Transmission of Emerging Pathogenic Leptospira Species from Fruit Bats in Northern Australia

Suhella Tulsiani (2010). Transmission of Emerging Pathogenic Leptospira Species from Fruit Bats in Northern Australia PhD Thesis, School of Veterinary Science, The University of Queensland.

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Author Suhella Tulsiani
Thesis Title Transmission of Emerging Pathogenic Leptospira Species from Fruit Bats in Northern Australia
School, Centre or Institute School of Veterinary Science
Institution The University of Queensland
Publication date 2010-12
Thesis type PhD Thesis
Supervisor Dr. Rowland N. Cobbold
Dr. Luke K. P. Leung
Dr. Hume E. Field
Mr. Lee D. Smythe
Total pages 301
Total colour pages 19
Total black and white pages 282
Subjects 07 Agricultural and Veterinary Sciences
Abstract/Summary Leptospirosis is an important, re-emerging, infectious disease of animals and humans. Leptospira, a spirochaete and the causative agent of leptospirosis, colonises the renal tissue of infected organisms where it can become endemic or result in clinical infection. Endemicity of a serovar is species specific and defines a reservoir or maintenance host, capable of renal shedding throughout life. Of the several mammalian maintenance hosts that have been described for the transmission of leptospires to humans, rodent reservoirs are the most significant. However, the sources of infection in rodents remain to be elucidated. The detection of antibodies and leptospiral DNA from fruit bats (Pteropus spp.) in Australia has alluded to a previously unrecognised role of fruit bats in the maintenance and transmission of leptospires to other species, in this case to associated ground-dwelling rodents. The primary aim of this thesis was to investigate the effect of the presence of Australian fruit bat (Pteropus conspicillatus) colonies on leptospiral prevalence and population dynamics of associated rodents in a leptospirosis endemic region of Australia. A subsequent aim was to advance the detection and description of leptospires from clinical and field samples. A lower rodent abundance but significantly higher prevalence of leptospiral infection was reported for rodents associated with fruit bat colonies when compared with control rodents not associated with fruit bat colonies. Isolation by culture, which by definition is the most confirmatory test for the detection of leptospires, detected four isolates, which could not be exhaustively compared across sites. On comprehensive analysis of sampling techniques employed for the study, it was evident that a high percentage of samples were contaminated, specifically those collected for examination by culture, which impacted on success of the surveillance study. Detection of leptospiral DNA by real-time Polymerase Chain Reaction (PCR) provided the highest sensitivity when compared to detection by culture, microscopic agglutination test and histopathology. This alluded to the robustness of molecular-based methods to contaminated samples. An additional benefit of collecting multiple samples from each specimen for detection of leptospires by multiple serological and molecular tests was highlighted. Random Amplified Polymorphic DNA (RAPD) markers have been previously used for the characterisation of leptospiral isolates, however, inconsistencies with reproducibility and subjective gel-scoring, have led to its disuse. PCR followed by high-resolution melt (HRM) curve analysis of amplicons from a number of bacterial and viral pathogens, including Campylobacter jejuni, have been successful at characterisation of pathogens. The two molecular methodologies were successfully combined for the serological characterisation of ten reference leptospiral isolates in a pilot trial. For the validation of this technique (RAPD-HRM), 23 strains of leptospires representing eight reference serovars, including the four strains isolated from rodents in this study, were tested. Intra-serovar divergence and inter-serovar convergence were reported during the course of this validation which may be indicative of discrepancies with serological classification. Further, evidence for genetic attenuation was noted in the strains representing the reference collection by the RAPD-HRM curve analysis. This trend was confirmed by analysis of hyper-immune antisera titre comparison between wild-type strains and corresponding reference strains. The attenuation may have occurred due to the high number of passages involved in the maintenance of the reference collections in agar-based, nutrient-rich medium. The data from the field surveillance of this study suggest that transmission of leptospires from fruit bats to rodents in Australia may not be influenced by the density of rodents at a site. Multiple samples collected for multiple tests from a single specimen increased chances of detecting leptospires. However, in the absence of isolation by culture of leptospires from renal samples from fruit bats, renal shedding, and therefore their role as reservoir hosts, cannot be confirmed. A high prevalence and low abundance in rodents associated with a fruit bat colony is suggestive of the potential for transmission which needs to be further evaluated. This surveillance constitutes the only study on the effect of the presence of a fruit bat colony on abundance and/ or leptospiral status in ground-dwelling, small mammals to date. To enhance detection and characterisation of leptospires from clinical and field isolates, the RAPD-HRM is presented here as a modern, closed-tube, reproducible, time and cost efficient method that is novel in bringing the knowledge of serovars to a molecular methodology. However, the diagnostic test could not be accurately validated due to evidence of attenuation in the reference strain collection. This thesis reports potential transmission dynamics from fruit bats to rodents in Australia, provides the foundation for future research into enhanced field-sampling techniques, presents a novel laboratory technique for characterisation of leptospiral isolates and provides a forum to highlight a gap in the quality control of reference strain collection, the effects of which can be mitigated by routine replacement with wild-type strains or in vitro challenge of collection.
Keyword Leptospira Spp.
Fruit Bats
disease transmission
High-resolution melt analysis
Quality Control
public health
emerging infectious diseases
Additional Notes Colour: 43,55,59,136,137,149,150,151,152,228,229,230,231,232,273,274,275,283,286 Landscape: 34, 233-272

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Created: Mon, 04 Apr 2011, 16:19:52 EST by Miss Suhella Tulsiani on behalf of Library - Information Access Service