Bulletin "Veterinary biotechnology"

Veterynarna biotehnologija – Veterinary biotechnology, 2022, 40, 9-20. [in Ukrainian]. https://doi.org/10.31073/vet_biotech40-01

AZYRKINA I.M., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., GARKAVENKO T.A.1, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., DOBROZHAN YU.V., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., KYIVSKA G.V., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise

1Research Institute of Organic Agriculture (FiBL)


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.


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