Protection From Mosquito Biting Provided by Permethrin-Treated Military Fabrics.

A study to evaluate the protection provided by permethrin-treated fabric following cold-water washing against biting by mosquitoes is reported. Australian Defense Force (ADF) disruptive pattern combat uniform (DPCU) shirt fabric and entire shirts were treated by dipping in a 0.6% emulsion (Perigen Defense, containing 500 g/liter permethrin), and commercial factory treatment in the United States (Factory A) and Europe (Factory B). Protection was recorded after 1, 3, 5, 10, 30, and 50 washes. The treated fabric provided 100% protection against bites of Anopheles farauti Laveran for at least 50 washes, although only 4.8-19.0% of this species fed through untreated DPCU. The protection provided by each type of permethrin treatment against Aedes aegypti (L.) biting was variable; however, there were no significant differences between the percentage of mosquitoes biting between 1 and 10 washes. A comparison between the two factory treatments for 1-50 washes also showed no statistical difference in Ae. aegypti feeding. Chemical analysis of fabric was conducted using gas chromatography and showed that the initial dose was 0.125 mg/cm(2) for Perigen-treated fabric, which fell to 0.004 mg/cm(2) after 10 washes. By contrast, factory treatments resulted in initial dose rates of 0.20 mg/cm(2) for Factory A and 0.19 mg/cm(2) for Factory B. After 10 washes, Factory A-treated fabric had 0.09 mg/cm(2) and Factory B 0.15 mg/cm(2) of permethrin. Despite the higher concentrations of permethrin in the fabric, there was not a commensurate increase in biting protection provided by the factory-treated fabric, compared with fabric treated by dipping in permethrin emulsion.

Field evaluation and user acceptability of repellent formulations containing DEET against mosquitoes in Australia.

Field efficacy trials comparing 2 formulations of deet against mosquitoes in Redcliffe, Queensland, Australia were conducted in February 2009. A formulation containing 35% deet in a gel (Australian Defence Force deet) provided > 95% protection for 3 h, while a formulation containing 40% deet in ethanol (Bushman) in a spray applicator provided > 95% for 6 h. A user acceptability study showed that 82% of soldiers using the Bushman formulation during contingency operations for 14-28 days in Timor-Leste would recommend this formulation to others and believed that the formulation provided protection against mosquitoes.

Influence of Orientia tsutsugamushi infection on the developmental biology of Leptotrombidium imphalum and Leptotrombidium chiangraiensis (Acari: Trombiculidae).

Leptotrombidium chiangraiensis Tanskul & Linthicum, and Leptotrombidium imphalum Vercammen-Grandjean are important vectors of scrub typhus in rice field habitats in northern Thailand. The developmental biology of all stages of the life cycle of two generations of these species of mites infected with Orientia tsutsugamushi (Hayashi) and uninfected mites is reported. The development of the infected lines of both F1 and F2 L. chiangraiensis were significantly longer than their respective uninfected lines (P < 0.05). The developmental times of uninfected and infected F1 lines of L. imphalum were not significantly different; however, F2 infected lines took significantly longer to develop (P < 0.05). Both F1 and F2 generations of infected L. imphalum and L. chiangraiensis oviposited on average >150 fewer eggs than uninfected mites.

Mosquito and Arbovirus Surveillance at Bradshaw Field Training Area, Northern Territory, Australia.

Mosquito surveillance and mosquito-borne arbovirus detections in the area of Bradshaw Field Training Area (BFTA) in Northern Territory, Australia, have not been previously documented. A survey to record the mosquito species within BFTA and determine if arboviruses are present in them was conducted in April 2013. A total of 8,530 mosquitoes were collected, using carbon dioxide-baited encephalitis virus surveillance traps, and the predominant species were Aedes normanensis, which was 74.2% of the mosquitoes collected, and Anopheles annulipes (11.1%), An. amictus (2.9%), and An. meraukensis (2.4%). The mean number of mosquitoes collected was 120.1 ± 13.7 per trap in the 71 traps placed. This study has shown mosquito abundance in the eastern area of BFTA during 2 wk of April 2013 was of high density, and therefore a heightened risk of arbovirus transmission. Reverse transcriptase-polymerase chain reaction and virus isolation identified a single Barmah Forest virus strain from a pool of Ae. normanensis, a positive rate of 1/174 (0.57%). This study has provided the 1st survey of mosquitoes and potential mosquito-borne virus transmission in BFTA.

Laboratory evaluation of the response of Aedes aegypti and Aedes albopictus uninfected and infected with dengue virus to deet.

Laboratory studies were conducted to compare the response of Aedes aegypti (L.) and Aedes albopictus (Skuse) adults, uninfected and infected with four serotypes of dengue virus to a repellent containing 5% N, N-diethyl-3-methylbenzamide (deet). The results showed that mosquitoes infected with the four serotypes of dengue responded similarly to uninfected mosquitoes.

Malaria vectors of Timor-Leste.

BACKGROUND: The island of Timor lies at the south-eastern edge of Indonesia on the boundary of the Oriental and Australian faunal regions. The country of Timor-Leste, which occupies the eastern part of the island, is malarious, but anopheline faunal surveys and malaria vector incrimination date back to the 1960 s. Over the last decade the malaria vectors of south-east Asia and the south-west Pacific have been intensely studied using molecular techniques that can confirm identification within complexes of isomorphic species. The aim of this study is to accurately identify the Anopheles fauna of Timor-Leste using these techniques. METHODS: The survey was carried out over the period February to June 2001. Standard entomological techniques--human landing collections, larval collections and CO2 baited light traps--were used to collect anophelines from the main geographical regions: coastal plains, inland plains, and highlands. Specimens were processed for identification by morphology and genotyped for the ribosomal DNA ITS2 by restriction analysis and/or DNA sequencing. Phylogenetic relationship of Anopheles sundaicus and Anopheles subpictus individuals was also assessed using DNA sequences from the ITS2 and mitochondrial cytochrome-b. All specimens, other than those from larval surveys, were processed to detect the presence of the Plasmodium parasite circumsporozoite protein by ELISA for vector incrimination. RESULTS: Of 2,030 specimens collected, seven species were identified by morphology: Anopheles barbirostris, Anopheles aconitus, Anopheles annularis, Anopheles maculatus, Anopheles peditaeniatus, An. sundaicus and Anopheles vagus. These were confirmed by molecular analysis with the addition of Anopheles flavirostris and an unidentified species designated here as An. vagus genotype B. This latter species was morphologically similar to An. vagus and An. subpictus and is likely to be the An. subpictus described by other workers for Timor. However, genetically this species showed strong affinities to the An. sundaicus complex. Anopheles vagus was the most common species but was rarely collected coming to bite humans; An. barbirostris and An. vagus genotype B were the two most common species collected in human landing catches and both were found positive for CS protein. CONCLUSIONS: The anopheline fauna of Timor-Leste is of Oriental origin with no evidence of elements from the Australian Region. The existence of species complexes will make the use of morphological markers problematic in the country. Using molecular analysis a number of issues regarding the anopheline fauna of Timor-Leste were resolved and nine putative species of Anopheles were identified; two species: An. barbirostris and An. vagus genotype B, were incriminated as malaria vectors.

Field Effectiveness of a Metofluthrin Fan-Based Emanator and Deet as Repellents Against Aedes vigilax in Southeast Queensland, Australia1.

A field study to compare a formulation containing 40% deet (N,N-diethyl-3-methyl benzamide) in ethanol (Bushman™) and a battery-powered fan emanator with a chemical strip containing 31.2% metofluthrin (OFF!® Clip-On™) was conducted at Redcliffe, Queensland, Australia, in February 2016. The 40% deet provided 100% protection against mosquitoes for 5 h until tests ceased, while the OFF! Clip-On device provided only 42.2-60.8% protection against Aedes vigilax during the same period.

Transstadial and transovarial transmission of Orientia tsutsugamushi in Leptotrombidium imphalum and Leptotrombidium chiangraiensis (Acari: Trombiculidae).

Transovarial transmission of Orientia tsutsugamushi (Hayashi) in laboratory colonies of Leptotrombidium chiangraiensis Tanskul & Linthicum and Leptotrombidium imphalum (Vercammen-Grandjean & Langston) (Acari: Trombiculidae) was studied for two generations. In L. chiangraiensis, the transovarial and filial infection rate was 100% in each generation. Only infected females were produced. In L. imphalum, the transovarial infection rate of the parental generation was 100% but declined to 93.3% in the F1 generation. The overall filial infection rate was 100% in the F1 but was only 62.3% in the F2 generation. In infected lines, only infected females were produced in the F1 generation, but 1.5% of the F2 progeny were infected males. Lower rates of transovarial transmission in L. imphalum may be the cause of the lower natural infection rates found in nature.

Laboratory and field evaluation of SS220 and deet against mosquitoes in Queensland, Australia.

Laboratory and field efficacy trials comparing deet (N,N-diethyl-3-methylbenzamide) and SS220 [(IS, 2'S)-2-methylpiperindinyl-3-cyclohexen-1-carboxamide] against mosquitoes in Queensland, Australia, were conducted. In the laboratory, both compounds provided between 150 and 195 min of protection against Aedes aegypti and between 18 and 80 min of protection against Anopheles farauti. In laboratory tests against Culex annulirostris, 20% SS220 provided 3 h of protection and 20% deet provided >6 h of protection. A field efficacy test was conducted at Redcliffe, Queensland in January 2008 and the predominant mosquito species collected was Cx. annulirostris (84.4% of collection). In the field, 20% SS220 provided significantly better protection against mosquitoes than 20% deet. Seven hours after application, SS220 provided greater than 96.0% protection against all mosquitoes, whereas 20% deet provided 58.9% protection.

Expectoration of Flaviviruses during sugar feeding by mosquitoes (Diptera: Culicidae).

Biological transmission of arboviruses to a vertebrate host occurs when virions are expelled along with saliva during blood feeding by a hematophagous arthropod. We undertook experiments to determine whether mosquitoes expectorate flaviviruses in their saliva while sugar feeding. Batches of Culex annulirostris Skuse and Culex gelidus Theobald (Diptera: Culicidae) were orally infected with Japanese encephalitis (family Flaviviridae, genus Flavivirus, JEV), Kunjin (family Flaviviridae, genus Flavivirus, KUNV; a subtype of West Nile virus), and Murray Valley encephalitis (family Flaviviridae, genus Flavivirus, MVEV) viruses. After a 7-d extrinsic incubation, these mosquitoes were offered sucrose meals via cotton pledgets, which were removed daily and processed for viral RNA by using real-time TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR) assays. JEV, MVEV, and KUNV RNA was detected in all pledgets removed from batches of Cx. gelidus on days 7-14 postexposure. In contrast, detection rates were variable for Cx. annulirostris, with KUNV detected in 0.3 M sucrose pledgets on all days postexposure, and JEV and MVEV detected on 57 and 50% of days postexposure, respectively. Higher concentrations of sucrose in the pledget did not increase virus detection rates. When individual JEV-infected Cx. gelidus were exposed to the sucrose pledget, 73% of mosquitoes expectorated virus with titers that were detectable by TaqMan RT-PCR. These results clearly show that flaviviruses are expectorated by infected mosquitoes during the process of sugar feeding on artificial pledgets. Potential applications of the method for arboviral bioassays and field surveillance are discussed.

Evaluation of bifenthrin and permethrin as barrier treatments for military tents against mosquitoes in Queensland, Australia.

A study comparing the effectiveness of barrier treatment of military tents with bifenthrin and permethrin in preventing entry of mosquitoes was conducted at Wide Bay Training Area, Queensland, Australia. Five military tents were erected at a site in bushland at Mosquito Creek, about 2 km from the coast. Two tents were sprayed with bifenthrin (Bistar 80SC, 0.1% mix, 12.5 ml/liter) and 2 with permethrin (Perigen 500, 1.2%, 24 ml/liter). One was left untreated and served as a control. Carbon dioxide-baited traps were placed inside each tent 0, 2, 4, 6, and 8 wk after treatment, and a single trap was placed in forest 50 m from the tents. The predominant mosquito species collected was Aedes vigilax (78% of collection). Compared to the untreated control, protection against mosquitoes entering treated tents was initially 78.6% for bifenthrin-treated tents and 84.3% for permethrin. At 4 wk, protection was 68.6% for bifenthrin and 50.7% for permethrin. After 6 wk, less than 34% protection was provided by either insecticide. There was no significant difference between the protection provided by either insecticide treatment. The study showed that barrier tent treatments provide a reasonable increase in preventing the entry of mosquitoes for at least 4 wk.

Protection of Military Personnel Against Vector-Borne Diseases: A Review of Collaborative Work of the Australian and US Military Over the Last 30 Years.

Australian and US military medical services have collaborated since World War II to minimize vector-borne diseases such as malaria, dengue, and scrub typhus. In this review, collaboration over the last 30 years is discussed. The collaborative projects and exchange scientist programs have resulted in mutually beneficial outcomes in the fields of drug development and personal protection measures against vector-borne diseases.

Deployable Molecular Detection of Arboviruses in the Australian Outback.

The most common causes of human infection from the arboviruses that are endemic in Australia are the arthritogenic alphaviruses: Ross River virus (RRV) and Barmah Forest virus (BFV). The most serious infections are caused by the neurotropic flaviviruses, Murray Valley encephalitis virus (MVEV) and the Kunjin subtype of West Nile virus. The greatest individual risk of arbovirus infection occurs in tropical/subtropical northern Australia because of the warm, wet summer conditions from December to June, where conventional arbovirus surveillance is difficult due to a combination of low population density, large distances between population centers, poor roads, and seasonal flooding. Furthermore, virus detection requires samples to be sent to Perth up to 2,000 km away for definitive analysis, causing delays of days to weeks before test results are available and public health interventions can be started. We deployed a portable molecular biology laboratory for remote field detection of endemic arboviruses in northern Queensland, then in tropical Western Australia and detected BFV, MVEV, and RRV RNA by polymerase chain reaction (PCR) assays of extracts from mosquitoes trapped in Queensland. We then used a field-portable compact real-time thermocycler for the samples collected in the Kimberley region of Western Australia. Real-time field PCR assays enabled concurrent endemic arbovirus distribution mapping in outback Queensland and Western Australia. Our deployable laboratory method provides a concept of operations for future remote area arbovirus surveillance.

Laboratory evaluation of methylated coconut oil as a larvicide for Anopheles farauti and Culex annulirostris.

The toxicity of methylated coconut oil (MCO) was compared with a commercially available oil larvicide (Golden Bear Oil GB-1111) in laboratory bioassays of 4th-stage larvae of Anopheles farauti Laveran and Culex annulirostris Skuse. Both larvicides were more toxic to Cx. annulirostris than to An. farauti and the LD50 (dose lethal to 50% of the test organisms) after 24 h exposure indicated that MCO was more toxic than GB-1111 for both An. farauti (LD50 = 8.6 microl versus 13.0 microl/156 cm2) and Cx. annulirostris (LD50 = 1.2 microl versus 3.6 microl/156 cm2). However, for LD95 GB-1111 was more toxic than MCO for both An. farauti (LD95 = 29 microl versus 48.0 microl/156 cm2) and Cx. annulirostris (LD95 = 7.2 microl versus 18.0 microl/156 cm2). Further study of MCO is warranted and may determine whether the addition of surfactant to MCO will lower the LD95. The possibility of community participation in the production and use of coconut products including MCO for malaria vector control is discussed.

Occurrence of Orientia tsutsugamushi in small mammals from Thailand.

Extensive sampling of small mammals was conducted in eight provinces of Thailand between September 9, 1992 and April 29, 2001. A total of 3,498 specimens representing 22 species were collected. Eighty-eight percent (3,089 of 3,498) of the animals were collected from a region in Chiangrai Province, which is commonly recognized as endemic for human scrub typhus. Blood and tissue samples from each animal were tested for the presence of Orientia tsutsugamushi, the etiologic agent of scrub typhus. The predominant species collected were Rattus rattus (53%, n = 1,863), R. losea (18%, n = 638), Bandicota indica (16%, n = 564), and R. exulans (4%, n = 146). Orientia tsutsugamushi was detected in 10 of the 22 species of mammals that included R. bukit (25% infected, 1 of 4), R. rattus (23%, 419 of 1,855), R. argentiventer (22%, 5 of 23), R. berdmorei (22%, 2 of 9), R. losea (13%, 82 of 638), B. indica (9%, 52 of 564), R. koratensis (8%, 1 of 12), B. savilei (3%, 1 of 30), R. exulans (1%, 2 of 146), and Tupaia glis (2%, 1 of 49). Infected animals were found in Chiangrai (18% infected, 563 of 3,084), Bangkok (11%, 1 of 9), Sukothai (3%, 1 of 30), and Nonthaburi (1%, 1 of 69) Provinces. The implications towards scrub typhus maintenance and transmission are discussed.

Efficiency of Leptotrombidium chiggers (Acari: Trombiculidae) at transmitting Orientia tsutsugamushi to laboratory mice.

Thirteen different laboratory colonies of Leptotrombidim chiggers [L. chiangraiensis Tanskul & Linthicum, L. deliense Walch and L. imphalum (Vercammen-Grandjean &Langston)] were evaluated for their ability to transmit Orientia tsutsugamushi (Hyashi) to mice. Of 4,372 transmission attempts using individual chiggers from all 13 colonies, 75% (n = 3,275) successfully infected mice. Transmission rates for the individual chigger colonies ranged from 7 to 80%. Increasing the number of chiggers that fed on a given mouse generally increased transmission rates. Transmission of O. tsutsugamushi to mice by different generations (F1-F11) of certain chigger colonies was stable; however, transmission rates varied greatly in other colonies. Transmission rates (both vertical and horizontal) of several L. changraiensis colonies and the L. deliense colony were the highest, suggesting that these colonies may be useful for the development of a chigger-challenge model that can be used to evaluate the efficacy of candidate scrub typhus vaccines or therapeutic agents in laboratory mice.

Survey of personal protection measures against mosquitoes among Australian defense force personnel deployed to East Timor.

A questionnaire was completed by 955 Australian Defense Force soldiers from two battalion groups to determine their usage of mosquito repellents and bed nets during peacekeeping duties in East Timor. The survey showed that most soldiers (84%) used repellents, but only 19% used them daily. The soldiers used a number of repellent formulations; however, few soldiers used the Australian Defense Force deet (diethyl methylbenzamide) formulation containing 35% deet in a gel. Most soldiers preferred several commercial formulations, which contained 7 to 80% deet. The occurrence of mosquito-borne disease in soldiers was not affected by repellent usage, as the use of repellents was comparable between infected and noninfected individuals. The overall frequency of bed net usage differed in the two battalion groups. The occurrence of malaria in soldiers from one battalion group who did not sleep under a bed net every night of their deployment was significantly (p = 0.007) higher than those who did.

Seroconversion to filarial antigens in Australian defence force personnel in Timor-Leste.

To investigate whether Australian soldiers were exposed to filarial parasites that cause lymphatic filariasis during a 6-month deployment to Timor-Leste, antifilarial antibody levels were measured in 907 soldiers using an enzyme linked immunosorbent assay (ELISA). Initial testing using Dirofilaria immitis antigen demonstrated that 49 of 907 (5.4%) soldiers developed antifilarial antibodies of the IgG1 subclass after deployment, whereas 1 of 944 (0.1%) seroconverted to the IgG4 subclass. When a sub sample of 88 D. immitis-reactive sera was subject to testing with an antifilarial antibody test using Brugia malayi antigen, 46 had elevated IgG antibodies, whereas 5 had elevated antibodies of the IgG4 subclass. A total of 24 soldiers seroconverted to B. malayi, as measured by parasite-specific IgG, whereas 1 seroconverted to IgG4. The relatively low number of seroconversions indicates a low but measurable risk of exposure to human filarial parasites among Australian soldiers deployed to Timor-Leste. However, to reduce the risk of exposure to these parasites, soldiers deploying to endemic areas should practice strict adherence to personal protective measures against mosquito bites.