G. Gale Wagner

A study of the systematics of Theileria spp. based upon small-subunit ribosomal RNA gene sequences

Abstract The systematics of benign and moderately pathogenic Theileria isolates from cattle and deer originating from different geographic regions was undertaken by small-subunit ribosomal RNA (SSU rRNA) gene nucleotide-sequence analysis. A maximum-likelihood phylogenetic tree constructed from these sequences resulted in two major divisions, each with a common ancestor. One major division branches into four relatively divergent groups, including (1) bovine Theileria sp. Type D (USA and Korea), (2) T. mutans Intona and Theileria sp. MSD (Africa), (3) T. cervi (USA), and (4) well-characterized pathogenic Theileria spp. (Africa). The other major division branches into two groups: (1) T. buffeli Warwick and T. buffeli Marula and (2) a second branch of closely related isolates with SSU rRNA gene Types B, B1, C, E, and H. Putative geographically associated diversity was noted only in the Korean bovine Theileria spp. with SSU rRNA gene types C and H and in African T. mutans Intona and Theileria sp. MSD. The current results show that the United States bovine Theileria isolates are not T. mutans because they have T. buffeli Marula (Type A) and/or Type D (species undesignated) SSU rRNA gene sequences. The taxonomic separation of T. buffeli Warwick from African T. mutans is confirmed in this study.

Identification of a point mutation in an esterase gene in different populations of the southern cattle tick, Boophilus microplus

Two esterase cDNA sequences were obtained from susceptible and organophosphorus resistant strains of Boophilus microplus. Both sequences have a high degree of homology to carboxylesterase B. One gene has identical sequences in both strains and the other showed two point mutations. One mutation produces an amino acid substitution when the amino acid sequence is deduced, this mutation was detected in six different populations susceptible and resistant to insecticides, but a pyrethroid resistant strain was the only one that showed only the mutant allele. Identification of this mutation and the strong signal detected in southern blot with this strain, suggest that esterases are contributing to detoxification of pyrethroid compounds, as a resistant mechanism in Mexican strains of the southern cattle tick.

Allele frequency and gene expression of a putative carboxylesterase-encoding gene in a pyrethroid resistant strain of the tick Boophilus microplus.

We utilized RNA Northern blot analysis and ribonuclease protection assays (RPA) to study the mRNA expression level of a putative carboxylesterase-encoding gene from several strains of Boophilus microplus (Canestrini). Both the Northern analysis and RPAs indicated that an esterase transcript was more abundant in the pyrethroid resistant strain, Coatzacoalcos (Cz), compared to a susceptible control strain and a resistant strain whose pyrethroid resistance is mediated through a target site insensitivity mechanism. A PCR-based assay was designed to identify the presence of a previously reported point mutation in this B. microplus esterase gene. The reported G-->A substitution at nucleotide 1120 creates an EcoR I site in the mutant allele which can be detected by EcoR I digestion of the amplification products. The PCR assays showed that the frequency of the mutant allele was highest in the Cz-resistant strain, which has been shown to have an esterase-mediated resistance mechanism. The PCR assay can be performed either on individual tick larvae or hemolymph from adults.

One Health approach to identify research needs in bovine and human babesioses: workshop report

BackgroundBabesia are emerging health threats to humans and animals in the United States. A collaborative effort of multiple disciplines to attain optimal health for people, animals and our environment, otherwise known as the One Health concept, was taken during a research workshop held in April 2009 to identify gaps in scientific knowledge regarding babesioses. The impetus for this analysis was the increased risk for outbreaks of bovine babesiosis, also known as Texas cattle fever, associated with the re-infestation of the U.S. by cattle fever ticks.ResultsThe involvement of wildlife in the ecology of cattle fever ticks jeopardizes the ability of state and federal agencies to keep the national herd free of Texas cattle fever. Similarly, there has been a progressive increase in the number of cases of human babesiosis over the past 25 years due to an increase in the white-tailed deer population. Human babesiosis due to cattle-associated Babesia divergens and Babesia divergens-like organisms have begun to appear in residents of the United States. Research needs for human and bovine babesioses were identified and are presented herein.ConclusionsThe translation of this research is expected to provide veterinary and public health systems with the tools to mitigate the impact of bovine and human babesioses. However, economic, political, and social commitments are urgently required, including increased national funding for animal and human Babesia research, to prevent the re-establishment of cattle fever ticks and the increasing problem of human babesiosis in the United States.

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.

Management factors associated with Babesia bovis seroprevalence in cattle from eastern Yucatán, Mexico.

The effect of management on the seroprevalence of Babesia bovis was studied in 399 Bos indicus cattle 1-2 years old from 92 farms in the eastern Yucatán, México. The management factors studied were: farm-type, production system, herd size, farm size, stocking density, vector control, dipping interval, type of dipping, type of acaricide and cattle introduction to the farm. A cross-sectional study was carried out (2-stage cluster sampling). The number of serum samples was proportionally distributed according to the number of farms in the nine locations of eastern Yucatán, México (399 animals from 92 farms). Antibody activity to B. bovis was tested using an indirect ELISA. The farms with a seroprevalence < or = 75% were considered as cases and those with seroprevalence > 75% were considered as controls. The variables with p < or = 0.20 were included in fixed effects logistic regression. The seroprevalence of the zone was 73.8% (66.3-81.3%). The following risk factors were found: Stocking density (< 1 head/ha, OR = 4.04, CI (OR) = 1.20-13.62) and dipping interval (> 60 days, OR = 5.07 CI (OR) = 1.26-20.48).

Babesia bovis: gene isolation and characterization using a mung bean nuclease-derived expression library

Genomic DNA prepared from erythrocyte cultures of Babesia bovis merozoites was digested with mung bean nuclease and used to construct a lambda gt11 expression library of B. bovis recombinants. Immunoscreening with two polyclonal antibody probes detected multiple recombinants from which two, designated Bb-1 and Bb-3, were chosen for further analysis. Monospecific immunoglobulins isolated from the screening sera using nitrocellulose-bound fusion proteins were employed to determine the native molecular weight and the intracellular location of the babesial proteins encoded by the recombinants. Clone Bb-1 encodes an antigen of 77,000 Da located at the apical end of the intraerythrocytic parasite. A protein of 75,000 Da encoded by clone Bb-3 is associated with the infected red blood cell cytoplasm and/or membrane but not with the merozoite.

Improved Enzyme-Linked Immunosorbent Assay Using C-Terminal Truncated Recombinant Antigens of Babesia bovis Rhoptry-Associated Protein-1 for Detection of Specific Antibodies

ABSTRACT An enzyme-linked immunosorbent assay (ELISA) based on a recombinant rhoptry-associated protein-1 (RAP-1) of Babesia bovis has been previously developed, but it was imperfect because some cross-reactions were still present in Babesia bigemina-infected bovine sera. To improve its accuracy for the specific detection of the antibodies to B. bovis, we constructed three C-terminal truncated recombinant antigens of the RAP-1—rCT1 (amino acids [aa] 301 to 408), rCT2 (aa 388 to 490), and rCT3 (aa 466 to 565)—by using a baculovirus expression system and evaluated their diagnostic potentials using ELISA. rCT1 and rCT2 were better diagnostic antigens in their sensitivities and diagnostic efficiencies than rCT3, although none of the recombinant antigens showed any cross-reactivity to B. bigemina-infected bovine sera. These results confirmed that the N-terminal 300-aa region caused cross-reactivity of the entire RAP-1 antigen, and the C-terminal truncated recombinant antigens were shown to be useful reagents for species-specific serodiagnosis.

Culture isolation and partial characterization of a Babesia sp. from a North American elk (Cervus elaphus).

Three North American yearling elk (Cervus elaphus) died with clinical symptoms suggestive of babesiosis. Babesia sp. organisms similar in morphology to B. odocoilei of white-tailed deer (Odocoileus virginianus) were observed in Giemsa-stained blood films from one of the elk. Continuous cultures of the parasite were established. Antiserum raised against the elk Babesia sp. isolate was compared to B. odocoilei specific antiserum in an immunofluorescent antibody assay; we found evidence of differences in reactivity to several Babesia spp. isolated from wildlife and domestic ruminants. Cultured parasites from the elk were not infective to either intact or splenectomized Bos taurus steers.

Comparative assessment of antibody isotypes to Brucella abortus by primary and secondary binding assays

Bovine isotypes IgM, IgG1, IgG2, and IgA, affinity purified via Brucella abortus lipopolysaccharide (LPS) attached covalently to agarose beads, were assessed for their antibody activity in various serological tests. In an enzyme linked immunosorbent test, using LPS as the antigen attached to a plastic matrix and goat anti-heavy chain specific IgG fractions conjugated to horseradish peroxidase, the limiting amount of antibody of each isotype detectable was 155 ng for IgM; 190 ng for IgG1; 220 ng for IgG2 and approximately 700 ng for IgA. In a hemolysis in gel test using LPS attached to bovine erythrocytes as the antigen, and guinea pig complement, the least amounts of detectable antibody were IgM:5500 ng; IgG1:60 ng; IgG2:4850 ng; IgA was not reactive. However, as preformed immune complexes all four isotypes were capable of activating bovine complement. For the indirect hemagglutination assay, both fresh sheep and chicken erythrocytes were sensitized with antigen. The least amounts of antibody detectable with LPS-coated sheep erythrocytes were IgG2: 625 ng; IgG1: 3750 ng; IgA: 4250 ng; and IgM: not reactive. Using LPS-coated chicken erythrocytes, only IgG2 was reactive to a limit of 1250 ng. Using isotypes as the antibody in an antibody-dependent cell-mediated cytotoxicity assay, normal bovine mononuclear cells as effector cells and chicken erythrocytes sensitized with LPS as target cells, only IgG2 was reactive. Specific activity was retained to approximately 1000 ng. The implication of these results in terms of diagnosis and protective immunity in brucellosis will be discussed.