Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
Veterinary Medicine & Surgery
Non Technical Summary
A significant proportion of U.S. dairy goat producers struggle to comply with the maximum regulatory limit for milk somatic cell count (SCC) in grade A milk. Many factors influence goats' milk SCC, including parity, days in milk (DIM), bacterial intramammary infection (IMI), and Caprine Arthritis Encephalitis Virus (CAEV) infection. Defining an accurate SCC threshold to predict bacterial IMI in goats has been hampered by these extraneous factors. Some bacterial pathogens, such as coagulasenegative staphylococci (CNS), can increase milk SCC and cause persistent IMI during lactation. However, this relationship has not been studied over the dry period. Current literature lacks scientific data to formulate recommendations regarding management of goats with high SCC at dry-off.The goal of this study is to determine risk factors (IMI status, parity, DIM, CAEV seropositivity) associated with elevations in SCC at dry-off and at the onset of the next lactation. Milk samples will be collected from both udder-halves from a convenience sample of 200 goats on a commercial goat dairy, twice before dry-off and twice after kidding. Milk cultures will be performed, bacterial isolates will be identified at the species level, and strain-typing will be performed to document persistence of IMI by the same strain. The herd is enrolled with the Dairy Herd Information Association (DHIA) and has monthly testing of individual goats for SCC, milk production, and milk components (fat and protein). Data from the two tests prior to dry-off and the first two tests in the subsequent lactation will be retrieved from the DHIA database using their publicly available software (PC-DART). Serum CAEV antibody titers will be determined and included as a covariate in the final data analysis to investigate potential influence of CAEV infection (because CAEV is a retrovirus serum antibodies titers are synonymous with infection) on either milk SCC or the persistence of IMI. Expected outcomes include a more detailed understanding of the factors that influence milk SCC before and after the dry (non-lactating) period and persistence of IMI over the dry period. In addition, these data will be used to establish a diagnostic algorithm (using SCC data and/or culture data) that can be used to guide management practices that will help reduce SCC in the subsequent lactation, e.g. intramammary antibiotic therapy of infected udder halves. Overall, this project is aimed at improving goat milk quality which will have positive impacts for dairy goat producer and consumers of goat dairy products.
Animal Health Component
90%
Research Effort Categories
Basic
10%
Applied
90%
Developmental
0%
Goals / Objectives
The long term goals of this work are to use evidence-based approaches to deliver practical information and solutions that can be used to improve udder health and milk quality on dairy goat operations.Specific Aims1) Determine risk factors (e.g. IMI status, parity, days in milk [DIM], Caprine Arthritis Encephalitis Virus [CAEV] seropositivity) associated with elevations in milk SCC at dry-off and at the onset of the next lactation.2) Design a diagnostic algorithm for goat producers to use to make management decisions at dry-off, e.g. intramammary antibiotic therapy or culling.
Project Methods
The study will be conducted on the same large commercial dairy goat herd where the preliminary data above were generated. The herd has no history of clinical signs of mycoplasma mastitis. The herd is enrolled with DHIA and has monthly testing of individual goats for SCC, milk production, and milk components (fat and protein). A convenience sample of 200 goats will be selected based on common dry-off dates so that goats can be enrolled at approximately the same time. Goats in this herd are dried off in cohorts of approximately 100 individuals. Goats will not receive intramammary antibiotic treatment during the study, and goats requiring parenteral antimicrobial therapy for bacterial disease during the study period will be excluded from the data analyses. Goat-level data (parity, DIM) will be retrieved from herd records. DHIA test day SCC, milk production, and milk components (fat and protein) for the two tests prior to dry-off and the first two tests in the subsequent lactation will be retrieved from PCDART (DHIA software), with the consent of the farm management.Sample CollectionUdder-half milk samples will be aseptically collected into sterile vials from all enrolled goats, approximately 15 days prior to dry-off (Sample 1), at dry-off (Sample 2), within 7 days of kidding (Sample 3), and approximately 15 days into lactation (Sample 4). Sample tubes will be indexed according to goat ID, transported on ice to the laboratory, and stored at -20°C until processed.On the day of the goats' enrollment in the study (day 15 before dry-off), a blood sample will be collected from each goat, allowed to clot, separated by centrifugation, and serum will be later used for determination of CAEV infection status. Serum will be stored at - 80°C until analysis. A commercial cELISA antibody test kit (VMRD Inc., Pullman WA) will be used to detect anti-SRLV antibodies as a proxy for CAEV infection. This test was reported to have a sensitivity of 100% and a specificity of 96.4% for the diagnosis of CAEV when compared to radioimmunoprecipitation assay (20). Because CAEV is a retrovirus, the presence of serum antibodies is considered diagnostic for infection.To help rule out the presence of mycoplasma mastitis in the herd, the bulk tank will be sampled at each farm visit and each sample cultured for Mycoplasma spp. Bulk tank milk testing is recommended to determine herd infection status and because shedding in milk can be intermittent, serial testing improves detection of Mycoplasma spp. (11). Culture of either fresh or frozen-thawed (-20°C for 14 days) milk samples has been reported to have a lower limit of detection than PCR, which is critical with bulk tank milk samples because lower concentrations of organisms are likely present than in individual milk samples from infected goats (21). Should bulk tank screening yield a positive Mycoplasma spp. isolation, individual milk samples from the enrolled goats will be cultured for Mycoplasma spp. using the same techniques as described for bulk milk samples.Bacteriological CulturePreviously frozen milk samples will be thawed at room temperature (~22°C) and 10 μl of milk will be plated on Columbia blood agar (CBA), incubated at 37°C for 24h, read at 24h, kept at room temperature, and read again at 48h. Colonies will be initially identified based on morphology, Gram staining, catalase test, and coagulase test. Cultures with ≥ 3 morphologically different colony types will be considered contaminated. Cultures with 2 morphologically different bacterial colonies will be considered mixed infections. All bacterial isolates identified will be sub-cultured onto CBA incubated for 18-24 hr at 37°C and stored in phosphate-buffered glycerol at -80°C for further identification.Culture of fresh (refrigerated) bulk tank milk samples for detection of Mycoplasma spp. will be performed on Hayflick medium at 35°C in 10% CO2 for up to 10 days. Should bulk tank screening yield a positive Mycoplasma spp. isolation, individual milk samples from the enrolled goats collected on the same day as the positive bulk tank sample will be cultured for Mycoplasma spp. using the same techniques.Speciation and Strain-typing of Bacterial IsolatesFor non-mycoplasma bacteria, initial species identification will be performed using MALDI-TOF mass spectrometry (MALDI Biotyper 3.1, Bruker Daltonics). This method uses species-specific spectra generated by ribosomal proteins based on their mass and charge. Spectra are compared to a proprietary or custom database and assigned a a score based on similarity with database spectra. The score is then used to determine genus and species. Briefly, each isolate will be recovered from storage media and grown on CBA at 37°C for 24 hr. A representative colony will be picked from the CBA plate and spotted in duplicate on the MALDI-TOF plate according to the manufacturer's instructions and run on the MALDI-TOF biotyper. Isolates with a score of ≥ 2 for a given genus and species on at least one of the duplicates will be considered adequate for species-level identification (22). Isolates not identified after initial screening will be rerun on the MALDI-TOF biotyper after using the ethanol/formic acid extraction method (22), and a score of ≥ 2 will likewise be required for species identification.Isolates that cannot be identified with MALDI- TOF will have lysates prepared for PCR amplification and partial sequencing of either the 16S rRNA gene, rpoB, or tuf gene as applicable to determine bacterial genus and species. Sequences will be compared with the NCBI database and species identification will be assigned with an identity of ≥ 99% homology and > 0.8% separation for 16S (23), ≥ 97% homology to a single species for rpoB (24), and ≥ 98% homology and > 0.8% separation for tuf (23).Goats with an IMI with the same bacterial species and strain-type in the two samples prior to dry-off, or at least one sample prior to dry-off and at least one sample after kidding, will be considered persistently infected prior to or over the dry period, respectively. Intramammary infections that do not meet these criteria will be considered transient. In cases of persistent IMI, the isolates recovered from the first and last samples will be subjected to strain-typing by pulsed-field gel electrophoresis (PFGE) using the method described by Middleton et al. (25). Briefly, DNA will be digested using an appropriate restriction endonuclease and separated by PFGE in a 1% agarose gel immersed in 0.5% TBE buffer at 14 °C for 20 h with a 130° ramp angle and a 5 to 50 s pulse time. Banding patterns of DNA fragments from pairs of isolates will be compared to one another both visually or using computer software (Bionumerics) to evaluate their genetic relatedness.