Vol. 30, No. 10
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1992, p. 2560-2566
0095-1137/92/102560-07$02.00/0 Copyright © 1992, American Society for Microbiology
Comparison of Four Different Enzyme-Linked Immunosorbent Assays for Serological Diagnosis of Salmonella enteritidis Infections in Experimentally Infected Chickens FRED G. vAN ZIJDERVELD,l* ANK M. vAN ZIJDERVELD-vAN BEMMEL,1 AND JULIUS ANAKOTIA2 Departments of Bacteriology1 and Immunology,2 DLO-Central Veternary Institute, Edelhertweg 15, P. O. Box 65, 8200AB Lelystad, The Netherlands Received 7 April 1992/Accepted 9 July 1992
The program for the eradication of SalmoneUa enteritidis from chickens in The Netherlands is based on bacteriological examination of breeding flocks. There is a great need for a specific and sensitive serological screening test. For that purpose, we developed four different enzyme-linked immunosorbent assays (ELISAs), i.e., an indirect ELISA with S. enteritidis flagellin, an indirect ELISA with S. enteritidis lipopolysaccharide, a double-antibody sandwich blocking ELISA that uses monoclonal antibodies against S. enteritidis flagellin (GM-DAS blocking ELISA), and a double-antibody sandwich ELISA that uses monoclonal antibodies against S. enteritidis lipopolysaccharide. In the present study, we compare the results of those ELISAs with sera from experimentally infected 1-day-old chickens and with sera and eggs from experimentally infected laying hens. Experimental infections were induced with strains of S. enteritidis phage types 1 and 2, S. typhimurium, and S. panama. Sera were collected up to days 44 and 39 after infection from 1-day-old chickens and laying hens, respectively. Only the GM-DAS blocking ELISA was able to discriminate between S. enteritidis infections and infections with the other serotypes. This ELISA had both a sensitivity and a specificity of 100%/ for all serum samples from experimentally infected chickens. A field study is in progress to evaluate whether this test can be implemented in the Dutch S. enteritidis eradication program.
The national Salmonella enteritidis monitoring and eradication program for poultry in The Netherlands became effective in September 1989. This program is based on periodic bacteriological examination of all primary and secondary breeding flocks in both the broiler and laying sectors and of hatcheries and imported poultry. Breeding flocks positive for S. entenitidis are slaughtered. Bacteriological examination for detecting flock infections on a practical basis is laborious, time-consuming, and expensive, especially when a statistically reliable number of fecal samples per flock is tested to allow the conclusion that a flock found bacteriologically negative for S. enteritidis is truly free of S. entenitidis. Bacteriological examination also may yield false-negative results when S. entenitidis is overgrown by other Salmonella serotypes present in the flock. Therefore, a suitable serological assay for use as a screening technique is needed. When samples in such a serological assay are found positive, a bacteriological culture examination should confirm the presence of S. entenitidis. An effective application of a serological assay for detecting flocks infected with S. enteritidis will be possible only if the assay is both sensitive and specific. Inadequate specificity as a result of reactions of the test antigen with antibodies to antigenically related Salmonella serotypes or other bacteria will result in too many flocks being falsely identified as positive for S. entenitidis. Many techniques for the serological identification of infected flocks have been described; these include agglutination tests (e.g., rapid whole-blood slide test, tube agglutina-
*
tion test, and antiglobulin agglutination test) and enzymelinked immunosorbent assays (ELISAs) with different antigens, such as lipopolysaccharide (LPS), flagellin, outer membrane proteins, and crude extracts (obtained by heat treatment) of S. entenitidis (1-3, 5-7, 9, 13, 17-19, 21). The conclusions of various studies are sometimes contradictory. According to Cooper et al. (5) and Nicholas and Cullen (18), the antiglobulin agglutination test and the LPS ELISA are more sensitive than the rapid slide test and the tube agglutination test. Chart et al. (3) concluded that screening chickens for evidence of infection with S. entenitidis with the LPS ELISA alone is inconclusive and that an agglutination test (whole-blood slide test) seems to be more definitive. Clear cross-reactions between salmonellae of the B and D groups in LPS ELISAs have been described by many authors (5, 18). Cross-reactions between different Salmonella serotypes also have been seen in flagellin ELISAs (21). The ELISA is a suitable technique for large-scale testing of flocks because, in general, it is quick, sensitive, and convenient for screening large numbers of sera. For those reasons, we preferred an ELISA as a screening technique. The present study compares the results of four different ELISAs with sera from experimentally infected 1-day-old chickens and with sera and eggs from experimentally infected laying hens. Experimental infections were induced with strains of S. enteritidis phage types 1 and 2, S. typhimurium, and S. panama. A double-antibody sandwich blocking ELISA that uses monoclonal antibodies (MAbs) against S. enteritidis flagellin (GM-DAS blocking ELISA) proved to be the most specific test, with a sensitivity adequate for detecting S. enteritidis-infected chickens.
Corresponding author. 2560
ELISAs FOR CHICKEN S. ENTERITIDIS INFECTIONS
VOL. 30, 1992
MATERIALS AND METHODS Bacterial strains. Four S. entertidis strains (CVI 1 to 4) were obtained from W. J. van Leeuwen and D. Pruys (National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands). Strains CVI 1 and 2 were typed as phage type 1 and strains CVI 3 and 4 were typed as phage type 2 with the Dutch phage typing system. These phage types correspond to phage types 4 and 8, respectively, of the United Kingdom phage typing system. Other Salmonella strains, i.e., S. enteritidis CVI 5 to 12, S. agona, S. derby, S. dublin, S. oranienburg, S. panama, S. strasbourg, and S. typhimurium (three strains), originated from the Central Veterinary Institute strain collection. Isolation and purification of flagellins. Highly motile S. entertidis CVI 1 and 4, S. derby, S. dublin, S. oranienburg, and S. typhimurium phage type 505 strains were obtained after several passages on heart infusion swarm agar (0.5% agar). Biphasic serotypes were rendered monophasic by elimination of the unwanted phases by immobilization with homologous salmonella H-agglutinating serum (Wellcome Diagnostics). Each strain was then inoculated into 10 ml of the medium described by Ibrahim et al. (11). After incubation for 16 h at 37°C, these cultures were used to inoculate 500-ml to 4-liter quantities of the same medium. After incubation for 8 h at 35°C, Salmonella cells were harvested by centrifugation at 10,000 x g for 30 min and then resuspended in saline solution. The isolation and purification of flagellins were essentially the same as those described by Ibrahim et al. (11). The purity of the flagellin preparations was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by use of the Tris-glycine discontinuous buffer system of Laemmli (14), universally supplemented with 0.5 M urea. Preparation of LPS. LPS was prepared from S. enteritidis CVI 1 and 4, S. strasbourg, and S. typhimurium CVI 23 by the hot water-phenol method of Westphal and Jann as described by Helander (8). Preparation of mouse hybridoma cell lines and production of MAbs. Female BALB/c mice were intraperitoneally immunized with purified flagellin from S. enteritidis CVI 1; per mouse, the inoculum, emulsified in Freund incomplete adjuvant, was 0.2 ml and contained 45 ,ug of protein. After 36 days, this procedure was repeated, and 18 days later, mice were inoculated intravenously with 22.5 p,g of protein in 0.01 M phosphate-buffered saline (pH 7.2) (PBS) without Freund incomplete adjuvant. The mice were sacrificed 3 days later. The preparation of mouse hybridoma cell lines, the detection of antiflagellin antibody-producing cell lines by an ELISA, the cloning procedure, and the production of MAbs were similar to those described previously (22). MAbs from selected stable cell lines were purified from ascitic fluid by ammonium sulfate precipitation (35% saturation) and dialysis against PBS. The purified MAb preparations (8 mg of protein per ml of PBS) were stored in aliquots of 1 ml at -70°C. The immunoglobulin isotype of each MAb was determined in immunodiffusion tests with mouse isotypespecific antisera. Hybridoma cell lines producing MAbs against S. enteritidis LPS were prepared as described above; the same immunization schedule was used, except that Formalin-killed bacteria were used (1 x 108 to 2 x 108 bacteria per mouse). ELISAs. The ELISAs used in this study were similar to those used in a study characterizing the F41 antigen of enterotoxigenic Escherichia coli (22). For details of the buffers and substrate used, the length of the incubation
2561
steps, and the conjugation of MAbs to horseradish peroxidase (HRPO), see reference 22. Optimal concentrations of antigens and HRPO conjugates were determined by checkerboard titrations. Appropriate controls were included on each test plate. (i) Indirect ELISA for screening of hybridoma culture supernatants and determination of titers of purified MAbs. The wells of microdilution plates were each coated with 100 pl of a solution of flagellins (containing 5 p,g of protein per ml) or LPSs (5 p,g/ml). After the plates were washed, 1:2 dilutions of hybridoma culture supernatants or serial twofold dilutions of purified MAb preparations were added. The plates were incubated and washed, and HRPO-labeled sheep anti-mouse immunoglobulin G (Fc specific; Serotec, Oxford, England) was added. After the plates were washed, a substrate solution was added, and the plates were read after 2 to 2.5 h of incubation. Titers of purified MAb preparations were expressed as the logarithm of the reciprocal of the highest dilution yielding an A450 that was 50% of the maximum obtainable absorbance value. (ii) Indirect ELISA with purified flagellin from S. enteritidis (GM ELISA). The GM ELISA was essentially the same as the ELISA described in (i), except that serial twofold dilutions of sera from experimentally infected chickens were tested and an HRPO-labeled mouse MAb (MAb CVI-ChIgG47.3) against chicken immunoglobulin G (12) was used as a conjugate. (iii) Indirect ELISA with LPS from S. enteritidis (LPS ELISA). The LPS ELISA was essentially the same as the ELISA described in (ii), except that the wells of microdilution plates were each coated with 100 p,l of a solution of LPS (5 p,g/ml) from S. entenitidis. (iv) Direct-competition ELISA for epitope analysis. Nonconjugated MAbs were added in serial twofold dilutions (range, 1:20 to 1:10,240) in volumes of 50 p1l to the wells of plates coated with purified S. entenitidis flagellin or LPS. After incubation for 30 min, 50 ,ul of the optimal dilution of each of the HRPO-conjugated MAbs was added per well; in this way, the dilutions of each nonconjugated MAb were allowed to compete with each conjugated MAb for its epitope on flagellin or LPS. Incubation was continued for 1 h, and the substrate solution was added after the plates were washed. The titers of nonconjugated MLAbs against a conjugated MAb were expressed as the logarithm of the reciprocal of the highest dilution yielding an A450 that was 50% of the absorbance value in wells to which only the conjugate was added. Epitopes were defined on the basis of the assumption that two MAbs showing no competition at all recognized two different epitopes. Two MAbs showing reciprocal competition with high titers were assumed to recognize the same epitope or two nearby epitopes. An epitope cluster was defined as a single epitope or a group of adjacent or overlapping epitopes that could not be distinguished as separate epitopes by epitope analysis with a competition ELISA because of steric hindrance. (v) GM-DAS blocking ELISA. Plates were coated with MAb gm 5 (8 mg/ml; 1:4,000 in 0.05 M bicarbonate-carbonate buffer [pH 9.6]) overnight at 37°C and used immediately or stored at -20°C. The crude antigen preparation containing native flagella was prepared from a 6-h brain heart infusion (1-liter) culture of highly motile S. entenitidis CVI 1. Bacteria were pelleted by centrifugation for 20 min at 10,000 x g and resuspended in 10 ml of PBS. This suspension was ultrasonically treated (MSE ultrasonic disintegrator) and
2562
J. CLIN. MICROBIOL.
vAN ZIJDERVELD ET AL.
centrifuged for 30 min at 10,000 x g. The supernatant was stored at -20°C in aliquots of 100 ,ul and used as a crude antigen preparation. After washing of the MAb gm 5-coated plates, the crude antigen preparation (1:5,000) was added, and the plates were incubated overnight at 4°C. The plates were washed, and the serum or egg yolk samples were added in serial twofold dilutions in PBS containing 0.05% Tween 80 (range, 1:5 to 1:2,560). The plates were incubated for 1 h at 37°C and washed, and HRPO-conjugated MAb gm 3 (3.4 mg/ml; 1:5,000) was added. After incubation for 1 h at 37°C, the plates were washed, the substrate solution was added and, after 2 h of incubation at room temperature, the plates were read at 450 nm. Titers were expressed as the logarithm of the reciprocal of the highest dilution yielding an A450 that was 50% of the absorbance value in wells to which only the conjugate was added. (vi) Double-antibody sandwich blocking ELISA that uses MAbs against S. enteritidis LPS (LPS-DAS blocking ELISA). Like the GM-DAS blocking ELISA, an LPS-DAS blocking ELISA with MAb LPS-Sent 15 (8 mg/ml; 1:4,000) as the coating antibody, LPS from S. enteritidis CVI 1 (1 mg/ml; 1:2,000) as the antigen, and MAb LPS-Sent 9 (2.7 mg/ml; 1:1,000) as the HRPO-conjugated MAb was developed. Experimental infections and animals. One-day-old chickens and laying hens were obtained from the specific-pathogen-free flock of the Central Veterinary Institute. Animals were housed in sterile isolators. The feed was sterilized by irradiation (1 Mrad). (i) Experimental infection of 1-day-old chickens. Four groups of 15 chickens (groups 2 to 5) and one group of 20 chickens (group 1) were housed in five sterile isolators on the day of hatching. On days 1, 2, and 3 after hatching, chickens in groups 2, 3, 4, and 5 were orally infected with approximately 200 cells of, respectively, S. panama CVI 51, S. typhimurium phage type 505 strain CVI 23, S. enteritidis phage type 2 strain CVI 4, and S. enteritidis phage type 1 strain CVI 1 per g of feed. Group 1 was the control group. On days 16, 30, and 44 after infection, five chickens per group (control group 6 or 7 chickens) were removed from the isolators and subjected to exsanguination. Serum samples were stored at -20°C. (ii) Experimental infection of laying hens. Four groups of five chickens and one group of four chickens (controls) were removed from the specific-pathogen-free flock at the age of 26 weeks and housed in isolators. Nine days later, chickens of groups 2, 3, 4, and 5 were orally infected with approximately 3 x 108 cells of, respectively, S. panama, S. typhimurium phage type 505, S. enteritidis phage type 2, and S. enteritidis phage type 1 per g of feed. Group 1 was the control group and consisted of four roosters. Blood samples were taken 9 days before infection and on days 7, 14, and 28 after infection. All chickens were subjected to exsanguination on day 39 after infection. Serum samples were stored at -20°C. Undamaged eggs were collected throughout the experiment, and the yolk of each egg was diluted 1:5 with PBS containing 0.05% Tween 80 and stored at -20°C. Bacteriological and postmortem examinations. Two or three times per week, fecal samples were taken for bacteriological examination throughout both experiments. Bacteriological examination for the presence of salmonellae was done by direct inoculation of brillant green agar and by inoculation and incubation of enrichment media (tetrathionate broth and brillant green selenite broth) followed by inoculation of brillant green agar.
conjugate of MAb number 2 3
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RESULTS Production of MAbs against S. enteritidis flagellin and epitope analysis. The cloning and selection procedure resulted in 23 stable hybridoma cell lines (MAbs gin 1 to gin 23). Seventeen lines were selected for ascitic fluid production; 14 of them produced sufficient ascitic fluid for further characterization. The results of the direct-competition ELISA clearly demonstrated that the 14 MAbs were directed against nine different epitope clusters (Fig. 1). We also concluded that MAbs against a particular epitope cluster barely interfered with the binding of other MAbs to their corresponding epitope clusters, although some MAbs showed nonreciprocal competition with MAbs against other epitope clusters. The 14 MAb preparations were tested in slide agglutination tests with strains of S. enteritidis, S. dublin (1,9,12;gp), S. derby (1,4,5,12;fg), S. agona (1,4,12;fgs), S. oranienburg (6,7;mt), and S. typhimurium (1,4,5,12;i:1,2). MAbs gm 2, 3, 5, 6, 7, 8, 10, and 12 agglutinated flagellated bacteria of one or more serotypes, but they did not agglutinate nonmotile or almost nonmotile bacteria of the same serotypes. Especially MAbs gin 2 and 3 were potentially the most promising for use in an ELISA, because they only reacted with S. enteritidis. MAbs gm S and 6 also agglutinated S. dublin; MAbs gin 7, 8, and 12 agglutinated S. enteritidis, S. dublin, and S. agona, and MAb gn 10 agglutinated S. enteritidis, S. dublin, S. agona, and S. derby. The other MAbs (gm 9, 13, 16, 18, 20, and 21) did not agglutinate any of the Salmonella serotypes tested. ELISA titers of the MAb preparations were determined against flagellins gm, gp, fg, and mt of, respectively, S. entertidis, S. dublin, S. derby, and S. oranienburg. MAbs gin 2 and 3 reacted only with gin flagellin, whereas MAbs gin 5 and 6 reacted similarly with gin and gp flagellins. MAbs gm 7, 8, 9, 10, 12, and 13 bound to gm and gp flagellins and also had similar titers to the fg flagellin of S. derby, although MAbs gin 7, 8, and 12 were not able to agglutinate the same S. derby strain with native flagella. MAbs gm 16, 18, 20, and 21 reacted with all flagellins tested. Production of MAbs against S. enteritidis LPS and epitope analysis. The cloning and selection procedure resulted in 16 stable cell lines. Epitope analysis (results not shown), slide agglutination tests, and the results of the indirect ELISA (data not shown) demonstrated that the MAbs could be
VOL. 30, 1992
divided into two groups. The first group, consisting of MAbs LPS-Sent 1, 2, 3, 4, 8, 9, 10, 14, and 15, only agglutinated Salmonella strains of the Dl group and were presumably directed against the 09 antigen; their ELISA titers indicated that they bound to LPS prepared from S. enteritidis only and not to S. typhimurium LPS. The other group, consisting of MAbs LPS-Sent 5, 6, 7, 11, 12, 13, and 16, did not agglutinate any of the Salmonella strains tested and bound in the ELISA to LPSs from both S. enteritidis and S. typhimurium. Development of the GM-DAS blocking ELISA and the LPS-DAS blocking ELISA. Because the HRPO-conjugated MAb in a double-antibody sandwich blocking ELISA is the crucial ingredient determining the specificity of the test, we decided to use MAb gm 2 or 3 as the conjugated MAb in the GM-DAS blocking ELISA. The coating antibody serves merely as an antibody that specifically catches the antigen from a crude antigen preparation; we first selected MAb gm 5 as the coating antibody. The Salmonella Dl group-specific MAbs LPS-Sent 15 and 9 were selected as, respectively, the coating antibody and the conjugated MAb in the LPS-DAS blocking ELISA. Clinical, bacteriological, and postmortem findings for experimental infections. One-day-old chickens did not show severe clinical signs after infection. Only during the first few days after infection did chickens of most groups show rather mild diarrhea and signs of a general health disturbance, especially the group infected with S. panama. None of the infected animals died, except for one chicken in the S. enteritidis phage type 1-infected group. All fecal samples taken during the entire experiment, except those from the controls, were positive for their respective Salmonella strains, and the postmortem bacteriological examination of the intestinal tracts of infected animals was always positive. Cross-contamination between groups was not observed. Laying hens did not show any signs of disease after infection. Most of the infected groups ceased to excrete salmonellae before the end of the experiment. Fecal samples from the control group were always negative for salmonellae. The bacteriological examination of fecal samples was positive for salmonellae up to days 22, 33, and 36 after infection for, respectively, the S. enteritidis phage type 1-, the S. typhimurium-, and the S. enteritidis phage type 2-infected groups. The bacteriological examination of fecal samples from the group infected with S. panama was positive until the end of the experiment. The postmortem (day 39 after infection) bacteriological examination of intestinal contents for the presence of salmonellae was negative for all groups, except for the group infected with S. panama. S. panama could be isolated from the intestinal contents of four of the five chickens in that group. Cross-contamination between groups was not observed. A bacteriological examination of all eggs collected during the experiment was negative for the presence of salmonellae. Serological response of 1-day-old chickens. The serum response of 1-day-old chickens, measured by the GM ELISA, the LPS ELISA, and the GM-DAS blocking ELISA, is shown in Fig. 2. Control animals did not show any response in these ELISAs. The mean titers in the S. enteritidis-infected groups against the gm flagellin (Fig. 2a) were already high at day 16 after infection and remained almost at the same level until day 44. Sera from S. panama- and S. typhimurium-infected chickens were negative in the GM ELISA at day 16, but their mean titers gradually increased from days 30 to 44. The titers in the S. typhimunium-infected chickens against the i flagellin of S. typhimurium (data not shown) were similar to those in
ELISAs FOR CHICKEN S. ENTERITIDIS INFECTIONS 3.5 3
2563
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the S. enteritidis-infected chickens against the gm flagellin of S. enteitidis. From Fig. 2a, we concluded that in very young chickens, it should be possible to discriminate between S. enteritidis infections and infections with the other Salmonella types by use of the GM ELISA. The results of the GM-DAS blocking ELISA (Fig. 2c) indicated that this ELISA was more specific than the GM ELISA. None of the S. typhimurium- or S. panama-infected chickens was positive in this ELISA during the course of the experiment. The titers in the S. enteritidis-infected chickens were at least equal to (early phase after infection) or 2- to 16-fold lower than (later phase after infection) those found in the GM ELISA. The response of S. enteritidis-infected chickens to S. enteritidis LPS was delayed compared with their response to S. enteritidis gm flagellin. Titers, if present, were very low (Fig. 2b) at day 16 but increased gradually and were maximal at day 44. Titers of individual serum samples against LPS of S. enteritidis phage type 1 were the same as those against LPS of S. enteritidis phage type 2 (results not shown). From the results of the LPS ELISA, it was impossible to discriminate between S. enteritidis- and S. panama-infected chickens; as expected (both are of the Salmonella Dl group), we
2564
vAN ZIJDERVELD ET AL.
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FIG. 3. Mean titers in serum per group of experimentally infected laying hens in the indirect ELISA with S. enteritidis gm flagellin (a), the indirect ELISA with S. enteritidis LPS (b), the GM-DAS blocking ELISA (c), and the LPS-DAS blocking ELISA (d). Symbols are as defined in the legend to Fig. 2.
observed no differences between the mean titers in these However, because of cross-antigenicity between S. enteritidis LPS and S. typhimurium LPS, we did not expect that the S. typhimunum-infected chickens would barely show a response to S. enteritidis LPS; only at day 44 after infection did some chickens show low titers. The response of S. typhimurium-infected chickens against S. typhimunum LPS (data not shown) was similar to that of S. enteritidisinfected chickens against S. enteritidis LPS; thus, the absence of an immune response against S. typhimunium LPS could not be the reason for the lack of reactivity in the S. enteritidis LPS ELISA. Serological response of laying hens. The serum response of 27.5-week-old laying hens (and roosters), measured by the GM ELISA, the LPS ELISA, the GM-DAS blocking ELISA, and the LPS-DAS blocking ELISA, is shown in Fig. 3. Control animals did not show any response in these ELISAs, except for low titers found in the GM ELISA for three chickens. The mean titers in the S. enteritidis-infected groups against the gm flagellin (Fig. 3a) were already high at day 7 after infection, reached a maximum at day 14, and gradually decreased thereafter. In contrast to the results for the 1-day-old chickens (Fig. 2a), the mean titers in the S. panama- and S. typhimurium-infected groups, although a little lower than those in the S. enteritidis-infected groups, showed the same pattern. We concluded that in older hens, it would be impossible to discriminate between S. enteritidis infections and infections with the other Salmonella types by use of the GM ELISA. The same conclusion was drawn for the LPS ELISA. The mean titers in the different groups were almost equal (Fig. 3b). In contrast to the 1-day-old chickens (Fig. 2b), older hens in all groups showed a much earlier response (within 7 days), and the hens infected with S. typhimurium showed high titers against S. enteritidis LPS. Theoretically, an groups.
ELISA with LPS from the Salmonella D2 group (09,46) should discriminate between infections with salmonellae of the Dl group (01,9,12) and salmonellae of the B group (01,4,5,12). For this reason, we tested all sera in an ELISA with LPS from S. strasbourg (09,46;d:1,7) as the antigen. The results of this ELISA (data not shown) were very disappointing; even S. enteritidis-infected chickens showed low titers. A double-antibody sandwich blocking ELISA based on MAbs has the potential advantage of being more specific (provided that the epitopes present on the antigen are not too close to each other or that the binding of an antibody to its epitope does not disturbs other epitopes for other reasons). This was not true for the LPS-DAS blocking ELISA, because this ELISA could not discriminate between S. enteritidis infections and infections with the other Salmonella types (Fig. 3d). The iesults of the GM-DAS blocking ELISA for laying hens (Fig. 3c) were in complete agreement with those of this ELISA for 1-day-old chickens. Again, this ELISA was specific, i.e., only S. ententidis-infected animals showed titers, and none of the S. typhimunum- or S. panamainfected chickens was positive. Titers in egg yolks. Because the GM-DAS blocking ELISA with serum samples was the only ELISA found specific for the detection of S. entenitidis-infected chickens, we used only this ELISA to determine titers in egg yolks. None of the eggs from S. typhimurium- or S. panama-infected chickens showed titers in this ELISA. Figure 4 shows the mean titers in egg yolks in the S. enteritidis phage type 1- and 2-infected in relation to their mean titers in serum. The first eggs containing measurable antibody levels were collected 9 and 11 days after infection for, respectively, phage type 2- and 1-infected hens; thus, eggs were positive later than serum. From day 12 after infection, almost all eggs had titers of 1:5
groups
to 1:640.
VOL. 30, 1992
ELISAs FOR CHICKEN S. ENTERITIDIS INFECTIONS TABLE 1. Number of serum samples positive in both experiments when tested in the GM-DAS blocking ELISA at a dilution of 1:5 or 1:10
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days after infection FIG. 4. Mean titers in egg yolks (... and - - -) per day for S. enteritidis phage type 1 (A and .)- and 2 (El and - - -)-infected laying hens in relation to the mean titers in serum (D and A) in the GM-DAS blocking ELISA.
GM-DAS blocking ELISA for routine screening. For S. enteritidis-specific routine screening of serum or egg yolk samples from poultry flocks, it is too laborious and too expensive to determine positive samples (flocks) by estimating titers by testing twofold serial dilutions of samples. A single sample or a pool of individual samples per well is preferred. To determine the optimal dilution to be tested and to estimate the positive cutoff ELISA absorbance value, we determined the mean absorbance values of the different dilutions for all the serum samples taken per group during the experiments (Fig. 5). From Fig. 5, we concluded that a safe optimal dilution of serum samples from both 1-day-old chickens and older hens was 1:5 or 1:10 and that the cutoff absorbance value was 0.6 or even higher. Using anA450 of 0.6 as the cutoff absorbance value, we found that the GM-DAS blocking ELISA had a
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JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1992, p. 2560-2566
0095-1137/92/102560-07$02.00/0 Copyright © 1992, American Society for Microbiology
Comparison of Four Different Enzyme-Linked Immunosorbent Assays for Serological Diagnosis of Salmonella enteritidis Infections in Experimentally Infected Chickens FRED G. vAN ZIJDERVELD,l* ANK M. vAN ZIJDERVELD-vAN BEMMEL,1 AND JULIUS ANAKOTIA2 Departments of Bacteriology1 and Immunology,2 DLO-Central Veternary Institute, Edelhertweg 15, P. O. Box 65, 8200AB Lelystad, The Netherlands Received 7 April 1992/Accepted 9 July 1992
The program for the eradication of SalmoneUa enteritidis from chickens in The Netherlands is based on bacteriological examination of breeding flocks. There is a great need for a specific and sensitive serological screening test. For that purpose, we developed four different enzyme-linked immunosorbent assays (ELISAs), i.e., an indirect ELISA with S. enteritidis flagellin, an indirect ELISA with S. enteritidis lipopolysaccharide, a double-antibody sandwich blocking ELISA that uses monoclonal antibodies against S. enteritidis flagellin (GM-DAS blocking ELISA), and a double-antibody sandwich ELISA that uses monoclonal antibodies against S. enteritidis lipopolysaccharide. In the present study, we compare the results of those ELISAs with sera from experimentally infected 1-day-old chickens and with sera and eggs from experimentally infected laying hens. Experimental infections were induced with strains of S. enteritidis phage types 1 and 2, S. typhimurium, and S. panama. Sera were collected up to days 44 and 39 after infection from 1-day-old chickens and laying hens, respectively. Only the GM-DAS blocking ELISA was able to discriminate between S. enteritidis infections and infections with the other serotypes. This ELISA had both a sensitivity and a specificity of 100%/ for all serum samples from experimentally infected chickens. A field study is in progress to evaluate whether this test can be implemented in the Dutch S. enteritidis eradication program.
The national Salmonella enteritidis monitoring and eradication program for poultry in The Netherlands became effective in September 1989. This program is based on periodic bacteriological examination of all primary and secondary breeding flocks in both the broiler and laying sectors and of hatcheries and imported poultry. Breeding flocks positive for S. entenitidis are slaughtered. Bacteriological examination for detecting flock infections on a practical basis is laborious, time-consuming, and expensive, especially when a statistically reliable number of fecal samples per flock is tested to allow the conclusion that a flock found bacteriologically negative for S. enteritidis is truly free of S. entenitidis. Bacteriological examination also may yield false-negative results when S. entenitidis is overgrown by other Salmonella serotypes present in the flock. Therefore, a suitable serological assay for use as a screening technique is needed. When samples in such a serological assay are found positive, a bacteriological culture examination should confirm the presence of S. entenitidis. An effective application of a serological assay for detecting flocks infected with S. enteritidis will be possible only if the assay is both sensitive and specific. Inadequate specificity as a result of reactions of the test antigen with antibodies to antigenically related Salmonella serotypes or other bacteria will result in too many flocks being falsely identified as positive for S. entenitidis. Many techniques for the serological identification of infected flocks have been described; these include agglutination tests (e.g., rapid whole-blood slide test, tube agglutina-
*
tion test, and antiglobulin agglutination test) and enzymelinked immunosorbent assays (ELISAs) with different antigens, such as lipopolysaccharide (LPS), flagellin, outer membrane proteins, and crude extracts (obtained by heat treatment) of S. entenitidis (1-3, 5-7, 9, 13, 17-19, 21). The conclusions of various studies are sometimes contradictory. According to Cooper et al. (5) and Nicholas and Cullen (18), the antiglobulin agglutination test and the LPS ELISA are more sensitive than the rapid slide test and the tube agglutination test. Chart et al. (3) concluded that screening chickens for evidence of infection with S. entenitidis with the LPS ELISA alone is inconclusive and that an agglutination test (whole-blood slide test) seems to be more definitive. Clear cross-reactions between salmonellae of the B and D groups in LPS ELISAs have been described by many authors (5, 18). Cross-reactions between different Salmonella serotypes also have been seen in flagellin ELISAs (21). The ELISA is a suitable technique for large-scale testing of flocks because, in general, it is quick, sensitive, and convenient for screening large numbers of sera. For those reasons, we preferred an ELISA as a screening technique. The present study compares the results of four different ELISAs with sera from experimentally infected 1-day-old chickens and with sera and eggs from experimentally infected laying hens. Experimental infections were induced with strains of S. enteritidis phage types 1 and 2, S. typhimurium, and S. panama. A double-antibody sandwich blocking ELISA that uses monoclonal antibodies (MAbs) against S. enteritidis flagellin (GM-DAS blocking ELISA) proved to be the most specific test, with a sensitivity adequate for detecting S. enteritidis-infected chickens.
Corresponding author. 2560
ELISAs FOR CHICKEN S. ENTERITIDIS INFECTIONS
VOL. 30, 1992
MATERIALS AND METHODS Bacterial strains. Four S. entertidis strains (CVI 1 to 4) were obtained from W. J. van Leeuwen and D. Pruys (National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands). Strains CVI 1 and 2 were typed as phage type 1 and strains CVI 3 and 4 were typed as phage type 2 with the Dutch phage typing system. These phage types correspond to phage types 4 and 8, respectively, of the United Kingdom phage typing system. Other Salmonella strains, i.e., S. enteritidis CVI 5 to 12, S. agona, S. derby, S. dublin, S. oranienburg, S. panama, S. strasbourg, and S. typhimurium (three strains), originated from the Central Veterinary Institute strain collection. Isolation and purification of flagellins. Highly motile S. entertidis CVI 1 and 4, S. derby, S. dublin, S. oranienburg, and S. typhimurium phage type 505 strains were obtained after several passages on heart infusion swarm agar (0.5% agar). Biphasic serotypes were rendered monophasic by elimination of the unwanted phases by immobilization with homologous salmonella H-agglutinating serum (Wellcome Diagnostics). Each strain was then inoculated into 10 ml of the medium described by Ibrahim et al. (11). After incubation for 16 h at 37°C, these cultures were used to inoculate 500-ml to 4-liter quantities of the same medium. After incubation for 8 h at 35°C, Salmonella cells were harvested by centrifugation at 10,000 x g for 30 min and then resuspended in saline solution. The isolation and purification of flagellins were essentially the same as those described by Ibrahim et al. (11). The purity of the flagellin preparations was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by use of the Tris-glycine discontinuous buffer system of Laemmli (14), universally supplemented with 0.5 M urea. Preparation of LPS. LPS was prepared from S. enteritidis CVI 1 and 4, S. strasbourg, and S. typhimurium CVI 23 by the hot water-phenol method of Westphal and Jann as described by Helander (8). Preparation of mouse hybridoma cell lines and production of MAbs. Female BALB/c mice were intraperitoneally immunized with purified flagellin from S. enteritidis CVI 1; per mouse, the inoculum, emulsified in Freund incomplete adjuvant, was 0.2 ml and contained 45 ,ug of protein. After 36 days, this procedure was repeated, and 18 days later, mice were inoculated intravenously with 22.5 p,g of protein in 0.01 M phosphate-buffered saline (pH 7.2) (PBS) without Freund incomplete adjuvant. The mice were sacrificed 3 days later. The preparation of mouse hybridoma cell lines, the detection of antiflagellin antibody-producing cell lines by an ELISA, the cloning procedure, and the production of MAbs were similar to those described previously (22). MAbs from selected stable cell lines were purified from ascitic fluid by ammonium sulfate precipitation (35% saturation) and dialysis against PBS. The purified MAb preparations (8 mg of protein per ml of PBS) were stored in aliquots of 1 ml at -70°C. The immunoglobulin isotype of each MAb was determined in immunodiffusion tests with mouse isotypespecific antisera. Hybridoma cell lines producing MAbs against S. enteritidis LPS were prepared as described above; the same immunization schedule was used, except that Formalin-killed bacteria were used (1 x 108 to 2 x 108 bacteria per mouse). ELISAs. The ELISAs used in this study were similar to those used in a study characterizing the F41 antigen of enterotoxigenic Escherichia coli (22). For details of the buffers and substrate used, the length of the incubation
2561
steps, and the conjugation of MAbs to horseradish peroxidase (HRPO), see reference 22. Optimal concentrations of antigens and HRPO conjugates were determined by checkerboard titrations. Appropriate controls were included on each test plate. (i) Indirect ELISA for screening of hybridoma culture supernatants and determination of titers of purified MAbs. The wells of microdilution plates were each coated with 100 pl of a solution of flagellins (containing 5 p,g of protein per ml) or LPSs (5 p,g/ml). After the plates were washed, 1:2 dilutions of hybridoma culture supernatants or serial twofold dilutions of purified MAb preparations were added. The plates were incubated and washed, and HRPO-labeled sheep anti-mouse immunoglobulin G (Fc specific; Serotec, Oxford, England) was added. After the plates were washed, a substrate solution was added, and the plates were read after 2 to 2.5 h of incubation. Titers of purified MAb preparations were expressed as the logarithm of the reciprocal of the highest dilution yielding an A450 that was 50% of the maximum obtainable absorbance value. (ii) Indirect ELISA with purified flagellin from S. enteritidis (GM ELISA). The GM ELISA was essentially the same as the ELISA described in (i), except that serial twofold dilutions of sera from experimentally infected chickens were tested and an HRPO-labeled mouse MAb (MAb CVI-ChIgG47.3) against chicken immunoglobulin G (12) was used as a conjugate. (iii) Indirect ELISA with LPS from S. enteritidis (LPS ELISA). The LPS ELISA was essentially the same as the ELISA described in (ii), except that the wells of microdilution plates were each coated with 100 p,l of a solution of LPS (5 p,g/ml) from S. entenitidis. (iv) Direct-competition ELISA for epitope analysis. Nonconjugated MAbs were added in serial twofold dilutions (range, 1:20 to 1:10,240) in volumes of 50 p1l to the wells of plates coated with purified S. entenitidis flagellin or LPS. After incubation for 30 min, 50 ,ul of the optimal dilution of each of the HRPO-conjugated MAbs was added per well; in this way, the dilutions of each nonconjugated MAb were allowed to compete with each conjugated MAb for its epitope on flagellin or LPS. Incubation was continued for 1 h, and the substrate solution was added after the plates were washed. The titers of nonconjugated MLAbs against a conjugated MAb were expressed as the logarithm of the reciprocal of the highest dilution yielding an A450 that was 50% of the absorbance value in wells to which only the conjugate was added. Epitopes were defined on the basis of the assumption that two MAbs showing no competition at all recognized two different epitopes. Two MAbs showing reciprocal competition with high titers were assumed to recognize the same epitope or two nearby epitopes. An epitope cluster was defined as a single epitope or a group of adjacent or overlapping epitopes that could not be distinguished as separate epitopes by epitope analysis with a competition ELISA because of steric hindrance. (v) GM-DAS blocking ELISA. Plates were coated with MAb gm 5 (8 mg/ml; 1:4,000 in 0.05 M bicarbonate-carbonate buffer [pH 9.6]) overnight at 37°C and used immediately or stored at -20°C. The crude antigen preparation containing native flagella was prepared from a 6-h brain heart infusion (1-liter) culture of highly motile S. entenitidis CVI 1. Bacteria were pelleted by centrifugation for 20 min at 10,000 x g and resuspended in 10 ml of PBS. This suspension was ultrasonically treated (MSE ultrasonic disintegrator) and
2562
J. CLIN. MICROBIOL.
vAN ZIJDERVELD ET AL.
centrifuged for 30 min at 10,000 x g. The supernatant was stored at -20°C in aliquots of 100 ,ul and used as a crude antigen preparation. After washing of the MAb gm 5-coated plates, the crude antigen preparation (1:5,000) was added, and the plates were incubated overnight at 4°C. The plates were washed, and the serum or egg yolk samples were added in serial twofold dilutions in PBS containing 0.05% Tween 80 (range, 1:5 to 1:2,560). The plates were incubated for 1 h at 37°C and washed, and HRPO-conjugated MAb gm 3 (3.4 mg/ml; 1:5,000) was added. After incubation for 1 h at 37°C, the plates were washed, the substrate solution was added and, after 2 h of incubation at room temperature, the plates were read at 450 nm. Titers were expressed as the logarithm of the reciprocal of the highest dilution yielding an A450 that was 50% of the absorbance value in wells to which only the conjugate was added. (vi) Double-antibody sandwich blocking ELISA that uses MAbs against S. enteritidis LPS (LPS-DAS blocking ELISA). Like the GM-DAS blocking ELISA, an LPS-DAS blocking ELISA with MAb LPS-Sent 15 (8 mg/ml; 1:4,000) as the coating antibody, LPS from S. enteritidis CVI 1 (1 mg/ml; 1:2,000) as the antigen, and MAb LPS-Sent 9 (2.7 mg/ml; 1:1,000) as the HRPO-conjugated MAb was developed. Experimental infections and animals. One-day-old chickens and laying hens were obtained from the specific-pathogen-free flock of the Central Veterinary Institute. Animals were housed in sterile isolators. The feed was sterilized by irradiation (1 Mrad). (i) Experimental infection of 1-day-old chickens. Four groups of 15 chickens (groups 2 to 5) and one group of 20 chickens (group 1) were housed in five sterile isolators on the day of hatching. On days 1, 2, and 3 after hatching, chickens in groups 2, 3, 4, and 5 were orally infected with approximately 200 cells of, respectively, S. panama CVI 51, S. typhimurium phage type 505 strain CVI 23, S. enteritidis phage type 2 strain CVI 4, and S. enteritidis phage type 1 strain CVI 1 per g of feed. Group 1 was the control group. On days 16, 30, and 44 after infection, five chickens per group (control group 6 or 7 chickens) were removed from the isolators and subjected to exsanguination. Serum samples were stored at -20°C. (ii) Experimental infection of laying hens. Four groups of five chickens and one group of four chickens (controls) were removed from the specific-pathogen-free flock at the age of 26 weeks and housed in isolators. Nine days later, chickens of groups 2, 3, 4, and 5 were orally infected with approximately 3 x 108 cells of, respectively, S. panama, S. typhimurium phage type 505, S. enteritidis phage type 2, and S. enteritidis phage type 1 per g of feed. Group 1 was the control group and consisted of four roosters. Blood samples were taken 9 days before infection and on days 7, 14, and 28 after infection. All chickens were subjected to exsanguination on day 39 after infection. Serum samples were stored at -20°C. Undamaged eggs were collected throughout the experiment, and the yolk of each egg was diluted 1:5 with PBS containing 0.05% Tween 80 and stored at -20°C. Bacteriological and postmortem examinations. Two or three times per week, fecal samples were taken for bacteriological examination throughout both experiments. Bacteriological examination for the presence of salmonellae was done by direct inoculation of brillant green agar and by inoculation and incubation of enrichment media (tetrathionate broth and brillant green selenite broth) followed by inoculation of brillant green agar.
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RESULTS Production of MAbs against S. enteritidis flagellin and epitope analysis. The cloning and selection procedure resulted in 23 stable hybridoma cell lines (MAbs gin 1 to gin 23). Seventeen lines were selected for ascitic fluid production; 14 of them produced sufficient ascitic fluid for further characterization. The results of the direct-competition ELISA clearly demonstrated that the 14 MAbs were directed against nine different epitope clusters (Fig. 1). We also concluded that MAbs against a particular epitope cluster barely interfered with the binding of other MAbs to their corresponding epitope clusters, although some MAbs showed nonreciprocal competition with MAbs against other epitope clusters. The 14 MAb preparations were tested in slide agglutination tests with strains of S. enteritidis, S. dublin (1,9,12;gp), S. derby (1,4,5,12;fg), S. agona (1,4,12;fgs), S. oranienburg (6,7;mt), and S. typhimurium (1,4,5,12;i:1,2). MAbs gm 2, 3, 5, 6, 7, 8, 10, and 12 agglutinated flagellated bacteria of one or more serotypes, but they did not agglutinate nonmotile or almost nonmotile bacteria of the same serotypes. Especially MAbs gin 2 and 3 were potentially the most promising for use in an ELISA, because they only reacted with S. enteritidis. MAbs gm S and 6 also agglutinated S. dublin; MAbs gin 7, 8, and 12 agglutinated S. enteritidis, S. dublin, and S. agona, and MAb gn 10 agglutinated S. enteritidis, S. dublin, S. agona, and S. derby. The other MAbs (gm 9, 13, 16, 18, 20, and 21) did not agglutinate any of the Salmonella serotypes tested. ELISA titers of the MAb preparations were determined against flagellins gm, gp, fg, and mt of, respectively, S. entertidis, S. dublin, S. derby, and S. oranienburg. MAbs gin 2 and 3 reacted only with gin flagellin, whereas MAbs gin 5 and 6 reacted similarly with gin and gp flagellins. MAbs gm 7, 8, 9, 10, 12, and 13 bound to gm and gp flagellins and also had similar titers to the fg flagellin of S. derby, although MAbs gin 7, 8, and 12 were not able to agglutinate the same S. derby strain with native flagella. MAbs gm 16, 18, 20, and 21 reacted with all flagellins tested. Production of MAbs against S. enteritidis LPS and epitope analysis. The cloning and selection procedure resulted in 16 stable cell lines. Epitope analysis (results not shown), slide agglutination tests, and the results of the indirect ELISA (data not shown) demonstrated that the MAbs could be
VOL. 30, 1992
divided into two groups. The first group, consisting of MAbs LPS-Sent 1, 2, 3, 4, 8, 9, 10, 14, and 15, only agglutinated Salmonella strains of the Dl group and were presumably directed against the 09 antigen; their ELISA titers indicated that they bound to LPS prepared from S. enteritidis only and not to S. typhimurium LPS. The other group, consisting of MAbs LPS-Sent 5, 6, 7, 11, 12, 13, and 16, did not agglutinate any of the Salmonella strains tested and bound in the ELISA to LPSs from both S. enteritidis and S. typhimurium. Development of the GM-DAS blocking ELISA and the LPS-DAS blocking ELISA. Because the HRPO-conjugated MAb in a double-antibody sandwich blocking ELISA is the crucial ingredient determining the specificity of the test, we decided to use MAb gm 2 or 3 as the conjugated MAb in the GM-DAS blocking ELISA. The coating antibody serves merely as an antibody that specifically catches the antigen from a crude antigen preparation; we first selected MAb gm 5 as the coating antibody. The Salmonella Dl group-specific MAbs LPS-Sent 15 and 9 were selected as, respectively, the coating antibody and the conjugated MAb in the LPS-DAS blocking ELISA. Clinical, bacteriological, and postmortem findings for experimental infections. One-day-old chickens did not show severe clinical signs after infection. Only during the first few days after infection did chickens of most groups show rather mild diarrhea and signs of a general health disturbance, especially the group infected with S. panama. None of the infected animals died, except for one chicken in the S. enteritidis phage type 1-infected group. All fecal samples taken during the entire experiment, except those from the controls, were positive for their respective Salmonella strains, and the postmortem bacteriological examination of the intestinal tracts of infected animals was always positive. Cross-contamination between groups was not observed. Laying hens did not show any signs of disease after infection. Most of the infected groups ceased to excrete salmonellae before the end of the experiment. Fecal samples from the control group were always negative for salmonellae. The bacteriological examination of fecal samples was positive for salmonellae up to days 22, 33, and 36 after infection for, respectively, the S. enteritidis phage type 1-, the S. typhimurium-, and the S. enteritidis phage type 2-infected groups. The bacteriological examination of fecal samples from the group infected with S. panama was positive until the end of the experiment. The postmortem (day 39 after infection) bacteriological examination of intestinal contents for the presence of salmonellae was negative for all groups, except for the group infected with S. panama. S. panama could be isolated from the intestinal contents of four of the five chickens in that group. Cross-contamination between groups was not observed. A bacteriological examination of all eggs collected during the experiment was negative for the presence of salmonellae. Serological response of 1-day-old chickens. The serum response of 1-day-old chickens, measured by the GM ELISA, the LPS ELISA, and the GM-DAS blocking ELISA, is shown in Fig. 2. Control animals did not show any response in these ELISAs. The mean titers in the S. enteritidis-infected groups against the gm flagellin (Fig. 2a) were already high at day 16 after infection and remained almost at the same level until day 44. Sera from S. panama- and S. typhimurium-infected chickens were negative in the GM ELISA at day 16, but their mean titers gradually increased from days 30 to 44. The titers in the S. typhimunium-infected chickens against the i flagellin of S. typhimurium (data not shown) were similar to those in
ELISAs FOR CHICKEN S. ENTERITIDIS INFECTIONS 3.5 3
2563
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the S. enteritidis-infected chickens against the gm flagellin of S. enteitidis. From Fig. 2a, we concluded that in very young chickens, it should be possible to discriminate between S. enteritidis infections and infections with the other Salmonella types by use of the GM ELISA. The results of the GM-DAS blocking ELISA (Fig. 2c) indicated that this ELISA was more specific than the GM ELISA. None of the S. typhimurium- or S. panama-infected chickens was positive in this ELISA during the course of the experiment. The titers in the S. enteritidis-infected chickens were at least equal to (early phase after infection) or 2- to 16-fold lower than (later phase after infection) those found in the GM ELISA. The response of S. enteritidis-infected chickens to S. enteritidis LPS was delayed compared with their response to S. enteritidis gm flagellin. Titers, if present, were very low (Fig. 2b) at day 16 but increased gradually and were maximal at day 44. Titers of individual serum samples against LPS of S. enteritidis phage type 1 were the same as those against LPS of S. enteritidis phage type 2 (results not shown). From the results of the LPS ELISA, it was impossible to discriminate between S. enteritidis- and S. panama-infected chickens; as expected (both are of the Salmonella Dl group), we
2564
vAN ZIJDERVELD ET AL.
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observed no differences between the mean titers in these However, because of cross-antigenicity between S. enteritidis LPS and S. typhimurium LPS, we did not expect that the S. typhimunum-infected chickens would barely show a response to S. enteritidis LPS; only at day 44 after infection did some chickens show low titers. The response of S. typhimurium-infected chickens against S. typhimunum LPS (data not shown) was similar to that of S. enteritidisinfected chickens against S. enteritidis LPS; thus, the absence of an immune response against S. typhimunium LPS could not be the reason for the lack of reactivity in the S. enteritidis LPS ELISA. Serological response of laying hens. The serum response of 27.5-week-old laying hens (and roosters), measured by the GM ELISA, the LPS ELISA, the GM-DAS blocking ELISA, and the LPS-DAS blocking ELISA, is shown in Fig. 3. Control animals did not show any response in these ELISAs, except for low titers found in the GM ELISA for three chickens. The mean titers in the S. enteritidis-infected groups against the gm flagellin (Fig. 3a) were already high at day 7 after infection, reached a maximum at day 14, and gradually decreased thereafter. In contrast to the results for the 1-day-old chickens (Fig. 2a), the mean titers in the S. panama- and S. typhimurium-infected groups, although a little lower than those in the S. enteritidis-infected groups, showed the same pattern. We concluded that in older hens, it would be impossible to discriminate between S. enteritidis infections and infections with the other Salmonella types by use of the GM ELISA. The same conclusion was drawn for the LPS ELISA. The mean titers in the different groups were almost equal (Fig. 3b). In contrast to the 1-day-old chickens (Fig. 2b), older hens in all groups showed a much earlier response (within 7 days), and the hens infected with S. typhimurium showed high titers against S. enteritidis LPS. Theoretically, an groups.
ELISA with LPS from the Salmonella D2 group (09,46) should discriminate between infections with salmonellae of the Dl group (01,9,12) and salmonellae of the B group (01,4,5,12). For this reason, we tested all sera in an ELISA with LPS from S. strasbourg (09,46;d:1,7) as the antigen. The results of this ELISA (data not shown) were very disappointing; even S. enteritidis-infected chickens showed low titers. A double-antibody sandwich blocking ELISA based on MAbs has the potential advantage of being more specific (provided that the epitopes present on the antigen are not too close to each other or that the binding of an antibody to its epitope does not disturbs other epitopes for other reasons). This was not true for the LPS-DAS blocking ELISA, because this ELISA could not discriminate between S. enteritidis infections and infections with the other Salmonella types (Fig. 3d). The iesults of the GM-DAS blocking ELISA for laying hens (Fig. 3c) were in complete agreement with those of this ELISA for 1-day-old chickens. Again, this ELISA was specific, i.e., only S. ententidis-infected animals showed titers, and none of the S. typhimunum- or S. panamainfected chickens was positive. Titers in egg yolks. Because the GM-DAS blocking ELISA with serum samples was the only ELISA found specific for the detection of S. entenitidis-infected chickens, we used only this ELISA to determine titers in egg yolks. None of the eggs from S. typhimurium- or S. panama-infected chickens showed titers in this ELISA. Figure 4 shows the mean titers in egg yolks in the S. enteritidis phage type 1- and 2-infected in relation to their mean titers in serum. The first eggs containing measurable antibody levels were collected 9 and 11 days after infection for, respectively, phage type 2- and 1-infected hens; thus, eggs were positive later than serum. From day 12 after infection, almost all eggs had titers of 1:5
groups
to 1:640.
VOL. 30, 1992
ELISAs FOR CHICKEN S. ENTERITIDIS INFECTIONS TABLE 1. Number of serum samples positive in both experiments when tested in the GM-DAS blocking ELISA at a dilution of 1:5 or 1:10
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days after infection FIG. 4. Mean titers in egg yolks (... and - - -) per day for S. enteritidis phage type 1 (A and .)- and 2 (El and - - -)-infected laying hens in relation to the mean titers in serum (D and A) in the GM-DAS blocking ELISA.
GM-DAS blocking ELISA for routine screening. For S. enteritidis-specific routine screening of serum or egg yolk samples from poultry flocks, it is too laborious and too expensive to determine positive samples (flocks) by estimating titers by testing twofold serial dilutions of samples. A single sample or a pool of individual samples per well is preferred. To determine the optimal dilution to be tested and to estimate the positive cutoff ELISA absorbance value, we determined the mean absorbance values of the different dilutions for all the serum samples taken per group during the experiments (Fig. 5). From Fig. 5, we concluded that a safe optimal dilution of serum samples from both 1-day-old chickens and older hens was 1:5 or 1:10 and that the cutoff absorbance value was 0.6 or even higher. Using anA450 of 0.6 as the cutoff absorbance value, we found that the GM-DAS blocking ELISA had a
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