Download MAMMARY GLAND RESPOND DURING THE INITIAL

MAMMARY GLAND RESPOND DURING THE INITIAL
STAGES OF AN INTRAMAMMARY INFECTION (IMI)
Kasey M. Moyes, James K. Drackley, Massimo Bionaz, Dawn E. Morin,
Sandra Rodriguez-Zas, Robin E. Everts, Harris A. Lewin, and Juan J. Loor
TAKE HOME MESSAGES
• IMI with S. uberis resulted in 1,972 differentially expressed genes in mammary tissue
compared with healthy mammary tissue.
•
Genes up-regulated (increased in activity) during infection were primarily associated with
immune system response, and genes down-regulated (decreased in activity) were
involved with milk fat synthesis.
•
This study provided new information on the early response factors of mammary tissue
during IMI with S. uberis; and may provide information to researchers on genes of
interest for genetic selection as well as new mastitis treatment and prevention strategies
for dairy farmers.
INTRODUCTION
The mechanisms involved in the initial stages of the immune system response of the mammary
gland to a mastitis-causing pathogen are not clear. The mammary gland has immune system
functions that contribute to the initial response to an intramammary infection (IMI). Studies have
determined that mammary cells secrete several immune proteins including cytokines such as
interleukin-8 (IL8) and tumor necrosis factor alpha (TNFA) and enzymes such as lysozyme
(LYZ) that help fight invading pathogens. S. uberis is one of the major environmental mastitiscausing pathogens that plague the dairy industry. A better understanding of the early response
factors of the mammary gland may help in the development of new genetic selection criteria as
well as new treatment and prevention strategies to combat S. uberis-associated mastitis. Dairy
researchers at the University of Illinois have access to some of the latest technological advances
in genomics with the use of the bovine microarray. This microarray can simultaneously analyze
thousands of different genes that may provide useful information on the signals produced by the
mammary gland during the initial stages of the immune system response. The objective of this
study was to determine the genomic-level responses in mammary tissue during IMI with S.
uberis in Holstein dairy cows during mid-lactation.
MATERIALS AND METHODS
Ten second-lactation or greater Holstein dairy cows past peak lactation (> 60 days post-partum)
were used. Cows were enrolled if composite milk somatic cell counts (SCC) < 200,000 cells/mL
and aseptically collected quarter foremilk samples were bacteriologically negative prior to IMI
challenge. Eligible cows were inoculated with 5,000 cfu of S. uberis (O140J; provided courtesy
of Dr. Joe Hogan, The Ohio State University) into one rear mammary quarter after the evening
milking. At 20 hours post-inoculation, before the expected peak in clinical signs, both rear
mammary quarters (infected and non-infected contralateral quarters) of all cows were biopsied
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for gene expression analysis. Aseptic quarter foremilk samples were collected periodically postinoculation to confirm infection by quantifying bacterial and SCC concentrations. A 13,257
oligonucleotide (70-mers) bovine array and quantitative PCR (qPCR) were used to measure gene
expression. The MIXED procedure of SAS was used for both array and qPCR comparing gene
expression responses between infected versus non-infected quarters within each cow. The
grouping of genes into certain functions and pathways involved in metabolism and/or immune
response were generated using Ingenuity Pathway Analysis (IPA).
RESULTS AND IMPLICATIONS
All cows developed clinical signs of mastitis during IMI challenge. Infection with S. uberis
affected the expression of 1,972 genes when compared to non-infected quarters. Of these, 1,031
genes were up-regulated (increased) and were primarily involved with the immune system
response. For example, the genes IL8 and TNFA were increased, which encode cytokine proteins
that are highly involved in the pro-inflammatory immune response as well as immune cell
recruitment to the site of infection. These responses could lead to an increase in other immune
cells, such as neutrophils, moving from the blood to the mammary gland, resulting in the
increase in milk SCC observed during mastitis. Within the 941 genes that were down-regulated
(decreased) during IMI with S. uberis, the primary function was milk fat synthesis (e.g.,
lipoprotein lipase [LPL], and CD36]). Very few genes involved in milk protein or lactose
synthesis were affected by S. uberis infection. This finding helps to explain the changes in milk
composition that occur in quarters infected with S. uberis when compared to other mastitiscausing pathogens. In addition, the genes encoding plasminogen (PLAU) and the plasminogen
receptor (PLAUR) were both increased in infected quarters. The activity of plasminogen has been
used as an indication of virulence of S. uberis associated mastitis.
Infection resulted in alterations of IL-10 signaling, IL-6 signaling, LXR/RXR signaling, and
glucocorticoid receptor signaling as the most-affected pathways. IL-10 is an immune systemrelated cytokine that has anti-inflammatory properties. IL-6 is another immune cytokine that has
both pro- and anti-inflammatory properties. Controlling the inflammatory response during
mastitis is beneficial to the mammary tissue since a severe or prolonged inflammatory response
can cause permanent damage to the mammary gland, leading to decreased milk production even
after milk secretions return to normal. Interestingly, LXR/RXR signaling pathways are involved
in metabolic regulations, indicating a relationship between immune system response and
metabolism during an IMI. Glucocorticoid receptor signaling was also a major pathway affected
by S. uberis infection. Glucocorticoids are stress-related hormones that increase in blood around
calving as well as during other periods of stress such as transportation and relocation.
Glucocorticoids have anti-inflammatory properties that contribute to immune system impairment
around calving, which partially explains the high incidence of mastitis during the first weeks of
lactation.
In conclusion, these results provide new insights into the early response mechanisms associated
with IMI from S. uberis. Our results may provide genes of interest for genetic selection as well
targets for development of new mastitis treatment and prevention strategies.
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