Cross-contamination with Salmonella on a broiler slaughterhouse line demonstrated by use of epidemiological markers

Journal of Applied Microbiology 2003, 94, 826 835 Cross-contamination with Salmonella on a broiler slaughterhouse line demonstrated by use of epidemiological markers J.E. Olsen 1, D.J. Brown 1, M. Madsen 2 and M. Bisgaard 1 1 Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark, and 2 Danish Veterinary Institute, Aarhus, Denmark 2002/175: received 17 April 2002, revised 26 July 2002 and accepted 9 January 2003 ABSTRACT J. E. O L S E N, D.J. B R O W N, M. M A D S E N A N D M. B I S G A A R D. 2003. Aims: To investigate contamination of surfaces on a poultry slaughter line from infected poultry and subsequent cross-contamination of non-infected poultry. Methods and Results: A broiler slaughterhouse was investigated for the presence of Salmonella on 17 defined points over two 1-week periods. Flocks supplied to slaughter and neck skin samples from processed chicken were likewise investigated. Salmonella was detected in 10 out of 18 flocks at ante-mortem inspection, while seven flocks tested positive in the finished products. Equipment at all but one control point at the slaughter line tested positive at least once during the study. The chicken receiving area was the most contaminated. By comparison of typing results from serotyping, plasmid profile typing and phage typing, direct evidence for cross-contamination with Salm. serotype Typhimurium, Salm. Serotype 4Æ12:b: and Salm. serotype Virchow on the slaughter line was obtained for four of the flocks. The cleaning procedure in place did not remove all Salmonella from the contaminated areas. Conclusions: Evidence for contamination of equipment on a slaughter line and subsequent cross-contamination to non-infected chicken was provided by typing methods. Significance and Impact of the Study: This study has provided detailed information on cross-contamination on a slaughter line by the use of phage typing and plasmid profiling. The study stresses the importance of controlling Salmonella in the primary production, as contamination of the equipment on the slaughter line will act as a vehicle to contaminate finished products. Cleaning procedures on slaughter lines cannot be expected to control this problem with the current equipment. Keywords: broiler production, cross-contamination, Salmonella, typing. INTRODUCTION The control of Salmonella in poultry and poultry products has been the goal of researchers and the poultry industry for many years. Several studies have shown that live poultry can introduce Salmonella into the processing plant (Bryan et al. 1968; Surkiewicz et al. 1969; Dougherty 1974). Such Correspondence to: J.E. Olsen, Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Stigbøjlen 4, DK-1870 Frederiksberg C., Denmark (e-mail: jeo@kvl.dk). contamination may result in widespread dissemination of salmonellae along the processing line and may lead to the contamination of the final product (Lillard 1990). Improving the microbiological quality of chickens in the processing plant is difficult. If, however, process control is built into the operation, cross-contamination can be minimized. Therefore, the identification of critical control points and development of techniques to control them is essential. It has been suggested that there are many stages during the poultry slaughtering process where cross-contamination of carcasses can occur. There is, however, only limited data ª 2003 The Society for Applied Microbiology

SALMONELLA CROSS-CONTAMINATION 827 available that demonstrates cross-contamination at given stages of the process. In order to define the critical control points and the routes of transmission, bacterial-typing methods with a high discriminatory power can be used (Olsen et al. 1993). The present study was undertaken retrospectively based on a collection of isolates from one slaughterhouse to determine Salmonella contamination on equipment at 17 defined points during the slaughter process and after cleaning and disinfection. Salmonella isolates from broiler flocks, on equipment in the abattoir and in finished products were compared by serotyping, phage typing and plasmid profiling to investigate cross-contamination on the slaughter line. MATERIALS AND METHODS Experimental design A large Danish poultry slaughterhouse was extensively investigated for the presence of Salmonella in 1994. All isolates obtained were stored in 15% glycerol at )80 C until typing was performed in the present study. Samples were collected over two periods of 6-day duration, which were separated by a period of 14 weeks and included ante-mortem (AM) control samples taken from each flock scheduled to be slaughtered, swabs taken from the slaughter line and neck skin samples taken from processed broilers. Ante-mortem control Caecal tonsils from 16 3-week-old broiler chicks per flock were examine 3 weeks prior to slaughter, supplemented with examination of 60 pools of five swab samples of faecal droppings per flock at a time closer to slaughter. Half a gram of two tonsils were pooled in selenite broth (1 : 9 v/v) (Oxoid, CM395, Basingstoke, UK) and incubated at 37 C for 18 h, followed by subculture onto modified brilliant green agar (Oxoid, CM328) supplemented with Lutensite 0Æ15% (Bie & Berntsen, Rødovre, Denmark) to suppress swarming bacteria. Incubations were carried out at 37 C for 18 20 h. Pooled swab samples of faecal material were incubated for pre-enrichment in phosphate buffered peptone water (Merck, 1-07228, Darmstadt, Germany) at 37 C for 18 20 h to recover injured cells. Samples were then added to Rappaport-Vassiliadis broth (Oxoid, CM669) at 1 : 100 and incubated at 41Æ5 C for a further 18 20 h. Then, samples were plated out as described above on Rambach agar (Merck, 1-07500) and incubated for a further 18 20 h at 37 C. evisceration equipment. The temperature of the scald tank was 51 52 C and cooling was obtained by water chilling. Samples were taken from equipment at 17 selected points of the slaughter line (Figure 1). Each sample consisted of five sterile, moistened, cotton wool swabs, which, after swabbing of the area (approximately 30 cm 50 cm), were processed as described for faecal samples above. Samples from scald tanks and disinfection baths were from the sides of the bath immediately over the water (three swabs) and from the equipments (two swabs). During the first week, samples were taken once on Sunday, once on Monday, and then twice each day until Friday. One sampling was performed in the morning before commencement of processing to check the cleaning procedures; the other was performed at the change of shift in the afternoon after a quick cleaning of the line had been carried out by hosing down the equipment. During the second week, samples were only taken once each day, from Sunday to Friday, in the morning before commencement of processing, except on Monday, when samples were taken during processing. The routine cleaning procedures at the slaughterhouse involved five separate steps: (i) hosing down with water; (ii) soaking with a detergent for 20 min, either Unirens (SFK A.m.b.A., Hvidovre, Denmark) or Kombinon (SFK A.m.b.A., Hvidovre, Denmark); (iii) hosing with highpressure water; (iv) disinfection treatment with 2% hypochlorite solution for 15 min; and (v) final hosing down with water. Transport crates and racks were cleaned in an automated washing facility with high-pressure water followed by disinfection treatment in 2% hypochlorite solution and housing down with water. Samples from processed broilers Neck skin samples (approximately 25 g) were collected, using sterile forceps and scissors, from 50 processed broilers per flock immediately before packaging. Samples were pooled in five groups of 10 and examined for the presence of Salmonella according to the Nordic Committee on Food Analysis (NMKL) Method No. 71 (Anonymous 1991). Briefly, one volume of sample was pre-enriched in phosphate buffered peptone water as above, 0Æ1 ml of this was enriched in pre-warmed Rapport Vassiliadis broth, and Salmonellasuspect colonies were identified on XLD agar plates. The slaughterhouse laboratories carried out the primary isolation with authorization from the Danish Central Veterinary Authorities, while identification and phenotyping were carried out at the Danish Veterinary Institute. Slaughterhouse line samples The slaughterhouse under study received approximately 100 000 broilers per day on one slaughter line with automated Identification of isolates From each plate with suspected Salmonella colonies, five typical colonies were selected and identified by biochemical

828 J.E. OLSEN ET AL. *17 Portioning and packing *16 * Control points 1. Crate washer 8. Plucker 2. Crate disinfect. bath 3. Rack washer 4. Cleaned crates 5. Cleaned crates racks 6. Neckskin slitter 7. Scalding bath 9. Transfer machinery 10. Neck breaker 11. Opening machine 12. Organ remover 13. In/Outside washing 14. Giblet system 15. Spray cooler 16. Collection bin 17. Packing machine Evisceration *10, 11, 12, 13, & 14 Vet control Organ vet control Plucking Scalding * 7 Crate cleaning area * 8 *1, 2, 3, 4, & 5 * 9 Stunning & bleeding * 6 Chilling *15 Fig. 1 Schematic presentation of the slaughterhouse under investigation with indication of the control points where Salmonella contamination was investigated and serological methods (Barrow and Feltham 1993). In the case of less than five typical colonies per plate, all colonies were examined. Epidemiological markers Phage typing. Strains of Salm. serotype Typhimurium were phage typed by the method of Anderson et al. (1977). Plasmid profile analysis. Isolates of Salmonella were screened for the presence of plasmid DNA using the method of Kado and Liu (1981). Gels were stained in ethidium bromide (Sigma) 2 mg l )1, illuminated with a 254-nm UV transilluminator (UVP; model TS-40) and the sizes of plasmids were calculated relative to the reference plasmid in Escherichia coli strains V517 (Macrina et al. 1978) and 39R861 (Threlfall et al. 1986) in accordance with the method of Rochelle et al. (1985). Coiled covalently closed (ccc) and open circular (oc) DNA molecules were differentiated according to Hintermann et al. (1981). RESULTS Isolation of Salmonella from live birds and neck skin samples A total of 18 flocks from six different farms were slaughtered during the sampling period. Table 1 summarizes the findings of Salmonella AM and in the finished products from these flocks. Eight flocks were negative at AM with both sampling methods, six flocks were positive by both methods and four flocks were positive by faecal sampling only. No flocks were found positive by analysis of caecal tonsils only. Three farms supplied more than one flock, originating in different houses on the farm. In farm A, six out of seven flocks were Salmonella positive with a total of four different serotypes. In farm D, both flocks supplied were Salmonella negative, while farm E supplied three positive and two negative flocks, and two different serotypes. Two out of the eight negative flocks and five out of the 10 positive flocks by AM analysis were positive by neck skin samples after processing. In the two negative (AM) flocks, the serotypes had been isolated from flocks slaughtered

SALMONELLA CROSS-CONTAMINATION 829 Table 1 Isolation of Salmonella from ante-mortem samples and from final product neck skin samples for the broiler flocks processed during the study (the number of positive samples/number of samples is given in parentheses) Salmonella isolation from Flock Slaughter Faecal samples Caecal tonsil samples Neck skin samples (finished product) A-1 Day 1, week 1 Salm. 4Æ12:b: (16/60) Negative Negative B-1 Day 1, week 1 Salm. Tm DT110 (56/60) Salm. Tm DT110 (3/7) Salm. Tm DT110 (2/5) A-2 Day 2, week 1 Negative Negative Negative C-1 Day 3, week 1 Negative Negative Salm. Tm DT110 (1/5) Salm. 4Æ12:b: (1/5) C-2 Day 3, week 1 Negative Negative Negative D-1 Day 3, week 1 Negative Negative Negative A-3 Day 4, week 1 Salm. Infantis (17/60) Negative Negative A-4 Day 5, week 1 Salm. Tm DT110 (19/59) Negative Negative Salm. Tm DT193 (8/59) Salm. Tm phage inf (5/59) Salm. 4Æ12:b: (4/59) D-2 Day 5, week 1 Negative Negative Negative E-1 Day 1, week 2 Salm. Tm DT110 (5/60) Salm. Tm DT110 (1/8) Negative E-2 Day 1, week 2 Salm. Tm DT110 (14/60) Salm. Tm rough (1/8) Negative E-3 Day 2, week 2 Negative Negative Negative E-4 Day 2, week 2 Negative Negative Negative E-5 Day 3, week 2 Salm. Tennesee (8/60) Salm. Tennessee (1/7) Salm. Tm DT110 (2/5) Salm. Tm DT110 (5/60) Salm. 4Æ12:b: (1/5) A-5 Day 3, week 2* Salm. Tm DT110 (9/59) Negative Salm. Tm DT110 (1/5) Salm. 4Æ12:b: (46/59) Salm. Tm DT135 (1/5) Salm. 4Æ12:b: (2/5) A-6 Day 4, week 2 Salm. Virchow (7/60) Negative Salm. 4Æ12:b: (2/5) Salm. 4Æ12:b: (45/69) A-7 Day 5, week 2 Salm. Infantis (5/60) Salm. Tm DT110 (1/8) Salm. Tm DT110 (4/5)à Salm. Tm DT110 (13/60) Salm. Infantis (1/5)à Salm. Virchow (1/5) F-1 Day 5, week 2 Negative Negative Salm. Tm DT110 (1/5) Salm. Virchow (1/5) Salm. 4Æ12:b: (1/5) *A proportion of this flock was not slaughtered until the morning of day 4, week 2. The isolation of Salm. typhimurium PT110 and PT135 were made from the same pooled neck skin sample. àone of the four Salm. typhimurium positive samples was also, at the same time, positive for Salm. infantis. previously in the same week. In the five positive (AM) flocks, both serotypes isolated at AM and additional serotype(s) (three flocks) were found. Isolation of Salmonella from the slaughterhouse processing line Sampling on Sunday in both weeks served to monitor the cleaning and disinfection procedures carried out while the plant was out of operation. In the first week, this showed that five points on the slaughter line were positive for Salmonella. Four isolates of Salm. Typhimurium and three isolates of Salm. serotype Hadar were recovered from the giblet handling system, scalding bath, crate washer and clean crates and from the crate racks. The only positive sample taken from the slaughter line on the Sunday of the second week was from the plucker, which yielded a plasmid-free strain of Salm. serotype Mbandaka, which was not observed again throughout the sampling period. Table 2 ranks the control points on the slaughterhouse line according to the prevalence of positive samples. The most frequently contaminated points were the crate washer, the cleaned crate racks, the rack washer and the plucker, all with more than 40% positive samples. Strains of Salm. Typhimurium DT110 and Salm. serotype 4Æ12:b: were demonstrated by plasmid profiling to persist on the slaughter line for 5 days (data not shown).

830 J.E. OLSEN ET AL. Table 2 Rank order of sample control points on the slaughter line as assessed by the number of samples positive for Salmonella Rank Control point (number in Figure1) Positive samples 1 Crate washer (1) 10/16 62Æ5 2 Cleaned crate racks (4) 8/16 50Æ0 3 Rack washer (3) 6/14 42Æ9 4 Plucker (8) 13/32 40Æ6 5 Opening machine (11) 6/16 37Æ5 6 Giblet system (14) 6/16 37Æ5 7 Cleaned crates (4) 11/32 34Æ4 8 Packing machine (17) 5/16 31Æ3 9 Organ remover (12) 5/16 31Æ3 10 Transfer machinery (9) 5/16 31Æ3 11 Collection bin (16) 4/15 26Æ7 12 Neck skin slitter (6) 4/16 25Æ0 13 Spray cooler (15) 4/32 12Æ5 14 Neck breaker (10) 2/16 12Æ5 15 Scalding bath (7) 2/16 12Æ5 16 In/outside washing (13) 1/16 6Æ3 17 Crate disinfect. bath (2) 0/16 0Æ0 % positive Demonstration of cross-contamination by plasmid profiling Flocks that were negative at the AM control and yielded positive samples in the finished product (C-1 and F-1), and flocks where new serotypes, or phage types of Salm. Typhimurium were observed in the finished products compared to the AM control (E-5, A-5 and A-7) were suspected of being contaminated on the slaughterhouse line. To provide a direct link between isolates from the previous flock(s) or contamination of the processing line and isolates obtained from the finished product, typing by plasmid profiling was performed. To demonstrate the complexity of results obtained with this approach, a summary of typing of isolates from the first week of sampling is shown in Table 3, while an overview of the results for both weeks in relation to possible crosscontamination is summarized in Table 4. Finished products of flock C-1 were positive for Salm. 4Æ12:b: and Salm. Typhimurium DT110 though the flock had been negative at AM control. These two serotypes/ phage types were also isolated from flocks A-1 and B-1 slaughtered previously the same week. Five different plasmid profiles were observed among the 16 Salm. 4Æ12:b: strain isolates from A-1. Eight control points on the slaughter line sampled positive for Salm. 4Æ12:b: on the afternoon when A-1 had been slaughtered (day 1 week 1). Strains with four different plasmid profiles were identified of which two: no plasmids (crate washer, clean crates, neck skin slitter, transfer machinery, opening machine, organ remover and packing machine) and 2Æ0 kbp (opening machine) corresponded to the profiles known to be harboured in flock A-1. Isolates of Salm. 4Æ12:b: were obtained from three points on the following day (day 2 week 1), all showing plasmid profiles known to be present in flock A-1. The isolates were obtained from the packing boxes, organ remover and the plucker. On the following day (day 3 week 1), two points were still positive with this serotype (one plasmid-free isolate and one with a 2Æ0 kbp profile). The Salm. 4Æ12:b: isolate from neck skin samples of flock C-1 on the same day had the 2Æ0-kbp plasmid profile. Plasmid profile analysis of the 59 isolates of Salm. Typhimurium DT110 recovered from flock B-1 at AM revealed eight different profiles. Some of these profiles were detected among strains isolated from equipment during the following days despite the fact that no other Salm. Typhimurium infected flocks were processed until day 5 week 1, and thus they probably originated from the processing of flock B-1. Isolation was made from one control point on day 1 week 1, in the afternoon after flock B-1 was slaughtered, from eight points on day 2 week 1, three points on day 3 week 1, and two control points on day 4 week 1. The single isolate of Salm. Typhimurium DT110 from the neck skin sample from flock C-1 had the plasmid profile 94;7Æ4 kbp. This profile, and, indeed, the 7Æ4-kbp plasmid was not detected in any other strain of Salmonella isolated from the slaughterhouse during the week or from any of the flocks being slaughtered. As mentioned previously, some points on the processing line had tested positive for Salm. Typhimurium DT110 on the Sunday preceding the first week of sampling. These isolates had the plasmid profile 94 kbp, the most common plasmid profile observed during the study or 100;94;2Æ9;2Æ kbp, a profile that was not observed again. Flock F-1 was likewise negative at AM while the finishing products were shown (day 5 week 2) to contain Salm. Typhimurium DT110, Salm. serotype Virchow and Salm. 4Æ12:b:. Salm. Typhimurium DT110 had been isolated from flocks E-1, E-2, E-5, A-5 and A-7 slaughtered earlier in the same week. Salm. Virchow and Salm. 4Æ12:b: were both detected in flock A-6 at AM control and Salm. 4Æ12:b: was also detected from flock A-5. The isolate of Salm. Typhimurium DT110 from flock F-1 was of profile 94;4Æ4;3Æ3;2Æ7 kb. In total, 14 different plasmid profiles were observed in strains of Salm. Typhimurium DT110 at AM control of flocks slaughtered in week 2, and 13 additional profiles were gathered among isolates from the control points on the slaughterhouse line. The particular profile of the isolate from flock F-1 finished products had not been isolated from the plant during the week or found in any of the flocks being slaughtered. A neck skin sample from flock F-1 was positive with Salm. Virchow, plasmid profile 6Æ0;3Æ0;2Æ7 kbp. This profile was

SALMONELLA CROSS-CONTAMINATION 831 Table 3 Summary of results of plasmid profiling from the first week of sampling Serotype (phage type)/plasmid profile [in kbp] (number of isolates) Day Flock ante-mortem control Slaughter line samples Finished product samples Sunday Salm. Typhm. DT110/94 (1) Salm. Typhm. DT110/100,94,3,2 (1) Monday Salm. Typhm. DT110/94 (33) Salm. Typhm. DT110/94 (1) Salm. Typhm. DT110/94 (1) Salm. Typhm. DT110/94,3 (17) Salm. Virchow/5Æ7 (1) Salm. Typhm. DT110/6Æ1 (1) Salm. Typhm. DT110/94,X 1 (10) Salm. Hadar/pl. free (1) Salm. 4Æ12:b: /pl. free (10) Salm. Hadar/2Æ5 (1) Salm. 4Æ12:b: /2Æ0 (3) Salm. 4Æ12:b: / pl. free (1) Salm. 4Æ12:b: /X 2 (3) Salm. 4Æ12:b: /2Æ0 (1) Salm. 4Æ12:b: /X 3 (2) Tuesday Salm. Typhm. DT110/94,X 4 (4) Salm. Typhm. DT110/100 (1) Salm. 4Æ12:b: /pl. free (1) Salm. 4Æ12:b: /2Æ1 (1) Wednesday Salm. Typhm. DT110/94,X 5 (2) Salm. Typhm. DT110/94,7 (1) Salm. Typhm. DT110/94 (1) Salm. 4Æ12:b: / pl. free (1) Salm. 4Æ12:b: /2Æ0 (1) Salm. 4Æ12:b: /2Æ0 (1) Thursday Salm. Infantis/5Æ9 (8) Salm. Typhm. DT110/94,X 6 (3) Salm. Infantis/100,6 (7) Salm. Typhm. DT110/94 (1) Salm. Infantis/X 5 (2) Salm. Virchow/5Æ7 (1) Salm. Virchow/X 7 (4) Salm. 4Æ12:b: /pl. free (1) Friday Salm. Typhm. DT110/94 (12) Salm. Typhm. DT110/94 (13) Salm. Typhm. DT110/94,2 (3) Salm. Typhm. DT110/94,X 9 (5) Salm. Typhm. DT110/94,X 8 (4) Salm. Typhm. DT193/94 (6) Salm. Typhm. DT193/94,2 (1) Salm. Typhm. DT193/94,X 10 (2) Salm. Typhm. NT/94 (3) Salm. Virchow/5Æ7 (1) Salm. Typhm. NT/94,4,3,3 (1) Salm. Virchow/X 11 (2) Salm. 4Æ12:b: /pl. free (4) 1 Six profiles with one or two isolates per type. 2 Three profiles with one isolate per type. 3 Two profiles with one isolate per type. 4 Four profiles with one isolate per type. 5 Two profiles with one isolate per type. 6 Three profiles with one isolate per type. 7 Four profiles with one isolate per type. 8 Four profiles with one isolate per type. 9 Four profiles with one or two isolates per type. 10 Two profiles with one isolate per type. 11 Two profiles with one isolate per type. not found among the isolates obtained at AM control in flock A-6, which had been slaughtered the day before. However, the same profile was obtained from the neck skins of flock A-7 slaughtered the same day as F-1 and also from cleaned crates in the processing plant on the same morning. The strain of Salm. 4Æ12:b:, found on 1/5 of the neck skin of F-1 was plasmid free. This plasmid profile had been obtained from the plucker the same morning, where also the neck breaker was found positive with a profile of 100;56;3Æ5;3Æ0, and from the plucker the day before (day 4 week 2). Plasmid-free Salm. 4Æ12:b: was also isolated from the neck slitter on the morning of day 3 week 2, before slaughter was started, and it was the most commonly observed profile (24 out of 46 isolates a total of 13 profiles) at AM control of flock A-5, which was slaughtered on day 3 week 2, and where a proportion of birds, due to delays, had not been slaughtered before the morning of day 4 week 2. Flock A-6 (day 4 week 2) was positive for Salm. 4Æ12:b: at AM control, but no isolates carried the particular profile. In total, seven profiles were identified in this flock. Flock E-5 was positive for Salm. serotype Tenessee and for Salm. Typhimurium DT110 at AM control. Salm. Tennessee was not isolated at any other point during the study. The finished products contained Salm. Typhimurium DT110, but with two plasmid profiles that were different from the two profiles observed at AM control. In

832 J.E. OLSEN ET AL. Table 4 Summary of investigation for cross-contamination on the slaughterhouse line in five flocks of broilers by use of plasmid profiling and phage typing Flock number (day of slaughter) Relevant type in neck skin samples Relevant types found on the process line (day positive) Relevant types found in flocks slaughtered previously the same week (day of slaughter) C-1 (3; week 1) Salm. 4Æ12:b: (2Æ0 kb) Same (1, 2, 3; week 1) Same, flock A-1 (1; week 1) Salm. Typhim. DT110 Salm. Typhim. DT110 other Salm. Typhim. DT110, other profiles, (94;7Æ4 kb) profiles (1, 2, 3; week 1) flock B-1 (1; week 1) F-1 (5; week 2) Salm. 4Æ12:b: (plasmid free) Same (3, 5; week 2) Same, flock A-5 (3; week 2) Salm. Typhim. DT110 Salm. Typhim. DT110 other Salm. Typhim. DT110 other profiles, (94;4Æ4;3Æ3;2Æ7) profiles (1, 2, 3, 4, 5; week 2) flocks E-1, E-2, E-5, A-5, A-7 (1, 3, 5; week 2) Salm. Virhow (6Æ0;3Æ0;2Æ7) Same (5; week 2) Same, flock A-7 (4; week 2) [neck skin sample] E-5 (3; week 2) Salm. 4Æ12:b: (plasmid free) Same (3; week 2) None A-5 (3; week 2) Salm. Typhim. DT135 None None A-7 (5; week 2) Salm. Virchow (6Æ0;3Æ0;2Æ7) Same (5; week 2) Same, flock A-7 (4; week 2) [neck skin sample] addition, a plasmid-free strain of Salm. 4Æ12:b: was isolated. An isolate of the same serotype and with the same profile had been obtained from the neck slitter prior to the start of the processing the same morning. Flock A-5 was positive for Salm. Typhimurium DT110 and Salm. 4Æ12:b: at AM control, but the finished products also contained an isolate of Salm. Typhimurium DT135. Salm. Typhimurium DT135 was only isolated this once during the study. None of the five plasmid profiles observed in strains of DT110 at AM control were subsequently observed in isolates obtained from the slaughter line or from finished products. However, one of the isolates from finished products from flock A-5 was of plasmid profile 94 kb, which was demonstrated at AM from flock E-2 slaughtered on day 1 week 2, from the opening machine, organ remover, transfer machine, neck skin slitter, crate washer and clean crates on the same day and from the crate washer and packing machine on day 2 week 2. Two of the Salm. 4Æ12:b: isolates from neck skin were of profile 3Æ3;2Æ8 kb and one isolate had the profile 90;3Æ5;3Æ3;3Æ0 kb. The AM control isolates contained 13 profiles of which none corresponded to the two profiles demonstrated in neck skin samples. However, all plasmid sizes included in the profiles of isolates in the finished products had been demonstrated in isolates at AM. Flock A-7 was positive for Salm. serotype Infantis and Salm. Typhimurium DT110 at AM. The neck skin samples contained the same serotypes/phage type in addition to an isolate of Salm. Virchow. The isolates of Salm. Typhimurium DT110 were of four plasmid profiles, two of which (94 and 94;2Æ1 kb) were recovered from all neck skin samples that tested positive with this serotype. The strains of Salm. Infantis were of two plasmid profiles at AM. The single isolate from neck skin carried a different profile from these. The isolate of Salm. Virchow was of plasmid profile 6Æ0;3Æ0;2Æ7 kb, which had been isolated from the cleaned crates the same morning. Isolation of Salmonella serotypes that were not found at AM control of birds slaughtered In both weeks, isolates of Salmonella serotypes that could not be explained from AM control of birds to be slaughtered were obtained. Thus, several isolations of Salm. Virchow were made from the slaughter line during week 1, although none of the flocks had tested positive at AM for this serotype, and isolation of Salm. Infantis was made from the plucker on days 2 and 3 of week 2, though the Salm. serotype Infantis positive flock A-7 was not slaughtered until day 5. DISCUSSION Several studies have concluded that the presence of Salmonella on live poultry can lead to the introduction of salmonella into the processing plant (Bryan et al. 1968; Lahellec and Collin 1985; Bailey et al. 1990; Corry et al. 2002), where the contamination of equipment can result in the contamination of the final products (Lillard 1990). Even spread from the slaughterhouse environment back to the farms through contaminated crates and vehicles may be seen, because inadequate cleaning and disinfection result in residual faecal soling and live bacteria (Rigby et al. 1980; Corry et al. 2002). Prevention of this spread is important to minimize the risk of consumers receiving contaminated broiler products. The present study has confirmed that the slaughter of Salmonella positive birds will lead to contamination of the

SALMONELLA CROSS-CONTAMINATION 833 processing line, and that standard cleaning procedures will not always eliminate this. The contamination of equipment was most heavy while slaughter was ongoing, as illustrated on day 1 of week 2, where 13 out of 17 control points tested positive while a Salm. Typhimurium infected flock was being slaughtered. Of the control points investigated, the chicken unloading area, which also included the crate washing facility, was the most heavily contaminated. It was especially noted that one-third of the samples taken from clean crates were Salmonella positive, which indicates that the slaughterhouse could be a significant risk factor for introduction of Salmonella back onto the farm. This observation confirms results from Corry et al. (2002), who observed salmonella contaminated transport crates after wash and disinfection. That transfer of Salmonella back to farms may happen has been indicated by use of epidemiological markers in a study where Salmonella were recovered from between 13 and 87% of disinfected crates at eight Danish poultry slaughterhouses (Brown et al., unpublished), and in a large outbreak of fowl typhoid in Denmark (Christensen et al. 1994). In an extensive analysis of risk factors for introduction of Salmonella into Danish broiler flocks, based on 7108 flocks slaughtered in 1992 and 1993, however, the slaughterhouse was only shown to be an important factor in a bivariable analysis. It was not significant in a multivariate model, possibly because the slaughterhouse is collinear with other area-related variables such as hatchery and feed-mill (Angen et al. 1996). By use of epidemiological markers it was possible to identify the same strains for a full week, despite cleaning procedures. Epidemiological markers have previously been used to demonstrate the persistence of a particular type in production systems over time, e.g. Salm. serotype Blockley on a farm for over a year (Limawongpranee et al. 1999) and to demonstrate that the Salmonella types present in feed mills were also the ones distributed throughout the broiler flocks that received the feed, and to demonstrate persistence of different serotypes over several years in different poultry production companies (Liebana et al. 2002). The number of positive samples from control points in the sections for killing, evisceration and giblet processing, cooling and the final processing and packaging area indicated that contamination decreases towards the end of the processing line. Though this result was based on relatively small differences in numbers of positive samples, it may indicate that cleaning is more effective towards the end of the processing line. However, it may also simply reflect that there are less Salmonella left to contaminate the equipment the longer the chicken has been on the processing line. In a study by Corry et al. (2002), on four abattoirs, evisceration machinery apparently was less frequently contaminated than in the present study and most contamination occurred in killing, bleeding trough and plucker. In the Danish programme for Salmonella control, the slaughtering of infected and non-infected broiler flocks are separated in time to prevent cross-contamination during processing. In order to ensure this, the AM control of broiler flocks for the presence of Salmonella must give a true reflection of the status of the flock, and the findings must be directly related to the status of the flock at slaughter. In the present study, the number of Salmonella positive flocks was twice as high when sampling was based on faecal droplets compared to caecal tonsils. This was also observed in the national surveillance programme, where the prevalence of Salmonella positive flocks increased considerably when the detection limit of the sampling method was reduced to a 5% flock prevalence (Anonymous 1995). This underlines the need to use sensitive methods in Salmonella control programmes. Recently, the sensitivity of the sampling method has been further improved by the introduction of a sock-sampling method (Skov et al. 1999). The present investigation was not designed to compare flock status at AM and at the time of slaughter, as birds were not sampled on arrival at the slaughterhouse, and the power of the sampling procedure was not the same at different stages in the study. With this in mind, it may not be surprising that there was only a moderate level of correlation between the status at AM and the isolation obtained after processing. Even with the same sampling procedure, several factors may cause discrepancy between AM and slaughterhouse samples. AM control is normally performed at the age of 3 weeks. Most flocks are not slaughtered until 2 weeks later, and during this period, the flock may become clear of infection or acquire a further infection. This has been shown in an investigation of 36 Salmonella positive flocks (method with a detection level of 20%) where only eight flocks sampled positive at both the age of approximately 1 week and at slaughter. Three flocks sampled negative at 1 week and positive before slaughter, while 25 flocks were positive at 1 week and negative at slaughter (Bisgaard et al. 1982). Infections with prevalence below the detection level at AM may also play a role. In a retrospective analysis based on the number of positive pools in the samples, Skov (2000) has estimated that approximately one-third of Danish Salmonella positive broiler flocks in the period from 1995 to 1997 had a flock prevalence at or below 5%. As demonstrated in the present study, cross-contamination at the slaughterhouse also contributes to the lack of correlation between AM and post-mortem results. Others have reported that abattoirs may be contaminated by salmonellas not being found in the flocks that are processed (Corry et al. 2002). Previous studies have shown that Salmonella serotypes, which are detected at different points in poultry production, may be recovered from the final products (McBride et al. 1980; Lahellec and Collin 1985; Jones et al. 1991). More detailed information has been obtained in studies that have

834 J.E. OLSEN ET AL. included typing methods for characterization of bacterial isolates. By use of epidemiological techniques, it was possible in the present investigation to trace the infection route more closely, and to provide a direct link between contamination of equipment and contamination of the final product. Thus, serotyping, plasmid profiling and phage typing (Salm. Typhimurium) provided evidence of crosscontamination in four out of five flocks that were suspected of being contaminated on the processing line. The confidence in this result, however, depends on the strength of the typing method, and based on experiences from a study of Salm. Enteritidis in poultry, is seems that the successful outcome of typing will depend on both geographical and animal origin of the isolates typed (Liebana et al. 2001). Phage typing did not provide good discrimination. Most Salm. Typhimurium isolates were of phage type DT110, and the few isolates of other phage types did not clarify the routes of cross-contamination. DT110 was the second most common type among broilers in Denmark in 1995, the first year where phage typing was reported from surveillance studies in Denmark (Anonymous 1996). The high prevalence makes conclusions based on phage typing weak. Plasmid profiling was chosen because it has been reported to give a high discrimination among isolates of Salmonella, not least in broiler flocks (Baggesen et al. 1992; Brown et al. 1992). The discriminatory power, may, however, depend on the serotype under study (Liebana et al. 2002). A high discriminatory power may account for the failure to recover isolates with the same plasmid profiles in flocks where the same serotype was demonstrated at AM control and in the final products. This was particularly the case with isolates of Salm. serotype Typhimurium DT110, where re-isolation was done at relevant points on the processing line, but where slightly different plasmid profiles were obtained. This situation is contrary to reports on typing of Salm. Enteritidis from poultry production, where a single common type was reported in an integrated poultry organization in UK (Davies et al. 1997). The use of chromosomally based methods like ribotyping or pulse field gel electrophoresis might have improved this. However, Danish isolates of Salm. Typhimurium DT110 from poultry show very little variation in any of these typing methods (Olsen et al. 1997; On and Baggesen 1997) probably because they come from a narrow source, i.e. few infected parent stocks. Where plasmid profiling proved too discriminatory for this serotype, chromosomally based methods would, therefore, most likely have provided too little discrimination. Plasmid profiling thus particularly formed the basis for demonstration of cross-contamination with Salm. Virchow and Salm. 4Æ12:b:. The use of this method to differentiate within Salm. 4Æ12:b: is well founded. In a study of 201 isolates of Salm. 4Æ12:b: from Danish broilers, 28 different plasmid profiles were detected, indicating a high discriminatory power of plasmid profiling (Chadfield et al. 2001). However, the plasmid-free profile, which was demonstrated in presumptive cross-contamination in the present study, accounted for 62% of the isolates, making conclusions based on this profile weak. A high prevalence of plasmid free strains of Salm. 4,12:b: has previously been reported in a study of Salmonella types isolated from British broiler production companies (Liebana et al. 2002). To the knowledge of the authors, no typing of a large collection of Danish strains of Salm. Virchow has been published. Although progress has been made in machine design in an effort to improve cleaning and disinfection, cross-contamination due to improper cleaning and disinfection still may represent a major problem as demonstrated during the present investigation. The findings of this study clearly underline the importance of the segregation of ÔcleanÕ and ÔdirtyÕ processes to reduce the possibility of cross-contamination within the slaughterhouse and the need to improve the hygienic standard of equipment used on the slaughter line. The use of epidemiological markers enabled firm conclusions as to the routes of contamination from the individual flock through the slaughterhouse process-line to the finished product. The use of such methods has proven valuable to elucidate the spread of salmonella during poultry processing. ACKNOWLEDGEMENTS The authors are indebted to Gitte Christensen, Charlotte Rasmussen and Anita Forslund for their technical assistance. We must also sincerely thank Dr Dorte L. 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