The effect of a combination of β(1-3) D-glucan and Propionibacterium granulosum on productive performance and immune modulation of immunocompromised and non-immunocompromised broiler chickens.

®Aim: The effect of a specific combination of a soluble ‚ (1-3) D-Glucan and Propionibacterium granulosum (Betamune ) was investigated on productive performance, immune response and immune dysfunction caused by cyclophosphamide (CP) in broiler chickens. Materials and Methods: Three hundred and sixty one-day-old broiler chicks were randomly allocated into four groups for 5 ® weeks. Betamune supplementation of 0.25 ml / L drinking water (presence or absence) for the first 7 days of age and CP (presence or absence) subcutaneous inoculation with 4 mg / chick for the first 3 days of life was done. ® Results: Treatment of broiler chicks with Betamune improved productive performance variables as compared with the blank control birds, where there were 10 points less in cumulative feed conversion ratio and significant increase (P<0.05) in final body weight, both intestinal length and diameter, and European production efficiency factor (EPEF). It also modulated the immune response, where there was non-significant improve in haemagglutination inhibition (HI) antibody titers against Newcastle disease (ND) virus vaccine and significant increase (P<0.05) in phagocytic % and phagocytic index. The lesion score after ND challenge reached only 70 in ‚ (1-3) D-Glucan group as compared with 80 in blank control group. The ® histomorphological examination of Betamune treated chickens at 5 weeks of age revealed lymphoid hyperplasia in bursal follicles, lymphoid cells of cortical portion of thymus glands and lymphoid cells in the white pulps of spleen. CP did affect ® bird's weight and suppressed immune system. Treatment CP suppressed birds with Betamune significantly increased (P<0.05) final body weight, dressing weight %, giblets weight %, intestinal diameter, improved FCR (28 points less than ® untreated group), decreased cumulative mortality and improved EPEF. Betamune counter attacked immune dysfunction caused by CP, where there was significant increase in HI antibody titer against ND vaccine, no significant increase in ® phagocytic % and phagocytic index and improve in the lesion score after ND challenge (99 as compared to 133). Betamune supplementation reduced microscopic lesion scores associated with CP immune dysfunction. Conclusion: It could be concluded that administration of a specific combination of soluble ‚1.3, D-Glucan and ® Propionibacterium granulosum (Betamune ) to broiler chickens improved chicken zootechnical performance response variables, had a potent immunomodulatory effect (potentiated immune response), evoked their immune response and enhanced their vaccination effectiveness.


Introduction
makers, the development of alternative methods to deal with the problems caused by bacteria in commercial Many diseases can be modified by direct adminianimal production is a high priority for both researchers stration of biological compounds that activate key pathways and farmers. A number of potential immunoin the immune system. The term immunosuppression is modulators may serve as antibiotic-alternatives for defined as a state of temporary or permanent dysfunction both the promotion of growth and disease resistance in of the immune response, which insults the immune animal production. Huff et al.
[2] have suggested that system leading to increased susceptibility to diseases yeast β-1, 3/1, 6-glucan may be useful as an alternative [1]. It is undoubtedly true that factors contributing to to the antibiotics due to its immuno-modulating function. immune-suppression would lead to immune-deficiency.
Immunodeficiency has led to increasing need for The latter is a hazard-anticipating causative agent the use of immunopotentiators (immunostimulants), of serious economic impacts in poultry industry all which are extrinsic or intrinsic substances that regulate over the world. As a result of the use of antibiotics or alter the scope, type, duration or competence of the many pathogenic bacteria have developed resistant immune response. Immunopotentiators strengthen the strains. This has led to extensive consideration for defense and immune mechanisms of the body and limiting the use of antibiotics in livestock breeding.
currently usable for stimulating the non-specific Given the demands of both consumers and the policy immune responsiveness in both the human and into 3 subgroups of 30 each. Those of groups 1 and 2 veterinary medical practice [3]. Potentiating of normal were immunosuppressed [11] by subcutaneous inoculation immune response in poultry occurs by alteration in any of cyclophosphamide (CP) in a dose of 4 mg / chick for step involved in the host's immunologic reaction either the first 3 days of life. Birds of groups 1 and 3 received ® in the classic humoral or in the cell-mediated systems. Betamune in a dose of 0.25 ml/liter drinking water for Eventually, it has already known that many diseases / the first 7 days of age and repeated from 22 to 28 days ® disorders, that have immunomodulated components, of age. Each liter of Betamune contained β1.3, Dcan be modified by administration of biological Glucan (3%) and Propionibacterium granulosum (0.17 compounds that activate key pathways in the immune gram). Chickens of group 2 were kept as positive system [4].
immunosuppressed controls while those of group 4 Beta-glucan (β-glucan) have been examined in were kept as blank control. several studies as antibiotic alternative, and have been Measured parameters: found to up regulate immune response and stimulate Productive performance: Chicken performance response growth in swine and poultry [2, [5][6][7]. The immuno variables were determined [12]. For body weight; all -modulating effects of β -glucan might result in an birds were weighed individually at 1st day and weekly increase in the functional activity of macrophage and for the entire period of the experiment (5 weeks). Feed heterophil cells [8 -10].
consumption was calculated, for each subgroup, This investigation was dedicated in an attempt to weekly; investigate the feed conversion ratio (FCR) (g investigate the possible effect of a specific combination feed / g live body weight). Feed conversion ratio (FCR) of a soluble β1.3, D-Glucan and Propionibacterium was calculated, for each subgroup, as follows: Feed granulosum produced by Kanzy medipharm, Canada, ® intake for the subgroup (kg) divided by the total body known under the trade name Betamune on weight gained for the same subgroup (kg). The total zootechnical performance, chicken immune response body weights included dead birds during the week. and immune dysfunction in broiler chickens.
Daily mortalities were recorded for each subgroup. The

Materials and Methods
European production efficiency factor (EPEF) was estimated at the end of the experimental period, for The present study was carried out at the Poultry each subgroup. The European production efficiency Research Center, Department of Animal Production, factor was calculated, for each subgroup, as follows: Faculty of Agriculture, Cairo University, Giza, Egypt.
(Average live body weight (Kg) * Livability (%))*100 / Experimental Birds: The experiment was carried out (Marketing age(day)*FCR) The carcass characteristics according to the National regulations on animal (dressing without Giblets (%), fleshing (%), giblets welfare and Institutional Animal Ethical Committee Weight (%), and intestine's length and diameter were (IAEC). Three hundred and sixty, one-day old measured on 10 birds of each subgroup, at 5 weeks of age. commercial Arbor Acres plus broiler chicks were used Immune status assessment: To investigate the possible in this study. Chicks were housed in semi closed house. ® effect of Betamune on the humoral immunity; an The chicks were provided with 24 hours light immunoassay was adopted. For this purpose, blood throughout the first three days, then 23 hours light and samples were collected from wing vein from 10 1 hour dark until slaughter time. Chicks were fed a randomly selected birds at weekly intervals (1-5 weeks commercial starter diet (23% crude protein and 3000 k of age) from each group. The serum samples were cal ME/kg diet) during the first two weeks of age, subjected to haemagglutination inhibition (HI) test for commercial grower diet (22% crude protein and 3150 determining antibody titers against ND vaccine kcal ME/kg diet) from 2-4 weeks of age, and then employing 8 haemagglutinating (HA) units [13]. To commercial finisher diet (19% crude protein and 3200 ® investigate the possible effect of Betamune on the cell kcal ME/kg diet). The diets compositions are indicated mediated immunity; measurement of phagocytic in Table-1. Semduramicin was added at a concentration activity of peripheral blood monocytes using Candida of 25 ppm as a coccidiostat, during the experimental albicans was adopted [14]. Ten chickens from each period. Feed and water were available ad libitum. All group were challenged with a velogenic viscerotropic chicks were vaccinated against Newcastle disease strain of ND (vVND) virus on the 35th day of age and vaccine (ND) at 7 and 21 day of age by using live kept under close observation for clinical signs and Hitchner B1 and La Sota strain vaccines, respectively. mortality for further 2 weeks. Dead as well as Live infectious bursal disease vaccine (IBD) was sacrificed birds at the end of observation period (49 administrated at 14 days of age. Drinking water method days) were subjected to post mortem examination for was used as rout of administration of live vaccines. On lesion scoring of ND. the day 10 of age, 0.5 ml inactivated Avian Influenza vaccine (AIV), (H5N1), was injected subcutaneously Histopathological assay: Specimens including bursa in the back of neck.
of Fabricius, thymus gland and spleen were collected Experimental design: One day-old Arbor Acres plus from randomly selected 5 sacrificed chickens / group at broiler chicks (n=360) were allotted into 4 equal 14 and 28 days of age and fixed in 15% buffered groups (1-4) of 90 birds each. Each group was divided formalin. Paraffin-embedded sections were routinely prepared from these fixed organs and stained with Statistical analyses: One-way analysis of variance was Hematoxylin and Eosin [15], and scored for histo-used using SAS software general liner models procedure pathological lesions [16]. [17]. The main factor was immunomodulator    Table 4). The results clearly showed that EPEF of were assessed for significance using Duncan's multiple ® range test [18] with significance set at P<0.05.
Betamune treated group was significantly the highest Results and following in descending order with control, CP+ ® Betamune and then CP suppressed group (Table 5). Productive Performance: The results presents in Table  Finally,    www.veterinaryworld.org ® samples (at 3 and 5 weeks of age, respectively) showed indicated a significant increase in CP+ Betamune no histopathological findings and normal histopathotreated group over CP suppressed group in the HI titers logical structure of lymphoid follicles in mucosal layer against ND vaccine at different examined intervals.
was recorded (Figs.1 and 13 respectively). In CP group However, non-significant improve in HI titers were ® lymphoid depletion was observed in the follicles with also recorded in Betamune treated group as compared to st nd edema in between at 1 sample (Fig.2). On the 2 blank control one. Cell mediated immunity parameters sample, moderate lymphoid depletion was noticed in (phagocytic % and phagocytic index) are reported in ® the central portion of the follicles (Fig.14). In Table ( Betamune group, the 1 sample (3 week-old) revealed significant increase (P<0.05) in phagocytic % and no histopathological alteration (Fig.3). However, the phagocytic index at 3 and 5 weeks of age as compared second sample showed lymphoid hyperplasia in the to blank control group. Chickens suppressed with CP ® st follicles (Fig.15). In CP + Betamune group, the 1 revealed significant decrease in phagocytic % at 3 and ® sample showed mild lymphoid depletion in the 5 weeks as compared to Betamune treated group. The follicles with edema in between (Fig.4), but only mild same group also revealed significant decrease in lymphoid depletion in the follicles was revealed in the phagocytic index at 3 and 5 weeks as compared to both nd ® 2 sample (Fig.16). Betamune and control group. Chickens suppressed with ® CP and treated with Betamune showed non-Thymus glands: In control group, no histopathological significant increase in phagocytic % and phagocytic findings were observed and the normal histopathoindex when compared to CP suppressed group at both logical structure of cortical and medullary portions st nd ages. Results of bioassay (Table 9) showed that the were recorded, in the 1 and 2 samples (Figs.5 and lesion score reached 133 in CP suppressed group as 17). In CP group lymphoid depletion was observed in ® st compared to 99 in CP suppressed + Betamune treated cortical and medullary portion at 1 sample (Fig.6). Bursa of Fabricious: In control group, 1 and 2 logical alteration at 1 sample (Fig.7). On 2 sample ®    hyperplasia was noticed in the lymphoid cells of the antibody titer against ND vaccine, phagocytic %, and ® phagocytic index. The lesion score after ND challenge cortical portion (Fig.19). In CP + Betamune treated st reached 70 as compared with 80 in blank controls. The group there was no histopathological alteration at 1 nd present results recorded also ten points in cumulative sample (Fig.8). While, on 2 sample there was FCR were less than untreated controls (1.85 vis. 1.95). lymphoid hyperplasia in cortical portion (Fig.20).
Neither histopathological lesion score could be detected Spleen: In control group, there was no histopathoin treated nor in blank control groups. However, logical findings observed and the normal histological lymphoid hyperplasia in bursa of Fabricius follicles ® structure of cortical and medullary portion was was noticed in Betamune treated group. Many recorded in samples taken at the two ages ( Fig. 9 and researchers agree with our results and established the 21). In CP group, lymphoid depletion was recorded in improvement in broiler performance when β-1, 3/1, 6st the follicles of the white pulps in 1 sample (Fig.10). glucan and Propionibacterium granulosum were Second sample showed mild depletion in white pulps supplemented in broiler diets [3, [19][20][21][22][23]. Zhang et al. while the red ones were congested (Fig. 22). In [3] interpreted this effect as β-1,3/1,6-glucan may play ® st Betamune group, 1 sample showed intact histological a role in the initiation of immune modulation; therefore, structure in white and red pulps (Fig. 11). Second suitable supplementation would be beneficial to the sample showed hyperplasia in the lymphoid cells in the performance of broiler chickens. In addition, supplewhite pulps associated with congestion in the red ones mentation increases performance by improving the nd ® at 2 sample (Fig. 23). In CP + Betamune treated average daily body weight gain and reducing the group, no histopathological alteration in both samples feed/weight gain ratio. While another researches were recorded ( Fig.12 and 24). Histopatho-logical showed that supplementation of β-1, 3/1, 6-glucan have lesion scores of major immune organs of immunoco-a physiological effects on intestinal digestive mucosa and increase the villus height of jejunal mucosa of mpromised and non-immuno-compromised broiler ® chickens [24][25][26]. chicken groups treated and untreated with Betamune Immunomodulators administered simultaneously are illustrated in Table (   the inhibition of early activation of tissue muscular dressing weight %, front part weight %, giblets weight th nuclear factor-KB (NF.KB) and NF-IL6 [29]. %, intestinal diameter, feed consumption (at 4 week of Many literatures showed that the immunoage) and EPEF. A significant decrease in total mortality modulating effects of β-glucan might result in an rate and gizzard weight was also obtained. While there increase in the functional activity of macrophage and was a 28 points decrease in FCR. On the other hand; heterophil cells [8][9][10], increased production of non significant increase in phagocytic %, phagocytic cytokines (such as IL-1, TNF-α and IL-6) and immunoindex and weight gain has been reported. For overall globulins [3]. judgment on immunomodulation of the studied In vitro, broiler macrophages from a cell line or preparation in immunosuppressed birds; a bioassay isolated from normal chickens when exposed to was carried out. Challenge with vVND virus resulted in ® various concentration of β-glucan get activated and lowered lesion score in Betamune treated as compared produce heightened levels of nitrite (a product of nitric with untreated group (99 vis. 133). This means that ® oxide syntheses gene activity) and cytokines such as administration of Betamune could partially counter IL-1. Furthermore, β-Glucan exposure also induced attacks immunosuppression and decreases lesions macrophage proliferation [30]. Moreover, in vivo attributed to ND challenge as well. The histomorphofeeding trials, the supplemented chickens with βlogical changes of major immune organs due to CP glucan had improved macrophage phagocytic suppression (expressed as lesion scores) reached 9 and ® functions, more persistent T-lymphoproliferative 5 in CP depressed group, 2 and 2 in their Betamune rd th response as measured by PHA-P mediated swelling ‹‹‹‹ treated control group at 3 and 5 week of age respectively in the toe web, much improved antibody response after Conclusion boost, and increased incidence of CD4 (T-helper) and CD8 (T-cytotoxic) positive lymphocyte subsets in the From the aforementioned results; it could be intestinal leukocyte population. Furthermore, dietary concluded that administration of a specific combination inclusion of β-glucan significantly improved the of soluble β1.3, D-Glucan and Propionibacterium ® growth of both primary and secondary lymphoid organ granulosum (Betamune ) to broiler chickens improved of chickens. chicken zootechnical performance response variables, The bacterial-killing and phagocytosis-stimulating had a potent immunomodulatory effect (potentiated effect of purified yeast β-1, 3/1, 6-glucan in four-dayimmune response), evoked their immune response and old male Leghorn chickens were reported [29].
enhanced their vaccination effectiveness. The present ® Significant increase in phagocytosis of peripheral study proved that Betamune played a positive role not blood cells and antibody production induced by oral only in non-immunocompromised broiler chickens but administration of β-glucans in balb / C mice were also also in immunocompromised birds by counter recorded [31]. Macrophages are part of the nonattacking their immune dysfunction caused by CP. specific first line of defense because of their ability to Author's contribution engulf and degrade invading microorganisms. Macrophages perform a variety of functions other than All authors contributed equally. All authors read and phagocytosis; they act as secretor cells, produce Nitric approved the final manuscript. oxide that kill intracellular microorganisms, secrete