Monoclonal antibody to a conserved epitope on proteins encoded by Babesia bigemina and present on the surface of intact infected erythrocytes

To define Babesia bigemina-specific antigens on the surface of infected erythrocytes, monoclonal antibodies (MAbs) were identified by live-cell immunofluorescence. As determined by live-cell immunofluorescence, two MAbs made to the Mexico strain reacted with the Mexico strain and three Kenya strains, while three MAbs made to the Kenya-Ngong strain reacted with the Kenya strains but not the Mexico strain. Binding of MAb 44.18 (made to the Mexico strain) to a strain-common epitope was confirmed by immunoelectron microscopy and by surface-specific immunoprecipitation of (35S) methionine-labeled proteins (200, 28 and 16 kDa in size), which also demonstrated that the MAb recognized an epitope on proteins encoded by B. bigemina. In immunoblots, the MAb bound to predominant antigens with sizes of 200 and 220 kDa in erythrocyte lysates infected with strains from Puerto Rico, St. Croix, Texcoco (Mexico), Kenya, and Mexico. Major antigens with sizes of antigens bound to erythrocytes infected with either the Mexico or Kenya strains as determined by live-cell immunofluorescence, allowing the conclusion that at least one conserved surface epitope was recognized. Calf precipitation, and infected erythrocytes sensitized with these antibodies were phagocytized by cultured bovine peripheral blood monocytes. These results provide a rationable for evaluating antigens identified by MAb 44.18 individually and as components of subunit vaccines

Comparison of a competitive inhibition ELISA and the card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in cattle

Objective: To compare a recently developed recombinant MSP-5 competitive inhibition ELISA with a card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centralein Australian cattle. Materials and methods: The ELISA was compared with the card agglutination test using 208 sera from cattle in Anaplasma-free herds, 86 sera from cattle experimentally infected with A marginale or A centrale and 757 sera from cattle in areas endemic for A marginale. Results: The specificity of the ELISA, based on testing 208 sera from cattle in Anaplasma-free areas, was 99.5%, and the sensitivities for detection of antibodies to A marginale and A centrale in sera from the experimentally infected cattle were 98.0% and 100%, respectively. For the same sets of sera, the specificity of the card agglutination test was 98.6% and the sensitivities for detection of antibodies to A marginale and A centrale were 98.0% and 100%, respectively. For the 757 sera collected from cattle in areas endemic for A marginale, the agreement between the ELISA and the card agglutination test depended on the positive threshold selected for the ELISA. The maximum achievable agreement was 91.5% (kappa = 0.73; 95% confidence interval 0.66, 0.79). Conclusion: We conclude that the competitive inhibition ELISA is a useful alternative to the card agglutination test for detection of A marginale or A centrale infection in cattle. The assay should be particularly useful for epidemiological applications such as prevalence studies and control programs

A Babesia bovis gene syntenic to Theileria parva p67 is expressed in blood and tick stage parasites

Completion of the Babesia bovis (T2Bo strain) genome provides detailed data concerning the predicted proteome of this parasite, and allows for a bioinformatics approach to gene discovery. Comparative genomics of the hemoprotozoan parasites B. bovis and Theileria parva revealed a highly conserved syntenic block of genes flanking the p67 gene of T. parva, a sporozoite stage-specific vaccine candidate against East Coast fever (ECF). The syntenic gene in B. bovis, designated bov57, encodes a protein of limited amino acid sequence identity (11.8%) to p67. Monoclonal antibodies were produced against recombinant BOV57 and were used to demonstrate expression of BOV57 in merozoite and kinete stages of the T2Bo strain of B. bovis. Transcript levels of bov57 in kinetes were increased 100-fold in comparison to msa-1, a previously identified gene encoding an erythrocyte stage surface protein. Amino acid sequence comparisons between the T2Bo strain and two attenuated and virulent strains from Argentina and Australia revealed a high degree of sequence conservation in BOV57 among these geographically and pathogenically divergent isolates (97% amino acid sequence identity). Additional genomic comparisons show that the bov57 gene locus is also conserved inB. bigemina and B. equi. While not identifiable through amino acid or nucleotide sequence similarity, the conserved gene order within this locus in multiple piroplasms may suggest a critical function adapted for each species’ unique host and life-cycle

Cloning, characterization and expression of a 200 Kilodalton diagnostic antigen of Babesia bigemina

Current serological tests for Babesia bigemina use semipurified merozoite antigens derived from infected erythrocytes. One of the major drawbacks of these tests is that antigen quality can vary from batch to batch. Since the quality of the antigen contributes to the sensitivity and specificity of serological tests, the use of standardized recombinant antigens should ensure consistency in assay quality. Previously, a 200-kDa merozoite antigen (p200) was identified as a candidate diagnostic antigen for use in a serological assay for the detection of B. bigemina antibodies in infected cattle. In this study, we have cloned, characterized, and expressed p200. A 3.5-kbp cDNA clone encoding p200 was isolated and shown to be almost full length, lacking approximately 300 bp at the 5' end. The predicted amino acid sequence shows that p200 consists of a long, highly charged central repeat region of an uninterrupted alpha helix, indicative of a fibrous protein. Immunoelectron microscopy localized p200 to the merozoite cytoplasm, suggesting that the antigen may be a structural protein involved in forming filament structures within the cytoskeleton. The 3.5-kbp cDNA was expressed in bacteria as a fusion protein with glutathione S-transferase (GST), but the yield was poor. To improve the yield, cDNA fragments encoding antigenic domains of p200 were expressed as fusions with GST. One of these fusion proteins, C1A-GST, is composed of a 7-kDa fragment of the p200 repeat region and contains epitopes that react strongly with sera from cattle experimentally infected with B. bigemina. Recombinant C1A-GST should permit the development of an improved enzyme-linked immunosorbent assay for the detection of antibodies against B. bigemina

The role of osmiophilic bodies and Pfg377 expression in female gametocyte emergence and mosquito infectivity in the human malaria parasite <i>Plasmodium falciparum</i>

Osmiophilic bodies are membrane-bound vesicles, found predominantly in Plasmodium female gametocytes, that become progressively more abundant as the gametocyte reaches full maturity. These vesicles lie beneath the subpellicular membrane of the gametocyte, and the release of their contents into the parasitophorous vacuole has been postulated to aid in the escape of gametocytes from the erythrocyte after ingestion by the mosquito. Currently, the only protein known to be associated with osmiophilic bodies in Plasmodium falciparum is Pfg377, a gametocyte-specific protein expressed at the onset of osmiophilic body development. Here we show by targeted gene disruption that Pfg377 plays a fundamental role in the formation of these organelles, and that female gametocytes lacking the full complement of osmiophilic bodies are significantly less efficient both in vitro and in vivo in their emergence from the erythrocytes upon induction of gametogenesis, a process whose timing is critical for fertilization with the short-lived male gamete. This reduced efficiency of emergence explains the significant defect in oocyst formation in mosquitoes fed blood meals containing Pfg377-negative gametocytes, resulting in an almost complete blockade of infectio

Survey of the livestock ticks of the North West province, South Africa

Ticks, as vectors of disease and damage agents, impact directly and indirectly on the economy of the livestock industry in southern Africa. This study surveyed the occurrence and distribution of ticks infesting livestock across the North West province, South Africa. During three phases in consecutive years, officers of the provincial Veterinary Department collected specimens monthly from livestock hosts at specified sites across the province. Data analysis constituted the fourth phase of the study. A total of 1090 collections from 265 sites yielded 42 566 tick specimens, comprising 22 different tick species (18 ixodids, 4 argasids). The specimens represent all of the major tick vectors of disease that occur in South Africa. The major tick-borne diseases (i.e. heartwater, both African and Asiatic bovine babesiosis and anaplasmosis) were found to be prevalent mainly in the north-eastern region of the province, which also displayed the highest tick species diversity. The central region appears transitory to some of the major vectors. Although some tick species were contained within specific regions, others were widespread across the province. Associated serology data show that most herds sampled in areas endemic for babesiosis and anaplasmosis in the north-eastern region are endemically unstable and at risk to these tick-borne diseases should vector control measures become ineffective