Scaled deployment of Wolbachia to protect the community from Aedes transmitted arboviruses

Community engagement

One of the key objectives of the Townsville project was establishing a community engagement framework that could be suitably scaled for a citywide deployment and could be used cross-culturally for future deployments. Previous deployments in Cairns had relied on obtaining individual consent from community members for the release activities, an approach that was unsuitable for the required scaling. Instead we developed a Public Acceptance Model (PAM) for our engagement that formed the basis for obtaining community support for the research activities. The PAM was based on a set of Public Participation Principles described in Table 1.

The PAM consisted of four key components:

Rearing

In order to establish the colony for release, wild mosquito eggs were collected from ovitraps set at 49 sites across Townsville and used to produce a wildtype colony. Material from this colony was stored as dried eggs and amplified only as required. Amplification of material from this colony was limited to F3 for use in outcrossing during colony maintenance. For stage 1 of the Townsville releases, eggs were produced from insectaries at Monash University, Melbourne or James Cook University, Cairns and shipped to the Townsville field office. For stages 2–4 all mosquito material was produced at Monash University.

The wildtype colony was backcrossed for three generations to a laboratory line infected with the wMel strain of Wolbachia¹¹. This new colony, TSV wMel.f was continuously maintained in order to produce ∼800,000 eggs per week. To maintain the material during mass production, the TSV wMel.f line was divided into two distinct colonies: ‘broodstock’ and ‘release material’. The ‘broodstock’ colony was reared under the more relaxed conditions described in 12 but kept at 26°C. Its purpose was to produce eggs for amplification and production of the ‘release material colony’. In order to prevent inbreeding, 10% wildtype males were added to each generation of the ‘broodstock’. The purpose of the ‘release material’ colony was to produce eggs for release; it did not provide any material for the next generation in the laboratory. In order to facilitate mass production, the ‘release material’ colony was maintained as described for the broodstock with the following modifications. No wild material was added to the ‘release material’ colony. Once eggs were hatched, first instar larvae were aliquoted into 500 ml plastic cups at a ratio of 150–180 larvae/400 ml of water. The larvae were fed once with half a fish food tablet (Tetramin Tropical Tablet, Tetra Holding (US) Inc., Germany) until pupation. Larval rearing cups were transferred to adult cages for emergence once 60% of larvae had pupated. Cages were stocked at a rate of ∼600 adults per (30 X 30 X 30cm) cage.

For both colonies, females (5–7 days old) were fed with human blood (Monash University Human Ethics approval CF11/0766 – 2011000387). They were provided the bloodmeal by introducing the arm of a volunteer into the selected cage. Females were fed until repletion (usually 10–15 minutes). Females were fed once per week, for one or two weeks depending on requirements. For safety, only one bloodfeeder was used per cage and bloodfeeders who showed any signs of fever or who were taking antibiotics were excluded.

Three 22 cm oviposition strips of red cotton duck cloth were placed in each cage three to five days after bloodfeeding. Oviposition strips were removed from cages four days later, and sandwiched between two double layers of 3mm thick kitchen sponge that had been covered with a single layer of paper towel, covered with a 3mm thick Perspex sheet and placed on a rack. Eggs were allowed to dry this way in an 80%RH controlled-temperature room for up to 24 hours before being placed in humidified containers. The humidity in these containers was maintained at ∼80%RH by providing a saturated KCl solution inside the containers.

After the oviposition strips had been dried, the density of eggs/cm on each strip was estimated to determine the length of egg strip to be cut for subsequent use in Mosquito Release Containers (MRCs). Eggs were then shipped to the Townsville field lab.

Hatch rate was tested for every batch of eggs produced. Matched sets of eggs were taken from a number of strips and photographed to assess desiccation and overall quality of the eggs. One portion of each matched set was shipped to the release site, and one set kept at the rearing facility. Once the eggs reached the release site, both sets of eggs were counted, hatched, and hatch rate determined by counting larvae. Hatch rate of 70% or above was considered acceptable. If hatch rate fell below 70%, the cause of this drop was investigated. In most cases, the cause was determined to be due to fluctuating environmental conditions or to slight changes made to the drying procedure, which was altered slightly throughout releases.

Wolbachia infection frequency was also tested each week of production. 80 females and 80 males were screened from each broodstock cohort using diagnostic qPCR as described below. If Wolbachia frequency fell below 97% in any broodstock cohort, the eggs from their resultant ‘release material colony’ would not be used for release, however this issue never arose.

The James Cook University rearing strategy differed slightly from the Monash rearing strategy. A single colony of ∼10,000 Townsville wMel-infected Ae. aegypti sourced from Monash was created in a semi-field flight cages¹³ in the Tropical Medicine Mosquito Research Facility located at James Cook University in Cairns. Based upon experience with earlier releases, we assumed that there is a loss of ∼50% of the colony per week. The colony was therefore refreshed with 2500 males and 2500 females each week. We also conducted backcrossing to maintain genetic diversity by adding males (10% of cage male population) sourced from an uninfected wildtype Townsville colony (< F4). To prevent introduction of wild females and potential loss of Wolbachia infection into the colony, we only added males. This was achieved by placing suspected male pupae based on size into cups of 10; any cups containing emerged females were discarded.

Females (5–7 days old) were fed with human blood on volunteers (JCU Human Ethics H4907). They were provided the bloodmeal by introducing 5 volunteer blood feeders into the field cage 3–5 times/week who let mosquitoes feed for 10 minutes. For safety bloodfeeders were screened at every feed for possible exposure to dengue infected mosquitoes using a questionnaire to access travel history, and their temperature was taken to detect fever. Any volunteers with fever, a possible exposure to dengue infected mosquitoes or who were taking antibiotics were excluded for a minimum of 2 weeks.

Eggs were harvested from partially flooded 10 L buckets containing 26 × 30 cm strips of red felt cloth placed in the semi-field cage. A perspex template 31cm in length with 12 1-cm holes drilled into it was placed over the cloth to limit oviposition to the exposed 1 cm area of the ovistrip. The ovistrips were collected 3 times/week, embryonated and dried three days later. Once removed from the cages, oviposition strips were placed on moist paper towel in a sealed plastic container, after 3 days the lid of the sealed container was removed and the eggs were allowed to dry this way in an 80%RH controlled temperature room for up to 24 hours before being placed in humidified containers. The humidity in these containers was maintained at ∼80%RH by providing a saturated KCl solution inside the containers. The cloth was then cut into individual eggstrips containing a single egg clump that could be deployed into egg release containers in the field. The number of eggs on each eggstrip was estimated by using reference photographs of eggstrips with known egg numbers as visual guides for fast estimation.

Mosquito releases

The municipal area of Townsville is ∼190km². However, within this area there were many areas where releases did not take place due to the lack of suitable Ae. aegypti habitat. Releases were restricted to residential and business areas within the city where Ae. aegypti breeding was likely to occur. This resulted in the actual area for release being reduced to approximately 66km² to effectively cover the city. The release program was divided into four stages (Figure 1).

Figure 1. Release site.

Map of Townsville city showing the boundaries of the four release stages.

Stage 1 covered a release area of 20km² and included the suburbs with known highest dengue transmission risk: South Townsville, Railway Estate, North Ward, Townsville City, Belgian Gardens, Castle Hill, West End, Garbutt, Currajong, Vincent, Gulliver, Aitkenvale, Mundingburra, Rosslea, Hyde Park, Pimlico, Mysterton and Hermit Park. In this stage, all releases were undertaken using bucket mosquito release containers (MRCs). These were 2.3L white polypropylene pails with lid (Peopleinplastic, Australia), with top 164mm diameter, base 145mm diameter, and height 147mm. Each bucket had four 6mm holes drilled 20mm apart in a square pattern in the side (Figure 2A). The inside of each bucket was roughened with sandpaper to allow mosquitoes to rest upon emergence. Into each bucket MRC was placed an egg strip containing approximately 100 viable eggs (estimated from hatch rate QA), 5 (summer) or 6 (winter) wafers of Aqua One vege wafer fish food (Aqua Pacific, UK) and 1L water. More food was provided in winter and the servicing cycle for these buckets was extended from 2 to 3 weeks to allow for longer emergence times.

Figure 2. Release containers.

Photos illustrating different mosquito release containers used in the deployment. (A) Bucket mosquito release containers (MRCs) used in stage 1 releases (B) Clear bucket MRC used in Wolbachia Warriors school program in stage 1 (C) Mozzie Box MRC that was used in stages 2–4 (D) Material given to school children as part of the Wolbachia Warriors program.

Bucket MRCs for stage 1 were placed by program staff in outdoor shaded areas at approximately 20% of all residential properties in a roughly evenly spaced arrangement in each suburb. They were serviced every two weeks by tipping out the water, cleaning the bucket and adding new food, water and eggs. An average of 88 adult mosquitoes were released from each bucket MRC in stage 1. Releases continued in each suburb until the frequency of Wolbachia in samples of field-caught mosquitoes from that suburb was above 50% for two consecutive weeks. For stage 1, it required between 7 and 19 weeks of releases for each suburb to reach that target

Stages 2, 3 & 4 covered release areas of 18, 18 and 10 km² respectively, and included the following suburbs. Stage 2: Cranbrook, Heatley, Kirwan/Thuringowa Central and Mount Louisa; stage 3: Condon, Pallarenda, Rowes Bay, Rasmussen and Kelso; stage 4: Idalia, Oonoonba, Wulguru/Stuart, Annandale and Douglas. Releases for these later stages did not rely on program team members to place all release containers. Instead, they utilised strategies that directly involved the community, such as the use of school students, direct community release, or through collaboration with local businesses. Releases for these stages also used Mozzie Box MRCs (Figure 2C) which consisted of a 775ml Food Pail (Detpak, Australia) without handle, and with measurements top 104×92mm, base 79×61mm, height 104mm. Four 5mm holes were punched into each MRC – one hole approximately 1cm from the top right and top left corners of each long-side face of the box. Each Mozzie Box MRC received 100 viable eggs (estimated from hatch rate QA), 4 (summer) or 5 (winter) wafers of Aqua One vege waters, and 400ml tap water. Mozzie Box MRCs were not re-used.

In stages 2–4 the goal was again to place MRCs at 20% of residences in the release area. This was done by using community engagement activities to identify participants who would agree to host an MRC. In areas where there were large spatial gaps in participation, the program team would then supplement coverage by visiting additional houses in these areas and obtaining consent to leave MRCs with residents at these locations. Finally, in the last two suburbs of stage 3 (Kelso & Rasmussen) and across stage 4, releases of adult mosquitoes⁷ were used to fill in gaps in MRC coverage.

During the 28 months of the release phase (stages 1–4), a total of approximately 4 million mosquitoes were released. Releases were undertaken with regulatory approval from the Australian Pesticides and Veterinary Medicines Authority (APVMA permit numbers PER14797 and PER82947).

School releases

The Wolbachia Warriors Program was developed both as a tool to engage children and their parents and make them aware of the program, and as an alternative channel to release mosquitoes. Five different primary schools were selected to run the program over the duration of the Townsville project. One school participated in each stage except for stage 2 where two schools participated. In total 943 students aged 6–12 participated in these programs.

School children were provided with a bucket MRC in stage 1 as used in operational releases in stage 1 but made of clear plastic to encourage student observation (Figure 2B) and Mozzie Box MRCs in stages 2–4 (Figure 2C), complete with mosquito eggs, food, instructions, a calendar to track progress, a magnifying glass, a badge for participation, and an educational booklet tailored for either lower (grade P-2) or upper primary (grade 3–6) students (Figure 2D). Each student was expected to undertake three consecutive releases with their MRC over a six-week period.

Materials were distributed at the schools by program communication and engagement staff, who gave presentations encouraging participation prior to each of the three mosquito release cycles. Students were asked to use their calendar to record the progress of the mosquito life cycle in their MRC, and to return it to program staff at the end of the release.

Direct community release

In these releases, a Mozzie Box MRC was provided directly to residents who set it up and reared the mosquitoes themselves at their place of residence. In stages 2-4, more than 6,000 households directly participated in establishing Wolbachia by managing their own release container. Almost half of these participants contacted the program team to receive an MRC, which was subsequently delivered to their house. The remaining participants were recruited through doorknocking, or through other recruitment methods such as community groups. Additional Mozzie Box MRCs were distributed through large local employers including the City Council, Telstra, The Townsville Hospital, James Cook University and Queensland Nickel. More than 200 people participated in these programs.

Quality assurance procedures

In stage 1, program staff checked 5–10% of all bucket MRCs to determine whether the bucket had failed or not, and if not to count pupal skins to obtain an estimate of adult emergence from which they could estimate release rates. In stage 2 – 4, a random selection of 5–15% of all MRCs were checked to determine if they were set correctly. Larvae, pupae and pupal skins were counted to estimate emergence rates in these stages (accounting for potential delayed development of mosquitoes at time of QA due to community members setting up MRCs later than day of delivery). This approach was supplemented in stage 3 with additional sentinel buckets that were set and checked by staff to determine average emergence rates. These data were then used to adjust numbers of eggs placed in MRCs.

Monitoring

Up to 172 Biogents Sentinal (BGS) traps were progressively rolled out across stage 1 during releases at a density of approximately 8 BGS traps per km². For stages 2–4 the BGS trap density was reduced to 4 per km², resulting in 74 – 115 traps being deployed per stage. Exact trap numbers fluctuated due to operational considerations (i.e. trap location no longer suitable, trap broken or missing, community request for trap to be removed or resident moved etc.).

Samples from BGS traps were collected weekly and returned to the field office for morphological identification. Ae. aegypti samples were stored in 70% ethanol and shipped to Monash University for diagnostic determination of Wolbachia infection status. After Feb 2016, samples were collected fortnightly instead of weekly as occurred in stage 1 until traps were finally removed from each suburb (Figure 3)

Figure 3. Wolbachia establishment by suburb.

For each stage (A–D) and suburb Wolbachia frequency is plotted against time. Yellow shading indicates periods when releases were undertaken. Bars show the number of mosquitos captured in Biogents Sentinel (BGS) traps and tested for Wolbachia.

Diagnostics

Adult Ae. aegypti samples collected from BGS traps in the field were screened for Wolbachia using Taqman qPCR on a Roche LightCycler 480 using a qualitative assay for presence or absence of Wolbachia as previously described¹⁴ but with the replacement of the Cy5-BHQ3 fluorophore-quencher pair in the wMel probe with the fluorophore-quencher LC640-IowaBlack (Integrated DNA technologies) to remove some of the Cy5 probe instability observed under varying light and ozone levels¹⁵.

Dengue case notification data

Dengue is a notifiable disease in Australia, which mandates clinicians and laboratories to report confirmed and suspected cases to local health authorities (See Queensland Dengue Management plan). Non-identifiable data was provided by Queensland Health Communicable Diseases Branch for all laboratory-confirmed and clinically diagnosed (probable) dengue cases with illness onset between 1 January 2000 and 30 June 2018, extracted from the Notifiable Conditions System (NOCS) on 3 July 2018. Case notifications within the Townsville local government authority were tabulated by month of illness onset and history of recent overseas travel during the 3 – 12 days prior to illness onset; a variable that is routinely captured in case notifications based on interview by public health teams (see 16 for interview protocol). The suburb of residence of four locally-acquired dengue cases notified in Townsville since Wolbachia deployments commenced in October 2014 was determined from situation reports published by the local public health unit.

Ethical considerations and consent

Ethics approval for human blood feeding mosquito colonies in Melbourne was issued from Monash University CF11/0766 a 2011000387 (Rearing mosquitoes using blood from human volunteers). All volunteers (no children involved) provided written consent.

In Cairns, Human Ethics approval for bloodfeeding (H6286) was provided by Human Research Ethics Committee, James Cook University. All adult subjects provided informed oral consent (no children were involved). Names of subjects providing oral consent were recorded in writing.

Townsville community mosquito releases were covered under Monash University ethics: MUHREC Approval CF16/763 - 2016000370 - Eliminate Dengue - Community based field releases of Wolbachia infected mosquitoes in Townsville, Queensland.

Surveys were undertaken under Monash ethics: MUHREC Approval CF13/2805 - 2013001515 - Eliminate Dengue - Community knowledge of dengue and Wolbachia based dengue control in Townsville, Queensland

Verbal and/or written consent from participants was obtained by phone, online or face-to-face to set BG traps, set MRCs (phase 1), or participate in Community Mosquito Releases.

Ethical approval was not required to access non-identifiable dengue case notification data collected as part of routine disease surveillance.