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.     

Molecular investigation of tick-borne infections in cattle from Xinjiang Uygur Autonomous Region,China

Tick-borne diseases cause significant losses to livestock production in tropical and subtropical regions. However, information about the tick-borne infections in cattle in Xinjiang Uygur Autonomous Region (XUAR), northwestern China, is scarce. In this study, nested polymerase chain reaction (PCR) assays and gene sequencing were used to detect and analyze epidemiological features of Babesia bovis, B. bigemina, Coxiella burnetii and Anaplasma bovis infections in XUAR. Out of 195 samples tested, 24 (12.3%), 67 (34.4%), 40 (20.5%) and 10 (5.1%) were positive for B. bovis, B. bigemina, C. burnetii and A. bovis, respectively. Sequencing analysis indicated that B. bovis SBP-4, B. bigemina Rap1a, C. burnetii htpB and A. bovis 16S rRNA genes from XUAR showed 99%–100% identity with documented isolates from other countries. Phylogenetic analyses revealed that B. bovis SBP-4, B. bigemina Rap1a, C. burnetii htpB and A. bovis 16S rRNA gene sequences clustered in the same clade with isolates from other countries. To the best of our knowledge, this is the first report of C. burnetii infection of cattle in XUAR. Furthermore, this study provides important data for understanding the distribution of tick-borne pathogens, and is expected to improve the approach for prevention and control of tick-borne diseases in China.     

Impact of using pyronaridine tetraphosphate- based combination therapy in the treatment of babesiosis caused by Babesia bovis,B. caballi,and B. gibsoni in vitro and B. microti in mice

The inhibitory efficacies of pyronaridine tetraphosphate (PYR), when used in combination with two novel and potent antibabesial drugs; clofazimine (CF), and MMV396693 were evaluated in the current study against the growth of Babesia bovis, B. caballi, and B. gibsoni in vitro and B. microti in mice. The in vitro study against the selected parasites was performed using combination of PYR with either CF or MMV396693 in ratios ranged from 0.75:0.75 to 0.25:0.25. Combined application of PYR/MMV396693 revealed additive and indifferent interactions against the in vitro growth of all screened Babesia parasites. PYR in combination with CF, achieved indifferent and antagonistic interactions with all used concentration ratios against the in vitro growth of B. bovis and B. caballi. Treatment with PYR-CF combination therapy caused significant inhibition (P < 0.05) of the fluorescence values at days 12, 14, 16, 18, and 22 p.i. in comparison with control mice. Of note, treatment with combination therapy exhibited inhibition in the growth of B. microti (23.16%) greater than those caused by PYR alone. In summary, the obtained results highlight the improvement in the in vivo antibabesial efficacy of PYR when used in combination with CF rather than using PYR alone but such inhibition is still lower than those caused by either DA or CF monotherapies.     

Modification of host erythrocyte membranes by trypsin and chymotrypsin treatments and effects on the in vitro growth of bovine and equine Babesia parasites

In the present study, we investigated the effects of protease pretreatments of host erythrocytes (RBC) on the in vitro growth of bovine Babesia parasites (Babesia bovis and B. bigemina) and equine Babesia parasites (B. equi and B. caballi). The selected proteases, trypsin and chymotrypsin, clearly modified several membrane proteins of both bovine and equine RBC, as demonstrated by SDS-PAGE analysis; however, the protease treatments also modified the sialic acid content exclusively in bovine RBC, as demonstrated by lectin blot analysis. An in vitro growth assay using the protease-treated RBC showed that the trypsin-treated bovine RBC, but not the chymotrypsin-treated ones, significantly reduced the growth of B. bovis and B. bigemina as compared to the control. In contrast, the growth of B. equi and B. caballi was not affected by any of these proteases. Thus, the bovine, but not the equine, Babesia parasites require the trypsin-sensitive membrane (sialoglyco) proteins to infect the RBC.     

Lymphocytes and Macrophages Are Infected by Theileria equi,but T Cells and B Cells Are Not Required to Establish Infection In Vivo

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.     

First description of Coxiella burnetii and Rickettsia spp. infection and molecular detection of piroplasma co-infecting horses in Xinjiang Uygur Autonomous Region,China

Q fever, spotted fever rickettsioses and equine piroplasmosis, are some of the most serious equine tick-borne diseases caused by Coxiella burnetii, Rickettsia spp., Babesia caballi and/or Theileria equi. This study surveyed and molecularly characterized these pathogens infecting horses in ten ranches from XUAR, China using molecular technology. Among 200 horse blood samples, 163 (81.5%) were infected with at least one of the pathogens. Rickettsia spp. was the most prevalent pathogen (n = 114, 57.0%), followed by C. burnetii (n = 79, 39.5%), T. equi (n = 79, 39.5%) and B. caballi (n = 49, 24.5%). Co-infections were observed in 61.3% of positive samples in this study. Statistically significant differences were observed between the sampling regions for C. burnetii, B. caballi and T. equi, and also in different age group for C. burnetii and T. equi. The genotype analysis indicated that C. burnetii htpB, Rickettsia spp. ompA, B. caballi rap-1, B. caballi 18S rRNA, T. equi EMA-1 and T. equi 18S rRNA gene sequences from horses in XUAR were variable. To the best of our knowledge, this study is the first report of C. burnetii and Rickettsia spp. infection and co-infected with piroplasma in horses in China. Overall, this study revealed the high infection rate of the pathogens in horses in XUAR, China. The current findings are expected to provide a basis for better tick-borne disease control in the region.     

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.     

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.     

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.     

Babesia bovis: effects of cysteine protease inhibitors on in vitro growth

In the present study, we examined the effects of four kinds of cysteine protease inhibitors (E64, E64d, leupeptin, and ALLN) on the in vitro asexual growth of Babesia bovis. Of these, only the lipophilic inhibitors, E64d and ALLN, were found to effectively inhibit the growth of B. bovis. In further experiments, E64d, but not ALLN, significantly suppressed the parasite’s invasion of host erythrocytes, while both chemicals, especially ALLN, inhibited the parasite’s replication within the infected erythrocytes. These data suggested the presence of cysteine protease(s) derived from B. bovis, in which the protease(s) would play important roles in the erythrocyte invasion and/or replication processes of the parasite.     

Discovery of a novel species,Theileria haneyi n. sp., infective to equids,highlights exceptional genomic diversity within the genus Theileria: implications for apicomplexan parasite surveillance

A novel apicomplexan parasite was serendipitously discovered in horses at the United States – Mexico border. Phylogenetic analysis based on 18S rDNA showed the erythrocyte-infective parasite to be related to, but distinct from, Theileria spp. in Africa, the most similar taxa being Theileria spp. from waterbuck and mountain zebra. The degree of sequence variability observed at the 18S rDNA locus also suggests the likely existence of additional cryptic species. Among described species, the genome of this novel equid Theileria parasite is most similar to that of Theileria equi, also a pathogen of horses. The estimated divergence time between the new Theileria sp. and T. equi, based on genomic sequence data, is greater than 33?million years. Average protein sequence divergence between them, at 23%, is greater than that of Theileria parva and Theileria annulata proteins, which is 18%. The latter two represent highly virulent Theileria spp. of domestic cattle, as well as of African and Asian wild buffalo, respectively, which differ markedly in pathology, host cell tropism, tick vector and geographical distribution. The extent of genome-wide sequence divergence, as well as significant morphological differences, relative to T. equi justify the classification of Theileria sp. as a new taxon. Despite the overall genomic divergence, the nine member equi merozoite antigen (EMA) superfamily, previously found as a multigene family only in T. equi, is also present in the novel parasite. Practically, significant sequence divergence in antigenic loci resulted in this undescribed Theileria sp. not being detectable using currently available diagnostic tests. Discovery of this novel species infective to equids highlights exceptional diversity within the genus Theileria, a finding with serious implications for apicomplexan parasite surveillance.     

Serological comparison of Australian and South American strains of Babesia argentina and Anaplasma marginale

The indirect fluorescent antibody test was used for the serological comparison of the parasites called Babesia argentina and Anaplasma marginale in Australia and Bolivia, South America. Cross-testing performed with antigens and antisera prepared in the two continents proved serological identity for both parasites. Because a similar study by Goldman & Rosenberg (1974) showed serological identity between B. bovis and B. argentina, by the law of priority the small Babesia of Australia and South America should be called B. bovis. The findings have implications in vaccination.     

Immunologic and molecular identification of Babesia bovis and Babesia bigemina in free-ranging white-tailed deer in northern Mexico

The suitability of white-tailed deer (Odocoileus virginianus) as hosts for the cattle ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus (Boophilus) annulatus, has been well documented. These ticks have a wide host range, and both transmit Babesia bovis and Babesia bigemina, the agents responsible for bovine babesiosis. Although this disease and its vectors have been eradicated from the United States and some states in northern Mexico, it still is a problem in other Mexican states. It is not known if wild cervids like white-tailed deer can act as reservoirs for bovine babesiosis. The purpose of this study was to determine if B. bovis and B. bigemina or antibodies against them occur in white-tailed deer in the states of Nuevo Leon and Tamaulipas, Mexico. Twenty blood samples from white-tailed deer from two ranches were collected and tested with a nested polymerase chain reaction (nested PCR) and indirect immunofluorescence antibody test (IFAT) for B. bovis and B. bigemina. Eleven samples were positive for B. bigemina and four for B. bovis by nested PCR; amplicon sequences were identical to those reported in GenBank for B. bovis (Rap 1) and B. bigemina. Results of the IFA test showed the presence of specific antibodies in serum samples. This is the first report of the presence of B. bovis and B. bigemina in white-tailed deer using these techniques and underscores the importance of cervids as possible reservoirs for bovine babesiosis.     

Closing the empty anti-Babesia gibsoni drug pipeline in vitro using fluorescence-based high throughput screening assay

In the current study, we evaluated the usefulness of a SYBR Green I (SG I) fluorescence assay for evaluation of the inhibitory effects of antibabesial drugs against the in vitro growth of Babesia gibsoni. Linearity and high-throughput screening (HTS) assays exhibited the validity of the SG I fluorescence assay for B. gibsoni parasite when performed at low hematocrits (HCTs) (2.5% and 5%) without daily changing of the medium. Interestingly, 5% HCT showed the highest value of the signal/noise ratio. Of note, there were no significant differences (P > .05) in the IC₅₀s of the commonly used antibabesial drugs (diminazene aceturate and/or imidocarb dipropionate) that calculated by either the SG I fluorescence assay with and without daily medium changing or by the fluorescence and microscopy methods at 2.5% and 5% HCTs. Such results confirmed that both HCTs are valid for mass drug screening against the in vitro growth of B. gibsoni. While the results of the HTS assay add merit to the assay when performed at 5% HCT especially when incubating the plates for 2 h in a dark after adding lysis buffer with SG I stain. Next, nine different drugs were screened to confirm the assay's usefulness. MMV396693, pyronaridine tetraphosphate and nerolidol drugs exhibited the highest effectiveness against the in vitro growth of B. gibsoni, next to diminazene aceturate. In summary, SG I fluorescence assay with 5% HCT without daily changing of the medium for B. gibsoni offers a novel approach for the large-scale screening of huge chemical libraries in in vitro cultures.     

Comparison of cattle responses to mono-specific or combined inoculations with Babesia bigemina and Babesia bovis vaccine strains

Combined inoculation of cattle with vaccine strains of Babesia bigemina and Babesia bovis induced lower antibody titers to B. bigemina than to B. bovis (previous study). Three groups of heifers were used to detect if the low antibody level was due to competition between Babesia species: individuals of G1 and G2 were inoculated with 10 million B. bigemina and B. bovis, respectively, and those of G3 with 10 million of each parasite. The prepatent periods, maximum parasitaemias and antibody titers (indirect immunofluorescent antibody test) were evaluated. The mean prepatent periods (days) for B. bigemina was of 5.6 (G1) and 5.2 (G3) and 7.0 (G2) and 6.7 (G3) for B. bovis (P > 0.05, "t" test). No differences were found in the parasitaemias. The only difference was found in the antibody titers to B. bovis, that were lower (P < 0.05 "t" test) from week 7 onwards when B. bovis was used in combination. The biological significance of this difference is unclear.     

Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites

A loop-mediated isothermal amplification (LAMP) technique has been used as a novel nucleic acid detection method, whereby the target DNA can be amplified with high specificity and sensitivity under an isothermal condition using a set of four specific primers. In this study, we designed two sets of the LAMP primers for rhoptry-associated protein-1 genes of Babesia bovis and B. bigemina, in which a restriction enzyme cleavage site was inserted into two pairs of species-specific primers to construct a multiplex LAMP (mLAMP) method by combining these two sets totaling eight primers. The mLAMP method was distinguishable between B. bovis and B. bigemina, simultaneously, due to the subsequent restriction enzyme analysis. The sensitivities of the mLAMP method were 10(3) and 10(5) times higher on the detection limits for B. bovis and B. bigemina, respectively, than those of the classical PCR methods. Of 40 blood samples collected from cattle living in Ghana, 12 and 27% were positively detected by the mLAMP for B. bovis and B. bigemina, respectively. Furthermore, 14 and 23% of 90 blood samples from cattle in Zambia showed mLAMP-positive reactions to B. bovis and B. bigemina, respectively. These findings indicate that this mLAMP method is a new convenient tool for simultaneous detection of the bovine Babesia parasites.     

Seroprevalence of Babesia caballi and Theileria equi in the Swiss horse population

In Switzerland, the prevalence and incidence of equine piroplasma parasite (EPP) infections are unknown. In order to obtain a first insight into the prevalence, a representative sample of 689 sera of horses from Switzerland was serologically tested for the presence of antibodies directed against T. equi and B. caballi using the Indirect Fluorescence Antibody Test (IFAT). A total of 50 (7.3%) horses were seropositive for EPP: overall, the seroprevalence of T. equi was significantly higher than that of B. caballi (p = 0.002). The seropositivities in indigenous horses (animals bred and raised in Switzerland) and in imported horses were 4.8% (11/230) and 8.5% (39/459), respectively. Unlike in indigenous horses, where no significant difference in seroprevalences could be observed between the two parasite species, the seroprevalence of T. equi was significantly higher (p < 0.001) than that of B. caballi in imported horses. Horses imported from France, Spain and Portugal exhibited a significantly higher seroprevalence, and horses imported from Germany a significantly lower seroprevalence of EPP compared to indigenous horses. There were no associations between sex, age, weight loss, surgery or blood transfusions with T. equi and B. caballi seroprevalences.The overall seroprevalence of 7.3% clearly shows that infection with EPP is a threat to the health of the horses in Switzerland. With the presumed expansion of permissive tick vectors, EPP infections will potentially increase in importance in the future. Therefore, continuous monitoring is mandatory.     

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.