Detection of multiple presence of antibiotic residues in slaughtered sheep at Duhok abattoir, Iraq

Article history: Received November 5, 2019 Accepted December 31, 2019 Available online September 7, 2020 The aim of this study is to investigate the presence and/or multi-presence of tetracycline, penicillin G, streptomycin, and gentamicin residues in slaughtered sheep carcasses at Duhok abattoir using ELISA technique. Eighty-eight samples were collected from different sites of twenty-two sheep carcasses. These samples included Longissimus dorsi, diaphragmatic muscles, liver and kidney. The samples were prepared, homogenized, extracted and assayed according to ELISA kit instructions. The results revealed that all 22 carcasses were contained at least one type of tested antibiotics. Multiple antibiotic residues were found in 17(77.27%) of examined carcasses. The study indicated that local ovine meat sold in Duhok province generally contained residues of antibiotics. Animals slaughtered might have been treated with veterinary drugs and the proper withdrawal period was not respected before slaughtering.


Introduction
Meat is an esteemed article of human diet as it provides proteins, energy, minerals and most B complex vitamins (1). Animal food industries are rapidly growing with consequent demand of healthy wholesome animal (2). In spite of their merits, meat could be a source of health hazards if it contains harmful material such as pathogens, toxins, residues of chemical agents as a parent compound or its metabolites (3). Residues in meat may result from many sources such as pesticides or from veterinary drugs which used to prevent or treat diseases, or to promote growth (4). Other disadvantages are that withdrawal time in relation to the maximum residue limit (MRL) is not taken into account (5)(6)(7)(8). Nowadays, antibiotics play a major role to rise agriculture and to enhance feed conversion in livestock industries (2). The most commonly used antimicrobials in food producing animals are the β-Lactames, aminoglycosides, macrolides, sulfonamides and lincosamides (3). Public health hazards associated with these residues include direct toxic effects on consumers like allergic reactions and indirect problems through induction of resistant strains of pathogenic bacteria (9). Residues from these antibiotics may be present in edible tissues, milk and eggs and may exert different levels of toxicity on consumers when consuming them (2). Thus, easy, rapid and sensitive tests are really needed for an effective at line use (10). At present, there are various ways for detection of antibiotic residues which include microbiological, instrumental and immunoassay methods (11). A great proportion of livestock in Duhok province is reared by the herdsmen who administer animal pharmaceutical agents particularly incorrect dosage of antibiotics without veterinary prescription and with ignorance of withdrawal periods (12,13).
The aims of this study is to detect and determine the residues of single or multiple presence of tetracycline, penicillin G, streptomycin and gentamicin in meat and various edible tissues of sheep slaughtered at Duhok province, Iraq.

Sampling
Eighty-eight samples have been collected from 22 sheep carcasses which were slaughtered within one week of August 2013 in Duhok abattoir to analyze for antibiotic residue by ELISA test. The samples were intentionally collected from organs and tissues suffering and affected by different pathological lesions. Four samples of 200 gm of Longissimus dorsi, diaphragmatic muscle, liver and kidney were taken and packed in clean plastic bags. Samples of the current study were intentionally taken from certain clinical cases and those suffering from pathological changes. The identified samples were transported immediately to the Faculty of Veterinary Medicine, Public Health laboratory, Duhok University in an ice box with a minimum period of delay. The samples were stored at -20°C in the freezer.

Sample preparation
Samples were finely homogenized to create a uniform form and were then extracted for each investigated antibiotic i.e. tetracycline, penicillin G, streptomycin and gentamicin according to the protocol recommended by the kit supplier (Shenzhen Lvshiyuan Biotechnology Co., Ltd, China). ELISA kits stored at 4°C. The product codes are LSY-10006, LSY-10019, LSY-100024-2 and LSY-10023 for tetracycline, penicillin g, streptomycin and gentamicin, respectively.

Calculation of antibiotics concentrations
The concentrations of antibiotics in the samples were calculated according to the following formula, Percentage of absorbance value = B/B0*100. In which B is the optical density value of samples and B0 is the optical density value of the standard solution. The values were then multiplying by the dilution factor as suggested by the kit manual. The standard and test absorbencies were entering manually to IPC software-version 4 from IPC Company, which then interpreted test results. Then the values were compare with the Maximum residue level (MRL) according EU standards (14).
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Discussion
In Iraq, the classic habit followed in which animal breeders administer a wide spectrum of veterinary drugs randomly to their animals without observing the withdrawal periods of these drugs before they are slaughtered for human consumption. Table 2 and 3 showed the percentage of samples containing tetracycline residues which referred that 50% of positive samples of Longissimus dorsi and diaphragm muscle exceeded recommended MRL 100 ppb as per EU standards for muscles (14).
Meat is considered as an essential feedstuff for human beings due to its high nutritional value, digestibility and palatability (1). In Iraq, livestock including sheep represent the main source of animal protein. Consequently, the emergent and increasing demand for animal products oblige many animal raisers to expand their scale of livestock production and to increase their profitable business by using different antibiotics indiscriminately and application of "Blanket treatment" for such animals without observing and attention of withdrawal periods of drugs to clinically diseased or apparently healthy sheep before being slaughtered (15,16).
Residual tetracycline levels of liver and kidney samples were below the maximum residual limit 300 ppb for liver and 600 ppb for kidney according to EU standards for both organs (14).
However, these values were not exceeding the MRL (14), suggesting that such animals had been treated with the drug and probably were not allowed adequate withdrawal period.
Tables 4-8 showed the percentage of Longissimus dorsi, liver, kidney diaphragmatic muscle samples containing penicillin G residues. The table also showed that 85% of positive samples of Longissimus dorsi and diaphragm contained which where over the MRL 50 ppb as per EU standards for all the examined organs (14). Tables 2-7 showed the percentage of Longissimus dorsi, diaphragmtic muscle, liver and kidney samples containing streptomycin residues referring that 47.6% of positive for the first two organs which contained residues levels over the MRL 500 ppb for liver and below the MRL 1000 ppb for kidney according to EU standard (14).
Regarding the percentage of gentamicin residues in different tissues, table 8 and 10 showed that 87.5% of both Longissimus dorsi and diaphragmatic muscles had residues over the MRL 50 ppb according to EU standard for muscles (14). On the other hand, these residues where 100% and 50% in the samples of liver and kidney, respectively according to the aforementioned standard (14).
It was found that, penicillin G and streptomycin 72.73% were present in a large number of the carcasses followed by tetracycline 63.64% and gentamicin 36.37%, which could be accounted to the availability of these antibiotics in local markets with low cost promoted their broad administration. It should be noted that penicillin G and streptomycin is dispersed in one combination solution called PS. Such occurrences of multiple residues in the inspected meats may be attributed to the fact that these samples were intentionally collected from animals suffered from several diseases reflected and manifested subsequently by abnormal and pathological lesions in their carcasses.
The lower percentage of gentamicin residue obtained is due to the fact that this drug has limited uses, since it is only used for urogenital infections which were actually found in the examined carcasses fulfilling those goals of the study.
In Iraq, there are limited numbers of studies on the detection of antibiotics in food of animal origin, (17) found that 52% of poultry meat samples were positive for various antibiotic residues. Subsequently, (11) reported the streptomycin residue rate as 50% and 60% in ovine and bovine meat samples, respectively. (18,19) found detectable levels of antibiotics in milk samples collected from Afak city, Al-Diwaniya and Mosul Province-Iraq, respectively. Multiple antibiotic residues 18% were found by (20) in meat from slaughtered cattle in Nigeria. However, a high percentage of antibiotic residues were found by (21) in Egypt, 58% of kidney samples and 14% of muscle in cattle samples were positive, while the percentage in buffaloes was 40% and 20%, for kidney and muscles, respectively. The lower percentage 2% of oxytetracycline residues was obtained by (22) in bovine carcasses slaughtered at Mansoura Abattoir, Egypt.
Our findings were in agreement with those of (23) from Thailand, who found that out of 130 samples, 51 (39 %) were positive for at least one of the tested antibiotic. Several authors from different developing countries detected higher proportion of antibiotic residues in various animal producing food (24)(25)(26)(27). A similar study carried out by (28) in Iran, showed that 14% of mutton and 22.8% of beef samples contained antibiotic residues. In Turkey, a study done by (29) revealed that 57.7% of beef samples and 45.7% of chicken meat samples collected from Ankara markets were positive for antibiotic residues. Similarly, (30) from Nigeria found that 46% of samples were positive to residues of tetracyclines. A study performed by (31) in Algeria, showed a strong presence of antibiotic residues in poultry meat, with 85.5% positive samples, most of them contained penicillin, tetracycline and aminoglycosides.
Generally, it is obvious that high rates of residues in various animal tissues and products are reported in developing countries in contrast to what is found in the developed world according to the European Community findings. Such reports revealed that positive samples for antibiotic residues in food of animal origin were less than 1%, exactly 0.19 % (32), which could be attributed to the type of animal husbandry, management and production system.

Conclusion
Consequently, in developing countries a greater proportion of animal breeding is done by uncultivated herdsmen who always purchase veterinary drugs over the counter for administration to their animals without a veterinary diagnosis, prescription and supervision, and it may still be regarded as a health hazards as they may cause allergic reactions or produce drug-tolerant bacteria. Unfortunately, this condition is an indication of lack of public health regulatory control in developing countries, which require urgent care and attention.

Acknowledgments
On behalf of the researchers, I extend my thanks and gratitude to the College of Veterinary Medicine, University of Mosul, for its continuous support for the research work

Conflict of Interest
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