Kyoko Hayashida

Evolution and genetic diversity of Theileria

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 parasites 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, Theileriaparva and Theileriaannulata. In contrast, genetic diversity in Theileriaorientalis, 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.

Comparative Genome Analysis of Three Eukaryotic Parasites with Differing Abilities To Transform Leukocytes Reveals Key Mediators of Theileria-Induced Leukocyte Transformation

ABSTRACT We sequenced the genome of Theileria orientalis, a tick-borne apicomplexan protozoan parasite of cattle. The focus of this study was a comparative genome analysis of T. orientalis relative to other highly pathogenic Theileria species, T. parva and T. annulata. T. parva and T. annulata induce transformation of infected cells of lymphocyte or macrophage/monocyte lineages; in contrast, T. orientalis does not induce uncontrolled proliferation of infected leukocytes and multiplies predominantly within infected erythrocytes. While synteny across homologous chromosomes of the three Theileria species was found to be well conserved overall, subtelomeric structures were found to differ substantially, as T. orientalis lacks the large tandemly arrayed subtelomere-encoded variable secreted protein-encoding gene family. Moreover, expansion of particular gene families by gene duplication was found in the genomes of the two transforming Theileria species, most notably, the TashAT/TpHN and Tar/Tpr gene families. Gene families that are present only in T. parva and T. annulata and not in T. orientalis, Babesia bovis, or Plasmodium were also identified. Identification of differences between the genome sequences of Theileria species with different abilities to transform and immortalize bovine leukocytes will provide insight into proteins and mechanisms that have evolved to induce and regulate this process. The T. orientalis genome database is available at http://totdb.czc.hokudai.ac.jp/. IMPORTANCE Cancer-like growth of leukocytes infected with malignant Theileria parasites is a unique cellular event, as it involves the transformation and immortalization of one eukaryotic cell by another. In this study, we sequenced the whole genome of a nontransforming Theileria species, Theileria orientalis, and compared it to the published sequences representative of two malignant, transforming species, T. parva and T. annulata. The genome-wide comparison of these parasite species highlights significant genetic diversity that may be associated with evolution of the mechanism(s) deployed by an intracellular eukaryotic parasite to transform its host cell. Cancer-like growth of leukocytes infected with malignant Theileria parasites is a unique cellular event, as it involves the transformation and immortalization of one eukaryotic cell by another. In this study, we sequenced the whole genome of a nontransforming Theileria species, Theileria orientalis, and compared it to the published sequences representative of two malignant, transforming species, T. parva and T. annulata. The genome-wide comparison of these parasite species highlights significant genetic diversity that may be associated with evolution of the mechanism(s) deployed by an intracellular eukaryotic parasite to transform its host cell.

Direct blood dry LAMP: a rapid, stable, and easy diagnostic tool for Human African Trypanosomiasis.

Loop-mediated isothermal amplification (LAMP) is a rapid and sensitive tool used for the diagnosis of a variety of infectious diseases. One of the advantages of this method over the polymerase chain reaction is that DNA amplification occurs at a constant temperature, usually between 60–65°C; therefore, expensive devices are unnecessary for this step. However, LAMP still requires complicated sample preparation steps and a well-equipped laboratory to produce reliable and reproducible results, which limits its use in resource-poor laboratories in most developing countries. In this study, we made several substantial modifications to the technique to carry out on-site diagnosis of Human African Trypanosomiasis (HAT) in remote areas using LAMP. The first essential improvement was that LAMP reagents were dried and stabilized in a single tube by incorporating trehalose as a cryoprotectant to prolong shelf life at ambient temperature. The second technical improvement was achieved by simplifying the sample preparation step so that DNA or RNA could be amplified directly from detergent-lysed blood samples. With these modifications, diagnosis of HAT in local clinics or villages in endemic areas becomes a reality, which could greatly impact on the application of diagnosis not only for HAT but also for other tropical diseases.

Amoebal endosymbiont Neochlamydia genome sequence illuminates the bacterial role in the defense of the host amoebae against Legionella pneumophila.

Previous work has shown that the obligate intracellular amoebal endosymbiont Neochlamydia S13, an environmental chlamydia strain, has an amoebal infection rate of 100%, but does not cause amoebal lysis and lacks transferability to other host amoebae. The underlying mechanism for these observations remains unknown. In this study, we found that the host amoeba could completely evade Legionella infection. The draft genome sequence of Neochlamydia S13 revealed several defects in essential metabolic pathways, as well as unique molecules with leucine-rich repeats (LRRs) and ankyrin domains, responsible for protein-protein interaction. Neochlamydia S13 lacked an intact tricarboxylic acid cycle and had an incomplete respiratory chain. ADP/ATP translocases, ATP-binding cassette transporters, and secretion systems (types II and III) were well conserved, but no type IV secretion system was found. The number of outer membrane proteins (OmcB, PomS, 76-kDa protein, and OmpW) was limited. Interestingly, genes predicting unique proteins with LRRs (30 genes) or ankyrin domains (one gene) were identified. Furthermore, 33 transposases were found, possibly explaining the drastic genome modification. Taken together, the genomic features of Neochlamydia S13 explain the intimate interaction with the host amoeba to compensate for bacterial metabolic defects, and illuminate the role of the endosymbiont in the defense of the host amoebae against Legionella infection.

Whole-Genome Sequencing of Theileria parva Strains Provides Insight into Parasite Migration and Diversification in the African Continent

The disease caused by the apicomplexan protozoan parasite Theileria parva, known as East Coast fever or Corridor disease, is one of the most serious cattle diseases in Eastern, Central, and Southern Africa. We performed whole-genome sequencing of nine T. parva strains, including one of the vaccine strains (Kiambu 5), field isolates from Zambia, Uganda, Tanzania, or Rwanda, and two buffalo-derived strains. Comparison with the reference Muguga genome sequence revealed 34 814–121 545 single nucleotide polymorphisms (SNPs) that were more abundant in buffalo-derived strains. High-resolution phylogenetic trees were constructed with selected informative SNPs that allowed the investigation of possible complex recombination events among ancestors of the extant strains. We further analysed the dN/dS ratio (non-synonymous substitutions per non-synonymous site divided by synonymous substitutions per synonymous site) for 4011 coding genes to estimate potential selective pressure. Genes under possible positive selection were identified that may, in turn, assist in the identification of immunogenic proteins or vaccine candidates. This study elucidated the phylogeny of T. parva strains based on genome-wide SNPs analysis with prediction of possible past recombination events, providing insight into the migration, diversification, and evolution of this parasite species in the African continent.

Preliminary Investigation of Trypanosomosis in Exotic Dog Breeds from Zambia's Luangwa and Zambezi Valleys Using LAMP

Abstract. Canine African trypanosomosis (CAT) is rarely reported in the literature. In this preliminary study, we evaluated the performance of loop-mediated isothermal amplification (LAMP) against microscopy to detect CAT in six exotic dog breeds naturally infected with trypanosomes from Zambias South Luangwa National Park and Chiawa Game Management Area. To our knowledge, this is the first report of CAT in Zambia. The patients exhibited a variety of aspecific clinical signs. The LAMP did not only confirm all six parasitologically positive CAT cases detected passively between April 2010 and January 2012, but was also critical in trypanosome speciation. According to LAMP, the majority of the dogs had monolytic infections with either Trypanosoma congolense or Trypanosoma brucei rhodesiense. The LAMP is thus a potential simple and cost-effective tool for trypanosome diagnosis in endemic regions. The rare report of zoonotic trypanosomes in dogs in Zambia has public health implications and justifies further investigations of CAT.

Genetic characterization of Babesia and Theileria parasites in water buffaloes in Sri Lanka

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.

The PCR detection and phylogenetic characterization of Babesia microti in questing ticks in Mongolia.

Babesia microti is a tick-transmitted zoonotic hemoprotozoan parasite. In the present study, we investigated B. microti infection in questing ticks in Mongolia. A total of 219 questing ticks were collected from three different Mongolian provinces (Bayan-Olgii, Khovsgol, and Selenge). Of these, 63 from Selenge were identified as Ixodes persulcatus, while the remaining 156 (from all three provinces) were identified as Dermacentor nuttalli. When the tick DNA samples were screened using a B. microti-specific nested PCR, 19 (30.2%) of the 63 I. persulcatus ticks were found to be B. microti-positive. The parasite was not detected in D. nuttalli. Subsequently, the 18S rRNA, cox1, and tufA sequences of B. microti were amplified, sequenced, and subjected to phylogenetic analyses. Sequencing analyses showed that the Mongolian 18S rRNA, cox1, and tufA sequences were 99.6-100%, 96.7-97.2%, and 94.7-95.3% homologous, respectively, with B. microti R1 strain US-type sequences from humans. In the phylogenetic analyses, the Mongolian cox1 and tufA sequences were found to be separate lineages, which formed sister-clades to the R1 strain sequences, while all of the Mongolian B. microti 18S rRNA sequences were clustered within US-type clade containing several other sequences of human origin. In conclusion, in addition to reporting the presence of B. microti for the first time in questing ticks in Mongolia, the present study found that Mongolian I. persulcatus ticks were infected with US-type B. microti. These findings warrant large-scale studies to detect and characterize B. microti in ticks, small mammals, and humans. Such studies should provide us with a better understanding of zoonotic Babesia epidemiology in Mongolia.

Genetic variations in merozoite surface antigen genes of Babesia bovis detected in Vietnamese cattle and water buffaloes

The genes that encode merozoite surface antigens (MSAs) in Babesia bovis are genetically diverse. In this study, we analyzed the genetic diversity of B. bovis MSA-1, MSA-2b, and MSA-2c genes in Vietnamese cattle and water buffaloes. Blood DNA samples from 258 cattle and 49 water buffaloes reared in the Thua Thien Hue province of Vietnam were screened with a B. bovis-specific diagnostic PCR assay. The B. bovis-positive DNA samples (23 cattle and 16 water buffaloes) were then subjected to PCR assays to amplify the MSA-1, MSA-2b, and MSA-2c genes. Sequencing analyses showed that the Vietnamese MSA-1 and MSA-2b sequences are genetically diverse, whereas MSA-2c is relatively conserved. The nucleotide identity values for these MSA gene sequences were similar in the cattle and water buffaloes. Consistent with the sequencing data, the Vietnamese MSA-1 and MSA-2b sequences were dispersed across several clades in the corresponding phylogenetic trees, whereas the MSA-2c sequences occurred in a single clade. Cattle- and water-buffalo-derived sequences also often clustered together on the phylogenetic trees. The Vietnamese MSA-1, MSA-2b, and MSA-2c sequences were then screened for recombination with automated methods. Of the seven recombination events detected, five and two were associated with the MSA-2b and MSA-2c recombinant sequences, respectively, whereas no MSA-1 recombinants were detected among the sequences analyzed. Recombination between the sequences derived from cattle and water buffaloes was very common, and the resultant recombinant sequences were found in both host animals. These data indicate that the genetic diversity of the MSA sequences does not differ between cattle and water buffaloes in Vietnam. They also suggest that recombination between the B. bovis MSA sequences in both cattle and water buffaloes might contribute to the genetic variation in these genes in Vietnam.

Genetic diversity among Trypanosoma (Duttonella) vivax strains from Zambia and Ghana, based on cathepsin L-like gene

Understanding the evolutionary relationships of Trypanosoma (Duttonella) vivax genotypes between West Africa and Southern Africa can provide information on the epidemiology and control of trypanosomosis. Cattle blood samples from Zambia and Ghana were screened for T. vivax infection using specie-specific PCR and sequencing analysis. Substantial polymorphism was obtained from phylogenetic analysis of sequences of cathepsin L-like catalytic domains. T. vivax from Ghana clustered together with West African and South American sequences, while T. vivax from Zambia formed one distinct clade and clustered with East African and Southern African sequences. This study suggests existence of distinct genetic diversity between T. vivax genotypes from West Africa and Zambia as per their geographical origins.