An experiment was conducted to study the protective role of herb Emblica officinalis in induced aflatoxicosis in broilers. Thirty day old broiler chicks divided into three groups. Birds of group T0 were fed with standard basal rations. Group T1 birds were fed with standard feed mixed with aflatoxin (AF) B1@150 ppb and group T2 birds were fed with standard feed mix with AFB1@150 ppb and E. officinalis @5 g/kg of feed. Skin thicknesses were measured in six birds from each group on 21st day for cell mediated immune response. Group T1 and T2 showed significant differences in weight gain and significant reduction in cell mediated immune response was noted as compared to T0 group. Gross examination of group T1 birds showed yellowish discoloration of liver and hepatomegaly, mild lesions in kidneys. Microscopically, liver parenchyma showed moderate hepatic derangement, focal formation of microgranuloma, multiple foci of hepato-cellular swelling, necrosis in some hepatocytes, congestion of blood vessels, mononuclear cells infiltrations around blood vessels, focal bile duct hyperplasia and sinusoidal aggregation of erythrocytes. In T2 group birds, microscopically liver, kidney, spleen and bursa of Fabricius showed mild changes as compared to T1. Present study suggest that supplementation of herb E. officinalis could reduce aflatoxin toxicity in broilers.
Indian poultry sector has made remarkable progress during last few decades growing from backyard farming to large commercial industry. Consequently the modern farming practices to fulfil the increasing demand of poultry birds are making birds prone to various infections and toxicities due to contamination of feed and water that produces great economic losses. Contamination of feed with mycotoxins is major problem (Mohanamba et al., 2007) encountered in poultry industry. Aflatoxin (AF) is most common mycotoxin produced by fungi Aspergillus flavus and Aspergillus parasiticus which contaminating the grains and cereals at any time before and or after or during harvesting, storage, transportation and processing of feed ingredients. Among aflatoxin B1, B2, G1, and G2, AF B1 is most toxic. Metabolites of AF are stable and resistant to degradation (Park, 2002; Desphande, 2002). Consumption of AF contaminated feed causes aflatoxicosis in poultry characterized by reduced feed intake, weight gain, feed utilization (Bailey et al., 2006; Shi et al., 2006, 2009), increased susceptibility to environmental, microbial stresses and mortality (Leeson et al., 1995 ; Jand et al., 2005). Aflatoxin is responsible for damage of liver, kidney and impaired immunity (Ibrahim et al., 2000; Oguz et al., 2003). AFB1 has immunosuppressive properties by affecting cell-mediated immunity (Meissonnier et al., 2008). Chronic exposures of chicken to aflatoxins depress the phagocytic efficiency and the delay hypersensitivity reactions in birds (Kadian et al., 1988). AFB1 at low dose decrease both mRNA and protein levels of lymphocytic IL-2, IFNγ and affected macrophage functions along with IL-1α, IL-6 and TNF production (Giambrone, 1978 and Dugyala and Sharma, 1996). Also AF causes deleterious effect by producing free radicals that is injurious to DNA, protein and lipid membrane (Yang et al., 2000). This deleterious effect of free radical could be effectively ameliorated by the antioxidant defence of the body and thus controlling aflatoxicosis (Yarru et al., 2009).
Use of chemical growth promoters and immunomodulator has been criticized due to adverse residual effects on consumers. There is an increase in demand for organic meat and eggs. Also practical and cost effective methods to detoxify mycotoxin present in feed stuffs on a larger scale basis are not available. In view of this, herbal and plant derivatives might be a valuable alternative to promote growth, immunomodulation and health in poultry as there is no residual toxicity. Wide use of E. officinalis in the Ayurveda is believed to increase defence against diseases. It has beneficial role in treatment of cancer, diabetes, hepatic and cardiac ailments, intestinal ulcers, anaemia and various other diseases. Also, it used as an antioxidant, immunomodulatory, antipyretic, analgesic, cytoprotective, antitussive and gastroprotective agent (Bhagat, 2014). Hence, considering the above facts, the present study was planned to study the preventive role of E. officinalis supplementation on Aflatoxicosis.
Material and Method
Chicken Management and Diet
Study was conducted in thirty day-old Ven-Cobb-400 broiler chicks. The chicks were divided into three equal groups with ten birds in each group having similar body weight within a group as well as between groups. Standard managemental procedures were followed throughout the course of experiment. The given diets were free of any growth promoter, toxin binders, antibiotic or known contaminants that would interfere with the study objectives. The ration was formulated as per BIS (2007) specification for broiler chicken.
The aflatoxin used in present study was procured from HiMedia lab, India. Pure crystalline AFB1 was incorporated into the diets by dissolving AFB1 in chloroform (1mg/10mL) followed by mixing the solution with appropriate quantities of ground feed in experimental groups (Denli et al., 2009; Kaoud, 2013). The premix feed was kept overnight at room temperature for the solvent to evaporate and was then mixed into the basal diet to provide the desired level of 150 ppb AF B1/ kg of feed.
Emblica officinalis (Amla) Powder
Commercially available E. officinalis powder was purchased from local ayurvedic market. The desire level of 5g E. officinalis /kg of feed was obtain by well mixing and homogenizing feed before offering to birds.
The birds of group T0 were kept as control group and was fed on basal diet without any supplementation throughout experimental period, birds of group T1 were fed basal diet supplemented with AF B1 @150 ppb per kg of feed from day eight onwards till the end of experiment and birds of group T2 were fed basal diet supplemented with E. officinalis @ 5 g/kg of feed +AF B1 @150 ppb per kg of feed from day eight onwards till the end of experiment. During experiment all the clinical signs were noted.
Organ Weights and % Organ Weight to Body Weight Ratios (Weight Index)
The live body weights (g) of six birds from each treatment were recorded just before slaughter. Six birds from each treatment were sacrificed by humane method at the end of experiment. Visceral organs viz. Liver, heart, bursa of Fabricius and spleen were dissected out from carcass and weighed and weight index was calculated as percentage of the weight of visceral organ to the total body weight of bird.
Cell Mediated Immune (CMI) Response by 2, 4-Dinitrochlorobenzene (DNCB)
Skin contact sensitivity by DNCB was used to asses CMI in different group. Test in each group was performed on 21st day described by More (1996) and Tiwari and Goel (1985). The skin thickness was measured before and 24, 48 and 72 hours after sensitization. One per cent (1%) solution of 1-Chloro-2, 4- dinitrobenzene (HiMedia) in acetone was prepared by dissolving one gram in 100 ml acetone and used to assay cell mediated immune response. DNCB (0.1 ml of 1 percent) was injected to six birds of each group intra-dermally at inter-digital space between third and fourth digit of right leg using one ml tuberculin syringe. This was allowed to dry immediately by blowing so as to avoid the solution running down the sides. The thickness of the skin at the site was measured using digital slide Vernier calliper before challenge (0 hour) and 24, 48 and 72 hours after challenge and expressed in millimetres.
Post Mortem Examination and Histopathology
Representative birds from each group were opened and all the visceral organs were examined for alteration if any. Representative samples of liver, kidneys, heart, spleen and bursa of Fabricius were collected from each group and fixed in 10 percent neutral buffered formalin (NBF). Tissue sections were cut at 3-5μ thickness and stained with routine Haematoxylin and Eosin method (Culling, 1974). Histo-pathological examination was done at the end of experiment.
The means and SE of different parameters in different treatment groups were calculated and the effects of the treatments were analyzed as per Snedecor and Cochran, (1994). The differences between the means were tested at P≤ 0.05 by ANOVAs.
The present experiment was conducted to study the effect of E. officinalis (Amla) on growth, immunomodulatory response and pathological changes in organs of birds fed with AF B1 in feed.
Birds from T1 showed slight reduced feed intake, poor growth and depression than control groupT0. T2 group showed improvement on feed intake than T1. No mortality was noted throughout the experimental period in any of the group studied. The birds in group T0 and T2 did not show any clinical signs and were healthy throughout the experiment.
Table 1: Effect of AF (150ppb/kg feed) and E. officinalis (5g/kg feed) on Mean live body weight (g) and % relative organ weights at 42 DPT
|I||Live wt of birds*||2029±1.0||1918±1.41||1965±2.17|
|V||Heart (%) **||0.53||0.51||0.522|
*Significant; **Non- significant
Growth, Live Body Weights (g) and Relative Organ Weights (%)
The average live body weights of broiler chicken at six weeks of age recorded for the treatment T0, T1 and T2 were 2029±1.0, 1918±1.41 and 1965±2.17 gm respectively (Table 1). The average live body weight of broiler chicken fed diet supplemented with AF B1 (T1) was significantly (P<0.05) lower than the broiler chicken fed on basal diet (T0) and diet supplemented with AF B1 and E. officinalis (T2). The relative weights (%) of liver, spleen, bursa of Fabricius and heart of broiler fed with basal diet (T0) were 2.562, 0.163, 0.139, and 0.530 while in birds fed with aflatoxin (T1) were 3.044, 0.135, 0.109, and 0.510 respectively. The corresponding values in birds supplemented with AF B1 and E. officinalis (T2) were 2.78, 0.151, 0.124, and 0.522 (Table 1). The results of present study indicated that the relative organ weights of liver increased significantly (P<0.05) in AFB1 treated group (T1) than other groups, while lymphoid organ weights viz. spleen and bursa of Fabricius were decreased significantly (P<0.05). The relative weight of heart was unaffected in all groups.
Cell Mediated Immune Response –Skin Contact Sensitivity
The skin thicknesses (mm) on 21st day of AF B1 (T1) and AF B1 + E. officinalis (T2) group birds were significantly lower (P<0.05) as compared to T0. There was no statistical significant difference (P<0.05) in the mean skin thickness across groups at time point zero. The difference in mean skin thickness between AF B1 (T1) and AF B1+ E. officinalis (T2) fed chicks was statistically non-significant at all other time points. The mean skin thickness between AF B1 (T1) or AF B1+ E. officinalis (T2) and or control (T0) group was significant (P<0.05) at 24, 48 and 72 hrs. Skin thickness increased slowly and reached maximum at 24 hrs after DNCB challenge and reduced subsequently. Histopathology of skin section at 24 hrs after DNCB challenge revealed congestion and oedema with mononuclear cell infiltration. The study clearly indicated that the AF B1 caused immunosuppression in birds.
Table 2: Mean± S.E of DNCB delayed skin hypersensitivity test result (mm) at 21st day
Means bearing different superscript within the same row differ significantly (P<0.05).
Alteration in Visceral Organs
The six birds were sacrificed randomly from each group on 42 day post treatment (DPT) of the study and the gross changes were noted by conducting systematic post mortem examination. The birds from group T0 and T2 did not reveal any appreciable gross changes in any of the organs studied. The birds from group T1 showed gross pathological changes in visceral organs viz. liver showed slight paleness and was icteric. However, heart muscles, spleen, kidneys and bursa of Fabricius were without any appreciable gross changes.
|Fig. 1: Normal liver of Control group (T0)||
Fig. 2: Icteric liver of AF fed group (T1)
The visceral organs viz., liver, heart, kidneys, spleen and bursa of Fabricius of the birds from all groups sacrificed on 42 day of age of experiment were subjected to histo-pathological examination. The microscopic examination of liver, kidney, spleen, heart and bursa from the birds of control group (T0) showed normal histological features. Mild to moderate histopathological changes were observed in the birds fed diet with aflatoxin (T1). The histopathology of liver showed moderate changes of derangement of hepatic parenchyma, focal formation of microgranuloma, multiple foci of hepato-cellular swelling, vacuolar cytoplasmic changes, loss of nuclei in some hepatocytes, congestion of blood vessels, mononuclear cells infiltrations around blood vessels, focal bile duct hyperplasia and sinusoidal congestion (Fig. 4.). Kidney of these birds showed cellular swelling with degenerative changes (Fig. 6.).
|Fig. 3: Group T0 (Control), Liver with normal parenchyma, H&E x 100
|Fig. 4: Group T1 (AFB1 150ppb) Liver showing degenerative, necrotic changes, cellular swelling and bile duct hyperplasia, H&E x 200|
Spleen and bursa of Fabricius showed lymphocytic depletion. Whereas, heart did not reveal any observable changes on histo-pathological examination. The histo-pathological examination of liver tissues from birds treated with AF B1 and E. officinalis (T2) showed mild histo-pathological changes as compared to T1. The hepatic parenchyma showed focal cellular swelling and congestion with mild degenerative changes in hepatocytes and occasional foci of necrosis (Fig. 5).
|Fig. 5: Group T2 (AFB1 + E. officinalis), Liver showing mild cellular swelling, H&E x 200||Fig. 6: Group T1 (AFB1), Kidney showing cellular swelling, 100 x H&E|
|Fig. 7: Group T2 (AFB1+ E. officinalis), Kidney with mild cellular swelling, 100 X H&E||Fig. 8: Group T0 (Control), Spleen showing normal cellular population in red and white pulp, 100 x H&E|
|Fig. 9: Group T1 (AFB1), Spleen showing mild depletion of cell population in red and white pulp and intermixing of pulps, 100 x H&E||Fig. 10: Group T2 (AFB1 + E. officinalis), Spleen showing focal minimal depletion of cell population in red and white pulp, 100 x H&E|
|Fig. 11: Group T1 (AFB1) Bursa of Fabricius showing cortico-medullary lymphoid depletion in bursal follicles, 100 x H&E|
The kidneys of T2 birds showed mild degenerative changes with cellular swelling (Fig. 7). Spleen and bursa of Fabricius showed mild depletion of cells from parenchyma. Heart sections did not show any observable changes.
In the present study body weight, CMI response and various alterations in different visceral organs were taken into consideration to investigate the effect of AF B1 (150ppb/kg of feed) and E. officinalis (5 g/kg of feed) supplementation.
The reduction in growth of broiler in AF B1 fed group were in agreement with Campbell et al. (1983) and Bakshi et al. (2000) who reported declined growth in birds fed with diet containing AF B1. Also Teleb et al. (2004) showed a significant decrease in live body weight of chicken fed on diet containing 30 ppb AF B1 for 45 days. The reduced weight gain is due to impaired liver function and decreased utilization of nutrients from feed affected the chicken weight gain and general health (Oguz and Kurtoglu, 2000). Addition of E. officinalis (5 g/kg) in feed found to improve the performance of broiler in present experiment was similar to the findings of Sapcota et al. (2006) by feeding of E. officinalis @ 2.5 g/kg in experimentally aflatoxin (300 ppb). The supplementation of E. officinalis in diet of broiler chicken might have improved the relative weights of liver, spleen and bursa of Fabricius due to improved feed efficiency (Sapcota et al., 2006), absolute body weight gain (Maini et al., 2007 and Mode et al., 2009); and free radicals scavenging effect (Kanchana et al., 2013).
The DNCB hypersensitivity test at 21st day showed the lower in skin thicknesses in both of AF B1 fed and AF B1+ E. officinalis fed groups indicating lowered CMI responses. Findings of the present study in agreement with the Kalorey et al. (2005) who found significant depression of delayed hypersensitivity in chicken fed AF B1@ 200ppb/kg of feed. Aflatoxin inhibits the chemotactic and phagocytic abilities of leucocytes and heterophils, respectively (Giambrone et al., 1978a; Ghosh et al., 1991) these immune cells are important in CMI. Aflatoxin also reacts with the T cells and affects the CMI (Thaxton et al., 1974).
AF mainly targets the liver, Kidney, Lymphoid organs viz. spleen and bursa of Fabricius. In this study the relative organ weight of liver was increased significantly (P<0.05) in AFB1 treated group than other groups, while lymphoid organ weights viz. spleen, bursa of Fabricius were decreased significantly (P<0.05) was similar to the finding of Kalorey et al. (2005) and Sakhare et al. (2007) feeding of AFB1 @ 0.2 ppm. The relative weight of heart was unaffected in all the groups. The increase in liver weight might be due its swelling caused by fatty infiltration and impaired lipid transport caused by aflatoxicosis. In aflatoxin fed group, the relative organ weights of lymphoid organs were decreased due to the lymphoid cells depletion.
In this study histopathology of liver, kidneys, spleen and bursa of Fabricius found significant moderate changes in the birds fed diet with AF B1 (150 ppb) similar to the finding of AF B1 feeding at lower level (50-100 ppb) Giambrone et al. (1985) and Ortatatli et al. (2005). Also the similar changes were noticed by Sakhare et al. (2007) and Rathod et al. (2013) of AF B1 feeding @200 ppb and @100-150ppb respectively. The histo-pathological examination of liver, kidney, spleen and bursa of Fabricius from birds treated with AF B1 and E. officinalis showed mild histo-pathological changes. Feeding of E. officinalis (5g/kg feed) for 42 day reduced the severity of AF lesions. Bhattacharya et al. (1999) reported that antioxidant property of tannoid principal of E. officinalis which had vitamin C like property. Kaleem et al. (2014) reported the effect of E. officinalis derived tannins on humoral immune responses and their protective efficacy against Eimeria infection in chickens.
Findings of the present study proved that Aflatoxin B1 at lower doses produces its detrimental effect on health of birds which reduces the production of poultry. Whereas feeding of E. officinalis through feed reduces the severity of toxin and losses in poultry. Hence E. Officinalis could be used in feed mix during unavoidable circumstances of aflatoxicosis in chickens.
We thank the Associate Dean and Department of Poultry science, KNP College of Veterinary Science, Shirwal, Satara for providing necessary facilities and support to carry out this work.
Conflict of Interest
Authors has declared no conflict of interest.