Bird-livestock interactions associated with increased cattle fecal shedding of ciprofloxacin-resistant Escherichia coli within feedlots in the United States

This research study was conducted to determine if bird depredation in feedlots is associated with the prevalence of ciprofloxacin-resistant Escherichia coli in cattle and to determine if removal of invasive bird species could be an effective management strategy to help reduce ciprofloxacin-resistant E. coli in cattle within the United States. European starlings (Sturnus vulgaris) were collected from feedlots within multiple geographic regions within the United States and European starlings within all regions tested positive for ciprofloxacin-resistant E. coli, but prevalence differed by region. Total number of birds on feedlots were positively associated with increased cattle fecal shedding of ciprofloxacin-resistant E. coli. Targeted control of invasive European starlings reduced bird numbers on feedlots by 70.4%, but decreasing populations of European starlings was not associated with corresponding reductions in bovine fecal prevalence of ciprofloxacin-resistant E. coli. These data provide evidence for the role of wild bird depredation in feedlots contributing to fecal shedding of ciprofloxacin-resistant E. coli, but a single month of European starling control in feedlots was not sufficient to impact the fecal carriage of this organism in cattle.

www.nature.com/scientificreports www.nature.com/scientificreports/ The objective of this study was twofold: (1) assess the relationship between bird abundance and ciprofloxacin-resistant Escherichia coli within cattle feedlots in the United States; (2) determine the efficacy of targeted invasive species management (removal of European starlings; Sturnus vulgaris) as a potential pre-harvest intervention strategy to reduce cattle fecal shedding of ciprofloxacin-resistant E. coli. Specifically, we wanted to determine if higher total bird numbers were associated with increased cattle fecal shedding of ciprofloxacin-resistant E. coli and if the removal of invasive European starlings would reduce cattle fecal shedding of ciprofloxacin-resistant E. coli, relative to comparable reference facilities not subjected to starling control operations.

Methods
We conducted this study from December 4, 2012 through March 12, 2013 with the cooperation of 35 feedlots. Feedlots were located within 4 regions; Eastern Colorado (n = 8), Kansas (n = 8), Texas panhandle (n = 8), Southern Iowa/Northern Missouri (n = 11). Feedlots experiencing bird damage (large foraging flocks of birds) were identified with the help of local cattlemen's associations. Bird damaged feedlots were randomly selected from a pool of commercial facilities, within each region, that had reported severe bird damage the previous year. Comparable reference feedlots were selected within each geographical region. A total of 18 treated and 17 reference facilities were included in the analysis. All participating facilities group housed animals in pens and produced feeder cattle as their primary commodity. Dairies, calving, or non-cattle livestock facilities were not included in the study.
This experimental protocol was approved by the USDA/APHIS/Wildlife Services, National Wildlife Research Center prior to data collection (Study Director James C Carlson; NWRC Protocol number QA-1945). Starling control operations were conducted by biologists from the United States Department of Agriculture/APHIS/ Wildlife Services. Starling control was conducted following agency policy as stated in USDA/APHIS/WS Directive 2.505. All methods were carried out in accordance with relevant guidelines and regulations.
Wildlife Services biologists initially established starling feeding sites within the 18 treatment feedlots using a bait preferentially selected by European starlings. Once starlings were observed to be consistently feeding on pre-bait, biologists used a 2% solution of DRC-1339 (3-chloro-p-toluidine hydrochloride) to reduce the number of depredating starlings. Technical DRC-1339 powder was mixed with water to create a 2% solution. Starling feed was soaked in the 2% solution and screen dried. The bait was applied at a concentration of 1:10 treated to untreated starling feed particles. All DRC-1339 applications were implemented consistent with directions "Compound DRC-1339 Concentrate -Feedlots"; EPA registration 56228-10.
Each feedlot was sampled twice, once before and once after starling control operations. During each sampling period we collected European starlings (n = 30) and cattle feces (n = 50) from within feedlots. Within each feedlot up to 10 pens were selected. These same pens were sampled before and after starling control operations. Within each pen we collected a minimum of five cattle fecal samples per visit. If a feedlot had fewer than 10 pens the total number of samples was distributed, as evenly as possible, among the available pens. For example, one facility housed animals in 2 large pens. Within this feedlot we collected 25 fecal samples per pen per visit. Within some feedlots fewer than 30 starlings were collected if birds could not be found.
Collection of cattle fecal samples followed methods that have been described previously 12 . Cattle fecal samples were collected from the floor of animal pens and only freshly voided fecal pats were sampled. In other words, the sample was collected from a fecal pat only after an animal was observed defecating. This procedure allowed us to standardize environmental exposure time among fecal samples and estimate herd prevalence of ciprofloxacin-resistant E. coli without confining animals for collection of rectal samples. Ten gram samples were scraped from the top of the fecal pat with disposable plastic spoons and stored in sterile Whirl-Paks (Nasco, Fort Atkinson, WI). We only collected fecal samples if we could reasonably determine, by visual inspection, that the sample was fresh and free of external environmental contaminants. All fecal samples were stored in coolers until they were shipped to the laboratory. Estimates of number of birds in animal pens were collected at the same time as fecal sample collection.
Number of birds on feedlots were estimated using counts of bird numbers on each pen's floor, feed bunkers, water troughs and feed lanes in front of the sampled pen. Estimates from these four locations were summed to calculate the total number of birds within pens. We averaged the total number of birds within pens among all the sampled pens within a feedlot. This mean bird estimate was multiplied by the total number of pens within the facility to produce a facility level bird estimate.
All starlings were collected with shotguns and no birds were collected off feedlots. All starlings were collected from within the animal pens and pen lanes. Starling samples were collected opportunistically and only done when it was safe to discharge firearms in feedlots. All specimens were individually bagged in sterile Whirl-Paks and stored in coolers until shipping.
Within each facility, diagnostic samples (starlings and cattle fecal samples) were collected on the same day and samples were shipped priority overnight to testing laboratories in Iowa and Colorado. All samples were shipped, in insulated boxes packed with Ice-Brix (Polar Tech Industries, Genoa, IL), to laboratories for isolation of ciprofloxacin-resistant E. coli. Only samples received by the laboratories within 24 hours of the date of collection were screened for ciprofloxacin-resistant E. coli. European starlings were shipped to the United States Department of Agriculture, National Wildlife Research Center (NWRC) in Fort Collins, Colorado, USA. Cattle fecal samples were shipped to Ohio State University, Food Animal Health Research Program in Wooster, OH, USA.
All European starling dissections occurred at the NWRC and were conducted using published methods 13 . Starling lower gastrointestinal tracts (GI, duodenum to the cloaca) were removed and placed in a sterile Whirl-Paks. To reduce risk of cross-contamination, we saturated the starling carcass, scissors, scalpels, and lab stations with 70% ethanol before removal of each starling GI tract. Lab mats and gloves were replaced after processing each starling. The starling GI samples were macerated for 120 sec at 200 rpm using a Stomacher 80 www.nature.com/scientificreports www.nature.com/scientificreports/ Biomaster (Seward Laboratory Systems, Bohemia, NY) paddle blender. Fecal material from the macerated starling GI tracts was squeezed by hand to one corner of the bag and an aliquot was extracted using sterile cotton swabs, making sure to completely saturate the tip of the swab. Starling fecal material, on the saturated cotton tipped swab, was then used for direct plating onto selective media.
Starling GI and cattle fecal samples were inoculated onto MacConkey agar (HiMedia, Mumbai, India) supplemented with 1 µg/ml ciprofloxacin (Sigma-Aldrich, St. Louis, MO) using sterile cotton-tipped applicators and incubated at 37 °C for 18-24 hr. Colonies displaying typical E. coli morphology were transferred to 10 ml of tryptic soy broth (TSB) and incubated overnight at 37 °C for 18-24 hours. Species confirmation for starling GI samples was achieved using the API 20E system (bioMérieux, Marcy-l'Étoile, France). E. coli susceptibility to ciprofloxacin was confirmed using the disk diffusion method following Clinical and Laboratory Standards Institute protocols and guidelines (CLSI, 2008). Species confirmation for cattle fecal samples was conducted using lactose and indole tests. All lactose and indole positive isolates were cultured on MacConkey agar supplemented with 2 µg/ ml ciprofloxacin. Colonies growing on the agar were isolated and tested for both gyrA and parC chromosomal mutations by PCR using previously reported primers 14 . PCR products were bi-directionally Sanger sequenced and the resulting data were aligned to the corresponding reference gene sequences available in NCBI Genbank (gyrA gene ID: 946614; parC gene ID: 947499). The gyrA and parC sequences were screened for combinations of chromosomal mutations expected to confer fluoroquinolone resistance 15,16 and if they were detected the E. coli isolate was classified as ciprofloxacin-resistant.
We tested efficacy of DRC-1339 as a control tool for invasive birds on feedlots using a Poisson model of count data in PROC GLIMMIX in SAS version 9.2 (SAS Institute, Cary, NC). The response variable was the estimated number of birds on feedlots. Fixed effects included treatment status (starling controlled feedlot/reference feedlot), sampling period (before/after starling control) and the interaction between treatment status and sampling period. Feedlots nested within treatment status were included as a random effect.
Separate mixed effects logistic regression models were created to test the association between total bird number and ciprofloxacin-resistant E. coli fecal shedding by cattle and to test the efficacy of starling control as a pre-harvest intervention strategy to reduce ciprofloxacin-resistant E. coli fecal shedding by cattle. Models were constructed using PROC GLIMMIX in SAS version 9.2. Both models, were fitted using a binomial distribution and the response variable was the number of positive ciprofloxacin-resistant E. coli samples divided by the total number of samples collected per pen. Model parameters were estimated using the maximum likelihood method and degrees of freedom were estimated using the between within option. Within both models, feedlots nested within treatment status, pens nested within feedlots, and the sampling period by feedlot interaction were all included as random effects.
To test for an association between total bird numbers and cattle fecal shedding of ciprofloxacin-resistant E. coli, we included region and the estimated number of birds on feedlots as fixed effects. To test the efficacy of starling control as a pre-harvest intervention strategy to reduce cattle fecal shedding of ciprofloxacin-resistant E. coli, we included region, treatment status (starling controlled feedlot/reference feedlot), sampling period (before/after starling control operations), and the interaction between treatment status and sampling period as fixed effects.
Additional explanatory variables for ciprofloxacin-resistant E. coli in feedlots were assessed in univariable analyses using PROC GLIMMIX in SAS version 9.2. The model was fitted using a binomial distribution and the response variable was the number of positive ciprofloxacin-resistant E. coli samples divided by the total number of samples collected per pen. Model parameters were estimated using the maximum likelihood method and degrees of freedom were estimated using the between within option. Feedlots nested within treatment status, pens nested within feedlots, and the sampling period by feedlot interaction were all included as random effects.
The additional explanatory variables were assessed to identify any potential wild bird, facility management, or environmental variables that may potentially be associated with cattle fecal shedding of ciprofloxacin-resistant E. coli in feedlots. The explanatory variables assessed in the analyses were selected because they have been identified as or suspected of contributing to bacterial contamination in feedlots 13,[17][18][19][20] . The variables assessed in these analyses occurred at two spatial scales (feedlots and pens within feedlots). The variables include birds (birds in feed bunkers, birds on water troughs, total number of birds in pens), cattle stocking (herd size, number of cattle within pen), environmental factors (temperature, time, and sampling period), and feedlot management factors (water troughs split pens, recycled water used in water troughs, cattle days in pen, cattle days on finishing ration, entry weight, exit weight and weight gained by cattle).
Most variables assessed within the univariable analyses are intuitively obvious, but some variables may need additional clarification. For example, weight gain was calculated by subtracting the pen averaged entry weight from the pen averaged exit weight data. Water troughs accessed by multiple pens identifies split-pen watering troughs that allow cattle from adjoining pens to drink from the same trough. Recycled water identifies facilities that recirculate the water provided to cattle within troughs. Total number of birds per pen reflects the sum of the estimates of birds from water troughs, pen floor, feed bunkers and pen lanes for each sampled pen.
A total of 15 additional univariable models were analyzed (m = 15). Because multiple tests were being conducted, we decided to control for false discoveries using the Benjamini Hochberg procedure 21 . For all univariable analyses the false discovery rate was set at α = 0.05. Models were ranked by p-values from smallest (1) to largest (m). Cutoff values for rejection of null hypotheses were calculated as (rank/m)*α. Reported odd ratios and their 95% confidence intervals were not adjusted for multiple testing.

Results
Targeted control of invasive European starlings using DRC-1339 was effective at reducing bird numbers on feedlots. Total number of birds on treatment facilities relative to the reference facilities not subjected to control operations decreased following DRC-1339 control operations (F 1 , 33 = 95,598, P = < 0.0001). Bird count data suggests targeted starling control operations reduced bird numbers by 70.4% on feedlots following DRC-1339 applications (Fig. 1). www.nature.com/scientificreports www.nature.com/scientificreports/ A total of 1,477 European starling specimens were collected for laboratory analysis. A total of 10.2% of starling GI tracts tested positive for ciprofloxacin-resistant E. coli and the probability of detection within starling GI tract samples appears to differ by geographical region (Fig. 2).
Targeted control of invasive European starlings was not an effective pre-harvest intervention strategy to reduce cattle fecal shedding of ciprofloxacin-resistant E. coli (F 1 , 33 = 0.60, P = 0.4454, Table 2). Based on LS-Means estimates of ciprofloxacin-resistant cattle fecal samples there does not appear to be any reduction in cattle fecal shedding of ciprofloxacin-resistant E. coli within starling controlled feedlots relative to reference feedlots (Fig. 4).
The analysis of the 15 univariable models of potential explanatory variables for cattle fecal shedding of ciprofloxacin-resistant E. coli did not reveal any statistically significant associations after Benjamini Hochberg adjustments were made (Table 3). www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
Wildlife incursions into animal agricultural operations have long been suspected as sources for diseases of concern to veterinary and human health [22][23][24] . For example, indistinguishable AMR S. enterica isolates were recovered from starlings, cattle, and the feed and water sources they share 13,20    www.nature.com/scientificreports www.nature.com/scientificreports/ reduced susceptibility to cefotaxime and ciprofloxacin were spatially correlated to starling night roosts in Ohio 25 . Proximity of starling night roosts was also shown to be spatially correlated with increased E. coli O157:H7 cattle fecal shedding in dairies 26 . These data are important because they provide indirect evidence that bird-livestock interactions may contribute to rates of cattle fecal shedding of E. coli 0157:H7 as well as S. enterica and E. coli with reduced susceptibilities to multiple antibiotics, including ciprofloxacin and cefotaxime. The data we present in this manuscript are the first to provide direct evidence to support the hypothesis that large foraging flocks of birds can contribute to increased cattle fecal shedding of ciprofloxacin-resistant E. coli.
Starling control operations reduced bird numbers on our treatment feedlots by an average of 70.4%. Yet, the time between pre treatment and post treatment sampling did not result in any significant change in cattle fecal shedding of ciprofloxacin-resistant E. coli. One would intuitively assume that significant reductions in bird   www.nature.com/scientificreports www.nature.com/scientificreports/ numbers on feedlots should translate to cattle harboring fewer organisms with reduced susceptibilities to ciprofloxacin. It is unclear why we did not see a significant reduction in the amount of ciprofloxacin-resistant E. coli isolated from cattle fecal pats, while seeing a positive correlation between bird numbers and cattle fecal shedding of ciprofloxacin-resistant E. coli. We suspect the time between starling control operations and post-treatment sampling may not have been long enough for these management actions to produce meaningful results. If so, starling control may have to occur year round or the moment starlings arrive on feedlots in the fall for it to be effective at reducing the amplification and spread of AMR organisms in animal agricultural operations.
It is important to note that other studies have shown that bird control was not an effective pre-harvest intervention strategy for reducing cattle fecal shedding of bacteria of concern to public health. Starling numbers were one of the strongest predictors for S. enterica contamination of cattle feed and water supplies, but starling numbers were not shown to be a good predictor for herd level prevalence of S. enterica 27 . Controlling starlings was associated with reduced S. enterica loads within cattle feed and water supplies, but starling control was not effective at reducing cattle fecal shedding of S. enterica over the time period of the study. Additionally, starling control programs were not an effective intervention strategy to reduce the overall prevalence of Campylobacter jejuni within feedlot cattle despite starlings harboring diverse C. jejuni strains including hypervirulent clone SA 28 . The totality of this information is discouraging. Bird numbers and bird depredation in feedlots and dairies is associated with higher herd level prevalence for multiple zoonotic and AMR organisms, but temporarily or transiently reducing bird numbers, after they have become established in animal agricultural operations, does not translate to quick reductions of herd level prevalence of those same organisms. In other words, once AMR organisms have been introduced by starlings, they may persist within cattle herds for considerable periods of time.
After population control programs were completed, approximately 30% of the pretreatment birds remained on feedlots. It is conceivable that the microbiological impact of birds is not additive and that only a few birds, moving between feedlots and dairies, are necessary for maintenance and amplification of ciprofloxacin-resistant E. coli in concentrated animal feeding operations. Additional studies are needed to better assess interactions between birds, cattle and the occurrence of AMR E. coli. For example, there is very little information related to antibiotic usage in agriculture, wildlife interactions and selective pressure on the maintenance of ciprofloxacin-resistance E. coli in livestock. It is conceivable that wildlife are contributing to these problems in complex and unforeseen ways. To adequately address public and environmental health concerns created through wildlife-livestock interactions we need to understand the specific risks created by wildlife so we can develop targeted and cost effective management strategies.
Bird-livestock interactions in animal agricultural operations may create an ecologically important link for the spread of ciprofloxacin-resistant E. coli to human populations. Synanthropic birds, especially European starlings, use feedlots in winter for food resources. Starlings typically quit using feedlots in spring when insects become abundant 29 . During the spring and summer, starlings are commonly found breeding in suburban and urban environments 30,31 . The ecological interactions of starlings suggest they could potentially move ciprofloxacin-resistant E. coli and other AMR organisms to environments dominated by people; human-bird transfer of E. coli has been documented before 32 . Birds seem to act as transporters, or as reservoirs, of resistant bacteria and could therefore have an important epidemiological role in the dissemination of resistance 33 . Thus, because of the unique ecology of invasive starlings in North America, they are a high risk species for the environmental dissemination of AMR organisms to environments and locations of concern to people.

Data availability
All raw data are archived at the National Wildlife Research Center (Study Director James C Carlson; NWRC Protocol number QA-1945) and are publicly available. Names and addresses of cooperating feedlots have been redacted from the raw data. All facilities were referenced by an alpha-numeric code and names and addresses of cooperating facilities will not be provided upon request as per the cooperator agreement established prior to data collection.