Orientador: Felix Guillermo Reyes Reyes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-22T02:54:44Z (GMT). No. of bitstreams: 1 Baptista_RafaeladeCarvalho_M.pdf: 1656875 bytes, checksum: e8e25ea957ad18b82ece74062e9bcd51 (MD5) Previous issue date: 2013 / Resumo: A ovinocultura vem despertando enorme interesse em todas as regiões do Brasil e sua produção destina-se, principalmente, na criação de cordeiros. Contudo, um dos grandes impasses nos sistemas de produção, especialmente em pastagens, são as parasitoses gastrointestinais. Os problemas associados com os parasitas acarretam danos à saúde dos animais, reduzindo o seu desempenho e os lucros do produtor. Como medida profilática, faz-se uso dos anti-parasitários, medicamentos veterinários com ação contra endo e ectoparasitas. Contudo, a utilização indiscriminada dos fármacos tem favorecido o desenvolvimento de estirpes resistentes responsáveis pela rápida ineficácia do composto. Como medida de controle dos nematoides gastrointestinais nas criações comerciais de ovinos, o fármaco costuma ser administrado baseando-se no resultado de duas formas de exame diagnóstico: contagem de ovos dos parasitas nas fezes e o teste FAMACHA® (exame da coloração da mucosa ocular) ou a aplicação do composto em períodos regulares de 28 dias, independente de exame. Na tentativa de determinar a melhor posologia (dose/frequência) do anti-helmíntico a ser empregado na prática, busca-se o melhor entendimento de suas propriedades farmacocinéticas, ou seja, determinar a quantidade do fármaco disponível no organismo para geração de um efeito terapêutico adequado. A moxidectina (MOX) é uma lactona macrocíclica que tem se destacado nos últimos anos pela sua eficiência anti-helmintica em baixas doses e pela longa persistência no organismo dos animais. Deste modo, o presente trabalho teve por objetivo desenvolver e validar um método analítico, empregando cromatografia líquida acoplada a espectrometria de massas (LC-ESI-MS/MS), avaliar o perfil farmacocinético da MOX no soro de cordeiros, assim como comparar as concentrações dos resíduos presentes no soro de animais submetidos a diferentes métodos de controle parasitários em condições de criação comercial em pastagens. O método analítico desenvolvido foi validado apresentando exatidão entre 80-107,3 % e valores de precisão entre 1,7-6,7 %. Os limites de detecção (LOD) e quantificação (LOQ) determinados foram de 2,0 ng mL-1 e 5,0 ng mL-1, respectivamente. Os resultados dos parâmetros de validação demonstraram que o método foi adequado para determinar a presença da MOX no soro de cordeiro. A disposição da MOX no soro foi avaliada em cordeiros da raça suffolk (n=7), após administração subcutânea de 0,2 mg kg-1 de peso corpóreo, em uma única dose. As concentrações de MOX no soro apresentaram valores de Cmáx 8,5 ± 2,81 ng mL-1, e Tmáx de 1 ± 0,7 dias. Os valores da AUC 0-8 e T1/2 foram 82,42 ± 8,02 ng dia.mL-1 e 7,66 ± 1,62 dias, respectivamente. Os resultados indicam uma absorção subcutânea lenta e, portanto, maior tempo de exposição sistêmica e menor disponibilidade do fármaco na circulação sanguínea, quando comparado com outras espécies como caprinos, bovinos ou com a ivermectina. Nenhum dos diferentes métodos de controle parasitários avaliados apresentou resíduos de MOX no soro superiores ao limite de quantificação do método / Abstract: The sheep breeding has aroused great deal of interest in all regions of Brazil and its production is mainly intended to the creation of lambs. However, the gastrointestinal parasitosis is one of the major difficulties in the production systems, especially in the field. Parasites cause animals¿ health damage, reducing their performance and the profits of the producer. As a consequence parasiticides are used as prophylactic measure. However, care on their use must be taken, since the indiscriminate use of veterinary drugs has favored the development of resistant strains, responsible for the loss of the drug effectiveness. As a control of gastrointestinal nematodes in the commercial sheep breeding, the veterinary drug is usually administered based on the result of two forms of diagnostic test: faecal egg count of parasites and the FAMACHA® test (examination of ocular mucosa color), or on the application of the veterinary drug at regular periods of 28 days, independently of any diagnostic test. In an attempt to determine the optimal dosage (dose / frequency) of the parasiticide to be used in practice, it is necessary the better understanding of their pharmacokinetic properties, in order to determine the amount of drug available in the body to generate an adequate therapeutic effect. Moxidectin (MOX) is a macrocyclic lactone that acts against endo and ecto-parasites, and has come to prominence in recent years due to its effectiveness at low doses and long persistence in the organism of the animals. Thus, the present study aimed to develop and validate an analytical method employing liquid chromatography coupled to mass spectrometry (LC-ESI-MS/MS), evaluate the pharmacokinetic profile of MOX in the serum of lambs, and to compare the MOX residue levels on the serum of animals subjected to different parasiticidal method of control in conditions of commercial breeding in the field. The validated analytical method presented between 80 to 107.3 % accuracy, and precision values between 1.7 - 6.7 %. The limits of detection (LOD) and quantitation (LOQ) were 2.0 ng mL-1 and 5.0 ng mL-1, respectively. The results of the validation parameters showed that the method was suitable to determine the presence of MOX in the lamb serum. The pharmacokinetic profile of MOX was evaluated in suffolk race lambs (n = 7), after a single subcutaneous dose administration of 0.2 mg kg-1 body weight. The serum concentrations of MOX showed Cmax value of 8.5 ± 2.81 ng mL-1, and Tmax of 1 day. The AUC0-8 and the T1/2 were 82.42 ± 8.02 ng day.mL-1 and 7.66 ± 1.62 days, respectively. The results indicated a slow MOX subcutaneous absorption and, therefore, increased systemic exposure time and low availability of the drug in blood circulation, when compared to other species such as goats or cattle, or to ivermectin. None of the different parasiticidal control methods evaluated showed MOX residue levels above the LOQ of the analytical method / Mestrado / Ciência de Alimentos / Mestra em Ciência de Alimentos

Σκοπός: Ο προσδιορισμός των επιπέδων συγκέντρωσης καταλοίπων κτηνιατρικών παρασιτοκτόνων ουσιών (ανθελμινθικών), σε νωπό γάλα μηρυκαστικών από περιοχές της Νοτίου Ελλάδος και η εκτίμηση της πρόσληψής τους από τον άνθρωπο. Συγκεκριμένα εξετάσθηκαν οι ουσίες, Albendazole, Febantel, Fenbendazole, Mebendazole και κάποιοι μεταβολίτες τους (Albendazole sulfoxide, Albendazole sulfone, Fenbendazole sulfone) στο νωπό γάλα προβάτων, αιγών και βοοειδών. Μέθοδοι: Χημική ανάλυση δειγμάτων γάλακτος, με τη βοήθεια Υγρής Χρωματογραφίας Υψηλής Απόδοσης και ανιχνευτή Συστοιχίας Διόδων, Υπεριώδους (UV). Χρήση βάσης δεδομένων σχετικά με την εκτίμηση της ημερήσιας κατανάλωσης γάλακτος και Φέτας (λήψη προσωπικών συνεντεύξεων, συμπλήρωση ερωτηματολογίων, συχνοτήτων κατανάλωσης τροφίμων, από δείγμα 723 μαθητών ηλικίας 10-12 ετών, από τη Νότια Ελλάδα). Αποτελέσματα: 34 από τα 123 δείγματα γάλακτος βρέθηκαν να περιέχουν κατάλοιπα των διερευνώμενων ουσιών, εκ των οποίων τα 11 υπερέβαιναν τα θεσπισμένα από την Ε.Ε. ανώτατα επιτρεπτά όρια καταλοίπων. Η Εκτιμώμενη Ημερήσια Πρόσληψη των ουσιών αυτών, από το εξετασθέν δείγμα πληθυσμού, κυμαίνεται από 0,4-15,9% της Αποδεκτής Ημερήσιας Πρόσληψης, ανάλογα με την ουσία και το τρόφιμο (νωπό αγελαδινό γάλα ή τυρί Φέτα). Υπάρχει γεωγραφική διακύμανση στη συχνότητα εμφάνισης των καταλοίπων η οποία είναι ιδιαίτερα αυξημένη σε περιοχές οι οποίες στερούνται επαρκών υπηρεσιών ελέγχου τροφίμων. Συμπεράσματα: Από την παρούσα εργασία προκύπτει ότι 11.4% των δειγμάτων γάλακτος, περιέχει κατάλοιπα ανθελμινθικών ουσιών, σε επίπεδα συγκεντρώσεων που υπερβαίνουν το ανώτατο επιτρεπτό όριο (έως και 7 φορές για τη Febantel). Το εύρημα αυτό, εγείρει ερωτήματα για την πλήρη εφαρμογή της Ο.Κ.Π.. Ωστόσο η κατανάλωση νωπού γάλακτος το οποίο περιέχει κατάλοιπα των υπό διερεύνηση ενώσεων, στα προαναφερθέντα επίπεδα συγκεντρώσεων, δεν οδηγεί σε υπέρβαση της Ανώτερης Ημερήσιας Πρόσληψης. Από την άλλη πλευρά, είναι απαραίτητο να τονιστεί ότι οι ουσίες αυτές είναι δυνατό να ανιχνευθούν και σε άλλα τρόφιμα ζωικής προέλευσης για τα οποία δεν έχουν θεσπιστεί MRLs και συνεπώς δε διεξάγονται οι αντίστοιχοι έλεγχοι. Κατά συνέπεια, είναι απαραίτητη η εκπαίδευση των κτηνοτρόφων σε θέματα Ο.Κ.Π., η εντατικοποίηση των ελέγχων και η στελέχωση των κρατικών υπηρεσιών, έτσι ώστε να αντισταθμίζονται οι ελλείψεις των νόμων και να αποφευγονται πιθανές δυσμενείς συνέπειες για τη δημόσια υγεία. / Aim: The determination of the concentration levels, of veterinary parasiticide drug (anthelmintics) residues, in ruminants’ raw milk, from regions of Southern Greece, and the residues’ intake estimation for humans. In particular, the investigated, residues were Albendazole, Febantel, Fenbendazole, Mebendazole and some of their metabolites (Albendazole sulfoxide, Albendazole sulfone, Fenbendazole sulfone), in sheep, goat and bovine raw milk. Methods: Chemical analysis of raw milk samples with High Performance Liquid Chromatography and UV Diode Array Detector. A databank concerning the daily consumption of milk and feta cheese was utilized (personal interviews and filling in of food frequency questionnaires, from a population sample of 723 pupils aged from 10-12 years old, in Southern Greece). Results: 34 out of the 123 milk samples, were found to contain residues of the investigated compounds and 11 of the contaminated samples, contained residues, exceeding the EU established MRLs. The Estimated Daily Intake for these residues resulting from the interviewed population sample, was ranging from 0,4 to 15,9% of the Acceptable Daily Intake, depending on the substance and the kind of food (raw bovine milk or feta cheese). There is a geographical variation concerning the residue occurrence, which is high in regions where food control agencies are poorly staffed. Conclusions: The present study indicates that 11.4% of milk samples analysed, contains concentration levels of anthelmintic residues above the maximum residue limit (up to 7 times for Febantel). This finding raises questions about the full implementation of Good Veterinary Practice. However, consumption of raw milk containing the aforementioned concentration levels of the compounds under investigation, does not result to exceedance of the Acceptable Daily Intake. On the other hand, it is necessary to emphasize that these substances are possible to be detected in other foods of animal origin for which no MRLs have been established and therefore the respective controls are not carried out. Consequently, training of stock-farmers on GVP, intensification of controls and staffing of government agencies, are needed to counterbalance deficiencies of laws and avoid potential adverse effects on public health.

The novel hypothesis that antimicrobial growth promoters (AGP) function by modulating the mammalian immune system was tested. Sampling methods to characterize the mucosa-associated microbiota of the murine intestine by terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that direct plug extraction was superior to wash methods. Using T-RFLP analysis, non-therapeutic administration of chlortetracycline (CTC) and sulfamethazine to beef cattle did not affect the composition of bacterial communities associated with intestinal mucosa and in digesta, with exception of those associated with mucosa of the proximal jejunum. Similarly, oral administration of non-therapeutic concentrations of CTC did not affect the mucosa-associated microbiota of the murine intestine. Oral administration of nontherapeutic concentrations of CTC prevented weight loss, reduced pathologic changes, modulated transcription levels of inflammatory cytokines in C. rodentium-infected mice, and did not consistently affect the colonic microbiota. These findings support the hypothesis that AGP primarily function by modulating the intestinal immune system. / xiv, 160 leaves ; 29 cm

Veterinary pharmaceuticals are routinely used in livestock production to treat diseases, prevent infections, and promote growth. However, the potential release of pharmaceuticals from agricultural activities has raised concerns because they may pose detrimental effects to the ecosystems and human health, for example fostering the evolution of antibiotic-resistant bacteria in the natural environment. A better understanding of the environmental fate of veterinary pharmaceuticals is critical to properly assess and mitigate their risks. This dissertation focuses on a major group of veterinary pharmaceuticals, ionophore antibiotics (IPAs), which is sold at over 4 million kilograms per year and constitutes more than one third of the total antibiotic consumption by the livestock industry in the U.S. Despite the extensive usage of IPAs, their environmental fate was not well-understood. Therefore, this study aimed at achieving a comprehensive understanding of the occurrence, persistence, and transformation of IPAs from poultry litter before and after applications to the agricultural lands. Three of the most commonly used members of IPAs were investigated in this study: monensin (MON), salinomycin (SAL), and narasin (NAR). Based on the common management practices of poultry litter, the potential abiotic and biotic transformation reactions of IPAs were examined under varying conditions relevant to the water-soil-litter systems. This dissertation consists of three sections. First, a robust analytical method was developed to quantify IPAs in various environmental compartments, especially in high organic-containing matrices such as poultry litter, and soil and runoff from litter-fertilized lands. Efforts were made to optimize the analytical method with respect to improving extraction recovery, reducing matrix effects, and validating a surrogate standard. Second, lab-scale experiments were set up to determine the chemical properties of IPAs in aqueous environments and to study the abiotic transformation of IPAs, including hydrolysis and photolysis. The results showed that IPAs are prone to hydrolytic transformation in acidic environments, which are likely to be encountered in acidic soils, alum-amended litter (alum: Al₂(SO₄)₃•12H₂O), and acidic runoff. Multiple transformation pathways were proposed based on the identified hydrolysis products. It is also noteworthy that the hydrolysis products of MON still exhibited a toxic effect on the selected microorganism (Bacillus subtilis). SAL and NAR were found to undergo direct photolysis under both UV light and sunlight irradiation. In natural water matrix, IPAs were also degraded by indirect photolysis with hydroxyl radicals generated by light-excited nitrate. Dissolved organic matter can shield IPAs from light and slow down their photolysis. Third, the biodegradation potential of IPAs was first tested in litter and soil microcosms. Factor analysis was conducted to delineate the interaction of water and temperature on IPA degradation in the litter. Litter-fertilized and non-fertilized soil microcosms were compared on the degradation of MON and SAL. Furthermore, the inhibition and biotransformation potential of IPAs were assessed under different redox conditions with litter-enriched cultures. Inhibition tests focused on examining IPAs’ impact on microbial community functions, including denitrification, sulfate-reduction, and methane production. Biodegradation tests were conducted with different electron acceptors, including oxygen, nitrate, sulfate, and organic carbons, with efforts to elucidate primary biotransformation products. On the basis of the results obtained in this study, several recommendations on litter management and IPA selection were made to help mitigate the release and transport of IPAs, as well as enhance their degradation. Overall, this study significantly improved the understanding of the environmental fate of IPAs and the obtained knowledge can aid proper selection of IPAs and management strategies in future applications to minimize the risks of antibiotic micropollutants in the environment.

The use of veterinary drugs continues to be a challenge since some farmers use drugs for medicinal purposes, while others use them as growth promoters without observing the set withdrawal times. In emerging economies, challenges are experienced in residue testing to ensure residue free animal products. It is therefore necessary to develop a simple, environmentally friendly and cost effective extraction method for veterinary drugs residues. A dispersive liquid-liquid micro-extraction method for simultaneous determination of chloramphenicol, florfenicol and thiamphenicol residues in water, bovine muscle and milk was developed optimised and validated. Analysis was performed with liquid chromatography coupled to electron spray ionization tandem mass spectrometry in negative ion multiple reaction monitoring mode. Dispersive liquid-liquid micro-extraction method development involved optimisation of parameters such as type and volume of extraction solvent, type and volume of disperser solvent and pH. The optimum extraction solvent was dichloromethane at a volume of 250 μL while the optimum disperser solvent was acetonitrile (1 mL) Extraction was found to be optimum at pH 7. The developed method was validated according to Commission decision 2002/657/EC. Linearity. Linearity was observed for water, bovine milk and muscle in the ranges of 0.3 μg kg-1 to 0.9 μg kg-1, 25 μg kg-1 to 100 μg kg-1 and 50 μg kg-1 to 200 μg kg-1 for chloramphenicol, thiamphenicol and florfenicol respectively with regression coefficients ranging between 0.9941 and 0.9999. Limit of detections for CAP, FFC & THAP were 0.082, 3.31, and 2.21 μg kg-1 respectively while Limit of quantifications were 0.163, 7.51 and 6.84 μg kg-1 respectively. The recoveries for water, bovine milk and bovine muscle ranged between 87.2% and 102%. In this study the validated dispersive liquid-liquid micro-extraction has been found to perform very well not only in the water matrix but also in the more complex matrices like bovine milk and muscle. / Chemistry / M. Sc. (Chemistry)