competitive enzyme immunoassay for the rapid identification of salmonella

COMPETITIVE ENZYME IMMUNOASSAY FOR THE RAPID IDENTIFICATION OF SALMONELLA

RAYMOND S.W. TSANG'*3, KLAUS H. NIELSEN2, SOPHIA BALSEVICIUS2. LINDA KELLY2, RASIK KHAKHRIA' and WENDY M. JOHNSON'

'National Laboratory for Entenc Pathogens Bureau of Microbiology

Laboratory Centre for Disease Control Health Canada

and

2Immunology Section Animal Diseases Research Institute Agriculture and Agn-Food Canada

Ottawa, Ontario, Canada

Accepted for Publication January 3, 1995

ABSTRACT

A cotnpetitive enzyme immunoassay using a murine monoclonal antibody M I 05 directed against a genus-speci'c epitope in the Salmonella Iipopolysaccharide wus used to identib over 200 strains of Salmonella submitted to the National Luboratory for Enteric Pathogens. R e itnmunoassay rapidly identi3ed 208 strains of Salmonella representative of subspecies I , I I , I&, I&, IV, and V , including 89 different seroQpesfrom 26 0 serogroups. The competitive enzyme immunoassay did not give positive results with 3 strains of Citrobacter freundii and 4 strains of Escherichia coli which were submitted to our laboratory as suspect Salmonella.

INTRODUCTION

At the present time there is no effective vaccine against the gastroenteric group of salmonellae for use in either human or animals. The most common preventive

Torresponding author: Dr. Raymond S . W. Tsang, National Laboratory for Enteric Pathogens, Bureau of Microbiology. Laboratory Centre for Disease Control. Health Canada, Ottawa, Ontario, Canada KIA OL2. Fax: (613) 941-2408, Phone: (613) 941-1747, e-mail: [email protected].

Journal of Rapid Methods and Automation in Microbiology 3 (1995) 203-214. All Rights Reserved. 0 Copyright 1995 by Food & Nutrition Press, Inc., Trumbull, Connecticut. 203

105 TSANG. NIELSEN. BALSEVICIUS. KELLY. KHAKHRIA and JOHNSON

control measure against foodborne salmonellosis is the application of the Hazard Analysis and Critical Control Point (HACCP) system to identify high risk areas for halting the transmission of salmonellae into the food chain (Simonsen et al. 1987). Constant vigilant measures such as mandatory reporting of Salmonella isolation and testing of animals. food, and feeds are also important components o f the control effort (e .g . , the Processed Animal Protein Order [I9891 and the Poultry Breeding Flocks and Hatcheries (Registration and Testing] Order [ 19891 in the UK (Baird-Parker 1990): D'Aoust, J.Y 1989).

The traditional method of detecting salmonellae involves isolation of the bacteria through a series of culture steps (reviewed by Varnam and Evans 1991) and identification of the isolated organisms by elaborate biochemical and serological methods (Ewing 1986; Kauffmann 1972). Such procedures are not only time- consuming. labor-intensive and costly. but are also dependent on the availability of technical expertise. The aim of the present work was to adapt a simple competitive enzyme immunoassay (EIA) that was developed for the detection of Sdmonella Iipopolysaccharide (LPS) antigen (Nielsen and Tsang 1992; Nielsen et ( I / . 1993) to the rapid identification of Salriiorzella strains. To evaluate whether the competitive EIA can identify common as well as rare Salmoriellu serotypes, wc tested over 300 Salmorzella strains submitted to the National Laboratory for Enteric Pathogens by the competitive EIA and compared the findings with those obtained by conventional biochemical and serological methods.

MATERIALS AND METHODS

Bacterial Strains

Bacterial strains used in this study included 215 isolates of suspected Sabnorzella submitted to the National Laboratory for Enteric Pathogens for identification and/or scrotyping. Bacteria were first plated on nutrient agar (Difco Lab., Detroit, Michigan) for confirmation of purity. and all subsequent studies were from growth (at 37C for 16-18 h) obtained from single isolated colonies.

Biochemical and Serological Identification of Bacteria

Biochemical identification of bacteria was performed using standard procedures as described by Ewing (1986) and Lennette et al. (1985). Serological identification of Salnioriella was done by detection of specific 0 and H antigens according to the Kauffmann-White scheme (Kauffniann 1972; Popoff and Le Minor 1992). Serotype names were maintained for strains within subspecies I as well as for those in other subspecies. For the Arizona strains, they were designated as S. urI':oriCie with their subspecies designations (111, or I&,) given in brackets. In ac-

COMPETITIVE EIA FOR SALMONELLA 205

cordance with the traditional nomenclature of Salmoriellu, the conventional writing of Salmonella ryphimuriurn was used instead of Salmoriella Typhimuriuni, which was adopted in the latest edition of Kauffinan-White Scheme (Popoff and Le Minor 1992).

Monoclonal Antibody

The production and characterization of a Salmonella genus-specific murine monoclonal antibody (MAb), M105, was described previously (Tsang et al. 1991). Mouse ascitic fluids, induced by the hybridoma cell line MI05 using standard procedures (Haagerraad et al. 1983). were used as sources of MAbs and were purified by high pressure liquid chromatography on a Bio-Gel TSK DEAE-5-PW column (BioRad Laboratories Ltd.. Mississauga, Ont.) equilibrated in 0.02 M Tris-HCI buffer, pH 8.5. MAb M105 dialyzed into 0.02 M Tris-HCI buffer, pH 8.5 was applied in 10 ml amounts onto the column. After adequate washing to remove unbound materials, MI05 was eluted from the column with a gradient consisting initially of a 0.02 M Tris-HCI buffer, pH 8.5 and finally of a 0.3 M Tris-HCI buffer, pH 7.0 containing 0.3 M NaCl at a flow rate of 5 ml per min. Fractions collected at 2 min intervals were monitored by absorbance at 280 nni as well as by indirect ELlSA using S. pphimurium LPS as antigens and horseradish peroxidase enzyme conjugated goat anti-mouse IgG (Jackson ImmunoResearch Lab., Bio/Can Scientific Inc., Mississauga, Ont.) for detection (Nielson et al. 1993).

Competitive Enzyme Immunoassay

This was performed as essentially described by Nielsen and Tsang (1992) and Nielsen e f t i / . (1993). Briefly, 96-well microtiter plates (Nunc 69620, Gibco-BRL, Burlington. Ontario) were coated overnight with 200 pl of 0.5 pg/ml of S. typhimurium LPS (Sigma Chem. Co., St. Louis, MO) in carbonate-bicarbonate buffer, pH 9.6. All incubation steps were at room temperature. Just before use, the microtiter plate was washed four times with 0.1 M Tris-buffer. pH 8.0, containing 0.15 M NaCl and 0.05% Tween 20. Samples, prepared by suspending test organisms in sterile phosphate buffered salone (PBS) to a cell density of ap- proximately MacFarland standard #3 and heating the cell suspension in a boiling water bath for 1 h, were premixed and incubated in a glass tube for 15 min with an equal volume of MAb MI05 suitably diluted (1:50,000) to give a limiting amount of reagent. The mixture was then added to duplicate wells of the antigen coated microtiter plate for 20 min incubation under constant shaking. Following four more washes as above, 200 pI of suitably diluted (1:6,OOO) horseradish peroxidase enzyme conjugated goat anti-mouse IgG (Jackson ImmunoResearch Lab.,


BioiCan Scientific Inc.. Mississauga. Ontario) was added to each well for 20 min reaction. After a final cycle of four more washes. 200 pI of substrate (4 niM H , 0 2 and 1 mM 2 . 2 '-Azino-bis[3-ethylbenzthiazoline-6-sulfonic acid) (Sigma Chem. Co.. St. Louis, MO) was added to each well for color development. Reading of the plate at a wavelength o f 305 nm by a microplate ELISA reader using computer software was done as described by Wright er a / . (1985). Dilu- tions of MAb MI05 and enzyme conjugated goat anti-mouse serum for use in the competitive EIA were chosen by checkerboard titrations as described previously (Niel\en ('[ a / . 1993).

RESULTS

Based on previous studies (Nielsen er N / . 1993) using a combination of different nonspecific interactions from either heterologous antigen (e.g.. E. coli LPS) or unrelated MAbs in place of MAb MI05 (e.g., anti-BruceIla abortus or anti- bovine immunoglobulins MAbs). 28.5% was found to be the maximum percentage of nonspecific inhibition obtained if 3 standard deviations (SD) were chosen to account for test-to-test variations. To allow for samples that may fall within an indeterminate grey zone. 35% was chosen arbitrarily as the cutoff point for calling the Competitive test positive.

Using such a criteria, all 208 strains of Sdnzonella comprising 89 different serotypcs troni 26 different 0 serogroups (Table 1) were correctly identified as S~i / rnnnr / l r i by the competitive EIA. Besides strains (n = 132) belonging to the 0 serogroups o f A to E that were reactive with MAb M105, 65 strains belonging to the higher 0 serogroups of F to 67 were also identified by the present im- niiinoassab , Similarly, not only strains belonging to subspecies I Salnionella were identified. 18 strains belonging to the other subspecies ( I strain of subspecies 11: I! strains of subspecies 111,; 8 strains of subspecies IIIb; 6 strains of subspecies IV: and I main of subspecies V ) were also positive in the competitive EIA using M A b MIOS.

All Sci/i?ionc/la strains gave more than 50% inhibition in the competitive EIA and the degree of inhibition demonstrated by the different Salmonella strains ranged from 52 to 100%. The majority of the Sril,nonel/a strains gave more than 80% inhibition. and there were only 1 I strains (about 5 % of all the Salmonelka strains tested) which gave less than 80% inhibition. Among those giving less than 80% inhibition. 7 gave levels of inhibition between 70 and 80% and only 4 gave levels of inhibition between 50 and 60%.

Seven \trains which had been submitted to our laboratory as suspected Sn/rnonc~//tr were identified by biochemical tests as either Citrobacrerfreundii (three


strains) or Escherichia coli (four strains). The degree of inhibition given by these non-Salmonella bacteria ranged from 0 to 29%. The three C. fieundii strains gave 29, 20, and 0% inhibition while the four E. coli strains gave 23, 7, 7, and 0% inhibition.

The mean percentage of inhibition given by the 208 Salmonella strains in the competitive EIA was 9 1.2 % with a SD of 7.4 % . Only 4 Salmonella strains tested gave levels of inhibition that fell below three SD from the mean level of inhibition given by all Salmonella strains tested. Despite the deviations of such strains from the majority of the Salmonella organisms tested, these four Salmonella strains still gave levels of inhibitions (more than 50%) which were much higher than those given by the non-Salmonella strains (less than 30%).

DISCUSSION

Previously, we described a murine MAb M105 against a genus-specific ep- tiope present in the Sabnonella LPS (Tsang et al. 1991), and developed a competitive EIA using this MAb for the detection of Salmonella LPS (Nielsen er al. 1993). Preliminary studies on the specificity of MI05 indicated that it reacted with serogroups A to E Salmonella, and that it did not react with a wide variety of different Gram-positive and Gram-negative bacteria with the exception of oc- casional strains of Enrerobacrer a,nglornerans (Todd et al. 1991). However extensive studies with known serotypes in the higher 0 serogroups and subspecies I1 to VI Salmonella had not been carried out to affirm the usefulness of this reagent as a polyvalent anti-Salmonella antibody.

Although the competitive EIA using MAb M105 was originally designed for the measurement of Salmonella LPS antigen and the detection of the organism, the lack of adequate sensitivity of such an enzyme immunoassay, like all other similar immunoassays using either monoclonal or polyclonal anti-Salmonella an- tibodies, to detect very low numbers of Salmonella organisms has prohibited them from direct application on clinical or food specimens. This is especially true for the zero level of tolerance of these organisms in our food and feeds. Therefore, to use enzyme immunoassay for the rapid detection of Salmonella, enrichment culture is a mandatory step to increase the number of organisms to a level detect- able by the current immunoassay technology (Tsang and Nielsen 1992).

In this study we investigated another application of the M105 competitive EIA: rapid identification of Salmonella without resorting to extensive biochemical and serological testings which require technical expertise for performance and inter- pretation. To show that EIA using MAb MI05 will identify common as well as

208 TSANG. NIELSEK. BALSEVICIUS. KELLY. KHAKHRIA and JOHNSON

TABLE I

USING MAb MI05 S,’lLMOiV€LL4 SEROTYPES* IDENTIFIED B Y COMPETITIVE ENZYME IMMUNOASSAY

No. of Serogroup Serotjpe Antigenic forniula isolates

A S. paratyphi A 2,12:a:- S. paratyphi A 1,2,12:a:-

3 1

B S. typhimurium 4,5,12:i:1,2 6 S. typhimurium 4,12:i:1,2 1

S. java 4,5,12:b:1,2 1

S. derby 4,12:f,g:- 1 S. sandiego 4,12 :e, h: e ,n, z,, 1 S. brandenburg 4,12 : 1, v: e , n, z,, 3 S. heidelberg 4,5,12:r:1,2 5 S. saintpaul 4,5,12:e,h:1,2 1 S. agona 4,12:f,g,~:- 4 S. California 4,12:g,m,t:- 1 Salmonella subsp. I’ 4,5,12:i:- 2 Salmonella subsp. I’ 4,5,12:-:1,2 2

Salmonella subsp. I‘ 4,12:d:- 1

S. bechuana (subsp. 11) 4,12:g,t:1,5 1

(var. Copenhagen)

(d-tartrate positive)

Salmonella subsp. I‘ 4,5,12:b:- 6

Salmonella subsp. I 4,12:-:- (non-motile) 2

S . S . S. S. S. S. S . S . S. S.

S. S . S .

inf antis irumu montevideo Ohio rissen oranienburg thompson virchow mbandaka eimsbuettel”

galiema livingstone kralendyk (subsp.

( S. livingstone

6,7:r:1,5 6,7:1,v:1,5 6,7:g,m,s:- 6,7: b: 1,w 6,7:f,g:- 6,7:m,t:- 6,7:k:1,5 6,7:r:1,2 6,7: Z,,:e,n, z,, 6,7,14:d:l,w

6,7:k:l, 2 6,7:d: 1,w

var. 14+)

IV) 6,7:2,,2,,:-

S. albany a: z , , z 2 , : - S. kentucky 8,20: i: 2 , S . hadar 6,8 : z,,: e, n, x S. newport 6,8:e,h:1,2 S. bovismorbificans 6,8:r:1,5

1 3 3 1 1 6 1 3 2 1

1 1 1

2 2 2 2 5

COMPETITIVE EIA FOR SALMONELLA

Serogroup Serotype Antigenic formula

E.

F

G

H

S . presov Salmonella subsp. I S. gatuni S . chailey S. goldcoast

S. javiana S. dublin S. enteritidis S . berta Salmonella subsp. I' Salmonella subsp. I' Salmonella subsp. I'

S . Uganda S. lexington S . muenster S . anatum S. weltevreden S . elisabethville" Salmonella subsp. I' Salmonella subsp. I' Salmonella subsp. I'

S . liverpool S. senftenberg S. senftenberg

S . taksony S. kref eld

S. aberdeen S . brijbhumi S. telhashomer S . arizonae"

(subsp. 111,)

S. poona S. telelkebir S. kintambo S . idikan

S . lindern" S . charity" S. Caracas"

6 I 8 : b:e, n, z,, 8 : - : - (non-motile) 6,8:b:e,n1x 6 I 8 : z,, z,, : e, n, z , , 6,8:r:l,w

9,12 9,12 9,12 9,12 9,12 9,12 9,12

1, z,, : 1 ,5 g1p:- g,m:- f ,g,t:-

1,v:- -:1,5

-.-

3 I 10: 1, z,,: 1 , 5 3 10 : z,, : 1 ,5 3,10:e1h:1,5 3,10:e,h:1,6 3 I 10 :r: 2, 3,10:r:117

3110:r:- 3,lO:-:1,6

3 115:Z1,,:-

lI3,19:d:e,n, z,,

(R phase of g,slt) 1,3 I 19: i: 2, lI3,19:y:l,w

1,3,19:g,s,t:- 1,3,19: Z2.,:-

11: i:1,2 1l:i: 1,5 11 : zlo: e n x 1 1 : k : z,,

13,22:2:1,6 13,23:d:e,n, z,, 13,23:m1t:- 13,23:i:115

6,14:d:e1n1x 1,6,14,25:d:e,n,x 6,14:g1m,s:-

209

No. of isolates

2 1 1 1 1

3 4 4 5 1 1 3

1 1 1 1

1 1 1

2 10 TSANG. NIELSEN. BALSEVICIUS. KELLY. KHAKHRIA and JOHNSON

TABLE I ( Cr>,lrrnwd)

No. of Serogroup Serotype Antigenic formula isolates

I S . mpouto" S. hvittingfoss s . hull S. Vancouver S . gaminara

16:m,t:- 16:b:e,n,x 16:b: 1 I 2 16:c: 1,5 16:d: 1,7

1 4 1 1 3

1

2

1

4 1

1 1

2

1

1 1

1

1

1 1

J

K

S. jangwani" 17:a: 1,5

S. arizonae

S. cerro (subsp. 111,)

18: z , , z ,z : -

18 :Z,, Z2,:-

M S. pomona S. vitkin

28:y: 1,7 28:l,v:e,n,x

S. freetown S. inverness

38:y: 1,5 38:k: 1,6

P

S. wandsworth 39:b: 1,2

S . johannesburg 1,40:b:e,n,x

S. kingabwa S. ahuza"

43:y:1,5 43:k: 1 , 5

V S. arizonae (subsp. 111,)

44: 2, , z , , :-

45:d:1,6 w

X

S. dugbe

S. bergen S. phoenix

(subsp. 11)

47 : i : e, n, z , , 47:b: 1 I 5

Salmonella- subsp. I' 47 : z,,z,, : - 1

Y S. marina (subsp. IV) 48:q,z,,:- S. arizonae" 48:1,v:1,5,7

S. arizonae 48:i:z (subsp. 111,)

(subsp. (111,)

6 2

1

COMPETITIVE EL4 FOR SALMONELLA 21 i

No. of Serogroup Serotype Antigenic formula isolates

S . arizonae 48 : i : z,, 1 (subsp. 111,)

Z S. bonaire (subsp. IV) 50:z,,z,,:- S. wassenaar (subsp. IV) 50:g,z,,:-

60 S. arizonae 60:r:z (subsp. 111,)

(subsp. 111,) S. arizonae" 60 : zs2: z,,

61 S. arizonae 61:i:z

S . arizonae 61:k:1,5,7 (subsp. 111,)

(subsp. 111,)

1 1

1

1

1

3

66 Salmonella subsp. V++ 66:?- 1

Rough Rough S. arizonae (subsp. 111,) Rough Salmonella subsp. IV

9 1 1

*Serotype names were retained in this table for serotypes belonging to subsp. I1 and IV; their subsp. designations were given in brackets. Arizona strains were described in this table as S. arizonae with their subsp. designations (I& or UIb) given in brackets. Also, the first letter of serotype name was not given in capital letter as Salmonella Typhimurium; instead the conventional writing of S. typhimurium was used. **Denotes serotypes which were identified for the first time from within Canada. #Denotes monophasic variants of subsp. I strains. ##Serotypes which were withdrawn from the 1992 Kauffrnann-White Scheme; S. eimsbuettel = S. livingstone var. 14+; S. charity = [1],6,14,[25]:d:e,n,x. +These isolates were not S. gallinarumlb. pullorum. + +Subsp. V = subsp. bongori which has been proposed to be elevated to the level of a species. Salmonella bongori (Reeves et al. 1989). - 0-antigen 66 and H-antigen under investigation.

rare serotypes of Salmonella from diverse serogroups and subspecies background, we subjected 215 strains of suspected Salmonella for testing by the M105 competitive EIA. Biochemical testings had identified 7 of the 215 strains as either E. coli or C. freundii. Of the remaining 208 strains identified as Salmonella, 132 belonged to the more common serogroups of A to E (Gardner and Provine 1987; Hargrett-Bean et al. 1988), while 65 were of the higher 0 serogroups and 11 strains were serologically rough. The majority of strains belonged to subspecies I, with only 18 stains belonging to five other subspecies. Besides the common


serotypes that are endemic in Canada (Lior and Khakhria 1985: Natl. Enteric Ref. Centre 1980-1988). 9 serotypes which have been identified for the first time from within Canada, as well as 18 monophasic subspecies I variants, 6 nonmotile 0 forms and 1 1 rough strains were among the Salmonella strains examined in this study.

Results presented in this study do indicate that competitive EIA using MAb M10S has potential for the rapid identification of Salmonella. Besides the common serotypes that the competitive EL4 can identify. rare and uncommon serotypes as well as serotypes that were identified for the first time in Canada were also detected. These findings further extend and confirm the genus-specificity of the MAb M105. Previous studies (Tsang er al. 1991; Todd et al. 1991) had only tested either unknown serotypes or a limited number of strains from the 0 serogroups of G. H. K , L. N , 0. R. V . Y. Z. and 66. Of the 578 strains of Snlt~iotiell~i examined. only two strains [a S. arizotiae. subsp. III,, 1 , 1 3 . 2 3 : ~ ~ , z ~ ~ : - (Tsang er (11. 1991) and a serogroup 0 strain (Todd et al. 1991)] were found to be nonreactive with M105. Specificity of M105 was demonstrated by its lack of reactivity with 30 different species of Gram-positive and Gram-negative non- Salmotiellci bacteria (Tsang et al. 199 1 1. In another independent study by Todd er (11. (1991 ), only two strains of Etiterobacter agglomeratzs out of a total of 282 strains of non-Salmonelln Grarn-negative bacteria tested were found to demonstrate reaction with M105.

The unique specificity of MAb M105 and the wide margin of difference in the results of the competitive EIA given by the Salmonella strains from non-Salmonella organisms (more than 50% inhibition given by the Salmonella strains versus less than 30% inhibition given by the non-Salmonella organisms) are critical factors for the success of such an assay. The simplicity of the competitive EIA is another advantage for easy implementation of the test in laboratories where technical expertise and experience with the organism is lacking. When coupled with recent developments in various isolation media for Subnoriella (Cox 1993 ; Poisson 1992; Rambach 1990). the competitive EIA may have a role in same-day detection, identification. and confirmation of suspicious Salmonella colonies growing on primary isolation medium.

ACKNOWLEDGMENTS

We wish to thank David Woodward. Margaret Bell, and Gail Christie for iden- tifying and serotyping the Sulmonella strains, and Sheila Masson for preparing the bacterial cell suspension for the immunoassay.


REFERENCES

BAIRD-PARKER, A.C. 1990. Foodborne salmonellosis. Lancet 336, 1231-1235. COX, J.M. 1993. Lysine-Mannitol-Glycerol Agar, a medium for the isolation

of Salmonella spp., including S. @phi and atypical strains. Appl. Environ. Microbiol. 59, 2602-2606.

D’AOUST, J.Y. 1989. Salmonella. In Foodborne Bacterial Pathogens, (M.P. Doyle, ed.) pp. 327445, Marcel Dekker, New York.

EWING, W.H. 1986. Identi’cation of Enterobacteriaceae, 4th Ed., Elsevier Publ., New York.

GARDNER. P. and PROVINE, H.T. 1987. Manual of Acute Bacterial Infec- tion. Early Diagnosis and Treatment, 2nd Ed., pp. 235, Little, Brown, and Co., BostonIToronto.

HAAGERRAAD, N., HELMAN, T. and HAAGERRAAD, J. 1983. The effect of preinjection of mice with pristane on ascites tumor formation and monoclonal antibody production. J. Immunol. Methods 61, 317-320.

HARGRETT-BEAN, N.T., PAVIA, A.T. and TAUXE, R.V. 1988. Salmonella isolates from humans in the United States, 1984-1986. MMWR 37, Suppl.

KAUFFMANN, F. 1972. Serological Diagnosis of Salmonella Species: KaufSmann- White Scheme, Munksgaard, Copenhagen.

LENNETTE, E.H., BALOWS, A., HAUSLER, Jr., W.J. and SHADOMY, H.J. 1985. Manual of Clinical Microbiology, 4th Ed., Am. Society for Microbiology, Washington, D . C.

LIOR, H. and KHAKHRIA, R. 1985. Prevalance of salmonella serotypes and phage types in Canada (1973-1982). In Proceedings of the International Sym- posium on Salmonella, (G.H. Snoeyenbos, ed.) pp. 332-333. Am. Assoc. of Avian Pathologists, New Orleans.

Ntl. Enteric Ref. Centre. 1980-1988. Salmonella, Shigella and enteropathogenic E. coli identified in Canadian Monthly Reports, Laboratory Centre for Disease Control, Ottawa, Canada.

NIELSEN, K.H. and TSANG, R.S.W. 1992. Comparison of analytical sen- sitivities of three enzyme immunoassay procedures for the detection of Salmonella lipopolysaccharide. J. Rapid Methods Automation Microbiol. I ,

NIELSEN, K.H., TSANG, R.S.W., GARCIA, M.M., SURBUJBALLI, 0. and NEMEC. M. 1993. Competitive enzyme immunoassay for the detection of Salmonella lipopolysaccharide. J. Rapid Methods Automation Microbiol. I ,

SS-2, 25-3 1.

227-239.

305-3 14.

214 TSANG. KIELSEN. BALSEVICIUS. KELLY. KHAKHRIA and JOHNSON

POISSON. D.M. 1992. Novobiocin. brillant green, glycerol, lactose agar: a new medium for the isolation of Scibnonelltr strains. Res. Microbiol. (Instit. Pasteur) 143. 21 1-216.

POPOFF. M.Y. and LE MINOR. L. 1992. Antigenic formulas of the Sulmonella serovars (Kauffmann-White scheme). WHO Collaborating Centre for Reference and Research on Sulrnoriella. Institute Pasteur. Paris.

RAMBACH. A. 1990. New plate medium for facilitated differentiation of Sultnonrlln spp. from Proteus spp. and other enteric bacteria. Appl. Environ. Microbiol. 56. 301-303.

REEVES. M .W.. EVINS, G.M . . HEIBA, A.A., PLIKAYTIS, R.D. and FARMER. J . J . 1989. Clonal nature of Sulmot ie lh f y h i and its genetic relatedness to other salmonellae as shown by multilocus enzyme elec- trophorscis. and proposal of Sulrtiorzell~ boripri comb. nov. J . Clin. Microbiol.

SIMONSEN. B.. BRYAN. F.L.. CHRISTIAN. J.H.B.. ROBEREI’S, T.A., TOMPKIN. R.B. and SILLIKER. J.H. 1987. Prevention and control of salmonellas through application of HACCP. Int. J . Food Microbiol. 4,

TODD, E.C.D. er (11. 1991. Evaluation of Sohnonrlla antisera for an optimum enzyme-linked antibody detection of Sulr?ioriello using hydrophobic grid mem- brane filters. Food Microbiol. 8. 3 1 1-324.

TSANG, R.S.W. and NIELSEN. K.H. 1992. Immunoassays for Salmonella. Genet. Eng. Biotechnologist 12. 14-1 8.

TSANG. R.S.W., NIELSEN, K.H.. HENNING. M.D., SCHLECHT, S. and ALEKSIC. S. 1991. A murine monoclonal antibody that recognizes a genus- specific epitope in the Sulmonella lipopolysaccharide outer core. Zbl. Bakt. 274. 446355 .

VARNAM. A.H. and EVANS. M.G. 1991. Snlriioriell~i. In Foodborne Pathogens. An Illusrrured Texr. pp. 5 1-85, Wolfe Publishin? Ltd.. Aylesbury, England. Englantl.

WRIGHT. P.F.. KELLY, W.A. and GALL. D.E.J. 1985. Application of a tim- ing protocol to the reduction of inter-plate variability in the indirect enzyme imniunoassay for detection of anti-Brucrlla antibody. J . Imrnunoassay 6,

27, 331-340.

227-241.

I89-20S.

competitive enzyme immunoassay for the rapid identification of salmonella

Documents