Search

Probes for affinity guided conjugation have shown great promise for the preparation of high-quality protein conjugates. However, such probes are often limited in their protein scope. Here, we demonstrate the synthesis and use of imidazole carbamate probes for affinity guided conjugation of an azide handle to a wide variety of metal binding proteins such as antibodies, enzymes, nanobodies and His6-tagged proteins. The azide handles were used to link dyes to the proteins for gel analysis and binding studies. In a comparative study we find that the probe containing three NTA groups show higher selectivity than the probe containing two. In a mixture of proteins, the probe shows preference for labelling His6-tagged proteins.

The present study had the purpose of demonstrating a positive correlation between enterococci and Salmonella in minced pork and beef. Data from 2001 to 2002 from retail minced pork and beef in Denmark were used and the association between concentration of enterococci and prevalence and concentration of Salmonella was examined. A total of 2187 and 2747 samples of minced pork and beef, respectively, were collected from butcher shops and supermarkets throughout the country. In pork, 2.1% of all samples were positive for Salmonella whereas 1.5% of beef samples were positive. Among samples with ≥100 CFU/g of enterococci, prevalence of Salmonella positive samples was 3.4%, which was significantly higher than 1.2% observed in minced meat with less than 100 CFU/g of enterococci (P < 0.001). A positive association between occurrence of enterococci and presence of Salmonella in retail minced meat was supported as both prevalence and concentration of Salmonella in positive samples increased with increasing concentrations of enterococci in minced meat. From our data, we suggest that minced meat containing more than 500 enterococci per gram is suspected of having been exposed to temperatures allowing growth of Salmonella. This is to our knowledge the first report, which links presence of an indicator to potential growth of Salmonella., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

To evaluate the effect of specific slaughter operations on the contamination of broiler carcasses with naturally occurring thermotolerant Campylobacter, experiments were carried out in two Danish commercial slaughter plants (Plant I and Plant II). Six broiler flocks determined Campylobacter positive prior to slaughter were investigated at four sampling locations within each slaughter plant. Quantification of thermotolerant Campylobacter in 30 neck skin samples per flock per sampling location showed that the evisceration operation in Plant I led to a significant increase in the Campylobacter concentration of 0.5 log(10) cfu/g in average, whereas no significant changes were observed during this operation in Plant II. Air chilling (Plant I) and water chilling (Plant II), both including a carcass wash prior to the chilling operation, caused similar, but significant reductions of 0.83 and 0.97 log(10) cfu/g, respectively. In packed frozen chickens (Plant II) an additional reduction of 1.38 log(10) cfu/g in average was obtained due to the freezing operation. In packed chilled chickens (Plant I), however, the number of thermotolerant Campylobacter per gram remained at the same level as after air chilling. Enumeration of thermotolerant Campylobacter in 30 intestinal samples per flock showed that in two of the six flocks examined the within flock colonization was very low (<3% and 27% positive samples). The remaining four flocks were colonized at percentages of 100 (three flocks) and 97 (one flock) and had intestinal mean counts ranging from 6.65 to 8.20 log(10) cfu/g. A correlation between Campylobacter concentrations in intestinal content and on chicken carcasses after the defeathering operation was documented. This finding indicates that a reduction in the Campylobacter concentration on chicken carcasses may also be obtained by interventions aimed at reducing the concentration of Campylobacter in the intestines of the living birds.

Campylobacter jejuni isolated from raw poultry meat collected at retail shops in Denmark in the period 1996-2003 were tested for susceptibility to seven antimicrobial agents. The food samples consisted of raw chicken meat and other raw poultry meat of domestic or imported origin. The highest levels of resistance among C. jejuni were observed for tetracycline, nalidixic acid and ciprofloxacin, whereas macrolide resistance was rarely detected. C. jejuni originating from other poultry meat (mainly duck and turkey meat) exhibited the highest occurrences of antimicrobial resistance monitored; approximately one third of the isolates were tetracycline resistant (N=100). Among chicken meat isolates, the occurrence of tetracycline resistance was significantly higher (P<0.005) in C. jejuni isolated from imported chicken meat (N=88) than in C. jejuni from Danish chicken meat (N=367). The same tendency was observed for chloramphenicol, nalidixic acid and ciprofloxacin (P<0.05). The trends in resistance in the period 1996-2003 among C. jejuni isolates from chicken meat indicate a decrease in the occurrence of resistance towards fluoroquinolones. This may be due to reduced application of fluoroquinolones for food animals. Monitoring of the occurrence of antimicrobial resistance in C. jejuni isolated from raw uncooked poultry has been performed on a yearly basis since 1996, thus providing useful insight into consumer exposure to antimicrobial-resistant C. jejuni.

A mathematical model is presented, which addresses individual hygiene practices during food preparation and consumption patterns in private homes. Further, the model links food preparers and consumers based on their relationship to household types. For different age and gender groups, the model estimates (i) the probability of ingesting a meal where precautions have not been taken to avoid the transfer of microorganisms from raw food to final meal (a risk meal), exemplified by the event that the cutting board was not washed during food preparation, and (ii) the probability of ingesting a risk meal in a private home, where chicken was the prepared food item (a chicken risk meal). Chicken was included in the model, as chickens are believed to be the major source of human exposure to the foodborne pathogen Campylobacter. Monte Carlo simulations showed that the probability of ingesting a risk meal was highest for young males (aged 18-29 years) and lowest for the elderly above 60 years of age. Children aged 0-4 years had a higher probability of ingesting a risk meal than children aged 5-17 years. This difference between age and gender groups was ascribed to the variations in the hygiene levels of food preparers. By including the probability of ingesting a chicken meal at home, simulations revealed that all age groups, except the group above 60 years of age, had approximately the same probability of ingesting a chicken risk meal, the probability of females being slightly higher than that of males. The simulated results show that the probability of ingesting a chicken risk meal at home does not only depend on the hygiene practices of the persons preparing the food, but also on the consumption patterns of consumers, and the relationship between people preparing and ingesting food. This finding supports the need of including information on consumer behavior and preparation hygiene in the consumer phase of exposure assessments.

Aims: To use amplified fragment length polymorphism (AFLP) analysis to evaluate the genetic relatedness among 254 Campylobacter jejuni reference and field strains of diverse origin representing all defined 'Penner' serotypes for this species., Methods and Results: Field strains (n = 207) from human diarrhoea and diverse animal and environmental sources were collected mainly through a National surveillance programme in Denmark and serotyped by use of the established 'Penner' scheme. Genetic relationships among these isolates, and the archetypal serotype reference strains, were assessed by numerical analysis of AFLP profiles derived from genomic DNA. Extensive genetic diversity was seen among the strains examined; however, 43 groups of isolates were identified at the 92% similarity (S-) level. Thirteen groups contained isolates from a single host, possibly representing genotypes of 'low risk' to human health. The remaining 30 groups contained isolates from humans, chickens and associated food products, cattle, sheep, turkeys, ostriches and/or dogs. Strains assigned to serotypes 2, 6/7, 11 and 12 formed major clusters at the 77.6% S-level. Most other serotypes did not form homogeneous clusters., Conclusions: High-resolution genotyping applied to strains from a comprehensive range of sources provides evidence for multiple sources of sporadic C. jejuni infection. The results suggest that public health protection measures should be directed at all foods of animal origin., Significance and Impact of the Study: The genetic relatedness among all 'Penner' serotypes of C. jejuni is assessed by AFLP analysis. In addition, further evidence of epidemic and host-specific clones of C. jejuni is provided.

A quantitative risk assessment comprising the elements hazard identification, hazard characterization, exposure assessment, and risk characterization has been prepared to assess the effect of different mitigation strategies on the number of human cases in Denmark associated with thermophilic Campylobacter spp. in chickens. To estimate the human exposure to Campylobacter from a chicken meal and the number of human cases associated with this exposure, a mathematical risk model was developed. The model details the spread and transfer of Campylobacter in chickens from slaughter to consumption and the relationship between ingested dose and the probability of developing campylobacteriosis. Human exposure was estimated in two successive mathematical modules. Module 1 addresses changes in prevalence and numbers of Campylobacter on chicken carcasses throughout the processing steps of a slaughterhouse. Module 2 covers the transfer of Campylobacter during food handling in private kitchens. The age and sex of consumers were included in this module to introduce variable hygiene levels during food preparation and variable sizes and compositions of meals. Finally, the outcome of the exposure assessment modules was integrated with a Beta-Poisson dose-response model to provide a risk estimate. Simulations designed to predict the effect of different mitigation strategies showed that the incidence of campylobacteriosis associated with consumption of chicken meals could be reduced 30 times by introducing a 2 log reduction of the number of Campylobacter on the chicken carcasses. To obtain a similar reduction of the incidence, the flock prevalence should be reduced approximately 30 times or the kitchen hygiene improved approximately 30 times. Cross-contamination from positive to negative flocks during slaughter had almost no effect on the human Campylobacter incidence, which indicates that implementation of logistic slaughter will only have a minor influence on the risk. Finally, the simulations showed that people in the age of 18-29 years had the highest risk of developing campylobacteriosis.

Campylobacter infection is one of the most common bacterial enteric pathogens. Campylobacter jejuni and Campylobacter coli infections are mostly food- and waterborne and especially poultry is often assumed to be an important source. The heat-stable serotyping system (the 'Penner' scheme) was used to study the serotype distribution of C. jejuni and C. coli isolated from different food products of poultry origin sampled from retail outlets in Denmark. A total of 156 isolates were serotyped, 85% of these were C. jejuni and 15% were C. coli. The most common C. jejuni serotypes were O:2 (30%), O:1,44 (12%) and the O:4-complex (8%). O:46 was the most frequent serotype among C. coli isolates. These serotypes are also common among Danish clinical isolates and isolates from broiler chickens and cattle. Differences in serotype distribution were seen for different kinds of poultry products. Isolates from chicken products covered a large selection of serotypes. In contrast, the majority of the isolates from other product groups (turkey, poussin, wild birds) were concentrated on 1-3 serotypes. Using the standard procedure for antigen preparation and serotyping, 25 of the 156 strains (16%) were nontypable. This rate of nontypable isolates is significantly higher than experienced for isolates from other sources than food products, i.e faecal samples from animals and humans. Subculturing and re-typing of the nontypable isolates improved the typability. After two, five and 10 subcultures 16, six and one isolate became typable, respectively. Only three isolates (2%) remained nontypable after 10 subcultures.