Paul Zborowski

Exploiting mosquito sugar feeding to detect mosquito-borne pathogens

Arthropod-borne viruses (arboviruses) represent a global public health problem, with dengue viruses causing millions of infections annually, while emerging arboviruses, such as West Nile, Japanese encephalitis, and chikungunya viruses have dramatically expanded their geographical ranges. Surveillance of arboviruses provides vital data regarding their prevalence and distribution that may be utilized for biosecurity measures and the implementation of disease control strategies. However, current surveillance methods that involve detection of virus in mosquito populations or sero-conversion in vertebrate hosts are laborious, expensive, and logistically problematic. We report a unique arbovirus surveillance system to detect arboviruses that exploits the process whereby mosquitoes expectorate virus in their saliva during sugar feeding. In this system, infected mosquitoes captured by CO2-baited updraft box traps are allowed to feed on honey-soaked nucleic acid preservation cards within the trap. The cards are then analyzed for expectorated virus using real-time reverse transcription-PCR. In field trials, this system detected the presence of Ross River and Barmah Forest viruses in multiple traps deployed at two locations in Australia. Viral RNA was preserved for at least seven days on the cards, allowing for long-term placement of traps and continuous collection of data documenting virus presence in mosquito populations. Furthermore no mosquito handling or processing was required and cards were conveniently shipped to the laboratory overnight. The simplicity and efficacy of this approach has the potential to transform current approaches to vector-borne disease surveillance by streamlining the monitoring of pathogens in vector populations.

Mosquito host-feeding patterns and implications for Japanese encephalitis virus transmission in northern Australia and Papua New Guinea.

Abstract.  Japanese encephalitis (JE) virus spread to northern Australia during the 1990s, transmitted by Culex annulirostris Skuse and other mosquitoes (Diptera: Culicidae). To determine the relative importance of various hosts for potential vectors of JE virus, we investigated the host‐feeding patterns of mosquitoes in northern Australia and Western Province of Papua New Guinea, with particular attention to pigs, Sus scrofa L. – the main amplifying host of JE virus in South‐east Asia. Mosquitoes were collected by CDC light traps baited with dry ice and 1‐octen‐3‐ol, run 16.00–08.00 hours, mostly set away from human habitations, if possible in places frequented by feral pigs. Bloodmeals of 2569 mosquitoes, representing 15 species, were identified by gel diffusion assay. All species had fed mostly on mammals: only <10% of bloodmeals were from birds. The predominant species was Cx. annulirostris (88%), with relatively few (4.4%) bloodmeals obtained from humans. From all 12 locations sampled, the mean proportion of Cx. annulirostris fed on pigs (9.1%) was considerably lower than fed on other animals (90.9%). Highest rates of pig‐fed mosquitoes (>30%) were trapped where domestic pigs were kept close to human habitation. From seven of eight locations on the Australian mainland, the majority of Cx. annulirostris had obtained their bloodmeals from marsupials, probably the Agile wallaby Macropus agilis (Gould). Overall proportions of mosquito bloodmeals identified as marsupial were 60% from the Gulf Plains region of Australia, 78% from the Cape York Peninsula and 64% from the Daru area of Papua New Guinea. Thus, despite the abundance of feral pigs in northern Australia, our findings suggest that marsupials divert host‐seeking Cx. annulirostris away from pigs. As marsupials are poor JE virus hosts, the prevalence of marsupials may impede the establishment of JE virus in Australia.

Entomological Investigations of an Outbreak of Japanese Encephalitis Virus in the Torres Strait, Australia, in 1998

Abstract Japanese encephalitis (JE) virus first appeared in Australia in 1995, when three clinical cases (two fatal) were diagnosed in residents on Badu Island in the Torres Strait, northern Queensland. More recently, two confirmed human JE cases were reported in the Torres Strait Islands and Cape York Peninsula, in northern Queensland in 1998. Shortly after JE virus activity was detected in humans and sentinel pigs on Badu Island in 1998, adult mosquitoes were collected using CO2 and octenol-baited CDC light traps; 43 isolates of JE virus were recovered. Although Culex sitiens group mosquitoes yielded the majority of JE isolates (42), one isolate was also obtained from Ochlerotatus vigilax (Skuse). Four isolates of Ross River virus and nine isolates of Sindbis (SIN) virus were also recovered from members of the Culex sitiens group collected on Badu Island in 1998. In addition, 3,240 mosquitoes were speciated and pooled after being anesthetized with triethylamine (TEA). There was no significant difference in the minimum infection rate of mosquitoes anesthetized with TEA compared with those sorted on refrigerated tables (2.8 and 1.6 per 1,000 mosquitoes, respectively). Nucleotide analysis of the premembrane region and an overlapping region of the fifth nonstructural protein and 3′ untranslated regions of representative 1998 Badu Island isolates of JE virus revealed they were identical to each other. Between 99.1% and 100% identity was observed between 1995 and 1998 isolates of JE from Badu Island, as well as isolates of JE from mosquitoes collected in Papua New Guinea (PNG) in 1997 and 1998. This suggests that the New Guinea mainland is the likely source of incursions of JE virus in Australia.

Blood sources of mosquitoes collected from urban and peri-urban environments in eastern Australia with species-specific molecular analysis of avian blood meals

To identify the hosts of mosquitoes collected from urban and peri-urban habitats in eastern Australia, 1,180 blood fed mosquitoes representing 15 species were analyzed by enzyme-linked immunosorbent assay and molecular techniques. Four common and epidemiologically important species could be classified according to their host-feeding patterns: Aedes aegypti was anthropophilic, Ae. vigilax was mammalophilic, Culex quinquefasciatus was ornithophilic, and Cx. annulirostris was opportunistic, readily feeding on birds and mammals. Mitochondrial cytochrome b DNA sequence data showed that more than 75% of avian blood meals identified from Cx. annulirostris collected from Brisbane, Newcastle, and Sydney originated from ducks (Order Anseriformes, Family Anatidae). More than 75% of avian blood meals from Cx. quinquefasciatus from Cairns belonged to one of three Passerine species, namely Sphecotheres vieilloti (figbird), Sturnus tristis (common myna), and Philemon buceroides (helmeted friarbird). This study demonstrates associations between vectors in Australia and vertebrate hosts of endemic and exotic arboviruses.

Arboviruses isolated from mosquitoes collected from urban and peri-urban areas of eastern Australia.

Abstract To determine the presence of arboviruses in mosquito populations from major urban areas of eastern Australia, a total of 67,825 mosquitoes, representing ∼60 species, was collected and tested from Cairns, Brisbane, and Sydney between January 2005 and April 2008. Mosquito pools were screened by inoculation onto mosquito cell cultures and the detection of viral antigen using a panel of flavivirus and alphavirus monoclonal antibodies in an enzyme-linked immunosorbent assay. Suspect positive samples were confirmed using virus-specific real-time reverse transcriptase–polymerase chain reaction assays. No flaviviruses were detected, but 2 alphaviruses were isolated from mosquito pools collected from Cairns, with 1 Barmah Forest virus isolate from a pool of 100 Aedes vigilax and 1 Ross River virus isolate from a pool of 83 Verrallina carmenti. In addition, a single Aedes alternans collected from Sydney yielded an isolate most similar to Stretch Lagoon virus, a newly described virus from the genus Orbivirus. These results suggest that during the study, arboviruses were circulating at a low level in the areas sampled. The findings from this study will promote public health awareness of the risk of arboviruses in urban areas, leading to more informative public health campaigns to safeguard the Australian public.

DOES 1-OCTEN-3-OL ENHANCE TRAP COLLECTIONS OF JAPANESE ENCEPHALITIS VIRUS MOSQUITO VECTORS IN NORTHERN AUSTRALIA?

ABSTRACT The responses of Japanese encephalitis virus (JEV) mosquito vectors to 1-octen-3-ol (octenol) and CO2 were evaluated using Centers for Disease Control (CDC) light traps at 3 sites in northern Australia. There was no significant difference between the number of Culex sitiens subgroup mosquitoes or Cx. gelidus collected in CDC light traps baited with either CO2 alone or CO2 + octenol on Badu Island. At both mainland locations, using octenol in combination with CO2 significantly increased collections of Cx. sitiens subgroup mosquitoes. Collections of nontarget species, such as Ochlerotatus spp., Anopheles spp., and Verrallina spp. were also significantly increased with the addition of octenol. At all 3 locations, reducing collections of nontarget mosquitoes by not using octenol increased the proportion of Culex spp. collected, thus potentially reducing the time and resources required to sort and process collections for JEV detection. Our results also indicate that trials into the efficacy of using octenol as an attractant should be carried out in each area prior to the implementation of a mosquito-based arbovirus surveillance system.

EFFICACY OF NOVEL UPDRAFT TRAPS FOR COLLECTION OF MOSQUITOES IN CAIRNS, AUSTRALIA

ABSTRACT We conducted trials in Cairns, Australia, to examine if novel updraft light traps collected significantly more mosquitoes than the Centers for Disease Control and Prevention (CDC) model 512 miniature light trap. Two new updraft traps, the Northern Australia Quarantine Strategy (NAQS) Mozzie Trap and a CDC updraft trap, both collected significantly more mosquitoes than the standard CDC light trap, with a mean CDC Trap Index (trap collections relative to paired standard CDC light trap collections) of 3.3 and 2.3, respectively. These traps both had large horizontal suction areas that increased the probability that attracted mosquitoes entered the trap updraft. However, if the CO2 source was located within the updraft of the CDC updraft trap, mosquito collections decreased considerably, indicating that placement of the bait is critical to trap performance. Creating an updraft by simply inverting the CDC trap body did not increase collections. The Mosquito Magnet X trap also did not collect significantly more mosquitoes than the CDC trap. Two CDC light traps sharing a 600 ml CO2/min gas line collected ca. 50% more mosquitoes than a single CDC trap baited with 600 ml CO2/min, suggesting that a single gas source could be used on a trap line consisting of multiple trap units. These studies suggest that the optimal trap design should incorporate a CO2 release system that lures mosquitoes to a large updraft within a bowl-shaped trap intake.