Original Scientific Article Feed contamination can occur at every stage of the feed chain, including manufacturing, storage, transport, and utilization. Ensuring feed safety is fundamental for livestock health, animal performance, and food safety throughout the feed-to-food chain. The study aimed to make qualitative and quantitative assessment of microbiological and chemical hazards in animal feed in North Macedonia from 2018 to 2022. A total of 1,629 feed samples were analysed for heavy metals, mycotoxins, and coccidiostats, whereas 598 samples were examined for microbial contamination. The results indicate a significant prevalence of heavy metals, with lead (Pb) detected in 100% of the samples, although mostly within regulatory limits. Mycotoxins, notably ochratoxin A (OTA) and aflatoxin B1 (AFB1 ), were also identified, with non-compliance in 10.88% of cattle feed for AFB1 . Coccidiostat residues exceeded the maximum levels in 4.6% of the samples. Microbiological analysis revealed that 1.34% of feed samples were contaminated with Salmonella spp., and 3.8% tested positive for sulphitereducing clostridia. The findings on both microbiological and chemical hazards indicate their potential to threaten the feed safety chain. Accordingly, this study emphasizes the need for continuous comprehensive feed safety monitoring and the enforcement of stringent safety regulations to safeguard animal and public health in North Macedonia. https://macvetrev.mk/LoadArticlePdf/413 2025-06-10 i xv https://doi.org/10.2478/macvetrev-2025-0023 feed safety contaminants coccidiostats microbiological hazards North Macedonia Elizabeta Dimitrieska Stojkovikj false 1 Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Biljana Stojanovska Dimzoska false 1 Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Vangelica Enimiteva false 1 Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Ljupco Angelovski false 1 Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Zehra Hajrulai Musliu false 1 Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Gordana Ilievska false 1 Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Dushica Koceva false 1 Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Aleksandra Angeleska false 1 Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Sandra Mojsova false 1 Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Sharma, V., Sharma, S., Datt, C. (2015). Potential hazards in animal feeds: safety and regulatory review. Indian J Anim Nutr. 32(3): 242-262. 1 Dorne, JLC, Mand Fink-Gremmels, J. (2013). Human and animal health risk assessment of chemicals in the food chain: comparative aspects and future perspectives. Toxicol Appl Pharmacol. 270(3): 187-195. PMid:22484160 2 https://doi.org/10.1016/j.taap.2012.03.013 D'Mello, JPF (2004). Microbiology of animal feeds assessing quality and safety of animal feeds, FAO, Rome, pp. 89 ̶105. 3 Radovanov-Pelagić, V., Jurić, V., Kunc, V., Ristić, M., Koljajić, V. (1999). Relationship between microflora and amount of mycotoxins in animal feed. Contemporary Agriculture, Novi Sad, 48(1-2): 281-284. [In Serbian] 4 Hinton, M. (1993). Spoilage and pathogenic microorganisms in animal feed. Int Biodeterior Biodegrad. 32(1-3): 67-74. 5 https://doi.org/10.1016/0964-8305(93)90040-9 Ricke, SC (2018). Chapter. 8. Feed Hygiene. In: J. Dewulf, F. Van Immerseel (Eds.), Biosecurity in animal production and veterinary medicine from principles to practice (pp. 177-209). Leuven, Belgium: ACCO (Academische Coöperative Vennootschap cvba) 6 https://doi.org/10.1079/9781789245684.0177 Đorđević, N., Dinić, B. (2007). Animal Feed. Cenzone tech-Europe, Aranđelovac [In Serbian] 7 WHO [World Health Organization]. (2020). Compendium of WHO and other UN guidance on health and environment. Chapter 5. Chemicals. c2023 [cited 2023 October 31]. https://cdn.who.int/media/docs/default-source/who-compendiumon-health-and-environment/who_compendium_chapter5_01092021.pdf?sfvrsn=20ca418_5 8 Wu, X., Cobbina, SJ, Mao, G., Xu, H., Zhang, Z., Yang, L. (2016). A review of toxicity and mechanisms of individual and mixtures of heavy metals in the environment. Environ Sci Pollut Res. 23(9): 8244-8259. PMid:26965280 9 https://doi.org/10.1007/s11356-016-6333-x Kim, JH (2023). Determination of safe levels and toxic levels for feed hazardous materials in broiler chickens: a review. J Anim Sci Technol. 65(3): 490-510. PMid:37332288 PMCid:PMC10271926 10 https://doi.org/10.5187/jast.2023.e26 EFSA Panel on Contaminants in the Food Chain (CONTAM). (2012). Scientific opinion on the risk for public health related to the presence of methylmercury and mercury in food. EFSA J. 10(12): 2985. 11 https://doi.org/10.2903/j.efsa.2012.2985 Kos, J., Anić, M., Radić, B., Zadravec M., Janić Hajnal, E., Pleadin, J. (2023). Climate change-a global threat resulting in increasing mycotoxin occurrence. Foods 12(14): 2704. PMid:37509796 PMCid:PMC10379110 12 https://doi.org/10.3390/foods12142704 Muñoz-Solano, B., Gonzáles-Peñas, E. (2023). Co-occurrence of mycotoxins in feed for cattle, pigs, poultry, and sheep in Navara, a region of Northern Spain. Toxins (Basel). 15(3): 172. PMid:36977063 PMCid:PMC10057204 13 https://doi.org/10.3390/toxins15030172 Leggieri, MC, Toscano, P., Battilani, P. (2021). Predicted aflatoxin B1 increase in Europe due to climate change: actions and reactions at global level. Toxins 13(4): 292. PMid:33924246 PMCid:PMC8074758 14 https://doi.org/10.3390/toxins13040292 Dimitrieska-Stojković, E., Stojanovska-Dimzoska, B., Ilievska, G., Uzunov, R., Stojković, G., Hajrulai-Musliu, Z., Jankuloski D. (2016), Assessment of aflatoxin contamination in raw milk and feed in Macedonia During 2013. Food Control. 59, 201-206. 15 https://doi.org/10.1016/j.foodcont.2015.05.019 Ilievska, G., Stojanovska-Dimzoska, B., Koceva, D., Stojković, G., Angeleska, A., Dimitrieska-Stojković, E. (2022). Dietary exposure and health risk assessment of aflatoxin M1 in dairy products consumed by the population of North Macedonia, J Food Qual Hazards Control. 9(1): 14-22. 16 https://doi.org/10.18502/jfqhc.9.1.9686 Pleadin, J., Lešić, T., Milićević D., Markov, K., Šarkanj, B., Vahčić, N., Kmetič, I., Zadravec, M. (2021). Pathways of mycotoxin occurrence in meat products: a review. Processes 9(12): 2122. 17 https://doi.org/10.3390/pr9122122 Pleadin, J., Jadrić, M., Kudumija, N., Zadravec, M., Kiš, G., Mihaljević, Ž., Škrivanko, M., Samardžija, M. (2024). Zearalenone in feed, urine and meat from three pig farms in Croatia. Vet Stanica 55(1): 1-11. 18 https://doi.org/10.46419/vs.55.1.10 Roila, R., Branciari, R., Pecorelli, I., Cristofani, E., Carloni, C., Ranucci, D., Fioroni, L. (2019). Occurrence and residues concentration of coccidiostats in feed and food of animal origin; Human exposure assessment. Foods 8(10): 447. PMid:31614486 PMCid:PMC6835225 19 https://doi.org/10.3390/foods8100477 Dorne, JLCM, Fernández-Cruz, ML, Bertelsen, U., Renshaw, DW, Peltonen, K., Anadon, A., Feil, A., Sanders, P., Wester, P., Fink-Gremmels, J. (2013). Risk assessment of coccidiostats during feed cross contamination: Animal and human health aspects. Toxicol Appl Pharmacol. 270(3): 196-208. PMid:21215766 20 https://doi.org/10.1016/j.taap.2010.12.014 Clarke, L., Fodey, TL, Crooks, SRH, Moloney, M., O'Mahony, J., Delahaut, P., O'Kennedy, R., Danaher, M. (2014). A review of coccidiostats and the analysis of their residues in meat and other food. Meat Sci. 97(3): 358-374. PMid:24534603 21 https://doi.org/10.1016/j.meatsci.2014.01.004 Rulebook for the list of undesirable substances in animal food and maximum permitted level as well as critical points for conducting source identification research and the reasons for exceeding the maximum permitted level, Official Journal of RNM No. 85 from 31.03.2020. 22 Rulebook for general and specific requirements for feed safety, Official Journal of RM No. 147 from 27.11.2012. 24. ISO 4833-1:2013 Microbiology of the food chain - Horizontal method for the enumeration of microorganisms Part 1: Colony count at 30 °C by the pour plate technique. https://www.iso.org/standard/53728.html 23 ISO 4833-1:2013 Microbiology of the food chain - Horizontal method for the enumeration of microorganisms Part 1: Colony count at 30 °C by the pour plate technique. https://www.iso.org/standard/53728.html 24 ISO 21527-2:2008 Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of yeasts and molds - Part 2: Colony count technique in products with water activity less than or equal to 0.95. https://www.iso.org/standard/38276.html 25 ISO 15213:2003 Microbiology of the food chain - Horizontal method for the detection and enumeration of Clostridium spp. - Part 1: Enumeration of sulfite-reducing Clostridium spp. by colony-count technique. https://www.iso.org/standard/26852.html 26 ISO 6579-1:2017 Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 1: Detection of Salmonella spp. https://www.iso.org/standard/56712.html 27 International Organization for Standardization (1999). ISO 6496:1999 - Animal feeding stuffs - Determination of moisture and other volatile matter content. https://www.iso.org/standard/12871.html 28 CEN 2003, EN 14084, Foodstuffs - Determination of trace elements - Determination of lead, cadmium, zinc, copper and iron by atomic absorption spectrometry (AAS) after microwave digestion, European Committee for Standardization, 2003. https://standards.iteh.ai/catalog/standards/cen/446d0bd2-4c65-4d15-a586-852f4a653f70/en-14084-2003?srsltid=AfmBOooMj_Dd74K3FwTQ2dZpTyc3YFpbQBYrSf7oxKgSCl3BvXx6EhiK 29 CEN 2002, EN 13086, Foodstuffs - Determination of trace elements - Determination of mercury by cold vapor atomic absorption spectrometry (CVAAS) after pressure digestion, European Committee for Standardization, 2002. https://standards.iteh.ai/catalog/standards/cen/a62f2c3c-cbea-4861-830f-448c7d80ae75/en-13806-2002?srsltid=AfmBOoo38ECsJEEJwAKvNH8lQvcUwdQbaLTTmIplEaYXLkAZj88ot6H4 30 Stojanovska-Dimzoska, B., Hajrulai-Musliu, Z., Uzunov, R., Angeleska, A., Blagoevska, K., Crceva Nikolovska, R., Ilievska, G., Dimitrieska-Stojkovikj, E. (2022). Study on the effectiveness of a multi-toxin immunoaffinity cleanup for reliable cost-effective HPLC-FLD analysis of mycotoxins in corn based food. Maced J Chem Chem Eng. 41(1): 77-88. 31 https://doi.org/10.20450/mjcce.2022.2422 Waters Corporation (2013). The analysis of coccidiostatic agents in feed using the ACQUITY UPLC I-class and XEVO TQ-S, application note 720004769en, August 2013. c2013 [cited 2019 December 27]. https://www.waters.com/waters/library.htm?cid=10160596&lid=134757454 32 Elliott, S., Frio, A., Jarman T. (2017). Heavy metal contamination of animal feedstuffs - a new survey. J Appl Anim Nutr. 5(8): 1-15. 33 https://doi.org/10.1017/jan.2017.7 Iqbal, H., Shafique, MA, Khan, MJ (2023). Evaluation of heavy metals concentration in poultry feed and poultry products. Saudi J Med Pharm Sci. 9(7): 489-495. 34 https://doi.org/10.36348/sjmps.2023.v09i07.019 Korish, MA, Attia, YA (2020). Evaluation of heavy metal content in feed, litter, meat, meat products, liver and table eggs. Animals 10(4): 727. PMid:32331361 PMCid:PMC7222721 35 https://doi.org/10.3390/ani10040727 Hejna, M., Moscatelli, A., Onelli, E., Baldi, A., Pilu, S., Rossi, L. (2019). Evaluation of concentration of heavy metals in animal rearing system. Ital J Anim Sci. 18(1): 1372-1384. 36 https://doi.org/10.1080/1828051X.2019.1642806 Wang, H., Dong, Y., Yang, YS, Toor, G., Zhang, X. (2013). Changes in heavy metal contents in animal feeds and manures in an intensive animal production region of China. J Environ Sci. 25(12): 2435-2442. PMid:24649675 37 https://doi.org/10.1016/S1001-0742(13)60473-8 Adamse, P., Van der Fels-Klerx, HJ, de Jong, J. (2017). Cadmium, lead, mercury and arsenic in animal feed and feed materials - trend analysis of monitoring results. Food Addit Contam: Part A. 34(8): 1298-1311. PMid:28278122 38 https://doi.org/10.1080/19440049.2017.1300686 Zhang, F., Li, Y., Yang, M., Li, W. (2012). Content of heavy metals in animal feeds and manures from farms of different scales in northeast China. Int J Environ Res Publ. Health. 9(8): 2658-2668. PMid:23066389 PMCid:PMC3447579 39 https://doi.org/10.3390/ijerph9082658 Sdogati, S., Pacini, T., Bibi, R., Caporali, A., Vardini, E., Orsini, S., Ortenzi, R., Pecorelli, I. (2024). Co-occurrence of aflatoxin B1, zearalenone and ochratoxin A in feed and feed materials in Central Italy from 2018-2022. Foods 13(2): 313. PMid:38254614 PMCid:PMC10815256 40 https://doi.org/10.3390/foods13020313 Santos Pereira, C., Cunha, SC, Fernandes, JO (2019). Prevalent mycotoxins in animal feed: occurrence and analytical methods. Toxins 11(5): 290. PMid:31121952 PMCid:PMC6563184 41 https://doi.org/10.3390/toxins11050290 Pietruk, K., Olejnik, M., Jedziniak, P., Szprengier-Juszkiewicz, T. (2015). Determination of fifteen coccidiostats in feed at carry-over levels using liquid chromatography-mass spectrometry. J Pharm Biomed Anal. 112, 50-59. PMid:25958138 42 https://doi.org/10.1016/j.jpba.2015.03.019 Delahaut, P., Pierret, G., Ralet, N., Dubois, M., Gillard, N. (2010). Multi-residue method for detecting coccidiostats at carry-over level in feed by HPLC-MS/MS. Food Addit Contam A Chem Anal Control Expo Risk Assess. 27(6): 801-809. PMid:20198524 43 https://doi.org/10.1080/19440040903552408 Annunziata, L., Visciano, P., Stramenga, A., Colagrande, MN, Campana, G., Scortichini, G., Migliorati, G., Compagnone, D. (2017). Determination of regulatory ionophore coccidiostat residues in feedstuffs at carry-over levels by liquid chromatography-mass spectrometry. PLoS ONE. 12(8): e0182831. PMid:28792977 PMCid:PMC5549955 44 https://doi.org/10.1371/journal.pone.0182831 Moretti, S., Fioroni, L., Giusepponi, D., Pettinacci, L., Saluti, G., Galarini, R. (2013). Development and validation of a multiresidue liquid chromatography/tandem mass spectrometry method for 11 coccidiostats in feed. J. AOAC Int. 96(6): 1245-1257. PMid:24645501 45 https://doi.org/10.5740/jaoacint.12-440 Wojdat, E., Kwiatek, K., Kozak, M. (2005). Microbiological quality of animal feeding stuffs in Poland. Bull Vet Inst Pulawy. 49(3): 315-318. 46 Čabarkapa, I., Kokić, B., Plavšić, D., Ivanov, D., Lević, J. (2009). Microbiological safety of animal feed. Biotechnol Anim Husb. 25(5-6): 1155-1162. 47 Maciorowski, KG, Herrera, P., Jones, FT, Pillai, SD, Ricke, SC (2007). Effects on poultry and livestock of feed contamination with bacteria and fungi. Anim Feed Sci Technol. 133(1): 109-136. 48 https://doi.org/10.1016/j.anifeedsci.2006.08.006 Udhayavel, S., Gopalakrishnamurthy, TR, Vasudevan, G., Shanmugasamy, M., Kandasamy, S. (2017). Occurrence of Clostridium Perfringens contamination in poultry feed ingredients: isolation, identification and its antibiotic sensitivity pattern. Anim Nutr. 3(3): 309-312. PMid:29767074 PMCid:PMC5941237 49 https://doi.org/10.1016/j.aninu.2017.05.006 Sapkota, AR, Lefferts, LY, McKenzie, S., Walker, P. (2007). What do we feed to food-producing animals? A review of animal feed ingredients and their potential impacts on human health. Environ Health Persp. 115(5): 663-670. PMid:17520050 PMCid:PMC1867957 50 https://doi.org/10.1289/ehp.9760 Kukier, E., Goldsztejn, M., Grenda, T., Krzysztof, K., Wasyl, D., Hoszowski, A. (2012). Microbiological quality of compound feed used in Poland. J Vet Res. 56(3): 349-354. 51 https://doi.org/10.2478/v10213-012-0061-x Liebana, E., Hugas, M. (2012). 5-Assessment of the microbiological risks in feedingstuffs for food-producing animals. In J. Fink-Gremmels (Ed.), Woodhead Publishing Series in Food Science, Technology and Nutrition, Animal Feed Contamination (pp. 66-93). Woodhead Publishing 52 https://doi.org/10.1533/9780857093615.1.66 Jones, FT (2011). A review of practical Salmonella control measures in animal feed. J Appl Poult Res. 20(1): 102-113. 53 https://doi.org/10.3382/japr.2010-00281 Vestby, LK, Møretrø, T., Langsrud, S., Heir, E., Nesse, LL (2009). Biofilm forming abilities of Salmonella are correlated with persistence in fish meal-and feed factories. BMC Vet Res. 5, 20. PMid:19473515 PMCid:PMC2693496 54 https://doi.org/10.1186/1746-6148-5-20