Table Of ContentCrombéetal.VeterinaryResearch2013,44:4
http://www.veterinaryresearch.org/content/44/1/4 VETERINARY RESEARCH
RESEARCH Open Access
Serological profiles in nursery piglets colonized
with Staphylococcus aureus
Florence Crombé1,2*, Wannes Vanderhaeghen1,2, Corné P de Vogel3, Willem J Van Wamel3, Kurt Barbé4,
Katleen Hermans2, Freddy Haesebrouck2 and Patrick Butaye1,2
Abstract
Atpresent, the immune response of pigs in relation to Staphylococcus aureus carriage is poorlyunderstood. This
study was aimed atinvestigating the dynamics of the anti-staphylococcalhumoral immune response in
methicillin-susceptibleS. aureus (MSSA)-positive piglets and atassessing the effect ofthe experimentalintroduction
ofa methicillin-resistant S.aureus (MRSA) Sequence Type (ST) 398strain. Therefore, serum samples were collected at
different times from 31 weaned piglets originating from four different sows. Twenty-four out of the 31 piglets were
challenged withMRSA ST398. The serum samples were analyzedfor IgG antibodies to 39S. aureus antigens,using a
multiplex bead-based assay (xMAP technology, Luminex Corporation). Though antibody responses showed broad
inter-individual variability, serological results appeared to be clustered bylitter oforigin.For most antigens, an
age-related response was observed with anapparentincrease inantibody titersdirected against staphylococcal
microbial surface componentsrecognizing adhesivematrix molecules (MSCRAMM),which have been shown to play
a rolein S.aureus colonization. In mostanimals,antibody titers directed against staphylococcal toxins or
immune-modulatingproteins decreased with age, possibly reflecting the absence of bacterial invasion. The
introduction of MRSA ST398 did not elicit a significant humoral immune reaction.
Thisstudy describes, for the first time, the humoral immuneresponse inweaned pigs colonized with S.aureus.
Introduction molecules (MSCRAMM) and against virulence factors
Staphylococcus aureusisan opportunistic pathogen resid- involvedinevadingordestroying hostdefenses[4-8].
ing on the skin and mucous membranes of humans and The extensive spread of livestock-associated methicillin-
severalanimalspecies[1,2].Inhumans,ithasbeenshown resistant S. aureus (LA-MRSA) clonal complex (CC) 398
thatnasalS.aureuscarriageincreasestheriskofinfection and CC9 among pigs has raised questions on the mechan-
by a factor three, with patients being mostly affected by isms establishing the interactions between MRSA and pigs
their autologous strain [3]. Conversely, a lower risk for [9-11]. As in humans, the pigs’ immune system might play
bacteremia-related death has been reported in S. aureus an important regulatory role. However, at present, know-
carrierscomparedtonon-carriers[3]. ledgeonthe(humoral)immuneresponseofpigstoS.aureus
Thedifferences inrisk andoutcomeofS.aureusinfec- is lacking. Therefore, our aim was to increase the under-
tions between human carriers and non-carriers have standingofthistopic.
been associated with the humoral immune response, In the present study, we examined the kinetics of anti-
protecting carriers from bacteremia mediated death and staphylococcal antibodies in weaned piglets colonized
non-carriers from S. aureus colonization. This response with S. aureus by determining the levels of IgG anti-
might include antibodies against staphylococcal micro- bodies to 9 MSCRAMM, 23 staphylococcal toxins and 7
bial surface components recognizing adhesive matrix immune-modulating proteins in serum samples. In
addition, we investigated the effect of the introduction
of a MRSA Sequence Type (ST) 398 strain on the pig-
*Correspondence:[email protected]
1VeterinaryandAgrochemicalResearchCentre(VAR),DepartmentofBacterial lets’humoralimmuneresponse.
Diseases,Groeselenberg99,Ukkel,Belgium
2GhentUniversity,FacultyofVeterinaryMedicine,DepartmentofPathology,
BacteriologyandAvianDiseases,Salisburylaan133,Merelbeke9820,Belgium
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Crombéetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Materials and methods Anti-staphylococcalantibodyassay
Animalsandserumsamples Levels of IgG directed against 39S. aureus proteins were
Venous blood samples were obtained from 31 healthy quantified simultaneously using a bead-based flow cyto-
weaned cross-bred piglets (Landrace × Piétrain, Malèves- metry technique (xMap; Luminex Corporation, Austin,
St.Marie,Belgium)previouslydescribedinatransmission TX, USA) based on a previously described method [5,6].
study with MRSA strain C26 (ST398, spa type t011, Briefly,serum sampleswerediluted 1:50inPBS-BN(PBS,
SCCmec type V) [12]. Briefly, at 21 days of age, piglets 1% bovine serum albumin, and 0.05% sodium azide
obtainedfromfourdifferent sows(1–4),weretransported [pH7.4]) for measurements of antigen-specific IgG. Per
to the experimentalsite and distributed into three experi- diluted sample, 50 μL was incubated with the different
mental groups (1–3) consisting of eight piglets, and one fluorescence-coloured antigen-coupled microspheres, i.e.
negative control group of seven piglets. All three experi- 3000 beads per antigen per well, in a 96-well filtermicro-
mentalgroupsincludedfourmaleandfemalepairsorigin- plate(Millipore,Amsterdam,TheNetherlands)for35min
ating from each of the four sows. The negative control at room temperature on a Thermomixer plate shaker
group was composed of three male and female pairs, ori- (Eppendorf, Nijmegen, The Netherlands). After washing
ginatingfromsows1–3,andasinglefemalepigletoriginat- the plate twice with PBS-BN and aspiration by vacuum
ing from the fourth sow. The transmission study was manifold, 50 μL of a 1:100 dilution of R-phycoerythrin
performed by introducing two MRSA-positive animals (RPE)-labelledanti-swineIgG(JacksonImmunoResearch,
(i.e. seeders that were inoculated with ~9 × 108 CFU of Westgrove, PA, USA) was added. Following a second in-
MRSA strain C26 two days before their introduction), at cubation(35minatroomtemperature)andwashingstep,
30daysofage,ineachexperimentalgroupcomposedofsix the microspheres were resuspended in 100 μL PBS-BN.
MRSA-negative piglets. Swab samples from all animals Measurements were performed on the Luminex 100 in-
werecollectedseparatelyfrombothanteriornares,theskin strument (BMD, Croissy-Beaubourg, France) using Lumi-
behind the ears and the perineum, during a period of six nex software (version 2.2). Tests were performed in
weeks to determine their S. aureus carriage state. For the independent duplicates and the median fluorescence in-
presentstudy,serumsamplesweretakenbeforethestartof tensity (MFI) values, reflecting semi-quantitative antibody
the transmission experiment (six days before introduction levels,wereaveraged.Duplicateswithacoefficientof vari-
oftheMRSApositiveanimals–age:24days)andat8,15, ance(CV)higherthan25%wereconsideredasnotrepro-
22,29and 36 days post introduction,correspondingto 38, ducible and deleted from the data set. If antibody binding
45,52,59and66daysofage. was observed in the serum samples incubated with the
Testingat24daysofagerevealedthatallanimalswere control beads (i.e. beads without protein coupled on their
positive for methicillin-susceptible S. aureus (MSSA) surface),thenonspecificMFIvaluesweresubtractedfrom
ST9 (spa type t3446 or t337). In all 24 animals that were theantigen-specificresults.Swinepooledserum,collected
included in the MRSA transmission study, MSSA and/or from 85 healthy pigs, incubated with protein-coupled
MRSA were detected until the end of the trial [12]. In beads was used as a positive control. Similarly, PBS-BN
the seven animals of the negative control group that wasincludedasanegativecontrol.
were not colonized with MRSA strain C26, MSSA was
not detected after the second sampling occasion, except Statisticalanalysis
once for one animal at the end [12]. To evaluate the ef- Independent-sample median test was performed to de-
fect of MRSA strain C26 introduction on the piglets’ termine the distribution across the animals grouped by
humoral response, animals were classified as persistent litter of origin with a 0.05 threshold at 24 days of age
MRSA carriers (n = 8) if ≥ 80% of the samples taken at (SPPSsoftware,version 19)[16].
different time points after the introduction of MRSA The kinetics of anti-staphylococcal antibodies were
were MRSA positive. Animals were classified as non- analyzed using linear regression during a 6-week period.
MRSA carriers (n = 7) if all samples were MRSA nega- A linear mixed model including fixed and random
tive; the other animals were classified as intermittent effects was used by means of the MIXED procedure of
MRSAcarriers (n=16). SPPS software, version 19. The model was fitted using
the Maximal Likelihood estimation (ML) method and
used to evaluate the effect of MRSA ST398 introduction
S.aureusstraincharacteristics on the antibody profiles of the piglets. Age (in days),
The presence of 39 virulence-associated genes (Table 1) group (non-MRSA carriers vs. persistent and intermit-
was determined in one MSSA isolate of each spa type tent MRSA carriers) and gender (male vs. female) were
originatingfromonerandomlyselectedanimalat24days introduced as fixed effects. To increase the degrees of
of age and in MRSA strain C26 by DNA microarray by freedom in the statistical analysis, the factor “age” was not
AlereTechnologiesGmbH,Jena,Germany [13-15]. included as a repeated measure, though the different
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Table1DNAmicroarrayresultsforthe39S.aureusantigensstudiedbyLuminexassay
AntigensofS.aureus Fullname DNAmicroarrayresults
ST398-V/t011 ST9/t3446 ST9/t337
MSCRAMM
ClfA clumpingfactorA + + +
ClfB clumpingfactorB + + +
FnbpA fibronectinbindingproteinA + + +
FnbpB fibronectinbindingproteinB + + +
IsdA iron-responsivesurfacedeterminantA + + +
IsdH iron-responsivesurfacedeterminantH ND ND ND
SasG surfaceproteinG - - -
SdrD serine-aspartatedipeptiderepeatD + + +
SdrE serine-aspartatedipeptiderepeatE ND ND ND
Immune-modulatingproteins
CHIPS chemotaxisinhibitoryproteinofS.aureus - - -
SCIN staphylococcalcomplementinhibitor - - -
SSL1 S.aureussurfaceproteinlike1 + + +
SSL3 S.aureussurfaceproteinlike3 -* + +
SSL5 S.aureussurfaceproteinlike5 + + +
SSL9 S.aureussurfaceproteinlike9 + + +
SSL11 S.aureussurfaceproteinlike11 - - -
Leucotoxins
HlgB haemolysinbeta +** + +
LukD leukocidinD - - -
LukE leukocidinE - - -
LukF leukocidinF + + +
LukS leukocidinS + + +
Staphylococcalenterotoxins
α-toxin alfa-toxin + + +
SEA staphylococcalenterotoxinA - - -
SEB staphylococcalenterotoxinB - - -
SEC staphylococcalenterotoxinC - - -
SED staphylococcalenterotoxinD - - -
SEE staphylococcalenterotoxinE - - -
SEG staphylococcalenterotoxinG - + +
SEH staphylococcalenterotoxinH - - -
SEI staphylococcalenterotoxinI - + +
SEJ staphylococcalenterotoxinJ - - -
SEM staphylococcalenterotoxinM - + +
SEN staphylococcalenterotoxinN - + +
SEO staphylococcalenterotoxinO - + +
SEQ staphylococcalenterotoxinQ - - -
SER staphylococcalenterotoxinR - - -
TSST-1 toxicshocksyndrometoxin-1 - - -
Exfoliativetoxins
ETA exfoliativetoxinA - - -
ETB exfoliativetoxinB - - -
ND:Notdetermined.
*-forStaphylococcalSuperantigenlikeprotein8/SSL3,+forStaphylococcalSuperantigenlikeproteinB3.
**+forun-truncatedHaemolysinBeta.
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sampling occasions are dependent, because of the limited Anti-staphylococcalantibodyresponse
dataofthestudy.However,theinteractionterm“age*group” Overallantibodyresponseovertime
wasaddedtocorrectforthetimeeffect.Inthiswaychanges IgG antibodies were detected in the positive control
between groups over time could be observed. The piglets sample (swine pooled serum) for all but one (i.e. SEB)
andtheirrespectivemothers(i.e.theinteractionterm“pig*- antigen. The negative control (PBS-BN) incubated with
sow”)wereintroducedasrandomeffecttoaccountforpig- protein-coupled beads resulted in low MFI values (< 9).
letandsowindividualeffectsanddisturbances. Serum incubated with control beads resulted in median
MFI values of 208 (range: 83–606). Two samples were
deleted from the data set, corresponding to serum of an
Results animal at 24 days of age and one at 59 days of age, be-
S.aureusstraincharacteristics cause the first one had high control bead levels (MFI-
Thepresenceofgenesencodingvirulencefactorsincluded value of 1969) and the other one showed MFI-values
in the antibody assay in the studied S. aureus strains is similar to the PBS-BN. Both piglets belong to the per-
mentionedinTable1. sistentMRSAcarriergroup.
Figure1Medianofthemedianfluorescenceintensity(MFI)valuesreflectingIgGlevelsfor33S.aureusantigensin31pigletsover
6weeks.Fourtrendswereobserved:increasingantibodylevels(A),increasingantibodylevelsfollowedbyaslowdecline(B,C,D),decreasing
antibodylevels(E,F)andnearlynovaryingantibodylevelsovertime(G).
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Median MFI values reflecting IgG levels against six trend, the levels of antibodies against α-toxin and SSL1
out of 39S. aureus antigens were nearly absent in the declined, it appeared that IgG levels increased for certain
piglets’serum samples, i.e. SEB, SEE, SEH, SEI, SEN and animalsat59and66daysofage(Figure4).
SEO (median MFI < 1) (data not shown). As a result,
the antibody response against these antigens will not be EffectofMRSAintroductiononantibodyprofiles
furtherdiscussed. The‘group’effectwassignificantforoneantigen(i.e.ClfB,
As shown in Figure 1, four antibody response trends p = 0.014), with mean IgG levels being significantly lower
could be observed over the 6-week period: i. continu- in persistent MRSA carriers compared with non-MRSA
ously increasing antibody levels over time (A), ii. in- carriers(p=0.031).MeanIgGlevelsdirectedagainstClfB
creasing antibody levels followed by a slow decline over werelower(thoughnot significant)inintermittentMRSA
time (B,C,D), iii. a high antibody level at the start that carriers compared with non-MRSA carriers (p = 0.09)
decreased over time (E,F) and iv. nearly no varying anti- (datanotshown).
body levels over time (G). In line with these observa- The humoral immune response to ClfA, ClfB, ETB and
tions, a significant “time” effect was observed (p < 0.05) FnbpB differed significantly between groups over time
for all but two of the 33 antigens (i.e. ETB and SEC) (age*groupinteraction;p<0.001,p=0.001,p=0.035and
(data not shown). p = 0.006, respectively). At the peak of the IgG response
(38daysof age),antibodylevelsdirected against ClfAand
Antigen-specificantibodylevelsclassifiedpersow ETBweresignificantlyhigherinnon-MRSAcarrierscom-
Although antibody levels varied among the animals, the pared to persistent MRSA carriers (p < 0.001; p = 0.003)
IgGlevelsdirectedagainstseveralantigensappearedtobe and intermittent MRSA carriers (p = 0.007; p = 0.006)
grouped by litter of origin (Figure 2). These data are pre- (Figure 5). For ClfB, antibody levels were significantly
sentedinanadditionaldatafile(seeAdditionalfile1).An higher in non-MRSA carriers compared to intermittent
independent-sample median test revealed that the distri- MRSA carriers (p < 0.001) though not compared to per-
butionsacrosstheanimalsgroupedbylitteroforiginwere sistent MRSA carriers (p = 0.12) (Figure 5). Conversely,
significantly different for 24 out of the 33 antigens tested for FnbpB, antibody levels were significantly higher in
at24daysofage(p<0.05)(Figure2).Also,whenfocusing non-MRSAcarrierscomparedtopersistentMRSAcarriers
on the antigens with decreasing global antibody profiles (p=0.003)thoughnotcomparedtoIC(p=0.058)(Figure5).
(i.e., α-toxin, HlgB, SSL1, SSL3, SSL5, SSL9, SSL11, LukE However,forETB,onenon-MRSAcarrierhadaveryhigh
and LukS), a litter-related decrease was perceived IgG level at 38 days of age and when deleting this record
over time (Figure 3). Remarkably though as a general from the dataset a significant difference was no longer
observed(age*groupinteraction;p=0.77).
Whendeletinganadditionalnon-MRSAcarrierfromthe
dataset, with high levels directed against ClfA and FnbpB
at38daysofage,asignificantdifferencewasalsonolonger
observed(age*groupinteraction;p=0.094andp=0.075).
For each sampling occasion, mean IgGlevels ± 95% CI
of intermittent MRSA carriers, persistent MRSA carriers
and non-MRSA carriers are presented in an additional
data file(see Additionalfile2).
Discussion
To our knowledge, this is the first study on humoral im-
muneresponseagainstS.aureusinweanedpigs.Weuseda
multiplex bead-based assay (xMAP technology, Luminex
Figure2Medianfluorescenceintensity(MFI)valuereflecting Corporation) to simultaneously quantify antibodies against
IgGlevelsfor33S.aureusantigensin30pigletsclassifiedper 39 staphylococcal proteins in small-volume serum samples.
litterat24daysofage.Eachdotrepresentsasinglepiglet,green This high-throughput immunological test is of particular
squaresrepresentlitter1,bluediamondsrepresentlitter2,red
interestsinceitgivestheopportunitytosimultaneouslystudy
trianglesrepresentlitter3andorangespheresrepresentlitter4.
thehumoralresponseof healthyordiseasedindividualstoa
MedianIgGlevelsareindicatedbyhorizontallines.*Significantwith
independent-samplemediantestacrossanimalsgroupedbylitterat specificpathogentowardslargesetsofantigens[5,6,17,18].
24daysofage(α-toxin,FnbpB,SED,SSL1,SSL3andSSL5,p<0.001; Antibodies against all but one of the 39 antigens (i.e.
ClfA,ClfB,ETA,HlgB,IsdA,IsdH,LukE,LukF,SdrE,SEJ,SSL9andSSL11, SEB) were detected in the swine pooled serum standard
p<0.005;ETB,LukD,p<0.01;SasG,SdrD,SECandTSST-1,p<0.05);
positive control. Therefore, the present study does not
†UnabletocomputeforSEQ.
allow drawing conclusions with regards to serological
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Figure3Medianfluorescenceintensity(MFI)valuesreflectingIgGlevelsagainstα-toxin,HlgB,SSL1,SSL3,SSL5,SSL9,SSL11,LukEand
LukSin31pigletsclassifiedperlitterovera6-weekperiod.Eachdotrepresentsasinglepiglet,greensquaresrepresentlitter1,bluediamonds
representlitter2,redtrianglesrepresentlitter3andorangespheresrepresentlitter4.MedianIgGlevelsareindicatedbyhorizontallines.
responses to SEB. However, given that SEB is generally synthesis, whereas antibodies with decreasing titers are
absent in S. aureus strains of animal origin [19], which most probably from maternal origin. Interestingly, most
was also the case for the investigated strains, it is un- antibodies with increasing levels were directed to antigens
likely thatpigsproducedantibodies againstthis antigen. belonging to the family of the MSCRAMM, which are
Fourtrendswereobservedamongthedynamicsofanti- knowntobeexpressedbyactivelydividingbacteriaandare
staphylococcal antibody response in all piglets colonized essential in the colonizationprocessof S.aureus[2,22-24].
withS.aureus:i.amainincreaseinresponseovertime,ii. Thisindicatesthatmaternalantibodiesdonotprotectpig-
an increase in response followed by a slow decline, iii. a lets from colonization. Indeed, it has been described that
main decrease in response over time or iv. almost no piglets derived from S. aureus-positive sows are more
changeinresponse.Giventhattheobservationalperiodof likely to become S. aureus colonized [25]. In contrast,
thepresentstudystartedatweaning,itislikelythatserum antibodies with decreasing levels were mostly directed
IgG directed against certain antigens are de novo synthe- against staphylococcal toxinsor immune-modulatingpro-
sized by the piglets whereas others are of maternal origin teinsinterferingwithhostdefencesandplayingaroledur-
[20,21]. Increasing antibody titers indicate de novo ingstaphylococcalinvasion[5,6].
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Figure4Medianfluorescenceintensity(MFI)valuesreflectingIgGlevelsagainstα-toxinandSSL1in31pigletsclassifiedperMRSA
carriergroupovera6-weekperiod.Eachdotrepresentsasinglepiglet,greensquaresrepresentintermittentMRSAcarriers(n=16);blue
diamondsrepresentpersistentMRSAcarriers(n=8),redtrianglesrepresentnon-MRSAcarriers(n=7).MedianIgGlevelsareindicatedby
horizontallines.
Figure5Meanofthemedianfluorescenceintensity(MFI)valuesreflectingIgGlevelsforClfA,ClfB,ETBandFnbpBperMRSAcarrier
groupovertime.GreensquaresrepresentintermittentMRSAcarriers(n=16);bluediamondsrepresentpersistentMRSAcarriers(n=8),red
trianglesrepresentnon-MRSAcarriers(n=7).Dataarepresentedasmean±95%CI.↑:MRSAintroduction.
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For certain antigens, antibody response was observed lower in persistent MRSA carriers and/or intermittent
thoughthe correspondinggeneswerenotdetectedonthe MRSAcarrierscomparedtonon-MRSAcarriersat38days
microarray in the investigated S. aureus strains. Hence, it of age. This might indicate that MRSA ST398 strain C26
must be noted that the microarray technology used here suppresses systemic antibody responses against certain S.
bears some limitation as it only detects genes matching aureus antigens thus possibly facilitating its colonization.
the probes printed on the array. Given that this composi- However, since extensive inter-individual differences were
te multi-strain array covers allelic variants of genes of observed, these significant effects appeared to result from
S. aureus genomes of diverse human and bovine lineages, few animals for ClfA and FnbpB. Future studies with
allelic variants specific for porcine ST398 and ST9 strains caesarean-derived colostrum-deprived piglets may help to
might be missing [14,15,26]. For example, Schijffelen [27] further elucidate the immune response associated with
reported that a scn gene homologue encoding SCIN was MRSAcolonization,althoughthisisamoreartificialmodel
present on a novel ‘animal-specific’staphylococcal patho- [39].Inaddition,theimmunogenicpotentialofhighlyviru-
genicityisland(SaPI-S0385),inMRSAST398strainS0385 lent MRSA clones such as the community-associated
that was not included on the array. Similarly, a chp gene MRSA-likeST8clone,knownasUSA300,recentlyisolated
homologue coding for Fprl1 inhibitory protein (Flipr) [28], frompigsinPeru[40]andUSA[41],areinterestingissues
a CHIPS homologue, has been described in MRSA ST398 to consider in further studies. In that way, the effect of
strainS0385.Anotherexplanationmightbethatothernon- stronglyimmunogenicextracellulartoxinssuchasPanton-
S. aureus staphylococcal species, present on the piglets’ Valentine leukocidin, a cytotoxin which is probably asso-
mucosae, express S. aureus like antigens. Several studies ciated with necrotizing lesion development, and additional
reported a high occurrence of non-S. aureus staphylococci enterotoxins on the humoral immune response of piglets
in the nares of pigs [29-32] and it has been shown that couldbestudied.
staphylococcal species share a common reservoir of viru- SincethefocushasbeensetonserumIgGwhichdomi-
lencefactors[33-35].Notably,S.aureusandStaphylococcus nates the systemic immunity, the possible role of IgA
hyicus, a pathogen causing exudative epidermitis in pigs involved in the mucosal immune system, remains to be
and a staphylococcal species commonly found in pigs’ en- investigated [20,42-44]. Also, the cellular immune re-
dogenousflora[29,36],carryrelatedETB[33],whichmight sponseafterS.aureuscolonizationshouldbeinvestigated.
explainthehighIgGlevelsobservedina38-day-oldanimal Inconclusion,thisisthefirstreportonthehumoralim-
inthepresent studythoughETBwasabsentintheinvesti- mune response in weaned pigs colonized with S. aureus.
gatedS.aureusstrains. Antibody titers directed against MSCRAMM increased
On the contrary, IgG levels against several enterotox- over time whereas antibody titers directed against
ins (i.e. SEI, SEO, SEN) – encoded on the enterotoxin staphylococcal toxins or immune-modulating proteins
gene cluster (egc) – were nearly absent though genes decreased, which might partly reflect the role of specific
coding for these factors were present in the investigated antigens in S. aureus colonization. The introduction of
S. aureus strains. Staphylococcal enterotoxins are likely MRSAST398didnotelicitasignificanthumoralimmune
to be mainly expressed during infection and less during reaction. The presence of maternally derived immunity
colonization, which might explain the absence of anti- andotherstaphylococcalspeciesinthepiglets’microbiota
bodies in sera from healthy pigs. Antibodies against egc mayhaveinfluencedtheresultsofthepresentstudy.
enterotoxinsarealsorarelydetectedinserafrom healthy
humans [37]. Additional files
The introduction of MRSA strain C26 at weaning
appeared not to increase the anti-staphylococcal antibody Additionalfile1:Medianfluorescentintensity(MFI)values
titers.Theseobservationsindicatealowimmunogenicpo- reflectingantigen-specificimmunoglobulin(Ig)Glevelsbetween
littersat24daysofage.
tential of strain C26 during colonization. However, since
Additionalfile2:Medianfluorescentintensity(MFI)values
the observational period is at weaning, maternal anti-
reflectingantigen-specificimmunoglobulin(Ig)Glevelsbetween
bodies might have interfered with the piglets’ response intermittentMRSAcarriers,persistentMRSAcarriersandnon-MRSA
[20,21]. Furthermore, conventional pigs naturally colo- carriersduring6weeks.
nized with S. aureus and having normal microbiota, in-
cluding various staphylococcal species [30,38], were used Competinginterests
Theauthorsdeclarethattheyhavenocompetinginterests.
in the present study. Though our trial resembles MRSA
colonization as noticed in the field situation on many pig Authors’contributions
herds [9,25], using such pigs complicates the interpret- Experimentaldesignandplanning:FC,KH,FH,PB;animalexperiments:FC;
ation of the immune response against the MRSA ST398 serologicalanalysisCPDV,WJVW;dataprocessingandstatisticalanalysisFC,
CPDV,WJVW,KB;draftingofthemanuscript:FC,WV;criticalrevisionofthe
strain used here. Indeed, in the present study, IgG levels
manuscript:CPDV,WJVW,KB,KH,FH,PB.Allauthorsreadandapprovedthe
directed against ClfA, ClfB and FnbpB were significantly manuscript.
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Acknowledgements 16. TheSPSSsoftwarewebsite[www.spss.com]
ThisstudywasapprovedbytheVeterinaryandAgrochemicalResearch 17. ShomaS,VerkaikNJ,deVogelCP,HermansPW,vanSelmS,MitchellTJ,van
Centre(VAR)animalcarecommitteeandfinancialsupportwasreceivedfrom RoosmalenM,HossainS,RahmanM,EndtzHP,vanWamelWJ,vanBelkum
theInstituteforthePromotionofInnovationbyScienceandTechnologyin A:Developmentofamultiplexedbead-basedimmunoassayforthe
Flanders(IWT)project070596. simultaneousdetectionofantibodiesto17pneumococcalproteins.EurJ
WethankWillemVanCampeandhisteamforexcellenttechnicalassistance. ClinMicrobiolInfectDis2011,30:521–526.
18. vandenBergS,BowdenMG,BosmaT,BuistG,vanDijlJM,vanWamelWJ,
Authordetails deVogelCP,vanBelkumA,Bakker-WoudenbergIA:Amultiplexassayfor
1VeterinaryandAgrochemicalResearchCentre(VAR),DepartmentofBacterial thequantificationofantibodyresponsesinStaphylococcusaureus
Diseases,Groeselenberg99,Ukkel,Belgium.2GhentUniversity,Facultyof infectionsinmice.JImmunolMethods2011,365:142–148.
VeterinaryMedicine,DepartmentofPathology,BacteriologyandAvian 19. MoneckeS,CoombsG,ShoreAC,ColemanDC,AkpakaP,BorgM,ChowH,
Diseases,Salisburylaan133,Merelbeke9820,Belgium.3ErasmusMedical IpM,JatzwaukL,JonasD,KadlecK,KearnsA,LaurentF,O’BrienFG,Pearson
Centre,DepartmentsofMedicalMicrobiologyandInfectiousDiseases, J,RuppeltA,SchwarzS,SciclunaE,SlickersP,TanHL,WeberS,EhrichtR:A
‘s-Gravendijkwal230,Rotterdam,TheNetherlands.4VrijeUniversiteitBrussel, fieldguidetopandemic,epidemicandsporadicclonesofmethicillin-
FacultyofEngeneering,Deptartmentoffundamentalelectricityand resistantStaphylococcusaureus.PLoSOne2011,6:e17936.
instrumentation(ELEC),Pleinlaan2,Brussels,Belgium. 20. BaileyM,HaversonK,InmanC,HarrisC,JonesP,CorfieldG,MillerB,Stokes
C:Thedevelopmentofthemucosalimmunesystempre-andpost-
Received:16July2012Accepted:11January2013 weaning:balancingregulatoryandeffectorfunction.ProcNutrSoc2005,
Published:22January2013 64:451–457.
21. ButlerJE,ZhaoY,SinkoraM,WertzN,KacskovicsI:Immunoglobulins,
antibodyrepertoireandBcelldevelopment.DevCompImmunol2009,
References 33:321–333.
1. GordonRJ,LowyFD:Pathogenesisofmethicillin-resistantStaphylococcus
aureusinfection.ClinInfectDis2008,46(Suppl5):S350–359. 22. WertheimHF,WalshE,ChoudhurryR,MellesDC,BoelensHA,MiajlovicH,
VerbrughHA,FosterT,vanBelkumA:KeyroleforclumpingfactorBin
2. HermansK,DevrieseLA,HaesebrouckF:Staphylococcus.InPathogenesisof
Staphylococcusaureusnasalcolonizationofhumans.PLoSMed2008,5:e17.
bacterialinfectionsinanimals.4thedition.EditedbyGylesCL,PrescottJF,
SongerJG,ThoenCO.USA:Wiley-Blackwell;2010:75–89. 23. BurianM,RautenbergM,KohlerT,FritzM,KrismerB,UngerC,Hoffmann
WH,PeschelA,WolzC,GoerkeC:Temporalexpressionofadhesion
3. WertheimHF,VosMC,OttA,vanBelkumA,VossA,KluytmansJA,van
factorsandactivityofglobalregulatorsduringestablishmentof
KeulenPH,Vandenbroucke-GraulsCM,MeesterMH,VerbrughHA:Riskand
Staphylococcusaureusnasalcolonization.JInfectDis2010,201:1414–1421.
outcomeofnosocomialStaphylococcusaureusbacteraemiainnasal
carriersversusnon-carriers.Lancet2004,364:703–705. 24. BurianM,WolzC,GoerkeC:RegulatoryadaptationofStaphylococcus
aureusduringnasalcolonizationofhumans.PLoSOne2010,5:e10040.
4. ClarkeSR,BrummellKJ,HorsburghMJ,McDowellPW,MohamadSA,Stapleton
25. WeeseJS,ZwambagA,RosendalT,Reid-SmithR,FriendshipR:Longitudinal
MR,AcevedoJ,ReadRC,DayNP,PeacockSJ,MondJJ,Kokai-KunJF,FosterSJ:
investigationofmethicillin-resistantStaphylococcusaureusinpiglets.
Identificationofinvivo-expressedantigensofStaphylococcusaureusand
ZoonosesPublicHealth2011,58:238–243.
theiruseinvaccinationsforprotectionagainstnasalcarriage.JInfectDis
2006,193:1098–1108. 26. McCarthyAJ,LindsayJA:GeneticvariationinStaphylococcusaureussurface
andimmuneevasiongenesislineageassociated:implicationsforvaccine
5. vanBelkumA,VerkaikNJ,deVogelCP,BoelensHA,VerveerJ,NouwenJL,
designandhost-pathogeninteractions.BMCMicrobiol2010,10:173.
VerbrughHA,WertheimHF:ReclassificationofStaphylococcusaureus
nasalcarriagetypes.JInfectDis2009,199:1820–1826. 27. SchijffelenMJ,BoelCH,vanStrijpJA,FluitAC:Wholegenomeanalysisofa
6. VerkaikNJ,deVogelCP,BoelensHA,GrumannD,HoogenboezemT,VinkC, livestock-associatedmethicillin-resistantStaphylococcusaureusST398
HooijkaasH,FosterTJ,VerbrughHA,vanBelkumA,vanWamelWJ:Anti- isolatefromacaseofhumanendocarditis.BMCGenomics2010,11:376.
staphylococcalhumoralimmuneresponseinpersistentnasalcarriers 28. PratC,BestebroerJ,deHaasCJ,vanStrijpJA,vanKesselKP:Anew
andnoncarriersofStaphylococcusaureus.JInfectDis2009,199:625–632. staphylococcalanti-inflammatoryproteinthatantagonizestheformyl
7. VerkaikNJ,LebonA,deVogelCP,HooijkaasH,VerbrughHA,JaddoeVW, peptidereceptor-like1.JImmunol2006,177:8017–8026.
HofmanA,MollHA,vanBelkumA,vanWamelWJ:Inductionofantibodies 29. DevrieseLA:Staphylococciinhealthyanddiseasedanimals.SocAppl
byStaphylococcusaureusnasalcolonizationinyoungchildren. BacteriolSympSer1990,19:S71–80.
ClinMicrobiolInfect2010,16:1312–1317. 30. BaeleM,ChiersK,DevrieseLA,SmithHE,WisselinkHJ,VaneechoutteM,
8. Colque-NavarroP,JacobssonG,AnderssonR,FlockJI,MollbyR:Levelsof HaesebrouckF:Thegram-positivetonsillarandnasalfloraofpiglets
antibodyagainst11Staphylococcusaureusantigensinahealthy beforeandafterweaning.JApplMicrobiol2001,91:997–1003.
population.ClinVaccineImmunol2010,17:1117–1123. 31. TulinskiP,FluitAC,WagenaarJA,MeviusD,vandevijverL,DuimB:
9. EFSA:Analysisofthebaseline-surveyontheprevalenceofmethicillin- Methicillin-resistantcoagulase-negativestaphylococcionpigfarmsasa
resistantStaphylococcusaureus(MRSA)inholdingswithbreedingpigs, reservoirofheterogeneousstaphylococcalcassettechromosomemec
intheEU,2008.PartA:MRSAprevalenceestimates;onrequestfromthe elements.ApplEnvironMicrobiol2012,78:299–304.
EuropeanCommission.EFSAJ2009,7:1376–82.doi:10.2903/j.efsa.2009.1376. 32. VanderhaeghenW,VandendriesscheS,CrombéF,DispasM,DenisO,
10. NeelaV,MohdZafrulA,MarianaNS,vanBelkumA,LiewYK,RadEG: HermansK,HaesebrouckF,ButayeP:Speciesandstaphylococcalcassette
PrevalenceofST9MRSAamongpigsandpighandlersinMalaysia.JClin chromosomemec(SCCmec)diversityamongmethicillin-resistantnon-
Microbiol2009,47:4138–4140. Staphylococcusaureusstaphylococciisolatedfrompigs.VetMicrobiol
11. WagenaarJA,YueH,PritchardJ,Broekhuizen-StinsM,HuijsdensX,Mevius 2012,158:123–128.
DJ,BoschT,vanDuijkerenE:Unexpectedsequencetypesinlivestock 33. AhrensP,AndresenLO:Cloningandsequenceanalysisofgenes
associatedmethicillin-resistantStaphylococcusaureus(MRSA):MRSAST9 encodingStaphylococcushyicusexfoliativetoxintypesA,B,C,andD.
andasinglelocusvariantofST9inpigfarminginChina.VetMicrobiol JBacteriol2004,186:1833–1837.
2009,139:405–409. 34. CunhaMLRS,CalsolariRAO:ToxigenicityinStaphylococcusaureusand
12. CrombéF,VanderhaeghenW,DewulfJ,HermansK,HaesebrouckF,ButayeP: coagulase-negativestaphylococci:epidemiologicalandmolecular
ColonizationandTransmissionofMethicillin-ResistantStaphylococcus aspects.MicrobiologyInsights2008,1:13–24.
aureusST398inNurseryPiglets.ApplEnvironMicrobiol2012,78:1631–1634. 35. OkeeMS,JolobaML,OkelloM,NajjukaFC,KatabaziFA,BwangaF,Nanteza
13. TheAlereTechnologiesGmbHwebsite[www.alere-technologies.com] A,KateeteDP:PrevalenceofvirulencedeterminantsinStaphylococcus
14. MoneckeS,KuhnertP,HotzelH,SlickersP,EhrichtR:Microarraybasedstudy epidermidisfromICUpatientsinKampala,Uganda.JInfectDevCtries
onvirulence-associatedgenesandresistancedeterminantsof 2012,6:242–250.
Staphylococcusaureusisolatesfromcattle.VetMicrobiol2007,125:128–140. 36. DevrieseLA,SchleiferKH,AdegokeGO:Identificationofcoagulase-
15. MoneckeS,JatzwaukL,WeberS,SlickersP,EhrichtR:DNAmicroarray- negativestaphylococcifromfarmanimals.JApplBacteriol1985,58:45–55.
basedgenotypingofmethicillin-resistantStaphylococcusaureusstrains 37. HoltfreterS,BauerK,ThomasD,FeigC,LorenzV,RoschackK,FriebeE,
fromEasternSaxony.ClinMicrobiolInfect2008,14:534–545. SellengK,LövenichS,GreveT,GreinacherA,PanzigB,EngelmannS,LinaG,
Crombéetal.VeterinaryResearch2013,44:4 Page10of10
http://www.veterinaryresearch.org/content/44/1/4
BrökerBM:egc-EncodedsuperantigensfromStaphylococcusaureusare
neutralizedbyhumanseramuchlessefficientlythanareclassical
staphylococcalenterotoxinsortoxicshocksyndrometoxin.InfectImmun
2004,72:4061–4071.
38. LoweBA,MarshTL,Isaacs-CosgroveN,KirkwoodRN,KiupelM,MulksMH:
Microbialcommunitiesinthetonsilsofhealthypigs.VetMicrobiol2011,
147:346–357.
39. ButlerJE,LagerKM,SplichalI,FrancisD,KacskovicsI,SinkoraM,WertzN,SunJ,
ZhaoY,BrownWR,DeWaldR,DierksS,MuyldermansS,LunneyJK,McCrayPB,
RogersCS,WelshMJ,NavarroP,KlobasaF,HabeF,RamsoondarJ:Thepiglet
asamodelforBcellandimmunesystemdevelopment.
VetImmunolImmunopathol2009,128:147–170.
40. ArriolaCS,GuereME,LarsenJ,SkovRL,GilmanRH,GonzalezAE,SilbergeldEK:
Presenceofmethicillin-resistantStaphylococcusaureusinpigsinPeru.PLoS
One2011,6:e28529.
41. LinY,BarkerE,KislowJ,KaldhoneP,StemperME,PantrangiM,MooreFM,
HallM,FritscheTR,NovickiT,FoleySL,ShuklaSK:Evidenceofmultiple
virulencesubtypesinnosocomialandcommunity-associatedMRSA
genotypesincompanionanimalsfromtheuppermidwesternand
northeasternUnitedStates.ClinMedRes2011,9:7–16.
42. BradleyPA,BourneFJ,BrownPJ:Therespiratorytractimmunesystemin
thepig.I.Distributionofimmunoglobulin-containingcellsinthe
respiratorytractmucosa.VetPathol1976,13:81–89.
43. BaileyM,HaversonK,InmanC,HarrisC,JonesP,CorfieldG,MillerB,Stokes
C:Theinfluenceofenvironmentondevelopmentofthemucosal
immunesystem.VetImmunolImmunopathol2005,108:189–198.
44. MacphersonAJ,SlackE:Thefunctionalinteractionsofcommensal
bacteriawithintestinalsecretoryIgA.CurrOpinGastroenterol2007,
23:673–678.
doi:10.1186/1297-9716-44-4
Citethisarticleas:Crombéetal.:Serologicalprofilesinnurserypiglets
colonizedwithStaphylococcusaureus.VeterinaryResearch201344:4.
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