Location: Ruminant Diseases and Immunology Research
2018 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 can be summed up with the completion and publication of an experiment examining the effect of pegylated granulocyte colony-stimulating factor (PEG-gCSF) treatment on experimental Escherichia coli infection in lactating Holsteins. PEG-gCSF treatment pre-infection resulted in reduced disease severity when administered before a bacterial challenge. We showed that PEG-gCSF treatment increased expression of a cell surface molecule that targets immune cells to the site of an infection. These cells quickly disappear from the blood shortly after infection, suggesting a mechanism for the reduced mastitis severity by priming immune cells for quick targeting to the site of infection. Further progress for Sub-objective 1.1 centers around the use of PEG-gCSF immediately prior to a cow calving, followed by experimental infection of the mammary gland. These studies have been started and will continue through the year as cows give birth. It is planned to use 9 cows per group, for a total of 18 cows where we will further explore the cell surface expression of an E-selectin ligand before infection on the mechanism of action of pegylated granulocyte colony-stimulating factor.
This year’s primary progress for Sub-objective 1.2 was data that allowed us to conclude that our hypothesis that fatty acid ester administration could induce accelerated mammary involution was incorrect under the conditions tested. However, the experiments did yield a wealth of dry secretion samples from the start of involution until 21 days dry. Using these samples, we confirmed the well-known fact the increase in lactoferrin in dry secretions is associated with dry secretion antimicrobial actions compared to milk. However, what is not appreciated and found by our research progress this year is that by day 21 dry, lactoferrin concentrations have very low correlations with the antimicrobial activity of dry secretions. This was found to be true for 6 mastitis inducing bacteria. These secretions at day 21 have antimicrobial activities that range from robust to none at all. The key antimicrobial factor(s) in dry secretions remains elusive and it is expected to be identified using a proteomics approach. The determination of the proteome of the dry secretions from several cows at various days after the cessation of milking has been initiated.
This year’s primary progress for Sub-objective 2.1. was sequencing 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, the expression changes of those genes when comparing bacterial strains that can cause persistent infections with those that cannot, and finally to analyze trends in the data that may indicate important functional trends of the genes that are changing for information that we can use to generate hypotheses. We showed that E. coli strains that cause persistent infections have genes that code for a protective shell for the bacteria (capsid), whereas, those that cause transient infections are missing those genes. We have constructed E. coli mutant to test the importance of these capsid genes and are currently testing them.
Accomplishments
1. Genetic and gene expression differences between E. coli strains that cause persistent versus transient mastitis in dairy cows. Infection of the mammary gland of cows with E. coli can result in a self resolving infection that last a few days or a persistent infection that can last months. It is important to determine the mechanisms that allows certain E. coli strain to persistently infect cows to allow for rapid detection and treatment of those cows likely to have a persistent infection. ARS researchers in Ames, Iowa, have published work on the genomic and transcriptome differences between E. coli strains that can cause a persistent infection and those strains that cause transient mammary gland infections in dairy cows. Research has identified genes that were only present in the strains that cause persistent disease, these genes help bacteria to generate a protective capsid that may protect them from the immune system. The loss of these genes in bacteria that are correlated with persistent disease will be tested for a corresponding loss of persistent mastitis in cows.
2. Discovery that a new biotherapeutic increases surface expression of an important adhesion molecule target on immune cells. Cows are immunosuppressed at the time of calving and therefore more susceptible to mastitis. Neutrophils are one of the cells functionally suppressed during the periparturient period. Increasing the number of neutrophils at the time of calving has been shown to reduce the incidence of mastitis, this can be accomplished with injections of a protein called granulocyte-colony stimulatory factor (G-CSF). ARS researchers at Ames, Iowa, have discovered a previously unrecognized effect of a commercial version of bovine G-CSF wherein cell surface expression of a recently discovered E-selectin ligand, myeloperoxidase, is increased on phagocytic cells called neutrophils and monocytes that are found in the blood. When G-CSF was administered to lactating dairy cows, their phagocytic cells were increased in number and in their apparent capacity to combat and reduce the severity of experimentally-induced mastitis in dairy cows. We found that the cows that had been treated with G-CSF produced more milk, ate more, had less bacteria in their milk, and had a more activated immune system compared to untreated cows. Increased expression of myeloperoxidase on their cell surface would enhance immune surveillance and may be part of the mechanism that allows the immune system to quickly respond to infection and thus reduce infection rates and severity of disease. Understanding the mechanisms of proteins given as treatments to mastitis is necessary for the development of future therapies with greater efficacy. This work defines that G-CSF helps get the cells at the site of infection, other treatments may be necessary to help the cells be more effective in destroying the pathogens.
Review Publications
Lippolis, J.D., Holman, D.B., Brunelle, B.W., Thacker, T.C., Bearson, B.L., Reinhardt, T.A., Sacco, R.E., Casey, T. 2017. Genomic and transcriptomic analysis of Escherichia coli strains associated with persistent and transient bovine mastitis and the role of colanic acid. Infection and Immunity. 86(1):e00566-17. https://doi.org/10.1128/IAI.00566-17.
Thacker, T.C., Lippolis, J.D., Brunelle, B.W., Casey, T., Reinhardt, T.A., Sacco, R.E., Holman, D.B. 2017. Genome sequences of Escherichia coli strains that cause persistent and transient mastitis. Genome Announcements. 5(34):e00775-17. https://doi.org/10.1128/genomeA.00775-17.
Dassanayake, R.P., Falkenberg, S.M., Register, K.B., Samorodnitsky, D., Nicholson, E.M., Reinhardt, T.A. 2018. Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis. PLoS One. 13(5):e0197677. https://doi.org/10.1371/journal.pone.0197677.
