Prevalence of Trichomoniasis by PCR in Women Attending Health Screening in Korea.

Trichomoniasis is the most common curable sexually-transmitted infection (STI) worldwide. There are few reports on the prevalence of Trichomonas vaginalis in Korea. The purpose of this study was to examine the prevalence of trichomoniasis by PCR in Guri city, Korea. All adult women who visited Hanyang University Guri Hospital for health screening within the National Health Care Service were invited to participate in the study, and 424 women were enrolled between March and June 2011. PCR was used to detect Trichomonas vaginalis using primers based on a repetitive sequence cloned from T. vaginalis (TV-E650). Fourteen women (3.3%) were found to have T. vaginalis. All were over 50, and they were significantly older on average than the 410 Trichomonas-negative women (mean ages 63.4 vs 55.3 years). It seems that T. vaginalis infection is not rare in women receiving health screening, especially among those over 50.

Comparison between mixed lysate antigen and α-actinin antigen in ELISA for serodiagnosis of trichomoniasis.

The aim of this study was to identify an antigen suitable for ELISA for serodiagnosis of Trichomonas vaginalis (T. vaginalis) infection. Mixed lysate antigen (Ag) from eight strains of T. vaginalis and recombinant α-actinin protein was compared. The sera of three groups were examined by ELISA: 73 women infected with trichomoniasis served as a positive control, 31 male volunteers as a negative control, and 424 women attending an outpatient health screening at Hanyang University Guri Hospital. Based on the cutoff optical density for each Ag obtained with a negative control, the serosensitivity of the mixed lysate Ag (79.5%) was significantly higher than that of the α-actinin (52.1%) in the 73 patients with trichomoniasis. The specificity using lysate Ag and α-actinin was 100% and 96.8%, respectively. On the other hand, the positivity rate in 424 outpatients was 39.2% and 11.8% with mixed lysate and α-actinin Ag, respectively. Taken together, mixed lysate Ag showed higher sensitivity and specificity than α-actinin. Therefore, mixed lysate may be a better Ag than α-actinin for ELISA for the diagnosis of trichomoniasis.

Allergenicity of two Anisakis simplex allergens evaluated in vivo using an experimental mouse model.

Anisakis (Anisakidae) is one of the most important causes of helminth-induced allergic reactions and elicits clinical responses that include urticaria, rhinitis, bronco-constriction, cough, and/or gastrointestinal symptoms. More than 13 reactive allergens have been identified in the serum of Anisakis allergy patients, but the allergenicity of only a few of these have been evaluated in vivo using a mouse model. To evaluate the allergenicity of two important allergens, Ani s 1 and Ani s 9, we induced experimental allergic airway inflammation in a mouse model by repeated intranasal administration of the allergens. Both recombinant proteins (rAni s 1 and rAni s 9) elicited increased airway hyperresponsivity, airway infiltration by inflammatory cells (especially eosinophils), bronchial epithelial cell hyperplasia, all of which are characteristic of allergic airway inflammation. These allergens significantly increased the levels of Th2-related cytokines (IL-4, IL-5, IL-13, and IL-25) and Th17 related cytokines (IL-6 and IL-17) in both splenocytes and airway (except IL-17 in airway by rAni s 9). OVA-specific IgE and total IgE were increased in rAni s 1 and rAni s 9 treated mice as compared with controls treated with OVA alone. In addition, these two allergens induced gene expression of thymic stromal lymphopoietin (TSLP) and IL-25 (initiators of the Th2 response), as well as CXCL1 (initiator of the Th17 response) in mouse lung epithelial cells. In conclusion, repeated intranasal treatments with rAni s 1 and rAni s 9 induced airway inflammation in mice by elevating of Th2 and Th17 responses in the lung.

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.

Alteration of helper T-cell related cytokine production in splenocytes during Trichinella spiralis infection.

Infection by Trichinella spiralis takes place in two distinct phases: one is the intestinal phase and the other is the muscle phase. To evaluate alterations in cytokine production during a T. spiralis infection, we periodically assessed the cytokine production of splenocytes in mice after infection (AI). The levels of Th2-related cytokines immediately increased after the initiation of T. spiralis larval intestinal invasion (1 week AI). These early elevations in the Th2 response might be associated with the innate immune responses of intestine epithelial cells against T. spiralis larval invasion. IL-4 and IL-13 levels reached a peak prior to the initiation of nurse cell formation (2 weeks AI). Additionally, all Th17-related cytokines, except for IL-17, increased slightly until 2 weeks AI. However, expression levels for all of the Th2 and Th17-related cytokines began to decrease after the initiation of nurse cell formation and reached basal levels at 4 weeks AI, except for IL-5. At the same time, the CD4(+)CD25(+)Foxp3(+) T (regulatory T, T(reg)) cell population increased significantly in the spleen. Additionally, the number of cells in the peripheral lymph nodes increased. In conclusion, T. spiralis larva intestinal invasion induced the production of Th2 and Th17 cell-related cytokines, and the cytokines decreased with T(reg) cell-related cytokine.

Endosymbionts of Acanthamoeba isolated from domestic tap water in Korea.

In a previous study, we reported our discovery of Acanthamoeba contamination in domestic tap water; in that study, we determined that some Acanthamoeba strains harbor endosymbiotic bacteria, via our molecular characterization by mitochondrial DNA restriction fragment length polymorphism (Mt DNA RFLP). Five (29.4%) among 17 Acanthamoeba isolates contained endosymbionts in their cytoplasm, as demonstrated via orcein staining. In order to estimate their pathogenicity, we conducted a genetic characterization of the endosymbionts in Acanthamoeba isolated from domestic tap water via 16S rDNA sequencing. The endosymbionts of Acanthamoeba sp. KA/WP3 and KA/WP4 evidenced the highest level of similarity, at 97% of the recently published 16S rDNA sequence of the bacterium, Candidatus Amoebophilus asiaticus. The endosymbionts of Acanthamoeba sp. KA/WP8 and KA/WP12 shared a 97% sequence similarity with each other, and were also highly similar to Candidatus Odyssella thessalonicensis, a member of the alpha-proteobacteria. The endosymbiont of Acanthamoeba sp. KA/WP9 exhibits a high degree of similarity (85-95%) with genus Methylophilus, which is not yet known to harbor any endosymbionts. This is the first report, to the best of our knowledge, to show that Methylophilus spp. can live in the cytoplasm of Acanthamoeba.

Acanthamoeba healyi: expressed gene profiles with enhanced virulence after mouse-brain passage.

The virulence of Acanthamoeba can be attenuated by long-term in vitro cultivation, and can be recovered by serial mouse-brain passage via intranasal inoculation. Recovery is concomitant with changes in expression of virulence-related genes. To investigate the virulence factors of Acanthamoeba, expressed sequence tags (ESTs) from two kinds of cDNA libraries-long-term in vitro cultivated A. healyi (OLD) and three times mouse-brain passaged A. healyi (MBP)-were compared using reciprocal BLAST analysis, eukaryotic orthologous groups (KOG) assignment, and gene annotation. A total of 938 (OLD) and 1033 (MBP) ESTs were sequenced and resulted in the assembling of 718 OLD and 833 MBP unique sequences. Comparison of the KOG analysis revealed a relatively higher percentage of MBP ESTs in genes related to transcription (K group), amino acid transport and metabolism (E group), coenzyme transport and metabolism (H group), and secondary metabolites biosynthesis, transport and metabolism (Q group). However, a higher percentage of unidentified MBP ESTs (57.9%) than OLD ESTs (28.9%) was evidence of the limited understanding of virulence-related factors of Acanthamoeba. Characterization of the genes expressed during brain passage in mice will be useful in clarifying the pathogenesis of granulomatous amoebic encephalitis by Acanthamoeba.

Identification of host immune regulation candidate genes of Toxascaris leonina by expression sequenced tags (ESTs) analysis.

Toxascaris leonina adult worms live in the gastrointestinal tract of dog, cat, and fox, releasing eggs which enter the environment by the fecal route. Previously, we reported that T. leonina adult worm derived protein was able to inhibit OVA-specific Th2 responses, and in particular, immunization with parasite proteins exerts a more profound protective effect than allergen treatment. In order to gain greater insight into the relevant immune evasion mechanisms as well as basic scientific information, we have generated ESTs of T. leonina adult female worm and investigated their functions using euKaryotic Orthologous Groups (KOG) database analysis. From the randomly selected plasmids containing DNA inserts, a total of 487 reads were collected from the T. leonina adult worm cDNA library. The annotated ESTs were classified into 25 KOG categories; the most of ESTs (7.90%) were annotated with energy production and conversion, and the second highly annotated category is translation, ribosomal structure and biogenesis related ESTs (7.69%). We also identified many host-parasite immune related genes including C-type lectin, galectin, SXP, and cathepsin L-like cysteine protease coding genes. It is necessary to get more information regarding these genes for understanding about the mechanisms of immune evasion of Toxascaris.

Construction of EST database for comparative gene studies of Acanthamoeba.

The genus Acanthamoeba can cause severe infections such as granulomatous amebic encephalitis and amebic keratitis in humans. However, little genomic information of Acanthamoeba has been reported. Here, we constructed Acanthamoeba expressed sequence tags (EST) database (Acanthamoeba EST DB) derived from our 4 kinds of Acanthamoeba cDNA library. The Acanthamoeba EST DB contains 3,897 EST generated from amebae under various conditions of long term in vitro culture, mouse brain passage, or encystation, and downloaded data of Acanthamoeba from National Center for Biotechnology Information (NCBI) and Taxonomically Broad EST Database (TBestDB). The almost reported cDNA/genomic sequences of Acanthamoeba provide stand alone BLAST system with nucleotide (BLAST NT) and amino acid (BLAST AA) sequence database. In BLAST results, each gene links for the significant information including sequence data, gene orthology annotations, relevant references, and a BlastX result. This is the first attempt for construction of Acanthamoeba database with genes expressed in diverse conditions. These data were integrated into a database (http://www.amoeba.or.kr).

Autophagy protein 8 mediating autophagosome in encysting Acanthamoeba.

Autophagy is an evolutionally conserved protein degradation pathway in eukaryotes. It plays essential roles during starvation, cellular differentiation, cell death, and aging by eliminating unwanted or unnecessary organelles and recycling the components for reuse. ATG8, a member of a novel ubiquitin-like protein family, is an essential component of the autophagic machinery. The present study identified and characterized autophagy protein 8 in Acanthamoeba castellanii an amphizoic amoeba causing granulomatous amoebic encephalitis and amoebic keratitis in humans. Real-time polymerase chain reaction demonstrated that the A. castellanii Atg8 (AcAtg8) gene encoding a 118 amino acid protein was highly expressed during encystation. Fluorescence microscopic analysis following transient transfection of enhanced green fluorescent protein-AcAtg8 revealed small or large vacuolar fluorescent structures in an encysting amoeba. The Atg8 fluorescent structures on the membrane were identified as autophagosomes by co-localization analysis with LysoTracker. Chemically synthesized small interfering RNA against AcAtg8 reduced the encystation efficiency and inhibited autophagosome formation in Acanthamoeba.

Keratitis by Acanthamoeba triangularis: report of cases and characterization of isolates.

Three Acanthamoeba isolates (KA/E9, KA/E17, and KA/E23) from patients with keratitis were identified as Acanthamoeba triangularis by analysis of their molecular characteristics, a species not previously recognized to be a corneal pathogen. Epidemiologic significance of A. triangularis as a keratopathogen in Korea has been discussed. Morphologic features of Acanthamoeba cysts were examined under a microscope with differential interference contrast (DIC) optics. Mitochondrial DNA (mtDNA) of the ocular isolates KA/E9, KA/E17, and KA/E23 were digested with restriction enzymes, and the restriction patterns were compared with those of reference strains. Complete nuclear 18S and mitochondrial (mt) 16S rDNA sequences were subjected to phylogenetic analysis and species identification. mtDNA RFLP of 3 isolates showed very similar patterns to those of SH621, the type strain of A. triangularis. 16S and 18S rDNA sequence analysis confirmed 3 isolates to be A. triangularis. 18S rDNA sequence differences of the isolates were 1.3% to 1.6% and those of 16S rDNA, 0.4% to 0.9% from A. triangularis SH621. To the best of our knowledge, this is the first report, confirmed by 18S and 16S rDNA sequence analysis, of keratitis caused by A. triangularis of which the type strain was isolated from human feces. Six isolates of A. triangularis had been reported from contaminated contact lens cases in southeastern Korea.

Characterization of a serine proteinase mediating encystation of Acanthamoeba.

Members of the genus Acanthamoeba, amphizoic protozoan parasites, are causative agents of granulomatous amoebic encephalitis and amoebic keratitis. Proteinases play a role in various biologic actions in Acanthamoeba, including host tissue destruction, pathogenesis, and digestion of phagocytosed food. Interestingly, we found that encystation of Acanthamoeba was inhibited by the serine proteinase inhibitor phenylmethanesulfonyl fluoride. In this study, we characterize a serine proteinase that is involved in mediating the encystation of Acanthamoeba. This encystation-mediating serine proteinase (EMSP) is shown to be highly expressed during encystation by real-time PCR and Western blot analysis. Chemically synthesized small interfering RNA against EMSP inhibited the expression of EMSP mRNA and significantly reduced the encystation efficiency of Acanthamoeba. An EMSP-enhanced green fluorescent protein fusion protein localized to vesicle-like structures within the amoeba. Using LysoTracker analysis, these vesicular structures were confirmed to be lysosomes. After incubation of the transfected amoeba in encystment media, small fluorescent vesicle-like structures gathered and formed ball-like structures, which were identified as colocalizing with the autophagosome. Taken together, these results indicate that EMSP plays an important role in the differentiation of Acanthamoeba by promoting autolysis.

Immunization of proteins from Toxascaris leonina adult worm inhibits allergic specific Th2 response.

Recently, the influence of parasitic infections on the incidence of allergic diseases has become the focus of increased attention. In order to ascertain whether parasite-derived proteins could inhibit the allergic specific Th2 response, we applied excretory-secretory protein (Tl-ES) or total protein (Tl-TP) of the adult worm Toxascaris leonina to asthma model mice prior to or simultaneously with OVA challenge, after which we assessed the OVA-specific Th2 responses. The group subjected to immunization with Tl-ES and Tl-TP (immunized group) evidenced a thinning of the bronchial epithelial and muscle layer, a disruption and shedding of epithelial cells, a reduction in the number of goblet cells, and a reduction in mucus production as compared to the group treated with Tl-ES coupled with OVA challenge (challenge with OVA groups) and the OVA-induced asthma group. The administration of Tl-ES and Tl-TP, regardless of injection time, was shown to inhibit the recruitment of inflammatory cells into the airway, and in particular, macrophages, neutrophils, and lymphocytes were significantly reduced as the result of the parasite proteins. However, the total number of eosinophils was slightly reduced as the result of the administration of parasite proteins. Sensitization and OVA challenge was shown to accelerate the secretion of Th2 cytokines (IL-4 and IL-5) within the lung, but in the immunized groups, those levels were lower. The administration of Tl-TP and OVA challenge group also evidenced a significant reduction in IL-4 levels as compared to the OVA-challenged group. The concentrations of Th2 cytokines in the Tl-ES and OVA challenge group were more similar to those observed in the OVA-challenged group. The concentration of IL-10 and TGF-beta in the lung was decreased substantially in the OVA-only challenge group, but the Tl-TP immunized group exhibited significantly induced IL-10 cytokine. OVA-specific IgG2a, IgG1, and IgE levels in the immunized groups were significantly lower than those detected in the OVA-challenged group. In conclusion, parasite-derived protein is able to inhibit OVA-specific Th2 responses, and in particular, immunization with parasite proteins exerts a more profound protective effect than is seen with the treatment of allergic reactions. The results of our study are encouraging in terms of our further understanding of the molecular basis of immune evasion by nematodes.

Expressed sequence tags of Trichinella spiralis muscle stage larvae.

In order to obtain greater insight into the relevant genomic expression patterns of Trichinella spiralis, 992 expressed sequence tags (ESTs) were collected from a cDNA library of T. spiralis muscle stage larvae and assembled into 60 clusters and 385 singletons. Of them, 445 (44.7%) ESTs were annotated to their homologous genes, and small fractions were matched to known genes of nematodes. The annotated ESTs were classified into 25 eukaryotic orthologous groups (KOG). Cytochrome C oxidase (34 clones) was found to be most frequent species.

Identification and characterization of Paragonimus westermani leucine aminopeptidase.

Paragonimus westermani is a tissue-invading trematode parasite that causes inflammatory lung disease as well as systemic infections including cerebral invasion in carnivorous mammals. While aminopeptidases play important roles in trematodes in the catabolism of host hemoglobin, an essential source of nutrient for the parasite, little is known about aminopeptidase in Paragonimus. Presently, we isolated a cDNA encoding a 58 kDa P. westermani leucine aminopeptidase (PwLAP). Deduced amino acid sequence of PwLAP exhibited significant sequence homology with LAP from Schistosoma spp. and Fasciola hepatica. Biochemical analysis of the recombinant PwLAP protein demonstrated preferential substrate specificity for Leu-NHMec and inhibition by EDTA, 1,10-phenanthroline, and bestatin, which are conserved characteristics of the M17 family of leucine aminopeptidase. PwLAP exhibited relatively higher enzyme activity in the presence of Mn2+ compared to Schistosoma mansoni LAP. Based on the biochemical properties and immunohistochemical analysis, PwLAP is concluded to represent a leucine aminopeptidase. The enzyme is most likely responsible for the catabolism of host hemoglobin, and, hence, represents a potential target of Paragonimus chemotherapy.

Acanthamoeba castellanii: gene profile of encystation by ESTs analysis and KOG assignment.

The trophozoite of Acanthamoeba transforms into a cyst, the resistant form under harmful environments such as starvation, cold and certain chemicals used in medical treatment. To investigate the factors mediating encystation, ESTs of encystation-induced A. castellanii were analysed and compared to those of trophozoites. Each EST was compared by the predicted proteins from the ESTs, to the cyst and the trophozoite by reciprocal BLAST analysis, KOG assignment, and gene annotation. In addition to the genes previously reported to encystation mediate such as cyst specific protein 21, protein kinase C, proteasome and heat shock protein, several genes like cullin 4, autophage protein 8 and ubiquitin-conjugating enzymes were identified to be related to encystation. Five kinds of proteinase genes were detected in cyst ESTs. The information of the genes expressed during encystation may open the way to further study on differentiation and resistance of cyst-forming pathogenic protozoa.

Molecular characterization of two myoglobins of Paragonimus westermani.

Myoglobins (Mbs), globin proteins, are present in high concentrations in trematodes. In Paragonimus westermani, 2 cDNAs were found to encode Mbs. The first clone, Pwmyo1, codes a total of 149 amino acids with a calculated mass of 16.6 kDa. The second, Pwmyo2, encodes a 146-amino acid protein with a calculated mass of 16.2 kDa. The predicted secondary structures showed the presence of 8 helices, which is the basic characteristic of Mbs. Sequence alignment revealed a high homology with the other trematode Mbs. The 2 clones contained the characteristic tyrosyl residues at helical positions B10 and distal E7, which are substitutions that have been previously shown to contribute to the high oxygen affinity of Mbs. Polyclonal antibodies against the recombinant Mbs were raised with no cross-reactivity observed. Immunolocalization revealed the proteins to be distributed generally throughout the parenchymal tissues, but absent from the tegument and reproductive organs. The cell mass of the eggs of the worm stained positive to Pwmyo2 but not Pwmyo1, suggesting the stage-specific expression of these Mbs.

Natural occurrence of Mycobacterium as an endosymbiont of Acanthamoeba isolated from a contact lens storage case.

Recent in vitro studies have revealed that a certain Mycobacterium can survive and multiply within free-living amoebae. It is believed that protozoans function as host cells for the intracellular replication and evasion of Mycobacterium spp. under harmful conditions. In this study, we describe the isolation and characterization of a bacterium naturally observed within an amoeba isolate acquired from a contact lens storage case. The bacterium multiplied within Acanthamoeba, but exerted no cytopathic effects on the amoeba during a 6-year amoebic culture. Transmission electron microscopy showed that the bacteria were randomly distributed within the cytoplasm of trophozoites and cysts of Acanthamoeba. On the basis of the results of 18S rRNA gene analysis, the amoeba was identified as A. lugdunensis. A 16S rRNA gene analysis placed this bacterium within the genus Mycobacterium. The bacterium evidenced positive reactivity for acid-fast and fluorescent acid-fast stains. The bacterium was capable of growth on the Middlebrook 7H11-Mycobacterium-specific agar. The identification and characterization of bacterial endosymbionts of free-living protozoa bears significant implications for our understanding of the ecology and the identification of other atypical mycobacterial pathogens.

Differentially expressed genes of Acanthamoeba castellanii during encystation.

To examine the expressed gene profile during encystation of Acanthamoeba castellanii Castellani, we used differentially expressed gene (DGE) screening by RT-PCR with 20 sets of random primers. From this analysis, we found that approximately 16 genes showed upregulation during encystation. We chose 6 genes, which had relatively higher expression levels, for further investigation. Based on homology search in database, DEG2 showed 55% of similarity with xylose isomerase, DEG9 showed 37% of similarity with Na P-type ATPase, and DEG14 showed 77% of similarity with subtilisin-like serine proteinase. DEG3 and DEG26 were identified as hypothetical proteins and DEG25 exhibited no significant similarity to any known protein. Encystation of Acanthamoeba has been suggested to be a process to resist adverse environmental or nutritional conditions. Further characterization studies of these genes may provide us with more information on the encystation mechanism of Acanthamoeba.

Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence.

The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.