Vol. 4, No. 4 Printed in U.S.A.
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1976, p. 349-353 Copyright 0 1976 American Society for Microbiology
Evaluation of Commercial Salmonella Antisera
0
Polyvalent and Vi
GRACIA M. EVINS,* LINDA L. LINNE, AND HENRY M. COLVIN
Center for Disease Control, Atlanta, Georgia 30333 Received for publication 18 June 1976
Salmonella somatic polyvalent antisera for groups A through E or A through I with or without Vi and Salmonella Vi antisera were purchased from four commerical producers and evaluated. Center for Disease Control (CDC) methods and specifications were used. The four polyvalent and two of the four Vi antisera did not meet CDC performance specifications. Suggestions are made for improving the laboratory usefulness of the reagents and the product labeling.
Commercial antisera for the serological identification of salmonellae have been available for years, but the ineffectiveness of these products in the identification of Salmonella isolates has been a continuing problem. Since 1971, the Center for Disease Control (CDC) has invited commercial producers to voluntarily submit samples of all lots of Salmonella antisera for evaluation before marketing. Many antisera that have not been submitted for evaluation are marketed. To determine the quality of Salmonella somatic polyvalent and Vi antisera available to consumer laboratories, as judged by CDC specifilcations, we ordered these antisera from the six commercial producers listing them in their catalogs and evaluated them. MATERIALS AND METHODS Antisera. Salmonella somatic polyvalent antisera, groups A through E or A through I, with or without Vi and Salmonella Vi antisera, were purchased from BBL, Lederle Diagnostics, Lee Laboratories, Inc., and Difco Laboratories. All products had expiration dates of 1977 or 1978. Grand Island Biological Co. and The Sylvana Co. replied that they had discontinued sales of Salmonella antisera. Grouping, subgrouping, and Vi antisera produced by the Biological Products Division, CDC, were used to verify the reactivity and smoothness of test antigens. Somatic (0) antigens. Antigens were produced from freeze-dried cultures of strains that had been originally serotyped by the Enteric Unit, National Salmonella Reference Center, CDC. The cultures were reconstituted, streaked on blood agar base plates, and incubated overnight (18 h) at 37°C. Smooth colonies were selected and streaked on blood agar base slants (12 ml of medium in 25- by 150-mm tubes). After 18 to 20 h of incubation at 37°C, the growth was scraped and emulsified in 1 ml of 0.85% NaCl. The dense suspensions were allowed to sit for at least 20 min for large particles to settle to the bottom. To avoid these particles, we withdrew the antigens from the top.
Each antigen preparation was tested with antisera containing homologous agglutinins for reactivity and with saline and groups A, B, C,, C2, D, El, E2, E3, and E4 heterologous antisera for smoothness. Only antigens giving 3+ or greater reactions with the routine test dilution of the homologous antisera and giving expected positive or negative reactions with heterologous antisera were selected for use. Unheated Vi antigens were negative with group D antisera. Each antigen listed in Table 1 was made from a different culture and used in serological assays performed on the same day. Flagellar (H) antigens. These antigens were stock formalinized infusion broth cultures prepared as described by the CDC specifications (1) and had been used as reference antigens for several years. They were used to assay the presence of H agglutinins in the 0 antisera. Normally, 0 antisera would be tested for the presence of H antigens that occur in the immunizing strains used to produce an 0 antiserum, but the immunizing strains were not identified in the literature accompanying the products. Therefore, 23 H antigens were selected to represent the serotypes that may have been used as immunizing strains. The volume of antisera purchased also limited the extent of testing for H agglutinins. Antiserum evaluation procedure. All testing was done by two persons having several years of experience evaluating commercially produced Salmonella antisera. Polyvalent antisera were tested with the 0 antigens listed in Table 1. Antigen serotypes used for assay of heterologous agglutinins in polyvalents were selected on the basis of known antigenic relationships with strains of groups A through I (3, 4). Vi antisera were tested with two antigens from each of four strains having Vi and with antigens from three strains selected especially for evaluation of Vi
antisera: a serotype paratyphi C having Vi antigen, and two S. typhi cultures. One of these two cultures has consistently exhibited only the Vi antigen; the other has exhibited only the 0. Antigens from 19 strains representing groups A through H, known not to contain Vi antigen, were used to assay for 0 agglutinins in the Vi antisera. These 19 strains are identified in Table 1. The commercial antisera were diluted according
349
350
EVINS, LINNE, AND COLVIN
J. CLIN. MICROBIOL.
TABLE 1. Slide agglutination reactions of Salmonella polyvalent antisera produced by four companies Salmonella
Antisera BBL lot
Difco lot
GrouSptyeFatrs6423 Factors Serotype Group 614203, 17GAEC, 0 polyvalent A
B
C,
C2
groupVi
Lederle lot
Lee lot
436-772, antigens 1-11, 13-16, 19, 22, 23, Vi 4 4 4 3 4
2155-13, antigens 1-10, 15, 19, 34,
CDCa ho-
4 4 4 3 2
3 3 3 3 3
mologous
Vi
1,2,12
4
1,2,12
4
1,2,12 1,2,12 1,2,12
4 3 3
3 4 3 3 3
paratyphi Bb typhimurium var. copenhagenb schleissheimb 4,5,12 nonmotile heidelberg st.-paul San-diego derby reading bredeney bredeney bredeney
4,5,12 4,12
2 3
3 1
2 3
3 3
3 3
1,4,12,27 4,5,12 1,4,5,12 1,4,5,12 4,5,12 1,4,[5],12 4,12 1,4,12,27 1,4,12,27
3 tr
1,4,12,27
3 4 3 4 4 3 4 3 3 3
tr 3 2 1 3 2 2 2
2 2 3 3 2 2 3 2 2 2
3 3 2 3 3 3 3 2 3 3
3 3 3 3 3 3 3 3 3 4
thompsonb
bornumb bareilly lille oranienburg oranienburg
6,7 6,7,14 6,7 6,7 6,7 6,7
2 3 3 4 4 4
2 2 2 2 2 3
2 3 2 3 3 3
1 2 2 3 2 3
3 3 3 4 3 3
2 1 2 2 3
2 2 1 2 3
2 2 3 3 3
3 3 3 3 3
paratyphi A paratyphi Ab paratyphi A paratyphi A paratyphi A
virginiab
8
kentuckyb
(8),20
blockley muenchen newport var. puerto-
6,8 6,8 6,8
3 2 3 3 3
rico
D
gallinarumb haarlemb berta enteritidis gallinarum javiana wangata
9,12 (9),46 9,12 1,9,12 9,12 9,12 1,9,12
4 3 4 3 4 3 4
3 3 2 2 tr 1 2
3 2 2 3 3 2 2
4 3 4 3 4 3 4
4 4 4 4 4 3 3
E,
anatumb muenster anatum london newingtonb binza binza drypool minneapolisb arkansas
3,10 3,10 3,10 3,10 3,15
4 4 4 4 4 3 4 4 4 4 4 4 4 4 4 4
4 3 4 4 4 4 4 4 4 4 4 4 4 4 4
4 4 4 3 4 3 4 4 4 4 4 4 4 4 4 4
3 3 3 3 4 3 4 4 3 3 3 3 4 3 4
-
2 tr
2 2
-
4
tr
3
E2
E3
E4
F
senftenbergb senftenberg senftenberg senftenberg senftenberg
1,3,19 1,3,19 1,3,19 1,3,19 1,3,19
4 4 4 4 4 2 3 3 4 4 4 4 4 4 4 4
Aberdeenb rubislaw
11 11
tr
(3),(15),34
3,15 3,15 3,15 (3),(15),34 (3),(15),34
(3),(15),34
4
3
EVALUATING SALMONELLA ANTISERA
VOL. 4, 1976
351
TABLE 1-Continued Salmonella
Antisera BBL lot
Difco lot
Group
Serotype
G
H
I
K M
0 U 51 54
Factors
Lederele lot an436-772, tigens 1-11, 13-16, 19, 22, 23, Vi
17GAEC, o polyvalent
614203, groups A-I,
-
2 1
2 2 2 2 2 3 3 3
3 2 3 3 3 . 2 2 2
Lee lot
2155-13, an-
tigens 1-10, 15, 19, 34, Vi
poonab poona poona poona poona worthingtonb cubana worthington
13,22,36 13,22,36 13,22,36 13,22,36 13,22,36 1,13,23,37 1,13,23,37 1,13,23,37
carraub carrau carrau
floridab sundsvall
6,14,24 6,14,24 6,14,24 1,6,14,25 1,6,14,25
2 3 2 2 2
2 2 2 3 2
3 4 4 2 4
gaminara gaminara mobeni var. S-
16 16 16
tr -
2 1 1
3 2 2
-
siegburg 28:y:adelaide berkeley treforest uccle
6,14,18 28 35 43 1,51 3,54
2
2
2
tr
-
-
-
-
-
-
-
1
-
-
-
-
2 1
2 1
CDCa homologous
-
3
-
4 4 4
tr -
1 1 -
1 -
2
3 3 3 3 3 3 3 4 4 3 4 4
3 4 3 4 3 3
S. typhi 2 (Felix) Vi 1 3 tr 4 S. typhi Vi 1 1 4 4 S. typhi Vi 1 1 4 3 S. typhi Vi 1 1 4 4 S. typhi Vi 3 4 a The reaction of the antigen with the routine test dilution of CDC homologous grouping or subgrouping antiserum is given. bStrains used for testing Vi antisera for the presence of 0 agglutinins.
to instructions accompanying each product and tested at this dilution only for the presence of 0 agglutinins. Droplets of approximately 0.01 ml of the routine test dilutions of each antiserum were distributed on divisions of a 50- by 75-mm glass slide. A droplet of equal volume of each appropriate antigen was added to a division containing the antiserum. With a loop, the antiserum and antigen within each division were mixed to form an area of approximately 5 by 20 mm. The slide was then tilted by hand continuously for 1 min, and the mixtures were observed for agglutination with a fluorescent lamp against a black background. The degree of agglutination was recorded as follows: 4+, all of the organisms appeared to be agglutinated and the supernatant fluid was clear; 3 +, approximately 75% of the organisms were agglutinated and the supernatant fluid was slightly cloudy; 2+, 50% of the organisms were agglutinated and the supernatant fluid was moderately cloudy; 1+, 25% of the organisms were agglutinated and the supernatant fluid was cloudy; tr (trace), less than 25% of the organisms
were agglutinated; - (negative), no apparent agglutination. To test for the presence of H agglutinins, each commercial antiserum was diluted 1:50 with 0.85% NaCl. In 13- by 100-mm tubes, 0.5-ml volumes of each diluted antiserum and equal volumes of each of the 23 stock H antigens were mixed. The mixtures were incubated in a 48 to 50°C water bath for 1 h. Each tube was observed for agglutination, and the degree of agglutination was recorded as described above. Performance criteria. CDC performance requirements, as summarized below, were used to assess the acceptability of each product evaluated (1). An acceptable polyvalent antiserum must give a minimum of 3+ agglutination within 1 min with antigens of homologous serotypes and must not react with serotypes of heterologous Salmonella 0 groups, except that cross-reactions not exceeding 1 + may occur with antigenically related salmonellae. In addition, a polyvalent antiserum for groups A through E or I may exceed 1 + agglutination with
352
EVINS, LINNE, AND COLVIN
strains having factor 1 and belonging to groups R, T, V, X, 51, and 53. A polyvalent antiserum having group E may exceed 1 + agglutination with group 54 strains that contain factor 03. The antisera must not contain flagellar agglutinins. An acceptable Vi antiserum must give a minimum of 3+ agglutination within 1 min with the V form ofS. typhi 2 (Felix) and several selected strains known to contain Vi antigen. The antiserum must agglutinate serotype paratyphi C having Vi; however, a reaction less than 3 + is acceptable. The antiserum must not produce agglutination within 1 min with the 0 form of S. typhi 901 or with antigens of Salmonella 0 groups A, B, C, (other than serotype paratyphi C), C2, and E.
J. CLIN. MICROBIOL.
product was unacceptable for the identification of Vi antigens. In addition, it reacted with 11 of the 23 H antigens used. Therefore, the product does not meet the CDC specifications. Lee polyvalent antiserum, lot 2155-13, was labeled, in part, "Poly Al antigens 1-10, 15, 19, 34, Vi." The package insert stated that it "contains somatic Group Antigens A through E and Vi." The product was unacceptable because of poor reactions with serotypes of group C,. Therefore, the product does not meet the CDC specifications. It was negative with the 23 H antigens used. Each of the Vi antisera gave 2+ reactions with ser paratyphi C and was negative with RESULTS somatic groups A through H and the 0 form of S. typhi 901. Lederle Vi antiserum, lot 437-721, Reactions of the polyvalent antisera, none of which met the CDC specifications outlined and Lee Vi antiserum, lot 2148-12, gave 3+ or above, are given in Table 1. Two of the four Vi 4+ reactions with all the homologous antigens and therefore meet CDC specifications. Vi antiantisera met the specifications. BBL lot K7GAGE and Difco lot 618805, sera, I7GAEC, lot BBL polyvalent antiserum, gave strong reactions with groups A through E gave 3+ reactions with only two of the five S. and moderate to poor reactions with groups G, typhi strains and therefore do not meet CDC H, K, and 54. This product was labeled "Salmo- specifications. nella 0 polyvalent antiserum" with no indicaDISCUSSION tion of which groups were included. The accomSerotyping of salmonellae is at best a confuspanying package insert intimated, but did not state, that the product was for groups A ing procedure, and inadequate labeling of antithrough E. If this coverage is assumed, the sera adds to the already cumbersome system. If product will incorrectly identify group H the antigens covered and not the groups are strains as belonging to groups A through E. stated on the label of a polyvalent antiserum Conversely, if the product is intended to include problems will arise; e.g., it is not known all Salmonella serotypes, as the vial label im- whether the 2 + reactions of the Lederle antiseplied, it will not identify groups F, G, I, M, and rum with group K strain serotype siegburg 0. Therefore, it does not meet the CDC specifi- 6,14,18 and serotype treforest 1,51 are homolocations. The product was negative with all of gous or heterologous reactions. The problems with the BBL label were stated the 23 H antigens used. The box of Difco polyvalent antiserum, lot previously. The Difco box label and vial label 614203, was labeled, in part, "Serotype groups are misleading as to the presence of Vi. The Lee A-I, Vi"; Vi was not on the vial label. Reactions labeling is satisfactory, since the package inwith serotypes of groups Cl, C2, G, and H sert contains the statement that groups A ranged from moderate to strong, but half of the through E and Vi are covered; however, label reactions within each group did not meet the space would be used more effectively if groups minimum 3+ potency requirement. The prod- A through E instead of "Poly Al" were stated. Polyvalent 0 antisera are difficult to produce uct was unacceptable for identifying Vi and strains of groups B, D, F, and I. In addition, it and evaluate. Reactions such as those in Table reacted with the 23 flagellar antigens used for 1 with groups A, E, and Vi are clear-cut, but testing for the presence of H agglutinins. problems arise in interpreting borderline reacTherefore, the product does not meet the CDC tions because of form variation prevalent in salmonellae. When moderate reactions are specifications. The vial of Lederle polyvalent antiserum, lot mixed with strong reactions within a group, 436-772, was labeled "Antigens 1-11, 13-16, 19, decisiveness is difficult; e.g., many of the 2+ 22, 23, Vi," but the groups covered were not reactions of Lederle antiserum with group B stated on the vial label or in the package insert. strains were rapid, strong 2+ reactions that Reactions with serotypes of groups B, C2, D, F, might be read 3 + if the antisera were tested and I ranged from moderate to strong, but half repeatedly. If this antiserum reacts with most of the reactions within each group did not meet group B strains to a degree that will identify the minimum 3 + potency requirement. The the isolates as salmonellae, the reagent has
VOL. 4, 1976
accomplished its purpose as a screening antiserum. The problem, however, is the possibility of not identifying isolates that have antigens poorly developed because of form variation as salmonellae. The antiserum giving rapid, strong 2+ reactions provides no margin for such cultures, and the cultures could be overlooked. Obviously, a polyvalent antiserum that gives 3+ or 4+ with almost all test strains A through E can be made, as shown by the BBL antiserum. All of the products, except Difco's, contain 50% glycerin and so are diluted 1:2 before saline is added. The addition of 2 ml of saline makes a dilution of 1:6. Less dilution might raise the borderline reactions to 3+ or 4+. However, decreasing the dilution might also increase cross-reactions, such as group H strains with antisera for A through E. Instead of absorbing the 6,14 factors, which jeopardizes the reactions with groups Cl and C2, the better solution would be to include groups H and K in the antiserum. Coping with factor 1 is more complex. Many of the strains of groups A through I have factor 1, so the presence of 01 agglutinins in a polyvalent antiserum for groups A through I is desirable. Strains from groups other than A, B, D, E, G, and H, in which factor 1 occurs, are rarely isolated (2); crosses with these create no real problems in the laboratory. A note in the package insert to the effect that the antiserum may cross-react with those salmonellae that have factor 1 and belong to groups R, T, V, X, 51, and 53 should be adequate. Likewise, a statement noting that 054 strains might react because of the relationship with factor 3 is appropriate. The presence of H agglutinins in an 0 polyvalent antiserum leads to confusion. The consumer, who finds an isolate positive with such an antiserum and then obtains only negative reactions with individual grouping antisera, has been misdirected. The living antigens may have reacted with the flagellar agglutinins in the reagent. H agglutinins were not said to be
EVALUATING SALMONELLA ANTISERA
353
present either on the labels or the package inserts of the two reagents that contained them. Meaningful evaluation of a polyvalent antiserum is too time consuming for most clinical laboratories. The actual testing of the reagent is the lesser part of the work. A culture collection must be maintained and antigens must be evaluated with homologous and heterologous antisera before being used. The practice of testing an antigen for smoothness by using only saline is inadequate. Many antigens that appear smooth with saline give, with heterologous antisera, erratic agglutination that identifies them as rough. This antigen evaluation is time consuming and requires a constant source of reliable antisera. Furthermore, the analyst must have detailed knowledge of serological relationships among salmonellae to correctly interpret results. It is unreasonable to expect this type of testing in any but the larger laboratories, where antiserum production is also feasible. In purchasing reagents, CDC advises consumers to specify that only reagents evaluated by CDC and found to meet CDC specifications will be accepted. Having reliable reagents readily available would do much to solve the problem. Only one of the antisera discussed in this report (Vi from Lee Laboratories) was voluntarily submitted for evaluation before marketing. LITERATURE CITED 1. Center for Disease Control. 1975. Specifications and evaluation methods for immunological and microbiological reagents, vol. 1: Bacterial, fungal, and parasitic, 4th ed. Center for Disease Control, Atlanta, Ga. 2. Edwards, P. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis, Minn. 3. Kauffmann, F. 1972. Serological diagnosis of Salmonella species Kauffmann-White schema, 1st ed. The Williams & Wilkins Co., Baltimore, Md. 4. LeMinor, L., R. Rohde, Ch. Chari6-Marsaines, and C. Coynault. 1971. etude sur les rapports antig6niques entre le groupe 0:54 et d'autres groupes 0 de Salmonella. Ann. Inst. Pasteur Paris 121:447-463.
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1976, p. 349-353 Copyright 0 1976 American Society for Microbiology
Evaluation of Commercial Salmonella Antisera
0
Polyvalent and Vi
GRACIA M. EVINS,* LINDA L. LINNE, AND HENRY M. COLVIN
Center for Disease Control, Atlanta, Georgia 30333 Received for publication 18 June 1976
Salmonella somatic polyvalent antisera for groups A through E or A through I with or without Vi and Salmonella Vi antisera were purchased from four commerical producers and evaluated. Center for Disease Control (CDC) methods and specifications were used. The four polyvalent and two of the four Vi antisera did not meet CDC performance specifications. Suggestions are made for improving the laboratory usefulness of the reagents and the product labeling.
Commercial antisera for the serological identification of salmonellae have been available for years, but the ineffectiveness of these products in the identification of Salmonella isolates has been a continuing problem. Since 1971, the Center for Disease Control (CDC) has invited commercial producers to voluntarily submit samples of all lots of Salmonella antisera for evaluation before marketing. Many antisera that have not been submitted for evaluation are marketed. To determine the quality of Salmonella somatic polyvalent and Vi antisera available to consumer laboratories, as judged by CDC specifilcations, we ordered these antisera from the six commercial producers listing them in their catalogs and evaluated them. MATERIALS AND METHODS Antisera. Salmonella somatic polyvalent antisera, groups A through E or A through I, with or without Vi and Salmonella Vi antisera, were purchased from BBL, Lederle Diagnostics, Lee Laboratories, Inc., and Difco Laboratories. All products had expiration dates of 1977 or 1978. Grand Island Biological Co. and The Sylvana Co. replied that they had discontinued sales of Salmonella antisera. Grouping, subgrouping, and Vi antisera produced by the Biological Products Division, CDC, were used to verify the reactivity and smoothness of test antigens. Somatic (0) antigens. Antigens were produced from freeze-dried cultures of strains that had been originally serotyped by the Enteric Unit, National Salmonella Reference Center, CDC. The cultures were reconstituted, streaked on blood agar base plates, and incubated overnight (18 h) at 37°C. Smooth colonies were selected and streaked on blood agar base slants (12 ml of medium in 25- by 150-mm tubes). After 18 to 20 h of incubation at 37°C, the growth was scraped and emulsified in 1 ml of 0.85% NaCl. The dense suspensions were allowed to sit for at least 20 min for large particles to settle to the bottom. To avoid these particles, we withdrew the antigens from the top.
Each antigen preparation was tested with antisera containing homologous agglutinins for reactivity and with saline and groups A, B, C,, C2, D, El, E2, E3, and E4 heterologous antisera for smoothness. Only antigens giving 3+ or greater reactions with the routine test dilution of the homologous antisera and giving expected positive or negative reactions with heterologous antisera were selected for use. Unheated Vi antigens were negative with group D antisera. Each antigen listed in Table 1 was made from a different culture and used in serological assays performed on the same day. Flagellar (H) antigens. These antigens were stock formalinized infusion broth cultures prepared as described by the CDC specifications (1) and had been used as reference antigens for several years. They were used to assay the presence of H agglutinins in the 0 antisera. Normally, 0 antisera would be tested for the presence of H antigens that occur in the immunizing strains used to produce an 0 antiserum, but the immunizing strains were not identified in the literature accompanying the products. Therefore, 23 H antigens were selected to represent the serotypes that may have been used as immunizing strains. The volume of antisera purchased also limited the extent of testing for H agglutinins. Antiserum evaluation procedure. All testing was done by two persons having several years of experience evaluating commercially produced Salmonella antisera. Polyvalent antisera were tested with the 0 antigens listed in Table 1. Antigen serotypes used for assay of heterologous agglutinins in polyvalents were selected on the basis of known antigenic relationships with strains of groups A through I (3, 4). Vi antisera were tested with two antigens from each of four strains having Vi and with antigens from three strains selected especially for evaluation of Vi
antisera: a serotype paratyphi C having Vi antigen, and two S. typhi cultures. One of these two cultures has consistently exhibited only the Vi antigen; the other has exhibited only the 0. Antigens from 19 strains representing groups A through H, known not to contain Vi antigen, were used to assay for 0 agglutinins in the Vi antisera. These 19 strains are identified in Table 1. The commercial antisera were diluted according
349
350
EVINS, LINNE, AND COLVIN
J. CLIN. MICROBIOL.
TABLE 1. Slide agglutination reactions of Salmonella polyvalent antisera produced by four companies Salmonella
Antisera BBL lot
Difco lot
GrouSptyeFatrs6423 Factors Serotype Group 614203, 17GAEC, 0 polyvalent A
B
C,
C2
groupVi
Lederle lot
Lee lot
436-772, antigens 1-11, 13-16, 19, 22, 23, Vi 4 4 4 3 4
2155-13, antigens 1-10, 15, 19, 34,
CDCa ho-
4 4 4 3 2
3 3 3 3 3
mologous
Vi
1,2,12
4
1,2,12
4
1,2,12 1,2,12 1,2,12
4 3 3
3 4 3 3 3
paratyphi Bb typhimurium var. copenhagenb schleissheimb 4,5,12 nonmotile heidelberg st.-paul San-diego derby reading bredeney bredeney bredeney
4,5,12 4,12
2 3
3 1
2 3
3 3
3 3
1,4,12,27 4,5,12 1,4,5,12 1,4,5,12 4,5,12 1,4,[5],12 4,12 1,4,12,27 1,4,12,27
3 tr
1,4,12,27
3 4 3 4 4 3 4 3 3 3
tr 3 2 1 3 2 2 2
2 2 3 3 2 2 3 2 2 2
3 3 2 3 3 3 3 2 3 3
3 3 3 3 3 3 3 3 3 4
thompsonb
bornumb bareilly lille oranienburg oranienburg
6,7 6,7,14 6,7 6,7 6,7 6,7
2 3 3 4 4 4
2 2 2 2 2 3
2 3 2 3 3 3
1 2 2 3 2 3
3 3 3 4 3 3
2 1 2 2 3
2 2 1 2 3
2 2 3 3 3
3 3 3 3 3
paratyphi A paratyphi Ab paratyphi A paratyphi A paratyphi A
virginiab
8
kentuckyb
(8),20
blockley muenchen newport var. puerto-
6,8 6,8 6,8
3 2 3 3 3
rico
D
gallinarumb haarlemb berta enteritidis gallinarum javiana wangata
9,12 (9),46 9,12 1,9,12 9,12 9,12 1,9,12
4 3 4 3 4 3 4
3 3 2 2 tr 1 2
3 2 2 3 3 2 2
4 3 4 3 4 3 4
4 4 4 4 4 3 3
E,
anatumb muenster anatum london newingtonb binza binza drypool minneapolisb arkansas
3,10 3,10 3,10 3,10 3,15
4 4 4 4 4 3 4 4 4 4 4 4 4 4 4 4
4 3 4 4 4 4 4 4 4 4 4 4 4 4 4
4 4 4 3 4 3 4 4 4 4 4 4 4 4 4 4
3 3 3 3 4 3 4 4 3 3 3 3 4 3 4
-
2 tr
2 2
-
4
tr
3
E2
E3
E4
F
senftenbergb senftenberg senftenberg senftenberg senftenberg
1,3,19 1,3,19 1,3,19 1,3,19 1,3,19
4 4 4 4 4 2 3 3 4 4 4 4 4 4 4 4
Aberdeenb rubislaw
11 11
tr
(3),(15),34
3,15 3,15 3,15 (3),(15),34 (3),(15),34
(3),(15),34
4
3
EVALUATING SALMONELLA ANTISERA
VOL. 4, 1976
351
TABLE 1-Continued Salmonella
Antisera BBL lot
Difco lot
Group
Serotype
G
H
I
K M
0 U 51 54
Factors
Lederele lot an436-772, tigens 1-11, 13-16, 19, 22, 23, Vi
17GAEC, o polyvalent
614203, groups A-I,
-
2 1
2 2 2 2 2 3 3 3
3 2 3 3 3 . 2 2 2
Lee lot
2155-13, an-
tigens 1-10, 15, 19, 34, Vi
poonab poona poona poona poona worthingtonb cubana worthington
13,22,36 13,22,36 13,22,36 13,22,36 13,22,36 1,13,23,37 1,13,23,37 1,13,23,37
carraub carrau carrau
floridab sundsvall
6,14,24 6,14,24 6,14,24 1,6,14,25 1,6,14,25
2 3 2 2 2
2 2 2 3 2
3 4 4 2 4
gaminara gaminara mobeni var. S-
16 16 16
tr -
2 1 1
3 2 2
-
siegburg 28:y:adelaide berkeley treforest uccle
6,14,18 28 35 43 1,51 3,54
2
2
2
tr
-
-
-
-
-
-
-
1
-
-
-
-
2 1
2 1
CDCa homologous
-
3
-
4 4 4
tr -
1 1 -
1 -
2
3 3 3 3 3 3 3 4 4 3 4 4
3 4 3 4 3 3
S. typhi 2 (Felix) Vi 1 3 tr 4 S. typhi Vi 1 1 4 4 S. typhi Vi 1 1 4 3 S. typhi Vi 1 1 4 4 S. typhi Vi 3 4 a The reaction of the antigen with the routine test dilution of CDC homologous grouping or subgrouping antiserum is given. bStrains used for testing Vi antisera for the presence of 0 agglutinins.
to instructions accompanying each product and tested at this dilution only for the presence of 0 agglutinins. Droplets of approximately 0.01 ml of the routine test dilutions of each antiserum were distributed on divisions of a 50- by 75-mm glass slide. A droplet of equal volume of each appropriate antigen was added to a division containing the antiserum. With a loop, the antiserum and antigen within each division were mixed to form an area of approximately 5 by 20 mm. The slide was then tilted by hand continuously for 1 min, and the mixtures were observed for agglutination with a fluorescent lamp against a black background. The degree of agglutination was recorded as follows: 4+, all of the organisms appeared to be agglutinated and the supernatant fluid was clear; 3 +, approximately 75% of the organisms were agglutinated and the supernatant fluid was slightly cloudy; 2+, 50% of the organisms were agglutinated and the supernatant fluid was moderately cloudy; 1+, 25% of the organisms were agglutinated and the supernatant fluid was cloudy; tr (trace), less than 25% of the organisms
were agglutinated; - (negative), no apparent agglutination. To test for the presence of H agglutinins, each commercial antiserum was diluted 1:50 with 0.85% NaCl. In 13- by 100-mm tubes, 0.5-ml volumes of each diluted antiserum and equal volumes of each of the 23 stock H antigens were mixed. The mixtures were incubated in a 48 to 50°C water bath for 1 h. Each tube was observed for agglutination, and the degree of agglutination was recorded as described above. Performance criteria. CDC performance requirements, as summarized below, were used to assess the acceptability of each product evaluated (1). An acceptable polyvalent antiserum must give a minimum of 3+ agglutination within 1 min with antigens of homologous serotypes and must not react with serotypes of heterologous Salmonella 0 groups, except that cross-reactions not exceeding 1 + may occur with antigenically related salmonellae. In addition, a polyvalent antiserum for groups A through E or I may exceed 1 + agglutination with
352
EVINS, LINNE, AND COLVIN
strains having factor 1 and belonging to groups R, T, V, X, 51, and 53. A polyvalent antiserum having group E may exceed 1 + agglutination with group 54 strains that contain factor 03. The antisera must not contain flagellar agglutinins. An acceptable Vi antiserum must give a minimum of 3+ agglutination within 1 min with the V form ofS. typhi 2 (Felix) and several selected strains known to contain Vi antigen. The antiserum must agglutinate serotype paratyphi C having Vi; however, a reaction less than 3 + is acceptable. The antiserum must not produce agglutination within 1 min with the 0 form of S. typhi 901 or with antigens of Salmonella 0 groups A, B, C, (other than serotype paratyphi C), C2, and E.
J. CLIN. MICROBIOL.
product was unacceptable for the identification of Vi antigens. In addition, it reacted with 11 of the 23 H antigens used. Therefore, the product does not meet the CDC specifications. Lee polyvalent antiserum, lot 2155-13, was labeled, in part, "Poly Al antigens 1-10, 15, 19, 34, Vi." The package insert stated that it "contains somatic Group Antigens A through E and Vi." The product was unacceptable because of poor reactions with serotypes of group C,. Therefore, the product does not meet the CDC specifications. It was negative with the 23 H antigens used. Each of the Vi antisera gave 2+ reactions with ser paratyphi C and was negative with RESULTS somatic groups A through H and the 0 form of S. typhi 901. Lederle Vi antiserum, lot 437-721, Reactions of the polyvalent antisera, none of which met the CDC specifications outlined and Lee Vi antiserum, lot 2148-12, gave 3+ or above, are given in Table 1. Two of the four Vi 4+ reactions with all the homologous antigens and therefore meet CDC specifications. Vi antiantisera met the specifications. BBL lot K7GAGE and Difco lot 618805, sera, I7GAEC, lot BBL polyvalent antiserum, gave strong reactions with groups A through E gave 3+ reactions with only two of the five S. and moderate to poor reactions with groups G, typhi strains and therefore do not meet CDC H, K, and 54. This product was labeled "Salmo- specifications. nella 0 polyvalent antiserum" with no indicaDISCUSSION tion of which groups were included. The accomSerotyping of salmonellae is at best a confuspanying package insert intimated, but did not state, that the product was for groups A ing procedure, and inadequate labeling of antithrough E. If this coverage is assumed, the sera adds to the already cumbersome system. If product will incorrectly identify group H the antigens covered and not the groups are strains as belonging to groups A through E. stated on the label of a polyvalent antiserum Conversely, if the product is intended to include problems will arise; e.g., it is not known all Salmonella serotypes, as the vial label im- whether the 2 + reactions of the Lederle antiseplied, it will not identify groups F, G, I, M, and rum with group K strain serotype siegburg 0. Therefore, it does not meet the CDC specifi- 6,14,18 and serotype treforest 1,51 are homolocations. The product was negative with all of gous or heterologous reactions. The problems with the BBL label were stated the 23 H antigens used. The box of Difco polyvalent antiserum, lot previously. The Difco box label and vial label 614203, was labeled, in part, "Serotype groups are misleading as to the presence of Vi. The Lee A-I, Vi"; Vi was not on the vial label. Reactions labeling is satisfactory, since the package inwith serotypes of groups Cl, C2, G, and H sert contains the statement that groups A ranged from moderate to strong, but half of the through E and Vi are covered; however, label reactions within each group did not meet the space would be used more effectively if groups minimum 3+ potency requirement. The prod- A through E instead of "Poly Al" were stated. Polyvalent 0 antisera are difficult to produce uct was unacceptable for identifying Vi and strains of groups B, D, F, and I. In addition, it and evaluate. Reactions such as those in Table reacted with the 23 flagellar antigens used for 1 with groups A, E, and Vi are clear-cut, but testing for the presence of H agglutinins. problems arise in interpreting borderline reacTherefore, the product does not meet the CDC tions because of form variation prevalent in salmonellae. When moderate reactions are specifications. The vial of Lederle polyvalent antiserum, lot mixed with strong reactions within a group, 436-772, was labeled "Antigens 1-11, 13-16, 19, decisiveness is difficult; e.g., many of the 2+ 22, 23, Vi," but the groups covered were not reactions of Lederle antiserum with group B stated on the vial label or in the package insert. strains were rapid, strong 2+ reactions that Reactions with serotypes of groups B, C2, D, F, might be read 3 + if the antisera were tested and I ranged from moderate to strong, but half repeatedly. If this antiserum reacts with most of the reactions within each group did not meet group B strains to a degree that will identify the minimum 3 + potency requirement. The the isolates as salmonellae, the reagent has
VOL. 4, 1976
accomplished its purpose as a screening antiserum. The problem, however, is the possibility of not identifying isolates that have antigens poorly developed because of form variation as salmonellae. The antiserum giving rapid, strong 2+ reactions provides no margin for such cultures, and the cultures could be overlooked. Obviously, a polyvalent antiserum that gives 3+ or 4+ with almost all test strains A through E can be made, as shown by the BBL antiserum. All of the products, except Difco's, contain 50% glycerin and so are diluted 1:2 before saline is added. The addition of 2 ml of saline makes a dilution of 1:6. Less dilution might raise the borderline reactions to 3+ or 4+. However, decreasing the dilution might also increase cross-reactions, such as group H strains with antisera for A through E. Instead of absorbing the 6,14 factors, which jeopardizes the reactions with groups Cl and C2, the better solution would be to include groups H and K in the antiserum. Coping with factor 1 is more complex. Many of the strains of groups A through I have factor 1, so the presence of 01 agglutinins in a polyvalent antiserum for groups A through I is desirable. Strains from groups other than A, B, D, E, G, and H, in which factor 1 occurs, are rarely isolated (2); crosses with these create no real problems in the laboratory. A note in the package insert to the effect that the antiserum may cross-react with those salmonellae that have factor 1 and belong to groups R, T, V, X, 51, and 53 should be adequate. Likewise, a statement noting that 054 strains might react because of the relationship with factor 3 is appropriate. The presence of H agglutinins in an 0 polyvalent antiserum leads to confusion. The consumer, who finds an isolate positive with such an antiserum and then obtains only negative reactions with individual grouping antisera, has been misdirected. The living antigens may have reacted with the flagellar agglutinins in the reagent. H agglutinins were not said to be
EVALUATING SALMONELLA ANTISERA
353
present either on the labels or the package inserts of the two reagents that contained them. Meaningful evaluation of a polyvalent antiserum is too time consuming for most clinical laboratories. The actual testing of the reagent is the lesser part of the work. A culture collection must be maintained and antigens must be evaluated with homologous and heterologous antisera before being used. The practice of testing an antigen for smoothness by using only saline is inadequate. Many antigens that appear smooth with saline give, with heterologous antisera, erratic agglutination that identifies them as rough. This antigen evaluation is time consuming and requires a constant source of reliable antisera. Furthermore, the analyst must have detailed knowledge of serological relationships among salmonellae to correctly interpret results. It is unreasonable to expect this type of testing in any but the larger laboratories, where antiserum production is also feasible. In purchasing reagents, CDC advises consumers to specify that only reagents evaluated by CDC and found to meet CDC specifications will be accepted. Having reliable reagents readily available would do much to solve the problem. Only one of the antisera discussed in this report (Vi from Lee Laboratories) was voluntarily submitted for evaluation before marketing. LITERATURE CITED 1. Center for Disease Control. 1975. Specifications and evaluation methods for immunological and microbiological reagents, vol. 1: Bacterial, fungal, and parasitic, 4th ed. Center for Disease Control, Atlanta, Ga. 2. Edwards, P. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis, Minn. 3. Kauffmann, F. 1972. Serological diagnosis of Salmonella species Kauffmann-White schema, 1st ed. The Williams & Wilkins Co., Baltimore, Md. 4. LeMinor, L., R. Rohde, Ch. Chari6-Marsaines, and C. Coynault. 1971. etude sur les rapports antig6niques entre le groupe 0:54 et d'autres groupes 0 de Salmonella. Ann. Inst. Pasteur Paris 121:447-463.
