Spherical Body Protein 4 from Babesia bigemina: A Novel Gene That Contains Conserved B-Cell Epitopes and Induces Cross-Reactive Neutralizing Antibodies in Babesia ovata.

Bovine babesiosis is a tick-transmitted disease caused by intraerythrocytic protozoan parasites of the genus Babesia. Its main causative agents in the Americas are Babesia bigemina and Babesia bovis, while Babesia ovata affects cattle in Asia. All Babesia species secrete proteins stored in organelles of the apical complex, which are involved in all steps of the invasion process of vertebrate host cells. Unlike other apicomplexans, which have dense granules, babesia parasites instead have large, round intracellular organelles called spherical bodies. Evidence suggests that proteins from these organelles are released during the process of invading red blood cells, where spherical body proteins (SBPs) play an important role in cytoskeleton reorganization. In this study, we characterized the gene that encodes SBP4 in B. bigemina. This gene is transcribed and expressed in the erythrocytic stages of B. bigemina. The sbp4 gene consists of 834 nucleotides without introns that encode a protein of 277 amino acids. In silico analysis predicted a signal peptide that is cleaved at residue 20, producing a 28.88-kDa protein. The presence of a signal peptide and the absence of transmembrane domains suggest that this protein is secreted. Importantly, when cattle were immunized with recombinant B. bigemina SBP4, antibodies identified B. bigemina and B. ovata merozoites according to confocal microscopy observations and were able to neutralize parasite multiplication in vitro for both species. Four peptides with predicted B-cell epitopes were identified to be conserved in 17 different isolates from six countries. Compared with the pre-immunization sera, antibodies against these conserved peptides reduced parasite invasion in vitro by 57%, 44%, 42%, and 38% for peptides 1, 2, 3, and 4, respectively (p < 0.05). Moreover, sera from cattle infected with B. bigemina cattle contained antibodies that recognized the individual peptides. All these results support the concept of spb4 as a new gene in B. bigemina that should be considered a candidate for a vaccine to control bovine babesiosis.

The GP-45 Protein, a Highly Variable Antigen from Babesia bigemina, Contains Conserved B-Cell Epitopes in Geographically Distant Isolates.

In B. bigemina, the 45 kilodaltons glycoprotein (GP-45) is the most studied. GP-45 is exposed on the surface of the B. bigemina merozoite, it is believed to play a role in the invasion of erythrocytes, and it is characterized by a high genetic and antigenic polymorphism. The objective of this study was to determine if GP-45 contains conserved B-cell epitopes, and if they would induce neutralizing antibodies. The comparative analysis of nucleotide and amino acids sequences revealed a high percentage of similarity between field isolates. Antibodies against peptides containing conserved B-cell epitopes of GP-45 were generated. Antibodies present in the sera of mice immunized with GP-45 peptides specifically recognize B. bigemina by the IFAT. More than 95% of cattle naturally infected with B. bigemina contained antibodies against conserved GP-45 peptides tested by ELISA. Finally, sera from rabbits immunized with GP-45 peptides were evaluated in vitro neutralization tests and it was shown that they reduced the percentage of parasitemia compared to sera from rabbits immunized with adjuvant. GP-45 from geographically distant isolates of B. bigemina contains conserved B-cell epitopes that induce neutralizing antibodies suggesting that this gene and its product play a critical role in the survival of the parasite under field conditions.

RON2, a novel gene in Babesia bigemina, contains conserved, immunodominant B-cell epitopes that induce antibodies that block merozoite invasion.

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.

Immunomolecular Characterization of MIC-1, a Novel Antigen in Babesia bigemina, Which Contains Conserved and Immunodominant B-Cell Epitopes that Induce Neutralizing Antibodies.

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.

Evaluación de la Patogenicidad y Capacidad Protectora de un Inmungeno Fresco Combinado de Babesia Bigemina y Babesia Bovis / Evaluación of pathogenicity and protección capability of a fresh combined immunogen of Babesia bigemina and Babesia bovis

El objetivo del trabajo fue evaluar la inocuidad y la capacidad inmunoprotectora de un inmunógeno combinado de Babesia bigemina y Babesia bovis a una dosis de 108 y 109, respectivamente, bajo un desafío controlado. En el primer experimento (inocuidad) se utilizaron 16 bovinos Holstein provenientes de una zona libre de garrapatas y negativos a anticuerpos contra Babesia spp, mediante la prueba de inmunofluorescencia indirecta (IFI), los cuales fueron inoculados, vía intramuscular, con 1 X 109 eritrocitos infectados (EI) con una cepa atenuada de B. bigemina y 108 EI de una clona irradiada de B. bovis, ambas derivadas de cultivo in vitro. Se observaron cambios en las constantes fisiológicas a partir del día 7 y hasta el día 14 posinoculación (PI) en los animales sin verse éstos físicamente afectados. El porcentaje de eritrocitos parasitados (PEP) fue <0.01 por ciento. El segundo experimento (inmunogenicidad), se realizó tres meses PI y consistió en el desafío de 8 de los animales del experimento 1 con cepas virulentas de ambas especies del protozoario a una dosis de 108 EI de cada una. Cuatro animales adicionales sirvieron como grupo testigo. El grupo inmunizado presentó ligera disminución en el volumen celular aglomerado (VCA) con temperatura rectal (TR) sin cambios y PEP de 0.06 y <0.01 para B. bigemina y B. bovis, respectivamente. El grupo testigo presentó TR mayor a 400C, disminución del VCA (29 por ciento) y el PEP fue de 0.5 para B. bigemina y 0.03 para B. bovis; estos animales requirieron tratamiento para evitar su muerte. Se concluye que el inmunógeno combinado de B. bigemina y B. bovis a dosis altas no provoca reacciones clínicas severas en animales susceptibles y proporciona una sólida protección al desafío controlado con cepas virulentas

Evaluación en Condiciones de Campo de la Vacuna inactivada de Anaplasma Marginale Denominada Plazvax / Evaluation in field conditions of inactivated vaccines of anaplasma marginale named Plazvax

Para determinar la efectividad de la vacuna Plazvax© contra la anaplasmosis bovina, se utilizaron 32 novillos susceptibles a la enfermedad. Un grupo de 24 novillos fue inmunizado contra Anaplasma marginale, inoculando subcutáneamente 1 ml (dos veces, 21 días aparte) de la vacuna. Ocho animales fungieron como testigos sin vacunar. Un mes después de la segunda inmunización, los animales fueron trasladados a una zona endémica de anaplasmosis para ser desafiados naturalmente. A partir del día ocho posintroducción al potrero (PIP) y hasta el día 105 se realizó un seguimiento que incluyó observación de manifestaciones clínicas, temperatura rectal (TR), hematocrito (HT) y parasitemia (PP). Tres bovinos murieron de babesiosis y los 29 animales restantes tuvieron seguimiento dos veces por semana a partir del día 42 PIP. El día 52 PIP se detectaron los primeros animales infectados con Anaplasma marginale (6, todos del grupo vacunado), y cuatro fueron tratados con oxitetraciclinas (días 52-64 PIP). A partir del día 84 PIP los animales testigo presentaron anaplasmosis severa con TR promedio de 40.50C, HT promedio de 13.2 por ciento y PP promedio de 9.4 por ciento hacia el día de tratamiento (días 87-95 PIP). Los animales vacunados presentaron los siguientes valores promedios de TR, 40.2ºC; HT, 16.3 por ciento; y PP 3.3 por ciento en el día de tratamiento (días 93-105 PIP). A pesar de existir diferencia en PP entre los animales vacunados y testigos, todos padecieron anaplasmosis clínica, requiriendo tratamiento. En conclusión, en su forma actual la vacuna no es recomendable para usarse en México; se sugiere el empleo o introducción en la vacuna Plazvax, de material antigénico de uno o más aislados de A. marginale provenientes de distintas regiones geográficas de México y realizar estudios sobre su capacidad inmunoprotectora en bovinos