Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes.

BACKGROUND: Theileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype. METHODS: A pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically. RESULTS: Each genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays. CONCLUSIONS: The genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes.

An epidemiological survey of vector-borne pathogens infecting cattle in Kyrgyzstan.

Cattle production is a major contributor to the national economy of Kyrgyzstan. Most cattle in Kyrgyzstan are managed via extensive systems and graze in communal pastures. As a result, infestations with ectoparasites are widespread, implying that various vector-borne diseases might be common in cattle. However, methods to control such infectious diseases are not available in Kyrgyzstan because the epidemiology of vector-borne pathogens (VBPs) infecting cattle remains unclear. The present study was therefore designed to survey Kyrgyz cattle for VBPs. We prepared blood DNA samples from 319 cattle in Kyrgyzstan and screened them with specific PCR assays for detecting Babesia bovis, Babesia bigemina, Babesia naoakii, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, and Anaplasma marginale infections. Our findings indicated that the surveyed cattle were infected with six of the eight pathogens targeted, with the exceptions being B. naoakii and Try. evansi. The most common pathogen was T. orientalis (84.3%), followed by B. bigemina (47.6%), T. annulata (16.6%), A. marginale (11.6%), Try. theileri (7.2%), and B. bovis (2.5%). Additional screening of the B. bovis- and B. bigemina-negative samples with a Babesia genus-specific 18S rRNA PCR identified two positive samples, and sequencing analysis confirmed that each of them was infected with either Babesia major or Babesia occultans. To the best of our knowledge, this is the first report of B. bovis, B. bigemina, B. occultans, Try. theileri, and A. marginale infections in cattle in Kyrgyzstan. Our findings suggest that cattle in Kyrgyzstan are at high risk of infectious diseases caused by VBPs.

Disruption of a DNA fragment that encodes the microneme adhesive repeat domain-containing region of the BBOV_III011730 does not affect the blood stage growth of Babesia bovis in vitro.

Babesia bovis, an intraerythrocytic hemoprotozoan parasite, causes the most pathogenic form of bovine babesiosis, negatively impacting the cattle industry. Comprehensive knowledge of B. bovis biology is necessary for developing control methods. In cattle, B. bovis invades the red blood cells (RBCs) and reproduces asexually. Micronemal proteins, which bind to sialic acid of host cells via their microneme adhesive repeat (MAR) domains, are believed to play a key role in host cell invasion by apicomplexan parasites. In this study, we successfully deleted the region encoding MAR domain of the BBOV_III011730 by integrating a fusion gene of enhanced green fluorescent protein-blasticidin-S-deaminase into the genome of B. bovis. The transgenic B. bovis, lacking the MAR domain of the BBOV_III011730, invaded bovine RBCs in vitro and grew at rates similar to the parental line. In conclusion, our study revealed that the MAR domain is non-essential for the intraerythrocytic development of B. bovis in vitro.

Clofazimine, a Promising Drug for the Treatment of Babesia microti Infection in Severely Immunocompromised Hosts.

BACKGROUND: Persistent and relapsing babesiosis caused by Babesia microti often occurs in immunocompromised patients, and has been associated with resistance to antimicrobial agents such as atovaquone. Given the rising incidence of babesiosis in the United States, novel drugs are urgently needed. In the current study, we tested whether clofazimine (CFZ), an antibiotic used to treat leprosy and drug-resistant tuberculosis, is effective against B. microti. METHODS: Mice with severe combined immunodeficiency were infected with 107B. microti-infected erythrocytes. Parasites were detected by means of microscopic examination of Giemsa-stained blood smears or nested polymerase chain reaction. CFZ was administered orally. RESULTS: Uninterrupted monotherapy with CFZ curtailed the rise of parasitemia and achieved radical cure. B. microti parasites and B. microti DNA were cleared by days 10 and 50 of therapy, respectively. A 7-day administration of CFZ delayed the rise of parasitemia by 22 days. This rise was caused by B. microti isolates that did not carry mutations in the cytochrome b gene. Accordingly, a 14-day administration of CFZ was sufficient to resolve high-grade parasitemia caused by atovaquone-resistant B. microti parasites. CONCLUSIONS: Clofazimine is effective against B. microti infection in the immunocompromised host. Additional preclinical studies are required to identify the minimal dose and dosage of CFZ for babesiosis.

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.

Highly sensitive nested PCR and rapid immunochromatographic detection of Babesia bovis and Babesia bigemina infection in a cattle herd with acute clinical and fatal cases in Argentina.

Bovine babesiosis is a tick-transmitted haemoparasitic disease caused by Babesia bovis and B. bigemina affecting cattle of tropical and subtropical regions around the world. Pathogens are transmitted by the tick vector Rhipicephalus microplus displaying a widespread distribution in northeastern Argentina. The disease is characterized by significant animal morbidity and mortality resulting in considerable economic loss. In this study, B. bovis and B. bigemina infection was investigated in a cattle herd of 150 adult bovines of pure Braford breed raised in a tick-hyperendemic field using molecular and serum antibody tests. A highly sensitive nested polymerase chain reaction (nPCR) assay targeting a species-specific region of the apocytochrome b gene resulted in direct B. bovis and B. bigemina detection in 27.3% and 54.7% of bovines, respectively. A recently developed immunochromatographic strip test (ICT) based on recombinant forms of spherical body protein 4 and the C-terminal region of rhoptry-associated protein 1 showed that 71.3% and 89.3% of bovines were seropositive for B. bovis and B. bigemina, respectively. The mixed infection rate as observed by direct (19.3%) and indirect detection (65.3%) coincided with those expected, respectively. Importantly, four months after sampling, nine bovines of the studied herd showed clinical signs of bovine babesiosis of which six animals eventually died. Microscopic detection of infected erythrocytes in Giemsa-stained blood smears confirmed B. bovis infection. Our study demonstrates that although animals showed a relatively high and very high rate of immunity against infection with B. bovis (71.3%) and B. bigemina (89.3%) parasites, respectively, clinical cases and fatalities due to the infection with B. bovis were observed. It is proposed that the most adequate control measure in the studied epidemiological situation is to vaccinate animals to prevent losses and/or an outbreak of bovine babesiosis.

Screening the Medicines for Malaria Venture Pathogen Box against piroplasm parasites.

Diminazene aceturate (DA) and imidocarb dipropionate are commonly used in livestock as antipiroplasm agents. However, toxic side effects are common in animals treated with these two drugs. Therefore, evaluations of novel therapeutic agents with high efficacy against piroplasm parasites and low toxicity to host animals are of paramount importance. In this study, the 400 compounds in the Pathogen Box provided by the Medicines for Malaria Venture foundation were screened against Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi. A fluorescence-based method using SYBR Green 1 stain was used for initial in vitro screening and determination of the half maximal inhibitory concentration (IC50). The initial in vitro screening performed using a 1 µM concentration as baseline revealed nine effective compounds against four tested parasites. Two "hit" compounds, namely MMV021057 and MMV675968, that showed IC50 < 0.3 µM and a selectivity index (SI)> 100 were selected. The IC50s of MMV021057 and MMV675968 against B. bovis, B. bigemina, T. equi and B. caballi were 23, 39, 229, and 146 nM, and 2.9, 3, 25.7, and 2.9 nM, respectively. In addition, a combination of MMV021057 and DA showed additive or synergistic effects against four tested parasites, while combinations of MMV021057 with MMV675968 and of MMV675968 with DA showed antagonistic effects. In mice, treated with 50 mg/kg MMV021057 and 25 mg/kg MMV675968 inhibited the growth of Babesia microti by 54 and 64%, respectively, as compared to the untreated group on day 8. Interestingly, a combination treatment with 6.25 mg/kg DA and 25 mg/kg MMV021057 inhibited B. microti by 91.6%, which was a stronger inhibition than that by single treatments with 50 mg/kg MMV021057 and 25 mg/kg DA, which showed 54 and 83% inhibition, respectively. Our findings indicated that MMV021057, MMV675968, and the combination treatment with MMV021057 and DA are prospects for further development of antipiroplasm drugs.

Inhibitory effects of fluoroquinolone antibiotics on Babesia divergens and Babesia microti, blood parasites of veterinary and zoonotic importance.

AIM: This study aimed to evaluate the inhibitory effects of fluoroquinolone antibiotics, including enrofloxacin, enoxacin, trovafloxacin, norfloxacin, and ofloxacin, on the in vitro and in vivo growth of Babesia divergens and Babesia microti parasites, respectively. MATERIALS AND METHODS: The in vitro and in vivo inhibitory effects of fluoroquinolone antibiotics against B. divergens and B. microti, respectively were evaluated using fluorescence-based assay. Additionally, combination therapies of highly effective fluoroquinolone antibiotics (enrofloxacin, enoxacin, and trovafloxacin) with diminazene aceturate, luteolin, or pyronaridine tetraphosphate were tested on the in vitro cultures of B. divergens. RESULTS: Enrofloxacin, trovafloxacin, and enoxacin were the most effective fluoroquinolones against the in vitro growth of B. divergens, followed by norfloxacin and ofloxacin. Furthermore, a combination of enoxacin or trovafloxacin with either diminazene aceturate, luteolin, or pyronaridine tetraphosphate significantly enhanced the inhibitory effect on the growth of B. divergens in in vitro cultures. In mice infected by B. microti, enoxacin and diminazene aceturate combination therapy exhibited a potential antibabesial effect. CONCLUSION: These results suggest that safe and cheap fluoroquinolone, such as enoxacin, might be used for the treatment of clinical cases caused by Babesia spp. in animals or humans.

Molecular epidemiology of Babesia species, Theileria parva, and Anaplasma marginale infecting cattle and the tick control malpractices in Central and Eastern Uganda.

East Coast fever, babesiosis, and anaplasmosis are the major tick-borne diseases affecting cattle productivity in Uganda. The emergence of acaricide-resistant ticks is suspected to have caused a rise in hemoparasites. This study sought to detect and characterize hemoparasites among farms in acaricide-failure hotspots of central as compared to the acaricide-failure naïve areas in Eastern Uganda. Nested PCR assays were performed to determine the prevalences of Babesia bovis, Babesia bigemina, Theileria parva, and Anaplasma marginale in cattle blood samples sourced from randomly selected farms. Randomly selected isolates were sequenced to determine the genetic diversity of the parasites using the following marker genes: B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein 1a, T. parva 104 kDa microneme-rhoptry antigen, and A. marginale major surface protein 5. Furthermore, partially and fully engorged adult ticks were collected for taxonomy, and tick-control practices were assessed using a semi-structured questionnaire. The prevalences of B. bigemina, T. parva, and A. marginale in cattle were 17.2, 65.1, and 22.0%, and 10.0, 26.5, and 3% in the central and eastern region, respectively. Whilst, B. bovis was not detected in the farms involved. The sequences for B. bigemina, T. parva, and A. marginale from the central region showed 99% identity with those from the eastern region. Of the 548 ticks collected, 319, 147, 76, and 6 were Rhipicephalus (Boophilus) decoloratus, Rhipicephalus appendiculatus, Amblyomma variegatum, and Rhipicephalus evertsi evertsi, respectively. The Rhipicephalus ticks were more abundant in the central region, whereas A. variegatum ticks were more abundant in the eastern region. Tick control malpractices were found in both Central and Eastern Uganda, and 42 of the 56 surveyed farms lacked appropriate restraining facilities and so they utilized either ropes or a 'boma' (enclosure). In summary, B. bigemina, T. parva, A. marginale and their co-infections were more prevalent in the central than eastern region; even though, tick control malpractices were observed in both regions. Therefore, an urgent tick and TBD control strategy is needed.

The effects of nitidine chloride and camptothecin on the growth of Babesia and Theileria parasites.

The treatment of bovine and equine piroplasmosis is limited to diminazene aceturate (DA) and imidocarb dipropionate. To address this challenge, we need to explore novel drug compounds and targets. Topoisomerases are potential drug targets because they play a vital role in solving topological errors of DNA strands during replication. This study documented the effectiveness of topoisomerase inhibitors, nitidine chloride (NC) and camptothecin (Cpt), on the growth of Babesia and Theileria parasites. The half maximal inhibitory concentrations (IC50s) against B. bovis, B. bigemina, B. caballi, and T. equi were 1.01 ±â€¯0.2, 5.34 ±â€¯1.0, 0.11 ±â€¯0.03, and 2.05 ±â€¯0.4 µM for NC and 11.67 ±â€¯1.6, 4.00 ±â€¯1.0, 2.07 ±â€¯0.6, and 0.33 ±â€¯0.02 µM for Cpt, respectively. The viability experiment revealed that 4, 10, and 4 µM treatments of NC or 48, 8, and 8 µM treatments of Cpt were sufficient to stop the in vitro regrowth of B. bovis, B. bigemina, and B. caballi, respectively. However, T. equi regrew in all of the concentrations used. Moreover, increasing the concentration of NC and Cpt to 16 µM and 1.2 µM (8 × IC50) did not eliminate T. equi. The micrographs of B. bigemina and B. caballi taken at 24 h and 72 h showed deformed merozoites and remnants of parasites within the red blood cell (RBC), respectively. The treatments of 25 mg/kg DA and 20 mg/kg NC administered intraperitoneally and 20 mg/kg NC given orally showed 93.7, 90.7, and 83.6% inhibition against Babesia microti (B. microti), respectively, compared to the untreated group on day 8. In summary, NC and Cpt were effective against Babesia and Theileria parasites in vitro. Moreover, 20 mg/kg NC administered intraperitoneally was as effective as 25 mg/kg DA against B. microti in mice and showed no toxic symptoms in mice. The results indicate that NC may, after further evaluations, prove to be an alternative drug against bovine and equine piroplasmoses.

17-DMAG inhibits the multiplication of several Babesia species and Theileria equi on in vitro cultures, and Babesia microti in mice.

Heat shock protein 90 (Hsp90) is a chaperone protein that stabilizes cells during stress or non-stress responses. Previous reports have shown that Hsp90 is a potential drug target to suppress the multiplication of several protozoan parasites. In this study, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an Hsp90 inhibitor, was evaluated for its inhibitory effect on five in vitro cultures of Babesia and Theileria species, including B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, and on the multiplication of a B. microti-infected mouse model. 17-DMAG showed the inhibitory effect in all of the species tested. The half maximum inhibition concentration (IC50) of 17-DMAG on B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi was 77.6 ±â€¯2.9, 62.4 ±â€¯1.9, 183.8 ±â€¯3.2, 88.5 ±â€¯9.6, and 307.7 ±â€¯7.2 nM, respectively. The toxicity assay on MDBK and NIH/3T3 cell lines showed that 17-DMAG affected the viability of cells with an IC50 of 15.5 ±â€¯4 and 8.8 ±â€¯2 µM, respectively. Since the IC50s were much lower on the parasites than on the host cell lines, the selectivity index were high for all tested species. Furthermore, the two-drug combination of 17-DMAG with diminazene aceturate (DA) and atovaquone (AV) showed synergism or addition on in vitro cultures of Babesia and Theileria parasites. In the mouse model, 17-DMAG at a concentration of 30 mg/kg BW effectively inhibited the multiplication of B. microti. Moreover, if combined with DA or AV, 17-DMAG showed a comparable inhibition at the half dose. Taken together, these results indicate that 17-DMAG is a potent drug for treating piroplamosis. The data warrant further evaluation of 17-DMAG as an antibabesial drug and as an option in combination with atovaquone for the treatment of human babesiosis.

Serological and molecular prevalence of equine piroplasmosis in Western Java, Indonesia.

Equine piroplasmosis is an economically significant disease caused by Theileria equi and Babesia caballi, which are tick-borne hemoprotozoan parasites. Infections with these parasite species had never been reported in horses in Indonesia. The aim of the present study was to investigate the prevalence of T. equi and B. caballi in horses reared in parts of Western Java, Indonesia. Blood samples were collected randomly from 235 horses in four different districts (Bandung, Depok, Tangerang, and Bogor) in Western Java, Indonesia. Thin blood smears prepared from the sampled animals were stained by Giemsa and observed under a light microscope. Serum samples prepared from blood were screened by enzyme-linked immunosorbent assays (ELISAs) based on recombinant forms of EMA-2 and BC48 antigens to determine the seroprevalence of T. equi and B. caballi, respectively. DNA samples extracted from the same blood samples were screened by EMA-2 and BC48 gene-based nested polymerase chain reaction (nPCR) assays for T. equi and B. caballi infections, respectively. Of 235 surveyed animals, five (2.1%) and 15 (6.4%) were seropositive for T. equi and B. caballi, respectively, whereas one and four horses were nPCR-positive for T. equi and B. caballi, respectively. All of the surveyed animals were negative for T. equi and B. caballi by microscopy. The T. equi EMA-2 and B. caballi BC48 gene fragments amplified by the nPCR assays were cloned, sequenced, and subjected to bioinformatic and phylogenetic analyses. The T. equi EMA-2 gene sequence from an Indonesian horse was identical to sequences from Florida and Washington strains and clustered together with these sequences in phylogeny. On the other hand, four Indonesian BC48 gene sequences shared 99.8-100% identity scores. This present study is the first to report T. equi and B. caballi in horses in Indonesia. Our findings highlight the need for monitoring horses in Indonesia for clinical piroplasmosis caused by T. equi and B. caballi.

Molecular and serological detection of bovine babesiosis in Indonesia.

BACKGROUND: Bovine babesiosis, mainly caused by Babesia bovis and B. bigemina, is a huge threat to the livestock industry. In Indonesia, the current distribution of the disease is unknown due to a lack of scientific study. METHODS: In the present study, 487 blood samples were collected from cattle with different breeding and age groups in a broad geographical area across the archipelago. The presence of antibodies and current infections of B. bovis and B. bigemina were determined using enzyme-linked immunosorbent assay (ELISA), immunochromatographic test (ICT), and nested PCR (nPCR) targeting B. bovis SBP-4 and B. bigemina RAP-1a genes. Sequence analysis was performed to the amplicon of B. bovis SBP-4, B. bigemina RAP-1a, and internal transcribed spacer (ITS) region of ribosomal RNA of both Babesia species. RESULTS: In total, B. bovis positives were detected by ELISA, single-ICT, dual-ICT and nPCR in 340 (69.8%), 317 (65.1%), 307 (63.0%) and 247 (50.7%) samples, respectively. For B. bigemina, the positive samples were detected in 134 (27.5%), 130 (26.7%), 127 (26.1%) and 93 (19.1%), respectively. Furthermore, mixed infections were found in 125 (25.7%), 113 (23.2%), 109 (22.4%) and 52 (10.7%) samples, respectively, which occurred only by chance and were not influenced by additional factors. The obtained nucleotide sequences of B. bovis SBP-4 and B. bigemina RAP-1a genes showed a high homology with other isolates from different countries. Further nucleotide sequence analysis using ITS region showed a great genetic diversity of B. bovis isolates among sampling locations; a lower diversity was found in B. bigemina ITS isolates. CONCLUSIONS: These data revealed the current distribution of B. bovis and B. bigemina infection in cattle in Indonesia. The rate of infection varied among sampling locations, cattle breeds and age groups. Furthermore, B. bovis ITS isolates from Indonesia were found to be more genetically diverse than B. bigemina ITS isolates. The data presented in this study are necessary to develop an effective strategy for controlling the disease in the country.

Chemotherapeutic efficacies of a clofazimine and diminazene aceturate combination against piroplasm parasites and their AT-rich DNA-binding activity on Babesia bovis.

Recently, we reported that clofazimine (CF) has an anti-piroplasm activity, but it could not completely eliminate parasites in the host. The currently available anti-piroplasm drug, diminazene aceturate (DA), has sometimes been reported to have toxic side effects. In the present study, we evaluated the combination treatment with CF and DA against piroplasms both in vitro and in vivo. Additionally, mRNA level and DNA amounts were analyzed in CF‒ and DA‒treated Babesia bovis by a qPCR. The CF-DA combination had additive effects on Babesia bovis, B. bigemina, and B. caballi and synergistic effects on Theileria equi. The CF-DA combination chemotherapies against B. microti in mice were more potent than their monotherapies. In the CF‒ and DA‒treated B. bovis, CF dose-dependently down-regulated mRNA level and DNA amounts of extranuclear genes (AT-rich featured), whereas DA down-regulated only DNA amounts of extranuclear genes, but those of nuclear genes were slightly down- or up-regulated by CF and DA. In conclusion, the CF-DA combination has a higher efficiency against piroplasms than CF or DA monotherapies. CF and DA might have an AT-rich DNA-binding activity. All results suggest that the CF-DA combination chemotherapy will be a better choice to treat piroplasmosis instead of DA monotherapy.

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.

Evaluation of immunochromatographic test (ICT) strips for the serological detection of Babesia bovis and Babesia bigemina infection in cattle from Western Java, Indonesia.

Three types of immunochromatographic test (ICT) strips were prepared for the detection of an antibody response against spherical body protein 4 (SBP-4) of Babesia bovis (bovICT), C-terminal-truncated rhoptry-associated protein 1 (rRAP1/CT17) of B. bigemina (bigICT), and the combination of both proteins (dual-ICT). The evaluation of their performance was conducted using a confirmed positive and negative serum panel for B. bovis and B. bigemina. Together with ELISA, the ICT strips were applied to determine the seroprevalence of bovine babesiosis in Western Java, Indonesia. Among 991 serum samples, 28.4%, 25.3%, and 24.5% of cattle were detected to be seropositive to B. bovis infection using ELISA, bovICT, and dual-ICT, respectively. B. bigemina seropositive was detected in 27.1%, 24.2%, and 22.8% of samples using ELISA, bigICT, and dual-ICT, respectively. The comparison of ICT strips and ELISA results using field serum samples showed good agreement with Kappa values >0.7 between all methods The application of ICT strips is preferable in the field situations where rapid diagnosis is required. Furthermore, the data showed the current seroprevalence of bovine babesiosis in Western Java, Indonesia, and efficient control strategies are needed to reduce economic losses due to the disease.

Seroprevalence of Babesia bovis, B. bigemina, Trypanosoma evansi, and Anaplasma marginale antibodies in cattle in southern Egypt.

Babesia bovis, B. bigemina, Trypanosoma evansi, and Anaplasma marginale infections cause serious diseases in cattle, and are primarily transmitted by arthropod vectors (ticks for B. bovis, B. bigemina, and A. marginale and various types of flies for T. evansi). In the last few years, there have been many reports of a high prevalence of certain protozoan infections in northern Egypt, but no accurate or adequate data are available for the southern regions. Therefore, in this study, we screened for evidence of such diseases in economically important cattle species using serum samples. The seroprevalence of protozoan infections in cattle was determined with enzyme-linked immunosorbent assays using species-specific diagnostic antigens. In a total of 301 cattle serum samples, 27 (9.0%), 100 (33.2%), and 127 (42.2%) were positive for specific antibodies against B. bovis, B. bigemina, and T. evansi, respectively. Sera from 90 cattle were also tested for antibodies against A. marginale, and 25 (28%) of them were positive. The highest coinfection rate occurred for B. bigemina and T. evansi with 10.6% (32/301). When age, sex, locality, and breeding system were investigated as predisposing factors, bulls and cattle <3 years old were more vulnerable to B. bovis infections than older animals, and geographic location affected the B. bigemina infection rate. The recorded seroprevalence of hemoprotozoan parasites and A. marginale in cattle suggests that these diseases have the potential capacity to detrimentally affect meat and milk production in southern Egypt.

Molecular characterization and phylogenetic analysis of Fasciola gigantica from western Java, Indonesia.

Fasciola gigantica and aspermic (hybrid) Fasciola flukes are thought to be distributed in Southeast Asian countries. The objectives of this study were to investigate the distribution of these flukes from unidentified ruminants in western Java, Indonesia, and to determine their distribution history into the area. Sixty Fasciola flukes from western Java were identified as F. gigantica based on the nucleotide sequences of the nuclear phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold) genes. The flukes were then analyzed phylogenetically based on the nucleotide sequence of the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene, together with Fasciola flukes from other Asian countries. All but one F. gigantica fluke were classified in F. gigantica haplogroup C, which mainly contains nad1 haplotypes detected in flukes from Thailand, Vietnam, and China. A population genetic analysis suggested that haplogroup C spread from Thailand to the neighboring countries including Indonesia together with domestic ruminants, such as the swamp buffalo, Bubalus bubalis. The swamp buffalo is one of the important definitive hosts of Fasciola flukes in Indonesia, and is considered to have been domesticated in the north of Thailand. The remaining one fluke displayed a novel nad1 haplotype that has never been detected in the reference countries. Therefore, the origin of the fluke could not be established. No hybrid Fasciola flukes were detected in this study, in contrast to neighboring Asian countries.

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.

Seroprevalence of antibody to NcSAG1 antigen of Neospora caninum in cattle from Western Java, Indonesia.

Neospora caninum can cause fetal abortion and neonatal mortality in cattle, and is a cause of economic concern worldwide. This study aimed to determine the prevalence of Neospora caninum-specific antibodies in cattle from Western Java, Indonesia. Serum samples from 991 cattle from 21 locations were tested for antibodies to N. caninum by using an enzyme-linked immunosorbent assay (ELISA) on the basis of recombinant NcSAG1. The overall seroprevalence was 16.6%, ranging from 0 to 87.5% in the sampled locations. The results of this study indicate latent infection rates of sampled animals were different in each location. Further studies are necessary to elucidate the relationship between N. caninum infection and abortion in cattle, and to identify risk factors for infection in high-prevalence environments.