VETERINARY JOURNAL ORIGINAL ARTICLE AUSTRALIA'S PREMIER VETERINARY SCIENCE TEXT ‘SMALL ANIMALS Ehrlichia canis rapid spread and possible enzooty in northern South Australia and distribution of its vector Rhipicephalus linnaei ‘ALL Chaber,* REasther,” B Cumming,”® R Irving," AL Keybur,! C Smart!” R OHandley’ © and L Lignerewx” Recent concerns have arisen in Australia regarding detections of the exotic bacterium Ehrlichia canis which has resulted in ehrlichi- (sis outbreaks. In Australia, itis spread by the tropical brown dog tick Rhipicephaluslinnael, formerly Rhipicephalus sanguineus sensu lato tropical lineage. Previously, the tick has been recorded in South Australia in the Coober Pedy and the Codnadatta areas. This study, which includes historical specimens data held in his- torical Australian arthropod collections, along with 10 sampled emote communities, confirms the wide distribution range of this species within the State. E. canis was detected by PCR inthe teks. ‘The percentage of dogs hosting PCR-positive ticks increased from 2.8% (95% confidence interval (Cl: 03 to 9.7) in. November December 2020 to 62:9% (95% Ck: 44.9 to 78.5) end of February 2021, initially in two then in seven Anangu Pitjantatjara Yankunytjatjara lands communities in the far northern regions of South Australia. Our results suggest a rapid spread of the patho- ‘gen. No evidence of E. canis was found in nine regional communi- ties. The extended tropical brown dog tick distribution indicates 2 ‘greater area where E. canis may occur and may require manage- ‘ment to minimise the impacts of ehrlichiosis outbreaks. Without the implementation of effective detection and control programs, this extended distribution of f.linnae’ is likely to result in the spread of the bacterlum to other regions Keywords Australia; Ehrlichia canis, communities; hipicephalus linaet ticks ehrlichiosis, remote Abbreviations APY, Anangu Pijantjatjara.Yankunysatjara; Cl confidence intervals; CME, canine monocytic ehrlichiosis, ‘use et 12022 oe 01 tayj13200 cks are obligatory haemstophagous ectoparasites found ‘worldwide. Ticks belonging to the Ripicephals sanguineus ‘complex have a global distribution. They are endophilic, meaning they reside in homes, dog kennels, and domestic animals! Vicinity.” They tend to prefer low-density housing with access 40 “School of Anal aed Veterinary Sciences Roseworthy Campus Te Univesity of porlnde Adele, South Asta stalls ane se chbersodelide cd. Feral iaragemert in Rl and emt mdigenus Cmenunes (AMR) 4 Sadgroves Crescent Vinele Laan Coury Norther Teta C82, Atos "Rael Herne Verena Grou, 834 Nor ot Rand, Mody South esa 509, Aol "ieassy esetch and Development Tear Commanmeath Scent and nds ‘esearch Organtaton, Austaan Cer for Dect roparedoes, Pate 8a) 24 Gecong tori 3220, Rtas ‘NL Chber and Rasher conve equal to his manuscript (© 202 The Auth Auton Veterinary Jounal pushed by J Wey Sons Astral Ld on behalf Ausaon Veterinary Ascii Aut gardens or open areas rather than high-density urban areas.” Although commonly found inside houses, they can be observed out side, crawling on walls and between rocks.” ‘They are monotropic, three-host tick species, with the domestic dog, Canis lupus familiar, boing the main host. However, they have been reported feeding on other mammal species including humans. Formerly referred to as the R_ sanguineus ‘ropical_ lineage, hipicephalus innaei has been a distinct taxonomic entity since 2021. It is the only species belonging to this group reported in “Austalia, This tick prefers tropical and semi-arid habitats but it was, found in many different climates, including teraperate parts of New South Wales, arid areas of the Northern ‘Territory and Western ‘Austealia, and tropical Queensland Houses and kennels protect the tick against extreme environmental conditions, and R. linac is likly to withstand a larger variety of envizonments.” ‘A.1965 Australian distribution map” shoved the enzootic presence of the tick throughout the northern regions of Western Australi, the majority ofthe Northern Territory and Queensland, and the very northeastem tip of New South Wales. In 2020, this range was expanded to include the entire western coast of Western Australia. and an increased range of northern inland New South Wales. ‘The southward spread is thought to be facilitated by inereases in the average number of warmer days and by human-asisted movements, allowing farther migration of brown dg, ticks to new areas which were previously inhospitable” R. linnact has been recorded in Oodinadatta and Coober Pedy" but otherwise published records of the tck in South Australia are limited. “The tick is a vector for Ehrlichia canis, a Gram-negative obligatory Intracellular bacterium of the order Ricktisiles, first described in 1935!” and which has emerged in Australia very recently." The dog constitutes the reservoir for this pathogen, which parasites eiculat- {ng monocytes causing a syndrome called canine monocytic ehrlil sis (CME). The bacterium circulates in macrophages and replicates, rmainly in the liver and spleen. Incubation in the dog is 8-20 days." It may be followed by an acute disease presenting with various cal signs ut certain cases may remain asymptomatic.” The clinical signs during the acute phase include petechae, ecchymoses, spleno- rmegaly, epistaxis and Iymphadenomegaly." ‘They are caused by autoimmune pancytopenia and lead to pale mucous membranes, peripheral oedema, lethargy, depression, anorexia and tachypnoza.!" Other associated clinical signs include pyrexia, oculonasal discharge, vomiting and weight loss!” Rarely, neurological signs such as cranial nerve dysfunction, seizures, ataxia and paresis wil occur ifthe Th can open access ale unde he terms ofthe iste Commons Aton NonComymetc NoDets Uc, which parts ie and tibution in ary med, ‘roid te ginal work prop hed the utes non conmercl and no masons da PontePore ‘SMALL ANIMALS bacterium enters the cerebrospinal fluid! Subeinical and chronic forms might then follow, lasting for months or years. The chronic phase is invariably terminal Death may occur due to haemorrhage and secondary infection’ before the chronic phase. A daly oral administration of doxyeycline at 10 mg/kg for one month isthe usual recommended antibiotic treatment for acute CME, although treat- ‘ment instituted afer the acute phase may be ineffective." ‘The three-host lifecycle of R.linnaei requires the larval, ayrmphal and adult stages each to ingest blood from diferent dogs, and each stage is susceptible to canis infection. This provides the tick with ‘opportunities to both become infected when feeding on infected logs, and to transmit the bacterium to multiple susceptible dogs.” In the absence of transovaran transmission, only nymphs and adult ticks transmit the bacterium. The adult ticks, especially male, can have more than one blood meal on multiple different hosts, further increasing the number of dogs that can be infected.” Adult ticks can transmit E. canis up to 155 days after becoming infected, allowing survival of the bacterium for extended periods while the tick is less active” Dogs can remain infectious with recurring houts of etic oss long aftr the inital phases of infection, which aids the mainte- nance of E, cans in the tick population." [hlichioss is a notifiable disease in dogs in Australia. Until 2020, i ‘was considered an exotic disease but it has since been reported in the towns of Halls Crock and Kununurra in the Kimberley region of ‘Western Australia in May 2020." In June 2020 it was diagnosed in ‘Territory, in the far northern regions of South Australia in March 2021," and then in the town of Mount Isa in northwest Queensland in January 2022." {In Australia in adlition to B.canis,R, Het is capable of transmit- ting other pathogens to dogs, such as Anaplasma platys and Babesia canis vogeli.” both causing syndromes mimicking CME. Materials and methods “The tick sampling happened in three campaigns during dog desexing and preventative health initiatives. The domestic dogs that were ‘owned in 10 remote Indigenous and nine regional communities in South Australia (sce Figure 1) were screened fo ticks. The screening consisted of manually checking the coat and ears ofthe dogs for ev- dence of attached adult ticks. The ticks were removed and placed in small plastic vials filed with 70% ethanol and kept at ambient temperature. “The fest campaign happened in August 2020 in seven regional com munities (Coober Pedy, Koonibba, Marla, Port Augusta, Oak Valley, odnadatta and Yalata), and the second campaign was done in October and November 2020 in two regional communities (Leigh Greek and Maree) and nine Anangu Pitjantatjra Yankunytjtjara (APY) lands communities. ‘The third and last campaign happened Figure 1. Distribution ofthe tick Rhipicephals ange in South Australia. The name ofthe regional communities where the specimens have been collected during ths study is indicated. A polygon represents the Anangu Ptjantjatjara Yankunytjatjara (APY) Lands. The date of collection and location of the historical specimens are mentioned. Map made with QGIS 3.24 (QGIS Development Team, 2021 QGIS Geographic Information System. Open-Source Geospatial Foundation Project, htp//wwcgis.ora), with free data sets for country boundaries and land borders from ‘Natural Earth. Source: htp/iwwrw.naturalearthdata conv. Australian Veterinary Journal © 2022 The thors Asai VetrinayJoual pushed by John Wik & Sons Austad on behalf of Austin Vteinary Asocaonaa ‘SMALL ANIMALS: luring the last week of February 2021 in 10 APY lands communities, Ticks from the three campaigns were sent to the Commonwealth including the nine communities already sampled. Sclentific and Industrial Research Organisation (Australian Centre [At the University of Adelaide the ticks from the ist wo campaigns cance PCR One ek pe dog font he Beene tan ‘were identified under a dissecting microscope according to the mor phological criteria (Figure 2) outlined by Roberts’ and Barker and Walker" canis by PCR. One tic per dog fromthe fist and the second eam paigns was tested, but whenever a tick elicited a positive reaction, the test was repeated on the other ticks found on the same dog, During the third campaign, the ticks collected from three dogs were pooled peri Figure 2. Unengorged adult female tropical brown dg tick Rhipicephaluslinael and morphological features (A) Dorsal view ofthe whole spect ‘men. The eyes are Indicated. (8) Ventral view ofthe same specimen. Note the hexagonal shape of the bass capitl, the bifid coxae 1 (ovals), and the anal groove posterior tothe anus (ae). (C) Detail of the bass capitull and coxae 7 (0) detall ofthe anal area and adanal plates. The size for teach bor is mentioned. Photographs taken with reversed Componon 28 mm enlarger lens (Schneider Kreuznach, Germany) and Wikon 40.1 Plan Infinity corrected microscope objective Uapan), mounted on Nikon 07200 DSLR. Assembled using focus stacking technique in Zerene stacker (Zerene Systems, Richland, WA, USA. Edited in GIMP 2.10 (the GIMP Development Team hitp//simp ora) © 2022 The Authors Autalion Veterinary Jounal pushed by hn Vey & Sons Ruslan Bea of Australian Veena Association. Astalian Veterinary Journal PoetPorat) ‘SMALL ANIMALS in the specimen vials, and the PCR was done on one tick from each vial. ‘The E canis PCR was performed as fllows: the DNA was extracted fo testing by placing a tick in a2 a bead beating tube (Fastrep. 24, MP Biomedicals, Santa Ana, CA, USA) containing 180 yL ATL Iyss butler (Qiagen, Germany) and two stainless stel 5 mm beads (Qiagen, Germany). The tick was homogenised at 6 m/s for 1 min and the tubes were then spun at 12,000 sf (relative central free) for 1 min, adding 20 pL. of proteinase K, before being incubated at 56°C frat least 3h The homogenate was spin at 1,000 ref for 1 min to pellet debris, the supernatant was careilly poured oto a new 1 ml tube, Equal volumes (200 i) of AL lysis buffer (Qiagen, Germany) and 100% ethanol was added, the tubes were vortexed and the supernatant was transferred to 2 spin column before con- tinuing with te standard Qiagen protocol (DNeasy Blood & Tissue Kit, Qiagen, Germany), The resultant DNA as used in an E canis specific probe-hased qPCR assay as described by Baneth tl." Historical tick collections were a Australia (htpsifwwowalaorgau the Online Zoological Collec- tions of Australian Muscums (hipssocamongasu/), and the Awstralian National Collection — (ps/datacsito sul collection/si641). Both queries “Rhipicephalus tinnae?” and “Rhipicephalus songuincus’ were entered. After downloading the records, the data were cleaned ip Excel (Micros, Redmond, WA, USA) and only the data regarding the specimens elected in South Australia wer retained, through the Atlas of Living Insect ‘The statistical difference between groups was evaluated with a Chi squared test, Differences were considered significant with P value -<005, 95% confidence intervals (95% CI) were calculated with the binomial method. Voucher specimens of R, linnaei collected during this study were posited at the South Australian Museum (Catalogue numbers J 25503-] 28507). Results ‘A total of 153 ticks were collected during the first two campaigns from all sampling sites: nine regional communities and ten APY Jands communities (see Figure 1). They were all R innaci ‘Among the 23 ticks collected on 23 dogs in the nine different regional communities during the first and second campaigns, none (0%) elicited a positive E camis-PCR result (ee Table 1) In the ten APY lands communities sampled during the second cam- palgn, 82 ticks were collected on 72 dogs. Seventy ticks from 70 dogs Jn seven communities elicited a negative PCR reaction, but ten ticks collected on one dog and two ticks collected on another dog in a dif {erent community were E. canis PCR-posiive From the 35 ticks collected on 35 dogs out of the 95 dogs handled daring the third campaign in February 2021, 22 were PCR-positive. [No evidence of E. canis was found in three communities among the 10 communities sampled, but only one or two ticks were tested in cach of those three communities The percentage of dogs hosting PCR-positve ticks in the APY lands was 28% (95% Cl: 0.3 to 97) and 62.9% (95% Cl: 44.9 t0 78.5) dur- ing the second and the third campaigns, respectively, This difference was significant X°(1, n = 107) = 4547, P < 0.0001, Different online resources led to the same list of specimens collected in South Australia. These specimens are part of the South Australian Museum Arachnology collection, held at the South Australian Table 1, Number of dogs with ticks and number of dogs with ticks PCR-posiive to hvichia canis Number of dogs with. canis-postve ticks/number of dogs with ticks Locations, ‘August 2020 October-November 2021 February 2021 Regional communities Yalata os (ak Valley on Koonibba on Oodnadata* ory Marla on Coober Pedy” on Maree 08 Leigh Creek on Port Augusta on emote Aboriginal communities APY lands 2m As Total ong 2176 As * Rhipicephaluslinnae has been previously recorded at these locations. All teks Identified were 8. innaet ‘APY, Anangu Pijantatjara Yankunytjtjara [Australian Veterinary Journal © 2022 The thors Asai Veterinary Jounal pubined by John Wik & Sons usa, id on behalf of Austaln Vteinay Asoc,‘Museum, The query “Rhiplephalus linnae?” did not return any result, but the query *Rhipicephalus sanguineus” provided the records for 114 historical specimens collected in South Australis. They were downloaded. Their collection date was kindly provided by the teres ‘rial invertebrates’ collection manager at the South Australian ‘Museum and ranged from 1975 to 2005. Geospatial information, ‘with accuracy dawn to the suburb, the livestock station or the coun- ‘ry town level was indicated for 108 specimens, Two specimens were collected from ‘the Adelaide suburbs’. The location of four speci ‘mens was lacking. A total of 54 specimens were collected in metro- politan Adelaide by at least 14 different persons between 1975 and 1997. The other 54 specimens were collected from different rural areas across the state between 1976 and 2005 (see Figure 1) Discussion 2. linnaet was found in all sampled locations in the state, nine of ‘which (eight regional communities and the APY lands) were not previously recorded. This result indicates that R.linnaei is more extensively distributed in South Australia than previously published Ir also suggests the enzooty of this tick in South Australian regional and Aboriginal communities t was the only tick species identified, This might only be a rediscovery as R.linnaei has indeed been col lected across the state and voucher specimens were deposited in arachnid historical collections sine the 1970s, This finding empha: sises the importance of such collections and calls for updated vectors surveys. This study does not constitute a formal survey. It difficult to mea: sure more accurately both the tick and the E, canis prevalences based ‘on our data, Nevertheless, no evidence of E. canis was found in the regional communities in 2020. The situation was diferent in the APY lands communities, with more communities being affected over time, going feom two communities affected out of nine, to seven communities affected out of 10. The prevalence of dogs hosting E. canis-positive ticks rose from 28% in October 2020 to 62.9% in February 2021, Our results suggest an important E. canis incidence increase in owned dogs within this four-month period, This finding {snot in agreement with an already established enzooty but it i in favour of the rapid spread of the bacterium, Both hypotheses were already suggested in an Australian E. canis phylogenetic study'" ‘without a clear conclusion. In the light ofthese findings and in the context of the emergence ofthis pathogen in Halls Creek, less than 9 months before our study and over 900 km away, we recommend an epidemiological enquiry to investigate both the movements of logs within communities and the chains of transmission. Our observation indicates that E. canis was limited in South ‘Australia to the APY lands, but its apparent rapid spread, associated ‘with the distribution of the vector across the state suggests that the ‘CME might become enzootic throughout South Australia. This is a concern for canine health. Factors influencing the reservoir for E. canis, such as the possiblity for dogs to roam fre, oF to move between locations, and factors influencing the vector such as the sub-optimal ectoparaste control, in particular R. limnaci, on dogs and in the environment, as well as favourable climatic conditions, are all lkely to amplify the spread of the pathogen. Those factors are ‘SMALL ANIMALS present in remote Aboriginal communities where dogs are valued ‘companions, and they often serve important cultural and/or spiritual roles. Unfortunately, the animal welfare authorities and veterinary services are scarce and infrequent in those communities which prevent rapid response to both acute and chronic cases of CME, As such, a high proportion of dags that develop chtlchiosis in remote communities are likely to go untreated. Due to the recent introduction of E. canis in the country, dog populations are likly still ave to this infection, and an important negative impact on dogs’ health and welfare as ‘well as on their owners welfare can therefore be forecast. Furthermore, the current funding deficits prohibit the implementa- tion of efficient anti-parastie programs in dogs and the control of environmental infestations. A repellent acaricide as well as a sys temic acarcie is recommended in order to prevent Ehrlichia trans- ‘mission, which may occur within hours ofan intial tick bite"? A systemic acaricde will assist in environmental tick control and reduce the rsk of infected ticks remaining in the environment. The role other cankls present in Australia, such as dingoes (Canis Jupus dingo) and foxes (Vulpes vulpes) might play as E, canis reser- voir isnot well documented nor understood, and requires scientific investigation, The detection of . canis calls for more viglance and screening of dogs with suggestive clinical signs, especially in remote areas. Ehrlichiosis should be included in the differential diagnosis ‘whenever compatible clinical signs are observed in dogs in, or from, South Australia, Whenever confirmed, the disease should be notified Lastly, relevant information should be provided to pet dog owners visitng those areas. Conclusion The triad relationship “canid ~ R, limnaei ~ E, canis’ is central in ‘managing chilichiosis in Australia. More research is needed 0 understand this disease and its impacts in Australia, including . linnaei surveys and further B. canis prevalence studies in areas where Rlinnaei is enzootic, both in domestic dogs and dingos, within a “One: Health” framework. Improved resources for remote community animal health programs are essential ‘Acknowledgments ‘We would like to acknowledge the traditional owners of the lands ‘where this study took place, and in particular acknowledge the com ‘munity residents and their dogs in the Anangu Pitjantjtjara Yankunytjajara (APY) Lands, Koonibba community, Oak Valley community and Yalata community. We would also hike to thank all veterinarians and staff involved in tick collection, in particular Dr Allison Crawley, Dr Kate Litchfield, Dr Roger Absalom and Dr Nat Wakeham, Furthermore, the authors sincerely appreciate and thank Yalata Anangu Aboriginal Corporation (YAAC), Tullawon Health Services (THS), Anangu PitjanjatjaraYankunytjajara and [Nganampa Health Council for asistance from and permission to Visit the communities involved. Open access publishing facilitated by © 2022 The Authors Autalion Veterinary Jounal pushed by hn Vey & Sons Ruslan Bea of Australian Veena Association. Astalian Veterinary Journal Es FiPorat) 6 ‘SMALL ANIMALS ‘The University of Adelaide, as part of the Wiley - The University of Adelaide agreement via the Council of Australian University Librarians Conflicts of interest and sources of funding “The authors declare no conflicts of interest or sources of funding for the work presented here, References 1. Tylor MA. Veterinary paastology. 4th edtion. 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