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.

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.

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.

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.

A gene expressed only in serum-resistant variants of Trypanosoma brucei rhodesiense.

The human infective African trypanosomes are host range variants of Trypanosoma brucei which are resistant to a lytic component in primate serum. T. b. rhodesiense occurs both as a form sensitive to lysis by normal human serum and as a form resistant to this lysis. Switching from one phenotype to the other has been observed in both directions. In the cloned T. b. rhodesiense ETAR1-repertoire we have detected 1.5-kb mRNAs only present in the resistant forms. In T. b. gambiense, which always occurs as a normal human serum-resistant form, no such transcript could be detected, indicating that another mechanism of resistance is involved here. Starting from an independent non-cloned T. b. rhodesiense population isolated from an infected patient, both resistant and sensitive trypanosomes have been prepared. Northern blot analysis of the total RNA prepared from these populations has revealed again the differential occurrence of the resistance-specific transcript, indicating that we are dealing with a general phenomenon associated with serum resistance in T. b. rhodesiense. As expected, Southern blot analyses have demonstrated that both serum-resistant and serum-sensitive forms of T. b. rhodesiense contain the gene coding for this transcript.

Sequences related to the major subunit gene fedA of F107 fimbriae in porcine Escherichia coli strains that express adhesive fimbriae.

Porcine Escherichia coli strains isolated from cases of postweaning diarrhea or edema disease were analysed for the presence of fedA, the major subunit gene of F107 fimbriae. The E. coli isolates were known to contain colonisation factor '8813', or to express F107, 2134P or other fimbriae, different from F4, F5, F6, and F41. PCR with fedA-specific primers, restriction enzyme digestion of the PCR product, and nucleotide sequence analysis demonstrated that 2134P pili, colonisation factor '8813' and fimbriae identified on Australian strains of the O141 serotype belong to one family of F107 fimbrial antigens.

The pathogenesis of edema disease in pigs. A review.

Edema disease is known to cause important losses in the period shortly after weaning. Although the disease is known for many decades, intensive studies with bacterial lysates of pathogenic E. coli, followed by biotechnological research the last ten years, has led to a better understanding of its pathogenesis. Especially the impact of the toxin is clearly established. Evidence also exists that adhesion factors play a crucial role in the pathogenesis of edema disease.

Chicken egg yolk antibodies against F18ab fimbriae of Escherichia coli inhibit shedding of F18 positive E. coli by experimentally infected pigs.

F18ab and F18ac are antigenic variants of a colonizing fimbria commonly found on E. coli associated with postweaning diarrhea and edema disease in pigs. Chicken F18ab antibodies were obtained by immunising hens with purified F18ab fimbriae. For their in vitro characterisation antibodies were isolated from diluted egg yolks by ammonium sulfate precipitation. In vitro adhesion tests demonstrated that the chicken F18ab antibodies inhibited attachment of F18ab positive E. coli bacteria to the intestinal mucosa. Just weaned piglets were experimentally infected with an F18ab positive edema disease strain of E. coli, or with an F18ac positive postweaning diarrhea E. coli strain. The animals were infected on the second day of a period during which chicken F18ab antibodies were added to their feed. During the same period, pigs of the control group received commercial eggs in which no F18 antibodies were detected. In both experimental infections the excretion of the F18 positive strain was reduced in pigs that received the F18ab antibodies as compared to the control animals. The F18ab antibodies diminished the cases of diarrhea and death in animals infected with F18ac positive E. coli.

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.

Prevalence of F107 fimbriae on Escherichia coli isolated from pigs with oedema disease or postweaning diarrhoea.

The study comprises fifty 4 to 12 weeks old pigs that died from oedema disease or severe diarrhoea. Smears were prepared from the mucosa of duodenum, jejunum and ileum, and by immunofluorescence F107 fimbrial antigens were detected. E. coli strains were isolated from the intestines and were characterised by slide agglutination (serogroup and F107 fimbriae production), by their cytotoxicity for Vero cells, and by gene amplification (genes coding for the major F107 subunit FedA, the toxin causing oedema disease SLT-IIv, and enterotoxins LTI, STIa and STII). F107 fimbriae were demonstrated in association with E. coli of serogroups O139:K12 and O141:K85a,b but not of serogroup O149:K91:F4a,c. Expression in culture of F107 fimbriae by some isolates gave additional evidence for production of these fimbriae by ETEC strains. The genetic determinant of SLT-Ilv was found in association with F107, and could not be detected in serogroup O149:K91:F4a,c. Gene fedA was demonstrated in two isolates which were devoid of SLT-IIv. Most isolates from cases of oedema disease belonged to serogroup O139:K12 and did not contain enterotoxin genes. Isolates from pigs that suffered from diarrhoea were serotyped O141:K85a,b or O149:K91:F4a,c, and carried at least two enterotoxin genes in their genomes. In a small proportion of the cases F107 antigens were demonstrated in intestinal smears although gene fedA was not detected in the corresponding isolates. The results confirm the importance of F107 fimbriae as virulence factor in oedema disease E. coli strains, but also demonstrate that F107 fimbriae can be found in association with postweaning diarrhoea isolates. In these latter strains enterotoxins were always demonstrated, irrespective of the presence of toxin SLT-IIv.

Designations F18ab and F18ac for the related fimbrial types F107, 2134P and 8813 of Escherichia coli isolated from porcine postweaning diarrhoea and from oedema disease.

The relatedness of the fimbriae produced by eight E. coli strains including type strains with F107 fimbriae, 2134P pili and colonization factor 8813 (preliminary F18), was examined. These strains had been isolated principally from pigs which were affected with postweaning diarrhoea or with oedema disease. The fimbriae were analyzed by means of electron microscopy, slide agglutination, immunofluorescence, immunogold labelling, immuno-diffusion, immunoelectrophoresis and western blot, molecular genetic techniques, and in vitro adhesion. The fimbriae of all the strains were long flexible filaments with a diameter not larger than 4.6 nm showing a zig-zag pattern. Results obtained by the serological techniques confirmed that the fimbriae possessed a common antigenic determinant designated 'a' in addition to a variant-specific determinant designated 'b' or 'c'. Immunoelectron microscopy demonstrated that the determinants 'a' and 'b' or 'a' and 'c' were localized along the same fimbrium. In immunoelectrophoresis, fimbrial extracts of selected strains yielded a single precipitation line towards the cathode. One single major subunit of approximately 15 kDa was recognised in western blots by antisera against the common antigenic determinant and the variant specific determinants. All strains possessed sequences related to gene fedA, coding for the major subunit of fimbriae F107. Two types of fedA-related subunit genes were differentiated, corresponding to the 'ab' and 'ac' types of fimbriae as defined by serological methods. The results demonstrated that F107 fimbriae, 2134P pili and colonization factor 8813 are related, and that two serological variants can be distinguished. We propose designations F18ab (for F107), and F18ac (for 2134P and 8813) in analogy to the nomenclature of F4 fimbriae.

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.

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.

Fimbrial colonisation factors F18ab and F18ac of Escherichia coli isolated from pigs with postweaning diarrhea and edema disease.

During the last 5 years at least four new types of colonisation factors have been described in association with porcine postweaning diarrhea and edema disease strains of E. coli. Recently, evidence was presented that these fimbrial factors are closely related to each other, and therefore the common denomination F18 was proposed. Until now, two variants F18ab and F18ac were identified that can be distinguished by serology. Alternatively, to circumvent elaborate growth conditions for the optimal expression of F18 fimbriae in vitro, PCR and subsequent restriction enzyme digestion of the amplification product can be used to differentiate F18ab from F18ac positive isolates. Reports that studied the prevalence of F18 positive E. coli show that this factor is present in about 30% to more than 50% of the PWD or ED strains negative for F4, F5, F6 or F41. Susceptibility of pigs to colonisation depends on the availability of intestinal receptors, and is under the control of a chromosomal locus. In young pigs susceptibility increases with age. Intestinal infection with F18 positive E. coli induces protection against repeated colonisation with E. coli bearing the homologous or the heterologous fimbrial variant of F18. Finally, preliminary passive protection studies suggest that F18 antibodies inhibit the colonisation of the pig's intestine by F18ab and F18ac positive strains.

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.

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.

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.

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.