Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance.

BACKGROUND NlmCategory: BACKGROUND content: "Benzimidazole resistance is associated with isotype-1 \xCE\xB2-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta." - Label: METHODS NlmCategory: METHODS content: "The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 \xCE\xB2-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L." - Label: RESULTS NlmCategory: RESULTS content: "The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r\xE2\x80\x89=\xE2\x80\x890.929, P\xE2\x80\x89<\xE2\x80\x890.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4-80.7% before treatment, and 82.3-92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r\xE2\x80\x89>\xE2\x80\x890.720, P\xE2\x80\x89<\xE2\x80\x890.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P\xE2\x80\x89<\xE2\x80\x890.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT." - Label: CONCLUSIONS NlmCategory: CONCLUSIONS content: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta

Characterization of a new albendazole resistant Fasciola hepatica isolate

Characterization of a new albendazole resistant Fasciola hepatica isolateMaría Martínez-Valladares11,2, Elora Valderas-García1,2, Verónica Castilla Gómez de Agüero1,2, Marta González-Warleta3, Laura Ceballos4, Juan P. Lirón4, Rodrigo Sanabria5, Cesar Pruzzo5, Luis I. Alvarez4.1Instituto de Ganadería de Montaña (CSIC-Universidad de León), Departamento de Sanidad Animal (Department of Animal Health), León, Spain.2Departamento de Sanidad Animal (Department of Animal Health), Facultad de Veterinaria, University of Leon, Leon, Spain.3Laboratorio de Parasitología, Centro de Investigaciones Agrarias de Mabegondo, AGACAL, Abegondo, A Coruña, Spain.4Laboratorio de Farmacología, Centro de Investigación Veterinaria de Tandil (CIVETAN), UNCPBA-CICPBA-CONICET, Facultad de Ciencias Veterinarias, Tandil, Argentina.5Laboratorio de Parasitología, Facultad de Ciencias Veterinarias, Universidad Nacional de la Plata (UNLP), La Plata, Argentina.The infection by Fasciola hepatica affects mainly ruminants although is present in a wide variety of species including humans. Fasciolosis control is mainly based on triclabendazole administration, the main drug indicated in humans. Albendazole (ABZ) is used against nematode and liver fluke infections in ruminants. The misuse of these drugs has led to the appearance of anthelmintic resistance. In this study, we characterized an ABZ resistant isolate and evaluate the use of a combined treatment to improve treatment efficacy. The isolate was collected from a slaughterhouse in Argentina, maintained under laboratory conditions and identified by means of the egg hatch test (EHT). Using these eggs metacercariae were produced to infect sheep artificially. When flukes reached the adult stage, animals were divided into two groups, one treated with ABZ (7.5 mg/kg bw) and another with a placebo. All sheep were slaughtered at day 14 post-treatment to collect and count the number of flukes. The resistance of ABZ was confirmed with a reduction of 44% of adult flukes in the treated group. At the necropsy, adult flukes from the livers and eggs from the gall bladders, both from sheep treated with the placebo, were collected. Adult flukes were maintained alive in RPMI medium to let them to excrete eggs for 24 hours. EHT was conducted and different results were observed with the two egg sets. The EHT performed with eggs collected directly from gall bladder confirmed the resistant status of the isolate. However, the EHT with eggs recovered from flukes resulted in a susceptible phenotype, showing that only previously laying eggs (in bile) can express the resistant phenotype. After producing more metacercariae from this resistant isolate, we are testing in vivo the efficacy of a combined treatment to improve its efficacy. The results will be presented during the WAAVP meeting.Fil: Martínez Valladares, María. Universidad de Leon. Facultad de Veterinaria; ArgentinaFil: Valderas García, Elora. Universidad de Leon. Facultad de Veterinaria; ArgentinaFil: Castilla Gómez de Agüero, Verónica. Universidad de Leon. Facultad de Veterinaria; ArgentinaFil: González Warleta, Marta. Universidad de Leon. Facultad de Veterinaria; ArgentinaFil: Ceballos, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Liron, Juan Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Sanabria, Rodrigo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Pruzzo, Cesar Ivan. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Alvarez, Luis Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina28th International Conference of tha World Association for the Advancement of Veterinary ParasitologyDublinIrlandaWorld Association for the Advancement of Veterinary ParasitologyUniversity College Dubli

Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance

[EN] BACKGROUND: Benzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta. METHODS: The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. RESULTS: The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P  0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. CONCLUSIONS: The E198L polymorphism can confer BZ resistance on its own in T. circumcinctaSIThis study was funded by the Spanish “Ramón y Cajal” Programme of the Ministry of Economy and Competitiveness (MMV, RYC‑2015‑18368), and the Cooperativa Bajo Duero, COBADU. EVG was funded by FPU16/03536, JG by Ramon Areces Foundation, VCGA by Junta de Castilla y León and Fondo Social Europeo (LE082‑18), MCP by the Stopping Transmission Of intestinal Parasites (STOP) project (EDCTP2 programme; RIA2017NCT‑1845) and MMV by the Spanish “Ramon y Cajal” Programme (RYC‑2015‑18368

Microbial community in resistant and susceptible Churra sheep infected by Teladorsagia circumcincta

[EN]Gastrointestinal nematodes (GIN) are a major threat to health and welfare in small ruminants worldwide. Teladorsagia circumcincta is a nematode that inhabits the abomasum of sheep, especially in temperate regions, causing important economic losses. Given that T. circumcincta and microbiome share the same niche, interactions between them and the host are expected. Although it is known that within a sheep breed there are animals that are more resistant than others to infection by GIN, it is not known if the microbiome influences the phenotype of these animals. Under this condition, 12 sheep were classified according to their cumulative faecal egg count (cFEC) at the end of a first experimental infection, 6 as resistant group (RG) and 6 as susceptible group (SG) to T. circumcincta infection. Then, all sheep were experimentally infected with 70,000 L3 of T. circumcincta and at day 7 days post-infection were euthanized. At necropsy, gastric mucosa and gastric content from abomasum were collected to extract bacterial DNA and sequence V3-V4 region from 16S rRNA gene using Ilumina technology. After bioanalysis performed, results showed that α-diversity and β-diversity remained similar in both groups. However, resistant phenotype sheep showed a higher number of bacteria butyrate-fermenting species as Clostridium sensu stricto 1 (abundance in RG: 1.29% and in SG: 0.069%; p = 0.05), and Turicibacter (abundance in RG: 0.31% and in SG: 0.027%; p = 0.07) in gastric content but also Serratia spp in gastric mucosa (abundance in RG: 0.12% and in SG: 0.041%; p = 0.07). A trend towards a significant negative correlation between cFEC and Clostridium sensu stricto 1 abundance in gastric content was detected (r = − 0.537; p = 0.08). These data suggest that microbiome composition could be another factor associated with the development of the resistant phenotype modifying the interaction with the host and the in last instance affecting the individual risk of infection.S

Benzimidazole and aminoalcohol derivatives show in vitro anthelmintic activity against Trichuris muris and Heligmosomoides polygyrus

[EN]Background: Infections by gastrointestinal nematodes cause significant economic losses and disease in both humans and animals worldwide. The discovery of novel anthelmintic drugs is crucial for maintaining control of these parasitic infections. Methods: For this purpose, the aim of the present study was to evaluate the potential anthelmintic activity of three series of compounds against the gastrointestinal nematodes Trichuris muris and Heligmosomoides polygyrus in vitro. The compounds tested were derivatives of benzimidazole, lipidic aminoalcohols and diamines. A primary screening was performed to select those compounds with an ability to inhibit T. muris L1 motility by > 90% at a single concentration of 100 µM; then, their respective IC50 values were calculated. Those compounds with IC50 < 10 µM were also tested against the adult stage of T. muris and H. polygyrus at a single concentration of 10 µM. Results: Of the 41 initial compounds screened, only compounds AO14, BZ6 and BZ12 had IC50 values < 10 µM on T. muris L1 assay, showing IC50 values of 3.30, 8.89 and 4.17 µM, respectively. However, only two of them displayed activity against the adult stage of the parasites: BZ12 killed 81% of adults of T. muris (IC50 of 8.1 µM) and 53% of H. polygyrus while BZ6 killed 100% of H. polygyrus adults (IC50 of 5.3 µM) but only 17% of T. muris. Conclusions: BZ6 and BZ12 could be considered as a starting point for the synthesis of further structurally related compounds.SIFinancial support came from MINECO: RETOS (AGL2016-79813-C2-1R/2R) and Junta de Castilla y León co-financed by FEDER, UE [LE020P17]. EVG was funded by FPU17/00627; VCGA and MAB are recipients of Junta de Castilla y Leon (JCyL) (LE082-18, LE051-18, respectively) and MMV by the Spanish “Ramon y Cajal” Programme (Ministerio de Economía y competitividad; MMV, RYC-2015-18368

Novel compound shows in vivo anthelmintic activity in gerbils and sheep infected by Haemonchus contortus

[EN] The control of gastrointestinal nematodes in livestock is becoming increasingly difficult due to the limited number of available drugs and the rapid development of anthelmintic resistance. Therefore, it is imperative to develop new anthelmintics that are effective against nematodes. Under this context, we tested the potential toxicity of three compounds in mice and their potential anthelmintic efficacy in Mongolian gerbils infected with Haemonchus contortus. The compounds were selected from previous in vitro experiments: two diamine (AAD-1 and AAD-2) and one benzimidazole (2aBZ) derivatives. 2aBZ was also selected to test its efficacy in sheep. In Mongolian gerbils, the benzimidazole reduced the percentage of pre-adults present in the stomach of gerbils by 95% at a dose of 200 mg/kg. In sheep, there was a 99% reduction in the number of eggs shed in faeces after 7 days at a dose of 120 mg/kg and a 95% reduction in the number of worm adults present in the abomasum. In conclusion, 2aBZ could be considered a promising candidate for the treatment of helminth infections in small ruminantsSIFinancial support came from MINECO: RETOS (AGL2016-79813-C2-1R/2R) and MICINN/AEI (PID2020- 119035RB-100). EVG was funded by FPU17/00627, FPU17/05346; VCGA, MAB, MCP and LGP are recipients of Junta de Castilla y León (JCyL) (LE082-18, LE051-18, LE135-19, LE096-20, respectively) and MMV by the Spanish “Ramon y Cajal” Programme (Ministerio de Economía y competitividad; MMV, RYC-2015-18368

Control of ovine trichostrongylidosis: design, synthesis and clinical efficacy trials of new anthelmintic molecules

232 p.[ES] Las infecciones producidas por parásitos helmintos, incluyendo nematodos, trematodos y cestodos, representan una importante causa de enfermedad y pérdida económica para la industria ganadera de pequeños rumiantes a nivel mundial. Dentro de los helmintos, los nematodos gastrointestinales son los más relevantes en términos de impacto sanitario y económico, tanto en países en desarrollo como en industrializados. En pequeños rumiantes, las especies responsables de producir las mayores pérdidas económicas son Haemonchus contortus, Teladorsagia circumcincta y Trichostrongylus spp. En condiciones de campo, la mayoría de las infecciones suelen ser mixtas, incluyendo diferentes especies de nematodos que se localizan en el abomaso e intestino. Se trata de procesos generalmente endémicos, de curso crónico y mortalidad baja. Sin embargo, el impacto de este tipo de parasitaciones en el animal va a depender no solo de la intensidad de la infección, si no también del estado fisiológico e inmunológico del hospedador. Los signos clínicos que suelen presentarse más comúnmente incluyen anorexia, diarrea, emaciación y anemia. Aunque, en casos de altos niveles de parasitación, sobre todo en animales jóvenes, se puede incluso producir la muerte. La administración de fármacos antihelmínticos ha sido el tratamiento de elección para el control de estas parasitosis durante muchos años debido a la disponibilidad de fármacos de alta eficacia y seguridad. Sin embargo, el creciente aumento de la prevalencia de la resistencia a los antihelmínticos a nivel mundial hace que los actuales programas de control sean costosos e insostenibles a largo plazo. Además hay que tener en cuenta que el cambio climático está provocando cambios en la incidencia e intensidad de estas infecciones, lo que agrava su impacto económico en la ganadería. Es por ello que la búsqueda de nuevos fármacos con actividad antihelmíntica y nuevos mecanismos de acción es de vital importancia en la actualidad. Para abordar esta problemática, el objetivo general de la presente Tesis Doctoral se enfoca en la búsqueda de nuevos compuestos con posible actividad antihelmíntica frente a las infecciones ovinas causadas por tricostrongílidos que puedan ayudar a resolver los problemas generados por la aparición de la resistencia. En este contexto, tres de los cinco capítulos en que se divide la presente Tesis Doctoral se centran en la evaluación de la posible actividad antihelmíntica de varias familias de compuestos sintetizados por el departamento de Química Farmacéutica de la Universidad de Salamanca. En primer lugar, los compuestos fueron sometidos a un cribado primario a una dosis de 50 μM para determinar su eficacia in vitro frente a distintos estadios parasitarios del nematodo T. circumcincta. Los compuestos con una eficacia superior al 90% fueron seleccionados para llevar a cabo estudios con una cepa resistente a los bencimidazoles, ivermectina y levamisol, así como para calcular la dosis eficaz 50 (DE50) de cada uno de ellos. Paralelamente se llevaron a cabo estudios in vitro de citotoxicidad para descartar aquellos más tóxicos. Los tres compuestos con mayor actividad antihelmíntica y menor toxicidad in vitro fueron seleccionados con el fin de continuar con los experimentos de toxicidad y eficacia en modelos animales. Dos derivados de diamina y un derivado bencimidazólico fueron los compuestos seleccionados como candidatos, todos ellos con una DE50 inferior a 3 μM en alguno de los ensayos llevados cabo. Una vez que se descartaron los posibles efectos adversos de la administración por vía oral de los tres candidatos en ratones, se evaluó su eficacia antihelmíntica utilizando para ello jerbos de Mongolia (Meriones unguiculatus) infectados con H. contortus. Tras observar una reducción del 95% en el número de pre-adultos presentes en el estómago de los animales del grupo tratado con el derivado bencimidazólico a una dosis de 200 mg/kg, se decidió probar la eficacia del compuesto en el hospedador definitivo, la oveja. En el último experimento de esta parte del trabajo se comprobó que el grupo de ovejas tratado con el derivado bencimidazólico a una dosis de 120 mg/kg, produjo una reducción del recuento de huevos en heces del 99% y del 95% en el número de adultos presentes en el abomaso, demostrando ser un candidato interesante para su desarrollo como fármaco antihelmíntico. Es por ello por lo que creemos necesario continuar realizando más estudios en futuro que permitan establecer una dosis mínima eficaz, así como determinar su eficacia en diferentes especies y cepas de nematodos gastrointestinales, y llevar a cabo estudios detallados de farmacocinética y farmacodinamia. En este contexto, durante la estancia internacional llevada a cabo en el “Swiss Tropical and Public Health Institute”, en Basilea (Suiza), parte de los compuestos utilizados en el cribado llevado a cabo en el nematodo T. circumcincta fueron evaluados en otro modelo de nematodo gastrointestinal que infecta ratones, Trichuris muris. De nuevo, tres compuestos fueron seleccionados por su potente actividad frente a larvas de primer estadio en un cribado inicial a una dosis de 100 μM, para posteriormente comprobar su eficacia frente a las formas adultas del parásito. Los tres candidatos consistían en dos derivados bencimidazólicos y un derivado de aminoalcohol, siendo distintos de los seleccionados en el estudio anterior. Adicionalmente, estos candidatos se testaron frente a las formas adultas del nematodo Heligmosomoides polygyrus. Como resultado final, dos de ellos mostraron eficacia en alguna de las dos formas adultas de los parásitos; uno de los derivados bencimidazólicos presentó una DE50 8,1 μM frente a T. muris y el el otro una DE50 de 5,3 μM frente a H. polygyrus. Es por ello que ambos compuestos podrían ser también potenciales candidatos para llevar a cabo ensayos de eficacia in vivo en un futuro utilizando animales infectados. Con la finalidad de combatir el problema de la falta de nuevos fármacos antihelmínticos, en la presente Tesis Doctoral se ha abordado otro enfoque relacionado con la búsqueda de técnicas de cribado mejoradas que permitan la evaluación de gran cantidad de compuestos en un corto periodo de tiempo. Así, durante la segunda estancia internacional realizada en el “Laboratory of Growth Regulators” de la Universidad de Palacký, en Olomouc (República Checa), se puso a punto un protocolo de cribado de compuestos de alto rendimiento utilizando el dispositivo wMicrotracker ONE, en T. circumcincta y H. contortus. Este dispositivo es capaz de medir de forma automatizada la movilidad de las distintas formas parasitarias tras la incubación con los compuestos a través de microhaces de luz infrarroja. Complementariamente, también hemos llevado a cabo estudios sobre el nivel de resistencia de los tricostrongílidos presente en los rebaños de ovino de la Comunidad Autónoma de Castilla y León frente a los fármacos bencimidazólicos mediante técnicas in vivo, in vitro y moleculares. Nuestro estudio confirmó un aumento de la presencia de resistencia antihelmíntica con respecto a los últimos datos analizados años anteriores, siendo T. circumcincta la especie más resistente. En este estudio el 35% de las explotaciones muestreadas resultaron resistentes al netobimín mediante el ensayo in vivo de reducción del número de huevos en heces y del 26% mediante el ensayo in vitro de eclosión de huevos, encontrándose una correlación muy alta entre ambas técnicas (r= -0.770; p 0,720; p < 0,0001).[EN] Infections caused by helminth parasites, including nematodes, trematodes and cestodes, represent an important cause of disease and economic loss for the small ruminant livestock industry. Within helminths, gastrointestinal nematodes are the most relevant in terms of health and economic impact in both developing and industrialised countries. Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus spp. are the three species responsible for the highest economic losses worldwide. Under field conditions, most infections are usually caused by more than one species of nematodes located in the abomasum and intestine. These are generally endemic processes, with chronic course and low mortality. However, the impact of this parasitism depends on the infection intessity, as well on the physiological and immunological state of the host. Most common clinical signs include anorexia, diarrhoea, emaciation and anaemia. However, in cases of high levels of infection, especially in young animals, death can take place. Administration of anthelmintic drugs has been the treatment of choice for the control of these parasites for many years due to the availability of highly effective and safe drugs. However, the increasing prevalence of anthelmintic resistance worldwide has led to control programmes that are costly and unsustainable in the long term. In addition, climate change is increasing the incidence and intensity of these infections, which exarcebates their economic impact on livestock farming. Therefore, searching for new drugs with anthelmintic activity and new mechanisms of action is vital. To address this problem, the general objective of the present Thesis is focused on the search for new compounds with potential anthelmintic activity against trichostrongylid infections in sheep which could alleviate the problems arising from the the emergence of resistance. In this context, three out of five chapters described in the present Thesis are focused on the evaluation of the possible anthelmintic activity of several families of compounds synthesized by the Department of Pharmaceutical Chemistry of the University of Salamanca. First, the compounds were subjected to a primary screening at a dose of 50 μM to determine their in vitro efficacy against different parasitic stages of the nematode T. circumcincta. Those compounds with an efficacy higher than 90% were selected to carry out studies with a triple resistant isolate (to benzimidazoles, ivermectin and levamisole), as well as to calculate their effective dose 50 (ED50). At the same time, in vitro cytotoxicity studies were carried out to discard the most toxic compounds. Therefore, the three compounds with the highest anthelmintic activity and lowest in vitro toxicity were selected in order to study their in vivo toxicity and efficacy in gerbils and sheep. Two diamine and one benzimidazole derivatives were selected as candidate compounds, all of them with an ED50 below 3 μM in one of the tests carried out. Once the potential adverse effects of oral administration of the candidates in mice were discarded, their anthelmintic efficacy was evaluated using Mongolian gerbils (Meriones unguiculatus) infected with H. contortus. After observing a 95% reduction in the number of pre-adults in the stomach of the gerbils in the group treated with the benzimidazole derivative at a dose of 200 mg/kg, we decided to evaluate the efficacy of the compound in the definitive host, the sheep. In the last experiment, the group of sheep treated with the benzimidazole derivative at a dose of 120 mg/kg produced a 99% reduction in the number of eggs in faeces 7 days after administration, and a 95% reduction in the number of adults present in the sheep abomasum. Under this context, the BZ derivative demonstrated to be an interesting candidate for the development of an anthelmintic drug. For this reason, we believe it is necessary to carry out further studies to establish a minimum effective dose, as well as to determine its efficacy in different GIN species and isolates, and to carry out detailed pharmacokinetic and pharmacodynamic studies. In this context, during the international stay at the “Swiss Tropical and Public Health Institute”, in Basel (Switzerland), part of the compounds previously tested in the nematode T. circumcincta, were evaluated against another model of GIN infecting mice: Trichuris muris. Three compounds were selected for their potent activity against the first larvae stage in an initial screening at a dose of 100 μM, and subsequently their efficacy against the adult forms of the parasite was tested. The three candidates consisted of two benzimidazole and one aminoalcohol derivatives, being different from those selected in the previous experiment. Additionally, these candidates were tested against adult forms of Heligmosomoides polygyrus. As a final result, two of them showed efficacy, at least, against one of the two parasites; one derivative showed an ED50 of 8.1 μM against T. muris and BZ12 showed an ED50 of 5.3 μM against H. polygyrus. Therefore, both compounds could also be potential candidates for future in vivo efficacy studies using infected animals. Another approach that we have considered in the present Thesis, in order to combat the problem of the lack of new anthelmintic drugs, is related to the search for improved screening techniques that allow the evaluation of large number of compounds in a short period of time. Thus, during the second international stay at the "Laboratory of Growth Regulators" of the University of Palacký, in Olomouc (Czech Republic), a high-throughput compound screening protocol was set up using the wMicrotracker ONE device on T. circumcincta and H. contortus. This device is able to automatically measure the mobility of the different parasite stages after their incubation with the compounds by means of infrared light microbeams. Complementarily, we have also carried out studies to determine the level of benzimidazole resistance of gastrointestinal nematodes present in sheep flocks in Castilla y León by means of in vivo, in vitro and molecular techniques. Our study confirmed an increase in the occurrence of anthelmintic resistance compared to the last data analysed in previous years, being T. circumcincta the most resistant species. In this study, 35% of the sampled farms were resistant to netobimin by the in vivo faecal egg reduction test and 26% by the in vitro egg hatching test, with a very high correlation between both techniques (r= -0.770; p 0.720; p < 0.0001).Ministerio de Universidade

A combined, automated high-throughput system for the quantification of the viability of the free-living nematode Caenorhabditis elegans

JOINT COMBAR - ACSRPC meeting: Anthelmintic Resistance in Ruminants: Who cares? Abstract Book, Poster Presentations, pp. 88. Gante (Bélgica). 27-29 agosto 2019.MINECO (AGL2016-79813-C2-1R, SAF2017-83575), FEDER, LE020P17, FPU16/03536 and RYC-2015-1836

New synthetic benzimidazole with potential anthelmintic activity

Póster presentado al: 2nd Working Groups Meeting COMBAR (Combatting Anthelmintic Resistance in Ruminants). León, 26-27 Septiembre 2018.Study funded by AGL2016-79813-C2-1/2-RPeer reviewe

Presente, pasado y futuro del diagnóstico de las infecciones por nematodos gastrointestinales en el ganado ovino (II)

3 páginas, 3 figuras