Location: Ruminant Diseases and Immunology Research
2020 Annual Report
Objectives
Objective 1. Develop non-antibiotic interventions to prevent and control mastitis, including developing and testing non-antibiotic immune modulators to prevent periparturient dairy cows from developing mastitis, and developing and testing dry cow therapy(s) that use natural, non-antibiotic strategies that accelerate the development of the cow’s natural antimicrobial dry secretions to prevent mastitis infections in subsequent lactations.
Sub-objective 1.1: Develop and test non-antibiotic immune modulators to prevent periparturient dairy cows from developing mastitis.
Sub-objective 1.2: Develop and test a dry cow therapy that uses natural, non-antibiotic strategies that accelerates the development of the cow’s natural antimicrobial dry secretions to prevent mastitis infections in the subsequent lactation.
Objective 2: Determine the interactions between mastitis-causing pathogens and the host innate immune mechanisms in the mammary gland, starting with determining the host-pathogen interaction associated with Escherichia coli strains linked to persistent mammary gland infections, and determining the host pathogen interaction associated with Staphylococcus aureus persistent infections.
Sub-objective 2.1: Determine the host-pathogen interaction associated with Escherichia coli strains linked to persistent mammary gland infections.
Sub-objective 2.2: Determine the host pathogen interaction associated with Staphylococcus aureus persistent infections.
Approach
Mastitis is the most prevalent infectious disease in dairy herds and the most costly disease for dairy producers. Older cost estimates for mastitis are in the neighborhood of $2 billion per year for producers. Newer estimates of the economic impact of mastitis on the dairy industry calculate the cost of a single case of clinical mastitis to be approximately $586 due to mammary gland damage, loss of milk production, discarded milk, and the costs of treatment and labor. Antibiotics are the mainstay for mastitis treatment and control and dairy cattle with mastitis receive more antibiotic therapy for its prevention and treatment than for all other dairy cattle diseases combined. Valid concerns by consumers regarding antibiotic usage need to be addressed by research on non-antibiotic alternatives. To achieve the goal of reducing the use of antibiotics we need a better understanding of how the immune system is failing to completely eliminate mastitis infections. Progress towards this goal can be achieved in two ways. First, is to manipulate the host in a way that optimizes the immune response to pathogens. Second, to gain a better understanding of the various mechanisms that allow bacteria to evade the host’s immune system. To achieve the goal of manipulating the immune system to optimize its response to pathogens we plan to develop non-antibiotic interventions to prevent and control mastitis. This approach would include developing and testing non-antibiotic immune modulators to prevent periparturient dairy cows from developing mastitis, and developing and testing dry cow therapy(s) that use natural, non-antibiotic strategies that accelerate the development of the cow’s natural antimicrobial dry secretions to prevent mastitis infections in subsequent lactations. To achieve the second goal of understanding the mechanisms of how bacteria can evade the immune system by studying the mechanisms that allow for persistent mammary gland infections. Knowledge of how bacteria escape the immune system and establish persistent infections is a necessary precursor to any therapeutic for these persistent infections. Successful manipulation of the host immune system that targets the pathogen at the site of the infection holds the potential of clearing an infection without the use of antibiotics.
Progress Report
This year’s progress for Sub-objective 1.1 was a continuation of our work examining the effect of pegylated granulocyte colony-stimulating factor (PEG-gCSF) treatment on experimental mastitis infection in lactating Holsteins. We have published our results describing the PEG-gCSF treatment of chronically infected cows with Staphylococcus aureus. However, the PEG-gCSF treatment does have a significant effect on the immune system by changing the cell surface expression of various proteins involved in targeting immune cells to the site of infection. To further this observation, we have started proteomics studies to determine the protein expression changes seen in cows treated with PEG-gCSF. Determination of protein expression changes will help us to understand the mechanisms that target immune cells to the site of infection, which is a critical part of the host immune process that needs to be understood.
This year’s primary progress for Sub-objective 1.2: We published that some dry secretion dogma is questionable. General dogma about the dry period and dry secretions role in managing new mastitis infections has been attributed to the ability of lactoferrin to slow the growth of certain mastitis causing bacteria. The increase in lactoferrin dry secretions while associated with dry secretion antimicrobial actions compared to milk but we found in our research is that by day 21 dry, lactoferrin concentrations have NO role antimicrobial activity of dry secretions. The lack of lactoferrin correlation was found to be valid for seven mastitis inducing bacteria. These day 21 dry secretions have antimicrobial activities that range from robust to none at all. The critical antimicrobial factor(s) in dry secretions remains a mystery that we hope to uncover with proteomics. The proteome of the dry secretion by days dry and cow has been completed and published and several proteins were correlated with the antimicrobial activities of day 21 dry secretions. Preliminary data suggest we may identify new and previously unrecognized critical protein components important to dry period mastitis prevention.
This year’s primary progress for Sub-objective 2.2: isolation and genome sequencing of a strain of Staphylococcus aureus found in a chronically infected cow on our facility. Preliminary data suggests that this strain is more resistant to clearance from the gland when treated with antibiotics when compared to our research strain of S. aureus. Further experiments to determine if there is a difference between S. aureus strains and their susceptibility to antibiotics are ongoing.
Accomplishments
1. The dry period in dairy cows is a critical time for the development on new mastitis infections that bloom during the first few weeks of the subsequent lactation. We know that components of the dry secretion can be antimicrobial but also vary from cow to cow. To understand and potentially regulate the antimicrobial components of the dry secretion we need an analysis of its components. The dry secretion proteome was completed for first 3 weeks of a dairy cow’s dry period. The proteome of the dry secretion by days dry and cow has been completed by ARS researchers at Ames, Iowa, and several proteins were correlated with the antimicrobial activities of day 21 dry secretions. Preliminary data suggest new and previously unrecognized critical protein components are important to dry-period mastitis prevention. The impact will come if we can improve drysection composition to help prevent new mastitis infections.
2. Genomic sequence of Staphylococcus aureus strain from chronically infected dairy cow. Staphylococcus aureus is a group of bacteria that causes mastitis in dairy cows that is often impossible to successfully treat with available antibiotics. In addition to the difficulty of curing Staphylococcus aureus mastitis in cows, these bacteria are contagious and therefore many of these animals are culled from the herd. A dairy cow with a spontaneous case of mastitis was shown to have a Staphylococcus aureus infection. Treatment with antibiotics did not result in a successful treatment outcome. ARS researchers at Ames, Iowa, have sequenced the genome of this strain of bacteria. At the time of our publication there was only one other bovine isolate of S. aureus with a sequenced genome. We are currently analyzing the sequence information for potential difference that may be important to pathogenesis. Greater understanding of the genomes of S. aureus strains that cause mastitis in dairy cows, especially those with differences in disease symptoms and presentation, will be important pieces of information to understand the pathology of this bacteria and how it causes a chronic infection.
Review Publications
Bannantine, J.P., Stabel, J.R., Lippolis, J.D., Reinhardt, T.A. 2018. Membrane and cytoplasmic proteins of Mycobacterium avium subspecies paratuberculosis that bind to novel monoclonal antibodies. Microorganisms. 6(4):127. https://doi.org/10.3390/microorganisms6040127.
Reinhardt, T.A., Lippolis, J.D. 2020. Characterization of bovine mammary gland dry secretions and their proteome from the end of lactation through day 21 of the dry period. Journal of Proteomics. https://doi.org/10.1016/j.jprot.2020.103831.
Powell, E.J., Eder, J.M., Reinhardt, T.A., Sacco, R.E., Casas, E., Lippolis, J.D. 2019. Differential phenotype of immune cells in blood and milk following pegylated granulocyte colony stimulating factor (PEG-gCSF) therapy during a chronic Staphylococcus aureus infection in lactating Holsteins. Journal of Dairy Science. 102(10):9268-9284. https://doi.org/10.3168/jds.2019-16448.
Dominguez-Perez, D., Lippolis, J.D., Dennis, M., Miller, B., Tiley, K., Vasconcelos, V., De Almeida, A.M., Campos, A. 2019. The Queen Conch (Lobatus gigas) proteome: a valuable tool for biological studies in marine gastropods. The Protein Journal. 38:628-639. https://doi.org/10.1007/s10930-019-09857-0.
Lippolis, J.D., Putz, E.J., Ma, H., Alt, D.P., Casas, E., Reinhardt, T.A. 2020. Genome sequence of a chronic Staphylococcus aureus isolated from a dairy cow that was non-responsive to antibiotic treatment. Microbiology Resource Announcements. 9(20). Article e00206-20. https://doi.org/10.1128/MRA.00206-20.
Dassanayake, R.P., Falkenberg, S.M., Stasko, J.A., Shircliff, A.L., Lippolis, J.D., Briggs, R.E. 2020. Identification of a reliable fixative solution to preserve complex architecture of bacterial biofilms for scanning electron microscopy evaluation. PLoS One. 15(5):e0233973. https://doi.org/10.1371/journal.pone.0233973.
Putz, E.J., Putz, A.M., Jeon, H., Lippolis, J.D., Ma, H., Reinhardt, T.A., Casas, E. 2019. MicroRNA profiles of dry secretions through the first three weeks of the dry period from Holstein cows. Scientific Reports. 9(19658). https://doi.org/10.1038/s41598-019-56193-5.
