West Nile and Usutu virus infections in wild birds admitted to rehabilitation centres in Extremadura, western Spain, 2017-2019.

West Nile virus (WNV) is an emerging flavivirus transmitted generally by mosquitoes of Culex genus. It is maintained in an enzootic life cycle where birds act as reservoir hosts. Humans and horses are also susceptible to infection, and occasionally, they suffer from neurological complications. However, they do not transmit the virus to other vectors, behaving as dead-end hosts. Sporadic WNV outbreaks observed in horses and wild birds from Extremadura (western Spain) during 2016 and 2017 seasons prompted to carry out this survey in wild birds, focused on specimens coming from two wildlife rehabilitation centres. Between October 2017 and December 2019, samples from 391 wild birds, belonging to 56 different species were collected and analysed in search of evidence of WNV infection. The analysis of serum samples for WNV-specific antibodies by ELISA, whose specificity was subsequently confirmed by virus-neutralisation test (VNT) showed positive results in 18.23 % birds belonging to 18 different species. Pelecaniformes (33.33 %), Accipitriformes (25.77 %) and Strigiformes (22.92 %) orders had the higher seroprevalences. Remarkably, WNV-specific antibodies were found in a black stork for the first time in Europe. Analysis by real time RT-PCR in symptomatic birds confirmed the presence of WNV lineage 1 RNA in griffon vulture and little owls. Specificity analysis of ELISA positive and doubtful sera was performed by differential VNT titration against WNV and two other cross-reacting avian flaviviruses found in Spain: Usutu virus (USUV) and Bagaza virus (BAGV). Only four samples showed USUV-specific antibodies (1.04 %) corresponding to three species: Eurasian eagle-owl, griffon vulture and great bustard (first detection in Europe) whereas no samples were found reactive to BAGV. Differential VNT yielded undetermined flavivirus result in 16 samples (4.17 %). This is the first study carried out on wild birds from Extremadura (western Spain). It highlights the widespread circulation of WNV in the region and its co-circulation with USUV.

The mosquito fauna of the western region of Spain with emphasis on ecological factors and the characterization of Culex pipiens forms.

This study updates the diversity, distribution, and seasonal trends of mosquitoes in a western region of Spain, assesses ecological determinants of Culex pipiens s.l., and determines form composition of Cx. pipiens s.s. POPULATIONS: A total of 1,495 mosquitoes of 16 species was collected during 2012-2013, of which Cx. pipiens s.l. and Cx. theileri were the most abundant. Five new records for An. maculipennis s.s., Orthopodomyia pulcripalpis, Aedes (Ochlerotatus) punctor, Cx. europaeus, and Cx. modestus were found for this region. Cx. pipiens density varied across weather and habitat patterns, correlating positively with high temperatures and with a preference for urbanized areas and rural areas within a proximity of ovine farms. Moreover, molecular identification by CQ11FL was performed in 467 Cx. pipiens s.s., detecting both pipiens (66%) and molestus (8.4%) forms coexisting in different habitats (urban, peri-urban and rural) aboveground with a high degree of hybridization (25.7%). The abundance of Cx. pipiens in urban areas and farms, with the presence of hybrids, may increase their capacity to act as bridge vectors for the transmission of arboviral infections. These data will be helpful for further implementation of entomological programs focused on risk assessment for arboviruses or other mosquito-borne pathogens.

Culex pipiens as a potential vector for transmission of Dirofilaria immitis and other unclassified Filarioidea in Southwest Spain.

Dirofilaria immitis is one of the most frequently detected mosquito-transmitted zoonotic filarioid nematode in mammals in Europe, being canine dirofilariosis a major animal health problem, endemic in the Mediterranean area. This study, focused on Southwest Spain, in order to bring new insights into (i) the epidemiology of Dirofilaria spp., (ii) the species of Culicid vectors possibly involved in their transmission and (iii) the genetic variability of those potential vectors. A total of 881 adult female mosquitoes from 11 different species, were captured during 2012-2013, and detection of filarioid DNA was attempted by PCR using specific primers (ITS-2 and COI), followed by DNA sequencing. In a single Culex pipiens specimen D. immitis DNA was detected both in the head-thorax and abdomen sections. Filarioid nematode DNA was also detected in eight additional Cx. pipiens specimens also in both the thorax and the abdomen, but analysis of sequence data did not allow unambiguous assignment of any of the obtained sequences to a previously defined species. All Cx. pipiens with filarioid DNA were individually analysed by CQ11 to discriminate between pipiens, molestus, and hybrid forms. Besides, rDNA ITS-2 sequence analysis revealed the presence of haplotype H1 and H2 of Cx. pipiens. To our knowledge this study revealed, for the first time in Spain, the occurrence of likely mature infection of D. immitis in Cx. pipiens, as well as with other yet uncharacterized nematodes, supporting its role as a potential vector of these filarids.

First molecular detection of Leishmania tarentolae-like DNA in Sergentomyia minuta in Spain.

Phlebotomine sand flies (Diptera, Psychodidae) are vectors of multiple Leishmania species, among which Leishmania infantum stands out as a being frequently pathogenic to humans and dogs in Mediterranean countries. In this study, Sergentomyia minuta sand flies were collected using CDC miniature light traps in different 431 biotopes from Southwest Spain. A total of 114 females were tested for the presence of Leishmania DNA by targeting ITS-1 and cyt-B sequences by PCR. Leishmania DNA was detected in one S. minuta. Characterization of the obtained DNA sequences by phylogenetic analyses revealed close relatedness with Leishmania tarentolae Wenyon, 1921 as well as with both human and canine pathogenic strains of Asian origin (China), previously described as Leishmania sp. To our knowledge, this is the first report of phlebotomine sand flies naturally infected with L. tarentolae-like in Spain. The possible infection of sand flies with novel Leishmania species should be taken into consideration in epidemiological studies of vector species in areas where leishmaniosis is endemic.

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.

First report of Thelazia callipaeda in wildlife from Spain.

We describe the first cases of infection by the nematode, Thelazia callipaeda (Spirurida, Thelaziidae) haplotype 1 in two red foxes (Vulpes vulpes) in Spain and discuss the potential role of red foxes as a reservoir for T. callipaeda.

Congenital toxoplasmosis in wild boar (Sus scrofa) and identification of the Toxoplasma gondii types involved.

Congenital toxoplasmosis has been little described in wild animals. We report a case of vertical transmission in wild boar (Sus scrofa). Necropsy and histopathologic examination of a pregnant female and her three fetuses revealed all to have lesions compatible with acute toxoplasmosis. Nested polymerase chain reaction B1 gene detected Toxoplasma gondii in maternal (heart and diaphragm) and fetal (central nervous system, retina, optic nerve, heart, lung, tongue, and diaphragm) samples. The mother had a mixed infection of T. gondii types I and III. One fetus with type III infection developed no malformations, but the others-one with type I infection and one infected by types I and III-showed bilateral ocular agenesis, prognathism, and agenesis of the nasal cartilage. These results suggest the pathogenicity of the various T. gondii types may differ in wild boars.

Seroprevalence of Oestrus ovis (Diptera, Oestridae) infestation and associated risk factors in ovine livestock from southwestern Spain.

This survey was carried out to determine the seroprevalence of nasal infestation by sheep bot fly, Oestrus ovis L., and to identify the risk factors associated with the disease in flocks in southwestern Spain. In total, 5,878 sera samples of adult sheep were collected at random in 551 farms from four provinces in the southwestern Spain: Badajoz, Cáceres, Córdoba, and Sevilla. Sera were tested by enzyme-linked immunosorbent assay for O. ovis antibodies, by using a crude L2 larval as antigen. The seropositive mean prevalence was 69.30%, and mean percentage of optical densities was 61.83%. There were significant differences between the provinces studied; C6rdoba and Sevilla were the provinces with more infested animals and higher seroprevalences. The correlation between seroprevalence and percentage of antibodies by farms was significant. There were only 18 farms free of seropositive animals, and 115 of the total 551 farms had all sampled animals seropositive, an indication of the high importance of this parasitosis in the investigated areas. Altitude, latitude, flock size, and ovine population density were the potential risk factors associated with the detection of O. ovis antibodies. Those animals breeding in regions located at low altitudes (<500 m), meridian latitudes (<39.5 degrees N), and on farms with medium-to-large flock size (>250 sheep) and high ovine population density (>100 sheep per km2) were more likely to be seropositive. These findings confirm that these studied factors should be considered as potential risk factors to the presence of O. ovis in ovines from southwestern Spain.