Large-scale performance assessment of the BG-Counter 2 used with two different mosquito traps.

BACKGROUND: Mosquitoes are important vectors of pathogens. They are usually collected with CO2-baited traps and subsequently identified by morphology. This procedure is very time-consuming. Automatic counting traps could facilitate timely evaluation of the local risk for mosquito-borne pathogen transmission or decision-making on vector control measures, but the counting accuracy of such devices has rarely been validated in the field. METHODS: The Biogents (BG)-Counter 2 automatically counts mosquitoes by discriminating the size of captured objects directly in the field and transmits the data to a cloud server. To assess the accuracy of this counting device, 27 traps were placed at 19 sampling sites across Germany and used in daily, weekly or bimonthly intervals from April until October 2021. The BG-Counter 2 was attached to a CO2-trap (BG-Pro trap = CO2-Pro) and the same trap was converted to also attract gravid mosquitoes (upside-down BG-Pro trap with a water container beneath = CO2-Pro-gravid). All captured mosquitoes were identified by morphology. The number of females (unfed and gravid), mosquito diversity and the number of identified specimens in relation to the counting data of the BG-Counter were compared between the two trapping devices to evaluate sampling success and counting accuracy. RESULTS: In total 26,714 mosquitoes were collected during 854 trap days. The CO2-Pro-gravid trap captured significantly more mosquitoes per trap day for all specimens, gravid females and non-gravid females, while there was no difference in the mosquito diversity. The linear model with the captured mosquitoes as a response and the counted specimens as a predictor explained only a small degree of the variation within the data (R2 = 0.16), but per individual trap the value could reach up to 0.62 (mean R2 = 0.23). The counting accuracy for the daily samples had a significant positive correlation with sample size, resulting in higher accuracy for the CO2-Pro-gravid trap and higher accuracy for sites and sampling months with high mosquito abundance. CONCLUSIONS: While the accuracy of the BG-Counter 2 is quite low, the device is able to depict mosquito phenology and provide information about local population dynamics.

Honey-baited FTA cards in box gravid traps for the assessment of Usutu virus circulation in mosquito populations in Germany.

Usutu virus (USUV) is becoming increasingly important to veterinary and human health in Germany. USUV has been implicated in mass die-off events of birds, especially of blackbirds (Turdus merula), and has experienced significant range expansion in the years since its first detection in 2010. Current detection methods rely primarily on dead bird surveillance or mass mosquito collection using CO2 as the main attractant. Dead bird surveillance can result in detection of disease circulation past the point at which control efforts would be most impactful. Vector surveillance offers the opportunity to detect disease circulation before significant outbreaks occur. However, current methods result in collections of extremely large numbers of predominantly nulliparous female mosquitoes who have not yet taken a blood meal. This study sought to test whether box gravid traps could successfully trap USUV infected gravid Culex mosquitoes, and if viral RNA could be successfully transferred and stabilised on an FTA card. During the month of August 2020, 18 Reiter-Cummings style box gravid traps with honey-baited FTA cards were set in a region of known USUV circulation around the southern border of Hesse, Germany. Four 48-hour trapping rounds were conducted. All mosquitoes and FTA cards were collected and stored during transport to the laboratory on dry ice. Samples and FTA cards were then transferred and stored in a freezer at -5 °C until identification. Identification was carried out on a chill plate before being sent with overnight courier in a styrofoam box with cooling elements for virus detection with a modified generic flavivirus RT-PCR. Mosquitoes were separated into pools by trap, date, species and feeding status. 2003 mosquitoes were caught in four rounds of trapping, 1834 or 88% of which were female Culex mosquitoes used for examination. 13 pools of mosquitoes and four FTA cards tested positive for USUV. No positive FTA cards were found in traps with positive mosquitoes and no positive mosquitoes were found in traps with positive FTA cards. Although fewer FTA cards than expected returned a positive result, this may have been a result of the extreme conditions experienced in the field and highlights the need to establish the temperature and humidity boundaries such a collection method can withstand. Box gravid traps however, provided a highly effective and targeted approach for capturing gravid female Culex mosquitoes, the most appropriate subpopulation for testing for USUV. Additionally, the simplicity and effectiveness of this trapping and surveillance method make it an attractive option for use as an early warning system, including for large scale surveillance programmes.

Rapid assessment of West Nile virus circulation in a German zoo based on honey-baited FTA cards in combination with box gravid traps.

BACKGROUND: For over a decade, monitoring of West Nile virus (WNV) in Germany has consisted of a bird monitoring programme as well as a mosquito-based surveillance programme employing CO2-baited encephalitis vector surveillance (EVS) traps for mass trapping and screening of mosquitoes. In contrast to the EVS traps, the Reiter/Cummings type box gravid trap collects gravid female mosquitoes, which have already taken a blood meal, increasing the likelihood of being infected with pathogens. The traps can be equipped with a honey-baited Flinders Technology Associates® (FTA) card to encourage sugar feeding by the trapped mosquitoes. FTA cards contain nucleic acid preserving substances, which prevent the degradation of viral RNA in the expectorated mosquito saliva and allows for testing the card for flavivirus RNA. This study aimed to assess the suitability of the method for WNV surveillance in Germany as an alternative to previous methods, which are expensive, time-consuming, and predominantly target host-seeking populations less likely to be infected with WNV. METHODS: In the Thüringer Zoopark Erfurt, snowy owls (Nyctea scandiaca) and greater flamingos (Phoenicopterus roseus) died of WNV infections in July and August 2020. In response, five Reiter/Cummings type box gravid traps were positioned during the daytime on the 10th, 13th, and 16th of September in five different locations. The FTA cards and mosquitoes in the chamber were collected, kept in a cool chain, and further processed for virus detection using a modified generic flavivirus reverse transcription PCR. RESULTS: A total of 15 trappings during September collected a total of 259 female mosquitoes, 97% of which were Culex pipiens sensu lato, as well as 14 honey-baited FTA cards. Eight mosquitoes tested PCR-positive for WNV. Four FTA cards tested PCR-positive for mosquito-borne flaviviruses, two of which were confirmed as WNV, and the remaining two confirmed as Usutu virus. CONCLUSION: The suitability of the FTA cards in preserving viral RNA in the field and rapid turnaround time from collection to result is combined with a simple, cost-effective, and highly specific trapping method to create an arbovirus surveillance system, which circumvents many of the difficulties of previous surveillance programmes that required the analysis of mosquitoes in the laboratory.

Experimental risk assessment for chikungunya virus transmission based on vector competence, distribution and temperature suitability in Europe, 2018.

BackgroundOver the last decade, the abundant distribution of the Asian tiger mosquito Aedes albopictus in southern Europe and the import of chikungunya virus (CHIKV) by infected travellers has resulted in at least five local outbreaks of chikungunya fever in France and Italy. Considering the ongoing spread of Ae. albopictus to central Europe, we performed an analysis of the Europe-wide spatial risk of CHIKV transmission under different temperature conditions. Methods:Ae. albopictus specimens from Germany and Italy were orally infected with CHIKV from an outbreak in France and kept for two weeks at 18 °C, 21 °C or 24 °C. A salivation assay was conducted to detect infectious CHIKV. Results: Analyses of mosquito saliva for infectious virus particles demonstrated transmission rates (TRs) of > 35%. Highest TRs of 50% for the mosquito population from Germany were detected at 18 °C, while the Italian population had highest TRs of 63% at 18 °C and 21 °C, respectively. Temperature data indicated a potential risk of CHIKV transmission for extended durations, i.e. sufficiently long time periods allowing extrinsic incubation of the virus. This was shown for areas already colonised by Ae. albopictus, as well as for large parts of central Europe that are not colonised. Conclusion: The current risk of CHIKV transmission in Europe is not primarily restricted by temperature, which allows extrinsic incubation of the virus, but rather by the vector distribution. Accordingly, all European countries with established populations of Ae. albopictus should implement respective entomological surveillance and monitoring systems, as basis for suitable control measures.