32 l KEYWORDS Fluoroquinolone residues; Ciprofloxacin; Norfloxacin; Ofloxacin; Consumer protection; Meat safety. PAGES 32. – .40 REFERENCES Vol. 2 No. 1 (2015) ARTICLE HISTORY Submitted: March 15, 2015 Revised: June 03, 2015 Accepted: June 24, 2015 Published July 11, 2015 CORRESPONDING AUTHOR Omotoso Adekunbi B., Animal Products and Processing Unit, Department of Animal Science, University of Ibadan, Ibadan, Nigeria e-mail: kunbiadeshiyan@yahoo.com phone: +234 08033900828 JOURNAL HOME PAGE riviste.unimi.it/index.php/haf Fluoroquinolone residues in raw meat from open markets in Ibadan, Southwest, Nigeria. Omotoso Adekunbi B.1* and Omojola Andrew B1 1 Animal Products and Processing Unit, Department of Animal Science, University of Ibadan, Ibadan, Nigeria. ABSTRACT. Misuse of fluoroquinolones in livestock production may lead to the presence of their residues in tissues of meat animals after slaughter, constituting health hazards to consumers. The present study was designed to screen for residues of three fluoroquinolones (ciprofloxacin, norfloxacin and ofloxacin) in raw meat. Microbiological assay, followed by High Performance Liquid Chromatography (HPLC) was used to screen three hundred and twenty samples of beef, chicken, pork and chevon purchased from open markets. Initial screening by microbiological assay revealed that 50%, 55%, 40% and 40% of beef, chicken, pork and chevon, respectively were positive for residues of antibiotics. Further analysis by HPLC with UV detection revealed the presence of ciprofloxacin, norfloxacin and ofloxacin at varying concentrations in the meat samples. Ofloxacin was the least in frequency and abundance in all meat types. Results obtained in this study have implications for public health and will lead to steps that will further enhance the safety of animal foods in order to protect consumers and the animal production industry. Omotoso a nd Omojola. - Int. J. of Health, Animal science a nd Food safety 2 (2015) 32.-.40 33 HAF © 2013 Vol. II, No. 1 ISSN: 2283-3927 1 Introduction Fluoroquinolones are syn thetic antimicrobials used in the treatment of severe and invasive infections in humans and animals. The presence of fluoroquinolone residues in meat has attracted extensive atten tion from national and international public health ag encies. This is because of th e risks associated with th e consumption of food con taminated with fluoroquinolone residues as they may be directly toxic or be a source of resistan t human pathogens, representing a possible risk to human health (Juan -García et al. 2006). Condition s such as phototoxic skin reactions in humans (Klecak et al., 1997) and chondrotoxic effects in young animals (Stahlmann et al., 2000) can be induced by fluoroquinolones. Probably the mos t importan t threat to human health as a result of fluoroquinolon e use in animal production is th e developm ent of fluoroquinolone-resistant bacteria as it has been discovered that a major route of transmission of resistant micro-organisms from animals to humans is through th e food chain (H ernández-Serrano, 2005). The abuse of veterinary drugs is one of the causes of drug residues in animal products (Saleh zadeh et al, 2006, Fagbamila et al., 2010). Drugs belonging to the fluoroquinolone class such as enrofloxacin, n orfloxacin, pefloxacin, ciprofloxacin and ofloxacin are often used in livestock production to con trol diseas es, leading to the production of healthy animals, thereby reducing farmers’ losses. Ofloxacin is a fluoroquinolon e of interest due to the resistance of a broad spectrum of microbes to it, especially as a result of its use as grow th promoter (Naeem et al., 2006). Low levels of ciprofloxacin, another fluoroquinolone, can strongly suppr ess facultative anaerobic human intestinal microflora (Cerniglia and Kotarski, 1999); it also leads to th e developmen t of resistant bacteria. Similarly, exposure to norfloxacin has been shown to induce resistance to other fluoroquinolon es and even to the structurally unrelated aminoglycosides in coagulase- negative staph ylococci (Deshmukh et al., 1997). Detection and control of fluoroquinolon e residues in meat is important due to mark et globalization and food safety issues. Methods developed for detecting f luoroquinolon e residues include microbiological assays, immunoassays, liquid chromatograph y, among others. Microbiological inhibitory tests, which rely on inhibition of g rowth of the test organism by th e residual drug, are inexpensive and simple; however, most lack specificity and few are quantitative. Microbiological assays are generally used for residue screening as part of an integrated s ystem with follow-up confirmatory analysis of suspicious samples (Choi et al., 1999). High performance liquid chromatography (HPLC) is used to separate, iden tify an d quantify different ch emical componen ts. It has been successfully applied to the detection of different drug residues in foods of animal origin (Reig et al., 2006). In order to protect human health, regulatory bodies such as the European Union and Codex Alim entarius Commission, have established maximum residue limits (MRLs) for veterinary drugs in differen t matrices of differen t animal species. Th e EU MRL for the sum of enrofloxacin and ciprofloxacin is adopted for comparison in this study. The World Health Organization has been cam paigning for an tibiotic resistance to be tackled from a food safety perspective (WH O, 2011). There is th erefore a need to establish a comprehensive database for residues of relevan t antibiotics in the m eat production industry as a step towards con trolling th e ris e in antimicrobial resistance in Africa. Presen tly, several countries cannot assure consumers of the safety of th e meat products th ey consume with Omotoso a nd Omojola. - Int. J. of Health, Animal science a nd Food safety 2 (2015) 32.-.40 34 HAF © 2013 Vol. II, No. 1 ISSN: 2283-3927 respect to drug residue con cen tration. This study was set up to investigate the presence of ciprofloxacin, norfloxacin and ofloxacin residues in four commonly consumed m eat types in Nigeria. 2 Materials and Methods A total of th ree hundred and twen ty raw meat samples (comprising 80 each of beef, chicken, chevon and pork ) were collected from four major markets in Ibadan, Nigeria. Th ey were collected on ice and kept frozen at -30C until analyzed. The analyses w ere carried out a t the Meat Science Laboratory of th e University of Ibadan and National Agency for Food an d Drug Administration and Control (NAFDAC) Laboratory, Agulu, Anambra State, Nigeria. Th e samples were screened for three fluoroquinolones, ciprofloxacin, norfloxacin and ofloxacin . 2.1 Qualitative analysis: Microbiological screening Initial screening was done using the One Plate Tes t (OPT) as described by Alla et al. (2011). The screening procedure has been previously reported in detail in a similar experimen t by Omotoso and Om ojola (2014). The tes t organism was Escherichia coli (ATCC 10536). Zone of inhibition was measured with mm graduated ruler. Negative samples were discarded while suspicious and positive samples were subjected to fu rther analysis for iden tification an d quantification of residual ciprofloxacin, norfloxacin and ofloxacin. 2.2 Identification and quantitative analysis Following residue extraction as described by Ovando et al. (2004), the fluoroquinolones, ciprofloxacin, norfloxacin and ofloxacin were identified using HPLC with UV detection. The HPLC equipmen t was Elite Lachrom VWR, Hitachi ch romatograph (Hitachi, Tokyo, Japan) comprising L2200 autosampler, L2130 pump and L2350 column oven, equipped with L2400 UV VIS detector and Ezchrom Elite software. Th e separating column was Elite C18 (250mmx4.6mmx5μm) (Hitachi, Tok yo, Japan). Reference standards of ciprofloxacin (99.6%), norfloxacin (99.5%) and ofloxacin (99.9) were provided by NAFDAC. Details of th e extraction and subsequent analysis have been reported by Omotoso and Omojola (2014). 2.3 Statistical analysis Statistical analysis was accomplished by analysis of variance using SPSS (2005). Treatm en t means were compared using Duncan Multiple Range Test and statis tical significance was set a t a probability of P ≤ 0.05. Omotoso a nd Omojola. - Int. J. of Health, Animal science a nd Food safety 2 (2015) 32.-.40 35 HAF © 2013 Vol. II, No. 1 ISSN: 2283-3927 3 Results Frequency of antibiotic contamination in the different meat types is shown in Figure 1. Initial microbiological screening revealed that more than half of the chicken samples tes ted positive for antimicrobial residues. This was followed by beef with 50% positive samples. Similarly, 40% of pork and chevon samples tes ted positive for antimicrobial residues. Figure 1. Percen tage of raw m eat samples positive for antibiotic residues . Percentages of meat samples positive for ciprofloxacin, norfloxacin and ofloxacin residues are shown in Figure 2. Mos t chicken and beef samples had detectable levels of ciprofloxacin; however, less than half of pork and chevon contained residues of this popular fluoroquinolone. The m eat type with the high est frequency of n orfloxacin residue was chick en, follow ed by beef. Ofloxacin occurred more frequen tly in beef (48.75%) than other meat types. It is worthy of note that pork, followed by chev on, had the least number of samples with detectable levels of an y of the th ree fluoroquinolon es. Levels of ciprofloxacin, norfloxacin and ofloxacin residues in the tes ted raw meat samples are shown in Table 1. In beef, m ean residual ciprofloxacin (231.08 ± 564.30) and norfloxacin (173.40 ± 154.57) were above th e adopted MRL of 100 µg/kg, while ofloxacin value (79.28 ± 183.70) was below the limit. In chicken, how ever, norfloxacin was the most abundan t fluoroquinolone, with a mean value that was sligh tly above 100µg/kg and significantly differen t from the levels of ciprofloxacin and ofloxacin. Although ciprofloxacin in pork was very high, levels of norfloxacin and ofloxacin in this meat- type were very low. High level of ciprofloxacin was also recorded in chevon (345.62 ± 796.35) but this had low er n orfloxacin and ofloxacin residues. Percen tages of meat samples containing residues of ciprofloxacin, norfloxacin an d ofloxacin above adopted MRL are shown in Table 2. 0 10 20 30 40 50 60 Beef Chicken Pork Chevon P e r c e n t (% ) p o s it iv e f o r a n ti b io ti c r e s id u e s Meat Type Omotoso a nd Omojola. - Int. J. of Health, Animal science a nd Food safety 2 (2015) 32.-.40 36 HAF © 2013 Vol. II, No. 1 ISSN: 2283-3927 Figure 2. Detection of ciprofloxacin, norfloxacin and ofloxacin in raw meat. Table 1. Mean concentration of fluoroquinolones in beef, chicken, pork and chevon. Meat type Mean concentrations (µg/kg) of fluoroquinolones P value Ciprofloxacin Norfloxacin Ofloxacin Beef 231.08 ± 564.30 173.40 ± 154.57 79.28 ± 183.70 0.536 Chicken 67.85 ± 125.42b 113.59 ± 158.32a 13.55 ± 22.46c 0.022 Pork 315.30 ± 834.35a 11.39 ± 28.31b 27.02 ± 64.48b 0.091 Chevon 345.62 ± 796.35a 30.93 ± 129.18b 12.37 ± 30.64b 0.047 a, b, c : means in the same row with different superscripts are significantly different (P ≤ 0.05) 4 Discussion Several reports have shown that th e presence of fluoroquinolone residues in human food obtained from animal sources has reduced the effectiveness of these agen ts in human medicine. In addition, direct toxicity and other side effects of fluoroquinolones are w ell established in animals and humans (Gru challa and Pirm ohamed, 2006; Khadra et al., 2012). Thus it is importan t to monitor residues of fluoroquinolones in frequ ently consumed meat and mea t products. 0 10 20 30 40 50 60 Ciprofloxacin Norfloxacin Ofloxacin P e r c e n t (% ) p o s it iv e s a m p le s Fluoroquinolones Beef Chicken Chevon Pork Omotoso a nd Omojola. - Int. J. of Health, Animal science a nd Food safety 2 (2015) 32.-.40 37 HAF © 2013 Vol. II, No. 1 ISSN: 2283-3927 Table 2. Percentag e of raw meat samples with fluoroquinolone residues above maximum residue limit. Fluoroquinolones Meat type n/N % above MRL* Ciprofloxacin Beef 40/80 23.75 Chicken 41/80 15.00 Pork 28/80 20.00 Chevon 23/80 38.75 Norfloxacin Beef 37/80 25.00 Chicken 44/80 35.00 Pork 24/80 6.25 Chevon 20/80 5.00 Ofloxacin Beef 39/80 15.00 Chicken 32/80 0.00 Pork 20/80 10.00 Chevon 11/80 0.00 n=number of positive samples; N = number of examined samples *100µg/kg- European Union MRL (Commission Regulation (EU) 37/2010) for the sum of ciprofloxacin and enrofloxacin in poultry muscle. In the present s tudy, raw samples of fou r meat types comm only consumed in Nigeria were tested for the pres ence of fluoroquinolones. Approximately 50% of th e three hundred an d twen ty samples were positive for residues of at least on e antibiotic active against the tes t organism, Escherichia coli. Fifty-five percen t of chicken meat and fifty percen t of beef were positive for antibiotic residues. In a related study using samples obtained from open markets in Sokoto, Nigeria, 44% of slaughtered cattle were found to con tain residues of antibiotics (Ibrahim et al., 2009). Residues have been reported in 21% of meat samples in Ghana (Novais et al., 2010), and 70% in Tanzania (Kurwijila et al., 2006). Tajick and Shohreh (2006) also found that more than 50% of poultry m eat tissues tes ted in Iran had residues of an timicrobials. In con trast, Alla et al. (2011) analyzed beef samples in Sudan and reported that only 3% of the muscles contained an tibiotic residue. Findings in this study rev ealed that fluoroquinolon es are frequently administered in meat - animal production in Nigeria. Results of microbiological screening were similar to findings in the screening of broiler m eat and beef sold in the markets of Ankara, T u rkey by Er et al., (2013 ) who discovered that 45.75% of chicken and 57.7% of beef were positive for quinolone residues; however, mean con centrations obtained in their study were below th e 100µg/kg mark (th e Omotoso a nd Omojola. - Int. J. of Health, Animal science a nd Food safety 2 (2015) 32.-.40 38 HAF © 2013 Vol. II, No. 1 ISSN: 2283-3927 mean lev els of quinolon e an tibiotic residue were found to be 30.81 ± 0.45 μg/kg in positiv e chicken samples and as low as 6.64±1.11μg/kg in positive beef samples ). In a similar experim en t to test for th e presence of fluoroquinolon es in chick en, Omotoso and Om ojola (2014) reported ciprofloxacin level of (354.83 ± 716.43 μg/kg) in imported chicken. The frequen cy of occurrence and high concentration of ciprofloxacin in th e present study may be due to the fact that ciprofloxacin is also a mark er residue for en rofloxacin. En rofloxacin is approved for use as a veterinary drug and when it is metabolized, its pharmacologically active metabolite, ciprofloxacin, is produced. Hence detection of ciprofloxacin during marke t screening often reflects the use of both enrofloxacin and ciprofloxacin (Navrátilová et al., 2011; Botsoglou and Fletouris 2001). Mean norfloxacin residues in beef and chick en were higher than the MRL. Ofloxacin concen tration was the lowes t in all tested m eat types except pork, which had higher ofloxacin than norfloxacin. Generally, ciprofloxacin was the most abundant in all except for chicken meat which had the highest mean norfloxacin (173.40 ± 154.57). The level of abundance of th e individual agents in each meat type is as follows Beef: Ciprofloxacin > Norfloxacin > Ofloxacin Chicken: Norfloxacin > Ciprofloxacin > Ofloxacin Pork: Ciprofloxacin > Ofloxacin > Norfloxacin Chevon: Ciprofloxacin > Norfloxacin > Ofloxacin The study shows that ofloxacin is not administered as frequ ently as the other tw o antimicrobial agents in livestock production in the study area. No individual chicken or chevon sample had ofloxacin lev els above MRL (Table 2). Howev er, 15% and 10% of beef and pork respectively contain ed ofloxacin at levels above MRL. This shows that ofloxacin may no t necessarily be a drug of con cern in chicken m eat and ch evon. Detection of fluoroquinolon e residues in meat samples from the city of Ibadan, Nigeria, corroborat es the findings of other researchers in other coun tries where residue monitoring is absent or inadequate. 5 Conclusion In the absence of official m onitoring programm e on drug residues in developing countries, misuse of antimicrobials, including fluoroquinolon es, is inevitable. Detection of residues of fluoroquinolones in half of tested meat samples shows the widespread use of these agen ts in cattle, poultry, hog and goat production in Nigeria during the period of th e study. The pres en t situation can be con trolled as less frequent contamination and reduced concentration in contaminated samples may result when steps are tak en towards encou raging prudent use of fluoroquinolones. Consumer protection can be ensured by screening food animals for residues of antibiotics before slaughter. 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