Keisuke Suganuma

Novel methods for the molecular discrimination of Fasciola spp. on the basis of nuclear protein-coding genes

Fasciolosis is an economically important disease of livestock caused by Fasciola hepatica, Fasciola gigantica, and aspermic Fasciola flukes. The aspermic Fasciola flukes have been discriminated morphologically from the two other species by the absence of sperm in their seminal vesicles. To date, the molecular discrimination of F. hepatica and F. gigantica has relied on the nucleotide sequences of the internal transcribed spacer 1 (ITS1) region. However, ITS1 genotypes of aspermic Fasciola flukes cannot be clearly differentiated from those of F. hepatica and F. gigantica. Therefore, more precise and robust methods are required to discriminate Fasciola spp. In this study, we developed PCR restriction fragment length polymorphism and multiplex PCR methods to discriminate F. hepatica, F. gigantica, and aspermic Fasciola flukes on the basis of the nuclear protein-coding genes, phosphoenolpyruvate carboxykinase and DNA polymerase delta, which are single locus genes in most eukaryotes. All aspermic Fasciola flukes used in this study had mixed fragment pattern of F. hepatica and F. gigantica for both of these genes, suggesting that the flukes are descended through hybridization between the two species. These molecular methods will facilitate the identification of F. hepatica, F. gigantica, and aspermic Fasciola flukes, and will also prove useful in etiological studies of fasciolosis.

Establishment of ATP-Based Luciferase Viability Assay in 96-Well Plate for Trypanosoma congolense

ABSTRACT Animal African trypanosomosis (AAT), caused by Trypanosoma congolense, is widespread throughout sub-Saharan Africa. There are significant concerns related to the current drugs available for the treatment of AAT due to their limited effectiveness across species and their adverse effects. Moreover, drug resistant trypanosomes have recently been reported in the field. High throughput screening (HTS) of large chemical compound library collections is a promising approach for identifying novel drug candidates. While HTS for Trypanozoon trypanosomes, T. brucei sspp. and T. evansi is well established, no assays have been developed for T. congolense. In the present study, the authors developed an ATP-based luciferase viability assay for T. congolense in a 96-well plate format. The calculated 50% inhibitory concentration (IC50) values for pentamidine and diminazene were 10–100 times higher in T. congolense than in T. brucei. This result suggests that the transporters for the 2 tested compounds differ between T. congolense and T. brucei. This assay could further be applied to screen novel chemical compounds for the treatment of AAT caused by T. congolense.

Molecular characterization of Cryptosporidium parvum from two different Japanese prefectures, Okinawa and Hokkaido

Abstract Infectious diarrhea is the most frequent cause of morbidity and mortality in neonatal calves. Cryptosporidium parvum is one of the main pathogens associated with calf diarrhea. Although diarrhea is a symptom of infection with various pathogens, investigations to detect the types of pathogens have never been performed in Japan. This study investigated the prevalence of four major diarrhea-causing pathogens in calves: C. parvum, rotavirus, coronavirus, and enterotoxigenic Escherichia coli (E. coli K99). Commercial immunochromatography testing of all four pathogens and molecular analysis of C. parvum with diarrhea in calves from southernmost Okinawa and northernmost Hokkaido, Japan, were conducted. The frequencies of C. parvum, rotavirus, coronavirus, and E. coli (K99) in Okinawa were 50%, 28%, 2.3%, and 4.7%, respectively. Watery fecal stools were significantly correlated with C. parvum (p <0.05). In oocyst calculations for C. parvum, no significant difference was observed between the single-infection cases and the mixed-infection cases with rotavirus. Interestingly, molecular analyses targeting small subunit ribosomal RNA as well as glycoprotein 60 (GP60) genes revealed that the C. parvum nucleotide sequences from the two prefectures were identical, indicating that C. parvum with a uniform characteristic is distributed throughout Japan. GP60 subtyping analysis identified C. parvum from Okinawa and Hokkaido as belonging to the IIaA15G2R1 subtype, a known zoonotic subtype. Hence, control of cryptosporidiosis is important not only for pre-weaned calves, but also for human health.

Plasmodium knowlesi thioredoxin peroxidase 1 binds to nucleic acids and has RNA chaperone activity.

Malaria parasites are under oxidative attack throughout their life cycle in human body and mosquito vector. Therefore, Plasmodium antioxidant defenses are crucial for its survival and being considered as interesting target for antimalarial drug design. Plasmodium knowlesi has emerged recently from its simian host to human in Southeast Asia and has been recognized as the fifth Plasmodium species that can cause human malaria. In this study, we cloned and characterized thioredoxin peroxidase 1 from P. knowlesi (PkTPx-1). PkTPx-1 gene was cloned, and recombinant protein was produced by heterologous overexpression in Escherichia coli. The recombinant protein was used for evaluation of enzymatic activity and polyclonal antibody production. Using the recombinant PkTPx-1 protein, its antioxidant activity was confirmed in a mixed-function oxidation assay where PkTPx-1 prevented nicking of DNA by hydroxyl radicals. PkTPx-1 was able to bind to double-strand DNA and RNA and had RNA chaperone activity in a nucleic acid melting assay indicating new function of PkTPx-1 other than antioxidant activity. Using specific polyclonal antibodies, it was indicated that PkTPx-1 is expressed in the cytoplasm of the parasite. Altogether, these results suggest that PkTPx-1 not only protects the parasite from the adverse effects of reactive oxygen species but also has RNA chaperone activity.

PCR detection of Babesia ovata from questing ticks in Japan.

Babesia ovata is a tick-transmitted hemoprotozoan parasite of cattle. In the present study, we analyzed tick DNA samples (n=1459) prepared from questing ticks collected from various cattle pastures in Hokkaido (Shibecha, Taiki, Otofuke, Memuro, and Shin-Hidaka districts) and Okinawa (Yonaguni Island) prefectures of Japan for B. ovata. When all the tick DNA samples were screened by a previously described B. ovata-specific apical membrane antigen-1 (AMA-1) gene-based polymerase chain reaction (PCR) assay, none of the DNA samples was positive. Therefore, we developed a PCR assay based on the protozoan beta-tubulin (β-tubulin) gene to detect B. ovata from ticks in Japan. In the specificity test, the PCR assay amplified the expected 444-bp target gene fragment from B. ovata DNA. No PCR products were amplified from DNA samples from other blood pathogens, bovine blood, or ticks. In addition, the PCR assay detected 100 fg of B. ovata-genomic DNA extracted from an in vitro culture of the parasites. Subsequently, when all the tick DNA samples were screened by this new PCR assay, 18 were positive for B. ovata. Positive samples were found only in the Yonaguni and Memuro areas. In Okinawa, where all the ticks were identified as Haemaphysalis longicornis, 9.7% of the samples were PCR-positive, while a single tick (Ixodes ovatus) from Memuro was infected with B. ovata. When the nucleotide sequences of the PCR amplicons were phylogenetically analyzed, they formed a separate clade containing a previously described β-tubulin gene sequence from B. ovata (Miyake strain), confirming that the PCR assay had detected only B. ovata from the tick DNA samples. This is the first report that describes the PCR detection of B. ovata in ticks. The findings warrant transmission experiments to evaluate I. ovatus as a potential vector of B. ovata.

Mycophenolic Acid and Its Derivatives as Potential Chemotherapeutic Agents Targeting Inosine Monophosphate Dehydrogenase in Trypanosoma congolense.

ABSTRACT This study aimed to evaluate the trypanocidal activity of mycophenolic acid (MPA) and its derivatives for Trypanosoma congolense. The proliferation of T. congolense was completely inhibited by adding <1 μM MPA and its derivatives. In addition, the IMP dehydrogenase in T. congolense was molecularly characterized as the target of these compounds. The results suggest that MPA and its derivatives have the potential to be new candidates as novel trypanocidal drugs.

Development of Monoclonal Antibodies That Target 1-Cys Peroxiredoxin and Differentiate Plasmodium falciparum from P. vivax and P. knowlesi

Prompt and accurate diagnosis of malarial patients is a crucial factor in controlling the morbidity and mortality of the disease. Effective treatment decisions require a correct diagnosis among mixed-species malarial patients. Differential diagnosis is particularly important in cases of Plasmodium vivax, a species that shares endemicity with P. falciparum in most endemic areas. Moreover, it is difficult to identify P. knowlesi on the basis of morphology alone, and rapid diagnostic tests are still not available for this malaria species. Therefore, the development of diagnostic tests applicable to the field is urgently needed. 1-Cys peroxiredoxin (1-Cys-Prx) in P. falciparum is abundantly expressed in the mature asexual stages, making it a promising candidate as a diagnostic antigen. In this study, we produced five monoclonal antibodies (mAbs) against P. falciparum 1-Cys-Prx (Pf1-Cys-Prx) by immunizing BALB/c mice with recombinant Pf1-Cys-Prx and subsequent hybridoma production. Cross reactivity of established mAbs with the orthologous molecule of Pf1-Cys-Prx in P. vivax (Pv1-Cys-Prx) and P. knowlesi (Pk1-Cys-Prx) was examined. Western blot analyses showed that three mAbs reacted with Pv1-Cys-Prx and Pk1-Cys-Prx but two mAbs did not. These results indicate that the two mAbs were effective in differentiating P. falciparum from P. vivax and P. knowlesi and could be used in differential diagnosis as well as comparative molecular studies of human Plasmodium species.

Trypanosoma vivax is the second leading cause of camel trypanosomosis in Sudan after Trypanosoma evansi

BackgroundThis study was conducted in response to recurring reports from eastern Sudan of camel trypanosomosis that can no longer be treated by currently available trypanocidal drugs. One hundred and eighty-nine blood samples were obtained from camels in different herds and local markets in the western part of Sudan, and a cross-sectional study was carried out between December 2015 and February 2016 to identify the causative agents and possible circulating genotypes.ResultsThe prevalence of trypanosomes detected using the conventional parasitological techniques of Giemsa-stained blood smears, wet blood smears and the microhematocrit centrifugation technique (MHCT) was 7% (13/189), 11% (21/189) and 19% (36/189), respectively. However, a multi-species KIN-PCR targeting the ITS region revealed that the prevalence of Trypanosoma evansi was 37% (70/189), while that of T. vivax was 25% (47/189). Consequently, we used a T. evansi-specific PCR (RoTat1.2 VSG gene) to analyse the KIN-PCR-positive samples and a T. vivax-specific PCR (Cathepsin L-like gene) to analyse all of the samples. The prevalence of T. evansi was 59% (41/70), while the prevalence of T. vivax was 31% (59/189). Mixed infections were detected in 18% (34/189) of the samples. These results were further confirmed by sequencing and a phylogenetic analysis of the complete internal transcribed spacer (ITS) region of T. evansi and the TviCatL gene of T. vivax.ConclusionWe conclude that T. vivax was newly introduced to the camel population and that T. evansi is no longer the single cause of camel trypanosomosis in Sudan. The presence of T. vivax in camels detected in this study is a challenge in the choice of diagnostic approaches, particularly serology, and PCRs. However, an analysis of drug resistance should be performed, and the genotypic variation should be verified. To our knowledge, this is the first molecular study on T. vivax and mixed-infection with T. vivax and T. evansi in Sudanese camels.

Characterisation of tabanid flies (Diptera: Tabanidae) in South Africa and Zambia and detection of protozoan parasites they are harbouring

Tabanids are haematophagous flies feeding on livestock and wildlife. In the absence of information on the relationship of tabanid flies and protozoan parasites in South Africa and Zambia, the current study was aimed at characterizing tabanid flies collected in these two countries as well as detecting protozoan parasites they are harbouring. A total of 527 tabanid flies were collected whereby 70·2% were from South Africa and 29·8% were from Zambia. Morphological analysis revealed a total of five different genera collected from the sampled areas namely: Ancala, Atylotus, Haematopota, Philoliche and Tabanus. DNA extracted from South African Tabanus par and Tabanus taeniola tested positive for the presence of Trypanosoma congolense (Savannah) and Trypanosoma theileri whilst one member from T. par was positive for Trypanosoma brucei species. DNA extracted from Zambian tabanid flies tested positive for the presence of Besnoitia species at 1·27% (2/157), Babesia bigemina 5·73% (9/157), Theileria parva 30·11% (30/157) and 9·82% (14/157) for Trypanosoma evansi. This study is the first to report on relationship of Babesia and Theileria parasites with tabanid flies. Further investigations are required to determine the role of tabanids in transmission of the detected protozoan parasites in livestock and wildlife in South Africa and Zambia.

Development of monoclonal antibodies against Plasmodium falciparum thioredoxin peroxidase 1 and its possible application for malaria diagnosis

Rapid diagnostic tests (RDTs) have been considered as an ideal alternative for light microscopy to detect malaria parasites especially in remote areas. The development and improvement of RDTs is an area of intensive research in the last decade. To date, few parasite proteins have been targeted in RDTs which are known to have certain deficiencies and made the researchers to look for other promising candidates to address this problem. Plasmodium falciparum thioredoxin peroxidase 1 (PfTPx-1) is abundantly expressed in the cytoplasm of the parasite and well conserved across Plasmodium species, making this antigen a promising target for malaria diagnosis. Several monoclonal antibodies (mAbs) were produced against PfTPx-1. The binding affinities of mAbs were measured. Several immunochromatographic tests (ICTs) were developed using different combination of mAbs. All mAbs showed promising affinities to be used for diagnosis. The sensitivities of ICTs were evaluated using recombinant PfTPx-1 whose results lead us to the preparation of 4 different ICTs. These tests showed positive reaction with P. falciparum in vitro culture supernatant indicating the release of PfTPx-1 during schizont rupture. Altogether, these findings suggest that PfTPx-1 is a promising biomarker to diagnose P. falciparum infection. However, the diagnostic performance of this antigen should be further validated using clinical samples.