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The detection of Salmonella serovars from animal feed and raw chicken using a combined immunomagnetic separation and ELISA method
Food Microbiology, 2001, 18, 361^366 Available online at http://www.idealibrary.com on
doi:10.1006/fmic.2001.0416
ORIGINAL ARTICLE
The detection of Salmonella serovars from animal feed and raw chicken using a combined immunomagnetic separation and ELISA method Lucielle P. Mans¢eld and S. J. Forsythe* The immunomagnetic separation technique was used in conjunction with an ELISA-based detection system to recover Salmonella enteritidis, Salmonella typhimurium and Salmonella virchow (2^26 103 cfu 25 g71) from raw chicken and animal feed. The detection time was less than 27 h and included the conventional pre-enrichment (18 h) step in bu¡ered peptone water and an enrichment step (6 h) in glucose nutrient broth. In contrast, the conventional method using Rappaport-Vassiliades # 2001Academic Press enrichment broth and selective plating on XLD agar required 72^96 h.
Introduction Conventional methods for Salmonella isolation and detection have three incubation periods: pre-enrichment, selective enrichment and differential agar detection (Blackburn 1993). Subsequently a minimum period of three days is required for presumptive Salmonella spp. isolation. In order to decrease the detection time, a number of selective enrichment broths and agars have been recommended (Mans¢eld and Forsythe 2000a). Ideally, selective enrichment should enable the multiplication of Salmonella spp. and suppress the growth of nonSalmonella cells. However the concentration of selective agents in the enrichment broth can be toxic to Salmonella cells (Fricker 1987, Flowers *Corresponding author: Dr S. J. Forsythe, Department of Life Sciences,The Nottingham Trent University, Clifton Lane, Nottingham, NG11 8NS. Fax: 0115 9486636. E-mail: [email protected] 0740 -0020/01/080361 + 06 $35.00/0
et al. 1992). Additionally, Chen et al. (1993) reported that 102^105 Salmonella cells ml71 pre-enrichment medium was necessary for Salmonella isolation using Rappaport-Vassilaides broth. The immunomagnetic separation (IMS) technique for Salmonella isolation has been used for a number of foods as an alternative to selective broths (Safar|¤ k et al. 1995, Mans¢eld and Forsythe 1996). The technique uses paramagnetic particles coated with anti-Salmonella speci¢c antibodies to capture the target cells from mixed cultures. The Salmonella cells can subsequently be detected using conventional end-detection methods such as XLD di¡erential agar. The IMS separation method has been successfully combined with the ELISA technique as the end-detection method for the recovery of Salmonella enteritidis from eggs and skimmed milk powder (Holt et al. 1995, Ma¤lkova¤ et al. 1998, Mans¢eld and Forsythe 2000b). However, these foods do not have a high intrinsic # 2001 Academic Press
Received: 22 September 2000 Department of Life Sciences, The NottinghamTrent University, Clifton Lane, Nottingham, NG118NS
362 L. P. Mans¢eld and S. J. Forsythe
microbial load or a particulate food matrix which could hinder certain detection methods. Previous studies of raw chicken using a combined IMS and ELISA technique gave a signi¢cant number of false negative results (Cudjoe et al. 1995). It was proposed that the high levels of intrinsic competing enterobacteriaceae inhibited the growth of Salmonella spp. or blocked the immunological binding between released Salmonella antigen and the immunomagnetic particles (Cudjoe et al. 1995). In the present study, IMS and the Locate1 ELISA method (Gangar et al. 1998) were used to replace the conventional selective enrichment broth and the detection agar steps respectively. The Locate1 method uses glucose nutrient broth as a post-enrichment step to increase the number of Salmonella cells to a detectable level (105^106 ml71 ). Hence the additional use of this medium with IMS and ELISA may overcome the previous di⁄culties, such as competing £ora, reported with raw chicken samples (Cudjoe et al. 1995). These combined methods were applied to the recovery of Salmonella enteritidis, Salmonella typhimurium and Salmonella virchow from arti¢cially contaminated raw chicken and animal feeds.
Methods Bacterial strains and culture conditions Cultures of S. enteritidis (serogroup D), S. typhimurium (serogroup B) and S. virchow (serogroup C) were maintained on tryptone soya agar (TSA, Unipath Ltd, Basingstoke, UK) slopes and grown overnight in 10 ml bu¡ered peptone water (BPW, Unipath Ltd) at 378C. Viable counts were determined using the standard plate procedure on tryptone soya agar (TSA, Unipath Ltd) at 378C.
Pre-enrichment and Salmonella spp. inoculation of food materials Raw chicken breast pieces and animal feed were purchased from local retailers. Samples (25 g) of animal feed were added to 225 ml BPW directly and mixed by swirling. Samples (25 g) of raw chicken breast were mixed with
50 ml BPW, homogenized (1 min, Colworth Stomacher, Seward, London, UK), added to 175 ml BPW and mixed by swirling. The pre-enrichment broths were inoculated with decimal dilutions of 18 h Salmonella cultures. Cell numbers were con¢rmed by plate count on TSA. All samples were incubated at 378C for 18 h. Uninoculated samples of animal feed and raw chicken were used as controls. The chicken and animal feed were analysed in duplicate.
Conventional selective enrichment method RV broth (9?9 ml) was inoculated with 0?1 ml aliquots of BPW pre-enriched samples and incubated at 428C. After 24 and 48 h, a loopful was streaked onto xylose lysine desoxycholate (XLD, Unipath Ltd) agar plates and incubated at 378C for 18 h.
Immunomagnetic separation Aliquots (1 ml) of BPW pre-enriched food samples were incubated with 20 ml of Salmonellaspeci¢c paramagnetic beads (Dynal UK Ltd, Wirral, UK) in 1?5 ml Eppendorf tubes and incubated at room temperature for 10 min. The beads were separated using a magnetic particle concentrator (MPC1-M, Dynal UK Ltd) for 3 min, suspended in 1 ml phosphate bu¡ered saline- 0?05% Tween 20 (PBS-T) washing buffer, separated and resuspended in 50 ml washing bu¡er. Aliquots (25 ml) were streaked on duplicate XLD plates for overnight incubation at 378C. The detection limit of IMS was determined by preparing decimal dilutions (161071^16107 cfu ml71) in triplicate of the Salmonella serovars in BPW and repeating the above IMS procedure with plating onto XLD agar (overnight incubation, 378C).
ELISA Samples (0?1^1 ml) were boiled for 20 min and cooled prior to analysis by ELISA (Locate1 Salmonella Immunoassay, Rho“ne-Diagnostics Technologies Ltd, Glasgow, UK) according to the manufacturer’s instructions. Samples (100 ml well71 ), including positive and negative controls provided by the manufacturer, were added to each well of the ELISA plate. The
Combined IMS and ELISA for Salmonella detection 363
plates were incubated at room temperature for 30 min. Unbound antigen was removed by aspiration. The wells were washed four times in wash bu¡er, once with distilled water (250 ml well71 ) and then dried by inversion. A 100 ml aliquot of the conjugated Salmonella-speci¢c antibody was added to each well and incubated at room temperature for 30 min. Unbound antibody was removed by washing. A 100 ml aliquot of 3, 3, 5, 5 -tetramethylbenzidine (TMB) substrate was added to each well and the plate incubated in the dark at room temperature for 30 min. The reaction was stopped with 2% sulphuric acid stopping solution (100 ml) and the absorbance immediately read at 450 nm using aTitertek Multiscan Plus reader (Flow Laboratories, Irvine, UK). A Salmonella-positive sample was recorded if the absorbance at 450 nm was 0?3. The detection limit of the ELISA technique was determined using decimal dilutions (36103^107 cfu ml71) in triplicate of the Salmonella serovars. To compare the a¡ect of grown medium on cell surface antigenicity, the Salmonella serovars were grown in BPW and glucose nutrient broth with 10 mg ml71 novobiocin (GN) for 18 h (37 and 428C respectively) and diluted in the respective medium. For the modi¢ed IMS-ELISA method, the IMS beads were resuspended in 100 ml tris bu¡ered saline (TBS) and incubated in 1 ml GN broth at 428C for 6 h prior to ELISA analysis.
Results Detection limit of assays The IMS method using plating on to XLD agar as the detection method, captured S. enteritidis, S. typhimurium and S. virchow cells at a minimum concentration of 1^10 cells ml71 (data not shown). In contrast, the detection limit of the combined IMS-ELISA methods was 106^109 Salmonella cells ml71 (Fig. 1). This was due to the detection limit of the ELISA method being equal to 106 Salmonella cells ml71 (Fig. 2). The sensitivity of the ELISA method was dependant upon the serovar and also the growth medium. Salmonella cells grown in GN gave higher absorbance values than those grown in BPW. For example, S.
Figure 1. Sensitivity of the combined IMS-ELISA method for the detection of S. enteritidis, S. typhimurium and S. virchow. S. enteritidis (-~-), S. typhimurium (-^-), S. virchow (-&-). Detection limit at Abs450nm = 0?3 (- - - - -). Where error bars for the standard deviation are not shown, the value was less than the area of the symbol (n = 3).
enteritidis grown in GN and BPW at cell densities of 16106 cfu ml71 gave absorbance values of 1?7 and 0?35 respectively (Fig. 2(a)).The same cell density of S. typhimurium and S. virchow cells (grown in GN broth) gave absorbance values of 1?3 and 0?9 respectively (Fig. 2(b) and (c)).
Recovery of Salmonella from raw chicken and feed samples Salmonella enteritidis, S. virchow and S. typhimurium were recovered from duplicate animal feed and raw chicken samples at all inoculation levels by enrichment in RV broth followed by plating on XLD agar (Table 1). The total test time was 3 days for most samples, but one sample was only positive after 48 h RV broth incubation giving a total test time of 4 days. In contrast, the IMS technique with plating on XLD agar recovered Salmonella spp. from all samples with a total detection time of 2 days. Proteus spp. were isolated on XLD from one uninoculated chicken sample.The combined IMSELISA method only recovered the three Salmonella serovars from animal feed inoculated at the highest level (103 cells 250 ml71). The IMSELISA total detection time was approx. 21 h. Neither S. enteritidis nor S. virchow were recovered from any of the chicken samples using the IMS-ELISA method (Table 1).
364 L. P. Mans¢eld and S. J. Forsythe
Figure 2. Detection of Salmonella enteritidis, S. typhimurium and S. virchow using the Locate1 ELISA method. Cells were grown in GN broth (-~-, 18 h, 428C) or BPW broth (-&-, 18 h, 378C) before dilution in respective broths. (a) S. enteritidis, (b) S. typhimurium, (c) S. virchow. Detection limit at Abs450nm=0?3 (- - - - -). Where error bars for the standard deviation are not shown, the value was less than the area of the symbol (n = 3). Table 1. The isolation of Salmonella strains from BPW pre-enrichment broths using RV-XLD, IMS-XLD and IMS-ELISA procedures Samplea
Animal feed Raw chicken
a
S. enteritidis
S. virchow
S. typhimurium
Inoculum size (cfu ml71 BPW)
RVXLDb
IMSXLD
IMSELISA
Inoculum size (cfu ml71 BPW)
RVXLD
IMSXLD
IMSELISA
Inoculum size (cfu ml71 BPW)
RVXLD
IMSXLD
IMSELISA
26103
+
+
+
46103
+
+
+
26103
+
+
+
26102 20 26103
+ 7 (+)c +
+ + +
+ + 7
46102 40 46103
+ + +
+ + +
+ 7 7
26102 20 26103
+ + +
+ + +
+ 7 +
26102 20
+ +
+ +
7 7
46102 40
+ +
+ +
7 7
26102 20
+ +
+ +
+ 7
Samples (25 g) homogenized in BPW (225 ml), inoculated with appropriate serovar and incubated 18 h at 378C. Salmonella serovars recovered on XLD agar from 24 h incubation of RV broth unless otherwise indicated. A positive result indicates the recovery of Salmonella from one or more samples (n = 2). c S. enteritidis recovered after 48 h incubation of RV broth from one sample. b
Combined IMS and ELISA for Salmonella detection 365
Table 2. Detection of Salmonella enteritidis during growth in GN broth (428C) using the combined IMSELISA procedure Incubation period (h) 2 a
Inoculum level (cfu ml7l) 26102 26103 26104
4
6
Absorbance (450 nm)b
Cell density (cfu ml71)
Absorbance (450 nm)
Cell density (cfu ml71)
Absorbance (450 nm)
Cell density (cfu ml71 )
0?055 (7)c 0?056 (7) 0?071 (7)
26103d 26104 26105
0?054 (7) 0?323 (+) 1?593 (+)
36104 36105 66106
0?608 (+) 1?689 (+) 1?712 (+)
26106 26107 16108
a
GN broth inoculated with S. enteritidis at prescribed levels and incubated at 428C. Average of duplicated experiments presented. c Only values 0?3 were recorded as positive. d S. enteritidis cell density (cfu ml71 ) enumerated on TSA (378C, 18 h). b
Table 3. The recovery of Salmonella enteritidis from triplet raw chicken pre-enrichment broths by RV-XLD and modi¢ed IMS-ELISA methods Number of replicates positive for S. enteritidis Inoculum size (cfu 250 ml71)a
RV-XLDb
Modi¢ed IMS-ELISAc
36103 36102 30 3 0?3
3 2 1 (2)d 2 1
3 3 3 2 1
Total
9 (10)
a
12 71
Number of S. enteritidis 250 ml BPW pre-enrichment broth (25 g raw chicken in 225 ml BPW), inoculated in triplicate and incubated for 18 h at 378C. b S. enteritidis recovered from 24 h incubation of RV broth unless otherwise indicated. c IMS samples incubated in GN broth (6 h, 428C) before ELISA analysis. d Positive only after 48 h RV broth incubation.
The IMS-ELISA method was modi¢ed by the inclusion of an incubation period in GN broth prior to ELISA analysis. Recovery of low cell numbers (26102 cfu 250 ml71) of S. enteritidis, in pure culture, was achieved by including a 6 h incubation in GN broth (Table 2). During the incubation period, the S. enteritidis multiplied to a detectable cell density (106 cfu ml71). Subsequently pre-enriched raw chicken samples spiked with S. enteritidis were analysed using the modi¢ed IMS-ELISA method (Table 3). Pre-enrichment broths of raw chicken (25 g in 225 ml BPW) in triplicate were inoculated
with 0?3^36103 S. enteritidis cells 250 ml71 before incubation (378C, 18 h). All broths inoculated with 36103 S. enteritidis cells were positive by both RV-XLD and modi¢ed IMSELISA methods. In addition, the modi¢ed IMS-ELISA technique also detected S. enteritidis cells in broths inoculated with 30 and 300 S. enteritidis cells 250 ml71. The RV-XLD method did not detect S. enteritidis in one replicate at these levels of inoculation (Table 3). Neither method detected S. enteritidis in one replicate inoculated with three S. enteritidis cells 250 ml71. Both methods detected S. enteritidis in one out of three broths inoculated with 0?3 S. enteritidis cells 250 ml71.
Discussion Although the detection limit of IMS was 1^10 Salmonella cells ml71, coupling the method with the Locate1 ELISA system increased the level of detection to 106^109 Salmonella cells ml71. This value is similar to previous Locate1 ELISA results reported by Gangar et al. (1996) and for a Salmonella ELISA system based on the genus speci¢c monoclonal antibody M105 (Mans¢eld and Forsythe 2000b). The detection level was dependent upon the serovar and also the growth medium (Figs 1 and 2). S. enteritidis gave higher absorbance values than S. typhimurium and S. virchow, especially when previously grown in GN broth (Fig. 2). The ELISA system uses antibodies against the Salmonella O-antigen and hence is dependent
366 L. P. Mans¢eld and S. J. Forsythe
upon the sequence of sugars of the lipopolysacharide (LPS) molecule in the Salmonella cell wall. It is known that growth conditions can affect the LPS structure (Nelson et al. 1991, Schlecht and Mayer 1994). Hence the changes in the ELISA detection levels may be due to growthinduced alterations in the LPS structure. Salmonella cells were not recovered as frequently from arti¢cially contaminated animal feed and raw chicken using the IMS-ELISA method compared with the conventional procedure of RV enrichment broth and XLD agar plates (Table 1). The Salmonella cells had been captured by the IMS technique as shown by the IMS-XLD results (Table 1). However compared with the IMS-ELISA method, the IMSXLD technique required an additional overnight incubation period for visible colony formation. Since it had been demonstrated (Fig. 2) that growth in GN broth enhanced the detection of Salmonella cells, aliquots of pre-enrichment broths (18 h) were incubated for 6 h prior to ELISA analysis. In addition, the use of novobiocin (10 mg ml71) in GN broth would also reduce the number of organisms such as Proteus spp. which might non-speci¢cally bind to the ELISA antibodies. The resultant modi¢ed IMS-ELISA method recovered S. enteritidis from arti¢cially contaminated chicken samples more frequently (12/15 compared with 10/15) than the conventional RV-XLD method (Table 3). The total test time was approx. 27 h. This included the standard 18 h pre-enrichment step in BPW, 30 min IMS, 6 h incubation in GN broth and the 2 h ELISA procedure. Hence Salmonella can be detected in material with high intrinsic microbial £ora in two successive days compared with the conventional 3^4-day period.
Acknowledgements The authors would like to thank Rho“ne-Diagnostics Technologies Ltd. for the provision of materials.
References Blackburn, C. de W. (1993) A review: rapid and alternative methods for the detection of Salmonellas in foods. J. Appl. Bacteriol. 75, 199^214.
Chen, H., Fraser, A. D. E. and Yamazaki, H. (1993) Evaluation of the toxicity of Salmonella selective media for shortening the enrichment period. Int. J. Food Microbiol. 18, 151^159. Cudjoe, K. S., Hagtvedt, T. and Dainty, R. (1995) Immunomagnetic separation of Salmonella from foods and their detection using immunomagnetic particle (IMP)-ELISA. Int. J. Food Microbiol. 27, 11^25. Flowers, R. S., D’Aoust, J.Y., Andrews,W. H. and Bailey, J. S. (1992) Salmonella. In Compendium of methods for the microbiological examination of Foods (Eds C. Vanderzant and D. F. Splittstoessert), pp. 371^422. American Public Health Association, Washington DC. Fricker, C. R. (1987) A review: the isolation of Salmonellas and campylobacters. J. Appl. Bacteriol. 63, 99^116. Gangar,V., Curiale, M. S., D’Onorio, A., Donnelly, C. and Dunnigan, P. (1996) LOCATE enzyme-linked immunosorbant assay of detection of Salmonella in food; Collaborative study. J. AOAC Int. 81, 419^437. Holt, P. S., Gast, R. P. and Greene, C. R. (1995) Rapid detection of Salmonella enteritidis in pooled liquid egg samples using magnetic bead-ELISA system. J. Food Protect. 58, 967^972. Ma¤lkova¤, K., Rauch, P.,Wyatt, G. M. and Morgan, M. R. A. (1998) Combined immunomagnetic separation and detection of Salmonella enteritidis in food samples. Food Agricult. Immunol. 10, 271^280. Mans¢eld, L. P. and Forsythe, S. J. (1996) Collaborative ring-trial of Dynabeads1 anti-Salmonella for immunomagnetic separation of stressed Salmonella cells from herbs and spices. Int. J. Food Microbiol. 29, 41^47. Mans¢eld, L. P. and Forsythe, S. J. (2000a) Detection of Salmonellae in food. Rev. Medical Microbiol. 11, 37^46. Mans¢eld, L. P. and Forsythe, S. J. (2000b) The detection of Salmonella using a combined immunomagnetic separation and ELISA enddetection procedure. Lett. Appl. Microbiol. 31, 279^283. Nelson, D., Bathgate, A. J. and Poxton, I. R. (1991) Monoclonal antibodies as probes for detecting lipopolysaccharide expression on Escherichia coli from di¡erent growth conditions. J. Gen. Microbiol. 137, 2741^2751. Safar|¤ k, I., Safrar|¤ kova¤, M. and Forsythe, S. J. (1995) The application of magnetic separations in applied microbiology. J. Appl. Microbiol. 78, 575^585. Schlecht, S. and Mayer, H. (1994) The in£uence of growth temperature on the amount of free R lipopolysaccharide, on the expression of R-core determinants and on O-chain lengths on Salmonella S forms. Zentral. Backteriol. Orig. Hyg. Prav. Med. 280, 448^457.
doi:10.1006/fmic.2001.0416
ORIGINAL ARTICLE
The detection of Salmonella serovars from animal feed and raw chicken using a combined immunomagnetic separation and ELISA method Lucielle P. Mans¢eld and S. J. Forsythe* The immunomagnetic separation technique was used in conjunction with an ELISA-based detection system to recover Salmonella enteritidis, Salmonella typhimurium and Salmonella virchow (2^26 103 cfu 25 g71) from raw chicken and animal feed. The detection time was less than 27 h and included the conventional pre-enrichment (18 h) step in bu¡ered peptone water and an enrichment step (6 h) in glucose nutrient broth. In contrast, the conventional method using Rappaport-Vassiliades # 2001Academic Press enrichment broth and selective plating on XLD agar required 72^96 h.
Introduction Conventional methods for Salmonella isolation and detection have three incubation periods: pre-enrichment, selective enrichment and differential agar detection (Blackburn 1993). Subsequently a minimum period of three days is required for presumptive Salmonella spp. isolation. In order to decrease the detection time, a number of selective enrichment broths and agars have been recommended (Mans¢eld and Forsythe 2000a). Ideally, selective enrichment should enable the multiplication of Salmonella spp. and suppress the growth of nonSalmonella cells. However the concentration of selective agents in the enrichment broth can be toxic to Salmonella cells (Fricker 1987, Flowers *Corresponding author: Dr S. J. Forsythe, Department of Life Sciences,The Nottingham Trent University, Clifton Lane, Nottingham, NG11 8NS. Fax: 0115 9486636. E-mail: [email protected] 0740 -0020/01/080361 + 06 $35.00/0
et al. 1992). Additionally, Chen et al. (1993) reported that 102^105 Salmonella cells ml71 pre-enrichment medium was necessary for Salmonella isolation using Rappaport-Vassilaides broth. The immunomagnetic separation (IMS) technique for Salmonella isolation has been used for a number of foods as an alternative to selective broths (Safar|¤ k et al. 1995, Mans¢eld and Forsythe 1996). The technique uses paramagnetic particles coated with anti-Salmonella speci¢c antibodies to capture the target cells from mixed cultures. The Salmonella cells can subsequently be detected using conventional end-detection methods such as XLD di¡erential agar. The IMS separation method has been successfully combined with the ELISA technique as the end-detection method for the recovery of Salmonella enteritidis from eggs and skimmed milk powder (Holt et al. 1995, Ma¤lkova¤ et al. 1998, Mans¢eld and Forsythe 2000b). However, these foods do not have a high intrinsic # 2001 Academic Press
Received: 22 September 2000 Department of Life Sciences, The NottinghamTrent University, Clifton Lane, Nottingham, NG118NS
362 L. P. Mans¢eld and S. J. Forsythe
microbial load or a particulate food matrix which could hinder certain detection methods. Previous studies of raw chicken using a combined IMS and ELISA technique gave a signi¢cant number of false negative results (Cudjoe et al. 1995). It was proposed that the high levels of intrinsic competing enterobacteriaceae inhibited the growth of Salmonella spp. or blocked the immunological binding between released Salmonella antigen and the immunomagnetic particles (Cudjoe et al. 1995). In the present study, IMS and the Locate1 ELISA method (Gangar et al. 1998) were used to replace the conventional selective enrichment broth and the detection agar steps respectively. The Locate1 method uses glucose nutrient broth as a post-enrichment step to increase the number of Salmonella cells to a detectable level (105^106 ml71 ). Hence the additional use of this medium with IMS and ELISA may overcome the previous di⁄culties, such as competing £ora, reported with raw chicken samples (Cudjoe et al. 1995). These combined methods were applied to the recovery of Salmonella enteritidis, Salmonella typhimurium and Salmonella virchow from arti¢cially contaminated raw chicken and animal feeds.
Methods Bacterial strains and culture conditions Cultures of S. enteritidis (serogroup D), S. typhimurium (serogroup B) and S. virchow (serogroup C) were maintained on tryptone soya agar (TSA, Unipath Ltd, Basingstoke, UK) slopes and grown overnight in 10 ml bu¡ered peptone water (BPW, Unipath Ltd) at 378C. Viable counts were determined using the standard plate procedure on tryptone soya agar (TSA, Unipath Ltd) at 378C.
Pre-enrichment and Salmonella spp. inoculation of food materials Raw chicken breast pieces and animal feed were purchased from local retailers. Samples (25 g) of animal feed were added to 225 ml BPW directly and mixed by swirling. Samples (25 g) of raw chicken breast were mixed with
50 ml BPW, homogenized (1 min, Colworth Stomacher, Seward, London, UK), added to 175 ml BPW and mixed by swirling. The pre-enrichment broths were inoculated with decimal dilutions of 18 h Salmonella cultures. Cell numbers were con¢rmed by plate count on TSA. All samples were incubated at 378C for 18 h. Uninoculated samples of animal feed and raw chicken were used as controls. The chicken and animal feed were analysed in duplicate.
Conventional selective enrichment method RV broth (9?9 ml) was inoculated with 0?1 ml aliquots of BPW pre-enriched samples and incubated at 428C. After 24 and 48 h, a loopful was streaked onto xylose lysine desoxycholate (XLD, Unipath Ltd) agar plates and incubated at 378C for 18 h.
Immunomagnetic separation Aliquots (1 ml) of BPW pre-enriched food samples were incubated with 20 ml of Salmonellaspeci¢c paramagnetic beads (Dynal UK Ltd, Wirral, UK) in 1?5 ml Eppendorf tubes and incubated at room temperature for 10 min. The beads were separated using a magnetic particle concentrator (MPC1-M, Dynal UK Ltd) for 3 min, suspended in 1 ml phosphate bu¡ered saline- 0?05% Tween 20 (PBS-T) washing buffer, separated and resuspended in 50 ml washing bu¡er. Aliquots (25 ml) were streaked on duplicate XLD plates for overnight incubation at 378C. The detection limit of IMS was determined by preparing decimal dilutions (161071^16107 cfu ml71) in triplicate of the Salmonella serovars in BPW and repeating the above IMS procedure with plating onto XLD agar (overnight incubation, 378C).
ELISA Samples (0?1^1 ml) were boiled for 20 min and cooled prior to analysis by ELISA (Locate1 Salmonella Immunoassay, Rho“ne-Diagnostics Technologies Ltd, Glasgow, UK) according to the manufacturer’s instructions. Samples (100 ml well71 ), including positive and negative controls provided by the manufacturer, were added to each well of the ELISA plate. The
Combined IMS and ELISA for Salmonella detection 363
plates were incubated at room temperature for 30 min. Unbound antigen was removed by aspiration. The wells were washed four times in wash bu¡er, once with distilled water (250 ml well71 ) and then dried by inversion. A 100 ml aliquot of the conjugated Salmonella-speci¢c antibody was added to each well and incubated at room temperature for 30 min. Unbound antibody was removed by washing. A 100 ml aliquot of 3, 3, 5, 5 -tetramethylbenzidine (TMB) substrate was added to each well and the plate incubated in the dark at room temperature for 30 min. The reaction was stopped with 2% sulphuric acid stopping solution (100 ml) and the absorbance immediately read at 450 nm using aTitertek Multiscan Plus reader (Flow Laboratories, Irvine, UK). A Salmonella-positive sample was recorded if the absorbance at 450 nm was 0?3. The detection limit of the ELISA technique was determined using decimal dilutions (36103^107 cfu ml71) in triplicate of the Salmonella serovars. To compare the a¡ect of grown medium on cell surface antigenicity, the Salmonella serovars were grown in BPW and glucose nutrient broth with 10 mg ml71 novobiocin (GN) for 18 h (37 and 428C respectively) and diluted in the respective medium. For the modi¢ed IMS-ELISA method, the IMS beads were resuspended in 100 ml tris bu¡ered saline (TBS) and incubated in 1 ml GN broth at 428C for 6 h prior to ELISA analysis.
Results Detection limit of assays The IMS method using plating on to XLD agar as the detection method, captured S. enteritidis, S. typhimurium and S. virchow cells at a minimum concentration of 1^10 cells ml71 (data not shown). In contrast, the detection limit of the combined IMS-ELISA methods was 106^109 Salmonella cells ml71 (Fig. 1). This was due to the detection limit of the ELISA method being equal to 106 Salmonella cells ml71 (Fig. 2). The sensitivity of the ELISA method was dependant upon the serovar and also the growth medium. Salmonella cells grown in GN gave higher absorbance values than those grown in BPW. For example, S.
Figure 1. Sensitivity of the combined IMS-ELISA method for the detection of S. enteritidis, S. typhimurium and S. virchow. S. enteritidis (-~-), S. typhimurium (-^-), S. virchow (-&-). Detection limit at Abs450nm = 0?3 (- - - - -). Where error bars for the standard deviation are not shown, the value was less than the area of the symbol (n = 3).
enteritidis grown in GN and BPW at cell densities of 16106 cfu ml71 gave absorbance values of 1?7 and 0?35 respectively (Fig. 2(a)).The same cell density of S. typhimurium and S. virchow cells (grown in GN broth) gave absorbance values of 1?3 and 0?9 respectively (Fig. 2(b) and (c)).
Recovery of Salmonella from raw chicken and feed samples Salmonella enteritidis, S. virchow and S. typhimurium were recovered from duplicate animal feed and raw chicken samples at all inoculation levels by enrichment in RV broth followed by plating on XLD agar (Table 1). The total test time was 3 days for most samples, but one sample was only positive after 48 h RV broth incubation giving a total test time of 4 days. In contrast, the IMS technique with plating on XLD agar recovered Salmonella spp. from all samples with a total detection time of 2 days. Proteus spp. were isolated on XLD from one uninoculated chicken sample.The combined IMSELISA method only recovered the three Salmonella serovars from animal feed inoculated at the highest level (103 cells 250 ml71). The IMSELISA total detection time was approx. 21 h. Neither S. enteritidis nor S. virchow were recovered from any of the chicken samples using the IMS-ELISA method (Table 1).
364 L. P. Mans¢eld and S. J. Forsythe
Figure 2. Detection of Salmonella enteritidis, S. typhimurium and S. virchow using the Locate1 ELISA method. Cells were grown in GN broth (-~-, 18 h, 428C) or BPW broth (-&-, 18 h, 378C) before dilution in respective broths. (a) S. enteritidis, (b) S. typhimurium, (c) S. virchow. Detection limit at Abs450nm=0?3 (- - - - -). Where error bars for the standard deviation are not shown, the value was less than the area of the symbol (n = 3). Table 1. The isolation of Salmonella strains from BPW pre-enrichment broths using RV-XLD, IMS-XLD and IMS-ELISA procedures Samplea
Animal feed Raw chicken
a
S. enteritidis
S. virchow
S. typhimurium
Inoculum size (cfu ml71 BPW)
RVXLDb
IMSXLD
IMSELISA
Inoculum size (cfu ml71 BPW)
RVXLD
IMSXLD
IMSELISA
Inoculum size (cfu ml71 BPW)
RVXLD
IMSXLD
IMSELISA
26103
+
+
+
46103
+
+
+
26103
+
+
+
26102 20 26103
+ 7 (+)c +
+ + +
+ + 7
46102 40 46103
+ + +
+ + +
+ 7 7
26102 20 26103
+ + +
+ + +
+ 7 +
26102 20
+ +
+ +
7 7
46102 40
+ +
+ +
7 7
26102 20
+ +
+ +
+ 7
Samples (25 g) homogenized in BPW (225 ml), inoculated with appropriate serovar and incubated 18 h at 378C. Salmonella serovars recovered on XLD agar from 24 h incubation of RV broth unless otherwise indicated. A positive result indicates the recovery of Salmonella from one or more samples (n = 2). c S. enteritidis recovered after 48 h incubation of RV broth from one sample. b
Combined IMS and ELISA for Salmonella detection 365
Table 2. Detection of Salmonella enteritidis during growth in GN broth (428C) using the combined IMSELISA procedure Incubation period (h) 2 a
Inoculum level (cfu ml7l) 26102 26103 26104
4
6
Absorbance (450 nm)b
Cell density (cfu ml71)
Absorbance (450 nm)
Cell density (cfu ml71)
Absorbance (450 nm)
Cell density (cfu ml71 )
0?055 (7)c 0?056 (7) 0?071 (7)
26103d 26104 26105
0?054 (7) 0?323 (+) 1?593 (+)
36104 36105 66106
0?608 (+) 1?689 (+) 1?712 (+)
26106 26107 16108
a
GN broth inoculated with S. enteritidis at prescribed levels and incubated at 428C. Average of duplicated experiments presented. c Only values 0?3 were recorded as positive. d S. enteritidis cell density (cfu ml71 ) enumerated on TSA (378C, 18 h). b
Table 3. The recovery of Salmonella enteritidis from triplet raw chicken pre-enrichment broths by RV-XLD and modi¢ed IMS-ELISA methods Number of replicates positive for S. enteritidis Inoculum size (cfu 250 ml71)a
RV-XLDb
Modi¢ed IMS-ELISAc
36103 36102 30 3 0?3
3 2 1 (2)d 2 1
3 3 3 2 1
Total
9 (10)
a
12 71
Number of S. enteritidis 250 ml BPW pre-enrichment broth (25 g raw chicken in 225 ml BPW), inoculated in triplicate and incubated for 18 h at 378C. b S. enteritidis recovered from 24 h incubation of RV broth unless otherwise indicated. c IMS samples incubated in GN broth (6 h, 428C) before ELISA analysis. d Positive only after 48 h RV broth incubation.
The IMS-ELISA method was modi¢ed by the inclusion of an incubation period in GN broth prior to ELISA analysis. Recovery of low cell numbers (26102 cfu 250 ml71) of S. enteritidis, in pure culture, was achieved by including a 6 h incubation in GN broth (Table 2). During the incubation period, the S. enteritidis multiplied to a detectable cell density (106 cfu ml71). Subsequently pre-enriched raw chicken samples spiked with S. enteritidis were analysed using the modi¢ed IMS-ELISA method (Table 3). Pre-enrichment broths of raw chicken (25 g in 225 ml BPW) in triplicate were inoculated
with 0?3^36103 S. enteritidis cells 250 ml71 before incubation (378C, 18 h). All broths inoculated with 36103 S. enteritidis cells were positive by both RV-XLD and modi¢ed IMSELISA methods. In addition, the modi¢ed IMS-ELISA technique also detected S. enteritidis cells in broths inoculated with 30 and 300 S. enteritidis cells 250 ml71. The RV-XLD method did not detect S. enteritidis in one replicate at these levels of inoculation (Table 3). Neither method detected S. enteritidis in one replicate inoculated with three S. enteritidis cells 250 ml71. Both methods detected S. enteritidis in one out of three broths inoculated with 0?3 S. enteritidis cells 250 ml71.
Discussion Although the detection limit of IMS was 1^10 Salmonella cells ml71, coupling the method with the Locate1 ELISA system increased the level of detection to 106^109 Salmonella cells ml71. This value is similar to previous Locate1 ELISA results reported by Gangar et al. (1996) and for a Salmonella ELISA system based on the genus speci¢c monoclonal antibody M105 (Mans¢eld and Forsythe 2000b). The detection level was dependent upon the serovar and also the growth medium (Figs 1 and 2). S. enteritidis gave higher absorbance values than S. typhimurium and S. virchow, especially when previously grown in GN broth (Fig. 2). The ELISA system uses antibodies against the Salmonella O-antigen and hence is dependent
366 L. P. Mans¢eld and S. J. Forsythe
upon the sequence of sugars of the lipopolysacharide (LPS) molecule in the Salmonella cell wall. It is known that growth conditions can affect the LPS structure (Nelson et al. 1991, Schlecht and Mayer 1994). Hence the changes in the ELISA detection levels may be due to growthinduced alterations in the LPS structure. Salmonella cells were not recovered as frequently from arti¢cially contaminated animal feed and raw chicken using the IMS-ELISA method compared with the conventional procedure of RV enrichment broth and XLD agar plates (Table 1). The Salmonella cells had been captured by the IMS technique as shown by the IMS-XLD results (Table 1). However compared with the IMS-ELISA method, the IMSXLD technique required an additional overnight incubation period for visible colony formation. Since it had been demonstrated (Fig. 2) that growth in GN broth enhanced the detection of Salmonella cells, aliquots of pre-enrichment broths (18 h) were incubated for 6 h prior to ELISA analysis. In addition, the use of novobiocin (10 mg ml71) in GN broth would also reduce the number of organisms such as Proteus spp. which might non-speci¢cally bind to the ELISA antibodies. The resultant modi¢ed IMS-ELISA method recovered S. enteritidis from arti¢cially contaminated chicken samples more frequently (12/15 compared with 10/15) than the conventional RV-XLD method (Table 3). The total test time was approx. 27 h. This included the standard 18 h pre-enrichment step in BPW, 30 min IMS, 6 h incubation in GN broth and the 2 h ELISA procedure. Hence Salmonella can be detected in material with high intrinsic microbial £ora in two successive days compared with the conventional 3^4-day period.
Acknowledgements The authors would like to thank Rho“ne-Diagnostics Technologies Ltd. for the provision of materials.
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