Naoaki Yokoyama

Obihiro University of Agriculture and Veterinary Medicine, Obibiro, Hokkaidō, Japan

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Publications (187)397.58 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: In the present study, we examined the contributions of macrophages to the outcome of infection with Babesia microti, the etiological agent of human and rodent babesiosis, in BALB/c mice. Mice were treated with clodronate liposome at different time courses of B. microti infection in order to deplete the macrophages. Notably, a depletion of host macrophages at the early and acute phases of infection caused a significant elevation of parasitemia associated with remarkable mortality in the mice. The depletion of macrophages at the resolving and latent phases of infection resulted in an immediate and temporal exacerbation of parasitemia coupled with mortality in mice. Reconstituting clodronate liposome-treated mice at the acute phase of infection with macrophages from naïve mice resulted in a slight reduction in parasitemia with improved survival, as compared to mice that received the drug alone. These results indicate that macrophages play a crucial role in the control of and resistance to B. microti infection in mice. Moreover, analyses of host immune responses revealed that macrophage-depleted mice diminished their production of Th1 cell-cytokines, including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Furthermore, depletion of macrophage at different time courses exaggerated the pathogenesis of the infection in deficient IFN-γ(-)/(-) and severe combined immunodeficiency (SCID) mice. Collectively, our data provides important clues about the role of macrophages in the resistance and control of B. microti and implies that the severity of the infection in immunocompromised patients might be due to impairment of the macrophages' function.
    Infection and immunity. 10/2014;
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    ABSTRACT: Cattle, buffaloes, and sheep are the main sources of meat and milk in Egypt, but their productivity is thought be greatly reduced by hemoprotozoan parasitic diseases. In this study, we analyzed the infection rates of Babesia bovis, B. bigemina, Theileria annulata, and T. orientalis, using parasite-specific PCR assays in blood-DNA samples sourced from cattle (n=439), buffaloes (n=50), and sheep (n=105) reared in Menoufia, Behera, Giza, and Sohag provinces of Egypt. In cattle, the positive rates of B. bovis, B. bigemina, T. annulata, and T. orientalis were 3.18%, 7.97%, 9.56%, and 0.68%, respectively. On the other hand, B. bovis and T. orientalis were the only parasites detected in buffaloes and each of these parasites was only found in two individual DNA samples (both 2%), while one (0.95%) and two (1.90%) of the sheep samples were positive for B. bovis and B. bigemina, respectively. Sequence analysis showed that the B. bovis Rhoptry Associated Protein-1 and the B. bigemina Apical Membrane Antigen-1 genes were highly conserved among the samples, with 99.3-100% and 95.3-100% sequence identity values, respectively. In contrast, the Egyptian T. annulata Merozoite Surface Antigen-1 gene sequences were relatively diverse (87.8-100% identity values), dispersing themselves across several clades in the phylogenetic tree containing sequences from other countries. Additionally, the T. orientalis Major Piroplasm Surface Protein (MPSP) gene sequences were classified as types 1 and 2. This is the first report of T. orientalis in Egypt, and of type 2 MPSP in buffaloes. Detection of MPSP type 2, which is considered a relatively virulent genotype, suggests that T. orientalis infection may have veterinary and economic significance in Egypt. In conclusion, the present study, which analyzed multiple species of Babesia and Theileria parasites in different livestock animals, may shed an additional light on the epidemiology of hemoprotozoan parasites in Egypt.
    Parasitology International 10/2014; · 2.30 Impact Factor
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    ABSTRACT: Bovine babesiosis is a livestock disease known to cause economic losses in endemic areas. The apicomplexan parasite Babesia bovis is able to invade and destroy the host's erythrocytes leading to the serious pathologies of the disease, such as anemia and hemoglobinuria. Understanding the egress mechanisms of this parasite is therefore a key step to develop new therapeutic strategies. In this study, the possible involvement of Ca(2+) in the egress of B. bovis merozoites from infected erythrocytes was investigated. Egress was artificially induced in vitro using calcium ionophore A23187 and thapsigargin to increase Ca(2+) concentration in the cytosol of the parasite cells. The increased intracellular Ca(2+) concentration following these treatments was confirmed using live cell Ca(2+) imaging with confocal laser scanning microscopy. Based on our findings, we suggest a Ca(2+) signalling pathway in the egress of B. bovis merozoites.
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science. 10/2014;
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    ABSTRACT: Babesia bovis is an apicomplexan parasite that causes babesiosis in infected cattle. Genomes of pathogens contain promising information that can facilitate the development of methods for controlling infections. Although the genome of B. bovis is publically available, annotated gene models are not highly reliable prior to experimental validation. Therefore, we validated a preproposed gene model of B. bovis and extended the associated annotations on the basis of experimentally obtained full-length expressed sequence tags (ESTs).
    BMC genomics. 08/2014; 15(1):678.
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    ABSTRACT: Theileria parasites infect a wide range of domestic and wild ruminants worldwide, causing diseases with varying degrees of severity. A broad classification, based on the parasite’s ability to transform the leukocytes of host animals, divides Theileria into two groups, consisting of transforming and non-transforming species. The evolution of transforming Theileria has been accompanied by drastic changes in its genetic makeup, such as acquisition or expansion of gene families, which are thought to play critical roles in the transformation of host cells. Genetic variation among Theileria parasites is sometimes linked with host specificity and virulence in the parasites. Immunity against Theileria parasites primarily involves cell-mediated immune responses in the host. Immunodominance and major histocompatibility complex class I phenotype-specificity result in a host immunity that is tightly focused and strain-specific. Immune escape in Theileria is facilitated by genetic diversity in its antigenic determinants, which potentially results in a loss of T cell receptor recognition in its host. In the recent past, several reviews have focused on genetic diversity in the transforming species, Theileria parva and Theileria annulata. In contrast, genetic diversity in Theileria orientalis, a benign non-transforming parasite, which occasionally causes disease outbreaks in cattle, has not been extensively examined. In this review, therefore, we provide an outline of the evolution of Theileria, which includes T. orientalis, and discuss the possible mechanisms generating genetic diversity among parasite populations. Additionally, we discuss the potential implications of a genetically diverse parasite population in the context of Theileria vaccine development.
    Infection Genetics and Evolution 08/2014; 27:250–263. · 2.77 Impact Factor
  • The Veterinary record 06/2014; · 1.80 Impact Factor
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    ABSTRACT: In vitro evaluation of chemotherapeutic agents against Babesia and Theileria parasites has become routine, and the effectiveness of these chemicals is usually determined by comparing the parasitemia dynamics of untreated and treated parasites. Although microscopy is widely used to calculate parasitemia, several disadvantages are associated with this technique. The present study evaluates a fluorescent-based method using SYBR Green I stain (SG I) to screen antibabesial agents in in vitro cultures of Babesia bovis. The linearity between relative fluorescence units (RFU) and parasitemia was found to be well correlated with a 0.9944 goodness of fit (r(2)) value. Subsequently, 50% inhibitory concentration (IC50) values were calculated for 3 antiprotozoan agents, diminazene aceturate, nimbolide, and gedunin, by this method. For diminazene aceturate and nimbolide, the IC50 values by fluorescence-based method (408 nM and 8.13 μM, respectively) and microscopy (400.3 nM and 9.4 μM, respectively) were in agreement. Furthermore, the IC50 value of gedunin by fluorescence-based method (19 μM) was similar to the recently described microscopy-based value (21.7 μM) for B. bovis. Additionally, the Z' factor (0.80-0.90), signal to noise (S/N) ratio (44.15-87.64), coefficient of variation at the maximum signal (%CVmax) (0.50-2.85), and coefficient of variation at the minimum signal (%CVmin) (1.23-2.21) calculated for the fluorescence method using diminazene aceturate were comparable to those previously determined in malaria research for this assay. These findings suggest that the fluorescence-based method might be useful for antibabesial drug screening and may have potential to be developed into a high-throughput screening (HTS) assay.
    Antimicrobial Agents and Chemotherapy 06/2014; · 4.57 Impact Factor
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    ABSTRACT: 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 99μM, respectively. Complete inhibition was observed at 200μM of miltefosine on the third day of culture for the three Babesia species and 400μM 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 30mg/kg resulted in a 71.7% inhibition of B. microti growth in BALB/c mice. Miltefosine might be used for drug therapy in babesiosis.
    Veterinary parasitology. 05/2014;
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    ABSTRACT: The 23-kDa piroplasm membrane protein of Theileria orientalis (p23) is an immunogenic protein expressed during the intraerythrocytic stage of the parasite; its function, however, remains unclear. To evaluate the host factor or factors that interact with p23, we examined the binding of p23 to components of the host cell surface. Recombinant p23 protein of the Ikeda genotype failed to bind to bovine red blood cells or to peripheral blood mononuclear cells, but did bind to Madin-Darby Bovine Kidney (MDBK) cells. A glycoarray assay showed that recombinant p23 proteins from the three genotypes bound to heparin, indicating that p23 is a heparin-binding Theileria surface molecule. Further analysis of heparin-binding molecules is useful for understanding attachment and invasion of T. orientalis merozoites.
    The Japanese journal of veterinary research 05/2014; 62(1-2):17-24. · 0.65 Impact Factor
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    ABSTRACT: A total of 658 cattle in 6 provinces in the Philippines were screened for Anaplasma marginale infection by using a diagnostic heat-shock operon (groEL) gene-PCR assay. The screening-positive samples were further tested using the major surface antigen protein 1a (Msp1a) gene-PCR assay. Screening PCR results showed 130 cattle (19.8%) were positive for the A. marginale infection. Subsequent amplification using the Msp1a gene only showed 93 samples (14.1%) to be positive. In addition, 37 tandem-repeat structures, including 20 novel structures, and 41 distinct genotypes were identified. Interestingly, multiple infections of 4 different genotypes were also observed in A. marginale-infected cattle. The present study demonstrated the prevalence and characterization of diverse genotypes of A. marginale in the Philippine cattle.
    Journal of Veterinary Medical Science 04/2014; · 0.88 Impact Factor
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    ABSTRACT: 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.
    Ticks and Tick-borne Diseases 01/2014; · 2.35 Impact Factor
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    ABSTRACT: Water buffaloes are thought to be the reservoir hosts for several hemoprotozoan parasites that infect cattle. In the present study, we surveyed Sri Lankan bred water buffaloes for infections with Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis using parasite-specific PCR assays. When 320 blood-derived DNA samples from water buffaloes reared in three different districts (Polonnaruwa, Mannar, and Mullaitivu) of Sri Lanka were PCR screened, B. bovis, B. bigemina, and T. orientalis were detected. While T. orientalis was the predominant parasite (82.5%), low PCR-positive rates were observed for B. bovis (1.9%) and B. bigemina (1.6%). Amplicons of the gene sequences of the Rhoptry Associated Protein-1 (RAP-1) of B. bovis, the Apical Membrane Antigen-1 (AMA-1) of B. bigemina, and the Major Piroplasm Surface Protein (MPSP) of T. orientalis were compared with those characterized previously in Sri Lankan cattle. While the B. bigemina AMA-1 sequences from water buffaloes shared high identity values with those from cattle, B. bovis RAP-1 sequences from water buffaloes diverged genetically from those of cattle. For T. orientalis, none of the MPSP sequence types reported previously in Sri Lankan cattle (types 1, 3, 5, and 7) were detected in the water buffaloes, and the MPSP sequences analyzed in the present study belonged to types N1 or N2. In summary, in addition to reporting the first PCR-based survey of Babesia and Theileria parasites in water buffaloes in Sri Lanka, the present study found that the predominant variants of water buffalo-derived B. bovis RAP-1 and T. orientalis MPSP sequences were different from those previously described from cattle in this country.
    Veterinary Parasitology 12/2013; · 2.38 Impact Factor
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    ABSTRACT: Hemotropic mycoplasmas (hemoplasmas) are cell-wall deficient, epierythrocytic bacteria that cause infectious anemia in several mammalian species. The prevalence of hemoplasma species was examined by screening and species-specific PCR using blood samples collected from 51 sika deer in Hokkaido, Japan. Molecular analyses were performed for the 16S rRNA, 23S rRNA and RNase P RNA (rnpB) gene sequences. A total of 23/51 (45%) deer DNA samples were positive for hemoplasmas in the screening PCR. Using species-specific PCR, 12 and 17 samples were positive for 'Candidatus Mycoplasma haemocervae' and 'Candidatus M. erythrocervae', respectively. Sequencing and phylogenetic trees of those three genes indicate that the 'Candidatus M. haemocervae' and 'Candidatus M. erythrocervae' detected in Japanese deer are potentially different species from the cervine hemoplasma found in deer from America and Brazil.
    Journal of Veterinary Medical Science 11/2013; · 0.88 Impact Factor
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    ABSTRACT: Allicin is an active ingredient of garlic that has antibacterial, antifungal, antiviral, and antiprotozoal activity. However, the inhibitory effects of allicin on Babesia parasites have not yet been examined. In the present study, allicin was tested as a potent inhibitor against the in vitro growth of bovine and equine Babesia parasites and the in vivo growth of Babesia microti in a mouse model. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia caballi, or Theileria equi was inhibited by allicin in a dose-dependent manner and had IC50 values of 818, 675, 470, and 742 μM, respectively. Moreover, allicin significantly inhibited (P < 0.001) invasion of B. bovis, B. bigemina, B. caballi, and T. equi into the host erythrocyte. Furthermore, mice treated with 30 mg/kg of allicin for 5 days significantly (P < 0.05) reduced the parasitemia of B. microti over the period of the study. To further examine the potential synergism of allicin with diminazene aceturate, growth inhibitory assays were performed in vitro and in vivo. Interestingly, combinations of diminazene aceturate with allicin synergistically potentiated its inhibitory effects in vitro and in vivo. These results indicate that allicin might be beneficial for the treatment of babesiosis, particularly when used in combination with diminazene aceturate.
    Parasitology Research 10/2013; · 2.85 Impact Factor
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    ABSTRACT: Apical membrane antigen-1 (AMA-1) is a microneme protein that exists in all apicomplexan parasites and plays an indispensable role in the invasion into host cell. Central region of ectodomains I and II of Babesia bovis apical membrane antigen-1 (BbAMA-1P) is highly conserved with these of Babesia species and may be beneficial for vaccine development against babesiosis. In the present study, recombinant protein encoding the central region of B. bovis AMA-1 (rBbAMA-1P) was produced in E. coli and its antiserum was prepared in mice for further molecular characterization. Anti-rBbAMA-1P serum specifically reacted with corresponding authentic protein of B. bovis as determined by Western blotting and IFAT. Cultured B. bovis treated with anti-rBbAMA-1P serum showed significant reduction in the in vitro growth of the parasites. Moreover, preincubated free merozoites with 1 mg/ml anti-rBbAMA-1P serum inhibited their efficiency in the invasion into erythrocytes (RBCs) by 61% and 70% at 3 hrs and 6 hrs, respectively. Our data suggest that the central region of domain I and II of BbAMA-1 may serve as a vaccine candidate against babesiosis.
    Experimental Parasitology 09/2013; · 2.15 Impact Factor
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    ABSTRACT: Babesia bovis is the causative agent of fatal babesiosis in cattle. In the present study, we investigated 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 Phillipines 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 Philippine 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.
    Parasitology International 09/2013; · 2.30 Impact Factor
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    ABSTRACT: 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.
    Veterinary Parasitology 07/2013; · 2.38 Impact Factor
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    ABSTRACT: Hemoprotozoan infections often cause serious production losses in livestock. In the present study, we conducted a PCR-based survey of Babesia bovis, Babesia bigemina, Theileria annulata, Theileria orientalis, Trypanosoma evansi and Trypanosoma theileri, using 423 DNA samples extracted from blood samples of cattle (n=202), water buffaloes (n=43), sheep (n=51) and goats (n=127) bred in the Hue and Hanoi provinces of Vietnam. With the exception of T. annulata and T. evansi, all other parasite species (B. bovis, B. bigemina, T. orientalis and T. theileri) were detected in the cattle populations, with B. bovis being the most common among them. Additionally, four water buffaloes and a single goat were infected with B. bovis and B. bigemina, respectively. The Hue province had more hemoprotozoan-positive animals than those from the Hanoi region. In the phylogenetic analyses, B. bovis-MSA-2b, B. bigemina-AMA-1 and T. theileri-CATL gene sequences were dispersed across four, one and three different clades in the respective phylograms. This is the first study in which the presence of Babesia, Theileria, and Trypanosoma parasites was simultaneously investigated by PCR in Vietnam. The findings suggest that hemoprotozoan parasites, some of which are genetically diverse, continue to be a threat to the livestock industry in this country.
    Journal of Veterinary Medical Science 07/2013; · 0.88 Impact Factor
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    ABSTRACT: In the present study, we screened blood DNA samples obtained from cattle bred in Brazil (n=164) and Ghana (n=80) for Babesia bovis using a diagnostic PCR assay and found prevalences of 14.6% and 46.3%, respectively. Subsequently, the genetic diversity of B. bovis in Thailand, Brazil and Ghana was analyzed, based on the DNA sequence of merozoite surface antigen-1 (MSA-1). In Thailand, MSA-1 sequences were relatively conserved and found in a single clade of the phylogram, while Brazilian MSA-1 sequences showed high genetic diversity and were dispersed across three different clades. In contrast, the sequences from Ghanaian samples were detected in two different clades, one of which contained only a single Ghanaian sequence. The identities among the MSA-1 sequences from Thailand, Brazil and Ghana were 99.0-100%, 57.5-99.4% and 60.3-100%, respectively, while the similarities among the deduced MSA-1 amino acid sequences within the respective countries were 98.4-100%, 59.4-99.7% and 58.7-100%, respectively. These observations suggested that the genetic diversity of B. bovis based on MSA-1 sequences was higher in Brazil and Ghana than in Thailand. The current data highlight the importance of conducting extensive studies on the genetic diversity of B. bovis before designing immune control strategies in each surveyed country.
    Journal of Veterinary Medical Science 07/2013; · 0.88 Impact Factor
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    ABSTRACT: Babesia bovis, the causative agent of severe bovine babesiosis, is endemic in Sri Lanka. The live attenuated vaccine (K-strain), which was introduced in the early 1990s, has been used to immunize cattle populations in endemic areas of the country. The present study was undertaken to determine the genetic diversity of merozoite surface antigens (MSAs) in B. bovis isolates from Sri Lankan cattle, and to compare the gene sequences obtained from such isolates against those of the K-strain. Forty-four bovine blood samples isolated from different geographical regions of Sri Lanka and judged to be B. bovis-positive by PCR screening were used to amplify MSAs (MSA-1, MSA-2c, MSA-2a1, MSA-2a2, and MSA-2b), AMA-1, and 12D3 genes from parasite DNA. Although the AMA-1 and 12D3 gene sequences were highly conserved among the Sri Lankan isolates, the MSA gene sequences from the same isolates were highly diverse. Sri Lankan MSA-1, MSA-2c, MSA-2a1, MSA-2a2, and MSA-2b sequences clustered within 5, 2, 4, 1, and 9 different clades in the gene phylograms, respectively, while the minimum similarity values among the deduced amino acid sequences of these genes were 36.8, 68.7, 80.3, 100, and 68.3%, respectively. In the phylograms, none of the Sri Lankan sequences fell within clades containing the respective K-strain sequences. Additionally, the similarity values for MSA-1 and MSA-2c were 40-61.8 and 90.9-93.2% between the Sri Lankan isolates and the K-strain, respectively, while the K-strain MSA-2a/b sequence shared 64.5-69.8, 69.3, and 70.5-80.3% similarities with the Sri Lankan MSA-2a1, MSA-2a2, and MSA-2b sequences, respectively. The present study has shown that genetic diversity among MSAs of Sri Lankan B. bovis isolates is very high, and that the sequences of field isolates diverged genetically from the K-strain.
    Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 07/2013; · 3.22 Impact Factor

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2k Citations
397.58 Total Impact Points


  • 2001–2014
    • Obihiro University of Agriculture and Veterinary Medicine
      • National Research Center for Protozoan Diseases
      Obibiro, Hokkaidō, Japan
  • 2012
    • Minoufiya University
      • Department of Parasitology
      Shabin al-Kum, Al Minūfīyah, Egypt
  • 2010
    • Dokkyo Medical University
      • Department of Tropical Medicine and Parasitology
      Totigi, Tochigi, Japan
  • 2008–2010
    • Tokai University
      • Institute of Glycoscience
      Hiratuka, Kanagawa, Japan
  • 2008–2009
    • National Institute of Advanced Industrial Science and Technology
      • Research Center for Medical Glycoscience
      Tsukuba, Ibaraki, Japan
  • 2004
    • Shenzhen Center for Disease Control and Prevention
      Bao'an, Guangdong, China
  • 2003
    • Jiangsu Institute of Parasitic Diseases
      Wu-hsi, Jiangsu Sheng, China
  • 2002
    • Kitasato University
      • Graduate School of Veterinary Medicine and Animal Sciences
      Edo, Tōkyō, Japan
  • 1999–2001
    • Aichi Cancer Center
      Ōsaka, Ōsaka, Japan
  • 1995–2001
    • The University of Tokyo
      • Faculty and Graduate School of Agriculture and Life Sceince
      Tokyo, Tokyo-to, Japan
  • 1997–1998
    • Yamaguchi University
      • Faculty of Agriculture
      Yamaguchi-shi, Yamaguchi-ken, Japan
    • Kagoshima University
      • Faculty of Agriculture
      Kagoshima-shi, Kagoshima-ken, Japan