479

Journal of Food Protection, Vol. 50, No. 6, Pages 479-484 (June 1987) Copyright1" International Association of Milk, Food and Environmental Sanitarians

Monoclonal Antibodies Directed Against the Flagellar Antigens of Listeria Species and Their Potential in EIA-Based Methods

JEFFREY M. FARBER* and JOAN I. SPEIRS

Bureau of Microbial Hazards, Food Directorate, Health Protection Branch, Health and Welfare Canada, Tunney's Pasture, Ottawa, Ontario, Canada K1A 0L2 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/6/479/1651168/0362-028x-50_6_479.pdf by guest on 23 September 2021

(Received for publication March 10, 1987)

ABSTRACT Enzyme immunoassay (EIA) methods have been widely used in food microbiology for detection of micro­ Monoclonal antibodies directed against antigens of Listeria organisms and their toxins (1,9,16). The EIA is a rapid spp. were produced. Three main classes of immunoglobulins and sensitive test which is simple and inexpensive. The were found that reacted with Listeria strains containing either antibody used in the EIA is critical to success of the pro­ the A, B, or C flagellar antigen. These antibodies reacted with Listeria monocytogenes, Listeria welshimeri, Listeria seeligeri, cedure, and must be very specific. This is especially true Listeria ivanovii and Listeria innocua, but not Listeria grayi, for Listeria, because it is known to cross-react with many Listeria murrayi or Listeria denitrificans. The monoclones gram-positive as well as gram-negative bacteria tested did not cross-react with any of the 30 non-Listeria cul­ (14,15,19). In an effort to develop a rapid EIA procedure tures examined, including Staphylococcus aureus and Strep­ for detection of Listeria spp. in foods, our initial goal tococcus faecalis. Cheese and milk samples naturally-contami­ was to develop a monospecific antibody which would nated with L. monocytogenes were found to be positive for Lis­ react only with Listeria spp. teria within two working days after initiation by using the monoclonal antibodies in an enzyme immunoassay. MATERIALS AND METHODS

Organisms Listeria monocytogenes is a pathogen of growing con­ L. monocytogenes serotypes 3a, 3b, 3c were obtained from cern to the food industry. It has caused at least four Dr. J. Rocourt, Institut Pasteur, France; strain V7 (serotype Via) major foodborne outbreaks in the last 6 years (3,5,8,18), from Dr. J. Lovett, FDA, Cincinnati, Ohio; and serotype Vic and its presence in dairy products has recently resulted (19112), 4c (19116), 4d (19117), 4e (19118), 5 {Listeria ivanovii; 19119), Listeria murrayi (25401), Listeria grayi in recalls of semi-soft cheeses, ice cream, sherbet and (19120) and Listeria denitrificans (14870) from the ATCC ice-milk products. (ATCC numbers in parentheses) in Rockville, MD. All other There is no one official method currently being used cultures (including non-Listeria strains) of Listeria (L. to isolate Listeria from foods. The cold enrichment pro­ monocytogenes serotype Vib (82-464), 4a (82-131), 4b (81- cedure (6), in which samples are kept at 4°C and subcul- 861), Listeria innocua (82-26), Listeria welshimeri and Listeria tured at weekly intervals for up to 6 months, still appears seeligeri were from collections maintained at the Health Protec­ to be the most sensitive method, although because of the tion Branch in Ottawa. Non-Listeria culture tested in this study length of time required, it is quite impractical. Thus there included: Staphylococcus aureus, Staphylococcus epidermidis, is a crucial need to develop a rapid and sensitive method Streptococcus faecalis, Streptococcus faecium, Streptococcus for detection of Listeria spp. in foods. durans, Streptococcus lactis, Streptococcus pyogenes, Strep­ tococcus faecalis subsp liquefaciens, Streptococcus faecalis A fluorescent-antibody technique for rapid identifica­ subsp. zymogenes, Streptococcus sanguis, Streptococcus tion of L. monocytogenes in meat and milk has been de­ salivarius, Bacillus cereus, Bacillus subtilis, Lactobacillus bre- scribed (10). However, because of cross-reactions of the vis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus antibody with micrococci, false-positive reactions oc­ helveticus, Lactobacillus plantarum, Lactobacillus acidophilus, curred when examining milk samples. A more promising Corynebacterium glutamicum, Rhodococcus equi, Erysipelothrix automated fluorescent-antibody technique for rapid de­ rhusiopathiae, Brochothrix thermosphacta, Escherichia coli, tection of L. monocytogenes in raw milk, uses flow Citrobacter freundii, Providencia sp., Shigella sonnet, Sal­ cytometry to permit the rapid characterization of Listeria, monella newport, Proteus rettgeri, and Pseudomonas based on parameters such as surface antigenicity, nucleic aeruginosa. acid content and cell morphology (4). However, this technique relies on commercially available polyclonal Preparation of flagellar extract antibodies which have proven to be non-specific (10). L. innocua strain 82-26 was transferred from a tryptose agar

JOURNAL OF FOOD PROTECTION, VOL. 50, JUNE 1987 480 FARBER AND SPEIRS

(Difco Laboratories, Detroit, MI) plate to 5-ml of tryptose tories, Mississauga, Canada). After incubation for 1 h at am­ phosphate broth (TPB; Difco Laboratories), which was incu­ bient temperature, 0.1 ml of hybridoma culture supernatant bated at 22°C for 8 h. Using this broth as inoculum, 50 flasks fluid was added and the plates incubated at 37°C for 1 h in

(500-ml Erlenmeyer) containing TPB (125 ml) plus 0.6% yeast 5% C02 in humid air. After washing the wells three times with extract and 0.2% glucose were inoculated and incubated for 3 PBS-Tween, 0.1 ml of peroxidase goat anti-mouse G + A + M d at 22°C. The cell suspensions were then centrifuged (10,200 (Zymed; supplier, Dimension Laboratories Inc., Mississauga, X g, 20 min) and each pellet resuspended in about 7 ml of Canada), diluted 1:1000 in PBS-FCS, was added and the plates 0.01 M phosphate buffered saline solution, pH 7.0 (PBS). The were incubated for 1 h at ambient temperature. The wells were resuspended pellets were pooled and then divided equally into washed three times with PBS-Tween and once with citrate buf­ two sterile 250-ml Erlenmeyer flasks. To remove flagella (22), fer pH 5.0 before addition of 0.1 ml of substrate consisting cells suspended in 85-90 ml of PBS were blended for 30 s in of 0.04% o-phenylenediamine (Sigma Chemical Co.) and a Polytron homogenizer (model PT 10-35; Brinkmann Instru­ 0.012% H202 in citrate buffer. After 15-30 min the reaction ments Canada, Ltd.) at low speed. The deflagellated cells were was blocked with addition of 0.05 ml of 2.5 M H2S04. Plates then centrifuged first at 3300 X g for 30 min and then at were read at 492 nm. Immunoglobulin classes were determined

16,000 x g for 20 min to remove remaining cells and debris. by coating wells with Zymed goat anti-mouse G + A + M and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/6/479/1651168/0362-028x-50_6_479.pdf by guest on 23 September 2021 The flagella were then recovered by centrifuging at 40,000 x using KPL (Kirkegaard and Perry Laboratories Inc., Gaith- g for 3 h in a Beckman model L8-70M ultracentrifuge ersburg, MD) peroxidase goat anti-mouse IgG (-y), IgA (a) or (Beckman Instruments Canada Inc., Toronto, Ontario). Pellets lgM (|x) as the detection antibody. were resuspended in sterile distilled water overnight at 4°C. Dot and blot assays. Dry nitrocellulose membranes (NCM) This fraction is referred to as semi-purified flagellar material. were obtained from either Bio-Rad Laboratories, Mississauga, Further purification was effected by running the semi-purified Canada, or as Sartorius 11306 cellulose nitrate, from BDH flagellar preparation through a DEAE-Sephacel column (Phar­ Chemical Ltd., Toronto, Canada. The NCM were either laid macia Fine Chemicals AB, Uppsala, Sweden). The proteins onto the surface of inoculated Modified MacBride's Agar were fractionated on the column by stepwise increases of NaCl (MMA; 13) or rolled over colonics lying on the surface of hy- concentration (0.1 M, 0.2 M, 0.4 M, 0.8 M,) in PBS (12). drophobic-grid membrane filters (HGMF; supplies as IsoGrad by QA Laboratories, Toronto, Canada). NCM were immersed Electron microscopy in 3% gelatin-tris buffered saline solution, pH 7.5 (TBS block­ Presence and purity of flagellar material was followed by ing solution) for 30 min with gentle agitation and transferred examining material under the electron microscope. Samples to undiluted hybridoma culture supernatant fluids containing were negatively stained by mixing with 3% potassium phos- Listeria anti-flagellar antibodies for 1 h. The membranes were photungstate, pH 7.5 and then were examined using a Siemens washed twice (10 min each) in 0.05% Tween 20-TBS, then Elmiscope 102 at 80 keV. placed in Zymed peroxidase goat anti-mouse G + A + M or KPL peroxidase goat anti-mouse IgG (-y), IgA (a) or lgM (|x) or Monoclonal antibody production HRP-Protein A (Bio-Rad Laboratories) for 1 h. The membranes BALB/c mice were immunized by intraperitoneal (200 |xg), were washed twice, then placed in HRP color development sol­ subcutaneous (200 |xg), and foot pad injections (25 |xg) of the ution consisting of 0.05% 2-chloro-l-naphthol, 0.015% H202 semi-purified flagellar preparation (1 mg/ml) in Freund com­ and 16% methanol in TBS (2). All reactions were carried out plete adjuvant. A further dose (200 ]xg) in Freund incomplete at room temperature. To terminate the reaction, membranes adjuvant was given by intraperitoneal and subcutaneous injec­ were rinsed in distilled water and air-dried. tion at day 14. On day 21, the mice received an intravenous Electrophoretic separation and immunoblotting injection of the same antigen; 3 d later the spleens were re­ SDS electrophoresis was performed on Phast System with moved. The spleen lymphocytes were mixed with SP2/0 cells Phast Gel"' gradient media 10-15, and SS Buffer Strips (Phar­ (GM 3569A, Institute for Medical Research, Camden, NJ) in macia AB, Uppsala, Sweden). a ratio of 1-10:1. Cells were fused with 50% (wt/vol) polyethylene glycol (1450 mol wt, Sigma Chemical Co., St. A lyphilized mixture of six protein standards (100 (xg each) Louis, MO) according to the procedure of Kohler and Milstein ranging in molecular weight from 14,400 to 94,000 daltons (//). Culturing was done in HAT medium in microtiter wells (Pharmacia) was applied to the gels along with the samples. (Falcon 3072). Hybridomas producing antibody to Listeria The electrophoresis was carried out at 250 V, 10.0 mA, 3.0 flagellar antigens were screened using the EIA procedure de­ W at 15°C for 65 Vh. Two gels were run simultaneously, with scribed below. only one of them being stained with Phast Gel Blue R (Coomassie R 350, Pharmacia). The unstained gel was transfer­ red onto a glass plate, a piece of NCM (Bio-Rad Laboratories) placed on top of the gel, and then a heavy weight applied. EIA procedures The NCM was left in contact with the gel overnight at 4°C, Microtiter plates. Hybridoma culture supernatant fluids were then lifted off and the immunoblot assay performed as described screened using an EIA procedure in microtiter plates (Immulon above. 1 flat bottom plates, Dynatech Laboratories, Inc; supplier, Fisher Scientific, Nepean, Canada). The EIA was performed using the Behring ELISA Processor II (Hoechst Canada Inc., EIA method with pure cultures Behring Diagnostics Division, Montreal, Canada). Wells were Cultures were grown up in 5 ml of brain heart infusion coated with 2-10 \i.g of flagellar antigen (DEAE-Sephacel (BH1), APT or TPB (Difco) at 30°C for 24-48 h. The cells purified fraction) in PBS and the plates incubated overnight at were spun down (10,200 X g, 20 min) and resuspended in 4°C. The wells were subsequently washed three times in PBS- PBS. Portions (5 |JL1 ) were then spotted onto dry NCM, the 0.5% Tween 20 (Sigma) and then filled with PBS containing membranes allowed to dry, and the EIA was performed. Addi­ 1% heat inactivated fetal ealf serum (PBS-FCS) (Flow Labora­ tionally, Iso-Grid membranes were inoculated with pure cultures

JOURNAL OF FOOD PROTECTION. VOL. 50, JUNE 1987 MONOCLONAL ANTIBODIES AGAINST LISTERIA SPP. 4g 1 of L. monocytogenes and then incubated on extra-modified the monoclonal antibodies, is shown in Table 1. The re­ MMA [MMA plus 1% Lab-Lemco powder (Oxoid Canada Inc., sults indicate reactivity with specific flagellar antigens as Nepean, Canada), 1% yeast extract (Difco Laboratories) and follows: monoclones 16-5 and E 10 with antigen A; C4, 0.5% Tryptone (Difco Laboratories)] for 48 h at 30°C. Blots D5 and 400 with antigen B; and 14-6 with antigen C. of the HGMF were taken with NCM, upon which the EI A was Figures 1 and 2 demonstrate the pattern of reactions performed. obtained with the anti-A and anti-B flagellar antigen monoclones. All serotypes of L. monocytogenes, L. EIA method with naturally-contaminated milk Milk samples, (five subsamples from one lot of milk) con­ ivanovii, L. innocua, L. welshimeri, and L. seeligeri (re­ taining about 103 L. monocytogenes/ml were filtered (0.1- and sults with latter two organisms not shown) reacted with 0.2-ml amounts) undiluted through Sartorius NCM which were the anti-B flagellar antigen monoclone C4 (Fig. 1). The placed on extra modified MMA and then incubated at 30°C for only species which did not react were L. grayi (results 48 h. The NCM was lifted off the agar plate and the dot EIA not shown), L. murrayi and L. denitrificans. The anti-A performed on the NCM. and anti-C flagellar antigen monoclones reacted similarly to the C4 monoclone except the anti-A monoclones did Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/6/479/1651168/0362-028x-50_6_479.pdf by guest on 23 September 2021 EIA method with naturally-contaminated cheese not react with serotypes '/2c and 3c (Fig. 2), and the anti- Naturally-contaminated cheeses from France, (five sub-sam­ C monoclone did not react with '/2a, '/2c, 3a, 3c and 5 ples each from two different brands of cheese) containing about (results not shown). None of the six monoclones reacted 104 L. monocytogenes/g, were decimally diluted in 0.1% (w/v) with any of the 30 non-Listeria cultures tested (Fig. 1 peptone water, stomached for 1 min, and then plated (0.1 ml) and 2; results only shown with 15 cultures). directly onto MMA. The plates were incubated at 30°C for 48 h and then a blot was taken with the Sartorius NCM from plates EIA reactions performed directly on HGMF, which had containing between 30 and 300 total colonies. The blot EIA previously been inoculated with a pure culture of L. reaction was performed on the NCM as described above. monocytogenes and incubated for 48 h at 30°C on double- strength BHI agar, were negative (results not shown). RESULTS However, when the EIA was performed on NCM blots of the HGMF, positive results (immunologic recognition) Initial experiments performed with polyclonal anti "H" were obtained (Fig. 3). antibodies made in rabbits or monoclonal antibodies against a mixture or whole cells and flagellar material The naturally-contaminated cheeses gave positive reac­ of L. innocua demonstrated cross-reactivity with other tions by the EIA method (Fig. 4) as evidenced by forma­ gram-positive bacteria (results not shown). Therefore at­ tion of purple spots on NCM paper. The patterns of posi­ tempts were made to produce monoclonal antibodies di­ tive dots correlated well with the pattern of blue colonies rected against the flagellar antigens of Listeria spp. SDS seen beforehand on MMA using the oblique lighting tech­ gel electrophoresis of the semi-purified flagellar extract, nique of Henry (7). demonstrated presence of one major band and four minor Naturally-contaminated raw milk samples were shown bands. After Western blotting, a positive reaction in the to be positive for Listeria by performing the EIA directly ELISA test was observed only with the major band (re­ on NCM which had various quantities of undiluted milk sults not shown). filtered through them (results not shown). Milk debris did The specificity to serovars of Listeria spp., of six of not obscure the reaction.

TABLE 1. Characteristics of some monoclonal antibodies produced against serovars of the genus Listeria and Listeria grayi, Listeria murrayi and Listeria denitrificans. Species of Flagellar ora Reaction with monoclones Listeria Serotype" "H" Antigens C4, D5, 400 16-5, E-10 14-6 L. monocytogenes '/2a, 3a A,B + L. monocytogenes Vic, 3c B,D + L. monocytogenes Vib, 3b, 4a, 4ab, A,B,C + 4b, 4c, 4d, 4e, 7 L. ivanovii 5 A,B,C + + + L. innocua 6a, 6b, 4ab A,B,C + + + L. welshimeri 6a, 6b A,B,C + + + L. seeligeri Vib, 4c, 4d, 6b A,B,C + + + L. grayi c E L. murrayi c E L. denitrificans _cl - "According to Seeliger and Jones (20). ''Monoclones C4 and 16-5 belong to the IgM mouse immunoglobulin class, D5 and 14-6 to the IgG class, and 400 and EIO to the IgA immunoglobulin class. CL. grayi and L. murrayi are both classified in the latest Bergey's manual (20) as species incertae sedis. AL. denitrificans is now considered not to be a member of the genus Listeria (20).

JOURNAL OF FOOD PROTECTION. VOL. 50, JUNE 1987 482 FARBER AND SPEIRS

ROW

1

JT\ 2

3

4 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/6/479/1651168/0362-028x-50_6_479.pdf by guest on 23 September 2021

Figure 1. Dot EIA reactions on NCM. Portions (5 \i.l) of bacterial suspensions in PBS were spotted onto NCM, air-dried and the EIA performed with monoclone C4 (anti-B flagellar antigen), and KPL peroxidase goat anti-mouse IgM. Organisms tested were as follows: LEFT: ROW 1 - 'ha, >hb, Vic, 3a; 2 - (L. monocytogenes serotypes) 3b, 3c, 4a, 4ab; 3-4b, 4c, 4d, 4e; 4-5, L. murrayi, L. denitrificans, S. aureus. RIGHT: ROW I - S. aureus. S. faecalis, S. durans, S. lactis; 2 - S. faecium, R. equi, L. acidophilus, E. coli; 3 - P. aeruginosa, B. cereus, B. subtilis, Providencia sp.; 4 - C. freundii, S. sonnei, P. rettgeri, L. monocytogenes.

ROW

mi'

Figure 2. Dot EIA reactions on NCM. Portions (5 [LI) of bacterial suspenisons in PBS were spotted onto NCM, air-dried and the EIA performed with monoclone E10 (anti-A flagellar antigen) and KPL peroxidase goat anti-mouse IgA. Organisms tested are identical to those listed in legend for Fig. 1.

DISCUSSION cently shown to have caused illness in humans (17). Al­ though L. welshimeri and L. innocua appear normally to The anti-flagellar monoclonal antibodies described here be avirulent, the latter organism has been shown to pos­ show promise for use in an EIA-based method for detect­ sess neurotropic properties when injected intracerebrally ing Listeria spp. in foods. These antibodies react with into suckling mice. L. monocytogenes, and other Listeria spp. that have an The excellent specificity of the antibodies is illustrated A, B or C type flagellar antigen. L. grayi and L. mur­ in Fig. 2. For example, an anti-A flagellar antigen anti­ rayi, which only have the E type flagellar antigen did body reacted with all serotypes tested, except for not react with any of the monoclonal antibodies. In addi­ serotypes Vic and 3c. Because the monoclones tested in tion, L. denitrificans which will probably be placed out this group reacted with Via. and 3a, they could not be of the genus entirely was non-reactive (22,24). These anti-C or anti-D (see Table 1), and therefore had to be reactivities are beneficial since none of the latter three either anti-A or anti-B flagellar antigen type antibodies. bacteria are pathogenic for man. L. seeligeri, L. welshim- It was deduced that they were anti-A because they did eri, L. ivanovii, and L. innocua as well as L. not react with serotypes Vic or 3c, both of which contain monocytogenes would probably be detected in an assay the B antigen. Only anti-flagellar antibodies (not anti- using these monoclonal antibodies. L. ivanovii and L. somatic) could have given this pattern of positive reac­ seeligeri are both potential pathogens, with the latter re­ tions (see reference 20). To further confirm that the

JOURNAL OF FOOD PROTFCTION. VOL. 50, JUNE 1987 MONOCLONAL ANTIBODIES AGAINST LISTERIA SPP. 483 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/6/479/1651168/0362-028x-50_6_479.pdf by guest on 23 September 2021

Figure 3. Detection by ElA of Listeria. Blots were taken from Figure 4. Detection by EIA of Listeria in foods. Decimal dilu­ colonies of L. monocytogenes on ISO-GRID membranes inocu­ tions of cheese naturally-contaminated with L. monocytogenes lated with pure cultures of the organism and incubated on were surface plated onto MM A, which was incubated at 30°C extra-modified MM A for 48 h at 30°C. The EIA was performed for 48 h. An imprint of the colonies was taken with NCM, upon on the NCM using monoclone C4 and KPL peroxidase goat which the EIA was performed using monoclone 400 and KPL anti-mouse IgM. peroxidase goat anti-mouse IgA.

monoclones were indeed anti-flagellar, Listeria spp. were tification of Listeria spp. was made within 2 d. The pat­ grown at either 22°C or 37°C and then tested by the EIA tern of positive dots obtained correlated perfectly with the method. The positive reactions obtained with organisms pattern of blue colonies observed by the oblique illumina­ grown at 22°C were much stronger than those grown at tion technique of Henry (7). Enough colony material re­ 37°C. Listeria spp. are known to be actively motile at mained on the agar after the blot was taken to permit 20-22°C, while having poor flagella development at the performing of sugar, hemolysin and pathogenicity 37°C. In addition, a positive EIA reaction was obtained tests. Although this method has good potential for foods only with the major protein band after Western blotting that have sufficiently high Listeria counts to enable detec­ of the semi-purified flagellar extract. As observed under tion on direct plating, it may not be suitable for foods the electron microscope, grid fields were covered with with low levels of contamination. Membrane filter flagellar filaments, and were by far the major proteins methods, however, might be attractive in permitting observed. It is not known why monoclone 14-6 (anti-C examination of larger quantities of food, by removing in­ antigen monoclone) did not react with L. ivanovii. It ap­ hibitors, and by allowing for a resuscitation step (21), pears that the C flagellar antigen of L. ivanovii may dif­ factors which may be important in increasing the sensitiv­ fer slightly in amino acid sequence from the C flagellar ity of detecting Listeria in foods. EIA reactions carried antigen of other Listeria spp. out directly on HGMF, which had been inoculated with The specificity of the monoclones was further demon­ a pure culture of L. monocytogenes and placed on double strated by the lack of cross-reactivity with the 30 bacte­ strength BHI agar, were negative. This was due to the rial strains tested. These included almost all of the gram- poor growth of Listeria on the HGMF as well as the poor positive bacteria and gram-negative bacteria which are protein binding properties of polysulfone membranes closely related to Listeria (23,25) and which have been (16). However, positive EIA reactions were obtained on shown to share common antigens with Listeria NCM blots of the HGMF, and this technique appears (14,15,19); most notably members of the staphylococci, promising. Equally promising was the positive result ob­ streptococci and bacilli. A cross-reaction of a strain of tained with naturally-contaminated milk, with the main S. durans with some of the monoclones (see Fig 1) was advantage of this EIA membrane filter technique being later found to be due to reaction with the IgG and IgM that no blotting step is required. The only disadvantage conjugate. with both of the above methods is that one cannot verify The potential of using these monoclones in an E1A- positive results. This will not be a problem if the monoc­ based assay for Listeria in foods was shown using natur­ lones show lack of cross-reactivity with other bacterial ally-contaminated cheese samples. A presumptive iden- genera.

JOURNAL OF FOOD PROTECTION. VOL. 50, JUNE 1987 484 FARBER AND SPE1RS

ACKNOWLEDGMENTS 12. Martinez, R. J. 1963. A method for the purification of bacterial flagella by ion exchange chromatography. J. Gen. Microbiol. We thank Dr. R. Pontrefact for performing the electron microscopy 33:115-120. work and Dr. N. Dickie for help in doing the gel chromatography of 13. McBride, M. E., and K. F. Girard. 1960. A selective method for flagellar extracts. the isolation of Listeria monocytogenes from mixed bacterial popu­ lations. J. Lab. Clin. Med. 55:153-157. 14. Minden, P., J. K. McClatchy, and R. S. Farr. 1972. Shared anti­ gens between heterologous bacterial species. Infect. Immun. 6:574- REFERENCES 582. 15. Neter, E., H. Anzei, and E. A. Gorzynski. 1960. Identification 1. Bartholomew, B. A., M. F. Stringer, G. N. Watson, and R. J. of an antigen common to Listeria monocytogenes and other bac­ Gilbert. 1985. Development and application of an enzyme linked teria. Proc. Soc. Exptl. Biol. Med. 105:131-134. immunosorbent assay for Clostridium perfringens type A enteroto- 16. Peterkin, P. I., and A. N. Sharpe. 1984. Rapid enumeration of xin. J. Clin. Pathol. 38:222-228. Staphylococcus aureus in foods by direct demonstration of en­ 2. Bio-Rad Laboratories. 1982. Immuno-Blot™ assay kit methods terotoxigenic colonies on membrane filters by enzyme immunoas­

manual. Bio-Rad Laboratories, Richmond, Calif. say. Appl. Environ. Microbiol. 47:1047-1053. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/6/479/1651168/0362-028x-50_6_479.pdf by guest on 23 September 2021 3. Centers for Disease Control. 1985. Listeriosis outbreak associated 17. Rocourt, J., H. Hof, A. Schrettenbrunner, R. Malinverni, and J. with Mexican-style cheese - California. MMWR 34:357-359. Bille. 1986. Meningite purulente aigue a Listeria seeligeri chez un 4. Donnelly, C. W., and G. J. Baigent. 1986. Method for flow adulte immunocompetent. Schweiz. med. Wschr. 116:248-251. cytometric detection of Listeria monocytogenes in milk. Appl. En­ 18. Schlech, W. F., P. M. Lavigne, R. A. Bortolussi, A. C. Allen, viron. Microbiol. 52:689-695. E. V. Haldane, A. J. Wort, A. W. Hightower, S. E. Johnson, 5. Fleming, D. W., S. L. Cochi, K. L. Macdonald, J. Brondum, S. H. King, E. S. Nicholls, and C. V. Broome. 1983. Epidemic P. S. Hayes, B. D. Plikaytis, M. B. Holmes, A. Audurier, C. listeriosis - evidence for transmission by food. New Eng. J. Med. V. Broome and A. L. Reingold. 1985. Pasteurized milk as a vehi­ 308:203-206. cle of infection in an outbreak of listeriosis. New Eng. J. Med. 19. Seeliger, H. P. R. 1961. Listeriosis. Hafner Publishing Company, 312:404-407. Inc., N.Y. 6. Gray, M. L., H. J. Stafseth, F. Thorp Jr., L. B. Sholl, and W. 20. Seeliger, H. P. R., and D. Jones. 1986. Genus Listeria Pirie. p. F. Riley Jr. 1948. A new technique for isolating Listerellae from 1235-1245. In P. H. A. Sneath, N. S. Mair and E. Sharpe (eds.), the bovine brain. J. Bacteriol. 55:471-476. Bergey's manual of systematic bacteriology, 7th ed., vol. 2. Wil­ 7. Henry, B. S. Dissociation in the genus Brucella. 1933. J. Infect. liams and Wilkins, Baltimore. Dis. 52:374-402. 21. Sharpe, A. N., P. I. Peterkin, and N. Malik. 1979. Improved de­ 8. Ho, J. L., K. N. Shands, G. Friedland, P. Eckind, and D. W. tection of coliforms and Escherichia coli in foods by a membrane Fraser. 1986. An outbreak of type 4b Listeria monocytogenes in­ filter method. Appl. Environ. Microbiol. 38:431-435. fection involving patients from eight Boston hospitals. Arch. In­ 22. Smith, R. W., and H. Koffler, 1971. Production and isolation of tern. Med. 146:520-524. flagella. p. 165-172. In J. R. Norris and D. W. Ribbons (eds.), 9. Ibrahim, G. F. 1986. A review of immunoassays and their applica­ Methods in microbiology, Vol. 5A. Academic Press, New York. tion to salmonellae detection in foods. J. Food Prot. 49:299-310. 23. Stuart, M. R., and P. E. Pease. 1972. A numerical study on the 10. Khan, M. A., A. Seaman, and M. Woodbine. 1977. Immuno- relationships of Listeria and Erysipelothrix. J. Gen. Microbiol. fluorescent identification of Listeria monocytogenes. Zentralbl. 73:551-565. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 24. Stuart, S. E., and H. J. Welshimer, 1973. Intrageneric relatedness 239:62-69. of Listeria Pirie. Int. J. Syst. Bacteriol. 23:8-14. 11. Kohler, G., and C. Milstein, 1975. Continuous cultures of fused 25. Stuart, S. E., and H. J. Welshimer, 1974. Taxonomic reexamina­ cells secreting antibody of predefined specificity. Nature 256:495- tion of Listeria Pirie and transfer of Listeria grayi and Listeria mur- 497. rayi to a new genus, Murraya. Int. J. Syst. Bacteriol. 24:177-185.

JOURNAL OF FOOD PROTECTION, VOL. 50, JUNE 1987