01-literature-review.Rmd

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  @guglielmoneIxodidaeAcariIxodoidea2020, @lohIdentificationTheileriaFuliginosalike2018, @cooperDetectionCoxiellaBurnetii2013, @greayIlluminatingBacterialMicrobiome2021, @brodyCaseTickTyphus1946, @popeIsolationRickettsiaResembling1955, @owenDetectionIdentificationNovel2006, @owenPotentiallyPathogenicSpotted2006, @sentausaGenomeSequenceRickettsia2013, @liHighPrevalenceRickettsia2010, @abdadRickettsiaGravesiiSp2017, @owenDetectionCharacterisationRickettsiae2007; @bennettCoxiellaBurnetiiWestern2011, @jonssonProductivityHealthEffects2008, @angusHistoryCattleTick1996
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# Literature Review{#litrev}
\chaptermark{Review}
\minitoc <!-- this will include a mini table of contents-->

## Preface {-}

\nocite{guglielmoneIxodidaeAcariIxodoidea2020}
\nocite{lohIdentificationTheileriaFuliginosalike2018}
\nocite{cooperDetectionCoxiellaBurnetii2013} \nocite{greayIlluminatingBacterialMicrobiome2021} \nocite{brodyCaseTickTyphus1946} \nocite{popeIsolationRickettsiaResembling1955} \nocite{owenDetectionIdentificationNovel2006} \nocite{owenPotentiallyPathogenicSpotted2006} \nocite{sentausaGenomeSequenceRickettsia2013} \nocite{liHighPrevalenceRickettsia2010} \nocite{abdadRickettsiaGravesiiSp2017} \nocite{owenDetectionCharacterisationRickettsiae2007} \nocite{bennettCoxiellaBurnetiiWestern2011}
\nocite{jefferiesTwoSpeciesCanine2003}
\nocite{jonssonProductivityHealthEffects2008}
\nocite{angusHistoryCattleTick1996}

Ticks (Acari: Ixodida) are a group of blood-feeding arthropods and represent one of the most important vector groups that affect human and animal health. Almost 900 species of tick have been described globally, and 10% have been documented to transmit a variety of pathogens to livestock, companion animals and humans [@jongejanGlobalImportanceTicks2004]. 
Ticks can carry a diverse range of infectious microbes such as viruses, piroplasms, spirochaetes and rickettsiales [@pfaffleEcologyTickborneDiseases2013]. 
Tick-borne diseases globally include Lyme borreliosis (*Borrelia burgdorferi* sensu lato), ehrlichiosis (*Ehrlichia chaffeensis)*, babesiosis (*Babesia* spp.), tick-borne encephalitis (Flavivirus) and Powassan disease (Powassan virus). 
A tick bite can cause local reactions, such as irritation and erythema migrans, and in some cases can induce severe systemic syndromes such as paralysis, anaphylaxis and mammalian meat allergy [@vannunenTickinducedAllergiesMammalian2015;@beamanNoninfectiousIllnessTick2018;@pienaarTickParalysisSolving2018]. 

Presently, there are 74 extant tick species in Australia that have been described  [@barkerList70Species2014;@ashMorphologicalMolecularDescription2017;@heathNewSpeciesTick2017;@kwakIxodesHeathiSp2018;@barkerIxodesBarkeriSp2019], with eight well known to bite humans [@barkerTicksAustraliaSpecies2014].  
To date only three human tick-borne pathogens (TBPs) are recognised as endemic in Australia [@gravesTickborneInfectiousDiseases2017]; *Rickettsia australis* and *Rickettsia honei* are the causative agents for Queensland tick typhus and Australian (Flinders Island) spotted fever, respectively.
The third TBP in Australia is *Coxiella burnetii* the causative agent of Q Fever, and while it is not usually associated with a tick-bite, ticks play a critical role in the life cycle of this bacterium. 
Despite endemic rickettsial infections being recognised in Australia, there has been an increasing pressure surrounding the presence of an unknown TBP causing illness in people bitten by Australian ticks. 
In particular, public concern around a ‘Lyme-like’ illnesses has gained considerable attention and a commonwealth senate inquiry was held in 2016 [@radcliffeGrowingEvidenceEmerging2016]. 

This literature review will provide a brief overview of tick systematics and tick biology. 
It will then explore some examples of tick-borne diseases recognised globally and reflect on the situation in Australia. 
In particular, it will review some of the recent discoveries over the past decade that have been made on the identification of novel microbes.
The review will then explore the role of hosts in the maintenance of ticks and tick-borne pathogens. 
This is given to lead to the idea of a One Health approach and the advantages of using a wildlife surveillance approach to understanding the dynamics of tick-borne diseases in Australia.
The review will then conclude with a brief insight into the technologies used to identify tick-borne microbes with a focus on new molecular approaches.
The overarching themes of this literature review include; ticks, zoonotic tick-borne pathogens, ecology of tick-borne pathogens, and use of new molecular tools and wildlife surveillance to investigate the potential the potential for a novel zoonotic tick-borne pathogen in Australia.

At the end of this literature review on outline of the thesis aims \@ref(thesis-aims) is provided. 

## Ticks 

### Tick systematics

Ticks belong to order Ixodida, which is split into three extant families; Ixodidae (hard ticks), Argasidae (soft ticks) and a single species belonging to the Nuttalliellidae family [@durdenModernTickSystematics2014].
The fourth family, Deinocrotonidae, is an extinct taxa that was described after discovery of the newly named species *Deinocroton draculi* in 99 million-year-old Cretaceous amber [@penalverTicksParasitisedFeathered2017].
The Ixodidae, or hard tick family, has at least 742 described species [@guglielmoneIxodidaeAcariIxodoidea2020] and represents the majority of species that are of veterinary and medical significance.
Ixodidae are characterised by the presence of a hard plate (scutum/conscutum) on the dorsal aspect and terminal mouth parts. 
In contrast Argasidae lack a hard plate (no scutum/conscutum) and have ventral mouth parts.
The Ixodidae family can further be divided into two clades.
The prostriata, which are characterised by the anal groove anterior to the anus and consists of a single genus, *Ixodes*.
The remainding Ixodidae genera belong to the metastriata clade which the anal groove (if present) posterior to the anus. 
The number of Argasidae (soft) ticks species has grown in recent years and there are over 200 species described [@mansArgasidIxodidSystematics2019]. 
Many of these species are cryptic and the full diversity of this family of ticks remains to be full described.

While the family level classification of ticks has remain stable for many years the phylogeny and systematics within these remains a point of contention; particularly for soft ticks [@mansArgasidIxodidSystematics2019]. 
Molecular tools have helped shed light on the evolutionary history of ticks and as such many changes to tick taxonomy have been made over the years. 
The current working hypothesis of tick phylogeny highlights the uniqueness of tick fauna in the Australasian region. 
Molecular and morphological analysis suggests *Ixodes* endemic to Australia, New Guinea and New Zealand belong to a unique *Ixodes* lineage with at least 28 extant species [@barkerSystematicsEvolutionTicks2004]. 
Although not all of these species have undergone molecular systematic analysis, there is a clear grouping of this distinct lineage (Figure \@ref(fig:F1treeixod)). 
The previously recognised genus *Aponomma* belonged to the hard tick family, Ixodidae, and was poorly defined.
To correct this a new genus was created called *Bothriocroton* and all indigenous Australian *Aponomma* species were moved to this new genus [@keiransAponommaBothriocrotonGlebopalma1994;@klompenNewSubfamilyBothriocrotoninae2002].
Since the creation of the genus *Bothriocroton* new species have been described and reinstated, bringing the total extant species to seven, all of which are endemic to the Australiasian region (Figure \@ref(fig:F1treeixod)) (*Bothriocroton tachyglossi*, *Bothriocroton auruginans*, *Bothriocroton concolor*, *Bothriocroton hydrosauri*, *Bothriocroton undatum*, *Bothriocroton glebopalma*, and *Bothriocroton oudemansi*) [@keiransAponommaBothriocrotonGlebopalma1994;@klompenNewSubfamilyBothriocrotoninae2002;@andrewsSystematicStatusAponomma2006;@beatiBothriocrotonOudemansiNeumann2008]. 
The remainding members of the former *Aponomma* genus were transferred to *Amblyomma*, however some issues of polyphyly within the genus *Amblyomma* remained [@burgerPhylogeneticAnalysisTicks2012;@burgerPhylogeneticAnalysisMitochondrial2013]. 
To correct this issue two new genera have since been established, *Robertsicus* and *Archaeocroton* [@barkerTwoNewGenera2018].

```{r F1treeixod, out.width='95%', out.align = 'left',fig.scap = "Phylogeny of hard tick genera (Ixodidae)", fig.cap = "Phylogeny of extant genera within the hard tick family (Ixodidae).", echo=FALSE}
knitr::include_graphics("figures/ms-figs/Ch1-treeixod.pdf")
```

To date Australia has 74 extant described tick species (Figure \@ref(fig:F1ausixod)). 
Five of these were introduced with domestic animals following European arrival in 1788: *Argas persicus*, *Otobius megnini*, *Haemaphysalis longicornis*, *Rhipicephalus sanguineus* (recently proposed as *Rhipicephalus linnaei*), and *Rhipicephalus australis* (*Boophilus microplus)* [@barkerTicksAustraliaSpecies2014]. 
The first new native Australian *Ixodes* tick was described in over 50 years, from woylies (*Bettongia penicillata*), a critically endangered marsupial in south-west Western Australia and was named *Ixodes woyliei* [@ashMorphologicalMolecularDescription2017]. 
The text ‘Australian Ticks’ by Roberts [-@robertsAustralianTicks1970] remains the foundation reference guide on ticks in Australia, in particular the detailed specimen descriptions and taxonomic keys used for morphological identification. 
The more recent guide, ‘Ticks of Australia’ by Barker and Walker [-@barkerTicksAustraliaSpecies2014], has provided a much needed update on the most common tick species that effect humans and animals in Australia.
As raised by Barker et al. [-@barkerList70Species2014] there are a number of issues surrounding the list of recognised ticks in Australia.
Since Barker et al. [-@barkerList70Species2014] there have also been a number of taxonomic changes and new information has been presented showing that the full picture of tick taxonomy in Australia is not yet complete.
In many cases host records of tick species in Australia are scarce and a large amount of time has passed since some records. 
This has resulted in a number of queries surrounding the complete list of tick species present in Australia. 
In some cases systematic classification remains elusive with biological data incomplete.
Issues relating to classification and/or presence of Australian ticks include: 
(i) *Amblyomma flavomaculatum* (yellow spotted monitor lizard tick) was listed in Roberts [-@robertsAustralianSpeciesAponomma1953;@robertsFurtherObservationsAustralian1964;@robertsAustralianTicks1970] and referred to as *Aponomma pulchrum*, which is now synonymous with *Am. flavomaculatum*. 
However, since these records by Roberts, there have been no positive identifications of this tick in Australia; 
(ii) the distinction between *Amblyomma australiense* and *Amblyomma echidnae* remains unclear [@guglielmoneCommentsControversialTick2009]. *Amblyomma australiense* was originally described from a museum specimens of the long-beaked echidna (*Zaglossus bruijnii*) in the Kimberly.
Roberts [-@robertsAustralianTicks1970] leaves open the possibility that *Am. echidnae* is a subspecies of *Am. australiense*. 
(iii) the status of *Bt. tachyglossi* was resurrected by Andrews [-@andrewsSystematicStatusAponomma2006], however prior to that it was considered a synonym of *Bt. hydrosauri*, therefore records prior to 2006 should be treated with caution;
(iv) records of *Ha. longicornis* were previously referred to as *Haemaphysalis bispinosa* [@robertsSystematicStudyAustralian1963], it is now recognised that *Ha. bispinosa* is a distinct species distributed across parts of Asia and early records were attributed.

Changes in tick nomenclature and taxonomy can cause disruption to ecologists, veterinarians and physicians.
In many instances changes in species names takes many years to filter through for use by the broader scientific community and for new names to be fully adopted.
For example, the reptile tick *Bt. hydrosauri* (formerly *Aponomma hydrosauri*) is an important vector of human disease (*R. honei*), and its changing species name can cause confusion to the public and medical professionals (e.g. @stenosAponommaHydrosauriReptileassociated2003). 
Additionally, the lack of accurate keys and morphological descriptions is particularly challenging in these instances. 
In the case of tick-borne diseases, accurate tick identification can greatly assist in a timely diagnosis. In areas where tick-borne disease knowledge is limited, such as Australia, accurate tick identification is of great benefit to researchers and clinicians attempting to untangle novel disease causes. 
In the case of *Ixodes holocyclus* and *Ixodes cornuatus*, only recently has sufficient keys and genetic data been published to allow the identification between these two morphologically very similar species [@songPhylogeneticPhylogeographicRelationships2011;@barkerTicksAustraliaSpecies2014;@kwakKeysMorphologicalIdentification2017]. 
*Ixodes holocyclus* remains the most important vector of illness to Australians being the cause of paralysis, mammalian meat allergy, significant local reactions and allergic responses and the vector of rickettsial diseases. 
There are many suspected misidentifications of *Ix. holocyclus*; for example Tasmanian devils (*Sarcophilus harrisii*) in Tasmania (TAS) [@vilcinsDetectionHepatozoonSpotted2009], likely *Ix. cornuatus*, and numbats (*Myrmecobius fasciatus*), in Western Australia (WA) [@calabyObservationsBandedAnteater1960], likely *Ixodes myrmecobii*.
More recently the Australian brown dog tick has been renamed to *Rh. linnaei* [@slapetaTropicalLineageBrown2021], previously known as *Rh. sanguineus* tropical lineage.
This name change is likely to create debate among taxonomist and the tick community, and therefore cause confusion for the general public. 
While taxonomy is the foundation of biological science and must be corrected were appropriate, it does cause unavoidable consequences in the short term. 

The unique Australian tick fauna is an important consideration in understanding the presence of tick-borne diseases, which are discussed in further detail in section three of this literature review. Tick-borne pathogens have coevolved in their vector host, and as such, the evolutionary history of Australian ticks means that it is likely that pathogens present elsewhere in the world would be different from those that have evolved with native Australian ticks.  

```{r F1ausixod, out.width='95%', out.align = 'left',fig.scap = "Tick species (Ixodida) of Australian.", fig.cap = "Ticks (Ixodida) present in Australian (74 extant species).", echo=FALSE}
knitr::include_graphics("figures/ms-figs/Ch1-ausixod.png")
```

### Tick biology and ecology

Ticks have a remarkably long life cycle compared to other vectors of infectious disease, such as mosquitoes (Culicidae) and lice (Phthiraptera). 
The life cycle of wild hard ticks is often measured in years and consists of four major stages; eggs, larva, nymph and adult. 
With the exemption of the egg stage, development requires a blood meal, which often involves several hours, or even days of engorgement [@cuppBiologyTicks1991]. 
The vast majority of hard ticks have a three-host life cycle, where each life stage feeds on a host and then detaches to spend some time in the environment before moulting to the next stage.
Adult females are able to detach and reattached multiple times to continue feeding. 
In the case of generalised tick species, current literature suggests that in most cases, immature life stages (e.g. larva and nymphs) feed on small to medium sized hosts and adult life stages feed on larger hosts [@apanaskevichLifeCyclesNatural2014].
Although this has been shown to occur in many cases such as *Ixodes ricinus* [@krasnovHostCommunityStructure2007], detailed studies on the life cycle and host dynamics of Australian ticks remains largely unknown. 

Host specificity is the association between a tick and vertebrate species that is critical for the ongoing survival and reproduction of the tick [@hoogstraalTickhostSpecificity1982]. 
There have been suggestions that up to 90% of tick species are considered 'host specific' [@hoogstraalTickhostSpecificity1982].
However, there has been suggestions that this finding is simply due to the incomplete sampling and reporting of tick-host records [@klompenEvolutionTicks1996]. 
In Australia, the primary reference for tick-host associations remains Roberts [-@robertsAustralianTicks1970], although Barker and Walker [-@barkerTicksAustraliaSpecies2014] has provided an updated reference for 16 species of veterinary and medical importance. 
Despite this update however, there is no single, up-to-date reference of tick-host associations. 
In many cases these findings remain buried in research laboratories, special interest groups and other niches. 
Since Roberts [-@robertsAustralianTicks1970] publication detailed studies on the biology, ecology and distribution of native Australian ticks have only been carried out on a handful of species most notably; *Ornithodoros gurenyi* (QLD, NT, SA, WA) [@doubeEcologyKangarooTick1972;@doubeTwoRacesKangaroo1975], *Amblyomma triguttatum* (SA) [@waudbySeasonalDensityFluctuations2007]; *Bt. hydrosauri* [@bullDispersalAustralianReptile1978;@andrewsMatingBehaviourAustralian1980;@belanHostDetectionFour1991;@chiltonInterspecificDifferencesMicrohabitat1993], *Ix. cornuatus* (VIC, TAS) [@jacksonGeneticVariationTicks2000;@jacksonDistributionsParalysisTicks2007;@songPhylogeneticPhylogeographicRelationships2011], *Ix. holocyclus* (QLD, NSW, VIC) [@doubeSeasonalPatternsAbundance1979;@jacksonGeneticVariationTicks2000;@jacksonDistributionsParalysisTicks2007;@songPhylogeneticPhylogeographicRelationships2011], *Ixodes tasmani* (NSW) [@murdochEcologyCommonMarsupial2005], *Haemaphysalis bancrofti* (VIC) [@laanOccuranceTickHaemaphysalis2011]. 
While these studies provide a solid foundation of Australian tick biology, in many cases they are largely limited to a relatively small study site. 
An experimental investigation of *Ixodes hirsti* placed 1600 larvae on rats and chickens, however they found that no larvae attached to either host species, and none survived more than 24 hours [@laanObservationsBiologyDistribution2011]. 
The authors described hosts removing the larvae by either eating or grooming.
The conditions of the experimental animals are not described in detail, however this perhaps suggests a host specificity of *Ixodes hirsti*, although the tick-host associations reviewed in the same paper show a wide range of taxa including kangaroos (*Macropus* spp.), Honeyeaters (*Phylidonyris* spp.), rats (*Rattus* spp.), and [dogs (*Canis lupus familiaris*)].{.correction} [@laanObservationsBiologyDistribution2011]. 
The life cycle of *Ix. tasmani* was shown to complete within 4 months in artificial laboratory models on rats [@murdochEcologyCommonMarsupial2005], which is comparatively short compared to other ixodids [@oliverBiologySystematicsTicks1989]. 
The nocturnal and nidicolous questing nature of *Ix. tasmani* coincides with the activity of many of its host marsupial species [@murdochEcologyCommonMarsupial2005].

## Tick-borne diseases in humans

This section of the review will briefly discuss some of the major human tick-borne diseases recognised globally, with an emphasis on bacterial pathogens. 
The summary provided is by no means exhaustive, it is given in the context to explain the types of microbes that have been associated with disease in humans.
The review will then reflect on human tick-borne pathogens currently documented and characterised in Australia.
Finally it will conclude by providing a summary of recent research that has been done in relation to novel microbes in Australian ticks and how they relate to potential causative agents of human disease.

This review is confined to infectious microorganisms transmitted by ticks. An in-depth and exhaustive review of each individual microbe is beyond the scope of this literature review. Instead the following is presented to provide context to microbes which are explored in more detail throughout the thesis. 
In the first instance, a general introduction of tick-associated organisms are introduced within a global context focusing on zoonotic significance. 
Following this, more detail is provided from an Australian context.

### Worldwide summary

Ticks are responsible for transmitting the greatest variety of pathogenic microbes of any arthropod vector. 
As a consequence they are important vectors of disease that affect humans, wildlife, livestock and companion animals [@jongejanGlobalImportanceTicks2004].
Incidence of human tick-borne diseases are steadily increasing, with recent reports from the Centre of Disease Control and Prevention (CDC, United States of America) describing a two-fold increase in the number of tick-borne disease cases; this accounts for 77% of all vector-borne diseases [@rosenbergVitalSignsTrends2018]. 

#### *Anaplasmataceae*

The family *Anaplasmataceae* are a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells. 
After a major taxonomic reorganisation in 2001, the phylogeny of the *Anaplasmataceae* family is now well accepted and includes *Ehrlichia*, *Anaplasma*, *Wolbachia*, *Neorickettsia* and the more recently described *Neoehrlichia* [@rarGeneticDiversityAnaplasma2021]. 
Well known human pathogens in this group include *Anaplasma phagocytophilum* and *E, chaffeensis* 
*Anaplasmataceae* are difficult to isolate and culture, and as such molecular tools are critical in the identification of members within this family. 
Conserved genes such as *16S rRNA*, *groEL* and *gltA* are used to identify and classify this group of bacteria  [@kawaharaUltrastructurePhylogeneticAnalysis2004].

*Anaplasma phagocytophilum* is the causative agent for granulocytic anaplasmosis in humans. The first case of human granulocytic anaplasmosis (HGA) was made in the United States in 1994 [@chenIdentificationGranulocytotropicEhrlichia1994].
However, it was not until 2001 that it reflected its current name (previously named *Ehrlichia phagocytophilum*) [@dumlerReorganizationGeneraFamilies2001]. 
*A. phagocytophilum* is also responsible for tick-borne fever in ruminants, equine anaplasmosis in horses and causes severe febrile diseases in dogs and cats [@rarAnaplasmaEhrlichiaCandidatus2011]. 
The vast majority of HGA cases are known from the United States, most notably in the northeastern and upper mid-western regions [@mmwrFinal2009Reports2010]; although present in Europe, the prevalence is significantly lower [@bakkenHumanGranulocyticAnaplasmosis2015]. 
A number of *A. phagocytophilum* vectors have been identified, including *Ixodes scapularis* [@telfordPerpetuationAgentHuman1996;@hodzicAcquisitionTransmissionAgent1998], *Ix. ricinus* [@lizPCRDetectionGranulocytic2000], *Ixodes persulcatus* [@eremeevaPrevalenceBacterialAgents2006], and *Ixodes ovatus* [@ohashiAnaplasmaPhagocytophilumInfected2005]. 
The vertebrate reservoirs of *A. phagocytophilum* remain unclear due to the presence of numerous diverse strains, however animals such as white-footed mice, white-tailed deer [@telfordPerpetuationAgentHuman1996;@ravynIsolationEtiologicAgent2001], dusty-footed woodrats [@nicholsonDuskyFootedWoodRats1999] and chipmunks [@foleyDistinctEcologicallyRelevant2009] have been highlighted as important reservoirs. 

Human ehrlichiosis is most notably caused by *E. chaffeensis*. 
It was first identified in the United States as causing human monocytic ehrlichiosis (HME) [@andersonEhrlichiaChaffeensisNew1991]. 
Since then 4,364 of confirmed HME have been reported to the CDC between 2003-2010 [@mmwrFinal2009Reports2010]. 
In North America the lone star tick (*Amblyomma americanum*) and white-tailed deer are regarded as the most important vector and vertebrate reservoir of the bacteria [@rarAnaplasmaEhrlichiaCandidatus2011]. 
To date, pathogen isolation has only been confirmed in the Unites States, however molecular and serological reports of the pathogen have been made throughout the world including Venezuela [@martinezEhrlichiaChaffeensisChild2008], Latin America [@gongora-biachiFirstCaseHuman1999;@dacostaMoreHumanMonocytotropic2006], South Korea [@parkDetectionAntibodiesAnaplasma2003], and Thailand [@heppnerHumanEhrlichiosisThailand1997]. 

Using molecular tools, reports of an *Ehrlichia*-like organism were made from *Ix. ricinus* ticks in the Netherlands [@schoulsDetectionIdentificationEhrlichia1999], and from rats (*Rattus norvegicus*) in China [@panEhrlichialikeOrganismGene2003]. 
Further studies showed there was no cross-reactivity of this organism with members of the genera *Anaplasma*, *Ehrlichia* or *Neorickettsia*, as such it was designated to be a new genus and was formally described by @kawaharaUltrastructurePhylogeneticAnalysis2004 as *Neoehrlichia mikurensis*. The first reports of *N. mikurensis* infection in humans were made in 2010 and 2011 [@fehrSepticemiaCausedTickborne2010;@vonloewenichDetectionCandidatusNeoehrlichia2010;@welinder-olssonFirstCaseHuman2010;@pekovaCandidatusNeoehrlichiaMikurensis2011] and were predominately from cases where patients were immunocompromised. 
Two decades after its initial discovery, the first study was published detailing the cultivation of the organism [@wassCultivationCausativeAgent2019]. 
Generally a rapid and full recovery is successful after treatment with antibiotics [@pekovaCandidatusNeoehrlichiaMikurensis2011]. 
*Neoehrlichia mikurensis* has been identified from ticks around the world including Sweden [@anderssonCoinfectionCandidatusNeoehrlichia2013], Germany [@dinizCandidatusNeoehrlichiaMikurensis2011], Austria [@glatzDetectionCandidatusNeoehrlichia2014], and Hungary [@hornokMolecularAnalysisIxodes2017]. 
Using transmission electron microscopy, *N. mikurensis* was recently identified from tick salivary glands [@ondrusPutativeMorphologyNeoehrlichia2020] providing evidence for transmission route via a tick bite.


#### *Borrelia*

Lyme borreliosis caused by a group of spirochaete bacteria (*B. burgdorferi* sensu lato) is endemic to North America and Europe. 
In North America the primary vector of *B. burgdorferi* s. l. is *Ix. scapularis*, and major reservoir hosts are well known to include white-footed mice and white-tailed deer [@halseyRoleIxodesScapularis2018]. 
In Europe the main vector responsible for Lyme Borreliosis is *Ix. ricinus* [@kirsteinLocalVariationsDistribution1997] with a number of vertebrates identified as reservoir hosts throughout the continent, most notably hedgehogs (*Erinaceus europaeus*) and voles (*Myodes glareolus*) [@jahfariMeltingPotTickborne2017;@coipanGeneticDiversityBorrelia2018;@estrada-penaHighThroughputSequencing2018]. 
Lyme borreliosis is typically manifested by an erythema migrans skin lesion (60-80% of cases) [@rizzoliLymeBorreliosisEurope2011], and known to develop into arthritis or various skin disorders [@stanekLymeBorreliosis2012]. 
Additional early symptoms may include fever, headaches, fatigue, and body aches and pains [@rizzoliLymeBorreliosisEurope2011;@clarkLymeBorreliosisHuman2013]. 
The development of neurological symptoms is another possible consequence of infection, and is known as Lyme neuroborreliosis, it has been documented in both North America and Europe [@clarkLymeBorreliosisHuman2013;@strleComparisonFindingsPatients2006].
A distinct, separate clade of *Borrelia* known as the relapsing fever (RF) group, are a cause of significant disease, and can be transmitted by argasid and ixodid ticks, and the human body louse [@lopezTickBorneRelapsingFever2016]. 
Acute symptoms of relapsing fever in humans are generally non-specific (e.g. fever, headache and nausea); however the disease is characterize by a unique cyclic nature, where acute episodes lasting a few days are followed by afebrile periods [@dworkinTickborneRelapsingFever2008]. 
In addition to the groups of Lyme borreliosis and relapsing fever *Borrelia*, there are a number of phylogenetically distinct genotypes which appear to be more host specific; these include '*Candidatus* Borrelia mahuryensis' from avian ticks [@munoz-lealCandidatusBorreliaIbitipoquensis2020], '*Candidatus* Borrelia tachyglossi' from echidna ticks [@lohMolecularCharacterizationCandidatus2017] and *Borrelia turcica* from reptiles ticks [@gunerBorreliaTurcicaSp2004].

#### *Coxiella*

An obligate intracellular bacteria, *Coxiella* has been isolated from a wide range of animals, and environmental samples throughout the world.
*C. burnetii* is the causative agent of Q fever that can cause disease in both humans and animals (mainly associated with livestock) [@gonzalez-barrioCoxiellaBurnetiiWild2018]. 
First discovered in Australia in 1935 after a cluster of abattoir workers became ill [@derrickFEVERNEWFEVER1937] the causative agent of the disease was not know, and the term Q fever was adapted meaning "query".
Since then it has been described in multiple countries, with incidences of human infection usually associated with direct livestock contact.
Despite the wide spread prevalence and economic impacts on the agriculture industry, the natural history of *C. burnetii* is not well understood. 
Q fever is usually acquired via inhalation route during close contact with infected animal material.
It has now been well established that ticks can become reservoirs and potential vectors of *C. burnetii* [@arricau-bouveryFeverEmergingReemerging2005], however the significance of ticks in transmission of the disease to humans is not yet fully understood.
Many other members of the *Coxiella* genus have been identified from ticks, and it is hypothesized that these species are mutualistic endosymbionts, which may provide nutritional advantages to the tick [@kobayashiMolecularDetectionGenotyping2021].


#### *Rickettsia*

The genus *Rickettsia* (Rickettsiaceae) is a group of obligate intracellular bacteria, that are among some of the oldest known vector-borne pathogens. 
Members of the genus *Rickettsia* can broadly be classified into (i) spotted fever group (SFG), (ii) typhus group (iii) *Rickettsia bellii* (ancestral) group and the (iv) *Rickettsia canadensis* group [@merhjRickettsialEvolutionLight2010]. 
The SFG is the most notable group of tick-borne disease, mainly transmitted by Ixodidae ticks [@parolaUpdateTickBorneRickettsioses2013]. 
Rocky-mountain spotted fever (RMSF), caused by *Rickettsia rickettsii* is the most well understood and studied SFG *Rickettsia*. 
Important vectors of *R. rickettsii* in North and Central America include the Rocky Mountain wood tick (*Dermacentor andersoni*), the American dog tick (*Dermacentor variabilis*), the Cayenne tick (*Amblyomma cajennense*) and the brown dog tick (*Rh. sanguineus*) [@dantas-torresRockyMountainSpotted2007;@lopez-perezDiversityRickettsiaeDomestic2021]. 
Although domestic dogs and wild mammals have been known to harbor the bacteria, the role of these reservoir hosts remains unclear.
Dogs have been implicated as the main reservoir for *Rickettsia felis*, a flea-borne spotted-fever *Rickettsia* most common in companion animals [@ng-nguyenDomesticDogsAre2020].
In other cases, small mammals, mainly rodents, are implicated in the life cycle of many *Rickettsia* species [@tomassoneNeglectedAspectsTickborne2018;@parisBriefHistoryMajor2020]

#### Viruses

At least 38 tick-transmitted viruses have been identified, with many more unclassified species [@labudaTickborneViruses2004]. With only one exception (African swine fever virus, family *Asfarviridae*) all tick-borne viruses recognised belong the RNA virus families (*Reoviridae*, *Rhabdoviridae*, *Orthomyxoviridae*, *Bunyaviridae* and *Flaviviridae*) [@labudaTickborneViruses2004]. 
Tick-borne flaviviruses represent some of the most medically important arboviruses around the world. 
Tick-borne encephalitis (TBE) (*Flaviviridae*: *Flavivirus*) is a growing public health issue in parts of Europe and Asia and highlights the complexity of the dynamics involved in tick-borne diseases [@gritsunTickborneEncephalitis2003]. 
Natural vectors of the disease involved in transmission of the virus to humans mainly include, *Ix. ricinus* and *Ix. persulcatus* [@labudaTickborneViruses2004;@sussTickborneEncephalitis20102011].
The virus has also been identified in field collected *Ixodes hexagonus* and studies have demonstrated that *Ixodes arboricola*, *Haemaphysalis concinna*, *Haemaphysalis inermis* and *Haemaphysalis punctata* are also competent vectors [@gresikovaTickborneEncephalitis1998].
Vertebrate hosts involved in the maintenance of TBE include voles (*M. arvalis*) and a variety of rodent species (*Apodemus* spp., *Microtus* spp., and *Myodes* spp.) [@achaziRodentsSentinelsPrevalence2011].

#### Eukaryotes

Compared to bacteria, the diversity of eukaryote organisms responsible for causing tick-borne disease is much more limited [@tokarzDiscoverySurveillanceTickBorne2021].
Ticks have also been associated in the transmission and life of a diverse range of organisms such as protozoa, fungi and nematodes.

Piroplasms are a group of single-celled, intracellular parasites that belong to the Apicomplexa phylum.
Characterised by two main genera, *Babesia* and *Theileria*, they are the primary agents of eukaryote tick-borne diseases in vertebrates.
Human babesiosis is a well-known infectious disease, recognised as an emerging public health issue in many parts of the world. 
There are over 100 species of *Babesia* (Apicomplexa: Piroplasmida) worldwide which have been identified in a wide range of wildlife and domestic animals [@kumarGlobalEmergenceHuman2021]. Six species of the *Babesia* are recognised as capable of infection humans. In North America human babesiosis is most commonly associated with *Babesia microti*, followed by *Babesia duncani*. While there has been evidence to show presence of human babesiosis in latin American countries, the true causative agents have not been as extensively studied [@kumarGlobalEmergenceHuman2021]. In Europe *Babesia divergens* is the main cause of human babesiosis followed by *Babesia venatorum*, only a small number of cases have been attributed to *B. microti* and *Babesia crassa*-like agent [@hildebrandtHumanBabesiosisEurope2021;@vannierHumanBabesiosis2012]. Cases of babesiosis have been reported from several Asian countries with reports including *Babesia crassa*-like agents, *B. divergens*, *B. microti*, *B. venatorum*, and *Babesia* spp. KO1 [@kumarGlobalEmergenceHuman2021].
*Ix. scapularis* is the primary vector of *B. microti* to humans, with most cases reportedly vectored by nymph ticks during late spring--early summer period [@spielmanEcologyIxodesDamminiborne1985;@swansonCoinfectionsAcquiredIxodes2006]. 
The primary vertebrate hosts identified in the transmission of the disease is the white-footed mouse (*Peromyscus leucopus*) [@spielmanEcologyIxodesDamminiborne1985]. 
Manifestations of babesiosis are diverse, and can range from asymptomatic to debilitating illness that can lead to death.
Most commonly patients experience fever, fatigue, chills and headaches, with symptoms appearing gradually 1--4 weeks after tick bite [@vannierHumanBabesiosis2008].

The genus *Theileria* is distinguished from *Babesia* by the presence of stages outside the red blood cell.
There are a number of species that infect and cause disease in animals, particularly ruminants, equids, rodents, and foxes [@almazanBabesiosisTheileriosisNorth2022].
Additionally, *Theileria* species have been described circulating in populations of wildlife and ticks worldwide [@mansReviewTheileriaDiagnostics2015;@wattsTheileriaOrientalisReview2016].
To date, there have been no reported cases of *Theileria* infecting people.

Trypanosomes are a group of flagellated protozoa that belong to phylum Euglenozoa.
Members of the genera *Leishmania* and *Trypanosoma* are known parasites of humans and animals and are widely distributed. 
Species that can cause severe human disease include *Trypanosoma cruzi*, responsible for Chagas disease in South and Central America, *Trypanosoma brucei gambiense* and *Trypanosoma brucei rhodesiense* which cause human African trypanosomiasis (HAT) (also known as sleeping sickness) and *Leishmania donovani* capable of causing cutaneous leishmaniasis [@kauferReviewSystematicsSpecies2020].
Although blood-sucking insects (class Insecta) are responsible for the majority of zoonotic transmission of trypanosomes, there is growing evidence to support that ticks may be involved in the life-cycle of these protozoans [@morzariaTransmissionTrypanosomaSp1986;@thekisoeTrypanosomeSpeciesIsolated2007].

Filarial nematodes have been described from a number of tick species worldwide.
Genetic characterisation has shown that similar species of filarial nematodes have recently been described from two widespread ticks in North America.
Separate genetic analysis showed that closely related *Monanema*-like filarial nematodes were identified from *Am. americanum* [@henningDiscoveryFilarialNematode2016] and *Ix. scapularis* [@tokarzCharacterizationMonanemaNematode2020].
However, there is currently no evidence to suggest that these tick-associated nematodes are common causes of human disease [@tokarzCharacterizationMonanemaNematode2020].

### Infectious human tick-borne pathogens in Australia

In comparison to the Northern Hemisphere, relatively few zoonotic tick-borne pathogens are recognised in Australia [@madison-antenucciEmergingTickBorneDiseases2020;@rochlinEmergingTickbornePathogens2020].
Queensland Tick Typhus (QTT) was first identified during World War II from soldiers training in Queensland. 
After a tick bite, individuals developed an eschar, fever and vesicular rash [@andrewTickTyphusNorth1946]. 
Although initial reports put it in the spotted fever group, the causative agent, *R. australis* was later shown to be genetically different [@stenosRickettsialOutermembraneProtein2000]. 
Early experimental work showed that *Ix. holocyclus* and *Ix. tasmani* were vectors of the bacteria [@campbellRickettsiosesAustraliaIsolation1974]. 
Since its discovery it has been identified along the east coast of Australia [@campbellQueenslandTickTyphus1979;@wilsonQueenslandTickTyphus2013;@fergieQueenslandTickTyphus2017] (Table \@ref(tab:T1ausTBD)). 
A recent study using real-time PCR identified that *R. australis* was present in 15.4% (23/149) of *Ix. holocyclus* in north-east New South Wales [@gravesIxodesHolocyclusTicktransmitted2016].

A spotted-fever-like illness was identified from a cluster of 26 patients from Flinders Island, Tasmania, a small community with a population of about 1000 [@stewartFlindersIslandSpotted1991].
A serological investigation found that while 46% of patients were positive for the detection of *R. australis*, evidence suggested the aetiological agent was different [@gravesSpottedFeverGroup1993]. 
It was later confirmed that patients were infected with *R. honei*, a pathogen originally isolated in Thailand [@gravesRickettsiaHonei2003]. 
An investigation into the tick reservoir on the Island found that 63% of the reptile-associated tick *Bt. hydrosauri* were positive for *R. honei* [@stenosAponommaHydrosauriReptileassociated2003] (Table \@ref(tab:T1ausTBD)). 
Human infection of Q Fever, caused by the bacteria *C. burnetii*, is usually acquired by inhalation of infectious aerosols from vertebrate hosts such as sheep, cattle and domestic pets.
*C. burnetii* has been identified in a number of ticks, including the human biting species *Ix. holocyclus* [@gravesIxodesHolocyclusTicktransmitted2016] and *Am. triguttatum* [@popeCoxiellaBurnetiKangaroos1960;@cooperSerologicalEvidenceCoxiella2012] (Table \@ref(tab:T1ausTBD)); however there is just a single published report of tick-borne Q fever in Australia (thought to be transmitted by *Am. triguttatum*) [@beamanPericarditisAssociatedTickborne1989].

\newpage

**Taxa of interest**

This next section will review recent research on tick-associated organisms that are related to taxa known to cause disease globally (i.e. family level relatedness) and 'endosymbiont' organisms from Australian ticks. 
The list of possible microbes is extensive and therefore this review will focus mainly on microbes that have been identified from ticks. 
Additionally as recent research has highlighted that microbes may have a broader vector range than previously thought (e.g. *Bartonella*), where relevant this review will include other vector-related microbes.

```{r message=FALSE, warning=FALSE, include=FALSE}
library(readxl)
library(tidyverse)
ch1_ausTBDs <- read_excel("tables/Ch1-ausTBDs.xlsx")
```

```{r T1ausTBD, echo=FALSE, message=FALSE, warning=FALSE}
library(kableExtra)
options(kableExtra.html.bsTable = T)
opts <- options(knitr.kable.NA = "")
knitr::kable(ch1_ausTBDs, booktabs = TRUE, linesep = "", caption = "Currently recognised Australian human tick-borne diseases.", 
  caption.short = "Australian human tick-borne diseases.") %>%
  kable_styling(font_size = 8.5) %>%
  column_spec(c(2,3), italic = T)
```

#### *Anaplasmataceae*

Through the recent use of molecular tools five novel species (or genotypes) of *Anaplasmataceae* have been described from native Australian ticks. 
Additionally, a number of organisms are known but remain to be formally described (Table \@ref(tab:T1anaplasm)).

Questing *Am. trigutattum* ticks were found to harbour Australia’s first endemic *Ehrlichia*, '*Ca*. Ehrlichia occidentalis' and a novel genotype of *Anaplasma bovis* Y11 [@goftonDetectionPhylogeneticCharacterisation2017]. 
Two species of *Neoehrlichia* were recently characterised from *Ix. holocyclus* along the east coast of Australia at a prevalence of 11.25% (44/391; 31 females, seven males and six nymphs) [@goftonPhylogeneticCharacterisationTwo2016].
The disease potential and transmission dynamics of these novel organisms remains unknown. 
A study on the microbiome of ticks parasitsing bandicoots confirmed the presence of both *Neoehrlichia* species and expanded the tick associations to include *Ix. tasmani* [@eganBacterialCommunityProfiling2020]. 
The same study also expanded the range of *A. bovis* Y11 to New South Wales and identified a novel *Neoehrlichia* and *Ehrlichia* species in ticks from quenda (*Isoodon fusciventer*) in south-west Western Australia. 
'*Candidatus* Ehrlichia ornithorhynchi' was described from the platypus and its tick *Ixodes ornithorhynchi* [@goftonNovelEhrlichiaSpecies2018].
A novel *Anaplasma* and *Ehrlichia* species has been identified in *Bothriocroton concolor* ticks from echidna in New South Wales and Queensland [@lohIdentificationCharacterisationMicroorganisms2018;@eganBacterialCommunityProfiling2020]. 
A novel *Neoehrlichia* and *Ehrlichia* were also identified from ticks (*Ixodes fecialis* and *Ixodes australiensis*) removed from quenda *Isoodon fusciventer* [@eganBacterialCommunityProfiling2020].

```{r message=FALSE, warning=FALSE, include=FALSE}
library(readxl)
ch1_tickmicrobes <-
  read_excel("tables/Ch1-austickmicrobes.xlsx")
```

```{r T1anaplasm, echo=FALSE, message=FALSE, warning=FALSE}
library(kableExtra)
ch1_anaplasm = ch1_tickmicrobes[23:36, , drop = TRUE]
knitr::kable(ch1_anaplasm, booktabs = TRUE, linesep = "", caption.short = "\\textit{Anaplasmataceae} species identified from Australian ticks.", 
  caption = "\\textit{Anaplasmataceae} species identified from Australian ticks using molecular methods.") %>%
  kable_styling(full_width = F) %>%
  kable_styling(font_size = 8.0) %>%
  column_spec(1, width = "10em") %>%
  column_spec(2, italic = T, width = "10em") %>%
  column_spec(3, width = "8em") %>%
  column_spec(4, width = "6em") %>%
  column_spec(5, width = "6em") %>%
  column_spec(6, width = "6em") %>%
  footnote(alphabet = c("Introduced species that are now considered endemic.", "Previously considered exotic, it was first identified in May 2020. Currently listed as a nationally notifiable disease and investigations into its origin are ongoing https://www.outbreak.gov.au/current-responses-to-outbreaks/ehrlichiosis-dogs."), threeparttable = T )
```

#### *Borrelia* 

At present four species of borreliae have been identified in Australia (Table \@ref(tab:T1borrelia)). 
Two species were introduced *Borrelia theileri* and *Borrelia anserina* with the importation of livestock. 
Avian spirochaetosis is associated with disease in poultry caused by *B. anserina* and is transmitted by the soft tick *Ar. persicus*. 
Bovine spirochaetosis is caused by *B. theileri* and is transmitted with the cattle tick, *Rh. australis* (formerly *Bo. microplus*) [@estrada-penaReinstatementRhipicephalusBoophilus2012]. 
The first native Australian *Borrelia* was described from long-haired rats (*Rattus villosissimus*) in north-western Queensland [@carleyNewSpeciesBorrella1962] and named *Borrelia queenslandica*. 
The authors suggested the spirochaete was transmitted by the soft kangaroo tick (*Or. gurneyi*), however this was never confirmed. 
To date *B. queensandica* has not been isolated since and as such no molecular data is available.
A recent discovery has presented the first molecular description of a native Australian *Borrelia* and brings the total number of (*Borrelia*) species present in Australia to four. '*Candidatus* Borrelia tachyglossi' was genetically described from echidna biting ticks *Bt. concolor* [@lohNovelBorreliaSpecies2016;@lohMolecularCharacterizationCandidatus2017]. 
A novel species of *Borrelia* that falls within the reptile *Borrelia* clade has also been identified in *Bothriocroton undatum* collected from the goanna in NSW [@panettaReptileassociatedBorreliaSpecies2017].


```{r T1borrelia, echo=FALSE}
library(kableExtra)
T1borrelia = ch1_tickmicrobes[37:42, , drop = TRUE]
knitr::kable(T1borrelia, booktabs = TRUE, linesep = "", caption.short = "\\textit{Borrelia} species identified from Australia.", 
  caption = "\\textit{Borrelia} species identified from Australian ticks and animals.") %>%
  kable_styling(full_width = F) %>%
#  kable_styling(latex_options = c("scale_down")) %>%
  kable_styling(font_size = 8.0) %>%
  column_spec(1, italic = T, width = "10em") %>%
  column_spec(2, italic = T, width = "10em") %>%
  column_spec(3, width = "8em") %>%
  column_spec(4, width = "6em") %>%
  column_spec(5, width = "6em") %>%
  column_spec(6, width = "6em") %>%
  footnote(alphabet = c("Introduced species with the important of livestock.", "Identification made by culture methods, isolated from rodent host - suspected tick vector listed."))
```

#### *Coxiella*

Studies on the presence of *C. burnetii* are particularly challenging due to the cross reactivity of serological assays and the conserved nature of the commonly used 16S rRNA gene (Table \@ref(tab:T1coxiella)). 
Therefore in this review I will only focus on molecular reports of *Coxiella* sp. from wildlife and ticks.
Sequences that were 98--99% similar to *C. burnetii* have been identified in
*Bothriocroton auruginans* from the common wombat (*Vombatus ursinus*) collected from Victoria [@vilcinsMolecularDetectionRickettsia2009;@beardMorphologicalIdentificationTicks2021].  
Recent descriptions of *C. burnetii* from native Australian tick species (*Ha. bancrofti*, and *Ix. holocyclus*) and the brown dog tick (*Rh. sanguineus*) are important in our understanding of the epidemiology of Q Fever in Australia [@chaladaMolecularSurveyTickBorne2018]. 
The positive detection of *C. burnetii* in these samples however has limited reliably due to the lack of DNA sequence results from the study. 
The authors also noted the presence of *Coxiella*-like symbionts in *Am. triguttatum*, *Ix. holocyclus*, *Rh. australis* and *Or. capensis*, which were also not sequenced. 
An investigation into the presence of *Coxiella* and *Coxiella*-like symbionts in Australian brown dog ticks (*Rh. linnaei* syn *Rh. sanguineus*) revealed the presence of a *Coxiella*-like symbiont (100% prevalence) through targeted PCR and sequencing of the 16S rRNA gene [@oskamMolecularInvestigationPresence2017]. 
Therefore without sequence results from PCR assays, the true presence of *C. burnetii* as determined by @chaladaMolecularSurveyTickBorne2018 remains questionable.  
*C. burnetii* was recently detected in kangaroo meat that was intended for companion animal consumption [@shapiroMolecularDetectionCoxiella2020] using qPCR.
One recent study stated that wildlife carers may be two times more likely to be infected with *C. burnetii* than the general public, however the study was hindered by a small sample size [@mathewsCoxiellaBurnetiiSeroprevalence2021]

```{r T1coxiella, echo=FALSE}
library(kableExtra)
ch1_coxiella = ch1_tickmicrobes[15:22, , drop = TRUE]
knitr::kable(ch1_coxiella, booktabs = TRUE, linesep = "", caption.short = "\\textit{Coxiella} species identified from Australian ticks.", 
  caption = "Identification of \\textit{Coxiella burnetii} within Australian ticks and wildlife using molecular methods. Serological tests were excluded from this summary due to the known cross-reactivity of \\textit{Coxiella}-like organisms with assays.") %>%
  kable_styling(full_width = F) %>%
  kable_styling(font_size = 8.0) %>%
  column_spec(1, italic = T, width = "10em") %>%
  column_spec(2, italic = T, width = "10em") %>%
  column_spec(3, width = "8em") %>%
  column_spec(4, width = "6em") %>%
  column_spec(5, width = "6em") %>%
  column_spec(6, width = "6em") %>%
  footnote(alphabet = c("Identified in a faecal sample using molecular assay.", "Records from Islands off Western Australia coastline."))
```


#### *Rickettsia*

**Spotted fever - Flinders Island Spotted or Australian Spotted fever?**

The identification of Flinders Island Spotted Fever (*R. honei*) was a significant breakthrough in the knowledge of tick-borne diseases in Australia. 
However, ongoing research into the distribution of the disease, and more recently genetic information, has meant the label of “Flinders Island” Spotted Fever causes significant issues. 
Only a few years after it was formally recognised, a new focus of *R. honei* spotted fever was identified in South Australia and Tasmania [@dyerNewFocusRickettsia2005;@unsworthNotOnlyFlinders2005]. 
Lane et al. [-@laneEvidenceSpottedFeverlike2005a] identified a *R. honei*-like sequence from a *Haemaphysalis novaeguineae* tick. 
The patient was bitten by the tick in north-east Queensland and become acutely unwell developing signs of rickettsial disease (later report by Unsworth et al. [-@unsworthThreeRickettsiosesDarnley2007]). 
Although molecular analysis did show similarities with *R. honei* strain TT-118 (Thai tick typhus), and *R. honei* (Flinders Island Spotted Fever), it was not able to fully resolve the relationships among the SFG rickettsia. 
A report of seven patients exhibiting similar symptoms to Flinders Island Spotted Fever was later published [@unsworthThreeRickettsiosesDarnley2007]. 
A combination of serological, molecular and culture techniques were used and *R. honei* subsp. marmionii was described in patients from Queensland, Tasmania and South Australia. 
It again confirmed the presence of the novel genotype and subsequent illness caused by a *Haemaphysalis novaeguineae* bite from Cape York Peninsula in far north Queensland. 
There have been no other identification of *R. honei* in a tick vector on mainland Australia. 
A recent case described a negative serological result from a patient, however subsequent shotgun sequencing of the blood showed it was positive for *R. honei* [@grahamDetectionSpottedFever2017]. 
The initial naming of the disease as "Flinders Island" Spotted Fever has potentially caused havoc on the diagnosis and treatment of the disease. 
Due to the non-specific acute symptoms, and at times unusual sequelae of spotted-fever, it is plausible that treating physicians may not consider FISF as a possible aetiology due to geographical restrictions. 
Scientifically sound and timely case reports are fundamental to ensure information is disseminated to the medical community.
A number of case reports of a *Rickettsia*-like illness have also been identified from patients in Western Australia. 
Molecular screening of a punch biopsy (taken at edge of eschar) sample taken from a patient bitten by *Ix. australiensis* in Walpole, south-west Western Australia showed the presence of *Rickettsia* sp. (unable to identify to species). 
Using a PCR assay the whole blood was negative, however serological testing showed evidence of acute infection with SFG *Rickettsia*; culture and PCR from the tick were negative [@rabyNewFociSpotted2016]. 
A female patient was bitten by a tick 150 km east of Esperance and DNA extracted from acute phase serum underwent PCR for the rickettsial 17kD antigen gene which generated a 429 bp sequence showed 100% similarity to *R. honei*, and 99.7% to *R. gravesii*.
It is unclear if *R. honei* has a larger geographical and vector range than previously thought or if in fact this was *R. gravesii* sequence showing a 1 bp mis-match to reference sequence [@rabyNewFociSpotted2016]. 
A serological study in Western Australia showed that those who frequented bushland had a higher risk of exposure to spotted fever group rickettsia compared to the reference population [@abdadSeroepidemiologicalStudyOutdoor2014]. 
In addition, there have also been a number of novel *Rickettsia* species recently described from native Australian ticks (Table \@ref(tab:T1rickettsia)).
Importantly many of these novel findings highlight the difficultly and ambiguity in species delimitation of the genus.  
A novel Australian *Rickettsia* was identified from the soft tick *Argas dewae* from bat roosting boxes in Victoria [@izzardRickettsialesRickettsialDiseases2010]. 
Gene sequences from five genes (*gltA*, *rOmpB*, *rOmpA*, *rrs* and *sca4*) totalling over 10 kb demonstrated that it fit the criteria for the designation of a novel species as per @fournierGeneSequenceBasedCriteria2003 and was tentatively named *Rickettsia dewae*.  
However, a recent study published by the same authors illustrates how whole genome sequence revealed that it is actually a divergent strain of *Rickettsia japonica* [@izzardIsolationDivergentStrain2018]. 
In that same study the authors also raise important questions on the classification of *Rickettsia* species as outlined in @fournierGeneSequenceBasedCriteria2003. 
With the growing trend towards whole genome sequencing it may in fact raise questions around descriptions of other *Rickettsia* species.  

```{r T1rickettsia, echo=FALSE}
library(kableExtra)
ch1_rickettsia = ch1_tickmicrobes[1:14, , drop = TRUE]
knitr::kable(ch1_rickettsia, booktabs = TRUE, linesep = "", caption.short = "\\textit{Rickettsia} species identified from Australian ticks.", 
  caption = "\\textit{Rickettsia} species identified from Australian ticks using molecular methods.") %>%
  kable_styling(full_width = F) %>%
  kable_styling(font_size = 8.0) %>%
  column_spec(1, italic = F, width = "10em") %>%
  column_spec(2, italic = T, width = "10em") %>%
  column_spec(3, width = "8em") %>%
  column_spec(4, width = "6em") %>%
  column_spec(5, width = "6em") %>%
  column_spec(6, width = "6em") %>%
  footnote(alphabet = c("Recognised human tick-borne pathogen."))
```

#### Viruses

Despite the presence and pathogenicity of tick-borne viruses being well described overseas, Australia does not currently recognise the presence of an endemic tick-borne virus. 
Virus-tick-vertebrate host relationships are highly specific [@labudaTickborneViruses2004]. 
A review of neglected arboviruses in Australia highlighted that despite a surge of research pioneered by the Commonwealth Scientific and Industrial Research Organisation (CSIRO), tick-borne virus research has largely remained undocumented [@gyawaliNeglectedAustralianArboviruses2017]. 
A summary of past and recent virus discoveries from Australian ticks is provided in Table \@ref(tab:T1virus).

During the 1960’s and 1970’s there was a strong research presence around novel tick-borne viruses in Australia led by CSIRO team. 
Gadgets Gully virus was described in 1976 from *Ixodes uriae* ticks at Macquarie Island [@st.georgeIsolationArbovirusesIncluding1985]. 
The virus was isolated by intra-cerebral inoculation of ground tick suspensions into neonatal mice, which resulted in mice developing neurological symptoms and died 5 days post infection. 
Serological characterisation of the isolate demonstrated that it was an unknown flavivirus however, there has been no further research and as such the ecology, transmission dynamics and its potential to cause disease in humans remains unknown. 
Saumerez Reef virus was described in 1974 from *Ornithorodoros capensis* ticks collected from nests of sooty terns (*Sterna fuscata*), Queensland [@st.georgeIsolationSaumarezReef1977]. 
Several other viral isolates were obtained from the hard tick *Ix. eudyptidis*, collected in Tasmania [@st.georgeIsolationSaumarezReef1977]. 
Intra-cerebral inoculation of neonatal mice caused death while intra-cerebral inoculation of weanling mice did induce antibody formation and no clinical illness. 
No data on potential human infection or human illness are available. 
Serological studies in silver gulls (*Larus novaehollandiae*) in Tasmania showed they had an antibody seroprevalence rates of 25%. Ticks and birds are thought to contribute to the natural transmission of Saumarez Reef virus, however, further studies are needed to elucidate the natural cycle of the virus. 
Vinegar Hill virus (VINHV) is a member of the Bunyavirales order (tentative member of the genus *Orthonairovirus*) and was isolated from the soft tick *Argas robertsi* collected from cattle egrets [@gauciGenomicCharacterisationVinegar2017].
Avian and human sera from local residents in the Coral Sea and Great Barrier Reef was used for testing against 19 known arboviruses. 
It found that antibodies were detected in 4% of avian and human sera and included Gadget’s Gully virus (flavivirus) and Murray Valley Encephalitis. 
It was noted however, that a number of antibodies were restricted to sea birds only.
Novel Phlebovirus with zoonotic potential was identified from a colony of shy albatross (*Thalassarche cauta*) on Albatross Island, northwest of Tasmania in the Hunter Island Group [@wangNovelPhlebovirusZoonotic2014]. 
Both ticks (*Ixodes eudyptidis*) and serum samples were collected, and sequences were obtaining via RNAseq using the 454 platform, however subsequent testing by ELISA or qPCR was unsuccessful. 

A unique iflavivirus has recently been identified from *Ix. holocyclus* in Queensland and New South Wales [@obrienDiscoveryNovelIflavirus2018]. 
Designed *Ix. holocyclus* iflavirus (IhIV), it represents the first virus sequence identified in *Ix. holocyclus* ticks. 
Members of the *Iflaviridae* family are considered 'arthropod-only' viruses, and to date have not been implicated in human disease.
More recently metatranscriptomic applications have characterised a number of novel viral sequences. 
Using this approach 19 novel RNA viruses were characterised which included members of the *Flaviviridae* and *Reoviridae* families [@harveyExtensiveDiversityRNA2019]. 
Members of these viral families include known human pathogens described in the northern hemisphere such as tick-borne encephalitis virus and Powassan virus (both belong to genus *Flavivirus*) and Colorado tick fever virus (genus *Coltivirus*).
Their isolation in the common human biting tick *Ix. holocyclus* makes them important candidates for future research into possible links to cases of human disease.

```{r T1virus, echo=FALSE}
library(kableExtra)
T1virus = ch1_tickmicrobes[43:76, , drop = TRUE]
knitr::kable(T1virus, booktabs = TRUE, longtable = T, linesep = "", caption.short = "Viruses identified from Australian ticks.", 
  caption = "Viruses identified from Australian ticks using molecular and culture based methods. Name of virus and genus or family in parentheses.") %>%
  kable_styling(full_width = F) %>%
  kable_styling(font_size = 8.0) %>%
  column_spec(1, italic = T, width = "10em") %>%
  column_spec(2, italic = T, width = "10em") %>%
  column_spec(3, width = "8em") %>%
  column_spec(4, width = "6em") %>%
  column_spec(5, width = "6em") %>%
  column_spec(6, width = "6em") %>%
  kable_styling(latex_options = c("repeat_header"))%>%
  footnote(alphabet = c("Antibodies identified from host blood samples.", "Records from Macquarie Island off Tasmania coastline.", "Records from Heron Island off Queensland coastline.", "Records from Hunter Island Group off Tasmania coastline.", "Records from Diamond Island off Tasmania coastline."))
```

#### Eukaryotes

Eukaryote organisms reviewed here were chosen based on their relationship with known agents responsible for tick-borne diseases globally.
Taxa included within this section include piroplasms (e.g. *Babesia* and *Theileria*), *Hepatozoon*, and *Trypanosoma*.
These organisms can broadly be classified as haemoprotozoa.
In Australia there is currently no recognised human eukaryote tick-borne pathogens.
So far studies have failed to provide evidence for agents of human tick-borne diseases that have been described in the northern hemisphere to be present in Australia.

Due to the recognised issues associated with morphological identification of haemoprotozoa [@zhuLooksCanDeceive2009;@lackPhylogenyEvolutionPiroplasmida2012;@kostygovEuglenozoaTaxonomyDiversity2021] this review will focus on molecular identifications of eukaryotes from Australian ticks.
A summary of selected eukaryote organisms that have been identified from Australian ticks are available in Table \@ref(tab:T1eukaryotes).

```{r T1eukaryotes, echo=FALSE}
library(kableExtra)
T1eukaryotes = ch1_tickmicrobes[77:108, , drop = TRUE]
knitr::kable(T1eukaryotes, booktabs = TRUE, longtable = T, linesep = "", caption.short = "Eukaryotes identified from Australian ticks.", 
  caption = "A selection of eukaryotes identified from Australian ticks using molecular based methods. Eukaryotes groups presented here represent organisms related to taxa associated with tick-borne diseases globally.") %>%
  kable_styling(full_width = F) %>%
  kable_styling(font_size = 8.0) %>%
  column_spec(1, italic = T, width = "10em") %>%
  column_spec(2, italic = T, width = "10em") %>%
  column_spec(3, width = "8em") %>%
  column_spec(4, width = "6em") %>%
  column_spec(5, width = "6em") %>%
  column_spec(6, width = "6em") %>%
  kable_styling(latex_options = c("repeat_header")) %>%
  footnote(alphabet = c("Imported species that are now considered endemic.", "Previously considered an exotic species to Australia, first identification made in 2018.", "Retrospective sequence analysis showed that sequences are more similar to the genus Hemolivia."))
```

There are currently no recognised endemic human protozoal tick-borne diseases in Australia.
To date there has been a single case of human babesiosis caused by *Babesia microti* in Australia from a patient without a travel history [@senanayakeFirstReportHuman2012].
Subsequent molecular investigation confirmed that the identification of *B. microti* and phylogenetic analysis showed that it grouped closely with other *B. microti* genotypes identified from the northern hemisphere [@papariniMolecularConfirmationFirst2014].
An epidemiological investigation was carried out at the time of the case and testing was conducted from a close relative and pet dog which all yielded negative results [@senanayakeFirstReportHuman2012].
Subsequently a widespread serological investigation was carried out on Australian blood donor samples for the presence of *B. microti* which included 7000 patients and did not identify any positives samples [@faddyNoEvidenceWidespread2019].

A number of piroplasms have been introduced to Australia, however they all related to livestock and domestic animals.
*Babesia canis* and *Theileria orientalis* are both associated with introduced tick species, the brown dog ticks (*Rh. sanguineus*) and the Asian longhorned ticks (*Ha. longicornis*).
Cattle tick fever is caused by *Babesia bovis* and *Babesia bigemina* and is present across the North of Australia. The two species were introduced to Australia with importation of livestock and are both vectored by the cattle tick (*Rh. australis*; syn. *Bo. microplus*) [@angusHistoryCattleTick1996].
While *Th. orientalis* has been identified in native Australian tick species such as *Ha. bancrofti* [@lakewEndemicInfectionCattle2021], the overwhelming evidence suggests the main vector is *Ha. longicornis* [@marendyHaemaphysalisLongicornisLifecycle2019].
However, there may be some variation of vector competence between the different genotypes of *Th. orientalis* [@forshawTheileriaOrientalisIkeda2020], widespread detection of this *Theileria* in native Australian ticks or animals has not yet been shown.

Genetic analysis of native species of piroplasms described from Australia has shown that they form a distinct clade [@barbosaSequenceAnalysesMitochondrial2019].
Of particular note is the group of *Babesia* species identified from Australian ticks and marsupials is phylogenticaly different from the human pathogen *B. microti* (belonging to the *Babesia microti* group.

While traditionally not associated with ticks, an early study identified that ticks may be involved in the life cycle of native Australian trypanosomes. 
Early studies using morphological tools identified trypanosomes within the Australian paralysis tick (*Ix. holocyclus*) collected from bandicoots infected with *Trypanosoma thylacis* [@mackerrasHaematozoaAustralianMammals1959].
Molecular evidence of the relationship between Australian trypanosomes and ticks followed 50 years later from the opposite side of the country in south-west Australia. 
Austen et al. [-@austenVectorTrypanosomaCopemani2011] identified *Trypanosoma copemani* from *Ixodes* sp. ticks collected from infected marsupials.
Motile trypanosomes were observed in *Ix. australiensis* collected from quokkas and the Gilbert's potoroo on Bald Island and the nature reserve Two Peoples Bay.

Australia was once considered free of *Leishmania*, however an endemic species has since been described after it was noted red kangaroos were showing signs of cutaneous leishmaniasis [@roseCutaneousLeishmaniasisRed2004].
The species has formally been described as *Leishmania macropodum* [@barrattIsolationNovelTrypanosomatid2017] and although studies are limited, current evidence suggests day-biting midges, *Forcipomyia* (*Lasiohelea*), as a likely vector [@dougallEvidenceIncriminatingMidges2011;@panahiUtilisingNovelSurveillance2020].
To date it has not been identified from any Australian ticks and or any other Australian state/territory outside the NT [@cleareRemainingVigilantExotic2014;@dybingGhostsChristmasAbsence2016;@thompsonExoticParasiteThreats2018]. 

Haemogregarines are a group of blood parasites that belong to the apicomplexa phylum, and are capable of infecting a range of vertebrates.
The classifications of haemogregarines is problematic, with the lack of a monophyletic group within the family Haemogregarinida [@al-quraishyHaemogregarinesCriteriaIdentification2021]. The group is likely to consist of at least three families. 
Recent molecular discoveries have shown that morphological identifications at the genus level may not be reliable for many haemoprotozoa. 
For example previous studies have shown that organisms considered to be *Hepatozoon* (-like) were actually members of distantly related groups [@merinoSarcocystidMisidentifiedHepatozoon2008;@zhuLooksCanDeceive2009]. 
Not only do these findings challenge taxonomic classification but they also show that microbes may not be as tissue-specific (e.g. blood, liver etc) as previously thought raising additional questions about the true life cycle of organisms. 

The genus *Hepatozoon* was first described following the identification of *Hepatozoon muris* (syn. *Hepatozoon perniciosum*) in laboratory rats and mites (*Laelaps echidninus*).
The genus is considered distinct from other haemogregarines due to two features of its life cycle; (i) the production of polysporocystic oocysts in hematophagous invertebrate definitive hosts, and (ii) transmission to vertebrate intermediate hosts via the ingestion of definitive hosts carrying sporulated oocysts [@smithGenusHepatozoonApicomplexa1996;@mathewPHYLOGENETICRELATIONSHIPSHEPATOZOON2000]. 
A review of *Hepatozoon* reported from Australian ticks is presented in Table \@ref(tab:T1eukaryotes).
Upon retrospective analysis of sequences generated from reptile ticks attributed to *Hepatozoon* [@vilcinsMolecularMorphologicalDescription2009] showed they were misclassified.
A BLAST analysis revealed the sequences were more closely related to *Hemoliva*, and only 94% similar to *Hepatozoon* sequences. 
*Hemoliva* has previously been reported from reptile ticks in Australia and to date has not been associated with disease in mammals.

Reports of associations between nematodes and Australian ticks are sparse in the literature. The most well documented is the parasitic filarial nematode *Cercopithifilaria johnstoni* association with the tick *Ixodes trichosuri* from Australian mammals, usually from bush rats [@sprattAspectsLifeHistory1988a;@mccannGenomeSequenceAustralian2021].

## Molecular tools

Tick microbiome studies began by using microscopy and cell culture techniques, these studies set the ground for work being done on the tick microbiome today.
Early studies by Cowdry [-@cowdryGroupMicroorganismsTransmitted1925] were limited by the technology at the time and as a result, were restricted by; (i) lack of distinguishing features between bacterial species and often pleomorphic taxa, and (ii) the presence of unculturable bacteria.
The advent of the polymerase chain reaction (PCR) [@mullisSpecificSynthesisDNA1987] and DNA sequencing technologies [@sangerDNASequencingChaintermination1977], overcame these early challenges and meant a definitive identification of microbes was now possible. 
The implementation of Sanger sequencing transformed the understanding bacterial pathogens with a vast increase in the sensitivity of detection methods [@chakravortyDetailedAnalysis16S2007;@macdonaldFrameworkDevelopingValidating2016].
In the context of tick-borne pathogens, PCR and Sanger sequencing shifted thinking from a single pathogen, to the idea that ticks can harbour a range of different microbes, which may co-occur in some tick species [@brouquiGuidelinesDiagnosisTickborne2004;@scolesPhylogeneticAnalysisFrancisellalike2004;@parolaTickFleaborneRickettsial2005].
Further advancements over time, such as quantitative PCR (qPCR), sophisticated methods for nucleic acid extraction, optimisation of PCR conditions (including inhibition and low copy number) have continued to change the way researchers investigate the microbiome [@hoffmannAnalysisTickSurface2020;@koloAnaplasmaPhagocytophilumOther2020].
The use of molecular barcodes has proven increasingly useful for the study of TBDs to elucidate not only the pathogenic microbes, but also the tick vector and potential host(s) species (through blood meal analysis). DNA barcoding is a powerful tool that can provide species identification by using standardised gene regions as internal taxonomy group tag [@hubertDNABarcodingSpecies2015]. Initially proposed as a tool for species identification, the joint application of DNA barcoding and high-throughput sequencing has shown to be a powerful tool in many contexts.
The advancement of molecular tools has highlighted the diversity of microbes harboured within ticks. 
Ticks removed from hosts contain nucleic acid material that consists of three main sources: (i) the tick itself; (ii) from the host (vertebrate) it was parasitising; and (iii) microbial organisms (e.g. bacteria, protozoa, viruses and helminths). 
The complex nature of the material from ticks lends itself well to platforms used in the characterisation of environment DNA. 

The introduction of high throughput sequencing has revolutionised the analysis of complex environmental samples.
Microbial diversity studies have adapted to high-throughput sequencing techniques using two broad appoaches by; (i) amplicon based - through targeting the hypervariable regions in the 16S rRNA gene; and (ii) shotgun sequencing - which sequences the entire suite of nucleic acid material in the extracted sample (variations can be made during library preparation) [@liuPracticalGuideAmplicon2020;@bhartiCurrentChallengesBestpractice2021]. 
Despite the presumption that shotgun sequencing remains superior to amplicon metabarcoding approaches, studies have shown targeted *16S rRNA* metabarcoding can yield up to 50% more phyla than shotgun based methods [@tesslerLargescaleDifferencesMicrobial2017]. 
The use of targeted *16S rRNA* metabarcoding is particularly favourable where the level of detection is low or where there is a large amount of host material.
The first application of high throughput sequencing methods to study the tick microbiome was in *Rhipicephalus* (*Bo.*) *microplus* [@andreottiAssessmentBacterialDiversity2011]. 
Since then, high-throughput methods have shed light on the diversity of the tick microbiome [@greayRecentInsightsTick2018] and seen a shift from the one tick - one pathogen understanding, towards the characterisation of the complete bacterial community present in ticks [@moutaillerCoinfectionTicksRule2016].
Bacterial metabarcoding approaches target one (or more) of the nine available hypervariable regions on the 16S rRNA gene; common regions sequenced include the V1-2 and V3-4 [@barbDevelopmentAnalysisPipeline2016;@yangSensitivityCorrelationHypervariable2016;@sperlingComparisonBacterial16S2017].
The short amplicons hinders accurate taxonomic and follow up molecular analysis is needed to provide greater phylogenetic resolution.
However, despite this, high throughput sequencing technologies is superior to traditional cloning and Sanger sequencing methods in characterising a diverse community of organisms. 
Studies of tick-borne diseases are often restricted in scope due to their narrow focus on known pathogens, by the use of species-specific or genus-specific primers, and as a result are at risk of overlooking potentially pathogenic agents or novel organisms.
Unbiased high-throughput sequencing provides an ideal method to identify new microbes that have the potential to cause disease in animals or humans.

Whilst the use of high-throughput sequencing (HTS) has been increasingly adapted by researchers worldwide, much less attention is given to its caveats and limitations. Methodological challenges of HTS include; (i) sequencing depth and short amplicon sequencing [@gihringMassivelyParallelRRNA2012;@houImpactNextgenerationSequencing2013;@simsSequencingDepthCoverage2014]; (ii) sequencing artefacts (errors and chimeric sequences) [@kuninWrinklesRareBiosphere2010;@haasChimeric16SRRNA2011]; and (iii) PCR amplification bias through the effect of *16S rRNA* copy number [@ahnEffectsPCRCycle2012] and annealing temperature [@suzukiBiasCausedTemplate1996].
To overcome some of the limitations associated with high-throughput sequencing a number of bioinformatic steps are introduced to provide quality control and assist in the interpretation of the large amount of data produced.
The steps can include primer trimming, removal of low quality reads, chimera detection (and removal), removal of low abundance reads and denoising [@edgarSearchClusteringOrders2010;@kuninWrinklesRareBiosphere2010;@haasChimeric16SRRNA2011;@edgarUNOISE2ImprovedErrorcorrection2016].
There are a number of pipelines and programs that have been developed to analyze 16S metabarcoding data, however they largely rely on similar underlying algorithms.
Widely used programs include mothur [@schlossIntroducingMothurOpensource2009], Quantitative Insights into Microbial Ecology (QIIME) [@caporasoQIIMEAllowsAnalysis2010], dada2 [@callahanDADA2HighresolutionSample2016], USEARCH [@edgarSearchClusteringOrders2010] and vsearch [@rognesVSEARCHVersatileOpen2016].
All programs listed are free, with the exception of 64-bit version of USEARCH (32-bit version is freely available). 
Generally, comparisons between these, and other pipelines, conclude that they remain more-or-less comparable – with the emphasis on customising the parameters to best suit each unique dataset [@nilakantaReviewSoftwareAnalyzing2014;@plummerComparisonThreeBioinformatics2015;@forsterComparisonThreeClustering2016].

Large curated reference *16S rRNA* datasets include GreenGenes [@desantisGreengenesChimerachecked16S2006], the Ribosomal Database project [@coleRibosomalDatabaseProject2009], SILVA [@pruesseSILVAComprehensiveOnline2007] and the EZ-Taxon [@chunEzTaxonWebbasedTool2007].
These datasets provide an additional point of stability between studies and generate more comparable results.
Bioinformatic pipelines and reference databases present an additional source of variation in analysis of HTS data, whereby a ‘one-size-fits-all’ approach is not appropriate. 
The development of freely available applications and pipelines has provided a fundamental basis for *16S rRNA* bioinformatic analyses [@schlossIntroducingMothurOpensource2009;@caporasoQIIMEAllowsAnalysis2010;@edgarSearchClusteringOrders2010]; however care must still be taken, particularly when forming comparisons between data sets analysed with different pipelines.

Estimates of abundance in microbiology are widely used to describe microbial community composition and diversity. 
The genomic copy number of the 16S rRNA gene varies considerably, from 1- 15 copies in some bacteria [@loucaCorrecting16SRRNA2018]. 
Therefore, the variation in abundance of 16S rRNA genes is due to both actual relative abundance differences in samples and variation in 16S rRNA gene copy number among bacteria present. 
In part this can be overcome through ecological models, such as an assessment of beta-diversity, which can be divided into two components; (i) turnover: difference between communities based on species presence/absence; and (ii) nestedness: differences in the abundance of species composition between communities [@baselgaPartitioningTurnoverNestedness2010;@baselgaBetapartPartioningBeta2017].

## One Health & Wildlife Surveillance

The final section of the review will provide some context to the One Health framework.
This is essential to understanding the rational for searching in wildlife for the causative agent(s) of human tick-borne disease.
The complex nature of tick-borne diseases means that they require a number of factors to overlap spatially and temporally.
Finding the pathogenic microbe in humans is only one part of the puzzle, and often the microbe(s) are more readily identified in the tick vector or wildlife reservoir host.
This section will introduce the concept of One Health, it will then briefly review the history of Lyme borreliosis in the United States and provide some insights into the potential cycle of similar tick-borne diseases in an Australian context.

### The One Health Concept

The One Health concept is based around a seamless interaction between veterinary and human medicine, bringing together clinicians, researchers, agencies and governments. 
The modern One Health trend usually focuses on zoonotic pathogens emerging from wildlife and production animal species [@dayOneHealthImportance2011]. 
Understanding of the role wildlife reservoirs have in the maintenance of pathogenic hosts has significantly shifted our view on human health. 
The use of wildlife surveillance as a tool for the detection of emerging zoonotic infectious disease is well established in some contexts. 
For example, the sylvatic life cycle of *Tr. cruzi* in South America [@denoyaEcologicalOverviewFactors2015], Ebola virus in Africa [@osterholmTransmissionEbolaViruses2015], Australian bat lyssavirus [@mayIdentificationFocusAreas2020]. 
Research has shown that compared to human infection, these zoonotic microbes can be readily identified in their respective wildlife reservoirs.
The mosquito-borne West Nile virus life cycle has been well documented and the sensitivity of detection is well understood.
It shows that surveillance of sentinel hosts and vectors provide a quicker and more sensitive method detection method than waiting for signs of infected people [@lemonGlobalInfectiousDisease2007]. 

Tick-borne diseases present a particularly challenging system to study, with a variety of complex factors influencing the epidemiology. 
The presence of tick-borne diseases involves the interplay between the pathogen, host, vector and the environment; with major contributors including climatic change, globalization, increased global travel and trade, urbanization and drug resistance [@dantas-torresClimateChangeBiodiversity2015;@kulesChallengesAdvancesDiagnosis2017;@gilbertImpactsClimateChange2021]. 
Examples of established monitoring programs include; tick surveillance in Great Britain [@jamesonTickSurveillanceGreat2011;@cullSurveillanceBritishTicks2018] and use of hunter-killed white-tailed deer (*Odocoileus virginianus*) in Canada [@bouchardHarvestedWhitetailedDeer2013]. 

Currently in Australia there are a number of arbovirus monitoring programs that are operated by governments or partner organisations.
For example sentinel ruminant herds are used to monitor the distribution of arboviruses and their vectors in Australia such as Bluetongue, Akabane and Bovine ephemeral fever (BEF) viruses [@nationalarbovirusmonitoringprogramNationalArbovirusMonitoring2019]. 
In Western Australia the Sentinel Chicken Surveillance Program is used as an early warning system to detect increases in flavivirus activity, mainly focused on the mosquito-borne Murray Valley encephalitis (MVE) and Kunjin (KUN) viruses [@departmentofhealthMedicalEntomologyAnnual2020].
However there is currently no state or national level surveillance of tick-borne pathogens in Australia.

### Lyme Borreliosis

Lyme arthritis was first described in the scientific literature in 1977 from a cluster of cases in eastern Connecticut [@steereLymeArthritisEpidemic1977]. 
The illness was characterized by recurrent attack of asymmetric swelling and joint paint. 
The authors described a geographical clustering of patients in sparsely settled areas with woody surrounds and peak disease occurrence in the summer months.
As a result doctors concluded the disease was best explained by transmission of an infectious agent by an arthropod vector. 
In 1982 a study demonstrated that *Ixodes dammini* (now recognised as *Ix. scapularis* [@sandersIxodesDamminiJunior1998]), was responsible for the transmission of a *Treponema*-like spirochaete causing long-lasting cutaneous lesions on rabbits 10-12 weeks after tick attachment [@burgdorferLymeDiseaseTickborne1982]. 
It was the first of its study to investigate to the role of vectors in the transmission of Lyme borreliosis in North America. 
A formal description of *B. burgdorferi* followed [@johnsonBorreliaBurgdorferiSp1984] and in 1997 [@fraserGenomicSequenceLyme1997] was the third bacteria to have its genome sequenced.

#### The ecology of *Borrelia* in North America

In most areas of North America the primary vector of *B. burgdorferi* s. l. is *Ix. scapularis*, and major reservoir hosts are well known to include white-footed mice and white-tailed deer [@halseyRoleIxodesScapularis2018]. 
However pine squirrels and deer mice have been identified as host sentinel hosts for *Borrelia hermsii* in western North America [@cadenasIdentificationHostBloodmeal2007]. 
Studies have identified significant difference in species diversity among sites, noting that islands have fewer species than mainland. 
Body mass has also been shown to have an effect on transmission dynamics.
A positive correlation between shown between host body mass and tick burdens for the different stages of *Ix. ricinus* [@hofmeesterFewVertebrateSpecies2016]. 
Nymphal burdens was positively correlated with increased infection of *B. burgdorgferi*.
The study also demonstrated that only a few hosts feed the majority of *Ix. ricinus* and they usually include the most widespread species in the environment.

### An Australian Framework to an endemic zoonotic tick-borne disease

The search for a novel zoonotic tick-borne disease in Australia faces a number of challenges. 
In order to robustly investigate potential novel pathogens it is important that detection methods are sufficiently broad, to limit biases. 
Using *16S rRNA* high-throughput sequencing it has revealed that Australian ticks have a unique suite of bacteria [@goftonBacterialProfilingReveals2015;@goftonInhibitionEndosymbiontCandidatus2015]. 
In particular a number of bacterial organisms, that are similar to, yet unique from global TBPs have since been described from Australian ticks [@goftonPhylogeneticCharacterisationTwo2016;@lohNovelBorreliaSpecies2016;@goftonDetectionPhylogeneticCharacterisation2017;@goftonNovelEhrlichiaSpecies2018].       
Even in the case of rickettsial diseases, which are recognised human tick-borne pathogens in Australia, the distribution and public health impact is poorly defined [@stewartRickettsiaAustralisQueensland2017]. 
There is currently no compulsory reporting of rickettsial infections in Australia. 
Additionally acute infections can be misdiagnosed or treated ineffectively. 
The genetic similarity of rickettsial organisms also makes accurate diagnosis difficult. 
Currently serological tests remain the ‘gold standard’ for the diagnosis of a rickettsial infection in Australia, however serological results often result in cross-reactivity with both other rickettsial species and nonrickettsial infectious diseases. 
In the search for a human disease(s) related to tick bites, records of human-tick associations are critical. 
Much of the research to date has centered around the paralysis tick (*Ix. holocyclus*), which is present along the east coast of Australia. 
However increasingly reports of tick-human associations have expanded geographically. 
A tissue punch biopsy or swab either at site of tick attachment or site of eschar has been shown to have a higher chance of identifying potential tick-borne pathogens than blood samples [@portilloGuidelinesDetectionRickettsia2017].  


# Thesis aims{-}

The overarching aims of this thesis are to understand the ticks parasitising urban wildlife and to characterise the microbial community associated with these ticks and their wildlife hosts. To address these aims, this thesis is divided into three major themes.

**1. Australian ticks**

Chapter \@ref(austicks) reviews records of Australian ticks in respect to distribution, hosts and genetic information.
This chapter reviews and curates records of Australian ticks collected from museum collections, public databases (e.g. living atlas Australia) and the literature.
Records of tick species from humans is also provided, and expands the number of tick species recorded biting humans in Australia. 
Updated distribution maps are provided for three common and wide spread species in Australia; the ornate kangaroo tick *Amblyomma triguttatum*, Australian paralysis tick *Ixodes holocyclus* and the marsupial tick *Ixodes tasmani*.
Genetic information of Australian ticks is synthesised and new data is presented.
In this chapter, a high-throughput sequencing approach using the 12S rRNA gene was developed and applied to the identification of hard ticks.

<!---
This is a common human biting tick with a wide distribution across Australia. 
The species is currently divided into four subspecies, however as noted by Roberts [-@robertsStatusMorphologicallyDivergent1962] "..the decision to allot subspecific status to the four forms is provisional...". 
Chapter \@ref(atrig) reviewed records of *Am. triguttatum*, providing an update on the distribution and hosts of this tick. 
In addition, morphological and molecular characterisation were also performed to inform the systematics of this tick. 
Chapter \@ref(wildlife-ticks) provided molecular insights into Australian ticks and a review of tick records from humans. 
-->

**2. Bacteria and haemoprotozoa present in wildlife and ticks**

For Chapters \@ref(wildlife-bacteria) and \@ref(wildlife-haemoprotozoa), samples were collected from Australian wildlife. 
Small mammal trapping was carried out in Perth, Western Australia and Sydney, New South Wales and blood, tissue and tick samples were collected from animals.
Amplicon metabarcoding on the Illumina MiSeq platform and targeted Sanger sequencing were used to characterise a suite of bacteria and selected haemoprotozoa.
This method provides an unbiased approach to investigate (i) the microbes present in these wildlife samples and (ii) uncover any overlap between sample types in order to identify microbes that may be transmitted by ticks into wildlife, which potentially act as reservoir hosts.

**3. Molecular characterisation microbes**

Chapters \@ref(black-rat) and \@ref(tas-devil) provide targeted information about selected haemoprotozoa from wildlife. 
*Trypanosoma lewisi* identified in Chapter \@ref(black-rat) was identified in blood samples. 
As Chapter \@ref(wildlife-haemoprotozoa) made use of data collected over an extended period of time, Chapter \@ref(black-rat) was written as a short communication to allow rapid dissemination of this result. 
Additionally, Chapter \@ref(black-rat) includes a near full length characterisation of the 18S rRNA gene that informs the phylogenetic position of this *Tr. lewisi*-like species. 
A collaborative research opportunity arose during my candidature to provide the first molecular screening of blood parasites from the iconic Tasmanian devil (Chapter \@ref(tas-devil)). 
While this paper presents a Tasmanian devil-centric view of haemoprotozoa, it also describes a surveillance method for potential zoonotic pathogens in previously neglected areas.