Abstract: Infectious bursal disease is still a challenging issue by posing a serious threat to the commercial poultry industry especially due to the emergence of highly Infectious Bursal Disease Virus (IBDV). In the present study, we evaluated the immunomodulatory effects of Tryptophan (Trp) on innate, humoral and cellular immune responses in chickens challenged by oral administration of intermediate plus strain of IBD virus at 28 days of age. A corn-soybean meal based diet containing different levels of Trp (0, 0.10 and 0.20) for the starter, (0, 0.07 and 0.15) for the grower and (0, 0.05 and 0.13) for the finisher has been utilized. In a completely randomized design with three treatments of five replicates each and 10 chickens per replicate, 150 Cobb 500 male broiler chickens from 0-49 days of age were subjected to Trp diet. To measure the innate, cellular and humoral immunity indicators (interferon-α, interferon-γ, immunoglobulin G, respectively) at 27, 35, 42 and 49 days of age, serum samples from each replicate of treatments were collected and subjected to ELISA. The result showed that Trp supplementation in the chickens basal diets significantly increased the serum levels of interferon-α at 35, 42 and 49 days of age (p<0.05), interferon-γ at 27, 35 and 49 days of age (p<0.05) and immunoglobulin G at 27, 35, 42 and 49 days of age (p<0.05). These results strongly suggest that tryptophan plays a vital role in modulation of protective immune response against IBDV.

INTRODUCTION

Infectious Bursal Disease (IBD) is an acute and contagious progressive disease of chickens that induces high morbidity and mortality in chickens at 3-6 weeks of age. The disease in younger chickens is usually sub-clinical and results in immunosuppression with subsequent poor immune response to different infections and vaccines. Therefore, the disease has a significant economic impact on poultray industry (Lukert and Saif, 2003).

Tryptophan, an essential amino acid has emerged as a regulator of many immunological and physiological processes. Its plasma concentration declines in animals suffering from different illnesses and inflammations (induced or natural), suggesting an increased utilization of the amino acid in such instances (Le Floc`h et al., 2004). IBDV primarily impairs the humoral immune response which is followed by sever immunosupression due to downregulation of T cells and macrophages. These two cells are the main cytokines producers which in turn regulate innate and cell mediated immunities. Cytokines are low-molecular-weight proteins or glycoproteins and sub-divided into various classes, one of the most significant of which is interferon family. Similar to mammalian interferons (IFN), there are two types of chicken IFNs; types I and II.

Type I IFN with antiviral activity, including IFNα produced by monocytes and macrophages and IFNβ produced by fibroblast and epithelial cells. IFNγ, a type II IFN, is produced by activated Natural Kill cells (NK) as well as T cells and considered to be a TH1-biased cytokine. Chicken IFNγ has many biological properties in common with mammalian IFNγ (Lowenthal et al., 2001). It activates macrophages to boost their antimicrobicidal activities, up-regulates the expression of Major Histocompatibility Complex (MHC) I molecules to increase antigen presentation and induces secretion of other cytokines such as interleukin-2 (IL-2) and IL-12 to develop Cell Mediated Immunity (CMI). IFNγ secreted by TH1 cells can also switch on the antibody classes to support phagocytosis and fixation of complement (such as IgG2a in mouse) (Tuting et al., 1999).

Various approaches are available to boost the immune response. An efficient one is manipulation of the basal diet by adding different nutrient. Studies have shown that the sever immunosupression and pathological symptoms caused by IBDV infection, could be reduced by supplementation of chicken’s basal diet with immunostimulator nutrients such as Trp. Therefore, the present study was aimed to investigate the effects of Trp on the systemic immune response. We assessed the serum levels of IFNα, IFNγ and total IgG as the indicators of innate, CMI and humoral immune responses, respectively.

MATERIALS AND METHODS

Three different dietary levels (A, B and C) of Trp for three age group periods (starter, grower and finisher) of broiler chickens were used. There were (Table 1) three levels of Trp for the starter (S) 0-21 days of age (S-A, 0-control; S-B, 0.10 and S-C, 0.20), three levels for the grower (G) 21-42 days of age (G-A, 0-control; G-B, 0.07 and G-C, 0.15) and three for the Finisher (F) 42-49 days of age (F-A, 0-control; F-B, 0.05 and F-C, 0.13). All diets met the National Research Council (1994) recommendations for broilers. One-day-old Cobb500 male broiler chickens (150) were utilized in the experiment consisting of 3 treatments with 5 replicates and 10 chickens per replicate each. Birds were housed randomly in pen, so that initially each bird occupied approximately 0.11 m² of floor space. The pens were floor pens with wood litter. Birds were maintained under continuous light and the environmental temperature in the barn was initially set at 31°C and was gradually reduced to 20°C by week 7. Feed and water were provided ad libitum throughout the experiment. Chickens were challenged by oral administration of intermediate plus strain of IBD virus (10-fold greater than normal vaccination doses) at 28 days of age. This strain was obtained from the poultry vaccine testing laboratory, Malaysian Vaccines and Pharmaceuticals. At 27 (Day Before Challenge: DBC), 35, 42 and 49 (Days After Challenge: DAC) days of age, two chicken was randomly selected from each replicate in each treatment and blood samples were collected from wing vein by Terumo Syringe with needle (0.7x32 mm). Blood samples (ten samples for each treatment) were centrifuged, serum was separated and stored at -20°C until use and analyzed in triplicate to assure that calculated and analyzed values were in agreement. Analysis of the immune response indicators including interferon α (INF α), interferon γ (INF γ) and total Immunoglobulin G (IgG)) were determined using appropriate Enzyme linked immunosorbent assay (ELIZA) (USCN, USA). INFα, INFγ and total IgG were evaluated as indicators of the effects of orally administered intermediate plus strain of IBDV with or without Trp on the systemic innate, cell-mediated and humoral immune responses, respectively.

Table 1:	Composition of experimental diets of broiler chickens during 0-49 days of age
1Supplied per kg of diet: vitamin A, 10000 IU; vitamin D3, 9790 IU; vitamin E, 121 IU; B12, 20 μg; riboflavin, 4.4 mg; calcium pantothenate, 40 mg; niacin, 22 mg; choline, 840 mg; biotin, 30 μg; thiamine, 4 mg; zinc sulphate, 60 mg; manganese oxide, 60 mg. 2Metabolizable energy

ELISA was performed according to the manufacturer’s instructions. Briefly, equal amounts (100 mL) of standard, negative controls and serum samples were added to the previously marked wells and the plates were incubated for 30 min at room temperature. After 3 washings, 100 mL of conjugate provided with the kit was added to each well and incubated for 30 min at room temperature. The plates were again washed and 100 mL of substrate solution was added and incubated for 15 min. Later, stop solution was added and plates were read at ELISA plate reader set at 450 nm.

At 49 days of age bursas and spleens were removed, sections of bursas and spleens tissues from the inoculated and control chickens were fixed in 10% formalin solution and stained with Hematoxylin and Eosin (H and E). Sections of bursas and spleens were examined microscopically and photographed by digital camera equipment (LEICA DMIRB, Leica Microsystems Wetzlar, Germany).

A completely randomized experimental design was used. All data were statistically analyzed using the General Linear Models (GLM). Procedure of SAS software (SAS Institute, 1996) was employed for the analysis of variance. To determine the differences among treatment means Duncan (1955)’s multiple range test was used. Means were considered different at p<0.05.

RESULTS AND DISCUSSION

Effects of different levels of Trp on the immune system indicators in broiler chickens are shown in Table 2-4. As the results indicate, tryptophan supplementation into the basal diets significantly increased interferon-γ, at 35, 42 and 49 DAC days of age (p<0.05) (Table 2), interferon-γ at 27 DBC, 35 and 49 DAC days of age (p<0.05) (Table 3) and immunoglobulin G at 27 DBC, 35, 42 and 49 DAC days of age of chickens (p<0.05) (Table 4). The different levels of Trp at 27 DBC days of age did not significantly affect interferon-γ. The different levels of Trp at 42 DAC days of age did not significantly affect interferon γ.

All the unimmunized control birds succumbed to infection by challenge virus. These chickens showed typical signs of IBD, as such some of them had gross bursal lesions characterized by pale color edema, point bleeding and significantly lower bursa to body weight ratio (p<0.05) compared to other chickens.

Spleen enlargement was also significantly higher in control group than the other groups. The bursal lesion scores are shown in Table 5 and 6 and histopathological lesion scores are shown in Fig. 1.

Table 2:	Effects of tryptophan on the serum level of interferon-α in broiler chickens (pg mL-1)

Table 3:	Effects of tryptophan on the serum level of interferon-γ in broiler chickens (pg mL-1)

Table 4:	Effects of tryptophan on the serum level of immunoglobulin G in broiler chickens (ug mL-1)
a-cMeans in each column with different superscripts are significantly different (p<0.05)

Table 5:	Effects of tryptophan on the bursa lesion scores in broiler chickens

The spleen lesion scores are shown in Table 7 and 8 and histological lesion scores are shown in Fig. 2.

As these data confirmed sever lesions were observed in group A whose diet was not supplemented with Trp and with the increase of Trp the lesions’ severity decreased (Fig. 1 and 2).

The results revealed that with an increase in Trp level, INFα, INFγ and IgG were enhanced linearly. This linear enhancement was continuous up to group C. As a whole, the highest level of all immunity indicators, were induced on DAC and DBC of age that is fitted in group C. Although, no statically responses were observed for INFα at 27 DBC and INFγ at 42 DAC, trends of their improvement were observed as Trp level was increased in diet. IBD is an acute, highly infectious and immunosuppressive disease caused by IBDV, a member of Birnaviridae family affecting mainly young chickens (Lukert and Saif, 1997). The primary target of the virus is actively dividing B cells in bursa of fabricius ultimately causing destruction of these lymphocytes, which leads to transient immunosuppression in affected chickens (Tayada et al., 2006).

Table 6:	Lesion scoring for Bursa of fabricus

Fig. 1:	Histopathology slides of bursa in different groups

The main objective of the study was to evaluate the immunostimulatory effects of high levels of Trp on the systemic immune response.

It is has been shown that Trp can reduce the immunosuppressive effects of live hot virus and amplify specific protective immune response against herpes simplex virus (Adams et al., 2004). In agreement with this finding, the results showed that dietary Trp indeed increased the serum levels of INFα, INFγ and IgG. The enhancement of immune response was further confirmed by histopathological examination of bursa and spleen. Innate immunity is anatomically positioned to serve as the first line of defense against variety of pathogens such as viral infections. One of the elements of innate immunity is IFNα, which is quickly upregulated after infection with viruses (Kawai and Akira, 2006). Mo et al. (2001) showed that recombinant IFNα suppressed IBDV and Newcastle disease infection in chickens. This finding is in line with the result in which the clinical signs of IBDV were reduced by increasing of IFNα in Trp fed group challenged with intermediate plus IBDV. Besides, Adams et al. (2004) demonstrated an enhancement of IFNα, IFNβ in chickens excessed with Trp and challenged by herpes simplex virus. As a result, virus replication was restricted.

Fig. 2:	Histological lesion of spleen in different groups

Table 7:	Effects of tryptophan on the spleen lesion scores in broiler chickens

In another study, the oral administration of Trp to rats enhanced phagocytosis by macrophages and could boost the innate immune response as well (Esteban et al., 2004).

IFNγ is associated with adaptive immune responses and as such would be expected to be upregulated later in the immune response (Takaoka and Kani, 2006). IFNγ is a pleiotropic cytokine secreted by Natural Killer (NK) cells and many T cells. This cytokine has the ability to direct the balance of TH cells to TH1 and induce cellular immune responses which leads to the lysis of virus-infected or tumor cells (Urban et al., 1996). Cellular immune response is centrally involved in the pathogenesis of various diseases. Within the immunological cascades of cellular immunity, interferon γ, among other cytokines, is critically involved (Winder et al., 2000). It has been reported that chicken IFNγ level is increased before the appearance of the first clinical signs. During the acute phase of IBD. The rise in antibody titres at 35 DAC indicates the booster effect of challenge virus on immune system; where in clonal proliferation and increased numbers of plasma cells lead to enhanced antibody production (Tizard, 2004) an increase in the levels of circulating cytokines like chicken IFNγ and TNFα occurs as demonstrated by the capture ELISA and cytotoxic bioassay, respectively (Saif, 1984). Co-administration of either IFNγ protein or plasmid DNA encoding IFNγ with DNA vaccine showed that the immune response was enhanced in felines (Flynn et al., 2000), ducks (Long et al., 2005) and chickens (Min et al., 2001) against challenge to feline immunodeficiency virus, hepatitis B virus and Eimeria acervulina, respectively. In another study, the increase of IFNγ in chickens whose diet enriched with Trp restricted the replication of herpes simplex virus (Adams et al., 2004). Consistent with these findings, the results showed that Trp supplementation into the basal diets significantly increased interferon γ, at 27 DBC and at 35 and 49 DAC days of age (p<0.05). In spite of the fact, the different levels of Trp at 42 DAC day of age did not significantly affect interferon γ. Available evidence suggests that Trp catabolism plays a role in immune responses by producing a local immunosuppressive environment that is able to control T-cell homeostasis and self-tolerance during inflammation (Platen et al., 2005).

Munn and Mellor (2002) showed that high affinity Trp transporter could be used to stimulate T cell mediated immune responses and increase T cell activation. Moffett and Namboodiri (2003) suggested that Trp can increase leukocyte in the spleen and immune reactivity. The levels of neutralizing antibodies determine the protection against IBDV in chickens (Van den Berg, 2000).

Table 8:	Lesion scoring for spleen

Binding of these antibodies with virus forms immune complexes, which are removed by complement-mediated lysis (Rautenschlein et al., 2002). Similar to other findings (Tayada et al., 2006), we observed a strong antibody response in all groups; however antibody titers were significantly higher in challenged groups which received Trp. The elevated antibody response and protection are attributed to the various immunoregulatory functions of Trp on the immune system. Antibodies specific to IBDV might be involved in neutralizing virus and the resultant immune complexes will be removed by complement-mediated lysis. The results of the study revealed that as well as adaptive immunity, innate immunity also plays an important role in the control of IBD infection (Rautenschlein et al., 2002).

CONCLUSION

The results demonstrate that full protection was achieved against intermediate plus IBDV in chickens supplemented with tryptophan. Tryptophan as a strong immunoregulator enhanced the innate immunity and the IBDV-specific antibody response. It also reduced the immunosuppressive effects of challenge virus on cell mediated immunity by increasing of IFNγ.

To the knowledge this is the first comprehensive study in which the stimulatory effects of tryptophan on innate, humoral and cellular immune responses of chickens challenged with live intermediate plus strain of IBDV have been investigated.

As the results revealed two times of NRC level of Trp supplementation in feed was safe and free from any side effects as well as having the optimum effects on all mentioned parameters which led to the protection of chickens against the intermediate plus strain of IBDV. We suggest that tryptophan as a safe, inexpensive, easy to administer in feed and valuable immunomodulator could be used not only for IBD but also for other viral infections of poultry.