Understanding and preventing mastitis in dairy cows – Immune regulation in the mammary gland

Dr. Anja Sipka collecting and evaluating milk samples at the dairy farm for milk leukocyte differentiation.

The immune system of the dairy cow is a much-studied subject but despite growing effort a lot of key mechanisms regulating mastitis remain enigmatic. It is well known that host factors have a large impact on the outcome of clinical mastitis in dairy cows. Depending on the stage of lactation, genetic predisposition, metabolic status and age of the animal, the unique composition of host derived mediators and immune cell subsets changes. This affects the cow’s immune performance and can lead to an increase in the susceptibility for intramammary infections and severity of the clinical response to pathogens.

At the Quality Milk Production Services (QMPS) research lab of Dr. Sipka in Ithaca, New York, the mission is to better understand the mechanisms shaping the immune system of the dairy cow. In a holistic approach that includes experimental mastitis trials and in vitro cell models, the group is working on identifying the immune cell subpopulations and host-derived mediators involved in the immune regulation in the

udder. The working hypothesis of the group is that the interplay of leukocyte subsets in the periphery and the mammary gland govern the outcome of mastitis. Host-derived mediators and signaling molecules are responsible for polarizing these leukocyte subpopulations and set the stage for the immune response. As an overall goal the group strives to find a way to modulate the immune system of the cow by priming bovine leukocytes and creating an environment that is optimally prepared to appropriately handle intramammary infections. The initial host-pathogen interaction seems to be most crucial in mastitis; therefore, the group draws a special focus on innate leukocyte cell populations and their functions. The Sipka lab’s most recent publications cover multivariate analyses on the count of leukocyte subsets in transition cows and the incidence of mastitis and metritis post-partum as well as studies on the presence of innate lymphocytes in the mammary gland and their antimicrobial properties against E. coli. In another line of work the lab collaborates with industrial partners on evaluating mastitis diagnostic assays. The assays take a novel approach on mastitis detection: an on-farm set up for the detection of bacterial DNA milk and an algorithm aiming to define stages of intramammary infections using milk leukocyte differentials.

The research facilities
Quality Milk Production Services is part of the New York State Animal Health Diagnostic Center at the College for Veterinary Medicine of Cornell University, Ithaca, New York. Established in 1946, the mission of the program is to promote the production of high quality milk by controlling mastitis through the assessment of udder health and milking procedures. The program uses a variety of field and laboratory services ranging from the detection of pathogens in milk and environmental samples to the evaluation of milking equipment and procedures, as well as housing facilities on farms. On a yearly basis QMPS performs on average 170,0000 cultures and other tests, as well as 2,600 farm visits. As a part of this strong team Dr. Sipka’s research group utilizes the resources of the diagnostic laboratory, including pathogen identification by MALDI-TOF and the comprehensive database of on-farm herd health data from a large range of different dairy farm types. Doctor Sipka’s research laboratories are equipped with facilities for cell culture, flow cytometry as well as bacteriology and molecular biology facilities. For experimental mastitis studies the large animal research and teaching unit of the Cornell University Animal Science Department provides housing and milking facilities for cows. The group is furthermore working with the Cornell University Teaching Dairy for smaller field studies and the establishment of new mastitis diagnostic tests.

Innate lymphocytes and regulation of the immune response in mastitis
The idea to study innate lymphocytes originated from an experimental mastitis trial where mid-lactating cows were infected with E. coli. In order to identify pathways involved in mastitis regulation the group studied the transcriptome of the mammary gland tissue. Analyzing the differentially expressed genes and pathways there was one result that sparked the curiosity of the group, because it involved a somewhat unusual cell type in the context of bovine mastitis. In humans, Natural Killer (NK) cells are known as early responders in bacterial infections. In bovines, however, their potential role in bacterial infections had not been studied so far. In tissue of the udder quarters infected with E. coli the NK cell mediated cytotoxicity pathway was among the top ten enriched gene expression pathways. While researchers have observed this before, there have not been any further studies on NK cells and their potential role in the inflamed mammary gland. Backed up by this preliminary data the research group set out to take a closer look at bovine NK cells in the mammary gland and their potential functions in the innate immune response.
The first step was to actually identify the cells in mammary gland tissue. Using immune histo-fluorescence, the group managed to detect small numbers of NK cells in tissue sections from infected quarters. A small number of NK cells were also detected in milk from quarters before being challenged by E. coli, as well as over the course of the infection. Although this was the first time NK cells were identified in mammary gland tissue and milk of cows, the low frequency of the cells raised the question of their biological importance. To address this aspect the second part of the project focused on studying NK cell function in vitro.
In an involved cell separation process including magnetic and laser assisted cell sorting, the group isolated NK cells from peripheral blood. Primary cultures of bovine mammary gland epithelial cells were used to create an environment close to the situation in the udder. The basic questions addressed in the in vitro study were 1) are NK cells attracted to the mammary gland in case of an infection and 2) do NK cells respond to contact with the pathogen? The cultures of primary bovine mammary gland epithelial cells were stimulated with UV irradiated E. coli, which mimics infection by presenting all the pathogen-associated patterns to the cultured cells. When NK cells were placed on top of the cultured mammary gland epithelial cells separated by a porous membrane, a significant migration of NK cells towards stimulated epithelial cells was observed within two hours. Furthermore, the researchers observed that the activation marker CD25 was up-regulated on the surface of the NK cells. In a second set of experiments, NK cells were incubated with live E.coli and were shown to significantly reduce counts of the bacteria within two hours of culture. The in vitro experiments showed that bovine NK cells have the potential to respond rapidly to a mastitis-causing E. coli isolate by migrating towards the infected quarter and likely produce antibacterial mediators once these cells have arrived at the site of infection. These findings introduce NK cells as a leukocyte population in the mammary gland with potential functions in the innate immune response in bovine mastitis. Despite the relatively small numbers of NK cells found in mammary gland tissue and milk, they could still be critical in shaping the immune response. It is expected that the mediators secreted by NK cells provide an immune modulatory potential. Taken together, the present study provides a first assessment of the potential role of bovine NK cells in the mammary gland during intramammary infections. Their presence in the microenvironment of the gland and the responsiveness to a mastitis E. coli strain observed in blood NK cells make them a worthwhile candidate for further research elucidating their immune regulatory properties in the context of bovine mastitis.

The idea to study innate lymphocytes originated from an experimental mastitis trial where midlactating cows were infected with E. coli.

Myeloid leukocyte subsets and their importance in mastitis post-partum
Following the same principle idea, the research group also focuses on monocytes, a heterogeneous myeloid cell population that was recently found to consist of three distinct subsets in cattle. The majority in peripheral blood are classical and intermediate monocytes, which express the LPS receptor CD14 on their surface and are characterized by their stronger pro-inflammatory response and less mature stage of differentiation. The third population, non-classical monocytes, are the most mature population and do not carry CD14 on their surface. It is yet not clear whether the three subpopulations are lineage specific, independent subsets or are maturation stages going from the most immature stage, the classical monocyte, to the most mature subset, non-classical monocytes.
Although the role and dynamics of the different bovine monocyte subsets in vivo remain unclear, it has been shown that cows developing infectious disease postpartum also have an altered monocyte response. The peripartal period is a particularly difficult time for the dairy cow. One of the many challenges is the transition of the immune system from tolerating the presence of the fetus to expressing the full range of inflammatory mediators in response to a pathogen after parturition. Failure to adapt smoothly to this change can lead to an inadequate immune response, which becomes apparent in the increased incidence of mastitis and metritis. This heightened susceptibility to infectious diseases postpartum is attributed to the overexpression of certain pro-inflammatory mediators and, at the same time, a decreased ability to clear the infection. It is unknown if this phenomenon also includes changes in monocyte subset composition or an altered function of individual monocyte subsets. In humans, however, it has been shown that there are associations between inflammatory conditions and monocyte subset composition. Based on this, the objective of the group was to investigate if there is a relationship between pre-partum blood monocyte subset composition and post-partum infectious disease in dairy cows.

Dr. Anja Sipka performing a flow cytometric analysis.

In collaboration with the immunology unit of Prof. Schuberth from the University for Veterinary Medicine of Hannover, and the Institute of Animal Nutrition of the Friedrich-Loeffler-Institute Braunschweig in Germany, the group analyzed a data set from a cohort of 27 Holstein cows. The data included counts of monocyte subsets and neutrophils at 42 and 14 days prior to expected calving date and the occurrence of metritis and mastitis within two weeks postpartum. The group used a multivariate logistic regression model to analyze the relationship between pre-partum cell counts of the leukocyte populations with post-partum disease. Half of the enrolled cows were found to either develop mastitis or metritis or both. The model revealed that higher counts of classical and intermediate monocytes at 14 days prior to the expected calving date were predictive of disease. In contrast, higher numbers of non-classical monocytes were negatively associated with disease. Interestingly, the neutrophil count pre-partum was not associated with the occurrence of mastitis or metritis post-partum. The results indicate that the number and composition of monocyte subsets before calving are related to the susceptibility to infectious disease within two weeks postpartum. Furthermore, the oppositional effect of immature, more pro-inflammatory subsets and the more mature subset strengthens the hypothesis that classical and non-classical monocytes have different functional roles in the inflammatory response in dairy cows. Another interesting aspect of the study is that the association of monocyte subset counts and post-partum disease was not found at the beginning of the dry period, 42 days prior to expected calving, but right at the time when the dairy cow is in transition at about two weeks before calving. This again emphasizes that the transition period spanning the transition period is of critical importance for the immune regulation of the dairy cow. Furthermore, the results can lead to the conclusion that there are mechanisms regulating the composition of monocyte subsets prior to parturition that render the dairy cow more susceptible to developing an infectious disease post-partum.

Future research projects
Based on the promising preliminary results the group is working on further projects to investigate the role of monocyte subsets in the immune regulation of mastitis and the overall immune competence of the dairy cow. Proposals and ongoing projects focus on investigating mechanisms that shape the composition of the different monocyte subsets in individual animals. The group aims to study leukocyte subsets in peripheral blood and samp of transition cows with different levels of negative energy balance. In collaboration with the research group of Dr. Mann, the effect of nutrient-sensing kinases on monocyte phenotype and function will be investigated ex vivo as well as in vitro co-cultures of monocytes and other leukocyte subsets. By being able to link a certain monocyte phenotype or composition to a heightened susceptibility for infectious disease the group envisions to evaluate the immune competence of individual cows. Identifying ‘special needs’ cows ahead of time will give the farmer an opportunity to support these cows and hopefully prevent disease and minimize losses.

Text and photos: Dr. Anja Sipka – Cornell University and Quality Milk Production Services

Scientific work that describes the research results

Sipka A, Pomeroy B, Hussen J, Eger M, Schukken Y, Schuberth HJ. Counts of bovine monocyte subsets prior to calving are predictive for postpartum occurrence of mastitis and metritis. Vet Res. 2017 Feb 21;48(1):13. doi: 10.1186/s13567-017-0415-8.

Hussen J, Duvel A, Sandra O, Smith D, Sheldon IM, Zieger P, Schuberth HJ: Phenotypic and functional heterogeneity of bovine blood monocytes. PLoS One 2013, 8(8):e71502.

Anja Sipka, Brianna Pomeroy, Suzanne Klaessig, Ynte Schukken. Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro Comparative Immunology, Microbiology and Infectious Diseases 2016 DOI: 10.1016/j.cimid.2016.08.001

Sipka A., Gurjar A., Klaessig S., Duhamel G.E., Skidmore A., Swinkles J., Cox P., Schukken Y., 2013. 
Prednisolone and Cefapirin act complementary in resolving experimental E. Coli mastitis. J of Dairy Sci. doi: 10.3168/jds.2012-6455. [Epub ahead of print]

Düvel A., Frank C., Schnapper A., Schuberth H.J., Sipka A. 2012 
Classicaly or alternatively activated bovine monocyte-derived macrophages in vitro do not resemble CD163/Calprotectin biased macrophage populations in the teat. Innate Immun. 18(6): 886-96