Wild Griffon Vultures (Gyps fulvus) as a Source of Salmonella and Campylobacter in Eastern Spain

Wild G i on Vul u es ( Gyps ul us ) as a Sou ce o Salmonella and Campylobac e in Eas e n Spain Cla a Ma in 1 *, Ma ia-Dolo es Palomeque 1 , F ancisco Ma co-Jime ´nez 2 , San iago Vega 1 1Ins i u o de Ciencias Biome ´dicas, Depa amen o de P oduccio ´n Animal, Sanidad Animal, Salud Pu ´blica Ve e ina ia y Ciencia y Tecnologı ´a de los Alimen os, Facul ad de Ve e ina ia, Uni e sidad CEU-Ca denal He e a, Al a a del Pa ia ca, Valencia, Spain, 2Ins i u o de Ciencia y Tecnologı ´a Animal, Uni e sidad Poli e ´cnica de Valencia, Valencia, Spain Abs ac The exis ence o Campylobac e and Salmonella ese oi s in wildli e is a po en ial haza d o animal and human heal h; howe e , he p e alence o hese species is la gely unknown. Un il now, only a ew s udies ha e e alua ed he p esence o Campylobac e and Salmonella in wild g i on ul u es and based on a small numbe o bi ds. The aim o his s udy was o e alua e he p esence o Campylobac e and Salmonella in wild g i on ul u es (n = 97) du ing he no mal inging p og amme a he Cinc o es Obse a o y in Eas e n Spain. In addi ion, he e ec o ages o indi iduals (ju eniles, subadul and adul ) on he p esence we e compa ed. Campylobac e was isola ed om 1 o 97 (1.0%) g i on ul u es and iden i ied as C. jejuni. Salmonella was isola ed om 51 o 97 (52.6%) g i on ul u es. No signi ican di e ences we e ound be ween he ages o indi iduals o he p esence o Salmonella. Se o yping e ealed 6 di e en se o a s among wo Salmonella en e ica subspecies; S. en e ica subsp. en e ica (n = 49, 96.1%) and S. en e ica subsp. salamae (n = 2, 3.9%). No mo e han one se o a was isola ed pe indi idual. The se o a s isola ed we e S. Typhimu ium (n = 42, 82.3%), S. Rissen (n = 4, 7.8%), S. Sen enbe g (n = 3, 5.9%) and S. 4,12:b[-] (n = 2, 3.9%). Ou esul s imply ha wild g i on ul u es a e a isk ac o o Salmonella ansmission, bu do no seem o be a ese oi o Campylobac e . We he e o e ule ou ul u es as a isk ac o o human campylobac e iosis. Ne e heless, u he s udies should be unde aken in o he coun ies o con i m hese esul s. Ci a ion: Ma in C, Palomeque M-D, Ma co-Jime ´nez F, Vega S (2014) Wild G i on Vul u es (Gyps ul us) as a Sou ce o Salmonella and Campylobac e in Eas e n Spain. PLoS ONE 9(4): e94191. doi:10.1371/jou nal.pone.0094191 Edi o : Axel Cloeckae , Ins i u Na ional de la Reche che Ag onomique, F ance Recei ed Janua y 24, 2014; Accep ed Ma ch 10, 2014; Published Ap il 7, 2014 Copy igh : ß2014 Ma in e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Funding: Funding o his p ojec came om he Conselle ia de In aes uc u as, Te i o io y Medio Ambien e (Regional Go e nmen /Gene ali a Valenciana). The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . * E-mail: [email p o ec ed]u.es In oduc ion Campylobac e iosis and salmonellosis a e he wo mos p e alen zoonoses wo ldwide [1]. These zoonoses ep esen an impo an public heal h p oblem and con olling he disease has become a i al challenge in mos coun ies [1–7]. Campylobac e - iosis and salmonellosis we e esponsible espec i ely o 212,064 and 99,020 cases o illnesses in he EU [1]. Mo eo e , campylobac e iosis is he mos common cause o acu e bac e ial gas oen e i is in he EU [1,8,9]. Apa om acu e gas oen e i is, campylobac e iosis may lead o mo e se e e, occasionally long- e m sequels such as Guillain-Ba e´ synd ome, eac i e a h i is and i i able bowel synd ome [10,11]. The high and apidly inc easing incidence and he capaci y o Campylobac e o cause conside able mo bidi y make campylobac e iosis a public heal h p oblem o conside able magni ude [2]. Howe e , compa ed o Salmonella, ew ou b eaks a e epo ed, and mos campylobac e - iosis cases a e conside ed o be ‘‘spo adic’’ a he han a pa o ecognised ou b eaks, wi h a seasonal peak in summe [12]. The exis ence o Campylobac e and Salmonella ese oi s in wildli e has been conside ed a po en ial haza d o animal and human heal h; howe e , he numbe o wildli e species ac ing as ese oi s is unknown [13]. Salmonella and Campylobac e species ha e been isola ed om a wide a ie y o wild bi ds, al hough he e is g ea a ia ion be ween axa [14–24]. Ne e heless, se e al s udies ha e sugges ed ha wild bi ds a e impo an in he epidemiology o human and li es ock salmonellosis [25–30]. In con as , o he s udies sugges ha hey do no p esen a majo public heal h haza d, gi en he low numbe s and sho du a ion o salmonella exc e ion [31], emphasising he con o e sial na u e o he subjec ma e . The ansmission pa hway o Campylobac e included di ec and indi ec con ac wi h in ec ed animals, people and en i onmen [2,25–28]. In his con ex , he shedding o wild bi ds aeces in o he en i onmen has been iden i ied as a signi ican ese oi o Campylobac e spp. [32]. Fo example, exposu e o con amina ed wild bi d aeces in playg ounds has been ecognised as a po en ial en i onmen al sou ce o campylobac e iosis, pa icula ly o child en [33]. A ian aecal ma e ound in child en’s playg ounds was posi i e o C. jejuni in bo h d ied and esh samples [34]. Recen ly, Waldens o¨m e al. [32] epo ed ha o be e unde s and Campylobac e biology in wild bi ds, a numbe o undamen al ques ions need o be add essed o elucida e he ole o wild bi ds as ese oi s o Campylobac e , o iden i y hos -speci ic s ains, how long wild bi ds a e colonised o and whe he he du a ion and in ensi y o bac e ial shedding a y depending on s ain o igin. Fu he mo e, does colonisa ion o C. jejuni ha e any measu able e ec s on body condi ion, o a e wild bi ds asymp oma ic ca ie s simila o chickens? PLOS ONE | www.plosone.o g 1 Ap il 2014 | Volume 9 | Issue 4 | e94191 Speci ically, in wild g i on ul u es bo h Molina-Lopez e al. [13] and Milla´n e al. [15] e alua e he p esence o Salmonella and, o ou bes knowledge, only Molina-Lopez e al. [13] e alua e he p esence o Campylobac e . In his con ex , Spain holds he mos impo an G i on ul u e popula ion in Eu ope [35–37]. P o ec- ion o b eeding colonies, limi ed di ec pe secu ion (shoo ing) and a educ ion in he use o poisons ha e assis ed he species’ eco e y [37]. This would no ha e been possible wi hou he a ailabili y o li es ock ca casses, abundan in Spain, and legisla ion which does no insis on he ca casses being bu ied o bu n [37]. The aim o his s udy was o in es iga e occu ence o Campylobac e and Salmonella in wild g i on ul u es and iden i y he isola es o species. Ma e ials and Me hods The E hics and Animal Wel a e Commi ee o he Uni e sidad CEU Ca denal He e a app o ed his s udy. All animals we e handled acco ding o he p inciples o animal ca e published by Spanish Royal Dec ee 1201/2005 (BOE, 2005; BOE = O icial Spanish S a e Gaze e). The Depa men o In as uc u e, Planning and En i onmen o he Valencian Regional Go e n- men (Gene ali a Valenciana) g an ed pe mission o ake samples. This s udy was conduc ed wi hin he conse a ion p ojec o endange ed species in he Valencia Region. The s udy popula ion is loca ed in eas e n Spain wi hin he p o ince o Cas ello´n. Wild g i on ul u es we e cap u ed in Sep embe and Oc obe 2013 a he Cinc o es Obse a o y (40u339N, 0u139W, Cas ello´n p o ince, Eas e n Spain), du ing he obse a o y’s no mal inging p og amme. A o al o 97 g i on ul u es we e cap u ed using a emo ely ac i a ed pu pose-buil cage (Figu e 1). The age o he animals was de e mined acco ding o he plumage cha ac e is ics and he colou o he bill and eye, classi ied as ju enile (less han 2 yea s), subadul (be ween 2 o 5 yea s) and adul (mo e han 5 yea s). F om each chick, wo cloacal samples we e aken using s e ile co on swabs (Ca y Blai s e ile anspo swabs, DELTALAB). We inse ed s e ile co on swabs 1 o 2 cm in o he cloaca o collec a sui able sample. Each sample was analysed o Campylobac e and o Salmonella isola ion. Addi ionally, blood samples we e collec ed om he b achial ein (abou 5 mL) and ans e ed o 200 ml p e- e ige a ed saline es ubes and s o ed a 0–4uC. Hema oc i concen a ion was measu ed using hepa inised mic o-hema oc i capilla y ubes cen i uged o 5 min a 10,000 pm and ead in a mic o hema oc i ube eade . De ec ion o Campylobac e spp. Bac e iological cul u e was pe o med in acco dance wi h ISO 10272-1:2006 o he de ec ion o Campylobac e spp. (Anonymous, 2006). Mo eo e , all samples we e es ed by di ec cul u e. P e- en iched samples we e cul u ed only i di ec cul u e was nega i e. Cloacal swabs we e di ec ly s eaked on o he wo selec i e aga pla es (mCCDA and P es on, AES labo a o ies, B uz Cedex, F ance) and incuba ed a 41.561uC o 4464 hou s as epo ed abo e. Mo eo e , cloacal swabs we e p e-en iched in 1:10 ol/ ol Bol on B o h (OXOID, Da dilly, F ance) and hen p e-incuba ed a 3761uC o 561 hou s. Then, he p e-en iched b o h was incuba ed a 41.561uC o 4361 hou s. A e wa ds, 10 ml o he sample was cul u ed on he wo selec i e aga pla es (mCCDA and P es on aga ) and incuba ed as desc ibed abo e. Campylobac e - like colonies we e pu i ied on blood aga and iden i ied o species le el on he basis o s anda d p ocedu es comp ising es s o hippu a e and indoxyl ace a e hyd olysis, ca alase p oduc ion, and suscep ibili y o cephalo in and nalidixic acid. De ec ion o Salmonella spp. The p ocedu e was based on ISO 6579: 2002 ecommenda ions (Annex D). Samples we e p e-en iched in 1:10 ol/ ol Bu e ed Pep one Wa e 2.5% (BPW, Scha lau, Ba celona, Spain) and hen incuba ed a 3761uC o 1862 hou s. The p e-en iched samples we e ans e ed on o Semi-Solid Modi ica ion Rappapo Vassi- liadis aga pla e (MSRV, Di co, Valencia, Spain) and incuba ed a 41.561uC o 24–48 hou s. The cul u e ob ained in MSRV was inocula ed on o Xylose–Lysine–Desoxychola e (XLD, Lio ilchem, Valencia, Spain) and Xylose–Lysine–Te gi ol-4 (XLT4, Bioka Diagnos ics, Pan in Cedex, F ance) and incuba ed a 3761uC o 24–48 hou s. A e incuba ion, 5 ypical colonies we e s eaked on o he su ace o p e-d ied nu ien aga pla es (Scha lab, Ba celona, Spain) 3761uC o 2463 hou s. Then, a biochemical es using API (API-20, bioMe ieux, Mad id, Spain) was pe o med o con i m Salmonella spp. Mo eo e , Salmonella s ains isola ed we e se o yped by he Minis y o Ag icul u e, Fishe ies and Food Re e ence Labo a o y (Alge e, Mad id, Spain) in acco dance wi h Kau man-Whi e-Le Mino echnique. S a is ical analyses We use a gene alised linea model (GLM) o compa e he body mass and hema oc i agains he ages o indi iduals (Ju eniles, subadul and adul ). As p e iously, a GLM which assumed a binomial dis ibu ion o Salmonella shedding was i ed o he da a o de e mine whe he he e is a main e ec o he ages o indi idual g i on ul u es. A P alue o less han 0.05 was conside ed o indica e a s a is ically signi ican di e ence. Da a a e p esen ed as leas squa es means 6s anda d e o o he leas squa es means. All s a is ical analyses we e ca ied ou using a comme cially a ailable so wa e p og am (SPSS 16.0 so wa e package; SPSS Inc., Chicago, Illinois, USA, 2002). Resul s All he g i on ul u es es ed in his s udy we e heal hy, acco ding o he hema oc i le el and body mass (Table 1). No signi ican di e ences we e ound be ween ages o indi iduals o body mass and hema oc i alue. Campylobac e was isola ed om 1 o 97 (1.0%) g i on ul u es and iden i ied as C. jejuni. Salmonella was isola ed om 51 o 97 (52.6%) g i on ul u es. No signi ican di e ences we e ound be ween he ages o indi iduals o he p esence o Salmonella (Table 2). Se o yping e ealed 6 di e en se o a s among wo Salmonella en e ica subspecies (Table 3); S. en e ica subsp. en e ica (n = 49, 96.1%) and S. en e ica subsp. salamae (n = 2, 3.9%). No mo e han one se o a was isola ed pe indi idual. The se o a s isola ed we e S. Typhimu ium (n = 42, 82.3%), S. Rissen (n = 4, 7.8%), S. Sen enbe g (n = 3, 5.9%) and S. 4,12:b[-] (n = 2, 3.9%). Discussion Ou expe imen was ca ied ou o examine colonisa ion o Salmonella and Campylobac e in wild g i on ul u es. To ou knowledge, his is he only s udy in he scien i ic li e a u e o e alua e a conside able sample size om a heal hy popula ion o wild g i on ul u es. Hema oc i alues a e a use ul indica o o he gene al s a e o heal h in ap o s [38,39]. The mean hema oc i alues ob ained we e simila o hose desc ibed p e iously in he li e a u e o g i on ul u es [40]. The shedding o wild bi d aeces in o he en i onmen has been iden i ied as a signi ican ese oi o Campylobac e spp. [32] and we es ed he hypo hesis ha wild g i on ul u es could also be a G i on Vul u es as Ca ie s o Salmonella and Campylobac e PLOS ONE | www.plosone.o g 2 Ap il 2014 | Volume 9 | Issue 4 | e94191 po en ial ese oi o Campylobac e . Wild bi ds may ac o in o bo h Campylobac e and Salmonella epidemiology by coming in o con ac wi h a m and ood p oduc ion animals and mo ing g ea dis ances h oughou he landscape [41,42]. Wild bi ds can unc ion as animal ec o s, sp eading diseases along mig a ion ou es and ans e ing zoono ic bac e ia h oughou la ge pa s o he wo ld in aecal d oppings [43]. In ou s udy, C. jejuni was iden i ied in g i on ul u e, in ag eemen wi h Molina-Lopez e al. [13] and consis en wi h obse a ions in o he species o wild bi ds [41,44]. Recen ly, G iekspoo e al. [45] demons a ed ha C. jejuni isola ed om wild bi ds a e in gene al dis inc om hose isola ed om human campylobac e iosis and ood animals. Thus, ou esul s sugges ha wild g i on ul u es a e ela i ely poo ly colonised by Campylobac e and a e likely o li le impo ance in human campylobac e iosis. To e i y his, u u e s udies will need o e alua e he geno ypes o Campylobac e isola es om wild g i on ul u es and hei ela ionship o hose isola ed om human campylobac e iosis. Ne e heless, he posi i e pe cen age o Salmonella de ec ed in his s udy among wild g i on ul u es was high (52.6%) and hese esul s a e inconsis en wi h hose o o he s udies, in which he de ec ion was lowe han 10% [13,15,23]. One possible explana- ion is due o he low numbe s o animals ha a e membe s o he same species examined in hese p e ious s udies (n = 9, [13]; n = 3, [15]; n = 12, [23]). Howe e , in gene al, he as majo i y o he s udies sugges a low p esence o Salmonella in wild ap o s [13,17,23,46,47]. The mos plausible explana ion o he high de ec ion a es o Salmonella could be associa ed wi h he eed. In ou s udy, 82.3% o Salmonella isola es belong o S. Typhimu ium, a se o a o pa icula public heal h signi icance. This is, oge he wi h S. En e i idis, one o he mos equen ly epo ed se o a s in ol ed in human salmonellosis [1,48]. Salmonella Typhimu ium has p e iously been desc ibed in g i on ul u e [13,15] and is conside ed he mos common se o ype ound in wild bi ds [49– 51]. In Spain, a e he appea ance o he i s cases o Bo ine Spongi o m Encephalopa hy, g i on ul u es almos exclusi ely ed on swine ca casses [52,53]. Acco ding o he la es s udies, S. Typhimu ium is he mos p e alen se o ype isola ed in swine p oduc ion [54–56]. Al hough i has p e iously been posi ed ha ap o s may ha e acqui ed he bac e ia by inges ing small passe ines, mice o pigeons [15,57], ou indings sugges he possibili y ha ul u es may be con amina ed by inges ing pig ca casses. In ap o s, i has been epo ed ha Salmonella may indica e in ec ion in o he animal popula ions (e.g. small mammals and passe ine bi ds) [58,59]. Milla´n e al. [15] sugges ha wild g i on ul u es could acqui e he bac e ia om sou ces such as li es ock ca casses o human esidues. Howe e , u he s udies a e necessa y o de e mine he o igin o he Salmonella epo ed he e and he exac ole, i any, o he pig in ansmi ing he in ec ion o g i on ul u es. Ne e heless, ou esul s unde line he need o conside he g i on ul u es as a po en ial in ec ion sou ce o his bac e ial pa hogen, and he zoono ic isk o he gene al Figu e 1. T ap used o cap u e he wild g i on ul u es ( Gyps ul us ) a Cinc o es Obse a o y (406339N, 06139W, Cas ello ´n p o ince, Eas e n Spain). (a) Cage buil ad hoc o ap ul u es wi h emo e con ol. (b) De ail o pig ca casses placed on he ap o a ac he ul u es (whi e a ows). (c) De ails o he la ge numbe o ul u es analysed. doi:10.1371/jou nal.pone.0094191.g001 Table 1. Measu emen s o body mass and hema oc i in he Spanish wild g i on ul u es (Gyps ul us) acco ding o age. Age n Body mass (g) Hema oc i (%) Ju eniles 5 8508676 45.260.4 Subadul 11 82506200 45.861.0 Adul 81 74986297 46.861.5 All 97 8447670 45.460.4 The age o he animals was de e mined acco ding o he plumage cha ac e is ics and he colou o he bill and eye (ju enile, less han 2 yea s; subadul , be ween 2 o 5 yea s; adul , mo e han 5 yea s). Da a a e p esen ed as leas squa es means 6s anda d e o o he leas squa es means. n: numbe o bi ds analysed. doi:10.1371/jou nal.pone.0094191. 001 G i on Vul u es as Ca ie s o Salmonella and Campylobac e PLOS ONE | www.plosone.o g 3 Ap il 2014 | Volume 9 | Issue 4 | e94191 popula ion should be conside ed. As an example, Da ies and Wales [42] ecen ly illus a ed he po en ial o domes ic ce eal c ops o pose a isk o Salmonella exposu e o li es ock ia compound eeds u s. All se o a s iden i ied ha e p e iously been associa ed wi h human salmonellosis. Al hough he o he se o a s may be conside ed ypes a ely associa ed wi h human disease, hey ha e also been in ol ed in human salmonellosis. Fo example, S. Rissen, which has p e iously been desc ibed in g i on ul u e [13], is commonly isola ed om pigs and po k p oduc s, whe eas i is a ely epo ed wo ldwide om human sou ces, excep o Thailand, whe e in ecen yea s a ema kable ise in in ec ion cases has been eme ging [60–63]. S. Sen enbe g has been isola ed om wild bi ds in no he n England [16]. Al hough S. Sen en- be g is no lis ed among he op 20 se o ypes implica ed in human illness [64], he o ganism is ou inely de ec ed in humans and has been ecognised in clinical non-human cases o disease ( anked #10 in 2006) and in non-clinical non-human cases ( anked #4), suppo ing he po en ial o he eme gence o his s ain ype in human disease [65]. In 2007, he Heal h P o ec ion Agency Labo a o y o En e ic Pa hogens epo ed 51 human isola es o Salmonella Sen enbe g in England and Wales [66]. In addi ion, o ou bes knowledge, Salmonella subsp. salamae has no been desc ibed in wild bi ds. I has been documen ed in ep iles and ed ox [17,67] and one case o S. salamae in ec ion associa ed wi h consump ion o ep ile mea in humans has been epo ed [68]. This s udy showed wild g i on ul u es as a isk ac o o Salmonella in ec ion, bu indica es ha wild g i on ul u es a e poo bac e ial ese oi s o Campylobac e and likely o minimal impo ance in human campylobac e iosis. Acknowledgmen s We wish o hank he echnical suppo o Ma in Su oca, Xema Gil and all he membe s o he esea ch g oup ‘‘Imp o emen o he Food Sa e y ela ed wi h he P oduc ion Sys em and Final P oduc s’’ (Ve e ina y Facul y, Uni e si y CEU-Ca denal He e a). Au ho Con ibu ions Concei ed and designed he expe imen s: CM FMJ SV. Pe o med he expe imen s: CM MDP. Analyzed he da a: CM FMJ. Con ibu ed eagen s/ma e ials/analysis ools: CM MDP FMJ SV. W o e he pape : CM FMJ. Re e ences 1. EFSA (2012) Eu opean Food Sa e y Au ho i y. The Eu opean Union Summa y Repo on T ends and Sou ces o Zoonoses, Zoono ic Agen s and Food-bo ne Ou b eaks in 2010. EFSA Jou nal 10: 2597. 2. Kappe ud G, Espeland G, Wahl E, Walde A, He iks ad H, e al. 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Lo´pez B, Ri as P, To o C, T e isi P, Baque o M (2012) Fi s case o Salmonella salamae in ec ion associa ed wi h consump ion o ep ile mea in humans. 22nd Eu opean Cong ess o Clinical Mic obiology and In ec ious Diseases, London, Uni ed Kingdom. p. 1578. G i on Vul u es as Ca ie s o Salmonella and Campylobac e PLOS ONE | www.plosone.o g 5 Ap il 2014 | Volume 9 | Issue 4 | e94191