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39 páginas, 3 figuras, 5 tablas., Helminthiasis is one of the gravest problems worldwide. There is a growing concern on less available anthelmintics and the emergence of resistance creating a major threat to human and livestock health resources. Novel and broad-spectrum anthelmintics are urgently needed. The free-living nematode Caenorhabditis elegans could address this issue through automated high-throughput technologies for the screening of large chemical libraries. This review discusses the strong advantages and limitations for using C elegans as a screening method for anthelmintic drug discovery. C elegans is the best model available for the validation of novel effective drugs in treating most, if not all, helminth infections, and for the elucidation the mode of action of anthelmintic candidates. This review also focuses on available technologies in the discovery of anthelmintics published over the last 15 years with particular attention to high-throughput technologies over conventional screens. On the other hand, this review highlights how combinatorial and nanomedicine strategies could prolong the use of anthelmintics and control resistance problems., Consejería de Educación, Junta de Castilla y León, Grant/Award Number: LE020P17; Ministerio de Economía y Competitividad, Grant/Award Numbers: AGL2016‐79813‐C2‐1R, RYC‐2015‐18368; European and Developing Countries Clinical Trials Partnership, Grant/Award Number: RIA2017NCT‐1845

17 páginas, 5 figuras, 3 tablas., Helminth infections are one of the most prevalent parasitic diseases affecting animals and humans worldwide. Anthelmintic resistance to the main drugs used to control these infections in animals, especially ruminants, is a major global problem that needs urgent solutions. The purpose of this study was to design and obtain new benzimidazole (BZ) derivatives to evaluate their in vitro ovicidal and larvicidal activities. Based on previous results from 2-phenylbenzimidazoles and new docking studies of different BZ-containing scaffolds in Teladorsagia circumcincta tubulin four structural groups of BZs were selected. In addition to several new members of those previously reported 2-phenylBZs (type I), some twenty 2-aminoBZ derivatives (type II) and twenty-five BZ amides (types III and IV) were prepared and evaluated for their ability to inhibit egg hatching and larval motility of T. circumcincta with results superior to those previously achieved. Nine of the fifty-five BZs tested displayed ovicidal activity higher than 90%, and fourteen induced more than 30% larval death in the assays at 50 µM. The benzamide BZ 42 showed the best ovicidal EC50 value of 0.92 µM with a selectivity index > 100 respecting HepG2 cells, while the benzylamine BZ 13 attained a higher than 50% larvicidal activity. Notably, the amide BZ 39 displayed both ovicidal (100%) and larvicidal (>50%) activities. The SAR analysis and the docking studies carried out led to the conclusion that, in addition to the effects on tubulin, pending experimental confirmation, there must be other mechanisms by which the evaluated BZs prevent hatching and limit the mobility of the parasitic larvae., Financial 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” Pro- gramme (Ministerio de Economía y competitividad; MMV, RYC- 2015-18368)

5 páginas, 1 figura., This 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. None of the authors have a conflict of interest.

15 páginas, 2 figuras., Diseases caused by trypanosomatids (Sleeping sickness, Chagas disease, and leishmaniasis) are a serious public health concern in low-income endemic countries. These diseases are produced by single-celled parasites with a diploid genome (although aneuploidy is frequent) organized in pairs of non-condensable chromosomes. To explain the way they reproduce through the analysis of natural populations, the theory of strict clonal propagation of these microorganisms was taken as a rule at the beginning of the studies, since it partially justified their genomic stability. However, numerous experimental works provide evidence of sexual reproduction, thus explaining certain naturally occurring events that link the number of meiosis per mitosis and the frequency of mating. Recent techniques have demonstrated genetic exchange between individuals of the same species under laboratory conditions, as well as the expression of meiosis specific genes. The current debate focuses on the frequency of genomic recombination events and its impact on the natural parasite population structure. This paper reviews the results and techniques used to demonstrate the existence of sex in trypanosomatids, the inheritance of kinetoplast DNA (maxi- and minicircles), the impact of genetic exchange in these parasites, and how it can contribute to the phenotypic diversity of natural populations., This research was partially funded by MINECO; SAF2017-83575-R. The first author CGC (LE255-16) as well as BDA (LE208-17) are recipients of Junta de Castilla y Leon (JCyL) and European Social Found (ESF)’s Fellowships Scheme for Doctoral Training Programs. MMV is funded by the Spanish “Ramon y Cajal” Programme (RYC-2015-18368).

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).

6 páginas, 3 figuras., This study describes early immunological mechanisms that underlie resistance to Teladorsagia circumcincta infection in adult Churra sheep. After a first experimental infection, 6 animals were classified as resistant (RG) and 6 as susceptible (SG) to T. circumcincta infection based on their cumulative faecal egg count (cFEC) at the end of the infection. RG showed higher IgA levels against somatic antigen of T. circumcincta fourth-larvae stage (L4) in serum at day 3 post-infection (pi) (p < 0.05) and close to significance at day 21 pi (p = 0.06). Moreover, a strong negative correlation between cFEC and specific IgA was only significant in RG at day 3 pi (r = − 0.870; p < 0.05), but absent in SG. At the end of this infection, sheep were treated with moxidectin and infected again 3 weeks later to be slaughtered at day 7 pi. At necropsy, the specific IgA levels in gastric mucosa were similar between groups; the absence differences at day 7 pi could be due to a previous increase in the IgA response, probably around day 3 pi, as described during the first infection. L4 burden, 68% lower in RG than in SG, was influenced by the specific IgA in gastric mucus and the number of γδ T cells. RG group showed a positive correlation between γδ T cells and eosinophils (r = 0.900; p = 0.037); however, this correlation was not found in SG. These results show that these two phenotypes show different early immune response pattern to T. circumcincta infection in Churra sheep., g Financial support for this project was received from the LE248U14 project of Junta de Castilla and León Government. VCGA is funded by Junta de Castilla y León and FSE (LE082-18), EVG by FPU grant (FPU17/00627) from Spanish Ministry of Education, BGG by the “Ramón y Cajal” Programme (RYC-2012-10230) from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO), MMV by the “Ramón y Cajal” Programme (RYC-2015-18368) from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and JNH by Agencia Canaria de Investigación, Innovación y Sociedad de la Información del Gobierno de Canarias y Fondo Social Europeo (ProID2017010109).

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.

12 páginas, 3 tablas, 3 figuras., Widespread use of antimicrobial drugs has led to high levels of drug-resistance in pathogen populations and a need for novel sources of anti-bacterial and anti-parasitic compounds. Macroalgae (seaweed) are potentially a rich source of bioactive compounds, and several species have traditionally been used as vermifuges. Here, we investigated the anti-parasitic properties of four common cold-water Nordic seaweeds; Palmaria palmata (Rhodophyta), Laminaria digitata, Saccharina latissima and Ascophyllum nodosum (Ochrophyta, Phaeophyceae). Screening of organic extracts against helminths of swine (Ascaris suum) and sheep (Teladorsagia circumcincta) revealed that S. latissima and L. digitata had particularly high biological activity. A combination of molecular networking and bio-guided fractionation led to the isolation of six compounds from extracts of these two species identified in both fermented and non-fermented samples. The six isolated compounds were tentatively identified by using MS-FINDER as five fatty acids and one monoglyceride: Stearidonic acid (1), Eicosapentaenoic acid (2), Alpha-Linolenic acid (3), Docosahexaenoic acid (4), Arachidonic acid (5), and Monoacylglycerol (MG 20:5) (6). Individual compounds showed only modest activity against A. suum, but a clear synergistic effect was apparent when selected compounds were tested in combination. Collectively, our data reveal that fatty acids may have a previously unappreciated role as natural antiparasitic compounds, which suggests that seaweed products may represent a viable option for control of intestinal helminth infections., This project was funded by the Green Development and Demonstration Program (GUDP) (Project No. 34009–17–1220). In vitro studies performed on T. circumcincta was funded by Short Term Scientific Mission (STSM) Grant provided by European Cooperation in Science and Technology (COST CA16230 - Combatting anthelmintic resistance in ruminants). MM-V funded by the Spanish “Ramon y Cajal” Program (Ministerio de Economía y Competitividad; MM-V, RYC-2015–18368).

17 páginas, 2 figuras, 1 tabla., M.C.-P. was funded by the “Stopping Transmission Of intestinal Parasites (STOP)” project (EDCTP2 program supported by the European Union; RIA2017NCT-1845—STOP), J.G. by Ramon Areces Foundation, and M.M.-V. by the Spanish “Ramon y Cajal” Program, Ministry of Economy and Competitiveness (RYC-2015-18368).

The genus Onchocerca includes several species associated with ungulates as hosts, although some have been identified in canids, felids, and humans. Onchocerca species have a wide geographical distribution, and the disease they produce, onchocerciasis, is generally seen in adult individuals because of its large prepatency period. In recent years, Onchocerca species infecting animals have been found as subcutaneous nodules or invading the ocular tissues of humans; the species involved are O. lupi, O. dewittei japonica, O. jakutensis, O. gutturosa, and O. cervicalis. These findings generally involve immature adult female worms, with no evidence of being fertile. However, a few cases with fertile O. lupi, O. dewittei japonica, and O. jakutensis worms have been identified recently in humans. These are relevant because they indicate that the parasite’s life cycle was completed in the new host—humans. In this work, we discuss the establishment of zoonotic Onchocerca infections in humans, and the possibility of these infections to produce symptoms similar to human onchocerciasis, such as dermatitis, ocular damage, and epilepsy. Zoonotic onchocerciasis is thought to be an emerging human parasitic disease, with the need to take measures such as One Health Strategies, in order to identify and control new cases in humans.

This study describes early immunological mechanisms that underlie resistance to Teladorsagia circumcincta infection in adult Churra sheep. After a first experimental infection, 6 animals were classified as resistant (RG) and 6 as susceptible (SG) to T. circumcincta infection based on their cumulative faecal egg count (cFEC) at the end of the infection. RG showed higher IgA levels against somatic antigen of T. circumcincta fourth-larvae stage (L4) in serum at day 3 post-infection (pi) (p 0.05) and close to significance at day 21 pi (p = 0.06). Moreover, a strong negative correlation between cFEC and specific IgA was only significant in RG at day 3 pi (r = - 0.870; p 0.05), but absent in SG. At the end of this infection, sheep were treated with moxidectin and infected again 3 weeks later to be slaughtered at day 7 pi. At necropsy, the specific IgA levels in gastric mucosa were similar between groups; the absence differences at day 7 pi could be due to a previous increase in the IgA response, probably around day 3 pi, as described during the first infection. L4 burden, 68% lower in RG than in SG, was influenced by the specific IgA in gastric mucus and the number of γδ T cells. RG group showed a positive correlation between γδ T cells and eosinophils (r = 0.900; p = 0.037); however, this correlation was not found in SG. These results show that these two phenotypes show different early immune response pattern to T. circumcincta infection in Churra sheep.

12 páginas, 4 figuras, 3 tablas., This 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).

42 páginas, 15 figuras, 1 tabla., Neglected Tropical Diseases (NTD) represent a serious threat to humans, especially for those living in poor or developing countries. Almost one-sixth of the world population is at risk of suffering from these diseases and many thousands die because of NTDs, to which we should add the sanitary, labor and social issues that hinder the economic development of these countries. Protozoan-borne diseases are responsible for more than one million deaths every year. Visceral leishmaniasis, Chagas disease or sleeping sickness are among the most lethal NTDs. Despite not being considered an NTD by the World Health Organization (WHO), malaria must be added to this sinister group. Malaria, caused by the apicomplexan parasite Plasmodium falciparum, is responsible for thousands of deaths each year. The treatment of this disease has been losing effectiveness year after year. Many of the medicines currently in use are obsolete due to their gradual loss of efficacy, their intrinsic toxicity and the emergence of drug resistance or a lack of adherence to treatment. Therefore, there is an urgent and global need for new drugs. Despite this, the scant interest shown by most of the stakeholders involved in the pharmaceutical industry makes our present therapeutic arsenal scarce, and until recently, the search for new drugs has not been seriously addressed. The sources of new drugs for these and other pathologies include natural products, synthetic molecules or repurposing drugs. The most frequent sources of natural products are microorganisms, e.g., bacteria, fungi, yeasts, algae and plants, which are able to synthesize many drugs that are currently in use (e.g. antimicrobials, antitumor, immunosuppressants, etc.). The marine environment is another well-established source of bioactive natural products, with recent applications against parasites, bacteria and other pathogens which affect humans and animals. Drug discovery techniques have rapidly advanced since the beginning of the millennium. The combination of novel techniques that include the genetic modification of pathogens, bioimaging and robotics has given rise to the standardization of High-Performance Screening platforms in the discovery of drugs. These advancements have accelerated the discovery of new chemical entities with antiparasitic effects. This review presents critical updates regarding the use of High-Throughput Screening (HTS) in the discovery of drugs for NTDs transmitted by protozoa, including malaria, and its application in the discovery of new drugs of marine origin., Financial support from the Ministerio de Economía y Competitividad (MINECO, AEI, FEDER, UE)[MINECO: AGL2016-79813-C2-1R and SAF2017-83575-R], the Junta de Castilla y León co-financed by FEDER, UE[LE020P17] to RBF and DBT, New Delhi [BT/IN/Spain/39/SM/2017-18] to MS are gratefully acknowledged. MAB(LE051-18) and DSC (LE020P17) are supported by a scholarship from the Junta de Castilla y León co-financed byFSE. MMV is supported by the Spanish “Ramon y Cajal” Programme Ministry of Economy and Competitiveness(Ministerio de Economia y Competitividad; MMV, RYC-2015-18368). We thank University of Leon for providingfunding to cover publication expenses.

5 páginas, 2 figuras., The distinction between Fasciola hepatica and Fasciola gigantica has been traditionally based on morphological criteria, although accurate recognition of the two flukes is usually difficult because of substantial variations in morphological features. The main aim of this study was to develop a PCR-based assay for discrimination between both species collected in sheep and cattle from Nigeria. A total of 47 animals, 33 cattle and 14 sheep, were sampled, and a single adult fluke was collected from each animal. DNA was extracted from flukes, and primers were designed based on mitochondrial DNA sequences to amplify a 304 bp fragment for the identification of F. hepatica and 752 bp for F. gigantica. PCR products from 12 flukes were sequenced for phylogenetic analysis. A total of 29 out of 47 flukes were identified as F. hepatica and 18 as F. gigantica. Within each host, the percentage of each fluke species was as follows: In cattle, 18/33 (54.5%) and 15/33 (45.5%) were F. hepatica and F. gigantica, respectively. In sheep, 11/14 (78.6%) were F. hepatica and 3/14 F. gigantica (21.4%). The phylogenetic analysis confirmed these results. Although the number of flukes collected in sheep was limited, it seems that F. hepatica is more prevalent in sheep than F. gigantica, whereas the percentage of each species was similar in cattle. This study confirms the presence of F. hepatica in Nigeria., This study was supported by the Spanish “Ramon y Cajal” Programme, Ministry of Economy and Competitiveness (Ministerio de Economia y Competitividad). VCG was funded by “Junta de Castilla y León” (LE082-18); JG was funded by Ramon Areces Foundation; EVG and MMV were funded by FPU16/03536 and the Spanish “Ramon y Cajal” Programme, Ministry of Economy and Competitiveness (Ministerio de Economia y Competitividad) (MMV, RYC-2015-18368), respectively.

The distinction between Fasciola hepatica and Fasciola gigantica has been traditionally based on morphological criteria, although accurate recognition of the two flukes is usually difficult because of substantial variations in morphological features. The main aim of this study was to develop a PCR-based assay for discrimination between both species collected in sheep and cattle from Nigeria. A total of 47 animals, 33 cattle and 14 sheep, were sampled, and a single adult fluke was collected from each animal. DNA was extracted from flukes, and primers were designed based on mitochondrial DNA sequences to amplify a 304 bp fragment for the identification of F. hepatica and 752 bp for F. gigantica. PCR products from 12 flukes were sequenced for phylogenetic analysis. A total of 29 out of 47 flukes were identified as F. hepatica and 18 as F. gigantica. Within each host, the percentage of each fluke species was as follows: In cattle, 18/33 (54.5%) and 15/33 (45.5%) were F. hepatica and F. gigantica, respectively. In sheep, 11/14 (78.6%) were F. hepatica and 3/14 F. gigantica (21.4%). The phylogenetic analysis confirmed these results. Although the number of flukes collected in sheep was limited, it seems that F. hepatica is more prevalent in sheep than F. gigantica, whereas the percentage of each species was similar in cattle. This study confirms the presence of F. hepatica in Nigeria.

8 páginas, 4 tablas, 2 figuras., Bacteria firmly attached to the gastrointestinal epithelium during the pre-weaning phase may show a significant impact on nutrient processing, immunity parameters, health and feed efficiency of lambs during post-weaning phases. Thus, the aim of this study was to describe the differences in the ileal epimural microbiota (e.g. total bacteria, Prevotella spp., Bifidobacterium spp. and Lactobacillus spp.) of fattening lambs promoted by early feed restriction during the suckling phase trying to elucidate some of the underlying mechanisms behind changes in feed efficiency during the fattening period. A total of 24 Merino lambs (average BW 4.81±0.256 kg) were used, 12 of them (ad libitum, ADL) kept permanently in individual pens with their mothers, whereas the other 12 lambs were separated from their dams for 9 h each day to be exposed to milk restriction (RES). After weaning (BW=15 kg) all the animals were penned individually, offered the same complete pelleted diet (35 g/kg BW per day) and slaughtered at a BW of 27 kg. During the fattening period, reduced gain : feed ratio (0.320 v. 0.261, P, This work was funded by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO, AGL2014-54124R). The authors would also like to thank MINECO (BFU2014-57964-R) for TLR quantification and CSIC (Proyecto Intramural Especial; Project 201540E084) for supporting J. Frutos with a training contract. Alba Santos gratefully acknowledges receipt of a pre-doctoral grant from the Spanish Ministry of Education, Culture and Sport (MECD, FPU15/01630). María Martínez-Valladares was funded by the Spanish ‘Ramón y Cajal’ Programme of the Ministry of Economy and Competitiveness (RYC-2015-18368).

6 páginas, 4 tablas., The aim of the present study was to determine the association between the presence of Ascaris suum at fattening pig farms, using different serological methods and the percentage of affected livers at slaughter, with performance and management indices. In total, 21 fattening pig farms from the North of Spain were included in the study. Serum samples were collected from pigs at slaughter and analysed for the presence of anti-Ascaris antibodies. For this, two different ELISAs were used. The first was based on the antibody recognition of the A. suum haemoglobin (As-Hb) molecule whereas the second test used the total extract of A. suum lung stage L3. The serological results were subsequently correlated with the percentage of condemned livers at slaughter, management practices and technical performance parameters including average daily gain (ADG) and feed conversion ratio (FCR). According to the data from the slaughterhouse, 12 out of the 21 farms had livers condemned due to liver white spots. A total of 10 farms (48%) had an average optical density ratio (ODr) exceeding the test cutoff when the As-Hb ELISA was used. This number increased to 18 farms (81%) when using the As-Lung-L3 ELISA. The average ODr of the farms on both ELISAs correlated positively with the percentage of affected livers (P < 0.01). Only the average ODr values obtained with the As-Lung-L3 ELISA were positively correlated with the FCR (P < 0.01). No correlation was found between percentage of affected livers or serology and the ADG. In relation to management practices, farms with greater than or equal to 50% slatted flooring and that applied the ‘all-in/all-out’ flow system showed a lower percentage of liver condemnations (P < 0.01), lower average ODr results on the As-Lung-L3 ELISA (P < 0.05) and lower FCR (P < 0.01) compared with those with less than 50% slatted flooring. This study emphasizes that serology is a promising diagnostic tool for diagnosing ascariasis at fattening pig farms. It also supports earlier findings that the presence of A. suum can have a significant negative impact on farm productivity and that stable infrastructure or management practices can have a considerable impact on the control of this parasite.

12 páginas, 4 tablas, 3 figuras., In dairy sheep flocks from Mediterranean countries, replacement and adult ewes are the animals most affected by gastrointestinal nematode (GIN) infections. In this study, we have exploited the information derived from an RNA-Seq experiment with the aim of identifying potential causal mutations related to GIN resistance in sheep. Considering the RNA-Seq samples from 12 ewes previously classified as six resistant and six susceptible animals to experimental infection by Teladorsagia circumcincta, we performed a variant calling analysis pipeline using two different types of software, gatk version 3.7 and Samtools version 1.4. The variants commonly identified by the two packages (high-quality variants) within two types of target regions – (i) QTL regions previously reported in sheep for parasite resistance based on SNP-chip or sequencing technology studies and (ii) functional candidate genes selected from gene expression studies related to GIN resistance in sheep – were further characterised to identify mutations with a potential functional impact. Among the genes harbouring these potential functional variants (930 and 553 respectively for the two types of regions), we identified 111 immune-related genes in the QTL regions and 132 immune-related genes from the initially selected candidate genes. For these immune-related genes harbouring potential functional variants, the enrichment analyses performed highlighted significant GO terms related to apoptosis, adhesion and inflammatory response, in relation to the QTL related variants, and significant disease-related terms such as inflammation, adhesion and necrosis, in relation to the initial candidate gene list. Overall, the study provides a valuable list of potential causal mutations that could be considered as candidate causal mutations in relation to GIN resistance in sheep. Future studies should assess the role of these suggested mutations with the aim of identifying genetic markers that could be directly implemented in sheep breeding programmes considering not only production traits, but also functional traits such as resistance to GIN infections., Financial support for this project was received from the LE248U14 project of Junta de Castilla and León Government. The research was also partially funded by the Spanish Ministry of Economy and Competitiveness (AGL2015- 66035-R project, co-funded by the European Regional Development Fund). P. K. Chitneedi is funded by a predoctoral fellowship from the Junta de Castilla and León Government and the European Social Fund. M. Martíınez-Valladares is also funded by the “Ramón y Cajal” Programme (RYC-2015-18368) from MINECO.

149 p. En el presente trabajo de Tesis Doctoral hemos pretendido establecer criterios y métodos de identificación de animales resistentes a la infección por Teladorsagia circumcincta en ovinos de raza Churra seleccionados en función de parámetros parasitológicos e inmunitarios. Durante el desarrollo del trabajo se han intentado establecer los siguientes aspectos: 1. Demostración de la relación existente entre el título de inmunoglobulina A tras una infección producida por T. circumcincta y la resistencia a la infección en ovejas de raza Churra. La resistencia a la infección se demostró mediante diversos parámetros: desarrollo del parásito en el hospedador; excreción de huevos con las heces; porcentaje de eosinófilos en sangre. 2. Influencia de la nutrición sobre la resistencia a la infección en ovejas Churras infectadas experimentalmente por T. circumcincta. La influencia de la proteína en la dieta sobre el desarrollo de la inmunidad se determinó experimentalmente administrando a los animales una dieta hipoproteica. 3. Desarrollo de una proteína recombinante con fines diagnósticos de la infección por T. circumcincta. El diagnóstico se realiza por medio de la detección de la inmunoglobulina A en ovejas resistentes y susceptibles a la infección. La finalidad es mejorar una prueba de ELISA a través de la expresión de un antígeno proteico del nematodo. 4. Comparación de dos técnicas de detección de animales resistentes a la infección por T. circumcincta. Mediante la técnica del ELISA se comparó el título de inmunoglobulina A frente antígeno somático de T. circumcincta o bien frente a un fragmento de la proteína recombinante disulfuro isomerasa en sangre, secreciones nasales y saliva

7 páginas, 5 tablas, 1 figura., In the current study, the egg hatch test (EHT) has been evaluated as an in vitro technique to detect albendazole (ABZ) resistance in Fasciola hepatica. The intra- and inter-assay variations of the EHT were measured by means of the coefficient of variation in different fluke isolates and over time; then, the results of the EHT were compared with the “gold standard” controlled efficacy test, which assesses the in vivo anthelmintic efficacy. The EHT was used later to evaluate the intra-herd variability regarding the level of ABZ resistance in calves infected by the same fluke isolate. Finally, several factors of the initial protocol were modified to improve the simplicity of the assay, including the incubation time of eggs with the drug and the use of eggs collected from faeces. The greatest uniformity between results within the assay and over time until 8 weeks after gallbladder collection (the deadline proposed for egg analysis) was obtained with an ABZ concentration of 0.5 μM. The length of exposure to ABZ was shown to be critical, as prolonged incubation (15 days) led to a change of ovicidal activity. The ABZ concentration of 0.5 μM is suggested as a possible discriminating dose to predict ABZ resistance, due to the close agreement between the results of the EHT at an ABZ concentration of 0.5 μM and those of the in vivo assays., This study was partially supported by CONICET and Agencia Nacional de Promoción Científica y Técnica (ANPCyT), from Argentina, and by the Spanish “Ramón y Cajal” Programme, Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad) (MMV, RYC-2015-18368).

20 páginas, 2 tablas, 4 figuras., Leishmaniasis is a group of zoonotic diseases caused by a trypanosomatid parasite mostly in impoverished populations of low-income countries. In their different forms, leishmaniasis is prevalent in more than 98 countries all over the world and approximately 360-million people are at risk. Since no vaccine is currently available to prevent any form of the disease, the control strategy of leishmaniasis mainly relies on early case detection followed by adequate pharmacological treatment that may improve the prognosis and can reduce transmission. A handful of compounds and formulations are available for the treatment of leishmaniasis in humans, but only few of them are currently in use since most of these agents are associated with toxicity problems such as nephrotoxicity and cardiotoxicity in addition to resistance problems. In recent decades, very few novel drugs, new formulations of standard drugs or combinations of them have been approved against leishmaniasis. This review highlights the current drugs and combinations that are used medical practice and recent advances in new treatments against leishmaniasis that were pointed out in the recent 2nd Conference, Global Challenges in Neglected Tropical Diseases, held in San Juan, Puerto Rico in June 2018, emphasizing the plethora of new families of molecules that are bridging the gap between preclinical and first-in-man trials in next future., Financial support from the Ministerio de Economía y Competitividad (MINECO, AEI, FEDER, UE) [MINECO, Funder Id: http://dx.doi.org/10.13039/501100003329, AGL2016-79813-C2-1R and Funder Id: http://dx.doi.org/10.13039/501100003329, SAF2017-83575-R], and the Junta de Castilla y León co-financed by FEDER, UE [LE020P17] is gratefully acknowledged. CGC and BGA are supported by scholarships from the Junta de Castilla y León co-financed by FEDER.

12 páginas, 2 tablas, 4 figuras., Gastrointestinal nematode infections are the main diseases in herds of small ruminants. Resistance to the main established drugs has become a worldwide problem. The purpose of this study is to obtain and evaluate the in vitro ovicidal and larvicidal activity of some 2-phenylbenzimidazole derivatives on susceptible and resistant strains of Teladorsagia circumcincta. Compounds were prepared by known procedures from substituted o-phenylenediamines and arylaldehydes or intermediate sodium 1-hydroxyphenylmethanesulfonate derivatives. Egg Hatch Test (EHT), Larval Mortality Test (LMT) and Larval Migration Inhibition Test (LMIT) were used in the initial screening of compounds at 50 μM concentration, and EC values were determined for the most potent compounds. Cytotoxicity evaluation of compounds was conducted on human Caco-2 and HepG2 cell lines to calculate their Selectivity Indexes (SI). At 50 μM concentration, nine out of twenty-four compounds displayed more than 98% ovicidal activity on a susceptible strain, and four of them showed more than 86% on one resistant strain. The most potent ovicidal benzimidazole (BZ) 3 showed EC = 6.30 μM, for the susceptible strain, while BZ 2 showed the lowest EC value of 14.5 μM for the resistant strain. Docking studies of most potent compounds in a modelled Teladorsagia tubulin indicated an inverted orientation for BZ 1 in the colchicine binding site, probably due to its fair interaction with glutamic acid at codon 198, which could justify its inactivity against the resistant strain of T. circumcincta., We would like to express our gratitude to Dr. Dave Bartley at Moredun Research Institute for providing the triple-resistant strainof T. circumcincta. Authors thank Bioinformatic & Molecular Design Research Centre (BMDRC, Yonsey University, Seoul, Korea) and Idorsia Pharmaceuticals Ltd. (Allschwil, Switzerland) for compli-mentary access to preADMET and Osiris Data Warrior databases, respectively. RE thanks financiation from RICET-USAL ProgramRD16/0027/0018. Financial 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 FPU16/03536, VCGA by Junta de Castilla y León and FSE (LE082-18), MAVby Junta de Castilla y León (LE051-18) and MMV by the Spanish “Ramon y Cajal” Programme (Ministerio de Economía y Competitividad; MMV, RYC-2015-18368), respectively.

6 páginas, 4 tablas., The present study was conducted to evaluate the in vitro anthelmintic activity and safety of methanolic and aqueous extracts of 9 plants against the ovine GIN Teladorsagia circumcincta. Initially the ovicidal efficacy of all extracts was tested at 50 mg/ml and at 1%, this last concentration for the methanolic extract of Elettaria cardamomum, by the Egg Hatch Assay. In those extracts with efficacy higher than 95%, the effective concentration required to inhibit 50% of egg hatching (EC) and their cytotoxicity, as the lethal dose 50 (LC), was also measured. The aqueous extracts of Aesculus hippocastanum, Isatis tinctoria, Chelidonium majus, E. cardamomum and Sisymbrium irio, with EC values ranging 1.30–2.88 mg/ml, and the aqueous extract of Jasminum polyanthum with a value of 6.41 mg/ml, showed the highest activities. The aqueous extract of J. polyanthum was the safest extract, followed by methanolic extract of E. cardamomum and aqueous extract of S. irio, all of them with a Selective Index higher than 1. According to our results, there is no correlation between the amount of total phenols or total tannins with the anthelmintic activity of the plants tested. Although results need to be interpreted with caution, as in vitro activity may not automatically translate into in vivo efficacy, those extracts with SI equal or higher than 1 and EC equal or lower than 6 mg/ml, could be taken into account for being used subsequently as feed or food additives in infected sheep., This study has been funded by the national project INIA (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria: RTA2013-00064-C02-02), Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad: AGL 2016-79813-C2-1R),the European Regional Development Fund (Fondos Feder) and the Spanish“Ramón y Cajal”Programme, Ministry of Economy and Competitiveness (MMV, RYC-2015-18368).

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.

13 páginas, 6 tablas., To elucidate the influence of dietary carnosic acid (CA) and vitamin E on animal performance, immune response indicators and haematological parameters before and after transport stress, 24 lambs were individually fed ad libitum with milk replacer (MR) using an auto-feeder. Once daily the lambs received MR alone (Group CON, n = 8), MR + 0.096 g CA/kg live weight (LW) (Group CARN, n = 8) or MR + 0.024 g of α-tocopheryl acetate per kg LW (Group VitE, n = 8). After reaching the target slaughter weight (12 ± 0.5 kg), blood samples were collected to measure haematological and immunological parameters. Then, lambs were subjected to 4-h road transport and blood samples were collected again for haematological assessment. The animals were subsequently slaughtered. Before road transport, dietary CA supplementation promoted a descent of circulating white blood cells (WBC), red blood cells (RBC), haematocrit and haemoglobin concentration when compared with Groups CON and VitE (p

8 páginas, 4 tablas., This 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).

A 22-month-old male Spanish water dog was hospitalized after its physical examination revealed fever and movement difficulty. After 24 h, the dog was found to have a high fever (39.5 °C) and was treated empirically with doxycycline/ciprofloxacin. At 48 h, after submission the fever rose to 41 °C and the animal presented with a stiff neck and dehydration. Peripheral blood and cerebrospinal fluid (CSF) were sampled and trophozoites with an Acanthamoeba-like morphology were observed in the CSF. PCR specific for Acanthamoeba, Naegleria fowleri and Balamuthia mandrillaris were performed and the CSF sample found positive for Acanthamoeba. Lungs, kidney, liver and spleen samples were collected post mortem. All collected organ samples were positive for Acanthamoeba by PCR, thus confirming a multisystemic infection. Water samples taken at a suspected site of infection yielded an almost identical PCR fragment to those of the clinical samples, indicating that this was probably where the infection originated. This is the first report of a fatal case of Acanthamoeba disseminated infection in a dog in Spain.

In the current study, Fasciola hepatica strains of sheep with different degrees of resistance to anthelmintics were analyzed by sequencing the cytochrome C oxidase (COX1) and the NADH dehydrogenase (NAD1) subunits. The strains were as follows: LS, susceptible to all drugs tested; CS, resistant to albendazole and triclabendazole; and SV, resistant to albendazole and clorsulon. The molecular characterization was done in eggs recovered from sheep infected by LS and CS. In relation to SV, eggs were recovered before (SV0) and after a treatment with albendazole (SVA) and clorsulon (SVC). Nested PCRs were carried out to amplify a fragment of 798 bp of the COX1 subunit and 870 bp of the NAD1 subunit. The pairwise sequence identity between eggs was analyzed for each strain. Population diversity indices, neutrality indices, and the degree of gene flow among the strains were evaluated. As a result, we have shown that there was homogeneity in the demographic expansion of the studied strains, and, according to the pairwise fixation index, these were not genetically differentiated. Although we found that the resistant strains had lower pairwise percentage similarities, higher haplotype diversity, and higher frequencies of specific SNPs, especially in the COX1 subunit, these differences were not very significant. Therefore, we conclude that the presence of adult flukes resistant to anthelmintics does not result in significant higher genetic diversity in the mtDNA of their eggs. © 2014 Springer-Verlag., National projects INIA-MEC; RTA2010-00094-C03-02; Consejo Superior de Investigaciones Cientificas (CSIC); European Social Fund

4 páginas., In the current study, we identified five sheep flocks with fasciolosis in the province of León (northwestern Spain) in order to determine the anthelmintic resistance status to three commonly used anthelmintics, namely albendazole (ABZ), triclabendazole (TCBZ) and clorsulon (CLOR). The identification of one flock resistant to ABZ and CLOR was shown after the faecal egg count reduction test (FECRT). The reductions in eggs per gram values were -17.6% and -68% against immature and adult flukes, respectively, after ABZ treatment; 85.15% and 44.91% against immature and adult flukes, respectively, after CLOR treatment; and 97.06% against both stages, after the administration of TCBZ. As an alternative to control the infection, two combinations of ABZ and CLOR were tested. In the first, both drugs were administered at the recommended dose of each; in this case, the efficiency reached values above 95% against both immature and adult flukes. However, when the combined drugs were administered at half the recommended dose of each, the efficiency of the combination was very low, i.e. 16.67% and -11.11% against mature and immature flukes, respectively. In conclusion, this preliminary report suggests a possible interaction between ABZ and CLOR after their joint administration. However, these results should be confirmed in other flocks., 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.

7 páginas, 5 tablas., Thirty-two lambs were divided in 4 groups with 2 replicates each (8 batches in total) according to their body weights. The lambs were fed a total mixed ration (TMR) formulated either with palm oil (CTRL, 2 replicates, 4 animals per replicate; 34gpalmoilkg-1 TMR) or flaxseed (FS, 2 replicates, 4 animals per replicate; 85gflaxseedkg-1 TMR). Four more batches were fed the same TMRs but enriched with quercetin (QCT, 2 replicates, 4 animals per replicate, 34g palm oil plus 2gquercetinkg-1 TMR; FS-QCT, 2 replicates, 4 animals per replicate, 85g flaxseed plus 2gquercetinkg-1 TMR). Three weeks after starting with the experimental diets, the animals were immunized by a subcutaneous injection of ovalbumin, and blood samples were collected at days 0, 4, 9, 14 and 17 post-immunization to measure antibodies against ovalbumin. Afterwards, all of the lambs were subjected to a 4-h transportation-stress period to study the evolution of haematological and biochemical parameters during road transport and, finally, slaughtered. Mean ova-specific IgG titres were significantly lower in the lambs fed FS on days 14 and 17 when compared to CTRL group (FS×DAY, P=0.033). Additionally, FS reduced white blood cells counts and tissue damage (creatine phosphokinase, P, Financialsupportreceivedfrom ‘Consejería de Educación de la Junta de Castilla y León’ (ProjectCSI185B11-2)is gratefullyacknowledged.Julio Benavides,MaríaMartinez- Valladares,MaríaL.Tejido,Raúl Bodas,and NuriaPrieto (JAE-Doccontracts)were supportedbytheprogramme ‘Junta para la Ampliación de Estudios’ (CSIC-European SocialFund).

8 páginas, 8 figuras, 1 tabla., Paramphistomosis and Fasciolosis caused by Calicophoron daubneyi and Fasciola hepatica, respectively, are frequent and important trematodoses in ruminant livestock worldwide. Both parasites use the same snail, Galba truncatula, as intermediate host. The aim of this study was to develop and validate an analytical method based on a mitochondrial DNA (mtDNA) multiplex PCR technique which would allow the early and specific identification, in one step, of C. daubneyi and F. hepatica infection in G. truncatula. First of all, a 1035 bp fragment of mtDNA from adult C. daubneyi worms was obtained. Then two pairs of specific mtDNA primers, which amplified a DNA fragment of 885 pb in the case of C. daubneyi, and of 425 pb in that of F. hepatica, were designed. By means of the multiplex PCR technique developed, there was always a specific amplification in samples from adult F. hepatica and C. daubneyi, but not from Calicophoron calicophorum, Cotylophoron cotylophorum, Cotylophoron batycotyle or Dicrocoelium dendriticum. Likewise, specific amplifications of the expected DNA fragments happened in all samples from snails harbouring larval stages of C daubneyi or F. hepatica, previously detected by microscopy. However, amplifications were not seen when DNA from snails harbouring other Digenea (Plagiorchiidae, Notocotylidae and furcocercous cercariae) was analysed. Moreover, DNA from G. truncatula molluscs free from infection was not amplified. The multiplex PCR assay permitted infection in the snails experimentally infected with 4 miracidia to be detected as early as day 1 p.i. in the case of F. hepatica and with only 2 miracidia from day 2 p.i. in both, C daubneyi and F. hepatica. Nevertheless it was necessary to wait until days 29 and 33 p.i. to see C. daubneyi and F. hepatica immature redia, respectively, using microscope techniques. The detection limit of the PCR technique was very low: 0.1 ng of DNA from C daubneyi and 0.001 ng of DNA from F. hepatica. This allowed infection by either F. hepatica or C daubneyi to be detected even when pools made up with only 1 mu l (60 ng of DNA) from infected snail plus 99 mu l from non-infected ones were analyzed. Moreover, simultaneous detection of both parasites was experimentally possible in pools made up with uninfected (98 mu l), C. daubneyi infected (1 mu l) and F. hepatica infected (1 mu l) snails. The most precise and early diagnosis of the infections using the multiplex PCR technique designed will allow more realistic epidemiological models of both infections to be established and consequently a better strategic control., This study was financially supported by the Spanish INIA, Project FAU2006-00021-C03-00 and by the European Social Funds, as well as by the Castile and León Autonomy (Spain) Project LE023A10-2.A.M. Martínez-Ibeas is supported by the ‘Castilla y León’ Autonomy (Spain) and the European Social Funds (Contract for young researchers). M. Martínez- Valladares is supported by the Spanish National Research Council (CSIC) and the European Social Funds (JAE Doc Contract). B. Mi˜nambres was supported by ‘Ramón y Cajal’ contract.

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.).

7 páginas, 5 figuras., The aim of this study was to compare three different techniques for the early diagnosis of the infection by Fasciola hepatica in experimentally and naturally infected sheep. The experimental group consisted of 7 sheep infected with 200 metacercariae; faecal samples were taken weekly until 12 week post-infection (wpi). Under natural conditions, 45 individual faecal samples and 2 pools of faeces were collected from three different flocks with a history of F. hepatica infection. The results obtained by a coprological Method were compared with a commercial immunoassay and with two PCR assays in faecal samples. Faecal eggs were detected by 9 wpi in experimental infection. On the other hand, only 24 out of 45 sheep were positive in naturally infected flocks. By means of a sandwich-ELISA kit, the infection was first detected by 4 wpi in the 57.1% experimentally infected sheep and this percentage reached 100% by 8 wpi. All naturally infected animals were positive with this method. Regarding PCR, a specific 423 bp fragment of mitochondrial DNA was amplified in faecal samples. The F. hepatica infection was detected from 3 wpi with a standard PCR, and from 2 wpi with a nested-PCR. Only 37 sheep out of 45 were positive by the standard PCR although the infection was diagnosed in all animals by the nested-PCR. In conclusion, the sensitivity of the nested-PCR described in our study is higher than the detection of eggs in faeces as well as the commercial immunoassay. Moreover, no cross reactions were described with gastrointestinal nematodes., This study has been funded by the National Project 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.

5 páginas, 2 figuras., The aim of this study was to evaluate the IgG activity in serum and milk samples of experimentally infected lactating ewes with the nematode Teladorsagia circumcincta as well as its relationship to the degree of infection. In a previous study 28 pregnant ewes were divided into two homogeneous groups, with high (H) and low (L) level of parasitism, respectively. Blood and milk samples were taken weekly after lambing until the end of the lactation, 126 days post-partum (pp). IgG against T. circumcincta were measured by means of an indirect ELISA. The kinetic of the immunoglubulins in serum samples showed a very low activity at the beginning but gradually increased throughout the lactation; H group showed higher values most of the sampled days than L group. Contrary, IgG in milk samples remained high during the first month pp, then decreased around 38% and by the end of the study rose again. Antibodies in both samples were correlated and especially during the second month of lactation (r = 0.3; p < 0.001). With the aim to correlate the immune response and the degree of infection we found an inverse relationship (r = −0.2; p < 0.05) during the second month of lactation between eggs and IgG in serum. However the correlation with immunoglobulins in milk was positive, mainly, on the last third of lactation (r = 0.2; p < 0.01). As a conclusion, the individual detection of total IgG antibodies against T. circumcincta in lactating ewes is highly dependent on the stage of lactation. Therefore, these associations should be confirmed under field conditions., This study has been funded by the CICYT national project, AGL2001-0314. The work of Martínez-Valladares M. has been supported by a postdoctoral Jae-Doc contract from Consejo Superior de Investigaciones Científicas (CSIC) and co-founded by the European Social Fund.

5 páginas, 1 tabla, 2 figuras, The aim of this work was to know the anthelmintic resistance (AR) status of a Spanish sheep flock infected by gastrointestinal nematodes (GIN) and the possible cross resistance among anthelmintics of the macrocyclic lactones (ML) family. The Faecal Egg Count Reduction Test (FECRT) was carried out to check the efficacy of albendazole (ZodalbenA (R)), levamisole (LEV) (EndexA (R)) and an oral formulation of ivermectin (IVM) (OramecA (R)), at the recommended dose rates. Then, the study was extended to check the cross resistance between drugs of the ML family: injectable IVM (IvomecA (R)), oral moxidectin (CydectinA (R)), injectable moxidectin (BiodectinA (R)) and doramectin (DectomaxA (R)), at the recommended dose rates. The GIN species were identified after faecal cultures in all groups. The FECRT showed the resistance of a Teladorsagia circumcincta isolate against LEV (39-58%), IVM (88-92%) and doramectin (85%). This study is the first report to confirm the side resistance between these MLs, which belong to the avermectin chemical group, in a Spanish sheep flock. The in vitro efficacy of LEV and IVM was measured by the Larval Feeding Inhibition Assay (LFIA) using the IC50 measurement (concentration needed to inhibit the ingestion of 50% L1). The values of the multidrug resistant isolate were 0.25 mu g/ml for LEV and 3 ng/ml for IVM. Both results were higher than the values obtained with a susceptible isolate, which could be indicative of AR. However, further research examining the response of a greater range of susceptible and resistant nematodes isolates should be carried out to establish a discrimination threshold., Fort Dodge Veterinaria, S.A. Madrid. Consejo Superior de Investigaciones Cientificas (CSIC). INIA-MEC, RTA2006-00183-C03-02