Location: Ruminant Diseases and Immunology Research
2019 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 determined that PEG-gCSF treatment of a chronically infected cow with Staphylococcus aureus does not have the ability to clear the infection. 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. This change in protein cell surface expression was observed on cells in the circulating blood cells and on the immune cells found in the milk. These observations are helping 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. Furthermore, these observations indicate that an increase of the mechanisms that target immune cells to the site of infection may not be sufficient to ensure full immune cell activation that would allow for clearance of the infection.
This year’s primary progress for Sub-objective 1.2: Basic mammary biology is not well understood and this lack of data limits our understanding of major dairy diseases such as mastitis. After giving birth, cows will lactate or produce milk for about a year at which time the cow will typically begin to decrease milk production and is then “dried off” as milking is ceased about 2 months before giving birth to another calf. The process of cessation of milk production is commonly referred to as drying off with mammary involution being the scientific term. The A calcium/hydrogen transporter implicated in mammary calcium transport and involution was discovered and studied. Both mammary cell calcium and involution are critical when a cow is dried off. The drying off process in considered critical to a healthy mammary gland in the next lactation. So any understanding of this process is vital. We found that our protein which is a calcium/hydrogen transporter plays a small role in cell death which is a part of the drying off process. Furthermore, we found that it is critical for normal lactose synthesis in lactation and therefore milk volume. While impacts are indirect to mastitis control the increased understanding of mammary biology give us the tools to further advance mammary health studies. As a part of 1.2 we also completed the proteome analyses from dry secretion samples described in last year’s report.
This year’s primary progress for Sub-objective 2.1. was publishing the data describing the DNA (genome) and all the messenger RNA (transcriptome) from several strains Escherichia (E.) coli, the most common environmental pathogen cause of bovine mastitis in the United States. Some of these bacteria cause persistent infections while others just a transient infection. Our goal was to determine what genes from very similar bacteria lead to some causing a more damaging and costly persistent infection. 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.
This year’s primary progress for Sub-objective 2.2. was infection of cows with Staphylococcus aureus and causing a chronic infection. Our goal was to isolate bacteria at various stages of infection from cows to determine the gene expression changes in bacteria through the various stages of the infection process. Although our preliminary data showed that it was possible to isolate sufficient numbers of bacteria from milk when growing the bacteria in whole milk in the laboratory, it was not possible to isolate enough bacteria from milk from an infected cow. We are currently exploring alternative methodologies to address questions about why some bacteria cause chronic infections.
Accomplishments
1. The effect of 50 years of breeding on the ability of Holsteins to fight mastitis. Dairy cow breeding and management has been able to dramatically increase milk production and at the same time reduce the number of cows in production. However, ever since the early 1970s, some researchers have expressed concern that the selective breeding for milk production could result in an associated increase in health problems. The University of Minnesota has maintained a herd of dairy cows with sires that reliably breed for the average milk yield seen in 1964. In collaboration with researchers at the University of Minnesota, ARS researchers at Ames, Iowa, have shown that Holsteins with genetics from 1964 were able to clear an experimental mastitis challenge with Escherichia coli better than modern Holsteins. This work shows that selective breeding for milk production is correlated with poorer immune defense against E. coli mastitis.
2. Discovery that a new biotherapeutic increases surface expression of an important adhesion molecule target on immune cells in milk. Cows are immunosuppressed at the time of calving and therefore more susceptible to mastitis. Targeting cells to the site of infection is a critical part of the process which the immune system successfully fights disease. ARS researchers at Ames, Iowa, have discovered a previously unrecognized effect of a commercial version of bovine G-CSF (PEG-gCSF). This effect is the increase of cell surface expression of a recently discovered protein called myeloperoxidase, that targets cells to the site of an infection. We found myeloperoxidase is increased on the surface of immune cells called neutrophils and monocytes that are found in the blood when animals are treated with PEG-gCSF. Although the immune cells traffic to the mammary gland, we have shown that PEG-gCSF administration is not sufficient to clear a chronic mastitis. Our findings provide evidence that PEG-gCSF therapy modifies cell surface expression of neutrophils and monocytes. However, while cells with higher surface myeloperoxidase accumulate in the mammary gland, the lack of bacterial control from these milk-derived cells suggest an incomplete role for PEG-gCSF treatment against chronic S. aureus infections. Although PEG-gCSF can target immune cells to the site of infection, other immune stimulators may be necessary to fully activate the immune system. This work is an important step forward in understanding the complex molecular signals that are responsible for targeting cells to the site of infection and understanding what is necessary for an effective immune response against a chronic infection.
Review Publications
Lippolis, J.D., Powell, E.J., Reinhardt, T.A., Thacker, T.C., Casas, E. 2019. Symposium review: omics in dairy and animal science-promise, potential, and pitfalls. Journal of Dairy Science. 102(5):4741-4754. https://doi.org/10.3168/jds.2018-15267.
Powell, E.J., Reinhardt, T.A., Casas, E., Lippolis, J.D. 2018. The effect of pegylated granulocyte colony-stimulating factor treatment prior to experimental mastitis in lactating Holsteins. Journal of Dairy Science. 101(9):8182-8193. https://doi.org/10.3168/jds.2018-14550.
Lippolis, J.D., Nally, J.E. 2018. Considerations for farm animal proteomics experiments: An introductory view gel based versus non-gel based approaches. In: de Almeida A., Eckersall D., Miller I., editors. Proteomics in Domestic Animals: from Farm to System Biology. Basel, Switzerland: Springer International Publishing AG. p. 7-16. https://doi.org/10.1007/978-3-319-69682-9_2.
