Infectious Bursal Disease (IBD) or Gumboro disease caused by IBD virus is one
of the most important immunosuppressive diseases affecting mainly young chickens
and considered as a threat to commercial poultry (Lukert and
Saif, 1997). Eradication of IBD virus is difficult because of its high stability
in the environment. Therefore, the principal approach for prevention is effective
vaccination against this disease. The current vaccines are killed or cell culture
adapted live, attenuated virus. Depending on the level of attenuation and virulence,
these live vaccines are further categorized as hot or intermediate plus, intermediate
and mild. The most effective strategies to control IBD include vaccination of
layer birds with inactivated oil-emulsified vaccines to provide maternal antibodies
in chickens or immunization of young chickens with live attenuated vaccines
followed with booster immunizations. After the emergence of very virulent IBDV,
mild vaccines are generally ineffective and presence of maternal antibodies
interferes with the efficacy of live vaccines. Another most important issue
associated with live vaccines is immunosuppression, which may cause lesions
similar to natural infection in vaccinated birds (Muller
et al., 2003). Thus, there is a definite requirement for a suitable
immunomodulator that can minimize the suppressive effects of live vaccines and
amplify specific protective response. Arginine, a semi-essential dibasic amino
acid has emerged as a regulator of many immunological and physiological processes.
Arginine attracted initial experimental attention in various animal tumor models
(Barbul et al., 1980) as a dietary supplement.
The most promising immunostimulatory effects were observed in immunocompromised
hosts after trauma, surgical stress or immunosuppression with HIV virus. Supplemental
Arg stimulates the functional activities of different cell types including Natural
Killer (NK) cells, macrophages, lymphokine activated killer cells, T and B cells
(De Jonge et al., 2002). Two pathways of arginine
metabolism have been identified as being critical to the immunomodulatory actions
of arginine in vivo. First, the arginase pathway, in which Arg is converted
to urea and ornithine, generating polyamines by the action of ornithine decarboxylase.
This route of polyamine synthesis may be the mechanism whereby Arg augments
lymphocyte mitogenesis. Induction of arginase has also been proposed as the
effector pathway in arginine-dependent macrophage-mediated tumor cell cytotoxicity
(Currie, 1978). Second, arginine is the sole substrate
for nitric oxide synthesis in biological systems. Nitric oxide is synthesized
from arginine by nitric oxide synthase resulting in the formation of nitric
oxide and citrulline. Nitric oxide is a ubiquitous molecule with important roles
in the maintenance of vascular tone, coagulation, the immune system and the
gastrointestinal tract and has been implicated as a factor in disease states
as diverse as sepsis, hypertension and cirrhosis (Billiar,
1995). Although, IBDV primarily impairs humoral immune response, sever immunosupression
is the result of downregulation of T cells and macrophages. These two cells
are the main producers of cytokines involved in innate and cell mediated immunities.
Cytokines are low-molecular-weight proteins or glycoproteins that are primarily
produced by immune cells and are able to direct and regulate immune responses.
Similar to mammalian Interferons (IFN), there are two types of chicken IFNs;
types I and II. Type I IFN with antiviral activity, includes 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 shares many biological properties with mammalian IFN-γ (Lowenthal
et al., 2001). It activates macrophages to enhance 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 the antibody
classes to support phagocytosis and fixation of complement (such as IgG2a in
mouse) (Tuting et al., 1999). The sever immunosupression
and pathological symptoms caused by IBDV infection, could be reduced by immunostimulator
nutrients such as Arg in the chickens basal diet. Therefore, the present
study was aimed to investigate the effects of Arg on the overall immune response.
The serum levels of IFNα, IFNγ and IgG were measured as the indicators
of innate, Cell mediate and humoral immune responses, respectively.
MATERIALS AND METHODS
Five different dietary levels (A-E) of Arg for three age group periods (starter,
grower and finisher) of broiler chickens were used. There were (Table
1) five levels for the starter (S) 0-21 days of age (S-A, 0-control; S-B,
0.67;S-C, 1.37; S-D, 2.07 and S-E 2.77), five levels for the grower (G) 21-42
days of age (G-A, 0-control; G-B, 0.53; G-C, 1.1; G-D, 1.68 and G-E, 2.25) and
five for the Finisher (F) 42-49 days of age (F-A, 0-control; F-B, 0.52; F-C,
1.04; F-D, 1.56 and F-E, 2.08).
of experimental diets of broiler chickens during 0-49 days of age
per kilogram of diet: vitamin A, 10,000 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;
All diets met the National Research Council (1994) recommendations
for broilers. One-day-old Cobb 500 male broiler chickens (250) were utilized
in the experiment consisting of 5 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 that was initially established on
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 orally 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), Immunoglobulin G (IgG) were determined using appropriate
Enzyme Linked Immunosorbent Assay (ELIZA) (USCN, USA). INF α, INF γ
and IgG were evaluated as indicators of the effects of orally administered intermediate
plus strain of IBDV with or without Arg on the systemic innate, cell-mediated
and humoral immune responses, respectively. ELISA was performed according to
the manufacturers instructions. Briefly, equal amounts (100 mL) of positive,
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. Duncan
(1955)`s multiple range test was used to determine differences among treatment
means. Means were considered different at p<0.05.
RESULTS AND DISCUSSION
Effects of different levels of Arg on the immune system indicators in broiler
chickens are shown in Table 2-4. Arginine
supplementation into the basal diets significantly increased interferon-α,
interferon-γ, immunoglobulin G at 35, 42 and 49 DAC days of age of chickens
of arginine on the serum level of interferon-α in broiler chickens
in each column with different superscripts are significantly different
of arginine on the serum level of interferon-γ in broiler chickens
bMeans in each column with different superscripts are significantly
of arginine on the serum level of immunoglobulin G in broiler chickens
in each column with different superscripts are significantly different
Interestingly, the different levels of Arg at 27 DBC day of age did not significantly
affect interferon-α, interferon-γ, whereas Arg at 27 DBC day of age
significantly increased immunoglobulin G (p<0.05).
All the unimmunized control birds succumbed to infection by intermediate plus 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. 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 Arg and with the increase of Arg the lesions severity decreased (Fig. 1 and 2).
The results revealed that with an increase in Arg level, INF α, INF γ and IgG were enhanced linearly. This linear enhancement was continuous up to group D but in group E, a negative deflection was observed. As a whole, the highest level of all immunity indicators, were induced on DAC and DBC of age that is fitted in group D. Although, no statically responses were observed for INF α, INF γ at 27 DBC, trends of their improvement were observed as Arg level was increased in diet.
Mass cull of chickens caused by Immunosuppressive effects of IBDV threatens
the poultry industry worldwide. The main objective of the study was to evaluate
high levels of Arg as an immunomodulator of systemic immunity. It is believed
that Arg can reduce the immunosuppressive effects of live hot vaccine and amplify
specific protective immune response against IBD (Mo
et al., 2001). The result showed that dietary Arg increased the serum
levels of INF α, INF γ and IgG. Two percentage of Arg supplementation
in feed was safe and free from any side effects (Tayada
et al., 2006). 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 IFN-α, which is quickly upregulated after infection
with viruses (Kawai and Akira, 2006). Mo
et al. (2001) showed that recombinant IFN-α suppressed IBDV
and Newcastle disease infection in chickens. This finding is in line with our
result in which the increase of IFNα in intermediate plus + Arg group reduced
the clinical signs of the infected chickens. In chickens, cellular innate immunity
constitutes NK and macrophages (Gobel et al., 2001).
Arginine supplementation has been shown to enhance NK (Brittenden
et al., 1994). Moreover, Reynolds et al.
(1990) observed interferon and macrophage cytotoxicity enhancement with
Arg enriched diet.
IFN-γ is associated with adaptive immune responses and as such would be
expected to be upregulated later in the immune response (Takaoka
and Kani, 2006). IFN-γ 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 TH1and induce cellular immune responses which leads
to the lysis of virus-infected or tumor cells (Urban et
of arginine on the bursa lesion scores in broiler chickens
scoring for Bursa of fabricus
slides of Bursa in different groups
It has been reported that chicken IFN-γ level is increased before the
appearance of the first clinical signs. During the acute phase of IBD infection
an increase in the levels of circulating cytokines like chicken IFN-γ and
TNF-α occur as demonstrated by the capture ELISA and cytotoxic bioassay,
respectively. The increased levels of IFN-γ and TNF-α correlated with
the progression of the disease and were highest in the animals that died of
co infection (Saif, 1984). Co-administration of either
IFN-γ protein or plasmid DNA encoding IFN-γ 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. Consistent with these findings, the results showed that Arg supplementation
into the basal diets significantly increased interferon-γ, at 35, 42 and
49 DAC days of age of chickens (p<0.05).
of arginine on the spleen lesion scores in broiler chickens
Interestingly, the different levels of Arg at 27 DBC day of age did not significantly
The levels of neutralizing antibodies determine the protection against IBDV
in chickens (Van den Berg, 2000). 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 Arg.
scoring for Spleen
slides of the spleen in different groups
The elevated antibody response and protection are attributed to the various
immunoregulatory functions of Arg on the immune system. It is believed that
Arg is required for the differentiation of pro B to pre B cells as well as being
involved in the release of these cells from bone marrow (De
Jonge et al., 2002).
Abdukalykova and Ruiz-Feria (2006) proved that high
levels of Arg (2.2%) and higher-than-industry levels supplementation of vitamin
E (80 IU kg-1) had an important immunomodulation effect on the cell-and
humoral-mediated immune responses of IBDV vaccinated broiler chickens by increasing
the amount of T cells and B cells and the CD4+ and CD8+ lymphocyte subpopulations.
Regardless of vitamin E, the results obtained from this study not only are complementary to their findings but also reveals that innate immunity is significantly enhanced by high level of Arg.
To the knowledge, this is the first comprehensive study of the effects of high levels of Arg on innate, humoral and cellular immune responses on chickens challenged with intermediate plus strain of IBDV. As the results revealed 2.5 times of NRC level of Arg 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. Therefore, Arg may be able to enhance the resistance of broilers to infectious diseases and may be useful in minimizing the stress associated with vaccination.