Association of Blastocystis ST6 with higher protease activity among symptomatic subjects.

BACKGROUND: Blastocystis sp. is an anaerobic intestinal protozoan parasite of humans and a wide range of animals worldwide. In the current study the correlation between the cysteine protease activity of clinical samples of Blastocystis sp. ST1-3 and 6 with the levels of pro-inflammatory cytokines was evaluated. METHODS: Stool samples were collected from subjects with or without clinical symptoms. All samples were cultivated in DMEM medium. The bacteria were eliminated or reduced in Blastocystis sp. positive samples subtypes 1-3 and 6 by a variety of antibiotics and consecutive sub-cultures. To prepare parasite lysate, 1 × 105 Blastocystis sp. from each isolate were harvested and lysed using freeze-thaw. Protease activity of each isolate was measured and the gene expression of pro-inflammatory biomarkers in HT-29 cell line sensed by isolates was investigated using quantitative Real-time PCR. RESULTS: Protease activity assay showed inter- and intra-subtype variations among subtypes regarding the presence of symptoms, while the protease activity of symptomatic isolates was higher than asymptomatic isolates. The highest and lowest levels of protease activity were seen in ST6 and ST2, respectively. However, patterns of the expression of pro-inflammatory biomarkers in HT-29 cell line was different regarding the presence of symptoms and time points. There was no significant correlation between protease activity of different subtypes with the expression levels of pro-inflammatory biomarkers. CONCLUSIONS: Our study indicated a higher protease activity among isolates from symptomatic compared to asymptomatic subjects, suggesting functional role for proteases in clinical symptoms due to Blastocystis sp. The lack of correlation between the levels of expression of pro-inflammatory biomarkers with subtypes regarding the presence of clinical symptoms proposes the importance of host-related factors in presentation of clinical symptoms.

New insights into the interactions between Blastocystis, the gut microbiota, and host immunity.

The human gut microbiota is a diverse and complex ecosystem that is involved in beneficial physiological functions as well as disease pathogenesis. Blastocystis is a common protistan parasite and is increasingly recognized as an important component of the gut microbiota. The correlations between Blastocystis and other communities of intestinal microbiota have been investigated, and, to a lesser extent, the role of this parasite in maintaining the host immunological homeostasis. Despite recent studies suggesting that Blastocystis decreases the abundance of beneficial bacteria, most reports indicate that Blastocystis is a common component of the healthy gut microbiome. This review covers recent finding on the potential interactions between Blastocystis and the gut microbiota communities and its roles in regulating host immune responses.

Epitope variances demonstrated by Blastocystis sp. ST3 symptomatic and asymptomatic isolates.

Blastocystis sp. is an enteric protozoan parasite of humans and many animals. Blastocystis sp. subtype 3 (ST3) proves to be the highest frequency case in most populations around the world and it is further distinguished into symptomatic and asymptomatic isolates based on the clinical symptoms exhibited by infected individuals. Phenotypic and genotypic studies implicate the distinctiveness of this parasite which may describe its pathogenesis. However, the antigenic distinctiveness which describes the antibody mediated cell lysis of this parasite has not been explored. This study was aimed to identify the cross-reactivity and cytotoxicity effect between three isolates of symptomatic and asymptomatic Blastocystis sp. ST3 respectively. Antigen specificity and diversity of this parasite was performed by coculturing sera (10-fold dilution) obtained from mice immunised with Blastocystis sp. symptomatic and asymptomatic antigens and the respective Blastocystis sp. ST3 live cells through complement dependant cell cytotoxicity (CDC) assay. The results obtained has shown that, the sera (at 10-fold diluted concentration) from symptomatic and asymptomatic solubilised antigen immunised mice were able to specifically lyse the respective live parasites with an average percentage of 82% and 86% respectively. There were almost 50% crossreactivity observed between the three isolates of Blastocystis sp. ST3 from symptomatic and asymptomatic group proving high antigen diversity or rather low antigen specificity within the same group. However, there was only 17% cross-reactivity observed between the mice sera and parasitic cells of different groups (symptomatic vs asymptomatic isolates) suggesting high specificity between these two groups. We, for the first time have proven that through CDC analysis there were epitopes dissimilarities between Blastocystis sp. ST3 symptomatic and asymptomatic isolates which may allow the parasite to set up diverse immune modulations such as imbalanced Th1/Th2 responses in an infected host.

Blastocystis sp., Parasite Associated with Gastrointestinal Disorders: An Overview of its Pathogenesis, Immune Modulation and Therapeutic Strategies.

Blastocystis sp. is a unicellular parasitic microorganism commonly found in the gastrointestinal tracts of humans and animals. It causes symptomatic or asymptomatic infection and its route of transmission is via fecal-oral. High prevalence of Blastocystis infection in developing countries is usually due to poor hygiene practices, exposure to animals infected with the parasite and intake of contaminated water or food. Blastocystis infected individuals often suffer from diarrhea, abdominal pain, nausea, and stomach bloating. Even though pathogenicity of Blastocystis is unclear, it is commonly associated with irritable bowel syndrome. In this review, we have analysed the evidence that shows the association between this microorganism and gastrointestinal disorders. There have been a number of studies which showed that the pathogenicity of Blastocystis is related to its different STs. The pathogenicity is speculated to be due to cysteine proteases formation which stimulates mucosal cells to release interleukin-8 which has been associated with extreme dehydration and gut inflammation. In vitro studies on human colonic epithelial cells revealed that incubation of Blastocystis modulated the host immune response by stimulating the formation of pro-inflammatory cytokines and granulocyte macrophage colonystimulating factor. Metronidazole is found to be the first-line drug of choice. Another treatment option is the combination therapy with trimethoprim/sulfamethoxazole.

Pathogenic mechanisms in Blastocystis spp. - Interpreting results from in vitro and in vivo studies.

Blastocystis spp. are commonly reported intestinal protists but whose clinical significance remains controversial. Infections have ranged from asymptomatic carriage to non-specific gastrointestinal symptoms and have also been linked to irritable bowel syndrome and urticaria in some patient populations. In vitro studies showed that both parasite and parasite lysates have damaging effects on intestinal epithelial cells causing apoptosis and degradation of tight junction proteins occludin and ZO1, resulting in increased intestinal permeability. Adhesion of trophic forms to the intestinal epithelium and release of cysteine proteases appear to be the major triggers leading to pathogenesis. Two putative virulence factors identified are cysteine proteases legumain and cathepsin B. Blastocystis spp. also have immuno-modulatory effects including degradation of IgA, inhibition of iNOS and upregulation of proinflammatory cytokines, IL8 and GM-CSF in intestinal epithelial cells and IL1ß, IL6 and TNFα in murine macrophages. Blastocystis spp. have also been reported to dampen response to LPS in intestinal epithelial cells and monocytes. Studies in rodent models and naturally infected pigs have shown that the parasite localizes to the lumen and mucosal surface of the large intestine mostly in the caecum and colon. The parasite has been found to cause mucosal sloughing, increase in goblet cell mucin, increased intestinal permeability and to induce a pro-inflammatory cytokine response with upregulation of TNFα, IFNγ and IL12. In this review, we summarize findings from in vitro and in vivo studies that demonstrate pathogenic potential but also show considerable inter and intra subtype variation, which provides a plausible explanation on the conflicting reports on clinical significance.

Immunopathological assessments of human Blastocystis spp. in experimentally infected immunocompetent and immunosuppresed mice.

Blastocystis spp., one of the most common parasites colonizing the human intestine, is an extracellular, luminal protozoan with controversial pathogenesis. The host's immune response against Blastocystis spp. infection has also not been defined yet. Therefore, this research aimed to assess the potential pathogenicity of this parasite and its ability to modulate the immune response in experimental infected immunocompetent and immunosuppresed mice. These results demonstrated that the infected immunosuppressed mice were more affected than infected immunocompetent mice. Histopathological examination of the small intestine in the infected immunosuppressed mice showed that Blastocystis spp. infiltrated all the layers. Moreover, the epithelia showed exfoliation and inflammatory cell infiltration in submucosa compared to that of the infected immunocompetent mice. As well, examination of the large intestine of the infected immunosuppressed group showed severe goblet cell hyperplasia. Blastocystis spp. infiltrated all the large intestine layers compared to that of the infected immunocompetent group. Furthermore, there was a significant upregulation of the expression of proinflammatory cytokines: interleukin 12 (IL-12) and tumor necrosis factor alpha (TNF-α) in the infected immunosuppressed mice compared to that of the infected immunocompetent ones (p ≤ 0.004 and p ≤ 0.002, respectively). However, the expression of anti-inflammatory cytokines (IL-4 and IL-10) was significantly downregulated in the infected immunosuppressed group compared to that of the infected immunocompetent group one at 10 days postinfection (p ≤ 0.002 and p ≤ 0.001, respectively). The results of this study revealed that Blastocystis spp. affected the production of pro- and anti-inflammatory cytokines in both groups of mice compared to healthy normal (naive) group. Additionally, these data showed that there was a significant upregulation (p ≤ 0.005) of the locally synthesized antibody: secretary IgA (sIgA) in the gut of the infected immunocompetent mice when compared to that of the infected immunosuppressed ones.

Eradication of Blastocystis in humans: Really necessary for all?

Blastocystis (initially named as Blastocystis hominis) has long been known as a protist without any clinical significance. However, there is now a huge pile of case reports where Blastocystis is blamed for the symptoms and the infection described in the patients. Introduction of the presence of as many as 17 Blastocystis subtypes while many infected individuals are non-symptomatic initially brought about the correlation between the subtypes and pathogenicity; however, the outcomes of these trials were not consistent and did not explain its pathogenicity. Today, it is mostly acknowledged that Blastocystis may colonize many individuals but the infection's onset depends on the interaction between the virulence of parasites and host's immune competence. Eradication of Blastocystis is essential in some cases where it is the only infectious agent and patient is suffering from some symptoms. In such cases, metronidazole is the drug of choice but its efficacy is relatively low in some cases. Other agents used include trimethoprim-sulfamethoxazole, paromomycin, and furazolidone. Recent studies on the interactions between human health and the role of gut microbiota introduces new data which may significantly change our point of view against some protists, which we tend to see as "parasites requiring urgent eradication for cure". May the presence or absence of some Blastocystis subtypes necessary for human health, or is the absence or presence of certain Blastocystis subtypes in human gut is associated with certain diseases/infections? The answers of these questions will surely guide us to select patients requiring treatment against Blastocystis infection in future.

Blastocystis specific serum immunoglobulin in patients with irritable bowel syndrome (IBS) versus healthy controls.

BACKGROUND: Blastocystis species are common enteric human parasites and carriage has been linked to Irritable Bowel Syndrome (IBS), particularly diarrhoea-predominant IBS. The spectrum of immune reactivity to Blastocystis proteins has been reported previously in symptomatic patients. We investigated differences in serum immunoglobulin profiles between patients with IBS, both positive and negative for Blastocystis carriage, and healthy controls (HC). METHODS: Forty diarrhoea-predominant IBS patients (26 patients positive for Blastocystis sp., 14 negative patients) and forty HC (24 positive, 16 Blastocystis-negative) were enrolled. Age, gender, ethnicity and serum immunoglobulin A (IgA) levels were recorded and faecal specimens were analysed using smear, culture and polymerase chain reaction amplification of ribosomal DNA. Sera were tested in Western blots and the reactivities compared to known targets using monoclonal antibodies Blastofluor® (Blastocystis specific antibody), MAb1D5 (cytopathicto Blastocystis cells), anti-promatrix metalloprotease-9 (anti-MMP-9) and SDS-PAGE zymograms. RESULTS: Levels of serum IgA were significantly lower in Blastocystis carriers (p < 0.001) but had no relationship to symptoms. Western blots demonstrated serum IgG antibodies specific for Blastocystis proteins of 17,27,37,50,60-65, 75-90, 95-105 and 150 kDa MW. Reactivity to the 27, 50 and 75-95 kDa proteins were found more frequently in the IBS group compared to the HC's (p < 0.001) and correlation was greater for Blastocystis-positive IBS patients (p < 0.001) than for negative IBS patients (p < 0.05). MAb1D5 reacted with proteins of 27 and 100 kDa, and anti-MMP-9 with 27, 50 and 75-100 kDa proteins. Bands were seen in zymograms around 100 kDa. CONCLUSIONS: Low serum IgA levels are associated with Blastocystis carriage. All IBS patients were more likely to demonstrate reactivity with Blastocystis proteins of 27 kDa (likely a cysteine protease), 50 and 75-95 kDa MW compared to HC. The presence of antibodies to these Blastocystis proteins in some Blastocystis-negative subjects suggests either prior exposure to Blastocystis organisms or antibody cross reactivities. The anti-proMMP-9 reaction at 50 and 75-100 kDa and the zymogram result suggest that metalloproteases may be important Blastocystis antigens. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials registry ACTRN: 12611000918921.

Subtype distribution of Blastocystis in Thai-Myanmar border, Thailand.

Blastocystis sp. is a common zoonotic intestinal protozoa which has been classified into 17 subtypes (STs). A cross-sectional study was conducted to determine the prevalence and subtype distribution of Blastocystis in villagers living on the Thai-Myanmar border, where the risk of parasitic infection is high. A total of 207 stool samples were collected and DNA was extracted. PCR and sequencing using primers targeting small-subunit ribosomal RNA (SSU rRNA) gene were performed. The prevalence of Blastocystis infection was 37.2% (77/207). ST3 (19.8%; 41/207) was the predominant subtype, followed by ST1 (11.6%; 24/207), ST2 (5.3%; 11/207), and ST4 (0.5%; 1/207). A phylogenetic tree was reconstructed using the maximum likelihood (ML) method based on the Hasegawa-Kishino-Yano + G + I model. The percentage of bootstrapped trees in which the associated taxa clustered together was relatively high. Some sequences of Blastocystis positive samples (TK18, 39, 46, 71, and 90) were closely related to animals (pig and cattle) indicating zoonotic risks. Therefore, proper health education in parasitic prevention for the villagers should be promoted to improve their personal hygiene. Further longitudinal studies are required to monitor the prevalence of parasitic infections after providing health education and to investigate Blastocystis ST in animals living in these villages.

A novel ELISA test for laboratory diagnosis of Blastocystis spp. in human stool specimens.

Detection of Blastocystis is routinely performed by microscopy, culture, and formyl-ether (ethyl acetate) concentration technique (FECT). Yet, these methods require special skilled personnel, are time consuming, and often involve processing that may cause misdiagnosis. The aim of this work is to demonstrate the usefulness of a newly introduced ELISA test for the detection of Blastocystis antigens in stool samples (CoproELISA(TM) Blastocystis, Savyon Diagnostics) as a proper alternative to currently used methods, especially microscopy. A cohort of 179 fresh/frozen clinical stool samples was tested by the ELISA test, and results were compared to consensus methods comprised of microscopic examination of Lugol's iodine staining, culture, and immunofluorescence assay (IFA). The new ELISA test was able to detect fewer than 10(3) cells, recognized subtypes 1, 2, 3, and 5 (comprising >95 % of human Blastocystis infections), and exhibited similar reactivity when comparing formalin-preserved samples to fresh/frozen samples. The test demonstrated 92 % sensitivity, 87 % specificity, and 89 % accuracy when culture, and IFA or microscopy consensus results were taken as reference. When the consensus was comprised of culture and IFA, the test demonstrated sensitivity, specificity, and accuracy of 82, 86, and 84 %, respectively. In contrast, the sensitivity of Lugol staining microscopy was only 18 %. This work presents a unique ELISA test that provides an alternative to the use of microscopy, currently most widely used method. The test enables high-throughput screening and diagnosis of Blastocystis, adaptation to automatic procedures.

[Extraenteric infection caused by Blastocystis spp. in a female patient with liver abscess].

The cases associated with the development of liver abscesses in a 64-year-old female patient after elective surgery for colon polyposis could form an opinion that extraenteric infection caused by Blastocystis spp. might develop in the immunocompromised host. The development of Blastocystis spp. in the presence of disintegrated liver tissue and inflammatory cells was verified by microscopic examination of liver abscess aspirates. The Romanovsky-Giemsa stained specimens exhibited typical amoeboid, vacuolar and, what is particularly important, dividing forms of Blastocystis spp. The patients full recovery after timely combination therapy with broad-spectrum antibiotics and imidazole group preparations also indirectly argues for the etiological role of Blastocystis spp. in the development of liver abscess with the signs of changes in both lungs (the signs of right lung compression and bilateral hydrothorax). Physicians' awareness of the potential clinical significance of Blastocystis spp. in immunodeficient patients is sure to expand the range of differential diagnostic studies of patients infected with Blastocystis spp.. particularly in case of gastrointestinal tract diseases of unknown etiology.

Differential regulation of proinflammatory cytokine expression by mitogen-activated protein kinases in macrophages in response to intestinal parasite infection.

Blastocystis is a common enteric protistan parasite that can cause acute, as well as chronic, infection and is associated with irritable bowel syndrome (IBS). However, the pathogenic status of Blastocystis infection remains unclear. In this study, we found that Blastocystis antigens induced abundant expression of proinflammatory cytokines, including interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α), in mouse intestinal explants, in mouse colitis colon, and in macrophages. Further investigation utilizing RAW264.7 murine macrophages showed that Blastocystis treatment in RAW264.7 macrophages induced the activation of ERK, JNK, and p38, the three major groups of mammalian mitogen-activated protein (MAP) kinases that play essential roles in the expression of proinflammatory cytokines. ERK inhibition in macrophages significantly suppressed both mRNA and protein expression of IL-6 and TNF-α and mRNA expression of IL-1ß. On the other hand, JNK inhibition resulted in reductions in both c-Jun and ERK activation and significant suppression of all three proinflammatory cytokines at both the mRNA and protein levels. Inhibition of p38 suppressed only IL-6 protein expression with no effect on the expression of IL-1ß and TNF-α. Furthermore, we found that serine proteases produced by Blastocystis play an important role in the induction of ERK activation and proinflammatory cytokine expression by macrophages. Our study thus demonstrated for the first time that Blastocystis could induce the expression of various proinflammatory cytokines via the activation of MAP kinases and that infection with Blastocystis may contribute to the pathogenesis of inflammatory intestinal diseases through the activation of inflammatory pathways in host immune cells, such as macrophages.

Statin pleiotropy prevents rho kinase-mediated intestinal epithelial barrier compromise induced by Blastocystis cysteine proteases.

Blastocystis is an enteric parasite that causes acute and chronic intestinal infections, often non-responsive to conventional antibiotics. The effects of Blastocystis infections on human epithelial permeability are not known, and molecular mechanisms of Blastocystis-induced intestinal pathology remain unclear. This study was conducted to determine whether Blastocystis species alters human intestinal epithelial permeability, to assess whether these abnormalities are rho kinase (ROCK)-dependent, and to investigate the therapeutic potential of the HMG-CoA reductase inhibitor Simvastatin in altered intestinal epithelial barrier function. The effect of metronidazole resistant (Mz(r)) Blastocystis isolated from a symptomatic patient on human colonic epithelial monolayers (Caco-2) was assessed. Modulation of enterocyte myosin light chain phosphorylation, transepithelial fluorescein isothiocyanate-dextran fluxes, transepithelial resistance, cytoskeletal F-actin and tight junctional zonula occludens-1 (ZO-1) by parasite cysteine proteases were measured in the presence or absence of HMG-CoA reductase and ROCK inhibition. Blastocystis significantly decreased transepithelial resistance, increased epithelial permeability, phosphorylated myosin light chain and reorganized epithelial actin cytoskeleton and ZO-1. These alterations were abolished by inhibition of enterocyte ROCK, HMG-CoA reductase and parasite cysteine protease. Our findings suggest that cysteine proteases of Mz(r) Blastocystis induce ROCK-dependent disruption of intestinal epithelial barrier function and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1. Simvastatin prevented parasite-induced barrier-compromise, suggesting a therapeutic potential of statins in intestinal infections.

Findings related to IL-8 and IL-10 gene polymorphisms in a Mexican patient population with irritable bowel syndrome infected with Blastocystis.

The intestinal protozoan parasite Blastocystis is one of the most common parasites worldwide in humans and, although its ability to cause human disease has been questioned, some reports have demonstrated that this microorganism is associated to the development of irritable bowel syndrome (IBS) and to a proinflammatory response, in which the expression of some cytokines is unregulated. Since inflammatory cytokine gene polymorphisms might have a role in the pathophysiology of IBS, we assessed the role of single nucleotide polymorphisms (SNPs) for interleukin (IL)-8 and IL-10, in previously collected DNA samples from IBS patients and controls, with or without Blastocystis infection. IL-8+396(G) and IL-10-1082 (A) alleles (p=0.0437 and p=0.0267, respectively), as well as their homozygous (p<0.0001 and p=0.0039, respectively) and IL-8+781(CT) (p=0.0248) genotypes were significantly overrepresented in patients with IBS in comparison with controls. IL-8+396(GG) genotype was relevant because it was associated to IBS (p<0.0001), to Blastocystis (p=0.0025), and to IBS­Blastocystis (p=0.0272). In the latter binomial association, this genotype presented a high contribution (etiological fraction =0.452) and a risk >fourfold to develop IBS. IL-8+781 (T) and IL-10-592 (C) alleles were also associated to Blastocystis and to IBS­Blastocystis, respectively (p=0.0448 and p=0.0166). Our results suggest that some IL-8 and IL-10 SNPs could change individual susceptibility increasing the relative risk in the development of IBS in Blastocystis carriers.

Expression of interferon gamma and proinflammatory cytokines in the cecal mucosa of rats experimentally infected with Blastocystis sp. strain RN94-9.

Blastocystis hominis is a zoonotic intestinal protozoan parasite whose pathogenic potential is still controversial. The aim of the present study was to clarify the pathogenicity of Blastocystis parasites in rats. Oral inoculation with 1 x 10(5) cysts of Blastocystis sp. strain RN94-9 in rats resulted in chronic infection in the cecum at least until 4 weeks after infection. Histological examination revealed neither mucosal sloughing nor inflammatory cell infiltration but showed a slight but significant increase in goblet cell numbers in the cecal mucosa 1-3 weeks post-infection. Differential staining of acidic and neutral mucins by the alcian blue-periodic acid-Schiff method showed that the predominantly increased cells were neutral mucin(+) but not acidic mucin(+) goblet cells. Reverse transcription real-time polymerase chain reaction studies demonstrated significant upregulation of the expression of interferon-gamma, interleukin (IL)-12, and tumor necrosis factor alpha, but not IL-6 or granulocyte-macrophage colony-stimulating factor, in the cecal mucosa at 2 and/or 3 weeks post-infection. The induction of local host responses, including mild goblet cell hyperplasia, and significant upregulation of type-1 and proinflammatory cytokines, suggest that Blastocystis sp. strain RN94-9 is a weakly pathogenic organism that could elicit proinflammatory as well as protective responses in local tissues.

Blastocystis ratti contains cysteine proteases that mediate interleukin-8 response from human intestinal epithelial cells in an NF-kappaB-dependent manner.

Blastocystis is a ubiquitous enteric protozoan found in the intestinal tracts of humans and a wide range of animals. Evidence accumulated over the last decade suggests association of Blastocystis with gastrointestinal disorders involving diarrhea, abdominal pain, constipation, nausea, and fatigue. Clinical and experimental studies have associated Blastocystis with intestinal inflammation, and it has been shown that Blastocystis has potential to modulate the host immune response. Blastocystis is also reported to be an opportunistic pathogen in immunosuppressed patients, especially those suffering from AIDS. However, nothing is known about the parasitic virulence factors and early events following host-parasite interactions. In the present study, we investigated the molecular mechanism by which Blastocystis activates interleukin-8 (IL-8) gene expression in human colonic epithelial T84 cells. We demonstrate for the first time that cysteine proteases of Blastocystis ratti WR1, a zoonotic isolate, can activate IL-8 gene expression in human colonic epithelial cells. Furthermore, we show that NF-kappaB activation is involved in the production of IL-8. In addition, our findings show that treatment with the antiprotozoal drug metronidazole can avert IL-8 production induced by B. ratti WR1. We also show for the first time that the central vacuole of Blastocystis may function as a reservoir for cysteine proteases. Our findings will contribute to an understanding of the pathobiology of a poorly studied parasite whose public health importance is increasingly recognized.

Exposure of Blastocystis species to a cytotoxic monoclonal antibody.

A recently described cytotoxic monoclonal antibody (mAb 1D5) raised against Blastocystis hominis isolate B, was tested for reactivity with 13 different isolates of Blastocystis. The isolates used were previously isolated from humans, rats and reptiles and were maintained as axenised cultures throughout the course of this study. Five B. hominis isolates (B, C, E, G and H) were found to react with mAb 1D5 in immunoblotting studies and the indirect fluorescence antibody test. The pattern of fluorescence observed for all five isolates was diffuse and patchy. Immunoblotting studies revealed that mAb 1D5 reacted with a 29-30-kDa protein found in all five isolates. Results of a cytotoxic assay showed that the mAb exhibited a complement-independent cytotoxic effect on all the exposed isolates. Microscopic observations showed differences in morphology between the Blastocystis cells exposed and unexposed to mAb. Acridine orange staining performed on both exposed and unexposed cells showed similar internal structures when viewed under fluorescence microscopy.

Characterization of protein profiles and cross-reactivity of Blastocystis antigens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis.

The protein profiles of Blastocystis hominis, B. lapemi, and B. ratti were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and species could be differentiated by this means as well as by Western-blot analysis with polyclonal antibodies. No intraspecies difference could be distinguished between the two B. hominis isolates or the three B. ratti isolates. Western-blot analysis showed extensive cross-reactivity of B. lapemi and B. hominis antigens with anti-B. ratti serum. Some of the cross-reactive antigens were glycoproteins as determined on the basis of their sensitivity to periodate treatment.