Key words: food hygiene; residues; salinomycin; broiler chicken s meat; microbiological

Transcription

1 Slov Vet Res 2002; 39 (2): UDC : : The determination of salinomycin residues... R. Cabadaj, J. Nagy, P. Popelka, D. Máté, A. Bugarský THE DETERMINATION OF SALINOMYCIN RESIDUES IN THE TISSUES OF BROILER CHICKENS BY USING MICROBIOLOGICAL DIFFUSION METHODS R. CABADAJ, J. NAGY, P. POPELKA, D. MÁTÉ, A. BUGARSKÝ Key words: food hygiene; residues; salinomycin; broiler chicken s meat; microbiological diffusion methods Two hundred three-day-old broiler chickens (ROSS hybrid) were used in the experiment. Salinomycin (Synvertas, Biotika Slovenska L up~a, The Slovak Republic) was orally administered to three groups of chickens in dosages of 600 mg/kg -1 of feed (60 mg salinomycin - group I); 900 mg/kg -1 of feed (90 mg salinomycin - group II); 1,800 mg/kg -1 of feed (180 mg salinomycin - group III). Sacox 120 (12 % salinomycin, Biotika Slovenska L up~a) was administered to the fourth group of chickens (500 mg/kg -1 feed; 60 mg of salinomycin). The birds were treated until they were 42 days old. Then 70 chickens were randomly selected and killed; 6 birds from each experimental group (I-IV) on the first day after the end of the treatment and two birds from each experimental group on the 2nd, 3rd, 4th, 5th, 6th and 7th days. Breast and thigh muscles, liver, gizzard and heart samples were analysed microbiologically, with Bacillus stearothermophillus var. calidolactis C 953 as a test micro-organism, and by the four-plate method. The four-plate method was more sensitive than the Bac. stearothermophillus microbiological method to the residues of salinomycin, which were detectable in the chicken tissues for between 4 and 6 days, depending on the dosage that they received. UGOTAVLJANJE OSTANKOV SALINOMICINA V TKIVIH BROJLERSKIH PI[^ANCEV Z MIKROBIOLO[KIMI DIFUZIJSKIMI METODAMI Klju~ne besede: higiena `ivil; rezidumi, salinomicin, meso brojlerskih pi{~ancev, mikrobiolo{ka difuzijska metoda Received: May 28, Address of author: Prof. Dr. Rudolf Cabadaj, DVM, The University of Veterinary Medicine, Department of Food Hygiene and Technology, Institute of Meat Hygiene and Technology, Komenskeho 73, Ko{ice, The Slovak Republic.

2 138 R. Cabadaj, J. Nagy, P. Popelka, D. Máté, A. Bugarský V poskusu smo uporabili dvesto tri dni starih brojlerskih pi{~ancev (kri`anci ross). Pi{~ance smo krmili z dodatkom salinomicina (synvertas, Biotika Slovenská L up~a, Slova{ka) v odmerkih 600 mg/kg krme (60 mg salinomicina - skupina I), 900 mg/kg krme (90 mg salinomicina - skupina II) in 1800 mg/kg krme (180 mg salinomicina - skupina III). ^etrti skupini pi{~ancev smo dodajali sacox 120 (12% salinomicina, Biotika Slovenská L up~a) v odmerku 500 mg/kg krme, 60 mg salinomicina). Pi{~ance smo tako tretirali do 42. dneva starosti. Nato smo naklju~no izbrali in `rtvovali 70 `ivali: dan po kon~anem eksperimentalnem krmljenju po 6 `ivali iz vsake skupine (I-IV), nato pa po dve `ivali iz vsake skupine 2., 3., 4., 5., 6. in 7. dan. Jemali smo vzorce prsnih in stegenskih mi{ic, jeter, mlin~ka in srca. Vzorce smo preiskali z mikrobiolo{ko metodo s testnim mikroorganizmom Bacillus stearothermophillus var. calidolactis C 953 in z metodo {tirih plo{~. Metoda {tirih plo{~ je bila ob~utljivej{a na ostanke salinomicina kot metoda z Bac. stearothermophillus. Ostanke salinomicina smo ugotovili v tkivih pi{~ancev od 4. do 6. dneva po eksperimentalnem krmljenju. Introduction In Slovakia, poultry meat, which has no substitute of equivalent nutritional value, ranks 2 nd in per capita meat consumption with each person consuming approximately 16 kg per year (1). Anticoccidial drugs currently have an important role in poultry farming, especially in the intensive conditions of poultry breeding. They are used for prevention, therapy and growth stimulation. They eliminate protozoal parasites in the birds, increase farm production and reduce economic losses caused by coccidiosis (2). This group of drugs is important primarily for the treatment of coccidiosis, especially that caused by Eimeria and Isospora. According to a financial analysis, antiprotozoic drugs are the fourth highest selling drugs worldwide with 11 % of the total market (3). During the last 10 years, the use of ionophore coccidiostats has spread significantly throughout the poultry industry. Their use has brought advantages such as increased productivity and reduced the incidence of coccidiosis. However, drugs with a narrow spectrum of therapeutic activity can be dangerous and with the effectiveness of anticoccidial drugs there is a possibility of drug toxicity. For this reason, drug administration must be precise as decreasing the drug content in feed risks an outbreak of coccidiosis as well as the problem of resistance, while increasing the content brings about the hazard of intoxication and economic costs. Salinomycin, monensin, and lasalocid are monovalent carboxyl-polyether ionophores. Salinomycin, produced by Streptomyces albus, destroys the cell membranes and causes their lysis (4). It is effective against sporozoides in the earlier and later stages of the coccidium life-cycle (5). On the farm a 6.6 % premix is principally used for the prevention of poultry coccidiosis caused by Eimeria tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti, and E. mivati. The use of salinomycin is also possible in other animals (cattle, pigs) as a growth stimulant and

3 The determination of salinomycin residues for improving feed utilization. In the digestive tract, salinomycin eliminates pathogenic strains of bacteria and coccidiosis, improves protein digestion, increases nitrogen utilization and reduces its elimination from the body (6). The adverse effects of salinomycin relate to exceeding the recommended doses in feed, to its administration to animals for which it is not prescribed, and to adverse interactions between salinomycin and simultaneously applied alternative ionophores. While it has no adverse effects on egg quality and production, its administration to laying hens is not recommended (7). It is compatible with all certified drugs, probiotics, and growth stimulators, except tiamulin. In combination with tiamulin it decreases productivity, and reversible ataxia and paralysis are found. The decrease in the effectiveness of anticoccidial drugs is related to the resistance of coccidia to them, and to increasing the elimination of anticoccidial drugs from animal tissues, especially by inductive mechanisms at the cell level (molecular pharmacology) (8). In recent years the presence of drug residues and their metabolites is supervised. In some cases there have been problems where, during drug elimination, more than one biotransformation product has been produced (9). Prolonged administration of anticoccidial drugs to a huge number of animals on farms, especially in the poultry industry, forced the authorities to make checks on the withdrawal periods, recommended doses and the maximum permitted limits of residues (10). Materials and methods Two hundred three-day-old chicks were used in our experiment. They were fed BR 1 feed mixtures for broiler chickens until they were 28 days old and BR 3 from then until they were 42 days old. At the beginning of the experiment the chicks were divided into five groups and two preparations of salinomycin were administered: Synvertas and Sacox 120 (12 % of salinomycin, Biotika Slovenska L up~a) as follows: I. 600 mg of Synvertas (60 mg of salinomycin)/1 kg of feed; II. 900 mg of Synvertas (90 mg of salinomycin)/1 kg of feed; III. 1,800 mg of Synvertas (180 mg of salinomycin)/1 kg of feed (three times the recommended dose); IV. 500 mg of Sacox 120 (60 mg of salinomycin)/1 kg of feed; V. a control group (fed with the same feed but without the coccidiostatic drugs). At the conclusion of the experimental feeding the chickens were killed as follows: on the first day after the conclusion, 6 chickens from each experimental group (I.-IV.) were killed; on the 2nd, 3rd, 4th, 5th, 6th, and 7th days after the end of the salinomycin administration, two chickens from each experimental group were killed; on the 7th day two chickens were killed from the control group (V ).

4 140 R. Cabadaj, J. Nagy, P. Popelka, D. Máté, A. Bugarský After they were killed the chickens were eviscerated and fresh samples of the breast and thigh muscles, liver, heart and the stomach were examined for the presence of drug residues using the microbiological diffusion screening methods: Bacillus stearothermophillus var. calidolactis C 953 method (11, 12, 13) Four-plate method (14) The samples of the breast and thigh muscles were examined in parallel, while the liver and heart samples were examined individually due to their small size. Results and discussion Based on our results we can declare that the inhibition zones of Bacillus stearothermophillus var. calidolactis C 953 microbiological diffusion method did not exceed 3 mm in the muscle samples and 5 mm in the viscera samples during the period after the conclusion of the experimental salinomycin administration. These permitted limits of inhibition zones relate to Addition I. of Directive 5200/1994. The following results were detected by the four-plate microbiological-diffusion method. On the first day after conclusion of the drug administration, inhibition zones reached 2 mm and more in four of six chickens in group I, 1 liver sample from group II, 2 liver samples from group III, and in all chicken samples from group IV. In this group the stomach of one chicken was positive, all the samples were highly positive in another one, and only the livers were positive in other chickens (Table 1). On the second day only one liver from group II was positive (Table 2) and on the third day one stomach sample from group I and liver samples from groups III and IV exceeded the permitted limits (Table 3). On the fourth day, amounts above the permitted limits were found in both liver samples from group II and in one liver sample from group III (Table 4). Liver samples from groups I, II, III, and IV were positive on the fifth day (Table 5). By the sixth day, a chicken s stomach from group III was the only positive sample found (Table 6) and on the seventh day there were no inhibition zones above the permitted limits found in any of the samples examined (Table 7). Akhtar et al. (15) administered hen feed medicated with various concentrations of salinomycin sodium salt to laying hens for 14 days following a 3-day withdrawal period. The highest concentrations were determined, by HPLC, in fatty parts such as egg yolk, ovarial egg and subcutaneous fat. In other tissues (liver, kidney, thigh and breast muscles) the concentrations were lower. The levels of the salinomycin residues depended on the salinomycin concentration in the feed, and decreased quickly after the end of the administration (15). Similar results were observed by Atef et al. (16) after administering a salinomycin premix (60 ppm) for 2 weeks. Birds administered pure salinomycin (20 mg/l kg of body weight) directly into the crop had higher concentrations in the blood serum and tissues than birds administered the premix salinomycin. The highest concentration of salinomycin residues occurred in the liver followed by the kidneys, muscles, fat, heart and skin. After 48 hours there were no detectable salinomycin residues remaining in the tissues except for the liver where the residues were eliminated after 72 hours.

5 The determination of salinomycin residues Table 1: Mean size of inhibition zones (mm) of chicken samples one day after finishing Breast muscle Thigh muscle Liver Stomach Heart Table 2: Mean size of inhibition zones (mm) of chicken samples two days after finishing Breast muscle Thigh muscle Liver Stomach Heart Table 3: Mean size of inhibition zones (mm) of chicken samples three days after finishing I II III IV I II III IV I I III IV I II III IV I II III IV Breast muscle Thigh muscle Liver Stomach Heart Table 4: Mean size of inhibition zones (mm) of chicken samples four days after finishing Breast muscle Thigh muscle Liver Stomach Heart

6 142 R. Cabadaj, J. Nagy, P. Popelka, D. Máté, A. Bugarský Table 5: Mean size of inhibition zones (mm) of chicken samples five days after finishing Breast muscle Thigh muscle Liver Stomach Heart Table 6: Mean size of inhibition zones (mm) of chicken samples six days after finishing Breast muscle Thigh muscle Liver Stomach Heart Table 7: Mean size of inhibition zones (mm) of chicken samples seven days after finishing Breast muscle Thigh muscle Liver Stomach Heart Conclusion The four plate microbiological diffusion method showed a higher sensitivity to the salinomycin residues than the Bacillus stearothermophillus var. calidolactis C 953 microbiological diffusion method. The four-plate method indicated the presence of salinomycin with a high probability in the 4-6 day period after the administration of the drug tested. Objectively, it should be possible to confirm with more sensitive, though more expensive physical and chemical methods. Acknowledgements This study was supported by VEGA grant 1/7020/20.

7 The determination of salinomycin residues References 1 Hadbavny M Sr., Korim P, Hadbavny M Jr., Korimova J. The conception of poultry farming development in The Slovak Republic in the years Slov Vet ^as 2001; 26(2): Ko`arova I, Mate D, Turek P, Nagy J. Anticoccidiostats from the view point of food hygiene. Slov Vet ^as 1997; 22, (4): [utiak V, [utiakova I. How and with what against coccidials? Infovet 1995;2 (3): Vander Kop PA, MacNeil JD. Separation and detection of monensin, lasalocid and salinomycin by thin - layer chromatography/bioautography. J Chromatography 1990; 508: Conway DP, Johnson JK, Guyonnet V, Long PL, Smothers CD. Efficacy of semduramicin and salinomycin against different stages of Eimeria tenella and E. acervulina in the chicken. Vet Parasitol 1993; 45: Kri` M. Salocin - use in pig farming. Veterinarstvi 1999; 5: Jones JE, Solis I, Hughes BL, Castaldo DJ, Toler JE. Production and egg - quality responses of white leghorn layers to anticoccidial agents. Poult Sci 1990; 69: [utiak V, [utiakova I. How and why the anticoccidiostat effect is falling down? Infovet 1995; 2 (4): [utiak V, [utiakova I. About fate of the drugs, pharmaceutical auxiliary substances, and other components in animal organism. Veterinarstvi 1998; 5: Ko`arova I, Mate D. Inhibitory effects of some anticoccidial drugs. In: Proceedings of lectures and posters. Hygiena alimentorum XIX. Ko{ice, 1998: Veterinary laboratory methods. Food hygiene. Bratislava: State Veterinary Administration of The Slovak Republic, The methodical direction of the uniform procedures for the evidence of inhibitory substances and drug residues in the food products of animal origin. Bratislava: State Veterinary Administration of The Slovak Republic, Supplement No. l to the Methodic directive No. 5200/1994. Bratislava: State Veterinary Administration of the Slovak Republic, Bogaerts R, Wolf P. A standardized method for the detection of residues of antibacterial substances in fresh meat. Fleischwirtschaft 1980; 60: Akhtar AH, Abou el-sooud K, Shehata MA. Concentrations of salinomycin in eggs and tissues of laying chickens fed medicated feed for 14 days followed by withdrawal for 3 days. Food Addit Contam 1996; 13 (8): Atef M, Ramadan A, Youssef SA, Abou el-sooud K. Kinetic disposition, systemic bioavailability and tissue distribution of salinomycin in chickens. Res Vet Sci 1993; 54 (2):

8 144 R. Cabadaj, J. Nagy, P. Popelka, D. Máté, A. Bugarský