Will L. Goff

Prospects for recombinant vaccines against Babesia bovis and related parasites

Babesial parasites infect cattle in tropical and temperate regions of the world and cause significant morbidity and mortality. Discovery of protective antigens that could be used in a killed vaccine has been slow and to date there are few promising vaccine candidates for cattle Babesia. This review describes mechanisms of protective innate and adaptive immune responses to babesial parasites and different strategies to identify potentially protective protein antigens of B. bovis, B. bigemina, and B. divergens. Successful parasites often cause persistent infection, and this paper also discusses how B. bovis evades and regulates the immune response to promote survival of parasite and host. Development of successful non‐living recombinant vaccines will depend on increased understanding of protective immune mechanisms and availability of parasite genomes.

A recombinant surface protein of Babesia bovis elicits bovine antibodies that react with live merozoites

Ten monoclonal antibodies (MoAbs) were generated against five surface-exposed proteins (16 kDa, 42 kDa, 44 kDa, 60 kDa, 225 kDa) on merozoites of Babesia bovis. A genomic library constructed in the lambda gt11 expression vector was screened with MoAbs in a plaque immunoassay for identification of clones expressing recombinant surface proteins. Two recombinant clones were identified (lambda Bo44-15 and lambda Bo44-16) that encoded a protein recognized by a MoAb specific for an epitope on the native 44-kDa surface protein. Southern blot analysis using radiolabeled Bo44-15 DNA (1.25 kb) against merozoite DNA and bovine leukocyte DNA confirmed the parasite-specificity of the cloned insert and revealed multiple bands of hybridization with merozoite DNA. Western blot analyses of lambda Bo44-15 lysogen preparations demonstrated that recombinant protein production in this clone was IPTG-induced and that the recombinant molecule was a beta-galactosidase fusion protein. Additionally, recombinant 44-kDa protein, purified by immunoaffinity chromatography, reacted with specific MoAb in Western blot assay indicating that the integrity of the epitope was retained during purification. Immune sera from calves immunized with purified recombinant Bo44-15 protein immunoprecipitated metabolically radiolabeled merozoite protein of 44 kDa indicating that antibody induced by recombinant Bo44-15 protein recognized native 44-kDa protein. Also, these sera reacted with the surface of live merozoites as evidenced by indirect immunofluorescence assay. Serum antibody titers determined by this assay had a wide range.

Molecular and Biological Characterization of a Newly Isolated Anaplasma Marginale Strain

Anaplasma marginale, a rickettsial hemoparasite of cattle and other ruminants, results in significant economic losses worldwide. Distinct strains of A. marginale have been identified based on differences in tick transmissibility, molecular size of surface proteins and DNA restriction fragments, and reactivity to a panel of monoclonal antibodies. These different strains vary considerably in their virulence, antigenic composition, and ability to protect against heterologous challenge. In this paper, we report on the molecular characterization of a newly isolated strain of A. marginale, designated St. Maries, recovered from an acutely infected cow in northern Idaho. Dermacentor andersoni ticks taken from the infected animal were tested for infection by RNA probe analysis. The infection rate of male ticks (as determined by midgut infection) was 100%, and the infection rate of female ticks was 83%. Infected male ticks were able to transmit the St. Maries strain to a susceptible calf. The high infection rate in male ticks may be particularly relevant, given that male ticks are believed to be epidemiologically important in transmission of A. marginale because of their intermittent feeding behavior, which promotes interhost transfer. The newly isolated strain differs from other US strains, including strains previously isolated in Idaho and Washington, based on reactivity to a panel of monoclonal antibodies and restriction fragment length polymorphisms. These results imply that antigenically distinct strains of A. marginale may arise within the same region. However, because epitopes previously demonstrated to be conserved in other A. marginale strains were also present in the St. Maries strain, it is possible to develop diagnostic tests against conserved epitopes that could be used for early identification of infected cows or ticks.

Evaluation of an enzyme-linked immunosorbent assay with recombinant rhoptry-associated protein 1 antigen against Babesia bovis for the detection of specific antibodies in cattle.

ABSTRACT The gene encoding Babesia bovis rhoptry-associated protein 1 (RAP-1) was used to develop an enzyme-linked immunosorbent assay (ELISA) to measure specific antibodies against B. bovis. The B. bovis RAP-1 gene was subcloned into a baculovirus transfer vector, and the RAP-1 protein was expressed in insect cells infected with a recombinant baculovirus. The recombinant B. bovis RAP-1 of 65 kDa was detected with anti-RAP-1 mouse serum by Western blotting, and this recombinant RAP-1 was used as an antigen in the ELISA. The ELISA was able to differentiate between B. bovis-infected sera and B. bigemina-infected sera or noninfected normal bovine sera. The results demonstrate that the recombinant RAP-1 expressed in insect cells might be a useful antigen for the detection of antibodies to B. bovis.

Comparison of a DNA probe, complement-fixation and indirect immunofluorescence tests for diagnosing Anaplasma marginale in suspected carrier cattle.

Most estimates of the prevalence of anaplasmosis have been based on serologic data using the complement-fixation (CF) and/or card agglutination tests. Since these tests are considered to be only about 50 percent reliable for detecting carrier cattle in enzootically stable herds, the need for more sensitive diagnostic tests is widely recognized. The objective in the present study was to compare the sensitivity of the CF test with that of the indirect immunofluorescence (IIF) test and a recently developed DNA probe in determining the prevalence of Anaplasma marginale infection in cattle from an enzootic area. The study herd consisted of 52 8-month-old steers and 13 3-year-old cows of mixed beef breed. All cattle were initially tested for this comparative purpose. All but one animal (one that was a positive reactor as assessed by all three tests, and served as a positive control), were treated with long-acting oxytetracycline in an attempt to clear any carrier infections. Each animal was then retested at 1 month and 2 months post-treatment (PT), in an effort to determine if the DNA probe could be used to evaluate the effectiveness of the drug. Six of the 65 (9.2%) initial serum samples were CF positive. In contrast, 60 (92.3%) and 64 (98.5%) of the samples were positive as assessed with the IIF test and the DNA probe, respectively. The DNA hybridization reactions varied in intensity within the sample population indicating different individual levels of infection. The DNA probe hybridized with two samples taken at 1 month PT, and with two different samples taken at 2 months PT. The mean IIF titers were reduced at both the 1 month and 2 month sampling times. These results suggest that the drug did not eliminate infections in all cattle. Some may have been cleared, but, in any event, the drug did reduce the level of infections below the sensitivity of the DNA probe and interrupted continuity of stimulation of antibody. Therefore, the DNA probe and the IIF test appear to be considerably more sensitive in detecting carrier infections than the CF test, and should be considered in future epidemiologic studies.

Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against Babesia bovis

ABSTRACT 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.

Competitive Enzyme-Linked Immunosorbent Assay Based on a Rhoptry-Associated Protein 1 Epitope Specifically Identifies Babesia bovis-Infected Cattle

ABSTRACT The competitive enzyme-linked immunosorbent assay (cELISA) format has proven to be an accurate, reliable, easily standardized, and high-throughput method for detecting hemoparasite infections. In the present study, a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of the rhoptry-associated protein 1 of the hemoparasite Babesia bovis was cloned and expressed as a histidine-tagged thioredoxin fusion peptide and used as antigen in a cELISA. The assay was optimized with defined negative and positive bovine sera, where positive sera inhibited the binding of the epitope-specific monoclonal antibody BABB75A4. The cELISA accurately differentiated animals with B. bovis-specific antibodies from uninfected animals and from animals with antibodies against other tick-borne hemoparasites (98.7% specificity). In addition, B. bovis-specific sera from Australia, Argentina, Bolivia, Puerto Rico, and Morocco inhibited the binding of BABB75A4, confirming conservation of the epitope. The assay first detected experimentally infected animals between 13 and 17 days postinfection, and with sera from naturally infected carrier cattle, was comparable to indirect immunofluorescence (98.3% concordance). The assay appears to have the characteristics necessary for an epidemiologic and disease surveillance tool.

SUSCEPTIBILITY OF ELK (CERVUS ELAPHUS) TO EXPERIMENTAL INFECTION WITH ANAPLASMA MARGINALE AND A. OVIS

Anaplasma ovis was experimentally transmitted from domestic sheep to elk (Cervus elaphus) and hack to splenectomized sheep. No rickettsemias were detected but serum from three of seven experimentally inoculated elk developed Anaplasma spp.-reactive antibody as measured by indirect immunofluorescence (IIF) or by the rapid card agglutination and complement fixation assays. Three elk were experimentally infected with A. marginale. The rickettsiae were detected in blood of these elk and caused disease in a splenectomized domestic bovine calf after subinoculation of blood from the elk. All three elk had positive titers with IIF. No clinical signs of illness were noted in any elk inoculated with either Anaplasma species.

The Isolation and Partial Characterization of A Babesia Sp. From Desert Bighorn Sheep (Ovis Canadensis Nelsoni)

ABSTRACT. A novel Babesia parasite of desert bighorn sheep was isolated. Its taxonomic description, host range, pathogenicity and antigenic relatedness were in vestigated. the parasite was infective for black‐tailed and white‐tailed deer, but with host‐specific differences compared to that of bighorn sheep. A splenectomized calf and domestic sheep were refractory to infection. A comparative immunofluorescence assay detected antigens cross‐reactive with Babesia odocoilei, B. divergens, B. equi and B. caballi, but not with B. bovis or, B. bigemina. Babesia odocoilei was also infective for bighorn sheep, allowing comparison by a cross‐challenge experiment, the results of which supported the conclusion that this parasite was not B. odocoilei. However, the bighorn sheep Babesia cannot currently be distinguished from B. capreoli described from roe deer in northern Germany. Data indicate that, while this parasite may not present a problem for domestic animals, it may cause disease in bighorn sheep and deer populations.

DNA probes distinguish geographical isolates and identify a novel DNA molecule of Babesia bovis.

A genomic DNA library of Babesia bovis was screened to identify DNA probe candidates for direct detection of the parasite. Two sequences, Bo6 and Bo25, had the highest sensitivity and further analysis revealed unique characteristics of each of these. Neither sequence hybridized detectably to bovine DNA. Bo6 detected 100 pg of both a Mexican and an Australian isolate of B. bovis, but Bo6 also detected 1.0 ng of Babesia bigemina DNA under identical conditions. A unique characteristic of Bo6 is that it hybridizes to an apparent 7.4-kilobase DNA in undigested genomic DNA of both B. bovis and B. bigemina. The sequence is well conserved between the 2 geographic isolates of B. bovis, but it is apparently divergent in B. bigemina. Bo25 did not hybridize detectably to bovine or B. bigemina DNA. This sequence detected 100 pg of homologous B. bovis Mexican isolate DNA, but the sensitivity was reduced to 1 ng for the Australian isolate DNA. The restriction enzyme profile of the Bo25 sequence in genomic DNA differed markedly in the number, size, and intensity of bands between the 2 B. bovis geographic isolates tested. Thus, the Bo25 sequence can distinguish geographic isolates of B. bovis.