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
Detection of antibodies to Anaplasma spp. using commercial competitive enzyme-linked immunosorbent assay (ccELISA) is based on the recombinant major surface protein 5 fused to maltose binding protein (MBP-MSP5) or glutathione S-transferase (GST-MSP5). To avoid false positive reactions due to the presence of antibodies against E. coli MBP in cattle, previous sera absorption is required. This study evaluated the replacement of MBP-MSP5 or GST-MSP5 antigens by the truncate MSP5 (residues 28-210) of A. marginale (tMSP5m), A. centrale (tMSP5c) and fusion protein MSP5 (tMSP5cm), expressed without N-terminus transmembrane helix in the ccELISA test. Immunoreactivity was evaluated by western blot using monoclonal antibodies against the tMSP5 and by in-house cELISA (hcELISA) with purified tMSP5m, tMSP5c or tMSP5cm using sera from cattle infected with A. marginale (n = 226) or vaccinated with A. centrale (n = 173) and uninfected cattle (n = 216). Results of hcELISA were compared with those of ccELISA. Recombinant protein was expressed highly soluble (> 95%) in E. coli without a molecular chaperone. Specificity of the hcELISA-tMSP5m, -MSP5c or -tMSP5cm was identical to (99.5%) and greater than that in ccELISA (96.3%). Sensitivity of hcELISA-tMSP5m and ccELISA was identical (95.5%), but lower than that of hcELISA-tMSP5cm (96.2%) and -tMSP5c (97.2%). The analysis of vaccinated cattle by hcELISA-tMSP5c showed sensitivity of 99.4%. In summary, the generation of fusion MSP5 A. marginale-A. centrale protein without transmembrane helix was a very effective method to express the recombinant protein highly soluble in the bacterial cytoplasm and contributed to an increased test performance for detecting antibodies in cattle naturally infected with A. marginale or vaccinated with A. centrale.
Assuntos
Anaplasma centrale , Anaplasma marginale , Anaplasmose , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/química , Doenças dos Bovinos , Anaplasmose/sangue , Anaplasmose/diagnóstico , Animais , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Recombinantes de Fusão/químicaRESUMO
A anaplasmose bovina é uma das principais causas de perdas produtivas e mortes no Rio Grande do Sul em rebanhos bovinos. O Anaplasma marginale é o principal agente causador da enfermidade e provoca hipertermia, anemia, prostração, abortos e perdas produtivas nos bovinos acometidos. Tendo em vista o controle deste hemoparasita em uma propriedade leiteira localizada no município de Eldorado do Sul no Rio Grande do Sul, na qual a incidência da doença era alta, 471 animais foram imunizados com Anaplasma centrale na busca de desenvolvimento cruzado para Anaplasma marginale. No experimento foi verificado que a incidência que normalmente era acima de 30% na propriedade passou para níveis inferiores a 5%. No entanto, foram verificados abortos decorrentes da imunização, principalmente nos animais que possuíam menos de 90 dias de prenhes. Já o número de mortes globais na fazenda caiu consideravelmente tendo em vista que a principal causa de morte era a anaplasmose bovina. Dos animais inoculados com A. centrale em torno de 15% apresentaram sintomatologia clínica da enfermidade e precisaram ser tratados com oxitetraciclina no período entre 15 e 30 dias após a imunização. O custo com tratamento empregado na propriedade posterior à imunização caiu em torno de 85% o que provocou impacto significativo economicamente na propriedade.(AU)
Bovine anaplasmosis is a major cause of production losses and deaths in Rio Grande do Sul cattle herds. Anaplasma sp. is the main causative agent of cattle disease. It causes hyperthermia, anemia, prostration, abortions and reduces milk production in affected animals. In order to control this hemoparasite on a dairy farm located in the municipality of Eldorado do Sul in Rio Grande do Sul, where the disease incidence was high, 471 animals were immunized with Anaplasma centrale in the search for cross-protectiv immunity for Anaplasma marginale. The property anaplasmosis incidence, which usually was above 30%, became 5% after the immunization. However, abortions were observed resulting from innoculaition, especially in animals that had less than 90 days of pregnancy. The global number of deaths on the farm dropped considerably given that the main cause of death was the bovine anaplasmosis. 15% of animals inoculated with A. centrale showed clinical symptoms of the disease between 15 and 30 days after immunization and had to be treated with oxytetracycline. The amount of money spent with anaplasmosis treatment decay 85% after the immunization, which caused significant economic impact on the property.(AU)
Assuntos
Animais , Bovinos , Anaplasma centrale/isolamento & purificação , Anaplasmose/terapiaRESUMO
A anaplasmose bovina é uma das principais causas de perdas produtivas e mortes no Rio Grande do Sul em rebanhos bovinos. O Anaplasma marginale é o principal agente causador da enfermidade e provoca hipertermia, anemia, prostração, abortos e perdas produtivas nos bovinos acometidos. Tendo em vista o controle deste hemoparasita em uma propriedade leiteira localizada no município de Eldorado do Sul no Rio Grande do Sul, na qual a incidência da doença era alta, 471 animais foram imunizados com Anaplasma centrale na busca de desenvolvimento cruzado para Anaplasma marginale. No experimento foi verificado que a incidência que normalmente era acima de 30% na propriedade passou para níveis inferiores a 5%. No entanto, foram verificados abortos decorrentes da imunização, principalmente nos animais que possuíam menos de 90 dias de prenhes. Já o número de mortes globais na fazenda caiu consideravelmente tendo em vista que a principal causa de morte era a anaplasmose bovina. Dos animais inoculados com A. centrale em torno de 15% apresentaram sintomatologia clínica da enfermidade e precisaram ser tratados com oxitetraciclina no período entre 15 e 30 dias após a imunização. O custo com tratamento empregado na propriedade posterior à imunização caiu em torno de 85% o que provocou impacto significativo economicamente na propriedade.(AU)
Bovine anaplasmosis is a major cause of production losses and deaths in Rio Grande do Sul cattle herds. Anaplasma sp. is the main causative agent of cattle disease. It causes hyperthermia, anemia, prostration, abortions and reduces milk production in affected animals. In order to control this hemoparasite on a dairy farm located in the municipality of Eldorado do Sul in Rio Grande do Sul, where the disease incidence was high, 471 animals were immunized with Anaplasma centrale in the search for cross-protectiv immunity for Anaplasma marginale. The property anaplasmosis incidence, which usually was above 30%, became 5% after the immunization. However, abortions were observed resulting from innoculaition, especially in animals that had less than 90 days of pregnancy. The global number of deaths on the farm dropped considerably given that the main cause of death was the bovine anaplasmosis. 15% of animals inoculated with A. centrale showed clinical symptoms of the disease between 15 and 30 days after immunization and had to be treated with oxytetracycline. The amount of money spent with anaplasmosis treatment decay 85% after the immunization, which caused significant economic impact on the property.(AU)
Assuntos
Animais , Bovinos , Anaplasma centrale/isolamento & purificação , Anaplasmose/terapiaRESUMO
Background: Anaplasma marginale ssp. centrale (A. centrale) exhibits low pathogenicity and therefore is used as a live vaccine against bovine anaplasmosis in several countries. During production of the vaccine, accidental contamination with Anaplasma marginale (A. marginale) is a risk that can jeopardize the entire batch of vaccine. Due to limitation of microscopic examination to detect low levels of parasitaemia, the present study aims to standardize a polymerase chain reaction assay using primers for the msp4 gene of Anaplasma sp. for detecting and differentiating with greater sensitivity and specificity the species of A. centrale and A. marginale in blood samples from experimentally infected cattle.Materials, Methods & Results: The DNA extraction was performed from frozen blood. Erythrocytes infected with known A. centrale, A. marginale served as positive control and the erytrocytes infected with Babesia bovis and Babesia bigemina served as the negative control polymerase chain reaction. PCR was standardized from annealing temperature variations of the primers, magnesium chloride concentration, amounts concentration of primers and DNA concentration of rickettsiae. By PCR method, it was analyzed the DNA from blood samples of 13 cattle positive to A. marginale by microscopic examination from smear stained with Giemsa. The PCR assay was specific for A. centrale and A. marginale, presented 100% identity without presenting cross-reactivity with other bovine hemoparasites. The detection limits of the PCR were 0.25 pg and 0.125 pg of DNA for detection of A. centrale and A. marginale DNA respectively.Discussion: A. marginale is an obligate intracellular bacterium that infects bovine erythrocytes causing extravascular hemolysis and anemia being considered the main agent of bovine anaplasmosis.[...](AU)
Assuntos
Anaplasma marginale/isolamento & purificação , Anaplasma centrale/isolamento & purificação , Fatores de Virulência/genética , Reação em Cadeia da Polimerase/métodosRESUMO
Background: Anaplasma marginale ssp. centrale (A. centrale) exhibits low pathogenicity and therefore is used as a live vaccine against bovine anaplasmosis in several countries. During production of the vaccine, accidental contamination with Anaplasma marginale (A. marginale) is a risk that can jeopardize the entire batch of vaccine. Due to limitation of microscopic examination to detect low levels of parasitaemia, the present study aims to standardize a polymerase chain reaction assay using primers for the msp4 gene of Anaplasma sp. for detecting and differentiating with greater sensitivity and specificity the species of A. centrale and A. marginale in blood samples from experimentally infected cattle.Materials, Methods & Results: The DNA extraction was performed from frozen blood. Erythrocytes infected with known A. centrale, A. marginale served as positive control and the erytrocytes infected with Babesia bovis and Babesia bigemina served as the negative control polymerase chain reaction. PCR was standardized from annealing temperature variations of the primers, magnesium chloride concentration, amounts concentration of primers and DNA concentration of rickettsiae. By PCR method, it was analyzed the DNA from blood samples of 13 cattle positive to A. marginale by microscopic examination from smear stained with Giemsa. The PCR assay was specific for A. centrale and A. marginale, presented 100% identity without presenting cross-reactivity with other bovine hemoparasites. The detection limits of the PCR were 0.25 pg and 0.125 pg of DNA for detection of A. centrale and A. marginale DNA respectively.Discussion: A. marginale is an obligate intracellular bacterium that infects bovine erythrocytes causing extravascular hemolysis and anemia being considered the main agent of bovine anaplasmosis.[...]