Stephen L. Doggett

Characterization of Virulent West Nile Virus Kunjin Strain, Australia, 2011

An encephalitis outbreak among horses was caused by a pathogenic variant of Kunjin virus.

Enhanced arbovirus surveillance with deep sequencing: Identification of novel rhabdoviruses and bunyaviruses in Australian mosquitoes

Viral metagenomics characterizes known and identifies unknown viruses based on sequence similarities to any previously sequenced viral genomes. A metagenomics approach was used to identify virus sequences in Australian mosquitoes causing cytopathic effects in inoculated mammalian cell cultures. Sequence comparisons revealed strains of Liao Ning virus (Reovirus, Seadornavirus), previously detected only in China, livestock-infecting Stretch Lagoon virus (Reovirus, Orbivirus), two novel dimarhabdoviruses, named Beaumont and North Creek viruses, and two novel orthobunyaviruses, named Murrumbidgee and Salt Ash viruses. The novel virus proteomes diverged by ≥ 50% relative to their closest previously genetically characterized viral relatives. Deep sequencing also generated genomes of Warrego and Wallal viruses, orbiviruses linked to kangaroo blindness, whose genomes had not been fully characterized. This study highlights viral metagenomics in concert with traditional arbovirus surveillance to characterize known and new arboviruses in field-collected mosquitoes. Follow-up epidemiological studies are required to determine whether the novel viruses infect humans.

Laboratory experiments on infection rates of Amblyospora dyxenoides (Microsporida: Amblyosporidae) in the mosquito Culex annulirostris.

Laboratory observations were made of the microsporidian parasite Amblyospora dyxenoides in its natural mosquito host, Culex annulirostris. There were no differences in the numbers of eggs laid and in the proportions which hatched between infected and uninfected females, indicating that the parasite did not affect fecundity. Unlike other species of Amblyospora which have been studied the development of binucleate spores in adult mosquitoes increase with age of the host in both sexes and in females it proceeds independently of egg development and blood feeding. The same trend is apparent for adult mosquitoes which acquired the infection in the larval stage by horizontal transmission from the intermediate copepod host as well as for mosquitoes which acquired oenocytic infections by transovarial transmission. There was considerable variation in the proportion of mosquitoes which became infected after exposure to A. dyxenoides infected copepods. Infections in larval progeny of female mosquitoes infected via spores produced in copepods ranged from 0 to 100% in individual batches and averaged 45.6% with meiospore infections, 19.3% with oenocytic infections, with the remaining 35.7% being uninfected. Similar variability was observed in the progeny of infected female mosquitoes in the second generation after exposure to infected copepods. During experiments in which the microsporidium was maintained in C. annulirostris through 9 successive transovarially transmitted cycles (by selectively rearing the progeny of females infected with binucleate spores after an initial exposure to infected copepods) the proportion of infected progeny with oenocytic infections increased from 25 to around 50% whereas the incidence of meiospore infections declined from 50 to 10%.(ABSTRACT TRUNCATED AT 250 WORDS)

Lyme disease: a search for a causative agent in ticks in south-eastern Australia

Attempts were made to identify the causative organism of Lyme disease in Australia from possible tick vectors. Ticks were collected in coastal areas of New South Wales, Australia, from localities associated with putative human infections. The ticks were dissected; a portion of the gut contents was examined for spirochaetes by microscopy, the remaining portion inoculated into culture media. The detection of spirochaetes in culture was performed using microscopy, and immunochemical and molecular (PCR) techniques. Additionally, whole ticks were tested with PCR for spirochaetes. From 1990 to 1992, approximately 12,000 ticks were processed for spirochaetes. No evidence of Borrelia burgdorferi or any other spirochaete was recovered from or detected in likely tick vectors. Some spirochaete-like objects detected in the cultures were shown to be artifacts, probably aggregates of bacterial flagellae. There is no definitive evidence for the existence in Australia of B. burgdorferi the causative agent of true Lyme disease, or for any other tick-borne spirochaete that may be responsible for a local syndrome being reported as Lyme disease.

Life cycle of Amblyospora indicola (Microspora: Amblyosporidae), a parasite of the mosquito Culex sitiens and of Apocyclops sp. copepods

The life cycle of Amblyospora indicola, a parasite of the mosquito Culex sitiens, was revealed by field observations and laboratory infection experiments conducted in Australia. In northern Queensland, infected C. sitiens larvae were often found breeding in association with two cyclopoid copepods: Apocyclops dengizicus and an undescribed species of the same genus. The latter species was found to be an intermediate copepod host of this microsporidium whereas A. dengizicus was not. One complete cycle of the parasite extends over two mosquito generations (by transovarial transmission from females with binucleate spores to their eggs) and by horizontal transmission between mosquitoes and copepods. The latter involves horizontal transmission from mosquitoes to copepods via meiospores produced in larval fat body infections and horizontal transmission from copepods to mosquitoes via uninucleate spores produced within infected copepods. Uninucleate clavate spores were formed in Apocyclops sp. nov. copepods 7-10 days after exposure to larval meiospores and were infectious to larvae of a microsporidian-free colony of C. sitiens. The development of A. indicola within mosquito larvae exposed to infected copepods is similar to that of A. dyxenoides infecting C. annulirostris. It proceeds from stages with a single nucleus to diplokaryotic binucleate cells in oenocytes. These stages persist through pupation to adult emergence after which time a proportion of male mosquitoes and female mosquitoes may develop binucleate spores without the need for a blood meal. A proportion of both male and female larval progeny of infected females with binucleate spores develop patent fat body infections via transovarial transmission and die in the fourth larval instar.(ABSTRACT TRUNCATED AT 250 WORDS)

Cuticle Thickening in a Pyrethroid-Resistant Strain of the Common Bed Bug, Cimex lectularius L. (Hemiptera: Cimicidae).

Thickening of the integument as a mechanism of resistance to insecticides is a well recognised phenomenon in the insect world and, in recent times, has been found in insects exhibiting pyrethroid-resistance. Resistance to pyrethroid insecticides in the common bed bug, Cimex lectularius L., is widespread and has been frequently inferred as a reason for the pest’s resurgence. Overexpression of cuticle depositing proteins has been demonstrated in pyrethroid-resistant bed bugs although, to date, no morphological analysis of the cuticle has been undertaken in order to confirm a phenotypic link. This paper describes examination of the cuticle thickness of a highly pyrethroid-resistant field strain collected in Sydney, Australia, in response to time-to-knockdown upon forced exposure to a pyrethroid insecticide. Mean cuticle thickness was positively correlated to time-to-knockdown, with significant differences observed between bugs knocked-down at 2 hours, 4 hours, and those still unaffected at 24 hours. Further analysis also demonstrated that the 24 hours survivors possessed a statistically significantly thicker cuticle when compared to a pyrethroid-susceptible strain of C. lectularius. This study demonstrates that cuticle thickening is present within a pyrethroid-resistant strain of C. lectularius and that, even within a stable resistant strain, cuticle thickness will vary according to time-to-knockdown upon exposure to an insecticide. This response should thus be considered in future studies on the cuticle of insecticide-resistant bed bugs and, potentially, other insects.

Converting Mosquito Surveillance to Arbovirus Surveillance with Honey-Baited Nucleic Acid Preservation Cards.

Spatially and temporally accurate information about infectious mosquito distribution allows for pre-emptive public health interventions that can reduce the burden of mosquito-borne infections on human populations. However, the labile nature of arboviruses, the low prevalence of infection in mosquitoes, the expensive labor costs for mosquito identification and sorting, and the specialized equipment required for arbovirus testing can obstruct arbovirus surveillance efforts. The recently developed techniques of testing mosquito expectorate using honey-baited nucleic acid preservation cards or sugar bait stations allows a sensitive method of testing for infectious, rather than infected, mosquito vectors. Here we report the results from the first large-scale incorporation of honey-baited cards into an existing mosquito surveillance program. During 4 months of the peak virus season (January-April, 2014) for a total of 577 trap nights, we set CO2-baited encephalitis vector survey (EVS) light traps at 88 locations in South Australia. The collection container for the EVS trap was modified to allow for the placement of a honey-baited nucleic acid preservation card (FTA™ card) inside. After collection, mosquitoes were maintained in a humid environment and allowed access to the cards for 1 week. Cards were then analyzed for common endemic Australian arboviruses using a nested RT-PCR. Eighteen virus detections, including 11 Ross River virus, four Barmah Forest virus, and three Stratford virus (not previously reported from South Australia) were obtained. Our findings suggest that adding FTA cards to an existing mosquito surveillance program is a rapid and efficient way of detecting infectious mosquitoes with high spatial resolution.

Encasing Mattresses in Black Plastic Will Not Provide Thermal Control of Bed Bugs, Cimex spp. (Hemiptera: Cimicidae)

The suggestion that bed bug (Cimex spp.; Hemiptera: Cimicidae)-infested mattresses wrapped in black plastic and exposed to sunlight will be heated sufficiently to kill the bed bugs was tested. Two types of mattresses were tested: a thin mattress of solid foam rubber and a thick multilayered inner spring mattress. Temperature probes were placed on both upper and lower sides of the mattresses, which were wrapped in black plastic and placed outside on a summer day for >9 h wherein the ambient temperature peaked at 36.5 degrees C. The maximum recorded temperature on the upper (sun-exposed) sides was 85 degrees C for both mattresses, whereas lower side temperatures for the thick mattress never exceeded 35 degreesC, and some areas of the thin mattress failed to exceed 36.50C. Therefore, with published thermal death points of 40-45 degrees C depending on exposure time, and opportunities for bed bugs to avoid lethal temperatures by retreating from hot zones, this technique seems to be not suitable for bed bug management.

Bacterial Profiling Reveals Novel “Ca. Neoehrlichia”, Ehrlichia, and Anaplasma Species in Australian Human-Biting Ticks

In Australia, a conclusive aetiology of Lyme disease-like illness in human patients remains elusive, despite growing numbers of people presenting with symptoms attributed to tick bites. In the present study, we surveyed the microbial communities harboured by human-biting ticks from across Australia to identify bacteria that may contribute to this syndrome. Universal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes in DNA samples from individual Ixodes holocyclus (n = 279), Amblyomma triguttatum (n = 167), Haemaphysalis bancrofti (n = 7), and H. longicornis (n = 7) ticks. The 16S amplicons were sequenced on the Illumina MiSeq platform and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomies. Nested PCR and Sanger sequencing were used to confirm the NGS data and further analyse novel findings. All 460 ticks were negative for Borrelia spp. by both NGS and nested PCR analysis. Two novel “Candidatus Neoehrlichia” spp. were identified in 12.9% of I. holocyclus ticks. A novel Anaplasma sp. was identified in 1.8% of A. triguttatum ticks, and a novel Ehrlichia sp. was identified in both A. triguttatum (1.2%) ticks and a single I. holocyclus (0.6%) tick. Further phylogenetic analysis of novel “Ca. Neoehrlichia”, Anaplasma and Ehrlichia based on 1,265 bp 16S rRNA gene sequences suggests that these are new species. Determining whether these newly discovered organisms cause disease in humans and animals, like closely related bacteria do abroad, is of public health importance and requires further investigation.

Confirmation of insecticide resistance in Cimex lectularius Linnaeus (Hemiptera: Cimicidae) in Australia

Insecticide resistance in the common bed bug, Cimex lectularius Linnaeus has been suspected in Australia with anecdotal reports of poor product performance. To investigate this possibility, LD50 values were determined via topical application of technical grade permethrin, deltamethrin, bendiocarb, pirimiphos‐methyl and imidacloprid serially diluted in acetone to a suspected resistant field‐collected strain and a susceptible laboratory strain. All compounds tested against the susceptible strain were efficacious. However, for the field strain, only pirimiphos‐methyl and imidacloprid showed high levels of activity. Resistance was confirmed in the field‐collected strain to the pyrethroids and bendiocarb, but not to pirimiphos‐methyl or imidacloprid. Resistance factors (‘resistant’ LD50/susceptible LD50) for each compound were: permethrin ≈ 1.235 million, deltamethrin ≈ 370 000, bendiocarb ≈ 250, pirimiphos‐methyl = 2.6, imidacloprid = 2.6. Bendiocarb, permethrin and deltamethrin all failed to return greater than 60% mortality at the maximum dose of 100 μg/μL. This research has significant operational implications for bed bug control and the registration process of new products in Australia.