Elevated IL-17 levels in semi-immune anaemic mice infected with Plasmodium berghei ANKA.

BACKGROUND: Alterations in inflammatory cytokines and genetic background of the host contribute to the outcome of malaria infection. Despite the promising protective role of IL-17 in infections, little attention is given to further understand its importance in the pathogenesis of severe malaria anaemia in chronic/endemic situations. The objective of this study, therefore, was to evaluate IL-17 levels in anaemic condition and its association with host genetic factors. METHODS: Two mice strains (Balb/c and CBA) were crossed to get the F1 progeny, and were (F1, Balb/c, CBA) taken through 6 cycles of Plasmodium berghei (ANKA strain) infection and chloroquine/pyrimethamine treatment to generate semi-immune status. Cytokine levels and kinetics of antibody production, CD4+CD25+T regulatory cells were evaluated by bead-based multiplex assay kit, ELISA and FACs, respectively. RESULTS: High survival with high Hb loss at significantly low parasitaemia was observed in Balb/c and F1. Furthermore, IgG levels were two times higher in Balb/c, F1 than CBA. While CD4+CD25+ Treg cells were lower in CBA; IL-4, IFN-γ, IL-12α and IL-17 were significantly higher (p < 0.05) in Balb/c, F1. CONCLUSIONS: In conclusion, elevated IL-17 levels together with high IL-4, IL-12α and IFN-γ levels may be a marker of protection, and the mechanism may be controlled by host factor (s). Further studies of F2 between the F1 and Balb/c will be informative in evaluating if these genes are segregated or further apart.

Prevalence and genotypic diversity of Entamoeba species in inhabitants in Kathmandu, Nepal.

In Nepal, gastrointestinal infections due to parasites including Entamoeba species are common. The main aim of this study was to identify species of Entamoeba using genotypic analysis. The prevalence of Entamoeba infections was examined by PCR in fecal samples from 143 inhabitants living close to wild rhesus macaques in Kathmandu, Nepal. The numbers of positive cases were one (0.7%) for E. histolytica, eight (5.6%) for E. dispar, seven (4.9%) for E. coli, and two (1.4%) for E. chattoni (E. polecki ST2). No infections with E. nuttalli, E. moshkovskii, and E. polecki ST1 were found. In E. dispar, at least seven different genotypes were detected from the eight samples by sequence analysis of tRNA-linked short tandem repeats. Different genotypes were found even in a couple from the same family. This is the first report demonstrating that E. dispar with high genotypic diversity is prevalent, rather than E. histolytica, in Kathmandu, and that zoonotic transmission of E. chattoni from rhesus macaques might occur in the inhabitants.

Isolation and Molecular Characterization of Entamoeba nuttalli Strains Showing Novel Isoenzyme Patterns from Wild Toque Macaques in Sri Lanka.

We have proposed the revival of the name Entamoeba nuttalli for a virulent ameba strain, P19-061405, from a rhesus macaque and located it phylogenetically between E. histolytica and E. dispar. As E. nuttalli was originally described for an ameba found in a toque macaque in Sri Lanka, the prevalence and characteristics of Entamoeba species in wild toque macaques were examined. PCR analysis of 227 stool samples from six locations showed positive rates for E. nuttalli, E. dispar, and E. histolytica of 18.5%, 0.4%, and 0%, respectively. Fifteen E. nuttalli strains were cultured successfully from five locations. The 18S ribosomal RNA gene showed only three nucleotide differences in comparison with P19-061405 strain. In isoenzyme analysis, the pattern of hexokinase in Sri Lankan strains was different from that of P19-061405 strains and the difference was confirmed by analysis of the genes. Hepatic inoculation of one of the Sri Lankan E. nuttalli strains in hamsters resulted in amebic abscess formation and body weight loss. These results demonstrate that E. nuttalli is prevalent in wild toque macaques and that several characteristics of the strains are unique. We conclude that use of the name E. nuttalli is appropriate for the new Entamoeba species found in nonhuman primates.

Splenic CD11c+ cells derived from semi-immune mice protect naïve mice against experimental cerebral malaria.

BACKGROUND: Immunity to malaria requires innate, adaptive immune responses and Plasmodium-specific memory cells. Previously, mice semi-immune to malaria was developed. Three cycles of infection and cure ('three-cure') were required to protect mice against Plasmodium berghei (ANKA strain) infection. METHODS: C57BL/6 J mice underwent three cycles of P. berghei infection and drug-cure to become semi-immune. The spleens of infected semi-immune mice were collected for flow cytometry analysis. CD11c(+) cells of semi-immune mice were isolated and transferred into naïve mice which were subsequently challenged and followed up by survival and parasitaemia. RESULTS: The percentages of splenic CD4(+) and CD11c(+) cells were increased in semi-immune mice on day 7 post-infection. The proportion and number of B220(+)CD11c(+)low cells (plasmacytoid dendritic cells, DCs) was higher in semi-immune, three-cure mice than in their naïve littermates on day 7 post-infection (2.6 vs 1.1% and 491,031 vs 149,699, respectively). In adoptive transfer experiment, three months after the third cured P. berghei infection, splenic CD11c(+) DCs of non-infected, semi-immune, three-cure mice slowed Plasmodium proliferation and decreased the death rate due to neurological pathology in recipient mice. In addition, anti-P. berghei IgG1 level was higher in mice transferred with CD11c(+) cells of semi-immune, three-cure mice than mice transferred with CD11c(+) cells of naïve counterparts. CONCLUSION: CD11c(+) cells of semi-immune mice protect against experimental cerebral malaria three months after the third cured malaria, potentially through protective plasmacytoid DCs and enhanced production of malaria-specific antibody.

Correlation between genotypes of tRNA-linked short tandem repeats in Entamoeba nuttalli isolates and the geographical distribution of host rhesus macaques.

Several polymorphic markers, including serine-rich protein genes, have been used for the genotyping of isolates from the morphologically indistinguishable protozoan parasites Entamoeba histolytica, Entamoeba dispar, and Entamoeba nuttalli. Genotypes of tRNA-linked short tandem repeats (STRs) are highly polymorphic, but the correlation with geographical distribution is unknown. We have recently isolated 15 E. nuttalli strains from wild rhesus macaques in four locations in Kathmandu Valley, Nepal. The sequences of the serine-rich protein genes of the E. nuttalli strains differed among the four locations. In this study, we analyzed tRNA-linked STRs in six loci of the 15 strains. Two genotypes were found in loci N-K2, R-R, and S(TGA)-D, three in locus S-Q, and five in locus D-A. In locus A-L, one major genotype and ten minor genotypes were found, resulting in mixtures of two to six genotypes in eight strains. By combination of the main genotypes in the six loci, the 15 strains were divided into nine genotypes. The genotypes observed in E. nuttalli strains were quite different from those in E. histolytica and E. dispar. A phylogenetic tree constructed from tRNA-linked STRs in the six loci reflected the different places of isolation. These results suggest that sequence diversity of tRNA-linked STRs in E. nuttalli occurs with relatively high frequency and might be a marker of geographical distribution of host rhesus macaques, even in limited areas.

Anti-erythropoietin antibody levels and its association with anaemia in different strains of semi-immune mice infected with Plasmodium berghei ANKA.

BACKGROUND: Malaria anaemia is still a major public health problem and its pathogenesis still unclear. Interestingly, the progression of anaemia is at relatively low parasitaemia with some mortality in the semi-immune individuals in the endemic areas despite adequate erythropoietin (EPO) synthesis. A recent study has shown that treatment with exogenous anti-erythropoietin (anti-EPO) antibodies (Ab) of infected mice gives protection against malaria infection, suggesting an important role for anti-EPO Ab in malaria. The objective of the study was to evaluate anti-EPO antibody levels in anaemic condition of different strains of semi-immune mice with malaria. METHODOLOGY: Semi-immune status was attained in four mice strains (Balb/c, B6, CBA and NZW) by repeated infections with 104Plasmodium berghei ANKA, and treatment with chloroquine/pyrimethamine. ELISA was used to measure anti-EPO Ab, transferrin and EPO while inflammatory cytokines measurement was done using bead-based multiplex assay kit. RESULTS: The mean anti-EPO Ab levels in the mice strains [Optical Density (OD) values at 450 nm: Balb/c (2.1); B6 (1.3); CBA (1.4) and NZW (1.7)] differed (p = 0.045), and were significantly higher when compared with uninfected controls, p < 0.0001, and mean anti-EPO Ab levels in the mice strains at recovery [OD values at 450 nm: Balb/c (1.8); B6 (1.1); CBA (1.5) and NZW (1.0) also differed (p = 0.0004). Interestingly, EPO levels were significantly high in NZW and low in Balb/c mice (p < 0.05), with those of B6 and CBA of intermediary values. Again, NZW were highly parasitaemic (20.7%) and the other strains (Balb/c, B6 and CBA) ranged between 2.2-2.8% (p = 0.015). Anti-EPO Ab correlated positively with extent of Hb loss (r = 0.5861; p = 0.003). Correlation of anti-EPO antibody with EPO was significant only in Balb/c mice (r = -0.83; p = 0.01). Significant levels of IL6 and IFNγ (p < 0.0001), both known to be associated with erythropoiesis suppression were observed in the Balb/c. Transferrin was significantly lower in Balb/c (p < 0.0001) when compared with the other mice strains (B6, CBA and NZW). CONCLUSION: This is the first ever report in estimating endogenous anti-EPO antibodies in malaria anaemia. The data presented here suggest that anti-EPO Ab is produced at infection and is associated with Hb loss. Host factors appear to influence anti-EPO antibody levels in the different strains of mice.

Anti-Trypanosoma cruzi activity of Coptis rhizome extract and its constituents.

BACKGROUND: Current therapeutic agents, including nifurtimox and benznidazole, are not sufficiently effective in the chronic phase of Trypanosoma cruzi infection and are accompanied by various side effects. In this study, 120 kinds of extracts from medicinal herbs used for Kampo formulations and 94 kinds of compounds isolated from medicinal herbs for Kampo formulations were screened for anti-T. cruzi activity in vitro and in vivo. METHODS: As an experimental method, a recombinant protozoan cloned strain expressing luciferase, namely Luc2-Tulahuen, was used in the experiments. The in vitro anti-T. cruzi activity on epimastigote, trypomastigote, and amastigote forms was assessed by measuring luminescence intensity after treatment with the Kampo extracts or compounds. In addition, the cytotoxicity of compounds was tested using mouse and human feeder cell lines. The in vivo anti-T. cruzi activity was measured by a murine acute infection model using intraperitoneal injection of trypomastigotes followed by live bioluminescence imaging. RESULTS: As a result, three protoberberine-type alkaloids, namely coptisine chloride, dehydrocorydaline nitrate, and palmatine chloride, showed strong anti-T. cruzi activities with low cytotoxicity. The IC50 values of these compounds differed depending on the side chain, and the most effective compound, coptisine chloride, showed a significant effect in the acute infection model. CONCLUSIONS: For these reasons, coptisine chloride is a hit compound that can be a potential candidate for anti-Chagas disease drugs. In addition, it was expected that there would be room for further improvement by modifying the side chains of the basic skeleton.

Histopathological studies in two strains of semi-immune mice infected with Plasmodium berghei ANKA after chronic exposure.

To mimic a human malaria infection in the endemic condition, two strains of mice (Balb/c and CBA) were infected and treated several times to generate so-called semi-immune status. As previously reported, neither mice (Balb/c and CBA) strain showed cerebral malaria, even in the susceptible C57BL/6 (B6). The significant difference between the mice strains in our previous study was the rate of destruction of uninfected red blood cells (uRBCs) at infection. After the established repeated cycles of infection and treatment and the final challenge with 10(4) Plasmodium berghei ANKA until minimum Hb, Balb/c and CBA mice were sacrificed. The spleen, liver, brain, kidney, lung, heart, and muscle were removed, stained with hematoxylin-eosin and analyzed with light microscopy. Previous observation suggested that Balb/c destroyed uRBC at much higher rate than the other strains although the parasitemia was very low. Pathological investigation carried out in this study revealed that this destruction was mainly contributed by the uRBCs as no parasite sequestration was observed in any of the organs. However, malaria pigment deposition was observed in spleen and liver of all the semi-immune mice strains. This histopathological study in the severe malaria anemia model, which is difficult to conduct in humans, will be helpful in taking into account different responses to malaria infection when designing therapeutic interventions and vaccine studies.

Genotyping of Entamoeba nuttalli strains from the wild rhesus macaques of Myanmar and comparison with those from the wild rhesus macaques of Nepal and China.

Entamoeba nuttalli found in macaques is phylogenetically the closest species to Entamoeba histolytica and is potentially pathogenic. In this study, the prevalence of Entamoeba infections was examined in wild rhesus macaques by examining 73 and 90 fecal samples collected from two sites, Popa Taung Kalat (PTK) and Pho Win Taung (PWT), in Myanmar. The positive rates of E. nuttalli detected using PCR were 49% and 31% in PTK and PWT, respectively, but no infections of E. histolytica and E. moshkovskii were found. Entamoeba dispar was detected in 6% of samples only from PWT. Positive rates of E. chattoni and E. coli were both 70% in PWT and 67% and 79% in PTK, respectively. Six E. nuttalli strains from PTK and eight from PWT were obtained in the culture with xenic medium and then, one and two strains, respectively, were axenized and finally cloned. The genotypic analysis of serine-rich protein genes revealed two genotypes each in both sites. The genotypes found in five of six strains from PTK were similar to those from the strains found in Nepal, whereas the remaining one from PTK and two from PWT were similar to those obtained from macaques in China. The sequence of the 18S rDNA of strains with these four genotypes was identical to that of the strains from China. Six loci of tRNA-linked short tandem repeats were analyzed for further genotyping of the strains. Although there were two types in locus A-L in PTK isolates, one of each type for PTK and PWT was found in the other loci, including locus A-L in PWT strains. These results demonstrated that the E. nuttalli strains from Myanmar are closer to the strains from macaques in China rather than those from macaques in Nepal.

Diagnosis of visceral leishmaniasis by polymerase chain reaction of DNA extracted from Giemsa's solution-stained slides.

Visceral leishmaniasis (VL) is caused by the protozoan parasite Leishmania donovani and is a potentially fatal disease in endemic areas of the world. Nepal is an endemic area in which VL causes major public health problems in the lowland areas of the southeast regions. The aim of the present study was to evaluate the sensitivity of polymerase chain reaction (PCR) amplification for the detection of Leishmania DNA from Giemsa's solution-stained bone marrow slides. Bone marrow samples were aspirated from a total of 115 VL suspected patients and used to prepare smears on glass slides and for the initiation of in vitro culture. Bone marrow slides were used for microscopic observation, DNA extraction, and subsequent PCR amplification. PCR analysis showed that all the positive samples were of Leishmania parasites. The PCR assay also showed a higher sensitivity (69%) than microscopic examination (57%) and culture (21%). In addition, PCR was able to detect VL in 12% of samples which were negative by microscopy. PCR of DNA extracted from Giemsa's solution-stained bone marrow slides is a suitable tool for confirming diagnosis in patients with VL and may also be useful in the diagnosis of difficult cases. Bone marrow smears are easily stored and can be easily sent to research centers where PCR is available. This makes PCR a good option for diagnosis in the field.

Rate of red blood cell destruction varies in different strains of mice infected with Plasmodium berghei-ANKA after chronic exposure.

BACKGROUND: Severe malaria anaemia in the semi-immune individuals in the holo-endemic area has been observed to occur at low parasite density with individual variation in the responses. Thus the following has been thought to be involved: auto-immune-mediated mechanisms of uninfected red blood cell destruction, and host genetic factors to explain the differences in individual responses under the same malaria transmission. In this study, the extent of red blood cell (RBC) destruction in different strains of semi-immune mice model at relatively low parasitaemia was studied. METHODOLOGY: To generate semi-immunity, four strains of mice were taken through several cycles of infection and treatment. By means of immunofluorescent assay and ELISA, sera were screened for anti-erythrocyte auto-antibodies, and their relationship with haematological parameters and parasitaemia in the strains of semi-immune mice was investigated. RESULTS: Upon challenge with Plasmodium berghei ANKA after generating semi-immune status, different mean percentage haemoglobin (Hb) drop was observed in the mice strains (Balb/c = 47.1%; NZW = 30.05%; C57BL/6 = 28.44%; CBA = 25.1%), which occurred on different days for each strain (for Balb/c, mean period = 13.6 days; for C57BL/6, NZW, and CBA mean period = 10.6, 10.8, 10.9 days respectively). Binding of antibody to white ghost RBCs was observed in sera of the four strains of semi-immune mice by immunofluorescence. Mean percentage Hb drop per parasitaemia was highest in Balb/c (73.6), followed by C57BL/6 (8.6), CBA (6.9) and NZW (4.0), p = 0.0005. Consequently, auto-antibodies level to ghost RBC were correlated with degree of anaemia and were highest in Balb/c, when compared with the other strains, p < 0.001. CONCLUSION: The results presented in this study seem to indicate that anti-RBC auto-antibodies may be involved in the destruction of uninfected RBC in semi-immune mice at relatively low parasite burden. Host genetic factors may also influence the outcome of auto-immune mediated destruction of RBC due to the variation in Hb loss per % parasitaemia and differences in antibody titer for each semi-immune mice strain. However, further studies at the molecular level ought to be carried out to confirm this.

Molecular characterization of Blastocystis isolates from children and rhesus monkeys in Kathmandu, Nepal.

To investigate the possible transmission of Blastocystis organisms between local rhesus monkeys and children in Kathmandu, Nepal, we compared the subtype (ST) and sequence of Blastocystis isolates from children with gastrointestinal symptoms and local rhesus monkeys. Twenty and 10 Blastocystis isolates were established from 82 and 10 fecal samples obtained from children and monkeys, respectively. Subtype analysis with seven sequence-tagged site (STS) primers indicated that the prevalence of Blastocystis sp. ST1, ST2 and ST3 was 20%, 20% and 60% in the child isolates, respectively. In contrast to human isolates, ST3 was not found in monkey isolates and the prevalence of ST1 and ST2 was 50% and 70%, respectively, including three mixed STs1 and 2 and one isolate not amplified by any STS primers, respectively. Since Blastocystis sp. ST2 has been reported as the most dominant genotype in the survey of Blastocystis infection among the various monkey species, sequence comparison of the 150bp variable region of the small subunit rRNA (SSU rRNA) gene was conducted among ST2 isolates of humans and monkeys. Sequence alignment of 24 clones developed from ST2 isolates of 4 humans and 4 monkeys showed three distinct subgroups, defined as ST2A, ST2B and ST2C. These three subgroups were shared between the child and monkey isolates. These results suggest that the local rhesus monkeys are a possible source of Blastocystis sp. ST2 infection of humans in Kathmandu.

DNA characterization of simian Entamoeba histolytica-like strains to differentiate them from Entamoeba histolytica.

Two simian Entamoeba histolytica-like strains, EHMfas1 and P19-061405, have been suggested to represent a new species based on genetic characterization. Sequence analyses of the hexokinase, glucose phosphate isomerase, and phosphoglucomutase genes supported the previous findings of isoenzyme analyses demonstrating a new zymodeme pattern. Phylogenetic studies of 18S rDNA, 5.8S rDNA, the chaperonin 60 gene, and the pyridine nucleotide transhydrogenase gene showed original clusters of simian E. histolytica-like strains below or near E. histolytica, respectively. Comparative studies of the chitinase and the serine-rich E. histolytica protein genes and locus 1-2 region revealed that most mutated units were shared among the simian E. histolytica-like strains. The similarities of each of the repeating units within the simian E. histolytica-like strains or E. histolytica and the differences of those between the both might be generated by concerted evolution. Our results indicate that EHMfas1 and P19-061405 should be considered to be the same species, despite that they were isolated from different monkey species and different habitats. Simian E. histolytica-like amebas may be endemic to macaque monkeys, as a counterpart to E. histolytica in humans, and should be differentiated from E. histolytica by the revival name Entamoeba nuttalli, as proposed for P19-061405.

Correlation between genotypes and geographic distribution of Entamoeba nuttalli isolates from wild long-tailed macaques in Central Thailand.

Entamoeba nuttalli found in non-human primates is the phylogenetically closest species to Entamoeba histolytica and is potentially pathogenic. However, infection of wild long-tailed macaques (Macaca fascicularis) with E. nuttalli has not been found. In this study, the prevalence of Entamoeba infections in wild long-tailed macaques was examined in seven locations in six provinces of Thailand. The positive rate for E. nuttalli in 214 fecal samples was 43.9% using PCR, but no infection with E. histolytica or Entamoeba dispar was found, demonstrating that long-tailed macaque is one of the natural hosts for E. nuttalli. Twenty-four E. nuttalli isolates were successfully cultured and four of them were axenized. The sequences of the 18S ribosomal RNA genes of E. nuttalli from long-tailed macaques differed from those of E. nuttalli isolates from other species of wild macaques. Eleven types of sequences in serine-rich protein genes were identified in the 24 isolates and these were specific for each location in Thailand. By analysis of six tRNA-linked short tandem repeat loci, these isolates were divided into 14 types, and each type was also location-specific. Phylogenetic analysis revealed correlation between genotypes of the parasite and the geographic distribution of the host macaques. Genetic distance and geographic distance correlated significantly in a Mantel test, with r values of 0.888 based on the tRNA-linked short tandem repeat loci and 0.815 based on the serine-rich protein genes. These results suggest that genetic divergence and co-evolution of the parasite occurred during dispersion and colonization of the host macaque, and that genotypic analysis of the parasite may enable identification of the geographic localization of the host.

Seasonal variation in the behavior of tailing wastes in Buyat Bay, Indonesia.

Seasonal variations in temperature, salinity, sigma-t and light transmittance were investigated in Buyat Bay, Indonesia, related to the dumping activity there. More than 2000 tons per day of tailings were disposed through a Submarine Tailing Disposal (STD) method at 82 m depth in Buyat Bay. An 80 m depth of the assumed pycnocline was not well confirmed. The seasonal variability of temperature, salinity and sigma-t showed the pycnocline of 40 m to 135 m at the deepest observation station (140 m) during 1997. Furthermore, the Mixed Layer Depth was in good agreement with the wind stress, and the wind stress affected the spreading of tailings there. The outlet of submarine tailing disposal must be below 135 m in Buyat Bay.

Analysis of change of red tide species in Yodo River estuary by the numerical ecosystem model.

Occurrence number of red tides in Osaka Bay in Japan is more than 20 cases every year. Diatom red tide was dominant in Osaka Bay, but the non-diatom red tide was dominant in early 1990s. Therefore, the material cycling in Yodo River estuary in Osaka Bay during August from 1991 to 2000 was analyzed by using the numerical ecosystem model and field observation data to clarify the reasons of change in red tide species. Year-to-year variation in calculated concentration ratio of diatom to non-diatom corresponds to the variation in observed ratio of red tide days of diatom to non-diatom. Limiting nutrient of primary production is phosphate over the period. Diatom dominated from 1991 to 1993, but it was difficult for non-diatom to grow due to the limitation by physical condition. Non-diatom was able to grow because of good physical and nutrient conditions from 1994 to 1996. And diatom dominated again under the good physical condition, and phosphorus supply was not enough for non-diatom to grow from 1998 to 2000. Phosphate concentration in the lower layer of Yodo River estuary was important to the variation in red tide species in the upper layer of Yodo River estuary.

NMR Biochemical Assay for Oxidosqualene Cyclase: Evaluation of Inhibitor Activities on Trypanosoma cruzi and Human Enzymes.

Oxidosqualene cyclase (OSC), a membrane-associated protein, is a key enzyme of sterol biosynthesis. Here we report a novel assay for OSC, involving reaction in aqueous solution, NMR quantification in organic solvent, and factor analysis of spectra. We evaluated one known and three novel inhibitors on OSC of Trypanosoma cruzi, a parasite causative of Chagas disease, and compared their effects on human OSC for selectivity. Among them, one novel inhibitor showed a significant parasiticidal activity.

An Entamoeba sp. strain isolated from rhesus monkey is virulent but genetically different from Entamoeba histolytica.

An Entamoeba sp. strain, P19-061405, was isolated from a rhesus monkey in Nepal and characterized genetically. The strain was initially identified as Entamoeba histolytica using PCR amplification of peroxiredoxin genes. However, sequence analysis of the 18S rRNA gene showed a 0.8% difference when compared to the reference E. histolytica HM-1:IMSS human strain. Differences were also observed in the 5.8S rRNA gene and the internal transcribed spacer (ITS) regions 1 and 2, and analysis of the serine-rich protein gene from the monkey strain showed unique codon usages compared to E. histolytica isolated from humans. The amino acid sequences of two hexokinases and two glucose phosphate isomerases also differed from those of E. histolytica. Isoenzyme analyses of these enzymes in the monkey strain showed different electrophoretic mobility patterns compared with E. histolytica isolates. Analysis of peroxiredoxin genes indicated the presence of at least seven different types of protein, none of which were identical to proteins in E. histolytica. When the trophozoites from the monkey strain were inoculated into the livers of hamsters, formation of amebic abscesses was observed 7 days after the injection. These results demonstrate that the strain is genetically different from E. histolytica and is virulent. Revival of the name Entamoeba nuttalli is proposed for the organism.

Structure and expression of three gp82 gene subfamilies of Trypanosoma cruzi.

The glycoprotein gp82 is a GPI-anchored cell surface protein of Trypanosoma cruzi and is involved in cell invasion. Gp82 is encoded by multiple genes. To investigate the genetic basis of its biological function, we analyzed structure and expression of gp82 multigene family members in the Peruvian and Guatemalan strains. Three major groups of gp82 genes (A, B and C) were categorized by analyzing multiple DNA clones from the genomic PCR products. Within each group, 95-97% homology was observed, whereas between the groups, homology was 67-79%. The copy numbers of groups A, B and C as determined by real-time PCR were 18, 8 and 7 copies, respectively, in the Peru-2 strain. Significant elevation of the mRNA expression levels (5-10 times more) of all the subfamily genes was observed in the metacyclic stage compared with the epimastigote stage. When we focused on the binding motif sequence reported previously, we found substantial difference between that of A and C. However, the peptide inhibition invasion assay showed no functional difference. Taken together, we demonstrated that three subfamilies of gp82 were in the genome of T. cruzi and maintained their functional structure, and that the mRNA expressions of those genes were equally controlled in a stage-specific manner.

Genotypic variation among lineages of Trypanosoma cruzi and its geographic aspects.

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb-e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb-e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb-e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.