Molecular differentiation of Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis by pyrosequencing.

Nematodes of the genus Trichinella which infect wildlife and domestic animals show a cosmopolitan distribution. These zoonotic parasites are the aetiological agents of a severe human disease, trichinellosis. Twelve taxa are recognized in the Trichinella genus, but they cannot be identified by morphology since they are sibling species/genotypes. For epidemiological studies, it is extremely important to identify each taxon since they have different distribution areas and host ranges. In the present study, polymerase chain reaction (PCR) amplification of the mitochondrial large subunit ribosomal RNA (lsu-RNA) gene coupled with a pyrosequencing technique was developed to distinguish among four Trichinella species: Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis. A PCR method was used to amplify the lsu-RNA of Trichinella sp. larvae in mouse muscles and single larvae collected from infected muscles by digestion. The results show that the four Trichinella species can be distinguished by using 26 nucleotides in the target region and the method is sensitive enough to identify individual larvae. The pyrosequencing provides a simple, rapid and high-throughput tool for the differentiation of Trichinella species.

Morphology of puparia of flesh flies in Thailand.

Puparia of five flesh fly species were investigated for forensic study. Boettcherisca nathani (Lopes, 1961), Boettcherisca peregrina (Robineau-Desvoidy, 1830), Lioproctia pattoni (Senior-White, 1924), Liopygia ruficornis (Fabricius, 1794) and Parasarcophaga (Liosarcophaga) dux (Thomson, 1869) were examined with a scanning electron microscopy (SEM). Differences between species were found in the number and arrangement of papillae in the anterior spiracle, the shape of intersegmental spines between the prothorax and mesothorax and the pattern of spiracular tufts at the posterior spiracle. The anterior spiracle of B. nathani had two rows, comprising 21-27 papillae; while those of B. peregrina and L. pattoni had one or two irregular rows with 24-26 and 20-28 papillae, respectively. Anterior spiracle of L. ruficornis and P. dux had one row of 10-15 papillae. Intersegmental spines between the prothorax and mesothorax and pattern of spiracular tufts at the posterior spiracle are morphologically different. L. ruficornis and P. dux puparia are similar, but the position of the interslit plate between the inner and middle spiracular slits was found to be an important attribute to separate both species. Morphometric analysis on the length and width of puparia of these species revealed statistically different among them. The key for identifying puparia of forensically important flesh flies has been provided.

Comparison of the quantitative formalin ethyl acetate concentration technique and agar plate culture for diagnosis of human strongyloidiasis.

The quantitative formalin ethyl acetate concentration technique (QFEC) was compared to agar plate culture (APC) for the detection of Strongyloides stercoralis larvae. QFEC could substitute for APC only when the parasite load was higher than 50 larvae per g of stool. This study serves as a good reminder to those conducting stool exams about the sensitivity and specificity limitations of both techniques.

Molecular cloning of a gene encoding matrix metalloproteinase-like protein from Gnathostoma spinigerum.

The advanced third-stage larvae (aL3) of Gnathostoma spinigerum contain a 24 kDa glycoprotein with diagnostic potential. Immunoscreening with the monoclonal antibody to the 24-kDa protein (mAb GN6/ 24) has identified a cDNA clone with an insert of 932 base pairs (bp). The insert contains a full-length gene of 732 bp encoding a protein that is 33-39% similar to matrix metalloproteinases (MMPs) of Caenorhabditis elegans and several lower and higher vertebrates. The MMP-like protein of G. spinigerum possesses the catalytic domain, but lacks the propeptide and hemopexin-like domains found in other MMPs. A signal peptide of 23 amino acids at its amino terminus indicates that it is a secretory protein, which is confirmed by Western blot analysis showing the presence of the 24 kDa protein in the excretory-secretory products of aL3.

Production of monoclonal antibodies to the cuticle of advanced third-stage larva of Gnathostoma spinigerum.

This study has demonstrated that sera from Balb/c mice infected with live advanced third-stage larvae (aL3), but not those immunized with crude larval extract, immunoprecipitated the 25-kDa protein from surface-iodinated extract of aL3. Hybridoma cell lines derived from spleen cells of an infected mouse secreted antibodies that reacted with several tissue of aL3 including the esophagus, intestine, muscle and cuticle by immunofluorescence assay. However, none of the cuticle-positive hybridoma cell lines produced antibodies that recognized surface-iodinated protein of aL3 by immunoprecipitation. Western blot analysis showed that monoclonal antibodies (MAbs) secreted by clones derived from one of the cuticle-positive hybridoma lines recognized proteins of molecular weights ranging from 55-96 kDa. The MAbs most likely reacted with the collagenous component of the cuticle.

Lack of efficacy of quinine and artemether against advanced third-stage larvae of Gnathostoma spinigerum in vitro.

The efficacy of quinine and artemether--the effective blood schizontocide in malarial treatment--has been in vitro tested with the advanced third-stage larvae of Gnathostoma spinigerum. All larvae were collected from freshwater eel (Fluta alba) and exposed to the culture medium, each containing either quinine dihydrochloride or artemether at a final concentration of 20 microg/ml and 0.5 microg/ml, respectively for 21 consecutive days. Larval motility was assessed daily and the topographical changes were assessed using scanning electron microscope after 21-days of drug exposure. All worms moved actively for 21 days of study period and no change in surface ultrastructure was observed. Quinine and artemether at these concentrations have no effect on movement and topographical changes on the advanced third-stage larvae of this parasite.

Gnathostoma spinigerum: analysis of protein patterns by two dimensional gel electrophoresis.

The protein extracts from male (MS) and female (FS) adults and advanced third-stage larvae (LS) of Gnathostoma spinigerum were separated by high resolution two-dimensional gel electrophoresis (2-DE). The polypeptide spots, as detected by silver staining, were subsequently identified. The spot patterns of LS, MS and FS were highly complex and consisted of more than 75, 44, 52 prominent spots, respectively. In addition, the stage-specific protein patterns were identified. This 2-DE database should provide an important reference for future biological and biochemical studies of G. spinigerum.

Antigenic components of Gnathostoma spinigerum recognized by infected human sera by two-dimensional polyacrylamide gel electrophoresis and immunoblotting.

Antigenic components of Gnathostoma spinigerum larval extract were revealed by two-dimensional gel electrophoresis (2-DE) and immunoblot analysis using sera from patients with 6 proven cases of gnathostomiasis, 5 presumptive cases of gnathostomiasis, 3 proven cases of angiostrongyliasis, 3 proven cases of cysticercosis, and pooled sera from healthy adults. By the 2-DE, the larval extract was highly complex and consisted of more than 75 polypeptides. Immunoblotting analysis of this larval extract after reaction with each of 6 proven gnathostomiasis sera revealed various numbers of antigenic spots ranging from 30 to 70 spots at the approximate molecular masses of less than 14.4 to more than 94 kDa with isoelectric points (pI) of less than 4.65 to 9.6. Antigenic spots at the approximate molecular mass of more than 30 kDa were recognized with the proven angiostrongyliasis, proven cysticercosis and healthy control sera but these sera did not react with the spots at approximate molecular masses of 23-25 kDa with pI of 8.3-8.5. The reacted spots, which consisted of at least 1 to 2 spots, were unique for the recognition of gnathostomiasis sera. Five out of 6 (83.3%) proven and 4 out of 5 (80%) presumptive gnathostomiasis sera reacted with these specific spots.

Recognition of deglycosylated larval proteins of Gnathostoma spinigerum by a monoclonal antibody and human gnathostomiasis antiserum.

The study on the recognition of 35S-labelled somatic antigens of Gnathostoma spinigerum advanced third-stage larva (aL3) has revealed that the mAb GN6/24 immunoprecipitated 26- and 24-kDa proteins from the undigested and N-glycosidase F-digested larval extracts, respectively. The recognition of the deglycosylated form of the glycoprotein indicated that the mAb reacted with the peptide epitope on the 26-kDa protein. Human gnathostomiasis antiserum immunoprecipitated most of the N-glycosidase F-digested larval proteins including the deglycosylated 26-kDa protein.

Fluctuations of larval excretion in Strongyloides stercoralis infection.

Follow-up stool examinations were carried out on two groups of the subjects who were screened negative (group 1) or positive (group 2) for Strongyloides stercoralis by the agar plate culture. This technique could detect S. stercoralis larvae in 87.5-96.4% of the subjects in group 2 and 0-5.9% of the subjects in group 1 on various days of the eight-week and four-week follow-up periods, respectively. The detection rate on each day of examination was not statistically different from that on the first day in both groups. Quantitative measurement of S. stercoralis larvae excreted in the feces of the subjects in group 2 by the standard direct smear method of Beaver and others revealed slight to marked fluctuations of the larval output in individual subjects. From the results of both stool examination methods, it could be implied that 52% of S. stercoralis-infected individuals had low-level infection.

Movability of advanced third-stage larva of Gnathostoma spinigerum exposed to albendazole sulphoxide in vitro.

Movability of advanced third-stage larvae of Gnathostoma spinigerum exposed to albendazole sulphoxide (AlbSO), the active metabolite of albendazole, was determined in vitro. Larvae in control groups moved actively with the whole body for all 21 days of the study period. In larvae treated with AlbSO 1 microg/ml, the movement was significantly reduced after 11 days exposed to the drug and to be only a part of body on the 15th-21st days. In larvae treated with AlbSO 2 microg/ml, the movement was initiated in decreasing after 9th days and to be only a part of body on the 12th-17th days. Finally, worms were immobile but not dead on the 20th-21st days. Although there was no larvae died at 21st days exposed to AlbSO in both concentrations; but all worms were sluggish and may die later. These lethargic worms may not be able to migrate in patients and leading to cure. Albendazole may not be benefit for acute symptom clearance; however, it can prevent the recurrent migratory swelling after the treatment of 21 day-course.

Specific gravity of Opisthorchis viverrini eggs.

The specific gravity of the eggs of the liver fluke Opisthorchisviverrini was determined using a sucrose gradient centrifugation and found to range from 1.2713 to 1.3043. The peak egg count was located at the sucrose fraction with a specific gravity of 1.2814. An attempt to float eggs in saturated sodium nitrate solution, sp.gr. 1.4, failed. Examination of human stool specimens for Oviverrini eggs by simple flotation in saturated sodium nitrate solution and the formol-ether sedimentation technique revealed that the flotation technique was not as efficient as the sedimentation technique. It was suggested that the flotation techniques were inappropriate for the detection of Oviverrini eggs in faeces or contaminated soil.

Indirect haemagglutination test using monoclonal antibody-affinity purified antigens for diagnosis of human paragonimiasis due to Paragonimus heterotremus.

An indirect haemagglutination test (IHA) using antigens purified by monoclonal antibody-affinity chromatography was developed for the diagnosis of human paragonimiasis caused by Paragonimus heterotremus. Sera from patients with paragonimiasis (n = 30) were evaluated, along with sera from other parasitic infections (n = 92), pulmonary tuberculosis (n = 18) and healthy controls (n = 30). The sensitivity, specificity as well as positive and negative predictive value of the IHA, calculated at the prevalence of disease at 17.6%, were all 100%.

Detection of Paragonimus heterotremus in experimentally infected cat feces by antigen capture-ELISA and by DNA hybridization.

An antigen capture enzyme-linked immunosorbent assay (antigen capture-ELISA) and DNA hybridization technique were developed and evaluated for their application in the detection of Paragonimus heterotremus infection in experimentally infected cats. An IgG fraction prepared from serum of a rabbit immunized with P. heterotremus excretory-secretory (ES) products was used as the capture antibody. An IgG1 monoclonal antibody specific to the 22- and 31.5-kDa ES products of P. heterotremus was used as the antigen probe. As little as 0.24 ng of the ES products could be detected by this technique. A specific P. heterotremus DNA probe derived from the P. heterotremus genomic DNA library containing 1,500 base pairs was used in a dot-blot hybridization assay for the detection of parasite DNA. The radioactively labeled probe could detect DNA released from as few as 2 P. heterotremus eggs. Both ELISA and DNA hybridization were found to have 100% specificity, with sensitivities of 73.7% and 100%, respectively.

Monoclonal antibodies to Paragonimus heterotremus and their potential for diagnosis of paragonimiasis.

Monoclonal antibodies (MAbs) specific to the lung fluke (Paragonimus heterotremus) were produced against the soluble metabolic products (excretory-secretory antigen). Three hybrids secreting MAbs specific for P. heterotremus antigens were identified by an indirect enzyme-linked immunosorbent assay (ELISA) against a panel of homologous and 24 heterologous parasite antigens and Mycobacterium tuberculosis. Of the three specific clones, clone 10F2, which was IgG1 producing and which gave immune complex bands with 31.5-kD and 22-kD polypeptides by gel electrophoresis and immunoblotting, was selected for further characterization and evaluation of its possible diagnostic potential. The result obtained from an indirect immunofluorescent antibody test suggested that MAb 10F2 reacted with mucosa and contents of the worm's intestine. The antibody could be readily used to prepare an affinity-purified antigen for use in an indirect ELISA that was highly sensitive and specific for the detection of circulating antibody in sera of paragonimiasis patients.

Growth and development of Gnathostoma spinigerum early third-stage larvae in vitro.

Early third-stage larvae of Gnathostoma spinigerum were cultured in RPMI-1640 supplemented with 25mM NaHCO3,100 units/ml penicillin G, 100 microg/ml of streptomycin, 5 microg/ml of amphotericin B and 10% foetal calf serum at 37 degrees C under 5% CO2 in air for 60 days. After 3 days of cultivation, the larvae moulted. Body sizes increased from 0.49 +/- 0.09 x 0.07 +/- 0.01 mm in length and width to 4.08 +/- 0.48 x 0.32 +/- 0.04 mm after 60 days of cultivation. The maximum body length and width of these larvae were 4.94 mm and 0. 35 mm, respectively. The survival rate (67.5 %) of the worms was observed at the end of cultivation. The addition of foetal calf serum was found to be essential for growth and development.

Detection of circulating Trichinella spiralis larvae by polymerase chain reaction.

Several investigators have successfully applied the polymerase chain reaction to the amplification of DNA from Trichinella spiralis muscle-stage larvae. We show herein that specific DNA can be amplified from T. spiralis migratory larvae in the blood of experimentally infected mice. The polymerase chain reaction detected the presence of migratory larvae in mouse blood from day 5 to day 14 of infection. The technique may be applied to human trichinosis, but its diagnostic value will depend on the severity and stage of the infection.