Effect of abrupt weaning at housing on leukocyte distribution, functional activity of neutrophils, and acute phase protein response of beef calves

Weaning is an inherent husbandry practice in cow-calf beef production systems that imposes physical, psychological, and nutritional stressors on calves. Integrated calf-to-beef production systems, such as seasonal grass-based systems, often combine weaning and housing [1], whereas non-integrated systems often combine weaning with additional stressors such as transportation and marketing, prior to entry into feedlots [2]. Following abrupt weaning, beef calves exhibit distress behaviours [3, 4], with alterations in hormonal mediators of stress [5, 6] and immune function [7–9] evident up to 7 d post-weaning. Furthermore, weaning is considered to be a predisposing factor to bovine respiratory disease (BRD) [10, 11].

Neutrophils provide the first line of cellular defence against pathogens, whereas lymphocytes are of pivotal importance in cell-mediated and humoral immunity [12, 13]. Although studies have examined neutrophil and lymphocyte function and distribution following transport [14, 15], and during natural and experimental cases of BRD [16–18], none have investigated the direct effects of weaning on these immune variables in beef calves. Additional information on the immune status of newly weaned calves at a time when pathogen exposure is heightened may be useful for identifying animals likely to succumb to infection.

Thus, the objectives of the study were to examine the effect of abrupt weaning at housing on i) peripheral leukocyte and differential counts, ii) granulocyte positive neutrophils and lymphocyte immunophenotypes, iii) phagocytic and oxidative burst activity, and surface expression of CD62L of neutrophils, and iv) the acute phase protein response in beef calves.

The immune variables measured in the present study showed that weaning together with movement of beef calves from a pasture environment to a housing environment elicited transient neutrophilia, impaired neutrophil phagocytic function, decreased peripheral lymphocyte count and altered immunophenotypes. To the authors' knowledge, this is the first study to collectively examine these immune responses to weaning and housing in the beef calf.

Stress-induced changes in immune function have been documented in cattle, with alterations to cell-mediated and humoral immunity having a significant impact on immunocompetence which may render an animal more susceptible to infection [19–22]. In the present study, neutrophil surveillance and phagocytic activity were affected by weaning, however bactericidal activity was unaffected. Post-weaning, peripheral neutrophil count increased which was most likely resultant of the decreased surface expression of L-selectin as defined by the reduced MFI CD62L on blood neutrophils. Reduction of this adhesion molecule from the surface of neutrophils, where it is constitutively expressed on resting cells prior to activation, suggests that weaning may have had a negative impact on the ability of these cells to roll along and adhere to the endothelium lining of blood vessels and thus, more cells remained in circulation as evidenced by neutrophilia. This phenomenon has been documented in physiologically stressed cattle, most notably coinciding with parturition [23–25] and following transportation [26].

Additionally, weaned calves had reduced percentage of neutrophils performing phagocytosis compared with control calves. This transient depression in phagocytosis was short lived with cells recovering this function by 7 d post-housing. This finding concurs with the other findings that report a transient decrease in neutrophil phagocytic activity in weaned foals [27]. Decreased phagocytosis has also been reported in horses following transportation [28], in cows that were milked only once-a-day [29], and in dairy cattle following administration of ACTH and dexamethasone [30–32]. Although phagocytic activity of neutrophils was impaired immediately post-weaning, the ability of these cells to perform oxidative burst activity was unaffected, implying that any internalised particles or bacteria could be efficiently degraded despite the depression in particle uptake by neutrophils. Unaltered respiratory burst activity was reported in 14 mo old Holstein-Friesian bulls that were subjected to either Burdizzo castration or hydrocortisone infusion [33]. Moreover, in vitro studies examining pharmacological high doses of infused hydrocortisone and therapeutic doses of glucocorticoids did not affect the oxidative burst activity of neutrophils isolated from high milk yielding Holsteins 2 - 5 weeks post-partum [34].

Appropriate lymphocyte responses provide immunoprotection against bacterial and viral antigens but can also critically influence immunopathogenesis of disease [35–37]. In agreement with other studies [6, 7, 38], weaning decreased peripheral lymphocyte count. Further investigation of the lymphocyte immunophenotypes showed that there was a greater reduction in the percentage of γδ T cells than αβ T cells post-weaning. Transient decreases in proportion of WC1⁺ lymphocytes were observed in both treatment groups, with the greatest degree of change observed when weaning and housing were combined. Calves held in conventional housing had significantly lower percentage WC1⁺ T cells compared with calves that were allowed free-range grazing and access to their dams [39]. Moreover, a decrease in WC1⁺ T cells in the free-ranging calves coincided with a change from an outdoor to indoor environment following a period of transportation. Contrary to the present findings, percentage WC1⁺ T cells did not return to baseline in that study, rather they remained decreased and after 3 weeks post-housing were not different from calves that were permanently housed [39]. The additional stressor of transportation may have contributed to the prolonged response observed in the latter study. Our findings are in agreement with other studies which have shown that γδ T cells are most sensitive to physiological and pharmacological stressors with reductions in percentage WC1⁺ lymphocytes observed following transportation of beef steers [14], parturition in dairy cows [40] and dexamethasone treatment in dairy and beef bulls [41, 42].

In the present study, the decrease in percentage αβ T cells post-weaning was evidenced by transient decreases in percentage CD4⁺ and CD8⁺ lymphocytes. Decreases in both peripheral CD4⁺ and CD8⁺ lymphocytes have been reported in calves at unloading after 14 h truck transportation, following which they returned to pre-transport values by 24 h [14], and in beef steers following induction into a feedlot environment [43]. Some studies examining the effect of stress in domestic animals have utilised the CD4⁺: CD8⁺ ratio as an indicator of stress-induced immunosuppression, however conflicting evidence exists in cattle [44–47]. Our findings showed that movement of beef calves from a pasture environment to a housing environment was able to elicit a transient increase in CD4⁺:CD8⁺ ratio. This increase may reflect recovery of immune competence in an attempt to restore homeostasis following the initial reaction to the onset of stress. An overall rise in CD4⁺: CD8⁺ ratio was reported in sheep that were housed either in isolation or in groups [47]. These authors attributed the rise in CD4⁺:CD8⁺ ratio to recovery of immune compensation following perturbation to lymphocyte subsets due to a stressful change in the environment. Thus, CD4⁺:CD8⁺ ratio may provide more information on the recovery of homeostasis rather than occurrence of immunosuppression. Further research into the utility of this ratio to describe a stress scenario in domestic animals is warranted.

In the present study, percentage MHC class II⁺ lymphocytes in peripheral blood increased significantly in weaned calves and to a lesser extent in control calves indicating that a change in environment in combination with and without weaning resulted in the activation of lymphocytes. This increase in percentage MHC class II⁺ lymphocytes may be due to increased B cells or activated T cells in circulation [48]. Increase in MHC class II⁺ lymphocytes may be due to expansion of NK cells bearing MHC class II molecules [49], however, this supposition cannot be confirmed by the findings of the present study as NK cells were not investigated. Our findings concur with Riondato et al. (2008) [14] who reported increased percentage MHC class II⁺ cells in transported beef steers, but in that study the response was more prolonged (> 7 d). Holstein bulls challenged with pharmacological doses of dexamethasone were reported to have increased percentage MHC class II⁺ cells compared with non-treated controls [50]. Furthermore, increased percentage MHC class II⁺ lymphocytes have been reported following repeated restraint and isolation stress in lambs [51].

The reduction in lymphocyte subsets is most likely attributable to a redistribution of these cells from the peripheral circulation to immune compartments or tissues of greater importance during a stressful event. Trafficking of cells is an important and dynamic factor for effective cell-mediated immunity and stress has been shown to influence this process [52–54].

It is important to note that despite the numerous changes in leukocyte counts, neutrophil function and lymphocyte immunophenotypes observed, generally these perturbations were transient and homeostasis was restored by 7 d post-weaning. Additionally, changes in some of the immune-related biomarkers, namely the proportion of γδ T cells and MHC class II⁺ cells in peripheral circulation and the phagocytic function of blood neutrophils, were evident following change in environment alone. Further research is warranted to investigate the potential use of these measures as prognostic biomarkers of stress sensitive and consequently, infection susceptible animals in response to other stressors.

In line with the previous study, total leukocyte count increased following weaning [6]. Monocyte, basophil and eosinophil counts were not sensitive to weaning or change in environment as evidenced by the unaltered profiles pre- and post-weaning.

Previous studies have shown that weaning induces an acute phase protein response in beef calves [9, 55, 56], whereas in the present study, although there was a numerical increase in fibrinogen and haptoglobin concentration, it was not statistically significant. Increased and unchanged concentration of fibrinogen and haptoglobin, respectively in weaned bull calves has been reported [7], whereas elevated responses in weaned steers have been reported for fibrinogen [6, 55] and haptoglobin [8, 9, 55].

Abrupt weaning at housing affected total leukocyte, neutrophil and lymphocyte counts, lymphocyte immunophenotypes and functional activity of neutrophils in beef calves. Neutrophilia was evident post-weaning, however, crucially, the functional capacity of these cells to effectively traffic from the periphery and phagocytose bacteria was temporarily decreased. Additionally, weaning resulted in a temporary reduction in lymphocyte subsets, most notably of γδ lymphocytes. Considered together, abrupt weaning transiently impaired immune function in beef calves and thus, in terms of immunocompetence, weaning management strategies with the potential to minimise stress in beef calves should be considered.

All animal procedures performed in this study were conducted under experimental licence from the Irish Department of Health and Children in accordance with the Cruelty to Animals Act 1876 and the European Communities (Amendment of Cruelty to Animals Act 1876) Regulation 2002 and 2005.