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Ticks are arthropod ectoparasites and vectors of pathogens affecting human and animal health worldwide. The exoskeleton is a structure that protect arthropods from natural threats such as predators and diseases. Both structural proteins and chemical elements are components of the exoskeleton. However, the chemical composition and effect of pathogen infection on tick exoskeleton properties has not been characterized. In this study, we characterized the chemical composition of tick exoskeleton and the effect of Anaplasma pathogen infection on the chemical elements of the exoskeleton and selected structural proteins. The chemical composition was characterized ventral, dorsal upper and dorsal lower regions of tick exoskeleton by scanning electron microscopy and energy dispersive spectroscopy and compared between infected and uninfected ticks. The levels of selected structural proteins were analyzed in infected and uninfected I. scapularis salivary glands by immunofluorescence analysis. The results showed that tick exoskeleton contains chemical elements also found in other arthropods. Some of the identified elements such as Mg and Al may be involved in tick exoskeleton stabilization through biomineralization of structural proteins that may be overrepresented in response to pathogen infection. These results suggested that pathogen infection alters the chemical composition of tick exoskeleton by mechanisms still to be characterized and with tick species and exoskeleton region-specific differences., This research was partially supported by the Consejería de Educación, Cultura y Deportes, JCCM, Spain, project CCM17-PIC-036 (SBPLY/17/180501/000185).

Rhipicephalus spp. (Acari: Ixodidae) ticks are obligate hematophagous arthropods, which constitute a model for the study of vector-host interactions. The chemical composition or elementome of salivary glands (SG) and cement provides information relevant for the study of protein-based complex multifunctional tissues with a key role in tick biology. In this study, we characterized the elementome of cement cones in Rhipicephalus sanguineus collected from naturally infested dogs and in SG and cement of R. bursa collected from experimentally infested rabbits at different feeding stages. The elementome was characterized using scanning electron microscopy (SEM) combined with energy dispersive X-ray spectroscopy (EDS). The results showed the identification of up to 14 chemical elements in the cement, and suggested tick/host-driven differences in the cement elementome between tick species and between SG and cement within the same species. By still unknown mechanisms, ticks may regulate cement elementome during feeding to affect various biological processes. Although these analyses are preliminary, the results suggested that N is a key component of the cement elementome with a likely origin in SG/salivary proteins (i.e., Glycine (C2H5NO2)-rich superfamily member proteins; GRPs) and other tick/host-derived components (i.e. NAPDH). Future research should be focused on tick elementome and its functional implications to better understand cement structure and function., This work was supported by the Consejería de Educación, Cultura y Deportes, JCCM, Spain, project CCM17-PIC-036 (SBPLY/17/180501/000185), and partially funded by Fundação para a Ciênciae Tecnologia (FCT) under the project PTDC/CVT-CVT/29073/2017 (UID/Multi/04413/2013). Margarita Villar was supported by the University of Castilla La 309 Mancha, UCLM, Spain, and the Fondo Europeo de Desarrollo Regional, FEDER, EU.

Humans evolved by losing the capacity to synthesize the glycan Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal), which resulted in the capacity to develop a protective response mediated by anti-α-Gal IgM/IgG antibodies against pathogens containing this modification on membrane proteins. As an evolutionary trade-off, humans can develop the alpha-Gal syndrome (AGS), a recently diagnosed disease mainly associated with allergic reactions to mammalian meat consumption. The etiology of the AGS is the exposure to tick bites and the IgE antibody response against α-Gal-containing glycoproteins and glycolipids. The objective of this study was to characterize the anti-α-Gal antibody response in association with the immune-mediated peripheral neuropathy, Guillain-Barré syndrome (GBS), and compare it with different factors known to modulate the antibody response to α-Gal such as exposure to tick bites and development of allergic reactions in response to tick bites. The results showed a significant decrease in the IgM/IgG response to α-Gal in GBS patients when compared to healthy individuals. In contrast, the IgM/IgG levels to α-Gal did not change in patients with allergic reactions to tick bites. The IgE response was not affected in GBS patients, but as expected, the IgE levels significantly increased in individuals exposed to tick bites and patients with tick-associated allergies. These results suggest that the immune pathways of anti-α-Gal IgM/IgG and IgE production are independent. Further studies should consider the susceptibility to allergic reactions to tick bites in GBS patients., This work was supported by the Consejería de Educación, Cultura y Deportes, JCCM, Spain, project CCM17-PIC-036 (SBPLY/17/180501/000185). MV was supported by the University of Castilla La Mancha (UCLM, Spain) and the Fondo Europeo de Desarrollo Regional, FEDER, EU. IGFM was supported by the UCLM. MC was funded by the Ministerio de Ciencia, Innovación y Universidades, Spain (grant FJC-2018-038277-I).

Animal tuberculosis (TB), which is caused by the Mycobacterium tuberculosis complex (MTC), is a zoonotic disease of global concern, and has a wide variety of wild and domestic reservoirs that can establish complex epidemiological systems. Of all the strategies employed to control TB, reducing the risks of interaction at the wildlife-livestock interface is a cornerstone. However, detailed protocols with which to assess and implement farm-specific preventive actions that can be employed against interactions with wildlife are lacking for extensive production systems. We describe an On-farm Wildlife Risk Mitigation Protocol that is applicable to beef cattle farming in Mediterranean environments in order to control the wildlife-livestock interaction and MTC transmission through the use of Farm-specific Action Plans (FsAP). We assessed the implementation and verification of FsAP in terms of its practical feasibility and acceptability by farmers (n=55 farms). Of the potential risk points, waterers (41.3 %) and waterholes (24.4 %) were the most common. Waterholes and springs were identified as the points with the greatest risks. Actions related to water management were essential on most farms (99 % of the high-risk points), as were those regarding wildlife management (36.4 % of the farms provided wild boar or cervids with supplementary food for hunting purposes). Overall, 75 % of the farmers adopted the plans to some extent, with an average of 31.8 % of actions implemented, but with high variability depending on the type of actions proposed. Farmers prioritised low-cost measures. Our results, in their entirety, indicate that the adoption of this On-farm Wildlife Risk Mitigation Protocol is practical and feasible in Mediterranean ecosystems, and can be easily transferred to professionals and adapted to other bioregions or epidemiological systems. The subsequent evaluation of FsAPs in terms of efficacy and cost-effectiveness, along with increasing their acceptance by farmers, are necessary steps for the further development of TB Risk Mitigation Programmes at a nationwide level., Funding was provided by projects AGL2013-48523-C3-1-R and AGL2016-76358-R (MINECO; AEI/FEDER, UE). This is also a contribution to the EU FEADER PDR projects “Alcudia” and “GOSTU” on farm biosafety. JMG holds a FPI pre-doctoral scholarship (BES-2015- 072206, MINECO). PA is supported by an extension of his “Ramón y Cajal” contract (RYC-2012-11970, MINECO-UCLM). SJR holds a PhD contract from the UCLM co-supported by the European Social Fund(2018/12504). JAB is supported by the European Union project (H2020 VACDIVA 862874).

This article belongs to the Special Issue Ectoparasite Biogenic Components in the Modulation of Vector-host-pathogen Interactions., Ticks are obligate hematophagous arthropods and vectors of pathogens affecting human and animal health worldwide. Cement is a complex protein polymerization substance secreted by ticks with antimicrobial properties and a possible role in host attachment, sealing the feeding lesion, facilitating feeding and pathogen transmission, and protection from host immune and inflammatory responses. The biochemical properties of tick cement during feeding have not been fully characterized. In this study, we characterized the proteome of Rhipicephalus microplus salivary glands (sialome) and cement (cementome) together with their physicochemical properties at different adult female parasitic stages. The results showed the combination of tick and host derived proteins and other biomolecules such as α-Gal in cement composition, which varied during the feeding process. We propose that these compounds may synergize in cement formation, solidification and maintenance to facilitate attachment, feeding, interference with host immune response and detachment. These results advanced our knowledge of the complex tick cement composition and suggested that tick and host derived compounds modulate cement properties throughout tick feeding., This research was funded by the Consejería de Educación, Cultura y Deportes, JCCM, Spain, project CCM17-PIC-036 (SBPLY/17/180501/000185). M.V. was supported by the University of Castilla La Mancha, UCLM, Spain, and the Fondo Europeo de Desarrollo Regional, FEDER, EU. I.G.F.M. was supported by the University of Castilla La Mancha, UCLM, Spain.

The poultry red mite (PRM), Dermanyssus gallinae, is a hematophagous ectoparasite considered as the major pest in the egg-laying industry. Its pesticide-based control is only partially successful and requires the development of new control interventions such as vaccines. In this study, we follow a vaccinology approach to identify PRM candidate protective antigens. Based on proteomic data from fed and unfed nymph and adult mites, we selected a novel PRM protein, calumenin (Deg-CALU), which is tested as a vaccine candidate on an on-hen trial. Rhipicephalus microplus Subolesin (Rhm-SUB) was chosen as a positive control. Deg-CALU and Rhm-SUB reduced the mite oviposition by 35 and 44%, respectively. These results support Deg-CALU and Rhm-SUB as candidate protective antigens for the PRM control., This work was partially supported by the European Cooperation in Science and Technology (COST), Improving current understanding and research for sustainable control of the poultry red mite Dermanyssus gallinae – COREMI project (Action FA1404). JFLB was supported by Ministerio de Ciencia, Innovación y Universidades (Spain), Doctorado Industrial contract (DI-14-06917) and Sabiotec SA. MV was supported by the University of Castilla- La Mancha (Spain).

Blood-feeding ectoparasites constitute a growing burden for human and animal health, and animal production worldwide. In particular, mites (Acari: Gamasida) of the genera Dermanyssus (Dermanyssidae) and Ornithonyssus (Macronyssidae) infest birds and cause gamasoidosis in humans. The tropical fowl mite, Ornithonyssus bursa, is commonly found in tropical and subtropical countries but rarely reported in Europe. In this research we characterized the first two cases in Spain of clinical gamasoidosis diagnosed in patients infested with O. bursa, and investigated the IgE, IgM and IgG antibody response to mite proteins and the carbohydrate Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) involved in the tick-bite associated alpha-Gal syndrome (AGS). The results suggested that O. bursa is establishing across Mediterranean countries, and may increase the risk for gamasoidosis. The immune antibody response to mite proteins was higher for IgM and similar for IgE and IgG antibodies between patients and non-allergic control individuals exposed to mite or tick bites. The anti-α-Gal antibody levels were similar between patients and controls, a result supported by the absence of this carbohydrate in mites. These results suggested that mite bites do not correlate with antibody response to acarine proteins or α-Gal, and are not associated with the AGS., This research was supported by the Consejería de Educación, Cultura y Deportes, JCCM, Spain, project CCM17-PIC-036 (SBPLY/17/180501/000185). JFLB was supported by Doctorado Industrial from Ministerio de Ciencia, Innovación y Universidades, Spain (contract DI-14-06917) and Sabiotec S.A., Spain. MV was funded by the Universidad de Castilla La Mancha, Spain.

The poultry red mite (PRM)

Dermanyssus gallinae, the poultry red mite, is a global threat to the commercial egg-laying industry. Control of D. gallinae is difficult, with only a limited number of effective pesticides and non-chemical treatments available. Here, we characterize the candidate vaccine antigen D. gallinae cathepsin D-1 (Dg-CatD-1) and demonstrate that purified refolded recombinant Dg-Cat-D1 (rDg-CatD-1) is an active aspartyl proteinase which digests haemoglobin with a pH optimum of pH 4. Soluble protein extracts from D. gallinae also have haemoglobinase activity, with a pH optimum comparable to the recombinant protein, and both proteinase activities were inhibited by the aspartyl proteinase inhibitor Pepstatin A. Enzyme activity and the ubiquitous localization of Dg-CatD-1 protein in sections of adult female mites is consistent with Dg-CatD-1 being a lysosomal proteinase. Using Dg-CatD-1 as a model vaccine antigen, we compared vaccine delivery methods in laying hens via vaccination with: (i) purified rDg-CatD-1 with Montanide™ ISA 71 VG adjuvant; (ii) recombinant DNA vaccines for expression of rDg-CatD-1 and (iii) transgenic coccidial parasite Eimeria tenella expressing rDg-CatD-1. In two independent trials, only birds vaccinated with rDg-CatD-1 with Montanide™ ISA 71 VG produced a strong and long-lasting serum anti-rDg-Cat-D1 IgY response, which was significantly higher than that in control birds vaccinated with adjuvant only. Furthermore, we showed that egg-laying rates of D. gallinae mites fed on birds vaccinated with rDg-CatD-1 in Montanide™ ISA 71 VG was reduced significantly compared with mites fed on unvaccinated birds., This work was supported by the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No 635408 (“PARAGONE: vaccines for animal parasites”).This article, which is included in the ‘Research update on Dermanyssus gallinae, the poultry red mite’ supplement, is sponsored and supported by Coventry University.

More than 70% of new human pathogens are zoonotic and many originate from the wildlife reservoir. Wildlife rehabilitation centres (WRC) are an easily accessible source for sample and data collection for preventive surveillance, but data collected this way may be biased. We use white storks (Ciconia ciconia) as a model to compare pathogen prevalence obtained in the field and WRC. We address factors that may affect disease prevalence data like origin, the age group and the “diseased” state of WRC admissions. In this study we compared prevalence of Escherichia coli and Salmonella spp. in the digestive tract; antibodies against West Nile virus, avian influenza and Newcastle disease virus, and antimicrobial resistance patterns of E. coli between nestling and adult wild storks established in different habitats (n = 90) and storks admitted to two different WRC (n = 30) in the same region. When age groups and colonies of origin were disregarded, the mean enterobacteria (E. coli, Salmonella) and viral antibody prevalence of the wild population (n = 90) were similar to prevalence observed in the individuals admitted to WRC (n = 30). However, in fledgling juvenile storks admitted to WRC, the prevalence of Salmonella spp. (13.3%), E. coli showing resistance to cefotaxime (37.9%) and against two antimicrobials at once (41.4%) were more similar to the prevalence in stork nestlings from landfill-associated colonies (7.9%, 37.1% and 48.6%, respectively for prevalence of Salmonella spp. and E. coli displaying, cefotaxime resistance and resistance against two antimicrobials), and significantly higher than in colonies located in natural habitats (0%; 10.5% and 15.8%, respectively). Thus, pathogen surveillance in individuals from an abundant species admitted to WRC is useful to monitor overall mean prevalence, but for certain pathogens may not be sufficient to detect differences between local populations. In addition, the ecology of the tested species and the specific temporal, spatial and age group distribution of WRC admissions have to be taken into account., This work was funded by the projects: RTA2011-00111-C03-02 and ERTA2013-0013 funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, INIA. María Cruz Camacho is a fellow of the Junta de Comunidades de Castilla – La Mancha (JCCM).

[EN]: There is a strong relationship between bird wing morphology and flight style. Wing shapes are related to manoeuvrability, flight speed, energetic costs during flight and take-off speed. Wing shape differences among species have been frequently studied but differences can also be found within species, between sexes and age groups. Many studies have assessed differences in wing shape between juveniles and adults in different passerine species but little is known about such differences in other bird orders. Performing a Size Constrained Components Analysis (SCCA) and a graphical approximation, we analysed the wing shape of juveniles and adults of eight species, including four passerines and four non-passerines of three different orders. According to a graphical approximation, we observed that wing shape differences between age groups differ among species. In the non-passerine species considered, juveniles have more pointed and concave wings than adults. In contrast, in the four passerine species, juveniles have more rounded wings than adults. However, the results for the C2 axis of SCCA (index of pointedness) do not completely agree with the graphical approximation. Our results showed that the C2 axis does not represent the same vector of wing shape variation in all species. The contribution of changes in C2 to variation in wing pointedness seemed to depend on the position of the wing tip, which is a good index of pointedness only when the wing tip is in the two most distal primary feathers. Surprisingly, the adults of some species do not have longer wings than the corresponding juveniles, because the feathers that define the wing tip do not grow longer during the first complete moult. We discuss the role of the first complete moult in changing the shape of juvenile wings into adult wings, and the implications of our results for the analysis of bird wing shape. [ES]: Existe una fuerte relación entre la morfología alar y el estilo de vuelo de un ave. La forma del ala está relacionada con la maniobrabilidad, la velocidad de vuelo, los costes energéticos durante el vuelo o la velocidad de despegue. Las diferencias en la forma del ala entre especies han sido estudiadas con frecuencia, pero también existen diferencias dentro de una especie, entre sexos y grupos de edad. Muchos estudios han evaluado las diferencias en la forma alar entre juveniles y adultos en diferentes especies de paseriformes, pero se sabe poco acerca de las diferencias en la forma alar entre grupos de edad en otros órdenes de aves. Realizando un análisis SCCA (‘Size Constrained Components Analysis’) y una aproximación gráfica, analizamos la forma alar de juveniles y adultos de ocho especies, cuatro paseriformes y cuatro no paseriformes de tres órdenes diferentes. De acuerdo con la aproximación gráfica, observamos que las diferencias en la forma alar entre grupos de edad difieren entre especies. En las especies de no paseriformes estudiadas, los juveniles tienen alas más puntiagudas y cóncavas que los adultos. En contraste, en las cuatro especies de paseriformes, los juveniles tienen alas más redondeadas que los adultos. Sin embargo, los resultados del eje C2 del SCCA no concuerdan completamente con la aproximación gráfica. nuestros resultados mostraron que el eje C2 no representa el mismo vector de variación de la forma del ala en todas las especies. La contribución de los cambios en C2 a la variación del apuntamiento del ala parece depender de la posición de la punta del ala, siendo un buen índice de apuntamiento solo cuando la punta del ala está en las dos plumas primarias más distales. Sorprendentemente, los adultos de algunas especies no tienen alas más largas que los respectivos juveniles, debido a que la pluma que define la punta del ala no tiene un incremento de tamaño durante la primera muda completa. Describimos cómo la primera muda completa es clave en el cambio de la morfología alar entre el ala de los juveniles y el ala de los adultos, y discutimos las implicaciones de nuestros resultados en relación con el análisis de la forma de las alas de las aves. Xabier Cabodevilla was supported by a PhD Grant, financed by the Basque Country Government (Grant no. PRE_2018_2_0273).

The poultry red mite (PRM), Dermanyssus gallinae (De Geer, 1778), is a worldwide distributed ectoparasite and considered a major pest affecting the laying hen industry in Europe. Based on available information in other ectoparasites, the mite microbiome might participate in several biological processes and the acquisition, maintenance and transmission of pathogens. However, little is known about the role of PRM as a mechanical carrier or a biological vector in the transmission of pathogenic bacteria. Herein, we used a metaproteomics approach to characterize the alphaproteobacteria in the microbiota of PRM, and variations in its profile with ectoparasite development (nymphs vs. adults) and feeding (unfed vs. fed). The results showed that the bacterial community associated with D. gallinae was mainly composed of environmental and commensal bacteria. Putative symbiotic bacteria of the genera Wolbachia, C. Tokpelaia and Sphingomonas were identified, together with potential pathogenic bacteria of the genera Inquilinus, Neorickettsia and Roseomonas. Significant differences in the composition of alphaproteobacterial microbiota were associated with mite development and feeding, suggesting that bacteria have functional implications in metabolic pathways associated with blood feeding. These results support the use of metaproteomics for the characterization of alphaproteobacteria associated with the D. gallinae microbiota that could provide relevant information for the understanding of mite-host interactions and the development of potential control interventions., This work was partially supported by the European Cooperation in Science and Technology (COST), Improving Current Understanding and Research for Sustainable Control of the Poultry Red Mite Dermanyssus Gallinae–COREMI Project (Action FA 1404) (https://www.coremi.eu/home.html). JFLB was supported by Doctorado Industrial contract (DI-14-06917) from Ministerio de Ciencia, Innovación y Universidades (Spain) and Sabiotec S. A. MV was supported by the University of Castilla-La Mancha (Spain).

Because of its ability to expedite specimen identification and species delineation, the barcode index number (BIN) system presents a powerful tool to characterize hyperdiverse invertebrate groups such as the Acari (mites). However, the congruence between BINs and morphologically recognized species has seen limited testing in this taxon. We therefore apply this method towards the development of a barcode reference library for soil, poultry litter, and nest dwelling mites in the Western Palearctic. Through analysis of over 600 specimens, we provide DNA barcode coverage for 35 described species and 70 molecular taxonomic units (BINs). Nearly 80% of the species were accurately identified through this method, but just 60% perfectly matched (1:1) with BINs. High intraspecific divergences were found in 34% of the species examined and likely reflect cryptic diversity, highlighting the need for revision in these taxa. These findings provide a valuable resource for integrative pest management, but also highlight the importance of integrating morphological and molecular methods for fine-scale taxonomic resolution in poorly-known invertebrate lineages., The study was carried out within the framework of the European Cooperation in Science and Technology (COST) Action (FA1404-COREMI) ‘Improving current understanding and research for sustainable control of the poultry red mite Dermanyssus gallinae’.

Objectives: The objective of this study was to analyse the prevalence of tracheal carriage of Staphylococcus aureus/MRSA in storks and to study the resistance and virulence genes in the obtained isolates. Methods: Tracheal samples from 92 stork nestlings of two landfill-associated and two natural-habitat colonies were inoculated in specific media for S. aureus and MRSA recovery. Antimicrobial susceptibility was tested, and the presence of resistance, virulence and immune evasion cluster (IEC) genes was analysed by PCR. S. aureus isolates were characterized by spa and agr typing. Staphylococcal cassette chromosome (SCC) mec type was determined for mecC-positive isolates, and MLST was performed for 17 selected S. aureus isolates. Results: S. aureus isolateswere identified in 32/92 samples (34.8%), and 38 isolateswere recovered. The prevalence of S. aureus was higher in nestlings from landfills (24/43, 55.8%) than in those fromnatural habitats (8/49, 16.3%). Three birds from landfill-associated colonies carried MRSA, two with mecA-positive strains [clonal complex (CC) 5-spa-t002 and CC398-spa-t011] and one with a mecC-positive strain [sequence type (ST) 3061-CC130-spat843- agr-III-SCCmecXI). None of the MRSA isolates presented IEC genes. Thirty-five MSSA isolates, which showed 18 different spa types (ascribed to CC5, CC7, CC22, CC30, CC45, CC59, CC133 and CC398), were obtained. The agr types detected were I (63%), II (29%) and III (8%). Resistance and virulence genes identified in MSSA were blaZ (n=25), erm(T) (n=9), erm(A) (n=1), tet(M) (n=2), fexA (n=3), str (n=2), tst (n=2), eta (n=1) and cna (n=15). The IEC types B, C, D and G were found in MSSA isolates, and two new STs were identified (ST3060 and ST3061). Conclusions: White storks are frequently tracheal carriers of S. aureus, including ST398 isolates. MRSA isolates of lineages CC398-mecA and CC130-mecC were detected in storks from landfill-associated colonies exposed to human residues. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

The poultry red mite (PRM), Dermanyssus gallinae, is a hematophagous ectoparasite of birds with worldwide distribution that causes economic losses in the egg-production sector of the poultry industry. Traditional control methods, mainly based on acaricides, have been only partially successful, and new vaccine-based interventions are required for the control of PRM. Vaccination with insect Akirin (AKR) and its homolog in ticks, Subolesin (SUB), have shown protective efficacy for the control of ectoparasite infestations and pathogen infection/transmission. The aim of this study was the identification of the akr gene from D. gallinae (Deg-akr), the production of the recombinant Deg-AKR protein, and evaluation of its efficacy as a vaccine candidate for the control of PRM. The anti-Deg-AKR serum IgY antibodies in hen sera and egg yolk were higher in vaccinated than control animals throughout the experiment. The results demonstrated the efficacy of the vaccination with Deg-AKR for the control of PRM by reducing mite oviposition by 42% following feeding on vaccinated hens. A negative correlation between the levels of serum anti-Deg-AKR IgY and mite oviposition was obtained. These results support Deg-AKR as a candidate protective antigen for the control of PRM population growth., This work was partially supported by COST Action FA 1404 (COREMI) (https://www.coremi.eu/home.html). JFLB was supported by Doctorado Industrial contract DI-14-06917 from Ministerio de Ciencia, Innovación y Universidades (Spain) and Sabiotec S.A. MV was supported by the Research Plan of the University of Castilla-La Mancha (Spain).

There is a surprisingly scarce amount of epidemiological and molecular data on the prevalence, frequency, and diversity of the intestinal protozoan parasites Giardia duodenalis and Cryptosporidium spp. in wildlife in general and mesocarnivore species in particular. Consequently, the extent of the cyst/oocyst environmental contamination attributable to these wild host species and their potential implications for public veterinary health remain largely unknown. In this molecular epidemiological survey a total of 193 individual faecal samples from badgers (Meles meles, n = 70), ferrets (Mustela putorius furo, n = 2), genets (Genetta genetta, n = 6), Iberian lynxes (Lynx pardinus, n = 6), beech martens (Martes foina, n = 8), mongooses (Herpestes ichneumon, n = 2), otters (Lutra lutra, n = 2), polecats (Mustela putorius, n = 2), red foxes (Vulpes vulpes, n = 87), wildcats (Felis silvestris, n = 2), and wolves (Canis lupus, n = 6) were obtained from road-killed, hunted, and accidentally found carcasses, and from camera-trap surveys or animals entering rescue shelters, during the period December 2003–April 2016. Investigated specimens were collected in five Spanish autonomous regions including Andalusia (n = 1), Asturias (n = 69), Basque Country (n = 49), Castile-La Mancha (n = 38), and Extremadura (n = 36). The presence of cysts/oocysts was confirmed by PCR-based methods targeting the small subunit (ssu) ribosomal RNA gene of these parasite species. Genotyping of the obtained isolates were attempted at appropriate markers including the glutamate dehydrogenase (G. duodenalis) and the 60-kDa glycoprotein (C. parvum and C. ubiquitum) loci. Overall, G. duodenalis was detected in 8% (7/87) of red foxes, a single beech marten, and a single wolf, respectively. Cryptosporidium was identified in 3% (2/70) of badgers, 8% (7/87) of red foxes, a single genet, and a single mongoose, respectively. None of the nine G. duodenalis isolates generated could be genotyped at the assemblage/sub-assemblage level. Out of the nine Cryptosporidium isolates successfully characterized, three were identified as C. canis (one in a mongoose and two in red foxes), and three as C. parvum (one in a badger and three in red foxes). The remaining three isolates were assigned to C. felis (in a red fox), C. hominis (in a badger), and C. ubiquitum (in a red fox), respectively. Two additional Cryptosporidium isolates infecting a badger and a genet, respectively, were untypable. The red fox was confirmed as a suitable host of potentially zoonotic Cryptosporidium species, mainly C. parvum and C. ubiquitum. The high mobility and wide home range of red foxes, together with their increasing presence in urban and peri-urban settings, may led to the overlapping of sylvatic and domestic cycles of the parasite, and consequently, to an increased risk of cryptosporidiosis in production animals and humans. The detection of C. hominis oocysts in a badger raises the question of whether this finding represents a true infection or a sporadic event of mechanical passage of C. hominis oocyst of anthroponotic origin., This study was funded by the Health Institute Carlos III, Spanish Ministry of Economy and Competitiveness under project CP12/03081. Additional funding was obtained from INIA under project RTA2014-00002-C02 (FEDER co-funded) and the Alfonso X El Sabio/Santander Foundation (Spain).

West Nile virus (WNV), a zoonotic pathogen naturally transmitted by mosquitoes whose natural hosts are birds, has spread worldwide during the last few decades. Resident birds play an important role in flavivirus epidemiology, since they can serve as reservoirs and facilitate overwintering of the virus. Herein, we report the first experimental infection of magpie (Pica pica) with two strains of West Nile virus, lineages 1 (NY-99) and 2 (SRB Novi-Sad/12), which are currently circulating in Europe. Magpies were highly susceptible to WNV infection, with similar low survival rates (30% and 42.8%) for both lineages. All infected magpies developed viremia detectable at 3 days post-infection with titers above those necessary for successful transmission of WNV to a mosquito. Neutralizing antibodies were detected at all time points analyzed (from 7 to 17 days post-infection). WNV genome was detected in the brains and hearts of all magpies that succumbed to the infection, and, in some of the surviving birds. WNV-RNA was amplified from swabs (oral and cloacal) at 3, 6 and 7 days post-infection and feather pulps, from 3 to 17 days post-infection, of infected animals. Even more, infectious virus was recovered from swabs up to 7 days post-infection and from feather pulps up to 10 days post infection. Sham-infected control animals were negative for viremia, viral RNA, and antibodies. These results suggest that the magpie, which is one of the most abundant corvid species in Europe, could represent a source of WNV transmission for birds and humans. Our observations shed light on the pathogenesis, transmission, and ecology of WNV and can benefit the implementation of surveillance and control programs., This work was supported by grants Recursos y Tecnologías Agrarias (E-RTA2013- 00013-C04, RTA2015-009, and E-RTA2017-00003-C02) from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA); and by grant P2013/ABI-2906 (PLATESA) from Comunidad Autónoma de Madrid.

The objectives were to evaluate the presence of vancomycin-resistant enterococci with acquired (VRE-a) and intrinsic (VRE-i) resistance mechanisms in fecal samples from different wild animals, and analyze their phenotypes and genotypes of antimicrobial resistance. A total of 348 cloacal/rectal samples from red-legged partridges (127), white storks (81), red kites (59), and wild boars (81) (June 2014/February 2015) were inoculated in Slanetz-Bartley agar supplemented with vancomycin (4 μg/mL). We investigated the susceptibility to 12 antimicrobials and the presence of 19 antimicrobial resistance and five virulence genes. In addition, we performed multilocus sequence typing, detection of IS16 and studied Tn1546 structure. One VRE-a isolate was identified in one wild boar. This isolate was identified as Enterococcus faecium, harbored vanA gene included into Tn1546 (truncated with IS1542/IS1216), and belonged to the new ST993. This isolate contained the erm(A), erm(B), tet(M), dfrG, and dfrK genes. Neither element IS16 nor the studied virulence genes were detected. Ninety-six VRE-i isolates were identified (89 Enterococcus gallinarum and seven Enterococcus casseliflavus), with the following prevalence: red kites (71.2 %), white storks (46.9 %), red-legged partridges (7.9 %), and wild boars (4.9 %). Most E. gallinarum isolates showed resistance to tetracycline (66.3 %) and/or erythromycin (46.1 %). High-level resistance to aminoglycosides was present among our VRE-i isolates: kanamycin (22.9 %), streptomycin (11.5 %), and gentamicin (9.4 %). In general, VRE-i isolates of red kites showed higher rates of resistance for non-glycopeptide agents than those of other animal species. The dissemination of acquired resistance mechanisms in natural environments could have implications in the global spread of resistance with public health implications., This work was supported by Project SAF2012-35474 from the Ministerio de Economía y Competitividad (MINECO) of Spain and the Fondo Europeo de Desarrollo Regional (FEDER), the project POIC-2014-001-P of the regional government of Castilla–La Mancha, and by the project RTA2011-00111-C03-02 from the National Institute for Research in Agricultural and Alimentary Technology (INIA), by CDTI (Centro para el Desarrollo Tecnológico Industrial, MINECO). Carmen Lozano has a contract associated with Project SAF2012-35474.

Orally delivered mycobacterial antigens may not sensitize the immunized animals causing a positive tuberculin skin test response. As the first step to address this critical issue, we characterized the response of farmed red deer (Cervus elaphus) to orally delivered heat-inactivated Mycobacterium bovis. Thirty-two adult red deer hinds from a farm known to be free of tuberculosis (TB) were randomly assigned to two different treatment groups, immunized (n = 24) and control (n = 8). Immunized hinds were dosed orally with 2 ml of PBS containing 6 × 10 heat-inactivated M. bovis. The mean skin test response of immunized deer to both avian purified protein derivative (aPPD) and bovine PPD (bPPD) was consistently lower in immunized than in control hinds. One year after immunization, immunized hinds had a significant reduction in the skin test response to aPPD and in the ELISA antibody levels against both aPPD and bPPD (24-36% reduction; P < 0.05). By contrast, no significant change was observed in the skin test response to phytohaemagglutinin, or in the ELISA antibody levels against the M. bovis specific antigen MPB70. The mRNA levels for C3, IFN-γ and IL-1β and serum protein levels for IFN-γ and IL-1β did not vary between immunized and control deer. However, serum C3 protein levels were significantly higher (P = 0.001) in immunized than in control deer six months after immunization. These results confirm that oral heat-inactivated M. bovis does not sensitize farmed red deer and therefore does not cause false-positive responses in the tuberculin skin test. The absence of sensitization in orally immunized deer opens the possibility of testing the vaccine in deer and possibly other ruminants without the risk of causing false-positive reactions in TB-tests. This study also provided the first evidence that orally-delivered inactivated mycobacterial antigens elicit some kind of immune response in a ruminant., Research funding is acknowledged to Plan Nacional grant AGL2014-56305 (MINECO, Spain and FEDER) and the EU FP7 grants WildTBvac #613779 and ANTIGONE #278976. VL was supported by a grant from Universidad Autónoma de Sinaloa, Mexico.