Analysis of the current risk of Leishmania infantum transmission for domestic dogs in Spain and Portugal and its future projection in climate change scenarios.

Canine leishmaniosis, caused by the protozoan parasite Leishmania infantum, is a cosmopolitan vector-borne zoonosis, transmitted principally by Phlebotomus perniciosus in Spain and Portugal, where it is considered an endemic disease. Ecoinformatics tools such as ecological niche models (ENM) have been successfully tested to model the distribution of the risk of infection of different parasitosis as they take into account environmental variables vital for their survival. The risk map proposed in this study combines the potential distribution of Ph. perniciosus in the Iberian Peninsula and the calculation of the infection rate of the parasite in the vector to model the risk of contracting the disease in a more realistic way. In fact, this weighting strategy improves the predictive power of the resulting model (R2 = 0.42, p = < 0.01) compared to the Ph. perniciosus ENM model alone (R2 = 0.13, p > 0.05). The places with the highest risk of transmission are the southwest and central peninsular area, as well as the Mediterranean coast, the Balearic Islands and the Ebro basin, places where the ideal habitat of Ph. perniciosus and the infection rate is also high. In the case of future projections under climate change scenarios, an increase in the risk of infection by L. infantum can be observed in most of the territory (4.5% in 2040, 71.6% in 2060 and 63% in 2080), mainly in the northern part of the peninsula. The use of ENMs and their weighting with the infection rate in Ph. perniciosus is a useful tool in predicting the risk of infection for L. infantum in dogs for a given area. In this way, a more complete model can be obtained to facilitate prevention and control.

Modelling the spatial risk of malaria through probability distribution of Anopheles maculipennis s.l. and imported cases.

Malaria remains one of the most important infectious diseases globally due to its high incidence and mortality rates. The influx of infected cases from endemic to non-endemic malaria regions like Europe has resulted in a public health concern over sporadic local outbreaks. This is facilitated by the continued presence of competent Anopheles vectors in non-endemic countries.We modelled the potential distribution of the main malaria vector across Spain using the ensemble of eight modelling techniques based on environmental parameters and the Anopheles maculipennis s.l. presence/absence data collected from 2000 to 2020. We then combined this map with the number of imported malaria cases in each municipality to detect the geographic hot spots with a higher risk of local malaria transmission.The malaria vector occurred preferentially in irrigated lands characterized by warm climate conditions and moderate annual precipitation. Some areas surrounding irrigated lands in northern Spain (e.g. Zaragoza, Logroño), mainland areas (e.g. Madrid, Toledo) and in the South (e.g. Huelva), presented a significant likelihood of A. maculipennis s.l. occurrence, with a large overlap with the presence of imported cases of malaria.While the risk of malaria re-emergence in Spain is low, it is not evenly distributed throughout the country. The four recorded local cases of mosquito-borne transmission occurred in areas with a high overlap of imported cases and mosquito presence. Integrating mosquito distribution with human incidence cases provides an effective tool for the quantification of large-scale geographic variation in transmission risk and pinpointing priority areas for targeted surveillance and prevention.

Geographic distribution of the V1016G knockdown resistance mutation in Aedes albopictus: a warning bell for Europe.

BACKGROUND: Colonization of large part of Europe by the Asian tiger mosquito Aedes albopictus is causing autochthonous transmission of chikungunya and dengue exotic arboviruses. While pyrethroids are recommended only to reduce/limit transmission, they are widely implemented to reduce biting nuisance and to control agricultural pests, increasing the risk of insurgence of resistance mechanisms. Worryingly, pyrethroid resistance (with mortality < 70%) was recently reported in Ae. albopictus populations from Italy and Spain and associated with the V1016G point mutation in the voltage-sensitive sodium channel gene conferring knockdown resistance (kdr). Genotyping pyrethroid resistance-associated kdr mutations in field mosquito samples represents a powerful approach to detect early signs of resistance without the need for carrying out phenotypic bioassays which require availability of live mosquitoes, dedicated facilities and appropriate expertise. METHODS: Here we report results on the PCR-genotyping of the V1016G mutation in 2530 Ae. albopictus specimens from 69 sampling sites in 19 European countries. RESULTS: The mutation was identified in 12 sites from nine countries (with allele frequencies ranging from 1 to 8%), mostly distributed in two geographical clusters. The western cluster includes Mediterranean coastal sites from Italy, France and Malta as well as single sites from both Spain and Switzerland. The eastern cluster includes sites on both sides of the Black Sea in Bulgaria, Turkey and Georgia as well as one site from Romania. These results are consistent with genomic data showing high connectivity and close genetic relationship among West European populations and a major barrier to gene flow between West European and Balkan populations. CONCLUSIONS: The results of this first effort to map kdr mutations in Ae. albopictus on a continental scale show a widespread presence of the V1016G allele in Europe, although at lower frequencies than those previously reported from Italy. This represents a wake-up call for mosquito surveillance programs in Europe to include PCR-genotyping of pyrethroid resistance alleles, as well as phenotypic resistance assessments, in their routine activities.

A Survey on Native and Invasive Mosquitoes and Other Biting Dipterans in Northern Spain.

PURPOSE: Haematophagous Diptera, such as mosquitoes (Culicidae), biting midges (Ceratopogonidae), and black flies (Simuliidae), are important insects for public and animal health due to their capacity to bite and transmit pathogens. Outdoor recreation areas are usually affected by biting species and provide suitable habitats to both adult and immature stages. This study aimed to determine the species diversity and larval sites of these Diptera groups in two golf courses. METHODS: A multi-method collection approach using ultraviolet-CDC traps, human landing catches, collection in breeding sites, and ovitraps was implemented during summer 2020 in northern Spain. Insects were determined by morphological features accompanied by DNA barcoding. RESULTS: A total of ten native mosquito species were recorded either as adults or as larval stages. The invasive species Aedes japonicus was collected only at egg or pupa stage in ovitraps. Culex pipiens s.l. and Culex torrentium were both common mosquito species accounting for 47.9% of the total larval site collections and their larvae might be found in a wide range of natural and artificial sites. Culiseta longiareolata specimens were also prominent (30.1% of the total) and occurred exclusively in man-made water-filled containers. A total of 13 Culicoides species were identified, 10 of which were captured by ultraviolet-CDC traps, particularly members of the Obsoletus complex (Culicoides obsoletus/Culicoides scoticus, 74.9%) and seven species by emergence traps, being the two most abundant C. kibunensis (44.8%) and C. festivipennis (34.9%). Simulium cryophilum was also collected hovering around the operator under field sampling. CONCLUSION: A comprehensive representation of the blood-sucking Diptera fauna and their larval sites was obtained by the multi-method approach in two Spanish golf courses.

Phlebotomine sand flies (Diptera, Psychodidae) from Spain: an updated checklist and extended distributions.

Phlebotomine sand flies (Diptera: Psychodidae) are the natural vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae) and phleboviruses (Bunyavirales: Phenuiviridae). In Spain, these vectors appear to be increasing their geographical distribution and have serious repercussions on public and veterinary health, encouraging studies of sand flies and their associated pathogens. An up-to-date and easily accessible compendium of current and historical data on their presence and detailed distribution is a crucial step towards the development and implementation of appropriate preventive strategies. A checklist on the presence and distribution of sand flies in Spain is compiled from data extracted from a comprehensive review of scientific literature published between 1909 and 2021 and our new records on the presence of sand flies specimens collected under the entomological surveillance of bluetongue vectors from the Spanish Ministry of Agriculture, Fishery and Food (MAPA) during the period 2004-2021. In total, 13 Spanish species of sand flies (two of them with controversial status) belonging to two genera and six subgenera are presented in this updated checklist, including new distribution data for seven species, among which several stand out as confirmed or suspected vectors of Leishmaniainfantum: Phlebotomusariasi, Ph.langeroni, Ph.mascittii, and Ph.perniciosus.

At the tip of an iceberg: citizen science and active surveillance collaborating to broaden the known distribution of Aedes japonicus in Spain.

BACKGROUND: Active surveillance aimed at the early detection of invasive mosquito species is usually focused on seaports and airports as points of entry, and along road networks as dispersion paths. In a number of cases, however, the first detections of colonizing populations are made by citizens, either because the species has already moved beyond the implemented active surveillance sites or because there is no surveillance in place. This was the case of the first detection in 2018 of the Asian bush mosquito, Aedes japonicus, in Asturias (northern Spain) by the citizen science platform Mosquito Alert. METHODS: The collaboration between Mosquito Alert, the Ministry of Health, local authorities and academic researchers resulted in a multi-source surveillance combining active field sampling with broader temporal and spatial citizen-sourced data, resulting in a more flexible and efficient surveillance strategy. RESULTS: Between 2018 and 2020, the joint efforts of administrative bodies, academic teams and citizen-sourced data led to the discovery of this species in northern regions of Spain such as Cantabria and the Basque Country. This raised the estimated area of occurrence of Ae. japonicus from < 900 km2 in 2018 to > 7000 km2 in 2020. CONCLUSIONS: This population cluster is geographically isolated from any other population in Europe, which raises questions about its origin, path of introduction and dispersal means, while also highlighting the need to enhance surveillance systems by closely combining crowd-sourced surveillance with public health and mosquito control agencies' efforts, from local to continental scales. This multi-actor approach for surveillance (either passive and active) shows high potential efficiency in the surveillance of other invasive mosquito species, and specifically the major vector Aedes aegypti which is already present in some parts of Europe.

Mosquito fauna in Extremadura (western Spain): Updated catalog with new records, distribution maps, and medical relevance.

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.

Characterization of the first autochthonous dengue outbreak in Spain (August-September 2018).

On October 3rd 2018, dengue virus (DENV) infection was confirmed in three family members (symptoms onset between August 18th and 27th) without travel history outside of Spain. They had been together in the Autonomous Communities (AC) of Murcia and Andalusia. By the end of October, a second cluster of two dengue cases (symptoms onset on September 27th and 30th) was confirmed in the AC of Murcia. DENV type 1 sequence was identical to the first cluster, and the epidemiological link was a visit from a case of the first cluster to a fruit-farm neighboring the small village of residence of the second cluster. The entomological investigation found Aedes albopictus activity in this area although all mosquitoes were PCR-negative for DENV. This is the first autochthonous dengue outbreak identified in Spain. This outbreak highlights challenges to timely detect and respond to DENV transmission and opens questions on dengue dynamics in a non-endemic context.

Evidence for infection but not transmission of Zika virus by Aedes albopictus (Diptera: Culicidae) from Spain.

BACKGROUND: A number of mosquito-borne viruses such as dengue virus (DENV), Usutu virus (USUV), West Nile virus (WNV) are autochthonously transmitted in Europe and six invasive mosquito species have been detected in this temperate region. This has increased the risk for the emergence of further mosquito-borne diseases. However, there is a paucity of information on whether European populations of invasive mosquito species are competent to transmit arboviruses. In this study, the susceptibility of Aedes albopictus originating from Spain and a laboratory-adapted colony of Aedes aegypti, was assessed for infection with, and transmission of Zika virus (ZIKV). Vertical transmission in both species was also assessed. METHODS: Aedes albopictus colonised from eggs collected in Spain and an existing colony of Ae. aegypti were fed infectious blood meals containing ZIKV (Polynesian strain) at 1.6 × 107 PFU/ml. Blood-fed mosquitoes were separated and maintained at 20 °C or 25 °C. Legs, saliva and bodies were sampled from specimens at 7, 14 and 21 days post-infection (dpi) in order to determine infection, dissemination and transmission rates. All samples were analysed by real-time RT-PCR using primers targeting the ZIKV NS1 gene. RESULTS: At 14 dpi and 21 dpi, ZIKV RNA was detected in the bodies of both species at both temperatures. However, live virus only was detected in the saliva of Ae. aegypti at 25 °C with a transmission rate of 44%. No evidence for virus expectoration was obtained for Ae. albopictus under any condition. Notably, ZIKV RNA was not detectable in the saliva of Ae. aegypti at 20 °C after 21 days. No vertical transmission of ZIKV was detected in this study. CONCLUSIONS: Experimental infection of Ae. albopictus colonized from Spain with ZIKV did not result in expectoration of virus in saliva in contrast to results for Ae. aegypti. No evidence of vertical transmission of virus was observed in this study. This suggests that this strain of Ae. albopictus is not competent for ZIKV transmission under the conditions tested.

First detection of Aedes japonicus in Spain: an unexpected finding triggered by citizen science.

BACKGROUND: Aedes japonicus is an invasive vector mosquito from Southeast Asia which has been spreading across central Europe since the year 2000. Unlike the Asian Tiger mosquito (Aedes albopictus) present in Spain since 2004, there has been no record of Ae. japonicus in the country until now. RESULTS: Here, we report the first detection of Ae. japonicus in Spain, at its southernmost location in Europe. This finding was triggered by the citizen science platform Mosquito Alert. In June 2018, a citizen sent a report via the Mosquito Alert app from the municipality of Siero in the Asturias region (NW Spain) containing pictures of a female mosquito compatible with Ae. japonicus. Further information was requested from the participant, who subsequently provided several larvae and adults that could be classified as Ae. japonicus. In July, a field mission confirmed its presence at the original site and in several locations up to 9 km away, suggesting a long-time establishment. The strong media impact in Asturias derived from the discovery raised local participation in the Mosquito Alert project, resulting in further evidence from surrounding areas. CONCLUSIONS: Whilst in the laboratory Ae. japonicus is a competent vector for several mosquito-borne pathogens, to date only West Nile virus is a concern based on field evidence. Nonetheless, this virus has yet not been detected in Asturias so the vectorial risk is currently considered low. The opportunity and effectiveness of combining citizen-sourced data to traditional surveillance methods are discussed.

How to increase the population of a Phlebotomus perniciosus (Diptera: Psychodidae) colony: a new method.

The sandfly Phlebotomus perniciosus is the most widespread vector of Leishmania infantum in Spain. Laboratory colonisation represents the most feasible source of information on the biology of these insects, but in conducting any study, the density of individuals in the colony may drop to such an extent that it is sometimes difficult to recover the initial population levels. A new technique was tested for the recovery of sandfly eggs in three different colonies; the recovery rate was studied by comparing the standard method of mass rearing with this new method of colony management. The results demonstrate a mean increase of 18.4% in adult production, a growth in colony productivity that justifies the inclusion of this process in the routine maintenance of any colony of sandflies.

How to increase the population of a Phlebotomus perniciosus (Diptera: Psychodidae) colony: a new method

The sandfly Phlebotomus perniciosus is the most widespread vector of Leishmania infantum in Spain. Laboratory colonisation represents the most feasible source of information on the biology of these insects, but in conducting any study, the density of individuals in the colony may drop to such an extent that it is sometimes difficult to recover the initial population levels. A new technique was tested for the recovery of sandfly eggs in three different colonies; the recovery rate was studied by comparing the standard method of mass rearing with this new method of colony management. The results demonstrate a mean increase of 18.4 percent in adult production, a growth in colony productivity that justifies the inclusion of this process in the routine maintenance of any colony of sandflies.