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.

The occurrence of alpha and flaviviruses from mosquitoes in the Northern Territory (NT), Australia, was first reported by Standfast et al. (1984), who collected mosquitoes between 1974 and 1976 at Beatrice Hill, 80 km from Darwin, NT. Later, Whelan and Weir (1993) collected over 340,000 mosquitoes between 1982 and 1992, and 117 alpha and flaviviruses were identified, which included 42 Ross River virus (RRV) and 17 Barmah Forest virus (BFV). The BFV was isolated from Aedes normanensis (Taylor) (6 occasions), Ae. vigilax Skuse (6), and Culex annulirostris (Skuse) (5). These collections were predominantly in the northern part of the territory (Whelan and Weir 1994). Relatively little mosquito surveillance has been conducted in the Southwestern area of the Top End of the Northern Territory.

Bradshaw Field Training Area (BFTA), is a large-scale military training facility for the Australian Defence Force (ADF) and coalition partners, located 600 km southwest of Darwin, NT. A desktop overview of potential mosquito-borne disease assessment was prepared by Whelan (1997). This assessment outlined the potential biting pest mosquitoes and biting midges (Family Ceratapogonidae) that may be found in BFTA. Whelan (1997) stated that field surveys in the training area, however, were needed for verification. Mosquito surveillance and arbovirus detections in the BFTA have not been previously undertaken; a survey was conducted in April 2013 and is reported here.

Study site

The BFTA is located in the midwest region of the Northern Territory, Australia (15°58′S, 130°47′E; Fig. 1), 600 km southwest of Darwin. It is bordered by the Victoria River in the south, the Fitzmaurice River in the north, to the west is the Joseph Bonaparte Gulf, and is bordered in the east by Coolibah and Innesvale Stations. The BFTA is over 8,700 km2 in area and is mainly composed of a coastal region, tidal and freshwater reaches of the Victoria and Fitzmaurice rivers, western dissected hills, internal draining valleys, a central sandstone plateau, and extensive braided floodplains (Whelan 1997). The vegetation at BFTA includes monsoon forest, riparian forest, eucalypt open forest woodland, low open forest woodland, shrubland, and grassland. The weather is tropical, with a wet monsoon season from October to April when most of the mean average of 940 ml of rain falls, and dry season from May to September each year. The temperature throughout the year is warm, with a mean monthly average of 28.7°C.

Fig. 1.

Map showing the traplines at Bradshaw Field Training Area, Northern Territory, Australia. Dark lines show navigable roads (Main Supply Routes). TFMA, Training Force Maintenance Area.

Fig. 1.

Map showing the traplines at Bradshaw Field Training Area, Northern Territory, Australia. Dark lines show navigable roads (Main Supply Routes). TFMA, Training Force Maintenance Area.

Close modal

Trap locations

Mosquito traps were set between 2 to 10 km apart in 3 main traplines within the BFTA. Trapline 1 (east line) was located on the Main Supply Route (MSR) in a northeasterly direction from the Training Force Maintenance Area (TFMA) to the road junction with the Ikymbon River. Trapline 2 (central line) was located along the MSR in a northwesterly direction from the TFMA to the Angalarri River crossing (Fig. 1). This also included Range Control, TFMA, and helicopter landing areas closer to the Victoria River entry gate area. Trapline 3 (west line) was located on the MSR west of the Angalarri River crossing to the Bradshaw Homestead area. Traps were placed along each trapline on each of 3 nights. These areas were selected as they were close to the only roads that could be navigated by vehicles.

Mosquito collections

Mosquitoes were collected using carbon dioxide (dry ice)–baited encephalitis virus surveillance traps. The traps were placed into locations within traplines between 1700 and 1900 h each night and retrieved before 0800 h the following morning. On return to the field laboratory, mosquito collection containers were placed into a −20°C freezer until the mosquitoes were immobilized. Mosquitoes were then identified to species using the keys of Lee et al. (1980–89), placed into species pools, and then stored in liquid nitrogen for return to our laboratory in Brisbane, Queensland, Australia.

Virus identification

In the laboratory pools of up to 25 mosquitoes each were placed into vials containing 5 glass beads. To each vial 2 ml of Roswell Park Memorial Institute medium was added, and the contents homogenized in a mechanical mixer (SPEX 8000; Spex Certiprep, Metuchen, NJ) for 2 min, then centrifuged at 3,000 rpm for 15 min at 4°C (Frances et al. 2004). The supernatant was then placed into vials and stored at −20°C until RNA extraction and virus identification could be conducted.

Viral RNA detection by reverse transcriptase–polymerase chain reaction

Alpha and flaviviruses were identified using a method described in detail elsewhere (Kisu et al. 2019; Liu et al. 2019a, 2019b). Mosquito homogenates were extracted for viral RNA by using the QIAamp Viral RNA Mini kit (Qiagen, Hiden, Germany) according to manufacturer instructions. The extracted mosquito RNA was reverse transcribed into cDNA by using random hexanucleotide primers (Promega, Madison, WI) and Superscript III Reverse Transcriptase (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Two pairs of nested polymerase chain reaction (PCR) pan-flavivirus primers binding to the envelope gene and 1 pan-flavivirus primer targeting nonstructural gene 5 were used to identify the presence of flaviviruses (Gaunt and Gould 2005, Maher-Sturgess et al. 2008), and 1 pair pan-alphavirus primers were used to identify the presence of alphavirus (Table 1; Pfeffer et al. 1997). The PCR was performed with 5 μl 10× Taq DNA polymerase buffer (Roche, Basel, Switzerland), 250 ng of both forward and reverse primers, 1 μl 10 mM deoxynucleoside triphosphates (Promega), 2.5 U Taq DNA polymerase (Roche), 5 μl of cDNA and RNase-free water to a final volume of 50 μl. The PCR thermocycler conditions were 94°C for 1 min, followed by 40 cycles of 94°C for 30 sec, 45°C for 30 sec, and 70°C for 1.5 min, and 70°C for 10 min. For nest PCR, 5 μl of 1st PCR product was diluted into 100 μl H2O, and 1 μl of diluted PCR product was used for nest PCR template. The PCR products were visualized on a 1% (w/v) agarose gel using ethidium 148 bromide and ultraviolet light.

Table 1.

Oligonucleotide primers used by reverse transcriptase–polymerase chain reaction to identify flaviviruses and alpha virus from mosquitoes.

Oligonucleotide primers used by reverse transcriptase–polymerase chain reaction to identify flaviviruses and alpha virus from mosquitoes.
Oligonucleotide primers used by reverse transcriptase–polymerase chain reaction to identify flaviviruses and alpha virus from mosquitoes.

Isolation of virus from mosquito homogenate

Fifty microliters of mosquito homogenate was inoculated onto monolayers of Aedes albopictus (Skuse) (C6/36) cells in 25 flasks and incubated at 30°C for 7 days. For reverse transcriptase–polymerase chain reaction (RT-PCR) assay, 140 μl of culture supernatant was collected from the inoculated cultures and then total RNA was extracted and tested by RT-PCR as described above. For immunofluorescence, mosquito supernatant infected cells or uninfected C6/36 cells were suspended, adhered to black immunofluorescence slide (Cel-Line/Thermo Fischer, Brisbane, Australia), fixed in acetone, and then blocked with 0.2% bovine serum albumin in phosphate-buffered saline (PBS) for 1 h. Following incubation with the anti-flavivirus or anti–alpha virus in monoclonal antibodies, the slides were washed with PBS and then stained with fluorescein isocyanate-conjugated goat anti–mouse IgG (H + L) (Dako, Giostrup, Denmark) for 1 h. Following another 3 washes in PBS, the slides were mounted onto glass microscope slides using ProLong Gold Anti-fade (Life Technology, Therma Fischer, Brisbane, Australia) and viewed under the Olympus fluorescence microscope. The genome sequences of the BFV strain isolated in this study were determined using the same method as that of Kizu et al. (2019) and Liu et al. (2019a, 2019b). The complete nucleotide sequence of glycoprotein E2 were aligned with available GenBank BFV sequences using ClustalW method, and a maximum clade credibility tree was generated using the Geneious (version 11.1.2; Lawrence, KS) Bayesian phylogenetic analysis method with an HKY85 substitution model, plus a gamma rate variation, with a Gamma molecular clock model of uniform Branch lengths, a chain length of 1 million, and a 10% burn-in length.

Mosquito species and numbers

The total number of mosquitoes and species collected during the survey is shown in Table 2. A total of 8,530 mosquitoes were collected, and the predominant species was Ae. normanensis, which was 74.2% of the mosquitoes collected, and Anopheles annulipes Walker (11.1%), An. amictus Edwards (2.9%), and An. meraukensis Venhuis (2.4%). There were 6,004 mosquitoes in 250 pools of 37 or fewer (usually 25 mosquitoes/pool) processed to determine the presence of alpha or flavivirus. The mean number of mosquitoes collected in each trapline is shown in Table 3. The results showed a mean of 120.1 ± 13.7 mosquitoes per trap in the 71 traps placed. The mean numbers in the east and west traplines were uniform and were not different. In the central line, significantly more mosquitoes were collected in traps 1 and 2, which were placed within 2 km of the Angalarri River. In the west line, the mean numbers in traps 5 and 6 were higher than the mean numbers from traps in the western trapline, but they were not statistically higher than other traps placed in this line. The mean trap numbers were of relatively high density and show that personal protection measures would have been needed by soldiers operating and living in this area of BFTA during the time of the surveys. The number of mosquitoes collected in traps placed at the TFMA, Airfield, and Range Control area was lower than the mean trap collection. Trap 1 placed at the Bradshaw Homestead (occupied by a family) had a mean of 133. 3 ± 13.7 mosquitoes per trap, and personal protection would be needed to be adhered to in this area.

Table 2.

Mosquito species collected and processed for detection of arbovirus from Bradshaw Field Training Area, Northern Territory, Australia, April 7–18, 2013.

Mosquito species collected and processed for detection of arbovirus from Bradshaw Field Training Area, Northern Territory, Australia, April 7–18, 2013.
Mosquito species collected and processed for detection of arbovirus from Bradshaw Field Training Area, Northern Territory, Australia, April 7–18, 2013.
Table 3.

Trap locations and mean number of mosquitoes collected.1

Trap locations and mean number of mosquitoes collected.1
Trap locations and mean number of mosquitoes collected.1

Identification of BFV

One strain of BFV was isolated from 250 mosquito pools (Table 2). The BFV strain was sequenced for the glycoprotein E2 gene and phylogenetic tree analysis classified the BFTA Strain (M/MIDI BFTA/2013) in Lineage III, which is currently circulating in eastern Australia. The phylogenetic analysis also showed that the BFTA Strain (M/MIDI BFTA/2013) was closely related to a strain isolated in Brisbane from a polyarthritis patient (H/MIDI4/2015) and other BFV strains isolated from mosquitoes collected in ADF training areas (Fig. 2).

Fig. 2.

Phylogenetic tree analysis of the single strain of Barmah Forest virus (BFV) (MIDI 409 BFTA 2013) isolated at Bradshaw Field Training Area (BFTA), and other strains of BFV, including a strain from a Brisbane Hospital patient (H/MIDI4/2015/MH618666 2). The naming convention of the strains is: name of host/strain/year of isolation/GenBank accession number. Hosts are H, humans; M, mosquito; BM, biting midge.

Fig. 2.

Phylogenetic tree analysis of the single strain of Barmah Forest virus (BFV) (MIDI 409 BFTA 2013) isolated at Bradshaw Field Training Area (BFTA), and other strains of BFV, including a strain from a Brisbane Hospital patient (H/MIDI4/2015/MH618666 2). The naming convention of the strains is: name of host/strain/year of isolation/GenBank accession number. Hosts are H, humans; M, mosquito; BM, biting midge.

Close modal

This study has shown mosquito abundance in the eastern area of BFTA during 2 wk of April 2013 was of moderately high density. The main mosquito species collected was Ae. normanensis, which was collected in relatively high numbers during this survey. This species has been associated with a number of arboviruses including RRV, BFV, Murray Valley encephalitis virus, Sindbis, Gan, Edge Hill, and Kokabera viruses (Russell 1998). In the arid zones of Western Australia and Northern Territory, virus activity often follows heavy seasonal rainfall sufficient to generate large populations of temporary ground-pool species such as Ae. normanensis (Russell 1998). Seventy percent of the mosquitoes collected in the current study were tested for the presence of arbovirus (Table 2). The BFV was identified in a single pool of Ae. normanensis, a positive rate of 1/250 (0.4%). Barmah Forest virus was first isolated in the Northern Territory from Beatrice Hill between 1974–76 (Standfast et al. 1984) and has been isolated occasionally since then (Whelan and Weir 1993, Jacups et al. 2008). The current collections were also made during a period when 229 human cases of BFV coincidentally increased in the Northern Territory (Kurucz et al. 2016). An investigation using the collection of mosquitoes in the Darwin area, BFV antibody testing of small animals, and review of the human cases was conducted. They collected and tested 4,641 mosquitoes, but none of these were infected, while none of the small animals trapped tested positive for BFV antibodies. Kurucz et al. (2016) concluded that the high number of human cases identified in 2012–13 were likely to be false positives, suggesting that the testing available during that time needed revision.

Detection of arboviruses in mosquitoes collected in remote areas in northern Australia requires samples to be sent to laboratories in capital cities (often several thousand kilometers) for definitive analysis (Inglis et al. 2016). The current study has shown that mosquitoes collected at BFTA, frozen and stored in liquid nitrogen, could be returned to Brisbane (ADFMIDI, approximate distance 3,500 km) and virus successfully identified. Also, samples of these collections were used in other laboratories to identify viruses. An insect-specific virus, Karumba virus, was identified from 8/15 pools of An. meraukensis (Colmant et al. 2017). Subsequently, a novel nodovirus, Culannivirus, was isolated from Cx. annulirostris (Warrilow et al. 2018) and Liao ning virus isolated from Ae. normanensis and Ae. vigilax (Prow et al. 2018).

A survey by Cooper et al. (1996) showed that the main vector of malaria in the Southwest Pacific region, Anopheles farauti Laveran s.s. was present on the coast just to the north (approximate distance 140 km) of BFTA. This species was not collected in the eastern area of BFTA during the current survey, and more collections closer to the western areas of BFTA are warranted in the future. However, there are no formed roads west of Bradshaw Homestead, so other transport methods are needed, such as the helicopters used by Cooper et al. (1996). A total of 16.8% of the overall collection of mosquitoes collected in this study were Anopheles spp., with the predominant species An. annulipes and An. amictus, both of which are common throughout northern Australia (Cooper et al. 1996). Cooper et al. (1995) did not find An. farauti south of 14°30′S latitude during extensive surveys in 1988–90 in the northwestern Northern Territory, and 15°30′S in the eastern part of the Northern Territory. Future surveys in BFTA will need to determine if An. farauti is present in the western area near to the coast, particularly if amphibious operations with coalition partners are conducted in this area.

This study has provided the 1st records of mosquitoes in the BFTA. The study has shown a high density of mosquitoes, and the main species collected were Ae. normanensis and Anopheles spp. More surveillance, particularly during the wetter months of the year, is warranted to determine risks of vector-borne disease in this training area. The study also found a single pool of Ae. normanensis mosquitoes positive for BFV. Phylogenetic tree analysis showed the isolated BFV strain is closely related to a virus strain isolated from an infected patient in Brisbane (Liu et al. 2019a). The information provided in this study will be essential for the conduct of risk assessments for military operations at BFTA and the development of effective vector-borne disease control measures in the area. The use of personal protection measures against mosquitoes (Frances and Debboun 2020) when military personnel are operating in BFTA is reinforced by this study.

We thank N. Lehmann and K. Zhang for assistance in the identification of arboviruses in mosquito homogenates, members of the ADF 1st Preventative Medicine Unit, especially N. Flindt, A. O'Shea, and I. Myles for field assistance, and R.D. Cooper for comments on an earlier draft of the manuscript. The opinions expressed herein are those of the authors and do not necessarily reflect those of the ADF or Defence Policy.

Colmant
AMG,
Hobson-Peters
J,
Bielefeldt-Ohmann
H,
van den Hurk
AF,
Hall-Mendelin
S,
Chow
WK,
Johansen
CA,
Fros
J,
Simmonds
P,
Watterson
D,
Cazier
C,
Kayvan Etebari
KK,
Asgari
S,
Schulz
BL,
Beebe
N,
Vet
LJ,
Piyasena
TBH,
Nguyen
H-D,
Barnard
RT,
Hall
RA.
2017
.
A new clade of insect-specific Flaviviruses from Australian Anopheles mosquitoes displays species-specific host restriction
.
mSphere
2
:
1
19
.
Cooper
RD,
Frances
SP,
Sweeney
AW.
1995
.
Distribution of members of the Anopheles farauti complex in the Northern Territory of Australia
.
J Am Mosq Control Assoc
11
:
66
71
.
Cooper
RD,
Frances
SP,
Waterson
DGE,
Piper
RG,
Sweeney
AW.
1996
.
Distribution of the anopheline mosquitoes in northern Australia
.
J Am Mosq Control Assoc
12
:
656
663
.
Frances
SP,
Cooper
RD,
Rowcliffe
KL,
Chen
N,
Cheng
Q.
2004
.
Occurrence of Ross River virus and Barmah Forest virus in mosquitoes at Shoalwater Bay Military Training Area, Queensland, Australia
.
J Med Entomol
41
:
115
120
.
Frances
SP,
Debboun
M.
2020
.
Personal protective measures against mosquitoes
.
In:
Debboun
M,
Nava
MR,
Rueda
LM,
eds.
Mosquitoes, communities, and public health in Texas
.
San Diego, CA
:
Academic Press
.
p
387
401
.
Gaunt
MW,
Gould
EA.
2005
.
Rapid subgroup identification of the flaviviruses using degenerate primer E-gene RT-PCR and site-specific restriction enzyme analysis
.
J Virol Methods
128
:
113
127
.
Inglis
TJJ,
Bradbury
RS,
McInnes
RL,
Frances
SP,
Merrit
AJ,
Levy
A,
Nicholsen
J,
Neville
PJ,
Lindsay
M,
Smith
DW.
2016
.
Deployable molecular detection of arboviruses in the Australian outback
.
Am J Trop Med Hyg
95
:
633
638
.
Jacups
SP,
Whelan
PI,
Currie
BJ.
2008
.
Ross River virus and Barmah Forest virus infections: a review of history, ecology, and predictive models, with implications for tropical northern Australia
.
Vector Borne Zoonotic Dis
8
:
283
297
.
Kizu
J,
Neuman
C,
Le Grand
L,
Liu
W.
2019
.
Discovery of cocirculating Ross River virus and Barmah Forest virus at Wide Bay Military Training Area, northeastern Queensland
.
J Am Mosq Control Assoc
35
:
220
223
.
Kurucz
N,
Markey
P,
Draper
A,
Melville
AL,
Weir
R,
Davis
S,
Warchot
A,
Boyd
AR,
Stokeld
D.
2016
.
Investigation into high Barmah Forest virus disease case numbers reported in the Northern Territory, Australia in 2012–2013
.
Vector Borne Zoonotic Dis
16
:
110
116
.
Lee
DJ,
Hicks
MM,
Griffiths
M,
Debenham
M,
Bryan
JH,
Russell
RC,
Marks
EN.
1980
–89. The Culicidae of the Australasian region. Entomology Monograph No. 2, Volumes 1–12
.
Canberra, Australia
:
Australian Government Publishing Service
.
Liu
W,
Kizu
J,
Hoare
C,
Mitchell
IR,
Gauci
PJ,
Gubala
AJ.
2019
a.
Genome sequences of Barmah Forest virus strains isolated from mosquitoes trapped in Australian Defence Force Training areas reveal multiple nucleotide insertions in the 3′ untranslated region
.
Microbiol Resour Announc
8
:
e00969
19
.
Liu
W,
Kizu
J,
Le Grand
L,
Mitchell
I,
Gauci
PJ,
Gubala
AJ.
2019
b.
Genome sequences of three Ross River virus isolates obtained from the Australian Defence Force
.
Microbiol Resour Announc
8
:
e00064
19
.
Maher-Sturgess
SL,
Forrester
NL,
Wayper
PJ,
Gould
EA,
Hall
RA,
Barnard
RT,
Gibbs
MJ.
2008
.
Universal primers that amplify RNA from all three flavivirus subgroups
.
Virol J
000
:
5
16
.
Pfeffer
M,
Proebster
B,
Kinney
RM,
Kaaden
OR.
1997
.
Genus-specific detection of alphaviruses by a semi-nested reverse transcription-polymerase chain reaction
.
Am J Trop Med Hyg
6
:
709
718
.
Prow
NA,
Mah
MG,
Deercain
JM,
Warrilow
D,
Colmant
AMG,
O'Brien
CA,
Harrison
JJ,
McLean
BJ,
Hewlett
EK,
Piyasena
TBH,
Hall-Mendelin
S,
Van Den Hurk
AF,
Watterson
D,
Huang
B,
Schulz
BL,
Webb
CE,
Johansen
CA,
Chow
WK,
Hobson-Peters
J,
Cazier
C.
2018
.
New genotypes of Liao ning virus (LNV) in Australia exhibit an insect-specific phenotype
.
J Gen Virol
99
:
596
609
.
Russell
RC.
1998
.
Vectors vs. humans in Australia: who is on top down under? An update on vector-borne disease and research on vectors in Australia
.
J Vector Ecol
23
:
1
46
.
Standfast
HA,
Dyce
AL,
St George
TD,
Muller
MJ,
Doherty
RL,
Carley
JG,
Fillipich
C.
1984
.
Isolation of arboviruses from insects collected at Beatrice Hill, Northern Territory of Australia, 1974–1976
.
Aust J Biol Sci
37
:
351
366
.
Warrilow
D,
Huang
B,
Newton
ND,
Harrison
JJ,
Johnson
KN,
Chow
WK,
Hall
RA,
Hobson-Peters
J.
2018
.
The taxonomy of an Australian nodovirus isolated from mosquitoes
.
PLoS ONE
13
:
e0210029
.
Whelan
PI.
1997
.
Potential biting insect pest and mosquito borne disease assessment: Bradshaw Field Training Area September 1997. Unpublished report for Connell Wagner for the proposed Bradshaw Field Training Area, environmental impact statement and environmental management plan
.
Darwin, Australia
:
NT Health
.
Available from NT Health, Darwin, Australia.
Whelan
PI,
Weir
R.
1993
.
The isolation of alpha and flaviviruses from mosquitoes in the Northern Territory 1982-1992
.
In:
Uren
MF,
Kay
BH,
eds.
Arbovirus research Australia. Volume 6
.
Brisbane, Australia
:
Commonwealth Scientific and Industrial Research Organisation and Queensland Institute of Medical Research
.
p
270
277
.