Phlebotomine sandflies and factors associated with their abundance in the leishmaniasis endemic area of Attiki, Greece.

Leishmaniasis is a parasitic disease of animals and humans caused by several Leishmania species and transmitted by phlebotomine sandflies. The aim of the present study was to identify the species of field collected phlebotomine sandflies in the endemic area of the Attiki during 4 consecutive years, to isolate the Leishmania parasites from the infected sandflies, and identify possible factors associated with sandfly abundance in the area. A total of 542 trappings were made in 46 collection sites, in purely urban areas, periurban areas, and purely rural areas in Attiki. Out of the 3254 sandflies trapped, 1448 (44.43%) were female and 241 (16.64%) of the females were blood fed while Leishmania infantum DNA was detected in the 0.41% of them. Regarding sandfly species, the most prevalent was Phlebotomus tobbi (41.52%) followed by Sergentomyia minuta (27.44%), P. neglectus (14.83%), P. simici (11.08%), P. papatasi (3.68%), P. similis (0.89%), and P. alexandri (0.56%). Periurban areas were found to have the highest density of sandfly populations.

Overwintering West Nile virus in active Culex pipiens mosquito populations in Greece.

The flavivirus West Nile Virus (WNV), which is transmitted by mosquitoes, poses a significant threat to both humans and animals, and its outbreaks often challenge public health in Europe and other continents. In recent years, there is an increasing trend of WNV incidence rates across several European countries. However, whether there is a year-round circulation or seasonal introduction has yet to be elucidated. Real-time polymerase chain reaction (PCR) identified WNV-positive Culex pipiens mosquitos in 6 out of 146 pools examined in winter 2022 that correspond to three out of the 24 study areas, located in two coastal regions units in Attica, Greece. Spatial dispersion of the six positive pools in the same region suggests a clustered circulation of WNV during the winter of 2022. This is the first study that documents the identification of WNV in Cx. pipiens populations, captured in adult traps during winter period. Our findings underscore the need to extend entomological surveillance programs to include the winter period, specifically in temperate climates and historically affected areas by WNV.

Field Evaluation of Commonly Used Adult Mosquito Traps in Greece.

Background: Entomological monitoring activities are a major part of mosquito and mosquito-borne diseases surveillance. Several trapping methods are implemented worldwide, aiming to gather data on species composition and their abundance in various study areas. Methods: Several methodological modifications, such as trapping systems baited with attractants or carbon dioxide, have been proposed to increase trap efficiency. The aim of this study was to test different trap types, commonly used in Greece to collect mosquitoes, with the addition of the Biogents Sentinel lure. Moreover, traps were placed in two distinct land types and two different heights above the ground to compare their efficacy. West Nile Virus is endemic in Greece, so we also aimed to detect viral presence and circulation in selected mosquito pools. Results: Adult mosquitoes of Aedes albopictus, Culex pipiens s.l., and Culiseta longiareolata were collected in both study areas. The trap type had a significant impact on the total collections, while the trap position and interaction between trap and position did not significantly affect mosquito catches. WNV was detected in Cx. pipiens s.l. pools examined from the two study areas. Conclusion: This study emphasizes the role of trapping methods as a key component for monitoring and surveillance of adult mosquito populations, reflecting that several trap types present with different mosquito species capture and catch rates.

Secondary vectors of Zika Virus, a systematic review of laboratory vector competence studies.

BACKGROUND: After the unprecedented Zika virus (ZIKV) outbreak in the western hemisphere from 2015-2018, Aedes aegypti and Ae. albopictus are now well established primary and secondary ZIKV vectors, respectively. Consensus about identification and importance of other secondary ZIKV vectors remain. This systematic review aims to provide a list of vector species capable of transmitting ZIKV by reviewing evidence from laboratory vector competence (VC) studies and to identify key knowledge gaps and issues within the ZIKV VC literature. METHODS: A search was performed until 15th March 2022 on the Cochrane Library, Lilacs, PubMed, Web of Science, WHOLIS and Google Scholar. The search strings included three general categories: 1) "ZIKA"; 2) "vector"; 3) "competence", "transmission", "isolation", or "feeding behavior" and their combinations. Inclusion and exclusion criteria has been predefined and quality of included articles was assessed by STROBE and STROME-ID criteria. FINDINGS: From 8,986 articles retrieved, 2,349 non-duplicates were screened by title and abstracts,103 evaluated using the full text, and 45 included in this analysis. Main findings are 1) secondary vectors of interest include Ae. japonicus, Ae. detritus, and Ae. vexans at higher temperature 2) Culex quinquefasciatus was not found to be a competent vector of ZIKV, 3) considerable heterogeneity in VC, depending on the local mosquito strain and virus used in testing was observed. Critical issues or gaps identified included 1) inconsistent definitions of VC parameters across the literature; 2) equivalency of using different mosquito body parts to evaluate VC parameters for infection (mosquito bodies versus midguts), dissemination (heads, legs or wings versus salivary glands), and transmission (detection or virus amplification in saliva, FTA cards, transmission to neonatal mice); 3) articles that fail to use infectious virus assays to confirm the presence of live virus; 4) need for more studies using murine models with immunocompromised mice to infect mosquitoes. CONCLUSION: Recent, large collaborative multi-country projects to conduct large scale evaluations of specific mosquito species represent the most appropriate approach to establish VC of mosquito species.

Wild Rabbit Exposure to Leishmania infantum, Toxoplasma gondii, Anaplasma phagocytophilum and Babesia caballi Evidenced by Serum and Aqueous Humor Antibody Detection.

Wild rabbits (Oryctolagus cuniculus) can be important sentinel species for the presence of zoonotic pathogens. Therefore, we collected blood samples from wild rabbits harvested by hunters during the hunting season 2019-2020 on the island of Lemnos, to determine exposure of wild rabbits to the zoonotic pathogens Leishmania infantum, Toxoplasma gondii, Anaplasma phagocytophilum and Babesia caballi, as well as aqueous humor to assess its diagnostic performance in terms of sensitivity, specificity, positive and negative likelihood ratios. Antibodies against these pathogens were detected by Indirect Immunofluorescence Antibody (IFA) assay. Out of the 72 wild rabbits included in the study, 4.2%, 5.5%, 18% and 9.7% were seropositive to L. infantum, T. gondii, A. phagocytophilum and B. caballi, respectively. Although less frequently, antibodies were also detected in aqueous humor of wild rabbits. The antibody detection in aqueous humor presented 100% specificity but decreased sensitivity compared to serum suggesting that aqueous humor could be successfully used in epidemiological studies to confirm exposure at the population level but has little diagnostic value at the individual level. This is the first report on the seropositivity of wild rabbits to A. phagocytophilum and B. caballi and the detection of antibodies against A. phagocytopylum, L. infantum, T. gondii and B. caballi in the aqueous humor.

A Comparison of Adult Mosquito Trapping Methods to Assess Potential West Nile Virus Mosquito Vectors in Greece during the Onset of the 2018 Transmission Season.

West Nile virus (WNV) threatens the health of humans and equines worldwide. Culex (Cx.) pipiens complex mosquitoes are major vectors but numerous other species have been implicated. Due to variations in blood-feeding behaviour, Cx. pipiens biotypes and hybrids influence transmission, from enzootic cycles (between mosquitoes and birds), to spill-over transmission to humans and equines. In this study, mosquitoes were collected in May-June 2018 during the early period of the transmission season from two regional units of Greece, where WNV cases had been reported in the previous four years (Palaio Faliro and Argolida). A total of 1062 mosquitoes were collected with Biogents Sentinel 2 traps collecting both a greater number of all mosquito species and the Cx. pipiens complex than CDC miniature light traps or Heavy Duty EVS traps. Molecular identification confirmed additional species including Aedes albopictus. The proportion of Cx. pipiens biotypes in Palaio Faliro was 54.5% pipiens, 20.0% molestus and 25.5% hybrids. In Argolida, the collection comprised 68.1% pipiens biotype, 8.3% molestus biotype and 23.6% hybrids. Screening resulted in WNV detection in three females of the pipiens biotype and in one hybrid. As hybrids play a role in spill-over transmission, these findings highlight the importance of entomological surveillance programs incorporating molecular xenomonitoring as an early warning before human cases at the onset of the transmission season.

Correction to: The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus complex (Diptera: Ceratopogonidae) at the European level.

An amendment to this paper has been published and can be accessed via the original article.

The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus Complex (Diptera: Ceratopogonidae) at the European level.

BACKGROUND: Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex. METHODS: Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex. RESULTS: Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus. CONCLUSIONS: To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region.