Angiostrongylus cantonensis Nematode Invasion Pathway, Mallorca, Spain.

Neural angiostrongyliasis is an emerging zoonosis caused by the rat lungworm, Angiostrongylus cantonensis. In humans, infection with this nematode often results in eosinophilic meningitis and other severe disorders of the central nervous system. Europe was deemed a nonendemic region until 2018, when A. cantonensis worms were detected on the Mediterranean island of Mallorca, Spain, a tourism hotspot. Since that time, a sentinel surveillance system and a molecular approach have been used to follow the invasion path of the rat lungworm on the island. A. cantonensis worms have been found in animals from 8 locations on the island over 3 consecutive years. Our preliminary results show a recognizable pattern of clinical signs in infected hedgehogs and a single mitochondrial haplotype circulating in Mallorca. We present strong evidence confirming that the rat lungworm has successfully established and colonized an island in Europe and discuss observations and possible strategies for its early detection across continental Europe.

Combined morphology and DNA-barcoding to identify Plagiorhynchus cylindraceus cystacanths in Atelerix algirus.

The acanthocephalan parasite Plagiorhynchus cylindraceus has a global distribution and utilizes isopods and birds as intermediate and definitive hosts, respectively. Occasionally, mammals of various orders can act as paratenic hosts. In hedgehogs, severe cases have been reported in juvenile specimens due to secondary infections, as a consequence of complete penetrations of the intestinal wall by cystacanths. In a 66-month study period, we found seven larvae of this parasite encysted in both, the peritoneal cavity and intestine of the Algerian hedgehog, Atelerix algirus in Majorca. Morphology alone was insufficient to identify the species, due to the lack of previous reports and taxonomy-informative characters. In the present report, we combined the use of morphology and the DNA-barcoding approach to confirm to identify cystacanths as P. cylindraceus. This is the first report of this parasite in this hedgehog species. The epidemiological implications will be discussed, including the risk of zoonosis and the importance of using modern approaches to identify immature acanthocephalan larvae in wildlife hosts.

Angiostrongylus cantonensis in North African hedgehogs as vertebrate hosts, Mallorca, Spain, October 2018.

In October 2018, two Atelerix algirus hedgehogs were admitted to the Wildlife Rehabilitation Hospital in Mallorca (Balearic Islands, Spain) with signs of acute neurological disease. Necropsy detected immature, fully developed nematodes in the subarachnoid space of both hedgehogs, including a gravid female worm. DNA-based molecular tools confirmed the nematode as Angiostrongylus cantonensis, an important aetiological agent of eosinophilic meningitis in humans. So far this zoonotic parasite in has not been reported in western European wildlife.

Modelling the range expansion of the Tiger mosquito in a Mediterranean Island accounting for imperfect detection.

BACKGROUNDS: Aedes albopictus (Diptera; Culicidae) is a highly invasive mosquito species and a competent vector of several arboviral diseases that have spread rapidly throughout the world. Prevalence and patterns of dispersal of the mosquito are of central importance for an effective control of the species. We used site-occupancy models accounting for false negative detections to estimate the prevalence, the turnover, the movement pattern and the growth rate in the number of sites occupied by the mosquito in 17 localities throughout Mallorca Island. RESULTS: Site-occupancy probability increased from 0.35 in the 2012, year of first reported observation of the species, to 0.89 in 2015. Despite a steady increase in mosquito presence, the extinction probability was generally high indicating a high turnover in the occupied sites. We considered two site-dependent covariates, namely the distance from the point of first observation and the estimated yearly occupancy rate in the neighborhood, as predicted by diffusion models. Results suggested that mosquito distribution during the first year was consistent with what predicted by simple diffusion models, but was not consistent with the diffusion model in subsequent years when it was similar to those expected from leapfrog dispersal events. CONCLUSIONS: Assuming a single initial colonization event, the spread of Ae. albopictus in Mallorca followed two distinct phases, an early one consistent with diffusion movements and a second consistent with long distance, 'leapfrog', movements. The colonization of the island was fast, with ~90% of the sites estimated to be occupied 3 years after the colonization. The fast spread was likely to have occurred through vectors related to human mobility such as cars or other vehicles. Surveillance and management actions near the introduction point would only be effective during the early steps of the colonization.

Improving the detection of Angiostrongylus cantonensis in the brain tissues of mammalian hosts.

Angiostrongylus cantonensis is an invasive nematode parasite that causes eosinophilic meningitis in many vertebrate hosts, including humans. This parasite is spreading rapidly through the six continents, with Europe being the final frontier. Sentinel surveillance may be a cost-effective strategy to monitor the pathogen's arrival to new geographical regions. Necropsy, followed by tissue digestion, is often used to recover helminth parasites from vertebrate host tissues, however, to detect brain parasites, this protocol is poorly utilized. Our brain digestion protocol is easily performed and 1) reduces false positivity and negativity, 2) provides accurate estimates of parasite burden and 3) helps establish a more precise prevalence. Early detection of A. cantonensis increases the efficacy of prevention, treatment, and disease control strategies for susceptible animal populations and humans.

Potential intermediate hosts of Angiostrongylus cantonensis in the European Mediterranean region (Mallorca, Spain).

Angiostrongylus cantonensis is the main etiological agent of eosinophilic meningitis, a disease that often leads to severe neurological manifestations in mammals and birds. In recent years, the prevalence of this zoonotic nematode has dramatically increased as it expands into new territories beyond its native range in Southeast Asia and the Pacific Basin.Its arrival to Europe, the last continent to be invaded, has raised many questions concerning the parasite's life cycle, particularly in the Mediterranean region, where it is now endemic. This study aims to collect evidence about intermediate hosts (snail and slug species) involved in the transmission of the A. cantonensis in Mallorca. We have conducted a systematic surveillance of gastropods within 2 km radius areas, surrounding the specific locations where infected hedgehogs were found. We employed a sequence-based detection approach that included a species-specific PCR amplification followed by DNA sequencing of the internal transcribed spacer I (ITS-1). This conservative approach was essential to avoid cross reactions with the numerous metastrongylid species known to be circulating in Mallorca. Overall, we collected and identified 398 gastropods belonging to 17 species, of which 11% tested positive for A. cantonensis. These gastropods were collected from touristic settlements to agricultural lands. Five gastropod species: Theba pisana, Cornu aspersum, Deroceras reticulatum, Milax gagates and Otala lactea, had been previously incriminated as A. cantonensis intermediate hosts, while 6 species: Papillifera papillaris, Cochlicella acuta, Eobania vermiculata, Ganula lanuginosa, Milax nigricans and Rumina decollata, are newly recorded hosts. The findings of this study have important epidemiological implications, and further measures are discussed to prevent neuroangiostrongylosis cases.

Multiple invasions, Wolbachia and human-aided transport drive the genetic variability of Aedes albopictus in the Iberian Peninsula.

The Asian tiger mosquito, Aedes albopictus, is one of the most invasive species in the world. Native to the tropical forests of Southeast Asia, over the past 30 years it has rapidly spread throughout tropical and temperate regions of the world. Its dramatic expansion has resulted in public health concerns as a consequence of its vector competence for at least 16 viruses. Previous studies showed that Ae. albopictus spread has been facilitated by human-mediated transportation, but much remains unknown about how this has affected its genetic attributes. Here we examined the factors that contributed to shaping the current genetic constitution of Ae. albopictus in the Iberian Peninsula, where the species was first found in 2004, by combining population genetics and Bayesian modelling. We found that both mitochondrial and nuclear DNA markers showed a lack of genetic structure and the presence of worldwide dominant haplotypes, suggesting regular introductions from abroad. Mitochondrial DNA showed little genetic diversity compared to nuclear DNA, likely explained by infection with maternally transmitted bacteria of the genus Wolbachia. Multilevel models revealed that greater mosquito fluxes (estimated from commuting patterns and tiger mosquito population distribution) and spatial proximity between sampling sites were associated with lower nuclear genetic distance, suggesting that rapid short- and medium-distance dispersal is facilitated by humans through vehicular traffic. This study highlights the significant role of human transportation in shaping the genetic attributes of Ae. albopictus and promoting regional gene flow, and underscores the need for a territorially integrated surveillance across scales of this disease-carrying mosquito.

Angiostrongylosis in Animals and Humans in Europe.

Lungworms in the genus Angiostrongylus cause disease in animals and humans. The spread of Angiostrongylus vasorum within Europe and the recent establishment of Angiostrongylus cantonensis increase the relevance of these species to veterinary and medical practitioners, and to researchers in parasitology, epidemiology, veterinary science and ecology. This review introduces the key members of the genus present in Europe and their impacts on health, and updates the current epidemiological situation. Expansion of A. vasorum from localized pockets to wide distribution across the continent has been confirmed by a rising prevalence in foxes and increasing reports of infection and disease in dogs, while the list of carnivore and mustelid definitive hosts continues to grow. The tropically distributed rat lungworm A. cantonensis, meanwhile, has been recorded on islands south of Europe, previously the Canary Islands, and now also the Balearic Islands, although so far with limited evidence of zoonotic disease. Other members of the genus, namely, A. chabaudi, A. daskalovi and A. dujardini, are native to Europe and mainly infect wildlife, with unknown consequences for populations, although spill-over can occur into domestic animals and those in zoological collections. The epidemiology of angiostrongylosis is complex, and further research is needed on parasite maintenance in sylvatic hosts, and on the roles of ecology, behaviour and genetics in disease emergence. Improved surveillance in animals and humans is also required to support risk assessments and management.

Aedes albopictus diversity and relationships in south-western Europe and Brazil by rDNA/mtDNA and phenotypic analyses: ITS-2, a useful marker for spread studies.

BACKGROUND: Aedes albopictus is a very invasive mosquito, which has recently colonized tropical and temperate regions worldwide. Of concern is its role in the spread of emerging or re-emerging mosquito-borne diseases. Ae. albopictus from south-western Europe and Brazil were studied to infer genetic and phenetic diversity at intra-individual, intra-population and inter-population levels, and to analyse its spread. METHODS: Genotyping was made by rDNA 5.8S-ITS-2 and mtDNA cox1 sequencing to assess haplotype and nucleotide diversity, genetic distances and phylogenetic networks. Male and female phenotyping included combined landmark-and outlined-based geometric morphometrics of wing size and shape. RESULTS: Specimens from seven populations from Spain, France and Brazil provided 12 cox1 and 162 5.8S-ITS-2 haplotypes, with great genetic variability difference between both markers (0.9% vs 31.2%). Five cox1 haplotypes were shared with other countries, mainly Italy, USA and China, but none was shared between Europe and Brazil. The 5.8S-ITS-2 showed 2-7 intra-individual (mean 4.7) and 16-34 intra-/inter-population haplotypes (24.7), including haplotypes shared between Spain, France and Brazil. A 4.3% of ITS-2 haplotypes were shared, mainly with Italy, USA and Thailand, evidencing worldwide spread and introductions from areas where recent outbreaks of Ae. albopictus-transmitted pathogens occurred. Wing size showed sex differences. Wing shape distinguished between Brazilian and European specimens. Both genetic and morphometric markers showed differences between insular Spain and continental Spain, France and Brazil. CONCLUSIONS: ITS-2 proves to be a useful marker to assess Ae. albopictus spread, providing pronouncedly more information than cox1, including intra-individual, intra-population and inter-population levels, furnishing a complete overview of the evolutionary exchanges followed by this mosquito. Wing morphometry proves to be a useful phenotyping marker, allowing to distinguish different populations at the level of both male and female specimens. Results indicate the need for periodic surveillance monitorings to verify that no Ae. albopictus with high virus transmission capacity is introduced into Europe.

Molecular Characterization of Mosquito Diversity in the Balearic Islands.

Several outbreaks of mosquito-borne diseases have taken place in Europe in recent years. In Spain, both active and passive surveillance have demonstrated that dengue and West Nile viruses are currently circulating, and seven autochthonous dengue cases have been reported in the last 2 yr. The effectiveness of vector control programs largely depends on the accuracy of the taxonomic identification of the species. However, in Spain, identification almost completely relies on the use of morphological keys to characterize the mosquito fauna. This study investigates the congruence between molecular and morphological species boundaries in 13 Spanish mosquito taxa. The Cytochrome c oxidase subunit I (COI) gene region was sequenced from 60 adult specimens collected in Mallorca, plus several representatives from other Spanish regions for comparative purposes. Phylogenetic relationships were established using Bayesian and maximum-likelihood approaches. Using three species delimitation algorithms (ABGD, mPTP, and GMYC), we found strong evidence for cryptic speciation within Anopheles algeriensis Theobald, a widespread mosquito in the Mediterranean basin. We also delimited the Mallorcan rock pool mosquito Aedes mariae (Sergent & Sergent), from mainland European populations. Finally, we found difficulties in the use of wing characters in species keys to distinguish Culiseta annulata (Schrk) from Culiseta subochrea (Edwards). Given that these species are vectors of pathogens of medical relevance and have veterinary importance, their accurate taxonomic identification is essential in European vector surveillance programs.

A molecular phylogeny of mosquitoes in the Anopheles barbirostris Subgroup reveals cryptic species: implications for identification of disease vectors.

The Barbirostris Subgroup of the genus Anopheles includes six mosquito species that are almost identical in adult morphology, but differ in their roles in the transmission of malaria and filariasis within Southeast Asia. The lack of robust, diagnostic morphological characters in adults has contributed to extensive misidentification of the species. Mosquitoes were collected from localities in Thailand and Indonesia, with an emphasis on specimens identified in the field as An. barbirostris and An. campestris. A 754 bp COI mitochondrial gene fragment was sequenced from 136 specimens and the rDNA ITS2 region (c.1600-1800 bp) from 51 specimens. Phylogenetic analyzes based on Bayesian methods, distance measures and Maximum-parsimony produced five clades (I-V) that are congruent between the nuclear and mitochondrial data sets. Based on adult female morphology, it is deduced that three of these clades, I-III, are members of the Barbirostris Complex whereas Clade V is An. campestris. The identity of Clade IV is as yet unknown. Using a haplotype network analysis, Clade III was found to have a star-like genealogy, suggesting population expansion. There were no shared haplotypes between clades. In Afrotropical anopheline mosquitoes, speciation has been linked to the expansion of human populations and the development of agriculture. We postulate that the radiation of species within the Barbirostris Subgroup in Southeast Asia may similarly be linked to human population expansion and the agrarian revolution. The development of a propensity for feeding on the blood of humans in some species of the Subgroup would have led to the transmission of malaria protozoa and filarial nematodes.

The impact of indoor residual spraying of deltamethrin on dengue vector populations in the Peruvian Amazon.

Dengue is an important public health problem in the Amazon area of Peru, resulting in significant morbidity each year. As in other areas of the world, ultra-low volume (ULV) application of insecticides is the main strategy to reduce adult populations of the dengue vector Aedes aegypti, despite growing evidence of its limitations as a single control method. This study investigated the efficacy of deltamethrin S.C. applied through indoor residual spraying (IRS) of dwellings in reducing A. aegypti populations. The residual effect of the insecticide was tested by monthly bioassays on the three most common indoor surfaces found in the Amazon area: painted wood, unpainted wood and brick. The results showed that in an area with moderate levels of A. aegypti infestation, IRS dramatically reduced all immature indices the first week after deltamethrin IRS application and the adult index from 18.5 to 3.1, four weeks after intervention (p<0.05). Even though housing conditions facilitated reinfestation with A. aegypti (100% of the houses have open roof eaves, 31.5% lack sewage systems, and 60.4% collected rain in open containers), indices remained low compared to baseline 16 weeks after insecticide application. Bioassays showed that deltamethrin S.C. caused mortalities >80% 8 weeks after application on all types of surfaces. The residual effect of the insecticide was greater on brick than on wooden walls (p<0.05). Our results demonstrate that IRS can have both an immediate and sustained effect on reducing adult and immature A. aegypti populations and should be considered as an adult mosquito control strategy by dengue vector control programs.

Functional constraints and evolutionary dynamics of the repeats in the rDNA internal transcribed spacer 2 of members of the Anopheles barbirostris group.

BACKGROUND: The Anopheles barbirostris group is widely distributed in Southeast Asia. Although seven species have been formally described, a molecular analysis of the rDNA ITS2 and the mitochondrial cytochrome oxidase I gene suggests that the group includes species that are morphologically very similar or identical.We have previously shown that species in the Anopheles barbirostris Subgroup have an exceptionally large ITS2 (>1.5 kb), greater than in any other Anopheline group. However, the molecular processes responsible for generating such a large ITS2 have not previously been explored. METHODS: To determine the processes by which this large ITS2 is generated, we examined the sequence and secondary structure of the ITS2 of 51 specimens from five species of the Anopheles barbirostris Subgroup. These include the anthropophilic species An. campestris and three morphospecies of the Barbirostris Complex: An. vanderwulpi, An. barbirostris I and III, together with a previously undescribed member of this group (Clade IV). RESULTS AND CONCLUSIONS: All the specimens were found to have an ITS2 greater than 1.5 kb in length. The possibility that the spacer sequences amplified were pseudogenes was examined and discarded. The large size of ITS2 in the species studied is due to the presence of internal repeats of approximately 110 bp in length, confined to the central region of the spacer. Repeats varied markedly between the species examined, with respect to their organization, number and sequence similarity. The nucleotide diversity increased in direct relation to size variation and the presence of non-repeated elements.A secondary structure analysis showed that the repeats form hairpin structures with a wide range of free energy values. These hairpin structures are known to facilitate the subsequent processing of mature rRNA. An analysis of the repeats from the different species suggests they originate from a common ancestor, with the repeats appearing before speciation of the Barbirostris Group.

The influence of sheep age group on the seasonal prevalence of oestrosis in the island of Majorca.

Oestrosis is highly prevalent in Mediterranean countries. Understanding the life cycle of Oestrus ovis is crucial to design effective control measures of this myiasis, largely based on the use of macrocyclic lactones. We carried out a survey of ovine oestrosis in the island of Majorca (Spain) and found that 46.03% of animals were infested in a 13-month period. Interestingly, we found significant differences in oestrosis prevalences in winter and autumn when separating the animals by group of age (P<0.001). Pearson correlation analysis showed that prevalence in lambs younger than 4 months was significantly affected by changes in air temperature (P<0.05), but this association was not significant in adult sheep (P=0.081). Chronic infestations or unsystematic treatments may explain confusing results in adult sheep. Observing the evolution of the disease in young lambs, we determined that the hypobiotic period took place from October to February and the beginning of fly activity occurred between May and June. Interannual variations in oestrosis prevalence indicate the need of monitoring the disease to establish the appropriate timing of treatments. We hypothesize that lambs are better indicators of the seasonality of oestrosis than their older counterparts. Furthermore, we propose that observing O. ovis infestations in young lambs can be used as an efficient early warning system of fly activity, to be applied in future control programs.

Entomological factors affecting the low endemicity of Chagas disease in Nazca, Southwestern Peru.

Chagas disease is prevalent in Peru. The province of Nazca, in the southwestern region of the country, shows a high intradomiciliary infestation rate of Triatoma infestans bugs. Although the vector is present, the number of Chagas disease cases appears to be much lower than those reported in the neighboring region of Arequipa. We examined 624 T. infestans from Nazca to determine the current Trypanosoma cruzi infection rates, and found that no bugs were infected with this parasite. These results contrast with those found in Arequipa, where 19-30% triatomines have been reported infected. To compare their vectorial capacity, we infected 30 T. infestans specimens, selected both from Nazca and Arequipa, by feeding bugs on T. cruzi-infected mice. The parasites developed all stages expected in the vector; furthermore, the infective stage, metacyclic trypomastigote, was found in both insect populations from the second week after infection. In addition, those insects that accepted to be fed with mice blood defecated immediately after finishing blood meal, indicating that they might be efficient vectors. We maintain that differences observed in infection rates between vectors from Nazca and Arequipa may be explained by differences in host availability. In Arequipa hosts are mainly small animals, whereas in Nazca the main blood source comes from birds, which are refractory to the infection.