Lesions of thymus and decreased percentages of the peripheral blood T-cell subsets in chickens fed on diets excess in selenium

Abstract

Selenium is an essential trace element possessing immune-stimulatory properties. The purpose of this 42-day study was to investigate the effects of excess dietary selenium on cellular immune function by determining morphological changes of thymus and peripheral blood T-cell subset. Three hundred 1-day-old avian broilers were fed on a basic diet (0.2 mg/kg selenium) or the same diet amended to contain 1, 5, 10, 15 mg/kg selenium supplied as sodium selenite (n = 60/group). Pathological lesions were progressed with the dietary Se level increased. Grossly, the volume of thymus was decreased. Histopathologically, lymphopenia and congestion were observed. Ultrastructurally, mitochondria injury was observed. In comparison with that of control group, 5, 10 and 15 mg/kg dietary Se decreased the percentage of CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells of the peripheral blood, as measured by flow cytometry. The results showed that excess selenium (more than 5 mg/kg) intake could cause lesions of thymus and decrease of T-cell subsets. The cellular immune function was finally impaired in broilers.

Introduction

Selenium (Se) is an essential trace element possessing immune-stimulatory properties. The effects of Se on the immune system have been reviewed in recent publication. ¹ Se compounds were reported to regulate the function of neutrophils, NK cells, B lymphocytes and T cells, ² and to increase lymphocyte GPx activity. ³ Diets specifically deficient in Se were found to reduce T-cell numbers and impair human lymphocyte proliferation in response to mitogen. ⁴ In chicken, Se supplements in the diet increased both antibody titers to Newcastle disease virus and antibody responses to Salmonella and aflatoxin vaccination. ⁵ However, the immune function was reduced by high level of dietary Se. Previous studies reported that excess Se-Met intake (2 mg/kg) resulted in B cell toxicity in mice. ⁶ An excess level of Se (1 μmol/L) inhibited Con A-induced T-cell mitogenesis in vitro. ⁷ Lymphocytic necrosis and atrophy of lymphoid organs (spleen, gut-associated lymphoid tissue, and lumbar lymph nodes) were observed in mallards that died from more than 20 mg/kg Se-Met. ⁸

As thymus is of vital importance in the differentiation and development of T cells, data showed that in young adult mice, about 1% of thymocytes migrate from thymus to periphery per day. ⁹ T lymphocytes mediate cellular immune function. The percentage of peripheral blood T-lymphocyte subsets is an important parameter that represents the composition of mature T cells in the body. The composition of mature T cells decides the biological function of mature T cells and finally relates to the cellular immune function of the body.

In poultry and livestock, additions of sodium selenium to feed stock are necessary, for prevention of Se-deficiency disease. However, Se poisonings have occurred because of errors in feed preparation. The clinical signs and pathological changes have been observed in Se toxic animals.^8,10–12 However, there have been no reports about the effect of dietary excess Se on the peripheral blood T-cell subsets and thymus in broilers so far. In the present research, the experiment was conducted with the objective of examining the effects of dietary excess Se on the percentages of peripheral blood T-lymphocyte subsets and thymus, and judging the cellular immune function of chickens by methods of experimental pathology and flow cytometry (FCM) and to provide helpful materials for the same or similar studies in both human and other animals in the future.

Materials and methods

Chicken and diets

All procedures were approved by the Si-Chuan Agricultural University Institutional Animal Care and Use Committee. Three hundred 1-day-old avian broilers were obtained from a commercial rearing farm (Wen-Jiang poultry farm, Sichuan province) and randomly assigned to five groups of 60 each. Chickens were placed in 20 one-square meter cages. The temperature inside the rearing house on arrival was 33–35°C and was decreased by 3°C each week until 22°C. The relative humidity was 65%–67%.The lighting program was 24 h of light. A corn-soybean basal diet was formulated. Sodium selenite was mixed into the corn-soybean basal diet to produce 1, 5, 10 and 15 mg/kg of Se (excess Se) diets. Nutritional requirements were adequate according to National Research Council. These diets and water were provided for ad libitum consumption throughout the 42 days of experimentation.

Relative weight of thymus

At 7, 14, 21, 28, 35 and 42 days of age during the experiment, after the body weight was weighed, five birds in each group were euthanized and necropsied. The macroscopic changes of thymuses were observed and recorded. Thymuses were dissected from each chick and weighed after dissecting connective tissue around the organ. Related weight of thymuses was calculated through the following formula:

$$\text{Related\hspace{0.5em}weight}=\text{organweight/bodyweight\hspace{0.5em}(g/kg)}$$

Pathological observation

At 7, 14, 21, 28, 35 and 42 days of age during the experiment, five birds in each group were euthanized and necropsied. Thymuses were took and fixed in 4% buffered formaldehyde and routinely processed in paraffin. Thin sections (5 μm) of each tissue were sliced from each block and mounted on glass. Slides were stained with hematoxylin and eosin Y (H&E). Histological slides were examined on an Olympus light microscope.

At the end of the experiment (42 days of age), three chickens in each group were euthanized and then immediately necropsied. Thymuses were sampled for ultrastructural observation, as described by Peng et al. ¹³

Determination of the peripheral blood CD4+ and CD8+ T-lymphocyte percentages

At 14, 28 and 42 days of age during the experiment, the peripheral blood of five birds in each group was taken to determine CD3⁺ CD4⁺, CD8⁺ T-cell percentages and CD4⁺/CD8⁺ ratio by the FCM method, as described by Chen et al. ¹⁴

Statistical analysis

Data were subjected to one-way analysis of variance using SPSS 11.0 software and presented as means ± standard deviation (M ± SD). Differences between means were assessed by t-test. A probability value <0.05 was considered to be significant.

Results

Clinical observation

From 3-day-old, broilers in 15 mg/kg Se group showed decreased feed intake and depression with fluffy feather. Broilers in 15 mg/kg Se group slept with a stand posture. Broilers in 10 mg/kg Se groups showed slightly decreased feed intake from 5-day old, but there were no obvious clinical symptoms in 1 and 5 mg/kg Se groups. After 14 days of age, chickens in 15 mg/kg Se group were sensitive to environmental factors. They were often crowded or flying when disturbed by noise or some other stress factors. At the end of the experiment, 26.38% and 49.23% loss of body weight were in 10 and 15 mg/kg Se groups, respectively, when compared with that of control group.

Changes of relative weight of thymus

Our data suggested that 10 mg/kg sodium selenite intake resulted in a decrease in the relative weight of thymus from 7 days of age to 42 days of age, compared to 0.2 mg/kg group. Differences were noted between 10 mg/kg group and 0.2 mg/kg group at 7, 21, 28, 35 and 42 days of age. The relative weight of thymus was decreased in 1 and 5 mg/kg groups only at 35 days of age. The results indicated that excess Se (≥10 mg/kg) intake markedly decreased relative weight of thymus especially in chickens. The results were shown in Figure 1.

Figure 1.

Effect of high Se on the relative weight of thymus in chickens (n = 5). Compared with 0.2 mg/kg group: *p < 0.05; **p < 0.01.

Pathological changes of the thymus

Macroscopically, thymuses were reduced in size especially in 10 and 15 mg/kg groups. Most obvious changes were observed at 35 days of age. Volume of the thymus was decreased with the dietary Se level increased (Figure 2).

Figure 2.

There are the thymuses (the first lobule on the left) at 35 days of age in 0.2, 1, 5, 10 and 15 mg/kg Se groups from left to right, respectively. The volume of the thymus is decreased with raising of the dietary Se content.

Histopathologically, lymphocytes in the medulla of thymus were decreased in number in 10 and 15 mg/kg groups from 14 days of age (Figure 3b). At the same time, capillaries had increased numbers because of congestion (Figure 3c) in comparison with those of 0.2 mg/kg group (Figure 3a). From 21 to 42 days of age, hypocellularity became gradually serious. Less tightly packed lymphocytes were appeared in the cortex and medulla (Figure 3d–f, h). At 42 days of age, the thymic lobules were involution with relatively thick medullar and thin cortex (Figure 3i) in 15 mg/kg Se group in comparison with those of 0.2 mg/kg group (Figure 3g).

Figure 3.

(a) Thymus of the 14-day-old chicken in 0.2 mg/kg group. (b) Thymus of the 14-day-old chicken in 10 mg/kg Se group. Lymphocytes in the medulla were slightly decreased in number. (c) Thymus of the 14-day-old chicken in 15 mg/kg Se group. Little veins and capillaries are obviously congested in thymic lobule. (d) Thymus of the 21-day-old chicken in 15 mg/kg Se group. Lymphocytes in the medulla were decreased in number. (e, f) Thymus of the 28-day-old chicken in 10 and 15 mg/kg Se groups. Lymphocytes in the cortex and medulla were decreased in number. (g) Thymus of the 42-day-old chicken in 0.2 mg/kg group. (h) Thymus of the 42-day-old chicken in 10 mg/kg Se group. Lymphocytes in the cortex and medulla were decreased in number. (i) Thymus of the 42-day-old chicken in 15 mg/kg Se group. The volume of thymic lobule is shrinked. HE (a–f, h) bars = 50 μm; g, (g, i) bars = 200 μm.

No ultrastructural changes were observed in 1 and 5 mg/kg group. The mitochondria of lymphocytes and reticulocytes from chickens fed on 10 and 15 mg/kg Se diets were enlarged and vacuolated with degenerating cristae (Figure 4b). More apoptotic lymphocytes were found in the thymus in 10 and 15 mg/kg Se groups. The apoptotic cells showed typical condensed nuclei with crescent shapes, cytoplasmic organelles with inconspicuous structure (Figure 4c).

Figure 4.

(a) Thymus of chicken in 0.2 mg/kg group. Bar = 3 μm. (b) Thymus of chicken in 10 mg/kg Se group. The mitochondria of lymphocytes are enlarged and vacuolated with degenerating cristae (→). Bar = 2 μm. (c)Thymus of chicken in 15 mg/kg Se group. The arrow is pointing to an apoptotic lymphocyte with condensed, crescent shaped nuclei. Bar = 2.5 μm.

Changes of the peripheral blood CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ T cells, and CD4⁺/CD8⁺ ratio

The percentage of CD3⁺ and CD3⁺CD4⁺ T cells was decreased with the dietary Se level increased. At 14 days of age, the percentage of CD3⁺ and CD3⁺CD4⁺ T cells in four high Se groups were markedly decreased (p < 0.01) in comparison with that of control group. The percentage of CD3⁺ T cells was lower (p < 0.01) in 10 and 15 mg/kg groups at 28 and 42 days of age, and the percentage of CD3⁺CD4⁺ T cells was lower in 15 mg/kg group at 28 and 42 days of age than in 0.2 mg/kg group. The percentages of CD3⁺CD8⁺ T cells in 5, 10 and 15 mg/kg groups were decreased when compared with that of 0.2 mg/kg group only at 14 days of age. However, there was no change about the CD3⁺CD4⁺/CD3⁺CD8⁺ ratio in four high Se groups. The results were shown in Figure 5.

Figure 5.

Effect of dietary Se on peripheral blood T-lymphocyte subsets in chickens (n = 5). Compared with 0.2 mg/kg group: *p < 0.05; **p < 0.01.

Discussion

The results of this study indicated that clinical signs in 10 and 15 mg/kg Se groups were associated with decreased feed intake, weight loss and irritability. Weight loss is also characteristic features of subchronic selenosis in adult mallard ducks and swine.^15,16 Sleeping with a stand posture and being sensitive to environment were the symptom of irritability. Previous report stated that symptoms of selenosis in human beings were fatigue, irritability and mild nerve damage. ¹⁷ Therefore, the future studies should focus on the biological and histological changes of nerve system in chicken fed on high dietary sodium selenite.

The thymus is the primary lymphoid organ which is responsible for the establishment and maintenance of the T cell in avian species. In the present study, the thymuses were greatly reduced in size and weight in 10 and 15 mg/kg groups. The result showed that the development of thymus was repressed by excess sodium selenite intake. Histologically, this reduction in weight was due to lymphopenia. The depleted lymphocytes were consistent with those described in a mallard duck fed on more than 20 mg/kg Met-Se diets. ⁸ Based on data available in animals and humans, it is generally believed that the volume of true thymic tissue attains maximum size at puberty, after which a disruption of thymic organization takes place. ¹⁸ Depletion of thymic cortex with an apparent loss of corticomedullary junction and decrease in the number of thymicepithelial and lymphocytic cells appeared in the involuting thymus. In the present study, the changes of thymus are similar to those occurring in physiological involution. Therefore, future studies should clarify the mechanisms of thymic atrophy caused by high dietary sodium selenite. Histological lesions became serious in a time-dependence manner. Gradually deteriorated lesions were related to the Se concentration, which was gradually accumulated in body. The histopathological lesions of thymus could explain the tissular basis of the decreased T-cell subsets.

The most obvious ultrastructural alterations in 10 and 15 mg/kg groups appeared in the mitochondria of the lymphocytes and reticulocytes in the thymus. Previous studies showed that exposure of isolated mitochondria to certain Se compounds resulted in a marked increase in superoxide generation.^19,20 Superoxide produced additional reactive oxygen species by cascading reactions. These oxidative radicals could trigger the aforementioned mitochondrial lesion and then release of proapoptotic proteins, such as cytochrome c, from mitochondria into the cytosol, ²¹ which induced the increase of apoptotic lymphocytes in thymus.

T lymphocytes are responsible for cell-mediated immunity and are further classified according to the presence of CD4 and CD8 proteins. ²² Most CD4⁺ T cells are helper T cells, which respond to exogenous antigen in association with major histocompatibility complex (MHC) class II molecules. CD8⁺ T cells respond to endogenous antigen in association with MHC class I molecules and generally function as cytotoxic T cells. ²³ In the present study, the percentage of CD3⁺ and CD3⁺CD4⁺ T cells was decreased in a dose-response manner. The percentages of CD3⁺CD8⁺ T cells were decreased in 5, 10 and 15 mg/kg groups. The results showed that the biological function of mature T cells was affected, and the cellular immune function was impaired accordingly in broilers when the dietary Se was more than 5 mg/kg. In the present study, there was no change about the CD3⁺CD4⁺/CD3⁺CD8⁺ ratio, which suggested that the relative proportion of these T cell subpopulations was maintained. Previous research suggested that Se concentrations are important in thymus development. ²⁴ Atrophy of thymus was observed in mallard ducks fed on more than 20 mg/kg Se. ⁸ In the present study, lymphopenia was observed in thymus of high Se groups too. T cell is derived from the thymus. Thus, lesions of thymus made us understand the reason of the changes of peripheral blood T-cells subsets in high Se groups.

According to the results in the present study and the aforementioned discussion, it is concluded that dietary Se excess (>5 mg/kg) reduces the percentages of peripheral blood T-cell subsets and impaired the histological structure of thymus in chickens. The cellular immune function was finally impaired in broilers.