Merozoite surface antigen 2 proteins of Babesia bovis vaccine breakthrough isolates contain a unique hypervariable region composed of degenerate repeats.
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The merozoite surface antigen 2 (MSA-2) proteins of Babesia bovis are members of the variable merozoite surface antigen (VMSA) family that have been implicated in erythrocyte invasion and are important targets for antibody-mediated blocking of invasion. Extensive sequence variation in another VMSA member, MSA-1, has been shown in all vaccine breakthrough isolates. To test the hypothesis that the msa-2 genes of vaccine breakthrough isolates would also encode a diverse set of proteins, the complete msa-2 locus was characterized from 12 Australian B. bovis strains and isolates, including two vaccine strains and eight vaccine breakthrough isolates, and compared to the loci in previously and newly characterized American strains. In contrast to American strains, the msa-2 loci of all Australian strains and isolates examined contain, in addition to msa-2c, only a solitary gene (designated msa-2a/b) closely related to American strain msa-2a and msa-2b. Nevertheless, the proteins encoded by these genes are quite diverse both between and within geographic regions and harbor evidence of genetic exchange among other VMSA family members, including msa-1. Moreover, all but one of the Australian breakthrough isolate MSA-2a/b proteins is markedly different from the vaccine strain from which immune escape occurred, consistent with their role in strain-specific protective immunity. The densest distribution of polymorphisms occurs in a hypervariable region (HVR) within the carboxy third of the molecule that is highly proline rich. Variation in length and content of the HVR is primarily attributable to differences in the order and number of degenerate nucleotide repeats encoding three motifs of unknown function. (+info)
Detection of Babesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA.
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Carrier cattle infected with Babesia bovis are difficult to detect because of the low numbers of parasites that occur in peripheral blood. However, diagnosis of low-level infections with the parasite is important for evaluating the efficacies of vaccines and in transmission and epidemiological studies. We used the polymerase chain reaction (PCR) to amplify a portion of the apocytochrome b gene from the parasite and tested the ability of this method to detect carrier cattle. The target sequence is associated with a 7.4-kb DNA element in undigested B. bovis genomic DNA (as shown previously), and the amplified product was detected by Southern and dot blot hybridization. The assay was specific for B. bovis, since no amplification was detected with Babesia bigemina, Trypanosoma brucei, Anaplasma marginale, or leukocyte DNA. The target sequence was amplified in DNA from B. bovis Mexico, Texas, and Australia S and L strains, demonstrating the applicability of the method to strains from different geographic regions. The sensitivity of the method ranged from 1 to 10 infected erythrocytes extracted from 0.5 ml of blood. This sensitivity was about 1,000 times greater than that from the use of unamplified parasite DNA. By the PCR method, six B. bovis carrier cattle were detected 86% of the time (range, 66 to 100%) when they were tested 11 times, while with microscopic examination of thick blood smears, the same carrier cattle were detected only 36% of the time (range, 17 to 66%). The method provides a useful diagnostic tool for detecting B. bovis carrier cattle, and the sensitivity is significantly improved over that of current methods. The results also suggest that characteristics of the apocytchrome b gene may make this a valuable target DNA for PCR-based detection of other hemoparasites. (+info)
Serological survey of Babesia bovis, Babesia bigemina, and Anaplasma marginale antibodies in cattle from the semi-arid region of the state of Bahia, Brazil, by enzyme-linked immunosorbent assays.
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The objectives of this work were to determine the prevalence of Babesia bovis, Babesia bigemina, and Anaplasma marginale detecting antibodies in cattle raised in the semi-arid region of the state of Bahia, Brazil, through indirect enzyme linked immunosorbent assays (ELISA) and to compare the performances of indirect enzyme-linked immunosorbent assays with crude (I-ELISA-CrAnaAg) and recombinant major surface protein-5 (I-ELISA-MSP-5Ag), as antigens to detect antibodies against A. marginale. An stable enzootic area was found in Senhor do Bonfim and Euclides da Cunha for B. bovis that showed 86 and 95.5% of prevalence, respectively, and for B. bigemina with 90.8 and 91.3%. On the other hand, Uaua and Juazeiro, were characterized as enzootically unstable areas, since prevalences were: B. bovis--63.7 and 56.4% and B. bigemina--53 and 54.8%, respectively. The prevalence of A. marginale in the four municipalities was above 97% with I-ELISA-CrAnaAg and 94.8% with I-ELISA-MSP-5Ag which is an indication of stable enzootic condition for the rickettsia. The I-ELISA-CrAnaAg and I-ELISA-MSP-5Ag showed a highly significant association (r = 0.977), which means that both ELISA tests are suitable for epidemiological studies of A. marginale. (+info)
The Babesia bovis merozoite surface antigen 1 hypervariable region induces surface-reactive antibodies that block merozoite invasion.
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A hypervariable region (HVR) previously identified in the carboxy-terminal one-third of the Babesia bovis variable merozoite surface antigen family was more extensively analyzed in merozoite surface antigen 1 (MSA-1) from 16 strains and isolates. The MSA-1 HVR is proline rich and contains three semiconserved motifs nearly identical to those described for the related family member MSA-2. Two MSA-1-specific monoclonal antibodies previously shown to be reactive with the merozoite surface bound to a recombinant construct encoding the HVR, indicating that the HVR is surface exposed and accessible to antibody binding. Importantly, these surface-reactive, HVR-specific monoclonal antibodies were capable of inhibiting merozoite infectivity of the host erythrocyte in vivo. The results indicate that the MSA-1 HVR is involved in erythrocyte invasion and suggest that selection of MSA-1 variants may be driven by invasion-blocking antibodies. (+info)
Validation of a competitive enzyme-linked immunosorbent assay for detection of antibodies against Babesia bovis.
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A previously developed competitive enzyme-linked immunosorbent assay (cELISA) based on a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of rhoptry-associated protein 1 of Babesia bovis was refined and validated for use internationally. Receiver operating characteristic analysis revealed an assay with a specificity and positive predictive value of 100% and a sensitivity of 91.1%, with various negative predictive values depending on the level of disease prevalence. The cELISA was distributed to four different laboratories, along with a reference set of 100 defined bovine sera, including known-positive, known-negative, and field samples. Pairwise concordances among the four laboratories ranged from 94% to 88%. Analysis of variance of the resulting optical densities and a test of homogeneity indicated no significant difference among the laboratories. Overall, the cELISA appears to have the attributes necessary for international application. (+info)
Transovarial transmission efficiency of Babesia bovis tick stages acquired by Rhipicephalus (Boophilus) microplus during acute infection.
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The protozoan parasite Babesia bovis, a reemerging threat to U.S. cattle, is acquired by adult female ticks of the subgenus Boophilus and is transovarially transmitted as the kinete stage to developing larval offspring. Sporozoites develop within larvae and are transmitted during larval feeding on a bovine host. This study evaluated the efficiency of B. bovis infection within Rhipicephalus (Boophilus) microplus following acquisition feeding on acutely parasitemic cattle. Parasite levels were quantified in blood from experimentally infected cattle and within hemolymph and larvae derived from acquisition-fed female B. microplus. There was a positive correlation between blood parasite levels in acutely parasitemic cattle and kinete levels in the hemolymph of adult female Boophilus ticks following acquisition feeding; however, there was no relationship between kinete levels in females and infection rates of larval progeny. Boophilus microplus females that acquisition fed produced larval progeny with infection rates of 12% to 48%. Importantly, larvae derived from replete females with very low levels of kinete infection, as demonstrated by microscopy and PCR, had infection rates of 22% to 30% and transmitted B. bovis during transmission feeding. These data demonstrate that although hemolymph infection may be undetectable, transmission to larval progeny occurs at a level which ensures transmission to the bovine host. (+info)
Induction of proliferative responses of T cells from Babesia bovis-immune cattle with a recombinant 77-kilodalton merozoite protein (Bb-1).
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A major portion of a Babesia bovis-specific gene encoding a 77-kDa merozoite protein (Bb-1) produced during natural infection in cattle and in microaerophilous culture was subcloned into the pGEX1N expression vector. Recombinant Bb-1 protein fused to glutathione S-transferase (Bb-1-GST) was used to examine cellular immune responses in B. bovis-immune cattle. Sera from rabbits immunized with Bb-1-GST reacted with fusion protein and with the native antigen present in crude B. bovis but not with B. bigemina merozoites. Bb-1-GST but not GST induced strong proliferation of T lymphocytes from these immune cattle, and Bb-1-reactive T-cell lines which consisted of a mixed population of either CD4+ and CD8+ cells or CD4+, CD8+, and "null" (gamma delta T) cells were established by in vitro stimulation of peripheral blood mononuclear cells with the recombinant fusion protein. Three CD4+ CD8- and three CD4- CD8+ Bb-1-specific T-cell clones were identified after limiting-dilution cloning of the cell lines. The studies described here demonstrate that the 77-kDa protein of B. bovis contains T-cell epitopes capable of eliciting proliferation of two types of T cells in immune cattle, an important consideration for the design of a recombinant subunit vaccine. (+info)
Persistently infected calves as reservoirs for acquisition and transovarial transmission of Babesia bovis by Rhipicephalus (Boophilus) microplus.
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Babesia bovis is a deadly disease of cattle resulting in severe economic losses in the vast regions of the world where it is endemic. If reintroduced into the United States, babesiosis would cause significant mortality in the naive cattle population. In order to address the risk to U.S. cattle, it is essential to quantify the transovarial transmission efficiency in adult female Boophilus microplus ticks following acquisition feeding on persistently infected cattle. This study tested the hypothesis that infection rates are the same for larval progeny derived from females fed to repletion during persistent or acute infection. Increasing parasite levels during acute infection correlated with an increasing number of females harboring kinetes detectable in hemolymph (r = 0.9). The percent infected larvae ranged from 0 to 20% when derived from females fed to repletion on persistently infected calves and from 4 to 6% when derived from females fed to repletion during acute parasitemia. There was no significant difference in infection rates of larval progeny, implying that the risk associated with the introduction of either persistently infected or acutely infected cattle is equal. Parasite levels ranged from 2.4 x 10(2) to 1.9 x 10(5) in 3-day-fed larvae derived from females fed to repletion on persistently infected cattle. One group of larvae failed to transmit the parasite, suggesting that a threshold level of parasites must be obtained by larval progeny via transovarial transmission in order for larvae to deliver sufficient parasites to infect a naive host. (+info)