Borrelia burgdorferi sensu lato (Bbsl) is spirochetes transmitted by ticks and known to cause Lyme disease. Chlamydia-like organisms (CLOs) comprise a large group of bacteria that can lead to serious health disorders, including miscarriage. Recently, CLOs have been found in ticks and patient skin biopsies. Due to the involvement of multiple potential vectors in the spread of these pathogens, the objective of this study was to confirm the presence of both organisms in the early developmental stages of selected vectors. Three potential vectors, Ixodes ricinus larvae, Culex pipiens larvae, and winged (unfed) adults of Lipoptena cervi, were collected in the Czech Republic in years 2019-2020. The presence of Bbsl and panchlamydial DNA was detected by PCR and positive samples were further analyzed by Sanger sequencing and phylogenetic tree construction. Bbsl DNA was proved in 1.5% (2/137) of I. ricinus larvae (identified as Borrelia afzelii and Borrelia garinii), in 1.7% (2/119) of C. pipiens larvae (both identified as B. garinii), and in 11% (3/27) of L. cervi (all identified as B. garinii). CLOs were identified in 0.7% (1/137) of I. ricinus larvae (Candidatus Protochlamydia) and in 7.4% (2/27) of L. cervi (unspecified genus), while C. pipiens larvae could not be evaluated (0%). This research represents the first investigation of the presence of CLOs in L. cervi. The detection of pathogen DNA in the early developmental stages of vectors suggests the potential for transgenerational transmission of Bbsl and CLOs in the selected vectors, although at a low rate.

Department of Biology and Wildlife Diseases Faculty of Veterinary Hygiene and Ecology University of Veterinary Sciences Brno Palackého tř 1946 1 612 42 Brno Czech Republic

Department of Biology Faculty of Education Masaryk University Poříčí 7 9 63900 Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Kamenice 5 625 00 Bohunice Brno Czech Republic

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Girschick H. J., Morbach H., Tappe D. Treatment of Lyme borreliosis. Arthritis Research & Therapy . 2009;11(6) doi: 10.1186/ar2853.258 PubMed DOI PMC

Manzoor K., Aftab W., Choksi S., Khan I. A. Lyme carditis: sequential electrocardiographic changes in response to antibiotic therapy. International Journal of Cardiology . 2009;137(2):167–171. doi: 10.1016/j.ijcard.2008.05.028. PubMed DOI

Margos G., Vollmer S. A., Ogden N. H., Fish D. Population genetics, taxonomy, phylogeny and evolution of Borrelia burgdorferi sensu lato. Infection, Genetics and Evolution . 2011;11(7):1545–1563. doi: 10.1016/j.meegid.2011.07.022. PubMed DOI PMC

Barbour A. G., Adeolu M., Gupta R. S. Division of the genus Borrelia into two genera (corresponding to Lyme disease and relapsing fever groups) reflects their genetic and phenotypic distinctiveness and will lead to a better understanding of these two groups of microbes (Margos, et al. 2016 There is inadequate evidence to support the division of the genus Borrelia. Int. J. Syst. Evol. Microbiol. doi: 10.1099/ijsem.0.001717) International Journal of Systematic and Evolutionary Microbiology . 2017;67(6):2058–2067. doi: 10.1099/ijsem.0.001815. PubMed DOI

Barbour A. G., Hayes S. F. Biology of Borrelia species. Microbiological Reviews . 1986;50(4):381–400. doi: 10.1128/mr.50.4.381-400.1986. PubMed DOI PMC

Sapi E., Bastian S. L., Mpoy C. M., et al. Characterization of biofilm formation by Borrelia burgdorferi in vitro. PLoS One . 2012;7(10) doi: 10.1371/journal.pone.0048277.e48277 PubMed DOI PMC

Meriläinen L., Herranen A., Schwarzbach A., Gilbert L. Morphological and biochemical features of Borrelia burgdorferi pleomorphic forms. Microbiology . 2015;161(Pt 3):516–527. PubMed PMC

McLean R. G., Ubico S. R., Hughes C. A., Engstrom S. M., Johnson R. C. Isolation and characterization of Borrelia burgdorferi from blood of a bird captured in the Saint Croix River Valley. Journal of Clinical Microbiology . 1993;31(8):2038–2043. doi: 10.1128/jcm.31.8.2038-2043.1993. PubMed DOI PMC

Kurtenbach K., Carey D., Hoodless A. N., Nuttall P. A., Randolph S. E. Competence of pheasants as reservoirs for Lyme disease spirochetes. Journal of Medical Entomology . 1998;35(1):77–81. doi: 10.1093/jmedent/35.1.77. PubMed DOI

Swanson K. I., Norris D. E. Detection of Borrelia burgdorferi DNA in lizards from Southern Maryland. Vector Borne Zoonotic Dis Larchmt N. . 2007;7(1):42–49. PubMed PMC

Levine J. F., Wilson M. L., Spielman A. Mice as reservoirs of the Lyme disease spirochete. American Journal of Tropical Medicine and Hygiene . 1985;34(2):355–360. doi: 10.4269/ajtmh.1985.34.355. PubMed DOI

Benach J. L., Bosler E. M., Hanrahan J. P., et al. Spirochetes isolated from the blood of two patients with Lyme disease. The New England Journal of Medicine . 1983;308(13):740–742. doi: 10.1056/NEJM198303313081302. PubMed DOI

Barbour A. G., Burgdorfer W., Hayes S. F., Péter O., Aeschlimann A. Isolation of a cultivable spirochete fromIxodes ricinus ticks of Switzerland. Current Microbiology . 1983;8(2):123–126.

Gern L. Life cycle of Borrelia burgdorferi sensu lato and transmission to humans. Skin Bioengineering . 2009;37:18–30. PubMed

Scoles G. A., Papero M., Beati L., Fish D. A relapsing fever group spirochete transmitted by Ixodes scapularis ticks. Vector Borne and Zoonotic Diseases (Larchmont, NY) . 2001;1(1):21–34. PubMed

Rijpkema S., Bruinink H. Detection of Borrelia burgdorferi sensu lato by PCR in questing Ixodes ricinus larvae from the Dutch North Sea island of Ameland. Experimental & Applied Acarology . 1996;20(7):381–385. PubMed

van Duijvendijk G., Coipan C., Wagemakers A., et al. Larvae of Ixodes ricinus transmit Borrelia afzelii and B. miyamotoi to vertebrate hosts. Parasit Vectors . 2016;9 doi: 10.1186/s13071-016-1389-5.97 PubMed DOI PMC

Rollend L., Fish D., Childs J. E. Transovarial transmission of Borrelia spirochetes by Ixodes scapularis: a summary of the literature and recent observations. Ticks Tick-Borne . 2013;4(1-2):46–51. doi: 10.1016/j.ttbdis.2012.06.008. PubMed DOI

Kahane S., Dvoskin B., Mathias M., Friedman M. G. Infection of Acanthamoeba polyphagawith Simkania negevensis and S. negevensis survival within amoebal cysts. Applied and Environmental Microbiology . 2001;67(10):4789–4795. doi: 10.1128/AEM.67.10.4789-4795.2001. PubMed DOI PMC

Thao M. L. L., Baumann L., Hess J. M., et al. Phylogenetic evidence for two new insect-associated Chlamydia of the family Simkaniaceae. Current Microbiology . 2003;47(1):46–50. doi: 10.1007/s00284-002-3953-9. PubMed DOI

Soldati G., Lu Z. H., Vaughan L., et al. Detection of mycobacteria and chlamydiae in granulomatous inflammation of reptiles: a retrospective study. Veterinary Pathology . 2004;41(4):388–397. doi: 10.1354/vp.41-4-388. PubMed DOI

Fehr A., Walther E., Schmidt-Posthaus H., et al. Candidatus Syngnamydia venezia, a novel member of the phylum Chlamydiae from the broad nosed pipefish, Syngnathus typhle. PloS One . 2013;8(8)e70853 PubMed PMC

Kahane S., Greenberg D., Friedman M. G., Haikin H., Dagan R. High prevalence of Simkania Z, a novel Chlamydia-like bacterium, in infants with acute bronchiolitis. Journal of Infectious Diseases . 1998;177(5):1425–1429. PubMed

Greub G., Boyadjiev I., La Scola B., Raoult D., Martin C. Serological hint suggesting that Parachlamydiaceae are agents of pneumonia in polytraumatized intensive care patients. Annals of the New York Academy of Sciences . 2003;990:311–319. doi: 10.1111/j.1749-6632.2003.tb07381.x. PubMed DOI

Baud D., Thomas V., Arafa A., Regan L., Greub G. Waddlia chondrophila, a potential agent of human fetal death. Emerging Infectious Diseases . 2007;13(8):1239–1243. doi: 10.3201/eid1308.070315. PubMed DOI PMC

Borel N., Ruhl S., Casson N., Kaiser C., Pospischil A., Greub G. Parachlamydia spp. and related Chlamydia-like organisms and bovine abortion. Emerging Infectious Diseases . 2007;13(12):1904–1907. doi: 10.3201/eid1312.070655. PubMed DOI PMC

Lamoth F., Jaton K., Vaudaux B., Greub G. Parachlamydia and Rhabdochlamydia: emerging agents of community-acquired respiratory infections in children. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America . 2011;53(5):500–501. doi: 10.1093/cid/cir420. PubMed DOI

Verweij S. P., Kebbi-Beghdadi C., Land J. A., Ouburg S., Morré S. A., Greub G. Waddlia chondrophila and Chlamydia trachomatis antibodies in screening infertile women for tubal pathology. Microbes and Infection . 2015;17(11-12):745–748. doi: 10.1016/j.micinf.2015.09.019. PubMed DOI

Somani J., Bhullar V. B., Workowski K. A., Farshy C. E., Black C. M. Multiple drug-resistant Chlamydia trachomatis associated with clinical treatment failure. Journal of Infectious Diseases . 2000;181(4):1421–1427. doi: 10.1086/315372. PubMed DOI

Sapi E., Gupta K., Wawrzeniak K., et al. Borrelia and Chlamydia can form mixed biofilms in infected human skin tissues. European Journal of Microbiology and Immunology . 2019;9(2):46–55. doi: 10.1556/1886.2019.00003. PubMed DOI PMC

Kousa M., Saikku P., Kanerva L. Erythema nodosum in chlamydial infections. Acta Dermato-Venereologica . 1980;60(4):319–322. doi: 10.2340/0001555560319322. PubMed DOI

Schuttelaar M. L., Laeijendecker R., Heinhuis R. J., Van Joost T. Erythema multiforme and persistent erythema as early cutaneous manifestations of Lyme disease. Journal of the American Academy of Dermatology . 1997;37(5):873–875. doi: 10.1016/S0190-9622(97)80015-1. PubMed DOI

Simakova A. I., Popov A. F., Dadalova O. B. Ixodes tick-borne borreliosis with erythema nodosum. Meditsinskaia parazitologiia i parazitarnye bolezni . 2005;4(4):31–32. PubMed

Valtonen V. V. Infection as a risk factor for infarction and atherosclerosis. Annals of Medicine . 1991;23(5):539–543. doi: 10.3109/07853899109150515. PubMed DOI

Völzke H., Wolff B., Lüdemann J., et al. Seropositivity for anti-Borrelia IgG antibody is independently associated with carotid atherosclerosis. Atherosclerosis . 2006;184(1):108–112. doi: 10.1016/j.atherosclerosis.2004.10.048. PubMed DOI

Schnarr S., Putschky N., Jendro M. C., et al. Chlamydia and Borrelia DNA in synovial fluid of patients with early undifferentiated oligoarthritis: results of a prospective study. Arthritis and Rheumatism . 2001;44(11):2679–2685. doi: 10.1002/1529-0131(200111)44:11<2679::aid-art447>3.0.co;2-c. PubMed DOI

Capinera J. L. Encyclopedia of Entomology . Springer Science & Business Media; 2008. p. p. 4411.

Stanek G., Wormser G. P., Gray J., Strle F. Lyme borreliosis. Lancet . 2012;379(9814):461–473. doi: 10.1016/S0140-6736(11)60103-7. PubMed DOI

Sonenshine D. E., Roe R. M. Biology of Ticks . Vol. 2. USA: OUP; 2013. p. p. 504.

Reisen W. K., Milby M. M., Presser S. B., Hardy J. L. Ecology of mosquitoes and St. Louis encephalitis virus in the Los Angeles Basin of California, 1987–1990. Journal of Medical Entomology . 1992;29(4):582–598. doi: 10.1093/jmedent/29.4.582. PubMed DOI

Sabatinelli G., Ranieri E., Gianzi F. P., Papakay M., Cancrini G. Role of Culex quinquefasciatus in the transmission of bancroftian filariasis in the Federal Islamic Republic of Comoros (Indian Ocean) Parasite Paris France . 1994;1(1):71–76. PubMed

Kimura M., Darbro J. M., Harrington L. C. Avian malaria parasites share congeneric mosquito vectors. Journal of Parasitology . 2010;96(1):144–151. doi: 10.1645/GE-2060.1. PubMed DOI

Vinogradova E. B. Pensoft; 2000. Culex Pipiens Pipiens Mosquitoes: Taxonomy, Distribution, Ecology, Physiology, Genetics, Applied Importance and Control; p. p. 264.

Foster W., Walker E. Medical and Veterinary Entomology . 3rd. Academic Press; 2002. Mosquitoes (Culicidae) pp. 203–262.

Hornok S., de la Fuente J., Biró N., et al. First molecular evidence of Anaplasma ovis and Rickettsia spp. in keds (Diptera: Hippoboscidae) of sheep and wild ruminants. Vector Borne and Zoonotic Diseases (Larchmont, NY) . 2011;11(10):1319–1321. doi: 10.1089/vbz.2011.0649. PubMed DOI

de Bruin A., van Leeuwen A. D., Jahfari S., et al. Vertical transmission of Bartonella schoenbuchensis in Lipoptena cervi. Parasit Vectors . 2015;8 doi: 10.1186/s13071-015-0764-y.176 PubMed DOI PMC

Buss M., Case L., Kearney B., Coleman C., Henning J. D. Detection of Lyme disease and anaplasmosis pathogens via PCR in Pennsylvania deer ked. Journal of Vector Ecology . 2016;41(2):292–294. doi: 10.1111/jvec.12225. PubMed DOI

Böse R., Petersen K. Lipoptena cervi (Diptera), a potential vector of Megatrypanum trypanosomes of deer (Cervidae) Parasitology Research . 2004;77(8):723–725. doi: 10.1007/BF00928691. PubMed DOI

Bezerra-Santos M. A., Otranto D. Keds, the enigmatic flies and their role as vectors of pathogens. Acta Tropica . 2020;209 doi: 10.1016/j.actatropica.2020.105521.105521 PubMed DOI

Kaitala A., Kortet R., Härkönen S., et al. Deer ked, an ectoparasite of moose in Finland: a brief review of its biology and invasion. Alces . 2009;45:85–88.

Paakkonen T., Mustonen A. M., Roininen H., Niemelä P., Ruusila V., Nieminen P. Parasitism of the deer ked, Lipoptena cervi, on the moose, Alces alces, in eastern Finland. Medical and Veterinary Entomology . 2010;24(4):411–417. doi: 10.1111/j.1365-2915.2010.00910.x. PubMed DOI

Korhonen E. M., Vera C. Pérez, Pulliainen A. T., et al. Molecular detection of Bartonella spp. in deer ked pupae, adult keds and moose blood in Finland. Epidemiology and Infection . 2015;143(3):575–585. doi: 10.1017/S0950268814001411. PubMed DOI PMC

Everett K. D., Bush R. M., Andersen A. A. Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. International Journal of Systematic Bacteriology . 1999;49(Pt 2):415–440. doi: 10.1099/00207713-49-2-415. PubMed DOI

Rijpkema S., Nieuwenhuijs J., Franssen F. F., Jongejan F. Infection rates of Borrelia burgdorferi in different instars of Ixodes ricinus ticks from the Dutch North Sea Island of Ameland. Experimental & Applied Acarology . 1994;18(9):531–542. PubMed

Hokynar K., Sormunen J. J., Vesterinen E. J., et al. Chlamydia-like organisms (CLOs) in finnish Ixodes ricinus ticks and human skin. Microorganisms . 2016;4(3) doi: 10.3390/microorganisms4030028.28 PubMed DOI PMC

Hauck D., Jordan D., Springer A., et al. Transovarial transmission of Borrelia spp., Rickettsia spp. and Anaplasma phagocytophilum in Ixodes ricinus under field conditions extrapolated from DNA detection in questing larvae. Parasit Vectors . 2020;13(1) doi: 10.1186/s13071-020-04049-7.176 PubMed DOI PMC

Žákovská A., Nejedla P., Holíková A., Dendis M. Positive findings of Borrelia burgdorferi in Culex (Culex) pipiens pipiens larvae in the surrounding of Brno city determined by the PCR method. Annals of Agricultural and Environmental Medicine (AAEM) . 2002;9(2):257–259. PubMed

Nejedla P., Norek A., Vostal K., Zakovska A. What is the percentage of pathogenic borreliae in spirochaetal findings of mosquito larvae? Annals of Agricultural and Environmental Medicine (AAEM) . 2009;16(2):273–276. PubMed

Melaun C., Zotzmann S., Santaella V. G., et al. Occurrence of Borrelia burgdorferi s.l in different genera of mosquitoes (Culicidae) in Central Europe. Ticks Tick-Borne Diseases . 2016;7(2):256–263. doi: 10.1016/j.ttbdis.2015.10.018. PubMed DOI

Gałęcki R., Jaroszewski J., Bakuła T., Galon E. M., Xuan X. Molecular detection of selected pathogens with zoonotic potential in deer keds (Lipoptena fortisetosa) Pathogens (Basel, Switzerland) . 2021;10(3)324 PubMed PMC

Žákovská A., Treml F., Nejezchlebová H., Nepeřený J., Budíková M., Bártová E. Leptospira interrogans Sensu Lato in wild small mammals in three moravian localities of the Czech Republic. Pathogens . 2022;11(8)888 PubMed PMC

Tamura K., Stecher G., Kumar S. Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution . 2021;38(7):3022–3027. doi: 10.1093/molbev/msab120. PubMed DOI PMC

Saitou N., Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution . 1987;4(4):406–425. PubMed

Tamura K., Nei M., Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences . 2004;101(30):11030–11035. doi: 10.1073/pnas.0404206101. PubMed DOI PMC