The bacterial biome of ticks and their wildlife hosts at the urban-wildland interface.

Advances in sequencing technologies have revealed the complex and diverse microbial communities present in ticks (Ixodida). As obligate blood-feeding arthropods, ticks are responsible for a number of infectious diseases that can affect humans, livestock, domestic animals and wildlife. While cases of human tick-borne diseases continue to increase in the northern hemisphere, there has been relatively little recognition of zoonotic tick-borne pathogens in Australia. Over the past 5 years, studies using high-throughput sequencing technologies have shown that Australian ticks harbour unique and diverse bacterial communities. In the present study, free-ranging wildlife (n=203), representing ten mammal species, were sampled from urban and peri-urban areas in New South Wales (NSW), Queensland (QLD) and Western Australia (WA). Bacterial metabarcoding targeting the 16S rRNA locus was used to characterize the microbiomes of three sample types collected from wildlife: blood, ticks and tissue samples. Further sequence information was obtained for selected taxa of interest. Six tick species were identified from wildlife: Amblyomma triguttatum, Ixodes antechini, Ixodes australiensis, Ixodes holocyclus, Ixodes tasmani and Ixodes trichosuri. Bacterial 16S rRNA metabarcoding was performed on 536 samples and 65 controls, generating over 100 million sequences. Alpha diversity was significantly different between the three sample types, with tissue samples displaying the highest alpha diversity (P<0.001). Proteobacteria was the most abundant taxon identified across all sample types (37.3 %). Beta diversity analysis and ordination revealed little overlap between the three sample types (P<0.001). Taxa of interest included Anaplasmataceae, Bartonella, Borrelia, Coxiellaceae, Francisella, Midichloria, Mycoplasma and Rickettsia. Anaplasmataceae bacteria were detected in 17.7% (95/536) of samples and included Anaplasma, Ehrlichia and Neoehrlichia species. In samples from NSW, 'Ca. Neoehrlichia australis', 'Ca. Neoehrlichia arcana', Neoehrlichia sp. and Ehrlichia sp. were identified. A putative novel Ehrlichia sp. was identified from WA and Anaplasma platys was identified from QLD. Nine rodent tissue samples were positive for a novel Borrelia sp. that formed a phylogenetically distinct clade separate from the Lyme Borrelia and relapsing fever groups. This novel clade included recently identified rodent-associated Borrelia genotypes, which were described from Spain and North America. Bartonella was identified in 12.9% (69/536) of samples. Over half of these positive samples were obtained from black rats (Rattus rattus), and the dominant bacterial species identified were Bartonella coopersplainsensis and Bartonella queenslandensis. The results from the present study show the value of using unbiased high-throughput sequencing applied to samples collected from wildlife. In addition to understanding the sylvatic cycle of known vector-associated pathogens, surveillance work is important to ensure preparedness for potential zoonotic spillover events.

Haemoprotozoan surveillance in peri-urban native and introduced wildlife from Australia.

Vector-borne haemoprotozoans comprise a diverse group of eukaryote single-celled organisms transmitted by haematophagous (blood-feeding) invertebrates. They can cause debilitating diseases that impact wildlife, livestock, companion animals and humans. Recent research has shown that Australian wildlife host a diverse range of haemoprotozoan species; however, to date this work has primarily been confined to a few host species or isolated populations in rural habitats. There has been little investigation into the presence of these blood parasites in wildlife inhabiting urban and peri-urban areas. In this study, blood and tissue samples and ticks were collected from wildlife in New South Wales and Western Australia. Extracted DNA samples were screened with pan-specific molecular assays to determine the presence of haemoprotozoans using amplicon metabarcoding and Sanger sequencing approaches. In addition, light microscopy was performed on blood films. Eight haemoprotozoans were identified in the present study, which included species of Babesia, Hepatozoon, Theileria and Trypanosoma. Blood samples were collected from 134 animals; 70 black rats (Rattus), 18 common brush-tailed possums (Trichosurus vulpecula), two bush rats (Rattus fuscipes), 22 chuditch (Dasyurus geoffroii), 20 long-nosed bandicoots (Perameles nasuta), one quenda (Isoodon fusciventer) and one swamp rat (Rattus lutreolus). Molecular screening of DNA extracted from blood samples identified 52.2% (95% CI: 43.8-60.5%) of individuals were positive for at least one haemoprotozoan species, with 19.4% (95% CI: 13.4-26.7%) positive for more than one species. The present study provides the first sequences of Theileria cf. peramelis from black rats and long-nosed bandicoots. Babesia lohae was identified from brush-tailed possums. Two Hepatozoon genotypes were identified from black rats and bush rats. Black rats showed the highest haemoprotozoan diversity, with five species identified. No known human pathogens that have been described in the northern hemisphere were identified in the present study, and future work is required to understand the zoonotic potential of these microbes in Australia. This work represents the first large-scale body of research using molecular tools to investigate haemoprotozoans in animals at the urban-wildland interface. Further research is needed to investigate potential consequences of infection in wildlife, particularly effects of pathogen spillover from invasive black rats to native wildlife.

Molecular identification of the Trypanosoma (Herpetosoma) lewisi clade in black rats (Rattus rattus) from Australia.

Invasive rodent species are known hosts for a diverse range of infectious microorganisms and have long been associated with the spread of disease globally. The present study describes molecular evidence for the presence of a Trypanosoma sp. from black rats (Rattus rattus) in northern Sydney, Australia. Sequences of the 18S ribosomal RNA (rRNA) locus were obtained in two out of eleven (18%) blood samples with subsequent phylogenetic analysis confirming the identity within the Trypanosoma lewisi clade.

Sequence analyses at mitochondrial and nuclear loci reveal a novel Theileria sp. and aid in the phylogenetic resolution of piroplasms from Australian marsupials and ticks.

The order Piroplasmida encompasses two main families: Babesiidae and Theileriidae, containing tick-borne pathogens of veterinary and medical importance worldwide. While only three genera (Babesia, Cytauxzoon and Theileria) comprising piroplasm parasites are currently recognised, phylogenetic studies at the 18S rRNA (18S) gene suggest that these organisms represent at least ten lineages, one of which comprises the relatively unique and highly diverse Theileria spp. from Australian marsupials and ticks. As an alternative to analysing 18S sequences alone, sequencing of mitochondrial genes has proven to be useful for the elucidation of evolutionary relationships amongst some groups of piroplasms. This research aimed to characterise piroplasms from Australian native mammals and ticks using multiple genetic markers (18S, cytochrome c, oxidase subunit III (cox3) and cytochrome B (cytB)) and microscopy. For this, nearly complete piroplasm-18S sequences were obtained from 32 animals belonging to six marsupial species: eastern bettong (Bettongia gaimardi), eastern quoll (Dasyurus viverrinus), eastern grey kangaroo (Macropus giganteus), swamp wallaby (Wallabia bicolor), quokka (Setonix brachyurus) and Gilbert's potoroo (Potorous gilbertii). The organisms detected represented eight novel Theileria genotypes, which formed five sub-clades within the main marsupial clade containing previously reported Australian marsupial and tick-derived Theileria spp. A selection of both novel and previously described Australian piroplasms at the 18S were also successfully characterised, for the first time, at the cox3 and cytB loci, and corroborated the position of Australian native theilerias in a separate, well-supported clade. Analyses of the cox3 and cytB genes also aided in the taxonomic resolution within the clade of Australian Piroplasmida. Importantly, microscopy and molecular analysis at multiple loci led to the discovery of a unique piroplasm species that clustered with the Australian marsupial theilerias, for which we propose the name Theileria lupei n. sp.

Long-term efficacy of an imidacloprid 10 % / flumethrin 4.5 % polymer matrix collar (Seresto®, Bayer) against the Australian paralysis tick (Ixodes holocyclus) in dogs.

Two placebo-controlled pen studies were conducted to assess the efficacy of an imidacloprid 10 %/flumethrin 4.5 % polymer matrix collar (Seresto®, Bayer; Investigational Veterinary Product (IVP)) against the Australian paralysis tick (Ixodes holocyclus). Dogs assigned to the placebo (n = 8) or IVP (n ≥ 8) groups had collars (placebo or IVP) attached on Day 0 and were infested with 30 unfed, adult, female I. holocyclus at 14­28 day intervals over 227 days. Ticks were counted 24, 48 and 72 h post infestation to determine the acaricidal efficacy of the IVP. The acaricidal efficacy of the IVP 72 h post infestation exceeded 95 % on Days 17 (99.3 %), 59 (99.7 %), 73 (96.6 %), 87 (100.0 %), 101 (96.4 %), 115 (99.1 %) and 171 (95.8 %), but dropped on Days 45 (94.0 %) and 143 (77.8 %), and declined from Day 199 (79.9 %) to 227 (65.5 %). No adverse events related to treatment were observed. This study has demonstrated the excellent acaricidal efficacy (97.9 %) of the IVP collar against I. holocyclus 72 h post infestation over 16 weeks.

The effects of formulation on the penetration and retention of budesonide in canine skin in vitro.

This study investigated the effects of formulation on the penetration and retention kinetics of budesonide through canine skin in vitro. Full thickness, thoracic, dog skin was mounted in Franz-type diffusion cells and randomly assigned to receive one of three 0.025% (0.25mg/mL) budesonide-containing formulations: Barazone (BZ, a novel formulation), isopropyl myristate (IPM) or propylene glycol (PG). At regular intervals over 84h, the amount of budesonide penetrating or retained within the skin was quantified using high performance liquid chromatography. The restricted (or residual) maximum likelihood mixed model predicted that the flux of budesonide from BZ was 9.2-fold (P<0.001) and 105-fold (P<0.001) greater than from IPM and PG, respectively. Similarly, the skin retention of budesonide from BZ was more than 3-fold (P<0.0001) and nearly 6-fold (P<0.0001) greater than from IPM and PG, respectively. This study has demonstrated that the formulation can greatly affect the skin penetration and retention of budesonide in dogs, and consequently could be selected to maximise drug concentration and retention at the site of action. This has the potential to improve the efficacy and safety of, and owner compliance with, topical glucocorticoid therapy in dogs.

The effects of skin disease on the penetration kinetics of hydrocortisone through canine skin in vitro.

This study investigated the effects of allergic skin disease on the penetration kinetics of hydrocortisone through canine skin in vitro. Full-thickness lesional and nonlesional (normal) skin was removed from the dorsal lumbosacral and dorsocaudal thoracic regions, respectively, of five canine cadavers. The dogs were suspected of having flea allergy dermatitis based on their distribution and types of skin lesions. Nonlesional skin was confirmed to be histologically normal, and the histopathology of the lesional skin was consistent with allergic dermatitis. Excised skin was clipped, mounted in Franz-type diffusion cells, and the transdermal penetration of a saturated, radiolabelled hydrocortisone solution was measured over 30 h. When the penetration data for all five dogs were pooled, a restricted (or residual) maximal likelihood mixed model predicted that the permeability coefficient and pseudosteady-state flux of hydrocortisone was more than twice as great (95% confidence interval 1.55-2.71 times as great; P < 0.0001) through lesional compared with nonlesional skin. There was no significant difference in the lag time for hydrocortisone penetration through lesional compared with nonlesional skin of the dogs. This study has confirmed that the transdermal penetration of hydrocortisone may be altered, typically increased twofold, but could be as high as 10-fold, through lesional compared with nonlesional skin of dogs with suspected flea allergy dermatitis. This is likely to be affected by variables such as disease severity, concurrent infections and interindividual differences in skin characteristics.

The effects of surface preparation on the penetration of hydrocortisone through canine skin.

This study investigated the effects of common skin surface preparations on the penetration kinetics of hydrocortisone through canine skin. Thoracic skin from five dogs was clipped of hair, divided between five treatment groups and prepared as follows: shaved (S); tape-stripped with adhesive bandage (TS); cleaned with aqueous chlorhexidine (Aq-C); cleaned with alcoholic chlorhexidine (Al-C); or allocated to the control group and had no further preparation performed (C). The skin samples were mounted in Franz-type diffusion cells and transdermal hydrocortisone penetration was measured over 30h. The pseudo-steady-state flux (J(SS)) of hydrocortisone through S, Al-C, Aq-C and TS skin was, respectively, 2.3 (P=0.021), 2.2 (P=0.037), 2.0 (P=0.070) and 1.5 (P=0.351) times greater than through the control skin, but there were no significant differences in the lag times (t(lag)) for hydrocortisone penetration between the groups. The study has shown that some skin surface preparations can significantly increase the subsequent penetration of hydrocortisone through canine skin in vitro.