David R. Allred

The ves Multigene Family of B. bovis Encodes Components of Rapid Antigenic Variation at the Infected Erythrocyte Surface

B. bovis, an intraerythrocytic protozoal parasite, establishes chronic infections in cattle in part through rapid variation of the polymorphic, heterodimeric VESA1 protein on the infected erythrocyte surface and sequestration of mature parasites. We describe the characterization of the ves1 alpha gene encoding the VESA1a subunit, thus providing a description of a gene whose product is involved in rapid antigenic variation in a babesial parasite. This three-exon gene, a member of a multigene family (ves), encodes a polypeptide with no cleavable signal sequence, a single predicted transmembrane segment, and a cysteine/lysine-rich domain. Variation appears to involve creation and modification or loss of a novel, transcribed copy of the gene.

Antigenic variation in Babesia bovis occurs through segmental gene conversion of the ves multigene family, within a bidirectional locus of active transcription

Antigenic variation in Babesia bovis is one aspect of a multifunctional virulence/survival mechanism mediated by the heterodimeric variant erythrocyte surface antigen 1 (VESA1) protein that also involves endothelial cytoadhesion with sequestration of mature parasitized erythrocytes. The ves1α gene encoding the VESA1a subunit was previously identified. In this study, we present the unique organization of the genomic locus from which ves1α is transcribed, and identify a novel branch of the ves multigene family, ves1β. These genes are found together, closely juxtaposed and divergently oriented, at the locus of active transcription. We provide compelling evidence that variation of both transcriptionally active genes occurs through a mechanism of segmental gene conversion involving sequence donor genes of similar organization. These results also suggest the possibility of epigenetic regulation through in situ switching among gene loci, further expanding the potential repertoire of variant proteins.

Characterization of a variant erythrocyte surface antigen (VESA1) expressed by Babesia bovis during antigenic variation

Babesia bovis, an intraerythrocytic, protozoal parasite of cattle, undergoes clonal antigenic variation (Allred DR, Cinque RM, Lane TJ, Ahrens KP. Infect Immun 1994;62:91-98). This ability could provide a mechanism by which the parasite escapes host immune defenses to establish chronic infection. Previous work identified two parasite-derived antigens of Mr 128,000 and 113,000 that were present on the surface of the infected erythrocyte and appeared to be associated with clonal antigenic variation (Allred DR, Cinque RM, Lane TJ, Ahrens KP. Infect Immun 1994;62:91 98). Two monoclonal antibodies (mAbs), 3F7.1H11 and 4D9.1G1, which recognize the variant erythrocyte surface antigen (VESA1) have been identified. These mAbs react only with the surface of erythrocytes infected with the B. bovis C9.1 clone in live-cell immunofluorescence assays. In both conventional and surface immunoprecipitations, the mAbs precipitate a variant antigen doublet that matches in mass the infected red blood cell (IRBC) surface antigens precipitated with bovine serum. In contrast, Western blot analysis revealed that only the Mr 128,000 polypeptide is recognized by the mAbs. Neither mAb recognizes antigenically variant progenitor or progeny parasite clones in any of the immunoassays, confirming the involvement of this antigen in rapid clonal antigenic variation. Failure to label this antigen with [9,10(n)-3H]myristic acid, [9,10(n)-3H]palmitic acid or D-[6-3H]glucosamine indicates that these polypeptides are neither N-glycosylated nor fatty acylated. Identity of the variant antigen recognized by the mAbs with that putatively identified with immune serum was confirmed by comparison of partial proteolytic digestion products. Unambiguous identification of the VESA1 antigen as a component of antigenic variation will facilitate characterization of the events leading to antigenic variation on the B. bovis-infected erythrocyte surface and its significance to parasite survival during chronic infection.

Isolate-specific parasite antigens of the Babesia bovis-infected erythrocyte surface.

Bovine erythrocytes taken from in vitro cultures of Babesia bovis parasites from Mexico and the United States were assayed for the presence of new epitopes on the erythrocyte surface. New surface-exposed epitopes were detected by means of a whole-cell antigen capture assay. These epitopes were subsequently demonstrated only on infected erythrocytes by immunofluorescence staining of intact, living cells. Parasite-synthesized antigens were identified on each isolate using a surface-specific immunoprecipitation technique to analyze metabolically-labeled infected erythrocytes. In the Mexico isolate these antigens were 120 kDa and 107 kDa, whereas in the United States isolate polypeptides of 135, 120 and 107 kDa were detected. In each of these assays, reaction of immune sera with the infected erythrocyte surface was found to be isolate-specific.

Babesiosis: persistence in the face of adversity

Many babesial parasites establish infections of long duration in immune hosts. Among different species, at least four mechanisms are known that could facilitate evasion of the host immune response, although no one species is (yet) known to use them all. This update strives to illustrate the ramifications of these mechanisms and the interplay between them.

The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5′ ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct.

Immune Evasion by Babesia bovis and Plasmodium falciparum: Cliff-dwellers of the Parasite World

Erythrocyte-dwelling parasites, such as Babesia bovis and Plasmodium falciparum, are not accessible to the host immune system during most of their asexual reproductive cycle because they are intracellular. While intracellular, the host immune response must be directed toward the surface of the infected erythrocyte. Immune individuals mount protective antibody and cell-mediated responses which eliminate most of the parasites, yet some survive to establish chronic infections. In this review, David Allred discusses some of the mechanisms used by these parasites to evade individual immune mechanisms targeting the infected erythrocyte to survive in the hostile environment of an effective immune response.

Outbreak of equine piroplasmosis in Florida

CASE DESCRIPTION A 7-year-old Quarter Horse gelding was hospitalized in Ocala, Fla, because of lethargy, fever, anorexia, and swelling of distal aspects of the limbs. A tentative diagnosis of equine piroplasmosis (EP) was made on the basis of examination of a blood smear. The case was reported to the Florida State Veterinarian, and infection with Babesia equi was confirmed. The subsequent investigation included quarantine and testing of potentially exposed horses for B equi and Babesia caballi infections, tick surveillance, and owner-agent interviews. CLINICAL FINDINGS 210 horses on 25 premises were tested for infection with EP pathogens. Twenty B equi-infected horses on 7 premises were identified; no horses tested positive for B caballi. Seven horses, including the index case, had clinical findings consistent with EP Dermacentor variabilis was considered the only potential tick vector for B equi collected, and all D variabilis specimens tested negative for Babesia organisms via PCR assay. Results of the epidemiological investigation suggested that B equi was spread by use of shared needles and possibly blood transfusions. All horses that tested positive were involved in nonsanctioned Quarter Horse racing, and management practices were thought to pose substantial risk of transmission of blood-borne pathogens. TREATMENT AND OUTCOME Final outcome of B equi-infected horses was euthanasia, death from undetermined causes, or shipment to a US federal research facility. CLINICAL RELEVANCE This investigation highlights the importance of collaboration between private veterinary practitioners, state veterinary diagnostic laboratories, and regulatory officials in the recognition, containment, and eradication of foreign animal disease.

Assocation of the 33 kDa extrinsic polypeptide (water-splitting) with PS II particles: immunochemical quantification of residual polypeptide after membrane extraction.

Various washing procedures were tested on Triton-prepared PS II particles for their ability to remove the 33 kDa extrinsic polypeptide (33 kDa EP) associated with the water-splitting complex. Residual 33 kDa EP was evaluated by Coomassie blue staining of SDS gels of washed particles and by Western blotting with an antibody specific for the 33 kDa EP. A wash with 16 mM Tris buffer, pH 8.3, inhibited water-splitting activity but did not remove all the 33 kDa EP. Sequential washes with 30 mM octyl glucoside (pH 8.0 and 6.8), and a single wash with 0.8 M Tris were also ineffective in removing all the 33 kDa EP. Washing with 1 M CaCl2 was more effective in removing 33 kDa EP; while only a faint trace of protein was detectable by Coomassie-staining, immunoblotting revealed a considerable remainder. The treated particles retained some water-splitting activity. The two step procedure of Miyao and Murata (1984) involving 1 M NaCl and 2.3 M urea was most effective, removing all but a trace of antibody positive protein. Our finding suggests that (1) the degree of depletion of the 33 kDa EP cannot be judged on the basis of Coomassie stain alone, and (2) this extrinsic protein is very tightly associated with the membrane, perhaps via a hydrophilic portion of this otherwise hydrophilic protein. The results also suggest that the presence or absence of the 33 kDa protein per se is not the primary determinant of residual water splitting activity.

ANTIGENIC VARIATION IN BABESIA BOVIS : HOW SIMILAR IS IT TO THAT IN PLASMODIUM FALCIPARUM?

Despite significant differences in some aspects of their life-cycles, the Apicomplexan parasites Babesia bovis and Plasmodium falciparum share many parallels. Significant among these are participation in rapid, clonal antigenic variation, and cyto-adherence and sequestration in the deep vasculature. Antigenic variation has long been thought to be primarily a mechanism of escape from antibody-mediated mechanisms of the hosts immune system. In each species, the components demonstrated to participate in antigenic variation are parasite-derived proteins expressed on the infected erythrocytes surface. Recently, the malarial component PfEMP1 has been found to be a multifunctional protein that is not only subject to antigenic variation, but also participates in cyto-adherence and rosetting (adhesion to uninfected erythrocytes). In the present review, the antigens elaborated on the surface of an erythrocyte infected with B. bovis, for immune evasion via antigenic variation, are described, and compared and contrasted with those from P. falciparum. The significance of the similarities between B. bovis and P. falciparum, and the potential for contributions to be made to our understanding of malaria through the study of babesiosis are discussed.