Loop-mediated isothermal amplification (LAMP) assay for the diagnosis of fasciolosis in sheep and its application under field conditions

[EN] Background: Loop-mediated isothermal amplification (LAMP) is a very specific, efficient, and rapid gene amplification procedure in which the reaction can run at a constant temperature. In the current study we have developed a LAMP assay to improve the diagnosis of Fasciola spp. in the faeces of sheep. Findings: After the optimisation of the LAMP assay we have shown similar results between this technique and the standard PCR using the outer primers of the LAMP reaction. In both cases the limit of detection was 10 pg; also, the diagnosis of fasciolosis was confirmed during the first week post-infection in experimental infected sheep by both techniques. In eight naturally infected sheep, the infection with F. hepatica was confirmed in all animals before a treatment with triclabendazole and on day 30 post treatment in two sheep using the LAMP assay; however, when we carried out the standard PCR with the outer primers, the results before treatment were the same but on day 30 post-treatment the infection was only confirmed in one out of the two sheep. On the other hand, the standard PCR took around 3 h to obtain a result, comparing with 1 h and 10 min for the LAMP assay. Conclusions: The LAMP assay described here could be a good alternative to conventional diagnostic methods to detect F. hepatica in faeces since it solves the drawbacks of the standard PCRSIWe thank Drs Manga-González and González-Lanza (Instituto de Ganadería de Montaña, CSIC-ULE, Spain) for providing DNA samples of D. dendriticum and C. daubneyi. This study has been funded by the national project INIA (Instituto Nacional de Investigaciones Agrarias: RTA2010-00094-C03-02), Ministry of Economy and Competitiveness (Ministerio de Economia y Competitividad), and also co-funded by the European Regional Development Fund (Fondos Feder

Critical experiments with albendazole in the treatment of protostrongylid infection of sheep

p. 543-553El trabajo da a conocer los resultados de una experiencia crítica con 60 ovejas. Divididas en 3 lotes: G-1 testigos; G-2 tratadas con una sola dosis de 10 mg/kg de peso vivo de ABZ; G-3, tratadas con dos dosis de 7,5 mg/kgN

Quantification of resistant alleles in the β-tubulin gene of field strains of gastrointestinal nematodes and their relation with the faecal egg count reduction test

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.[EN]Background: Benzimidazole (BZ) resistance in gastrointestinal nematodes is associated with a single nucleotide polymorphism (SNP) at codons 167, 198 and 200 in the isotype 1 of beta-tubulin gene although in some species these SNPs have also been associated with resistance to macrocyclic lactones. In the present study we compared the levels of resistance in Teladorsagia circumcincta and Trichostrongylus colubriformis by means of the faecal egg reduction test (FECRT) and the percentage of resistant alleles obtained after pyrosequencing. The study was conducted in 10 naturally infected sheep flocks. Each flock was divided into three groups: i) group treated with albendazole (ABZ); ii) group treated with ivermectin (IVM); iii) untreated group. The number of eggs excreted per gram of faeces was estimated at day 0 and 14 post-treatment. Results: Resistance to ABZ was observed in 12.5% (1/8) of the flocks and to IVM in 44.4% (4/9) of them. One flock was resistant to both drugs according to FECRT. Coprocultures were performed at the same dates to collect L3 for DNA extraction from pooled larvae and to determine the resistant allele frequencies by pyrosequencing analysis. In T. circumcincta, SNPs were not found at any of the three codons before treatment; after the administration of ABZ, SNPs were present only in two different flocks, one of them with a frequency of 23.8% at SNP 167, and the other 13.2% % at SNP 198. In relation to T. colubriformis, we found the SNP200 before treatment in 33.3% (3/9) of the flocks with values between 48.5 and 87.8%. After treatment with ABZ and IVM, the prevalence of this SNP increased to 75 and 100% of the flocks, with a mean frequency of 95.1% and 82.6%, respectively. Conclusion: The frequencies observed for SNP200 in T. colubriformis indicate that the presence of resistance is more common than revealed by the FECRT.SIThis study has been funded by the national project INIA (Instituto Nacional de Investigaciones Agrarias: RTA2013-00064-C02-02) of the Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad), the European Regional Development Fund (Fondos Feder), the Spanish “Ramón y Cajal” Programme of the Ministry of Economy and Competitiveness (MMV, RYC-2015-18368), and the Cooperativa Bajo Duero, COBADU. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). The authors would like to thank all animal owners for their willingness to collaborate in the study

Implementation of an extended ZINB model in the study of low levels of natural gastrointestinal nematode infections in adult sheep

[EN] Background: In this study, two traits related with resistance to gastrointestinal nematodes (GIN) were measured in 529 adult sheep: faecal egg count (FEC) and activity of immunoglobulin A in plasma (IgA). In dry years, FEC can be very low in semi-extensive systems, such as the one studied here, which makes identifying animals that are resistant or susceptible to infection a difficult task. A zero inflated negative binomial model (ZINB) model was used to calculate the extent of zero inflation for FEC; the model was extended to include information from the IgA responses. Results: In this dataset, 64 % of animals had zero FEC while the ZINB model suggested that 38 % of sheep had not been recently infected with GIN. Therefore 26 % of sheep were predicted to be infected animals with egg counts that were zero or below the detection limit and likely to be relatively resistant to nematode infection. IgA activities of all animals were then used to decide which of the sheep with zero egg counts had been exposed and which sheep had not been recently exposed. Animals with zero FEC and high IgA activity were considered resistant while animals with zero FEC and low IgA activity were considered as not recently infected. For the animals considered as exposed to the infection, the correlations among the studied traits were estimated, and the influence of these traits on the discrimination between unexposed and infected animals was assessed. Conclusions: The model presented here improved the detection of infected animals with zero FEC. The correlations calculated here will be useful in the development of a reliable index of GIN resistance that could be of assistance for the study of host resistance in studies based on natural infection, especially in adult sheep, and also the design of breeding programs aimed at increasing resistance to parasitesSIThis work was supported by a competitive grant from the Castilla and León regional government (Junta de Castilla y León) (Ref. LE245A12-2); the EC-funded Innovative Training Network (ITN) NematodeSystemHealth (M. A. and J. M. P., P7-KBBE-2009-3-245140); the BBSRC grant BB/L004004070/1; the Spanish “Ramón y Cajal” Programme from the Spanish Ministry of Economy and Competitiveness (B. G. G., RYC-2012-10230) and a postdoctoral Jae-Doc contract from the Consejo Superior de Investigaciones Científicas (CSIC) and co-funded by the European Social Fund (M. M. V.

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

Quantitative trait loci for resistance to trichostrongylid infection in Spanish Churra sheep

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Anthelmintic activity of aminoalcohol and diamine derivatives against the gastrointestinal nematode Teladorsagia circumcincta

11 páginas, 5 figuras, 4 tablas.Gastrointestinal nematodes (GIN) infections are a serious problem in livestock production due to the great economic losses they cause. Their control is increasingly difficult because of the rapid development of drug resistance and the limited number of available drugs. Therefore, this study evaluated 18 aminoalcohol and 16 diamine derivatives against eggs, first and third stage larvae from a susceptible and a resistant isolate of Tela-dorsagia circumcincta collected from sheep. The effectiveness of the in vitro anthelmintic activity of the com-pounds was evaluated using three different procedures: Egg Hatch Test (EHT), Larval Mortality Test (LMT) and Larval Migration Inhibition Test (LMIT). Those compounds with activities higher than 90 % in the initial screening at 50 μM were selected to determine their half maximal effective concentration (EC50). In parallel, cytotoxicity assays were conducted on Caco2 and HepG2 cell lines to calculate Selectivity Indexes (SI) for each compound. The diamine 30 presented the best results in preventing egg hatching, displaying the lowest EC50 value (1.01 ±0.04 μM) of all compounds tested and the highest SI (21.21 vs. Caco-2 cells). For the LMIT, the diamine 34 showed the highest efficacy, with EC50 values of 2.67 ±0.08 and 3.02 ±0.09 μM on the susceptible and resistant isolate of the parasite, respectively.We are grateful to Dave Bartley, from Moredun Research Institute for providing the triple-resistant isolate of T. circumcincta. RE thank the RICET contract (E07D401988BR) EC Programs. Financial support came from MINECO: RETOS (AGL2016-79813-C2-1R/2R) and Junta de Castilla y León (JCyL) co-financed by FEDER, UE (LE020P17). EVG was funded by FPU16/03536; JV, VCGA and MAB are recipients of Junta de Castilla y Leon and European Social Found (ESF)’s Fellowships Scheme for Doctoral Training Programs. (JCYL-USAL35B, LE082-18, LE051-18, respectively) and MMV by the Spanish “Ramon y Cajal” Programme (Ministerio de Economía y Competitividad; RYC-2015-18368).Peer reviewe

Modelos de crecimiento y producción en España: historia, ejemplos contemporáneos y perspectivas

En el presente trabajo se presenta una revisión sobre los modelos forestales desarrollados en España durante los últimos años, tanto para la producción maderable como no maderable y, para la dinámica de los bosques (regeneración, mortalidad). Se presentan modelos tanto de rodal completo como de clases diamétricas y de árbol individual. Los modelos desarrollados hasta la fecha se han desarrollado a partir de datos procedentes de parcelas permanentes, ensayos y el Inventario Forestal Nacional. En el trabajo se muestran los diferentes submodelos desarrollados hasta la fecha, así como las plataformas informáticas que permiten utilizar dichos modelos. Se incluyen las principales perspectivas de desarrollo de la modelización forestal en España.In this paper we present a review of forest models developed in Spain in recent years for both timber and non timber production and forest dynamics (regeneration, mortality). Models developed are whole stand, size (diameter) class and individual-tree. The models developed to date have been developed using data from permanent plots, experimental sites and the National Forest Inventory. In this paper we show the different sub-models developed so far and the friendly use software. Main perspectives of forest modeling in Spain are presented.The models described in this paper were funded by different regional, national and European projects, and some of them were elaborated by the authors. This work was funded by the Spanish Government by the SELVIRED network (code AGL2008-03740) and the strategic project «Restauración y Gestión Forestal» (code PSE-310000-2009-4)