Coxiella burnetii infection persistence in a goat herd during seven kidding seasons after an outbreak of abortions: the effect of vaccination.

Coxiella burnetii infection was monitored during seven kidding seasons (2017-2023) in a dairy goat herd that after an outbreak of Q fever abortions was vaccinated with an inactivated phase I vaccine. Due to the high infection rate just after the outbreak, only the replacement stock was vaccinated during the first three kidding seasons, and when the average herd immunity had decreased (fourth kidding season onwards), the whole herd was vaccinated. Vaginal swabs, feces, and milk were analyzed by PCR to monitor infection, and dust and aerosols were analyzed to measure C. burnetii environmental contamination. One year after the onset of the outbreak, a significant reduction in C. burnetii shedding loads was observed, but the percentage of shedding animals remained high until the third kidding season. By the seventh kidding season, no shedders were detected. The bacterial load excreted was significantly lower in vaccinated compared with unvaccinated animals, and in yearlings compared with multiparous. C. burnetii was detected by PCR in aerosols collected inside the animal premises throughout the study period except in the last season; whereas, aerosols collected outdoors tested negative in the last three kidding seasons. Viable C. burnetii was detectable in environmental dust collected inside the barn until the third kidding season following the outbreak. These results indicate that after an outbreak of Q fever, the risk of infection for humans and susceptible animals can remain high for at least three kidding seasons when the number of C. burnetii animal shedders is still high, even when bacterial excretion is low. IMPORTANCE: Q fever is a zoonosis distributed worldwide. Ruminants are the main reservoir, and infection can cause high rates of abortion. After entering a farm, Coxiella burnetii infection can persist in the animal population over several lambing/kidding periods. Once infection is established in a herd, vaccination with the inactivated Phase I vaccine significantly reduces bacterial shedding, but although at low levels, excretion may continue to occur for several lambing/kidding seasons. The time that C. burnetii remains viable in the farm environment after an outbreak of Q fever determines the period when risk of infection is high for the people in close contact. This work showed that this period extends at least three kidding seasons after the outbreak. These results provided valuable information on the epidemiology of C. burnetii infection in goat herds and may help to develop guidelines for controlling the disease and reducing infection risk for susceptible people and animals.

Mosquito community composition in two major stopover aquatic ecosystems used by migratory birds in northern Spain.

Mosquitoes (Diptera: Culicidae) are common bloodsucking Diptera frequently found in aquatic environments, which are valuable ecosystems for many animal species, particularly migrating birds. Therefore, interactions between these animal species and mosquitoes may play a critical role in pathogen transmission. During 2018-2019, mosquitoes were collected from two aquatic ecosystems in northern Spain using different methodologies and identified using classical morphology and molecular tools. A total of 1529 males and females of 22 native mosquito species (including eight new records for the region) were trapped using CO2 -baited Centers for Disease Control and Prevention (CDC) traps and sweep netting. Among the blood-fed female mosquitoes, 11 vertebrate host species-six mammals and five birds-were identified using DNA barcoding. The developmental sites of eight mosquito species were determined across nine microhabitats, and 11 mosquito species were caught landing on humans. The flight period varied among mosquito species, with some peaking in the spring and others in the summer. Our study highlights the advantages of mosquito sampling using various techniques to comprehensively characterise species composition and abundance. Information on the trophic preferences, biting behaviour and influence of climatic variables on the ecology of mosquitoes is also provided.

Spirocerca lupi in the stomach of two Andean foxes (Lycalopex culpaeus) from Chile.

The genus Spirocerca includes nematodes that parasitize the stomach and the oesophagus of carnivores, chiefly canids. Herein, we provide new data about the morphological, histopathological, and molecular characterization of Spirocerca sp. in Andean foxes (Lycalopex culpaeus) in Chile. Intact immature worms, identified as Spirocerca sp., were recovered in the lumen of the stomach from two foxes. Histologically, worms morphologically consistent with spirurid nematodes were present within the wall of the stomach and surrounded by nodular areas of inflammation with central necrotic debris. Molecular analysis of the cox1 gene yielded 19 sequences and 5 nucleotide sequence types with 99.95 to 99.98% similarity, being shared between both foxes. Nucleotide similarity ranged from 93.1 (with genotype 2 of S. lupi and S. vulpis) to 95.8% (with genotype 1 of S. lupi), a higher similarity than noted from sequences of S. lupi from an Andean fox from Peru (91.0 to 93.3%). However, the Poisson Tree Processes for species delineation did not support the existence of a new species Spirocerca. Phylogenetic and nucleotide analyses suggest that these specimens belong to a new variant or genotype of S. lupi or to a cryptic species. Whether the presence of the worms in the stomach has to do with genotypic differences in parasites or host or some combination is uncertain. Spirocerca lupi has never been found in Chilean dogs and must be investigated.

A Q fever outbreak among visitors to a natural cave, Bizkaia, Spain, December 2020 to October 2021.

We describe a large Q fever outbreak reported in Spain, including 108 cases, 53 with pneumonia and 27 requiring hospitalisations. The first cases were detected in February 2021 among rock climbers visiting a cave in Bizkaia, and the last case was detected in October 2021. Most cases were notified after the Easter holiday (April-May 2021). More males (63.9%) than females (36.1%) were infected (median ages: 42 (1-68) and 39 years (6-61), respectively). We detected Coxiella burnetii by PCR in faecal, dust and/or aerosol samples taken inside the cave in March 2021, and in dust and aerosol samples collected between March 2021 and February 2023. Coxiella burnetii from dust samples were cultured on Vero cells, showing viability for 24 months. Based on serological and genotyping data, goats sheltering in the cave were the most likely source of infection. The cave was closed on 29 April 2021, movements of goats and sheep in the area were restricted (March-July 2021), and the animals were vaccinated in October 2021. Investigation of Q fever outbreaks requires a multidisciplinary One Health approach as these outbreaks can occur in unexpected places like natural sites where animals are present.

High seroprevalence of feline morbilliviruses in free-roaming domestic cats in Chile.

Feline morbillivirus infections have gained increased attention due to repeated reports of their association with urinary tract disease in cats. In the present study, 112 serum samples from free-roaming domestic cats in Chile were tested for antibodies against feline morbillivirus genotypes 1 and 2 (FeMV-1 and FeMV-2) using an indirect immunofluorescence assay. In total, 63% of the animals showed antibodies against one or both FeMV genotypes. Antibodies directed exclusively against FeMV-2 were significantly more prevalent in male cats. The correlation of sex and FeMV-2 infection might give insight into potential routes of transmission. We provide, for the first time, serological data on FeMV in Chile.

Hemoplasmas Are Endemic and Cause Asymptomatic Infection in the Endangered Darwin's Fox (Lycalopex fulvipes).

Mycoplasma haemocanis is prevalent in the endangered Darwin's fox (Lycalopex fulvipes) in its main stronghold, Chiloé Island (Chile). The origin of the infection, its dynamics, its presence in other fox populations and the potential consequences for fox health remain unexplored. For 8 years, hemoplasmal DNA was screened and characterized in blood from 82 foxes in Chiloé and two other fox populations and in 250 free-ranging dogs from Chiloé. The prevalence of M. haemocanis in foxes was constant during the study years, and coinfection with "Candidatus Mycoplasma haematoparvum" was confirmed in 30% of the foxes. Both hemoplasma species were detected in the two mainland fox populations and in Chiloé dogs. M. haemocanis was significantly more prevalent and more genetically diverse in foxes than in dogs. Two of the seven M. haemocanis haplotypes identified were shared between these species. Network analyses did not show genetic structure by species (foxes versus dogs), geographic (island versus mainland populations), or temporal (years of study) factors. The probability of infection with M. haemocanis increased with fox age but was not associated with sex, season, or degree of anthropization of individual fox habitats. Some foxes recaptured years apart were infected with the same haplotype in both events, and no hematological alterations were associated with hemoplasma infection, suggesting tolerance to the infection. Altogether, our results indicate that M. haemocanis is enzootic in the Darwin's fox and that intraspecific transmission is predominant. Nevertheless, such a prevalent pathogen in a threatened species represents a concern that must be considered in conservation actions.IMPORTANCEMycoplasma haemocanis is enzootic in Darwin's foxes. There is a higher M. haemocanis genetic diversity and prevalence in foxes than in sympatric dogs, although haplotypes are shared between the two carnivore species. There is an apparent tolerance of Darwin's foxes to Mycoplasma haemocanis.

High prevalence of chigger mite infection in a forest-specialist frog with evidence of parasite-related granulomatous myositis.

Amphibians are hosts for a wide variety of micro- and macro-parasites. Chigger mites from the Hannemania genus are known to infect a wide variety of amphibian species across the Americas. In Chile, three species (H. pattoni, H. gonzaleacunae and H. ortizi) have been described infecting native anurans; however, neither impacts nor the microscopic lesions associated with these parasites have been described. Here, we document 70% prevalence of chigger mite infection in Eupsophus roseus and absence of infection in Rhinoderma darwinii in the Nahuelbuta Range, Chile. Additionally, we describe the macroscopic and microscopic lesions produced by H. ortizi in one of these species, documenting previously undescribed lesions (granulomatous myositis) within the host's musculature. These findings highlight that further research to better understand the impacts of chigger mite infection on amphibians is urgently required in Chile and elsewhere.

Differences in the ectoparasite fauna between micromammals captured in natural and adjacent residential areas are better explained by sex and season than by type of habitat.

We compared the ectoparasite fauna in 608 micromammals (chiefly 472 wood mice Apodemus sylvaticus, 63 Algerian mice Mus spretus, and 51 greater white-toothed shrews Crocidura russula) captured in natural and adjacent residential areas in spring and autumn during three consecutive years in four areas in periurban Barcelona (NE Spain). We found little support for an association of urbanization with differences in infestation by ectoparasites. Prevalence of Rhipicephalus sp. tick in wood mice and shrews was significantly higher in residential than in natural habitats, and the opposite was found for the flea Ctenophtalmus andorrensis catalanensis in shrews. Marked differences in the prevalence of the flea Leptopsylla taschenbergi amitina in wood mice between seasons were observed in natural but not in residential habitats, probably due to enhanced flea survival probabilities in the latter. However, as a rule, males were more frequently and heavily infested than females, and the prevalence was higher in autumn than in spring. Our results suggest that the ectoparasite fauna of periurban micromammals is shaped more by other factors than by habitat modification. People living in residential areas are at risk of contact with the arthropods borne by non-commensal micromammals and the pathogens transmitted by them.

Using a top predator as a sentinel for environmental contamination with pathogenic bacteria: the Iberian wolf and leptospires.

The Iberian wolf (Canis lupus) is the top predator in the Iberian environments in which it lives, feeding on a wide range of species, thus encountering a wide range of disease agents. Therefore, the wolf can serve as sentinel of environmental contamination with pathogens. We investigated the exposure of free-living wolves to 14 serovars of Leptospira interrogans sensu lato. Kidney samples from 49 wolves collected from 2010-2013 in northwestern Spain were analysed by culture, direct immunofluorescence and polymerase chain reaction. Tissue fluids were analysed for antibodies by a microscopic agglutination test. Ten wolves (observed prevalence: 20%, 95% confidence interval = 11-33%) showed evidence of contact with leptospires, eight through direct detection and nine through serology (7 wolves were positive according to both techniques). Titres below the cut-off level were also detected in seven cases. Serovars confirmed were Canicola (n = 4), Icterohaemorrhagiae (n = 3) and Sejroë, Ballum and Grippotyphosa (n = 1 each), indicating that wolves were infected with serovars for which dogs, rodents and ungulates, are the natural hosts and supporting the utility of the wolf and other large predators as environmental sentinels for pathogens.

Mosquitoes in urban green spaces and cemeteries in northern Spain.

BACKGROUND: Mosquitoes inhabiting urban green spaces and cemeteries in Europe represent a crucial facet of public health concern and contribute to the ecological balance. As urbanization intensifies, these areas increasingly serve as vital habitats for various mosquito species, fostering breeding grounds and increasing the risk of disease transmission. METHODS: A study was conducted in the three main cities (inland, coastal, and estuarine) of the Basque Country, northern Spain, to investigate the species composition, abundance, dynamic populations, larval habitats, and host preferences of mosquitoes in urban green spaces and cemeteries. CDC traps and dipping were used to collect mosquitoes for 2 years (2019-2020). RESULTS: A total of 21 mosquito species were identified, with Culex pipiens s.l. being the most abundant and widespread. The three ecological forms of Cx. pipiens were found, and Cx. pipiens pipiens was the most common in both green areas and cemeteries. Morphological identification together with molecular tools identified 65 COI sequences with high homology. The highest species richness was found in the inland city, followed by the coastal city and the estuarine city. Mosquito abundance was significantly higher in green areas compared to cemeteries and in the coastal and estuarine cities compared to the inland city. The investigation of larval breeding sites highlighted the dominance of Cx. pipiens s.l., particularly in semi-artificial ponds, diverse water-holding containers (tyres and buckets) and drainage systems in green areas; in cemeteries, most of the larvae were found in flowerpots and funerary urns. Seasonal activity exhibited variable peaks in mosquito abundance in the different cities, with a notable increase in July or August. Additionally, blood meal analysis revealed that Cx. pipiens s.l. fed on several common urban avian species. CONCLUSIONS: Studies on mosquitoes are essential to understand their role in disease transmission and to design targeted and sustainable management strategies to mitigate the associated risks.

Epizootiology and Pathology of Sarcoptic Mange in Two Species of Fox (Lycalopex spp.) in Human-Dominated Landscapes of Central Chile.

Sarcoptic mange caused by Sarcoptes scabiei can have catastrophic consequences for wildlife. We inspected 122 Andean foxes (Lycalopex culpaeus), collected by active (n=66) or passive (n=56) surveillance, and 28 South American gray foxes (Lycalopex griseus; all from passive surveillance) for mange in Chile (2015-19). In Andean foxes, gross lesions of mange were diagnosed in 24% of passively and 9% of actively collected foxes, although observed prevalences might be underestimated. Seroprevalence was 37 and 18%, respectively, indicating that some individuals recovered from infection or were developing the disease. No differences were found between age and sex groups. Comparing data from passive surveillance, occurrence of gross lesions was lower in gray foxes (5%). Body condition was significantly better in Andean foxes without lesions than in diseased foxes, which had significantly lower albumin concentrations than healthy individuals. Among the 12 foxes with gross lesions, four, six and two individuals were categorized as having type I, type II, and type III lesions, respectively, based on clinical severity. Histologic severity correlated with gross lesions and included irregular epidermal hyperplasia with hyperkeratosis, which was marked in type II and III infections. Conventional PCR targeting of the cox1 gene fragment revealed four nucleotide sequence types, showing 99-100% identity among them and between 99% and 100% identity with previously published sequences of S. scabiei. A significant association between the occurrence of mange in foxes and distance to the nearest house was found. We speculate that diseased foxes tended to approach human settlements, perhaps in search of food. Visual inspection of 211 rural dogs from the study area did not reveal gross mange lesions in any animal. Sarcoptic mange is enzootic in the Andean fox in the study area and should be considered in the management of the species.

Mapping the risk of exposure to Crimean-Congo haemorrhagic fever virus in the Iberian Peninsula using Eurasian wild boar (Sus scrofa) as a model.

Crimean-Congo haemorrhagic fever (CCHF) virus (CCHFV) is a tick-borne zoonotic pathogen that can cause a lethal haemorrhagic disease in humans. Although the virus appears to be endemically established in the Iberian Peninsula, CCHF is an emerging disease in Spain. Clinical signs of CCHFV infection are mainly manifested in humans, but the virus replicates in several animal species. Understanding the determinants of CCHFV exposure risk from animal models is essential to predicting high-risk exposure hotspots for public health action. With this objective in mind, we designed a cross-sectional study of Eurasian wild boar (Sus scrofa) in Spain and Portugal. The study analysed 5,291 sera collected between 2006 and 2022 from 90 wild boar populations with a specific double-antigen ELISA to estimate CCHFV serum prevalence and identify the main determinants of exposure probability. To do so, we statistically modelled exposure risk with host- and environment-related predictors and spatially projected it at a 10 × 10 km square resolution at the scale of the Iberian Peninsula to map foci of infection risk. Fifty-seven (63.3 %) of the 90 populations had at least one seropositive animal, with seroprevalence ranging from 0.0 to 88.2 %. Anti-CCHFV antibodies were found in 1,026 of 5,291 wild boar (19.4 %; 95 % confidence interval: 18.3-20.5 %), with highest exposure rates in southwestern Iberia. The most relevant predictors of virus exposure risk were wild boar abundance, local rainfall regime, shrub cover, winter air temperature and soil temperature variation. The spatial projection of the best-fit model identified high-risk foci as occurring in most of western and southwestern Iberia and identified recently confirmed risk foci in eastern Spain. The results of the study demonstrate that serological surveys of CCHFV vector hosts are a powerful, robust and highly informative tool for public health authorities to take action to prevent human cases of CCHF in enzootic and emergency settings.

Co-infection patterns of vector-borne zoonotic pathogens in owned free-ranging dogs in central Chile.

We investigated the co-occurrence of the nine of the most relevant canine vector-borne pathogens (CVBP) using conventional and real-time PCR and evaluated risk factors and potential non-apparent haematological alterations associated with co-infection in 111 rural, owned, free-ranging dogs in the Metropolitan Region of Chile.At least one pathogen was detected in 75% of the dogs. DNA of Anaplasma platys (Ap; 36%), Candidatus Mycoplasma haematoparvum (CMhp; 31%), Mycoplasma haemocanis (Mhc; 28%), Trypanosoma cruzi (17%), Leishmania spp. (4.5%), and Acanthocheilonema reconditum (1%) was detected. All dogs were negative for Ehrlichia spp., Rickettsia spp., Bartonella spp., Piroplasmida, and Hepatozoon spp. Thirty-eight dogs (34%) were coinfected. CMhp was involved in 71%, Mhc in 58%, and Ap in 50% of the co-infections. The most common co-infection pattern was CMhp-Mhc (37% of the cases). The prevalence of Ap was higher in juvenile than in adult dogs, whereas the opposite was found for CMhp and Mhc. Adult dogs were four times more likely of being co-infected than juveniles. Co-infected animals showed higher white blood cell count, segmented neutrophil count, and GGT levels than non-co-infected dogs. Clinically healthy but infected dogs may act as reservoirs of CVBP, and their free-ranging behavior would facilitate the spread of these pathogens to other dogs as well as human beings or wild carnivores.

Molecular identification of Bartonella spp. and Rickettsia felis in fox fleas, Chile.

Seventy-five flea pools (one to ten fleas per pool) from 51 Andean foxes (Lycalopex culpaeus) and five South American grey foxes or chillas (Lycalopex griseus) from the Mediterranean region of Chile were analyzed for the presence of DNA of Bartonella spp. and Rickettsia spp. through quantitative real-time PCR for the nouG and gltA genes, respectively. Positive samples were further characterized by conventional PCR protocols, targeting gltA and ITS genes for Bartonella, and gltA, ompA, and ompB genes for Rickettsia. Bartonella was detected in 48 % of the Pulex irritans pools (B. rochalimae in three pools, B. berkhoffii in two pools, B. henselae in one pool), and 8 % of the Ctenocephalides felis felis pools (B. rochalimae, one pool). Rickettsia was confirmed in 11 % of P. irritans pools and 92 % of the Ct. felis pools. Characterization confirmed R. felis in all sequenced Rickettsia-positive pools. All Ct. canis pools were negative. A Ct. felis pool from a wild-found domestic ferret (Mustela putorius furo) also resulted positive for R. felis. Although opportunistic, this survey provides the first description of zoonotic pathogens naturally circulating in fleas parasitizing Chilean free-living carnivores.

Invasive Aedes mosquitoes in an urban-peri-urban gradient in northern Spain: evidence of the wide distribution of Aedes japonicus.

BACKGROUND: The expansion of invasive mosquitoes throughout Europe has increased in recent decades. In northern Spain, Aedes albopictus was detected for the first time in 2014, and Aedes japonicus was detected in the three Basque provinces in 2020. This study aimed to evaluate the distribution of these mosquito species and their association with factors related to urbanization. METHODS: In 2021, a total of 568 ovitraps were deployed in 113 sampling sites from 45 municipalities with > 10,000 inhabitants. Oviposition substrate sticks were replaced each fortnight and examined for Aedes eggs from June to November. Aedes eggs were counted, and the eggs from a selection of positive oviposition sticks, encompassing at least one stick from each positive ovitrap, were hatched following their life cycle until the adult stage. When egg hatching was not successful, PCR targeting the COI gene and sequencing of amplicons were carried out. RESULTS: Eggs were detected in 66.4% of the sampling sites and in 32.4% of the ovitraps distributed in the three provinces of the Basque Country. Aedes albopictus and Ae. japonicus were widespread in the studied area, confirming their presence in 23 and 26 municipalities, respectively. Co-occurrence of both species was observed in 11 municipalities. The analysis of the presence of Aedes invasive mosquitoes and the degree of urbanization (urban, suburban, peri-urban) revealed that Ae. albopictus showed a 4.39 times higher probability of being found in suburban areas than in peri-urban areas, whereas Ae. japonicus had a higher probability of being found in peri-urban areas. Moreover, the presence of Ae. albopictus was significantly associated with municipalities with a higher population density (mean = 2983 inh/km2), whereas Ae. japonicus was associated with lower population density (mean = 1590 inh/km2). CONCLUSIONS: The wide distribution of Ae. albopictus and Ae. japonicus observed confirmed the spread and establishment of these species in northern Spain. A new colonization area of Ae. japonicus in Europe was confirmed. Due to the potential impact of Aedes invasive mosquitoes on public health and according to our results, surveillance programs and control plans should be designed considering different urbanization gradients, types of environments, and population density.

Efficacy of Protein Baits with Fipronil to Control Vespa velutina nigrithorax (Lepeletier, 1836) in Apiaries.

The yellow-legged hornet (Vespa velutina nigrithorax), outside its natural range, has become a major threat to domestic bees. Several control methods have been used to fight against V. velutina, but the results achieved are not satisfactory. The use of protein baits with biocides has shown to be an effective method to control invasive wasp populations, but they have not been used to control V. velutina. Thus, the efficacy of protein baits containing fipronil to reduce the presence of hornets in apiaries was evaluated in this study. After laboratory determination of the optimal efficacy of a protein bait at a 0.01% concentration of fipronil, field trials were conducted involving 222 beekeepers. The data reported by the 90 beekeepers who completed the requested questionnaire demonstrated that in the groups of apiaries with the highest pressure of hornets (groups with 10-30 and >30 hornets), there was a significant decrease in the presence of V. velutina, lasting at least two weeks. The reduction in the number of hornets was positively correlated with bait consumption, and bait consumption was positively correlated with the number of hornets present at the time of treatment. Although the method used has shown good efficacy and the concentration of fipronil used was very low; possible negative effects on the environment should also be evaluated.

Stable prevalence of Coxiella burnetii in wildlife after a decade of surveillance in northern Spain.

Coxiella burnetii is an obligate intracellular zoonotic bacterium widespread in nature that causes Q fever in animals and humans. The most common sources of human infection are domestic ruminants, but wildlife can also act as reservoir. Here, spleen samples from 652 wild ungulates and 218 wild birds collected in 2011-2019 in the Basque Country (northern Spain) were analysed by real-time PCR (IS1111 gene) and the results compared with data from a past study in 2001-2006. Among wild ungulates, C. burnetii DNA was detected in 7.0% (6/86) of roe deer (Capreolus capreolus), 1.9% (9/484) of wild boar (Sus scrofa) and 2.4% (2/82) of red deer (Cervus elaphus). The prevalence in roe deer was significantly higher compared to wild boar (p = 0.006). Among wild birds, only one white stork (Ciconia ciconia) tested positive. SNP-typing of C. burnetii-positive samples showed that wild ungulates shared SNP 2, SNP 6 and SNP 8 genotypes with domestic ruminants of the region. However, the white stork harboured a C. burnetii genotype (SNP 3) never identified in the studied area before. Comparing these results with those obtained in the same area a decade before (2001-2006), no significant differences were observed in the prevalence of C. burnetii in any of the wildlife species, indicating stability in C. burnetii prevalence. Nevertheless, continuous surveillance is needed to monitor any future changes in the reservoir role of roe deer and wild boar considering the increase in density of both species observed in Europe in the last decades.

Culicoides species community composition and feeding preferences in two aquatic ecosystems in northern Spain.

BACKGROUND: Aquatic ecosystems provide breeding sites for blood-sucking insects such as Culicoides biting midges (Diptera: Ceratopogonidae), but factors affecting their distribution and host choice are poorly understood. A study was undertaken at two nature reserves in northern Spain to examine the abundance, species composition, population dynamics and feeding patterns of biting midges between 2018 and 2019. METHODS: Culicoides were captured by light suction traps baited with CO2 and by sweep netting vegetation. Blood meals and species identification of blood-fed specimens were determined using cytochrome c oxidase I subunit (COI) DNA barcoding. Multivariate generalized linear models were used to evaluate the associations between the abundance of Culicoides, the species richness and other parameters. RESULTS: The 4973 identified specimens comprised 28 species of Culicoides. These included two species reported for the first time in northern Spain, thus raising to 54 the number of Culicoides species described in the region. Specimens of all 28 species and 99.6% of the total specimens collected were caught in suction traps, while sweep netting vegetation revealed just 11 species and 0.4% of the total specimens. Midge abundance peaked in June/early July, with five species comprising > 80% of the captures: Culicoides alazanicus (24.9%), Culicoides griseidorsum (20.3%), Culicoides poperinghensis (16.2%), Culicoides kibunensis (10.7%) and Culicoides clastrieri (9.6%). DNA barcode analysis of blood meals from eight Culicoides species revealed that they fed on 17 vertebrate species (3 mammals and 14 birds). Species in the subgenus Avaritia were primarily ornithophilic, except for C. griseidorsum and C. poperinghensis. Host DNA from blood meals was successfully amplified from 75% of blood-fed females. A pictorial blood meal digestion scale is provided to accurately assess the blood-fed status of female Culicoides. CONCLUSIONS: The large number of different blood meal sources identified in the midges captured in this study signals the likely importance of wild birds and mammals (e.g. red deer and wild boar) as reservoir/amplifying hosts for pathogens. Available hosts are more exposed to being bitten by biting midge populations in aquatic ecosystems in late spring and early summer.

SEROSURVEY FOR SELECTED PARASITIC AND BACTERIAL PATHOGENS IN DARWIN'S FOX (LYCALOPEX FULVIPES): NOT ONLY DOG DISEASES ARE A THREAT.

The Darwin's fox (Lycalopex fulvipes) is one of the most endangered carnivores worldwide, with the risk of disease spillover from domestic dogs being a major conservation threat. However, lack of epidemiologic information about generalist, non-dog-transmission-dependent protozoal and bacterial pathogens may be a barrier for disease prevention and management. To determine the exposure of some of these agents in Darwin's fox populations, 54 serum samples were collected from 47 Darwin's foxes in Southern Chile during 2013-18 and assessed for the presence of antibodies against Brucella abortus, Brucella canis, Coxiella burnetii, pathogenic Leptospira (serovars Grippotyphosa, Pomona, Canicola, Hardjo, and Copehageni), Toxoplasma gondii, and Neospora caninum. The highest seroprevalence was detected for T. gondii (78%), followed by pathogenic Leptospira (14%). All the studied Leptospira serovars were confirmed in at least one animal. Two foxes seroconverted to Leptospira and one to T. gondii during the study period. No seroconversions were observed for the other pathogens. No risk factors, either intrinsic (sex, age) or extrinsic (season, year, and degree of landscape anthropization), were associated with the probability of being exposed to T. gondii. Our results indicate that T. gondii exposure is widespread in the Darwin's fox population, including in areas with minimal anthropization, and that T. gondii and pathogenic Leptospira might be neglected threats to the species. Further studies identifying the causes of morbidity and mortality in Darwin's fox are needed to determine if these or other pathogens are having individual or population-wide effects in this species.

Pitfalls in Tick and Tick-Borne Pathogens Research, Some Recommendations and a Call for Data Sharing.

An understanding of the relationships of ticks and tick-borne pathogens can only be achieved by integrating data from multiple studies. The publication of raw material is a necessary step for wide-area meta-analyses and study design, data collection and reporting require harmonization. This is an opinion paper, not a consensus position, and is open to debate. This work reflects our view about how data should be communicated in mainstream journals. We indicate rules that should be observed in recording weather data, to avoid serendipitous correlations between the density of ticks and climate variables and recommend the inclusion of raw data in reports. We stress the need for standardized methods to collect ticks that cannot be obtained by standard flagging. The reporting of infection rates of pathogens in ticks should avoid conclusions based on pure molecular findings in feeding ticks. Studies demonstrating the vectorial capacity of ticks should not be supported only by molecular surveys of feeding ticks. Vacuous conclusions about vectorial or reservoir status based solely on the finding of genomic material of a pathogen should be discouraged. We stress that phylogenetic studies based on random selection of sequences from GenBank are unsuitable. We firmly support the development of a dedicated server of curated sequences of ticks and pathogens as a standard for future studies.