Diagnostics and molecular epidemiology of the Sarcoptes scabiei mite infesting Australian wildlife

Parasitic infestations have always been a noteworthy topic for human and animal health globally, with many considered a result of spill-over and zoonosis. One such parasite, Sarcoptes scabiei, is known to infest over 300 million humans per year and has been documented in over 104 mammals. It has recently been classified as a neglected tropical disease and is among the top 50 most prevalent human diseases. With uncertainty over (i) the global epidemiology of S. scabiei and (ii) the reliability of current diagnostics methods, much still needs to be understood if stakeholders are to successfully develop strategies to control this parasite. The overall objective of this thesis was to study the molecular epidemiology and genetic typing of S. scabiei infesting Australian animals and assess the diagnostic methods for sarcoptic mange. At the global scale, numerous genetic studies have attempted to reveal how the host species and host geographic location influence S. scabiei phylogenetics. By performing an analysis of the global literature (Chapter 2), I was able to reveal that there were inconsistencies in gene loci and phylogenetic conclusions used in these previous studies. Furthermore, by executing a contemporary analytical approach employing molecular markers on existing S. scabiei sequences, it was apparent that (i) new S. scabiei samples, (ii) appropriate gene loci targets, and (iii) advanced phylogenetic approaches are necessary to more confidently comprehend the origins of mange in Australia. As there were only a limited number of Australian marsupial-derived S. scabiei sequences, and that three of the most commonly used gene loci used for typing are located within the mitochondria, I performed mitochondrial genome sequencing of mites collected from koalas and wombats (Chapter 3). It was revealed that there is a high sequence similarity not just within marsupial S. scabiei mites, but also to the only human-derived S. scabiei mitochondrial genome. Furthermore, by examining individual gene phylogenies, I concluded that cox1 is the most informative gene as the cox1 phylogeny inferred was consistent with the complete mitochondrial genome phylogeny with the highest resolution of ancestral lineages. Building on the identification of cox1 as an informative gene target, I greatly expanded the molecular typing of S. scabiei within Australia (Chapter 4). I identified that mites collected from koalas, wombats, foxes and dogs across five states of Australia were unable to be phylogenetically separated by their host or location. Thus, I considered it highly plausible that multiple spill-over events may have occurred in Australia, as many haplotypes are identical to European and non-European sequences. Furthermore, I suggested that it is likely that canids are the source for transmission of mange throughout Australian wildlife as dogs and foxes share identical haplotypes to wombats and koalas. Finally, I detected a distinguishable human-specific lineage, distinct from the dominant mixed animal clade. Clinical diagnosis of mange/scabies typically involves the collection of skin scrapings followed by microscopic detection of the mite. This method yields results with a high risk of false negatives, however. I performed the first comparative S. scabiei diagnostic study on a unique sample set collected from bare-nosed wombats. Here, I assessed a variety of putatively useful approaches including observational scoring, microscopy, PCR on skin scraping DNA and PCR on skin swab DNA (Chapter 5). I concluded that: (i) observational scoring positively correlated with counts from microscopy, however this approach tended to under-diagnose early mange; (ii) species-specific S. scabiei PCR enhanced the sensitivity of mite detection in relation to microscopy and; (iii) swabs as a method for sample collection is questionable due to inadequate host cell uptake and likelihood of producing false negatives. Finally, I sought to improve the use of molecular techniques for S. scabiei diagnosis (Chapter 6). I developed a novel rapid diagnostic tool using a Loop Mediated Isothermal Amplification assay, which I demonstrated to be specific to S. scabiei and able to produce a rapid diagnostic result within 30 minutes. Since this method can be performed without advanced laboratory equipment, this development has potential direct roles as an ancillary method with microscopy at the point-of-care to reduce the number of potential S. scabiei false-negative results obtained by microscopy alone in both human and veterinary settings. In summary, this thesis has contributed to: (i) the expansion of S. scabiei phylogeny by highlighting the high genetic variability of the single mite species; (ii) suggested multiple spill-over events may be the consequence of inadequate screening of imports/exports possibly globally and; (iii) has demonstrated the incompatible variety of different diagnostic methods for S. scabiei which may be supplemented with the development of a new highly sensitive and specific molecular technique. The contributions I have made in S. scabiei research will aid in future conservation efforts to aid in understanding transmission risks to threatened populations and enhance diagnostic procedures in clinical, field and remote settings

Nano- and Micro-materials in the Treatment of Internal Bleeding and Uncontrolled Haemorrhage

Internal bleeding is defined as the loss of blood that occurs inside of a body cavity. After a traumatic injury, haemorrhage accounts for over 35% of pre-hospital deaths and 40% of deaths within the first 24hours. Coagulopathy, a disorder in which the blood is not able to properly form clots, typically develops after traumatic injury and results in a higher rate of mortality. The current methods to treat internal bleeding and coagulopathy are inadequate due to the requirement of extensive medical equipment that is typically not available at the site of injury. To discover a potential route for future research, several current and novel treatment methods have been reviewed and analysed. The aim of investigating different potential treatment options is to expand available knowledge, while also call attention to the importance of research in the field of treatment for internal bleeding and haemorrhage due to trauma

The health of mothers of children with a life-limiting condition; a comparative cohort study

ObjectivesThis study aimed to quantify the incidence rates of common mental and physical health conditions in mothers of children with a life-limiting condition.MethodsComparative national longitudinal cohort study using linked primary and secondary care data from the Clinical Practice Research Datalink in England. Maternal-child dyads were identified in these data. Maternal physical and mental health outcomes were identified in the primary and secondary care datasets using previously developed diagnostic coding frameworks. Incidence rates of the outcomes were modelled using Poisson regression adjusting for deprivation, ethnicity and age and accounting for time at risk.ResultsA total of 35,683 mothers, 8,950 had a child with a life-limiting condition, 8,868 had a child with a chronic condition and 17,865 had a child with no long-term condition.The adjusted incidence rates of all of the physical and mental health conditionswere significantly higher in the mothers of children with a life-limiting condition when compared to those mothers with a child with no long-term condition. (e.g. depression IRR 1.21 (95%CI 1.13 to 1.30) cardiovascular disease IRR 1.73 (95%CI 1.27 to 2.36), death in mothers IRR 1.59 (95%CI 1.16 to 2.18).ConclusionsThis study clearly demonstrates the higher incidence rates of common and serious physical and mental health problems and death in mothers of children with a life limiting condition. Further research is required to understand how best to support these mothers, but healthcare providers should consider how they can target this population to provide preventative and treatment services

The Value of Participating in British Exploring Society Expeditions: A Three Year Multi Cohort Study

A primary aim of many expeditions is to facilitate personal development of young people and 15 while there is much anecdotal evidence to support this aim, there is limited empirical work of 16 varied quality that explores the specific nature of such benefits. This research examined nine 17 summer BES expeditions (Norway, Namibia & Amazon in 2012; Finnmark, Ladakh and 18 Namibia in 2013 and 2014) involving 58 young people (aged between 15 and 22) who completed 19 three on-line questionnaires to collect qualitative (open ended questions) and quantitative (likert 20 scale) data. Measurement of four psychological attributes associated with effective character 21 development were used: mental toughness, coping skills, GRIT and leadership skills. Surveys 22 were completed at three stages; 1) pre expedition, 2) immediately post expedition and 3) three 23 months post expedition. Results indicated that the expeditions impacted positively on the 24 psychological attributes of young people, with short term effects (three months). 25 For the 58 participants, there were statistically significant improvements and small positive 26 effect sizes in mental toughness (P=.006; ηp 2 .167), leadership skills (P=.004; ηp 2 .18), and 27 GRIT (P=.001; ηp 2 .218). There was no significant difference (P>0.05) or effect size for the 28 application of coping strategies. Qualitative data provided insights into how the learning took 29 place and individual perspectives on the overall value of the expedition in relation to narrative 30 understandin

Comparative genomics of koala, cattle and sheep strains of Chlamydia pecorum

© 2014 Bachmann et al.; licensee BioMed Central Ltd. Background: Chlamydia pecorum is an important pathogen of domesticated livestock including sheep, cattle and pigs. This pathogen is also a key factor in the decline of the koala in Australia. We sequenced the genomes of three koala C. pecorum strains, isolated from the urogenital tracts and conjunctiva of diseased koalas. The genome of the C. pecorum VR629 (IPA) strain, isolated from a sheep with polyarthritis, was also sequenced.Results: Comparisons of the draft C. pecorum genomes against the complete genomes of livestock C. pecorum isolates revealed that these strains have a conserved gene content and order, sharing a nucleotide sequence similarity > 98%. Single nucleotide polymorphisms (SNPs) appear to be key factors in understanding the adaptive process. Two regions of the chromosome were found to be accumulating a large number of SNPs within the koala strains. These regions include the Chlamydia plasticity zone, which contains two cytotoxin genes (toxA and toxB), and a 77 kbp region that codes for putative type III effector proteins. In one koala strain (MC/MarsBar), the toxB gene was truncated by a premature stop codon but is full-length in IPTaLE and DBDeUG. Another five pseudogenes were also identified, two unique to the urogenital strains C. pecorum MC/MarsBar and C. pecorum DBDeUG, respectively, while three were unique to the koala C. pecorum conjunctival isolate IPTaLE. An examination of the distribution of these pseudogenes in C. pecorum strains from a variety of koala populations, alongside a number of sheep and cattle C. pecorum positive samples from Australian livestock, confirmed the presence of four predicted pseudogenes in koala C. pecorum clinical samples. Consistent with our genomics analyses, none of these pseudogenes were observed in the livestock C. pecorum samples examined. Interestingly, three SNPs resulting in pseudogenes identified in the IPTaLE isolate were not found in any other C. pecorum strain analysed, raising questions over the origin of these point mutations.Conclusions: The genomic data revealed that variation between C. pecorum strains were mainly due to the accumulation of SNPs, some of which cause gene inactivation. The identification of these genetic differences will provide the basis for further studies to understand the biology and evolution of this important animal pathogen

Meaning behind measurement : self-comparisons affect responses to health related quality of life questionnaires

Purpose The subjective nature of quality of life is particularly pertinent to the domain of health-related quality of life (HRQOL) research. The extent to which participants’ responses are affected by subjective information and personal reference frames is unknown. This study investigated how an elderly population living with a chronic metabolic bone disorder evaluated self-reported quality of life. Methods Participants (n = 1,331) in a multi-centre randomised controlled trial for the treatment of Paget’s disease completed annual HRQOL questionnaires, including the SF-36, EQ-5D and HAQ. Supplementary questions were added to reveal implicit reference frames used when making HRQOL evaluations. Twenty-one participants (11 male, 10 female, aged 59–91 years) were interviewed retrospectively about their responses to the supplementary questions, using cognitive interviewing techniques and semi-structured topic guides. Results The interviews revealed that participants used complex and interconnected reference frames to promote response shift when making quality of life evaluations. The choice of reference frame often reflected external factors unrelated to individual health. Many participants also stated that they were unclear whether to report general or disease-related HRQOL. Conclusions It is important, especially in clinical trials, to provide instructions clarifying whether ‘quality of life’ refers to disease-related HRQOL. Information on selfcomparison reference frames is necessary for the interpretation of responses to questions about HRQOL.The Chief Scientist Office of the Scottish Government Health Directorates, The PRISM funding bodies (the Arthritis Research Campaign, the National Association for the Relief of Paget’s disease and the Alliance for Better Bone Health)Peer reviewedAuthor final versio

An Estimation of the Entomological Inoculation Rate for Ifakara: A Semi-Urban Area in a Region of Intense Malaria Transmission in Tanzania.

An entomological study on vectors of malaria and their relative contribution to Plasmodium falciparum transmission in the semi-urban area of Ifakara, south-eastern Tanzania, was conducted. A total of 32 houses were randomly sampled from the area and light trap catches (LTC) performed in one room in each house every 2 weeks for 1 year. A total of 147 448 mosquitoes were caught from 789 LTC; 26 134 Anopheles gambiae s.l., 615 A. funestus, 718 other anophelines and 119 981 culicines. More than 60% of the total A. gambiae s.l. were found in five (0.6%) LTCs, with a maximum of 5889 caught in a single trap. Of 505 A. gambiae s.l. speciated by polymerase chain reaction, 91.5% were found to be A. arabiensis. Plasmodium falciparum sporozoite enzyme-linked immunosorbent assay tests were performed on 10 108 anopheles mosquitoes and 39 (0.38%) were positive. Entomological inoculation rate (EIR) estimates were generated using a standard method and an alternative method that allows the calculation of confidence intervals based on a negative binomial distribution of sporozoite positive mosquitoes. Overall EIR estimates were similar; 31 vs. 29 [95% confidence interval (CI): 19, 44] infectious bites per annum, respectively. The EIR ranged from 4 (95% CI: 1, 17) in the cool season to 108 (95% CI: 69, 170) in the wet season and from 54 (95% CI: 30, 97) in the east of the town to 15 (95% CI: 8, 30) in the town centre. These estimates show large variations over short distances in time and space. They are all markedly lower than those reported from nearby rural areas and for other parts of Tanzania

A single sub-km Kuiper Belt object from a stellar Occultation in archival data

The Kuiper belt is a remnant of the primordial Solar System. Measurements of its size distribution constrain its accretion and collisional history, and the importance of material strength of Kuiper belt objects (KBOs). Small, sub-km sized, KBOs elude direct detection, but the signature of their occultations of background stars should be detectable. Observations at both optical and X-ray wavelengths claim to have detected such occultations, but their implied KBO abundances are inconsistent with each other and far exceed theoretical expectations. Here, we report an analysis of archival data that reveals an occultation by a body with a 500 m radius at a distance of 45 AU. The probability of this event to occur due to random statistical fluctuations within our data set is about 2%. Our survey yields a surface density of KBOs with radii larger than 250 m of 2.1^{+4.8}_{-1.7} x 10^7 deg^{-2}, ruling out inferred surface densities from previous claimed detections by more than 5 sigma. The fact that we detected only one event, firmly shows a deficit of sub-km sized KBOs compared to a population extrapolated from objects with r>50 km. This implies that sub-km sized KBOs are undergoing collisional erosion, just like debris disks observed around other stars.Comment: To appear in Nature on December 17, 2009. Under press embargo until 1800 hours London time on 16 December. 19 pages; 7 figure

PhOTO Zebrafish: A Transgenic Resource for In Vivo Lineage Tracing during Development and Regeneration

Background: Elucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo. Methodology: PhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ~100µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin. Conclusions/Significance: PhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior

The cascading pathogenic consequences of Sarcoptes scabiei infection that manifest in host disease

Sarcoptic mange, caused by the parasitic mite Sarcoptes scabiei, causes a substantive burden of disease to humans, domestic animals and wildlife, globally. There are many effects of S. scabiei infection, culminating in the disease which hosts suffer. However, major knowledge gaps remain on the pathogenic impacts of this infection. Here, we focus on the bare-nosed wombat host (Vombatus ursinus) to investigate the effects of mange on: (i) host heat loss and thermoregulation, (ii) field metabolic rates, (iii) foraging and resting behaviour across full circadian cycles, and (iv) fatty acid composition in host adipose, bone marrow, brain and muscle tissues. Our findings indicate that mange-infected V. ursinus lose more heat to the environment from alopeciaaffected body regions than healthy individuals. Additionally, mange-infected individuals have higher metabolic rates in the wild. However, these metabolic demands are difficult to meet, because infected individuals spend less time foraging and more time inactive relative to their healthy counterparts, despite being outside of the burrow for longer