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.
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.
Daily intramuscular injection with thyroxine (T4) at a dose of 2.5 micrograms/100 g body weight decreased the larvae and adult worm burden of Parastrongylus malaysiensis in the brain and pulmonary arteries of male Sprague-Dawley albino rats. In contrast, rats treated with propyl thiouracil (PTU), an antithyroid drug, at a dose of 3.75 mg/100 g body weight retained greater numbers of larvae and adult worms. The results may reflect the contrasting immunomodulatory effects of T4 and PTU that influence the susceptibility of the host.
This work investigated the anti-amoebic activity of two samarium (Sm) complexes, the acyclic complex [bis(picrato)(pentaethylene glycol)samarium(III)] picrate-referred to as [Sm(Pic)2(EO5)](Pic)-and the cyclic complex [bis(picrato)(18-crown-6)samarium(III)] picrate-referred to as [Sm(Pic)2(18C6)](Pic). Both Sm complexes caused morphological transformation of the protozoa Acanthamoeba from its native trophozoite form carrying a spine-like structure called acanthopodia, to round-shaped cells with loss of the acanthopodia structure, a trademark response to environmental stress. Further investigation, however, revealed that the two forms of the Sm complexes exerted unique cytotoxicity characteristics. Firstly, the IC50 of the acyclic complex (0.7 μg/mL) was ~ 10-fold lower than IC50 of the cyclic Sm complex (6.5 μg/mL). Secondly, treatment of the Acanthamoeba with the acyclic complex caused apoptosis of the treated cells, while the treatment with the cyclic complex caused necrosis evident by the leakage of the cell membrane. Both treatments induced DNA damage in Acanthamoeba. Finally, a molecular docking simulation revealed the potential capability of the acyclic complex to form hydrogen bonds with profilin-a membrane protein present in eukaryotes, including Acanthamoeba, that plays important roles in the formation and degradation of actin cytoskeleton. Not found for the cyclic complex, such potential interactions could be the underlying reason, at least in part, for the much higher cytotoxicity of the acyclic complex and also possibly, for the observed differences in the cytotoxicity traits. Nonetheless, with IC50 values of
Gonadectomized male laboratory rats were given 0.06 mg/kg estradiol benzoate daily for 14 days before being inoculated with 50 third-stage larvae of Parastrongylus malaysiensis. Hormone treatment was continued until the rats were killed. The numbers of larvae in the brain and of adult worms in the pulmonary area of the rats were determined every 7 days after the inoculation. It was found that the rats treated daily with estradiol benzoate had significantly and consistently higher numbers of larvae and adult worms as compared with the controls. The number of total leukocytes increased significantly after the rats were infected. The results show that estradiol-treated rats become susceptible to P. malaysiensis infection, which may indicate that the immunosuppressive effects of testosterone observed in earlier studies may partly be caused by estradiol that was peripherally aromatized from testosterone.
Gonadectomized male albino rats aged 7 weeks were given 1.5 mg/kg testosterone propionate daily and inoculated with 50 third-stage larvae of Angiostrongylus malaysiensis. The treatment significantly increased the number of larvae and adult worms recovered from the brain and pulmonary arteries, respectively, and the rats exhibited smaller thymus glands. The total numbers of leukocytes, monocytes, neutrophils, and especially eosinophils increased significantly post-infection, but the counts were higher in the untreated infected controls. Presumably, immunosuppressive effects of testosterone may at least partly be responsible for the higher loads of A. malaysiensis worms found in male rats as compared with females in the field.
Malaria remains a major public health problem due to the emergence and spread of Plasmodium falciparum strains resistant to chloroquine. There is an urgent need to investigate new and effective sources of antimalarial drugs. This research proposed a novel method of fern-mediated synthesis of silver nanoparticles (AgNP) using a cheap plant extract of Pteridium aquilinum, acting as a reducing and capping agent. AgNP were characterized by UV-vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, energy-dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD). Phytochemical analysis of P. aquilinum leaf extract revealed the presence of phenols, alkaloids, tannins, flavonoids, proteins, carbohydrates, saponins, glycosides, steroids, and triterpenoids. LC/MS analysis identified at least 19 compounds, namely pterosin, hydroquinone, hydroxy-acetophenone, hydroxy-cinnamic acid, 5, 7-dihydroxy-4-methyl coumarin, trans-cinnamic acid, apiole, quercetin 3-glucoside, hydroxy-L-proline, hypaphorine, khellol glucoside, umbelliferose, violaxanthin, ergotamine tartrate, palmatine chloride, deacylgymnemic acid, methyl laurate, and palmitoyl acetate. In DPPH scavenging assays, the IC50 value of the P. aquilinum leaf extract was 10.04 μg/ml, while IC50 of BHT and rutin were 7.93 and 6.35 μg/ml. In mosquitocidal assays, LC50 of P. aquilinum leaf extract against Anopheles stephensi larvae and pupae were 220.44 ppm (larva I), 254.12 ppm (II), 302.32 ppm (III), 395.12 ppm (IV), and 502.20 ppm (pupa). LC50 of P. aquilinum-synthesized AgNP were 7.48 ppm (I), 10.68 ppm (II), 13.77 ppm (III), 18.45 ppm (IV), and 31.51 ppm (pupa). In the field, the application of P. aquilinum extract and AgNP (10 × LC50) led to 100 % larval reduction after 72 h. Both the P. aquilinum extract and AgNP reduced longevity and fecundity of An. stephensi adults. Smoke toxicity experiments conducted against An. stephensi adults showed that P. aquilinum leaf-, stem-, and root-based coils evoked mortality rates comparable to the permethrin-based positive control (57, 50, 41, and 49 %, respectively). Furthermore, the antiplasmodial activity of P. aquilinum leaf extract and green-synthesized AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of P. falciparum. IC50 of P. aquilinum were 62.04 μg/ml (CQ-s) and 71.16 μg/ml (CQ-r); P. aquilinum-synthesized AgNP achieved IC50 of 78.12 μg/ml (CQ-s) and 88.34 μg/ml (CQ-r). Overall, our results highlighted that fern-synthesized AgNP could be candidated as a new tool against chloroquine-resistant P. falciparum and different developmental instars of its primary vector An. stephensi. Further research on nanosynthesis routed by the LC/MS-identified constituents is ongoing.
Mosquito vectors (Diptera: Culicidae) are responsible for transmission of serious diseases worldwide. Mosquito control is being enhanced in many areas, but there are significant challenges, including increasing resistance to insecticides and lack of alternative, cost-effective, and eco-friendly products. To deal with these crucial issues, recent emphasis has been placed on plant materials with mosquitocidal properties. Furthermore, cancers figure among the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012. It is expected that annual cancer cases will rise from 14 million in 2012 to 22 million within the next two decades. Nanotechnology is a promising field of research and is expected to give major innovation impulses in a variety of industrial sectors. In this study, we synthesized titanium dioxide (TiO2) nanoparticles using the hydrothermal method. Nanoparticles were subjected to different analysis including UV-Vis spectrophotometry, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), zeta potential, and energy-dispersive spectrometric (EDX). The synthesized TiO2 nanoparticles exhibited dose-dependent cytotoxicity against human breast cancer cells (MCF-7) and normal breast epithelial cells (HBL-100). After 24-h incubation, the inhibitory concentrations (IC50) were found to be 60 and 80 μg/mL on MCF-7 and normal HBL-100 cells, respectively. Induction of apoptosis was evidenced by Acridine Orange (AO)/ethidium bromide (EtBr) and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining. In larvicidal and pupicidal experiments conducted against the primary dengue mosquito Aedes aegypti, LC50 values of nanoparticles were 4.02 ppm (larva I), 4.962 ppm (larva II), 5.671 ppm (larva III), 6.485 ppm (larva IV), and 7.527 ppm (pupa). Overall, our results suggested that TiO2 nanoparticles may be considered as a safe tool to build newer and safer mosquitocides and chemotherapeutic agents with little systemic toxicity.
Malaria, the most widespread mosquito-borne disease, affects 350-500 million people each year. Eco-friendly control tools against malaria vectors are urgently needed. This research proposed a novel method of plant-mediated synthesis of silver nanoparticles (AgNP) using a cheap seaweed extract of Ulva lactuca, acting as a reducing and capping agent. AgNP were characterized by UV-vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), and X-ray diffraction (XRD). The U. lactuca extract and the green-synthesized AgNP were tested against larvae and pupae of the malaria vector Anopheles stephensi. In mosquitocidal assays, LC50 values of U. lactuca extract against A. stephensi larvae and pupae were 18.365 ppm (I instar), 23.948 ppm (II), 29.701 ppm (III), 37.517 ppm (IV), and 43.012 ppm (pupae). LC50 values of AgNP against A. stephensi were 2.111 ppm (I), 3.090 ppm (II), 4.629 ppm (III), 5.261 ppm (IV), and 6.860 ppm (pupae). Smoke toxicity experiments conducted against mosquito adults showed that U. lactuca coils evoked mortality rates comparable to the permethrin-based positive control (66, 51, and 41%, respectively). Furthermore, the antiplasmodial activity of U. lactuca extract and U. lactuca-synthesized AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. Fifty percent inhibitory concentration (IC50) values of U. lactuca were 57.26 μg/ml (CQ-s) and 66.36 μg/ml (CQ-r); U. lactuca-synthesized AgNP IC50 values were 76.33 μg/ml (CQ-s) and 79.13 μg/ml (CQ-r). Overall, our results highlighted out that U. lactuca-synthesized AgNP may be employed to develop newer and safer agents for malaria control.
A main challenge in parasitology is the development of reliable tools to prevent or treat mosquito-borne diseases. We investigated the toxicity of magnetic nanoparticles (MNP) produced by Magnetospirillum gryphiswaldense (strain MSR-1) on chloroquine-resistant (CQ-r) and sensitive (CQ-s) Plasmodium falciparum, dengue virus (DEN-2), and two of their main vectors, Anopheles stephensi and Aedes aegypti, respectively. MNP were studied by Fourier-transform infrared spectroscopy and transmission electron microscopy. They were toxic to larvae and pupae of An. stephensi, LC50 ranged from 2.563 ppm (1st instar larva) to 6.430 ppm (pupa), and Ae. aegypti, LC50 ranged from 3.231 ppm (1st instar larva) to 7.545 ppm (pupa). MNP IC50 on P. falciparum were 83.32 μg ml(-1) (CQ-s) and 87.47 μg ml(-1) (CQ-r). However, the in vivo efficacy of MNP on Plasmodium berghei was low if compared to CQ-based treatments. Moderate cytotoxicity was detected on Vero cells post-treatment with MNP doses lower than 4 μg ml(-1). MNP evaluated at 2-8 μg ml(-1) inhibited DEN-2 replication inhibiting the expression of the envelope (E) protein. In conclusion, our findings represent the first report about the use of MNP in medical and veterinary entomology, proposing them as suitable materials to develop reliable tools to combat mosquito-borne diseases.
Blastocystis sp. is known to be the most commonly found intestinal protozoan parasite in human fecal surveys and has been incriminated to cause diarrhea and abdominal bloating. Binary fission has been widely accepted as the plausible mode of reproduction for this parasite. The present study demonstrates that subjecting the parasites in vitro to higher temperature shows the proliferation of parasite numbers in cultures. Transmission electron microscopy was used to compare the morphology of Blastocystis sp. subtype 3 isolated from a dengue patient having high fever (in vivo thermal stress) and Blastocystis sp. 3 maintained at 41 °C (in vitro thermal stress) and 37 °C (control). Fluorescence stains like acridine orange (AO) and 4',6'-diamino-2-phenylindole (DAPI) were used to demonstrate the viability and nuclear content of the parasite for both the in vitro and in vivo thermal stress groups of parasites. Blastocystis sp. at 37 °C was found to be mostly vacuolar whereas the in vitro thermal stressed isolates at 41 °C were granular with electron dense material seen to protect the granules within the central body. Parasites of the in vivo thermal stressed group showed similar ultrastructure as the in vitro ones. AO and DAPI staining provided evidence that these granules are viable which develop into progenies of Blastocystis sp. These granular forms were then observed to rupture and release progenies from the mother cells whilst the peripheral cytoplasmic walls were seen to degrade. Upon exposure to high temperature both in vitro and in vivo, Blastocystis sp. in cultures show higher number of granular forms seen to be protected by the electron dense material within the central body possibly acting as a protective mechanism. This is possibly to ensure the ability to survive for the granules to be developed as viable progenies for release into the host system.
Blastocystis sp., an intestinal organism is known to cause diarrhea with metronidazole regarded as the first line of treatment despite reports of its resistance. The conflicting reports of variation in drug treatment have been ascribed to subtype differences. The present study evaluated in vitro responses due to metronidazole on ST3 isolated from three symptomatic and asymptomatic patients, respectively. Symptomatic isolates were obtained from clinical patients who showed symptoms such as diarrhea and abdominal bloating. Asymptomatic isolates from a stool survey carried out in a rural area. These patients had no other pathogens other than Blastocystis. Ultrastructural studies using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed drug-treated ST3 from symptomatic patients were irregular and amoebic with surface showing high-convoluted folding when treated with metronidazole. These organisms had higher number of mitochondrion-like organelle (MLO) with prominent cristae. However, the drug-treated ST3 from asymptomatic persons remained spherical in shape. Asymptomatic ST3 showed increase in the size of its central body with the MLO located at the periphery.
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.
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
Dientamoeba fragilis, a trichomonad parasite is usually found in the gastrointestinal tract of human, and it is known to be the cause for gastrointestinal disease. The parasite is globally distributed and mostly found in rural and urban areas. The parasite is found in humans and nonhuman primates such as the macaques, baboons, and gorillas. Often, the parasite is confused with another largely found organism in stools called Blastocystis sp. especially when seen directly under light microscopy on culture samples containing both parasites. Both sometimes are seen with two nuclei with sizes tending to be similar which complicates identification. Stools were collected fresh from nine previously diagnosed persons infected with D. fragilis who also were found to be positive for Blastocystis sp. Samples were then cultured in Loeffler's medium and were stained with Giemsa, iron hematoxylin, and modified Fields' (MF) stain, respectively. D. fragilis was differentiated from Blastocystis sp. when stained with MF stain by the presence of a thinner outer membrane with clearly demarcated nuclei in the center of the cell whilst Blastocystis sp. had a darker and thicker stained outer membrane with the presence of two nuclei. The staining contrast was more evident with modified Fields' stain when compared with the other two. The simplicity in preparing the stain as well as the speed of the staining procedure make MF stain an ideal alternate. The modified Fields' stain is faster and easier to prepare when compared to the other two stains. MF stain provides a better contrast differentiating the two organisms and therefore provides a more reliable diagnostic method to precisely identify one from the other especially when cultures show mixed infections.
Established criteria for morphological typing of sarcocysts was applied to a large series of cases of human skeletal muscle sarcocystosis in Malaysia to determine the type of sarcocyst present. We also wanted to test the general usefulness of this classification and to determine if there are any new cyst types. Three-dimensional (3-D) reconstruction was done to see if the sarcocyst has a distinct 3-D morphology. A total of 66 sarcocysts from 21 cases of human muscle sarcocystosis obtained from a previous prevalence study were examined. Tissue sections (5 microns thick) were stained with haematoxylin and eosin and studied under the light microscope. For 3-D reconstruction, an image analyser was used to align and reconstruct the sarcocyst after microscopic images had been captured with a charge-coupled device (CCD) camera. All the cysts best fit into the type 4 category. This classification is generally useful, although cyst wall characteristics and zoite size appear to be the most reliable criteria for classification. The cyst width averaged 77 microns (range, 30-137.5 microns). Cyst walls were smooth, had no cytophaneres and were less than 1 micron thick. No secondary cyst wall or surrounding inflammation was evident. Numerous cyst merozoites with diameters averaging 1 micron filled the cyst lumen. Although septa were not apparent, in many cysts, zoites were arranged in a unique, curvilinear fashion that suggested their presence. 3-D reconstruction showed the sarcocyst to be a long, tortuous "cylinder" with no branching or other distinguishing feature.
In February 2013, forty-seven Notched threadfin bream, the Nemipterus peronii, were sampled from the eastern coastal waters of the South China Sea. The concentration of various elements, namely cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), strontium (Sr), manganese (Mn), selenium (Se), Lead (Pb), nickel (Ni), aluminum (Al), arsenic (As), iron (Fe), and Zinc (Zn) were analyzed in the liver, muscle, and kidney organs of the host, as well as in their parasites Hysterothalycium reliquens (nematode) and the Paraphilometroides nemipteri (nematode), using inductively coupled plasma mass spectrometry (ICP-MS). The former group of parasites showed highest accumulation capacity for Cr, Cu, Fe, Mn, Se, Ni, and Zn while the latter group had high accumulation potential of As, Hg, Cd, Al, Pb, and Sr. The divergence in heavy-metal accumulation profiles of both nematodes is linked with the specificity of microhabitats, cuticle morphology, and interspecific competition. The outcome of this study indicates that both parasite models can be used for biomonitoring of metal pollution in marine ecosystems.
The ornamental fish trade provides a pathway for the global translocation of aquatic parasites. We examined a total of 1020 fish imported from Singapore, Malaysia, Thailand, or Sri Lanka to Australia (including freshwater and marine fish species) for monogenean ectoparasites. Fish were received following veterinary certification that they showed no clinical signs of pests and diseases from the exporting country and visual inspection at Australian border control. Australian import conditions require mandatory treatment for goldfish with parasiticides (e.g. trichlorfon, formaldehyde, sodium chloride) for the presence of gill flukes (Dactylogyrus vastator Nybelin, 1924 and Dactylogyrus extensus Mueller and Van Cleave, 1932) prior to export. Over 950 individual parasites were detected in five imported fish species, representing 14 monogenean species. Seven Dactylogyrus spp. including D. vastator and three Gyrodactylus spp. infected goldfish, Carassius auratus Linnaeus, 1758, from Malaysia, Singapore, and Thailand. Dactylogyrus ostraviensis Řehulka, 1988, infected rosy barb, Pethia conchonius Hamilton, 1822, from Singapore, Sri Lanka, and Thailand while two Trianchoratus spp. infected three spot gourami, Trichopodus trichopterus Pallas, 1970 and pearl gourami Trichopodus leerii Bleeker, 1852, from Sri Lanka. Urocleidoides reticulatus Mizelle & Price, 1964, infected guppy, Poecilia reticulata Peters, 1859, from Sri Lanka. The discovery of D. vastator in goldfish, as well as 13 other monogenean species, shows that pre-export health requirements, which include chemical treatment of goldfish, and inspection of all ornamental fish species did not prevent infection by monogeneans. Inspection prior to exportation and at border control must account for the highly cryptic nature of monogenean parasites and consider alternatives to current pre-export conditions and visual inspection at border control.
Gonadectomized male mice aged 5 weeks were given 5 mg testosterone propionate daily for 14 days. The treatment significantly decreased the number of blood leukocytes. The number of all individual types of leukocytes except basophils in vehicle-treated gonadectomized mice was increased. Testosterone-treated mice consistently had a lower number of leukocytes after being infected with Plasmodium berghei than did vehicle-treated mice. The results suggest that testosterone suppresses the production of leukocytes and that testosterone-treated mice become more susceptible to parasite infection.