Jose Ma. M. Angeles

Utilization of ELISA using thioredoxin peroxidase-1 and tandem repeat proteins for diagnosis of Schistosoma japonicum infection among water buffaloes.

Background The presence of animal reservoirs in Schistosoma japonicum infection has been a major obstacle in the control of schistosomiasis. Previous studies have proven that the inclusion of control measures on animal reservoir hosts for schistosomiasis contributed to the decrease of human cases. Animal surveillance should therefore be included to strengthen and improve the capabilities of current serological tests. Methodology/Principal Findings Thioredoxin peroxidase-1 (SjTPx-1) and four tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, Sj7TR) were initially evaluated against human sera. The previous test showed high sensitivity and specificity for antibody detection against SjTPx-1 and Sj7TR. In this study, the immunodiagnostic potential of these recombinant proteins was evaluated using enzyme-linked immunoassay on 50 water buffalo serum samples collected in Cagayan, the Philippines as compared with the soluble egg antigen (SEA). For specificity, 3 goat serum samples positive with Fasciola hepatica were used and among the antigens used, only SEA showed cross-reaction. Stool PCR targeting the S. japonicum 82 bp mitochondrial NAD 1 gene was done to confirm the true positives and served as the standard test. Twenty three samples were positive for stool PCR. SjTPx-1 and Sj1TR gave the highest sensitivity among the recombinant proteins tested for water buffalo samples with 82.61% and 78.26% respectively which were higher than that of SEA (69.57%). Conclusions/Significance These results prove that SjTPx-1 works both for humans and water buffaloes making it a good candidate antigen for zoonotic diagnosis. Sj1TR showed good results for water buffaloes and therefore can also be used as a possible candidate for detecting animal schistosome infection.

Human Antibody Response to Thioredoxin Peroxidase-1 and Tandem Repeat Proteins as Immunodiagnostic Antigen Candidates for Schistosoma japonicum Infection

Schistosomiasis continues to be a public health problem in many tropical and subtropical countries. Improving the diagnostic tools for surveillance and monitoring in areas that have reached elimination level will help hasten the possible elimination of this disease. This study therefore aims to develop enzyme-linked immunosorbent assay through the use of recombinant proteins such as thioredoxin peroxidase-1 (SjTPx-1) and four tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, and Sj7TR). Cutoff values were calculated using 38 serum samples from healthy Japanese volunteers. Sera from 35 schistosomiasis-confirmed patients, four cured from the disease by chemotherapy, and 15 endemic negative controls were used to assess these antigens. SjTPx-1 and Sj7TR both had 85.71% sensitivity. Furthermore, these antigens were also tested against human sera positive for other parasitic infections and showed no or very minimal cross-reaction. These results suggest the potential defined antigens for development of an accurate diagnostic test for schistosomiasis.

Plasmodium vivax and Plasmodium knowlesi: cloning, expression and functional analysis of 1-Cys peroxiredoxin.

Malaria parasites like other aerobes need to detoxify the reactive oxygen species (ROS) that are mainly produced from hemoglobin degradation in the food vacuole. Since Plasmodium lacks catalase and genuine glutathione peroxidase, they are highly dependent on peroxiredoxins (Prxs) and superoxide dismutases for ROS detoxification. Prxs are protective antioxidant enzymes that act through reduction of hydrogen peroxides. In recent years, several studies have been done on Prx family of human malaria parasites mainly on Plasmodium falciparum but not much on the other human malaria species. In this study 1-Cys peroxiredoxin (1-Cys-Prx) from Plasmodium vivax and Plasmodium knowlesi were cloned and characterized. The complete genes coding for 1-Cys-Prx of P. vivax (Pv1-Cys-Prx) and P. knowlesi (Pk1-Cys-Prx) were PCR amplified and the recombinant proteins were produced by heterologous over-expression in Escherichia coli. Both recombinant proteins showed antioxidant activity with the mixed function oxidation assay. Using specific polyclonal antibodies, it was indicated that Pv1-Cys-Prx and Pk1-Cys-Prx are expressed in the cytoplasm of the parasite. Altogether, the results suggested that 1-Cys-Prxs protect the parasites from oxidative damages.

Cloning and characterization of Plasmodium vivax thioredoxin peroxidase-1

Reactive oxygen species produced from hemoglobin digestion and the host immune system could have adverse effects on malaria parasites. To protect themselves, malaria parasites are highly dependent on the antioxidant enzymes, including superoxide dismutases and thioredoxin-dependent peroxidases. To date, several thioredoxin peroxidases (TPx) have been characterized in Plasmodium falciparum, but the TPx in Plasmodium vivax has not yet been characterized. The complete sequence of gene coding for thioredoxin peroxidase-1 of P. vivax (PvTPx-1) was amplified by PCR and cloned. Using the recombinant PvTPx-1 (rPvTPx-1), polyclonal antibody was produced in mice for immunolocalization of the enzyme in the parasite. The antioxidant activity of rPvTPx-1 was evaluated by mixed-function oxidation assay. PvTPx-1 has two conserved cysteine residues in the amino acid sequence at the positions 50 and 170 which formed a dimer under a non-reducing condition. Using a thiol mixed-function oxidation assay, the antioxidant activity of rPvTPx-1 was revealed. Indirect immunofluorescence microscopy with the specific antibody indicated that PvTPx-1 was expressed in the cytoplasm of the erythrocytic stage of the parasite in a dots-like pattern. The results suggest that P. vivax uses TPx-1 to reduce and detoxify hydrogen peroxides in order to maintain their redox homeostasis and proliferation in the host body.

Establishment of ATP-Based Luciferase Viability Assay in 96-Well Plate for Trypanosoma congolense

ABSTRACT Animal African trypanosomosis (AAT), caused by Trypanosoma congolense, is widespread throughout sub-Saharan Africa. There are significant concerns related to the current drugs available for the treatment of AAT due to their limited effectiveness across species and their adverse effects. Moreover, drug resistant trypanosomes have recently been reported in the field. High throughput screening (HTS) of large chemical compound library collections is a promising approach for identifying novel drug candidates. While HTS for Trypanozoon trypanosomes, T. brucei sspp. and T. evansi is well established, no assays have been developed for T. congolense. In the present study, the authors developed an ATP-based luciferase viability assay for T. congolense in a 96-well plate format. The calculated 50% inhibitory concentration (IC50) values for pentamidine and diminazene were 10–100 times higher in T. congolense than in T. brucei. This result suggests that the transporters for the 2 tested compounds differ between T. congolense and T. brucei. This assay could further be applied to screen novel chemical compounds for the treatment of AAT caused by T. congolense.

Water buffalo as sentinel animals for schistosomiasis surveillance

Introduction About 75% of human pathogens are zoonotic, meaning that they are communicated from animals to humans. (1) Schistosomiasis is a zoonotic disease with a complex transmission cycle involving aquatic snails and least 40 species of mammals, which serve as reservoir hosts. Worldwide, more than 700 million people are at risk of schistosomiasis and over 240 million people are infected with the parasite. (2) Schistosomiasis has been eliminated in Japan and the coastal plains of China by a combination of medical treatment, health education, improved water quality and sanitation and snail control through environmental modification, molluscicide and new farming methods. The World Health Organization (WHO) targets elimination of schistosomiasis in the Eastern Mediterranean, the Caribbean, Indonesia and the Mekong river basin by 2015, and in the Western Pacific, the Americas and selected African countries by 2020. (3) Here we argue that elimination guidelines for schistosomiasis should include surveillance of the animal reservoir. Transmission patterns Mapping of the transmission patterns in humans and animals can lead to a better understanding of transmission of schistosomiasis between different host species. Parts of China, Indonesia and the Philippines are endemic for schistosomiasis caused by Schistosoma japonicum. Cattle, water buffalo, goats, dogs, pigs and rats are potentially important reservoir hosts for this parasite because of their contact with humans. Other animals including cats and horses are not significant contributors because of their limited contact with contaminated water. (4) Grazing ruminants including goats, cattle and water buffalo are exposed to the parasite in transmission sites such as rice paddies. However, goats do not contribute much to transmission as they are uncommon in areas where schistosomiasis is endemic. Cattle and water buffalo are the major animal reservoirs contributing to human transmission of S. japonicum. (4-7) In China, bovine defecation is thought to be the main cause of environmental contamination. (6) Molecular studies have also suggested that transmission is through bovines rather than other domesticated animals. Cattle are less capable of recovering from infection than water buffalo. (8) Despite the fact that buffalo are capable of clearing the parasite, they are a major reservoir host because they are repeatedly exposed to the parasite when pulling ploughs in rice paddies. (9) In rice-growing regions, water buffalo are a good choice of sentinel animal. In Anhui and Sichuan provinces of China, re-emergence of schistosomiasis in humans was attributed to the high prevalence of schistosome infection among cows and water buffalo. (10,11) These human infections could be avoided by setting up a sensitive animal surveillance system to detect infection in animals and, by treating them promptly, preventing further contamination of the environment. Diagnostic tests Microscopic examination of stool samples is a standard test for several parasitic diseases, including schistosomiasis. However, microscopy has low sensitivity in diagnosing schistosomiasis among water buffalo due to the large volume of bovine excreta. Therefore, a more sensitive and specific diagnostic test is needed to identify schistosomiasis-free areas and prevent re-emergence of the disease. In previous studies, polymerase chain reaction (PCR) (12) and recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) (13) have been found to be useful in identifying S. japonicum in stool samples. Surveillance can also be carried out in abattoirs where cattle and buffalo are slaughtered. Documentation of the original source of livestock then allows schistosome-infested sites to be located. Medical and veterinary approaches The complexity of zoonotic schistosomiasis transmission has been a major hindrance to elimination of the disease. …

Plasmodium knowlesi thioredoxin peroxidase 1 binds to nucleic acids and has RNA chaperone activity.

Malaria parasites are under oxidative attack throughout their life cycle in human body and mosquito vector. Therefore, Plasmodium antioxidant defenses are crucial for its survival and being considered as interesting target for antimalarial drug design. Plasmodium knowlesi has emerged recently from its simian host to human in Southeast Asia and has been recognized as the fifth Plasmodium species that can cause human malaria. In this study, we cloned and characterized thioredoxin peroxidase 1 from P. knowlesi (PkTPx-1). PkTPx-1 gene was cloned, and recombinant protein was produced by heterologous overexpression in Escherichia coli. The recombinant protein was used for evaluation of enzymatic activity and polyclonal antibody production. Using the recombinant PkTPx-1 protein, its antioxidant activity was confirmed in a mixed-function oxidation assay where PkTPx-1 prevented nicking of DNA by hydroxyl radicals. PkTPx-1 was able to bind to double-strand DNA and RNA and had RNA chaperone activity in a nucleic acid melting assay indicating new function of PkTPx-1 other than antioxidant activity. Using specific polyclonal antibodies, it was indicated that PkTPx-1 is expressed in the cytoplasm of the parasite. Altogether, these results suggest that PkTPx-1 not only protects the parasite from the adverse effects of reactive oxygen species but also has RNA chaperone activity.

Effect of thioredoxin peroxidase-1 gene disruption on the liver stages of the rodent malaria parasite Plasmodium berghei.

Phenotypic observation of thioredoxin peroxidase-1 (TPx-1) gene-disrupted Plasmodium berghei (TPx-1 KO) in the liver-stage was performed with an in vitro infection system in order to investigate defective liver-stage development in a mouse infection model. Indirect immunofluorescence microscopy assay with anti-circumsporozoite protein antibody revealed that in the liver schizont stage, TPx-1 KO parasite cells were significantly smaller than cells of the wild-type parent strain (WT). Indirect immunofluorescence microscopy assay with anti-merozoite surface protein-1 antibody, which was used to evaluate late schizont-stage development, indicated that TPx-1 KO schizont development was similar to WT strain development towards the merozoite-forming stage (mature schizont). However, fewer merozoites were produced in the mature TPx-1 KO schizont than in the mature WT schizont. Taken together, the results suggest that TPx-1 may be involved in merozoite formation during liver schizont development.

Development of Monoclonal Antibodies That Target 1-Cys Peroxiredoxin and Differentiate Plasmodium falciparum from P. vivax and P. knowlesi

Prompt and accurate diagnosis of malarial patients is a crucial factor in controlling the morbidity and mortality of the disease. Effective treatment decisions require a correct diagnosis among mixed-species malarial patients. Differential diagnosis is particularly important in cases of Plasmodium vivax, a species that shares endemicity with P. falciparum in most endemic areas. Moreover, it is difficult to identify P. knowlesi on the basis of morphology alone, and rapid diagnostic tests are still not available for this malaria species. Therefore, the development of diagnostic tests applicable to the field is urgently needed. 1-Cys peroxiredoxin (1-Cys-Prx) in P. falciparum is abundantly expressed in the mature asexual stages, making it a promising candidate as a diagnostic antigen. In this study, we produced five monoclonal antibodies (mAbs) against P. falciparum 1-Cys-Prx (Pf1-Cys-Prx) by immunizing BALB/c mice with recombinant Pf1-Cys-Prx and subsequent hybridoma production. Cross reactivity of established mAbs with the orthologous molecule of Pf1-Cys-Prx in P. vivax (Pv1-Cys-Prx) and P. knowlesi (Pk1-Cys-Prx) was examined. Western blot analyses showed that three mAbs reacted with Pv1-Cys-Prx and Pk1-Cys-Prx but two mAbs did not. These results indicate that the two mAbs were effective in differentiating P. falciparum from P. vivax and P. knowlesi and could be used in differential diagnosis as well as comparative molecular studies of human Plasmodium species.

Development of monoclonal antibodies against Plasmodium falciparum thioredoxin peroxidase 1 and its possible application for malaria diagnosis

Rapid diagnostic tests (RDTs) have been considered as an ideal alternative for light microscopy to detect malaria parasites especially in remote areas. The development and improvement of RDTs is an area of intensive research in the last decade. To date, few parasite proteins have been targeted in RDTs which are known to have certain deficiencies and made the researchers to look for other promising candidates to address this problem. Plasmodium falciparum thioredoxin peroxidase 1 (PfTPx-1) is abundantly expressed in the cytoplasm of the parasite and well conserved across Plasmodium species, making this antigen a promising target for malaria diagnosis. Several monoclonal antibodies (mAbs) were produced against PfTPx-1. The binding affinities of mAbs were measured. Several immunochromatographic tests (ICTs) were developed using different combination of mAbs. All mAbs showed promising affinities to be used for diagnosis. The sensitivities of ICTs were evaluated using recombinant PfTPx-1 whose results lead us to the preparation of 4 different ICTs. These tests showed positive reaction with P. falciparum in vitro culture supernatant indicating the release of PfTPx-1 during schizont rupture. Altogether, these findings suggest that PfTPx-1 is a promising biomarker to diagnose P. falciparum infection. However, the diagnostic performance of this antigen should be further validated using clinical samples.