Biodetoxification of Aflatoxin-Contaminated Chick Feed

METABOLISM AND NUTRITION Biodetoxification of Aflatoxin-Contaminated Chick Feed ´ vila-Gonzalez,† M. T. Casaubon-Huguenin,‡ R. A. Cervantes-Olivares,§ Z. I. Tejada-Castan˜eda,*1 E. A C. Va´squez-Pela´ez,# E. M. Herna´ndez-Baumgarten,储 and E. Moreno-Martı´nez¶ *Ciencias de la Produccio´n y de la Salud Animal, Universidad Nacional Auto´noma de Me´xico (UNAM), FES-Cuautitla´n (FESC), Carretera Cuautitla´n-Teoloyucan Km 2.5, Cuautitla´n Izcalli, Edo. de Me´xico, C. P. 54714, Me´xico; †Facultad de Medicina Veterinaria y Zootecnia (FMVZ), Centro de Ensen˜anza, Investigacio´n y Extensio´n en Produccio´n Avı´cola, Calle Salvador Dı´az Miro´n S/N, Col. Zapotitla´n, Tla´huac, Me´xico; ‡Departamento de Produccio´n de Aves, §Departamento de Bacteriologı´a e Inmunologı´a, and #Departamento de Gene´tica y Bioestadı´stica, UNAM, FMVZ, Ciudad universitaria, Coyoaca´n, C.P. 04510, Me´xico; 储Laboratorio de Microscopı´a Electro´nica. UNAM-FESC, Av 1° de mayo s/n, Cuautitla´n-Izcalli, Edo. de Me´xico. C. P. 54740, Me´xico; and ¶Unidad de Investigacio´n en Granos y Semillas, UNAM-FESC, Av. Dr. Jorge Jime´nez Cantu´ s/n, Cuautitla´n-Izcalli, C. P. 54740, Me´xico ABSTRACT Two studies were done to study detoxification of aflatoxin (AF)-contaminated chick feed with Nocardia corynebacteroides (NC). In the first study, pathogenicity of the bacteria was studied; in the second, the nutritional value of detoxified feed was evaluated. Commercial corn was divided into 2 sublots, one of which was contaminated with AF. Both lots were divided into 2 parts; the first was inoculated with NC. Four corn-soybean diets were prepared from the 4 corn lots. A completely randomized design was used with 2 × 2 factorial arrangement in which the factors were AF contaminated or not and NC inoculated or not. One hundred Ross 308 chicks (1-d-old, male) were used in 4 treatments with 5 repetitions and 5 chickens per cage. Bird weight and feed consumption were recorded weekly. Each week, 1 chick per treatment repetition was killed for histopathologic analysis of liver, kidney, bursa of Fabricius, pancreas, and small intestine

(duodenum, jejunum, and ileum) and for analysis by scanning electron microscopy of the 3 sections of the intestine. At 21 d (the end of both experiments), 1 chick per treatment repetition was killed, and moisture, lipid content, and residual AF in liver were detected. Results at 3 wk did not show differences between treatments (P > 0.05) in any of the variables. In the second study, the same methodology was used except that greater levels of AF were used (800 and 1,200 ␮g of AFB1/kg of feed). Results showed differences (P < 0.05) in body weight, lipid content, and residual AF in liver. Histopathologic studies showed statistical differences in lesion severity in liver, duodenum, and kidney. Scanning electron microscopy analysis showed severe lesions of intestinal mucosa that mainly affected tight junctions in AF treatments. It can be concluded that NC is safe for chicks and may be used to partly detoxify chicken feed contaminated with AF.

Key words: aflatoxin, biodetoxification, Nocardia corynebacteroides 2008 Poultry Science 87:1569–1576 doi:10.3382/ps.2007-00304

INTRODUCTION Aflatoxins (AF) constitute a group of heterocyclic metabolites synthesized mainly by Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius. At least 18 different AF have been identified, including AFB1, B2, G1, G2, B2a, G2a, M1, M2, P1, Q1, aflatoxicol A and B, D1, of which only the first 4 are found naturally; the others are metabolic products of animal or microbial systems or are produced spontaneously in response to environmental chemical products (Cole and Cox, 1981). Toxic effects of

©2008 Poultry Science Association Inc. Received July 20, 2007. Accepted March 31, 2008. 1 Corresponding author: [email protected]

AF commonly observed in animals include poor absorption of nutrients sometimes leading to death, reduced tissue integrity, lower growth rates and poor feed conversion, reduced immune response, reproductive problems in males and females, and increasing sensitivity to extreme temperatures (Leeson et al., 1995; Davegowda and Murthy, 2005). Different physical, chemical, and biological feed detoxification methods have been developed; physical methods range from simple sifting to separate fungi contaminated material to UV light or gamma radiation with 60Co application; chemical methods include the application of acidic, alkaline, oxidizing, and reducing substances, and aluminosilicate as a fixative agent. Biological methods use microorganisms such as Pseudomonas maltophilia, Rhizopus oryzae, Tetrahymena pyriformis, Trichoderma viride, Flavobac-

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terium aurantiacum, or glucomannans of Saccharomyces cerevisiae cell walls (Moerck et al., 1980; Doyle et al., 1982; Roob, 1993; Ma´rquez and Tejada, 1995; Singh, 1995; Basappa and Shanta, 1996; Grant and Phillips, 1998; Afzali and Davegowda, 1999; Galvano et al., 2001; Aravind et al., 2003; Davegowda and Murthy, 2005). Ciegler et al. (1966) studied approximately 1,000 microorganisms (fungi, bacteria, and algae) to observe if they were capable of degrading AF. Some fungi and their spores transformed aflatoxin B1 into new fluorescent compounds; of the bacteria, only Flavobacterium aurantiacum (NRRL-B 184) reduced AF in solution. The test was done by adding aflatoxin B1 (AFB1) to the bacteria culture medium and analyzing residual AFB1 after incubation. Doyle et al. (1982) observed that F. aurantiacum, A. niger, A. parasiticus, and A. flavus were capable of transforming AFB1 into aflatoxicol; the degree of degradation depends on the strain, pH, temperature, and AF concentration. Different researchers have used F. aurantiacum to degrade AFB1; Hao and Brackett (1988) observed 23% elimination of AFB1 in nondefatted peanut milk and 40% from a phosphate buffer at 24 h. As part of a program of our institution that deals with detoxification by microorganisms of animal feed contaminated with mycotoxins, we selected F. aurantiacum (NRRL B-184) to be used to reduce AF contamination of growing chicks’ diets. For this purpose, a strain of F. aurantiacum (NRRL B-184) was requested from the National Center for Agricultural Utilization Research (ARS, USDA). Alejandro Rooney kindly sent us the strain and David Labeda (Northern Regional Research Laboratory Culture Collection, Peoria, IL; personal communication) informed us that it had changed name and classification and that now it is known as Nocardia corynebacteroides (NRRL 24037). The objective of this project was to study the effect of AF detoxification by N. corynebacteroides (NRRL 24037). In experiment 1 we determined the pathogenicity and in experiment 2 we established the nutritional value of corn that was contaminated with AF and detoxified with N. corynebacteroides (NC) when used in growing chick diets.

MATERIALS AND METHODS Preparation of Diets Commercial corn, washed in running water and autoclaved at 103 kPa for 30 min, was divided into 2 parts. One part was placed in 3-L jars, moisture was adjusted to 18%, and the corn was inoculated with a strain of A. parasiticus (ATCC 26691; ATCC, 1989). The culture was grown in agar-dextrose-peptone medium, incubated at 24°C with stirring for 2 wk, until analysis of jar samples showed levels of AFB1>1,900 ␮g per kg of corn (AOAC, 2000; method 972.26–968.22). After incubation, the corn was autoclaved to stop the fungal growth (68.9 kPa for 15 min), and both lots of corn (AF contaminated, AFC, and not contaminated, NAF) were divided into 2 portions. The first portion of each one was left without change. The second portion of each had the moisture level in-

Table 1. Basal diet composition Item Ingredient, kg/100 kg Corn Soybean meal Other1 Calculated analysis Crude protein Metabolizable energy, kcal

55.18 37.21 7.61 21.5 3,0102

1 Other (kg/100 kg): vegetable oil, 2.71; calcium orthophosphate, 1.89; calcium carbonate, 1.50; methionine (65%), 0.43; sodium chloride, 0.33; L-LysineⴢHCl, 0.30; L-threonine, 0.10; trace minerals, 0.10; vitamins mix, 0.10; choline chloride, 0.10; antioxidant, 0.015 (NRC,1994). 2 Equivalent to 12,593 MJ/kg.

creased to 38% with distilled sterilized water, and was placed in 3-L glass jars and inoculated with a suspension of NC. The bacterial inoculum was cultured in bloodagar medium, incubated at 37°C for 72 h, and harvested with a phosphate buffer (0.1 M, pH 7.0). Tests were performed to estimate the colony-forming units (FAD, 1995) that were added to the jars (2.43 × 107 − 2.57 × 108); then, the cultures were incubated at 28°C for 72 h. Corn colonization by NC was observed at 24, 48 and 72 h, taking samples of the inoculated corn in an aseptic way. After washing with phosphate buffer in an amount in milliliters equivalent to the weight in grams of the sample taken, dilutions were made and spread in agar-blood dishes, following the method described previously. After incubation with the bacteria, both lots were autoclaved to stop the bacterial growth. The 4 corn lots were placed in stainless steel trays in a forced-air oven at 50°C for 24 h. All lots were kept at room temperature for 24 h, and then ground and mixed well. The contents of AFB1, AFB2, AFG1, AFG2 and aflatoxicol B (AFOH) in the 4 lots of corn were determined in quadruplicate (AOAC, 2000; methods 972.26, 968.22). The average amount of AFB1 that was detected was 1 ␮g of AFB1/kg of corn in NAF and 5 ␮g of AFB1/kg of corn in NAF treated with NC. Aflatoxins B2, G1, G2, and AFOH were not detected (ND) in noncontaminated corn; corn contaminated with aflatoxins and treated with NC (AFC-NC) had a total amount of aflatoxins of 1,717 ␮g/ kg of corn (1,310, 355, 52, ND, and ND ␮g/kg, equivalent to 76.3, 20.71, 3.0, 0, and 0% of the total). In contaminated corn (AFC), total aflatoxins were 2,279 ␮g/kg of corn (1,900, 76, 266, 9, and 28 ␮g/kg, equivalent to 83.4, 3.3, 11.7, 0.4, and 1.2% of the total) for AFB1, B2, G1, G2, and AFOH, respectively. Then, an AFP1 analysis was run by HPLC on the AFC and AFC-NC samples. For both trials with the 4 corn lots, 4 corn-soybean meal diets were prepared for starting growing chicks that covered satisfactorily the requirements suggested by the NRC (1994). Table 1 describes the diet composition.

Pathogenicity–Trial 1 To study if low levels of AFB1 in feed affected the pathogenesis of NC, 2 treatments with low AF levels were

BIODETOXIFICATION OF AFLATOXIN

studied. In this trial, AFC corn was diluted with NAF corn, and AFC-NC was diluted with NAF-NC corn. Treatments had the following AFB1 contents (␮g/kg of feed): NAF: 1; NAF-NC: 5; AFC-NC: 120; and AFC: 140.

Nutritional Value of Detoxified Corn–Trial 2 The AFB1 levels in the diets were (␮g/kg of feed) NAF: 2; NAF-NC: 3; AFC-NC: 800; and AFC: 1,200.

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1964). Samples were dried to critical point (critical point dryer Samdri-780a, Tousimis Research Co., Rockville, MD) and observed by scanning electron microscope (JEOL-6360LV, Tokyo, Japan) under low vacuum conditions. Images were obtained with retrodispersed electrons. After each sample was observed, it was covered with a fine gold coat (Fine Coat ION Sputter-JFC-1100, Tokyo, Japan) and observed again under high vacuum conditions with the same electron microscope (Kessel and Kardon, 1979; Watt, 1997).

Experimental Design and Birds In each study, 100 male Ross 308 chicks (1-d-old) were used. They were divided into 4 groups of similar weight. A totally randomized design was used with a 2 × 2 factorial arrangement, the factors being with and without AF and treated or not treated with NC, as described earlier. Four treatments with 5 repetitions and 5 chickens per cage were carried out. The chicks were lodged in an electrically controlled breeder (Petersime Inc., Gettysburg, OH) and were vaccinated at 10 d for Newcastle disease virus. Bird weight and feed consumption was recorded weekly.

Histopathology One chicken was randomly chosen from each pen each week and killed by cervical dislocation for the pathogenicity study. For the nutritive value study, 1 chick per pen was selected at 21 d. Liver, kidney, bursa of Fabricius, pancreas, and intestine (duodenum, jejunum, and ileum) were studied. The 3 intestinal sections were studied by using scanning electronic microscopy. Gross lesions were recorded before taking organs for analysis. From each bird, a 3- to 4-mm section of the central portion of the bursa of Fabricius was taken, a similar section was taken from the left lobe of the liver and from the middle lobe of the right kidney. Finally, 1-cm-long sections were taken from each portion of the intestine. The duodenum sample was taken at the same time as the pancreas sample; the jejunum sample was taken between the duodenum and Meckel’s diverticulum, and the ileum sample was taken from the area between Meckel’s diverticulum and the beginning of the cecum. All samples were fixed in 10% buffered neutral formalin for 24 h, using histology techniques and standard staining procedures (Luna, 1968). Evaluation was performed in a double blind study. The degree of severity of each of the different types of lesions was expressed as 0 (no lesions), 1 (slight), 2 (moderate) or 3 (severe). The sum of the numerical values of all lesions together for each organ that was studied was used in the statistical analysis to compare treatments. Intestinal samples for scanning electron microscopy were taken at the same time as the samples for histopathology, immediately cut and opened to place on a small piece of filter paper, and then immediately submerged in Karnovsky’s fixative agent (Karnovsky, 1965). Then, the samples were postfixed in osmium tetroxide, dehydrated in different ethanol solutions from lesser to greater concentrations until absolute alcohol was reached (Pease,

Chemical and Serological Analysis of Liver At the end of the study (3 wk), 1 chicken was killed per treatment repetition as previously indicated to determine moisture and total lipids in liver (AOAC, 2000; methods 934.01 and 923.07, respectively) and residual AF in liver (Trucksess et al., 1983). For serology, 2 chickens per treatment were bled by brachial vein puncture; whole blood was placed in vials without anticoagulant. Serum was obtained and serum albumin, gamma-glutamyl transferase (GGT, EC 2.3.2.2), aspartate aminotransferase (AST, EC 2.6.1.1) and alanine aminotransferase (ALT) were analyzed (using diagnostic kits of Diagnostic Chemicals Ltd., Charlestown, Prince Edward Island, Canada). After bleeding, the chickens were euthanized and autopsied, and samples were collected for histopathology.

Ethical Approval Ethical approval was obtained from the Institutional Subcommittee for Experimental Animal Care of the Masters’ and Doctorates’ Degree Program of the National Autonomous University of Mexico.

Statistical Analysis The results were analyzed by ANOVA, with later mean comparison by the Student-Newman-Keuls method. To fulfill assumed normality and data independence of total organ lesions, histopathology analyses were transformed to their square root (SAS Institute, 2001).

RESULTS AND DISCUSSION Pathogenicity–Trial 1 Results obtained from N. corynebacteroides colonization in NAF and AFC corn are shown in Table 2. As can be seen, the bacterial population increased at least 2 to 3 log times during the first 24 h and remained without change or was reduced after 48 and 72 h. Hao and Brackett (1988) observed that NC growth was slightly reduced after 24 h of incubation in phosphate buffer (pH 7.0) but even more at lower pH. Lillehoj et al. (1967) observed that NC in the presence of 5 ppm or more of AF developed morphologically aberrant forms and partly inhibited growth. In this study, the levels of AF were not as high as those used by Lillehoj et al. (1967) and aberrant forms

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Table 2. Colonization of noncontaminated and aflatoxin (AF)-contaminated corn by Nocardia corynebacteroides (cfu/g) used in both trials (data for 2 replicates shown) Autoclaved noncontaminated corn,1 cfu/g

Incubation time, h Initial 24 48 72 Initial 24 48 72

2.43 3.44 3.50 3.04 2.57 3.34 4.38 4.23

× × × × × × × ×

107 1010 1010 1010 108 1010 109 109

Autoclaved aflatoxincontaminated corn,2 cfu/g 2.43 × 107 1.17 × 1010 2.77 × 1010 2.0 × 1010 2.57 × 108 6.48 × 1010 5.41 × 109 4.46 × 109

AFB1 content = 1 ␮g/kg of corn. 2 AF content: AFB1 = 1,900; AFB2 = 76; AFG1 = 266; AFG2 = 9; and AFOH = 28 ␮g/kg of corn. 1

were not observed. Results of animal performance at 21 d of age did not show significant differences between treatments (P > 0.05): average BW was 735 ± 4.83 g (mean ± SE), average feed intake was 982 ± 17.97 g, and average feed efficiency was 0.75 ± 0.028. Average liver weight/ BW ratio (g/g) was 2.24 ± 0.039; moisture (g/100 g) was 71.4 ± 0.53, and lipids (g/100 g) were 16.4 ± 0.145. Aflatoxin consumed in the period (␮g/kg of feed) was 1, 4.8, 119, and 139 for NAF, NAF-NC, AFC-NC, and AFC treatments, respectively. Differences were detected (P < 0.05) in liver content of residual AFB1 (␮g/kg of liver): ND, ND, 69.8, and 108.3 ± 5.39 for treatments NAF, NAFNC, AFC-NC, and AFC, respectively. Total damage of the studied organs and analysis by scanning electron microscopy during the study period did not detect differences between treatments in the pathogenicity study (P > 0.05). The results obtained show that under the study conditions, NC is apparently nonpathogenic for the broiler chicken; the level of AFB1 in treatments AFC-NC and AFC did not negatively affect their performance. Few studies have been done to determine NC safety; most have studied their AF detoxifying capacity in vitro, without studying if their presence in the diet could cause damage to the consumer. Ciegler et al. (1966) did a test with ducklings, feeding them an NC cell suspension that had been incubated under different conditions with AFB1 and AFG1, to determine if all the

AF had been eliminated or if some product formed during AF degradation was toxic. Tuason (1983) observed that copra meal treated with NC caused low mortality in chicken embryos. Nevertheless, in a later study Dalmacio et al. (1996) observed a slight increase in chicken embryo mortality when chickens were treated with oil and water of copra meal treated with NC; forced feeding of chickens with bacteria-treated coconut meal caused reduction in dry matter digestibility.

Nutritional Value of Detoxified Corn–Trial 2 Table 3 describes chicken performance results and consumed AF at 21 d. Weight gain shows differences between treatments (P < 0.05). Differences were not detected between this treatment and treatments NAF and NAF-NC, even though chickens in treatment AFC-NC consumed 825 ␮g of AFB1/kg of feed. Treatment AFC showed differences and consumed AFB1 was 1,155 ␮g of AFB1/kg of feed during the study period. Patterson (1973) considers that chickens are somewhat resistant to AF intoxication, and reports that the LD50 (lethal dose 50) for chickens is 6.5 to 16.5 mg/kg of BW, whereas for ducklings it is 0.34 to 0.56 mg/kg of BW, because ducks are very susceptible. In Table 4, values in liver for moisture, lipids, and residual AFB1, ALT, AST, GGT, and serum albumin are shown. Moisture did not show differences between treatments (P > 0.05). Statistical analysis of pathological lesions caused by AF showed differences between studied organs (P < 0.01), treatments (P < 0.06), and interactions between organs and treatment (P < 0.01). Organs and lesions that showed differences at 21 d are shown in Table 5. Significant differences were detected in lesion severity in liver, disperse lymphoid clumps (P < 0.023), biliary duct proliferation (P < 0.048), duodenum, lymphoid infiltration (P < 0.0181), and kidney hemorrhages (P < 0.0359). The toxic effect of AF on the broiler depends on the doses and time of exposure, so that 2 types of aflatoxicosis can be distinguished: acute and chronic. Acute poisoning may be recognized as an acute hepatotoxic disease characterized clinically by depression, anorexia, jaundice, and hemorrhages. Experimentally, levels of 0.5 ppm of AFB1 in the diet when consumed over 4 wk may cause a reduction in feed consumption and weight loss in the animals, while 1.0 ppm of AF in the diet for 1 or 2 wk does not affect

Table 3. Performance of chicks at 21 d1 in trial 2 evaluating the nutritive value of aflatoxin (AF)-decontaminated chick diets Treatment2 Item Body weight, g Feed intake, g Feed efficiency, g/g Consumed aflatoxin, ␮g/kg of feed

NAF

NAF-NC

AFC-NC

AFC

SE

790.2a 1,062.2a 0.75a 2

782.4a 1,083.6a 0.72a 3

762a 1,032.1a 0.74a 825

680.3b 962.8a 0.71a 1,155

11.49 14.33 0.009

Within a row, means with different letters are significantly different (P < 0.05). Average of 5 replicates. 2 NAF = noncontaminated corn (AFB1 = 2 ␮g/kg); NAF-NC = noncontaminated corn treated with Nocardia corynebacteroides corn (AFB1 = 3 ␮g/kg of feed); AFC-NC = AF-contaminated corn treated with N. corynebacteroides corn (AFB1 = 800 ␮g/kg of feed); AFC = AF-contaminated corn (AFB1 = 1,200 ␮g/kg of feed). a,b 1

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BIODETOXIFICATION OF AFLATOXIN 1

Table 4. Moisture, lipid content, and residual aflatoxin (AF) in liver and serum profile of chicks at 21 d in trial 2 evaluating the nutritive value of AF-decontaminated chick diets Treatment2 Item

NAF

NAF-NC

AFC-NC

AFC

SE

Moisture, g/100 g Lipids, g/100 g (dry basis) Residual AFB1 in liver, ␮g/kg Serum profile Alanine aminotransferase, IU/L Aspartate aminotransferase, IU/L Gamma-glutamyl tranferase, IU/L Albumin, g/L

71.6 15.4c ND3

72.4 14.1c ND

68.3 32.71b 220 b

73.3 53.71a 388 a

0.53 0.46 24.53

15 b 222 a 15.5b 12 ab

21 a 236 a 23 a 7c

11 c 201 b 12 b 14 a

11 c 202 b 12 b 13 ab

0.50 2.76 0.64 0.35

Within a row, means with different letters are significantly different (P < 0.05). Average of 2 replicates. 2 NAF = noncontaminated corn (AFB1 = 2 ␮g/kg); NAF-NC = noncontaminated corn treated with Nocardia corynebacteroides corn (AFB1 = 3 ␮g/kg of feed); AFC-NC = AF-contaminated corn treated with N. corynebacteroides corn (AFB1 = 800 ␮g/kg of feed); AFC = AF-contaminated corn (AFB1 = 1,200 ␮g/kg of feed). 3 ND = not detected. a–c 1

performance (Kubena et al., 1993a,b), although Merkley et al. (1987) stated that broilers are more susceptible to AF during the first 3 wk. Hsieh (1979) describes an effect of AF on lipid metabolism that affects transport resulting in an increase in liver size and states that lipid transport is affected first before growth rate, RNA synthesis, and fatty liver induction. Dafalla et al. (1987), in a study where chickens were fed with diets that had a 0.5 ppm AFB1 content over 1, 2, and 3 wk, observed petechial or ecchymotic hemorrhages in thighs, hematomas in liver and spleen, and kidney congestion. Birds that received 0.5 ppm of AFB1 during 4 wk showed pale yellow liver, bile vesicle edema, and multifocal congestion areas in kidneys and perirenal edema. Different researchers (Tung et al., 1975; Dalvi and McGowan, 1984; Chattopadbyay et al., 1985; Aravind et al., 2003) have described that AF at 0.5,

1.0, and 2 mg/kg of feed levels cause increases of ALT, AST, and GGT. Protein reduction has been studied by several researchers (Hamilton et al., 1973; Huff et al., 1986; Jessar and Singh, 1993; Kubena et al., 1993a,b), who found that 1 to 3 mg of AF/kg of feed causes significant depression of total protein, albumin, and globulin. Results obtained in this experiment in relation to animal behavior, hepatic metabolism damage, and histopathology results analysis indicate that these effects are due to AF in the diet (Ergun et al., 2006). Figure 1 (panels A, B, C, and D) shows SEM images of the brush border of the epithelial cells of the duodenum. Panel A is from treatment NAF and shows normal microvilli (MV) and tight junction (TJ). In this image, the intestinal lumen lies to the left and the evenness of the MV can be seen. Panel B is from treatment NAF-NC and normal

Table 5. Organ and lesions that showed significant differences between treatments in 21 d1 in trial 2 evaluating the nutritive value of aflatoxin (AF)-decontaminated chick diets Organ and lesion Liver; disperse lymphoid clumps

Liver; proliferation of biliary ducts

Duodenum; lymphoid infiltration

Kidney; hemorrhages

a,b

Treatment2 NAF NAF-NC AFC-NC AFC-NC NAF NAF-NC AFC-NC AFC NAF NAF-NC AFC-NC AFC NAF NAF-NC AFC-NC AFC

Severity of damage3 b

1.20 1.00b 2.40ab 3.00a 1.00b 1.20b 2.60a 2.80a 2.00b 2.00b 2.00b 2.60a 0.80b 0.40b 1.40ab 1.80a

P≤ 0.023

0.048

0.0181

0.0359

Means with different letters within each organ/lesion are significantly different. Average of 5 replicates. 2 NAF = noncontaminated corn (AFB1 = 2 ␮g/kg); NAF-NC = noncontaminated corn treated with Nocardia corynebacteroides corn (AFB1 = 3 ␮g/kg of feed); AFC-NC = AF-contaminated corn treated with N. corynebacteroides corn (AFB1 = 800 ␮g/kg of feed); AFC = AF-contaminated corn (AFB1 = 1,200 ␮g/kg of feed). 3 Severity of lesions: slight = 1; medium = 2; severe = 3. 1

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Figure 1. Scanning electron micrographs of duodenum samples of chicks fed aflatoxin (AF)-decontaminated diets. A) Treatment NAF (noncontaminated corn, AFB1 = 2 ␮g/kg) showing duodenum, normal microvilli (MV), and normal tight junction (TJ); B) treatment NAF-NC (noncontaminated corn treated with Nocardia corynebacteroides corn, AFB1 = 3 ␮g/kg of feed) showing duodenum, normal microvilli (MV), and normal tight junction (TJ); C) treatment AFC-NC (AF-contaminated corn treated with N. corynebacteroides corn, AFB1 = 800 ␮g/kg of feed) showing duodenum with partly affected microvilli (AMV) and partly affected tight junction (ATJ); D) treatment AFC (AF-contaminated corn, AFB1 = 1,200 ␮g/kg of feed) showing duodenum with uniformly affected MV; the tight junctions have disappeared and denatured proteins (ATJ) are observed.

MV can also be seen. The lumen in this case is to the right, and the TJ to the left shows a few irregular globules, which differ from the flat surface seen in panel A. Even so, we do not consider that Nocardia alone causes damage. Panel C from treatment AFC-NC shows slight damage of the affected microvilli (AMV) and some interruptions of the tight junction (ATJ). Panels A and B show the layer of microvilli separated from the epithelial cells, whereas panels C and D show the MV still attached to the epithelial cells, which broke during the processing of the samples and the breakage did not occur at the union of the cells. Panel D from treatment AFC shows the AMV uniformly affected. Even though panels were taken at a lower magnification, it can be seen that the MV are shortened and fused in what appears to be 2 or 3 MV fused into 1. The tight junction (ATJ) on the epithelium from panel D has completely disappeared and only irregular masses of denatured proteins remain. The damage observed is interpreted as loss of the polarity of the epithelial cells. The TJ separates the epithelium on a luminal side, where the MV are found, and a basolateral side, where lymph and absorbed nutrients are separated from the luminal side. The loss of polarity can alter the absorption of nutrients. In lower magnification pictures of the epithelial surface

(not included) the MV are detached from the epithelial cells creating bald spots, and dead cells are rounded and loosened from the epithelium leaving a hole. These changes were present in all samples, but are more abundant in treatments AFC-NC and AFC, to the extent that some of the holes coalesce to form a groove that runs the thickness of the epithelium. Sometimes groups of 20 cells or more come off the epithelium simultaneously as a mass. None of these changes have been reported before because pathology studies are conducted on 5- to 10␮m sections, stained with hematoxylin and eosin and observed on an optical microscope, where the brush border is barely discernible. Interference in the nutrient absorption process because of altered MV and ruptured mucosa could explain the lesser weight gain of the animals fed AFB1. The fact that damage observed in treatment AFC-NC was less than in treatment AFC is consistent with the rest of this experiment’s data. Similar damage was observed in jejunum and ileum samples. The importance of the small intestine in xenobiotic phase I and II metabolism is based on the presence of numerous metabolic enzymes in the organ, and it is the first exposure site of xenobiotics to metabolic systems.

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Moreover, there is a large surface available for xenobiotic absorption (Kaminsky and Zheng, 2003) and its morphology has an important role in metabolic competence of the organ, with diverse anatomical and physiological characteristics. The life of the enterocyte is short; it is estimated that their life span is 1 to 2 cells per 100 per hour (Lin et al., 1999); in humans, enterocytes life span is 2 to 6 d (Kaminsky and Zheng, 2003), in chickens the life span is 2 d and a little longer in the duodenum and jejunum (Moran, 1982). Enzymes within the intestinal lumen come from 2 sources: from gastrointestinal tract secretions of gastric, pancreatic, and intestinal origin and from bacteria, the latter concentrated in the ileum and colon (Doherty and Charman, 2002). By the manner in which phase I xenobiotic metabolism is carried out, it is difficult to separate the contributions of the liver and the intestine. The short duration of enterocyte life reduces its enzymatic potential and any lesion that is caused during the covalent union between the activated xenobiotic and some macromolecule of the enterocyte is also short lived (Kaminsky and Zheng, 2003). The most common form of xenobiotic absorption is passive diffusion through the intestinal wall against a concentration gradient. Nevertheless, saturation and competitive inhibition can affect absorption rate, which would indicate that facilitated and active transport systems play a role (Doherty and Charman, 2002). Transport proteins in the intestinal brush border and basolateral membranes allow the xenobiotic to penetrate the enterocyte cytosol and be metabolized (Doherty and Charman, 2002). Because all epithelial cells have unions that maintain cells together and to the matrix, occluding and tight junctions, and anchoring ones have a very important role in xenobiotic absorption and metabolism functions by maintaining polarity in epithelia (Alberts et al., 2002). Tight junctions work in 2 ways: 1) they seal cell membranes of adjacent cells to create an impermeable or semipermeable barrier to diffusion along the cell layer, and 2) they act as barriers in the double lipid layer to restrict membrane transport protein diffusion between the apical zone and the basolateral zone of the membrane of each epithelial cell. Epithelial cells may transiently alter the tight junctions to increase the flow of solutes and water through breaches in the unions. In our study, we observed significant damage to the tight junctions, probably because of the passage of toxins. Aflatoxins are transported in blood together with a protein, usually albumin (Dirr and Schabort, 1986). It can be deduced that AF absorption in the intestine may be carried out by several possible routes: 1) AF penetrate the enterocyte by passive diffusion, are biotransformed by Cyt-P450, and are joined to a protein and, as adducts, arrive at the liver through the portal vein; 2) because AF are liposoluble compounds, they enter through membranes by a paracellular route, and perhaps this damages the tight junctions (Klaassen and Roozman, 1991); and 3) in the liver, blood passes through the hepatocyte leaving by way of the hepatic vein that empties into

the vena cava of the general circulation. The effect of AF on intestinal mucosa tight junctions should be studied in greater detail. The results observed in this study show that NC is safe for broilers. Its use to detoxify AF-contaminated feed is possible, because it reduces the amount of AFB1 by forming other compounds that have a lower toxicity such as AFB2 and by making others disappear such as AFG2 and AFOH, probably by transforming them into other compounds with a different solubility. According to Cole and Cox (1981), the toxicity of AFB2 is approximately 15 times less, that of AFP1 is 20 times less, and that of aflatoxicol is 18 times less than AFB1 for ducklings. Galtier (2003) describes that hydroxylated or dealkylated products such as AFM1, AFP1, or AFQ1 are quickly conjugated to glucuronic acid or sulfate and then are excreted with bile or urine in several animal species. The HPLC analysis of AFC-NC detected 480 ␮g of AFP1/kg of corn, confirming the possibility of lesser toxicity in this treatment. By the results that were obtained it can be concluded that N. corynebacteroides may be used to partly detoxify AF-contaminated feed given to growing broilers.

ACKNOWLEDGMENTS The authors thank Genoveva Garcı´a and Magda Carvajal (Biology Institute) and Benjamin Fuentes (CEIEPAV) for their technical support; Yolanda Hornelas (Instituto de Ciencias del Mar, Ciudad Universitaria, Me´xico) for electron micrography; and Sofia Gonza´lez and Rosario Ruiz (FES-Cuautitla´n, Cuautitla´n-Izcalli, Universidad Nacional Auto´noma de Me´xico) for their aid in the preparation of samples for scanning electron microscopy.

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