Proteomic and immunomic analysis of Schistosoma mekongi egg proteins.

Schistosomiasis remains a global health problem. In the Mekong river basin, approximately 80,000 people are at risk of infection by Schistosoma mekongi. The parasite's eggs become entrapped in the host's organs and induce massive inflammation, contributing to the pathogenesis of schistosomiasis. In addition, egg antigens are important in circumoval precipitin tests (COPTs) and other diagnostic techniques. Little is known regarding the egg proteins of S. mekongi, and so we applied immunoblotting and mass spectrometry-based proteomic approaches to study these proteins and their antigenicity. A total of 360 unique proteins were identified in S. mekongi eggs using proteomic analyses. The major protein components of S. mekongi eggs were classified into several groups by functions, including proteins of unknown function, structural proteins, and regulators of transcription and translation. The most abundant proteins in S. mekongi eggs were antioxidant proteins, potentially reflecting the need to neutralize reactive oxidative species released from host immune cells. Immunomic analyses revealed that only DNA replication factor Cdt1 and heat shock protein 70 overlap between the proteins recognized by sera of infected mice and humans, illustrating the challenges of knowledge transfer from animal models to human patients. Forty-one immunoreactive protein bands were recognized by either mouse or patient sera. Phosphoglycerate kinase, fructose-1,6-bisphosphate aldolase and elongation factor 1 appeared to be interesting immunogens of S. mekongi eggs as these proteins were recognized by polyclonal IgMs and IgGs in patient sera. Our findings provide new information on the protein composition of S. mekongi eggs as well as the beginnings of a S. mekongi immunogen dataset. These data may help us better understand the pathology of schistosomiasis as well as natural antibody responses against S. mekongi egg proteins, both of which may be useful in including S. mekongi to other schistosoma diagnostic, vaccine and immunotherapy development.

Excretory-secretory product of third-stage Gnathostoma spinigerum larvae induces apoptosis in human peripheral blood mononuclear cells.

Human gnathostomiasis caused by third-stage Gnathostoma spinigerum larvae (G. spinigerum L3) is an important zoonotic disease in tropical areas of the world. The excretory-secretory products (ES) that are excreted by infective larva play a significant role in host immune evasion and tissue destruction. To investigate the poorly understood mechanisms of G. spinigerum L3 pathogenesis, we focused on the potential effect of ES on inducing apoptosis in human immune cells by using human peripheral blood mononuclear cells (PBMCs) as a model. Early and late apoptosis of PBMCs were assessed following the exposure of these cells to G. spinigerum L3 ES (0.1, 0.5, and 1.0 µg/ml) for 6-48 h. The apoptotic cells were identified by flow cytometric staining of PBMC with FITC-annexin V and propidium iodide. The expression of regulatory genes related to apoptosis mechanisms in ES-treated PBMCs was investigated using a Human Apoptosis RT2 Profiler™ PCR Array. The results showed significant levels of early phase apoptosis at 18 h and of late phase apoptosis at 24 h. We speculate that this apoptosis in PBMCs occurs via the extrinsic pathway. Apoptosis in the ES-induced PBMCs was observed as quickly as 90 min after exposure, and the highest effect was observed at 18-24 h. Furthermore, ES can trigger apoptosis lasting for 48 h. Our findings expand the understanding of one of the mechanisms involved, immune-evasive strategy mechanism used by G. spinigerum larvae during human gnathostomiasis.

Molecular and immunological characterization of cathepsin L-like cysteine protease of Paragonimus pseudoheterotremus.

Cathepsin L is a cysteine protease belonging to the papain family. In parasitic trematodes, cathepsin L plays essential roles in parasite survival and host-parasite interactions. In this study, cathepsin L of the lung fluke Paragonimus pseudoheterotremus (PpsCatL) was identified and its molecular biological and immunological features characterized. A sequence analysis of PpsCatL showed that the gene encodes a 325-amino-acid protein that is most similar to P. westermani cathepsin L. The in silico three-dimensional structure suggests that PpsCatL is a pro-enzyme that becomes active when the propeptide is cleaved. A recombinant pro-PpsCatL lacking the signal peptide (rPpsCatL), with a molecular weight of 35 kDa, was expressed in E. coli and reacted with P. pseudoheterotremus-infected rat sera. The native protein was detected in crude worm antigens and excretory-secretory products and was localized in the cecum and in the lamellae along the intestinal tract of the adult parasite. Enzymatic activity of rPpsCatL showed that the protein could cleave the fluorogenic substrate Z-Phe-Arg-AMC after autocatalysis but was inhibited with E64. The immunodiagnostic potential of the recombinant protein was evaluated with an enzyme-linked immunosorbent assay (ELISA) and suggested that rPpsCatL can detect paragonimiasis with high sensitivity and specificity (100 and 95.6 %, respectively). This supports the further development of an rPpsCatL-ELISA as an immunodiagnostic tool.

Molecular characterization of serine protease inhibitor isoform 3, SmSPI, from Schistosoma mansoni.

Serine protease inhibitors, known as serpins, are pleiotropic regulators of endogenous and exogenous proteases, and molecule transporters. They have been documented in animals, plants, fungi, bacteria, and viruses; here, we characterize a serpin from the trematode platyhelminth Schistosoma mansoni. At least eight serpins have been found in the genome of S. mansoni, but only two have characterized molecular properties and functions. Here, the function of S. mansoni serpin isoform 3 (SmSPI) was analyzed, using both computational and molecular biological approaches. Phylogenetic analysis showed that SmSPI was closely related to Schistosoma haematobium serpin and Schistosoma japonicum serpin B10. Structure determined in silico confirmed that SmSPI belonged to the serpin superfamily, containing nine α-helices, three ß-sheets, and a reactive central loop. SmSPI was highly expressed in schistosomules, predominantly in the head gland, and in adult male and female with intensive accumulation on the spines, which suggests that it may have a role in facilitating intradermal and intravenous survival. Recombinant SmSPI was overexpressed in Escherichia coli; the recombinant protein was of the same size (46 kDa) as the native protein. Immunological analysis suggested that mice infected with S. mansoni responded to rSmSPI at 8 weeks postinfection (wpi) but not earlier. The inhibitory activity of rSmSPI was specific to chymotrypsin but not trypsin, neutrophil elastase, and porcine pancreatic elastase. Elucidating the biological and physiological functions of SmSPI as well as other serpins will lead to further understanding of host-parasite interaction machinery that may provide novel strategies to prevent and control schistosomiasis in the future.

Non-encapsulated Trichinella spp., T. papuae, diminishes severity of DSS-induced colitis in mice.

BACKGROUND: Helminths use various mechanisms to avoid host immunity and protect themselves from being eliminated. Despite evading host immune responses, immunosuppression and regulation mechanisms elicit functions that diminish the adverse effects of unrelated inflammatory diseases. OBJECTIVE: We investigated whether helminthic infections can ameliorate inflammatory diseases. METHODS: Mice were infected with Trichinella papuae and then subjected to induced colitis through the oral administration of dextran sulfate sodium (DSS). Macroscopic and microscopic examinations measured weight loss, stool consistency, gross bleeding, colon length, and tissue inflammation. In addition, cytokine expression was observed in colon tissue by SYBR real-time RT-PCR to investigate the Th1, Th2, and regulatory cytokines. RESULT: The results showed that T. papuae infection decreased the severity of DSS-inducedcolitis, including weight loss, bloody diarrhea, shortening of colon, and colon tissue damage in mice (p <0.05). The expression level of IL-4 was high in the colons of DSS-treated mice without helminthic infection, while infected mice with DSS treatment had lower IL-4 levels (p <0.05). Uninfected DSS-treated mice failed to produce IL-10 mRNA in colon tissue, which may cause more severe colitis. In contrast, prior T. papuae infection DSS-treated mice had IL-10 levels in the colon significant lower than the normal and infected control groups. CONCLUSION: Our data provide the evidence that prior T. papuae infection can ameliorate DSS-induced colitis in mice and may be considered for a novel therapeutic strategy against immunological diseases in the future.

Genetic diversity of Taenia asiatica from Thailand and other geographical locations as revealed by cytochrome c oxidase subunit 1 sequences.

Twelve 924 bp cytochrome c oxidase subunit 1 (cox1) mitochondrial DNA sequences from Taenia asiatica isolates from Thailand were aligned and compared with multiple sequence isolates from Thailand and 6 other countries from the GenBank database. The genetic divergence of T. asiatica was also compared with Taenia saginata database sequences from 6 different countries in Asia, including Thailand, and 3 countries from other continents. The results showed that there were minor genetic variations within T. asiatica species, while high intraspecies variation was found in T. saginata. There were only 2 haplotypes and 1 polymorphic site found in T. asiatica, but 8 haplotypes and 9 polymorphic sites in T. saginata. Haplotype diversity was very low, 0.067, in T. asiatica and high, 0.700, in T. saginata. The very low genetic diversity suggested that T. asiatica may be at a risk due to the loss of potential adaptive alleles, resulting in reduced viability and decreased responses to environmental changes, which may endanger the species.

Evaluation of recombinant serine protease inhibitor from Trichinella spiralis for immunodiagnosis of swine trichinosis.

Serine protease inhibitors, known as serpins, are mainly expressed in newborn and early-stage Trichinella spiralis larvae, suggesting that T. spiralis serpin (TsSERP) could be used as antigen for the immunodiagnosis of swine trichinosis. We produced His-tagged recombinant TsSERP (rTsSERP) in Escherichia coli and purified it using a Co(2+)-affinity column. Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) were performed to determine T. spiralis-infected swine sera samples (n = 5), negative controls (n = 26), and other parasite-infected samples (n = 83). WB showed that T. spiralis-infected sera initially reacted with rTsSERP at day 6 post-infection (dpi), and more strongly in late infection (62 and 84 dpi). However, other parasite-infected sera also elicited cross-reactivity to rTsSERP. On the other hand, indirect ELISA showed that TsSERP was an appropriate antigen for detecting late (> 60 dpi) but not early infection. No cross-reaction was observed with other parasite-infected sera. Sensitivity and specificity of TsSERP-ELISA at 62 dpi was 80% and 100%, respectively, and at 84 dpi 100% and 100%, respectively. These preliminary results show that TsSERP-ELISA method is suitable for the diagnosis of swine trichinosis, and could become the standard test for diagnosis of trichinosis in several hosts, including humans.

Degradation of human matrix metalloprotease-9 by secretory metalloproteases of Angiostrongylus cantonensis infective stage.

Angiostrongylus cantonensis infection is the major cause of eosinophilic meningitis. Successful migration and evasion of the immune system by infective-stage larvae (L3) rely heavily on secreted proteases, which activate human pro-matrix metalloprotease (MMP-9) into active MMP-9. This study showed that the proteases in excretory-secretory (ES) products of A. cantonensis third stage larvae degraded recombinant and native human proMMP-9 in a dose- and time-dependent manner. Protease inhibitory assays showed that metalloproteases were the key enzymes involved in the degradation of human proMMP-9. To assess the effects of ES products on inflammation, ES products were incubated with THP-1 human monocytic cells, which showed induction of MMP-2 and not MMP-9 production. These results indicated that degradation of human MMP-9 was due to metalloproteases present in ES of A. cantonensis L3, which may be involved in suppressing the host's immune response to allow parasite migration to the host central nervous system.

Population structure of Angiostrongylus cantonensis (Nematoda: Metastrongylidae) in Thailand based on PCR-RAPD markers.

Angiostrongylus cantonensis is the causative agent of angiostrongyliasis, which is widely distributed throughout the world. It can specifically infect many species of intermediate and definitive hosts. This study examined the genetic differentiation and population structure using the RAPD-PCR method of parasites obtained from 8 different geographical areas of Thailand. Based on 8 primers, high levels of genetic diversity and low levels of gene flow among populations were found. Using genetic distance and neighbor-joining dendrogram methods, A. cantonensis in Thailand could be divided into two groups with statistically significant genetic differentiation of the two populations. However, genotypic variations and haplotype relationships need to be further elucidated using other markers.

Haplorchis taichui as a possible etiologic agent of irritable bowel syndrome-like symptoms.

The aim of this study is to clarify the clinical features of Haplorchis taichui infection in humans in Nan Province, Thailand, and to correlate the clinical features with irritable bowel syndrome (IBS)-like symptoms. In this study area, only H. taichui, but neither other minute intestinal flukes nor small liver flukes were endemic. The degree of infection was determined by fecal egg counts and also by collecting adult worms after deworming. The signs and symptoms of individual patients together with their hematological and biochemical laboratory data were gathered to evaluate the relationship between the clinical features and the severity of infection. Special emphasis was made to elucidate the possible similarities of the clinical features of H. taichui infection and IBS-like symptoms. The results showed useful clinical information and the significant (> 50%) proportion of haplorchiasis patients complained of abdominal pain, lassitude, and flatulence, which were the important diagnostic symptoms of IBS. This study has reported a possible link between H. taichui and IBS, and H. taichui might probably play a role in the etiology of these IBS-like symptoms.

Pila ampullacea and Pomacea canaliculata, as new paratenic hosts of Gnathostoma spinigerum.

Aquatic snails, Pila ampullacea and Pomacea canaliculata were experimentally found to be suitable paratenic hosts for advanced third-stage larvae (L3) of the nematode Gnathostoma spinigerum, the causative parasite of gnathostomiasis in humans. G. spinigerum (L3) were found to be encapsulated in the tissue of the snail's foot and its internal organs. The infection, intensity and survival of third-stage larvae of G. spinigerum in both species of aquatic snails are described. This is the first evidence to reveal that not only vertebrates but also invertebrates (snails) can serve as paratenic hosts to this parasite. Aquatic snails are one of several sources of human gnathostomiasis in Thailand.

Transcriptome and excretory-secretory proteome of infective-stage larvae of the nematode Gnathostoma spinigerum reveal potential immunodiagnostic targets for development.

BACKGROUND: Gnathostoma spinigerum is a harmful parasitic nematode that causes severe morbidity and mortality in humans and animals. Effective drugs and vaccines and reliable diagnostic methods are needed to prevent and control the associated diseases; however, the lack of genome, transcriptome, and proteome databases remains a major limitation. In this study, transcriptomic and secretomic analyses of advanced third-stage larvae of G. spinigerum (aL3Gs) were performed using next-generation sequencing, bioinformatics, and proteomics. RESULTS: An analysis that incorporated transcriptome and bioinformatics data to predict excretory-secretory proteins (ESPs) classified 171 and 292 proteins into classical and non-classical secretory groups, respectively. Proteins with proteolytic (metalloprotease), cell signaling regulatory (i.e., kinases and phosphatase), and metabolic regulatory function (i.e., glucose and lipid metabolism) were significantly upregulated in the transcriptome and secretome. A two-dimensional (2D) immunomic analysis of aL3Gs-ESPs with G. spinigerum-infected human sera and related helminthiases suggested that the serine protease inhibitor (serpin) was a promising antigenic target for the further development of gnathostomiasis immunodiagnostic methods. CONCLUSIONS: The transcriptome and excretory-secretory proteome of aL3Gs can facilitate an understanding of the basic molecular biology of the parasite and identifying multiple associated factors, possibly promoting the discovery of novel drugs and vaccines. The 2D-immunomic analysis identified serpin, a protein secreted from aL3Gs, as an interesting candidate for immunodiagnosis that warrants immediate evaluation and validation.

Capillariasis: chronic watery diarrhea--not only from microorganisms.

A 54-year-old male Thai patient from Prachin Buri Province presented with a history of chronic watery diarrhea for many years. He passed stool five to ten times per day with occasionally colicky pain, abdominal distension, nausea and vomiting. He had visited hospitals and private clinics and received treatment but with no improvement. He presented to the Hospital for Tropical Diseases, Bangkok, Thailand, where on physical examination, he had moderate dehydration, weakness, abdominal distension and a gurgling abdomen. The eggs, larvae and adult worms of Capillaria philippinensis were found on stool examination. The patient was admitted and treated with Mebendazole for 20 days, whereupon his symptoms resolved. Two months previously, he had ingested a raw small fresh-water fish dish called "Phra-Pla Siw/Soi". Small fresh-water fish near the patient's home were collected and examined for Capillaria philippinensis larva. The results were negative for parasitic organisms.

Transcriptomic analysis of male and female Schistosoma mekongi adult worms.

BACKGROUND: Schistosoma mekongi is one of five major causative agents of human schistosomiasis and is endemic to communities along the Mekong River in southern Lao People's Democratic Republic (Laos) and northern Cambodia. Sporadic cases of schistosomiasis have been reported in travelers and immigrants who have visited endemic areas. Schistosoma mekongi biology and molecular biology is poorly understood, and few S. mekongi gene and transcript sequences are available in public databases. RESULTS: Transcriptome sequencing (RNA-Seq) of male and female S. mekongi adult worms (a total of three biological replicates for each sex) were analyzed and the results demonstrated that approximately 304.9 and 363.3 million high-quality clean reads with quality Q30 (> 90%) were obtained from male and female adult worms, respectively. A total of 119,604 contigs were assembled with an average length of 1273 nt and an N50 of 2017 nt. From the contigs, 20,798 annotated protein sequences and 48,256 annotated transcript sequences were obtained using BLASTP and BLASTX searches against the UniProt Trematoda database. A total of 4658 and 3509 transcripts were predominantly expressed in male and female worms, respectively. Male-biased transcripts were mostly involved in structural organization while female-biased transcripts were typically involved in cell differentiation and egg production. Interestingly, pathway enrichment analysis suggested that genes involved in the phosphatidylinositol signaling pathway may play important roles in the cellular processes and reproductive systems of S. mekongi worms. CONCLUSIONS: We present comparative transcriptomic analyses of male and female S. mekongi adult worms, which provide a global view of the S. mekongi transcriptome as well as insights into differentially-expressed genes associated with each sex. This work provides valuable information and sequence resources for future studies of gene function and for ongoing whole genome sequencing efforts in S. mekongi.

Third-stage Gnathostoma spinigerum larva excretory secretory antigens modulate function of Fc gamma receptor I-mediated monocytes in peripheral blood mononuclear cell culture.

BACKGROUND: Third (infective)-stage Gnathostoma spinigerum larvae (L3) mainly cause human gnathostomiasis. G. spinigerum L3 migrate throughout the subcutaneous tissues, vital organs, and central nervous system and can cause various pathogenesis including sudden death. Interestingly, G. spinigerum L3 can survive and evade host cellular immunity for months or years. The effects of G. spinigerum excretory-secretory (ES) products involved in larval migration and immune-evasive strategies are unknown. Monocytes are innate immune cells that act as phagocytic and antigen-presenting cells and also play roles against helminthic infections via a complex interplay between other immune cells. Fc gamma receptor I (FcγRI) is a high-affinity receptor that is particularly expressed on monocytes, macrophages, and dendritic cells. The cross-linking of FcγRI and antigen-antibody complex initiates signal transduction cascades in phagocytosis, cytokine production, and antibody-dependent cell-mediated cytotoxicity (ADCC). This study investigated whether ES antigen (ESA) from G. spinigerum L3 affects monocyte functions. RESULTS: Cultures of normal peripheral blood mononuclear cells (PBMC) separated from healthy buffy coats were used as a human immune cell model. ESA was prepared from G. spinigerum L3 culture. Using Real-Time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the effect of ESA to down-regulate FcγRI mRNA expression in monocytes during 90 min of observation was not well delineated. Flow cytometry analysis revealed a significant phenotypic-decreased FcγRI expression on the monocyte surface at 12 hours (h) of cultivation with the ESA (p = 0.033). Significantly reduced monocyte-mediated phagocytosis capacity was consistently observed after 12 h of ESA pretreatment (p = 0.001). CONCLUSIONS: Our results suggest that G. spinigerum ESA modulates monocyte function via depletion of FcγRI expression. This study provides preliminary information for future in-depth studies to elucidate mechanisms of the immune-evasive strategy of G. spinigerum larvae.

Host-finding behavior of Strongyloides stercoralis infective larvae to sodium cation, human serum, and sweat.

The host-finding behavior of Strongyloides stercoralis infective larvae was examined by in vitro agarose assay method. As human body fluid contains 0.85% (ca 0.15 molar) NaCl, various concentrations of sodium chloride, from 0.5M to 0.01M (7 steps), were examined. Many larvae were attracted at concentrations between 0.5 and 0.05M of sodium chloride. The concentration of 0.05M attracted the most larvae. The concentration of 0.02M of sodium chloride showed greatly reduced larval attraction compared with 0.05M. Therefore, the threshold concentration was determined as 0.05M. Then, 0.05M of chemicals were examined in a further experiment. Chloride compounds (NaCl, KCl, CaCl2, MgCl2) were investigated. These chemicals are components of human body fluids. Distilled water was used as the control in all experiments. Only sodium chloride attracted the larvae. Next, alkaline compounds were examined [NaOH, KOH, Ca(OH)2, and Mg(OH)2]. Larvae accumulated only at the NaOH site. The results suggested that the Na cation is important for larval attraction. A high pH value did not influence attraction at all. Next, human serum was tested. The human serum used was from normal serum to 1:32 diluted sera by distilled water (7 steps). Hierarchical attraction was seen according to serum concentration. Next, human sweat was collected from a limited zone of chest skin where only eccrine glands were distributed. Non-diluted sweat attracted the most larvae. Sweat might act as one of the most probable factors for infection by this skin-penetrating nematode.

Gnathostoma infection in fish caught for local consumption in Nakhon Nayok Province, Thailand I. Prevalence and fish species.

Between August 2000 and August 2001, 12,216 fish of 73 species were purchased from several local markets in Nakhon Nayok Province, Thailand, and examined for the presence of Gnathostoma larvae. Almost all species were fresh-water fish that had grown naturally, rather than raised commercially. Eight species were found to be infected with gnathostome larvae. The overall prevalence was 5.1% (626/12,216) and a total of 5,969 larvae was recovered. The highest rate of infection (30.1 %) was found in Monopterus albus (swamp eel). The rates in the remaining infected fish were as follows: Anabas testudineus (climbing perch) 7.7%, Channa striata (striped snake-head fish) 7.4%, Clarius macrocephalus (Gunther's walking catfish) 6.7%, Channa micropeltes (giant snake-head fish) 5.1%, Channa lucius (blotched snake-head fish) 4.0%, Clarius batrachus (Batrachian walking catfish) 1.4%, and Ompok krattensis (butter sheatfish) 0.6%. The mean number of larvae/fish was highest in swamp eels (10.0 larvae/eel), and the maximum number of 698 larvae was recovered from one eel. The body sizes of the recovered G. spinigerum advanced third-stage larvae were 2.70-5.10 mm in length (average, 3.97+/-0.50 mm) and 0.29-0.60 mm in width (average, 0.40+/-0.04 mm). The average number of cephalic hooklets of the larvae from rows 1 to 4 were 41.8+/-0.5 (range, 40-43), 43.6+/-0.6 (range, 42-45), 46.1+/-0.9 (range, 44-48) and 49.3+/-0.7 (range, 48-51), respectively.

Soil-transmitted helminthiases and health behaviors among schoolchildren and community members in a west-central border area of Thailand.

The prevalence of soil-transmitted helminthic infections and health behaviors related to infections in schoolchildren and villagers of a community (4 hamlets) was studied in Hauy Kayeng subdistrict, Thong Pha Phum district, in the north of Kanchanaburi Province. The intestinal helminth infection rate of the schoolchildren was 15.6%. Hookworm infection was the most prominent (9.8%), followed by Trichuris trichiura (6.2%), and Ascaris lumbricoides (2.2%). The community showed higher prevalence rates and was infected with more types of intestinal helminths than the schoolchildren. Thirty-five point two percent (35.2%) of the residents were infected with soil-transmitted helminths, 30.5% with hookworm, 3.4% with A. lumbricoides and 2.2% with T. trichiura. Almost all hookworm cases (94.3%) were light intensity infections, while only 1.3% were heavy infections. Moreover, the hookworm infection rate in the community was found to be much higher when a stool culture method was used (39.1%). With this technique, 2.3% Strongyloides stercoralis infections were detected in the community population. Examination of the health behavior of the study samples showed that approximately 75% always defecated in a toilet. Schoolchildren who always wore shoes comprised 67%, which was lower than the community, at 85%.

Gnathostoma infection in Nakhon Nayok and Prachin Buri, Central Thailand.

Gnathostoma infection in Nakhon Nayok and Prachin Buri Provinces, Central Thailand, was investigated. The prevalence and intensity of infection of swamp eels were determined; dog fecal samples and fresh-water copepods were examined for evidence of infection. The overall prevalence of eel infection was 38.1% (117/307) in Nakhon Nayok and 24.0% (74/308) in Prachin Buri--the former rate being significantly higher than the latter. Most of the positive Nalkhon Nayok eels (53.8%) harbored only 1-9 larvae; only one eel bore more than 50 larvae. In Prachin Buri, 67.6% of the positive eels harbored 1-9 larvae; again, only one eel bore more than 50 larvae. The mean number of 11.0 +/- 10.4 larvae/eel in Nakhon Nayok was not significantly different from that of Prachin Buri (9.3 +/- 11.4). A total of 1,292 gnathostome larvae were recovered from 307 eels in Nakhon Nayok. Of these, 52.3% had accumulated in the liver and 47.7% had spread throughout the muscles. In eels from Prachin Buri, 50.6% and 49.4% of the total of 688 larvae (from 308 eels) were found in the liver and muscles, respectively. The larvae preferred encysting in ventral of muscles rather than dorsal part; they preferred the middle portion to the anterior and posterior portions. The average length of gnathostome larvae recovered from Nakhon Nayok eels was 4.0 +/- 0.5 mm (range 2.5-5.1 mm) and the average body width was 0.40 +/- 0.05 mm (range 0.29-0.51 mm). Those from eels in Prachin Buri were 3.9 +/- 0.5 mm (range 2.2-5.1 mm) and 0.34 +/- 0.05 mm (range 0.20-0.48 mm), respectively. The mean body length and width of the larvae from eels in Nakhon Nayok were significantly greater than those of the larvae from eels in Prachin Buri. In Ban Phrao, Nakhon Nayok, none of the first 44 fecal specimens examined was positive. Of the second (68) and the third (70) specimens, one (1.5%) and two (2.9%) samples were positive. However, six months after the third fecal collection, no eggs were found. In Tha Ngam, Prachin Buri, no eggs were found in all three batches (109, 115, and 100 fecal samples). A cyclops survey of 4,000-5,000 crustacea from each of two areas (Ban Phrao and Tha Ngam) found no evidence of natural cyclops infection.

Improved antigens for IgG-ELISA diagnosis of strongyloidiasis.

Two preparations of antigens for the diagnosis of strongyloidiasis were prepared from an extract of the infective larvae of Strongyloides stercoralis: a crude antigen (CA) and a molecular weight cut-off antigen (MWCOA). Both antigens were analysed by indirect ELISA against the sera of strongyloidiasis (26 cases), other helminthiases (167) and normal controls (30). The larvae were obtained from fecal culture by a modified polyethylene tube technique after screening tests by triple simple smears per case. The larvae were extracted with distilled water and further sonicated to obtain a supernatant, the CA. A part of the CA was separated for an antigen containing molecules of lower than 30 kDa by an ultrafree-MC centrifugal filter tube (PLTK): this was designed as the MWCOA. The CA gave 96.15% sensitivity and 40.12% (67/167) specificity at a cut-off value of 0.980 (5SD); false positives were produced by 19 of 20 different helminthiases. The MWCOA produced 96.15% sensitivity at cut-off value of 0.71 (4SD); the specificity of the test was 78.44% (131/167), higher than that of CA. False positives also appeared with 15 other helminthic infections. This study suggests that MWCOA is more specific than CA. A purified MWCOA will be necessary in order to reduce cross-reactivity and provide the suitable diagnosis of strongyloidiasis.