Detection of tick-borne bacterial DNA (Rickettsia sp.) in reptile ticks Amblyomma moreliae from New South Wales, Australia

Ticks are major arthropod vectors of disease, transmitting tick-borne pathogens during blood meal episodes. Rickettsia spp. and Borrelia spp. are two tick-borne pathogens of zoonotic concern previously identified in DNA isolates from the tick genera Amblyomma and Bothriocroton associated with reptilian hosts in Australia. Some reports suggest that these reptile ticks bite and attach to humans via accidental parasitism and transmit disease, with the tick Bothriocroton hydrosauri known to transmit Rickettsia honei or Flinders Island Spotted Fever Rickettsia to humans. This descriptive study aims to identify the ticks collected from wild reptiles submitted to veterinary clinics and captured by snake rescuers from New South Wales (NSW), Australia, and detect the presence of tick-borne bacterial DNA using quantitative polymerase chain reaction (qPCR) to detect Rickettsia spp. and Bartonella spp. and conventional nested-PCR to detect Borrelia spp. Morphological identification revealed ticks removed from one eastern blue-tongued lizard (Tiliqua scincoides scincoides) from North-Eastern NSW (Lismore), one eastern blue-tongued lizard from the Greater Sydney area (Canley Heights), one diamond python (Morelia spilota spilota) from the Greater Sydney area (Woronora Heights) and one red-bellied black snake (Pseudechis porphyriacus) from the Greater Sydney Area (Cronulla) in New South Wales were Amblyomma moreliae. No ticks were positive for Bartonella spp. and Borrelia spp. DNA using real-time PCR targeting ssrA gene and nested PCR targeting Borrelia-specific 16S rRNA gene, respectively. Real-time PCR targeting gltA, ompA, ompB and 17kDa gene of Rickettsia spp. revealed 14 out of 16 ticks were positive. The undescribed Rickettsia sp. DNA was identical to that previously recovered from reptile ticks in Australia and closely related to Rickettsia tamurae and Rickettsia monacensis, both of which are aetiologic pathogens of the Spotted Fever Group Rickettsiosis (SFGR). These results accentuate the ongoing need for increased study efforts to understand zoonotic potential of bacteria from reptile ticks and the tick-reptile-human relationship. Supplementary Information The online version contains supplementary material available at 10.1007/s00436-023-08108-7.


Introduction
Ticks are major arthropod vectors of disease, with various vertebral hosts including humans.With the increasing global incidence of tick-borne disease, there is an emerging need to understand the tick-host relationship to mitigate the risks of such diseases impacting communities (Dantas-Torres et al. 2012).Rickettsioses and borrelioses are tick-borne diseases caused by obligate intracellular bacteria of the genus Rickettsia, and spirochaete bacteria of the genus Borrelia, respectively (Ostfeld and Keesing 2000;Whiley et al. 2016).Such tick-borne diseases include the Spotted Fever Group Rickettsiosis (SFGR) and Lyme Borreliosis (LB) which threaten the health and well-being of humans (Ostfeld and Keesing 2000;Whiley et al. 2016).In Australia, the main aetiological species of SFGR are Rickettsia australis and Rickettsia honei 89 Page 2 of 7 and there is no convincing evidence that Lyme disease nor the causative species Borrelia burgdorferi occurs in Australia (Collignon et al. 2016;Graves et al. 2006).
Some Australian reports demonstrate that there are Amblyomma and Bothriocroton species associated with reptile hosts which bite and attach on to humans, although such cases remain rare and noteworthy (Egan et al. 2022;Roberts 1970).Patients reported clinical presentations such as headaches, local cutaneous inflammation, tenderness of superficial inguinal lymph nodes, but no evidence of prolonged symptoms (Egan et al. 2022;Norval et al. 2020).Although further studies to screen the patient and the tick for the presence of tick-borne pathogens were not pursued, it is evident through these case reports that reptile ticks have the potential to parasitise humans.
The aim of this study was to detect the presence reptileborne tick pathogen DNA in a small collection of ticks from reptiles from NSW, Australia.The study utilised quantitative PCR (qPCR) and nested PCR diagnostic assays to detect Rickettsia spp., Bartonella spp.and Borrelia spp. in ticks collected from wild reptiles submitted to veterinary clinics and captured by snake rescuers.

Materials and methods
Between January 2022 to June 2022, reptile ticks (n = 16) acquired from reptiles (n=4) found across NSW, Australia were donated to us the University of Sydney by veterinary hospitals and snake catchers (Supplementary Table 1, Table 1).The ticks were stored in 70% (w/v) ethanol.All ticks were examined under stereomicroscope (5-200X Olympus, Macquarie Park, Australia) and digital microscope (Keyence, United States of America) for identification using published dichotomous keys and descriptions (Roberts 1970).Total DNA was isolated as previously described and stored at -20 °C (Chandra et al. 2022).A selection of ticks was processed for molecular identification using conventional PCR, targeting a ~650-nt fragment of cytochrome c oxidase subunit 1 (cox1) as previously described using S0725 and S0726 primers (Panetta et al. 2017).PCR products were sequenced (Macrogen Ltd., Seoul, South Korea) and sequences were assembled and compared to related cox1 sequences using CLC Main Workbench 22 (CLC bio, Qiagen, Chadstone, Australia).In addition, we assembled full cox1 from a single RNAseq dataset (SRR8074777) generated by Harvey et al. (2019) from 20 adults of Amblyomma moreliae collected from Blue-tongue lizard in Sydney, NSW.
DNA from all sixteen ticks was screened using Rickettsia spp./ Bartonella spp.multiplex real-time PCR utilising Luna Universal Probe qPCR Master Mix (New England Biolabs, Victoria, Australia) targeting gltA citrate synthase gene (~75bp amplicon) for Rickettsia, and a ssrA transfer-messenger RNA (~300-bp amplicon) for Bartonella as previously adopted (Diaz et al. 2012;Šlapeta and Šlapeta 2016;Stenos et al. 2005).Each run included a positive control carrying Bartonella and Rickettsia target DNA insert, a negative control with sterile PCR-grade water to control for contamination during PCR, and an additional blank template (NTC) to control for contamination during DNA extraction.The arbitrary real-time PCR threshold was set to a single threshold at 100rfu.Results were considered positive if samples yielded a C t value <36 and negative if C t > 40, and any samples that yielded C t values ≥ 36 were suspect positive results (Huang et al. 2021).
Detection of Borrelia spp.spirochetes were performed using a 16S rRNA gene nested-PCR by amplifying a ~1250nt fragment using previously published Borrelia-specific 16S rRNA primers as previously adopted (Loh et al. 2016).Each run included a blank negative control filled with sterile PCR-grade water.PCR was run on T100 Thermal Cycler (BioRad), as previously described (Panetta et al. 2017).
The Rickettsia spp.and Bartonella spp.multiplex realtime PCR assay revealed the presence of Rickettsia spp.DNA in fourteen out of sixteen ticks with an average C t value of 25.3 (min. 24.4, max. 35.5).Fourteen ticks DNA samples that were Rickettsia spp.DNA positive using the real-time PCR assay were characterised by multilocus typing using nested PCR at four Rickettsia spp.DNA loci.Seven tick samples yielded identical ~650-nt Rickettsia gltA sequences (OR537304-10), and multiple sequence alignment with phylogenetic analysis of gltA sequences from representative Rickettsia spp.revealed a high identity (100%) to six sequences of R. cf.tamurae from Bothriocroton undatum (Fabricius, 1775) (MG004673-78) from New South Wales, Australia (Panetta et al. 2017).Nine samples yielded identical ~488-nt Rickettsia ompA amplification products (OR537324-32) which revealed high identity (99.6%, 2/488) to a sequence from R. sp.801a from Amblyomma fimbriatum Koch, 1844 (EU283837) from the Northern Territory, Australia (Vilcins et al. 2009).Eleven ticks yielded identical ~379-nt ompB products (OR537333-43) with 96% similarity to a sequence from Rickettsia sp. from Ixodes boliviensis Neumann, 1904 (MW699710).Thirteen ticks yielded identical ~410-nt 17kDa amplification products (OR537311-23) with high identity (100%) to a sequence from Rickettsia sp.777c from A. fimbriatum (EU283838) from the Northern Territory, Australia (Vilcins et al. 2009).At least one tick from each of the four host reptiles were positive for Rickettsia spp.Sequence identity and phylogenetic position suggest that this is a novel species within the same clade as Rickettsia tamurae and Rickettsia monacensis (Fig. 2).
No ticks were positive for Bartonella spp.DNA (C t value > 40) using the Rickettsia spp.and Bartonella spp.multiplex real-time PCR assay.DNA amplification of 16S rRNA using conventional nested PCR revealed no ticks positive for Borrelia DNA.

Discussion
Ticks are amongst the most significant arthropod vectors of zoonotic disease, with a diverse pool of documented reptilian hosts.In Australia, Amblyomma and Bothriocroton are recognised as the main genera of ticks with reptiles documented as the main hosts for some of their species (Barker and Barker 2023).The identification of tick species is a fundamental task in the epidemiological studies of tick-born disease.
Here, the morphological identification enabled us to develop a molecular marker for future genetic identification and comparison to other related species (Mediannikov and Fenollar 2014).Key features which distinguish A. moreliae from other Australian reptile tick species, previously ascribed to the genera Amblyomma and Aponomma, are well-described in published dichotomous keys and descriptions (Roberts 1970).However, the generic taxonomy used by Roberts has subsequently been revised, with some species of Aponomma transferred to the new genus Bothriocroton Keirans, King & Sharrad, 1994, and the remaining Aponomma species, including the type species, transferred to Amblyomma, and thus the genus name Aponomma is no longer used (Barker and Murrell 2004;Klompen et al. 2002).
To the best of our knowledge, this study is the first to sequence A. moreliae.Currently, only A. moreliae transcriptomes (SRR8074777) are published and available (Harvey et al. 2019;Uribe et al. 2020).Whilst the purpose of the phylogenetic tree is to demonstrate the relationship between our Australian A. moreliae with other Amblyomma species, it only incorporates 1 of 2 Australasian Amblyomma species -A.fimbriatum -and none of the 11 other endemic Australian Amblyomma species (Barker and Barker 2023) (Fig. 1).Therefore, with the lack of endemic Australian Amblyomma representation in the phylogenetic tree, the relationship between A. moreliae, A. dissimile, A. testudinarium and A. geoemydae cannot be conclusive.
One notable diseases transmitted by ticks is SFGR, a zoonotic disease with a wide spectrum of severity ranging from fever, headache, eschar, rash, nausea, vomiting, to death even in young, healthy people (Mahajan 2012).The spotted fever rickettsial pathogens previously reported from Australia are Rickettsia australis, Rickettsia honei and Rickettsia honei subspecies marmionii (Graves et al. 2006;Stenos et al. 2003;Unsworth et al. 2007).In Australia, R. honei is the rickettsial species associated with both SFGR and reptile ticks but emerging studies are beginning to find evidence of novel rickettsial species closely related to R. tamurae (Panetta et al. 2017;Vilcins et al. 2009;Whiley et al. 2016).Similar to these studies, trees derived from analysis of the sequences of all four target genes (gltA,ompA,ompB,17kDa) show that the novel rickettsial DNA sequenced from our ticks lies closely within the same clade as R. tamurae and R. monacensis, which are part of the SFGR documented to cause clinical rickettsial disease in East Asian and European countries, respectively (Chao et al. 2017;Dobler et al. 2009;Kim et al. 2010).
Although the outcome of this study is preliminary, the presence of rickettisal DNA closely related to SFGR pathogens adds to the growing number of species being found in Australia.This Rickettisal DNA appears to be indiscriminate against the reptilian host species involved in this study, thus warranting its potential to harbour in various reptiles.This warrants further efforts to characterise these rickettsiae and assess disease prevalence in a larger scale in Australian ticks and their hosts (Vilcins et al. 2009).Novel reptile-associated Borrelia spp.identified in B. undatum, Amblyomma calabyi Roberts, 1963, A. fimbriatum, and nymph Amblyomma limbatum Neumann, 1899 in Australia represents a distinct Borrelia clade which is evolutionarily, ecologically, and genetically unique to Lyme borreliosis and relapsing fever Borrelia groups (Gofton et al. 2023).The lack of Borrelia positive ticks in this study along with the lacking evidence of Lyme borreliosis and relapsing fever Borrelia groups and the existence of their ticks in Australia suggests that it is unlikely that Australian A. moreliae is an unlikely vector for Borrelia-related disease (Collignon et al. 2016;Jakab et al. 2022).
Using multilocus genotyping, we confirm the existence of an unnamed Australian species of Rickettsia which has previously been reported from the reptile ticks A. fimbriatum in the Northern Territory, in all stages of reptile ticks Bothriocroton hydrosauri (Denny, 1843) recovered from 89 Page 6 of 7 Tiliqua rugosa in South Australia, as well as B. undatum recovered from Varanus varius in New South Wales (Panetta et al. 2017;Vilcins et al. 2009;Whiley et al. 2016).We consider our recovered species a novel species that was previously labelled as Rickettsia cf.tamurae (MG004673 to MG004678) or Rickettsia sp.777c from A. fimbriatum (EU283838) (Panetta et al. 2017;Vilcins et al. 2009).It will be important to establish an in vitro culture of this rickettsial bacterium before its formal description.Isolation and genomic characterisation will be required to confirm the species status of this novel yet widely distributed Rickettsia sp.Australia.
Our tick-A.moreliae-is an endemic Australian reptile tick species widespread throughout eastern states, NSW, Queensland, and Victoria, that is only occasionally reported from mammals (Barker and Barker 2023;Roberts 1964;Roberts 1970;Shea 1983).Its potential to transmit zoonotic tick-borne diseases is unknown, but evidence of it harbouring Rickettsia with high phylogenetic identity to species with known zoonotic potential and its presence in various reptilian species warrants further investigation to better understand the risks posed by reptilian ticks in Australia.

Conclusion
In this study, utilised samples from reptiles in the Sydney area, were dominated by A. moreliae.Ticks in the sample tested positive for rickettsial DNA but not for Bartonella spp.and Borrelia spp.DNA.We confirm the existence of an unnamed Australian species of Rickettsia which has previously been originally reported from the reptile ticks in Australia.Future study efforts to identify the pathogenicity of this rickettsial pathogen can complement this study.

Fig. 1
Fig.1The evolutionary history of Amblyomma cox1 gene sequences.The tree was inferred using the Minimum Evolution (ME) method, with the evolutionary distances computed using the Kimura 2-parameter method conducted in MEGA11.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches (>50%).All posi-

Fig. 2
Fig.2The evolutionary history of Rickettsia gltA, ompA, ompB and 17kDa gene sequences.The trees were inferred using the Minimum Evolution (ME) method, with the evolutionary distances computed using the Kimura 2-parameter method conducted in MEGA11.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to

Table 1
Amblyomma moreliae collected from reptiles in New South Wales, Australia