Milk fever in dairy cows is preceded by activation of innate immunity and alterations in carbohydrate metabolism prior to disease occurrence.

The objective of this study was to search for potential alterations in innate immunity reactants and carbohydrate and lipid metabolism in the blood of transition dairy cows before, during, and after clinical occurrence of milk fever (MF) and identify potential predictive biomarkers of disease. One hundred pregnant multiparous Holstein dairy cows were involved in the study starting from -8wks before the expected day of parturition until +8wks postpartum as part of a large retrospective longitudinal study. Health status, DMI, milk yield, and milk composition were monitored during the whole experimental period. Six healthy cows (CON) and 6 cows that showed clinical signs of MF were selected for blood analyses. Serum concentrations of lactate, non-esterified fatty acids (NEFA), ß-hydroxybutyric acid (BHBA), interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor (TNF), haptoglobin (Hp), and serum amyloid A (SAA) were determined. Results indicated that concentrations of serum lactate, IL-6, TNF, SAA, and Hp were greater in cows with MF than those in the CON group at different time points. Moreover, serum lactate, TNF, SAA, and Hp were greater in cows with MF starting at -8 and -4wks prior to parturition. Both principal component analysis (PCA) and partial least squares - discriminant analysis (PLS-DA) showed separated clusters between MF and CON cows at -8, -4, and disease diagnosis weeks. Overall DMI and milk production were lower in MF-affected cows. Additionally milk fat and fat:protein ratio were greater in MF. In conclusion, cows affected by MF showed alterations in some of the innate immunity reactants and metabolites related to carbohydrate metabolism several weeks prior to appearance of clinical signs of MF. Variable importance in projection plots demonstrated that TNF and SAA in the serum were the strongest discriminators between MF cows and CON ones, which might be useful as predictive biomarkers of the disease.

Parturition and hypocalcemia blunts calcium signals in immune cells of dairy cattle.

The stress of parturition in the dairy cow is associated with increased susceptibility to infectious disease. During the periparturient period the demands for calcium are increased; these increased demands for calcium can result in subclinical or clinical hypocalcemia. Periparturient cows also experience significant immune suppression. Because intracellular calcium signaling is a key early feature in immune cell activation, we have hypothesized that the increased demand for calcium in periparturient cows may adversely affect intracellular calcium stores of immune cells. This reduction in intracellular calcium stores in immune cells could blunt intracellular calcium release following an activating stimulus, contributing to the immune suppression seen in these animals. To test this hypothesis, peripheral mononuclear cells were obtained from 27 multiparous dairy cows spanning a period of 2 wk before and 2 wk after parturition. Following activation of these cells by anti-CD3 antibodies plus secondary antibodies, intracellular calcium release from intracellular stores was measured. The intracellular calcium released in response to the activation signal declined as calcium demand for lactation became more intense and recovered as plasma calcium normalized. Intracellular calcium stores in peripheral mononuclear cells, estimated by pretreating cells with pervanadate and ionomycin, significantly decreased at parturition and returned to normal levels as the cows' blood calcium returned to normal levels. Hypocalcemia, which is common in periparturient dairy cows, is associated with decreased intracellular calcium stores in peripheral mononuclear cells. Our data suggest that this is the cause of a blunted intracellular calcium release response to an immune cell activation signal. It is concluded that intracellular Ca stores decrease in peripheral blood mononuclear cells (PBMC) before parturition and development of hypocalcemia. This suggests that systemic calcium stress precedes measurable hypocalcemia, particularly in cows that will develop milk fever. Therefore, PBMC intracellular Ca stores are a more sensitive measure of calcium stresses in transition cow. This decrease in PBMC intracellular Ca stores before parturition and the development of hypocalcemia contributes to periparturient immune suppression.

Effects of extracellular Ca2+ on phagocytosis and intracellular Ca2+ concentrations in polymorphonuclear leukocytes of postpartum dairy cows.

The aim of this study was to determine the effects of extracellular Ca(2+) concentration ([Ca(2+)](e)) on phagocytosis and intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine polymorphonuclear leukocytes (PMNs). The experiments were performed by using blood samples from parturient paretic and clinically normal parturient cows and manipulating the [Ca(2+)](e) in vitro. Phagocytosis by PMNs (with and without stimulation with phorbol myristate acetate and inhibition with cytochalasin B) and resting [Ca(2+)](i) were significantly lower in parturient paretic cows. Repletion of Ca(2+) in the extracellular media for the samples from these animals increased phagocytosis and resting [Ca(2+)](i). In the blood of clinically normal parturient cows, decreasing the [Ca(2+)](e) decreased phagocytosis and resting [Ca(2+)](i) in PMNs, but increasing the [Ca(2+)](e) did not affect phagocytosis. These results suggest that the hypocalcemic condition of parturient paretic cows in vivo causes decreased phagocytosis and resting [Ca(2+)](i) in PMNs, which may partly contribute to greater susceptibility to infection.

Phagocytosis of bovine blood and milk polymorphonuclear leukocytes after ozone gas administration in vitro.

To determine the effects of ozone on the phagocytosis of bovine polymorphonuclear leukocytes (PMNs), ozone gas was administered in vitro on the blood and milk of healthy lactating cows, cows with acute mastitis, and cows with milk fever. In the blood of healthy dairy cattle, although there was no significant effect of ozone gas on the viability of the leukocytes, phagocytosis of PMNs significantly decreased. In contrast, ozone gas administration in vitro significantly increased phagocytosis of PMNs from the blood of cows with acute mastitis and milk fever, and from mastitic milk. These findings showed that ozone administration in vitro has positive and negative effects on bovine PMN phagocytosis, depending on the health status of the animal.

Study of immunological dysfunction in periparturient Holstein cattle selected for high and average milk production.

Data from twenty assays of traits associated with innate and adaptive immunity were evaluated from 137 periparturient Holstein cows. These cows had been selected through planned matings for four different levels of milk production (high and average pounds of milk, and high and average pounds of milk fat plus protein). For up to seven generations, the genetic lines were produced by mating females of each line to sires of corresponding merit. With the exceptions of neutrophil ingestion of Staphylococcus aureus and directed migration, all assays measuring neutrophil functions were depressed beginning 2 to 3 weeks before calving through 3 weeks after calving. Serum concentrations of immunoglobulin G1 decreased while those of immunoglobulin G2 increased around calving time. Serum complement and conglutinin concentrations decreased before calving and reached a minimum around calving time. Cows selected for high milk production (pounds of milk and pounds of milk fat plus proteins) had significantly higher (P < 0.10) numbers of circulating neutrophils and mononuclear cells, had higher (P < 0.10) neutrophil resting chemiluminescence and higher (P < 0.10) neutrophil directed migration than cows with average production potentials. There were significant (P < 0.001) sire progeny group differences for most traits associated with the immune system that we tested. These results can be considered encouraging, in that selection for high milk yield did not produce unfavorable correlated responses in the functional capacity of immune function traits, and that there is sufficient genetic variation in these immunological traits among sires of high genetic merit for milk production to potentially improve the immunocompetence of periparturient cows through planned mating experiments.

IgG from cows with parturient paresis changes the thermodynamic behaviour of bovine erythrocyte acetylcholinesterase.

Parturient paresis affects about 10% of all cows. The most common treatment is injection of various Ca-salts and MgCl2 and about 80% recover after repeated injections. The effect of IgG on the Arrhenius plot of bovine erythrocyte AChE was studied. The plot was normal, i.e. biphasic (a broken line) in the presence of IgG from healthy cows. The highest concentration tested was 10 mg/ml. Before one injection of the Ca-Mg solution 5 mg/ml IgG from sera of paretic cows (n = 10) changed the Arrhenius plots to monophasic (linear). Thus, paretic cows have antibodies against AChE. After injection the change to linear plots was observed already with 1 mg/ml IgG. Apparently, the level of antibodies in serum affecting AChE increased about 5-fold. This is considered to be due to dissociation of bound antibodies from various AChEs. All cows recovered after one injection and release of AChE antibodies might be important for the recovery. Incubation of blood from paretic cows (n = 3) with Ca-salts and MgCl2 increased the amount of free AChE-antibodies about twofold; the Arrhenius plots became linear at 2.5 mg/ml IgG compared with 5 mg/ml before the incubation. This should be due to release from AChEs on blood corpuscles as erythrocytes and lymphocytes. But the increase of antibodies after incubation of blood does not account for the whole increase following the treatment of paretic cows. AChE antibodies are probably also dissociated from other sites, such as neuromuscular junctions.

Periparturient hypocalcemia in cows: effects on peripheral blood neutrophil and lymphocyte function.

Periparturient dairy cows are quite susceptible to intramammary infections and clinical mastitis, epidemiologic evidence indicates that parturient paresis (milk fever) greatly increases the risk of mastitis, although a causal relationship has not been established. In the present experiment the effects of hypocalcemia at parturition on the immune status of dairy cows were investigated. Ten healthy, multiparous Holstein cows were fed a high Ca diet prepartum to induce hypocalcemia at parturition. Five of these cows received intramuscular parathyroid hormone (crude synthetic N-terminus 1-34) to prevent hypocalcemia at parturition. Effects of hypocalcemia on various neutrophil and lymphocyte functions were determined during the periparturient period, ranging from 6 wk prepartum to 5 wk postpartum. All cows exhibited severe loss of immune cell function in the weeks surrounding parturition. Hypocalcemia or the development of parturient paresis did not exacerbate the immune cell dysfunction. This implies that the degree of hypocalcemia observed did not have a large or irreversible influence on neutrophil and lymphocyte function in periparturient cows.