Preventive Veterinary Medicine 117 (2014) 542–553
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Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed
Abattoir based survey of Salmonella in finishing pigs in the United Kingdom 2006–2007 E.A. Marier a,∗ , L.C. Snow a , T. Floyd a , I.M. McLaren b , J. Bianchini a , A.J.C. Cook c , R.H. Davies b a Epidemiology, Surveillance and Risk Group, Animal Health and Veterinary Laboratories Agency, Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom b Department of Bacteriology, Animal Health and Veterinary Laboratories Agency, Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom c School of Veterinary Medicine, Faculty of Health & Medical Sciences, University of Surrey, Guildford, Surrey GU2 7TE, United Kingdom
a r t i c l e
i n f o
Article history: Received 15 December 2013 Received in revised form 22 August 2014 Accepted 6 September 2014 Keywords: Pig Salmonella Risk factors Slaughter
a b s t r a c t All European Union (EU) Member States (MSs) were required to conduct a baseline survey from October 2006 to September 2007 to estimate the prevalence of Salmonella infection in finisher pigs at slaughter. In the United Kingdom (UK), samples for microbiological culture were collected from the ileo-caecal lymph nodes, from carcass swabs and from caecal contents. Meat juice samples were also collected for testing in an enzyme-linked immunosorbent assay (MJE) to detect a serological response. Salmonella was isolated from 22% (CI95% 19–25%) of ileo-caecal lymph nodes, 15% (CI95% 12–18%) of carcass swabs and 22% (CI95% 19–26%) of caecal contents. A quarter (25%, CI95% 22–29%) of MJE samples were positive for Salmonella antibodies (cut-off, S/P ≥ 0.25). The most frequently identified serovars were Salmonella typhimurium (57%) and Salmonella derby (26%). The results were used to investigate some factors associated with Salmonella infection in slaughter pigs. The weight of the carcass was significant, with a lower probability of obtaining a positive meat juice result from pigs with a weight greater than 75 kg (p = 0.03). The weight of the lymph node sample was significantly associated with Salmonella status, with a heavier sample of lymph nodes being more likely to be positive (OR = 2.16 CI95% 1.07–4.39). ‘Carcass weight’ in two of the classes – 75–79 kg and 80–84 kg – (OR = 0.44 CI95% 0.28–0.70; OR = 0.64 CI95% 0.49–0.85) and ‘fewer-than-1500-pigs’ scheduled for slaughter on the day of sampling’ (OR = 0.41 CI95% 0.24–0.71) were also associated with a reduced risk of isolating Salmonella from lymph node. The model for carcass swabs showed a positive association with ‘time-elapsed-since-thestart-of-the-line’ indicating a higher risk of contamination as the day progressed (p < 0.01). This model also showed positive association between isolation of Salmonella from a carcass swab and the occurrence of Salmonella in caecal contents from the same pig (OR = 2.22 CI95% 1.38–3.59) and a negative association with time in singeing units (OR = 0.88 CI95% 0.79–0.98). Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author. Tel.: +44 01932 357618; fax: +44 01932 359429. E-mail address: [email protected] (E.A. Marier).
Pigs have been identified as a reservoir and carrier of Salmonella (Fedorka-Cray et al., 2000). In recent years, pork has been increasingly recognised in a number of countries
http://dx.doi.org/10.1016/j.prevetmed.2014.09.004 0167-5877/Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.
E.A. Marier et al. / Preventive Veterinary Medicine 117 (2014) 542–553
as a potential source of human salmonellosis (Hald et al., 2003; Boyen et al., 2008). Salmonella-positive carrier pigs represent a constant source of Salmonella contamination for the abattoir and processing environment, and contaminated pig meat products are a human health hazard (Boyen et al., 2008). Various studies to estimate the prevalence of Salmonella in slaughter pigs have been previously carried out in Great Britain (Davies et al., 2004; Milnes et al., 2008, 2009) as well as in a number of other countries including Germany (Kasbohrer et al., 2000), Italy (Bonardi et al., 2003) and Canada (Letellier et al., 1999). Also, to monitor the level of Salmonella exposure in the UK pig production, meat juice samples (MJE) were tested periodically from each batch of pigs sent to the abattoir, as part of the Zoonoses National Control Plan (ZNCP, 2008–2012, BPEX). Results from these studies have identified that about a quarter of pigs’ caecal contents were contaminated with Salmonella at slaughter in the UK (Davies et al., 2004; Clough et al., 2009). In Denmark, an association between Salmonella sero-prevalence in slaughter pigs and carcass contamination was identified (Sorensen et al., 2004). Other studies which have investigated risks factors at slaughter identified evisceration practices, polishing equipment, splitting, dressing and meat inspection as potential contributors to carcass contamination when slaughtering Salmonella-positive pigs (Berends et al., 1997; Botteldoorn et al., 2003; Baptista et al., 2010). In 2006, to provide the scientific basis for setting a community reduction target for Salmonella in slaughter pigs (Anon, 2008a,b), each EU Member State (MS) was required to carry out a survey of the prevalence of Salmonella infection in finisher pigs at slaughter. This paper reports the results of the EU baseline survey in the UK and of the data on aspects of abattoir practices collected and investigated for evidence of any association with Salmonella status in commercially slaughtered pigs. To enable comparison with previous surveys and the national monitoring programme for Salmonella in pigs (Davies et al., 2000, 2004; Twomey et al., 2007), Defra (Department for Environment, Food & Rural Affairs) agreed to add the collection of caecal contents and meat juice samples to the sampling protocol. The aim of the study was to attempt to link available potential risk factors recorded during the baseline survey with the Salmonella status of the pigs. As this was an anonymous survey, few farm level risk factors were available. Such risk factors were therefore not included in the analysis. 2. Materials and methods 2.1. Sampling plan The United Kingdom’s baseline survey was carried out over a 12-month period from 1 October 2006 to 30 September 2007. The method followed the technical specifications document (SANCO/40162/2006) annexed to Commission Decision 2006/668/EC. The calculation was based on an assumption of an infinite population (>100,000 pigs slaughtered per year) and an estimated prevalence of 50% at a minimum accuracy of 4%, with 95% confidence.
543
In 2006, the UK had an estimated population of 4.7 million pigs, which corresponded to a minimum sample size of 600 pig carcasses plus a further 10% (60 carcasses) to account for any issues during the survey. Therefore, 660 pigs kept in the UK for at least three months preceding slaughter were chosen at random. The sampling was performed to ensure that the largest capacity slaughterhouses, which together represented at least 80% of the slaughtered finisher pig population in the UK, were included. The number of carcasses to be sampled per abattoir was proportional to their annual throughput during the previous year and was obtained by multiplying the sample size by the proportion of carcasses processed. Sampling was equally distributed between months to cover all seasons, starting with October 2006 as month 1. A sampling schedule was produced monthly for each abattoir to indicate which day and which pig on a specific day it had to sample. The days that each abattoir had to sample in each month were selected at random using a random selector sheet in Microsoft® Excel. The rank of the carcass to be sampled on a specific day was also selected at random using this method, with a maximum that reached the highest possible number of finisher pigs slaughtered in the slaughterhouse on a given day. If it was not possible to sample the pig selected for logistical reasons, a second number was picked using the same method of random selection. Casualties and condemned carcasses were excluded from the survey. As carcasses with a live weight of less than 50 kg or more than 170 kg were atypical for the commercial market and were likely to be substantially older or younger than the majority of pigs, they were also excluded from the survey as per the baseline survey’s technical specifications (SANCO/40162/2006). If the selected carcass was excluded, the next suitable carcass on the line was selected instead. 2.2. Sample collection A total of four samples were collected from each carcass: a carcass swab, a muscle sample, an aggregate of ileo-caecal lymph nodes and, to allow comparison with previous GB surveys, a sample of caecal contents. Each sample category was expected to provide evidence of specific types of exposure. The contamination of a carcass swab may come from the sampled pig, from cross-contamination by other pigs or from the abattoir environment; the meat juice sample provides evidence of prior exposure to Salmonella but not from the slaughter process; the lymph node usually provides evidence of an infected pig at the moment of sampling while contaminated caecal contents may be from ingestion of contaminated material and passive transit in gut or infection and could be farm or abattoir associated. The carcass swab was taken on the left or right side of the carcass using one single sponge for all four sites described in Annex A of Standard ISO 17604 (Anon, 2003, hind limb, abdomen, mid-dorsal region, jowl). Two sites were swabbed with one side of the sponge, which was turned over for swabbing the remaining two sites. A muscle sample (approximately 4 cm3 ) was taken from the diaphragm (avoiding fat, gristle or membrane), or occasionally from the neck muscle (if the diaphragm was unsuitable) and frozen after collection. A minimum of five
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E.A. Marier et al. / Preventive Veterinary Medicine 117 (2014) 542–553
ileo-caecal lymph nodes – easy to collect tissues most often colonised by Salmonella (Carlson and Blaha, 2001) – were bluntly harvested with gloved fingers to collect a total sample weight of at least 25 g – as per the standard sample size for ‘EN-ISO 6579:Annex D’ methodology – and these were put into a small pot which was then sealed as required by the technical specifications. In some cases where insufficient ileo-caecal lymph nodes were present, adjacent jejunal lymph nodes may have been collected to reach 25 g of material. After the lymph nodes had been removed, the whole caecum was collected at the abattoir, tied up and sent in leak-proof plastic bags to the laboratory. Upon receipt of the caecum, its surface was sterilised by using a palate knife heated by Bunsen flame, then an incision was made using a disposable sterile scalpel and the content of the caecum was milked into a screw capped pot. Ten grams of caecal contents were used for testing. The samples were taken by government officials from the Meat Hygiene Service (MHS) in Great Britain and officials from the Department of Agriculture and Rural Development (DARD) in Northern Ireland. They were packed in an insulated box together with an ice pack and sent on the same day by first-class post to the national Salmonella Reference laboratory at the Veterinary Laboratories Agency (VLA) (now Animal and Plant Health Agency (APHA)). 2.3. Laboratory analysis Bacterial examination of samples began within 24 h of arrival at the laboratory and no later than 96 h after collection at the abattoir. The isolation method used for Salmonella was performed as described in ISO6579 2002 Annexe D. This involved preparing pre-enrichment culture in Buffered Peptone Water (BPW) (sample to BPW ratio 1:10) at 37 ◦ C for 18 h. The BPW culture was then sub-cultured to Modified Semi Solid Rappaport Vassiliadis medium (MSRV); Difco 1868-17 – 0.1 ml as 3 equal and equidistant drops on the surface of the MSRV plate and incubated at 41.5 ◦ C for 24 h and 48 h. After incubation, at 24 h and 48 h, MSRV plates were examined for typical Salmonella-like growth and subcultured on to Xylose Lysine Deoxycholate agar plates (XLD); Difco 278850 and on to Brilliant Green agar BGAN; Oxoid CM329 plus 20 g/ml Novobiocin; Merck 44206 and incubated at 37 ◦ C for 24 h. Incubated XLD and BGAN plates were examined for characteristic Salmonella colonies. Up to five suspected colonies were checked for Salmonella and one positive colony per plate was confirmed serologically or biochemically. Confirmed positive isolates were serotyped according to the Kauffman–White scheme (Popoff, 2001). Salmonella typhimurium and Salmonella enteritidis isolates were ‘phage-typed according to the Health Protection Agency, Colindale and Ward schemes (Anderson et al., 1977; Ward et al., 1987). A serological test was carried out to detect Salmonella antibodies in the meat samples. On receipt at the laboratory, the chilled meat samples were stored at −20 ◦ C until analysis. After thawing, the meat-juice extracts were tested using a commercially available Salmonella lipopolysaccharide (LPS) ELISA kit for detection of antibodies to invasive
serovars within serogroups B and C1 (Guildhay Vetsign kit). Test optical density (OD) results were converted to sample-to-positive (S/P) ratios, and samples were defined as negative or positive according to the S/P ratio. Within this categorisation S/P ratios of less than or equal to 0.25 were regarded as negative and >0.25 as positive. 2.4. Data collection and analysis The samplers were asked to collect information about the abattoir process (slaughtering speed, singeing time and scalding temperature), the sampling date and time and the carcass (carcass weight, detail on farm of origin) at the time of sampling using the structured questionnaire provided. All data obtained from the questionnaires and the test results from the four samples were entered into a bespoke database developed in Microsoft® Access. Data were subject to manual cleaning and checking and consideration of logical consistency (e.g. carcass weight within accepted limits, time of slaughter must be later than the time at which line started). When needed, abattoir officials were contacted to confirm anomalies (and outliers) or unclear details from the questionnaire. Statistical analyses were carried out using Stata 10® . Univariable analysis was carried out to determine whether any of the recorded variables were associated with the Salmonella status of the pig using each of three different bacteriological sampling methods (carcass swab, lymph node and caecal contents). Associations between abattoir level factors and the serological status of the pig as measured by meat juice ELISA was considered to be less informative and thus no further analysis of this outcome was carried out apart from the variable ‘carcass weight’. A p-value of ≤0.25 was taken to indicate a possible association. Potentially associated explanatory variables were entered into three different multivariable logistic regression models (one model for each type of sample). Using a forward stepwise procedure, the variables were included or excluded in the final model based on the Wald test p-value of 0.25) were detected in 152 out of 599 meat juice samples tested, which yielded a seroprevalence of 25% (CI95% 22–29%). A total of 23 serovars were identified from the 350 positive samples, isolated from 242 pigs. The most frequently isolated serovars were S. typhimurium and S. derby (57% and 26% of all isolates, respectively) (Table 1). Two other serovars designated as of particular public health significance by the EC were isolated: S. enteritidis and S. infantis. Monophasic Salmonella was also detected in this survey. The S. typhimurium ‘phage types that were most frequently isolated were U288 and DT193. 3.2. Risk factors for Salmonella infection 3.2.1. Univariable and multivariable models The results from the univariable and multivariable models that estimate the association between the isolation of Salmonella and abattoir level variables in the three different types of sample (caecal contents, lymph nodes and carcass swabs) are shown in Tables 3–5 respectively. There was no evidence of any important association between caecal contents status and abattoir level variables. The estimated standard errors in relation to bacteriological tests were adjusted to account for clustering within the abattoir. 3.2.2. Throughput effect Multivariable models using lymph node culture as an outcome showed evidence of an association between the number of pigs scheduled for slaughter and Salmonella status. As throughput (number of pigs scheduled for slaughter per day) decreased, the risk of a pig being positive was also reduced. Abattoirs with a throughput of less than 1500 pigs per day had a significantly reduced risk of isolation of Salmonella from lymph nodes (p < 0.001) (OR = 0.41, CI95% 0.24–0.71). The variable ‘throughput’ had an apparently significant effect (p < 0.01) on the carcass swab contamination but only in the univariate analysis. However, the risk of carcass contamination is likely to be associated with a number of factors including line speed, number of pigs slaughtered prior to the sampled one, time of day, slaughterhouse hourly throughput and operator competence (Letellier et al., 2009; Arguello et al., 2013). A number of these factors are likely to be correlated at the abattoir level. Therefore, an aggregated single variable was defined to assess the risk of contamination of the carcass: the ‘time-elapsed-since-thestart-of-the-line’. This was estimated from the recorded time that the line started and the recorded time of sampling base. Thus, it took into account the speed of the line (hourly throughput) and the time that elapsed until the selected pig
Fig. 1. Carcass swabs results versus time since start of the line (hours) with logit transformed lowess smoother.
was sampled. This variable was independent of the time at which the abattoir had started to operate. This new variable showed an important association with the risk of a positive carcass swab when considered both as categorical and continuous. To determine whether ‘time-elapsed-sincestart-of-the-line’ should be included as a categorical or a continuous variable, the smoothed lowess plot of predicted carcass swab result (Fig. 1) against the ‘time-elapsed-sincestart-of-the-line’ was examined and appeared to show a smooth increase. The categories chosen – based on quartile – for the univariate were dismissed in favour of using the continuous variable for the final model. Fig. 1 shows that the relationship is roughly linear beyond the first 3–4 h, indicating an increased risk of a positive carcass swab as the day progressed. This significant association (p < 0.01) supports the conclusion that the greater the time elapsed between start and slaughter, the greater the potential of carcass contamination (Table 5). 3.2.3. Carcass weight effect There was an association between the odds of obtaining a positive meat juice result or positive lymph node result and the weight of the carcass. For the meat juice univariate outcome, pigs weighing over 75 kg were less likely to have a Salmonella-positive meat juice result than those under 75 kg (p = 0.03). A similar pattern was observed for the lymph node outcome where pigs weighing over 75 kg were half as likely to have a Salmonella-positive lymph node; except with the carcass weight greater than 84 kg where the evidence for an association between the weight and isolation of Salmonella from lymph nodes was very weak (OR = 0.77, p = 0.29) (Table 4). 3.2.4. Lymph node sample weight effect The multivariable model results for lymph node Salmonella status also showed an association with the weight of the lymph nodes sampled. The weights obtained varied between 15 and 45 g, although no more than 25 g of macerated material was taken for testing. Pigs from which a heavier lymph node sample was taken were more likely to be positive, but this was only statistically significant for the highest two categories where samples of more than 25 g
546 Table 1 Serovars identified from each sample type and total number of pigs that yielded each serovar from either carcass swab, lymph node or caecal content. Also shown are the percentage of positive samples and percentage of total samples belonging to each serovar. Serovar
Caecal content
4,12:i:4,5,12:-1,2 4,5,12:i:S. agama S. anatum S. bovismorbificans S. coeln S. derby S. enteritidis 1 S. Give S. Goldcoast S. Heidelberg S. infantisa S. Kedougou S. London S. Mbandaka O Rough:l,v:1,5 O-Rough:fg:O-Rough:i:1,2 S. Panama S. Reading S. Rissen S. typhimuriuma Total a b
% of Total Positive (n = 129)
% of total tested (n = 586)
2
1.6
0.3
3 1 1 4
2.3 0.8 0.8 3.1
0.5 0.2 0.2 0.7
37 1 2 4 1 1 7 4 2
28.7 0.8 1.6 3.1 0.8 0.8 5.4 3.1 1.6
6.3 0.2 0.3 0.7 0.2 0.2 1.2 0.7 0.3
6 1 52 129
4.7 0.8 40.3 22.0%
1.0 0.2 8.9
Total number samples positive
Lymph nodes
% of total positive (n = 91)
% of total tested (n = 599)
1 1 4
1.1 1.1 4.4
0.2 0.2 0.7
1 1
1.1 1.1
0.2 0.2
20
22.0
3.3
1
1.1
0.2
4 1
4.4 1.1
0.7 0.2
1.1 2.2 1.1 2.2 5.5 1.1 49.5 15.2%
0.2 0.3 0.2 0.3 0.8 0.2 7.5
1 2 1 2 5 1 45 91
Salmonella serovar of public health significance as designated by the EU. There was no Hadar or Virchow found. Note: more than one serovar may be isolated from a single pig.
Total number samples positive
Total (carcass swabs, lymph nodes, or caecal content) % of Total Positive (n = 130)
% of total tested (n = 599)
4 1 1 1 1 27 2 1 1
3.1 0.8 0.8 0.8 0.8 20.8 1.5 0.8 0.8
0.7 0.2 0.2 0.2 0.2 4.5 0.3 0.2 0.2
2 3
1.5 2.3
0.3 0.5
1 5 0 80 130
0.8 3.9 0 61.5 21.7%
0.2 0.8 0 13.4
Total no. pigs
% of total positive (n = 244)
% of total tested (n = 599)
2 1 9 2 1 5 1 63 2 3 5 1 1 11 6 2 1 2 1 3 13 1 140 276b
0.8 0.4 3.7 0.8 0.4 2.1 0.4 25.8 0.8 1.2 2.1 0.4 0.4 4.5 2.5 0.8 0.4 0.8 0.4 1.2 5.3 0.4 57.4
0.3 0.2 1.5 0.3 0.3 0.8 0.2 9.7 0.5 0.5 0.3 0.2 0.2 1.3 1.0 0.2 0.2 0.2 0.2 0.3 2.3 0.2 19.9
E.A. Marier et al. / Preventive Veterinary Medicine 117 (2014) 542–553
Total number samples positive
Carcass swab
Table 2 ‘Phage types identified from each sample type and total number of pigs yielding each ‘phage type from caecum content, carcass swab or lymph node. Also shown are the percentage of positive samples and percentage of total samples belonging to each ‘phage type. ‘phage type
S. typhimurium
104 104B 120 170 193 194 208 56 56A 85 97 RDNCa U288 U302 U308 UNTY
S. Enteritidis
13A 4b
Total no. caecum samples positive
% of total positive (n = 137)
% of total tested (n = 626)
Total no. swab samples positive
% of total positive (n = 97)
% of total tested (n = 641)
1.0 3.1 1.0 1.0 11.3
0.2 0.5 0.2 0.2 1.7
6 3
4.4 2.2
1.0 0.5
9
6.6
1.4
1 3 1 1 11
2
1.5
0.3
3
3.1
0.5
1
0.7
0.2
17 9 1 8
12.4 6.6 0.7 5.8
2.7 1.4 0.2 1.3
1 16 2
1.0 16.5 2.1
8
8.3
1
0.7
0.2
Total no. lymph samples positive
% of total positive (n = 139)
% of total tested (n = 639)
Total no. pigs
% of total pig positive (n = 259)
% of total pig tested (n = 641)
4.3 4.3 0.4 0.4 13.9 0.4 1.5 0.8 0.4 0.4 0.4 1.2 19.7 6.2 0.4 7.7
1.7 1.7 0.2 0.2 5.6 0.2 0.6 0.3 0.2 0.2 0.2 0.5 8.0 2.5 0.2 3.1
0.4 0.4
0.2 0.2
5 8
3.6 5.8
0.8 1.3
20 1 2 2 1 1
14.4 0.7 1.4 1.4 0.7 0.7
3.1 0.2 0.3 0.3 0.2 0.2
0.2 2.5 0.3
2 28 9
1.4 20.1 6.5
0.3 4.4 1.4
1.3
7
5.0
1.1
11 11 1 1 36 1 4 2 1 1 1 3 51 16 1 20
1 1
0.7 0.7
0.2 0.2
1 1
E.A. Marier et al. / Preventive Veterinary Medicine 117 (2014) 542–553
Serovars
Note: more than one ‘phage type may be isolated from a single pig. a RDNC means that the isolate reacts with the ‘phages but does not conform to a recognised ‘phage type.
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Table 3 Univariate analysis concerning the isolation of Salmonella spp from caecal contents. Variable
Level
n
Positive (%)
p-value
Carcass weight (kg)
84
162 142 162 120
22 22 21 24
0.93
Due kill order
Up to 500 501–1000 1001–2000 >2000
182 140 188 76
21 21 20 30
0.30
Method of cleaning lairage
Water only Pressure hose Disinfectant Pressure hose with disinfectant
50 269 36 220
28 19 19 25
0.37
Number of pigs scheduled for slaughter on day of sampling (Throughput)
>2900 2501–2900 1501–2500 Up to 1500
150 143 142 145
19 22 12 19
0.50
Time elapsed since start of line (hours)
≤1.7 1.71/3.40 3.41/5.60 >5.6 Continuous
140 140 143 144 567
24 18 22 24
Sample month (quarter)
January–March April-June July-September October-December
150 139 148 149
25 24 23 16
Time of day
am pm
255 331
22 22
0.86
Hourly Throughput on day of sampling (pigs/hour)
100–200 200–300 300–500 >500
70 194 273 49
19 24 20 27
0.66
>11 8–10 5–7 ≤4
67 172 126 138
22 17 23 25
0.31
2000
185 143 192 79
21 20 23 24
0.77
Water only Pressure hose Disinfectant Pressure hose with disinfectant
50 271 38 229
22 20 16 25
0.55
>2900
150
22
2501–2900 1501–2500 Up to 1500
143 142 145
28 25 10
≤1.7 1.71/3.40 3.41/5.60 >5.6 Continuous
143 143 147 147
22 18 25 20
Sample month (quarter)
January–Machr April–June July–September October–December
153 141 156 149
22 23 23 18
0.67
Time of day
am pm
261 338
20 23
0.35
100–200
71
13
200–300 300–500 >500
197 281 50
20 23 34
67
24
8–10 5–7 ≤4 Continuous
177 129 140
23 20 26
32 g
84 115 226 171
12 19 24 26
p-value
Multivariable OR
Due kill order
Method of cleaning lairage
Number of pigs scheduled for slaughter on day of sampling (Throughput)
Time elapsed since start of line (hours)
Hourly throughput on day of sampling (pigs/hour)
Time carcase spent in the singeing unit (seconds)
Temperature of scalding tank (◦ C)
Weight of lymph node sample
>11
Contents lists available at ScienceDirect
Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed
Abattoir based survey of Salmonella in finishing pigs in the United Kingdom 2006–2007 E.A. Marier a,∗ , L.C. Snow a , T. Floyd a , I.M. McLaren b , J. Bianchini a , A.J.C. Cook c , R.H. Davies b a Epidemiology, Surveillance and Risk Group, Animal Health and Veterinary Laboratories Agency, Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom b Department of Bacteriology, Animal Health and Veterinary Laboratories Agency, Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom c School of Veterinary Medicine, Faculty of Health & Medical Sciences, University of Surrey, Guildford, Surrey GU2 7TE, United Kingdom
a r t i c l e
i n f o
Article history: Received 15 December 2013 Received in revised form 22 August 2014 Accepted 6 September 2014 Keywords: Pig Salmonella Risk factors Slaughter
a b s t r a c t All European Union (EU) Member States (MSs) were required to conduct a baseline survey from October 2006 to September 2007 to estimate the prevalence of Salmonella infection in finisher pigs at slaughter. In the United Kingdom (UK), samples for microbiological culture were collected from the ileo-caecal lymph nodes, from carcass swabs and from caecal contents. Meat juice samples were also collected for testing in an enzyme-linked immunosorbent assay (MJE) to detect a serological response. Salmonella was isolated from 22% (CI95% 19–25%) of ileo-caecal lymph nodes, 15% (CI95% 12–18%) of carcass swabs and 22% (CI95% 19–26%) of caecal contents. A quarter (25%, CI95% 22–29%) of MJE samples were positive for Salmonella antibodies (cut-off, S/P ≥ 0.25). The most frequently identified serovars were Salmonella typhimurium (57%) and Salmonella derby (26%). The results were used to investigate some factors associated with Salmonella infection in slaughter pigs. The weight of the carcass was significant, with a lower probability of obtaining a positive meat juice result from pigs with a weight greater than 75 kg (p = 0.03). The weight of the lymph node sample was significantly associated with Salmonella status, with a heavier sample of lymph nodes being more likely to be positive (OR = 2.16 CI95% 1.07–4.39). ‘Carcass weight’ in two of the classes – 75–79 kg and 80–84 kg – (OR = 0.44 CI95% 0.28–0.70; OR = 0.64 CI95% 0.49–0.85) and ‘fewer-than-1500-pigs’ scheduled for slaughter on the day of sampling’ (OR = 0.41 CI95% 0.24–0.71) were also associated with a reduced risk of isolating Salmonella from lymph node. The model for carcass swabs showed a positive association with ‘time-elapsed-since-thestart-of-the-line’ indicating a higher risk of contamination as the day progressed (p < 0.01). This model also showed positive association between isolation of Salmonella from a carcass swab and the occurrence of Salmonella in caecal contents from the same pig (OR = 2.22 CI95% 1.38–3.59) and a negative association with time in singeing units (OR = 0.88 CI95% 0.79–0.98). Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author. Tel.: +44 01932 357618; fax: +44 01932 359429. E-mail address: [email protected] (E.A. Marier).
Pigs have been identified as a reservoir and carrier of Salmonella (Fedorka-Cray et al., 2000). In recent years, pork has been increasingly recognised in a number of countries
http://dx.doi.org/10.1016/j.prevetmed.2014.09.004 0167-5877/Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.
E.A. Marier et al. / Preventive Veterinary Medicine 117 (2014) 542–553
as a potential source of human salmonellosis (Hald et al., 2003; Boyen et al., 2008). Salmonella-positive carrier pigs represent a constant source of Salmonella contamination for the abattoir and processing environment, and contaminated pig meat products are a human health hazard (Boyen et al., 2008). Various studies to estimate the prevalence of Salmonella in slaughter pigs have been previously carried out in Great Britain (Davies et al., 2004; Milnes et al., 2008, 2009) as well as in a number of other countries including Germany (Kasbohrer et al., 2000), Italy (Bonardi et al., 2003) and Canada (Letellier et al., 1999). Also, to monitor the level of Salmonella exposure in the UK pig production, meat juice samples (MJE) were tested periodically from each batch of pigs sent to the abattoir, as part of the Zoonoses National Control Plan (ZNCP, 2008–2012, BPEX). Results from these studies have identified that about a quarter of pigs’ caecal contents were contaminated with Salmonella at slaughter in the UK (Davies et al., 2004; Clough et al., 2009). In Denmark, an association between Salmonella sero-prevalence in slaughter pigs and carcass contamination was identified (Sorensen et al., 2004). Other studies which have investigated risks factors at slaughter identified evisceration practices, polishing equipment, splitting, dressing and meat inspection as potential contributors to carcass contamination when slaughtering Salmonella-positive pigs (Berends et al., 1997; Botteldoorn et al., 2003; Baptista et al., 2010). In 2006, to provide the scientific basis for setting a community reduction target for Salmonella in slaughter pigs (Anon, 2008a,b), each EU Member State (MS) was required to carry out a survey of the prevalence of Salmonella infection in finisher pigs at slaughter. This paper reports the results of the EU baseline survey in the UK and of the data on aspects of abattoir practices collected and investigated for evidence of any association with Salmonella status in commercially slaughtered pigs. To enable comparison with previous surveys and the national monitoring programme for Salmonella in pigs (Davies et al., 2000, 2004; Twomey et al., 2007), Defra (Department for Environment, Food & Rural Affairs) agreed to add the collection of caecal contents and meat juice samples to the sampling protocol. The aim of the study was to attempt to link available potential risk factors recorded during the baseline survey with the Salmonella status of the pigs. As this was an anonymous survey, few farm level risk factors were available. Such risk factors were therefore not included in the analysis. 2. Materials and methods 2.1. Sampling plan The United Kingdom’s baseline survey was carried out over a 12-month period from 1 October 2006 to 30 September 2007. The method followed the technical specifications document (SANCO/40162/2006) annexed to Commission Decision 2006/668/EC. The calculation was based on an assumption of an infinite population (>100,000 pigs slaughtered per year) and an estimated prevalence of 50% at a minimum accuracy of 4%, with 95% confidence.
543
In 2006, the UK had an estimated population of 4.7 million pigs, which corresponded to a minimum sample size of 600 pig carcasses plus a further 10% (60 carcasses) to account for any issues during the survey. Therefore, 660 pigs kept in the UK for at least three months preceding slaughter were chosen at random. The sampling was performed to ensure that the largest capacity slaughterhouses, which together represented at least 80% of the slaughtered finisher pig population in the UK, were included. The number of carcasses to be sampled per abattoir was proportional to their annual throughput during the previous year and was obtained by multiplying the sample size by the proportion of carcasses processed. Sampling was equally distributed between months to cover all seasons, starting with October 2006 as month 1. A sampling schedule was produced monthly for each abattoir to indicate which day and which pig on a specific day it had to sample. The days that each abattoir had to sample in each month were selected at random using a random selector sheet in Microsoft® Excel. The rank of the carcass to be sampled on a specific day was also selected at random using this method, with a maximum that reached the highest possible number of finisher pigs slaughtered in the slaughterhouse on a given day. If it was not possible to sample the pig selected for logistical reasons, a second number was picked using the same method of random selection. Casualties and condemned carcasses were excluded from the survey. As carcasses with a live weight of less than 50 kg or more than 170 kg were atypical for the commercial market and were likely to be substantially older or younger than the majority of pigs, they were also excluded from the survey as per the baseline survey’s technical specifications (SANCO/40162/2006). If the selected carcass was excluded, the next suitable carcass on the line was selected instead. 2.2. Sample collection A total of four samples were collected from each carcass: a carcass swab, a muscle sample, an aggregate of ileo-caecal lymph nodes and, to allow comparison with previous GB surveys, a sample of caecal contents. Each sample category was expected to provide evidence of specific types of exposure. The contamination of a carcass swab may come from the sampled pig, from cross-contamination by other pigs or from the abattoir environment; the meat juice sample provides evidence of prior exposure to Salmonella but not from the slaughter process; the lymph node usually provides evidence of an infected pig at the moment of sampling while contaminated caecal contents may be from ingestion of contaminated material and passive transit in gut or infection and could be farm or abattoir associated. The carcass swab was taken on the left or right side of the carcass using one single sponge for all four sites described in Annex A of Standard ISO 17604 (Anon, 2003, hind limb, abdomen, mid-dorsal region, jowl). Two sites were swabbed with one side of the sponge, which was turned over for swabbing the remaining two sites. A muscle sample (approximately 4 cm3 ) was taken from the diaphragm (avoiding fat, gristle or membrane), or occasionally from the neck muscle (if the diaphragm was unsuitable) and frozen after collection. A minimum of five
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ileo-caecal lymph nodes – easy to collect tissues most often colonised by Salmonella (Carlson and Blaha, 2001) – were bluntly harvested with gloved fingers to collect a total sample weight of at least 25 g – as per the standard sample size for ‘EN-ISO 6579:Annex D’ methodology – and these were put into a small pot which was then sealed as required by the technical specifications. In some cases where insufficient ileo-caecal lymph nodes were present, adjacent jejunal lymph nodes may have been collected to reach 25 g of material. After the lymph nodes had been removed, the whole caecum was collected at the abattoir, tied up and sent in leak-proof plastic bags to the laboratory. Upon receipt of the caecum, its surface was sterilised by using a palate knife heated by Bunsen flame, then an incision was made using a disposable sterile scalpel and the content of the caecum was milked into a screw capped pot. Ten grams of caecal contents were used for testing. The samples were taken by government officials from the Meat Hygiene Service (MHS) in Great Britain and officials from the Department of Agriculture and Rural Development (DARD) in Northern Ireland. They were packed in an insulated box together with an ice pack and sent on the same day by first-class post to the national Salmonella Reference laboratory at the Veterinary Laboratories Agency (VLA) (now Animal and Plant Health Agency (APHA)). 2.3. Laboratory analysis Bacterial examination of samples began within 24 h of arrival at the laboratory and no later than 96 h after collection at the abattoir. The isolation method used for Salmonella was performed as described in ISO6579 2002 Annexe D. This involved preparing pre-enrichment culture in Buffered Peptone Water (BPW) (sample to BPW ratio 1:10) at 37 ◦ C for 18 h. The BPW culture was then sub-cultured to Modified Semi Solid Rappaport Vassiliadis medium (MSRV); Difco 1868-17 – 0.1 ml as 3 equal and equidistant drops on the surface of the MSRV plate and incubated at 41.5 ◦ C for 24 h and 48 h. After incubation, at 24 h and 48 h, MSRV plates were examined for typical Salmonella-like growth and subcultured on to Xylose Lysine Deoxycholate agar plates (XLD); Difco 278850 and on to Brilliant Green agar BGAN; Oxoid CM329 plus 20 g/ml Novobiocin; Merck 44206 and incubated at 37 ◦ C for 24 h. Incubated XLD and BGAN plates were examined for characteristic Salmonella colonies. Up to five suspected colonies were checked for Salmonella and one positive colony per plate was confirmed serologically or biochemically. Confirmed positive isolates were serotyped according to the Kauffman–White scheme (Popoff, 2001). Salmonella typhimurium and Salmonella enteritidis isolates were ‘phage-typed according to the Health Protection Agency, Colindale and Ward schemes (Anderson et al., 1977; Ward et al., 1987). A serological test was carried out to detect Salmonella antibodies in the meat samples. On receipt at the laboratory, the chilled meat samples were stored at −20 ◦ C until analysis. After thawing, the meat-juice extracts were tested using a commercially available Salmonella lipopolysaccharide (LPS) ELISA kit for detection of antibodies to invasive
serovars within serogroups B and C1 (Guildhay Vetsign kit). Test optical density (OD) results were converted to sample-to-positive (S/P) ratios, and samples were defined as negative or positive according to the S/P ratio. Within this categorisation S/P ratios of less than or equal to 0.25 were regarded as negative and >0.25 as positive. 2.4. Data collection and analysis The samplers were asked to collect information about the abattoir process (slaughtering speed, singeing time and scalding temperature), the sampling date and time and the carcass (carcass weight, detail on farm of origin) at the time of sampling using the structured questionnaire provided. All data obtained from the questionnaires and the test results from the four samples were entered into a bespoke database developed in Microsoft® Access. Data were subject to manual cleaning and checking and consideration of logical consistency (e.g. carcass weight within accepted limits, time of slaughter must be later than the time at which line started). When needed, abattoir officials were contacted to confirm anomalies (and outliers) or unclear details from the questionnaire. Statistical analyses were carried out using Stata 10® . Univariable analysis was carried out to determine whether any of the recorded variables were associated with the Salmonella status of the pig using each of three different bacteriological sampling methods (carcass swab, lymph node and caecal contents). Associations between abattoir level factors and the serological status of the pig as measured by meat juice ELISA was considered to be less informative and thus no further analysis of this outcome was carried out apart from the variable ‘carcass weight’. A p-value of ≤0.25 was taken to indicate a possible association. Potentially associated explanatory variables were entered into three different multivariable logistic regression models (one model for each type of sample). Using a forward stepwise procedure, the variables were included or excluded in the final model based on the Wald test p-value of 0.25) were detected in 152 out of 599 meat juice samples tested, which yielded a seroprevalence of 25% (CI95% 22–29%). A total of 23 serovars were identified from the 350 positive samples, isolated from 242 pigs. The most frequently isolated serovars were S. typhimurium and S. derby (57% and 26% of all isolates, respectively) (Table 1). Two other serovars designated as of particular public health significance by the EC were isolated: S. enteritidis and S. infantis. Monophasic Salmonella was also detected in this survey. The S. typhimurium ‘phage types that were most frequently isolated were U288 and DT193. 3.2. Risk factors for Salmonella infection 3.2.1. Univariable and multivariable models The results from the univariable and multivariable models that estimate the association between the isolation of Salmonella and abattoir level variables in the three different types of sample (caecal contents, lymph nodes and carcass swabs) are shown in Tables 3–5 respectively. There was no evidence of any important association between caecal contents status and abattoir level variables. The estimated standard errors in relation to bacteriological tests were adjusted to account for clustering within the abattoir. 3.2.2. Throughput effect Multivariable models using lymph node culture as an outcome showed evidence of an association between the number of pigs scheduled for slaughter and Salmonella status. As throughput (number of pigs scheduled for slaughter per day) decreased, the risk of a pig being positive was also reduced. Abattoirs with a throughput of less than 1500 pigs per day had a significantly reduced risk of isolation of Salmonella from lymph nodes (p < 0.001) (OR = 0.41, CI95% 0.24–0.71). The variable ‘throughput’ had an apparently significant effect (p < 0.01) on the carcass swab contamination but only in the univariate analysis. However, the risk of carcass contamination is likely to be associated with a number of factors including line speed, number of pigs slaughtered prior to the sampled one, time of day, slaughterhouse hourly throughput and operator competence (Letellier et al., 2009; Arguello et al., 2013). A number of these factors are likely to be correlated at the abattoir level. Therefore, an aggregated single variable was defined to assess the risk of contamination of the carcass: the ‘time-elapsed-since-thestart-of-the-line’. This was estimated from the recorded time that the line started and the recorded time of sampling base. Thus, it took into account the speed of the line (hourly throughput) and the time that elapsed until the selected pig
Fig. 1. Carcass swabs results versus time since start of the line (hours) with logit transformed lowess smoother.
was sampled. This variable was independent of the time at which the abattoir had started to operate. This new variable showed an important association with the risk of a positive carcass swab when considered both as categorical and continuous. To determine whether ‘time-elapsed-sincestart-of-the-line’ should be included as a categorical or a continuous variable, the smoothed lowess plot of predicted carcass swab result (Fig. 1) against the ‘time-elapsed-sincestart-of-the-line’ was examined and appeared to show a smooth increase. The categories chosen – based on quartile – for the univariate were dismissed in favour of using the continuous variable for the final model. Fig. 1 shows that the relationship is roughly linear beyond the first 3–4 h, indicating an increased risk of a positive carcass swab as the day progressed. This significant association (p < 0.01) supports the conclusion that the greater the time elapsed between start and slaughter, the greater the potential of carcass contamination (Table 5). 3.2.3. Carcass weight effect There was an association between the odds of obtaining a positive meat juice result or positive lymph node result and the weight of the carcass. For the meat juice univariate outcome, pigs weighing over 75 kg were less likely to have a Salmonella-positive meat juice result than those under 75 kg (p = 0.03). A similar pattern was observed for the lymph node outcome where pigs weighing over 75 kg were half as likely to have a Salmonella-positive lymph node; except with the carcass weight greater than 84 kg where the evidence for an association between the weight and isolation of Salmonella from lymph nodes was very weak (OR = 0.77, p = 0.29) (Table 4). 3.2.4. Lymph node sample weight effect The multivariable model results for lymph node Salmonella status also showed an association with the weight of the lymph nodes sampled. The weights obtained varied between 15 and 45 g, although no more than 25 g of macerated material was taken for testing. Pigs from which a heavier lymph node sample was taken were more likely to be positive, but this was only statistically significant for the highest two categories where samples of more than 25 g
546 Table 1 Serovars identified from each sample type and total number of pigs that yielded each serovar from either carcass swab, lymph node or caecal content. Also shown are the percentage of positive samples and percentage of total samples belonging to each serovar. Serovar
Caecal content
4,12:i:4,5,12:-1,2 4,5,12:i:S. agama S. anatum S. bovismorbificans S. coeln S. derby S. enteritidis 1 S. Give S. Goldcoast S. Heidelberg S. infantisa S. Kedougou S. London S. Mbandaka O Rough:l,v:1,5 O-Rough:fg:O-Rough:i:1,2 S. Panama S. Reading S. Rissen S. typhimuriuma Total a b
% of Total Positive (n = 129)
% of total tested (n = 586)
2
1.6
0.3
3 1 1 4
2.3 0.8 0.8 3.1
0.5 0.2 0.2 0.7
37 1 2 4 1 1 7 4 2
28.7 0.8 1.6 3.1 0.8 0.8 5.4 3.1 1.6
6.3 0.2 0.3 0.7 0.2 0.2 1.2 0.7 0.3
6 1 52 129
4.7 0.8 40.3 22.0%
1.0 0.2 8.9
Total number samples positive
Lymph nodes
% of total positive (n = 91)
% of total tested (n = 599)
1 1 4
1.1 1.1 4.4
0.2 0.2 0.7
1 1
1.1 1.1
0.2 0.2
20
22.0
3.3
1
1.1
0.2
4 1
4.4 1.1
0.7 0.2
1.1 2.2 1.1 2.2 5.5 1.1 49.5 15.2%
0.2 0.3 0.2 0.3 0.8 0.2 7.5
1 2 1 2 5 1 45 91
Salmonella serovar of public health significance as designated by the EU. There was no Hadar or Virchow found. Note: more than one serovar may be isolated from a single pig.
Total number samples positive
Total (carcass swabs, lymph nodes, or caecal content) % of Total Positive (n = 130)
% of total tested (n = 599)
4 1 1 1 1 27 2 1 1
3.1 0.8 0.8 0.8 0.8 20.8 1.5 0.8 0.8
0.7 0.2 0.2 0.2 0.2 4.5 0.3 0.2 0.2
2 3
1.5 2.3
0.3 0.5
1 5 0 80 130
0.8 3.9 0 61.5 21.7%
0.2 0.8 0 13.4
Total no. pigs
% of total positive (n = 244)
% of total tested (n = 599)
2 1 9 2 1 5 1 63 2 3 5 1 1 11 6 2 1 2 1 3 13 1 140 276b
0.8 0.4 3.7 0.8 0.4 2.1 0.4 25.8 0.8 1.2 2.1 0.4 0.4 4.5 2.5 0.8 0.4 0.8 0.4 1.2 5.3 0.4 57.4
0.3 0.2 1.5 0.3 0.3 0.8 0.2 9.7 0.5 0.5 0.3 0.2 0.2 1.3 1.0 0.2 0.2 0.2 0.2 0.3 2.3 0.2 19.9
E.A. Marier et al. / Preventive Veterinary Medicine 117 (2014) 542–553
Total number samples positive
Carcass swab
Table 2 ‘Phage types identified from each sample type and total number of pigs yielding each ‘phage type from caecum content, carcass swab or lymph node. Also shown are the percentage of positive samples and percentage of total samples belonging to each ‘phage type. ‘phage type
S. typhimurium
104 104B 120 170 193 194 208 56 56A 85 97 RDNCa U288 U302 U308 UNTY
S. Enteritidis
13A 4b
Total no. caecum samples positive
% of total positive (n = 137)
% of total tested (n = 626)
Total no. swab samples positive
% of total positive (n = 97)
% of total tested (n = 641)
1.0 3.1 1.0 1.0 11.3
0.2 0.5 0.2 0.2 1.7
6 3
4.4 2.2
1.0 0.5
9
6.6
1.4
1 3 1 1 11
2
1.5
0.3
3
3.1
0.5
1
0.7
0.2
17 9 1 8
12.4 6.6 0.7 5.8
2.7 1.4 0.2 1.3
1 16 2
1.0 16.5 2.1
8
8.3
1
0.7
0.2
Total no. lymph samples positive
% of total positive (n = 139)
% of total tested (n = 639)
Total no. pigs
% of total pig positive (n = 259)
% of total pig tested (n = 641)
4.3 4.3 0.4 0.4 13.9 0.4 1.5 0.8 0.4 0.4 0.4 1.2 19.7 6.2 0.4 7.7
1.7 1.7 0.2 0.2 5.6 0.2 0.6 0.3 0.2 0.2 0.2 0.5 8.0 2.5 0.2 3.1
0.4 0.4
0.2 0.2
5 8
3.6 5.8
0.8 1.3
20 1 2 2 1 1
14.4 0.7 1.4 1.4 0.7 0.7
3.1 0.2 0.3 0.3 0.2 0.2
0.2 2.5 0.3
2 28 9
1.4 20.1 6.5
0.3 4.4 1.4
1.3
7
5.0
1.1
11 11 1 1 36 1 4 2 1 1 1 3 51 16 1 20
1 1
0.7 0.7
0.2 0.2
1 1
E.A. Marier et al. / Preventive Veterinary Medicine 117 (2014) 542–553
Serovars
Note: more than one ‘phage type may be isolated from a single pig. a RDNC means that the isolate reacts with the ‘phages but does not conform to a recognised ‘phage type.
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Table 3 Univariate analysis concerning the isolation of Salmonella spp from caecal contents. Variable
Level
n
Positive (%)
p-value
Carcass weight (kg)
84
162 142 162 120
22 22 21 24
0.93
Due kill order
Up to 500 501–1000 1001–2000 >2000
182 140 188 76
21 21 20 30
0.30
Method of cleaning lairage
Water only Pressure hose Disinfectant Pressure hose with disinfectant
50 269 36 220
28 19 19 25
0.37
Number of pigs scheduled for slaughter on day of sampling (Throughput)
>2900 2501–2900 1501–2500 Up to 1500
150 143 142 145
19 22 12 19
0.50
Time elapsed since start of line (hours)
≤1.7 1.71/3.40 3.41/5.60 >5.6 Continuous
140 140 143 144 567
24 18 22 24
Sample month (quarter)
January–March April-June July-September October-December
150 139 148 149
25 24 23 16
Time of day
am pm
255 331
22 22
0.86
Hourly Throughput on day of sampling (pigs/hour)
100–200 200–300 300–500 >500
70 194 273 49
19 24 20 27
0.66
>11 8–10 5–7 ≤4
67 172 126 138
22 17 23 25
0.31
2000
185 143 192 79
21 20 23 24
0.77
Water only Pressure hose Disinfectant Pressure hose with disinfectant
50 271 38 229
22 20 16 25
0.55
>2900
150
22
2501–2900 1501–2500 Up to 1500
143 142 145
28 25 10
≤1.7 1.71/3.40 3.41/5.60 >5.6 Continuous
143 143 147 147
22 18 25 20
Sample month (quarter)
January–Machr April–June July–September October–December
153 141 156 149
22 23 23 18
0.67
Time of day
am pm
261 338
20 23
0.35
100–200
71
13
200–300 300–500 >500
197 281 50
20 23 34
67
24
8–10 5–7 ≤4 Continuous
177 129 140
23 20 26
32 g
84 115 226 171
12 19 24 26
p-value
Multivariable OR
Due kill order
Method of cleaning lairage
Number of pigs scheduled for slaughter on day of sampling (Throughput)
Time elapsed since start of line (hours)
Hourly throughput on day of sampling (pigs/hour)
Time carcase spent in the singeing unit (seconds)
Temperature of scalding tank (◦ C)
Weight of lymph node sample
>11
