PlexProbes enhance qPCR multiplexing by discriminating multiple targets in each fluorescent channel.

The probe technology described in this paper facilitates detection and discrimination of multiple targets in a single fluorescent channel during PCR. This provides a strategy for doubling the number of targets that can be analysed simultaneously on existing PCR instruments. These probes are referred to as PlexProbes and produce fluorescence that can be switched 'on' or 'off' in the presence of target by manipulating the temperature. During PCR, fluorescence can be measured at multiple temperatures allowing discrimination of specific targets at defined temperatures. In a single fluorescent channel, a model duplex assay allowed either real-time or endpoint detection of Chlamydia trachomatis (CT) at 52°C and end-point detection of Neisseria gonorrhoeae (GC) at 74°C. Using this model system, as few as 40 copies of each specific target could be detected as single infection or co-infection, regardless of the presence or absence of the other target. A PlexProbe prototype assay for sexually transmitted infections (PP-STI) which simultaneously enables detection and differentiation of six targets using only three fluorescent channels was then constructed and evaluated. The PP-STI assay detects GC (2 gene targets), CT, Mycoplasma genitalium (MG), Trichomonas vaginalis (TV) and an internal control (IC). To evaluate assay performance, a panel of archived clinical samples (n = 337) were analysed using PP-STI and results compared to those obtained with a commercially available diagnostic assay. The overall agreement between results obtained with the PP-STI assay and the reference test was greater than 99.5%. PlexProbes offer a method of detecting more targets from a single diagnostic test, empowering physicians to make evidence-based treatment decisions while conserving time, labour, sample volume and reagent costs.

Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades.

The development of Subzymes demonstrates how the catalytic activity of DNAzymes can be controlled for detecting nucleic acids; however, Subzymes alone lack the sensitivity required to detect low target concentrations. To improve sensitivity, we developed a feedback system using a pair of cross-catalytic Subzymes. These were individually tethered to microparticles (MP) and separated by a porous membrane rendering them unable to interact. In the presence of a target, active PlexZymes® cleave a first Subzyme, which separates a first DNAzyme from its MP, allowing the DNAzyme to migrate through the membrane, where it can cleave a second Subzyme. This releases a second DNAzyme which can now migrate through the membrane and cleave more of the first Subzyme, thus initiating a cross-catalytic cascade. Activated DNAzymes can additionally cleave fluorescent substrates, generating a signal, and thereby, indicating the presence of the target. The method detected 1 fM of DNA homologous to the ompA gene of Chlamydia trachomatis within 30 min, demonstrating a 10,000-fold increase in sensitivity over PlexZyme detection alone. The Subzyme cascade is universal and can be triggered by any target by modifying the target sensing arms of the PlexZymes. Further, it is isothermal, protein-enzyme-free and shows great potential for rapid and affordable biomarker detection.

Identification of diverse arthropod associated viruses in native Australian fleas.

Fleas are important vectors of zoonotic disease. However, little is known about the natural diversity and abundance of flea viruses, particularly in the absence of disease associations, nor the evolutionary relationships among those viruses found in different parasitic vector species. Herein, we present the first virome scale study of fleas, based on the meta-transcriptomic analysis of 52 fleas collected along the eastern coast of Australia. Our analysis revealed 18 novel RNA viruses belonging to nine viral families with diverse genome organizations, although the majority (72%) possessed single-stranded positive-sense genomes. Notably, a number of the viruses identified belonged to the same phylogenetic groups as those observed in ticks sampled at the same locations, although none were likely associated with mammalian infection. Overall, we identified high levels of genomic diversity and abundance of viruses in the flea species studied, and established that fleas harbor viruses similar to those seen to other vectors.

Out-of-Africa, human-mediated dispersal of the common cat flea, Ctenocephalides felis: The hitchhiker's guide to world domination.

The cat flea (Ctenocephalides felis) is the most common parasite of domestic cats and dogs worldwide. Due to the morphological ambiguity of C. felis and a lack of - particularly largescale - phylogenetic data, we do not know whether global C. felis populations are morphologically and genetically conserved, or whether human-mediated migration of domestic cats and dogs has resulted in homogenous global populations. To determine the ancestral origin of the species and to understand the level of global pervasion of the cat flea and related taxa, our study aimed to document the distribution and phylogenetic relationships of Ctenocephalides fleas found on cats and dogs worldwide. We investigated the potential drivers behind the establishment of regional cat flea populations using a global collection of fleas from cats and dogs across six continents. We morphologically and molecularly evaluated six out of the 14 known taxa comprising genus Ctenocephalides, including the four original C. felis subspecies (Ctenocephalides felis felis, Ctenocephalides felis strongylus, Ctenocephalides felis orientis and Ctenocephalides felis damarensis), the cosmopolitan species Ctenocephalides canis and the African species Ctenocephalides connatus. We confirm the ubiquity of the cat flea, representing 85% of all fleas collected (4357/5123). Using a multigene approach combining two mitochondrial (cox1 and cox2) and two nuclear (Histone H3 and EF-1α) gene markers, as well as a cox1 survey of 516 fleas across 56 countries, we demonstrate out-of-Africa origins for the genus Ctenocephalides and high levels of genetic diversity within C. felis. We define four bioclimatically limited C. felis clusters (Temperate, Tropical I, Tropical II and African) using maximum entropy modelling. This study defines the global distribution, African origin and phylogenetic relationships of global Ctenocephalides fleas, whilst resolving the taxonomy of the C. felis subspecies and related taxa. We show that humans have inadvertently precipitated the expansion of C. felis throughout the world, promoting diverse population structure and bioclimatic plasticity. By demonstrating the link between the global cat flea communities and their affinity for specific bioclimatic niches, we reveal the drivers behind the establishment and success of the cat flea as a global parasite.

Accurate identification of Australian mosquitoes using protein profiling.

Australian mosquito species significantly impact human health through nuisance biting and the transmission of endemic and exotic pathogens. Surveillance programmes designed to provide an early warning of mosquito-borne disease risk require reliable identification of mosquitoes. This study aimed to investigate the viability of Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a rapid and inexpensive approach to the identification of Australian mosquitoes and was validated using a three-step taxonomic approach. A total of 300 mosquitoes representing 21 species were collected from south-eastern New South Wales and morphologically identified. The legs from the mosquitoes were removed and subjected to MALDI-TOF MS analysis. Fifty-eight mosquitoes were sequenced at the cytochrome c oxidase subunit I (cox1) gene region and genetic relationships were analysed. We create the first MALDI-TOF MS spectra database of Australian mosquito species including 19 species. We clearly demonstrate the accuracy of MALDI-TOF MS for identification of Australian mosquitoes. It is especially useful for assessing gaps in the effectiveness of DNA barcoding by differentiating closely related taxa. Indeed, cox1 DNA barcoding was not able to differentiate members of the Culex pipiens group, Cx. quinquefasciatus and Cx. pipiens molestus, but these specimens were correctly identified using MALDI-TOF MS.

Cat fleas (Ctenocephalides felis) carrying Rickettsia felis and Bartonella species in Hong Kong.

Fleas are commonly recorded on stray as well as domestic dogs and cats in Hong Kong. Fleas can be a major cause of pruritus in dogs and cats and also vectors of potentially zoonotic bacteria in the genera Rickettsia and Bartonella. Morphological examination of 174 fleas from dogs and cats living in Hong Kong revealed only cat fleas (Ctenocephalides felis). Cytochrome c oxidase subunit 1 gene (cox1) genotyping of 20 randomly selected specimens, revealed three cox1 haplotypes (HK-h1 to HK-h3). The most common haplotype was HK-h1 with 17 specimens (17/20, 85%). HK-h1 was identical to cox1 sequences of fleas in Thailand and Fiji. HK-h1 and HK-h2 form a distinct cat flea cox1 clade previously recognized as the Clade 3. HK-h3 forms a new Clade 6. A multiplex Bartonella and Rickettsia real-time PCR of DNA from 20 C. felis found Bartonella and Rickettsia DNA in three (15%) and ten (50%) C. felis, respectively. DNA sequencing confirmed the presence of R. felis, B. clarridgeiae and Bartonella henselae. This is the first reported study of that kind in Hong Kong, and further work is required to expand the survey of companion animals in the geographical region. The sampling of fleas on domestic cats and dogs in Hong Kong revealed them to be exclusively infested by the cat flea and to be harbouring pathogens of zoonotic potential.

The Role of Heterotypic DENV-specific CD8+T Lymphocytes in an Immunocompetent Mouse Model of Secondary Dengue Virus Infection.

Dengue is the most prevalent arthropod-borne viral disease worldwide and is caused by the four dengue virus serotypes (DENV-1-4). Sequential heterologous DENV infections can be associated with severe disease manifestations. Here, we present an immunocompetent mouse model of secondary DENV infection using non mouse-adapted DENV strains to investigate the pathogenesis of severe dengue disease. C57BL/6 mice infected sequentially with DENV-1 (strain Puerto Rico/94) and DENV-2 (strain Tonga/74) developed low platelet counts, internal hemorrhages, and increase of liver enzymes. Cross-reactive CD8+ T lymphocytes were found to be necessary and sufficient for signs of severe disease by adoptively transferring of DENV-1-immune CD8+T lymphocytes before DENV-2 challenge. Disease signs were associated with production of tumor necrosis factor (TNF)-α and elevated cytotoxicity displayed by heterotypic anti-DENV-1 CD8+ T lymphocytes. These findings highlight the critical role of heterotypic anti-DENV CD8+ T lymphocytes in manifestations of severe dengue disease.

Isolation of Novel Trypanosomatid, Zelonia australiensis sp. nov. (Kinetoplastida: Trypanosomatidae) Provides Support for a Gondwanan Origin of Dixenous Parasitism in the Leishmaniinae.

The genus Leishmania includes approximately 53 species, 20 of which cause human leishmaniais; a significant albeit neglected tropical disease. Leishmaniasis has afflicted humans for millennia, but how ancient is Leishmania and where did it arise? These questions have been hotly debated for decades and several theories have been proposed. One theory suggests Leishmania originated in the Palearctic, and dispersed to the New World via the Bering land bridge. Others propose that Leishmania evolved in the Neotropics. The Multiple Origins theory suggests that separation of certain Old World and New World species occurred due to the opening of the Atlantic Ocean. Some suggest that the ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia evolved on Gondwana between 90 and 140 million years ago. In the present study a detailed molecular and morphological characterisation was performed on a novel Australian trypanosomatid following its isolation in Australia's tropics from the native black fly, Simulium (Morops) dycei Colbo, 1976. Phylogenetic analyses were conducted and confirmed this parasite as a sibling to Zelonia costaricensis, a close relative of Leishmania previously isolated from a reduviid bug in Costa Rica. Consequently, this parasite was assigned the name Zelonia australiensis sp. nov. Assuming Z. costaricensis and Z. australiensis diverged when Australia and South America became completely separated, their divergence occurred between 36 and 41 million years ago at least. Using this vicariance event as a calibration point for a phylogenetic time tree, the common ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia appeared in Gondwana approximately 91 million years ago. Ultimately, this study contributes to our understanding of trypanosomatid diversity, and of Leishmania origins by providing support for a Gondwanan origin of dixenous parasitism in the Leishmaniinae.

Cat fleas (Ctenocephalides felis) from cats and dogs in New Zealand: Molecular characterisation, presence of Rickettsia felis and Bartonella clarridgeiae and comparison with Australia.

The cat flea (Ctenocephalides felis) is the most common flea species parasitising both domestic cats and dogs globally. Fleas are known vectors of zoonotic pathogens such as vector borne Rickettsia and Bartonella. This study compared cat fleas from domestic cats and dogs in New Zealand's North and South Islands to Australian cat fleas, using the mitochondrial DNA (mtDNA) marker, cytochrome c oxidase subunit I and II (cox1, cox2). We assessed the prevalence of Rickettsia and Bartonella using genus specific multiplexed real-time PCR assays. Morphological identification confirmed that the cat flea (C. felis) is the most common flea in New Zealand. The examined fleas (n=43) at cox1 locus revealed six closely related C. felis haplotypes (inter-haplotype distance 1.1%) across New Zealand. The New Zealand C. felis haplotypes were identical or near identical with haplotypes from southern Australia demonstrating common dispersal of haplotype lineage across both the geographical (Tasman Sea) and climate scale. New Zealand cat fleas carried Rickettsia felis (5.3%) and Bartonella clarridgeiae (18.4%). To understand the capability of C. felis to vector zoonotic pathogens, we determined flea cox1 and cox2 haplotype diversity with the tandem multiplexed real-time PCR and sequencing for Bartonella and Rickettsia. This enabled us to demonstrate highly similar cat fleas on cat and dog populations across Australia and New Zealand.

Evaluation of the bacterial microbiome of two flea species using different DNA-isolation techniques provides insights into flea host ecology.

Fleas (Siphonaptera) are ubiquitous blood-sucking pests of animals worldwide and are vectors of zoonotic bacteria such as Rickettsia and Bartonella. We performed Ion Torrent PGM amplicon sequencing for the bacterial 16S rRNA gene to compare the microbiome of the ubiquitous cat flea (Ctenocephalides f. felis) and the host-specific echidna stickfast flea (Echidnophaga a. ambulans) and evaluated potential bias produced during common genomic DNA-isolation methods. We demonstrated significant differences in the bacterial community diversity between the two flea species but not between protocols combining surface sterilisation with whole flea homogenisation or exoskeleton retention. Both flea species were dominated by obligate intracellular endosymbiont Wolbachia, and the echidna stickfast fleas possessed the endosymbiont Cardinium. Cat fleas that were not surface sterilised showed presence of Candidatus 'Rickettsia senegalensis' DNA, the first report of its presence in Australia. In the case of Rickettsia, we show that sequencing depth of 50 000 was required for comparable sensitivity with Rickettsia qPCR. Low-abundance bacterial genera are suggested to reflect host ecology. The deep-sequencing approach demonstrates feasibility of pathogen detection with simultaneous quantitative analysis and evaluation of the inter-relationship of microbes within vectors.

Evidence for a specific host-endosymbiont relationship between 'Rickettsia sp. genotype RF2125' and Ctenocephalides felis orientis infesting dogs in India.

BACKGROUND: Fleas of the genus Ctenocephalides serve as vectors for a number of rickettsial zoonoses, including Rickettsia felis. There are currently no published reports of the presence and distribution of R. felis in India, however, the ubiquitous distribution of its vector Ctenocephalides felis, makes it possible that the pathogen is endemic to the region. This study investigates the occurrence of Rickettsia spp. infection in various subspecies of C. felis infesting dogs from urban areas of Mumbai, Delhi and Rajasthan in India. METHODS: Individual fleas collected off 77 stray dogs from Mumbai, Delhi and Rajasthan were screened for Rickettsia spp. by a conventional PCR targeting the ompB gene. Further genetic characterisation of Rickettsia-positive fleas was carried out using nested PCR and phylogenetic analysis of partial DNA sequences of the gltA and ompA genes. Ctenocephalides spp. were morphologically and genetically identified by PCR targeting a fragment of cox1 gene. RESULTS: Overall, 56/77 fleas (72.7%), including 22/24 (91.7%) from Delhi, 32/44 (72.7%) from Mumbai and 2/9 (22.2%) from Rajasthan were positive for Rickettsia DNA at the ompB gene. Sequences of gltA fragments confirmed the amplification of Rickettsia sp. genotype RF2125. The ompA gene of Rickettsia sp. genotype RF2125 was characterised for the first time and shown 96% identical to R. felis. Three species of Ctenocephalides were identified, with the Ctenocephalides felis orientis being the dominant flea species (69/77; 89.6%) in India, followed by Ctenocephalides felis felis (8/77; 10.4%). CONCLUSIONS: High occurrence of Rickettsia sp. genotype RF2125 in C. felis orientis and the absence of R. felis suggests a specific vector-endosymbiont adaptation and coevolution of the Rickettsia felis-like sp. within subspecies of C. felis.

Integrated morphological and molecular identification of cat fleas (Ctenocephalides felis) and dog fleas (Ctenocephalides canis) vectoring Rickettsia felis in central Europe.

Fleas of the genus Ctenocephalides are the most common ectoparasites infesting dogs and cats world-wide. The species Ctenocephalides felis and Ctenocephalides canis are competent vectors for zoonotic pathogens such as Rickettsia felis and Bartonella spp. Improved knowledge on the diversity and phylogenetics of fleas is important for understanding flea-borne pathogen transmission cycles. Fleas infesting privately owned dogs and cats from the Czech Republic (n=97) and Romania (n=66) were subjected to morphological and molecular identification and phylogenetic analysis. There were a total of 59 (60.82%) cat fleas (Ctenocephalides felis felis), 30 (30.93%) dog fleas (Ctenocephalides canis), 7 (7.22%) European chicken fleas (Ceratophyllus gallinae) and 1 (1.03%) northern rat flea (Nosopsyllus fasciatus) collected in the Czech Republic. Both C. canis and C. felis felis were identified in Romania. Mitochondrial DNA sequencing at the cox1 gene on a cohort of 40 fleas revealed the cosmopolitan C. felis felis clade represented by cox1 haplotype 1 is present in the Czech Republic. A new C. felis felis clade from both the Czech Republic and Romania is also reported. A high proportion of C. canis was observed from dogs and cats in the current study and phylogeny revealed that C. canis forms a sister clade to the oriental cat flea Ctenocephalides orientis (syn. C. felis orientis). Out of 33 fleas tested, representing C. felis felis, C. canis and Ce. gallinae, 7 (21.2%) were positive for R. felis using diagnostic real-time PCR targeting the gltA gene and a conventional PCR targeting the ompB gene. No samples tested positive for Bartonella spp. using a diagnostic real-time PCR assay targeting ssrA gene. This study confirms high genetic diversity of C. felis felis globally and serves as a foundation to understand the implication for zoonotic disease carriage and transmission by the flea genus Ctenocephalides.

TLR4 genotype and environmental LPS mediate RSV bronchiolitis through Th2 polarization.

While 30%-70% of RSV-infected infants develop bronchiolitis, 2% require hospitalization. It is not clear why disease severity differs among healthy, full-term infants; however, virus titers, inflammation, and Th2 bias are proposed explanations. While TLR4 is associated with these disease phenotypes, the role of this receptor in respiratory syncytial virus (RSV) pathogenesis is controversial. Here, we evaluated the interaction between TLR4 and environmental factors in RSV disease and defined the immune mediators associated with severe illness. Two independent populations of infants with RSV bronchiolitis revealed that the severity of RSV infection is determined by the TLR4 genotype of the individual and by environmental exposure to LPS. RSV-infected infants with severe disease exhibited a high GATA3/T-bet ratio, which manifested as a high IL-4/IFN-γ ratio in respiratory secretions. The IL-4/IFN-γ ratio present in infants with severe RSV is indicative of Th2 polarization. Murine models of RSV infection confirmed that LPS exposure, Tlr4 genotype, and Th2 polarization influence disease phenotypes. Together, the results of this study identify environmental and genetic factors that influence RSV pathogenesis and reveal that a high IL-4/IFN-γ ratio is associated with severe disease. Moreover, these molecules should be explored as potential targets for therapeutic intervention.

Perlecan domain V inhibits α2 integrin-mediated amyloid-ß neurotoxicity.

Amyloid-ß (Aß) peptide is a key component of amyloid plaques, one of the pathological features of Alzheimer's disease. Another feature is pronounced cell loss in the brain leading to an enlargement of the ventricular area and a decrease in brain weight and volume. Aß plaque deposition and neuronal toxicity can be modeled by treating human cortical neuronal cultures with Aß and showing robust Aß deposition and neurotoxicity that is mediated by α2ß1 and αvß1 integrins. The current study expands on these findings by showing that the domain V of perlecan, a known α2 integrin ligand, inhibits Aß neurotoxicity in an α2 integrin-dependent manner. Additionally, Aß binds more efficiently to cells expressing activated α2 integrin. Finally the inhibition of Aß neurotoxicity with domain V is synergistic with inhibitors of αv integrin and ß1 integrin. We propose that domain V and potentially other α2 integrin ligands could be a new therapeutic approach for inhibiting the Aß plaque deposition and neurotoxicity observed in Alzheimer's disease.