J Arthropod-Borne Dis, 2011, 6(1): 36–44 A Nikpay et al.: Analysis of Immunogenic Relevant ... 35 Original Article Analysis of Immunogenic Relevant Proteins in Rhipicephalus (Boophilus) annulatus Tick *A Nikpay 1, S Nabian 2, M Taheri 2 1Department of Pathobiology, Faculty of Veterinary Medicine, University of Mazandaran, Amol, Iran 2Department of Parasitology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran (Received 19 May 2010; accepted 6 Nov 2011) Abstract Background: Considering the importance of ticks as a main group transmitting pathogen organisms, this study de- signed to recognize immunogenic proteins in different tissues of Rhipicephalus (Boophilus) annulatus tick and to find out if there are common proteins in these tissues. Methods: Seven cattle were experimentally infested with about 10000 R. annulatus larvae and their humoral im- mune response to extracts of salivary gland and ovary of adult ticks and larval extracts during infestation were deter- mined by ELISA and Western blot analysis. Measurements of serum antibodies level recorded weekly, from week 0 to week 9. Results: Using Western blot analysis, 15 fractions from soluble antigens extracted from salivary gland and larvae, and 14 fractions in the larval extracts were recognized. These findings illustrate the recognition of common antigens with molecular weight of 170, 117, 100, 70, 37, 33 and 30 kDa from different antigens by resistant cattle sera. Conclusion: Common antigens are present in different tissues of Rhipicephalus (Boophilus) annulatus, which can be used as a target in immunization against ticks. Keywords: Rhipicephalus (Boophilus) annulatus, Salivary gland, Ovary, Larva, Immunoblot, ELISA Introduction Cattle develop resistance to ticks following natural infestation (Reik 1962, Dipeolu et al. 1992, Cruz et al. 2008) and vaccination (Op- debeek et al. 1989, Willadsen et al. 1988, 1999, 2006, Wikel 1999a, b, Ogden et al. 2002, Trimnel et al. 2005, Leal et al. 2006, Perez 2010). It has been shown that infested animals with Boophilus microplus induce an immune response and acquire a partial resistance to subsequent infestations, in which the internal organs of the ectoparasite become damaged, leading to an increase of feeding time, a de- crease in the numbers of engorged females, less uptake of blood meal, a decrease in the number and viability of eggs (Wikel 1988). There is a general view, however, that a vac- cine with only one antigen will not be sufficient to generate an effective immune response able to control the proliferation of the ticks under field conditions (Willadsen 1990). Salivary gland, gut, embryo, and larval extracts of various ticks have been studied as possible target for vaccine. However, these tissues have been examined independently (Wikel 1984, Wiladsen et al. 1988, Wong and Opdebeek 1989). We examined the cattle immune response to salivary gland, ovary, and larval extracts of R. annulatus tick at the same time with the aim of finding common antigens, which in vaccination protocol could be advantageous upon stage, or organ specific antigens. The tick R. annulatus is an important ectoparasite *Corresponding author: Dr Ali Nikpay, E-mail: ali.nikpay@gmail.com 36 J Arthropod-Borne Dis, 2011, 6(1): 36–44 A Nikpay et al.: Analysis of Immunogenic Relevant ... 2 of cattle that present in West Africa, Central Africa, Asia, certain parts of Southern Sudan and Europe. In this study, for the first time, salivary gland, ovary, and larval extracts of this spe- cies have been studied at the same time as possible target antigens to determine the cat- tle immune response to them. Materials and Methods Tick infestation Seven healthy 3-5 months old Holstein cattle being negative sera for tick infestation were provided from Tehran Veterinary Medi- cine Faculty Research Institute (Amin abad). They were housed in tick-proof pens in- dependently. Each cattle was infested with about 10000 R. annulatus larvae as described by Brown (1988). Briefly, tubes containing larvae fas- tened to a shaved flank of cattle with adhe- sive tape. Then, the surface of the infestation area was covered with a piece of cloth. Antigen preparation Partly engorged female ticks were washed with 70% ethanol and then washed three times with sterile distilled water. After drying, they were maintained at 28o C and 85% re- lative humidity. The antigens used in the ELISA were extracts of salivary glands, ova- ries, and larvae. Salivary glands and ovaries were dissected with fine-tipped forceps in cold PBS, rinsed in fresh PBS and then each of these organs and tick larvae were ho- mogenized in a glass tissue grinder sepa- rately in PBS containing 1 mM phenyl methyl sulphonyl fluoride at 4o C following sonicated for 30 minnutes on ice with 40 W. The ho- mogenates centrifuged at 20000 g for 30 min at 4o C, the supernatant were stored at -70o C. The proteins of the extracts were deter- mined using the method of Warburg (Tietz 1986, Hudson and Hay 1994). Sera collection Positive and negative references sera col- lected from infested and uninfested cattle were used to standardize the ELISA test. The posi- tive sera were obtained by infesting seven three months-old calves with 10,000 of R. annulatus larvae. Weekly for 12 consecutive weeks, blood was collected by jugular vein and allowed to clot for 2 hour at room tem- perature and then centrifuged at 800 g for 15 min and extracted serum aliquoted and kept at -20o C. ELISA ELISA plates were coated with 4 µg per well of three antigens in 20 mM carbonate buffer (pH= 9.6) by incubation overnight at 4o C (Harlow and Lane 1988). Having been washed three times, the plates were incu- bated for 1 hour at 37o C with 5% skim milk- PBS. Then, test sera diluted 1/200 were incubated at 37o C. After 1 hour, the plates were washed three times and 100 µl of 1/2000 diluted sheep anti –bovine IgG con- jugated with peroxidase were added to the individual wells. After incubation at 37o C for 1 hour, the plates were washed again and the enzyme substrate was added. The sub- strate was prepared by dissolving 62 mg of (2, 2, Azino-bis 3-ethyl benz- thiazoline-6- sulfonic acid) in 50 ml of distilled water and the optical density (OD) was determined at 405 nm. Sera from cattle were tested against anti R. annulatus antibodies at 1-week inter- vals from week 0 (the week of infestation) to week 9. SDS-PAGE electrophoresis of different tissues Tick extract preparations (20 µg/lane) were resolved by sodium dodecyl sulfate polyacryla- mid gel electrophoresis (SDS-PAGE) using a discontinuous gel system in reducing condi- tion (Laemmli 1970) with an acrylamide con- centration of 5% in stacking gel and 12% in 37 J Arthropod-Borne Dis, 2011, 6(1): 36–44 A Nikpay et al.: Analysis of Immunogenic Relevant ... 3 the separation gel. A high molecular weight protein ladder (Fermentas) was used as mo- lecular weight markers. Finally, the gel was stained with Coomassie brilliant blue. Western blot analysis Soluble proteins extracted from salivary glands, ovaries and larvae of R. annulatus (20 µg/lane) were resolved by SDS-PAGE using a discontinuous gel system as described previously (Laemmli 1970). The antigens se- parated were transferred to nitrocellulose pa- per (NCP) (Wang and Nuttal 1994), and non- specific reactive sites on the NCP were blocked for 1h in Tris-buffered saline with 0.5% tween 20 (TBS-T pH8), containing 3% bovine skim milk on a shaking plate at room temperature. The NCP was incubated for 1 h at 37o C with bovine sera diluted 1/50 in PBS (pH= 7.2). The NCP was incubated for a further 1 h at 37oC with sheep anti- bovine IgG (conjugated with HRPO) diluted 1/2000 in PBS-tween20. After 1 hour, the paper was washed and reacted with Di amino benzidine (DAB) (Sigma) as a substrate. The NCP was washed three times between each step of the assay with PBS-Tween. Results Characterization of humoral immune re- sponse The experimental infestation of seven cattle with approximately 10000 larvae of R. an- nulatus was performed to characterize the humoral immune response to tick extracts. Furthermore, approximately 2500±252 ticks were collected per animal between Days 21 and 26. Figure 1 shows changes of antibody levels measured by ELISA in the sera of the ex- perimentally infested animals against sali- vary glands, ovaries, and larvae antigens. All extracts showed positive reactions with all sera tested starting from the first week of in- festation. A steady increase of the antibody level was observed during the experiment from week 0 to week 9, however, a slight decrease antibody production occurred in the week 9 of infestation. SDS-PAGE electrophoresis of different tissues Coomassie brilliant blue stain differentiates a vast profile of proteins in tick’s antigens. In salivary gland, ovary and larval extracts 22, 18, and 25 fractions were more dominant respectively. Protein bands ranging in mo- lecular weight from 18 to more than 200 KDa .The bands >200, 170, 117, 100, 93, 70, 55, 50, 44, 37, 30 and 27 kDa were common in all extracts (See in Fig. 2). Western blot analysis of salivary, ovary antigens and larva extracts In order to analyze the peptides reacting with the bovine antibodies, Western blot as- says were carried out using the sera with higher ELISA OD that belonged to 21st day after infestation. The profile of three differ- ent antigens recognized by cattle positive sera are presented in Fig.3. Serum from ex- perimentally infested cattle recognized ap- proximately 15 fractions from soluble anti- gens extracted from salivary gland and lar- vae. Challenged cattle also recognized 14 protein bands in the ovary extract. The negative control sera recognized no band. These findings illustrate the recognition of common proteins with molecular weight of 170, 117, 100, 70, 37, 33 and 30 kDa from different antigens by sera of infested cattle. Discussion It is extremely important to have defined immunogenic molecules in order to dissect the events involved in acquisition and expres- sion of tick resistance. Some studies clearly show that a number of tick tissues can be used to induce artificial resistance to tick feeding, an approach that holds significant 38 J Arthropod-Borne Dis, 2011, 6(1): 36–44 A Nikpay et al.: Analysis of Immunogenic Relevant ... 4 promise as an alternative method for tick control (Wickel et al. 1988, Turni et al. 2004, Leal et al. 2006, Willadsen 2006, Cruz et al. 2008). Antibodies reactive with tick extracts have been described by several re- searchers (Willadsen 1980, Wikel 1984). Immunoelectrophoresis, immunofluorescence, ELISA (Wikel and Osburn 1982, Brossard and Wikel 1997, Cruz et al. 2008, Perez 2010, Kopp et al. 2010) have been used to examine serological responses against ticks. Many investigators have used the Western blot technique to identify relevant antigens of several tick species using sera collected from different animal hosts (Whelen et al. 1986, Brown 1988, Trimnel 2005, Cruz et al. 2008). Salivary glands, gut and larval ex- tracts have been tested as immunogens to control tick infestation (Opdebeeck et al. 1989, Barriga et al. 1991). Da Silva Vaz et al. (1994) also indicated that Rhipicephalus (Boophilus) microplus infested cattle de- velop antibodies to components of tick sali- vary gland, gut, embryo and larvae during the first infestation. Fig. 1. The comparison of serum antibody response changes (OD value changes) in seven calves infested with R. annulatus using different tissues antigens. Results expressed as mean of seven infested bovine sera 39 J Arthropod-Borne Dis, 2011, 6(1): 36–44 A Nikpay et al.: Analysis of Immunogenic Relevant ... 2 Fig. 2. SDS-PAGE analysis of proteins derived from salivary glands, ovary and larvae of Rhipicephalus (Boophilus) annulatus. The numbers in left side of each column indicate the molecular weight (kD) of each fraction 40 J Arthropod-Borne Dis, 2011, 6(1): 36–44 A Nikpay et al.: Analysis of Immunogenic Relevant ... 3 Fig. 3. Western blot analysis of R. annulatus antigens by tick infested bovine sera (positive serum). The numbers in left side of each column indicate the molecular weight of each fraction In the present study, ELISA was used to compare the antibody levels developed against R. annulatus salivary gland, ovary and lar- vae protein extracts of sera from cattle sub- mitted to experimental infestation. The results of this part of our study showed the impact of approximately high-density tick infestation on humoral immune responses induced in experimentally infested cattle suggesting the antibody level increased during the period of infestation and as mentioned in Fig. 1, a sig- nificant increase was observed in third week after infestation that the greatest amount of ticks were harvested at this time. These re- sults are in agreement with our previous study and some other researchers (Oegden et al. 41 J Arthropod-Borne Dis, 2011, 6(1): 36–44 A Nikpay et al.: Analysis of Immunogenic Relevant ... 2 2002, Cruz et al. 2008, Nikpay et al. 2008, Perez 2010). Using Western blot analysis, some basic information have provided about antigens extracted from R. annulatus tick recognized by sera collected from cattle in- fested with this species. The recognized frac- tions could be used as target for subsequent antigen characterization studies, and points out some probable antigenic sites in specific tick tissues. ELISA and Western blot data indicate that R. annulatus infested cattle de- velop antibodies to components of tick sali- vary glands, ovaries and larvae during the infestation. In our experiment, all extracts showed the same absorbance value changes. One possi- ble explanation of the similarities in the re- activity of different tissues is that the bovine is inoculated with various tissue antigens dur- ing the tick feeding process, or that there are common antigens in different tissues of R. annulatus and according to these results it seemed to be a correct assumption. These findings further illustrate the presence of common crucial antigens, which are be- lieved to be 170, 117, 100, 70, 37, 33 and 30 kDa depending upon the assay system em- ployed because they were recognized in three extracts by anti tick sera. Electeropho- retic comparison of salivary gland extracts of R. annulatus specimens demonstrated some similar proteins with salivary gland extracts of Hyalomma specimens (Nabian et al. 2005). Also it was shown that some proteins of salivary gland of R. annulatus tick changed in different temperatures but protein bands of 29, 36, 55 and 97 were constant at all temperatures (Nabian et al. 2005). For the first time in the current study salivary glands, ovary and larvae extracts have been used as antigenic sources at the same time and it was showed that ovaries could be a source of immunogenic proteins such as the other tissues of tick studied previously. It should be mentioned that there was not any similar study about R. annulatus tick and this investigation was conducted for the first time in Iran to characterize the immunogenic antigens in this species. The findings of the present study estab- lish the presence of common antigens be- tween ovary, larvae, and salivary glands of R. annulatus ticks. Further studies will be aimed at the isolation of common proteins and test- ing for antigenicity and immunogenicity fol- lowed by purification and subsequent in vitro expression for potential vaccine development. Vaccination of hosts with antigenic tis- sues and secretions of ticks may provide a new method to control ticks. Extracts of whole tick body homogenates and various organs especially have been tested as to their ability to induce tick resistance in the host (Mc- Gowan et al. 1980). The significance of these observations is with regard to the induction of host immunity to ticks and the develop- ment of tick vaccines using common proteins. Acknowledgments Authors wish to acknowledge the finan- cial support of the research council of Te- hran University and Veterinary Medicine Fac- ulty of Tehran University. The authors de- clare that there is no conflict of interests. References Barriga OO, Andujar F, Andrzejewski WJ (1991) Manifestation of immunity in sheep repeatedly infested with Am- blyomma americanum ticks. J Parasi- tol. 77: 703–709. Birk HW, Koepsell H (1987) Reaction of monoclonal antibodies with plasma membrane proteins after binding on ni- trocellulose: renaturing of antigenic sites and reduction of non-specific antibody binding. Anal Biochem. 164: 12–22. Brossard M, Wikel SK (1997) Immunology of interaction between ticks and hosts, Med Vet Entomol. 11: 270–276. 42 J Arthropod-Borne Dis, 2011, 6(1): 36–44 A Nikpay et al.: Analysis of Immunogenic Relevant ... 3 Brown SJ (1988) Western blot analysis of Amblyomma americanum-derived stage- specific and shared antigens using se- rum from guinea pigs expressing resis- tance. Vet Parasitol. 28: 163–171. Cruz APR, Silva SS, Mattos RT, Da Silva Vaz Jr I, Masuda A, Ferreira CAS (2008) Comparative IgG recognition of tick extracts by sera of experimen- tally infested bovines. Vet Parasitol. 158: 152–158. Da Silva Vaz JrI, Shozo Ozki L, Masuda A (1994) Serum of Boophilus microplus infested cattle reacts with different tick tissues. Vet Parasitol. 52: 71–78. Dipeolu OO, Mongi AO, Essuman S, Amoo AO, Ndungu JN (1992) Studies on naturally acquired immunity to African ticks. II. Observations on cattle ex- posed to Rhipicephalus appendiculatus under varying periods of repeated in- festations. Vet Parasitol. 41: 293–320. Harlow E, Lane D (1988) Antibodies: A laboratory Manual. Cold Spring Har- bor Laboratory, New York. Hudson L, Hay FC (1994) Practical Immu- nology, 3rd edition. Blackwell Sci- entific Publications. Oxford. Kopp N, Diaz D, Amacker M, Odongo D, Beierb K, Nitschb C, Bishopc R, Daubenbergera C, (2010) Identification of a synthetic peptide inducing cross- reactive antibodies binding to Rhipice- phalus (Boophilus) decoloratus, Rhi- picephalus (Boophilus) microplus, Hya- lomma anatolicum anatolicum and Rhipicephalus appendiculatus BM86 homologues. Vaccine 28: 261–269. Laemmli EK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680– 685. Leal A, Seixas A, Pohl P, Ferreira C, Logullo C, Oliveira P, Farias S, Termignoni C, Itabajara da Silva Vaz Jr, Masuda A (2006) Vaccination of bovines with re- combinant Boophilus Yolk pro-Cathep- sin. Vet Immunol Immunopathol. 114: 341–345. McGowan MJ, Homer JT, O'Dell GV, McNew RW, Barker RW (1980) Performance of ticks fed on rabbits inoculated with extracts derived from homogenized ticks Amblyomma macu- latum Koch (Acarina: Ixodidae) J parasitol. 66(1): 42–48. Nabian S, Rahbari S, Haddadzadeh H, Alidadi N (2005) A study on the effects of different temperatures on protein changes in the salivary glands of Hya- lomma anatolicum anatolicum and Rhipicephalus (Boophilus) annulatus. Iranian J Vet Res Univ Shiraz. 6: 35– 39. Nikpay A, Nabian S, Rahbari S, Shayan P, Alidadi N (2008) Detection of humoral immune response of calves to Boophi- lus Annulatus by ELISA. Iranian J Parasitol. 3(1): 26–30. Ogden NH, Casey ANJ, Lawrie CH, French NP, Woldehiwet Z, Carter SD (2002) IgG responses to salivary gland extract of Ixodes ricinus ticks vary inversely with resistance in naturally exposed sheep. Med Vet Entomol. 16: 186–192. Opdebeeck JP, Wong JYM, Dobson C (1989) Hereford cattle protected against Boophilus micropolus with antigens purified by immunoaffinity chromatog- raphy from larval and adult ticks. Immunol. 67: 388–393. Riek RF (1962) Studies on the reactions of animals to infestation with ticks.VI. Re- sistance of cattle to infestation with the tick Boophilus microplus (Canestrini). Aust J Agric Res. 13: 532–550. Steen NA, Barker SC, Alewood PF (2006) Proteins in the saliva of the Ixodida (ticks): Pharmacological features and bio- logical significance. Toxicon 47: 1–20. Tietz NW (1986) Textbook of Clinical Chemistry. Saunders Company. 43 J Arthropod-Borne Dis, 2011, 6(1): 36–44 A Nikpay et al.: Analysis of Immunogenic Relevant ... 4 Turni C, Lee RP, Jackson LA (2004) A com- parison of the immunosuppressive ef- fects of salivary gland extracts from two laboratory strains of Boophilus mi- croplus. Int J Parasitol. 34: 833–838. Wang H, Nuttal PA (1994) Comparison of the proteins in salivary glands, saliva and haemolymph of Rhipicephalus ap- pendiculatus female ticks during feed- ing. Parasitol. 109: 517–523. Whelen SK, Richardson LK, Wikel SK (1986) Dot- ELISA assessment of guinea pig antibody responses to repeated Dermacentor andersoni infestation. J Parasitol. 72(1): 155–162. Wikel SK, Osburn RL (1982) Immune re- sponsiveness of the bovine host to re- peated low level infestations with Der- macentor andersoni. Ann Trop Med Parasitl. 76: 405–414. Wikel SK (1984) Immunomodulationof host responses to ectoparasite infestation. Vet Parasitol. 14(3–4): 321–339. Wikel SK (1988) Immunological control of hematophagus arthropod vectors: Utiliza- tion of novel antigens. Vet Parasitol. 29: 235–264. Wikel SK (1999a) Tick modulation of host immunity: an important factor in pa- thogen transmission. Int J Parasitol. 29: 851–859. Wikel SK (1999b) Modulation of the host immune system by ectoparasitic arthro- pods: blood-feeding and tissue- dwell- ing arthropods manipulate host defenses to their advantages. Am Inst Biol Sci. 94(4): 45–49. Willadsen P (1980) Immunity to ticks. Adv Parasitol. 18: 293–313. Willadsen P (1990) Perspectives for subunit vaccines for the control of ticks. Para- sitologia. 32: 195–200. Willadsen P (2006) Tick control: thoughts on a research agenda. Vet Parasitol. 138: 161–168. Willadsen P, Jongejan F (1999) Immunology of the tick–host interaction and the control of ticks and tick-borne dis- eases. Parasitol Today. 15(7): 258–262. Willadsen P, McKenna RV, Riding GA (1988) Isolation from the cattle tick, Boophilus microplus, of antigenic ma- terial capable of eliciting a protective immunological response in the bovine host. Int J Parasitol. 18: 183–189. Wong JYM, Opdebeeck JP (1989) Protec- tive efficacy of antigens solubilized from gut membranes of the cattle tick, Boophi- lus microplus. Immunol. 66: 149–155. 44