Aflatoxin B₁ (AFB₁) is a strong mutagenic, carcinogenic and teratogenic compound (Mohamed & Metwally, 2009) and can be found in foods and fodder in the world as a common contaminant. The inhibitory effects of AFB₁ on cytokines expression and T-cell subsets have been demonstrated in various studies. The dietary AFB₁ can induce inhibitory effects on interleukin-1 (IL-1), interleukin-2 (IL-2) and interleukin-6 (IL-6) production in rats’ splenocytes (Hinton et al., 2003). There is also a decrease in contents of serum IL-2 and interferon gamma (IFN-γ) in broilers exposed to AFB₁ (Chen et al., 2013a). However, Meissonnier et al. (2008) found linear increases in IFN-γ, IL-6 and interleukin-10 (IL-10) mRNA with increase in dietary levels of AFB₁ in pigs. In addition, the increase of IFN-γ, IL-6 and tumor necrosis factor alpha (TNF-α) mRNA (proinflammatory cytokines) has been observed in chickens exposed to AFB₁ (Li et al., 2014). The percentage of splenic CD8+ T-cells in rats shows dose-dependent decreases with dietary AFB₁ increasing (Qian et al., 2014). Intoxicated mice with AFB₁ also exhibit significant decreases in the percentage of CD4+ T-cells in the spleen and in the number of CD3+ T-cells in the intestinal T-cells (Hatori et al., 1991; Tomková et al., 2002). Chen et al. (2014) observed reduced percentages of CD3+, CD3+CD4+ and CD3+CD8+ T-cells in the spleen of broilers administered AFB₁. Contrary to these reports, Qian et al. (2014) reported a significant increase in the percentages of CD3+ and CD8+ T-cells in the spleen when rats are exposed to AFB₁. These findings indicate that the effects of AFB₁ on the cytokines and T-cell subsets are not always similar in different settings.

In poultry, the cecal tonsil is the largest lymphoid organ of the gut-associated lymphoid tissue (Deng et al., 2012). This organ is involved in both antibody production and cell-mediated immune functions and plays an important role in intestinal mucosal immunity (Olah et al., 1984; Humberd et al., 2007). However, there are no systematic reports regarding the effect of dietary AFB₁ on cytokines and T-cell subsets in the cecal tonsil of poultry. The purpose of this paper was to investigate the effects of dietary AFB₁ at different time points on the cytokines mRNA expression and T-cell subsets in the cecal tonsil of broiler chickens by quantitative real-time PCR (qRT-PCR) and flow cytometry (FCM) analyses in order to provide helpful information for future studies in immunosuppressive effects induced by AFB₁ in poultry.

One hundred and fifty six 1-day-old healthy male broiler chickens were divided into control group (0 mg AFB₁/kg feed) and AFB₁ group (0.6 mg pure AFB₁/kg feed). Table 1 shows the control diet which was formulated according to National Research Council (NRC) (Shini & Kaiser, 2009) and Chinese Feeding Standard of Chicken (NY/T33-2004) recommendations. Broilers were housed in cages with electrically heated units and were provided with water as well as aforementioned diets (Table 1) ad libitum for 21 days. At 7, 14 and 21 days of age during the experiment, the cecal tonsils of six birds in each group were taken to determine CD3+, CD3+CD4+, CD3+CD8+ T-cell percentages by FCM as described by Liu et al. (2012) and the levels of IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ and TNF-α mRNA expression by qRT-PCR analysis according to the method reported by Chen et al. (2013b). The animal protocols used in this work and all procedures of the experiment were performed in compliance with the laws and guidelines of Sichuan Agricultural University Animal Care and Use Committee (Approval No: 2012-024). Statistical analyses were performed using the Mann-Whitney U test, a non-parametric test for two independent samples, by SPSS software for Mac v.20.0 (IBM Corp, Armonk, NY, USA). A value of p< 0.05 was considered significant.

Table 2 shows that the expression levels of all cytokines in the AFB₁ group significantly decreased (p<0.01) at 14 and 21 days in comparison with those of the control group. Furthermore, Table 2 and Figure 1 revealed that the percentages of CD3+, CD3+CD4+ and CD3+CD8+ T-cells significantly decreased (p<0.05 or p<0.01) in the AFB₁ group at 14 and 21 days in comparison to the control group. These results indicate that both cytokines mRNA expression and T-cell subsets population in the cecal tonsil were reduced when broilers were fed with 0.6 mg/kg dietary AFB₁. There is a close relationship between the thymus and differentiation of T lymphocytes (Kidd et al., 1996). Thymus is the place where T-cells are activated and differentiated to CD4 or CD8 T-cells, and then mature T-cells migrate from thymus to the peripheral blood and secondary immune organs (Erf et al., 1998). According to the report of Guo et al. (2012), AFB₁ induces suppression of thymus development. Therefore, the decrease of T-cell subsets of the cecal tonsils observed in this study could be simultaneously associated to the suppressed development of thymus. Moreover, it had been confirmed that AFB₁ could cause depressed synthesis of DNA and RNA (Erf et al., 1998), which may also contribute to the reduced percentages of T-cell subsets. In addition, it is known that the differentiation of T-cells is closely related to the cytokines (Huang et al., 2014) and many cytokines are secreted by T-cells (Scott, 1993).

The CD4+/CD8+ ratio can be used to provide a convenient standard for changes in cellular immune status during disease, nutritional stress, and auto-immune problems (Kantrow et al., 1997). In the present study, the CD4+/CD8+ ratio in the AFB₁ group showed a decreased tendency compared with the control group, but without apparent difference (p> 0.05) (Table 2) in the AFB₁ group during the experiment. These results indicate that the relative proportion of T-cell subpopulations was maintained in the two groups during the experiment.

Our results (Table 2) show that the levels of cytokines mRNA expression in the AFB₁ group decreased progressively from 7 to 21 days of age, and the values (except for IL-6 and IL-10) in the AFB₁ group were lower at 14 (p<0.01) and 21 (p<0.01) days of age than those in the control group. In addition, the percentages of CD3+, CD3+CD4+, CD3+CD8+ T-cells showed a significant decline already by 14 (p<0.05 or p<0.01) and 21 (p<0.05 or p<0.01) days of age (Table 2). Finally, the CD4+/CD8+ ratio fell during the experiment, but not in statistically significant manner (Table 2). These results suggest that AFB₁-induced reduction of cytokines mRNA expression and the percentages of T-cell subsets in the cecal tonsil differed with progression of the exposure. The mechanism for this is worth of further study.

It is concluded that the dietary exposure to 0.6 mg/kg of AFB₁ reduced the levels of cytokines (IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α and IFN-γ) mRNA expression and the percentages of T-cell subsets (CD3+, CD3+CD4+ and CD3+CD8+) in the cecal tonsils of broiler chicken.