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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

ELISAweredetermined

by

statistically

evaluating false-positives and false-negatives. Ideal values of 90%orbetterwereachieved in nearlyallinstances. Eachantigenwas alsotestedwithheterologous antiserain an efforttodevelopapolyvalentassay for Salmonellaspecies.Nosingle antigen detected allpositive

heterologous

antisera.

Therefore,

apolyvalent antigen com-posed of the three serogroup antigens was tested. The results suggested that Salmonella infections can be detected by

measuring

serum IgG levels with a polyvalent ELISA6 to 9

days

postinfection.

In vitrocultureis the

predominant

meansfor

isolating

and

identifying

Salmonella

species

fromfecalsamples.This is timeconsuming,

usu-ally

requiring

72 to96h for the

organism

tobe

definedbyitscultural,

biochemical,

and

serolog-ical

properties.

Analternative method isto

de-tect serumantibodies formed

against infecting

Salmonella

species.

The antibodiescanbe de-tected

by

the Widal

(tube

agglutination)

(16) or

passive

hemagglutination

test

(10).

These tests

are notused

routinely

dueto

technique-related

insensitivity and

nonspecific

cross-reactions. However, detection ofserumantibodiesto Sal-monella infections is still

theoretically

more de-sirable for mass screening than in vitro cul-ture. The

enzyme-linked

immunosorbent assay

(ELISA)

has been

developed

forawide

variety

of

antigen-antibody

reactions

(4,

6,

15),

indicat-ing

promise

for determination of

antibody

titers

toSalmonella infections.

An ELISA test for detection of Salmonella antibodieswasdescribed

by

Carlsson etal. (5). Antibody levels were compared both quantita-tivelyand

qualitatively

byELISA,Widal agglu-tination,passive

agglutination,

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 been

diagnosedby 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.

281

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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. Animals

gavaged

with live S.

typhimurium

had positive

IgG

ELISA tests at7daysandpositive IgMtests at 9

days

post-inoculation (p.i.) (Fig. 1). Between

days

19and

26,

allantiserawerepositiveonthe

IgG

and

IgM

ELISA. On the finalbleeding

day

(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 6

days p.i. (Fig. 2).

All seracollected fromdays6

through

28

p.i.

hadpositiveIgGELISA titers for

S. 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 19

through

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,

theweeklytrendwastoward

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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 99.3 100

S. 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.14,1981

0

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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-

0

0o0

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

A

1.0

-0

E

, o .00

-o0

0.1,.

012

0 0

1 C)

0 0

0 0

. . . 11 0 a I I 9

20

°1

A

0 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. rubislaw

murium

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 a

polyvalent

Salmonella

ELISA.

An obvious question of cross-reactions with normalgutflora

resulting

ininvalidtests, poor

sensitivity,

andpoor

specificity

wasanswered in

this 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) useda

polyspecific

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,

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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

usedtoextract

lipopolysaccharide. 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 were

recognized

in highfrequency byheterologous antisera,no

an-tigenwas100%effective. Thepolyvalent antigen providedahigher degreeofsensitivitythan any of the threehomologoustestsystems

(Table

4), probably as a result of the

cross-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.

LITERATURE CITED

1. Bourne, F. J., J. W. Honour, and J. Pickup. 1971. Natural antibodiestoEscherichia coli in the pig. Im-munology20:433-436.

2. Brown, W. R.,and E. Lee. 1974.Radioimmnunological

measurementsof bacterial antibodies. II. Human serum antibodiesare reactive with Bacteriodes fragilis and Enterococcusingastrointestinalandimmunological dis-orders.Gastroenterology 66:1145-1153.

3. Bullock, S. L.,and K. W. Walls. 1977. Evaluation of someof the parameters of theenzyme-linked immuno-specificassay.J. Infect. Dis. 136:279-285.

4. Carlsson,H.E.,andA. A.Lindberg. 1978. Immuno-chemistry ofsalmonella0-antigens.ActaPathol. Mi-crobiol. Scand. Sect. C86:237-244.

5.Carlsson, H. E., A. A. Lindberg, and S. Hammar-strom. 1972. Titration of antibodiestosalmonella 0 antigens byenzyme-linked immunosorbent assay. In-fect. Immun. 6:703-708.

6. Engval, E.,andP.Perlman.1971. Enzyme-linked im-munosorbent assay(ELISA). Quantitativeassay of

im-munoglobulinG.Immunochemistry8:871-874. 7. Horowitz, S. A.,and G. H. Cassell. 1978. Detection of

antibodies to Mycoplasma pulmonis by an enzyme-linked immunosorbent assay. Infect. Immun. 22:161-170.

8.Karlsson, K.,H. E.Carlsson,R.Merenger,and A. A. Lindberg. 1980.Applicationandusefulnessof enzyme

immunoassayfordiagnosisof Salmonellatyphimurium

infection.Scand. J. Infect. Dis. 12:41-47.

9. Mavgard,W.L.,and J. H.Litchfield. 1963. Occurrence ofunusualsalmonellaeinlaboratorymice. J. Bacteriol. 85:1851-1852.

10. Neter,E. 1956.Bacterialhemagglutinationand hemoly-sis.Bacteriol.Rev. 20:166-168.

11. Quick,J.D.,H. S.Goldberg,and A.C. Sonnenwirth. 1972.Humanantibodytobacteroidaceae.Am.J.Clin.

CLIN. MICROBIOL.

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VOL. 14,1981 DETECTION OF SALMONELLA SPECIES BY ELISA 287

Nutr.25:1351-1356. indirectimmunofluorescence forrapid identificationof 12. Roberts,R.S. 1949. The endotoxin of Bact. coli. J.Comp. SalmoneUa enteritidisandusefulness ofenzyme-linked

Pathol. 59:284-304. immunosorbent assay. J. Infect. Dis.140:927-936. 13. Schwabe, C.W., H. P.Riemann, and C. E. Franti. 15.Volier,A.D.,E.Bidwell,and A.Bartlett. 1976.

En-1977.Epidemiologyinveterinarypractice. Lea & Febi- zyme immunoassays in diagnostic medicine. Bull.

ger,Philadelphia. W.H.O.53:55-65.

14. Svenungsson, B., H.Jorbeck,and A. A.Lindberg. 16.Widal,F. 1896.Serodiagnosisde la fievre typhoide. Bull. 1979.Diagnosis of salmonella infections: specificity of Mem. Soc. Hop. Paris13:561-566.

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