Veterynarna biotehnologija – Veterinary biotechnology, 2022, 40, 9-20. [in Ukrainian]. https://doi.org/10.31073/vet_biotech40-01
State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise
1Research Institute of Organic Agriculture (FiBL)
COMPARISON OF THE EFFICIENCY OF THE MICROBIOLOGICAL METHOD FOR DETERMINING THE RESIDUAL CONTENT OF ANTIBIOTICS OF THE TETRACYCLINE GROUP IN EGGS WITH THE ARBITRATE METHOD OF LIQUID CHROMATOGRAPHY WITH A DUAL MASS-SPECTROMETRIC DETECTOR
Introduction. In recent decades, antibiotics have been widely used in poultry for preventive and curative purposes, and a third of them used in Europe are intended for veterinary use.For poultry farming, antibiotics are important for the prevention and control of infectious diseases.
Antibiotics of the tetracycline group are used to treat bronchopneumonia, dysentery, gastroenteritis, alimentary toxic dyspepsia, sepsis, urinary tract infections, coccidiosis, pulorosis, pasteurellosis and other diseases. Improper use of tetracycline antibiotics in poultry, leads to the accumulation of its residues in poultry products causing digestive disorders, creates a risk of adverse effects on the intestinal microflora in humans, which further leads to toxic allergic reactions and reduced immunity.Therefore, the residual content of tetracycline antibiotics in eggs is considered a danger to the health of the population of Ukraine.
The goal of the work was to compare the effectiveness of the developed microbiological method for detecting the residual content of tetracycline antibiotics in eggs with the arbitration method of liquid chromatography with a double mass spectrometric detector.
Materials and methods.The study was performed in production conditions on Leghorn laying hens aged 35 weeks at the peak of egg laying (90%), weighing up to 2 kg. The chickens were fed with complete feed. Doxycycline was administrated with water individually to each bird. According to the principle of analogue groups, chickens were divided into control and experimental groups of 10 heads each. The experimental group was fed doxycycline with water for therapeutic purposes at a dose of 100 mg / kg for 7 days, the control group of laying hens was kept on the main diet under the same conditions, but without antibiotics. For 21 days, three eggs (pooled sample) were collected daily from three different locations in the henhouse (three parallels) from laying hens from each group, which were tested in parallel by microbiological and LC-MS / MS for doxycycline residues.
For the study, the developed microbiological method was used, the principle of which is the ability of an antibiotic from a sample to diffuse into agar, therefore, if it is present, the Bacillus cereus ATCC 11778 test culture sensitive to this group of antibiotics does not grow on a nutrient medium, but forms zones of inhibition. As for the arbitration method for the detection of antibiotics a liquid chromatograph with a two-quadrupole mass spectrometric detector, positive ionization electrospray, and Xevo Waters Corporation's MasiLynx 4.1 software was used. The chromatograph was equipped with an ACQUITY UPLC® BEH C18 analytical column, 1.7 μm, 2.1 mm x 100 mm, which allowed to determine the exact concentration of doxycycline residues in the tested egg samples.
Results of research and discussion. After analyzing the results of studies of egg samples obtained from the experimental group of hens, it was found that during the first week the residual content of doxycycline was detected by the microbiological method in all egg samples (yolks), since clear zones of inhibition of test cultures of Bacillus cereus ATCC 11778 were observed around the disks soaked in the liquid of the samples, which diameter ranged from 18.0 to 23.0 mm, which fully complied with the established parameters for evaluating this method for a positive result. The LC-MS/MS method also confirmed the residual content of doxycycline in the same samples at a level of 208.681 µg/kg to 2025.783 µg/kg, which indicated that the maximum allowable levels (MAL) established by the regulatory documents of Ukraine were exceeded.
The lowest level of doxycycline residues by LC-MS/MS was detected on the 13th day of the experiment (45.597 μg/kg), results below the detection limit of the method (≤29.0 μg/kg) were obtained on the 14th day of the experiment, however, Bacillus cereus ATCC 11778 still gave clear zones of growth inhibition with a diameter of 15.0 mm, which indicated the presence in the studied egg samples of the residual content of antibiotics of the tetracycline group.
Conclusion and prospects further research:
1. 100% agreement was confirmed between the results of studies obtained using the microbiological method for detecting the residual content of tetracycline antibiotics in eggs (yolks) and the LC-MS/MS arbitration method.
2. It has been proven that the developed microbiological method for detecting the residual content of antibiotics of the tetracycline group at the level of ½ of the MAL established by regulatory legal acts meets the requirements for screening methods and can be used for the purposes of state control.
Keywords: antibiotics of the tetracycline group, doxycycline, microbiological method, liquid chromatography method with a double mass spectrometric detector, screening method.
- Thiele-Bruhn, S. (2003). Pharmaceutical antibiotic compounds in soils – A review. J Plant Nutr. Soil, 166, 145-167. doi: 10.1002/jpln.200390023.
- Kantiani, L., Farré, M., Grases i Freixiedas, J.M., & Barceló, D. (2010). Development and validation of a pressurised liquid extraction liquid chromatography-electrospray-tandem mass spectrometry method for β-lactams and sulfonamides in animal feed. J. Chromatogr. A., 1217, 4247-4254. doi: 10.1016/j.chroma.2010.04.029.
- Laxminarayan, R., Duse, A., Wattal, C., Zaidi A.K., Wertheim, H.F., Sumpradit, N., Vlieghe, E., Hara, G.L., Gould, I.M., Goossens, H., et al. (2013). Antibiotic resistance–the need for global solutions. Lancet Infect. Dis., 13, 1057-1098. doi: 10.1016/S1473-3099(13)70318-9.
- Piątkowska, M., Jedziniak, P., & Żmudzki, J. (2012). Residues of Veterinary Medicinal Products and Coccidiostats in Eggs: Causes, Control and Results of Surveillance Program in Poland. Polish Journal of Veterinary Sciences, 15(4), 803-812.
- Yunus, A.W., Nasir, M.K., Aziz, T., & Böhm, J. (2009). Prevalence of Poultry Diseases in District Chakwal and Their Interaction with Mycotoxicosis: 2. Effects of Season and Feed. Journal of Animal and Plant Sciences, 19(1), 1-5.
- Chapman, H.D., & Jeffers, T.K. (2014). Vaccination of Chickens Against Coccidiosis Ameliorates Drug Resistance in Commercial Poultry Production. International JournInt J Parasitol Drugs Drug Resist., Dec. 4(3), 214-217. doi: 10.1016/j.ijpddr.2014.10.002.
- Goetting, V., Lee, K.A., & Tell, L.A. (2011). Pharmacokinetics of Veterinary Drugs in Laying Hens and Residues in Eggs: A Review of the Literature. Journal of Veterinary Pharmacology and Therapeutics, 34, 521-556.
- Rokka, M., Jestoi, M., & Peltonen, K. (2012). Trace Level Determination of
Polyether Ionophores In Feed. BioMedResearchInternational, 2013, 1–12. http://doi.org/10.1155/2013/151363.
- Seri, H.I. (2013). Introduction to Veterinary Drug Residues: Hazards and Risks. Paper Presented at the Workshop: Veterinary Drug Residues in Food Derived from Animals (Our Goal of Protecting Consumers). The National Medicinal and Poisons Board, Khartoum, Sudan May 26 – 27th, 2013. http://www.sustech.edu/staff_publications/2013070315212363.pdf.
- Kempe, M., & Verachtert, B. (2000). Cartridges with Molecularly Imprinted Recognition Elements for Antibiotic Residues Monitoring in Milk Cream. Pure and Applied Biochemistry. Lunds Universitét Centre for Chemistry and Chemical Engineering:Getingevagen, Lund., 1-10.
- Marazuela, M.D., & Bogialli, S.A. (2009). Review of novel strategies of sample preparation for the determination of antibacterial residues in foodstuffs using liquid chromatography-based analytical methods. Anal. Chim. Acta., 645, 5-17. doi: 10.1016/j.aca.2009.04.031.
- Bera, A.K., Bhattacharya, D., Pan, D., Dhara, A., Kumar, S., & Das, S.K. (2010). Evaluation of Economic Losses Due to Coccidiosis in Poultry Industry in India. Agriculture Economic Research, 23, 91-96.
- Marshall, B.M., & Levy, S.B. (2011). Food Animals and Antimicrobials: Impacts on Human Health. Clinical Microbiology Reviews, 24(4), 718-733.
- Haller, M.Y., Müller, S.R., McArdell, C.S., Alder, A.C., & Suter, M.J.F. (2002). Quantification of veterinary antibiotics (sulfonamides and trimethoprim) in animal manure by liquid chromatography – mass spectrometry. J. Chromatogr. A., 952, 111-120. doi: 10.1016/S0021-9673(02)00083-3.
- Barton, M.D. (2005). Antibiotic use in animal feed and its impact on human healt. Nutr. Res. Rev., 13, 279-299. doi: 10.1079/095442200108729106.
- Commission E.Euopean Commission Regulation Commission Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. (2010). European Union; Brussels, Belgium.
- Nakaz MOZ Ukrainy vid 06.08.2013 No. 695 “Pro zatverdzhennia Parametriv bezpechnosti miasa ptytsi” [Order of the Ministry of Health of Ukraine dated 06.08.2013 No. 695 “Approval of the Safety Parameters of Poultry Meat”]. (2013). zakon.rada.gov.ua. Retrieved from https://zakon.rada.gov.ua/laws/show/z1364-13#Text [in Ukrainian].
- Guidelines No 3049–84. Metodicheskie ukazania po opredeleniu ostatochnyh kolichestv antibiotikov v produktah zhyvotnovodstva [Guidelines for the determination of antibiotic residues in animal products]. (1984). docs.cntd.ru. Retrieved from https://docs.cntd.ru/document/1200049289 [in Russian].
- Garkavenko, T.O., Azyrkina, I.M., & Kyivska, G.V. (2016). Porivnialnyi analiz mikrobiolohichnyh metodiv shchodo vyznachennia zalyshkovyh kilkostei antymikrobnyh preparative u produktsii ptahivnytstva [Comparative analysis of microbiological methods for determining the residual amounts of antimicrobials in poultry products]. Veterynarnabiotehnologia – VeterinaryBiotechnology, 28, 28-41 [in Ukrainian].
- Garkavenko, T.O., Azyrkina, I.M., & Shalimova, L.O. (2019). Vyznachenniamikrobiolohichnymskryning-metodomzalyshkovyhkil’kostei antybiotykiv tetracyklinovoi grupy u miasi ptytsi, yaitsiah ta yayechnyh produktah [Determination by microbiological screening method of residual amounts of antibiotics of the tetracycline group in poultry meat, eggs and egg products]. Kyiv: DNDILDVSE [in Ukrainian].
- Garkavenko, T.O., & Azyrkina, I.M. (2015). Metody vyznachennia zalyshkovyh kilkostei antymikrobnyh preparative v produktah ptahivnytstva [Methods for determining the residual amounts of antimicrobial drugs in poultry products]. Veterynarnabiotehnologia – VeterinaryBiotechnology, 26, 33-41 [in Ukrainian].
- Europeiska Konventsia pro zahyst hrebetnyh tvaryn, shcho vykorystovuyut’sia dlia doslidnyh ta inshyh naukovyh tsilei. Strasburg, 18 bereznia 1986 roku [European Convention for the Protection of Vertebrate Animals used for Research and Other Scientific Purposes. Strasbourg, 18 March 1986]. https://zakon.rada.gov.ua/laws/show/994_137#Text [in Ukrainian].
- Kozhemiakin, Yu.M., Hromov, O.S., Boldyrieva, N.Ye., et al. (2017). Naukovo-praktychnirekomendatsiizutrymannialaboratornyhtvaryntarobotyznymy[Scientific and practical recommendations for the maintenance of laboratory animals and work with them]. Kyiv [in Ukrainian].
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