A preliminary study through lymphocyte immunophenotyping of the effects of different forms of phosphorus deficiency on dairy cattle blood haematological parameters and immune status

Metabolic diseases, especially macronutrient deficiencies, represent a significant health problem in high-yielding dairy cattle. Interest in phosphorus has increased significantly since the Council of the European Community passed a directive concerning the protection of waters against pollution caused by nitrates from agricultural sources (the Nitrates Directive). European Union Member States have a choice of two ways to implement the provisions of the Nitrates Directive, either by designating vulnerable areas or by drawing up an Action Programme (2). The effect of complying with the Directive is also to limit phosphorus content in the soil, which contributes to a decrease in its concentration in feed for ruminants. Research indicates that the amount of phosphorus in the diet of dairy cattle covers the nutritional requirements of these animals and ensures normal reproductive processes and the skeletal system structure. As dairy production increases, the amount of the available element in the diet does not increase in parallel, which can very often lead to a reduction in phosphorus blood concentration (hypophosphataemia) (4).

Phosphorus is involved in numerous biochemical processes, including energy reactions and mineralisation of the skeletal system, and is responsible for normal nerve conduction. Hypophosphataemia is a common problem on high-producing dairy cow farms, especially during the periparturient and early lactation periods. Phosphorus deficiency can occur in clinical, subclinical and atypical forms. The clinical signs of phosphorus deficiency include periparturient recumbency and postparturient haemoglobinuria (PPH). In the initial stage, periparturient recumbency is characterised by difficulty in movement (lameness, soreness and swelling of the joints) and a fluctuating appetite. In the final stage, the animal cannot maintain a standing position, but its appetite is maintained. Post-parturient haemoglobinuria, manifested by anaemia and the presence of haemoglobin in the urine, is often indicative of the presence of hypophosphataemia in the herd. An increasingly common form of low blood inorganic phosphorus (Pi) levels in dairy cattle is the atypical form. The most common symptoms of it include intermittent appetite disorders, a fall in milk production, increasingly pale mucous membranes and lameness. Such symptoms do not clearly indicate a specific cause and often lead to misguided treatment because the underlying disorder is misdiagnosed (7, 11, 15).

During the periparturient period, in addition to phosphorus deficiency, an efficiency loss in the immune system also affects cows, which increases their susceptibility to infectious diseases. The causes of the development of periparturient immunosuppression have not been fully explained and are therefore considered to be a multifactorial process (18). The literature data show that in animals, the phosphorus level has a significant impact on the immune system function (4, 18). Dairy cattle’s granulocyte counts decline concomitantly with (especially chronic) hypophosphataemia, but this does not interfere with phagocytosis activity (4, 5). In addition, it was demonstrated that the phosphorus status in the mammalian body affected the function of both the cellular and humoral immune responses (12, 15). Cellular immunity is associated with T helper cells (CD4⁺) and cytotoxic T cells (CD8⁺), whereas humoral immunity is associated with B cells. The main functions of the aforementioned cells are to recognise antigens that lead to apoptosis of an infected cell and to create an immune memory of the body (9).

The aim of the study was to determine the effects of different forms of phosphorus deficiency on white blood cell parameters in cows during the periparturient period and on their immune status. These effects were investigated through lymphocyte immunophenotyping by flow cytometry.

The flow cytometry gating strategy is presented in Fig. 1. The first step was selecting single cells for analysis on the FSC-A (pulse area) vs FSC-H (pulse height) plot (Fig. 1A). Next, the lymphocyte population was gated based on cell size (FSC-A) and granularity (SSC-A) parameters (Fig. 1B). Within the lymphocyte population, the percentage of cells positive for the expression of the analysed antigen was determined on the corresponding fluorescence vs SSC-A plots (Fig. 1C–F)

Fig. 1.

Gating strategy for immunophenotyping of bovine peripheral blood lymphocytes. A – single-cell gating; B – lymphocyte gating; C–F – fluorescence cytograms of positive cells

The blood morphology results are presented in Table 1. In all the animal groups under study, a reduction in Pi concentration in relation to the accepted reference standards according to Meyer and Harvey (17) was observed, with the lowest Pi levels noted in animal group III (with PPH) and group IV (with periparturient recumbency).

The haematological test showed a reduction in the leukocyte count in all diseased cows. In group IV, the lowest WBC value, neutrophilia (an increase in the percentage of neutrophils) and lymphopaenia (a decrease in the percentage of lymphocytes) were noted. The percentage of leukocytes forming the TCD4⁺ lymphocyte subpopulation was observed to have increased in diseased cows compared to the control group. The highest value was obtained for group IV animals. The percentage which was the TCD8⁺ lymphocyte subpopulation decreased, with the lowest value noted for cows with atypical hypophosphataemia (group II). The CD4: CD8 lymphocyte ratio in all diseased animals was higher than that in the control group, the highest value having been determined in group II. Expression of the MHC II protein in the cows under study was higher than in healthy animals. In addition, it was noted that the BCD21⁺ lymphocyte subpopulation was larger in diseased animals compared to healthy cows. Anaemia was observed in groups II, III and IV. Normocyte hypochromic anaemia was noted in all the animals with atypical phosphorus deficiency, whereas normocytic normochromic anaemia with a more severe course than that in group II was noted in groups III and IV.

Phosphorus supplementation in animal diets may affect the stimulation of peripheral lymphocyte proliferation and function in many species. In a narrative review, Heyer et al. (9) showed that in pigs in which the effect of using different levels of phosphorus in the feed on lymphocyte proliferation was examined, there was a positive linear relationship between the amount of phosphorus in the diet and the proliferation of these cells. In our study, we found that phosphorus-deficient cows had a lower percentage of CD8⁺ lymphocytes compared to control animals, and the percentage of CD4⁺ lymphocytes was higher than average. In addition, noted research confirming the results obtained by Engstrom et al. (6) that a low-phosphorus diet increased the concentration of 1,25 dihydroxyvitamin D3, which inhibits some lymphocyte functions (including proliferation reactions). It was also observed that the phosphorus supply in feed may affect the B and T lymphocyte counts in the blood (9). Heyer et al. (9) incorporated existing assessments of the percentage of T cells by flow cytometry and found that the CD4 : CD8 lymphocyte ratio in broilers fed a phosphorus-rich feed was higher than that in birds with a phosphorus-poor diet. These results contradict our observations in cows, as higher CD4 : CD8 lymphocyte ratios were observed in all phosphorus-deficient groups than in healthy animals. The contradiction may simply reflect species differences, and in addition, the administration of high doses of phosphorus may affect the body differently to the use of normal doses of this element. In cattle studies, the CD4 : CD8 lymphocyte ratio was shown to be >1 (13). However, it can vary depending on the health status of the animal. In cattle with infectious diseases, this ratio was <1. In the present study, in healthy Holstein-Friesian dairy cattle the average CD4 : CD8 ratio was 1.93, whereas Denholm et al. (3) demonstrated a 2.38 ratio in the same cow breed from Scotland. In addition, they found that the percentage of T cells and the CD4 : CD8 ratio were largely heritable traits in cattle and could be improved through breeding and manipulation of the animal’s living environment. The CD4 : CD8 lymphocyte ratio is indicative of the functioning of the animal and human immune system. It was observed that decreasing values of this parameter indicated an immune system dysfunction and could be considered an indicator of severe infections and malignant neoplasms (3). An almost twice as high CD4 : CD8 ratio was observed in the present investigation in the atypical form of phosphorus deficiency, than was measured in healthy animals. The authors found no information in the available literature on how phosphorus deficiency may affect the cellular response of the body. Mehrzad and Zhao (16) observed that a CD4 : CD8 ratio of over 4 may be indicative of immune dysregulation in animals. Denholm et al. (3) demonstrated that cows with a higher CD4 : CD8 ratio were often prone to developing lameness. In the authors’ own study, the highest CD4 : CD8 ratio was noted in group II animals, in which lameness was also observed at different times. The correlation between immune dysfunction and raised CD4 : CD8 ratios seems to be confirmed by the studies conducted by Denholm et al. (3) and Malafaia et al. (14) in cattle, in which the consequences of subclinical phosphorus deficiency often included problems associated with reduced animal immunity and increased susceptibility to infectious diseases such as mastitis, metritis, brucellosis and other diseases affecting cow reproduction. In our study, at the time of sample collection, no infectious diseases had been recorded. However, based on the anamnesis and clinical examination carried out at a later stage, it emerged that all animals in group II had suffered disorders such as paronychia, mastitis or metritis, or even respiratory disorders within the six months previous to the identification of the Pi deficiency. In the other groups (III and IV), the percentages of animals with these disease histories were lower and ranged from 40 to 50%. This is consistent with the observations of Denholm et al. (3), Malafaia et al. (15), and Mehrzad and Zhao (16). However, in the control group, only one cow developed mastitis at a later stage, and one cow developed paronychia.

Our results indicate an immune system dysregulation, as confirmed by a significant increase in the expression of MHC II on lymphocytes in groups III and IV, suggesting immune system activation. This activation may have multiple causes; in this particular case, it may have been the presence of chronic inflammation or immune stress potentially resulting from environmental and dietary factors, as supported by the findings of Jurewicz and Stern (10). Additionally, the observed increase in the expression of the CD21⁺ antigen present on the surface of B lymphocytes in phosphorus-deficient groups may indicate stimulation of the humoral immune response in these animals, which aligns with the observations of Chen et al. (1).

It should be noted that long-term (even minor) phosphorus deficiency in an atypical form clearly affects the cellular immunity of the body and is seen in the CD4: CD8 ratio. In formerly phosphorus-deficient cows, infectious diseases are more likely to be observed, even after the Pi level have been restored. Despite the less marked symptoms in animals with atypical hypophosphataemia, increased susceptibility of cows to infectious agents and greater disturbance in parameters determining immunity are observed. However, the cellular and humoral response of the body to phosphorus deficiency requires further research.