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Functional bovine immunoglobulins in Boophilus microplus hemolymph
veterinary parasitology ELSEVIER
Veterinary Parasitology62 (1996) 155-160
Functional bovine immunoglobulins in Boophilus microplus hemolymph Itabajara da Silva Vaz Jr. a, Regina Helena Moraes Martinez a, Alexandre Oliveira a, Augusto Heck a, Carlos Logullo b, Jogo Carlos Gonzales c, Homero Dewes a,d, Aoi Masuda a.e,* a Centro de Biotecnologia do Estado do Rio Grande do Sul, Universidade Federal do Rio Grande do Sul, Av. Bento Gongalves 9500, C.P. 15005, 91501-970, Porto Alegre, RS, Brazil b Departamento de BioqMmica M~dica, Universidade Federal do Rio de Janeiro, Cidade Universitdria, 21941-590, Rio de Janeiro, RJ, Brazil c Faculdade de Veterindria, Universidade Federal do Rio Grande do Sul, Av. Bento Gon~2alves 9500, C.P. 15005, 91501-970, Porto Alegre, RS, Brazil a Departamento de Bioffsica, Universidade Federal do Rio Grande do Sul, Av. Bento Gon~2alves 9500, C.P. 15005, 91501-970, Porto Alegre, RS, Brazil e Departamento de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gon~alves 9500, C.P. 15005, 91501-970, Porto Alegre, RS, Brazil
Received 22 February 1995; accepted 29 May 1995
Abstract The aim of the present work was to quantify the passage of bovine immunoglobulins into the hemolymph of the tick Boophilus microplus during the feeding process and to determine their antibody activity. This knowledge is of paramount importance when vector control or blocking of disease transmission is attempted by vaccination of cattle. Approximately 2% of bovine immunoglobulin present in the serum as determined by competitive ELISA was demonstrated in hemolymph of B. microplus and antibody activity against an antigen of B. microplus in the hemolymph of ticks fed on bovine immunized with the antigen purified from tick eggs was detected by Western blot assay. The antibody reactivity detected against the B. microplus antigen showed that functional antibodies are present in the hemolymph of fully engorged ticks for at least 48 h after completing the parasitic life cycle. Keywords: Boophilus microplus; Cattle-Arthropoda;Immunity-Arthropoda
' Correspondingauthor at Centro de Biotecnologiado Estado do Rio Grande do Sul, UFRGS C.P. 15005, Campus do Vale, 91501-970. Porto Alegre, RS, Brasil. 0304-4017/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0304-401 7(95)0085 1-9
156
1. da Silva Vaz Jr. et al. / Veterinary Parasitology 62 (1996) 155-160
1. Introduction
The tick Boophilus microplus is an ectoparasite of the bovine and it is the vector to tick-borne disease agents Babesia bigemina, Babesia boris and Anaplasma marginale (Willadsen et al., 1988). Bovines exposed repeatedly to tick Boophilus microplus infestation or immunized with tick tissues acquire a partial resistance to subsequent infestations by damaging internal organs of the ectoparasite, which causes reduction in the volume of ingested blood, decrease in number and viability of eggs and death. This protection is based on humoral and cellular immune responses (Opdebeeck, 1994; Kimaro and Opdebeeck, 1994). However, the mechanisms by which antibodies and cells involved in immune response damage the ticks is not fully understood. A variety of tick extract antigens has been tested for the development of an effective vaccine (Roberts, 1968; Willadsen et al., 1988; Wong and Opdebeeck, 1989). The presence of immunoglobulins of the host animal in the hemolymph (Minoura et al., 1985), and antibody activities against tick tissues (Schlein et al., 1976) and protozoan parasites (Fujisaki et al., 1984) have been observed in different species of ticks. There is a possibility of antibodies against stages of parasites developing in arthropods having the capacity of blocking disease transmission to the vertebrate host. This approach has been extensively studied in Plasmodium spp./mosquito complex (Quakyi et al., 1987; Moelans and Schoenmakers, 1992). The objectives of the present work were to quantify the passage of bovine immunoglobulins into the hemolymph of Boophilus microplus and to assay the antibody activity against Boophilus microplus antigen. 2. Materials and methods
2.1. Antigen One 50000 molecular weight glycoprotein purified from eggs of Boophilus microplus by means of ammonium sulphate precipitation, chromatography on DEAEToyopearl and on Sephadex G-100 columns (Logullo et al., unpublished data) was used as immunogen and as antigen in the Western blot.
2.2. Immunization and infestation Two calves from a tick-free area were housed in individual tick-proof pens on slatted floors. One was immunized with Boophilus microplus antigen by four inoculations of 100 /zg of purified protein with saponin, as adjuvant, 2 weeks apart and the other one was inoculated with saponin, as control. After 30 days both were infested with 30000 Boophilus microplus larvae.
2.3. Hemolymph samples Samples of 2-10 /xl of hemolymph from fully engorged female ticks were collected by amputation of the legs up to 48 h after completing the parasitic life cycle, centrifuged, and kept frozen at - 2 0 °C until use.
I. da Silva Vaz Jr. et al. / Veterinary Parasitology 62 (1996) 155-160
157
2.4. Competitive ELISA Microtitration plates were coated with 0.1 ~g per well of bovine IgG in 20 mM carbonate buffer (pH 9.6) by incubation overnight at 4°C, washed three times, and incubated for 1 h at 37 °C with 5% cow non-fat dry milk-phosphate buffered saline (PBS) (pH 7.2) (BLOTTO) (Harlow and Lane, 1988). Fifty microliters of rabbit anti-bovine IgG-peroxidase conjugate (1 : 4000) plus 50 /xl of diluted tick hemolymph (1 : 100, 1 : 200 or 1 : 400) in BLOTTO were incubated for 1 h at 37 °C. After washing the plates three times with PBS, the chromogen and the substrate were added (3.4 mg o-phenylenediamine, 5 /xl H 2 0 2 in 0.1 M citrate-phosphate buffer, pH 5.0). Reaction was stopped after 15 min with 12.5% H2SO 4 and the optical density (OD) was determined at 492 nm. The standard curve was obtained by incubating the rabbit anti-bovine IgG-peroxidase conjugate with the known concentration of bovine IgG (0.06-3 /zg ml- 1) in the same plate and conditions. Diluted bovine sera (1 : 5000) were used as reference. Incubation of conjugate without soluble IgG and incubation of conjugate without coated IgG in the plate, were used as positive and negative controls, respectively. Results were expressed as mean + standard error of two independent experiments tested in triplicate.
2.5. Western blot Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 3% acrylamide in the stacking gel and 12.5% in the running gel was used. Samples of l 0 / x g of protein per cm of gel in sample buffer containing 5 M urea, 0.1% SDS and 10% fl-mercaptoethanol were used (Laemmli, 1970). The transfers were performed at 70 V for 1 h at 4°C in 12 mM carbonate buffer, pH 9.9 (Dunn, 1986). Strips 4 mm wide were blocked with 5% BLOTTO for 2 h at room temperature. Diluted sera (1:50) and hemolymph (1: 10) in 5% BLOTTO were incubated overnight at room temperature. Rabbit anti-bovine IgG-biotin conjugate was incubated for 1 h at room temperature before avidin-peroxidase was added, following incubation for 40 rain in 5% BLOTrO. After three washes with 1% BLOTTO and one with PBS the chromogen and the substrate were added (5 mg 3,3'-diaminobenzidine in 30 ml PBS plus 150/~l HE0 2 and 100 /.tl CoC12) (Towbin et al., 1979; Burnette, 1981).
3. Results
By competitive ELISA the concentration of immunoglobulins was determined in sera of calves and in hemolymph of ticks. It was observed that the immunoglobulin content in serum was 15.88 _+ 1.73 mg m1-1, in hemolymph 0.21 _ 0.05 mg ml -~ for Boophilus microplus fed on the non-immunized calf and 0 . 4 4 _ 0.11 mg ml -l for Boophilus microplus fed on the immunized calf (Fig. 1). The data showed that the passage of bovine immunoglobulins is approximately 2% of the concentration present in the bovine serum.
1. da Silva Vaz Jr. et al./ Veterinary Parasitology 62 (1996) 155-160
158 20 18
15.88 ± 1.73
16 14 12 10 Z
8 6 4 2
0.21 ± 0.05
0.44 ± 0.11
HemolymphA
Hemolymph B
0 Serum
Fig. 1. Determination of bovine immunoglobulin concentration in tick hemolymph by competitive ELISA. Serum: pool of four non-infested bovine sera; Hemolymph A: hemolymph of Boophilus microplus fed on non-immunized bovine; Hemolymph B: hemolymph of Boophilus microplus fed on immunized bovine.
97_
66-
~Y ~ i ~ /ii}!~ 45-
31-
I
2S4
Fig. 2. Western-blot analysis of Boophilus microplus antigen probed with antibodies from non-immunized bovine serum (Strip 1), immunized bovine serum (Strip 2), hemolymph of tick fed on non-immunized bovine (Strip 3) and hemolymph of tick fed on immunized bovine (Strip 4). Molecular weight markers × 103.
I. da Silva Vaz Jr. et al./ VeterinaryParasitology62 (1996) 155-160
159
Western blot indicated that host immunoglobulins present in vector hemolymph retained antibody activity against Boophilus microplus antigens for at least 48 h after completing the parasitic life cycle. Because of the carbohydrate content of this antigen a sharp band could not be obtained on the gel (Fig. 2).
4. Discussion The presence of functional immunoglobulins in the hemolymph of Boophilus microplus supports earlier studies showing passage of bovine serum proteins including immunoglobulin from the gut into the tick hemocel (Tracey-Patte et al., 1987). Intact IgG molecules were found in lxodes ricinus (Brossard and Rais, 1984), Ornithodorus moubata (Minoura et al., 1985), as well as in Hyalomma excavatum and Rhipicephalus sanguineus (Ben-Yakir, 1989). These studies used immunogenic proteins such as ovalbumin and cytochrome c to raise antibodies to be measured in the hemolymph. Antibody activity against relevant bovine disease agents was detected in Haemaphysalis longicornis and O. moubata fed on calves immunized against Theileria sergenfi (Fujisaki et al., 1984). Our work shows that approximately 2% of the bovine immunoglobulin concentration present in the serum passes to the tick hemocel and that antibody activity against one Boophilus microplus antigen in the hemolymph of fully engorged ticks is present for at least 48 h after completing the parasitic life cycle. The detection of antibody activity against Boophilus microplus antigens in hemolymph suggests that antibodies against internal tick organs other then gut tissues could be used to control tick infestation by interfering in physiologic functions not related to the digestive system.
Acknowledgments This work was supported by grants from Funda~5o de Amparo h Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Financiadora de Estudos e Projetos (FINEP), Conselho Nacional de Desenvolvimento Cientffico e Tecnol6gico (CNPq) and Programa de Apoio ao Desenvolvimento Cienfffico e Tecnol6gico (PADCT).
References Ben-Yakir, D., 1989. Quantitative studies of host immunoglobulinsG in the hemolymphof ticks (Acari). J. Med. Entomol., 244: 243-246. Brossard, M. and Rais, O., 1984. Passage of hemolysins through the midgut epithelium of female lxodes ricinus L. fed on rabbits infested or reinfested with tick. Experientia, 40: 561-563. Burnette, W.N., 1981. Western blotting: Electrophoretic transfer of proteins from sodium dodecyl sulfatepolyacrylamidegels to unmodified nitrocellulose and radiographic detection with antibody and radioiodihated protein. Anal. Biochem., 112: 195-203. Duma,S.D., 1986. Effectsof the modificationof transferbuffer compositionand the renaturation of proteins in gels on the recognition of proteins on Western-blot by monocional antibodies. Anal. Biochem., 157: 144-153.
160
I. da Silva Vaz Jr. et aL / Veterinary Parasitology 62 (1996) 155-160
Fujisaki, K., Kamio, T. and Kitaoka, S., 1984. Passage of host serum components, including antibodies specific for Theileria sergenti, across the digestive tract of argasid and ixodid ticks. Ann. Trop. Med. Parasitol., 78: 449-450. Harlow, E. and Lane, D., 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 726 pp. Kimaro, E.E. and Opdebeeck, J.P., 1994. Tick infestations on cattle vaccinated with extracts from eggs and the gut of Boophilus microplus. Vet. Parasitol., 52: 61-70. Laemmli, E.K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227: 680-685. Minoura, H., Chinzei, Y. and Kitamura, S., 1985. Ornithodoros moubata: Host lmmunoglobulin G in Tick Hemolymph. Exp. Parasitol., 60: 355-363. Moelans, I.I.M.D. and Schoenmakers, J.G.G., 1992. Crossreactive antigens between life cycle stages of Plasmodiumfalciparum. Parasitol. Today, 8: 118-123. Opdebeeck, J.P., 1994. Vaccines against blood-sucking arthropods. Vet. Parasitol., 54: 205-222. Quakyi, I.A., Carter, R., Rener, J., Kumar, N., Good, M.F. and Miller, L.H., 1987. The 230-KDA gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking. J. lmmunol., 139: 4213-4217. Roberts, J.A., 1968. Acquisition by the host of resistance to the cattle tick, Boophilus microplus (Canestrini). J. Parasitol., 54: 657-662. Schlein, Y., Spira, D.T. and Jacobson, R.L. 1976. The passage of serum immunoglobulins through the gut of Sarcophaga falculata Pand. Ann. Trop. Med. Parasitol., 70: 227-230. Towbin, H., Staehelin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Tracey-Patte, P.D., Kemp, D.H. and Johnston, L.A.Y., 1987. Boophilus microplus: passage of bovine immunoglobulins and albumin across the gut of cattle ticks feeding on normal or vaccinated cattle. Res. Vet. Sci., 43: 287-290. Willadsen, P., McKenna, R.V. and Riding, G.A., 1988. Isolation from the cattle tick, Boophilus microplus, of antigenic material capable of eliciting a protective immunological response in the bovine host. Int. J. Parasitol., 18: 183-189. Wong, J.Y.M. and Opdebeeck, J.P., 1989. Protective efficacy of antigens solubilized from gut membranes of the cattle tick, Boophilus microplus. Immunology, 66: 149-155.
Veterinary Parasitology62 (1996) 155-160
Functional bovine immunoglobulins in Boophilus microplus hemolymph Itabajara da Silva Vaz Jr. a, Regina Helena Moraes Martinez a, Alexandre Oliveira a, Augusto Heck a, Carlos Logullo b, Jogo Carlos Gonzales c, Homero Dewes a,d, Aoi Masuda a.e,* a Centro de Biotecnologia do Estado do Rio Grande do Sul, Universidade Federal do Rio Grande do Sul, Av. Bento Gongalves 9500, C.P. 15005, 91501-970, Porto Alegre, RS, Brazil b Departamento de BioqMmica M~dica, Universidade Federal do Rio de Janeiro, Cidade Universitdria, 21941-590, Rio de Janeiro, RJ, Brazil c Faculdade de Veterindria, Universidade Federal do Rio Grande do Sul, Av. Bento Gon~2alves 9500, C.P. 15005, 91501-970, Porto Alegre, RS, Brazil a Departamento de Bioffsica, Universidade Federal do Rio Grande do Sul, Av. Bento Gon~2alves 9500, C.P. 15005, 91501-970, Porto Alegre, RS, Brazil e Departamento de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gon~alves 9500, C.P. 15005, 91501-970, Porto Alegre, RS, Brazil
Received 22 February 1995; accepted 29 May 1995
Abstract The aim of the present work was to quantify the passage of bovine immunoglobulins into the hemolymph of the tick Boophilus microplus during the feeding process and to determine their antibody activity. This knowledge is of paramount importance when vector control or blocking of disease transmission is attempted by vaccination of cattle. Approximately 2% of bovine immunoglobulin present in the serum as determined by competitive ELISA was demonstrated in hemolymph of B. microplus and antibody activity against an antigen of B. microplus in the hemolymph of ticks fed on bovine immunized with the antigen purified from tick eggs was detected by Western blot assay. The antibody reactivity detected against the B. microplus antigen showed that functional antibodies are present in the hemolymph of fully engorged ticks for at least 48 h after completing the parasitic life cycle. Keywords: Boophilus microplus; Cattle-Arthropoda;Immunity-Arthropoda
' Correspondingauthor at Centro de Biotecnologiado Estado do Rio Grande do Sul, UFRGS C.P. 15005, Campus do Vale, 91501-970. Porto Alegre, RS, Brasil. 0304-4017/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0304-401 7(95)0085 1-9
156
1. da Silva Vaz Jr. et al. / Veterinary Parasitology 62 (1996) 155-160
1. Introduction
The tick Boophilus microplus is an ectoparasite of the bovine and it is the vector to tick-borne disease agents Babesia bigemina, Babesia boris and Anaplasma marginale (Willadsen et al., 1988). Bovines exposed repeatedly to tick Boophilus microplus infestation or immunized with tick tissues acquire a partial resistance to subsequent infestations by damaging internal organs of the ectoparasite, which causes reduction in the volume of ingested blood, decrease in number and viability of eggs and death. This protection is based on humoral and cellular immune responses (Opdebeeck, 1994; Kimaro and Opdebeeck, 1994). However, the mechanisms by which antibodies and cells involved in immune response damage the ticks is not fully understood. A variety of tick extract antigens has been tested for the development of an effective vaccine (Roberts, 1968; Willadsen et al., 1988; Wong and Opdebeeck, 1989). The presence of immunoglobulins of the host animal in the hemolymph (Minoura et al., 1985), and antibody activities against tick tissues (Schlein et al., 1976) and protozoan parasites (Fujisaki et al., 1984) have been observed in different species of ticks. There is a possibility of antibodies against stages of parasites developing in arthropods having the capacity of blocking disease transmission to the vertebrate host. This approach has been extensively studied in Plasmodium spp./mosquito complex (Quakyi et al., 1987; Moelans and Schoenmakers, 1992). The objectives of the present work were to quantify the passage of bovine immunoglobulins into the hemolymph of Boophilus microplus and to assay the antibody activity against Boophilus microplus antigen. 2. Materials and methods
2.1. Antigen One 50000 molecular weight glycoprotein purified from eggs of Boophilus microplus by means of ammonium sulphate precipitation, chromatography on DEAEToyopearl and on Sephadex G-100 columns (Logullo et al., unpublished data) was used as immunogen and as antigen in the Western blot.
2.2. Immunization and infestation Two calves from a tick-free area were housed in individual tick-proof pens on slatted floors. One was immunized with Boophilus microplus antigen by four inoculations of 100 /zg of purified protein with saponin, as adjuvant, 2 weeks apart and the other one was inoculated with saponin, as control. After 30 days both were infested with 30000 Boophilus microplus larvae.
2.3. Hemolymph samples Samples of 2-10 /xl of hemolymph from fully engorged female ticks were collected by amputation of the legs up to 48 h after completing the parasitic life cycle, centrifuged, and kept frozen at - 2 0 °C until use.
I. da Silva Vaz Jr. et al. / Veterinary Parasitology 62 (1996) 155-160
157
2.4. Competitive ELISA Microtitration plates were coated with 0.1 ~g per well of bovine IgG in 20 mM carbonate buffer (pH 9.6) by incubation overnight at 4°C, washed three times, and incubated for 1 h at 37 °C with 5% cow non-fat dry milk-phosphate buffered saline (PBS) (pH 7.2) (BLOTTO) (Harlow and Lane, 1988). Fifty microliters of rabbit anti-bovine IgG-peroxidase conjugate (1 : 4000) plus 50 /xl of diluted tick hemolymph (1 : 100, 1 : 200 or 1 : 400) in BLOTTO were incubated for 1 h at 37 °C. After washing the plates three times with PBS, the chromogen and the substrate were added (3.4 mg o-phenylenediamine, 5 /xl H 2 0 2 in 0.1 M citrate-phosphate buffer, pH 5.0). Reaction was stopped after 15 min with 12.5% H2SO 4 and the optical density (OD) was determined at 492 nm. The standard curve was obtained by incubating the rabbit anti-bovine IgG-peroxidase conjugate with the known concentration of bovine IgG (0.06-3 /zg ml- 1) in the same plate and conditions. Diluted bovine sera (1 : 5000) were used as reference. Incubation of conjugate without soluble IgG and incubation of conjugate without coated IgG in the plate, were used as positive and negative controls, respectively. Results were expressed as mean + standard error of two independent experiments tested in triplicate.
2.5. Western blot Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 3% acrylamide in the stacking gel and 12.5% in the running gel was used. Samples of l 0 / x g of protein per cm of gel in sample buffer containing 5 M urea, 0.1% SDS and 10% fl-mercaptoethanol were used (Laemmli, 1970). The transfers were performed at 70 V for 1 h at 4°C in 12 mM carbonate buffer, pH 9.9 (Dunn, 1986). Strips 4 mm wide were blocked with 5% BLOTTO for 2 h at room temperature. Diluted sera (1:50) and hemolymph (1: 10) in 5% BLOTTO were incubated overnight at room temperature. Rabbit anti-bovine IgG-biotin conjugate was incubated for 1 h at room temperature before avidin-peroxidase was added, following incubation for 40 rain in 5% BLOTrO. After three washes with 1% BLOTTO and one with PBS the chromogen and the substrate were added (5 mg 3,3'-diaminobenzidine in 30 ml PBS plus 150/~l HE0 2 and 100 /.tl CoC12) (Towbin et al., 1979; Burnette, 1981).
3. Results
By competitive ELISA the concentration of immunoglobulins was determined in sera of calves and in hemolymph of ticks. It was observed that the immunoglobulin content in serum was 15.88 _+ 1.73 mg m1-1, in hemolymph 0.21 _ 0.05 mg ml -~ for Boophilus microplus fed on the non-immunized calf and 0 . 4 4 _ 0.11 mg ml -l for Boophilus microplus fed on the immunized calf (Fig. 1). The data showed that the passage of bovine immunoglobulins is approximately 2% of the concentration present in the bovine serum.
1. da Silva Vaz Jr. et al./ Veterinary Parasitology 62 (1996) 155-160
158 20 18
15.88 ± 1.73
16 14 12 10 Z
8 6 4 2
0.21 ± 0.05
0.44 ± 0.11
HemolymphA
Hemolymph B
0 Serum
Fig. 1. Determination of bovine immunoglobulin concentration in tick hemolymph by competitive ELISA. Serum: pool of four non-infested bovine sera; Hemolymph A: hemolymph of Boophilus microplus fed on non-immunized bovine; Hemolymph B: hemolymph of Boophilus microplus fed on immunized bovine.
97_
66-
~Y ~ i ~ /ii}!~ 45-
31-
I
2S4
Fig. 2. Western-blot analysis of Boophilus microplus antigen probed with antibodies from non-immunized bovine serum (Strip 1), immunized bovine serum (Strip 2), hemolymph of tick fed on non-immunized bovine (Strip 3) and hemolymph of tick fed on immunized bovine (Strip 4). Molecular weight markers × 103.
I. da Silva Vaz Jr. et al./ VeterinaryParasitology62 (1996) 155-160
159
Western blot indicated that host immunoglobulins present in vector hemolymph retained antibody activity against Boophilus microplus antigens for at least 48 h after completing the parasitic life cycle. Because of the carbohydrate content of this antigen a sharp band could not be obtained on the gel (Fig. 2).
4. Discussion The presence of functional immunoglobulins in the hemolymph of Boophilus microplus supports earlier studies showing passage of bovine serum proteins including immunoglobulin from the gut into the tick hemocel (Tracey-Patte et al., 1987). Intact IgG molecules were found in lxodes ricinus (Brossard and Rais, 1984), Ornithodorus moubata (Minoura et al., 1985), as well as in Hyalomma excavatum and Rhipicephalus sanguineus (Ben-Yakir, 1989). These studies used immunogenic proteins such as ovalbumin and cytochrome c to raise antibodies to be measured in the hemolymph. Antibody activity against relevant bovine disease agents was detected in Haemaphysalis longicornis and O. moubata fed on calves immunized against Theileria sergenfi (Fujisaki et al., 1984). Our work shows that approximately 2% of the bovine immunoglobulin concentration present in the serum passes to the tick hemocel and that antibody activity against one Boophilus microplus antigen in the hemolymph of fully engorged ticks is present for at least 48 h after completing the parasitic life cycle. The detection of antibody activity against Boophilus microplus antigens in hemolymph suggests that antibodies against internal tick organs other then gut tissues could be used to control tick infestation by interfering in physiologic functions not related to the digestive system.
Acknowledgments This work was supported by grants from Funda~5o de Amparo h Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Financiadora de Estudos e Projetos (FINEP), Conselho Nacional de Desenvolvimento Cientffico e Tecnol6gico (CNPq) and Programa de Apoio ao Desenvolvimento Cienfffico e Tecnol6gico (PADCT).
References Ben-Yakir, D., 1989. Quantitative studies of host immunoglobulinsG in the hemolymphof ticks (Acari). J. Med. Entomol., 244: 243-246. Brossard, M. and Rais, O., 1984. Passage of hemolysins through the midgut epithelium of female lxodes ricinus L. fed on rabbits infested or reinfested with tick. Experientia, 40: 561-563. Burnette, W.N., 1981. Western blotting: Electrophoretic transfer of proteins from sodium dodecyl sulfatepolyacrylamidegels to unmodified nitrocellulose and radiographic detection with antibody and radioiodihated protein. Anal. Biochem., 112: 195-203. Duma,S.D., 1986. Effectsof the modificationof transferbuffer compositionand the renaturation of proteins in gels on the recognition of proteins on Western-blot by monocional antibodies. Anal. Biochem., 157: 144-153.
160
I. da Silva Vaz Jr. et aL / Veterinary Parasitology 62 (1996) 155-160
Fujisaki, K., Kamio, T. and Kitaoka, S., 1984. Passage of host serum components, including antibodies specific for Theileria sergenti, across the digestive tract of argasid and ixodid ticks. Ann. Trop. Med. Parasitol., 78: 449-450. Harlow, E. and Lane, D., 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 726 pp. Kimaro, E.E. and Opdebeeck, J.P., 1994. Tick infestations on cattle vaccinated with extracts from eggs and the gut of Boophilus microplus. Vet. Parasitol., 52: 61-70. Laemmli, E.K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227: 680-685. Minoura, H., Chinzei, Y. and Kitamura, S., 1985. Ornithodoros moubata: Host lmmunoglobulin G in Tick Hemolymph. Exp. Parasitol., 60: 355-363. Moelans, I.I.M.D. and Schoenmakers, J.G.G., 1992. Crossreactive antigens between life cycle stages of Plasmodiumfalciparum. Parasitol. Today, 8: 118-123. Opdebeeck, J.P., 1994. Vaccines against blood-sucking arthropods. Vet. Parasitol., 54: 205-222. Quakyi, I.A., Carter, R., Rener, J., Kumar, N., Good, M.F. and Miller, L.H., 1987. The 230-KDA gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking. J. lmmunol., 139: 4213-4217. Roberts, J.A., 1968. Acquisition by the host of resistance to the cattle tick, Boophilus microplus (Canestrini). J. Parasitol., 54: 657-662. Schlein, Y., Spira, D.T. and Jacobson, R.L. 1976. The passage of serum immunoglobulins through the gut of Sarcophaga falculata Pand. Ann. Trop. Med. Parasitol., 70: 227-230. Towbin, H., Staehelin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Tracey-Patte, P.D., Kemp, D.H. and Johnston, L.A.Y., 1987. Boophilus microplus: passage of bovine immunoglobulins and albumin across the gut of cattle ticks feeding on normal or vaccinated cattle. Res. Vet. Sci., 43: 287-290. Willadsen, P., McKenna, R.V. and Riding, G.A., 1988. Isolation from the cattle tick, Boophilus microplus, of antigenic material capable of eliciting a protective immunological response in the bovine host. Int. J. Parasitol., 18: 183-189. Wong, J.Y.M. and Opdebeeck, J.P., 1989. Protective efficacy of antigens solubilized from gut membranes of the cattle tick, Boophilus microplus. Immunology, 66: 149-155.