Changes in Cecal Mic obio a and Mucosal Gene
Exp ession Re ealed New Aspec s o Epizoo ic Rabbi
En e opa hy
Ch is ine Ba
¨ue l
1.
, M. Ca men Collado
1.
, Manuel Zu
´n
˜iga
1
, En ique Blas
2
, Gaspa Pe
´ ez Ma ı
´nez
1
*
1Labo a o io de Bac e ias La
´c icas y P obio
´ icos, Depa amen o de Bio ecnologı
´a, Ins i u o de Ag oquı
´mica y Tecnologı
´a de Alimen os (IATA), Consejo Supe io de
In es igaciones Cien ı
´ icas (Spanish Na ional Resea ch Council) (CSIC), Valencia, Spain, 2Animal Nu i ion Resea ch G oup, Ins i u e o Animal Science and Technology,
Poly echnic Uni e si y o Valencia (UPV), Valencia, Spain
Abs ac
Epizoo ic Rabbi En e opa hy (ERE) is a se e e disease o unknown ae iology ha mainly a ec s pos -weaning animals. I s
incidence can be p e en ed by an ibio ic ea men sugges ing ha bac e ial elemen s a e c ucial o he de elopmen o
he disease. Mic obial dynamics and hos esponses du ing he disease we e s udied. Cecal mic obio a was cha ac e ized in
h ee abbi g oups (ERE-a ec ed, heal hy and heal hy p e ea ed wi h an ibio ics), ollowed by ansc ip ional analysis o
cy okines and mucins in he cecal mucosa and e mix by q- PCR. In heal hy animals, cecal mic obio a wi h o wi hou
an ibio ic p e ea men was e y simila and domina ed by Alis ipes and Ruminococcus. P opo ions o bo h gene a
dec eased in ERE abbi s whe eas Bac e oides,Akke mansia and Rikenella inc eased, as well as Clos idium,c-P o eobac e ia
and o he oppo unis ic and pa hogenic species. The ERE g oup displayed ema kable dysbiosis and educed axonomic
di e si y. T ansc ip ion a e o mucins and in lamma o y cy okines was e y high in ERE abbi s, excep IL-2, and i s analysis
e ealed he exis ence o wo clea ly di e en gene exp ession pa e ns co esponding o In lamma o y and (mucin)
Sec e o y P o iles. Fu he mo e, hese p o iles we e associa ed o di e en bac e ial species, sugges ing ha hey may
co espond o di e en s ages o he disease. O he da a ob ained in his wo k ein o ced he no ion ha ERE mo bidi y and
mo ali y is possibly caused by an o e g ow h o di e en pa hogens in he gu o animals whose immune de ence
mechanisms seem no o be adequa ely esponding.
Ci a ion: Ba
¨ue l C, Collado MC, Zu
´n
˜iga M, Blas E, Pe
´ ez Ma ı
´nez G (2014) Changes in Cecal Mic obio a and Mucosal Gene Exp ession Re ealed New Aspec s o
Epizoo ic Rabbi En e opa hy. PLoS ONE 9(8): e105707. doi:10.1371/jou nal.pone.0105707
Edi o : Mo
´nica V. Cunha, INIAV, I.P.- Na ional Ins i u e o Ag icul u e and Ve e ina y Resea ch, Po ugal
Recei ed Ap il 3, 2014; Accep ed July 22, 2014; Published Augus 22, 2014
Copy igh : ß2014 Ba
¨ue l 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.
Da a A ailabili y: The au ho s con i m ha all da a unde lying he indings a e ully a ailable wi hou es ic ion. Da a a e a ailable om MG-RAST as he
Rabbi -ERE-p ojec using he accession numbe 4543253.3.
Funding: This wo k was suppo ed by he g an s AGL2010-22211, AGL2006-07596 and by he Fun-C-Food CSD2007-00063 p ojec om he Consolide -Ingenio
p og amme, bo h om he Spanish Minis y o Science and Inno a ion. 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 .
* Email: [email p o ec ed]ic.es
.These au ho s con ibu ed equally o his wo k.
In oduc ion
The 19
h
cen u y wi nessed he eme gence o animal ancy and
abbi s (O yc olagus cuniculus) began o be aised also as pe s, and
a p esen days hey a e among he mos impo an labo a o y
models. Rabbi s a e hindgu e men e s, hei cecum has a la ge
size and, wi h he s omach, i has a e y ele an ole in he
diges i e unc ion. Cop ophagy (caeco ophy) is a e y cha ac e -
is ic habi o his species ha aids o comple e he diges ion o
ege able componen s, acili a es he assimila ion o p o eins and
o he nu ien s syn hesized by cecal bac e ia and main ains gu
bac e ial popula ions. In es inal heal h o domes ic abbi s is qui e
delica e and any dis up ion o he diges i e p ocess esul s in
gas oin es inal diseases, mos equen ly ela ed o die o s ess
[1]. A high incidence o diges i e diseases o unknown ae iology in
young mammals is o en ela ed o dis o ions in mic obio a
composi ion [2]. The con ibu ion o he gas oin es inal ac
mic obio a owa ds mammalian hos heal h and pe o mance is
now widely accep ed. P e ious s udies on abbi cecal mic obio a
pe o med using classical cul u e-based echniques [3] and
ecen ly, by molecula echniques [4], showed ha bac e ial
species a e mainly s ic anae obes wi h p edominance o he
phylum Fi micu es o e Bac e oides-P e o ella.
Epizoo ic abbi en e opa hy (ERE) is a se ious a m abbi
disease ha eaches 30–95% mo ali y, independen o he b eed,
wi h a pa icula incidence in he pos -weaning pe iod [5]. The
main symp oms a e cecal impac ion, abdominal dis ension wi h
gas and luid accumula ion in s omach and in es ines, le ha gy,
hunched pos u e, subno mal empe a u e and copious clea
mucus dia hoea some imes p eceded by cons ipa ion, o which
i is also called Mucoid En e opa hy. In acu e o m, clinical signs
p ecede dea h by 1–3 days, o 7–9 days in a longe e m condi ion.
The mos ele an his ological ea u e is a ema kable goble cells
hype plasia o ileum, cecum, e mix, sacula ed colon and colon
[6]. In p ac ice, exhaus i e hygienic measu es a e equi ed o
cons ain his epizoosis and, in some cases, only sus ained
an ibio ic medica ion p e en s ERE mo ali y. A g ea e o has
been de o ed o s udy he ae iology o ERE and i is now accep ed
PLOS ONE | www.plosone.o g 1 Augus 2014 | Volume 9 | Issue 8 | e105707
ha bac e ial elemen s a e in ol ed in he pa hogenesis, since
an ibio ic ea men s a e usually e ec i e in he p e en ion o
ERE, and also because he cecal con en il a e o ERE abbi s
inocula ed o heal hy animals can ep oduce he disease [7].
Howe e , o he iden i ied and uniden i ied ac o s ela ed o he
en i onmen , husband y condi ions and du a ion o he weaning
pe iod ce ainly pa icipa e in he de elopmen o ERE [8]. Die is
impo an , as he disease has been ep oduced p o iding eed wi h
a educed ib e con en and liga ion o he cecum has been shown
o be an in asi e, bu e ec i e o m o ep oduce he disease [6,8].
Exhaus i e bac e iological analyses ca ied ou in he pas could
no esol e he mic oo ganisms in ol ed, al hough clos idial
species and coli o ms ha e been mos equen ly ound in diseased
abbi s [6,8–10].
A e y ele an ea u e o ERE is he abundan p oduc ion o
mucus h oughou he small, bu ema kably, in he la ge in es ine
and cecum [8]. Gas oin es inal mucins p oduced by goble cells
a e he main s uc u al componen s o he mucus laye ha has a
c ucial lub ica ing and p o ec i e ole. Mucins a e syn hesized as a
i s line o de ence and accumula e di e en ial e ec o and
egula o y esponses agains a ple ho a o commensal and
pa hogen mic oo ganisms. Thei syn hesis is induced by p o-
in lamma o y s imuli s emming om pa hogen in asion o
chemical agg ession o he epi helia, as pa o he inna e de ence
sys em, bu also mic oo ganisms and i uses e icien ly ecognize
mucins as binding a ge s [11]. ERE his opa hological signs ha e
also been desc ibed [7,8], bu u he biochemical, immunological
o molecula echniques ha e no been implemen ed, and
he e o e, a deepe pe spec i e o he disease is missing.
In his s udy, qPCR and py osequencing o 16S DNA
amplicons we e used o desc ibe and compa e he cecal mic obio a
in heal hy abbi s and o analyse he impac o an ibio ic ea men
and ERE on he mic obio a composi ion. In addi ion, gene
exp ession o mucins and p o-in lamma o y cy okines was also
de e mined and co ela ions wi h a ia ions in cecal mic obio a
we e es ima ed. This s udy also e ealed new bac e ial g oups
signi ican ly ela ed o ERE and new insigh s on he pa hology o
his disease.
Ma e ials and Me hods
Animals, eeding and housing condi ions
Animals used in he expe imen we e h ee-way c ossb eed
abbi s om ma e nal lines A and V and pa e nal line R,
de eloped by he Poly echnic Uni e si y o Valencia (Animal
B eeding Uni ), Spain. They we e g own in an expe imen al a m
ha kep s anda d comme cial handling condi ions a he
Poly echnic Uni e si y o Valencia. Lac a ion las ed 30 days and
animals we e sepa a ed in g oups o en pe cage in he a ening
a ea. A e weaning a g oup o abbi s (an ibio ic ea men ) was
ed ad libi um wi h comme cial medica ed pelle ed abbi eed
(Nan a, Nu eco, Spain) con aining lyncomycin (29 ppm), spec i-
nomycin (29 ppm), iamu in (40 ppm) and neomycin (121 ppm). A
second g oup o abbi s was ed wi h a ba ch o he same eed
wi hou an ibio ics. In o de o inc ease he incidence o ERE
hese abbi s we e housed in a a ening sec ion o he a m ed
wi hou an ibio ics, ba ch a e ba ch, du ing 4 mon hs p e ious o
he expe imen . A he age o 40 days, en heal hy animals and en
animals su e ing ypical ERE symp oms, like apa hy, yellowish
pe ianal ecal s ain (mucus), cecal impac ion and wa e y sound in
he gu we e sampled a andom and om di e en cages o he
sec ion wi hou an ibio ics. Also en abbi s wi h an ibio ic
ea men we e selec ed andomly in he a m. All abbi s we e
collec ed on he same day. Heal hy o ERE animals (gas in he
s omach and impac ion o he cecum) ha upon nec opsy could
no be unequi ocally diagnosed we e elimina ed om he s udy
( o al o 2). The comple e clean cecum was ex ac ed a e liga ion
a ileo-cecal al e and he comple e o gan, including he e mix,
was immedia ely ozen a 280uC un il use.
This s udy was ca ied ou in s ic acco dance wi h he Spanish
na ional ules (RD223/1988 and RD1201/2005) ha p o ec
animals used in expe imen a ion and o he scien i ic pu poses.
The p o ocol was app o ed by he Commi ee on he E hics o he
Poly echnic Uni e si y o Valencia (UPV). Due o hei small size,
animals we e eu hanized by ce ical disloca ion and hen
dissec ed.
Nucleic acids ex ac ion om cecal con en , cecal
mucosa and e mix
The ozen cecum con en was agmen ed in pieces o
app oxima ely 5 g and quickly de os ed. Then, o al DNA was
ex ac ed om 200–300 mg samples o cecal con en using a
Qiagen s ool DNA ex ac ion ki (QIAgen, Hilden, Ge many)
acco ding o manu ac u e ’s ins uc ions wi h a p e ious dis up-
ion wi h a bead bea ing s ep.
RNA samples o q-RT-PCR we e ob ained as ollows. Cecal
mucosa ci cula agmen s o 10–15 mm diame e and ans e sal
e mix sec ions o 5 mm we e ca e ully excised and immedia ely
subme ged in TRIzol Reagen (In i ogen). The issue was
homogenized wi h a Poly on de ice and o al RNA was isola ed
using he RNeasy Mini Ki (Qiagen) acco ding o he manu ac-
u e s’ ins uc ions. Con amina ing genomic DNA was diges ed
using Deoxy ibonuclease I (Sigma) and hen, RNA quan i y and
quali y was e alua ed using he Agilen 2100 Bioanalyse (Agilen ).
Re e se ansc ip ion eac ions we e pe o med using he T an-
sc ip o Fi s S and cDNA Syn hesis Ki (Roche) acco ding o he
ins uc ions o he manu ac u e .
Quan i a i e eal- ime PCR (qPCR) analysis
The qPCRs o speci ic bac e ial g oups we e conduc ed as
p e iously desc ibed [12]. qPCR ampli ica ion and de ec ion was
pe o med in a Ligh Cycle 480 Real-Time PCR Sys em (Roche).
Each eac ion mix u e o 10 ml consis ed o SYBR G een PCR
Mas e Mix (Roche), 0.5 ml o each o he speci ic p ime s [12] a a
concen a ion o 0.25 mM, and 1 ml o empla e DNA. We also
analysed me hanobac e ia using Me hanob e ibac e genus-speci -
ic p ime s (MET-105 and MET-386 ) and M. smi hii ni H gene
speci ic p ime s (Mni -342 and Mni -363 ) as desc ibed elsewhe e
[13]. A mel ing cu e analysis was made a e ampli ica ion o
assess he speci ici y o he ampli ica ion eac ion. The bac e ial
concen a ion in each sample was calcula ed by in e pola ion o
he ob ained C
alues o s anda d cu es. These we e c ea ed
using se ial 10- old dilu ion o pu e cul u e-speci ic DNA
agmen s o known size co esponding o 10 o 10
9
numbe o
agmen gene copies/ml.
Tagged PCR ampli ica ion o bac e ial 16S ibosomal
genes o py osequencing
A ba coded p ime se based on uni e sal p ime s 27F (59-
GAGTTTGATCMTGGCTCAG-39), and 518R (59-
WTTACCGCGGCTGCTGG-39), we e used o ampli y a
500 bps o he 16S RNA genes encompassing he V3 egion.
The PCR was ca ied ou using a high- ideli y KAPA-HiFi
polyme ase (KappaBiosys ems, US) wi h an annealing empe a-
u e o 52uC and 20 cycles o minimize PCR biases. Pu i ied PCR
p oduc s we e pooled in equimola amoun s, as 454 Roche
p o ocols desc ibe, and submi ed o py osequencing using he
Rabbi Cecal Mic obio a and Gene Exp ession in ERE
PLOS ONE | www.plosone.o g 2 Augus 2014 | Volume 9 | Issue 8 | e105707
Genome Sequence FLX Ti anium Se ies (454 Li e Science,
B an o d, USA). All o he p ocedu es ollowed he manu ac u e ’s
di ec ions (454 Li e Science) and we e conduc ed a Mac ogen
(Seoul, Sou h Ko ea). Bo h chains o all amplicons we e sequenced
o assu e high quali y da a. Sequence da a ob ained in his wo k
a e a ailable in MG-RAST (h p://me agenomics.anl.go /) as
Rabbi -ERE p ojec wi h accession Numbe 4543253.3.
Gene exp ession analysis o abbi genes h ough eal
ime qPCR
Fo e e se ansc ip ion 5 mg o o al RNA we e used. The
eac ions we e pe o med using he T ansc ip o Fi s S and
cDNA Syn hesis Ki (Roche) acco ding o he ins uc ions o he
manu ac u e . Quan i a i e eal- ime PCR was pe o med on a
Ligh Cycle 480 Real Time PCR Sys em using SYBR G een I
Mas e Mix (Roche). PCR cycling condi ions comp ised an ini ial
polyme ase ac i a ion s ep a 95uC o 10 min ollowed by 40
cycles o 10 s a 95uC, 10 s a 60uC and 12 s a 72uC. qPCR
p ime s we e designed using he P ime -Blas ool (h p://www.
ncbi.nlm.nih.go /) and a e lis ed in Table S14. qPCR p ime s
we e alida ed o con i m e iciency h ough se ial dilu ions. PCR
p oduc s we e con i med by aga ose gel elec opho esis o yield a
unique band and, addi ionally, a e each qPCR un a dissocia ion
cu e was pe o med.
S a is ical analysis
Fo he analysis o qPCR da a, IBM-SPSS 19.0 so wa e (SPSS
Inc., Chicago, IL, USA) was used. Due o non-no mal dis ibu-
ion, mic obial da a a e exp essed as medians wi h in e qua ile
anges (IQR). Compa isons among da a o mo e han wo g oups
o abbi s we e done by applying he Wilcoxon/K uskal-Wallis
es , and compa isons be ween da a o wo g oups we e done by
applying he Mann-Whi ney U es . The Bon e oni adjus men
es was also applied o co ec he signi icance o mul iple es
compa isons among h ee g oups. The x-squa e es was used o
es ablish di e ences in he bac e ial p e alence be ween he
s udied g oups. A P,0.050 was conside ed s a is ically signi ican .
The possible co ela ion be ween a iables was s udied by
applying Pea son’s co ela ion coe icien , and signi icance was
es ablished a 0.05%.Rega ding py osequencing, low quali y
sequences we e il e ed ou o emo e sequences ha ing a leng h
sho e han 100 nucleo ides om aw da a se s. Sequences we e
aligned and classi ied agains he SILVA comp ehensi e RNA
da abase (h p://www.a b-sil a.de/). A de eplica e eques on he
pipeline was used o iden i ying he ep esen a i e sequences o
each ope a ional axonomic uni (OTU) gene a ed om he
comple e linkage clus e ing wi h a 97% simila i y. A e axono-
mical assignmen o py osequencing da a, ela i e equencies o
di e en axonomic ca ego ies ob ained we e calcula ed using he
S a is ical Analysis o Me agenomic P o iles p og am (STAMP
.2.0.0) [14]. S a is ical di e ences be ween expe imen al abbi
g oups we e es ima ed by ANOVA analysis wi h he Games-
Howell pos -hoc es and he mul iple es co ec ion o Benjamini-
Hochbe g as implemen ed in STAMP. Ra e ac ion cu es we e
calcula ed using he Ra e ac Win p og am (h p://www.uga.edu/
,s a a/so wa e/So wa e.h ml) and also, alpha di e si y indexes
we e de e mined wi h he QIIME pipeline om a e ied ables
using he Shannon-Wiene index o di e si y, he Chao1 index
o ichness and also, Obse ed Species (numbe o unique OTUs)
and Phylogene ic Dis ance (PD_whole). A be a di e si y dis ance
ma ix was compu ed om he p e iously cons uc ed OTU able
using UniF ac analysis. Unweigh ed (p esence/absence me ix)
and weigh ed (p esence/absence/abundance me ix) UniF ac
dis ances we e used o cons uc wo- and h ee-dimensional
P incipal Coo dina es Analyses (PCoA) plo s. Biplo s we e
gene a ed as pa o he be a di e si y analysis in Qiime using
genus le el OTU ables showing p inciple coo dina e clus e ing o
samples alongside weigh ed axonomic g oup da a. Da a on
assigned sequences a he genus le el sha ed be ween samples we e
used o gene a e a Venn diag am.
Rela i e gene-exp ession was quan i ied acco ding o he
e iciency-co ec ed me hod [15] using he REST 2009 so wa e
ool. Di e ences in inpu cDNA we e no malized o glyce alde-
hyde-3-phospha e dehyd ogenase (GAPDH) and b-ac in (ACTB)
exp ession.
Finally, co ela ion analysis was pe o med wi h he S a -
g aphics p og amme, as well as he P incipal Componen Analysis
(PCA) o he sequences and exp ession da a, wi h ecip ocal
p ojec ions, which we e hen d awn wi h SigmaPlo 10.0.
Resul s
Bac e ial popula ions in he cecum o heal hy abbi s
A o al o 89,091 16S RNA double s anded sequence eads
we e ob ained om 10 cecal samples o heal hy indi iduals ed
wi h non-medica ed eed. The a e age ead leng h was 492.5 bp.
Using he SILVA da abase o axonomic assignmen , he mos
common phyla we e Fi micu es we e Fi micu es (78.25% o o al
eads), Bac e oide es (15.75%), Ve ucomic obia (2.40%), and
Tene icu es (2.39%). Wi hin Fi micu es, in he o de Clos idiales,
he mos abundan amilies we e Ruminococcaceae (42.48% o
o al eads) and Lachnospi aceae (34.85%) while in he o de
Bac e oidales, Rikenellaceae (6.38%) was he mos abundan
amily. We ound 90 di e en gene a in cecal samples o heal hy
abbi s om which he p edominan ones we e Alis ipes (5.63% o
o al eads), Ruminococcus (4.02%), Akke mansia (2.40%) and
Subdolig anulum (2.28%). Howe e , he 20 mos abundan
sequences (37.76% o o al eads) could no be classi ied a he
species le el: 15 o hem belonged o he class Clos idia ( amilies
Lachnospi aceae and Ruminococcaceae), including he mos
abundan sequence in heal hy abbi s EF445173 al eady epo ed
[3] (8.34% o o al eads) ( amily Lachnospi aceae); 4 o he class
Bac e oidia ( amily Rikenellaceae); and one belonged o he class
Ve ucomic obia (genus Akke mansia; Tables S1, S2, S3, S4).
Impac o an ibio ic ea men and ERE on he cecal
mic obio a
Fu he 20 cecal samples o abbi s belonging o wo addi ional
g oups, heal hy ea ed wi h an ibio ics (A) and ERE abbi s (E),
we e collec ed in o de o be compa ed o he p e ious heal hy
abbi s wi hou an ibio ic ea men , used as con ol g oup (C).
One sample ailed in he an ibio ic g oup (A) so ha a o al o 19
samples we e analysed. A e quali y il e ing and leng h imming
199,217 16S DNA eads we e analysed, wi h an a e age numbe
o axonomically assigned high-quali y double s anded eads o
10,485 pe abbi wi h an a e age size o 492 bp (Tables S1, S2,
S3, S4).
The domina ing Phyla in all abbi s we e Fi micu es ollowed by
Bac e oide es. Class Clos idia (Fi micu es) was mo e abundan in
heal hy g oups (A = 85.49%, C = 78.06%, E = 54.07%; co ec ed
p,0.001) and class Bac e oidia in he ERE g oup (A = 8.83%,
C = 15.73%, E = 22.18%; co ec ed p = 0.013). ERE abbi s
showed a ema kable inc ease in P o eobac e ia, pa icula ly class
c-P o eobac e ia (A = 0.016%, C = 0.023%, E = 10.03%; co ec -
ed p = 0.003). The numbe o Ve ucomic obiae was also highe
in ERE (A = 0.84%, C = 2.40% E = 8.40%, co ec ed p = 0.009)
mos ly due o a g ea inc ease o eads o he genus Akke mansia
(see below). O he axons wi h lowe o al numbe s showed
Rabbi Cecal Mic obio a and Gene Exp ession in ERE
PLOS ONE | www.plosone.o g 3 Augus 2014 | Volume 9 | Issue 8 | e105707
ema kably di e en p opo ions in he ERE g oup: class Bacilli
was mo e abundan in he ERE g oup (A = 0.07%, C = 0.02%,
E = 1.1%), and Tene icu es (class Mollicu es; A = 3.68%,
C = 2.39%, E = 0.44%) less equen in diseased animals.
A e he sc u iny o o al coun s o bac e ial amilies in he h ee
g oups impo an di e ences we e de ec ed (Figu e 1). An ibio ic
ea men (A) educed he numbe o eads in he amilies
Clos idiaceae and Ruminococcaceae. A numbe o bac e ial
amilies we e in high numbe s o speci ically p esen only in he
ERE g oup, such as Ve ucomic obiaceae, En e obac e iaceae
and Bac e oidaceae (Figu e 1). A a lowe axonomical le el, he
mos equen gene a in heal hy g oups (A,C) we e Ruminococcus
and Alis ipes, and in ERE abbi s Bac e oides (12,45%), Akke -
mansia (8,40%), Esche ichia (8,25%), Rikenella (3.40%) as well as
Clos idium (1.24%) showed a majo p esence (Figu e 2, Table
S4). The compa ison o unclassi ied sequences showed ha wo
Lachnospi aceae eads o unknown genus al eady epo ed obese
humans DQ799912.1.1389 [16] and anae obic sludge
CU919535.1.1344 [17] accoun ed o 8.96% and 4.87% espec-
i ely o o al iden i ied sequences in ERE abbi s. In addi ion,
po en ial oxin p oducing Fi micu es ha e also been ound, such as
species o he genus Lysinibacillus, wi h 0.99% o o al eads in
ERE cecal samples.
The h ee g oups showed a ema kable di e si y, wi h
a e ac ion cu es indica ing a numbe o Ope a ional Taxonomic
Uni s (OTUs) o e 10,000 when eads we e clus e ed a 97%
sequence iden i y ( he consensus alue o de e mining species
bounda ies). Ra e ac ion cu es, a OTU le el, show ha he ERE
g oup had lowe bac e ial di e si y han he con ol and an ibio ic
g oups (Figu e 3). The slope a he end o he cu es o An ibio ic
and Con ol g oups sugges s ha mic obio a is e y di e se and a
new e o s would be needed o ep esen he comple e abbi cecal
mic obiome. The 16S sequences o all indi iduals in each g oup
we e pooled, wi h an ope a ional axonomic-uni de ini ion se a
97% sequence iden i y. An OTU was a clus e o 16S RNA
sequences which we e o e 95% iden ical, a conse a i e es ima e
o he bounda y be ween species, es ablished a 97% o ull-
leng h 16S gene sequences (Each a e ac ion cu e is plo ed,
along wi h i s 95% con idence in e al). As was seen in he p ima y
analysis, he o e all di e si y in ERE g oup was signi ican ly lowe
han ha in con ol subjec s and an ibio ic ea men . O e all
di e si y o axa wi hin he samples was de e mined by alpha
di e si y me ics (Chao1 me ic es ima es he species ichness;
Shannon es ima es he di e si y; he Obse ed Species; Phyloge-
ne ic Dis ance (PD_whole_ ee)) showed lowe di e si y, phyloge-
ne ic ichness and e enness in ERE g oup compa ed o hose
obse ed in he o he wo g oups: con ol and an ibio ic g oup
(Figu e 3). Venn diag am shows a b oade pe spec i e, whe e
Con ol and An ibio ic g oups sha e mos o he bac e ial amilies
and gene a. A co e o 13 amilies and 24 gene a we e p esen in
ERE, an ibio ic and con ol g oup (Figu e 4).
P incipal Coodina es Analysis (PCoA) o he samples clea ly
sepa a ed he ERE abbi s om he es o g oup samples in bo h,
weigh ed (Figu e 5, panel A) and unweighed (Figu e 5, panel B)
plo s (PC1 accoun ing o 14% o he a iance in unweigh and
50% in weigh Uni ac analysis), indica ing ha he ERE
mic obio a is composi ionally dis inc . Heal hy indi iduals wi h
o wi hou an ibio ic ea men (An ibio ic,Con ol) clus e ed
oge he and sepa a ely om he ERE g oup o animals. This
indica ed ha mic obio a om g oups An ibio ic and Con ol a e
no signi ican ly di e en , bu a ema kable dysbiosis is a ec ing
ERE abbi s. In o de o explo e axonomic d i ing ac o s o
pa e ns in a PCoA bac e ial gene a we e plo ed in he same
diag am o he unweigh ed PCoA o all samples (Biplo ), whe e
ela i e abundance o hese axons we e also ep esen ed by he
sphe e sizes. The genus Bac e oides clea ly clus e ed wi h ERE
samples, clus e ing o heal hy samples was d i en by wo
uncha ac e ized gene a o he Ruminococcaceae amily, one
Figu e 1. To al eads o bac e ial amilies iden i ied by SILVA
a e py osequencing o he 16S RNA o he cecal con en o
abbi s. The g aph ep esen s he a e age o each o he h ee
g oups: con ol (whi e), an ibio ic ea men (g ey) and ERE (black).
doi:10.1371/jou nal.pone.0105707.g001
Figu e 2. P opo ional piled up column g aph ep esen ing he
a e age abundance o he di e en bac e ial gene a o he
con ol g oup (heal hy, no an ibo ics) (C), abbi s ea ed wi h
an ibio ics (A) and ERE animals (E). Mos abundan gene a a e
ma ked on he ba .
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Rabbi Cecal Mic obio a and Gene Exp ession in ERE
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genus o Lachnospi aceae and a less abundan genus o he
Clos idia class (Figu e 5, panel C).
Finally, qPCR was pe o med o analyse he p esence o ce ain
species and o con i m signi ican di e ences in he p opo ion o
speci ic axonomic g oups be ween ERE, an ibio ic and heal hy
con ol g oup samples (Table S5). Signi ican ly highe le els o
Akke mansia muciniphila,Bac e oides-P e o ella and Clos idium
coccoides g oup we e obse ed in ERE g oups compa ed o hose
obse ed in con ol (p = 0.001, p = 0.042 and p = 0.050, espec-
i ely) and an ibio ic (p = 0.002, p = 0.002 and p = 0.049, espec-
i ely). Fu he mo e, we de e mined he p esence o bac e ia om
he genus Me hanob e ibac e as example o A chaea in abbi
cecal samples. A signi ican ly highe le el o he bac e ia o he
genus Me hanob e ibac e was obse ed in he ERE g oup
compa ed o he heal hy con ol and an ibio ic g oups (p,
0.0001), al hough he species M. smi hii was de ec ed mo e o en
in heal hy con ols (4/10, 40%) and an ibio ic g oup (3/10, 30%)
han in ERE g oup (1/10, 10%). Howe e , o e all p opo ions o
me anobac e ia (Me hanob e ibac e ) a e e y small, as ampli ied
molecules o 16S DNA we e wo (C g oup) o i e (E g oup)
o de s o magni ude below o al bac e ia.
Mucin and cy okine exp ession in he cecum
One o Epizoo ic Rabbi En e opa hy main physiological
symp oms is he abundan sec e ion o mucins a he cecum and
colon [18], o which moni o ing hei syn hesis du ing he cou se o
he disease, compa ed o ha in heal hy animals, was conside ed a
p io i y issue. Mucin sec e ion by he goble cells is known o be
induced by in lamma o y signals, equen ly a e pa hogen challeng-
es [11], hence we sea ched in he abbi genome p ojec (h p://
www.ncbi.nlm.nih.go /genome? e m = o yc olagus%20cuniculus) in
o de o design sui able p ime s o q-RT-PCR o mucin and
cy okine encoding genes. MUC1, MUC4 and MUC13 we e
success ully es ed and also he SAM-poin ed domain-con aining
E s-like ac o gene (SPDEF), an ac i a o o goble cells which is
induced by p o-in lamma o y signals and by pa hogen s imuli, ha
also egula es mucine exp ession [19]. Homologous gene o MUC2,
he dominan in es inal mucin in humans, was no a ailable a he
onse o his s udy. Also p ime s o abbi p o-in lamma o y
cy okines and lymphocy e di e en ia ion p ecu so s we e designed
(TNF-a,IL-4,IL-6,IL-8,IFN-cand IL-2). Gene exp ession o all he
genes was de e mined in cecal mucosa samples o all 30 abbi s
(Table S7), and exp ession da a could be success ully calcula ed wi h
he excep ion o IL-4 whose C
was oo high o allow eliable
calcula ions in cecal mucosa. T ansc ip ion da a we e calcula ed as
ela i e exp ession o ERE (E) and an ibio ic g oups (A) agains he
mean alue o he heal hy con ol (C). Rabbi s su e ing ERE
displayed an inc eased ansc ip ion le el o all mucins, especially
MUC1 (Figu e 6, panel A). The a e age exp ession alues o
SPDEF had no signi ican inc ease in any o he h ee g oups,
howe e , pa icula di e ences in he ERE g oup will be dissec ed
below. Exp ession o cy okines IL-8, TNF-aand IL-6 we e also
signi ican ly up egula ed (Figu e 6, panel A). This esul showed a
di ec linkage be ween he disease s a us and mucin syn hesis in he
cecal epi helium o ERE abbi s, indica ing he p esence o a s ong
p o-in lamma o y s imulus in he cecal con en s. IFN-cshowed a
small induc ion, whe eas IL-2 had a mode a e dec ease in
ansc ip ion. Howe e , a g ea a iabili y was no iced in he
ansc ip ion a es o he cy okines wi hin he ERE g oup, o his
eason we decided o u he analyse his g oup (see below). Li le
di e ences we e ound be ween he ansc ip ion a es o abbi s
ea ed wi h an ibio ics (A) and he con ol g oup (C), besides a sligh
endency o o e p oduce mucins in he A g oup (Figu e 6, panel A).
Figu e 3. Ra e ac ion cu es ep esen an app oxima ion o he
numbe o species iden i ied o he o al numbe o sequences
ob ained (A). The numbe o species was ep esen ed by he numbe o
ope a ional axonomic uni s (OTUs). Cu es we e calcula ed wi h
Ra e ac Win so wa e a 97% simila i y le el co esponding o species-
le el phylo ypes. Ra e ac ion analysis also included he es ima ion o
he di e si y o bac e ial axa in each sample wi h di e en me ics
wi hin he QIIME pipeline, such as he alpha di e si y indices: Chao1 (B),
Shannon (D), Phylogene ic dis ances (C) and Obse ed Species (E).
doi:10.1371/jou nal.pone.0105707.g003
Figu e 4. Venn diag am showing he ela i e abundance a he
genus le el based on he classi ica ion o he pa ial 16S DNA
bac e ial sequences om cecal samples o an ibio ic, heal hy
con ol and ERE g oups using he RDP classi ie so wa e o
axonomy assignmen : con ol g oup (heal hy, no an ibio ics)
(Con ol), abbi s ea ed wi h an ibio ics (An ibio ic) and ERE
animals (ERE).
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Fu he mo e, gene exp ession da a in cecal mucosae o ERE was
analysed in de ail. A ema kable and highly signi ican co ela ion
was de ec ed be ween TNF-aand IL-6 and IL-8, as expec ed om
hei di ec connec ion wi h canonical p o-in lamma o y signaling
pa hways (i.e. NF-kB), and also connec ion o IFN-cwi h TNF-a
and IL-6 and IL-8 could be con i med. Rega ding mucin
exp ession, we ound a posi i e co ela ion (low signi icance, p#
0.1) o SPDEF exp ession wi h MUC13 sugges ing egula o y
connec ions, and a nega i e co ela ion wi h IL-2 and INFc,
sugges ing a e e se ( ep essing) egula o y connec ion, bu no
ela ionship o SPDEF wi h he wo o he mucins MUC1 and
MUC4. Howe e , MUC13 nega i e co ela ion o INFcand TNF-
awas g ea e and mo e signi ican , and also ha o MUC1 wi h IL-
2 (Table S6).
Then, PCA o gene exp ession da a in he cecal mucosa was
pe o med and d awn (Figu e 7). Va iables clea ly g ouped in wo
clus e s, one including cy okines (IFN-c, TNF-a, IL-6 and IL-8)
and he second, mucins (MUC13, MUC1, MUC4 and he
egula o SPDEF). They we e sepa a ed along he o dina e axis
(second componen ) and IL-2 emained dis an om ei he g oup,
unde lining an independen exp ession pa e n. In ac , exp ession
o his cy okine showed e y small a ia ion be ween heal hy and
ERE abbi s. When da a om all abbi s we e p ojec ed on he
gene exp ession PCA, heal hy indi iduals (Con ol and An ibio ic
g oups) clus e ed igh ly a he nega i e (le ) side o he abcise axis
(PC1), while ERE animals we e all p ojec ed in he posi i es alues
( igh ). In e es ingly, abbi s su e ing he disease we e sepa a ed in
wo subse s along he o dina e axis (PC2) wi h abbi s E5, E6, E8
(n = 3) and E1, E2, E3, E4, E7, E9, E10 (n = 7) close o he clus e
Figu e 5. P incipal Coo dina es Analysis (PCoA) 3D plo s gene a ed wi h UniF ac showing clus e ing o OTUs bac e ial g oups o
cecal samples om ERE, and Heal hy (Con ol and An ibio ic) g oups. PCoA is a Mul idimensional Scaling Me hod ha allows o explo e and
o isualize dissimila i ies o phylogene ic da a om a dis ance ma ix impo ed om RDP, assigning a loca ion o each sample in a 3D g aphics. Panel
A, unweigh ed plo ; panel B, weigh ed plo ( aking in o accoun he ela i e abundance o each axon in he samples); panel C, Biplo ep esen a ion
o PCoA o unweigh ed, pai wise Uni ac dis ances showing clus e ing o bac e ial g oups cecal samples om ERE, Con ol and An ibio ic g oups. Blue
sphe es a e Con ol abbi s, ed sphe es a e he An ibio ic g oup and ligh b own sphe es a e he ERE abbi s. In panel C, he Biplo shows he
p ojec ion o axa posi ions (g ey sphe es) ha ep esen weigh ed a e ages o he coo dina es o all samples. The size o he sphe es is p opo ional
o he ela i e abundance o he axon.
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o in lamma o y cy okines and mucins, espec i ely. In ac he
exp ession p o iles o cy okines and mucins o hese wo subg oups
we e e y di e en . Samples o he i s g oup (E5, E6, E8) had
e y high exp ession o p o-in lamma o y cy okines, IL-6x16, IL-
8x8, TNF-ax3 and IFN-cx11 old g ea e han he o he g oup,
while samples in he second g oup (E1, E2, E3, E4, E7, E9, E10)
had highe exp ession o mucins (Table 1, Table S8), o which
hey we e assigned o In lamma o y P o ile and Sec e o y P o ile,
espec i ely. Un o una ely he no he In lamma o y P o ile was
low (n = 3) o u he compa isons.
Mucin and cy okine exp ession in he e mix
T ansc ip ion was also assayed wi h e mix samples, as hese
samples could be en iched in lymphocy es (Figu e 6, panel B).
In e es ingly, he e we e signi ican di e ences be ween ansc ip-
ion le els o Con ol samples and ERE in all genes s udied, excep
IFN-cand TNF-a, and mucin genes (MUC1, MUC13, MUC4)
and cy okine genes IL-4, IL-6, IL-8 ollowed a simila endency o
ha in he cecal mucosa, al hough IL-4 could no be quan i ied in
he cecal mucosa. Induc ion o mucins mRNA was much lowe
han in he cecum (Figu e 6, panel A). No signi ican di e ences
we e ound be ween an ibio ic and he con ol g oups (Figu e 6,
panel B).
When compa ing only gene exp ession o e mix and cecal
samples o ERE animals, we de ec ed no up egula ion o IFN-c
and TNF-ain e mix, bu a mode a e o e exp ession o IL-2 ( wo
Figu e 6. G aphs ep esen ing ela i e gene exp ession da a de e mined by q-RT-PCR. Values a e calcula ed ela i e o he Con ol g oup
and no malized o endogenous ACTB and GAPDH exp ession. Box plo s show he median alue (solid line inside boxes), he limi s o box ep esen
he 25 h and 75 h pe cen ile, and whiske s depic he 5 h and 95 h pe cen ile (*P,0.05 s. Con ol; **P,0.001 s. Con ol).
doi:10.1371/jou nal.pone.0105707.g006
Figu e 7. PCA o cy okine gene exp ession, whe e axonomic
da a om each abbi sample a e p ojec ed. Red ci cles indica e
g oupings o gene exp ession da a. Blue ci cles indica e he posi ion o
indi idual abbi s.
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Rabbi Cecal Mic obio a and Gene Exp ession in ERE
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Table 1. Mean alues and s anda d de ia ion o gene exp ession alues, ela i e o con ol abbi s, o he wo likely p o iles (In lamma o y and Sec e o y) ound in abbi s
su e ing ERE.
Cecum Ve mix
In lamma o y p o ile (n = 3) Sec e o y p o ile (n = 7) In lamma o y p o ile (n = 3) Sec e o y p o ile (n = 7)
Mean SDe Mean SDe P alue Mean SDe Mean SDe P alue
MUC1 21.62 4.07 31.75** 11.66 0.039 6.84 1.95 8.80 4.46 0.180
MUC13 7.04 2.60 17.35** 5.96 0.003 7.90 2.55 8.47 3.39 0.390
IL2 1.07 0.48 0.62 0.38 0.120 3.88 2.56 1.81 1.10 0.146
IFN-c17.24** 4.02 1.54 1.14 0.009 1.84 1.65 1.11 0.66 0.263
MUC4 22.15 17.57 12.62 10.52 0.227 4.89 2.30 2.71 1.06 0.119
IL8 123.20 113.26 15.63 16.93 0.120 22.35 19.25 3.95 3.88 0.119
TNF-a20.81** 6.06 6.47 6.72 0.013 1.88* 0.87 0.94 0.32 0.097
IL6 341.28* 229.66 21.24 27.43 0.068 4.74 2.57 2.45 1.12 0.130
IL4 - - - - - 4.17** 0.88 2.74 1.08 0.040
SPDEF 0.74 0.32 1.71** 0.79 0.013 3.47 1.76 4.29 1.82 0.269
As e isks ep esen a e ages signi ican ly g ea e in one p o ile han he o he , wi h signi icance:
(*)p#0.1;
(**)p#0.05.
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Rabbi Cecal Mic obio a and Gene Exp ession in ERE
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old) and SPDEF ( ou old). Exp ession da a om e mix wi hin
he ERE g oup we e u he analysed in o de o asce ain i he e
could also be wo p o iles, as hose ound in he cecum samples.
In e es ingly, IL-4 exp ession in e mix samples could be
quan i ied and i was signi ican ly g ea e in he In lamma o y
P o ile han in he Sec e o y P o ile.
Cecal mic obio a and gene exp ession ela ionships
Di e en s a is ical analysis we e un o de e mine likely
co ela ions be ween indi idual gene exp ession da a and bac e ial
equencies, wi h he aim o ela e possible o ganisms o bac e ial
axons o he pa hogenesis o ERE. As expec ed, global co ela ion
analysis o ela i e gene exp ession and mic obial g oups in all
abbi s showed ha high exp ession o MUC1, MUC13, MUC4,
IL-8 and IL-6 and TNF-awas in all cases signi ican ly bound o
high eads o hose bac e ial axons mos abundan in ERE, such
as o de s Ve ucomic obiales, En e obac e iales and Bac e oi-
dales, and on he con a y, high exp ession o hose ma ke s we e
co ela ed o low le els o Clos idiales eads belonging o he class
Clos idia ( amilies Lachnospi aceae and Ruminococcaceae),
desc ibed as associa ed o heal hy abbi samples (see abo e). Also
a lowe axon le el (genus), wi h he excep ion o Alis ipes and
Anae o uncus, all mos abundan gene a we e signi ican ly
co ela ed o high o low mucins and p o-in lamma o y cy okines
exp ession, depending whe he hey we e mos abundan in he
ERE o Heal hy g oups, espec i ely (Tables S9, S10, S11, S12).
Also we ied o ind, h ough co ela ion analysis, o ganisms
signi ican ly bound o he gene exp ession P o iles iden i ied
(In lamma o y and Sec e o y) wi hin he ERE g oup, bu signi ican
in o ma ion could no be ob ained due o he low numbe o
abbi s in he In lamma o y P o ile. Howe e , plain PCA a
species (o ganisms) le el pe o med wi h mos abundan sequences
in he ERE g oup, wi h p ojec ion o gene exp ession da a,
indica ed ha uncul u ed and uncha ac e ized isola es o he
genus Akke mansia,Alis ipes and Bac e iodes clus e ed wi h IL-6,
IL-8, TNFaand IFNcand in he p oximi y o abbi samples 5, 6
and 8 (In lamma o y P o ile) (Figu e S1). Di e se sequences we e
posi ioned nex o MUC1 and MUC13 in he PCA plo wi h
ep esen a i es o Akke mansia,Esche ichia ( ou sequences),
Bac e oides ( wo sequences), Clos idium and h ee uniden i ied
species and genus o he amily Lachnospi aceae. Howe e , he
ou sequences o Esche ichia and one Lachnospi aceae we e close
o he co e o MUC1 and MUC13 p ojec ion. This s esses he
di e ences be ween he In lamma o y and Sec e o y P o iles and
sugges s hey may be bound o speci ic mic obial en i onmen /
elemen s.
Discussion
S udies using molecula me hods o he analysis o he gu
mic obio a in abbi s a e no abundan . Capilla y elec opho esis
single-s anded con o ma ion polymo phism (CE-SSCP) and
dena u ing g adien gel elec opho esis (DGGE) we e use ul o
de e mine he simila i y be ween bac e ial popula ions o he cecal
con en and so aeces [20] on die s wi h di e en ib e con en
[21] and o d aw a gene al quan i a i e map o he e olu ion o he
mic obio a along he abbi ’s li e [4]. Cloning o 16S RNA genes
in Esche ichia coli o e ed a mo e p ecise iden i ica ion o he
bac e ial inhabi an s o abbi ’s gu , howe e , he p ocedu e
in oduced quali a i e and quan i a i e biases. In a i s in en o y,
Abecia e al. (2005) [21] showed he ele ance in abbi ’s cecal
con en o Clos idia (Lachnospi aceae, Ruminococcaceae) and
Ve ucomic obia, and epo ed a numbe o new uniden i ied
sequences. Au ho s desc ibed he absence o sequences o he
P e o ella–Bac e oides g oup, howe e , nowadays iden i ica ion
da abases ha e been en iched and we could classi y all hose
sequences (G oup 5, in Abecia e al, 2005), in he Bac e oide es
phylum (Table S13). In a la e wo k, a la ge numbe o sequences
(228) we e analysed and dis ibu ed in 70 OTUs in nine clus e s,
whe e he majo i y o sequences belonged o he phylum
Fi micu es (94%), only h ee we e Bac e oide es (4%) and one
Ve ucomic obia [3]. Py osequencing has been ecen ly used o
in es iga e he mic obial composi ion o a a ie y o cecal il a es
and ac ions ha ep oduced ERE in abbi s, bu no s a is ical
di e ences we e ound be ween samples in ha wo k [22]. Ou
wo k showed ha cecal mic obio a in heal hy abbi s is domina ed
by Fi micu es (78.25% o o al OTUs), Bac e oide es (15.75%),
Ve ucomi obia (2.40%) and Tene icu es (2.39%) and his
quan i a i ely simila o ha ound in o he monogas ic
he bi o es (hindgu e men e s) [23] and in mouse, as he closes
model, in which also Fi micu es domina e o e Bac e oide es
[24,25].
In his wo k, we ob ained 289,000 good quali y sequences (bo h
s ands) om 29 samples o acili a e he compa a i e analysis o
heal hy abbi s ea ed o no wi h an ibio ics and abbi s su e ing
ERE. The i s su p ising esul was ha he e we e no signi ican
di e ences due o he an ibio ic ea men , despi e ha i was
epo ed ha an ibio ics educed he mic obial di e si y in abbi s
[22,26], as hey did in mice [27] and humans [28]. The ac ha
we ound no signi ican di e ences be ween con ol abbi s and he
an ibio ic g oup migh be due o he p olonged p e en i e
medica ion in he expe imen al a m, a si ua ion ha could
gene a e a uni o m en i onmen al popula ion ins alled in he a m
ha ul ima ely colonize abbi s. Howe e , his uni o m mic obi-
ological en i onmen could acili a e he de ec ion o bac e ial
pa hogens in ol ed in ERE, o a leas o es ablish he
mic obiological scena io in he cecum o abbi s su e ing ERE.
The mos ele an ea u e o ERE cecal mic obio a is a
ema kable dysbiosis and educed axonomic di e si y. An
ex eme en i onmen mus be gene a ed du ing ea ly pa hogen-
esis, as occu s in a numbe o o he pa hologies wi h a ma ked
in lamma o y p o ile, whe e pa icula ly c-P o eobac e ia p oli -
e a e, such as human in lamma o y bowel disease (IBD),
nec o izing en e ocoli is in human in an s [29] o pigle sho
bowel synd ome and mu ine model o C ohn’s disease [30–33].
Quan i a i e and quali a i e di e ences be ween heal hy and ERE
abbi s ha e been ound, such as he abundance o Ve ucomi-
c obiae, pa icula ly he genus Akke mansia.A. muciniphila is a
ypical in es inal anae obe equen ly associa ed o human heal hy
mucosa [34]. I s high p e alence in ERE cecal samples could be
ela ed o hei mucin sca enge cha ac e [35,36], howe e , A.
muciniphila can agg a a e gu in lamma ion when he e is
p e alen a Salmonella in ec ion [37]. Ruminococcus is he mos
ele an genus o he Fi micu es phylum dominan in heal hy
indi iduals, ha no ably dec eases in ERE, despi e he ac ha
his is gene ally an e icien mucin deg ading genus [30,31].
Ano he componen o he heal hy mic obio a is he genus
Alis ipes. Bo h play an impo an ole in he deg ada ion o
ege able eed componen s and he p oduc ion o sho chain a y
acids [38,39], hence con olling Bac e oide es numbe s [40], and
he e o e, could cons i u e essen ial componen s o abbi cecal
mic obio a and candida es as p obio ics. Me hanobac e ia (A -
chaea) we e epo ed in low numbe s in he cecal con en o
abbi s, only p esen a e day 7 o li e [4]. Ou da a ob ained by
qPCR con i med i . P oduc ion o me hane has been shown o be
in e sely p opo ional o he size o animals. Ca bon exc e ed as
me hane is ep esen s a me abolic loss, hence in he case o small
he bi o es like abbi s, his may ep esen a deg ee o e men a i e
Rabbi Cecal Mic obio a and Gene Exp ession in ERE
PLOS ONE | www.plosone.o g 9 Augus 2014 | Volume 9 | Issue 8 | e105707