Animal welfare and microbiological safety of poultry meat: Impact of different at-farm animal welfare levels on at-slaughterhouse Campylobacter and Salmonella contamination

Food Control 109 (2020) 106921

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Animal welfare and microbiological safety of poultry meat: Impact of different at-farm animal welfare levels on at-slaughterhouse Campylobacter and Salmonella contamination

T

Luigi Iannetti∗, Diana Neri, Gino Angelo Santarelli, Giuseppe Cotturone, Michele Podaliri Vulpiani, Romolo Salini, Salvatore Antoci, Gabriella Di Serafino, Elisabetta Di Giannatale, Francesco Pomilio, Stefano Messori Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise “G. Caporale”, National Reference Laboratory for Campylobacter, Campo Boario, 64100, Teramo, Italy

A R T I C LE I N FO

A B S T R A C T

Keywords: Animal welfare Campylobacter Farm Food safety Salmonella Slaughterhouse

Stress factors and poor animal welfare can increase the susceptibility of food-producing animals to diseases, posing microbial risks to consumers. Animal welfare levels, objectively measured with the application of the Welfare Quality® protocol, were assessed in thirteen broiler flocks, including organic ones, to evaluate the presence of statistically significant differences in relation to Campylobacter and Salmonella faecal shedding and consequent microbiological contamination of broiler carcases at slaughterhouse. Each flock underwent animal welfare evaluation the day before slaughtering, followed by Campylobacter and Salmonella detection in faeces (caecal content) and neck skin at slaughterhouse. A total of 1040 samples (520 caecal contents; 520 neck skins) were included in the study. Campylobacter enumeration and Salmonella serotyping were also carried out. The highest welfare scores were reported in organic flocks. Significantly lower Campylobacter concentrations both in caecal content and neck skins (P < 0.05) were reported in organic batches, compared to high welfare conventional batches. Low-welfare batches showed higher prevalence of Salmonella both in neck skins and caecal content, with a statistically significant difference compared to high-welfare batches (43.6% versus 2.9% in neck skins; 19.3% versus 0% in caecal content; P < 0.00001). Salmonella Infantis and Salmonella Bredeney were the most common serotypes, while Campylobacter jejuni and Campylobacter coli were the detected species. This study provides new evidence that high animal welfare standards at farms, other than an ethical issue and a value-add for the end product, could also improve the microbiological safety of poultry meat, ultimately contributing to the protection of consumers.

1. Introduction Animal welfare and food safety are major issues in the production of food of animal origin. There is scientific evidence to support the fact that animal welfare should not be considered only as an ethical issue, but also from a food safety point of view: stress factors and poor welfare can increase the susceptibility of food-producing animals to diseases, posing risks to consumers, for example through common food-borne infections like Salmonella, Campylobacter and Escherichia coli STEC (EFSA, 2019). Campylobacter and Salmonella are the most important pathogens in relation to the poultry meat production chain, and they are both included among zoonotic agents listed in the European Union directive 2003/99 on the monitoring of zoonoses and zoonotic agents (Anonymous, 2003). Campylobacter is the causative agent of the most

∗

frequent zoonosis in Europe since 2005. Campylobacteriosis alone represents 70% of all cases of zoonosis, with more than 200,000 cases per year (EFSA/ECDC, 2018). It is a gastrointestinal disease mostly due to the consumption of contaminated poultry meat or other cross-contaminated food. The broiler chicken is the main reservoir of this pathogen, and current strategies to control Campylobacter infection in poultry, mainly based on farm biosecurity, often prove insufficient. Salmonella is the second most prevalent zoonotic agent in Europe, and also demonstrates a strong association with products from the poultry sector, particularly meat and eggs (EFSA/ECDC, 2018). Healthy carrier animals of different farm species are considered the most important source of microbiological contamination of carcases at abattoir (Botteldoorn, Heyndrickx, Rijpens, Grijspeerdt, & Herman, 2003; Malher et al., 2011; Rasschaert et al., 2008) and shedding could be

Corresponding author. Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, via Campo Boario, 64100, Teramo, Italy. E-mail address: [email protected] (L. Iannetti).

https://doi.org/10.1016/j.foodcont.2019.106921 Received 30 May 2019; Received in revised form 24 September 2019; Accepted 25 September 2019 Available online 26 September 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.

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levels from hatchlings to slaughtering. Although farming conditions were quite standardised, they vary from one farm to another. In particular, two out of the 13 batches were farmed according to European Union legislation for organic production. For broiler chickens this is ruled by regulations 834/2007 (Anonymous, 2007) and 889/2008 (Anonymous, 2008). Among other requirements, these foresee for broiler chickens:

increased after stress factors are applied, further contributing to carcass contamination (Traub-Dargartz et al., 2006). Studies of poultry have shown that shedding of pathogens can increase after transport and feed withdrawal (FAO/WHO, 2009; Rostagno, 2009). Other research demonstrates that some farm and slaughterhouse characteristics, as well as management practices, may affect prevalence of Campylobacter and Salmonella (Alpigiani et al., 2017; Corrier et al., 1999; Rostagno, Wesley, Trampel, & Hurd, 2006; Traub-Dargatz, Ladely, Dargatz, & Fedorka-Cray, 2006; Wesley, Rostagno, Hurd, & Trampel, 2009, 2005; Whyte, Collins, McGill, Monahan, & O’Mahony, 2001). However, there has been neither evaluation of the effects of different management systems on carcass contamination, nor comprehensive measurement of the level of at-farm stress in any of these studies. In general, there is a lack of knowledge about the effects of different management systems on poultry meat microbiological contamination. To develop mitigation strategies, more research is needed to deepen our understanding of the contribution of on-farm stress to carcass contamination. Management factors should be included in these investigations, and particular interest given to organic farming, currently governed by specific regulations in the European Union (Anonymous, 2007, 2008). Organic farming could strongly influence the welfare of animals bred under these conditions, including lower stocking densities, access to open-air areas, longer life, environmental enrichment, and reduced use of antibiotics. In fact, there is very limited literature concerned with the effects of this kind of management on animal welfare and on the microbiological quality of meat. Studies of organic vegetables have been more frequently carried out (Kuan et al., 2017; Lücke, 2017; Szczech et al., 2018). The aim of the present study, carried out in the framework of a research project funded by the Italian Ministry of Health, was to compare levels of animal welfare associated with different on-farm practices and evaluate their impact on Campylobacter and Salmonella faecal shedding and consequent microbiological contamination of broiler carcases at slaughterhouse. The Welfare Quality® broiler protocol (2009), an internationally recognised protocol for the integration of animal welfare in the food quality chain, produced from a research project financed by the European Commission, was used for an objective and animal-based measurement of animal welfare at farms. The scope was to give scientific evidence that there is a statistically significant relationship between the use of ‘animal-welfare friendly’ management methods and the level of faecal shedding of pathogens and consequent carcass contamination. This would shed light on the connections between animal welfare and microbiological quality of meat.

• Access to an open-air area through pop holes for at least one-third of the life; • Minimum life of 81 days; • Presence of perches of size and number commensurate with the size of the group; • No more than 4800 broiler chickens per each poultry house, or more if separated in groups of a minimum 4800 broilers each.

All batches were tested for Campylobacter at 30 days of age to ensure that only batches definitely contaminated by Campylobacter were included in the study. This was necessary to evaluate if the animal welfare level could make a difference when it comes to the Campylobacter concentration in broiler carcass skins. Batches were identified with a letter indicating the farm (from A to F) followed by a number indicating the batch: A1, A2, B1, B2, B3, C1, C2, D1, D2, E1, E2, F1, F2. Two batches per farm were examined, except farm B for which three batches were tested, as it changed from conventional (B1) to organic (B2 and B3) production during the study. Evaluations were carried out from May 2016 to September 2018. Animals from organic batches were slaughtered when they were between 83 and 84 days old, while the other batches were slaughtered when they were between 42 and 52 days old. The genotype was Ross 308 for all batches; the number of animals per batch ranged from 5973 to 24,355. The animals were slaughtered in two slaughterhouses, both located in the Marche region.

2.2. Animal welfare assessment The day before slaughtering, each batch underwent animal welfare evaluation with the Welfare Quality® protocol (2009) slightly modified according to De Jong et al. (2015) for faster data collection. According to the protocol, data were mostly collected on farms and then completed at the slaughterhouses with some information relating to possible diseases and lesions reported from carcass inspection. The protocol was produced as part of the 6th Framework Research programme of the European Commission, and is based on the internationally recognised ‘five freedoms’, which according to the World Organisation for Animal Health (OIE) should provide valuable guidance regarding animal welfare (OIE, 2018). In particular, the animal welfare measures used in the protocol are generated from four animal welfare principles (good feeding, good housing, good health, appropriate behaviour), and further divided into 12 criteria. These are mostly animal-based (observations of the animal response to the environment) and partially resource-based (evaluation of the structures and the environment where the animals are kept and evaluation of their management). A list

2. Materials and methods 2.1. Animals Thirteen batches of broiler chickens from six farms located in four regions of northern and central Italy (Veneto, Emilia-Romagna, Marche and Abruzzi) were included in the study. All belonged to the same integrated poultry company that manages the production chain at all

Table 1 The principles and the criteria (with examples of measures) that are the basis for the Welfare Quality® broiler protocol. Welfare Principle

Welfare Criterion

Measure

Good feeding

Absence of prolonged hunger Absence of prolonged thirst Comfort around resting Thermal comfort Ease of movement Absence of injuries Absence of disease Good human-animal relationship Positive emotional state

Suitable and appropriate diet (evaluation of carcases at slaughterhouse) Number of drinkers available per animal Plumage cleanliness, litter quality, air dust test Panting, huddling Stocking density Lameness, foot pad dermatitis, hock burns On farm mortality, culls on farm, evaluation of carcases at slaughterhouse Avoidance test (ADT) Qualitative Behaviour Assessment (QBA)

Good housing

Good health Appropriate behaviour

2

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total score. Seven batches were included in the LW group, four batches in the HW group (HW) and two batches in the O group. Both HW and O groups showed ‘excellent’ mean scores for the good feeding principle (89.7 ± 7.0 and 101.3 ± 1.7, respectively), while LW batches were all just ‘acceptable’ (43.7 ± 19.6). The other three Welfare Quality® principles scored lower on average. In fact, only the O group was very close to ‘enhanced’ for both good housing (47.4 ± 2.8) and good health (49.3 ± 1.3), while LW groups were just over ‘acceptable’, particularly for good housing (20.5 ± 23.4). Appropriate behaviour was never more than ‘acceptable’ in all groups, with organic flocks showing values sharply higher than others (39.4 ± 12.1 in O, versus 21.6 ± 1.49 in LW and 19.9 ± 4.3 in HW). For microbial contamination of carcass skins at slaughterhouse, Salmonella was found in all LW batches, with prevalence in single batches ranging from 20 to 70%, while it was absent or rare in HW (from 0 to 7.5%) and O (from 0 to 5%) batches. Table 2 reports the details of Salmonella prevalence in all tested batches, clustered according to welfare level. Salmonella prevalence in LW batches was 43.6% (122/280; 95% CI 37.9–49.4), while in the HW and O batches it was 2.9% (7/240; 95% CI 1.4–5.9). The statistical difference (P < 0.00001) for Salmonella prevalence between LW and HW/O welfare groups was highly significant. All batches were positive for Campylobacter on skin (only Campylobacter-positive batches had been included in the study), with prevalence ranging from 15% to 100%. In total, 453 out of 520 samples (87.1%, 95% CI 84.0–90.0) tested positive for Campylobacter detection. Mean Campylobacter concentrations in each batch are shown in Table 3. Unlike Salmonella, very low concentrations of Campylobacter were found in all LW batches. However, a statistically significant difference (P < 0.05) was found between Campylobacter contamination levels detected in carcases from batches assigned to HW and contamination levels reported in O groups, the latter showing much lower mean concentrations (1419 ± 511 CFU/g versus 262 ± 109 CFU/g). In Fig. 2A the distribution of Campylobacter concentrations in skin of carcases belonging to animals from HW (160 animals) and O (80 animals) groups are reported, highlighting the statistically significant difference (P < 0.05). In Fig. 2B the distribution of Campylobacter concentrations in each batch is also reported. In particular it should be noted that a statistically significant difference was found between batches from the same farm after it changed from the conventional (B1) to the organic system (B2 and B3); this change resulted in a sharp improvement of welfare score (from 176 to 250.1) and a statistically significant reduction of Campylobacter contamination (from 1833 CFU/ g to 185 CFU/g, P < 0.05). There were no statistically significant differences between batches assigned to the same welfare group (HW or O), even if they had been sampled in different seasonal periods. For caecal contents, the results reflected what was found for carcass skins, with Salmonella consistently detected only in LW batches (19.3%, 54/280; 95% CI 15.1–24.3) and never in HW and O batches (Table 2). A statistically significant difference was found between LW and HW/O batches (P < 0.05). Campylobacter concentrations (Table 3) were usually lower in LW batches in the presence of high Salmonella prevalence but, just as reported on carcass skins, a difference was noted between HW and O groups (9.24 log10 CFU/g versus 8.50 log10 CFU/g, mean values). Only one LW batch (C2) tested negative for caecal contents (0/40), but positive to Campylobacter detection in some skin samples (6/40) and below the limit of detection of the Campylobacter enumeration method (< 10 CFU/g). This is consistent with findings from other batches where the frequency of contamination on carcass skins was often higher than in caecal contents (Table 3), probably due to the spreading of Campylobacter through cross-contamination of carcases during processing. Statistically significant improvement was found in batches B2 and B3, compared to batch B1, after the change of the farm to organic production (from 9.29 to 8.27 log10 CFU/g, P < 0.00001). Serotyping showed that Salmonella Infantis (77/129; 59.7%) and

of the criteria in relation to the principles is presented in Table 1. The evaluation of each batch required about 2 h and produced a score for each animal welfare principle; a general animal welfare score was then calculated according to Tuyttens et al. (2015) and assigned to each batch. This score is composed of the sum of the scores assigned to each of the four animal welfare principles (maximum score 100) and can range from 0 to 400. Batches were classified into HW (high welfare) or LW (low welfare) depending on whether their welfare score was above or below the median value of 157.10. As the two organic batches showed welfare scores higher than all the others, a third group, O (organic) was identified with welfare scores above 224. 2.3. Microbiological analysis Each of the thirteen batches, previously assessed for animal welfare, was then followed to the slaughterhouse to be subjected to sampling for microbiological analysis. Forty samples of faeces (caecal contents) after evisceration, and forty samples of carcass skin (neck skin) after cooling were taken for each batch, with a total of 1040 samples. Samples were refrigerated at 2–8 °C and carried to the laboratory within 12 h for examination. Samples were tested for detection and enumeration of Campylobacter according to ISO 10272-1: 2006 and ISO 10272–2:2006, respectively, and for detection of Salmonella according to AFNOR NF V 03–100 Systemé BAX Salmonella. Campylobacter strains were tested for species identification with multiplex-PCR (Wang et al., 2002); Salmonella strains were serotyped for somatic antigen O and flagellar antigen H according to ISO/TR 6579-3: 2014. 2.4. Data analysis Collected data were exported to Excel (Version 2010; Microsoft, Richmond, USA) and then analysed with the statistical software XLstat (Addinsoft, Belmont, USA), to highlight statistically significant differences between the three animal welfare groups (HW, LW, O) in relation to the presence of Salmonella and the concentration of Campylobacter in faeces (caecal contents) and carcases. In particular, the analysis of variance (ANOVA) was used to evaluate the differences in Campylobacter concentrations using log transformed data and with the level of significance set at P < 0.05. The conditions for the applicability of ANOVA were verified; data in the groups were normally distributed and there was variance homogeneity. Campylobacter concentrations below the limit of detection of the enumeration method were considered as 0 in cases of a negative result at the detection method; as 5 in cases of positive results at the detection method as 5 CFU/g was the intermediate value between the limits of detection of the two methods (0.04 CFU/g for the detection method, 10 CFU/g for the enumeration method). Prevalence of contamination of Salmonella was calculated, setting a confidence interval (CI) at 95%. The χ2 test was used to evaluate differences between welfare groups with regard to Salmonella prevalence (significance set at P < 0.05). In particular, differences between the results (Salmonella detected or not in skin or caeca) reported from LW batches (lower welfare scores) were compared to the results reported from HW and O batches (higher welfare scores). 3. Results Twelve animal-based indicators (‘criteria’) referring to the four Welfare Quality® principles (good feeding, good housing, good health, appropriate behaviour) were assessed. Therefore for each batch a score for each principle was available. According to the Welfare Quality® protocol, principle scores could be ‘excellent’ if they were over the threshold of 80; ‘enhanced’ between 55 and 80; and, ‘acceptable’ over 20. The sum of the four principles was considered as the total welfare score and ranged from 77.3 to 250.1. In Fig. 1 the results for each batch are graphically represented, including the scores for each principle and 3

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Fig. 1. Results of animal welfare assessment at farm in all batches, including the results for each of the 4 principles and the total welfare score. Black arrows point out the border values between LW (Low Welfare) and HW (High Welfare) batches and between HW and O (Organic) batches.

contaminated by both species was much more frequent on carcass skin (21.9% of positive samples, 99/453). C. jejuni alone was found in 48.6% (220/453) of positive samples, while C. coli alone was found in 29.6% (134/453) of positive samples. No statistically significant differences in relation to Campylobacter species were noted between batches classified in different animal welfare groups.

Table 2 Salmonella prevalence (with 95% confidence interval) in carcass skins and caecal content in all batches, clustered according to the welfare level (LW: Low Welfare; HW: High Welfare; O: Organic). Welfare Group

Batch

SKIN

CAECAL CONTENT

Prevalence

95% CI

Prevalence

95% CI

C1 C2 D1 E1 E2 F1 F2 LW batches

57.5% 70% 52.5% 20% 20% 20% 65% 43.6%

42.1–71.6 54.5–81.9 37.4–67.1 10.6–34.9 10.6–34.9 10.6–34.9 49.4–77.9 37.9–49.4

27.5% 52.5% 5% 2.5% 2.5% 5% 40% 19.3%

16.1–42.9 37.4–67.1 1.5–16.5 0.6–12.9 0.6–12.9 1.5–16.5 26.3–55.5 15.1–24.3

HW

A1 A2 B1 D2 HW batches

0% 7.5% 2.5% 2.5% 3.12%

0–8.6 3.7–19.9 0.6–12.9 0.6–12.9 1.4–7.1

0% 0% 0% 0% 0%

0–8.6 0–8.6 0–8.6 0–8.6 0–8.6

O

B2 B3 O batches

5% 0% 2.5%

1.5–16.5 0–8.6 0.7–8.6

0% 0% 0%

0–8.6 0–8.6 0–8.6

24.8%

21.3–28.7

10.4%

8–13.3

LW

Total

4. Discussion The main aim of this research was to verify, in different broiler batches, the presence of a significant correlation between the welfare score measured at farm and the microbiological contamination of carcases at slaughterhouse. Particular interest was focused on Campylobacter concentrations on carcass neck skins after cooling in slaughterhouse. For this matrix, a hygiene criterion process has become mandatory according to European Union legislation since January 2018, viz. the presence of Campylobacter is tolerated only when it is below the concentration of 1000 CFU/g (Anonymous, 2017). Moreover, several studies have demonstrated that a reduction of Campylobacter concentration on carcass skin could significantly reduce the risk posed by this microbe to consumer health (Nauta et al., 2009; VinuezaBurgos, Cevallos, Cisneros, Van Damme, & De Zutter, 2018), and the European Food Safety Authority (EFSA) stated that containing Campylobacter levels on carcass skin below 1000 CFU/g could reduce the health risk by 50% (EFSA, 2010). This limit is a great challenge for poultry companies, particularly in southern European countries, where Campylobacter is often found over these levels, e.g. 44.2% of carcases in Spain, 79.6% in Malta, 24.3% in Portugal, and 12.5% in Italy according to the last European baseline study (EFSA, 2010). Our results support the hypothesis that carcass contamination at slaughterhouse is influenced by the welfare or stress which broilers experience during their life. Some studies have indicated that stress at farm and pre-slaughtering could affect the shedding of Campylobacter and Salmonella (Rostagno, 2009), but this has never been correlated with comprehensive at-farm animal welfare evaluations. More recently, a pilot study from Alpigiani et al. (2017) reported correlations between animal welfare indicators at slaughterhouse and the presence of Campylobacter in broiler flocks (caecal content and carcass skin), concluding that severe lesions on foot pad and arthritis, usually associated with poor at-farm welfare, digestive troubles and litter humidity, can be effectively used to predict Campylobacter infected flocks. It is known that the composition of the intestinal microbiota can highly impact on

Salmonella Bredeney (49/129; 38.0%) were the most frequent serotypes on carcass skins. A similar situation was reported in caecal contents, but with Salmonella Bredeney more frequently found (33/54; 61.1%) than Salmonella Infantis (21/54; 38.9%). Noticeably, these two serotypes were highly prevalent in LW batches, representing almost the whole of Salmonella strains isolated in this category, both considering skins and caecal contents (175/176; 99.4%). The few Salmonella strains belonging to other serotypes were mostly (3/4; 75%) found in HW and O batches: S. Agona – two strains in skin from one O batch; S. Typhimurium – one strain on skin from one HW batch; S. Coeln – one strain in caecal contents from one LW batch. The other four strains of S. Infantis were isolated from HW batches (all were found on skin from two HW batches). As concerns Campylobacter species, in caecal contents C. jejuni was found in 55.8% (230/412) of positive samples, while C. coli was found in 44.7% (184/412) of positive samples. In two caecal contents (0.48%, 2/412) both species were reported. The presence of samples 4

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Table 3 Campylobacter concentration and frequency of contamination in carcass skins and caecal contents in all batches, clustered according to the welfare level (LW: Low Welfare; HW: High Welfare; O: Organic). Welfare Group

Batch

SKIN

CAECAL CONTENT

Mean Concentration CFU/g

Frequency of contamination

Mean Concentration Log10 CFU/g

Frequency of contamination

LW

C1 C2 D1 E1 E2 F1 F2 LW batches

30 1 1687 88 1326 90 54 468

60% 15% 100% 92.5% 100% 85% 97.5% 78.6%

6.34 0 8.66 6.63 8.99 6.82 6.74 7.89

72.5% 0% 97.5% 87.5% 100% 52.5% 92.5% 71.8%

HW

A1 A2 B1 D2 HW batches

1892 987 1833 968 1419

85% 100% 100% 100% 96.2%

8.44 9.41 9.29 9.34 9.24

57.5% 80% 97.5% 100% 83.7%

O

B2 B3 O batches

340 185 262

97.5% 100% 98.7%

8.27 8.66 8.50

92.5% 100% 96.2%

729

87.1%

8.83

79.2%

Total

very high Salmonella prevalence. It is possible to speculate that the competition between these two pathogens could have advantaged Salmonella, to the detriment of Campylobacter. Similarly, it may be that the particular environmental conditions found in low-welfare batches and the consequent stress response from the animals could have selected a specific microbiota not favourable to Campylobacter, as the sensitivity of this microbe to the changes in the intestinal microflora of poultry is well known (Baffoni et al., 2017; Han et al., 2017). However, further studies about the microbiota composition in these different conditions should be carried out to clarify if low Campylobacter counts in LW batches could be linked to their specific microbiota and/or to the competitive action of Salmonella. No relevant statistical differences were reported between different welfare groups in relation to Campylobacter species (C. jejuni and C. coli). However, a much higher frequency of the contemporary presence in the same sample of strains from these two Campylobacter species was reported on carcass skins compared to caecal contents; this was most probably due to cross-contamination during processing, frequently cited as a relevant source of Campylobacter in poultry meat (Di Giannatale et al., 2010; Marotta et al., 2015; Sasaki et al., 2013). Crosscontamination during processing is a factor that might impact on carcass contamination and which could be independent from animal welfare of farms. Campylobacter, in particular, is considered unable to multiply outside the animal host and its survival in the processing environment is usually poor (Jones, 2001). Therefore cross-contamination is more likely to happen inside the same batch or between batches slaughtered on the same day, making the phenomenon of Campylobacter persistence inside the slaughterhouse-processing environment quite rare. However our findings of relevant differences between different welfare groups for Salmonella and Campylobacter were reported not only on carcass skins but also in caecal contents, sustaining the presence of a direct correlation between good animal welfare at farm and final carcass contamination at slaughterhouse. In conclusion, this study provided new evidence that ‘animal-welfare friendly’ poultry meat could not only be more attractive to increasing numbers of consumers for ethical reasons, but also safer from a microbiological point of view, and therefore twice as valuable for poultry companies. More studies could be usefully focussed on the gut microbiota composition in relation to the animal welfare scores of different batches, to shed light on the biological mechanisms that ultimately influence bacterial shedding and microbiological

pathogen microorganisms, particularly as concerns Campylobacter in broilers, through competitive exclusion, metabolites, or modification of the immune response (Han et al., 2017). Recent studies have demonstrated that environmental factors such as litter, feed access, and climate – all impacting on the general animal welfare level of a flock – can affect the composition of intestinal microbiota in chickens (Kers et al., 2018). According to our results, a significant relationship can be noted between the Welfare Quality® score and faecal shedding and carcass contamination from certain foodborne bacterial pathogens, especially Salmonella. The widespread presence of Salmonella in low-welfare batches, in sharp contrast with high-welfare flocks, is an objective demonstration of the positive impact of welfare-friendly management on microbiological safety. The almost-exclusive presence of two serotypes (Infantis and Bredeney) in these highly contaminated low-welfare batches, in contrast with the serotypes found in high-welfare flocks, could also be related to the effect of stress on the microbiota composition. Campylobacter levels also seem to be influenced by the welfare score, but this was less evident than Salmonella. Our results could be suggestive of a mitigating effect of high welfare on Campylobacter concentrations particularly on skin concentrations, but this has not always been confirmed by caecal content. Moreover, this effect was only highlighted comparing organic batches (O) and high welfare conventional batches (HW). In particular, different flocks from the same farm showed a sharp reduction in faecal and carcass contamination after the change to organic production and the consequent increasing of the welfare score. However, we cannot exclude that the significant reduction in Campylobacter contamination after the shift to organic production could also be linked to the prolonged rearing time and possible changes in the immune response to this microorganism, as organic and conventional batches have clearly different lifespans. Certainly, a higher welfare score for organic batches has been demonstrated in our study, and also a significantly lower Salmonella prevalence has been highlighted compared to lower welfare score batches. Consumer demand for organic products is usually mostly based on the desire for more healthy food in relation to chemical contamination and with less environmental impact (Kranjac, Vapa-Tankosić, & Knežević, 2017). Based on our results, animal welfare and microbiological safety could also benefit from this kind of farm management, adding further value for producers. It was expected to find high concentrations of Campylobacter, particularly in low-welfare flocks. This was not observed, in contrast to the 5

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Fig. 2. A. Distribution of Campylobacter concentrations (log10 CFU/g) in skin of carcases included in HW and O groups. B. Distribution of Campylobacter concentrations (log10 CFU/g) in skin of carcases from batches A1, A2, B1, D2 (HW) and from batches B2 and B3 (O).

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contamination of poultry meat.

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Acknowledgements This study was funded by the Italian Ministry of Health, in the framework of the Ricerca Finalizzata Project n. GR-2011-02349917 entitled “Farm Animal Welfare and Food Safety: Effect of on-farm and pre-slaughtering stress factors on microbiological contamination of poultry meat”. The authors are grateful to Renzo Galli and Silvia Pergola for their assistance at slaughterhouse, to Cristina Marfoglia, Violeta Di Marzio, Gabriella Centorotola, Alessandra Alessiani, Tiziana Persiani, Romina Romantini for their lab activities, and to Emanuele D'Erasmo, Daniela D'Angelantonio, Francesca Lombardi, Cristina Rapagnà for their help in animal welfare evaluations at farm and sampling at slaughterhouse. References Alpigiani, I., Abrahantes, J. C., Michel, V., Huneau-Salaün, A., Chemaly, M., Keeling, L. J., et al. (2017). Associations between animal welfare indicators and Campylobacter spp. in broiler chickens under commercial settings: A case study. Preventive Veterinary Medicine, 147, 186–193. Anonymous (2003). Directive 2003/99/EC of the european parliament and of the council of 17 november 2003 on the monitoring of zoonoses and zoonotic agents, amending council decision 90/424/EEC and repealing council directive 92/117/EEC. Official Journal of the European Union, L325, 31–40. Anonymous (2007). Council Regulation (EC) No 834/2007 of 28 June 2007 on organic production and labelling of organic products and repealing Regulation (EEC) No 2092/91. Official Journal of the European Union, L189, 1–23. Anonymous (2008). Commission Regulation (EC) No 889/2008 of 5 September 2008 laying down detailed rules for the implementation of Council Regulation (EC) No 834/2007 on organic production and labelling of organic products with regard to organic production, labelling and control. Official Journal of the European Union, L250, 1–84. Anonymous (2017). Commission Regulation (EU) 2017/1495 of 23 August 2017 amending Regulation (EC) No 2073/2005 as regards Campylobacter in broiler carcases. Official Journal of the European Union, L218, 1–6. Baffoni, L., Gaggia, F., Garofolo, G., Di Serafino, G., Buglione, E., Di Giannatale, E., et al. (2017). Evidence of Campylobacter jejuni reduction in broilers with early synbiotic administration. International Journal of Food Microbiology, 251, 41–47. Botteldoorn, N., Heyndrickx, M., Rijpens, N., Grijspeerdt, K., & Herman, L. (2003). Salmonella on pig carcasses: Positive pigs and cross contamination in the slaughterhouse. Journal of Applied Microbiology, 95, 891–903. Corrier, D. E., Byrd, J. A., Hargis, B. M., Hume, M. E., Bailey, R. H., & Stanker, L. H. (1999). Presence of Salmonella in the crop and ceca of broiler chickens before and after preslaughter feed withdrawal. Poultry Science, 78, 45–49. De Jong, I. C., Hindle, V. A., Butterworth, A., Engel, B., Ferrari, P., Gunnik, H., et al. (2015). Simplifying the Welfare Quality® assessment protocol for broiler chicken welfare. Animal, 10, 117–127. Di Giannatale, E., Prencipe, V., Colangeli, P., Alessiani, A., Barco, L., Staffolani, M., et al. (2010). Prevalence of thermotolerant Campylobacter in broiler flocks and broiler carcasses in Italy. Veterinaria Italiana, 46, 415–423. European Food Safety Authority (EFSA) (2010). Analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU, 2008. Part A: Campylobacter and Salmonella prevalence estimates. EFSA Journal, 8, 1503. European Food Safety Authority (EFSA) (2019). Animal welfare: Introduction. https:// www.efsa.europa.eu/en/topics/topic/animal-welfare last, Accessed date: 15 May 2019. European Food Safety Authority and European Centre for Disease Prevention and Control (EFSA/ECDC) (2018). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017. EFSA Journal, 16, 5500. Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) (2009). Risk assessment of Campylobacter spp. in broiler chickens: Technical Report. Microbiological risk assessment series No 12. Geneva (pp. 132pp). . Han, Z., Willer, T., Li, L., Pielsticker, C., Rychlik, I., Velge, P., et al. (2017). Influence of the gut microbiota composition on Campylobacter jejuni colonization in chickens.

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