MapCAST : Real-Time Collaboration With Concept Maps

This thesis describes the development of the application mapCAST, a computer-based concept-mapping tool that allows synchronous collaboration via TCP/IP networks, such as the Internet The useability and feasibility of mapCAST as a computer-based tool was examined and analysed in a real world situation. Results indicate that mapCAST is successful as a collaborative tool in a situations involving knowledge organisation, but lacks certain functionality that many Macintosh users are accustomed to

An investigation of Coxiella burnetii and Coxiella-like bacteria in the brown dog tick (Rhipicephalus sanguineus)

There are a wide variety of animal reservoirs of the zoonotic bacterium Coxiella burnetii (C. burnetii), and ticks may play a significant role in the natural transmission cycle of this pathogen. Recently, domestic dogs have been implicated as reservoirs of C. burnetii. Dogs are the primary hosts of Rhipicephalus sanguineus (R. sanguineus), and C. burnetii has previously been detected in these ticks. The objectives of this study were to identify and record R. sanguineus ticks collected from dogs in Australia, and to investigate the prevalence of C. burnetii in these ticks. Subsequent to this, the bacterial microbiome of R. sanguineus ticks was investigated. The IS1111a transposase element gene was targeted using qPCR to detect C. burnetii DNA in R. sanguineus. The Ion Torrent™ Next-Generation Sequencing platform was used to sequence bacterial 16S rDNA in the ticks. In this study, 2,577 R. sanguineus ticks were morphologically identified and recorded in the Northern Territory, South Australia and Western Australia. There was no positive detection of C. burnetii in a subset of 31 R. sanguineus ticks by qPCR. Next-generation sequencing of the universal bacterial 16S rRNA gene revealed that a Coxiella sp. was present in 53/59 (90%) tick pools. The sequences were compared to GenBank submissions and a 100% match was obtained to a Coxiella sp. from R. sanguineus in the Philippines. A phylogenetic analysis of this Coxiella sp. showed that it does not group with the pathogenic C. burnetii. This Coxiella sp. may be a non-pathogenic endosymbiont of R. sanguineus, and future investigations could aim to assess the role of Coxiella endosymbionts in R. sanguineus, and whether this bacterium causes cross-reactivity in immunologic assays used for the diagnosis of Q fever in people

Recent insights into the tick microbiome gained through next-generation sequencing

The tick microbiome comprises communities of microorganisms, including viruses, bacteria and eukaryotes, and is being elucidated through modern molecular techniques. The advent of next-generation sequencing (NGS) technologies has enabled the genes and genomes within these microbial communities to be explored in a rapid and cost-effective manner. The advantages of using NGS to investigate microbiomes surpass the traditional non-molecular methods that are limited in their sensitivity, and conventional molecular approaches that are limited in their scalability. In recent years the number of studies using NGS to investigate the microbial diversity and composition of ticks has expanded. Here, we provide a review of NGS strategies for tick microbiome studies and discuss the recent findings from tick NGS investigations, including the bacterial diversity and composition, influential factors, and implications of the tick microbiome

Illuminating the bacterial microbiome of Australian ticks with 16S and Rickettsia-specific next-generation sequencing

Next-generation sequencing (NGS) studies show that mosquito and tick microbiomes influence the transmission of pathogens, opening new avenues for vector-borne pathogen control. Recent microbiological studies of Australian ticks highlight fundamental knowledge gaps of tick-borne agents. This investigation explored the composition, diversity and prevalence of bacteria in Australian ticks (n = 655) from companion animals (dogs, cats and horses). Bacterial 16S NGS was used to identify most bacterial taxa and a Rickettsia-specific NGS assay was developed to identify Rickettsia species that were indistinguishable at the V1-2 regions of 16S. Sanger sequencing of near full-length 16S was used to confirm whether species detected by 16S NGS were novel. The haemotropic bacterial pathogens Anaplasma platys, Bartonella clarridgeiae, “Candidatus Mycoplasma haematoparvum” and Coxiella burnetii were identified in Rhipicephalus sanguineus (s.l.) from Queensland (QLD), Western Australia, the Northern Territory (NT), and South Australia, Ixodes holocyclus from QLD, Rh. sanguineus (s.l.) from the NT, and I. holocyclus from QLD, respectively. Analysis of the control data showed that cross-talk compromises the detection of rare species as filtering thresholds for less abundant sequences had to be applied to mitigate false positives. A comparison of the taxonomic assignments made with 16S sequence databases revealed inconsistencies. The Rickettsia-specific citrate synthase gene NGS assay enabled the identification of Rickettsia co-infections with potentially novel species and genotypes most similar (97.9–99.1%) to Rickettsia raoultii and Rickettsia gravesii. “Candidatus Rickettsia jingxinensis” was identified for the first time in Australia. Phylogenetic analysis of near full-length 16S sequences confirmed a novel Coxiellaceae genus and species, two novel Francisella species, and two novel Francisella genotypes. Cross-talk raises concerns for the MiSeq platform as a diagnostic tool for clinical samples. This study provides recommendations for adjustments to Illuminaʼs 16S metagenomic sequencing protocol that help track and reduce cross-talk from cross-contamination during library preparation. The inconsistencies in taxonomic assignment emphasise the need for curated and quality-checked sequence databases

Detection and phylogenetic characterisation of novel Anaplasma and Ehrlichia species in Amblyomma triguttatum subsp. from four allopatric populations in Australia

Anaplasma and Ehrlichia spp. are tick-borne pathogens that can cause severe disease in domestic animals, and several species are responsible for emerging zoonoses in the northern hemisphere. Until recently, the only members of these genera reported in Australia (A. marginale, A. centrale, and A. platys) were introduced from other continents, through the importation of domestic animals and their associated ticks. However, unique Anaplasma and Ehrlichia 16S rRNA gene sequences were recently detected for the first time in native Australian ticks, particularly in Amblyomma triguttatum subsp. ticks from southwest Western Australia (WA). We used molecular techniques to survey Am. triguttatum subsp. ticks from four allopatric populations in southern and western Australia for Anaplasma and Ehrlichia species, and described here the phylogeny of these novel organisms. An A. bovis variant (genotype Y11) was detected in ticks from two study sites; Yanchep National Park (12/280, 4.3%) and Barrow Island (1/69, 1.4%). Phylogenetic analysis of 16S rRNA and groEL gene sequences concluded that A. bovis genotype Y11 is a unique genetic variant, distinct from other A. bovis isolates worldwide. Additionally, a novel Ehrlichia species was detected in Am. triguttatum subsp. from three of the four study sites; Yanchep National Park (18/280, 6.4%), Bungendore Park (8/46, 17.4%), and Innes National Park (9/214, 4.2%), but not from Barrow Island. Phylogenetic analysis of 16S, groEL, gltA, and map1 gene sequences revealed that this Ehrlichia sp. is most closely related to, but clearly distinct from, E. ruminantium and Ehrlichia sp. Panola Mountain. We propose to designate this new species '. Candidatus Ehrlichia occidentalis'. Anaplasma bovis genotype Y11 and 'Candidatus E. occidentalis' are the first Anaplasma and Ehrlichia species to be recorded in native Australian ticks

A survey of ticks (Acari: Ixodidae) of companion animals in Australia

Background: Ticks are among the most important vectors of pathogens affecting companion animals, and also cause health problems such as tick paralysis, anaemia, dermatitis, and secondary infections. Twenty ixodid species have previously been recorded on dogs, cats, and horses in Australia, including Rhipicephalus sanguineus, Ixodes holocyclus and Haemaphysalis longicornis, which transmit tick-borne diseases. A survey of hard ticks (Acari: Ixodidae) was conducted during 2012-2015 to investigate tick species that infest dogs, cats, and horses in Australia. Methods: Individual tick specimens were collected from dogs, cats and horses across Australia and sample collection locations were mapped using QGIS software. Ticks were morphologically examined to determine species, instar and sex. The companion animal owners responded to questionnaires and data collected were summarised with SPSS software. Results: A total of 4765 individual ticks were identified in this study from 7/8 states and territories in Australia. Overall, 220 larvae, 805 nymphs, 1404 males, and 2336 females of 11 tick species were identified from 837 companion animal hosts. One novel host record was obtained during this study for Ixodes myrmecobii, which was found on Felis catus (domestic cat) in the town of Esperance, Western Australia. The most common tick species identified included R. sanguineus on dogs (73 %), I. holocyclus on cats (81 %) and H. longicornis on horses (60 %). Conclusions: This study is the first of its kind to be conducted in Australia and our results contribute to the understanding of the species and distribution of ticks that parasitise dogs, cats, and horses in Australia. Records of R. sanguineus outside of the recorded distribution range emphasise the need for a systematic study of the habitat range of this species. Several incomplete descriptions of ixodid species encountered in this study hindered morphological identification

Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification

Recycled wastewater can carry human-infectious microbial pathogens and therefore wastewater treatment strategies must effectively eliminate pathogens before recycled wastewater is used to supplement drinking and agricultural water supplies. This study characterised the bacterial composition of four wastewater treatment plants (WWTPs) (three waste stabilisation ponds and one oxidation ditch WWTP using activated sludge treatment) in Western Australia. The hypervariable region 4 (V4) of the bacterial 16S rRNA (16S) gene was sequenced using next-generation sequencing (NGS) on the Illumina MiSeq platform. Sequences were pre-processed in USEARCH v10.0 and denoised into zero-radius taxonomic units (ZOTUs) with UNOISE3. Taxonomy was assigned to the ZOTUs using QIIME 2 and the Greengenes database and cross-checked with the NCBI nr/nt database. Bacterial composition of all WWTPs and treatment stages (influent, intermediate and effluent) were dominated by Proteobacteria (29.0-87.4%), particularly Betaproteobacteria (9.0-53.5%) and Gammaproteobacteria (8.6-34.6%). Nitrifying bacteria (Nitrospira spp.) were found only in the intermediate and effluent of the oxidation ditch WWTP, and denitrifying and floc-forming bacteria were detected in all WWTPs, particularly from the families Comamonadaceae and Rhodocyclales. Twelve pathogens were assigned taxonomy by the Greengenes database, but comparison of sequences from genera and families known to contain pathogens to the NCBI nr/nt database showed that only three pathogens (Arcobacter venerupis, Laribacter hongkongensis and Neisseria canis) could be identified in the dataset at the V4 region. Importantly, Enterobacteriaceae genera could not be differentiated. Family level taxa assigned by Greengenes database agreed with NCBI nr/nt in most cases, however, BLAST analyses revealed erroneous taxa in Greengenes database. This study highlights the importance of validating taxonomy of NGS sequences with databases such as NCBI nr/nt, and recommends including the V3 region of 16S in future short amplicon NGS studies that aim to identify bacterial enteric pathogens, as this will improve taxonomic resolution of most, but not all, Enterobacteriaceae species

Molecular investigation into the presence of a Coxiella sp. in Rhipicephalus sanguineus ticks in Australia

Q fever is an infectious disease with a global distribution caused by the intracellular bacterium, Coxiella burnetii, which has been detected in a large number of tick species worldwide, including the brown dog tick, Rhipicephalus sanguineus. Recent reports of a high seroprevalance of C. burnetii in Australian dogs, along with the identification of additional Coxiella species within R. sanguineus ticks, has prompted an investigation into the presence and identification of Coxiella species in R. sanguineus ticks in Australia. Using a combination of C. burnetii species-specific IS1111a transposase gene and Coxiella genus-specific 16S rRNA PCR assays, a Coxiella sp. was identified in 100% (n = 199) of R. sanguineus ticks analysed, and C. burnetii was not detected in any R. sanguineus ticks studied. Phylogenetic analysis of the 16S rRNA gene revealed the Coxiella sequences were closely related to Coxiella sp. identified previously in R. sanguineus and R. turanicus ticks overseas. This study illustrates the value of using genus specific PCR assays to detect previously unreported bacterial species. Furthermore, the presence of an additional Coxiella sp. in Australia requires further investigation into its potential for contributing to serological cross-reactions during Q fever testing

Increased genetic diversity and prevalence of co-infection with Trypanosoma spp. in koalas (Phascolarctos cinereus) and their ticks identified using next-generation sequencing (NGS)

Infections with Trypanosoma spp. have been associated with poor health and decreased survival of koalas (Phascolarctos cinereus), particularly in the presence of concurrent pathogens such as Chlamydia and koala retrovirus. The present study describes the application of a next-generation sequencing (NGS)-based assay to characterise the prevalence and genetic diversity of trypanosome communities in koalas and two native species of ticks (Ixodes holocyclus and I. tasmani) removed from koala hosts. Among 168 koalas tested, 32.2% (95% CI: 25.2-39.8%) were positive for at least one Trypanosoma sp. Previously described Trypanosoma spp. from koalas were identified, including T. irwini (32.1%, 95% CI: 25.2-39.8%), T. gilletti (25%, 95% CI: 18.7-32.3%), T. copemani (27.4%, 95% CI: 20.8-34.8%) and T. vegrandis (10.1%, 95% CI: 6.0-15.7%). Trypanosoma noyesi was detected for the first time in koalas, although at a low prevalence (0.6% 95% CI: 0-3.3%), and a novel species (Trypanosoma sp. AB-2017) was identified at a prevalence of 4.8% (95% CI: 2.1-9.2%). Mixed infections with up to five species were present in 27.4% (95% CI: 21-35%) of the koalas, which was significantly higher than the prevalence of single infections 4.8% (95% CI: 2-9%). Overall, a considerably higher proportion (79.7%) of the Trypanosoma sequences isolated from koala blood samples were identified as T. irwini, suggesting this is the dominant species. Co-infections involving T. gilletti, T. irwini, T. copemani, T. vegrandis and Trypanosoma sp. AB-2017 were also detected in ticks, with T. gilletti and T. copemani being the dominant species within the invertebrate hosts. Direct Sanger sequencing of Trypanosoma 18S rRNA gene amplicons was also performed and results revealed that this method was only able to identify the genotypes with greater amount of reads (according to NGS) within koala samples, which highlights the advantages of NGS in detecting mixed infections. The present study provides new insights on the natural genetic diversity of Trypanosoma communities infecting koalas and constitutes a benchmark for future clinical and epidemiological studies required to quantify the contribution of trypanosome infections on koala survival rates

Inhibition of the endosymbiont “Candidatus Midichloria mitochondrii” during 16S rRNA gene profiling reveals potential pathogens in Ixodes ticks from Australia

Background: The Australian paralysis tick (Ixodes holocyclus) is of significant medical and veterinary importance as a cause of dermatological and neurological disease, yet there is currently limited information about the bacterial communities harboured by these ticks and the risk of infectious disease transmission to humans and domestic animals. Ongoing controversy about the presence of Borrelia burgdorferi sensu lato (the aetiological agent of Lyme disease) in Australia increases the need to accurately identify and characterise bacteria harboured by I. holocyclus ticks. Methods: Universal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes present in DNA samples from I. holocyclus and I. ricinus ticks, collected in Australia and Germany respectively. The 16S amplicons were purified, sequenced on the Ion Torrent platform, and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomy. Initial analysis of I. holocyclus and I. ricinus identified that > 95 % of the 16S sequences recovered belonged to the tick intracellular endosymbiont “Candidatus Midichloria mitochondrii” (CMM). A CMM-specific blocking primer was designed that decreased CMM sequences by approximately 96 % in both tick species and significantly increased the total detectable bacterial diversity, allowing identification of medically important bacterial pathogens that were previously masked by CMM.Results: Borrelia burgdorferi sensu lato was identified in German I. ricinus, but not in Australian I. holocyclus ticks. However, bacteria of medical significance were detected in I. holocyclus ticks, including a Borrelia relapsing fever group sp., Bartonella henselae, novel “Candidatus Neoehrlichia” spp., Clostridium histolyticum, Rickettsia spp., and Leptospira inadai. Conclusions: Abundant bacterial endosymbionts, such as CMM, limit the effectiveness of next-generation 16S bacterial community profiling in arthropods by masking less abundant bacteria, including pathogens. Specific blocking primers that inhibit endosymbiont 16S amplification during PCR are an effective way of reducing this limitation. Here, this strategy provided the first evidence of a relapsing fever Borrelia sp. and of novel “Candidatus Neoehrlichia” spp. in Australia. Our results raise new questions about tick-borne pathogens in I. holocyclus ticks