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Agriculture and Agri-Food Canada, Guelph, Canada N1G 5C9. ABSTRACT Bacillus subtilis ANSB060 from the fish gut had strong ability to detoxify aflatoxins.

Protective effect of Bacillus subtilis ANSB060 on egg quality, biochemical and histopathological changes in layers exposed to aflatoxin B1 Q. G. Ma,*1 X. Gao,*1 T. Zhou,† L. H. Zhao,* Y. Fan,* X. Y. Li,* Y. P. Lei,* C. Ji,* and J. Y. Zhang*2,3 *State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China 100193; and †Guelph Food Research Center, Agriculture and Agri-Food Canada, Guelph, Canada N1G 5C9 ABSTRACT Bacillus subtilis ANSB060 from the fish gut had strong ability to detoxify aflatoxins. The aim of this research was to investigate the protective effect of B. subtilis ANSB060 (ANSB060) on egg quality and biochemical and histopathological changes of liver and kidney in laying hens when exposed to aflatoxin B1. Treatments (C20, C40, and C60) were prepared by substituting corn contaminated by aflatoxin B1 (AFB1) at different proportions (20, 40, and 60%) for normal corn in basic diets. The aflatoxin degradation enzyme (E) treatments (E20, E40, and E60) were mixed with the fermentation liquor of ANSB060 with C20, C40, and C60, respectively. The results showed that ANSB060 can improve the eggshell strength in E60 compared with C60 (P ≤ 0.05), and toxin reduced the content of total protein (in groups C20, C40, and C60) and

albumin (in C20 and C40; P < 0.05) and heightened the activities of GPT (in C60) and GOT (in C40 and C60) in serum (P < 0.05). In the liver, AFB1 inhibited the activity of superoxide dismutase and glutathione peroxidase (C40 and C60; P < 0.05) and increased the content of malonaldehyde (in C40 and C60), which induced the damage in the liver and kidney as shown in the photomicrographs of hematoxylin and eosin-stained sections. The addition of ANSB060 can enhance the activity of antioxidant enzymes, and it recovered the protein synthesis in liver. Moreover, ANSB060 also ameliorated the damage of liver and kidney tissue and restored them to normal. Hence, ANSB060 had the ability to inhibit the damage induced by AFB1; it will have a great potential in industrial applications.

Key words: aflatoxin B1, Bacillus subtilis ANSB060, dwarf layer, protection 2012 Poultry Science 91:2852–2857 http://dx.doi.org/10.3382/ps.2012-02474

INTRODUCTION Aflatoxins are secondary metabolites predominantly produced by Aspergillus flavus, Aspergillus nomius, and Aspergillus parasiticus (Kurtzman et al., 1987; Cotty and Bhatnagar, 1994). They can harm the health of humans and animals because of carcinogenicity, mutagenicity, teratogenicity, and immunosuppressive effects (Gao et al., 2011). Various symptoms have been related with aflatoxicosis in animals, including lethargy, anorexia, lower growth rate, and poor feed utilization (Magnoli et al., 2011a). For humans, it will cause liver cancer when they are exposed to aflatoxins long-term (Pitt, 2000; Alberts et al., 2006). ©2012 Poultry Science Association Inc. Received May 15, 2012. Accepted July 17, 2012. 1 Coauthor: Q. G. Ma and X. Gao are the co-first authors. 2 Corresponding author: [email protected] 3 Present address: State Key Laboratory on Animal Nutrition, China Agricultural University, No. 2. West Road Yuanmingyuan, Beijing 100193, P.R. China.

China is one country with the most serious contamination of aflatoxins (Williams et al., 2004). Foodstuffs are easily contaminated by aflatoxins during harvesting, transportation, and storage. Moreover, because aflatoxins are stable, physical and chemical methods to degrade them have many limitations, such as loss of product nutrition, organoleptic qualities, undesirable health effects, and high cost of equipment (Basappa and Shantha, 1996; Teniola et al., 2005). These disadvantages encouraged the development of biological methods to degrade aflatoxins. Many microorganisms selected from the environment and the feces of animals were able to degrade aflatoxins. For example, Mycobacteriumis, Stenotrophomonas maltophilia, Aspergillus parasiticus, Trichoderm viride, and Mucor ambiguous showed the ability to decrease the aflatoxins (Huynh et al., 1984; Liu et al., 2001; Hormisch et al., 2004; Guan et al., 2008). However, most of them were not allowed to be applied in food or feed under the Direct of Ministry of Agriculture, People’s Republic of China. In this case,

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PROTECTION OF ANSB060 ON AFLATOXICOSIS LAYERS Table 1. Composition of basal diets during the experiment Ingredient Maize Soybean meal Cottonseed meal Wheat bran CaHPO4 Limestone Salt Choline chloride Sodium bicarbonate Methionine Lysine Mineral premix1 Vitamin premix2 Total

% 62.34 22.00 3.00 2.00 1.60 8.23 0.25 0.08 0.20 0.09 0.07 0.11 0.03 100

Nutrition component

Content

ME (kcal/kg) CP (%) Lysine (%) Methionine (%) Methionine + cystine (%) Calcium (%) Phosphorus (%)              

2,650 16.5 0.87 0.42 0.66 3.48 0.58  

           

1Provided per kilogram of diet: iron, 60 mg; zinc, 66 mg; manganese, 65 mg; iodine, 1.0 mg; copper, 8 mg; selenium, 0.3 mg. 2Provided per kilogram of diet: vitamin A, 12,500 IU; vitamin D , 4,125 IU; vitamin E, 12 IU; vitamin K, 2 mg; 3 thiamine, 1 mg; riboflavin, 8.5 mg; pyroxidine, 8 mg; vitamin B12, 5 mg; pantothenic acid, 50 mg; niacin, 32.5 mg; biotin, 2 mg; folic acid, 5 mg; choline, 500 mg.

our laboratory had selected one strain of Bacillus subtilis ANSB060 from the fish gut which had strong ability to detoxify toxins, and percentages of aflatoxin B1 (AFB1), M1, and G1 degradation were 81.5, 60, and 80.7%, respectively. At the same time, ANSB060 showed high antimicrobial activities against pathogenic bacteria and resistance to the simulated gut environments (Gao et al., 2011). Therefore, the aim of this research was to investigate the effect of ANSB060 on egg quality and biochemical parameters of laying hens when exposed to AFB1.

MATERIALS AND METHODS Microorganisms and Culture The probiotic strain, B. subtilis ANSB060, was originally isolated from the fish gut (Gao et al., 2011). The B. subtilis ANSB060 was cultured in Luria-Bertani medium at 37°C for 20 h using batch fermentation.

Experimental Birds In total, seventy 60-wk-old vaccinated dwarf layers, with a mean BW of 1.6 kg were randomly assigned into 7 experimental groups, with 10 replicate pens per treatment and 1 hen per pen. Throughout the study (6 wk), feed and water were provided ad libitum. The birds were housed at average 18°C under 16 h of lighting. Before the beginning of the experimental study, all hens were given 2 wk to adapt to the diets and surroundings.

Experimental Design The formula of basic diets for the control group is presented in Table 1. The basal diet (C0: control) was tested by HPLC to ensure that it contained no residual mycotoxins (aflatoxins, deoxynivalenol, zearalenone, and ochratoxin). Treatments (C20, C40, and C60) were prepared by substituting corn contaminated by AFB1

at different proportions (20, 40, and 60%) for normal corn in basic diets. The aflatoxin degradation enzyme (E) treatments (E20, E40, and E60) were mixed with the fermentation liquor of B. subtilis ANSB060 with C20, C40, and C60, respectively (fermentation liquor of B. subtilis ANSB060: moldy corn = 0.4 L:1 kg). The content of AFB1 in moldy corn was 70 μg/kg and without other mycotoxins. The detection method for aflatoxin was according to Trucksess et al. (1994). In the last week of the whole study, 6 eggs from every treatment were used to measure the egg quality, including yolk weight, yolk color, egg white height, egg shape index, eggshell color, eggshell strength, shell thickness, and Haugh unit. The test methods of these indices followed Zhang et al. (2005). On 42nd day, 6 birds per treatment were slaughtered by dislocation of the neck vertebrae and bleeding. Blood samples were collected for biochemical study. Within one hour of collection the serum was separated. The serum was then analyzed for total protein (TP) and albumin (ALB) content, glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), and alkaline phosphatase (ALP) activities. About 0.5 g of liver of every bird was homogenated and analyzed for GOT, GPT, ALP, superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity and malonaldehyde (MDA) content. These were determined with the clinical chemistry analyzer (Commercial Kit, Nanjing Jiangcheng Bioengineering Institute, China) according to the manufacturer’s recommended procedure. The same position of kidney and liver of every bird was removed and fixed in 10% neutral-buffered formalin solution, dehydrated in graded alcohol, and embedded in paraffin. Sections of 3 to 5 μm were obtained and stained with hematoxylin and eosin, and then to valuate portal and periportal necrosis, congestion, fatty change, dysplasia, and neoplastic transformation by light microscopy.

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Table 2. Effect of ANSB060 fed to laying hens on egg quality when exposed to aflatoxin B1 (n = 6)

Group1

Egg weight (g)

Yolk weight (g)

Yolk color

Egg white height (mm)

Egg shape index

Eggshell color

Eggshell strength (kg/cm2)

Shell thickness (mm)

Haugh unit

C0 C20 E20 C40 E40 C60 E60 SEM P-value

56.71 56.48 56.58 53.95 54.31 53.32 53.39 0.46 0.19

16.97 15.58 16.17 15.47 16.50 15.40 16.20 0.74 0.17

7.33 7.00 7.50 6.75 7.50 7.33 7.40 0.70 0.71

5.43 5.20 5.67 6.70 5.96 5.38 6.60 0.82 0.13

1.43 1.37 1.36 1.35 1.36 1.42 1.38 0.04 0.10

79.50 82.80 83.22 84.28 79.33 83.70 83.15 4.1 0.41

2.39ab 2.42ab 2.31ab 2.14b 2.42ab 2.07b 2.78a 0.30 0.05

0.33 0.34 0.32 0.30 0.30 0.29 0.32 0.02 0.07

73.77 72.97 80.00 81.40 80.98 76.37 82.01 4.61 0.09

a,bMeans

within column with different superscripts differ significantly (P ≤ 0.05). control; C20: contains 20% moldy corn; C40: contains 40% moldy corn; C60: contains 60% moldy corn; E20: contains 20% moldy corn and ANSB060; E40: contains 40% moldy corn and ANSB060; E60: contains 60% moldy corn and ANSB060. 1C0:

All data were subjected to analysis by ANOVA for a complete randomized design, using the GLM procedure of SAS software (version 9; SAS Institute Inc., Cary, NC). Statements of statistical significance were based on P ≤ 0.05.

RESULTS Egg Quality The quality parameters of the eggs collected during the experimental period are summarized in Table 2. Eggshell strength decreased in groups C40 and C60, but the effects were not significant (P > 0.05). And an increasing trend appeared in E40 and E60, especially, the eggshell strength in E60 was significantly stronger than C60 (P ≤ 0.05). There were no significant differences in the egg white height, egg shape index, eggshell color, and Haugh unit among treatment groups (P > 0.05).

Blood Metabolic Enzyme Activity Effects of ANSB060 on the blood metabolic enzyme index of laying hens exposed to AFB1are shown in Table 3. The TP content decreased significantly in groups C20, C40, and C60 compared with C0 (P < 0.05); ANSB060 can increase the content of TP in serum, especially, in group E20 and E40, the concentration was significantly higher than C20 and C40, respectively (P < 0.05). However, E60 was not significantly different from C60. The content of ALB in C20 and C40 reduced significantly compared with C0 (P < 0.05). Adding ANSB060 in C20 and C40 can enhance the ALB content in the blood, and the content in E20 increased significantly compared with C20 (P < 0.05). The GPT activity heightened significantly in C60 compared with C0; ANSB060 appears to spar the effect of aflatoxin on this parameter with the E60 treatment being significantly lower than the C60 treatment and not significantly different from the control treatment. The AFB1 in the diets can increase the GOT activity, especially C40 and C60 showed significantly higher activity than C0, whereas ANSB060 reduced GOT activity signifi-

cantly in E40 and E60 compared with their matched C40 and 60 treatments, and the E40 and E60 treatments did not significantly differ from the control treatment (P > 0.05).

Liver Metabolic Enzyme and Antioxidant Index Effects of ANSB060 on liver metabolic enzyme and antioxidant index of laying hens exposed to AFB1 are shown in Table 4. The SOD and GSH-Px activities in different treatments had similar trends. The AFB1 showed the ability to decrease the activities of SOD and GSH-Px. As AFB1 content in the diets increased, the SOD and GSH-Px activities decreased. In groups C40 and C60, the activity was significantly lower than C0 (P < 0.05). Though the effect was not significant (P > 0.05), ANSB060 can raise the activities of SOD and GSH-Px, keeping them to the normal level. The AFB1 in the diets enhanced the MDA content. Among different treatments, C40 and C60 were significantly higher than C0. In groups E40 and E60, ANSB060 can decrease the content of MDA in liver (P < 0.05).

Histopathology Figure 1 showed the photomicrographs of hematoxylin and eosin-stained liver sections of layers fed different treatments. As the AFB1 in the diets increased, the damage of liver tissue was more serious. Especially, the section from C60 showed macrovesicular steatosis, nucleolus visible, central necrosis of hepatic lobule, and diffuse liver necrosis, and it was followed by C40 and C20. Figure 2 shows the photomicrographs of hematoxylin and eosin-stained kidney sections of broilers from different treatments. The AFB1 can induce the damage of kidney, and these harms will be worse as the moldy corn in diets increases. The C20, C40, and C60 showed the dilatation of tubules, degeneration of renal tubular epithelial cells, damage and loss of brush border, vacuolation of plasmacyte, shedding of necrotic tissue from tubules, formation of tube-type debris, and interstitial infiltration of inflammatory cells. Whereas

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PROTECTION OF ANSB060 ON AFLATOXICOSIS LAYERS Table 3. Effects of ANSB060 on blood protein and metabolic enzyme index of laying hens exposed to aflatoxin B1 for 6 wk (n = 6)1 Group2

TP content (g/L)

ALB content (g/L)

GPT activity (IU/L)

GOT activity (IU/L)

ALP activity (IU/L)

C0 C20 E20 C40 E40 C60 E60 SEM P-value

33.03a 31.40c 32.52ab 31.72bc 32.90a 31.08c 31.73bc 0.66 0.04

25.73a 20.41c 25.21ab 21.86b 24.54ab 27.56a 26.10a 2.6