INFECTION AND IMMUNITY, May 2007, p. 2476–2483 Vol. 75, No. 5
0019-9567/07/$08.00⫹0 doi:10.1128/IAI.01908-06
Copy igh © 2007, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Immuniza ion o Pigs To P e en Disease in Humans: Cons uc ion
and P o ec i e E icacy o a Salmonella en e ica Se o a
Typhimu ium Li e Nega i e-Ma ke Vaccine
䌤
†
Ma in Selke,
1
Jochen Meens,
1
S en Sp inge ,
2
Ronald F ank,
3
and Ge ald-F. Ge lach
1
*
Ins i u e o Mic obiology, Depa men o In ec ious Diseases, Uni e si y o Ve e ina y Medicine Hanno e ,
Hanno e , Ge many
1
; Imp s o we k Dessau-To nau GmbH, PSF 214, Rodleben OT To nau, Ge many
2
;
and Depa men o Chemical Biology, Helmhol z Cen e o In ec ion Resea ch (HZI),
Masche ode Weg 1, D-38124 B aunschweig, Ge many
3
Recei ed 4 Decembe 2006/Re u ned o modi ica ion 21 Janua y 2007/Accep ed 4 Feb ua y 2007
Zoono ic in ec ions caused by Salmonella en e ica se o a Typhimu ium pose a cons an h ea o
consume heal h, wi h he pig being a pa icula ly majo sou ce o mul id ug- esis an isola es. Vacci-
na ion, as a p omising app oach o educe coloniza ion and shedding, has been sca cely used, as i
in e e es wi h cu en con ol p og ams elying on se ology as a means o he d classi ica ion. In o de o
o e come his p oblem, we se ou o de elop a nega i e-ma ke accine allowing he di e en ia ion o
in ec ed om accina ed animals (DIVA). Applying an immunop o eomic app oach wi h wo-dimensional
gel elec opho esis, Wes e n blo , and quad upole ime-o - ligh andem mass spec ome y, we iden i ied
he OmpD p o ein as a sui able nega i e ma ke . Using allelic exchange, we gene a ed an isogenic mu an
o he licensed li e accine s ain Salmopo c and showed ha i ulence o Salmopo c and ha o he
mu an s ain, Salmopo c⌬ompD, we e indis inguishable in BALB/c mice. In a pig in ec ion expe imen
including wo o al immuniza ions wi h Salmopo c⌬ompD and challenge wi h a mul i esis an S.en e ica
se o a Typhimu ium DT104 clinical isola e, we con i med he p o ec i e e icacy o Salmopo c⌬ompD in
pigs, showing a signi ican educ ion o bo h clinical symp oms and coloniza ion o lymph nodes and
in es inal ac . OmpD immunogenic epi opes we e de e mined by pep ide spo a ay analyses. Upon
es ing o se e al 9-me pep ides, each including an immunogenic epi ope, one pep ide (posi ions F
100
o
Y
108
) ha acili a ed he de ec ion o in ec ed animals independen o hei accina ion s a us (DIVA
unc ion) was iden i ied. The app oach desc ibed o e comes he p oblems cu en ly limi ing he use o
bac e ial li e accines and holds conside able po en ial o u u e de elopmen s in he ield.
Bac e ial ood-bo ne pa hogens a e o inc easing conce n
o consume s and policy make s wo ldwide, as hey e-
quen ly cause epidemic in es inal disease ou b eaks and
he eby a e esponsible o high economic losses. One o he
mos impo an ood-bo ne pa hogens is Salmonella en e ica
se o a Typhimu ium, which is esponsible o 1.9% (Czech
Republic) o 64.7% (New Zealand) o he non yphoid Sal-
monella in ec ions epo ed wo ldwide o he yea s 2000 o
2001 (16). In he Uni ed S a es, non yphoidal Salmonella
s ains a e esponsible o an es ima ed 1.41 million cases o
human in ec ions and mo e han 500 human dea hs annually
(24). In Eu ope, he numbe o o icially epo ed cases
amoun s o mo e han 175,000 acco ding o he epo o he
Eu opean Food Sa e y Au ho i y (EFSA) o he yea 2005
(h p://www.e sa.eu opa.eu/e c/medialib/e sa/science/moni o ing
_zoonoses/ epo s/zoonoses_ epo _2005.Pa .0001.File.da
/Zoonoses_ epo _2005.pd ), wi h he es ima ed numbe o
un epo ed cases being se e al imes highe .
The eme gence and apid sp ead o mul id ug esis an Sal-
monella spp. (especially phage ype DT104) (1, 3, 30, 34) limi
he he apeu ic al e na i es in cases o in asi e in ec ions and
ha e been shown o be associa ed wi h an inc eased bu den o
illness (16). Thus, in Eu ope, 9.1% o human Salmonella in ec-
ions a e caused by S.en e ica se o a Typhimu ium, wi h 21.4%
o hese in ec ions being due o phage ype DT104 (EFSA epo
o 2005 [see abo e]). Mul id ug- esis an S.en e ica se o a
Typhimu ium s ains a e ound in pigs wi h pa icula ly high
equency (15) and can be isola ed om po k and po k p od-
uc s (7, 21) (EFSA epo o 2005 [see abo e]). In Ge many,
o example, 3.2% o samples aken om minced po k mea
we e Salmonella posi i e: 67% o hese isola es we e S.en e ica
se o a Typhimu ium, and 69% o hese we e esis an o mo e
han ou an ibio ics (EFSA epo o 2005 [see abo e]). In
addi ion, non- ood-bo ne animal- o-human (17, 51) and hu-
man- o-human (33) ansmissions o sys emically sp eading
mul id ug- esis an S.en e ica se o a Typhimu ium DT104
s ains ha e been epo ed, emphasizing he po en ial isk o
his pa hogen o human heal h.
In o de o educe he po en ial isk o consume s, i is
c ucial o minimize pa hogen in ake in o he ood chain. In he
Eu opean Union, su eillance sys ems a e equi ed ( egula ion
[EC] 2160/2003; h p://eu opa.eu.in /eu -lex/p i/en/oj/da /2003
/l_325/l_32520031212en00010015.pd ), and a numbe o coun-
ies a e speci ically moni o ing he occu ence o Salmonella
in he po k p oduc ion chain (2, 11, 19, 45, 48). In pigs, Sal-
* Co esponding au ho . Mailing add ess: Ins i u u¨ Mik obiologie,
Zen um u¨ In ek ionsmedizin, S i ung Tie a¨ z liche Hochschule Han-
no e , Bischo shole Damm 15, 30173 Hanno e , Ge many. Phone: 49
511 856 7598. Fax: 49 511 856 7697. E-mail: [email p o ec ed].
† Supplemen al ma e ial o his a icle may be ound a h p://iai
.asm.o g/.
䌤
Published ahead o p in on 12 Feb ua y 2007.
2476
on Janua y 20, 2017 by GESELLSCHAFT FUR BIOTECHNO-h p://iai.asm.o g/Downloaded om
monella moni o ing is based on mea juice se ology (26).
The e o e, immuniza ion wi h con en ional accines canno be
used as a means o educe in ec ion and shedding o he pa ho-
gen, bu o mula ions o accines acili a ing he di e en ia ion
o in ec ed and accina ed animals (DIVA) (4, 9, 46) would be
equi ed.
DIVA li e accines ha e been used wi h good success o he
elimina ion o i al in ec ions such as Aujeszky’s disease in pigs
(42, 43) o bo ine he pes i us in ec ions (46). Fo bac e ial
diseases, only expe imen al DIVA li e accines ha e been de-
sc ibed (22), and no DIVA accines agains ood-bo ne pa ho-
gens ha e been cons uc ed o da e. This likely is due o he
easy use o an ibio ics in li es ock o he apy and as eed
addi i es un il he ecen pas (5, 47), he cos s and expe imen-
al di icul ies in ol ed in bac e ial DIVA li e accine cons uc-
ion, and, as gene ically modi ied o ganisms a e in ol ed, he
necessi y o license hese accines a he Eu opean Agency o
he E alua ion o Medicinal P oduc s. This licensing p oce-
du e equi es ex ensi e expe imen al es ing and he e o e is
p o i able only i he ma ke demand is su icien .
He e, we p esen he de elopmen o an S.en e ica se o a
Typhimu ium DIVA li e accine o pigs. We desc ibe a gen-
e al app oach o cons uc and es a bac e ial DIVA li e
accine in ol ing (i) he iden i ica ion a o nonessen ial immu-
nogenic an igen by wo-dimensional polyac ylamide gel elec-
opho esis (2D-PAGE), Wes e n blo ing, and quad upole
ime-o - ligh (Q-TOF) andem mass spec ome y (MS/MS),
(ii) he cons uc ion o an isogenic in- ame mu an lacking
o eign DNA by allelic exchange, (iii) he de elopmen o a
disc imina o y enzyme-linked immunoso ben assay (ELISA)
upon epi ope mapping by pep ide spo a ay analyses, and (i )
an exempla y immuniza ion and challenge expe imen .
MATERIALS AND METHODS
Bac e ial s ains, plasmids, and p ime s. The bac e ial s ains, plasmids, and
p ime s used in his wo k a e lis ed in Table 1.
P epa a ion o ou e memb ane-associa ed p o eins. Memb ane-associa ed
p o eins we e p epa ed as desc ibed p e iously (14). B ie ly, S.en e ica se o a
Typhimu ium cells we e g own wi h shaking in 150 ml o Lu ia-Be ani (LB)
b o h a 37°C o an op ical densi y a 600 nm (OD
600
) o 0.7. Cells we e ha es ed
by cen i uga ion a 8,000 ⫻g o 10 min a 4°C, esuspended in 30 ml o
de e gen wash bu e (NaCl [150 mM], T is-HCl [10 mM, pH 8.0], sodium
deoxychola e [0.05%], and sodium azide [0.04%]), incuba ed o 30 min in a
shaking incuba o a 37°C, and cen i uged o 10 min a 7,000 ⫻g. This ea -
men e icien ly solubilizes memb ane-associa ed lipop o eins while lea ing he
cells mos ly in ac . The cell- ee supe na an con aining ou e memb ane-asso-
cia ed p o eins as well as con amina ing in eg al ou e memb ane p o eins was
il e ed (0.22-␮m po e size) and s o ed a ⫺20°C un il u he use. P o ein
concen a ions we e de e mined by Mic o-BCA (Pie ce Bio echnology, Rock-
o d, ILL). Fo 2D-PAGE, he cell- ee supe na an was p ecipi a ed o e nigh
wi h ichlo oace ic acid (10% inal concen a ion). A e cen i uga ion a
15,000 ⫻g o 15 min a 4°C, pelle s we e washed wice wi h pu e ace one,
esuspended in 500 o 1,000 ␮l bidis illed wa e , and s o ed a ⫺20°C.
P epa a ion o ou e memb ane p o eins. Bac e ia g own and ha es ed as
desc ibed abo e we e esuspended in 2 ml T is-HCl (50 mM, pH 8.0) wi h
suc ose (25%, w / ol) and ozen a ⫺70°C. A e hawing, bac e ia we e incu-
ba ed wi h lysozyme (2 mg/ml) and sa cosyl (2%, w / ol) o 1 h, ollowed by
sonica ion (B anson-Soni ie B-30; Heinemann, Schwa¨bisch Gmu¨nd, Ge many)
using a mic o ip a he maximum-s eng h se ing o h ee cycles o 30 s and a
du y cycle o 60%. The lysa e was cen i uged (15,000 ⫻g o 30 min), ollowed
by ul acen i uga ion o he supe na an (100,000 ⫻g o 90 min). The sonica-
ion in combina ion wi h sa cosyl disin eg a es he cy oplasmic memb ane and
emo es ou e memb ane-associa ed p o eins. The esul ing pelle con aining
he ou e memb ane ac ion wi h in eg al memb ane p o eins was esuspended
in 100 ␮lH
2
O and s o ed a ⫺20°C.
De ec ion o immunogenic ou e memb ane-associa ed p o eins. Aliquo s o
500 ␮g o su ace-associa ed p o eins we e sepa a ed using 2D-PAGE wi h
ImmobilineD yS ips (24 cm, pH 4 o 7). Sepa a ed p o eins we e ans e ed
on o a ni ocellulose memb ane using a semid y-p o ein ans e sys em (No aBlo ;
Ame sham Pha macia Bio ech AB, Uppsala, Sweden) and sc eened by Wes e n
blo ing wi h h ee po cine se a (a hype immune se um om a pig accina ed
h ee imes wi h Salmopo c, a pool o ield se a om pigs posi i e by he
Salmo ype PigSc een ELISA, and a pool o se a om pigs expe imen ally in-
ec ed wi h S.en e ica se o a De by), each used a a 1:200 dilu ion. Blo s we e
de eloped using an alkaline phospha ase-conjuga ed goa an i-swine immuno-
globulin G (Diano a, Hambu g, Ge many) and BCIP (5-b omo-4-chlo o-3-in-
dolylphospha e) and ni oblue e azolium as a ch omogenic subs a e. P o ein
spo s eac ing s ongly wi h all h ee se a we e selec ed o iden i ica ion ia
Q-TOF MS/MS.
Iden i ica ion o immunogenic p o eins. Coomassie blue-s ained spo s co e-
sponding o immunogenic p o eins we e cu om a 2D PAGE gel, ypsina ed,
and elu ed om he gel by using a me hod sligh ly modi ied om ha desc ibed
p e iously by Wilm e al. (50). B ie ly, he gel pieces we e dehyd a ed wi h 100
␮l ace oni ile, ehyd a ed wi h 30 ␮l ehyd a ion bu e (100 mM NH
4
HCO
3
con aining 10 mM di hio h ei ol), and hen ea ed wi h 100 mM iodoace amide
in 30 ␮l 100 mM NH
4
HCO
3
. Dehyd a ion and ehyd a ion we e epea ed, and
he dehyd a ed gel pieces we e hen ehyd a ed wi h 3 o 15 ␮l ehyd a ion bu e
con aining 20 ng/␮l ypsin (sequencing g ade) o 12 o 16 h a 37°C. Pep ides
we e ex ac ed using 50 mM NH
4
HCO
3
ollowed by a solu ion con aining 50%
( ol/ ol) ace oni ile and 5% ( ol/ ol) o mic acid. A e e apo a ion o all liquid
in a acuum cen i uge, pep ides we e esuspended in a solu ion con aining 50%
( ol/ ol) ace oni ile and 0.1% ( ol/ ol) o mic acid. Pep ide sequences we e
de e mined om MS/MS agmen a ion da a eco ded on an ESI Q-TOF mass
spec ome e (Q-To Ul ima; Wa e s, Mil o d, MA). P o eins we e iden i ied by
using he p og am P o einLynx Globals Se e ( e sion 2.1; Wa e s) and by
sea ching he Na ional Cen e o Bio echnology In o ma ion (NCBI) comple e
da abase ( p:// p.ncbi.nlm.nih.go ./BLAST/db/FASTA/).
Iden i ica ion and syn hesis o immunogenic epi opes. Epi ope mapping was
pe o med by pep ide spo a ay analysis using o e lapping 15-me pep ides
ini ia ing a e e y hi d amino acid (12, 13). The pep ide-coa ed memb ane was
we ed wi h e hanol o enhance he sol a ion o hyd ophobic pep ide spo s and
hen washed h ee imes wi h T is-bu e ed saline (TBS) (10 mM T is [pH 7.0],
154 mM NaCl [pH 7.0]) o 10 min and inally incuba ed o e nigh a 4°C in 10
ml o memb ane blocking solu ion (pH 7.0) (80% [ ol/ ol] TBS–0.05% Tween 20
[T-TBS; pH 8.0], 20% [ ol/ ol] casein-based blocking bu e concen a e [Geno-
sys Bio echnology, Camb idge, England], 5% [w / ol] suc ose). A e washing
wi h 10 ml T-TBS (pH 7.0), he memb ane was incuba ed wi h po cine se um
aised agains he S. en e ica se o a Typhimu ium accine s ain Salmopo c o
2 h (dilu ed 1:200 in memb ane blocking solu ion). The blo was de eloped as
desc ibed abo e. The ch omogenic eac ion was s opped by washing blo s wice
wi h phospha e-bu e ed saline. The memb ane was s ipped using 20 ml o
N,N-dime hyl o mamide wice o 10 min o dissol e he blue colo o spo
signals, ollowed by washing h ee imes wi h wa e , s ipping mix A (8 M u ea,
1% sodium dodecyl sul a e, and 0.5% me cap oe hanol in phospha e-bu e ed
saline [pH 7.0]), s ipping mix B (10% ace ic acid, 50% e hanol, 40% wa e ), and
e hanol, espec i ely. A e wa ds, blo ing was epea ed wi h po cine nega i e
se um (a pool o Salmo ype PigSc een ELISA-nega i e se a om pigs om a
Salmonella- ee he d) in o de o exclude unspeci ic epi opes.
Pu a i e immunogenic epi opes (noname s) we e syn hesized wi h an amino-
e minal bio inyla ion linked by a 2-aminohexanoic acid linke and pu i ied by
high-pe o mance liquid ch oma og aphy (Pep ide Special y Labo a o ies
GmbH, Heidelbe g, Ge many). Lyophilized pep ides we e esuspended in dis-
illed wa e o ob ain s ock solu ions o 5 and 10 mg/ml, espec i ely. Pu a i e
immunogenic epi opes and su ace-exposed domains we e p edic ed using he
algo i hms “An igenic” and “B2TMR-HMM” (18, 20, 23, 29), espec i ely.
Cons uc ion o an isogenic S. en e ica se o a Typhimu ium Salmopo c⌬ompD
s ain. A unca ed ompD gene wi h an in- ame dele ion was cons uc ed using
a class IIs es ic ion endonuclease app oach (38). Two PCR p oduc s o 983 bp
and 792 bp we e gene a ed using p ime s oDWST5_ou a/oOMPDKO1 and
oOMPDKO2/oDWST5_ou b, espec i ely (Table 1). Bo h agmen s we e e-
s ic ed wi h BsmBI, liga ed, eampli ied, and cloned in o pTOPO2.1 (In i o-
gen, Heidelbe g, Ge many). The inse was con i med by nucleo ide sequence
analysis, emo ed by SacI es ic ion, and liga ed in o he mu agenesis ec o
pROKB2 es ic ed wi h SacI, esul ing in plasmid pSOM14666. The plasmid was
ans o med in o he dono s ain Esche ichia coli ␤2155 and mobilized in o S.
en e ica se o a Typhimu ium Salmopo c by il e ma ing. The dono was g own
on LB aga pla es supplemen ed wi h kanamycin (50 ␮g/ml) and diaminopimelic
acid (10 mM) a 37°C o 16 h, and he ecipien was g own on Columbia sheep
VOL. 75, 2007 S. ENTERICA SEROVAR TYPHIMURIUM DIVA VACCINE 2477
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blood aga (Oxoid GmbH, Wesel, Ge many) a 37°C o 44 h. Bac e ia we e
ha es ed wi h s e ile co on swabs and esuspended in TNM bu e (1 mM
T is-HCl [pH 7.2], 100 mM NaCl, 10 mM MgSO
4
) oanOD
600
o 1. Fil e
ma ing was pe o med as desc ibed p e iously (28); he conjuga ion mix u e was
pla ed on o LB aga pla es supplemen ed wi h kanamycin (40 ␮g/ml) and incu-
ba ed a 37°C o e nigh . Colonies we e subcul u ed and sc eened ia PCR wi h
ompD-speci ic p ime s oOMPD1 and oOMPD2. Colonies con aining bo h he
wild ype and he unca ed gene we e conside ed o be po en ial genomic
coin eg a es and we e used o coun e selec ion.
Coun e selec ion. A single colony o S. en e ica se o a Typhimu ium Salmo-
po c coin eg a e was inocula ed o e nigh in LB b o h. Sal - ee LB b o h (2.5
ml) con aining 10% suc ose was inocula ed wi h 2.5 ␮l o he cul u e g own
o e nigh and kep a 37°C in a shaking incuba o (200 pm) o 4 h. In o de o
imp o e coun e selec ion e icacy, he cul u e was subsequen ly s o ed a 4°C o
72 h, and 50-␮l aliquo s we e pla ed on o LB aga . Single colonies we e eplica
pla ed on o LB aga wi h (40 ␮g/ml) and wi hou kanamycin. The geno ype o
kanamycin-sensi i e colonies was de e mined by PCR wi h p ime s oOMPD1
and oOMPD2 and e i ied by Sou he n blo analysis, nucleo ide sequencing
analysis, and pulsed- ield gel elec opho esis.
Vi ulence s udy in BALB/c mice. Vi ulence o S. en e ica se o a Typhimu ium
Salmopo c⌬ompD was assessed in an in ec ion model by de e mining he 50%
le hal dose (LD
50
) in BALB/c mice in compa ison o he pa en s ain Salmo-
po c. The animals, 32 emale 17- o 20-g BALB/c mice, we e in ec ed pai wise
wi h 1 ⫻10
2
o1⫻10
8
CFU o ei he he pa en o he mu an s ain and
TABLE 1. Bac e ial s ains, plasmids, and p ime s used in his s udy
S ain, plasmid, o p ime Cha ac e is ic(s) Sou ce o e e ence
S ains
E. coli ␤2155 h B1004 p o hi s A hsdS lacZ⌬M15 (F⬘lacZ⌬M15 laqI
q
aD36 p oA
⫹
p oB
⫹
)⌬dapA::e m (E m
) ecA::RPA-2- e (Tc
)::Mu-Km (Km
)␭pi
8
E. coli TOP10 F
⫺
mc A ⌬(m -hsdRMS-mc BC)␾80lacZ⌬M15 ⌬lacX74 ec A1 deoR
a aD139 ⌬(a a leu)7697 galU galK psL (S
)endA1 nupG
TOPO TA cloning;
In i ogen
S. en e ica se o a Typhimu ium
421/125
S. en e ica subsp. en e ica se o a Typhimu ium li e accine s ain p esen
in he licensed accine Salmopo c
IDT, Dessau-To nau,
Ge many
S. en e ica se o a Typhimu ium
421/125⌬ompD
Unma ked ompD-nega i e knockou mu an o S. en e ica se o a
Typhimu ium 421/125
This wo k
S. en e ica se o a Typhimu ium
DT104 27/96
S.en e ica se o a Typhimu ium ield isola e ca ying phage ype DT104
wi h Amp
Clin
E y
Pen
Te
44
Plasmids
pROCKB2 8.9-kb ansconjuga ion ec o based on pROKB1 wi h o iR6K and
mobRP4,Ap
Km
, polycloning si e, sacB
This wo k
pSOM14666 T ansconjuga ion ec o pROCKB2 ca ying a unca ed e sion o ompD
wi h o iR6K,mobRP4,Ap
Km
, polycloning si e, sacB
This wo k
pTOPO 2.1 Topoisome ase I-“enhanced” E. coli cloning ec o ca ying ampicillin and
kanamycin esis ance de e minan s as well as a lacZ gene o blue-whi e
selec ion
TOPO TA cloning;
In i ogen
pSOM810 Vec o pTOPO 2.1 ca ying PCR p oduc o p ime s pOMPDKOMP1
and pOMPDKOMP2, s a ing 516 bp ups eam o he ompD s a
codon and ending 31 bp downs eam o he s op codon, o
complemen a ion o S. en e ica se o a Typhimu ium ⌬ompD
This wo k
P ime s
oDWST5ou a 5⬘-TGC TCTAGA CCC GGA GAA ATT ATC AGC AA-3⬘( o wa d
p ime con aining an XbaI es ic ion si e on he 5⬘end si ua ed 974 bp
ups eam o he ompD s a codon)
This wo k
oDWST5ou b 5⬘-GCC GAGCTC AGA GAT TGC CAG AGC GTC AT-3⬘( e e se
p ime con aining a SacI es ic ion si e on he 5⬘end si ua ed 753 bp
downs eam o he ompD open eading ame)
This wo k
oOMPDKO1 5⬘-CGTCTCGAAGTTTCATTTTAATAATCCTTAT-3⬘( e e se p ime
wi h BsmBI es ic ion ecogni ion si e on 5⬘end si ua ed 9 bp down-
s eam o he ompD s a codon; e e se p ime o oDWST5_ou a o
ampli ica ion o he ups eam DNA agmen used o cons uc a un-
ca ed ompD)
This wo k
oOMPDKO2 5⬘-CGTCTCGACTTCTGAACTACCAGTTCTAATT-3⬘( o wa d p ime
wi h BsmBI es ic ion ecogni ion si e on 5⬘end si ua ed 2 bp down-
s eam o he ompD s op codon; o wa d p ime o oDWST5_ou b o
ampli ica ion o he downs eam DNA- agmen used o cons uc a
unca ed ompD)
This wo k
oOMPD1 5⬘-CCATACCAGGATTGCGCTG-3⬘( o wa d p ime si ua ed 393 bp
ups eam o he ompD s a codon)
This wo k
oOMPD2 5⬘-CGGTAAGCCGAAACCACAG-3⬘( e e se p ime si ua ed 317 bp
downs eam o he ompD open eading ame)
This wo k
oOMPDKOMP1 5⬘-GGCGGGCCGATATTGATATT-3⬘( o wa d p ime o complemen a-
ion o S. en e ica se o a Typhimu ium ⌬ompD si ua ed 516 bp up-
s eam o he ompD s a codon; he 516-bp nucleo ide sequence was
also analyzed o p omo e s using he Web-based p og am BPROM
[www.so be y.com] o ensu e ha he p omo e o he ompD gene was
included)
This wo k
oOMPDKOMP2 5⬘-GGACTGGCTTTGTATTCAGAC-3⬘( e e se p ime o complemen-
a ion o S. en e ica se o a Typhimu ium ⌬ompD si ua ed 31 bp down-
s eam o he ompD s op codon)
This wo k
2478 SELKE ET AL. INFECT.IMMUN.
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obse ed o 19 days pos in ec ion. Spleens and li e s o animals dying du ing he
ime o obse a ion we e cul u ed o possible eisola ion o he espec i e s ain;
isola ed Salmonella colonies we e iden i ied using he IDT Salmonella diagnos ic
ki and a PCR wi h ompD-speci ic p ime s, acili a ing he di e en ia ion o
Salmopo c⌬ompD and Salmopo c. The LD
50
was calcula ed using P obi analysis
so wa e (SPSS Inc., Chicago, IL).
Vaccina ion ial in pigs. The Salmopo c⌬ompD s ain cons uc ed in his
s udy was used as an o al accine in a accina ion s udy in pigs (Ge man Land-
ace) and compa ed o he pa en s ain (Salmopo c) and a placebo g oup. The
ial was pe o med essen ially as desc ibed p e iously (41); an S.en e ica se o a
Typhimu ium DT104 clinical isola e (s ain 27/96) was used as a challenge s ain.
The animal expe imen included h ee g oups o six pigs each, 4 weeks o age,
accina ed o ally wi h placebo, S.en e ica se o a Typhimu ium Salmopo c⌬ompD
(6 ⫻10
8
CFU/ml pe accina ion), o S.en e ica se o a Typhimu ium Salmo-
po c (acco ding o manu ac u e ’s ins uc ions), espec i ely. Th ee addi ional
pigs we e accina ed wi h S.en e ica se o a Typhimu ium Salmopo c⌬ompD o
in es iga e he de elopmen o an ibody i e s un il 3 weeks a e in ec ion. Two
ounds o accina ion we e pe o med 3 weeks apa . Blood samples we e aken
p io o he i s (day 0) and second (day 21) immuniza ions, 1 week be o e
challenge, and a nec opsy. Fo he h ee pigs kep un il 3 weeks a e in ec ion,
addi ional blood samples we e aken 1, 2, and 3 weeks pos challenge (see Table
S4 in he supplemen al ma e ial). Animals we e ca ed o in acco dance wi h he
p inciples ou lined in he Eu opean Con en ion o he P o ec ion o Ve eb a e
Animals Used o Expe imen al and O he Scien i ic Pu poses (Eu opean T ea y
Se ies no. 123 [h p://con en ions.coe.in / ea y/EN/Menup incipal.h m]; pe mi
no. 06/1066).
In ec ion o pigs. Fo o al in ec ion on day 43, a 50-ml cul u e (LB medium)
was inocula ed wi h 5 ml o a liquid cul u e o S.en e ica se o a Typhimu ium
DT104 s ain 27/96 (LB medium) g own o e nigh and was g own wi h shaking
a 37°C o an OD
600
o 0.9. The cul u e was placed on ice o 10 min and kep
on ice un il use o a maximum o 2 h. Pigle s we e in ec ed wice wi hin 2 hou s,
and inoculum doses we e de e mined immedia ely a e in ec ion ia se ial di-
lu ion and subsequen pla ing on o Rambach aga pla es supplemen ed wi h
ampicillin (100 ␮g/ml). In ec ions we e ca ied ou using a ben -knob cannula
and sy inge o adminis e he app op ia e dose o S.en e ica se o a Typhi-
mu ium DT 104 s ain 27/96 o he pigle s 3 weeks a e he second immuniza ion
ia he o al ou e (5 ⫻10
8
CFU/ml pe ound, esul ing in a o al dose o 1 ⫻10
9
CFU/ml).
Su eillance o animals. Body empe a u e, eeding beha io , and clinical
symp oms we e eco ded a leas daily o each indi idual pig o as needed. A
clinical sco ing sys em was employed o assess he clinical condi ion o each
indi idual animal as ollows. A sco e o 1 each was gi en o he occu ence o
e e ( ec al empe a u e ⬎40°C), lack o appe i e, and dia hea/ omi us/le h-
a gy, esul ing in a minimum clinical sco e o 0 and a maximum sco e o 4 pe
day; he added daily clinical sco es o days 1 o 7 we e designa ed he o al
clinical sco e. S a is ical analysis o he o al clinical sco e was pe o med using
he Wilcoxon es . Animals we e bled on days 0, 36, 50, 57, and 65 du ing he
cou se o he expe imen .
Bac e iological examina ion o o gan samples. In o de o de e mine he
p o ec i e e icacy o he accine wi h espec o coloniza ion, o gan samples
(ileocecal lymph nodes, ileum [app oxima ely 10 cm c anial o he ileocecal
al e], and cecum [apex]) we e aken a pos mo em examina ion. In es inal
o gan samples we e washed, and 0.1 g o mucosa was emo ed by sc aping wi h
a scalpel, aken up o 1 ml wi h saline, and homogenized using a bead bea e
(speed o 5.0, 40 s/ ound; The moSa an ) wi h h ee glass beads (diame e , 3
mm). Ten old se ial dilu ions we e pla ed on o Rambach aga con aining 100
␮g/ml o ampicillin in o de o supp ess he g ow h o o he En e obac e iaceae
and incuba ed o e nigh a 37°C. he Salmonella colonies g own we e coun ed,
he numbe o CFU/ml was calcula ed, and indi idual colonies we e con i med o
be exempla y by an ibio ic esis ance yping and S.en e ica se o a Typhimu ium
DT104-speci ic mul iplex PCR (32).
ELISAs. Fo he OmpD-speci ic pep ide-based ELISA, Nunc Immobilize
S ep a idin F96 mic o i e pla es we e incuba ed o 1 h wi h 100 ␮l o OmpD-
de i ed syn he ic, bio inyla ed pep ide (posi ions F
100
o Y
108
o he OmpD
p o ein; 25 ␮g/ml) as a solid-phase an igen. Po cine se a we e p eabso bed wi h
a whole-cell lysa e o S.en e ica se o a Typhimu ium Salmopo c⌬ompD o 1h
in o de o emo e possible c oss- eac i e an ibodies di ec ed agains simila
epi opes o o he po ins. Se ial wo old dilu ions o he p eabso bed se a (s a -
ing wi h a dilu ion o 1:10) we e added and incuba ed o 1ha oom empe -
a u e. The ELISA was de eloped using goa an i-pig pe oxidase conjuga e
(Diano a) and 2,2-azino-di-[3-e hylbenzi hiazoline sul ona e] (ABTS; Roche Di-
agnos ics, Mannheim, Ge many) as a subs a e. To de e mine he i e o an i-
Salmonella LPS an ibodies, he comme cial Salmo ype PigSc een ELISA (Labo
Diagnos ik GmbH, Leipzig, Ge many) was ca ied ou acco ding o he manu-
ac u e ’s ins uc ions.
Nucleo ide sequence accession numbe . The nucleo ide sequence o plasmid
pRouB2 has been submi ed o GenBank unde accession numbe AM180348.
RESULTS
Iden i ica ion o a sui able ma ke p o ein. To iden i y su -
ace-exposed, immunogenic p o eins o he licensed li e ac-
cine Salmopo c, exponen ially g owing bac e ia we e ha es ed
and ea ed wi h sodium deoxychola e o en ich ou e mem-
b ane p o eins in he supe na an ia de e gen wash. By sep-
a a ing he en iched ou e memb ane p o eins by 2D-PAGE
and sc eening hei immunogenici y by Wes e n blo analyses
(see Fig. S1 in he supplemen al ma e ial), we iden i ied i e
highly immunogenic p o eins (see Table S1 in he supplemen-
al ma e ial). They eac ed s ongly wi h a pool o se a om
animals accina ed h ee imes wi h Salmopo c as well as wi h
pools o se a om animals in ec ed wi h S.en e ica se o a
Typhimu ium o S.en e ica se o a De by, espec i ely. The
p o eins co esponding o he immuno eac i e spo s we e cu
om a Coomassie blue-s ained 2D-PAGE gel and iden i ied
a e ypsin diges ion using Q-TOF MS/MS. Fou p o eins
could be iden i ied unambiguously as he majo po ins OmpC
and OmpD, a homolog o an ou e memb ane p o ein o
Acine obac e spp., and elonga ion ac o Tu.
Cha ac e iza ion o OmpD as a pu a i e nega i e ma ke . In
silico analyses o he candida e p o eins esul ed in he selec-
ion o ou e memb ane po in D (OmpD) as a pu a i e nega-
i e ma ke . By pe o ming a mapping o con inuous an igenic
epi opes o he OmpD p o ein by pep ide spo a ay analysis
(see Fig. S2 in he supplemen al ma e ial) wi h o e lapping
15-me s, we iden i ied ou highly immunogenic epi opes
h oughou he p o ein. These ma ched only pa ially wi h pu-
a i e immunogenic epi opes and su ace-exposed domains
p edic ed using he algo i hms “An igenic” (see Table S2 in
he supplemen al ma e ial) and “B2TMR-HMM” (see Table
S3 in he supplemen al ma e ial), espec i ely. All ou
epi opes we e syn hesized as bio inyla ed pep ides and es ed
as solid-phase an igens o hei applicabili y in an OmpD-
speci ic ELISA, wi h pep ide 2 acili a ing he bes disc imina-
ion be ween OmpD an ibody-posi i e and -nega i e se a (Fig. 1).
Gene a ion o an ompD-de icien mu an s ain. An isogenic
S.en e ica se o a Typhimu ium Salmopo c⌬ompD mu an was
cons uc ed by allelic exchange using he suicide plasmid
pSOM14666 (con aining an in- ame dele ion o ompD) (see
Fig. S3 in he supplemen al ma e ial) upon conjuga ion wi h E.
coli ␤2155 as he dono s ain. Plasmid coin eg a es we e se-
lec ed on kanamycin, con i med by PCR, and esol ed by su-
c ose coun e selec ion. Suc ose- esis an and kanamycin-sen-
si i e colonies we e con i med by PCR, nucleo ide sequencing,
and Sou he n blo analysis as well as pulsed- ield gel elec o-
pho esis (see Fig. S4 in he supplemen al ma e ial). The phe-
no ype was con i med by one-dimensional PAGE (Fig. 2) and
subsequen Q-TOF MS/MS. The mu an gene a ed is ee o
o eign DNA and was designa ed S.en e ica se o a Typhi-
mu ium Salmopo c⌬ompD.
Vi ulence in BALB/c mice. In o de o ensu e an unchanged
i ulence o he mu an s ain, we challenged 16 g oups o wo
BALB/c mice each wi h eigh di e en doses o Salmopo c and
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Salmopo c⌬ompD and de e mined he LD
50
o be app oxi-
ma ely 1.3 ⫻10
8
CFU o bo h s ains (see Table S4 in he
supplemen al ma e ial). These esul s demons a ed ha he
lack o ompD does no addi ionally a enua e S.en e ica se o-
a Typhimu ium Salmopo c o BALB/c mice.
P o ec i e e icacy in pigs. Fou clinical symp oms ( e e
[ⱖ40.0°C], le ha gy, educed ood up ake, and en e i is [ om-
i us/dia hea]) we e added o esul in a clinical sco e wi h a
maximum o 4 pe animal and day. A signi ican di e ence (P⫽
0.046; Wilcoxon es ) be ween ei he o he accina ed g oups
and he placebo g oup was obse ed (Fig. 3). All animals in he
placebo g oup de eloped e e 2 days pos in ec ion and
showed educed eed up ake, whe eas none o he accina ed
animals de eloped e e , and only wo accina ed animals
showed hesi an eed up ake. No signi ican di e ence be ween
he g oups accina ed wi h S.en e ica se o a Typhimu ium
Salmopo c and S.en e ica se o a Typhimu ium Salmopo c-
⌬ompD was obse ed.
One week pos in ec ion, animals we e nec opsied. All pigs
showed eac i e hype emia in he ileum and cecum, wi h some
o he placebo- accina ed animals showing highly eac i e hy-
pe emia in he cecal mucosa. Salmonella coloniza ion was de-
e mined o h ee de ined loca ions (ileal and cecal mucosa
and ileocecal lymph node). Fo he cecum (P⫽0.042; F ied-
mann es ) and ileocecal lymph nodes (P⫽0.007; F iedmann
es ), accina ion wi h ei he accine signi ican ly educed col-
oniza ion (Table 2). Howe e , he educ ion o coloniza ion
was abou 10- old lowe upon accina ion wi h S.en e ica se -
o a Typhimu ium Salmopo c⌬ompD han upon accina ion
wi h he S.en e ica se o a Typhimu ium Salmopo c pa en
s ain (Table 2).
Ma ke p ope ies o he OmpD p o ein. In addi ion o he
p o ec i e e icacy, he unc ionali y o he nega i e ma ke
OmpD was in es iga ed in he accina ion s udy. In o de o
unc ion as a se ologically de ec able ma ke , an ibodies di-
FIG. 1. Disc imina o y e icacy o OmpD-de i ed immunogenic
epi opes. Bio inyla ed epi opes 1 o 4 we e used as solid-phase an i-
gens on s ep a idin-coa ed ELISA pla es and incuba ed wi h in e nal
posi i e and nega i e con ol se a, espec i ely, ob ained om animals
in he accina ion ial. The ba s show he means and s anda d de i-
a ions o h ee independen ELISA expe imen s. OD%, ela i e op i-
cal densi y o he nega i e con ol se um compa ed o ha o he
posi i e con ol se um (de ined as 100 OD%) o each coa ing pep ide.
FIG. 2. Cha ac e iza ion o S.en e ica se o a Typhimu ium
Salmopo c⌬ompD. Pheno ypes we e cha ac e ized using memb ane
p epa a ions o Salmopo c (lane 1), Salmopo c⌬ompD (lane 2), and
Salmopo c⌬ompD complemen ed in ans wi h plasmid pSOM810
(lane 3). P o ein bands A o C we e sequenced using Q-TOF MS/MS
and iden i ied as being OmpC (A), OmpD (B), and OmpF (C), e-
spec i ely. LMW, low molecula weigh ma ke (Ame sham Pha mcia
Bio ech AB, Uppsala, Sweden).
FIG. 3. Clinical sco es o pigs in he accina ion and challenge ial.
The o al sco es o indi idual animals o days 1 o 7 pos in ec ion and
he a i hme ic means (ho izon al ba s) calcula ed o g oups 1 o 3 (six
animals each) a e gi en. The as e isks indica e s a is ical signi icance
(Wilcoxon es ), and he P alues a e gi en.
TABLE 2. Reisola ion o he challenge s ain om ileocecal
lymph nodes, cecum, and ileum
Tissue Reisola ion o challenge s ain (CFU/g issue)
a
Non accina ed Salmopo c⌬ompD Salmopo c
Ileocecal lymph
nodes
1.21 ⫾10
4
*5.13 ⫾10
2
*6.67 ⫾10
1
*
Cecum 6.79 ⫾10
4
*2.67 ⫾10
2
*1.67 ⫾10
1
*
Ileum 7.30 ⫾10
4
1.67 ⫾10
3
6.67 ⫾10
2
a
An ⴱindica es ha he alues we e signi ican ly lowe (P⬍0.05; F iedmann
es ) han hose in he non accina ed con ol g oup.
2480 SELKE ET AL. INFECT.IMMUN.
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ec ed agains he OmpD p o ein ha e o be de eloped by he
hos upon in ec ion wi h S.en e ica se o a Typhimu ium o
accina ion wi h a comme cial S.en e ica se o a Typhi-
mu ium accine bu no upon accina ion wi h he ma ke
accine s ain S.en e ica se o a Typhimu ium Salmopo c-
⌬ompD. Thus, a se ological di e en ia ion o in ec ed and ac-
cina ed pigs is made possible (DIVA p inciple).
Salmonella an ibody i e s as assessed in he Salmo ype Pig-
Sc een ELISA s eadily ose du ing he s udy in bo h accina ed
g oups, whe eas i e s in he placebo g oup s ayed nega i e
(see Fig. S5 in he supplemen al ma e ial). An ibody i e s in
he OmpD-speci ic pep ide-based ELISA de eloped in he S.
en e ica se o a Typhimu ium Salmopo c- accina ed g oup bu
emained nega i e be o e challenge in he S.en e ica se o a
Typhimu ium Salmopo c⌬ompD- and placebo- accina ed
g oups (Fig. 4) (day 0 and day 36). Sho ly a e challenge,
OmpD-speci ic se ocon e sion was obse ed in he S.en e ica
se o a Typhimu ium Salmopo c⌬ompD- and placebo- acci-
na ed g oups (Fig. 4) (day 50). Th ee animals accina ed wi h
S.en e ica se o a Typhimu ium Salmopo c ⌬ompD be o e
in ec ion we e moni o ed se ologically up o 3 weeks pos chal-
lenge. OmpD-speci ic an ibody i e s emained posi i e un il
he end o he expe imen (Fig. 4) (days 57 and 65).
DISCUSSION
In his epo , we desc ibe he iden i ica ion o a DIVA
ma ke , i s in oduc ion in o he con en ionally a enua ed li e
accine s ain Salmopo c, and he de elopmen o a disc imi-
na o y ELISA sys em. Salmopo c was chosen o his wo k
because i has been shown o educe bo h shedding and colo-
niza ion o he po cine in es inal ac (41). The app oach
de eloped is s aigh o wa d and migh be gene ally applicable
o he cons uc ion o li e bac e ial DIVA accines.
The adap a ion o a widely used allelic exchange s a egy
wi h sacB as he coun e selec able ma ke allowed he con-
s uc ion o a Salmonella accine s ain ca ying an in- ame
dele ion o he ompD gene no con aining o eign DNA. Al-
hough his me hod is mo e labo ious han he commonly used
PCR-based allelic exchange desc ibed p e iously by Da senko
and Wanne (6), his app oach migh be ad an ageous o
accine de elopmen and subsequen licensing. Thus, he e-
sul ing s ain is indis inguishable om a spon aneously occu -
ing dele ion mu an and he e o e is no conside ed a gene ically
modi ied o ganism acco ding o Eu opean egula ions on gene ic
enginee ing (di ec i e [EC] 2001/18; h p://eu opa.eu/eu -lex/p i
/en/oj/da /2001/l_106/l_10620010417en00010038.pd ).
The p o ein OmpD, chosen as selec able ma ke , is one o
he mos abundan p o eins in he ou e memb ane o Salmo-
nella, ep esen ing abou hal o he 1 ⫻10
5
o2⫻10
5
po in
molecules pe cell unde a o able g ow h condi ions (27, 36).
I is p esen in all S.en e ica se o a s wi h he excep ion o S.
en e ica se o a Typhi (35, 37). In con as o he majo po in
OmpC, howe e , OmpD is no ound in o he g am-nega i e
bac e ia (40). The p esence o OmpD and o he po ins in he
de e gen wash ac ion is likely due o he p esence o mem-
b ane esicles (blebs) commonly o med by g am-nega i e bac-
e ia (31, 52). On he one hand, i s high abundance makes
OmpD a highly a ac i e choice as a nega i e ma ke . Thus,
se ocon e sion is likely o occu upon in ec ion o pigs wi h any
Salmonella se o a (excep S.en e ica se o a Typhi), he eby
ac i a ing he DIVA unc ion. This b oad-spec um DIVA
unc ion is manda o y o a po cine Salmonella accine, as
legisla ion is no limi ed o ce ain se o a s. The e o e, con-
en ional ma ke s ains, such as ough mu an s (39, 49), can-
no be used. On he o he hand, OmpD is homologous o o he
po ins common among En e obac e iaceae. The e o e, in o de
o ob ain a su icien ly disc imina o y ELISA e icacy, no he
en i e OmpD p o ein bu an OmpD-speci ic pep ide selec ed
by pep ide spo a ay analyses had o be used as a solid-phase
an igen.
In addi ion o he mo e di icul se up o a disc imina o y
se ological es , he use o a majo immunogenic p o ein as a
DIVA an igen has wo o he possible d awbacks. Thus, he
dele ion migh (i) cause an addi ional a enua ion o he mu-
an s ain and (ii) diminish p o ec i e e icacy. Fo he dele-
ion o OmpD om S.en e ica se o a Typhimu ium, epo s
wi h espec o a enua ion a e con o e sial (10, 25). Bo h
s udies, howe e , ha e been ca ied ou using an ompD mu an
ob ained by Tn10-based mu agenesis (10, 25). In o de o en-
su e unchanged i ulence o he S.en e ica se o a Typhi-
mu ium Salmopo c⌬ompD s ain, we ca ied ou an LD
50
de-
e mina ion in BALB/c mice. This expe imen unambiguously
showed equal i ulence o bo h s ains.
The accina ion s udy pe o med demons a ed ha pigs
accina ed wi h ei he accine we e p o ec ed equally well
om clinical symp oms, he eby con i ming he esul s om
he mouse expe imen . On he o he hand, he DIVA accine
showed a educed e icacy in compa ison o he Salmopo c
pa en s ain wi h espec o educing o gan coloniza ion.
Thus, he numbe o S.en e ica se o a Typhimu ium DT104
cells o be isola ed om ileocecal lymph node, cecum, and
FIG. 4. Se ological esponses o pigs in a disc imina o y ELISA
upon immuniza ion and challenge. Animals we e immunized on days 1
and 21 and challenged on day 43. Blood was aken on days 0, 36, and
50 (six animals pe g oup) and on days 57 and 65 ( h ee animals).
An i-OmpD an ibody i e s we e de e mined using an ELISA wi h
OmpD-de i ed pep ide 2 as a solid-phase an igen. The ho izon al line
ma ked wi h “⫹” (OD% ⫽100) indica es he posi ion o he in e nal
posi i e con ol consis ing o he pooled se a om day 50. OD%,
ela i e op ical densi y o he ex se a compa ed o ha o he in e nal
posi i e con ol se um (de ined as 100 OD%).
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ileum was 10- old highe in pigs immunized wi h Salmopo c-
⌬ompD han in hose immunized wi h he Salmopo c pa en
s ain. These esul s imply ha a dele ion o he OmpD p o-
ein, al hough o no consequence wi h espec o causing sep-
icemia in mice upon in ape i oneal applica ion, migh educe
coloniza ion and su i al upon o al applica ion in he po cine
gas oin es inal ac . Howe e , he DIVA accine s ain s ill
educes S.en e ica se o a Typhimu ium eisola ion a es sig-
ni ican ly (10- o 100- old).
As an in ec ion occu ing a e accina ion is no masked,
he S.en e ica se o a Typhimu ium Salmopo c⌬ompD ac-
cine s ain, despi e i s educed e icacy in educing coloniza-
ion, migh be a aluable ool in se osu eillance-based Salmo-
nella con ol p og ams aimed a educing he isk o human
in ec ion. The ac ha S.en e ica se o a Typhimu ium
Salmopo c⌬ompD does no con ain o eign DNA and ha i s
pa en s ain, S.en e ica se o a Typhimu ium Salmopo c, has
been used ex ensi ely in he ield should acili a e he ealiza-
ion o u u e ield s udies and subsequen licensing p oce-
du es. Fu he mo e, he S.en e ica se o a Typhimu ium
Salmopo c⌬ompD accine s ain migh be usable as a ca ie o
o eign an igens and migh he e o e open new ways o he
cons uc ion o mul i alen li e accines o li es ock.
ACKNOWLEDGMENT
This s udy was suppo ed by he Biop o ile p ojec PTJ-BIO/
0313037 (BMBF), Bonn, Ge many.
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