Genomic DNA from 16 Blastocystis hominis isolates comprising of eight asymptomatic isolates (A1-A8) and eight symptomatic isolates (S1-S8) was amplified by arbitrarily primed polymerase chain reaction (AP-PCR) using 38 arbitrary 10-mer primers. Six primers (A10, B5, C20, D1, F6, and F10) generated reproducible DNA fingerprints. AP-PCR amplification revealed similar DNA fingerprints among all symptomatic isolates (S1-S8) with common bands at 850 bp using primer A10, 920 bp using primer B5, and 1.3 kbp using primer D1. Isolates A1, A3, A4, A5, A6, and A7 showed similar DNA banding patterns and all asymptomatic isolates (A1-A8) shared a major band at 1 kbp using primer B5. Isolates A2 and A8 showed distinct DNA banding patterns that differed from the remainder of the isolates. The results of the phylogenetic analyses showed that all symptomatic isolates (S1-S8) formed a clade with >70% similarity among the isolates and which were clearly separate from asymptomatic isolates A1, A3, A4, A5, A6, and A7. Asymptomatic isolates A2 and A8 formed two distinct and separate clades. AP-PCR revealed higher genetic variability within the asymptomatic isolates than within the symptomatic isolates. The present study suggests that AP-PCR can be a valuable method for differentiating between isolates of B. hominis and our results support the hypothesis that our asymptomatic and symptomatic B. hominis isolates may represent two different strains/species with varying pathogenic potential.
Non-isotopic polymerase chain reaction (PCR)-based single-strand conformation polymorphism and sequence analyses of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) were utilized to genetically characterise ascaridoids from dogs and cats from China by comparison with those from other countries. The study showed that Toxocara canis, Toxocara cati, and Toxascaris leonina from China were genetically the same as those from other geographical origins. Specimens from cats from Guangzhou, China, which were morphologically consistent with Toxocara malaysiensis, were the same genetically as those from Malaysia, with the exception of a polymorphism in the ITS-2 but no unequivocal sequence difference. This is the first report of T. malaysiensis in cats outside of Malaysia (from where it was originally described), supporting the proposal that this species has a broader geographical distribution. The molecular approach employed provides a powerful tool for elucidating the biology, epidemiology, and zoonotic significance of T. malaysiensis.
The amoeboid form of Blastocystis hominis has been reported infrequently, and its morphological descriptions have yielded conflicting and confusing reports. In the present study, we used the amoeboid forms seen predominantly in symptomatic patients infected with Blastocystis to provide detailed descriptions on the fine surface structure and intracellular morphology. Scanning electron microscopy revealed the irregular shape of the amoeboid form, with an intercalated fibrillar structure and a highly convoluted surface with deep indentations and projected pseudopodia. Transmission electron microscopy showed the existence of two types of amoeboid forms of B. hominis in in vitro culture, one with a large central vacuole containing tiny electron-dense particles while the other contains multiple small vacuoles in the cytoplasm. A surface coat with varying thickness surrounded the amoeboid form, which also showed prominent, extended pseudopodia of varying shape. Irregularly shaped mitochondrion-like organelles with prominent cristae, lipid inclusions, and multiple vacuoles were frequently seen in close proximity with the pseudopodia. The characteristic nucleus with a crescentic band of electron-dense chromatin material was also seen.
Blastocystis hominis has been regarded as an enigmatic parasite as many aspects of its basic biology remain uncertain. Many reproductive processes have been suggested for the organism; however, to date, only the binary fission has been proven. Plasmotomy is one of the modes of reproduction previously suggested to be seen in in vitro cultures. The present study provides trichrome and acridine orange staining evidence for the existence of nucleic acid suggestive of division of nucleus into multinucleate forms with the respective cytoplasm dividing giving rise to two or three progeny B. hominis. Transmission electron micrographs further confirmed that these daughter cells had respective surrounding surface coat, mitochondria, and vacuoles.
This article is a review of the latest information on the prevalence of G. lamblia in South Asia, South East Asia and Far East, characterizing the current endemic situation within these regions. Around 33 published papers from 2002-2007 were collected on G. lamblia. The included countries were Nepal, Bangladesh, India, Cambodia, Vietnam, Malaysia, Philippines, Indonesia, Thailand, Republic of Korea, and China. Only five published papers were discarded because data was extracted before 2002-2007 or they are not included within our regions, emphasizing more on G. lamblia in animals, or performed at extensive molecular level. The prevalence of G. lamblia varied markedly between studies illustrating higher levels in the urban than in the rural areas, more among poor communities, slightly higher in males than in females with age range of 2-5-year-old children, and among university students, old-aged people, HIV-positive patients, and gastric carcinoma patients. Though G. lamblia is not a life-threatening parasite, nevertheless, it is still considered as the most common water-borne diarrhea-causing disease. It is important to understand the etiology, frequency, and consequences of acute diarrhea in children. Routine surveillance such as bi-annual follow-up treatments, treating G. duodenalis cysts and other protozoa oocysts detected in ground water sources, and continuous health education are the most preventive measures.
Despite frequent reports on the presence of Blastocystis hominis in human intestinal tract, its pathogenicity remains a matter of intense debate. These discrepancies may be due to the varying pathogenic potential or virulence of the isolates studied. The present study represents the first to investigate both phenotypic and genotypic characteristics of B. hominis obtained from symptomatic and asymptomatic individuals. Symptomatic isolates had a significantly greater size range and lower growth rate in Jones' medium than asymptomatic isolates. The parasite cells of symptomatic isolates exhibited rougher surface topography and greater binding affinity to Canavalia ensiformis (ConA) and Helix pomatia (HPA). The present study also identifies further phenotypic characteristics, which aided in differentiating the pathogenic forms from the non-pathogenic forms of B. hominis. Blastocystis subtype 3 was found to be correlated well with the disease.
Blastocystis from infected stools of a person who showed chronic symptoms of abdominal discomfort and diarrhea were examined over a 6-month period, using transmission electron microscopy, for the ultrastructural changes from vacuolar to cystic stage. The study confirms the irregular shedding phenomenon of the organism previously reported, and for the first time, records sequential changes in encystation in stools collected over a time period. The study also confirms the existence of a precystic stage which has an immature cell wall consisting of a layer of a homogenous electron-dense mass surrounding the cell which acts as a intermediatory stage between the vacuolar and cystic stage.
This represents the first study to determine the genetic diversity of Blastocystis sp. among cancer and HIV/AIDS patients. Forty Blastocystis sp. isolates obtained from 20 cancer and 20 HIV/AIDS patients were genotyped by PCR using seven pairs of known sequenced-tagged site primers. Out of the 40 isolates, 38 were identified as one of the known genotypes and two isolates were negative with all the STS primers. Blastocystis sp. subtype 3 which is reported to be associated with disease was found to be predominant among the study subjects.
Nine 50-l surface water samples from a Malaysian recreational lake were examined microscopically using an immunomagnetisable separation-immunofluorescent method. No Cryptosporidium oocysts were detected, but 77.8% of samples contained low numbers of Giardia cysts (range, 0.17-1.1 cysts/l), which were genetically characterised by SSU rRNA gene sequencing. Genotype analyses indicated the presence of Giardia duodenalis assemblage A suggesting potential risk to public health. The present study represents the first contribution to our knowledge of G. duodenalis assemblages in Malaysian recreational water.
Blastocystis hominis is one of the most common intestinal protozoan parasites in humans, and reports have shown that blastocystosis is coupled with intestinal disorders. In the past, researchers have developed an in vitro model using B. hominis culture filtrates to investigate its ability in triggering inflammatory cytokine responses and transcription factors in human colonic epithelial cells. Studies have also correlated the inflammation by parasitic infection with cancer. The present study provides evidence of the parasite facilitating cancer cell growth through observing the cytopathic effect, cellular immunomodulation, and apoptotic responses of B. hominis, especially in malignancy. Here we investigated the effect of solubilized antigen from B. hominis on cell viability, using peripheral blood mononuclear cells (PBMCs) and human colorectal carcinoma cells (HCT116). The gene expressions of cytokines namely interleukin 6 (IL-6), IL-8, tumor necrosis factor alpha, interferon gamma, nuclear factor kappa light-chain enhancer of activated B cells (a gene transcription factor), and proapoptotic genes namely protein 53 and cathepsin B were also studied. Results exhibited favor the fact that antigen from B. hominis, at a certain concentration, could facilitate the growth of HCT116 while having the ability to downregulate immune cell responses (PBMCs). Therefore, there is a vital need to screen colorectal cancer patients for B. hominis infection as it possesses the ability to enhance the tumor growth.
The fact whether Blastocystis hominis can invade has always been in question. Apart from a few sporadic studies such as that done on gnotobiotic guinea pigs which showed surface invasion and mucosal inflammation of the host's intestine caused by B. hominis infection, no real documentation of invasion has been proven. Studies have shown that hyaluronidase is secreted during the penetration into the host's skin and gut by nematode parasites. Hyaluronidase activity in protozoa namely Entamoeba histolytica has also been described previously. This study attempts to determine hyaluronidase in urine samples of B. hominis-infected rats. The presence of hyaluronidase in urine provides an indirect evidence of invasion by B. hominis into colonic epithelium causing the degradation of extracellular matrix proteins namely hyaluronic acid (HA). HA is depolymerized by hyaluronidase which may be used by organisms to invade one another. In this study, the levels of urinary hyaluronidase of Sprague-Dawley rats infected with B. hominis were monitored for 30 days. Hyaluronidase levels in the infected rats were significantly higher on days 28 and 30 compared to the day before inoculation (P < 0.01 and P < 0.05, respectively). During this stage, parasitic burden in infected stools was also at a high level. Proinflammatory cytokines, interleukin-6 and interleukin-8, were also significantly higher (P < 0.05) in the serum of infected rats. The study demonstrates that since no other pathogen was present and that amoeboid forms of the parasites have been shown to exist previously, the elevated levels of hyaluronidase in this preliminary finding suggests that the organism is capable of having invasion or penetration activity in the hosts' intestine.
Entamoeba histolytica is the etiologic agent for amoebiasis. The excretory-secretory (ES) products of the trophozoites contain virulence factors and antigens useful for diagnostic applications. Contaminants from serum supplements and dead trophozoites impede analysis of ES. Therefore, a protein-free medium that can sustain maximum viability of E. histolytica trophozoites for the longest time duration will enable collection of contaminant-free and higher yield of ES products. In the present study, we compared the efficacy of four types of media in maintaining ≥ 95% trophozoite viability namely Roswell Memorial Park Institute (RPMI-1640), Dulbecco's Modified Eagle Medium (DMEM), phosphate-buffered saline for amoeba (PBS-A), and Hank's balanced salt solution (HBSS). Concurrently, the effect of adding L: -cysteine and ascorbic acid (C&A) to each medium on the parasite viability was also compared. DMEM and RPMI 1640 showed higher viabilities as compared to PBS-A and HBSS. Only RPMI 1640 showed no statistical difference with the control medium for the first 4 h, however the ≥ 95% viability was only maintained for the first 2 h. The other protein-free media showed differences from the serum- and vitamin-free TYI-S-33 control media even after 1 h of incubation. When supplemented with C&A, all media were found to sustain higher trophozoite viabilities than those without the supplements. HBSS-C&A, DMEM-C&A, and RPMI 1640-C&A demonstrated no difference (P>0.05) in parasite viabilities when compared with the control medium throughout the 8-h incubation period. DMEM-C&A showed an eightfold increment in time duration of sustaining ≥ 95% parasite viability, i.e. 8 h, as compared to DMEM alone. Both RPMI 1640-C&A and HBSS-C&A revealed fourfold and threefold increments (i.e., 8 and 6 h, respectively), whereas PBS-A-C&A showed only one fold improvement (i.e., 2 h) as compared to the respective media without C&A. Thus, C&A-supplemented DMEM or RPMI are recommended for collection of ES products.
Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) may play an important role in host-cell invasion by the Eimeria species, protozoan parasites which can cause severe intestinal disease in livestock. Here, we report the structural organization of the PIP5K gene in Eimeria maxima (Weybridge strain). Two E. maxima BAC clones carrying the E. maxima PIP5K (EmPIP5K) coding sequences were selected for shotgun sequencing, yielding a 9.1-kb genomic segment. The EmPIP5K coding region was initially identified using in silico gene-prediction approaches and subsequently confirmed by mapping rapid amplification of cDNA ends and RT-PCR-generated cDNA sequence to its genomic segment. The putative EmPIP5K gene was located at position 710-8036 nt on the complimentary strand and comprised of 23 exons. Alignment of the 1147 amino acid sequence with previously annotated PIP5K proteins from other Apicomplexa species detected three conserved motifs encompassing the kinase core domain, which has been shown by previous protein deletion studies to be necessary for PIP5K protein function. Phylogenetic analysis provided further evidence that the putative EmPIP5K protein is orthologous to that of other Apicomplexa. Subsequent comparative gene structure characterization revealed events of intron loss/gain throughout the evolution of the apicomplexan PIP5K gene. Further scrutiny of the genomic structure revealed a possible trend towards "intron gain" between two of the motif regions. Our findings offer preliminary insights into the structural variations that have occurred during the evolution of the PIP5K locus and may aid in understanding the functional role of this gene in the cellular biology of apicomplexan parasites.
Vaccine development against the blood-stage malaria parasite is aimed at reducing the pathology of the disease. We constructed a recombinant Mycobacterium bovis bacille Calmette Guerin (rBCG) expressing the 19 kDa C-terminus of Plasmodium falciparum merozoite surface protein-1 (MSP-1(19)) to evaluate its protective ability against merozoite invasion of red blood cells in vitro. A mutated version of MSP-1(19), previously shown to induce the production of inhibitory but not blocking antibodies, was cloned into a suitable shuttle plasmid and transformed into BCG Japan (designated rBCG016). A native version of the molecule was also cloned into BCG (rBCG026). Recombinant BCG expressing the mutated version of MSP-1(19) (rBCG016) elicited enhanced specific immune response against the epitope in BALB/c mice as compared to rBCG expressing the native version of the epitope (rBCG026). Sera from rBCG016-immunized mice contained significant levels of specific IgG, especially of the IgG2a subclass, against MSP-1(19) as determined by enzyme-linked immunosorbent assay. The sera was reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA) and inhibited merozoite invasion of erythrocytes in vitro. Furthermore, lymphocytes from rBCG016-immunized mice demonstrated higher proliferative response against the MSP-1(19) antigen as compared to those of rBCG026- and BCG-immunized animals. rBCG expressing the mutated version of MSP-1(19) of P. falciparum induced enhanced humoral and cellular responses against the parasites paving the way for the rational use of rBCG as a blood-stage malaria vaccine candidate.
In this study, we successfully expressed a chimerical surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii in Pichia pastoris. Eighty human serum samples, including 60 from confirmed cases of toxoplasmosis, were tested against the purified recombinant SAG1/2 in Western blots. Results of Western blots targeted at Toxoplasma IgG and IgM showed that the recombinant SAG1/2 reacted with all sera from the toxoplasmosis cases but none with the Toxoplasma-negative serum samples. These results showed that the P. pastoris-derived recombinant SAG1/2 was sensitive and specific and suitable for use as antigen for detecting anti-Toxoplasma antibodies. To further investigate the immunological characteristic of the recombinant protein, the recombinant SAG1/2 was injected subcutaneously into BALB/c mice, and their serum was tested against total protein lysate of T. gondii. Mice immunized with the recombinant SAG1/2 reacted specifically with the native SAG1 and SAG2 of T. gondii. Significant proliferation of splenocytes stimulated with tachyzoite total protein lysate was observed in vaccinated BALB/c mice but not in those from negative control mice. Specific production of IFN-γ, the Th1-type cytokines, was also found in stimulated splenocytes from vaccinated mice. These results show that the chimeric protein recombinant SAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice. Finally, vaccinated mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P
Blastocystis sp. is a common intestinal parasite. To date, there have been sporadic and scanty studies on Blastocystis sp. carried out in rural communities in Nepal. We surveyed the prevalence of Blastocystis sp. and its possible associated risk factors, and reported the predominant Blastocystis sp. subtype in two rural communities, Bolde Phediche and Bahunipati, in Nepal. Human faecal samples were collected from 241 participants, cultured using in vitro cultivation and examined for Blastocystis sp. The presence of Blastocystis sp. in faecal samples was further confirmed by polymerase chain reaction (PCR) and subsequently genotyped using subtype-specific sequence tagged site (STS) primers. There were 26.1% (63/241) of the participants that were infected by Blastocystis sp. We detected 84.1% (53/63) of Blastocystis sp. subtype 4 infections in these rural communities. The unusually high prevalence of Blastocystis sp. subtype 4 can be attributed to the rearing of family-owned animals in barns built close to their houses. Eighty one percent (51/63) of the Blastocystis sp. infected participants drank not boiled or unfiltered water. The present study revealed that Blastocystis sp. could pose a health concern to the communities and travellers to the hilly area in Nepal. Infection may be transmitted through human-to-human, zoonotic and waterborne transmissions. We provide recommendations to ensure good public health practices.
Trichomonas vaginalis, a flagellated protozoan parasite, is commonly found in the genitourinary tract of humans. Its mode of reproduction has always been reported to be binary fission. The high parasite numbers seen in a relatively short period in in vitro cultures led us to believe that there must be other modes of reproduction. The present study for the first time provides transformational evidence at the ultrastructural level seen in tropohozoites of T. vaginalis undergoing a multiple asexual mode of reproduction. The findings show that the single cell with a nucleus is capable of dividing to as many as eight nuclei within the cytoplasmic body. Before the commencement of division, the nucleus remained round or ovoid in shape with condensed chromatin masses and only a few endoplasmic reticula surrounding the nucleus. During the division, the nucleus started to elongate and become irregular in shape with visible chromatin masses condensing with the accumulation of numerous endoplasmic reticula. Nuclear division gave rise to as many as eight nuclei within a cell, which could be seen to be connected by numerous endoplasmic reticula. In addition, a high number of hydrogenosomes and vacuoles can be seen in multinucleated T. vaginalis compared with single nucleated T. vaginalis. This study confirms that multiple modes of nuclear division do exist in T. vaginalis and are a precursor to progeny formation.
The pathogenesis of Blastocystis hominis in human hosts has always been a matter of debate as it is present in both symptomatic and asymptomatic individuals. A recent report showed that B. hominis isolated from an asymptomatic individual could facilitate the proliferation and growth of existing cancer cells while having the potential to downregulate the host immune response. The present study investigated the differences between the effects of symptomatic and asymptomatic derived solubilized antigen of B. hominis (Blasto-Ag) on the cell viability and proliferation of colorectal cancer cells. Besides that, the gene expression of cytokine and nuclear transcriptional factors in response to the symptomatic and asymptomatic B. hominis antigen in HCT116 was also compared. In the current study, an increase in cell proliferation was observed in HCT116 cells which led to the speculation that B. hominis infection could facilitate the growth of colorectal cancer cells. In addition, a more significant upregulation of Th2 cytokines observed in HCT116 may lead to the postulation that symptomatic Blasto-Ag may have the potential in weakening the cellular immune response, allowing the progression of existing tumor cells. The upregulation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) was observed in HCT116 exposed to symptomatic Blasto-Ag, while asymptomatic Blasto-Ag exhibited an insignificant effect on NF-κB gene expression in HCT116. HCT116 cells exposed to symptomatic and asymptomatic Blasto-Ag caused a significant upregulation of CTSB which lead to the postulation that the Blasto-Ag may enhance the invasive and metastasis properties of colorectal cancer. In conclusion, antigen isolated from a symptomatic individual is more pathogenic as compared to asymptomatic isolates as it caused a more extensive inflammatory reaction as well as more enhanced proliferation of cancer cells.
Trichomonas vaginalis, a flagellated protozoan parasite causes a variety of adverse health consequences in both men and women. The parasite exists in the trophozoite and the pseudocystic stage. The study reports for the first time that pseudocyst forms of T. vaginalis isolated from cervical neoplasia (CN) patients demonstrated distinct, different and significant in vitro growth profiles when grown in vitro cultures from day 1 up to day 5 (p<0.05, Mann-Whitney test) when compared with the same life cycle stages isolated from non-cervical neoplasia but symptomatic patients (NCN). Pseudocysts from CN and NCN isolates remained viable in distilled water until 3 h 10 min and 2 h 10 min, respectively. The nucleus of pseudocysts in CN isolates using acridine orange and DAPI showed more intense staining revealing higher nuclear content. The FITC-labeled Concanavalin A stained stronger green fluorescence with surface of pseudocysts in CN isolates showing more rough and creased surface with higher numbers of deep micropores with larger numbers of chromatin masses, vacuoles, and hydrogenosomes. The study confirms that pseudocystic stage from CN, despite the uniformity in appearance of being rounded and showing no motility without a true cyst wall under light microscopy, demonstrated different biochemical, surface, and ultrastructural properties. The study provides evidence that phenotypic variant forms of pseudocysts does exist and possibly does play a role in exacerbating cervical cancer.
Domestic dogs, Canis lupus, have been one of the longest companions of humans and have introduced their own menagerie of parasites and pathogens into this relationship. Here, we investigate the parasitic load of 212 domestic dogs with fleas (Siphonaptera) chewing lice (Phthiraptera), and ticks (Acarina) along a gradient from rural areas with near-natural forest cover to suburban areas in Northern Borneo (Sabah, Malaysia). We used a spatially-explicit hierarchical Bayesian model that allowed us to impute missing data and to consider spatial structure in modelling dog infestation probability and parasite density. We collected a total of 1,968 fleas of two species, Ctenocephalides orientis and Ctenocephalides felis felis, from 195 dogs (prevalence, 92 %). Flea density was higher on dogs residing in houses made of bamboo or corrugated metal (increase of 40 % from the average) compared to timber or stone/compound houses. Host-dependent and landscape-level environmental variables and spatial structure only had a weak explanatory power. We found adults of the invasive chewing louse Heterodoxus spiniger on 42 dogs (20 %). The effect of housing conditions was opposite to those for fleas; lice were only found on dogs residing in stone or timber houses. We found ticks of the species Rhipicephalus sanguineus as well as Haemaphysalis bispinosa gp., Haemaphysalis cornigera, Haemaphysalis koenigsbergi, and Haemaphysalis semermis on 36 dogs (17 %). The most common tick species was R. sanguineus, recorded from 23 dogs. Tick infestations were highest on dogs using both plantation and forest areas (282 % increase in overall tick density of dogs using all habitat types). The infestation probability of dogs with lice and ticks decreased with elevation, most infestations occurred below 800 m above sea level. However, the density of lice and ticks revealed no spatial structure; infestation probability of dogs with these two groups revealed considerable autocorrelation. Our study shows that environmental conditions on the house level appeared to be more influential on flea and lice density whereas tick density was also influenced by habitat use. Infestation of dogs with Haemaphysalis ticks identified an important link between dogs and forest wildlife for potential pathogen transmission.