The influence of probiotic administration on selected leukocyte subpopulations and the serum amyloid A concentration in the peripheral blood of dairy cows during different lactation periods

In dairy cow breeding, maintaining a high milk yield and the profitability of its production depends on many factors: mainly the fertility and health of cows, their genetic predisposition, the appropriacy of their maintenance, the feeding method and the quality and balance of the feeding ration. The cycling of cows through pregnancy, delivery and milk production determines consecutive, correspondingly cyclical lactation periods, i.e. the dry period, perinatal period and lactation. Cows in a herd are functionally distributed to appropriate sections in accordance with these periods. This facilitates milking and provision of nutrition strictly adapted to the given lactation period and thus to the current needs of the cows (24). The health of cows is determined by many internal and environmental factors that may come into play in different periods of lactation (29). However, it is important to emphasise the importance for dairy cow health of the perinatal period, which includes the last weeks of the lactation period until about 60 days postpartum (DPP). Within this time, the transition period – from three weeks before parturition to approximately three weeks after it – merits the closest attention. The numerous physiological and hormonal changes that occur in the bodies of dairy cows during the perinatal period allot a fundamental role to this period in determining the course of all subsequent lactations (14). The perinatal period has the highest prevalence of metabolic and deficiency diseases such as postpartum hypocalcaemia, hypomagnesaemia, fatty liver, ketosis, mastitis, metritis and abomasal displacement, as confirmed by numerous studies (23, 30). The cow’s immune system is also temporarily suppressed during this time (19), and this state in dairy cows during the peripartum period is closely related to the metabolic diseases cited above or predisposes cows to mastitis, metritis and placental retention (15). Although constantly evolving, our knowledge of the dynamics and pathophysiology of immunosuppression during this period remains unsatisfactory. Effective strategies to improve defence mechanisms while maintaining cows’ health and well-being during this critical period, as well as during other periods of lactation, remain to be devised (1).

There has been a growing interest in the use of probiotics to enhance milk production and cow health in recent years. Probiotics are natural strains of intestinal bacteria specific to the digestive tract of an animal species. Probiotic products contain live or dead microorganisms and substances produced by them. When administered orally, they colonise the intestines and prevent the overgrowth of pathogenic microorganisms, thereby improving digestion and optimising feed utilisation (5). By helping to stabilise the balance of the microbial population and enzymatic activity in the gastrointestinal tract, they have a positive effect on animal growth, development and productivity (3, 31). Probiotics may contain one or several strains of microorganisms and may be administered in the form of microbial additives, separately or in combination with other substances (32). The mechanism of action of probiotic microorganisms introduced to the gastrointestinal tract of an animal is complex and multifaceted. Primarily, these organisms compete for adhesion on the intestinal epithelium, for nutrients and for bacteriostatic substances. In addition, they inhibit the development of pathogens and stimulate systemic immunity (41). The precise mechanism underlying the immunomodulatory effects of probiotics remains poorly understood. The impact of whole probiotic bacteria cells or their fragments on T and B lymphocyte function as mediators of an immune response to antigens has been demonstrated in humans and mice. This effect is believed to occur via a complex formed between the bacteria and cells of the gut-associated lymphoid tissue. Probiotics also stimulate immunocompetent cells to secrete cytokines that suppress, modulate and regulate the intensity of systemic and local immune responses (12). It is currently unclear what impact probiotics have on immune system function in dairy cows; however, the majority of published research, predominantly in the form of review articles, has demonstrated a positive effect (8, 40).

The objective of the study was to conduct a comparative assessment of selected leukocyte subpopulations and the SAA concentration in peripheral blood mononuclear cells of cows administered a probiotic as a nutritional additive and these subpopulations and concentration in this blood of cows fed without the probiotic addition, during different lactation periods.

The aim of the present study was to determine whether probiotic supplementation has an effect on the function of the immune system in dairy cows and the magnitude of any effect at different stages of lactation. Selected indices of the systemic immune response in cows were assessed during the period from the last week of lactation, through the dry period and the peripartum period to the peak of the following lactation (i.e. 90 DPP). The effects of probiotics on specific parameters of the immune system in dairy cows have rarely been described in detail in the literature. The majority of studies on cattle made general statements regarding the immunomodulatory effect of probiotics in this animal species. Furthermore, the majority of research undertakings on probiotic support of the dairy cow immune system spanned a limited time of probiotic use and observed a short period of their effect. Additionally, this research has often been limited to the transition period (8, 28). It is evident that this is the most crucial phase of the entire lactation process, as it demands adaptation to complex metabolic transformations caused by parturition and the commencement of milk production (14, 37). However, changes in feeding conditions occur several times during lactation: firstly, before drying off, then besides the change during transition, also when adjusting the postpartum feed ration to a cow’s current milk yield. The authors maintain that knowledge of the use of probiotics at different stages of lactation is important, because it allows us to decide whether probiotic use is reasonable in terms of supporting the activity of the immune system. It may be learned that the limitation of their use to the transition period is sufficient (5).

An increase in the percentage of Foxp3⁺ regulatory lymphocytes and activated B lymphocytes (CD25⁺) and a simultaneous decrease in the percentage of nonactivated B lymphocytes (CD21⁺) was observed in the assessed subpopulations of leukocytes in cows from the experimental group. This may mean that the increase in the percentage of regulatory lymphocytes results in the activation of B lymphocytes, which is manifested by the heavier presence of the CD25⁺ activation receptor on these lymphocytes. This is evidence for not only the potentiated activity of cellular mechanisms (phagocytosis and phagocytic killing) previously described by Brodzki et al. (5), but also the activation of humoral immune mechanisms in the group of cows under study. These changes may result from the triggering of regulatory processes in the immune systems of cows receiving the probiotic. Regulatory lymphocytes contribute to the stabilisation of the cow’s immune system through secretion of anti-inflammatory cytokines, such as IL-10. The appropriate mechanisms necessary to eliminate a possible threat and at the same time maintain homeostasis are activated. Lymphocytes which are CD4⁺ Th cells, the task of which is to support cellular activities and humoral mechanisms of immunity, also contribute to this phenomenon. Specific subpopulations of activated Th lymphocytes redirect the immune response by secreting appropriate cytokines to Th1, Th2 or T17 type (16). In this study, the Th lymphocytes and hence the direction of the immune response were not precisely differentiated; however, a higher percentage of T CD4⁺ lymphocytes in cows receiving the probiotic than in cows in the control group was demonstrated throughout the postpartum period. In addition, a rise in the percentage of lymphocytes, granulocytes and monocytes with β2 (CD18+) and αM (CD11b⁺) integrin receptors and the persistence of the percentage at an elevated level were also observed, which should be associated with a primed leukocyte state and the possibility of their adherence to the vascular endothelium blood vessels in the target tissues. The next stages of the diapedesis process initiated by integrins are the penetration through the wall of blood vessels into the tissues and combat against or removal of possible threats at the site of exposure in peripheral tissues.

Special attention should be paid to the immune mechanisms occurring in cows in the control group. The course and direction of the immune phenomena were similar to those in the experimental group, taking into account the proportions of individual leukocyte subpopulations. However, based on the obtained results, the immune system activity in this group of cows was significantly lower. Additionally, the increasing percentage of CD8⁺ cytotoxic/suppressor lymphocytes in cows from the control group at three examination dates before and after parturition may indicate periodic switching of the immune response mechanisms from humoral to cellular, and shows fluctuations in the activity of these mechanisms. There are too little data for a more precise assessment, and further research should be conducted; however, the immune system activity of the cows in the control group was less stable than that of the experimental group. This finding is of significant importance, as it allows the assumption that cows supplemented with the probiotic had a superior immune status prior to parturition and were more capable of adapting in the postpartum period. This is in accordance with the available literature, which indicates that during this period, the immune response of cows is weakened, increasing their susceptibility to infections, particularly those affecting the uterus and udder (30). The precise causes of immunosuppression during this period remain unclear.

A contributive factor to postpartum dairy cow immunosuppression may be the elevated levels of nonesterified fatty acids (NEFA) and ketone bodies during peripartum negative energy balance, this having been indicated by investigations of bovine blood (14, 17). Levels of NEFA and β-hydroxybutyrate acid (BHBA – a ketone body) were not evaluated in this study, but it cannot be excluded that concentrations of these metabolites were increased in these cows. The study presented by Nocek et al. (26) seemed to confirm these hypotheses, as it showed that the concentration of NEFA was lower in cows that were fed a diet containing Enterococcus faecium. Similar results with Bacillus subtilis natto as a dietary supplement were obtained by other authors (27). Cows supplemented with Enterococcus faecium had lower BHBA concentrations in the postpartum period in the study by Nocek and Kautz (25). Furthermore, Luan et al. (22) observed reduced postpartum ketone concentrations in cows supplemented with Bacillus pumilus. During periods of high energy demand at the beginning of lactation, lower concentrations of NEFA and ketones suggest that cows mobilise less energy from adipose tissue. In addition, increased glucose and insulin concentrations were observed in the blood serum of cows fed Enterococcus faecium in the postpartum period, in contrast to animals not fed probiotics (26). There are at least two reasons why higher blood glucose levels and lower adipose tissue mobilisation (lower NEFA and BHBA) in the postpartum period are very beneficial for cows. Firstly, more glucose can be directed to the udder for milk production, which is important for the maintenance of high milk yield. Secondly, glucose is an essential source of energy for ongoing physiological processes, for the immune system and especially for phagocytes. It may also affect the ratio of leukocyte subpopulations (10, 18).

The present study was not able to show a direct effect of the probiotics on the energy status of the cows, as the indices mentioned above (NEFA, BHBA and glucose) were not evaluated. However, the possible mode of action of probiotics as it relates to what our study did assess can be partially explained by the relationships referred to above and presented in the cited publications. The results suggest that the probiotic applied in the trial was able to improve the energy balance of the cow and that it also indirectly changed the assessed leukocyte subpopulations. Probiotics also significantly improve the digestibility of the feed consumed by cows and at the same time increase the systemic concentration of energy components, proteins, vitamins and minerals (42), which may also be important for immune cell function. According to Galdeano et al. (12), probiotics may also stimulate the immune system in animals through the mechanism of altering the composition of the gastrointestinal microbiota, which may shift the direction of the immune response from Th2 (humoral) to Th1 (cellular), thereby maintaining immune homeostasis in the intestines. This would mean that probiotics can stimulate appropriate immune cell subpopulations, mainly dendritic cells, T lymphocytes and plasma cells, to produce cytokines, thanks to which they can modulate the immune response (9, 20). The direction of the response depends on the probiotic used, as different probiotic strains can stimulate the secretion of different cytokines (11, 33). The mechanism for the maintenance of immune homeostasis is not fully understood. However, it is believed that it is by stimulating cytokine secretion that probiotics promote the adaptive immune response, in which T regulatory lymphocytes (CD4⁺, CD25⁺ and Foxp3⁺) play a major role and producing an anti-inflammatory cytokine – IL-10 – which modulates the immune response (21, 35).

The full mechanism responsible for the changes in leukocyte subpopulations in the blood of the experimental cows cannot be demonstrated on the basis of our research. Similar research conducted by Galdeano et al. (13) also did not indicate a specific mechanism of immunomodulatory activity exerted by probiotics. It cannot be excluded that the modulating effect of probiotics was imparted in our research in the same way as it was in the research referred to above. In particular, the previously described increase in the number of Foxp3⁺ regulatory lymphocytes in cows treated with the probiotic seems to be the leading mechanism regulating these changes. The increase in the percentage of Foxp3⁺ lymphocytes in our study resulted in the presence of a greater number of CD4⁺ Th lymphocytes, activated B CD25⁺ lymphocytes and β2 CD18+ and αM CD11b+ integrins. This arrangement in leukocyte subpopulations presumably enabled the cows to use of both cellular and humoral mechanisms simultaneously. Therefore, it can be concluded that the immune system of a cow that received the probiotic was better prepared to react in the event of an emergency than the immune system of a cow that did not receive the probiotic. At the same time, despite the readiness of the immune system, its stabilisation and even equilibration was also observed. In this context, note should be taken of the constantly low level of SAA throughout the experiment in the cows in the probiotic group compared to the control cows. Blood APPs characteristic of cattle, as SAA is, are mainly produced by hepatocytes in the liver in response to pro-inflammatory cytokines (e.g. IL-6 and tumour necrosis factor (TNF) and glucocorticoids (2, 39). Serum amyloid A is an apolipoprotein that appears as a first-line protein up to 24–48 h after the onset of the inflammatory factor, e.g. after infection, and its secretion is dependent on IL-1 and/or TNF-a (38). Changes in the secretion of SAA are observed in many conditions related to both systemic and local infections of the body, stressful situations, deficiencies, and even during ongoing physiological changes occurring in various periods of lactation (6, 7, 38). The persistently high concentration of SAA and its significant fluctuations during the study in cows that did not receive the probiotic may be manifestations of the continuous increased activity of immune cells participating in nonspecific immune mechanisms, which results in the production of APPs in liver cells. This may mean that the immune system of the cows in the experimental group activated the appropriate regulatory processes preventing the immune cells from reacting too rapidly to the factors, despite their readiness. In other words, the tolerance of immune system effector cells may be much higher in cows that received the probiotic than in cows which did not. Recent reports indicate that the phenomenon of immune system tolerance is very important in maintaining homeostasis in cows (34).

The presented research showed an increase in the percentages of Foxp3⁺, CD4⁺ Th and activated B CD25⁺ lymphocytes and β2 CD18+ and αM CD11b+ integrins in cows after probiotic treatment. Activation of regulatory processes in the immune system of cows receiving the probiotic allowed the simultaneous use of both cellular and humoral mechanisms. This specific pattern of leukocyte subpopulations may indicate the activation of mechanisms that are necessary to eliminate possible harmful factors and, at the same time, to stabilise the immune system. This was confirmed by the persistence of a constant, low level of SAA throughout the course of the experiment in the cows receiving the probiotic. Therefore, it can be assumed that the immune system of cows treated with the probiotic was better prepared to react to infectious or harmful agents, and that it more easily adapted to changes in conditions in different lactation periods, especially in the postpartum period. The use of probiotics can reduce the incidence of infectious diseases in dairy cows.