Laboratory colonization and maintenance of Anopheles atroparvus from the Ebro Delta, Spain.

BACKGROUND: Historically, Anopheles atroparvus has been considered one of the most important malaria vectors in Europe. Since malaria was eradicated from the European continent, the interest in studying its vectors reduced significantly. Currently, to better assess the potential risk of malaria resurgence on the continent, there is a growing need to update the data on susceptibility of indigenous Anopheles populations to imported Plasmodium species. In order to do this, as a first step, an adequate laboratory colony of An. atroparvus is needed. METHODS: Anopheles atroparvus mosquitoes were captured in rice fields from the Ebro Delta (Spain). Field-caught specimens were maintained in the laboratory under simulated field-summer conditions. Adult females were artificially blood-fed on fresh whole rabbit blood for oviposition. First- to fourth-instar larvae were fed on pulverized fish and turtle food. Adults were maintained with a 10% sucrose solution ad libitum. RESULTS: An An. atroparvus population from the Ebro Delta was successfully established in the laboratory. During the colonization process, feeding and hatching rates increased, while a reduction in larval mortality rate was observed. CONCLUSIONS: The present study provides a detailed rearing and maintenance protocol for An. atroparvus and a publicly available reference mosquito strain within the INFRAVEC2 project for further research studies involving vector-parasite interactions.

Plasmodium vivax Malaria Viewed through the Lens of an Eradicated European Strain.

The protozoan Plasmodium vivax is responsible for 42% of all cases of malaria outside Africa. The parasite is currently largely restricted to tropical and subtropical latitudes in Asia, Oceania, and the Americas. Though, it was historically present in most of Europe before being finally eradicated during the second half of the 20th century. The lack of genomic information on the extinct European lineage has prevented a clear understanding of historical population structuring and past migrations of P. vivax. We used medical microscope slides prepared in 1944 from malaria-affected patients from the Ebro Delta in Spain, one of the last footholds of malaria in Europe, to generate a genome of a European P. vivax strain. Population genetics and phylogenetic analyses placed this strain basal to a cluster including samples from the Americas. This genome allowed us to calibrate a genomic mutation rate for P. vivax, and to estimate the mean age of the last common ancestor between European and American strains to the 15th century. This date points to an introduction of the parasite during the European colonization of the Americas. In addition, we found that some known variants for resistance to antimalarial drugs, including Chloroquine and Sulfadoxine, were already present in this European strain, predating their use. Our results shed light on the evolution of an important human pathogen and illustrate the value of antique medical collections as a resource for retrieving genomic information on pathogens from the past.

Genetic affinities of an eradicated European Plasmodium falciparum strain.

Malaria was present in most of Europe until the second half of the 20th century, when it was eradicated through a combination of increased surveillance and mosquito control strategies, together with cross-border and political collaboration. Despite the severe burden of malaria on human populations, it remains contentious how the disease arrived and spread in Europe. Here, we report a partial Plasmodium falciparum nuclear genome derived from a set of antique medical slides stained with the blood of malaria-infected patients from Spain's Ebro Delta, dating to the 1940s. Our analyses of the genome of this now eradicated European P. falciparum strain confirms stronger phylogeographical affinity to present-day strains in circulation in central south Asia, rather than to those in Africa. This points to a longitudinal, rather than a latitudinal, spread of malaria into Europe. In addition, this genome displays two derived alleles in the pfmrp1 gene that have been associated with drug resistance. Whilst this could represent standing variation in the ancestral P. falciparum population, these mutations may also have arisen due to the selective pressure of quinine treatment, which was an anti-malarial drug already in use by the time the sample we sequenced was mounted on a slide.

Arbovirus surveillance: first dengue virus detection in local Aedes albopictus mosquitoes in Europe, Catalonia, Spain, 2015.

Dengue has emerged as the most important viral mosquito-borne disease globally. The current risk of dengue outbreaks in Europe appeared with the introduction of the vector Aedes albopictus mosquito in Mediterranean countries. Considering the increasing frequency of dengue epidemics worldwide and the movement of viraemic hosts, it is expected that new autochthonous cases will occur in the future in Europe. Arbovirus surveillance started in Catalonia in 2015 to monitor imported cases and detect possible local arboviral transmission. During 2015, 131 patients with a recent travel history to endemic countries were tested for dengue virus (DENV) and 65 dengue cases were detected. Twenty-eight patients with a febrile illness were viraemic, as demonstrated by a positive real-time RT-PCR test for DENV in serum samples. Entomological investigations around the viraemic cases led to the detection of DENV in a pool of local Ae. albopictus captured in the residency of one case. The sequence of the DENV envelope gene detected in the mosquito pool was identical to that detected in the patient. Our results show how entomological surveillance conducted around viraemic travellers can be effective for early detection of DENV in mosquitoes and thus might help to prevent possible autochthonous transmission.

Mitochondrial DNA from the eradicated European Plasmodium vivax and P. falciparum from 70-year-old slides from the Ebro Delta in Spain.

Phylogenetic analysis of Plasmodium parasites has indicated that their modern-day distribution is a result of a series of human-mediated dispersals involving transport between Africa, Europe, America, and Asia. A major outstanding question is the phylogenetic affinity of the malaria causing parasites Plasmodium vivax and falciparum in historic southern Europe-where it was endemic until the mid-20th century, after which it was eradicated across the region. Resolving the identity of these parasites will be critical for answering several hypotheses on the malaria dispersal. Recently, a set of slides with blood stains of malaria-affected people from the Ebro Delta (Spain), dated between 1942 and 1944, have been found in a local medical collection. We extracted DNA from three slides, two of them stained with Giemsa (on which Plasmodium parasites could still be seen under the microscope) and another one consisting of dried blood spots. We generated the data using Illumina sequencing after using several strategies aimed at increasing the Plasmodium DNA yield: depletion of the human genomic (g)DNA content through hybridization with human gDNA baits, and capture-enrichment using gDNA derived from P. falciparum Plasmodium mitochondrial genome sequences were subsequently reconstructed from the resulting data. Phylogenetic analysis of the eradicated European P. vivax mtDNA genome indicates that the European isolate is closely related to the most common present-day American haplotype and likely entered the American continent post-Columbian contact. Furthermore, the European P. falciparum mtDNA indicates a link with current Indian strains that is in agreement with historical accounts.

Novel flaviviruses detected in different species of mosquitoes in Spain.

We report the characterization of three novel flaviviruses isolated in Spain. Marisma Mosquito virus, a novel mosquito borne virus, was isolated from Ochlerotatus caspius mosquitoes; Spanish Ochlerotatus flavivirus and Spanish Culex flavivirus, two novel insect flaviviruses, were isolated from Oc. caspius and Culex pipiens, respectively. During this investigation, we designed a sensitive RT-nested polymerase chain reaction method that amplifies a 1019bp fragment of the flavivirus NS5 gene and could be directly used in clinical or environmental samples for flavivirus characterization and surveillance. Analysis of the sequence generated from that amplicon contains enough phylogenetic information for proper taxonomic studies. Moreover, the use of this tool allowed the detection of additional flavivirus DNA forms in Culex, Culiseta, and Ochlerotatus mosquitoes.

Malaria resurgence risk in southern Europe: climate assessment in an historically endemic area of rice fields at the Mediterranean shore of Spain.

BACKGROUND: International travel and immigration have been related with an increase of imported malaria cases. This fact and climate change, prolonging the period favouring vector development, require an analysis of the malaria transmission resurgence risk in areas of southern Europe. Such a study is made for the first time in Spain. The Ebro Delta historically endemic area was selected due to its rice field landscape, the presence of only one vector, Anopheles atroparvus, with densities similar to those it presented when malaria was present, in a situation which pronouncedly differs from already assessed potential resurgence areas in other Mediterranean countries, such as France and Italy, where many different Anopheles species coexist and a different vector species dominates. METHODS: The transmission risk was assessed analysing: 1) climate diagrams including the minimum temperature for Plasmodium falciparum and Plasmodium vivax development; 2) monthly evolution of the Gradient Model Risk (GMR) index, specifying transmission risk period and number of potential Plasmodium generations; 3) ecological characteristics using remote sensing images with the Eurasia Land Cover characteristics database and the monthly evolution of the Normalized Difference Vegetation Index (NDVI); 4) evaluation of A. atroparvus population dynamics. RESULTS: Climatological analyses and GMR index show that a transmission risk presently exists, lasting from May until September for P. falciparum, and from May until October for P. vivax. The GMR index shows that the temperature increase does not actually mean a transmission risk increase if accompanied by a precipitation decrease reducing the number of parasite generations and transmission period. Nevertheless, this limitation is offset by the artificial flooding of the rice fields. Maximum NDVI values and A. atroparvus maximum abundance correspond to months with maximum growth of the rice fields. CONCLUSIONS: The Ebro Delta presents the ecological characteristics that favour transmission. The temperature increase has favoured a widening of the monthly potential transmission window with respect to when malaria was endemic. The combined application of modified climate diagrams and GMR index, together with spatial characterization conforms a useful tool for assessing potential areas at risk of malaria resurgence. NDVI is a good marker when dealing with a rice field area.

Surveillance of arboviruses in Spanish wetlands: detection of new flavi- and phleboviruses.

The presence of viruses in arthropods in Spain has been studied over 5 years. Flaviviruses similar to cell-fusing agent, sequences of a flavivirus related to those transmitted by mosquitoes, and a phlebovirus similar to Naples and Toscana viruses were detected. Their potential human or animal pathogenicity should be studied.

RDNA sequences of Anopheles species from the Iberian Peninsula and an evaluation of the 18S rRNA gene as phylogenetic marker in anophelinae.

The complete 18S rDNA and internal transcribed spacer (ITS)-2 rDNA sequences were obtained from Anopheles atroparvus Van Thiel and Anopheles plumbeus Stephens from two areas of Spain. The number of nucleotide differences in the 18S rDNA of the two species is high compared with differences in the same gene of other invertebrate vectors. In Anopheles, short 18S rDNA sequences are richer in AT than the longer sequences, which are richer in GC and include extremely GC-biased expanded regions. Four small regions in the variable regions V4 and V7 contain the majority of nucleotide differences. The results did not support the use of partial sequences for relationship analyses. Genetic distances and phylogenetic analyses supported the most recent classification of Anopheles. The complete 18S rDNA sequence is better for studying anopheline phylogenetics.