J C Audru
email: jc.audru@brgm.fr

J L Vernier

B Capdeville

J J Salindre

É Mouly

Natural Hazards and Earth System Sciences / Research

Keywords: seismic, preparedness, public, campaign, Antilles, Martinique, French Earthquake

Martinique is a French island in the Lesser Antilles, with a high seismic hazard. In 2006, Martinican stakeholders involved in seismic safety formed the "Réplik" working group ("Aftershock" in French), the first of its kind in this region. This paper addresses a mid-term appraisal of the first seismic awareness campaign organized by Réplik from 2006 to 2011, and how it has modified, or not, local earthquake and tsunami preparedness. Despite efforts from Réplik to assess its efficiency through surveys, a growing gap is noted between the observed awareness and the actual preparedness of the public. As usual, gender, age, educational level, then boredom and saturation contribute to this discrepancy; strong cultural items may also influence the perception of actions. To remain efficient and respond to public's expectations, Réplik must redirect its actions towards a cultural congruence of information: consideration of religion and local beliefs, comprehensive messages on TV and radio, use of Creole language, participatory experiences and drills, with a little bit of science. So that, the Réplik stakeholders can hope to increase Martinicans' involvement into the preparedness process, to cope quickly with a strong earthquake and this know-how can be shared with other seismically active islands in the Caribbean.

Introduction

Martinique Island is a French Overseas island of the Lesser Antilles archipelago, in the eastern Caribbean Sea. Martinique is located at a plate boundary (Fig. 1), above the North American plate subduction beneath the Caribbean plate at 2cm/yr. [START_REF] Lopez | Is there a northern Lesser Antilles forearc block?[END_REF]; it was formed over 25 million years by the joining together of several volcano complexes [START_REF] Westercamp | 1/50 000 geological map of Martinique and its notice[END_REF][START_REF] Germa | The volcanic evolution of Martinique Island: Insights from K-Ar dating into the Lesser Antilles arc migration since the Oligocene[END_REF]. This geodynamic position on top of a descending slab implies a high seismic hazard (Fig. 1): around 1 000 events are detected each year, located at the subduction interface, within the Atlantic slab and the deforming Caribbean plate [START_REF] Beauducel | Empirical model for rapid macroseismic intensities prediction in Guadeloupe and Martinique[END_REF] but only a very few of these events are felt. The French SisFrance catalogue of historical earthquakes (BRGM, 2009) illustrates several strong MSK intensity events that have hit the island in the past: 1727 (VIII), 1827 (VII), 1839 (IX), 1946 (VII-VIII), 1999 (Mb 5.5, VII) and the recent 2007 earthquake (Mw 7.4 and EMS98 int.VI-VII, see Fig. 2). Historical tsunamis are also reputed to have submerged the Atlantic coastline of Martinique (BRGM, 2010). These submersions had several origins [START_REF] O'loughlin | Carribbean tsunamis, a 500-Year History from 1498-1998[END_REF][START_REF] Lambert | Historical tsunami database for France and its overseas territories[END_REF]: the 1755 Lisbon earthquake, the 1767 Barbados earthquake, the 1867 Virgin Islands earthquake and the 1902 Mount Pelée volcanic eruption. There is still a high concern in Martinique within public services regarding an eventual close, strong earthquake rupturing the oceanic floor at the subduction front 250 km off the eastern coast of Martinique.

Mainland Martinique includes a 1128-km² mountainous island (Fig. 2). The highest point is Mount Pelée standing at 1397 m, a large explosive volcano which last erupted in 1929 and previously in 1902, destroying the then main city of Saint-Pierre. The current population of Martinique is around 403,000 inhabitants (INSEE, 2011), with one fourth living in or around Fort-de-France, the rest mostly in coastal towns (Fig. 2).

In 2005, France reviews its national seismic zonation. The highest peak ground acceleration of the territory is attributed to Martinique and Guadeloupe (PGA over 0,3g for a 475 yr.-return period). In 2007, the National Earthquake Plan is launched; it includes the Antilles Earthquake Plan, to dedicate specific actions to Martinique and Guadeloupe, owing to their high hazard level and island specificity. The objective was to reduce the vulnerability of people and buildings in the French West Indies.

The objective of this paper is to present a mid-term appraisal of this recent and short seismic educational campaign in Martinique. A number of original actions were carried out for the first time from 2006 to 2011, and annual surveys were used to check the assimilation of actions and how these modified, or not, the preparedness of the general public [START_REF] Audru | Seismic hazard status and mitigation actions in Guadeloupe and Martinique, French West Indies[END_REF]. Successes and failures Departmental Agency for Housing Information, the Departmental Fire and Rescue Service and several private consultants specialized in media, communication and social psychology. This diversity allows a wide array of sensibilities and ideas and favours the diffusion of messages agreed on by all partners.

Réplik's actions relating to earthquake and tsunami preparedness are characterised by events and innovations, taking place all year long but particularly in November (end of cyclonic season). These actions target residents (adults, pupils, employees, construction professionals etc.) and non-residents [START_REF] Audru | Los canales de información pública sobre el riesgo sísmico en Martinica, Antillas Francesas[END_REF].

Actions in public areas include travelling theatre skits (how to talk about seismic preparedness with humour in various situations of contemporary life, in Creole and French), a prevention caravan and the earthquake simulator of the General Council (which allows people to learn about earthquakes' origins and to experience shaking), scientific conferences about the effects and consequences of earthquakes and tsunamis, art exhibitions related to earthquakes, information stands in annual housing fairs or hardware stores, and participation in Carnival parades. The "Réplik for Companies" action is specifically dedicated to employees and is very successful. It consists in information which is given to employees in their workplace during working hours, which is of major importance.

Seismic preparedness presentations are given at the request of companies, and are adapted to workplaces and workmates with leaflets tailored specifically to companies' working practices then being distributed.

Actions also take place in schools. Booklets are adapted to pupils, preparedness and evacuation plans are carried out by teachers and pupils under the authority of the Local Education Authority. In five high schools, an original project ("seismometer at school") installed seismometers for educational purposes (www.edusismo.org). The seismometers monitor the local and worldwide seismicity; recordings are operated by pupils and transmitted for computing to seismologists. The signals, downloadable for all, are then used by pupils within the framework of their curriculum.

Various goodies are distributed to remind people how to behave in the event of an earthquake or evacuate if a tsunami occurs: tee-shirts, caps, whistles, magnets, posters and brochures drawn by children, mouse pads, etc. Leaflets with instructions for safety during an earthquake have been sent by post to all families in Martinique with electricity bills. Local TV channels, cinema or radio stations are also involved in the campaign; they broadcast short spots, reality shows and cartoons of families facing earthquake situations.

Tourists arriving to Martinique are informed through short notes regarding dos and don'ts during earthquakes and tsunamis, printed on free roadmaps which are offered by hotels, tourism information desks and rental cars.

With time, the Réplik leaflets and messages gradually benefit from a wider partnership and dissemination network, usually for free: buses, pharmacies, medical offices, insurance firms, post offices, petrol outlets, commercial centres, tourism offices etc. (Audru et al., 2011a). Examples of printed materials and media are shown in Fig. 3. Eventually, all advice, videos, leaflets, dos and don'ts related to seismic and tsunami safety from Réplik are compiled on a Martinique-specific website (www.replik972.fr). From August 2011 to July 2012, the site recorded 262,000 short hits and around 22,500 thirty-minute visits, which is actually not a high result.

On the neighbouring French Island of Guadeloupe, a website dedicated to the self-evaluation of (Bengoubou-Valerius, pers. Comm.). This confirms that in Martinique, many private buildings do not meet the mandatory seismic building rules in force since 1996 (French PS-92 codes), despite strong site effects have been evidenced since 1995 by microzonations [START_REF] Gagnepain-Beyneix | Experimental study of site effects in the Fort-de-France area (Martinique Island)[END_REF]Chassagneux et al., 1996 among others). This is why Réplik has also funded technical sheets and simplified building codes dedicated to traditional small-scale builders. As a complement, specific training courses are dedicated to masons regarding paraseismic building practices, based on recent and neighbours experiences [START_REF] Spence | Saving lives in earthquakes: successes and failures in seismic protection since 1960[END_REF][START_REF] Adams | The safer building programme in the UK Virgin Islands[END_REF]. However, despite their high interest, the courses do not appeal to lots of self-employed masons (100 a year), probably due to the perceived loss of time involved in attending. For public buildings, done and current seismic diagnoses underline a high level of vulnerability and a large number of retrofit and reconstruction programs have been implemented.

From a scientific point of view, the Antilles Earthquake Plan encourages applied scientific studies.

The regional evaluation of tsunami hazards is completed for the Caribbean French islands, using simulations based on the compilation historical scenarios [START_REF] Pedreros | Étude préliminaire de l'aléa tsunami aux Antilles françaises[END_REF]. Precise tsunami submersion maps are in project, to help municipalities to organize their evacuation plan.

Seismic microzonations are already in progress in several districts of Martinique following a homogeneous methodology [START_REF] Monge | Seismic risk assessment at both urban and regional scales in the French Lesser Antilles, methods and results[END_REF][START_REF] Vanoudheusden | Seismic microzonation of a municipality in French West Indies[END_REF], as well as a predictive earthquake-induced damage evaluation for the whole island [START_REF] Belvaux | Cartographie départementale du risque sismique en Martinique[END_REF]. In 2009, the SeisMCaRe® (Seismic Mitigation in the Caribbean Region) symposium (www.seismcare.com) gathered together twenty Caribbean countries in order to share know-hows, successes and failures in mitigation and education experiences between Caribbean neighbours.

Alarming results obtained from initial simulations of strong earthquake consequences [START_REF] Monge | Seismic risk assessment at both urban and regional scales in the French Lesser Antilles, methods and results[END_REF] encouraged the authorities to organize, within the AEP framework, a wide simulation drill in 2008 which took place simultaneously in Martinique and Guadeloupe. The "Richter Antilles" drill lasted 24 hours and tested several topics [START_REF] Cova | Earthquake crisis simulation exercise in the French Antilles: methods and experience feedback[END_REF] such as the coordination of rescue services, management dysfunctions (population, lifelines, and casualties), national reinforcements, and deceased people management. The drill marked a turning point for such simulation exercises in France [START_REF] Winter | Méthodologie pour la réalisation d'un exercice de crise sismique de type Richter[END_REF].

Despite this wide array of preparedness actions from 2006 to 2011, a question rapidly arises: what is the actual impact and effectiveness of this educational campaign among the population? The question is important, almost vital, given the historical records and the high seismic and tsunami hazard threatening Martinique.

The public's general perception of preparedness actions

Methodology

The perception of preparedness actions by the public does not benefit from a strong history and feedback. It is approached through the analysis of five short surveys conducted in Martinique in various conditions in 1999, then from 2006 to 2010 (Fig. 4). The 1999 survey was conducted by the University of the French West Indies after the 1999 earthquake; the last four were organized by a specialized polling company under Réplik request. The surveys structures remained quite simple;

detailed information about the data collection process, questionnaires and analysis is available from the author on request.

Results

The 1999 survey occurred within ten days following the 5.5 earthquake. The authors [START_REF] Léone | Le séisme du 8 juin 1999 de la Martinique, résultats de l'enquête postsismique sur les personnes se trouvant dans les bâtiments. Département GEODE, Note technique de l'Université des Antilles et de la Guyane[END_REF] telephoned 224 people who were standing in buildings during the event. The analysis of their answers outlined that 75% of those surveyed described inappropriate behaviour during the event and 64% after the event; later on, only 22% of those surveyed took preparedness measures. The people surveyed had then expressed high expectations in terms of information and preparation advice.

In 2006, a second survey was conducted among 334 Martinican visitors to the itinerant earthquake simulator. Colbeau-Justin et al. ( 2007) had outlined that most of the visitors (72.5%) proved interested in safety measures, especially in terms of technical aspects (49%) rather than scientific aspects; the latter being considered useless for individual protection. Women showed more interest (60%) in the correct behaviour to adopt during an earthquake, in the reliability of the home interior and in the preparation of survival kits, while men (40%) were interested in technical skills (construction, first aid certificates).

The survey also evidenced the public's preference for an immediate response organization (as opposed to post-crisis planning) via TV (65%), rather than through written brochures (31.5%), insets in newspapers, scientific conferences or neighbourhood meetings, which was indeed common to several countries [START_REF] Spence | Saving lives in earthquakes: successes and failures in seismic protection since 1960[END_REF]. On this basis, Colbeau-Justin et al. ( 2007) suggested that Martinicans preferred personal learning experiences and visual demonstrations, which explains the continuing success of the earthquake simulator and of the itinerant theatre. This use of a few media for addressing prevention actions is also preferred in Turkey for instance [START_REF] Tekeli-Yeşil | Earthquake awareness and perception of risk among the residents of Istanbul[END_REF].

The male-female dichotomy is observed elsewhere [START_REF] Mulilis | Gender and earthquake preparedness. A research study of gender issues in disaster management: differences in earthquake preparedness due to traditional stereotyping or cognitive appraisal[END_REF]; [START_REF] Solberg | The social psychology of seismic hazard adjustment: reevaluating the international literature[END_REF], from a detailed lecture of the related psychological literature, suggest that experience, gender and also age can shape risk perception and thus a campaign's successes or failures. The above analysis encouraged Réplik to prepare TV spots and cartoons featuring a woman preparing her family for a potential earthquake.

The 2007 survey followed the 7.4 earthquake (which occurred one month before). This poll (computer-assisted telephone interviews) of 1050 Martinicans (Ipsos Antilles, 2008) aged over 15 who felt the earthquake, revealed that 63% of people judged themselves sufficiently informed, while 68% had heard about Réplik's actions. The analysis showed that 62% knew how to behave during the earthquake and 70% believed they had appropriate behaviour during the event. Indeed, only 20% of people had immediately exited buildings, 21% moved away from building facades, 19% had listened to the radio and 2-3% had cut electricity and gas. On the contrary, 42% of people inside buildings had inadequate responses: 41% used stairs to exit buildings during the shaking, 38% remained inside buildings after the first shock, 39% used a cellular phone and 36% entered back into the buildings shortly after the main shock. More educated people and those younger than 36 were much more aware of the reality of risk (89%) than elderly or less educated people (69%), who were less affected by communication campaigns. The survey showed that appropriate instructions given by Réplik were mostly known, but were rarely enforced. People's knowledge was affected by surprise, fear and even panic for some, preventing them from behaving in an appropriate way (Ipsos Antilles, 2008). Finally, only 3 to 18% of Martinicans, mainly those under the age of 36, spontaneously said they would enhance their preparedness, these values went up to 71% when people were told what sort of action to take (survival kits, home security, seismic drills etc.).

In 2009, a fourth survey (Ipsos Antilles, 2009) tested the response to actions developed through Réplik in 2008, in order to assess the impact of the modified campaign. The survey of 503 people living in Martinique (computer-assisted telephone interviews) showed that awareness of Replik increased to 79% of the public, especially among women. This was mainly due to the strong emphasis placed on TV cartoons (seen by 79.9%) and radio spots (heard by 64.6%) with the involvement of local popular personalities (singers, writers, the archbishop etc.) and of the prevention caravan (9%). The perception of dos and don'ts in building codes increased in up to 54% of the public. TV and radio remained the best dissemination channels even if their audience decreased by 10% compared to 2007. The toll-free information phone number got very few calls (1%) and was then abandoned. This survey revealed that the 2007 earthquake experience increased preparedness for a while, as observed in other studies [START_REF] Dooley | Earthquake preparedness: predictors in a community survey[END_REF][START_REF] Nguyen | Exploring the causal relationship between exposure to the 1994 Northridge Earthquake and pre-and post-earthquake preparedness activities[END_REF], but with variable impact, may be depending on 2007 material losses, as observed elsewhere [START_REF] Lindell | Household adjustment to earthquake hazard: a review of the research[END_REF], or most probably on individuals' unrealistic estimations of their own ability to cope with the consequences of an earthquake [START_REF] Colbeau-Justin | How to deal with the safety needs of people at seismic risk?[END_REF].

The last Réplik survey occurred in 2010 (Ipsos Antilles, 2010). This poll (computer-assisted telephone interviews) of 509 Martinicans outlined a decrease in Réplik awareness to 74% of respondents (Fig. 4). The participation in prevention actions decreased considerably with the exception of the theatre skit (11%), illustrating either a good knowledge of correct behaviours or saturation or even public boredom regarding preparedness actions (Audru et al., 2011a). Respondents found the actions' content less relevant than previous years. The TV (79%) and the radio (49%) remained the best communication vectors, far above printed materials (11%) and internet information channels (3%). This latter information highlights the difference of culture to cope with, in earthquake prevention, with internet being a much more promising dissemination technique for Turk people for instance [START_REF] Tekeli-Yeşil | Earthquake awareness and perception of risk among the residents of Istanbul[END_REF]) than for Martinicans. The observed decrease in people's interest through this survey suggests that inadequate actions and messages were presented at some time during the 2010 Réplik campaign: these factors decrease people's knowledge [START_REF] Johnston | Measuring Tsunami Preparedness in Coastal Washington, United States[END_REF], as does the frequency of the messages, [START_REF] Mileti | The causal sequence of risk communication in the Parkfield earthquake prediction experiment[END_REF] which can alter risk awareness and contribute to the persistence of inadequate behaviour.

A parallel survey conducted through the housing self-evaluation website confirmed that in both Guadeloupe and Martinique, 75% of people know the dos and don'ts regarding earthquake safety; however, only 20% say that they are "earthquake-ready" and 32% have prepared a survival-kit (Bengoubou-Valerius, pers. comm.). These values are close to the 2008 post-earthquake survey and may reflect an inappropriate self-estimation of preparedness.

Discussion

Martinican respondents to the surveys generally have a high level of knowledge about the possible occurrence of strong earthquakes or tsunamis. As an encouraging result, Réplik's actions have significantly raised an interest and its set points were known by 80% of the public in Martinique in 2010. However, the surveys outline a discrepancy between those who are aware and those who actually prove able to cope with earthquake or tsunami consequences. Considerable efforts must still be made to reinforce people's involvement. Which hypotheses can be made to explain the obstacles and what are the perspectives?

A first hypothesis is based on daily life in Martinique, which is strongly influenced by fatalism. It is anchored in religion but also in magical beliefs or superstitions which have been inherited from the population's African, European and Amerindian origins [START_REF] Léti | L'univers magico-religieux antillais, ABC des croyances et des superstitions d'hier et d'aujourd'hui[END_REF]: "Quimbois", for instance, comprises practices related to magic and sorcery very similar to "Voodoo" in Haiti [START_REF] Revert | La magie antillaise[END_REF].

Indeed, beliefs like "earthquakes occur during the hot season", "talking about earthquakes makes them occur", "the island will sink" and "the island will be cut in half" are still common [START_REF] Léone | Le séisme du 8 juin 1999 de la Martinique, résultats de l'enquête postsismique sur les personnes se trouvant dans les bâtiments. Département GEODE, Note technique de l'Université des Antilles et de la Guyane[END_REF][START_REF] Sarant | Satisfaction résidentielle et perceptions du risque sismique chez les résidents d'habitat social en Guadeloupe, DDE Guadeloupe, Secteur pilote d'innovation outre-mer[END_REF][START_REF] Colbeau-Justin | Le risque sismique en Martinique, de sa perception à son intégration dans des pratiques préventives[END_REF]. Regarding religion, up to 13% of people interviewed for the 2008 survey attribute a divine origin to earthquakes, mainly those over 55 years of age and people not having been to high school (Ipsos Antilles, 2008). Magical beliefs and religion, as an important element of the Martinican culture, have not been explored through surveys yet. At this stage, but no one knows to what extent yet, one can make the hypothesis that such popular beliefs and religion decrease the actual perception of the threat by misrepresenting the actual consequences of an earthquake in Martinique, as observed in other regions [START_REF] Turner | Waiting for Disaster: Earthquake Watch in California[END_REF]. It implies that nothing can be done to protect oneself and it clearly influences individual's involvement;

this inhibits or slows down citizens' engagement in preventative practices and preparedness actions [START_REF] Turner | Waiting for Disaster: Earthquake Watch in California[END_REF][START_REF] Paton | When good intentions turn bad: Promoting natural hazard preparedness[END_REF]. This is probably the reason why, in survey answers, the possible consequences of earthquakes are never clearly described by people and therefore are not clearly anticipated and, even less, prepared for. These cultural and religious considerations have to be much more included in the next Réplik phase, in order to increase the effectiveness of prevention [START_REF] Chester | Theology and disaster studies: The need for dialogue[END_REF][START_REF] Chester | The importance of religion in shaping volcanic risk perception in Italy, with special reference to Vesuvius and Etna[END_REF]. [START_REF] Solberg | The social psychology of seismic hazard adjustment: reevaluating the international literature[END_REF] argue that the preparedness sources of information have to be strongly culturally congruent with the general public in order to be fully trusted and accepted. A role may be given to the clergy (Chester, 1985) to help overcome the perception of the divine and inevitable consequences of earthquakes and tsunamis. The inclusion of much more local culture into the preventative actions of an efficient preparedness plan is also supported by [START_REF] Tanaka | The impact of disaster education on public preparation and mitigation for earthquakes: a cross-country comparison between Fukui, Japan and the San Francisco Bay Area, California, USA[END_REF] or either the United Nations (2005).

Another hypothesis is that messages are not correctly formatted. Martinican and Turkish surveys for instance [START_REF] Tekeli-Yeşil | Earthquake awareness and perception of risk among the residents of Istanbul[END_REF] highlight similar knowledge and preparedness attitudes according to socioeconomic factors and educational level. [START_REF] Tekeli-Yeşil | Earthquake awareness and perception of risk among the residents of Istanbul[END_REF] or [START_REF] Barooah | Capacity building for earthquake safety and mitigation through awareness and community education[END_REF] in India both favour preparedness programmes carried out by the media to target weaker people characterized by lower educational and socio-economic levels. [START_REF] Nathe | Public education for earthquake hazards[END_REF], then [START_REF] Olshansky | Making a difference: stories of successful seismic safety advocates[END_REF], give simple guides to reaching these ambitious objectives as part of an efficient long haul campaign: clear messages using common, comprehensive words, tailored for specific audiences, sent through modern media and a wide partnership network. An unusual approach is given by [START_REF] Paton | When good intentions turn bad: Promoting natural hazard preparedness[END_REF] who define three successive stages of preparedness: motivation to prepare, formation of intentions, and the conversion of intentions into actions. People progress to the next phase under relatively high (but appropriate, see [START_REF] Lamontagne | Earth scientists can help people who fear earthquakes[END_REF] levels of hazard anxiety. Specific strategies form a step from one stage to another, for example information targeted to a specific community, as cited by Tekeli-Yesil et al. (2011) above. [START_REF] Weiss | Les comportements de protection face au risque naturel: de la résistance à l'engagement[END_REF] promote the perspective of a more participatory communication for the prevention of natural hazards.

A third hypothesis states that science is not in a good position in the campaign. People show very little interest in science because it does not help them in practical prevention actions (rescue kits, furniture etc.). Despite the low levels of audience interest, science may still have a role to play.

According to [START_REF] Lamontagne | Earth scientists can help people who fear earthquakes[END_REF], seismologists should follow psychological courses to help and support the public's emotional reactions before or after an earthquake. Scientists should also participate in communication plans which include concrete facts about earthquakes, to anticipate the event and its associated anxiety [START_REF] Lamontagne | Seismologists can help attenuate postearthquake public vibrations[END_REF][START_REF] Mcclure | When earthquake damage is seen as preventable: attributions, locus of control and attitudes to risk[END_REF]. Science can at least promote up-to-date scientific ideas instead of fakes and rumours disseminated by local beliefs or the internet.

These hypotheses regarding local and cultural factors may explain the failures in Réplik's campaign.

Our set of observations, despite it is quite short in time and despite the surveys are simple, favours new prevention axes for Martinique. The second phase of the Antilles Earthquake Plan will begin in 2013 and will offer the opportunity to test these hypotheses and to redirect Réplik's actions.

First, a survey would specifically explore the weight of beliefs and religion in the representations of earthquakes and tsunamis in Martinique. This will allow a tailored response to Martinicans' demands and needs, in accordance with most recent UN advice (United Nations, 2005). The use of much more Creole language in media and messages could help overcoming the cultural beliefs, the barrier of fatalism and the disinterest in basic science. Such a redirection may help people to take an extra step towards the preparedness process of Paton and overcome their subjective representations.

Then emphasis should be put on the answer to growing demand for practical skills: individual short training courses for adults and pupils (first aid courses, appropriate behaviour at home, outside, at work, at the seaside etc.), home security (furniture organisation and securing, rescue kits for homes, work and cars etc.). TV and radio will be the privileged vectors.

In Martinique, the community network is very dense, due to the small space formed by the island and to the numerous interconnections between families. This community strength should be used to increase hazard preparedness, following [START_REF] Paton | Predicting community earthquake preparedness: a cross-cultural comparison of Japan and New Zealand[END_REF] who suggest that mainstream community activities can increase the likelihood of developing preparedness among neighbours. The organisation of participatory experiences (neighbourhood solidarity, earthquake simulators, seismic and tsunami drills in suburbs, towns or at work, etc.) is to be encouraged. The creation of a special day dedicated to an historical or recently felt earthquake would be another opportunity for community actions, following the model of "shakeout" drills (www.shakeout.org) initially organized by the Earthquake Country Alliance of California ( 2003).

On a technical point of view, the training courses and booklets for building professionals have to be strongly simplified for small builders or individual masons in order to attract more such artisans, using another similar experience [START_REF] Adams | The safer building programme in the UK Virgin Islands[END_REF]. Simplifications should help to avoid misunderstandings and comply with vernacular traditions [START_REF] Spence | Saving lives in earthquakes: successes and failures in seismic protection since 1960[END_REF], demonstrations of the techniques in the field would be extended to ensure a more successful course [START_REF] Leslie | Think before you build: an earthquake reconstruction project in Yemen[END_REF].

Conclusions

At the beginning of the Réplik campaign in 2006, public awareness was quite low in Martinique despite historical events and recent earthquakes. The Réplik actions significantly raised an interest and its set points are now known by most of Martinicans, especially through TV and radio which prove the most efficient vectors. However, despite efforts on the part of the Réplik group to assess the efficiency of educational actions, this paper outlines a growing gap between the observed awareness and the actual preparedness of the public. As usual, gender, age, educational level, boredom, saturation, but also Martinique's culture may explain this discrepancy.

To remain attractive and efficient and to respond to this public's expectations and needs, Réplik has to upgrade its appeal. An opportunity lies in the coming second phase of the Antilles Earthquake Plan, to anchor existing actions or successful overseas experiences much more into local culture: consideration of cultural beliefs and religion to maintain the congruence with information, use of Creole language, specific education to specific people, participatory experiences, seismic and tsunami drills,, with a little bit of science to maintain a moderate level of knowledge. This is thought to increase people's involvement in the construction of preparedness. Thus, one can hope that Martinique will rapidly be able to cope with a strong earthquake, and this know-how will benefit other seismically active islands in the Caribbean.

Figures

Fig. 1: Sketch-map of Martinique Island within the Caribbean-Atlantic plate-boundary setting. The main regional faults and earthquakes are illustrated. The large arrow represents the motion of the Atlantic plate relative to the Caribbean plate (from [START_REF] Lopez | Is there a northern Lesser Antilles forearc block?[END_REF].

Fig. 2 :

 2 Fig. 2: The Martinique mountainous topography culminates at Mount Pelée volcano (1397 m; 4 583 ft). The island is divided into 34 municipalities; Roman letters plotted into municipalities areas represent the EMS98 intensities evaluated after the M7.4, November 2007 slab earthquake (data from BCSF, 2008).

Fig. 3 :

 3 Fig. 3: Examples of Réplik information supports. A) The Réplik logo since 2006; B) Tsunami leaflet; C) Arts exhibition "haz'arts"; D) Video series featuring a Martinican family; E) Simplified technical brochure for construction; F) Magnet with dos and don'ts; G) Instructions poster drawn by children; H) theatre skit of "Tranblad" which means "shaking" in Creole (featuring an authentic childbirth during the 2007 earthquake).

Fig. 4 :

 4 Fig. 4: Evolution of awareness and of several preparedness topics among successive polls within the Martinique population from 1999 to 2010. A) Notoriety of Réplik actions; B) Perception of prevention and preparedness actions.

Acknowledgements

The authors wish to thank the French Ministry for Environment, the Martinique Directorate for Environment, Planning and Housing and the French Geological Survey (BRGM) for supporting and funding this paper. This study is a part of the long-term Réplik joint venture gathering Martinican communities and stakeholders; both are also thanked for their implication in seismic safety in Martinique. The authors are finally grateful to their referees, to D. Paton, K. Tanaka and S. Tekeli-Yeşil for their highly constructive comments on our initial manuscript. All cited open file BRGM reports are freely downloadable from www.brgm.fr and numerical matter from Réplik is available from the authors on request. The paper is dedicated to Jean-Pierre Doudic.

implemented and validated by Réplikthe French Geological Survey, the Civil Defence, the French Army, the Academy, the Seismological and Volcanological Observatory, the Architects Council, the


Leen Timbermont
email: leen.timbermont@ugent.be

Freddy Haesebrouck

Richard Ducatelle

Filip Van Immerseel

Necrotic enteritis in broilers: an updated review on the pathogenesis

Keywords: clostridium perfringens, necrotic enteritis, pathogenesis, broilers

published or not. The documents may come

Introduction

Enteric diseases are an important concern to the poultry industry because of production losses, increased mortality, reduced welfare of birds and increased risk of contamination of poultry products for human consumption. Necrotic enteritis was first described by [START_REF] Parish | Necrotic enteritis in fowl (Gallus Gallus domesticus). 1. Histopathology of the disease and isolation of a strain of Clostridium welchii[END_REF]. It is a widespread disease in broilers imposing a significant economic burden on the poultry industry worldwide. The total global economic loss as a consequence of necrotic enteritis outbreaks in broiler farms is estimated to be over 2 billion dollars annually (Van der Sluis, 2000a,b). The causative agent of necrotic enteritis is Clostridium perfringens, a Gram-positive spore-forming anaerobe.

Growth-promoting antibiotics have been banned from animal feed in the European Union and, worldwide, the use of antimicrobial growth promoters is being reduced because of concern for the spread of antimicrobial resistance. This has contributed to the higher prevalence of economically important diseases such as necrotic enteritis (Van Immerseel et al., 2009).

Clinical signs and lesions. Necrotic enteritis usually occurs in broiler chickens at about 4 weeks after hatching and is found in all poultry-growing areas of the world [START_REF] Long | Necrotic enteritis in broiler chickens. I. A review of the literature and the prevalence of the disease in Ontario[END_REF][START_REF] Dahiya | Potential strategies for controlling necrotic enteritis in broiler chickens in post-antibiotic era[END_REF]. It may present as acute clinical disease or be a subclinical condition.

Clinical necrotic enteritis.

The classical acute clinical form of the disease is characterized by a sudden increase in flock mortality, often without premonitory signs, although wet litter is sometimes an early indicator of disease. The course is often peracute, with death in 1 to 2 hours. Mortality rates may sometimes rise up to 50% [START_REF] Helmboldt | The pathology of necrotic enteritis in domestic fowl[END_REF][START_REF] Wijewanta | Bacteriological studies of fatal Clostridium perfringens type-A infection in chickens[END_REF]Kohler, 1973;[START_REF] Riddell | The influence of diet on necrotic enteritis in broiler chickens[END_REF].

Subclinical form of Clostridium perfringens infection. In the past few years, a subclinical form has become more prevalent. In this form of necrotic enteritis, no overt clinical signs are present and usually there is no peak mortality. Chronic intestinal mucosal damage leads to production losses due to poor digestion and absorption, reduced weight gain and increased feed-conversion ratio [START_REF] Elwinger | Factors affecting the incidence of necrotic enteritis, caecal carriage of Clostridium perfringens and bird performance in broiler chicks[END_REF][START_REF] Kaldhusdal | Reduced incidence of Clostridium perfringens-associated lesions and improved performance in broiler chickens treated with normal intestinal bacteria from adult fowl[END_REF]. During subclinical infection, the intestinal damage can allow the bacteria to reach the bile duct and portal blood stream. Colonization of the liver by high numbers of C. perfringens results in cholangiohepatitis. Diseased livers are enlarged and have a pale appearance with red or white foci [START_REF] Onderka | Fibrosing cholehepatitis in broiler chickens induced by bile duct ligations or inoculation of Clostridium perfringens[END_REF]Løvland & Kaldhusdal, 1999;[START_REF] Sasaki | Cholangiohepatitis in broiler chickens in Japan: histopathological, immunohistochemical and microbiological studies of spontaneous disease[END_REF]. Liver lesions are found during meat inspection at slaughter, often without any sign of clinical disease in the flock. This increases the number of condemnations at processing. Taken together, there is a general consensus that, although clinical outbreaks of necrotic enteritis may cause high levels of mortality, the subclinical form of the disease is more important than the clinical form because it may persist in broiler flocks without overt clinical manifestations. Since the disease is undetected and birds remain untreated, subclinical necrosis causes the greatest economic losses in the poultry production industry (Kaldhusdal & Hofshagen, 1992;[START_REF] Dahiya | Potential strategies for controlling necrotic enteritis in broiler chickens in post-antibiotic era[END_REF].

Intestinal pathology.

Gross lesions are usually restricted to the small intestine, but lesions can also occur in other organs, such as liver and kidney. Upon necropsy, the duodenum, jejunum and ileum are usually thin walled and filled with gas. Clinical necrotic enteritis is characterized by confluent mucosal necrosis of large parts of the small intestine, covered with 1A) (Helmbold & Bryant, 1971;[START_REF] Long | Necrotic enteritis in broiler chickens. II. Pathology and proposed pathogenesis[END_REF][START_REF] Broussard | Necrotic enteritis in cagereared commercial layer pullets[END_REF][START_REF] Olkowski | Responses of broiler chickens orally challenged with Clostridium perfringens isolated from field cases of necrotic enteritis[END_REF]. Typical subclinical cases have ulcers in the form of a depression in the mucosal surface, with discolored, amorphous material adhering to the mucosal surface (Figure 1B) (Kaldhusdal & Hofshagen, 1992).

Microscopic examination of early stages of necrotic enteritis show strong inflammatory reactions to C. perfringens. The lamina propria is hyperaemic and infiltrated with numerous inflammatory cells, mainly heterophilic granulocytes. Most significant early changes are seen at the interface of the basal domain of enterocytes and lamina propria. These areas are extensively oedematous, allowing for the substantial disturbance of the structural integrity between the lamina propria and the enterocytes [START_REF] Olkowski | Responses of broiler chickens orally challenged with Clostridium perfringens isolated from field cases of necrotic enteritis[END_REF]. Microscopic examination of later stages of necrotic enteritis lesions show diffuse and severe coagulative necrosis of the mucosa, involving the luminal third to half of the mucosa. In these areas necrosis of enterocytes is apparent on the villi. A clear line of demarcation between necrotic and viable tissue and an accumulation of heterophilic granulocytes at the junction is seen. If present, the pseudomembrane consists of masses of tissue fragments, necrotic cells, cell debris, and numerous bacterial colonies suspended in mucus. Congestion of blood vessels is detected in the lamina propria and submucosa. Large, Gram-positive rods are associated with areas of necrosis but do not invade the epithelium or are not found to be attached to the viable mucosal epithelial cells, despite their massive presence on the tissue debris in the lumen [START_REF] Helmboldt | The pathology of necrotic enteritis in domestic fowl[END_REF][START_REF] Long | Necrotic enteritis in broiler chickens. II. Pathology and proposed pathogenesis[END_REF][START_REF] Al-Sheikhly | Role of coccidia in the occurrence of necrotic enteritis of chickens[END_REF][START_REF] Broussard | Necrotic enteritis in cagereared commercial layer pullets[END_REF][START_REF] Olkowski | Responses of broiler chickens orally challenged with Clostridium perfringens isolated from field cases of necrotic enteritis[END_REF]. enteritis. Thus, wheat, rye, oats and barley are risk factors for necrotic enteritis, whereas maize is not [START_REF] Branton | Influence of a wheat diet on mortality of broiler chickens associated with necrotic enteritis[END_REF]Hofshagen & Kaldhusdal, 1992;Kaldhusdal & Hofshagen, 1992;[START_REF] Kaldhusdal | Association between cereal contents in the diet and incidence of necrotic enteritis in broiler chickens in Norway[END_REF][START_REF] Riddell | The influence of diet on necrotic enteritis in broiler chickens[END_REF][START_REF] Craven | Colonization of the intestinal tract by Clostridium perfringens and fecal shedding in diet-stressed and unstressed broiler chickens[END_REF][START_REF] Jia | Effect of diet type and enzyme addition on growth-performance and gut health of broiler chickens during subclinical Clostridium perfringens challenge[END_REF]. Some of these effects may be related to differences in digesta viscosity, decreased nutrient digestibility and prolonged intestinal transit time [START_REF] Choct | Increased small intestinal fermentation is partly responsible for the anti-nutritive activity of non-starch polysaccharides in chickens[END_REF]. High dietary concentrations of animal protein, such as fishmeal, have also been reported to increase the incidence of necrotic enteritis (Truscott & Al-Sheikhly, 1977;[START_REF] Drew | Effects of dietary protein source and level on intestinal populations of Clostridium perfringens in broiler chickens[END_REF][START_REF] Gholamiandehkordi | Quantification of gut lesions in a subclinical necrotic enteritis model[END_REF]. In general, protein-rich diets containing relatively high concentrations of poorly digestible proteins lead to high concentrations of protein in the gastrointestinal tract and thus act as substrates for the bacteria.

The dietary fat source may also have an effect on the C. perfringens population.

Animal fat increases C. perfringens counts compared to vegetable oil [START_REF] Knarreborg | Effects of dietary fat source and subtherapeutic levels of antibiotic on the bacterial community in the ileum of broiler chickens at various ages[END_REF]. Even the physical form of the feed may influence the incidence of necrotic enteritis.

Feed containing some large-sized and many small-sized particles predispose more to necrotic enteritis than feed containing particles uniform in size [START_REF] Branton | Influence of a wheat diet on mortality of broiler chickens associated with necrotic enteritis[END_REF][START_REF] Engberg | The influence of grinding and pelleting of feed on the microbial composition and activity in the digestive tract of broiler chickens[END_REF].

Stress.

Apart from the feed, any factor that causes stress in broiler chickens could predispose them to necrotic enteritis because it could alter the intestinal environment in such a way that the risk of induction of necrotic enteritis is elevated [START_REF] Mcdevitt | Necrotic enteritis; a continuing challenge for the poultry industry[END_REF]. Programmed alterations in the feeding regime (moving from starter diets to grower diets), are frequently associated with necrotic enteritis. Furthermore, immunosuppressive agents such as chick anaemia virus, Gumboro disease or Marek's disease, reduce resistance to gut infections and may increase the severity of disease. Also increases in stocking density predispose to necrotic et al., 2006). However, the latter is restricted in the European Union by legislation to 33 kg/m² and can be, under certain conditions, raised to 42 kg/m² (directive 2007/43/EG).

Coccidiosis. The best-known predisposing factor for necrotic enteritis is mucosal damage caused by coccidial pathogens (Williams, 2005). Coccidiosis is often seen to precede or occur concurrently with field outbreaks of necrotic enteritis [START_REF] Long | Necrotic enteritis in broiler chickens. I. A review of the literature and the prevalence of the disease in Ontario[END_REF][START_REF] Broussard | Necrotic enteritis in cagereared commercial layer pullets[END_REF][START_REF] Gazdzinski | Necrotic enteritis in turkeys[END_REF][START_REF] Porter | Bacterial enteritides of poultry[END_REF]. Moreover, it has been shown in experimental infection studies that C. perfringens and Eimeria spp. act synergistically in inducing necrotic enteritis lesions. Co-infection with C. perfringens and Eimeria oocysts or an overdose of commercial coccidiosis vaccines containing attenuated Eimeria strains result in more animals with lesions or in higher mortality rates compared with birds receiving only Eimeria or only C. perfringens [START_REF] Al-Sheikhly | Role of coccidia in the occurrence of necrotic enteritis of chickens[END_REF][START_REF] Shane | Etiology and pathogenesis of necrotic enteritis[END_REF][START_REF] Baba | Clostridial population and the intestinal lesions in chickens infected with Clostridium perfringens and Eimeria necatrix[END_REF][START_REF] Gholamiandehkordi | Quantification of gut lesions in a subclinical necrotic enteritis model[END_REF][START_REF] Park | Immunopathology and cytokine responses in broiler chickens coinfected with Eimeria maxima and Clostridium perfringens with the use of an animal model of necrotic enteritis[END_REF][START_REF] Pedersen | Reproducible infection model for Clostridium perfringens in broiler chickens[END_REF]. Eimeria parasites colonize the small intestine and kill epithelial cells as a consequence of the intracellular stages of their life cycle (Williams, 2005). Plasma proteins leak into the gut lumen through the resulting gaps in the epithelial lining of the intestinal lumen, and these can be used as growthsubstrate by C. perfringens strains [START_REF] Van Immerseel | Clostridium perfringens in poultry: an emerging threat of animal and public health[END_REF]. Moreover, coccidial infection induces a T-cell mediated inflammatory response that enhances intestinal mucogenesis. This enhanced mucin production provides a growth advantage to C. perfringens due to its ability to use mucus as a substrate [START_REF] Collier | Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth[END_REF].

Presence of poultry pathogenic strains. The intestine of birds suffering from necrotic enteritis contains large numbers of C. perfringens organisms, up to 10 6 -10 8 cfu/g of the intestinal contents, whereas in healthy broilers, counts from 0-10 5 cfu/g of the intestinal [START_REF] Long | Necrotic enteritis in broiler chickens. II. Pathology and proposed pathogenesis[END_REF][START_REF] Baba | Clostridial population and the intestinal lesions in chickens infected with Clostridium perfringens and Eimeria necatrix[END_REF][START_REF] Si | Quantification of cell proliferation and alpha-toxin gene expression of Clostridium perfringens in the development of necrotic enteritis in broiler chickens[END_REF]. However, the presence of C. perfringens in the intestinal tract of broiler chickens, even at high numbers, is not sufficient to produce necrotic enteritis [START_REF] Long | Necrotic enteritis in broiler chickens. III. Reproduction of the disease[END_REF][START_REF] Cowen | Experimentally induced necrotic enteritis in chickens[END_REF]1987, Kaldhusdal et al., 1999;;[START_REF] Craven | Colonization of the intestinal tract by Clostridium perfringens and fecal shedding in diet-stressed and unstressed broiler chickens[END_REF][START_REF] Pedersen | Experimental infections with rifampicin-resistant Clostridium perfringens strains in broiler chickens using isolator facilities[END_REF][START_REF] Nauerby | Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens[END_REF]. Therefore, C. perfringens cell counts alone have not been directly associated with necrotic enteritis. Indeed, not all C. perfringens strains are able to induce necrotic enteritis; they need to possess host specific virulence factors in order to be pathogenic for poultry.

It is pertinent to note that C. perfringens is auxotrophic for 13 amino acids [START_REF] Shimizu | Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater[END_REF][START_REF] Myers | Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens[END_REF]. An increase in available nutrients would thus allow C. perfringens to proliferate extensively, leading to increased intestinal toxin levels. It can thus be speculated that when the conditions for inducing lesions are present, a higher number of bacteria might result in more severe lesions but only when poultry pathogenic strains are present. Predisposing factors are thus important but it is essential that a poultry pathogenic strain is present to induce disease.

Bacteriocins. Bacteriocins may have an important role in the pathogenesis of necrotic enteritis. Genetic characterization by PFGE or amplified fragment length polymorphism (AFLP) has revealed that in healthy flocks, different genotypes of C. perfringens type A can be found, even within individual birds and within the same gut segment. In contrast, isolates from a flock with necrotic enteritis are generally clonal within one flock, regardless of the animal or the organ of isolation [START_REF] Engström | Molecular typing of isolates of Clostridium perfringens from healthy and diseased poultry[END_REF][START_REF] Nauerby | Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens[END_REF][START_REF] Gholamiandehkordi | Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status[END_REF]. These results were confirmed by multilocus sequence typing analysis (MLST) [START_REF] Chalmers | Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations[END_REF]. After natural recovery or treatment, birds again start carrying multiple genetic types [START_REF] Nauerby | Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens[END_REF]. The fact that this single strain dominance has been described in several studies indicates that it is an essential step in [START_REF] Barbara | Necrotic enteritis-producing strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks[END_REF]. Finally, the secretion of factors inhibiting growth of other C. perfringens strains is shown to be more prevalent in outbreak strains than in normal microbiota strains in vitro [START_REF] Barbara | Necrotic enteritis-producing strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks[END_REF]Timbermont et al., 2009b). Proteinaceous toxic compounds produced by bacteria that generally inhibit the growth of closely related strains are called bacteriocins [START_REF] Jack | Bacteriocins of Gram-positive bacteria[END_REF]. Taken together, these results suggest that this is a trait associated with virulence.

Although there is no information on in vivo bacteriocin production by C. perfringens, production of bacteriocins by a virulent strain in the gut would allow this strain to displace the C. perfringens strains present. Inhibiting the growth of other C. perfringens strains in the gut can also be important as part of the competition for nutrients.

Collagenolytic enzymes. [START_REF] Olkowski | Responses of broiler chickens orally challenged with Clostridium perfringens isolated from field cases of necrotic enteritis[END_REF][START_REF] Olkowski | Sub-clinical necrotic enteritis in broiler chickens: novel etiological consideration based on ultra-structural and molecular changes in the intestinal tissue[END_REF] suggested that the initial pathological changes are due to the activity of collagenolytic enzymes. It was shown that damage to the villi initially occurs at the level of the basement membrane and lateral domain of the enterocytes, spreading throughout the lamina propria, while epithelial damage occurs later in the process. The nature of the morphological changes indicates that the initiation of the pathological process leading to necrotic enteritis involves proteolytic factors affecting the extracellular matrix and cellular junctions. Indeed, in broilers undergoing necrotic enteritis, the extracellular matrix is disorganized and can even be completely absent. It was shown that thus suggested that the pathology may be the result of bacterial collagenases, whose action is enhanced when mucosal damage (e.g. induced by coccidia) is present, or of host matrix metalloproteinases that are activated by the host-pathogen interaction [START_REF] Olkowski | Sub-clinical necrotic enteritis in broiler chickens: novel etiological consideration based on ultra-structural and molecular changes in the intestinal tissue[END_REF].

Toxin production. For a long time, alpha toxin was proposed to be the main virulence factor for necrotic enteritis in poultry, although C. perfringens outbreak strains as well as normal broiler microbiota isolates are type A and thus express alpha toxin [START_REF] Nauerby | Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens[END_REF][START_REF] Gholamiandehkordi | Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status[END_REF]. Moreover, no apparent difference in the levels of alpha toxin was found when the alpha toxin production in vitro was compared between strains associated with necrotic enteritis and isolates derived from the microbiota of normal broilers [START_REF] Gholamiandehkordi | Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status[END_REF]). Yet another study found that the intestinal level of alpha toxin was not correlated with disease lesion scores [START_REF] Wilkie | The effect of henegg antibodies on Clostridium perfringens colonization of the gastrointestinal tract of broiler chickens[END_REF]. Finally, an alpha toxin null mutant, constructed from a virulent chicken isolate, was equally able to cause necrotic lesions in broiler chickens compared to the wild-type strain [START_REF] Keyburn | Alpha-toxin of Clostridium perfringens is not an essential virulence factor in necrotic enteritis in chickens[END_REF].

Another observation that argues strongly against the role of alpha toxin in necrotic enteritis is the massive heterophil, lymphocyte, and plasma cell infiltration in infected tissues (Al-Sheikhly & Truscott, 1977b;[START_REF] Shane | Etiology and pathogenesis of necrotic enteritis[END_REF]Gazdzinsky & Julian, 1992). In gas gangrene, a disease proved to be mediated by alpha toxin, marked leukostasis and lack of inflammatory infiltrate is commonly observed in the tissues infected by C. perfringens cells [START_REF] Flores-Díaz | Role of Clostridium perfringens phospholipase C in the pathogenesis of gas gangrene[END_REF]. Alpha toxin-negative mutants of C. perfringens are not able to cause gas gangrene in mice but do promote profound inflammatory responses [START_REF] Awad | Virulence studies on chromosomal alpha-toxin and theta-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of alpha-toxin in Clostridium perfringens-mediated gas gangrene[END_REF].

Thus, the massive immune-cell influx in necrotic enteritis lesions seems to be inconsistent with the known effects of alpha toxin on the innate immune system.

Recently, a novel toxin has been described that is associated with necrotic enteritis in broilers [START_REF] Keyburn | NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens[END_REF]. The C. perfringens necrotic enteritis B-like toxin (NetB) is a 11 member of the ß-barrel pore-forming toxin family. The toxin causes cell rounding and lysis in a chicken Leghorn male hepatoma cell line (LMH), and it was shown that NetB forms plasma membrane pores with an estimated pore diameter of 1.6 -1.8 nm [START_REF] Keyburn | NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens[END_REF]. [START_REF] Lepp | Identification of novel pathogenicity loci in Clostridium perfringens strains that cause avian necrotic enteritis[END_REF] found that the netB gene is located on an approximately 85 kb plasmid. The VirSR two-component signal transduction system regulates the expression of netB. The NetB toxin production is upregulated when the VirSR system is activated as a result of a high C. perfringens population density. This is an efficient environmental adaptation since NetB would only be produced when the C. perfringens concentration is high and there is an increased probability of subsequent nutrient limitation. Damage of the host cells caused by NetB might provide enough nutrients for C. perfringens [START_REF] Cheung | The VirSR two-component signal transduction system regulates NetB toxin production in Clostridium perfringens[END_REF].

A netB null mutant of C. perfringens failed to cause necrotic lesions in the gut of experimentally infected broilers, but a mutant complemented with the wild-type netB gene was as virulent as the wild-type strain [START_REF] Keyburn | NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens[END_REF]. Confirmation of the role of NetB in disease came from the finding that most necrotic enteritis outbreak strains carry the netB gene, whereas non-necrotic enteritis derived C. perfringens isolates lack this gene [START_REF] Keyburn | NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens[END_REF][START_REF] Keyburn | Association between avian necrotic enteritis and Clostridium perfringens strains expressing NetB toxin[END_REF]. This gene is only found in poultry C. perfringens strains, except for one isolate that was recovered from a cow (Martin & Smyth, 2009). Moreover, several groups screened C. perfringens isolates derived from different countries for the occurrence of netB. In a Canadian study, 95% (39/41) of C. perfringens isolates from broilers with necrotic enteritis were positive for netB while only 35% (7/20) isolates from healthy boilers were netB positive [START_REF] Chalmers | Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations[END_REF]. A screening of isolates from the US reported a carriage rate of 58% (7/12) for the necrotic enteritis isolates while only 8.75% (7/80) normal microbiota strains carried the netB gene (Martin and Smyth, 2009). Despite the fact that the netB gene is strongly associated with necrotic enteritis-derived strains, in every study a minor fraction of netB negative isolates from diseased birds and a 12 minor fraction of netB positive isolates from healthy birds are found. However, because necrotic enteritis is a multifactorial disease, the presence of netB positive strains without disease may be due to the lack of predisposing factors. The netB negative isolates from birds suffering from necrotic enteritis may indicate that other, yet to be determined virulence factors are produced by these strains. However, netB negative strains do not induce necrotic enteritis in experimental animal models. To determine if netB is essential for virulence, it is important to test the disease producing capability of both netB positive strains recovered from healthy chickens, and netB negative strains recovered from chickens suffering from necrotic enteritis. Recent studies, using a large number of NetB positive and NetB negative C. perfringens strains to inoculate broiler chickens, showed a perfect match between necrotic enteritis induction and the ability to produce NetB [START_REF] Keyburn | Association between avian necrotic enteritis and Clostridium perfringens strains expressing NetB toxin[END_REF]Smyth & Martin, 2010).

Adhesion. Recently, it has been shown that C. perfringens is capable of binding to extracellular matrix molecules (Martin & Smyth, 2010;[START_REF] Wade | Clostridium perfringens genes with implications for both virulence and colonization during necrotic enteritis[END_REF]. The ability to adhere to the host's intestinal epithelium and to extracellular matrix molecules (ECMMs) in the gut are strategies used by numerous bacterial enteropathogens. Although, ECMMs are not normally exposed on healthy intestinal epithelium, they can be exposed as a consequence of tissue damage by Eimeria species, which are an important predisposing factor for necrotic enteritis development, or by toxins and collagenolytic enzymes secreted by C. perfringens.

It has been shown that disease producing strains bind better to collagen type III and IV and fibrinogen than non-disease producing strains. It has also been shown that netB positive severe disease producing strains were capable of binding to collagen type III, IV, and V, fibrinogen, laminin and vitronectin at higher levels than netB positive mild disease producing strains. This suggests that the presence of netB, a critical factor in the development of necrotic enteritis, and the ability to adhere to ECMMs work synergistically (Martin & Smyth, 2010). This hypothesis is supported by the work of [START_REF] Wade | Clostridium perfringens genes with implications for both virulence and colonization during necrotic enteritis[END_REF]: a gene, strongly associated with disease-causing isolates, was mutated in a virulent strain background and the mutant showed an altered ability to bind to immobilized ECMMs in vitro. But more importantly, the isogenic mutant strain was less able to colonize the gut environment and displayed a seriously reduced virulence in a disease induction model. The ability to adhere to ECMMs thus has a significant role in the virulence and is an important factor for colonization of the gastrointestinal tract [START_REF] Wade | Clostridium perfringens genes with implications for both virulence and colonization during necrotic enteritis[END_REF].

General discussion.

After it was shown that alpha toxin is not an essential virulence factor in necrotic enteritis in broiler chickens [START_REF] Keyburn | Alpha-toxin of Clostridium perfringens is not an essential virulence factor in necrotic enteritis in chickens[END_REF]Timbermont et al., 2009a), new C. perfringens virulence factors have been proposed and new insight has been gained into the pathogenesis of C. perfringens induced necrotic enteritis. The current knowledge is summarized in Figure 2.

In short, Eimeria parasites induce leakage of plasma proteins by killing epithelial cells as a consequence of the intracellular stages of their life cycle. Moreover, according to [START_REF] Collier | Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth[END_REF], a coccidial infection enhances mucus production in the intestine. Both effects provide an increase in available nutrients and create an environment favourable for proliferation of C. perfringens. Indeed, C. perfringens lacks the machinery to produce 13 amino acids [START_REF] Shimizu | Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater[END_REF] but is able to utilize mucus as a substrate [START_REF] Collier | Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth[END_REF]. Taken together, Eimeria infection is an important predisposing factor as it may result [START_REF] Barbara | Necrotic enteritis-producing strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks[END_REF]Timbermont et al., 2009b). By inhibiting other C. perfringens strains, the virulent strains are able to obtain the maximum benefit of the increased nutrient availability due to the Eimeria infection. The virulent strain is thus able to proliferate massively and secretes virulence factors playing a role in the induction of necrotic lesions. [START_REF] Olkowski | Responses of broiler chickens orally challenged with Clostridium perfringens isolated from field cases of necrotic enteritis[END_REF]2008) suggested that the nature of the morphological changes seen in the early stages of necrotic enteritis is consistent with the action of collagenolytic enzymes. Indeed, the pathological changes start at the basal and lateral domains of enterocytes and then invade progressively the entire lamina propria. Both host collagenases and collagenolytic enzymes secreted by proliferating pathogens may play a role. It has been shown that virulent C. perfringens strains are able to secrete collagenolytic enzymes and high levels of different collagenolytic enzymes are found in intestinal tissue of broilers challenged with C. perfringens, in comparison to controls [START_REF] Olkowski | Sub-clinical necrotic enteritis in broiler chickens: novel etiological consideration based on ultra-structural and molecular changes in the intestinal tissue[END_REF]. Furthermore, the NetB toxin is crucial for the induction of avian necrotic enteritis [START_REF] Keyburn | NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens[END_REF]. It is, with one exception, only found in chicken C. perfringens isolates (Martin & Smyth, 2009). A 100% correlation has been found between NetB production and the ability to produce disease in a necrotic enteritis induction model [START_REF] Keyburn | Association between avian necrotic enteritis and Clostridium perfringens strains expressing NetB toxin[END_REF]Smyth & Martin, 2010). NetB forms pores in a chicken epithelial cell line in vitro. Its activity in vivo is not yet been shown, but it may induce necrotic lesions by forming pores in enterocytes leading to cell death [START_REF] Keyburn | NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens[END_REF][START_REF] Keyburn | Association between avian necrotic enteritis and Clostridium perfringens strains expressing NetB toxin[END_REF]. Whether this is involved in the early or later stages of disease progression is not clear. Because of the damage induced by the Eimeria parasites, the C. perfringens toxins and the collagenolytic enzymes, virulent C. perfringens strains are able to bind to exposed ECMMs, to colonize the intestine and thus to induce more (Martin & Smyth, 2010;[START_REF] Wade | Clostridium perfringens genes with implications for both virulence and colonization during necrotic enteritis[END_REF]. Not only an environment favourable for the growth of C. perfringens but also the presence of strains with host specific virulence factors are required to induce necrotic enteritis in broilers.

  brown or bile-stained pseudomembrane (Figure

  Predisposing factors. Nutrition. The key risk factor for the development of necrotic enteritis is an intestinal environment that favours growth of C. perfringens. The nature of the diet is an important non-bacterial factor that influences the incidence of necrotic enteritis. Diets with indigestible, water-soluble non-starch polysaccharides predispose to necrotic

  necrotic enteritis. Moreover, this dominance of one virulent C. perfringens isolate could be reproduced in an experimental infection model after administration of a combined inoculum comprising different necrotic enteritis strains or a combination of virulent and avirulent C. perfringens strains

C

  . perfringens strains isolated from field cases of necrotic enteritis secrete several potent collagenolytic enzymes and that broilers challenged with C. perfringens show elevated levels of several collagenolytic enzymes in the intestinal tissue in comparison to controls. It was

  of C. perfringens in the gut. C. perfringens not only produces toxins but also bacteriocins. A significantly higher proportion of virulent C. perfringens strains produce bacteriocins compared to normal microbiota C. perfringens strains

FiguresFigure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. Summary of the pathogenesis of C. perfringens induced necrotic enteritis.

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E-mail: cavanagh@metronet.co.uk URL: http://mc.manuscriptcentral.com/cavp Avian Pathology