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The brown dog tick Rhipicephalus sanguineus sensu Roberts, 1965 across Australia: Morphological and molecular identification of R. sanguineus s.l. tropical lineage
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The brown dog tick Rhipicephalus sanguineus sensu Roberts, 1965 across Australia: Morphological and molecular identification of R. sanguineus s.l. tropical lineage Shona Chandraa, Gemma C. Maa, Alex Burleighb, Graeme Browna, Jacqueline M. Norrisa, ⁎ Michael P. Wardc, David Emerya, Jan Šlapetaa, a b c
Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, Sydney, 2006, New South Wales, Australia Northern Territory Veterinary Services, Katherine, 0850, Northern Territory, Australia Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, Camden, 2570, New South Wales, Australia
A R T I C LE I N FO
A B S T R A C T
Keywords: cox1 Brown dog tick Mitochondrial DNA Remote locations Survey Rhipicephalus sanguineus
The brown dog tick Rhipicephalus sanguineus (Latreille, 1806) is the most widely distributed tick species globally. Throughout the world there are at least two divergent lineages on dogs that are traditionally grouped into what was known as R. sanguineus. The species R. sanguineus was recently redescribed using a neotype reported from countries with a temperate climate. The second lineage distributed in countries with primarily tropical climates is currently designated R. sanguineus s.l. tropical lineage. Here, we present a comprehensive genetic evaluation of Australian brown dog ticks from across the continent that complements the morphological study of R. sanguineus sensu Roberts (1965). A total of 294 ticks were collected from dogs around Australia ― including New South Wales, Queensland, the Northern Territory and Western Australia ― for morphological identification. All ticks were morphologically identified as R. sanguineus sensu Roberts (1965). DNA was isolated from a single leg from morphologically characterised individuals from New South Wales (n = 14), Queensland (n = 18), Northern Territory (n = 7) and Western Australia (n = 13), together with ticks from Fiji (n = 1) and the Seychelles (n = 1) for comparison with Australian ticks. The study revealed three cox1 haplotypes clustered only with R. sanguineus s.l. tropical lineage’. An updated distribution of R. sanguineus sensu Roberts (1965) is compared to the 1965 distribution. In the Australian context, R. sanguineus s.l. has appeared in north-western New South Wales but remains absent from coastal New South Wales. Despite both temperate and tropical climates being present in Australia, only R. sanguineus s.l. tropical lineage was found. The evidence does not support the presence of the strictly defined brown dog tick, R. sanguineus by Nava et al. (2018) in Australia, because the examined ticks are genetically and morphologically distinct. We recommend using the term brown dog tick, R. sanguineus sensu Roberts (1965) for specimens from Australia.
1. Introduction The brown dog tick Rhipicephalus sanguineus (Latreille, 1806) is an important parasite to the medical and veterinary communities due to vector competence for several pathogens (Seneviratna et al., 1973; Walker et al., 2005; Otranto et al., 2009). Found in all but one
continent, this endophilic tick predominantly parasitises canids, contributing to its globally distributed status (Walker et al., 2005). Two divergent lineages – temperate and tropical – have been recognised and are the subject of numerous taxonomic and biological studies (MoraesFilho et al., 2011; Dantas-Torres et al., 2013; Zemtsova et al., 2016; Jones et al., 2017; Low and Prakash, 2018; Nava et al., 2018). A
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Corresponding author. E-mail addresses: [email protected] (S. Chandra), [email protected] (G.C. Ma), [email protected] (A. Burleigh), [email protected] (G. Brown), [email protected] (J.M. Norris), [email protected] (M.P. Ward), [email protected] (D. Emery), [email protected] (J. Šlapeta).
https://doi.org/10.1016/j.ttbdis.2019.101305 Received 24 May 2019; Received in revised form 13 August 2019; Accepted 21 September 2019 1877-959X/ © 2019 Published by Elsevier GmbH.
Please cite this article as: Shona Chandra, et al., Ticks and Tick-borne Diseases, https://doi.org/10.1016/j.ttbdis.2019.101305
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neotype belonging to the ‘temperate lineage’ of the brown dog tick was used to redescribe R. sanguineus1 (Nava et al., 2018). Larger countries spanning both temperate and tropical zones ― such as the United States of America (USA) and Brazil ― are known to possess both lineages of brown dog ticks (Moraes-Filho et al., 2011; Jones et al., 2017). Similarly, Australia is a continent with tropical to temperate climatic zones, where the brown dog tick is well described morphologically (Roberts, 1965, 1970; Greay et al., 2016). No comprehensive molecular survey of Australian brown dog ticks exists to complement the detailed morphological survey of Roberts (1965). In Australia, the brown dog tick R. sanguineus sensu lato (s.l.) was first described from a dog in Queensland (QLD) (Neumann, 1897). In Australia, Roberts (1965) is the definitive morphological study for this species and the first and only distributional analysis across the continent. The only molecular identification based on a specimen from New South Wales (NSW) demonstrated the presence of R. sanguineus ‘tropical lineage’ (Dantas-Torres et al., 2013). This study surveyed R. sanguineus s.l. found Australia-wide to elucidate its morphological and molecular identity. Ticks were collected from dogs from NSW, QLD, the Northern Territory (NT) and Western Australia (WA) and were identified morphologically by conventional taxonomy and confirmed with scanning electron microscopy (SEM). Molecular identification was determined at the cytochrome c oxidase subunit I (cox1) mitochondrial DNA level. The study enabled us to provide an update of the current distribution and lineage status of the brown dog ticks in Australia.
2.2. Morphological identification and scanning electron microscopy (SEM) All ticks were morphologically identified using a stereo microscope (SMZ-2B, Nikon, Australia) using various keys and guides (Roberts, 1965, 1970; Walker et al., 2003). Morphologically important features were more closely visualised and photographed using a digital microscope (VHX-6000, KEYENCE Inc., Japan). The ticks used for SEM were R. sanguineus s.l. “Coominya” strain, reared by the von Berky Veterinary Services (VBVS), as colony ticks on dogs. Ticks of this strain were sourced from an undisclosed greyhound breeding and training property in Coominya, South East QLD and from the NT between 2004 and 2008. The colony ticks have been independently maintained on dogs at the study site and the strain has progressed through at least 20 life cycles, since it was initially established in 2009. Ticks of this strain have not been introduced to new genetic material since being established. Key morphological features were excised from male and female colony tick samples, identified as R. sanguineus s.l. “Coominya” strain, using a clean no. 11 scalpel blade and was adhered to a 12.7 mm mounting SEM pin stub (Ted Pella Inc., USA) with double sided carbon tape. It was then placed on a rotary planetary specimen stage within a K550X Sputter Coater unit (Quorum Technologies, Kent, UK) and was coated in gold (Au) with the following parameters: current 25 mA, time 2:00 min and coating 15 nm. Once coated, the specimen was placed in a JEOL Neoscope, JCM-6000 (JEOL Inc., Nikon Inc., Japan) for SEM observation.
2.3. DNA isolation and amplification of the cytochrome c oxidase subunit I (cox1)
2. Materials and methods
One to five ticks were randomly selected per sample and one leg at the fourth coxae was excised for DNA isolation using a sterile single use no. 11 scalpel blade. The remainder of the tick was kept for morphological identification. The excised leg was used for total tick genomic DNA (gDNA) isolation with the ISOLATE II Genomic DNA Kit (Bioline, Australia), as described by Chandra et al. (2019). A 525 nucleotide (nt) 5′ fragment of the cytochrome c oxidase subunit 1 (cox1) gene was amplified in a conventional PCR using forward primer S0725 (F1) (5′-TAC TCT ACT AAT CAT AAA GAC ATT GG3′) and reverse primer S0726 (R1) (5′−CCT CCT CCT GAA GGG TCA AAA AAT GA-3′) as previously described by Chandra et al. (2019). MyTaq™ Red Mix (Bioline, Australia) was used for cox1 amplification in 30 μL reactions using 2 μL template genomic DNA and the PCR was run with the cycling conditions as described by Chandra et al. (2019). All reactions contained a positive control and PCR grade water was used for a no template control. All conventional PCRs were conducted in an Applied Biosystems Veriti™ Thermal Cycler (Life Sciences, Australia) or a T100™ Thermal Cycler (BioRad, Australia). PCR products were sequenced at Macrogen Ltd. (Seoul, South Korea).
2.1. Study site and material collection Australia is a large country and continent that encompasses a land area of 7,682,300 km2 and is situated in the southern hemisphere, between latitudes 10 °S and 43 °S and between longitudes 113 °E and 153 °E (Australian Government, 2018a, b). The climate is highly variable, with the northern aspects of Australia experiencing more tropical climate, whereas the southern localities have more of a temperate climate (Australian Government, 2018b). Central Australia, which makes up a significant portion of the country, is predominantly arid dessert or semi-arid environments (Australian Government, 2018b) (Fig. 1). Ticks from dog hosts (n = 48), Canis lupus familiaris, were collected between 2012–2018 from remote locations across four Australian states and territories (NSW, QLD, NT, WA; Figs. 1 and 2). Most of the ticks from each location were collected from one dog, however ticks from three locations were pooled from multiple dogs (SC0927-SC0930) and were stored in 70% ethanol after collection. Ticks collected were of varying developmental stages (eggs, 46 nymphs, 126 adult males, 117 adult females) and varying levels of engorgement (Fig. 1). The samples were collected from dogs in Aboriginal communities in the NT, WA and QLD. The ticks from NSW were collected from dogs in remote communities (Ma et al., 2019). Additionally, as a comparative measure, we collected one tick from a dog in Victoria, Seychelles in 2013 and one tick from a dog in Viseisei, Fiji in 2011.
2.4. DNA sequence analysis and phylogeny Sequences were assembled using CLC Main Workbench 6.8.1 (CLCbio, Qiagen, Denmark). Phylogenetic analysis of Rhipicephalus spp. DNA and the composition of the nucleotide sequences were determined using MEGA 7.0 (Kumar et al., 2016). Phylogenetic comparison was made between available brown dog tick haplotypes on GenBank (National Center for Biotechnology Information, NCBI) using MEGA 7.0 to determine the haplotypes within the Australian tick population (Kumar et al., 2016). The evolutionary history was inferred using the maximum likelihood (ML) and minimum evolution (ME) method and distances were computed using the Tamura-3 and Kimura-2 methods, respectively, in MEGA 7.0 (Tajima and Nei, 1984; Rzhetsky and Nei, 1992; Kumar et al., 2016).
1 For the purpose of this study, we will use the term Rhipicephalus sanguineus ‘temperate lineage’ as the label for Rhipicephalus sanguineus sensu stricto as redescribed according to the neotype by Nava et al. (2018), and Rhipicephalus sanguineus ‘tropical lineage’ as the label for material genetically identifiable as R. sanguineus sensu lato ‘tropical lineage’. The brown dog tick species complex, which includes the above-mentioned lineages, as well as several other named and unnamed species, will be henceforth referred to as Rhipicephalus sanguineus sensu lato.
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Fig. 1. Map of tick localities and an updated distribution map for Rhipicephalus sanguineus sensu Roberts (1965) in Australia (a). Areas surveyed for the presence of brown dog ticks from Western Australia (b), the Northern Territory (c) and New South Wales (d). Data from Western Australia (b) and New South Wales (d) are anecdotal evidence gathered via phone and email surveys, while data from the Northern Territory (c) was surveyed and visually identified by one of the authors, AB.
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Fig. 2. Degree of tick burden and infestations on dogs in remote locations of Australia. (a)-(c) depicts degree of R. sanguineus sensu Roberts (1965) infestation in dogs from an Aboriginal community in Goodooga, New South Wales. (d) a dog from Walgett, New South Wales with many R. sanguineus sensu Roberts (1965) in the cutaneous marginal pouch of the dog’s ear.
rounded, never spurlike” and spiracular plates were long, narrow and comma-shaped (Figs. 3 and 4) (Roberts, 1965). Comparatively, male R. sanguineus sensu Nava et al. (2018) has quad-triangular festoons with the median festoon being distinctly wider than long, hexagonal profile of the basis capitulum, the ventral adanal plates are fairly broad, long and sub-triangular, the adanal shields are “conspicuous, pointed, shorter than adanal plates, posterior end narrower than the width of adjacent festoon”, the spiracular plates are long and narrow with the “dorsal prolongation narrower than the width of the adjacent festoon” (Nava et al., 2018). Female ticks displayed the morphological features described, including hexagonal dorsal profile of basis capitulum, scutum heavily punctuated with long, pronounced cervical grooves that were “bowshaped”, scutum “posterolateral margins obtusely angled, posterior angle with a small marginal expansion”, spiracular plate is a wide comma-shape and the genital aperture resembled a broad U-shape that is “widely open and rounded, wider than long” located between coxae II (Figs. 3 and 5) (Roberts, 1965). Comparatively, female R. sanguineus sensu Nava et al. (2018) displayed the same hexagonal dorsal profile of the basis capitulum, and the scutum was unornate with moderately punctuated scutum, and pronounced, broad but shallow cervical grooves, the spiracular plate was “broadly elongated with narrow dorsal prolongation”, and the genital aperture was “broadly U-shaped, lateral margins diverging anteriorly, located between coxae II” (Nava et al., 2018).
2.5. Availability of data and material Nucleotide sequence data from this study are available in the GenBank (NCBI) database under accession numbers MK967890MK967943. Sequence alignments and raw SEM images are available at LabArchives: https://doi.org/10.25833/r2sv-qa85. Voucher specimens were deposited in the CSIRO Australian National Insect Collection (ANIC) in Canberra, ACT, Australia.
3. Results 3.1. Rhipicephalus sanguineus s.l. from dogs in remote Aboriginal communities A total of 294 ticks were collected from 48 dogs in remote, Aboriginal communities across the NT, NSW, QLD and WA (Fig. 2). One tick (SC099) had been collected from a puppy at the Sydney Animal Hospitals Inner West (Sydney, NSW) that was recently rehomed from Alice Springs, NT. Morphological analysis using stereo and digital microscopes revealed that all ticks found were R. sanguineus sensu Roberts (1965). Male ticks displayed the same morphological features described, including distinct festoons, usually with protruding median festoon, hexagonal profile of basis capitulum, “adanal shields subtriangular, the internal margin mildly concave anteriorly, its posterior angle usually 4
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Fig. 3. Key morphological features of un-engorged adult male (a, SC0922-1) and female (b, SC0923-1) Rhipicephalus sanguineus sensu Roberts (1965). The following morphological features are displayed for the male specimen (a): (i) subtriangular shape of the adanal shields “the internal margin mildly concave anteriorly, its posterior angle usually rounded, never spurlike” (Roberts, 1965), (ii) long, narrow and commashaped spiracular plate, and (iii) distinct festoons, with protruding median festoon. The following morphological features are displayed for the female specimen (b): (i) the wide, comma-shaped spiracular plate, (ii) the heavily punctuated scutum with long, pronounced, bowshaped cervical grooves, and (iii) broad U-shape of the genital aperture that is “widely open and rounded, wider than long” (Roberts, 1965).
common haplotype (haplotype 1, 34/52, 64.2%) was identical to a R. sanguineus s.l. specimen from a dog in the Seychelles. Haplotype 2 (17/ 52, 34.0%) was identical to R. sanguineus s.l. “Coominya” strain originally from a dog in QLD and R. sanguineus s.l. from a dog in Fiji. All 52 ticks from Australia were molecularly R. sanguineus ‘tropical lineage’. Phylogenetic analysis (ML, ME) revealed a high bootstrap support (99–100%) that the ticks analysed were in the same clade as R. sanguineus ‘tropical lineage’ available on GenBank (NCBI) from Australia, South Africa, Brazil, India and China (Fig. 6). Localities of the DNA characterised ticks were plotted on a map (Fig. 1). An updated distribution map of R. sanguineus s.l. was created based on present records of R. sanguineus s.l. found on dogs in this study and two additional surveys (Fig. 1). Firstly, veterinary health records (2011–2015) of one of us (AB) from the NT and WA were collated for the records of presence of brown dog ticks (Aboriginal communities, n = 56) and included on the map (Fig. 1). Secondly, to determine the anecdotal presence of brown dog ticks in coastal northern NSW and coastal WA. For the coastal NSW survey, 33 veterinary clinics from Port Macquarie to Byron Bay and one clinic in Tamworth and one in Armidale were contacted and six reported the sporadic presence of brown dog ticks (Fig. 1). In WA, 33 veterinary clinics were contacted from Port Hedland to Albury and 11 clinics reported the presence of brown dog ticks on dogs from Port Hedland to the Margaret River. The problem
Nymph ticks had a similar hexagonal shape of the basis capitulum as seen in adult males and females, scutum without obvious punctuations, bifid coxae I and an ovular spiracular plate, as per the literature (Roberts, 1965). Comparatively, nymph R. sanguineus sensu Nava et al. (2018) displayed similar characteristic features as those described by Roberts (1965) including the unornate scutum with “few and shallow punctuations”, the bifid coxae I with triangular spurs (Nava et al., 2018). To pair the morphology of the R. sanguineus s.l. from Australia, we performed SEM to observe key morphological features of the dorsal and ventral views of male and female R. sanguineus s.l. “Coominya” strain laboratory raised colony ticks (Figs. 4 and 5). Our findings confirm that morphologically, male and female brown dog tick specimens were identical to those described by Roberts (1965), hence referred to as R. sanguineus sensu Roberts (1965).
3.2. Rhipicephalus sanguineus ‘tropical lineage’ across Australia For the molecular identity of R. sanguineus sensu Roberts (1965), 52 ticks from Australia (48 from 46 individual dogs, four from four pools of ticks from dogs) were selected for DNA analysis at the cox1 mitochondrial DNA marker (525 nt). Three unique haplotypes were observed (haplotype 1–3; range: 99.43–100.00% identity). The most 5
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Fig. 4. Distinctive characteristics of male Rhipicephalus sanguineus sensu Roberts (1965) “Coominya” strain reared by the von Berky Veterinary Services (VBVS) as colony ticks on dogs from Coominya, Queensland, Australia as seen with SEM and genetically typed as the R. sanguineus ‘tropical lineage’. Dorsal aspect of male R. sanguineus ‘tropical lineage’ (a), ventral aspect of male R. sanguineus ‘tropical lineage’ (b), elongated spiracular plate (c), size proportion and location of spiracular plate relative to adanal plate and accessory shield (d), mouth parts (e), ventral plates of male specimen depicting adanal plates, accessory shields, spiracular plate and festoons (f) are displayed for the “Coominya” strain colony specimens by VBVS.
introduction of the domestic dog during European settlement in 1788 (Roberts, 1965; Van Dyck and Strahan, 2008). Notably, ships from England travelled via Tenerife, Cape Town and Rio De Janeiro, so it is possible that R. sanguineus s.l. was picked up along the way (Martins, 2006). Australia has been heavily involved with the global trade of merchandise and goods and services, with imports and exports, since before Federation in 1901 (Bingham, 2016). It is possible that the brown dog ticks migrated to Australia via the global trade routes, which serviced the Asia-Pacific regions, Europe and North and South America (Bingham, 2016). It has been suggested that the spread and distribution of brown dog ticks across remote and central Australia is associated with nomadic Aboriginal communities (Roberts, 1965; Barker and Walker, 2014). The degree of parasitism is linked to factors including overpopulation and nutritional stresses and is compounded by limited access to veterinary services and acaricide products, features often associated with remote Aboriginal communities. The correlation between tick-borne diseases, and by extension ticks, with low socio-economics has been well documented (Godfrey and Randolph, 2011; Stefanoff et al., 2012), and this applies to both Aboriginal and non-Aboriginal populations in Australia. Until the recent designation of the neotype for R. sanguineus using
areas that were surveyed in WA seemed to be from Port Hedland to Geraldton. Clinics from Perth to the Margaret River only reported the sporadic presence of brown dog ticks.
4. Discussion Historically in Australia, the presence of brown dog ticks, R. sanguineus s.l., was reported initially, from a dog parasitised by six males and five females in QLD, Australia in 1896 (Neumann, 1897). Since then, R. sanguineus s.l. was reported in Australia as a common parasite of dogs (Tryon, 1917, 1919; Seddon, 1947, 1951). Later, Roberts (1965) published a comprehensive taxonomic study on R. sanguineus s.l. in Australia based on material collected from dogs and heavily infested buildings with dogs. To date, it is the most detailed taxonomical publication of brown dog ticks in Australian literature and to authors knowledge, the Southern Hemisphere. Yet, there is only one cox1 sequence of an Australian R. sanguineus s.l. on GenBank (NCBI; accession KC243880) collected from NSW, which is genetically R. sanguineus ‘tropical lineage’ (Dantas-Torres et al., 2013). Since R. sanguineus s.l. are commonly associated with dogs and other wild canids, it is likely that the tick migrated to Australia with the 6
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Fig. 5. Distinctive characteristics of female Rhipicephalus sanguineus sensu Roberts (1965) “Coominya” strain reared by the von Berky Veterinary Services (VBVS) as colony ticks on dogs from Coominya, Queensland, Australia as seen with SEM and genetically typed as the R. sanguineus ‘tropical lineage’. Dorsal aspect of female R. sanguineus ‘tropical lineage’ (a), ventral aspect of female R. sanguineus ‘tropical lineage’ (b), spiracular plate (c), U-shaped posterior lips of genital aperture (d), mouth parts (e), posterior margin of the dorsal scutum (f) are displayed for the “Coominya” strain colony specimens by VBVS.
material genetically typed as R. sanguineus ‘temperate lineage’, there was confusion surrounding the taxonomic status of this species (Nava et al., 2018). Taxonomic ambiguity continues to surround the other ticks within the species complex, notably R. sanguineus ‘tropical lineage’. This study confirms the presence of R. sanguineus ‘tropical lineage’ on dogs across remote, Aboriginal communities of Australia. The areas surveyed were of different climates, ranging from a cooler temperate environment in NSW to dry, arid dessert in the NT and WA and the warm tropics in QLD (Australian Government, 2018a, b). These specimens are identical to those descriptions of Australian brown dog ticks in 1965 and we have confirmed this with scanning electron microscopy (Roberts, 1965; Dantas-Torres et al., 2013). While Nava et al. (2018) defines what the former so-called ‘temperate lineage’ of the brown dog tick is, Roberts (1965) coupled with our current work defines both morphologically and genetically what is known as the ‘tropical lineage’ of the brown dog tick. In Australia, the areas surveyed had a mean temperature between 18 °C and 33 °C during 1961–1991 (Australian Bureau of Meteorology, 2018). Despite the range of environments surveyed the evidence did not support the presence of the re-described R. sanguineus ‘temperate
lineage’ in Australia. Notably, Australia’s temperature (based on ten year averages) has warmed considerably since 1950, and continues to increase (CSIRO and Australian Bureau of Meteorology, 2016). Australia’s monthly temperature anomalies also indicate that there is a marked increase in the maximum average monthly day-time temperatures, from 2.20% observed between 1951–1980 to more than 11.45% from 2001 to 2015 (CSIRO and Australian Bureau of Meteorology, 2016). Since 1965, the range of R. sanguineus sensu Roberts (1965) in Australia has expanded considerably (Roberts, 1965; Greay et al., 2016).The increase in extreme temperatures and the increase incidence of warmer than average days may facilitate the migration and survival of brown dog ticks in Australia in areas where it was previously inhospitable for its life and biological reproduction (CSIRO and Australian Bureau of Meteorology, 2016; Australian Bureau of Meteorology, 2018). Based on our findings, brown dog ticks in Australia do not thrive in coastal NSW. While there is anecdotal evidence for localised cases of sightings of brown dog ticks along coastal NSW, they do not appear to reproduce at the same rates as known endemic sites. Similarly, sightings of brown dog ticks on dogs in Sydney, NSW should 7
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Fig. 6. Molecular phylogenetic analysis of Rhipicephalus sanguineus s.l. at the cytochrome c oxidase subunit I (cox1) ribosomal DNA loci. The evolutionary history, evolutionary distance and evolutionary rate differences were inferred by using the following methods: maximum likelihood method based on the Tamura-3 model and Gamma distribution was used to model the evolutionary rate differences among sites (4 categories) (ML T92 + G) and minimum evolution with Kimura-2 Parameters and Gamma distribution (4 categories) (ME K2P + G). Test of phylogeny was implicated through Bootstrap method (500 replications). Codon positions included were 1st and 2nd. The bootstrap confidence intervals (%) have been grouped as follows: ML T92 + G / ME K2P + G, and values < 50% have been hidden. The evolutionary analyses were conducted in MEGA 7 (Kumar et al., 2016). The cox1 sequence of the recently designated neotype for R. sanguineus (MH630346) by Nava et al. (2018) from Montpellier, France has been indicated in bold.
the target organism by international and national regulatory bodies (Australian Pesticides and Veterinary Medicines Authority, 2018). The designation ‘R. sanguineus’ for efficacy studies is not further specified, and thus is ambiguous, because of the existence of multiple distinct genetic, biological and ecological lineages (Moraes-Filho et al., 2015; Jones et al., 2017; Dantas-Torres et al., 2018). One of those lineages, R. sanguineus ‘temperate lineage’, includes the neotype of R. sanguineus from Montpellier, France (Nava et al., 2018). If we would accept a restricted view that only the ‘temperate lineage’ is called R. sanguineus, we immediately put in question most (if not all) efficacy studies, because it is not straightforward to review which lineage(s) were used in the clinical trials. Most studies and trials use morphological identification only, but the unequivocal, morphological identification of R. sanguineus from all other related lineages is challenging. Even in the redescription of R. sanguineus authors conclude that “morphological diagnoses of ticks from this group will continue to be difficult” (Nava et al., 2018). The present study demonstrates the presence of R. sanguineus sensu Roberts (1965) (= ‘tropical lineage’) across Australia at the mtDNA cox1 level. This contrasts with the USA and Brazil, which have both R.
be considered imports from endemic regions. It is likely that coastal NSW does not provide the ideal environmental conditions as there is no biomass to encourage proliferation and it is warm enough for dogs to live outdoors, whereas brown dog ticks have adapted to be endophilic. In Spain, R. sanguineus ‘temperate lineage’ was not reported along the coastal, Atlantic ecosystem, but present in the inland, Mediterranean ecosystems (Sobrino et al., 2012). It was suggested that the microclimates in the Atlantic ecosystem were too wet and unfavourable for the endophilic tick (Sobrino et al., 2012). However, with continued climate change, it is possible that brown dog ticks will continue to spread further south in Australia to areas where people and dogs coexist. The veterinary implications of their migration include the spread of vectored canine tick-borne diseases and dogs with high tick burdens can present with clinical anaemia (Hii et al., 2015; Shapiro et al., 2017; Greay et al., 2018). The brown dog tick is a well-recognised parasite of veterinary significance and the object of multiple experimental trials to demonstrate efficacy of parasiticides (Rohdich et al., 2014; Beugnet et al., 2015; Burgio et al., 2016; McTier et al., 2016). Efficacy data included in registration documents and eventual drug labels claim ‘R. sanguineus’ as 8
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sanguineus ‘temperate lineage’ and R. sanguineus ‘tropical lineage’, despite having a similar geographical size and temperature gradient to Australia (Moraes-Filho et al., 2011; Jones et al., 2017). In validating previous descriptions (Roberts, 1965) we propose that in Australia, brown dog ticks be referred to as R. sanguineus sensu Roberts (1965).
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J. 93, 58–66. https://doi.org/10.1111/avj. 12293. Jones, E.O., Gruntmeir, J.M., Hamer, S.A., Little, S.E., 2017. Temperate and tropical lineages of brown dog ticks in North America. Vet. Parasitol. Reg. Stud. Reports 7, 58–61. https://doi.org/10.1016/j.vprsr.2017.01.002. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. https://doi. org/10.1093/molbev/msw054. Low, V.L., Prakash, B.K., 2018. First genetic characterization of the brown dog tick Rhipicephalus sanguineus sensu lato in Peninsular Malaysia. Exp. Appl. Acarol. 75, 299–307. https://doi.org/10.1007/s10493-018-0279-2. Ma, G.C., Worthing, K.A., Ward, M.P., Norris, J.M., 2019. Commensal Staphylococci including methicillin-resistant Staphylococcus aureus from dogs and cats in remote New South Wales, Australia. Microb. Ecol. https://doi.org/10.1007/s00248-019-01382-y. Martins, L., 2006. A bay to be dreamed of: british visions of Rio de Janeiro. Portuguese Stud. Rev. 22, 19–38. McTier, T.L., Chubb, N., Curtis, M.P., Hedges, L., Inskeep, G.A., Knauer, C.S., Menon, S., Mills, B., Pullins, A., Zinser, E., Woods, D.J., Meeus, P., 2016. Discovery of sarolaner: a novel, orally administered, broad-spectrum, isoxazoline ectoparasiticide for dogs. Vet. Parasitol. 222, 3–11. https://doi.org/10.1016/j.vetpar.2016.02.019. Moraes-Filho, J., Marcili, A., Nieri-Bastos, F.A., Richtzenhain, L.J., Labruna, M.B., 2011. Genetic analysis of ticks belonging to the Rhipicephalus sanguineus group in Latin America. Acta Trop. 117, 51–55. https://doi.org/10.1016/j.actatropica.2010.09.006. Moraes-Filho, J., Krawczak, F.S., Costa, F.B., Soares, J.F., Labruna, M.B., 2015. Comparative evaluation of the vector competence of four south american populations of the rhipicephalus sanguineus group for the bacterium Ehrlichia canis, the agent of canine monocytic ehrlichiosis. (Report). PLoS One 10. https://doi.org/10.1371/ journal.pone.0139386. Nava, S., Beati, L., Venzal, J.M., Labruna, M.B., Szabó, M.P.J., Petney, T., SarachoBottero, M.N., Tarragona, E.L., Dantas-Torres, F., Silva, M.M.S., Mangold, A.J., Guglielmone, A.A., Estrada-Peña, A., 2018. Rhipicephalus sanguineus (Latreille, 1806):
5. Conclusions We propose and recommend that brown dog ticks collected from Australia, be referred to as R. sanguineus sensu Roberts (1965). Morphologically, the Australian brown dog tick is identical to those originally and comprehensively described by Roberts (1965) and genetically it represents the ‘tropical lineage’ of the brown dog tick. We report that only R. sanguineus ‘tropical lineage’ was found in Australia despite the presence of both temperate and tropical climates in the country. Funding The study was in part funded by the Dugdale Guy Peele Bequest (Sydney School of Veterinary Science, University of Sydney) and the Australian Companion Animal Health Foundation grant 001/2017. Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. Sample submissions were provided by veterinary practitioners and approval was not applicable. Declaration of Competing Interest The authors declare that they have no competing interests. Acknowledgements The authors thank and acknowledge all Aboriginal communities and the traditional custodians of the communities involved in this study. We acknowledge and thank Andrew and Janet von Berky, VBVS for supplying us with the laboratory-raised “Coominya” strain of the brown dog tick, maintained by the Investigators VBVS as a colony, using dogs as hosts. We thank Dr. Robert (Bob) Irving, Dr. Anne Fawcett (University of Sydney), Dr. Megan Lui (Zoetis Australia), Dr. Victoria Morin-Adeline (Ortus Laboratories) and Martha Morin for aiding in tick collection and the various veterinary nurses, veterinarians and staff who were involved in the collection of the ticks for this study, including the Northern Territory Veterinary Services, Seadog Vets and Sydney Animal Hospital Inner West. Emma Louise McTavish, Hamish Kadrian, Holly Dawson and Riley Jane Anderson assisted with morphological identification and DNA isolation in this study as part of an undergraduate, self-directed project for the unit of study, AVBS3002 Laboratory Disease Investigations, at the University of Sydney. The authors acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy & Microanalysis, University of Sydney. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.ttbdis.2019.101305. 9
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north-west New South Wales and the Northern Territory, Australia. BMC Vet. Res. 13, 238. https://doi.org/10.1186/s12917-017-1169-2. Sobrino, R., Millán, J., Oleaga, Á., Gortázar, C., de La Fuente, J., Ruiz-Fons, F., 2012. Ecological preferences of exophilic and endophilic ticks (Acari: ixodidae) parasitizing wild carnivores in the Iberian Peninsula. Vet. Parasitol. 184, 248–257. https://doi. org/10.1016/j.vetpar.2011.09.003. Stefanoff, P., Rosinska, M., Samuels, S., White, D.J., Morse, D.L., Randolph, S.E., 2012. A national case-control study identifies human socio-economic status and activities as risk factors for tick-borne encephalitis in Poland. PLoS One 7, e45511. https://doi. org/10.1371/journal.pone.0045511. Tajima, F., Nei, M., 1984. Estimation of evolutionary distance between nucleotide sequences. Mol. Biol. Evol. 1, 269–285. Tryon, H., 1917. Report of the Entomologist and Vegetable Pathologist. Ann. Rep. Qld. Dep. Agric. Stock 1916-1917, Queensland, Australia, pp. 49–63. Tryon, H., 1919. Report of the Entomologist and Vegetable Pathologist. Annual Report of the Queensland Department of Agriculture and Stock 1918-1919, Queensland, Australia, pp. 37–49. Van Dyck, S., Strahan, R., 2008. The Mammals of Australia, 3rd ed. Reed New Holland, Sydney. Walker, A.R., Bouattour, A., Camicas, J.L., Estrada-Peña, A., Horak, I., Latif, A.A., Pegram, R.G., Preston, P.M., 2003. Ticks of Domestic Animals in Africa: a Guide to Identification of Species. 1st ed. Bioscience Reports, Edinburgh, Scotland. Walker, J.B., Horak, I., Keirans, J.E., 2005. The Genus Rhipicephalus (Acardi, Ixodidae): a Guide to the Brown Ticks of the World, 2nd ed. Cambridge University Press, New York, USA. Zemtsova, G.E., Apanaskevich, D.A., Reeves, W.K., Hahn, M., Snellgrove, A., Levin, M.L., 2016. Phylogeography of Rhipicephalus sanguineus sensu lato and its relationships with climatic factors. Exp. Appl. Acarol. 69, 191–203. https://doi.org/10.1007/ s10493-016-0035-4.
Neotype designation, morphological re-description of all parasitic stages and molecular characterization. Ticks Tick. Dis. 9, 1573–1585. https://doi.org/10.1016/j. ttbdis.2018.08.001. Neumann, G., 1897. Revision de la famille des ixodidés. In: Société zoologique de France (Ed.), Mémoires de la Société zoologique de France. Au Siege de la Societe, Paris, pp. 324–423. Otranto, D., Dantas-Torres, F., Breitschwerdt, E.B., 2009. Managing canine vector-borne diseases of zoonotic concern: part one. Trends Parasitol. 25, 157–163. https://doi. org/10.1016/j.pt.2009.01.003. Roberts, F.H.S., 1965. The taxonomic status of the species of the genera Rhipicephalus Koch and Boophilus curtice (Acarina: ixodidae) occurring in Australia. Aust. J. Zool. 13, 491–524. https://doi.org/10.1071/ZO9650491. Roberts, F.H.S., 1970. Australian Ticks, 1st ed. Commonwealth Scientific and Industrial Research Organisation, Melbourne, Australia. Rohdich, N., Roepke, R., Zschiesche, E., 2014. A randomized, blinded, controlled and multi-centered field study comparing the efficacy and safety of Bravecto(TM) (fluralaner) against Frontline(TM) (fipronil) in flea- and tick-infested dogs. Parasit. Vectors 7, 83. https://doi.org/10.1186/1756-3305-7-83. Rzhetsky, A., Nei, M., 1992. A simple method for estimating and testing minimum-evolution trees. Mol. Biol. Evol. 9, 945–967. https://doi.org/10.1093/oxfordjournals. molbev.a040771. Seddon, H.R., 1947. Host Check List of Helminth and Arthropod Parasites Present in Domesticated Animals in Australia, 1st ed. Division of Veterinary Health, Department of Health, Canberra. Seddon, H.R., 1951. Diseases of Domestic Animals in Australia: Tick and Mite Infestations, 1st ed. Division of Veterinary Health, Department of Health, Canberra. Seneviratna, P., Weerasinghe, Ariyadasa, S., 1973. Transmission of Haemobartonella canis by the dog tick, Rhipicephalus sanguineus. Res. Vet. Sci. 14, 112–114. Shapiro, A.J., Brown, G., Norris, J.M., Bosward, K.L., Marriot, D.J., Balakrishnan, N., Breitschwerdt, E.B., Malik, R., 2017. Vector-borne and zoonotic diseases of dogs in
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Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
The brown dog tick Rhipicephalus sanguineus sensu Roberts, 1965 across Australia: Morphological and molecular identification of R. sanguineus s.l. tropical lineage Shona Chandraa, Gemma C. Maa, Alex Burleighb, Graeme Browna, Jacqueline M. Norrisa, ⁎ Michael P. Wardc, David Emerya, Jan Šlapetaa, a b c
Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, Sydney, 2006, New South Wales, Australia Northern Territory Veterinary Services, Katherine, 0850, Northern Territory, Australia Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, Camden, 2570, New South Wales, Australia
A R T I C LE I N FO
A B S T R A C T
Keywords: cox1 Brown dog tick Mitochondrial DNA Remote locations Survey Rhipicephalus sanguineus
The brown dog tick Rhipicephalus sanguineus (Latreille, 1806) is the most widely distributed tick species globally. Throughout the world there are at least two divergent lineages on dogs that are traditionally grouped into what was known as R. sanguineus. The species R. sanguineus was recently redescribed using a neotype reported from countries with a temperate climate. The second lineage distributed in countries with primarily tropical climates is currently designated R. sanguineus s.l. tropical lineage. Here, we present a comprehensive genetic evaluation of Australian brown dog ticks from across the continent that complements the morphological study of R. sanguineus sensu Roberts (1965). A total of 294 ticks were collected from dogs around Australia ― including New South Wales, Queensland, the Northern Territory and Western Australia ― for morphological identification. All ticks were morphologically identified as R. sanguineus sensu Roberts (1965). DNA was isolated from a single leg from morphologically characterised individuals from New South Wales (n = 14), Queensland (n = 18), Northern Territory (n = 7) and Western Australia (n = 13), together with ticks from Fiji (n = 1) and the Seychelles (n = 1) for comparison with Australian ticks. The study revealed three cox1 haplotypes clustered only with R. sanguineus s.l. tropical lineage’. An updated distribution of R. sanguineus sensu Roberts (1965) is compared to the 1965 distribution. In the Australian context, R. sanguineus s.l. has appeared in north-western New South Wales but remains absent from coastal New South Wales. Despite both temperate and tropical climates being present in Australia, only R. sanguineus s.l. tropical lineage was found. The evidence does not support the presence of the strictly defined brown dog tick, R. sanguineus by Nava et al. (2018) in Australia, because the examined ticks are genetically and morphologically distinct. We recommend using the term brown dog tick, R. sanguineus sensu Roberts (1965) for specimens from Australia.
1. Introduction The brown dog tick Rhipicephalus sanguineus (Latreille, 1806) is an important parasite to the medical and veterinary communities due to vector competence for several pathogens (Seneviratna et al., 1973; Walker et al., 2005; Otranto et al., 2009). Found in all but one
continent, this endophilic tick predominantly parasitises canids, contributing to its globally distributed status (Walker et al., 2005). Two divergent lineages – temperate and tropical – have been recognised and are the subject of numerous taxonomic and biological studies (MoraesFilho et al., 2011; Dantas-Torres et al., 2013; Zemtsova et al., 2016; Jones et al., 2017; Low and Prakash, 2018; Nava et al., 2018). A
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Corresponding author. E-mail addresses: [email protected] (S. Chandra), [email protected] (G.C. Ma), [email protected] (A. Burleigh), [email protected] (G. Brown), [email protected] (J.M. Norris), [email protected] (M.P. Ward), [email protected] (D. Emery), [email protected] (J. Šlapeta).
https://doi.org/10.1016/j.ttbdis.2019.101305 Received 24 May 2019; Received in revised form 13 August 2019; Accepted 21 September 2019 1877-959X/ © 2019 Published by Elsevier GmbH.
Please cite this article as: Shona Chandra, et al., Ticks and Tick-borne Diseases, https://doi.org/10.1016/j.ttbdis.2019.101305
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neotype belonging to the ‘temperate lineage’ of the brown dog tick was used to redescribe R. sanguineus1 (Nava et al., 2018). Larger countries spanning both temperate and tropical zones ― such as the United States of America (USA) and Brazil ― are known to possess both lineages of brown dog ticks (Moraes-Filho et al., 2011; Jones et al., 2017). Similarly, Australia is a continent with tropical to temperate climatic zones, where the brown dog tick is well described morphologically (Roberts, 1965, 1970; Greay et al., 2016). No comprehensive molecular survey of Australian brown dog ticks exists to complement the detailed morphological survey of Roberts (1965). In Australia, the brown dog tick R. sanguineus sensu lato (s.l.) was first described from a dog in Queensland (QLD) (Neumann, 1897). In Australia, Roberts (1965) is the definitive morphological study for this species and the first and only distributional analysis across the continent. The only molecular identification based on a specimen from New South Wales (NSW) demonstrated the presence of R. sanguineus ‘tropical lineage’ (Dantas-Torres et al., 2013). This study surveyed R. sanguineus s.l. found Australia-wide to elucidate its morphological and molecular identity. Ticks were collected from dogs from NSW, QLD, the Northern Territory (NT) and Western Australia (WA) and were identified morphologically by conventional taxonomy and confirmed with scanning electron microscopy (SEM). Molecular identification was determined at the cytochrome c oxidase subunit I (cox1) mitochondrial DNA level. The study enabled us to provide an update of the current distribution and lineage status of the brown dog ticks in Australia.
2.2. Morphological identification and scanning electron microscopy (SEM) All ticks were morphologically identified using a stereo microscope (SMZ-2B, Nikon, Australia) using various keys and guides (Roberts, 1965, 1970; Walker et al., 2003). Morphologically important features were more closely visualised and photographed using a digital microscope (VHX-6000, KEYENCE Inc., Japan). The ticks used for SEM were R. sanguineus s.l. “Coominya” strain, reared by the von Berky Veterinary Services (VBVS), as colony ticks on dogs. Ticks of this strain were sourced from an undisclosed greyhound breeding and training property in Coominya, South East QLD and from the NT between 2004 and 2008. The colony ticks have been independently maintained on dogs at the study site and the strain has progressed through at least 20 life cycles, since it was initially established in 2009. Ticks of this strain have not been introduced to new genetic material since being established. Key morphological features were excised from male and female colony tick samples, identified as R. sanguineus s.l. “Coominya” strain, using a clean no. 11 scalpel blade and was adhered to a 12.7 mm mounting SEM pin stub (Ted Pella Inc., USA) with double sided carbon tape. It was then placed on a rotary planetary specimen stage within a K550X Sputter Coater unit (Quorum Technologies, Kent, UK) and was coated in gold (Au) with the following parameters: current 25 mA, time 2:00 min and coating 15 nm. Once coated, the specimen was placed in a JEOL Neoscope, JCM-6000 (JEOL Inc., Nikon Inc., Japan) for SEM observation.
2.3. DNA isolation and amplification of the cytochrome c oxidase subunit I (cox1)
2. Materials and methods
One to five ticks were randomly selected per sample and one leg at the fourth coxae was excised for DNA isolation using a sterile single use no. 11 scalpel blade. The remainder of the tick was kept for morphological identification. The excised leg was used for total tick genomic DNA (gDNA) isolation with the ISOLATE II Genomic DNA Kit (Bioline, Australia), as described by Chandra et al. (2019). A 525 nucleotide (nt) 5′ fragment of the cytochrome c oxidase subunit 1 (cox1) gene was amplified in a conventional PCR using forward primer S0725 (F1) (5′-TAC TCT ACT AAT CAT AAA GAC ATT GG3′) and reverse primer S0726 (R1) (5′−CCT CCT CCT GAA GGG TCA AAA AAT GA-3′) as previously described by Chandra et al. (2019). MyTaq™ Red Mix (Bioline, Australia) was used for cox1 amplification in 30 μL reactions using 2 μL template genomic DNA and the PCR was run with the cycling conditions as described by Chandra et al. (2019). All reactions contained a positive control and PCR grade water was used for a no template control. All conventional PCRs were conducted in an Applied Biosystems Veriti™ Thermal Cycler (Life Sciences, Australia) or a T100™ Thermal Cycler (BioRad, Australia). PCR products were sequenced at Macrogen Ltd. (Seoul, South Korea).
2.1. Study site and material collection Australia is a large country and continent that encompasses a land area of 7,682,300 km2 and is situated in the southern hemisphere, between latitudes 10 °S and 43 °S and between longitudes 113 °E and 153 °E (Australian Government, 2018a, b). The climate is highly variable, with the northern aspects of Australia experiencing more tropical climate, whereas the southern localities have more of a temperate climate (Australian Government, 2018b). Central Australia, which makes up a significant portion of the country, is predominantly arid dessert or semi-arid environments (Australian Government, 2018b) (Fig. 1). Ticks from dog hosts (n = 48), Canis lupus familiaris, were collected between 2012–2018 from remote locations across four Australian states and territories (NSW, QLD, NT, WA; Figs. 1 and 2). Most of the ticks from each location were collected from one dog, however ticks from three locations were pooled from multiple dogs (SC0927-SC0930) and were stored in 70% ethanol after collection. Ticks collected were of varying developmental stages (eggs, 46 nymphs, 126 adult males, 117 adult females) and varying levels of engorgement (Fig. 1). The samples were collected from dogs in Aboriginal communities in the NT, WA and QLD. The ticks from NSW were collected from dogs in remote communities (Ma et al., 2019). Additionally, as a comparative measure, we collected one tick from a dog in Victoria, Seychelles in 2013 and one tick from a dog in Viseisei, Fiji in 2011.
2.4. DNA sequence analysis and phylogeny Sequences were assembled using CLC Main Workbench 6.8.1 (CLCbio, Qiagen, Denmark). Phylogenetic analysis of Rhipicephalus spp. DNA and the composition of the nucleotide sequences were determined using MEGA 7.0 (Kumar et al., 2016). Phylogenetic comparison was made between available brown dog tick haplotypes on GenBank (National Center for Biotechnology Information, NCBI) using MEGA 7.0 to determine the haplotypes within the Australian tick population (Kumar et al., 2016). The evolutionary history was inferred using the maximum likelihood (ML) and minimum evolution (ME) method and distances were computed using the Tamura-3 and Kimura-2 methods, respectively, in MEGA 7.0 (Tajima and Nei, 1984; Rzhetsky and Nei, 1992; Kumar et al., 2016).
1 For the purpose of this study, we will use the term Rhipicephalus sanguineus ‘temperate lineage’ as the label for Rhipicephalus sanguineus sensu stricto as redescribed according to the neotype by Nava et al. (2018), and Rhipicephalus sanguineus ‘tropical lineage’ as the label for material genetically identifiable as R. sanguineus sensu lato ‘tropical lineage’. The brown dog tick species complex, which includes the above-mentioned lineages, as well as several other named and unnamed species, will be henceforth referred to as Rhipicephalus sanguineus sensu lato.
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Fig. 1. Map of tick localities and an updated distribution map for Rhipicephalus sanguineus sensu Roberts (1965) in Australia (a). Areas surveyed for the presence of brown dog ticks from Western Australia (b), the Northern Territory (c) and New South Wales (d). Data from Western Australia (b) and New South Wales (d) are anecdotal evidence gathered via phone and email surveys, while data from the Northern Territory (c) was surveyed and visually identified by one of the authors, AB.
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Fig. 2. Degree of tick burden and infestations on dogs in remote locations of Australia. (a)-(c) depicts degree of R. sanguineus sensu Roberts (1965) infestation in dogs from an Aboriginal community in Goodooga, New South Wales. (d) a dog from Walgett, New South Wales with many R. sanguineus sensu Roberts (1965) in the cutaneous marginal pouch of the dog’s ear.
rounded, never spurlike” and spiracular plates were long, narrow and comma-shaped (Figs. 3 and 4) (Roberts, 1965). Comparatively, male R. sanguineus sensu Nava et al. (2018) has quad-triangular festoons with the median festoon being distinctly wider than long, hexagonal profile of the basis capitulum, the ventral adanal plates are fairly broad, long and sub-triangular, the adanal shields are “conspicuous, pointed, shorter than adanal plates, posterior end narrower than the width of adjacent festoon”, the spiracular plates are long and narrow with the “dorsal prolongation narrower than the width of the adjacent festoon” (Nava et al., 2018). Female ticks displayed the morphological features described, including hexagonal dorsal profile of basis capitulum, scutum heavily punctuated with long, pronounced cervical grooves that were “bowshaped”, scutum “posterolateral margins obtusely angled, posterior angle with a small marginal expansion”, spiracular plate is a wide comma-shape and the genital aperture resembled a broad U-shape that is “widely open and rounded, wider than long” located between coxae II (Figs. 3 and 5) (Roberts, 1965). Comparatively, female R. sanguineus sensu Nava et al. (2018) displayed the same hexagonal dorsal profile of the basis capitulum, and the scutum was unornate with moderately punctuated scutum, and pronounced, broad but shallow cervical grooves, the spiracular plate was “broadly elongated with narrow dorsal prolongation”, and the genital aperture was “broadly U-shaped, lateral margins diverging anteriorly, located between coxae II” (Nava et al., 2018).
2.5. Availability of data and material Nucleotide sequence data from this study are available in the GenBank (NCBI) database under accession numbers MK967890MK967943. Sequence alignments and raw SEM images are available at LabArchives: https://doi.org/10.25833/r2sv-qa85. Voucher specimens were deposited in the CSIRO Australian National Insect Collection (ANIC) in Canberra, ACT, Australia.
3. Results 3.1. Rhipicephalus sanguineus s.l. from dogs in remote Aboriginal communities A total of 294 ticks were collected from 48 dogs in remote, Aboriginal communities across the NT, NSW, QLD and WA (Fig. 2). One tick (SC099) had been collected from a puppy at the Sydney Animal Hospitals Inner West (Sydney, NSW) that was recently rehomed from Alice Springs, NT. Morphological analysis using stereo and digital microscopes revealed that all ticks found were R. sanguineus sensu Roberts (1965). Male ticks displayed the same morphological features described, including distinct festoons, usually with protruding median festoon, hexagonal profile of basis capitulum, “adanal shields subtriangular, the internal margin mildly concave anteriorly, its posterior angle usually 4
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Fig. 3. Key morphological features of un-engorged adult male (a, SC0922-1) and female (b, SC0923-1) Rhipicephalus sanguineus sensu Roberts (1965). The following morphological features are displayed for the male specimen (a): (i) subtriangular shape of the adanal shields “the internal margin mildly concave anteriorly, its posterior angle usually rounded, never spurlike” (Roberts, 1965), (ii) long, narrow and commashaped spiracular plate, and (iii) distinct festoons, with protruding median festoon. The following morphological features are displayed for the female specimen (b): (i) the wide, comma-shaped spiracular plate, (ii) the heavily punctuated scutum with long, pronounced, bowshaped cervical grooves, and (iii) broad U-shape of the genital aperture that is “widely open and rounded, wider than long” (Roberts, 1965).
common haplotype (haplotype 1, 34/52, 64.2%) was identical to a R. sanguineus s.l. specimen from a dog in the Seychelles. Haplotype 2 (17/ 52, 34.0%) was identical to R. sanguineus s.l. “Coominya” strain originally from a dog in QLD and R. sanguineus s.l. from a dog in Fiji. All 52 ticks from Australia were molecularly R. sanguineus ‘tropical lineage’. Phylogenetic analysis (ML, ME) revealed a high bootstrap support (99–100%) that the ticks analysed were in the same clade as R. sanguineus ‘tropical lineage’ available on GenBank (NCBI) from Australia, South Africa, Brazil, India and China (Fig. 6). Localities of the DNA characterised ticks were plotted on a map (Fig. 1). An updated distribution map of R. sanguineus s.l. was created based on present records of R. sanguineus s.l. found on dogs in this study and two additional surveys (Fig. 1). Firstly, veterinary health records (2011–2015) of one of us (AB) from the NT and WA were collated for the records of presence of brown dog ticks (Aboriginal communities, n = 56) and included on the map (Fig. 1). Secondly, to determine the anecdotal presence of brown dog ticks in coastal northern NSW and coastal WA. For the coastal NSW survey, 33 veterinary clinics from Port Macquarie to Byron Bay and one clinic in Tamworth and one in Armidale were contacted and six reported the sporadic presence of brown dog ticks (Fig. 1). In WA, 33 veterinary clinics were contacted from Port Hedland to Albury and 11 clinics reported the presence of brown dog ticks on dogs from Port Hedland to the Margaret River. The problem
Nymph ticks had a similar hexagonal shape of the basis capitulum as seen in adult males and females, scutum without obvious punctuations, bifid coxae I and an ovular spiracular plate, as per the literature (Roberts, 1965). Comparatively, nymph R. sanguineus sensu Nava et al. (2018) displayed similar characteristic features as those described by Roberts (1965) including the unornate scutum with “few and shallow punctuations”, the bifid coxae I with triangular spurs (Nava et al., 2018). To pair the morphology of the R. sanguineus s.l. from Australia, we performed SEM to observe key morphological features of the dorsal and ventral views of male and female R. sanguineus s.l. “Coominya” strain laboratory raised colony ticks (Figs. 4 and 5). Our findings confirm that morphologically, male and female brown dog tick specimens were identical to those described by Roberts (1965), hence referred to as R. sanguineus sensu Roberts (1965).
3.2. Rhipicephalus sanguineus ‘tropical lineage’ across Australia For the molecular identity of R. sanguineus sensu Roberts (1965), 52 ticks from Australia (48 from 46 individual dogs, four from four pools of ticks from dogs) were selected for DNA analysis at the cox1 mitochondrial DNA marker (525 nt). Three unique haplotypes were observed (haplotype 1–3; range: 99.43–100.00% identity). The most 5
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Fig. 4. Distinctive characteristics of male Rhipicephalus sanguineus sensu Roberts (1965) “Coominya” strain reared by the von Berky Veterinary Services (VBVS) as colony ticks on dogs from Coominya, Queensland, Australia as seen with SEM and genetically typed as the R. sanguineus ‘tropical lineage’. Dorsal aspect of male R. sanguineus ‘tropical lineage’ (a), ventral aspect of male R. sanguineus ‘tropical lineage’ (b), elongated spiracular plate (c), size proportion and location of spiracular plate relative to adanal plate and accessory shield (d), mouth parts (e), ventral plates of male specimen depicting adanal plates, accessory shields, spiracular plate and festoons (f) are displayed for the “Coominya” strain colony specimens by VBVS.
introduction of the domestic dog during European settlement in 1788 (Roberts, 1965; Van Dyck and Strahan, 2008). Notably, ships from England travelled via Tenerife, Cape Town and Rio De Janeiro, so it is possible that R. sanguineus s.l. was picked up along the way (Martins, 2006). Australia has been heavily involved with the global trade of merchandise and goods and services, with imports and exports, since before Federation in 1901 (Bingham, 2016). It is possible that the brown dog ticks migrated to Australia via the global trade routes, which serviced the Asia-Pacific regions, Europe and North and South America (Bingham, 2016). It has been suggested that the spread and distribution of brown dog ticks across remote and central Australia is associated with nomadic Aboriginal communities (Roberts, 1965; Barker and Walker, 2014). The degree of parasitism is linked to factors including overpopulation and nutritional stresses and is compounded by limited access to veterinary services and acaricide products, features often associated with remote Aboriginal communities. The correlation between tick-borne diseases, and by extension ticks, with low socio-economics has been well documented (Godfrey and Randolph, 2011; Stefanoff et al., 2012), and this applies to both Aboriginal and non-Aboriginal populations in Australia. Until the recent designation of the neotype for R. sanguineus using
areas that were surveyed in WA seemed to be from Port Hedland to Geraldton. Clinics from Perth to the Margaret River only reported the sporadic presence of brown dog ticks.
4. Discussion Historically in Australia, the presence of brown dog ticks, R. sanguineus s.l., was reported initially, from a dog parasitised by six males and five females in QLD, Australia in 1896 (Neumann, 1897). Since then, R. sanguineus s.l. was reported in Australia as a common parasite of dogs (Tryon, 1917, 1919; Seddon, 1947, 1951). Later, Roberts (1965) published a comprehensive taxonomic study on R. sanguineus s.l. in Australia based on material collected from dogs and heavily infested buildings with dogs. To date, it is the most detailed taxonomical publication of brown dog ticks in Australian literature and to authors knowledge, the Southern Hemisphere. Yet, there is only one cox1 sequence of an Australian R. sanguineus s.l. on GenBank (NCBI; accession KC243880) collected from NSW, which is genetically R. sanguineus ‘tropical lineage’ (Dantas-Torres et al., 2013). Since R. sanguineus s.l. are commonly associated with dogs and other wild canids, it is likely that the tick migrated to Australia with the 6
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Fig. 5. Distinctive characteristics of female Rhipicephalus sanguineus sensu Roberts (1965) “Coominya” strain reared by the von Berky Veterinary Services (VBVS) as colony ticks on dogs from Coominya, Queensland, Australia as seen with SEM and genetically typed as the R. sanguineus ‘tropical lineage’. Dorsal aspect of female R. sanguineus ‘tropical lineage’ (a), ventral aspect of female R. sanguineus ‘tropical lineage’ (b), spiracular plate (c), U-shaped posterior lips of genital aperture (d), mouth parts (e), posterior margin of the dorsal scutum (f) are displayed for the “Coominya” strain colony specimens by VBVS.
material genetically typed as R. sanguineus ‘temperate lineage’, there was confusion surrounding the taxonomic status of this species (Nava et al., 2018). Taxonomic ambiguity continues to surround the other ticks within the species complex, notably R. sanguineus ‘tropical lineage’. This study confirms the presence of R. sanguineus ‘tropical lineage’ on dogs across remote, Aboriginal communities of Australia. The areas surveyed were of different climates, ranging from a cooler temperate environment in NSW to dry, arid dessert in the NT and WA and the warm tropics in QLD (Australian Government, 2018a, b). These specimens are identical to those descriptions of Australian brown dog ticks in 1965 and we have confirmed this with scanning electron microscopy (Roberts, 1965; Dantas-Torres et al., 2013). While Nava et al. (2018) defines what the former so-called ‘temperate lineage’ of the brown dog tick is, Roberts (1965) coupled with our current work defines both morphologically and genetically what is known as the ‘tropical lineage’ of the brown dog tick. In Australia, the areas surveyed had a mean temperature between 18 °C and 33 °C during 1961–1991 (Australian Bureau of Meteorology, 2018). Despite the range of environments surveyed the evidence did not support the presence of the re-described R. sanguineus ‘temperate
lineage’ in Australia. Notably, Australia’s temperature (based on ten year averages) has warmed considerably since 1950, and continues to increase (CSIRO and Australian Bureau of Meteorology, 2016). Australia’s monthly temperature anomalies also indicate that there is a marked increase in the maximum average monthly day-time temperatures, from 2.20% observed between 1951–1980 to more than 11.45% from 2001 to 2015 (CSIRO and Australian Bureau of Meteorology, 2016). Since 1965, the range of R. sanguineus sensu Roberts (1965) in Australia has expanded considerably (Roberts, 1965; Greay et al., 2016).The increase in extreme temperatures and the increase incidence of warmer than average days may facilitate the migration and survival of brown dog ticks in Australia in areas where it was previously inhospitable for its life and biological reproduction (CSIRO and Australian Bureau of Meteorology, 2016; Australian Bureau of Meteorology, 2018). Based on our findings, brown dog ticks in Australia do not thrive in coastal NSW. While there is anecdotal evidence for localised cases of sightings of brown dog ticks along coastal NSW, they do not appear to reproduce at the same rates as known endemic sites. Similarly, sightings of brown dog ticks on dogs in Sydney, NSW should 7
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Fig. 6. Molecular phylogenetic analysis of Rhipicephalus sanguineus s.l. at the cytochrome c oxidase subunit I (cox1) ribosomal DNA loci. The evolutionary history, evolutionary distance and evolutionary rate differences were inferred by using the following methods: maximum likelihood method based on the Tamura-3 model and Gamma distribution was used to model the evolutionary rate differences among sites (4 categories) (ML T92 + G) and minimum evolution with Kimura-2 Parameters and Gamma distribution (4 categories) (ME K2P + G). Test of phylogeny was implicated through Bootstrap method (500 replications). Codon positions included were 1st and 2nd. The bootstrap confidence intervals (%) have been grouped as follows: ML T92 + G / ME K2P + G, and values < 50% have been hidden. The evolutionary analyses were conducted in MEGA 7 (Kumar et al., 2016). The cox1 sequence of the recently designated neotype for R. sanguineus (MH630346) by Nava et al. (2018) from Montpellier, France has been indicated in bold.
the target organism by international and national regulatory bodies (Australian Pesticides and Veterinary Medicines Authority, 2018). The designation ‘R. sanguineus’ for efficacy studies is not further specified, and thus is ambiguous, because of the existence of multiple distinct genetic, biological and ecological lineages (Moraes-Filho et al., 2015; Jones et al., 2017; Dantas-Torres et al., 2018). One of those lineages, R. sanguineus ‘temperate lineage’, includes the neotype of R. sanguineus from Montpellier, France (Nava et al., 2018). If we would accept a restricted view that only the ‘temperate lineage’ is called R. sanguineus, we immediately put in question most (if not all) efficacy studies, because it is not straightforward to review which lineage(s) were used in the clinical trials. Most studies and trials use morphological identification only, but the unequivocal, morphological identification of R. sanguineus from all other related lineages is challenging. Even in the redescription of R. sanguineus authors conclude that “morphological diagnoses of ticks from this group will continue to be difficult” (Nava et al., 2018). The present study demonstrates the presence of R. sanguineus sensu Roberts (1965) (= ‘tropical lineage’) across Australia at the mtDNA cox1 level. This contrasts with the USA and Brazil, which have both R.
be considered imports from endemic regions. It is likely that coastal NSW does not provide the ideal environmental conditions as there is no biomass to encourage proliferation and it is warm enough for dogs to live outdoors, whereas brown dog ticks have adapted to be endophilic. In Spain, R. sanguineus ‘temperate lineage’ was not reported along the coastal, Atlantic ecosystem, but present in the inland, Mediterranean ecosystems (Sobrino et al., 2012). It was suggested that the microclimates in the Atlantic ecosystem were too wet and unfavourable for the endophilic tick (Sobrino et al., 2012). However, with continued climate change, it is possible that brown dog ticks will continue to spread further south in Australia to areas where people and dogs coexist. The veterinary implications of their migration include the spread of vectored canine tick-borne diseases and dogs with high tick burdens can present with clinical anaemia (Hii et al., 2015; Shapiro et al., 2017; Greay et al., 2018). The brown dog tick is a well-recognised parasite of veterinary significance and the object of multiple experimental trials to demonstrate efficacy of parasiticides (Rohdich et al., 2014; Beugnet et al., 2015; Burgio et al., 2016; McTier et al., 2016). Efficacy data included in registration documents and eventual drug labels claim ‘R. sanguineus’ as 8
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sanguineus ‘temperate lineage’ and R. sanguineus ‘tropical lineage’, despite having a similar geographical size and temperature gradient to Australia (Moraes-Filho et al., 2011; Jones et al., 2017). In validating previous descriptions (Roberts, 1965) we propose that in Australia, brown dog ticks be referred to as R. sanguineus sensu Roberts (1965).
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5. Conclusions We propose and recommend that brown dog ticks collected from Australia, be referred to as R. sanguineus sensu Roberts (1965). Morphologically, the Australian brown dog tick is identical to those originally and comprehensively described by Roberts (1965) and genetically it represents the ‘tropical lineage’ of the brown dog tick. We report that only R. sanguineus ‘tropical lineage’ was found in Australia despite the presence of both temperate and tropical climates in the country. Funding The study was in part funded by the Dugdale Guy Peele Bequest (Sydney School of Veterinary Science, University of Sydney) and the Australian Companion Animal Health Foundation grant 001/2017. Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. Sample submissions were provided by veterinary practitioners and approval was not applicable. Declaration of Competing Interest The authors declare that they have no competing interests. Acknowledgements The authors thank and acknowledge all Aboriginal communities and the traditional custodians of the communities involved in this study. We acknowledge and thank Andrew and Janet von Berky, VBVS for supplying us with the laboratory-raised “Coominya” strain of the brown dog tick, maintained by the Investigators VBVS as a colony, using dogs as hosts. We thank Dr. Robert (Bob) Irving, Dr. Anne Fawcett (University of Sydney), Dr. Megan Lui (Zoetis Australia), Dr. Victoria Morin-Adeline (Ortus Laboratories) and Martha Morin for aiding in tick collection and the various veterinary nurses, veterinarians and staff who were involved in the collection of the ticks for this study, including the Northern Territory Veterinary Services, Seadog Vets and Sydney Animal Hospital Inner West. Emma Louise McTavish, Hamish Kadrian, Holly Dawson and Riley Jane Anderson assisted with morphological identification and DNA isolation in this study as part of an undergraduate, self-directed project for the unit of study, AVBS3002 Laboratory Disease Investigations, at the University of Sydney. The authors acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy & Microanalysis, University of Sydney. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.ttbdis.2019.101305. 9
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