Insights into the microbiome of 1Vector and Water-Borne Pathogen Research Group, School of Veterinary and Life Sciences, from Australian companion Murdoch University, Perth, Western Australia, Australia Telleasha L Greay1, Alireza Zahedi1, Anna-Sheree Krige1, Jadyn M Owens1, Andrea Paparini1, Alexander W Gofton1, Robert L Rees2, Una M Ryan1, Charlotte L Oskam1, and Peter J Irwin1 2Bayer Health, Brisbane, Queensland, Australia

In Australia, only three -borne pathogens (TBP) of pets are known; these include Babesia gibsoni [1], Babesia vogeli [2, 3], and Anaplasma platys [4, 5] transmitted by the introduced brown dog tick (). However, during a 2012-15 tick survey of companion animals (627 dogs, 153 cats, and 57 horses), 77% of tick species identi ed were native ticks [6] whose vector potential is largely unknown. Recent next- generation sequencing (NGS) studies on native Australian ticks have demonstrated the capabilities of NGS to broadly screen for pathogens and novel species [7-9], enabling the characterisation of the diversity and composition of targeted aspects (e.g. bacteria) of the tick microbiome. Furthermore, there is a need for surveillance in Australian ticks for pathogens that occur overseas as pets and their owners returning from international travels, and imported animals, pose a risk of introducing overseas pathogens, which has occurred in the past [10]. This study aimed to conduct a comprehensive survey of bacteria and piroplasms, as well as other Apicomplexa species (Hepatozoon spp., Cytauxzoon spp., and Toxoplasma spp.) within the microbiome of ticks collected from dogs, cats, and horses.

Observed ZOTUs Chao1 Shannon Simpson Figure A. α-diversity comparisons between tick species using observed A. 300 number of ZOTUs, Chao1, Shannon diversity index, and Simpson’s diversity 1.00 index species richness estimators; Figure B. principal coordinate analysis (PCoA) of

tick microbiomes based on Weighted UniFrac distance between samples. 300 Sample selection Figures were produced in R Studio using the phyloseq R package [11]. 1 75 493 Figure Legend 3 140 0.75 Tick Species Haemaphysalis sp. R. australis Collection 200 Tick Hosts A. t. triguttatum I. cornuatus R.sanguineus Bothriocroton sp. I. hirsti Host Species 200 All states and territories (except ACT) H. bancrofti I. holocyclus Dog H. lagostrophi I. myrmecobii Horse 2 0.50

H. longicornis I. tasmani Cat ersity Measure

Identification Table 2. NCBI BLAST results for 18S rRNA sequences obtained from 17 PCR- positive ticks Alpha Di v 100 Percent Tick species and Collection 100 Instars Top BLAST hit similarity instar Host location 1 0.25 Babesia canis 100% Rhipicephalus Dogs (2) NT: Katherine Females: 2,336 vogeli for 300 bp sanguineus QLD: Torres (HQ662635.1) female (1) and Strait larva (1) Males: 1,404 Theileria 100% Haemaphysalis Dogs (2) NSW: orientalis for 309 bp longicornis Missabotti and (XR_696404.1) Nymphs: 805 nymphs (2) Verona 0 0 0 0.00 Hepatozoon sp. 99% tasmani Dogs (2) TAS: B. (KC342523.1) for 303 bp females (3) & Devonport (2) Larvae: 220 Cat (1) and Port Sorell (1) 0.6

0 500 1000 1500 2000 km 97% Haemaphysalis Horse (1) NSW: Eungai Species for 323 bp bancrofti male (1) Creek Theileria sp. 97% Ixodes tasmani Dogs (2) TAS: (JQ682879.1) for 303 bp females (2) Devonport (1) 10 Native and Lower 0.4 Wilmot (1) 98% Ixodes tasmani Dogs (2) TAS: Port for 310 bp females (2) Sorell (1) and 3 Introduced (87% query Lower Wilmot coverage) (1) DNA extraction 0.2 Babesia sp. 98% Cat (1) QLD: Park

2 (JQ682876.1) .2 [16%] for 299 bp female (1) Ridge Axi s Table 1. Subset of ticks extracted from companion animals Babesia sp. 98% Haemaphysalis Horse (1) NSW: Tanja (JQ437265.1) for 319 bp sp. female (1) Tick species Dogs Cats Horses Total (with 87% query 0.0 t. triguttatum 11 0 13 24 coverage) Bothriocroton sp. 6 0 1 7 Toxoplasma 98% Ixodes tasmani Dog (1) NSW: Seaforth gondii for 304 bp female (1) Haemaphysalis bancrofti 1 1 3 5 (XR_00197435 6.1) Haemaphysalis lagostrophi 0 0 1 1 Hepatozoon 100% Ixodes holocyclus Dog (1) QLD: Sarina −0.2 canis for 303 bp female (1) Haemaphysalis longicornis* 51 0 24 75 (KX712128.1) Haemaphysalis sp. 0 0 4 4 −0.2 0.0 0.2 0.4 0.6 Ixodes cornuatus 9 1 0 10 Piroplasma & Axis.1 [29.9%] Ixodes hirsti 0 1 0 1 Apicomplexa spp.18S Sanger Bacterial 16S gene Ixodes holocyclus 182 126 26 334 gene (V8) amplicons NGS (V1-2) amplicons Ixodes myrmecobii 4 1 0 5 sequencing i) 17 PCR positive samples sequenced on the Ixodes tasmani 48 10 1 59 sequenced on the ABI Prism 96-capillary 3730xl Rhipicephalus australis* 1 0 2 3 DNA analyzer Illumina MiSeq platform Rhipicephalus sanguineus* 180 0 0 180 ii) Sequences were BLAST searched against the NCBI i) 38.5 million sequences and 3,766 zero-radius Grand Total 493 140 75 708 database OTUs (ZOTUs) obtained after sequence pre- processing in USEARCH v10 *Introduced species Sequencing 3 ii) ZOTUs were compared to the SILVA and NCBI NR database for taxonomic assignment Key Findings & Future Research Directions

References 1. Muhlnickel CJ, Jeeries R, Morgan-Ryan UM, Irwin PJ. Babesia gibsoni infection in three dogs in Victoria. Aust Vet J. 2002;80:606-10. Two tick-borne pathogens were identi ed: Anaplasma including Babesia spp., Hepatozoon spp., Theileria spp., 2. Jeeries R, Ryan UM, Muhlnickel CJ, Irwin PJ. Two species of canine Babesia in Australia: detection and characterization by PCR. J Parasitol. 2003;89:409-12. 3. Brown GK, Can eld PJ, Dunstan RH, Roberts TK, Martin AR, Brown CS et al. platys in 13/180 brown dog ticks from dogs in NT, QLD, and Toxoplasma sp. Detection of Anaplasma platys and Babesia canis vogeli and their impact on platelet numbers in free-roaming dogs associated with remote Aboriginal communities in Australia. Aust Vet J. 2006;84:321-5. and WA, and Coxiella burnetii in a single paralysis tick The overseas canine pathogen Hepatozoon canis was 4. Brown GK, Martin AR, Roberts TK, Aitken RJ. Detection of Ehrlichia platys in dogs in Australia. Aust Vet J. 2001;79:554-8. 5. Brown GK, Martin AR, Roberts TK, Dunstan RH. Molecular detection of (Ixodes holocyclus) from a cat in QLD. detected in an engorged paralysis tick removed from a Anaplasma platys in lice collected from dogs in Australia. Aust Vet J. 2005;83:101-2. 6. Greay TL, Oskam CL, Gofton AW, Rees RL, Ryan UM, Irwin PJ. A survey of ticks Recently described potential pathogens Candidatus dog in QLD. (: ) of companion animals in Australia. Parasit Vectors. 2016;9:1-10. 7. Gofton AW, Oskam CL, Lo N, Beninati T, Wei H, McCarl V et al. Inhibition of the endosymbiont "Candidatus Midichloria mitochondrii" during 16S rRNA gene Neoehrlichia australis and Ca. Neoehrlichia arcana were pro ling reveals potential pathogens in Ixodes ticks from Australia. Parasit Genetic characterisation and phylogenetic comparisons of the Vectors. 2015;8:345. 8. Gofton AW, Doggett S, Ratchford A, Oskam CL, Paparini A, Ryan U et al. detected in paralysis ticks from cats and dogs in QLD Bacterial pro ling reveals novel “Ca. Neoehrlichia”, Ehrlichia, and Anaplasma novel bacterial and Apicomplexa species are required to further species in Australian human-biting ticks. PLoS ONE. 2015;10:e0145449. 9. Barbosa AD, Gofton AW, Paparini A, Codello A, Greay T, Gillett A et al. Increased and NSW. understand their pathogenic potential. genetic diversity and prevalence of co-infection with Trypanosoma spp. in koalas (Phascolarctos cinereus) and their ticks identi ed using next-generation sequencing (NGS). PLOS ONE. 2017;12:e0181279. Novel bacterial species identi ed in native Australian The identi cation of the overseas pathogen H. canis 10. Churchill RC, Best DR. of a horse in Australia. Aust Vet J. 1976;52:487. 11. McMurdie and Holmes. An R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE. 2013;8(4):e61217 ticks include Coxiella sp., Francisella spp., and Rickettsia sp. emphasises the need for continued pathogen surveillance in Thank you to Murdoch University, Bayer Animal Health (Germany), Bayer Australia, and the Australian Research Council for nancially supporting our research. We also thank Dr. Sam Abraham and Dr. Mark O’Dea for their assistance with access to ticks. sequencing facilities at the Western Australian State Agricultural Biotechnology Seven novel Apicomplexa species were identi ed, Centre (SABC), and Ms. Frances Brigg at the SABC for her assistance with Sanger sequencing. Lastly, thank you to Bayer Australia area managers and the general public for their enormous tick collection eorts. Dog, cat, horse, and microscope vector images designed by Freepik (www.freepik.com)

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