Detection of antimicrobial and anthelmintic residues in bulk tank milk from four different mesoregions of Minas Gerais State-Brazil

Milk is one of the most consumed foods worldwide and its production has shown a continuous increase (Oliveira et al., 2010). Brazil is one of the largest producers and consumers of cow milk in the world, with about 30.7 billion liters of milk produced in 2010. Minas Gerais State is the major milk producer in Brazil, and it is responsible for approximately 30% of the national milk production. The Central Mineira, Oeste of Minas, Triângulo Mineira/Alto Paranaíba and Metropolitana de Belo Horizonte mesoregions are important milk producing regions in Minas Gerais State, Brazil, with a settlement of several important dairy industries (Figure 1). Raw milk collected in these regions supplies dairies with cheese, dry milk, yogurt, milk beverages, dessert cream, pasteurized and UHT milk production (Borges et al., 2013). 

Milk is one of the most consumed foods worldwide and its production has shown a continuous increase (Oliveira et al., 2010).Brazil is one of the largest producers and consumers of cow milk in the world, with about 30.7 billion liters of milk produced in 2010.Minas Gerais State is the major milk producer in Brazil, and it is responsible for approximately 30% of the national milk production.The Central Mineira, Oeste of Minas, Triângulo Mineira/Alto Paranaíba and Metropolitana de Belo Horizonte mesoregions are important milk producing regions in Minas Gerais State, Brazil, with a settlement of several important dairy industries (Figure 1).Raw milk collected in these regions supplies dairies with cheese, dry milk, yogurt, milk beverages, dessert cream, pasteurized and UHT milk production (Borges et al., 2013).

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Antimicrobial and anthelmintic drugs are widely used in veterinary medicine and the residues of these drugs constitute a risk for human health since they can cause allergic reactions in hypersensitive individuals and may lead to the appearance of drugresistant bacteria, nematodes or trematodes.Thus, analysis of these residues plays a key role in ensuring food safety, and to accomplish this objective, maximum residue levels for these drugs have been established by regulatory agencies in many countries, including Brazil (Brazil, 1999).Failure to observe stipulated withdrawal periods for these drugs has resulted in several reports of their residues in milk.However, few data are available in Brazil, mainly when considering the anthelmintic drugs (Lobato et al., 2006;Nero et al., 2007;Bando et al., 2009;Fonseca et al., 2009;Oliveira et al., 2010;Zanella et al., 2010).
Thus, the present study aimed to evaluate the residues of antimicrobial and anthelmintic drugs in bulk tank milk samples from four different mesoregions of Minas Gerais State, Brazil.For the present study 83 bulk tank milk samples were collected for anthelmintic residue detection from four different mesoregions (22 cities) of Minas Gerais State (Triângulo Mineiro/Alto Paranaíba, n = 37; Oeste de Minas, n = 26; Central Mineira, n = 15; and Metropolitana de Belo Horizonte, n = 5) during January, 2012 (Fig. 1a), which represent the most important dairy regions of Minas Gerais State, and those 70 milk samples were also analyzed for antimicrobial residues.The antimicrobial residue detection included quinolones, ceftiofur, thiamphenicol, streptomycin, tylosin and tetracyclines, while antithelmintics detected included benzimidazoles, amino benzimidazoles, levamisole, avermectins, thiabendazole, moxidectin and triclabendazole.The preparation of the milk samples, the detection of the antimicrobials (Anti Microbial Array II, cat n°.EV3524A; Randox Laboratories Ltd., UK) and of the anthelmintics (Anthelmintics Array, cat n°.EV3770; Randox Laboratories Ltd., UK) were performed with a biochip array platform, using competitive antibody-capture immunoassay as recommended by the manufacturer's protocol.The amino benzimidazoles assay included detection of amino-mebendazole, albendazole 2-amino sulphone, and amino-fluobendazole; the avermectins assay included emamectin benzoate, epinomectin, abamectin, ivermectin, and doramectin; the thiabendazole assay included detection of cambendazole, thiabendazole, and 5hydroxythiabendazole; and the triclabendazole assay included keto-triclabendazole, triabendazole, and triclabendazole sulphoxide.
http://dx.doi.org/10.1590/1678-41627274The results are showed in Table 1, and were divided in: 1) samples between the limit level of detection of the kits and the Brazilian maximum residue levels (BMRLs) (quinolones 40μg/L; ceftiofur 100 μg/L; thiamphenicol 50μg/L; streptomycin 200μg/L; tetracyclines 100μg/L; avermectins 10μg/L; and 2) samples with levels above the BMRLs.Although most of the drug residue concentrations were below the current BMRLs (Brazil, 1999), this survey indicates that the withdraw periods for antimicrobials, and especially for anthelmintic drugs has not been fully respected by all dairy farmers.
Reports have shown that some anthelmintics found in milk are stable during storage and are not affected by some fermentation processes.
Sometimes even an increase in concentration may occur, as showed in ripened cheese (Whelan et al., 2010).Furthermore, since the use of anthelmintic drugs in lactating cows is prohibited in Brazil, these results demonstrate the need for more strict control of their use to ensure milk safety, quality, and integrity (Brazil, 2002).
Findings similar to the present study were reported by Nero et al. (2007) It is noteworthy that the occurrence of anthelmintc residues reported in this work are slightly higher than those described by Brazil (2011) which described none of milk samples above the BMRLs (Table 1 and Figure 2).Lobato et al. (2006) also reported that 17.8% of the retailed milk samples contained ivermectin levels above the detection limit by highperformance liquid chromatography with fluorescence detector, but none of these samples had levels above the BMRLs.Therefore, the findings in the current report of anthelmintic residues in raw milk are similar to those described by the Program of Analysis of Veterinary Drugs Residues in Food from Animal Origin (ANVISA, 2009) for UHT milk and dry milk, respectively: 2.80%, and 7.25% of positive samples for abamectin residue; 41.29% and 52.17% of positive samples for ivermectin residue; and 3.87% and 5.80% of positive samples for doramectin residue.One sample of UHT milk (0.22%) presented doramectin levels above the BMRLs.All of these reported anthelmintics belong to the abamectins group.
Although fast screening methods (i.e.immunological or microbial inhibition assays) are commonly used to detect the presence of antimicrobial and anthelmintics in foods, more accurate chromatographic methods are required by the governmental regulatory agencies to identify and confirm the presence of these compounds (Oliveira et al., 2010).

Figure 1
Figure 1.a) Geographical origin of raw milk samples submitted for anthelmintic and antimicrobial residue detection from four different mesoregions of Minas Gerais State -Brazil.b) Milk production from the different mesoregions of Minas Gerais State -Brazil in 2010.

Table 1
*BMRLs not established for milk LLD: limit level of detection BMRLs: Brazilian maximum residue levels