A laboratory framework for ongoing optimization of amplification-based genomic surveillance programs.

IMPORTANCE: This study provides a laboratory framework to ensure ongoing relevance and performance of amplification-based whole genome sequencing to strengthen public health surveillance during extended outbreaks or pandemics. The framework integrates regular reviews of the performance of a genomic surveillance system and highlights the importance of ongoing monitoring and the identification and implementation of improvements to whole genome sequencing methods to enhance public health responses to pathogen outbreaks.

SABRes: in silico detection of drug resistance conferring mutations in subpopulations of SARS-CoV-2 genomes.

The emergence of resistance to antiviral drugs increasingly used to treat SARS-CoV-2 infections has been recognised as a significant threat to COVID-19 control. In addition, some SARS-CoV-2 variants of concern appear to be intrinsically resistant to several classes of these antiviral agents. Therefore, there is a critical need for rapid recognition of clinically relevant polymorphisms in SARS-CoV-2 genomes associated with significant reduction of drug activity in virus neutralisation experiments. Here we present SABRes, a bioinformatic tool, which leverages on expanding public datasets of SARS-CoV-2 genomes and allows detection of drug resistance mutations in consensus genomes as well as in viral subpopulations. We have applied SABRes to detect resistance-conferring mutations in 25,197 genomes generated over the course of the SARS-CoV-2 pandemic in Australia and identified 299 genomes containing resistance conferring mutations to the five antiviral therapeutics that retain effectiveness against currently circulating strains of SARS-CoV-2 - Sotrovimab, Bebtelovimab, Remdesivir, Nirmatrelvir and Molnupiravir. These genomes accounted for a 1.18% prevalence of resistant isolates discovered by SABRes, including 80 genomes with resistance conferring mutations found in viral subpopulations. Timely recognition of these mutations within subpopulations is critical as these mutations can provide an advantage under selective pressure and presents an important step forward in our ability to monitor SARS-CoV-2 drug resistance.

Rhipicephalus linnaei (Audouin, 1826) recognised as the "tropical lineage" of the brown dog tick Rhipicephalus sanguineus sensu lato: Neotype designation, redescription, and establishment of morphological and molecular reference.

We re-describe the adult stages of Rhipicephalus linnaei (Audouin, 1826), and characterise its diagnostic molecular traits. A male R. linnaei collected in Esna City, Luxor Governorate, Egypt is designated as the neotype. Rhipicephalus linnaei is re-established as a valid tick name and removed from the synonymy list of Rhipicephalus sanguineus (Latreille, 1806). Rhipicephalus linnaei is most similar to R. sanguineus and Rhipicephalus camicasi Morel, Mouchet & Rodhain, 1976 because they share similar elongated comma-like spiracula that are narrowly visible dorsally, and the dorsal prolongation is narrower than the width of the adjacent festoon. The male of R. camicasi is distinguished from R. linnaei by the non-tapering caudal widening of the spiracula. The male of R. sanguineus is distinguished from R. linnaei by shorter extension that does not taper into a long narrow extension of the spiracula. The genital pore atrium of female R. linnaei is broadly U-shaped, while it is a narrower U-shape in R. sanguineus. The remaining species within the R. sanguineus species complex - Rhipicephalus sulcatus Neumann, 1908, Rhipicephalus turanicus Pomerantsev, 1940, Rhipicephalus guilhoni Morel & Vassilades, 1963, Rhipicephalus secundus Feldman-Muhsam, 1952 and Rhipicephalus afranicus Bakkes, 2020, all exhibit spiracula with the dorsal prolongation as wide as the adjacent festoon. The DNA sequence of R. linnaei is most closely related to R. guilhoni. The phylogenetic analysis of mitogenome (mtDNA) sequences including assembled mtDNA from whole genome sequencing of the neotype supports R. linnaei as a well-defined taxon when compared with DNA sequences of other species of the R. sanguineus species complex, in particular: R. sanguineus, R. turanicus, R. secundus and R. camicasi. Molecularly, R. linnaei belongs to the so-called R. sanguineus s.l. "tropical lineage" distributed globally including the Americas, Africa, Europe, Asia and is the only species from R. sanguineus species complex in Australia.

Mitochondrial genome of Rhipicephalus cf. camicasi Morel, Mouchet et Rodhain, 1976 from a camel (Camelus dromedarius Linnaeus) in Riyadh, Saudi Arabia.

Rhipicephalus camicasi Morel, Mouchet et Rodhain, 1976 is thought to be distributed across Africa, Arabian Peninsula and the Mediterranean region. It belongs to the Rhipicephalus sanguineus (Latreille, 1806) species complex. Mitochondrial genome sequences are becoming frequently used for the identification and differentiation of tick species. In the present study, the entire mitochondrial genome of R. cf. camicasi (~15 kb) collected from a camel in Saudi Arabia was sequenced and compared with mitogenomes of two species of Rhipicephalus Koch, 1844. The mitochondrial genome is 87.8% and 91.7% identical to the reference genome of R. sanguineus (sensu stricto, former "temperate lineage") and Rhipicephalus linnaei (Audouin, 1826) (former "tropical lineage"). The current study delivers a molecular reference for material that resembles R. camicasi. We propose to consider the current material, including the complete mitogenome, as the reference for R. camicasi, until a revision using topotypical material is available.

Rapid Spread of Severe Fever with Thrombocytopenia Syndrome Virus by Parthenogenetic Asian Longhorned Ticks.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is spreading rapidly in Asia. This virus is transmitted by the Asian longhorned tick (Haemaphysalis longicornis), which has parthenogenetically and sexually reproducing populations. Parthenogenetic populations were found in ≥15 provinces in China and strongly correlated with the distribution of severe fever with thrombocytopenia syndrome cases. However, distribution of these cases was poorly correlated with the distribution of populations of bisexual ticks. Phylogeographic analysis suggested that the parthenogenetic population spread much faster than bisexual population because colonization is independent of sexual reproduction. A higher proportion of parthenogenetic ticks was collected from migratory birds captured at an SFTSV-endemic area, implicating the contribution to the long-range movement of these ticks in China. The SFTSV susceptibility of parthenogenetic females was similar to that of bisexual females under laboratory conditions. These results suggest that parthenogenetic Asian longhorned ticks, probably transported by migratory birds, play a major role in the rapid spread of SFTSV.

Unbiased Characterization of the Microbiome and Virome of Questing Ticks.

Due to their vector capacity, ticks are ectoparasites of medical and veterinary significance. Modern sequencing tools have facilitated tick-associated microbiota studies, but these have largely focused on bacterial pathogens and symbionts. By combining 16S rRNA gene sequencing with total RNA-sequencing methods, we aimed to determine the complete microbiome and virome of questing, female Ixodes holocyclus recovered from coastal, north-eastern New South Wales (NSW), Australia. We present, for the first time, a robust and unbiased method for the identification of novel microbes in ticks that enabled us to identify bacteria, viruses, fungi and eukaryotic pathogens. The dominant bacterial endosymbionts were Candidatus Midichloria sp. Ixholo1 and Candidatus Midichloria sp. Ixholo2. Candidatus Neoehrlichia australis and Candidatus Neoehrlichia arcana were also recovered, confirming that these bacteria encompass I. holocyclus' core microbiota. In addition, seven virus species were detected-four previously identified in I. holocyclus and three novel species. Notably, one of the four previously identified virus species has pathogenic potential based on its phylogenetic relationship to other tick-associated pathogens. No known pathogenic eukaryotes or fungi were identified. This study has revealed the microbiome and virome of female I. holocyclus from the environment in north-eastern NSW. We propose that future tick microbiome and virome studies utilize equivalent methods to provide an improved representation of the microbial diversity in ticks globally.

The "tropical lineage" of the brown dog tick Rhipicephalus sanguineus sensu lato identified as Rhipicephalus linnaei ().

The brown dog tick (Rhipicephalus sanguineus) parasitises dogs. Over the past decade, two distinct lineages have been recognised - R. sanguineus sensu lato "temperate lineage" and R. sanguineus sensu lato "tropical lineage". The nominal taxon R. sanguineus (Latreille, 1806) was recently associated with the "temperate lineage". We here identify the "tropical lineage" as Rhipicephalus linnaei (Audouin, 1826) using material from Australia, where no other Rhipicephalus species parasitises dogs. Whole genome sequencing of R. linnaei from Australia, Fiji and Laos, and assembly of their complete mitochondrial DNA (~15 kb) confirms the genetic identity and distinctness from all other known species within the brown dog tick species complex. Designation of the species R. linnaei is unequivocally supported by material available through the Australian National Insect Collection, Australia. Accordingly, we are formally justified in using R. linnaei for the "tropical lineage".

Biotic Factors Influence Microbiota of Nymph Ticks from Vegetation in Sydney, Australia.

Ticks are haematophagous ectoparasites of medical and veterinary significance due to their excellent vector capacity. Modern sequencing techniques enabled the rapid sequencing of bacterial pathogens and symbionts. This study's aims were two-fold; to determine the nymph diversity in Sydney, and to determine whether external biotic factors affect the microbiota. Tick DNA was isolated, and the molecular identity was determined for nymphs at the cox1 level. The tick DNA was subjected to high throughput DNA sequencing to determine the bacterial profile and the impact of biotic factors on the microbiota. Four nymph tick species were recovered from Sydney, NSW: Haemaphysalis bancrofti, Ixodes holocyclus, Ixodes trichosuri and Ixodes tasmani. Biotic factors, notably tick species and geography, were found to have a significance influence on the microbiota. The microbial analyses revealed that Sydney ticks display a core microbiota. The dominating endosymbionts among all tick species were Candidatus Midichloria sp. Ixholo1 and Candidatus Midichloria sp. Ixholo2. A novel Candidatus Midichloria sp. OTU_2090 was only found in I. holocyclus ticks (nymph: 96.3%, adult: 75.6%). Candidatus Neoehrlichia australis and Candidatus Neoehrlichia arcana was recovered from I. holocyclus and one I. trichosuri nymph ticks. Borrelia spp. was absent from all ticks. This study has shown that nymph and adult ticks carry different bacteria, and a tick bite in Sydney, Australia will result in different bacterial transfer depending on tick life stage, tick species and geography.

New insights on the epidemiology of Coxiella burnetii in pet dogs and cats from New South Wales, Australia.

Q fever is considered one of the most important zoonoses in Australia. Whilst ruminants are the primary reservoirs for Coxiella burnetii, and the major source of human infection, human cases have also been reported following contact with pet dogs and cats. This study aimed to estimate the prevalence of seropositivity to, and bacterial shedding of, C. burnetii by pet dogs and cats in a region with a high human Q fever incidence and explore risk factors for C. burnetii exposure. Samples (serum, whole blood, reproductive tissue, reproductive swabs) and questionnaires (completed by the pet's owner) were collected from dogs and cats from eight communities across remote New South Wales (NSW), Australia. Overall 86/330 dogs (26.1%, 95% CI 21.3-30.8%) and 19/145 cats (13.1%, 95% CI 7.6-18.6%) were seropositive to C. burnetii. Seroprevalence varied significantly between communities and was highest in communities within 150 km of a 2015 human Q fever outbreak. Feeding raw kangaroo was identified as a risk factor for seropositivity (adjusted OR 3.37, 95% CI 1.21-9.43). Coxiella burnetii DNA was not detected from any dog or cat whole blood, reproductive tissue or vaginal/preputial swab using qPCR targeting the IS1111 and com1 genes. Our findings suggest that companion animals are frequently exposed to C. burnetii in western NSW. Geographical variation in C. burnetii seroprevalence amongst companion animals - which corresponds with a human Q fever outbreak - suggests a shared environmental source of infection is likely with important consequences for public and animal health. The lack of detection of C. burnetii DNA from healthy companion animals suggests that pet dogs and cats are not an important reservoir for human Q fever infection outside a narrow periparturient window.

The brown dog tick Rhipicephalus sanguineus sensu Roberts, 1965 across Australia: Morphological and molecular identification of R. sanguineus s.l. tropical lineage.

The brown dog tick Rhipicephalus sanguineus (Latreille, 1806) is the most widely distributed tick species globally. Throughout the world there are at least two divergent lineages on dogs that are traditionally grouped into what was known as R. sanguineus. The species R. sanguineus was recently redescribed using a neotype reported from countries with a temperate climate. The second lineage distributed in countries with primarily tropical climates is currently designated R. sanguineus s.l. tropical lineage. Here, we present a comprehensive genetic evaluation of Australian brown dog ticks from across the continent that complements the morphological study of R. sanguineus sensu Roberts (1965). A total of 294 ticks were collected from dogs around Australia - including New South Wales, Queensland, the Northern Territory and Western Australia - for morphological identification. All ticks were morphologically identified as R. sanguineus sensu Roberts (1965). DNA was isolated from a single leg from morphologically characterised individuals from New South Wales (n = 14), Queensland (n = 18), Northern Territory (n = 7) and Western Australia (n = 13), together with ticks from Fiji (n = 1) and the Seychelles (n = 1) for comparison with Australian ticks. The study revealed three cox1 haplotypes clustered only with R. sanguineus s.l. tropical lineage'. An updated distribution of R. sanguineus sensu Roberts (1965) is compared to the 1965 distribution. In the Australian context, R. sanguineus s.l. has appeared in north-western New South Wales but remains absent from coastal New South Wales. Despite both temperate and tropical climates being present in Australia, only R. sanguineus s.l. tropical lineage was found. The evidence does not support the presence of the strictly defined brown dog tick, R. sanguineus by Nava et al. (2018) in Australia, because the examined ticks are genetically and morphologically distinct. We recommend using the term brown dog tick, R. sanguineus sensu Roberts (1965) for specimens from Australia.

Rhipicephalus sanguineus sensu lato from dogs and dromedary camels in Riyadh, Saudi Arabia: low prevalence of vector-borne pathogens in dogs detected using multiplexed tandem PCR panel.

Despite the global distribution of the brown dog tick, Rhipicephalus sanguineus (Latreille, 1806) sensu lato (s.l.), limited information exists about their identity from the Arabian Peninsula. Ticks from free roaming urban dogs and dromedary camels in Riyadh, Saudi Arabia were morphologically identified, confirmed with scanning electron microscopy and characterised at mitochondrial DNA (cox1, 12S rDNA and 16S rDNA). A total of 186 ticks were collected from 65 free roaming dogs (n = 73) and 84 dromedary camels (n = 113). Morphologically, 5.9% (11/186) were R. sanguineus s.l. and Hyalomma spp. (93.5%, 174/186). From within R. sanguineus s.l., the presence of Rhipicephalus cf. camicasi Morel, Mouchet et Rodhain, 1976 (1 dog, 2 camels) and Rhipicephalus turanicus Pomerantsev, 1936 (1 camel) is reported. The examined R. cf. camicasi form a sister group to R. sanguineus s.l. tropical lineage at all DNA markers. Dogs were parasitised by Hyalomma dromedarii Koch, 1844 (n = 59), Hyalomma impeltatum Schulze et Schlottke, 1930 (n = 1), Hyalomma excavatum Koch, 1844 (n = 2), Hyalomma turanicum Pomerantsev, 1946 (n = 1) and Hyalomma rufipes Koch,1844 (n = 1). DNA from dog blood (n = 53) from Riyadh confirmed a low prevalence of canine vector-borne pathogens that does not exceed 5.7% for Babesia spp., Mycoplasma spp., Anaplasma platys, Hepatozoon canis and Ehrlichia canis using multiplexed tandem PCR (MT-PCR) and diagnostic PCR. Low prevalence of R. sanguineus s.l. on dogs likely contributed to the low level of canine vector-borne pathogens in Saudi Arabia. We demonstrate that dogs in the central Arabian Peninsula are more commonly parasitised by Hyalomma spp. than R. sanguineus s.l.

Reptile-associated Borrelia species in the goanna tick (Bothriocroton undatum) from Sydney, Australia.

BACKGROUND: Knowledge on the capacity of Australian ticks to carry Borrelia species is currently limited or missing. To evaluate the potential of ticks to carry bacterial pathogens and their DNA, it is imperative to have a robust workflow that maximises recovery of bacterial DNA within ticks in order to enable accurate identification. By exploiting the bilateral anatomical symmetry of ticks, we were able to directly compare two DNA extraction methods for 16S rRNA gene diversity profiling and pathogen detection. We aimed to assess which combination of DNA extraction and 16S rRNA hypervariable region enables identification of the greatest bacterial diversity, whilst minimising bias, and providing the greatest capacity for the identification of Borrelia spp. RESULTS: We collected Australian endemic ticks (Bothriocroton undatum), isolated DNA from equal tick halves using two commercial DNA extraction methods and sequenced samples using V1-V3 and V3-V4 16S rRNA gene diversity profiling assays. Two distinct Borrelia spp. operational taxonomic units (OTUs) were detected using the V1-V3 16S rRNA hypervariable region and matching Borrelia spp. sequences were obtained using a conventional nested-PCR. The tick 16S rRNA gene diversity profile was dominated by Rickettsia spp. (98-99%), while the remaining OTUs belonged to Proteobacteria (51-81%), Actinobacteria (6-30%) and Firmicutes (2-7%). Multiple comparisons tests demonstrated biases in each of the DNA extraction kits towards different bacterial taxa. CONCLUSIONS: Two distinct Borrelia species belonging to the reptile-associated Borrelia group were identified. Our results show that the method of DNA extraction can promote bias in the final microbiota identified. We determined an optimal DNA extraction method and 16S rRNA gene diversity profile assay that maximises detection of Borrelia species.

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.