JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1978, p. 360-3680095-1137/78/0008-0360$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 8, No. 4

Printed in U.S.A.

Assessment of Availability and Efficacy of CommercialSalmonella Grouping Antisera

GRACIA M. EVINS,I* LINDA L. LINNE,' AND HENRY M. COLVIN2Diagnostic Products Evaluation Branch, Biological Products Division,' and Office of the Director7 Bureau

of Laboratories, Center for Disease Control, Atlanta, Georgia 30333

Received for publication 3 May 1978

Salmonella somatic antisera for groups A to E were purchased from fourcommercial producers directly by the Center for Disease Control (CDC) andindirectly through two hospitals. CDC specifications and methods were used toevaluate antisera shipped directly to CDC. To assess the performance of theproducts under simulated user conditions, we used the commercial antiserapurchased indirectly through the hospitals to group coded cultures. Of the 23antisera ordered by CDC and the hospitals, the CDC received all 23, a largemedical complex received 20, and a private hospital received 9. Similar resultswere obtained with producer and CDC test methods. Forty-five different lots ofantisera were evaluated, ofwhich 20% did not meet CDC specifications. The CDCspecifications and latest revisions are discussed.

The Diagnostic Products Evaluation Branch,Center for Disease Control (CDC), evaluatescommercial Salmonella reagents submitted vol-untarily by producers before they are marketedand also evaluates marketed reagents. Most ofthe consumer problems conveyed to the Diag-nostic Products Evaluation Branch have con-cerned the reactivity of antisera, but difficultiesin obtaining reagents have also been reported.To assess their quality, as judged by CDC spec-ifications, and availability, CDC purchased Sal-monella somatic (0) antisera (i) directly fromcommercial producers, (ii) through a large uni-versity teaching medical center, and (iii) througha large private hospital.Commercial reagents are also tested by the

Diagnostic Products Evaluation Branch at therequest of the Food and Drug Administration.These evaluations are done according to theproducers' instructions, which are packaged withthe reagents. The differences between thesemethods and those in the CDC specificationsvary depending upon the particular reagent andthe producer. The results obtained by usingCDC and commercial testing procedures werecompared by using the antisera purchased di-rectly from the producers.

In addition to evaluating with CDC methodsthe antisera purchased through the hospitals,we used these products to group coded salmo-nella cultures. The latter procedure providedresults which (i) could be used to assess thetypes of identification that would be obtainedwith these products in clinical laboratories and

(ii) could confirm the regular evaluation proce-dure results.

MATERIALS AND METHODSAntisera. Purchase orders for Salmonella somatic

antisera groups A, B, CI, C2, D, and E were submittedby CDC and the two laboratories directly to BBL,Lederle Diagnostics, Lee Laboratories, Inc., and DifcoLaboratories. Group D antiserum was not orderedfrom Difco because it was not listed in the Difcocatalog. Orders not filled within 2 months were can-celled. The products received were stored at 4°C atthe hospitals for this 2-month period and then wereshipped to CDC under the same conditions as theywould have been by the producer. Forty-three of the52 reagents thus obtained had 1978 through 1980expiration dates, with the other 9 expiring in 1977. Allwere evaluated before their expiration dates. Reagentsare identified by group, company, and lot number inTable 1.

Grouping and subgrouping antisera produced by theBiological Products Division, CDC, were used to verifythe reactivity and smoothness of test antigens.

Somatic antigens. Antigens used to evaluate theantisera by CDC test methods were produced fromfreeze-dried cultures of strains that had been originallyserotyped in the Enteric Unit, National SalmonellaReference Center, CDC. The coded cultures were re-cent isolates from groups A to H, K, N, O, R, T, 51,and 54, serotyped in the Enteric Unit, CDC. All anti-gens were dense suspensions of living organisms in0.85% NaCI as described previously (2). These antigenswere used for both CDC and producer test methodsbecause they were specified in the instructions accom-panying the commercial antisera purchased directlyby CDC in 1975. In addition, Lederle package insertssuggested using boiled organisms suspended in saline,

360

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COMMERCIAL SALMONELLA GROUPING ANTISERA 361

and Lee literature gave instructions for emulsifying a

colony in saline directly on the slide.Four different inserts accompanied the Difco re-

agents. The three dated May 1970, October 1974, andJune 1975 accompanied reagents purchased directlyby CDC and recommended using a dense suspensionof the organisms in saline. The April 1976 packageinsert, which accompanied the reagents purchasedthrough the hospitals, recommended that a loopful ofgrowth be emulsified directly in the antiserum on a

slide, and that antigen suspensions be reserved fortesting that required boiled organisms. Furthermore,the 1976 Difco brochure stated "They [Difco antisera]have not been tested employing antigen suspension insaline or alcohol-treated cultures."Each antigen listed in Tables 1 and 2 was made

from a different culture and used in serological assays

performed the day it was prepared. Since the sero-

groups of the cultures shown in Table 2 were unknown,criteria for selecting antigen suspensions differed fromthose for antigens shown in Table 1. Antigens in Table1 were first tested with various dilutions of CDChomologous grouping or subgrouping antiserum.Those that agglutinated at 3+ or stronger with theroutine test dilution of homologous antiserum were

further tested for smoothness with saline and groupsA, B, CI, C2, D, E1, E2, E3, and E4 heterologous antisera.Those antigens that reacted normally were used. Theantigen suspensions of the coded cultures were pre-

tested only with saline to simulate the process ofserogrouping salmonella isolates in the clinical labo-ratory.

Flagellar (H) antigens. These antigens were stockFormalinized infusion broths prepared as described inthe CDC specifications (2). Somatic antisera pur-

chased directly by CDC were tested for the presenceof agglutinins to the H antigens of the immunizingstrains used to produce the O antiserum. The immu-nizing strains were identified for us by each company

except BBL, whose antisera were tested with H anti-gens of serotypes most likely to have been used as

immunizing strains. The commercial antisera pur-chased indirectly through the hospitals were not as-

sayed for H agglutinins because inadequate quantitieswere available.Antiserum evaluation procedure. Two persons

experienced in evaluating commercially produced Sal-monella antisera did ail the testing. Antisera weretested with ail homologous antigens listed in Table 1,and limited heterologous antigens of groups A to 67were selected on the basis of known antigenic relation-ships. The coded cultures (Table 2) were used onlywith those antisera purchased through the hospitalsand with CDC antisera.The commercial antisera were diluted according to

manufacturers' instructions and tested, at this dilutiononly, for the presence of O agglutinins. The major

difference between the CDC testing procedure andthose of the producers was the amount of reagentsused. Difco and Lederle literature specified using 1drop (0.05 ml) each of antigen and antiserum. The LeeLaboratories brochure specified that the dropper sup-plied with the vial be used to dispense 1 drop ofantiserum and stressed that the drop of antigen mustbe of comparable volume. BBL instructions specified

using a small drop of each reagent. Since the 0.05-mlamount of each reagent satisfied the requirements ofeach company except Lee Laboratories, it was used forall antisera except theirs. A Clay Adams Micro-Selec-tapette (set at 50 1l) was used to dispense the 0.05-mlamounts, and the Lee Laboratories droppers, whichdispensed about 0.05 ml, were used with their antisera.

Droplets of 0.01 ml of each reagent were used in theCDC method for evaluating all antisera by CDC spec-ifications and in grouping coded cultures.

All reactions were read at 1 min, and Difco antiserareactions were also read at 2 min as specified in theirinstructions. The remainder of the slide test procedure,reading reactions, and testing for H agglutinins wasdone as described previously (2), except that largerareas on the slides had to be marked off because ofthe larger volumes of reagents specified in the com-mercial procedures.Performance criteria. CDC performance require-

ments (see below) were used to judge the acceptabilityof each reagent evaluated (2).An acceptable group-specific antiserum must agglu-

tinate at 3+ or greater with homologous antigenswithin 1 min. It must not react within 1 min withheterologous Salmonella O groups. Cross-reactionswith antigenically related salmonellae may occur, butmust not exceed 1+ agglutination within 1 min. How-ever, group H antisera may agglutinate at higher than1+ with strains from other groups which contain factor14, and group E antisera may agglutinate at higherthan 1+ with group 54 strains which contain factor 3.Many antigenic relationships are not expressed in theschema. An antiserum for a subgroup of group C mustnot react with heterologous members of group C. Asomatic antiserum must not contain flagellar aggluti-nins.

RESULTSOf the 23 reagents ordered by each institution,

CDC received 23, the medical complex 20, andthe private hospital 9. Two lots of antisera pur-chased directly by CDC were also obtainedthrough the medical complex. Of the nine lotsbought through the private hospital, four fromDifco and one from Lederle were from the samelots obtained by the medical complex. The pri-vate hospital received no reagents from BBLand Lee Laboratories. Since sets of duplicatesreacted comparably, only the results on the du-plicate products sold directly to CDC and to themedical complex are shown in Table 1. Threereagents of the 23 (13%) purchased directly fromthe producer by CDC, 5 of the 20 (25%) pur-chased through the medical complex, and 4 ofthe 9 (44%) purchased through the private hos-pital did not meet CDC specifications. Of 45different lots tested, 9 (20%) were unacceptable.Fifteen of the 45 (33%) had been submitted toCDC for premarket evaluation. No H agglutininswere detected in the antisera.Only results obtained with the CDC method

are presented in Table 1, because the reactions

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COMMERCIAL SALMONELLA GROUPING ANTISERA 365

with the CDC and the producer's method dif-fered only slightly. These minor variations af-fected the final assessment only of Lederle groupCi as described below. Reactions with Difcoantisera read at 1 min usually remained thesame or intensified after 2 min (e.g., a 3+ becamea stronger 3+ or a weak 2+ became a routine2+), an insufficient change to have any effect onthe acceptability of the Difco reagents.Group A. Among the group A antisera (see

Table 1), the Lederle product lot 395-267 was ofbetter quality than the others, since it reactedstrongly with all homologous strains and hadonly one weak, but acceptable, heterologous re-action. BBL group A antiserum lot 17GAEDcross-reacted weakly with many group B strains,a fact which is acceptable because factors 1 and12 are common to both groups.Group B. The 2+ cross-reaction of Difco

group B lot 615879 with S. typhi did not renderthe product unacceptable, because it did notcross-react with sevèn other group D strains.The bulk product from which Lederle group Blot 433-749 was packaged had been previouslysubmitted to CDC for premarket evaluation anddid not meet specifications because of heterolo-gous reactions with three of five group C2 strains.The bulk product had cross-reacted at strongerthan the 1+, whereas the purchased packagedproduct reacted only faintly. Although these twoweak heterologous reactions do not render theproduct unacceptable, stronger cross-reactionsmight occur with some group C2 isolates.Group C. When tested with the CDC method,

Lederle antiserum group C1 lot 418-794 reactedstrongly with three of the six homologousstrains. In addition, one 3+ reaction with theCDC method was only 2+ with the Lederlemethod, making the product unacceptable withfour of the six homologous strains.Group D. Lederle group D lot 418-795 anti-

serum was inadequate because it did not identifythe (9),46 strain, a problem which was correctedin subsequent lots submitted voluntarily forevaluation.Group E. Difco antiserum group E lot 624848

reacted at barely acceptable levels with 3,10 and3,15 strains and did not identify (3),(15),34strains. Lederle antiserum group E lot 475-717reactions with E3 strains were weaker than de-sirable, but the product was classified as accept-able because the 3 and 15 factors in these strainsare partial antigens. Two lots ofgroup E antiserareacted strongly with a group 54 strain becausesome of the 054 strains possess factor 3 (8).Because group 54 strains are isolated infre-quently relative to group E isolates, it is impract-ical to require the absorption of factor 3 agglu-tinins from group E antisera.

Table 2 shows the results obtained when setsof group A to E antisera from each producerwere used to identify coded cultures. Two anti-gens reacted with saline and could not begrouped. The CDC antisera readily identifiedgroups A, B, Ci, D, and E strains but not the C2strains. BBL antisera detected groups A, B, C1,C2, D, and E strains, but group D antiserum didnot distinguish between B and D strains.No group C1 and D Difco antisera were avail-

able for testing. The Difco set identified A, B,and C2 cultures, but group A antiserum misiden-tified six strains of groups B, D, E, G, and R.The Difco C2 antiserum reacted in a misleadingmanner with a C1 strain, and group E antiserundid not detect an E3 strain.The variety of lots of antisera received from

Lederle allowed two sets of tests to be run withcoded samples. The sets identified group B, C1,D, and E strains, but reacted inconsistently withgroups A and C2.The Lee Laboratories set of antisera correctly

identified all strains.

DISCUSSIONPractical considerations for production and

use were the basis for the definitions and thepotency and specificity requirements for Sal-monella reagents in the CDC specifications (2).Antisera for some O antigens may be specificwithout absorption, whereas antisera for otherssuch as 2 or 19 require extensive absorption,which generally lowers the homologous titer. Byseparating these, we can require strong homol-ogous reactions for antisera that are usable withlimited absorption and permit weaker homolo-gous reactions for the others. This approachprovides reliable reagents for the clinical labo-ratory without imposing unnecessary productionrequirements.

In grouping cultures, it is unnecessary to useantisera that have been absorbed so that theyreact with only one antigenic factor in the slideagglutination test. Single-factor antisera areneeded in determining serotype rather than se-rogroup. The immunizing strain for a groupingantiserum should be carefully selected to containas little as possible of antigens that are commonto other groups, to render the resulting antise-rum more specific (3). Although cross-reactionsmay occur, they should not be as strong as thehomologous reaction (e.g., an unabsorbed groupA antiserum at the routine test dilution reacted4+ with a 1,2,12 strain, but reacted 1+ withstrains of groups D, G, and H and 2+ with an E4strain, as shown by Ewing [6]).The Difco package insert accompanying group

A lot 622771 dated April 1977 explained, "A

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366 EVINS, LINNE, AND COLVIN

serum prepared from a 1,2,12 culture, if notabsorbed, will react with cultures of serogroupsB and D in varying degrees, depending upon theconcentration of the commonly shared 1 and 12factors." This same insert recommended using asingle-factor antiserum in serogrouping. Thepackage insert received with Difco group A lot615979 antiserum, which expired in February1978, is dated October 1974 and does not havethis explanation. It is interesting that the twolots of Difco group B antisera evaluated do notcross-react with strains of groups A and D.In 1975 the CDC specifications (1) for Sal-

monella antisera were revised as follows: Sal-monella O subgroups except Ci and C2 weredeleted from the reagents covered. The properevaluation of subgrouping antisera dependsupon the interaction of the antisera within a set.For example, antiserum for subgroup El (3,10)may have enough factor 3 agglutinins present toreact with E2 (3,15), E3 [(3),(15),34], and E4(1,3,19) strains. This crossing is acceptable if theEl product reacts more rapidly and strongly withEl strains. If the antiserum has been absorbedso that it reacts only with 3,10 strains, it shouldbe called a single-factor 10 antiserum. This mu-tual dependency within the subgroup makes itimpractical to test subgrouping antisera in theCDC premarket evaluation program.Groups C1, C2, C3, and C4 can be treated

differently. Edwards and Ewing (4) separatedthese strains but did not label 8,20 and 6,7,14strains as C3 and C4. Kauffmann (7) denoted 8and 8,20 strains as group C3 and 6,7,14 strains asC4, with the notation that the latter are C1lysogenized by phage 14. In the context of pro-ducing and using grouping antiserum, it is prac-tical to retain 6,7,14 agglutinins in group C1 and8 and 8,20 in group C2 antisera, thus allowingcultures without factor 6 to be used as immuniz-ing strains for group C2 antiserum. For groupsCi and C2 (if a 6,8 strain is used), immunizingstrains that have minimal amounts of the 6,factor should be used so that cross-reactions arenot apparent in the slide agglutination test (3).

In the 1975 CDC specifications, antisera wereno longer required to meet potency specifica-tions with antigens made by heating the orga-nisms in alcohol, centrifuging, and resuspendingin saline. If the manufacturer's instructions spec-ify using living organisms only, the product isevaluated only with living antigens. In general,more dilute antisera can be used with livingantigens than with alcohol-treated antigens (4).All U.S. commercial producers recommend us-ing living antigens.

Realizing that some antisera are more difficultto produce than others, in the 1972 specifications(1) we had eliminated requirements for mini-

J. CLIN. MICROBIOL.

mum homologous O titers so that each productcould be diluted appropriately. Many commer-cial Salmonella antisera give neither very strongnor very weak reactions at the routine test di-lution. A less diluted product might provide anantiserum of good rather than mediocre quality.However, commercial producers have chosen torestrict themselves, since all recommend a 1:3dilution for every somatic antiserum, irrespec-tive of the difficulties peculiar to the productionof individual reagents.Three antisera that met CDC specifications

failed to identify the coded cultures adequately.They were Lederle group A lot 451-704, CDCgroup C2, and Lederle group C2 lot 431-739.Lederle group C1 lot 457-758 met specificationsin premarket testing but was reported to have"borderline homologous reactions." These dis-crepant results probably result from form vari-ation of the cultures and mediocre antisera.

Culture variations make evaluation resultsand clinical laboratory results difficult to inter-pret. Consider, for example, the homologous re-sults for group A in Tables 1 and 2. Lederlegroup A lot 466-730 did not meet CDC potencyspecifications when evaluated, but lot 451-704did. Yet, when used with antigens selected asthey would be in a clinical setting, the lotsreacted comparably (Table 2). Although lot 451-704 did meet CDC specifications, Lederle lot395-267 would be used with more confidencebecause it allows margin for form variation. Theother two lots reacted at levels ranging frombarely meeting specifications with one set ofantigens to not quite meeting with another.

Failure to identify the Lederle group C2 anti-serum as unacceptable probably stems from fail-ure to recognize CDC group C2 as unacceptable.The C2 antigens used in evaluating the antiserawere selected by testing them with various di-lutions of CDC group C2 antiserum. Only thosethat reacted at 3+ with the routine test dilutionof the antiserum were used. The coded cultureswere not screened in this manner, and only onereacted at an adequate level. To prevent theseproblems recurring, we made three changes:CDC group C2 antiserum is no longer beingdistributed; to meet potency specifications, ho-mologous reactions must be definite rather thanmarginal 3+ reactions; and routine test dilutionsfor several reference antisera have been loweredso that evaluation antigens will be more likeclinical isolates and not a select population. Evenwith these changes, a product that causes prob-lems in the clinical laboratory may meet CDCspecifications because testing cannot be all-en-compassing, and the antigenic composition ofisolates cannot be foreseen.When Salmonella O antisera are evaluated,

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in addition to form variation, roughness of cul-tures is an inherent problem that is difficult tohandle in a manner equitable to both producerand consumer. Kauffmann (7) noted that roughSalmonella cultures do not always react withsaline and do not contain the complete O anti-gen. Obviously, rough cultures should not beused to evaluate antisera, since they may havestrong homologous reactions with poor-qualityantisera or, conversely, weak reactions withgood-quality antisera. Therefore, despite thefact that clinical laboratories use only salinecontrols in detecting rough cultures, CDC meth-ods include testing the evaluation antigen forroughness with nine antisera (groups A to E4)before using it. This careful screening of evalu-ation antigens may prevent detecting rough ag-glutinins in the antiserum being tested.

In the clinical laboratory the unknown cultureis tested with group A to E antisera as in theCDC screening method. Therefore, when theantigen-saline control is negative, the clinicallaboratorian must distinguish between reactionscaused by expected antigenic relationships andthose caused by roughness of the culture, aprocess which is, at best, educated guessing.According to Kauffmann (7), the only way todetermine whether the strain is rough is to im-munize a rabbit and test the resulting antiserumfor the presence of rough agglutinins, a proce-dure which would not be performed routinely.Even the CDC method of testing is quite time-consuming and limits the number of culturesprocessed because each antigen is made, evalu-ated, and used in 1 day.

It would be helpful for the laboratorian toestablish the adequacy of a set of antisera andthen use it over a long period so that the idio-syncrasies of the individual antisera are familiar.Unfortunately, expiration dates interfere withthis approach. Many Salmonella antisera dis-tributed by CDC were made over 15 years agoand still retain their potency. The extension ofexpiration dates on Salmonella antisera shouldbe considered to avoid wasting antisera and thetime spent evaluating them.The part of the study comparing CDC and

producer methods was completed in 1976 beforethe use of antigen suspensions was excludedfrom Difco recommended methods. In additionto saline suspensions of living organisms, otherantigens such as saline suspensions of boiledorganisms were recommended in the commercialcompany brochures, but it was not feasible touse each procedure. Emulsifying a colony inantiserum on a slide may be practical for prelim-inary work with a polyvalent antiserum, butwhen testing with six or more grouping antisera,making an antigen suspension is logical. How-

ever, using a drop of antigen suspension insteadof only bacterial growth might dilute the anti-serum to the extent that homologous reactionsare diminished. Not only is the method of emul-sifying bacterial growth on a slide tedious, butusing different colonies as antigens would haveintroduced bias into comparing reagents.Using 0.05 ml of a reagent when less will

suffice not only increases the cost of the test butis more hazardous, because of the increased riskof spills when the slide is rocked and the reac-tions read.

In some laboratories, it would be difficult todo basic Salmonella serogrouping because ofthe difficulty of obtaining satisfactory reagents.The question of the extent of serotyping of sal-monellae that should be done in a particularlaboratory has been debated for years. Accord-ing to Edwards and Ewing (4), the personnel oflarge or small laboratories should be able toidentify completely S. typhi, S. cholerae-suis,and S. enteritidis bioser. Paratyphi A, ser. Par-atyphi B, and ser. Typhimurium. For this pur-pose, they recommended using a basic set ofantisera consisting of a polyvalent antiserum, Oantisera for groups A to E, H antisera for anti-gens a, b, c, d, i, and 1 (one) complex, single-factor H antisera for factors 2 and 5, and a Viantiserum.As stated previously by Evins et al. (5), it is

ludicrous to expect the clinical laboratory to dothe type of reagent evaluation presented here,yet the careful checking of test antigens makesthe evaluation meaningful. Use of a preeval-uated reagent should allay concern over its ini-tial sensitivity and specificity, but does not annulthe obligation of the user to control quality.The most elemental quality control for Sal-

monella antisera for groups A to E requires themaintenance of 14 cultures in the smooth fonn.The proper use ofeven 14 strains is an unrealisticrequirement in most clinical laboratories sinceserogrouping of salmonella is only one taskamong many.When purchasing reagents, it is advisable for

consumers to request reagents that have beenevaluated by CDC and found to meet CDCspecifications. Even though the scarcity of theantisera impedes the effectiveness of this policy,the requirement should eventually improve thequality of the reagents that are available. Labo-ratory Licensure and Food and Drug Adminis-tration regulations that recognize realistic exten-sion of expiration dates would help to alleviatethe availabiity problem.Because of the current emphasis on preven-

tive medicine, and on the epidemiological signif-icance of salmonellosis and the fact that Sal-monella serotyping has been a practical labora-

367VOL. 8, 1978

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368 EVINS, LINNE, AND COLVIN

tory procedure for over 30 years, it seems rea-sonable to expect that a basic set of Salmonellaantisera be easily obtained and consistently re-liable.

LITERATURE CITED

1. Center for Disease Control. 1972. Specifications forSalmonella antisera. Center for Disease Control, At-lanta, Ga.

2. Center for Disease Control. 1975. Specifications andevaluation methods for immunological and microbiolog-ical reagents, vol. 1. Bacterial, fungal, and parasitic, 4thed. Center for Disease Control, Atlanta, Ga.

3. Edwards, P. R., and W. H. Ewing. 1962. Identificationof Enterobacteriaceae, 2nd ed., p. 105. Burgess Publish-

J. CLIN. MICROBIOL.

ing Co., Minneapolis.4. Edwards, P. R., and W. H. Ewing. 1972. Identification

of Enterobacteriaceae, 3rd ed. Burgess Publishing Co.,Minneapolis, Minn.

5. Evins, G. M., L. L. Linne, and H. M. Colvin. 1976.Evaluation of commercial Salmonella O polyvalent andVi antisera. J. Clin. Microbiol. 4:349-353.

6. Ewing, W. H. 1972. Isolation and identification of Sal-monella and Shigella, p. 31. Center for Disease Control,Atlanta, Ga.

7. Kauffmann, F. 1972. Serological diagnosis of Salmonellaspecies, Kauffmann-White schema, 1st ed. The Williams& Wilkins Co., Baltimore.

8. LeMinor, L., R. Rohde, Ch. Charié-Marsaines, and C.Coynault. 1971. Étude sur les rapports antigéniquesentre le groupe 0:54 et d'autres groupes O de Salmo-nella. Ann. Inst. Pasteur Paris 121:447-463.

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