Symposium review: Environmental effects on mammary immunity and health*

Environmental effects on pathogen abundance and access are precursors to mastitis. Indeed, high heat and humidity, and unsanitary housing and equipment, are associated with greater pathogen load and exposure. Although less is known about effects of environment on a cow’s ability to resist infection, several indicators suggest that it can affect pathogen responses. Mastitis incidence and bulk tank somatic cell count vary with season, typically peaking in summer. Recent controlled studies have revealed that heat stress exposure results in changes in the microbiome of the cow and her environment, which may relate to negative effects on milk quality and cow health. Alternatively, specific pathogen loads may vary based on housing dynamics rather than associations with physical environment. Indeed, hous-ing-related stressors, such as overcrowding and social group challenge, influence secretion of glucocorticoids, thus affecting pathogen resistance in the cow. Two key seasonal variables are photoperiod and temperature, specifically the heat stress consequent to elevated temperature and humidity. Shifts in light duration regulate immune function in other species, but apparently have limited effect on udder health of lactating cows. In contrast, in dry cows, short days increase peripheral blood mononuclear cell number and are associated with lower somatic cell count in the next lactation, compared with long days. With heat stress, elevated body temperature directly affects expression of immune-related genes in mammary tissue. Responses depend on duration of exposure and feature acute upregulation of immune-signaling pathways, followed by enrichment of other immune-related pathways after prolonged exposure. Most responses are transient and recover within 1 wk. Functionally, heat stress impairs some aspects of acquired immunity in dry cows, including antigen responses and lymphocyte proliferation, but apparently not innate immune function. However, heat stress in late gestation reduces neutrophil phagocytosis and killing in vitro, and neutrophils in circulation are reduced in vivo as are responses to pathogen challenge in the subsequent lactation. A holistic understanding of the complex interplay of environment, pathogens, and host is needed to inform advances in this area.


SEASONAL EFFECTS ON MAMMARY HEALTH
Even in intensively managed animal production systems such as modern dairy operations, several seasonal effects on cow health and productivity can be observed.Examples include disease incidence, variation in reproductive performance, and altered milk component levels throughout the year.Whereas some of these effects involve environmental impacts on the exposure to pathogens, others are more related to specific cow responses.Our objective in this review was to focus on the effects of the 2 primary environmental influences on cows during their life cycle, specifically photoperiod and heat stress, to assess the potential for effects on pathogen exposure and on the cow's response to a pathogen, with specific reference to mammary gland immunity.
Diseases such as mastitis result from a complex interplay among the components of the host-pathogenenvironment triad.Numerous studies have described the fluctuations that occur seasonally in broad indicators of mammary health such as bulk tank SCC (Gillespie et al., 2012;Ferreira and Devries, 2015).These studies generally detail an inverse relationship between milk yield and SCC with the increase in SCC following summer peaks in ambient temperature.For example, Tao et al. (2018) reported that bulk tank SCC in Georgia (USA) reach a peak in September and a nadir in May, consistent with the idea that high ambient temperatures induce greater pathogen exposure of the cow, followed by increases in clinical and subclinical intramammary infections.More controlled studies indicate that variation in the environment alters the SCC within a herd, supporting the idea that seasonal shifts in temperature, humidity, or even photoperiod might influence pathogen loads and exposure.Zeinhom et al. (2016) tracked SCC at the cow level in the same herd across seasons in a subtropical environment to assess the effect of shifts in pathogen load (i.e., the amount of pathogens in the environment) on production and mammary health.Milk samples were collected from cows during summer, fall, and winter with corresponding maximum temperature-humidity index (THI) of >78, 72 to 78, and <72 at each sampling in those seasons.An inverse relationship of milk yield and milk fat concentration was observed with THI, and milk protein concentration was lower in summer relative to the other seasons.Milk SCC increased as THI increased, and the number of milk samples that were positive for Staphylococcus aureus and Escherichia coli increased with THI.These observations at the cow level support the idea that pathogen exposure increases with ambient temperatures and humidity.However, a coincident effect on host resistance, or how the animal responds to a pathogen, cannot be excluded.
Given the effect of season on pathogen load, housing might be expected to affect the exposure and thus response at the mammary gland.Lambertz et al. (2014) examined 4 variations of loose housing to investigate the effect of barn temperature and access to grazing on mammary health, as indicated by SCC.Cows were held in loose housing that was either warm or cold, and in each temperature barn, the animals had access to pasture or no pasture access.Thus, the design was a 2 × 2 factorial of barn temperature and grazing.Somatic cell count was measured throughout the year at monthly intervals, and the local THI at the farm level was determined 3 d before each test day.Across all treatments, THI was positively correlated with SCC, but the housing system did not substantially affect the response.This lack of substantial effect of housing environment on SCC suggests that pathogen exposure, although present, likely does not explain all of the variation in mammary health responses across the year.
Evidence of a direct host response of immune responses with season comes from both in vivo and in vitro studies.Li et al. (2021) examined basal antioxidant and cytokine activity in lactating cows across seasons, comparing basal 8 cows in groups across spring, summer, fall, and winter seasons.Because the responses in spring and fall were not different, those data were combined and compared with extremes of summer and winter.Whereas both low THI (winter) and high THI (summer) affected antioxidant capacity and immune responses relative to the more moderate seasons, the high THI of summer was more of a negative factor on immune responses relative to cold stress effects in the winter.Although any effect of photoperiod changes cannot be excluded, these observations support the concept that heat stress affects cows' ability to respond to pathogen insult.

HEAT STRESS AND MAMMARY HEALTH
In an effort to tease apart the relative effect of elevated temperature versus light exposure on immune function, Lecchi et al. (2016) collected blood from lactating cows and then exposed PMN from those cows to heat stress in vitro to assess the effect of temperature on multiple indicators of immune function.Relative to a baseline temperature of 39°C, overnight culture of PMN at 41°C reduced phagocytic capacity by about 40%.Superoxide production, an indicator of killing ability of the same PMN, was lower in the heat-stressed PMN in both resting and phorbol 12-myristate 13-acetatestimulated states.The responses did not appear to be a function of overall loss of cells, as apoptosis was unaffected by temperature in vitro.These observations are strong evidence that heat stress has direct effect on the function of immune cells.
In a subsequent study, heat stress effects were examined in isolated monocytes rather than the broader PMN population (Catozzi et al., 2020).Using a similar model with blood from lactating cows subjected to basal (39°C) or heat stress (41°C) in vitro, heat stress depressed monocyte viability and increased apoptosis, suggesting that some portion of immune suppression with heat stress is mediated by loss of cell number.Gene expression for heat shock proteins (HSF 1 and HSP-70) were stimulated by the 41°C treatment, consistent with these monocytes attempting to protect cellular function in response to the temperature insult.Expression of the transcription factors STAT 1 and STAT 2 were depressed with heat stress, as was the cytokine IL-10, indicating a direct effect of heat on the relative immune capacity of monocytes in vitro.
Heat stress results in similar decreases in immune status in cows during the dry period.For example, do Amaral et al. ( 2011) compared the IgG response to an innocuous antigen (chicken ovalbumin) in heat-stressed versus cooled cows both during the dry period and after parturition when all cows were managed under active cooling.After an initial immunization at dry off, heat stress cows had lower IgG responses to booster immunizations at 2 and 4 wk when under heat stress.After calving, when all cows were cooled, no difference in IgG response was noted.In addition, a direct, negative effect of heat stress was observed on lymphocyte proliferation during the dry period.Collectively, these data suggest a Dahl and McFadden: LACTATION BIOLOGY SYMPOSIUM direct effect of heat stress to suppress adaptive immune function in the dry period, with potentially significant implications for the transition period.
Additional carryover effects of dry period heat stress on innate immune function have been observed in the following lactation.do Amaral et al. ( 2011) monitored neutrophil phagocytosis and oxidative burst activity during the dry period under heat stress and cooling, and subsequently during lactation in the same cows when all were cooled.There was no difference in neutrophil function between groups in the dry period, but in lactation both phagocytosis and oxidative burst were improved in the animals that had been previously cooled when dry compared with those that were under heat stress.Thompson et al. (2014) found that heat stress exposure in the dry period resulted in lower circulating neutrophil number and activity in lactation despite the fact that all cows were cooled at that time.Further, responses to a Streptococcus uberis challenge were improved by prior dry period cooling, along with greater milk yield and overall performance.Heat stress reduces DMI in dry cows, and could thus explain some of the observed reductions in immune response.However, the DMI reduction in dry cows under heat stress does not affect overall metabolic status or drive animals into negative energy balance prepartum (do Amaral et al., 2011;Thompson et al., 2014).Additionally, after parturition, the cows with the improved neutrophil status (i.e., cooled in the dry period) exhibit elevated concentrations of nonesterified fatty acids and BHB, an observation directly opposite of an expected negative effect on immune function (do Amaral et al., 2009).These observations support the concept that heat stress has direct and persistent indirect effects on immune function in cattle, and these changes in host responsiveness contribute to seasonal changes in mammary health.
If heat stress alters immune function, one expected outcome would be an increase in disease incidence in cows that experience a dry period under hot ambient conditions.Thompson and Dahl (2012) tested that hypothesis by comparing disease incidence in a large herd on a commercial dairy in Florida between cows that were dry in the summer versus those that were dry in winter, when all cows were housed on pasture during the dry period.Cows dry during the summer (June, July, August) had higher incidences of mastitis, respiratory illness, and retained fetal membranes relative to genetically similar herdmates managed under the same conditions in winter (December, January, February).These observations are consistent with the effect of heat stress on immune function translating to depressed disease responses, even after the heat stress insult has been removed.

PHOTOPERIOD EFFECTS ON MAMMARY HEALTH
The preceding discussion does not eliminate photoperiod as a potential effector of environmental effects on mammary health.Indeed, there is strong evidence, particularly in late gestation cows, that manipulation of light exposure alters immune response.Auchtung et al. (2004) exposed cows at dry off to either short (8L:16D) or long days (16L:8D) and the animals remained on those treatments until parturition.Photoperiod exposure produced the expected differences in circulating prolactin (PRL), with increases in PRL under long days.A concomitant inverse relationship to prolactin receptor (PRL-R) expression on leukocytes was observed, with greater PRL-R mRNA in short-day cows relative to long days throughout the dry period.
Prolactin emerges as a potential endocrine mediator of the observed effects of photoperiod on immune function.Auchtung and Dahl (2004) manipulated circulating PRL by exposing calves to long or short days, and then replaced PRL under short-day treatment with daily injections or constant infusion via osmotic minipumps.Effect of photoperiod and exogenous PRL treatments on lymphocyte proliferation and neutrophil chemotaxis indicated that the increase in PRL and reduced PRL-R under long days resulted in lower immune responsiveness.Further, the replacement of PRL to calves under short days completely reversed the effects of short-day photoperiod.Therefore, the hypothesis that photoperiod mediates effects on immune function via shifts in PRL sensitivity is supported.
Another study that supports the concept of influence of photoperiod on mammary immune function comes from a comparison of the interaction of photoperiod and time on the mammary transcriptome during the dry period.Bentley et al. (2015) collected mammary biopsies, at 24 and 9 d before calving, after exposure to a short or long day since dry off at approximately 60 d prepartum.Photoperiod exposure during the dry period affects genes associated with cell proliferation and immune function during lactogenesis stage I and lactogenesis stage II.Time relative to parturition affected genes associated with milk synthesis and immune function, with expression increasing as lactation approached.Functional analysis of differentially expressed genes in the interaction of photoperiod and time indicates that the genes associated with the IGF-1 signaling pathway may mediate the effects of photoperiod on mammary function.

SUMMARY
Seasonal shifts in temperature and light affect the growth and survival of pathogens in the environment, and likely lead to greater pathogen exposure.However, increased pathogen load is not the only reason for seasonal differences in disease.Considering mammary health in the pathogen-host-environment triad, it is clear that exposure to elevated ambient temperatures and humidity with heat stress, and shifts in the duration of light exposure on an annual basis, affect the cow's ability to mount an immune response.Both the acquired and innate arms of the immune system are negatively affected by heat stress, particularly during late gestation and early lactation.Although evidence supports an effect of thermal and photoperiodic cues on several endocrine systems, PRL and IGF-1 emerge as possible mediators of the negative outcomes associated with heat stress and long days on mammary health.

Symposium review :
Environmental effects on mammary immunity and health* G. E. Dahl 1 † and T. B. McFadden 2