Original Article

Metagenomics Characterization of Ixodes ricinus Intestinal Microbiota as Major Vector of Tick-Borne Diseases in Domestic Animals


Background: Understanding the microbiota of disease vectors can help for developing new strategies to prevent the transmis­sion of vector pathogens. Ixodes ricinus is one of the most notorious tick vectors with increasing importance in Iran and other parts of the world while there is limited data on its microbiota. This study aimed to use metagenomics for identifying the I. ricinus tick’s microbiota of Iran.

Methods: A total of 39 adult ticks were collected from Mazandaran (21 females), Gilan (17 females), and Golestan (1 male). Five tick pools prepared from 39 adults of I. ricinus were subjected to metagenomics analysis. The data were analyzed by targeting the V6 region of the 16S rRNA gene by Illumina 4000 Hiseq sequencing.

Results: Among hundreds of intestinal microbiota identified by metagenomics, various pathogenic microorganisms distributed in 30 genera and species including those responsible for tick-borne diseases resided in the genera Coxiella, Rickettsia, and Burkholderia were found.

Conclusion: Our results indicated that metagenomics identifies bacteria genera and species which cannot be easily rec­ognized by routine methods. The presence of such pathogenic bacteria indicates the importance of possible zoonotic diseases in this region which could affect public health. These results further substantiate the importance of advanced metagenomics analyses to identify neglected tick-borne pathogens which enable researchers to provide efficient mapping roads for the management of emerging and re-emerging infectious diseases.

1. Walter DE, Proctor HC (1999) Mites: ecol-ogy, evolution and behaviour.
2. Sharifdini M, Norouzi B, Azari-Hamidian S, Karamzadeh N (2021) The first rec-ord of ectoparasites of raccoons (Procy-on lotor) (Carnivora, Procyonidae) in Iran. Persian J Acarol. 10(1): 41–54.
3. Paules CI, Marston HD, Bloom ME, Fauci AS (2018) Tickborne Diseases-Confront¬ing a Growing Threat. N Engl J Med. pmid: 30044925.
4. Černý J, Lynn G, Hrnková J, Golovchenko M, Rudenko N, Grubhoffer L (2020) Man¬agement options for Ixodes ricinus-as¬so¬ciated pathogens: a review of prevention strategies. Int J Environ Res Publ Health. 17(6): 1830.
5. James M, Bowman A, Forbes K, Lewis F, McLeod J, Gilbert L (2013) Environ-men¬tal determinants of Ixodes ricinus ticks and the incidence of Borrelia burgdor¬feri sensu lato, the agent of Lyme bor¬reliosis, in Scotland. Parasitology. 140 (2): 237–246.
6. Alfredsson M, Olafsson E, Eydal M, Unnsteinsdot¬tir ER, Hansford K, Wint W, Alexander N, Medlock JM (2017) Surveil¬lance of Ixodes ricinus ticks (Ac-ari: Ixodi¬dae) in Iceland. Parasit Vec-tors. 10(1): 466.
7. Mooney PA, Rechid D, Davin EL, Kat-ragkou E, de Noblet-Ducoudré N, Breil M, Cardoso RM, Daloz AS, Hoffmann P, Lima DCA, Meier R, Soares PMM, Sofiadis G, Strada S, Strandberg G, Toelle MH, Lund MT (2022) Land–at-mosphere interactions in sub-polar and alpine cli¬mates in the CORDEX Flag-ship Pilot Study Land Use and Climate Across Scales (LUCAS) models–Part 2: The role of changing vegetation, The Cryosphere. 16: 1383–1397.
8. Bellato A, Pintore MD, Catelan D, Pau¬tas-so A, Torina A, Rizzo F, Mandola ML, Man¬nelli A, Casalone C, Tomassone L (2021) Risk of tick-borne zoonoses in urban green areas: A case study from Turin, north¬west¬ern Italy. Urban Forest Urban Green. 64: 127297.
9. Choubdar N, Oshaghi MA, Rafinejad J, Pour¬mand MR, Maleki-Ravasan N, Salehi-Vaziri M, Telmadarraiy Z, Karim-ian F, Koosha M, Rahimi-Foroushani A, Masoo¬mi S, Arzamani K, Nejati J, Karami M, Mozaffari E, Salim-Abadi Y, Moradi-Asl E, Taghilou B, Shirani M (2019) Effect of meteorologi¬cal factors on Hyalomma species com¬position and their host preference, sea¬sonal preva-lence and infection status to crimean-congo haemorrhagic fe¬ver in Iran. J Ar-thropod Borne Dis. 13 (3): 268–283.
10. Seedorf H, Griffin NW, Ridaura VK, Reyes A, Cheng J, Rey FE, Smith MI, Simon GM, Scheffrahn RH, Woebken D, Spor¬mann AM, Van Treuren W, Ursell LK, Pirrung M, Robbins-Pianka A, Cantarel BL, Lombard V, Henrissat B, Knight R, Gordon JI (2014) Bacteria from diverse habitats colonize and com-pete in the mouse gut. Cell. 159(2): 253–266.
11. Eybpoosh S, Haghdoost AA, Mostafavi E, Bahrampour A, Azadmanesh K, Zolala F (2017) Molecular epidemiology of in¬fectious diseases. Electron Phys. 9(8): 5149.
12. Lange A (2021) Interactions between in-tes¬ti¬nal commensal bacteria and differ-ent hosts: two sides of the same story: PhD Thesis, Universität Tübingen.
13. Tabasi M, Eybpoosh S, Siadat SD, El-yasinia F, Soroush A, Bouzari S (2021) Modulation of the gut microbiota and se¬rum biomarkers after laparoscopic sleeve gastrectomy: a 1-year follow-up study. Obes Surg. 31: 1949–1956.
14. Narasimhan S, Fikrig E (2015) Tick mi¬cro-biome: the force within. Trends Par-asitol. 31(7): 315–323.
15. Dantas-Torres F, Chomel BB, Otranto D (2021) Ticks and tick-borne diseases: a One Health perspective. Trends Parasi-tol. 28(10): 437–446.
16. Wilhelmsson P, Jaenson TG, Olsen B, Wal¬denström J, Lindgren P-E (2020) Migra¬tory birds as disseminators of ticks and the tick-borne pathogens Borrelia bacte¬ria and tick-borne encephalitis (TBE) vi¬rus: a seasonal study at Ottenby Bird Ob¬ser¬va¬tory in South-eastern Sweden. Par¬a¬sit Vectors. 13: 1–17.
17. Rizzoli A, Silaghi C, Obiegala A, Rudolf I, Hubálek Z, Földvári G, Plantard O, Vayssier-Taussat M, Bonnet S, Spit¬alská E, Kazimírová M (2014) Ixodes ricinus and Its transmitted pathogens in urban and peri-urban areas in Europe: New Hazards and relevance for public health. Front Public Health. 2: 251.
18. Wilhelmsson P, Pawełczyk O, Jaenson TG, Waldenström J, Olsen B, Forsberg P, Lind¬gren PE (2021) Three Babesia species in Ixodes ricinus ticks from migratory birds in Sweden. Parasit Vectors. 14(1): 183.
19. Pettersson JH-O (2013) The Origin of the Genus Flavivirus and the Ecology of Tick-Borne Pathogens, PhD Thesis, Acta Uni¬ver¬sitatis Upsaliensis.
20. Blanda V, Torina A, La Russa F, D'Ago-stino R, Randazzo K, Scimeca S, Giu-dice E, Caracappa S, Cascio A, de la Fuente J (2017) A retrospective study of the char¬acterization of Rickettsia species in ticks collected from humans. Ticks Tick Borne Dis. 8(4): 610–614.
21. Cutler SJ, Ruzic-Sabljic E, Potkonjak A (2017) Emerging borreliae–Expanding be¬yond Lyme borreliosis. Mol Cell Probes. 31: 22–27.
22. Mansfield KL, Cook C, Ellis RJ, Bell-Sakyi L, Johnson N, Alberdi P, de la Fuente J, Fooks AR (2017) Tick-borne pathogens induce differential expression of genes pro¬moting cell survival and host resistance in Ixodes ricinus cells. Parasit Vectors. 10(1): 81.
23. Reis C, Cote M, Le Rhun D, Lecuelle B, Levin ML, Vayssier-Taussat M, Bonnet SI (2011) Vector competence of the tick Ixodes ricinus for transmission of Bar-tonella birtlesii. PLoS Negl Trop Dis. 5(5): e1186.
24. Pajoro M, Pistone D, Varotto Boccazzi I, Mereghetti V, Bandi C, Fabbi M, Scat-torin F, Sassera D, Montagna M (2018) Molecular screening for bacterial path-ogens in ticks (Ixodes ricinus) collected on migratory birds captured in northern Italy. Folia Parasitol. 65: 2018.008.
25. Laaksonen M, Klemola T, Feuth E, Sormunen JJ, Puisto A, Mäkelä S, Penttinen R, Ruohomäki K, Hänninen J, Sääksjärvi IE, Vuorinen I, Sprong H, Hytönen J, Vesterinen EJ (2018) Tick-borne pathogens in Finland: comparison of Ixodes ricinus and I. persulcatus in sympatric and parapatric areas. Parasit Vectors. 11(1): 556.
26. Song X, Zhong Z, Gao L, Weiss BL, Wang J (2022) Metabolic interactions between disease-transmitting vectors and their mi¬crobiota. Trends Parasitol. 38(8): 697–708.
27. Fukuda K, Ogawa M, Taniguchi H, Saito M (2016) Molecular approaches to stud-ying microbial communities: targeting the 16S ribosomal RNA gene. J UOEH. 38 (3): 223–232.
28. Mathison BA, Pritt BS (2014) Laboratory identification of arthropod ectoparasites. Clin Microbiol Rev. 27(1): 48–67.
29. Salehi Z, Shams-Ghahfarokhi M, Fattahi A, Ghazanfari M, Yazdanparast SA (2018) A head-to-head comparison of four cry¬o¬preservation protocols of dermatophyte species. Infect Epidemiol Microbiol. 4(3): 109–114.
30. Patel RK, Jain M (2012) NGS QC Toolkit: a toolkit for quality control of next gen-eration sequencing data. PloS One. 7(2): e30619.‏
31. Okonechnikov K, Conesa A, García-Al-cal¬de F (2016) Qualimap 2: advanced multi-sample quality control for high-through¬put sequencing data. Bioinfor-matics. 32(2): 292-294.
32. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson A. JA, Holmes SP (2016) DADA2: high-resolution sample inference from Illumina amplicon data. Nature Meth¬ods. 13(7): 581–583.
33. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO (2013) The SILVA ribosomal RNA gene database project: improved data pro¬cessing and web-based tools. Nucleic Acids Res. 41(Database issue): D590–596.
34. Bolyen E, Rideout JR, Dillon MR, Boku-lich NA, Abnet CC, Al-Ghalith GA, Al-exander H, Alm EJ, Arumugam M, As-nicar F, Bai Y, Bisanz JE, Bittinger K, Brejnrod A, Brislawn CJ, Brown CT, Cal¬lahan BJ, Caraballo-Rodríguez AM, Chase J, Cope EK, Da Silva R, Diener C, Dorrestein PC, Douglas GM, Durall DM, Duvallet C, Edwardson CF, Ernst M, Es¬taki M, Fouquier J, Gauglitz JM, Gibbons SM, Gibson DL, Gonzalez A, Gorlick K, Guo J, Hillmann B, Holmes S, Holste H, Huttenhower C, Huttley GA, Janssen S, Jarmusch AK, Jiang L, Kaehler BD, Kang KB, Keefe CR, Keim P, Kelley ST, Knights D, Koester I, Kosciolek T, Kreps J, Langille MGI, Lee J, Ley R, Liu YX, Loftfield E, Lo-zupone C, Maher M, Marotz C, Martin BD, McDonald D, McIver LJ, Melnik AV, Metcalf JL, Mor¬gan SC, Morton JT, Naimey AT, Navas-Molina JA, No-thias LF, Orchanian SB, Pear¬son T, Peo-ples SL, Petras D, Preuss ML, Pruesse E, Rasmussen LB, Rivers A, Robeson MS, Rosenthal P, Segata N, Shaf¬fer M, Shiffer A, Sinha R, Song SJ, Spear JR, Swafford AD, Thompson LR, Torres PJ, Trinh P, Tripathi A, Turnbaugh PJ, Ul-Hasan S, Van der Hooft JJJ, Vargas F, Vázquez-Baeza Y, Vogtmann E, von Hip¬pel M, Walters W, Wan Y, Wang M, War¬ren J, Weber KC, Williamson CHD, Wil¬lis AD, Xu ZZ, Zaneveld JR, Zhang Y, Zhu Q, Knight R, Caporaso JG (2019) Re¬producible, interactive, scala-ble and exten¬sible microbiome data sci-ence using QIIME 2. Nat Biotechnol. 37(8): 852–857.
35. Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinfor-matics. 30(9): 1312–1313.
36. Carpi G, Cagnacci F, Wittekindt NE, Zhao F, Qi J, Tomsho LP, Drautz DI, Rizzoli A, Schuster SC (2011) Meta¬genomic profile of the bacterial com¬munities as-sociated with Ixodes ricinus ticks. PLoS One. 6(10): e25604.
37. Portillo A, Palomar AM, de Toro M, San-tibáñez S, Santibáñez P, Oteo JA (2019) Exploring the bacteriome in anthro-pophilic ticks: To investigate the vectors for di¬ag¬nosis. Plos One. 14(3): e0213384.
38. Choubdar N, Karimian F, Koosha M, Oshaghi MA (2021) An integrated over-view of the bacterial flora composition of Hyalomma anatolicum, the main vec-tor of CCHF. PLoS Negl Trop Dis. 15(6): e0009480.
39. Batool M, Blazier JC, Rogovska YV, Wang J, Liu S, Nebogatkin IV, Rogov-skyy AS (2021) Metagenomic analysis of individ¬ually analyzed ticks from East-ern Europe demonstrates regional and sex-de¬pend¬ent differences in the mi¬cro-biota of Ixodes rici¬nus. Ticks Tick Borne Dis. 12(5): 101768.
40. Kmeť V, Čaplová Z (2019) An update on the Ixodes ricinus microbiome. J Micro-bi¬ol Biotechnol Food Sci. 8(6): 1340–1342.
41. Sacchi L, Bigliardi E, Corona S, Beninati T, Lo N, Franceschi A (2004) A sym-biont of the tick Ixodes ricinus invades and con¬sumes mitochondria in a mode similar to that of the parasitic bacterium Bdel¬lovib¬rio bacteriovorus. Tissue Cell. 36(1): 43–53.
42. Lo N, Beninati T, Sassera D, Bouman EA, Santagati S, Gern L, Sambri V, Masu¬za-wa T, Gray JS, Jaenson TG, Bouat¬tour A, Kenny MJ, Guner ES, Khari¬tonenkov IG, Bitam I, Bandi C (2006) Wide-spread dis¬tribution and high preva¬lence of an alpha-proteobacterial symbi¬ont in the tick Ixodes ricinus. Environ Microbi-ol. 8(7): 1280–1287.
43. Lejal E, Chiquet J, Aubert J, Robin S, Es-trada-Peña A, Rue O, Midoux C, Mari-adas¬sou M, Bailly X, Cougoul A, Gasqui P, Cosson JF, Chalvet-Monfray K, Vayssier-Taussat M, Pollet T (2021) Tem¬poral patterns in Ixodes ricinus mi-crobi¬al communities: an insight into tick-borne mi¬crobe interactions. Microbi¬ome. 9(1): 153.
44. Porter SR, Czaplicki G, Mainil J, Guattéo R, Saegerman C (2011) Q Fever: current state of knowledge and perspectives of research of a neglected zoonosis. Int J Microbiol. 2011: 248418.
45. Mostafavi E, Rastad H, Khalili M (2012) Q fever: an emerging public health con-cern in Iran. Asian J Epidemiol. 5(3): 66–74.
46. Hosseini-Chegeni A, Tavakoli M, Telma-darraiy Z, Faghihi F (2020) Molecular de¬tection of spotted fever group Rick-ettsia (Rickettsiales: Rickettsiaceae) in ticks of Iran. Arch Razi Inst. 75(3): 317–325.
47. Eybpoosh S, Fazlalipour M, Baniasadi V, Pouriayevali MH, Sadeghi F, Ahmadi Vasme¬hjani A, Karbalaie Niya MH, Hew¬son R, Salehi-Vaziri M (2019) Epi-de¬miology of West Nile Virus in the Eastern Mediterranean region: A sys-tematic review. PLoS Negl Trop Dis. 13(1): e0007081.
48. Ghaderi E, Salehi Vaziri M, Mostafavi E, Moradi G, Rahmani K, Zeinali M, Shir-zadi MR, Erfani H, Afrasiabian SH, Eybpoosh S (2019) Evaluation of Cri-mean-congo Hemorrhagic Fever (CCHF) Surveillance System in Iran. Iran J Ep-idemiol. 15(3): 300–312.
49. Duval BD, Elrod MG, Gee JE, Chantratita N, Tandhavanant S, Limmathurotsakul D, Hoffmaster AR (2014) Evaluation of a latex ag¬glutination assay for the identi-fication of Burkholderia pseudomallei and Burkhold¬eria mallei. Am J Trop Med Hyg. 90(6): 1043–1046.
50. Pal M (2005) Importance of zoonoses in public health. Indian J Anim Sci. 75(5): 586–591.
51. Brinkerhoff RJ, Clark C, Ocasio K, Gauthi¬er DT, Hynes WL (2020) Factors af¬fect¬ing the microbiome of Ixodes scapularis and Amblyomma americanum. PloS One. 15(5): e0232398.
52. Van Treuren W, Ponnusamy L, Brinker-hoff RJ, Gonzalez A, Parobek CM, Juli-ano JJ, Andreadis TG, Falco RC, Zieg¬ler LB, Hathaway N, Keeler C, Emch M, Bai¬ley JA, Roe RM, Apperson CS, Knight R, Meshnick SR (2015) Varia-tion in the microbiota of Ixodes ticks with regard to ge¬ography, species, and sex. Appl Environ Microbiol. 81(18): 6200–6209.
53. Cronin P, Joyce SA, O’Toole PW, O’Connor EM (2021) Dietary fibre modulates the gut microbiota. Nutrients. 13(5): 1655.
IssueVol 17 No 2 (2023) QRcode
SectionOriginal Article
DOI https://doi.org/10.18502/jad.v17i2.13620
Tick-borne disease; Microbiota; Ixodes ricinus; Next-generation sequencing; Emerging pathogens

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Yousefi Behzadi M, Mehrabi A, Ahmadinezhad M, Rohani M, Naddaf SR, Bagheri A, Shams-Ghahfarokhi M, Maghsoudlou E, Mojahed N, Mounesan L, Tahmasebi Z, Sohrabi A, Salehi-Vasiri M, Salehi Z, Razzaghi-Abyaneh M. Metagenomics Characterization of Ixodes ricinus Intestinal Microbiota as Major Vector of Tick-Borne Diseases in Domestic Animals. J Arthropod Borne Dis. 2023;17(2):152–164.