Campylobacter presence on Dutch broiler farms and associated risk factors.

Campylobacter is the most reported zoonotic pathogen in humans in the European Union. Poultry is a major source of human infection with Campylobacter. Although many studies are done on the presence of Campylobacter in broilers and theoretically effective control measures are known, their relative importance at broiler farms remains poorly understood. Therefore, the aim of this study was to investigate the presence of Campylobacter on selected broiler farms in the Netherlands, to determine the moment of introduction, and associated risk factors. A longitudinal study on 25 broiler farms was carried out between June 2017 and December 2020. Fecal samples were collected weekly from 43 broiler houses. In total 497 flocks were sampled. Putative variables on flock and farm characteristics for a risk factor analysis were gathered through questionnaires. Risk factors associated with the presence of Campylobacter in a broiler flock were determined using regression models. In total 30% of the flocks included in the study were positive for Campylobacter. Factors associated with presence of Campylobacter at slaughter age included: season, mowing lawns and presence of agricultural side activities. While summer/autumn and mowing lawns were associated with an increase in Campylobacter presence in flocks, the farmer having agricultural side activities other than poultry production was associated with a decrease. Analysis of the age at which flocks first tested Campylobacter positive revealed that slower growing breeds became positive on average 1 wk later compared to regular growers. This study revealed a delayed introduction of Campylobacter in slower grower vs. regular grower broiler flocks reared indoors. In addition, it confirmed importance of season as major risk factor. The relevance of mowing and preceding positive flocks as risk factors needs further investigation.

Environmental Sampling Methods for Detection of Salmonella Infections in Laying Hens: A Systematic Review and Meta-Analysis.

Salmonellosis is the second most commonly reported foodborne gastrointestinal infection in humans in the European Union (EU). Most outbreaks are caused by Salmonella Enteritidis, present in contaminated food products, particularly in egg and egg products. In recent years, an increase in the prevalence of Salmonella in laying hen flocks in the EU has been observed. For the effective control of infection, adequate detection is key. In laying hen flocks, the occurrence of Salmonella in the EU is monitored by the culture of environmental samples (dust, faeces, and boot swabs). The performance of sampling procedures described in the literature for the detection of Salmonella in laying hens was reviewed. In total, 924 abstracts were screened, resulting in the selection of 87 abstracts and 18 publications for qualitative and quantitative analyses, respectively. Sample sizes and sampling locations of faecal material and dust were variable and poorly described. Microbiological culture methods used to detect Salmonella were variably described in the literature and were often incomplete. Overall, the available literature indicates higher sensitivity of environmental versus individual hen matrices and points to differences in sensitivity between environmental matrices. For non-cage housing systems, boot swabs are the preferred samples, while for cage housing systems dust might be a more reliable sample.

Assessing potential determinants of the stagnating trend in Salmonella Enteritidis human infections in Europe and options for intervention: A multi-criteria decision analysis.

Background: After years of significant decline, the incidence of Salmonella enterica serotype Enteritidis (SE) human infections in Europe has started stagnating in recent years. The reasons for this stagnation remain largely unclear and are possibly multifactorial and interconnected in nature. We assessed and ranked several potential determinants of the stagnating SE trend in Europe, as well as different options for intervention at the level of poultry health and production, public health (infra)structure, and pathogen biology. Methods: A Multi-Criteria Decision-Analysis (MCDA) approach based on the Analytical Hierarchy Process was used. Through two separate surveys, a European panel of Salmonella experts first provided weights for several pre-defined criteria and subsequently scored different potential determinants and options for intervention (i.e. alternatives) against the criteria, during 2020-21. The weighting and scoring were based on Saaty's pairwise comparisons. The final ranking of the alternatives was derived from the summation of the products of each criterion weight with the score of the corresponding alternative. Sensitivity analyses were performed to assess the impact of different methodological choices, including European regions, and domains of expertise on the ranking of the determinants and options for intervention. Results: The first and second-ranked determinants of the stagnated trend in human SE infections were related to poultry health and production, namely "inadequacies of sampling programmes" and "premature relaxation of control measures". This ranking agreed with the ranking of the options for intervention, which were also those at the poultry health and production level, specifically "stricter biosecurity", "improving sampling", and "better/increased vaccination". Differences in rankings were observed among European regions and domains of expertise. Conclusions: The rankings of potential determinants and options for intervention for the stagnating SE trend in Europe pointed to the level of poultry health and production. Salmonella-control activities in poultry in Europe are harmonized across countries since many years, but the results of this study suggest that further improvements may be necessary for some countries. A multidisciplinary collaboration among veterinarians, public health professionals, and microbiologists is needed to further understand the origins of the stagnating SE trend and to identify effective interventions in order to reverse the trend, contextually in a given country, following a One Health approach.

Challenging the "gold standard" of colony-forming units - Validation of a multiplex real-time PCR for quantification of viable Campylobacter spp. in meat rinses.

Campylobacter jejuni is the leading bacterial food-borne pathogen in Europe. Despite the accepted limits of cultural detection of the fastidious bacterium, the "gold standard" in food microbiology is still the determination of colony-forming units (CFU). As an alternative, a live/dead differentiating qPCR has been established, using propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from dead, membrane-compromised cells. The PMA treatment was combined with the addition of an internal sample process control (ISPC), i.e. a known number of dead C. sputorum cells to the samples. The ISPC enables i), monitoring the effective reduction of dead cell signal by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses during processing. Here, we optimized the method for routine application and performed a full validation of the method according to ISO 16140-2:2016(E) for the quantification of live thermophilic Campylobacter spp. in meat rinses against the classical enumeration method ISO 10272-2:2017. In order to render the method applicable and cost-effective for practical application, the ISPC was lyophilized to be distributable to routine laboratories. In addition, a triplex qPCR was established to simultaneously quantify thermophilic Campylobacter, the ISPC and an internal amplification control (IAC). Its performance was statistically similar to the two duplex qPCRs up to a contamination level of 4.7 log10Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR reaction, i.e. 2.3 log10 live Campylobacter per ml of sample. The alternative method passed a relative trueness study, confirming the robustness against different meat rinses, and displayed sufficient accuracy within the limits set in ISO 16140-2:2016(E). Finally, the method was validated in an interlaboratory ring trial, confirming that the alternative method was fit for purpose with a tendency of improved repeatability and reproducibility compared to the reference method for CFU determination. Campylobacter served as a model organism, challenging CFU as "gold standard" and could help in guidance to the general acceptance of live/dead differentiating qPCR methods for the detection of food-borne pathogens.

Internal sample process control improves cultivation-independent quantification of thermotolerant Campylobacter.

Quantification of Campylobacter is challenging and one major reason is the fact that bacteria lose cultivability due to cold or oxygen stress during storage at retail. Alternative live/dead discriminatory qPCR currently lacks standardization and might overestimate live cells in the presence of dead cells. In this study an internal sample process control (ISPC) was developed. The ISPC consists of a specified number of peroxide-killed C. sputorum cells to be added to each sample in order to monitor (i) the level of reduction of the signal from dead cells and (ii) DNA losses during sample processing. A species-specific fragment of the 16S rRNA gene of C. sputorum was selected as real-time PCR target, based on its similar size and gene copy number compared to the C. jejuni/coli/lari target and confirmed in an exclusivity study. Extension of the amplification oligonucleotides for the target of thermotolerant Campylobacter improved real-time PCR efficiency, rendering the method suitable for quantification according to international standards. Concordant PCR signal variation of both C. jejuni and C. sputorum targets in co-inoculated chicken rinses verified the suitability of the ISPC. This provides a crucial step towards implementation of cultivation-independent quantification for improved food safety of fastidious bacteria.

Reviewing Interventions against Enterobacteriaceae in Broiler Processing: Using Old Techniques for Meeting the New Challenges of ESBL E. coli?

Extended-spectrum beta-lactamase- (ESBL-) producing Enterobacteriaceae are frequently detected in poultry and fresh chicken meat. Due to the high prevalence, an impact on human colonization and the spread of antibiotic resistance into the environment is assumed. ESBL-producing Enterobacteriaceae can be transmitted along the broiler production chain but also their persistence is reported because of insufficient cleaning and disinfection. Processing of broiler chickens leads to a reduction of microbiological counts on the carcasses. However, processing steps like scalding, defeathering, and evisceration are critical concerning fecal contamination and, therefore, cross-contamination with bacterial strains. Respective intervention measures along the slaughter processing line aim at reducing the microbiological load on broiler carcasses as well as preventing cross-contamination. Published data on the impact of possible intervention measures against ESBL-producing Enterobacteriaceae are missing and, therefore, we focused on processing measures concerning Enterobacteriaceae, in particular E. coli or coliform counts, during processing of broiler chickens to identify possible hints for effective strategies to reduce these resistant bacteria. In total, 73 publications were analyzed and data on the quantitative reductions were extracted. Most investigations concentrated on scalding, postdefeathering washes, and improvements in the chilling process and were already published in and before 2008 (n=42, 58%). Therefore, certain measures may be already installed in slaughterhouse facilities today. The effect on eliminating ESBL-producing Enterobacteriaceae is questionable as there are still positive chicken meat samples found. A huge number of studies dealt with different applications of chlorine substances which are not approved in the European Union and the reduction level did not exceed 3 log10 values. None of the measures was able to totally eradicate Enterobacteriaceae from the broiler carcasses indicating the need to develop intervention measures to prevent contamination with ESBL-producing Enterobacteriaceae and, therefore, the exposure of humans and the further release of antibiotic resistances into the environment.

Molecular relatedness of ESBL/AmpC-producing Escherichia coli from humans, animals, food and the environment: a pooled analysis.

Background: In recent years, ESBL/AmpC-producing Escherichia coli (ESBL/AmpC-EC) have been isolated with increasing frequency from animals, food, environmental sources and humans. With incomplete and scattered evidence, the contribution to the human carriage burden from these reservoirs remains unclear. Objectives: To quantify molecular similarities between different reservoirs as a first step towards risk attribution. Methods: Pooled data on ESBL/AmpC-EC isolates were recovered from 35 studies in the Netherlands comprising >27 000 samples, mostly obtained between 2005 and 2015. Frequency distributions of ESBL/AmpC genes from 5808 isolates and replicons of ESBL/AmpC-carrying plasmids from 812 isolates were compared across 22 reservoirs through proportional similarity indices (PSIs) and principal component analyses (PCAs). Results: Predominant ESBL/AmpC genes were identified in each reservoir. PCAs and PSIs revealed close human-animal ESBL/AmpC gene similarity between human farming communities and their animals (broilers and pigs) (PSIs from 0.8 to 0.9). Isolates from people in the general population had higher similarities to those from human clinical settings, surface and sewage water and wild birds (0.7-0.8), while similarities to livestock or food reservoirs were lower (0.3-0.6). Based on rarefaction curves, people in the general population had more diversity in ESBL/AmpC genes and plasmid replicon types than those in other reservoirs. Conclusions: Our 'One Health' approach provides an integrated evaluation of the molecular relatedness of ESBL/AmpC-EC from numerous sources. The analysis showed distinguishable ESBL/AmpC-EC transmission cycles in different hosts and failed to demonstrate a close epidemiological linkage of ESBL/AmpC genes and plasmid replicon types between livestock farms and people in the general population.

Pre-scald brushing for removal of solids and associated broiler carcass bacterial contamination.

The aim of the study was to investigate the effect of brushing prior to scalding on reducing the E. coli and Enterobacteriaceae concentrations on carcasses. Three visits were arranged to a commercial slaughterhouse in which carcasses were cleaned in a separate line. Ten batches were sampled to compare the E. coli and Enterobacteriaceae concentrations on carcasses before and after a stand-alone brushing unit. Per batch, 8 carcasses before and 8 after brushing were sampled by the whole-carcass rinse method. Furthermore, the dry matter content and the pH were determined in these samples, as these parameters indirectly (dry matter) or directly (pH) influence the scalding lethality. Results revealed a small but statistically significant reduction (P < 0.001) in E. coli and Enterobacteriaceae concentrations on the brushed carcasses. The concentrations on whole carcasses were reduced on average by 0.3 log for both E. coli and Enterobacteriaceae. Rinse samples from treated carcasses had significantly less dry matter on average by 2.5 g (P < 0.001) and significantly higher pH by 0.08 units (P < 0.001). Although these differences are statistically significant, they might have rather low biological relevance; thus, further optimization of brushes is needed for more relevant results. This study confirms that brushing reduces bacterial concentrations on carcasses, which may be increased potentially by enlarging the brushed surface of the carcass. Further in-line investigations are needed to observe the effect of brushing on bacterial concentrations in scalding water and on carcasses after scalding and at the end of processing.

Explanatory Variables Associated with Campylobacter and Escherichia coli Concentrations on Broiler Chicken Carcasses during Processing in Two Slaughterhouses.

This study aimed at identifying explanatory variables that were associated with Campylobacter and Escherichia coli concentrations throughout processing in two commercial broiler slaughterhouses. Quantative data on Campylobacter and E. coli along the processing line were collected. Moreover, information on batch characteristics, slaughterhouse practices, process performance, and environmental variables was collected through questionnaires, observations, and measurements, resulting in data on 19 potential explanatory variables. Analysis was conducted separately in each slaughterhouse to identify which variables were related to changes in concentrations of Campylobacter and E. coli during the processing steps: scalding, defeathering, evisceration, and chilling. Associations with explanatory variables were different in the slaughterhouses studied. In the first slaughterhouse, there was only one significant association: poorer uniformity of the weight of carcasses within a batch with less decrease in E. coli concentrations after defeathering. In the second slaughterhouse, significant statistical associations were found with variables, including age, uniformity, average weight of carcasses, Campylobacter concentrations in excreta and ceca, and E. coli concentrations in excreta. Bacterial concentrations in excreta and ceca were found to be the most prominent variables, because they were associated with concentration on carcasses at various processing points. Although the slaughterhouses produced specific products and had different batch characteristics and processing parameters, the effect of the significant variables was not always the same for each slaughterhouse. Therefore, each slaughterhouse needs to determine its particular relevant measures for hygiene control and process management. This identification could be supported by monitoring changes in bacterial concentrations during processing in individual slaughterhouses. In addition, the possibility that management and food handling practices in slaughterhouses contribute to the differences in bacterial contamination between slaughterhouses needs further investigation.

Reduction of extended-spectrum-ß-lactamase- and AmpC-ß-lactamase-producing Escherichia coli through processing in two broiler chicken slaughterhouses.

Whilst broilers are recognised as a reservoir of extended-spectrum-ß-lactamase (ESBL)- and AmpC-ß-lactamase (AmpC)-producing Escherichia coli, there is currently limited knowledge on the effect of slaughtering on its concentrations on poultry meat. The aim of this study was to establish the concentration of ESBL/AmpC producing E. coli on broiler chicken carcasses through processing. In addition the changes in ESBL/AmpC producing E. coli concentrations were compared with generic E. coli and Campylobacter. In two slaughterhouses, the surface of the whole carcasses was sampled after 5 processing steps: bleeding, scalding, defeathering, evisceration and chilling. In total, 17 batches were sampled in two different slaughterhouses during the summers of 2012 and 2013. ESBL/AmpC producing E. coli was enumerated on MacConkey agar with 1mg/l cefotaxime, and the ESBL/AmpC phenotypes and genotypes were characterised. The ESBL/AmpC producing E. coli concentrations varied significantly between the incoming batches in both slaughterhouses. The concentrations on broiler chicken carcasses were significantly reduced during processing. In Slaughterhouse 1, all subsequent processing steps reduced the concentrations except evisceration which led to a slight increase that was statistically not significant. The changes in concentration between processing steps were relatively similar for all sampled batches in this slaughterhouse. In contrast, changes varied between batches in Slaughterhouse 2, and the overall reduction through processing was higher in Slaughterhouse 2. Changes in ESBL/AmpC producing E. coli along the processing line were similar to changes in generic E. coli in both slaughterhouses. The effect of defeathering differed between ESBL/AmpC producing E. coli and Campylobacter. ESBL/AmpC producing E. coli decreased after defeathering, whereas Campylobacter concentrations increased. The genotypes of ESBL/AmpC producing E. coli (blaCTX-M-1, blaSHV-12, blaCMY-2, blaTEM-52c, blaTEM-52cvar) from both slaughterhouses match typical poultry genotypes. Their distribution differed between batches and changed throughout processing for some batches. The concentration levels found after chilling were between 10(2) and 10(5)CFU/carcass. To conclude, changes in ESBL/AmpC producing E. coli concentrations on broiler chicken carcasses during processing are influenced by batch and slaughterhouse, pointing to the role of both primary production and process control for reducing ESBL/AmpC producing E. coli levels in final products. Due to similar changes upon processing, E. coli can be used as a process indicator of ESBL/AmpC producing E. coli, because the processing steps had similar impact on both organisms. Cross contamination may potentially explain shifts in genotypes within some batches through the processing.

A comparison of fluctuations of Campylobacter and Escherichia coli concentrations on broiler chicken carcasses during processing in two slaughterhouses.

The causes of differences in Campylobacter and Escherichia coli concentrations on broiler chicken carcasses after chilling between slaughterhouses are not fully identified. Therefore, it is a challenge for slaughterhouses to comply with Process Hygiene Criteria for broiler meat. The aim of the study was to identify which processing steps contribute to increases or decreases in Campylobacter and E. coli concentrations within and between two slaughterhouses. Identifying the processing steps with variable performance could explain the differences in bacterial concentrations after chilling between slaughterhouses. Thermotolerant Campylobacter and E. coli concentrations on carcasses during broiler processing were measured during the summer period in 21 trials after bleeding, scalding, defeathering, evisceration and chilling. In two slaughterhouses with comparable Campylobacter and E. coli concentrations in the incoming batches (after bleeding), the mean log10 concentrations are found to be significantly different after chilling. Campylobacter concentrations decreased by 1.40 log10 in Slaughterhouse 1 and by 1.86 log10 in Slaughterhouse 2, whereas E. coli decreased by 2.19 log10 in Slaughterhouse 1 and by 2.84 log10 in Slaughterhouse 2. Higher concentrations of Campylobacter and E. coli on carcasses after chilling were observed in Slaughterhouse 1 in which an increase in concentrations was observed after evisceration. The effect of processing on Campylobacter and E. coli concentrations in Slaughterhouse 1 did not differ between batches. In Slaughterhouse 2, the effect of processing on the concentrations of both bacteria varied over batches. Changes in E. coli concentration levels during processing were similar to Campylobacter except for defeathering. E. coli concentration significantly decreased after defeathering in both slaughterhouses, whereas Campylobacter increased in Slaughterhouse 2 and in Slaughterhouse 1 no significant changes were observed. The patterns of increases and decreases in bacterial concentrations during processing are specific for each slaughterhouse. Inhomogeneous patterns potentially explain the differences in concentrations after chilling between slaughterhouses. Critical processing steps should be validated in each slaughterhouse by longitudinal studies and potentially based on E. coli. E. coli has a potential to be used as an indicator of processing hygiene, because the impact of most of the studied processing steps was similar as for Campylobacter.

Propidium monoazide does not fully inhibit the detection of dead Campylobacter on broiler chicken carcasses by qPCR.

A real time quantitative PCR combined with propidium monoazide (PMA) treatment of samples was implemented to quantify live C. jejuni, C. coli and C. lari on broiler chicken carcasses at selected processing steps in the slaughterhouse. The samples were enumerated by culture for comparison. The Campylobacter counts determined with the PMA-qPCR and the culture method were not concordant. We conclude that the qPCR combined with PMA treatment of the samples did not fully reduce the signal from dead cells.