Molecular epidemiological survey of Babesia bovis,Babesia bigemina,and Babesia sp. Mymensingh infections in Mongolian cattle

Bovine babesiosis caused by Babesia species is an economically significant disease of cattle. Severe clinical babesiosis in cattle is caused by Babesia bovis, B. bigemina, and the recently discovered Babesia sp. Mymensingh. Mongolia is an agricultural country with a large cattle inventory. Although previous studies have detected active infections of B. bovis and B. bigemina in Mongolian cattle, only a few provinces were surveyed. Additionally, the endemicity of Babesia sp. Mymensingh in Mongolia remains unknown. We screened blood DNA samples from 725 cattle reared in 16 of the 21 Mongolian provinces using B. bovis-, B. bigemina-, and Babesia. sp. Mymensingh-specific PCR assays. The overall positive rates of B. bovis, B. bigemina, and Babesia sp. Mymensingh were 27.9% (n = 202), 23.6% (n = 171), and 5.4% (n = 39), respectively. B. bovis and B. bigemina were detected in cattle in all surveyed provinces; whereas Babesia sp. Mymensingh was detected in 11 of the 16 surveyed provinces. On a per province basis, the B. bovis- B. bigemina-, and Babesia sp. Mymensingh-positive rates were 5.9–52.0%, 9.1–76.3%, and 0–35.7%, respectively. In conclusion, this is the first report of Babesia sp. Mymensingh in Mongolia. In addition, we found that species of Babesia that are capable of causing bovine clinical babesiosis, including B. bovis, B. bigemina, and Babesia sp. Mymensingh, are widespread throughout the country.     

Gliding motility of Babesia bovis merozoites visualized by time-lapse video microscopy

Background

Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed “gliding motility”. However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite.

Methodology/Principal Findings

Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion.

Conclusions/Significance

This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding.     

Artemisone inhibits in vitro and in vivo propagation of Babesia bovis and B. bigemina parasites

Artemisone was evaluated, in in vitro and in vivo, for control of bovine babesiosis caused by Babesia bigemina and Babesiabovis parasites. In vitro, artemisone reduced parasitemia in a dose-dependent manner: the inhibitory effects increased gradually, reaching a maximum inhibition of 99.6% and 86.4% for B. bigemina and B. bovis, respectively 72 h after initiation of treatment with initial parasitemia of 0.5%. In calves infected with either B. bigemina or B. bovis artemisone treatment was well tolerated and prevented development of acute babesiosis in all animals except for one B. bovis-infected calf. The treatment did not eliminate all blood parasites, and recovered animals carried a persistent low-level infection. Treatment with artemisone may be useful as an alternative drug for preventing the pathology that results from babesiosis, without interfering with acquired immune protection following recovery from an acute babesiosis infection or vaccination.     

Evaluation of the inhibitory effect of Zingiber officinale rhizome on Babesia and Theileria parasites

The effect of Zingiber officinale rhizome methanolic extract (ZOR) on the in vitro growth of bovine Babesia (B. bovis, B. bigemina, and B. divergens) and equine piroplasm (B. caballi, and Theileria equi) parasites and on the growth of B. microti in mice was evaluated in this study. The possible in vitro synergistic interaction between ZOR and either diminazene aceturate (DA) or potent Medicines for Malaria Venture (MMV) hits from the malaria box was also investigated. In vitro, ZOR reduced the growth of B. bovis, B. bigemina, T. equi, and B. caballi in a dose-dependent manner. B. divergens was the most susceptible parasite to the in vitro inhibitory effect of ZOR. DA and MMV compounds enhanced the in vitro inhibitory antibabesial activity of ZOR. 12.5 mg/kg DA when administrated in combination with ZOR in mice exhibited a significant inhibition (P < 0.05) in B. microti growth better than those observed after treatment with 25 mg/kg DA monotherapy. These findings suggest that ZOR could be a viable medicinal plant for babesiosis treatment, particularly when combined with a modest dose of either DA or powerful anti-B. bigemina MMV hits.     

Babesia rodhaini, Babesia bovis, and Babesia bigemina: analysis and sorting of red cells from infected mouse or calf blood by flow fluorimetry using 33258 Hoechst.

The DNA of Babesia spp. parasites within host intact red blood cells was labeled using the fluorescent bisbenzimidazole dye 33258 Hoechst. The labeled cells were sorted on a fluorescence activated cell sorter on the basis of cell fluorescence (proportional to DNA content) and the intensity of light scattered from the cells at low angles (related to cell size). The optimal conditions for dye uptake were established for the murine parasite Babesia rodhaini and the bovine parasites B. bovis and B. bigemina. Uninfected cells were nonfluorescent after incubation with the dye and could be completely separated from infected fluorescent cells. The fluorescence of cells infected with B. rodhaini was proportional to the number of parasite nuclei per cell. With saturation levels of dye, samples infected with B. bovis or B. bigemina in which erythrocytes contained one or two parasites, both exhibited only one fluorescent cell peak. Cell sorting did not eliminate the infectivity of B. rodhaini. The method may be used to separate populations of uninfected blood cells and cells infected with Babesia spp. for biochemical and immunochemical experiments.     

Comparative analysis of gene expression between Babesia bovis blood stages and kinetes allowed by improved genome annotation

Throughout their life cycle, Babesia parasites alternate between a mammalian host, where they cause babesiosis, and the tick vector. Transition between hosts results in distinct environmental signals that influence patterns of gene expression, consistent with the morphological and functional changes operating in the parasites during their life stages. In addition, comparing differential patterns of gene expression among mammalian and tick parasite stages can provide clues for developing improved methods of control. Hereby, we upgraded the genome assembly of Babesia bovis, a bovine hemoparasite, closing a 139 kbp gap, and used RNA-Seq datasets derived from mammalian blood and tick kinete stages to update the genome annotation. Of the originally annotated genes, 1,254 required structural changes, and 326 new genes were identified, leading to a different predicted proteome compared to the original annotation. Next, the RNA-Seq data was used to identify B. bovis genes that were differentially expressed in the vertebrate and arthropod hosts. In blood stages, 28% of the genes were upregulated up to 300 fold, whereas 26% of the genes in kinetes, a tick stage, were upregulated up to >19,000 fold. We thus discovered differentially expressed genes that may play key biological roles and serve as suitable targets for the development of vaccines to control bovine babesiosis.     

Molecular epidemiology of bovine Babesia spp. and Theileria orientalis parasites in beef cattle from northern and northeastern Thailand

Beef cattle production represents the largest cattle population in Thailand. Their productivity is constrained by tick-borne diseases such as babesiosis and theileriosis. In this study, we determined the prevalence of Babesia bigemina, Babesia bovis and Theileria orientalis using polymerase chain reaction (PCR). The genetic markers that were used for detection of the above parasites were sequenced to determine identities and similarity for Babesia spp. and genetic diversity of T. orientalis. Furthermore the risk factors for the occurrence of the above protozoan parasites in beef cattle from northern and northeastern parts of Thailand were assessed. A total of 329 blood samples were collected from beef cattle in 6 provinces. The study revealed that T. orientalis was the most prevalent (30.1%) parasite in beef cattle followed by B. bigemina (13.1%) and B. bovis (5.5%). Overall, 78.7% of the cattle screened were infected with at least one of the above parasites. Co-infection with Babesia spp. and T. orientalis was 30.1%. B. bigemina and T. orientalis were the most prevalent (15.1%) co-infection although triple infection with the three parasites was observed in 3.0% of the samples. Sequencing analysis revealed that B. bigemina RAP1 gene and B. bovis SBP2 gene were conserved among the parasites from different cattle samples. Phylogenetic analysis showed that the T. orientalis MPSP gene from parasites isolated from cattle in north and northeast Thailand was classified into types 5 and 7 as reported previously. Lack of tick control program was the universal risk factor of the occurrence of Babesia spp. and T. orientalis infection in beef cattle in northern and northeastern Thailand. We therefore recommend training of farmers on appropriate tick control strategies and further research on potential vectors for T. orientalis and elucidate the effect of co-infection with Babesia spp. on the pathogenicity of T. orientalis infection on beef in northern and northeastern Thailand.     

Detection of Babesia bovis Using DNA Hybridization1

ABSTRACT. Plasmids containing inserts of Babesia bovis DNA were prepared and clones suitable for use in the diagnosis of B. bovis infections were isolated. Dot blot hybridization with DNA from these plasmids, which probably contain repetitive sequences, can detect after an overnight exposure 100 pg of B. bovis DNA, which corresponds to the amount of DNA present in 50 μl of 0.01% parasitemic erythrocytes. No detectable cross-hybridization was observed with Babesia microti, Plasmodium falciparum, Plasmodium vivax, Boophilus, or cow DNA. A small amount of cross-hybridization was observed with 10 ng Babesia bigemina DNA. Use of these probes in a hybridization assay may be helpful in the diagnosis of babesiosis in cattle and ticks, in the confirmation of strain identities, and in correlating virulence with particular strains of Babesia.     

qPCR estimates of <Emphasis Type="Italic">Babesia bovis</Emphasis> and <Emphasis Type="Italic">Babesia bigemina</Emphasis> infection levels in beef cattle and <Emphasis Type="Italic">Rhipicephalus microplus</Emphasis> larvae

Babesia spp. are tick-transmitted intraerythrocytic apicomplexan parasites that infect wild and domestic animals. Babesia bovis and B. bigemina are endemic and responsible for enormous economic losses to the livestock industry in most of the Brazilian territory, wherein the tick Rhipicephalus microplus is the unique vector. Better understanding of epidemiology and parasite–host interactions may improve the tools for disease control and genetic management for selection of resistant animals. This study aimed to detect, quantify and measure the correlation between B. bigemina and B. bovis infection levels in bovine blood and into tick, by absolute quantification of hemoparasite DNA using qPCR. Blood bovine samples and larvae pools from 10 engorged R. microplus females were collected from each Canchim heifers (5/8 Charolais?+?3/8 zebu, n?=?36). All evaluated samples were positive for both Babesia species tested. Correlations of B. bovis and B. bigemina levels between cattle and tick host were 0.58 and 0.66, respectively. These high positive correlation coefficients indicate that parasitemia load in the bovine may be dependent on or may determine the parasitemia load in the ticks.     

Babesia bigemina,Babesia argentina, and Anaplasma marginale: Coinfectious immunity in bovines

Forty-eight intact and eight splenectomized cattle were used to evaluate different systems of coinfectious immunization against Babesia bigemina, Babesia argentina, and Anaplasma marginale. Coinfectious immunity was induced by two methods: (1) blood of cattle acutely infected with B. bigemina, B. argentina and A. marginale was used as the source of inoculum and the post vaccination reactions were chemotherapeutically controlled with Imidocarb, Ganaseg, Gloxazone, and Liquamycin, and (2) by artificially inducing babesiosis with the blood of carrier cattle with chronic infections of B. bigemina and B. argentina without chemotherapy. The degree of resistance was determined by bloodborne and tick-borne challenges. Ticks were collected from cattle and identified as Boophilus microplus and Dermacentor nitens. Vaccinated cattle demonstrated a high degree of resistance to babesiosis and anaplasmosis; however, cattle without coinfectious immunity were treated chemotherapeutically to prevent death losses.     

Diversity of Babesia bovis merozoite surface antigen genes in the Philippines

Babesia bovis is the causative agent of fatal babesiosis in cattle. In the present study, we investigated the genetic diversity of B. bovis among Philippine cattle, based on the genes that encode merozoite surface antigens (MSAs). Forty-one B. bovis-positive blood DNA samples from cattle were used to amplify the msa-1, msa-2b, and msa-2c genes. In phylogenetic analyses, the msa-1, msa-2b, and msa-2c gene sequences generated from Philippine B. bovis-positive DNA samples were found in six, three, and four different clades, respectively. All of the msa-1 and most of the msa-2b sequences were found in clades that were formed only by Philippine msa sequences in the respective phylograms. While all the msa-1 sequences from the Philippines showed similarity to those formed by Australian msa-1 sequences, the msa-2b sequences showed similarity to either Australian or Mexican msa-2b sequences. In contrast, msa-2c sequences from the Philippines were distributed across all the clades of the phylogram, although one clade was formed exclusively by Philippine msa-2c sequences. Similarities among the deduced amino acid sequences of MSA-1, MSA-2b, and MSA-2c from the Philippines were 62.2–100, 73.1–100, and 67.3–100%, respectively. The present findings demonstrate that B. bovis populations are genetically diverse in the Philippines. This information will provide a good foundation for the future design and implementation of improved immunological preventive methodologies against bovine babesiosis in the Philippines. The study has also generated a set of data that will be useful for futher understanding of the global genetic diversity of this important parasite.     

New Molecules in Babesia gibsoni and Their Application for Diagnosis,Vaccine Development,and Drug Discovery

Babesia gibsoni is an intraerythrocytic apicomplexan parasite that causes piroplasmosis in dogs. B. gibsoni infection is characterized clinically by fever, regenerative anemia, splenomegaly, and sometimes death. Since no vaccine is available, rapid and accurate diagnosis and prompt treatment of infected animals are required to control this disease. Over the past decade, several candidate molecules have been identified using biomolecular techniques in the authors'' laboratory for the development of a serodiagnostic method, vaccine, and drug for B. gibsoni. This review article describes newly identified candidate molecules and their applications for diagnosis, vaccine production, and drug development of B. gibsoni.     

Bovine babesiosis: Cattle protected in the field with a frozen vaccine containing Babesia bovis and Babesia bigemina cultured in vitro with a serum-free medium

An attenuated live vaccine containing Babesia bovis and B. bigemina cultured in vitro with a serum-free medium was assessed for its clinical protection conferred of naïve cattle, under natural tick-challenge in a high endemicity zone to Babesia spp. Three groups of six animals were treated as follows: group I (GI) received a vaccine derived from parasites cultured with a free-serum medium; group II (GII) were immunized with the standard vaccine, with parasites cultured in a medium supplemented with 40% (v/v) bovine serum; and a control group (GIII) inoculated with non-infected bovine erythrocytes. Inocula were administered by IM route. Experimental animals were kept during 23 days after vaccination in a cattle farm free of ticks and Babesia spp. Thereafter, cattle were moved to a high endemicity farm for natural exposure to Babesia spp. transmitted by Rhipicephalus microplus ticks. Protection against clinical babesiosis was observed in bovines belonging to GI (100%) and GII (83.33%), while the control animals (GIII) were not protected, and showed severe clinical signs, closely related to babesiosis, were observed for at least three consecutive days during the challenge. These were fever, anemia, which were measured simultaneously, and circulating parasites were detected by optic light microscopy. All cattle showed B. bovis and B. bigemina in stained blood films during the challenge; B. bovis antibody titers were higher than those to B. bigemina in GI and GII, and lower titers were determined in GIII. The protective capacity of the vaccine derived from B. bovis and B. bigemina cultured in vitro in a serum-free medium was demonstrated.     

A library of recombinant Babesia microti cell surface and secreted proteins for diagnostics discovery and reverse vaccinology

Human babesiosis is an emerging tick-borne parasitic disease and blood transfusion-transmitted infection primarily caused by the apicomplexan parasite, Babesia microti. There is no licensed vaccine for B. microti and the development of a reliable serological screening test would contribute to ensuring the safety of the donated blood supply. The recent sequencing of the B. microti genome has revealed many novel genes encoding proteins that can now be tested for their suitability as subunit vaccine candidates and diagnostic serological markers. Extracellular proteins are considered excellent vaccine candidates and serological markers because they are directly exposed to the host humoral immune system, but can be challenging to express as soluble recombinant proteins. We have recently developed an approach based on a mammalian expression system that can produce large panels of functional recombinant cell surface and secreted parasite proteins. Here, we use the B. microti genome sequence to identify 54 genes that are predicted to encode surface-displayed and secreted proteins expressed during the blood stages, and show that 41 (76%) are expressed using our method at detectable levels. We demonstrate that the proteins contain conformational, heat-labile, epitopes and use them to serologically profile the kinetics of the humoral immune responses to two strains of B. microti in a murine infection model. Using sera from validated human infections, we show a concordance in the host antibody responses to B. microti infections in mouse and human hosts. Finally, we show that BmSA1 expressed in mammalian cells can elicit high antibody titres in vaccinated mice using a human-compatible adjuvant but these antibodies did not affect the pathology of infection in vivo. Our library of recombinant B. microti cell surface and secreted antigens constitutes a valuable resource that could contribute to the development of a serological diagnostic test, vaccines, and elucidate the molecular basis of host-parasite interactions.     

Enzydmatic Characterization of Babesia bovis1

ABSTRACT. Agarose gel electrophoresis was used to identify metabolic enzymes in Babesia bovis and B. bigemina. Glutamate dehydrogenase, lactate dehydrogenase, glucose phosphate isomerase, and hexokinase were identified in B. bovis- and B. bigemina-infected erythrocytes and B. bovis merozoite preparations. A specific electrophoretic mobility was observed for each enzyme. Malate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and adenylate kinase were only detected in normal erythrocyte preparations. Inter-species, but not intra-species, variation was noted when comparing electrophoretograms of both species. Kinin-activating activity was not detected in B. bovis-infected erythrocyte or merozoite preparations at pH 4.2 or 7.6.     

The effect of chemotherapy on Babesia bigemina in the tick vector Boophilus microplus

Percentages of feeding ticks in which Babesia bigemina could be detected (infection rates) were determined following treatment of bovine hosts with each of four babesicides. Infection rates were suppressed by imidocarb dipropionate, quinuronium sulphate and amicarbalide, reaching minimum levels 3–4 days after treatment, but imidocarb dihydrochloride had comparatively little effect. Total elimination of the parasite from ticks was not achieved. Treatment of tick infested hosts with imidocarb dipropionate or quinuronium sulphate failed to prevent transmission of B. bigemina by transovarian passage or by transfer of adult male ticks. These findings indicate that the use of babesicides for chemotherapy is unlikely to have a significant effect on the rate of transmission of B. bigemina.