Recipient Organization
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES,IA 50011
Performing Department
VDPAM
Non Technical Summary
The use of antimicrobials in food animals continues to cause concern regarding development of antimicrobial resistance (AMR). There is little data on AMR development in small ruminants. Data suggests that most dairy goats have an intramammary infection at dry-off. Without treatment, most of these infections persist into the following lactation. Almost no evidence-based data exists to inform the development of effective antibiotic stewardship recommendations and educational campaigns towards dairy goats. Our goal is to improve goat milk quality through prudent treatment programs that maintain food safety, and to disseminate an Extension program to stakeholders. The central hypothesis is that application of approaches demonstrated to benefit antimicrobial stewardship and minimize AMR development in dairy cattle can be effective in goats. We will attain the overall goal via these objectives:1. Determine the antimicrobial residue depletion profile in milk and meat of goats following administration of dry cow intramammary antimicrobials.2. Utilizing a selective approach, determine the efficacy of dry-off antimicrobial products against intramammary infections.3. Determine whether administration of dry cow antimicrobials results in the development of antimicrobial resistance in dairy goats.4. Develop a comprehensive Extension program that demonstrates how established mastitis prevention strategies, coupled with a judicious antimicrobial approach, can minimize antimicrobial resistance and preserve food safety. We will obtain our goals through a sequential series of trials that follow the above objectives, culminating with a comprehensive, sustainable Extension program that provides dairy goat producers with resources for efficacious antimicrobial use that minimizes AMR and preserves food safety.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Our goal in this proposed project is to improve goat milk quality through efficacious and prudent treatment programs that maintain food safety, and to create and disseminate an educational outreach program to stakeholders on how to implement these strategies. The central hypothesis is that application of approaches demonstrated to benefit antimicrobial stewardship and minimize AMR development in dairy cattle can be foundational for similarly effective strategies in dairy goats. This hypothesis is based on previous research results showing successful use of dry-off antimicrobial therapy in dairy goatsand the successful implementation of selective treatment programs in dairy cattle. Additionally, despite widespread use of antimicrobials in the treatment of mastitis in dairy cattle, development of antimicrobial resistance has been minimal. Lastly, while no dry-off antimicrobial products are labeled for use in lactating dairy goats, residue detection test kits are available that are labeled for use with goat milk that will be able to detect antimicrobial residues to levels below US Food and Drug Administration (FDA) tolerances for the drug residues in dairy cattle. This will reduce the risk for antimicrobial residues in does treated with intramammary (IMM) antimicrobials. We will attain the overall goal of this project via implementation of the following objectives:Objective 1. Determine the antimicrobial residue depletion profile in milk and meat of goats following the administration of dry cow intramammary antimicrobials. Our working hypothesis is that the time for the residues to deplete will be longer than the time required for the drugs to deplete in dairy cows.Objective 2. Utilizing a selective approach, determine the efficacy of dry cow antimicrobial products for the treatment of infections present at the end of the lactating period in the goat. Our working hypothesis is that selective utilization of dry-off intramammary antimicrobial programs will be efficacious towards improving milk quality and decreasing antimicrobial use compared to blanket dry-off therapy of all does.Objective 3. Determine whether the implementation of dry cow antimicrobial programs result in the development of antimicrobial resistance in dairy goats. Our working hypothesis is that the implementation of dry cow antimicrobial programs will not increase AMR in dairy goats and decrease the selective pressure for resistance.Objective 4. Develop a comprehensive producer education program to be delivered via Extension offerings that demonstrates how established on farm mastitis prevention strategies, coupled with a judicious antimicrobial approach, can minimize antimicrobial resistance and preserve food safety. Our working hypothesis is that we can impact implementation of best management practices (BMP's) through centralized distribution of educational materials on milk quality improvement and efficacious therapy based on research completed on modern goat operations.
Project Methods
Determination of milk residue depletion. As there are no IMM drugs approved for use in dairy goats, products currently labeled for dry-off therapy in dairy cows (cephapirin and cloxacillin). In order to determine an appropriate milk withdrawal, we will follow the protocol outlined by the US FDA. At the last milking prior to dry-off treatment, milk will be collected in floor pails, then well-stirred, and duplicate 50 mL composite milk samples will be taken and frozen for future analysis. For each of the two drugs, twenty does will be infused with one tube of the dry-off antimicrobial in each udder half following complete milk out of the udder and aseptic teat preparation. After kidding, does will have milk sampled two times per day for a period of ten days for drug residue analysis. At each milking, does will be prepped using the farm's written protocol, except does will not be fore-stripped. Clean milking equipment fitted with a floor pail will be used to collect all harvested milk. Following milking unit attachment, milk flow will be observed until milk flow completely ceases from all udder halves for a minimum of 15 seconds and then the unit will be removed. Following milking unit removal, duplicate, well-stirred composite 50-mL milk samples and a single 10-mL milk sample will be collected from the floor pails.Milk concentrations of CEPH and CLOX will be determined using LC-MS by the ISU VDL Analytical Chemistry Lab using a validated method adopted from cow milk.Determination of meat residue depletion. For both CEPH and CLOX, an estimated meat withdrawal will be determined following US FDA guidance. For each antimicrobial product, twenty does will be selected and treated as described in the previous experiment. Starting at 21 days after dry treatment, four does per treatment group will be humanely euthanized every week for five weeks. Following euthanasia, 200 g each of kidney, liver, and loin skeletal muscle will be collected and frozen at -80 °C until analyzed for drug concentration via LC-MS.Meat residue concentrations of CEPH and CLOX will be determined using LC-MS by the ISU VDL Analytical Chemistry Lab using a validated method.Determining the efficacy of IMM antimicrobial therapy. Study personnel will travel to participating dairies on the same day each week to collect dry-off (DO) samples, administer dry therapy, and collect post-kidding (PK) samples. Seven days prior to expected dry-off, study personnel will travel to participating farms to identify animals for study participation. Records from all does due to be dried off in the next week will be evaluated for recent disease treatments, DHIA test day SCC data, and previous clinical mastitis treatment events, with the data being captured for analysis following completion of the study. Animals that match study criteria will be screened via milk culture for determination of study enrollment. Upon entry of potential study animals into the milking parlor, animals will be assessed to assure that both halves are functional, that clinical mastitis is not present, and there is no systemic illness indicated. Prior to milking, all animals that meet inclusion criteria will have duplicate, aseptic DO milk samples collected from each half using procedures described by the National Mastitis Council. Between each of the duplicate samples collected from each half, the teat end will be re-sanitized. Following sample collection, does will be milked according to the farm's protocol and released from the parlor. Milk samples will immediately be placed on ice and transported to the laboratory for culture.One week after the initial culture is collected, study technicians will return to the farm to administer dry-off therapies according to randomized allocation. Does will be brought to the parlor as a group for their final milking of the lactation and milked according to the farm's protocol. Study personnel will again assure that the doe fits the enrollment criteria. After completion of milking, does assigned to the NC group will be post-milking teat dipped and receive no further treatment. Does assigned to one of the antibiotic therapy groups (CEPH or CLOX) will be treated with one IMM tube per half following disinfecting the teat. Following IMM infusion, does will be post-milking teat dipped.As does begin kidding following their dry period, study personnel will travel to study farms weekly to collect duplicate post-kidding (PK) samples between 1-7 days in milk using the same milk collection and handling procedures for collection of DO samples. At subsequent weekly visits, samples will continue to be collected on does that kidded within the last week and any health events that occurred during the previous week in study does that previously kidded will be captured.Milk samples will undergo bacteriologic culture at the veterinary diagnostic laboratory of the respective study personnel. Initially the S1 sample for DO or PK will be cultured using accepted procedures for mastitis microbiology. To complete the bacterial culture, samples will be warmed to room temperature and then 10 μL of milk will be transferred to 5% sheep blood agar and MacConkey agar using sterile transfer devices (loop or pipette). Agar plates will be incubated at 37°C and examined for bacterial growth by laboratory technicians at 24 and 48 hours after plate inoculation. For the DO and PK samples, a presumptive diagnosis of potential IMM pathogens will be based on colony morphology and other testing. All bacteria that meet the definitions of IMM pathogens will undergo confirmatory identification. While only does with IMM infections caused by CNS bacteria will be included in the efficacy study, all isolates from DO samples will undergo confirmation of identification to be used later to calculate the potential differences in quantity of antimicrobial exposure between full blanket dry-off therapy compared to a selective approach. Confirmatory identification of suspect IMM pathogens will be made with use of Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-ToF MS Biotyper. Bruker Corp. Bremen, Germany).Following completion of culturing, DO and PK sample results from each udder half of study animals will be compared to determine bacteriological cure and development of a new IMM infection. An udder half will have achieved a bacteriological cure if the bacteria present in the DO sample are not present in the PK sample. A new IMI infection in an udder half will be determined if a pathogen is isolated from the PK sample that was not present in the DO sample (36).Antimicrobial susceptibility testing will be completed on DO and PK bacterial isolates from udder halves that do not achieve a bacteriological cure and on bacterial isolates present in PK samples from udder halves that acquire a new IMI during the dry period. All AST will be completed at the ISU VDL. A standard broth microdilution method will be performed following manufacturer's instructions using commercially available Sensititre veterinary mastitis CMV1AMAF plates (ThermoFisher Scientific, Waltham, MA).Bacterial isolates from DO and PK CNS infections that fail to cure, as defined above, and new IMM infections in the PK samples will undergo genomic sequencing. Genomic DNA of isolates will be extracted using the Wizard Genomic DNA kit (Promega). Indexed Illumina sequencing libraries will be prepared and sequenced on the Illumina platform according to the manufacturer's instructions at ISU's DNA Facility. For the DO CNS isolates that fail to cure, strain similarity and verification will be completed by aligning the DO and PK isolates to determine phylogenetic similarity. Additionally, DO and PK CNS isolates that fail to cure and new IMM infections, the antibiotic resistance genes will be identified by aligning the genomes against available databases.