Reduced levels of serum 25(OH)D₃ are associated with tuberculosis positive cattle under conditions of high natural exposure to Mycobacterium bovis

Highlights

• Reduced levels of serum 25(OH)D₃ are associated to bovine TB.

• Reduced levels of serum 25(OH)D₃ are associated to low mycobactericidal capacity in bovine macrophages model.

• Levels of serum > 80 ng / mL 25 (OH) D₃ are necessary to counteract infection by M. bovis.

Abstract

Serum vitamin D (25(OH)D₃) concentrations of < 30 ng/mL in cattle are insufficient to induce an adequate immune response against intracellular pathogens, which suggests that the efficacy of the immune response may be highly dependent on the bioavailability of 25(OH)D₃. This study shows an overview of both in vitro and in vivo 25(OH)D3-mediated immune modulation amongst dairy cattle naturally exposed to M. bovis. Tuberculin status was confirmed by interferon gamma release assay (IGRA), and natural exposure was demonstrated by polymerase chain reaction (PCR). Tuberculin (-) cattle have a higher serum concentration of 25(OH)D₃ ($\overline{X}$= 87.12 ng/mL) when compared to tuberculin (+) cattle ($\overline{X}$ = 45.86 ng/mL). Reduced serum 25(OH)D₃ levels are associated with the presence of bovine TB, and serum 25(OH)D₃ levels of > 80 ng/mL are necessary to counteract infection by M. bovis. Kill assays were performed to evaluate in vitro 25(OH)D₃ modulation of intracellular M. bovis growth in bovine macrophages, which showed that reduced serum 25(OH)D₃ levels are associated with diminished mycobactericidal capacity in this experimental model. On the other hand, increased 25(OH)D₃ in culture media enhances phagocytosis and nitric oxide production, which in turn improves capacity to combat M. bovis.

Introduction

Tuberculosis is one of the most studied infectious diseases in the world. However, certain aspects of the disease are still not fully understood [1], [2]. Mycobacterium bovis is an important pathogen belonging to the M. tuberculosis complex (MTBC), which affects a wide variety of both wild and domesticated hosts. As the causative agent of bovine tuberculosis (bTB), M. bovis unquestionably has worldwide zoonotic potential and represents a serious threat to animal and human health. bTB is present mainly in developing countries, causing considerable economic losses and restrictions on beef/cattle commercialization [3]. None of the existing official control and prevention policies focus on bolstering affected animals' immune response to M. bovis [4], [5].

Calcitriol (1,25(OH)₂D₃), the bioactivated form of vitamin D₃, in addition to its well-known role in calcium and phosphorus homeostasis [6], is a powerful immunomodulator. It is a fat-soluble hormone derived from cholesterol whose main source is the photochemical conversion of 7-dihydrocholesterol to vitamin D₃ (cholecalciferol) in the skin through exposure to UV rays; it is then hydroxylated twice- first in the liver, where it is converted to 25(OH)D₃, and subsequently in the kidneys to produce calcitriol [7]. Immune system cells express the genetic codes for 25-hydroxyvitamin D₃ 1α-hydroxylase (CYP27B1), which then allows for intracrine calcitriol synthesis with two essentially opposing effects: while in innate immunity calcitriol participates in macrophage activation and subsequently more efficient microbicidal function [8], [9], [10], [11], [12], in the adaptive immune response it acts on lymphocytes, down-regulating interferon gamma (IFN-γ) production in chronic inflammation [13], [14].

1,25(OH)₂D₃ has long been associated with resistance and immunity to tuberculosis [15], [16], [17], [18], and its role in controlling mycobacterial infection has been proven in recent years. In cattle experimentally infected with M. bovis, a rapid increase in serum calcitriol concentration is observed, along with heightened intracellular calcitriol production in mononuclear cells present in lymph nodes and within the granuloma itself [19]. Macrophages are the main actors involved in the immune response against M. bovis infection, and their mycobactericidal activity depends on the vitamin D signaling pathway [20], [21], [22], [23]. Macrophages stimulated with 25(OH)D₃ and IFN-γ have been shown to reduce intracellular growth of M. tuberculosis by 60% [24]. In addition, recognition of PAMP by macrophages is necessary to activate CYP27B1 expression and the signaling pathway from the vitamin D receptor (VDR), with the subsequent expression of genes related to the production of antimicrobial molecules [22], [25], [26] and an increased amount of cathelicidins, beta defensins, and nitric oxide (NO) [27], [28], [29]. The intracellular production of NO in the bovine macrophage, which has been proposed as a tuberculosis-resistant mechanism, is also dependent on calcitriol [30], [31], [32].

Bioavailability of 25(OH)D₃ must therefore play an important role in defense against tuberculosis infection. Reduced levels of serum 25(OH)D₃ (<30 ng/mL) delay cell differentiation, phagosome maturation, and phagolysosomal fusion, resulting in poor control of the bacteria [12], [33], [34], [35], [36], [37], [38]. The role of vitamin D₃ in the immune response to tuberculosis has been documented via the phenomenon of racial predisposition to the disease. African Americans are more susceptible to clinical illness when infected with tuberculosis, which parallels lower serum 25(OH)D₃ levels. Mycobacterium elimination becomes more efficient in human macrophages derived from peripheral blood mononuclear cells (PMBC) infected with M. tuberculosis and cultured with serum from Caucasians with 25(OH)D₃ concentrations > 28 ng/mL, associated with an increase in cathelicidin mRNA expression (LL37). On the other hand, when monocytes are cultured with serum from African Americans (<10 ng/mL of 25(OH)D₃), cathelicidin expression decreases, favoring mycobacterial survival. Interestingly, the microbicidal activity of macrophages is re-established when they are cultured with serum supplemented with 40 ng/mL of 25(OH)D₃ [35].

A similar situation occurs with other mycobacteria. For example, a dairy herd with natural exposure to M. avium subsp paratuberculosis (MAP) presented an average blood 25(OH)D₃ concentration of 62.5 ng/mL. Animals that were serology positive for MAP had a lower concentration (5.3 ng/mL) of 25(OH)D₃ when compared to MAP-negative individuals [39]. The effect of paratuberculosis infection on serum 25(OH)D₃ concentration has also been evaluated, with clinically ill cows presenting a significantly lower 25(OH)D₃ concentration (58.8 ng/mL) than either subclinical (97 ng/mL) or uninfected cows (84 ng/mL). Thus, serum 25(OH)D₃ concentration is linked to the ability of animals to fight infection [40]. In general, it has been observed that immune system cells are potentially susceptible to changes in serum 25(OH)D₃ concentrations, affecting their initial capacity to control pathogens [28], [36]. Currently, experimental studies specifying optimal 25(OH)D₃ concentrations for an adequate immune response to infectious agents are lacking.

The present study was carried out in a herd of dairy cattle with natural exposure to M. bovis and a bTB prevalence of greater than 20% as demonstrated by the presence of DNA in nasal fluids from both negative and positive reactors to the Single Intradermal Tuberculin (SIT) test. This indicates exposure to mycobacteria, active transmission, and a high probability of becoming infected. When diagnosing bTB by SIT, only 20–30% of the cows were positive, while the rest remained negative. In this herd, bTB management consisted in separating the SIT (+) cows in an adjoining barn, with a fence as the sole division between both structures. Both groups were kept in dairy production with the same diet and husbandry practices, and the same caretakers worked in both barns. To complement the study, animals from a Holstein Friesian bTB-free herd were included. The goal of this experiment was to evaluate a possible association between serum 25(OH)D₃ concentrations and a negative or positive result for bTB on a tuberculin test and an IFN-γ release assay in a herd with high natural exposure to M. bovis. In addition, the mycobactericidal activity of bovine macrophages was assessed in vitro in cultures supplemented with serum from SIT (+) or SIT (-) cows with different 25(OH)D₃ concentrations.