สััตวแพทย ์มหานครสาร · 2014. 8. 19. · column (iac;...

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การปนเปื้อนสารพิษอะฟลาทอกซินและออกคราทอกซิน เอ ในอาหารสัตว์เลี้ยงในประเทศไทย

ณัฐสิทธิ์ ตันสกุล1,# กมลชัย ตรงวานิชนาม1 ปารียา อุดมกุศลศรี1 และศศธิร ลิ้มสุวรรณ2

1ภาควิชาเภสัชวิทยา คณะสัตวแพทยศาสตร์ มหาวิทยาลัยเกษตรศาสตร์ กรุงเทพฯ

2บริษัท ศูนย์วิทยาศาสตร์เบทาโกร จํากัด คลองหลวง ปทุมธานี

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คําสําคัญ: อะฟลาทอกซิน ออกคราทอกซิน เอ อาหารสัตว์ อาหารสัตว์เลี้ยง ประเทศไทย #ผู้รับผิดชอบบทความ สัตวแพทย์มหานครสาร. 2557. 9(1): 1-9. E-mail address: [email protected]

Natthasit Tansakul et al. / J. Mahanakorn Vet. Med. 2014. 9(1): 1-9.

2

A Survey of Aflatoxins and Ochratoxin A Contamination in Pet Food in Thailand

Natthasit Tansakul1,#, Kamolchai Trongvanichnam1, Pareeya Udomkusonsri1 and

Sasithorn Limsuwan2

1Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, THAILAND 2Betagro Science Center Co., Ltd., KlongNueng, KlongLuang, Pathumthani, THAILAND

Abstract: Presence of mycotoxin in feed ingredient is commonly known. Exposure of mycotoxin may generate a serious concern for animal health. In tropical zone, mycotoxin that produced by post harvest fungi such as aflatoxin and ochratoxin A is frequently found in animal feed. In general, mycotoxin contamination in animal feed are mostly report specific on livestock feed rather than pet food. This study was aimed to monitor the mycotoxins contamination in commercial dry dog and cat food in Thailand. The mycotoxins were determined by the HPLC system with post-column bromination and fluorescence detection. As presented, the result clearly showed that there was no sample free from aflatoxin contamination. The total AF level was found at trace level. In addition, 7 samples of dog food and 9 samples of cat food were contaminated with OTA. The mean values of OTA-positive pet food samples were 2.46-14.04 μg/kg for dog and cat food.

Keywords: Aflatoxin, Ochartoxin A, Feed, Pet food, Thailand #Corresponding author J. Mahanakorn Vet. Med. 2014. 9(1): 1-9. E-mail address: [email protected]

Introduction

Mycotoxin, secondary fungal metabolite, is naturally and unavoidable toxicant. It has significant impact on human and animal health. Hence, consumers are pay attention to food and feed safety. Mycotoxin contamination in animal feeding stuff is of interested worldwide and has

been well reviewed (Leung et. al., 2006; Tansakul, 2012). In tropical zone, fungal is commonly present in agricultural commodities and thus could be a contaminant in food and feed (Martins et al., 2003). Mycotoxin in which produced by post-harvest fungi such as aflatoxin (AF) and ochratoxin A (OTA) is usually found in


3

foodstuff. Aflatoxin is a carcinogenic and mutagenic metabolite produced by the fungal strains of Aspergillus spp. As known, A. flavus produces aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2), whereas A. parasiticus produces those aflatoxin B serie as well as aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) (Shephard, 2009).

Aflatoxin is metabolized extensively by the liver where it is the majority of the toxic effects. Exposure can be acute or chronic depend on toxin concentrations and show variable clinical signs such as weakness, diarrhea, alternate liver function and blood chemistry profiles. Dog is one particularly specie sensitive to the aflatoxin effect (Scudamore, et al., 1997). Another interested toxin produced by post-harvest fungi is ochratoxin which has 3 members designated as A, B and C. Among those, OTA is considered to be the most toxic. It has wide range of toxicological effects associated with the inhibition of the protein synthesis due to its competition with phenylalanine. Kidney is the main target of OTA toxicity in mammalian species. Moreover, OTA is classified as a possible carcinogen in category Group 2B. As known, acute or chronic exposure of mycotoxin may generate a serious concern for animal health. Hence, monitoring on feeding stuff is necessary to prevent a cause of animal health hazard.

Contamination of AF and OTA in pet food have been reported worldwide e.g. Austria (Razzazi et al., 2001; Böhm et al., 2010), Turkey (Gunsen et al., 2002), Brazil (Maia et al., 2002; Campos et. al., 2009; Wouters et al., 2013). However, little scientific information is available for prevalence of mycotoxin in pet food in Thailand. Therefore, AF contamination in 30 samples from ten different market brands of dog food was tested. In addition, total of 60 commercial market samples of dog and cat foods were monitored for OTA contamination. The mycotoxins levels were carried out by the HPLC fluorescence detection system couple with post-column bromination.

Materials and Methods

Total of 90 dry pet food samples were collected for 2 separated analysis; AF contamination in dry dog food (n=30) and OTA contamination in dry dog food (n=30) and dry cat food (n=30). All samples were blind sampling taken from Pet shop’s shelf in central region of Thailand during April 2011- February 2012, kept in -80°C and tested within 3 months after collected. Mixed aflatoxin standard (HPLC grade) of AFB1, AFB2, AFG1 and AFG2 from Supelco® (Bellefonte PA, USA) and OTA standard from Sigma® were used. The method was partially validated. Briefly, the calibration of five


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standard solutions concentration range from 0.25-10 ng/ml was prepared and duplicate injection daily. The detection limit (LOD) and the limit of quantification (LOQ) were determined at signal-to-noise ratio of 3:1 and signal-to-noise ratio of 10:1, respectively. Repeatability, reproducibility and recovery were conducted in triplicate (n=3) with control spiked with 10 μg/kg of AFs and 5 μg/kg of OTA. Both mycotoxins were carried out by HPLC fluorescence detector with post-column bromination (a signal enhancement of Kobracell) and separated on analytical column (ACE C-18, 150 mm×4.6 mm, 5 μm, ACE, Aberdeen, UK). Analysis functions were obtained by linear regression (R2) of peak areas on standard concentrations.

For AF analysis, HPLC was performed under an excitation and emission wavelengths at 365 and 440 nm, respectively. Extraction and clean-up procedure were modified following Böhm’s method (Böhm et al., 2010).The analysis was run at flow rate of 1 ml/min using mixture of water:methanol:acetonitrile (62:22:16, v/v/v) where 119 mg of potassium bromide (KBr) and 350 μl of 4 M HNO3 were added to 1 L of mobile phase. Sample preparation for AF analysis was done by adding 30 ml of acetonitrile:deionized water (60:40, v/v) into 10 g of each sample and mixed for 30 min. Then, the matrix was filtrated though paper

filters and kept into polypropylene test tube. Thereafter, the immuno-affinity column (IAC; AflaCLEAN™, LCTech GmbH, Germany) was used for clean-up procedure by dilute 3 ml of extracted solution with 30 ml PBS. Then the diluted samples were applied on IAC following the construction. Each eluate was put into glass test tube and allowed to dry under gentle nitrogen stream. The sample was reconstituted with 1 ml mobile phase and kept in an aliquot at -20°C pending injected into the HPLC system.

For OTA analysis, the method was performed following Reiter et al., 2011. The system was done by running with mobile phase consisted of methanol : acetonitrile : 3.3% aqueous solution of glacial acetic acid (35:35:30, v/v/v) at flow rate 0.8 ml/min. OTA was detected by the HPLC with fluorescence detector at an excitation wavelength of 333 nm and an emission wavelength of 467 nm. For extraction and clean-up process, added 100 ml of acetonitrile : deionized water (60:40, v/v)into 25 g of sample then mixed by stirring for 30 min and filtered through a filterpaper. Thereafter, 4 ml of the liquid was diluted with 50 ml PBS containing 0.5%Tween 20 and applied on the IAC (OtaCLEAN™, LCTech GmbH, Germany). Then passed 10 ml distilled water to wash the column and eluted the substance with 2 ml of methanol. After well evaporated, the

sample mobile at -20analysis

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7

OTA is typically found not only in cereal grains but also in animal tissue because of its long half-life and bound tightly with plasma proteins. A former report, Razzazi et al., (2001), detected mean value of OTA residue at 0.85 μg/kg from 16 out of 26 cat kidney. In Korea, it was documented that 3 dogs died from renal failure that feed contaminated with OTA might be involved (Jeong et al., 2006). Moreover, there was evidence in Thailand that 4 out of 50 dogs died from chronic renal failure related to OTA exposure (Pothimongkolkul et al., 2006).

The current results showed incidence of OTA remains in cat food more than in dog food. However, dog food appears to be contaminated with OTA higher level than in cat food. The present study showed lower incidence of OTA contamination in pet food but higher level than that found in previous reports (González et al., 1997; Razzazi et al., 2001). In the EU, the maximum level for OTA in complementary and complete feeding stuffs for pigs is set at 50 μg/kg. So far, no level limit stipulated for OTA in animal feed in Thailand. Unfortunately, the samples did not simultaneously analyze the co-occurrence of both mycotoxins. However, no information concerning the synergistic or chronic effects of low mycotoxin exposure on health hazard for dog and cat (Böhm, 2006). Therefore, detection of aflatoxin and

OTA in pet food and majority organ such as liver and kidney samples should further be investigated. Regarding the fact that mycotoxin is naturally contaminated in agricultural products including pet food, the effectiveness of its control depends on a rigorous monitoring program.

Acknowledgements A study funding was partially

supported by UNOVET NETWORK CO.LTD., Thailand. Chemical agents were kindly provided by the University of Veterinary Vienna, Vienna, Austria. The authors declare no conflict of interest.

References Böhm, J. 2006. Effects of mycotoxins on

domestic pet species. In: Laue, D.K. and Tucker, L.A. (eds). Recent advances in pet nutrition. Nottingham University Press, Nottingham, U.K, 169-192.

Böhm, J., Koinig, L., Razzazi-Fazeli, E., Blajet-Kosicka, A., Twaruzek, M., Grajewski, J. and Lang, C. 2010. Survey and risk assessment of the mycotoxins deoxynivalenol, zearalenone, fumonisins, ochratoxin A, and aflatoxins in commercial dry dog food. Mycotox. Res. 26: 147-153.


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Campos, S.G., Keller, L.M., Cavaglieri, L.R., Krüger, C., Juri, M.G.F., Dalcero, A.M., Magnoli, C.E. and Rosa, C.A.R. 2009. Aflatoxigenic fungi and aflatoxin B1 in commercial pet food in Brazil. World Mycotox. J. 2(1): 85-90.

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González, H.H.L., Martinez, E.J. and Resnik, S.L. 1997. Fungi associated with sorghum grain from Argentina. Mycopathol. 139: 35-41.

Gunsen, U. and Yaroglu, T. 2002. Aflatoxin in dog and horse feeds in Turkey. Vet. Hum. Toxicol. 44: 113-114.

Jeong, W.I., Do, S.H., Jeong, D.H., Chung, J.Y., Yang, H.J., Yuan, D.W., Hong, I.H., Park, J.K., Goo, M.J. and Jeong, K.S. 2006. Canine renal failure syndrome in three dogs. J. Vet. Sci. 7: 299-301.

Leung, M.C.K., Dı´az, L.G. and Smith, T.K. 2006. Mycotoxins in Pet Food: A Review on Worldwide Prevalence and Preventative Strategies. J. Agric. Food Chem. 54: 9623-9635

Maia, P.P. and Bastos de Siqueira, P. 2002. Occurrence of aflatoxins B1, B2, G1 and G2 in some Brazilian pet foods. Food Addit. Contam. 19(12): 1180-1183.

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Pothimongkolkul, S., Sailasuta, A. and Pattamalai, B. 2006. The Relationship Between Ochratoxin a Residue in Kidneys and Nephropathic Lesions in Canine Chronic Renal Failure. The proceeding of 32nd annual conference of Thai Veterinary Medical Association. 31-32.

Razzazi, E., Böhm, J., Grajewski, J., Szczepaniak, K., Kübber-Heiss, A.J. and Iben, C.H. 2001. Residues of ochratoxin A in pet foods, canine and feline kidneys. J. Anim. Physiol. Anim. Nutr. (Berl). 85(7-8): 212-216.

Reiter, E.V., Cichna-Markl, M., Tansakul, N., Shim, W.B., Chung, D.H., Zentek, J. and Razzazi-Fazeli, E. 2011. Sol-gel immunoaffinity chromatography for the cleanup of ochratoxin A contaminated grains. J. Chromatogr. A. 1218(42): 7627-7633.

Scudamore, K.A., Hetmanski, M.T., Nawaz, S., Naylor, J. and Ainbird, S. 1997. Determination of mycotoxins in pet foods sold for domestic pets and wild birds using linked- column immunoassay clean-up and HPLC. Food Addit. Contam. 14: 175-182.


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Sharma, M. and Marquez, C. 2001. Determination of aflatoxins in domestic pet foods (dog and cat) using immunoaffinity column and HPLC. Anim. Feed Sci. Technol. 93: 109-114.

Shephard, G.S. 2009. Aflatoxin analysis at the beginning of the twenty-first century. Anal. Bioanal. Chem. 395(5): 1215-1224.

Streit, E., Schatzmayr, G., Tassis, P., Tzika, E., Marin, D., Taranu, I., Tabuc, C., Nicolau, A., Aprodu, I., Puel, O. and Oswald, I.P. 2012. Current situation of mycotoxin contamination and co-occurrence in animal feed--focus on Europe. Toxins. 4(10): 788-809.

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