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

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

Molecular Characterization of Haemaphysalis Species and a Molecular Genetic Key for the Identification of Haemaphysalis of North America

Haemaphysalis longicornis (Acari: Ixodidae), the Asian longhorned tick, is native to East Asia, but has become established in Australia and New Zealand, and more recently in the United States. In North America, there are other native Haemaphysalis species that share similar morphological characteristics and can be difficult to identify if the specimen is damaged. The goal of this study was to develop a cost-effective and rapid molecular diagnostic assay to differentiate between exotic and native Haemaphysalis species to aid in ongoing surveillance of H. longicornis within the United States and help prevent misidentification. We demonstrated that restriction fragment length polymorphisms (RFLPs) targeting the 16S ribosomal RNA and the cytochrome c oxidase subunit I (COI) can be used to differentiate H. longicornis from the other Haemaphysalis species found in North America. Furthermore, we show that this RFLP assay can be applied to Haemaphysalis species endemic to other regions of the world for the rapid identification of damaged specimens. The work presented in this study can serve as the foundation for region specific PCR-RFLP keys for Haemaphysalis and other tick species and can be further applied to other morphometrically challenging taxa

Bacterial tick-associated infections in Australia: current studies and future directions

It may seem perplexing that there is any uncertainty in Australia about the existence of zoonotic tick-associated infections1–3. Outside this country, particularly in the northern hemisphere, tick-borne diseases such as human granulocytic anaplasmosis, babesiosis, Boutonneuse fever, ehrlichiosis, Lyme borreliosis, and tick-borne encephalitis, have well documented aetiologies, epidemiology, diagnostic methods, and treatments. Why is Australia different and what research is being conducted to address this issue? This article briefly addresses these questions and explains how high-throughput metagenomic analysis has started to shed light on bacterial microbiomes in Australian ticks, providing new data on the presence and distribution of potentially zoonotic microbial taxa

Rethinking Coxiella infections in Australia

Coxiellaburnetii is the causative agent of coxiellosis in animals and Q fever in humans. Despite being a vaccine preventable disease, Q fever remains a frequently reported zoonotic infection in Australia. Recently, a Coxiella species was identified in brown dog ticks (Rhipicephalus sanguineus) in urban and rural regions of Australia. Further molecular characterisation revealed that it is genetically identical to ‘Candidatus Coxiella massiliensis’ (KM079627) described in R. sanguineus ticks removed from humans with eschars in France and serologic cross-reactivity among ‘Ca. Coxiella massiliensis’ and C.burnetii may occur. This report highlights the need for molecular testing of seropositive companion animals and humans to determine which species of Coxiella they are infected with, in order to further assess Coxiella species associated with Coxiella infections in Australia

Comparison of morphological and molecular methods to identify the diet of a generalist omnivore

Context: Ecologists need robust and effective methods to quantify the diet of animals. However, assessing dietary composition can be challenging because most animals are seldom observed eating, especially when studying rare or cryptic species. Aims: Morphological analysis of scats has been extensively used previously, and recent advances in the accessibility of DNA barcoding techniques have also made molecular approaches a viable alternative for diet analysis from scats. We compared the results from two methods of scat analysis, to trial the use of contemporary approaches in scat analysis. Methods: In the present study, we used morphological analysis and DNA barcoding of matter in scats to catalogue the diet of a generalist omnivore, the greater bilby (Macrotis lagotis Thylacomyidae), in the West Kimberley. The composition and diversity of diet items, as well as the taxonomic identification level, were compared between methods. Key results: Each method provided complimentary results; morphological analysis uncovered the type of matter consumed (e.g. root, seed) and relative proportion of the total undigested content, whereas DNA barcoding could assign such matter to a taxon. Even though dietary DNA could be extracted from only 38% of scats, DNA barcoding identified a greater diversity of taxa in scats than did morphological analyses. Barcoding could detect the presence of highly-digestible items such as cossid moths (Cossidae) and spiders (Araneae). Conclusions: Morphological analysis was useful for quantifying relative abundance of diet categories; however, DNA barcoding detected a greater diversity of dietary items within scats. Despite the expense of DNA barcoding, the method can more accurately identify the taxa consumed, whereas morphology can greatly underestimate dietary species diversity. However, the technical requirements for performing DNA analysis make it expensive, while resource-limited field ecologists can generally perform morphological analysis with appropriate training. Implications: Researchers and land managers will benefit from using both approaches in concert to gain a robust understanding of the local bilby diet. However, the cost and limitations of DNA barcoding (particularly when dealing with degraded DNA) mean that this approach should only be employed when the quality of the genetic material within samples is suitable. We recommend conducting exploratory analysis using morphological analysis (potentially in the field), with follow-up DNA barcoding to detect highly digestible items in fresh scats

Cryptosporidium and Giardia in dam water on sheep farms – An important source of transmission?

Cryptosporidium and Giardia infections can negatively impact livestock health and reduce productivity, and some species and genotypes infecting livestock have zoonotic potential. Infection occurs via the faecal-oral route. Waterborne infections are a recognised source of infection for humans, but the role of livestock drinking water as a source of infection in livestock has not been described. This study aimed to determine whether contaminated drinking water supplies, such as farm dams, are a likely transmission source for Cryptosporidium and Giardia infections for extensively managed sheep. Dam water samples (n = 47) were collected during autumn, winter and spring from 12 farm dams located on six different farms in south west Western Australia, and faecal samples (n = 349) were collected from sheep with access to these dams. All samples were initially screened for Cryptosporidium spp. at the 18S locus and Giardia spp. at the gdh gene using qPCR, and oocyst numbers were determined directly from the qPCR data using DNA standards calibrated by droplet digital PCR. Cryptosporidium-positive sheep faecal samples were typed and subtyped by sequence analysis of 18S and gp60 loci, respectively. Giardia-specific PCR and Sanger sequencing targeting tpi and gdh loci were performed on Giardia- positive sheep faecal samples to characterise Giardia duodenalis assemblages. To identify Cryptosporidium and Giardia spp. in dam water samples, next-generation sequencing analysis of 18S and gdh amplicons were performed, respectively. Two species of Cryptosporidium (Cryptosporidium xiaoi and Cryptospordium ubiquitum (subtype family XIIa)) were detected in 38/345 sheep faecal samples, and in water from 9/12 farm dams during the study period, with C. xiaoi the species most frequently detected in both faeces and dam water overall. Giardia duodenalis assemblages AI, AII and E were detected in 36/348 faecal samples and water from 10/12 farm dams. For dam water samples where oo/cysts were detected by qPCR, Cryptosporidium oocyst concentration ranged from 518–2429 oocysts/L (n = 14), and Giardia cyst concentration ranged from 102 to 1077 cysts/L (n = 17). Cryptosporidium and Giardia with zoonotic potential were detected in farm dam water, including C. ubiquitum, C. hominis, C. parvum, C. cuniculus, C. xiaoi, and G. duodenalis assemblages A, B and E. The findings suggest that dam water can be contaminated with Cryptosporidium species and G. duodenalis assemblages that may infect sheep and with zoonotic potential, and farm dam water may represent one source of transmission for infections

Further characterisation of Leucocytozoon podargii in wild tawny frogmouths (Podargus strigoides) in Western Australia

The present study assessed the prevalence and morphology of Leucocytozoon podargii from wild tawny frogmouths (Podargus strigoides) in Western Australia (WA) and genetically characterised the cytochrome b gene (cyt b) of L. podargii in wild tawny frogmouths from WA and Queensland (QLD). The prevalence of L. podargii in wild tawny frogmouths from WA was 93.3% (14/15; 95% CI, 68.1–99.8%). The morphological characters of L. podargii from WA were similar to L. podargii from QLD: the gametocytes were round-oval shape, approximately 8–12 μm in diameter; the macrogametocytes were 12.4 μm in diameter; microgametocytes were 10.4 μm in diameter; and the ratio of macrogametocytes and microgametocytes was 3:2. Sequence analysis of partial cyt b gene fragments revealed that L. podargii sequences isolated from wild tawny frogmouths in WA shared the highest similarity (99.8% at nucleotide level and 100% at protein level) with L. podargii isolated from wild tawny frogmouths in QLD. The mitochondrial 18S rRNA gene of L. podargii gametocytes was quantified using droplet digital PCR (ddPCR), and the highest gametocyte load was detected in the lung. This finding corresponds to the results of the histological study. Based on the morphological and molecular studies, it was concluded that the Leucocytozoon parasite identified from wild tawny frogmouths in WA is consistent with L. podargii from wild tawny frogmouths in QLD, and the present study has genetically characterised two different L. podargii genotypes (QLD and WA) for the first time

Profiling the diversity of Cryptosporidium species and genotypes in wastewater treatment plants in Australia using next generation sequencing

Wastewater recycling is an increasingly popular option in worldwide to reduce pressure on water supplies due to population growth and climate change. Cryptosporidium spp. are among the most common parasites found in wastewater and understanding the prevalence of human-infectious species is essential for accurate quantitative microbial risk assessment (QMRA) and cost-effective management of wastewater. The present study conducted next generation sequencing (NGS) to determine the prevalence and diversity of Cryptosporidium species in 730 raw influent samples from 25 Australian wastewater treatment plants (WWTPs) across three states: New South Wales (NSW), Queensland (QLD) and Western Australia (WA), between 2014 and 2015. All samples were screened for the presence of Cryptosporidium at the 18S rRNA (18S) locus using quantitative PCR (qPCR), oocyst numbers were determined directly from the qPCR data using DNA standards calibrated by droplet digital PCR, and positives were characterized using NGS of 18S amplicons. Positives were also screened using C. parvum and C. hominis specific qPCRs. The overall Cryptosporidium prevalence was 11.4% (83/730): 14.3% (3/21) in NSW; 10.8% (51/470) in QLD; and 12.1% (29/239) in WA. A total of 17 Cryptosporidium species and six genotypes were detected by NGS. In NSW, C. hominis and Cryptosporidium rat genotype III were the most prevalent species (9.5% each). In QLD, C. galli, C. muris and C. parvum were the three most prevalent species (7.7%, 5.7%, and 4.5%, respectively), while in WA, C. meleagridis was the most prevalent species (6.3%). The oocyst load/Litre ranged from 70 to 18,055 oocysts/L (overall mean of 3426 oocysts/L: 4746 oocysts/L in NSW; 3578 oocysts/L in QLD; and 3292 oocysts/L in WA). NGS-based profiling demonstrated that Cryptosporidium is prevalent in the raw influent across Australia and revealed a large diversity of Cryptosporidium species and genotypes, which indicates the potential contribution of livestock, wildlife and birds to wastewater contamination

Metagenomic analysis of Cryptosporidium species and genotypes in wastewater treatment plants

No abstract availabl