S E Biotechnol.Agron.Soc.Environ.200812(3),317-334 Le
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ointsur:1.INTRODUCTION
Eventhoughpigmeat,withaworldwideshareofabout 50%, is by far the most preferred meat by European Union (EU) consumers, the poultry meat production has shown the more favourable progression, with a meanannualincreaserateof2.5%from1992to2002 (EuropeanCommission,2005);itrecordedaworldwide share of around 26% in 2005 (i.e. 70million tons). Moreover,worldpoultryproductionandconsumption arepredictedtostillincreaseoverthenextsevenyears by more than 20%, i.e. an average annual growth of approximately2.5%.Thisexpansionismainlydriven bylowpoultrymeatproductioncosts(relativetobeef and pork), strong consumer preference, increased use in food preparations and a high demand for low priceproteinsontheworldwidemarket.Furthermore, poultrymeathasgenerallybenefitedfromtheBovine
Spongiform Encephalopathy and Foot-and-Mouth Diseaseoutbreaks,inthepastfewyears.
Nevertheless,theaviansectorhasalsofacedseveral sanitaryproblemstowhichmediacoveragewasgiven since a few years. In June 1999, the dioxin crisis in Belgium was caused by dioxin-contaminated food components.Thewidespreadavianinfluenzaepidemic since 2003 has completely disrupted production and tradeinmanyareasoftheworld,notablySouthEast AsiabutalsotheUSandCanada.Besidethesetime-limitedoutbreaks,poultryproductionisconfrontedwith amajorpermanentproblemthatismuchlessknown. Poultry remains an important vehicle of bacterial human pathogens, leading to foodborne diseases by contaminated poultry products consumption and incriminated by epidemiological reports all over the world.ThemostreportedpathogenagentisSalmonella spp. but, over the last three decades,Campylobacter
Contaminationofpoultryflocksbythehumanpathogen
Campylobacter
spp.andstrategiestoreduceitsprevalence
atthefarmlevel
SabrinaVandeplas
(1),ChristopherMarcq
(1),RobinDuboisDauphin
(2),YvesBeckers
(1),
PhilippeThonart
(2),AndréThéwis
(1)(1)GemblouxAgriculturalUniversity–FUSAGx.UnitédeZootechnie.PassagedesDéportés,2.B-5030Gembloux (Belgium).E-mail:vandeplas.s@fsagx.ac.be
(2)GemblouxAgriculturalUniversity–FUSAGx.UnitédeBio-industries.PassagedesDéportés,2.B-5030Gembloux (Belgium).
ReceivedonJuly28,2007,acceptedonJanuary15,2008.
EntericCampylobacterspp.bacteriaarehumanpathogensthatfrequentlycontaminatepoultryflocks.Consumptionofproducts frompoultryoriginmaythenleadtoacutebacterialenteritiscalledcampylobacteriosisofwhichprevalenceisincreasingfor abouttenyearsinEurope.ThisreviewsummarizesCampylobacterepidemiologicaldata,riskfactorsforcontaminationin poultryflocksandconceivablestrategiestocontrolthispathogen.
Keywords.Campylobacter,epidemiology,poultry,prevalencereduction,prevention.
Contaminationdesélevagesdevolaillesparl’agentpathogènehumainCampylobacterspp.etlesstratégiespourréduire saprévalenceauniveaudesélevages.Campylobacterspp.estunebactérieentériquepathogènepourl’hommequicontamine fréquemmentlesélevagesdevolailles.Laconsommationdeproduitsd’origineaviairepeutainsientraînerunegastro-entérite bactérienne aiguë appelée campylobactériose, dont la prévalence augmente depuis une dizaine d’années en Europe. Cette synthèsebibliographiquerésumelesdonnéesépidémiologiquessurCampylobacter,lesfacteursderisquedecontamination danslesélevagesdevolaillesetlesstratégiesenvisagéespourluttercontrecepathogène.
spp.hasemergedasanincreasingconcernalloverthe world.Itisamajorcauseofahumanacutebacterial enteritis called campylobacteriosis (van Vliet etal., 2001). UnlikeSalmonella,Campylobacter is mainly aprobleminextensivepoultryproduction,withupto 100%oforganicfarmsbeingcontaminated(Engvall, 2001). This review will focus on prophylactic measuresandcurativetreatmentsdevelopedtoreduce the incidence ofCampylobacter infections in broiler flocks,attheprimaryproductionlevel.
2.CAMPYLOBACTERIOSIS
Campylobacter spp. have been recognized as a causeofdiarrhoealillnessinhumansince1972.The Campylobacterspeciesassociatedwithfoodpoisoning includeCampylobacterjejuni,Campylobactercoli, CampylobacterlariandCampylobacterupsaliensis.C. jejuniispredominantwhileC.coliaccountsformost oftheremainder(Hariharanetal.,2004).Accordingto aFrenchstudy,C.jejuniwasfoundinca.68%ofthe isolatesfromhumancampylobacteriosiscases(Dachet etal.,2004).
Dose-responsestudieshaveshownthatingestionof about10(Ridleyetal.,2004)to500cells(Rosenquist etal., 2003) could already be sufficient to infect the human host. Pathogens invade epithelial cells in the ileum and large intestine thanks to chemotaxis and high motility, which causes inflammatory diarrhoea withusuallymoderateuncharacteristicsymptoms(van Vlietetal.,2001).
ComplicationsfollowingCampylobacterinfection are uncommon, but an association with certain neurological disorders is noteworthy (Butzler, 2004). It is estimated that one on 1000Campylobacter infections lead to the Guillain-Barré syndrome, an acutedemyelinatingdiseasecharacterizedbymuscular paralysisandleadingto2-3%mortality(Allos,1997). This syndrome is usually confined to very young or elderlypatientsortoimmuno-compromisedsuffering people(Altekruseetal.,1999).
2.1.Publichealthimpact
Inmostindustrializedcountries,thereportedincidence of campylobacteriosis has increased during the last decade.In2004,atotalof183,961casesoflaboratory confirmed campylobacteriosis were recorded in the EU-25,comparedto120,462casesin1999.Theoverall incidence was 47.6 per 100,000 population, which is slightly higher than forSalmonella (42.2). This makesCampylobacter the most commonly reported gastrointestinal bacterial pathogen in humans in the EU (EFSA, 2006). On the other hand, in Belgium, Campylobacter infections represent the second cause
offoodborneillness,justafterSalmonella(CSH,2005), withanestimatedannualnumberofcasesofabout65per 100,000 population (Ducoffre, 2006). In 1984, the sentinel laboratories network recorded only just 1,703 cases of infection. During the nineties, campylobacteriosisincidencehascontinuallyincreased toreach7,473casesin2000,althoughtheincreasein the number ofCampylobacter infections cases until 1996 could mainly be attributed to problems at the surveillance level (van Dessel, 2005). From 2000 to 2003, the illness incidence was reduced. However, it tends to increase again since 2004, without reaching thelevelsobservedin2000.Itisusuallyestimatedthat 90%ofCampylobactercontaminationareduetomeat consumptionand80%specificallycomefrompoultry meat.Nevertheless,theriseofCampylobacterincidence observed for more than 20years may also be partly due to an increase of the poultry meat consumption duringthisperiod,ratherthanonlyanincreaseinthe proportionofcontaminatedpoultry(ICGFI,1999).
The high incidence of Campylobacter spp. diarrhoea, its duration and possible sequelae, make campylobacteriosis important from a socio-economic perspective.
2.2.Economicandsocialimportance
Campylobacteriosis affects each year a significant proportion of humans worldwide. Foodborne gastrointestinaldiseasesaremajorburdensonsociety causingconsiderablesufferingandlossofproductivity. Besides the discomfort felt by sick people, these infections have major economic repercussions by directillnesscosts(laboratorydiagnosis,consultations, medical cares, hospitalization, etc.) and indirect costs (work inefficacy, days lost work, etc.) (ICGFI, 1999;Bogaardtetal.,2004).InTheNetherlands,the economic costs of campylobacteriosis are estimated at21million€peryearforapopulationof16million (Mangen etal., 2005a). Costs for campylobacteriosis are difficult to estimate becauseof differences in the simulationmodelsused.Differencesinonecasecost according to two recent studies, i.e. 465€ in the United Kingdom (Roberts etal., 2003) and 77€ in TheNetherlands(vandenBrandhofetal.,2004)show thecomplexityofestimatingthesecosts.
3.CAMPYLOBACTERANDTHEANIMAL HOSTS
3.1.CharacteristicsofCampylobacterspecies
the cells can change to spherical or coccal forms. Their polar flagellum conferred them a characteristic darting,andcorkscrew-likemotility.Theyareunable to oxidize or ferment carbohydrates but they reduce nitrate and nitrite.C. jejuni is the most frequent of the four thermophilic Campylobacter species that isisolatedfromhuman,andisoneof20speciesand subspecies within the genus Campylobacter and familyCampylobacteraceae. The other thermophilic speciesincludeC.coli,C.upsaliensisandC.lari.The thermophilicspeciesarecharacterizedbytheirability togrowbestbetween37and42°Candtheirinabilityto growat25°C.Forthemostpart,Campylobacterrequire amicroaerobicatmosphereforgrowthandcanbevery difficulttoworkwithinlaboratorysettings,duetotheir fragilenature.However,isolatesareextremelydiverse, compared to some other enteropathogens. There are morethan60differentheat-stableserotypes,morethan 100heat-labile serotypes, differences in adherence properties, invasive properties, toxin production, serumresistance,colonizationpotential,aerotolerance andtemperaturetolerance.Thisdiversitymaybepartly explainedbythegenomicplasticityofCampylobacter. The high levels of multiple-strain colonization and high frequency of incidence in mammals and birds meanthereissubstantialopportunityforexchangeof geneticmaterialandexplaintheabilityofbacteriato surviveinextremeconditions.
3.2.Transmissionvectors
Campylobacter, as Salmonella, may be carried asymptomatically, as commensal organism, in the alimentary tract of all warm-blood animals. Because thispathogencanbetransferredfromanimalstoman, Campylobacter is considered as a zoonotic bacteria (WHO,2000).Humaninfectionmaybecausedbydirect contactwithcontaminatedanimalsoranimalcarcasses. In the case of domesticated animals as cattle, sheep, goats,pigsandespeciallypoultry,pathogenscanspread viatheslaughterprocesstorawandfinishedproducts. Campylobactermayalsobetransferredtohumansby consumptionofundercookedorrecontaminatedmeat, orthehandlingofrawproducts(Bryanetal.,1995).Itis noteworthythat,despitethemeatimportance,thisdoes notrepresenttheonlyfoodvehicleforCampylobacter and large campylobacteriosis outbreaks are usually associatedwithcontaminateddrinkingwaterorrawor contaminatedmilk(Friedmanetal.,2004).According to Mead etal. (1999), food contamination could originate for 80% ofCampylobacter infection cases. Regardinginter-humanstransmission,itisconsidered toberelativelyexceptional(Adaketal.,1995;Studahl etal.,2000;Winquistetal.,2001).
As mentioned above, the most important Campylobacterspeciesknowntocausehumanillness
are the thermophilic species:C. jejuni,C. coli and C. lari. Birds, especially breeding poultry, appear to be the main reservoir for these pathogens, their internaltemperatureof41-42°Cbeingfavourablefor thermophilicCampylobacter proliferation (Hariharan etal., 2004). Therefore, foods of poultry origin have been identified as significant sources of human campylobacteriosis. In Belgium, more than 40% of campylobacteriosiscaseswouldbeassociatedtopoultry meatconsumption(Vellingaetal.,2002).In1999,the finding of dioxin-contaminated feeding stuffs caused the Belgian authorities to withdraw all poultry meat andeggsfromthemarket.Theestimatedreductionin campylobacteriosis cases during the following crisis monthswas40%withoutanyotherexplicativeevent thathappenedinthisperiod.Furthermore,theBelgian poultryreintroduction4weekslateronthemarketlead back to the previous campylobacteriosis incidence situation.
Another factor that could link together chicken consumption and human pathogen acquisition is the important similarity between human and poultry serotypes (Petersen etal., 2001). Nevertheless, it is advisable to relativize this affirmation. Several studieshaveshownthatsomeCampylobacterstrains colonizing chicken are not human pathogens while some human isolated strains are unable to colonize poultry(Corryetal.,2001).
3.3.Poultrycolonisation
Colonized chickens usually show no observable clinical symptoms of infection even when young animalsareexposedtohighdosesunderexperimental conditions (Newell etal., 2003). Corry etal. (2001) reportedfurthermorepossibleobservationofenteritis andhepatitissymptomsorexcessivemortalityofvery youngchicks.
Experimentally, the dose of viable C. jejuni requiredtocolonizechicksandchickenscanbeaslow as40cfuevenifnumbersfrom104to107cfucanbe frequently found in literature (Udayamputhoor etal., 2003;Bjerrum,2005).Furthermore,astrainvariability concerningtheabilitytocolonizethechickendigestive tract is also reported (Stas etal., 1999). Infection pattern in poultry is also age-dependent. Actually, Campylobacterisnotdetectedinchickslessthan2to 3weeks of age under commercial broiler production conditions, and that may be related to high levels of circulatingCampylobacter-specificmaternalantibodies in young chickens, which gradually decrease to undetectablelevelsat2to3weeksofage(Sahinetal., 2003).
thegutandfromspleenandliver(Beeryetal.,1988; Achenetal.,1998).Themicroorganismremainsinthe intestinallumenatthecryptslevel,withoutadhesion. Once colonization is established,Campylobacter can rapidly reach extremely high numbers in the cæcal contents, from 105 to 109 cfu.g-1 of content (Schoeni etal.,1992;Achenetal.,1998;Woodalletal.,2005).
3.4.Poultryflockprevalence
The reported proportion of Campylobacter-positive broiler chickens flocks (the flock prevalence) varies betweencountries,rangingfrom5%tomorethan90% (EFSA,2005),assummarizedintable1.Thisapparent variationintheflockprevalencemayreflectsignificant differences between countries, but is affected by samplingtime,duringthebreedingperiod,andageand typeofbirds(conventional,free-range,organic).The method of detection (direct plating vs. enrichment), and type of sample (cæcal contents, fresh droppings, litter) also influence the detection ofCampylobacter spp.(Jørgensenetal.,2002;Oyarzabaletal.,2005).
Table2 shows more frequently contaminated broilersflocksinextensiverearingsystems,especially those allowing access of the birds to an open-air range (organic, “Label Rouge”, etc.). According to Heuer etal. (2001), the higher contamination rate of free-range broiler production could be explained by an unimpeded access to soil and water in the
open-air range, a longer rearing period and differences in chickenhostlineages.
Distribution of Campylobacter species is also dependentontherearingsystem,asshownintable2 and on the country.C. jejuni is the most frequently isolated species in poultry farms, whatever the productionsystem.C.coliislesscommonalthoughit ispredominantinsomeEUMemberStatesasSlovenia (Zormanetal.,2006).Moreover,thisspeciestendsto become more frequent from a few years (Desmonts etal.,2004).Finally,althoughrelativelyscarce,C.lari canalsobeisolatedfrompoultrysamples(Denisetal., 2001;Haldetal.,2001).
3.5.Riskfactorsforcontaminationatfarmlevel Althoughseveralriskfactorsforinfectionofbroilers with Campylobacter spp. have been identified,
Table1.Review ofCampylobactercontamination prevalence in broiler chickens—Synthèse de la prévalence de la contaminationparCampylobacterchezlepouletdechair.
Country Numberofanalysedflocks Contaminationrate Additionalinformation Reference
Denmark 4286(standard) 46% Oneyearstudy; Wedderkoppetal.,2001
cloacalswabs
Denmark 10(8standardpoultryfarms) 50% Cloacalswabsjust Haldetal.,2001
beforeslaughtering
Denmark 160(39farms)
standard:79 36.7% Studyleadingfrom Heueretal.,2001
organic:22 100.0% 1998to1999
extensiveindoor:59 49.2%
UnitedStates 3farmswithopen-airrange 32.0to68.0% McCreaetal.,2006
*«LabelRouge»:Frenchextensiverearingbroilerproductionwithaccesstoanopen-airrange—LabelRouge:productionextensive françaisedepouletsdechair,avecaccèsàunparcoursextérieur.
GreatBritain 100(standard) 4weeksoldbroilers: Evansetal.,2000
40%
7weeksoldbroilers:
>90%
France 24(standard) 79.2% Freshdroppingsfrom Denisetal.,2001
35to40daysofage
France 620ofwhich:
standard:403 56.6% Monitoringprogram Avrainetal.,2001 *«LabelRouge»:62 80.0% leadingin1999
export:155 51.3%
Table2.Campylobacterspecies distribution according to thepoultryproductionsystem—Distributiondesespèces deCampylobacter en fonction du système de production aviaire(Heueretal.,2001).
Poultryproduction Campylobacter Campylobacter
system jejuni coli
Standard 86.2% 10.3%
knowledge about the various routes by which flocks become infected and their relative importance is still incomplete.Theriskfactorsthathaverepeatedlybeen identifiedaresummarizedbelow.
VerticalTransmission.Campylobactercanbepresent in the poultry reproductive system. Nevertheless, several authors dismiss the assumption that vertical transmissionisamajorsourceofpathogentransmission (van de Giessen etal., 1992; Jacobs-Reitsma, 1995; Chuma etal., 1997; Sahin etal., 2003). The main reasons proposed are a poorCampylobacter survival oneggshellsandinabilitytopenetrate,tosurviveand tomultiplyintoeggsinnaturalconditions.Meanwhile, someevidencecouldbefoundforverticaltransmission ofCampylobacter(Callicottetal.,2006).
Horizontal transmission from the outer environment:
Flock-to-flock transmission and litter role. Campylobactertransmissionfromacontaminatedflock tothefollowingflockseemstobenotveryimportant. Campylobacterisactuallysensitivetodetergentsand disinfectants as well as dry conditions found in the poultry house during the empty period, although a little number of bacteria could survive during flocks intervals(Evansetal.,2000;Petersenetal.,2001).
Dry and aerobic conditions of clean fresh litter are considered harmful toC. jejuni as reported by Newell etal. (2003) and Hutchinson etal. (2005). On the other hand, litter can be contaminated by broiler fæcal droppings and then favours pathogen transmission through the flock. Nevertheless, in Belgium,theproblemoflitterascontaminationvector isnotrecognizedbecausehousesaregenerallycleaned anddisinfectedandthelitterisreplacedbetweentwo subsequentflocks.
Dirty contaminated litter spread over the land can scatter the microorganism in the environment. Contaminated sewage are attractive for wild birds and insects that can be infected and then become Campylobacter vectors (Jones, 2001; Stanley etal., 2003).
Environment and open-air range. Campylobacter is able to survive in the house surroundings soil (Bull etal., 2006) and the farmer can therefore act as a pathogen vector forCampylobacter entrance in the broilerhouse,forinstanceviafarmer’sboots(Newell etal.,2003).Theopen-airrangetowhichbroilershave accessinfree-rangepoultryproductioncouldalsobea major environmental source for flock contamination. WhenCampylobacter is isolated from the open-air range soil or from stagnant water, before the birds go out, the precedent flock may be responsible for the contamination. Furthermore, even if the open-air
range seems to beCampylobacter-free, it is possible thatCampylobacterispresentunderaViablebutNon Culturable (VNC)-form. Induced through cell stress, particularly in drastic soil conditions (Rivoal etal., 2005), VNC represents a resting or dormant stage extremely difficult to detect, which could return to virulent form under appropriate conditions (Moore, 2001).
This transmission route seems yet not negligible as shown by Rivoal etal. (2005). Among seven poultry farms sampled from 1996 to 1999, four had gotinformationabouttherespectofstrictbiosecurity measures aimed at preventing the introduction of Campylobacter into flocks. In these farms, flock contaminationappearedfromsixweeksofage,atthe timeofoutdoorrearingperiod.Inthethreefarmsfor which no biosecurity measures were applied, broiler contaminationappearedfromtwoweeksofage,then beforetheaccesstotheopen-airrange.Theinfluence of the open-air range on the contamination is yet not fully understood.According to a recent study of the “Agence Française de Sécurité Sanitaire des Aliments” (AFSSA), access to an open-air range is not the mainCampylobacter contamination route of free-rangebroilerproduction.Among73farms,close to three quarters of flocks were contaminated before accesstotheopen-airrange.Attheendoftherearing period, all the flocks wereCampylobacter-positive, andconcernedmainlyC.jejuni(Huneau-Salaünetal., 2005).
Feed and drinking water. Campylobacter can not surviveinpoultryfeedbecauseofatoolowmoisture rate (Altekruse etal., 1999; Newell etal., 2003) althoughfeed,asdrinkingwater,canbecontaminated byfæcaldroppingsduringtherearingperiodandcan serveastransmissionroute(Bulletal.,2006).Onthe otherhand,watercanbearealcontaminationvector forbroilerchickens,asshownbyShankeretal.(1990) who succeeded to infect broilers with artificially contaminatedwater.
Wildanddomesticatedanimal.Becauseofthepathogen unability to multiply outside warm-blooded animals, farmanimalslikepoultry,cattle,pigs,sheepandgoats (Oportoetal.,2007),petslikecatsanddogs,andwild animals like birds and rodents, are often considered as importantCampylobacter reservoir. Although the broilercontaminationbywildanddomesticatedanimals does not seem to be direct, except for the free-range broilerproductions,animalbearingandfæcalshedding ofthebacteriahavebeenactuallypointedoutinseveral studies(Stanleyetal.,2003;Hutchinsonetal.,2005) as a potential origin of environmental contamination (Nicholsonetal.,2005).
Because of their microaerophilic metabolism and theirinabilitytogrowthattemperaturesbelow31°C, the presence of Campylobacter in streams, rivers andpondscanthenbetakenasasignofrecentfæcal contaminationbylivestockorwildanimal(Friedman etal., 2000) but can last up to four months (Rollins etal., 1986; Hazeleger etal., 1998).Campylobacter serotypes and genotypes are not systematically corresponding, and the wild animals role should be relativelylimitedafterall(Petersenetal.,2001). Insects.Someauthorshavemadetheassumptionthat insectslikefliescouldplayapartintheCampylobacter epidemiology (Skov etal., 2004). They could act as mechanical vectors, transmitting pathogens from reservoir environment or animals to broiler flocks (Ekdahl etal., 2005; Nichols, 2005). Nevertheless, insects seem to be contaminated by the broilers and mayactaspathogenvectoronlyafterwards.
4.EUROPEANLEGISLATION
Following several different sanitary crises, the Community legislation on food hygiene has been progressivelyrestructuredandstrengthenedinorderto establishacoherentandconsistentnetworkofhygiene rules based on an integrated approach covering the whole food chain “from stable to table”. The new legal instrument on food hygiene ensures that the Member States comply with the Good Hygiene/ Farming Practices (GHP) in livestock production, as appliedinBelgium.ThereflectionoftheCommission onthenewapproachtofoodsafety,coveringtheentire productionchainofallfoodstuffs,bothofanimaland ofplantorigin,resultedintheadoptionoftheWhite PaperonfoodsafetyinJanuary2000.
The main principles depicted in the White Paper are: the assurance of a high standard of food safety; the responsibility for food safety primarily upon food businesses, including feed manufacturers and farmers; the assurance of a “farm to table” policy; thepossibilitiesfortraceabilityandtransparencyand
thepossibilitiestotakeintoaccounttheprecautionary principle and other legitimate factors, where appropriate.
These rules would be essential to prevent contamination and spread of zoonotic agents in farms and are the basis of the European legislation concernedwiththemonitoringandcontrolofzoonoses and zoonotic agents at the primary production, transformation and distribution levels. With the aim of decreasing the incidence of zoonoses in humans, of improving the control of zoonoses in the primary production and of strengthening the collection of relevantdatatosupportriskassessmentactivitiesand risk management decisions, the European Union has decidedmorerecentlytointegrateandtostandardize thedifferentnationalmonitoringandsurveyplansby theestablishmentoftheDirective2003/99/CEandthe Regulations(EC)n°2160/2003andn°1003/2005.
The specific purpose of these Regulations is “to ensurethatproperandeffectivemeasuresaretakento detect and to controlSalmonella and other zoonotic agents,particularlyatthelevelofprimaryproduction, inordertoreducetheirprevalenceandtherisktheypose topublichealth”.Salmonellaistheprimaryzoonotic agenttargetedatprimaryproductionasitrepresentsan importantburdentopublichealth.From2010,poultry meatcontainingSalmonellain25gshallnotbeplaced onthemarketwithoutanyindustrialtreatmentableto eliminateSalmonella.
Such measures are not yet implemented for Campylobacteratthistimebutareactuallyexamined byTheCommunityEconomicandSocialCommittee, asmallnumberofMemberStatesandatapreliminary stage the European Parliament (Kremer, 2005). It is within this context that the European Food Safety Authority (EFSA) has formulated several recommendations in its Scientific Report in 2005 (EFSA, 2005). They concern particularly the intensification of epidemiological studies about Campylobacter and the reduction of the proportion of Campylobacter-infected poultry farms, by the applicationofstrictbiosecuritymeasures.
evaluatethesecostsbymeansofaneconomicmodel. AccordingtoMangenetal.(2005b),theannualincome ofbroilerfarmerscouldnotbearincreasedproduction costswithoutanyadditionalbonus,andthissituation isallthemoreactualforextensivesmall-sizedpoultry farms.
5.INTEGRATEDAPPROACHTOREDUCE FLOCKCONTAMINATION
Given the public health and economic problem representedbyCampylobacter,andthestrengthening oftheEuropeanlegislationrelatingtoanimalproducts contamination by zoonotic agents, it is important to take measures in order to reduceCampylobacter prevalence throughout the poultry production chain leadingtoareducedincidenceofthehumanillness.
Inarecentriskevaluation,theCSH(2005)showed thattherisktocontractillnessdecreasessignificatively if the proportion of contaminated meat-based preparationsmaybelimitedoreliminatedinthefood distribution chain. Moreover, it is not just presence or absence of pathogenic bacteria that is important, butalsotheamountsinwhichtheyarepresent.Dutch (Nauta etal., 2007) and Danish (Rosenquist etal., 2003)studieshaveparticularlydevelopedquantitative microbiological risk assessment models based on mathematical dose-response model to estimate the relationshipbetweeningesteddoseandtheprobability ofdevelopingcampylobacteriosis.
Many broiler flocks can become infected with Campylobacter spp. at many stages of the poultry production chain. Therefore, the only intervention strategy to reduce the exposure of humans to Campylobacterspp.seemstobeanintegratedapproach (Snijdersetal.,2002),withmultiplecontrolmeasures along the poultry production chain, for instance at farm level, during transport, at the slaughterhouse and/orattheproducttransformationstep(Line,2002; Hariharanetal.,2004;Whitaker,2006).
Risk factors and sanitary measures for contamination during catching and transportation have been presented by Ramabu etal. (2004) and Rasschaert etal. (2007). The risk factors associated with the slaughter operations on the contamination of carcasses have been studied by Rosenquist etal. (2006) and EFSA (2005) have reviewed the risk management options available at this level. Furthermore,techniquesofpreventingcontamination or decontaminating raw meat and poultry meat products in the food processing industry have been discussedbyseveralauthors(Huffman,2002;Woteki etal.,2003;Dinçeretal.,2004).Wotekietal.(2003) havealsopresentedindetailsnecessarystrategiesat theconsumerlevel.
6.USUALPREVENTIONMETHODS
6.1.Hygienemeasures
Practical biosecurity measures at the farm level have been determined as the primary strategy to prevent colonisation of housed broiler flocks with Campylobacterenteringtheprocessingplantandhence thefoodchain(vandeGiessenetal.,1992;ICGFI,1999; Gibbensetal.,2001;Rivoaletal.,2005).Nevertheless, many authors have shown that biosecurity measures areonlypartlyeffectiveincontrollingCampylobacter contamination(Pattison,2001;Sahinetal.,2003;Van Gerweetal.,2005).
Measures that are important to protect the flock includethewashingofhands,thewearingofprotective clothinganddedicatedfootwear,therespectofhouse cleaning and disinfection protocoles, provision of Campylobacter-free water, feed and the removal of spentlitterbetweentwoflocks.Detailsaboutbiosecurity measures designed to control Campylobacter have beenreportedbyAllenetal.(2005).
Thelimitedactionofhygieneproceduresisbased on the fact that in conditions where broilers are confrontedwithenvironmentalfactorsthatarescarcely controllable(open-airrange,wildbirds,domesticated animalsfaeces,etc.),i.e.organicandfree-rangeflocks, biosecurity is difficult to apply. In these production systems, Rivoal etal. (2005) have shown that, even if strict hygiene measures allow broiler flocks to be Campylobacter-negativeduringthefirstweeksofage (theindoorperiod),birdsarealmostalwayscolonized at slaughter, after the access of birds to the open-air range.
Nevertheless,evenifhighlevelsofenvironmental exposure to Campylobacter may overwhelm best practicebiosecuritymeasuresandthatthesepractices cannotguaranteeinfectionprevention,theycanhelp todelaytheonsetofCampylobactercolonizationand areconsequentlyessential.
6.2.Antibioticsuse
various veterinary practices in antimicrobial usage. Whatever the opinion we have in this debate, these antibioticshavebeenbannedintheEUsinceJanuary 2006,accordingtothe“PrecautionaryPrinciple”.
6.3.Acidification
It is generally acknowledged thatCampylobacter is sensitive to acid conditions (AFSCA, 2006). Several strategies developed to reduce Campylobacter populations are based on the acidification of the pathogenenvironment.
Drinkingwaterandfeedacidification.Theinvitro studies realized by Chaveerach etal. (2002) have pointed out the bactericidal activity of organic acids usedindividuallyorincombination.Thefourstudied acids (formic, acetic, propionic and hydrochloric), aloneorincombinationatdifferentformulationratios, weremixedwithacommercialbroilerfeedintobottles containing250mloftapwater.Theacidcombinations have shown an interesting bactericidal activity at pH 4.0 with Campylobacter numbers declining below 1logcfu.ml-1within1h,andthereductionwashigher thanthedecreasingeffectobservedwiththedifferent acidsusedindividually.
Water being an efficientCampylobacter vector, Chaveerach etal. (2004) studiedinvivo the drinking wateracidificationbythesamefourorganicacidsas a prophylactic measure. During all the experiment, noCampylobacter was found in acidified drinking water.Althoughacidificationseemstobeaneffective measuretocontrolwaterasaprominentcontamination vector, most chickens were infected at the end of the experiment, demonstrating the impact of other contamination ways. Byrd etal. (2001) have also studied drinking water acidification during pre-slaughterfeedwithdrawal.Theadditionof0.5%lactic acid in drinking water significantly reduced crop contaminationwithCampylobacterascomparedwith thecontrols(62.3%vs85.1%).
Another study by Heres etal. (2004) has tested fermented feed containing high concentrations of organic acids (5.7% lactic and 0.7% acetic) on susceptibility of chickens to Campylobacter and Salmonella. Broilers fed with fermented feed until 21days of age needed a ten times higher dose of Campylobacter to achieve the same proportion of infected chickens as the control population. Nevertheless, the protective effects seem relatively limitedanddependentontheinfectiondoseaccording tothepathogeninoculated.
Litteracidification.Acidificationofpoultrylitterhas also been suggested as a method to limit pathogen
proliferationinbreedingflocks.Line(2002)assessed twocommerciallyavailablelittertreatments(aluminium sulfate and sodium bisulfate) on Campylobacter prevalence and cæcal colonization of broilers. For example, treatment of pine shavings litter with the lowest level of aluminium sulfate, i.e. 3.63kg per 4.6m²littersignificantlyreducedcæcalCampylobacter colonizationfrequencyby65%andeffecteda3.4log reductionincæcalpathogenpopulations.Nevertheless, itisnoteworthythat,evenatthelowesttreatmentlevel, suchhighconcentrationsaredifficulttoincludeinan environmental-respectfulrearingsystem.
7.COMPLEMENTARYDEVELOPING STRATEGIES
7.1.Nonantagonism-basedstudies
Activeandpassiveimmunity.Vaccinationofpoultry againstCampylobacter has been considered to be a more effective measure than strict hygiene practices by some studies (de Zoete etal., 2007), because of the observation of aCampylobacter-specific immune responseinchickens(Riceetal.,1997).
So,thestudyofWyszynskaetal.(2004)hasshown thatchickenimmunizationwithavirulentSalmonella vaccinestraincarryingC.jejunicjaAgene,encoding highlyimmunogenicproteins,maybeanattractiveand efficientapproachforbirdvaccination.
About the passive immunization, Sahin etal. (2003)haveobservedthatC.jejuni-specificmaternal antibodieshavearoleinprotectionagainstcolonization inyoungCampylobacter-negativechicks.Furthermore, Tsubokura etal. (1997) showed a prophylactic and therapeuticeffectsagainstC.jejuni,foratleast5days post-infection, by oral administration of bovine and chicken immunoglobulin preparations to 22-days-old chickens.Nevertheless,theuseofmaternalantibodies could be hindered by their short protection period, unabletocoverthewholerearingperiod.Wilkie(2006) purifiedandconcentratedeggyolkantibodiesfromC. jejunivaccinatedhens.Threehoursafterexperimentally infecting day-of-hatch broiler chicks with 5.107 cfu C. jejuni, yolk antibodies were administered via oral gavageorinthefeedatafinalconcentrationof0.5% (w/w)untilday11post-challenge.Despitemeasurable antibody activity invitro, no significant reduction in the intestinal colonization byC.jejuni could be demonstrated.
betweenthepresenceofnaturalenvironmentalphage and a reduction in the Campylobacter population colonizing broiler chicken caeca. Although it is a relatively new developing technique, it has already givensomeinterestingresults.However,Goodeetal. (2003)emphasizethelimitationofphageuseatfarm level i.e. the potential for fast selection of resistant Campylobacter following the simultaneous pathogen andbacteriophagerelease.Theseauthorswouldlimit consequently the bacteriophages use at the slaughter stage. On the other side, Wagenaar etal. (2005) considerthereleaseofphage-infectedCampylobacter intheenvironmenttobeacceptable,sincephageshave been shown to reside inCampylobacter populations presentonnaturallyinfectedpoultry.
Diet modification.Heres etal. (2003) have studied theeffectoffeedfermentationontheCampylobacter contamination of broiler chickens. They used a moistenedcommercialstandardbroilerfeed(feed:water ration= 1:1.4) supplemented with aLactobacillus plantarumstraintofermentthemixture.Theresulting product,namedFLF(fermentedliquidfeed),leadtoa significant reduction ofCampylobacter susceptibility inchickens.Thisreportedeffectwasparticularlydueto thehighorganicacidsconcentrationsandtheresulting pHdecreaseinthefeed.FLFhadalsoaneffectonthe chickenintestinalmicroflora(Heres,2004).
Cereal-basedbroilerdietscontainanti-nutritiveNon-Starch Polysaccharides (NSP) that increase intestinal viscosity, impairing digestion and reducing broiler performances(Bedford,2001).Additionofexogenous enzymes,inparticularxylanasesandglucanases,reduces anti-nutritiveeffectsofNSPandimproveszootechnical poultry performance. Moreover, growth-promoting enzymeshavealsoshowninterestingantagonisticeffect againstCampylobacter. By reducing viscosity of the intestinalcontents,xylanasescaninducemodifications ofthechickensflora(Vahjenetal.,1998)andreduce C.jejunicontaminationwhentheseenzymesareadded tothebroilerdiet,asshownbyFernandezetal.(2000). These authors have found significant reductions of theC. jejuni cæcal colonization (from 0.3 to 0.5log cfu.g-1 cæcal content on average) by 0.1% xylanases supplementationofthediet.Thisreductioncanbedue toalowerintestinalviscosityaswellastothereduction ofthedigestatransittime,leadingtoatooshorttime for the pathogen establishment. Viscosity reduction could stimulate mucin production in the small and largeintestinesandinthecæca,aswellaschangesin themucincomposition.Somemucinglycoproteinsare responsiblefortheprotectivepropertiesofthemucus gelsinthegastrointestinaltract.
It is however important to point out that the use of feed additives is subjected to strict European legislations. Regulation (EC) n°1831/2003 of the
EuropeanParliamentandoftheCouncilofSeptember 22, 2003 on additives for use in animal nutrition, including enzymes, lays down rules governing the Community authorization of the additives and, in particular, defines the conditions that a substance or a product should meet to be granted authorization, and the labelling conditions for these additives. Authorization of the additive needs to pass the risk assessment by EFSA. To be legally placed on the marketandused,feedadditivesmustbeprovedtohave afavourableeffectonthecharacteristicsofthefeedto whichitisaddedoronanimalproduction,tohaveno harmfuleffectonanimalhealth,humanhealthorthe environmentandthatthepresentationoftheadditive oralterationofthefeaturesoftheproductstowhichit isaddeddoesnotharmormisleadtheconsumer.All these procedures are expensive and time-consuming so that enzymes approach may only be attractive if thepurposeofpathogenpreventioniscombinedwith performanceimprovement.
7.2.Microbiologicalcompetition
Competitive exclusion flora.Competitive exclusion (CE) is a concept taking advantage of bacterial antagonism to reduce animal intestinal colonization by pathogenic microorganisms.The study of defined or undefined flora acting by competitive exclusion mechanismswasfirstinitiatedinthe1970sbyNurmi etal. (1973). They observed that introduction of gut contents originating from adult cocks to 1-2d old chicks can protect young birds againstSalmonella infantis infection. Figure1, adapted from van der Wielen (2002), summarizes possible interactions between competitive exclusion flora and potential pathogensinbroilercæca.Atwofoldcompetitionmay operate in the gastrointestinal tract, i.e. competition for nutriments and for adhesion sites. Moreover, CE bacterialformulationsmayhaveadirectantimicrobial effect by the production of lactic acid, volatile fatty acid,hydrogenperoxideorbacteriocins.
average incidence colonization reduction observed inCE-andMCE-treatedbirdswas2.2%and15.6%, respectively.Ontheotherhand,Laisneyetal.(2003) failed to show beneficial effect of cæcal CE flora on broilerinfectionwith102-103cfuC.jejuniat15days ofage.Becauseofthelimitedadvantageforthepoultry producers,thepracticalapplicationofCEhasonlya greatsuccessinFinland.
Furthermore, it is difficult to ensure the absence of potentially pathogen organisms in the bacterial compositions.ItisnoteworthythatChenetal.(2001) aimed to preventCampylobacter colonization of the chickens intestinal tract by early inoculation in these chickens of non-pathogenicC. jejuni strains used as defined CE preparation. Nevertheless, some authors predict a promising future for CE (Schneitz, 2005), among others owing to the ban of growth-promoting antibiotics in animal production and sanitary requirementsthatbecomemoreandmorestrict. Acidifyingbacteria.BecauseoftheCEdisadvantages, the current trend is now the development of defined floraalthoughtheworkismadecomplicatedbylack of knowledge of the mechanism of CE and of the type of bacteria involved in the process (Chaveerach etal., 2004; Bjerrum, 2005). Acidifying bacteria, particularly lactic acid bacteria (LAB), contribute
since several thousand years to preserve food. Nevertheless, their antimicrobial properties are not limitedtothefoodindustryfield.Severalinvitroand invivo studies, summarized intables3 and4, have investigatedthebacterialantagonisticactivitiesagainst Campylobacter.
Lactobacilli are frequently used in theseinvitro studies. Chaveerach etal. (2004) have assessed the inhibitoryactivityofaLactobacillusfermentum(P93) strainisolatedfromthechickengutontenC.jejuni/ coli strains by diffusion agar assay and co-culture in anaerobic conditions. The experiment revealed an antagonisticeffectoftheL.fermentumstrainagainst all the ten Campylobacter tested strains, which decreased of 4.10± 2.15logcfu.ml-1 during 24h of co-culture incubation. The authors have suggested that the inhibitory effect ofLactobacillus (P93) on Campylobactergrowthcouldbeexplainedmainlyby organic acids production, resulting in pH reduction. Furthermore,theinhibitoryeffectwasenhancedwhen thepHlevelintheculturemediawaslow.Levelsand types of organic acids produced depend on bacterial species or strains, culture composition and growth conditions (Ammor etal., 2006). According to van derWielenetal.(2000)andChaveerachetal.(2004), the acid dissociation stage is an essential factor for antagonismeffect.vanderWielenetal.(2000)stated
Direct antimicrobial
activity Production of
bactericidal substances
Digestion of nutrients Production of beneficial ingredients
Competition for nutrients
Competition for space and adhesion
sites
CE-bacteria
Epithelium
Host mediated effects: epithelial cell functioning peristalsis immunomodulation
thattheundissociatedformoftheseshort-chainacids can diffuse freely across the bacterial membrane and dissociates inside the cell, thereby reducing the internalpHandcausinginternalpathogencelldamage. Someauthorsmentionalsothedamagecausedbythe anionitselfaswell,andinparticulartheinhibitionof fundamentalmetabolicfunctions(vanderWielenetal., 2000;Chaveerachetal.,2002).
Theinvitro study realised by Fooks etal. (2002) aimedtoinvestigateantagonisticeffectsoflactobacilli (L.plantarum,L.pentosus,L.acidophilus,L.reuteri). L. plantarum 0407 showed the most promising inhibitory activity onCampylobacter growth, both using plate assays and co-culture. This antimicrobial activityappearedtodependonthecarbohydratesource suppliedinvitro,suggestingthatasuitablecarbohydrate substrate supplementation may enhance competitive exclusion by lactobacilli. The experiment of Chang etal.(2000)triedtogetclosertoinvivoconditions,by investigatingtheimpactofaselectedlactobacillimixed
culture (L. acidophilus,L. fermentum,L. crispatus, L.brevis)onC.jejuniinsimulatedchickendigestive tract.TheC.jejuni and lactobacilli were mixed with sterilepoultryfeedandincubatedat41.1°Cforvarious lenghtsoftimeandpHvalues,simulatingfivesegments ofthedigestivetract.AllthetestedLactobacillusspp. showed an antagonistic effect onCampylobacter in individualsectionsandthewholesimulateddigestive tractmodels.
Then, several studies have pointed out the bactericidal activity of hydrogen peroxide (H2O2) produced by LAB in the presence of oxygen (Felten etal., 1999; Strus etal., 2006). Hydrogen peroxide may inhibit growth of bacteria that do not possess protectivemechanismslikecatalaseorperoxidase.Its antimicrobialeffectmayresultmainlyfromoxidation phenomenons causing denaturing of a number of enzymes and from the peroxidation of membrane lipidsandproteinsleadingtoanincreasedmembrane permeability(Edens,2003;Ammoretal.,2006).Zhao
Table4.InvivostudiesofprobioticsshowingantagonismagainstCampylobacter—Étudesinvivodebactériesprobiotiques présentantunantagonismevis-à-visdeCampylobacter.
Antagonisticmicroorganisms Observedeffects Reference
CombinationofCitrobacterdiversus, Flockcolonizationrate:-62% Schoenietal.,1994 Klebsiellapneumoniae,Escherichiacoli,
withmannose
Lactobacillusacidophilus FrequencyofCampylobacterjejunishedding:-70% Morishitaetal.,1997 +Streptococcusfaecium Jejunalcolonization:-27%
Enterococcusfaecium Nosignificantdifference Netherwoodetal.,1999
Purifiedbacteriocinof Significantreductionofintestinal Sternetal.,2005 Paenibacilluspolymyxa contaminationrateandfrequency
Table3. Invitro studies assessing the antagonism of microorganisms against Campylobacter—Études invitro de l’antagonismedemicro-organismesvis-à-visdeCampylobacter.
Antagonisticmicroorganisms Principaltests Observedorassumedantagonisticeffects Reference
Lactobacillusplantarum Agardiffusion SignificantinhibitionofCampylobacter Fooksetal.,2002
Bifidobacteriumbifidum Co-culturewith jejunigrowth;increasednumberof
Campylobacterjejuni probioticafter24h;lactateandacetate
production
Bacilluscirculans Spottest Productionofbacteriocinsinhibiting Svetochetal.,2005
Paenibacilluspolymyxa Campylobacter
Mixtureoflactobacilli: Campylobacterjejuni Campylobacterjejuniandlactobacilli Changetal.,2000
Lactobacillusacidophilus andlactobacilli enumeration:absenceofCampylobacter
Lactobacillusfermentum mixedwithsterilepoultry jejuniforthelastincubation
Lactobacilluscrispatus feedfollowedbysuccessive
Lactobacillusbrevis incubationsat41.1°Cin
invitrotestssimulatingthe
poultrydigestivetract
Lactobacillusspp. Agardiffusion Productionofformicandaceticacids; Chaveerachetal., Co-culturewith productionofanantimicrobialpeptide 2004
etal. (2006) showed that incubation of 7.0logcfu. ml-1C.jejuniwith0.1and0.2%H
2O2insuspension reducedC.jejunipopulationsbyca.2.0and4.5logcfu. ml-1,respectively.Furthermore,someauthorsstudied the efficacy of broiler carcasses decontamination with H2O2during the slaughter processing.Although Wagenaar etal. (2004) observed that immersion of carcassesin1,2,3and4%H2O2solutionscontaining glycerol resulted in average reductions of 0.3 up to 1.4logcfu for the mesophilic aerobic counts, they did not measureCampylobacter loads on carcasses. Moreover, Dickens etal. (1997) demonstrated that additionofupto1.5%H2O2tosprayswatersduring defeatheringhadnoeffectontotalaerobicplatecounts ofpickedunevisceratedcarcasseswhencomparedto thewatercontrol.
Besides organic acids and H2O2, bacteriocins are thethirdkindofcompoundsthatmayhelptoinhibit Campylobactergrowth,asshownbySternetal.(2006) forabacteriocinproducedbyaLactobacillussalivarius strain. Bacteriocins are peptidic compounds with antimicrobial properties produced by some bacteria. Their target is mainly the cytoplasmic membrane, forming pores that allow the unregulated outflow of essential ions, leading to bacteria death (Papagianni, 2003). The bacteriocins have often a relatively restrictedspectrumofactivityagainstbacteriastrains closelyrelatedtotheproducingstrain.Particularly,the genusPaenibacillushasbeenpointedoutbyRussian and American researchers. Svetoch etal. (2005) haverevealedtheproduction,bythreePaenibacillus polymyxa strains, of bacteriocins effective against Campylobacter. One of these bacteriocins, secreted by P. polymyxa NRRL-B-30509, was purified and microencapsulated to evaluate a bacteriocin-based treatment to reduceC. jejuni colonization in poultry (Stern etal., 2005). The purified preparation was incorporated in chicken feed at the rate of 0.25g.kg-1. One day old chicks were orally infected with108cfuC.jejuniandwereprovidedfromdayseven tochickenfeedcontainingornot(control)bacteriocin.
Ten days after C. jejuni challenge, comparison of cæcalcontaminationratebetweencontrolandtreated chickens showed that bacteriocin treatment reduced levelsofintestinalcolonizationbyC.jejunifrom4.6 to6.3logcfu.g-1offæces(P ≤0.05).
Probiotics. The probiotic notion derives directly from the competitive exclusion concept. Unlike the CE treatments, probiotics are compositions containingoneorseveralwell-definedstrains.Several descriptions have been proposed for probiotics (Jin etal.,1997)buttheymaygloballybedefinedasliving microorganismsthat,onceingested,beneficiallyaffect the host animal by improving its microbial balance (Fuller,1989).Themainexpectedcharacteristicsand functions for an efficient probiotic strain in poultry production,presentedintable5,includemaintaining normalintestinalmicroflorabycompetitiveexclusion and antagonism, altering metabolism by increasing digestive enzyme activity, improving feed intake and digestion and neutralizing enterotoxins and stimulatingtheimmunesystem(Ghadban,2002).The useofprobioticmicroorganismsinanimalproduction iswellcontrolledandisconsidered,asenzymesand feedadditives,byRegulation(EC)n°1831/2003ofthe EuropeanParliamentandoftheCouncilofSeptember 22,2003.
About theinvivo studies, Morishita etal. (1997) have assessed the antagonistic effect of probiotic containing aL. acidophilus strain combined with a Streptococcus faecium. This avian-specific probiotic was given to chicks from day one to day three; moreover, birds were challenged withC. jejuni 6h after the first oral administration of probiotic. At 40daysofage,theprobiotic-treatedgrouphada70% (P= 0.0001) decreased number of birds shedding C.jejuniwhencomparedwiththecontrolgroupgiven distilledwaterinsteadofprobiotic.Theyalsofounda 27%(P=0.0001)reductioninthenumberofchickens that were colonized in the jejunum at slaughter in comparisonwiththecontrolledbirds.
Table5.Expected characteristics and functions of probiotics in animal production (adapted from Edens, 2003)— Caractéristiquesetfonctionssupposéesdesprobiotiquesenproductionanimale(adaptédeEdens,2003).
Characteristics Functions
Nonpathogenic Productionofinhibitorysubstancesagainstotherbacteria
Rapiditytocolonizeintestinalepitheliumandmucus Activecompetitionforadhesionsites
Tolerancewithindustrialmanufacturingandstorage Stimulationoftheimmunesystem
Improvementofnutrimentsabsorption
Improvementofanimalperformances
Reductionofpathogenexcretioninfæces
Hostadaptedbycreationofabeneficialmicroecology Exclusion(colonizationprevention)orbactericidaleffect
againstpathogens
Resistancestogastricandbiliaryacids Alterationofmicrobialmetabolism
