RESUMO
Babesia bovis and Theileria annulata are tick-borne hemoprotozoans that impact bovine health and are responsible for considerable fatalities in tropical and subtropical regions around the world. Both pathogens infect the same vertebrate host, are closely related, and contain similar-sized genomes; however, they differ in invertebrate host specificity, absence vs. presence of a schizont stage, erythrocyte invasion mechanism, and transovarial vs. transstadial transmission. Phylogenetic analysis and bidirectional best hit (BBH) identified a similar number of aspartic, metallo, and threonine proteinases and nonproteinase homologs. In contrast, a considerably increased number of S54 serine rhomboid proteinases and S9 nonproteinase homologs were identified in B. bovis, whereas C1A cysteine proteinases and A1 aspartic nonproteinase homologs were found to be expanded in T. annulata. Furthermore, a single proteinase of families S8 (subtilisin-like protein) and C12 (ubiquitin carboxyl-terminal hydrolase), as well as four nonproteinase homologs, one with dual domains M23-M23 and three with S9-S9, were exclusively present in B. bovis. Finally, a pronounced difference in species-specific ancillary domains was observed between both species. We hypothesize that the observed degradome differences represent functional correlates of the dissimilar life history features of B. bovis and T. annulata. The presented improved classification of piroplasmid proteinases will facilitate an informed choice for future in-depth functional studies.
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Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host−pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen.
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Babesia bovis and B. bigemina are tick-transmitted parasites causing bovine babesiosis, characterized by significant morbidity and mortality leading to economic losses to the livestock industry in tropical and subtropical regions worldwide. Animals that recover from acute infection remain carriers with low parasitemia acting as a source of transmission, and often escape detection. An improved diagnosis of a B. bovis and/or B. bigemina infection of carrier animals is enabled by the availability of detection methods with high sensitivity. To this end, two nested PCR assays targeting the cytochrome b (cytb) genes of B. bovis and B. bigemina (cytb-nPCR), have been recently developed and an increased sensitivity with respect to reference protocols has been shown (Romero-Salas et al., 2016). In this study, the specificity against a panel of hemoparasites that potentially co-occur with B. bovis and B. bigemina was demonstrated to ensure applicability of the cytb-nPCR assays in a wide range of regions where bovine babesiosis is endemic. Furthermore, we compared both reported cytb-nPCR assays with reference nPCR and qPCR protocols for (i) their capability to detect carrier animals in the field, and (ii) their reproducibility when performed in different laboratories by independent operators. We show that, in a panel of bovine field samples (n = 100), the cytb-nPCR assays detected a considerably higher number of 25% B. bovis and 61% B. bigemina-positive animals compared to 7% and 20% B. bovis and 55% and 49% B. bigemina-positive animals when tested by reference nPCR and qPCR protocols, respectively. Cytb-nPCRs were also found superior in the detection of carrier animals when field samples from Africa were analyzed. In addition, both the B. bovis and B. bigemina cytb-nPCR assays were independently validated in a single blinded study in three laboratories. Importantly, no significant differences in the number/percentage of infected animals was observed using cytb-nPCR assays. In summary, the cytb-nPCR assays detected a considerably higher number of chronically infected B. bovis and B. bigemina carrier animals compared to reference nPCR and qPCR protocols, when applied in different epidemiological field situations. Furthermore, a high reproducibility between laboratories could be demonstrated.
Assuntos
Babesia bovis , Babesia , Babesiose , Doenças dos Bovinos , Carrapatos , Animais , Babesia/genética , Babesia bovis/genética , Babesiose/parasitologia , Bovinos , Doenças dos Bovinos/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reprodutibilidade dos Testes , Carrapatos/genéticaRESUMO
The indirect fluorescent antibody test (IFAT) is the most frequently used test to conduct seroepidemiological studies so far, and it is regarded as the "gold standard" test for the serological diagnosis of bovine babesiosis. The aim of the present study was to compare the enzyme-linked immunosorbent assay (ELISA) and the rapid immunochromatography test (ICT) for use in the serological diagnosis of cattle exposed to B. bovis in Mexico. The evaluation of test performance was carried out with 30 positive and 30 negative reference sera. A total of 72 bovine sera samples collected from cattle in a region with endemic bovine babesiosis were analyzed by ELISA and ICT, and the results were compared with those of IFAT. Kappa value (k) was also calculated to determine the agreement between tests. The sensitivity and specificity of ELISA for detecting antibodies against B. bovis were 87% (26/30) and 80% (24/30), respectively. The sensitivity and specificity of ICT for detecting antibodies against B. bovis were 90% (27/30) and 83.3% (25/30), respectively. The overall concordance determined for ELISA and ICT was 94.4% (68/72) and 98.6% (71/72), respectively, when the results were compared with those of IFAT. ICT was more sensitive and specific in this comparative study, showing good strength of agreement (k = 0.79) with respect to IFAT. ICT combines a strip-based assay system that is fast, practical, and sensitive for detection of antibodies to B. bovis, which suggests that it could be applied in the field without requiring any laboratory equipment for its use and interpretation of test results.
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Cattle fever ticks, Rhipicephalus microplus and R. annulatus have been eradicated from the United States and inspectors from the U.S. Department of Agriculture (USDA), Animal Plant Health Inspection Service (APHIS), Cattle Fever Tick Eradication Program (CFTEP) monitor the quarantine zone along the Texas border to prevent the introduction of livestock carrying cattle fever ticks from Mexico. Stray livestock apprehended by CFTEP in the zone are checked for ticks and tested for infectious disease-causing pathogens but are not evaluated for evidence of infection with tick-borne pathogens. We tested blood samples collected from stray cattle by CFTEP inspectors for evidence of infection with tick-borne pathogens. As a comparison group representing U.S. resident cattle, we tested blood samples that had been sent to the Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL) for unrelated testing. Both sets of blood samples were evaluated using the same specific and broad-spectrum PCR assays. For the border cattle the overall prevalence of infection with one or more tick-borne pathogen was 58.5 % (79/135) with many co-infections, including 30 cattle positive for Babesia bovis and/or Babesia bigemina (22.2 %) and 77 cattle positive for Anaplasma marginale (57 %), three of these animals were also positive for Borrelia theileri. No resident cattle represented by the TVMDL samples were infected with either of the Babesia spp., or with Borrelia theileri, but three were positive for Theileria orientalis and 7.3 % (7/96) were positive for A. marginale. These data show that cattle originating in Mexico have a higher prevalence of infection with tick-borne pathogens relative to resident U.S. cattle and specifically, a proportion are infected with bovine Babesia, which is absent from U.S. cattle populations. Consequently, these stray cattle may be a reservoir of tick-borne pathogens and if populations of Boophilus ticks become reestablished in areas where they had previously been eradicated, could pose a significant risk to the U.S. Cattle industry.
Assuntos
Anaplasmose/epidemiologia , Babesiose , Doenças dos Bovinos/epidemiologia , Coccidiose/veterinária , Doenças Transmitidas por Carrapatos/epidemiologia , Anaplasma/isolamento & purificação , Anaplasma marginale/isolamento & purificação , Animais , Vetores Aracnídeos/microbiologia , Vetores Aracnídeos/parasitologia , Babesia/isolamento & purificação , Babesiose/epidemiologia , Borrelia/isolamento & purificação , Bovinos , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/parasitologia , Coccidiose/epidemiologia , Reservatórios de Doenças/microbiologia , Reservatórios de Doenças/parasitologia , Vetores de Doenças , México , Reação em Cadeia da Polimerase/veterinária , Infecções Protozoárias em Animais/epidemiologia , Rhipicephalus/microbiologia , Rhipicephalus/parasitologia , Texas , Theileria/isolamento & purificação , Theileriose/epidemiologiaRESUMO
Surface proteins bound to the cell membrane by glycosylphosphatidylinositol (GPI) anchors are considered essential for the survival of pathogenic protozoans. In the case of the tick-transmitted hemoparasite Babesia bovis, the most virulent causative agent of bovine babesiosis, the GPI-anchored proteome was recently unraveled by an in silico approach. In this work, one of the identified proteins, GASA-1 (GPI-Anchored Surface Antigen-1), was thoroughly characterized. GASA-1 is 179 aa long and has the characteristic features of a GPI-anchored protein, including a signal peptide, a hydrophilic core and a hydrophobic tail that harbors a GPI anchor signal. Transcriptomic analysis shows that it is expressed in pathogenic and attenuated B. bovis strains. Notably, the gasa-1 gene has syntenic counterparts in B. bigemina and B. ovata, which also encode GPI-anchored proteins. This is highly unusual since all piroplasmid GPI-anchored proteins described so far have been found to be species-specific. Sequencing of gasa-1 alleles from B. bovis geographical isolates originating from Argentina, USA, Brazil, Mexico and Australia showed over 98 % identity in both nucleotide and amino acid sequences. A recombinant form of GASA-1 (rGASA-1) was generated in E. coli and anti-rGASA-1 antibodies were raised in mice. Fixed and live immunofluorescence assays showed that GASA-1 is expressed in in vitro cultured B. bovis merozoites and surface-exposed. Moreover, incubation of B. bovis in vitro cultures with anti-GASA-1 antibodies partially, but significantly, reduced erythrocyte invasion, indicating that this protein bears neutralization-sensitive antibody epitopes. Splenocytes of rGASA-1-inoculated mice showed a specific proliferative response when exposed to the recombinant protein, indicating that GASA-1 bears T-cell epitopes. Finally, sera from a group of B. bovis-infected cattle reacted with the recombinant protein, demonstrating that GASA-1 is expressed during natural infection of bovines with B. bovis, and suggesting that it is immunodominant. The high degree of conservation among B. bovis isolates and the presence of syntenic genes in other Babesia species suggest a relevant role of GASA-1 and GASA-1-like proteins for parasite survival, especially considering that, due to their surface location, they are exposed to the selection pressure of the host immune system. The highlighted features of GASA-1 make it an interesting candidate for the development of vaccines against bovine babesiosis.
RESUMO
Babesia bovis and Babesia bigemina are tick-transmitted piroplasms that cause severe damage to the livestock industry in tropical regions of the world. Recent studies demonstrated differences in infection levels of these haemoparasites among bovine breeds and variation between individual cows regarding resistance to these diseases. This study aimed to estimate the repeatability and correlations between B. bovis and B. bigemina using two cattle breeding systems, an individual system (IS) and a collective paddock system (CPS). All animals were Holstein breed, and the levels of B. bovis and B. bigemina in blood samples were estimated by quantitative polymerase chain reaction (qPCR). The estimated correlations for the B. bigemina and B. bovis DNA copy number for IS and CPS were moderate and high, respectively, whereas repeatability estimates for both systems and both Babesia species were moderate. Although we cannot infer that the type of rearing system directly influenced the correlation and repeatability coefficients, it appears that the bovine parasitemia burden may be dependent on (or determine) the parasitemia burden on ticks because the bovines remained in the same place for a longer time in both systems. Thus, the babesiosis infection levels of the ticks may have been uniform, a phenomenon that also ensures greater uniformity in cattle infection. This factor may have favored the occurrence of infected ticks leading to higher repeatability estimates and correlations. Our study confirms high variability in resistance/susceptibility between breeds, and the high correlations found may be linked to this characteristic and the most intensive breeding type of dairy cattle. Besides, under the present study conditions, the estimated correlations suggest that measuring an infection level of one Babesia species can predict the level of infection of the other.
Assuntos
Babesia bovis , Babesia , Babesiose/epidemiologia , Doenças dos Bovinos , Bovinos/parasitologia , Animais , Babesia/isolamento & purificação , Babesia bovis/isolamento & purificação , Cruzamento , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/parasitologia , DNA de Protozoário/isolamento & purificação , Indústria de Laticínios , ParasitemiaRESUMO
The apical membrane antigen 1 (AMA-1) is a protein of the micronemes that is present in all organisms of the phylum Apicomplexa; it has been shown that AMA-1 plays an essential role for parasite invasion to target cells. It has been reported that AMA-1 is conserved among different isolates of Babesia; however, it is unknown whether the protein contains conserved B-cell epitopes and whether these epitopes are recognized by antibodies from cattle in endemic areas. In this research, using an in silico analysis, four peptides were designed containing exposed and conserved linear B-cell epitopes from the extracellular region of Babesia bovis AMA-1. The selected peptides were chemically synthesized, and then each peptide was emulsified and used to immunize two bovines per peptide. The antibodies produced against these peptides were able to recognize intra-erythrocytic parasites in an IFAT, except peptide 4, which was insoluble. The synthetic peptides were covalently fixed to the wells of an ELISA plate and incubated with sera from B. bovis naturally infected cattle. Peptides P2AMA and P3AMA were recognized by the sera of naturally infected cattle from different regions of Mexico. Statistical analysis showed that the ELISA test for peptides P2AMA and P3AMA had a concordance of 91.2% and 61.1% compared to the IFAT, a sensitivity of 94.56% and 71.74%, and a specificity of 76.19% and 14.2%, respectively. The presence of antibodies in bovine sera from endemic areas that bind to the identified peptides indicates that AMA-1 from B. bovis has conserved B-cell epitopes involved in the immune response under natural conditions. However, to propose their use as vaccine or diagnostics candidates, a further characterization of the humoral immune response elicited in cattle by these peptides is needed.
Assuntos
Babesia bovis/imunologia , Babesiose/imunologia , Doenças dos Bovinos/imunologia , Epitopos de Linfócito B/imunologia , Proteínas de Membrana/imunologia , Peptídeos/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Bovinos , Simulação por Computador , Ensaio de Imunoadsorção Enzimática/veterinária , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Imunidade Humoral , Imunização/veterinária , México , Vacinação/veterináriaRESUMO
Bovine babesiosis is a tick-transmitted haemoparasitic disease caused by Babesia bovis and B. bigemina affecting cattle of tropical and subtropical regions around the world. Pathogens are transmitted by the tick vector Rhipicephalus microplus displaying a widespread distribution in northeastern Argentina. The disease is characterized by significant animal morbidity and mortality resulting in considerable economic loss. In this study, B. bovis and B. bigemina infection was investigated in a cattle herd of 150 adult bovines of pure Braford breed raised in a tick-hyperendemic field using molecular and serum antibody tests. A highly sensitive nested polymerase chain reaction (nPCR) assay targeting a species-specific region of the apocytochrome b gene resulted in direct B. bovis and B. bigemina detection in 27.3% and 54.7% of bovines, respectively. A recently developed immunochromatographic strip test (ICT) based on recombinant forms of spherical body protein 4 and the C-terminal region of rhoptry-associated protein 1 showed that 71.3% and 89.3% of bovines were seropositive for B. bovis and B. bigemina, respectively. The mixed infection rate as observed by direct (19.3%) and indirect detection (65.3%) coincided with those expected, respectively. Importantly, four months after sampling, nine bovines of the studied herd showed clinical signs of bovine babesiosis of which six animals eventually died. Microscopic detection of infected erythrocytes in Giemsa-stained blood smears confirmed B. bovis infection. Our study demonstrates that although animals showed a relatively high and very high rate of immunity against infection with B. bovis (71.3%) and B. bigemina (89.3%) parasites, respectively, clinical cases and fatalities due to the infection with B. bovis were observed. It is proposed that the most adequate control measure in the studied epidemiological situation is to vaccinate animals to prevent losses and/or an outbreak of bovine babesiosis.
Assuntos
Babesia/isolamento & purificação , Babesiose/epidemiologia , Doenças dos Bovinos/epidemiologia , Rhipicephalus/parasitologia , Animais , Argentina/epidemiologia , Babesia/genética , Babesia/imunologia , Babesia bovis/genética , Babesia bovis/imunologia , Babesia bovis/isolamento & purificação , Babesiose/parasitologia , Bovinos , Doenças dos Bovinos/parasitologia , Cromatografia de Afinidade/veterinária , Feminino , Masculino , Reação em Cadeia da Polimerase/veterinária , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
Abstract Serum and DNA samples from 15 naturally infected calves in Seropédica, Brazil, were obtained quarterly from birth to 12 months of age, in order to longitudinally evaluate their humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis. Anti-B. bovis IgG antibodies were detected by an indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Using DNA amplification, sequencing and phylogenetic analysis, the genetic diversity of B. bovis was assessed based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 0, 3 and 5 sequences of the msa-1, msa-2b and msa-2c genes were obtained, respectively. The present study demonstrated that the msa-2b and msa-2c gene sequences amplified from blood DNA of B. bovis-positive calves were genetically diversified. These data emphasize the importance of conducting deeper studies on the genetic diversity of B. bovis in Brazil, in order to design diagnostic antigens and vaccines in the future.
Resumo Para avaliar longitudinalmente a resposta imune humoral anti-B. bovis e a diversidade genética de antígenos de superfície de merozoítos de B. bovis, entre bezerros naturalmente infectados em Seropédica, Brasil, amostras de soro e DNA de 15 bezerros foram obtidas trimestralmente, desde o nascimento até 12 meses de idade. Anticorpos IgG para B. bovis foram detectados pelos testes de Imunofluorescência Indireta e Ensaio de Imunoadsorção Enzimático Indireto. Usando-se amplificação de DNA, sequenciamento e análises filogenéticas, a diversidade genética de B. bovis, com base nos genes que codificam antígenos de superfície de merozoítos (MSA-1, MSA-2b e MSA-2c) foi investigada. Os resultados da sorologia demonstraram que, até os seis meses de idade, todos os bezerros desenvolveram imunidade ativa contra B. bovis. Entre as 75 amostras de DNA avaliadas, foram obtidas 0, 3 e 5 sequências dos genes msa-1, msa-2b e msa-2c. O presente estudo demonstrou que sequências dos genes msa-2b e msa-2c amplificadas a partir de amostras de sangue positivas para B. bovis de bezerros de Seropédica, foram geneticamente distintas. O presente trabalho realça a importância de se realizar estudos aprofundados sobre a diversidade genética de B. bovis no Brasil, objetivando o desenvolvimento de antígenos para o diagnóstico e vacinas no futuro.
Assuntos
Animais , Babesiose/parasitologia , Babesiose/transmissão , Doenças dos Bovinos/parasitologia , Doenças dos Bovinos/transmissão , Babesia bovis/genética , Babesia bovis/imunologia , Filogenia , Variação Genética , Brasil , BovinosRESUMO
Serum and DNA samples from 15 naturally infected calves in Seropédica, Brazil, were obtained quarterly from birth to 12 months of age, in order to longitudinally evaluate their humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis. Anti-B. bovis IgG antibodies were detected by an indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Using DNA amplification, sequencing and phylogenetic analysis, the genetic diversity of B. bovis was assessed based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 0, 3 and 5 sequences of the msa-1, msa-2b and msa-2c genes were obtained, respectively. The present study demonstrated that the msa-2b and msa-2c gene sequences amplified from blood DNA of B. bovis-positive calves were genetically diversified. These data emphasize the importance of conducting deeper studies on the genetic diversity of B. bovis in Brazil, in order to design diagnostic antigens and vaccines in the future.(AU)
Para avaliar longitudinalmente a resposta imune humoral anti-B. bovis e a diversidade genética de antígenos de superfície de merozoítos de B. bovis, entre bezerros naturalmente infectados em Seropédica, Brasil, amostras de soro e DNA de 15 bezerros foram obtidas trimestralmente, desde o nascimento até 12 meses de idade. Anticorpos IgG para B. bovis foram detectados pelos testes de Imunofluorescência Indireta e Ensaio de Imunoadsorção Enzimático Indireto. Usando-se amplificação de DNA, sequenciamento e análises filogenéticas, a diversidade genética de B. bovis, com base nos genes que codificam antígenos de superfície de merozoítos (MSA-1, MSA-2b e MSA-2c) foi investigada. Os resultados da sorologia demonstraram que, até os seis meses de idade, todos os bezerros desenvolveram imunidade ativa contra B. bovis. Entre as 75 amostras de DNA avaliadas, foram obtidas 0, 3 e 5 sequências dos genes msa-1, msa-2b e msa-2c. O presente estudo demonstrou que sequências dos genes msa-2b e msa-2c amplificadas a partir de amostras de sangue positivas para B. bovis de bezerros de Seropédica, foram geneticamente distintas. O presente trabalho realça a importância de se realizar estudos aprofundados sobre a diversidade genética de B. bovis no Brasil, objetivando o desenvolvimento de antígenos para o diagnóstico e vacinas no futuro.(AU)
Assuntos
Animais , Bovinos , Bovinos/parasitologia , Babesiose/classificação , Babesiose/genética , Babesia bovis/genética , Variação GenéticaRESUMO
Bovine babesiosis is the most important protozoan disease transmitted by ticks. In Plasmodium falciparum, another Apicomplexa protozoan, the interaction of rhoptry neck protein 2 (RON2) with apical membrane antigen-1 (AMA-1) has been described to have a key role in the invasion process. To date, RON2 has not been described in Babesia bigemina, the causal agent of bovine babesiosis in the Americas. In this work, we found a ron2 gene in the B. bigemina genome. RON2 encodes a protein that is 1351 amino acids long, has an identity of 64% (98% coverage) with RON2 of B. bovis and contains the CLAG domain, a conserved domain in Apicomplexa. B. bigemina ron2 is a single copy gene and it is transcribed and expressed in blood stages as determined by RT-PCR, Western blot, and confocal microscopy. Serum samples from B. bigemina-infected bovines were screened for the presence of RON2-specific antibodies, showing the recognition of conserved B-cell epitopes. Importantly, in vitro neutralization assays showed an inhibitory effect of RON2-specific antibodies on the red blood cell invasion by B. bigemina. Therefore, RON2 is a novel antigen in B. bigemina and contains conserved B-cell epitopes, which induce antibodies that inhibit merozoite invasion.
Assuntos
Anticorpos Antiprotozoários/sangue , Babesia/genética , Epitopos de Linfócito B/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Animais , Babesia/imunologia , Babesiose/parasitologia , Bovinos , DNA de Protozoário/imunologia , Eritrócitos/parasitologia , Genoma de Protozoário , Masculino , Merozoítos/genética , Merozoítos/imunologia , Testes de NeutralizaçãoRESUMO
Protection against the intraerythrocytic protozoan parasite Babesia bovis depends on both strong innate and adaptive immune response, this latter involving the presentation of parasite antigens to CD4+ T-lymphocytes by professional antigen-presenting cells. Secretion of Th1 cytokines by CD4+ T cell is also very important for isotype switching to IgG2, the best opsonising antibody isotype in cattle, to target extracellular parasites and parasite antigens displayed at the erythrocyte surface. In the field of vaccinology, heterologous prime-boost schemes combining protein-adjuvant formulations with a modified vaccinia Ankara vector expressing the same antigen have demonstrated the induction of both humoral and cellular immune responses. It has been previously demonstrated that MVA-infected dendritic cells can present antigens in the context of MHC II and activate CD4+ T cell. These results support the use of the MVA viral vector for a pathogen like Babesia bovis, which only resides within erythrocytes. In this study, 13-15-months-old Holstein-Friesian steers were immunised with a subunit vaccine as a prime and a modified vaccinia Ankara vector as a boost, both expressing a chimeric multi-antigen (rMABbo - rMVA). This antigen includes the immunodominant B and T cell epitopes of three B. bovis proteins: merozoite surface antigen - 2c (MSA - 2c), rhoptry associated protein 1 (RAP - 1) and heat shock protein 20 (HSP20). Responses were compared with the Babesia bovis live attenuated vaccine used in Argentina (R1A). Eleven weeks after the first immunisation, all bovines were challenged by the inoculation of a virulent B. bovis strain. All groups were monitored daily for hyperthermia and reduction of packed cell volume. Both the rMABbo - rMVA and R1A vaccinated animals developed high titters of total IgG antibodies and an antigen-specific Th1 cellular response before and after challenge. However, all rMABbo - rMVA steers showed clinical signs of disease upon challenge. Only the R1A live vaccine group developed an immune response associated with in vitro neutralising antibodies at a level that significantly inhibited the parasite invasion. The lack of protection observed with this recombinant formulation indicates the need to perform further basic and clinical studies in the bovine model in order to achieve the desired effectiveness. This is the first report in which a novel vaccine candidate against Babesia bovis was constructed based on a recombinant and rationally designed viral vector and evaluated in the biological model of the disease.
Assuntos
Babesia bovis/imunologia , Babesiose/prevenção & controle , Doenças dos Bovinos/prevenção & controle , Vacinas Protozoárias/imunologia , Vacinação/veterinária , Animais , Anticorpos Neutralizantes/imunologia , Babesiose/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Epitopos/imunologia , Imunidade Celular , Imunidade Humoral , Masculino , Proteínas Recombinantes/imunologia , Células Th1/imunologia , Vacinas Atenuadas/imunologia , Vaccinia virus/imunologiaRESUMO
Babesia bovis is the etiological agent of bovine babesiosis, a disease transmitted by Rhipicephalus microplus, which affects cattle herds in tropical and subtropical regions of the world, causing significant economic losses due to decreasing meat and milk yield. This study used molecular techniques to determine the occurrence and genetic diversity of B. bovis, based on the genes encoding the spherical body protein (sbp-2) and the merozoite surface antigens (MSAs) genes, in a herd of 400 Nellore (Bos indicus) sampled from beef cattle farms in the Pantanal region, state of Mato Grosso do Sul, Midwestern Brazil. The results of the nested PCR assays based on the sbp-2 gene indicated that 18 (4.5%) calves were positive for B. bovis; out of them, while 77.7% (14/18) were positive for the B. bovis msa-2b fragment, 66.6% (12/18) were positive for the msa-2c fragment. The phylogenetic analysis based on the maximum likelihood method using 14 sequences from msa-2b clones and 13 sequences from msa-2c clones indicated that the sequences detected in this study are clearly distributed in different cladograms. These findings corroborated the diversity analysis of the same sequences, which revealed the presence of 14 and 11 haplotypes of the msa-2b and msa-2c genes, respectively. Furthermore, the entropy analyses of amino acid sequences revealed 78 and 44 high entropy peaks with values ranging from 0.25 to 1.53 and from 0.27 to 1.09 for MSA-2B and MSA-2C, respectively. Therefore, the results indicate a low molecular occurrence of B. bovis in beef cattle sampled in the Brazilian Pantanal. Despite this, a high degree of genetic diversity was found in the analyzed B. bovis population, with possibly different haplotypes coexisting in the same animal and/or in the same studied herd.
Assuntos
Antígenos de Protozoários/genética , Babesia bovis/genética , Babesiose/parasitologia , Doenças dos Bovinos/parasitologia , Variação Genética , Sequência de Aminoácidos , Animais , Antígenos de Superfície/genética , Babesiose/epidemiologia , Brasil , Bovinos , Doenças dos Bovinos/epidemiologia , Entropia , Merozoítos , Filogenia , Reação em Cadeia da Polimerase/veterinária , Áreas AlagadasRESUMO
BACKGROUND: Babesia bovis belongs to the phylum Apicomplexa and is the major causal agent of bovine babesiosis, the most important veterinary disease transmitted by arthropods. In apicomplexan parasites, the interaction between AMA1 and RON2 is necessary for the invasion process, and it is a target for vaccine development. In B. bovis, the existence of AMA1 has already been reported; however, the presence of a homolog of RON2 is unknown. The aim of this study was to characterize RON2 in B. bovis. RESULTS: The B. bovis ron2 gene has a similar synteny with the orthologous gene in the B. bigemina genome. The entire ron2 gene was sequenced from different B. bovis strains showing > 99% similarity at the amino acid and nucleotide level among all the sequences obtained, including the characteristic CLAG domain for cytoadherence in the amino acid sequence, as is described in other Apicomplexa. The in silico transcription analysis showed similar levels of transcription between attenuated and virulent B. bovis strains, and expression of RON2 was confirmed by western blot in the B. bovis T3Bo virulent strain. Four conserved peptides, containing predicted B-cell epitopes in hydrophilic regions of the protein, were designed and chemically synthesized. The humoral immune response generated by the synthetic peptides was characterized in bovines, showing that anti-RON2 antibodies against peptides recognized intraerythrocytic merozoites of B. bovis. Only peptides P2 and P3 generated partially neutralizing antibodies that had an inhibitory effect of 28.10% and 21.42%, respectively, on the invasion process of B. bovis in bovine erythrocytes. Consistently, this effect is additive since inhibition increased to 42.09% when the antibodies were evaluated together. Finally, P2 and P3 peptides were also recognized by 83.33% and 87.77%, respectively, of naturally infected cattle from endemic areas. CONCLUSIONS: The data support RON2 as a novel B. bovis vaccine candidate antigen that contains conserved B-cell epitopes that elicit partially neutralizing antibodies.
Assuntos
Antígenos de Protozoários/imunologia , Babesia bovis/imunologia , Babesiose/imunologia , Epitopos de Linfócito B/imunologia , Imunidade Humoral , Peptídeos/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/administração & dosagem , Babesia bovis/patogenicidade , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , Simulação por Computador , Epitopos de Linfócito B/genética , Eritrócitos/parasitologia , Imunização , Peptídeos/administração & dosagem , Peptídeos/síntese química , Peptídeos/genética , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/genéticaRESUMO
Bovine babesiosis caused by protozoan parasites Babesia bovis and B. bigemina is one of the most important causes of losses for the livestock industry in tropical and subtropical regions of the world. Therefore, highly sensitive and specific tools for these hemoparasites detection and monitoring are required, especially in carrier animals, in which low parasite levels were usually present. In this context, qPCR assays have been successfully and fairly used in last years. Aiming to improve the performance of Babesia levels monitoring by qPCR, some of main aspects of this methodology that may influence results were tested: DNA extraction kits, whole blood EDTA pre-treatment, blood source (tip of tail or jugular vein), erythrocytes isolation, FTA card interference and qPCR system of detection. Under our experimental conditions, both EDTA pre-treatment and FTA card application have no influence on the sensitivity of detection, and two DNA extraction kits provided higher sensitivity compared to others. As expected, blood samples collected from the tip of tail vessels presented higher levels of B. bovis DNA compared to those obtained from the jugular vein, and erythrocytes processed isolated has also improved the sensitivity compared to whole blood. Moreover, both qPCR assays here developed using hydrolysis probes for B. bovis and B. bigemina detection, presented enhanced reproducibility compared to qPCR assays using intercalating dye system. Even, qPCR for B. bigemina using hydrolysis probe here developed presented higher sensitivity compared to intercalating dye system. This study has contributed to the improvement of molecular diagnosis of bovine babesiosis, which may improve epidemiological studies related to these pathogens.
Assuntos
Babesia bovis/genética , DNA/isolamento & purificação , Monitoramento Ambiental/métodos , Animais , Babesia/genética , Babesiose/genética , Bovinos , Doenças dos Bovinos/genética , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos TestesRESUMO
Water buffaloes (Bubalus bubalis) are raised in tropical and subtropical regions of the world, and act as hosts of Babesia bovis parasites and the tick vector Rhipicephalus microplus. As no clinical cases of B. bovis-infection have been reported, we hypothesized that, unlike bovines, water buffaloes respond asymptomatically to an acute infection. To test this hypothesis, we inoculated two groups of 24-month-old Mediterranean breed water buffaloes with 108 erythrocytes infected with two Argentine B. bovis isolates: BboM2P (nâ¯=â¯5) or BboS2P (nâ¯=â¯5). These strains displayed mild (BboM2P) or high (BboS2P) pathogenicity in Bos taurus calves of the same age (nâ¯=â¯5 and nâ¯=â¯1, respectively), when tested in parallel. In water buffaloes, no changes in body temperature were observed with both strains, and no hematocrit changes were detected in BboM2P-inoculated animals. In contrast, in the BboS2P-inoculated water buffalo group significant but relatively minor reductions in haematocrit values were noted compared to the infected bovine. The parasitemia attained in water buffaloes was considerably lower than in bovines and could only be detected by nested PCR, or indirectly via serology, whereas in most bovines, it could also be detected in Giemsa-stained smears under the light microscope. Our results show that water buffaloes present no or significantly mitigated clinical symptoms to B. bovis infections and suggest that they are able to substantially reduce and/or eliminate B. bovis parasites from circulation by an efficient innate immune mechanism.
Assuntos
Babesia bovis/isolamento & purificação , Babesiose/parasitologia , Doenças dos Bovinos/diagnóstico , Parasitemia/diagnóstico , Animais , Babesia bovis/genética , Babesia bovis/imunologia , Babesia bovis/patogenicidade , Babesiose/diagnóstico , Babesiose/imunologia , Búfalos/imunologia , Búfalos/parasitologia , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/parasitologia , DNA de Protozoário/genética , Ensaio de Imunoadsorção Enzimática/veterinária , Eritrócitos/parasitologia , Hematócrito , Imunidade Inata , Masculino , Parasitemia/epidemiologia , Reação em Cadeia da Polimerase/veterinária , Rhipicephalus/microbiologia , Testes SorológicosRESUMO
Babesia bigemina is one of the most prevalent species causing bovine babesiosis around the world. Antigens involved in host cell invasion are vaccine targets for this disease but are largely unknown in this species. The invasion process of Babesia spp. into erythrocytes involves membrane proteins from the apical complex. A protein stored in the micronemes, called Micronemal Protein 1 (MIC-1), contains a sialic acid binding domain that participates in the invasion process of host cells and is a vaccine candidate in other apicomplexan parasites. It is not known if there is a homologous gene for mic-1 in B. bigemina. Therefore, the aim of this study was to characterize the mic-1 gene homologue in Babesia bigemina. A gene was found with a microneme adhesive repeat (MAR) domain in the predicted amino acid sequence. Transcription was determined by reverse transcription polymerase chain reaction (RT-PCR). Subsequently, antibodies against peptides containing conserved B-cell epitopes were used to confirm the expression of MIC-1 in intraerythrocytic merozoites. The presence of anti MIC-1 antibodies in cattle naturally infected with B. bigemina was determined and up to 97.4% of the cattle sera (113 out of 116) identified MIC-1 using enzyme-linked immunosorbent assay (ELISA) methods. Finally, antibodies against MIC-1 were able to block 70% merozoite invasion in-vitro.
RESUMO
The current method for Babesia spp. serodiagnosis based on a crude merozoite antigen is a complex and time-consuming procedure. An indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant multi-antigen of Babesia bovis (rMABbO) was developed for detection of antibodies in bovines suspected of infection with this parasite. The multi-antigen comprises gene fragments of three previously characterized B. bovis antigens: MSA-2c, RAP-1 and the Heat Shock protein 20 that are well-conserved among geographically distant strains. The cutoff value for the new rMABbo-iELISA was determined using 75 known-positive and 300 known-negative bovine sera previously tested for antibodies to B. bovis by the gold-standard ELISA which uses a merozoite lysate. A cutoff value of ≥35% was determined in these samples by receiver operator characteristic (ROC) curve analysis, showing a sensitivity of 95.9% and a specificity of 94.3%. The rMABbo-iELISA was further tested in a blind trial using an additional set of 263 field bovine sera from enzootic and tick-free regions of Argentina. Results showed a good agreement with the gold standard test with a Cohen's kappa value of 0.76. Finally, the prevalence of bovine babesiosis in different tick enzootic regions of Argentina was analyzed where seropositivity values among 68-80% were obtained. A certain level of cross reaction was observed when samples from B. bigemina infected cattle were analyzed with the new test, which can be attributed to shared epitopes between 2 of the 3 antigens. This new rMABbo-iELISA could be considered a simpler alternative to detect anti Babesia spp. antibodies and appears to be well suited to perform epidemiological surveys at the herd level in regions where ticks are present.
RESUMO
Babesiosis is an economically important infectious disease affecting cattle worldwide. In order to longitudinally evaluate the humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis among naturally infected calves in Taiaçu, Brazil, serum and DNA samples from 15 calves were obtained quarterly, from their birth to 12 months of age. Anti-B. bovis IgG antibodies were detected by means of the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). The polymerase chain reaction (PCR) was used to investigate the genetic diversity of B. bovis, based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 2, 4 and 5 sequences of the genes msa-1, msa-2b and msa-2c were obtained. The present study demonstrated that the msa-1 and msa-2b genes sequences amplified from blood DNA of calves positive to B. bovis from Taiaçu were genetically distinct, and that msa-2c was conserved. All animals were serologically positive to ELISA and IFAT, which used full repertoire of parasite antigens in despite of the genetic diversity of MSAs.(AU)
A babesiose é uma doença infecciosa economicamente importante que afeta o gado bovino em todo o mundo. Para avaliar longitudinalmente a resposta imune humoral contra B. bovis e a diversidade genética de antígenos de superfície de merozoítos de B. bovis, entre bezerros naturalmente infectados em Taiaçu, Brasil, amostras de soro e DNA de 15 bezerros, foram obtidos trimestralmente, desde o nascimento até aos 12 meses de idade. Os anticorpos IgG para B. bovis foram detectados pelos testes de Imunofluorescência Indireta e Ensaio de Imunoadsorção Enzimático Indireto. A Reação em Cadeia da Polimerase foi utilizada para investigar a diversidade genética de B. bovis, com base em genes que codificam antígenos de superfície de merozoítos (MSA-1, MSA-2b e MSA-2c). Os resultados da sorologia demonstraram que até seis meses de idade todos os bezerros desenvolveram imunidade ativa contra B. bovis. Entre as 75 amostras de DNA avaliadas, foram obtidas 2, 4 e 5 sequências dos genes msa-1, msa-2b e msa-2c. O presente trabalho demonstrou que as sequências dos genes msa-1 e msa-2b amplificadas do DNA do sangue de amostras positivas a B. bovis de bezerros de Taiaçu foram geneticamente distintas, e msa-2c conservadas. Todos os animais foram soropositivos ao ELISA e ao IFAT, os quais utilizaram o repertório completo de antígenos parasitários, apesar da diversidade genética dos MSAs.(AU)