Selection and transfer of an IncI1-tet(A) plasmid of Escherichia coli in an ex vivo model of the porcine caecum at doxycycline concentrations caused by crosscontaminated feed.

AIMS: The aim of this study was to investigate the effect of subtherapeutic intestinal doxycycline (DOX) concentrations (4 and 1 mg l-1 ), caused by cross-contamination of feed, on the enrichment of a DOX-resistant commensal Escherichia coli and its resistance plasmid in an ex vivo model of the porcine caecum. METHODS AND RESULTS: A DOX-resistant, tet(A)-carrying, porcine commensal E. coli strain (EC 682) was cultivated for 6 days in the porcine caecum model under different conditions (0, 1 and 4 mg l-1 DOX). EC 682, other coliforms and anaerobic bacteria were enumerated daily. A selection of isolated DOX-resistant coliforms (n = 454) was characterized by rep-PCR clustering, PCR assays (Inc1 and tet(A)) and micro broth dilution susceptibility tests (Sensititre). Both 1 and 4 mg l-1 DOX-enriched medium had a significantly higher selective effect on EC 682 and other resistant coliforms than medium without DOX. Transconjugants of EC 682 were isolated more frequently in the presence of 1 and 4 mg l-1 DOX compared to medium without DOX. CONCLUSIONS: Subtherapeutic intestinal DOX concentrations have the potential to select for DOX-resistant E. coli, and promote the selection of transconjugants in a porcine caecum model. SIGNIFICANCE AND IMPACT OF THE STUDY: Cross-contamination of feed with antimicrobials such as DOX likely promotes the spread of antimicrobial resistance. Therefore, it is important to develop or fine-tune guidelines for the safe use of antimicrobials in animal feed and its storage.

Phage and MLVA typing of Salmonella enteritidis isolated from layers and humans in Belgium from 2000-2010, a period in which vaccination of laying hens was introduced.

The aim of the study was to characterize isolates of Salmonella enterica serovar Enteritidis (S. Enteritidis) obtained from humans and layer farms in Belgium collected during 2000-2010. Three periods were compared, namely (i) before implementation of vaccination (2000-2004), (ii) during voluntary vaccination (2005-2006) and (iii) during implementation of the national control program (NCP) for Salmonella including mandatory vaccination against S. Enteritidis (2007-2010). The characteristics compared across time periods were distributions of phage type and multiple-locus variable number tandem-repeat assay (MLVA). While PT4 and PT21 were predominantly isolated in Belgium in layers and humans before 2007, a significant reduction of those PTs was observed in both populations in the period 2007-2010. The relative proportion of PT4b, PT21c and PT6c was found to have increased considerably in the layer population since 2007. In the human population, PT8, PT1 and the group of 'other' PTs were more frequently isolated compared to the previous periods. When comparing the proportion of the predominant MLVA types Q2 and U2, no significant difference was found between the layer and human population in the three periods and between periods within each category (layer and human). A significant difference in isolate distribution among MLVA clusters I and II was found between human and layer isolates recovered during Period 3 and in the human population between Period 1 and 3. Results suggest that the association between S. Enteritidis in layers and the occurrence of the pathogen in humans changed since implementation of the NCP in 2007.

Assessment of human exposure to 3rd generation cephalosporin resistant E. coli (CREC) through consumption of broiler meat in Belgium.

Acquired resistance of Escherichia coli to 3rd generation cephalosporin antimicrobials is a relevant issue in intensive broiler farming. In Belgium, about 35% of the E. coli strains isolated from live broilers are resistant to 3rd generation cephalosporins while over 60% of the broilers are found to be carrier of these 3rd generation cephalosporin resistant E. coli (CREC) after selective isolation. A model aimed at estimating the exposure of the consumer to CREC by consumption of broiler meat was elaborated. This model consists of different modules that simulate the farm to fork chain starting from primary production, over slaughter, processing and distribution to storage, preparation and consumption of broiler meat. Input data were obtained from the Belgian Food Safety agencies' annual monitoring plan and results from dedicated research programs or surveys. The outcome of the model using the available baseline data estimates that the probability of exposure to 1000 colony forming units (cfu) of CREC or more during consumption of a meal containing chicken meat is ca. 1.5%, the majority of exposure being caused by cross contamination in the kitchen. The proportion of CREC (within the total number of E. coli) at primary production and the overall contamination of broiler carcasses or broiler parts with E. coli are dominant factors in the consumer exposure to CREC. The risk of this exposure for human health cannot be estimated at this stage given a lack of understanding of the factors influencing the transfer of cephalosporin antimicrobial resistance genes from these E. coli to the human intestinal bacteria and data on the further consequences of the presence of CREC on human health.

Defining European preparedness and research needs regarding emerging infectious animal diseases: results from a Delphi expert consultation.

Emerging and major infectious animal diseases can have significant international impact on social, economic and environmental level, and are being driven by various factors. Prevention and control measures should be prepared at both national and international level to mitigate these disease risks. Research to support such policy development is mostly carried out at national level and dedicated transnational research programmes are still in its infancy. This research reports on part of a process to develop a common strategic research agenda on emerging and major infectious diseases of livestock in Europe, covering a 5-15-year time span. A two round online Delphi study was conducted to explore the views of experts on issues relating to research needs on emerging infectious diseases of livestock in Europe. Drivers that may influence the incidence of emerging infectious animal diseases in both the short (next 5 years) and medium term (10-15 years) were identified. Drivers related to regulatory measures and biological science developments were thought to decrease the incidence, and socio-economic factors to increase the incidence of emerging infectious animal diseases. From the first round a list of threats to animal health was compiled and participants combined these threats with relevant drivers in the second round. Next to identifying threats to animal health, also possible mitigatory actions to reduce the negative impact of these threats were identified. Participants emphasised that interdisciplinary research is needed to understand drivers of emerging infectious animal diseases, as well as to develop prevention and control measures which are both socio-economic and technical. From this it can be concluded that interdisciplinary research combining both natural and social research themes is required. Some of the European member states research budget needs to be allocated so that effective prevention and mitigation strategies can be developed.

Investigation of an excess of Salmonella enteritidis phage type 14b and MLVA type 4-7-3-13-10-2-2 in Luxembourg, Belgium and Germany during 2010.

We investigated an increase of human cases of Salmonella Enteritidis occurring from August until November 2010 in Belgium, Luxembourg and Germany involving an estimated three hundred laboratory confirmed cases. Molecular typing indicated that the increase in Luxembourg and Belgium was due a particular strain having phage type 14b, MLVA pattern 4-7-3-13-10-2-2 and fully susceptible to the Enternet panel of antibiotics. MLVA and phage typing were found to have similar discriminatory power on a collection of 40 Belgian and Luxembourg strains isolated during 2010. Epidemiological investigations in Luxembourg suggested eggs as a possible source for some cases, although supermarket eggs tested were negative. No other EU countries observed a substantial increase of cases, although three smaller outbreaks in Germany were also due to a strain with the same phage type and MLVA pattern. In 2010 the EU directive banning battery cages came into force in Germany followed by a dioxin food scare incident. Given that the EU Laying Hens Directive will come into force across all Member States in 2012, a closer monitoring of Salmonella contamination of imported eggs at retail and wholesale level is recommended.

Detection and characterization of Salmonella in lairage, on pig carcasses and intestines in five slaughterhouses.

In this study, conducted at five slaughterhouses, individual pigs were sampled and followed up from stunning to cooling down of the carcasses. In this way, Salmonella prevalence and possible risk points were described. At the lairage area, pens were sampled using overshoes. At stunning and bleeding, pigs were individually identified and subsequently swabs were taken of the oral cavity and the carcass after polishing, splitting and forced chilling. Additionally, duodenum, ileum, rectum and mesenteric lymph nodes were extracted and samples were taken of the scalding water. All samples were submitted to Salmonella isolation and Salmonella isolates were serotyped and genotyped by pulsed-field gel electrophoresis (PFGE). Of all samples taken (n = 1953), 14.1% were Salmonella positive. The prevalence of S. in the lairage area varied widely (from 0 to 100%) between the slaughterhouses. Of the sampled pigs (n = 226), 48.2% were positive in at least one sample. Statistical analysis revealed that the contamination of the lairage area was related to a higher amount of positive carcasses after polishing. Furthermore, the contamination of the carcasses after splitting and forced chilling was related to the contamination level of the carcass after polishing. A relation between the outer (carcass) contamination and the inner (gut content and lymph nodes) contamination of a pig could not be established. The predominant serotypes were S. Typhimurium (58.7%) and S. Derby (17.4%). Genotyping revealed 46 different PFGE profiles among the 276 Salmonella isolates. The same genotype at the lairage area as in the oral cavity of the pigs was found in 95%. The results indicate that the lairage area is a primary source of Salmonella in slaughter pigs and that carcass contamination originates from the environment rather than from the pig (inner contamination) itself. It further shows that slaughterhouses vary in their capability of dealing with Salmonella positive pigs. A slaughterhouse specific approach is needed, however, general guidelines should be provided to decrease the contamination level of the lairage area and the slaughter environment.

Drastic decrease of Salmonella Enteritidis isolated from humans in Belgium in 2005, shift in phage types and influence on foodborne outbreaks.

In Belgium, non-typhoidal salmonellosis and campylobacteriosis are the two most frequently reported foodborne illnesses. During 2005, a 71% decrease of Salmonella Enteritidis infections compared with the average annual number cases in the period 2000-2004 was recorded by the Belgian National Reference Centre for Salmonella and Shigella. After the peak of 1999, the total number of salmonellosis cases decreased gradually, with the exception of 2003 when an increase was again recorded due to the rise of isolates belonging to the serotype Enteritidis. PT4, the predominant phage type of serotype Enteriditis over recent years (except in 2003), became the second most prevalent phage type in 2005 after PT21. We present in this paper the epidemiology (incidence and trends) of human salmonellosis in Belgium and assess the role of the vaccination programme in layer flocks on the decline of the incidence of human salmonellosis and foodborne outbreaks due to S. Enteritidis.

Sanitary control in bovine embryo transfer. How far should we go? A review.

Embryo transfer is a globally executed technique which, when properly done, has both economic and sanitary advantages. International guidelines are available to prevent infection of the embryo with pathogens, both originating from the donor animals as from the environment. This manuscript describes the bacteria, viruses, protozoa, fungi and prions that are of major concern in the context of embryo transfer in cattle. In addition, the actual scientific knowledge on these pathogens is evaluated in terms of the current international and national guidelines and legislation.

Uncertainty of measurement for competitive and indirect ELISAs.

A method for the estimation of the uncertainty of measurements for Gaussian outcomes of enzyme-linked immunosorbent assay (ELISA) is described using competitive and indirect foot and mouth disease (FMD) ELISAs. Assay repeatability was determined by random effects analysis of variance, and the normality of the residuals was checked. The standard errors of the individual predicted values were transformed into confidence intervals around the corresponding observed values and further transformed into probabilities of being above/below a cut-off. Logistic regression models were subsequently used to interpolate probability values for the whole range of possible assay values. The uncertainty of measurement of a test result was finally defined as the probability of not observing the same qualitative test result when retesting the same sample. For the competitive ELISA any sample with a percent inhibition 4% above the cut-off value had an uncertainty level (probability of a negative result in the case of retest) below 5%. In the indirect ELISA with a cut-off OD of 0.1, the uncertainty was below 5% for any sample with a normalised OD value above 0.22.

A multiplex PCR to identify porcine mycoplasmas present in broth cultures.

Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculare can be present in the lungs of pigs at the same time. These three mycoplasma species all require similar growth conditions and can be recovered from clinical samples using the same media. We have developed a multiplex PCR as a helpful tool for rapid differentiation of these three species in the course of isolation. Based on the 16S ribosomal DNA sequences, three different forward primers and a single reverse primer were selected. Each forward primer was compared to available mycoplasma sequences, showing the primers to be specific. The three amplification products observed of 1129 bp (M. hyorhinis), 1000 bp (M. hyopneumoniae) and 754 bp (M. flocculare) were clearly distinguishable on a 1% agarose gel. In addition, no cross-reaction with Mycoplasma hyosynoviae, another porcine mycoplasma, was noted. This multiplex PCR using the proposed set of primers is the first reported assay that allows the simultaneous identification of the different Mycoplasma species isolated from the lungs of pigs.

Comparison of five repetitive-sequence-based PCR typing methods for molecular discrimination of Salmonella enterica isolates.

Five repetitive-element PCR (rep-PCR) techniques [primer sets ERIC1R-ERIC2 and REP1R-REP2I and primers ERIC2, BOXA1R, and (GTG)5] were evaluated for the discrimination of Salmonella enterica isolates at the serotype level. On the basis of number, even distribution over the whole fingerprint, and clarity of bands in the fingerprints, the enterobacterial repetitive intergenic consensus (ERIC) primer set and the (GTG)5 primer were chosen for use in the following experiments. For these two primer sets, reproducibility was tested on different lysates of five selected serotypes of Salmonella in the same PCR by using three different PCR runs. Reproducibility was poor between different PCR runs but high within the same PCR run. Furthermore, 80 different serotypes and five isolates which were not typeable by serotyping were fingerprinted. All strains were typeable by the ERIC primer set and the (GTG)5 primer and generated unique fingerprints, except for some strains with incomplete antigenic codes. Finally, 55 genetically different strains belonging to 10 serotypes were fingerprinted to examine the genetic diversity of the rep-PCR within serotypes. This experiment showed that one serotype did not always correlate to only one ERIC or (GTG)5 fingerprint but that the fingerprint heterogeneity within a serotype was limited. In epidemiological studies, ERIC- and/or (GTG)5-PCR can be used to limit the number of strains that have to be serotyped. The reproducibility of isolates in one PCR run, the discriminatory power, and the genetic diversity (stability) of the fingerprint were similar for the Eric primer set and the (GTG)5 primer, so both are equally able to discriminate Salmonella serotypes.

Risk factors for the herd-level bacteriologic prevalence of Salmonella in Belgian slaughter pigs.

Herd-level risk factors for salmonellosis in pigs were investigated in a cross-sectional study on 62 Belgian farrow-to-finish pig herds belonging to one slaughterhouse cooperative. Data concerning housing and ventilation, management, hygiene, biosecurity, production parameters, feeding, disease control and transport to the slaughterhouse were collected during a herd visit by means of a questionnaire. The percentage of positive animals in a slaughterhouse delivery, as determined by qualitative Salmonella isolation in the mesenteric lymph nodes taken from 30 slaughter pigs, was the outcome variable. All samples were taken in 4 different slaughterhouses. Variables first were submitted to a univariable analysis using a logistic mixed regression model, with herd as random effect. Variables which were related to the Salmonella prevalence (P < 0.05) were analysed further in a multivariable model. The clustering of Salmonella infection within a pen also was studied in a generalised mixed model with pen as random effect. Salmonella isolates were identified by serotype. In 57 (92%) of the herds, at least one sample was found positive for Salmonella. The median percentage of positive Salmonella samples per delivery was 64% (range: 0-100%). In the multivariable model, only type of floor was related significantly to the prevalence: 100% (95% CI 88-100) for herds with <50% slatted floors to 54% (36-70) for herds with fully slatted floors. The results from the analysis should be interpreted with care because only 62 herds were included in the study. Clustering between pigs from the same pen could not be demonstrated (variance +/- S.D.: 0.11 +/- 0.16). S. typhimurium (30%) and S. derby (20%) were most common among the 23 different serotypes that were found.

Conserved regions in the sequence of the F4 (K88) fimbrial adhesin FaeG suggest a donor strand mechanism in F4 assembly.

Oral immunization of newly weaned piglets with recombinant F4 (K88) fimbrial adhesin FaeG induces a F4-specific immune response, significantly reducing F4+ Escherichia coli excretion following challenge. In order to use FaeG subunits in an oral vaccine against F4+ enterotoxigenic E. coli, it is necessary to determine the conservation of the adhesin subunit. Hereto, the faeG sequence was determined of 21 F4ac+ E. coli field isolates from piglets with diarrhoea and subsequently compared with these of the reference strain GIS26 and previously reported FaeG sequences from F4ab, F4ac and F4ad antigenic variant strains. The FaeG amino acid sequence was 96-100% homologous within each F4 serotype, but only 92 and 88% when the F4ab and F4ad antigenic variants were compared with the F4ac antigenic variant. Furthermore, the conserved regions of the adhesin suggest a donor strand mechanism in F4 fimbriae assembly as reported for type 1 and P pili. In conclusion, the results of the reported experiments support the usefulness FaeG in an oral subunit vaccine against F4+ E. coli infections or as a mucosal carrier since the adhesin is conserved among F4+ E. coli field isolates.

Characterization of the adhesin of Escherichia coli F18 fimbriae.

Previous research has suggested that the adhesin encoded by the F18 fimbrial operon in Escherichia coli is either the FedE or FedF protein. In this work, we show that anti-FedF antibodies, unlike anti-FedE serum, were able to inhibit E. coli adhesion to porcine enterocytes. Moreover, specific adhesion to enterocytes was shown with purified FedF-maltose binding protein.

Complete nucleotide sequence of a 43-kilobase genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104 and its identification in phage type DT120 and serovar Agona.

This study describes the characterization of the recently described Salmonella genomic island 1 (SGI1) (D. A. Boyd, G. A. Peters, L.-K. Ng, and M. R. Mulvey, FEMS Microbiol. Lett. 189:285-291, 2000), which harbors the genes associated with the ACSSuT phenotype in a Canadian isolate of Salmonella enterica serovar Typhimurium DT104. A 43-kb region has been completely sequenced and found to contain 44 predicted open reading frames (ORFs) which comprised approximately 87% of the total sequence. Fifteen ORFs did not show any significant homology to known gene sequences. A number of ORFs show significant homology to plasmid-related genes, suggesting, at least in part, a plasmid origin for the SGI1, although some with homology to phage-related genes were identified. The SGI1 was identified in a number of multidrug-resistant DT120 and S. enterica serovar Agona strains with similar antibiotic-resistant phenotypes. The G+C content suggests a potential mosaic structure for the SGI1. Emergence of the SGI1 in serovar Agona strains is discussed.

Automated 5' nuclease assay for detection of virulence factors in porcine Escherichia coli.

This paper describes the development of a 5' nuclease assay for detection of virulence factor genes responsible for colonization factors and toxins in Escherichia coli isolated from pigs. Colonization factors were F4, F5, F6, F18, F41 and the outer membrane protein intimin. Toxins were heat stable (STa, STb, EAST1) and heat labile (LT) enterotoxins and the verocytotoxin variant 2e (VT2e). To correctly identify false negative results, an endogenous internal control targeting the E. coli 16 S rRNA gene was incorporated in each test tube. The assay was evaluated using a collection of E. coli reference strains which have previously been examined with phenotypical assays or DNA hybridization. Furthermore, the assay was evaluated by testing porcine E. coli field strains, previously characterized. The 5' nuclease assay correctly detected the presence of virulence genes in all reference strains. When testing field strains there was generally excellent agreement with results obtained by laboratories in Belgium and Germany. In conclusion, the 5' nuclease assay developed is a fast and specific tool for detection of E. coli virulence genes in the veterinary diagnostic laboratory.

Polyclonal sandwich ELISAs to detect F18 fimbriated Escherichia coli in pigs in Northern Ireland.

F18 fimbriated Escherichia coli are a newly described cause of postweaning diarrhea in pigs. Polyclonal rabbit antisera were raised to the antigenic variants, F18ab and F18ac, of these fimbriae and were used to develop monospecific sandwich enzyme-linked immunosorbent assays (ELISAs). The ELISAs were standardized with type cultures characterized by polymerase chain reaction techniques (PCR) and then used to conduct a study of the prevalence of F18 fimbriated E. coli in pigs in Northern Ireland. A total of 176 isolates were tested by ELISA and PCR. Eight isolates were positive for F18 by ELISA, of which 2 were shown to be false positives by PCR and one was PCR positive but ELISA negative. Of the 6 confirmed ELISA positives, all produced VT2 toxin and 3 produced ST toxin. Four positives were from serogroups O138 and O139, previously associated with porcine diarrhea.

Occurrence of a Salmonella enterica serovar typhimurium DT104-like antibiotic resistance gene cluster including the floR gene in S. enterica serovar agona.

Recently a chromosomal locus possibly specific for Salmonella enterica serovar Typhimurium DT104 has been reported that contains a multiple antibiotic resistance gene cluster. Evidence is provided that Salmonella enterica serovar Agona strains isolated from poultry harbor a similar gene cluster including the newly described floR gene, conferring cross-resistance to chloramphenicol and florfenicol.

Analysis of foodborne disease in Belgium in 1997.

Foodborne disease represents a major problem for public health in industrialized countries, albeit with a low lethality. Foodborne diseases are defined as a group of viral, bacterial or parasitic gastrointestinal infections transmitted by means of food. Proper food-hygiene practices and surveillance of individual diseases and in particular outbreaks are the first steps in targeting their prevention. The incidence of this illness is difficult to estimate. In the Netherlands a yearly incidence of gastrointestinal infections of 500 per 1,000 inhabitants is estimated, of which most are foodborne. To set up priorities in the actions to undertake, to establish the most frequent risks, to develop preventive efforts and to answer to international requirements, accurate data on foodborne disease from Belgium are required. In order to co-ordinate the initiatives in the Belgian context, a working group was set up in 1995. In 1997 a total of 2,013 persons with foodborne disease were identified as part of 140 outbreaks, 22 of which occurred with 10 cases or more. Salmonella Enteritidis (88 outbreaks) was identified as the main pathogen in foodborne disease, followed by S. Typhimurium (11), S. Hadar (4). Eggs and meat products were identified as the main food-items involved, although it remains difficult to obtain proper intervention studies allowing to identify the specific cause(s). In 1997, a total of 12,732 human Salmonella isolates and 5,617 Campylobacter isolates were identified by the respective national reference laboratories. Salmonella isolates from Belgium accounted in 1997 for more than a fifth of all Salmonella isolates in the EU. The final objective of the working group is the implementation of a surveillance system for all risk factors concerned with the development of food-related illness, including an early warning system and an efficient analysis of microbiological criteria relating to human health, food and food production, including livestock. An essential element of this surveillance is communication of the results, risks and measures for prevention between all the departments, institutions and public health authorities concerned.