Nucleic Acid Preservation Card Surveillance Is Effective for Monitoring Arbovirus Transmission on Crocodile Farms and Provides a One Health Benefit to Northern Australia.

The Kunjin strain of West Nile virus (WNVKUN) is a mosquito-transmitted flavivirus that can infect farmed saltwater crocodiles in Australia and cause skin lesions that devalue the hides of harvested animals. We implemented a surveillance system using honey-baited nucleic acid preservation cards to monitor WNVKUN and another endemic flavivirus pathogen, Murray Valley encephalitis virus (MVEV), on crocodile farms in northern Australia. The traps were set between February 2018 and July 2020 on three crocodile farms in Darwin (Northern Territory) and one in Cairns (North Queensland) at fortnightly intervals with reduced trapping during the winter months. WNVKUN RNA was detected on all three crocodile farms near Darwin, predominantly between March and May of each year. Two of the NT crocodile farms also yielded the detection of MVE viral RNA sporadically spread between April and November in 2018 and 2020. In contrast, no viral RNA was detected on crocodile farms in Cairns during the entire trapping period. The detection of WNVKUN and MVEV transmission by FTATM cards on farms in the Northern Territory generally correlated with the detection of their transmission to sentinel chicken flocks in nearby localities around Darwin as part of a separate public health surveillance program. While no isolates of WNVKUN or MVEV were obtained from mosquitoes collected on Darwin crocodile farms immediately following the FTATM card detections, we did isolate another flavivirus, Kokobera virus (KOKV), from Culex annulirostris mosquitoes. Our studies support the use of the FTATM card system as a sensitive and accurate method to monitor the transmission of WNVKUN and other arboviruses on crocodile farms to enable the timely implementation of mosquito control measures. Our detection of MVEV transmission and isolation of KOKV from mosquitoes also warrants further investigation of their potential role in causing diseases in crocodiles and highlights a "One Health" issue concerning arbovirus transmission to crocodile farm workers. In this context, the introduction of FTATM cards onto crocodile farms appears to provide an additional surveillance tool to detect arbovirus transmission in the Darwin region, allowing for a more timely intervention of vector control by relevant authorities.

Waterproof, low-cost, long-battery-life sound trap for surveillance of male Aedes aegypti for rear-and-release mosquito control programmes.

BACKGROUND: Sterile male rear-and-release programmes are of growing interest for controlling Aedes aegypti, including use an "incompatible insect technique" (IIT) to suppress transmission of dengue, Zika, and other viruses. Under IIT, males infected with Wolbachia are released into the suppression area to induce cytoplasmic incompatibility in uninfected populations. These and similar mosquito-release programmes require cost-effective field surveys of both sexes to optimize the locations, timing, and quantity of releases. Unfortunately, traps that sample male Ae. aegypti effectively are expensive and usually require mains power. Recently, an electronic lure was developed that attracts males using a 484 Hz sinusoidal tone mimicking the female wingbeat frequencies, broadcast in a 120 s on/off cycle. When deployed in commercially available gravid Aedes traps (GATs), the new combination, sound-GAT (SGAT), captures both males and females effectively. Given its success, there is interest in optimizing SGAT to reduce cost and power usage while maximizing catch rates. METHODS: Options considered in this study included use of a smaller, lower-power microcontroller (Tiny) with either the original or a lower-cost speaker (lcS). A 30 s on/off cycle was tested in addition to the original 120 s cycle to minimize the potential that the longer cycle induced habituation. The original SGAT was compared against other traps incorporating the Tiny-based lures for mosquito capture in a large semi-field cage. The catch rates in waterproofed versions of this trap were then compared with catch rates in standard [BG-Sentinel 2 (BGS 2); Biogents AG, Regensburg, Germany] traps during an IIT field study in the Innisfail region of Queensland, Australia in 2017. RESULTS: The system with a low-power microcontroller and low-cost speaker playing a 30 s tone (Tiny-lcS-30s) caught the highest proportion of males. The mean proportions of males caught in a semi-field cage were not significantly different among the original design and the four low-power, low-cost versions of the SGAT. During the IIT field study, the waterproofed version of the highest-rated, Tiny-lcS-30s SGAT captured male Ae. aegypti at similar rates as co-located BGS-2 traps. CONCLUSIONS: Power- and cost-optimized, waterproofed versions of male Ae. aegypti acoustic lures in GATs are now available for field use in areas with sterile male mosquito rear-and-release programmes.

A low-cost, battery-powered acoustic trap for surveilling male Aedes aegypti during rear-and-release operations.

The Aedes aegypti mosquito is a primary vector of several serious arboviruses throughout the world and is therefore of great concern to many public health organizations. With vector control methodology pivoting towards rearing and releasing large numbers of genetically modified, sterilized, or Wolbachia-infected male mosquitoes to control vector populations, economical surveillance methods for release tracking becomes increasingly necessary. Previous work has identified that male Ae. aegypti are attracted to female wingbeat frequencies and can be captured through artificial playback of these frequencies, but the tested systems are cost-prohibitive for wide-scale monitoring. Thus, we have developed a simple, low-cost, battery-powered, microcontroller-based sound lure which mimics the wingbeat frequency of female Ae. aegypti, thereby attracting males. We then tested the efficacy of this lure in combination with a passive (non-powered) gravid Aedes trap (GAT) against the current gold-standard, the Biogents Sentinel (BGS) trap, which requires main power (household power) and costs several times what the GAT does. Capture rates of male Ae. aegypti in sound-baited GATs (Sound-GATs) in these field tests were comparable to that of the BGS with no inhibitory effects of sound playback on female capture. We conclude that the Sound-GAT is an effective replacement of the costly BGS for surveillance of male Ae. aegypti mosquitoes, particularly in the developing countries where funding is limited, and has the potential to be adapted to target males of other medically important species.

Assessment of synthetic floral-based attractants and sugar baits to capture male and female Aedes aegypti (Diptera: Culicidae).

BACKGROUND: The viruses transmitted by Aedes aegypti, including dengue and Zika viruses, are rapidly expanding in geographic range and as a threat to public health. In response, control programs are increasingly turning to the use of sterile insect techniques resulting in a need to trap male Ae. aegypti to monitor the efficacy of the intervention. However, there is a lack of effective and cheap methods for trapping males. Thus, we attempted to exploit the physiological need to obtain energy from sugar feeding in order to passively capture male and female Ae. aegypti (nulliparous and gravid) in free-flight attraction assays. Candidate lures included previously identified floral-based (phenylacetaldehyde, linalool oxide, phenylethyl alcohol, and acetophenone) attractants and an attractive toxic sugar bait-based (ATSB) solution of guava and mango nectars. A free-flight attraction assay assessed the number of mosquitoes attracted to each candidate lure displayed individually. Then, a choice test was performed between the best-performing lure and a water control displayed in Gravid Aedes Traps (GAT). RESULTS: Results from the attraction assays indicated that the ATSB solution of guava and mango nectars was the most promising lure candidate for males; unlike the floral-based attractants tested, it performed significantly better than the water control. Nulliparous and gravid females demonstrated no preference among the lures and water controls indicating a lack of attraction to floral-based attractants and sugar baits in a larger setting. Although the guava-mango ATSB lure was moderately attractive to males when presented directly (i.e. no need to enter a trap or other confinement), it failed to attract significantly more male, nulliparous female, or gravid female Ae. aegypti than water controls when presented inside a Gravid Aedes Trap. CONCLUSIONS: Our findings suggest that the use of volatile floral-based attractants and sugar mixtures that have been identified in the literature is not an effective lure by which to kill Ae. aegypti at ATSB stations nor capture them in the GAT. Future trapping efforts would likely be more successful if focused on more promising methods for capturing male and female Ae. aegypti.

Assessment of Local Mosquito Species Incriminates Aedes aegypti as the Potential Vector of Zika Virus in Australia.

BACKGROUND: Within the last 10 years Zika virus (ZIKV) has caused unprecedented epidemics of human disease in the nations and territories of the western Pacific and South America, and continues to escalate in both endemic and non-endemic regions. We evaluated the vector competence of Australian mosquitoes for ZIKV to assess their potential role in virus transmission. METHODOLOGY/PRINCIPAL FINDINGS: Mosquitoes were exposed to infectious blood meals containing the prototype African ZIKV strain. After 14 days incubation at 28°C and high relative humidity, infection, dissemination and transmission rates were assessed. Infection in Culex annulirostris and Cx. sitiens could not be detected. 8% of Cx. quinquefasciatus were infected, but the virus did not disseminate in this species. Despite having infection rates > 50%, Aedes notoscriptus and Ae. vigilax did not transmit ZIKV. In contrast, Ae. aegypti had infection and transmission rates of 57% and 27%, respectively. In susceptibility trials, the virus dose required to infect 50% (ID50) of Ae. aegypti was106.4 tissue culture infectious dose50 (TCID50)/mL. Additionally, a threshold viral load within the mosquito of at least 105.1 TCID50 equivalents/mL had to be reached before virus transmission occurred. CONCLUSIONS/SIGNIFICANCE: We confirmed Ae. aegypti to be the most likely mosquito vector of ZIKV in Australia, although the restricted distribution of this species will limit the receptive zone to northern Queensland where this species occurs. Importantly, the role in ZIKV transmission of Culex and other Aedes spp. tested will be negligible. Despite being the implicated vector, the relatively high ID50 and need for a high titer disseminated infection in Ae. aegypti suggest that high mosquito population densities will be required to facilitate epidemic ZIKV transmission among the currently immunologically naïve human population in Australia.

A critical assessment of vector control for dengue prevention.

Recently, the Vaccines to Vaccinate (v2V) initiative was reconfigured into the Partnership for Dengue Control (PDC), a multi-sponsored and independent initiative. This redirection is consistent with the growing consensus among the dengue-prevention community that no single intervention will be sufficient to control dengue disease. The PDC's expectation is that when an effective dengue virus (DENV) vaccine is commercially available, the public health community will continue to rely on vector control because the two strategies complement and enhance one another. Although the concept of integrated intervention for dengue prevention is gaining increasingly broader acceptance, to date, no consensus has been reached regarding the details of how and what combination of approaches can be most effectively implemented to manage disease. To fill that gap, the PDC proposed a three step process: (1) a critical assessment of current vector control tools and those under development, (2) outlining a research agenda for determining, in a definitive way, what existing tools work best, and (3) determining how to combine the best vector control options, which have systematically been defined in this process, with DENV vaccines. To address the first step, the PDC convened a meeting of international experts during November 2013 in Washington, DC, to critically assess existing vector control interventions and tools under development. This report summarizes those deliberations.

Unforeseen costs of cutting mosquito surveillance budgets.

A budget proposal to stop the U.S. Centers for Disease Control and Prevention (CDC) funding in surveillance and research for mosquito-borne diseases such as dengue and West Nile virus has the potential to leave the country ill-prepared to handle new emerging diseases and manage existing ones. In order to demonstrate the consequences of such a measure, if implemented, we evaluated the impact of delayed control responses to dengue epidemics (a likely scenario emerging from the proposed CDC budget cut) in an economically developed urban environment. We used a mathematical model to generate hypothetical scenarios of delayed response to a dengue introduction (a consequence of halted mosquito surveillance) in the City of Cairns, Queensland, Australia. We then coupled the results of such a model with mosquito surveillance and case management costs to estimate the cumulative costs of each response scenario. Our study shows that halting mosquito surveillance can increase the management costs of epidemics by up to an order of magnitude in comparison to a strategy with sustained surveillance and early case detection. Our analysis shows that the total costs of preparedness through surveillance are far lower than the ones needed to respond to the introduction of vector-borne pathogens, even without consideration of the cost in human lives and well-being. More specifically, our findings provide a science-based justification for the re-assessment of the current proposal to slash the budget of the CDC vector-borne diseases program, and emphasize the need for improved and sustainable systems for vector-borne disease surveillance.

Field validation of a transcriptional assay for the prediction of age of uncaged Aedes aegypti mosquitoes in Northern Australia.

BACKGROUND: New strategies to eliminate dengue have been proposed that specifically target older Aedes aegypti mosquitoes, the proportion of the vector population that is potentially capable of transmitting dengue viruses. Evaluation of these strategies will require accurate and high-throughput methods of predicting mosquito age. We previously developed an age prediction assay for individual Ae. aegypti females based on the transcriptional profiles of a selection of age responsive genes. Here we conducted field testing of the method on Ae. aegypti that were entirely uncaged and free to engage in natural behavior. METHODOLOGY/PRINCIPAL FINDINGS: We produced "free-range" test specimens by releasing 8007 adult Ae. aegypti inside and around an isolated homestead in north Queensland, Australia, and recapturing females at two day intervals. We applied a TaqMan probe-based assay design that enabled high-throughput quantitative RT-PCR of four transcripts from three age-responsive genes and a reference gene. An age prediction model was calibrated on mosquitoes maintained in small sentinel cages, in which 68.8% of the variance in gene transcription measures was explained by age. The model was then used to predict the ages of the free-range females. The relationship between the predicted and actual ages achieved an R(2) value of 0.62 for predictions of females up to 29 days old. Transcriptional profiles and age predictions were not affected by physiological variation associated with the blood feeding/egg development cycle and we show that the age grading method could be applied to differentiate between two populations of mosquitoes having a two-fold difference in mean life expectancy. CONCLUSIONS/SIGNIFICANCE: The transcriptional profiles of age responsive genes facilitated age estimates of near-wild Ae. aegypti females. Our age prediction assay for Ae. aegypti provides a useful tool for the evaluation of mosquito control interventions against dengue where mosquito survivorship or lifespan reduction are crucial to their success. The approximate cost of the method was US$7.50 per mosquito and 60 mosquitoes could be processed in 3 days. The assay is based on conserved genes and modified versions are likely to support similar investigations of several important mosquito and other disease vectors.

Operational trials of remote mosquito trap systems for Japanese encephalitis virus surveillance in the Torres Strait, Australia.

Japanese encephalitis virus (JEV) appears nearly annually in the Torres Strait in far northern Queensland, Australia, and is a threat to invade the Australian mainland. Surveillance has involved the use of sentinel pigs that develop detectable viremias and antibody titers to JEV. However, pigs are amplifying hosts for JEV, and thus pose a health risk to the public and to pig handlers who bleed the pigs. A remote mosquito trap system would not have these risks. We report on trials using a remote mosquito trap system for the surveillance of JEV in the Torres Strait. The Mosquito Magnet (MM) Pro, MM Liberty Plus, and a novel updraft trap, the NAQS Mozzie Trap, were run at Badu and Moa islands in the Torres Strait and at Bamaga in the northern Cape York Peninsula from 2002-2005. TaqMan real-time polymerase chain reaction (PCR) was used to detect JEV nucleic acid in weekly mosquito collections. Sentinel pigs located at Badu were also bled and the serum processed by reverse transcriptase (RT)-PCR for JEV antigen and enzyme-linked immunosorbent assay (ELISA) for anti-JEV antibodies. JEV was detected in mosquito collections each year but not in each trap. No JEV was detected in trapped mosquitoes before detection in sentinel pigs. The mosquito trap system cost ca. AU$10,000 per site, about AU$5,000 less than a pig-based system. However, trap failures caused by mosquito-clogged motors, electrical faults, and blocked gas lines reduced the efficacy of some mosquito traps. Nonetheless, a remote mosquito trap system, employing stand alone traps and PCR for viral antigen detection, can be a safe, economical way to detect arbovirus activity in remote areas.

Laboratory and field assessment of some kairomone blends for host-seeking Aedes aegypti.

Using laboratory Y-tube olfactometers, the attractiveness of lactic acid and 2 kairomone blends from the United States Department of Agriculture (USDA) and BioGents GmbH (BG) was assessed for attractiveness to Aedes aegypti. Four geographically disparate populations were assessed: North Queensland Australia (NQA), Florida USA, Minas Gerais Brazil (MGB), and Singapore. In descending order, populations were attracted to USDA, BG blends, and lactic acid. MGB was poorly attracted to lactic acid alone. The blends were less attractive than human odor. Proprietary blends were modified, and their attractiveness was assessed to find the optimum attractive mixture for NQA. Adding acetone to BG, and ammonia and caproic acid to USDA, improved attractiveness in the laboratory. Field attractiveness was assessed by coupling the blends with a newly developed BG-Sentinel Ae. aegypti trap. Trials were carried out using the BG blend, BG blend plus acetone, USDA blend, USDA blend plus ammonia and caproic acid, and a control trap with no kairomones. The traps were highly effective, with mean 24-h collections up to 11.15 Ae. aegypti per trap, and this species made up 91.7% of collections. However, the effectiveness of the unbaited control trap indicated that the BG-Sentinel has visual attractive properties for Ae. aegypti and that the kairomone lures added little to trap performance in NQA.