JOURNAL OF CLINICAL MICROBIOLOGY, June 1991, p. 1243-1248

Vol. 29, No. 6

0095-1137/91/061243-06$02.00/0

Class-Specific Immune Response to Yersinia enterocolitica Serotype 09 Antigens as Determined by Enzyme-Linked Immunosorbent Assay LUIS FERNANDEZ-LAGO,* F. JAVIER SANTOYO, NIEVES VIZCAINO, AND ANDRES CHORDI Department of Microbiology, Faculty of Pharmacy, University of Salamanca, 37007 Salamanca, Spain

Received 9 November 1990/Accepted 27 March 1991

An enzyme-linked immunosorbent assay (ELISA) using lipopolysaccharide (S-LPS) as the antigen was used to analyze the antibody response in rabbits orogastrically and intravenously infected with virulent (plasmidbearing) Yersinia enterocolitica 09 strains (pYV+) and with the avirulent (plasmid-cured) derivatives (pYV-). A significative response of immunoglobulin G (IgG), IgA, and IgM antibodies against the S-LPS antigen was evident in sera from the rabbits orogastrically infected with pYV+ strains. This immune response was stronger and persisted longer than those obtained with the corresponding pYV- strains. In contrast, few differences were observed in the titers and evolution of IgG, IgA, and IgM antibodies against the S-LPS antigen in rabbits intravenously infected with pYV+ and pYV- strains. These results suggest that the necessity of the virulence plasmid for the establishment of infection by Y. enterocolitica serotype 09 is conditioned by the infection route used. When the S-LPS ELISA was compared with the radial immunodiffusion test using the native hapten as the antigen, the results showed that the ELISA technique was more sensitive. However, only those sera obtained between 2 and 8 weeks postinfection from rabbits intravenously infected with plasmid-bearing strains were positive in the radial immunodiffusion test. In recent years several authors have reported a marked increase in the frequency of infections due to Yersinia enterocolitica (12). Symptoms commonly ascribed to these infections include acute ileitis and mesenteric lymphadenitis, in rare cases complicated by septicemia (36). The pathogenic potential of this microorganism is partially due to the expression of determinants encoded on a 40- to 50-MDa virulenceassociated plasmid. Bacterial strains harboring this plasmid express a series of different temperature-dependent phenotypes: calcium-dependent growth (13), autoagglutination activity (24), production of V and W antigens (31), production of Yersinia outer membrane proteins (YOMPs) (4, 5, 33), increased cell surface hydrophobicity (23), and resistance to the bactericidal effect of serum (30). In addition to these plasmid-mediated virulence factors, other chromosomally encoded determinants also appear to be of importance in the pathogenicity of Y. enterocolitica (27, 28). Infections caused by Y. enterocolitica are generally associated with a high humoral response. Isolation of the bacteria from feces is not always feasible. Accordingly, diagnosis often depends on serological assays (1, 16, 17). By the immunoblotting technique, specific antibodies directed against plasmid-encoded YOMPs and/or released proteins have been found both in sera from patients (25) and in sera obtained from animals experimentally infected with this micoorganism (19). However, with the enzyme-linked immunosorbent assay (ELISA), no great differences have been observed between results with extracts obtained for use as antigens from plasmid-positive and plasmid-cured derivatives of the same strain, indicating that antibodies to chromosomally encoded structures dominate in the course of Y. enterocolitica infection (15). One of these is the lipopolysaccharide (S-LPS) antigen. Antibodies against the S-LPS molecule have been detected by ELISA in the sera of patients

*

with yersiniosis, and the usefulness of the ELISA using S-LPS as the antigen, in terms of specificity and sensitivity, has been well established (14, 16, 18). In this study, by using a rabbit experimental model, the ELISA was used to examine the kinetics of antibodies of the immunoglobulin G (IgG), IgA, and IgM classes against Y. enterocolitica serotype 09 S-LPS antigen in sera obtained at different stages from rabbits orogastrically and intravenously infected with three Y. enterocolitica 09 plasmid virulenceassociated and plasmid-cured derived strains. A further objective was to compare these results with those recorded by studying the sera by the radial immunodiffusion (RID) test with the polysaccharide native hapten (NH) obtained from Y. enterocolitica 09 as the antigen. MATERIALS AND METHODS

Bacterial cultures. Plasmid-bearing virulent (pYV+) Y. enterocolitica serotype 09 strains W830, W836, and W22708 were kindly provided by G. Wauters, Brussels, Belgium. Their characteristics and culture conditions have been described previously (2, 26). Plasmid-cured (pYV-) isogenic derivatives were obtained by repeated subcultivation of pYV+ parents on magnesium oxalate agar at 37°C (31). pYV+ strains were characterized with respect to their ability to undergo spontaneous autoagglutination (24) and calcium-dependent growth (20) and to bind crystal violet (3) and by plasmid screening via agarose gel electrophoresis (32). Cultures were stored as cell suspensions at -20°C in 50% glycerol and cultivated in tryptic soy broth and tryptic soy agar (BBL Microbiology Systems, Cockeysville, Md.) enriched with 0.3% yeast extract (Difco). Extraction and purification of S-LPS. S-LPS extraction from the smooth Y. enterocolitica serotype 09 strain W22708 with hot phenol-water was done by the method of Westphal et al. (39) with the modifications of Hurvell (21). Purification of crude S-LPS isolate in the phenolic phase (f5)

Corresponding author. 1243

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FERNANDEZ-LAGO ET AL.

was performed as follows. Fifteen-milligram batches of f5 were dissolved in 2.5 ml of double-distilled water (ddH2O)

and subjected to ultracentrifugation at 105,000 x g for 9 h at 4°C to yield S-LPS as a clear deposited gel. The pellet was dissolved in ddH20, which was then dialyzed and lyophilized. To reduce the protein content, 100 mg of this antigen was dissolved in 10 ml of 0.15 M phosphate-buffered saline (PBS), pH 7.1, and 5 mg of proteinase K (Sigma Chemical Co., St. Louis, Mo.) was added (9). The mixture was held at 50°C for 2 h and at 25°C for 48 h. Five volumes of methanol consisting of 1% methanol saturated with sodium acetate were added, and the S-LPS was sedimented by centrifugation at 10,000 x g for 20 min. The pellet was redissolved in PBS, and the procedure was repeated once. The precipitate finally obtained was dissolved in ddH20, dialyzed against ddH20, and lyophilized. A 50-mg portion of this preparation was dissolved in ddH20 and fractionated on a PM-30 membrane (Diaflo). The retained volume containing S-LPS, detected by double gel diffusion in agarose borate containing 10% NaCl (11), was dialyzed against ddH20 and then lyophilized. All preparations were analyzed for protein by the Bradford method (6) and for 2-keto-3-deoxyoctulosonic acid (KDO) by the method described by Warren (37). Preparations of S-LPS used in this work contained, as a percentage of dry weight, 1.17% KDO and 1.86% protein. NH extraction and purification. To obtain NH from Y. enterocolitica serotype 09 strain W22708, the technique described previously by Fernandez-Lago et al. (11) was followed. Purification of the second precipitate, the NHenriched fraction, was carried out by a previously described procedure (10). Preparations of NH used in the RID test contained, as a percentage of dry weight, 0.03% protein. Experimental infection of rabbits. In the present study, 24 New Zealand White rabbits (mean weight + standard deviation, 2.25 + 0.25 kg) were used. They were divided into two animals per group in accordance with the infection route used and the strain employed (Y. enterocolitica serotype 09 strains W22708, W830, and W836). All infections were initiated with freshly thawed Y. enterocolitica stock. For inoculum preparation, bacteria (pYV+ or pYV- derivatives) were grown with agitation in tryptic soy broth enriched with 0.3% yeast extract at 25°C for 16 h. Next, an equal volume of the same medium was added, and the culture was incubated for 4 h at 37°C. For orogastric

infection, bacterial cells were harvested by centrifugation, suspended in NaHCO3 (10%, wt/vol), and adjusted to a final concentration of 2 x 109 CFU/ml. Next, 10 ml of this suspension was given by the orogastric route through a feeding tube (diameter, 4 mm) passed into the stomach. Two days later, the inoculation was repeated. The rabbits were deprived of food for 18 h before infection. For intravenous infection, bacterial cells were suspended in sterile physiological saline (0.9%) at a concentration of 108 CFU/ml, and 2 ml of this suspension was injected into the marginal vein. One week later, the inoculation of each rabbit was repeated under same conditions. In both infection models, blood was collected weekly over a 16-week period. The rabbits did not show any agglutinins before infection. Immune sera. Immunization of rabbits with strain W22708(pYV+) and its pYV-derivative was performed as follows (38). Bacteria were grown in tryptic soy broth at 25°C for 14 to 18 h, washed once in saline, and heated at 120°C for 1.0 h. The antigens were adjusted to 109 cells per ml, and 1.0, 1.0, 1.5, 1.5, and 2.0 ml were injected intrave-

J. CLIN. MICROBIOL.

nously at 3- to 4-day intervals. The animals were bled weekly and at 6 days after the final injection. Serological tests. For double gel diffusion, the gels were prepared with 1% agarose in borate buffer (pH 8.3) containing 10% NaCI (11). The RID test was performed by a previously described technique (10, 11, 22); in each case, five different concentrations of purified NH (2, 4, 8, 16, and 32 ,ug/ml) were used. In this test, sera which developed a precipitation ring in less than 3 to 4 h were considered positive. ELISA procedure. The ELISA was done as described previously (10). The test was performed with disposable polystyrene microtiter plates with flat-bottomed wells (Linbro; Flow Laboratories Inc., McLean, Va.). As the antigen, S-LPS purified from Y. enterocolitica serotype 09 strain W22708 at a concentration of 1 ,ug (dry weight) per ml was used. Goat anti-rabbit peroxidase conjugates with anti-IgG, anti-IgA, and anti-IgM (Fc) specificity (Nordic Immunological Laboratories, Tilburg, The Netherlands) were used at a 1:3,000 dilution for anti-IgG and anti-IgA and a 1:1,500 dilution for anti-IgM. o-Phenylenediamine (Sigma) was used as a substrate. After 10 min the enzymatic reaction was stopped with 3 M H2SO4, and the A492 was read on an STL-Labinstruments EAR 400 FT. As negative controls, sera obtained from 20 healthy rabbits were used, and the optical density values were subtracted from those of the sera of infected animals. Antibody titers in serial dilutions of the test sera were determined. RESULTS ELISA titers of antibody to Y. enterocolitica serotype 09 S-LPS antigen in orogastrically infected rabbits. Figure 1 shows the means of the reciprocal ELISA titers of antibody against S-LPS antigen of two rabbits orogastrically infected with the Y. enterocolitica serotype 09 pYV+ strain W22708. Antibodies of the IgG class at a significative titer (640) were demonstrated at 2 weeks postinfection, reached a maximal level (1,280) between 3 and 4 weeks after infection, and thereafter decreased progressively. Antibodies of the IgA and IgM classes specific for this antigen were also demonstrated (Fig. 1B and C) at 2 weeks postinfection. Maximal titers of 40 and 20, respectively, were observed. In contrast, a weak humoral response was observed in rabbits orogastrically infected with plasmid-cured derivatives (pYV-) of the same strain. Maximal titers of antibody of the IgG class equal to or less than 40 were found only between 2 and 6 weeks postinfection. Specific antibodies of the IgA and IgM classes were practically negligible (Fig. 1). Similar behavior in the dynamics of the immune response was also observed in the rabbits orogastrically infected with strains W830 and W836. ELISA titers of antibody to Y. enterocolitica serotype 09 S-LPS antigen in intravenously infected rabbits. Experimental intravenous infection of rabbits with virulent (pYV+) and plasmid-cured derived (pYV-) Y. enterocolitica serotype 09 strains W22708, W830, and W836 gave a similar immune response against the S-LPS antigen (Fig. 2). Virulent Y. enterocolitica serotype 09 induced a maximal titer of specific IgG class antibodies (40,960) at 3 weeks postinfection and maintained this level over the following 5 weeks. Starting 4 weeks after this period, the titer slowly declined to about 2,560 (Fig. 2A). Specific IgA and IgM class antibodies against the S-LPS antigen were also detected. Maximal antibody titers of 1,280 and 640 were observed between the second and sixth and the second and fourth week postinfec-

VOL. 29, 1991 4

'I) 0. -I 0

IMMUNE RESPONSE TO Y. ENTEROCOLITICA ANTIGENS

1500

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1200 Co

-I

(0%

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0

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4

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6 8 10 12 14 16 18 20 Weeks after Infection

2

4

6 8 10 12 14 16 18 20 Weeks after Infection

4

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qc

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FIG. 1. Kinetics of IgG (A), IgA (B), and IgM (C) class antibodies to Y. enterocolitica serotype 09 S-LPS antigen determined by ELISA with sera of two rabbits orogastrically infected with 2 x 109 cells of the plasmid-positive (*) or plasmid-cured (Wg) Y. enterocolitica 09 strain W22708.

FIG. 2. Kinetics of IgG (A), IgA (B), and IgM (C) class antibodies to Y. enterocolitica serotype 09 S-LPS antigen determined by ELISA with sera of two rabbits intravenously infected with 108 cells of the plasmid-positive (*) or plasmid-cured (El) Y. enterocolitica 09 strain W22708.

tion, respectively (Fig. 2B and C). Finally, when these results were compared with those obtained with the intravenously injected rabbits infected with a plasmid-cured Y. enterocolitica 09 strain, a similar evolution was observed over time for the antibodies of the IgG, IgA, and IgM classes. However, the observed titers were 1 or 2 dilutions lower than those detected for experimental infection with virulence-associated plasmid-bearing cells (Fig. 2).

weeks postinfection from rabbits infected with plasmidbearing strains were positive (Table 1). In all of these sera, titers of specific antibodies of the IgG class (determined by ELISA) against the S-LPS antigen ranging between 10,240 and 40,960 were observed. In contrast, all of the sera obtained at different times from rabbits infected intravenously with plasmid-cured strains were negative in the RID test. Additionally, sera obtained from orogastrically infected rabbits were all negative in the RID test, regardless of whether the virulence plasmid was present in these strains (Table 1). Finally, the presence of antibodies reactive in the RID test

Determination of antibodies

against

NH from Y. enteroco-

litica serotype 09 by the RID test. When sera from intravenously infected rabbits were studied by the RID test using NH as the antigen, only those obtained between 2 and 8

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TABLE 2. RID results obtained with rabbit sera obtained after immunization with heat-killed cells of plasmid-bearing or plasmidnegative Y. enterocolitica serotype 09 strain W22708a

TABLE 1. RID results obtained with rabbit sera obtained after infection with plasmid-bearing or plasmid-negative Y. enterocolitica serotype 09 strain W22708' ELISA titer of antiPresence of virulence

plasmid + -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

+ -

Infection

n beb

i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. ORO ORO ORO ORO ORO ORO ORO ORO

No. of wks postinfection

1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 10 10 13 13 2 2 3 3 4 4 5 5

Reaction in RID testc -

-

+ -

+ -

+ -

+ -

+ -

+ -

+ -

-

-

-

-

-

-

body against S-LPS antigen d

IgG

640 80

IgA

IgM

40 10 640 160 1,280 320

80 10 640

10,240 2,560 20,480 10,240 40,960 1,280 640 20,480 40,960 1,280 320 20,480 40,960 1,280 160 20,480 320 40,960 160 20,480 320 40,960 80 20,480 160 20,480 80 10,240 160 20,480 80 10,240 640 20

80

1,280 20 1,280 40 640 20

80 80 40

MICROBIOL.

320 640

640 640 640 320 320 320 160 80 80 80 80 40 20 40 20 20 10 20 20 10 10 10 10

In the RID test, NH from Y. enterocolitica serotype 09 strain W22708 used as the antigen. b See Materials and Methods. i.v., intravenous; ORO, orogastric. ' Each serum sample was tested with five concentrations of NH (2, 4, 8, 16, and 32 ,ug/ml). Positive reactions (+) were recorded in less than 4 h. d Titers are expressed as reciprocals. a

was

against the NH from Y. enterocolitica serotype 09 was studied with four serum samples obtained from rabbits immunized with plasmid-bearing or plasmid-cured heatkilled cells of Y. enterocolitica W22708. ELISA titers of antibody of the IgG class against S-LPS antigen in these sera were found to range between 40,960 and 81,920. No antibodies against the NH in the RID test were observed in either case (Table 2). DISCUSSION The pathogenicity of Y. enterocolitica strains is determined by the presence of a virulence plasmid; loss of this plasmid has been associated with a significant decrease in the level of virulence (36). Plasmidic genes are thought to encode a series of YOMPs which are essential virulence determinants (7). By using animal models, it has been demonstrated that mutants defective in the expression of one or several of the plasmid-encoded YOMPs lose their capacity to cause disease (29). Nevertheless, in addition to such plasmidic genes, some loci present in the bacterial chromosome are also involved in the pathogenesis of these bacteria (27, 28). Antibodies directed against YOMPs and/or released proteins have been found in convalescent-phase sera from patients infected with Y. enterocolitica (25) as well as in

Presence of

virulence plasmid

+ +

No. of wks after immunizationb

4 4 5 5

Reaction in RID testc -

-

ELISA titer of antibody against S-LPS

antigen'

IgG

IgA

IgM

40,960 81,920 81,920 81,920

1,280 2,560 2,560 1,280

160 640 320 640

a In the RID test, NH from Y. enterocolitica serotype 09 was used as the antigen. b For description of immunization protocol, see Materials and Methods. c Each serum sample was tested at five concentrations of NH (2, 4, 8, 16, and 32 jig/ml). Positive reactions were recorded in less than 4 h. d Titers are expressed as reciprocals.

rabbits experimentally infected with enteropathogenic Y. enterocolitica strains (19). Still, little is known at present about the kinetics of the immune response against the S-LPS antigen. The results obtained with orogastrically infected rabbits demonstrated that the presence and the absence of the virulence plasmid in these bacterial strains are associated with a significant difference in the antibody response against the S-LPS antigen. Maximal titers of IgG class antibodies were observed between 2 and 9 weeks postinfection with orogastrically infected plasmid-bearing cells. In contrast, a weak antibody response was observed after orogastric infection with plasmid-negative cells. These findings suggest that both plasmid-positive and plasmid-cured cells are able to penetrate into the intestinal mucosa. However, only the plasmid-bearing cells multiply and spread into lymphatic organs, resulting in a prolonged immune response. Recently (27), two chromosomal loci (inv and ail) in Y. enterocolitica that promote invasion of epithelial cells have been characterized. Moreover, it is possible that the ail locus encodes a Y. enterocolitica invasion factor that may be involved in pathogenesis (28). However, the results obtained in the present study show that possibly both chromosomally encoded and plasmid-encoded genes are necessary in orogastrically infected rabbits for penetration of the intestinal wall, proliferation in the host tissue, and establishment of infection. Slight differences in the levels of IgG, IgA, and IgM class antibodies generated in rabbits intravenously infected with plasmid-bearing cells and their plasmid-negative derivatives have been observed by S-LPS ELISA. Such findings point to the possibility that virulence factors not associated with plasmids are also responsible for bacterial growth in tissues in vivo. A similar picture has been observed recently (35, 36) in intravenously infected mice with plasmid-positive and plasmid-cured strains of Y. pseudotuberculosis. Loss of the virulence plasmid was associated in this study with a significant decrease in the level of virulence, although the bacteria were still capable of multiplying in host tissues during the early phase of infection (35, 36). In this respect it is necessary to point out that natural infections due to Y. enterocolitica do not usually involve systemic development (34). The intravenous route that we used in this study bypasses the intestinal step and would reflect the stage of systemic dissemination by virulent bacteria. Accordingly, the different inoculation routes used in this study can in part

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explain the different behavior observed with orogastrically and intravenously infected rabbits. The results obtained in the RID test using the polysaccharide NH from Y. enterocolitica serotype 09 as the antigen demonstrate that only the sera obtained between 2 and 4 weeks postinfection from intravenously infected rabbits with plasmid-positive cells produced a ring of precipitation around the wells after 3 to 4 h of incubation. In the ELISA, titers of the S-LPS antibody of the IgG class in these sera ranged between 10,240 and 40,960. However, similar antibody levels have been observed in sera from rabbits intravenously infected with plasmid-cured cells and in sera obtained from rabbits immunized with heat-killed Y. enterocolitica 09 plasmid-bearing and plasmid-negative cells. In contrast, no positive reactions against the NH antigen in these sera were demonstrated in the RID test. Nevertheless, with certain differences, a similar pattern has been observed in sera from cattle infected with Brucella abortus by using B. melitensis polysaccharide hapten isolated from either the rough B115 (PB) or the smooth 16M (NH) strain in the RID test. Positive RID reactions were observed only in sera from infected animals. On the other hand, sera obtained from vaccinated cattle were negative (22). With regard to the subject under discussion, it is also necessary to note that the polysaccharide haptens from Y. enterocolitica 09 and B. melitensis 16M are immunologically indistinguishable (11), yielding similar results in the RID test with sera from bacteriologically positive cattle (8), and that the presence of precipitating antibodies to NH correlates with the intensity of the antigenic stimulus elicited by a Brucella spp. (8). However, it remains to be explained why only the sera obtained from rabbits intravenously infected with plasmid-bearing cells were positive in the RID test. A possible interpretation might lie in the presence or absence of the virulence plasmid in this bacterial strain and the nature of the immune response generated in the course of the infection. It is possible that a different antigenic stimulus would have elicited antibodies with different affinities which would have reacted in a different fashion in the RID test against the polysaccharide hapten from Y. enterocolitica 09. Nevertheless, further studies are necessary to shed further light on this possibility. Finally, the results of the present work also demonstrate the utility of the S-LPS antigen for carrying out the serological diagnosis of infections caused by Y. enterocolitica. ACKNOWLEDGMENT This work was supported by grant PA86-0107 from the Spanish CICYT. REFERENCES

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