PROBIOTICS AS AN IMMUNE ENHANCER IN BROILERS

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1 Sarhad J. Agric. Vol.25, No.3, 2009 PROBIOTICS AS AN IMMUNE ENHANCER IN BROILERS ALTAF-UR-RAHMAN*, SARZAMIN KHAN*, DAULAT KHAN *, NAILA CHAND*, MUNIB HUSSAIN**, SOHAIL AHMED*, SAYED MOHAMMAD SOHAIL*, MOHSIN ALI*, NAZIR AHMAD*, ISHTIAQ AHMED***, IKRAM-UL-HAQ**** and IJAZ AHMAD* * Faculty of Animal Husbandry & Veterinary Sciences, NWFP Agricultural University, Peshawar, Pakistan ** Animal Science Division, National Agricultural Research Centre, Islamabad, Pakistan *** Veterinary Diagnostic and Research Centre, Mandia, Abbotabad, Pakistan **** Department of Agricultural Extension Education & Communication, NWFP Agricultural University, Peshawar, Pakistan ABSTRACT Present study was carried out at MS poultry farm Baffa, Mansehra during The main objective of this study was to investigate the effect of different levels of probiotics named as Organic Green Culture on the immune response of broiler chicks, using Newcastle Disease Haemagglutination Inhibation (HI) antibody titer as indicator. For this purpose, 448 Hubbard day-old broiler chicks were randomly divided into four groups viz. A, B, C and D. Each group comprised 4 sub groups of 28 chicks each. Four experimental rations, I (control), II, III and IV containing probiotic at 0, 1, 2 and 4 g/kg ration were randomly allotted to these groups. The experiment lasted for 42 days. The mean Hemagglutinin Inhibition (HI) antibody titer values were 2.37, 2.25, 2.25 and 2.12, for groups A, B, C and D, respectively at day 1 st. A non significant (P>0.05) difference was observed among all the groups. At day 10 th the mean HI antibody titer was 3.18, 3.25, 3.93 and 3.56 for groups A, B, C and D, respectively. Groups C and D showed significantly (P<0.001) high HI antibody titer values than groups A and B. For day 20 th the mean HI antibody titer values were 5.25, 5.43, 8.00 and 7.62 for groups A, B, C and D, respectively. There was significant (P<0.001) difference among groups A, B, C and D. Groups C and D was significantly (P<0.001) higher in HI values than groups A and B. On day 35 th the mean HI antibody titer was 7.31, 7.62, 9.81 and for groups A, B, C and D, respectively. The groups C and D presented significantly (P<0.001) higher HI antibody titer values than groups C and D. It was concluded that Probiotics 2 g/kg rations successfully improved the immune status of broiler chicks Key Words: Probiotics, Immune Response, Broilers Citation: Altaf-ur-Rahman, S. Khan, D. Khan, M. Hussain, S. Ahmed, S.M. Sohail, I. Ahmed, Ikram-ul-Haq and I. Ahmad Probiotics as an immune enhancer in broilers. Sarhad J. Agric. 25(3): INTRODUCTION The successful poultry production is based on feeding, breeding, marketing, management and the well developed immune status of the birds. Immunity means the power of resistance, against the pathogenic microorganisms. Such power of defense has two main sources, natural and induced. Schat and Myers (1991) stated that the natural immunity in the avian is concentrated in egg yolk, which discharges its antibodies directly into the intestinal lumen and assures protection of the young bird against infections. Efforts have been made to compensate the natural immune status of the broilers through the artificial means, like vaccinating or feeding antibiotics. Both of these practices are conventional or traditional. The residues of both the treatments are mostly inheritable in the broiler meat, which are hazardous for the human health. During the process of preparation, the improper isolation of the antibodies could result high mortality. It is difficult for the layman to do the proper vaccination or to follow all the conditions needed for mixing the antibiotics with the other macro or micro feed ingredients during ration preparation. Vaccines and antibiotics are highly sensitive to storage capacity, temperature, humidity and aerobic or anaerobic conditions. To overcome such problems, the Probiotics might be proved as an effective alternative for vaccine as well as for antibiotics. The Probiotics are non nutrient feed additives. They are a natural organic extracts, obtainable mostly from medicinal plants or from self grown forest herbs. These extracts contain microorganisms, which have the ability to produce lactic acid, enzymes, free amino acids and short-chain peptide. When the Probiotics are given to the chicks along with the feeds, they could help to improve the intestinal flora (Andreatti-Filho and Silva, 1999), which is beneficial to the animal and antagonistic to

2 Altaf-Ur-Rahman et al. Probiotics as an immune enhansor in broilers 458 harmful microbes (Green and Sainbury, 2001; Honma et al., 1987). Studies with broiler chicks have indicated a positive response to dietary supplementation of probiotic (Midilli and Tuncer, 2001). Yeo and Kim (1997) observed significant improvements in daily gain, feed intake and immune status in broiler chicks fed Probiotics. Moreover, Jin (1997) reviewed the recent advances on the use and mode of action of Probiotics (direct-fed microbials) in enhancing poultry production. The addition of Probiotics to the diet has been found to improve growth performance and feed conversion in broilers, and egg mass, egg weight and egg size in layers. The mode of action of Probiotics in poultry includes (i) maintaining normal intestinal micro flora by competitive exclusion and antagonism, (ii) altering metabolism by increasing digestive enzyme activity and decreasing bacterial enzyme activity and ammonia production, (iii) improving feed intake and digestion, and (iv) neutralizing enterotoxins and stimulating the immune system. Keeping in view the beneficial effects of Probiotics, the present experiment was designed to investigate the effect of various feed added levels of probiotic named as Organic Green Culture available in the local market (imported from Korea) on the immune response of commercial broiler chicks using Newcastle Disease (ND) titer as an indicator. The present study would also be helpful to confirm the manufacturer's claims about the efficacy of the product improving the immune response in the broiler chicks. MATERIALS AND METHODS The trial was carried out at a commercial poultry farm at Baffa, district Mansehra, North West Frontier Province, Pakistan. The experiment was conducted in Completely Randomized Design (CRD), feeding 4 rations to 4 groups, with 112 birds per group. Each group was replicated in four with 28 birds per replicate. For such purpose, 600 Hubbard day- old broiler chicks were purchased from the local hatchery. Four hundred and forty-eight birds were selected on the basis of nearly the same live weight and soundness of the general health. These 448 chicks were randomly distributed into four groups A, B, C and D fed with four experimental rations I, II, III, and IV, respectively. The experiment lasted for 42 days. The required quantity of each feed ingredient was purchased from the local market and feed was prepared on the farm, which was divided into four equal lots. Ration I was kept as control. For ration II, III and IV the probiotic was added at the rate of 1, 2 and 4 g/kg of basal ration in starter as well as in finisher rations (Table I-II). Table I. Composition of the starter rations, using each ingredient g/kg Ingredients Ration I (control) Ration II Ration III Ration IV Probiotic Maize Wheat Canola meal Corn gluten meal Soybean meal Blood meal Fish meal Rice polishing Molasses Rock phosphate L-Lysine DL-Metheonine Vit. Min. Premix Total Table II. Proximate composition of the experimental starter rations Parameters Ration I Ration II Ration III Ration IV ME (Kcal/kg) Crude protein Crude fiber Dry matter Ash E.Extracts

3 Sarhad J. Agric. Vol.25, No.3, A representative sample of one kg of feed from each experimental ration was stored in freezer for the subsequent analyses such as dry matter, ash, crude protein, and crude fat and crude fiber (Tables III - IV), according to the procedures given in AOAC (1990). During the experimental period, feed and water were provided ad-libitum to the birds. Table III. Composition of the finisher rations, using each ingredient g/kg Ingredients Ration I (control) Ration II Ration III Ration IV Probiotic Maize Wheat Canola meal Corn gluten meal Soybean meal Blood meal Fish meal Rice polishing Molasses Rock phosphate L-Lysine DL-Metheonine Vit.Min.Premix Total Table IV. Proximate composition of the experimental finisher rations Parameters Ration I Ration II Ration III Ration IV ME (Kcal/kg) Crude protein Crude fiber Dry matter Ash E.Extracts The birds were raised adopting standard housing procedure on conventional deep litter system, with open sided house. All the pens were located in one house and each pen was provided with feeders, drinkers and electric bulbs (100 watts). During experiment strict biosecurity measures were taken to prevent any chance of disease occurrence. The birds were vaccinated according to normal routine broiler farming practices. The antibody titer against Newcastle Disease was measured by Hemagglutination inhibition (HI) test as described by Allan et al. (1978). To study the effect of probiotic on the immune status of the experimental birds, blood samples were collected from 4 birds per replication (Table VI). The blood collection was made on different age days during experiment like, 1, 10, 20 and 35. After collection, the blood samples were allowed to clot. The serum was separated after over night refrigeration and was stored at -10 o C. The hemagglutination inhibition (HI) test was carried out in micro-titration plates. Eight samples were titrated in one plate at a time. Fifty micro liters of PBS was added in each well of the plate. The serum samples of 100 micro liters were placed in the first row of the wells with the help of four-channel pipette. After mixing, standard volume (50 micro liters) from each row was transferred to the next row, this procedure was repeated till column number 10 th. While 50 micro liters of the suspension was discarded from the 10 th well. The wells of column 11 th and 12 th were kept without serum samples in order to maintain them as control. Fifty micro liters, four hemagglutinating units of antigen was placed in each well and the plates were incubated at room temperature for 30 minutes. Fifty micro liters of one percent chicken RBCs suspension was added with the help of four channel pipette into each well of the plate and the plate was gently tapped to ensure even suspension of RBCs in the mixture. The plate was kept undisturbed at room temperature until a clear pattern of hemagglutination inhibition was seen. The wells showing button formation at the bottom was taken as positive, while those with characteristic reticulum settling of RBCs at the bottom was considered as negative. The HI antibody titer of each serum sample was narrated as reciprocal of its end point dilution. Thus, the HI antibody titers of all the serum samples were recorded in this way.

4 Altaf-Ur-Rahman et al. Probiotics as an immune enhansor in broilers 460 The data were statistically analyzed for antibody titer against ND with the standard procedures of analysis of variance using Completely Randomized Design, as described by Steel and Torrie (1981). The means were compared for significance of difference with the LSD procedure (Steel and Torrie, 1981). The statistical package (SAS, 2000) was used to perform the above analysis on computer. Statistical Model Yij = µ+tj+єij Yij = ith observation of jth treatment. Tj = Effects due to treatment. Єij = Experimental or random error. µ = Over all mean (mean effect). Table V. Experimental design for measurement of antibody titer against ND Treatments Replicates Rations Groups R 1 R 2 R 3 R 4 I A II B III C IV D RESULTS AND DISCUSSION Table VI is presenting the overall effect of feeding different levels of probiotic added in the broiler rations, on their immune status. For this purpose, the data for HI antibody titer against ND were recorded on different age days 1 st, 10 th, 20 th and 35 th. On day 1 st, mean HI antibody titer values were 2.37, 2.25, 2.25 and 2.12 for groups A, B, C and D, respectively. There was no significant (P>0.05) difference between all the groups. On day 10 th mean HI antibody titer was 3.18, 3.25, 3.93 and 3.56, receptively. There was significant (P<0.001) difference among all the groups. Whereas, the mean HI antibody titer values were 5.25, 5.43, 8.00 and 7.62 for groups A, B, C and D on day 20 th, respectively. Groups C and D showed significantly (P<0.001) higher values than groups A and B. On day 35 th, mean values for HI antibody titer were 7.31, 7.62, 9.81 and This comparison showed that groups C and D exhibited the highest mean HI antibody titer against ND than groups A and B. It was postulated that the increasing level of probiotic in the experimental diets would improve the immune status of the experimental birds, consistently as compared to the chicks fed control ration. The overall view of the results confirmed the hypothesis in general. Table VI. Effect of various levels of probiotic on HI antibody titer in broiler chicks HI Day 1 st Day 10 th Day 20 th Day35 th I A a 3.18 c 5.25 c 7.31 b II B a 3.25 c 5.43 c 7.62 b III C a 3.93 a 8.00 a 9.81 a IV D a 3.56 b 7.62 b a LSD values P Values Mean values with the same letter are not significantly different. Moreover, these investigations are further authenticated by the work of many researchers such as Jin et al. (1997) and Shoeib et al. (1997) reported improved immunity in the commercial broiler chickens by continuous feeding of the probiotic. Moreover, the results are also supported by the findings of Panda et al. (2000) who tested the immune response to Escherichia (E) coli of broilers fed diets with various levels of probiotic. There was significantly higher antibody production in the 1 g probiotic supplementation at 10 days and 5 days post-inoculation in response to SRBC antigen when injected at 14 days and 21 days of age respectively, compared to control. The birds fed probiotic were less susceptible to E. coli challenge than controls. Probiotics might be considered more effective, during the early life period of the chicks, where the micro flora is progressing or under pathogenic stresses. Such concept was favored by Schat and Myres (1991) also reported that immune protection in poultry could concentrate in the egg yolk, which transmit its antibodies directly into the intestinal lumen and assured protection of the young bird against infections during first four days after hatching.

5 Sarhad J. Agric. Vol.25, No.3, REFERENCES Allan, W.H., J.E. Lancaster and B. Toth Newcastle disease vaccines, their production and use. FAO (UN), Rome. pp AOAC Official methods of analysis (13 th Ed). Assoc. of Analytical Chemists. Washigton D.C., USA. Andreatti-Filho, R.L. and E.N. Silva Effect of carbohydrates and anaerobic cecal microflora on intestinal colonization by S. typhimurium and S. enteritidis in broiler chicks. Revista Brasileira de Ciencia Avicola. 1: Green, A.A. and D.W.B. Sainsbury The role of probiotic in producing quality poultry products. XV Europ. Symp. Quality of Poultry Products. XV Europ. Symp. Quality of Poultry Meat, 9-12 Sep Kusadasi/Turkey. pp Honma, N., K. Ohatani and H. Kikuchi On effects of lactic acid bacteria. Part-II, Clinical effects. New Med. Clinic. pp.36: 75. Jin, L.Z Probiotics in poultry: modes of action. World's Poult. Sci. J. 53: Jin, L.Z., Y.W. Ho, N. Abdullah and S. Jalaludin Probiotics in poultry: modes of action. World's Poult. Sci. J. 4(53): Midilli, M. and S.D. Tuncer The effects of enzyme and probiotic supplementaiton to diets on broiler performance. Turk. J. Vet. Anim. Sci Panda, A.K., M.R. Reddy, S.V.R. Rao, M.V.L.N. Raju and N.K. Praharaj Growth, carcass characteristics, immunocompetence and response to Escherichia coli of broilers fed diets with various levels of probiotic. Archiv fur Geflugelkunde. 64: Schat, K.A. and T.J. Myres Avian intestinal immunity. Crit. Rev. Poult. Biol. 3: (quoted from the book Biotecnology in Animal Feeds and Animal Feeding: An overview.chapter 10, 213p). Statistical Analysis System A handbook of statistical analysis system. ver Instt. Inc. Cary, New California. Steel, R.G.D. and J.H. Torrie Principle and procedures of statistics. McGraw Hill Book. Co. Inc. New York. Shoeib, H.K., A.N. Sayed, S.A. Sotohy and S.K. Abdel-Ghaffar Response of broiler chicks to probiotic (pronifer) supplementation. Assiut Vet Med. J. 36: Yeo, J. and K.I. Kim Effect of feeding diets containing an antibiotic, a probiotic or yucca extract on growth and intestinal urease activity in broiler chicks. Poult. Sci. 76:

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