SerodiagnosisofListeriamonocytogenes.doc

BAM: Serodiagnosis of Listeria monocytogenesJanuary 2001Bacteriological Analytical ManualChapter 11Serodiagnosis ofListeriamonocytogenesAuthors:Reginald W. Bennettand Robert E. WeaverAlthough serological confirmation is not necessary for regulatory identification ofListeriamonocytogenes, it is useful for determining the prevalence of specific serotypes in epidemiological studies and for environmental recontamination tracking. Early attempts to confirmL.monocytogenesserologically were generally not successful because of cross-reactions with other organisms (1). Serology, therefore, should always follow cultural and biochemical identification (see Chapter 10). Serological assays ofListeria, reviewed by Gray and Killinger (4), include agglutination, precipitation, and complement fixation assays as well as automated fluorescent antibody techniques, using flow cytometry (3) and rapid method ELISA kits, which are based on monoclonal and polyclonal antibodies (5). However, these serological systems were developed to detect allListeriaspp. They have been used successfully to screen foods for the genus (see Chapter 10), but they do not differentiateL.monocytogenesfrom otherListeriaspecies.This chapter presents procedures for the serological identification ofL.monocytogenesby flagellar and somatic antigenic profiles (2), using agglutination as the serological tool.1. A. Equipment and materials1. Centrifuge2. Refrigerator (4-6C)3. Steamer4. Inoculating needle5. Tubes, 6 x 50 mm6. Test tube rack (for 6 x 50 mm tubes)7. Dispenser, 25, 50, 100 l, or comparable8. Water bath (48C)9. Microscope slides1. B.Mediaandreagents1. EB motility medium (M48)2. Tryptose phosphate broth (TPB) (M168)3. Formaldehyde, 37% solution4. Formal saline, 0.5%5. Physiological saline, 0.85% (R63)6. McFarland No. 3 turbidimetric standard (R42)7. Somatic (O) antisera8. Flagellar (H) antisera1. C. Preparation of materials and media1. EB motility medium. Prepare as specified. Distribute 10 m1 amounts of medium in 18 x 125 mm or comparable size screw-cap tubes. Refrigerate medium until used.2. Tryptose phosphate broth (TPB). Prepare as directed. Distribute 8.0 ml amounts in 16 x 125 mm or comparable size screw-cap tubes.3. Formal saline (0.5%). Add formaldehyde in a concentration of 0.5% to 0.85% physiological saline.4. No. 3 McFarland standard. Prepare turbidity standard No. 3 of the McFarland nephelometer scale. Mix 3.0 ml 1% BaCl2solution with 97.0 ml 1% H2SO4solution.5. H and O antisera. Dilute according to manufacturers directions.6. Cells (antigens to be tested). PrepareL.monocytogenescells as specified under procedures.1. D. ProceduresThe uniform approach to serotyping is first to determine the flagellar (H) serotype, and then to determine the somatic (O) serotype and/or subserovar, depending on the refinement of the antisera. Specific somatic types are associated with specific flagellar serogroups. The relationship of somatic and flagellar antigenic factors forL.monocytogenesis shown in Table 1.Table 1. Diagnostic scheme showing relationship of somatic (O) and flagellar (H) antigenic factors ofL.monocytogenesH factorsO factorsA1a (1/2a)3a1a(1); 1a(1,2);3a (4)(b)C1b (1/2b)a;3b4a(7,9); 4b(5,6); 4b(6); 4d(8)(b)D2 (1/2c)(a)3CaSeeliger and Donker-Voet designations.bBrackets indicate that antisera to somatic antigens are available.Antisera to H antigens are also available.Scheme for routine serodiagnosis of L. monocytogenes, based on H and O antigenic factors.2. Routine serological typing for flagellar antigensRemove growth from agar slant with straight inoculating needle and stab tube of EM motility agar if both flagellar and somatic antigens are required for serodiagnosis. See scheme for growth and treatment events. Incubate inoculated motility agar at 25C for 24-48 h. Pick colonies from outer edge of motile growth on EB motility agar (Fig. 1) and inoculate tube of TPB or similar agar. Incubate inoculated TPB for 18-24 h at 25C.Fig. 1. Typical umbrella-like growth ofL.monocytogeneson motility agar.Add formaldehyde solution (37%) for final volume of 0.5% to broth culture (0.04 ml of formaldehyde to 8.0 ml of broth culture). Allow formaldehyde-treated broth to incubate 4 h at 25C or treat as live culture. Collect cells by centrifugation (1600 xg, 30 min); then resuspend cells in 0.5% formal saline to turbidity equal to McFarland No. 3 standard. The broth cell suspension works equally as well as the washed standardized cells for the agglutination test.In 6 x 50 mm tubes, mix cells (antigen) to be tested with equal volumes (100 l) of predetermined dilutions of H antisera with serological factors, A, C, and D. To prepare negative control, place 100 l of test cells in equal volume of saline. Incubate tubes containing bacterial cells and antisera as well as negative control in water bath preset at 48C.Observe tubes for agglutination after 1 h incubation. If agglutination occurs, a sediment will form and the supernatant will be as clear as the negative control (or clearer). Agitate tubes slightly (execute gently with finger) to resuspend sediment. Typical tube agglutination reaction is shown in Fig. 2.Fig. 2. Comparative tube agglutination test showing positive agglutination reaction (tube on left with granular appearance) and typical negative reaction (tube on right with smooth, homogenous appearance in the serodiagnosis ofL.monocytogenes.2. Routine serological typing for somatic antigensRemove growth from agar slant or comparable media with inoculating needle and inoculate tube(s) of TPB if flagellar serodiagnosis is not being done. If both flagellar and somatic antigen profiles are being determined, see scheme for routine serodiagnosis ofL.monocytogenesbased on H and O factors. Collect cells (antigen) by centrifugation (1600 g, 30 min). Wash cells once in TPB for slide test or once with 0.5% formal saline for tube agglutination testing.For slide testing, resuspend cells in minimal amount of 0.5% formal saline to prepare heavy cell suspension (cell turbidity equal to or greater than that of McFarland No. 3). Place 25 l of factor serum on slide with equal volume of cells (antigen). To prepare negative control, mix 25 l of cell suspension with 25 l of saline. Mix antiserum and cells together while rocking slide back and forth.To observe agglutination, hold slide against black background near a desk lamp. Typical positive (agglutination) and negative (smooth) reactions are shown in Fig. 3. If reaction is not smooth or fails to give good observable agglutination, test cells by tube agglutination, which is considerably more sensitive. The tube test for routine serotyping of O antigens is the same as that used for testing H antigen factors. For somatic serotyping, incubate tubes for 2 h in 48C water bath and refrigerate overnight, or incubate overnight in 48C water bath.Fig. 3. Comparative slide agglutination test showing typical positive agglutination reaction (left section of slide) and negative serological reaction (right section of slide) in the serological typing ofL.monocytogenes.References1. Bennett, R.W. 1986. Detection and quantitation