Development of a Gaussia luciferase immunoprecipitation assay for detecting Schistosoma japonicum infection.

Timely and accurate diagnosis of Schistosoma infection is important to adopt effective strategies for schistosomiasis control. Previously, we demonstrated that Schistosoma japonicum can secret extracellular vesicles and their cargos may serve as a novel type of biomarkers for diagnosing schistosomiasis. Here, we developed a Gaussia luciferase immunoprecipitation assay combined with S. japonicum extracellular vesicle (SjEV) protein to evaluate its potential for diagnosing schistosomiasis. A saposin-like protein (SjSLP) identified from SjEVs was fused to the Gaussia luciferase as the diagnostic antigen. The developed method showed good capability for detecting S. japonicum infection in mice and human patients. We also observed that the method could detect Schistosoma infection in mice as early as 7 days of post-infection, which showed better sensitivity than that of indirect ELISA method. Overall, the developed method showed a good potential for detecting Schistosoma infection particularly for early stage, which may provide an alternative strategy for identify Schistosoma infection for disease control.

Microscopic and molecular detection of Deraiophoronema evansi (Lewis, 1882) in domestic Bactrian camels (Camelus bactrianus) of Mongolia.

Cameline filarosis is an important parasitic disease having an economic impact on the camel industry around the world. However, there has been no study on filarosis in Bactrian camels of Mongolia. Therefore, the aim of the present study was to detect and identify microfilariae of Deraiophoronema evansi (D. evansi) in Bactrian camels from three provinces, located in southern and southwestern Mongolia. Blood samples were obtained from 400 healthy two-humped camels of different ages and both sexes. All blood samples were analysed using a variety of diagnostic techniques. Microfilariae were detected in 30 Bactrian camels (7.5%) by the Knott technique, while 13 Bactrian camels (3.3%) tested positive in a direct smear test. D. evansi was detected in 18 Bactrian camels (4.5%) by PCR assay. Prevalence was shown to be high among Bactrian camels in the age group up to 5 years, while the lowest positive results were obtained for Bactrian camels in the 5-10-year age group and the over 10-year age group. To confirm the morphological identification, D. evansi-COI gene sequences were subjected to phylogenetic analyses. The D. evansi-COI gene sequences from Mongolian two-humped camels were identical to sequences from Iranian one-humped camels and were clustered together with these sequences in the phylogeny. This is the first report of molecular detection and identification of microfilariae of D. evansi in Bactrian camels of Mongolia.

Phytochemical Characterization and Chemotherapeutic Potential of Cinnamomum verum Extracts on the Multiplication of Protozoan Parasites In Vitro and In Vivo.

Cinnamomum verum is a commonly used herbal plant that has several documented properties against various diseases. The existing study evaluated the inhibitory effect of acetonic extract of C. verum (AECV) and ethyl acetate extract of C. verum (EAECV) against piroplasm parasites in vitro and in vivo. The drug-exposure viability assay was tested on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3) and human foreskin fibroblast (HFF) cells. Qualitative phytochemical estimation revealed that AECV and EAECV containing multiple bioactive constituents namely alkaloids, tannins, saponins, terpenoids and remarkable amounts of polyphenols and flavonoids. AECV and EAECV inhibited B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi multiplication at half-maximal inhibitory concentrations (IC50) of 23.1 ± 1.4, 56.6 ± 9.1, 33.4 ± 2.1, 40.3 ± 7.5, 18.8 ± 1.6 µg/mL, and 40.1 ± 8.5, 55.6 ± 1.1, 45.7 ± 1.9, 50.2 ± 6.2, and 61.5 ± 5.2 µg/mL, respectively. In the cytotoxicity assay, AECV and EAECV affected the viability of MDBK, NIH/3T3 and HFF cells with half-maximum effective concentrations (EC50) of 440 ± 10.6, 816 ± 12.7 and 914 ± 12.2 µg/mL and 376 ± 11.2, 610 ± 7.7 and 790 ± 12.4 µg/mL, respectively. The in vivo experiment showed that AECV and EAECV were effective against B. microti in mice at 150 mg/kg. These results showed that C. verum extracts are potential antipiroplasm drugs after further studies in some clinical cases.

Flavonoids isolated from the flowers of Pulsatilla flavescens and their anti-piroplasm activity.

Pulsatilla species are known as "Yargui", and their flowers are traditionally used in Mongolia as a tonic and for the treatment of inflammatory diseases. By chemical investigation of P. flavescens flowers, 21 flavonoids, including a new chalcone C-glucoside, chalconaringenin 2'-O-ß-D-glucopyranosyl-5'-ß-D-glucopyranoside, and two new flavanone C-glucosides, (2R)- and (2S)-naringenin 8-ß-D-glucopyranosyl-4'-O-ß-D-glucopyranoside, were isolated. The absolute configurations of the seven flavanone glucosides were elucidated by ECD spectra. For the isolated compounds, inhibitory activity against Babesia caballi and Theileria equi, which cause fatal diseases in horses, was estimated. Although most of the isolated chalcone and flavanone derivatives did not show any anti-piroplasm activity, all the isolated flavone and flavonol derivatives showed moderate effects against B. caballi and/or T. equi.

Flavonoid and Galloyl Glycosides Isolated from Saxifraga spinulosa and Their Antioxidative and Inhibitory Activities against Species That Cause Piroplasmosis.

Eight new flavonoid-based 3'-O-ß-d-glucopyranosides (1-8) and three new galloyl glucosides (9, 11, 12), were isolated from the aerial parts of Saxifraga spinulosa, along with 25 known compounds. The structures of the new compounds were elucidated by spectroscopic methods. Most of the isolated compounds exhibited potent DPPH radical-scavenging activities. Further, their inhibitory activities were evaluated against Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi, protozoan parasites that cause piroplasmosis in livestock. The results indicated that several of these compounds showed growth-inhibitory effects on such organisms that cause piroplasmosis.

Babesia bovis BOV57, a Theileria parva P67 homolog, is an invasion-related, neutralization-sensitive antigen.

Babesia bovis BOV57, which is a homolog of the Theileria parva vaccine candidate antigen P67, is expressed in both the tick and blood stages of the life cycle of this parasite. However, the vaccine potential of BOV57 remained to be investigated. In the present study, we generated recombinant BOV57 (rBOV57) and prepared polyclonal antibodies against rBOV57 in mice and rabbits. Indirect immunofluorescence assays conducted with the mouse anti-rBOV57 antibody demonstrated that BOV57 localized at the apical end of paired merozoites in infected bovine red blood cells, whereas the antigen was found in the parasite membrane around the apical end of intraerythrocytic single and extracellular merozoites. In an invasion-inhibition assay, the rabbit anti-rBOV57 antibody potentially inhibited RBC invasion of B. bovis merozoites in vitro. In addition, the invasion inhibition mediated by rabbit anti-rBOV57 antibody resulted in a reduced growth rate of B. bovis in the in vitro culture. These findings indicated that B. bovis BOV57 plays a critical role in the invasion of merozoites into red blood cells, suggesting its potential as a subunit vaccine candidate against B. bovis infection in cattle. Furthermore, we analyzed the genetic diversity of bov57 gene sequences isolated from Sri Lanka, Mongolia, the Philippines, and Vietnam. The bov57 gene sequences derived from Mongolia, the Philippines, and Vietnam were conserved, whereas insertion and/or deletion mutations resulted in sequence diversity among the Sri Lankan samples. In summary, BOV57 is an invasion-related, neutralization-sensitive antigen encoded by the bov57 gene, which displays higher sequence diversity than previously reported.

Recombinant methionine aminopeptidase protein of Babesia microti: immunobiochemical characterization as a vaccine candidate against human babesiosis.

Human babesiosis is the most important zoonotic protozoan infection in the world. This is the first report of the cloning, expression, purification, and immunobiochemical characterization of a methionine aminopeptidase 1 (MetAP1) protein from Babesia microti (B. microti). The gene encodes a MetAP1 protein of B. microti (BmMetAP1) of approximately 66.8 kDa that includes glutathione S-transferase (GST) tag and shows MetAP activity. BmMetAP1 was detected in a lysate of B. microti and further localized in cytoplasm of the B. microti merozoite. rBmMetAP1 was found to be immunogenic, eliciting a high antibody titer in mice. Moreover, rBmMetAP1 stimulated the production of IFN-γ and IL-12 but not IL-4. Finally, rBmMetAP1 was able to provide considerable protection to mice against a B. microti challenge infection based on a reduction in peak parasitemia levels and earlier clearance of the parasite as compared with control mice. Taken together, these results suggest that rBmMetAP1 confers significant protection against experimental B. microti infection and might be considered a potential vaccine target against human babesiosis.

Molecular and biochemical characterization of methionine aminopeptidase of Babesia bovis as a potent drug target.

Aminopeptidases are increasingly being investigated as therapeutic targets in various diseases. In this study, we cloned, expressed, and biochemically characterized a member of the methionine aminopeptidase (MAP) family from Babesia bovis (B. bovis) to develop a potential molecular drug target. Recombinant B. bovis MAP (rBvMAP) was expressed in Escherichia coli (E. coli) as a glutathione S-transferase (GST)-fusion protein, and we found that it was antigenic. An antiserum against the rBvMAP protein was generated in mice, and then a native B. bovis MAP was identified in B. bovis by Western blot assay. Further, an immunolocalization assay showed that MAP is present in the cytoplasm of the B. bovis merozoite. Analysis of the biochemical properties of rBvMAP revealed that it was enzymatically active, with optimum activity at pH 7.5. Enhanced enzymatic activity was observed in the presence of divalent manganese cations and was effectively inhibited by a metal chelator, ethylenediaminetetraacetic acid (EDTA). Moreover, the enzymatic activity of BvMAP was inhibited by amastatin and bestatin as inhibitors of MAP (MAPi) in a dose-dependent manner. Importantly, MAPi was also found to significantly inhibit the growth of Babesia parasites both in vitro and in vivo; additionally, they induced high levels of cytokines and immunoglobulin (IgG) titers in the host. Therefore, our results suggest that BvMAP is a molecular target of amastatin and bestatin, and those inhibitors may be drug candidates for the treatment of babesiosis, though more studies are required to confirm this.

Identification and characterization of profilin antigen among Babesia species as a common vaccine candidate against babesiosis.

We have characterized a member of the profilin (PROF) family protein as a common antigen in three pathogens-Babesia bovis (B. bovis), Babesia bigemina (B. bigemina), and Babesia microti (B. microti)-and evaluated its immunogenic and cross-protective properties against a challenge infection with B. microti in BALB/c mice. The recombinant PROF proteins of B. bovis, B. bigemina, and B. microti were successfully expressed in Escherichia coli (E. coli) as soluble GST fusion proteins (rBboPROF, rBbigPROF, and rBmPROF, respectively), and they were found to be antigenic. On probing with mouse anti-rPROF serum, green fluorescence was observed on the parasites' cytosols by confocal laser microscopy. Immunization regimes in BALB/c mice using rPROFs induced cross-protective immunity against B. microti infection based on high levels of cytokines and immunoglobulin (IgG) titers, a reduction in peak parasitemia levels, and earlier clearance of the parasite as compared with control mice. The findings of the present study indicate that PROF is a common antigen among bovine and murine Babesia parasites, and it might be used as a common vaccine candidate against babesiosis.

A longitudinal study of Babesia and Theileria infections in cattle in Sri Lanka.

Throughout the world, infections with the Babesia and Theileria parasites often result in economically significant clinical disease in cattle. We conducted a longitudinal survey of Babesia and Theileria infections in cattle from the Polonnaruwa (n=75; dry zone) and Nuwara Eliya (n=161; wet zone) districts of Sri Lanka. DNA from blood samples collected in June, September, and December 2014 and March 2015 was screened for Babesia bovis, Babesia bigemina, Theileria annulata and Theileria orientalis using specific polymerase chain reactions (PCRs). Additionally, serum samples collected from the animals were screened using enzyme-linked immunosorbent assays (ELISAs) to detect B. bovis- and B. bigemina-specific antibodies. All of the animals surveyed in Polonnaruwa and 150 (93.2%) of the animals surveyed in Nuwara Eliya were PCR-positive for Babesia and/or Theileria at least once during the study period. A greater percentage of the cattle in Polonnaruwa were positive for T. annulata and T. orientalis than B. bovis or B. bigemina at all time points. T. orientalis was the most common infection in Nuwara Eliya. Additionally, more cattle were seropositive for B. bigemina than B. bovis in both districts. Although significant variations were sometimes observed in the rates of animals that were positive for B. bigemina, T. annulata, and T. orientalis at the different sampling time points, the rates of new infections with these parasites (by PCR or ELISA) on second, third, and fourth time points among the parasite-negative samples at the first, second, and third time points, respectively, did not differ between the sampling in either district-suggesting that the parasite species infected cattle at a constant rate in these locations. However, in Polonnaruwa, the rates of new infection with T. annulata were higher than the rates of new infection with T. orientalis. The rates were also higher than those in Nuwara Eliya. In Nuwara Eliya, the rates of new infection with T. orientalis were higher than the rates of new infection with T. annulata. The rates were also higher than those in T. orientalis in Polonnaruwa. These differences might be due to variations in the density and activity of the specific tick vectors within and between the districts. Our findings suggest the need for year-round control measures against bovine Babesia and Theileria infection in Sri Lanka. Further studies to determine the densities of the vector tick species in the different geographical areas of the country are warranted.

Evaluation of the inhibitory effects of miltefosine on the growth of Babesia and Theileria parasites.

Miltefosine, a membrane-active synthetic ether-lipid analogue, has antiproliferative and antiparasitic effects. In this study, the inhibitory effects of miltefosine were evaluated against three Babesia species and Theileria equi in vitro and against Babesia microti in mice. The drug showed significant growth inhibition from an initial parasitemia of 1% for Babesia bovis, Babesia bigemina, Babesia caballi, and T. equi with IC50 values of 25, 10.2, 10.4, and 99 µM, respectively. Complete inhibition was observed at 200 µM of miltefosine on the third day of culture for the three Babesia species and 400 µM on the fourth day for T. equi. Reverse-transcription PCR (RT-PCR) showed that miltefosine inhibited the transcription of choline-phosphate cytidylyltransferase in B. bovis. Miltefosine at a dose rate of 30 mg/kg resulted in a 71.7% inhibition of B. microti growth in BALB/c mice. Miltefosine might be used for drug therapy in babesiosis.

The molecular prevalence and MSA-2b gene-based genetic diversity of Babesia bovis in dairy cattle in Thailand.

Bovine babesiosis is an economically significant disease that affects dairy farming operations in Thailand. In the present study, 1824 blood-DNA samples prepared from cattle bred in 4 different regions of the country (North, Northeast, Central, and South) were screened using a nested PCR for the specific detection of Babesia bovis. While the overall prevalence of B. bovis was 8.8%, the Central region of Thailand was found to be a high-risk area of the country, as the prevalence of the parasite was 15.0%. The positive rate was relatively higher among the animals of 1-5 years of age. The genetic diversity among the B. bovis parasites was also studied based on their MSA-2b gene, and the findings showed that the Thai sequences were dispersed across 8 of 13 total clades observed in the phylogram. Three of these clades were formed only of Thai sequences. Similarity among the deduced MSA-2b amino acid sequences determined in the present study was 68.3-100%. In conclusion, the present study found that all the locations surveyed were infected with B. bovis and that the parasite populations in Thailand were genetically diverse. Our findings highlight the need for further studies in Thailand to generate more information before a sound control strategy could be implemented against B. bovis.

Prevalence and genetic diversity of equine piroplasms in Tov province, Mongolia.

Equine piroplasmosis represents a serious problem in horse industry. Although, researchers suggested the possible use of sub-unit vaccines to control equine piroplasmosis, the genetic diversity of vaccine candidate antigens was not properly investigated. In the present study, we screened 250 horses reared in three different districts of Tov province, Mongolia, for Babesia caballi and Theileria equi using ELISA and nested PCR (nPCR) assays. Among these animals, piroplasms were detected in 128 (51.2%) horses by nPCR assays (B. caballi, 42.4%; T. equi, 6.4%; and mixed infections, 2.4%), while 204 (81.6%) were positive by ELISA (B. caballi, 51.6%; T. equi, 19.6%; and mixed infections, 10.4%). Male and middle-aged horses showed higher positive rates than female and younger or older horses. The findings also suggested that a combination of nPCR and ELISA techniques might be useful to detect horses that were chronically or subclinically infected with piroplasms. B. caballi-BC48 and T. equi-EMA-1 gene sequences, in addition to 18S rRNA, were subjected to phylogenetic analyses, and the findings suggested the presence of genetically diverse populations of equine piroplasms in Mongolia. BC48 sequences were separated into four clades in phylogram, and all the Mongolian sequences determined in the present study were found in a single clade. However, a single BC48 sequence previously isolated from a tick in Mongolia formed a separate branch. Similarly, EMA-1 sequences formed four clades, and Mongolian sequences were observed in two different clades, one of which was formed only of Mongolian sequences and is suggested as a new clade. This is the first report that describes the genotypes of equine piroplasms in Mongolia. The findings also emphasized the need for further investigations to study the effect of genetic diversity observed among BC48 as well as EMA-1 sequences on host's immune responses.

Inhibitory effects of pepstatin A and mefloquine on the growth of Babesia parasites.

We evaluated the inhibitory effects of pepstatin A and mefloquine on the in vitro and in vivo growths of Babesia parasites. The in vitro growth of Babesia bovis, B. bigemina, B. caballi, and B. equi was significantly inhibited (P < 0.05) by micromolar concentrations of pepstatin A (50% inhibitory concentrations = 38.5, 36.5, 17.6, and 18.1 µM, respectively) and mefloquine (50% inhibitory concentrations = 59.7, 56.7, 20.7, and 4 µM, respectively). Furthermore, both reagents either alone at a concentration of 5 mg/kg or in combinations (2.5/2.5 and 5/5 mg/kg) for 10 days significantly inhibited the in vivo growth of B. microti in mice. Mefloquine treatment was highly effective and the combination treatments were less effective than other treatments. Therefore, mefloquine may antagonize the actions of pepstatin A against babesiosis and aspartic proteases may play an important role in the asexual growth cycle of Babesia parasites.

Cloning and characterization of histone deacetylase from Babesia bovis.

The effect of inhibitors of histone deacetylase (HDAC) on Apicomplexa has been previously reported with the discovery of apicidin, a cyclic tetrapeptide having broad-spectrum antiparasitic activity. In the current study, we expressed Babesia bovis (B. bovis) recombinant-HDAC 3 (rBbHDAC3) as a GST-fusion protein in Escherichia coli (E. coli) and found that it was antigenic. An antiserum against the recombinant protein was generated in mice. The mice serum demonstrated the presence of HDAC in B. bovis by a Western blot assay. The murine anti-rBbHDAC3 reacted with B. bovis, Babesia bigemina (B. bigemina), Theileria equi (T. equi), and Babeisa caballi (B. caballi) merozoites in the indirect fluorescent antibody test (IFAT). Furthermore, the HDAC-enzymatic activity of the rBbHDAC3 protein was evaluated by a colorimetric assay. The enzymatic activity of rBbHDAC3 was inhibited by 100 ng/ml of apicidin, and the inhibitory effect of apicidin was dose-dependent. The inhibition of BbHDAC3 by apicidin was confirmed by Western blot, IFAT, and reverse transcription-polymerase chain reaction (RT-PCR). Finally, apicidin potentially inhibited the in vitro growth of Babesia parasites. The lower IC(50) values of apicidin against apicomplexan parasites than those of mammalian cells point to HDAC as an excellent drug target. The findings of the present study indicate that BbHDAC3 is a potential target for apicidin and might be a promising target for the development of novel anti-babesial drugs.

Apicoplast-targeting antibacterials inhibit the growth of Babesia parasites.

The apicoplast housekeeping machinery, specifically apicoplast DNA replication, transcription, and translation, was targeted by ciprofloxacin, thiostrepton, and rifampin, respectively, in the in vitro cultures of four Babesia species. Furthermore, the in vivo effect of thiostrepton on the growth cycle of Babesia microti in BALB/c mice was evaluated. The drugs caused significant inhibition of growth from an initial parasitemia of 1% for Babesia bovis, with 50% inhibitory concentrations (IC(50)s) of 8.3, 11.5, 12, and 126.6 µM for ciprofloxacin, thiostrepton, rifampin, and clindamycin, respectively. The IC(50)s for the inhibition of Babesia bigemina growth were 15.8 µM for ciprofloxacin, 8.2 µM for thiostrepton, 8.3 µM for rifampin, and 206 µM for clindamycin. The IC(50)s for Babesia caballi were 2.7 µM for ciprofloxacin, 2.7 µM for thiostrepton, 4.7 µM for rifampin, and 4.7 µM for clindamycin. The IC(50)s for the inhibition of Babesia equi growth were 2.5 µM for ciprofloxacin, 6.4 µM for thiostrepton, 4.1 µM for rifampin, and 27.2 µM for clindamycin. Furthermore, an inhibitory effect was revealed for cultures with an initial parasitemia of either 10 or 7% for Babesia bovis or Babesia bigemina, respectively. The three inhibitors caused immediate death of Babesia bovis and Babesia equi. The inhibitory effects of ciprofloxacin, thiostrepton, and rifampin were confirmed by reverse transcription-PCR. Thiostrepton at a dose of 500 mg/kg of body weight resulted in 77.5% inhibition of Babesia microti growth in BALB/c mice. These results implicate the apicoplast as a potential chemotherapeutic target for babesiosis.

Genetic detection of Babesia bigemina from Mongolian cattle using apical membrane antigen-1 gene-based PCR assay.

We developed a new nested PCR (nPCR) assay based on the Babesia bigemina apical membrane antigen-1 (AMA-1) gene sequence for parasite-specific detection. The primers were designed to amplify 738-bp and 211-bp fragments of the AMA-1 gene by primary and nested PCRs, respectively. The assay was proven to be specific for the B. bigemina, whereas the previously established SpeI-AvaI nPCR assay amplified not only the target fragment of B. bigemina but also a homologous one from Babesia ovata. The AMA-1 nPCR assay was also evaluated using field DNA samples extracted from 266 bovine blood samples collected from Mongolia in 2010. In a comparative evaluation, 90 (33.8%) and 25 (9.4%) of the blood samples showed positive reactions for B. bigemina by the SpeI-AvaI nPCR and AMA-1 nPCR assays, respectively. The sequencing analysis of the nPCR products confirmed that the AMA-1 nPCR method had specifically detected the target B. bigemina DNA. However, 4 different kinds of sequences were determined among the SpeI-AvaI nPCR amplicons. Two of them were derived from B. bigemina and B. ovata, while the origins of the others were unknown. In the current study, the presence of B. bigemina was clearly demonstrated among Mongolian cattle populations by the current nPCR assay for the first time. Furthermore, our findings also indicate that the AMA-1 nPCR assay may be a useful diagnostic tool for the specific detection of B. bigemina.