A unique Babesia bovis spherical body protein is conserved among geographic isolates and localizes to the infected erythrocyte membrane.

Using monoclonal antibody (mAb) 70/52.9, generated from a Babesia bovis fraction enriched for spherical body organelles, we have identified a 135-kDa protein containing an epitope conserved in B. bovis strains from Texas, Mexico, and Australia. The protein was localized to the spherical bodies of the babesial apical complex and was designated spherical body protein 3 (SBP3), according to the established nomenclature. Immunofluorescence studies showed binding of the 70/52.9 mAb to the infected-erythrocyte membrane region but not to their uninfected counterparts, demonstrating a host-cell association shared with the previously isolated B. bovis spherical body proteins, SBP1 and SBP2. Using mAb 70/52.9, the full-length cDNA encoding SBP3 was isolated from an expression library, sequenced, and oligonucleotide primers synthesized to amplify the genomic copy by polymerase chain reaction. The genomic copy contained no introns and was identical to the cDNA sequence with each containing a single, large open reading frame encoding a protein of 1089 residues. Analysis of the SBP3 amino acid sequence revealed no significant amino acid identity to SBP1 and SBP2 and a lack of repeated epitopes, a notable feature of the other two spherical body proteins. Labeled probes derived from the coding region of SBP3 hybridized to single fragments on Southern blots containing B. bovis genomic DNA indicating a single copy gene. With the identification of this third spherical body protein, which associates with the cytoplasmic face of the infected-erythrocyte membrane, a complement of distinct B. bovis proteins have been identified that are likely to contribute to intracellular survival, growth, and development for this parasite. The encoded protein should be valuable for functional investigations and evaluation of potential targets for host immunity.

A Babesia bovis 225-kilodalton spherical-body protein: localization to the cytoplasmic face of infected erythrocytes after merozoite invasion.

A 225-kDa Babesia bovis protein occurs on the cytoplasmic side of infected-erythrocyte membranes. Here it is demonstrated that the 225-kDa protein localizes to spherical-body organelles of merozoites. Organelles consistent in size and shape with spherical bodies were isolated between 1.17 and 1.21 g/cm(3) in a sucrose density gradient. Organelles consistent with rhoptries and micronemes were also present in fractions from 1.17 to 1.19 g/cm(3). Antisera generated by immunizing mice with the fraction (1.20 to 1.21 g/cm(3)) most enriched for spherical bodies reacted predominantly with spherical bodies in B. bovis merozoites. A monoclonal antibody generated from this immunization (70/97.14) recognized an epitope that occurs in the repeat region of the 225-kDa protein (now referred to as SBP2). Monoclonal antibody 70/97.14 bound to merozoite spherical bodies, vesicles in infected-host cytoplasm, and the cytoplasmic face of the infected-erythrocyte membrane. These results indicate that spherical-body proteins become associated with the host membrane via transport through the erythrocyte cytoplasm after intracellular invasion.

Isotypic antibody responses in cattle infected with Haemophilus somnus.

Bovine antibody responses to Haemophilus somnus were compared on the basis of clinical and bacteriological findings. Serum IgG1 and IgM antibody titres were significantly increased in clinically normal cattle that were bacteriologically positive for H somnus from the nasal or vaginal mucosae compared with clinically normal, negative cows. IgG2 titres did not differ significantly between these two groups. However, IgG2 antibody was significantly higher in animals with H somnus disease (pneumonia or abortion) than in clinically normal cattle (whether bacteriologically positive or negative), while IgG1 and IgM titres did not differ between diseased and bacteriologically positive, clinically normal cattle. These antibody trends were duplicated in experimental H somnus abortion or pneumonia, with the greatest response occurring within the IgG2 subclass. Cattle vaccinated systemically with killed whole H somnus produced a predominant IgG2 response with minimal IgG1 and IgM responses. These results demonstrate that IgG2 antibody is consistently elevated in H somnus disease, and suggest that this response may be useful in discriminating diseased from asymptomatic cattle.

Hemoperitoneum caused by rupture of a juvenile granulosa cell tumor in an equine neonate.

A neonatal foal was examined because of apparent abdominal pain and distention, anemia, and hemoperitoneum. Exploratory laparotomy was performed, and a large spherical mass, which had ruptured, was found in the area of the left ovary. Left salpingo-oophorectomy was performed. The mass was determined to be a juvenile granulosa cell tumor.