The sensitivity of larval paralysis assay (LPA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-formazan (MTT-formazan) assay was compared to evaluate the anthelmintic activity of plant extracts. In this study, the methanolic extract of Azadirachta indica (neem) was evaluated for its activity against the infective-stage larvae (L(3)) of susceptible and resistant Haemonchus contortus strains using the two aforementioned assays. In both in vitro assays, the same serial concentrations of the extract were used, and the median lethal concentrations were determined to compare the sensitivity of both assays. The results revealed a significant difference (P < 0.05) in the sensitivity of the LPA and the MTT-formazan assay. The MTT-formazan assay is more feasible for practical applications because it measured the L(3) mortality more accurately than LPA. This study may help find a suitable assay for investigating the anthelmintic activity of plant extracts against trichostrongylid nematodes.
Blastocystis sp. is a common intestinal parasite found in humans and animals. The possibility of zoonotic transmission to humans from livestock especially goats led us to investigate the genetic diversity of caprine Blastocystis sp. obtained from five different farms in Peninsular Malaysia. Moreover, there is a lack of information on the prevalence as well as genetic diversity of Blastocystis sp. in goat worldwide. Results showed that 73/236 (30.9 %) of the goats were found to be positive for Blastocystis infection. The most predominant Blastocystis sp. subtype was ST1 (60.3 %) followed by ST7 (41.1 %), ST6 (41.1 %), and ST3 (11.0 %) when amplified by PCR using sequenced-tagged site (STS) primers. Four farms had goats infected only with ST1 whereas the fifth showed mixed infections with multiple STs. The proximity of the fifth farm to human dwellings, nearby domesticated animals and grass land as opposed to a sterile captive environment in the first four farms may account for the multiple STs seen in the fifth farm. Since ST1, ST3, ST6 and ST 7 were previously reported in human infection worldwide in particular Malaysia, the potential of the zoonotic transmission of blastocystosis should not be disregarded. The implications of different farm management systems on the distribution of Blastocystis sp. STs are discussed.
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.
Tor tambroides, a common and appreciated cyprinid fish of the Tasik Kenyir water reservoir in Malaysia, is one of the species selected for propagation. This fish was first successfully propagated in Malaysia by the Department of Agriculture, Sarawak, Malaysia, and the breeding program continued throughout the country. The gills were frequently infected by a Myxobolus species to be described as Myxobolus tambroides sp. n. The small, 50 to 70 μm, round plasmodia of this species is located intralamellarly. Plasmodia were filled with pyriform myxospores, 9.9 and 7.4 μm wide. In sutural view, the caudal end of the myxospores had a distinctive valvular groove, parallel with the suture. Plasmodia caused deformations on the affected and the neighbouring gill lamellae. The 18S rDNA sequence of M. tambroides sp.n. did not show a close relationship with any other Myxobolus spp., represented in the GenBank. This might be an emerging parasite likely to impact the propagation of this fish.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Detection of Strongyloides stercoralis infection particularly in asymptomatic individuals is often hampered due to the lack of standard diagnostic tools. In this study, the use of serological and molecular approaches were investigated for the detection of S. stercoralis infection among an Orang Asli (indigenous) community following a preliminary detection by microscopic examination of faecal samples. Out of 54 individuals studied, 17/54 (31.5%) were detected to be positive for S. stercoralis infection by enzyme-linked immunosorbent assay (ELISA), compared to 0/54 (0%) by faecal examination. Further confirmation performed by a nested polymerase chain reaction (PCR) using DNA extracted from faecal samples of these 17 individuals yielded 3/17 (17.6%) positives for S. stercoralis DNA amplification. No amplification was seen with the other 37 faecal samples, which were negative by microscopy and ELISA. As the high ELISA positive results were suspected to be false-positives, ELISA is not recommended for use as a detection tool but may be beneficial for evaluating the effectiveness of anti-Strongyloides drugs. The present finding indicated that PCR should be considered as an alternative diagnostic tool for the detection of S. stercoralis infection.
This study focuses on the larvicidal, oviposition, and ovicidal effects of a crude extract of Artemisia annua against Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus. Dried cells of Artemisia annua from cell suspension cultures were extracted using hexane. The extract showed moderate larvicidal effects against mosquitoes. At 24-h post treatment, the LC50 values for Anopheles sinensis, Aedes aegypti, and Culex quinquefasciatus were recorded as 244.55, 276.14, and 374.99 ppm, respectively. The percentage mortality of larvae was directly proportional to the tested concentration. Anopheles sinensis was found to be the most susceptible species, whereas Culex quinquefasciatus was the most tolerant to the Artemisia annua extract. The results indicated that the Artemisia annua extract showed concentration-dependent oviposition deterrent activity and had a strong deterrent effect. At 500 ppm, the percentage effective repellency was more than 85% compared with the control group for all the species, with oviposition activity index values of -0.94, -0.95, and -0.78 for Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus, respectively. In the ovicidal assay, the percentage hatchability of eggs after treatment with 500 ppm of Artemisia annua extract was significantly lower than the control, with values of 48.84 ± 4.08, 38.42 ± 3.67, and 79.35 ± 2.09% for Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus, respectively. Artemisia annua was found to be more effective against Aedes aegypti and Anopheles sinensis compared with Culex quinquefasciatus. This study indicated that crude extract of A. annua could be a potential alternative for use in vector management programs.