The role of immune suppression in the battle for quality milk (Proceedings)

The adult dairy cow is in a constant battle with infectious agents trying to invade her body and establish disease.  The mammary gland is especially vulnerable to infection because of its location in relationship to bedding materials that are contaminated with fecal material.  Pol and Ruegg (2007) documented a clinical mastitis incidence in Wisconsin dairy cattle of 40% per year. 

Based on somatic cell count (SCC) analysis and culture data, we know clinical mastitis represents only 25-35% of all intramammary (IMM) infections.  Given that SCC analysis is typically done on a monthly basis, it would seem likely that we do not detect many new IMM infections.  Therefore, it would seem logical to assume that nearly every cow in a milking herd experiences at least one or more IMM infections per lactation, many for a very short duration.  Even though mastitis is one of the most common diseases on dairy farms, these numbers would be much higher would it not be for a strong immune system to adequately deal with mild infections. 

It is not uncommon to see dairy farms dealing with poor cow health to also battle with high SCC's.  This is most likely related to stress on the immune system which impairs its functionality.  Additionally, antibacterial agents alone cannot control an IMM infection without the assistance of a functional immune system.  Depending on the stage of lactation and presence of IMM infections, the immune system has varying levels of functionality.  This paper will provide a brief review on immune protection of the mammary gland, issues relating to immune suppression and how this suppression impacts udder health.

Immune protection of the mammary gland

The primary means of protection for the mammary gland are the physical barriers provided by the skin that covers the gland and the muscular sphincters that close the teat canal between milkings.  In addition, the cells lining the teat duct produce keratin that traps bacteria.  This keratin has natural antibacterial properties.  During the milking process, keratin sloughs off and is flushed out of the gland.  An additional physical barrier is provided by the process of milking, which serves a protective function in that it flushes the contents out of the mammary gland on a regular basis.

If a foreign agent gets past the physical barriers, a reactive protective mechanism must be initiated.  The innate immune system becomes the most important defense system of the mammary gland.  The innate system serves a role to detect and kill an array of pathogens.  A decrease in the function of even one component of the innate immune system can have detrimental effects on the cow's immune response. 

Polymorphonuclear cells (PMN) including neutrophils, basophils, and eosinophils, play a key role in the innate immune system by detecting and containing infectious agents.  While basophils and eosinophils have important functions, the neutrophil becomes the most important PMN for the mammary gland in dealing with IMM bacterial infections. 

Neutrophils make up only 20-30% of the leukocytes in the blood pool in ruminant animals which is a much smaller percentage than other animal species, such as carnivores.  An 800 kg cow has approximately 1.4 x 1011 neutrophils circulating in the blood with an average half-life of approximately six hours.  This means that a cow is replacing one-half of its neutrophils every six hours from bone marrow stores which require a large amount of dietary energy and protein.       

Neutrophils are attracted into the mammary gland when bacterial invasion occurs by a process called chemotaxis.  Chemotaxis is initiated by local changes in the mammary tissues due to bacterial invasion or tissue damage caused by the bacteria.  This tissue damage serves to initiate the complement system which produces substances such as C5a which act as chemoattractants for neutrophils.  Chemotaxis initiates changes to the endothelial lining of local blood vessels causing neutrophils to selectively adhere to the vessel wall and slow neutrophil passage through the vessel. 

The neutrophil then migrates through the vessel wall and into the infected area.   While neutrophils have the ability to be stand-alone killers, they work much more effectively with the assistance of the acquired immune system.  IgG2 and IgM, produced by lymphoid cells, are the primary antibodies in the mammary gland to assist the neutrophil by opsonizing bacteria to promote bacterial uptake by the neutrophil.  Once ingested, the neutrophil kills bacterium through generation of reactive oxygen species (ROS) and release of lytic enzymes and antimicrobial peptides from intracellular granules.

 Periparturient immune suppression

Cows in the periparturient period have the most profound amount of immune suppression experienced throughout lactation.  Not coincidentally, the majority of metabolic diseases, such as milk fever, retained placenta, ketosis and displaced abomasums, occur within the first two to four weeks after calving.  In a study looking at the correlation between metabolic disease and mastitis incidence, milk fever increased the risk of development of mastitis by 8 times (Curtis et al, 1983).  Other studies have demonstrated an increase in mastitis risk in cows that have experienced ketosis, retained placenta, twinning, dystocia, and lameness before the first breeding (Oltenacu and Ekesbo, 1994; Emanuelson et al, 1994; Peeler et al, 1994). 

It is well understood that the process of parturition is a highly stressful process.  In addition to this stress, the dairy cow must initiate colostrogenesis and lactogenesis.  Kehrli et al (1989 a, b) demonstrated that neutrophil and lymphocyte function is depressed in animals during the periparturient period.  This depression is especially prevalent in the dairy cow.   A more recent trial utilized proteomic techniques to compare pre-partum versus post-partum neutrophil function (Lippolis et al, 2006).  In this study, there was a change in protein expression for more than 300 proteins between the two groups.

USDA researchers (Kimura et al, 1999) undertook a trial to determine whether periparturient immune suppression was related to parturition or the initiation of lactation.  The group utilized mastectomized cows versus a control group of intact cows.  All intact cows developed milk fever at parturition and experienced a significant increase in non-esterified fatty acid (NEFA) concentration for the first ten days of lactation.  They determined that mastectomized, post-partum cows underwent similar decreases in neutrophil myeloperoxidase activity at parturition compared to intact controls. 

The difference between the two groups was that mastectomized cows returned to normal neutrophil function more quickly, thus proving that both parturition and colostrogenesis/lactogenesis have independent effects on immune suppression.  When looking at lymphocyte populations though, they found that mastectomized cows had minimal decrease in lymphocyte function while intact cows suffered significant depression in lymphocyte function. 

Clinical and sub-clinical hypocalcemia commonly occurs at parturition.  Goff et al (1996) demonstrated that cows can remain sub-clinically hypocalcemic throughout the first week of lactation.  Depression of contraction rate and strength of smooth muscle is directly proportional to serum calcium concentration.  Decreased function of smooth muscle directly impacts IMM infection status as the teat sphincter will be less completely closed.  In addition, hypocalcemic cows spend more time lying down thus increasing teat end exposure to bacterial invasion.  Both of these factors increase the risk for the development of new IMM infections.

Calcium metabolism in the periparturient period has a direct effect on immune cell function.  Kimura, et al (2006) determined that intracellular calcium plays a vital role in peripheral blood mononuclear cell (PBMC) activation in that lower intracellular calcium level slows PMBC activation.  In cows, intracellular calcium in PMBC decreases before parturition and the development of hypocalcemia.  This systemic calcium stress indicates that the cow is experiencing PMBC immune suppression before demonstrating symptoms of sub-clinical or clinical hypocalcemia.

Energy metabolism is equally important in the development of immune suppression around parturition.  Cows are in negative energy balance for 45-75 days postpartum.  They are also in negative protein balance for a similar period of time.  The development of an immune response against an infection is not without expense to energy metabolism. 

While no work has been done in the bovine, extrapolation of human data from individuals suffering from severe infections indicates that maintenance energy demands increased by 40% above normal. This extra expenditure of energy lasted three weeks beyond the onset of the illness.  Incorporating maintenance energy needs of the cow into this situation, the cow would need to increase her maintenance energy needs nearly 4 Mcal/day. 

If a cow was consuming a lactating diet containing 1.65 Mcal of net energy per kilogram (kg), she would need to consume an additional 2.4 kg of dry matter per day (Goff and Kimura, 2002).  Thus, if a cow is already in negative energy and protein balance at the time she acquired an infection, she would be expected to drive herself in a larger negative energy and protein deficit or produce a less effective immune response with the energy and protein she has available. 

Several studies have looked at the relationship between negative energy balance and immune function.  Dutch researchers (Suriyasathaporn et al, 2000) found that negative energy balance and ketosis in early lactation causes polymorphonuclear cells (PMN's) to react slower to new IMM infections.  In cows that have slower reacting PMN's to new IMM's, the severity of the mastitis is worse.  The reasons for the slower reaction are multi-factorial including:

·         Inhibited phagocytosis and killing due to lower production of ROS.

·         Reduced ability to attract new PMN's to the area via chemotaxis.

·         Reduced capacity to migrate to the infected gland independent of the reduced chemotaxis listed in step 2.

While Suriyasathaporn's group suggested that phagocytosis was actually decreased, several other research groups have found neutrophil phagocytosis to be increased, but the production of ROS is decreased leading to poorer neutrophil function.  This phenomenon is not fully understood but may be explained by a selective depression of the activation pathway of the oxidative burst or that energy is not available for the initiation of the oxidative burst in the presence of large number of pathogens (Burvenich, Bannerman et al. 2007).  Nonetheless, no matter what the reason for the slower response by neutrophils, the severity of IMM infections during this period is worse.

Hoeben et al (1999) found that beta-hydroxybutyrate (BHBA) and acetoacetate, in similar concentrations as found after parturition, inhibited in vitro hematopoietic cells.  They also demonstrated a negative relationship between BHBA and PMN chemiluminescence.

Kremer et al (1993) undertook a study looking at the relationship between in vitro neutrophil chemotaxis and the severity of induced E coli mastitis between ketotic and non-ketotic cows.  In non-ketotic cows, mastitis cases ranged from mild to severe with the severity of the infection negatively correlated with the degree of pre-calving neutrophil chemotaxis.  The experimental infection in the ketotic group resulted in all cows developing severe mastitis regardless of the state of pre-calving neutrophil chemotaxis. 

Immune suppression in other stages of lactation

The length of the negative energy balance in early lactation appears to have an effect on immune suppression in that cows that have minimal periods of negative energy balance have less profound effects on immune suppression as compared to cows that undergo longer periods of negative energy balance.  However, in a separate trial, Perkins et al (2002) found that feed restriction in mid-lactation, regardless of length, did not have an effect on the immune response when cows were subjected to IMM endotoxin injections.  The lack of a demonstrated effect on immune response in mid-lactation most likely was related to the stage of lactation in which the cows were exposed to the treatment and underscores the multi-factorial nature of immune suppression.

As cows age, there appears to be a decrease in immune function as evidenced by older animals being more severely affected by infectious agents.  This reduced function is related to impaired function of neutrophils along with a reduced production of ROS in older animals (Burvenich, Bannerman et al. 2007).

In a recent review of immune function and its relationship to E coli mastitis (Burvenich, Bannerman et al. 2007), the reviewers detailed the results of several papers looking at genetic control of immune function.  In their review, the authors state that high milk yield will not have a genetically related negative impact as related to immune function compared to lower producing cows.  However, it is well known that as milk production has increased over time, so has the incidence of clinical mastitis.  This may be associated with the degree of negative energy and protein balance high producing cows experience and changes in teat anatomy of high producing cows rather than genetic changes to the innate immune system.

Summary

Immune suppression relating to poor performance of quality milk programs is often overlooked by dairymen and veterinarians. The most apparent component of IMM immune suppression is related to activities that occur during the periparturient period.  This immune suppression is multi-factorial, thus reduction of immune suppression must have multi-pronged approach. 

While many research groups are looking at ways to alter the immune response of the cow to IMM infections, those breakthroughs may be years in coming.  The most practical approaches for today's dairyman include a combination of improving and maintaining cow hygiene, managing diets around the periparturient period to minimize dry matter intake and hypocalcemia, and implementing procedures to minimize cow stress. 

Veterinary practitioners can play an important role in helping dairy farmers monitor and improve milk quality.  In situations where immune suppression is evident, it may be prudent to concentrate the resources of farm personnel at improving immune function of the herd rather than concentrating on controlling the mastitis problem.  In addition, implementing monitoring programs, as simple as monitoring disease incidence and feed consumption during key periods of lactation, will help indentify situations where excessive immune suppression is occurring.