Veterinary World

     Open access and peer reviewed journal  

ISSN (Online): 2231-0916


Home l Editorial board l Instructions for authors l Reviewer guideline l Open access policy l Archives l FAQ

Open Access

Copyright: The authors. This article is an open access article licensed under the terms of the Creative Commons Attribution License

( which permits unrestricted use, distribution and reproduction in any medium, provided the work is properly cited.

Research (Published online: 24-06-2015)

15.  Partial characterization of a novel anti-inflammatory protein from salivary gland extract of Hyalomma anatolicum anatolicum (Acari: Ixodidae) ticks - Mayukh Ghosh, Nirmal Sangwan and Arun K. Sangwan

Veterinary World, 8(6): 772-776



   doi: 10.14202/vetworld.2015.772-776


Mayukh Ghosh: Department of Veterinary Physiology and Biochemistry, College of Veterinary Sciences, Lala Lajpat Rai University of

Veterinary and Animal Sciences, Hisar, Haryana, India;

Nirmal Sangwan: Department of Veterinary Physiology and Biochemistry, College of Veterinary Sciences, Lala Lajpat Rai University of

Veterinary and Animal Sciences, Hisar, Haryana, India;

Arun K. Sangwan: Department of Veterinary Parasitology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana, India;


Received: 22-01-2015, Revised: 16-05-2015, Accepted: 23-05-2015, Published online: 24-06-2015


Corresponding author: Nirmal Sangwan, e-mail:

Citation: Ghosh M, Sangwan N, Sangwan AK (2015) Partial characterization of a novel anti-inflammatory protein from salivary gland extract of Hyalomma anatolicum anatolicum (Acari: Ixodidae) ticks, Veterinary World 8(6):772-776.

Aim: Hyalomma anatolicum anatolicum ticks transmit Theileria annulata, causative agent of tropical theileriosis to cattle and buffaloes causing a major economic loss in terms of production and mortality in tropical countries. Ticks have evolved several immune evading strategies to circumvent hosts’ rejection and achieve engorgement. Successful feeding of ticks relies on a pharmacy of chemicals located in their complex salivary glands and secreted saliva. These chemicals in saliva could inhibit host inflammatory responses through modulating cytokine secretion and detoxifying reactive oxygen species. Therefore, the present study was aimed to characterize anti-inflammatory peptides from salivary gland extract (SGE) of H. a. anatolicum ticks with a view that this information could be utilized in raising vaccines, designing synthetic peptides or peptidomimetics which can further be developed as novel therapeutics.

Materials and Methods: Salivary glands were dissected out from partially fed adult female H. a. anatolicum ticks and homogenized under the ice to prepare SGE. Gel filtration chromatography was performed using Sephadex G-50 column to fractionate the crude extract. Protein was estimated in each fraction and analyzed for identification of anti-inflammatory activity. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) was run for further characterization of protein in desired fractions.

Results: A novel 28 kDa protein was identified in H. a. anatolicum SGE with pronounced anti-inflammatory activity.

Conclusion: Purification and partial characterization of H. a. anatolicum SGE by size-exclusion chromatography and SDSPAGE depicted a 28 kDa protein with prominent anti-inflammatory activity.

Keywords: anti-inflammatory, Hyalomma anatolicum anatolicum, size-exclusion chromatography, sodium dodecyl sulfate - polyacrylamide gel electrophoresis, tropical theileriosis.

1. Brown, C.G.D. (1997) Dynamics and impact of tick borne diseases of cattle. Trop. Anim. Health Prod., 29 Suppl 4: 1-3.
2. Preston, P.M. (2001) The Encyclopedia of Arthropod Transmitted infections. 1st ed. CABI Publishing, Wallingford (UK). p487-504.
3. ICAR Vision 2030. (2011) Project Director, Directorate of Knowledge Management in Agriculture. Indian Council of Agricultural Research, Krishi Anusandhan Bhavan, Pusa, New Delhi.
4. Minjauw, L. and McLeod, A. (2003) Tick borne diseases and poverty. The impact of tick and tick borne diseases on the livelihoods of small-scale and marginal livestockowners in India and eastern and southern Africa. In: Research Report. DFID Animal Health Programme. UK: Centre for Tropical Veterinary Medicine, University of Edinburgh.
5. Ribeiro, J.M.C. and Francischetti, I.M.B. (2003) Role of arthropod saliva in blood feeding: Sialome and post-sialome perspectives. Annu. Rev. Entomol., 48: 7388.
6. Islam, M.K., Tsuji, N., Miyoshi, T., Alim, M.A., Huang, X., Hatta, T. and Fujisaki, K. (2009) The Kunitz-like modulatory protein haemangin is vital for hard tick blood-feeding success. PLoS Pathog., 5: e1000497.
7. Fontaine, A., Diouf, I., Bakkali, N., Missé, D., Pagčs, F., Fusi, T., Rogier, C. and Almeras, L. (2011) Implication of haematophagous arthropod salivary proteins in host-vector interactions. Parasit. Vectors, 4: 187.
PMid:21951834 PMCid:PMC3197560
8. Guo, X., Booth, C.J., Paley, M.A., Wng, X., De Ponte, K., Fikrig, E., Narasimhan, S. and Montgomery, R.R. (2009) Inhibition of neutrophil function by two tick salivary proteins. Infect. Immun., 77: 2320-2329.
PMid:19332533 PMCid:PMC2687334
9. Randolph, S.E. (2009) Tick-borne disease systems emerge from the shadows: The beauty lies in molecular detail, the message in epidemiology. Parasitology, 136: 1403-1413.
10. Mori, A., Konnai, S., Yamada, S., Hidano, A., Murase, Y., Ito, T., Takano, A., Kawabata, H., Onuma, M. and Ohashi, K. (2010) Two novel salp15-like immunosuppressant genes from salivary glands of ixodespersulcatus schulze tick. Insect. Mol. Biol., 19: 359-365.
11. Francischetti, I.M.B., Sá-Nunes, A, Mans, B.J, Santos, I.M. and Ribeiro, J.M.C. (2009) The role of saliva in tick feeding. Front Biosci., 14: 2051-2088.
12. Anisuzzaman, M., Islam, M.K., Alim, M.A., Miyoshi, T., Hatta, T., Yamaji, K., Matsumoto, Y., Fujisaki, K. and Tsuji, N. (2011) Longistatin, a plasminogen activator, is key to the availability of blood-meals for ixodid ticks. PLoS Pathog., 7: e1001312.
13. Kazimírová, M. and Štibrániová, I (2013) Tick salivary compounds: their role in modulation of host defences and pathogen transmission. Front Cell Infect. Microbiol., 3: 43.
PMid:23971008 PMCid:PMC3747359
14. Déruaz, M., Bonvin, P., Severin, I.C., Johnson, Z., Krohn, S., Power, C.A. and Proudfoot, A.E.I. (2013) Evasin-4, a tick-derived chemokine-binding protein with broad selectivity can be modified for use in preclinical disease models. FEBS. J., 280: 4876-4887.
PMid:23910450 PMCid:PMC4240464
15. Tirloni, L., Reck, J., Terra, R.M.S., Martins, J.R., Mulenga, A., Sherman, N.E., Fox, J.W., Yates, J.R. 3rd., Termignoni, C., Pinto, A.F. and Vaz Ida S, Jr. (2014) Proteomic analysis of cattle tick Rhipicephalus (Boophilus) microplus saliva: A comparison between partially and fully engorged females. PLoS One, 9(4): e94831.
16. Radulović ŽM, Kim TK, Porter LM, Sze SH, Lewis L, Mulenga A. (2014) A 24-48 h fed Amblyomma americanum tick saliva immuno-proteome. BMC Genomics, 15(1): 518.
17. Garcia, G.R., Gardinassi, L.G., Ribeiro, J.M.C., Anatriello, E., Ferreira, B.R., Moreira, H.N.S., Mafra, C., Martins, M.M., Szabó, M.P.J., de Miranda-Santos, I.K.F. and Maruyama, S.R. (2014) The sialotranscriptome of Amblyomma triste, Amblyomma parvum and Amblyomma cajennense ticks, uncovered by 454-based RNA-seq. Parasit Vectors, 7(1): 430.
18. Paesen, G.C., Adams, P.L., Harlos, K., Nuttall P.A. and Stuart, D.I. (1999) Tick histamine-binding proteins: Isolation, cloning, and three-dimensional structure. Mol. Cell, 3: 661-671.
19. Leboulle, G., Crippa, M., Decrem, Y., Mejri, N., Brossard M., Bollen A. and Godfroid, E. (2002) Characterization of a novel salivary immunosuppressive protein from ixodes ricinus ticks. J. Biol. Chem., 277: 10083-10089.
20. Kotsyfakis, M., Sa-Nunes, A., Francischetti, I.M.B., Mather, T.N., Andersen, J.F. and Ribeiro, J. M.C. (2006) Antiinflammatory and immunosuppressive activity of sialostatin L, a salivary cystatin from the tick Ixodes scapularis. J Biol Chem., 281: 26298-26307.
21. Wu, J., Wang, Y., Liu, H., Yang, H., a, D., Li, J., Li, D., Lai, R. and Yu, H. (2010) Two immunoregulatory peptides with antioxidant activity from tick salivary glands. J. Biol. Chem., 285(22): 1-19.
22. Kaufman, R. (2010) Ticks: Physiological aspects with implications for pathogen transmission. Ticks Tick Borne Dis., 1: 11-22.
23. Fialová, A., Cimburek, Z., Iezzi, G. and Kopecký, J. (2010) Ixodes ricinus tick saliva modulates tick-borne encephalitis virus infection of dendritic cells. Microbes Infect., 12: 580-585.
24. Chmelar, J., Oliveira, C.J., Rezacova, P., Francischetti, I.M.B., Kovarova, Z., Pejler, G., Kopacek, P., Ribeiro, J.M.C., Mares, M., Kopecky, J. and Kotsyfakis, M. (2011) A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation. Blood, 117: 736-744.
PMid:20940421 PMCid:PMC3031492
25. Schuijt, T.J., Coumou, J., Narasimhan, S., Dai, J., Deponte, K., Wouters, D., Brouwer, M., Oei, A., Roelofs, J.J., van Dam, A.P., van der Poll, T., Van't Veer, C., Hovius, J.W. and Fikrig, E. (2011) A tick mannose-binding lectin inhibitor interferes with the vertebrate complement cascade to enhance transmission of the Lyme disease agent. Cell Host Microbes, 10: 136-146.
PMid:21843870 PMCid:PMC3170916
26. Randolph, S.E. (2011) Transmission of tick-borne pathogens between co-feeding ticks: Milan labuda's enduring paradigm. Ticks Tick Borne Dis., 2: 179-182.
27. Radolf, J.D., Caimano, M.J., Stevenson, B. and Hu, L.T. (2012) Of ticks, mice and men: Understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat. Rev. Microbiol., 10: 87-99.
28. Valdes, J.J. (2014) Antihistamine response: A dynamically refined function at the host-tick interface. Parasit Vectors, 7(1): 491.
PMid:25358914 PMCid:PMC4226919
29. Ghosh, M., Sangwan, N. and Sangwan, A.K. (2014) Variations in free radical scavenging activities and antioxidant responses in salivary glands of Hyalomma anatolicum anatolicum and Hyalomma dromedarii (Acari: Ixodidae) ticks. Vet. World, 7(10): 876-881.
30. Oliveira, C.J., Anatriello, E., de Miranda-Santos, I.K., Francischetti, I.M., Sá-Nunes, A., Ferreira, B.R. and Ribeiro, J.M.C. (2013) Proteome of Rhipicephalus sanguineus tick saliva induced by the secretagogues pilocarpine and dopamine. Ticks Tick Borne Dis., 4(6): 469-477.
PMid:24029695 PMCid:PMC3917560
31. Tirloni, L., Seixas, A., Mulenga, A., da Silva, VI Jr., Termignoni, C. (2014) A family of serine protease inhibitors (serpins) in the cattle tick Rhipicephalus (Boophilus) microplus. Exp. Parasitol., 137: 25-34.
32. Cotte, V., Sabatier, L., Schnell, G., Carmi-Leroy, A., Rousselle, J.C., Arsene-Ploetze, F., Malandrin, L., Sertour, N., Namane, A., Ferquel, E. and Choumet, V. (2014) Differential expression of Ixodes ricinus salivary gland proteins in the presence of the Borrelia burgdorferi sensu lato complex. J. Proteomics, 96: 29-43.
33. Lewis, L.A., Radulović, Z.M., Kim, T.K., Porter, L.A. and Mulenga, A. (2015) Identification of 24h Ixodes scapularis immunogenic tick saliva proteins. Ticks Tick Borne Dis., 6(3): 424-434.
PMid:25825233 PMCid:PMC4415496
34. Tan, A.W., Francischetti, I.M., Slovak, M., Kini, R.M. and Ribeiro, J.M.C. (2015) Sexual differences in the sialomes of the zebra tick, Rhipicephalus pulchellus. J. Proteomics, 117: 120-144.
35. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Potein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275.
36. Shinde, U.A., Phadke, A.S., Nair, A.M., Mungantiwar, A.A., Dikshit, V.J. and Saraf, V.O. (1999) Membrane stabilizing activity–a possible mechanism of action for the anti-inflammatory activity of Cedrus deodara wood oil. Fitoterapia, 70: 251-257.
37. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.
38. Morrissey, J.H. (1981) Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal. Biochem., 117: 307-310.
39. Vancova, I., Hajnická, V., Slovák, M., Kocáková, P., Paesen, G.C. and Nutall, P.A. (2010) Evasin-3-like anti-chemokine activity in salivary gland extracts of ixodid ticks during blood-feeding: A new target for tick control. Parasite Immunol., 32: 460-463.
40. Kern, A., Collin, E., Barthel, C., Michel, C., Jaulhac, B. and Boulanger, N. (2011) Tick saliva represses innate immunity and cutaneous inflammation in a murine model of lyme disease. Vector Borne Zoonotic Dis., 11: 1343-1350.
41. Oliveira, C.J., Sa-Nunes, A., Francischetti, I.M., Carregaro, V., Anatriello, E., Silva, J.S., Santos, I.K., Ribeiro, J.M.C. and Ferreira, B.R. (2011) Deconstructing tick saliva: Non-protein molecules with potent immunomodulatory properties. J. Biol. Chem., 286: 10960-10969.
42. Chmelar, J., Calvo, E., Pedra, J.H.F., Francischetti, I.M.B. and Kotsyfakis, M. (2012) Tick salivary secretion as a source of antihemostatics. J. Proteomics, 75: 3842-3854.
PMid:22564820 PMCid:PMC3383439
43. Mudenda, L., Pierlé, S.A., Turse, J.E., Scoles, G.A., Purvine, S.O., Nicora, C.D., Clauss, T.R., Ueti, M.W., Brown, W.C. and Brayton, K.A. (2014) Proteomics informed by transcriptomics identifies novel secreted proteins in Dermacentor andersoni saliva. Int J Parasitol., 44(13): 1029-37.
44. Páleníková, J., Lieskovská, J., Langhansová, H., Kotsyfakis, M., Chmelař, J., Kopecký, J. (2015) Ixodes ricinsus salivary Serpin IRS-2 Affects Th17 differentiation via inhibition of the interleukin-6/STAT-3 signaling pathway. Infect. Immun., 83(5): 1949-56.
45. Porter, L., Radulović, Ž., Kim, T., Braz, G.R., Da Silva Vazm, I, Jr. and Mulenga, A. (2015) Bioinformatic analyses of male and female Amblyomma americanum tick expressed serine protease inhibitors (serpins). Ticks Tick Borne Dis., 6(1): 16-30.