This study aimed to investigate the antibody responses in mice immunized with Gnathostoma spinigerum crude antigen (GsAg) incorporated with the combined adjuvant, a synthetic oligonucleotide containing unmethylated CpG motif (CpG ODN 1826) and a stable water in oil emulsion (Montanide ISA720). Mice immunized with GsAg and combined adjuvant produced all antibody classes and subclasses to GsAg except IgA. IgG2a/2b/3 but not IgG1 subclasses were enhanced by immunization with CpG ODN 1826 when compared with the control groups immunized with non-CpG ODN and Montanide ISA or only with Montanide ISA, suggesting a biased induction of a Th1-type response by CpG ODN. After challenge infection with live G. spinigerum larvae, the levels of IgG2a/2b/3 antibody subclasses decreased immediately and continuously, while the IgG1 subclass remained at high levels. This also corresponded to a continuous decrease of the IgG2a/IgG1 ratio after infection. Only IgM and IgG1 antibodies, but not IgG2a/2b/3, were significantly produced in adjuvant control groups after infection. These findings suggest that G. spinigerum infection potently induces a Th2-type biased response.
Adjuvants, Immunologic/*administration & dosage ; Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/*administration & dosage/*immunology ; Gnathostoma/*immunology ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Male ; Mannitol/administration & dosage/*analogs & derivatives ; Mice ; Oleic Acids/*administration & dosage ; Oligodeoxyribonucleotides/*administration & dosage ; Th1 Cells/immunology ; Th2 Cells/immunology

Paragonimiasis is an important food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. Of the 7 members of the genus known in Thailand until recently, only P. heterotremus has been confirmed as causing human disease. An 8th species, P. pseudoheterotremus, has recently been proposed from Thailand, and has been found in humans. Molecular data place this species as a sister species to P. heterotremus, and it is likely that P. pseudoheterotremus is not specifically distinct from P. heterotremus. In this study, we collected metacercariae of both nominal species (identification based on metacercarial morphology) from freshwater crabs from Phetchabun Province in northern Thailand, Saraburi Province in central Thailand, and Surat Thani Province in southern Thailand. In addition, we purchased freshwater crabs imported from Myanmar at Myawaddy Province, western Thailand, close to the Myanmar-Thailand border. The DNAs extracted from excysted metacercariae were PCR-amplified and sequenced for ITS2 and cox1 genes. The ITS2 sequences were nearly identical among all samples (99-100%). Phylogenies inferred from all available partial cox1 sequences contained several clusters. Sequences from Indian P. heterotremus formed a sister group to sequences from P. pseudoheterotremus-type metacercariae. Sequences of P. heterotremus from Thailand, Vietnam, and China formed a separate distinct clade. One metacercaria from Phitsanulok Province was distinct from all others. There is clearly considerable genetic variation in the P. heterotremus complex in Thailand and the form referred to as P. pseudoheterotremus is widely distributed in Thailand and the Thai-Myanmar border region.
Animals ; Cluster Analysis ; DNA, Ribosomal Spacer/chemistry/genetics ; Electron Transport Complex IV/genetics ; *Genetic Variation ; Metacercariae/genetics/isolation & purification ; Molecular Sequence Data ; Myanmar ; Paragonimus/*classification/*genetics/isolation & purification ; Phylogeny ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Sequence Homology ; Shellfish/parasitology ; Thailand

Members of the genus Trichinella are small nematodes that can infect a wide range of animal hosts. However, their infectivity varies depending on the parasite and host species combination. In this study, we examined the susceptibility of 4 species of laboratory rodents, i.e., mice, rats, hamsters, and gerbils to Trichinella papuae, an emerging non-encapsulated Trichinella species. Trichinella spiralis and Trichinella pseudospiralis were also included in this study for comparison. Fifteen animals of each rodent species were infected orally with 100 muscle larvae of each Trichinella species. Intestinal worm burden was determined at day 6 and 10 post-inoculation (PI). The numbers of muscle larvae were examined at day 45 PI. The reproductive capacity index (RCI) of the 3 Trichinella species in different rodent hosts was determined. By day 6 PI, 33.2-69.6% of the inoculated larvae of the 3 Trichinella species became adult worms in the small intestines of the host animals. However, in rats, more than 96% of adult worms of all 3 Trichinella species were expelled from the gut by day 10 PI. In gerbils, only 4.8-18.1% of adult worms were expelled by day 10 PI. In accordance with the intestinal worm burden and the persistence of adults, the RCI was the highest in gerbils with values of 241.5+/-41.0 for T. papuae, 432.6+/-48 for T. pseudospiralis, and 528.6+/-20.6 for T. spiralis. Hamsters ranked second and mice ranked third in susceptibility in terms of the RCI, Rats yielded the lowest parasite RCI for all 3 Trichinella species. Gerbils may be an alternative laboratory animal for isolation and maintenance of Trichinella spp.
Animals ; *Animals, Laboratory ; Cricetinae ; *Disease Susceptibility ; Gerbillinae ; Intestines/parasitology ; Male ; Mice ; Muscles/parasitology ; Parasite Load ; Rats ; Rodent Diseases/*parasitology/pathology ; Trichinella/*growth & development ; Trichinellosis/parasitology/pathology/*veterinary

Gnathostoma spinigerum can cause subarachnoid hemorrhage (SAH). The detection of specific antibodies in serum against G. spinigerum antigen is helpful for diagnosis of neurognathostomiasis. There is limited data on the frequency of G. spinigerum infection in non-traumatic SAH. A series of patients diagnosed as non-traumatic SAH at the Srinagarind Hospital, Khon Kaen University, Thailand between January 2011 and January 2013 were studied. CT or MR imaging of the brain was used for diagnosis of SAH. Patients were categorized as aneurysmal subarachnoid hemorrhage (A-SAH) or non-aneurysmal subarachnoid hemorrhage (NA-SAH) according to the results of cerebral angiograms. The presence of specific antibodies in serum against 21- or 24-kDa G. spinigerum antigen was determined using the immunoblot technique. The detection rate of antibodies was compared between the 2 groups. Of the 118 non-traumatic SAH patients for whom cerebral angiogram and immunoblot data were available, 80 (67.8%) patients had A-SAH, whereas 38 (32.2%) had NA-SAH. Overall, 23.7% were positive for specific antibodies against 21- and/or 24-kDa G. spinigerum antigen. No significant differences were found in the positive rate of specific antibodies against G. spinigerum in both groups (P-value=0.350).
Adult ; Aged ; Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/diagnostic use ; Brain/radiography ; Female ; Gnathostoma/immunology/*isolation & purification ; Gnathostomiasis/*diagnosis/*parasitology ; Humans ; Immunoblotting ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Serum/immunology ; Subarachnoid Hemorrhage/*diagnosis/*etiology ; Thailand ; Tomography, X-Ray Computed

We compared the efficacy and applicability of a modified formalin-ether concentration technique (M-FECT) to the conventional FECT (C-FECT) and the agar plate culture (APC) method for the detection of Strongyloides stercoralis larvae. For this purpose, we used 600 human fecal specimens collected in an endemic area of southern Thailand. In the M-FECT, we used 2 layers of wire meshes, instead of gauze, to avoid the loss by absorption/adhesion of larvae to the gauze during filtration, and we reduced the exposure time of S. stercoralis larvae in stool samples to formalin. By such simple modifications, the efficacy of M-FECT has become comparable to APC and was much better than that of C-FECT for the diagnosis of strongyloidiasis.
Animals ; Ether ; Feces/parasitology ; Formaldehyde ; Humans ; Parasitology/*methods ; Specimen Handling/*methods ; Strongyloides stercoralis/*isolation & purification ; Strongyloidiasis/*diagnosis ; Thailand

A synthetic peptide was prepared based on the antigenic region of Paragonimus westermani pre-procathepsin L, and its applicability for immunodiagnosis for human paragonimiasis (due to Paragonimus heterotremus) was tested using an ELISA to detect IgG4 antibodies in the sera of patients. Sera from other helminthiases, tuberculosis, and healthy volunteers were used as the references. This peptide-based assay system gave sensitivity, specificity, accuracy, and positive and negative predictive values of 100%, 94.6%, 96.2%, 100%, and 88.9%, respectively. Cross reactivity was frequently seen against the sera of fascioliasis (75%) and hookworm infections (50%). Since differential diagnosis between paragonimiasis and fascioliasis can be easily done by clinical presentation and fascioliasis serology, this cross reaction is not a serious problem. Sera from patients with other parasitoses (0-25%) rarely responded to this synthetic antigen. This synthetic peptide antigen seems to be useful for development of a standardized diagnostic system for paragonimiasis.
Adult ; Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/*diagnostic use ; Clinical Laboratory Techniques/*methods ; Cross Reactions ; Enzyme-Linked Immunosorbent Assay/methods ; Humans ; Immunoglobulin G/*blood ; Paragonimiasis/*diagnosis ; Paragonimus/*immunology ; Parasitology/*methods ; Predictive Value of Tests ; Sensitivity and Specificity

The 2 principal species of hookworms infecting humans are Necator americanus and Ancylostoma duodenale. Case studies on zoonotic hookworm infections with Ancylostoma ceylanicum and/or Ancylostoma caninum are known mainly from Asian countries. Of these 2 zoonotic species, only A. ceylanicum can develop to adulthood in humans. In the present study, we report a molecular-based survey of human hookworm infections present in southern and northeastern Thailand. Thirty larval hookworm samples were obtained from fecal agar plate cultures of 10 patients in northeastren Thailand and 20 in southern Thailand. Partial ITS1, 5.8S, and ITS2 regions of the ribosomal DNA genes were amplified using PCR. The amplicons were sequenced, aligned, and compared with other hookworm sequences in GenBank database. The results showed that, in Thailand, N. americanus is more prevalent than Ancylostoma spp. and is found in both study areas. Sporadic cases of A. ceylanicum and A. duodenale infection were seen in northeastern Thailand.
Ancylostoma/classification/genetics/*isolation & purification ; Ancylostomiasis/*epidemiology ; Animals ; Cluster Analysis ; DNA, Ribosomal Spacer/chemistry/genetics ; Feces/parasitology ; Humans ; Molecular Sequence Data ; Necator americanus/classification/genetics/*isolation & purification ; Necatoriasis/*epidemiology ; Phylogeny ; Polymerase Chain Reaction ; Prevalence ; RNA, Ribosomal, 5.8S/genetics ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Thailand/epidemiology

Lung fluke, Paragonimus heterotremus, is a flatworm causing pulmonary paragonimiasis in cats, dogs, and humans in Southeast Asia. We examined the ultrastructure of the testis of adult P. heterotremus with special attention to spermatogenesis and spermiogenesis using scanning and transmission electron microscopy. The full sequence of spermatogenesis and spermiogenesis, from the capsular basal lamina to the luminal surface, was demonstrated. The sequence comprises spermatogonia, spermatocytes with obvious nuclear synaptonemal complexes, spermatids, and eventual spermatozoa. Moreover, full steps of spermatid differentiation were shown which consisted of 1) early stage, 2) differentiation stage representing the flagella, intercentriolar body, basal body, striated rootlets, and electron dense nucleus of thread-like lamellar configuration, and 3) growing spermatid flagella. Detailed ultrastructure of 2 different types of spermatozoa was also shown in this study.
Animals ; Male ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Paragonimus/*physiology/*ultrastructure ; Spermatogenesis ; Spermatozoa/ultrastructure ; Testis/ultrastructure

Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to generate PCR products of 156 base pairs for both species. The melting points of S. japonicum and S. mekongi PCR products were 84.5+/-0.07degrees C and 85.7+/-0.07degrees C, respectively. The method permits amplification from a single cercaria or an egg. The HRM real-time PCR is a rapid and simple tool for differentiation of S. japonicum and S. mekongi in the intermediate and final hosts.
Animals ; DNA Primers/genetics ; Mice ; Parasitology/*methods ; RNA, Ribosomal, 18S/genetics ; Real-Time Polymerase Chain Reaction/*methods ; Schistosoma/*classification/*genetics ; Snails ; Time Factors ; Transition Temperature

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5+/-0.2degrees C, 79.0+/-0.3degrees C, 76.8+/-0.1degrees C, and 79.9+/-0.1degrees C, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.
Animals ; Blood/*parasitology ; Brugia/classification/genetics/*isolation & purification ; Cats ; Culicidae/*parasitology ; Dirofilaria immitis/classification/genetics/*isolation & purification ; Dogs ; Humans ; Male ; Parasitology/*methods ; RNA, Helminth/genetics ; RNA, Ribosomal, 5S/genetics ; Real-Time Polymerase Chain Reaction/*methods ; Sensitivity and Specificity ; Transition Temperature ; Wuchereria bancrofti/classification/genetics/*isolation & purification