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Biliary ochratoxin A as a biomarker of ochratoxin exposure in laying hens: An experimental study after administration of contaminated diets
Research in Veterinary Science 100 (2015) 265–270
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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Biliary ochratoxin A as a biomarker of ochratoxin exposure in laying hens: An experimental study after administration of contaminated diets Sara Armorini a, Khaled Mefleh Al-Qudah b, Alberto Altafini a, Anna Zaghini a, Paola Roncada a,* a Department of Veterinary Medical Sciences, School of Agriculture and Veterinary Medicine- Alma Mater Studiorum, University of Bologna, via Tolara di Sopra 50, Ozzano Emilia (BO) 40064, Italy b Department of Veterinary Clinical Sciences, Jordan University of Science and Technology, Irbid 22110, Jordan
A R T I C L E
I N F O
Article history: Received 8 September 2014 Accepted 1 March 2015 Keywords: Ochratoxin A Bile HPLC Laying hens
A B S T R A C T
To evaluate the levels of ochratoxin (OTA) in kidney, liver and bile of laying hens, forty-five laying hens were enrolled in this study and divided into three equal groups: a control group D0, and two experimental groups, D1 fed with 10 μg/kg OTA diet and D2 fed with 200 μg/kg OTA diet for 6 weeks. Kidneys, livers, and bile from all hens were collected and analyzed by HPLC method for the presence of OTA. Eggs collected 2 days before the start of the experiment and 2 days after its end were also analyzed for the presence of OTA. Results show a relevant biliary excretion of the mycotoxin, with high levels of OTA in the bile after administration of the toxin. OTA level in eggs was below the limit of detection (LOD). These results suggest the suitability of using bile as a matrix for screening measurements of OTA in laying hens. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Ochratoxin A is a natural poison contaminating a large variety of plants and leading to the appearance of residues in animal origin products (Gourama and Bullerman, 1995; Stoev, 2010). Ochratoxins are fungal secondary metabolites produced by several species of Aspergillus and Penicillium, predominantly Aspergillus ochraceus and Penicillium verrucosum. The most important toxin is ochratoxin A (OTA), which is more toxic and more frequently found than ochratoxin B or ochratoxin C (Ringot et al., 2006). Crops used for animal feed can be easily contaminated by fungi during growth, harvest, or storage, resulting in the occurrence of mycotoxins (Monbaliu et al., 2010). OTA occurs as a common contaminant of cereals, peanuts, as well as soya, coffee and cocoa beans (Birò et al., 2002). In poultry as well as in other animal species, OTA appears in different body tissues including kidneys, liver, muscles, gastrointestinal organs, lymphoid tissues, skeletal system, blood and reproductive tract (Dwivedi and Burns, 1984; European Food Safety Authority (EFSA), 2004; Pozzo et al., 2013a, 2013b). The feed of monogastric animals such as poultry and pigs can be monitored for the presence of mycotoxins and allow
* Corresponding author. Department of Veterinary Medical Sciences, School of Agriculture and Veterinary Medicine- Alma Mater Studiorum, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia (BO), Italy. Tel.: +39 0512097511; fax: +39 051799511. E-mail address: [email protected] (P. Roncada). http://dx.doi.org/10.1016/j.rvsc.2015.03.004 0034-5288/© 2015 Elsevier Ltd. All rights reserved.
the formulation of diets that are well tolerated by animals (Fink-Gremmels, 2008). The European Commission (2006) recommendation 2006/576/EC indicates that the maximum tolerable level of ochratoxin A in poultry feeds is 0.1 mg/kg. Several experimental feeding trials with OTA contaminated feeds have indicated its harmful effects on growing chicks and its potential risk for the poultry industry. Avian nephropathy associated with OTA exposure was reported in Denmark by Elling et al. (1975), which detected microscopical renal changes in four out of 14 kidneys. In the USA five independent episodes of ochratoxicosis in turkeys, two episodes in laying hens, and two episodes in broiler chickens were reported by Hamilton et al. (1982); the histopathology of affected birds always reveals on edema and necrosis of the proximal tubules of the kidneys with no changes in the liver or other organs. A lethal gastroenteritis was reported in Italy due to feeding chickens, rabbits and dogs with moldy bread (Visconti and Bottalico, 1983). Mycotoxicosis in farm animals and laying hens were reported in western Canada during a 3 year study (Abramson et al., 1983). Other reports of ochratoxicosis in lying birds came from Hungary (Bata et al., 1996), the USA (Howell, 1981), and Great Britain (Page et al., 1980). The common clinical symptoms observed in ochratoxicosis are retardation of growth, dullness, huddling, decrease appetite, reduced feed intake, weakness and diarrhea (Dwivedi and Burns, 1984); OTA also affects egg production, fertility and hatchability (Page et al., 1980). However, there are no pathognomonic symptoms of ochratoxicosis and its diagnosis seems to be difficult.
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Few works in literature focused on OTA toxicokinetics and residues in chicken (Bozzo et al., 2008; Dietrich et al., 2005; Galtier, 1978; Galtier et al., 1981; Pozzo et al., 2013b), but to date, the biliary route of excretion was not considered to detect the level of OTA; this might be because urinary excretion seems to be a primary route of elimination, beside that bile is not an edible tissue. Shephard et al. (1994) reported that biliary excretion is a major route of elimination of the mycotoxin fumonisin B1 from blood circulation and only small amount of toxin appeared to be absorbed from the intestinal tract of rats. The enterohepatic circulation of OTA has been demonstrated in studies in rodents (Kumagai, 1985; Roth et al., 1988) and newborn calves (Sreemannarayana et al., 1988). All these studies showed secondary distribution peaks of OTA in the serum and intestinal contents and concluded to a biliary secretion of the toxin followed by its reabsorption from the intestine. Reabsorption of OTA from the intestine back to the blood circulation, as a result of biliary recycling, promotes the systemic redistribution of OTA to different tissues (Ringot et al., 2006). The aim of our study was to evaluate the levels of OTA in bile, kidney, liver, and eggs in laying hens after the administration of 10 and 200 μg/kg of controlled diets. Secondly, we try to demonstrate the suitability of using bile for the early detection of OTA in poultry.
Table 1 Ingredients of diet fed to laying hens for 6 weeks during the entire experiment.a Ingredient
%
Maize ground Soya bean ground meal Barley ground Dry alfa-alfa Calcium carbonate Corn gluten Soya oil Calcium phosphate Sodium chloride Premixb
52.60 20.00 6.00 5.00 8.40 4.00 1.50 1.60 0.40 0.50
a Chemical composition of basal diet: dry matter 88.8%, crude protein 16.4%, crude fat 5%, crude fiber 3.81%, ash 12.65%, calcium (Ca) 3.70%, phosphorus (P) 0.6%, sodium (Na) 0.18%. b Composition per kilogram of premix: 2,600,000 IU vitamin A; 600,000 IU vitamin D3; 4000 mg α-tocopherol acetate; 400 mg vitamin K3; 1000 mg vitamin B1; 6000 mg vitamin B2; 4 mg vitamin B6; 400 mg vitamin B12; 6000 mg vitamin PP; 20 mg biotin; 100 mg folic acid; 2000 mg pantothenic acid; 100,000 mg choline; 10,000 mg Zn; 10,000 mg Fe; 12,000 mg Mn; 1000 mg Cu; 200 mg I; 100 mg Co; 40 mg Se; 6000 mg total xanthophyll; 1000 mg BHT (butylated hydroxytoluene).
2. Materials and methods
Committee of the Alma Mater Studiorum-Università di Bologna, in accordance with specific legislation concerning protection of animals used for scientific purposes.
2.1. Animals and sampling
2.2. Liver and kidney extraction
Forty-five Warren (Isa Brown) 30-week-old laying hens obtained from a commercial facility, with an average weight of 1.80 ± 0.035 kg, were randomly divided into 2 experimental groups (D1 and D2), and the control group (D0), each comprising of 15 animals. All the birds were housed in individual cages with separate feeders, kept under identical environmental conditions (temperature, humidity) and held under 16 h of light and 8 h of dark per day. All birds were monitored daily for any adverse clinical symptoms. After a period of acclimatization (45 days) and 20 days after eggs laying beginning, hens received the following diets for 6 weeks: a control group (D0) was fed with a control diet free of OTA; an experimental group (D1) was fed with OTA 10 μg/kg experimentally contaminated feed; the third experimental group (D2) was fed with OTA 200 μg/kg experimentally contaminated feed. Before feeding, the basal diet was tested by means of an ELISA (Veratox® Ochratoxin, Neogen Corporation, Lansing, Michigan, USA) to ensure that it contained no residual ochratoxin A. The experimental diets were obtained by mixing the basal diet (Table 1) with naturally contaminated ground maize containing 280 mg/kg of OTA. Ten or 0.5 g of contaminated maize was mixed with 14 kg of basal diet to obtain OTA levels of 200 μg/kg and 10 μg/kg respectively. The two experimental groups were fed with 100 g/day/animal of contaminated diet. Consequently each animal of the D1 group received a total of 1 μg of OTA daily, while each animal of the D2 group received 20 μg of OTA daily. For evaluation of OTA, 20 eggs randomly collected from each experimental group, 10 eggs collected the first 2 days before the start of treatment and 10 eggs at the last 2 days of the experiment were analyzed. White and yolk were separately collected and subjected to freeze drying process. Finally, after being vacuum-packed, the samples were kept at −20 °C until their analysis by using HPLC-FD method. At the end of the experimental period, laying hens were euthanized by vertebral dislocation followed by exsanguination. From all the birds (controls and OTA administered groups) kidney, liver and samples of bile (directly from gallbladder) were collected and kept frozen (−20 °C) until their HPLC-FD analysis. The plan of this study was authorized by Italian Ministry of Health on the basis of a previous judgment issued by the Ethic Scientific
Ochratoxin A extraction from liver and kidney was executed on 2.5 g of tissue thawed, acidified with 2.5 ml citric acid 30% water solution and extracted with 15 ml dichloromethane. This mixture was homogenized (3 min) with Ultra-Turrax T25 (IKA Labortechnik, Staufen, Germany) then rinsed with 2.5 ml dichloromethane. The samples were sonicated (30 min), cooled on ice (10 min) and, finally, centrifuged (2600g for 10 min). Fourteen milliliters of organic layer (equal to 2 g of liver or kidney) was subjected to clean up. 2.3. Liver and kidney clean up The organic extracts were loaded onto Isolute® Si 500 mg/10 ml (Biotage AB, Uppsala, Sweden) previously conditioned with 5 ml dichloromethane. The cartridges were washed with 5 ml hexane. OTA in liver and kidney samples was eluted differently. In liver samples, OTA was eluted with 5 ml tetrahydrofuran/ hexane 1:3 (v/v) followed by 5 ml diethyl ether/hexane 1:2 (v/v), while in kidney samples, 5 ml tetrahydrofuran/hexane 1:3 (v/v) was used only for washing the SPE columns and the elution took place with 5 ml diethyl ether/hexane 1:2 (v/v) followed by 5 ml acetone/ dichloromethane 1:4 (v/v). For both matrices, the fraction collected was reduced to dryness by means Univapo (Univapo Martinsried/Munich, Germany), the residue was dissolved in 500 μl methanol and 20 μl was injected into HPLC. 2.4. Bile extraction OTA extraction was performed on 200 μl of bile added to 200 μl of citric acid 30% water solution. After the addition of 2 ml of dichloromethane, the mixture was extracted in a blender for 30 minutes, cooled on ice (10 min) and thereafter centrifuged (2600g for 10 min). The lower organic layer (1600 μl) was transferred into a conical tube and evaporated under vacuum (Univapo Martinsried/Munich, Germany). The dry residue was reconstituted with 800 μl of methanol and 20 μl injected into HPLC.
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2.5. Egg white and egg yolk extraction OTA extraction from egg white and egg yolk was executed on 1 g of lyophilised sample, rehydrated with 8 ml water, acidified with 5 ml citric acid 30% water solution and extracted with 20 ml dichloromethane in a blender for 30 minutes. The samples were sonicated (15 min), cooled on ice (10 min) and, finally, centrifuged (2600g for 10 min). From this step on, white egg samples and yolk egg samples were differently processed. Fifteen milliliters of organic layer (equal to 0.75 g of sample) was collected, and subject to clean up (yolk egg samples) or evaporated under vacuum (white egg samples). In the last case, the dry residue was reconstituted with 750 μl of methanol and 20 μl injected into HPLC. 2.6. Egg yolk clean up The organic extracts were loaded onto Isolute® Si 500 mg/10 ml (Biotage AB, Uppsala, Sweden) previously conditioned with 5 ml dichloromethane. The cartridges were washed with 5 ml hexane, and OTA was eluted with 5 ml tetrahydrofuran/hexane 1:3 (v/v). Finally the fraction collected was evaporated under vacuum (Univapo Martinsried/Munich, Germany), the dry residue was reconstituted with 750 μl of methanol and 20 μl injected into HPLC. 2.7. Solvents and reagents The chemicals and solvents used for OTA extraction and for sample extract clean up were ACS grade (citric acid, dichloromethane, hexane, diethyl ether, acetone) or HPLC grade (water, tetrahydrofurane, methanol). The solvents used for HPLC analysis (water, acetonitrile, isopropyl alcohol, acetic acid) were HPLC grade. All chemicals and solvents were purchased from Mallinckrodt Baker B.V. (Deventer, The Netherlands). OTA standard was purchased from Sigma-Aldrich Co. (St Louis, MO, USA). 2.8. Chromatographic apparatus The HPLC system was consisted of a 126 Solvent Delivery System and a 507 autosampler (Beckman, San Ramon, CA, USA) fitted with a 20 μl loop, equipped with a fluorometric detector (Jasco 821 FP, USA). Fluorescence excitation and emission wavelengths were 340 nm and 460 nm, respectively. The system was interfaced, via “32 Karat” software (Beckman, San Ramon, CA, USA), to a personal computer for the control of instruments, data acquisition, and processing. The HPLC column was a C18 Onyx Monolithic column 100 mm × 4.6 mm (Phenomenex, Torrance, CA, USA) (white egg and yolk egg analyses) coupled in sequence to a C18 Chromolith Performance RP-18e column 100 mm × 4.6 mm (Merck, Darmstad, Germany) for liver, kidney, and bile analyses. The chromatographic conditions were the same for all the considered matrixes. Chromatographic separation was achieved in isocratic condition. The mobile phase (flow rate 1 ml/min) was a mixture of 68% of water/isopropyl alcohol/acetonitrile/acetic acid 1% (79:7:7:7 v/v) and 32% of acetonitrile. 2.9. Method validation Starting from 1 mg OTA (Sigma-Aldrich Co., St. Louis, MO, USA) dissolved in 2 ml ethanol, a stock standard solution 500 μg/ml was set up and stored at −20 °C. From stock standard solutions, working standard solutions in methanol were set up to generate reference curves. These curves were composed of several standards (1, 5, 10, 20, 50 ng/ml) for bile quantification and 1, 2, 5, 8, 10 ng/ml for liver, kidney, white egg, and yolk egg.
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In order to prepare the calibration curves for bile, liver, kidney, white egg, and yolk egg, aliquots of blank samples were spiked with OTA stock standard solution at the same concentrations reached in reference curves. The samples were extracted and purified using the protocol described earlier for unknown samples. Calibration curves were analyzed using the least squares linear regression analysis and determination coefficient (R2) was calculated. The OTA concentrations in the unknown samples were calculated from their peak area using the slope and intercept of the calibration curves. A complete validation of the analytical procedure for extraction and quantification of OTA was performed in accordance with the guidelines on validation of analytical procedures suggested by the European Agency for Evaluation of the Medicinal Products (EMEA) and Committee for Veterinary Medicinal Products (CVMP) (1998). Specificity, related to the absence of interfering substances under the experimental conditions, was determined by calculating the mean values (±standard deviation) of the retention time of OTA present in direct standards and in unknown samples. Mycotoxin recovery was established by comparison of the peak areas obtained from blank samples spiked with OTA and the peak areas of reference standards at the same concentration level. Percentages of recovery, accuracy and precision were obtained in the range 1–50 ng/ml of OTA for bile and 1–10 ng/ml for liver, kidney, white egg, and yolk egg. The limit of quantification (LOQ) of the methods was the lowest concentration of the calibration curves of the mycotoxin. The limit of detection (LOD) was established on the basis of a signal-tonoise ratio of 3 at the mycotoxin retention time. This study was performed according to ISO 9001 requirements. 3. Results During the 6 weeks of the experiment all hens were healthy without observable adverse clinical symptoms. The calibration curves were linear by the high determination coefficient of the regression line for OTA in the considered concentration range (R2 always >0.99). The OTA mean recovery percentages were 78.25 ± 7.12%, 91.95 ± 7.89%, 82.21 ± 3.75%, 65.82 ± 2.66% and 69.70 ± 9.36% for bile, kidney, liver, white egg and yolk egg respectively. At the considered experimental conditions the specificities of the methods were acceptable. Retention time for OTA was 10.19 ± 0.30 minutes in liver and kidney analysis, 9.55 ± 0.13 minutes in bile analysis, 5.93 ± 0.08 in white egg analysis and 5.46 ± 0.08 in yolk egg analysis. No interfering substances in any of the matrices were observed at the OTA retention time. Representative HPLC-FD chromatograms of a blank bile sample (A), a bile spiked sample (50 ng/ml) (B), and a bile sample after administration of contaminated feed (C) are shown in Fig. 1. LOD were 0.5 ng/g for liver, kidney, white egg, yolk egg, and 0.3 ng/ml for bile; the LOQ was 1.0 ng/g for all matrices. OTA bile concentrations are summarized in Table 2. The mean (±S.D.) levels of OTA detected in D1 and D2 groups were respectively 6.37 ± 5.45 ng/ml and 139.47 ± 83.45 ng/ml for bile, 0.45 ng/g and 1.04 ± 0.63 ng/g for kidney; in D2 group only one liver sample resulted positive (0.47 ng/g). No OTA residues were detected in egg white and egg yolk samples from both experimental and control groups. 4. Discussion The results of egg analyses in the two experimental groups D1 and D2 show that levels of OTA were below LOD. Similar results were reported in Italy after the administration of higher dose of OTA (Bozzo et al., 2011). Detectable levels of OTA were found in eggs of
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Fig. 1. Chromatogram of (A) blank bile sample, (B) bile spiked sample (50 ng/ml) and (C) bile sample after administration of contaminated feed.
Table 2 Ochratoxin A levels in bile, kidney and liver of laying hens (mean values ± S.D.). Hens No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 mean ±S.D.
Bile (ng/ml)
Kidney (ng/g)
Liver (ng/g)
D0
D1
D2
D0
D1
D2
D0
D1
D2
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. – –
5.96 7.08 4.97 9.76 3.01 n.d. 4.21 5.96 2.59 4.71 21.78 3.12 14.02 1.73 6.71 6.37 ±5.45
183.53 346.84 288.09 99.73 138.01 50.08 112.27 100.07 94.39 65.27 179.27 170.44 99.13 68.12 96.95 139.47 ±83.45
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. – –
n.d. n.d. n.d. n.d. 0.45 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.45 –
0.96 n.d. n.d. 0.58 1.05 n.d. 0.55 0.92 0.88 0.62 2.71 1.79 0.93 0.98 0.48 1.04 ±0.63
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. – –
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. – –
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.47 n.d. n.d. n.d. n.d. n.d. 0.47 –
D0: control group; D1: first experimental group, received 10 μg/kg diet of OTA; D2: second experimental group, received 200 μg/kg diet of OTA. n.d.: not detectable (below LOD). LOD was 0.5 ng/g for liver and kidney and 0.3 ng/ml for bile; LOQ was 1.0 ng/g for all matrixes.
hens fed 10 mg/kg (Juszkiewicz et al., 1982) but not 1 mg/kg (Piskorska-Pliszczyn´ska and Juszkiewicz, 1990). Denli et al. (2008) did not detect residues of OTA above the detection limit (0.05 μg/kg) in the eggs after administration of 2 mg/kg of OTA in feed. A natural incidence of ochratoxin A in eggs was recently reported in Pakistan (Iqbal et al., 2014), where 28 out of 35 eggs had a high level of OTA ranged from LOD (0.06 ng/g) to 2.98 ng/g. However, a conflicting report regarding the presence of OTA in eggs was found in literature (European Food Safety Authority (EFSA), 2004). A strong correlation between OTA concentration in feed and its residues in different animal tissues was reported by several authors (Bozzo et al., 2008; Duarte et al., 2011). Some studies were conducted
in poultry and pigs for the measurements of OTA in different tissue samples, mainly the edible tissues such as kidney, liver and muscle (Birò et al., 2002; Denli and Perez, 2010; Malagutti et al., 2005). However, no single study used the bile of poultry to estimate the presence and the level of OTA. Some studies mentioned the excretion of OTA in the bile of rats (Kumagai, 1985; Li et al., 2000). In the present study, feeding laying hens two diets contaminated with 10 and 200 μg OTA/kg caused higher levels of OTA in bile than in kidney and liver (Table 2). In fact, our results show that feeding a low concentration 10 μg/kg diet of OTA to laying hens, the mycotoxin can be detected only in bile (6.37 ± 5.45 ng/ml), while the levels of OTA in kidney and liver are below LOD. Feeding laying hens a higher level of OTA (200 μg/kg) caused a very high concentration of OTA in bile (139.47 ± 83.45 ng/ml) versus lower levels found in kidney (1.04 ± 0.63 ng/g) and in the only one positive sample of liver (0.47 ng/g). Both doses of OTA in the two experimental groups showed no clinical or subclinical signs in laying hens. Several studies reported that urinary and fecal excretion of OTA are the main routes of plasma clearance for the toxin, and the contribution of each excretory route is influenced by the dose, the route of administration and the enterohepatic circulation of OTA (Ringot et al., 2006). Some authors suggested that other routes of OTA elimination such as biliary excretion in poultry should be considered (Birò et al., 2002). Nip and Chu (1979) reported that in goat 53% of OTA is excreted in feces. In calves more than one-half of the intravenously administered OTA was excreted in feces (Sreemannarayana et al., 1988). While Xiao et al. (1991) demonstrate that less than 10% of intravenously administered OTA was excreted in the feces of sheep and that was hydrolyzed to OTα. Some data about the excretion of OTA in milk of cows and sows (Mortensen et al., 1983; Ribelin et al., 1978) are available, but no data about biliary excretion of OTA in laying hens or in poultry are reported. Our study shows a high level of biliary excretion in laying hens since high concentrations of OTA were found in the bile after administration of two different doses of the toxin. Our data also show that in the group of laying hens that received the 200 μg/kg diet (D2 group), the
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average level of OTA in bile (139.47 ng/ml) was 0.697% of the daily dose (20 μg) of the administered toxin, while in kidney the average level of OTA (1.04 ng/g) was only 0.005% of the daily dose of the administered toxin, and OTA concentration in the only positive liver sample (0.47 ng/g) was 0.002% of the daily dose of the administered toxin. In the group of laying hens which received 10 μg/kg diet (D1 group), the average level of OTA in bile (6.37 ng/ ml) was 0.637% of the daily dose (1 μg) of the administered toxin, while only 0.045% appeared in the only positive kidney sample (0.45 ng/ml) and no residues of OTA in liver. Giving the fact that the overall rate of absorption of ochratoxin A in chickens after oral administration is approximately 40% (Galtier et al., 1981) and comparing the concentrations detected in the analyzed matrices with the daily dose of the adsorbed toxin, in the D2 group, the average level of OTA in bile is 1.743% of the daily dose of the adsorbed toxin (8 μg), while 0.013% appears in kidney and 0.005% in liver. In D1 group, the average levels of OTA in bile and in kidney are 1.592% and 0.112% respectively of the daily dose of the adsorbed toxin (0.4 μg). Data obtained in this experiment suggest that OTA biliary excretion plays an important role in laying hens. The average OTA levels detected in bile samples were higher than in other matrices analyzed or even observed in similar study (Bozzo et al., 2008) and resulted more obvious and reliable than clinical or biochemical signs of ochratoxicosis. Therefore, these results suggest the suitability of using bile as a matrix for screening and diagnosing measurements of OTA in laying hens. Firstly because OTA was found highly concentrated in bile, more than in kidney and liver which are generally considered the tissues of choice for the evaluation of animal tissues intended for human consumption. Moreover, the removal of gallbladder during the slaughtering process is easy, fast, and it does not entail any economic loss due to the sampling of edible tissues. Finally we can state that the HPLC-FD analysis of bile for the detection of OTA is fast and cheap. In particular, the sample preparation procedure used in our laboratory is a simple liquid– liquid extraction that does not provide a passage of clean up with SPE or immunoaffinity columns. On the contrary, the same analysis of other tissues needs more steps (homogenization, filtration, and clean up) and consequently the procedure takes longer and is more expensive. 5. Conclusion Our results show that the ratio between OTA concentration in bile of laying hens and the quantity of ingested OTA is constant. If this is confirmed by further studies, the analysis of bile might be useful to predict the amount of ingested OTA by poultry. For the future we propose to study the residual kinetics of OTA in bile and in edible tissues during and after administration of different levels of contaminated diets. References Abramson, D., Mills, J.T., Boycott, B.R., 1983. Mycotoxins and mycoflora in animal feedstuff in Western Canada. Canadian Journal of Comparative Medicine 47, 23–26. Bata, A., Glavits, R., Vanyi, A., Salyi, G., 1996. Important mycotoxicosis in poultry. Hungarian Veterinary Journal 5, 395–408. Birò, K., Solti, L., Barna-Vetrò, I., Bagò, G., Glàvits, R., Szabò, E., et al., 2002. Tissue distribution of ochratoxin A as determined by HPLC and ELISA and histopathological effects in chickens. Avian Pathology 31, 141–148. Bozzo, G., Ceci, E., Bonerba, E., Desantis, S., Tantillo, G., 2008. Ochratoxin A in laying hens: high-performance liquid chromatography detection and cytological and histological analysis of target tissues. Journal of Applied Poultry Research 1, 151–156. Bozzo, G., Bonerba, E., Ceci, E., Colao, V., Tantillo, G., 2011. Determination of ochratoxin A in eggs and target tissues of experimentally drugged hens using HPLC–FLD. Food Chemistry 126, 1278–1282.
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Iqbal, S.Z., Nisar, S., Asi, M.R., Jinap, S., 2014. Natural incidence of aflatoxins, ochratoxin A and zearalenone in chicken meat and eggs. Food Control 43, 98–103. Juszkiewicz, T., Piskorska-Pliszczynska, J., Wisniewska, H., 1982. Ochratoxin A in laying hens: Tissue deposition and passage into eggs. In: Mycotoxins and Phycotoxins. Proceedings of the V International IUPAC Symposium. Technical University, Vienna, pp. 122–125. Kumagai, S., 1985. Ochratoxin A: plasma concentration and excretion into bile and urine in albumin-deficient rats. Food and Chemical Toxicology 23, 941–943. Li, S., Marquardt, R.R., Frohlich, A.A., 2000. Identification of ochratoxins and some of their metabolites in bile and urine of rats. Food and Chemical Toxicology 38, 141–152. Malagutti, L., Zanotti, M., Scampini, A., Sciaraffia, F., 2005. Effects of ochratoxin A on heavy pig production. Animal Research 54, 179–184. Monbaliu, S., Van Poucke, C., Detavernier, C., Dumoulin, F., Van De Velde, M., Schoeters, E., et al., 2010. Occurrence of mycotoxins in feed as analyzed by a multi-mycotoxin LC-MS/MS method. Journal of Agricultural and Food Chemistry 58, 66–71. Mortensen, H.P., Hald, B., Larsen, A.E., Madsen, A., 1983. Ochratoxin a contaminated barley for sows and piglets. Pig performance and residues in milk and pigs. Acta Agriculturae Scandinavica 33, 349–352. Nip, W.K., Chu, F.S., 1979. Fate of ochratoxin A in goats. Journal of Environmental Science and Health Part B 14, 319–333. Page, R.K., Stewart, G., Wyatt, R., Buch, P., Fletcher, O.J., Brown, J., 1980. Influence of low levels of ochratoxin A on egg production, egg shell stains and serum uric-acid levels in leghorn type hens. Avian Diseases 24, 777–780. Piskorska-Pliszczyn´ska, J., Juszkiewicz, T., 1990. Tissue deposition and passage into eggs of ochratoxin A in Japanese quail. Journal of Environmental Pathology, Toxicology and Oncology 10, 8–10. Pozzo, L., Salamano, G., Mellia, E., Gennero, M.S., Doglione, L., Cavallarin, L., et al., 2013a. Feeding a diet contaminated with ochratoxin A for broiler chickens at the maximum level recommended by the EU for poultry feeds (0.1 mg/kg). 1. Effects on growth and slaughter performance, haematological and serum traits. Journal of Animal Physiology and Animal Nutrition 97 (Suppl. 1), 13–22. Pozzo, L., Cavallarin, L., Antoniazzi, S., Guerre, P., Biasibetti, E., Capucchio, M.T., et al., 2013b. Feeding a diet contaminated with ochratoxin A for broiler chickens at the maximum level recommended by the EU for poultry feeds (0.1 mg/kg). 2. Effects on meat quality, oxidative stress, residues and histological traits. Journal of Animal Physiology and Animal Nutrition 97 (Suppl. 1), 23–31. Ribelin, W.E., Fukushima, K., Still, P.E., 1978. The toxicity of ochratoxin A to ruminants. 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Ringot, D., Changoa, A., Schneider, Y.J., Larondelle, Y., 2006. Toxicokinetics and toxicodynamics of ochratoxin A, an update. Chemico-Biological Interactions 159, 18–46. Roth, A., Chakor, K., Creppy, E.E., Kane, A., Röschenthaler, R., Dirheimer, G., 1988. Evidence of an enterohepatic circulation of ochratoxin A in mice. Toxicology 48, 293–308. Shephard, G.S., Thiel, P.G., Sydenham, E.W., Alberts, J.F., 1994. Biliary excretion of the mycotoxin fumonisin B1 in rats. Food and Chemical Toxicology 32, 489–491. Sreemannarayana, O., Frohlich, A.A., Vitti, T.G., Marquardt, R.R., Abramson, D., Phillips, G.D., 1988. Studies of the tolerance and disposition of ochratoxin A in young calves. Journal of Animal Science 66, 1703–1711.
Stoev, S.D., 2010. Studies on some feed additives and materials giving partial protection against the suppressive effect of ochratoxin A on egg production of laying hens. Research in Veterinary Science 88, 486–491. Visconti, A., Bottalico, A., 1983. High levels of ochratoxin A and B in moldy bread responsible for mycotoxicosis in farm animals. Journal of Agricultural and Food Chemistry 31, 1122–1123. Xiao, H., Marquardt, R.R., Frohlich, A.A., Phillips, G.D., Vitti, T.G., 1991. Effect of hay and a grain diet on the bioavailability of ochratoxin A in the rumen of sheep. Journal of Animal Science 69, 3715–3723.
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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Biliary ochratoxin A as a biomarker of ochratoxin exposure in laying hens: An experimental study after administration of contaminated diets Sara Armorini a, Khaled Mefleh Al-Qudah b, Alberto Altafini a, Anna Zaghini a, Paola Roncada a,* a Department of Veterinary Medical Sciences, School of Agriculture and Veterinary Medicine- Alma Mater Studiorum, University of Bologna, via Tolara di Sopra 50, Ozzano Emilia (BO) 40064, Italy b Department of Veterinary Clinical Sciences, Jordan University of Science and Technology, Irbid 22110, Jordan
A R T I C L E
I N F O
Article history: Received 8 September 2014 Accepted 1 March 2015 Keywords: Ochratoxin A Bile HPLC Laying hens
A B S T R A C T
To evaluate the levels of ochratoxin (OTA) in kidney, liver and bile of laying hens, forty-five laying hens were enrolled in this study and divided into three equal groups: a control group D0, and two experimental groups, D1 fed with 10 μg/kg OTA diet and D2 fed with 200 μg/kg OTA diet for 6 weeks. Kidneys, livers, and bile from all hens were collected and analyzed by HPLC method for the presence of OTA. Eggs collected 2 days before the start of the experiment and 2 days after its end were also analyzed for the presence of OTA. Results show a relevant biliary excretion of the mycotoxin, with high levels of OTA in the bile after administration of the toxin. OTA level in eggs was below the limit of detection (LOD). These results suggest the suitability of using bile as a matrix for screening measurements of OTA in laying hens. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Ochratoxin A is a natural poison contaminating a large variety of plants and leading to the appearance of residues in animal origin products (Gourama and Bullerman, 1995; Stoev, 2010). Ochratoxins are fungal secondary metabolites produced by several species of Aspergillus and Penicillium, predominantly Aspergillus ochraceus and Penicillium verrucosum. The most important toxin is ochratoxin A (OTA), which is more toxic and more frequently found than ochratoxin B or ochratoxin C (Ringot et al., 2006). Crops used for animal feed can be easily contaminated by fungi during growth, harvest, or storage, resulting in the occurrence of mycotoxins (Monbaliu et al., 2010). OTA occurs as a common contaminant of cereals, peanuts, as well as soya, coffee and cocoa beans (Birò et al., 2002). In poultry as well as in other animal species, OTA appears in different body tissues including kidneys, liver, muscles, gastrointestinal organs, lymphoid tissues, skeletal system, blood and reproductive tract (Dwivedi and Burns, 1984; European Food Safety Authority (EFSA), 2004; Pozzo et al., 2013a, 2013b). The feed of monogastric animals such as poultry and pigs can be monitored for the presence of mycotoxins and allow
* Corresponding author. Department of Veterinary Medical Sciences, School of Agriculture and Veterinary Medicine- Alma Mater Studiorum, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia (BO), Italy. Tel.: +39 0512097511; fax: +39 051799511. E-mail address: [email protected] (P. Roncada). http://dx.doi.org/10.1016/j.rvsc.2015.03.004 0034-5288/© 2015 Elsevier Ltd. All rights reserved.
the formulation of diets that are well tolerated by animals (Fink-Gremmels, 2008). The European Commission (2006) recommendation 2006/576/EC indicates that the maximum tolerable level of ochratoxin A in poultry feeds is 0.1 mg/kg. Several experimental feeding trials with OTA contaminated feeds have indicated its harmful effects on growing chicks and its potential risk for the poultry industry. Avian nephropathy associated with OTA exposure was reported in Denmark by Elling et al. (1975), which detected microscopical renal changes in four out of 14 kidneys. In the USA five independent episodes of ochratoxicosis in turkeys, two episodes in laying hens, and two episodes in broiler chickens were reported by Hamilton et al. (1982); the histopathology of affected birds always reveals on edema and necrosis of the proximal tubules of the kidneys with no changes in the liver or other organs. A lethal gastroenteritis was reported in Italy due to feeding chickens, rabbits and dogs with moldy bread (Visconti and Bottalico, 1983). Mycotoxicosis in farm animals and laying hens were reported in western Canada during a 3 year study (Abramson et al., 1983). Other reports of ochratoxicosis in lying birds came from Hungary (Bata et al., 1996), the USA (Howell, 1981), and Great Britain (Page et al., 1980). The common clinical symptoms observed in ochratoxicosis are retardation of growth, dullness, huddling, decrease appetite, reduced feed intake, weakness and diarrhea (Dwivedi and Burns, 1984); OTA also affects egg production, fertility and hatchability (Page et al., 1980). However, there are no pathognomonic symptoms of ochratoxicosis and its diagnosis seems to be difficult.
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Few works in literature focused on OTA toxicokinetics and residues in chicken (Bozzo et al., 2008; Dietrich et al., 2005; Galtier, 1978; Galtier et al., 1981; Pozzo et al., 2013b), but to date, the biliary route of excretion was not considered to detect the level of OTA; this might be because urinary excretion seems to be a primary route of elimination, beside that bile is not an edible tissue. Shephard et al. (1994) reported that biliary excretion is a major route of elimination of the mycotoxin fumonisin B1 from blood circulation and only small amount of toxin appeared to be absorbed from the intestinal tract of rats. The enterohepatic circulation of OTA has been demonstrated in studies in rodents (Kumagai, 1985; Roth et al., 1988) and newborn calves (Sreemannarayana et al., 1988). All these studies showed secondary distribution peaks of OTA in the serum and intestinal contents and concluded to a biliary secretion of the toxin followed by its reabsorption from the intestine. Reabsorption of OTA from the intestine back to the blood circulation, as a result of biliary recycling, promotes the systemic redistribution of OTA to different tissues (Ringot et al., 2006). The aim of our study was to evaluate the levels of OTA in bile, kidney, liver, and eggs in laying hens after the administration of 10 and 200 μg/kg of controlled diets. Secondly, we try to demonstrate the suitability of using bile for the early detection of OTA in poultry.
Table 1 Ingredients of diet fed to laying hens for 6 weeks during the entire experiment.a Ingredient
%
Maize ground Soya bean ground meal Barley ground Dry alfa-alfa Calcium carbonate Corn gluten Soya oil Calcium phosphate Sodium chloride Premixb
52.60 20.00 6.00 5.00 8.40 4.00 1.50 1.60 0.40 0.50
a Chemical composition of basal diet: dry matter 88.8%, crude protein 16.4%, crude fat 5%, crude fiber 3.81%, ash 12.65%, calcium (Ca) 3.70%, phosphorus (P) 0.6%, sodium (Na) 0.18%. b Composition per kilogram of premix: 2,600,000 IU vitamin A; 600,000 IU vitamin D3; 4000 mg α-tocopherol acetate; 400 mg vitamin K3; 1000 mg vitamin B1; 6000 mg vitamin B2; 4 mg vitamin B6; 400 mg vitamin B12; 6000 mg vitamin PP; 20 mg biotin; 100 mg folic acid; 2000 mg pantothenic acid; 100,000 mg choline; 10,000 mg Zn; 10,000 mg Fe; 12,000 mg Mn; 1000 mg Cu; 200 mg I; 100 mg Co; 40 mg Se; 6000 mg total xanthophyll; 1000 mg BHT (butylated hydroxytoluene).
2. Materials and methods
Committee of the Alma Mater Studiorum-Università di Bologna, in accordance with specific legislation concerning protection of animals used for scientific purposes.
2.1. Animals and sampling
2.2. Liver and kidney extraction
Forty-five Warren (Isa Brown) 30-week-old laying hens obtained from a commercial facility, with an average weight of 1.80 ± 0.035 kg, were randomly divided into 2 experimental groups (D1 and D2), and the control group (D0), each comprising of 15 animals. All the birds were housed in individual cages with separate feeders, kept under identical environmental conditions (temperature, humidity) and held under 16 h of light and 8 h of dark per day. All birds were monitored daily for any adverse clinical symptoms. After a period of acclimatization (45 days) and 20 days after eggs laying beginning, hens received the following diets for 6 weeks: a control group (D0) was fed with a control diet free of OTA; an experimental group (D1) was fed with OTA 10 μg/kg experimentally contaminated feed; the third experimental group (D2) was fed with OTA 200 μg/kg experimentally contaminated feed. Before feeding, the basal diet was tested by means of an ELISA (Veratox® Ochratoxin, Neogen Corporation, Lansing, Michigan, USA) to ensure that it contained no residual ochratoxin A. The experimental diets were obtained by mixing the basal diet (Table 1) with naturally contaminated ground maize containing 280 mg/kg of OTA. Ten or 0.5 g of contaminated maize was mixed with 14 kg of basal diet to obtain OTA levels of 200 μg/kg and 10 μg/kg respectively. The two experimental groups were fed with 100 g/day/animal of contaminated diet. Consequently each animal of the D1 group received a total of 1 μg of OTA daily, while each animal of the D2 group received 20 μg of OTA daily. For evaluation of OTA, 20 eggs randomly collected from each experimental group, 10 eggs collected the first 2 days before the start of treatment and 10 eggs at the last 2 days of the experiment were analyzed. White and yolk were separately collected and subjected to freeze drying process. Finally, after being vacuum-packed, the samples were kept at −20 °C until their analysis by using HPLC-FD method. At the end of the experimental period, laying hens were euthanized by vertebral dislocation followed by exsanguination. From all the birds (controls and OTA administered groups) kidney, liver and samples of bile (directly from gallbladder) were collected and kept frozen (−20 °C) until their HPLC-FD analysis. The plan of this study was authorized by Italian Ministry of Health on the basis of a previous judgment issued by the Ethic Scientific
Ochratoxin A extraction from liver and kidney was executed on 2.5 g of tissue thawed, acidified with 2.5 ml citric acid 30% water solution and extracted with 15 ml dichloromethane. This mixture was homogenized (3 min) with Ultra-Turrax T25 (IKA Labortechnik, Staufen, Germany) then rinsed with 2.5 ml dichloromethane. The samples were sonicated (30 min), cooled on ice (10 min) and, finally, centrifuged (2600g for 10 min). Fourteen milliliters of organic layer (equal to 2 g of liver or kidney) was subjected to clean up. 2.3. Liver and kidney clean up The organic extracts were loaded onto Isolute® Si 500 mg/10 ml (Biotage AB, Uppsala, Sweden) previously conditioned with 5 ml dichloromethane. The cartridges were washed with 5 ml hexane. OTA in liver and kidney samples was eluted differently. In liver samples, OTA was eluted with 5 ml tetrahydrofuran/ hexane 1:3 (v/v) followed by 5 ml diethyl ether/hexane 1:2 (v/v), while in kidney samples, 5 ml tetrahydrofuran/hexane 1:3 (v/v) was used only for washing the SPE columns and the elution took place with 5 ml diethyl ether/hexane 1:2 (v/v) followed by 5 ml acetone/ dichloromethane 1:4 (v/v). For both matrices, the fraction collected was reduced to dryness by means Univapo (Univapo Martinsried/Munich, Germany), the residue was dissolved in 500 μl methanol and 20 μl was injected into HPLC. 2.4. Bile extraction OTA extraction was performed on 200 μl of bile added to 200 μl of citric acid 30% water solution. After the addition of 2 ml of dichloromethane, the mixture was extracted in a blender for 30 minutes, cooled on ice (10 min) and thereafter centrifuged (2600g for 10 min). The lower organic layer (1600 μl) was transferred into a conical tube and evaporated under vacuum (Univapo Martinsried/Munich, Germany). The dry residue was reconstituted with 800 μl of methanol and 20 μl injected into HPLC.
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2.5. Egg white and egg yolk extraction OTA extraction from egg white and egg yolk was executed on 1 g of lyophilised sample, rehydrated with 8 ml water, acidified with 5 ml citric acid 30% water solution and extracted with 20 ml dichloromethane in a blender for 30 minutes. The samples were sonicated (15 min), cooled on ice (10 min) and, finally, centrifuged (2600g for 10 min). From this step on, white egg samples and yolk egg samples were differently processed. Fifteen milliliters of organic layer (equal to 0.75 g of sample) was collected, and subject to clean up (yolk egg samples) or evaporated under vacuum (white egg samples). In the last case, the dry residue was reconstituted with 750 μl of methanol and 20 μl injected into HPLC. 2.6. Egg yolk clean up The organic extracts were loaded onto Isolute® Si 500 mg/10 ml (Biotage AB, Uppsala, Sweden) previously conditioned with 5 ml dichloromethane. The cartridges were washed with 5 ml hexane, and OTA was eluted with 5 ml tetrahydrofuran/hexane 1:3 (v/v). Finally the fraction collected was evaporated under vacuum (Univapo Martinsried/Munich, Germany), the dry residue was reconstituted with 750 μl of methanol and 20 μl injected into HPLC. 2.7. Solvents and reagents The chemicals and solvents used for OTA extraction and for sample extract clean up were ACS grade (citric acid, dichloromethane, hexane, diethyl ether, acetone) or HPLC grade (water, tetrahydrofurane, methanol). The solvents used for HPLC analysis (water, acetonitrile, isopropyl alcohol, acetic acid) were HPLC grade. All chemicals and solvents were purchased from Mallinckrodt Baker B.V. (Deventer, The Netherlands). OTA standard was purchased from Sigma-Aldrich Co. (St Louis, MO, USA). 2.8. Chromatographic apparatus The HPLC system was consisted of a 126 Solvent Delivery System and a 507 autosampler (Beckman, San Ramon, CA, USA) fitted with a 20 μl loop, equipped with a fluorometric detector (Jasco 821 FP, USA). Fluorescence excitation and emission wavelengths were 340 nm and 460 nm, respectively. The system was interfaced, via “32 Karat” software (Beckman, San Ramon, CA, USA), to a personal computer for the control of instruments, data acquisition, and processing. The HPLC column was a C18 Onyx Monolithic column 100 mm × 4.6 mm (Phenomenex, Torrance, CA, USA) (white egg and yolk egg analyses) coupled in sequence to a C18 Chromolith Performance RP-18e column 100 mm × 4.6 mm (Merck, Darmstad, Germany) for liver, kidney, and bile analyses. The chromatographic conditions were the same for all the considered matrixes. Chromatographic separation was achieved in isocratic condition. The mobile phase (flow rate 1 ml/min) was a mixture of 68% of water/isopropyl alcohol/acetonitrile/acetic acid 1% (79:7:7:7 v/v) and 32% of acetonitrile. 2.9. Method validation Starting from 1 mg OTA (Sigma-Aldrich Co., St. Louis, MO, USA) dissolved in 2 ml ethanol, a stock standard solution 500 μg/ml was set up and stored at −20 °C. From stock standard solutions, working standard solutions in methanol were set up to generate reference curves. These curves were composed of several standards (1, 5, 10, 20, 50 ng/ml) for bile quantification and 1, 2, 5, 8, 10 ng/ml for liver, kidney, white egg, and yolk egg.
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In order to prepare the calibration curves for bile, liver, kidney, white egg, and yolk egg, aliquots of blank samples were spiked with OTA stock standard solution at the same concentrations reached in reference curves. The samples were extracted and purified using the protocol described earlier for unknown samples. Calibration curves were analyzed using the least squares linear regression analysis and determination coefficient (R2) was calculated. The OTA concentrations in the unknown samples were calculated from their peak area using the slope and intercept of the calibration curves. A complete validation of the analytical procedure for extraction and quantification of OTA was performed in accordance with the guidelines on validation of analytical procedures suggested by the European Agency for Evaluation of the Medicinal Products (EMEA) and Committee for Veterinary Medicinal Products (CVMP) (1998). Specificity, related to the absence of interfering substances under the experimental conditions, was determined by calculating the mean values (±standard deviation) of the retention time of OTA present in direct standards and in unknown samples. Mycotoxin recovery was established by comparison of the peak areas obtained from blank samples spiked with OTA and the peak areas of reference standards at the same concentration level. Percentages of recovery, accuracy and precision were obtained in the range 1–50 ng/ml of OTA for bile and 1–10 ng/ml for liver, kidney, white egg, and yolk egg. The limit of quantification (LOQ) of the methods was the lowest concentration of the calibration curves of the mycotoxin. The limit of detection (LOD) was established on the basis of a signal-tonoise ratio of 3 at the mycotoxin retention time. This study was performed according to ISO 9001 requirements. 3. Results During the 6 weeks of the experiment all hens were healthy without observable adverse clinical symptoms. The calibration curves were linear by the high determination coefficient of the regression line for OTA in the considered concentration range (R2 always >0.99). The OTA mean recovery percentages were 78.25 ± 7.12%, 91.95 ± 7.89%, 82.21 ± 3.75%, 65.82 ± 2.66% and 69.70 ± 9.36% for bile, kidney, liver, white egg and yolk egg respectively. At the considered experimental conditions the specificities of the methods were acceptable. Retention time for OTA was 10.19 ± 0.30 minutes in liver and kidney analysis, 9.55 ± 0.13 minutes in bile analysis, 5.93 ± 0.08 in white egg analysis and 5.46 ± 0.08 in yolk egg analysis. No interfering substances in any of the matrices were observed at the OTA retention time. Representative HPLC-FD chromatograms of a blank bile sample (A), a bile spiked sample (50 ng/ml) (B), and a bile sample after administration of contaminated feed (C) are shown in Fig. 1. LOD were 0.5 ng/g for liver, kidney, white egg, yolk egg, and 0.3 ng/ml for bile; the LOQ was 1.0 ng/g for all matrices. OTA bile concentrations are summarized in Table 2. The mean (±S.D.) levels of OTA detected in D1 and D2 groups were respectively 6.37 ± 5.45 ng/ml and 139.47 ± 83.45 ng/ml for bile, 0.45 ng/g and 1.04 ± 0.63 ng/g for kidney; in D2 group only one liver sample resulted positive (0.47 ng/g). No OTA residues were detected in egg white and egg yolk samples from both experimental and control groups. 4. Discussion The results of egg analyses in the two experimental groups D1 and D2 show that levels of OTA were below LOD. Similar results were reported in Italy after the administration of higher dose of OTA (Bozzo et al., 2011). Detectable levels of OTA were found in eggs of
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Fig. 1. Chromatogram of (A) blank bile sample, (B) bile spiked sample (50 ng/ml) and (C) bile sample after administration of contaminated feed.
Table 2 Ochratoxin A levels in bile, kidney and liver of laying hens (mean values ± S.D.). Hens No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 mean ±S.D.
Bile (ng/ml)
Kidney (ng/g)
Liver (ng/g)
D0
D1
D2
D0
D1
D2
D0
D1
D2
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. – –
5.96 7.08 4.97 9.76 3.01 n.d. 4.21 5.96 2.59 4.71 21.78 3.12 14.02 1.73 6.71 6.37 ±5.45
183.53 346.84 288.09 99.73 138.01 50.08 112.27 100.07 94.39 65.27 179.27 170.44 99.13 68.12 96.95 139.47 ±83.45
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. – –
n.d. n.d. n.d. n.d. 0.45 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.45 –
0.96 n.d. n.d. 0.58 1.05 n.d. 0.55 0.92 0.88 0.62 2.71 1.79 0.93 0.98 0.48 1.04 ±0.63
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. – –
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. – –
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.47 n.d. n.d. n.d. n.d. n.d. 0.47 –
D0: control group; D1: first experimental group, received 10 μg/kg diet of OTA; D2: second experimental group, received 200 μg/kg diet of OTA. n.d.: not detectable (below LOD). LOD was 0.5 ng/g for liver and kidney and 0.3 ng/ml for bile; LOQ was 1.0 ng/g for all matrixes.
hens fed 10 mg/kg (Juszkiewicz et al., 1982) but not 1 mg/kg (Piskorska-Pliszczyn´ska and Juszkiewicz, 1990). Denli et al. (2008) did not detect residues of OTA above the detection limit (0.05 μg/kg) in the eggs after administration of 2 mg/kg of OTA in feed. A natural incidence of ochratoxin A in eggs was recently reported in Pakistan (Iqbal et al., 2014), where 28 out of 35 eggs had a high level of OTA ranged from LOD (0.06 ng/g) to 2.98 ng/g. However, a conflicting report regarding the presence of OTA in eggs was found in literature (European Food Safety Authority (EFSA), 2004). A strong correlation between OTA concentration in feed and its residues in different animal tissues was reported by several authors (Bozzo et al., 2008; Duarte et al., 2011). Some studies were conducted
in poultry and pigs for the measurements of OTA in different tissue samples, mainly the edible tissues such as kidney, liver and muscle (Birò et al., 2002; Denli and Perez, 2010; Malagutti et al., 2005). However, no single study used the bile of poultry to estimate the presence and the level of OTA. Some studies mentioned the excretion of OTA in the bile of rats (Kumagai, 1985; Li et al., 2000). In the present study, feeding laying hens two diets contaminated with 10 and 200 μg OTA/kg caused higher levels of OTA in bile than in kidney and liver (Table 2). In fact, our results show that feeding a low concentration 10 μg/kg diet of OTA to laying hens, the mycotoxin can be detected only in bile (6.37 ± 5.45 ng/ml), while the levels of OTA in kidney and liver are below LOD. Feeding laying hens a higher level of OTA (200 μg/kg) caused a very high concentration of OTA in bile (139.47 ± 83.45 ng/ml) versus lower levels found in kidney (1.04 ± 0.63 ng/g) and in the only one positive sample of liver (0.47 ng/g). Both doses of OTA in the two experimental groups showed no clinical or subclinical signs in laying hens. Several studies reported that urinary and fecal excretion of OTA are the main routes of plasma clearance for the toxin, and the contribution of each excretory route is influenced by the dose, the route of administration and the enterohepatic circulation of OTA (Ringot et al., 2006). Some authors suggested that other routes of OTA elimination such as biliary excretion in poultry should be considered (Birò et al., 2002). Nip and Chu (1979) reported that in goat 53% of OTA is excreted in feces. In calves more than one-half of the intravenously administered OTA was excreted in feces (Sreemannarayana et al., 1988). While Xiao et al. (1991) demonstrate that less than 10% of intravenously administered OTA was excreted in the feces of sheep and that was hydrolyzed to OTα. Some data about the excretion of OTA in milk of cows and sows (Mortensen et al., 1983; Ribelin et al., 1978) are available, but no data about biliary excretion of OTA in laying hens or in poultry are reported. Our study shows a high level of biliary excretion in laying hens since high concentrations of OTA were found in the bile after administration of two different doses of the toxin. Our data also show that in the group of laying hens that received the 200 μg/kg diet (D2 group), the
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average level of OTA in bile (139.47 ng/ml) was 0.697% of the daily dose (20 μg) of the administered toxin, while in kidney the average level of OTA (1.04 ng/g) was only 0.005% of the daily dose of the administered toxin, and OTA concentration in the only positive liver sample (0.47 ng/g) was 0.002% of the daily dose of the administered toxin. In the group of laying hens which received 10 μg/kg diet (D1 group), the average level of OTA in bile (6.37 ng/ ml) was 0.637% of the daily dose (1 μg) of the administered toxin, while only 0.045% appeared in the only positive kidney sample (0.45 ng/ml) and no residues of OTA in liver. Giving the fact that the overall rate of absorption of ochratoxin A in chickens after oral administration is approximately 40% (Galtier et al., 1981) and comparing the concentrations detected in the analyzed matrices with the daily dose of the adsorbed toxin, in the D2 group, the average level of OTA in bile is 1.743% of the daily dose of the adsorbed toxin (8 μg), while 0.013% appears in kidney and 0.005% in liver. In D1 group, the average levels of OTA in bile and in kidney are 1.592% and 0.112% respectively of the daily dose of the adsorbed toxin (0.4 μg). Data obtained in this experiment suggest that OTA biliary excretion plays an important role in laying hens. The average OTA levels detected in bile samples were higher than in other matrices analyzed or even observed in similar study (Bozzo et al., 2008) and resulted more obvious and reliable than clinical or biochemical signs of ochratoxicosis. Therefore, these results suggest the suitability of using bile as a matrix for screening and diagnosing measurements of OTA in laying hens. Firstly because OTA was found highly concentrated in bile, more than in kidney and liver which are generally considered the tissues of choice for the evaluation of animal tissues intended for human consumption. Moreover, the removal of gallbladder during the slaughtering process is easy, fast, and it does not entail any economic loss due to the sampling of edible tissues. Finally we can state that the HPLC-FD analysis of bile for the detection of OTA is fast and cheap. In particular, the sample preparation procedure used in our laboratory is a simple liquid– liquid extraction that does not provide a passage of clean up with SPE or immunoaffinity columns. On the contrary, the same analysis of other tissues needs more steps (homogenization, filtration, and clean up) and consequently the procedure takes longer and is more expensive. 5. Conclusion Our results show that the ratio between OTA concentration in bile of laying hens and the quantity of ingested OTA is constant. If this is confirmed by further studies, the analysis of bile might be useful to predict the amount of ingested OTA by poultry. For the future we propose to study the residual kinetics of OTA in bile and in edible tissues during and after administration of different levels of contaminated diets. References Abramson, D., Mills, J.T., Boycott, B.R., 1983. Mycotoxins and mycoflora in animal feedstuff in Western Canada. Canadian Journal of Comparative Medicine 47, 23–26. Bata, A., Glavits, R., Vanyi, A., Salyi, G., 1996. Important mycotoxicosis in poultry. Hungarian Veterinary Journal 5, 395–408. Birò, K., Solti, L., Barna-Vetrò, I., Bagò, G., Glàvits, R., Szabò, E., et al., 2002. Tissue distribution of ochratoxin A as determined by HPLC and ELISA and histopathological effects in chickens. Avian Pathology 31, 141–148. Bozzo, G., Ceci, E., Bonerba, E., Desantis, S., Tantillo, G., 2008. Ochratoxin A in laying hens: high-performance liquid chromatography detection and cytological and histological analysis of target tissues. Journal of Applied Poultry Research 1, 151–156. Bozzo, G., Bonerba, E., Ceci, E., Colao, V., Tantillo, G., 2011. Determination of ochratoxin A in eggs and target tissues of experimentally drugged hens using HPLC–FLD. Food Chemistry 126, 1278–1282.
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