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13 Pág. Centro de Investigación en Sanidad Animal (CISA), West Nile virus (WNV) is a mosquito-borne emerging virus in Europe with capacity to cause neurological complications such as encephalitis or meningoencephalitis in humans, birds or equids. In Spain, WNV is actively circulating in mosquitoes, birds and horses in different regions, but never has been deeply studied in Extremadura. Therefore, the aim of this study was to evaluate the seroprevalence of WNV in equids of those areas and to analyse the risk factors associated with exposure to the virus. A total of 199 out of 725 equids presented antibodies against WNV by competition ELISA (27.45%), while 22 were doubtful (3.03%). Anti-WNV IgM antibodies were detected in 16 equids (2.21%), and 3 animals were doubtful (0.41%). All ELISA-reactive positive/doubtful sera (N = 226) were further tested by micro-virus neutralization test (VNT), and a total of 143 horses were confirmed as positive for WNV, obtaining a seroprevalence of 19.72% in equids of western Spain. In addition, specific antibodies against USUV were confirmed in 11 equids. In 24 equids, a specific flavivirus species (detected by ELISA test) could not be determined. The generalized linear mixed-effects models showed that the significant risk factors associated with individual WNV infection in equids were the age (adults) and hair coat colour (light), whereas in USUV infections, it was the breed (pure). Data demonstrated that WNV and USUV are circulating in regions of western Spain. Given the high WNV seroprevalence found in equids from the studied areas, it is important to improve the surveillance programmes of public health to detect undiagnosed human cases and to establish a vaccination programme in equid herds in these regions., This research was supported financially by grant IB16135 funded by the Regional Ministry of Economy and Infrastructure, Extremadura Government and the European Regional Development Fund ‘A way to make Europe’ and grant E-RTA2015-00002-C02-00 funded by INIA. FG is supported by Ayudas a la Formación Investigador Predoctoral 2018 Formation contract (PD18056) from the Extremadura Government. PAS is funded by a FPI predoctoral fellowship from INIA. MF is currently funded by a Juan de la Cierva 2017 Formación contract (FJCI-2017-34394) from the Ministry of Science, Innovation and Universities.

Animal sanctuaries are important organisations that promote animal welfare and health as well as social awareness. Following the appearance of several donkeys with severe and distinctive skin lesions in a sanctuary from western Spain in 2021, a multicomponent study was performed to rule out the possible causes. The lesions were mainly concentrated on the extremities and, to a lesser extent, on the face and/or chest. The use of cotton leggings on their extremities as an external barrier to prevent them from bites and treatments with antiparasitic, antiseptic washes and dermatitis lotion showed to be effective measures to improve the donkey lesions. Skin scraping was negative for any relevant causative agents. Histopathological examination of the lesions showed an inflammatory infiltrate at the superficial dermal level, compatible with diffuse chronic dermatitis. A field entomological study was conducted from May to October 2021 to ascertain if any Diptera was responsible for these lesions. Considerable numbers of the stable fly Stomoxys calcitrans were recorded in both sticky traps and decomposing straw bedding. Several species of hematophagous Diptera were also recorded in lower numbers in other traps placed on the farm facilities. According to the entomological data, the location of the bites and clinical signs, it was concluded that S. calcitrans was the main cause of the skin lesions in the donkeys. To our best knowledge, this is the first clinical case of donkeys affected by the stable fly in Spain. For differential diagnosis, a brief discussion of the evidence caused by other biting Diptera groups is provided.

The large diversity of new tick-borne phleboviruses, and the negative impacts of the virulent viruses on human/animal health have led to a growing interest in their analysis. In this report, new insights are brought out into the diversity of putative phleboviruses circulating in Portugal (both the continental territory and the islands of Sao Miguel, in the Azores, and Madeira), as well as in the Spanish western regions of Extremadura and Castilla and Leon. Phlebovirus sequences were frequently detected (L-segment) from both questing and feeding ticks, but especially in Rhipicephalus sanguineus sensu lato (s.l.) specimens. These sequences were detected in adult ticks, as well as nymphs and eggs, supporting the hypothesis of viral maintenance by vertical transmission. Though multiple genetic groups could be identified in phylogenetic trees (AnLuc, KarMa, RiPar virus 1, and Spanish group 1 and 2), all the sequences from Portugal and Spain shared common ancestry with other viral sequence obtained from samples collected over a large geographic coverage. Spatiotemporal analysis placed Middle-East as the geographic origin of the most recent common ancestor (MRCA) of all phleboviruses analysed in the present study. More recent viral transitions might include migrations from Spain to continental Portugal, and from there to the Portuguese Islands. Our findings suggest that the time of the MRCA of phleboviruses was dated around 225 years ago [95% HPD: 124–387 year before the last sampling date].

An important element of vector control and surveillance of mosquito-borne diseases is updated information on vector species distribution. The aim of this study was to collect available information about mosquito species reported in Extremadura between 1920 and 2020 and create a catalog that would combine both published data and our recent field identifications. An exhaustive list is hereby presented, including species status and detailed distribution maps at a municipal level as well as their importance for public health. A total of 33 species, classified into five genera: Anopheles (five species), Aedes (14), Culex (nine), Culiseta (four), and Orthopodomyia (one) has been recorded, including 31 autochthonous, one invasive, Aedes (Stegomyia) albopictus, and one disappeared since 1953, Aedes (Stegomyia) aegypti. For the first time in Extremadura, we report the presence of important vectors such as Aedes (Aedimorphus) vexans vexans and Culex (Culex) perexiguus, and the new record of six species in the province of Badajoz, namely: Aedes (Dahliana) echinus, Aedes (Fredwardsius) vittatus, Aedes (Ochlerotatus) berlandi, Aedes (Ochlerotatus) pulcritarsis, Culex (Culex) mimeticus, and Culiseta (Culiseta) subochrea. Nineteen of these species are potential vectors of medical and veterinary relevance.

10 Pág. Centro de Investigación en Sanidad Animal (CISA), 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., Research funded by Consejería de Economía e Infraestructura of the Junta de Extremadura and the European Regional Development Fund, a Way to Make Europe, through the research project (grant numbers IB16135 and GR18148) and the National Institute for Agricultural and food Research and Technology (INIA-CISA) (grant number E-RTA2015-00002-C02-00)

The detection of three Gongylonema sp. infective larvae in two specimens of the dung beetle Geotrupes mutator (Marsham, 1802) from western Spain is reported here for the first time in Europe. Scanning electron microscopy confirmed that the analyzed specimens belong to the genus Gongylonema, but it was not possible to determine the species identity by the lack of morphological information in the literature and because many of the phenotypic characteristics had not yet fully developed at this juvenile stage. Nevertheless, a phylogenetic analysis using amplified cox1 nucleotide sequences has revealed that the studied larvae could be clearly discriminated (< 89% identity) from all the other Gongylonema cox1 sequences available in public genetic databases. While our results are limited by the scarcity of genetic information available for this genus, the possibility that the analyzed specimens might correspond to a new species should not be ruled out, and more studies are needed. The results provided in this report indicate that G. mutator is involved in the transmission cycle of Gongylonema sp. to vertebrates in Europe., The Authors also appreciate the technical and human support provided by the facility of analysis and characterization of solids and surfaces of SAIUEx (financed by UEX, Junta de Extremadura, MICINN, FEDER, and FSE).

Spirocerca lupi infection in dogs (Canis domesticus) is associated with esophageal lesions that may evolve to a neoplastic stage in the form of esophageal sarcoma. In the red fox (Vulpes vulpes) infected with the closely related Spirocerca vulpis, similar lesions may occur in the stomach, but neoplastic forms have not been reported. We characterize Spirocerca vulpis-induced lesions in the fox, using pathology and immunohistochemical (IHC) techniques. Seventy-one out of 163 Spirocerca vulpis-positive red foxes were selected and subjected to histopathological study. Lesions were classified as patchy or diffuse. Ten patchy and 10 diffuse lesion samples were studied using three IHC markers (CD68, CD3, and CD79α for macrophages, T lymphocytes, and B lymphocytes, respectively) and H&E stain for neutrophils and eosinophils. Intensity of necrosis, hemorrhages, and the presence of collagen was also analyzed. Of the S. vulpis-positive red foxes, 96.9% had S. vulpis nodules localized in the gastric area (wall and/or omentum), and 3.1% had nodules in the small intestine. All the samples had a moderate to severe lymphoplasmacytic infiltrate. Mild eosinophil infiltration was observed in both types of lesions, while neutrophil infiltration was significatively higher in the patchy than in the diffuse lesions. Fibrosis with mature collagen fibers was also predominant in the patchy lesions along with the presence of T lymphocytes and macrophages. Both the patchy and diffuse patterns had very few B lymphocytes. These findings suggest that the diffuse form is an earlier stage of the lesion, which eventually evolves into patchy forms. Neoplastic forms were not seen. Although more studies are necessary, this study describes the lesions, characterizes the inflammatory infiltrates, and establishes a possible evolution of the different pathological forms of S. vulpis infection in the red fox.

Changes in environmental conditions, whether related or not to human activities, are continuously modifying the geographic distribution of vectors, which in turn affects the dynamics and distribution of vector-borne infectious diseases. Determining the main ecological drivers of vector distribution and how predicted changes in these drivers may alter their future distributions is therefore of major importance. However, the drivers of vector populations are largely specific to each vector species and region. Here, we identify the most important human-activity-related and bioclimatic predictors affecting the current distribution and habitat suitability of the mosquito Culex pipiens and potential future changes in its distribution in Spain. We determined the niche of occurrence (NOO) of the species, which considers only those areas lying within the range of suitable environmental conditions using presence data. Although almost ubiquitous, the distribution of Cx. pipiens is mostly explained by elevation and the degree of urbanization but also, to a lesser extent, by mean temperatures during the wettest season and temperature seasonality. The combination of these predictors highlights the existence of a heterogeneous pattern of habitat suitability, with most suitable areas located in the southern and northeastern coastal areas of Spain, and unsuitable areas located at higher altitude and in colder regions. Future climatic predictions indicate a net decrease in distribution of up to 29.55%, probably due to warming and greater temperature oscillations. Despite these predicted changes in vector distribution, their effects on the incidence of infectious diseases are, however, difficult to forecast since different processes such as local adaptation to temperature, vector-pathogen interactions, and human-derived changes in landscape may play important roles in shaping the future dynamics of pathogen transmission. info:eu-repo/semantics/acceptedVersion

The main aim of this study was to not only establish the prevalence of the recently described Spirocerca vulpis parasite in the wild-life cycle of carnivores in western Spain but to also elaborate a model to explain the risk of infestation based on 16 topo-climatic and habitat variables. During the period from June 2016 to November 2017, 1644 carcasses of red foxes (Vulpes vulpes) and another 105 wild mammals, legally hunted or killed in car accidents, were analyzed. Parasitic nodules of Spirocerca were found in 6% of the foxes, and the molecular analyses established a homology of our samples with the species S. vulpis. There were no differences in the occurrence of the infestation between sexes, but there were differences in terms of age, such that infestation was proportionally more frequent among young individuals. In terms of temporality, a higher percentage of positive cases was observed during the late-autumn and winter months, especially between December and February. This study provides new data on the factors that predispose S. vulpis infection in the red fox. Model results indicate that a spatial pattern exists in the occurrence and prevalence of this species in the studied area (higher probabilities to the west), and that this pattern seems to mainly be associated with topo-climatic variables., This research did notreceive any specific grant from funding agencies of the public, commercial, or not-for-profit sectors.

Leishmaniosis caused by Leishmania infantum is present in Mediterranean countries, with high prevalence in areas of the center and south of Spain. However, in some regions such as Extremadura (in southwest of Spain), data has not been updated since 1997. The aim of this work was (i) to provide information about the distribution of phlebotomine sand fly species in western of Spain (Extremadura region), (ii) to determine risk factors for the presence of sand fly vectors and (iii) to detect Leishmania DNA and identify blood meal sources in wild caught females. During 2012–2013, sand flies were surveyed using CDC miniature light-traps in 13 of 20 counties in Extremadura. Specimens were identified morphologically and females were used for molecular detection of Leishmania DNA by kDNA, ITS-1 and cyt-B. In addition, blood meals origins were analyzed by a PCR based in vertebrate cyt b gene. A total of 1083 sand flies of both gender were captured and identified. Five species were collected, Phlebotomus perniciosus (60.76%), Sergentomyia minuta (29.92%), P. ariasi (7.11%), P. papatasi (1.48%) and P. sergenti (0.74%). The last three species constitute the first report in Badajoz, the most southern province of Extremadura region. Leishmania DNA was detected in three out of 435 females (one P. pernicious and two S. minuta). Characterization of obtained DNA sequences by phylogenetic analyses revealed close relatedness with Leishmania tarentolae in S. minuta and L. infantum in P. perniciosus. Haematic preferences showed a wide range of hosts, namely: swine, humans, sheep, rabbits, horses, donkeys and turkeys. The simultaneous presence of P. perniciosus and P. ariasi vectors, the analysis of blood meals, together with the detection of L. infantum and in S. minuta of L. tarentolae, confirms the ideal conditions for the transmission of this parasitosis in the western of Spain. These results improve the epidemiological knowledge of leishmaniosis and its vectors in this part of Spain, highlighting the need for ongoing entomological and parasitological surveillance.

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.

Estudio financiado por la Junta de Extremadura y el Fondo Euro-peo FEDER “Una manera de hacer Europa”, mediante el proyectocon ref. IB16135.

A study was performed to evaluate the implication of Protophormia terraenovae larvae as a surgical therapy for wounded skin. Three groups of sheep (n = 25) were considered based on larval doses. Groups 1 and 2 were artificially infested with low and high concentrations of L1 stage P. terraenovae, respectively, and group 0 served as a control. Skin biopsies were taken at 4 and 14 d postinfestation (D.P.If). A histopathological study was carried out to evaluate the lesions with a score, numbers of eosinophils and mast cells, and an immunohistochemical analysis of CD3, CD79α, and CD68 as T lymphocytes, B lymphosytes, and macrophages, respectively. The results indicated that higher larval doses led to faster regeneration by 14 D.P.If. Furthermore, the higher larval doses showed a high number of the CD68 marker and eosinophils and a low number of CD3 and CD79α markers and mast cells. In addition, the number of mast cells, T lymphocytes, and macrophage markers increased when the lesion progressed; however, a low number of immunolabeled CD79α cells and eosinophils were observed. The results indicate a possible positive effect of larvae in the healing of certain wounds.

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.

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.

The aim of this study was to investigate possible influence of different helmintosis in the development of Trichinella spiralis in experimental infected pigs. Forty-two Iberian pigs were allocated to six groups. Three groups were single inoculated with Ascaris suum, Metastrongylus apri or T. spiralis, respectively. Two groups were co-infected with T. spiralis and A. suum or T. spiralis and M. apri, respectively, while the last group included uninfected control pigs. Clinical signs were only observed in pigs with single or concurrent M. apri infections, with more severe respiratory symptoms in pigs with mixed M. apri infection. The number of A. suum and M. apri lung larvae, intestinal larvae of A. suum and adult M. apri were reduced in pigs with mixed Trichinella infections compared to pigs with single infections. In contrast, the number of liver white spots was higher in pigs with mixed infections. While T. spiralis muscular larval burdens were increased in pigs concomitantly infected with M. apri, they were reduced in pigs concomitantly infected with A. suum, compared to pigs receiving single infections with either of these helminths. Pigs with single or mixed A. suum infections showed higher eosinophil levels compared to the remaining groups. IgGt, IgG1, IgG2 and IgM against T. spiralis antigen could not be detected in pigs with single Ascaris or Metastrongylus infections, indicating that no cross-antibodies were produced. IgGt, IgG1 and IgM antibodies were detected earlier and generally at higher levels in mixed T. spiralis infections compared to single T. spiralis infections. The results suggest that T. spiralis had a low synergistic interaction with M. apri in concomitantly infected pigs, and an antagonistic interaction in concurrent infection with A. suum.

Food safety regulations require the control of the presence of protozoa in meats destined for human consumption. Wild boar (Sus scrofa) meat may constitute a source of zoonoses. A 23.8% (688/2881) seroprevalence of anti-Toxoplasma gondii antibodies and 72.2% (662/910) Sarcocystis sarcocysts prevalence were detected among wild boars hunted in Southwestern areas of Spain. Identity of Sarcocystis spp. was performed by RFLP-PCR and sequencing, detecting S. miescheriana (7/8) and the zoonotic S. suihominis (1/8). Risk assessment studies of these coccidian in meats destined to human consumption are needed.

A serodiagnostic test for the diagnosis of infestation by the sheep nasal bot fly, Oestrus ovis (Linne) was examined. The enzyme-linked immunosorbent assay (ELISA) technique was used to analyze and compare the production of immunoglobulin G (IgG) antibodies against excretory-secretory products (ESP) and crude extract (CE) antigens from all the different larval stages of O. ovis in the sera of 276 adult sheep sampled in summer (n = 135) and winter (n = 141). ESP from first stage larvae was the most sensitive, coating antigen in winter and ESP from second stage larvae during summer. The most specific values were obtained by ESP against L1 in winter and by CE against L3 in summer. These results show that the stage of larval development has a significant impact on the humoral immune response over the course of a season. A significant correlation (P < 0.001) was found between the number of O. ovis larvae and the serum antibody levels using all differents antigens, except L3 CE. In Spain, where a long favourable period exists for the evolution and development of the different stage larvae between March and November, the ELISA test using L1 ESP antigen during winter and L2 ESP antigen in summer may be used for ovine oestrosis immunodiagnosis.

This survey was conducted to determine the chronobiology and seroprevalence of nasal bot infestation ( Oestrus ovis ) in Spain and to identify the risk factors associated with this disease in caprine herds. A total of 1590 sera from adult goats were collected at random on 175 farms in southwestern Spain. Sera were tested by ELISA, using crude protein from second stage larvae as antigen. The mean seroprevalence was 46.04% and mean percentage of optical densities was 41.83. These data indicate a high prevalence of this parasite in the investigated areas. The serological survey revealed that goats managed at higher altitudes, at meridians latitudes and on farms with small herds had a smaller probability of infestation. Eighty goat heads, obtained from abattoirs in the central region of Spain, were collected and examined for nasal botflies from February to October 2002. O. ovis larval stages were recovered from the nasal-sinus cavities of 23 goats, reaching a prevalence of 34.94%. The mean larval burden was 3.9 larvae per infested head. No first instars were found during February and March, when the second instar reached its larger count. The third instar was observed in very small number during the whole period of study, with one peak occurring in July–August. These data show the existence of a favourable period for the development of larval instars of O. ovis in goats that starts in February and finishes in September.

Adult Ascaris suum were dissected to obtain different worm components (body wall, body fluid, ovaries, uterus and oesophagus) which were used as antigens when testing 95 sera of naturally A. suum-infected Iberian pigs by enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). Pigs with patent Ascaris infections had significantly lower ELISA optical density values than pigs without adult worms when using the body fluid and the body wall as antigens. A poor negative correlation was found between adult intestinal worm burden or eggs in faeces and specific antibody responses, measured by ELISA and WB using all antigens. By WB, the recognition of specific bands was variable, but three groups of bands with molecular weights of 97 kDa, 54–58 kDa and 42–44 kDa were generally recognized by sera from naturally infected pigs as well as from hyperimmunized pigs when using the five antigen extracts. The ELISA and WB techniques may be used for immunodiagnosis, using somatic adult worm antigens, to declare young pigs to be Ascaris-free but cannot be used for individual Ascaris-diagnosis in adult Iberian pigs.

We used PCR to detect Toxoplas- ma 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% )w ere 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.

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.

5 páginas, 2 tablas., The objective of the current study was to evaluate the efficacy of a single treatment with a long-acting injectable formulation of moxidectin (MOX) at 1.0 mg/kg bodyweight (b.w.) against natural infection by nasal bats (Oestrus ovis) in sheep with special attention to first instar larvae (L1). Firstly, a local farm with clinical history of oestrosis was chosen to conduct the assay. A total of 49 sheep were pre-selected at the end of the summer according to the presence of evident clinical signs of infection and confirmed later by means of an indirect ELISA against excretory-secretory products from L1 to detect IgG antibodies. After that, 24 sheep were chosen to carry out the study on the basis of positive serology and age since the oldest ones were selected. The day 0 of the assay, the treatment group was administered with the MOX formulation by subcutaneous injection at the base of the left ear and the control group was administered with a saline solution in the same way. All sheep were slaughtered on day 28 post-treatment (pt). At the necropsy, the head of all sheep were cut off and split into two sagital sections and all larvae from nasal passages, septum, middle meatus, conchae and sinuses were recovered. After the necropsy, a significant number of L1 was only found in the control group and therefore the efficacy of the MOX formulation was only calculated against this stage. As a result, the formulation was 90.2% effective against L1 for sheep slaughtered at day 28 pt., This study has been funded by Fort Dodge Veterinaria. The work of Martínez-Valladares M. has been supported by a postdoctoral Jae-Doc contract from Consejo Superior de Investigaciones Científicas (CSIC) and co-funded by the European Social Fund.

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.

A study has been carried out with the aim to determine possible interactions between Ascaris suum and Metastrongylus apri under experimentally infected pigs. Twenty-eight Iberian pigs were allocated into four groups. Group 1 was inoculated with 5000 infective A. suum eggs; group 2 received concurrently 5000 infective A. suum eggs and 5000 infective M. apri larvae; group 3 received 5000 infective M. apri larvae; group 4 served as uninfected controls. In each group, pigs were necropsied on day 7 (n = 4) and day 28 (n = 3) post-infection (p.i.). Pigs with single M. apri infections showed earlier and more severe respiratory symptoms compared to pigs with mixed infection, while no clinical signs were observed in pigs single infected with A. suum. Mean burdens of immature A. suum and immature and adult M. apri were reduced in pigs with concomitant infection both on day 7 and 28 p.i., respectively. In contrast, the number of white spots was significantly increased on day 7 in pigs with mixed infection. In addition, pigs of group 1 showed the highest eosinophil levels in blood compared to pigs in groups 2 (intermediate levels) and 3 (moderate levels). The results suggest an antagonistic interaction between A. suum and M. apri in concomitantly infected pigs.

A chronobiological study of oestrosis was conducted for larval instars of Oestrus ovis from November 2000 to September 2002 with the examination of 477 adult sheep of the southwest region of Spain. Skulls from slaughtered sheep were examined and the different O. ovis larval stages (L1, L2, L3) were recovered from the nasal-sinus cavities. O. ovis larvae were detected in 339 sheep, reaching a prevalence of 71.1%. Only one farm was free of infested sheep indicating a prevalence of the 97.91% among studied flocks. The mean larval burden was 18.54 larvae per infested head during the coldest months in the southwest of Spain when the larval burden reached its highest levels, especially of the first larval stage (L1). However, the maximum percentage of L1 coincided with the minimum percentage of the second larval stage (L2). The third larval stage (L3) was observed in relatively low levels during the entered study period, but two peaks occurred in April–May and in September–October. During the 2 years of sampling, all the different larval stages were simultaneously recovered throughout the year, indicating the existence of a long favourable period for the evolution and development of the larval instars, which would start between February and March and finishing in November.

A total of 35 pigs aged 15 weeks old, and 21 pigs aged 8 weeks old were divided into 7 groups. Groups 1 and 2 were uninfected and challenge control groups, respectively. Groups 3 and 4 were infected weekly with 6 increasing doses ofAscaris suumeggs, and group 4 was additionally treated with pyrantel. Groups 5, 6, and 7 were immunized weekly with the 14, 42, or 97 kDa fractions from adult worms, respectively. Animals of groups 2–7 were challenged with 10 000A. suumeggs 7 days after the last infection/immunization. Serum was sampled weekly and specific IgG1, IgG2, and IgM responses were measured. Pigs of groups 5, 6, and 7 showed high IgG1 and IgG2 responses especially against adult worms antigens, while infected groups had high IgG1 and IgM responses, especially against larva. The IgG1 responses were negatively correlated to the numbers of larvae in the lungs, and positively associated with the liver white spot numbers. There was a positive correlation between IgG2 and the numbers of white spots and lung larvae, while IgM was negatively correlated with these parasitological measures. These findings are discussed and it is suggested that acquired resistance againstA. suumlarvae is correlated with the induction of IgG1 and IgM, and not with IgG2, and that future vaccination protocols may focus on inducing the Th2 activity.

In the present work, we carry out an immunopathological study of the swine ascariosis, under different conditions (control, infection and immunization). Twenty-one Iberian pigs were used and divided in seven groups. Groups 1 and 2 were the uninfected and challenged controls, respectively. Groups 3 and 4 were weakly infected with increasing doses of Ascaris suum eggs and treated with pyrantel (Group 4). Groups 5-7 were immunized with 14, 42 and 97 kDa proteins from the parasite, respectively. Groups 2-7 were challenged with 10,000 infective eggs 7 days before the sacrifice. The focal parasitic granulomata with eosinophils and lymphocytes were the main histopathological lesions in the liver of reinfected pigs, while more marked cellular infiltrate and abundant connective tissue were seen in the livers of immunized animals. There were important deposits of antigens in the livers of immunized and infected pigs. Antigens were mainly located in the connective tissue, with positive staining detection of the somatic larvae antigen, the body wall from the adult worms and the 14-, 42- and 97-kDa proteins. However, cholangiols, biliary ducts and macrophages presented an immunohistochemical positive stain against excretory-secretory and somatic antigens from the larvae and the body fluid antigen from the adult parasite. The detection of A. suum antigens in the liver of infected pigs improves the diagnosis of swine ascariosis. It may be possible to apply these procedures for diagnosis of human ascariosis in liver biopsies since A. suum from swine have been previously used as a substitute for the study of the human parasite Ascaris lumbricoides.

Resistance to Ascaris suum infections was investigated in 8- and 15-week-old Iberian pigs. Groups of 3 or 5 pigs were immunized weekly for 6 weeks with antigens of adult A. suum: a 97 kDa body wall (BW) fraction, a 42 kDa fraction of pseudocoelomic fluid (PF) or a 14 kDa PF-fraction; or were inoculated with increasing doses of infective eggs (500-20 000), with or without abbreviation by pyrantel pamoate. All immunized pigs and unimmunized control pigs, were challenged with 10 000 infective eggs 7 days after the last immunization. The number of liver lesions and lung larvae was substantially lower in the older pigs than in the younger tines 7 days after challenge, but the resistance in immunized pigs of both age groups was similar in comparison to the challenge controls of the same age. The highest degree of resistance against lung larvae was observed in pigs immunized with A. suum eggs (97.99%). The pigs immunized with the 14 kDa and 42 kDa PF-fractions were also well protected (67-93%), while no protection was produced by the 97 kDa BW fraction (0-49%). The reduction of white spots following immunization was less evident, with a maximum of 82% reduction in egg-inoculated young pigs.