Transmission of Calicophoron daubneyi and Fasciola hepatica in Galicia (Spain): Temporal follow-up in the intermediate and definitive hosts

Background Paramphistomosis caused by Calicophoron daubneyi and fasciolosis caused by Fasciola hepatica are common parasitic diseases of livestock animals. Transmission of the diseases depends on the presence of intermediate hosts, i.e. freshwater gastropods such as lymnaeids. We carried out a 2-year-long study of the dynamics of the snail population acting as the intermediate host for these parasites, considering the population structure in terms of size/age and infection status. In addition, we determined the kinetics of trematode egg excretion in grazing cows. Generalized Additive Models (GAMs) were used to analyze the associations between different response variables and snail size, sampling month and weather-related variables. Results Of the molluscan species examined, Galba truncatula, Radix peregra, Anisus (Anisus) leucostoma and Pisidium casertanum (n = 2802), only G. truncatula was infected with C. daubneyi or F. hepatica, at prevalence rates of 8.2% and 4.4% respectively. The probability of infection with C. daubneyi or F. hepatica was linearly related to snail size, although in different ways (negative for C. daubneyi and positive for F. hepatica). The total snail population increased in winter, when specimens of all size classes were found. Infected snails were more abundant during spring-autumn. Mature cercariae of both parasites were found in most seasons. In the statistical models, the sampling month accounted for a high percentage (71.9–78.2%) of the observed variability in snail abundance. The inclusion of climatic variables in the models moderately increased the percentage of deviance explained (77.7–91.9%). Excretion of C. daubneyi eggs in cow faeces was always higher than that of F. hepatica eggs. Conclusions Particular care should be taken to prevent pastures and the surrounding environment being contaminated with parasite eggs during winter-spring, when the number of snails susceptible to miracidial infections is maximal. This is therefore the optimal time for treating grazing animals. Nevertheless, control of trematodosis based only on chemotherapy is difficult in an area such as the study area, where environmental factors favour the regular appearance of snail populations harbouring mature cercariaeThe present study was financially supported by the Ministerio de Economía y Competitividad (AGL2011-30563-C03-03). The funders did not have any role in the study design, data collection and analyses, decision to publish or preparation of the manuscriptS

Fasciola spp: Mapping of the MF6 epitope and antigenic analysis of the MF6p/HDM family of heme-binding proteins

MF6p/FhHDM-1 is a small cationic heme-binding protein which is recognized by the monoclonal antibody (mAb) MF6, and abundantly present in parenchymal cells and secreted antigens of Fasciola hepatica. Orthologs of this protein (MF6p/HDMs) also exist in other causal agents of important foodborne trematodiasis, such as Clonorchis sinensis, Opisthorchis viverrini and Paragonimus westermani. Considering that MF6p/FhHDM-1 is relevant for heme homeostasis in Fasciola and was reported to have immunomodulatory properties, this protein is expected to be a useful target for vaccination. Thus, in this study we mapped the epitope recognized by mAb MF6 and evaluated its antigenicity in sheep. The sequence of the MF6p/FhHDM-1 ortholog from F. gigantica (MF6p/FgHDM-1) was also reported. By means of ELISA inhibitions with overlapping synthetic peptides, we determined that the epitope recognized by mAb MF6 is located within the C-terminal moiety of MF6p/FhHDM-1, which is the most conserved region of MF6p/HDMs. By immunoblotting analysis of parasite extracts and ELISA inhibitions with synthetic peptides we also determined that mAb MF6 reacted with the same intensity with F. hepatica and F. gigantica, and in decreasing order of intensity with C. sinensis, O.viverrini and P. westermani orthologs. On the contrary, mAb MF6 showed no reactivity against Dicrocoelium dendriticum and Schistosoma mansoni. The study of the recognition of peptides covering different regions of MF6p/FhHDM-1 by sera from immunized sheep revealed that the C-terminal moiety is the most antigenic, thus being of potential interest for vaccination. We also demonstrated that the production of antibodies to MF6p/FhHDM-1 in sheep infected by F. hepatica occurs relatively early and follows the same pattern as those produced against L-cathepsins.This work was funded by the Ministerio de Ciencia e Innovación, Spain (Grants AGL2011-30563-C03-01, AGL2011-30563-C03-02 and AGL2011-30563-C03-03); Xunta de Galicia, Spain (Grants GPC 2014/058 and ED431B 2017/18); Network of Biomedical Research on Tropical Diseases (RICET), Spain (Grant RD12/0018/0013) and the European Fund for Regional Development (FEDER). VMS holds a predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (Programa de Formación del Profesorado Universitario). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA

Recombinant proteins expressed in E. coli are frequently purified by immobilized metal affinity chromatography (IMAC). By means of this technique, tagged proteins containing a polyhistidine sequence can be obtained up to 95% pure in a single step, but some host proteins also bind with great affinity to metal ions and contaminate the sample. A way to overcome this problem is to include a second tag that is recognized by a preexistent monoclonal antibody (mAb) in the gene encoding the target protein, allowing further purification. With this strategy, the recombinant protein can be directly used as target in capture ELISA using plates sensitized with the corresponding mAb. As a proof of concept, in this study we engineered a Trichinella-derived tag (MTFSVPIS, recognized by mAb US9) into a His-tagged recombinant Fasciola antigen (rFhLAP) to make a new chimeric recombinant protein (rUS9-FhLAP), and tested its specificity in capture and indirect ELISAs with sera from sheep and cattle. FhLAP was selected since it was previously reported to be immunogenic in ruminants and is expressed in soluble form in E. coli, which anticipates a higher contamination by host proteins than proteins expressed in inclusion bodies. Our results showed that a large number of sera from non-infected ruminants (mainly cattle) reacted in indirect ELISA with rUS9-FhLAP after single-step purification by IMAC, but that this reactivity disappeared testing the same antigen in capture ELISA with mAb US9. These results demonstrate that the 6XHis and US9 tags can be combined when double purification of recombinant proteins is required.This work was supported by: Ministerio de Economía y Competitividad (Spain) [grant number AGL2011-30563-C03 and AGL2014-57125R], Ministerio de Economía, Industria y Competitividad (INIA, Spain) [grants numbers RTA2017-00010-C02-01 and RTA2017-00010-C02-02] and the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia, Spain) [grant number ED431B 2017/18]. RAOM holds a predoctoral fellowship from the Spanish Ministerio de Economía y Competitividad (Programa de Formación de Personal Investigador). VMS is supported by a contract under the grant ED431B 2017/18. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Calicophoron daubneyi (Paramphistomidae) in slaughtered cattle in Castilla y León (Spain)

The prevalence and aetiology of natural paramphistomosis was investigated in cattle slaughtered in the Castilla y León region (Spain) over a 3 year-period. The overall prevalence of positive animals was 6.20%. The parasite burden per animal ranged from 8 to 8005 (median = 144) and the ruminal atrium had the highest parasite burden whereas the ruminal dorsal sac the lowest. The prevalence and parasite burden increased with age while these parameters were lower in cattle under intensive management. Calicophoron daubneyi was the only Paramphistomidae species identified using morphoanatomical, histological and molecular methods in the studied animals.This work was supported by grant LE023A10-2 fromJunta de Castilla y León (JCyL). A.M. Martínez-Ibeas was supported by the JCyL andthe European Social Funds (ESF) and J. Benavides by theJAE-Doc programme (CSIC-ESF).Peer Reviewe

Development and Evaluation of a New Lateral Flow Immunoassay for Serodiagnosis of Human Fasciolosis

Fasciolosis is an important plant-borne trematode zoonosis. This disease is of both clinical and veterinary relevance and, according to the WHO, is considered a re-emerging disease that is spreading around the world. Fasciolosis has a serious impact on health because of the large size of the parasite and the effects of the parasite in down-regulating the host immune response. Human fasciolosis can be distinguished by an acute phase, in which the parasite migrates through different tissues, and a chronic phase in which it invades the bile ducts. Here we describe the development of a rapid, simple and inexpensive immunochromatographic diagnostic method, based on the use of a recombinant cathepsin L1 protein, which performs better than other more complex indirect methods, providing similar specificity and higher sensitivity. The simplicity of the method represents a great advantage for the intervention systems applied in different endemic areas by WHO, such as passive case finding (e.g. Vietnam) and selective treatment (e.g. Egypt). Because of its characteristics, the system can be applied to both phases of the disease, and in holo, meso and hyperendemic areas where point-of-care testing is required

Animal health aspects of adaptation to climate change: beating the heat and parasites in a warming Europe

Weather patterns in northern European regions have changed noticeably over the past several decades, featuring warmer, wetter weather with more extreme events. The climate is projected to continue on this trajectory for the foreseeable future, even under the most modest warming scenarios. Such changes will have a significant impact on livestock farming, both directly through effects on the animals themselves, and indirectly through changing exposure to pests and pathogens. Adaptation options aimed at taking advantage of new opportunities and/or minimising the risks of negative impacts will, in themselves, have implications for animal health and welfare. In this review, we consider the potential consequences of future intensification of animal production, challenges associated with indoor and outdoor rearing of animals and aspects of animal transportation as key examples. We investigate the direct and indirect effects of climate change on the epidemiology of important livestock pathogens, with a particular focus on parasitic infections, and the likely animal health consequences associated with selected adaptation options. Finally, we attempt to identify key gaps in our knowledge and suggest future research priorities.</p

Preliminary proteomic analysis of excretory-secretory and tegumental antigens from adult worms of Dicrocoelium dendriticum

2 páginas.-- Poster presentado al XI Congresso Ibérico de Parasitología (Lisboa, Portugal, 15-18 de septiembre de 2009)[ES]Dicrocoelium dendriticum, trematodo hepático causante de la dicroceliosis, afecta al hígado, vesícula biliar y conductos biliares de rumiantes domésticos y silvestres y, ocasionalmente, infecta al hombre. Aunque dicha parasitosis está ampliamente distribuida, su impacto sanitario y económico no ha sido cuantificado convenientemente, por una parte, debido a la naturaleza subclínica de la enfermedad y, por otra, a la carencia de un método inmunológico estandarizado, sensible y específico de diagnóstico in vivo. En este sentido, la identificación y caracterización de proteínas antigénicas específicas, implicadas en la interacción parásito-hospedador definitivo, constituye un requisito indispensable para el diagnóstico inmunológico y para su posible utilización como dianas vacunales. El objetivo del presente trabajo es el análisis de los proteomas de extractos de excreción-secreción y tegumento de vermes adultos de D. dendriticum. El antígeno excretor-secretor se obtuvo a partir de la incubación de adultos vivos (40 vermes/ml) en RPMI-1640 durante 24 horas, según el protocolo establecido por Revilla-Nuín et al. (2005). El extracto tegumentario se recolectó tras la incubación de un número elevado de vermes vivos (700 a 1000) en TBS-Triton X-100 1%, durante 30 min, a 4º C y 70 rpm. Con el fin de mejorar la resolución de las proteínas y eliminar posibles moléculas contaminantes, ambos extractos se trataron de dos maneras diferentes: 1/ Precipitación con TCA-acetona; 2/ Utilización del kit ReadyPrepTM 2-D Cleanup (Bio-Rad). Posteriormente, se sometieron a electroforesis 2D. En la primera dimensión se utilizaron tiras IPG de 7 cm, en gradiente de pH 3-10, cargadas con 60 a 100 µg proteína/tira. La segunda dimensión se realizó en minigeles de poliacrilamida al 10 ó 12%, teñidos posteriormente con Coomassie Colodial. Se seleccionaron 15 spots mayoritarios en cada uno de los extractos antigénicos y se analizaron mediante espectrometría de masas MALDI TOF/TOF. Para la identificación de las proteínas se empleó el motor de búsqueda MASCOT y la base de datos NCBInr.El extracto de excreción-secreción se resolvió en numerosos spots con pesos moleculares entre 9 y 84 kDa y puntos isoeléctricos (pI) entre 4 y 9. De los 15 spots analizados, mediante MALDI, únicamente se ha podido establecer homología para 8 de ellos, que se corresponden con 6 proteínas diferentes. Una de ellas, identificada en 3 spots, presenta similitud en el 36% de su secuencia con la mioglobina de D. dendriticum. El mapa proteómico del extracto de tegumento reveló que la mayoría de las proteínas presentaban pIs de 5,5 a 9, en un rango de pesos moleculares de 10 a 110 kDa. De los 15 spots analizados por MALDI TOF/TOF se pudieron identificar 12, que presentaban homología con 7 proteínas distintas. De ellas una se corresponde con 1 proteína del hospedador (pre-pro albúmina sérica) y otra fue identificada como mioglobina en 4 spots, 3 de los cuales estaban también presentes en el extracto de excreción-secreción. En conclusión, los proteomas de antígeno excretor-secretor y de tegumento revelan similitud, tanto en lo que respecta al número de spots como a su distribución en el rango de pesos moleculares y puntos isoeléctricos. Por espectrometría de masas MALDI TOF/TOF se identificaron 12 proteínas diferentes, de las cuales al menos 1 procede del hospedador. Finalmente, hay que señalar que, es necesario profundizar en los estudios proteómicos, tanto para completar la identificación de las proteínas como para determinar las de mayor poder antigénico e inmunogénico.[EN]Dicrocoelium dendriticum, the hepatic trematode responsible for dicrocoeliosis, affects the liver, gall bladder and bile ducts of domestic and wild ruminants and, also occasionally, infects humans. Although this parasitosis is widely distributed, its sanitary and economic impact has not been properly quantified, on the one hand due to its subclinical nature and, on the other hand, due to the lack of a standardized immunological, sensitive and specific diagnostic method in vivo. The identification and characterization of specific antigenic proteins involved in the parasite-definitive host interplay becomes an essential requirement for immunological diagnosis and for their possible use as vaccination targets. The objective of the present work is the proteomic analysis of the excreted-secreted proteins and the tegumental proteins of D.dendriticum adult worms. The excretory-secretory antigen was obtained after incubation of live adults (40 worms/ml) in RPMI-1640 during 24 h, according to the protocol established by Revilla-Nuín et al. (2005). The tegumental extract was collected after incubating of a higher number (700 to 1000) of live worms in TBS-Triton X-100 1% during 30 min at 4º C and 70 rpm. To improve protein resolution and remove possible contaminant particles, both extracts were treated in two different ways: 1/ TCA-acetone precipitation; 2/ ReadyPrepTM2-D Cleanup kit (Bio-Rad). When this process was completed, 2-D electrophoresis was applied. On the first dimension we used 7 cm IPG strips, with a pH gradient from 3 to 10, charged with 60-100 µg protein/strip. The second dimension was performed on 10-12% polyacrylamide minigels which were later stained with Coloidal Coomassie. The 15 major spots were chosen and analyzed by MALDI TOF/TOF mass spectrometer. In order to identify different proteins, MASCOT software and NCBInr database were used.The excretory-secretory extract revealed numerous spots located between 9 to 84 kDa and isoelectric points (pI) from 4 to 9. From the 15 spots analyzed by MALDI, homology could only be established for 8 of them, which were matched with 6 different proteins. One of them, which was identified in 3 spots, showed similarity on 36% of its sequence with D. dendriticum myoglobin. The proteomic map of tegumental extract revealed that most of the proteins showed pI from 5.5 to 9 with a molecular weight between 10 and 110 kDa. Fifteen spots were analyzed by MALDI TOF/TOF, and only 12 were identified, which showed homology with 7 different proteins. One of them was matched with a host protein (pre-pro serum albumin) and another was identified as myoglobin in 4 spots, 3 of them were also found in the excretion-secretion extract. In conclusion, the proteomes of excretory-secretory and tegument antigens revealed similarity both in the number of spots and in their distribution on the molecular weight and isoelectric point ranges. Twelve different proteins were identified by MALDI TOF/TOF mass spectrometry, at least one of them had a host origin. Finally, proteomic studies need to be deeper as well to complete the protein identifications and to determine those with the higher antigenic or immunogenic power.Estudio financiado por la Junta de Castilla y León (Proyecto CSI01A06) y la CICYT (Proyecto AGL2007-62824).Peer reviewe

Bovine paramphistomosis in Galicia (Spain): Prevalence, intensity, aetiology and geospatial distribution of the infection

12 páginas, 5 figuras, 4 tablas.The present study explored various basic aspects of the epidemiology of paramphistomosis in Galicia, the main cattle producing region in Spain. In total, 589 cows from different farms located across the region were selected at random in the slaughterhouse for examination of the rumens and reticula for the presence of Paramphistomidae flukes. Paramphistomes were found in 111 of 589 necropsied cows (18.8%; 95% CI: 15.7-21.9%), with higher prevalences of infection in beef cows than in dairy cows (29.2% vs 13.9%). Although the number of flukes per animal was generally low (median = 266 flukes), some cows harboured large parasite burdens (up to 11,895 flukes), which may have harmful effects on their health or productivity. Cows with higher parasite burdens also excreted greater numbers of fluke eggs in their faeces, which suggests that heavily parasitized mature cows play an important role in the transmission of paramphistomosis. This role may be particularly important in Galicia, where the roe deer, which is the only wild ruminant in the study area, was found not to be a reservoir for the infection. The use of morpho-anatomical and molecular techniques applied to a large number of fluke specimens provided reliable confirmation that Calicophoron daubneyi is the only species of the family Paramphistomidae that parasitizes cattle in Galicia. The environmental data from the farms of origin of the necropsied cows were used in Bayesian geostatistical models to predict the probability of infection by C. daubneyi throughout the region. The results revealed the role of environmental risk factors in explaining the geographical heterogeneity in the probability of infection in beef and dairy cattle. These explanatory factors were used to construct predictive maps showing the areas with the highest predicted risk of infection as well as the uncertainty associated with the predictions.Ministerio de Economia y Competitividad FAU2006-00021-C03-00. Junta de Castilla y Leon (Spain). European Social FundsPeer reviewe

Development and validation of a mtDNA multiplex PCR for identification and discrimination of Calicophoron daubneyi and Fasciola hepatica in the Galba truncatula snail

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.Peer reviewe