Inhibition of the endosymbiont "Candidatus Midichloria mitochondrii" during 16S rRNA gene profiling reveals potential pathogens in Ixodes ticks from Australia

Parasites and Vectors

Volumn 8, Issue 1, 2015, Pages

  Gofton, Alexander W ^a   Oskam, Charlotte L ^a   Lo, Nathan ^b   Beninati, Tiziana ^b   Wei, Heng ^b   McCarl, Victoria ^b   Murray, Dáithí C ^c   Paparini, Andrea ^a   Greay, Telleasha L ^a   Holmes, Andrew J ^b   Bunce, Michael ^c   Ryan, Una ^a   Irwin, Peter ^a  

^a MURDOCH UNIVERSITY   (Australia)

^b UNIVERSITY OF SYDNEY   (Australia)

^c CURTIN UNIVERSITY   (Australia)

Author keywords

16s community profiling; Borrelia; Candidatus Midichloria; Candidatus Neoehrlichia; Ixodes holocyclus; Ixodes ricinus; Metagenomics; Tick; Vector borne disease; Zoonoses

Indexed keywords

BACTERIAL RNA; BLOCKING PRIMER; GENOMIC DNA; PRIMER DNA; RNA 16S; UNCLASSIFIED DRUG; BACTERIAL DNA;

AMPLICON; ARTICLE; AUSTRALIA; BACTERIUM; BACTERIUM DETECTION; BACTERIUM IDENTIFICATION; BARTONELLA HENSELAE; BORRELIA BURGDORFERI; CANDIDATUS MIDICHLORIA MITOCHONDRII; CANDIDATUS NEOEHRLICHIA; CLOSTRIDIUM HISTOLYTICUM; CONTROLLED STUDY; ENDOSYMBIONT; FEMALE; GENE AMPLIFICATION; GENE EXPRESSION PROFILING; GENUS; GERMANY; IXODES; IXODES HOLOCYCLUS; IXODES RICINUS; LEPTOSPIRA; LEPTOSPIRA INADAI; LYME DISEASE; MALE; MICROBIAL DIVERSITY; MICROBIOME; NEXT GENERATION SEQUENCING; NONHUMAN; NUCLEOTIDE SEQUENCE; NYMPH; POLYMERASE CHAIN REACTION; POPULATION ABUNDANCE; RICKETTSIA; RNA PURIFICATION; RNA SEQUENCE; SPECIES; ALPHAPROTEOBACTERIA; ANIMAL; ARACHNID VECTOR; BORRELIA; CLASSIFICATION; GENETICS; ISOLATION AND PURIFICATION; MICROBIOLOGY; MOLECULAR GENETICS; PHYLOGENY; PHYSIOLOGY; SYMBIOSIS;

ANIMALIA; ARTHROPODA; BACTERIA (MICROORGANISMS); BARTONELLA HENSELAE; BORRELIA; BORRELIA BURGDORFERI GROUP; BORRELIA SP.; CLOSTRIDIUM HISTOLYTICUM; IXODES; IXODES HOLOCYCLUS; IXODES RICINUS; IXODIDA; LEPTOSPIRA INADAI; RICKETTSIA;

ALPHAPROTEOBACTERIA; ANIMALS; ARACHNID VECTORS; AUSTRALIA; BORRELIA; DNA, BACTERIAL; FEMALE; IXODES; MALE; MOLECULAR SEQUENCE DATA; PHYLOGENY; POLYMERASE CHAIN REACTION; RNA, RIBOSOMAL, 16S; SYMBIOSIS;

EID: 84935508692     PISSN: None     EISSN: 17563305     Source Type: Journal    
DOI: 10.1186/s13071-015-0958-3     Document Type: Article

References (97)

1. Dantas-Torres F, Chomel BB, Otranto D. Ticks and tick-borne diseases: a One health perspective. Trends Parasitol. 2012;28(10):437-46.
2. Githeko AK, Lindsay SW, Confalonieri UE, Patz JA. Climate change and vector-borne diseases: a regional analysis. Bull World Health Organ. 2000;78(9):1136-47.
3. Parola P, Raoult D. Ticks and tickborne bacterial diseases in humans: an emerging infectious threat. Clin Infect Dis. 2001;32(6):897-928.
4. Hartelt K, Oehme R, Frank H, Brockmann SO, Hassler D, Kimmig P. Pathogens and symbionts in ticks: prevalence of Anaplasma phagocytophilum (Ehrlichia sp.), Wolbachia sp., Rickettsia sp., and Babesia sp. in Southern Germany. Intl J Med Microbio Sup. 2004;293(37):86-92.
5. Sanogo YO, Zeaiter Z, Caruso G, Merola F, Shpynov S, Brouqui P, et al. Bartonella henselae in ixodes ricinus ticks (acari: ixodida) removed from humans, belluno province. Italy Emerg Infect Dis. 2003;9(3):329-32.
6. Burgdorfer W, Lane RS, Barbour AG, Gresbrink RA, Anderson JR. The western black-legged tick, Ixodes pacificus: a vector of Borrelia burgdorferi. Am J Trop Med Hyg. 1985;34(5):925-30.
7. Dumler JS, Bakken JS. Ehrlichial diseases of humans: emerging tick-borne infections. Clin Infect Dis. 1995;20(5):1102-10.
8. Petersen JM, Mead PS, Schriefer ME. Francisella tularensis: an arthropod-borne pathogen. Vet Res. 2009;40(2):07.
9. Azad AF, Beard CB. Rickettsial pathogens and their arthropod vectors. Emerg Infect Dis. 1998;4(2):179-86.
10. Richter D, Matuschka F-R. "Candidatus Neoehrlichia mikurensis", Anaplasma phagocytophilum and Lyme disease spirochetes in questing European vector ticks and in feeding ticks removed from people. J Clin Microbiol. 2011;JCM:05802-11.
11. Colwell DD, Dantas-Torres F, Otranto D. Vector-borne parasitic zoonoses: emerging scenarios and new perspectives. Vet Parasitol. 2011;182(1):14-21.
12. Shaw SE, Day MJ, Birtles RJ, Breitschwerdt EB. Tick-borne infectious diseases of dogs. Trends Parasitol. 2001;17(2):74-80.
13. Welc-Faleciak R, Hildebrandt A, Sinski E. Co-infection with Borrelia species and other tick-borne pathogens in humans: two cases from Poland. Ann Agric Environ Med. 2010;17(2):309-13.
14. Hofhuis A, Harms M, van den Wijngaard C, Sprong H, van Pelt W. Continuing increase of tick bites and Lyme disease between 1994 and 2009. Ticks Tick-Borne Dis. 2015;6(1):69-74.
15. Stewart A, Glass J, Patel A, Watt G, Cripps A, Clancy R. Lyme arthritis in the hunter valley. Med J Aust. 1982;1(3):139.
16. Russell R. Vectors vs. Humans in Australia - who is on top down under? an update on vector-borne disease and research on vectors in Australia. J Vector Ecol. 1998;23(1):1-46.
17. Russell RC, Doggett SL, Munro R, Ellis J, Avery D, Hunt C, et al. Lyme disease: a search for a causative agent in ticks in south-eastern Australia. Epidemiol Infect. 1994;112(2):375-84.
18. Wills MC, Barry RD. Detecting the cause of Lyme disease in Australia. Med J Aust. 1991;155(4):275.
19. Mayne PJ. Emerging incidence of Lyme borreliosis, babesiosis, bartonellosis, and granulocytic ehrlichiosis in Australia. Int J Gen Med. 2011;4:845-52.
20. Barker SC, Walker AR, Campelo D. A list of the 70 species of Australian ticks; diagnostic guides to and species accounts of Ixodes holocyclus (paralysis tick), Ixodes cornuatus (southern paralysis tick) and Rhipicephalus australis (Australian cattle tick); and consideration of the place of Australia in the evolution of ticks with comments on four controversial ideas. Int J Parasitol. 2014;44(12):941-53.
21. Barker SC, Walker AR. Ticks of Australia. The species that infest domestic animals and humans. Zootaxa. 2014;3816:1-144.
22. Song S, Shao R, Atwell R, Barker S, Vankan D. Phylogenetic and phylogeogra.phic relationships in Ixodes holocyclus and Ixodes comuatus (Acari: Ixodidae) inferred from COX1 and ITS2 sequences. Int J Parasitol. 2011;41(8):871-80.
23. Campbell RW, Domrow R. Rickettsioses in Australia: isolation of Rickettsia tsutsugamushi and R. australis from naturally infected arthropods. Trans R Soc Trop Med Hyg. 1974;68(5):397-402.
24. Klyachko O, Stein BD, Grindle N, Clay K, Fuqua C. Localization and visualization of a coxiella-type symbiont within the lone star tick. Amblyomma americanum. Appl Environ Microbiol. 2007;73(20):6584-94.
25. Lalzar I, Harrus S, Mumcuoglu KY, Gottlieb Y. Composition and seasonal variation of Rhipicephalus turanicus and Rhipicephalus sanguineus bacterial communities. Appl Environ Microbiol. 2012;78(12):4110-6.
26. Liu L, Li L, Liu J, Hu Y, Liu Z, Guo L, et al. Coinfection of Dermacentor silvarum olenev (acari: Ixodidae) by Coxiella-Like, Arsenophonus-like, and Rickettsia-like symbionts. Appl Environ Microbiol. 2013;79(7):2450-4.
27. Scoles GA. Phylogenetic analysis of the Francisella-like endosymbionts of Dermacentor ticks. J Med Entomol. 2004;41(3):277-86.
28. Sun LV, Scoles GA, Fish D, O'Neill SL. Francisella-like endosymbionts of ticks. J Invertebr Pathol. 2000;76(4):301-3.
29. Venzal JM, Estrada-Peña A, Castro O, de Souza CG, Félix ML, Nava S, et al. Amblyomma triste Koch, 1844 (Acari: Ixodidae): hosts and seasonality of the vector of Rickettsia parkeri in Uruguay. Vet Parasitol. 2008;155(1-2):104-9.
30. Andreotti R, de León AAP, Dowd SE, Guerrero FD, Bendele KG, Scoles GA. Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing. BMC Microbiol. 2011;11(1):6.
31. Benson MJ, Gawronski JD, Eveleigh DE, Benson DR. Intracellular symbionts and other bacteria associated with deer ticks (ixodes scapularis) from Nantucket and Wellfleet, cape Cod. Massachusetts. Appl Environ Microbiol. 2004;70(1):616-20.
32. Hirunkanokpun S, Kittayapong P, Cornet J-P, Gonzalez J-P. Molecular evidence for novel tick-associated spotted fever group rickettsiae from Thailand. J Med Entomol. 2003;40(2):230-7.
33. Niebylski ML, Schrumpf ME, Burgdorfer W, Fischer ER, Gage KL, Schwan TG. Rickettsia peacockii sp. nov., a New species infecting wood ticks, dermacentor andersoni, in western Montana. Int J Syst Bacteriol. 1997;47(2):446-52.
34. Reis C, Cote M, Paul REL, Bonnet S. Questing ticks in suburban forest are infected by at least six tick-borne pathogens. Vector Borne Zoonotic Dis. 2011;11(7):907-16.
35. Satta G, Chisu V, Cabras P, Fois F, Masala G. Pathogens and symbionts in ticks: a survey on tick species distribution and presence of tick-transmitted micro-organisms in Sardinia, Italy. J Med Microbiol. 2011;60(1):63-8.
36. Sassera D, Beninati T, Bandi C, Bouman EAP, Sacchi L, Fabbi M, et al. "Candidatus Midichloria mitochondrii", an endosymbiont of the tick Ixodes ricinus with a unique intramitochondrial lifestyle. Int J Syst Evol Microbiol. 2006;56(11):2535-40.
37. Beninati T, Riegler M, Vilcins I-ME, Sacchi L, McFadyen R, Krockenberger M, et al. Absence of the symbiont "Candidatus Midichloria mitochondrii" in the mitochondria of the tick Ixodes holocyclus. FEMS Microbiol Lett. 2009;299(2):241-7.
38. Harrus S, Perlman-Avrahami A, Mumcuoglu KY, Morick D, Eyal O, Baneth G. Molecular detection of Ehrlichia canis, Anaplasma bovis, Anaplasma platys, Candidatus Midichloria mitochondrii and Babesia canis vogeli in ticks from Israel. Clin Microbiol Infect. 2011;17(3):459-63.
39. Williams-Newkirk AJ, Rowe LA, Mixson-Hayden TR, Dasch GA. Presence, genetic variability, and potential significance of "Candidatus Midichloria mitochondrii" in the lone star tick Amblyomma americanum. Exp Appl Acarol. 2012;58(3):291-300.
40. Ahantarig A, Trinachartvanit W, Baimai V, Grubhoffer L. Hard ticks and their bacterial endosymbionts (or would be pathogens). Folia Microbiol (Praha). 2013;58(5):419-28.
41. Haine ER. Symbiont-mediated protection. Proc Biol Sci. 2008;275(1633):353-61.
42. Walker T, Johnson PH, Moreira LA, Iturbe-Ormaetxe I, Frentiu FD, McMeniman CJ, et al. The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations. Nature. 2011;476(7361):450-3.
43. Epis S, Sassera D, Beninati T, Lo N, Beati L, Piesman J, et al. Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species. Parasitology. 2008;135(4):485-94.
44. Dergousoff SJ, Chilton NB. Novel genotypes of Anaplasma bovis, "Candidatus Midichloria" sp. and Ignatzschineria sp. in the Rocky Mountain wood tick, Dermacentor andersoni. Vet Microbiol. 2011;150(1-2):100-6.
45. Montagna M, Sassera D, Epis S, Bazzocchi C, Vannini C, Lo N, et al. "Candidatus Midichloriaceae" fam. nov. (Rickettsiales), an ecologically widespread clade of intracellular alphaproteobacteria. Appl Environ Microbiol. 2013;79(10):3241-8.
46. Granquist EG, Kristiansson M, Lindgren P-E, Matussek A, Nødtvedt A, Okstad W, et al. Evaluation of microbial communities and symbionts in Ixodes ricinus and ungulate hosts (Cervus elaphus and Ovis aries) from shared habitats on the west coast of Norway. Ticks Tick Borne Dis. 2014;5(6):780-4.
47. Williams-Newkirk AJ, Rowe LA, Mixson-Hayden TR, Dasch GA. Characterization of the bacterial communities of life stages of free living lone star ticks (amblyomma americanum). PLoS One. 2014;9(7):e102130.
48. Hornok S, Földvári G, Elek V, Naranjo V, Farkas R, de la Fuente J. Molecular identification of Anaplasma marginale and rickettsial endosymbionts in blood-sucking flies (Diptera: Tabanidae, Muscidae) and hard ticks (Acari: Ixodidae). Vet Parasitol. 2008;154(3-4):354-9.
49. Richard S, Seng P, Parola P, Raoult D, Davoust B, Brouqui P. Detection of a new bacterium related to "Candidatus Midichloria mitochondrii" in bed bugs. Clin Microbiol Infect. 2009;15:84-5.
50. Reeves WK, Dowling APG, Dasch GA. Rickettsial agents from parasitic dermanyssoidea (Acari: Mesostigmata). Exp Appl Acarol. 2006;38(2-3):181-8.
51. Lo N, Beninati T, Sassera D, Bouman EA, Santagati S, Gern L, et al. Widespread distribution and high prevalence of an alpha-proteobacterial symbiont in the tick Ixodes ricinus. Environ Microbiol. 2006;8(7):1280-7.
52. Mariconti M, Epis S, Gaibani P, Valle CD, Sassera D, Tomao P, et al. Humans parasitized by the hard tick Ixodes ricinus are seropositive to Midichloria mitochondrii: Is Midichloria a novel pathogen, or just a marker of tick bite? Pathogens Global Health. 2012;106(7):391-6.
53. Skarphédinsson S, Jensen PM, Kristiansen K. Survey of tickborne infections in Denmark. Emerging Infect Dis. 2005;11(7):1055-61.
54. Carpi G, Cagnacci F, Wittekindt NE, Zhao F, Qi J, Tomsho LP, et al. Metagenomic profile of the bacterial communities associated with ixodes ricinus ticks. Plos One. 2011;6(10):e25604.
55. Melničáková J, Derdáková M, Barák I. A system to simultaneously detect tick-borne pathogens based on the variability of the 16S ribosomal genes. Parasit Vectors. 2013;6:269.
56. Menchaca AC, Visi DK, Strey OF, Teel PD, Kalinowski K, Allen MS, et al. Preliminary Assessment of Microbiome Changes Following Blood-Feeding and Survivorship in the Amblyomma americanum Nymph-to-Adult Transition using Semiconductor Sequencing. PLoS ONE. 2013;8(6).
57. Nakao R, Abe T, Nijhof AM, Yamamoto S, Jongejan F, Ikemura T, et al. A novel approach, based on BLSOMs (Batch Learning Self-Organizing Maps), to the microbiome analysis of ticks. ISME J. 2013;7(5):1003-15.
58. Bonnet S, Michelet L, Moutailler S, Cheval J, Hébert C, Vayssier-Taussat M, et al. Identification of parasitic communities within European ticks using next-generation sequencing. PLoS Negl Trop Dis. 2014;8(3):e2753.
59. Budachetri K, Browning RE, Adamson SW, Dowd SE, Chao C-C, Ching W-M, et al. An insight into the Microbiome of the Amblyomma maculatum (Acari: Ixodidae). J Med Entomol. 2014;51(1):119-29.
60. Ponnusamy L, Gonzalez A, Van Treuren W, Weiss S, Parobek CM, Juliano JJ, et al. Diversity of rickettsiales in the microbiome of the lone star tick, Amblyomma americanum. Appl Environ Microbiol. 2014;80(1):354-9.
61. Zhang X-C, Yang Z-N, Lu B, Ma X-F, Zhang C-X, Xu H-J. The composition and transmission of microbiome in hard tick, Ixodes persulcatus, during blood meal. Ticks Tick Borne Dis. 2014;5(6):864-70.
62. Vayssier-Taussat M, Moutailler S, Michelet L, Devillers E, Bonnet S, Cheval J, et al. Next generation sequencing uncovers unexpected bacterial pathogens in ticks in western Europe. PLoS One. 2013;8(11):e81439.
63. Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. PNAS. 1985;82(20):6955-9.
64. Liu Z, DeSantis TZ, Andersen GL, Knight R. Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers. Nucl Acids Res. 2008;36(18):e120.
65. Klappenbach JA, Dunbar JM, Schmidt TM. rRNA operon copy number reflects ecological strategies of bacteria. Appl Environ Microbiol. 2000;66(4):1328-33.
66. Větrovský T, Baldrian P. The variability of the 16S rRNA gene in bacterial genomes and its consequences for bacterial community analyses. PLoS One. 2013;8(2):e57923.
67. Jensen PM. Host seeking activity of ixodes ricinus ticks based on daily consecutive flagging samples. Exp Appl Acarol. 2000;24(9):695-708.
68. Arthur DR. British ticks. London: Butterworths; 1963.
69. Roberts FHS. Australian ticks. Melbourne: Commonwealth Scientific and Industrial Research Organisiation; 1970.
70. Vestheim H, Jarman SN. Blocking primers to enhance PCR amplification of rare sequences in mixed samples - a case study on prey DNA in Antarctic krill stomachs. Front Zoology. 2008;5(1):12.
71. Turner S, Pryer KM, Miao VPW, Palmer JD. Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence Analysis1. J Eukaryot Microbiol. 1999;46(4):327-38.
72. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Meth. 2010;7(5):335-6.
73. Chakravorty S, Helb D, Burday M, Connell N, Alland D. A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. J Microbiol Methods. 2007;69(2):330-9.
74. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28(12):1647-9.
75. Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26(19):2460-1.
76. Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Meth. 2013;10(10):996-8.
77. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 2011;27(16):2194-200.
78. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol. 2006;72(7):5069-72.
79. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215(3):403-10.
80. Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 2004;5(1):113.
81. Boessenkool S, Epp LS, Haile J, Bellemain E, Edwards M, Coissac E, et al. Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA. Mol Ecol. 2012;21(8):1806-15.
82. Khanna M, Park P, Zirvi M, Cao W, Picon A, Day J, et al. Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors. Oncogene. 1999;18(1):27-38.
83. Liles MR, Manske BF, Bintrim SB, Handelsman J, Goodman RM. A census of rRNA genes and linked genomic sequences within a soil metagenomic library. Appl Environ Microbiol. 2003;69(5):2684-91.
84. Rauter C, Hartung T. Prevalence of Borrelia burgdorferi sensu lato genospecies in Ixodes ricinus ticks in Europe: a metaanalysis. Appl Environ Microbiol. 2005;71(11):7203-16.
85. Carley JG, Pope JH. A new species of Borrelia (B. queenslandica) from Rattus villosissimus in Queensland. Aust J Exp Biol Med Sci. 1962;40:255-61.
86. Mackerras M. The haematozoa of Australian mammals. Aust J Zoo. 1959;7(2):105-35.
87. Pekova S, Vydra J, Kabickova H, Frankova S, Haugvicova R, Mazal O, et al. Candidatus Neoehrlichia mikurensis infection identified in 2 hematooncologic patients: benefit of molecular techniques for rare pathogen detection. Diagn Microbiol Infect Dis. 2011;69(3):266-70.
88. Grankvist A, Andersson P-O, Mattsson M, Sender M, Vaht K, Höper L, et al. Infections with the tick-borne bacterium "Candidatus Neoehrlichia mikurensis" mimic non-infectious conditions in patients with B cell malignancies or autoimmune diseases. Clin Infect Dis. 2014;18:ciu189.
89. Li H, Jiang J-F, Liu W, Zheng Y-C, Huo Q-B, Tang K, et al. Human Infection with Candidatus Neoehrlichia mikurensis. China Emerg Infect Dis. 2012;18(10):1636-9.
90. Obiegala A, Pfeffer M, Pfister K, Tiedemann T, Thiel C, Balling A, et al. Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum: prevalences and investigations on a new transmission path in small mammals and ixodid ticks. Parasites & Vectors. 2014;7(1):563.
91. Yabsley MJ, Murphy SM, Luttrell MP, Wilcox BR, Howerth EW, Munderloh UG. Characterization of "Candidatus Neoehrlichia lotoris" (family Anaplasmataceae) from raccoons (Procyon lotor). Int J Syst Evol Microbiol. 2008;58(0 12):2794-8.
92. Graves S, Unsworth N, Stenos J. Rickettsioses in Australia. Ann N Y Acad Sci. 2006;1078:74-9.
93. Brown GK, Canfield PJ, Dunstan RH, Roberts TK, Martin AR, Brown CS, et al. 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(9):321-5.
94. Wójcik-Fatla A, Zajac V, Cisak E, Sroka J, Sawczyn A, Dutkiewicz J. Leptospirosis as a tick-borne disease? detection of leptospira spp. In ixodes ricinus ticks in eastern Poland. Ann Agric Environ Med. 2012;19(4):656-9.
95. Schmid GP, Steere AC, Kornblatt AN, Kaufmann AF, Moss CW, Johnson RC, et al. Newly recognized Leptospira species ("Leptospira inadai" serovar lyme) isolated from human skin. J Clin Microbiol. 1986;24(3):484-6.
96. Wójcik-Fatla A, Zajac V, Sawczyn A, Cisak E, Sroka J, Dutkiewicz J. Occurrence of Francisella spp. in Dermacentor reticulatus and Ixodes ricinus ticks collected in eastern Poland. Ticks Tick Borne Dis. 2015;6(3):253-7.
97. Kugeler KJ, Gurfield N, Creek JG, Mahoney KS, Versage JL, Petersen JM. Discrimination between Francisella tularensis and Francisella-Like Endosymbionts when screening ticks by PCR. Appl Environ Microbiol. 2005;71(11):7594-7.