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Use of ferrous sulphate and immunomagnetic separation to recover Salmonella enteritidis from raw eggs
International Journal ELSEVIER
International Journal of Food Microbiology 23 (1994) 149-158
of Food Microbiology
Use of ferrous sulphate and immunomagnetic separation to recover Salmonella enteritidis from raw eggs K o f i t s y o S. C u d j o e *, R a g n h i l d K r o n a , B e r i t G r 0 n , E g i l O l s e n Dynal Microbiology R&D, c / o Department of Pharmacology, Microbiology and Food Hygiene, Norwegian College of Veterinary Medicine, P.O. Box 8146 Dep, 0033 Oslo, Norway Received 25 November 1993; revision received 31 January 1994; accepted 10 March 1994
Abstract Contaminated eggs or foods containing eggs have been a source of food borne salmonellosis, with a significant proportion of these outbreaks being attributed to Salmonella enteritidis. Since the level of contamination in individual eggs or a pool of such eggs may be low, enrichment to increase cell numbers can take several days. Pre-enrichment of raw blended eggs which have been supplemented with ferrous sulphate at a concentration of 35 mg/1, for 6 h at 37°C, significantly enhanced the growth of Salmonella. Using Dynabeads ® Anti-Salmonella (Dynal, Osio), a new commercial product for selective enrichment of Salmonella from food samples, we have defined a protocol based on immunomagnetic separation, specially for raw eggs. The application of this protocol enables definitive detection of Salmonella enteritidis from eggs within 30 h. Keywords: Immunomagnetic separation; Salmonella enteritidis; Raw eggs
I. Introduction
C o n t a m i n a t e d eggs o r foods c o n t a i n i n g eggs have b e e n a s o u r c e of f o o d b o r n e salmonellosis, with a significant p r o p o r t i o n of t h e s e o u t b r e a k s b e i n g a t t r i b u t e d to Salmonella enteritidis. (St. Louis et al., 1988; Stevens et al., 1989; C e n t e r s for D i s e a s e C o n t r o l , 1990). A l t h o u g h s t o r a g e t e m p e r a t u r e a b u s e over long p e r i o d s o f t i m e can n a t u r a l l y l e a d to high n u m b e r s (Lin et al., 1988; T e l z a k et al., 1990), the level o f S. enteritidis c o n t a m i n a t i o n in n a t u r a l l y i n f e c t e d eggs is g e n e r a l l y r e g a r d e d
* Corresponding author. Tel. + 47 (22) 964835. Fax + 47 (22) 964814. 0168-1605/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1605(94)00029-6
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as low (Humphrey et al., 1991). A direct and rapid assessment of freshly laid eggs in an effort to reduce the risk of contamination posed to the public would be beneficial to both egg producers and consumers. However, the feasibility of such a proposition is yet to be proven because lysozyme, ovotransferrin and the alkaline pH in egg white are known to kill or inhibit the growth of a wide variety of microorganisms (Haines, 1939; Brooks, 1960). Salmonellae are known to secrete their own chelators during extended storage which enables them to compete with ovotransferrin, and therefore are able to survive and grow in eggs (Garibaldi, 1970). Humphrey et al. (1991), indicated that S. enteritidis is able to survive in the albumen and grow depending on the age of the egg and the proximity of the organism to the yolk membrane. Humphrey et al. (1989) diluted the egg contents prior to enrichment, thus indirectly reducing the effect of inhibitors before proceeding with selective enrichment followed by plating. Gast (1993), on the other hand, took partial advantage of salmonella's own mechanisms to circumvent the effect of ovotransferrin by incubating the egg contents for 2 or 5 days at 37°C or 25°C respectively. Thereafter, a ten-fold dilution of the eggs in a non-selective broth medium was incubated for 24 h at 37°C followed by a further ten-fold dilution in a selective broth; which was also incubated for 24 h at 37°C before plating. Thus enrichment to increase cell numbers has consequently generally taken 48 h or in some instances, days before plating for presumptive identification. In an attempt to reduce the time used for enrichment, immunomagnetic separation (Skjerve et al., 1990; Skjerve and Olsvik, 1991; Cudjoe et al., 1993) was employed using Dynabeads ® Anti-Salmonella (Dynal, Oslo, Norway). This is a new kind of selective enrichment product, which enables the detection of low numbers (2 log units) of salmonellae per ml of food samples containing high numbers ( > 10 6) of background flora. Iron in the form of ferrous sulphate has been demonstrated to promote the growth of Gram-negative bacteria in eggs (Garibaldi, 1960; Clay and Board, 1991) and its successful use at levels of 35 m g / l in a non-selective broth to isolate Salmonella from raw eggs has been reported (Gast and Beard, 1992; Gast, 1993). A protocol for the isolation and detection of Salmonella enteritidis in raw eggs based on the phenomenon of growth promotion using ferrous sulphate together with immunomagnetic separation is described in this paper.
2. Material and methods Preparation o f inoculum. Salmonella enteritidis phage type 8 (Strain number 64),
obtained from the National Institute of Public Health (Oslo, Norway), was grown for 20 h in Brain Heart Infusion broth (Difco Laboratories, Detroit, MI) and the optical density of the culture adjusted in a spectrophotometer to OD620n m = 0.036 corresponding to ca. 5 × 107 cfu/ml. Decimal dilutions in buffered peptone water (BPW; Difco), were used in the inoculation experiments described below.
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Immunomagnetic separation followed by plating. Twenty microlitre aliquots of Dynabeads ® Anti-Salmonella (Dynal) were dispensed into either 1.5 ml Eppendorf tubes or 15 ml conical tubes with a screw-cap. One or 10 ml of samples to be analyzed were then added to the respective tubes and the capped tubes incubated for 10 min at ambient temperature on a rotating device (Rotamix RK, Heto Lab Equipment, Aller0d, Denmark). Gentle rotation during incubation prevents particles from settling and improves the binding kinetics. At the end of incubation, particles were sedimented using the appropriate magnetic particle concentrator (MPC®-M or MPC®-6, Dynal), and the sample supernatant aspirated and discarded. After two washes with 1 ml of phosphate buffered saline, pH 7.5, containing 0.05% Tween 20 (PBS-T), the beads-bacteria complex was taken up with 50 ~1 of PBS-T and streaked onto XLD agar. All plates were incubated at 37°C for 24 h before reading. Particle loss during IMS of Salmonella from eggs. To study the extent of particle loss in eggs during IMS, a mouse monoclonal antibody was I125-1abelled and immunoadsorbed onto Dynabeads ® M-280 sheep anti-mouse IgG. These iodinelabelled antibody coated particles were then used in a standard IMS procedure with undiluted and three-, five- and ten-fold dilutions of blended raw egg mixtures and particle recovery in each case monitored by a Gamma counter (Packard Auto-Gamma-5650, Packard Instrument Company. Illinois, USA). Growth of Salmonella during a short enrichment in egg. The contents of one egg was mixed and blended by homogenizing in a stomacher (IUL Instruments, Barcelona, Spain) and two-, five, and ten-fold dilutions in BPW prepared. Ten ml volumes of undiluted and diluted samples were seeded with ca. 10 cells of S. enteritidis and incubated at 37°C for 4 h followed by direct spread-plating of 100 /~1 of each sample onto XLD medium, and IMS analysis of 1 ml aliquots in triplicate. In a further experiment, a mixed pool of ten eggs (ca. 500 ml), was seeded with ca. 10 S. enteritidis cells/ml and blended as above. Two-, five- and ten-fold dilutions of the seeded egg pool were made in BPW to obtain ca. 5, 2 and 1 cells/ml respectively, incubated at 37°C for 4 h and examined by direct spread-plating and IMS analysis as above. Effect of ferrous sulphate on the growth of Salmonella in eggs. S. enteritidis was seeded into the blended contents of two sets of undiluted and three-, five and ten-fold dilutions of eggs, to give ca. 15 cells/ml. Ferrous sulphate was added to one set of samples to a final concentration of 35 mg/l. After 4 h at 37°C, 100 ~l of each sample was directly spread-plated onto XLD agar and the plates incubated for 24 h at 37°C. Recovery of Salmonella from pooled raw eggs supplemented with ferrous sulphate. To simulate very low levels of natural contamination of only Salmonella, the contents of ten eggs were pooled and each pool seeded with 1-2 cells of S. enteritidis on three occasions. Ferrous sulphate solution was added to each pool to a final
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concentration of 35 mg/1 before incubation at 37°C. After 4 and 6 and 24 h, 10 ml of the egg mixture was removed and diluted five-fold. IMS analysis as well as direct plating of 100/xl of sample aliquots was carried out in duplicate on 1 ml aliquots of the diluted egg mixture as described previously. In a subsequent analysis of 12 pools of 10 eggs, the volume of particles used was increased from 20 to 40 pA and incubation time extended from 10 to 30 min. Ferrous sulphate supplemented samples were incubated for 4, 6 and 24 h. At each sampling time, 10 ml of the egg mixture was removed and two- and five-fold dilutions made. Both one and 10 ml aliquots of the two- and five-fold diluted samples were subjected to IMS analysis whilst 100/xl of the undiluted samples was directly plated at each time interval.
3. Results
Particle loss during IMS of Salmonella from eggs. Table 1 depicts the extent of particle loss in different dilutions of raw egg mixtures. The more viscous, undiluted mixtures recorded the highest particle loss of 78% compared with 8% for ten-fold diluted samples. Three- and five-fold dilutions of the egg samples resulted in 47 and 16% particles losses respectively. Growth of Salmonella in raw egg during a short enrichment. Growth of S. enteritidis in different dilutions of raw eggs as measured by direct plating after 4 h enrichment, was greatest (3.5 log cycles) in the ten-fold diluted sample and lowest (ca. 2.5 log cycles) in the undiluted egg sample (Fig. 1). For all dilutions, absolute counts obtained using IMS analysis were lower than in the corresponding direct plated samples. The difference was greatest for the undiluted samples which is consistent with the lower recovery of particles from viscous samples (Table 1). Effect of ferrous sulphate on the growth of Salmonella in eggs. The addition of ferrous sulphate at levels of 35 m g / l to counteract the effect of ovotransferrin as a chelating agent (Garibaldi, 1960; Clay and Board, 1991; Gast, 1993), significantly enhanced the growth of S. enteritidis seeded into the blended contents of undiluted raw eggs compared to unsupplemented samples (Table 2). Over 1 log unit
Table 1 Level of iodine-labelled particles recovered from dilutions of blended egg mixtures after IMS Dilutions of egg
Undiluted 1 :3 1:5 1 : 10
1125 labelle d particle counts (cpm) Initial
Final
25 458 25 600 25814 26360
5 579 13 648 21 754 24126
% Particle recovered
22.0 53.0 84.0 92.0
K.S. Cudjoe et al. / International Journal of Food Microbiology 23 (1994) 149-158
153
Log cfu/ml 4
o
Undiluted
1:2
1:5
1:10
Dilutions of raw eggs Log cfu/ml 4
0
BI~ Colonycounts
Undiluted
1:2
1:5
m
Recoveredby IMS
--
Initialinoculum
1:I0
Dilutions of raw eggs
Fig. l. Salmonella enteritidis colony counts using direct and IMS plating after 4 h enrichment at 37°C when initially seeded into undiluted and different dilutions of blended raw eggs at constant (A) and different (B) levels.
more of growth was achieved in the supplemented undiluted raw eggs. Two and five-fold supplemented dilutions achieved 0.7 and 0.6 log units more growth respectively than the non-supplemented dilutions of eggs. No significant growth
Table 2 Growth measurement of seeded Salmonella enteritidis (15 cells/ml) after a 4 h enrichment with and without ferrous sulphate supplementation in undiluted and dilutions of raw egg Dilutions of egg
Undiluted 1:3 1:5 1 : 10 a
Mean of three results.
Mean a colony counts (log c f u / m l ) from egg samples With ferrous sulphate
Without ferrous sulphate
3.0 3.1 3.2 2.7
1.8 2.4 2.6 2.8
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Table 3 Effect of incubation time on recovery of Salmonella enteritidis (seeded at levels of 1-2 cells/500 ml) from iron-supplemented raw eggs Raw egg p o o l samples
1
2
Sampling period
Total
Direct plating of undiluted egg pool
IMS b analysis from 1 ml of five-fold diluted egg pool
4
m
m
6
m
__
+++ -
+++ -
-
-
+++
+++ -
+++ 0 0 3
+++ 0 0 3
24 4 6
3
Level a of Salmonella and samples detected by method
24 4 6 24 4 6 24
a Plates with typical Salmonella colonies between 1 and l0 were recorded with a plus sign (+), between 10 and 100 colonies with a two plus sign (+ +), and more than 100 colonies with a three-plus sign (+ + +). b IMS analysis was done using 20 txl of particles with 10 min of incubation.
difference was observed in ten-fold d i l u t e d samples with or without ferrous sulphate supplementation.
Recovery o f Salmonella from pooled raw eggs supplemented with ferrous sulphate. O n t h r e e s e p a r a t e occasions, w h e n a pool of t e n eggs was seeded with 1 - 2
S.
enteritidis cells a n d e n r i c h e d for up to 24 h with ferrous s u l p h a t e s u p p l e m e n t a t i o n , IMS analysis (20 /xl of particles, 10 m i n i n c u b a t i o n ) of five-fold dilutions of aliquots a n d direct p l a t i n g of 100 /xl of u n d i l u t e d samples after 4 a n d 6 h gave negative results ( T a b l e 3). However, IMS a n d direct plating of the same samples after 24 h e n r i c h m e n t were positive. T a b l e 4 shows the results of IMS analysis using 4 0 / x l of particles a n d 30 m i n i n c u b a t i o n d u r i n g IMS as well as direct plating of the 12 e n r i c h e d ferrous s u l p h a t e s u p p l e m e n t e d ten-egg pools i n o c u l a t e d with 1 - 2 s a l m o n e l l a e . A f t e r 6 h e n r i c h m e n t , IMS b a s e d o n 1 a n d 10 ml sample volumes recovered Salmonella from 11 a n d 12 respectively out of 12 such egg pools, whilst direct p l a t i n g of the u n d i l u t e d samples yielded only six as positive for Salmonella. All samples were positive for Salmonella after 24 h e n r i c h m e n t irrespective of m e t h o d used in this study.
4. Discussion A c c o r d i n g to G a r i b a l d i (1960) lysozyme, c o n a l b u m i n (ovotransferrin) a n d alkaline p H in egg white kill or inhibit the growth of a wide variety of microorganisms,
K.S. Cudjoe et al. / International Journal of Food Microbiology 23 (1994) 149-158
155
but the yolk, or a mixture of the yolk and albumen has very little inhibitory effect (Haines, 1939; Brooks, 1960). The level of Salmonella enteritidis contamination in naturally infected eggs are low (Humphrey et al., 1991) and enrichment of raw eggs to increase the numbers of salmonellae generally takes several days before plating for presumptive identification (Humphrey et al. 1989; Gast and Beard, 1992; Gast, 1993). The growth enhancing abilities of ferrous sulphate on Gram-negative bacteria has been demonstrated (Garibaldi, 1960) but its application to Salmonella detection from raw eggs has been limited to its use only in enrichment broths (Gast and Beard, 1992; Gast, 1993), without any overall saving in time. In our approach, unlike that of Gast (1993), ferrous sulphate was added directly to raw, blended and undiluted eggs prior to enrichment. This significantly enhanced the growth of seeded S. enteritidis in eggs and eliminated the need for dilution of raw eggs prior to enrichment. This enhanced growth enabled subsequent significant recovery of Salmonella after 6 h enrichment by IMS using Dynabeads ® Anti-Salmonella. After 24 h enrichment both IMS analysis and direct plating recorded high levels of Salmonella starting from an initial inoculum of 1-2 cells in ca. 500 ml raw blended eggs. Our results show clearly that during abbreviated enrichment of 4 h duration, the mixture of the yolk and albumen still had a significant inhibitory effect on the growth of Salmonella. This inhibition, however, appears to be completely neutralized by the addition of ferrous sulphate to allow sufficient growth within 6 h for subsequent detection. The level of target bacteria recovered when using Dynabeads ® Anti-Salmonella, is directly correlated to the extent of the particle-bacteria complex recovered after IMS. Particle loss during IMS due to the influence of certain types of sample matrices can be sufficient to significantly affect the results of the analysis (Skjerve and Olsvik, 1991). The more viscous the sample matrix, for example undiluted raw blended eggs, the higher the loss of particles used in the analysis (Table 1). Whilst a ten-fold dilution of raw egg samples resulted in only 8% loss of particles, such a high dilution would concomitantly reduce the level of Salmonella present per ml of sample for analysis. If raw egg samples with an initial low level of natural contamination were supplemented with ferrous sulphate prior to enrichment, a 16% particle loss due to a five-fold dilution of sample would not significantly affect the sensitivity of the system. Samples must be thoroughly mixed, preferably by homogenizing in a stomacher before taking aliquots for dilutions and subsequent analysis. This is very important for all sampling and detection methods since lumps a n d / o r colloidal particles of egg white not properly blended with the yolk, could harbour Salmonella that are growing in localized sites in the blended pools and could be missed. Since the presence of one Salmonella cell in 25 g or a pooled contents of raw eggs must be detected, it is imperative that some form of pre-enrichment be undertaken when examining pooled samples. For pooled raw eggs (> 500 ml), supplemented with ferrous sulphate as described above, IMS alone enables the concentration of low levels of the target bacteria from larger volumes of the diluted samples for detection. IMS-analysis on five-fold dilutions of pooled eggs samples using 20 /~1 of Dynabeads ® Anti-Salmonella and incubating for 10 min
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Table 4 Effect of incubation time on recovery of from iron-supplemented Raw egg pool
Sampling
samples
p e r i o d (h)
1
24 2
+
++
+++
+++
-
-
4 -
+
++
+++
+++
+++
4
-
+
+
+
++
+++
+++
+++
4
-
+
+
6
+
+
++
+++
+++
+++ ++
4
-
6
-
+
+++
+++
4
+
+
+++
+++
-
-
-
+
++
+++
+++
+++
-
-
+
+
++
+++
+++
+++
-
+++
4 6 24 4 6
+++ +
4 24
-
+
+
+++
+++
+++
4
-
--
-
6
+
+
+
+++
+++
+++ +
24
24 4
-
6
-
-
+++
+++
24 Totals
++
+++
6
12
--
+
+
6
11
--
+
6
24
ll/
diluted egg pool
+++
24
9
l0 ml 5-fold
diluted egg pool
-
24
8
1 ml 2-fold
egg pool
-
24
7
IMS b analysis from:
of undiluted
+++
6
6
Direct plating
a n d s a m p l e s d e t e c t e d by m e t h o d
-
6
5
Salmonella
+++
24 4
( s e e d e d at l e v e l s o f 1 - 2 c e l l s / 5 0 0
4 24
3
Level a of
4
6
Salmonella enteritidis
raw eggs
+++
4
[I
2
3
6
6
11
12
24
12
12
12
a See Table 3 for legend. b I M S a n a l y s i s w a s d o n e u s i n g 4 0 ~1 o f p a r t i c l e s w i t h 30 rain o f i n c u b a t i o n ,
ml)
K.S. Cudjoe et al. / International Journal of Food Microbiology 23 (1994) 149-158
157
with continuous mixing did not, however, recover the seeded Salmonella (Table 3). When the analysis was subsequently repeated using 40 instead of 20 Fzl of the beads and incubating for 30 instead of 10 min, recovery of the seeded Salmonella cells was significantly improved. There was no significant difference between the number of positive samples detected using either 1 ml of two- and 10 ml of five-fold dilutions for IMS (Table 4). The increased particle volume compensated for the particles lost, especially in the two-fold diluted sample matrix, while the extended incubation improved the binding efficiency of the particles to the target bacteria in both instances. On the basis of the above results, we propose the following protocol for detection of S. enteritidis from raw eggs: 1. Aseptically crack open the egg or eggs and mix/blend thoroughly both white and yolk and add ferrous sulphate solution to a final concentration of 35 rag/1. 2. Pre-incubate the egg mixture at 37°C. 3. After 6 h of pre-incubation, mix the egg mixture thoroughly, remove and dilute an aliquot five-fold in BPW and further remove either 1 or 10 ml from the diluted aliquot for IMS. 4. Reincubate the egg mixture for a further 18 h. 5. For IMS analysis, pipette 40 /zl of Dynabeads ® Anti-Salmonella into either an Eppendorf tube or 15 ml tube with screw cap. Add either 1 or 10 ml diluted sub-sample depending on the tube of choice. 6. Incubate with constant rotation for 30 min and wash once in PBS-T with the help of a suitable magnetic particle concentrator (MPC®-M, MPC®-I or MPC®-6; Dynal). 7. Resuspend the particle-bacteria complex in 50 ~I of wash buffer and streak plate 25 pA onto two different Salmonella differential diagnostic media, for example, xylose lysine desoxycholate agar (XLD) and brilliant green agar (BGA). 8. Incubate plates at 37°C for 24 h and pick out presumptive Salmonella colonies for serological and biochemical confirmation. 9. If further confirmation of the results are needed, repeat steps 5 - 7 on the 24 h enriched culture and directly streak-plate a loopful of the undiluted egg onto each of the two Salmonella diagnostic media. Extended storage of cracked eggs or egg-containing foods at warm temperatures provide ample opportunity for extensive bacterial growth (Lin et al., 1988; Telzak et al., 1990). For such samples, IMS analysis should be carried out on 1 ml of ten-fold diluted sample aliquots after 24 h pre-enrichment with ferrous sulphate supplementation. In such a scenario a reversion to the original protocol using 20 pA of Dynabeads ® Anti-Salmonella and incubating only for 10 min with continuous or occasional mixing is necessary to avoid unnecessary non-specific binding of diverse background flora to the particles. References Brooks, J. (1960) Mechanism of the multiplication of Pseudomonas in the hen's egg. J. Appl. Bacteriol., 23, 499-509.
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Centers for Disease Control (1990) Update: Salmonella enteritidis infections and shell-eggs, United States, 1990. Morbid. Mortal. Weekly Rep. 37, 490-496. Clay, C.E. and Board, R.G. (1991) Growth of Salmonella enteritidis in artificially contaminated hens' shells eggs. Epidemiol. lnfect. 106, 271-281. Cudjoe, K.S., Patel, P.D., Olsen, E., Skjerve, E. and Olsvik, O (1993) Immunomagnetic separation techniques for detection of pathogenic bacteria in foods. In: R.G. Kroll, A. Gilmour and M. Sussman (editors), New Techniques in Food and Beverage Microbiology. Society for Applied Bacteriology Technical Series, 31. Blackwell, London, pp. 17 29. Garibaldi, J.A. (1960) Factors in egg white which control growth of bacteria. Food Res. 25, 337 344. Garibaldi, J.A. (1970) Role of microbial iron transport compounds in the bacterial spoilage of eggs. Appl. Microbiol. 20, 558-560. Gast, R.K. and Beard, C.W. (1992) Detection and enumeration of Salmonella enteritidis in fresh and stored eggs laid by experimentally infected hens. J. Food Prot. 55, 152-156. Gast, R.K. (1993) Recovery of Salmonella enteritidis from inoculated pools of egg contents. J. Food Prot. 56, 21 24. Haines, R.B. (1939) Microbiology in the preservation of the hen's egg. G.P. Dep. Sci. Ind. Res. Food Invest. Board, Spec. Rep. 47. Humphrey, T.J., Greenwood, M., Gilbert, R.J., Rowe, B. and Chapman, P.A. (1989) The survival of salmonellas in shell eggs cooked under simulated domestic conditions. Epidemiol. Infect. 103. 35 -45. Humphrey, T.J., Whitehead, A., Gawler, A.H.L., Henley, A. and Rowe, B. (1991) Numbers of Salmonella enteritidis in the contents of naturally contaminated hens' eggs. Epidemiol. Infect. 103,489 496. Lin, F.-Y., Morris, J.G., Trump, D., Jr., Tilghman, D., Wood, P.K., Jackman, N., Israel, E. and Libonati, J.P. (1988) Investigation of an outbreak of Salmonella enteritidis gastroenteritidis associated with consumption of eggs in a restaurant chain in Maryland. Am. J. Epidemiol. 128, 839-844. Skjerve, E., R~rvik, L.M. and Olsvik, 0. (1990) Detection of Listeria monocytogenes in foods using immunomagnetic separation. Appl. Environ. Microbiol. 56, 3478-3481. Skjerve, E. and Olsvik, ~. (1991) Immunomagnetic separation of Salmonella from foods. Int. J. Food Microbiol. 14, 11-18. St. Louis, M.E., Morse, D.L., Potter, E., DeMelfi, T.M., Guzewich, J.J., Tauxe, R.V. and Blake, P.A. (1988) The emergence of grade A eggs as a major source of Salmonella enteritidis infections: new implications for control of salmonellosis. J. Am. Med. Assoc. 259, 2103-2107 Stevens, A., Joseph, C., Bruce, J., Fenton, D., O'Mahony, M., Cunnigham, D., O'Conner, B. and Rowe, B. (1989) A large outbreak of Salmonella enteritidis phage type 4 associated with eggs from overseas. Epidemiol. Infect. 103, 25-433. Telzak, E.E., Budnick, L.D., Greenberg, M.S.Z., Blum, S., Shayegani, M., Benson, C.E. and Schultz, S. (1990) A nosocomial outbreak of Salmonella enteritidis infection due to the consumption of raw eggs. N. Engl. J. Med. 323, 394-397.
International Journal of Food Microbiology 23 (1994) 149-158
of Food Microbiology
Use of ferrous sulphate and immunomagnetic separation to recover Salmonella enteritidis from raw eggs K o f i t s y o S. C u d j o e *, R a g n h i l d K r o n a , B e r i t G r 0 n , E g i l O l s e n Dynal Microbiology R&D, c / o Department of Pharmacology, Microbiology and Food Hygiene, Norwegian College of Veterinary Medicine, P.O. Box 8146 Dep, 0033 Oslo, Norway Received 25 November 1993; revision received 31 January 1994; accepted 10 March 1994
Abstract Contaminated eggs or foods containing eggs have been a source of food borne salmonellosis, with a significant proportion of these outbreaks being attributed to Salmonella enteritidis. Since the level of contamination in individual eggs or a pool of such eggs may be low, enrichment to increase cell numbers can take several days. Pre-enrichment of raw blended eggs which have been supplemented with ferrous sulphate at a concentration of 35 mg/1, for 6 h at 37°C, significantly enhanced the growth of Salmonella. Using Dynabeads ® Anti-Salmonella (Dynal, Osio), a new commercial product for selective enrichment of Salmonella from food samples, we have defined a protocol based on immunomagnetic separation, specially for raw eggs. The application of this protocol enables definitive detection of Salmonella enteritidis from eggs within 30 h. Keywords: Immunomagnetic separation; Salmonella enteritidis; Raw eggs
I. Introduction
C o n t a m i n a t e d eggs o r foods c o n t a i n i n g eggs have b e e n a s o u r c e of f o o d b o r n e salmonellosis, with a significant p r o p o r t i o n of t h e s e o u t b r e a k s b e i n g a t t r i b u t e d to Salmonella enteritidis. (St. Louis et al., 1988; Stevens et al., 1989; C e n t e r s for D i s e a s e C o n t r o l , 1990). A l t h o u g h s t o r a g e t e m p e r a t u r e a b u s e over long p e r i o d s o f t i m e can n a t u r a l l y l e a d to high n u m b e r s (Lin et al., 1988; T e l z a k et al., 1990), the level o f S. enteritidis c o n t a m i n a t i o n in n a t u r a l l y i n f e c t e d eggs is g e n e r a l l y r e g a r d e d
* Corresponding author. Tel. + 47 (22) 964835. Fax + 47 (22) 964814. 0168-1605/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1605(94)00029-6
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as low (Humphrey et al., 1991). A direct and rapid assessment of freshly laid eggs in an effort to reduce the risk of contamination posed to the public would be beneficial to both egg producers and consumers. However, the feasibility of such a proposition is yet to be proven because lysozyme, ovotransferrin and the alkaline pH in egg white are known to kill or inhibit the growth of a wide variety of microorganisms (Haines, 1939; Brooks, 1960). Salmonellae are known to secrete their own chelators during extended storage which enables them to compete with ovotransferrin, and therefore are able to survive and grow in eggs (Garibaldi, 1970). Humphrey et al. (1991), indicated that S. enteritidis is able to survive in the albumen and grow depending on the age of the egg and the proximity of the organism to the yolk membrane. Humphrey et al. (1989) diluted the egg contents prior to enrichment, thus indirectly reducing the effect of inhibitors before proceeding with selective enrichment followed by plating. Gast (1993), on the other hand, took partial advantage of salmonella's own mechanisms to circumvent the effect of ovotransferrin by incubating the egg contents for 2 or 5 days at 37°C or 25°C respectively. Thereafter, a ten-fold dilution of the eggs in a non-selective broth medium was incubated for 24 h at 37°C followed by a further ten-fold dilution in a selective broth; which was also incubated for 24 h at 37°C before plating. Thus enrichment to increase cell numbers has consequently generally taken 48 h or in some instances, days before plating for presumptive identification. In an attempt to reduce the time used for enrichment, immunomagnetic separation (Skjerve et al., 1990; Skjerve and Olsvik, 1991; Cudjoe et al., 1993) was employed using Dynabeads ® Anti-Salmonella (Dynal, Oslo, Norway). This is a new kind of selective enrichment product, which enables the detection of low numbers (2 log units) of salmonellae per ml of food samples containing high numbers ( > 10 6) of background flora. Iron in the form of ferrous sulphate has been demonstrated to promote the growth of Gram-negative bacteria in eggs (Garibaldi, 1960; Clay and Board, 1991) and its successful use at levels of 35 m g / l in a non-selective broth to isolate Salmonella from raw eggs has been reported (Gast and Beard, 1992; Gast, 1993). A protocol for the isolation and detection of Salmonella enteritidis in raw eggs based on the phenomenon of growth promotion using ferrous sulphate together with immunomagnetic separation is described in this paper.
2. Material and methods Preparation o f inoculum. Salmonella enteritidis phage type 8 (Strain number 64),
obtained from the National Institute of Public Health (Oslo, Norway), was grown for 20 h in Brain Heart Infusion broth (Difco Laboratories, Detroit, MI) and the optical density of the culture adjusted in a spectrophotometer to OD620n m = 0.036 corresponding to ca. 5 × 107 cfu/ml. Decimal dilutions in buffered peptone water (BPW; Difco), were used in the inoculation experiments described below.
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151
Immunomagnetic separation followed by plating. Twenty microlitre aliquots of Dynabeads ® Anti-Salmonella (Dynal) were dispensed into either 1.5 ml Eppendorf tubes or 15 ml conical tubes with a screw-cap. One or 10 ml of samples to be analyzed were then added to the respective tubes and the capped tubes incubated for 10 min at ambient temperature on a rotating device (Rotamix RK, Heto Lab Equipment, Aller0d, Denmark). Gentle rotation during incubation prevents particles from settling and improves the binding kinetics. At the end of incubation, particles were sedimented using the appropriate magnetic particle concentrator (MPC®-M or MPC®-6, Dynal), and the sample supernatant aspirated and discarded. After two washes with 1 ml of phosphate buffered saline, pH 7.5, containing 0.05% Tween 20 (PBS-T), the beads-bacteria complex was taken up with 50 ~1 of PBS-T and streaked onto XLD agar. All plates were incubated at 37°C for 24 h before reading. Particle loss during IMS of Salmonella from eggs. To study the extent of particle loss in eggs during IMS, a mouse monoclonal antibody was I125-1abelled and immunoadsorbed onto Dynabeads ® M-280 sheep anti-mouse IgG. These iodinelabelled antibody coated particles were then used in a standard IMS procedure with undiluted and three-, five- and ten-fold dilutions of blended raw egg mixtures and particle recovery in each case monitored by a Gamma counter (Packard Auto-Gamma-5650, Packard Instrument Company. Illinois, USA). Growth of Salmonella during a short enrichment in egg. The contents of one egg was mixed and blended by homogenizing in a stomacher (IUL Instruments, Barcelona, Spain) and two-, five, and ten-fold dilutions in BPW prepared. Ten ml volumes of undiluted and diluted samples were seeded with ca. 10 cells of S. enteritidis and incubated at 37°C for 4 h followed by direct spread-plating of 100 /~1 of each sample onto XLD medium, and IMS analysis of 1 ml aliquots in triplicate. In a further experiment, a mixed pool of ten eggs (ca. 500 ml), was seeded with ca. 10 S. enteritidis cells/ml and blended as above. Two-, five- and ten-fold dilutions of the seeded egg pool were made in BPW to obtain ca. 5, 2 and 1 cells/ml respectively, incubated at 37°C for 4 h and examined by direct spread-plating and IMS analysis as above. Effect of ferrous sulphate on the growth of Salmonella in eggs. S. enteritidis was seeded into the blended contents of two sets of undiluted and three-, five and ten-fold dilutions of eggs, to give ca. 15 cells/ml. Ferrous sulphate was added to one set of samples to a final concentration of 35 mg/l. After 4 h at 37°C, 100 ~l of each sample was directly spread-plated onto XLD agar and the plates incubated for 24 h at 37°C. Recovery of Salmonella from pooled raw eggs supplemented with ferrous sulphate. To simulate very low levels of natural contamination of only Salmonella, the contents of ten eggs were pooled and each pool seeded with 1-2 cells of S. enteritidis on three occasions. Ferrous sulphate solution was added to each pool to a final
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concentration of 35 mg/1 before incubation at 37°C. After 4 and 6 and 24 h, 10 ml of the egg mixture was removed and diluted five-fold. IMS analysis as well as direct plating of 100/xl of sample aliquots was carried out in duplicate on 1 ml aliquots of the diluted egg mixture as described previously. In a subsequent analysis of 12 pools of 10 eggs, the volume of particles used was increased from 20 to 40 pA and incubation time extended from 10 to 30 min. Ferrous sulphate supplemented samples were incubated for 4, 6 and 24 h. At each sampling time, 10 ml of the egg mixture was removed and two- and five-fold dilutions made. Both one and 10 ml aliquots of the two- and five-fold diluted samples were subjected to IMS analysis whilst 100/xl of the undiluted samples was directly plated at each time interval.
3. Results
Particle loss during IMS of Salmonella from eggs. Table 1 depicts the extent of particle loss in different dilutions of raw egg mixtures. The more viscous, undiluted mixtures recorded the highest particle loss of 78% compared with 8% for ten-fold diluted samples. Three- and five-fold dilutions of the egg samples resulted in 47 and 16% particles losses respectively. Growth of Salmonella in raw egg during a short enrichment. Growth of S. enteritidis in different dilutions of raw eggs as measured by direct plating after 4 h enrichment, was greatest (3.5 log cycles) in the ten-fold diluted sample and lowest (ca. 2.5 log cycles) in the undiluted egg sample (Fig. 1). For all dilutions, absolute counts obtained using IMS analysis were lower than in the corresponding direct plated samples. The difference was greatest for the undiluted samples which is consistent with the lower recovery of particles from viscous samples (Table 1). Effect of ferrous sulphate on the growth of Salmonella in eggs. The addition of ferrous sulphate at levels of 35 m g / l to counteract the effect of ovotransferrin as a chelating agent (Garibaldi, 1960; Clay and Board, 1991; Gast, 1993), significantly enhanced the growth of S. enteritidis seeded into the blended contents of undiluted raw eggs compared to unsupplemented samples (Table 2). Over 1 log unit
Table 1 Level of iodine-labelled particles recovered from dilutions of blended egg mixtures after IMS Dilutions of egg
Undiluted 1 :3 1:5 1 : 10
1125 labelle d particle counts (cpm) Initial
Final
25 458 25 600 25814 26360
5 579 13 648 21 754 24126
% Particle recovered
22.0 53.0 84.0 92.0
K.S. Cudjoe et al. / International Journal of Food Microbiology 23 (1994) 149-158
153
Log cfu/ml 4
o
Undiluted
1:2
1:5
1:10
Dilutions of raw eggs Log cfu/ml 4
0
BI~ Colonycounts
Undiluted
1:2
1:5
m
Recoveredby IMS
--
Initialinoculum
1:I0
Dilutions of raw eggs
Fig. l. Salmonella enteritidis colony counts using direct and IMS plating after 4 h enrichment at 37°C when initially seeded into undiluted and different dilutions of blended raw eggs at constant (A) and different (B) levels.
more of growth was achieved in the supplemented undiluted raw eggs. Two and five-fold supplemented dilutions achieved 0.7 and 0.6 log units more growth respectively than the non-supplemented dilutions of eggs. No significant growth
Table 2 Growth measurement of seeded Salmonella enteritidis (15 cells/ml) after a 4 h enrichment with and without ferrous sulphate supplementation in undiluted and dilutions of raw egg Dilutions of egg
Undiluted 1:3 1:5 1 : 10 a
Mean of three results.
Mean a colony counts (log c f u / m l ) from egg samples With ferrous sulphate
Without ferrous sulphate
3.0 3.1 3.2 2.7
1.8 2.4 2.6 2.8
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Table 3 Effect of incubation time on recovery of Salmonella enteritidis (seeded at levels of 1-2 cells/500 ml) from iron-supplemented raw eggs Raw egg p o o l samples
1
2
Sampling period
Total
Direct plating of undiluted egg pool
IMS b analysis from 1 ml of five-fold diluted egg pool
4
m
m
6
m
__
+++ -
+++ -
-
-
+++
+++ -
+++ 0 0 3
+++ 0 0 3
24 4 6
3
Level a of Salmonella and samples detected by method
24 4 6 24 4 6 24
a Plates with typical Salmonella colonies between 1 and l0 were recorded with a plus sign (+), between 10 and 100 colonies with a two plus sign (+ +), and more than 100 colonies with a three-plus sign (+ + +). b IMS analysis was done using 20 txl of particles with 10 min of incubation.
difference was observed in ten-fold d i l u t e d samples with or without ferrous sulphate supplementation.
Recovery o f Salmonella from pooled raw eggs supplemented with ferrous sulphate. O n t h r e e s e p a r a t e occasions, w h e n a pool of t e n eggs was seeded with 1 - 2
S.
enteritidis cells a n d e n r i c h e d for up to 24 h with ferrous s u l p h a t e s u p p l e m e n t a t i o n , IMS analysis (20 /xl of particles, 10 m i n i n c u b a t i o n ) of five-fold dilutions of aliquots a n d direct p l a t i n g of 100 /xl of u n d i l u t e d samples after 4 a n d 6 h gave negative results ( T a b l e 3). However, IMS a n d direct plating of the same samples after 24 h e n r i c h m e n t were positive. T a b l e 4 shows the results of IMS analysis using 4 0 / x l of particles a n d 30 m i n i n c u b a t i o n d u r i n g IMS as well as direct plating of the 12 e n r i c h e d ferrous s u l p h a t e s u p p l e m e n t e d ten-egg pools i n o c u l a t e d with 1 - 2 s a l m o n e l l a e . A f t e r 6 h e n r i c h m e n t , IMS b a s e d o n 1 a n d 10 ml sample volumes recovered Salmonella from 11 a n d 12 respectively out of 12 such egg pools, whilst direct p l a t i n g of the u n d i l u t e d samples yielded only six as positive for Salmonella. All samples were positive for Salmonella after 24 h e n r i c h m e n t irrespective of m e t h o d used in this study.
4. Discussion A c c o r d i n g to G a r i b a l d i (1960) lysozyme, c o n a l b u m i n (ovotransferrin) a n d alkaline p H in egg white kill or inhibit the growth of a wide variety of microorganisms,
K.S. Cudjoe et al. / International Journal of Food Microbiology 23 (1994) 149-158
155
but the yolk, or a mixture of the yolk and albumen has very little inhibitory effect (Haines, 1939; Brooks, 1960). The level of Salmonella enteritidis contamination in naturally infected eggs are low (Humphrey et al., 1991) and enrichment of raw eggs to increase the numbers of salmonellae generally takes several days before plating for presumptive identification (Humphrey et al. 1989; Gast and Beard, 1992; Gast, 1993). The growth enhancing abilities of ferrous sulphate on Gram-negative bacteria has been demonstrated (Garibaldi, 1960) but its application to Salmonella detection from raw eggs has been limited to its use only in enrichment broths (Gast and Beard, 1992; Gast, 1993), without any overall saving in time. In our approach, unlike that of Gast (1993), ferrous sulphate was added directly to raw, blended and undiluted eggs prior to enrichment. This significantly enhanced the growth of seeded S. enteritidis in eggs and eliminated the need for dilution of raw eggs prior to enrichment. This enhanced growth enabled subsequent significant recovery of Salmonella after 6 h enrichment by IMS using Dynabeads ® Anti-Salmonella. After 24 h enrichment both IMS analysis and direct plating recorded high levels of Salmonella starting from an initial inoculum of 1-2 cells in ca. 500 ml raw blended eggs. Our results show clearly that during abbreviated enrichment of 4 h duration, the mixture of the yolk and albumen still had a significant inhibitory effect on the growth of Salmonella. This inhibition, however, appears to be completely neutralized by the addition of ferrous sulphate to allow sufficient growth within 6 h for subsequent detection. The level of target bacteria recovered when using Dynabeads ® Anti-Salmonella, is directly correlated to the extent of the particle-bacteria complex recovered after IMS. Particle loss during IMS due to the influence of certain types of sample matrices can be sufficient to significantly affect the results of the analysis (Skjerve and Olsvik, 1991). The more viscous the sample matrix, for example undiluted raw blended eggs, the higher the loss of particles used in the analysis (Table 1). Whilst a ten-fold dilution of raw egg samples resulted in only 8% loss of particles, such a high dilution would concomitantly reduce the level of Salmonella present per ml of sample for analysis. If raw egg samples with an initial low level of natural contamination were supplemented with ferrous sulphate prior to enrichment, a 16% particle loss due to a five-fold dilution of sample would not significantly affect the sensitivity of the system. Samples must be thoroughly mixed, preferably by homogenizing in a stomacher before taking aliquots for dilutions and subsequent analysis. This is very important for all sampling and detection methods since lumps a n d / o r colloidal particles of egg white not properly blended with the yolk, could harbour Salmonella that are growing in localized sites in the blended pools and could be missed. Since the presence of one Salmonella cell in 25 g or a pooled contents of raw eggs must be detected, it is imperative that some form of pre-enrichment be undertaken when examining pooled samples. For pooled raw eggs (> 500 ml), supplemented with ferrous sulphate as described above, IMS alone enables the concentration of low levels of the target bacteria from larger volumes of the diluted samples for detection. IMS-analysis on five-fold dilutions of pooled eggs samples using 20 /~1 of Dynabeads ® Anti-Salmonella and incubating for 10 min
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Table 4 Effect of incubation time on recovery of from iron-supplemented Raw egg pool
Sampling
samples
p e r i o d (h)
1
24 2
+
++
+++
+++
-
-
4 -
+
++
+++
+++
+++
4
-
+
+
+
++
+++
+++
+++
4
-
+
+
6
+
+
++
+++
+++
+++ ++
4
-
6
-
+
+++
+++
4
+
+
+++
+++
-
-
-
+
++
+++
+++
+++
-
-
+
+
++
+++
+++
+++
-
+++
4 6 24 4 6
+++ +
4 24
-
+
+
+++
+++
+++
4
-
--
-
6
+
+
+
+++
+++
+++ +
24
24 4
-
6
-
-
+++
+++
24 Totals
++
+++
6
12
--
+
+
6
11
--
+
6
24
ll/
diluted egg pool
+++
24
9
l0 ml 5-fold
diluted egg pool
-
24
8
1 ml 2-fold
egg pool
-
24
7
IMS b analysis from:
of undiluted
+++
6
6
Direct plating
a n d s a m p l e s d e t e c t e d by m e t h o d
-
6
5
Salmonella
+++
24 4
( s e e d e d at l e v e l s o f 1 - 2 c e l l s / 5 0 0
4 24
3
Level a of
4
6
Salmonella enteritidis
raw eggs
+++
4
[I
2
3
6
6
11
12
24
12
12
12
a See Table 3 for legend. b I M S a n a l y s i s w a s d o n e u s i n g 4 0 ~1 o f p a r t i c l e s w i t h 30 rain o f i n c u b a t i o n ,
ml)
K.S. Cudjoe et al. / International Journal of Food Microbiology 23 (1994) 149-158
157
with continuous mixing did not, however, recover the seeded Salmonella (Table 3). When the analysis was subsequently repeated using 40 instead of 20 Fzl of the beads and incubating for 30 instead of 10 min, recovery of the seeded Salmonella cells was significantly improved. There was no significant difference between the number of positive samples detected using either 1 ml of two- and 10 ml of five-fold dilutions for IMS (Table 4). The increased particle volume compensated for the particles lost, especially in the two-fold diluted sample matrix, while the extended incubation improved the binding efficiency of the particles to the target bacteria in both instances. On the basis of the above results, we propose the following protocol for detection of S. enteritidis from raw eggs: 1. Aseptically crack open the egg or eggs and mix/blend thoroughly both white and yolk and add ferrous sulphate solution to a final concentration of 35 rag/1. 2. Pre-incubate the egg mixture at 37°C. 3. After 6 h of pre-incubation, mix the egg mixture thoroughly, remove and dilute an aliquot five-fold in BPW and further remove either 1 or 10 ml from the diluted aliquot for IMS. 4. Reincubate the egg mixture for a further 18 h. 5. For IMS analysis, pipette 40 /zl of Dynabeads ® Anti-Salmonella into either an Eppendorf tube or 15 ml tube with screw cap. Add either 1 or 10 ml diluted sub-sample depending on the tube of choice. 6. Incubate with constant rotation for 30 min and wash once in PBS-T with the help of a suitable magnetic particle concentrator (MPC®-M, MPC®-I or MPC®-6; Dynal). 7. Resuspend the particle-bacteria complex in 50 ~I of wash buffer and streak plate 25 pA onto two different Salmonella differential diagnostic media, for example, xylose lysine desoxycholate agar (XLD) and brilliant green agar (BGA). 8. Incubate plates at 37°C for 24 h and pick out presumptive Salmonella colonies for serological and biochemical confirmation. 9. If further confirmation of the results are needed, repeat steps 5 - 7 on the 24 h enriched culture and directly streak-plate a loopful of the undiluted egg onto each of the two Salmonella diagnostic media. Extended storage of cracked eggs or egg-containing foods at warm temperatures provide ample opportunity for extensive bacterial growth (Lin et al., 1988; Telzak et al., 1990). For such samples, IMS analysis should be carried out on 1 ml of ten-fold diluted sample aliquots after 24 h pre-enrichment with ferrous sulphate supplementation. In such a scenario a reversion to the original protocol using 20 pA of Dynabeads ® Anti-Salmonella and incubating only for 10 min with continuous or occasional mixing is necessary to avoid unnecessary non-specific binding of diverse background flora to the particles. References Brooks, J. (1960) Mechanism of the multiplication of Pseudomonas in the hen's egg. J. Appl. Bacteriol., 23, 499-509.
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Centers for Disease Control (1990) Update: Salmonella enteritidis infections and shell-eggs, United States, 1990. Morbid. Mortal. Weekly Rep. 37, 490-496. Clay, C.E. and Board, R.G. (1991) Growth of Salmonella enteritidis in artificially contaminated hens' shells eggs. Epidemiol. lnfect. 106, 271-281. Cudjoe, K.S., Patel, P.D., Olsen, E., Skjerve, E. and Olsvik, O (1993) Immunomagnetic separation techniques for detection of pathogenic bacteria in foods. In: R.G. Kroll, A. Gilmour and M. Sussman (editors), New Techniques in Food and Beverage Microbiology. Society for Applied Bacteriology Technical Series, 31. Blackwell, London, pp. 17 29. Garibaldi, J.A. (1960) Factors in egg white which control growth of bacteria. Food Res. 25, 337 344. Garibaldi, J.A. (1970) Role of microbial iron transport compounds in the bacterial spoilage of eggs. Appl. Microbiol. 20, 558-560. Gast, R.K. and Beard, C.W. (1992) Detection and enumeration of Salmonella enteritidis in fresh and stored eggs laid by experimentally infected hens. J. Food Prot. 55, 152-156. Gast, R.K. (1993) Recovery of Salmonella enteritidis from inoculated pools of egg contents. J. Food Prot. 56, 21 24. Haines, R.B. (1939) Microbiology in the preservation of the hen's egg. G.P. Dep. Sci. Ind. Res. Food Invest. Board, Spec. Rep. 47. Humphrey, T.J., Greenwood, M., Gilbert, R.J., Rowe, B. and Chapman, P.A. (1989) The survival of salmonellas in shell eggs cooked under simulated domestic conditions. Epidemiol. Infect. 103. 35 -45. Humphrey, T.J., Whitehead, A., Gawler, A.H.L., Henley, A. and Rowe, B. (1991) Numbers of Salmonella enteritidis in the contents of naturally contaminated hens' eggs. Epidemiol. Infect. 103,489 496. Lin, F.-Y., Morris, J.G., Trump, D., Jr., Tilghman, D., Wood, P.K., Jackman, N., Israel, E. and Libonati, J.P. (1988) Investigation of an outbreak of Salmonella enteritidis gastroenteritidis associated with consumption of eggs in a restaurant chain in Maryland. Am. J. Epidemiol. 128, 839-844. Skjerve, E., R~rvik, L.M. and Olsvik, 0. (1990) Detection of Listeria monocytogenes in foods using immunomagnetic separation. Appl. Environ. Microbiol. 56, 3478-3481. Skjerve, E. and Olsvik, ~. (1991) Immunomagnetic separation of Salmonella from foods. Int. J. Food Microbiol. 14, 11-18. St. Louis, M.E., Morse, D.L., Potter, E., DeMelfi, T.M., Guzewich, J.J., Tauxe, R.V. and Blake, P.A. (1988) The emergence of grade A eggs as a major source of Salmonella enteritidis infections: new implications for control of salmonellosis. J. Am. Med. Assoc. 259, 2103-2107 Stevens, A., Joseph, C., Bruce, J., Fenton, D., O'Mahony, M., Cunnigham, D., O'Conner, B. and Rowe, B. (1989) A large outbreak of Salmonella enteritidis phage type 4 associated with eggs from overseas. Epidemiol. Infect. 103, 25-433. Telzak, E.E., Budnick, L.D., Greenberg, M.S.Z., Blum, S., Shayegani, M., Benson, C.E. and Schultz, S. (1990) A nosocomial outbreak of Salmonella enteritidis infection due to the consumption of raw eggs. N. Engl. J. Med. 323, 394-397.