Epidemiological survey on pet dog ticks (Rhipicephalus turanicus sensu stricto) and their blood samples in Korla, northwestern China

Epidemiological survey on pet dog ticks (Rhipicephalus turanicus sensu stricto) and their blood samples in Korla, northwestern China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Epidemiological survey on pet dog ticks (Rhipicephalus turanicus sensu stricto) and their blood samples in Korla, northwestern China Jianhui Chen, Shen Shi, Nannan Cui, Lixin Su, Ziqi Wang, Fang Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4730842/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Pet dogs pose a potential risk to transmitting zoonotic pathogens by ticks. however information about the prevalence status in pet dog of Tick-borne diseases is currently limited. Result In the study, 196 blood samples and 223 parasitic ticks were collected from pet dogs in Korla, northwestern China. Based on morphological and molecular characteristics, all ticks were identified as Rhipicephalus turanicus sensu stricto.We used primers targeting the 16S ribosomal( 16S rRNA ) for detection of Anaplasma bovis species, targeting the small subunit 18S ribosomal RNA gene ( 18S rRNA ) for detection of Hepatozoon canis species and targeting htpAB-associated repetitive element gene ( IS111 ) for detection of Coxiella burnetii species The nPCR-positive products were sequenced, aligned, and phylogenetically analyzed. Three tick-borne pathogenic bacteria were detected in the samples. Coxiella burnetii were detected both in parasitic ticks and in blood samples with a detection rate of 17.93% (40/233) in ticks and 79.1% (155/196) in blood samples, followed by 21.52% Hepatozoon canis (48/233) in tick, 2.5% Anaplasma bovis (5/196) in blood samples. Conclusion This study provided molecular evidence for the occurrence of A. bovis , H . canis and C. burnetii circulating in pet dogs and their parasitic ticks in northwestern China. Understanding the prevalence of Tick-borne diseases in pet dog is essential for developing effective strategies for disease control and management. Pet dogs Parasitic ticks Anaplasma bovis Coxiella burnetii Hepatozoon canis northwestern China Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Tick-borne diseases (TBDs) are relatively common in pet dogs, and dog-human relationships may facilitate the spread of tick-borne pathogens among people [ 1 ]. Many factors can affect the spread of tick-borne diseases in pet dogs, such as the pet dog's living environment, parasite control, and the health awareness of pet dog owners [ 2 ]. However, parasite control in pet dogs is often neglected due to the lack of knowledge about parasite hazards by dog owners. [ 3 ]. Due to the increasing number of pet dogs in China and their close relationship with humans, there is a need to study the epidemiological status of tick-borne zoonotic pathogens[ 4 ]. Korla City is listed as important transportation hub and material distribution center of Xinjiang Uygur Autonomous Region (XUAR, northwestern China) with more than 477,000 registered population. It is located in the northeast edge of the Taklamakan Desert, which is the second largest desert in the world. Extreme dryness with an average annual precipitation of 58.6 mm and an annual maximum evaporation of 2788.2 mm is its climatic characteristics [ 5 ]. Rhipicephalus turanicus , Dermacentor marginatus and Hyalomma asiaticum were previously reported as dominant tick species in oasis of Taklamakan desert [ 6 ]. TBPs in pet dogs and their parasitic ticks, such as Candidatus Rickettsia barbariae , Rickettsia massiliae , Rickettsia conorii , Rickettsia sibirica , Ehrlichia chaffeensis , Anaplasma phagocytophilum , Anaplasma ovis and Brucella spp ., were previously reported in north region of XUAR [ 7 , 8 ]. In present study, we further investigated A. bovis , C. burnetii and H. canis in blood samples of pet dogs and their ticks in Korla City, northwestern China. Material and methods Sample Collection Two shelters for stray dogs and five pet hospitals close to pastures in Korla City (934 m above sea level; 41°14′N 85°11′E) in Tarim Basin, XUAR were selected between late April to mid-May 2021(coinciding with the peak activities of adult ticks), blood and tick samples were collected from pet dogs based on clinical symptoms that include depression, weight loss, and anorexia. All samples were collected with the permission of the pet owner and sample collection was carried out by a local veterinarian. The blood samples are collected into a vacuum tube containing ethylene diamine tetraacetic acid (EDTA) anticoagulant while we collect ticks from the dog's whole body, placing them in tubes containing 75% ethanol and 5% glycerol Identification of Ticks Extract total DNA from 200 µL whole blood samples using a blood DNA extraction kit (Omega Bio-tek, Norcross, USA) and genomic DNA from whole ticks using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) with an overnight following the manufacturer’s instructions. All ticks are identified based on morphology before DNA is extracted, as previously described.[ 9 , 10 ]. Subsequently, molecular taxonomic analysis based on partial mitochondrial 16S rRNA (460 bp) gene sequences was performed on 30 representative ticks (4–6 ticks per veterinary clinic) to confirm tick species.Subsequently, 30representative ticks, with 4–6 ticks at each veterinary clinic,were subjected to molecular classification analysis based on partial mitochondrial 16S rRNA (460bp) gene sequences to confirm tick species[ 11 ]. Detection of Tick-Borne Pathogens We used a partial 16S rRNA (450bp) gene to detect Anaplasma bovis [ 12 ]. The molecular detection of Coxiella burnetii was performed using the IS111 (260bp) [ 13 ]. Hepatozoon canis were detected targeting 18S rRNA [ 14 ]. DNA from our laboratory was used as positive controls for Anaplasma bovis , Coxiella burnetii and Hepatozoon canis . Double distilled water was used as a negative control (Dongsheng, Guangzhou, China). Use the basic local alignment search tool (BLAST) to compare sequence results with reference sequences available in a centralized database ( http://www.ncbi.nlm.nih.gov/BLAST/ ). Phylogenetic trees were constructed using the maximum-likelihood method using MEGA X software ( https://www.megasoftware.net).Characteristic s of the amplified fragments and corresponding primer sequences are provided in Table S1 of the Supplementary Information. RESULTS All ticks (72 male ticks and 151 female ticks) were collected and morphologically identified as Rhipicephalus turanicus sensu stricto ( s.s. )(Fig. 1 ). The obtained sequences of Rh.turanicus s.s. have been deposited in the GenBank database.Phylogenetic trees analysis further confirmed these results (Fig. 2 ). Three tick-borne pathogens were detected in this test, among which the highest detection rate was Coxiella burnetii , which was detected in both ticks and blood samples, with a detection rate of 17.93% (40/233) in ticks and 79.1% (155/196) in blood samples, followed by Hepatozoon canis 21.52% (48/233) in ticks and 21.52% (48/233)Three tick-borne pathogenic bacteria were detected in the samples. Among them, co-infection with H . canis and C . burnetii was detected in 12 blood samples. All ticks and blood samples were also tested for borrelia burgdorferi and Leptospira . However, none of the samples were infected with these pathogens. Among all the positive ticks and blood samples, A. bovis and C. burnetii showed 99.78% and 99.63 identity to the corresponding sequences of A. bovis (MH255939) from Shaanxi Province and C. burnetii (KX852471) from XUAR, respectively. H. canis showed 99.84% identity to the corresponding sequences from Czech Republic (KX712129). Phylogenetic trees analysis further confirmed these results (A. bovis Fig. 3 ; H. canis Fig. 4 ; C. burnetii Fig. 5 ). All sequences from this study were deposited in the GenBank ( http://www.ncbi.nlm.nih.gov ) database ( Rh.turanicus s.s PP346393; A . bovis PP267323; C . burnetii PP348678-PP472476; H . canis PP267259-PP267260). DISCUSSION Tick-borne zoonotic diseases have been increasing in humans and pet dog cases [ 15 ]. In this study, C . burnetii, H . canis and A . bovis were screened out in pet dogs and their parasitic ticks. To the best of our knowledge, this is the first report of C . burnetii and H . canis in Rh. turanicus s.s. in China. Anaplasma is transmitted by ticks and contains seven proven species. Two of these species, A. phagocytophilum and A. capra , commonly cause disease in humans[ 16 ]. A . bovis was initially thought to be just an animal pathogen until the first patient case was reported in 2019[ 17 , 18 ]. In this study, A . bovis was detected both in dog ticks and blood samples. This finding suggests that it is vital to further survey A . bovis among pet dogs, ticks and dog owners especially in oasis of Taklamakan Desert in the future. C . burnetii can infect a variety of domestic and wild animals, including mammals, birds, and reptiles. Previously, cattle, sheep, and goats were considered the primary hosts[ 19 ]. At the same time, dogs and cats are classified as mammals susceptible to C . burnetii [ 20 ]. Pet animals, especially those in close contact with their owners, play an important role in reservoirs of C . burnetii , which causes urban Q fever and sporadic Q fever [ 21 ]. In this study, it was not only found in the samples of dog ticks, but also in dog blood samples with 79.1% (155/196) positive rate. This result gives a strong warning to public health security against Q fever. To date, Hepatozoon includes at least 340 species and can infect a wide range of vertebrate hosts, such as mammals, reptiles, birds, fish, and invertebrates. In terms of its primary vectors, Amblyomma ovale , Rhipicephalus microplus , Haemaphysalis longicornis and Haemaphysalis flava ticks have been identified as definite hosts for Hepatozoon [ 22 – 24 ]. In this study, we found H . canis with 21.52% (48/233) positivity in Rh.turanicus s.s ticks. Although this study doesn’t confirm Rh.turanicus s.s being vectored as H . canis , we still believe H. canis pose a potential risk to dogs and dog owners in local people. Co-infection is common in tick-bitten mammals. Previously, Some scholars have discovered that Co-infections were identified in 16.7% I. ricinus ticks (89/534), which accounted for 64.5% (89/138) of all infected ticks. Co-infection prevalence was 14.3% (11/77) in adults and 17.1% (78/457) in nymphs[ 25 ]. Meanwhile, ticks can acquire a variety of pathogenic species (such as parasites, bacteria or viruses) through blood-sucking to different vertebrate hosts or through systemic transmission of common feeding,Meanwhile, ticks can acquire multiple pathogenic species (such as parasites, bacteria or viruses) through systemic transmission during blood feeding on their different vertebrate hosts, or through co-feeding[ 26 ]. In this study, H . canis and C . burnetii were simultaneously detected in 12 dog blood samples. This study extends tick-borne co-infection in pet dogs. Conclusions With the number of pet dogs increasing in China, it is necessary to strengthen the supervision of pet dogs and stray dogs in order to control tick-borne zoonotic diseases in the horizon of “One World One Health”. Declarations Acknowledgements The author would like to thank all the veterinarians who participated in the study as well as all the colleagues who contributed to sample collecting and sample preparation. Funding This research was funded by the Construction of tick-borne infectious disease early warning system in Xinjiang Uygur Autonomous Region [grant numbers 2022B03014-4]. Competing interests The authors declare no competing interests. Author contributions Conceptualization JC, YW, XW,SS,GZ; methodology JC,NC,LS,ZW; data curation JC,FL,SZ,SS,GZ; writing—original draft preparation JC,SS,YW, XW,NC; writing—review and editing: all authors. All authors have read and agreed to the published version of the manuscript. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethical approval and consent to participate All the protocols, experimentation, and animal manipulation were thoroughly reviewed and approved by the Animal Welfare Committee of Shihezi University (Approval numbers A2020-113-01). Consent for publication Not applicable. References Otranto D, Dantas-Torres F, Mihalca AD, Traub RJ, Lappin M, Baneth G. Zoonotic Parasites of Sheltered and Stray Dogs in the Era of the Global Economic and Political Crisis. Trends Parasitol. 2017;33(10):813–25. Katagiri S, Oliveira-Sequeira TC. Prevalence of dog intestinal parasites and risk perception of zoonotic infection by dog owners in São Paulo State, Brazil. Zoonoses Public Health. 2008;55(8–10):406–13. Irwin PJ. It shouldn't happen to a dog … or a veterinarian: clinical paradigms for canine vector-borne diseases. Trends Parasitol. 2014;30(2):104–12. Chomel B. Tick-borne infections in dogs-an emerging infectious threat. Vet Parasitol. 2011;179(4):294–301. Zibibula·SiMaYi, Zhang Y, Ying Z. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4730842","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":328670048,"identity":"dace2df2-3317-4523-8476-d816cf99a40e","order_by":0,"name":"Jianhui Chen","email":"","orcid":"","institution":"School of Public Health, Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianhui","middleName":"","lastName":"Chen","suffix":""},{"id":328670049,"identity":"5881fe94-da40-42b1-8da3-d9b3cfb744a6","order_by":1,"name":"Shen Shi","email":"","orcid":"","institution":"Key Laboratory of Vector-borne Infectious 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14:07:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4730842/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4730842/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62222623,"identity":"5009bdb6-bc09-43a0-afe3-b1bd83c486bc","added_by":"auto","created_at":"2024-08-11 12:38:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2243097,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological analysis of \u003cem\u003eRhipicephalus turanicus sensu stricto\u003c/em\u003e collected from pet dogs. a Male, dorsal; b male, ventral\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4730842/v1/9d604f77e5bf49c5d0bd5c1e.png"},{"id":62222624,"identity":"a64b9ae4-a340-4080-bdda-352057a46c66","added_by":"auto","created_at":"2024-08-11 12:38:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1100695,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on partial gene of the \u003cem\u003e16S rRNA\u003c/em\u003e of \u003cem\u003eRhipicephalus turanicus sensu stricto\u003c/em\u003e(▲) collected from pet dogs obtained in this study in northwestern China. The evolutionary history was inferred using the neighbor-joining method (bootstrap replicates: 1000) with MEGA 7.0.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4730842/v1/651d8299ba40dc6c7fe2a017.png"},{"id":62223258,"identity":"e1c0ab2f-c383-48ab-9dd8-d6e35e0b88da","added_by":"auto","created_at":"2024-08-11 12:46:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":607287,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on partial gene of the \u003cem\u003e16S rRNA\u003c/em\u003e of \u003cem\u003eAnaplasma bovis \u003c/em\u003e(▾)from blood samples obtained in this study in northwestern China. The evolutionary history was inferred using the neighbor-joining method (bootstrap replicates: 1000) with MEGA 7.0.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4730842/v1/9babdd124a5de58051c306f8.png"},{"id":62223503,"identity":"75196c0b-ddb1-476a-8404-130e91a73e46","added_by":"auto","created_at":"2024-08-11 12:54:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":643427,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on partial gene of the \u003cem\u003e18S rRNA\u003c/em\u003e sequence of \u003cem\u003eHepatozoon canis\u003c/em\u003e(•) from \u003cem\u003eRhipicephalus turanicus sensu stricto\u003c/em\u003e obtained in this study in northwestern China. The evolutionary history was inferred using the neighbor-joining method (bootstrap replicates: 1000) with MEGA 7.0.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4730842/v1/354d9b5637be1489ac956e8f.png"},{"id":62222620,"identity":"262a45cc-ca55-451c-8cba-8be449147f5d","added_by":"auto","created_at":"2024-08-11 12:38:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1107140,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on partial gene of the htpABgenesequence of \u003cem\u003eCoxiella burnetii\u003c/em\u003e (◼) from \u003cem\u003eRhipicephalus turanicus sensu stricto\u003c/em\u003e and blood samples obtained in this study in northwestern China. The evolutionary history was inferred using the neighbor-joining method (bootstrap replicates: 1000) with MEGA 7.0.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4730842/v1/24796a85233835043a0b9fad.png"},{"id":65841518,"identity":"a6793243-51ae-4574-89c3-9c75b0ce8ce7","added_by":"auto","created_at":"2024-10-03 12:17:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7897029,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4730842/v1/40e261c1-e4e7-4632-be27-e0217e6bbb89.pdf"},{"id":62223256,"identity":"29be38aa-b3ad-4f76-8ffd-bd4206d08e96","added_by":"auto","created_at":"2024-08-11 12:46:49","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":21349,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-4730842/v1/5815593ee90d34475f216c35.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Epidemiological survey on pet dog ticks (Rhipicephalus turanicus sensu stricto) and their blood samples in Korla, northwestern China","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTick-borne diseases (TBDs) are relatively common in pet dogs, and dog-human relationships may facilitate the spread of tick-borne pathogens among people [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Many factors can affect the spread of tick-borne diseases in pet dogs, such as the pet dog's living environment, parasite control, and the health awareness of pet dog owners [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, parasite control in pet dogs is often neglected due to the lack of knowledge about parasite hazards by dog owners. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Due to the increasing number of pet dogs in China and their close relationship with humans, there is a need to study the epidemiological status of tick-borne zoonotic pathogens[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eKorla City is listed as important transportation hub and material distribution center of Xinjiang Uygur Autonomous Region (XUAR, northwestern China) with more than 477,000 registered population. It is located in the northeast edge of the Taklamakan Desert, which is the second largest desert in the world. Extreme dryness with an average annual precipitation of 58.6 mm and an annual maximum evaporation of 2788.2 mm is its climatic characteristics [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. \u003cem\u003eRhipicephalus turanicus\u003c/em\u003e, \u003cem\u003eDermacentor marginatus\u003c/em\u003e and \u003cem\u003eHyalomma asiaticum\u003c/em\u003e were previously reported as dominant tick species in oasis of Taklamakan desert [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. TBPs in pet dogs and their parasitic ticks, such as \u003cem\u003eCandidatus Rickettsia barbariae\u003c/em\u003e, \u003cem\u003eRickettsia massiliae\u003c/em\u003e, \u003cem\u003eRickettsia conorii\u003c/em\u003e, \u003cem\u003eRickettsia sibirica\u003c/em\u003e, \u003cem\u003eEhrlichia chaffeensis\u003c/em\u003e, \u003cem\u003eAnaplasma phagocytophilum\u003c/em\u003e, \u003cem\u003eAnaplasma ovis\u003c/em\u003e and \u003cem\u003eBrucella spp\u003c/em\u003e., were previously reported in north region of XUAR [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In present study, we further investigated \u003cem\u003eA. bovis\u003c/em\u003e, \u003cem\u003eC. burnetii and H. canis\u003c/em\u003e in blood samples of pet dogs and their ticks in Korla City, northwestern China.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample Collection\u003c/h2\u003e \u003cp\u003eTwo shelters for stray dogs and five pet hospitals close to pastures in Korla City (934 m above sea level; 41\u0026deg;14\u0026prime;N 85\u0026deg;11\u0026prime;E) in Tarim Basin, XUAR were selected between late April to mid-May 2021(coinciding with the peak activities of adult ticks), blood and tick samples were collected from pet dogs based on clinical symptoms that include depression, weight loss, and anorexia. All samples were collected with the permission of the pet owner and sample collection was carried out by a local veterinarian. The blood samples are collected into a vacuum tube containing ethylene diamine tetraacetic acid (EDTA) anticoagulant while we collect ticks from the dog's whole body, placing them in tubes containing 75% ethanol and 5% glycerol\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of Ticks\u003c/h2\u003e \u003cp\u003eExtract total DNA from 200 \u0026micro;L whole blood samples using a blood DNA extraction kit (Omega Bio-tek, Norcross, USA) and genomic DNA from whole ticks using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) with an overnight following the manufacturer\u0026rsquo;s instructions. All ticks are identified based on morphology before DNA is extracted, as previously described.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Subsequently, molecular taxonomic analysis based on partial mitochondrial 16S rRNA (460 bp) gene sequences was performed on 30 representative ticks (4\u0026ndash;6 ticks per veterinary clinic) to confirm tick species.Subsequently, 30representative ticks, with 4\u0026ndash;6 ticks at each veterinary clinic,were subjected to molecular classification analysis based on partial mitochondrial \u003cem\u003e16S\u003c/em\u003e rRNA (460bp) gene sequences to confirm tick species[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDetection of Tick-Borne Pathogens\u003c/h2\u003e \u003cp\u003eWe used a partial \u003cem\u003e16S rRNA\u003c/em\u003e (450bp) gene to detect \u003cem\u003eAnaplasma bovis\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The molecular detection of \u003cem\u003eCoxiella burnetii\u003c/em\u003e was performed using the IS111 (260bp) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eHepatozoon canis\u003c/em\u003e were detected targeting \u003cem\u003e18S rRNA\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. DNA from our laboratory was used as positive controls for \u003cem\u003eAnaplasma bovis\u003c/em\u003e, \u003cem\u003eCoxiella burnetii\u003c/em\u003e and \u003cem\u003eHepatozoon canis\u003c/em\u003e. Double distilled water was used as a negative control (Dongsheng, Guangzhou, China). Use the basic local alignment search tool (BLAST) to compare sequence results with reference sequences available in a centralized database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/BLAST/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/BLAST/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Phylogenetic trees were constructed using the maximum-likelihood method using MEGA X software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.megasoftware.net).Characteristic\u003c/span\u003e\u003cspan address=\"https://www.megasoftware.net).Characteristic\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003es of the amplified fragments and corresponding primer sequences are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e of the Supplementary Information.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eAll ticks (72 male ticks and 151 female ticks) were collected and morphologically identified as \u003cem\u003eRhipicephalus turanicus sensu stricto\u003c/em\u003e (\u003cem\u003es.s.\u003c/em\u003e)(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The obtained sequences of Rh.turanicus s.s. have been deposited in the GenBank database.Phylogenetic trees analysis further confirmed these results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThree tick-borne pathogens were detected in this test, among which the highest detection rate was \u003cem\u003eCoxiella burnetii\u003c/em\u003e, which was detected in both ticks and blood samples, with a detection rate of 17.93% (40/233) in ticks and 79.1% (155/196) in blood samples, followed by \u003cem\u003eHepatozoon canis\u003c/em\u003e 21.52% (48/233) in ticks and 21.52% (48/233)Three tick-borne pathogenic bacteria were detected in the samples. Among them, co-infection with \u003cem\u003eH\u003c/em\u003e. \u003cem\u003ecanis\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eburnetii\u003c/em\u003e was detected in 12 blood samples. All ticks and blood samples were also tested for \u003cem\u003eborrelia burgdorferi\u003c/em\u003e and \u003cem\u003eLeptospira\u003c/em\u003e. However, none of the samples were infected with these pathogens.\u003c/p\u003e \u003cp\u003eAmong all the positive ticks and blood samples, \u003cem\u003eA. bovis\u003c/em\u003e and \u003cem\u003eC. burnetii\u003c/em\u003e showed 99.78% and 99.63 identity to the corresponding sequences of \u003cem\u003eA. bovis\u003c/em\u003e (MH255939) from Shaanxi Province and C. burnetii (KX852471) from XUAR, respectively. \u003cem\u003eH. canis\u003c/em\u003e showed 99.84% identity to the corresponding sequences from Czech Republic (KX712129). Phylogenetic trees analysis further confirmed these results (A. bovis Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; H. canis Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; C. burnetii Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll sequences from this study were deposited in the GenBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database (\u003cem\u003eRh.turanicus s.s\u003c/em\u003e PP346393;\u003cem\u003eA\u003c/em\u003e. \u003cem\u003ebovis\u003c/em\u003e PP267323; \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eburnetii\u003c/em\u003e PP348678-PP472476; \u003cem\u003eH\u003c/em\u003e. \u003cem\u003ecanis\u003c/em\u003e PP267259-PP267260).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eTick-borne zoonotic diseases have been increasing in humans and pet dog cases [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In this study, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eburnetii, H\u003c/em\u003e. \u003cem\u003ecanis\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ebovis\u003c/em\u003e were screened out in pet dogs and their parasitic ticks. To the best of our knowledge, this is the first report of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eburnetii\u003c/em\u003e and \u003cem\u003eH\u003c/em\u003e. \u003cem\u003ecanis\u003c/em\u003e in \u003cem\u003eRh. turanicus s.s.\u003c/em\u003e in China.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnaplasma\u003c/em\u003e is transmitted by ticks and contains seven proven species. Two of these species, \u003cem\u003eA. phagocytophilum\u003c/em\u003e and \u003cem\u003eA. capra\u003c/em\u003e, commonly cause disease in humans[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ebovis\u003c/em\u003e was initially thought to be just an animal pathogen until the first patient case was reported in 2019[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this study, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ebovis\u003c/em\u003e was detected both in dog ticks and blood samples. This finding suggests that it is vital to further survey \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ebovis\u003c/em\u003e among pet dogs, ticks and dog owners especially in oasis of Taklamakan Desert in the future.\u003c/p\u003e \u003cp\u003e \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eburnetii\u003c/em\u003e can infect a variety of domestic and wild animals, including mammals, birds, and reptiles. Previously, cattle, sheep, and goats were considered the primary hosts[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. At the same time, dogs and cats are classified as mammals susceptible to \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eburnetii\u003c/em\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Pet animals, especially those in close contact with their owners, play an important role in reservoirs of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eburnetii\u003c/em\u003e, which causes urban Q fever and sporadic Q fever [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this study, it was not only found in the samples of dog ticks, but also in dog blood samples with 79.1% (155/196) positive rate. This result gives a strong warning to public health security against Q fever.\u003c/p\u003e \u003cp\u003eTo date, \u003cem\u003eHepatozoon\u003c/em\u003e includes at least 340 species and can infect a wide range of vertebrate hosts, such as mammals, reptiles, birds, fish, and invertebrates. In terms of its primary vectors, \u003cem\u003eAmblyomma ovale\u003c/em\u003e, \u003cem\u003eRhipicephalus microplus\u003c/em\u003e, \u003cem\u003eHaemaphysalis longicornis\u003c/em\u003e and \u003cem\u003eHaemaphysalis flava\u003c/em\u003e ticks have been identified as definite hosts for \u003cem\u003eHepatozoon\u003c/em\u003e [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this study, we found \u003cem\u003eH\u003c/em\u003e. \u003cem\u003ecanis\u003c/em\u003e with 21.52% (48/233) positivity in \u003cem\u003eRh.turanicus s.s\u003c/em\u003e ticks. Although this study doesn\u0026rsquo;t confirm \u003cem\u003eRh.turanicus s.s\u003c/em\u003e being vectored as \u003cem\u003eH\u003c/em\u003e. \u003cem\u003ecanis\u003c/em\u003e, we still believe \u003cem\u003eH. canis\u003c/em\u003e pose a potential risk to dogs and dog owners in local people.\u003c/p\u003e \u003cp\u003eCo-infection is common in tick-bitten mammals. Previously, Some scholars have discovered that Co-infections were identified in 16.7% I. ricinus ticks (89/534), which accounted for 64.5% (89/138) of all infected ticks. Co-infection prevalence was 14.3% (11/77) in adults and 17.1% (78/457) in nymphs[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Meanwhile, ticks can acquire a variety of pathogenic species (such as parasites, bacteria or viruses) through blood-sucking to different vertebrate hosts or through systemic transmission of common feeding,Meanwhile, ticks can acquire multiple pathogenic species (such as parasites, bacteria or viruses) through systemic transmission during blood feeding on their different vertebrate hosts, or through co-feeding[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this study, \u003cem\u003eH\u003c/em\u003e. \u003cem\u003ecanis\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eburnetii\u003c/em\u003e were simultaneously detected in 12 dog blood samples. This study extends tick-borne co-infection in pet dogs.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWith the number of pet dogs increasing in China, it is necessary to strengthen the supervision of pet dogs and stray dogs in order to control tick-borne zoonotic diseases in the horizon of \u0026ldquo;One World One Health\u0026rdquo;.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author would like to thank all the veterinarians who participated in the study as well as all the colleagues who contributed to sample collecting and sample preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Construction of tick-borne infectious disease early warning system in Xinjiang Uygur Autonomous Region [grant numbers 2022B03014-4].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization JC, YW, XW,SS,GZ; methodology JC,NC,LS,ZW; data curation JC,FL,SZ,SS,GZ; writing\u0026mdash;original draft preparation JC,SS,YW, XW,NC; writing\u0026mdash;review and editing: all authors. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the protocols, experimentation, and animal manipulation were thoroughly reviewed and approved by the Animal Welfare Committee of Shihezi University (Approval numbers A2020-113-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOtranto D, Dantas-Torres F, Mihalca AD, Traub RJ, Lappin M, Baneth G. Zoonotic Parasites of Sheltered and Stray Dogs in the Era of the Global Economic and Political Crisis. Trends Parasitol. 2017;33(10):813\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatagiri S, Oliveira-Sequeira TC. Prevalence of dog intestinal parasites and risk perception of zoonotic infection by dog owners in S\u0026atilde;o Paulo State, Brazil. Zoonoses Public Health. 2008;55(8\u0026ndash;10):406\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIrwin PJ. It shouldn't happen to a dog \u0026hellip; or a veterinarian: clinical paradigms for canine vector-borne diseases. Trends Parasitol. 2014;30(2):104\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChomel B. Tick-borne infections in dogs-an emerging infectious threat. Vet Parasitol. 2011;179(4):294\u0026ndash;301.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZibibula\u0026middot;SiMaYi, Zhang Y, Ying Z. 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Ticks Tick-borne Dis. 2021;12(1).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pet dogs, Parasitic ticks, Anaplasma bovis, Coxiella burnetii, Hepatozoon canis, northwestern China","lastPublishedDoi":"10.21203/rs.3.rs-4730842/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4730842/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePet dogs pose a potential risk to transmitting zoonotic pathogens by ticks. however information about the prevalence status in pet dog of Tick-borne diseases is currently limited.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e \u003cp\u003eIn the study, 196 blood samples and 223 parasitic ticks were collected from pet dogs in Korla, northwestern China. Based on morphological and molecular characteristics, all ticks were identified as Rhipicephalus turanicus sensu stricto.We used primers targeting the 16S ribosomal(\u003cem\u003e16S rRNA\u003c/em\u003e) for detection of \u003cem\u003eAnaplasma bovis\u003c/em\u003e species, targeting the small subunit 18S ribosomal RNA gene (\u003cem\u003e18S rRNA\u003c/em\u003e) for detection of \u003cem\u003eHepatozoon canis\u003c/em\u003e species and targeting htpAB-associated repetitive element gene (\u003cem\u003eIS111\u003c/em\u003e) for detection of \u003cem\u003eCoxiella burnetii\u003c/em\u003e species The nPCR-positive products were sequenced, aligned, and phylogenetically analyzed. Three tick-borne pathogenic bacteria were detected in the samples. \u003cem\u003eCoxiella burnetii\u003c/em\u003e were detected both in parasitic ticks and in blood samples with a detection rate of 17.93% (40/233) in ticks and 79.1% (155/196) in blood samples, followed by 21.52% \u003cem\u003eHepatozoon canis\u003c/em\u003e (48/233) in tick, 2.5% \u003cem\u003eAnaplasma bovis\u003c/em\u003e (5/196) in blood samples.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study provided molecular evidence for the occurrence of \u003cem\u003eA. bovis\u003c/em\u003e, \u003cem\u003eH\u003c/em\u003e. \u003cem\u003ecanis\u003c/em\u003e and \u003cem\u003eC. burnetii\u003c/em\u003e circulating in pet dogs and their parasitic ticks in northwestern China. Understanding the prevalence of Tick-borne diseases in pet dog is essential for developing effective strategies for disease control and management.\u003c/p\u003e","manuscriptTitle":"Epidemiological survey on pet dog ticks (Rhipicephalus turanicus sensu stricto) and their blood samples in Korla, northwestern China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-11 12:38:44","doi":"10.21203/rs.3.rs-4730842/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"06a1e28d-dc9e-4f6e-8943-0250ecb282db","owner":[],"postedDate":"August 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-03T12:08:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-11 12:38:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4730842","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4730842","identity":"rs-4730842","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}