Action Mechanism and Immunity Status of Broiler Chickens after Spray and Drinking Water Administration of Kanne-Brottrunk®

Poultry producers are challenged to improve production while using fewer antibiotics due to increased restriction on antimicrobial usage so prebiotics and probiotics are being tested under different experimental conditions to study the pathways used by these substances to assist in the prevention of carcass contamination and in the elimination of pathogens present in the bird's organisms. The current study aimed to investigate the effect of spray and orally administered Kanne-Brottrunk® which contain Lactobacillus and Bifidobacterium at which lactic acids bacteria concentration is 5x10 cfu/ml in addition to vitamins, minerals, essential amino acids and trace elements on the mucosal trachea IgA, IL-6 and INF-γ as well as healthiness of intestinal and tracheal mucosa using electron microscopy. Six hundred one day old Cobb broilers chickens were reared on litter in three separated groups each one contain 200 chicks, 1 group "Drinking Water Administrated", 2group "Spray Administrated" and "control non Kanne-Brottrunk® medicated group". Serum, trachea and intestine collected for evaluation of immune status and epithelium healthiness at 14, 21, 28 and 35 day old. Immunological Parameters measured were IL-6, INF-γ and Mucosal IgA concentration using EISA kits accompanied by tracheal and intestinal electron microscopy histomorphology. The results proved that Kanne-Brottrunk® administrated in water stimulate immune response by increased level of IL-6, INF-γ and Mucosal IgA and also increase immune cells infiltration as plasma cell, lymphoid cells, macrophages, and heterophile in trachea and intestine with protection of their tracheal cilia and intestinal villi more than aerosol administration.


Introduction
Sub-therapeutic use of antibiotics as growth promoters in animal feed has evoked wide spread concern, with their use banned in many countries due to the potential to develop antibiotic resistance in microbial populations associated with human and animal diseases.Probiotics are becoming one of the alternative to antibiotic growth promoters (AGP).The most important objectives of using probiotics are maintain and improve animal performance and prevent and control enteric pathogens.Probiotics are live microorganism when administered in adequate amount confer a health benefit on the host [1].There are several types of probiotics available to be used in poultry, with arrange of microorganism present with different metabolic activities and actions.They present variations as to the capacity of colonizing the intestine or not, which justifies variations on the results of their use.The bacterial contents of the animal intestine contain over 400 bacterial species; 99 % of these species are obligatory anaerobic and about 10% are Lactobacilli and Bifidobacteria [2].Lactobacilli have the longest history as probiotics and are still among the most common ingredients used in animal feed.In chickens the passage of feed and other ingested material though GI-tract is relatively fast and this is important when using lactobacilli with probiotic characteristics, because of this short passage time probiotics in chickens have a very short time to exert their actions and therefore good adhesion or even colonization characteristic play an important role.Lactobacillus and Bifidobacterium are probably the safest microorganisms used as probiotics.Microorganisms used as probiotics are selected on the bases of their survival in the gastrointestinal environment and ability to withstand low PH and high concentration of bile acids and another desirable characteristic is the ability to adhere to the intestinal epithelium [3].This work was planned to investigate the effect of spray and orally administered Kanne-Brottrunk® which contain Lactobacillus and Bifidobacterium which are still alive in the gut as they are resistant against the gastric acid and digestive enzymes, lactic acids bacteria concentration is 5x10 6 cfu/ml in addition to vitamins, minerals, essential amino acids and trace elements on the mucosal trachea IgA, humoral immune response, cellular immune response as well as healthiness of cilia of upper respiratory tract and intestinal mucosa using electron microscopy.

Chickens
Six hundred one day old Cobb broilers chickens were reared on litter in three separated groups, balanced rations and clean drinking water were given continuously, the following vaccination program was applied ;Hitcher B1 at 6 day of age, IBD "intermediate plus strain" at 13 days of age and Lasota strain at 18 day of age.Anti-mycoplasma drug (Tilmycosin phosphate, 275 mg) was given in the first 3 days for 8 hours daily.

Experimental Design
1 st group "Drinking Water Administrated: Comprising 200 chicks; Kanne-Brottrunk® was given orally according to the producer recommended dose as follow (Table 1).

Statistical Analysis
The results were presented as means± SE.All given parameters were compared between the control group and the experimental groups using the one way ANOVA with fixed effects of the factors using statistica 6.0 (Start Soft INC.).Differences were considered significant at p<0.05.

Histomorphology
Trachea and intestine were admitted to Electron Microscope unit, Assiut University for Histomorphological study according to [7].

Performance Parameters
a. Feed intake and feed efficiency according to [8]. b.Body weight gain according to [9].

Results of Histomorphology
As showed in Figs (A, 1, 2, 3, 4, 5, 6, 7 and 8) for drinking water treated group there was an increase in immune cells infiltration and epithelium healthiness of intestinal villi, while Figs (B, 9, 10, 11, 12, 13, 14, 15, and 16) for aerosol treated group, there were dramatic stimulation of mucosal immune cells in both trachea and intestine with increase in proliferation of plasma cells, macrophages, lymphoid cells, endocrine cells, goblet cell and heterophiles.At the same time, aerosol treated group (G2-Tr-21,28 and 35 days of age) showed deciliation of moderate areas of the covering epithelium with rupture of goblet cells on the surface sometimes with swelling of the surface epithelium especially when compared with normal trachea at 21 day old chickens (Figure 12b).

Results of Performance Parameters
Feed intake and feed efficiency: feed intake in in 1 st group was 680 kg, while in 2 nd group 690 kg and 702 in control group.While Body weight gain: mean body weight of the control group was 1.950 kg (1.8 FCR), while in 1 st group was 2.100 kg (1.6 FCR) and 2 kg for 2 nd group (1.7 FCR).

Discussion
A prebiotic is a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of normal flora.Among the most commonly studied substances in the family of prebiotics are the oligosaccharides, which are non-digestible carbohydrates, particularly fructo-ligosaccharides (FOS), gluc-oligosaccharides (GOS), and mannano-ligosaccharides (MOS) [10].Prebiotics may change gut microbiota in two ways: (i) by supplying the nutrients for beneficial bacteria, or (ii) by leading pathogenic bacteria to acknowledge attachment sites in oligosaccharides as intestinal mucosa sites, thus reducing undesired intestinal colonization, which in turn decreases the incidence of infections and improves the integrity of the mucosal surface of the gut wall [11].On the other hand, the mode of action of probiotics is related to the competition for attachment sites, or competitive exclusion.The bacteria present in the probiotic attach to the intestinal mucosa, thus forming a physical barrier that blocks the attachment of pathogenic bacteria [12].
In chicken, the passage of feed and other ingested materials through GIT is relatively fast 2-5 hours [13], this is not only importance for the digestion and absorption but it is also of importance with respect to the administration of Lactobacilli with probiotic characteristic therefore good adhesion or even colonization characteristics play an important role.According to [14] and [15], The bacterium genera present in probiotics that are directly related to the increase in immunity of poultry are Lactobacillus and Bifidobacterium mainly when related to diseases affecting the gastrointestinal tract, while according to [16,17].The antigens (Lipopolysaccharides and Peptidoglycans) are constantly released in intestinal lumen and are fundamental in the development and maintenance of local immune response since they have chemotactic effect on epithelial cells and cells related to mucosa immunity and induce changes in intestinal epithelium of the host.The mean IL6 concentration in orally administrated group (1 st Gp) were 21,26,30 and 35 pg/ml compared with 19,24,27 and 28 pg/ml in sprayed group(2 nd Gp) in sera collected at 14,21,28 and 35 days respectively, compared with 10,12,14,16 pg/ml in control (3 rd gp).Concerning IFN-γ mean concentration in orally and spray treated groups, the following concentration were recorded 196,344,494 and 510 pg/ml in (Gp1) and 180,318,436 and 492 pg/ml in (Gp2) at 14,21,28 and 35 days respectively, compared with 75,75,85,120 pg/ml in control group.GIT component of the immune system protecting the host from different types of antigens in the lumen of the GIT is affected by probiotics.Both innate and adaptive immunity are affected by probiotics.Probiotics formulations prevent chronic inflammation of the GIT through stimulation of innate immunity [18,19].Several studies have demonstrated immunostimulatory effects of probiotics.Bai et al., [3] demonstrated that a probiotic containing L.fermentum and S.cervisae stimulated intestinal T-cell immune system, indicated by increased production of CD3+, CD4+ and CD8+ T-lymphocytes in the GIT broiler chickens.Expression of CD3+, IL2 and IFN-γ genes was significantly greater in the small intestine of neonatal chicks (day 3 and 7) fed with Lactobacillus spp.[20].Concerning results of mucosal IgA concentrations in Gp1 which recorded 14,39,110 and 192 ng/ml compared with 24, 54,116 and 242 ng/ml in Gp2 at 14, 21, 28 and 35 days respectively, also control group values were 10, 18,45 and 90 ng/ml Probiotics increase serum immunoglobulins levels [21], studied the effects of probiotics on serum level of IgA and IgM in chickens and recorded a significant increased levels, also Landy and Kavyani [8]., noticed increase in antibody against the common poultry diseases Newcastle, infectious bronchitis and infectious bursal disease after treatment with probiotics.
Probiotics maintain the proper balance of useful microbial population in the intestine of bird, which is important for the efficient feed conversion, growth, productivity and stimulation of birds' immune mechanisms to combat pathogens [22].It has been proved that effective probiotics help to accelerate development of normal micro flora in chicks and poults (Bansal, et al., 2011).Even spray applications of probiotic have been found to be effective against Salmonella infection in chicks [23].As feed additive, probiotics show a good impact on the poultry performance [24].Establishing and maintaining healthy gut microflora is essential, which improves the microbial environment of a bird's intestinal tract by displacing harmful bacteria.Especially, in newly hatched chicks, the gut is sterile and starts acquiring microflora from environment.Since pathogenic microbes may multiply faster than the beneficial bacteria, chicks may get infected at this time.Successful colonization of probiotics depends on the survival and stability of the microbial strain used, their relationship with the host, dose and usage frequency, and host health, nutritional status, age, stress and genetics [25].In the present study application of Kanne-Brottrunk® by aerosol method accompanied by dramatic stimulation of mucosal immune cells in both trachea and intestine which gather by increase proliferation of plasma cells, macrophages, lymphoid cells, endocrine cells, goblet cell and heterophiles.At the same time, aerosol treated group (G2-Tr-21, 28 and 35 days of age) showed deciliation of moderate areas of the covering epithelium with rupture of goblet cells on the surface sometimes with swelling of the surface epithelium.

Figure 4 .
Figure 4. Micrograph of trachea sub epithelial showed congestion of the vasculature, edema and presence of plasma and macrophage cells

Figure 5 .Figure 6 .Figure 7 .
Figure 5. Micrograph of the villous core contain numerous plasma cells, lymphocytes and macrophages with inter cellular edema

Figure 8 .
Figure 8. Micrograph of trachea sub epithelial lamina of the trachea showing cellular infiltration of lymphoid and macrophages with edema in between