(Boophilus) Annulatus Tick

J Arthropod-Borne Dis, 2012, 6(1): 36–44 A Nikpay et al.: Analysis of Immunogenic Relevant ...

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 immune response to extracts of salivary gland and ovary of adult ticks and larval extracts during infestation were determined 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 cine with only one antigen will not be sufficient natural infestation (Reik 1962, Dipeolu et al. to generate an effective immune response 1992, Cruz et al. 2008) and vaccination (Op- able to control the proliferation of the ticks debeek et al. 1989, Willadsen et al. 1988, 1999, under field conditions (Willadsen 1990). 2006, Wikel 1999a, b, Ogden et al. 2002, Salivary gland, gut, embryo, and larval Trimnel et al. 2005, Leal et al. 2006, Perez extracts of various ticks have been studied as 2010). possible target for vaccine. However, these It has been shown that infested animals tissues have been examined independently with Boophilus microplus induce an immune (Wikel 1984, Wiladsen et al. 1988, Wong response and acquire a partial resistance to and Opdebeek 1989). subsequent infestations, in which the internal We examined the cattle immune response organs of the ectoparasite become damaged, to salivary gland, ovary, and larval extracts of leading to an increase of feeding time, a de- R. annulatus tick at the same time with the crease in the numbers of engorged females, aim of finding common antigens, which in less uptake of blood meal, a decrease in the vaccination protocol could be advantageous number and viability of eggs (Wikel 1988). upon stage, or organ specific antigens. The There is a general view, however, that a vac- tick R. annulatus is an important ectoparasite

*Corresponding author: Dr Ali Nikpay, E-mail: 3356 [email protected] J Arthropod-Borne Dis, 2012, 6(1): 36–44 A Nikpay et al.: Analysis of Immunogenic Relevant ...

of cattle that present in West Africa, Central Sera collection Africa, Asia, certain parts of Southern Sudan Positive and negative references sera col- and Europe. lected from infested and uninfested cattle were In this study, for the first time, salivary used to standardize the ELISA test. The posi- gland, ovary, and larval extracts of this spe- tive sera were obtained by infesting seven cies have been studied at the same time as three months-old calves with 10,000 of R. possible target antigens to determine the cat- annulatus larvae. Weekly for 12 consecutive tle immune response to them. weeks, blood was collected by jugular vein and allowed to clot for 2 hour at room tem- Materials and Methods perature and then centrifuged at 800 g for 15 min and extracted serum aliquoted and kept o Tick infestation at -20 C. Seven healthy 3-5 months old Holstein cattle being negative sera for tick infestation ELISA were provided from Tehran Veterinary Medi- ELISA plates were coated with 4 µg per cine Faculty Research Institute (Amin abad). well of three antigens in 20 mM carbonate They were housed in tick-proof pens in- buffer (pH= 9.6) by incubation overnight at o dependently. 4 C (Harlow and Lane 1988). Having been Each cattle was infested with about 10000 washed three times, the plates were incu- o R. annulatus larvae as described by Brown bated for 1 hour at 37 C with 5% skim milk- (1988). Briefly, tubes containing larvae fas- PBS. Then, test sera diluted 1/200 were o tened to a shaved flank of cattle with adhe- incubated at 37 C. After 1 hour, the plates sive tape. Then, the surface of the infestation were washed three times and 100 µl of area was covered with a piece of cloth. 1/2000 diluted sheep anti –bovine IgG conjugated with peroxidase were added to the Antigen preparation individual wells. After incubation at 37o C Partly engorged female ticks were washed for 1 hour, the plates were washed again and with 70% ethanol and then washed three the enzyme substrate was added. The sub- times with sterile distilled water. After drying, strate was prepared by dissolving 62 mg of they were maintained at 28o C and 85% re- (2, 2, Azino-bis 3-ethyl benz- thiazoline-6- lative humidity. The antigens used in the sulfonic acid) in 50 ml of distilled water and ELISA were extracts of salivary glands, ova- the optical density (OD) was determined at ries, and larvae. Salivary glands and ovaries 405 nm. Sera from cattle were tested against were dissected with fine-tipped forceps in anti R. annulatus antibodies at 1-week inter- cold PBS, rinsed in fresh PBS and then each vals from week 0 (the week of infestation) to of these organs and tick larvae were ho- week 9. mogenized in a glass tissue grinder sepa- rately in PBS containing 1 mM phenyl methyl SDS-PAGE electrophoresis of different sulphonyl fluoride at 4o C following sonicated tissues for 30 minnutes on ice with 40 W. The ho- Tick extract preparations (20 µg/lane) were mogenates centrifuged at 20000 g for 30 min resolved by sodium dodecyl sulfate polyacryla- at 4o C, the supernatant were stored at -70o mid gel electrophoresis (SDS-PAGE) using a C. The proteins of the extracts were deter- discontinuous gel system in reducing condi- mined using the method of Warburg (Tietz tion (Laemmli 1970) with an acrylamide con- 1986, Hudson and Hay 1994). centration of 5% in stacking gel and 12% in

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the separation gel. A high molecular weight level was observed during the experiment from protein ladder (Fermentas) was used as mo- week 0 to week 9, however, a slight decrease lecular weight markers. Finally, the gel was antibody production occurred in the week 9 stained with Coomassie brilliant blue. of infestation.

Western blot analysis SDS-PAGE electrophoresis of different Soluble proteins extracted from salivary tissues glands, ovaries and larvae of R. annulatus Coomassie brilliant blue stain differentiates (20 µg/lane) were resolved by SDS-PAGE a vast profile of proteins in tick’s antigens. using a discontinuous gel system as described In salivary gland, ovary and larval extracts previously (Laemmli 1970). The antigens se- 22, 18, and 25 fractions were more dominant parated were transferred to nitrocellulose pa- respectively. Protein bands ranging in mo- per (NCP) (Wang and Nuttal 1994), and lecular weight from 18 to more than 200 non- specific reactive sites on the NCP were KDa .The bands >200, 170, 117, 100, 93, 70, blocked for 1h in Tris-buffered saline with 55, 50, 44, 37, 30 and 27 kDa were common 0.5% tween 20 (TBS-T pH8), containing 3% in all extracts (See in Fig. 2). bovine skim milk on a shaking plate at room temperature. The NCP was incubated for 1 h Western blot analysis of salivary, ovary at 37o C with bovine sera diluted 1/50 in antigens and larva extracts PBS (pH= 7.2). The NCP was incubated for In order to analyze the peptides reacting a further 1 h at 37oC with sheep anti- bovine with the bovine antibodies, Western blot as- IgG (conjugated with HRPO) diluted 1/2000 says were carried out using the sera with st in PBS-tween20. After 1 hour, the paper was higher ELISA OD that belonged to 21 day washed and reacted with Di amino benzidine after infestation. The profile of three differ- (DAB) (Sigma) as a substrate. The NCP was ent antigens recognized by cattle positive washed three times between each step of the sera are presented in Fig.3. Serum from ex- assay with PBS-Tween. perimentally infested cattle recognized approximately 15 fractions from soluble antigens extracted from salivary gland and lar- Results vae. Challenged cattle also recognized 14 Characterization of humoral immune re- protein bands in the ovary extract. sponse The negative control sera recognized no The experimental infestation of seven cattle band. These findings illustrate the recognition with approximately 10000 larvae of R. an- of common proteins with molecular weight nulatus was performed to characterize the of 170, 117, 100, 70, 37, 33 and 30 kDa from humoral immune response to tick extracts. different antigens by sera of infested cattle. Furthermore, approximately 2500±252 ticks were collected per animal between Days 21 Discussion and 26. Figure 1 shows changes of antibody levels It is extremely important to have defined measured by ELISA in the sera of the ex- immunogenic molecules in order to dissect perimentally infested animals against sali- the events involved in acquisition and expres- vary glands, ovaries, and larvae antigens. All sion of tick resistance. Some studies clearly extracts showed positive reactions with all show that a number of tick tissues can be sera tested starting from the first week of in- used to induce artificial resistance to tick festation. A steady increase of the antibody feeding, an approach that holds significant 383 J Arthropod-Borne Dis, 2012, 6(1): 36–44 A Nikpay et al.: Analysis of Immunogenic Relevant ...

promise as an alternative method for tick of several tick species using sera collected control (Wickel et al. 1988, Turni et al. from different animal hosts (Whelen et al. 2004, Leal et al. 2006, Willadsen 2006, Cruz 1986, Brown 1988, Trimnel 2005, Cruz et al. et al. 2008). Antibodies reactive with tick 2008). Salivary glands, gut and larval ex- extracts have been described by several re- tracts have been tested as immunogens to searchers (Willadsen 1980, Wikel 1984). control tick infestation (Opdebeeck et al. Immunoelectrophoresis, immunofluorescence, 1989, Barriga et al. 1991). Da Silva Vaz et ELISA (Wikel and Osburn 1982, Brossard al. (1994) also indicated that Rhipicephalus and Wikel 1997, Cruz et al. 2008, Perez (Boophilus) microplus infested cattle de- 2010, Kopp et al. 2010) have been used to velop antibodies to components of tick sali- examine serological responses against ticks. vary gland, gut, embryo and larvae during Many investigators have used the Western the first infestation. blot technique to identify relevant antigens

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

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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

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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 experimentally infested cattle suggesting the compare the antibody levels developed against antibody level increased during the period of R. annulatus salivary gland, ovary and lar- infestation and as mentioned in Fig. 1, a sig- vae protein extracts of sera from cattle sub- nificant increase was observed in third week mitted to experimental infestation. The results after infestation that the greatest amount of of this part of our study showed the impact ticks were harvested at this time. These re- of approximately high-density tick infestation sults are in agreement with our previous study on humoral immune responses induced in and some other researchers (Oegden et al.

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2002, Cruz et al. 2008, Nikpay et al. 2008, this investigation was conducted for the first Perez 2010). Using Western blot analysis, time in Iran to characterize the immunogenic some basic information have provided about antigens in this species. antigens extracted from R. annulatus tick The findings of the present study estab- recognized by sera collected from cattle in- lish the presence of common antigens be- fested with this species. The recognized frac- tween ovary, larvae, and salivary glands of R. tions could be used as target for subsequent annulatus ticks. Further studies will be aimed antigen characterization studies, and points at the isolation of common proteins and test- out some probable antigenic sites in specific ing for antigenicity and immunogenicity fol- tick tissues. ELISA and Western blot data lowed by purification and subsequent in vitro indicate that R. annulatus infested cattle de- expression for potential vaccine development. velop antibodies to components of tick sali- Vaccination of hosts with antigenic tis- vary glands, ovaries and larvae during the sues and secretions of ticks may provide a infestation. new method to control ticks. Extracts of whole In our experiment, all extracts showed the tick body homogenates and various organs same absorbance value changes. One possi- especially have been tested as to their ability ble explanation of the similarities in the re- to induce tick resistance in the host (Mc- activity of different tissues is that the bovine Gowan et al. 1980). The significance of these is inoculated with various tissue antigens dur- observations is with regard to the induction ing the tick feeding process, or that there are of host immunity to ticks and the develop- common antigens in different tissues of R. ment of tick vaccines using common proteins. annulatus and according to these results it seemed to be a correct assumption. These Acknowledgments findings further illustrate the presence of common crucial antigens, which are be- Authors wish to acknowledge the finan- lieved to be 170, 117, 100, 70, 37, 33 and 30 cial support of the research council of Te- kDa depending upon the assay system em- hran University and Veterinary Medicine Fac- ployed because they were recognized in ulty of Tehran University. The authors de- three extracts by anti tick sera. Electeropho- clare that there is no conflict of interests. retic comparison of salivary gland extracts of R. annulatus specimens demonstrated some References similar proteins with salivary gland extracts of Hyalomma specimens (Nabian et al. 2005). Barriga OO, Andujar F, Andrzejewski WJ Also it was shown that some proteins of (1991) Manifestation of immunity in salivary gland of R. annulatus tick changed sheep repeatedly infested with Am- in different temperatures but protein bands blyomma americanum ticks. J Parasi- of 29, 36, 55 and 97 were constant at all tol. 77: 703–709. temperatures (Nabian et al. 2005). Birk HW, Koepsell H (1987) Reaction of For the first time in the current study monoclonal antibodies with plasma salivary glands, ovary and larvae extracts have membrane proteins after binding on ni- been used as antigenic sources at the same trocellulose: renaturing of antigenic sites time and it was showed that ovaries could be and reduction of non-specific antibody a source of immunogenic proteins such as binding. Anal Biochem. 164: 12–22. the other tissues of tick studied previously. It Brossard M, Wikel SK (1997) Immunology should be mentioned that there was not any of interaction between ticks and hosts, similar study about R. annulatus tick and Med Vet Entomol. 11: 270–276. 422 J Arthropod-Borne Dis, 2012, 6(1): 36–44 A Nikpay et al.: Analysis of Immunogenic Relevant ...

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