In forensic entomology, breeding of fly larvae in a controlled laboratory environment using animal tissue is a common technique to obtain insect developmental time for the estimation of postmortem interval. Previous studies on growth media are mostly on the effect of different diets on fly development. However, the interaction effects between temperature and food type used have not been explored. The objective of this study was to compare the use of cow's liver agar and raw liver on the development of a forensically important fly, Megaselia scalaris (Loew). This study also determined the interaction between different temperatures and different food types on the growth of this species. A total of 100 M. scalaris eggs were transferred into each of the two media mentioned above. Liver agar was prepared by adding dried ground liver into nutrient agar, whilst raw liver was naturally prepared from the same animal source. This experiment was conducted at 27, 30 and 33 °C in an incubator in a continuously dark condition. Length and weight of larvae, puparia and adult samples were determined. Total developmental times for larvae feeding on liver agar at each temperature were approximately 7-15 h slower than those feeding on raw liver. Survival rates were almost equal in both diets but were lower at 33 °C. Mean larva length in both diets did not differ significantly at all temperatures, but larvae feeding on liver agar had lower mean weight values than those in raw liver at 30 and 33 °C. The effect of temperature was significant in female puparia weight and male adult weight whereas the effect of diet types was significant in both male and female puparia size and weight. Interaction effects of temperature and food type on M. scalaris puparium size and adult weight were significant, indicating that puparium size and adult weight depended on both food type and temperature. This experiment highlighted the use of cow's liver agar as an alternative diet to breed M. scalaris in the laboratory and the importance of considering the interaction effect between temperatures and food types when deciding the most suitable medium in fly larva rearing.
Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the food industry. Commonly used serological tests involve preparation of whole Toxoplasma lysate antigens from tachyzoites which are costly and hazardous. An alternative method for better antigen production involving the prokaryotic expression system was therefore used in this study. Recombinant dense granular protein, GRA2, was successfully cloned, expressed, and purified in Escherichia coli, BL21 (DE3) pLysS. The potential of this purified antigen for diagnosis of human infections was evaluated through western blot analysis against 100 human serum samples. Results showed that the rGRA2 protein has 100 and 61.5 % sensitivity towards acute and chronic infection, respectively, in T. gondii-infected humans, indicating that this protein is useful in differentiating present and past infections. Therefore, it is suitable to be used as a sensitive and specific molecular marker for the serodiagnosis of Toxoplasma infection in both humans and animals.
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.
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.
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 is one of the most common human parasites that inhabit the intestinal tract. Conflicting reports continue to exist regarding the existence and the functional role of the amoeboid forms in the life cycle of the parasite. The present study investigates the presence of these forms in 20 isolates obtained from ten symptomatic and asymptomatic patients respectively. A total of 10,000 parasite cells per ml from each isolate were inoculated into three culture tubes each containing 3 ml of Jones' medium supplemented with 10% horse serum, incubated at 37 degrees C. The contents were examined daily for 10 days. Irregular and polymorphic amoeboid forms with multiple extended pseudopodia were observed in all isolates from symptomatic patients, while none of the isolates from asymptomatic patients showed the presence of the amoeboid forms. The amoeboid forms were initially noted on day 2 and the percentages increased from 2% to 28%, with peak percentages from day 3 to day 6. Transmission electron microscopy revealed two types of amoeboid forms; one containing a large central vacuole completely filled with tiny electron-dense granules, and the other which revealed multiple small vacuoles within the central body. The cytoplasm contained strands of electron-dense granules resembling rough endoplasmatic reticulum, which is suggestive of active protein synthesis. The surface coat of the amoeboid form surrounding the parasite showed uneven thickness. Acridine orange stained the central body yellow and the periphery orange, indicating activity at the level of nucleic acids. The amoeboid form could either be an indicator of pathogenicity of B. hominis, or the form likely to contribute to pathogenicity and be responsible for the symptoms seen in patients.
A gene encoding the larval excretory-secretory antigen TES-120 of the dog ascarid worm Toxocara canis was cloned into the methylotrophic yeast Pichia pastoris. Specificity of the recombinant TES-120 antigen produced by the yeast was investigated. Forty-five human serum samples from patients infected with different()parasitic organisms, including 8 cases of toxocariasis, were tested against the recombinant antigen in immunoblot assays. Results from the assays showed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only. This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis.
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.
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.
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.
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
In this study, the genome of the Plasmodium falciparum Gombak A strain was examined for the presence of a gene encoding falcipain-2, a cysteine protease, using homology-based polymerase chain reaction cloning. The nucleotide sequence obtained from the gene cloned (designated pFG1) is approximately 99% homologous to other falcipain-2 genes from different strains. Comparatively, it is 69% homologous to falcipain-3 genes. Direct cloning of the falcipain-2 gene and its resemblance to the reported corresponding mRNA transcript suggests the absence of introns in this gene. Sequence alignment and comparison revealed four amino acid differences at positions 15, 51, 59 and 414 in the falcipain-2 from P. falciparum Gombak A as compared to other falcipain-2 proteins from different strains.
Acanthamoeba sp. is a free-living amoeba known to cause chronic central nervous system infection or eye infection in humans. Many cases remain undetected for want of a good detection system. We report for the first time a rapid staining method to facilitate the identification of Acanthamoeba sp. using the modified Field's staining technique. A. castellanii, which was used in the present experiment, is maintained in our laboratory in mycological peptone medium (Gibco). The cultures were pooled together and smears were made on glass slides for staining purposes. Different types of stains such as Field's stain, modified Field's stain, Wright's stain, Giemsa stain, Ziehl-Neelsen stain, and trichrome stain were used to determine the best stain for the identification of this amoeba. The concentration of various stains and the duration of staining were varied to provide the best color and contrast for each stain. Acanthamoeba was also obtained from the brain of experimentally infected mice and was stained with various stains as mentioned above to determine the best stain for use in identifying the presence of this parasite in experimentally infected animals. The modified Field's stain gives a very good color contrast as compared with other stains. Furthermore, it takes only 20 s to be carried out using the least number of reagents, making it suitable for both laboratory and field use.
The shedding pattern of the protozoan parasite, Blastocystis hominis, is investigated in man and in experimental animal infections. The shedding pattern of the vacuolar and cystic forms of Blastocystis hominis in infected individuals have been shown in the present study to be irregular. The study shows that there is marked fluctuation in the shedding of the parasite from day to day, varying from as high as 17 to 0 per x40 microscopic field. The cystic stages when estimated in 8 Blastocystis-infected individuals ranged from as high as 7.4x10(5) cysts per gram of stool to 0. The shedding of cystic and vacuolar forms observed over a period of 20 days in experimentally-infected Wistar rats were not only shown to be irregular but the amount varied from host to host. The study has important diagnostic implications in that the stool samples must be collected more than once from patients showing clinical signs and symptoms to eliminate the cause of it to Blastocystis. The study also shows that there are asymptomatic individuals who pass a large amount of cysts as such individuals should be treated to prevent transmission to others.
A total of 20 isolates of Blastocystis were characterized using a single set of polymerase chain reaction (PCR) primers. The amplification product revealed five types of pattern. All four isolates from Singapore yielded PCR products quite different from those of the local isolates. However, most of the local isolates showed a major product at either 280 or 500 bp, or both. We also suspected that the amplification product detected at 280 bp might be an indicator of the pathogenicity of this parasite. One isolate (M12) obtained from a monkey showed patterns similar to those of human isolates (10203 and KP1) and probably belongs to the same strain. The results indicate that the intraspecific or interstrain variations in these 20 Blastocystis isolates belong to 5 different patterns. The differences among isolates of the same strain revealed by the presence or absence of certain amplification products showed further intrastrain variations in this parasite.
The present study investigated whether people working closely with animals were at higher risk of getting infected with Blastocystis hominis. The prevalence of the parasite was determined in two population groups, i.e., animal handlers and normal healthy individuals who did not work with animals. In all, 105 stool samples were collected from animal handlers from 2 local research institutions, a local zoo, and a local abattoir and 163 stool samples were collected from normal healthy individuals residing in high-rise flats in the city. The in vitro culture method used in the study detected that 41% of 105 animal handlers and 17% of 163 flat-dwellers in the city were positive for Blastocystis. This statistically significant finding (P = 0.0000313) shows that people who work closely with animals do stand at risk of acquiring Blastocystis infection.
For elucidation of the taxonomic status of the Japanese Fasciola species, whole mitochondrial DNA of Fasciola hepatica from Australia, F. gigantica from Malaysia, and Fasciola sp. from Japan was digested with three four-base-cutting endonucleases: HinfI, MspI, and RsaI. The resulting digestion patterns showed that for each enzyme there were some bands specific for each geographical isolate and that the Japanese Fasciola sp. shared more bands with F. gigantica than with F. hepatica. Nucleotide sequences of two regions, the second internal transcribed spacer (ITS2) of the nuclear ribosomal RNA cluster and mitochondrial cytochrome c oxidase subunit I (COI), were also compared among them. The ITS2 sequence was highly conserved among the three isolates. F. gigantica and the Japanese Fasciola sp. were identical, but they differed from the Australian F. hepatica at six sites, one of which was a deletion. The COI sequence was less conserved but implied a similar relationship between the isolates. There seems no reason to regard the Japanese Fasciola sp. as anything other than a strain of F. gigantica.