The treatment of Acanthamoeba infections remains problematic, suggesting that new targets and/or chemotherapeutic agents are needed. Bioassay-guided screening of drugs that are clinically-approved for non-communicable diseases against opportunistic eukaryotic pathogens is a viable strategy. With known targets and mode of action, such drugs can advance to clinical trials at a faster pace. Recently Bortezomib (proteasome inhibitor) has been approved by FDA in the treatment of multiple myeloma. As proteasomal pathways are well known regulators of a variety of eukaryotic cellular functions, the overall aim of the present study was to study the effects of peptidic and non-peptidic proteasome inhibitors on the biology and pathogenesis of Acanthamoeba castellanii of the T4 genotype, in vitro. Zymographic assays revealed that inhibition of proteasome had detrimental effects on the extracellular proteolytic activities of A. castellanii. Proteasome inhibition affected A. castellanii growth (using amoebistatic assays), but not viability of A. castellanii. Importantly, proteasome inhibitors affected encystation as determined by trophozoite transformation into the cyst form, as well as excystation, as determined by cyst transformation into the trophozoite form. The ability of proteasome inhibitor to block Acanthamoeba differentiation is significant, as it presents a major challenge in the successful treatment of Acanthamoeba infection. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic infections.
Balamuthia mandrillaris is a protist pathogen that can cause encephalitis with a mortality rate of more than 95%. Early diagnosis followed by aggressive treatment is a pre-requisite for successful prognosis. Current methods for identifying this organism rely on culture and microscopy, antibody-based methods using animals, or involve the use of molecular tools that are expensive. Here, we describe the isolation of antibody fragments that can be used for the unequivocal identification of B. mandrillaris. B. mandrillaris-specific antibody fragments were isolated from a bacteriophage antibody display library. Individual clones were studied by enzyme-linked immunosorbent assay, and immunofluorescence. Four antibody clones showed specific binding to B. mandrillaris. The usefulness of phage antibody display technology as a diagnostic tool for isolating antibody fragments against B. mandrillaris antigens and studying their biological role(s) is discussed further.
Thirty snakehead fish, Channa micropeltes (Cuvier, 1831) were collected at Lake Kenyir, Malaysia. Muscle, liver, intestine and kidney tissues were removed from each fish and the intestine was opened to reveal cestodes. In order to assess the concentration of heavy metal in the environment, samples of water in the surface layer and sediment were also collected. Tissues were digested and the concentrations of manganese (Mn), zinc (Zn), copper (Cu), cadmium (Cd) and lead (Pb) were analysed by using inductively-coupled plasma mass-spectrometry (ICP-MS) equipment. The results demonstrated that the cestode Senga parva (Fernando and Furtado, 1964) from fish hosts accumulated some heavy metals to a greater extent than the water and some fish tissues, but less than the sediment. In three (Pb, Zn and Mn) of the five elements measured, cestodes accumulated the highest metal concentrations, and in remaining two (Cu and Cd), the second highest metal accumulation was recorded in the cestodes when compared to host tissues. Therefore, the present study indicated that Senga parva accumulated metals and might have potential as a bioindicator of heavy-metal pollution.
Molecular methods are used increasingly for the detection of Toxoplasma gondii infection. This study developed a rapid, sensitive, and specific conventional triplex PCR for the detection of the B1 gene and ITS1 region of T. gondii using newly designed primers and an internal control based on the Vibrio cholerae HemM gene. The annealing temperature and concentrations of the primers, MgCl(2), and dNTPs were optimized. Two sets of primers (set 1 and 2) were tested, which contained different segments of the T. gondii B1 gene, 529 repeat region and ITS1 region. A series of sensitivity tests were performed using parasite DNA, whole parasites, and spiked human body fluids. Specificity tests were performed using DNA from common protozoa and bacteria. The newly developed assay based on set 2 primers was found to be specific and sensitive. The test was capable of detecting as little as 10 pg T. gondii DNA, 10(4) tachyzoites in spiked body fluids, and T. gondii DNA in the organ tissues of experimentally infected mice. The assay developed in this study will be useful for the laboratory detection of T. gondii infection.
The protozoan Trichomonas vaginalis a sexually transmitted protozoan parasite causes vaginitis, urethritis and cervicitis in humans. The present study highlights phenotypic 'variant' forms of trophozoites isolated from patients suffering from cervical neoplasia condition. The growth curve of 10 isolates i.e., four non-cervical neoplasia (NCN) isolates (NCN1-NCN4) and six cervical neoplasia (CN) isolates (CN1-CN6) showed two distinct and different in vitro growth profiles. The parasite count and growth rates were significantly higher in trophozoites from CN isolates in cultures of day 2 up to day 8 (p<0.05, Mann-Whitney test). The average generation time was 1.84±0.40 and 3.38±0.55h for NCN and CN isolates respectively. The nucleus of trophozoites 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 trophozoites in CN isolates showing more rough and creased surface with numerous deep micropores. Transmission electron microscopy studies revealed that there was higher numbers of vacuoles and hydrogenosomes in these forms. The study mounted staining techniques, growth profiles, morphology, morphometry studies using scanning and transmission electron microscopy and confirms that the trophozoites from cervical neoplasia proliferates at a higher rate, shows higher FITC-labeled Concanavalin A binding with rough and creased surface implying that these are virulent forms which can aggravate or exacerbate cervical neoplasia conditions. The large numbers of hyrogenosomes and vacuoles implies that these forms are active and implicates a possible role in such conditions.
Toxoplasma gondii is an intra-cellular parasite that infects humans through vertical and horizontal transmission. The cysts remain dormant in the brain of infected humans and can reactivate in immunocompromised hosts resulting in acute toxoplasmic encephalitis which may be fatal. We determined the onset and progression of brain cysts generation in a mouse model following acute toxoplasmosis as well as the ability of brain cysts to reactivate in vitro. Male Balb/c mice, (uninfected control group, n = 10) were infected orally (study group, n = 50) with 1000 tachyzoites of T. gondii (ME49 strain) and euthanized at 1, 2, 4, 8 and 16 weeks post infection. Brain tissue was harvested, homogenized, stained and the number of brain cysts counted. Aliquots of brain homogenate with cysts were cultured in vitro with confluent Vero cells and the number of cysts and tachyzoites counted after 1 week. Brain cysts but not tachyzoites were detected at week 2 post infection and reached a plateau by week 4. In vitro Vero cells culture showed similar pattern for cysts and tachyzoites and reactivation of cyst in vitro was not influenced by the age of the brain cysts.
The protozoan parasite Eimeria tenella has a complex life cycle that includes two major asexual developmental stages, the merozoite and the sporozoite. The expressed sequence tag (EST) approach has been previously used to study gene expression of merozoites. We report here the generation and analysis of 556 ESTs from sporozoites. Comparative analyses of the two datasets reveal a number of transcripts that are preferentially expressed in a specific stage, including previously uncharacterised sequences. The data presented indicate the invaluable potential of the comparative EST analysis for providing information on gene expression patterns in the different developmental stages of E. tenella.
BmR1 recombinant antigen has previously been shown to demonstrate high sensitivity and specificity in the serological diagnosis of brugian filariasis in humans. In this study, the pattern of recognition of antibody to BmR1 during Brugia malayi infection was investigated by employing Meriones unguiculatus as the experimental model. Thirty two gerbils were infected subcutaneously with 120 L(3); and two control groups each comprising 25 animals were employed. ELISA using BmR1 was used to detect filaria-specific IgG antibodies elicited by the gerbils; using sera collected from the day 1 until day 150 post-inoculation (p.i.). The results showed that BmR1 detected B. malayi infection in gerbils harboring adult worms irrespective of the presence of circulating microfilaria, and was exemplified by positive ELISA results in nine a microfilaraemic animals that harbored live adult worms. The initial time of the antibody recognition was at day 8 p.i. and the antibody titre showed some correlation with adult worm burden.
A cross-sectional study was carried out to identify species and determine the prevalence of Cryptosporidium sp. shedding in pre-weaned and post-weaned dairy calves and to identify management factors that may be contributing to disease. A total of 240 calf faecal samples were collected from 16 farms in two districts in Johor, Malaysia, and screened by PCR. The overall Cryptosporidium prevalence was 27.1%. The prevalence of Cryptosporidium species in pre-weaned calves was 32.4% for C. parvum, 26.5% for C. bovis, followed by C. andersoni (20.6%), C. ryanae (11.8%) and mixed sp. (8.8%). The prevalence of Cryptosporidium species in post-weaned calves was 35% for C. bovis followed by C. andersoni and C. ryanae (30% each) and mixed sp. (5%). Subtyping analysis of 8 of the 11 C. parvum isolates at the gp60 locus identified five isolates as IIdA15G1, one as IIa18A3R1 and two isolates as IIa17G2R1. Management factors that increased the risk of Cryptosporidium infection included having other cattle farms close by, feeding calves with saleable milk, keeping pre-weaned calves in pens with slatted floors and keeping post-weaned calves in pens with a sand floor.
The presently reported study provides a detailed morphological description of the female and the male of a new species of the genus Parabrachiella-Parabrachiella jarai sp. nov. The parasites were sampled from marine fish, silver sillago, Sillago sihama (Perciformes: Sillaginidae), captured in Malaysia in 1994 and Hong Kong in 1995. The new species bears some resemblance to Parabrachiella lata (Song et Chen, 1976) but differs from it in details of second antenna, mandible, and maxilliped. The genus Parabrachiella currently covers 67 species including those recently transferred from Neobrachiella Kabata, 1979. An amended generic diagnosis is proposed for Parabrachiella and Thysanote. Some members of Parabrachiella are herewith transferred to Thysanote and some Thysanote are now placed in Parabrachiella.
The gene encoding the 195,000-Da major merozoite surface antigen (gp195) of the FUP (Uganda-Palo Alto) isolate of Plasmodium falciparum, a strain widely used for monkey vaccination experiments, has been cloned and sequenced. The translated amino acid sequence of the FUP gp195 protein is closely related to the sequences of corresponding proteins of the CAMP (Malaysia) and MAD-20 (Papua New Guinea) isolates and more distantly related to those of the Wellcome (West Africa) and K1 (Thailand) isolates, supporting the proposed allelic dimorphism of gp195 within the parasite population. The prevalence of dimorphic sequences within the gp195 protein suggests that many gp195 epitopes would be group-specific. Despite the extensive differences in amino acid sequence between gp195 proteins of these two groups, the hydropathy profiles of proteins representative of both groups are very similar. The conservation of overall secondary structure shown by the hydropathy profile comparison indicates that gp195 proteins of the various P. falciparum isolates are functionally equivalent. This information on the primary structure of the FUP gp195 protein will enable us to evaluate the possible roles of conserved, group-specific and variable epitopes in immunity to the blood stage of the malaria parasite.
Malaria remains one of the world's most important infectious diseases and is responsible for enormous mortality and morbidity. Human infection with Plasmodium knowlesi is widely distributed in Southeast Asia. Merozoite surface protein-1₁₉ (MSP-1₁₉), which plays an important role in protective immunity against asexual blood stage malaria parasites, appears as a leading immunogenic antigen of Plasmodium sp. We evaluated the sensitivity and specificity of recombinant P. knowlesi MSP-1₁₉ (rMSP-1₁₉) for detection of malarial infection. rMSP-1₁₉ was expressed in Escherichia coli expression system and the purified rMSP-1₁₉ was evaluated with malaria, non-malaria and healthy human serum samples (n = 215) in immunoblots. The sensitivity of rMSP-1₁₉ for detection of P. knowlesi, Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale infection was 95.5%, 75.0%, 85.7% and 100%, respectively. rMSP-1₁₉ did not react with all the non-malaria and healthy donor sera, which represents 100% specificity. The rMSP-1₁₉ could be used as a potential antigen in serodiagnosis of malarial infection in humans.
We here describe the novel finding that brain endothelial cells in vitro can stimulate the growth of Plasmodium falciparum through the production of low molecular weight growth factors. By using a conditioned medium approach, we show that the brain endothelial cells continued to release these factors over time. If this mirrors the in vivo situation, these growth factors potentially would provide an advantage, in terms of enhanced growth, for sequestered parasitised red blood cells in the brain microvasculature. We observed this phenomenon with brain endothelial cells from several sources as well as a second P. falciparum strain. The characteristics of the growth factors included: <3 kDa molecular weight, heat stable, and in part chloroform soluble. Future efforts should be directed at identifying these growth factors, since blocking their production or actions might be of benefit for reducing parasite load and, hence, malaria pathology.
To accelerate the discovery of novel leads for the treatment of Human African Trypanosomiasis (HAT), it is necessary to have a simple, robust and cost-effective assay to identify positive hits by high throughput whole cell screening. Most of the fluorescence assay was made in black plate however in this study the HTS assay developed in 384-well format using clear plate and black plate, for comparison. The HTS assay developed is simple, sensitive, reliable and reproducible in both types of plates. Assay robustness and reproducibility were determined under the optimized conditions in 384-well plate was well tolerated in the HTS assay, including percentage of coefficient of variation (% CV) of 4.68% and 4.74% in clear and black 384-well plate, signal-to-background ratio (S/B) of 12.75 in clear 384-well plate and 12.07 in black 384-well plate, Z' factor of 0.79 and 0.82 in clear 384-well plate and black 384-well plate, respectively and final concentration of 0.30% dimethylsulfoxide (DMSO) in both types of plate. Drug sensitivity was found to be comparable to the reported anti-trypanosomal assay in 96-well format. The reproducibility and sensitivity of this assay make it compliant to automated liquid handler use in HTS applications.
Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS-PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ∼14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band.
The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n=20), acute (IgM positive, IgG positive; n=20) and chronic (IgM negative, IgG positive; n=20) toxoplasmosis patients, and toxoplasmosis negative control patients (n=20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients' serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P<0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.
Plasmodium knowlesi is a malaria parasite of Old World monkeys and is infectious to humans. In this study Macaca fascicularis was used as a model to understand the host response to P. knowlesi using parasitological and haematological parameters. Three M. fascicularis of either sex were experimentally infected with P. knowlesi erythrocytic parasites from humans. The pre-patent period for P. knowlesi infection in M. fascicularis ranged from seven to 14 days. The parasitemia observed was 13,686-24,202 parasites per microL of blood for asexual stage and 88-264 parasites per microL of blood for sexual stage. Periodicity analysis adopted from microfilaria periodicity technique of asexual stage showed that the parasitemia peak at 17:39h while the sexual stage peaked at 02:36 h. Mathematical analysis of the data indicates that P. knowlesi gametocytes tend to display periodicity with a peak (24:00-06:00) that coincides with the peak biting activity (19:00-06:00) of the local vector, Anopheles latens. The morphology of P. knowlesi resembled P. falciparum in early trophozoite and P. malariae in late trophozoite. However, it may be distinguishable by observing the appliqué appearance of the cytoplasm and the chromatin lying inside the ring. Haematological analysis on macaques with knowlesi malaria showed clinical manifestations of hypoglycaemia, anaemia and hyperbilirubinemia. Gross examination of spleen and liver showed malaria pigments deposition in both organs.
Giardia duodenalis is an important intestinal protozoan in Yemen with infection rates ranging from 18% to 27%. To date, there has been no genotyping study to provide a better understanding of the transmission dynamic. This study was conducted to genotype and subtype G. duodenalis in Yemen. Stool samples were collected from 503 Yemeni outpatients between 1 and 80 years old, including 219 males and 284 females. Giardia cysts were detected via microscopy after the formal-ether concentration. Genotyping of Giardia was carried out using PCR and sequence analysis of the 16s rRNA and b-giardin genes. Of the 89 microscopy-positive Giardia samples, 65 were successfully sequenced, of which 66% (43 of 65) were identified as G. duodenalis assemblage A and 34% (22 of 65) as assemblage B. Further subtyping analysis based on b-giardin gene identified the presence of subtypes A2 and A3, which belong to the anthroponotic sub-assemblage AII. Data of the study suggest that anthroponotic transmission played a potential role in the transmission of giardiasis in the community. However, further genotyping and subtyping studies of specimens from humans and animals living in the same households are needed for a more definitive understanding of giardiasis transmission in Yemen.
Chloroquine (CQ) and mefloquine (MQ) are no longer potent antimalarial drugs due to the emergence of resistant Plasmodium falciparum. Combination therapy has become the standard for many regimes in overcoming drug resistance. Roxithromycin (ROM), a known p-glycoprotein inhibitor, is reported to have antimalarial activity and it is hoped it will potentiate the effects of both CQ/MQ and reverse CQ/MQ-resistance. We assayed the effects of CQ and MQ individually and in combination with ROM on synchronized P. falciparum (Dd2 strain) cultures. The IC(50) values of CQ and MQ were 60.0+/-5.0 and 16.0+/-3.0 ng/ml; these were decreased substantially when combined with ROM. Isobolograms indicate that CQ-ROM combinations were relatively more synergistic (mean FICI 0.70) than MQ-ROM (mean FICI 0.85) with their synergistic effect at par with CQ-verapamil (VRP) (mean FICI 0.64) and MQ-VRP (mean FICI 0.60) combinations. We conclude that ROM potentiates the CQ/MQ response on multidrug-resistant P. falciparum.
Acanthamoeba is an opportunistic protist pathogen that is responsible for serious human and animal infection. Being one of the most frequently isolated protists from the environment, it is likely that it readily encounters microaerophilic environments. For respiration under anaerobic or low oxygen conditions in several amitochondriate protists, decarboxylation of pyruvate is catalyzed by pyruvate ferredoxin oxidoreductase instead of pyruvate dehydrogenase. In support, Nitazoxanide, an inhibitor of pyruvate ferredoxin oxidoreductase, is effective and non-mutagenic clinically against a range of amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. The overall aim of the present study was to determine in vitro efficacy of Nitazoxanide against Acanthamoeba castellanii. At micromolar concentrations, the findings revealed that Nitazoxanide neither affected A. castellanii growth or viability nor amoeba-mediated host cell monolayer damage in vitro or extracellular proteolytic activities. Similarly, microaerophilic conditions alone had no significant effects. In contrast, microaerophilic conditions together with Nitazoxanide showed amoebicidal effects and inhibited A. castellanii-mediated host cell monolayer damage as well as extracellular proteases. Using encystation assays, it was observed that Nitazoxanide inhibited trophozoite transformation into cysts both under aerophilic and microaerophilic conditions. Furthermore, pre-treatment of cysts with Nitazoxanide inhibited A. castellanii excystation. These findings are important in the identification of potential targets that could be useful against parasite-specific respiration as well as to understand the basic biology of the life cycle of Acanthamoeba.