Sains Malaysiana 52(3)(2023): 821-835

http://doi.org/10.17576/jsm-2023-5203-11

Molecular Characterization of Ochratoxin a Producing Indigenous Aspergillus Strains from Poultry Feed in Pakistan

(Pencirian Molekul Okratoksin Penghasil Strain Aspergillus Asli daripada Makanan Poltri di Pakistan)

GULL NAZ¹, AFTAB AHMAD ANJUM^1*, TEHREEM ALI¹, MUHAMMAD NAWAZ¹, SANAULLAH IQBAL², MATEEN ABBAS³ & RABIA MANZOOR¹

¹Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan

²Department of Food Science and Human Nutrition, University of Veterinary and Animal Sciences, Lahore, Pakistan

 ³Quality Operations Laboratory, Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan

Received: 12 July 2022/Accepted: 17 January 2023

Abstract

Ochratoxin A (OTA) is nephrocarcinogenic and immunosuppressive toxin and OTA producing molds contaminate the food crops. Isolation and identification of ochratoxin producing fungi was carried out from poultry feed samples (n=120) followed by preliminary confirmation through macroscopic and microscopic characteristics. Purified fungal isolates identified as Aspergillus 1842(91.68%) followed by Penicillium 91 (4.53%), Mucor 52 (2.58), Alternaria 7 (0.35%), Cladosporium 6 (0.29%), Fusarium 4 (0.199%) and un-identified (07). OTA production was confirmed through thin layer chromatography (TLC) followed by high performance liquid chromatography (HPLC). Only 41 isolates (2.22%) out of 1842 Aspergillus isolates were able to produce toxin. At genetic level, characterization was performed through polymerase chain reaction (PCR) using species specific gene primers. From 41 isolates 27, 9 and 5 were characterized as Aspergillus terreus, Aspergillus parasiticus, and Aspergillus ochraceus respectively. Physical and chemical factors were optimized for OTA production. Under the effect of 37 °C temperature and 7.5 pH of Sabouraud dextrose broth (SDB) medium, higher toxin (969.45±.03 µg/mL) production was observed from ASPO-6 isolate. ASPO-4 isolate produce higher toxin amount in SDB medium with supplementation of maize 5%, wheat 1% and rice 3%. OTA stability was determined by adjusting standard concentration of 100 µg/mL in organic solvents (chloroform, acetonitrile and methanol) and organic solids. Least percentage log reduction in OTA concentration and stability of OTA was observed in opaque vials with chloroform and sucrose and transparent vials with sucrose after 6 months. OTA can be used as indigenous standard for identification of OTA from field samples.

Keywords: Aspergillus; Ochratoxin A; polymerase chain reaction; stability; thin layer chromatography

Abstrak

Okratoksin A (OTA) ialah toksin nefrokarsinogenik dan imunosupresif dan kulapuk penghasilan OTA mencemari tanaman makanan. Pengasingan dan pengenalpastian kulat penghasilan okratoksin telah dijalankan daripada sampel makanan poltri (n=120) diikuti dengan pengesahan awal melalui ciri makroskopik dan mikroskopik. Pengasingan kulat tulen dikenal pasti sebagai Aspergillus 1842(91.68%) diikuti oleh Penicillium 91(4.53%), Mucor 52(2.58), Alternaria 7(0.35%), Cladosporium 6(0.29%), Fusarium 4(0.199%) dan tidak dikenal pasti (07). Pengeluaran OTA telah disahkan melalui kromatografi lapisan nipis (TLC) diikuti oleh kromatografi cecair prestasi tinggi (HPLC). Hanya 41 pencilan (2.22%) daripada 1842 pencilan Aspergillus dapat menghasilkan toksin. Pada peringkat genetik, pencirian dilakukan melalui tindak balas rantai polimerase (PCR) menggunakan primer gen khusus spesies. Daripada 41 pencilan 27, 9 dan 5 masing-masing dicirikan sebagai Aspergillus terreus, Aspergillus parasiticus dan Aspergillus ochraceus. Faktor fizikal dan kimia telah dioptimumkan untuk pengeluaran OTA. Di bawah kesan suhu 37 °C dan 7.5 pH medium Sabouraud dextrose broth (SDB), pengeluaran toksin yang lebih tinggi (969.45±.03 µg/mL) diperhatikan daripada pencilan ASPO-6. Pengasingan ASPO-4 menghasilkan jumlah toksin yang lebih tinggi dalam medium SDB dengan tambahan jagung 5%, gandum 1% dan beras 3%. Kestabilan OTA ditentukan dengan melaraskan kepekatan piawai 100 µg/mL dalam pelarut organik (kloroform, asetonitril dan metanol) dan pepejal organik. Peratusan log pengurangan paling sedikit dalam kepekatan OTA dan kestabilan OTA diperhatikan dalam botol legap dengan kloroform dan sukrosa dan botol lutsinar dengan sukrosa selepas 6 bulan. OTA boleh digunakan sebagai piawaian asli untuk mengenal pasti OTA daripada sampel lapangan.

Kata kunci: Aspergillus; kestabilan; kromatografi lapisan nipis; Okratoksin A; tindak balas rantai polimerase

REFERENCES

Aboagye-Nuamah, F., Kwoseh, C.K. & Maier, D.E.  2021. Toxigenic mycoflora, aflatoxin and fumonisin contamination of poultry feeds in ghana. Toxicon 198(2): 164-170.

Algammal, A.M., Elsayed, M.E., Hashem, H.R., Ramadan, H., Sheraba, N.S., El-Diasty, E.M., Abbas, S.M. & Hetta, H.F. 2021. Molecular and HPLC-based approaches for detection of aflatoxin B 1 and ochratoxin A released from toxigenic Aspergillus species in processed meat. BMC Microbiology 21(1): 1-10.

Antonio-Bautista, A., Vazquez-Badillo, M., Galindo-Cepeda, M.E., Flores-Naveda, A., Benítez-Neri, E., Sánchez-Arizpe, A. & Arispe-Vázquez, J. 2021. Effect of the temperature and relative humidity in stored sotol (Dasylirion cedrosanum Trel.) seeds on fungi biodiversity. Journal of Phytology 13: 16-20.

Armutcu, C., Uzun, L. & Denizli, A. 2018. Determination of Ochratoxin A traces in foodstuffs: Comparison of an automated on-line two-dimensional high-performance liquid chromatography and off-line immunoaffinity-high-performance liquid chromatography system. Journal of Chromatography A 1569: 139-148.

Becker, P., Normand, A.C., Vanantwerpen, G., Vanrobaeys, M., Haesendonck, R., Vercammen, F., Stubbe, D., Piarroux, R. & Hendrickx, M. 2019. Identification of fungal isolates by MALDI-TOF mass spectrometry in veterinary practice: Validation of a web application. Journal of Veterinary Diagnostic Investigation 31(3): 471-474.

Bhat, R., Rai, R.V. & Karim, A.A. 2010. Mycotoxins in food and feed: Present status and future concerns. Comprehensive Reviews in Food Science and Food Safety 9(1): 57-81.

Cervini, C., Verheecke-Vaessen, C., Ferrara, M., García-Cela, E., Magistà, D., Medina, A., Gallo, A., Magan, N. & Perrone, G. 2021. Interacting climate change factors (CO₂ and temperature cycles) effects on growth, secondary metabolite gene expression and phenotypic ochratoxin A production by Aspergillus carbonarius strains on a grape-based matrix. Fungal Biology 125(2): 115-122.

Danbappa, A.A.R., Alhassan, K.A. & Shah, M.M. 2018. Isolation and identification of microbial contaminants associated with commercial poultry feeds. Journal of Applied and Advanced Research 3(5): 142-147.

Dudakova, A., Spiess, B., Tangwattanachuleeporn, M., Sasse, C., Buchheidt, D., Weig, M., Groß, U. & Bader, O. 2017. Molecular tools for the detection and deduction of azole antifungal drug resistance phenotypes in Aspergillus species. Clinical Microbiology Reviews 30(4): 1065-1091.

Ehrlich, K.C., Kobbeman, K., Montalbano, B.G. & Cotty, P.J. 2007. Aflatoxin-producing Aspergillus species from Thailand. International Journal of Food Microbiology 114(2): 153-159.

Freire, L., Passamani, F.R.F., Thomas, A.B., Nassur, R.D.C.M.R., Silva, L.M., Paschoal, F.N., Pereira, G.E., Prado, G. & Batista, L.R. 2017. Influence of physical and chemical characteristics of wine grapes on the incidence of Penicillium and Aspergillus fungi in grapes and ochratoxin A in wines. International Journal of Food Microbiology 241: 181-190.

Gaur, A. & Adholeya, A. 2000. Effects of the particle size of soil-less substrates upon AM fungus inoculum production. Mycorrhiza 10(1): 43-48.

Ghaemmaghami, S.S., Modirsaneii, M., Khosravi, A.R. & Razzaghi-Abyaneh, M. 2016. Study on mycoflora of poultry feed ingredients and finished feed in Iran. Iranian Journal of Microbiology 8(1): 47.

Ghaemmaghami, S.S. & Nowroozi, H. 2018. Toxigenic fungal contamination for assessment of poultry feeds: Mashed vs. Pellet. Iranian Journal of Toxicology 12(5): 5-10.

Gherbawy, Y.A., El-Dawy, E.G. & Mohamed, H.M. 2019. Molecular characterization of ochratoxin A-producing fungi isolated from poultry feed. Journal of Experimental and Applied Animal Sciences 3(1): 15-27.

Greco, M.V., Franchi, M.L., Golba, S.L.R., Pardo, A.G. & Pose, G.N. 2014. Mycotoxins and mycotoxigenic fungi in poultry feed for food-producing animals. The Scientific World Journal 2014: 1-9.

Hassan, Z.U., Al-Thani, R.F., Migheli, Q. & Jaoua, S. 2018. Detection of toxigenic mycobiota and mycotoxins in cereal feed market. Food Control 84: 389-394.

Hendrix, J.D., Zhang, X., Campbell, Y.L., Zhang, L., Siberio, L., Cord, C.L., Silva, J.L., Goddard, J., Kim, T., Phillips, T.W. & Schilling, M.W. 2018. Effects of temperature, relative humidity, and protective netting on Tyrophagus putrescentiae (Schrank) (Sarcoptiformes: Acaridae) infestation, fungal growth, and product quality of dry cured hams. Journal of Stored Products Research 77: 211-218.

Henry, T., Iwen, P.C. & Hinrichs, S.H. 2000. Identification of Aspergillus species using     internal     transcribed     spacer regions 1 and 2. Journal of Clinical Microbiology 38: 1510-1515. 

Hussaini, A.M., Timothy, A.G., Olufunmilayo, H.A., Ezekiel, A.S. & Godwin, H.O. 2009. Fungi and some mycotoxins found in mouldy Sorghum in Niger State, Nigeria. World Journal of Agricultural Sciences 5(1): 5-17.

IARC. 1993. Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans 56. Geneva: WHO.

Ibrahim, M., Kabir, J., Kwanashie, C., Salawudeen, M. & Joshua, Z. 2017. Occurrence of mycotoxigenic fungi in poultry feeds at live-bird markets, Zaria, Nigeria. Sokoto Journal of Veterinary Sciences 15: 53-59.

Illueca, F., Vila-Donat, P., Calpe, J., Luz, C., Meca, G. & Quiles, J.M. 2021. Antifungal activity of biocontrol agents in vitro and potential application to reduce mycotoxins (Aflatoxin B1 and Ochratoxin A). Toxins 13(11): 752.

Kehinde, M., Oluwafemi, F., Itoandon, E., Orji, F. & Ajayi, O. 2014. Fungal profile and aflatoxin contamination in poultry feeds sold in Abeokuta, Ogun State, Nigeria. Nigerian Food Journal 32(1): 73-79.

Kholife, M., Moawad, A., Diab, A.M. & El-keredy, A. 2019. Mycological examination of fish feed stuff with special reference to mycotoxin production. Slovenian Veterinary Research 56(22): 303-312.

Kiseleva, M., Chalyy, Z., Sedova, I. & Aksenov, I. 2020. Stability of mycotoxins in individual stock and multi-analyte standard solutions. Toxins 12(2): 94.

Koh, C.Y., Modahl, C.M., Kulkarni, N. & Kini, R.M. 2018. Toxins are an excellent source of therapeutic agents against cardiovascular diseases. Seminars in Thrombosis and Hemostasis 6: 1-15.

Lappa, I.K., Kizis, D. & Panagou, E.Z. 2017. Monitoring the temporal expression of genes involved in ochratoxin a production of Aspergillus carbonarius under the influence of temperature and water activity. Toxins 9(10): 296.

Lasram, S., Barketi, A., Mliki, A. & Ghorbel, A. 2012. Growth and ochratoxin A production by Aspergillus carbonarius at different pHs and grape maturation stages. Letters in Applied Microbiology 54(5): 418-424.

Liazid, A., Palma, M., Brigui, J. & Barroso, C.G. 2007. Investigation on Ochratoxin A stability using different extraction techniques. Talanta 71(2): 976-980.

Maciorowski, K., Herrera, P., Jones, F., Pillai, S. & Ricke, S. 2007. Effects on poultry and livestock of feed contamination with bacteria and fungi. Animal Feed Science and Technology 133(1-2): 109-136.

Mantle, P. 2002. Risk assessment and the importance of ochratoxins. International Biodeterioration & Biodegradation 50(3-4): 143-146.

Moez, E., Noel, D., Brice, S., Benjamin, G., Pascaline, A. & Didier, M. 2020. Aptamer assisted ultrafiltration cleanup with high performance liquid chromatography-fluorescence detector for the determination of OTA in green coffee. Food Chemistry 310: 125851.

Nasaruddin, N., Jinap, S., Samsudin, N.I., Kamarulzaman, N.H. & Sanny, M. 2021. Prevalence of mycotoxigenic fungi and assessment of aflatoxin contamination: A multiple case study along the integrated corn‐based poultry feed supply chain in Malaysia. Journal of the Science of Food and Agriculture 101(5): 1812-1821.

Nwiyi, P.O., Nwabuko, C.N., Ndubueze, A. & Christain, O. 2019. Isolation and identification of mycotoxigenic organisms in poultry feed from selected locations in Abia State, Nigeria. Asian Journal of Research in Animal and Veterinary Sciences 3(4): 1-9.

Oliveira, G.,  Evangelista, S.R., Passamani, F.R.F., Santiago, W.D., Cardoso, M. das-G. & Batista, L.R. 2019. Influence of temperature and water activity on Ochratoxin A production by Aspergillus strain in coffee south of Minas Gerais/Brazil. Lebensmittel-Wissenschaft & Technologie 102: 1-7.

Pardo, E., Marín, S., Ramos, A. & Sanchis, V. 2005. Effect of water activity and temperature on mycelial growth and ochratoxin A production by isolates of Aspergillus ochraceus on irradiated green coffee beans. Journal of Food Protection 68(1): 133-138.

Pena, A., Cerejo, F., Lino, C. & Silveira, I. 2005. Determination of ochratoxin A in Portuguese rice samples by high performance liquid chromatography with fluorescence detection. Analytical and Bioanalytical Chemistry 382(5): 1288-1293.

Petzinger, E. & Weidenbach, A. 2002. Mycotoxins in the food chain: the role of ochratoxins. Livestock Production Science 76(3): 245-250.

Pleadin, J. 2015. Mycotoxins in grains and feed: Contamination and toxic effect in animals. Biotechnology in Animal Husbandry 31(4): 441-456.

Rafique, A., Javed, M. & Ali, S.S. 2018. Contamination of commercial poultry feeds and feed ingredients with aspergillus flavus and aflatoxin b1 in punjab pakistan. Journal of Animal and Plant Sciences 28(4): 1021-1026.

Ren, P., Ahearn, D. & Crow, S.J. 1999. Comparative study of Aspergillus mycotoxin production on enriched media and construction material. Journal of Industrial Microbiology and Biotechnology 23(3): 209-213.

Rosa, C.D.R., Palacios, V., Combina, M., Fraga, M., Rekson, A.D.O., Magnoli, C. &  Dalcero, A.M. 2002. Potential ochratoxin A producers from wine grapes in Argentina and Brazil. Food Additives & Contaminants 19(4): 408-414.

Saddozai, A.A., Raza, S. & Saleem, S.A. 2012. Microbial count and shelf life of strawberry juice. Pakistan Journal of Agricultural Research 25(3): 218-223.

Saleemi, M.K., Khan, M.Z., Khan, A. & Javed, I. 2010. Mycoflora of poultry feeds and mycotoxins producing potential of Aspergillus species. Pakistan Journal of Botany 42(1): 427-434.

Sana, S., Anjum, A.A., Yaqub, T., Nasir, M., Ali, M.A. & Abbas, M. 2019. Molecular approaches for characterization of aflatoxin producing Aspergillus flavus isolates from poultry feed. Pakistan Veterinary Journal 39(2): 169-174.

Sivakumar, V., Singaravelu, G. & Sivamani, P. 2014. Isolation, characterization and growth optimization of toxicogenic molds from different animal feeds in Tamilnadu. International Journal of Current Microbiology and Applied Sciences 3(9): 430-445.

Trail, F., Mahanti, N. & Linz, J. 1995. Molecular biology of aflatoxin biosynthesis. Microbiology 141(4): 755-765.

Wang, Y., Liu, F., Wang, L., Wang, Q., Selvaraj, J.N., Zhao, Y., Wang, Y., Xing, F. & Liu, Y. 2018. pH-signaling transcription factor AopacC regulates ochratoxin A biosynthesis in Aspergillus ochraceus. Journal of Agricultural and Food Chemistry 66(17): 4394-4401.

Zeng, D., Lin, Z., Zeng, Z., Fang, B., Li, M., Cheng, Y-H. & Sun, Y. 2019. Assessing global human exposure to T-2 toxin via poultry meat consumption using a lifetime physiologically based pharmacokinetic model. Journal of Agricultural and Food Chemistry 67(5): 1563-1571.

^*Corresponding author; email: aftab.anjum@uvas.edu.pk