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[Background] Persistence of gastrointestinal nematode (GIN) infection and the related control methods have major impacts on the sheep industry worldwide. Based on the information generated with the Illumina OvineSNP50 BeadChip (50 K chip), this study aims at confirming quantitative trait loci (QTL) that were previously identified by microsatellite-based genome scans and identifying new QTL and allelic variants that are associated with indicator traits of parasite resistance in adult sheep. We used a commercial half-sib population of 518 Spanish Churra ewes with available data for fecal egg counts (FEC) and serum levels of immunoglobulin A (IgA) to perform different genome scan QTL mapping analyses based on classical linkage analysis (LA), a combined linkage disequilibrium and linkage analysis (LDLA) and a genome-wide association study (GWAS). ; [Results] For the FEC and IgA traits, we detected a total of three 5 % chromosome-wise significant QTL by LA and 63 significant regions by LDLA, of which 13 reached the 5 % genome-wise significance level. The GWAS also revealed 10 significant SNPs associated with IgAt, although no significant associations were found for LFEC. Some of the significant QTL for LFEC that were detected by LA and LDLA on OAR6 overlapped with a highly significant QTL that was previously detected in a different half-sib population of Churra sheep. In addition, several new QTL and SNP associations were identified, some of which show correspondence with effects that were reported for different populations of young sheep. Other significant associations that did not coincide with previously reported associations could be related to the specific immune response of adult animals. ; [Discussion] Our results replicate a FEC-related QTL located on OAR6 that was previously reported in Churra sheep and provide support for future research on the identification of the allelic variant that underlies this QTL. The small proportion of genetic variance explained by the detected QTL and the large number of functional candidate genes identified here are consistent with the hypothesis that GIN resistance/susceptibility is a complex trait that is not determined by individual genes acting alone but rather by complex multi-gene interactions. Future studies that combine genomic variation analysis and functional genomic information may help elucidate the biology of GIN disease resistance in sheep. ; This work was supported by a competitive grant from the Castilla and León regional government (Junta de Castilla y León) (Ref. LE245A12-2) and the AGL2012-34437 project funded by the Spanish Ministry of Economy and Competitiveness (MINECO). M Atlija is a grateful grantee of a Marie Curie fellowship funded by the EC-funded Initial Training Network (ITN) NematodeSystemHealth (FP7-PEOPLE-2010-ITN Ref. 264639). B Gutiérrez-Gil is funded through the Spanish "Ramón y Cajal" Programme (RYC-2012-10230) from the MINECO. ; USD 1,755 APC fee funded by the EC FP7 Post-Grant Open Access Pilot ; Peer reviewed

8 páginas, 2 figuras. ; Individual mutations (e.g. L256F) and polymorphisms in the avr-14B gene, a glutamate-gated chloride channel subunit, have been associated with ivermectin (IVM) resistance in Caenorhabditis elegans and Cooperia oncophora. The aim of the present study was to determine the full-length coding sequence of the avr-14B subunit homologue in Teladorsagia circumcincta and determine the presence/absence of the putative L256F SNP or any other potential SNPs of interest. Subsequently, we investigated sequence polymorphisms and transcription patterns between four different T. circumcincta isolates: two from Scotland (MTci1 susceptible and MTci5 triple resistant to benzimidazoles, levamisole and IVM) and two from Spain (S-Sp susceptible and R-Sp double resistant to levamisole and IVM). The complete amino acid sequence of the T. circumcincta avr-14B subunit comprises 438 amino acids. Pyrosequencing analysis failed to detect the presence of the L256F mutation in any of the MTci5 or Sp-R samples tested. However, we revealed significant allele frequency changes by means of SSCP analysis of a 106 bp region encompassing the L256F SNP. Allele E showed the greatest change, following IVM exposure in vitro and in vivo, although sequence analysis did not reveal any coding changes. Sequence analysis of the fulllength avr-14B coding sequence showed that two SNPs exclusively found in the resistant strain McTi5 (I270F and T305A) are situated in codons involved in the interaction of the receptor with IVM. Moreover, other potentially significant SNPs (K361E and L364M) were identified between transmembrane regions 3 and 4. However, due to the low frequency of all these SNPs, we cannot conclude they confer IVM resistance in T. circumcincta. Moreover, a modest increase in expression of the avr-14B in both resistant isolates has been shown although these differences were not sufficiently great to consider avr-14B to be the sole or even a major determinant of IVM resistance in this species. ; This study has been funded by the national projects INIA-MEC, RTA2006-00183-C03-02 and RTA2010-00094-C03-02. The work of María Martínez-Valladares has been supported by a postdoctoral Jae-Doc contract from the Consejo Superior de Investigaciones Científicas (CSIC) and co-funded by the European Social Fund. The authors would also like to thank Dr. Jeff Eng, McGill University, Montreal, QC for his help with the SSCP analysis and Dr Fiona Kenyon of the Moredun Research Institute, Edinburgh, for her help with the LFIA analysis. The financial support of the Scottish Government Rural and Environmental Research and Analysis Directorate (RERAD) is gratefully acknowledged. ; Peer reviewed

11 páginas, 2 Figuras, 2 Tablas. ; The present study exploited the RNA-seq technology to analyze the transcriptome of target tissues affected by the Teladorsagia circumcincta infection in two groups of adult ewes showing different statuses against gastrointestinal nematode (GIN) infection with the aim of identifying genes linked to GIN infection resistance in sheep. For this, based on the accumulated faecal egg count of 18 adult Churra ewes subjected to a first experimental infection with T. circumcincta, six ewes were classified as resistant and six others as susceptible to the infection. These 12 animals were dewormed and infected again. After humanitarian sacrifice of these 12 animals at day 7 post-infection, RNA samples were obtained from abomasal mucosa and lymph node tissues and RNA-Seq datasets were generated using an Illumina HiSeq 2000 sequencer. The distribution of the genes based on their expression level were very similar among the two different tissues and conditions. The differential expression analysis performed with two software (DESeq and EdgeR) only identified common differentially expressed genes (DEGs), a total of 106, in the lymph node samples which were considered as GIN-activated. The enrichment analysis performed for these GIN-activated genes identified some pathways related to cytokine-mediated immune response and the PPARG signaling pathway as well as disease terms related to inflammation and gastro-intestinal diseases as enriched. A systematic comparison with the results of previous studies confirmed the involvement of genes such as ITLN2, CLAC1 and galectins, in the immune mechanism activated against T. circumcincta in resistant sheep. ; Financial support for this project was received from the LE248U14 project of Junta de Castilla and León Government. P. K. Chitneedi is funded by a predoctoral fellowship from the Junta de Castilla and León Government and the European Social Fund. B Gutiérrez-Gil is funded by the "Ramón y Cajal" Programme (RYC-2012-10230) from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO). M. Martínez-Valladares is also funded by the "Ramón y Cajal" Programme (RYC-2015-18368) from MINECO. ; Peer Reviewed

18 páginas, 6 figuras, 2 tablas. ; Several recent studies have demonstrated the role of long non-coding RNAs (lncRNAs) in regulating the defense mechanism against parasite infections, but no studies are available that investigated their relevance for immune response to nematode infection in sheep. Thus, the aim of the current study was to (i) detect putative lncRNAs that are expressed in the abomasal lymph node of adult sheep after an experimental infection with the gastrointestinal nematode (GIN) Teladorsagia circumcincta and (ii) to elucidate their potential functional role associated with the differential host immune response. We hypothesized that putative lncRNAs differentially expressed (DE) between samples from animals that differ in resistance to infection may play a significant regulatory role in response to nematode infection in adult sheep. To obtain further support for our hypothesis, we performed co-expression and functional gene enrichment analyses with the differentially expressed lncRNAs (DE lncRNAs). In a conservative approach, we included for this predictive analysis only those lncRNAs that are confirmed and supported by documentation of expression in gastrointestinal tissues in the current sheep gene atlas. We identified 9,105 putative lncRNA transcripts corresponding to 7,124 gene loci. Of these, 457 were differentially expressed lncRNA loci (DELs) with 683 lncRNA transcripts. Based on a gene co-expression analysis via weighted gene coexpression network analysis, 12 gene network modules (GNMs) were found significantly correlated with at least one of 10 selected target DE lncRNAs. Based on the principle of "guilt-by-association," the DE genes from each of the three most significantly correlated GNMs were subjected to a gene enrichment analysis. The significant pathways associated with DE lncRNAs included ERK5 Signaling, SAPK/JNK Signaling, RhoGDI Signaling, EIF2 Signaling, Regulation of eIF4 and p70S6K Signaling and Oxidative Phosphorylation pathways. They belong to signaling pathway categories like Cellular Growth, Proliferation and Development, Cellular Stress and Injury, Intracellular and Second Messenger Signaling and Apoptosis. Overall, this lncRNA study conducted in adult sheep after GIN infection provided first insights into the potential functional role of lncRNAs in the differential host response to nematode infection. ; Financial support for the experimental work of this project was received from the LE248U14 project of the Junta de Castilla and León regional government, whereas the storage and processing of the generated sequencing datasets have been funded by the RTI2018-093535-B-I00 project from the Spanish Ministry of Science and Innovation (MICINN). PC was funded by a Short-Term Scientific Missions of the Functional Annotation of Animal Genomes-European network (FAANG-Europe) COST Action CA15112 to do a short research stay at Leibniz Institute for Farm Animal Biology (FBN). MM-V was also funded by the "Ramón y Cajal" Programme (RYC-2015-18368) from MICINN. ; Peer reviewed

9 páginas, 4 Figuras, 1 Tabla. ; 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 parasites. ; This 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.). ; Peer Reviewed

7 páginas, 2 tablas, 1 figura. ; The Stopping Transmission Of intestinal Parasites (STOP) project is part of the EDCTP2 programme supported by the European Union (grant number RIA2017NCT-1845 — STOP). JG is personally supported by Ramon Areces Foundation, Spain; MMV by the Spanish 'Ramón y Cajal' Programme, Ministry of Economy and Competitiveness (RYC-2015-18368); and SK is supported by DELTAS Africa Initiative grant # DEL15-011 to THRiVE-2. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

12 páginas, 3 tablas, 3 figuras. ; There is an urgent need for an extensive evaluation of benzimidazole efcacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequenc‑ ing, and standardized it for large-scale benzimidazole efcacy screening use. ; This study was funded by the 'Stopping Transmission Of intestinal Parasites (STOP)' project (EDCTP2 programme supported by the European Union; RIA2017NCT-1845-STOP). JG was personally supported at the beginning of the work by the Ramón Areces Foundation and is now funded by the Spanish 'Juan de la Cierva' Programme, Ministry of Economy and Competitiveness (FJC-2018-38305). MCP is supported by the Junta de Castilla y León and Fondo Social Europeo, and MMV by the Spanish 'Ramón y Cajal' Programme, Ministry of Economy and Competitiveness (RYC-2015-18368). ; Peer reviewed

10 pages, 1 table , 2 figures, 1 additional file. ; Background: For ruminants reared on grazing systems, gastrointestinal nematode (GIN) parasite infections represent the class of diseases with the greatest impact on animal health and productivity. Among the many possible strategies for controlling GIN infection, the enhancement of host resistance through the selection of resistant animals has been suggested by many authors. Because of the difficulty of routinely collecting phenotypic indicators of parasite resistance, information derived from molecular markers may be used to improve the efficiency of classical genetic breeding. ; Methods: A total of 181 microsatellite markers evenly distributed along the 26 sheep autosomes were used in a genome scan analysis performed in a commercial population of Spanish Churra sheep to detect chromosomal regions associated with parasite resistance. Following a daughter design, we analysed 322 ewes distributed in eight half-sib families. The phenotypes studied included two faecal egg counts (LFEC0 and LFEC1), anti-Teladorsagia circumcincta LIV IgA levels (IgA) and serum pepsinogen levels (Peps). ; Results: The regression analysis revealed one QTL at the 5% genome-wise significance level on chromosome 6 for LFEC1 within the marker interval BM4621-CSN3. This QTL was found to be segregating in three out of the eight families analysed. Four other QTL were identified at the 5% chromosome-wise level on chromosomes 1, 10 and 14. Three of these QTL influenced faecal egg count, and the other one had an effect on IgA levels. ; Conclusion: This study has successfully identified segregating QTL for parasite resistance traits in a commercial population. For some of the QTL detected, we have identified interesting coincidences with QTL previously reported in sheep, although most of those studies have been focused on young animals. Some of these coincidences might indicate that some common underlying loci affect parasite resistance traits in different sheep breeds. The identification of new QTL may suggest the existence of complex host-parasite relationships that have unique features depending on the host-parasite combination, perhaps due to the different mechanisms underlying resistance in adult sheep (hypersensitivity reactions) and lambs (immunity). The most significant QTL identified on chromosome 6 for LFEC1 may be the target for future fine-mapping research efforts. ; This work was supported by the Spanish Ministry of Education and Science (Projects 1FD97-0225 and 1FD97-0427) and by the European Union through the project genesheepsafety (QLK5-2000-00656). Financial support from the Castilla and León regional government (Junta de Castilla y León) by a grant for research groups of excellence (Project GR43) is acknowledged. Beatriz Gutiérrez-Gil is funded by the "Juan de la Cierva Program" of the Spanish Ministry of Education and Science. ; Peer reviewed

19 páginas, 4 figuras, 2 tablas. ; World Health Organization goals against soil-transmitted helminthiases (STH) are pointing towards seeking their elimination as a public health problem: reducing to less than 2% the proportion of moderate and heavy infections. Some regions are reaching WHO goals, but transmission could rebound if strategies are discontinued without an epidemiological evaluation. For that, sensitive diagnostic methods to detect low intensity infections and localization of ongoing transmission are crucial. In this work, we estimated and compared the STH infection as obtained by different diagnostic methods in a low intensity setting. We conducted a cross-sectional study enrolling 792 participants from a district in Mozambique. Two stool samples from two consecutive days were collected from each participant. Samples were analysed by Telemann, Kato-Katz and qPCR for STH detection. We evaluated diagnostic sensitivity using a composite reference standard. By geostatistical methods, we estimated neighbourhood prevalence of at least one STH infection for each diagnostic method. We used environmental, demographical and socioeconomical indicators to account for any existing spatial heterogeneity in infection. qPCR was the most sensitive technique compared to composite reference standard: 92% (CI: 83%– 97%) for A. lumbricoides, 95% (CI: 88%– 98%) for T. trichiura and 95% (CI: 91%– 97%) for hookworm. qPCR also estimated the highest neighbourhood prevalences for at least one STH infection in a low intensity setting. While 10% of the neighbourhoods showed a prevalence above 20% when estimating with single Kato-Katz from one stool and Telemann from one stool, 86% of the neighbourhoods had a prevalence above 20% when estimating with qPCR. In low intensity settings STH estimated prevalence of infection may be underestimated if based on Kato-Katz. qPCR diagnosis outperformed the microscopy methods. Thus, implementation of qPCR based predictive maps at STH control and elimination programmes would disclose hidden transmission and facilitate targeted interventions for transmission interruption. ; BGP and JM received financial support for this study from Mundo Sano Foundation (www.mundosano.org). JG was personally supported at the beginning of the work by the Ramón Areces Foundation and is now funded by the Spanish 'Juan de la Cierva' Programme, Ministry of Economy and Competitiveness (FJC-2018-38305). MMV is personally supported by the Spanish 'Ramón y Cajal' Programme, Ministry of Economy and Competitiveness (RYC-2015-18368). MCP is personally supported by Junta de Castilla y León and Fondo Social Europeo (LE-135-19). ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya. CISM is supported by the Government of Mozambique and the Spanish Agency for International Development (AECID). Prof. Dr. P.C. Flu Foundation also founded this project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ; Peer reviewed

16 páginas, 2 tablas, 6 figuras. ; Helminth infections are ubiquitous in grazing ruminant production systems, and are responsible for significant costs and production losses. Anthelmintic Resistance (AR) in parasites is now widespread throughout Europe, although there are still gaps in our knowledge in some regions and countries. AR is a major threat to the sustainability of modern ruminant livestock production, resulting in reduced productivity, compromised animal health and welfare, and increased greenhouse gas emissions through increased parasitism and farm inputs. A better understanding of the extent of AR in Europe is needed to develop and advocate more sustainable parasite control approaches. A database of European published and unpublished AR research on gastrointestinal nematodes (GIN) and liver fluke (Fasciola hepatica) was collated by members of the European COST Action "COMBAR" (Combatting Anthelmintic Resistance in Ruminants), and combined with data from a previous systematic review of AR in GIN. A total of 197 publications on AR in GIN were available for analysis, representing 535 studies in 22 countries and spanning the period 1980–2020. Reports of AR were present throughout the European continent and some reports indicated high within-country prevalence. Heuristic sample size-weighted estimates of European AR prevalence over the whole study period, stratified by anthelmintic class, varied between 0 and 48%. Estimated regional (country) prevalence was highly heterogeneous, ranging between 0% and 100% depending on livestock sector and anthelmintic class, and generally increased with increasing research effort in a country. In the few countries with adequate longitudinal data, there was a tendency towards increasing AR over time for all anthelmintic classes in GIN: aggregated results in sheep and goats since 2010 reveal an average prevalence of resistance to benzimidazoles (BZ) of 86%, macrocyclic lactones except moxidectin (ML) 52%, levamisole (LEV) 48%, and moxidectin (MOX) 21%. All major GIN genera survived treatment in various studies. In cattle, prevalence of AR varied between anthelmintic classes from 0–100% (BZ and ML), 0–17% (LEV) and 0–73% (MOX), and both Cooperia and Ostertagia survived treatment. Suspected AR in F. hepatica was reported in 21 studies spanning 6 countries. For GIN and particularly F. hepatica, there was a bias towards preferential sampling of individual farms with suspected AR, and research effort was biased towards Western Europe and particularly the United Kingdom. Ongoing capture of future results in the live database, efforts to avoid bias in farm recruitment, more accurate tests for AR, and stronger appreciation of the importance of AR among the agricultural industry and policy makers, will support more sophisticated analyses of factors contributing to AR and effective strategies to slow its spread. ; This review is based upon work from COST Action COMBAR CA16230, supported by COST (European Cooperation in Science and Technology) and from the Livestock Helminth Research Alliance (LiHRA). HRV and ERM are supported by UK Research and Innovation (UKRI) through BBSRC grant BB/ M003949/1; HRV is also supported by the University of Liverpool's Institute of Infection and Global Health, and ERM by UKRI BBSRC project BB/R010250/1 and the UK Department of Food, Environment and Rural Affairs, Veterinary Medicines Directorate grant VM0543. DJB is supported by the Scottish Government's Rural and Environmental Science and Analytical Services (RESAS) division. MMV was funded by the Spanish "Ramón y Cajal" Programme of the Ministry of Economy and Competitiveness (MMV, RYC2015-18368). MK and JV were supported by the Czech Republic Ministry of Education, Youth and Sports INTER-COST project (LTC19018). The authors would like to thank all COMBAR consortium members who contributed to the development of the database but who did not meet the ICMJE criteria for co-authorship. Thanks are also due to Carine Paraud (ANSES Niort) and Philippe Jacquiet (National Veterinary School, Toulouse) for providing unpublished data from sheep and goat surveys for France and to Vasile Cozma (University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania) for providing data from surveys for Romania.