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A study was conducted to evaluate the efficacy of ivermectin and albendazole sulfoxide for the control of gastrointestinal nematodes. Thirty Pelibuey ewes were randomly assigned to three experimental groups: Ivermectin 4% (10.2 mg/kg), albendazole sulfoxide 17% (3.75 mg/kg) and an untreated control group. Faecal samples were collected before drug administration (day 0) and on days 7, 14, 21, 28, 35, and 42 post-treatment. The quantitative diagnosis of the eggs was carried out using the McMaster technique and cultures were carried out to recover larvae by the migration method with the Baermann apparatus. The anthelmintic efficacy of the treatments was determined by the Abbott's formula and the effect of anthelmintics on the number of eggs per gram of faeces (EPG) by an analysis of variance with a completely randomized design. The most abundant species were Haemonchus contortus and Strongyloides papillosus. A significant decrease in the amount of EPG was observed from day 7 of treatment with both anthelmintics, with albendazole sulfoxide and ivermectin showing efficacy above 95% from day 7 and 14 post-treatment, respectively, until day 21. At 24 h post-treatment, ivermectin showed lower efficacy than albendazole sulfoxide (69.5 vs 90.9% respectively), but efficacy was similar and above 90% for both drugs from day 7. The efficacy of lbendazole sulfoxide against Haemonchus contortus larvae was 88.2% at 24 h post-treatment compared to 69.5% for ivermectin, but only the latter showed an efficacy greater than 95% on days 14 and 21 post-administration. It is concluded that albendazole sulfoxide has a faster effect and a longer efficacy for the control of gastrointestinal nematode eggs, while ivermectin showed greater efficacy for the control of Haemonchus contortus larvae without evidence of resistance to both drugs., Se realizó un estudio para evaluar la eficacia de la ivermectina y del albendazol sulfóxido para el control de nematodos gastrointestinales. Treinta ovejas Pelibuey fueron asignadas aleatoriamente a tres grupos experimentales: ivermectina al 4% (10.2 mg/kg), albendazol sulfóxido al 17% (3.75 mg/kg) y un grupo control no tratado. Se recolectaron muestras fecales antes de la administración de los fármacos (día 0) y en los días 7, 14, 21, 28, 35 y 42 pos-tratamiento. Se realizó el diagnóstico cuantitativo de los huevos mediante la técnica de McMaster y se realizaron cultivos para recuperar larvas por el método de migración con el aparato de Baermann. La eficacia antihelmíntica de los tratamientos se determinó por medio de la fórmula de Abbott y el efecto de los antihelmínticos sobre la cantidad de huevos por gramos de heces (HPG) por medio de un análisis de varianza con un diseño completamente al azar. Las especies más abundantes fueron Haemonchus contortus y Strongyloides papillosus. Se observó una disminución significativa en la cantidad de HPGH a partir del día 7 del tratamiento con ambos fármacos, mostrando el albendazol sulfóxido y la ivermectina una eficacia por encima del 95% a partir del día 7 y 14 postratamiento, respectivamente, hasta el día 21. La eficacia del albendazol sulfóxido contra larvas de Haemonchus contortus fue de 88.2% a las 24 h pos-tratamiento en comparación con el 69.5% de la ivermectina, pero solo esta última mostró una eficacia superior al 95% los días 14 y 21 postadminstración. Se concluye que el albendazol sulfóxido tiene un efecto más rápido y una eficacia más prolongada para el control de huevos de nemátodos gastrointestinales, mientras que la ivermectina mostró una mayor eficacia para el control de larvas de Hamonchus contortus sin indicios de resistencia a ambos fármacos.

RESUMEN Se realizó un estudio para evaluar la eficacia de la ivermectina y del albendazol sulfóxido para el control de nematodos gastrointestinales. Treinta ovejas Pelibuey fueron asignadas aleatoriamente a tres grupos experimentales: ivermectina al 4% (10.2 mg/kg), albendazol sulfóxido al 17% (3.75 mg/kg) y un grupo control no tratado. Se recolectaron muestras fecales antes de la administración de los fármacos (día 0) y en los días 7, 14, 21, 28, 35 y 42 pos-tratamiento. Se realizó el diagnóstico cuantitativo de los huevos mediante la técnica de McMaster y se realizaron cultivos para recuperar larvas por el método de migración con el aparato de Baermann. La eficacia antihelmíntica de los tratamientos se determinó por medio de la fórmula de Abbott y el efecto de los antihelmínticos sobre la cantidad de huevos por gramos de heces (HPGH) por medio de un análisis de varianza con un diseño completamente al azar. Las especies más abundantes fueron Haemonchus contortus y Strongyloides papillosus. Se observó una disminución significativa en la cantidad de HPGH a partir del día 7 del tratamiento con ambos fármacos, mostrando el albendazol sulfóxido y la ivermectina una eficacia por encima del 95% a partir del día 7 y 14 postratamiento, respectivamente, hasta el día 21. La eficacia del albendazol sulfóxido contra larvas de Haemonchus contortus fue de 88.2% a las 24 h pos-tratamiento en comparación con el 69.5% de la ivermectina, pero solo esta última mostró una eficacia superior al 95% los días 14 y 21 postadminstración. Se concluye que el albendazol sulfóxido tiene un efecto más rápido y una eficacia más prolongada para el control de huevos de nemátodos gastrointestinales, mientras que la ivermectina mostró una mayor eficacia para el control de larvas de Hamonchus contortus sin indicios de resistencia a ambos fármacos. ABSTRACT A study was conducted to evaluate the efficacy of ivermectin and albendazole sulfoxide for the control of gastrointestinal nematodes. Thirty Pelibuey ewes were randomly assigned to three experimental groups: Ivermectin 4% (10.2 mg/kg), albendazole sulfoxide 17% (3.75 mg/kg) and an untreated control group. Faecal samples were collected before drug administration (day 0) and on days 7, 14, 21, 28, 35, and 42 post-treatment. The quantitative diagnosis of the eggs was carried out using the McMaster technique and cultures were carried out to recover larvae by the migration method with the Baermann apparatus. The anthelmintic efficacy of the treatments was determined by the Abbott's formula and the effect of anthelmintics on the number of eggs per gram of faeces (EPG) by an analysis of variance with a completely randomized design. The most abundant species were Haemonchus contortus and Strongyloides papillosus. A significant decrease in the amount of EPG was observed from day 7 of treatment with both anthelmintics, with albendazole sulfoxide and ivermectin showing efficacy above 95% from day 7 and 14 post-treatment, respectively, until day 21. At 24 h post-treatment, ivermectin showed lower efficacy than albendazole sulfoxide (69.5 vs 90.9% respectively), but efficacy was similar and above 90% for both drugs from day 7. The efficacy of albendazole sulfoxide against Haemonchus contortus larvae was 88.2% at 24 h post-treatment compared to 69.5% for ivermectin, but only the latter showed an efficacy greater than 95% on days 14 and 21 post-administration. It is concluded that albendazole sulfoxide has a faster effect and a longer efficacy for the control of gastrointestinal nematode eggs, while ivermectin showed greater efficacy for the control of Haemonchus contortus larvae without evidence of resistance to both drugs.

Las principales rutas de administración de los fármacos utilizados en medicina veterinaria son la vía oral (PO), la intramuscular(IM), la subcutánea (SC) y la intravenosa (IV). Aun así, la administración tópica cutánea ha adquirido gran aceptación, particularmente para lograr un efecto local en diferentes regiones anatómicas de la piel, lo cual ya es una práctica común en medicina humana y, cada vez más frecuente, en veterinaria. Sin embargo, durante la última década, se han logrado grandes avances en la formulación y en el uso de la administración tópica cutánea (pour-on, spot-on,spray) de diferentes fármacos con el objetivo de lograr una absorción transdermal y, así, un efecto terapéutico sistémico. En este sentido, cabe destacar que cualquier sustancia aplicada sobre la piel tiene la potencialidad de ser absorbida, teniendo siempre en consideración que la capacidad de penetrar y atravesar la barrera cutánea dependerá de factores inherentes a la piel, a la formulación y a los fármacos. El presente trabajo de tesis doctoral es de índole farmacológico, aunque sus resultados tienen una alta relación con aspectos del tratamiento antiparasitario en caninos. Como resultado de la experiencia adquirida en nuestro lugar de trabajo y de la búsqueda bibliográfica realizada, este trabajo de tesis se basa en la postulación de la siguiente hipótesis general de trabajo: La relación entre las características fisiológicas de la piel (fisiología cutánea) y las propiedades físico-químicas del fármaco administrado tópicamente afecta el proceso de absorción percutánea de lactonas macrocíclicas. El desarrollo y la validación de las metodologías para la extracción físico-química e identificación y cuantificación cromatográfica de las moléculas endectocidas bajo estudio en las diferentes matrices fue un aspecto importante para el desarrollo del presente trabajo de tesis. A partir de un método altamente sensible, como es la cromatografía líquida de alto rendimiento (HPLC), se logró determinar las concentraciones de moxidectin (MXD) y doramectina (DRM) en las matrices estudiadas. Los resultados obtenidos de las validaciones aseguran que los métodos de extracción y de cuantificación por HPLC sean analíticamente adecuados para detectar y cuantificar MXD y DRM en plasma canino y medio receptor (in vitro) desde concentraciones iguales o superiores a 0,1 ng/ml. Con el propósito de caracterizar profundamente el comportamiento de estas moléculas en caninos, se propuso estudiar la farmacocinética de las vías endovenosa, oral y tópica cutánea. Para ello, se utilizaron seis (6) caninos clínicamente sanos, de raza Beagle, cuatro (4) hembras y dos (2) machos, entre 9,8 y 15,5 kg de peso y entre 12 y 36 meses de edad. Los animales se encontraban alojados en la sección caniles de la Facultad de Ciencias Veterinarias de Universidad de Buenos Aires (FCV-UBA) bajo estrictas normas de bienestar animal. Los resultados obtenidos reflejaron el comportamiento esperable para cada una de las vías, pero con algunas características particulares. Tanto el MXD como la DRM fueron administrados de manera endovenosa, con éxito, con el objetivo de eliminar la influencia del proceso de absorción a las disposiciones de los fármacos. Esto permitió caracterizar con mayor certeza el comportamiento de la absorción de las otras vías bajo estudio. En este sentido, para el MXD, observamos que la biodisponibilidad luego de la administración oral fue superior al 100%. Si bien es teóricamente imposible encontrar una biodisponibilidad mayor a la endovenosa (100%), se pueden hipotetizar algunas explicaciones basadas en las características del fármaco. Para la vía tópica en particular, detectamos un tiempo de absorción prolongado, esto puede observarse no sólo por los casi 10 días requeridos para alcanzar la concentración máxima (Tmax),sino también en los casi 13 días de tiempo medio de absorción (TMA), lo que demuestra la presencia de grandes obstáculos, al ser aplicado sobre la piel, para poder alcanzar la circulación sistémica. Con un comportamiento similar, del análisis comparativo de la DRM por las tres vías, puede verse que la fase de absorción tras la administración tópica difiere significativamente de la correspondiente a la administración oral. Estas diferencias están claramente demostradas por los parámetros farmacocinéticos que caracterizan la absorción, donde el TMA obtenido es significativamente mayor para la vía tópica en comparación con la oral (6,3 ± 2,6 y 1,3 ± 1,9 días, respectivamente). Asimismo, también puede observarse que las diferencias entre los valores del Cl de la administración tópica son estadísticamente significativas cuando se comparan con los valores de Cl de las administraciones endovenosa y oral. Cuando comparamos los perfiles entre ambas drogas, puede verse que el MXD presenta una mayor velocidad de absorción que la DRM, tanto por la vía tópica (0,6 ± 0,4 d y 1,1 ± 0,8 d, respectivamente) como por la oral (0,05 ± 0,02 d y 0,06 ± 0,02 d, respectivamente). Para la vía oral se destaca que, mientras que la DRM no logra alcanzar el 85% de absorción de la dosis, el MXD supera ampliamente el 100%. Esto puede explicarse por las magnitudes de los procesos de distribución y de re-distribución que presenta dicho fármaco. La rápida y total absorción de la dosis oral permite que haya en una fracción corta de tiempo una gran cantidad plasmática de fármaco disponible para distribuirse a los tejidos (Vss 8,2 ± 1,3 L y 1,9 ± 0,4 L para el MXD y la DRM, respectivamente). Esta gran distribución, junto, en parte, con una recirculación entero-hepática, en la cual podría tener una importante participación la actividad de la glucoproteína P (Ward et al., 2004a; 2004b; Lespine et al., 2012), determina que, al iniciarse la fase franca de eliminación, ocurra un fenómeno de re-distribución de una gran magnitud. De los resultados obtenidos y de lo expuesto anteriormente, es importante destacar que la vía tópica demuestra ser beneficiosa en muchos aspectos técnicos, pero requiere de un trabajo farmacotécnico mayor (como ocurre en medicina humana) para lograr una mejor permeación a través de los estratos de la piel y, como consecuencia, alcanzar concentraciones más altas en la circulación sistémica y obtener así mayor eficacia clínica. Para ahondar aún más en el estudio de absorción tópica en piel, se trabajó en un modelo in vitro con cámaras de Franz modificadas. La comparación de las permeaciones cutáneas de ambos fármacos arrojó diferencias estadísticamente significativas en la mayoría de los parámetros estudiados. Así, el flujo de la DRM fue significativamente mayor que el del MXD. Lo mismo ocurrió con el coeficiente de permeación aparente y con el porcentaje de fármaco permeado. Tal como se ha visto en experiencias previas en nuestro Laboratorio, el pasaje de fármacos a través de las capas superficiales de la piel es un proceso dificultoso, lo cual se evidencia claramente, en estos estudios, por las bajas cantidades totales acumuladas en el medio receptor en relación a la dosis total aplicada (aproximadamente 0,01%). Más allá de la originalidad de los datos generados, también se tuvo como objetivo la construcción de un modelo predictor utilizando y combinando la información de ambas fases experimentales, in vivo e in vitro. Para ello, se realizó un análisis de correlación in vivo-in vitro (CIVIV). Así, la presente tesis doctoral presenta alternativas que, con los datos propios generados, ha alcanzado un nivel aceptable de predicción del comportamiento de los fármacos estudiados en los animales experimentales. Esta metodología aún requiere mayores estudios y ser desafiada en distintas pruebas en diferentes escenarios en búsqueda de una mayor robustez. Fil: Nejamkin, Pablo. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Fil: Sallovitz, Juan Manuel. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Fil: Albarellos, Gabriela A. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. In veterinary medicine, the main routes of drug administration are orally (PO), intramuscularly (IM), subcutaneously (SC) and intravenously (IV). Even though, skin topical administration has recently gained great acceptance, especially when looking for a local effect in different anatomical regions. This a common practice in human medicine and is becoming more frequent in veterinary medicine. However, during the last decade, great advancements have been achieved in formulating different drugs for their topical administration (pour-on, spot-on, spray) aiming to a systemic therapeutic effect. It is noteworthy that any drug, when administered on the skin, has the potential to be absorbed, depending on its capability of penetrating into and pass through the skin layers. This will depend on many factors related to the skin, the formulation and the drug. In spite of the present thesis work is mainly pharmacological, its results can be related to many aspects of antiparasitic treatment in dogs. As a result of our own experience and works of other authors, this thesis work is based on the following hypothesis: After their topical administration, the interactions between physiological characteristics of canine skin and physico-chemical properties of the antiparasitic macrocyclic lactones effect the cutaneous absorption process. The development and validation of the analytical chromatographic methods for quantification of the endectocides molecules under study were an important feature of the thesis work. This sensitive and reliable method allowed to determine moxidectin (MXD) and doramectin (DRM) concentrations of or higher than 0.1 ng/mL in the different matrices studied (plasma and receptor medium) in vivo and in vitro. In order to thoroughly characterize the pharmacological behavior of these molecules in canines, the pharmacokinetics after their intravenous, oral and topical administration were studied. Six clinically sound Beagle dogs, 4 females and 2 males, of 9.8-15.5 kg of bogy weight and between 12- to 16-month-old were used. Animals were housed at the small animal facilities of the Facultad de Ciencias Veterinarias, Universidad Nacional de Buenos Aires. Animals’ health and welfare were monitored along the whole experimental period. Observed results showed pharmacokinetic profiles as expected; however, there were unexpected own features. Both MXD and DRM were successfully intravenously administered in order to characterize drugs’ disposition avoiding biases due to the absorption process. In turn, the observed pure drug disposition allowed to confidently characterize the absorption processes of each of the other two routes of administration (oral and topical). Hence, MXD showed an oral bioavailability in excess of 100%, that is not possible, since only after the intravenous administration a plasma bioavailability of 100% can be reached. Many causes can be responsible of this, such as artifactual and non-stationary or non-lineal pharmacokinetics. However, a more plausible explanation is the role played by transportermediated elimination and absorption processes, which are a characteristic of the antiparasitic macrocyclic lactones. As to the topical administration, a prolonged absorption time was observed (Tmax = 10 days; MAT = 13 days), which suggests that passage through the skin to reach the systemic circulation is a difficult process. Similarly, when comparing oral and topical absorption of DRM, significant differences can be observed. These differences clearly demonstrated by the pharmacokinetic parameters of absorption (TMA 6.3 ± 2.6 y 1.3 ± 1.9 for the topical and oral administration, respectively). When comparing both drugs, MXD presents a faster absorption compared to DRM both after topical (t½ab 0.6 ± 0.4 days and 1.1 ± 0.8 days, respectively) as well after the oral administration (0.05 ± 0.02 days and 0.06 ± 0.02 days, respectively). The oral availability showed marked differences, hence, while DRM oral bioavailability reached to 85%, MXD largely exceeded 100%. The latter case can be explained by an extremely rapid drug absorption, determining high amounts of drug available for distribution, re-distribution and saturation of transporter-mediated elimination. A higher distribution was observed for MXD compared to DRM (Vss 8.2 ± 1.3 L/kg and 1.9 ± 0.4 L/kg para el MXD y la DRM, respectively). Thus, a large distribution along with a saturated elimination and, possibly, increased reabsorption (entero-hepatic circulation) could have determined that, when elimination begun, a huge re-distribution and/or re-absorption took place at the same time. Albeit the topical administration presents many benefits, it needs more pharmacotechnical work (as done in human medicine) in order to achieve a better and more controlled percutaneous permeation and, hence, achieve more predictable plasma concentration profiles and efficacies. To further characterize topical absorption, an in vitro model was developed by utilizing the Franz diffusion cell technique. As with previous experiences in our laboratory, drug passage through the superficial skin layers (particularly, the stratum corneus) is a difficult process and was evidenced for both drugs by the low percentages of permeation observed (approximately 0.01%). The comparison of the in vitro percutaneous absorption of both drugs showed statistically significant differences. DRM flux was higher than MXD flux, as well as the apparent permeation coefficient and the percentage of drug permeated. Although the low skin permeation observed in the in vitro models, this technique has a great potential for the veterinary pharmaceutical industries in the development and testing of topical formulations. Data generated by the present work are quite original, nevertheless, another objective was the development of a mathematical model by combining both in vitro and in vivo data by the convolution technique to predict in vivo plasma profile from in vitro data. An in vivo-in vitro correlation analysis was performed for both drugs. Even though an acceptable predictive level was achieved, further work is needed and predicted profiles have to be challenged under different scenarios.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES Soybeans and beans are considered oleaginous plants of great economic importance in Brasil. Maize is a cereal that also generates profitability for the economy, but its use, to a large extent, is as animal feed. In the last years, this crops suffered with infestations of the caterpillar Helicoperva Armigera, being the main problem the resistance of this pest to most pesticides. The indiscriminate use of pesticides and the lack of knowledge and use of good agricultural practices are the main causes of this, as well as undermining food security. Proof of this is the increased presence of pesticides in soil and agricultural crops. The aim of this work was to develop and validate a method based on QuEChERS extraction for the determination of residues of the avermectins: abamectin, emamectin benzoate, eprinomectin, ivermectin and doramectin in soybean, bean and maize samples by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Avemerctins were selected for the study because of the recent release of emamectin as a pesticide in the three matrices under study. Thus, a method for soybean, bean and maize was developed, where 5 g of sample and 10 mL of ultrapure water were used, as well as a 10 mL mixture of acetonitrile:isopropanol solvents in a ratio of 9:1 (v/v), with subsequent addition of partition salts. In the cleaning step five different sorbents were evaluated: C18 + PSA, Florisil®, silica, Supel™ QuE Z-Sep+ and EMR-Lipid, in their activated and non-activated form. With the clean-up step using EMR-Lipid, limits of detection of 1.2 μg kg-1 for abamectin, doramectin, emamectin benzoate and ivermectin, and of 2.4 μg kg-1 for eprinomectin were achieved. Accuracy and precision evaluated at the levels 4, 8, 20, 40 and 80 μg kg-1 presented satisfactory results. The method was successfully applied in real samples and is a good option for routine analysis Soja e feijão são consideradas oleaginosas de grande importância econômica no Brasil. O milho é um cereal que também gera lucratividade para a economia, porém seu uso, em grande parte, é para alimentação animal. Nos últimos anos, essas culturas sofreram com infestações da largarta Helicoperva Armigera, sendo o principal problema associado a essa praga, a resistência a maioria dos agrotóxicos. O uso de forma indiscriminada de agrotóxicos e a falta de conhecimento e do emprego das boas práticas agrícolas são as principais causas desse fato, além de prejudicar a segurança alimentar. Prova disso, é o aumento da presença de agrotóxicos em solos e culturas agrícolas. Assim, o presente trabalho teve por objetivo desenvolver e validar um método baseado na extração pelo método QuEChERS para a determinação de resíduos de avermectinas: abamectina, benzoato de emamectina, eprinomectina, ivermectina e doramectina em amostras de soja, feijão e milho empregando a cromatografia líquida da ultra-alta eficiência acoplada à espectrometria de massas em série (UHPLC-MS/MS). As avemerctinas foram selecionadas para o estudo devido a recente liberação do emprego da emamectina como agrotóxico nas três matrizes em estudo. Com isso, desenvolveu-se um método para soja, feijão e milho, onde foi empregado 5 g de amostra e 10 mL de água, além de uma mistura de 10 mL dos solventes acetonitrila:isopropanol na proporção 9:1 (v/v), com posterior adição de sais de partição. Na etapa de limpeza avaliou-se cinco diferentes sorventes, sendo eles: C18+PSA, Florisil®, sílica, Supel™ QuE Z-Sep+ e EMR-Lipid, em sua forma ativada e não ativada. Com a etapa de limpeza usando EMR-Lipid, foram obtidos os limites de detecção de 1,2 μg kg-1 para abamectina, doramectina, benzoato de emamectina e ivermectina, e de 2,4 μg kg-1 para eprinomectina. Precisão e exatidão avaliadas nos níveis 4, 8, 20, 40 e 80 μg kg-1 apresentaram resultados satisfatórios. O método foi aplicado com sucesso em amostras reais e é uma boa opção para análises de rotina.

La parasitosis gastrointestinal continúa siendo una de las principales causas de pérdidas económicas en la producción pecuaria y dentro de ella, la haemonchosis es sin duda la enfermedad más patógena y la que mayores pérdidas genera en la producción ovina. El control de las parasitosis debiera consistir en diversas estrategias de manejo de los animales y las pasturas junto con la administración de antihelmínticos, luego del diagnóstico parasitológico y terapéutico adecuado. Sin embargo, en las últimas tres décadas el control se basó casi exclusivamente en el utilización masiva de drogas antihelmínticas de amplio espectro cada vez más eficaces y persistentes. El incremento de la resistencia antihelmíntica junto con las nuevas tendencias de producción orgánica y disminución de los residuos de drogas en los alimentos generó interés en el estudio de nuevas estrategias de control. En este contexto el presente trabajo de Tesis Doctoral abordó la resistencia antihelmíntica desde un enfoque integrador fármaco-parasitológico, cuyo objetivo fue estudiar la relación que existe entre las concentraciones de diferentes lactonas macrocíclicas en plasma, tejidos/fluidos de localización parasitaria del ovino y en el parásito “blanco” y la eficacia sobre Haemonchus contortus resistente. Para ello se dividió la Tesis en 3 secciones experimentales. En la Primera se realizó la caracterización in vivo de factores que afectan la relación entre concentraciones en el sitio de localización parasitaria, concentraciones en el parásito “blanco” (H. contortus) y eficacia antihelmíntica para diferentes lactonas macrocíclicas en corderos infestados con parásitos resistentes y bajo condiciones estandarizadas. Dentro de esta sección se relizaron 3 experimentos en donde se comparó la utilización de dos vías de administración (subcubtánea e intraruminal), las diferentes lactonas macrocíclicas (ivermectina-abamectina-moxidectina) y la duplicación de la dosis como estrategias para aumentar la eficacia antihelmíntica. Se encontró que si bien la disposición plasmática es mayor al utilizar la vía subcutánea, las concentraciones son mayores en los tejidos y contenidos del tracto gastrointestinal a los 2-3 días post-tratamiento por vía intraruminal y esto produce un efecto relevante cuando se trata de nematodos resitentes, de tal forma que la eficacia contra H. contortus fue un 40 % mayor al utilizar la vía intraruminal, coincidentemente con las mayores concentraciones de ivermectina halladas dentro del parásito. Esto demuestra que la incorporación de la droga al parásito por vía transcuticular es relevante para la eficacia clínica, aún para aquellos parásitos que son hematófagos. En el segundo experimento se encontró que a la dosis terapéutica, moxidectina fue más efectiva frente a nematodos resistentes a ivermectina. Las concentraciones de los 3 fármacos encontradas en los tejidos del tracto gastrointestinal durante los primeros 2 días post tratamiento es relevante para su acción sobre los nematodos resistentes. Sin embargo, a pesar de que moxidectina obtuvo las menores concentraciones acumuladas en los parásitos adultos, el total de parásitos recuperados (al día 2) post tratamiento y su eficacia antihelmíntica fue mayor que la de ivermectina y abamectina. En el tercer experimento, donde se comparó la eficacia y la acumulación de fármacos en los tejidos de los corderos y en el H. contortus, utilizando ivermectina y moxidectina a la dosis terapéutica y al doble de la dosis terapéutica, debido al extremo grado de resistencia, sólo la administración de doble dosis de moxidectina fue efectiva. En la Sección Experimental II, se caracterizó in vitro la actividad de las lactonas macrocíclicas contra H. contortus resistente, mediante el desarrollo del test de inhibición de motilidad larval utilizando estadios larvarios de H. contortus. Esta prueba resultó ser una prueba eficaz para establecer diferencias entre aislamientos susceptibles de resistentes. Sin embargo, es una prueba difícil de implementar para el diagnóstico de resistencia de rutina. También se desarrolló un método ex vivo, para caracterizar el ingreso del fármaco dentro de los parasitos adultos. El incremento de la exposición a ivermectina y moxidectina se reflejan como un incremento en la cantidad de droga recuperada en los H. contortus. En la Sección Experimental III se estudiaron los cambios génicos en H. contortus asociados al fenómeno de resistencia. Se validó una metodología para cuantificar la expresión de glicoproteína-P2 en aislamientos de H. contortus susceptibles y resistentes a LMs. Se encontró una sobreexpresión en los aislamientos de parásitos resistentes. Además, el tratamiento con ivermectina, a diferencia de abamectina y moxidectina, fue el único que indujo un incremento significativo de la expresión de glicoproteína-P2 en los especímenes de H. contortus resistentes recuperados de corderos tratados a los 0,5 y 1 días post-tratamiento comparado con los H. contortus recuperados de animales sin tratamiento. Como conclusión, el presente trabajo de Tesis Doctoral a través de estudios in vivo, in vitro y utilizando herramientas de la biología molecular aporta información que es de utilidad para diseñar estrategias para prologar la vida útil de las lactonas macrocíclicas siempre que sean usados de manera racional y con una base de evaluación profesional por el veterinario. Fil: Lloberas, María Mercedes. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Fil: Lifschitz, Adrián L. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Fil: Entrocasso, Carlos M. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Gastrointestinal parasitic diseases are the main causes of economic losses in livestock. Haemonchus contortus infection is undoubtedly the most pathogenic disease that causes the greatest losses in sheep production. The parasite control should be based on management strategies of animals and pastures together with the rational administration of anthelmintics after the appropriate parasitological diagnostic. However, in the last three decades, parasitic control has been based almost exclusively on the massive use of broad-spectrum and persistent anthelminthic drugs. The increase in anthelminthic resistance along with the new tendency of organic production and reduction of drug residues in food generated interest in the study of new control strategies. In this context, the present work of Doctoral Thesis focused on the anthelmintic resistance from an integrative pharmaco-parasitological approach. The objective of the currern work was to study the relationship that exists between the concentrations of different macrocyclic lactones in plasma, tissues / fluids of parasitic location of sheep and in the target parasite and the efficacy against resistant H. contortus. The Thesis was divided into 3 experimental sections. In the first, the in vivo characterization of factors affecting the relationship between concentrations at the site of parasitic localization, concentrations in the target parasite and anthelmintic efficacy for different macrocyclic lactones in lambs infested with resistant nematodes was evaluated under standardized conditions. Three experiments were carried out within this section, comparing the use of two administration routes (subcutaneous and intra-ruminal), the different macrocyclic lactones (ivermectin-abamectin-moxidectin) and the duplication of the dose as strategies to increase the anthelmintic efficacy. The results showed that although the plasma disposition is greater after the ivermectin administration by the subcutaneous route, higher drug concentrations were measured in the tissues and contents of the gastrointestinal tract after 2-3 days post-treatment by intraruminal route. The drug increment in the target tissues was reflected in a higher concentrations of ivermectin found within the parasite and a higher efficacy against H. contortus after the intraruminal administration. The current results corroborated that the drug entry to the parasite by the transcuticular route is relevant for clinical efficacy, even for those parasites that are hematophagous. In the second experiment, it was found that moxidectin administered at the therapeutic dose was more effective vi against nematodes resistant to ivermectin. Although moxidectin showed the lowest accumulated concentrations in adult parasites, lower number of parasites were recovered (at day 2) after moxidectin treatment and its anthelmintic efficacy was greater compared to those obtained after ivermectin and abamectin administration. In the third experiment, the influence of the dose rate on the efficacy and accumulation of drugs in the target tissues of lambs and in H. contortus was evaluated. Due to the high level of resistance, only the administration of double dose of moxidectin was effective. In Experimental Section II, the activity of macrocyclic lactones against resistant H. contortus was characterized in vitro by the development of the larval motility inhibition test using larval stages of H. contortus. This test demonstrated to be an effective test to establish differences between resistant and susceptible isolates. However, it is a difficult test to implement for the routinely diagnostic of anthelmintic resistance. An ex vivo method was also developed to characterize the entry of the drug into the adult parasites. The increased exposure to ivermectin and moxidectin enhanced the amount of drug recovered in H. contortus. In Experimental Section III, the genetic changes in H. contortus associated with the phenomenon of resistance were studied. The expression of glycoprotein-P2 in susceptible and resistant H. contortus isolates was determined. Overexpression was found in isolates of resistant parasites. In addition, treatment with ivermectin, unlike abamectin and moxidectin, was the only one that induced a significant increase in the expression of glycoprotein-P2 in resistant H. contortus recovered from treated lambs at 0.5 and 1 day post-administration. In conclusion, the present Doctoral Thesis validated in vivo and in vitro studies and molecular tools to improve the parasitic control strategies and to extend the lifespan of macrocyclic lactones. To obtain this in the field, it is necessary the rational use of these drugs in each farm with a previous professional evaluation by the veterinarians.

Resumo: Os nematoides gastrintestinais são um dos principais fatores limitantes na criação de pequenos ruminantes no mundo. Os benzimidazóis (BZ), as lactonas macrocíclicas (LM) e os imidazotiazóis são os anti-helmínticos mais utilizados para o controle desses parasitas. Contudo, o uso de fármacos leva inevitavelmente ao desenvolvimento de resistência anti-helmíntica (RAH). O diagnóstico de RAH é realizado principalmente por meio de métodos fenotípicos com baixa sensibilidade. Dessa forma, métodos moleculares, são necessários para melhorar a capacidade de detecção da RAH. Os objetivos desse trabalho foram avaliar o estado da RAH em nematoides gastrintestinais de ovinos no Ceará, investigar a relação entre a resistência a BZ e LM e padronizar técnica de diagnóstico de resistência molecular a levamisol (LEV) em H. contortus. Para tanto, o trabalho foi dividido em três experimentos. No experimento I foi realizado um levantamento da situação da resistência a BZ por meio do teste de eclosão de ovos (TEO) e da PCR em tempo real (qPCR) para identificar os polimorfismos de nucleotídeo único (SNPs) F167Y, F200Y e E198A. No experimento II foi realizada a seleção para resistência do isolado de H. contortus Inbred-Susceptible Edinburgh (ISE) com doses crescentes de oxifendazol (OXF), ivermectina (IVM) e oxifendazol mais ivermectina (IVMOXF). O desenvolvimento da resistência foi avaliado por qPCR, TEO para BZ e teste de desenvolvimento larva (TDL) para ivermectina. No terceiro experimento foi realizado a qPCR para o diagnóstico de resistência a LEV, baseando-se na deleção de 63pb no gene Hco-acr-8b codificante para os receptores nicotínicos de acetilcolina. Foram testadas 26 populações de H. contortus: Kokstad (LEV resistente), ISE (LEV susceptível) e 24 populações obtidas de rebanhos no estado do Ceará. Foi realizado o TDL para LEV em cinco isolados de campo. No experimento I a concentração efetiva média para inibir 50% da eclosão de ovos (CE50) no TEO foi de 2,46 ?g/mL (± 0,58 ?g/mL) e a resistência a BZ foi detectada em todas as fazendas pesquisadas. As frequências médias de alelos resistentes nos códons F200Y, F167Y e E198A foram 34,16% (± 12,13%), 58,31% (± 18,89%) e 0,016 (±0,012), respectivamente. No experimento II todos os tratamentos levaram ao aumento da frequência de alelos resistentes para os SNP F200Y e F167Y (p

Haemonchus contortus is one of the most common and economically significant causes of disease in small ruminants worldwide, and the control programs of parasitic nematodes - including H. contortus - rely mostly on the use of anthelmintic drugs. The consequence of the use of this, as the sole sanitary strategy to avoid parasite infections, was the reduction of the efficacy of all chemotherapeutic products with a heavy selection for resistance. The widespread of anthelmintic resistance and the difficulty of its early diagnosis has been a major concern for the sustainable parasite management on farms. The objective of this research was to determine and compare the ivermectin (IVM) and moxidectin (MOX) effect in a selected field strain of H. contortus with a known resistance status, using the in vitro larval migration on agar test (LMAT). Third stage larvae of the selected isolate were obtained from faecal cultures of experimentally infected sheep and incubated in eleven increasing diluted concentrations of IVM and MOX (6, 12, 24, 48, 96, 192, 384, 768, 1536, 3072 and 6144µg/mL). The dose-response sigmoidal curves were obtained using the R² value of >0.90 and the lethal concentration (LC50) dose for the tested anthelmintic drugs using a four-parameter logistic model. The LC50 value for MOX was significantly lower than IVM (1.253µg/mL and 91.06µg/mL), identifying the H. contortus isolate as considerably less susceptible to IVM compared to MOX. Furthermore, the LMAT showed a high consistency (p0,90 e a dose de concentração letal (CL50) para as drogas anti-helmínticas testadas, utilizando um modelo logístico de quatro parâmetros. O valor de CL50 para MOX foi significativamente menor do que para IVM (1,253µg/mL e 91,06µg/mL), identificando o isolado de H. contortus como consideravelmente menos suscetível à IVM em comparação à MOX. Além disso, o LMTA mostrou uma alta consistência (p

Resumen Se realizó una prueba de establo para evaluar la eficacia residual de ivermectina 3,15% y los niveles plasmáticos en animales tratados con Ivomec GOLD® y con una formulación genérica frente a desafíos larvarios de Rhipicephalus (B.) microplus. En ambas formulaciones la eficacia residual presentó una gran dispersión en los días sin caída de garrapatas teleoginas post tratamiento. Descontando el ciclo parasitario la residualidad se presentó en un rango de 35 a 63 días. El análisis estadístico de los resultados de ambas formulaciones estudiadas a través de la mediana, demostró una residualidad de 52 días frente a larvas infestantes de R. (B) microplus. Los parámetros farmacocinéticos presentaron valores medios de: área bajo la curva (ABC) 1795±188 y 1351±118, picos plasmáticos (Tmax) a los 13,4±4,1 y 15,0±3,6 días post inoculación con concentración máxima (Cmax) de 65,4±1,5 y 41,1±0,3 ppb para la formulación de Ivomec GOLD® y la genérica respectivamente. Concentraciones inferiores a 10 ppb pueden representar el umbral por debajo del cual se logró desarrollar el ciclo parasitario hasta obtener garrapatas teleoginas. Los resultados obtenidos permitirán tener un mejor conocimiento para evaluar el comportamiento de las formulaciones en pruebas de establo y de campo tanto en el estudio de la eficacia, la residualidad y en el tiempo de espera para la faena. Summary A stall test was performed to evaluate two ivermectin formulations (Ivomec Gold® and a generic one at 3.15%). Residual efficacy against larval challenges of Rhipicephalus (B.) microplus and plasmatic levels of ivermectin were studied. In both formulations residual efficacy presented a large scatter in the number of days of detachment of engorged females after treatment, discounting the residual parasitic cycle a range of 35 to 63 days was presented. Statistical analysis of the results of both formulations showed a residual period of 52 days (median) before reinfestation with larvae of R. (B) microplus. The average pharmacokinetic parameters area under the curve (AUC), represented values of 1795±188 and 1351±118, whereas plasma peaks (Tmax) occurred at 13.4±4.1 and 15.0±3.6 days post inoculation reaching maximum concentration (Cmax) of 65.4±1.5 and 41.1±0.3 ppb in Ivomec GOLD ® and generic formulation respectively. Concentrations below 10 ppb may represent the threshold below the parasitic cycle could develop and engorged ticks obtained. These results allow a better understanding to evaluate the performed of new formulations in stall and field trials to evaluate efficacy, residual efficacy and the withholding period.

Background: in Colombia, the control of tick infestation in cattle is almost exclusively performed with chemical acaricides. It is important to determine the degree of resistance of Riphicephalus microplus field populations to ivermectins (IVM) as the first step to design strategies to increase the useful life of acaricides and decrease the reliance on chemical controls. Objective: to test the degree of resistance to IVM. Methods: three cattle farms in Antioquia (Colombia) where IVM had previously failed to control infestations of R. microplus ticks were studied. Ticks were collected several months apart in 2013, and the larval immersion test (LIT) was performed on the progeny of the adult females. Concentration-mortality data were subjected to probit analysis. Results: the three populations showed lethal concentrations (LC) 50 and 99 of ≥ 30 ppm and ≥ 400 ppm, respectively. There was no difference observed in the LC at different times of collection for each population studied. Such high values for LC, together with very low regression slopes (≤ 2), indicated a very heterogeneous response to increasing concentrations of IVM, which is common for resistant populations. At two farms, subcutaneous injection with a long-acting formulation of IVM at a dosage of 630 μg/Kg was ineffective at eliminating existing infestations and protecting against reinfestations, confirming farmer suspicions of lost efficacy. Conclusion: epidemiological studies are necessary to assess the current status of resistance to IVM in this region of Colombia, and it is likely that the intensive use of IVM will aggravate this situation in the future. Antecedentes: en Colombia, el control de la infestación de garrapatas en el ganado se ha realizado casi exclusivamente con acaricidas químicos y es importante determinar el grado de resistencia de las poblaciones de campo de Riphicephalus microplus a ivermectinas (IVM) como el primer paso para diseñar tácticas que aumenten su vida útil y disminuyan la dependencia de los controles químicos. Objetivo: probar el grado de resistencia a IVM. Métodos: se evaluó la resistencia de R. microplus a IVM en tres fincas ganaderas en Antioquia (Colombia) donde se sospechaba que productos con IVM estaban fracasando para controlar infestaciones por garrapatas. Se recolectaron garrapatas con varios meses de diferencia durante el año 2013 y se empleó la prueba de inmersión de larvas (LIT) con la descendencia de las hembras adultas. Los datos de concentraciónmortalidad fueron sometidos a análisis probit. Resultados: los resultados para las tres poblaciones estudiadas mostraron concentraciones letales (LC) 50 y 99 de ≥ 30 ppm y ≥ 400 ppm, respectivamente. No se observó diferencia en las LC obtenidas en diferentes momentos de recogida para cada población estudiada. Los altos valores de LC, junto con pendientes muy bajas en las rectas de regresión (≤ 2), indicaron una respuesta muy heterogénea a concentraciones crecientes de IVM, propio de poblaciones resistentes. En dos de las granjas, la administración de inyecciones subcutáneas con una formulación de acción prolongada de la IVM a dosis de 630 μg/Kg fue incapaz de eliminar las infestaciones y proteger frente a reinfestaciones, confirmando las sospechas de los ganaderos de pérdida de eficacia. Conclusión: estudios epidemiológicos son necesarios para evaluar el estado actual de la resistencia a IVM en esta región de Colombia, y es probable que el uso intensivo de IVM agrave esta situación en el futuro Antecedentes: na Colômbia, o controle de infestação com carrapatos em bovinos tem sido realizado quase exclusivamente com acaricidas químicos pelo qual se faz importante para determinar o grau de resistência de populações de campo de Rhipicephalus microplus a uma das drogas antiparasitárias de maior uso no manejo de gado, a ivermectina (IVM), como o primeiro passo para projetar táticas para aumentar a sua vida útil e diminuir a dependência de controles químicos. Objetivo: testar o grau de resistência à ivermectina. Método: foi avaliada a resistência de R. microplus à ivermectina em três fazendas de gado leiteiro localizadas no departamento de Antioquia (Colômbia) com suspeita de que o controle dos carrapatos com a utilização deste produto não estava sendo efetivo. Coletaram-se carrapatos em diferentes meses durante o ano 2013, e foi utilizado o teste de imersão de larvas (LIT) na descendência das fêmeas adultas, como método para detecção da resistência. Os dados de concentração-mortalidade foram submetidos à análise Probit. Resultados: todas as três populações avaliadas mostraram concentrações letais (LC) 50 de ≥ 30 ppm, e LC 99 de ≥ 400 ppm. Não foram observadas diferenças na LC entre as coletas feitas nos diferentes meses, em cada população amostrada. Os altos valores de LC encontrados junto com uma baixa regressão linear (≤ 2), indicam uma resposta muito heterogênea as concentrações crescentes de ivermectina, próprio de populações resistentes. Em duas das fazendas, a administração de injeções subcutâneas de ivermectina com uma formulação de longa ação, em doses de 630 μg/Kg, não foram capazes de eliminar as infestações e proteger contra a reinfestação, confirmando as reclamações dos fazendeiros para a perda de eficácia da droga antiparasitária. Conclusões: os estudos epidemiológicos são necessários para avaliar o estado atual da resistência à ivermectina nesta região da Colômbia, e é provável que o uso intensivo da ivermectina agrave ainda mais esta situação no futuro.

Summary Background: in Colombia, the control of tick infestation in cattle is almost exclusively performed with chemical acaricides. It is important to determine the degree of resistance of Riphicephalus microplus field populations to ivermectins (IVM) as the first step to design strategies to increase the useful life of acaricides and decrease the reliance on chemical controls. Objective: to test the degree of resistance to IVM. Methods: three cattle farms in Antioquia (Colombia) where IVM had previously failed to control infestations of R. microplus ticks were studied. Ticks were collected several months apart in 2013, and the larval immersiontest (LIT) was performed on the progeny of the adult females. Concentration-mortality data were subjected to probit analysis. Results: the three populations showed lethal concentrations (LC) 50 and 99 of ≥ 30 ppm and ≥ 400 ppm, respectively. There was no difference observed in the LC at different times of collection for each population studied. Such high values for LC, together with very low regression slopes (≤ 2), indicated a very heterogeneous response to increasing concentrations of IVM, which is common for resistant populations. At two farms, subcutaneous injection with a long-acting formulation of IVM at a dosage of 630 μg/Kg was ineffective at eliminating existing infestations and protecting against reinfestations, confirming farmer suspicions of lost efficacy. Conclusion: epidemiological studies are necessary to assess the current status of resistance to IVM in this region of Colombia, and it is likely that the intensive use of IVM will aggravate this situation in the future. Keywords: low efficacy, macrocyclic lactones, ticks. Resumen Antecedentes: en Colombia, el control de la infestacion de garrapatas en el ganado se ha realizado casi exclusivamente con acaricidas quimicos y es importante determinar el grado de resistencia de las poblaciones de campo de Riphicephalus microplus a ivermectinas (IVM) como el primer paso para disenar tacticas que aumenten su vida util y disminuyan la dependencia de los controles quimicos. Objetivo : probar el grado de resistencia a IVM. Metodos: se evaluo la resistencia de R. microplus a IVM en tres fincas ganaderas en Antioquia (Colombia) donde se sospechaba que productos con IVM estaban fracasando para controlar infestaciones por garrapatas. Se recolectaron garrapatas con varios meses de diferencia durante el ano 2013 y se empleo la prueba de inmersion de larvas (LIT) con la descendencia de las hembras adultas. Los datos de concentracionmortalidad fueron sometidos a analisis probit. Resultados: los resultados para las tres poblaciones estudiadas mostraron concentraciones letales (LC) 50 y 99 de ≥ 30 ppm y ≥ 400 ppm, respectivamente. No se observo diferencia en las LC obtenidas en diferentes momentos de recogida para cada poblacion estudiada. Los altos valores de LC, junto con pendientes muy bajas en las rectas de regresion (≤ 2), indicaron una respuesta muy heterogenea a concentraciones crecientes de IVM, propio de poblaciones resistentes. En dos de las granjas, la administracion de inyecciones subcutaneas con una formulacion de accion prolongada de la IVM a dosis de 630 μg/Kg fue incapaz de eliminar las infestaciones y proteger frente a reinfestaciones, confirmando las sospechas de los ganaderos de perdida de eficacia. Conclusion : estudios epidemiologicos son necesarios para evaluar el estado actual de la resistencia a IVM en esta region de Colombia, y es probable que el uso intensivo de IVM agrave esta situacion en el futuro. Palabras clave: garrapatas, lactonas macrociclicas, perdida de eficacia. Resumo Antecedentes: na Colombia, o controle de infestacao com carrapatos em bovinos tem sido realizado quase exclusivamente com acaricidas quimicos pelo qual se faz importante para determinar o grau de resistencia de populacoes de campo de Rhipicephalus microplus a uma das drogas antiparasitarias de maior uso no manejo de gado, a ivermectina (IVM), como o primeiro passo para projetar taticas para aumentar a sua vida util e diminuir a dependencia de controles quimicos. Objetivo: testar o grau de resistencia a ivermectina. Metodo: foi avaliada a resistencia de R. microplus a ivermectina em tres fazendas de gado leiteiro localizadas no departamento de Antioquia (Colombia) com suspeita de que o controle dos carrapatos com a utilizacao deste produto nao estava sendo efetivo. Coletaram-se carrapatos em diferentes meses durante o ano 2013, e foi utilizado o teste de imersao de larvas (LIT) na descendencia das femeas adultas, como metodo para deteccao da resistencia. Os dados de concentracao-mortalidade foram submetidos a analise Probit. Resultados: todas as tres populacoes avaliadas mostraram concentracoes letais (LC) 50 de ≥ 30 ppm, e LC 99 de ≥ 400 ppm. Nao foram observadas diferencas na LC entre as coletas feitas nos diferentes meses, em cada populacao amostrada. Os altos valores de LC encontrados junto com uma baixa regressao linear (≤ 2), indicam uma resposta muito heterogenea as concentracoes crescentes de ivermectina, proprio de populacoes resistentes. Em duas das fazendas, a administracao de injecoes subcutâneas de ivermectina com uma formulacao de longa acao, em doses de 630 μg/Kg, nao foram capazes de eliminar as infestacoes e proteger contra a reinfestacao, confirmando as reclamacoes dos fazendeiros para a perda de eficacia da droga antiparasitaria. Conclusoes: os estudos epidemiologicos sao necessarios para avaliar o estado atual da resistencia a ivermectina nesta regiao da Colombia, e e provavel que o uso intensivo da ivermectina agrave ainda mais esta situacao no futuro. Palavras chave: carrapatos, lactonas macrociclicas, perda de eficacia.

Avaliou-se a eficácia de lactonas macrocíclicas (ivermectina e moxidectina) sobre a eventual ocorrência de efeitos colaterais e acompanharam-se, após a alta parasitológica, por 12 meses, os cães tratados, visando detectar a recidiva do quadro dermatopático. Dos 63 animais, 59% eram fêmeas, 76% apresentavam precisa definição racial e 67% tinham pelame curto. A ivermectina (0,6mg/kg/dia) foi administrada por via oral a 31 cães, e a moxidectina (0,5mg/kg/cada 72 horas), pela mesma via, a 32 animais. Os tempos médios para a obtenção da primeira negativação do exame parasitológico do raspado cutâneo e para a consecução da alta foram, respectivamente, de 90 e 130 dias para a ivermectina e de 108 e 147 dias para a moxidectina. A ivermectina acarretou menos (16,1%) efeitos colaterais em relação à moxidectina (37,5%) (P=0,03). As recidivas foram, respectivamente, 10,3% e 13% para ivermectina e moxidectina. Não houve diferença entre os dois protocolos de terapia quanto aos percentuais de recidiva (P=0,67) e eficácia (P=0,61). Ambas as lactonas macrocíclicas mostraram-se eficazes: ivermectina 89,7% e moxidectina 87%. The efficacy of ivermectin and moxidectin for treatment of generalized canine demodicosis, was evaluated to detect the eventual occurrence of side effects caused by the use of these drugs, and to follow the treated dogs for 12 months after obtaining parasitologic cure. Of 63 dogs, 59% were females, 76% were defined as purebred and 67% had short hair. Ivermectin (0.6mg/kg/daily) was orally administered to 31 dogs and moxidectin (0.5mg/kg/every 72 hours) to 32 dogs. The average number of days to obtain the first negative skin scraping results and the parasitologic cure were, respectively, 90 and 130 days for ivermectin, and 108 and 147 days for moxidectin. Ivermectin caused fewer side effects (16.1%) than moxidectin (37.5%) (P0.67) and efficacy (P>0.61). Both drugs were effective and safe to treat generalized canine demodicosis: ivermectin 89.7% and moxidectin 87.0%.

O amplo espectro de ação dos endectocidas e a praticidade de aplicação das suas formulações pour-on são fatores que têm estimulado uma maior frequência de utilização destes produtos na criação de bovinos. Neste estudo, avaliou-se a eficácia anti-helmíntica da ivermectina, administrada via pour-on, 500mcgkg-1, contra nematódeos de bovinos provenientes de diferentes rebanhos. Foram adquiridos 12 bezerros de cada um dos quatro rebanhos selecionados. Todos os 48 bezerros utilizados apresentavam médias de ovos por grama de fezes (OPG) maiores que 500, considerando-se três contagens consecutivas. Na sequencia, os animais de cada rebanho foram divididos em dois grupos de seis bovinos cada, sendo um tratado com ivermectina 500mcgkg-1 via pour-on e outro mantido como controle. Quatorze dias depois do tratamento, os bovinos foram submetidos à eutanásia para contagem de endoparasitas. A ivermectina demonstrou resultados nulos de eficácia contra H. placei nos quatro rebanhos. Contra C. punctata, tal formulação teve eficácia nula nos rebanhos provenientes de Jaboticabal,SP, e de Formiga,MG, e eficácias de 75,8% e 58,4% nos rebanhos provenientes de São José do Rio Pardo,SP, e de São Sebastião do Paraíso,MG, respectivamente. Valores de eficácia de 94,2% (Jaboticabal), 0,0% (São José do Rio Pardo), 94,2% (Formiga) e 39,2% (São Sebastião do Paraíso) foram detectados contra O. radiatum. Com base nos resultados encontrados, pode-se concluir que as quatro populações de Haemonchus placei e de Cooperia punctata avaliadas foram resistentes à ivermectina, administrada via pour-on, 500mcgkg-1. Foram observadas cepas de Oesophagostomum radiatum resistentes à ivermectina em dois dos quatro rebanhos testados. The broad spectrum of endectocides and the easy use mode of their pour-on formulations are factors that have stimulated a higher frequency of use these drugs in cattle. In this study was evaluated the efficacy of ivermectin pour-on using the dose of 500mcgkg-1, against nematodes in naturally infected cattle from different herds. Twelve calves were brought from each of the four farms selected. All the 48 calves used showed mean of eggs per gram of feces (EPG) greater than 500 considering three consecutive counts. Subsequently, animals from each herd were divided into two groups of six animals each, one treated pour-on with ivermectin 500mcgkg-1 and other kept as untreated control. Calves were euthanized 14 days after treatment for counting the endoparasites. Ivermectin showed null effect against H. placei in all the herds evaluated. The drug was also ineffective against C. punctata in the herds from Jaboticabal,SP and Formiga,MG, and reached efficacy of 75.8% and 58.4% in herds from São José do Rio Pardo,SP and São Sebastião do Paraíso,MG, respectively. Efficacies of 94.2% (Jaboticabal), 0.0% (São José do Rio Pardo), 94.2% (Formiga) and 39.2% (São Sebastião do Paraíso) were detectedagainst O. radiatum. Based on these results obtained on the present study, the four populations of Haemonchus placei and Cooperia punctata evaluated were resistant to ivermectin pour-on using a dose of 500mcgkg-1. Ivermectin-resistant strains of Oesophagostomum radiatum were found in two of the four herds evaluated.

El presente estudio se desarrolló en las sabanas de Casanare, a fin de determinar los grados de susceptibilidad o resistencia antihelmíntica que presentan las poblaciones de pequeños Strongylus de esta región del país, frente a las lactonas macrocíclicas. Se muestrearon cuatro municipios del departamento: Aguazul, Paz de Ariporo, Maní y El Yopal, entre junio del 2006 y abril del 2007, recolectando en cada uno de los municipios diez muestras de materia fecal fresca, directamente de los potreros y completamente al azar. Las muestras fueron procesadas mediante la técnica coprológica de MacMaster, estableciendo el número de hpg de materia fecal por municipio. A las seis muestras con el conteo de hpg de materia fecal más alto, de cada predio se extrajeron larvas L3 mediante las técnicas de coprocultivo y Baermann-Wetzel con las cuales se corrió la prueba análisis del desarrollo de larvas (LDA), detectando poblaciones de pequeños Strongylus altamente susceptibles a la acción de las lactonas macrocíclicas (ivermectina). This study was conducted in the grasslands of the Department of Casanare, seeking to determine the degree of anthelmintic susceptibility or resistance against macrocyclic lactones in small populations of Strongylus in this region of the country. Samples were taken from four municipalities in the department: Aguazul, Paz de Ariporo, Maní and El Yopal, between June, 2006 and April, 2007, where ten fresh fecal samples were collected in each municipality, directly from the field and in a completely random way. The samples were processed using the MacMaster coprological technique, thus determining the highest count of fecal epg by municipality. L3 larvae were extracted from each of the six samples with the highest count of fecal epg through the coprological and Baermann-Wetzel tests with which the Larvae Development Analysis (LDA) test was run, detecting Small Strongylus highly susceptible to the action of macrocyclic lactones (ivermectin).

Eighty-four half-blood Gir × Holstein (F1) calves aged six months who were naturally infected by gastrointestinal helminths and maintained in rotational grazing received different anthelmintic treatments. Group A received anthelmintics according to the usual management in the property (eight treatments, seven including a macrocyclic lactone agent). Group B received strategic treatment (ivermectin 3.15%) at the beginning and at the end of the rainy period. Eggs per gram of feces (EPG) counts and genus of larvae from fecal cultures were determined on a monthly basis from April 2002 to December 2003. There was no significant reduction (p > 0.05) in EPG counts in any group after anthelminthic treatment, and the larvae in fecal cultures observed were Cooperia, Haemonchus, Oesophagostomum and a few Trichostrongylus. Cooperia was the most prevalent genus in the first four months of the experiment and Haemonchus in the following months. In 2003, tracer calves were introduced onto the pastures monthly and they showed high nematode burden many times throughout the year, and Cooperia punctata and Haemonchus contortus were the main species identified. The results suggest that there is anthelminthic resistance in this farm, mainly to macrocyclic lactones, and the development of immunity by crossbred animals was vital to reduce nematode burden. Oitenta e quatro bezerras meio sangue Gir × holandês (F1) com seis meses de idade, naturalmente infectadas por helmintos gastrintestinais e mantidas em pastejo rotacionado receberam diferentes tratamentos anti-helmínticos. O grupo A recebeu anti-helmínticos segundo manejo empregado na propriedade (oito tratamentos, sete com produtos à base de lactonas macrocíclicas). O grupo B recebeu tratamento estratégico (ivermectina 3,15%) no inicio e final de período chuvoso. Mensalmente, no período de abril de 2002 a dezembro de 2003, foram realizadas contagens de ovos por grama de fezes (OPG) e coproculturas. Não houve redução significativa (p > 0,05) nas contagens de OPG em nenhum dos grupos após os tratamentos anti-helmínticos, e as larvas encontradas na coprocultura foram Cooperia, Haemonchus, Oesophagostomum e poucos Trichostrongylus, com predominância de Cooperia nos quatro meses iniciais e Haemonchus nos meses seguintes. No ano de 2003, bezerros traçadores foram alocados mensalmente nos pastos, apresentando altas cargas parasitárias na maioria dos meses do ano, sendo as principais espécies identificadas: Cooperia punctata e Haemonchus contortus. Os resultados indicam que os nematódeos da fazenda apresentam resistência anti-helmíntica, principalmente às lactonas macrocíclicas, e o desenvolvimento de imunidade foi primordial para reduzir a carga parasitária dos animais mestiços.

An overview of the macrocyclic lactones (ML), with recent results and their effect on the control of Rhipicephalus (Boophilus) microplus, is presented in this paper. This review describes in detail the chemistry, pharmacokinetics and dose presentation, security and time withdrawal and environmental safety of the groups belonging to ML: avermectins (ivermectin, doramectin, eprinomectin, abamectin) and milbemycin (moxidectin). There is also a discussion on their efficacy and the problem of resistance of R. (B.) microplus to ML. Finally, it is concluded that due to their high lipid solubility ML are widely distributed in tissues, being effective in the control of R. (B.) microplus in cattle. The efficacy of short-acting ivermectin, doramectin, eprinomectin, abamectin and moxidectin are similar ( 90% effective after four weeks post treatment, PT) to control adult stages of R. (B.) microplus, however, in long-acting LM are available the market and present efficiency of 95% with persistence up to 70 days PT.

An overview of the macrocyclic lactones (ML), with recent results and their effect on the control ofRhipicephalus(Boophilus)microplus, is presented in this paper. This review describes in detail the chemistry, pharmacokinetics and dose presentation, security and time withdrawal and environmental safety of the groups belonging to ML: avermectins (ivermectin, doramectin, eprinomectin, abamectin) and milbemycin (moxidectin). There is also a discussion on their efficacy and the problem of resistance ofR.(B.)microplusto ML. Finally, it is concluded that due to their high lipid solubility ML are widely distributed in tissues, being effective in the control ofR.(B.)microplus in cattle. The efficacy of short-acting ivermectin, doramectin, eprinomectin, abamectin and moxidectin are similar (> 90% effective after four weeks post treatment, PT) to control adult stages ofR. (B.)microplus, however, in long-acting LM are available the market and present efficiency of > 95% with persistence up to 70 days PT. &nbsp, En esta revisión se describe en detalle la composición química, farmacocinética, presentación y dosis, seguridad y tiempo de retiro y la seguridad ambiental de los grupos que componen a las LM: avemectinas (ivermectina, doramectina, eprinomectina, abamectina) y milbemicinas (moxidectina). Además se presenta una discusión sobre su eficacia y el problema de resistencia deRhipicephalus(Boophilus)microplusa las LM. Se concluye que las LM por su alta liposolubilidad se distribuyen ampliamente en los tejidos, siendo eficaces para el control deR. (B.)microplus en el ganado bovino. La eficacia de la ivermectina, doramectina, eprinomectina, abamectina y moxidectina de corta acción es similar (> 90% de eficacia a las cuatro semanas postratamiento, PT) para el control de fases adultas deR. (B.)microplus; sin embargo, existen en el mercado LM de larga acción que presentan eficacia > 95% con persistencia hasta 70 días PT. &nbsp

Por intermédio de avaliações preliminares a campo em bovinos naturalmente infestados pelo carrapato Boophilus microplus, foi identificada uma população de carrapatos Boophilus microplus que apresentou sobrevivência a um programa de controle estratégico com uma lactona macrocíclica, no município de São Gabriel, RS. Esta cepa foi reproduzida em laboratório e submetida a testes in vitro e em estábulo. Testes de imersão com teleóginas, frente à ivermectina e a moxidectina mostraram sobrevivência e postura em concentrações quevariaram entre 25 e 1000ppm para ambas as moléculas, enquanto que em uma população sensível, cepa Porto Alegre, houve mortalidade de 100% nas concentrações entre 200 e 1000ppm. Um primeiro teste em estábulo, mostrou variabilidade de eficácia diária, entre os dias 3 e 24 pós-tratamento (p.t.), de 14,02% a 93,33%, para doramectina, de 30,25% e 89,00% para moxidectina e de 46,64% e 99,53% para ivermectina. Em um segundo teste em estábulo, utilizando-se unicamente doramectina em comparação com um grupo controle, a eficácia média observada foi de 64,15% entre os dias 1 e 23 p.t., sendo que a maior eficácia registrada foi de 82,68% no dia 8 p.t. Por outro lado, um terceiro teste frente a doramectina em estábulo, registrou variações de eficácia entre 10,73% e 74,22% nos dias 2 e 23 p.t. Em um outro teste comparativo com uma cepa sensível de referência (cepa Porto Alegre), frente a doramectina, a eficácia média entre os dias 1 e 23 p.t. foi de 80,49% para a cepa São Gabriel e de 91,23% para a cepa Porto Alegre. Esses resultados, além de mostrarem uma variabilidade na eficácia das lactonas macrocíclicas testadas, alertam para os riscos da diminuição da eficácia carrapaticida com o uso intensivo e sistemático de um mesmo ingrediente ativo no campo e, por outro lado, enfatizam a necessidade do desenvolvimento de testes in vitro que possamdiagnosticar precocemente e com menores custos, o surgimento de eventuais populações resistentes as lactonas macrocíclicas Trough preliminary field evaluation on naturally infested cattle it was identified one population of Boophilus microplus ticks which showed survival to a strategic control program with a macrocyclic lactone in the locality of São Gabriel, RS. This strain was maintained in laboratoryconditions and submitted to in vitro and stall tests. Immersion tests with adult females against ivermectin and moxidectin showed survival and egg laying in concentrations that range from 25 to 1000ppm for both active ingredients while in a susceptible tick population strain (Porto Alegre strain) there was 100% of mortality at 200 e 1000ppm. A first stall test showed variability in the daily efficacy, between 3 e 24 post treatment (p.t.), from 14.02% to 93.33%, against doramectin, from 30.25% to 89.00% against moxidectin and from 46.64% to 99.53% against ivermectin. In a second stall test, only with doramectin in comparison with a control group, average of efficacy was 64.15% between 1 and 23 days p.t., while the best efficacy was observed in the day 8 p.t.(82.68%). However, a third stall test against doramectin, showed variations in the efficacy between 10.73% and 74.22% between days 2 and 23 p.t. In adition, a comparative stall test with a susceptible reference strain (strain Porto Alegre) against doramectin, average of efficacybetween days 1 and 23 p.t. was 80.49% for the São Gabriel strain and 91.23% for the Porto Alegre strain. These results beyond showed a variability in the efficacy of some macrocyclic lactones, draw attention to the risks of decreasing acaricide efficiency with the intensive andsystematic use of a same active ingredient in the field, and, give emphasis to the needs of further in vitro tests in order to early detect arising of lactones macrocyclic resistant populations with minor cost and major confidence

A eficácia comparativa da três aplicações de formulações injetáveis de uma ou duas marcas comerciais de ivermectina 1% (IVMa e IVMb), ivermectina L.A. 1% (IVMc), doramectina (DRM) e de um produto endectocida experimental (PEE), em comparação com um grupo controle não tratado, foi avaliada contra os parasitos Boophilus microplus, larvas de Dermatobia hominis e helmintos gastrintestinais, em bovinos de corte da raça Canchim, naturalmente infestados. Setenta e nove animais foram distribuídos, de acordo com o peso, em quatro grupos de 16 animais e um de 15 animais cada, sendo T1 controle, T2 IVMa, T3 IVMb ou PEE, T4 IVMc e T5 DRM. Os animais foram submetidos a contagem dos ectoparasitos, coleta de fezes e passagem, no dia 0 e cada 28 dias, durante os 252 dias de experimento. Amostras de fezes de cada grupo eram utilizadas na realização de coprocultura para determinar as gêneros dos helmintos envolvidos. Houve diferença entre os pesos dos grupos tratados e o grupo não tratado, mas não houve diferença entre os grupos tratados. Os produtos estudados foram eficientes no controle do Boophilus mocriplus até a quarta abaliação, a partir da quinta avaliação houve um aumento nas infestações de todos os grupos e foi necessário fazer um controle com banhos carrapaticidas. The comparative efficacy of three applications of injectable formulations of one or two brands of ivermectin 1% (IVMa and IVMb) LA 1% ivermectin (IVMc), doramectin (DRM) and an experimental endectocid product (ESP), compared with an untreated control group, was assessed against the parasites Boophilus microplus larvae of Dermatobia hominis and gastrointestinal helminths in beef cattle Canchim naturally infested. Seventy-nine animals were divided according to weight in four groups of 16 animals and one of 15 animals each: T1 - control, T2 - IVMa, T3 - or PEE IVMb, T4 - T5 and IVMc - DRM. The animals were subjected to counting of ectoparasites, feces, and passing on day 0 and every 28 days during the 252 days of experiment. Stool samples from each group were used in performing stool culture to determine the genera of nematodes involved. There were differences between the weights of the treated groups and untreated group, but no difference between the treated groups. The products studied were effective in controlling the Boophilus mocriplus abaliação until the fourth, fifth from the evaluation there was an increase in infestations of all groups and was needed to make a control with acaricide.

El trabajo se realizó de abril a julio del 2001 en un predio del Sur de Chile con 84 bovinos de aproximadamente 280 kg, infectados naturalmente, que se distribuyeron en 7 grupos de 12 animales: Un grupo Control tratado con placebo y seis grupos tratados subcutáneamente con 0.2 mg/kg de Ivermectina (3 productos comerciales diferentes), Moxidectina, Abamectina y Doramectina. Materia fecal se obtuvo del recto de todos los animales 3 días antes, el día del tratamiento, y posteriormente cada semana hasta los 77 días post-tratamiento (p.t.). Recuentos de huevos (hpg) se realizaron a cada muestra. Se realizó la prueba de reducción de la oviposición (FECRT) para determinar resistencia antihelmíntica. Cultivos de larvas acumulativos se realizaron a cada grupo en cada fecha de muestreo. Los días 0, 14, 56 y 77 p.t. se determinó el número de larvas por kg de pasto seco de los 4 potreros utilizados. El peso vivo de los animales se obtuvo los días -3 y 63 p.t. Los resultados se analizaron considerando significativo un p < 0.05. El grupo control tuvo una oviposición que fluctuó entre los 140 y 500 hpg. Los 6 grupos tratados presentaron una reducción significativa de la oviposición hasta el día 42 p.t.; en los grupos tratados con Moxidectina, Abamectina y Doramectina hubo reducción significativa hasta los días 63 y 77 p.t. El día 21 p.t. se comprobó resistencia antihelmíntica a todos los productos, exceptuando la Doramectina. Las Ivermectinas tuvieron persistencia de efectividad de 14, 28 y 35 días, la Moxidectina y Abamectina de 42 días y la Doramectina de 49 días. Al día 63 p.t., los animales tratados pesaron, en promedio, 15. kg más que los animales del grupo control (p < 0.05). Se concluye que los productos tuvieron efectividad y persistencia de efectividad diferentes, pero que no se diferenciaron al analizar el rendimiento productivo The study was carried out from April to July 2001 on a cattle farm located in the south of Chile (lat. 40º- 53’S, long. 70º - 44’O). Naturally infected bovine of 280 kg average body weight were distributed in 7 groups (n = 12): control group (placebo), and 6 groups treated subcutaneously with 0.2 mg/kg of Ivermectin (3 groups with different commercial products), Moxidectin, Abamectin and Doramectin. Individual faecal samples were obtained from all animals 3 days prior to, on the treatment day and then, weekly until the 77 days post-treatment (p.t.). Individual egg-counts (epg), faecal egg reduction test and accumulative larvae cultures per group were carried out.. The pasture contamination was determined on day’s 0, 14, 56 and 77 p.t. Individual body weight on days -3 and 63 p.t. was recorded. The results were analysed considering significant p < 0.05. The control group presented mean egg counts fluctuating between 140 and 500 epg. The 6 treated groups showed a significant reduction of the epg until day 42 p.t. In the groups treated with Moxidectin, Abamectin and Doramectin there was a significant reduction up until days 63 and 77 p.t. Anthelminthic resistance were found for all products, excepting Doramectin at day 21 p.t. Persistence of effectiveness until 14, 28 and 35 days were found for the products with Ivermectin, 42 days for Moxidectin and Abamectin and 49 days for Doramectin. Until day 63 p.t., all treated animals weighed 15.1 kg more than the control animals (p < 0.05). It was concluded that the products showed differences in their effectiveness and persistence