Molecular detection of Usutu virus in pools of Culex pipiens mosquitoes in Greece.

Usutu virus (USUV) is a mosquito-borne flavivirus originating from Africa, that belongs to the Japanese encephalitis virus (JEV) complex. In nature, USUV involves Culex spp. mosquitoes acting as vectors and birds as amplifying hosts. The virus has recently spread in Europe and is considered an emerging human pathogen. This is the first research study performed in Greece revealing the presence and circulation of USUV in Culex spp. mosquito populations. Out of the 1,500 mosquito pools tested with real-time RT-PCR, four (Roesch et al., 2019) were positive for USUV. All four pools were collected from the region of Central Macedonia, Northern Greece.

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.

Mitigating the Threat of Invasive Mosquito Species Expansion: A Comprehensive Entomological Surveillance Study on Kastellorizo, a Remote Greek Island.

The expansion of the tiger mosquito, a vector that can transmit diseases such as dengue, chikungunya, and Zika virus, poses a growing threat to global health. This study focuses on the entomological surveillance of Kastellorizo, a remote Greek island affected by its expansion. This research employs a multifaceted approach, combining KAP survey (knowledge, attitude, practices), mosquito collection using adult traps and human landing catches, and morphological and molecular identification methods. Results from questionnaires reveal community awareness and preparedness gaps, emphasizing the need for targeted education. Mosquito collections confirm the presence of the Aedes albopictus, Aedes cretinus, and Culex pipiens mosquitoes, highlighting the importance of surveillance. This study underscores the significance of community engagement in entomological efforts and proposes a citizen science initiative for sustained monitoring. Overall, this research provides essential insights for developing effective mosquito control programs in remote island settings, thereby emphasizing the importance of adopting a One Health approach to mitigate the spread of vector-borne diseases.

Winter survival of adults of two geographically distant populations of Aedes albopictus in a microclimatic environment of Athens, Greece.

Overwintering capacity is a major step towards establishment of invasive mosquitoes from the tropics in temperate zone areas and one of the main elements determining next seasons' population size that regulates disease transmission of competent invasive vector species. The Asian tiger mosquito, Aedes albopictus (Skuse) (Diptera: Culiciidae) is an aggressive invasive species that has greatly expanded its geographical boundaries over the last few decades. The species' ability to induce photoperiodic-based egg diapause allows its overwintering in temperate regions, which favors its establishment in higher latitudes. In warmer temperate areas winter survival can be accomplished in the adult stage as well especially in human-made shelters. Aedes albopictus is already showing signs of adaptation to colder climates which may result in disease transmission in new areas. Although the Asian tiger mosquito has invaded Greece since 2003-4, little is known regarding its overwintering capacity in the country, especially as far as adults are regarded. We studied the survival of Ae. albopictus adults during winter in a protected shelter in Athens, the capital city of Greece. The study involved two geographically isolated populations originating from Chania (Crete, most southern part of Greece), and Palaio Faliro (Athens, Central Greece). We exposed different cohorts of F3 adults of the above two populations that emerged from field collected eggs to "winter condition" from November 2018 to beginning of January 2019. in a protected microclimatic environment. Adult mortality was recorded systematically until the death of the last individual in the cohort. Results demonstrated evidence of winter survival of adults for both populations. Longevity of both females and males of the Palaio Faliro population was longer than that of the Chania population for almost all installation dates. Survival curves, regardless of the date of installation, were steeper for females of the Chania population compared to those from Palaio Faliro. In conclusion, overwinter survival of both populations in the adult stage highlights the need for future studies, considering local and microclimatic condition that vary significantly between regions. Winter climatic conditions play vital role in adult survival of this highly important public health species concerning distribution limits and knowledge on winter survival of Ae. albopictus transmitted viruses.

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.

Entomological Surveillance Activities in Regions in Greece: Data on Mosquito Species Abundance and West Nile Virus Detection in Culex pipiens Pools (2019-2020).

Human cases of West Nile virus (WNV) infections have been recorded in Greece since 2010, with seasonal outbreaks (summer-autumn) on an almost annual basis, caused mainly by the WNV lineage 2 strain (Nea Santa-Greece-2010). National Public Health Organization (NPHO) in Greece is annually implementing enhanced surveillance of human WNV infection, in order to promptly identify human cases of WNV infection and monitor distribution in terms of time and place. Entomological surveillance activities were carried out on a national basis in 2019 and 2020, under NPHO coordination and the collaboration of several private subcontractors, along with the Unit of Medical Entomology, Laboratory for Surveillance of Infectious Diseases (LSID). The aim was to monitor mosquito species composition, abundance, and WNV circulation in mosquito pools of Culex pipiens s.l. species. Adult mosquito traps were placed in selected sites; collected samples were morphologically characterized and pooled by date of collection, location, and species types. Female Culex pipiens s.l. pools were tested for WNV and WNV infection rates (MIR and MLE) were estimated. Highest mean number of female Culex pipiens s.l. species was recorded in Central Macedonia both for 2019 and 2020. Six hundred and fifty-nine mosquito pools (147 in 2019 and 512 in 2020) of female Culex pipiens s.l. were examined for WNV presence. The highest MLE was detected in Western Macedonia in 2019 and in Thessaly in 2020. Here, we present data on the mosquito species composition in the studied areas and WNV detection in mosquitoes from areas in Greece where the specific national mosquito surveillance program was implemented, for two years, 2019 and 2020.

Molecular Evidence of a Broad Range of Pathogenic Bacteria in Ctenocephalides spp.: Should We Re-Examine the Role of Fleas in the Transmission of Pathogens?

The internal microbiome of common cat and dog fleas was studied for DNA evidence of pathogenic bacteria. Fleas were grouped in pools by parasitized animal. DNA was extracted and investigated with 16S metagenomics for medically relevant (MR) bacteria, based on the definitions of the International Statistical Classification of Diseases and Related Health Problems (WHO). The MR bacterial species totaled 40, were found in 60% of flea-pools (N = 100), and included Acinetobacterbaumannii, Bacteroidesfragilis, Clostridiumperfringens, Enterococcusfaecalis, E. mundtii, Fusobacteriumnucleatum, Haemophilusaegyptius, Kingellakingae, Klebsiellapneumoniae, Leptotrichiabuccalis, L. hofstadii, Moraxellalacunata, Pasteurellamultocida, Propionibacteriumacnes, P. propionicum, Proteusmirabilis, Pseudomonasaeruginosa, Rickettsiaaustralis, R. hoogstraalii, Salmonellaenterica, and various Bartonella, Staphylococcus, and Streptococcus species. B. henselae (p = 0.004) and B. clarridgeiae (p = 0.006) occurred more frequently in fleas from cats, whereas Rickettsiahoogstraalii (p = 0.031) and Propionibacteriumacnes (p = 0.029) had a preference in fleas from stray animals. Most of the discovered MR species can form biofilm, and human exposure may theoretically occur through the flea-host interface. The fitness of these pathogenic bacteria to cause infection and the potential role of fleas in the transmission of a broad range of diseases should be further investigated.

Molecular detection of Rickettsia felis in common fleas in Greece and comparative evaluation of genotypic methods.

INTRODUCTION: Rickettsia felis is the causative agent of flea-borne spotted fever (FBSF), an emerging zoonosis. Although there is evidence of FBSF in Greece, fleas, the classic vectors of R. felis, have not been adequately studied. Thus, the aim of this study was to detect and characterize bacteria of genus Rickettsia and especially R. felis from common fleas parasitizing domestic cats and dogs in Greece and evaluate the efficiency of established molecular techniques. MATERIALS AND METHODS: DNA of flea-pools (samples) by animal-host was investigated by quantitative real-time PCRs (qPCR), and 16S metagenomics (16S). Determination of Rickettsia spp., Rickettsia felis-like organisms (RFLOs), and R. felis was based on a combination of qPCRs targeting gltA and ompB genes, 16S automated metagenomics and manual comparison of 16S sequences for >99% similarity with the publicly available 16S R. felis GenBank sequences using the Basic Local Alignment Search Tool (BLAST>99). Information for the animal-hosts was available and statistically analyzed. RESULTS: Among 100 flea-pools, R. felis was detected in 14 samples with a combination of six, five and three assays in 10, two and two samples, respectively. The sensitivity of the assays for Rickettsia genus (16S, and genus specific qPCRs) ranged from 62.5% to 93.8% and the specificity from 65.0% to 100%. R. felis-targeting qPCRs for gltA and ompB demonstrated sensitivity and specificity of 92.9% and 100%, and 100.0% and 87.5%, respectively. 16S metagenomics using the assay software was not able to identify R. felis positive specimens, although manual BLAST>99 did identify the species, but demonstrated sensitivity of 92.9% and specificity of 65.0%. No association of the detection rate of Rickettsia genus or R. felis, with the epidemiological data collected, was identified. CONCLUSIONS: These observations suggest the occurrence of R. felis in fleas from pets in Attica, Greece, but PCR and sequencing assays varied considerably in sensitivity and specificity and a consensus methodology for assigning the positivity status is required to be established.

Seasonal Phenological Patterns and Flavivirus Vectorial Capacity of Medically Important Mosquito Species in a Wetland and an Urban Area of Attica, Greece.

Seasonal patterns of mosquito population density and their vectorial capacity constitute major elements to understand the epidemiology of mosquito-borne diseases. Using adult mosquito traps, we compared the population dynamics of major mosquito species (Culex pipiens, Aedes albopictus, Anopheles spp.) in an urban and a wetland rural area of Attica Greece. Pools of the captured Cx. pipiens were analyzed to determine infection rates of the West Nile virus (WNV) and the Usutu virus (USUV). The data provided were collected under the frame of the surveillance program carried out in two regional units (RUs) of the Attica region (East Attica and South Sector of Attica), during the period 2017-2018. The entomological surveillance of adult mosquitoes was performed on a weekly basis using a network of BG-sentinel traps (BGs), baited with CO2 and BG-Lure, in selected, fixed sampling sites. A total of 46,726 adult mosquitoes were collected, with larger variety and number of species in East Attica (n = 37,810), followed by the South Sector of Attica (n = 8916). The collected mosquitoes were morphologically identified to species level and evaluated for their public health importance. Collected Cx. pipiens adults were pooled and tested for West Nile virus (WNV) and Usutu virus (USUV) presence by implementation of a targeted molecular methodology (real-time PCR). A total of 366 mosquito pools were analyzed for WNV and USUV, respectively, and 38 (10.4%) positive samples were recorded for WNV, while no positive pool was detected for USUV. The majority of positive samples for WNV were detected in the East Attica region, followed by the South Sector of Attica, respectively. The findings of the current study highlight the WNV circulation in the region of Attica and the concomitant risk for the country, rendering mosquito surveillance actions and integrated mosquito management programs as imperative public health interventions.

Sand Fly (Diptera: Psychodidae: Phlebotominae) Population Dynamics and Natural Leishmania Infections in Attica Region, Greece.

A 2-yr sand fly (Diptera: Psychodidae) seasonality study was performed in Attica Region, Greece, from June 2017 until November 2018, aiming also to detect the presence of Leishmania infection in the collected sand flies. In total, 701 sand flies were collected from urban areas within the Attica Region using BG-Sentinel traps, set weekly in eight fixed sites. Five species were identified morphologically and molecularly, namely Phlebotomus tobbi (Adler and Theodor), which was most the most commonly collected species, followed by P. Neglectus (Tonnoir), P. papatasi (Scopoli), P. simici (Theodor), and Sergentomyia minuta (Rondani). During both survey years sand fly populations peaked in late August to early September. Fifty-nine monospecific pools were examined for Leishmania detection by analyzing the ITS1 nuclear region using both RFLPs and sequencing, seven of which were found positive. Leishmania DNA was identified as L. infantum in six pools (five P. papatasi and one P. tobbi), whereas in one P. papatasi pool Leishmania DNA was identified as L. tropica. This is the first time that L. tropica has been detected in naturally infected sand flies from the Attica Region as well as in central Greece, while previously it has only been detected in sand flies collected from Central Macedonia (Northern Greece).

Entomological Data and Detection of West Nile Virus in Mosquitoes in Greece (2014-2016), Before Disease Re-Emergence in 2017.

West Nile virus (WNV) cases were seasonally recorded in humans and animals in Greece, from 2010 to 2014, and circulation of the virus was detected in different Regional Units of the country. Small scale entomological surveillance activities were carried out by several regions and regional units in Greece, during 2014-2016, with the participation of subcontractors for the vector control programs aiming to record presence/absence of mosquito species, and monitor and control mosquito populations. Mosquito traps were placed in rural and urban sites; specimens were collected, morphologically characterized, and pooled by date of collection, location, and species types. Mosquito pools containing Culex pipiens, Aedes caspius, and Aedes albopictus were examined for the presence of WNV and positive pools were detected in different areas of the country. Sequencing of a selected number of amplicons revealed WNV lineage 2 partial NS5 gene sequences. In this study, we present data on the mosquito species composition in the areas of study and WNV detection from several parts of Greece, in 6, 11, and 26 mosquito pools corresponding to the years 2014, 2015, and 2016, respectively. A total of 15 WNV human infections were reported to the public health authorities of the country in 2014, whereas no human cases were detected for 2015-2016. Taking into consideration the complex epidemiological profile of WNV and unforeseen changes in its circulation, re-emergence of WNV human cases in Greece was possible and expected, thus rendering surveillance activities imperative.

Evidence of Brucella melitensis DNA in the Microbiome of Ctenocephalides felis from Pet Cats in Greece.

Cat fleas (Ctenocephalides felis) are the most prevalent ectoparasites of pet animals with cosmopolitan distribution, obligatory hematophagous, and may prey on humans to receive bloodmeals. We studied the microbiota of 100 flea-pools, containing C. felis, and collected from equal number of cats and dogs in the region of Attica, Greece, including Athens. The 16S metagenomics technique detected Brucella spp. nucleotide sequence that was identified as Brucella melitensis DNA by a real-time PCR, in five flea-pools, corresponding to five cats, one owned and the remaining four stray, residing in semiurban and urban areas, respectively. No definite conclusions can be drawn as to the pathway that led to the presence of B. melitensis in common fleas parasitizing cats. We suspect flea or cat contact with wild rodents, ubiquitous in various environments, which participate in the B. melitensis biology. The proximity of the cats and their fleas with humans and previous observations of flea potential to transmit B. melitensis in laboratory animals warrant a more elaborate research as to the vectorial dynamics, the ecological pathways resulting in pathogen carriage, and the risk for public health.

Entomological Study of the Mosquito Fauna in the Regional Unit of Drama, Region of East Macedonia-Thrace, Greece (2015 to 2016).

This study reports the mosquito collections conducted from June to September of 2015 and 2016, in Regional Unit (R.U.) of Drama, East Macedonia-Thrace Region, in Northeastern Greece. A total of 923 specimens were examined based on their morphological characteristics and identified to the species level. Medically important taxa were recognized among the 15 mosquito species recorded belonging to seven genera. All data presented here comprise new distribution records due to lack of previous mosquito faunal surveys in the R.U. of Drama.

Records of Aedes albopictus (Skuse, 1894) (Diptera; Culicidae) and Culex tritaeniorhynchus (Diptera; Culicidae) Expansion in Areas in Mainland Greece and Islands.

Annual entomological surveillance programs aiming to monitor mosquito populations and record presence and absence of mosquito species have been performed in Greece. We report, in this study, new records and expansion of Aedes albopictus in the islands of Lesvos (region of North Aegean), Crete (region of Crete), and the regional units of Rodopi in East Macedonia-Thrace. Furthermore, Culex tritaeniorhynchus was recorded for the first time in Arta (region of Epirus) in northwestern Greece.

West Nile Virus Circulation in Mosquitoes in Greece (2010-2013).

Background of the Study. Following a large West Nile virus (WNV) epidemic in Northern Greece in 2010, an active mosquito surveillance system was implemented, for a 3-year period (2011, 2012, and 2013). Description of the Study Site and Methodology. Using mainly CO2 mosquito traps, mosquito collections were performed. Samples were pooled by date of collection, location, and species and examined for the presence of WNV. Results. Positive pools were detected in different areas of the country. In 2010, MIR and MLE values of 1.92 (95% CI: 0.00-4.57) and 2.30 (95% CI: 0.38-7.49) were calculated for the Serres Regional Unit in Central Macedonia Region. In 2011, the highest MIR value of 3.71(95% CI: 1.52-5.91) was recorded in the Regions of Central Greece and Thessaly. In 2012, MIR and MLE values for the whole country were 2.03 (95% CI: 1.73-2.33) and 2.15 (95% CI: 1.86-2.48), respectively, for Cx. pipiens. In 2013, in the Regional Unit of Attica, the one outbreak epicenter, MIR and MLE values for Cx. pipiens were 10.75 (95% CI: 7.52-13.99) and 15.76 (95% CI: 11.66-20.65), respectively. Significance of Results/Conclusions. The contribution of a mosquito-based surveillance system targeting WNV transmission is highlighted through the obtained data, as in most regions positive mosquito pools were detected prior to the date of symptom onset of human cases. Dissemination of the results on time to Public Health Authorities resulted in planning and application of public health interventions in local level.

West Nile virus state of the art report of MALWEST Project.

During the last three years Greece is experiencing the emergence of West Nile virus (WNV) epidemics. Within this framework, an integrated surveillance and control programme (MALWEST project) with thirteen associate partners was launched aiming to investigate the disease and suggest appropriate interventions. One out of seven work packages of the project is dedicated to the State of the Art report for WNV. Three expert working groups on humans, animals and mosquitoes were established. Medical databases (PubMed, Scopus) were searched together with websites: e.g., WHO, CDC, ECDC. In total, 1,092 relevant articles were initially identified and 258 of them were finally included as references regarding the current knowledge about WNV, along with 36 additional sources (conference papers, reports, book chapters). The review is divided in three sections according to the fields of interest: (1) WNV in humans (epidemiology, molecular characteristics, transmission, diagnosis, treatment, prevention, surveillance); (2) WNV in animals (epidemiological and transmission characteristics concerning birds, horses, reptiles and other animal species) and (3) WNV in mosquitoes (control, surveillance). Finally, some examples of integrated surveillance programmes are presented. The introduction and establishment of the disease in Greece and other European countries further emphasizes the need for thorough research and broadening of our knowledge on this viral pathogen.