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Immunoblot analysis of IgG antibody responses to Rhipicephalus (Boophilus) microplus in resistant and susceptible bovines
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Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
Immunoblot analysis of IgG antibody responses to Rhipicephalus (Boophilus) microplus in resistant and susceptible bovines
that are differentially recognized by resistant hosts may target components of tick saliva that are essential for blood feeding and constitute useful antigens for a vaccine.
Wanessa A. Carvalho 1,∗ , Alexandre Firmino 2 , Daniela D. Moré 1 , Gervasio H. Bechara 3 , Beatriz F. de Miranda Santos Rossetti, K.F. Isabel 1
doi:10.1016/j.vetimm.2008.10.158
1
Department Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil 2 EMBRAPA Genetics Resources, Brasília, Brazil 3 Department Veterinary Pathology, School of Veterinary and Agronomical Sciences, São Paulo State University, Jaboticabal, Brazil Keywords: Rhipicephalus microplus; Antibody; Lectin; Saliva; Immunoblot E-mail address: [email protected] (W.A. Carvalho). Species: Ruminants Bovines express breed-specific, heritable, contrasting phenotypes when exposed to the cattle tick, R. microplus. Breeds of Bos indicus (R) are significantly more resistant than those of B. Taurus (S). Different breeds develop qualitative and quantitative different immune responses against ticks including production immunoglobulin and antibodies specific for tick saliva. This study was performed to determine the frequencies and specificities of bovine IgG antibodies binding to components of saliva, egg extracts (EE) and unfed larvae extracts (UFLE) of R. microplus and also to determine whether there are different immunological patterns of recognition of proteins from these materials by R (n = 5; B. indicus, Nelore breed) and S (n = 5; B. taurus, Holstein breed) bovines undergoing high and low natural tick infestations. In order to determine the level of infestation in each breed ticks were counted on one side of S cattle at weekly intervals for the duration of the experiment and serum was collected at different time points. Proteins from tick saliva, EE and UFLE were separated by SDS-PAGE and were electrophoretically transferred onto nitrocellulose (NC) paper. IgG-binding proteins were detected in individual sera from infested animals and as a control we used the sera from these animals before they have any contact with the ectoparasite. A total of fifteen protein bands with molecular weights ranging from 200 to 10 kDa were recognized in tick saliva, but resistant cattle recognized less protein bands whether exposed to high or low numbers of ticks. EE and UFLE are recognized with the same pattern by R and S bovines and this pattern is consistent across all levels of infestation. Salivary proteins were separated in a 2D gel and transferred to NC paper. A spot differentially recognized by R sera was eluted from a replicate gel and its amino acid sequence revealed 100% homology with bovine hemoglobin, however its pI was not as expected for the native protein, suggesting it had been modified in the tick salivary gland. Immunoblots of saliva with lectins and antiLewisx antibody indicate that the modification is due to fucosylated carbohydrates. Our results indicate that there is a difference in the reactivity to antigenic proteins in saliva from R. microplus and that the level of infestation is related to the antibody response of the host. In addition, antigens
Kinetics of leukocyte responses of bovines immunized with SBbo23290 (Babesia bovis) and SBm7462 (Rhipicephalus (Boophilus) microplus) Diogo Coelho de Padua Oliveira, Hugo Guieiro Ribeiro Rocha, Bruna Alves Devéns, Marilia Nogueira da Gama, Anna Paula B. Ribeiro Ferreira, Carla Leite Medeiros, Joaquín Hernan Patarroyo ∗ , Marlene Isabel Vargas Viloria Laboratory of Biology and Control of Haematozoa and Vectors, Institute of Biotechnology Applied to Agriculture and Animal Science (BIOAGRO/Veterinary Departament), Federal University of Vic¸osa, 36571-000 Vic¸osa, MG, Brazil Keywords: Leukocyte kinetic; Babesia bovis; Riphicephalus (Boophilus) microplus; Vaccine E-mail address: [email protected] (J.H. Patarroyo). Species: Ruminants The tick Riphicephalus (Boophilus) microplus is responsible for the great economical damages in the cattle breeding, because it causes reduction in the production of milk and meat and it provokes damages to the leather. This parasite is the transmitter of the protozoa Babesia bovis and Babesia bigemina and of the rickettsiae Anaplasma marginale, which cause the Bovine Tick Fever. Nowadays these parasites present high incidence and prevalence in Brazil and in others countries of tropical climate. The present work had as objective to analyze the leukocyte kinetic of bovine immunized with the synthetic immunogens SBm7462 and SBbo23290, in the monovalent and polyvalent forms. These peptides were prepared using saponina as adjuvant, being applied via the subcutaneous route, individually in the left and right cervical area (treatment 1) and in associated doses in the same side of the cervical area (treatment 2). Each treatment was applied three times with intervals of 30 days. The challenge was made 30 and 34 days after the third inoculation, being put ±2000 larvaes of R. (B.) microplus (strain free from haemoparasites) and inoculating the B. bovis (strain UFV1 – 9th passage in the concentration of 1 × 106 babesias/mL), respectively. Through the analysis of the results, was observed that all the vaccinated animals, independently of the inoculation form of the immunogen (monovalent and polyvalent), presented alterations in the population of lymphocytes present in the circulatory blood. Among the sub-populations of lymphocytes analyzed, there was more prominence for the B cells CD21+ and T cells WC1+ , which have important functions in the control of the babesioses. It was also verified by the analysis of the samples, accomplished five days after the second vaccination, a significant increase in the number of T cells CD4+ . Through the evaluation of the cellular immune response presented by the animals, it can be concluded that the synthetic peptides SBbo23290 and SBm7462 in the treatments I and II, using saponina as adjuvant, were capable to
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
Immunoblot analysis of IgG antibody responses to Rhipicephalus (Boophilus) microplus in resistant and susceptible bovines
that are differentially recognized by resistant hosts may target components of tick saliva that are essential for blood feeding and constitute useful antigens for a vaccine.
Wanessa A. Carvalho 1,∗ , Alexandre Firmino 2 , Daniela D. Moré 1 , Gervasio H. Bechara 3 , Beatriz F. de Miranda Santos Rossetti, K.F. Isabel 1
doi:10.1016/j.vetimm.2008.10.158
1
Department Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil 2 EMBRAPA Genetics Resources, Brasília, Brazil 3 Department Veterinary Pathology, School of Veterinary and Agronomical Sciences, São Paulo State University, Jaboticabal, Brazil Keywords: Rhipicephalus microplus; Antibody; Lectin; Saliva; Immunoblot E-mail address: [email protected] (W.A. Carvalho). Species: Ruminants Bovines express breed-specific, heritable, contrasting phenotypes when exposed to the cattle tick, R. microplus. Breeds of Bos indicus (R) are significantly more resistant than those of B. Taurus (S). Different breeds develop qualitative and quantitative different immune responses against ticks including production immunoglobulin and antibodies specific for tick saliva. This study was performed to determine the frequencies and specificities of bovine IgG antibodies binding to components of saliva, egg extracts (EE) and unfed larvae extracts (UFLE) of R. microplus and also to determine whether there are different immunological patterns of recognition of proteins from these materials by R (n = 5; B. indicus, Nelore breed) and S (n = 5; B. taurus, Holstein breed) bovines undergoing high and low natural tick infestations. In order to determine the level of infestation in each breed ticks were counted on one side of S cattle at weekly intervals for the duration of the experiment and serum was collected at different time points. Proteins from tick saliva, EE and UFLE were separated by SDS-PAGE and were electrophoretically transferred onto nitrocellulose (NC) paper. IgG-binding proteins were detected in individual sera from infested animals and as a control we used the sera from these animals before they have any contact with the ectoparasite. A total of fifteen protein bands with molecular weights ranging from 200 to 10 kDa were recognized in tick saliva, but resistant cattle recognized less protein bands whether exposed to high or low numbers of ticks. EE and UFLE are recognized with the same pattern by R and S bovines and this pattern is consistent across all levels of infestation. Salivary proteins were separated in a 2D gel and transferred to NC paper. A spot differentially recognized by R sera was eluted from a replicate gel and its amino acid sequence revealed 100% homology with bovine hemoglobin, however its pI was not as expected for the native protein, suggesting it had been modified in the tick salivary gland. Immunoblots of saliva with lectins and antiLewisx antibody indicate that the modification is due to fucosylated carbohydrates. Our results indicate that there is a difference in the reactivity to antigenic proteins in saliva from R. microplus and that the level of infestation is related to the antibody response of the host. In addition, antigens
Kinetics of leukocyte responses of bovines immunized with SBbo23290 (Babesia bovis) and SBm7462 (Rhipicephalus (Boophilus) microplus) Diogo Coelho de Padua Oliveira, Hugo Guieiro Ribeiro Rocha, Bruna Alves Devéns, Marilia Nogueira da Gama, Anna Paula B. Ribeiro Ferreira, Carla Leite Medeiros, Joaquín Hernan Patarroyo ∗ , Marlene Isabel Vargas Viloria Laboratory of Biology and Control of Haematozoa and Vectors, Institute of Biotechnology Applied to Agriculture and Animal Science (BIOAGRO/Veterinary Departament), Federal University of Vic¸osa, 36571-000 Vic¸osa, MG, Brazil Keywords: Leukocyte kinetic; Babesia bovis; Riphicephalus (Boophilus) microplus; Vaccine E-mail address: [email protected] (J.H. Patarroyo). Species: Ruminants The tick Riphicephalus (Boophilus) microplus is responsible for the great economical damages in the cattle breeding, because it causes reduction in the production of milk and meat and it provokes damages to the leather. This parasite is the transmitter of the protozoa Babesia bovis and Babesia bigemina and of the rickettsiae Anaplasma marginale, which cause the Bovine Tick Fever. Nowadays these parasites present high incidence and prevalence in Brazil and in others countries of tropical climate. The present work had as objective to analyze the leukocyte kinetic of bovine immunized with the synthetic immunogens SBm7462 and SBbo23290, in the monovalent and polyvalent forms. These peptides were prepared using saponina as adjuvant, being applied via the subcutaneous route, individually in the left and right cervical area (treatment 1) and in associated doses in the same side of the cervical area (treatment 2). Each treatment was applied three times with intervals of 30 days. The challenge was made 30 and 34 days after the third inoculation, being put ±2000 larvaes of R. (B.) microplus (strain free from haemoparasites) and inoculating the B. bovis (strain UFV1 – 9th passage in the concentration of 1 × 106 babesias/mL), respectively. Through the analysis of the results, was observed that all the vaccinated animals, independently of the inoculation form of the immunogen (monovalent and polyvalent), presented alterations in the population of lymphocytes present in the circulatory blood. Among the sub-populations of lymphocytes analyzed, there was more prominence for the B cells CD21+ and T cells WC1+ , which have important functions in the control of the babesioses. It was also verified by the analysis of the samples, accomplished five days after the second vaccination, a significant increase in the number of T cells CD4+ . Through the evaluation of the cellular immune response presented by the animals, it can be concluded that the synthetic peptides SBbo23290 and SBm7462 in the treatments I and II, using saponina as adjuvant, were capable to