Ameliorative Efficacy of Mycodetox B2 on Liveability, Immune Response and Liver Pathology During Aflatoxicosis in Broiler Chickens

To establish the efficacy of Mycodetox B2 in alleviating aflatoxicosis, day-old broiler chicks (n=200) were divided into 5 treatment groups (T1-control; T2-T1+200ppb aflatoxin B1 (AFB1); T3-T1+300ppb AFB1; T4-T2+Mycodetox B2 and T5-T3+Mycodetox B2). Each diet was fed to 5 replicates of 8 birds each for 42 days of age. Overall liveability percentage (LP) in T3 was lower (P<0.05) than T1. The LP in T4 and T5 was higher than T2 and T3 and statistically similar to T1. The cell mediated immunity (CMI) and haemagglutination (HA) titre values in T2 and T3 were lower (P<0.05) than T1. The CMI and HA titre values in T4 and T5 were higher (P<0.05) than T2 and T3; and statistically similar to T1. The haemoglobin (Hb) values in T2 and T3 were lower (P<0.05) than T1. The Hb values of T4 and T5 were higher (P<0.05) than T2 and T3. The Hb value of T2 was higher (P<0.05) than T3. The Hb value of T5 was higher (P<0.05) than T1. The heterophil/lymphocyte (H/L) ratio of T1 was lower (P<0.05) than T2 and T3. The H/L ratio of T4 and T5 was lower (P<0.05) than T2 and T3; and similar to T1. The liver in T2 and T3, was enlarged, pale, and having petechial haemorrhages with rounded borders. The liver of T4 and T5 was normal as T1. The liver histopathology of T2 and T3 revealed marked destruction of hepatic cords, dilated and congested central veins, adenomatous necrotic foci, fatty change, severe degenerative changes and marked infiltration of mononuclear cells around the portal veins, hypertrophy of the hepatocytes and hyperplasia of the bile ducts with destruction of portal vein. It was concluded that dietary AFB1 at 200 or 300 ppb level resulted in reduced liveability, suppression of immunity, decreased haemoglobin, increased heterophil/lymphocyte ratio and; gross and histopathological alterations in liver. Moreover, the inclusion of Mycodetox B2 during aflatoxicosis alleviated these adverse effects in broiler chickens.

Mycotoxicosis, which is a worldwide problem, is characterized by its nephrotoxic, immunosuppressive, hepatotoxic, carcinogenic, mutagenic and teratogenic effects in animals and poultry (Patil et al., 2005, 2006, 2014, 2017a, b; Katole et al., 2013; Patial et al., 2013; Patel et al., 2015; Patil and Degloorkar, 2016a, b, 2018; Singh, 2019a-g; Singh et al., 2019a, b, c; Sharma et al., 2019c). Aflatoxicosis in poultry causes lowered performance in terms of reduced body weight gain, feed intake and feed efficiency (Singh et al., 2011; Khatke et al., 2012b; Patil et al., 2013; Silambarasan et al., 2013; Singh and Mandal, 2013; Singh et al., 2013a, b; Shamsudeen et al., 2013; Sharma et al., 2014a; Sharma et al., 2015; Singh et al., 2015, 2016; Sharma et al., 2016; Singh, 2019a, b, e; Sharma et al., 2019c), reduced nutrient utilisation (Silambarasan et al., 2013), increased mortality (Singh et al., 2011; Khatke et al., 2012b; Shamsudeen et al., 2013; Sharma et al., 2014a; Sharma et al., 2015; Silambarasan et al., 2015; Singh, 2019c, d, f), anemia (Singh et al., 2015, 2016), hepatotoxicosis and haemorrhage (Singh et al., 2013a, b; Singh et al., 2015, 2016), gross lesions in organs (Singh et al.,2013a, b; Singh et al., 2015, 2016), altered relative weight of organs (Khatke et al., 2012c; Patil et al., 2013; Singh et al., 2013b; Shamsudeen et al., 2014; Sharma et al., 2015; Silambarasan et al., 2015; Singh, 2019a, b, e; Sharma et al., 2019a), altered carcass quality traits (Shamsudeen et al., 2014; Silambarasan et al., 2015; Sharma et al., 2019a; Sharma et al., 2019d), altered biochemistry (Singh et al., 2011; Khatke et al., 2012c; Patil et al., 2013; Singh and Mandal, 2013; Singh et al., 2013a, b; Sharma et al., 2014b; Silambarasan et al., 2016; Singh, 2019a, b, e; Sharma et al., 2019b) and histopathological lesions in organs (Khatke et al., 2012a; Sharma et al., 2014b; Silambarasan et al., 2016; Singh, 2019c, d, f; Sharma et al., 2019b). It impairs humoral and cellular immune responses in poultry and increases susceptibility to environmental and infectious agents (Khatke et al., 2012a; Patil et al., 2013; Sharma et al., 2014b; Silambarasan et al., 2016; Sharma et al., 2016; Singh, 2019c, d, f) leading to severe economic losses. For aflatoxicosis amelioration, Silambarasan et al. (2013) reported that due to differences in binding capacity of adsorbents, some portion of the toxins gets absorbed and causes deleterious effects in poultry. Diatomaceous earth, sodium bentonite and zeolite either at 0.5% or 1% level were partially effective in ameliorating aflatoxicosis. Mannan oligosaccharides and Saccharomyces cerevisiae (@ 0.05%, 0.1%, 0.2%) alone and their combination moderately ameliorated the adverse effects of 300 ppb aflatoxin (Khatke et al., 2012b). Methionine (as DL-methionine) at 500 ppm or methionine hydroxy analogue (MHA) at 769 ppm level in aflatoxin (500 ppb) contaminated diet ameliorated aflatoxicosis in growing Japanese quails (Singh et al., 2016). 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 aflatoxin contaminated diet provided partial protection from aflatoxicosis in broiler chickens. Butylated hydroxyanisole (BHA) at 1000 and 2000 ppm levels provided partial amelioration of aflatoxicosis caused by 1 ppm total aflatoxin in broiler chickens (Singh and Mandal, 2013). Inclusion of 40 mg Zn/kg to the aflatoxin contaminated diet ameliorated the ill effects of aflatoxicosis on performance of the birds (Sharma et al., 2014a). Based on the previous research on mycotoxicosis, Mycodetox B2 was formulated and the objective of the present investigation was to establish the effectiveness of this toxin binder in alleviating aflatoxicosis in broiler chickens.

Aflatoxin 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 B1 was produced on maize substrate as per the method of Singh and Shrivastav (2012). The extraction and estimation of aflatoxin B1 was done as per Pons et al. (1966). Aqueous acetone was used for extraction of the toxin. Aflatoxin contents were finally quantified using a UV spectrophotometer.

Experimental design was completely randomized design. There were five dietary treatments viz. T1-control; T2-T1+200ppb aflatoxin B1 (AFB1); T3- T1+300ppb AFB1; T4-T2+Mycodetox B2 and T5- T3+Mycodetox B2. Each dietary treatment had 5 replicates and each replicate had 8 chicks. The experiment was conducted in broiler chickens for 6 weeks of age. The various dietary treatments were prepared by mixing mouldy maize to get the desired concentration of 200 and 300 ppb AFB1 and the Mycodetox B2 at the rate of 132g per quintal of feed. The Mycodetox B2 formulated 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%).

Day-old broiler chicks (n=200) were obtained from experimental hatchery, CARI, Izatnagar. The chicks were wing banded, weighed individually and distributed randomly into five groups. All birds were reared under standard management conditions for 6 weeks. All birds were fed with broiler starter ration from 1-21 days and broiler finisher ration from 22 to 42 days. The composition of broiler starter and finisher ration was as below: The starter diet with maize 55.5, Deoiled rice bran 1.88, soybean meal 31.0, guar corma 4, rapeseed meal 4, fish meal 4.5, limestone 0.7, dicalcium phosphate 1.6, common salt 0.2, DLmethionine 0.07, lysine 0.125, TM premix 0.11, vitamin premix 0.15, B complex 0.015, choline chloride 0.05 and coccidiostat 0.05%; and finisher diet with maize 62.42, Deoiled rice bran 2.01, soybean meal 20.5, guar corma 4, rapeseed meal 4, fish meal 4, limestone 0.5, dicalcium phosphate 1.6, common salt 0.25, DL-methionine 0.03, lysine 0.07, TM premix 0.10, vitamin premix 0.15, B complex 0.015, choline chloride 0.05 and coccidiostat 0.05% were formulated. TM premix supplied Mg, 300; Mn, 55; I, 0.4; Fe, 56; Zn, 30; Cu, 4 mg/kg diet. Vitamin premix supplied VitA, 8250 IU; Vit. D3, 1200 ICU; Vit. K, 1mg per kg diet. B complex supplied Vit. B1, 2mg; Vit. B2, 4mg; Vit. B12, 10mcg; niacin, 60mg; pantothenic acid, 10mg; choline, 500mg per kg diet. The starter diet contained 22.3% crude protein, 2,807 Kcal ME/kg, lysine 1.28%, methionine 0.51%, calcium 1.09% and available P 0.50%. The corresponding values in finisher diet were 20.06%, 2,876 Kcal/kg, 1.04%, 0.43%, 1.09% and 0.42%. The protein as per AOAC (1990) and calcium contents as per Talapatra et al. (1940) were estimated, while the concentration of lysine, methionine, available P and metabolizable energy value 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 in chickens. At the end of the experiment, blood and liver samples were collected and the liver samples were fixed in 10% formal saline. The fixed liver tissues 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.

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.

The results on liveability percentage calculated on the basis of week-wise mortality of broiler chickens fed on different dietary treatments was statistically analysed and presented in Table 1. The data on cell mediated immune response to PHA-P measured as foot web index and humoral immune response measured as haemagglutination titre (HA) against SRBCs; and haematological parameters of broilers fed on different dietary treatments was statistically analyzed and presented in Table 2.

At first week of age, no mortality was recorded. During second week, the liveability percentage was numerically lower in aflatoxin fed groups i.e. T2 and T3 compared to other treatment groups. During third week, the liveability percentage in group T3 was lower (P<0.05) than that of control (T1) indicating that 300 ppb level of AFB1 contamination caused significantly reduced liveability. At fourth week, the liveability percentage did not vary significantly among various dietary treatments. During fifth and sixth week, the liveability percentage of group T3 was lower (P<0.05) than that of T1. The liveability percentage of group T2 was lower by 10 percent compared to that of control; however it did not vary significantly. The liveability percentage of groups T4 and T5 was higher than those of AFB1 fed groups (T2 and T3) and statistically similar to that of control. This finding of the present investigation revealed that dietary AFB1 at 200 ppb level caused higher mortality as compared to that of control. However, this level of dietary AFB1 did not produce heavy mortality, which might be due to the low level of toxin in the feed. Similar result was also reported by Sharma et al. (2014a) where dietary AFB1 at 250 ppb level was reported to reduce the liveability percentage but did not produce heavy mortality. The results of the present study further indicated that 300 ppb AFB1 contamination in diet resulted in reduced (P<0.05) liveability percentage in broiler chickens. This result was in agreement with Silambarasan et al. (2015); Khatke et al. (2012c); Raju and Devegowda (2000) who also reported significant reduction in liveability percentage due to 300 ppb level AFB1 in the diet of broiler chickens. Significant increase in mortality due to AFB1 contamination in feed has also been reported by earlier researchers (Sharma et al., 2015; Shamsudeen et al., 2013; Singh et al., 2011; Singh, 2019c; Singh, 2019d; Singh, 2019f; Kubena et al., 1998; Denli et al., 2009; Reddy et al., 1982; Gopi, 2006).

Immune system is highly sensitive indicator of aflatoxicosis in poultry. It suppresses both humoral and cell mediated immunity (Silambarsan et al., 2016; Khatke et al., 2012a). Aflatoxins are reported to affect the immune system because of their ability to inhibit protein synthesis (Singh et al., 2013a). At the cellular level, cell-mediated immune functions are impaired by AFB1 exposure in poultry (Hoerr, 2010). The present study revealed the CMI value of aflatoxin fed groups (T2 and T3) was lower (P<0.05) than that of control group (T1). This indicated that inclusion of 200 and 300 ppb aflatoxin in diet decreased (P<0.05) the CMI as 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. Khatke et al. (2012a); Silambarasan et al. (2016) also reported a –significant decrease in the CMI response at 300 ppb level of dietary aflatoxin in broiler chickens. Giambrone et al. (1978) also observed marked effect of aflatoxicosis on cell mediated immunity in the chicken, as measured by graft-versus-host and delayed hypersensitivity reaction. Since CMI plays a major role in resistance coccidiosis, a reduction in this immunologic function by AFB1 could make chicks more susceptible to this disease. Ghosh and Chauhan (1991) also reported that 300 ppb AFB1 in diet resulted in immune suppression with no apparent clinical effects, but can result in flock morbidity and/or mortality caused by secondary infections in broiler chickens. Yunus et al. (2011) also reported that addition of 400 ppb AFB1 in the diet of broiler chickens caused adverse effects of aflatoxicosis on CMI. Suppression of CMI response could be due to impaired lymphoblastogenesis (Chang et al., 1976) and impairment of lymphokine production (Ghosh et al., 1991). Reduced CMI during aflatoxicosis in chickens was also earlier reported by several researchers (Bakshi, 1991; Kadian et al., 1988; Deo et al., 1998; Hoerr, 2010; Patil et al., 2013; Sharma et al., 2014b; Sharma et al., 2016; Silambarasan et al., 2016; Singh, 2019c, d, f; Sharma et al., 2019c). In case of humoral immunity, the HA titre value of AFB1 fed groups (T2 and T3) was lower (P<0.05) than that of control (T1). The HA titre value of groups T4 and T5 was higher (P<0.05) than those of T2 and T3 and statistically similar to that of T1. This finding was in agreement with Khatke et al. (2012a); Silambarasan et al. (2016) also reported a significant decrease in the humoral immune response at 300 ppb level of dietary AFB1 in broiler chickens. Oguz et al. (2003) also reported decreased humoral immunity at lower dose (50 ppb) of 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). Nonspecific factors such as complement, interferon and nonspecific serum protein concentrations also decrease due to liver damage (Tung et al., 1975). Antibody titers are often reduced, whether measured as total serum levels of IgA, IgG and IgM (Tung et al., 1975; Giambrone et al., 1978; Chen et al., 2014), as production of specific antibodies in response to sheep red blood cells (Taxton et al., 1974; Ghosh et al., 1991; Verma et al., 2004) or as exposure response to infectious bronchitis virus, infectious bursal disease virus, Newcastle disease virus or Pasteurella multocida (Azzam and Gabal, 1997; Azzam and Gabal, 1998; Gabal and Azzam, 1998). AFB1 can also impair the effectiveness of vaccination for these poultry diseases (Boulton et al., 1982; Azzam and Gabal, 1998; Ibrahim et al., 2000). Reduced humoral immune response due to experimental aflatoxicosis has also been reported by earlier researchers (Virdi et al., 1989; Bakshi, 1991; Patil et al., 2013; Sharma et al., 2014b; Sharma et al., 2016; Silambarasan et al., 2016; Singh, 2019c; Singh, 2019d; Singh, 2019f; Sharma et al., 2019c).

Table 1: Liveability percentage in broiler chickens as influenced by various dietary treatments

 

Table 2: Cell mediated and humoral immunity, and haematological parameters of broiler chickens fed on various dietary treatments

 

It was concluded that dietary AFB1 at 200 or 300 ppb level resulted in reduced liveability, suppression of immunity, decreased haemoglobin content, increased heterophil/lymphocyte ratio and; gross and histopathological alterations in liver. However, dietary inclusion of Mycodetox B2 alleviated these adverse effects in broiler chickens.