Seroprevalence of Schistosoma japonicum Infection Among Dogs and Water Buffaloes Using Recombinant Antigen ELISA in New Corella, Davao del Norte, Philippines.

PURPOSE: Animal reservoirs are important targets for controlling and eliminating schistosomiasis. Prevalence studies showed that dogs (Canis familiaris) and water buffaloes (Bubalus bubalis) are important reservoirs of S. japonicum. Previous studies have validated the use of the recombinant proteins Sj1TR, Sj7TR, and SjTPx-1 in ELISA as diagnostics for dogs and water buffaloes from schistosomiasis-endemic areas. In this study, we aimed to determine the seroprevalence of S. japonicum among dogs and water buffaloes in New Corella, Davao del Norte, Philippines, using the recombinant proteins Sj1TR, Sj7TR, and SjTPx-1 in ELISA format. METHODS: Fecal and serum samples were collected from dogs (n = 63) and water buffaloes (n = 57). Schistosome-positive samples were detected using formalin-ether concentration technique (FECT), stool polymerase chain reaction, and enzyme-linked immunosorbent assay (ELISA) using soluble egg antigen (SEA), rSj1TR, rSj7TR, and rSjTPx-1. Positivity rates, sensitivity, specificity, predictive values, accuracy, and kappa values were calculated. RESULTS: Mean positivity rates for schistosome infection were high for both dogs (x = 15.40%) and water buffaloes (x = 6.32%). On dogs, the sensitivity and specificity of the tests are as follows: 66.67% and 100% for rSj7TR-ELISA, while 100% and 93.33% for rSjTPx-1-ELISA, respectively. rSjTPx-1-ELISA showed the highest agreement with stool PCR among all diagnostic tests, with an overall kappa value of 0.824. On water buffaloes, the sensitivity and specificity of both rSj1TR-ELISA and rSjTPx-1-ELISA are 100.0% and 98.15%. Both tests also had an overall kappa value of 0.84. CONCLUSION: To ensure elimination and prevention of schistosomiasis in humans, the use of validated serological diagnostics such as recombinant antigen ELISA is preferable for field detection in animals, especially in resource-limited areas.

Malarial Antibody Detection with an Engineered Yeast Agglutination Assay.

Malaria, a disease caused by the Plasmodium parasite carried by Anopheles mosquitoes, is commonly diagnosed by microscopy of peripheral blood smears and with rapid diagnostic tests. Both methods show limited detection of low parasitemia that may maintain transmission and hinder malaria elimination. We have developed a novel agglutination assay in which modified Saccharomyces cerevisiae cells act as antigen-displaying bead-like particles to capture malaria antibodies. The Epidermal Growth Factor-1 like domain (EGF1) of the Plasmodium falciparum merozoite surface protein-1 (PfMSP-119) was displayed on the yeast surface and shown to be capable of binding antimalaria antibodies. Mixed with a second yeast strain displaying the Z domain of Protein A from Staphylococcus aureus and allowed to settle in a round-bottomed well, the yeast produce a visually distinctive agglutination test result: a tight "button" at a low level of malarial antibodies, and a diffuse "sheet" when higher antibody levels are present. Positive agglutination results were observed in malaria-positive human serum to a serum dilution of 1:100 to 1:125. Since the yeast cells are inexpensive to produce, the test may be amenable to local production in regions seeking malaria surveillance information to guide their elimination programs.

Serological evaluation of the schistosome's secretory enzyme phytochelatin synthase and phosphoglycerate mutase for the detection of human Schistosoma japonicum infection.

Secretory enzymes from Schistosoma japonicum are promising candidate antigens in the diagnosis of schistosomiasis. Our previous studies have proven that thioredoxin peroxidase-1 (SjTPx-1) is useful for the detection of this parasitic disease in humans, water buffaloes, and dogs. In this study, we evaluated two more secretory enzymes namely phosphoglycerate mutase (SjPGM) and phytochelatin synthase (SjPCS) with SjTPx-1 as the reference antigen. SjPGM was shown to have good diagnostic potentials in animal samples in previous studies, whereas SjPCS was chosen because of its absence in the mammalian hosts. Serum samples including 96 endemic negative controls, 107 schistosomiasis japonica positive samples, and 31 samples positive for other parasitic trematode infections (Clonorchis sinensis, Opisthorchis viverrini, Paragonimus westermani) were tested with the antigens using enzyme-linked immunosorbent assay. Results showed that SjPCS detected more positive samples and had fewer cross-reactions than SjPGM. With 85.05% sensitivity and 93.55% specificity, SjPCS can therefore be used in the detection of human schistosomiasis.

Field Evaluation of Recombinant Antigen ELISA in Detecting Zoonotic Schistosome Infection Among Water Buffaloes in Endemic Municipalities in the Philippines.

In this study, we investigated the use of recombinant antigens thioredoxin peroxidase-1 (rSjTPx-1) and tandem repeat rSj1TR in evaluating the antibody positivity rates of Schistosoma japonicum infection among water buffaloes from four endemic areas in the Philippines, two municipalities with high endemicity (Calatrava, Negros Occidental and Catarman, Northern Samar) and two municipalities nearing elimination with no cases of human schistosomiasis (Talibon and Trinidad, Bohol). These recombinant antigen ELISA assays were compared with other diagnostic tests including SEA-ELISA, FECT, and fecal-based PCR. Results showed that rSj1TR-ELISA has the highest agreement with PCR in all study areas. Furthermore, significant positivity rates among water buffaloes were seen in Talibon and Trinidad, indicating that water buffaloes are maintaining the schistosome parasites in transmission areas even in the absence of human infection. Hence, serological assay using a more sensitive and specific rSj1TR-ELISA can be used for animal surveillance to prevent emergence and re-emergence of human schistosomiasis.

Behind Enemy Lines: Immunomodulatory Armamentarium of the Schistosome Parasite.

The deeply rooted, intricate relationship between the Schistosoma parasite and the human host has enabled the parasite to successfully survive within the host and surreptitiously evade the host's immune attacks. The parasite has developed a variety of strategies in its immunomodulatory armamentarium to promote infection without getting harmed or killed in the battlefield of immune responses. These include the production of immunomodulatory molecules, alteration of membranes, and the promotion of granuloma formation. Schistosomiasis thus serves as a paradigm for understanding the Th2 immune responses seen in various helminthiases. This review therefore aims to summarize the immunomodulatory mechanisms of the schistosome parasites to survive inside the host. Understanding these immunomodulatory strategies not only provides information on parasite-host interactions, but also forms the basis in the development of novel drugs and vaccines against the schistosome infection, as well as various types of autoimmune and inflammatory conditions.

Schistosoma japonicum cathepsin B as potential diagnostic antigen for Asian zoonotic schistosomiasis.

In this study, the diagnostic value of Schistosoma japonicum cathepsin B (SjCatB) was evaluated as an antigen for the early detection of S. japonicum infection. SjCatB is a key protease used by the cercaria to penetrate the intact skin of the host for transdermal infection. The early exposure of the host's immune system to this enzyme may elicit early production of antibodies against this molecule. Therefore, the recombinant SjCatB (rSjCatB) was expressed in Escherichia coli with N-terminal 6xHis-tag. rSjCatB was tested for its performance as a diagnostic antigen using indirect enzyme-linked immunosorbent assay (ELISA) with sera from experimentally infected mice collected at > 8 weeks post-infection. Showing 100% sensitivity and 95.0% specificity in the ELISA, rSjCatB was then evaluated with sera from experimentally infected mice collected at 1-7 weeks post-infection to determine how early the antibodies can be detected. Results showed that as early as 6 weeks post-infection, 2 of the 3 infected mice were found to be positive with the antibodies against SjCatB. Furthermore, the potential of the recombinant antigen in detecting human schistosomiasis was evaluated with archived serum samples collected from individuals who had been diagnosed with S. japonicum infection by stool examination. Results showed 86.7% sensitivity and 96.7% specificity suggesting its high diagnostic potential for human schistosomiasis. In addition, SjCatB showed minimal cross-reaction with the sera collected from patients with other parasitic diseases. In conclusion, the results of this study suggest that SjCatB will be useful in the development of a sensitive and specific early detection test for S. japonicum infection.

Detection of canine Schistosoma japonicum infection using recombinant thioredoxin peroxidase-1 and tandem repeat proteins.

Humans and dogs live very close together and share various pathogens causing zoonotic parasitoses like schistosomiasis. A previous population genetics study done for schistosomes in the Philippines suggested that there is a high transmission level of Schistosoma japonicum among humans and dogs proving that the latter are important reservoirs for this zoonotic parasite. A more sensitive and specific test detecting schistosome infection in dogs will therefore strengthen the zoonotic surveillance, which might help in the possible elimination of this ancient disease. In this study, recombinant thioredoxin peroxidase-1 (SjTPx-1) and tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, Sj7TR) previously tested on human and water buffalo samples were used to assess its diagnostic applicability to dogs. Fifty-nine dog serum and stool samples were collected in the schistosomiasis-endemic municipalities of Calatrava, Negros Occidental and Catarman, Northern Samar in the Philippines and examined using the ELISA as compared to microscopy and fecal sample-based PCR. Samples positive for Babesia gibsoni and Dirofilaria immitis were also used to check for cross-reaction. Results showed that SjTPx-1 (80% sensitivity, 92.3% specificity) and Sj7TR (73.3% sensitivity, 92.3% specificity) have good potentials for diagnosing S. japonicum infection in dogs. These diagnostic antigens will therefore improve the surveillance in the transmission of the parasites from dogs to humans.

Integrative taxonomic approach of trypanosomes in the blood of rodents and soricids in Asian countries, with the description of three new species.

Trypanosoma lewisi (Kinetoplastea: Trypanosomatida: Trypanosomatidae) with a cosmopolitan distribution is the type species of the subgenus Herpetosoma, which includes ca. 50 nominal species isolated mainly from rodents. Since members of Herpetosoma in different host species have an almost identical morphology of bloodstream forms, these trypanosomes are referred to as 'T. lewisi-like', and the molecular genetic characterization of each species is necessary to verify their taxonomy. In the present study, we collected blood samples from 89 murid rodents of 15 species and 11 soricids of four species in Indonesia, Philippines, Vietnam, Taiwan, and mainland China for the detection of hemoprotozoan infection. T. lewisi and T. lewisi-like trypanosomes were found in the blood smears of 10 murid animals, which included Bandicota indica (two rats), Rattus argentiventer (one rat), and Rattus tiomanicus (two rats) in Indonesia; Rattus rattus (one rat) in the Philippines; and Niviventer confucianus (four rats) in mainland China. Furthermore, large- or medium-sized non-T. lewisi-like trypanosomes were detected in two soricids, Crocidura dracula in Vietnam and Anourosorex yamashinai in Taiwan, respectively. Molecular genetic characterization of the small subunit (SSU) ribosomal RNA gene (rDNA) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene indicated that the trypanosomes from all the murid hosts had identical SSU rDNA or gGAPDH gene nucleotide sequences except for those in N. confucianus in mainland China. These N. confucianus-infecting trypanosomes also showed several unique morphological features such as smaller bodies, anteriorly positioned nuclei, and larger rod-shaped kinetoplasts when compared with T. lewisi trypomastigotes. Trypanosoma (Herpetosoma) niviventerae n. sp. is erected for this new species. Similarly, based on morphological and molecular genetic characterization, Trypanosoma sapaensis n. sp. and Trypanosoma anourosoricis n. sp. are proposed for the trypanosomes in C. dracula in Vietnam and A. yamashinai in Taiwan, respectively. More effort directed toward the morphological and molecular genetic characterization of the trypanosomes of rodents and soricids is required to fully understand the real biodiversity of their hemoflagellates.

Evaluation of Schistosoma japonicum thioredoxin peroxidase-1 as a potential circulating antigen target for the diagnosis of Asian schistosomiasis.

Asian schistosomiasis caused by Schistosoma japonicum is a serious zoonotic disease endemic in China, the Philippines and parts of Indonesia. Mass drug administration in endemic areas resulted to decline in disease severity and intensity. The low intensity of infection limits the use of current parasitological methods for schistosomiasis diagnosis. Detection of parasite circulating antigens might provide more informative result as it may indicate the true status of infection. In this study, S. japonicum thioredoxin peroxidase-1 (SjTPx-1) a 22 kDa secreted antioxidant enzyme expressed throughout the life stages of the parasite was evaluated for its potential use as a biomarker for schistosomiasis japonica infection. Rabbit polyclonal antibody and mouse monoclonal antibodies (mAbs) were raised against the recombinant SjTPx-1 (rSjTPx-1). The antibodies produced against the recombinant antigen was confirmed to detect the native SjTPx-1 in crude adult worm lysate. Likewise, the specific binding of mAbs to parasite TPx-1 and not to mammalian peroxiredoxin-1 orthologues was also confirmed. The double antibody sandwich ELISA developed in this study was able to detect at least 1 ng/ml of rSjTPx-1. In addition, this method was able to detect the antigen from all serum samples of experimentally infected rabbit and mice. The diagnostic potential of SjTPx-1 in human clinical samples was also evaluated, in which 4 out of 10 stool-confirmed serum samples had detectable levels of the antigen. The results suggest that SjTPx-1 can be a potential biomarker for Asian zoonotic schistosomiasis.

Geographic strain differentiation of Schistosoma japonicum in the Philippines using microsatellite markers.

BACKGROUND: Microsatellites have been found to be useful in determining genetic diversities of various medically-important parasites which can be used as basis for an effective disease management and control program. In Asia and Africa, the identification of different geographical strains of Schistosoma japonicum, S. haematobium and S. mansoni as determined through microsatellites could pave the way for a better understanding of the transmission epidemiology of the parasite. Thus, the present study aims to apply microsatellite markers in analyzing the populations of S. japonicum from different endemic areas in the Philippines for possible strain differentiation. METHODOLOGY/ PRINCIPAL FINDINGS: Experimental mice were infected using the cercariae of S. japonicum collected from infected Oncomelania hupensis quadrasi snails in seven endemic municipalities. Adult worms were harvested from infected mice after 45 days of infection and their DNA analyzed against ten previously characterized microsatellite loci. High genetic diversity was observed in areas with high endemicity. The degree of genetic differentiation of the parasite population between endemic areas varies. Geographical separation was considered as one of the factors accounting for the observed difference between populations. Two subgroups have been observed in one of the study sites, suggesting that co-infection with several genotypes of the parasite might be present in the population. Clustering analysis showed no particular spatial structuring between parasite populations from different endemic areas. This result could possibly suggest varying degrees of effects of the ongoing control programs and the existing gene flow in the populations, which might be attributed to migration and active movement of infected hosts from one endemic area to another. CONCLUSIONS/ SIGNIFICANCE: Based on the results of the study, it is reasonable to conclude that genetic diversity could be one possible criterion to assess the infection status in highly endemic areas. Genetic surveillance using microsatellites is therefore important to predict the ongoing gene flow and degree of genetic diversity, which indirectly reflects the success of the control program in schistosomiasis-endemic areas.