Bacterial association observations in Lucilia sericata and Lucilia cuprina organs through 16S rRNA gene sequencing.

Blowfly (Diptera: Calliphoridae) species Lucilia sericata (Meigen) and related species Lucilia cuprina (Wiedmann) are important agricultural pests, assist in forensic fields and also have a therapeutic role in medicine. Both species (though predominantly L. sericata) are utilised in a clinical setting for maggot debridement therapy (MDT) where the larvae ingest necrotic tissue and bacteria from non-healing wounds. Conversely, larvae of L. cuprina feed invasively, as major initiators of sheep myiasis in Australia, New Zealand, and the UK, among other regions. Both species exhibit larval and adult interactions with bacterially rich environments, but the significance of this in the composition of their microbiome has yet to be considered. This study utilised dissected samples of digestive and reproductive organs from both disinfected and non-disinfected adults and larvae of both species for bacterial DNA extraction, followed by 16S rRNA gene sequencing. Sequencing data indicated unsurprisingly that digestive tracts of both genders and female salivary glands from all non-disinfected samples carry the most concentrated amounts of bacteria. Genera Pseudomonas and Corynebacterium were also highly represented within all organs and species analysed. Comparison of bait lures to sample sequence read output of insect specimens showed no correlation with genera such as Pseudomonas present in insects, while absent from wild bait, and in reduced amounts from fleece bait profiles. With this information, future work can focus on key organs such as the spermathecae and salivary glands, while also providing the potential to identify the role these bacteria may play in the blowfly life cycle. KEY POINTS: Genera Pseudomonas appears consistently in the microbiome of Lucilia species. Female spermathecae and salivary glands show the highest microbial diversity. Bacterial profiles of L. sericata and L. cuprina have similar composition.

Murray Valley encephalitis virus surveillance and control initiatives in Australia. National Arbovirus Advisory Committee of the Communicable Diseases Network Australia.

Mechanisms for monitoring Murray Valley encephalitis (MVE) virus activity include surveillance of human cases, surveillance for activity in sentinel animals, monitoring of mosquito vectors and monitoring of weather conditions. The monitoring of human cases is only one possible trigger for public health action and the additional surveillance systems are used in concert to signal the risk of human disease, often before the appearance of human cases. Mosquito vector surveillance includes mosquito trapping for speciation and enumeration of mosquitoes to monitor population sizes and relative composition. Virus isolation from mosquitoes can also be undertaken. Monitoring of weather conditions and vector surveillance determines whether there is a potential for MVE activity to occur. Virus isolation from trapped mosquitoes is necessary to define whether MVE is actually present, but is difficult to deliver in a timely fashion in some jurisdictions. Monitoring of sentinel animals indicates whether MVE transmission to vertebrates is actually occurring. Meteorological surveillance can assist in the prediction of potential MVE virus activity by signalling conditions that have been associated with outbreaks of Murray Valley encephalitis in humans in the past. Predictive models of MVE virus activity for south-eastern Australia have been developed, but due to the infrequency of outbreaks, are yet to be demonstrated as useful for the forecasting of major outbreaks. Surveillance mechanisms vary across the jurisdictions. Surveillance of human disease occurs in all States and Territories by reporting of cases to health authorities. Sentinel flocks of chickens are maintained in 4 jurisdictions (Western Australia, the Northern Territory, Victoria and New South Wales) with collaborations between Western Australia and the Northern Territory. Mosquito monitoring complements the surveillance of sentinel animals in these jurisdictions. In addition, other mosquito monitoring programs exist in other States (including South Australia and Queensland). Public health control measures may include advice to the general public and mosquito management programs to reduce the numbers of both mosquito larvae and adult vectors. Strategic plans for public health action in the event of MVE virus activity are currently developed or being developed in New South Wales, the Northern Territory, South Australia, Western Australia and Victoria. A southern tri-State agreement exists between health departments of New South Wales, Victoria and South Australia and the Commonwealth Department of Health and Aged Care. All partners have agreed to co-operate and provide assistance in predicting and combatting outbreaks of mosquito-borne disease in south-eastern Australia. The newly formed National Arbovirus Advisory Committee is a working party providing advice to the Communicable Diseases Network Australia on arbovirus surveillance and control. Recommendations for further enhancement of national surveillance for Murray Valley encephalitis are described.

Barmah Forest virus epidemic on the south coast of New South Wales, Australia, 1994-1995: viruses, vectors, human cases, and environmental factors.

In 1995, the largest recorded outbreak of human disease resulting from infection with the mosquito transmitted alphavirus Barmah Forest (BF) virus occurred along the south coast of New South Wales, Australia. The virus was first isolated in early January from mosquitoes collected at Batemans Bay and predisposed the recognition of 135 human clinical cases. The cases of BF virus were identified initially from Batemans Bay during late January, and the majority (30%) of all cases came from this town. After 5 wk, all major centers on the south coast had clinical patients. Aedes vigilax (Skuse) were especially abundant at Batemans Bay, with levels up to 8 times greater than normal. This species yielded 111 isolates and appeared to be the major vector of BF virus. Attempts to examine if BF virus was maintained in the field by vertical transmission within Ae. vigilax populations were unsuccessful; no evidence of vertical transmission with BF virus, nor any other arbovirus, was found in > 17,000 adults emerging from field-collected larvae from the region following peak virus activity. In addition to BF virus, other viruses were recovered from field-collected adult mosquitoes, including Ross River (10 isolates), Edge Hill (21), and Stratford (10). Ae. vigilax again yielded the majority of these viral isolates. The BF virus outbreak appeared to be associated with several factors. A lack of recent BF virus activity in the region provided a highly susceptible human population, and unusual weather conditions of above average rainfall coupled with high tides resulted in extraordinarily large populations of Ae. vigilax.

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 a new species of Duboscqia (Microsporida: Thelohaniidae) infecting the mosquito Anopheles hilli and an intermediate copepod host, Apocyclops dengizicus.

A new species of Microsporida, Duboscqia dengihilli, was found infecting larvae of the mosquito Anopheles hilli in northern Queensland, Australia. Laboratory experiments showed that binucleate spores formed within infected female mosquitoes were responsible for transovarial transmission to the next generation. Sporogony within the larval fat body was initiated by two diplokarya, one at each end of the cell, which undergo meiosis within a single sporophorous vesicle to form 16 meiospores. These spores are responsible for horizontal transmission to the copepod Apocyclops dengizicus. The microsporidium is transmitted back to the mosquito host via uninucleate pyriform spores formed within the copepod host which are infectious to larvae of A. hilli. The meronts within larvae infected by horizontal transmission ultimately develop into the binucleate spores within adult females to complete the life cycle. Thus, the development of this microsporidium involves vertical transmission between successive mosquito generations and horizontal transmission between mosquitoes and copepods similar to the life cycles of Amblyospora and Parathelohania.

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)

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)