Rabbits and Humans. serotype 4d (J. Donker-Voet), and Staphylococcus. aureus strain 1641y (Center for Disease Control

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1 APPLIED MICROBIOLOGY, June 1974, p Vol. 27, No. 6 Copyright ( 1974 American Society for Microbiology Printed in U.S.A. Immune Response to Listeria monocytogenes in Rabbits and Humans SANDRA A. LARSEN, JOHN C. FEELEY, AND WALLIS L. JONES Center for Disease Control, Atlanta, Georgia 3333 Received for publication 11 March 1974 Rabbits were immunized with listeria antigens, staphylococcus antigen, or with both, and the course of their immune response was monitored. Antibodies to Listeria and Staphylococcus were produced in both immunoglobulin M (IgM) and immunoglobulin G (IgG) classes in response to inoculation with the specific antigen. Cross-responses occurred in rabbits injected only with Listeria or only with Staphylococcus, as well as in rabbits injected with both antigens. L. monocytogenes serotype 4d appeared to be immunologically distinct from L. monocytogenes serotype 2 and its cross-reaction with. Human sera from bacteriologically confirmed cases of listeriosis were examined to determine the nature of the immunological response of man to Listeria. In the sera studied, IgM was the predominant antibody produced to Listeria, whereas cross-reactions with Staphylococcus were observed in both the IgM and the IgG antibody classes. The nature of the antibody response of humans and animals to Listeria monocytogenes has been difficult to ascertain. Complications are encountered because of serological crossreactions with other organisms (1, 15-18, 2, 26) and perhaps because of a difference in response to listeria infections between man and animals. In 1955, Osebold and Sawyer (14) found that 77% of all human sera they tested agglutinated L. monocytogenes. They speculated that these agglutinins resulted from a cross-reaction from some other antigenic stimulus, probably Staphylococcus. Since then, many investigators have attempted to devise a specific serological test for listeriosis (8, 12, 18, 19, 24) Ȯsebold and Aalund (12) and Szatalowicz et al. (24) suggested that reduction of the immunoglobulin M (IgM) of sera with 2-mercaptoethanol (2-ME) would solve the problems of cross-reactions and still enable detection of all listeria-positive sera. In our previous study (8), we found this not to be the case, as have Brandis et al. (5) and Seeliger and Emmerling (19). This present study was begun to extend our previous findings and to further examine the antibody response to Listeria of rabbits under controlled conditions and of man with naturally acquired infections. MATERIALS AND METHODS. L. monocytogenes strain 5348, serotype 2 (H. P. R. Seeliger), L. monocytogenes strain 21, 5 serotype 4d (J. Donker-Voet), and Staphylococcus aureus strain 1641y (Center for Disease Control [CDC]) were used to prepare the trypsin- and nontrypsin-treated antigens used in the immunization study. Strain 7973, serotype la (H. P. R. Seeliger), and strain F4, serotype 4b (J. Donker-Voet), were used to prepare the trypsinized listeria antigens used in the study of human responses. Trypsinized antigens were prepared by the method of Osebold et al. (13), as modified by Larsen and Jones (8). The non-trypsinized antigens were prepared in the same manner but with the trypsin treatment omitted. were stored at a concentration that, when diluted 1:2 in.85% saline, would read 5 to 53% transmission on a Coleman Junior spectrophotometer at 43 nm. These antigens diluted 1: 2 in saline were used both for rabbit inoculation and the agglutination tests. Animals and immunizations. New Zealand white rabbits weighing from 3 to 4 kg were used for immunization; essentially, the inoculation schedule of Seeliger (18) was followed. All rabbits were prebled, and only those having a titer of 2 or less with the listeria or staphylococcus antigens were immunized. Three groups composed of four rabbits each were inoculated as shown in Table 1. Rabbits were injected every other day and were bled before each injection. During the periods when the rabbits were not immunized, they were bled weekly. Human sera. The human sera studied were submitted to CDC for listeriosis serology from patients with bacteriologically confirmed cases. Gel filtration on Sephadex G-. A 4-ml volume of rabbit serum from selected bleedings was passed through a column (1.5 by 9 cm) of Sephadex G- (6) and eluted with a.1 M tris(hydroxymethyl)- aminomethane (Tris)-hydrochloric acid buffer (ph

2 6 LARSEN, FEELEY, AND JONES APPL. MICROBIOL. Dosage TABLE 1. Inoculation schedule Group I Group II Group III 1 and 2a 3 and 4 5 and 6 7 and 8 9 and 1 11 and 12 Antigen-first series, graduated,,.,., doses from.5 ml to 5. ml trypsin no trypsin trypsin no trypsin no trypsin no trypsin 5-Month rest Antigen-second series, 2 doses.,,,. serotype 4d, of 1. ml no trypsin no trypsin no trypsin no trypsin no trypsin no trypsin 2-Month rest Antigen-third series, 5 doses,,,,. serotype 4d of 1. ml no trypsin no trypsin no trvpsin no trypsin no trypsin no trypsin a Rabbit numbers. 7.5). Fractions of 1 ml were collected. The protein sodium barbital buffer as electrolyte. Immunodiffusion after electrophoresis was developed against the concentration was monitored at 28 nm with a Uvicord spectrophotometer (LKB, Sweden). Gel filtration of human serum involved passing 2.5 ml of serum Human serum fractions were diffused against goat above antisera. through the above column and eluting again with the anti-human IgM, H chain-specific (CDC), goat antihuman IgG, Fc fragment-specific (Hyland Labo- Tris-hydrochloric acid buffer Agglutination tests. Agglutinins were determined ratories, Costa Mesa, Calif.,) antisera in immunodiffusion tests and after electrophoresis. on serum and serum fractions by combining the method of Osebold and Aalund (12) with the microtiter method of Gaultney et al. (7). In brief,.85% sera were absorbed with packed cells of L. monocyto- Absorption. Various fractions of rabbit and human saline containing.5% safranin dye was used as genes serotypes la, 2, or 4d or with. serum diluent in.5-ml amounts. For whole serum, a Fractions were absorbed at 5 C for 2 h and then 1:6. dilution was prepared, and.1 ml of this overnight at 4 C; they were then centrifuged and dilution was placed in the first well of the V-bottom tested for agglutinins. polystyrene microtiter plate. For serum fractions, a.1-ml volume of each fraction was placed in the first RESULTS well. Fractions 28 through 36, then alternate fractions from 38 through 7, were tested in this manner. All All rabbits responded to the listeria and dilutions were made with.5-ml loops on a Cooke staphylococcus antigens immediately after the automatic diluter (Cooke Engineering Co., Alexandria, Va.). The listeria and staphylococcus antigens trypsin- and nontrypsin-treated antigens of L. first injection. Sera were tested with both the were diluted 1:2 in.85% saline containing safranin monocytogenes serotypes 2 and 4d and. Whether trypsinized or nontrypsinized ; then.5 ml of the antigen was added to each well. Therefore, the final dilution for the whole serum in the first well was 1:, and the final dilution for the antigens were used in the agglutination test serum fraction in the first well was 1: 2. All plates made no substantial difference in the final titer were incubated at 37 C overnight, and then refrigerated for 1 h at 4 C before being read. tiserum. The antiserum of rabbits immunized or in the degree of cross-reactivity of the an- 2-ME treatment. For 2-ME treatment of whole with the trypsinized listeria antigens had serum and serum fractions, the agglutination test was slightly higher titer (usually one well) and less performed as described above, except that a.1 M cross-reactivity than did the sera of rabbits dilution of 2-ME made in.85% saline containing immunized with the nontrypsinized listeria antigens. Titers with the homologous antigen safranin was used for serum diluent. Antigen for the first well row was also diluted 1:2 in.85% saline containing 2-ME and safranin. Antigen for all other reached a peak level of to 6,4 after the wells was diluted 1:2 in.85% saline containing fourth injection (Table 2). safranin without 2-ME. A.5-ml drop of antigen The distribution of the immune response to was added to each well as above. the homologous listeria antigen after the initial, Immunodiffusion and immunoelectrophoresis. second, and final injections of the first immunization series is shown in Fig. 1. As determined All rabbit serum fractions were checked for homogeneity by immunodiffusion in 1.5% Noble agar against by immunodiffusion and immunoelectrophoresis, fractions 3, 31, and 32 were the only ones goat class-specific anti-rabbit immunoglobulin G (IgG), anti-rabbit IgM, and polyvalent anti-igg, where only IgM antibodies were detected. Fractions 33 through 44 contained mixtures of both -IgM, and -IgA antisera (Cappel Laboratories, Downingtown, Pa.). The Noble agar was prepared in a solution of 75 ml of.5 M sodium barbital buffer (ph IgM and IgG antibodies. In fractions 45 through 8.6) and ml of distilled water. Selected fractions 55 only IgG antibodies were detected, whereas were also checked for homogeneity by immunoelectrophoresis in the above Noble agar with the.5 M immunoglobulin A (IgA) fractions 56 through 78 contained both IgG and antibodies.

3 VOL. 27, 1974 IgM antibodies predominated in the response after the initial injection as confirmed by reduction of the fractions with 2-ME. The antibody response after the second injection was still mainly IgM, but IgG antibodies were beginning to appear. After the final injection, the response was about equal in both IgM and IgG antibody fractions (Table 3). IgM antibodies accounted for the cross-reactions in this first immunization series (Table 3); they were limited to fractions 32 through 4. Cross-reactions appeared after the initial injection at agglutination titer levels less than or equal to in whole sera and remained at this IMMUNE RESPONSE TO L. MONOCYTOGENES 7 level throughout the first series (Table 2). All cross-reactions could be removed by absorptions with without altering the homologous titer. Rabbits immunized in this first series with S. aureus responded as did those immunized with Listeria (Table 2). Cross-reactions with the serotype 2 strain were limited to the IgM antibody fractions, and no cross-reactions were detected with the serotype 4d strain (Tables 2 and 3). During a 5-month period in which the rabbits were not immunized, the homologous titers of the sera from all rabbits began to drop steadily 2 TABLE 2. Agglutination titers for untreated and 2-ME-treated sera after final inoculation of each series Determinations + a III + + serotype 4d Series 1 Untreated sera: 2-ME-reduced sera: Series 2 Untreated sera: 2-ME-reduced sera: Series 3 Untreated sera: 2-ME-reduced sera: a Immunogen. < < 5 < < < < 5 8 < < < 5 < < < 8 Titers < < < 4 <

4 8 LARSEN, FEELEY, AND JONES APPL. MICROBIOL. gous agglutination titers increased to levels of to. Fractionation and 2-ME reduction of serum fractions obtained before the first immunization after the 5-month rest revealed that cross-reactions with Staphylococcus were now 1.- Initial weeks after the final injection and continued to drop until reaching a minimal titer of or at 1 month after the last inoculation. This titer remained stable during the remainder of the period. However, during this period, heterolo E.6-5-, a _ Second g.6 xo X Final co E.6- N.5-,, -- absorption at 28 nm - titer of whole fractions --- titer of 2-me reduced fractions 128 * Fraction Number FIG. 1. Antibody response to serotype 2 after initial, second, and final injections. 74

5 VOL. 27, 1974 TABLE 3. Determinations IMMUNE RESPONSE TO L. MONOCYTOGENES Specific antibody responses after final injection in each series as determined on serum fractions Antibody response ' Serotype 4D serotype 4d Series 1 IgM, serotype IgM, serotype 4d + IgM, 4 _ + IgG, serotype 2 + _ IgG, serotype 4d _ + IgG, + Series 2 IgM, serotype IgM, serotype 4d + + ± IgM, IgG, serotype _ + _ ± IgG, serotype 4d _ IgG, Series 3 IgM, serotype IgM, serotype 4d IgM, IgG, serotype IgG, serotype 4d + + _ IgG, a Immunogen. in both the IgM and IgG antibody fractions of the sera. IgM antibody predominated in the cross-reactions with the heterologous listeria antigen. The first injection with the homologous listeria antigen in the second immunization series resulted in an immediate rise in titer. This titer was at a level comparable to the titer after the fourth injection of the first immunization series ( to ). Cross-reactions remained at the level established during the rest period or rose one dilution (Table 2). After fractionation of the sera and reduction of the serum fractions with 2-ME, the homologous reactions were found to be in both the IgM and IgG antibody fractions (Table 3). IgM antibody continued to predominate in cross-reactions with the heterologous listeria antigens, whereas both IgM and IgG antibodies were detected in response to Staphylococcus (Table 3). The sera of the rabbits injected in the first series with Listeria and at this point with Staphylococcus demonstrated a rise in titer to. The titer increased to a level no greater than originally seen in rabbits inoculated for the first time with Staphylococcus (Table 2). Separation of the serum on Sephadex G- and reduction of the fractions with 2-ME indicated that IgM and IgG antibodies predominated in responses to Staphylococcus and to 9 the original antigen, whereas IgM antibodies predominated in the response to the heterologous listeria antigen (Table 3). The sera of rabbits injected only with Staphylococcus demonstrated a rise in titer to S. aureus to a level of to (Table 2). A rise in cross-reaction titers to both listeria antigens was observed, and the antibody was found to be IgM (Tables 2 and 3). Rabbits injected in the first immunization series with and now with L. monocytogenes serotype 4d responded immediately with agglutinins to both serotype 4d and to Staphylococcus (Table 2). When these sera were reduced with 2-ME, IgM antibodies were found to predominate in the responses to both listeria antigens, whereas the IgM and IgG antibodies to the original staphylococcus antigen remained constant (Table 3). None of the rabbits were immunized for a period of 7 weeks. The new homologous reactions dropped to titers of to 4, whereas cross-reactions and titers remaining from the first immunization series were constant. Rabbits injected in the first and second series with homologous antigen responded in the third series with the homologous antigen as they had in the second series (Table 2). The response was now in both IgM and IgG antibody fractions, but with IgG antibodies predominant (Table 3).

6 11 LARSEN, FEELEY, AND JONES APPL. MICROBIOL. An increase in the cross-reaction to the heterologous listeria antigen and to the staphylococcus antigen was observed after several injections (Table 2). IgM antibody initially predominated in the cross-reaction to the heterologous antigen, whereas both IgM and IgG antibodies were detected in staphylococcus cross-reactions. After the fifth injection of this third series, IgG antibody was also detected in the cross-reactions to the heterologous listeria antigens (Table 3). Absorption of these sera, which were produced only to L. monocytogenes, with S. aureus removed the heterologous listeria crossreactions as well as the staphylococcus crossreaction without reducing the homologous titer (Table 4A and B.). Rabbits injected in the first series with Listeria and in the second and third series with Staphylococcus responded initially in the third series by an increase in titer to both Staphylococcus and the original listeria antigen. By the fifth injection with Staphylococcus, crossreaction to the heterologous listeria antigen TABLE 4. appeared (Table 2). All responses were in both IgM and IgG antibody classes. Results of crossabsorptions of serum fractions exhibiting the greatest degree of cross-reactivity are shown in Table 4C and D. Rabbits injected in all three series with S. aureus responded by the final injection of the third series with IgM antibodies to serotype 4d, IgM and IgG antibodies to serotype 2, and IgM but predominantly IgG antibodies to (Tables 2 and 3). Cross-absorption results are shown in Table 4E. Rabbits injected in the first series with S. aureus and then in the second and third series with L.' monocytogenes serotype 4d responded with IgM and IgG antibodies to both listeria antigens and to Staphylococcus (Tables 2 and 3). Results of cross-absorptions are shown in Table 4F. Fractionation of human sera on Sephadex G- and immunodiffusion and immunoelectrophoresis of the fractions indicated that fractions 28 through 35 contained IgM antibodies; Absorption of serum fractions from bleedings after final injections of the third series Agglutination titer Titer after absorption with: Determination Dnbefore absorption beforenabsrtion te A. Serum against L. monocytogenes serotype < < 2 < <2 B. Serum against L. monocytogenes serotype 4d 8 <2 8 <2 1,24 1,24 < 2 1, <2 C. Serum against L. monocytogenes serotype 2 and 64 < <2 64 1, ,24 32 D. Serum against L. monocytogenes serotype 4d and 32 <2 32 < <2 64 1, ,24 8 E. Serum against 32 < < ,48 1,24 2,48 64 F. Serum against and L. monocytogenes serotype 4d 6 <2 6 <2 2,48 2,48 < 2 2,48 1, <2

7 VoL 27, 1974 fractions 36 through 41 contained predominantly IgM antibodies with slight IgG antibody contamination; and fractions 42 through 52 contained mainly IgG antibodies (Fig. 2). All of the 1 human sera examined exhibited a similar agglutination pattern. With the listeria antigen, the agglutination peak was reached in fractions 32 through 37. A biphasic pattern of agglutination was observed with the staphylococcus antigen. The first peak occurred in fractions 32 through 37 and the second peak occurred in fractions 47 through 56 (Fig. 2). Of the two sets of paired sera examined, no differences in agglutination titers could be observed between the serum obtained from the first and second bleedings. The serum fractions of three of the sera examined exhibited agglutination with the listteria antigens after 2-ME reduction. This agglutination reaction appeared in fractions 47 through 58. Two of the sera with IgG antibodies were from one patient. All titers to the listeria antigens of the reduced serum fractions were at levels no greater than 4. All 1 sera agglutinated with the staphylococcus antigen after reduction with 2-ME. Agglutination began in fraction 42 and ended in fraction 66. Titers were usually 16 or less. Absorption of serum fractions with S. aureus completely removed the antibodies to S. aureus, whereas the titer to L. monocytogenes remained unaltered. DISCUSSION Rabbits responded to both the trypsinized and nontrypsinized L. monocytogenes antigens after only one injection. The IgM antibody l.6- ' IMMUNE RESPONSE TO L. MONOCYTOGENES response appeared after the initial inoculation, and within a week it was followed by IgG agglutinins. However, the IgM antibody was not transient, as it can be in response to some antigens (3, 4, 22, ), but remained the dominant antibody throughout the study. Armstrong and Sword (2) reported similar results with their hyperimmunized rabbits, whereas Aalund et al. (1) reported that in sheep 5 to 66% of the agglutination activity resulted from the presence of IgG antibodies. From cross-immunizations and cross-absorptions of these sera, we hoped to better understand the antigenic relationship between L. monocytogenes and. All rabbits immunized only with Listeria developed both IgM and IgG antibodies to Staphylococcus. Absorption of these sera with Staphylococcus removed the staphylococcus agglutinins without reducing the homologous titer (Table 4A and 4B). Welshimer (26) also found this to be true of L. monocytogenes antisera absorbed with Staphylococcus epidermidis. Aalund et al. (1) found that sheep immunized with L. monocytogenes and then with responded with antibodies to after the first injection with. Welshimer (27) found that rabbits immunized with L. monocytogenes and then with S. epidermidis responded immedi-, ately with agglutinins to S. epidermidis. Our rabbits injected first with listeria antigens and then with staphylococcus antigen responded with both IgM and IgG antibodies to Staphylococcus. Absorption of these sera with Staphylococcus removed only the staphylococcus agglutinins, whereas absorption with listeria -absorption at 28 nm -titer to la antigen - titer to 4b antigen -.o- titer to staphylococcus antigen i /t--128 ; *D 5 R!6 5; S %~~~~~~~~~d [28 ~ Fraction Number FIG. 2. Absorption and agglutination patterns of a Listeria-positive human serum.

8 112 LARSEN, FEELEY, AND JONES cells removed only the listeria agglutinins (Table 4C and 4D). Welshimer (27) found that absorption of sera produced against S. epidermidis with L. monocytogenes significantly reduced the titer to S. epidermidis. Although we did not have this occur in the absorption of our antisera to S. aureus (Table 4E), we did find that antisera produced to and then to L. monocytogenes serotype 4d when absorbed with had both the agglutinins to and to L. monocytogenes serotype 2 eliminated. Conversely, absorption of these sera with serotype 2 cells removed all agglutinins to serotype 2 and reduced the staphylococcus agglutination titer significantly without affecting the titer to serotype 4d (Table 4F). Aalund et al. (1) found serotype 4b to be immunologically independent of the cross-reaction between serotypes 1 and 2 and -in the IgG antibody class; we agree with this finding and extend it to cover serotype 4d. In our previous study of human sera (8), we found that the staphylococcus cross-reaction was more prevalent with the sera of patients with serotype la or lb infections than with the sera of patients with serotype 4b infections. Absorption of these sera with Staphylococcus eliminated the agglutinins for Staphylococcus but did not alter the listeria titer. With normal human sera, we found that 5.4% agglutinated with Listeria serotype lb and 6.% agglutinated with Staphylococcus. Absorption of these sera with Staphylococcus completely eliminated agglutinins for both Staphylococcus and Listeria serotype lb. Also in our previous study (8), we found the human response to Listeria to be almost exclusively an IgM antibody response. Our closer examination of 1 human sera in this study confirmed our previous finding. We also found the human response to Staphylococcus to be in both immunoglobulin classes. Therefore, reduction of sera with 2-ME did not eliminate the staphylococcus cross-reaction but probably did inactivate true IgM antibodies to Listeria. To explain the predominance of IgM antibodies in listeria antisera, Armstrong and Sword (2) theorized that, since Listeria grow within macrophages (9, 11), small amounts of antigen are constantly released to stimulate the IgM antibody response. Uhr and Finkelstein () suggested that antigenic presence is necessary for continued synthesis of IgM antibodies. Svehag and Mandel (22, 23) reported that continued synthesis of IgM antibodies appears to depend on the size of the antigen dose, APPL. MICROBIOL. therefore reaffirming the opinion of Armstrong and Sword (2). However, Bauer and Stavitsky (3) postulated that, since somatic (polysaccharide) antigens stimulate the formation of easily detectable IgM antibodies, these antibodies are most commonly seen. Since heat-killed antigens injected in rather large doses were used in our rabbit study, we feel that the Bauer and Stavitsky theory (3) may be more correct in this instance. Perhaps in human infections with Listeria the explanation of Armstrong and Sword (2) is more applicable. Findings from this study substantiate those from previous studies. First, we reaffirmed that there is a definite antigenic relationshp between L. monocytogenes serotypes la, lb, and 2, and. Perhaps one antigenic site is responsible for the cross-reaction with Staphylococcus, and another antigenic site is responsible for Listeria-specific antibody production. Second, we confirmed that IgM antibodies predominate in the human agglutination response to Listeria throughout the course of the disease. Therefore, the presence of IgM or the absence of IgG antibodies to Listeria should not be considered indicative of the disease stage as it is for some other diseases (21). ACKNOWLEDGMENTS We would like to express our appreciation to Edith A. Hambie and to Fred T. McKinney for their valuable technical assistance. LITERATURE CITED 1. Aalund, O., J. W. Osebold, F. A. Murphy, and R. A. DiCapua Antibody heterogeneity in experimental listeriosis. J. Immunol. 97: Armstrong, A. S., and C. P. Sword Antibody responses in experimental infections with Listeria monocytogenes. J. Immunol. 98: Bauer, D. C., and A. B. Stavitsky On the different molecular forms of antibody synthesized by rabbits during the early response to a single injection of protein and cellular antigens. Proc. Nat. Acad. Sci. U.S.A. 47: Benedict, A. A., R. J. Brown, and R. Ayengar Physical properties of antibody to bovine serum albumin as demonstrated by hemagglutination. J. Exp. Med. 115: Brandis, H., H. Werner, and A. Viebahn Erfahrungen mit der chemischen Immunoglobulin-Differenzierung bie der Serodiagnostik der Listeriose. Klin. Wochenschr. 49: Flodin, P Dextran gels and their applications in gel filtration. Pharmacia, Uppsala, Sweden. 7. Gaultney, J. B., R. D. Wende, and R. P. Williams Microagglutination procedures for febrile agglutination tests. Appl. Microbiol. 22: Larsen, S. A., and W. L. Jones Evaluation and standardization of an agglutination test for human listeriosis. Appl. Microbiol. 24: Mackaness, G. B The immunological basis of acquired cellular resistance. J. Exp. Med. 12:15-12.

9 VOL. 27, 1974 IMMUNE RESPONSE TO L. MONOCYTOGENES 1. Neter, E., H. Anzai, and E. A. Gorzynski Identification of an antigen common to L. monocytogenes and other bacteria. Proc. Soc. Exp. Biol. Med. 15: Njoku-Obi, A. N., and J. W. Osebold Studies on mechanism of immunity in listeriosis. I. Interaction of peritoneal exudate cells from sheep with Listeria monocytogenes in vitro. J. Immunol. 89: Osebold, J. W., and O. Aalund Interpretation of serum agglutinating antibodies to Listeria monocytogenes by immunoglobulin differentiation. J. Infect. Dis. 118: Osebold, J. W.,. Aalund, and C. E. Chrisp Chemical and immunological composition of surface structures of Listeria monocytogenes. J. Bacteriol. 89: Osebold, J. W., and M. T. Sawyer Agglutinating antibodies for Listeria monocytogenes in human serum. J. Bacteriol. 7: Potel, J Wo stehen wir im Wissen (iber die Listeriose? Medizinische (Stuttgart) 28: Rantz, L. A., E. Randall, and A. Zuckerman Hemolysis and hemagglutination by normal and immune serums of erythrocytes treated with a non-species specific bacterial substance. J. Infect. Dis. 98: Seeliger, H. P. R Serologische Kreuzreaction zwischen Listeria monocytogenes und Enterokokken. Z. Hyg. Infectionskr. 141: Seeliger, H. P. R., Listeriosis. Hafner Publishing 113 Co., Inc., New York. 19. Seeliger, H. P. R., and P. Emmerling Zum Vorkommen 2-Mercaptoathanol-resistenter und empfindlicher Listeria-Agglutinine in Human- und Tierseren. Z. Med. Mikrobiol. Immunol. 155: Seeliger, H. P. R., and F. Sulzbacher Antigenic relationships between Listeria monocytogenes and Staphylococcus aureus. Can. J. Microbiol. 2: Spink, W. W Host-parasite relationship in brucellosis. Lancet 2: Svehag, S., and B. Mandel The formation and properties of poliovirus-neutralizing antibody. I. 19S and 7S antibody formation: differences in kinetics and antigen dose requirement for induction. J. Exp. Med. 119: Svehag, S., and B. Mandel The formation and properties of polio-virus-neutralizing antibody. II. 19S and 7S antibody formation: differences in antigen dose requirement for sustained synthesis, anamnesis, and sensitivity to X-irradiation. J. Exp. Med. 119: Szatalowicz, F. T., D. C. Blenden, and M. S. Khan Occurrence of listeria antibodies in select occupational groups. Can. J. Publ. Health. 61: Uhr, J. W., and M. S. Finkelstein Antibody formation. IV. Formation of rapidly and slowly sedimenting antibodies and immunological memory to bacteriophage ox 174. J. Exp. Med. 117: Welshimer, H. J Staphylococcal antibody production in response to injections with Listeria monocytogenes. J. Bacteriol. 79: Welshimer, H. J Some serological reactions observed with Listeria monocytogenes p Second Symposium on Listeria Infections. Bozeman, Montana. Montana State College, Bozeman. Downloaded from on May 8, 218 by guest