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In humans, Entamoeba histolytica is the main pathogen causing various amoebiases, while E. moshkovskii falls between being a pathogen and non-pathogen. The two species have similar behavior patterns but differ significantly in pathogenicity, with previous studies and clinical data indicating that E. moshkovskii has a low level of pathogenicity. Meaningfully, the biological characteristics of E. moshkovskii make it a potential model organism and a protein display platform for studying the functions of important Entamoeba proteins. Here, an Amoeba-pcDNA3.1 vector capable of overexpressing E. histolytica-sourced Igl-C protein was constructed and successfully transfected into E. moshkovskii. High levels of expression of the Igl-C, EGFP, and NeoR genes were identified in Igl-C-transfected trophozoites using qRT-PCR, and they were subsequently confirmed using immunoblotting. Transfection of Igl-C protein improved the adherence and phagocytosis of E. moshkovskii, demonstrating that E. histolytica Igl mediated amoebic adhesion. Moreover, as a manifestation of protein virulence, the ability of post-transfected trophozoites to induce inflammation in host macrophages was also enhanced. In conclusion, this study utilizing the characteristics of E. moshkovskii confirmed its potential to serve as a model organism. E. moshkovskii could replace E. histolytica as the target of gene editing, allowing more efficient study of amoebic pathogenicity.

Entamoeba nuttalli infection is highly prevalent in captive and wild macaques. A recent study suggested that the genetic factor of host macaques was correlated with the genotypes of E. nuttalli isolates. This study focused on the correlation between the rhesus macaque host major histocompatibility complex gene and E. nuttalli infection. Thirty-nine stool samples were obtained from Mount Qing-ling (Guizhou Province, China). Polymerase chain reaction analysis detected the infection rate of E. nuttalli, Entamoeba coli, and Entamoeba chattoni as 69.23%, 69.23%, and 87.18%, respectively. A new Serine-rich Protein genotype was detected, and the rRNA of E. nuttalli isolates from Mount Qian-ling was completely identical to the GY4 strain. In the distance-based neighbor-joining tree, Mamu-DRB1, not Mamu-DPB or Mamu-B gene, was related to E. nuttalli infection. Mamu-DRB1 genes of rhesus macaques in Mounts Qian-ling and Long-hu were highly polymorphic, and the rhesus macaques with two major types of Mamu-DRB1 showed susceptibility to E. nuttalli infection. The Mamu-DRB1 gene analysis in this study indicated that the Mamu-DRB1 gene is an important factor that influences the susceptibility of E. nuttalli infection in Chinese Macaca mulatta. This study contributes to a better understanding of host susceptibility to Entamoeba., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Entamoeba nuttalli infection is prevalent in captive and wild macaques. Recent studies have suggested that genotypes of E. nuttalli isolates are correlated with the geographical distribution of host macaques. Correlation of amoebic genotypes with genetic diversity of host macaques was analyzed in present study. Sixty fresh stool samples were obtained from wild Tibetan macaques living in Mount Huang (HS) of the An-hui Province in China. PCR analysis revealed that the most prevalent Entamoeba species was E. chattoni (E. polecki ST2) (86.7%) followed by E. nuttalli (58.3%) and E. coli (25%). Six E. nuttalli HS isolates were successfully cultured. The tRNA-linked short tandem repeat (STR) loci and serine-rich protein gene of E. nuttalli isolates from four different regions of China (Mount Long-hu, Gui-yang, Mount E-mei, and HS, the former three isolates were obtained in previous studies) were studied and high numbers of polymorphisms were detected. When genetic diversity of different populations of E. nuttalli isolates was compared with geographical distance, an r 2 value of 0.919 was assigned by a Mantel test based on the tRNA-STR loci. In host macaques, the mtDNA HVS-I gene was also highly polymorphic in each of the genomes. Multiple regression analysis using E. nuttalli tRNA-STR loci genetic, macaque mtDNA HVS-I gene, and geographic distances showed an r 2 value of 0.943, indicating that a higher relevance was demonstrated when geographic and host gene factors were considered. Analysis of genetic factor of host would benefit for better understanding of the evolution of E. nuttalli.

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.

Background: Entamoeba nuttalli is an intestinal protozoan with pathogenic potential that can cause amebic liver abscess. It is highly prevalent in wild and captive macaques. Recently, cysts were detected in a caretaker of nonhuman primates in a zoo, indicating that E. nuttalli may be a zoonotic pathogen. Therefore, it is important to evaluate the pathogenicity of E. nuttalli in detail and in comparison with that of E. histolytica., Methodology/principal Findings: Trophozoites of E. nuttalli GY4 and E. histolytica SAW755 strains were inoculated into liver of hamsters. Expression levels of proinflammatory factors of hamsters and virulence factors from E. histolytica and E. nuttalli were compared between the two parasites. Inoculations with trophozoites of E. nuttalli resulted in an average necrotic area of 24% in liver tissue in 7 days, whereas this area produced by E. histolytica was nearly 50%. Along with the mild liver tissue damage induced by E. nuttalli, expression levels of proinflammatory factors (TNF-α, IL-6 and IL-1β) and amebic virulence protein genes (lectins, cysteine proteases and amoeba pores) in local tissues were lower with E. nuttalli in comparison with E. histolytica. In addition, M2 type macrophages were increased in E. nuttalli-induced amebic liver abscesses in the late stage of disease progression and lysate of E. nuttalli trophozoites induced higher arginase expression than E. histolytica in vitro., Conclusions/significance: The results show that differential secretion of amebic virulence proteins during E. nuttalli infection triggered lower levels of secretion of various cytokines and had an impact on polarization of macrophages towards a M1/M2 balance. However, regardless of the degree of macrophage polarization, there is unambiguous evidence of an intense acute inflammatory reaction in liver of hamsters after infection by both Entamoeba species.

Unique species of macaques are distributed across Sulawesi Island, Indonesia, and the details of Entamoeba infections in these macaques are unknown. A total of 77 stool samples from Celebes crested macaques (Macaca nigra) and 14 stool samples from pigs were collected in Tangkoko Nature Reserve, North Sulawesi, and the prevalence of Entamoeba infection was examined by PCR. Entamoeba polecki was detected in 97% of the macaques and all of the pigs, but no other Entamoeba species were found. The nucleotide sequence of the 18S rRNA gene in E. polecki from M. nigra was unique and showed highest similarity with E. polecki subtype (ST) 4. This is the first case of identification of E. polecki ST4 from wild nonhuman primates. The sequence of the 18S rRNA gene in E. polecki from pigs was also unique and showed highest similarity with E. polecki ST1. These results suggest that the diversity of the 18S rRNA gene in E. polecki is associated with differences in host species and geographic localization, and that there has been no transmission of E. polecki between macaques and pigs in the study area., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

We have recently demonstrated the potentially virulent species Entamoeba nuttalli as one of the highly prevalent parasites in rhesus macaques (Macaca mulatta) in Mount Long-hu and Gui-yang in China. Tibetan macaque (Macaca thibetana) is a unique species living in China. To evaluate the prevalence of Entamoeba species in wild Tibetan macaques, we obtained 89 stool samples in Mount E-mei of Si-chuan Province in China. PCR analysis detected E. nuttalli, Entamoeba coli, and Entamoeba polecki ST2 in 17%, 42%, and 66% of the samples, respectively, whereas Entamoeba histolytica and Entamoeba dispar were undetected. This study is the first to report on the detection of E. nuttalli from Tibetan macaques. Six E. nuttalli isolates were obtained, 18S rRNA gene and six tRNA-linked short tandem repeat (STR) loci of the isolates were sequenced. The Mantel test results gave an r value of 0.97 of relationships between geographical distance and genetic diversity of Chinese E. nuttalli populations, indicating a significant isolation-by-distance effect in Chinese E. nuttalli according to the tRNA-STR loci sequences. Structural analysis of E. nuttalli isolates based on tRNA-linked STR loci demonstrated three Chinese E. nuttalli populations with their respective features, but the Gui-yang population was located in the middle. In the distance-based NJ tree, E. nuttalli isolates were divided into five different branches, and E-mei isolates were attributed to an independent branch to distinguish them from Gui-yang and Long-hu isolates. Genetic analysis in this study provided clues of the genetic differences between E. nuttalli isolates from Tibetan macaques and rhesus macaques in China., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.)

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.

We have recently resurrected the name Entamoeba nuttalli Castellani, 1908 for a potentially virulent ameba isolate, P19-061405, obtained from a rhesus macaque in Kathmandu, Nepal. The ameba was morphologically indistinguishable from Entamoeba histolytica/Entamoeba dispar/Entamoeba moshkovskii, but located phylogenetically between E. histolytica and E. dispar. To evaluate the prevalence of E. nuttalli infection in wild rhesus macaques, 112 fecal samples were collected in four locations of the Kathmandu Valley. PCR analysis of DNA extracted from the feces showed positive rates of E. nuttalli, E. dispar, E. histolytica and E. moshkovskii of 51%, 12%, 0% and 0%, respectively. A total of 14 E. nuttalli isolates were obtained from four locations, of which 6 were established as axenic cultures. The sequences of the serine-rich protein gene of E. nuttalli isolates differed among four locations although no differences were found in the composition of sequence motifs. Isoenzyme pattern was analyzed in 8 isolates obtained from three locations. In hexokinase, the mobility of the slower migrating band was located between E. histolytica and E. dispar regardless of the culture conditions. These results demonstrate that E. nuttalli is highly prevalent in wild rhesus macaques in Nepal. Rhesus macaques appear to be one of the natural hosts and heterogeneity of the serine-rich protein gene might be useful for geographical typing of isolates., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Long-tailed macaques (Macaca fascicularis) are bred in China for export and for use in experiments. Entamoeba infections in captive long-tailed macaques were surveyed in one of the biggest colonies located in Guangxi Province, China. One stool sample was obtained from each of the 152 different cages representing >3,000 macaques in the colony. The samples were examined by PCR for five Entamoeba species. The number of detected Entamoeba coli infections comprised 94% of the samples, 93% for Entamoeba chattoni, and 83% for Entamoeba dispar. In contrast, Entamoeba histolytica and Entamoeba nuttalli were not detected. Six isolates of E. dispar were obtained by culture in Tanabe-Chiba medium. Analysis of serine-rich protein genes in these isolates showed two genotypes, one of which is identical to that of the E. dispar SAW760 strain in humans. This suggests transmission of E. dispar between humans and nonhuman primates. These results demonstrate that Entamoeba infections are common, but virulent Entamoeba species are absent in this colony. This work also confirms the need for monitoring with PCR-based identification of Entamoeba species for captive macaques in breeding colonies to ensure animal health and protection of humans from zoonotic hazards.

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. [ABSTRACT FROM AUTHOR]

Many colonies of macaques ( Macaca fascicularis and Macaca mulatta) are maintained in China, especially in Guangxi and Guizhou. A total of 803 fresh stool samples infected with Entamoeba were obtained from three big colonies of macaques located in southwest China. The samples were examined for the presence of five Entamoeba species using PCR. Entamoeba nuttalli, Entamoeba dispar, Entamoeba coli, and Entamoeba chattoni infections were detected, but Entamoeba histolytica infection was not. This study is the first to report on the prevalence of E. nuttalli in wild macaques from China. Eighteen E. nuttalli isolates and five E. dispar isolates were obtained by culturing the samples in Tanabe-Chiba medium. The serine-rich protein (SRP), ribosomal RNA (rRNA), hexokinase (HXK), glucose-6-phosphate isomerase (GPI), and phosphoglucomutase (PGM) genes of E. nuttalli isolates were compared with other reported isolates. The results showed clear differences among the Chinese E. nuttalli isolates and other isolates based on the SRP gene sequences. However, HXK, GPI, and PGM genes of these strains were similar to those of other isolates. The rRNA genes of E. coli and E. chattoni were also amplified and analyzed from these samples. The results suggested that host species might be a more important factor than geographic location in amebic genetic diversity. [ABSTRACT FROM AUTHOR]