Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
Veterinary Medicine & Surgery
Non Technical Summary
Mastitis, inflammation of the mammary gland, is the most common disease impacting the health and productivity of adult dairy cows. Mastitis significantly impacts dairy productivity costing the industry in excess of $2 billion annually. Mastitis is primarily caused by an intra mammary infection (IMI) with bacteria or other microbial pathogens. Mastitis can be clinical (with changes in the character of the milk or physical changes in the mammary gland} or subclinical (with no overtly obvious manifestations). Subclinical mastitis is the most common type of mastitis on dairy farms, and is diagnosed by measuring the milk somatic cell count {SCC), which serves as a proxy for the inflammatory cells (primarily neutrophils) in milk (Sargeant et al., 2001}.Jersey cattle contribute significantly to the US dairy economy with approximately 1.3 million head making up about 14% of the total US dairy cattle population, and numbers have been increasing over the last few years. Currently, there is a paucity of data regarding pathogen-specific associations with elevated milk SCC in Jersey cattle with subclinical mastitis, the most common form of the disease. While Jersey cattle anecdotally seem to be less susceptible to some forms of mastitis, they on average seem to have higher milk sec concentrations than Holstein cattle. Hence, the aim of this study is to improve our understanding of the causes of elevated milk SCC in Jersey cattle with subclinical mastitis. By understanding the potential drivers for elevated milk sec in Jersey cattle with subclinical mastitis, dairy producers and veterinarians can work to develop targeted mastitis control strategies to reduce the prevalent bacterial populations and minimize their impact on cow-level and herd-level (bulk tank milk) SCC.Approximately 750 cows on five Jersey confinement dairy operations in Missouri will be studied by collecting mammary quarter foremilk samples once monthly for three months from all lactating cows. Milk samples will be cultured for bacterial pathogens and submitted for milk SCC enumeration. Bacterial pathogens will be identified to the genus and species level using MALDI-TOF mass spectrometry or 16S rRNA gene sequence analysis as applicable. Milk SCC will be performed by a commercial laboratory using a flow cytometric counter.Data will be recorded in a spreadsheet to include herd, cow, mammary quarter, culture results and milk SCC. Descriptive data will include prevalence of the various bacterial pathogens among mammary quarters and cows within herd. The association between milk sec and IMI will be evaluated using a multivariate mixed modelling with sec as the dependent variable and herd, cow, mammary quarter, sample period (1-3} and IMI status (no growth or bacterial IMI categorized by pathogen genus and species) as the independent variables to understand the association between milk sec, bacterial pathogens, and herd.Results will be reported in peer-reviewed and non-peer-reviewed publications and presented at local, state, and national meetings. If in-person meetings are not possible, virtual formats will be used for technology transfer.
Animal Health Component
100%
Research Effort Categories
Basic
0%
Applied
100%
Developmental
0%
Goals / Objectives
Long-term GoalsJersey cattle contribute significantly to the US dairy economy with approximately 1.3 million head making up about 14% of the total US dairy cattle population. Mastitis significantly impacts dairy productivity costing the industry in excess of $2 billion annually. Currently, there is a paucity of data regarding pathogen-specific associations with elevated milk sec in Jersey cattle with subclinical mastitis. While Jersey cattle anecdotally seem to be less susceptible to some forms of mastitis, they on average seem to have higher milk sec concentrations than Holstein cattle. Hence, the long-term goal of this research is to improve our understanding of the causes of elevated milk SCC in Jersey cattle with subclinical mastitis, the most common form of the disease worldwide. By understanding the potential drivers for elevated milk sec in Jersey cattle with subclinical mastitis, dairy producers and veterinarians can work to develop targeted mastitis control strategies to reduce the prevalent bacterial populations and minimize their impact on cowlevel and herd-level (bulk tank milk) SCC. These efforts will in turn be translated into more milk produced, longer shelf life, and improved dairy product yields thus benefiting dairy farmers and addressing the societal need for increased amounts of wholesome animal-source foods.Specific Obiectives1. Assess associations between mammary quarter milk sec and bacterial IMI in Jersey cattle.This work will allow for in-depth characterization of subclinical mastitis in Jerseys and will build on a parallel project funded by the American Jersey Cattle Club Research Foundation to evaluate the prevalence of nonaureus Staphylococcus (NAS) species in Jersey herds in Missouri. The parallel project proposed herein will expand on the NAS prevalence inquiry to include all bacterial pathogens isolated from cows' mammaryquarters and will provide additional funding to investigate the association between mammary quarter IMI and subclinical inflammation as measured by milk SCC.
Project Methods
Sampling MethodsThis prospective study will involve enrollment of five all-Jersey confinement dairy herds in Missouri. The average expected herd size will be approximately 150 lactating cows/herd, yielding a total animal enrollment of 750 cows. Mammary quarter foremilk samples will be collected from all functional quarters of all lactating cows once monthly for three months for bacterial culture and sec enumeration.Sample CollectionTeats will be disinfected and dried according to each farm's pre-milking hygiene protocol. Next, the apex and barrel of each teat will be scrubbed with a 70% isopropyl alcohol-soaked cotton swab whilst wearing disposable gloves. Each quarter will be stripped of two streams of milk before aseptically collecting 5-10 ml into a sterile plastic vial for bacterial culture (Middleton et al., 2017; Wattenburger et al., 2020). Immediately after, a sample for sec enumeration will be collected from each quarter into a separate vial containing 2-bromo-2-nitropropane-1, 3-diol preservative tablets (Broad Spectrum Microtabs fl, D & F Control Systems, Inc., Dublin, CA, USA). All vials will be labelled with individual cow identification and mammary quarter sampled.Laboratory MethodsBacterial Isolation and Identification -All milk samples will be frozen (-20°C) within eight hours of collection. Milk samples will be thawed at room temperature (~22°C) prior to plating on Columbia blood agar for culturing. Plates will be incubated at 37°C and read at 24 and 48 hours. Colony counts will be recorded and primary bacterial identification will be performed according to the National Mastitis Council guidelines (Middleton et al., 2017}. All bacterial isolates will be sub-cultured and speciated using MALDITOF mass spectrometry per the manufacturer's instructions (Bruker). Species-level bacterial identification will be considered reliable for MALDI-TOF analysis scores >2.0 for all non-Staphylococcus bacteria and considered reliable for all Staphylococcus for analysis scores >1.7 (Cameron et al., 2017 and 2018, Adkins et al., 2018). If an isolate scores below this cut-off, a new colony will be analyzed. Isolates not identified by MALDI-TOF, will be subjected to polymerase chain reaction amplification of a portion 165 rRNA gene followed by sequence analysis of the amplicon to identify the bacterial genus and species.A mammary quarter will be defined as having an IMI if an individual organism is isolated at a concentration of;::: 100 CFU/ml (Dohoo et al., 2011) except for Bacillus species that will have a threshold of?? 500 CFU/ml. Culture samples yielding two different colony types will be defined as "mixed IMI" and samples with?? 3colony types will be defined as "contaminated." Mammary quarters with mixed and contaminated results will be treated as missing data when evaluating the association between milk sec and IMI.Somatic Cell Count- SCC milk samples will be shipped to Mid-South Dairy Records in Springfield, Missouri for enumeration on a flow cytometric counter (Bentley Somacount FCM, Bentley Instruments, Chaska, MN, USA). Results will be reported to the investigators by e-mail.Data AnalysisData will be recorded in a spreadsheet to include herd, cow, mammary quarter, culture results and milk SCC. Descriptive data will include prevalence of the various bacterial pathogens among mammary quarters and cows within herd. The association between milk sec and IMI will be evaluated using a multivariate mixed modelling with SCC as the dependent variable and herd, cow, mammary quarter, sample period {1-3) and IMI status {no growth or bacterial IMI categorized by pathogen genus and species) as the independent variables.The sample size for this study is a convenience sample premised on the average size of Jersey herds in Missouri and the number of herds expected to participate in the study based on phone conversations with each producer and input from Dr. Scott Poock, MU Dairy Extension Veterinarian. The sample size is expected to be sufficient to define within herd prevalence and provide a sufficient number of herds from different locales to assess herd-to-herd differences.Anticipated Results and Potential PitfallsThese data will provide an estimate of the prevalent bacterial causes of IMI in Jersey cattle and will provide insight into the association between IMI status (infected versus no growth as well as differences between different pathogens) and mammary gland inflammation. These data can be compared with published data in Holstein cattle to determine where breed differences may exist and perhaps explain why Jersey cattle have higher milk sec, but fewer cases of bacterial mastitis.Potential pitfalls include not being able to enroll a sufficient number of cattle, but given Dr. Poock's relationship with multiple Jersey herds and the influence of the American Jersey Cattle Club having funded the mastitis pathogen prevalence portion of the work, it is anticipated that we can achieve the study enrollment targets. Given that this is an observational study, it is also possible that sufficient numbers of mammary quarters with different bacterial causes of IMI or no IMI may not be enrolled leading to insufficient replicates to draw meaningful conclusions in the multivariate analysis. While there is always this risk, prior work by our group in Holstein dairy cattle and dairy goats suggests that, given our projected sample size, we should have sufficient numbers of mammary quarters with at least the predominant pathogens to conduct the proposed analyses.