J. H. Smid

Risk Factors for Campylobacteriosis of Chicken, Ruminant, and Environmental Origin: A Combined Case-Control and Source Attribution Analysis

Background Campylobacteriosis contributes strongly to the disease burden of food-borne pathogens. Case-control studies are limited in attributing human infections to the different reservoirs because they can only trace back to the points of exposure, which may not point to the original reservoirs because of cross-contamination. Human Campylobacter infections can be attributed to specific reservoirs by estimating the extent of subtype sharing between strains from humans and reservoirs using multilocus sequence typing (MLST). Methodology/Principal Findings We investigated risk factors for human campylobacteriosis caused by Campylobacter strains attributed to different reservoirs. Sequence types (STs) were determined for 696 C. jejuni and 41 C. coli strains from endemic human cases included in a case-control study. The asymmetric island model, a population genetics approach for modeling Campylobacter evolution and transmission, attributed these cases to four putative animal reservoirs (chicken, cattle, sheep, pig) and to the environment (water, sand, wild birds) considered as a proxy for other unidentified reservoirs. Most cases were attributed to chicken (66%) and cattle (21%), identified as the main reservoirs in The Netherlands. Consuming chicken was a risk factor for campylobacteriosis caused by chicken-associated STs, whereas consuming beef and pork were protective. Risk factors for campylobacteriosis caused by ruminant-associated STs were contact with animals, barbecuing in non-urban areas, consumption of tripe, and never/seldom chicken consumption. Consuming game and swimming in a domestic swimming pool during springtime were risk factors for campylobacteriosis caused by environment-associated STs. Infections with chicken- and ruminant-associated STs were only partially explained by food-borne transmission; direct contact and environmental pathways were also important. Conclusion/Significance This is the first case-control study in which risk factors for campylobacteriosis are investigated in relation to the attributed reservoirs based on MLST profiles. Combining epidemiological and source attribution data improved campylobacteriosis risk factor identification and characterization, generated hypotheses, and showed that genotype-based source attribution is epidemiologically sensible.

Increased risk for Campylobacter jejuni and C. coli infection of pet origin in dog owners and evidence for genetic association between strains causing infection in humans and their pets.

We compared Campylobacter jejuni/coli multilocus sequence types (STs) from pets (dogs/cats) and their owners and investigated risk factors for pet-associated human campylobacteriosis using a combined source-attribution and case-control analysis. In total, 132/687 pet stools were Campylobacter-positive, resulting in 499 strains isolated (320 C. upsaliensis/helveticus, 100 C. jejuni, 33 C. hyointestinalis/fetus, 10 C. lari, 4 C. coli, 32 unidentified). There were 737 human and 104 pet C. jejuni/coli strains assigned to 154 and 49 STs, respectively. Dog, particularly puppy, owners were at increased risk of infection with pet-associated STs. In 2/68 cases vs. 0.134/68 expected by chance, a pet and its owner were infected with an identical ST (ST45, ST658). Although common sources of infection and directionality of transmission between pets and humans were unknown, dog ownership significantly increased the risk for pet-associated human C. jejuni/coli infection and isolation of identical strains in humans and their pets occurred significantly more often than expected.

Practicalities of Using Non-Local or Non-Recent Multilocus Sequence Typing Data for Source Attribution in Space and Time of Human Campylobacteriosis

In this study, 1208 Campylobacter jejuni and C. coli isolates from humans and 400 isolates from chicken, collected in two separate periods over 12 years in The Netherlands, were typed using multilocus sequence typing (MLST). Statistical evidence was found for a shift of ST frequencies in human isolates over time. The human MLST data were also compared to published data from other countries to determine geographical variation. Because only MLST typed data from chicken, taken from the same time point and spatial location, were available in addition to the human data, MLST datasets for other Campylobacter reservoirs from selected countries were used. The selection was based on the degree of similarity of the human isolates between countries. The main aim of this study was to better understand the consequences of using non-local or non-recent MLST data for attributing domestically acquired human Campylobacter infections to specific sources of origin when applying the asymmetric island model for source attribution. In addition, a power-analysis was done to find the minimum number of source isolates needed to perform source attribution using an asymmetric island model. This study showed that using source data from other countries can have a significant biasing effect on the attribution results so it is important to carefully select data if the available local data lack in quality and/or quantity. Methods aimed at reducing this bias were proposed.

A biotracing model of Salmonella in the pork production chain.

In biotracing systems, downstream chain information and model-based approaches are used to trace the sources of microbial contamination in a food chain. This article includes the results of a biotracing model for Salmonella in the pork slaughter process chain. A Bayesian belief network model was used in which information on the Salmonella level at different locations in the slaughterhouse were used in combination with prior knowledge about the dynamics of Salmonella throughout the slaughter line. Data collected in a Dutch slaughterhouse were used to specify prior beliefs about the model inputs and to iteratively refine the distributions of the parameters in the model to obtain an optimal description of that specific slaughterhouse. The primary purpose of the model is to trace the sources of contamination for individual Salmonella-positive carcasses at the end of the slaughter line. The model results indicated that house flora on or in the carcass splitter was the source of contamination for many carcasses, especially for those that carried contamination on the cutting side. The results also indicated that the parameter values of the model may be subject to temporal variation and can be used as a tool to provide estimates of such trends. This model illustrates the concept of biotracing, gives insight into the dynamics of Salmonella in the slaughter line, and indicates the sites in the line where data collection is most effective for biotracing. This biotracing model was implemented as an interactive computer application, which is a step in the process toward an operational biotracing system by which a stakeholder can initiate immediate responses to Salmonella contamination and other hazards in the pork slaughterhouse.

Attribution of human Salmonella infections to animal and food sources in Italy (2002-2010): adaptations of the Dutch and modified Hald source attribution models.

The Dutch and modified Hald source attribution models were adapted to Italian Salmonella data to attribute human infections caused by the top 30 serotypes between 2002 and 2010 to four putative sources (Gallus gallus, turkeys, pigs, ruminants), at the points of animal reservoir (farm), exposure (food), and both combined. Attribution estimates were thus compared between different models, time periods and sampling points. All models identified pigs as the main source of human salmonellosis in Italy, accounting for 43-60% of infections, followed by G. gallus (18-34%). Attributions to turkeys and ruminants were minor. An increasing temporal trend in attributions to pigs and a decreasing one in those to G. gallus was also observed. Although the outcomes of the two models applied at farm and food levels essentially agree, they can be refined once more information becomes available, providing valuable insights about potential targets along the production chain.

Campylobacteriosis in returning travellers and potential secondary transmission of exotic strains.

SUMMARY Multilocus sequence types (STs) were determined for 232 and 737 Campylobacter jejuni/coli isolates from Dutch travellers and domestically acquired cases, respectively. Putative risk factors for travel-related campylobacteriosis, and for domestically acquired campylobacteriosis caused by exotic STs (putatively carried by returning travellers), were investigated. Travelling to Asia, Africa, Latin America and the Caribbean, and Southern Europe significantly increased the risk of acquiring campylobacteriosis compared to travelling within Western Europe. Besides eating chicken, using antacids, and having chronic enteropathies, we identified eating vegetable salad outside Europe, drinking bottled water in high-risk destinations, and handling/eating undercooked pork as possible risk factors for travel-related campylobacteriosis. Factors associated with domestically acquired campylobacteriosis caused by exotic STs involved predominantly person-to-person contacts around popular holiday periods. We concluded that putative determinants of travel-related campylobacteriosis differ from those of domestically acquired infections and that returning travellers may carry several exotic strains that might subsequently spread to domestic populations even through limited person-to-person transmission.

Tracing the sources of human salmonellosis: A multi-model comparison of phenotyping and genotyping methods

Salmonella source attribution is usually performed using frequency-matched models, such as the (modified) Dutch and Hald models, based on phenotyping data, i.e. serotyping, phage typing, and antimicrobial resistance profiling. However, for practical and economic reasons, genotyping methods such as Multi-locus Variable Number of Tandem Repeats Analysis (MLVA) are gradually replacing traditional phenotyping of salmonellas beyond the serovar level. As MLVA-based source attribution of human salmonellosis using frequency-matched models is problematic due to the high variability of the genetic targets investigated, other models need to be explored. Using a comprehensive data set from the Netherlands in 2005-2013, this study aimed at attributing sporadic and domestic cases of Salmonella Typhimurium/4,[5],12:i:- and Salmonella Enteritidis to four putative food-producing animal sources (pigs, cattle, broilers, and layers/eggs) using the modified Dutch and Hald models (based on sero/phage typing data) in comparison with a widely applied population genetics model - the asymmetric island model (AIM) - supplied with MLVA data. This allowed us to compare model outcomes and to corroborate whether MLVA-based Salmonella source attribution using the AIM is able to provide sound, comparable results. All three models provided very similar results, confirming once more that most S. Typhimurium/4,[5],12:i:- and S. Enteritidis cases are attributable to pigs and layers/eggs, respectively. We concluded that MLVA-based source attribution using the AIM is a feasible option, at least for S. Typhimurium/4,[5],12:i:- and S. Enteritidis. Enough information seems to be contained in the MLVA profiles to trace the sources of human salmonellosis even in presence of imperfect temporal overlap between human and source isolates. Besides Salmonella, the AIM might also be applicable to other pathogens that do not always comply to clonal models. This would add further value to current surveillance activities by performing source attribution using genotyping data that are being collected in a standardized fashion internationally.

Comparative Exposure Assessment of ESBL-Producing Escherichia coli through Meat Consumption

The presence of extended-spectrum β-lactamase (ESBL) and plasmidic AmpC (pAmpC) producing Escherichia coli (EEC) in food animals, especially broilers, has become a major public health concern. The aim of the present study was to quantify the EEC exposure of humans in The Netherlands through the consumption of meat from different food animals. Calculations were done with a simplified Quantitative Microbiological Risk Assessment (QMRA) model. The model took the effect of pre-retail processing, storage at the consumers home and preparation in the kitchen (cross-contamination and heating) on EEC numbers on/in the raw meat products into account. The contribution of beef products (78%) to the total EEC exposure of the Dutch population through the consumption of meat was much higher than for chicken (18%), pork (4.5%), veal (0.1%) and lamb (0%). After slaughter, chicken meat accounted for 97% of total EEC load on meat, but chicken meat experienced a relatively large effect of heating during food preparation. Exposure via consumption of filet americain (a minced beef product consumed raw) was predicted to be highest (61% of total EEC exposure), followed by chicken fillet (13%). It was estimated that only 18% of EEC exposure occurred via cross-contamination during preparation in the kitchen, which was the only route by which EEC survived for surface-contaminated products. Sensitivity analysis showed that model output is not sensitive for most parameters. However, EEC concentration on meat other than chicken meat was an important data gap. In conclusion, the model assessed that consumption of beef products led to a higher exposure to EEC than chicken products, although the prevalence of EEC on raw chicken meat was much higher than on beef. The (relative) risk of this exposure for public health is yet unknown given the lack of a modelling framework and of exposure studies for other potential transmission routes.

Quantifying the sources of Salmonella on dressed carcasses of pigs based on serovar distribution

Salmonella serotyping data, qualitatively described by van Hoek et al. (2012), were used to quantify potential sources of Salmonella in a Dutch pig slaughterhouse. Statistical tests to compare per-day Salmonella prevalence and serotyping data from multiple points in the chain were used to find transmission pathways. A statistical model based on serotyping data was developed to attribute Salmonella on dressed carcasses to the most likely source. Approximately two-third of dressed carcasses carrying Salmonella on the medial surface had been contaminated by house flora. For carcasses carrying Salmonella on the distal surface, transient Salmonella from incoming pigs was a more important source. The relevance of the different sources of Salmonella varied within and between sampling days. Results were compared to those of another modeling approach, in which Salmonella concentration data from the same samples were used (Smid et al., 2012). They mostly agreed. The approach chosen by an individual slaughterhouse depends on the data that are collected.

Variability and uncertainty analysis of the cross-contamination ratios of salmonella during pork cutting.

The transfer ratio of bacteria from one surface to another is often estimated from laboratory experiments and quantified by dividing the expected number of bacteria on the recipient surface by the expected number of bacteria on the donor surface. Yet, the expected number of bacteria on each surface is uncertain due to the limited number of colonies that are counted and/or samples that can be analyzed. The expected transfer ratio is, therefore, also uncertain and its estimate may exceed 1 if real transfer is close to 100%. In addition, the transferred fractions vary over experiments but it is unclear, using this approach, how to combine uncertainty and variability into one estimate for the transfer ratio. A Bayesian network model was proposed that allows the combination of uncertainty within one experiment and variability over multiple experiments and prevents inappropriate values for the transfer ratio. Model functionality was shown using data from a laboratory experiment in which the transfer of Salmonella was determined from contaminated pork meat to a butchers knife, and vice versa. Recovery efficiency of bacteria from both surfaces was also determined and accounted for in the analysis. Transfer ratio probability distributions showed a large variability, with a mean value of 0.19 for the transfer of Salmonella from pork meat to the knife and 0.58 for the transfer of Salmonella from the knife to pork meat. The proposed Bayesian model can be used for analyzing data from similar study designs in which uncertainty should be combined with variability.