Location: Ruminant Diseases and Immunology Research
2021 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 is the final report for the project 5030-32000-115-00D terminating September 30, 2021. This year’s progress for Sub-objective 1.1 continued our work examining the effect of pegylated granulocyte colony-stimulating factor (PEG-gCSF) treatment on experimental mastitis infection in lactating Holsteins. We also detailed that 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, this year, we have completed sample collection for a proteomics study to determine the protein expression changes seen in cows treated with PEG-gCSF. Determination of protein expression changes will help us 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. During the life cycle of this project, we have published two manuscripts detailing the use of PEG-gCSF as a mastitis treatment. The first paper showed that PEG-gCSF reduced Escherichia (E.) coli mastitis severity in dairy cows. The second paper showed that PEG-gCSF was an ineffective treatment for chronic Staphylococcus aureus mastitis.
This year's primary progress for Sub-objective 1.2: General dairy science dogma about the dry period and dry secretion's role in managing and/or preventing new mastitis infections has been attributed to the ability of lactoferrin to slow the growth of certain mastitis-causing bacteria. The increase in lactoferrin in dry secretions is associated with dry secretion antimicrobial activity. However, by day 21 dry, we show that lactoferrin concentrations have little to no antimicrobial activity in dry secretions. The lack of lactoferrin correlation was found to be valid for seven mastitis pathogens. This 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 the cow has been completed and published. 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. During the life cycle of this project, we published 3 papers on this objective that have appeared in annual reports.
This year’s primary progress for Sub-objective 2.2, we determined that Staphylococcus (S.) aureus chronically infected cows could not be super-infected with E. coli. Mixed infections are uncommon, and this may be due to the first pathogen causing an immune response. Chronic S. aureus infections may not themselves be cleared by the immune system, but they do prevent the introduction of some secondary infections. During the life cycle of this project, we have isolated, sequenced the genome of a strain of Staphylococcus aureus found in a chronically infected cow in our facility.
During the life cycle of this project, we have done sequencing of the DNA (genome) and all the messenger RNA (transcriptome) from several strains Escherichia (E.) coli. Some of these bacteria cause persistent infections while others just a transient. Our goal was to determine what genes from very similar bacteria lead to some causing a more damaging and costly persistent infection. We have completed the analysis of the transcriptome, which includes both identification of the genes and the expression changes of those genes when comparing bacterial strains that can cause persistent infections. We showed that E. coli strains that cause persistent infections have genes that code for a protective shell for the bacteria (capsule), whereas, those that cause transient infections are missing those genes. In addition, E. coli strains that cause persistent infections have genes that code for motility. Collaborators in Israel showed that this motility defective bacterial strain had a reduced ability to cause disease in mice. We have completed an experimental mastitis study in cows using the motility defective bacterial strain and showed no reduction in mastitis in dairy cows.
Accomplishments
1. A gene thought to participate in ion transport (TMEM165) controls milk production by regulating the milk sugar lactose concentration. Lactose is an essential component of milk and is the primary controller of milk's water content. ARS researchers in Ames, Iowa, have demonstrated that ion transporter identified in the mammary gland is a crucial regulator of lactose concentration in milk and, therefore, normal milk production. We tested whether the gene participates in normal milk production using mice with the ion transport gene knocked out. It is crucial in the lactating mammary gland for normal lactose biosynthesis and normal growth rates of nursing pups. The milk of TMEM165-deficient mice contained elevated concentrations of fat, protein, iron, and zinc, which are likely caused by decreased water transport into milk due to the reduced lactose biosynthesis. These findings suggest that the regulation of ion transport sustains the production of lactose. A basic understanding of critical mammary function in support of lactation is lacking and will be needed to make future advances in milk production. These results are primarily used for dairy geneticists and mammary biologists' basic understanding of which genes are essential for milk production.
2. Chronic Staphylococcal aureus mastitis (S. aureus) is a significant problem for the dairy industry and is assumed to occur due to immune suppression or immune avoidance. Therefore, the question arose whether chronic S. aureus mastitis might increase as cows’ susceptibility to another infection. Chronic S. aureus mastitis prevents secondary infection by Escherichia coli (E. coli). ARS researchers in Ames, Iowa, have demonstrated that cows with a chronic S. aureus infection cannot be given a secondary infection with E. coli. These two classes of bacteria elicit different immune responses. However, the immune response directed by a chronic S. aureus infection is sufficient to prevent subsequent infection. This fascinating finding requires much more research to understand the immunological changes induced by chronic Staphylococcal aureus mastitis, which prevents an E. coli infection. This research will lead to a greater understanding of the immune system and its ability to fight mammary gland infections, the most significant health concern for dairy farmers. This knowledge will be of great use to mastitis researcher's understanding of disease processes in mastitis.
Review Publications
Almeida, A.M., Ali, A., Ceciliani, F., Eckersall, P., Hernandez-Castellano, L.E., Han, R., Hodnik, J.J., Jaswal, S., Lippolis, J.D., McLaughlin, M., Miller, I., Mohanty, A.K., Mrljak, V., Nally, J.E., Nanni, P., Plowman, J.E., Poleti, M.D., Ribeiro, D.M., Rodrigues, P., Roschitzki, B.,Schlapbach,R., Staric, J., Yang, Y., Zachut, M. 2021. Domestic animal proteomics in the 21st century: a global retrospective and viewpoint analysis. Journal of Proteomics. 241. Article 104220. https://doi.org/10.1016/j.jprot.2021.104220.
Reinhardt, T.A., Lippolis, J.D. 2020. Dataset of bovine mammary gland dry secretion proteome from the end of lactation through day 21 of the dry period. Data in Brief. 31. Article 105954. https://doi.org/10.1016/j.dib.2020.105954.
