Spatial Clustering of Escherichia coli with Reduced Susceptibility to Cefotaxime and Ciprofloxacin among Dairy Cattle Farms Relative to European Starling Night Roosts.

European starlings (Sturnus vulgaris) have been implicated in the dispersal of zoonotic enteric pathogens. However, their role in disseminating antimicrobial-resistant organisms through their home range has not been clearly established. The aim of this study was to determine whether starling night roosts served as foci for spreading organisms with reduced susceptibility to antimicrobials among dairy cattle farms. Bovine faecal pats were collected from 150 dairy farms in Ohio. Each farm was visited twice (in summer and fall) between 2007 and 2009. A total of 1490 samples (10 samples/farm over two visits) were tested for Escherichia coli with reduced susceptibility to cefotaxime and ciprofloxacin. Using a spatial scan statistic, focal scans were conducted to determine whether clusters of farms with a high prevalence of organisms with reduced susceptibility to cefotaxime and ciprofloxacin surrounded starling night roosts. Faecal pats 13.42% and 13.56% of samples carried Escherichia coli with reduced susceptibility to cefotaxime and ciprofloxacin, respectively. Statistically significant (P < 0.05) spatial clusters of faecal pats with high prevalence of Escherichia coli showing reduced susceptibility to cefotaxime and ciprofloxacin were identified around these night roosts. This finding suggests that the risk of carriage of organisms with reduced susceptibility to antimicrobials in cattle closer to starling night roosts was higher compared to cattle located on farms further from these sites. Starlings might have an important role in spreading antimicrobial-resistant E. coli to livestock environments, thus posing a threat to animal and public health.

A longitudinal study of feed contamination by European starling excreta in Ohio dairy farms (2007-2008).

The objectives of this study were to understand the temporal pattern of contamination of cattle feed by starling excrement on dairy farms and to evaluate the temporal pattern in recovering Escherichia coli O157:H7 or Salmonella in relation to the absolute mass of excrement recovered. A longitudinal study was conducted on 15 dairy farms in Ohio from July 2007 to October 2008. One open-topped tray filled with bird feed was placed near a cattle feeding site; bird excrement from the tray was weighed monthly for 12 consecutive months. Linear regression models with a random intercept for farm were computed to examine the association between the absolute weight of excrement recovered each month or the farm-specific standard score for weight of excrement, and month or season. Exact logistic regression was used to determine whether an association between recovering E. coli O157:H7 or Salmonella was present and the amount of excrement recovered and season. A spatial scan statistic was used to test for evidence of space-time clustering of excrement, based on the standard score for the weight of the excrement, among our study farms. A total of 5 of 179 excrement samples (2.79%) were positive for E. coli O157:H7 and 2 (1.12%) were positive for Salmonella. A significantly higher level of contamination with excrement was observed during the winter. The odds of recovering a pathogen increased with the amount of excrement recovered and decreased if the excrement was collected in the winter. A spatio-temporal cluster of contamination with excrement was detected. These findings provide basic information for future quantitative microbial risk assessments concerning the role of starlings in spreading enteric pathogens on dairy farms.

Spatial epidemiology of Escherichia coli O157:H7 in dairy cattle in relation to night roosts Of Sturnus vulgaris (European Starling) in Ohio, USA (2007-2009).

The goal of our study was to use spatial scan statics to determine whether the night roosts of European starlings (Sturnus vulgaris) act as point sources for the dissemination of Escherichia coli O157:H7 among dairy farms. From 2007 to 2009, we collected bovine faecal samples (n = 9000) and starling gastrointestinal contents (n = 430) from 150 dairy farms in northeastern Ohio, USA. Isolates of E. coli O157:H7 recovered from these samples were subtyped using multilocus variable-number tandem repeat analysis (MLVA). Generated MLVA types were used to construct a dendrogram based on a categorical multistate coefficient and unweighted pair-group method with arithmetic mean (UPGMA). Using a focused spatial scan statistic, we identified statistically significant spatial clusters among dairy farms surrounding starling night roosts, with an increased prevalence of E. coli O157:H7-positive bovine faecal pats, increased diversity of distinguishable MLVA types and a greater number of isolates with MLVA types from bovine-starling clades versus bovine-only clades. Thus, our findings are compatible with the hypothesis that starlings have a role in the dissemination of E. coli O157:H7 among dairy farms, and further research into starling management is warranted.

Use of dairies by postreproductive flocks of European starlings.

Knowledge of the behavior and movement patterns of European starlings (Sturnus vulgaris L.) is important to wildlife managers that seek to resolve conflicts at livestock facilities. We captured and radio tagged 10 starlings at each of 5 dairies in northeastern Ohio. From September 19 to October 31, 2007, we obtained sufficient data from 40 birds to study their behavior and movements. The birds visited the dairies where they were initially captured (home sites) on 85% of the days, spending 58% of each day at the dairies. Onsite arrival and departure times were 2.5h after sunrise and 3.1h before sunset. Daily visits by radio-tagged cohorts from the other dairies were greatest for the 2 most proximate dairies (1.3 km apart), with number of visits between this pairing >7× that of the 9 other pairings combined (4.1-6.5 km apart). Two birds used their home sites intermittently as roosts, arriving 3.8h before sunset and departing 0.2h after sunrise. In addition to using home-site roosts, these birds also used a distant roost (22km) that was used by 36 of the 40 birds. The efficacy of starling management programs, especially lethal management, depends on degree of site fidelity, use of other facilities, and roosting behavior. For example, starlings that use dairies as roosting sites may require a different management strategy than required at dairies used as daytime sites because of differences in arrival and departure behavior. Our research will help resource managers evaluate current management strategies already in place and change them, if needed, to fit the behavior profile of starlings using dairies and other types of livestock facilities.

Association of wild bird density and farm management factors with the prevalence of E. coli O157 in dairy herds in Ohio (2007-2009).

Our objective was to determine the role that European starlings (Sturnus vulgaris) play in the epidemiology of Escherichia coli O157:H7 in dairy cattle. We visited 150 dairy farms in Ohio twice during summer and fall months from 2007 to 2009. Fresh faecal pats from 30 lactating cows were collected during each visit. Information on farm management and environmental variables was gathered through a questionnaire administered to the farm owner. The number of starlings observed on the farm was also recorded. Approximately 1% of dairy cattle and 24% of farms were positive for E. coli O157. Risk factors associated with the presence of E. coli O157 in faecal pats included contact between adult cattle and calves, types or number of ventilation and manure management systems and number of birds per milking cow.

Multiple-locus variable-nucleotide tandem repeat subtype analysis implicates European starlings as biological vectors for Escherichia coli O157:H7 in Ohio, USA.

AIMS: To provide molecular epidemiological evidence of avian transmission of Escherichia coli O157:H7 between dairy farms in Ohio, this study was designed to identify genetic relatedness between isolates originating from bovine faecal samples and intestinal contents of European starlings captured on these farms. METHODS AND RESULTS: During a three-year period (2007-2009), cattle (n = 9000) and starlings (n = 430) on 150 different dairy farms in northern Ohio were sampled for the presence of E. coli O157:H7. Isolates were subjected to multiple-locus variable-nucleotide tandem repeat analysis (MLVA). Distinct allelic groups were identified on most farms; however, isolates clustering into three MLVA groups originated from both cattle and birds on different farms. CONCLUSIONS: Sharing of indistinguishable epidemiologically linked E. coli O157 MLVA subtypes between starlings and cattle on different farms supports the hypothesis that these birds contribute to the transmission of E. coli O157:H7 between dairy farms. SIGNIFICANCE AND IMPACT OF STUDY: A continued need exists to identify and to improve preharvest measures for controlling E. coli O157:H7. Controlling wildlife intrusion, particularly European starlings, on livestock operations, may be an important strategy for reducing dissemination of E. coli O157:H7 between farms and thereby potentially decreasing the on-farm prevalence of E. coli O157:H7 and enhancing the safety of the food supply.

Risk factors associated with Escherichia coli O157:H7 in Ontario beef cow-calf operations.

The aim of this study was to identify farm management factors associated with the prevalence of Escherichia coli O157:H7 among cattle in Ontario beef cow-calf operations. A total of 119 cow-calf operations with more than 50 cows in southern Ontario were visited between June and December 2002. From each farm, 65 fresh fecal samples were collected and cultured for E. coli O157:H7. Colonies of E. coli O157:H7 were isolated using immunomagnetic separation and standard microbiological techniques. Final confirmation of suspected colonies was based on identifying E. coli O157:H7-specific genes by PCR and serotyping of representative isolates. A questionnaire was administered to collect information on farm size, cattle demographics, farm management practices, the presence of other livestock and wildlife, and other aspects of the farm environment. Associations between the prevalence of E. coli O157:H7 in cattle feces and management factors were determined using a multivariable logistic regression model that included random effects for farm and county. The presence of pigs on farm, use of corn silage supplementation in winter, number of times cattle were taken to a show in the previous 12 months and the percentage of cows on farm were significant risk factors for the presence of E. coli O157:H7 in fecal pat samples, after controlling for region and the age group of the sampled animals. These findings highlight the potential roles of biosecurity and avoiding mixed animal agriculture in controlling the prevalence of E. coli O157:H7 in beef cow-calf operations.

Diversity of staphylococcal cassette chromosome in coagulase-negative staphylococci from animal sources.

AIMS: To type the staphylococcal cassette chromosome (SCC) in coagulase-negative staphylococci (CoNS) from animal sources. METHODS AND RESULTS: A total of 92 CoNS isolates recovered from farm animals was analysed. The top three staphylococcal species were Staphylococcus lentus (34), S. sciuri (31), and S. xylosus (13). The presence of the cassette chromosome recombinase (ccr) genes ccrA1, ccrB1, ccrA2, ccrB2, ccrA3, ccrB3 and ccrC, the mec regulatory genes mecI and mecR1, and Tn554 was used to differentiate the SCC. A total of 60 of the 92 isolates were methicillin resistant. Among the 60 methicillin-resistant Staphylococcus spp. isolates, SCCmec (mecA-carrying SCC) types I, III, IV and V were identified in 24 isolates based on the combinations of the ccr genes and the mec regulatory genes, with type III being predominant. The single S. epidermidis carried SCCmec type IV. SCC type III was also identified in two of 32 methicillin-susceptible isolates. Identical SCCmec types were present in different species of CoNS. Pulsed-field gel electrophoresis (PFGE) generated 64 patterns out of 81 PFGE typeable isolates. Indistinguishable clones were detected in animals from different farms. CONCLUSIONS: Heterogeneous SCC existed in CoNS of diverse genetic background. Both clonal transmission of methicillin-resistant CoNS and horizontal transfer of SCCmec occurred in the animal production environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study adds to our knowledge of SCCmec type and the diversity of SCC in CoNS.

Bovine veterinarians' knowledge, beliefs, and practices regarding antibiotic resistance on Ohio dairy farms.

The perceptions, knowledge, and gaps in the knowledge of Ohio bovine veterinarians about antibiotic resistance (ABR) issues were systematically determined. In-depth interviews of 10 bovine practitioners in Ohio were completed to inform the development of a survey that was subsequently distributed to all Ohio members of the American Association of Bovine Practitioners and the bovine-specialized members of the Ohio Veterinary Medical Association (n = 174). The 43 respondents (26% response rate) included practitioners with a cross-section of clinical experience and sizes of dairy herds served. Respondents' knowledge regarding selection pressures that cause bacteria to acquire resistance was positively correlated with their knowledge of transmission routes of ABR organisms to dairy cows (r = 0.60) and potential consequences of ABR with respect to animal health (r = 0.52). Veterinarians believed that antibiotics are used by producers to treat a variety of illnesses and that specific antibiotics (e.g., gentamicin) are used without veterinary consultation. More than 75% of the veterinarians thought that one-on-one meetings and handouts containing good management practices, diagnosis descriptions, and appropriate dosages for antibiotics would be effective ways to educate their clients about ABR. However, only 23% of veterinarians reported that they consistently provide treatment protocols for antibiotic use. By first assessing the information needs of the audience, these results indicated that, rather than expending resources to develop educational materials directed at improving bovine practitioner knowledge of the subject, communication and outreach efforts that encourage and facilitate information flow from veterinarians to dairy producers may be more effective tools to affect prudent antibiotic use on dairy farms.

Isolation of Escherichia coli O157:H7 strains that do not produce Shiga toxin from bovine, avian and environmental sources.

AIMS: To determine the potential for naturally occurring Shiga toxin-negative Escherichia coli O157 to acquire stx(2) genes. METHODS AND RESULTS: Multiple E. coli O157:H7 isolates positive for eae and ehxA, but not for stx genes, were isolated from cattle, water trough sediment, animal bedding and wild bird sources on several Ohio dairy farms. These isolates were experimentally lysogenized by stx(2)-converting bacteriophage. CONCLUSIONS: Shiga toxin-negative strains of E. coli O157 are present in multiple animal and environmental sources. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-negative strains of E. coli O157 present in the food production environment are able to acquire the stx genes, demonstrating their potential to emerge as new Shiga toxin-producing E. coli strains.

Preharvest control of Escherichia coli O157 in cattle.

Bovine manure is an important source of Escherichia coli O157 contamination of the environment and foods; therefore, effective interventions targeted at reducing the prevalence and magnitude of fecal E. coli O157 excretion by live cattle (preharvest) are desirable. Preharvest intervention methods can be grouped into 3 categories: 1) exposure reduction strategies, 2) exclusion strategies, and 3) direct antipathogen strategies. Exposure reduction involves environmental management targeted at reducing bovine exposure to E. coli O157 through biosecurity and environmental niche management such as feed and drinking water hygiene, reduced exposure to insects or wildlife, and improved cleanliness of the bedding or pen floor. In the category of exclusion, we group vaccination and dietary modifications such as selection of specific feed components; feeding of prebiotics, probiotics, or both; and supplementation with competitive exclusion cultures to limit proliferation of E. coli O157 in or on exposed animals. Direct antipathogen strategies include treatment with sodium chlorate, antibiotics, bacteriophages, in addition to washing of animals before slaughter. Presently, only 1 preharvest control for E. coli O157 in cattle has been effective and has gained widespread adoption-the feeding probiotic Lactobacillus acidophilus. More research into the effectiveness of parallel and simultaneous application of 1 or more preharvest control strategies, as well as the identification of new pre-harvest control methods, may provide practical means to substantially reduce the incidence of human E. coli O157-related illness by intervening at the farm level.

Longitudinal study of fecal shedding of Escherichia coli O157:H7 in feedlot cattle: predominance and persistence of specific clonal types despite massive cattle population turnover.

Identification of the sources and methods of transmission of Escherichia coli O157:H7 in feedlot cattle may facilitate the development of on-farm control measures for this important food-borne pathogen. The prevalence of E. coli O157:H7 in fecal samples of commercial feedlot cattle in 20 feedlot pens between April and September 2000 was determined throughout the finishing feeding period prior to slaughter. Using immunomagnetic separation, E. coli O157:H7 was isolated from 636 of 4,790 (13%) fecal samples in this study, with highest prevalence earliest in the feeding period. No differences were observed in the fecal or water trough sediment prevalence values of E. coli O157:H7 in 10 pens supplied with chlorinated drinking water supplies compared with nonchlorinated water pens. Pulsed-field gel electrophoresis of XbaI-digested bacterial DNA of the 230 isolates obtained from eight of the pens revealed 56 unique restriction endonuclease digestion patterns (REDPs), although nearly 60% of the isolates belonged to a group of four closely related genetic subtypes that were present in each of the pens and throughout the sampling period. The other REDPs were typically transiently detected, often in single pens and on single sample dates, and in many cases were also closely related to the four predominant REDPs. The persistence and predominance of a few REDPs observed over the entire feeding period on this livestock operation highlight the importance of the farm environment, and not necessarily the incoming cattle, as a potential source or reservoir of E. coli O157:H7 on farms.

Livestock drinking water microbiology and the factors influencing the quality of drinking water offered to cattle.

The microbial quality of livestock drinking water was evaluated in 473 cattle water troughs located at 99 different cattle operations. The mean log10-transformed coliform and Escherichia coli concentrations per milliliter of trough water were 1.76 +/- 1.25 (SD) and 0.98 +/- 1.06 (SD), respectively. The degree of E. coli contamination was positively associated with the proximity of the water trough to the feedbunk, protection of the trough from direct sunlight, lower concentrations of protozoa in the water, and warmer weather. Salmonella sp. were isolated from 2/235 (0.8%) troughs and shigatoxigenic-E. coli O157 was recovered from 6/473 (1.3%) troughs. Four experimental microcosms simulating cattle water troughs were used to further evaluate the effects of protozoal populations on the survival of E. coli O157 in cattle water troughs. Escherichia coli O157 of bovine fecal origin proliferated in all microcosms. Reduction of protozoal populations by treatment with cycloheximide was associated with increased persistence of E. coli O157 concentrations in the microcosms. Water troughs are a major source of exposure of cattle to enteric bacteria, including a number of foodborne pathogens, and this degree of bacterial contamination appeared to be associated with potentially controllable factors.

Cattle water troughs as reservoirs of Escherichia coli O157.

Environmental survival of Escherichia coli O157 may play an important role in the persistence and dissemination of this organism on farms. The survival of culturable and infectious E. coli O157 was studied using microcosms simulating cattle water troughs. Culturable E. coli O157 survived for at least 245 days in the microcosm sediments. Furthermore, E. coli O157 strains surviving more than 6 months in contaminated microcosms were infectious to a group of 10-week-old calves. Fecal excretion of E. coli O157 by these calves persisted for 87 days after challenge. Water trough sediments contaminated with feces from cattle excreting E. coli O157 may serve as a long-term reservoir of this organism on farms and a source of infection for cattle.

Methods for the isolation of water-borne Escherichia coli O157.

AIMS: To develop improved methods for the detection of Escherichia coli O157 from water and sediments. METHODS AND RESULTS: The effects of different broth enrichment media (unsupplemented tryptic soya broth, tryptic soya broth with antibiotics, and gram-negative broth), incubation durations (5 and 24 hrs), incubation temperatures (37 and 44.5 degrees C) and the use of immunomagnetic separation (IMS) on the sensitivity of E. coli O157 detection were evaluated on artificially and naturally-contaminated water and sediment samples. The sensitivity of recovery of E. coli O157 from samples was dependent upon the media composition, temperature duration of incubation and the use of IMS. CONCLUSION: Use of high temperature (44.5 degrees C) incubation for 24 hrs in unsupplemented tryptic soya broth and the use of IMS improved the sensitivity of E. coli O157 culture from water and sediment samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods described can be used to increase the sensitivity of E. coli O157 detection from water and sediments.

Polymerase chain reaction for definitive identification of Yersinia ruckeri.

Yersinia ruckeri causes enteric red mouth (ERM) disease in salmonids. Serologic identification of Y. ruckeri is hampered by cross-reactivity with other bacterial isolates of fish origin. Oligonucleotide primers incorporating Y. ruckeri unique sequences were designed to amplify a 409 bp fragment of Y. ruckeri 16S rDNA. The primers did not amplify other genetically related Yersinia or a wide variety of other aquatic or piscine bacteria. This assay provides a rapid, definitive identification of Y. ruckeri that is not subject to the variability inherent in serologic methods.