Accelerated Bioconversion of Chemically Solubilized Lignite Solution to Methane by Methanogenic Consortium: Experimental Results and Their Application to the Subsurface Cultivation and Gasification Method.

Lignite is an obsolete and less commercially circulated natural resource due to its low calorific value worldwide. The effective conversion of lignite into methane is important considering the global energy crunch. This study reported the effective bioconversion of organic matter released from chemically solubilized lignite to methane using two methanogenic consortia types: mixed methanogenic enrichment culture (mMEC) and SAL25-2. We demonstrated in a microcosm study that the start of methane generation was observed within seven days. Furthermore, the methane yield increased as the total organic carbon concentration of the chemically solubilized lignite solution increased. Surprisingly, methane production using mMEC was drastically enhanced by approximately 50-fold when pulverized lignite was added as conductive material (CM) to the microcosms. To the best of our knowledge, this is the highest number of times methane production increased relative to the control. Our results demonstrated that bioaugmentation using a methanogenic consortium and adding pulverized lignite as CM could facilitate the bioconversion of chemically solubilized lignite solution to methane and lead to effective utilization of subterranean lignite, regarded as a neglected natural resource, without any further excavation processes.

Desulfovibrio subterraneus sp. nov., a mesophilic sulfate-reducing deltaproteobacterium isolated from a deep siliceous mudstone formation.

A novel mesophilic sulfate-reducing bacterium, strain HN2T, was isolated from groundwater sampled from the subsurface siliceous mudstone of the Wakkanai Formation located in Horonobe, Hokkaido, Japan. The bacterium was Gram-negative and vibrio-shaped, and its motility was conferred by a single polar flagellum. Cells had desulfoviridin. Catalase and oxidase activities were not detected. It grew in the temperature range of 25-40 °C (optimum, 35 °C) and pH range of 6.3-8.1 (optimum, pH 7.2-7.6). It used sulfate, thiosulfate, dimethyl sulfoxide, anthraquinone-2,6-disulfonate, Fe3+, and manganese oxide, but not elemental sulfur, nitrite, nitrate, or fumarate as electron acceptors. The strain showed weak growth with sulfite as the electron acceptor. Fermentative growth with pyruvate, lactate and cysteine was observed in the absence of sulfate, but not with malate or fumarate. NaCl was not required, but the strain tolerated up to 40 g l-1. Strain HN2T did not require vitamins. The major cellular fatty acids were iso-C15 : 0 (23.8 %), C18 : 1 ω9t (18.4 %), C18 : 0 (15.0 %), C16 : 0 (14.5 %), and anteiso-C17 :0 (10.1 %). The major respiratory quinone was menaquinone MK-6(H2). The G+C content of the genomic DNA was 56.7 mol%. Based on 16S rRNA gene sequence analysis, the closest phylogenetic relative of strain HN2T is Desulfovibrio psychrotolerans JS1T (97.0 %). Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values of the strains HN2T and D. psychrotolerans JS1T were 22.2 and 79.8 %, respectively. Based on the phenotypic and molecular genetic evidence, we propose a novel species, D. subterraneus sp. nov. with the type strain HN2T (=DSM 101010T=NBRC 112213T).

Evaluation of novel oocyst wall protein candidates of Toxoplasma gondii.

Oocyst stage of Toxoplasma gondii is characterized by a durable wall that confers a strong protection to this protozoan parasite in face of harsh environmental conditions. Thus, it is considered the key for transmission of T. gondii. Analysis of oocyst wall composition is mandatory therefore; the aim of this study was to identify novel T. gondii oocyst wall proteins and test their use in detection of these oocysts in environmental samples. Five candidates of novel T. gondii oocyst wall proteins (TgOWPs) were identified and named TgOWP8 through TgOWP12. Recombinant protein of TgOWP8 was expressed in E. coli using glutathione S-transferase as fusion protein. Polyclonal antibody was produced and validated by indirect immunofluorescence antibody assay (IFA). For detection by IFA, we used different methods for fixation and permeabilization of oocysts to improve the antigen-antibody detection. Specificity to wall of T. gondii oocyst was confirmed and revealed absence of cross reactivity with bradyzoite cyst wall and tachyzoites. Although some TgOWPs were identified previously, our study represents a continuation of molecular investigations of oocyst wall proteins as an essential structure for the longevity and infectivity of this stage and also provided new trial to improve T. gondii oocysts detection.

Effects of Aerobic Growth on the Fatty Acid and Hydrocarbon Compositions of Geobacter bemidjiensis BemT.

Geobacter spp., regarded as strict anaerobes, have been reported to grow under aerobic conditions. To elucidate the role of fatty acids in aerobiosis of Geobacter spp., we studied the effect of aerobiosis on fatty acid composition and turnover in G. bemidjiensis BemT. G. bemidjiensis BemT was grown under the following different culture conditions: anaerobic culture for 4 days (type 1) and type 1 culture followed by 2-day anaerobic (type 2) or aerobic culture (anaerobic-to-aerobic shift; type 3). The mean cell weight of the type 3 culture was approximately 2.5-fold greater than that of type 1 and 2 cultures. The fatty acid methyl ester and hydrocarbon fraction contained hexadecanoic (16:0), 9-cis-hexadecenoic [16:1(9c)], tetradecanoic (14:0), tetradecenoic [14:1(7c)] acids, hentriacontanonaene, and hopanoids, but not long-chain polyunsaturated fatty acids. The type 3 culture contained higher levels of 14:0 and 14:1(7c) and lower levels of 16:0 and 16:1(9c) compared with type 1 and 2 cultures. The weight ratio of extracted lipid per dry cell was lower in the type 3 culture than in the type 1 and 2 cultures. We concluded that anaerobically-grown G. bemidjiensis BemT followed by aerobiosis were enhanced in growth, fatty acid turnover, and de novo fatty acid synthesis.

Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use.

The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.

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

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

Anaerobic decomposition of humic substances by Clostridium from the deep subsurface.

Decomposition of humic substances (HSs) is a slow and cryptic but non-negligible component of carbon cycling in sediments. Aerobic decomposition of HSs by microorganisms in the surface environment has been well documented; however, the mechanism of anaerobic microbial decomposition of HSs is not completely understood. Moreover, no microorganisms capable of anaerobic decomposition of HSs have been isolated. Here, we report the anaerobic decomposition of humic acids (HAs) by the anaerobic bacterium Clostridium sp. HSAI-1 isolated from the deep terrestrial subsurface. The use of (14)C-labelled polycatechol as an HA analogue demonstrated that the bacterium decomposed this substance up to 7.4% over 14 days. The decomposition of commercial and natural HAs by the bacterium yielded lower molecular mass fractions, as determined using high-performance size-exclusion chromatography. Fourier transform infrared spectroscopy revealed the removal of carboxyl groups and polysaccharide-related substances, as well as the generation of aliphatic components, amide and aromatic groups. Therefore, our results suggest that Clostridium sp. HSAI-1 anaerobically decomposes and transforms HSs. This study improves our understanding of the anaerobic decomposition of HSs in the hidden carbon cycling in the Earth's subsurface.

Characterization of Toxoplasma gondii glyoxalase 1 and evaluation of inhibitory effects of curcumin on the enzyme and parasite cultures.

BACKGROUND: The glyoxalase pathway, which includes two enzymes, glyoxalase 1 and 2 (Glo1 and Glo2), is a ubiquitous cellular system responsible for the removal of cytotoxic methylglyoxal produced during glycolysis. Protozoan parasites, including Toxoplasma gondii (T. gondii) tachyzoites, produce methylglyoxal because of increased glycolytic fluxes. A Glo1 inhibitor such as curcumin could be considered a drug candidate for anti-protozoan, anti-inflammatory, and anti-cancer therapy. METHODS: The T. gondii Glo1 gene (TgGlo1) was cloned and the recombinant protein was produced. Enzyme kinetics of TgGlo1 and five mutants were evaluated by adding methylglyoxal and glutathione to a reaction mixture. Finally, the inhibitory effects of various concentrations of curcumin on recombinant TgGlo1 were evaluated using in vitro cultures of T. gondii. RESULTS: Active recombinant TgGlo1 was successfully produced and the active sites (E166 and E251) of TgGlo1 were verified by point mutagenesis. Curcumin at the tested doses inhibited the enzymatic activity of recombinant TgGlo1 as well as the parasitic propagation of in vitro-cultured T. gondii. The Ki and IC50 were 12.9 ± 0.5 µM and 38.3 ± 0.9 µM, respectively. CONCLUSION: The inhibitory effect of curcumin on the enzymatic activity of TgGlo1 and parasitic propagation of T. gondii could be explored in the potential development of a potent drug for the treatment of toxoplasmosis. However, considering the fact that curcumin is known to have many effects on other molecules in the micromolar range, further elucidation of curcumin's direct inhibition of the glyoxalase system of T. gondii will be needed.

Methanosarcina subterranea sp. nov., a methanogenic archaeon isolated from a deep subsurface diatomaceous shale formation.

A methanogenic archaeon, strain HC-2(T), was isolated from a deep diatomaceous shale formation. The strain grew on methanol, monomethylamine, dimethylamine, trimethylamine and dimethylsulphide, but not on acetate, H2/CO2, formate, 2-propanol, 2-butanol or cyclopentanol. Cells were Gram-stain-negative, non-motile, and coccus-like, 0.9-1.4 µm in diameter, and occurred singly, in pairs, or as aggregates. The strain grew at 10-40 °C (optimum 35 °C), pH 5.9-7.4 (optimum pH 6.6-6.8) and in 0-0.6 M NaCl (optimum 0.1-0.2 M). The genomic DNA G+C content was 41.5 mol% and the 16S rRNA gene sequence was closely related to those of Methanosarcina lacustris DSM 13486(T) (99.1%) and Methanosarcina siciliae DSM 3028(T) (98.3%). Values for DNA-DNA hybridization with these strains were less than 30%. The phenotypic and phylogenetic features of HC-2(T) indicate that it represents a novel species of the genus Methanosarcina , for which the name Methanosarcina subterranea sp. nov. is proposed. The type strain is HC-2(T) ( = DSM 22503(T) = JCM 15540(T) = NBRC 102578(T)).

Molecular characterization of a new Babesia bovis thrombospondin-related anonymous protein (BbTRAP2).

A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite's growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.

Methanoculleus horonobensis sp. nov., a methanogenic archaeon isolated from a deep diatomaceous shale formation.

A methanogenic organism from the domain Archaea, designated strain T10(T), was isolated from groundwater sampled from a deep diatomaceous shale formation located in Horonobe, Hokkaido, Japan. The strain utilized H2/CO2 and formate as substrates for methanogenesis. Cells were strictly anaerobic, Gram-negative-staining, flagellated, irregular coccoids, 0.7-1.6 µm in diameter, and occurred singly. The strain grew at 25-45 °C (optimum 37-42 °C), at pH 5.8-8.2 (optimum pH 6.7-6.8) and in the presence of 0-1.3 M NaCl (optimum 0.1-0.2 M NaCl). The G+C content of the genomic DNA was 62.9 mol%. 16S rRNA gene sequencing revealed that, although the strain is a member of the genus Methanoculleus, it clearly differed from all described species of this genus (95.5-98.3 % sequence similarity). Values for DNA-DNA hybridization with type strains of closely related Methanoculleus species were less than 50 %. Phenotypic and phylogenetic features of strain T10(T) clearly indicate that it represents a novel species of the genus Methanoculleus, for which the name Methanoculleus horonobensis sp. nov. is proposed. The type strain is T10(T) ( = DSM 21626(T) = JCM 15517(T)).

Actin polymerization mediated by Babesia gibsoni aldolase is required for parasite invasion.

Host cell invasion by apicomplexan parasites driven by gliding motility and empowered by actin-based movement is essential for parasite survival and pathogenicity. The parasites share a conserved invasion process: actin-based motility led by the coordination of adhesin-cytoskeleton via aldolase. A number of studies of host cell invasion in the Plasmodium species and Toxoplasma gondii have been performed. However, the mechanisms of host cell invasion by Babesia species have not yet been studied. Here, we show that Babesia gibsoni aldolase (BgALD) forms a complex with B. gibsoni thrombospondin-related anonymous protein (BgTRAP) and B. gibsoni actin (BgACT), depending on tryptophan-734 (W-734) in BgTRAP. In addition, actin polymerization is mediated by BgALD. Moreover, cytochalasin D, which disrupts actin polymerization, suppressed B. gibsoni parasite growth and inhibited the host cell invasion by parasites, indicating that actin dynamics are essential for erythrocyte invasion by B. gibsoni. This study is the first molecular approach to determine the invasion mechanisms of Babesia species.

A novel dense granule protein, GRA22, is involved in regulating parasite egress in Toxoplasma gondii.

The intracellular protozoan parasite Toxoplasma gondii is capable of invading any nucleated cell and replicates within a parasitophorous vacuole (PV). This microenvironment is modified by secretory proteins from organelles named rhoptries and dense granules. In this report, we identify a novel dense granule protein, which we refer to as GRA22. GRA22 has no significant homology to any other known proteins. GRA22 possesses a signal peptide at the N-terminal end which is responsible for dense granule and PV localization. The RH strain GRA22 contains 12 copies of tandem repeats consisting each of 21 amino acids located between the 42nd and 293rd amino acid residues from a full length of 624 amino acids. On the other hand, ME49 strain GRA22 has 10 copies of tandem repeats. The Neospora caninum GRA22 ortholog completely lacks this repetitive sequence. GRA22 knock out parasites show a similar growth rate as the parental strain. However, the timing of egress is earlier than that of the parental strain. These results suggest that GRA22 is involved in regulating parasite egress in T. gondii.

Farsenyl pyrophosphate synthase is a potential molecular drug target of risedronate in Babesia bovis.

A cDNA encoding farnesyl pyrophosphate synthase of Babesia bovis (BbFPPS) has been isolated, cloned and characterized as molecular drug target. Sequence analysis revealed that BbFPPS contains an open reading frame of 1011bp with predicted 336 amino acids and molecular mass of 38kDa. Antiserum raised in mice against recombinant BbFPPS expressed in Escherichia coli specifically reacted with native protein of B. bovis parasites by Western blot analysis and indirect immunofluorescent test. Enzymatic assay using recombinant BbFPPS revealed that the Km value of the enzyme for isopentenyl pyrophosphate and dimethylallyl pyrophosphate was 2.494±1.536µM. Risedronate inhibited the activity of BbFPPS yielding IC50 value of 8.4±1.2nM. Furthermore, the in vitro growth of B. bovis was significantly inhibited in the presence of a micromolar concentration of risedronate (IC50=4.02±0.91µM). No regrowth of B. bovis was observed at 10µM of risedronate in the subsequent viability test. These results demonstrate that BbFPPS is the molecular target of risedronate, which could inhibit the in vitro growth of B. bovis.

Cloning and characterization of histone deacetylase from Babesia bovis.

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

Apicoplast-targeting antibacterials inhibit the growth of Babesia parasites.

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

C-Terminal region of 48-kDa rhoptry protein for serological detection of Babesia caballi antibodies in horses.

A recombinant C-terminal antigen derived from Babesia caballi 48-kDa rhoptry protein (rBc48/CT) was made for the development of a serologically diagnostic test. Antiserum raised against the rBc48/CT reacted specifically with the corresponding native protein by Western blotting and the indirect fluorescent antibody test (IFAT). Next, an indirect enzyme-linked immunosorbent assay (Bc48/CT-ELISA) and an immunochromatographic test based on the Bc48/CT (Bc48/CT-ICT) were constructed and employed for the detection of an antibody to B. caballi in a variety of equine sera. The results of Bc48/CT-ELISA and Bc48/CT-ICT were highly concordant with those of IFAT and ELISA, with full-length protein of Bc48 used as the reference tests. Our results demonstrate the success of Bc48/CT as antigen for the serological diagnosis of B. caballi infection in horses.

Phylogenetic relationships of Mongolian Babesia bovis isolates based on the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c genes.

We conducted a molecular epidemiological study on Babesia bovis in Mongolia. Three hundred blood samples collected from cattle grazed in seven different districts were initially screened using a previously established diagnostic polymerase chain reaction (PCR) assay for the detection of B. bovis-specific DNA. Positive samples were then used to amplify and sequence the hyper-variable regions of three B. bovis genes encoding the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c. The diagnostic PCR assay detected B. bovis among cattle populations of all districts surveyed (4.4-26.0%). Sequences of each of the three genes were highly homologous among the Mongolian isolates, and found in a single phylogenetic cluster. In particular, a separate branch was formed only by the Mongolian isolates in the MSA-2b gene-based phylogenetic tree. Our findings indicate that effective preventative and control strategies are essential to control B. bovis infection in Mongolian cattle populations, and suggest that a careful approach must be adopted when using immunization techniques.

Microbial communities associated with acetate-rich gas-petroleum reservoir surface facilities.

We evaluated the microbial communities in acetate-rich production waters from separators of a high-temperature gas-petroleum reservoir in Higashi-Niigata, Japan. Bacterial and archaeal 16S rRNA gene libraries constructed from these waters were dominated by Acetobacterium-, Methanofollis-, and Methanosarcina-related sequences. The libraries constructed from enrichment cultures of the production waters were dominated by sequences related to the Acetobacterium- and Methanofollis-related sequences.

Molecular prevalence of different genotypes of Theileria orientalis detected from cattle and water buffaloes in Thailand.

Here we report on an epidemiological study regarding the molecular prevalence of different genotypes of Theileria orientalis present among domestic cattle and water buffalo populations bred in Thailand. A phylogenetic analysis based on the parasitic gene encoding a major piroplasm surface protein revealed the presence of 5 genotypes (Types 1, 3, 5, 7, and N-3) in cattle and 7 genotypes (Types 1, 3, 4, 5, 7, N-2, and N-3) in water buffaloes. Types 4, 7, and N-3 of T. orientalis were reported for the first time in water buffaloes. The previously reported C and Thai types from Thailand clustered as types 7 and 6, respectively, in the present analysis. Great similarities were observed among nucleotide sequences of isolates of the same genotype from cattle and water buffaloes, and, therefore, water buffaloes were considered to serve as a reservoir for these genotypes of T. orientalis in Thailand. In conclusion, T. orientalis parasites circulating in Thailand are more diverse in their genetic characters than previously anticipated.