Ameliorating Effect of Immunostimulants on Ochratoxicated Broiler Chickens. By Abdel-Alim. G. A., K. Madian., Kawkab, A. Ahmed.*, A. El- Nabraway., M. H. H. Awad. Faculty of Veterinary Medicine, UniversityCairo Department of Poultry Diseases * Department of Pathology

Presented by Dr. Kawkab, A. Ahmed

Mycotoxins are often found as natural contaminants in grains. Ochratoxin-A (OA) is the major component of a group of secondary metabolites produced by several fungi. Ochratoxin in poultry decreases growth rate performance, has nephrotoxic effect, immunosuppressive effect and cause reproductive failure. Introduction

Inhibition of protein synthesis, formation of DNA adducts, and provocations of DNA single-strand breaks are suggested mode of actions of ochratoxin as a result of oxidative stress. The immunosuppressive effect of ochratoxin on chickens immune system can not be ignored which would lead to immune dysfunction that is of major interest because of the widespread occurrence in commercial chickens. Administration of biological compounds that activate key pathways in the immune system can strengthen the defense and immune mechanisms of the body and currently can be used for stimulating the non-specific immune responsiveness in both the human and veterinary medical practice.

Eventually, it has already known that many diseases / disorders, that have immunomodulated components, can be modified by administration of biological compounds that activate key pathways in the immune system. They strengthen the defense and immune mechanisms of the body and currently can be used for stimulating the non-specific immune responsiveness in both the human and veterinary medical practice.

Objective This study was conducted to study the ameliorating effect of some commercial immuostimulant compounds on the toxic effects induced by ochratoxicosis in broiler chickens.

Material and Methods Ration : Naturally Ochratoxin-A (OA) contaminated commercial ration containing 5 ppm Ochratoxin A was used from one day of age. Immunostimulants : 1.1- Nutrilac, I. G. A; liquid acidifier containing 90% lactic acid and 10% formic acid was used in drinking water in a dose of 3 ml / Liter during first 1-5 days and repeated at 15th-17th days of age as recommended by the manufacturer company.

1. Immunoaid dry: powder contains a mixture of yeast mannanooligosacccharides and yeast fermentation extracts of Saccharomyces cervesiae on mineral carrier was used in ration in a dose of one kg / Ton from one day-old till the end of the experiment as recommended by the manufacturer company. Vaccines: a- Newcastle disease virus (NDV) vaccines Both live (B1 type, B1 strain) ND vaccine were used for vaccination of birds at 7 and 21 days of age respectively by eye drop routes.

b- Infectious bursal disease (IBDV) vaccine A freeze-dried live vaccine Noblis Gumboro 228E was given by eye drop route as recommended by the manufacturer company. Newcastle disease virus (NDV) challenge strain characterized by Velogenic viscerotropic (VVND) strain Sheble and Reda (1976) was used for challenging chickens using a dose of EID50/ml/bird by intramuscular injection. Sheep reed blood cells suspension (SRBcs) A 7% suspension of the washed RBCs was prepared in P.B.S and each bird was injected with 1 ml at 19 days of age.

Methods used for immunological study: 1- Haemagglutination inhibition (HI) test: Serum samples collected weekly from one to five of age from different groups were subjected to HI tests for determination of haemagglutinating antibodies titer against NDV. 2- Response to sheep red blood cells: Blood samples collected at 3, 6, and 9 days post injection of sheep RBCs were subjected to Haemagglutination test (HA) for determination of haemagglutinating antibodies titer to sheep RBCs.

3- Serum protein electrophoresis: Electrophoretic profiles of sera collected at the 5th week of age were tested using cellulose polyacetate strips. 4- Bioassay: Challenge with the velogenic viscerotropic strain of Newcastle disease virus (VVND) was carried out at the 5 th week of age and birds were kept for two weeks observation period for clinical signs, mortality and post mortem lesions of ND. 5- Lymphoid organ weight ratio: Lymphoid organs; bursa of Fabricius (BF), thymus and spleen were collected at 5th week of age for determination of relative lymphoid organs weight ratio.

Histopathology : Specimens from liver, kidneys, bursa of Fabricius, spleen, thymus and caecal tonsils were collected weekly from two randomized chickens from all studied groups, fixed in 10%neutral buffered formalin and stained with Hematoxylin and Eosin Statistical analysis: Data were grouped and expressed as means ± standard errors of the means. The obtained data (group means for all response variable in each experiment) were analyzed by analysis of variance (ANOVA).

Experimental design One hundred and eighty, one-day-old; meat-type broiler chickens (Hubbard breed) were allotted into 4 groups (1-4) by 3 replicates consisting of 15 each as follow: G1 : fed on diet containing 5 ppm OA and treated with Nutrilac, I. G.A. G2 : fed on diet containing 5 ppm OA and treated with immunoaid dry. G3 : fed on diet containing 5 ppm OA without any treatment (+VE). G4 : fed on diet and served as a blank negative control.

Birds from each groups were weighted weekly for determination of weekly mean body weight. Organs and blood samples were collected from all groups in different intervals for histpathology, lymphoid organ weight/ body weight ratio and measurement of immunological parameters as described before in material and methods. Ten, 5-weeks old chickens were inoculated with VVND in a dose of EID50/ml/bird by IM injection to assess the pathogenicity of the virus and 90% mortality obtained.

Table (1) Mortality and protection percentage of broiler chickens fed on diet containing 5 ppm ochratoxin A and treated with immunostimulatns. Deaths after NDV challenge Deaths before NDV challenge OA TreatmentGroup % Dead/Total % / /45 + Im- Dry / /45 + Nutrilac / /45 + +VE3 209/ /45 + Blank4 +ve = positive control

Table (2) The effect of the immunostimulatns on body weight of broiler chickens fed on diet containing 5 ppm OA. Age OATreat. Grp. 5 wk4 wk3 wk2 wk1 wk 291.0±17.1 c 268.7±16.3 c 194.7±13.90 b 115.7±10.7 c 76.66±8.7 bc + Im-dry ±21.3 d 339.3±18.4 d 221.0±14.8 c 148.3±12.1 d 79.0±8.8 c +IGA ±19.1 b 288.3±16.9 b 183.0±13.5 b 132.7±115 b 69.0±8.3 b + + VE ±32.1 a 689.0±262 a 398.5±19.9 a 270.5±16.4 a 105.7±10.2 a - Blank L.S.D. (Least significant difference) * Values are expressed as group means ± SEM of 3 replicates of 15 broiler chicks (n=15). a- d : Means within a column without superscripts are significantly different (p < 0.05). L.S.D.: least significant difference as determined by Fisher’s protected L.S.D procedure.

Table (3). The effect of Nutrilac, I.G.A., and immunoaid-dry on the lymphoid organs weight of chickens fed on diet containing 5 ppm OA for 5 weeks. S/B Wt index T/B Wt index BF/B Wt index B. wt. OA Treat. Grp ac ± b ± a ± c ±291 + Im-dry c ± a ± ab ± d ±456 + I.G.A ac ± b ± a ± b ± ve a ± a ± a ± a ± Blank L.S.D. (Least significant difference) * Values are expressed as group means ± SD of 3 replicates of 15 broiler chicks (n=15). a- d : Means within a column without superscripts are significantly different (p < 0.05). L.S.D.: least significant difference as determined by Fisher’s protected L.S.D procedure

Table (4) HI geometric means against ND vaccination of broiler chickens fed on diet containing 5 ppm ochratoxin A and treated with immunostimulants. HI Geometric means against NDV / (Age) OA TreatmentGroups 5 wk4 wk3 wk2 wk 1 wk Im- dry I.G.A Ve Blank 4

Table (5) HA geometric means against sheep red blood cells of broiler chickens fed on diet containing 5 ppm ochratoxin A and treated with immunostimulants. HA Geometric means against S.R.B.Cs. OATreatmentGroup 9 ds PI6 ds PI3 ds PI * Im-dry I.G.A Ve Blank4 * Days post S.R.B.Cs. injection.

Table (6) Serum electrophoretic analysis of broiler chickens fed on diet containing 5 ppm ochratoxin A and treated with immunostimulants. A/G ratio Serum protein fractionsOATreat.Group Globulin%Albumin % Gam ma BetaAlpha 2 Alpha Im-dry I.G.A Ve Blank4

Table (7) The effect of the immunopotentiators on immune cell activation score of broiler chickens fed on diet containing 5 ppm ochratoxin A Groups Histopathological changes 4 Blank 3 + Ve 2 I.G.A. 1 Im-dry Bursa of Fabricius: Lymphoblasts activation Lymphocytic hyperplasia Sum score Spleen: Lymphoblasts activation Sum score 0073Total immune cell activation score Histopathologic assessments of the experimental parameters were graded as follows: 0 - showing no changes - 1, 2, and 3 indicating mild, moderate and severe changes respectively.

Table (8) The effect of the immunopotentiators on histopathological changes in broiler chickens fed on diet containing 5 ppm ochratoxin A. Group 4Group 3Group 2Group 1Histopathological changes Bursa of Fabricius Congestion of interfollicular B.V Lymohocytic necrosis & depletion Sum lesion score Spleen: Lymphocytic depletion Reticular cells hyperplasia Sum lesion score Thymus gland: Lymphocytic necrosis & depletion Thymic hemorrhage Sum lesion score Histopathologic assessments of the experimental parameters were graded as follows: (0) no changes, (1) mild, (2) moderate, (3) severe changes.

Group 4Group 3 + Ve Group 2 I.G.A. Group 1 Im-dry Histopathological changes Caecal tonsils: Lymphocytic necrosis & depletion Sum lesion score Kidney: Tubular nephrosis Tubular epithelial necrosis Calculi & cellular casts formation * chronic interstitial nephritis Chronic uretritis Sum lesion score Liver: Kupffer cell activation *Sinusoidal dilatation Hepatocellula vaculation Periacinar hepatic necrosis Hepatic hemorrhage Portal infiltration with heterophil * portal connective tissue proliferation * Bile duct hyperplasia Sum lesion score Total Lesion Score

Liver A : G1-one week PT showing hepatocelluar vacculation together with focal hepatic hemorrhages B : G1 - three weeks PT showing focal area of hepatic necrosis associated with mononuclaer cells infiltration ( small arrow) as well as portal infiltration with heterophil (large arrow) C : G2 - three weeks PT showing focal heterophilic cells aggregations (arrow) D : G3 - three weeks PT showing portal connective tissue prolifiration associated with hyperactivation and hyperplasia of epithelial lining bile ducts (arrow). A B C D

E : G3; 5 weeks PT showing hyperactivation and hyperplasia of the epithelial lining bile duct with formation of newly formed ductless bile duct (arrow). F : G4 ; 4 weeks PT showing no hitopathological changes. E F

Caecal Tonsils: A : G1; three weeks PT showing mild lymphocytic depletion in the lymphatic follicle in the submucosa of the caecum (arrow). B : G3: three weeks PT showing marked lymphocytic necrosis and depletion of the lymphatic nodule of the lamina propria as well as submucosa of the caecum (arrow). C : G4; three weeks PT showing no histopathological changes. AB C

Kidney A : G1; one week Pt showing tubular nephrosis associated with pyknotic nuclei of their epithelial lining (arrow). B : G2; three weeks PT showing distention of some renal tubules with heterophilic cell casts (arrow). C : G2; three weeks PT showing marked tubular necrosis completely replaced with mononuclear cell infiltration together with intratubular basophilic lamillated calculi formation D : G3; three weeks PT showingchronic interstitial nephritis together with chronic uretritis characterized by desquamation and exfoliation of urothelium as well as intraluminal distention with cellular debris, exudates,and calculi. A B CD

E : G3 ; three weeks PT showing chronic interstitial nephritis together with chronic uretritis characterized by desquamation and exfoliation of urothelium as well as intraluminal distention with cellular debris, exudates,and calculi. F : G4 ; 4 weeks PT showing no histopathological changes. E F

Bursa of Fabricius A: G1; three wks PT showing congestion of intrafollicular blood vessels (arrow). B: G1; three wks PT showing lymphoblasts activation associated with slight lymphocytic depletion C: G2: five wks PT showing interfollicular C.T. prolifiration (arrow) and lymphocytic hyperplasia D: G3: 4 wks PT showing lymphocytic necrosis & depletion especially in the medullary portion of the follicle. A B C D

E: G4; 4 wk PT showing no pathological changes ( H & E 66) E

Spleen A: G2; 2wk Pt showing mild lymphocytic depletion of some lymphoid follicle as well as lymphoblasts activation. B: G3; 2wk PT showing reticular cells hyperplasia (arrow). C: G4; three wks PT showing no pathological changes A B C

Thymus gland D: G2; 4 wk PT showing no histopathological changes. E: G3; 4 wks PT showing lymphocytic necrosis and depletion. D E

The used immunostimulants, especially Nutrilac I.G.A., not only ameliorate the toxic effect of ochratoxin "A" but also counteracted its detrimental effects on the immune system as indicated by: 1- Reduction in mortality of broiler chicken fed on diet containing 5 ppm Ochratoxin-A from 33.3% in non treated group to 24.4% in Nutrilac treated group and to 13.3% in immunoaid-dry treated group. 2- Reduction in the mortality from 50% to 29.4% in ochratoxicated birds that challenged with VVNDV was recorded in Nutrilac I.G.A treated group. 3- Both studied immunostimulants potentiate antibody responses to parentrally delivered antigens (NDV and sheep red blood cells).

4- Serum electrophoretic patterns revealed an albumin/globulin ratio of 0.45 in blank control group as compared with 0.89 and 1.10 in Nutrilac I. G. A. treated and untreated ochratoxicated chicken groups, respectively. 5- Severe histopathological alterations were observed in kidneys, liver, and lymphoid organs (bursa of Fabricius, thymus glands, spleen and caecal tonsils) of ochratoxicated birds while Mild histopathological alterations were recorded in Nutrilac I.G.A. and Immune dry treated group.

6- The used immunopotentiators reduced the total lesion scores from 58 in ochratoxicated chicken group to 40 and 47 in Nutrilac I.G.A. and immunoaid-dry treated groups, respectively. Moreover; they developed scores of 7 and 3 on immune cell activation of bursa of Fabricius and spleen manifested as lymphoblasts activation and lymphocytic hyperplasia as compared with 0 score in ochratoxicated chicken group

The actual mechanism by which Nutrilac I.G.A ameliorate the toxic effect of OA is not known, However, many speculations were recorded. First speculation based on that organic acid content of this product which has a possible metabolic regulatory effect those potentates the process of detoxification. Second speculation is based on the presence of weak acid in that Nutrilac that might act as a synergist to enhance the effectiveness of antioxidant at a cellular level which in turn reduce the lipid peroxidation caused by Ochratoxin A.