CLINICALMICROBIOLOGY, Sept.1981,p.281-287 0095-1137/81/090281-07$02.00/0
Detection of Salmonella
Infections
by
Polyvalent
Enzyme-Linked Immunosorbent Assay
RONALD H. LENTSCH,* ROSANNE P. BATEMA,ANDJOSEPH E. WAGNER Veterinary MedicalDiagnostic Laboratory, UniversityofMissouri, Columbia, Missouri65211
Received2February1981/Accepted30April1981
Serum immunoglobulin G (IgG) and IgMwere measured forindividual Sal-monellaspecies bytheenzyme-linkedimmunosorbent assay(ELISA).F344 rats were experimentally infected with Salmonella typhimurium (serogroup B), S. enteritidis (serogroup D),and S. rubislaw (serogroup G). Endotoxin extracted fromeachserogroup served as theantigenin aclassical indirectELISA. Antibody specificfor each SalmonellaserogroupwasdetectedbyELISA.Normal gut flora fromcontrol animals
appeared
not to causecross-reactionsintheELISA.Speci-ficity andsensitivity of the
IgG
ELISAweredeterminedby
statistically
evaluating false-positives and false-negatives. Ideal values of 90%orbetterwereachieved in nearlyallinstances. Eachantigenwas alsotestedwithheterologous antiserain an efforttodevelopapolyvalentassay for Salmonellaspecies.Nosingle antigen detected allpositiveheterologous
antisera.Therefore,
apolyvalent antigen com-posed of the three serogroup antigens was tested. The results suggested that Salmonella infections can be detected bymeasuring
serum IgG levels with a polyvalent ELISA6 to 9days
postinfection.In vitrocultureis the
predominant
meansforisolating
andidentifying
Salmonellaspecies
fromfecalsamples.This is timeconsuming,
usu-ally
requiring
72 to96h for theorganism
tobedefinedbyitscultural,
biochemical,
andserolog-ical
properties.
Analternative method istode-tect serumantibodies formed
against infecting
Salmonella
species.
The antibodiescanbe de-tectedby
the Widal(tube
agglutination)
(16) orpassive
hemagglutination
test(10).
These testsare notused
routinely
duetotechnique-related
insensitivity and
nonspecific
cross-reactions. However, detection ofserumantibodiesto Sal-monella infections is stilltheoretically
more de-sirable for mass screening than in vitro cul-ture. Theenzyme-linked
immunosorbent assay(ELISA)
has beendeveloped
forawidevariety
of
antigen-antibody
reactions(4,
6,15),
indicat-ing
promise
for determination ofantibody
titerstoSalmonella infections.
An ELISA test for detection of Salmonella antibodieswasdescribed
by
Carlsson etal. (5). Antibody levels were compared both quantita-tivelyandqualitatively
byELISA,Widal agglu-tination,passiveagglutination,
andquantitative precipitation tests.ELISA was the most sensi-tive test. Later, group-specific antigens were usedtostimulateanimmuneresponse in rabbits (4), and serum antibody was measured by the ELISA method. Recently, Salmonella enteriti-dis and S.typhimurium
outbreaks have beendiagnosedby ELISA (8, 14). As yet, researchers anddiagnosticians in general have not used the ELISAas aclinical polyvalent test to efficiently screenlargenumbers ofanimals forSalmonella antibodytiters.
Theobjective ofthisinvestigationwas to de-velop an ELISA for detection of Salmonella speciesserumantibodies inrodentsand evaluate apolyvalent antigen foruse inthe ELISA.Anin vitro serological test of this nature would be highly desirable for diagnostic screening of large numbers of rats and mice in commercial colonies orin researchfacilities. SinceSalmonella sero-groups B and D represent ahighpercentageof Salmonella strains isolated from humans (14) and these serogroups contain nearly all of the strainscausingdisease inmice andrats (9), the test was directed against these serogroups. In addition,these studies wereintended to examine thepossibility of detecting, by ELISA,
subclini-cal infectionsor carrierstates. Furthermore, the potential for false-positives due to cross-reac-tions with other gut flora was explored and shown to be
negligible.
MATERIALS AND METHODS
Bacterial strains. S.typhimuriumLT2 wild type, 0:4,5,12 (ATCC 15277),S.enteritidis, 0:1,9,12 (ATCC 13076), andS. rubislaw, 0:11 (ATCC 10717), were used. An isolate ofS.enteritidisfrom a porcine source wasused to infectexperimental animals.
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Antigen and endotoxin preparation. Each strain was grown inTrypticasesoybroth (TSB)(BBL Microbiology Systems, Cockeysville, Md.) at 37°C overnight. The cells forantigen preparationwere har-vestedby centrifugationat10,000xg.Endotoxinwas extracted by the method of Roberts (12).Twograms ofdriedcells wassuspended in 200mlofdistilledwater heatedto80°C for60min. The mixturewasallowed tocool to roomtemperature and then centrifugedat 6,000 xgfor 30 min. The residuewasextracted once morewith100ml ofdistilledwater.Thepooled super-natants werefilteredthroughaWhatmanno.42 filter and concentrated to approximately '/2o the original volume. The concentrated solutionwasdialyzed over-nightat4°Cagainst distilledwater.Itwasthen recon-centrated toapproximately 25 ml anddialyzed over-nightagainstrunningtapwater.Thiswasfollowedby overnight dialysis againstdistilledwaterat4°C. The endotoxinsolutionswerethen lyophilized and stored atroomtemperature.
Sera.(i)Hyperimmunesera.Hyperimmunesera wereprepared for eachofthethreeserotypesof Sal-monella. Each of three groups offive 3-month-old, conventionally housed male F344 ratsreceiveda dif-ferent serotype intramuscularly. Formalinized cells were prepared from 12-h cultures in TSB. The 12-h cultureswerecentrifuged andwashed twice in phos-phate-buffered saline. The packed cells were then resuspendedinFormalin.A0.2-mlsuspension of for-malinized cells in Freund incomplete adjuvant (1:1) was used. This was followed by an intraperitoneal injection (0.2ml) of heat-killed organisms (from12-h cultures) in TSB 16 and 30 days later. Blood was drawn onday37. Uninoculated cohort animalswere bled foruseasreferencenegatives.
(ii) Experimental infection.Tosimulateanatural infection, three groups of nine 4-week-old maleF344 rats wereinoculated by gastricgavage with 0.5 ml of TSBcontaining 1.0x 109to3.5x 109 live organisms. Each group was inoculated witha different Salmo-nella serogroup. Additionally, there was a cohort group of nine uninoculatedrats. Bloodwasdrawnby cardiacpuncturefromtwoanimals in eachgroup three timesweeklythrough 28days post-inoculation. Just before initial inoculation and on days animals were bled, fecal pellets were cultured in gram-negative broth. Uninoculated control animals were bled and culturedonthesamescheduleasinoculated animals.
ELISA. The ELISAtest wasperformedessentially asdescribedbyCarlssonetal.(5).However,microtiter plates (MABioproducts,Walkersville, Md.)wereused insteadofpolystyrene tubes.Theplateswere coated with endotoxin (10 Lg/ml)for4hat37°C. Serawere diluted 1:20 and 1:100, and all tests were done in duplicate. Anti-ratimmunoglobulin (IgG)andanti-rat IgMconjugates wereprepared according to the pro-cedureoutlinedbyHorowitz andCassell(7). Substrate (p-nitrophenylphosphate)wasincubatedat37°Cfor 30 min in IgG assays and for60 min in IgM assays. The reaction was stopped by the addition of 1 N NaOH followed byrefrigeration forat least 20 min beforereadingat 400 nm on aBeckmanDB spectro-photometer. Each sample was transferred from the microtiterplatewithaRainin P200(Rainin,Woburn,
J. CLIN. MICROBIOL.
Mass.)toamicrocuvette (HellmaCells, ForestHills, N.Y.). As previouslydescribed (7), substrate, conju-gate, and antisera controls, plus positive (hyperim-mune)andnegativesera,wereused.
Analysis of data. The ELISA value was deter-mined by subtractingthe conjugate control absorb-ancevalue from the averageabsorbance valueof each test serum dilution. Initial minimal positive ELISA values (MPEV) for eachSalmonella assay were cal-culatedby adding2 standarddeviations to the mean of tested reference negative sera. A positive ELISA value was defined as greater than or equal to the
MPEV, and anegative ELISA valuewasdefinedas lessthan the MPEV. Sera from infected animals could bedivided intotwogroups:those which gaveapositive ELISA value (true-positive) andthose which gave a
negativeELISA value (false-negative). Sera from un-infected control animals could also be divided intotwo groups:those withanegativeELISA value (true-neg-ative) andthose with a positiveELISA value (false-positive). Thesensitivity andspecificity (13) ofeach assaywerecalculatedwithinitial MPEV andseveral
higher minimal absorbance values. Sensitivity was calculatedbythefollowingformula:sensitivity=XEV oftrue-positives/XEVoftrue-positives+XEVof false-negatives, where EV is ELISA value. Specificity equaledMEVoftrue-negatives/XEVoftrue-negatives +MEVoffalse-positives. Table 1 gives the resultant sensitivitiesandspecificitiesforoptimal minimal pos-itiveabsorbance values.
RESULTS
Experimental
infection. Animalsgavaged
with live S.
typhimurium
had positiveIgG
ELISA tests at7daysandpositive IgMtests at 9
days
post-inoculation (p.i.) (Fig. 1). Betweendays
19and26,
allantiserawerepositiveontheIgG
andIgM
ELISA. On the finalbleedingday
(day28p.i.),negative titerswereobtained from someinfected animals:oneofeight IgG and five
of
eight
IgM.
Animals infected with S.typhi-murium were cultured positive on day 2, and manyremained sothroughouttheexperiment.
S. enteritidis
IgG
antibody was detected 6days p.i. (Fig. 2).
All seracollected fromdays6through
28p.i.
hadpositiveIgGELISA titers forS. enteritidis.
IgM
titersweredetected in 12 of 19seratestedondays11through28p.i. (Fig.2).
S. enteritidis was recovered from the feces of mostanimalsondays6through 28.
TiterstobothIgGandIgMweredetectedon
days
9, 14,
and 19through
28for S.rubislaw-infected animals (Fig. 3). S. rubislaw was cul-turedfrom the feces ofmostanimalsondays 2
through
28.Table 2 isacomparison of theIgG ELISA and culture results for each
Salmonella-infected
group ofanimals. As expected, numerous
posi-tive culture results and few posiposi-tive ELISA re-sultswereobserved in the 1st week forallthree groups.
Thereafter,
theweeklytrendwastowardon February 7, 2020 by guest
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DETECTION OF SALMONELLA SPECIES BY ELISA 2.0 _ A
1.0 0E c0c
.00.1;
0.1 .
0 0
0
0 0 0
00 0
2.0- B
1.0
-occE m o
o
-0 _u
0.1 _
0 0
c) n0
0 0 0
0 0
0
012 1 v
O012 5 7 9 12
1*4
16 19 21 23 26 28 Time(Days)0 0
0
0
0
02 I 7I 9
012 5 7 9 12 14 16 19 21 23 26 28 Time(Days)
FIG. 1. Serum ELISA valuesforS.typhimurium-infectedrats at1:20dilution. Base lineswerecalculated from30reference negativesera.Allpointsabove the base linesareconsideredpositive.Points may represent morethanoneanimal.(A)SerumIgGvalues.The base line is0.962,which is2standarddeviations above the meanofreferencenegativeseraplus0.010.(B)SerumIgMvalues. The base line is0.009,which is 2 standard deviations above themeanof referencenegativesera.
TABLE 1. Sensitivity,specificity,andminimal absorbancefor eachSalmonellaELISAa
Minimal
Sensitiv
Assay positive ity
Specificity
Assay
~absorb-
it (%ance
S.
tphimurium'
0.062 99.3 100S. enteritidis' 0.072 95.66 100
S.rubislaw" 0.151 93.8 87.7
aIgG ELISAat 1:20antiseradilution.
b Calculated from30negative sera. 'Calculated from53negativesera. d Calculatedfrom 25 negative sera.
moreELISApositives.Inthe finalweek, nearly all infected animals were ELISA positive and
only a small fraction were culture positive for
Salmonella.
Antisera-antigencross-reactions. (i) An-tisera versus heterologous antigens. Posi-tivesera for eachSalmonella species from
ex-perimentallyinfectedanimalswerereacted with
the othertwoheterologousantigens.
Hyperim-mune serafor eachagentwere similarly tested.
Different degrees of cross-reactivity were
ob-servedamongthe three antigens, with S. typhi-muriumbeingthe most cross-reactive(Table 3). S. enteritidis was the least cross-reactive. No single antigendetected all of thepositivesin the
heterologoussystem.
(ii) Antisera versus polyvalent antigen.
Ten positive serum samples from rats
experi-mentally infected with each of the three Sal-monella strains were incubated in microtiter platescoated with a mixture ofthe three anti-gens (10 ,ug/ml per antigen). The polyvalent ELISA antigen detectedeverypositiveserum as determined by that serum's homologous reac-tion.Negative controlsera werealso tested with thepolyvalent antigen.Inthreecases,the poly-valent antigen detected positive titers in sera which were negative by the ELISA using the homologous antigen (Table4). Twoofthesesera were collected on day 28 fromanimals inocu-lated withaSalmonella species.
DISCUSSION
An ELISA for Salmonella infections of ro-dents was described before this study (8, 14). However, thetestwas notdesignedas aclinical assayformassscreeningof rodents for Salmo-nella
species.
The study reported herein pre-sents data onthe development of a polyvalent ELISA test fordetecting clinicalinfections and possiblyforscreeningrodentcoloniesforlatent infections. S. typhimurium and S. enteritidis were selected to representserogroups B and D, which are the mostcommonserogroups isolated inhumans (14) and the mostimportant Salmo-nellapathogensin ratsand mice. Includingan antigenically unrelated (not cross-reactive) S. rubislaw (serogroup F) provides evidence for 283 VOL.14,19810
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284 LENTSCH, BATEMA, AND WAGNER
2.0..
° 1.0_0
m
o .00
-0
o0t
o t
0
0
0.1_
B
0 o o
I-
00o0
o~~~~ 0
00~~~~
I I. . .
4 6 8 111214 1719 21 242628 Time(Days)
0 4 6 8 1112 14
Time
(days)1719 21 242628
FIG. 2. Serum ELISA valuesforS. enteritidis-infectedrats at 1:20dilution. Base lineswerecalculated from53referencenegativesera.Allpointsabove thebase linesareconsideredpositive.Pointsmayrepresent morethanoneanimal. (A)SerumIgGvalues. The base line is0.072,which is2standard deviations above the meanofreferencenegativeseraplus0.010.(B)SerumIgMvalues. The base line is0.172,whichis2standard deviations above themeanofreferencenegativesera.
o 0 2.0 1 B
o 1.0_
o o o 0
10 E Coo
.00
o qe
.0.1
0.1
-S
8 O
.- . . . . I I a I ITX
01 2 5 7 9 1214 16 192123 2628
Time(Days)
X .I I I I I . I. I
5 7 9 12 1416 19 2123 2628
Time (Days)
FIG. 3. Serum ELISAvaluesforS.rubislaw-infectedrats at 1:20 dilution. Base lineswerecalculatedwith
25reference negativesera.Allpointsabove the base linesareconsideredpositive.Pointsmayrepresentmore
thanoneanimal.(A)SerumIgGvalues. The base lineis0.151,whichis 2standard deviations above themean
of reference negativesera.(B)SerumIgMvalues. The base line is0.000.
1.0_
01
0 E co8X
.00
.0
0.1_
0
2.0_1
A1.0
-0
E
, o .00
-o0
0.1,.
012
0 0
1 C)
0 0
0 0
. . . 11 0 a I I 9
20
°1
A0 0
0 0
0 0
0
0 0 0 0
0
0
0 0
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TABLE 2. Culture andELISA resultsa No.positive/no. tested
Days S.typhimurium S.enteritidis S. rubislaw
p.i.
Cul- ELISA Cul- ELISA Cul- ELISA
ture ture ture
7 4/8 1/8 3/6 3/6 6/8 0/8
14 3/6 5/6 4/6 5/6 4/6 2/6
21 0/6 5/6 2/6 6/6 2/6 4/6
28 3/12 11/12 3/9 9/9 3/7 7/7
aIgGELISA results at 1:20 serum dilution.
TABLE 3. Positive Salmonellaantisera'versus homologous and heterologous antigens
No.positive/no. tested
Antiserum S.typhi-
S.
enteriti- S. rubislawmurium
dis
1:20 1:100 1:20 1:100 1:20 1:100 S.typhimurium 6/6 6/6 4/6 4/6 6/6 6/6 S.enteritidis 1/2 1/2 2/2 2/2 1/2 1/2 S. rubislaw 5/7 5/7 2/7 2/7 7/7 7/7
a Serum wasassayedforIgG antibody.
TABLE 4. Polyvalent ELISA afor threeSalmonella species
No.positive
Antiserum Dilution Polyva- Homolo-lent an- gous an-tigen tigen
S. antimurium 1:20 9 8
1:100 8 8
S. enteritidis 1:20 10 9
1:100 9 9
S. rubislaw 1:20 10 10
1:100 10 9
aPlates coated witha
mix
of the threeserogroup antigens. Ten animalsweretested per assay.the
development
of apolyvalent
SalmonellaELISA.
An obvious question of cross-reactions with normalgutflora
resulting
ininvalidtests, poorsensitivity,
andpoorspecificity
wasanswered inthis study through use of
experimentally
in-fected andhype unized animals. These an-imals were reared in conventional facilities carrying "normal" intestinal flora (Escherichia coli,Bacteriodes, Pseudomonas, etc.)and dem-onstrated no cross-reactive interference. The controlanimalsin these studieshadabase-line absorbancenear zero.The effectivenessof anELISAwas tested by
monitoring experimental infections (Fig.1-3). In all three groups, titers to the organisms were observed between 6 and 11 days for both IgM andIgG,as wouldbeexpected.
A simple explanation does not exist for the simultaneousrise ofIgGandIgM.Typically, the IgM riseshouldoccur 3 to 5days p.i. However, the introduction of live Salmonella by gastric gavage into normalrats maynotinvokea class-ical immune response. Rats seem to be more resistant thanmice toSalmonella infections and generally do notdevelop clinical disease unless under stress.They frequently become carriers. Datapresented inTable2substantiateacarrier stateor subclinical disease. Many animals con-tinued toshedSalmonellathroughout the study without clinical disease, also suggesting that ELISA has the potentialto detectSalmonella carriers. The immuneresponseprovokedby nor-malgutflora and carrierstateshould be similar. Researchers (1,2, 11)investigating the humoral responsetonornalflora admit that the immune response inthe gut isnotwell understood. At-temptshaveyet tobe made inacomplete study determining the antibody response ofa nonin-vasiveorganism.Although the researchersagree that IgM,IgG, and IgAresponses are involved (1,2, 11),the degreetowhich each immunoglob-ulin classassertsitself is controversial. Further-more,
Svenungsson
etal.(14) usedapolyspecific
antiserun andreportedverylowrelative titers to anactive infection of S. enteritidisup to 11 days in duration.Itisdifficultto compareresults from anotherlaboratory, but this finding sup-ports our data.These researchers didnot specif-ically determinetheinitialIgMresponse, mak-ing it difficulttocorrelate their results withours. One other
possibility
foralateIgMresponseis that the enzyme conjugate was less sensitive. Although possible, this is unlikely since the anti-IgMconjugatewaspreparedsimilarly and from thesame source as the IgGconjugate; in addi-tion, high absorbance values (>1.0) were ob-tained fromseraofinfectedanimals, suggesting ahighly sensitive conjugate.Averyimportantpart ofdeveloping aclinical testissettingtheminimalvaluetobeconsidered apositivetest.ELISAvaluesfornegativeserum, 25 or more for each Salmonella species, were accumulated, and 2 standard deviations from the mean of thesevalueswas at firstdetermined tobeour base line. Toascertain the success of the test and the base
line,
sensitivity and speci-ficity valueswerecalculated. Sensitivity consid-ersthenumberoffalse-negatives,andspecificity 14,on February 7, 2020 by guest
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considers the number offalse-positives. Ideally, both values should be 90%orabove.By moving the base line 0.01 ofanabsorbance unit higher than2standard deviations above the mean, both the sensitivity and specificity were above 95% forS. enteritidis and S. typhimurium. S. rubis-law achieved nearthe ideal values. Fewer sera weretestedin theS. rubislaw system. As more seraareexamined,thespecificityandsensitivity valuesfor S.rubislaw should rise.Furthermore, the strictestguidelines weremaintained for de-termining the number oftrue-positives.All sera wereconsideredpositiveif theanimalhad been inoculated with theorganism, even thoughthe animal at notime had symptoms of disease or shed Salmonella. These animals probably will notseroconvert, yetbyourdefinition they would be considered false-negatives. Due to the
ex-pected and observed decline of IgM titers to near zero, specificity and sensitivity values would be of little value andwere notdetermined. In reference to Table 2, a strong case can be made forELISAas moresensitive thanculture for detection of Salmonella-infected animals. Forscreening purposes, once the animals sero-convert, the ELISA seems to be superior to
culture. However, one must keepin mind that theELISAisanindirecttechnique, and confir-mation of positives by other methods is sug-gested.
Antigen was extracted by the watermethod reported by Roberts (12). This method is not oneof thetechniques
commonly
usedtoextractlipopolysaccharide. Roberts did an extensive study demonstrating the recovery of the lipo-polysaccharidefraction. At any rate, theextract inourwork served very wellastheantigenand did not seem to cross-react with antibodies formedagainst normalflora.
Next,a polyvalent
antigen
was evaluated for useinthe ELISA. Each Salmonellaantigenwas testedagainsttwoother Salmonellaspecies an-tiseratodetermine ifoneantigencouldprovide adequate cross-reactivity and thus avoid the need for a polyvalent antigen (Table 3). Al-though all three antigens wererecognized
in highfrequency byheterologous antisera,noan-tigenwas100%effective. Thepolyvalent antigen providedahigher degreeofsensitivitythan any of the threehomologoustestsystems
(Table
4), probably as a result of thecross-reactivity
of heterologousantisera(Table
3).Considering
this information, we areassumingthat with the cor-rect combination of serogroupantigens, a truly polyvalentSalmonella ELISA will result.In conclusion, the ELISA described herein provides a high level of sensitivity and specificity and has the potential to become a valuable clinical test. Intestinal flora other than Salmo-nella species appear not to have interfered with the assay. Our results indicated that an ELISA for quantitating IgG would be superior to mea-suring IgM levels. Measurable IgG titers oc-curred as early as IgM titers and would not decrease in long-term infections. Finally, these initial results provide strong evidence that a single polyvalent ELISA test may be capable of identifying natural infections or carrier states produced by a variety of Salmonellaspecies. A polyvalenttestwould be of great significance for long-termresearch projects or commercial sup-pliersof research animals. The test would reduce expenses for shipping of animals or media to diagnostic laboratories and would greatly reduce thetime necessary to establish a diagnosis.
ACKNOWLEDGMENTS
Wethank Phyllis J. Mitchell for her competent technical assistance.
This project was partially supported by Public Health ServicegrantsNOI CM97211,NOI CM87157, andRR00471 from the National Institutesof Health.
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