Parasitology Research

, Volume 113, Issue 11, pp 4189–4197 | Cite as

Besnoitia besnoiti tachyzoites induce monocyte extracellular trap formation

  • Tamara Muñoz-Caro
  • Liliana M. R. Silva
  • Christin Ritter
  • Anja Taubert
  • Carlos HermosillaEmail author
Original Paper


Extracellular trap (ET) formation has been demonstrated as an important novel effector mechanism of polymorphonuclear neutrophils (PMN), eosinophils, mast cells and macrophages acting extracellularly against pathogens. In the present study, we show that tachyzoites of the emerging apicomplexan parasite Besnoitia besnoiti, that have recently been reported as potent inducers of PMN-derived ETosis, also trigger the release of ETs in an additional cell type, namely in monocytes. Fluorescence illustrations as well as scanning electron microscopy analyses (SEM) showed monocyte-promoted ET formation to be rapidly induced upon exposure to viable tachyzoites of B. besnoiti. Classical characteristics of ETs were confirmed by the co-localization of extracellular DNA with histones (H3) or myeloperoxidase (MPO) in parasite-entrapping structures. Monocyte-derived ETs were efficiently abolished by DNase I treatment and significantly reduced by treatments with inhibitors of MPO and NADPH oxidase, thus strengthening the key roles of reactive oxygen species (ROS) and MPO in monocyte ET formation. For comparative reasons, we additionally tested sporozoite stages of the closely related parasite Eimeria bovis for their capacity to induce monocyte-derived ETs and showed that these stages indeed induce ETs. To our best knowledge, we here report for the first time on monocyte ETs against the apicomplexan parasites B. besnoiti and E. bovis. Our results indicate that monocyte-triggered ETs may represent an important effector mechanism of the host early innate immune response against B. besnoiti and add a new cell type to the list of cells capable to release ETs.


Besnoitia besnoiti Eimeria bovis Extracellular traps Monocytes 



We would like to acknowledge Brigitte Hofmann, Davinia Pérez and Klaus Becker for their excellent work in cell culture, parasite preparation and monocyte isolation as well as the collection of blood samples. We extend further our thanks to Gerd Magdowski, Institute of Anatomy and Cell Biology, Justus Liebig University, for his excellent contribution in scanning electron microscopy analyses. TMC is a PhD student of the International Giessen Graduate Centre for Life Sciences (GGL) at the JLU Giessen, Germany, and LMRS is a PhD student (grant BD 72032/2010) of the Foundation for Science and Technology (FCT; Portugal).


  1. Abi Abdallah DS, Denkers EY (2012) Neutrophils cast extracellular traps in response to protozoan parasites. Front Immunol 3:382. doi: 10.3389/fimmu.2012.00382 PubMedGoogle Scholar
  2. Abi Abdallah DS, Lin C, Ball CJ, King MR, Duhamel GE, Denkers EY (2012) Toxoplasma gondii triggers release of human and mouse neutrophil extracellular traps. Infect Immun 80(2):768–777. doi: 10.1128/IAI.05730-11 PubMedCrossRefPubMedCentralGoogle Scholar
  3. Altincicek B, Stotzel S, Wygrecka M, Preissner KT, Vilcinskas A (2008) Host-derived extracellular nucleic acids enhance innate immune responses, induce coagulation, and prolong survival upon infection in insects. J Immunol 181(4):2705–2712PubMedCrossRefGoogle Scholar
  4. Alvarez-Garcia G, Frey CF, Mora LM, Schares G (2013) A century of bovine besnoitiosis: an unknown disease re-emerging in Europe. Trends Parasitol 29:407–415. doi: 10.1016/ PubMedCrossRefGoogle Scholar
  5. Aulik NA, Hellenbrand KM, Czuprynski CJ (2012) Mannheimia haemolytica and its leukotoxin cause macrophage extracellular trap formation by bovine macrophages. Infect Immun 80:1923–1933. doi: 10.1128/IAI.06120-11 PubMedCrossRefPubMedCentralGoogle Scholar
  6. Behrendt JH, Ruiz A, Zahner H, Taubert A, Hermosilla C (2010) Neutrophil extracellular trap formation as innate immune reactions against the apicomplexan parasite Eimeria bovis. Vet Immunol Immunopathol 133:1–8. doi: 10.1016/j.vetimm.2009.06.012 PubMedCrossRefGoogle Scholar
  7. Branzk N, Papayannopoulos V (2013) Molecular mechanisms regulating NETosis in infection and disease. Semin Immunopathol 35:513–530. doi: 10.1007/s00281-013-0384-6 PubMedCrossRefPubMedCentralGoogle Scholar
  8. Brinkmann V, Zychlinsky A (2007) Beneficial suicide: why neutrophils die to make NETs. Nat Rev Microbiol 5:577–582. doi: 10.1038/nrmicro1710 PubMedCrossRefGoogle Scholar
  9. Brinkmann V, Zychlinsky A (2012) Neutrophil extracellular traps: is immunity the second function of chromatin? J Cell Biol 198:773–783. doi: 10.1083/jcb.201203170 PubMedCrossRefPubMedCentralGoogle Scholar
  10. Brinkmann V et al (2004) Neutrophil extracellular traps kill bacteria. Science 303:1532–1535. doi: 10.1126/science.1092385 PubMedCrossRefGoogle Scholar
  11. Cortes H, Leitao A, Vidal R, Vila-Vicosa MJ, Ferreira ML, Caeiro V, Hjerpe CA (2005) Besnoitiosis in bulls in Portugal. Vet Rec 157:262–264PubMedGoogle Scholar
  12. Dubey JP, Shkap V, Pipano E, Fish L, Fritz DL (2003) Ultrastructure of Besnoitia besnoiti tissue cysts and bradyzoites. J Eukaryot Microbiol 50:240–244PubMedCrossRefGoogle Scholar
  13. Dubey JP, van Wilpe E, Blignaut DJ, Schares G, Williams JH (2013) Development of early tissue cysts and associated pathology of Besnoitia besnoiti in a naturally infected bull (Bos taurus) from South Africa. J Parasitol 99:459–466. doi: 10.1645/12-128.1 PubMedCrossRefGoogle Scholar
  14. EFSA (2010) Bovine Besnoitiosis: An emerging disease in Europe EFSA Journal 8Google Scholar
  15. Fernandez-Garcia A et al (2009) First Isolation of Besnoitia besnoiti From a Chronically Infected Cow in Spain. J Parasitol 95:474–476. doi: 10.1645/Ge-1772.1 PubMedCrossRefGoogle Scholar
  16. Fiege N, Klatte D, Kollmann D, Zahner H, Burger HJ (1992) Eimeria bovis in cattle: colostral transfer of antibodies and immune response to experimental infections. Parasitol Res 78:32–38PubMedCrossRefGoogle Scholar
  17. Gentile A, Militerno G, Schares G, Nanni A, Testoni S, Bassi P, Gollnick NS (2012) Evidence for bovine besnoitiosis being endemic in Italy-first in vitro isolation of Besnoitia besnoiti from cattle born in Italy. Vet Parasitol 184:108–115. doi: 10.1016/j.vetpar.2011.09.014 PubMedCrossRefGoogle Scholar
  18. Gollnick NS, Gentile A, Schares G (2010) Diagnosis of bovine besnoitiosis in a bull born in Italy. Vet Rec 166:599. doi: 10.1136/vr.c2314 PubMedCrossRefGoogle Scholar
  19. Guimarães-Costa AB, Nascimento MT, Froment GS, Soares RP, Morgado FN, Conceicao-Silva F, Saraiva EM (2009) Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps. Proc Natl Acad Sci USA 106:6748–6753. doi: 10.1073/pnas.0900226106 PubMedCrossRefPubMedCentralGoogle Scholar
  20. Harker KS, Ueno N, Wang T, Bonhomme C, Liu W, Lodoen MB (2013) Toxoplasma gondii modulates the dynamics of human monocyte adhesion to vascular endothelium under fluidic shear stress. J Leukoc Biol 93:789–800. doi: 10.1189/jlb.1012517 PubMedCrossRefGoogle Scholar
  21. Hermosilla C, Barbisch B, Heise A, Kowalik S, Zahner H (2002) Development of Eimeria bovis in vitro: suitability of several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal, Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells. Parasitol Res 88:301–307PubMedCrossRefGoogle Scholar
  22. Hermosilla C, Ruiz A, Taubert A (2012) Eimeria bovis: an update on parasite-host cell interactions. Int J Med Microbiol 302:210–215. doi: 10.1016/j.ijmm.2012.07.002 PubMedCrossRefGoogle Scholar
  23. Hermosilla C, Muñoz-Caro T, Silva LMR, Ruiz A, Taubert A (2014) The intriguing host innate immune response: novel anti-parasitic defence by neutrophil extracellular traps. Parasitology:1–10. doi: 10.1017/S0031182014000316
  24. Hornok S, Fedak A, Baska F, Hofmann-Lehmann R, Basso W (2014) Bovine besnoitiosis emerging in Central-Eastern Europe, Hungary. Parasitol Vectors 7:20. doi: 10.1186/1756-3305-7-20 CrossRefGoogle Scholar
  25. Jackson AR (1964) The isolation of viable coccidial sporozoites. Parasitology 54:87–93PubMedCrossRefGoogle Scholar
  26. Jacquiet P et al. (2009) Besnoitiose bovine : enquêtes épidémiologiques dans le Sud-Ouest de la France. Recueil des Journées Nationales :8Google Scholar
  27. Lesser M, Braun U, Deplazes P, Gottstein B, Hilbe M, Basso W (2012) First cases of besnoitiosis in cattle in Switzerland. Schweiz Arch Tierheilkd 154:469–474. doi: 10.1024/0036-7281/a000389 PubMedCrossRefGoogle Scholar
  28. Lippolis JD, Reinhardt TA, Goff JP, Horst RL (2006) Neutrophil extracellular trap formation by bovine neutrophils is not inhibited by milk. Vet Immunol Immunopathol 113:248–255. doi: 10.1016/j.vetimm.2006.05.004 PubMedCrossRefGoogle Scholar
  29. Martinelli S et al (2004) Induction of genes mediating interferon-dependent extracellular trap formation during neutrophil differentiation. J Biol Chem 279:44123–44132. doi: 10.1074/jbc.M405883200 PubMedCrossRefGoogle Scholar
  30. Mehlhorn H, Klimpel S, Schein E, Heydorn AO, Al-Quraishy S, Selmair J (2009) Another African disease in Central Europa: besnoitiosis of cattle. I. Light and electron microscopical study. Parasitol Res 104:861–868. doi: 10.1007/s00436-008-1267-y PubMedCrossRefGoogle Scholar
  31. Muñoz-Caro T, Hermosilla C, Silva LMR, Cortes H, Taubert A (2014) Neutrophil extracellular traps as innate immune reaction against the emerging apicomplexan parasite Besnoitia besnoiti. PLoS One 9:e91415. doi: 10.1371/journal.pone.0091415 PubMedCrossRefPubMedCentralGoogle Scholar
  32. Ng TH, Chang SH, Wu MH, Wang HC (2013) Shrimp hemocytes release extracellular traps that kill bacteria. Dev Comp Immunol 41:644–651. doi: 10.1016/j.dci.2013.06.014 PubMedCrossRefGoogle Scholar
  33. Olias P, Schade B, Mehlhorn H (2011) Molecular pathology, taxonomy and epidemiology of Besnoitia species (Protozoa: Sarcocystidae) infection, genetics and evolution. Infect Genet Evol 11:1564–1576. doi: 10.1016/j.meegid.2011.08.006 PubMedCrossRefGoogle Scholar
  34. Papayannopoulos V, Zychlinsky A (2009) NETs: a new strategy for using old weapons. Trends Immunol 30:513–521. doi: 10.1016/ PubMedCrossRefGoogle Scholar
  35. Pilsczek FH et al (2010) A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol 185:7413–7425. doi: 10.4049/jimmunol.1000675 PubMedCrossRefGoogle Scholar
  36. Quan JH, Zhou W, Cha GH, Choi IW, Shin DW, Lee YH (2013) Kinetics of IL-23 and IL-12 secretion in response to Toxoplasma gondii antigens from THP-1 monocytic cells. Korean J Parasitol 51:85–92. doi: 10.3347/kjp.2013.51.1.85 PubMedCrossRefPubMedCentralGoogle Scholar
  37. Rinaldi L, Maurelli MP, Musella V, Bosco A, Cortes H, Cringoli G (2013) First cross-sectional serological survey on Besnoitia besnoiti in cattle in Italy. Parasitol Res 112:1805–1807. doi: 10.1007/s00436-012-3241-y PubMedCrossRefGoogle Scholar
  38. Rose ME, Hesketh P (1979) Immunity to coccidiosis: T-lymphocyte- or B-lymphocyte-deficient animals. Infect Immun 26:630–637PubMedPubMedCentralGoogle Scholar
  39. Schares G et al (2009) First in vitro isolation of Besnoitia besnoiti from chronically infected cattle in Germany. Vet Parasitol 163:315–322. doi: 10.1016/j.vetpar.2009.04.033 PubMedCrossRefGoogle Scholar
  40. Schares G et al (2011) Evaluation of a commercial ELISA for the specific detection of antibodies against Besnoitia besnoiti. Vet Parasitol 175:52–59PubMedCrossRefGoogle Scholar
  41. Schares G et al (2013) Novel tools for the diagnosis and differentiation of acute and chronic bovine besnoitiosis. Int J Parasitol 43:143–154. doi: 10.1016/j.ijpara.2012.10.011 PubMedCrossRefGoogle Scholar
  42. Silva LM, Muñoz-Caro T, Gerstberger R, Vila-Vicosa MJ, Cortes HC, Hermosilla C, Taubert A (2014) The apicomplexan parasite Eimeria arloingi induces caprine neutrophil extracellular traps. Parasitol Res. doi: 10.1007/s00436-014-3939-0 Google Scholar
  43. Taubert A, Hermosilla C, Behrendt J, Zahner H (2006) Reaction of bovine endothelial cells in vitro to coccidia (Eimeria bovis, Toxoplasma gondii, Neospora caninum) infections as the expression of a non-adaptive immune response. Berl Munch Tierarztl Wochenschr 119:274–281PubMedGoogle Scholar
  44. Taubert A, Behrendt JH, Suhwold A, Zahner H, Hermosilla C (2009) Monocyte- and macrophage-mediated immune reactions against Eimeria bovis. Vet Parasitol 164:141–153. doi: 10.1016/j.vetpar.2009.06.003 PubMedCrossRefGoogle Scholar
  45. Ueno N, Harker KS, Clarke EV, McWhorter FY, Liu WF, Tenner AJ, Lodoen MB (2014) Real-time imaging of Toxoplasma-infected human monocytes under fluidic shear stress reveals rapid translocation of intracellular parasites across endothelial barriers. Cell Microbiol 16:580–595. doi: 10.1111/cmi.12239 PubMedCrossRefGoogle Scholar
  46. von Kockritz-Blickwede M, Nizet V (2009) Innate immunity turned inside-out: antimicrobial defense by phagocyte extracellular traps. J Mol Med 87:775–783. doi: 10.1007/s00109-009-0481-0 CrossRefGoogle Scholar
  47. von Kockritz-Blickwede M, Goldmann O, Thulin P, Heinemann K, Norrby-Teglund A, Rohde M, Medina E (2008) Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation. Blood 111:3070–3080. doi: 10.1182/blood-2007-07-104018 CrossRefGoogle Scholar
  48. Yipp BG et al (2012) Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat Med 18:1386–1393. doi: 10.1038/nm.2847 PubMedCrossRefGoogle Scholar
  49. Yousefi S et al (2008) Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense. Nat Med 14:949–953. doi: 10.1038/nm.1855 PubMedCrossRefGoogle Scholar
  50. Yousefi S, Mihalache C, Kozlowski E, Schmid I, Simon HU (2009) Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps. Cell Death Differ 16:1438–1444. doi: 10.1038/cdd.2009.96 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Tamara Muñoz-Caro
    • 1
  • Liliana M. R. Silva
    • 1
  • Christin Ritter
    • 1
  • Anja Taubert
    • 1
  • Carlos Hermosilla
    • 1
    Email author
  1. 1.Institute of Parasitology, BFSJustus Liebig University GiessenGiessenGermany

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