Hepatoprotective and Nephroprotective Effect of Aegle marmelos and Andrographis paniculata in Aflatoxicosis of Broiler Chicken

Toxigenic strains of the fungi, Aspergillus flavus and A. parasiticus in food grains produces Aflatoxins (AF). These microbes invade and grow in feed ingredients and thrive under conducive environment especially under hot humid conditions to release their secondary metabolites. The level of contamination of the toxin in feed was brought under permissible limit through strict control measures. Addition of various hepatoprotectants and toxin binders during feed mixing help to minimise the deleterious effect of AF. Aegle marmelos and Andrographis paniculata are two well-known hepatoprotective plants (Shakya, 2020; Bhar et al., 2019). Aegle marmelos belongs to the family Rutaceae and  Andrographis paniculata belonging to family Acanthaceae, grows abundantly throughout tropical and sub-tropical regions. AL-Zuhariy and Hassan (2017) studied about hepatoprotective and immunostimulatory effect of andrographolide against aflatoxicosis in broiler chicken. The authors could not access any study on the effect of Aegle marmelos among poultry. The objective of this study was to evaluate the hepatoprotective and nephroprotective effect of Aegle marmelos and Andrographis paniculata individually and in combination in sub lethal aflatoxicosis.

The study was conducted at Department of Veterinary Pathology, College of Veterinary and Animal Sciences, Mannuthy, Thrissur, during Apr 2018. A. flavus NRRL 6513 culture was sub cultured every 15 days and maintained at room temperature to ensure availability of fresh spores. Maize was used as substrate for producing aflatoxins (Shotwell et al., 1966). Aflatoxin B1 content was estimated to be 63.77 ppm using TLC method at AFAQAL Namakkal. A. marmelos mature leaves and A. paniculata whole plants were collected locally and were authenticated. The plant materials were dried under shade and powdered using a pulverizer. Forty-eight day-old Vencobb 400 strain broiler chicks were procured locally, which were randomly divided into eight groups of six birds each and fed with the following diets for 21 days viz. standard feed (T1), T1 with 100 ppb AF (T2), T1 with 0.1 per cent A. marmelos powder and 0.1 per cent A. paniculata powder (T3), T1 with 100ppb AF and 0.10 per cent A. marmelos powder (T4), T1 with 100ppb AF and 0.10 per cent A. paniculata powder (T5), T1 with 100 ppb AF and 0.20 per cent A. marmelos powder (T6), T1 with 100ppb AF and 0.20 per cent A. paniculata powder (T7) and T1 containing 100 ppb AF, 0.1 per cent A. marmelos powder and 0.1 per cent A. paniculata powder (T8).All the experimental birds were sacrificed on 21st day of the experiment. Liver, kidney, thymus, spleen, bursa of Fabricius, gizzard and intestine were examined for gross lesions and observations were recorded. Samples of liver (500 mg) were taken from all birds in cold Tris-HCL buffer (pH 7.5) and refrigerated. The levels of lipid peroxidation in liver was estimated by the method described by Fraga et al., (1988) and expressed in terms of nano moles of MDA/g of tissue. Tissue samples of liver, kidney spleen, bursa of Fabricius, thymus, gizzard and intestine of all the birds were fixed in 10 per cent neutral buffered formalin, processed and embedded in paraffin. Tissue sections were cut at 4-5 m m thickness and stained with haematoxylin and eosin (Suvarna et al., 2018). Data was analysed statistically using SPSS version 24.0.

Mean (±SE) lipid peroxidation levels of all treatment birds recorded at 21st day are presented in Table 1. Broiler chicks of T2 group showed a significant increase in lipid peroxidation levels compared with other groups at 21st day of experiment. No significant difference existed between other groups of birds compared to the control. Lipid peroxidation levels of broiler chicks fed with 100 ppb AF alone were significantly increased compared to that of control. The significant increase in MDA concentration of T2 broilers could be due to AFB1 induced release of epoxides, which conjugated readily with glutathione enzymes of lipid peroxidation and was in accordance with Verma and Mathuria (2008). Lipid peroxidation levels of T3, T4, T5, T6, T7 and T8 were statistically similar to T1. The beneficial effect of these plant materials in this present study could be due to the anti-oxidant potential of their active components (Abhishek et al., 2010; Aneesh et al., 2018a; Aneesh et al., 2018b).
 
In the present study, no clinical signs and pathological gross lesions could be observed among all the groups, in any of the organs except yellowish discoulouration in liver of T2 group (Fig 1). Similar findings were previously observed among birds exposed to AF by Gholami-Ahangaran (2015), Kumar and Balachandran (2009) and Yavuz et al., (2017). Gross appearance of organs such as kidney, spleen, thymus, bursa of Fabricius, gizzard and intestine of all other treatments were identical to that of T1. Ortatatli et al., (2005) observed that absence of gross lesions in T2 group could be attributed to low levels of AF and short duration used in the present experiment. Histopathological observations are summarized in Table 2. Lesions were observed in liver, kidney, spleen, thymus, bursa of fabricius and intestine of T2 group (Fig 2 to 7). No lesions were observed in the organs of T1 and T3 groups. The histological appearance of the organs among T4, T5, T6, T7 and T8 groups showed improvement compared with that of T2 group and in most cases were comparable to T1.
 

 

 

 

 

 

 

 
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Liver of T2 group of birds revealed the presence of cytoplasmic vacuolations, Kupffer cell hyperplasia, focal accumulation of mononuclear cells and acinar pattern of parenchyma compared to normal histological appearance of T1 and T3 groups. Similar lesions were also observed with 100 to 150 ppb AF exposure among broilers by Ramdas et al., (2013). Daly (2010) and Gholami-Ahangaran et al., (2016) observed that cytoplasmic vacuolations of liver due to macromolecular damages of cells and oxidative damage of deoxyribonucleic acid in aflatoxicosis. Histopathological lesions of Kupffer cell hyperplasia, accumulation of mononuclear cells caused by hepatotoxic action of AF which was in accordance with Kumar and Balachandran (2014) and Nithya (2016). Upregulation of genes responsible for cell proliferation could be the cause of proliferative changes (Yarru, 2009). Acinar pattern could be the result of accumulation of calcium in hepatocytes leading to mitochondrial dysfunction and reduced production of adenosine triphosphate (Lakkawar, 2015).
 
Microscopic picture of T4 birds were closer to that of T1 with presence of a few cytoplasmic vacuolations and mild accumulation of mononuclear cells. In T6 birds, the microscopic appearance of liver was better than that of T4 with normal appearance as that of T1. Histologically the liver of T5 birds was similar to that of T1 except for the presence of a few cytoplasmic vacuolations. The microscopical features of T7 liver were identical to that of T1. Liver of T8 birds was identical to that of T1. The hepatoprotective ability of plants could be due to anti-oxidant potential of flavonoids component of the plant (Aneesh et al., 2018a; Aneesh et al., 2018b). A. paniculata enhances hepatic glutathione peroxidase, superoxide dismutase and glutathione-S-transferase activities due to its antioxidative potential. Similar effect of polyherbal preparations on liver toxicity was also observed previously by Singh et al., (2015).

Kidney of T2 birds revealed epithelial degeneration, mononuclear accumulation, desquamation of proximal convoluted tubule (PCT) and vacuolation of distal convoluted tubule (DCT). Similar observations were previously observed among broilers exposed to AF by Ortatatli (2005), George (2007) and Nithya (2016). Inflammatory and degenerative changes caused by the accumulated AF in the tubules could be the cause of epithelial degeneration and cytoplasmic vacuolations (Benjamin, 1978). Mononuclear infiltration and tubular damage could be due to lipid peroxidative damage of renal tubules caused by AF (Darwish, 2011). Histopathological lesion of mild epithelial degeneration was observed in kidney of T4 birds. Apparently normal appearance of kidney was observed among birds of T5, T6 and T7. Mild accumulation of mononuclear cells, few vacuolations in DCT, mild desquamation of PCT were observed in T8. Aneesh et al., (2018a) and Sivakumar and Rajeshkumar (2015) respectively interpreted that the nephroprotective action of A. marmelos and A. paniculata could be due to its anti-oxidant properties.

Microscopic examination of spleen revealed significant reduction of white pulp indicating deficiency of lymphocytes. Immunosuppression of AF could be the reason for lymphocyte depletion (Ortatatli, 2005). Microscopic appearance was improved and was returning to normal as that of T1 in T3 and apparently normal in T4, T5, T6, T7 and T8. Protective efficacy of A. marmelos could be due to the presence of phytoconstituents with antioxidant properties like quercetin and related flavonoids. As per Seth et al., (2016), quercetin acts by blocking the enzyme xanthine oxidase, chelating iron and directly scavenging hydroxyl, peroxy and superoxide radicals inhibits lipid peroxidation. It was proven previously by Neogy et al., (2008) that A. paniculata stimulates glutathione levels thus prevents the oxidative damage.
       
Thymus of T2 birds revealed severe depletion of lymphocytes indicating immunosuppressive effect of AF. Bursa of Fabricius of T2 birds was characterized by presence of cytoplasmic vacuolations, thinning of cortical area and lymphocyte depletion. The histopathology of bursa of Fabricus in this study could be due to immunosuppressive effect of AF which was in accordance with Arulmozhi (1999). Histological lesions of bursa of Fabricius were significantly improved in T4, T5, T6, T7 and T8 and were apparently normal as that of control. Positive effect of A. paniculata could be due to immunomodulatory property of andrographolide present in the plant (Jayakumar et al., 2013).
 
Intestines of T2 broilers revealed catarrhal enteritis characterised by mild hyperplasia of goblet cells. Similar results were reported previously by Calnek et al., (1997), Jakhar and Sadana (2004) and Kumar and Balachandran (2009). Kashyap et al., (2017) reported that the enteritis could have originated due to secondary bacterial infection initiated by immunosuppression of AF. Intestines of all other treatments revealed identical histology as that of control. Gizzard of birds of all treatments including T2 showed no abnormality. This result was contrary to reports of George (2007). The variation could be due to very low concentration of AF and short duration in this study.

The present experiment showed hepatoprotective and nephroprotective effect of A. marmelos leaf powder and A. paniculata plant powder. The concentrations of these plant materials did not cause any significant variation in the hepatoprotective and nephroprotective effects. Both the plant materials at concentrations of 0.1 per cent and 0.2 per cent were found to be equally effective. The combination of plant powders at 0.1 per cent used each without AF did not elicit any negative effect and its response was identical to control. The combination of plant materials at 0.1 per cent levels also elicited similar response as that of plant materials used at individual concentrations. It could be concluded that A. marmelos leaf powder and A. paniculata plant powder could be added to the broiler diet in the above concentrations to effectively counter the deleterious effects of subclinical aflatoxicosis.

This study was funded by Kerala Veterinary and Animal Sciences University, Pookode.

The authors declare that they have no conflict of interest.