First description of Sarcocystis species infecting Barbary sheep (Ammotragus lervia).

Barbary sheep (Ammotragus lervia) is a North African native wild Caprinae, introduced in the 1970s in new territories such as Spain, the USA, and Mexico. Here, we describe Sarcocystis species in Barbary sheep. Sarcocysts were found in 19 out of 56 adult A. lervia in Southern Spain and characterized morphologically and molecularly. By light microscopy, sarcocysts had thin (< 1 µm) or thick (> 2 µm) walls. By transmission electron microscopy, sarcocysts with thick walls had Type 14 villar protrusions corresponding to S. tenella/S. capracanis of domestic sheep (Ovis aries) or goats (Capra hircus). Sarcocysts with thin walls had Type 7b villar protrusions that corresponded to S. arieticanis/S. hircicanis of domestic sheep or goats. Molecular analyses allowed the identification of only thick-walled Sarcocystis species. Six sarcocysts were assigned to S. tenella (99.2-100% and 95.6-100% sequence similarity within 18S rRNA and COI, respectively) and 19 sarcocysts were assigned to S. capracanis (98.5-99.8% and 97.9-99.0% sequence similarity within 18S rRNA and COI, respectively). Further studies are needed for taxonomic identification of sarcocysts in Barbary sheep because Sarcocystis species in sheep and goats are not cross transmissible despite morphological similarities.

Molecular survey of Besnoitia spp. (Apicomplexa) in faeces from European wild mesocarnivores in Spain.

Numerous studies have unsuccessfully tried to unravel the definitive host of the coccidian parasite Besnoitia besnoiti. Cattle infections by B. besnoiti cause a chronic and debilitating condition called bovine besnoitiosis that has emerged in Europe during the last two decades, mainly due to limitations in its control associated with the absence of vaccines and therapeutical tools. Although the exact transmission pathways of B. besnoiti is currently unknown, it is assumed that the parasite might have an indirect life cycle with a carnivore as definitive host. Current lack of studies in wildlife might underestimate the importance of free-living species in the epidemiology of B. besnoiti. Thus, the aim of the present study is to assess the presence of Besnoitia spp. in free-ranging mesocarnivores in Spain. DNA was searched by PCR on faeces collected from wild carnivores as a first approach to determine which species could be considered as potential definitive host candidates in further research. For this purpose, a total of 352 faecal samples from 12 free-living wild carnivore species belonging to the Canidae, Felidae, Herpestidae, Mustelidae, Procyonidae and Viverridae families were collected in seven Spanish regions. PCR testing showed that Besnoitia spp. DNA was present in four faecal samples from red foxes collected in western Spain, an area with the greatest density of extensively reared cattle and associated with high incidence of bovine besnoitiosis in the country. To date, this is the first report of a B. besnoiti-like sequence (99.57% homology) from carnivore faeces in a worldwide context. Red foxes might contribute to the epidemiology of B. besnoiti, although further studies, mostly based on bioassay, would be needed to elucidate the accuracy and extent of these interesting findings.

Parasites of the Reintroduced Iberian Lynx (Lynx pardinus) and Sympatric Mesocarnivores in Extremadura, Spain.

The Iberian lynx (Lynx pardinus) is one of the most endangered felid species in the world. Conservation efforts have increased its population size and distribution and reinforced their genetic diversity through captive breeding and reintroduction programmes. Among several threats that the Iberian lynx faces, infectious and parasitic diseases have underlined effects on the health of their newly reintroduced populations, being essential to identify the primary sources of these agents and assess populations health status. To achieve this, 79 fresh faecal samples from Iberian lynx and sympatric mesocarnivores were collected in the reintroduction area of Extremadura, Spain. Samples were submitted to copromicroscopic analyses to assess parasite diversity, prevalence, and mean intensity of parasite burden. Overall, 19 (24.1%, ±15.1-35.0) samples were positive for at least one enteric parasite species. Parasite diversity and prevalence were higher in the Iberian lynx (43.8%) compared with the others mesocarnivores under study (e.g., the red fox Vulpes vulpes and the Egyptian mongoose Herpestes ichneumon). Ancylostomatidae and Toxocara cati were the most prevalent (15.6%) parasites. Obtained results revealed that Iberian lynx role as predator control might have reduced parasite cross-transmission between this felid and mesocarnivores due to their decreasing abundances. Surveillance programs must include regular monitoring of this endangered felid, comprising mesocarnivores, but also domestic/feral and wild cat communities.

Microbial community of Hyalomma lusitanicum is dominated by Francisella-like endosymbiont.

Exploring tick associations with complex microbial communities and single-microbial partners, especially intracellular symbionts, has become crucial to understand tick biology. Of particular interest are the underlying interactions with biological consequences i.e. tick fitness, vector competence. In this study, we first sequenced the 16S rRNA bacterial phylogenetic marker in adult male ticks of Hyalomma lusitanicum collected from 5 locations in the province of Cáceres to explore the composition of its microbial community. Overall, 16S rRNA sequencing results demonstrated that the microbial community of H. lusitanicum is mostly dominated by Francisella-like endosymbionts (FLEs) (ranging from 52% to 99% of relative abundance) suggesting it is a key taxon within the microbial community and likely a primary endosymbiont. However, further research is required to explore the mechanisms underlying the interaction between FLEs and H. lusitanicum.

Detection of new Crimean-Congo haemorrhagic fever virus genotypes in ticks feeding on deer and wild boar, Spain.

Crimean-Congo haemorrhagic fever virus (CCHFV) is the causative agent of the severe tick-borne, often fatal, zoonotic Crimean-Congo haemorrhagic fever (CCHF), which is widely distributed worldwide. The CCHFV transmission to humans occurs through tick bite, crushing of engorged ticks or contact with infected host blood. Previously, CCHFV genotype Africa III was reported in Spain. Given the emergence of CCHF and the role of ticks in pathogen maintenance and transmission, we investigated the presence and genotype identity of the virus in tick species parasitizing abundant wild host species in south-western Spain. A total of 613 adult ticks were collected from hunter-harvested wild ungulates in twenty locations throughout south-western Spain. Ticks were identified, nucleic acids were extracted, RNA was analysed by a nested RT-PCR targeting CCHFV S segment, and the amplicons were sequenced. According to the 212-bp sequence amplified, the presence of CCHFV human genotype Europe V was detected in Hyalomma lusitanicum and Dermacentor marginatus ticks collected from red deer, fallow deer and Eurasian wild boar in different locations from south-western Spain. Genotype Africa IV was also detected in a H. lusitanicum tick collected from a red deer. The detection of CCHFV in different tick species collected from various wild hosts and localities provided strong evidence of widespread CCHFV presence in the region, suggesting that the circulation of the virus in Spain requires more attention. Additionally, the identification of the CCHFV genotype Europe V in ticks suggested that its introduction in Spain was probably from Eastern Europe.

Blastocystis sp. Subtype Diversity in Wild Carnivore Species from Spain.

The occurrence and molecular diversity of the stramenopile eukaryote Blastocystis sp. was investigated by PCR and sequencing (Sanger and NGS) methods in 380 faecal specimens of free-living carnivores in Spain. Blastocystis sp. was confirmed in 1.6% (6/380) of the specimens analysed. Two samples from a common genet and a fox were successfully subtyped as ST7 by Sanger. Using NGS, ST14 was found in a fox and a European polecat, ST7 in a fox, and two additional foxes presented mixed infections of ST1/ST2/ST4 and ST1/ST2/ST7, respectively. Wild carnivore species could act as carriers of zoonotic Blastocystis subtypes.

Sarcocystis morae (Apicomplexa) in Fallow Deer (Dama dama) from Spain: Ultrastructure and New Host Record.

Members of the genus Sarcocystis are frequently found infecting members of the family Cervidae. Although Sarcocystis species are generally host specific for their intermediate hosts, species in cervids appear to be less host specific. Here, we report fallow deer (Dama dama) as a new host for Sarcocystis morae, originally described from the red deer (Cervus elaphus). Tongues of 69 legally hunted animals in Spain were tested for sarcocysts, and the species were characterized by light microscopy, ultrastructurally and molecularly. Sarcocysts were identified in 66.7% of D. dama. Sarcocysts had thin (<2 µm thick) cyst wall with hair-like villar protrusions bifurcated at their tips resembling type 8a. Genetic sequences obtained for 18S rRNA and COI reached 99.6-100% and 97.9-98.7% similarity, respectively, to those of S. morae from the red deer. The present study provides new data concerning lower level of host specificity within Sarcocystis genus for cervids.

Genomic Characterization of Crimean-Congo Hemorrhagic Fever Virus in Hyalomma Tick from Spain, 2014.

Crimean-Congo hemorrhagic fever (CCHF) is a severe tick-borne disease caused by CCHF virus (CCHFV). Ticks in the genus Hyalomma are the main vectors and reservoirs of CCHFV. In Spain, CCHFV was first detected in Hyalomma ticks from Cáceres in 2010. Subsequently, two autochthonous CCHF cases were reported in August 2016. In this study, we describe the characterization of the CCHFV genome directly from Hyalomma lusitanicum collected in Cáceres in 2014. Phylogenetic analyses reveal a close relationship with clade III strains from West Africa, with an estimated divergence time of 50 years. The results of this work suggest that CCHFV has been circulating in Spain for some time, and most likely originated from West Africa.

Sarcoptes scabiei infestation does not alter the stability of ectoparasite communities.

BACKGROUND: The host represents a heterogeneous ecosystem where multiple parasite species co-occur and interact with each other for space and resources. Although these interactions may rule the features of an infracommunity and may shape the infracommunity response to external perturbations, the resilience of ectoparasite communities to new infestations remains poorly explored. METHODS: We analysed the composition of the ectoparasite communities found on 214 individual Iberian ibexes (Capra pyrenaica) inhabiting the Sierra Nevada Natural Space, southern Spain. Using classification and regression trees, we explored how the presence of Sarcoptes scabiei (a highly contagious mite), the off-host environment and the host sex govern the prevalence and abundance of lice and ticks. Null model analysis was applied to assess the impact of S. scabiei on the structure of the ectoparasite communities. RESULTS: Our results suggest that S. scabiei infestation acts in tandem with off-host environment and host sex to define the prevalence and abundance of lice and ticks. We also provided evidence for differences in species co-occurrence only at the early stages of S. scabiei infestation. Regarding species diversity, we recorded that ectoparasite communities in scabietic ibexes reached a high richness faster than those in healthy individuals. CONCLUSIONS: Even though we show that ectoparasite burden is correlated with S. scabiei infestation, off-host environment and host sex, the species response to S. scabiei infestation and climate seem to be highly variable and influenced by ectoparasite life-history traits. Ectoparasite communities also appear resilient to perturbations which is in agreement with what was previously reported for endoparasites. Future refinement of sample collection and the incorporation of ecological and epidemiological-related variables may allow us to establish causal effects and deepen the knowledge about the mechanisms and consequences of ectoparasite interactions.

Molecular characterization of bot flies, Oestrus spp., (Diptera, Oestridae), from domestic and wild Bovidae hosts.

The identification of Oestrus spp. larvae from Bovidae hosts is a difficult task due to the great morphological similarity between species. The lack of unambiguous identification criteria could have also serious epidemiological implications since domestic and wild hosts are sympatric in many natural areas. In order to accurately identify the Oestrus parasitizing hosts, we characterized two different genetic markers, 28S (rRNA) and COI, in larvae collected from domestic sheep and goats, European mouflon and Iberian ibex. Our sequence analyses demonstrate that all samples, except those from Iberian ibex, greatly resembles O. ovis and so we conclude that the species parasitizing this ibex is not O. ovis. Further studies will be needed to confirm whether it is in fact O. caucasicus, as previously suggested, or even a new species.

Prevalence and genotype identification of Toxoplasma gondii in wild animals from southwestern Spain.

We used PCR to detect Toxoplasma gondii in the principal game species in southwestern Spain. We detected T. gondii in 32.2% of animals tested. Prevalences varied from 14.7% in wild boar (Sus scrofa) to 51.2% in red fox (Vulpes vulpes). The most prevalent genotype was type II (50.0%), followed by type III (20.6%) and type I (5.9%). Mixed infections (11.8%) were detected in wild boar (types I+III) and red fox (types II+III). Polymorphic strains (11.8%) were detected in several species. The high prevalence and the genetic variability shown could have implications for infection of farm animals and humans.