Effect of aflatoxin, ochratoxin and their interaction on growth performance, immunity and jejunal morphometry of broiler chickens

INTRODUCTION

Presence of mycotoxins in feed is one of the major constraints in maintaining feed quality, especially in tropical countries. The Food and Agriculture Organization has estimated that 25% of the world’s crops are affected by mycotoxins each year (Das, 2004). The most widespread mycotoxin i.e., aflatoxins are of great concern in warm and humid climatic conditions like India (Singh et al., 2010). Swamy et al. (2012) found that South Asian feeds are contaminated with multiple mycotoxins, along with aflatoxin. Singh et al. (2010) reported that 90% of the maize samples were positive for aflatoxin B₁ and the values ranged from non-detectable to 0.80 ppm, with an average of 0.14 ppm of aflatoxin B₁ . In poultry, aflatoxin ingestion leads to “Aflatoxicosis” syndrome that is characterized by retardation in growth (Singh et al., 2013; Sharma etal., 2014), decreased feed consumption and feed conversion efficiency (Silambarasan et al., 2013; Singhet al., 2013; Sharma et al., 2014), immunosuppression and the increase of mortality (Sharma, 2013). Ochratoxin A causes significant losses and reduction in the profitability of poultry industry due to its effects on performance and health (Agawane and Lonkar, 2004). It causes a reduction in productive performance (growth rate, feed consumption, poorer feed conversion) and increased mortality (Singh et al. 2015; Singh et al. 2016). The risk associated with ochratoxin residues in poultry meat represents a public health concern. It is three times more toxic than aflatoxin to the birds. Under field conditions, mycotoxicosis in poultry is most commonly caused by exposure to a group of mycotoxins, rather than exposure to individual mycotoxins. Different types of mycotoxins produced by a single or several fungal species may be occurring simultaneously on various agriculture commodities, thereby adding more potential risk of mixed mycotoxicosis. The objective of the present study was to evaluate the impact of aflatoxin, ochratoxin and their interaction on growth performance, immunity and gut morphometry of broiler chickens.

MATERIALS AND METHODS

Production of mycotoxins: Aflatoxin was produced by growing Aspergillus flavus NRRL 6513 on maize. Fermentations were carried out in batches as per the method described by Shotwell et al. (1966). The extraction and estimation of aflatoxin was done as per the procedure of Pons et al. (1966). Aflatoxin contents were finally quantified using a UV spectrophotometer. Ochratoxin was produced as per the method described by Singh et al. (2013a ) using Aspergillus westerdijkiae NRRL 3174.The extraction and estimation of aflatoxin was done as per the procedure of AOAC (1995).

Experimental design: Experimental design was completely randomized design (CRD). There were nine dietary treatments. Each dietary treatment had 4 replicates and each replicate had 8 chicks. The experiment was conducted in broiler chickens from day-old to 6 weeks of age. The various dietary treatments were prepared by mixing the required quantity of mycotoxins to get their desired concentration in basal diet (Table 1).

Effect of aflatoxin, ochratoxin and their interaction on growth performance, immunity and jejunal morphometry of broiler chickens - Image 1

Biological experiment and analysis: Day-old broiler chicks were obtained from experimental hatchery, CARI, Izatnagar. The chicks were wing banded, weighed individually and distributed randomly into nine groups. All birds were reared under standard management conditions from 0-6 weeks of age. All birds were fed with broiler starter ration for 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 53.93, soybean meal 42.70, limestone 0.9, dicalcium phosphate 1.70, common salt 1.30, DLmethionine 0.16, TM premix 0.10, vitamin premix 0.15, B complex 0.015, choline chloride 0.05; and finisher diet with maize 63.63, soybean meal 33.40, limestone 0.95, dicalcium phosphate 1.40, common salt 0.20, DLmethionine 0.11, TM premix 0.10, vitamin premix 0.15, B complex 0.015, choline chloride 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 Vit A, 8250 IU; Vit. D₃ , 1200 ICU; Vit. K, 1mg; vitamin E 40 IU 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 22.09% crude protein, 2,848 Kcal ME/kg, calcium 0.99%, available P 0.45%, lysine 1.27%, methionine 0.53% and threonine 1.01%.The corresponding values in finisher diet were 19.04%, 2,946 Kcal/kg, 0.90%, 0.38%, 1.05%, 0.44% and 0.87%.The protein as per AOAC (1995) and calcium contents as per Talapatra et al. (1940) were estimated, while the concentrations of lysine, methionine, threonine, available P and metabolizable energy values were calculated. Weekly individual body weight and feed consumption of each group were recorded and the FCR was calculated. Humoral immune response to sheep red blood cells and cellular immunity to PHA-P was observed on 30th day and 24th day of experimental trial, respectively. At 14th, 28th and 42nd days post-hatch intestine samples were collected and fixed in 10% formal saline and histopathological slides were prepared. Slides were read for morphometric study by microscop-OLYMTUS, BH41 using software Crog Rex C5.The statistical analysis was done using SPSS 16.0 version. Results were considered as significant at the level of 95% (P < 0.05) for comparison.

RESULTS AND DISCUSSION

Growth Performance: The data pertaining to effect of various dietary treatments on average body weight gain, feed intake and feed conversion ratio of broilers in different growth phases is presented in Table 2.

Effect of aflatoxin, ochratoxin and their interaction on growth performance, immunity and jejunal morphometry of broiler chickens - Image 2

Body weight gain (BWG): During starter phase (0-3 weeks),finisher phase (4-6 weeks)and overall growth period (0-6 weeks) of trial, the BWG of birds in control group (T₁ ) was higher (P<0.05) than those of all other treatment groups. The lowest BWG was observed in group T₉ . The BWG in group T₂ was lower (P<0.05) than that of control (T₁ ), indicating that AFB₁ contamination of feed at 150 ppb level reduced (P<0.05) the BWG of broiler chickens. Significant depression in BWG due to dietary aflatoxin was earlier reported in several studies (Singh et al., 2013; Silambarasan et al., 2013; Sharma, 2014; Verma et al., 2004). The BWG in group T₄ was lower (P<0.05) than that of control, indicating that OTA contamination of diet at 150 ppb level resulted in reduction (P<0.05) in BWG. This finding was in agreement with previous investigations with dietary ochratoxin level of 50 -100 ppb (Stove et al., 2004; ElBarkouky and Abu Taleb, 2008), 200 ppb (El- Barkouky et al., 2010; Singh et al., 2015; Singh et al., 2016).In the present study, the BWG in T₂ was higher (P<0.05) than that of T₄ , indicating that OTA was more deleterious than AFB₁ in reducing the BWG of broiler chickens. The average BWG in groups T₃ and T₄ was statistically similar, suggesting that OTA was almost two times more toxic than AFB₁ in reducing the BWG of broiler chickens. When aflatoxin and ochratoxin were given individually (T₂ , T₃ , T₄ and T₅ ) caused decrease in body weight gain by 6.14, 12.03, 12.94 and 14.76%, respectively. However, when these mycotoxins were given in combination (T₆ , T₇ , T₈ and T₉ ) the decrease in BWG was 19.82, 22.80, 23.83 and 28.69%, respectively. Thus, the combination of both toxins caused severe depression in BWG, which lead to the synergistic toxicity of these two mycotoxins on BWG. A similar synergistic effect of mycotoxins was observed by Campbell et al. (1983) and Huff et al. (1992). Decreased BWG might be attributed to the decrease in protein absorbtion and/or utilization or inhibition of protein synthesis caused by mycotoxins (Kubena et al., 1983, 1988, 1989). Pathological changes in the liver also cause a reduction in hepatic protein synthesis as both mycotoxins are known to inhibit hepatic protein synthesis.

Feed intake (FI):During starter phase (0-3 weeks) of growth trial, the FI among groups T₁ , T₂ , and T₃ was statistically similar but higher (P<0.05) than that of other treatment groups. The FI of aflatoxin alone fed groups (T₂ and T₃ ) was statistically similar to that of control. However, significant reduction in feed consumption of broilers due to aflatoxicosis was earlier reported by several workers (Verma et al., 2004; Singh et al., 2011; Singh et al., 2013; Sharma et al., 2014). The FIof OTA alone fed groups (T₄ and T₅ ) was lower (P<0.05) than that of control, indicating that OTA was more potent than AFB₁ in exerting feed refusal effect on broilers. These findings were in agreement with earlier reports by various workers (Sawale et al., 2009; Singh et al., 2015;Singh et al., 2016). With regards to finisher phase (4-6 weeks) of growth trial, the feed intake in control group (T₁ ) was higher (P<0.05) than T₂ and T₃ . Several researchers (Kubena et al., 1990, 1998) also reported a decrease in feed consumption due to aflatoxin contamination of feed. With regards to cumulative feed intake (0-6 weeks), the feed consumption in control group was higher (P<0.05) than any other treatment groups. Decreasing trend of FI from T₁ to T₉ was observed in all the three growth phases. The result indicated that when AFB₁ and OTA given individually in treatments T₂ , T₃, T₄ and T₅ , there was a decrease in FI by 4.18, 7.01, 9.25 and 9.74%, respectively. However, when the two mycotoxins were given in combination (groups T₆ to T₉ ) the decrease in feed consumption was 11.45, 13.80, 14.25 and 15.29%, respectively. The decrease in feed consumption indicated additive effect of mycotoxins in lowering the feed consumption of broiler chickens.

Feed conversion ratio(FCR): During starter phase (0-3 weeks), finisher phase (4-6 weeks) and overall period (0- 6 weeks) of growth trial, the FCR of groups T₁ to T₅ was statistically similar to that of control. During starter phase, the FCR of groups T₆ , T₇ and T₉ was higher (P<0.05) than that of control. During finisher phase and overall growth period, the FCR in group T₆ , T₇ , T₈ and T₉ was higher (P<0.05) than that of control, indicating that combine feeding of AFB₁ and OTA deteriorated the feed efficiency compared to individual feeding of these mycotoxins. Also, the increase in FCR of groups T₆ to T₉ was higher than the sum of the increase caused by individual mycotoxin. Therefore, combine feeding of AFB₁ and OTA led to the synergistic toxicity of these two mycotoxins on feed conversion efficiency. Poor feed efficiency due to dietary AF (0.3 ppm) and ochratoxin (50-200 ppb) contaminated feed was earlier reported in several studies (Singh et al., 2013, Silambarsan et al., 2013; Singh et al., 2015; Singh et al., 2016). Similarly, Huff and Doerr (1981) observed a significant decrease in feed efficiency in broiler chickens fed AF (2.5 µg/g) and OTA (2µg/g) simultaneously.

Immune response: The data pertaining to CMI response to PHA-P measured as foot web index and humoral immune response measured as haemagglutination titre (HA) against SRBC’s in broiler chickens fed various dietary treatments is presented in Table 3. The CMI value and HA titre value of control group (T₁ ) was higher (P<0.05) than any other treatment groups. The CMI value and HA titre value among groups T₂ , to T₉ was statistically similar. The CMI of group T₉ was the lowest among various dietary treatment groups. The result indicated that the combined feeding of AFB₁ and OTA showed an additive effect on immune response (both CMI and humoral immunity). The present study revealed that aflatoxin caused significant reduction in CMI and humoral immune response of broilers. The immunosuppressive effects of aflatoxin on cell mediated immunity and HA titre value has been well demonstrated in various animal species (Sharma, 1993). Yunus et al. (2011) reported that 0.4 ppm aflatoxin in the diet of broiler chickens showed negative results on CMI. Silambarasan et al. (2013); Abaji (2012) and Sharma (2013) also reported a significant decrease in the CMI response and HA titre value at a concentration of 250 to 300 ppb aflatoxin in broiler chickens. Elaroussi et al. (2008) also reported that feeding OTA @ 400 to 800 µg/kg feed to day-old broiler chicks until 5th week of age significantly reduced the CMI response as determined by wattle response to PHA-P antigen. Singh et al. (1990) found significant depression on delayed type hypersensivity reaction to DNCB in broiler chicks given 0.5ppm AF and 2 ppm OTA. A synergistic effect of the combine toxicity of AF and OTA was evident from a marked reduction in MST as compared to MST of individual toxicity. CMI in terms of MST sensitive to DNCB was significantly reduced in chicks given the combination of 2 mg/kg aflatoxin and 4 mg/kg ochratoxin adversely affect immunoglobulin production (Campbell et al., 1983; Effat, 1989). In the present study, combine feeding of AF and OTA caused an additive effect on HA titre value. A significant reduction in HA titre was recorded in chickens given 4 ppm OTA or a combination of AF (1or 2 ppm) and OTA (2 or 4 ppm) (Verma et al., 2004). Reduction in antibody titre against Newcastle disease and Infectious Bursal disease was recorded by feeding 300 ppb AF and 2ppm OTA in diet of broiler chickens and condition was further aggravated in combination of both mycotoxins in the diet (Raju and Devegowda, 2000).

Intestinal (distal jejunal) morphometry: The parameters pertaining to morphometry of distal jejunum of GIT as influenced by various dietary treatments are given in Table 4.Results related to villus length indicated that the mean villus length of three periods of control group (T₁ ) was higher and crypt depth was lowest compared to all other treatment groups. Group T₉ showed the minimum villi length (697.62 µm) and highest (195.69 µm) crypt depth as compared to control group. There was gradual and significant increase in villus length to crypt depth ratio from 2nd week (4.00 µm) to 6th week (5.81 µm) of age. The mean villus length to crypt depth ratio of control group was 6.99 µm, which was significantly higher than those of any other treatment groups. Significantly lowest (3.6µm) villus length to crypt depth ratio value was observed in group T₉ compared to that of control (6.99 µm). Decreased villus length, increased crypt depth and decreased VH: CD was observed due to feeding 0.5 and 2 ppm aflatoxin in diet of broiler chickens (Jahanian et al., 2016). Sharma (2013) also found reduced (P<0.05) villus length/ crypt depth ratio in aflatoxin fed group compared to control in broiler chickens. Applegate et al. (2009) found that intestinal crypt depth, but not villus length (thus influencing the villus: crypt ratio), increased linearly with increasing AF concentration in the diet (0, 0.6, 1.2, and 2.5 mg/kg) in layer hens.

It was concluded that co-contamination of aflatoxin and ochratoxin caused severe reduction in growth performance compared to individual mycotoxins and combine feeding of aflatoxin(300 ppb)and ochratoxin (250 ppb) led to the synergistic toxicity on body weight gain, feed intake, feed efficiency and immunity in broiler chickens.