Performing Department
Vet Population Medicine & Diagnostic Science
Non Technical Summary
Our long-term objective is to ensure the soundness of the environment and address the uncertainty in the climate change while sustaining the economic viability of dairy production systems in NYS. The transport of pathogens of pathogens from livestock production systems contribute significantly to the degradation of the environment and the uncertainty associated with climate change.We are planning to carryout epidemiologic studies targeting dairy operations, both traditional and organic, to identify the zoonotic pathogens that are transported from these farms, shed light on the pathways by which they are transported, identify the factors that promote their presence and transportation, and determine the management practices that mitigate their associate risk.Knowledge developed through these studies will be incorporated in tools and educational materials and shared with the stakeholders.
Animal Health Component
0%
Research Effort Categories
Basic
0%
Applied
100%
Developmental
0%
Goals / Objectives
Characterize and develop management practices to reduce GHG emissions and transport of nutrients, pathogens, pharmaceuticals, and VOCs from livestock production systems. This will include management of feed, manure collection, manure storage, and manure application. (Miller, Harrison, Herbert, Moriera, Miller, Powell, Rotz, Wattiaux, Hashemi)
Develop science-based tools and educational materials to promote environmental stewardship on US dairy and beef industries. (Harrison, Herbert, Powell, Rotz, Westendorf, Westra)
Project Methods
We are planning to carry out a longitudinal epidemiologic study to determine the incidence of these foodborne pathogens in dairy and fresh produce organic operations in NYS. Animals, fresh produce, and environmental samples will be collected quarterly and analyzed for the presences of E. coli O157:H7, non-O157 food adulterant serotypes (O11, O26, O45, O111, O121, and O145) and Campylobacter (C. jejuni, C. coli, and C. lari) using a combination of bacterial enrichment and real-time PCR detection system. A twostep approach will be implemented to identify farms that housing animals with these pathogens and has potential to contribute to environmental quality degradation: First milk filters will be collected from these farms and examined for the presence of these pathogens using a combination of bacteriological and molecular techniques. Second, farms that are positive in the milk filter examination will be followed up with a thorough investigation that includes both animal and environmental sampling. The number of animals and environmental samples will be proportional to the size of the herd and the farm. The proportional sampling will be stratified by the age of the animals. Two types of samples will be collected: animals and environment samples. Fecal samples will be collected per rectum from three age groups of cattle: newborns, heifer, and adult cows.The first sample from the newborn will be collected at from animals up to 30 days of age.Heifers and cows will be sampled once each season for a total of 90 samples from animals on the farm. Calves will be sampled throughout the year.c. In addition to the animal sampling, we are planning to collect environmental samples on each farm following our previous sampling strategy. A multidimensional scale will be used to identify hydrologically sensitive sites along the pathway from animal housing and manure handling areas to the stream edge. Composite samples will be collected from each site at the time of animal sampling. The concept is to shed light on the pathway by which the pathogens moves from the source and contaminate the environment.Sample processing and Detection E. coli O157:H7Samples (animal and environmental) will be screened for E. coli O157:H7 using the BAX® System (http://www2.dupont.com/Qualicon/en_US/products/BAX_System/bax_realtime_EcoliH7.html). All samples will be inoculated into a primary enrichment medium consisting of modified E. coli broth (MEC broth) supplemented with novobiocin (16 mg/L) at a ratio of 1:10 (W/V). The enriched broth will be incubated for 24 hr at 37 oC. An inoculum of 20 µl of the incubated enriched will be transferred into 1 ml of secondary enrichment medium consisting of Brain Heart Infusion (BHI) broth without antibiotics and incubated for three hours at 37 oC. The inoculated BHI media will then be incubated for three hours at 37 oC before testing with the real-time PCR.Non-O157 Shiga Toxin Producing E. coli O GroupsAll samples will be screened for the presence of the STEC virulence genes (stx1, stx2 and eae) using a Real-time PCR assay (http://www2.dupont.com/Qualicon/en _US/products /BAX_System /bax_ecoli_testing.html) and will be performed on the bacterial lysate prepared after the secondary enrichment. The protocol calls for a two-step process. All samples will be further screened for the food adulterant serogroups of E. coli which include O26, O45, O103, O 111, O121, and O145.PCR detectionPCR detection will performed using the BAX Automated System. A 20 µl aliquot of the secondary enrichment (BHI) was added to 200 µl of the prepared lysing reagent (mixture of protease and lysis buffer) provided by the manufacturer. The samples will then be heated at 37°C and 95°C in a lysis reagent solution to rupture the bacterial cell wall and release the DNA. PCR tablets, which contain all the reagents necessary for PCR, will be hydrated with 30 µl of the lysed sample and processed in the cycler/detector provided by the manufacturer (AB 7500 FAST). A table of results that includes cfu/ml values for each target will be displayed, along with graphs of amplification curves.Campylobacter spp.Samples will be screened for C. jejuni, C. coli, and C. lari using the BAX® System (http://www2.dupont.com/Qualicon/en_US/products/BAX_System/bax_realtime_campy.html). All samples will be enriched in BHI, at a ration 1:10 (W/V), containing 6mg of cefoperazone, 6mg of vancomycin, and 2 mg of amphotericin each dissolved in 80 ml of distilled water. The inoculated media will be incubated at a microaerophilic atmosphere at 37°C for 24 hrs. A total of 20 µl of the incubated enriched inoculum will be transferred into 1 ml of the secondary enrichment medium (BHI without antibiotics) and incubated for 24 hrs at 37oC before processing by real-time PCR.PCR detectionAs in E. coli.We are planning to collect data on factors that are hypothesized to associate with the likelihood of introduction and permutation of these pathogens among these operations. Animals' factors may include source of feed, size of herd, biosecurity measures, and sanitary practices. Environmental factors may include type of soil, pH of soil, microbial concentration per gram, the source of fertilizer, biosecurity, sanitary measures. Hierarchal data analysis statistical techniques will be used to identify factors associated with the likelihood of these pathogens in animals and products from organic operations. In this Aim we will integrate the interaction between the animal and the environment on the likelihood of transmission and perpetuation of these pathogens to the environment. Using a combination of deterministic and stochastic methods, scenario path models will be developed describing the transmission and fate of the most prevalent species and genotypes, identified in Aim 1, from the source to environmental waters. For example, equations 1 through 3 describe the indirect transmission between cattle and the environment. The rate of change in the environmental concentration with a particular pathogen genotype (Wi) depends on the rate of contamination by an infected animal, (Ic), rate of environmental contamination by the other sources (gi); and the loss of viability of the pathogen (a). The described scenario relies on the rate of contamination of the environment (q) by cattle, the probability of contact with pathogens (j).The notations S, I, n, r, and R, indicates the number of susceptible cattle, number shedding the particular pathogen, total number of animals, rate of recovery, and number of recovered animals, respectively. Because of the uncertainty associated with some of the parameters several iterations will be run using the Monte Carlo simulations in @RISK software to determine whether the environmental transmission and fate rate would be altered if reasonable variations in each of the above parameters were made. We will also perform sensitivity analysis. Our model will also include information on management practice at these farms that promote environmental stewardship. Practice that reduce this microbial load will be identified in the analyses.The proposed research activities outlined in Aim 1 and 2 will generate improved understanding of the process of introduction, transmission, and fade of these pathogens. In collaboration with agricultural and regulatory stakeholders, we will develop beneficial management practices that are science-based to maximize the effectiveness of these practices in mitigating the associated risk at the farm level. The findings will be integrated into extension and educational programs for the purpose of closing the knowledge gap.