Effect of Supplementation of Toxin Binder (Mycodetox B1) on Liveability, Immune Response and Organ Pathology in Induced Aflatoxicosis in Japanese Quails

1. Introduction

Mycotoxins are found world-wide as natural contaminants of feed and feedstuffs (Katole et al., 2013). The toxicity of mycotoxins i.e. mycoxicosis, is characterized by their nephrotoxic, immunosuppressive, hepatotoxic, carcinogenic, mutagenic and teratogenic effects in animals and poultry (Patil et al., 2005, 2006, 2014, 2017a, b; Patial et al., 2013; Patel et al., 2015; Patil and Degloorkar, 2016a, b, 2018). Aflatoxicosis in poultry causes lowered performance in terms of reduced body weight gain, feed intake and feed efficiency (Silambarasan et al., 2013; Singh et al., 2015; Singh et al., 2016), reduced nutrient utilisation (Silambarasan et al., 2013), increased mortality (Khatke et al., 2012b; Sharma et al., 2014), anemia (Singh et al., 2015; Singh et al., 2016), hepatotoxicosis and haemorrhage (Churchil et al., 2014; Singh et al., 2015; Singh et al., 2016; Pathak et al., 2017) and altered biochemistry (Singh and Mandal, 2013; Singh et al., 2013a). It impairs humoral and cellular immune responses in poultry and increases susceptibility to environmental and infectious agents (Khatke et al., 2012a) leading to severe economic losses. In literature, several approaches for decontamination of aflatoxin contaminated feed are available, but they are very expensive and not applicable under practical conditions. Therefore, a series of experiments was conducted. Among the various aflatoxin adsorbents, diatomaceous earth, sodium bentonite and zeolite either at 0.5% or 1% level were partially effective in ameliorating the different adverse effects of aflatoxin in broiler chickens. Among three mycotoxin adsorbents tested, diatomaceous earth was less effective in comparison to sodium bentonite and zeolite. However, combination of the binders at a time was the most effective in ameliorating the adverse effects of AFB1 in broiler chickens (Silambarasan et al., 2013). Use of mannan oligosaccharide (MOS) and Saccharomyces cerevisiae (SC) (at the rate 0.05%, 0.1%, 0.2%) alone and their combination moderately ameliorated the adverse effects of 300 ppb aflatoxin (Khatke et al., 2012b). The 0.2% level of MOS and SC was more effective than 0.05% and 0.1% level in counteracting the 300 ppb of aflatoxin in the feed. MOS appeared to be more efficacious than SC in counteracting aflatoxicosis in broiler chickens. However, the combination of SC and MOS did not show any synergistic effect in counteracting aflatoxicosis (Khatke et al., 2012c). Singh et al. (2013a) reported that inclusion of butylated hydroxytoluene in the diet of broiler chickens at 1000 ppm provided moderate protection against the adverse effects of aflatoxicosis. Singh et al. (2016) reported that supplementation of methionine (as DL-methionine) at 500 ppm or its analogue methionine hydroxy analogue at 769 ppm level in AFB1 (500 ppb) contaminated diet ameliorated the adverse effects on growth performance, feed conversion efficiency, yield of different organs and blood biochemicals in growing Japanese quails. In another study, Singh et al. (2013b) reported that inclusion of methionine at additional 0.025 and 0.05% levels over the prescribed requirements in the 1 ppm total AFB1 contaminated diet provided partial protection from the adverse effects of aflatoxicosis in broiler chickens. Based on the previous research on mycotoxicosis in various avian species, toxin binder (Mycodetox B1) was formulated at ICAR-CARI, Izatnagar and the objective of the present investigation was to test the efficacy of it in ameliorating aflatoxicosis in Japanese quails.

2. Materials and Methods

2.1 Production and Analysis of Aflatoxin

Aflatoxin B1 was produced using the fungal strain Aspergillus flavus NRRL 6513 that was obtained from U.S. Department of Agriculture, Illinois, USA. To get the fresh spores, the culture was regularly subcultured on potato dextrose agar medium slants and stored at 5°C. Aflatoxin was produced on maize substrate and the fermentations were carried out in batches as per Shotwell et al. (1966). The extraction and estimation of AFB1 was done as per Pons et al. (1966). Aqueous acetone was used for extraction and the toxin contents were finally quantified using a spectrophotometer.

2.2 Experimental Design

Experimental design was completely randomized design (CRD). There were five dietary treatments (Table 1). Each dietary treatment had 3 replicates and each replicate had 15 chicks. The experiment was conducted in Japanese quails from dayold to 5 weeks of age. The various dietary treatments were prepared by mixing mouldy maize to get the desired concentration of 400 and 600 ppb AFB1 and the toxin binder was used at the rate of 145 g per quintal of feed. The toxin binder developed at ICAR-CARI, Izatnagar after a decade of intensive research efforts in mycotoxicosis consisted of sodium bentonite (20.69%), zeolite (27.59%), activated charcoal (6.90%) mannan oligosaccharide (13.79%), methionine (17.24%), butylated hydroxytoluene (3.35%) and choline (10.34%).

2.3 Ingredients and Chemical Composition of Basal Feed (%)

A basal diet with maize 54.2, rice bran (deoiled) 2, soybean meal (solvent extracted) 31.15, sunflower meal 2, rapeseed meal 4, fish meal 4, limestone 0.75, dicalcium phosphate 1.4, salt 0.15, DL-methionine 0.06, trace mineral (TM) premix 0.1, vitamin premix 0.165, and choline chloride 0.03% was formulated. The TM premix supplied Mg 300, Mn 55, I 0.4, Fe 56, Zn 30 and Cu 4 mg/kg diet. The vitamin premix supplied vit. A 8250 IU, vit. D3 1200 IU, vit. K1 mg, vit. B1 2 mg, vit. B2 4 mg, vit. B12 10 mcg, niacin 60 mg, pantothenic acid 10 mg, choline 500 mg, vit. E 40 IU per kg diet. The control diet so formulated contained crude protein 23.95%, metabolisable energy 2795 kcal/kg, calcium 1.05%, available phosphorus 0.47%, lysine 1.2% and methionine 0.50%. The crude protein content as per AOAC (1995) and calcium content as per Talapatra et al. (1940) were estimated, while the concentrations of lysine, methionine, available P and metabolizable energy values were calculated. Mortality was recorded as and when occurred. The cell mediated immune response to PHA-P antigen was evaluated by the method described by Corrier and DeLoach (1990). The microtitre haemagglutination procedure as described by Siegel and Gross (1980) was followed to measure total HA antibody titres. At the end of the experiment, organ samples were collected and fixed in 10% formal saline. The formal saline fixed samples were cut into pieces of 2-3 mm thickness and washed thoroughly in tap water overnight before dehydrating the tissues in ascending grades of alcohol (50%, 60%, 70%, 80%, 90% absolute alcohol I and II). The dehydrated tissues were cleared in benzene and embedded in paraffin blocks. Serial sections of 5- micron thickness were cut and stained with hematoxyline and eosin (Culling, 1968) and examined for various histopathological changes.

2.4 Statistical Analysis

The collected data was subjected to statistical analysis using Statistical Package for Social Sciences (SPSS Version 16.0). The recorded data were subjected to one-way analysis of variance with comparison among means was made by Duncan‘s multiple range test with significance level of P<0.05.

3. Results and Discussion

The results on week-wise liveability percentage of Japanese quails fed on various dietary treatments are presented in Table 2. The data pertaining to cell mediated response to PHA-P measured as foot web index and humoral immune response measured as haemagglutination titre (HA) against SRBCs, and haematological parameters in Japanese quails fed on various dietary treatments was statistically analyzed and presented in Table 3.

3.1 Liveability Percentage

At first week of age, no mortality was recorded. During second week of age, the liveability percentage was numerically lower in 600 ppb aflatoxin fed group (T3). During third week of age, the liveability percentage in group T3 was lower (P<0.05) than that of control (T1). During fourth week of age, the liveability percentage varied from 86.67 in T3 to 95.56 in control. However, there was no significant difference in liveability percentage among various dietary treatments. During fifth week of age, the liveability percentage varied from 86.67 in T3 to 95.56 in control. The overall liveability percentage in T3 was lower (P<0.05) than that of control. The overall liveability percentage in T2 was lower (P<0.05) than that of T4. The overall liveability percentage in T2 was numerically lower than that of T1. The overall liveability percentage in T4 and T5 was higher (P<0.05) than that of T3 and statistically similar to that of control. The result of present study revealed that 600 ppb aflatoxin level in the feed resulted in higher (P<0.05) mortality compared to that of control. However, inclusion of aflatoxin at 400 ppb did not because heavy mortality, which might be due to the low level of dietary aflatoxin. This finding was in agreement with that of Sharma et al. (2014) where in 250 level of aflatoxin contamination in feed was reported to reduce the liveability percentage but did not produce heavy mortality. The results further showed that aflatoxin contamination of feed at 600 ppb (T3) level resulted in reduced (P<0.05) liveability percentage in Japanese quail. Khatke et al. (2012c); Silambarasan et al. (2015) also reported an increased mortality due to 300 ppb level of aflatoxin in the diet of broiler chickens. Reduced liveability percentage due to aflatoxin contamination in feed has also been reported by earlier researchers (Reddy et al., 1982; Gopi, 2006; Denli et al., 2009). In the present study, the overall liveability percentage of T4 and T5 was statistically similar to that of control. This result suggested that inclusion of toxin binder to the aflatoxin contaminated feed ameliorated the adverse effects of aflatoxicosis on liveability percentage in Japanese quails.

3.2 Cell Mediated and Humoral Immune Response

Immune system is highly sensitive indicator of the aflatoxicosis in poultry. Aflatoxinimpairs the humoral and cellular immune responses (CMI) and increase susceptibility to some environmental and infectious agents (Azzam and Gabal, 1998). In the present study, the CMI value of aflatoxin fed groups (T2 and T3) was lower (P<0.05) than that of control group (T1). This result showed that contamination of dietary aflatoxin at 400 and 600 ppb levels indiet decreased (P<0.05) the CMI response compared to that

Effect of Supplementation of Toxin Binder (Mycodetox B1) on Liveability, Immune Response and Organ Pathology in Induced Aflatoxicosis in Japanese Quails - Image 1

of control. The CMI value in T4 and T5 was higher (P<0.05) than those of T2 and T3 and statistically similar to that of control. The CMI value between T4 and T5 was statistically similar. A significant decrease in the CMI response at 300 ppb level of dietary aflatoxin in broiler chickens was also reported by Silambarasan et al. (2016); Khatke et al. (2012a). Ghosh and Chauhan (1991) observed that 300 ppb AFB1 in broiler feed caused immune suppression with no apparent clinical effects, but can result in flock morbidity and/or mortality caused by secondary infections. Yunus et al. (2011) also reported ill effects of aflatoxicosis on cell mediated immunity at 400 ppb aflatoxin level in the diet of broiler chickens. Giambrone et al. (1978) indicated that aflatoxin had marked effect on cell mediated immunity in the chicken, as measured by graft-versus-host and delayed hyper sensitivity reaction. Since CMI plays a major role in resistance coccidiosis, are ductions in this immunologic function by aflatoxin could make chicks more susceptible to this disease. Suppression of CMI response may be due to impaired lymphoblastogenesis (Chang et al., 1976) and impairment of lymphokine production (Ghosh et al., 1991). Decreased CMI response in chickens due to AFB1 feeding were also earlier reported by Kadian et al. (1988); Deo et al. (1998); Bakshi (1991). In the present study, inclusion of toxin binder to the aflatoxin contaminated feed ameliorated the adverse effects of aflatoxicosis on CMI in Japanese quails. In the case of humoral immunity, the HA titre value of AFB1 fed groups (T2 and T3) was lower (P<0.05) than that of control group (T1). This result showed that contamination of dietary AFB1 at 400 and 600 ppb levels in feed decreased (P<0.05) the HA titre as compared to that of control. The HA titre value in T4 and T5 was higher (P<0.05) than those of T2 and T3 and statistically similar to that of control. The HA titre value between groups T4 and T5 was statistically similar. This result revealed that the inclusion of dietary AFB1 at 400 and 600 ppb levels in feed decreased (P<0.05) the humoral immune response compared to that of control. This result was in agreement with Thaxton et al. (1974) who also recorded reduced antibody production following injection of sheep red blood cells in chickens experiencing aflatoxicosis. Oguz et al. (2003) also reported decreased humoral immunity at 50 ppb AFB1 contamination in feed. Aflatoxin depresses protein synthesis via inhibition of RNA polymerase, which results in suppression of specific immunoglobulin synthesis (Giambrone et al., 1985). Aflatoxin is an immunosuppressant by virtue of its ability to stimulate lysosomal degradation of immunoglobulins (DeDuve and Wattiaux, 1966). Non-specific factors such as complement, interferon and nonspecific serum protein concentrations also decrease due to liver damage (Tung et al., 1975). During experimental aflatoxicosis, reduced humoral immune response has also been reported by earlier researchers (Virdi et al., 1989; Bakshi, 1991). Silambarasan et al. (2016); Khatke et al. (2012a) also reported a significant decrease in the humoral immune response at 300 ppb level of dietary AFB1 in broiler chickens. In the present study, inclusion of toxin binder to the AFB1 contaminated diet ameliorated the adverse effects of aflatoxocosis on humoral immunity in Japanese quails.

3.3 Haematological Parameters

The haemoglobin (Hb) value in aflatoxin fed groups (T2 and T3) was lower (P<0.05) than that of control group (T1). The Hb value in T4 and T5 was higher (P<0.05) than those of T2 and T3; and statistically similar to that of control. The Hb value in T2 was statistically similar to that of T3. The Hb value of T4 was statistically similar to that of T5. The results revealed that AFB1 at 400 and 600 ppb levels resulted in reduced (P<0.05) Hb concentration in Japanese quails. Sharma (2013) also reported that aflatoxin contamination at 250 ppb level resulted in reduced haemoglobin level in broiler chickens. This finding was also in agreement with that of Kececi et al. (1998); Basmacioglu et al. (2005) who reported reduced Hb level at 2.5 and 2.0 ppm aflatoxin, respectively in broiler chickens. In the present study, inclusion of toxin binder to the aflatoxin contaminated feed ameliorated the adverse effects of aflatoxicosis in Japanese quails. In case of heterophil/lymphocyte (H/L) ratio, the H/L ratio of control (T1) was lower (P<0.05) as compared to those of aflatoxin fed groups (T2 and T3). The H/L ratio of groups T4 and T5 was lower (P<0.05) than those of T2 and T3 and statistically similar to that of control.

In the present study, dietary contamination of aflatoxin at 400 and 600 ppb levels resulted in increased (P<0.05) H/L ratio in Japanese quails. Sharma (2013) also reported an increased (P<0.05) H/L ratio due to 250 ppb dietary aflatoxin in the feed of broiler chickens. Also, Basmacioglu et al. (2005) reported elevated H/L ratio in broilers due to feeding 2 ppm aflatoxin. This result is also in agreement with those of Huff et al. (1986); Oguz et al. (2003), where in the suppressive effects of aflatoxin on haematopoiesis and immune responses were reported. The increase in heterophil counts suggested that the toxin elicited the inflammatory response (Kececi et al., 1998). In the present study, inclusion of toxin binder to the 400 or 600 ppb AFB1 contaminated diet alleviated the adverse effects of aflatoxicosis on haematological parameters of Japanese quails.

3.4 Gross and Histopathology of Organs

Gross and histopathological alterations of organs are a common feature of aflatoxicosis. Morphologically, the organs of T1 group, receiving basal feed were normal in size, colour and border marking, and served as reference.

In organs of groups T2 and T3, petechiae were seen in the kidneys and lungs. Ecchymotic haemorrhages were seen in the liver and leg muscles of birds. Petechial haemorrhages were also seen on the thymus and bursa of Fabricius. The liver was enlarged, fatty and pale in appearance and the bursa was atrophied. Congestion and haemorrhages were seen over the heart, lungs, and small intestines and proventriculus. Muscular haemorrhages were seen over the leg and breast muscles in T2 and T3 groups. The livers of group T3 were enlarged and having rounded borders. The organs of T4 and T5 groups were as normal in appearance as that of T1 group. In the present study, 400 and 600 ppb of AFB1 contamination in feed resulted in altered gross pathology of organs. Similar gross legions on morphology of liver due to aflatoxicosis were also reported by Singh et al. (2015); Singh et al. (2016). The results of the present study revealed that addition of toxin binderat 400 ppb (T4) and 600 ppb (T5) AFB1 contaminated feed ameliorated the adverse effects of aflatoxicosis on morphology of organs in Japanese quails.

Liver is the primary organ for the metabolism of AFB1, therefore, the alterations were observed in the liver parenchyma as liver is the main target organ in aflatoxicosis.

With regard to histopathology, in control group (T1), the architectural and cellular organization of liver was normal and this group served as a reference standard for comparison with other groups. Aflatoxicosis caused by 400 (T2) and 600 ppb (T3) levels of AFB1 contamination resulted in severe degenerative changes in hepatocytes with greater disorganisation in tissue marking the hepatotoxicity. In groups T2 and T3, aflatoxicosis resulted in marked destruction of hepatic cords, dilated and congested vasculature, fatty change, hepatocytic degeneration, necrosis and marked infiltration of the mononuclear cells (MNCs) around the portal veins.

Aflatoxicosis also caused hepatocytic hypertrophy and hyperplasia of the bile ducts in the liver. In groups T4 and T5, the architectural and cellular organization of liver was normal and was almost comparable to that of T1. Histopathology of intestine revealed severe necrosis, degenerative changes and infiltration of inflammatory cells in groups T2 and T3.

Similar histopathological alterations due to aflatoxicosis caused by 300 ppb level of AFB1 were also reported by Khatke et al. (2012a); Silambarasan et al. (2016). In the present study, inclusion of toxin binder in the 400 ppb (T4) and 600 ppb (T5) aflatoxin contaminated diet ameliorated the adverse effects on histopathology of liver and intestine in Japanese quails.

4. Conclusion

It was concluded that the dietary inclusion of AFB1 at 400 and 600 ppb levels resulted in increased mortality, suppression of immunity, decreased haemoglobin concentration, increased heterophil/lymphocyte ratio; and morphological and histological alterations in the internal organs. Inclusion of toxin binder (Mycodetox B1) in AFB1 contaminated diet ameliorated the ill effects in terms of mortality, immunity, haematological parameters, and gross and histopathology of organs in Japanese quails.

This article was originally published in Livestock Research International | January-March, 2019 | Volume 07 | Issue 01 | Pages 05-11.

Effect of Supplementation of Toxin Binder (Mycodetox B1) on Liveability, Immune Response and Organ Pathology in Induced Aflatoxicosis in Japanese Quails

Afla V ONE 2 - 80 ppb Method For Aflatoxins In Complete Feeds and Pet Foods

Effects of Deoxynivalenol-Contaminated Diets on Productive, Morphological, and Physiological Indicators in Broiler Chickens