An analysis of cattle farmers' perceptions of drivers and barriers to on-farm control of Escherichia coli O157.

Structural equation modelling and survey data were used to test determinants' influence on farmers' intentions towards Escherichia coli O157 on-farm control. Results suggest that farmers more likely to show willingness to spend money/time or vaccinate to control Escherichia coli O157 are those: who think farmers are most responsible for control; whose income depends more on opening farms to the public; with stronger disease control attitudes; affected by outbreaks; with better knowledge and more informed; with stronger perceptions of biosecurity measures' practicality; using a health plan; who think farmers are the main beneficiaries of control; and whose farms are dairy rather than beef. The findings might suggest that farmers may implement on-farm controls for E. coli O157 if they identify a clear hazard and if there is greater knowledge of the safety and efficacy of the proposed controls.

High prevalence of paratuberculosis in rabbits is associated with difficulties in controlling the disease in cattle.

There is increasing evidence that the European wild rabbit (Oryctolagus cuniculus) is a wildlife reservoir for paratuberculosis and infected populations may contribute to the persistence of infection in livestock. The aim of this study was to test the hypothesis that farms with difficulties controlling paratuberculosis in their cattle herds have a higher prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) infection in their rabbit populations. A total of 281 rabbits from 13 beef farms in the East of Scotland were randomly sampled in early spring 2007. Participating farms were in paratuberculosis control programmes under the Premium Cattle Health Scheme (PCHS), and were classified as 'responder' (paratuberculosis under control) or 'low responder' (a persistent number of paratuberculosis-positive cattle despite control measures in place) farms. Of the rabbits sampled, 23.8% tested positive for MAP, with those on 'low responder' farms having a greater probability of being infected (0.4) relative to rabbits on 'responder' farms (0.1). The association suggests that MAP-infected rabbits may contribute to the persistence of paratuberculosis in domestic livestock and undermine control strategies that focus on livestock alone. This study provides the first evidence of an association between the persistence of paratuberculosis in livestock despite the implementation of disease control strategies, and MAP-infected sympatric wild rabbit populations.

Associations between the presence of virulence determinants and the epidemiology and ecology of zoonotic Escherichia coli.

The severity of human infection with pathogenic Escherichia coli depends on two major virulence determinants (eae and stx) that, respectively, produce intimin and Shiga toxin. In cattle, both may enhance colonization, but whether this increases fitness by enhancing cattle-to-cattle transmission in the field is unknown. In E. coli O157, the almost uniform presence of the virulence determinants in cattle isolates prevents comparative analysis. The availability to this study of extensive non-O157 E. coli data, with much greater diversity in carriage of virulence determinants, provides the opportunity to gain insight into their potential impact on transmission. Dynamic models were used to simulate expected prevalence distributions for serogroups O26 and O103. Transmission parameters were estimated by fitting model outputs to prevalence data from Scottish cattle using a Bayesian Markov chain Monte Carlo (MCMC) approach. Despite similar prevalence distributions for O26 and O103, their transmission dynamics were distinct. Serogroup O26 strains appear well adapted to the cattle host. The dynamics are characterized by a basic reproduction ratio (R(0)) of >1 (allowing sustained cattle-to-cattle transmission), a relatively low transmission rate from environmental reservoirs, and substantial association with eae on transmission. The presence of stx(2) was associated with reduced transmission. In contrast, serogroup O103 appears better adapted to the noncattle environment, characterized by an R(0) value of <1 for plausible test sensitivities, a significantly higher transmission rate from noncattle sources than serogroup O26, and an absence of fitness benefits associated with the carriage of eae. Thus, the association of eae with enhanced transmission depends on the E. coli serogroup. Our results suggest that the capacity of E. coli strains to derive fitness benefits from virulence determinants influences the prevalence in the cattle population and the ecology and epidemiology of the host organism.

Failure to transmit scrapie infection by transferring preimplantation embryos from naturally infected donor sheep.

The objective of the study was to examine whether or not the preimplantation embryo can act as a carrier of classic scrapie infection. The study was carried out on quarantined premises with sheep of highly susceptible scrapie genotypes. Uninfected embryos, collected from New Zealand-derived Suffolk ewes, were surgically transferred into recipient ewes that were also of New Zealand origin. Seventeen negative control lambs were born on the study premises from these embryo transfers. Thirty-nine experimental lambs were from embryos collected from naturally infected donor ewes. The experimental lambs were also born on the study premises after their surgical transfer into recipient ewes of New Zealand origin. These embryos had been collected from donor ewes in a scrapie-infected flock where the ewes were clinically sick with scrapie or developed clinical scrapie after embryo collection. All lambs were confirmed as scrapie susceptible of the ARQ/ARQ genotype. Twenty-eight experimental animals survived to the end point of the study at 5 yr of age with a mean survival of 1579 d. In the negative control group, 12 of 17 sheep survived to 5 yr of age with a mean survival of 1508 d. Postmortem examinations were carried out on all animals derived by embryo transfer, and in none was histologic or immunohistochemical evidence of scrapie found. In contrast, in the originating flock the majority of scrapie cases occurred in ARQ/ARQ genotyped animals where a 56% mortality from scrapie had been recorded in animals of this genotype. Thus, the study provides no evidence for transmission of scrapie and reinforces published evidence that vertical transmission of scrapie may be circumvented by embryo transfer procedures.

Exploiting strain diversity to expose transmission heterogeneities and predict the impact of targeting supershedding.

When a few individuals generate disproportionately many secondary cases, targeted interventions can theoretically lead to highly efficient control of the spread of infection. Practical exploitation of heterogeneous transmission requires the sources of variability to be quantified, yet it is unusual to have empirical data of sufficient resolution to distinguish their effects. Here, we exploit extensive data on pathogen shedding densities and the distribution of cases, collected from the same population within the same spatio-temporal window, to expose the comparative epidemiology of independent Escherichia coli O157 strains. For this zoonotic pathogen, which exhibits high-density shedding (supershedding) and heterogeneous transmission in its cattle reservoir, whether targeting supershedding could be an effective control depends critically on the proposed link between shedding density and transmissibility. We substantiate this link by showing that our supershedder strain has nearly triple the R(0) of our non-supershedder strain. We show that observed transmission heterogeneities are strongly driven by superspreading in addition to supershedding, but that for the supershedder strain, the dominant strain in our study population, there remains sufficient heterogeneity in contribution to R(0) from different shedding densities to allow exploitation for control. However, in the presence of substantial within-host variability, our results indicate that rather than seek out supershedders themselves, the most effective controls would directly target the phenomenon of pathogen supershedding with the aim of interrupting or preventing high shedding densities. In this system, multiple sources of heterogeneity have masked the role of shedding densities-our potential targets for control. This analysis demonstrates the critical importance of disentangling the effects of multiple sources of heterogeneity when designing targeted interventions.

Evaluation of a survey approach to estimating the prevalence of cattle carrying antimicrobial-resistant Escherichia coli.

A random survey of farms in the Highlands and Islands of Scotland provides estimated of the prevalence of calves, finishers and cows carrying ampicillin, apramycin and/or nalidixic acid resistant Escherichia coli. While the survey provides information on the geographical variation in risk, the results are of limited value for interpreting causality.

Cefixime-tellurite rhamnose MacConkey agar for isolation of Vero cytotoxin-producing Escherichia coli serogroup O26 from Scottish cattle and sheep faeces.

AIMS: To compare rhamnose MacConkey agar supplemented with cefixime and tellurite (CT-RMac) and tryptone bile X-glucuronide (TBX) agars as isolation media for Vero cytotoxin-producing Escherichia coli (VTEC) serogroup O26 from animal faeces. METHODS AND RESULTS: Nine VTEC O26 were isolated from sheep faeces; out of which six were isolated only on CT-RMac and one was isolated only on TBX. One hundred and twelve VTEC O26 were isolated from calf faeces; out of which 97% were from CT-RMac and 52% were from TBX. In a study of E. coli O26 strains, 84% of VT-positive O26 did not ferment rhamnose when compared with 16% of VT-negative O26. VT-positive (19%) and VT-negative (39%) E. coli O26 strains did not grow on CT-RMac agar. CONCLUSIONS: It is important to consider that VTEC O26 strains either may ferment rhamnose or may be sensitive to the CT supplement of CT-RMac agar. SIGNIFICANCE AND IMPACT OF THE STUDY: This work compares CT-RMac and TBX agars as isolation medium for VTEC O26 from Scottish animal faeces and highlights that VTEC O26 may be missed if only CT-RMac agar is used.

Modelling the epidemiology and transmission of Verocytotoxin-producing Escherichia coli serogroups O26 and O103 in two different calf cohorts.

Mathematical models are constructed to investigate the population dynamics of Verocytotoxin-producing Escherichia coli (VTEC) serogroups O26 and O103 in two different calf cohorts. We compare the epidemiological characteristics of these two serogroups within the same calf cohort as well as the same serogroups between the two calf cohorts. The sources of infection are quantified for both calf cohort studies. VTEC serogroups O26 and O103 mainly differ in the rate at which calves acquire infection from sources other than infected calves, while infected calves typically remain infectious for less than 1 week regardless of the serogroups. Fewer than 20% of VTEC-positive samples are the result of calf-to-calf transmission. PFGE typing data are available for VTEC-positive samples to further subdivide the serogroup data in one of the two calf cohort studies. For serogroup O26 but not O103, there is evidence for unequal environmental exposure to infection with different PFGE types.

Sensitivity of an immunomagnetic-separation-based test for detecting Escherichia coli O26 in bovine feces.

The sensitivity of a test for cattle shedding Escherichia coli serogroup O26 was estimated using several fecal pats artificially inoculated at a range of concentrations with different E. coli O26 strains. The test involves the enrichment of fecal microflora in buffered peptone water, the selective concentration of E. coli O26 using antibody-coated immunomagnetic-separation beads, the identification of E. coli colonies on Chromocult tryptone bile X-glucuronide agar, and confirmation of the serogroup with E. coli serogroup O26-specific antisera using slide agglutination. The effective dose of E. coli O26 for an 80% test sensitivity (ED(80)) was 1.0 x 10(4) CFU g(-1) feces (95% confidence interval, 4.7 x 10(3) to 2.4 x 10(4)). Differences in test sensitivity between different E. coli O26 strains and fecal pats were also observed. Individual estimates of ED(80) for each strain and fecal pat combination ranged from 4.2 x 10(2) to 4.8 x 10(5) CFU g(-1). These results suggest that the test is useful for identifying individuals shedding a large number of E. coli O26 organisms or, if an appropriate number of individuals in a herd are sampled, for identifying affected herds. The study also provides a benchmark estimate of sensitivity that can be used to compare alternative tests for E. coli O26 and a methodological approach that can be applied to tests for other pathogenic members of the Enterobacteriaceae and other sample types.

Heterogeneous shedding of Escherichia coli O157 in cattle and its implications for control.

Identification of the relative importance of within- and between-host variability in infectiousness and the impact of these heterogeneities on the transmission dynamics of infectious agents can enable efficient targeting of control measures. Cattle, a major reservoir host for the zoonotic pathogen Escherichia coli O157, are known to exhibit a high degree of heterogeneity in bacterial shedding densities. By relating bacterial count to infectiousness and fitting dynamic epidemiological models to prevalence data from a cross-sectional survey of cattle farms in Scotland, we identify a robust pattern: approximately 80% of the transmission arises from the 20% most infectious individuals. We examine potential control options under a range of assumptions about within- and between-host variability in infection dynamics. Our results show that the within-herd basic reproduction ratio, R(0), could be reduced to <1 with targeted measures aimed at preventing infection in the 5% of individuals with the highest overall infectiousness. Alternatively, interventions such as vaccination or the use of probiotics that aim to reduce bacterial carriage could produce dramatic reductions in R(0) by preventing carriage at concentrations corresponding to the top few percent of the observed range of counts. We conclude that a greater understanding of the cause of the heterogeneity in bacterial carriage could lead to highly efficient control measures to reduce the prevalence of E. coli O157.

Prevalence and virulence factors of Escherichia coli serogroups O26, O103, O111, and O145 shed by cattle in Scotland.

A national survey was conducted to determine the prevalence of Escherichia coli O26, O103, O111, and O145 in feces of Scottish cattle. In total, 6,086 fecal pats from 338 farms were tested. The weighted mean percentages of farms on which shedding was detected were 23% for E. coli O26, 22% for E. coli O103, and 10% for E. coli O145. The weighted mean prevalences in fecal pats were 4.6% for E. coli O26, 2.7% for E. coli O103, and 0.7% for E. coli O145. No E. coli O111 was detected. Farms with cattle shedding E. coli serogroup O26, O103, or O145 were widely dispersed across Scotland and were identified most often in summer and autumn. However, on individual farms, fecal shedding of E. coli O26, O103, or O145 was frequently undetectable or the numbers of pats testing positive were small. For serogroup O26 or O103 there was clustering of positive pats within management groups, and the presence of an animal shedding one of these serogroups was a positive predictor for shedding by others, suggesting local transmission of infection. Carriage of vtx was rare in E. coli O103 and O145 isolates, but 49.0% of E. coli O26 isolates possessed vtx, invariably vtx1 alone or vtx1 and vtx2 together. The carriage of eae and ehxA genes was highly associated in all three serogroups. Among E. coli serogroup O26 isolates, 28.9% carried vtx, eae, and ehxA-a profile consistent with E. coli O26 strains known to cause human disease.

Modelling the epidemiology of Verocytotoxin-producing Escherichia coli serogroups in young calves.

We investigate the epidemiology of 12 Verocytotoxin-producing Escherichia coli (VTEC) serogroups observed in a calf cohort on a Scottish beef farm. Fitting mathematical models to the observed time-course of infections reveals that there is significant calf-to-calf transmission of VTEC. Our models suggest that 40% of all detected infections are from calf-to-calf transmission and 60% from other sources. Variation in the rates at which infected animals recover from infection by different VTEC serogroups appears to be important. Two thirds of the observed VTEC serogroups are lost from infected calves within 1 day of infection, while the rest persist for more than 3 days. Our study has demonstrated that VTEC are transmissible between calves and are typically lost from infected animals in less than 1 week. We suggest that future field studies may wish to adopt a tighter sampling frame in order to detect all circulating VTEC serogroups in similar animal populations.

Phenotypic and functional characterisation of follicle-associated epithelium of rectal lymphoid tissue.

Lymphoid follicles cluster in the terminal rectum of various animal species and of man and hence this site may be important in the development of immune responses to pathogens. For the induction of immune responses at mucosal sites, interplay is required between various cell types performing functions ranging from antigen-sampling cells via antigen-presenting cells to antigen-specific lymphocytes. Therefore, we have characterised the cell populations and relevant functioning of follicle-associated epithelium (FAE) and associated follicles in the terminal portion of rectum in cattle as a representative mammal. Immunohistochemical studies of this region identified immune cell subsets (CD4+, CD8+, WC 1+gammadelta, CD2+, CD 21+ and CD 40+ cells) characteristic of an immune-inductive site. Examination of FAE identified a subset of cells with structural and functional features of antigen-sampling M-cells. Cells of the FAE and adjacent follicle-associated crypts expressed vimentin and a subset of these cells internalised microparticles, a further attribute of M-cells. The FAE cells were phenotypically heterogeneous and therefore the function and phenotype of these cell subsets requires further characterisation, particularly with respect to their potentially important role in the interaction of hosts with pathogens and the development of immune responses.

Distribution of Escherichia coli O157 in bovine fecal pats and its impact on estimates of the prevalence of fecal shedding.

The distribution of Escherichia coli O157 in bovine feces was examined by testing multiple samples from fecal pats and determining the density of E. coli O157 in immunomagnetic separation (IMS)-positive fecal samples. The density of E. coli O157 in bovine feces was highly variable, differing by as much as 76,800 CFU g(-1) between samples from the same fecal pat. The density in most positive samples was <100 CFU g(-1), the limit of reliable detection by IMS. Testing only one 1-g sample of feces per pat with IMS may result in a sensitivity of detection as low as 20 to 50%. It is therefore probable that most surveys have greatly underestimated the prevalence of E. coli O157 shedding in cattle and the proportion of farms with shedding cattle. The sensitivity of the detection of E. coli O157 in bovine feces can be as much as doubled by testing two 1-g samples per pat rather than one 1-g sample.

Transfer of antibiotic resistance between commensal and pathogenic members of the Enterobacteriaceae under ileal conditions.

AIM: To determine the rate of antibiotic resistance transmission between commensal and pathogenic representatives of the Enterobacteriaceae. METHODS AND RESULTS: Through the use of a validated in vitro simulation of the porcine ileum, the transmission of antibiotic resistance was detected between commensal Escherichia coli, E. coli O157 and Salmonella spp. Countable transconjugant populations arose readily and, in one example, proved capable of indefinite persistence. CONCLUSIONS: Genetic material conferring antibiotic resistance is readily transmissible between members of the Enterobacteriaceae under ileal conditions. Recipient phenotype influences the persistence of multi-resistant transconjugants. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation that the conjugal transmission of antibiotic resistance is commonplace under ileal conditions impacts primarily on the risk of food contamination by multi-resistant bacteria. The establishment of a multi-resistant transconjugant population as a dominant member of the microflora maintains a genetic reservoir of antimicrobial resistance.

Influence of tetracycline exposure on tetracycline resistance and the carriage of tetracycline resistance genes within commensal Escherichia coli populations.

AIMS: To assess the influence of incremental tetracycline exposure on the genetic basis of tetracycline resistance within faecal Escherichia coli. METHODS AND RESULTS: Through the adoption of a novel combination of multiple breakpoint selection, phenotypic characterization and the application of a polymerase chain reaction based gene identification system it proved possible to monitor the influence of antibiotic exposure on resistance gene possession. Using tetracycline as a case study a clear hierarchy was revealed between tet genes, strongly influenced by host antimicrobial exposure history. CONCLUSIONS: The antimicrobial exposure regime under which an animal is produced affects both the identity and magnitude of resistance gene possession of a selected bacterial population within its enteric microflora. Among the ramifications associated with such resistance gene selection is the degree of resistance conferred and the carriage of linked resistance determinants. This selection is applied by exposure to antibiotic concentrations well below recognized minimum inhibitory tetracycline concentration breakpoints widely adopted to characterize bacterial 'susceptibility'. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the ability of minimal antibiotic exposure to select for the continued persistence of resistance genes within the enteric microflora. It is clearly demonstrated that different antimicrobial regimes select for different resistance genes, the implications of which are discussed.

The quantitative measurement of antimicrobial resistance in Escherichia coli at the meta-population level (meta-population analysis).

AIMS: To define a method that describes antimicrobial resistance of meta-populations of bacteria in both a quantitative and biologically meaningful way. METHODS AND RESULTS: Using spiral plating and colony counting technology we obtained, from animal faecal samples, the density of Escherichia coli that grow at different concentrations of antibiotic. A mathematical description of this dose-response curve fitted the data well. The parameters of this model have biological meaning and the model allowed subtle differences between meta-populations to be detected. CONCLUSIONS: This method, termed MPA (meta-population analysis), is practical and provides a useful quantitative description of antimicrobial resistance in a bacterial meta-population. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that resistance can be defined quantitatively. The method may be used in many epidemiological and clinical studies of antimicrobial resistance in animals and humans.

What is antibiotic resistance and how can we measure it?

Antibiotic resistance is being found with increasing frequency in both pathogenic and commensal bacteria of humans and animals. Quantifying resistance within and between bacterial and host populations presents scientists with complex challenges in terms of laboratory methodologies and sampling design. Here, we discuss, from an epidemiological perspective, how antibiotic resistance can be defined and measured and the limitations of current approaches.