presented any clinical symptom suggestive of Lyme disease in the past 12 months. Secondly we assessed the diagnostic value of the two most commonly used serologic tests ELISA and IF, by comparing the test results of 100 serums with the results of a western blot test, used for the purpose as reference standard. In the third part we evaluated the impact of 4 different diagnostic strategies and evaluated the outcome with emphasize on preventing unnecessary use of antibiotics.

## Results

A seroprevalence of 22% was observed in our sample with an evidence of age related effect (p-value=0.02) on the presence of *Borrelia burgdorferi* antibodies. When evaluating diagnostic value of serological tests we could observe that IF and ELISA tests both perform well regarding to sensitivity and are adapted tests when used as first step testing. Their specificity is too low to be able to be confident in these tests to confirm Lyme disease in horses. A second more specific test (western blot) is necessary to confirm the positive results. With regard to rational use of antibiotics we could observe a possible decrease ranging from 17 to 47% of non-appropriate antibiotic use when choosing for a two-step strategy compared to the use of a single serology.

## Conclusion

This study argues for a necessary change in the management of horses suspected of Lyme disease. Taking in account the epidemiologic datas of seroprevalence collected for Wallonia, as well as the diagnostic values of the two most commonly used serologic tests we illustrated the urgent need to convince veterinary practitioners to confirm the positive results of *Borrelia burgdorferi* before administration of antibiotic treatment.

This study on Lyme disease is thus an example of a "one health" driven study.

**Keywords:** Lyme disease/Borrelia burgdorferi/seroprevalence/horses/diagnostic testing/rational use of antibiotics

<footnote n="1">¹ Laboratoires Réunis, Junglinster, Luxembourg</footnote>

<footnote n="2">² Faculty of Veterinary Medicine, Department of Companion Animals and Equids, University of Liège, Liège, Belgium</footnote>

<footnote n="3">³ Labéo Frank Duncombe, Caen, France</footnote>

<footnote n="4">⁴ AVIA-GIS, Beerse, Belgium</footnote>
# Effectiveness and cost efficiency of syndromic surveillance compared to conventional surveillance: Bluetongue serotype 8 across countries as case study

Sarah Welby<fnref n="1" />, Gerdien Van Schaik<fnref n="2" />, Anouk Veldhuis<fnref n="2" />, Henriette Brouwer<fnref n="2" />, Carole Peroz<fnref n="3" />, Inge Berends<fnref n="2" />, Christine Fourichon<fnref n="3" />, Paul Wever<fnref n="2" />, Yves Van Der Stede<fnref n="1" />

<figure><img src="image_30.png" /></figure>

Sarah.Welby@coda-cerva.be

Following the Bluetongue serotype 8 outbreaks that occurred in Europe during the years 2006 and 2007, European legislation prescribed active surveillance components and passive surveillance components to be carried out in the cattle population. The present study aimed at comparing the relative efficacy of each surveillance component in proving the absence of infection or early detection in different countries and to evaluate if syndromic surveillance components could be of added value in terms of efficacy of detection and in terms of cost efficiency.

In a first stage, bluetongue surveillance data from France, The Netherlands and Belgium for the surveillance components in place since 2006-2007 in each country were collected and analyzed. In terms of substantiating freedom from infection, given a design prevalence of 2%, all components performed well in all countries.

In a second stage, it was investigated how effective those conventional surveillance components were for early detection and if syndromic surveillance could be of added value. For this purpose, sensitivity of detection was estimated based on a model that combined outputs of scenario tree models and reed frost models in order to account for the timeliness of detection. Preliminary findings showed that active serological surveillance (cross sectional and sentinel) did not perform as well. The sensitivity dropped down from 99% to 40% depending on the expected within herd prevalence and countries. In contrast; clinical reporting and syndromic surveillance data (using fertility data), performed much better than the active components with sensitivity ranging around 99% and 70% for passive clinical surveillance and syndromic surveillance (using fertility data) respectively. No alerts were triggered using rendering plant and milk production data in Belgium, therefore no output sensitivity values were available for those syndromic surveillance components. Whereas in the Netherlands milk production data provided relatively good sensitivity values and rendering plant data triggered alerts but of very low sensitivity value. The delay in triggering alerts using rendering plant data, explained the low sensitivity of detection values with this component in the Netherlands. If direct costs are taken into account, the active surveillance components (cross sectional and sentinel) were the cheapest. However for early detection the cost efficiency ratio was lower for passive and syndromic surveillance.

From the present study it can be concluded that implementing syndromic surveillance on a routine base for early detection and in addition implement the existing traditional surveillance methods once suspicions are raised would probably be the most effective and best cost efficient option.

**Keywords:** efficiency; efficacy; surveillance; early detection

<footnote n="1">¹ Veterinary and Agrochemical Research Centre, National Reference Laboratory (CODA-CERVA)</footnote>

<footnote n="2">² GD Animal Health Service, Animal Health Monitoring & Surveillance team, Deventer, the Netherlands, Brussels, Belgium</footnote>

<footnote n="3">³ ONIRIS-INRA National College of Veterinary Medicine, Food Science and Engineering, Research Unit Biology, Epidemiology and Risk Analysis in Animal Health - BIOEPAR, Nantes, France</footnote>
# Serological monitoring 2008-2015 of Walloon dairy herds exposition to Leptospira hardjo based on BTM Elisa

Guy Czaplicki <fnref n="1" />, Laurent Delooz <fnref n="1" />,<fnref n="2" />, Christian Quinet <fnref n="1" />, Jean-Yves Houtain <fnref n="1" />, Claude Saegerman <fnref n="2" />

<figure><img src="image_31.png" /></figure>

guy.czaplicki@arsia.be

Bovine leptospirosis is a worldwide often neglected zoonosis mainly caused by Leptospira hardjo. In a serological study on bulk tank milk, Dom et al (1991) found that 9.2% of Belgian dairy herds were exposed to this pathogen with a higher apparent prevalence in Wallonia.

During the second half of 2014, Wallonia faced an emergence of abortion cases in Walloon bovine herds mainly linked to serovars grippotyphosa and australis but not hardjo (Delooz et al., 2015): the question was whether an increase in exposure was also observed with respect to this particular serovar hardjo.

The objective of this study was therefore to investigate the apparent herd-level prevalence of specific exposure to *Leptospira hardjo* and incidence among 728 randomly selected Walloon dairy herds by serological testing of bulk tank milk in a period of time covering 2008 to 2015 and examine a possible association between both testing positive for and incidence of exposure with some risk factors such as herd size, location of the herd, presence and use of breeding bull. Samples of 2008, 2009, 2014 and 2015 were tested with Linnodee *Leptospira hardjo ELISA®* according to the manufacturer's recommendations.

The results confirm that *Leptospira hardjo* remains endemic in Walloon dairy herds, but with apparent prevalence much lower in 2014-2015 than in 2008-2009, and a very low incidence in 2015. This decline contrasts sharply with the concomitant emergence of other serotypes (mainly *L. grippotyphosa* and *L. australis*) than hardjo and suggests that the dynamics of infection and maintenance with *Leptospira hardjo* is quite different from the other serotypes in the Walloon conditions.

The geographical location, the size of the herd or the use of breeding bulls could affect the risk of exposure and infection in a herd but have yet to be confirmed.

**Keywords:** Leptospirosis; Hardjo; Dairy herds; Bulk Tank Milk

<footnote n="1">¹ Association Régionale de Santé et d'Identification Animales (ARSIA)</footnote>

<footnote n="2">² Faculty of Veterinary Medicine, University of Liège (UREAR-ULg, FARAH)</footnote>
<figure><img src="image_32.png" /></figure>

Association d'Epidémiologie et de Santé Animale

# Biosecurity in livestock, pets and horses

7th - 9th Sept. 2016 - Liège (Belgium)
Congress 25 years

Animal epidemiology
Past, present and future
# Keynote lecture

## Ensuring continuous biosecurity for high health high performance (HHP) horses: a public-private collaboration

**Morgane Domínguez** DVM, MPH, PhD, S. Munstermann, L. Weber-Vintzel

Organisation Mondiale de la Santé Animale (OIE), Paris, France

Morgane is a veterinary epidemiologist at the World Organisation for Animal Health (OIE), experienced in the field of disease surveillance, alert and response at national and international levels. Morgane currently works on the facilitation of international movements of sport horses for competition events, as part of the OIE's partnership agreement with the International Federation of Horseracing Authorities (IFHA) and the Federation Equestre Internationale (FEI).

<figure><img src="image_33.png" /></figure>

m.dominguez@oie.int

<figure><img src="image_34.png" /></figure>

## The HHP framework

### Establishment and maintenance of a high health status

#### Pillars

The HHP principles were approved by the 180 OIE Member Countries in 2014 and laid down in the OIE *Terrestrial Animal Health Code* (1). They only apply to the temporary importation (i.e. not exceeding 90 days) of international athletes registered by the FEI or the IFHA for competition purposes (not for breeding purposes or for permanent residency).

The HHP framework is based on already well established OIE principles such as certification, traceability and identification, as well as compartimentalisation which aims at separating an animal subpopulation with a distinct health status by practices primarily related to biosecurity (2, 3).

Based on continuous biosecurity, veterinary supervision, traceability and contingency planning, the HHP pillars are designed to provide assurance of the low health risk associated with the temporary importation of a certified HHP horse.

#### Stringent biosecurity

A compartment has to be approved by the Veterinary Authority and registered by the horse industry (FEI, IFHA) before it can qualify.

The establishment and maintenance of the high health status of horses in a compartment rely on continuous and fully documented application of HHP biosecurity measures detailed in specific OIE guidelines (*OIE Handbook for the Management of High Health, High Performance Horses*) and applicable at all times (home stable, places of temporary residence, during travel, at competitions) (4).

The HHP biosecurity measures tackle possible routes of direct or indirect disease introduction into the compartment (other animals, people, vehicles, equipment, feed, water or pests). The risk of venereal transmission is also addressed by the prohibition of breeding activities in the compartment. Relevant biosecurity provisions should also be considered to ensure appropriate vector protection, depending on the local situation in the country/zone of residence, place of competition, transport routes and lay-over points.

#### Continuous veterinary supervision, early detection and contingency planning

From the start of the qualification period and throughout the HHP membership, horses in the compartment are under continuous veterinary supervision (i.e., daily observation, twice daily temperature check, and at least one veterinary visit per week). This should allow for the rapid detection of any sign of disease.

Contingency planning is an integral part of the HHP framework and a contingency plan should be developed for all places where HHP horses are held (home stable, places of temporary residence, transport vehicles, event venue).

The early detection, in conjunction with sound contingency planning, allow for appropriate measures to be promptly taken to minimise any risk of spread.

#### International traceability

Traceability is of extreme importance for the integrity of the HHP framework. HHP horses should bear a permanent unique identifier (microchip) and individual HHP records should be kept and stored in an international database.

Compliance with the HHP requirements should be continuously documented and monitored and should be regularly audited by the Veterinary Authority. Non-compliance can result in suspension of the high health status of the compartment or in the suspension of the horse's HHP membership.
**<u>Simplified requirements for temporary importation</u>**

The HHP pillars are intended to mitigate the risk of spread for most OIE-listed diseases of relevance to equines, however, for a few diseases, a risk, albeit at a variable level, may persist: HHP disease-specific mitigation measures (such as laboratory testing, vaccinations) have been established to mitigate this risk (5).

Disease-specific mitigation measures have been included in a model HHP Veterinary Certificate for six diseases (African horse sickness, equine influenza, equine infectious anaemia, equine piroplasmosis, glanders and Venezuelan equine encephalomyelitis) (4).

**<u>Enhanced public-private collaboration</u>**

The importance of the roles that private operators (persons responsible for a compartment), private veterinarians, the horse industry (FEI and IFHA) and national Veterinary Services have to play jointly in the operationalisation of the HHP framework cannot be overemphasized.

At national level, it is essential to foster closer collaborations between National Veterinary Authorities and the equine private sector (National Equestrian Federations; National Racing Authorities) to ensure a safe and trustworthy management of the HHP horses.

**<u>Conclusion</u>**

The HHP framework is designed to provide assurance of the low health risk associated with the temporary importation of certified HHP horses. Meanwhile it offers a harmonised, practically feasible, globally applicable framework based on simplified certification requirements.

The HHP framework offers an alternative to horse movement systems already in place under bi-lateral and regional arrangements and is intended to enable more nations to benefit from the expansion of the sport horse industry.

**<u>References</u>**

1. World Organisation for Animal Health (OIE) (2015). – Chapter 4.16. High health status horse subpopulation. In Terrestrial Animal Health Code. OIE, Paris. Available at: www.oie.int/index.php?id=169&L=0&htmfile=chapitre_high_level.htm (accessed on 10 August 2016).

2. World Organisation for Animal Health (OIE) (2015). – Chapter 4.3. Zoning and compartmentalisation. In Terrestrial animal health Code. OIE, Paris. Available at: www.oie.int/index.php?id=169&L=0&htmfile=chapitre_zoning_compartment.htm (accessed on 10 August 2016).

3. World Organisation for Animal Health (OIE (2015). – Chapter 4.4. Application of compartmentalisation. In Terrestrial Animal Health Code. OIE, Paris. Available at: www.oie.int/index.php?id=169&L=0&htmfile=chapitre_application_compartment.htm (accessed on 10 August 2016).

4. World Organisation for Animal Health (OIE) (2015). – Handbook for the management of high health, high performance horses. OIE, Paris. Available at: http://www.oie.int/fileadmin/Home/eng/Our_scientific_expertise/docs/pdf/Chevaux/HHP_Handbook_May_2016_V2.pdf (accessed on 10 August 2016).

5. Dominguez M., Munstermann S., Murray G. & Timoney P. 'High-health, high-performance' horses: risk mitigation strategies for OIE-listed diseases. Rev. Sci. Tech. Off. Int. Epiz., 2015, 34 (3), 837-848
Selected oral presentations

Biosecurity measures applied in the United Arab Emirates: a comparative study between
livestock and wildlife sectors

Anne-Lise Chaber <fnref n="1" />, <fnref n="2" />, Claude Saegerman <fnref n="1" />

<figure><img src="image_35.png" /></figure>

alchaber@hotmail.com; claude.saegerman@ulg.ac.be

In 2013, the livestock population in the UAE exceeded 4.3 million heads with sheep and goats accounting for 90% of this. The overall number of captive wild ungulates (gazelle types) is difficult to assess as there is no registration system in place or enforced in the UAE with regard to the possession of wildlife. Those animal collections, mainly owned by high-ranking families, are therefore not registered and kept far from public viewing. Nonetheless, some collections are housing more than 30 000 ungulates in one location.

The primary objective of this study was to describe the biosecurity measures currently applied in UAE ungulate facilities for different wildlife and livestock sectors. A secondary objective was to use the output from this biosecurity survey to investigate which sector could be categorized into risk groups for disease introduction and spread.

Between October 2014 and May 2015, biosecurity questionnaire data were collected in the Emirates of Abu Dhabi, Dubai, Ras Al Khaimah, Fujeirah, Ajman, Umm al Quwain and Sharjah from 14 wildlife collections, 30 livestock farms and 15 mixed (wildlife and livestock farms). These investigations through questionnaires allowed us to quantify and assess statistically biosecurity practices and levels for both livestock and wildlife sectors.

In both sectors, biosecurity measures could be improved and only a few facilities had high biosecurity scores. The group of small un-registered farms (Ezba) represented the highest risk of disease transmission to other animals due to their lack of biosecurity awareness.

Keywords: Biosecurity ; Wildlife ; Collection ; Livestock ; Farms ; UAE

<footnote n="1">¹ Faculty of Veterinary Medicine, University of Liège (UREAR-ULg, FARAH)</footnote>

<footnote n="2">² Wildlife Consultant L.L.C, Al Ain.</footnote>
# How to assess the biosecurity level in stud farms? Survey among 70 Belgian and French horse breeders

Héloïse Oudin<fnref n="1" />, Marie-France Humblet<fnref n="1" />, Claude Saegerman<fnref n="2" />

<figure><img src="image_36.png" /></figure>

mfhumblet@ulg.ac.be

## Objective

The European horse industry is not safe from infectious diseases, as evidenced by several events that occurred over the last twenty years (e.g. West Nile fever, equine viral arteritis or infectious equine anaemia). These events have given rise to a demand on biosecurity from the sector despite few evidence-based data on the usefulness of a biosecurity program in horse farms. However, given its special features, the horse industry needs a suitable biosecurity program. The main objective of the study was to propose and test a method for assessing the biosecurity level of equine farms, with a view to develop biosecurity tools for the whole sector.

## Methods

A questionnaire was conducted in 70 Belgian (Wallonia; N = 30) and French (Normandy; N = 40) horse farms, between July 2015 and January 2016. The questionnaire included 45 closed and opened questions, grouped in 12 sections. Horse farms were prospected by several means (direct or indirect contacts, equestrian events, directories provided by breeders and internet contact search). Their selection was based on their location and breeders were invited to answer the questionnaire in a face-to-face interview. The sample size was based on the principle of saturation data. Horse breeders, mainly professional, were grouped in four categories: trot (N = 15), gallop (N = 13), saddle horses (N = 31) and other (N = 11). Practices were compared to an “ideal” model (noting scale), in order to assess their risk and biosecurity levels. Each horse breeder was allocated a risk score (maximal score = 45) and a biosecurity ratio (biosecurity note/maximal reachable note). Regarding statistical analyses, a Poisson regression was used at first intention for count variables and a negative binomial regression in case of extra-binomial variability. A linear regression was applied to analyse biosecurity ratios. Pearson and Spearman correlation coefficients investigated the relationships between two variables. Fisher exact and Shapiro tests compared two proportions and tested the adequacy with normal distribution. Any result ≤ 0.05 was considered as significant.

## Results

The proposed questionnaire seemed appropriate to assess the different types of farms. The type of farming did not influence the risk and biosecurity levels of farms. The risk score was higher among professionals than among non-professionals. French farms have a significantly higher biosecurity ratio than Belgian farms, probably due to better awareness of French breeders. Breeders should primarily work on reducing the risk. The breeders' perception of the utility of biosecurity measures for the control of infectious diseases in the equine population and the impellent level associated with biosecurity does not depend neither on the risk score nor on the biosecurity ratio. Furthermore, there is no relationship between the utility score and the impellent score associated to biosecurity. Yet, biosecurity is often synonymous with constraints.

## Conclusions

There is significant room for improvement in terms of biosecurity in equine farms. A biosecurity protocol requires planning, implementation and monitoring of targeted and prioritized measures, based on the risk profile. Given the potential impacts on economy, public and environment health, as well as on animal welfare, it is important to raise horse breeders' awareness and to develop appropriate biosecurity tools.

**Keywords:** horse farm ; questionnaire ; risk score ; biosecurity ratio ; awareness ; biosecurity tool

<footnote n="1">¹ Department for Occupational Safety and Health, Biosafety and Biosecurity Unit, University of Liege, Liege</footnote>

<footnote n="2">² Faculty of Veterinary Medicine, University of Liège (UREAR-ULg, FARAH)</footnote>
# A descriptive study to assess the perception of cattle farmers towards the implementation of biosecurity

Bert Damiaans<fnref n="1" />, Steven Sarrazin<fnref n="1" />, Evelien Heremans<fnref n="1" />, Jeroen Dewulf<fnref n="1" />

<figure><img src="image_37.png" /></figure>

bert.damiaans@ugent.be

## Objective

The shift from cure towards prevention in veterinary medicine involves the implementation of biosecurity. In a recent study it was shown that that few biosecurity measures were undertaken by Belgian cattle farmers, although basic biosecurity measures are present in the majority of the farms. Therefore the aim of this study was to assess the perception of cattle farmers towards the implementation of biosecurity.

## Methods

The study consisted of a qualitative part in the form of a focus group and a quantitative part in the form of a questionnaire. Eight female Flemish cattle farmers participated in the focus group discussion, during which several topics concerning biosecurity were discussed. Based on the results of the focus group a questionnaire with 18 semi-closed (5-level Likert scale) and 5 open questions was developed and conducted face-to-face during the fair for Flemish agriculture in January 2015. The results (91 respondents) were analysed using basic descriptive analysis in Microsoft Excel and SPSS.

## Results

Although Flemish cattle farmers are familiar with several specific biosecurity measures, such as quarantine and the use of herd-specific clothing, they associate these measures with 'disease prevention' rather than with the term 'biosecurity'. They tend to relate this term to food safety (23%) and ecological farming (21%) or have no idea what this concept is about (48%). Forty-four percent of the farmers feels threatened by the term. The majority of the farmers (87%) feels responsible for the implementation of biosecurity on their farm and 52% indicates to have sufficient information to implement biosecurity adequately, while the focus group emphasized the need for additional information. Nearly all farmers (98%) identified their herd veterinarian as the main source of this information, but the focus group stressed that a lack of time of the herd veterinarian is a major threshold. Furthermore, only 45% of the farmers is willing to pay their herd veterinarian for such preventive advice and 47% believes that it is more difficult to implement biosecurity for cattle farmers than for pig and poultry farmers. Nonetheless, only 20% and 32% of the respondents is convinced that the implementation of biosecurity will cost them money and will require more time and effort. Finally, 80% sees room for improvement of the biosecurity level in their herd. Although extensive information concerning biosecurity may currently be available, farmers do not associate disease prevention with 'biosecurity' and indicate the need of more information on the level of the farmers in a practical manner. Farmers believe their herd veterinarian should be their main source of information, but are reluctant to pay for this information and raise a lack of time of the herd veterinarian as barrier. By analogy with pigs and poultry, in a first step farmers could be provided with accessible and practical information through a biosecurity testing tool to explain the concept of biosecurity and to increase their awareness. Secondly, a preventive strategy could be developed in collaboration with the herd veterinarian who knows the herd structure and can inform the farmer on the critical points for that specific farm.

## Conclusion

The ultimate goal should be to consider biosecurity a collective responsibility, with farmers taking initiative to improve their biosecurity level and herd veterinarians as coaches.

**Keywords:** biosecurity; cattle; perception; disease prevention

<footnote n="1">¹ Veterinary Epidemiology Unit Department of Reproduction, Obstetrics and Herd Health Faculty of Veterinary Medicine Ghent University - Salisburylaan 133, 9820 Merelbeke - Belgium</footnote>
# Assessing motivators and obstacles for implementation of biosecurity measures in animal production in Europe

Bert Damiaans¹, Steven Sarrazin¹, Jeroen Dewulf¹

¹ Veterinary Epidemiology Unit Department of Reproduction, Obstetrics and Herd Health Faculty of Veterinary Medicine Ghent University - Salisburylaan 133, 9820 Merelbeke - Belgium

<figure><img src="image_38.png" /></figure>

bert.damiaans@ugent.be

## Objective

Farm biosecurity includes the prevention of disease transmission within and between farms and is important to establish the shift from cure to prevention in veterinary medicine. This concept is well established in pig and poultry production and also in cattle production the potential to implement biosecurity is present. However, there is still room for improvement in all species. To identify key elements of known biosecurity measures and the motivators and obstacles for their implementation, a workshop was organized by European Innovation Partnership for Agricultural Productivity and Sustainability (EIP-AGRI).

## Methods

Sixty-one participants involved in animal production including farmers or their representatives, researchers, farmer advisors, practicing veterinarians, animal health services, representatives of trade, industry, government or EU from 22 European countries attended the workshop in Brussels in January 2015. Respectively 22, 26 and 13 of the attendees represented the cattle, pig and poultry sector. For their species of interest they evaluated 21 biosecurity measures by considering for each measure the assumed application in their country, expected efficacy in disease prevention, expected feasibility and expected return on investment (ROI) on a 5-level Likert scale. The results were analysed using basic descriptive analysis in Microsoft Excel.

## Results

Scoring well for feasibility and efficacy across the species are biosecurity measures meant to limit infection pressure, such as cleaning and disinfection of transport vehicles before entering the farm, of the stables after every production round and a carcass storage separated from the stable (top 3 ranking across the species). Preventing spread between groups by using holding specific equipment scores well, except for poultry where it has one of the lowest scores of all measures (rank 17/21). Likely explained by the fact that in a poultry stable little equipment is used during a production round. Scoring among the top in two of the animal species (pig and poultry) are measures which prevent disease entrance on the farm by use of herd specific clothes and boots and a hygiene lock before entering the stable. Also rodent control is considered important. All of these biosecurity measures are expected to have a high ROI. They contain measures of internal and external biosecurity, showing it is considered important to adhere to both.

The biosecurity measures scoring lowest (rank 20 and 21 in each species) were showering and a 24 hour animal free period before entering the stable. Both measures scored consistently low on feasibility. Also their ROI is lowest of all, since high investments may be required to implement them. Moreover, limiting a veterinarian to perform only one visit a day seems unrealistic.

It is striking that the answers for pigs and poultry are on average substantially higher than for cattle (pigs 17,5%; 15%; 20%, poultry 15%; 22,5%; 15% higher for efficacy, feasibility and ROI, respectively). People working in cattle seem to have a less positive view on the expected effects of biosecurity in comparison to pig or poultry production.

## Conclusion

Across species the same trends were noticed though for cattle the scores for efficacy, feasibility and expected ROI were substantially lower than for the other species. Measures pertaining to both internal and external biosecurity were considered important. Biosecurity measures receiving low scores were those considered unfeasible.

**Keywords:** biosecurity; cattle; poultry; pig husbandry
# Posters

## Influence of the purchase policy on the risk of PI birth, a retrospective study in Southern Belgium

Marie-Pierre Michiels<fnref n="1" />, Laurent Delooz<fnref n="1" />,<fnref n="2" />, Elise Dion<fnref n="1" />, Jean-Yves Houtain<fnref n="1" />

<figure><img src="image_39.png" /></figure>

jeanyves.houtain@arsia.be

## Introduction

Since January 2015, a compulsory control plan aiming the eradication of the bovine viral diarrhea virus (BVDV) is implemented in Belgium. Purchases are considered as a major cause of between herds transmission. This study assesses the influence of the purchases on the risk of PI birth in walloon herds, based on the results obtained during the 1st year of the national control plan.

## Material and methods

Based on the informations retrieved in the belgian national database for cattle identification and traceability (SANITEL), the number of calves born during the year 2015 has been determined for each herd located in Wallonia.

For each herd with at least 1 calf born during 2015, the average number of purchased animals during the last four years (from 2012 to 2015) was calculated.

The results of the laboratory analyses and the official individual BVD statuses of born and purchased animals have been obtained from the national BVD database.

An animal was considered as "PI" if it obtained at least one positive result with no negative result afterwards, to a test approved for the direct detection of the BVD virus according to the belgian legislation.

The pregnancy status and the stage of pregnancy of purchased cows was extrapolated using the number of days separating the date of introduction in the herd and the calving date. A cow was considered as purchased while pregnant if it calved less than 280 days after the introduction date.

The incidence of BVDV at birth was estimated by dividing the number of PI calves born during the examined period by the number of calves born and tested during the same period.

A herd was defined as "infected" if at least one PI was born during the year 2015.

Herd prevalence and birth incidence were estimated with 95 % confidence intervals (95% CI) assuming a binomial exact distribution.

Risk factors associated with BVDV infection at individual or herd level, were expressed in odds-ratios which statistical significance was assessed calculating the 95 % confidence interval.

## Results

0.5 % (95%CI:0.49-0.53) of the 466 156 calves born in 2015 were PI. Among calves whose mother was purchased while being pregnant, 1.35 % (95%CI:1.11-1.59) were PI. Cows purchased while being pregnant were nearly 3 times (OR 2.73; 95%CI:2.27-3.28;) more at risk to give birth to a PI calf compared to cows inseminated on the farm.

The birth of at least 1 PI calf was observed in 948 of the 8899 breeding herds representing an apparent herd prevalence (HPa) of 10.7 % (95%CI:10.0-11.3).

Compared to herds not having bought any animal during last 4 years, the risk of being infected was 2.64 higher (95%CI: 2.25-3.10) for herds having purchased at least 1 animal per year during the same period and 3.58 higher (95%CI: 2.99-4.28) in herds having purchased more than 5 animals per year.

## Conclusions

Our results confirm what was already known about the risk of BVDV transmission between herds via purchased animals and particularly highlights a higher risk when introducing pregnant cows. Concerning the purchase policy at herd level, our data suggest that the more a farmer buys animals, the more his herd risks to be infected.

Given these results, not having included measures on biosecurity in animal trade can be considered a major pitfall in the current BVDV control national plan jeopardizing the targeted sanitation of the Walloon livestock.

**Keywords:** BVD ; Purchase policy; southern Belgium

<footnote n="1">¹ Association Régionale de Santé et d'Identification Animales (ARSIA) - Allée des Artisans 2, B-5590 Ciney - Belgium</footnote>

<footnote n="2">² Faculty of Veterinary Medicine, University of Liège (UREAR-ULg, FARAH)</footnote>
# Analytical Hierarchy Process for the evaluation of biosecurity levels in fattening rabbit farms.

Tiziano Dorotea ¹, Katia Capello ¹, Matteo Mazzucato ¹, Filippo Buniolo, Romina Brunetta ¹, Fabrizio Agnoletti ¹, Lebana Bonfanti ¹

¹ Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe) - Viale dell'Università', 10 - 35020 Legnaro (Padua) - Italy

<figure><img src="image_40.png" /></figure>

tdorotea@izsvenezie.it

## Objectives

Italy is globally the second highest producer country for rabbit meat, with the north-eastern regions accounting for the 38% of total National rabbit meat production. Nevertheless, the rabbit production sector in north-eastern Italy is highly heterogeneous when considering the holding and the managerial characteristics, hampering a reliable definition of the overall biosecurity and farm hygiene situation. The present study aims at defining a framework to identify and rank structural and managerial elements at farm-level, to obtain a clearer picture of the rabbit meat production sector in north-eastern Italy. This would also indicate whether actions are needed to improve the overall biosecurity level in the territory.

## Material and methods

A structured questionnaire was designed to collect information on the farm structures and management procedures. An Analitycal Hierarchy Process (AHP) approach was used to structure the elements of the questionnaire into two levels, to enable scoring each of the observed characteristics, allowing ranking the farms according to their biosecurity level. The first level of the structure consisted of five macro-variables (Farm characteristics, Barriers, Documentation, Biosecurity measures, and Farm data), which grouped the second-level elements (Farming type, Cage type, Ambient temperature control systems, Storage area, Chain/bar, Draining wells, Water source, Loading bay, Fences, Filter zone, Animal movement, Disinfection area, Disinfection equipment, Barriers at shed level, Growth chart, Mortality register, Cleanliness/disinfection protocols, Drug stocking, Closable shed, Contacts with other animals, Dedicated clothes, Period of fallowing, Semen origin, Disinfectants used alternatively, Farmer's age, Year of farm construction, Year of farm renovation). Six experts on rabbit sector were asked to express a preference in a series of pairwise comparisons between the variables included in the two levels separately. The relative importance of each variable was scored using a numerical scale from 1 to 9, where 1 meant that the two variables were similarly important and 9 that the one variable was much more important than the other. The evaluations of the experts were aggregated into a single set of weights using a geometric mean.

## Results

Overall the experts evaluated the 'Biosecurity measures' as the most important (i.e. highest score) in the definition of the overall biosecurity level, followed by the 'Barriers' variable, with a contribution to the total score equal to 37% and 25%, respectively. The most important second-level elements within these two macro-variables were: 'Contacts with other animals' (28% of relative contribution) and 'Period of fallowing' (19%) for 'Biosecurity measures', and 'Barriers at shed level' (25%) and 'Animal movement' (19%) for 'Barriers'.

## Conclusion

This study highlighted the structural and managerial factors that primarily influence the biosecurity level of rabbit farms in north-eastern Italy. Moreover, we explored the practical application of an objective tool to rank farms (and/or other holdings) accordingly to their biosecurity levels, allowing the identification of the elements that could be potential candidate for dedicated actions to improve the overall condition of fattening rabbit sector, also to limit the risk of rabbit infectious disease introduction.

**Keywords:** rabbit ; biosecurity measures ; AHP
<figure><img src="image_41.png" /></figure>

Association d'Epidémiologie et de Santé Animale

Vector borne diseases
Keynote lecture

# The German Arbovirus Surveillance and Mosquito Monitoring Program, 2009 – 2015

## Jonas Schmidt-Chanasit (MD)

Department of Arbovirology, Bernhard Nocht Institute for Tropical Medicine, Hamburg

Jonas is the Professor of Arbovirology at University of Hamburg and the Deputy Director of the WHO Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research at Bernhard-Nocht-Institute for Tropical Medicine. He has acquired a long-lasting experience both at national and international level, working for WHO and the European Union. He is member of several national committees of the German Ministry of Health. He has carried out epidemiological investigations in Germany and abroad, and is the national coordinator and principal investigator of the German Arbovirus Surveillance Program. His area of expertise includes arboviruses and other emerging viruses, such as hantaviruses and bornaviruses. He has conducted outbreak investigations on Usutu virus and other arboviruses in Germany and in Developing Countries; in particular, he investigated the recent Zika virus epidemic in the Americas.

jonassi@gmx.de

<figure><img src="image_42.png" /></figure>

Up to now, four different mosquito-borne viruses (moboviruses) have been found in Germany. In 1968, Tahyna virus (TAHV) was isolated from mosquitoes that were trapped around Baunach, south-west Germany. TAHV is the causative agent of Valtice fever, an influenza-like illness occurring in summer and early autumn. Moreover, meningitis cases due to TAHV infection were also reported. Therefore, TAHV is the first human pathogenic mobovirus that was isolated from mosquitoes in Germany. Since these early discoveries in the late 60s of the last century, mobovirus surveillance in mosquitoes, humans and animals was not performed regularly and therefore, longitudinal data sets are missing, especially from Germany. Thus, we initiated a program that compiles and analyses mobovirus and vector data collected over a number of successive years. This provided a solid base to determine the underlying causes of the seasonal fluctuations in mobovirus activity and the relative abundance of the mosquito vector species. This information can be used as a basis for vector control programs and might provide an early warning of the presence of moboviruses in Germany. Since 2009, mobovirus surveillance was performed in cooperation with the German Mosquito Control Association (KABS), mostly in south-west Germany. So far, more than one million mosquitoes were captured and assayed for the presence of moboviruses.

In 2009, Sindbis virus (SINV) was isolated from *Culex* and *Anopheles* mosquitoes that were exclusively trapped in the city of Weinheim, south-west Germany (Jöst et al., 2010). SINV is the causative agent of a febrile illness in humans associated with maculopapular rash and joint pain. Consequently, a study was initiated together with the Robert Koch Institute (RKI), to investigate the medical importance of SINV in that area (Jöst et al., 2011c). Only four out of 3389 investigated blood donor samples were tested positive for SINV-specific-IgG antibodies and all samples from 355 patients with clinically suspected acute SINV infections were tested negative for SINV-specific antibodies or SINV RNA, thus demonstrating the low medical importance of SINV in south-west Germany (Jöst et al., 2011c).

In 2009, Batai virus (BATV) was isolated from *Anopheles maculipennis* mosquitoes trapped around the village of Waghäusel (Jöst et al., 2011a). Until this survey, there was no evidence for circulation of BATV in Germany. Analysis of partial S, M, and L segments showed that the sequences from all three segments were most closely related to BATV, indicating that the virus has not undergone reassortment (Jöst et al., 2011a). Phylogenetic analysis revealed a close relationship of the isolated BATV strain from Germany with strains from Slovakia, Ukraine, and Russia (Jöst et al., 2011a). BATV may cause a mild illness among sheep and cattle. Thus, to estimate the veterinary importance of BATV, we investigated the presence of BATV-specific antibodies and BATV RNA in 548 bovines from southwest Germany, and we demonstrated that 3 cattle serum samples contained BATV-neutralizing antibodies, resulting in a seroprevalence of 0.55%. Thus, our results confirm local transmission and indicate cattle as potential hosts of BATV in southwest Germany (Hoffmann et al., 2015).

In 2010, Usutu virus (USUV) was isolated from *Culex pipiens pipiens* mosquitoes trapped in the city of Weinheim (Jöst et al., 2011b). Since June 2011, considerable mortality in wild and captive bird species was observed in south-west Germany. Consequently, since June 2011, 223 dead birds were collected and tested for the presence of viral pathogens (Becker et al., 2012). USUV RNA was detected by real-time RT-PCR in 86 birds representing 6 species. The virus was isolated in cell culture from the heart of 18 Blackbirds (Turdus merula). USUV-specific antigen was demonstrated by immunohistochemistry in brain, heart, liver, and lung of infected Blackbirds (Becker et al., 2012). The complete polyprotein coding sequence was obtained by deep sequencing of liver and spleen samples of a dead Blackbird from Mannheim (BH65/11-02-03). Phylogenetic analysis of the German USUV strain BH65/11-02-03 revealed a close relationship with strain Vienna that caused mass mortality among birds in Austria in 2001. Wild birds from lowland river
valleys in southwest Germany were mainly affected by USUV, but also birds kept in aviaries. Our data suggest that after the initial detection of USUV in German mosquitoes in 2010, the virus spread in 2011 and caused epizootics among wild and captive birds in southwest Germany (Becker et al., 2012). The data also indicated an increased risk of USUV infections in humans in Germany. Therefore, from September 2011 until November 2012, 31 serum samples from German patients with clinically suspected acute USUV infections were tested for USUV-specific antibodies (Allering et al., 2012). All samples tested negative. In addition, 4,200 serum samples from healthy blood donors from south-west Germany were collected in January 2012 and also analysed for the presence of specific antibodies. One sample tested positive for USUV-IgG and -IgM (Allering et al., 2012). Thus, the seroprevalence of USUV antibodies in healthy blood donors from south-west Germany was low in January 2012. The emergence of USUV in Europe allows us to understand how an arbovirus spreads, adapts, and evolves in a naive environment. Thus, understanding the epidemiological and evolutionary processes that contribute to the emergence, maintenance, and further spread of viral diseases is the *sine qua non* to develop and implement surveillance strategies for their control. Thus, we analyzed the phylogeny and evolutionary history of all available USUV strains, including 77 newly sequenced complete genomes from a variety of host species at a temporal and spatial scaled resolution (Engel et al., 2016). The results showed that USUV can be classified into six distinct lineages and that the most recent common ancestor of the recent European epizootics emerged in Africa at least 500 years ago. We demonstrated that USUV was introduced regularly from Africa into Europe in the last 50 years, and the genetic diversity of European lineages is shaped primarily by in situ evolution, while the African lineages have been driven by extensive gene flow (Engel et al., 2016). Most of the amino acid changes are deleterious polymorphisms removed by purifying selection, with adaptive evolution restricted to the NS5 gene and several others evolving under episodic directional selection, indicating that the ecological or immunological factors were mostly the key determinants of USUV dispersal and outbreaks (Engel et al., 2016). Host-specific mutations have been detected, while the host transition analysis identified mosquitoes as the most likely origin of the common ancestor and birds as the source of the recent European USUV lineages. Our results suggest that the major migratory bird flyways could predict the continental and intercontinental dispersal patterns of USUV and that migratory birds might act as potential long-distance dispersal vehicles (Engel et al., 2016).

## References

Jöst H, Bialonski A, Storch V, Günther S, Becker N, Schmidt-Chanasit J: Isolation and phylogenetic analysis of Sindbis viruses from mosquitoes in Germany. **J Clin Microbiol** 48 (2010) 1900-1903.

Jöst H, Bialonski A, Schmetz C, Günther S, Becker N, Schmidt-Chanasit J: Isolation and phylogenetic analysis of Batai virus, Germany. **Am J Trop Med Hyg** 84 (2011a) 241-243.

Jöst H, Bialonski A, Maus D, Sambri V, Eiden M, Groschup MH, Günther S, Becker N, Schmidt-Chanasit J: Isolation of Usutu virus in Germany. **Am J Trop Med Hyg** 85 (2011b) 551-553.

Jöst H, Bürck-Kammerer S, Hütter G, Lattwein E, Lederer S, Litzba N, Bock-Hensley O, Emmerich P, Günther S, Becker N, Niedrig M, Schmidt-Chanasit J: Medical importance of Sindbis virus in south-west Germany. **J Clin Virol** 52 (2011c) 278-279.

Hofmann M, Wiethölter A, Blaha I, Jöst H, Heinemann P, Lehmann M, Miller T, Cadar D, Yanase T, Kley N, Eiden M, Groschup M, Schmidt-Chanasit Surveillance of Batai virus in bovines from Germany. **J Clin Vaccine Immunol** 22 (2015) 672-673.

Becker N, Jöst H, Ziegler U, Eiden M, Höper D, Emmerich P, Fichet-Calvet E, Ehichioya DU, Czajka C, Gabriel M, Hoffmann B, Beer M, Tenner-Racz K, Racz P, Günther S, Wink M, Bosch S, Konrad A, Pfeffer M, Groschup MH, Schmidt-Chanasit J. Epizootic emergence of Usutu virus in wild and captive birds in Germany. **PLoS One** 7 (2012) e32604.

Allering L, Jöst H, Emmerich P, Günther S, Lattwein E, Schmidt M, Seifried E, Sambri V, Hourfar K, Schmidt-Chanasit J. Detection of Usutu virus infection in a healthy blood donor from south-west Germany, 2012. **Euro Surveill** 17 (2012) pii: 20341.

Engel D, Jöst H, Wink M, Börstler J, Bosch S, Garigliany MM, Jöst A, Czajka C, Lühken R, Ziegler U, Groschup MH, Pfeffer M, Becker N, Cadar D, Schmidt-Chanasit J. Reconstruction of the Evolutionary History and Dispersal of Usutu Virus, a Neglected Emerging Arbovirus in Europe and Africa. **MBio** 7 (2016) e01938-15.
Selected oral presentations

Qualitative spatial assessment of human TBE risk factors in Belgium

Sophie Roelandt <fnref n="1" />, Els Ducheyne <fnref n="2" />, Vanessa Suin <fnref n="3" />, Steven Van Gucht <fnref n="3" />, Stefan Roels <fnref n="1" />, Guy Hendrickx <fnref n="4" />,
Yves Van Der Stede <fnref n="5" />

<figure><img src="image_43.png" /></figure>

Sophie.Roelandt@coda-cerva.be

Mapping is a risk-based method that has been used particularly in vector-borne diseases to identify the areas and time periods in which surveillance is more likely to successfully detect emerging health threats at an early stage. This greatly benefits governments and public health agencies and should be performed in interdisciplinary collaboration. Mapping cases and risk factors to study TBE(V) tick borne encephalitis (virus) emergence and epidemiology has been used in many endemic countries.

In QGIS vector layers, we first mapped the sampling frames and gold standard seroneutralisation seropositive sample results collected from three previously published TBEV studies in veterinary sentinel: in dogs (Roelandt et al., 2011), in cattle (Roelandt et al., 2014) and in wild boar (Roelandt et al., 2016). Since Belgium currently has no confirmed autochthonous human TBE cases, 9 clinically suspected human patients with inconclusive serological results were also added. The vaccination and exposure history of these patients is unfortunately unknown (Source data: WIV-ISP; 2009-2013). From the very abundant literature on TBE risk factors, Belgian raster data (1 km2 pixel resolution) was acquired on eight frequently mentioned TBE risk factors (RF). This included roe deer and wild boar predicted densities, cattle and small ruminant actual densities, an *I. ricinus* tick distribution/density layer, Corine Land Cover 2006 forest presence/absence, human proximity indices to forests ("human exposure"), and the monthly cooling rate during Autumn (August to December).

The TBE-risk of each pixel was then defined by defining a cut-off using the distribution of each RF-layer. We selected the most extreme 20% (upper or lower) quantiles for each layer, except for the forests (presence/absence). Each selection of 20% represents the more extreme cases at the higher end of the TBE risk scale (risk present). All other quantiles were considered to be not at risk or at very low risk (risk absent). In a third step, the number of risk factors per 1 km2 was calculated by simply summing the number of risk factors present per pixel. The resulting qualitative TBE risk factor presence map with RF color code (Range: 0-6 RF's present) will be presented, including the serologically positive municipalities ("Cases"). Finally, the average number of TBEV risk factors for tested infected municipality was calculated and compared to the average number in the tested free municipalities by the one-sided Mann-Whitney-U test (significance: p<0.05).

The final map showed a subjective overlap of animal sentinel seropositive communities in areas with higher numbers of risk factors, as well as overlaps between species seropositivity in all Belgian sentinel studies (dog-human-roe deer-cattle-wild boar). This association, however, was only statistically significant for the combined data of cattle and wild boar (MWU: p<0.05). We will critically discuss our general approach to this first and qualitative TBE risk (factor) mapping exercise for Belgium, the results obtained and the potential improvements that should allow for future predictive modelling and spatial risk mapping. In conclusion, TBEV does seem to be present in Belgium, where it would be expected to emerge based on TBE risk factors, despite previous negative predictions.

Keywords: TBE(V) ; tick ; borne encephalitis virus ; mapping ; risk factors ; case data ; sentinel surveillance ; medical surveillance

<footnote n="1">¹ Centre for Veterinary and Agrochemical Research (CODA-CERVA) - Groeselenbergstraat 99 1180 Ukkel Belgium - Belgium</footnote>

<footnote n="2">² Agriculture and Veterinary Information and Analysis (Avia-GIS) - Risschotlei 33 2980 Zoersel Belgium - Belgium</footnote>

<footnote n="3">³ Institute of Public Health (WIV-ISP) - Rue Juliette Wytsman 14, 1050 Ixelles Belgium - Belgium</footnote>

<footnote n="4">⁴ Agriculture and Veterinary Information and Analysis (Avia-GIS)</footnote>

<footnote n="5">⁵ Ghent University (Ugent) - Salisburylaan 133 9820 Merelbeke - Belgium</footnote>
# Resurgence of Schmallenberg virus in Belgium after 3 years of epidemiological silence

**Laurent Delooz**<sup>1,2</sup>, **Guy Czaplicki**<sup>1</sup>, **Jean-Yves Houtain**<sup>1</sup>, **Nick De Regge**<sup>3</sup>, **Ann-Brigitte Caij**<sup>3</sup>, **Christian Quinet**<sup>1</sup>,
**Thierry Petitjean**<sup>1</sup>, **Claude Saegerman**<sup>2</sup>

<figure><img src="image_44.png" /></figure>

laurent.delooz@arsia.be

## Objective

Since November 2009, the surveillance of brucellosis is performed through a passive monitoring of mandatory reported bovine abortions. In addition to brucellosis, the Regional Association for Animal Registration and Health (ARSIA) also monitored many other diseases including Schmallenberg virus (SBV). Since January 2012, the SBV was detected by RT-PCR performed at the National Reference Laboratory (NRL) in Brussels on a large number of malformed bovine aborted foetuses from all regions from Belgium. Between 2012 and 2013, this virus caused a significant increase in the number of abortion or calves at birth term with defects. In April 2016, this virus is once again detected by RT-PCR on a case of bovine abortion. This work describes the reemergence of SBV in Belgium.

## Methods

From January 2008 to June 2016, more than 20 000 abortions cases were analyzed in the framework of brucellosis monitoring in the southern part of Belgium. In this area, approximately 10 000 cattle herds with 1 200 000 bovines are registered. Informations issued from the anamnesis were recorded in the laboratory information management system. The geographical localization of each case of abortion was possible using the Lambert coordinates and the Belgian cattle identification and movement traceability system (SANITRACE).

In the laboratory, depending on the availability and quality of material, all analyses specified in the standardized panel were made. In the necropsy room, blood was sampled on Whatman 3MM CHRO blotting paper in the heart or the femoral vein of foetus. The necropsy systematically included an examination, a sampling of the brain and a classification of the congenital abnormalities. All fetuses with suspicious congenital defects were analyzed for Schmallenberg virus. Brain and blood were sampled and respectively analysed by RT-PCR performed at the Belgian NRL (since 2012) and SBV specific ELISA test performed at ARSIA (since 2016).

## Results

After three years of silence, the Schmallenberg virus is once again detected by PCR on a case of bovine abortion. A two-year old Holstein heifer, not vaccinated against SBV, aborted on the 11th April 2016 and her eight-month-old malformed foetus was autopsied at ARSIA. This foetus showed hydrocephalus, torticollis and arthrogryposis. The RT-PCR confirmed the presence of viral RNA of SBV in the brain sample while the ELISA antibody test on maternal serum and foetal blood demonstrated that both were seropositive for SBV antibodies. Knowing abortion date and age of aborted foetus, we conclude fertilisation occurred in August 2015. In addition, as gross lesions observed are compatible with an infection between 60 and 100 days of pregnancy, we can expect the dam was thus probably infected in October or November, 2015. The virus was probably circulating during this period, which is consistent with the period of activity of the midge vector. After these first cases, others cases were detected during the spring 2016.

## Conclusion

The demonstration of SBV circulation in Belgium during autumn 2015 is important to anticipate future events and advice veterinarians about the risks associated with calving, where unborn calves have been infected during this period of virus circulation.

**Keywords:** Schmallenberg virus ; SBV ; Resurgence ; Abortion ; Cattle ; Congenital ; Malformations ; Belgium

<footnote n="1"><sup>1</sup> Association Régionale de Santé et d'Identification Animales (ARSIA) - Allée des Artisans 2 5590 Ciney - Belgium</footnote>

<footnote n="2"><sup>2</sup> Faculty of Veterinary Medicine, University of Liège (UREAR-ULg, FARAH)</footnote>

<footnote n="3"><sup>3</sup> Unit enzootic and (re)emerging viral diseases; Veterinary and Agrochemical Research Centre (CODA-CERVA) - Groeselenberg 99, B-1180 Brussels - Belgium</footnote>
# What are the vector-borne pathogens present in the blood of French cattle?
## Results of a field study on 130 samples

Claire Pelletier¹, Muriel Vayssier², Christelle Roy³, Jean-Luc Zonderland⁴, Eric Collin⁵, Etienne Petit⁶, Pierre Mathevet⁷, Eric Sellal⁸

¹ BioDev - 317 avenue Jean Jaurès LYON - France

² Ecole Nationale Vétérinaire d'Alfort (ENVA) - 14 rue Pierre et Marie Curie 94700 Maisons-Alfort - France

³ Groupement de Défense Sanitaire de Corrèze (GDS19) - 30 Puy Pinçon 19001 TULLE - France

⁴ Laboratoire Départemental de Corrèze (LDA19) - Le Treuil 19012 TULLE - France

⁵ Pôle Vétérinaire du Gouet au Lie - 30 rue des Ecoles 22150 PLOUEC SUR LIE - France

⁶ Fédération Régionale des Groupements de Défense Sanitaire de Bourgogne (FRGDS Bourgogne) - DIJON - France

⁷ Tirsev - LYON - France

⁸ BioSellal - 317 avenue Jean Jaurès LYON - France

<figure><img src="image_45.png" /></figure>

claire.pelletier@biodev-diag.com

## Objective

With global warming and environmental changes, the population of bloodsucking arthropods is changing and with it the nature of potentially transmitted pathogens. If studies were conducted in France on pathogens found in wildlife or on ticks, no study had been conducted to date on cattle.

We therefore undertook to study, the apparent prevalence of major bacteria and parasites transmitted primarily by ticks, by analyzing 130 cattle showing clinical signs of hyperthermia and / or anemia with real-time PCR tests developed by BioSellal. In parallel, Ticks collected from sick animals have also been analyzed using a High-throughput Fluidigm screening system developed by Muriel Vayssier team.

This first descriptive study is also part of a One Health project given that some sought pathogens are also zoonotic agents as *A. phagocytophilum* or *Borrelia*.

## Material and method

To get a more representative view, four French regions were chosen for their variety of climates, types of breeds and production:

- Auvergne, middle mountain, mixed farms, hardy local breeds

- Burgundy, continental climate, meat cattle, Charolais breed

- Brittany, oceanic climate, dairy herds, Prim 'Holstein breed

- Reunion Island, tropical climate, mixed farms with local breeds

A total of 130 cattle in the meadow, having unexplained hyperthermia (> 39.5 ° C) and no antibiotic or piroplasmicides treatment since at least 15 days have been selected. A blood sample in EDTA tube was taken and nucleic acid extraction was performed using extraction kits BioExtract® Superball® or BioExtract® Columns (BioSellal).Duplex Real-time PCR analyzes (pathogenic + endogenous IPC) was performed according to Table 1.

<table><caption>**Table 1**</caption><thead><tr><td>Period of analysis</td><td>List of Pathogens</td></tr></thead><tbody><tr><td>Between July 1 and December 1, 2015<br>Broad Screening on the first 43 samples received</td><td>A.phagocytophilum, A marginale, A centrale, A platys, A ovis, Babesia spp-Theileria spp, and in case of positivity B divergens, B bigemina, B bovis, B microti, B venatorum, T annulata; Mycoplasma wenyonii, Mycoplasma ovis, Chlamydiales spp, Bartonella spp., Rickettsia spp, Coxiella burnetii, Francisella tularensis, Candidatus Neoehrlichia mikurensis, Borelia burgderfori sensu lato, Borrelia lusitaniae, Borrelia myamotoi</td></tr><tr><td>Between 1 December 2015 and 1 July 2016<br>Targeted screening according to the first results for the 87 samples</td><td>A.phagocytophilum, A marginale, Babesia spp-Theileria spp, and in case of positivity B divergens, B bigemina, B bovis, B microti, B venatorum, T annulata; Mycoplasma wenyonii</td></tr></tbody></table>

## Results (Table 2)

In Auvergne, Burgundy and Brittany, we found a high prevalence for *A.phagocytophilum* (33%), *Babesia* spp. (23%), *Babesia divergens* (15%). These two agents are often detected simultaneously with a potential synergistic effect. Note the detection of *A marginale*, responsible for bovine Anaplasmosis, pathogen usually associated with tropical or hot climate which seems to emerge in more temperate zones. This pathogen was, not surprisingly, found on the island of La Réunion (66%) in association with *Babesia* spp (83%) and mainly *B.bigemina* (50%). Concordant results were obtained on Ticks.

This study also described for the first time on part of French territory the prevalence of an emerging pathogen, *Mycoplasma wenyonii*, with a significant prevalence of 27%. This pathogen has recently been described in
Japan, Switzerland and Brittany, associated with various intensity clinical manifestations. If its pathogenicity remains to strengthen, it is noteworthy that in our study, this pathogen has been detected sole in 30% of cases.

Finally, for different pathogens identified in our study, we obtained variables Ct values with definitely an effect of bacterial or parasitic load on the intensity of the symptoms observed. Also an estimate of the amount of pathogen present could be interesting to monitor the effectiveness of treatment.

<caption>**Table 2**</caption>

**2a. First screening: number of positive results**

<table><thead><tr><th>Origin</th><th>Total of samples analyzed</th><th>A.phagocytophilum</th><th>Amarginale</th><th>Babesia spp</th><th>B.divergens</th><th>M.wenyonii</th><th>Others</th></tr></thead><tbody><tr><td>Burgundy</td><td>14</td><td>9</td><td>3</td><td>8</td><td>6</td><td>4</td><td>0</td></tr><tr><td>Auvergne</td><td>29</td><td>12</td><td>0</td><td>14</td><td>9</td><td>7</td><td>0</td></tr><tr><td>Total</td><td>43</td><td>21</td><td>3</td><td>22</td><td>15</td><td>11</td><td>0</td></tr></tbody></table>

**2b. Second screening: number of positive results**

<table><thead><tr><th>Origin</th><th>Total of samples analyzed</th><th>A.phagocytophilum</th><th>Amarginale</th><th>Babesia spp</th><th>B.divergens</th><th>B.bigemina</th><th>M.wenyonii</th></tr></thead><tbody><tr><td>Burgundy</td><td>2</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Auvergne</td><td>43</td><td>9</td><td>0</td><td>6</td><td>3</td><td>0</td><td>8</td></tr><tr><td>Brittany</td><td>30</td><td>9</td><td>1</td><td>0</td><td>0</td><td>0</td><td>13</td></tr><tr><td>Reunion Island</td><td>12</td><td>0</td><td>8</td><td>10</td><td>0</td><td>6</td><td>4</td></tr><tr><td>Total</td><td>87</td><td>18</td><td>10</td><td>16</td><td>3</td><td>6</td><td>25</td></tr></tbody></table>

**Conclusion**

This study has described for the first time the apparent prevalence of major vector-borne pathogens in blood on French cattle, without distinction of climate (except the tropics), type of production or cattle breed.

This study led to the development of a range of real-time PCR kits by the BioSellal Company, the Bio-T Blood kits, validated according to the French standard NF U47-600-2 to be used for relative quantification.

**Keywords:** Ticks ; Cattle ; real ; time PCR ; Anaplasma ; Babesia ; Mycoplasma wenyonii
# Schmallenberg virus infection in sheep: a model of within-herd seroprevalence evolution

François Claine¹, Laetitia Wiggers¹, Damien Coupeau¹, Benoît Muylkens¹, Nathalie Kirschvink¹

¹ University of Namur

<figure><img src="image_46.png" /></figure>

francois.claine@unamur.be

## Objective

The research sheep flock of the University of Namur underwent Schmallenberg virus (SBV) infection in autumn 2011 and SBV re-emergence between July and October 2012, leading to high within-flock seroprevalence. Since late 2012, no serological evidence of SBV novel episode has been detected thus increasing yearly the number of seronegative animals. The objective of this study was to modelize the evolution of SBV seroprevalence in this sheep flock based on duration of colostral and active immunity.

## Methods

To evaluate the decay of colostral antibodies against SBV, lambs (n=30) born from SBV infected ewes were blood collected at birth, 48h after first colostrum intake and then bimonthly until 150 days after birth (DAB). To assess the persistence of active immunity against SBV, ewes (n=50) infected in 2011 and 2012 were quarterly blood collected from early 2013 until late 2015. Anti-SBV antibody titers were evaluated in animals' serum by virus neutralization tests (VNT) and expressed as the dilution neutralizing 50% of the challenge virus (ED50). Animals were considered seropositive if the log2 ED50 was > 3.49.

## Results

In lambs, the median (min – max) length of time between birth and the first detection of VNT negative result was 122 days (74 – 144). In adult sheep, all the 50 animals remained seropositive from early 2013 until late 2015. Median (min-max) log2 ED50 anti-SBV antibody titers in early 2013 equaled 8.97 (6.48 – 10.96) and were significantly higher than titers obtained at the following sampling times that equaled 8.47 (6.47 – 8.47) in early 2014, 8.47 (6.47 – 8.47) in early 2015 and 8.47 (6.47 – 8.47) in late 2015 respectively (p<0.05). Based on these results, any lamb born from SBV seropositive ewe was supposed to be protected by colostral antibodies until 4 months of age. Moreover, any animal older than 4 months was considered to be SBV infected if born before late 2012. Lambs under 4 months and born from seronegative ewes as animals born after late 2012 were considered seronegative. Regarding these data and the renewal in this research sheep flock, the evolution of SBV seroprevalence was modelized. In early 2013, the estimated within-flock SBV seroprevalence equaled 91% (669/735) while it equaled 76% (509/669) and 50% (235/474) in early 2014 and late 2014 respectively. In autumn 2015, the SBV seroprevalence dropped to 37% (152/410). The accuracy of this model was confirmed by VNT performed on serum samples collected among the entire sheep flock.

## Conclusion

Based on calculated duration of colostral immunity in lambs and active immunity in adult sheep, the evolution of SBV seroprevalence was modelized at a herd scale. The absence of novel SBV circulation since late 2012 and the flock renewal have clearly increased the number of seronegative animals as soon as colostral protection against SBV disappears. This could promote novel SBV episode.

**Keywords:** Schmallenberg virus ; sheep ; seroprevalence ; dynamics
## Posters

# Optimisation of an ELISA immunoenzymatic technique for the detection of IgG antibodies against Trypanosomo sp. in cattle population of Ecuador

Zulay González-Martínez<fnref n="1" />, Andrea Rodrígue-Cabezas<fnref n="1" />, Armando Reyna-Bello<fnref n="2" />, Jorge Ron-Román<fnref n="1" />, Lucinda Tavares-Marques<fnref n="2" />, María-Augusta Chávez-Larrea<fnref n="1" />

<figure><img src="image_47.png" /></figure>

machavez@espe.edu.ec

## Introduction

Bovine trypanosomiasis, caused by the protozoan *Trypanosoma* sp., is characterised by anaemia, fever, progressive weight loss, reduced milk production, infertility and the death of the animal. It is widely distributed in tropical and subtropical areas, where vectors such as flies belonging to *Stomoxys* and *Tabanus* genus are endemic. In Ecuador, the only report of *Trypanosoma* sp. mentioned a 22.7% prevalence by IFAT (Wells et al., 1977). The aim of the present study was to standardize an ELISA test, using soluble extract of *T. evansi* as antigen (Ag).

## Materials and methods

In order to obtain Ag, Sprague Dawley rats were inoculated with 1x10⁶ *Trypanosomas* (*T. evansi* TEVA strain). Antigens were obtained after DEAR-cellulose chromatography and sonication of centrifuged blood, once a 1x10⁹ parasitaemia was measured in rats. For the ELISA test, 96-well polystyrene plates (Costar™), soy milk powder, conjugated HRP anti-bovine antibody (Bethyl®) and ABTS Chromogen (Invitrogen®) were used. In order to determine the optimal conditions of serum and conjugated Ag, serial dilutions of Ag with ABTS chromogen (5, 10 and 20 µg/mL) were performed, with two negative controls sera and one positive in serial dilutions between 1/10 until 1/2,800 and 4 dilutions of conjugated anti-bovine (1/5,000, 1/10,000, 1/20,000 and 1/40,000). Negative control sera were obtained from 27 Holstein cows (negative PCR), belonging to "El Prado" ESPE Farm (altitude of 2,748 m) located in an area free of the main disease vectors. The standardised ELISA was used to test 216 bovine sera from the Quito Metropolitan Slaughterhouse and 178 sera from 16 dairy farms located in the Province of Santo Domingo de los Tsáchilas.

## Results

At first, was set a blocking buffer 5% soy milk in PBS, then we performed dilutions of serum and conjugated Ag mentioned above. The higher rate between positive and negative was obtained with Ag impregnation with a concentration of 10µg/mL in PBS, serum dilution of 1/100 in PBS-T and conjugated 1/15,000 in PBS-T. Once the optimal working conditions were reached, the cut-off point was established, based on negative sera. A cut-off of 0.336 was set, as the average optical density [+ three times its standar deviation] of 27 sera.

A 47.68% prevalence (103/216) was measured in the slaughterhouse vs. 69.66% (124/178) in Santo Domingo dairy farms (range: 41%-100% per farm).

## Conclusions

Due to the wide diversity of animals slaughtered in Quito Metropolitan Slaughterhouse, the prevalence we measured could better reflect the prevalence of the whole Ecuadorian territory while the high value registered in dairy farms could be related to the hot and humid climate typical of Santo Domingo. The high *Trypanosoma* prevalence identified with the ELISA test confirms the presence of *T. vivax* and/or *T. evansi* in cattle farms in Ecuador, including dairy cows, due to the antigenic community of these species. For these reasons, it is crucial to set molecular or serological diagnostic techniques that could differentiate both parasites, in order to implement reasoned treatments to control the disease in Ecuadorian cattle farms and limit associated losses.

## References

Wells EA, Betancourt A, Ramirez LE (1977). Serological evidence for the geographical distribution of Trypanosoma vivax in the New World. Trans. R. Soc. Trop. Med. Hyg. 71:448-9.

**Keywords:** Tripanosomiasis; Trypanosoma sp.; vector; ELISA; antigen; seroprevalence

<footnote n="1">¹ Carrera de Ingeniería en Biotecnología, Departamento de Ciencias de la Vida y la Agricultura, Universidad de las Fuerzas Armadas (GISAH - ESPE) - Ecuador</footnote>

<footnote n="2">² Grupo de Inmunobiología, Centro de estudios Biomédicos y Veterinarios, Universidad Nacional Experimental Simón Rodríguez, Caracas, Venezuela</footnote>
# Polymerase Chain Reaction in the diagnosis of Anaplasma marginale in cattle populations of Ecuador and its further molecular identification by 16S ribosoma sequencing

Leandro Tana-Hernández<fnref n="1" />, Armando Reyna-Bello<fnref n="2" />, Jorge Ron-Román³, María-Augusta Chávez-Larrea<fnref n="1" />

<figure><img src="image_48.png" /></figure>

machavez@espe.edu.ec

## Introduction

Bovine anaplasmosis, caused by the bacterium *Anaplasma marginale*, is endemic worldwide in tropical and subtropical areas. In cattle, resulting socio-economic losses are: a decreased production, quarantine, treatment, vector control, and deaths of sick animals. The main vectors of *A. marginale* are ticks and blood-sucking flies, including horse flies and stable flies. The first line diagnosis is based on observation of blood smears, but sensitivity and specificity of this technique are poor, compared to other methods such as molecular biology. No study has been published on molecular diagnosis or identification of this haemotropic bacterium in Ecuador so far, nevertheless, diagnosis through blood smear was used in several undergraduate tesis.

## Materials and methods

The present study was conducted to determine the prevalence, based on molecular identification, of *A. marginale* in dairy cattle of Santo Domingo de los Tsachilas. This province of Ecuador, with an altitude below 1,300 m, has a humid climate appropriate for the development of disease vectors. The cattle population of study included 133 adult lactating cows, crossbred *Bos indicus-Bos taurus*, and 18 bulls. Two polymerase chain reaction (PCR) tests were used to amplify specific regions of *A. marginale* DNA, isolated from cattle blood samples. The first PCR was elaborated with primer to amplify MSP5, as previously described, and the second PCR test used primer for amplification of Rickettsias 16S, in order to identify the specific *A. marginale* circulating in the Ecuadorian province of interest. The products of 16S PCR were previously cloned, in order to ensure only one fragment was sequenced at a time, which provides a more reliable and complete sequence.

## Results

A total of 130 blood samples were positive, equivalent to a 86.1% prevalence: the specific MSP5 gene of *A. marginale* ranged between 40 and 100%, confirming the presence of the bacterium in all these samples. Thirty-six positive samples were randomly selected to identify *A. marginale* isolate by amplification of the 16S gene conserved region; 80.6% (29/36) were positive. Three isolates were cloned (SD1, SD5 and SD18) and sequenced to identify circulating *A. marginale*: we observed 100% similarity between SD1 and SD18, in accordance with GenBank access code samples CP006847.1, CP000030.1, AF414874 1, AF309868.1 and AF309866.1. The SD5 sample presented a 99.9% similarity with sequences mentioned above, because of two modified nucleotide bases, TC and GA, in positions 249 131 and 248 401, respectively.

## Conclusions

Out of all samples analyzed, 86.1% were infected with *A. marginale*. The species of *A. marginale* was identified, thanks to a 99.99% similarity in the three isolates, two of which being identical to isolates previously registered in GenBank (100% and 99.9% undescribed similarity) but never described in Ecuador to date. Such high prevalence is mainly linked to two factors: (i) the presence of vectors in the area and (ii) the lack of treatment and control of the disease. Our results also indicate, on the basis of modified nucleotide bases identified in SD5, the presence of a different *A. marginale* strain in Ecuador, which should pave the way for further investigations.

**Keywords:** Anaplasmosis ; bovine ; Anaplasma marginale ; polymerase chain reaction ; MSP5 ; 16S ; Santo Domingo de los Tsáchilas ; Ecuador.

<footnote n="1">¹ Carrera de Ingeniería en Biotecnología, Departamento de Ciencias de la Vida y la Agricultura, Universidad de las Fuerzas Armadas (GISAH - ESPE) - Ecuador</footnote>

<footnote n="2">² Grupo de Inmunobiología, Centro de estudios Biomédicos y Veterinarios, Universidad Nacional Experimental Simón Rodríguez, Caracas, Venezuela</footnote>
<figure><img src="image_49.png" /></figure>

Association d'Epidémiologie et de Santé Animale

One health :
antimicrobial resistance

7th - 9th Sept. 2016 - Liège (Belgium)
Congress 25 years

**Animal epidemiology**
Past, present and future
Keynote lecture

# Animal-human-environment continuum in the emergence and spread of antibiotic resistance in human pathogens

Axel Cloeckaert Ph.D. <fnref n="1" />,<fnref n="2" />

Axel is Director of Research at the Infectiology and Public Health Unit of INRA Nouzilly. His research focuses on the genetics of antimicrobial resistance in enterobacterial pathogens and on the biodiversity of bacterial species belonging to the family Brucellaceae.

<figure><img src="image_50.png" /></figure>

Axel.Cloeckaert@tours.inra.fr

<figure><img src="image_51.png" /></figure>

Emergence and spread of antibiotic resistance is a major problem that concerns both animal and human health. Indeed, most antibiotic classes are used to treat either animal or human infections. Although within these classes some antibiotics have been developed to be used only in veterinary medicine they select in any case for resistance mechanisms that concern the corresponding antibiotic used in human medicine. In addition, resistance genes cluster together on mobile elements such as plasmids or genomic islands which means that the use of the one antibiotic to treat animal infections may coselect for resistance to another antibiotic to treat human infections. There are nowadays numerous examples of spread of multiple drug resistance (MDR) in both Gram-positive and Gram-negative bacteria, including the emergence of new plasmid-mediated resistance mechanisms such as those recently and increasingly reported for polymyxins (1). Polymyxins were introduced in the 1950s and have been considered until recently to be too nephrotoxic and too neurotoxic for their regular use for treating infections in humans (2). Their large usage was restricted to animals (3). However, due to the paucity of remaining antibiotics for treating human infections, polymyxins (colistin, polymyxin B) have become the last resort, in particular for treating infections due to carbapenem-resistant *Enterobacteriaceae*. The emergence and global spread of plasmid-mediated resistance mechanisms to polymyxins is at present thus a major concern for both animal and human health.

Among *Enterobacteriaceae* spreading antibiotic resistance, *Salmonella* spp. are major zoonotic bacteria and foodborne pathogens, and among the most frequently isolated bacteria from various sources, i.e. environment, plant, feed, animal, food and finally human. They thus represent a good example for animal-human-environment continuum in the emergence and spread of antibiotic resistance in human pathogens.

From a historical point of view, international spread of multiple antibiotic resistance in *Salmonella* spp. may have started during the 1980s, with the emergence of a particular clone of *Salmonella enterica* serovar Typhimurium definitive phage type 104, commonly named DT104 (4, 5). This clone initially displayed resistance to 5 commonly used antimicrobials, namely ampicillin (Ap), phenicolics (chloramphenicol (Cm)/florfenicol (Ff)), streptomycin (Sm)/spectinomycin (Sp), sulfonamides (Su), and tetracyclines. The ApCmFfStSpSuTc multiple-antibiotic resistance profile of S. *enterica* Typhimurium DT104 was shown to be conferred by an antibiotic resistance gene cluster carried by a chromosomal genomic island called *Salmonella genomic island 1* (SGI1) (5). The resistance gene cluster consists of a complex class 1 integron, of which many variants have been described. Variant antibiotic resistance gene clusters are generated through recombination events or through gene cassette replacement events (5). In addition, some insertion sequence (IS) elements have been shown to be involved in genetic variation of SGI1 (6, 7, 8). Variants of SGI1 have been named alphabetically in the order of their discovery from SGI1-A to SGI1-Z (9). Since the 1990s, SGI1 has spread globally through several S. *enterica* serovars and it has since 2007 also been reported in clinical, environmental and animal isolates of *Proteus mirabilis* (10-16). Some SGI1 variants carrying critical resistance genes that may compromise treatment of human infections have also emerged since 2006. Some examples are the *qnr* gene(s) conferring resistance to quinolones and extended-spectrum beta-lactamase (ESBL) genes such as *bla*<sub>VEB-6</sub> (12). Integration of the *bla*<sub>NDM-1</sub> carbapenemase gene into a related island, namely *Proteus* genomic island 1 (PGI1-PmPEL), in a *Proteus mirabilis* clinical isolate has also been reported (17).

Fluoroquinolones (e.g. ciprofloxacin), together with extended-spectrum cephalosporins (ESC), are the treatment of choice for nontyphoid salmonellosis, as stable resistance to the most common members of different families of antimicrobial agents mentioned above has developed during the 1990s with the DT104 epidemic. Considering threat to human health, of particular concern is nowadays the international spread of ciprofloxacin-resistant S. *enterica* serovar Kentucky ST198, firstly detected in travel-associated human cases (18). This clone is not only highly resistant to ciprofloxacin but also multidrug-resistant due to the presence of a variant of SGI1, mostly SGI1-K or variants derived of it (6). This variant besides antibiotic resistance genes carry also a mercury resistance genes cluster. The clonal MDR ST198 population was mostly detected in Egypt before 2005, but has now rapidly spread throughout Africa and the Middle East (18-21). Another matter of concern is its expansion in livestock. It was initially identified in autochthonous poultry but was then found in

<footnote n="1">¹ INRA, UMR1282 Infectiologie et Santé Publique, Nouzilly, France</footnote>

<footnote n="2">² Université François Rabelais de Tours, UMR1282 Infectiologie et Santé Publique, Tours, France</footnote>
various animals and foods in France, in the US, in Germany and in Poland (19). Several isolates give rise to considerable concern, as they have become producers of various carbapenemases and/or cephamycinase and/or ESBL (20, 21).

This clone was probably selected in 3 steps in Egypt during the 1990s and the early 2000s, with poultry being a potential major vehicle (18). Intensive aquaculture reliant on large amounts of antimicrobial agents may have played an initial role through the acquisition of the genomic island SGI1- K. Although there is ongoing debate on the potential aquacultural origin of the SGI1 in *S. enterica* Typhimurium DT104, the origin of *S. enterica* Paratyphi B variant Java isolates carrying SGI1 has been clearly associated with tropical fish farmed in Asia (22, 23). The common use of fluoroquinolones, including ciprofloxacin, enrofloxacin, and ofloxacin, in chicken and turkey production in Nigeria and Morocco may have further contributed to its spread with the acquisition of fluoroquinolone resistance mechanisms, consisting mainly of target modifications (mutations in the *gyrA* and *parC* genes). Shellfish, possibly contaminated through the aquatic environment, itself contaminated by humans or poultry, may constitute a secondary food reservoir in areas of endemicity, as observed in Morocco.

SGI1 has been shown to be an integrative mobilizable element, requiring conjugative plasmids of the IncA/C family for its mobilization (24). In 2005, we reported that SGI1 could be conjugally transferred from *S. enterica* donor strains to non-SGI1 *S. enterica* and *E. coli* recipient strains where it integrated into the recipient chromosome in a site-specific manner (24). Briefly, after excision of SGI1 from the *Salmonella* chromosome, the conjugative mobilization in trans by the conjugative helper IncA/C plasmid pR55 occurs between donor and recipient strains. In the recipient cell, the circular form of SGI1 integrates in a site-specific manner at the 3' end of the chromosomal *trmE* gene (24). The first gene of SGI1 coding for an integrase (*int*) was shown to be essential for this mobilization mechanism. SGI1 appeared to be a non-self-transmissible but mobilizable element and was thus classified within the group of site-specific integrative mobilizable elements (IMEs) that are related to integrative conjugative elements (ICEs) (24). Mobilization of SGI1 was later found to be exclusively mediated by plasmids of the IncA/C family (25). The IncA/C-encoded transcriptional activator complex AcaCD was further shown to trigger the SGI1 excision and *in trans* conjugative mobilization (26).

Besides multidrug resistance mechanisms and genetic supports presented above, of particular concern since the 2000s is also the worldwide development of plasmid-mediated resistance to other key antimicrobials, namely extended-spectrum cephalosporins (27). The use of newer β-lactams in animal husbandry and veterinary medicine may have facilitated the spread of extended-spectrum cephalosporin-resistant *Salmonella* strains in livestock. Numerous papers have reported the spread through plasmids, of several Inc groups, of either ESBL or AmpC genes conferring resistance to ESC. In EU countries, such as Belgium or France, such resistances emerged mostly in poultry and most likely poultry products were at the origin of human infections reported (28-30). The most prevalent plasmids were of the IncI1 or IncH12 groups, responsible for the spread of the *bla*~TEM-52~ and *bla*~CTX-M-2~ resistance genes, respectively (28-30). These resistances have spread in several *S. enterica* serovars such as Agona, Derby, Infantis, Paratyphi B, Typhimurium, and Virchow. Besides SGI1 was also detected in some of these isolates conferring an additional MDR phenotype (29).

In conclusion, several critical resistance mechanisms are cumulating in *Salmonella* spp. since the 1990s, through diverse genetic acquisition modes, e.g. genomic islands, plasmids, mutations. Whereas the first important epidemic MDR *S. enterica* Typhimurium DT104 clone did not seem to be able to cumulate all resistance mechanisms together, more recent clones such as *S. enterica* serovar Kentucky ST198 are able to do so and to spread globally. The genetics behind this cumulation ability remains to be investigated. The colistin resistance gene *mcr-1* has also been reported in *Salmonella* spp. but to my knowledge not yet in a strain with concomitant fluoroquinolone and ESC resistance. Given the spread of the MDR ST198 clone we may also expect the emergence of colistin resistance in this clone. Therefore, from a One Health perspective, coordinated epidemiological surveillance and control measures should be implemented, from the environmental source to human contamination, to limit the spread of new resistant genes and clones.

## References

1. Schwarz S, Johnson AP. 2016. Transferable resistance to colistin: a new but old threat. J Antimicrob Chemother pii: dkw274.

2. Nordmann P, Poirel L. 2016. Plasmid-mediated colistin resistance: an additional antibiotic resistance menace. Clin Microbiol Infect 22:398-400.

3. Poirel L, Nordmann P. 2016. Emerging plasmid-encoded colistin resistance: the animal world as the culprit? J Antimicrob Chemother pii: dkw074.

4. Cloeckaert A, Schwarz S. 2011. Molecular characterization, spread and evolution of multidrug resistance in *Salmonella enterica* Typhimurium DT104. Vet Res 2001 32:301-310.

5. Mulvey MR, Boyd DA, Olson AB, Doublet B, Cloeckaert A. 2006. The genetics of *Salmonella* genomic island 1. Microbes Infect 8:1915-1922.

6. Doublet B, Praud K, Bertrand S, Collard JM, Weill FX, Cloeckaert A. 2008. Novel insertion sequence- and transposon-mediated genetic rearrangements in genomic island SGI1 of *Salmonella enterica* serovar Kentucky. Antimicrob Agents Chemother 52:3745-3754.
7. Doublet B, Praud K, Weill FX, Cloeckaert A. 2009. Association of IS26-composite transposons and complex In4-type integrons generates novel multidrug resistance loci in *Salmonella* genomic island 1. J Antimicrob Chemother 63:282-289.

8. Le Hello S, Weill FX, Guibert V, Praud K, Cloeckaert A, Doublet B. 2012. Early strains of multidrug-resistant *Salmonella enterica* serovar Kentucky sequence type 198 from Southeast Asia harbor *Salmonella* genomic island 1-J variants with a novel insertion sequence. Antimicrob Agents Chemother 56:5096-5102.

9. Qin S, Qi H, Zhang Q, Zhao D, Liu ZZ, Tian H, Xu L, Xu H, Zhou M, Feng X, Liu HM. 2015. Emergence of extensively drug-resistant *Proteus mirabilis* harboring a conjugative NDM-1 plasmid and a novel *Salmonella* Genomic Island 1 variant, SGI1-Z. Antimicrob Agents Chemother 59:6601-6604.

10. Ahmed AM, Hussein Al, Shimamoto T. 2007. *Proteus mirabilis* clinical isolate harbouring a new variant of *Salmonella* genomic island 1 containing the multiple antibiotic resistance region. J Antimicrob Chemother 59:184-190.

11. Boyd DA, Shi X, Hu QH, Ng LK, Doublet B, Cloeckaert A, Mulvey MR. 2008. *Salmonella* genomic island 1 (SGI1), variant SGI1-I, and new variant SGI1-O in *Proteus mirabilis* clinical and food isolates from China. Antimicrob Agents Chemother 52:340-3404.

12. Siebor E, Neuwirth C. 2011. The new variant of *Salmonella* genomic island 1 (SGI1-V) from a *Proteus mirabilis* French clinical isolate harbours *bla*~VEB-6~ and *qnrA1* in the multiple antibiotic resistance region. J Antimicrob Chemother 66:2513-2520.

13. Siebor E, Neuwirth C. 2013. Emergence of *Salmonella* genomic island 1 (SGI1) among *Proteus mirabilis* clinical isolates in Dijon, France. J Antimicrob Chemother 68:1750-1756.

14. Lei CW, Zhang AY, Liu BH, Wang HN, Guan ZB, Xu CW, Xia QQ, Cheng H, Zhang DD. 2014. Molecular characteristics of *Salmonella* genomic island 1 in *Proteus mirabilis* isolates from poultry farms in China. Antimicrob Agents Chemother 58:7570-7572.

15. Lei CW, Zhang AY, Liu BH, Wang HN, Yang LQ, Guan ZB, Xu CW, Zhang DD, Yang YQ. 2015. Two novel *Salmonella* genomic island 1 variants in *Proteus mirabilis* isolates from swine farms in China. Antimicrob Agents Chemother 59:4336-4338.

16. Schultz E, Haenni M, Mereghetti L, Siebor E, Neuwirth C, Madec JY, Cloeckaert A, Doublet B. 2015. Survey of multidrug resistance integrative mobilizable elements SGI1 and PGI1 in *Proteus mirabilis* in humans and dogs in France, 2010-13. J Antimicrob Chemother 70:2543-2546.

17. Girlich D, Dortet L, Poirel L, Nordmann P. 2015. Integration of the *bla*~NDM-1~ carbapenemase gene into *Proteus genomic* island 1 (PGI1-PmPEL) in a *Proteus mirabilis* clinical isolate. J Antimicrob Chemother 70:98-102.

18. Le Hello S, Hendriksen RS, Doublet B, Fisher I, Nielsen EM, Whichard JM, Bouchrif B, Fashae K, Granier SA, Jourdan-Da Silva N, Cloeckaert A, Threlfall EJ, Angulo FJ, Aarestrup FM, Wain J, Weill FX. 2011. International spread of an epidemic population of *Salmonella enterica* serotype Kentucky ST198 resistant to ciprofloxacin. J Infect Dis 204:675-684.

19. Le Hello S, Bekhit A, Granier SA, Barua H, Beutlich J, Zając M, Münch S, Sintchenko V, Bouchrif B, Fashae K, Pinsard JL, Sontag L, Fabre L, Garnier M, Guibert V, Howard P, Hendriksen RS, Christensen JP, Biswas PK, Cloeckaert A, Rabsch W, Wasyl D, Doublet B, Weill FX. 2013. The global establishment of a highly-fluoroquinolone resistant *Salmonella enterica* serotype Kentucky ST198 strain. Front Microbiol 4:395.

20. Le Hello S, Harrois D, Bouchrif B, Sontag L, Elhani D, Guibert V, Zerouali K, Weill FX. 2013. Highly drug-resistant *Salmonella enterica* serotype Kentucky ST198-X1: a microbiological study. Lancet Infect Dis 13:672-679.

21. Ktari S, Le Hello S, Ksibi B, Courdavault L, Mnif B, Maalej S, Fabre L, Hammami A, Weill FX. 2015. Carbapenemase-producing *Salmonella enterica* serotype Kentucky ST198, North Africa. J Antimicrob Chemother 70:3405-3407.

22. Cabello FC. 2009. Aquaculture and florfenicol resistance in *Salmonella enterica* serovar Typhimurium DT104. Emerg Infect Dis 15:623; author reply 623-4. doi: 10.3201/eid1504.081171.

23. Levings RS, Lightfoot D, Hall RM, Djordjevic SP. 2006. Aquariums as reservoirs for multidrug-resistant *Salmonella* Paratyphi B. Emerg Infect Dis 12:507-510.

24. Doublet B, Boyd D, Mulvey MR, Cloeckaert A. 2005. The *Salmonella* genomic island 1 is an integrative mobilizable element. Mol Microbiol 55:1911-1924.

25. Douard G, Praud K, Cloeckaert A, Doublet B. 2010. The *Salmonella* genomic island 1 is specifically mobilized in trans by the IncA/C multidrug resistance plasmid family. PLoS One 5:e15302.

26. Carraro N, Matteau D, Luo P, Rodrigue S, Burrus V. 2014. The master activator of IncA/C conjugative plasmids stimulates genomic islands and multidrug resistance dissemination. PLoS Genet 10:e1004714.

27. Arlet G, Barrett TJ, Butaye P, Cloeckaert A, Mulvey MR, White DG. 2006. *Salmonella* resistant to extended-spectrum cephalosporins: prevalence and epidemiology. Microbes Infect 8:1945-1954.
28. Bertrand S, Weill FX, Cloeckaert A, Vrints M, Mairiaux E, Praud K, Dierick K, Wildemauve C, Godard C, Butaye P, Imberechts H, Grimont PA, Collard JM. 2006. Clonal emergence of extended-spectrum beta-lactamase (CTX-M-2)-producing *Salmonella enterica* serovar Virchow isolates with reduced susceptibilities to ciprofloxacin among poultry and humans in Belgium and France (2000 to 2003). J Clin Microbiol 44:2897-2903.

29. Cloeckaert A, Praud K, Doublet B, Bertini A, Carattoli A, Butaye P, Imberechts H, Bertrand S, Collard JM, Arlet G, Weill FX. 2007. Dissemination of an extended-spectrum-beta-lactamase *bla*TEM-52 gene-carrying Incl1 plasmid in various *Salmonella enterica* serovars isolated from poultry and humans in Belgium and France between 2001 and 2005. Antimicrob Agents Chemother 51:1872-1875.

30. Doublet B, Praud K, Nguyen-Ho-Bao T, Argudín MA, Bertrand S, Butaye P, Cloeckaert A. 2014. Extended-spectrum β-lactamase- and AmpC β-lactamase-producing D-tartrate-positive *Salmonella enterica* serovar Paratyphi B from broilers and human patients in Belgium, 2008-10. J Antimicrob Chemother 69:1257-1264.
# Selected oral presentations

## A quantitative exposure model for the cross-contamination of pig feeding-stuffs as a result of antimicrobial residues

Marilena Filippitzi<fnref n="1" />, Steven Sarrazin<fnref n="1" />, Jeroen Dewulf<fnref n="1" />

✉ mariaeleni.filippitzi@ugent.be

## Introduction

The cross-contamination of non-medicated feed with residues of antimicrobials causes a public and animal health concern associated with the potential for selection and dissemination of resistance in commensal bacteria and potentially zoonotic bacteria. To identify the extent of the situation, we built a model that provides a way to estimate the percentage of cross-contaminated pig feed in total and per different levels at which cross-contamination may occur (i.e. the feed mill, the transport truck, the farm), for different levels of antimicrobial medicated feed (AB MF) produced in a country per year.

## Materials and methods

The potential carry-over of AB traces was assessed- from the feed mill to the transport truck, and the storage and distribution at the farm. The model was thus subdivided in three modules, one for each level, and five exposure pathways were considered. It was built using @Risk® software (Palisade Corporation®) and was run at 10,000 iterations per simulation.

## Results

The model estimated that, given our assumptions, when a hypothetical level of xi=2% of the feed produced in a country per year is AB MF, C1i= 5.5% (95%CI 3.4%; 11.4%) of the total feed produced in a year (T1i) could be cross-contaminated with different levels of AB due to practices related to MF. In detail, 1.80% (95%CI 0.2%; 7.7%) of T1i in such a country would be due to cross-contamination occurring at the feed mill, 1.83% (95%CI 1.3%; 2.0%) at the transport truck and 1.84% (95%CI 1.2%; 2.0%) at the farm level. The model also demonstrated that, even in cases where MF would be produced in end-of-line mixers or fine dosing system trucks would be used, the risk would not be completely removed; the percentage of cross-contaminated feed produced in a country (where xi=2%) per year would be C2i=3.7% (95%CI 2.9%; 4.0%) and C3i=2.4% (95%CI 1.6%; 2.7%), respectively.

## Discussion

The model showed a real risk of cross-contamination of feed due to practices related to MF. The risk is hard to be fully removed and thus, the use of MF should be avoided as much as possible. The model demonstrated that a considerable risk of cross-contamination can be avoided when MF is not produced in the main mixing line of feed mills and when FDS trucks are used. But, even in cases where these scenarios would be implemented, the risk would not be completely removed given that sources of cross-contamination would still exist at the truck and farm levels and hence should not be overlooked. The model can be used to provide estimations for different levels of AB MF produced and for different policies applied and should then be informed with data after carefully considering the specific situation.

**Keywords:** Medicated feed ; cross ; contamination ; antimicrobials ; residues ; risk assessment

<footnote n="1">¹ Veterinary Epidemiology Unit, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University - Belgium</footnote>
# The influence of the introduction of antimicrobial use guidelines on prescription habits in first-line, small animal veterinarians

Steven Sarrazin<fnref n="1" />, Marjolijn Schlepers<fnref n="1" />, Femke Vandael<fnref n="1" />, Evelyne De Graef<fnref n="2" />, Hilde De Rooster<fnref n="3" />, Jeroen Dewulf<fnref n="1" />

steven.sarrazin@ugent.be

## Objective

The use of antimicrobials in small animals, as in any other animal species including humans, can promote the selection of less susceptible and resistant bacteria. An expert group in human medicine defined strategic goals to optimize antimicrobial use among which the development and implementation of antimicrobial use guidelines. For small animals is Belgium, the Belgian Centre of Expertise on Antimicrobial Consumption and Resistance in Animals (AMCRA) developed guidelines for prudent use of antimicrobials to support veterinarians in the choice for an antimicrobial agent when treatment is necessary. The aim of this study was to evaluate the impact of introducing these antimicrobial use guidelines on prescription habits of veterinarians in small animal practices.

## Methods

During this prospective study the prescription habits of fifteen first-line, small animal veterinarians during first consultations of cats and dogs, one month prior to implementation of antimicrobial use guidelines and at least 20 days thereafter were followed for six groups of clinical conditions (related to skin, respiratory tract, digestive tract, urogenital tract, ear or other). A first outcome of interest was whether a veterinarian had prescribed antimicrobials during a consultation. A second outcome of interest was, when antimicrobials were used or prescribed during a consultation, whether they were selected according to the antimicrobial use guidelines. Additionally, changes in choice of active substance were critically evaluated against the introduced antimicrobial use guidelines. The guidelines listed first, second and/or third choice antimicrobials per condition based on scientific literature regarding efficacy.

## Results

During cat consultations, antimicrobials were prescribed in 72% (143/199) and in 60% (150/249) of the consultations before and after the introduction of antimicrobial use guidelines, respectively. A similar decrease was noticed for dog consultations: 80% (280/350) of antimicrobial prescriptions before and 68% (358/525) afterwards. Furthermore, there was an increase of consultations of cats (+13%) and dogs (+11%) where veterinarians handled according to the AMCRA guidelines: 1) antimicrobials prescribed only when indicated and 2) only a first (or second) choice antimicrobial was prescribed. Amoxicillin clavulanate, cefovecin and cephalexin were the most commonly prescribed antimicrobials before and after the introduction of the antimicrobial use guidelines. The relative number of second choice antimicrobials in cats (-16%) and off-label products in dogs (-10%) decreased significantly (*P* < 0.05). Although not significant, the prescription of third choice antimicrobials increased in cats (+8%) and dogs (+4%). The results of this study suggest that antibacterial use guidelines may be useful tools to partially change the prescription habits of small animal veterinarians. The next step is to develop communication strategies to inform large groups of veterinarians about the need of responsible use of antibiotics and about the benefits of using the antimicrobial use guidelines.

## Conclusion

Veterinarians were less prone to prescribe antimicrobials and handled in more occasions according to the antimicrobial use guidelines. However, the unexpected increase of third choice antimicrobials showed that more emphasis on public health needs to be put in future guidelines to avoid this unwanted shift.

**Keywords:** small animals ; antimicrobial use ; guidelines

<footnote n="1">¹ Veterinary Epidemiology Unit, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University - Belgium</footnote>

<footnote n="2">² Centre of Expertise on Antimicrobial Consumption and Resistance in Animals</footnote>

<footnote n="3">³ Medicine and Clinical Biology of Small Animals, Faculty of Veterinary Medicine, Ghent University</footnote>
# Identification of bovine methicillin resistant staphylococci from Europe, Africa and North America by colony hybridization, PCR and antibiotic sensitivity

Cyrille Ngassam-Tchamba¹, Damien Thiry¹, Marjorie Bardiau¹, Jean-Noël Duprez¹, Maria Agurdin², Olivier Denis², Jacques Mainil¹

¹ Bacteriology, Department of Infectious and Parasitic Diseases, FVM and FARAH, ULg, Liège

² NRC S. aureus, Microbiology, Erasmus Hospital, Free University of Brussels

damien.thiry@ulg.ac.be

Mastitis is the costliest pathology in dairy cattle and staphylococci are the most prevalent bacterial mastitis pathogens worldwide. Antimicrobial treatment of mastitis has led to the selection of resistant staphylococci, of which the Methicillin Resistant S. aureus (MRSA) are the most studied ones. Still, MR has also been described for non-aureus staphylococci (MRS) species. Bovine MRS(A) represent not only a problem in the treatment of mastitis, but also a potential hazard in public health via the inter-Staphylococcus transferability of the mobile "Staphylococcal Cassette Chromosome" (SCC) carrying the mec genes encoding MR and the zoonotic potential of some Staphylococcus species. The aim of this study is the comparison of genetic and phenotypic methods for the identification of MRS(A) isolated from bovine mastitis in European, African and North-American countries.

A total of 1168 mastitis-associated staphylococci were isolated between 2005 and 2014 in Belgium, Italy, Switzerland, Senegal, Niger and Canada, and kept at -80°C until further use. Out of them, 867 isolates were identified to S. aureus while 301 isolates were non aureus staphylococci. All 1168 staphylococci were tested genetically by the dot blot hybridization assay on positively charged nylon membranes (Roche) after DNA extraction with 32P-radioactively labelled probes derived from the *mecA* and *mecC* genes and phenotypically by growth on "Chrom MRSA ID®" agar plates. Isolates positive at both or either tests were further studied by PCR targeting the same two genes and by the disk diffusion assay to oxacillin and cefoxitin.

A total of 265 isolates (23%) were positive at both or either tests. Out of them, 27 S. aureus (10%) but no non-aureus (0%) tested positive both for DNA hybridization with the *mecA* probe and for growth on "Chrom MRSA ID®" plates. No isolate tested positive with the *mecC* probe. In addition, 32 S. aureus (12%) and 15 non aureus (6%) were positive with the *mecA* probe only and 169 S. aureus (64%) and 22 non aureus (8%) grew on "Chrom MRSA ID®" plates only. The S. aureus originate from Belgium (105), Italy (6), Canada (31), Senegal (38) and Niger (48) whereas the non-aureus originate from Belgium (25), Italy (1) and Niger (11). All of them are being tested with the PCR targeting the *mecA* gene and by the disk diffusion assay to oxacillin and cefoxitin.

Most isolates (72%) grew on "Chrom MRSA ID®" plates only while few (18%) were positive to the hybridization with the *mecA* probe only. This high difference between the results of both tests could be explained by the weak specificity of phenotypic tests comparing to genetic tests. The others 10% of the isolates (S. aureus) which are positive with the two methods (dot blot hybridization and "Chrom MRSA ID®") can be considered as MRSA mediated by the *mecA* gene. However, results of PCR and disk diffusion assay will confirm respectively the presence of *mec* genes and which of the two methods is the most suitable for identifying MRS from mastitis cases in cattle. Comparison of the results of phenotypic and genetic assays will indicate whether other variant(s) than *mecA* and *mecC* may be present in MRS. Further genetic and phenotypic studies are needed to (i) identify the non-aureus isolates to the species level; (ii) compare the MRS(A) isolated in the different countries by their biotypes, serotypes, lysotypes, and virulotypes, without forgetting their SCC*mec* and their clonal complex; and (iii) identify the *mec* gene variant present in hybridization-positive PCR-negative isolates, if any.

**Keywords:** Staphylococcus aureus ; MRSA ; bovine ; mastitis
# Identification of bovine and porcine colistin-resistant mcr1-positive Escherichia coli

Jacques Mainil<fnref n="1" />, Adeline Muylaert<fnref n="1" />, Marc Saulmont<fnref n="2" />, Mia Vanrobaeys<fnref n="3" />, Jean-Noël Duprez<fnref n="1" />, Cyrille Ngassam-Tchamba<fnref n="1" />, Katelijn Dierick<fnref n="4" />, **Damien Thiry**<fnref n="1" />, Nadine Botteldoorn<fnref n="4" />

jg.mainil@ulg.ac.be

Polymyxins, especially colistin, have been used for years in veterinary medicine and were rediscovered a few years ago as last resort antibiotics in human medicine against multi-resistant Gram negative bacterial pathogens. For years, only chromosome-mediated resistance to colistin was identified as a consequence of mutation(s) in lipid A-encoding genes. Recently, however, a plasmid-located gene (mcr1) was identified in Gram-negative enterobacteria and has since been found by PCR in several, but not all, bovine, human, porcine and poultry colistin-resistant Escherichia coli (Liu YY et al. Lancet Infect Dis, 2016, 16(2), 161-168; Nordmann P and Poirel L. Clin Microbiol Infect, 2016, 22, 398-400; Schwarz S and Johnson AP. J Antimicrob Chemother, 2016, in press, doi: 10.1093/jac/dkw274).

The purpose of this study was to compare phenotypic and genetic for the detection of resistance to colistin and of the mcr1 gene in a collection of Escherichia coli isolated from different animal species and from humans.

More than 3000 *E. coli* isolates from cattle, pigs, dogs, cats, horses, rabbits, chickens, ducks and humans were tested for resistance to colistin by growing them on agar plates with 1mg/ml of colistin. The Minimal Inhibitory Concentrations (MIC) of and the presence of the *mcr1* gene in all growing isolates were determined using the E test® and colony hybridization assay with a *mcr1* specific gene probe, respectively. The probe-positive isolates were further tested with the *mcr1* gene specific PCR.

A total of 410 *E. coli* isolated grew on 1mg/ml colistin-containing agar plates. The majority of isolates grew well, but several grew sparsely with only few isolated colonies. As determined by the E test®, MIC of 273 isolates (67%) was 1mg/ml of colistin and higher; conversely, MIC of 137 isolates (33%) was lower than 1mg/ml of colistin.

Of those 410 *E. coli* isolates, 34 from pigs and bovines (9% of isolates growing on colistin-containing agar plates; 25% of isolates with MIC higher than 1mg/ml) hybridized with the *mcr1* gene-derived probe: 5 from pigs and 11 from bovines gave black spots (including five from the same calf), while 18 from pigs and one from bovine gave grey spots. All but one pig isolate had a MIC between 1.5 and 16 mg/ml of colistin.

Fifteen “black spot” probe-positive isolates tested positive with the *mcr1* gene specific PCR as did 3 porcine “grey spot” probe-positive isolates, while the remaining 16 isolates repeatedly tested negative even after lowering the annealing temperature.

This study confirms that (i) the results of phenotypic assays for the detection of colistin resistance can not be always trusted; (ii) the *mcr1* gene is not the only one mechanism of resistance to colistin; (iii) *mcr1* variants may exist that can not be detected by the classical PCR.

Phenotypic assays like growth on colistin-containing agar plates can still represent a first base screening assay, although the MIC determination using the E test® confirms a >1mg/ml MIC for only 2 out of 3 growing isolates.

Presence of *mcr1* gene and putative variants (like the most recently described *mcr2* gene; Xavier BB et al., Eurosurveillance, 21, 7 July 2016) in all probe-positive isolates will be confirmed after Whole Genome Sequencing that will also allow comparing the *mcr1*-positive plasmids and isolates from pigs and cattle to similar human *E. coli* isolates. Further studies should also be performed to identify the colistin resistance mechanism in *mec*-negative isolates.

**Keywords:** Escherichia coli; colistin; antibioresistance; bovine; porcine

<footnote n="1">¹ Bacteriology, Department of Infectious and Parasitic Diseases, FVM and FARAH, ULg, Liège</footnote>

<footnote n="2">² Association Régionale de Santé et d'Identification Animales (ARSIA) - Allée des Artisans 2 5590 Ciney - Belgium</footnote>

<footnote n="3">³ DGZ Vlaanderen, 8820 Torhout</footnote>

<footnote n="4">⁴ Dienst Voedselpathogenen, Operationele Directie Overdraagbare en Besmettelijke Ziekten, WIV-ISP, 1050 Bruxelles</footnote>
Posters

# Isolation of bacteriophages against Klebsiella pneumoniae and in vivo activity

Damien Thiry<fnref n="1" />, Virginie Passet<fnref n="2" />, Nicolas Dufour<fnref n="3" />, Luisa De Sordi<fnref n="3" />, Jacques Mainil<fnref n="1" />, Laurent Debarbieux<fnref n="3" />, Sylvain Brisse<fnref n="2" />

<figure><img src="image_52.png" /></figure>

damien.thiry@ulg.ac.be

Klebsiella pneumoniae is a bacterial pathogen able to induce severe healthcare-associated or community-acquired infections in humans and animals. The constant emergence of antibiotic resistant strains reinforces the need to find alternatives to antibiotic treatments. The use of bacteriophages is a promising approach. The aim of this study was to isolate bacteriophages directed against K. pneumoniae strains and to test their efficacy in a murine model.

Bacteriophages against five different K. pneumoniae (2 of capsular type K1 and K2 and 1 undetermined) were isolated and purified from waste water collected in Paris area. The morphology of plaques (zones of bacterial killing) was recorded and several of them were purified three times by successive replating. Phage titers were determined by serial dilutions on their respective hosts as well as on 18 other Klebsiella strains to identify their host range. Kinetics of bacterial lysis were monitored during 15h at 3 multiplicities of infection, in triplicates. For in vivo experiment, a total of 10 mice were inoculated with 200 µl of K. pneumoniae (4.6E+07 CFU) by oral gavage and the level of K. pneumoniae in fecal samples was monitored for 10 days. Five mice did not receive any treatment and 5 other mice received a cocktail of three bacteriophages (8E+07 PFU) at day 4 post-inoculation.

A total of 54 bacteriophages were isolated and purified with titers ranging from 2E+5 to 3.6E+10 PFU/ml. The host range study showed that bacteriophages against K. pneumoniae have a specificity related to the capsular type of their bacterial host. Lysis kinetics of bacteria suggested that different phages were isolated. Despite difficulties with the murine intestinal model, evidence was obtained that bacteriophages are able to reduce intestinal carriage.

Our results show that bacteriophages isolated against K. pneumoniae are specific for a given capsular type, although further studies are necessary to provide more details on this capsular specificity and its molecular determinants. To fully address the in vivo potential of phages, a reliable mouse model of intestinal carriage of K. pneumoniae strains needs to be established.

**Keywords:** Klebsiella pneumoniae; Bacteriophage; murine model

<footnote n="1">¹ Bacteriology, Department of Infectious and Parasitic Diseases, FVM and FARAH, ULg, Liège</footnote>

<footnote n="2">² Microbial Evolutionary Genomics Unit - Institut Pasteur de Paris</footnote>

<footnote n="3">³ Molecular Biology of Gene in Extremophiles Unit - Institut Pasteur de Paris</footnote>