RESUMO
Immunization of calves with Anaplasma centrale is used to prevent acute anaplasmosis caused by A. marginale. Natural and vaccine-acquired immunity is detected through serologic tests based primarily on A. marginale recombinant major surface protein 5 (MSP5m) because it has 91% identity with MSP5 from A. centrale (MSP5c). We developed a displacement, double-antigen, sandwich ELISA (ddasELISA) to detect antibodies against A. marginale or A. centrale. For ddasELISA validation, we analyzed serum samples positive for antibodies against Anaplasma spp. from cattle naturally infected with A. marginale (n = 300) or vaccinated with A. centrale (n = 255). Species-specific nested PCR (nPCR) assays were used to confirm infection. The optical density (OD) values obtained from antibodies directed at unique epitopes of A. marginale (ODAm) or A. centrale (ODAc) were used in the formula ODAm/ODAc. If the derived ratio was >0.38, the serum sample was considered positive for antibodies against A. marginale, with 98.9% sensitivity and 98.0% specificity. In a field evaluation, we analyzed 702 Anaplasma spp. antibody-positive serum samples from 34 herds by ddasELISA and nPCR; 571 were classified by ddasELISA as A. marginale-infected or A. centrale-vaccinated, with 84% agreement (κ = 0.70) between ddasELISA and nPCR. Our results indicate that ddasELISA could be used as a cost-effective alternative to molecular techniques to confirm infection with A. marginale in countries in which prevention is based on vaccination with A. centrale.
Assuntos
Anaplasma centrale , Anaplasma marginale , Anaplasmose , Doenças dos Bovinos , Bovinos , Animais , Anaplasmose/diagnóstico , Anaplasmose/prevenção & controle , Anaplasma , Ensaio de Imunoadsorção Enzimática/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Recombinantes , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/prevenção & controleRESUMO
BACKGROUND: Cattle fever ticks (CFT), Rhipicephalus (Boophilus) annulatus and R. (B.) microplus, are vectors of microbes causing bovine babesiosis and pose a threat to the economic viability of the US livestock industry. Efforts by the Cattle Fever Tick Eradication Program (CFTEP) along the US-Mexico border in south Texas are complicated by the involvement of alternate hosts, including white-tailed deer (Odocoileus virginianus) and nilgai (Boselaphus tragocamelus). METHODS: In the present study, we use a spatially explicit, individual-based model to explore the potential effects of host species composition and host habitat use patterns on southern cattle fever ticks (SCFT, R. (B.) microplus) infestation dynamics and efficacy of eradication schemes. RESULTS: In simulations without eradication efforts, mean off-host larval densities were much higher when cattle were present than when only white-tailed deer and nilgai were present. Densities in mesquite and meadows were slightly higher, and densities in mixed brush were much lower, than landscape-level densities in each of these scenarios. In eradication simulations, reductions in mean off-host larval densities at the landscape level were much smaller when acaricide was applied to cattle only, or to cattle and white-tailed deer, than when applied to cattle and nilgai. Relative density reductions in mesquite, mixed brush, and meadows depended on host habitat use preferences. Shifting nilgai habitat use preferences increasingly toward mixed brush and away from mesquite did not change mean off-host larval tick densities noticeably at the landscape level. However, mean densities were increased markedly in mesquite and decreased markedly in mixed brush, while no noticeable change in density was observed in meadows. CONCLUSIONS: Our results suggest that continued integration of field data into spatially explicit, individual-based models will facilitate the development of novel eradication strategies and will allow near-real-time infestation forecasts as an aid in anticipating and preventing wildlife-mediated impacts on SCFT eradication efforts.
Assuntos
Dinâmica Populacional/estatística & dados numéricos , Rhipicephalus , Infestações por Carrapato/veterinária , Anaplasmose/prevenção & controle , Animais , Animais Selvagens/parasitologia , Antílopes/parasitologia , Vetores Artrópodes , Babesiose/prevenção & controle , Bovinos , Doenças dos Bovinos/prevenção & controle , Simulação por Computador/estatística & dados numéricos , Cervos/parasitologia , Reservatórios de Doenças/veterinária , Interações Hospedeiro-Parasita , Gado/parasitologia , México , Texas , Controle de Ácaros e Carrapatos/métodosRESUMO
Cattle tick fever (CTF) causes significant economic losses in the livestock sector. The pathogenic action of the hemoparasites is associated with anemia, weight loss, abortion and reduced productivity, which result with animal death. Programs to prevent CTF involve several procedures, including immunization, chemoprophylaxis and use of ectoparasiticides, together with the vector control in the environment. The objective of this study was to report an acute outbreak of CTF in a group of 157 Hereford cattle from a farm without presence of the vector, that were moved to a farm in the same state with a high tick infestation (Rhipicephalus microplus). On the day before the transportation, the animals received a chemoprophylaxis with imidocarb dipropionate (3 mg/kg, SC), which was repeated 21 days after the first application. After 42 days, some animals showed signs compatible with CTF, which was confirmed through clinical examination, necropsy, histopathological and hemoparasitological analyses. The morbidity rate was 37.6% and the mortality rate was 24.8%. Calves that were recently weaned were the group most affected with the tick fever, morbidity (100% and mortality (73%). Chemoprophylaxis in association with use of ectoparasiticides was not sufficient to control the outbreak of the disease.
Assuntos
Anaplasmose , Babesiose , Doenças dos Bovinos , Quimioprevenção/veterinária , Infestações por Carrapato , Anaplasmose/diagnóstico , Anaplasmose/epidemiologia , Anaplasmose/prevenção & controle , Animais , Babesiose/diagnóstico , Babesiose/epidemiologia , Babesiose/prevenção & controle , Bovinos , Doenças dos Bovinos/prevenção & controle , Rhipicephalus , Infestações por Carrapato/prevenção & controle , Infestações por Carrapato/veterináriaRESUMO
Anaplasma marginale is the causative agent of the severe bovine anaplasmosis. The tick Rhipicephalus microplus is one of the main vectors of A. marginale in tropical and subtropical regions of the world. After the tick bite, the bacterium invades and proliferates within the bovine erythrocytes leading to anemia, impairment of milk production and weight loss. In addition, infection can cause abortion and high mortality in areas of enzootic instability. Immunization with live and inactivated vaccines are employed to control acute bovine anaplasmosis. However, they do not prevent persistent infection. Consequently, infected animals, even if immunized, are still reservoirs of the bacterium and contribute to its dissemination. Antimicrobials are largely employed for the prophylaxis of bovine anaplasmosis. However, they are often used in sublethal doses which may select pre-existing resistant bacteria and induce genetic or phenotypic variations. Therefore, we propose a new standardized in vitro assay to evaluate the susceptibility of A. marginale strains to different antimicrobials. This tool will help health professionals to choose the more adequate treatment for each herd which will prevent the selection and spread of resistant strains. For that, we initially evaluated the antimicrobial susceptibility of two field isolates of A. marginale (Jaboticabal and Palmeira) infecting bovines. The least susceptible strain (Jaboticabal) was used for the standardization of an antimicrobial assay using a culture of Ixodes scapularis-derived tick cell line, ISE6. Results showed that enrofloxacin (ENRO) at 0.25, 1 or 4 µg/mL and oxytetracycline (OTC) at 4 or 16 µg/mL are the most efficient treatments, followed by OTC at 1 µg/mL and imidocarb dipropionate (IMD) at 1 or 4 µg/mL. In addition, this proposed tool has technical advantages compared to the previously established bovine erythrocyte culture. Thereby, it may be used to guide cattle farmers to the correct use of antimicrobials. The choice of the most suitable antimicrobial is essential to eliminate persistent infections, prevent the spread of resistant strains and help controlling of bovine anaplasmosis.
Assuntos
Anaplasma marginale/efeitos dos fármacos , Anaplasmose/prevenção & controle , Antibacterianos/farmacologia , Vetores Aracnídeos/citologia , Doenças dos Bovinos/prevenção & controle , Rhipicephalus/citologia , Anaplasmose/tratamento farmacológico , Anaplasmose/microbiologia , Animais , Antibacterianos/uso terapêutico , Vetores Aracnídeos/parasitologia , Brasil , Bovinos , Doenças dos Bovinos/tratamento farmacológico , Doenças dos Bovinos/microbiologia , Linhagem Celular , Enrofloxacina/farmacologia , Eritrócitos/microbiologia , Imidocarbo/análogos & derivados , Imidocarbo/farmacologia , Imidocarbo/uso terapêutico , Masculino , Testes de Sensibilidade Microbiana , Oxitetraciclina/farmacologia , Oxitetraciclina/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Rhipicephalus/parasitologiaRESUMO
Abstract Cattle tick fever (CTF) causes significant economic losses in the livestock sector. The pathogenic action of the hemoparasites is associated with anemia, weight loss, abortion and reduced productivity, which result with animal death. Programs to prevent CTF involve several procedures, including immunization, chemoprophylaxis and use of ectoparasiticides, together with the vector control in the environment. The objective of this study was to report an acute outbreak of CTF in a group of 157 Hereford cattle from a farm without presence of the vector, that were moved to a farm in the same state with a high tick infestation (Rhipicephalus microplus). On the day before the transportation, the animals received a chemoprophylaxis with imidocarb dipropionate (3 mg/kg, SC), which was repeated 21 days after the first application. After 42 days, some animals showed signs compatible with CTF, which was confirmed through clinical examination, necropsy, histopathological and hemoparasitological analyses. The morbidity rate was 37.6% and the mortality rate was 24.8%. Calves that were recently weaned were the group most affected with the tick fever, morbidity (100% and mortality (73%). Chemoprophylaxis in association with use of ectoparasiticides was not sufficient to control the outbreak of the disease.
Resumo A "tristeza parasitária bovina" (TPB) gera importantes perdas econômicas na bovinocultura mundial. A ação patogênica dos hemoparasitas resulta em anemia, perda de peso, abortos e diminuição da produtividade, culminando, muitas vezes, em óbito dos animais. Um programa de prevenção para TPB necessita de medidas integradas, como a imunização, quimioprofilaxia e utilização de ectoparasiticidas, em conjunto com ações que visem ao controle ambiental dos vetores. Este artigo tem em vista o relato de um surto de TPB em uma fazenda de produção de bovinos de corte e com alta infestação do carrapato (Rhipicephalus microplus). A fazenda adquiriu 157 animais puros de origem, da raça Hereford, proveniente de uma fazenda sem presença do vetor. No dia anterior ao transporte, os animais receberam quimioprofilaxia com dipropionato de imidocarb (3mg/Kg/SC), repetindo-se 21 dias após a primeira aplicação. Aos 42 dias, alguns bezerros manifestaram sinais clínicos compatíveis com TPB, sendo confirmado pelo exame clínico, necropsia, análises histopatológicas e hemoparasitológicas. A morbidade foi de 37,6% (59/157), e a letalidade de 24,8% (39/157). A categoria de bezerros recém desmamados foi a mais acometida, com morbidade de 100% (52/52) e letalidade de 73% (38/52). A quimioprofilaxia associada à utilização de ectoparasiticidas foram insuficientes para evitar a ocorrência do surto da enfermidade.
Assuntos
Animais , Babesiose/diagnóstico , Babesiose/prevenção & controle , Babesiose/epidemiologia , Anaplasmose/diagnóstico , Anaplasmose/prevenção & controle , Anaplasmose/epidemiologia , Infestações por Carrapato/prevenção & controle , Infestações por Carrapato/veterinária , Bovinos , Doenças dos Bovinos/prevenção & controle , Quimioprevenção/veterinária , RhipicephalusRESUMO
In Uruguay, control of Rhipicephalus microplus began in 1910. In 1941 the eradication of R. micoplus throughout the country was declared mandatory, although this attempt was unsuccessful. Since 2008 the country was divided into two regions: the south-western region, which is free of ticks; and a region of tick control that includes all departments to the north of the Rio Negro and five departments in the eastern region. In Uruguay, investigations on R. microplus, babesiosis and anaplasmosis started in 1921, and in the 1970s, studies of the epidemiology of R. microplus determined that from 2 to 3.5 generations can be produced annually and that the country is in an area of enzootic instability for babesiosis and anaplasmosis. Knowledge of tick epidemiology and of tick resistance to different acaricides led to the development of efficient methods of control or eradication, including integrated control and generational treatment. Although research results have led to a legal framework regarding R. microplus control, these measures have had variable results. This can be attributed to several factors, such as the discontinuation of the control measures, variable financial resources, changes in the dynamics of livestock movement, failure to adopt available technology for tick control by farmers, climate change, environmental alterations such as forestation and the increasing resistance of ticks to acaricides, which led to the development of multiresistant ticks. This paper reviews the history of R. microplus, babesiosis and anaplasmosis in Uruguay and proposes alternatives for their control.
Assuntos
Anaplasmose/prevenção & controle , Babesiose/prevenção & controle , Rhipicephalus/parasitologia , Infestações por Carrapato/veterinária , Acaricidas , Anaplasmose/economia , Animais , Babesiose/economia , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/prevenção & controle , Mudança Climática , Resistência a Medicamentos , Rhipicephalus/efeitos dos fármacos , Rhipicephalus/microbiologia , Controle de Ácaros e Carrapatos , Infestações por Carrapato/epidemiologia , Infestações por Carrapato/prevenção & controle , UruguaiRESUMO
Abstract Vaccination against Anaplasma marginale has been considered an important control strategy for bovine anaplasmosis. Recently, mice immunized with rMSP1 a linked to carbon nanotubes (MWNT) showed significant immune responses, generating a new possibility for use of an inactivated vaccine. The objective of this study was to investigate the cellular and humoral responses in calves immunized with MWNT+rMSP1a , associated with inactivated vaccine of A. marginale produced in vitro, and evaluate the toxic effects of the MWNT on renal and hepatic function. rMSP1a was covalently linked to MWNT. Inactivated vaccine (AmUFMG2) was produced by cultivating A. marginale in IDE8 cells. Twenty-four Holstein calves were divided (four groups) and immunized subcutaneously with PBS and non-carboxylated MWNT (control, G1), AmUFMG2 (G2), MWNT+rMSP1a (G3), and AmUFMG2 with MWNT+rMSP1a (G4). Blood samples were collected for total leukocyte counts, biochemical profiling and evaluation of the cellular and humoral response. Immunization with MWNT+rMSP1a induced increase in the total number of leukocytes, NK cells, in the lymphocyte populations and higher levels of antibodies compared to calves immunized only with AmUFMG2. Furthermore, MWNT did not induce changes in the biochemical profile. These data indicate that MWNT+rMSP1a were able to induce the immune responses more efficiently than AmUFMG2 alone, without generating toxicity.
Resumo Vacinação contra Anaplasma marginale tem sido considerada uma importante estratégia de controle da anaplasmose bovina. Recentemente, camundongos imunizados com rMSP1a funcionalizada à nanotubos de carbono (MWNT) apresentaram resposta imune significante, gerando nova possibilidade para o uso da vacina inativada. O objetivo desse estudo foi investigar a resposta celular e humoral em bezerros imunizados com MWNT+rMSP1a, associado com a vacina inativada de A. marginale produzida in vitro, e avaliar os efeitos tóxicos dos MWNT nas funções hepática e renal. rMSP1 a foi ligada covalentemente aos MWNT. Vacina inativada (AmUFMG2) foi produzida através do cultivo de A. marginale em células IDE8. Vinte e quatro bezerros Holandeses foram divididos (quatro grupos) e imunizados subcutaneamente com: PBS e MWNT não-carboxilados (controle, G1), AmUFMG2 (G2), MWNT+rMSP1 a (G3), e AmUFMG2 com MWNT+rMSP1a (G4). Amostras de sangue foram coletadas para contagem de leucócitos, perfil bioquímico e avaliação da resposta celular e humoral. Imunização com MWNT+rMSP1a induziu aumento dos leucócitos totais, células NK, na população de linfócitos e altos níveis de anticorpos comparado com animais imunizados apenas com AmUFMG2. Além disso, MWNT não induziu alterações no perfil bioquímico. Esses dados indicam que MWNT+rMSP1a foram capazes de induzir eficientemente a resposta imune comparado com AmUFMG2 sozinho, sem gerar toxicidade.
Assuntos
Animais , Bovinos , Portadores de Fármacos , Vacinas Bacterianas/imunologia , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/prevenção & controle , Nanotubos de Carbono , Anaplasma marginale/imunologia , Imunogenicidade da Vacina , Anaplasmose/prevenção & controle , Imunidade Humoral , Imunidade CelularRESUMO
Vaccination against Anaplasma marginale has been considered an important control strategy for bovine anaplasmosis. Recently, mice immunized with rMSP1 a linked to carbon nanotubes (MWNT) showed significant immune responses, generating a new possibility for use of an inactivated vaccine. The objective of this study was to investigate the cellular and humoral responses in calves immunized with MWNT+rMSP1a , associated with inactivated vaccine of A. marginale produced in vitro, and evaluate the toxic effects of the MWNT on renal and hepatic function. rMSP1a was covalently linked to MWNT. Inactivated vaccine (AmUFMG2) was produced by cultivating A. marginale in IDE8 cells. Twenty-four Holstein calves were divided (four groups) and immunized subcutaneously with PBS and non-carboxylated MWNT (control, G1), AmUFMG2 (G2), MWNT+rMSP1a (G3), and AmUFMG2 with MWNT+rMSP1a (G4). Blood samples were collected for total leukocyte counts, biochemical profiling and evaluation of the cellular and humoral response. Immunization with MWNT+rMSP1a induced increase in the total number of leukocytes, NK cells, in the lymphocyte populations and higher levels of antibodies compared to calves immunized only with AmUFMG2. Furthermore, MWNT did not induce changes in the biochemical profile. These data indicate that MWNT+rMSP1a were able to induce the immune responses more efficiently than AmUFMG2 alone, without generating toxicity.
Assuntos
Anaplasma marginale/imunologia , Anaplasmose/prevenção & controle , Vacinas Bacterianas/imunologia , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/prevenção & controle , Portadores de Fármacos , Imunogenicidade da Vacina , Nanotubos de Carbono , Animais , Bovinos , Imunidade Celular , Imunidade HumoralRESUMO
Anaplasma marginale (A. marginale) has a remarkable impact on livestock production, and an effective vaccine is not currently available due to the inexistence of a small animal model. Recently, BALB/c mice were successfully infected with A. marginale, resulting in an acute and persistent anaplasmosis infection. Here, we designed a hybrid protein containing repeats of polypeptide 1a from major surface protein-1 complex (MSP1a) repeats and common epitopes of outer membrane proteins (OMPs) OMP7, OMP8 and OMP9 expressed in Escherichia coli. Our proof-of-concept assessed vaccinal effectiveness against a challenge with live bacteria. The MSP1a/OMP7/8/9 immunized BALB/C mice exhibited a strong reduction in rickettsemia and had no signs of anaplasmosis or hepatic lesions. In contrast, the non-immunized mice exhibited signs of anaplasmosis and a body weight loss associated with increases in monocyte and neutrophil counts. Furthermore, the non-immunized mice displayed atrophies with chronic inflammatory infiltrates in the spleen and increased binucleation and hydropic degeneration in the hepatocytes. Our findings demonstrated that immunization with our hybrid protein induced a strong reduction in rickettsemia and conferred protection against anaplasmosis. Therefore, given the strong evidence of the protective effect against anaplasmosis, hybrid protein designs are potential candidates for the rational design of vaccinal subunits.
Assuntos
Anaplasmose/prevenção & controle , Proteínas da Membrana Bacteriana Externa/imunologia , Epitopos/imunologia , Sequência de Aminoácidos , Anaplasma marginale/fisiologia , Anaplasmose/imunologia , Anaplasmose/microbiologia , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/prevenção & controle , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos BALB C , RatosRESUMO
In order to understand the genetic diversity of A. marginale, several efforts have been made around the world. This rickettsia affects a significant number of ruminants, causing bovine anaplasmosis, so the interest in its virulence and how it is transmitted have drawn interest not only from a molecular point of view but also, recently, some genomics research have been performed to elucidate genes and proteins with potential as antigens. Unfortunately, so far, we still do not have a recombinant anaplasmosis vaccine. In this review, we present a landscape of the multiple approaches carried out from the genomic perspective to generate valuable information that could be used in a holistic way to finally develop an anaplasmosis vaccine. These approaches include the analysis of the genetic diversity of A. marginale and how this affects control measures for the disease. Anaplasmosis vaccine development is also reviewed from the conventional vaccinomics to genome-base vaccinology approach based on proteomics, metabolomics, and transcriptomics analyses reported. The use of these new omics approaches will undoubtedly reveal new targets of interest in the near future, comprising information of potential antigens and the immunogenic effect of A. marginale proteins.
Assuntos
Anaplasma marginale , Anaplasmose , Vacinas Bacterianas , Doenças dos Bovinos , Variação Genética/imunologia , Genoma Bacteriano/imunologia , Anaplasma marginale/genética , Anaplasma marginale/imunologia , Anaplasmose/genética , Anaplasmose/imunologia , Anaplasmose/prevenção & controle , Animais , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/uso terapêutico , Bovinos , Doenças dos Bovinos/genética , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/prevenção & controleRESUMO
Anaplasma marginale is an intraerythrocytic vector-borne infectious agent of cattle. Immunization with the current vaccine, based on parasitized erythrocytes with live Anaplasma centrale, shows some constraints and confers partial protection, suggesting the feasibility for the development of new generation of vaccines. The aim of the present study was to assess the effect of sequential immunization of BALB/c mice, with herpesvirus amplicon vector-based vaccines combined with protein-based vaccines, on the quality of the immune response against the major surface protein 5 of A. marginale. The highest antibody titers against MSP5 were elicited in mice that received two doses of adjuvanted recombinant protein (p < 0.0001). Mice treated with a heterologous prime-boost strategy generated sustained antibody titers at least up to 200 days, and a higher specific cellular response. The results presented here showed that sequential immunization with HSV-based vectors and purified antigen enhances the quality of the immune response against A. marginale.
Assuntos
Anaplasma marginale/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Imunidade Inata , Anaplasma marginale/genética , Anaplasma marginale/metabolismo , Anaplasmose/prevenção & controle , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Vacinas Bacterianas/virologia , Bovinos , Doenças dos Bovinos/prevenção & controle , Linhagem Celular Tumoral , Chlorocebus aethiops , Vetores Genéticos/genética , Herpesvirus Humano 1/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células VeroRESUMO
Bovine anaplasmosis is a disease caused by the intraerythrocytic rickettsia Anaplasma marginale. Surface proteins (MSPs) of A. marginale are important in the interaction of the pathogen with the host and constitute potential vaccine targets against this pathogen. Currently, there is no commercial inactivated vaccine against bovine anaplasmosis that can generate a protective immune response that effectively prevents the development of clinical disease. The objective of this study was to evaluate the humoral and cellular immune responses of BALB/c mice immunized with the recombinant fragment of rMSP1a from A. marginale using carbon nanotubes as a carrier molecule. The fragment of rMSP1a comprising the N-terminal region of the protein was expressed in Escherichia coli BL21, purified by nickel affinity chromatography and covalently linked to multiwalled carbon nanotubes (MWNTs). After this functionalization, thirty BALB/c mice were divided into five groups, G1 (rMSP1a), G2 (MWNT+rMSP1a), G3 (MWNT), G4 (adjuvant) and G5 (unimmunized). The mice were immunized subcutaneously at days 0, 21 and 42. Blood samples were collected on day 11 after immunization. The spleens were collected, and the splenocytes were cultured for cell proliferation assays and cell immunophenotyping. Mice immunized with rMSP1a (G1 and G2) produced high levels of anti-rMSP1a IgG, demonstrating that the functionalization to carbon nanotubes did not interfere with protein immunogenicity. Immunization with MWNT+rMSP1a significantly induced higher percentages of CD4(+)CD44(+) and CD4(+)CD62L(+) lymphocytes, high levels of TNF-α, and a higher proliferative rate of splenocytes compared to mice immunized with rMSP1a alone (G1 group). Therefore, additional experiments using cattle should be performed to determine the efficacy, safety, immunogenicity and protection induced by rMSP1a associated with MWNT.
Assuntos
Anaplasmose/prevenção & controle , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Nanotubos de Carbono/química , Anaplasma marginale , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/química , Células Cultivadas , Citocinas/imunologia , Portadores de Fármacos/química , Epitopos/imunologia , Feminino , Imunidade Celular , Imunidade Humoral , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Baço/citologia , Baço/imunologia , Linfócitos T/imunologiaRESUMO
Bovine anaplasmosis is a hemoparasitic disease that causes considerable economic loss to the dairy and beef industries. Cattle immunized with the Anaplasma marginale MSP1 outer membrane protein complex presents a protective humoral immune response; however, its efficacy is variable. Immunodominant epitopes seem to be a key-limiting factor for the adaptive immunity. We have successfully demonstrated that critical motifs of the MSP1a functional epitope are essential for antibody recognition of infected animal sera, but its protective immunity is yet to be tested. We have evaluated two synthetic vaccine formulations against A. marginale, using epitope-based approach in mice. Mice infection with bovine anaplasmosis was demonstrated by qPCR analysis of erythrocytes after 15-day exposure. A proof-of-concept was obtained in this murine model, in which peptides conjugated to bovine serum albumin were used for immunization in three 15-day intervals by intraperitoneal injections before challenging with live bacteria. Blood samples were analyzed for the presence of specific IgG2a and IgG1 antibodies, as well as for the rickettsemia analysis. A panel containing the cytokines' transcriptional profile for innate and adaptive immune responses was carried out through qPCR. Immunized BALB/c mice challenged with A. marginale presented stable body weight, reduced number of infected erythrocytes, and no mortality; and among control groups mortality rates ranged from 15% to 29%. Additionally, vaccines have significantly induced higher IgG2a than IgG1 response, followed by increased expression of pro-inflammatory cytokines. This is a successful demonstration of epitope-based vaccines, and protection against anaplasmosis may be associated with elicitation of effector functions of humoral and cellular immune responses in murine model.
Assuntos
Anaplasma marginale/imunologia , Anaplasmose/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Epitopos/imunologia , Imunidade Celular , Imunidade Humoral , Motivos de Aminoácidos/imunologia , Anaplasmose/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/química , Bovinos , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Epitopos/genética , Eritrócitos/imunologia , Eritrócitos/virologia , Feminino , Imunização , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Mediadores da Inflamação/imunologia , Camundongos , Peptídeos/síntese química , Peptídeos/imunologia , Baço/citologia , Baço/imunologia , Transcrição GênicaRESUMO
The rickettsia Anaplasma marginale causes the haemolytic disease bovine anaplasmosis, an economic problem in tropical and subtropical areas worldwide. The closely related but less pathogenic Anaplasma centrale is commonly used as a live vaccine to prevent anaplasmosis, but it can only be produced from infected blood. UFMG1 is a low pathogenic Brazilian strain of A. marginale, which has been shown to protect cattle against a high pathogenic Brazilian isolate. As UFMG1 can be grown in tick cells, the strain was proposed as a possible cell culture-derived vaccine. We have evaluated whether UFMG1 could protect cattle against a geographically distant heterologous strain, using A. centrale vaccination as a standard for comparison. Trial calves were infected with UFMG1, A. centrale or PBS. UFMG1-infected animals were more symptomatic than those infected with A. centrale, but none required treatment. All calves were then challenged with the Israeli A. marginale Gonen strain (one of the most prevalent strain in Israel). The A. centrale group had the mildest symptoms, while UFMG1 and control groups both had a more severe response. Nevertheless, the challenge did not cause life-threatening disease in any group. Animals infected with A. centrale had a significantly higher IgG response than UFMG1, when measured in an ELISA against initial bodies from their homologous strain or Gonen. The level of cross-reactivity of the response to initial infection correlated significantly with reduced symptoms after challenge. In conclusion, UFMG1 had limited effect in preventing disease by the geographically distant heterologous Gonen strain. While the low pathogenicity of the Gonen strain in this trial makes it impossible to conclusively state that UFMG1 would have given no protective effect against more serious disease, the comparatively low IgG response to UFMG1 suggests it would not have been as effective as A. centrale.
Assuntos
Anaplasma marginale/imunologia , Anaplasmose/microbiologia , Anticorpos Antibacterianos/imunologia , Vacinas Bacterianas/administração & dosagem , Doenças dos Bovinos/microbiologia , Bovinos/microbiologia , Vacinação/métodos , Anaplasma marginale/genética , Anaplasma marginale/isolamento & purificação , Anaplasmose/imunologia , Anaplasmose/prevenção & controle , Animais , Formação de Anticorpos , Brasil , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Seguimentos , Carrapatos/microbiologia , Resultado do Tratamento , Vacinação/veterináriaRESUMO
The protective efficacy of an inactivated vaccine from Anaplasma marginale that was cultured in tick cells (IDE8) for use against bovine anaplasmosis was evaluated. Five calves (Group 1) were inoculated subcutaneously, at 21-day intervals, with three doses of vaccine containing 3 × 10(9) A. marginale initial bodies. Five control calves received saline solution alone (Group 2). Thirty-two days after the final inoculation, all the calves were challenged with approximately 3 × 10(5) erythrocytes infected with A. marginale high-virulence isolate (UFMG2). The Group 1 calves seroconverted 14 days after the second dose of vaccine. After the challenge, all the animals showed patent rickettsemia. There was no significant difference (p > 0.05) between the Group 1 and 2 calves during the incubation period, patency period or convalescence period. All the animals required treatment to prevent death. The results suggest that the inactivated vaccine from A. marginale produced in IDE8 induced seroconversion in calves, but was not effective for preventing anaplasmosis induced by the UFMG2 isolate under the conditions of this experiment.
Assuntos
Anaplasma marginale/imunologia , Anaplasmose/prevenção & controle , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/prevenção & controle , Carrapatos/microbiologia , Vacinas de Produtos Inativados/imunologia , Animais , Bovinos , Técnicas de Cultura de Células , MasculinoRESUMO
The protective efficacy of an inactivated vaccine from Anaplasma marginale that was cultured in tick cells (IDE8) for use against bovine anaplasmosis was evaluated. Five calves (Group 1) were inoculated subcutaneously, at 21-day intervals, with three doses of vaccine containing 3 × 10(9) A. marginale initial bodies. Five control calves received saline solution alone (Group 2). Thirty-two days after the final inoculation, all the calves were challenged with approximately 3 × 10(5) erythrocytes infected with A. marginale high-virulence isolate (UFMG2). The Group 1 calves seroconverted 14 days after the second dose of vaccine. After the challenge, all the animals showed patent rickettsemia. There was no significant difference (p > 0.05) between the Group 1 and 2 calves during the incubation period, patency period or convalescence period. All the animals required treatment to prevent death. The results suggest that the inactivated vaccine from A. marginale produced in IDE8 induced seroconversion in calves, but was not effective for preventing anaplasmosis induced by the UFMG2 isolate under the conditions of this experiment.(AU)
Foi avaliada a eficácia de uma vacina protetora para Anaplasma marginale cultivada em células de carrapato (IDE8) para uso contra a anaplasmose bovina. Cinco bezerros (Grupo 1) foram inoculados por via subcutânea com três doses, intervalados de 21 dias, de vacina contendo 3 × 10(9) corpúsculos iniciais de A. marginale inicial. Cinco bezerros do grupo controle receberam apenas solução salina (Grupo 2). Trinta e dois dias após a inoculação final, todos os bezerros foram desafiados com aproximadamente 3 × 10(5) eritrócitos infectados com isolado de A. marginale alta virulência (UFMG2). Os bezerros do Grupo 1 soroconverteram-se 14 dias após a segunda dose da vacina. Após o desafio, todos os animais mostraram riquestsemia patente. Não houve diferença significativa (p > 0,05) entre bezerros do Grupo 1 e 2 em período de incubação, período de patência, ou período de convalescença. Todos os animais necessitaram de tratamento para prevenir a morte. Os resultados sugerem que uma vacina inativada de A. marginale, produzida em IDE8, induz soroconversão em bezerros, mas não é eficaz na prevenção de anaplasmose induzida pelo isolado UFMG2 nas condições deste experimento.(AU)
Assuntos
Animais , Masculino , Bovinos , Anaplasma marginale/imunologia , Anaplasmose/prevenção & controle , /microbiologia , Doenças dos Bovinos/prevenção & controle , Carrapatos/microbiologia , Vacinas de Produtos Inativados/imunologia , Técnicas de Cultura de CélulasRESUMO
The protective efficacy of an inactivated vaccine from Anaplasma marginale that was cultured in tick cells (IDE8) for use against bovine anaplasmosis was evaluated. Five calves (Group 1) were inoculated subcutaneously, at 21-day intervals, with three doses of vaccine containing 3 × 10(9) A. marginale initial bodies. Five control calves received saline solution alone (Group 2). Thirty-two days after the final inoculation, all the calves were challenged with approximately 3 × 10(5) erythrocytes infected with A. marginale high-virulence isolate (UFMG2). The Group 1 calves seroconverted 14 days after the second dose of vaccine. After the challenge, all the animals showed patent rickettsemia. There was no significant difference (p > 0.05) between the Group 1 and 2 calves during the incubation period, patency period or convalescence period. All the animals required treatment to prevent death. The results suggest that the inactivated vaccine from A. marginale produced in IDE8 induced seroconversion in calves, but was not effective for preventing anaplasmosis induced by the UFMG2 isolate under the conditions of this experiment.
Foi avaliada a eficácia de uma vacina protetora para Anaplasma marginale cultivada em células de carrapato (IDE8) para uso contra a anaplasmose bovina. Cinco bezerros (Grupo 1) foram inoculados por via subcutânea com três doses, intervalados de 21 dias, de vacina contendo 3 × 10(9) corpúsculos iniciais de A. marginale inicial. Cinco bezerros do grupo controle receberam apenas solução salina (Grupo 2). Trinta e dois dias após a inoculação final, todos os bezerros foram desafiados com aproximadamente 3 × 10(5) eritrócitos infectados com isolado de A. marginale alta virulência (UFMG2). Os bezerros do Grupo 1 soroconverteram-se 14 dias após a segunda dose da vacina. Após o desafio, todos os animais mostraram riquestsemia patente. Não houve diferença significativa (p > 0,05) entre bezerros do Grupo 1 e 2 em período de incubação, período de patência, ou período de convalescença. Todos os animais necessitaram de tratamento para prevenir a morte. Os resultados sugerem que uma vacina inativada de A. marginale, produzida em IDE8, induz soroconversão em bezerros, mas não é eficaz na prevenção de anaplasmose induzida pelo isolado UFMG2 nas condições deste experimento.
Assuntos
Animais , Bovinos , Masculino , Anaplasma marginale/imunologia , Anaplasmose/prevenção & controle , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/prevenção & controle , Carrapatos/microbiologia , Vacinas de Produtos Inativados/imunologia , Técnicas de Cultura de CélulasRESUMO
This study investigated whether a low pathogenicity isolate of Anaplasma marginale with an appendage (UFMG1) could protect calves from infection with a pathogenic A. marginale isolate (UFMG2). Two groups of five Friesian calves were each inoculated with UFMG1 by intravenous injections of either A. marginale-infected tick cell cultures (group 1) or blood stabilates (group 2); a third (control) group was injected with saline. All animals were inoculated with a blood stabilate containing a high pathogenicity A. marginale isolate (UFMG2) 75 days after the UFMG1 inoculation. After infection with UFMG2, animals in groups 1 and 2 presented low rickettsaemia, but no clinical signs and no reduction in packed cell volume (PCV). Control animals became sick, with high rickettsaemia (16% infected erythrocytes) and a reduction in PCV (71%), resulting in 60% deaths. Up to 2 weeks after the UFMG2 inoculation, msp1α UFMG1 sequences were detected in groups 1 and 2. Four weeks after UFMG2 inoculation, UFMG2 sequences were detected in these animals, along with a new msp1α genotype sequence, closely related to that of the UFMG2 isolate. Control animals had UFMG2 msp1α sequences up to 4weeks after inoculation with UFMG2 and the new msp1α genotype sequence could be detected on the sixth week. The origin of the new A. marginale genotype was unknown, but may represent the first example of MSP1a antigenic variation in infected cattle. The results confirmed the low pathogenicity of the UFMG1 isolate, which provided clinical protection against the highly pathogenic A. marginale UFMG2. Infection with UFMG1 did not prevent the establishment of a second isolate, suggesting protection without infection-exclusion among A. marginale isolates.
Assuntos
Anaplasma marginale/patogenicidade , Anaplasmose/microbiologia , Doenças dos Bovinos/microbiologia , Anaplasma marginale/genética , Anaplasma marginale/imunologia , Anaplasmose/prevenção & controle , Animais , Brasil , Bovinos , Doenças dos Bovinos/prevenção & controle , Eritrócitos/imunologia , Eritrócitos/microbiologia , Genótipo , Masculino , Distribuição AleatóriaRESUMO
The outer membrane proteins of Anaplasma marginale have been the focus of research to obtain an improved vaccine against bovine anaplasmosis. We evaluated the capacity of the recombinant plasmids pcDNA-msp1alpha, pcDNA-msp1beta, and pcDNA-mp5 to express MSP1a, MSP1b, and MSP5 proteins, and to determine the immunogenicity of BALB/c mice immunized with these plasmids individually or in association. Expression of proteins was confirmed in Vero cells by IFA. The combination of recombinant plasmids showed high antibodies response, produced better induction of Th1 response than individual plasmids, and induced significant proliferation of splenocytes. The mice sera immunized with A. marginale showed seroconversion and reacted with all native MSPs, but demonstrated predominance of the humoral IgG1 isotype and did not induce significant proliferation of splenocytes. The use of association of recombinant plasmid can be an effective strategy for the immunoprophylaxis of anaplasmosis.
Assuntos
Anaplasma marginale/imunologia , Anaplasmose/prevenção & controle , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas de DNA/imunologia , Anaplasma marginale/genética , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Bovinos , Doenças dos Bovinos/prevenção & controle , Proliferação de Células , Feminino , Imunoglobulina G/sangue , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Baço/imunologia , Vacinas de DNA/genéticaRESUMO
Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.