Research Communication Determination of oxytetracycline residues in cattle
meat marketed in the Kilosa district, Tanzania
Oxytetracycline is used to treat various diseases in cattle. However, its use may be associated Zuhura I. Kimera1 with unacceptable residue levels in food. Oxytetracycline residues in tissues from indigenous Robinson H. Mdegela2 cattle were determined in a cross-sectional study conducted in the Kilosa district, Tanzania, Consolatha J.N. Mhaiki3Esron D. Karimuribo2 between November 2012 and April 2013. A total of 60 tissue samples, including muscle, liver and kidney, were collected from slaughterhouses and butchers and analysed for oxytetracycline using high-performance liquid chromatography. Oxytetracycline residues were found in 71.1% of the samples, of which 68.3% were above acceptable regulatory levels. The mean concentration of oxytetracycline across tissues was 3401.1 µg/kg ± 879.3 µg/kg; 1Department of Fisheries concentrations in muscle, liver and kidney were 2604.1 µg/kg ± 703.7 µg/kg, 3434.4 µg/kg ± Development, Ministry 606.4 µg/kg and 3533.1 µg/kg ± 803.6 µg/kg, respectively. High levels of oxytetracycline of Livestock and Fisheries residue in meat from indigenous cattle may pose a health threat to consumers in Kilosa. Development, Tanzania The findings possibly reflect a general lack of implementation of recommended withdrawal 2Department of Veterinary periods, ignorance about drug use and lack of extension services. Strict regulation of the use Medicine and Public Health, of antimicrobial drugs in the livestock industry and associated testing of animal-derived food Sokoine University of sources prior to marketing are required.
Agriculture, Tanzania 3Department of Soil Science, Sokoine University of Agriculture, Tanzania In Tanzania, livestock farming generally follows traditional practices whereby pastoralists and agro-pastoralists rear mostly local breeds for meat and milk production (Ministry of Livestock Department of Physical Science, Sokoine University and Fisheries Development 2010). Livestock farming faces several constraints, including diseases, of Agriculture, Tanzania poor genetic potential of animals, poor management and nutrition, and drought (Mellau, Nonga & Karimuribo 2010). Owing to limited extension services and poor animal health delivery systems, farmers buy veterinary drugs from veterinary shops and treat their livestock themselves. However, when drugs are administered by non-professionals, correct dosages and withdrawal periods are unlikely to be observed, which poses a potential hazard to human health (Barton 2000). The possible effects include toxic or allergic reactions, development of bacterial resistance and disturbance of normal intestinal microflora composition (Abbasi et al. 2011; Pena et al. 2005; PO Box 3021, Morogoro Uekane, Neto & Gomes 2011).
The use of antimicrobial agents in food-producing animals has become a notable public health Dates:
Received: 24 Nov. 2014
concern, especially in developing countries where such drugs are administered indiscriminately Accepted: 27 Aug. 2015 (Bedada & Zewde 2012; Muriuki et al. 2001; Olatoye & Basiru 2013). The increased use of Published: 27 Nov. 2015 antimicrobials in animal production is due to their being applied both therapeutically and prophylactically; some are also routinely added to animal feeds at sub-therapeutic levels for How to cite this article:
Kimera, Z.I., Mdegela, R.H.,
growth promotion (Bedada & Zewde 2012; Nonga et al. 2010).
Mhaiki, C.J.N., Karimuribo, E.D., Mabiki, F., Nonga, H.E. Oxytetracycline is one of the most commonly used antibiotics in livestock production in Tanzania et al., 2015, ‘Determination and other African countries (Katakweba et al. 2012; Olufemi & Agboola 2009). Apart from being of oxytetracycline residues in cattle meat marketed in a broad-spectrum antibiotic, oxytetracycline is also cheap, readily available from veterinary the Kilosa district, Tanzania', shops and accessed easily, without restrictions, by farmers (Nonga et al. 2009; Olatoye & Basiru Onderstepoort Journal of 2013). Katakweba et al. (2012) reported that a number of drugs, such as oxytetracycline, are used Veterinary Research 82(1), abusively to treat and protect cattle against various diseases. Moreover, informal vendors are Art. #911, 5 pages. often seen selling oxytetracycline and other tetracycline-based drugs at informal markets and along the road, without any prescription being required or restrictions imposed (Bedada & Zewde 2012; Karimuribo et al. 2013).
To protect humans from harmful effects of veterinary drug residues in animal-derived food Read online:
sources, the United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO) have set standards for maximum residue limits in foods. These limits apply to read online.
Copyright: 2015. The Authors. Licensee: AOSIS OpenJournals. This work is licensed under the Creative Commons Attribution License.
Research Communication to the parent drug or chemical and its metabolites that may standard (Sigma, St Louis), oxalic acid dihydrate, citric acid accumulate and be deposited or stored within the cells, tissues monohydrate and disodium ethylenediaminetetraacetate or organs following administration of the compound. The (Na EDTA) (Techno Pharmachem, India), anhydrous acceptable maximum residue limits for tetracycline-based disodium hydrogen phosphate (Carlo Ebra, Milan), methanol, compounds, including chlortetracycline and oxytetracycline, acetonitrile and HPLC water (Carlo Ebra, Milan). Whatman are set at 200 µg/kg, 600 µg/kg and 1200 µg/kg for cattle- membranes, microsyringe membrane filters (Chromafil CA derived muscle, liver and kidney, respectively.
20/25s), nylon membranes (P/N 0235-0301) and Cronus C-18 solid-phase extraction cartridges (200 mg/3 mL, Withdrawal periods of 5–20 days are recommended before Labhut) were used for the chromatography steps.
slaughter, depending on the species and the nature of the food products (Blanchflower et al. 1997). However, regulatory Each tissue sample (5 g) was homogenised three times in a bodies in Tanzania have not yet set withdrawal periods for Mcllvaine buffer–EDTA solution (20 mL, 20 mL and 10 mL) veterinary drugs and farmers rely only on the directions and collected in a 50-mL polypropylene centrifuge tube. The given in the package insert. This information is always mixture was then centrifuged at 4000 g for 10 min and the written in English, a language the majority of Tanzanian supernatant was filtered through a single Whatman filter, farmers do not understand. Therefore, farmers rarely comply pre-moistened with 2 mL Mcllvaine buffer–EDTA solution, with the recommendations and usage is also not monitored into a 250-mL sidearm flask. The solid-phase extraction was by the responsible regulatory authorities. Consequently, conducted by conditioning the extraction cartridge with veterinary drug residues are likely to be present in food of 20 mL methanol followed by 20 mL HPLC-grade water. The animal origin. This study sought to investigate the presence final sample extract was applied to an 18-carbon cartridge, of excessive concentrations of oxytetracycline residues in which was subsequently washed with 20 mL HPLC-grade cattle meat marketed in the Kilosa district, Tanzania.
water. Oxytetracycline was eluted with 6 mL methanolic oxalic acid solution into a 10-mL volumetric flask, which was Materials and methods
then filled with water to volume.
Muscle, liver and kidney sample extracts were analysed for This study was conducted in the Kilosa district (5°55'–7°53'S, oxytetracycline residues according to AOAC Official Method 36°30'–37°30'E), which is located approximately 300 km west 995.09 (AOAC International 2000), with some modifications. of Dar es Salaam in east central Tanzania. The district is The HPLC instrument (Shimadzu 20AD) was fitted with divided into three zones, namely Kilosa, Gairo and Mikumi, an autosampler (SIL-20 AHT) and a UV detector at 350 nm and spans a land area of 19 056 km2. The human population was used for analysis. A reversed-phase 18-carbon column of the district is documented as 438 175 (National Bureau of (150 mm × 4.60 mm; particle size, 5 µm; Supelco) was used Statistics 2012).
at 25 °C for separation. The sample injection volume was 1 µL at a flow rate of 0.8 mL/min. A low-pressure gradient Samples were obtained from cattle slaughtered at the Kilosa, system, consisting of water and methanol, acetonitrile and Gairo and Mikumi slaughter slabs and at the Parakuyo aqueous oxalic acid (10:30:60) as the mobile phase, was and Chakwale livestock markets. Records obtained during applied for a retention time of 15 min. To determine residues sampling indicated that the animals originated from ten in the samples, they were analysed concurrently with the different villages across the district.
oxtetracycline standard solutions (0.05 µg/mL, 0.1 µg/mL, 0.25 µg/mL, 0.5 µg/mL and 1.0 µg/mL). The extract from Animals were selected for sampling using simple random each sample was injected in duplicate to obtain an average sampling techniques. Information such as the name of the peak height of positive samples. Samples were considered owner or supplier, the village of origin, any pathological positive for oxytetracycline residue if their retention time lesions at the time of sampling and inspection status prior and peak corresponded to that of the reference standards. to slaughter was collected before sampling. Three samples The retention time of the reference standard was 4.3 min.
(muscle, liver and kidney) of 100 g – 200 g each were obtained from each of 20 animals, yielding 60 tissue samples Oxytetracycline residues in tissue sample extracts were in total. All samples were collected in separate polythene quantified against the aforementioned concentrations of bags and transported on ice to the analytical laboratory at the oxytetracycline reference standards. The standards the Sokoine University of Agriculture (Faculty of Veterinary were analysed in duplicate and the peak areas appropriate Medicine). The samples were stored in a freezer at -20 °C for to specific standard concentrations were measured. These approximately 1 week and thawed at room temperature for were used to calculate the residue concentrations in sample eight hours before analysis.
High-performance liquid chromatography (HPLC) was used As it was not possible to obtain cattle that had not been to analyse the samples. All the reagents and chemicals were of treated with oxytetracycline or other veterinary drugs prior HPLC or analytical grade. Reagents included oxytetracycline to slaughter, three oxytetracycline-free guinea pigs (all of the Research Communication same age, sex and weight) raised at the Faculty of Veterinary Medicine, Sokoine University of Agriculture, were used as controls. One guinea pig served as negative control, whereas the other two were injected with 20% oxytetracycline (Laprovet, Indre-et-Loire) at 10 mg/kg body weight and 20 mg/kg body weight, respectively. After 24 h the three control animals were placed in a gas chamber (61.4% CO , 20.3% O and 18.29% N ) for 5 min before being humanely killed. Muscle, liver and kidney tissue samples (5 g each) were subjected separately to the extraction, clean-up and elution procedures as described for the test samples derived from cattle. Two samples of each of the three tissue types were taken from each of the control animals. The control samples were run through the HPLC column under similar Retention time (min)
conditions to the oxytetracycline standard solutions. A blank sample eluted from the solid-phase extraction cartridge was FIGURE 1: Combined chromatogram of the negative control sample (without
included to check for the analytical column efficiency during oxytetracycline) and the sample spiked with 10 mg/kg 20% oxytetracycline.
Operational conditions of the HPLC instrument were tested to ensure the robustness of the method. These checks included y = 15405x + 5123.3 varying the percentage of solvents in the mobile phase R² = 0.9227 (methanol: 10% – 30%; acetonitrile: 10% – 30%; oxalic acid: 50% – 70%), the column temperature (from 25 °C to 40 °C) and the flow rate (from 0.6 mL/min to 1 mL/min). The pH Area 10 000
of the buffer was changed from 2.0 to 7.0. Stability of the sample and standard solutions at room temperature was also tested.
The data were analysed using Epi Info (version 7) (Centre for Disease Control, Atlanta, USA). The Chi-square statistic FIGURE 2: Calibration curve depicting the best-fit line from analytical
and confidence intervals were used to compare proportions; a probability of P < 0.5 was considered statistically significant. Descriptive statistics were used to compute and 83% oxytetracycline was recovered from the samples. means, standard deviations and range. Analysis of variance The limit of detection and the limit of quantification were (ANOVA) was used to compare differences in means of 1.936 mg/kg and 6.7 mg/kg, respectively. The correlation coefficients associated with the linear regression for the analytical oxytetracycline standard (Figure 1) and test samples (Figure 2) were R2 = 0.92 and R2 = 0.94, respectively. (The linear regression equation is shown in Figure 2). For Permission for this study was granted by the Executive quantification purposes, the best-fit line was expected to Directors of the Kilosa District Council and ethical approval be 99%, but based on the local environment, nature of the for the study was obtained from the Ethical Committee of the equipment and the laboratory used, the obtained fits were Sokoine University of Agriculture. The university issued a research permit letter on behalf of the Tanzanian Commission for Science and Technology.
Of the 20 animals sampled, 17 (85%) tested positive for oxytetracycline residues. Moreover, 68.3% of the positive tissue samples contained oxytetracycline residues above the acceptable levels for muscle, liver and kidney (Food and Agriculture Organization/World Health Organization The non-spiked samples from the control animals peaked at a 2014). The mean concentration of oxytetracycline residues different time from that of the analytical standards, whereas across all tissues was 3401.1 µg/kg ± 879.3 µg/kg. For the both oxytetracycline-spiked samples peaked at 4.3 min respective tissue types, the mean concentrations were as expected. The higher concentration of oxytetracycline 2604.1 µg/kg ± 703.7 µg/kg for muscle, 3434.4 µg/kg ± was associated with a higher peak. The peak for extracted 606.4 µg/kg for liver and 3533.1 µg/kg ± 803.6 µg/kg for oxytetracycline was also detected at 4.3 min and was of kidney tissue. Oxytetracycline concentrations were higher similar height to the spiked control sample. Between 79% than the acceptable levels in all tissue types sampled from Research Communication TABLE 1: Oxytetracycline concentration in cattle tissue from the Kilosa district,
is associated with high levels of residues in edible tissues of food-producing animals. The high incidence of Zone of sample Number of
Antibiotic concentrations (µg/kg)
oxytetracycline residues observed in the current study probably reflects cattle being sold for slaughter whilst under 1548.0 ± 708.3 1484.8 ± 493.2 1126.7 ± 204.3 a therapeutic or prophylactic regimen of oxytetracycline or 1553.2 ± 957.1 1307.6 ± 660.1 1083.3 ± 282.7 animals being slaughtered before the end of the withdrawal 388.3 ± 183.5 582.4 ± 220.6 357.8 ± 192.8 period (5–7 days when the antibiotic has been administered at a dose of 10 mg/kg for 7 days [Aiello & Moses 2010]). It animals from the Kilosa and Mikumi zones (Table 1). Lower is also of concern that these levels of oxytetracycline were concentrations of oxytetracycline were found in samples found in tissue from indigenous cattle, because more than collected from cattle slaughtered in the Gairo zone (Table 1).
98% of the cattle population in Tanzania (approximately 21 million) are indigenous breeds and the main source of meat consumed in Tanzania (National Bureau of Statistics 2012). The purpose of this study was to determine the presence of In addition, there is no official monitoring programme oxytetracycline residues in beef from indigenous cattle in the and consumer response towards the dangers posed by Kilosa district, Tanzania. The results showed a high residual drug residues is passive. Thus, there is a risk of sustained presence of oxytetracycline (71.1%), with a notable number consumer exposure to antibiotic residues and the associated of positive samples being above the acceptable maximum effects on human health.
residue levels recommended for meat by the WHO and FAO. The finding can probably be attributed to widespread The high levels of antibiotic residues found may be due use of oxytetracycline for treatment and prevention of cattle to insufficient knowledge about drug use and the lack of diseases and is possibly exacerbated by failure to observe extension services. Livestock keepers in Kilosa are mostly withdrawal periods (Karimuribo et al. 2013). Higher levels Maasai, Mang'ati or Sukuma, who are known pastoralists of oxytetracycline residue were found in liver and kidney in Tanzania, and livestock extension officers' access to tissue than in muscle, which can be attributed to their being these farmers is problematic. There are few livestock field organs of metabolism and excretion and therefore they are at officers available and the majority are found in the vicinity of town centres such as Gairo. In the Kilosa and Mikumi greater risk of exposure to residues (Olatoye & Ehinmowo zones, no veterinary services were offered because of the 2010). After administration, oxytetracycline enters all tissues areas' remoteness and poor infrastructure. The higher and body fluids, but higher concentrations are found in the oxytetracycline residue levels found in samples from villages kidney, liver, bile, lungs and bones (Aiello & Moses 2010). in the latter zones may be due to the lack of veterinary Oxytetracycline is excreted mainly via urine and bile, which services, including extension services. The role of livestock explains the high concentrations of residue observed in extension officers is to advise farmers on proper animal kidney and liver tissues in this study. As liver and kidney management systems and disease control programmes, are considered luscious offal from cattle and are popular including vaccination. Our findings support the conclusion amongst most meat consumers in Tanzania, the detection of of Muriuki et al. (2001) that variation in residue levels – high levels of oxytetracycline residues in these tissues is of even from the same district – reflects the variation in animal importance to public health.
husbandry practices as used by different livestock keepers and in different areas.
The proportions of oxytetracycline-positive samples found in this study were higher than reported in other studies Easy access to antibiotics such as oxytetracycline, together (Olufemi & Agboola 2009), although Bedada and Zewde with a lack of awareness, insufficient extension activities and (2012) reported a comparable proportion of oxytetracycline- inadequate usage guidelines from manufacturers, may lead positive muscle tissue samples (71.3%; n = 384) from cattle to misuse and overuse of the drug and possibly failure to in Ethiopia. However, in a study analysing muscle tissue observe withdrawal periods. These actions may contribute from cattle in the Morogoro and Dodoma municipalities, to the presence of high levels of antibiotic residues in meat Tanzania, only 41.2% of samples tested positive for (Nisha 2008).
oxytetracycline residues (Mmbando 2004). Oxytetracycline residues in muscle tissue were reported in 45.6% and 54.4% The lack of farmers' awareness of the possible side-effects of samples in studies from Kenya (Muriuki et al. 2001) and of antimicrobials and other drugs in humans also has to Nigeria (Olufemi & Agboola 2009), respectively, which are be considered (Karimuribo et al. 2013). Administration of both relatively low compared to levels seen in the current drugs to food-producing animals requires consideration not study. This difference may be due to different sample types, only of effects on the animal but also of effects in humans laboratory methods and possible variation in oxytetracycline who consume food from these animals. The high levels of use depending on the animal management system of the antibiotic residues found in this study suggest that the public consuming animal products originating from the Kilosa district may have been exposed to antimicrobial Nisha (2008) reported that indiscriminate use of antibiotics residues. Our results, together with those of Mmbando to treat pyrexia, inflammation, wounds and viral diseases (2004) and Nonga et al. (2009) about antibiotic residues in Research Communication broiler chickens, suggest that some communities are exposed to small doses of antimicrobials from various animal food sources. This practice may contribute to the development of Abbasi, M.M., Babaei, H., Ansarin, M., Nourdadgar, A. & Nemati, M., 2011, ‘Simultaneous determination of tetracyclines residues in bovine milk samples by solid phase extraction and HPLC-FL method', Advanced Pharmaceutical Bulletin Aiello, S.E. & Moses, M.A. (eds.), 2010, The Merck Veterinary Manual for veterinary professionals, Merck, Sharp and Dohme, Whitehouse Station.
AOAC International, 2000, AOAC Official method 995.09 Chlorotetracycline, The findings of our study may be indicative of the oxytetracycline and tetracycline in edible animal tissues: Liquid chromatographic method, AOAC International, viewed 20 March 2013, from inappropriate use and management of veterinary drugs by livestock keepers in the Kilosa district specifically, but Barton, M.D., 2000, ‘Antibiotic use in animal feed and its impact on human also more generally in Tanzania. We therefore recommend health', Nutrition Research Reviews 13, 279–299. stricter regulation of the use of veterinary drugs in the livestock industry as well as the inspection of livestock Bedada, A.H. & Zewde, B.M., 2012, ‘Tetracycline residue levels in slaughtered beef cattle from three slaughterhouses in central Ethiopia', Journal of Global products prior to marketing. Furthermore, livestock keepers Veterinaria 8(6), 546–554.
need to be educated on the importance of adhering to the Blanchflower, W.J., McCracken, R.J., Haggan, A.S. & Kennedy, D.G., 1997, ‘Confirmatory assay for the determination of tetracycline, oxytetracycline, chlortetracycline and recommended drug withdrawal periods and possible its isomers in muscle and kidney using liquid chromatography-mass spectrometry', Journal of Chromatography. B, Biomedical Sciences and Applications 692, 351– human health effects associated with presence of veterinary drug residues in food of animal origin. Veterinarians Food and Agriculture Organization/World Health Organization, 2014, Residue and livestock officers should also promote alternative evaluation of certain veterinary drugs: Joint FAO/WHO Expert Committee on Food Additives, 78th meeting 2013, FAO JECFA Monographs no. 15, Food and Agriculture management options aimed at good animal husbandry and Organization, Rome.
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Katakweba, A.A.S., Mtambo, M.M.A., Olsen, J.E. & Muhairwa, A.P., 2012, ‘Awareness of human health risks associated with the use of antibiotics among livestock keepers The authors acknowledge financial support from the and factors that contribute to selection of antibiotic resistance bacteria within livestock in Tanzania', Livestock Research for Rural Development 24(10), article no. project Enhancing Pro-poor Innovations in Natural 170, viewed 06 June 2013, fr Resources and Agriculture Value Chains (EPINAV). We Mellau, L.S.B., Nonga, H.E. & Karimuribo, E.D., 2010, ‘A slaughterhouse survey of lung appreciate the support and cooperation from the livestock lesions in slaughtered stocks at Arusha, Tanzania', Preventive Veterinary Medicine 97, 77–82. PMID: 2087 keepers and field officers in the study area. Technical Ministry of Livestock and Fisheries Development, 2010, Livestock sector development support rendered by M. Mugusi and J. Fitwangile is strategy, Ministry of Livestock and Fisheries Development, Dar es Salaam.
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A Survey of Barriers to Treatment Access in Rheumatoid Arthritis Country Annex Report: Argentina August 2010 Barriers to RA treatment access across Latin America: Argentina Barriers to RA treatment access across Latin America: Argentina 1 Interviews Six respondents were interviewed in the Argentinean part of the study. Four of the respondents were doctors whilst two were patient representatives. All doctors were hospital based, three from public Hospital and one from a private Hospital. The two patient representatives were from an NGO which provides counsel to RA patients.
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