Risk factors for Escherichia coli O157 on beef cattle ranches located near a major produce production region.

Our goal was to identify climate variables and management practices associated with the presence of E. coli O157 in rangeland cow-calf operations located in a major leafy green production region in the California Central Coast. E. coli O157 was present in 2·6% (68/2654) of faecal, 1·5% (3/204) of water and 1·1% (1/93) of sediment samples collected on eight ranches over 2.5 years. Five (62·5%) ranches were positive at least once during the study. The odds of detecting E. coli O157 in faecal samples was higher during periods of higher maximum soil temperature, higher maximum relative humidity, and larger herd sizes, but decreased as wind speed increased. Molecular subtyping of isolates from cattle faeces and streams/sediments suggested minimal movement of strains between ranches. The findings suggest that E. coli O157 prevalence is relatively low on cow-calf ranches in this region, spatially constrained, but may vary by weather conditions and herd size.

Risk factors for elevated Enterococcus concentrations in a rural tropical island watershed.

Associations were examined between riparian canopy cover, presence of cattle near streams, and month of year with the concentration of Enterococcus (Most Probable Number (MPN)/100 ml) in surface water at Waipa watershed on the North Side of the Hawaiian island Kaua'i. Each one percent decrease in riparian canopy cover was associated with a 3.6 MPN/100 ml increase of waterborne Enterococcus. Presence of cattle near monitoring sites was associated with an increase of 99.3 MPN/100 ml of Enterococcus in individual grab samples. Lastly, summer samples (July) were substantially higher in concentration of Enterococcus than winter collected samples (February) in Enterococcus in sampled streams. These results suggest that reducing canopy cover and introduction of cattle into riparian zones may contribute to increases of Enterococcus concentrations in stream water.

Reducing microbial contamination in storm runoff from high use areas on California coastal dairies.

High use areas are a fundamental part of California coastal dairies and grazing livestock ranches as feeding areas, nurseries, and sick pens. High stocking densities and daily use in these areas lead to soil surfaces devoid of vegetation and covered in manure, with high potential for manure transport during winter rains to receiving waters regulated for shellfish harvesting and recreation. We characterized the association between California's Mediterranean climate and a series of existing and proposed management practices on fecal coliform bacteria (FCB) transport from high use areas on dairies and ranches. Results from 351 storm runoff samples collected below 35 high-use areas indicate that removal of cattle during winter, locating high use areas on level ground, application of straw and seeding, and vegetative buffer strip implementation were significantly associated with FCB concentration and load reductions. These results complement our findings for reductions of specific pathogens in runoff from these areas. These findings have practical significance because they document surface water quality benefits that the studied management practices provide in application on working farms and ranches. This direction is critical and timely for on-farm management efforts seeking to reduce microbial pollution in runoff and comply with indicator bacteria water quality criteria.

Efficacy of natural wetlands to retain nutrient, sediment and microbial pollutants.

Wetlands can improve water quality through natural processes including sedimentation, nutrient transformations, and microbial and plant uptake. Tailwater from irrigated pastures may contribute to nonpoint source water pollution in the form of sediments, nutrients, and pathogens that degrade downstream water quality. We examined benefits to water quality provided by a natural, flow-through wetland and a degraded, channelized wetland situated within the flood-irrigation agricultural landscape of the Sierra Nevada foothills of Northern California. The non-degraded, reference wetland significantly improved water quality by reducing loads of total suspended sediments, nitrate, and Escherichia coli on average by 77, 60, and 68%, respectively. Retention of total N, total P, and soluble reactive P (SRP) was between 35 and 42% of loads entering the reference wetland. Retention of pollutant loads by the channelized wetland was significantly lower than by the reference wetland for all pollutants except SRP. A net export of sediment and nitrate was observed from the channelized wetland. Decreased irrigation inflow rates significantly improved retention efficiencies for nitrate, E. coli, and sediments in the reference wetland. We suggest that maintenance of these natural wetlands and regulation of inflow rates can be important aspects of a best management plan to improve water quality as water runs off of irrigated pastures.

Farm factors associated with reducing Cryptosporidium loading in storm runoff from dairies.

A systems approach was used to evaluate environmental loading of Cryptosporidium oocysts on five coastal dairies in California. One aspect of the study was to determine Cryptosporidium oocyst concentrations and loads for 350 storm runoff samples from dairy high use areas collected over two storm seasons. Selected farm factors and beneficial management practices (BMPs) associated with reducing the Cryptosporidium load in storm runoff were assessed. Using immunomagnetic separation (IMS) with direct fluorescent antibody (DFA) analysis, Cryptosporidium oocysts were detected on four of the five farms and in 21% of storm runoff samples overall. Oocysts were detected in 59% of runoff samples collected near cattle less than 2 mo old, while 10% of runoff samples collected near cattle over 6 mo old were positive. Factors associated with environmental loading of Cryptosporidium oocysts included cattle age class, 24 h precipitation, and cumulative seasonal precipitation, but not percent slope, lot acreage, cattle stocking number, or cattle density. Vegetated buffer strips and straw mulch application significantly reduced the protozoal concentrations and loads in storm runoff, while cattle exclusion and removal of manure did not. The study findings suggest that BMPs such as vegetated buffer strips and straw mulch application, especially when placed near calf areas, will reduce environmental loading of fecal protozoa and improve stormwater quality. These findings are assisting working dairies in their efforts to improve farm and ecosystem health along the California coast.

Detection of Campylobacter jejuni from the skin of broiler chickens, ducks, squab, quail, and guinea fowl carcasses.

Campylobacter jejuni is often found on broiler carcasses and can cause gastroenteritis in humans. Both carcass rinses and swabs of the skin have been utilized to ascertain the prevalence of C. jejuni in the processing plant. Not all poultry commodities are equally capable of carrying C. jejuni on the carcass skin. Our objective was to measure the probability of C. jejuni detection (sensitivity) for the skin swabbing method followed by enrichment in semisolid media, and to ascertain the sensitivity of this method for commercial broiler, duck, squab, quail, and guinea fowl. The probability of detecting skin contaminated with C. jejuni was significantly higher for broiler chicken compared to retail duck, squab, quail, or guinea fowl for 10 or 100 colony-forming units (CFU)/in2 of skin (1 in2 = 1 square inch = 2.5 x 2.5 cm). Thirty-three percent (10 CFU/in2) and 100% (100 CFU/in2) of skin samples from broilers were positive for C. jejuni at the levels inoculated while 7-20% and 47-80% of skin samples were detected as contaminated with C. jejuni at 10 or 100 CFU/in2 for retail duck, squab, quail, and guinea fowl, respectively. Our method of using skin swabs and enrichment with semisolid media generated a sensitivity of almost 100% for detecting C. jejuni at 1000 or 10,000 CFU/in2 skin regardless of poultry species. The level of contamination that our method could detect with 50% and 90% reliability (DT50 and DT90) was 14 and 79 (broilers); 67 and 406 (squab); 39 and 226 (quail); 69 and 400 (guinea fowl); 69 and 400 (duck) CFU/in2 of skin, respectively.

Cryptosporidium oocyst persistence in agricultural streams -a mobile-immobile model framework assessment.

Rivers are a means of rapid and long-distance transmission of pathogenic microorganisms from upstream terrestrial sources. Pathogens enter streams and rivers via overland flow, shallow groundwater discharge, and direct inputs. Of concern is the protozoal parasite, Cryptosporidium, which can remain infective for weeks to months under cool and moist conditions, with the infectious stage (oocysts) largely resistant to chlorination. We applied a mobile-immobile model framework to assess Cryptosporidium transport and retention in streams, that also accounts for inactivation. The model is applied to California's Central Valley where Cryptosporidium exposure can be at higher risk due to agricultural and wildlife nonpoint sources. The results demonstrate that hyporheic exchange is an important process to include in models characterizing pathogen dynamics in streams, delaying downstream transmission and allowing for immobilization processes, such as reversible filtration in the sediments, to occur. Although in-stream concentrations decrease relatively quickly (within hours), pathogen accumulation of up to 66% of the inputs due to immobilization processes in the sediments and slower moving surface water could result in long retention times (months to years). The model appropriately estimates baseflow pathogen accumulation and can help predict the potential loads of resuspended pathogens in response to a storm event.

Colonizing capability of Campylobacter jejuni genotypes from low-prevalence avian species in broiler chickens.

Genetic variations in Campylobacter jejuni or host factors result in low prevalence rates among nonchicken poultry species. The objective of this study was to determine the colonizing potential, in broiler chickens, of C. jejuni that was recovered from low-prevalence avian species. Twenty-day-old Campylobacter-negative broiler chicks were inoculated by oral gavage with genetically different primary isolates of C. jejuni recovered from squab, duck, or chicken. Serial sampling and microbiologic testing of ceca were used to determine the level of colonization and the prevalence of positive chickens. All isolates were recovered from chickens by 10 days postinoculation. The C. jejuni strains recovered from challenged birds were genetically identical to the inoculated strains. By 10 days postinoculation, treatment groups inoculated with duck or control chicken isolates were 100% positive. The level of colonization by the squab isolate on day 2 postinoculation was significantly less than the duck or chicken isolates and had not colonized all birds by day 10 postinoculation.

Prevalence of Campylobacter and Salmonella species on farm, after transport, and at processing in specialty market poultry.

The prevalence of Campylobacter and Salmonella spp. was determined from live bird to prepackaged carcass for 3 flocks from each of 6 types of California niche-market poultry. Commodities sampled included squab, quail, guinea fowl, duck, poussin (young chicken), and free-range broiler chickens. Campylobacter on-farm prevalence was lowest for squab, followed by guinea fowl, duck, quail, and free-range chickens. Poussin had the highest prevalence of Campylobacter. No Salmonella was isolated from guinea fowl or quail flocks. A few positive samples were observed in duck and squab, predominately of S. Typhimurium. Free-range and poussin chickens had the highest prevalence of Salmonella. Post-transport prevalence was not significantly higher than on-farm, except in free-range flocks, where a higher prevalence of positive chickens was found after 6 to 8 h holding before processing. In most cases, the prevalence of Campylobacter- and Salmonella-positive birds was lower on the final product than on-farm or during processing. Odds ratio analysis indicated that the risk of a positive final product carcass was not increased by the prevalence of a positive sample at an upstream point in the processing line, or by on-farm prevalence (i.e., none of the common sampling stations among the 6 commodities could be acknowledged as critical control points). This suggests that hazard analysis critical control point plans for Campylobacter and Salmonella control in the niche-market poultry commodities will need to be specifically determined for each species and each processing facility.

New genotypes and factors associated with Cryptosporidium detection in mussels (Mytilus spp.) along the California coast.

A 3 year study was conducted to evaluate mussels as bioindicators of faecal contamination in coastal ecosystems of California. Haemolymph samples from 4680 mussels (Mytilus spp.) were tested for Cryptosporidium genotypes using PCR amplification and DNA sequence analysis. Our hypotheses were that mussels collected from sites near livestock runoff or human sewage outflow would be more likely to contain the faecal pathogen Cryptosporidium than mussels collected distant to these sites, and that the prevalence would be greatest during the wet season when runoff into the nearshore marine environment was highest. To test these hypotheses, 156 batches of sentinel mussels were collected quarterly at nearshore marine sites considered at higher risk for exposure to livestock runoff, higher risk for exposure to human sewage, or lower risk for exposure to both faecal sources. Cryptosporidium genotypes detected in Haemolymph samples from individual mussels included Cryptosporidium parvum, Cryptosporidium felis, Cryptosporidium andersoni, and two novel Cryptosporidium spp. Factors significantly associated with detection of Cryptosporidium spp. in mussel batches were exposure to freshwater outflow and mussel collection within a week following a precipitation event. Detection of Cryptosporidium spp. was not associated with higher or lower risk status for exposure to livestock faeces or human sewage sources. This study showed that mussels can be used to monitor water quality in California and suggests that humans and animals ingesting faecal-contaminated water and shellfish may be exposed to both host-specific and anthropozoonotic Cryptosporidium genotypes of public health significance.

Animal and farm influences on the dynamics of antibiotic resistance in faecal Escherichia coli in young dairy calves.

It is believed that the intensive use of antibiotics in the management of disease in pre-weaned calves contributes to high levels of antibiotic resistance in commensal and pathogenic bacteria. We described the temporal dynamics of antibiotic-susceptibility patterns seen in bovine enteric Escherichia coli in pre-weaned calves on dairy farms and dedicated calf-rearing facilities. Cohorts of 30 calves at each of six farms were sampled at 2-week intervals during the pre-weaning period. Faecal E. coli isolates were analyzed for antibiotic susceptibility to 12 antibiotics with the disk-diffusion method and grouped using cluster analysis of inhibition-zone patterns. The influences of calf age, farm-type, and individual-calf antibiotic therapy on the clusterings were assessed using stratified analyses and cumulative multinomial logistic regression using generalized estimating equation with antibiotic-resistance cluster as an ordinal-dependent variable. The model controlled for farm and cohort by a nested design and included a repeated measure on calf at each sampling occasion. E. coli from calves 2 weeks of age were more likely to be increasingly multiply resistant than E. coli from day-old calves (OR = 53.6), as were 4- and 6-week-old calves (OR = 29.8 and 16.4, respectively). E. coli from calves on dedicated calf-rearing facilities were more likely to be increasingly multiply resistant than E. coli from dairy-reared calves (OR = 2.4). E. coli from calves treated with antibiotics within 5 days prior to sampling were also more likely to be increasingly multiply resistant than E. coli from calves not exposed to individual antibiotic therapy (OR = 2.0).

Association of minimum inhibitory concentration cluster patterns with dairy management practices for environmental bacteria isolated from bulk tank milk.

Environmental bacteria have emerged over the past few years to become significant causes of mastitis. Bacteria in this group are often reported by practicing veterinarians to be increasingly resistant to intramammary therapy and responsible for elevated bulk tank somatic cell counts. The purpose of this study was to determine the extent of association of the minimum inhibitory concentrations for selected antimicrobial agents with environmental bacteria isolated from bulk tank milk on California dairies and their housing facilities, husbandry practices, and antimicrobic-use strategies. Bulk tank milk samples were collected from 2 dairy cooperatives that had their milk cultured at the Milk Quality Laboratory, University of California Davis, Veterinary Medicine Teaching and Research Center in Tulare, CA. Samples were collected from July 2001 through March 2002 on 88 d; and 404 environmental bacteria isolated from 93 dairies were found. Minimum inhibitory concentrations were determined on 337 of the isolates for 10 antimicrobial agents. Cluster analysis was performed on the minimum inhibitory concentration values for each organism, and 4 antimicrobial clusters with varying degrees of resistance were found.A 69-question survey questionnaire was completed on-farm for 49 of the 73 dairies that had at least 3 environmental bacterial isolates. The questionnaire sought information on housing facilities, milking management, mastitis prevention, antimicrobial usage strategies, and owner/veterinary involvement in disease control and prevention. Multinomial logistic regression analysis found significant associations between the antimicrobial agent-resistance cluster groups and some of the housing and bedding practices, failure to dry udders before milking, and antimicrobial treatment of nonmastitis conditions. No association was noted for antimicrobial agent treatment of mastitis and the resistance cluster patterns.

Persistence of cellulitis-associated Escherichia coli DNA fingerprints in successive broiler chicken flocks.

Avian cellulitis in broiler chickens is primarily caused by Escherichia coli. Previous research found that the E. coli isolates of cellulitis origin were unique to each ranch, suggesting that these E. coli were endemic within the ranch environment. To test the hypothesis that the E. coli associated with cellulitis are endemic in the litter of the broiler house, we designed a study to determine whether E. coli DNA fingerprints associated with cellulitis persist over successive flocks that are grown in the same house. In addition, we assessed the impact of different cleaning and disinfection strategies on this persistence. Two broiler houses were followed on each of five farms over 3-4 flocks. A total of 353 E. coli isolates from cellulitis lesions were analyzed in this study, and 314 of these isolates (89%) were DNA fingerprinted by PFGE. In each ranch, there were several DNA fingerprint patterns that were present over successive flocks, regardless of the cleaning and disinfection strategy utilized. Isolates persisted as long as 191 days, implying that these E. coli are capable of persisting in the broiler house environment for long periods of time. In addition, these E. coli isolates were associated with cellulitis lesions in successive flocks. Thus, the isolates of E. coli that are associated with cellulitis in broiler chickens appear to be endemic in the litter environment of the broiler house.

The prevalence of shedding of Cryptosporidium and Giardia spp. based on a single fecal sample collection from each of 91 horses used for backcountry recreation.

Cryptosporidium parvum and Giardia duodenalis are now recognized as primary enteric pathogens in animals and humans. Regulatory agencies, such as the Environmental Protection Agency are under increasing pressure to reduce the concentration of these protozoa in surface waters. Given the popularity of recreational riding of horses on public land in California backcountry, concerns have been raised by various regulatory agencies as to whether horses used for backcountry recreation are a significant source of C. parvum and G. duodenalis for the environment. The prevalence of fecal shedding of Cryptosporidium and Giardia in horses with a history of being ridden in California backcountry during 1993 and 1994 was estimated. Using both direct fluorescent antibody and levitation centrifugation tests, none of 91 single-collection fecal samples from throughout California had Cryptosporidium oocysts or Giardia cysts. Horses ranged from 4 to 24 years of age. Because none of the 91 samples, collected 1 time from each horse, were positive and assuming that the sensitivity and specificity of the test methods employed were 100%, the highest probable prevalence of shedding for either protozoal pathogen was < 3.2% for the cohort of horses studied.

A statistical model for assessing sample size for bacterial colony selection: a case study of Escherichia coli and avian cellulitis.

A general problem for microbiologists is determining the number of phenotypically similar colonies growing on an agar plate that must be analyzed in order to be confident of identifying all of the different strains present in the sample. If a specified number of colonies is picked from a plate on which the number of unique strains of bacteria is unknown, assigning a probability of correctly identifying all of the strains present on the plate is not a simple task. With Escherichia coli of avian cellulitis origin as a case study, a statistical model was designed that would delineate sample sizes for efficient and consistent identification of all the strains of phenotypically similar bacteria in a clinical sample. This model enables the microbiologist to calculate the probability that all of the strains contained within the sample are correctly identified and to generate probability-based sample sizes for colony identification. The probability of cellulitis lesions containing a single strain of E. coli was 95.4%. If one E. coli strain is observed out of three colonies randomly selected from a future agar plate, the probability is 98.8% that only one strain is on the plate. These results are specific for this cellulitis E. coli scenario. For systems in which the number of bacterial strains per sample is variable, this model provides a quantitative means by which sample sizes can be determined.

Assessing antibiotic resistance in fecal Escherichia coli in young calves using cluster analysis techniques.

This study uses cluster analysis techniques to describe the antibiotic susceptibility patterns seen in calf fecal Escherichia coli (E. coli). Cohorts of 30 dairy calves at six farms were sampled at 2-week intervals during the pre-weaning period. At each sampling occasion five fecal E. coli isolates per calf were analyzed for antibiotic susceptibility to 12 antibiotics using the disk diffusion method. All isolates had a profile consisting of the aggregate measured inhibition zone size for each of the evaluated antibiotics. Several cluster analytic algorithms were assessed to partition the E. coli isolates. For our data, Ward's minimum variance method met the objectives of the study. Relative to the number of possible combinations of resistance clusters, a parsimonious set of 14 patterns was developed. This set of E. coli isolates exhibited a limited set of resistance patterns to the different antibiotics indicating that certain resistance genes may be linked.

Cryptosporidia on dairy farms and the role these farms may have in contaminating surface water supplies in the northeastern United States.

The prevalence and risk factors for shedding of cryptosporidia by dairy cattle and calves and the prevalence and risk factors for cryptosporidia in surface waters associated with dairy farms were determined for a well-defined watershed in the northeastern United States. Eleven dairy farms were enrolled in the study and subjected to monthly sampling over a 6-month period. Animal-, water-, and manure-management practices were determined by survey and fecal, on-farm water, and stream samples were obtained monthly and evaluated for the presence of cryptosporidia. Ninety-one percent of the dairy farms in our study had Cryptosporidium on their premises. Fifteen percent of the sampled calves 0-3 weeks of age were shedding cryptosporidia. The risk factors for calves shedding cryptosporidia were contact between calves and frequent bedding changes. The probability of shedding cryptosporidia decreased with age. Nine percent of farm-associated stream samples were cryptosporidia-positive. The single risk factor for detecting cryptosporidia in surface water was increasing frequency of spreading of manure on fields. The probability of detecting cryptosporidia in streams decreased as 5-day cumulative precipitation increased. There were no animal-associated or barnyard-management features associated with detecting cryptosporidia in farm-impacted streams.

Prevalence of and risk factors for shedding of Cryptosporidium parvum in Holstein Freisian dairy calves in central México.

A total of 31 dairy farms from three states in central México were selected for this study in order to determine the prevalence of and risk factors for Cryptosporidium parvum shedding in young Holstein Freisian calves. Fecal samples were obtained once from each calf for acid-fast staining for detection of C. parvum oocysts. Information on each calf and on each dairy's management practices regarding the maternity pen, calf hutches and calf feeding was obtained by personal interview using a standardized questionnaire. Of the 31 dairies, 29 had one or more calves shedding C. parvum oocysts. The overall point prevalence was 25% (128/512). Dairy calves from the states of Hidalgo, Jalisco, and México had overall point prevalences of 28% (51/185), 29% (33/112) and 20% (44/215), respectively. Day of age was strongly associated with the risk of shedding C. parvum oocysts, with a maximum risk of shedding at approximately 15 days of age. Using mixed-effects logistic regression with herd as the random effect, feeding starter grain to calves, sweeping out the maternity pen, and using hay bedding in the maternity pen were significantly associated with increased odds of shedding C. parvum oocysts. We speculate that the association between feeding starter grain to calves and the higher odds of shedding C. parvum is linked to an increased duration rather than a higher incidence density of shedding. In addition, the association between sweeping the maternity pen and the increased odds of shedding C. parvum may be attributable to dairy personnel using the same broom for cleaning calf hutches and the maternity pen, thereby cross-contaminating oocysts from infected to newborn calves.

A prospective study of management and litter variables associated with cellulitis in California broiler flocks.

Cellulitis has emerged as an economically important disease of broiler chickens. The impact of environmental risk factors on the incidence of cellulitis has not been evaluated in the United States. Escherichia coli (E. coli), the causative agent, is introduced through skin scratches during the grow out. Our previous work suggested that the litter was an important reservoir for cellulitis-associated E. coli. We hypothesized that factors contributing to a positive environment for E. coli growth would increase the opportunity for exposure of a broiler to an infectious dose of E. coli, capable of initiating a cellulitis lesion. This prospective study of 304 flocks on five farms from two integrated broiler companies was conducted to determine the effect of environmental factors on the prevalence of cellulitis in California broiler flocks. Environmental variables included temperature, wind velocity, and relative humidity (RH) at the litter surface. Litter variables measured included E. coli and total gram-negative bacteria load (colony forming units/g dry matter), water activity, and pH. Management variables such as clean out, the number of flocks reared on the same litter (litter run, LR), and downtime (DT) between flocks were also evaluated. Cellulitis ranged from 0.197% to 6.04%. Significant associations were identified using linear regression between farm, LR, DT, ambient temperature during the brooding period, gram-negative bacteria load in the litter during the brooding period, RH mid-grow out, and E. coli load late in the grow out. The significant variation in the rate of cellulitis between farms combined with the strong association of LR and DT with cellulitis demonstrated that management choices were highly influential in this disease syndrome. Based on these data and our previous findings, managers would be advised to increase DT between flocks and perform a total clean out of the house when a flock processes with a high incidence of cellulitis.

Prevalence of pathogenic Escherichia coli in the broiler house environment.

Matched sampling of Escherichia coli from broiler house litter and bird lesions of either cellulitis or colibacillosis was conducted to investigate the relationship of pathogenic E. coli to those found in the environment. Isolates were collected from six broiler flocks representing six geographically disparate ranches. Isolates were compared by flock for similarity in serotype and genotyped by pulsed-field gel electrophoresis. Serotyping revealed a considerable dissociation between the two groups of isolates. The prevalence of pathogenic E. coli that matched the environmental isolates from the same house was 0 to 3%. Statistical analysis of the serotype data showed a strong dependence of serotype on isolate source, indicating a high probability that a particular serotype would be found among lesions or litter but not in both groups. Genotyping of isolates on two farms supported the results of serotyping and provided differentiation of isolates that could not by typed by serology. These results suggested that the prevalence of pathogenic E. coli in the broiler house was independent of the prevalence of other commensal or environmental E. coli. Understanding the composition of E. coli populations in commercial poultry production may have bearing on the epidemiology and control of E. coli related diseases.