Effect of Supplementation of Toxin Binder (Mycodetox B2) on Liveability, Immune Response and Pathology of Organs During Aflatoxicosis in Japanese Quails

1. Introduction

Mycotoxins are found worldwide as natural contaminants or co-contaminants of feed and feedstuffs (Katole et al., 2013). The toxicity of mycotoxins i.e. mycotoxicosis, is characterized by its 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; Silambarasan et al., 2016) leading to severe economic losses in poultry industry. Aflatoxin contamination of feedstuffs has been reported to be of a wide range from 1 to 900 μg/kg in commonly used ingredients as well as mixed feed samples in developing countries (Mohanamba et al., 2007; Singh et al., 2010).

To combat the aflatoxicosis in poultry, a series of experiments were conducted in our laboratory. Due to differences in binding capacity of adsorbents, some portion of the toxins gets absorbed and causes deleterious effects in poultry (Silambarasan et al., 2013). Among the various aflatoxin adsorbents, diatomaceous earth, sodium bentonite and zeolite either at 0.5% or 1% level were partially effective in ameliorating the adverse effects of 300 ppb dietary aflatoxin in broiler chickens. Among three mycotoxin adsorbents tested, diatomaceous earth was least 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). Khatke et al. (2012b) reported that use of mannan oligosaccharide (MOS) and Saccharomyces cerevisiae (SC) (at 0.05%, 0.1%, 0.2% level) alone and their combination moderately ameliorated the adverse effects of 300 ppb aflatoxin. The 0.2% level of MOS and SC was more effective than 0.05% and 0.1% level in counteracting aflatoxicosis. 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. (2015, 2016) reported that supplementation of methionine (as DL-methionine) at 500 ppm or its analogue methionine hydroxy analogue (MHA) at 769 ppm level in aflatoxin (500 ppb AFB1) 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 inclusion of methionine at additional 0.025 and 0.05% levels over the prescribed requirements in the 1 ppm total aflatoxin contaminated diet provided partial protection from the adverse effects in broiler chickens. Singh and Mandal (2013) reported that butylated hydroxyanisole (BHA) at 1000 and 2000 ppm levels provided partial amelioration of aflatoxicosis caused by 1 ppm total aflatoxin in broiler chickens. Sharma et al. (2014) reported that supplementation of 40 mg Zn/kg to the aflatoxin contaminated diet ameliorated the ill effects of aflatoxicosis on performance of the birds. Based on the previous research on mycotoxicosis in various avian species, toxin binder (Mycodetox B2) was formulated and the objective of the present study was to establish its efficacy in ameliorating the 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 (PDA) medium slants and stored at 5°C. AFB1 was produced on maize substrate. 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 of the toxin 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 three replicates and each replicate had 15 chicks. The experiment was conducted in Japanese quail from day old 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 at the rate of 132 g per quintal of feed. The toxin binder (Mycodetox B2) developed at ICAR-CARI, Izatnagar after a decade of intensive research efforts in mycotoxicosis consisted of sodium bentonite (30.30%), zeolite (30.30%), mannan oligosaccharide (15.15%), methionine (18.94%), butylated hydroxyanisole (3.74%) and zinc (1.52%).

Effect of Supplementation of Toxin Binder (Mycodetox B2) on Liveability, Immune Response and Pathology of Organs During Aflatoxicosis in Japanese Quails - Image 1

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. K 1 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 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 data pertaining to week-wise liveability percentage of Japanese quails fed on various dietary treatments are presented in Table 2. The results 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

During first week of age, no mortality was recorded. At second week of age, the liveability percentage was numerically lower in T3 as compared to other treatment groups. During third week of age, the liveability percentage in group T3 was lower (P<0.05) than that of control (T1). The liveability percentage in other treatment groups was statistically similar to that of control. Similar trend was observed during fourth week of age where liveability percentage varied from 91.99 in groups T2 and T3 to 95.56 in control (T1). During fifth week of age, the overall liveability percentage varied from 86.67 in T3 to 95.56 in control (T1). 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 present study revealed that aflatoxin (600 ppb) contamination in the diet resulted in higher (P<0.05) mortality compared to that of control. However, aflatoxin (400 ppb) contamination of feed did not cause 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) wherein aflatoxin (250 ppb) 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 quails. Silambarasan et al. (2015); Khatke et al. (2012c) 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 (Denli et al., 2009; Reddy et al., 1982; Gopi, 2006). The results of the present study - showed that overall liveability percentage of T4 and T5 was statistically similar to that of control. Thus, addition of Mycodetox B2 to the aflatoxin contaminated feed ameliorated the adverse effects on liveability percentage in Japanese quails.

3.2 Cell Mediated and Humoral Immune Response

Reduced immunity is a common feature of aflatoxicosisin poultry. Aflatoxinimpairs the humoral and cellular immune responses and increase susceptibility to some environmental and infectious agents (Patil et al., 2014). In the present study, the CMI value of control group (T1) was higher (P<0.05) than that of aflatoxin fed groups (T2 and T3). This result showed that aflatoxicosis caused by 400 and 600 ppb levels of aflatoxin in feed decreased (P<0.05) the CMI response compared to that 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. This result was in agreement with those of Silambarasan et al. (2016); Khatke et al. (2012a) who also reported significant decrease in the CMI response at 300 ppb level of dietary aflatoxin in broiler chickens. Ghosh and Chauhan (1991) observed that 300 ppb AFB1 in broiler feed caused immuno-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 CMI at 400 ppb aflatoxin in the diet of broiler chickens. Giambrone et al. (1978) indicated that aflatoxin had marked effect on CMI in the chicken, as measured by graft-versus-host and delayed hypersensitivity 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 aflatoxin feeding were reported earlier by Kadian et al. (1988); Bakshi (1991); Deo et al. (1998). The results of the present study further revealed that the addition of Mycodetox B2 to the AFB1 contaminated diet ameliorated the adverse effects on cell mediated immunity in Japanese quails. With regard to humoral immunity, the HA titre value of control group (T1) was higher (P<0.05) than those of aflatoxin fed groups (T2 and T3). 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 indicated that contamination of aflatoxin 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 SRBCs in chickens experiencing aflatoxicosis. Khatke et al. (2012a); Silambarasan et al. (2016) also reported a significant decrease in the humoral immune response at 300 ppb level of dietary aflatoxin in broiler chickens. Oguz et al. (2003) also reported decreased humoral immunity at 50 ppb of aflatoxin 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 B1 is an immunosuppressant by virtue of its ability to stimulate lysosomal degradation of immunoglobulins (DeDuve and Wattiaux, 1966). Nonspecific factors such as complement, interferon and non-specific 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). The present study revealed that incorporation of toxin binder to the 400 and 600 ppb AFB1 contaminated feed ameliorated the adverse effects on humoral immunity in Japanese quails.

3.3 Haematological Parameters

The haemoglobin (Hb) value of control group (T1) was higher (P<0.05) than those of aflatoxin fed groups (T2 and T3). 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 present study revealed that aflatoxin contamination of feed at 400 and 600 ppb levels resulted in decreased (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 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, addition of toxin binder II to the 400 and 600 ppb aflatoxin contaminated diet ameliorated the adverse effects on haemoglobin concentration in Japanese quails. With regard to heterophil/lymphocyte (H/L) ratio, the H/L ratio of aflatoxin fed groups (T2 and T3) was higher (P<0.05) than that of control (T1). 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. The H/L ratio between groups T4 and T5 was statistically similar. In the present study, aflatoxicosis caused by 400 and 600 ppb aflatoxin resulted in increased (P<0.05) H/L ratio in Japanese quail. Similarly, Sharma (2013) also reported increase (P<0.05) in the H/L ratio due to 250 ppb dietary aflatoxin in the feed of broiler chickens. Basmacioglu et al. (2005) also reported increased 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) wherein 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 aflatoxin contaminated diet ameliorated the adverse effects on haematological parameters in Japanese quails.

3.4 Gross and Histopathology of Organs

Gross and histopathological lesions in organs are a common feature of aflatoxicosis. Grossly, the organs of T1 group, receiving basal feed were normal in size, shape and colour, and taken as reference. In AFB1 fed groups (T2 and T3), petechiae were seen in the kidneys and lungs, ecchymotic haemorrhages was seen in the liver, leg muscles, thymusand bursa of Fabricius. The liver was enlarged, fatty and pale in appearance however the bursa of Fabricius was atrophied. Congestion and a few haemorrhagic spots were also seen on the heart, lungs, small intestine and proventriculus. Muscular haemorrhages were seen on the leg as well as breast muscles of birds of groups T2 and T3.

The liver of birds in group T3 showed enlargement with rounded borders. The organs of T4 and T5 groups were normal in appearance as that of T1 group. The present study revealed that AFB1 at 400 and 600 ppb levels resulted in altered gross pathology of organs. Similar gross pathological legions on liver due to aflatoxicosis caused by 500 ppb aflatoxin in feed were also reported earlier (Singh et al., 2015, 2016). In the present study, inclusion of Mycodetox B2 to the 400 ppb or 600 ppb aflatoxin contaminated feed ameliorated the adverse effects on gross pathology of organs in Japanese quails. Liver, acts as primary organ for the metabolism of aflatoxin, is the main target organ for aflatoxicosis in poultry. With regard to histopathology, in control group (T1), the architectural and cellular organization of liver was normal and this group was taken reference standard for comparison with other groups.

Aflatoxicosis in groups T2 and T3 resulted in severe degenerative changes in hepatic cells with greater disorganisation in tissue marking the hepatotoxicity. In groups T2 and T3, aflatoxicosis resulted in marked destruction of hepatic cords, and dilatation and congestion of central veins, adenomatous necrotic foci, fatty change, hepatocytic degeneration and marked infiltration of mononuclear cells around the portal area. Aflatoxicosis also caused hypertrophy of the hepatocytes and hyperplasia of the bile duct with destruction of portal vein in the liver.

In groups T4 and T5, the architectural and cellular organization of liver was almost normal and comparable to that of T1. Tissue sections of intestine of birds from groups T2 and T3 exhibited severe necrosis, degenerative changes and infiltration of inflammatory cells due to aflatoxicosis.

Similar histopathological alterations due to aflatoxicosis caused by 300 ppb level of aflatoxin were also reported by earlier workers (Khatke et al., 2012a; Silambarasan et al., 2016).

The results of present study revealed that the addition of toxin binder to the 400 ppb and 600 ppb AFB1 contaminated diet ameliorated the adverse effects on tissue architecture of Japanese quails.

4. Conclusion

It was concluded that experimentally induced aflatoxicosis at 400 or 600 ppb dietary level resulted in reduced liveability, suppression of immunity, decreased haemoglobin concentration, increased heterophil/lymphocyte ratio; and gross and histopathological alterations in organs. The inclusion of toxin binder (Mycodetox B2) to the AFB1 contaminated feed ameliorated the adverse effects on liveability, immunity, haematological parameters, and gross and histopathology of internal organs in Japanese quails.

This article was originally published in Journal of Poultry Science and Technology, January-March 2019, Volume 07, Issue 01, Pages 01-07.

Effect of Supplementation of Toxin Binder (Mycodetox B2) on Liveability, Immune Response and Pathology of Organs During Aflatoxicosis in Japanese Quails

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