Optimizing honey-card arbovirus surveillance to rapidly detect transmission of mosquito-borne virus transmission in Florida PI: Nathan Burkett-Cadena Florida Medical Entomology Laboratory University of Florida – IFAS 200 9th St. SE Vero Beach, FL 32962 772-778-7200 nburkettcadena@ufl.edu Co-PI: Thomas R. Unnasch Department of Global Health University of South Florida 3720 Spectrum Blvd. IDRB suite 304 Tampa, FL 33604 tunnasch@health.usf.edu 813-974-0507 FL DACS Contract # 22397 Final Report Sept. 10, 2016

Abstract This project constitutes field evaluations of prototype traps and the commercially available Passive Box Trap (SMACK trap) for arbovirus surveillance in Florida using the honey-card techniques. The project was a collaborative effort between the Florida Medical Entomology Laboratory, Anastasia Mosquito Control District, and the University of South Florida. This project was completed with successful establishment and trapping at each of five field sites in Indian River and St. Johns Counties. Each site consisted of 3 traps being sampled twice weekly over 10 months (Sep-Nov, 2015 & Mar-Aug 2016) for 1,800 total trap nights. Nearly 180,000 female mosquitoes were captured and identified representing 41 species. Arbovirus activity in both counties was relatively low during the study period. Three EEEV-positive samples were recovered from honey cards. Nineteen total arbovirus seroconversions were detected in sentinel chickens during the same period in the two counties, and these were not at sites of honey-card trap studies. The relatively low arbovirus activity in the two counties during the study period complicates the drawing of meaningful conclusions regarding the effectiveness of honey-cards for arbovirus surveillance. Results of the current project suggest that the methods of mosquito sampling and arbovirus detection may be effective for detecting arbovirus transmission, yet additional research and development are needed before honey cards are relied upon as an indicator of arbovirus risk in Florida.

INTRODUCTION Zoonotic mosquito-borne zoonotic viruses, such as West Nile virus, eastern

equine encephalitis virus and St. Louis encephalitis virus, present major challenges to public health infrastructure of Florida and elsewhere (Zohrabian et al. 2004; Sejvar et al. 2006; Sejvar 2007). These debilitating and often deadly viruses are transmitted from wild avian reservoir hosts (mostly songbirds) to humans and domestic animals by a number of mosquito species in Florida. Because the virus first develops in wild birds, outbreaks arboviral disease in humans and domestic animals are notoriously difficult to predict. In Florida, sentinel chicken arbovirus surveillance is perhaps the most widely utilized early detection system. The sentinel chicken program is operated primarily by mosquito control districts around the state, and involves the regular collection and testing of blood samples from chickens, to detect transmission of arboviruses in advance of human cases (Nelson et al. 1983). The placement of sentinel flocks was based primarily on the locations of human cases of SLE from a widespread epidemic of that virus in 1978. Despite the decline of human SLE cases in Florida, and the increase of EEE cases and the arrival of WNV, the locations of sentinel flocks are rarely changed, due to their large size and expense. The sentinel chicken arbovirus surveillance program has had mixed success since its inception. While the sentinel program provided timely early warnings SLE amplification in Florida (Day 1991), this was not the case for WNV, such that human cases of WNV infection peak before sentinel chicken infections (USGS, 2012). In the case of EEE, sentinel chickens often fail to provide early warning of amplification, such that EEEV infections in sentinel birds, horses, and humans often occur synchronously during periods of rapid EEEV amplification and spillover (Day and Stark 1996). The objective of this work was to evaluate and optimize and efficient system for detection of arbovirus transmission in Florida utilizing traps that are much easier to maintain and relocate than sentinel chicken flocks. The pivotal component of the system is honey-infused nucleic acid-preserving substrates (honey-cards). Honey-cards are placed within the collection chambers of modified mosquito traps (Figure 1-a,b,c). Trapped mosquitoes within the chambers feed upon the honey for their nourishment. As the mosquitoes feed upon the honey, they salivate. Virus particles are found in the saliva of infectious mosquitoes and is inactivated and preserved by chemicals found in the honey-card. The honey-cards can be tested by PCR for arboviruses. This technique allows for collection and testing of very large numbers of mosquitoes, without the time intensive and expensive process of mosquito identification and pool screening. The methods of honey-card deployment were initially developed in Australia, for surveillance of arboviruses in remote areas of the country (Hall-Medelin et al. 2010). Ritchie et al. (2013) and have developed passive traps (Figure 2-c) that do not require batteries and capture large numbers of mosquitoes that readily feed on honey cards. Several Australian arboviruses have been detected using these passive traps (van den Hurk et al. 2014). Three traps were evaluated in Florida, with mixed results (Burkett-Cadena et al. 2016). Early detection of arbovirus transmission is our best weapon against these pathogens, giving mosquito control districts a critical window of action to impact the vector population. The surveillance system proposed here will add critical days, or perhaps even weeks, to the temporal window that mosquito control

districts have to effectively combat arbovirus vectors in epidemic situations. Pool-screening of mosquitoes, has the potential of providing the most timely indicator of viral activity. However pool-screening is an extremely time consuming and labor intensive process. Field-collected females must be identified to species, sorted into pools of 50 individuals by species, location and date, and then each pool subjected to a screening assay. Extraction of genetic material and RT-PCR amplification of each pool (species, location and date) is expensive and time consuming. New techniques for minimizing these procedures are thus needed. METHODOLOGY

Field studies were replicated at sites in Indian River and St. Johns Counties, FL. Field sites were selected based upon historical records of mosquito and arbovirus activity maintained by AMCD. Traps were sampled twice weekly at each of five total surveillance sites in Indian River and St. Johns County. One modified one honey-card gravid trap, and one honey-card resting trap, and one honey-card passive box trap “SMACK trap” (Figure 1) were operated at peridomestic and sylvatic (forest habitat) sites selected from historic data maintained by AMCD and IRMCD. The traps were run 7 days per week with two site visits per week to retrieve collections and honey cards for virus screening. At the time of trap retrieval, honey-cards were removed from the collection chamber, labeled with site and date then shipped overnight to USF, where they were screened for presence of EEEV and WNV by RT-PCR using protocols employed by the FDOH arbovirus laboratory. A subset of the mosquitoes (100 females) were identified to collect data on species composition of each trap and site. The subset was weighed extrapolate the abundance and species composition of the entire trap sample. Fieldwork was conducted in two periods, a late summer/fall period (Sep 1 - Nov 31) and spring/summer period (May 1 – Aug 31).

Laboratory experiments were also conducted to test different preservatives for resisting degradation of samples (mold growth) and gustatory responses of mosquitoes. Filter papers were first treated with Sodium Bicarbonate (0.1%), EDTA (0.1%; food preservative), FTA (Whattman), USTOP (proprietary) or untreated. These preservative cards were then soaked in a drop of blue (dyed) honey (0.2g). One of each of the honey-card types were then placed into paper cages containing n=5 females of Culex quinquefasciatus (4 per treatment). At 48-hr females were freeze-killed to examine for evidence of honey-feeding (blue coloration in abdomen). The honey-cards were then placed in Petri dishes and monitored for 8 days for mold growth. Mold growth was

Figure 1. Honey-card traps utilized in past and proposed fieldwork. Automated gravid trap (A), Automated resting trap (B) and passive box trap (C).

quantified as the proportion of 56 subdivisions of the treated filter papers that had mold growth. Honey-feeding was quantified as the proportion of females that showed evidence of feeding on honey from the treated filter papers.

The distributions of mosquitoes, by trap type were compared using Chi-square test of independence. The distributions of arbovirus detection, by county were compared using Chi-square test of independence. ANOVA was used to test for differences in mold inhibition and mosquito feeding for various honey card types. RESULTS AND DISCUSSION

Combined, 179,085 female mosquitoes were collected using traps in Indian River and St. Johns Counties. By far, more females were sampled using the SMACK trap (passive box trap) than other traps, in both counties (Table 1; Figure 2). Culex nigripalpus was the most common or second most common mosquito species in all trap types in both counties (Table 1, Figure 2), constituting 26-61% of resting trap collections, 30-50% of gravid trap collections, and 74-91% of SMACK trap collections. Other very common mosquitoes in traps included Culex erraticus, Psorophora columbiae, Aedes atlanticus, Anopheles crucians, Culex salinarius, Culex coronator and Culex quinquefasciatus. Less common of the major medically important vector species included Culiseta melanura.

Figure 2. Total mosquitoes captured by Honey-card traps gravid trap and resting trap in two Florida counties.

Figure 3. Total mosquitoes captured by Honey-card traps gravid trap and resting trap in two Florida counties, by species.

Table 1. Total numbers of female mosquitoes collected from SMACK, Gravid and Resting traps at 5 sites in Indian River and St. Johns Counties, FL. Others includes species for which <50 specimens were collected (Ae. aegypti, Ae. fulvus pallens, Ae. mitchellae, Ae. pertinax, Ae. sollicitans, Ae. triseriatus, Ae. vexans, An. atropos, An. perplexens, An. punctipennis, Cq. perturbans,Cs. melanura, Cx. interrogator, Cx. territans, Or. signifera, Ps. ciliata, Ps. howardii, Ur. sapphirina, Wy. mitchellii). St. Johns Indian River Species SMACK Gravid Resting SMACK Gravid Resting Aedes albopictus 2 23 12 51 89 3 Aedes atlanticus 1,491 1,530 79 333 47 5 Aedes canadensis 2 0 0 513 0 0 Aedes infirmatus 120 39 2 90 14 2 Aedes taeniorhynchus 0 0 0 69 0 0 Anopheles crucians 2,419 100 4 465 30 17 An. quadrimaculatus 10 2 1 131 17 13 Culex coronator 0 0 0 1,331 449 2 Culex erraticus 114 26 11 3,853 269 150 Culex nigripalpus 12,920 883 65 139,694 1827 361 Culex pilosus 0 0 0 68 163 6 Culex quinquefasciatus 30 128 65 719 406 6 Culex restuans 10 34 0 1 2 0 Culex salinarius 4 0 0 1,717 97 8 Culiseta inornata 93 4 0 0 0 0 Deinocerites cancer 0 0 0 134 23 5 Mansonia dyari 2 2 0 212 0 0 Mansonia titillans 0 0 0 1,281 7 1 Psorophora columbiae 200 11 1 3,303 21 2 Psorophora cyanescens 0 0 0 13 2 0 Psorophora ferox 14 50 1 98 11 1 Uranotaenia lowii 0 4 0 4 183 3 Total 17,506 2,871 249 154,200 3,673 586

Results of Chi-square test of independence suggests that the numbers of total

mosquitoes are not distributed uniformly between St. Johns and Indian River Counties (chi-square=7,290.79; d.f.=2; P<0.001). Chi-square test of independence also suggests that the numbers of arbovirus detections were not significantly different for sentinel chickens and honey-card traps in the two counties (chi-square=0.368; d.f.=1; P=0.544).

Low numbers of arbovirus detections were made using honey-cards and sentinel chickens in the tow counties where sampling was performed. In Indian River County, just 3 arbovirus seroconversions were detected. All 3 of these were on a single day (Dec. 2), from a single flock (Lockwood), all were West Nile virus (Table 2). Unfortunately, mosquito sampling using honey-card traps was not conducted during December in Indian River County, as per the original proposed methods. In St. Johns County, 16 arbovirus seroconversions were detected in sentinel chickens, EEEV (n=11) and WNV (n=5), during the study period. Nine of eleven EEEV seroconversion were at a single site (Cartwheel Bay) while WNV seroconversions were split amongst four sites (flocks).

Table 2. Sentinel chicken seroconversions to EEEV and WNV in Indian River and St. Johns Counties, May 2015 – August 2016).

County Date Location Virus Conversions St. Johns 6/15/20 Cartwheel Bay EEEV 3 St. Johns 6/22/20 Cartwheel Bay EEEV 1 St. Johns 6/29/20 Cartwheel Bay EEEV 1 St. Johns 7/7/20 Joe Ashton EEEV 2 St. Johns 8/27/20 Cartwheel Bay EEEV 1 St. Johns 8/31/20 Cartwheel Bay EEEV 3 St. Johns 11/2/15 Flagler estates WNV 1 St. Johns 11/2/15 Davis Park WNV 2 St. Johns 11/9/15 Joe Ashton WNV 1 St. Johns 11/9/15 Don Manuel WNV 1 Indian River 12/2/15 Lockwood WNV 3

Mold was prevalent on papers treated with Sodium Bicarbonate and untreated

filter paper. Mold was not detected on EDTA, FTA or USTOP-treated filter papers. The percentage of honey-card squares with mold was significantly greater for control (untreated filter paper) filter than sodium bicarbonate (P<0.001), EDTA (P<0.001), and USTOP (P<0.001). These three treated cards (sodium bicarbonate, EDTA and USTOP) were not different than one another, with respect to mold inhibition (P>0.05).

Mosquitoes generally would not feed on honey-soaked FTA cards, but would feed on EDTA and USTOP honey-cards. The percentage of honey-fed females was significantly greater for sodium bicarbonate (P<0.001), EDTA (P=0.014), and USTOP (P<0.001), compared to FTA honey-cards. These three cards (sodium bicarbonate, EDTA and USTOP) were not different than untreated cards, with respect to honey feeding by mosquitoes (P>0.05).

Despite the very large numbers of mosquitoes sampled in the current project, very few arbovirus detections (n=3 EEEV) were made via the honey-card method. This may be due to the relatively low arbovirus activity, that was observed in general, in the two counties where the fieldwork was conducted (n=19 total sero-conversions). In addition, relatively small numbers of key vector species (Culiseta melanura, Culex quinquefasciatus) were collected during the current research, as compared to previous years, when arbovirus seroconversions and detections from honey-cards were encountered in greater numbers (Burkett-Cadena et al. 2016).

Figure 4. Total mosquitoes captured by Honey-card traps gravid trap and resting trap in two Florida counties.

Cost analysis of sentinel chickens and honey-cards

SENTINEL CHICKENS: HAI screening is estimated to cost $6.00 per test (FDOH estimate). However, if a specimen goes for confirmatory testing (ELISA or PRNT), then the cost for testing increases substantially. Using approximate figures from AMCD and FDOH, the following estimates were produced: At 33 weeks per year, 60 vials (chickens) per week, FDOH spends approximately $11,880 per season in sample testing (materials only) for AMCD chickens. AMCD spends $8,000 per season for cage supplies/feed/birds and sample shipping. Therefore, materials costs for maintaining sentinel chickens, shipping samples, and arbovirus screening is approximately $20,000 for AMCD.

HONEY-CARDS: PCR screening is estimated to cost $6.00 per test (FMEL estimate). Using approximate figures from AMCD and FMEL, the following estimates were produced: At 33 weeks per year, 60 samples (honey-cards) per week, sample testing (materials only) for honey-cards at AMCD would cost approximately $11,880 per season. Depending upon trap type used, consumable such as carbon-dioxide, batteries, honey, cards, sponges etc would likely require a similar budget ($8,000) as materials and shipping for sentinel chickens, such that the total costs (materials only) for the two methods are roughly equal. Personnel and travel costs for the two methods (sentinel chicken and honey-cards) are likely to be very similar, as two visits per site per week are required for either method. CONCLUSIONS Evaluations of prototype traps and the commercially available Passive Box Trap (SMACK trap) for arbovirus surveillance in Florida using the honey-card techniques suggests that the SMACK trap is an effective passive trap for collecting a limited diversity of vector mosquito species, particularly Cx. nigripalpus, vector of SLE and WNV in Florida. Nearly 180,000 female mosquitoes of 41 species were sampled and putatively assayed by honey-cards for this project. Overall arbovirus transmission in both counties where research was conducted was very low during the study period, a situation that complicates the drawing of meaningful conclusions regarding the effectiveness of honey-cards for arbovirus surveillance. Honey-cards may be effective for detecting arbovirus transmission, although additional research and development are needed before being widely relied upon.

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