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.
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ul u es om di e en ages.
Age n
Salmonella
(%)
Ju eniles 5 80.0
Subadul 11 54.5
Adul 81 50.6
All 97 52.6
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).
n: numbe o bi ds analysed.
doi:10.1371/jou nal.pone.0094191. 002
Table 3. Salmonella se o a s isola ed om wild g i on
ul u es (Gyps ul us).
Subspecies Se o a n
en e ica Monophasic Typhimu ium 4,5,12:i: - 40
en e ica Monophasic Typhimu ium 4,12:i: - 1
salamae 4,12:b: - 2
en e ica Sen enbe g 1,3,19: g,s, : - 3
en e ica Rissen 6,7: ,g: - 4
en e ica Typhimu ium 4,12:i: 1,2 1
n: numbe o s ains isola ed.
doi:10.1371/jou nal.pone.0094191. 003
G i on Vul u es as Ca ie s o Salmonella and Campylobac e
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