Comparison of the expression of cytokine genes in the bursal tissues of the chickens following challenge with infectious bursal disease viruses of varying virulence

Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), is an acute, highly contagious and immunosuppressive disease in young chickens, resulting in great economic loss in the poultry industry [1]. IBDV can be differentiated into two serotypes (serotype 1 and 2) [2]. Serotype 1 shows different degrees of pathogenicity and mortality in chickens, whereas serotype 2 is avirulent in chickens [3]. Based on virulence, serotype 1 strains are classified as classically, intermediate, very or hypervirulent virulent [1].

IBDV is a non-enveloped, double-stranded (ds) RNA virus consisting of two segments, segment A (3.2 kb) and B (2.9 kb), encoding five proteins and belongs to the Birnaviridae family [4‐6]. IBDV mainly affects young chickens from 3-6 weeks of age [7]. Although viral antigen has been detected in other organs within the first few hours of infection, the most extensive virus replication takes place primarily in the bursa of Fabricius [6]. Activated dividing B lymphocytes that secrete IgM⁺ serve as target cells for the virus [8, 9]. Viral infection results in lymphoid depletion of B cells and the destruction of bursal tissues [10], leading to an increased susceptibility to other infectious diseases and poor immune response to vaccines [5].

Replication of IBDV in the bursa is accompanied by an influx of T cells [8, 11]. The marked influx of T cells into the infected bursa indicates that cell-mediated immunity plays important roles in the clearance of virus particles [12, 13]. The T cells in the bursa of chickens infected by virus are activated, with up-regulated expression of a number of cytokine genes, such as interleukin (IL)-1β, IL-6 and interferon (IFN)-γ [14]. The change in the level of cytokine expression is closely associated with organizational destruction, inflammation and apoptosis [13]. The direct immunosuppressive effects of IBDV on T cells and their function remain unclear. Chickens infected with IBDV resulted in suppression of cellular immune responses and a subsequently reduction in the ability to respond to secondary infections [15].

The CD4⁺ helper T (Th) cells play crucial roles in immune responses. The CD4⁺ T cells have been classified as either Th1 or Th2 based on their cytokine profiles [16]. Th1 cells have evolved to enhance clearance of intracellular pathogens and are defined on the basis of their production of IFN-γ [17]. Th2 cells are critical for the control of certain parasitic infections through the production of the clustered group of cytokines IL-4, IL-5 and IL-13 [18]. Chickens are able to mount a typical Th1 or Th2 biased cytokine response after experimental viral and helminth parasitic infections, respectively [19]. Cytokines are important mediators and regulators of both types of host responses. However, little is known about the role of cytokine following IBDV infection. It is extremely important to gain an understanding of pathological changes and immunosuppression caused by IBDV infection and the immune responses evoked.

In the present study, our objective was to further investigate the Th1/Th2 paradigm by examining the transcriptional profile of cytokines in the bursal tissues of chickens infected with either vvIBDV H strain or the cell-adapted virus Ts strain at 1, 3 and 5 days post-infection (dpi) and also to test the hypothesis that the IBDVs with varying virulence induces different cytokine profiles during the course of infection.

Avian cytokines, like their mammalian counterparts, are influential in host immune response to pathogenic infection [20]. The cytokine responses to IBDV in the bursa of chickens are poorly described and their role in the pathogenesis of such infections has not yet been extensively studied. Of the seven genes examined in this study, the levels of expression of IFN-γ, IL-2 and IL-12P40 genes in the bursa tissues following H strain infection were increased compared with the IL-4, IL-5, IL-13 and IL-10 genes; the expression levels of IL-4, IL-5, IL-13 and IL-10 genes in Ts-infected chicken bursa had a higher fold-change than the IFN-γ, IL-2 and IL-12p40 genes. The results obtained from gene expression analysis of Th1 and Th2 cytokines revealed that the vvIBDV, H strain induces an immune response characteristic of the Th1 pathway; In contrast, the cell-adapted virus, Ts strain induced an immune response characteristic of the Th2 pathway. The results also revealed the early activation of a variety of antiviral host defenses after infection, for example activation of innate immune responses, cell-mediated immune responses and modulation of host transcription.

Our results showed that the viral load in bursal tissues increased approximately 1000-fold after infection with the H strain compared with the Ts strain (Figure 1). The vvIBDV, H strain had a stronger capability of replication and spread in the bursa of birds than cell-adapted Ts strain. Based on earlier work from our laboratory [21], the doses used in this study contained 10^3.4 egg infectious dose₅₀ (EID₅₀) H strain and 10^6.5 tissue culture infectious dose₅₀ (TCID₅₀) Ts strain and should have demonstrated a positive signal at approximately the bursal tissues the same time. These date indicate that the load and replication of IBDV in the bursa is closely related to clinical symptoms and pathology, which is in accordance with Eldaghayes's study [14].

The present studies showed that there was a trend of up- or down-regulation in the expression levels of several cytokine genes in the bursa following either H strain or Ts strain infection. A higher viral load in the bursa was associated with significantly higher expression of cytokine genes. This is in agreement with the previous reports made by Abel and Abdul-Careem [22, 23] who studied virus replication and cytokine gene expression following virus infection and found a significant association between higher viral RNA levels and cytokine transcript concentration in various tissues. These results demonstrate that the difference in the expression levels of cytokines was possibly influenced by the different degree of viral replication. However, factors influencing the timing of cytokine regulation in bursal tissues and the cause and effect relationship between host response and viral replication are not clear from the present observations. Future experiments need to be conducted to examine more cytokines during the course of infection with variously virulent IBDVs.

Our results suggest that the H strain tends to up-regulate the Th1 cytokines response. Th1 cells are characterized by the secretion of IFN-γ, IL-2 and IL-12p40, and a strong cell-mediated immunity that is geared towards effective elimination of intracellular pathogens such as viruses [17]. IL-2 stimulates proliferation of chicken T lymphocytes and NK cells [24‐26]. Production of IL-12 and IFN-γ is critical to host defense against intracellular pathogens [27], indicating that it is possible to observe simultaneous up-regulation of IFN-γ and IL-10 in response to IBDV infection. The observed increase in IFN-γ expression in IBDV-infected bursa presumably reflects the inflammatory response and is consistent with earlier published results [14, 28], suggesting that cell-mediated responses are initiated to resolve infections. IFN-γ-induced activation of macrophages (Mφ) results in the stimulation of nitric oxide synthase (iNOS), which in turn leads to the production of apoptotic mediators such as nitric oxide (NO) or tumor necrosis factor-α (TNF-α) [29]. Previous studies from our laboratory demonstrated that apoptosis was induced by the vvIBDV H strain in the chicken bursa [30]. Furthermore our observations suggest that enhanced IFN-γ expression was associated with disease progression in IBDV- infected chickens.

However the Ts strain tends to up-regulate effects of the Th2 cytokine response. Th2 cells secrete IL-4, IL-5, IL-13 and IL-10, and are geared towards a humoral immune response against parasites and allergic reactions [18]. IL-4 has been shown to direct B cells to produce the anti-allergen IgE, to inhibit Th1 cell function and to prevent the production of IL-2, IL-12 and IFN-γ that are necessary for development of cytotoxic T cells [31]. However our results did not observe that up-regulation in the expression of Th2 cytokines suppressed transcriptional activities of Th1 cytokines. Previous reports by Heidari [32] have shown that because of the substantial level of IL-4 mRNA expression in the Marek's disease virus (MDV)-infected birds. It is not unusual to observe transcriptional activities of IL-2 being severely suppressed. A change in cytokine expression levels is closely related to virulence and replication of IBDV, but the mechanism by which this occurs is still not fully understood.

As expected, IBDV infection caused cell-mediated immune-related cytokine responses in the bursa, as shown (Figure 2 and 3) by the up-regulation or down-regulation of detected cytokines expression. This is in accordance with the pathology and clinical signs of two distinctly virulent IBDV. Infection with vvIBDV results in lymphoid depletion, marked atrophy of the bursal tissues and high rates of mortality [33, 34], but Ts strain infection does not cause obviously clinical signs [21].

Of the evaluated cytokines, prolonged expression of IFN-γ and IL-10 genes was up-regulated due to IBDV infection. IL-10 is a potent stimulator of NK cells [35, 36], a function that might contribute to the clearance of the pathogen and facilitate antigen acquisition from dead cells for cross-priming activated antigen-presenting cells (APCs), providing a link between the innate and the adaptive immune responses [37]. The expression of IL-10 in the bursa following IBDV infection has not been studied previously. In the present study our results indicated that IL-10 expression was markedly increased and similar to the extent of up-regulated expression of IFN-γ following infection by the H or Ts strain. This is consistent with the fact that IL-10 plays a dual role in infectious diseases [37] and is in agreement with the observation made recently by Abdul-Careem [22] who recorded that the expression of the IL-10 gene followed the pattern of expression of the IFN-γ gene to a certain extent in both pre- and post-hatched herpesvirus of turkey (HVT)-immunized chickens. In general, both IL-10 and IFN-γ are known to be important cytokines in the cell-mediated immune response and evoke host responses to the pathogen in chickens [38, 39]. This also suggests that IL-10 may play a role as an immunostimulatory cytokine similar to IFN-γ after IBDV infection.

In summary, we have shown that infection with IBDV induces changes in the level of expression related to Th1 and Th2 in the chicken bursa and that the vvIBDV, H strain strongly induced an increase in cytokine expression. It is clear that changes in the extent of cytokine expression were closely associated with virulence of the virus and viral replication. Further studies are necessary to elucidate the function of the cytokines in pathogenesis and immunity against IBDV.