Nitaya Thammapalerd

Geographic diversity among genotypes of Entamoeba histolytica field isolates

ABSTRACT It has been known that only 5 to 10% of those infected with Entamoeba histolytica develop symptomatic disease. However, the parasite and the host factors that determine the onset of disease remain undetermined. Molecular typing by using polymorphic genetic loci has been proven to aid in the close examination of the population structure of E. histolytica field isolates in nature. In the present study, we analyzed the genetic polymorphisms of two noncoding loci (locus 1-2 and locus 5-6) and two protein-coding loci (chitinase and serine-rich E. histolytica protein [SREHP]) among 79 isolates obtained from different geographic regions, mainly Japan, Thailand, and Bangladesh. When the genotypes of the four loci were combined for all isolates that we have analyzed so far (overlapping isolates from mass infection events were excluded), a total of 53 different genotypes were observed among 63 isolates. The most remarkable and extensive variations among the four loci was found in the SREHP locus; i.e., 34 different genotypes were observed among 52 isolates. These results demonstrate that E. histolytica has an extremely complex genetic structure independent of geographic location. Our results also show that, despite the proposed transmission of other sexually transmitted diseases, including human immunodeficiency virus infection, from Thailand to Japan, the spectra of the genotypes of the E. histolytica isolates from these two countries are distinct, suggesting that the major E. histolytica strains prevalent in Japan at present were likely introduced from countries other than Thailand. Although the genetic polymorphism of the SREHP locus was previously suggested to be closely associated with the clinical presentation, e.g., colitis or dysentery and liver abscess, no association between the clinical presentation and the SREHP genotype at either the nucleotide or the predicted amino acid level was demonstrated.

Enzyme-linked immunosorbent assay based on monoclonal and polyclonal antibodies for the detection of Entamoeba histolytica antigens in faecal specimens

A murine monoclonal antibody (MAb) (Eh208C2-2) raised against crude lysate of the pathogenic HM-1:IMSS strain of Entamoeba histolytica was used in an enzyme-linked immunosorbent assay for the detection of E. histolytica antigens in faecal specimens. The detection limit of the assay was 110 and 280 amoebae/ml of the HM-1:IMSS and HK-9 strains in phosphate-buffered saline, respectively. The assay was applied to single stool samples from 3 groups of individuals comprising 40 patients whose stools were positive for E. histolytica trophozoites and/or cysts (group I), 48 patients whose stools were negative for E. histolytica but positive for other parasites (group II), and 36 parasitologically-negative healthy controls (group III). Positivity rates of 77.5%, 2.1% and 2.7% were found in samples from groups I, II and III respectively. Specificity, positive and negative predictive values, and efficiency of the assay were 97.6%, 93.9%, 90.1% and 91.1% respectively. When group I samples were further divided into a trophozoite-positive subgroup IA (13 samples) and a cyst-positive subgroup IB (27 samples), the positive rates were 100% and 66.7%, respectively (P < 0.025).

Characterization of vitellin and vitellogenin of giant tiger prawn Penaeus monodon using monoclonal antibodies specific to vitellin subunits

Summary Monoclonal antibodies specific to Penaeus monodon vitellin subunits were produced from mouse immunized with sodium dodecyl sulfate (SDS) treated ovarian extract prepared from gravid ovaries. After fusion of mouse spleen cells with P3X myeloma, hybridomas were selected by indirect immunoperoxidase ELISA against P. monodon ovarian extract. This was followed by dot-blotting against native and denatured proteins from ovarian extract, female haemolymph, and male haemolymph, then by dot-blotting against each vitellin subunit. Hybridoma clones producing antibodies specific to each of vitellin subunits with a molecular mass of 83, 74, 104 and 58, 104 and 45 kD, antibodies specific to the 215 kD protein, an oocyte-specific protein, and one monoclonal antibody specific to haemocyanin were isolated. All monoclonal antibodies could bind to both native and denatured proteins. Western blot analysis of ovarian extract and female haemolymph from gravid ovary prawns separated by PAGE and SDS-PAGE revealed five vitellin subunits, molecular mass of 104, 83, 74, 58 and 45 kD in ovarian extract, and four vitellogenin related polypeptides, molecular mass of 200, 104, 83 and 74 kD in the female haemolymph. From the immunoreactive relationships among these proteins, it could be assumed that vitellogenin may be released into the haemolymph in two forms, 200 and 74 kD, then the 200 kD polypeptide was either processed into the 104 and 83 kD polypeptides, or directly taken up into the oocyte. In the oocyte, the 104 kD protein would be further cleaved into 58 and 45 kD polypeptides while the 74 kD protein would undergo slight modification or remained unchanged. Western blot analysis of vitellin subunits at various stages of ovarian development revealed that the 200 kD protein appeared in the oocyte during early ovarian development and the 45 and 58 kD proteins appeared during the late development.

Monoclonal antibody-based immunohistochemical demonstration of Entamoeba histolytica in liver tissues of experimentally infected hamster (Mesocricetus auratus)

Histopathological changes and the presence of Entamoeba histolytica trophozoites was sequentially followed after intrahepatic inoculation of the parasites in 42 hamsters, 35 of which received no treatment whereas the remaining seven were treated with metronidazole. The liver tissues were examined for amoebic trophozoites by a monoclonal antibody (mAb)-based immunofluorescence assay (IFA), a mAb-based immunoperoxidase (IPx) and H & E staining. The number of hamsters developing abscesses was increased with time and was highest on day 30. Cellular infiltration with inflammatory cells and glycogen depletion were observed as early as day 5, followed thereafter by more intense inflammation of portal canals, periportal fibrosis, bile duct proliferation and hepatocyte degeneration. In 7 metronidazole-treated hamsters, no obvious pathological damage was seen. In a group of seven hamsters each, both IPx and IFA were positive in 3, 3, 4, 5 and 4 hamsters and in 3, 4, 3, 3 and 5 hamsters on days 5, 10, 15, 20 and 30, respectively. In 18 control hamsters, IPx, IFA and H & E were all negative. If the result from H & E was used as a gold standard, agreement between H & E and IFA and H & E and IPx were 91.4%, and 88.6%, respectively. Sensitivity and specificity were 93.8% and 89.5%, respectively for IFA, and 93.8% and 84.2%, respectively for IPx.

Rapid identification of Acanthamoeba from contact lens case using loop-mediated isothermal amplification method

A method employing loop-mediated isothermal amplification (LAMP) of 18S ribosomal RNA gene was developed to detect Acanthamoeba in contact lens cases. A prevalence of 7% (10/150) was detected, with 100% sensitivity and 100% specificity when compared with the standard culture technique. Using visual inspection of turbidity a minimum of 10pg of Acanthamoeba DNA could be detected, 10 times more sensitive than quantitative PCR employing two of the LAMP primers. The production of LAMP amplicons was confirmed by gel-electrophoresis and ethidium bromide staining. The LAMP procedure takes less than 2h to perform and will be useful for incorporation into a point-of-care screening of suspected Acanthamoeba infection.

Mouse IgE response to an allergen from Entamoeba histolytica.

Attempts were made to demonstrate the presence of allergenic components in an extract of axenically cultured Entamoeba histolytica (HK 9). Various strains of mice were immunized i.p. with a low dose of the extract mixed with Al(OH)3 and boosted with the same mixture 4 wk later. A high titer of IgE antibody response was demonstrated in BALB/c, DBA/2 (H-2d), CBA (H-2k), and SWM strains but not in C3H/He (H-2k) strain. The extract was fractionated by gel filtration with Sephadex G-200 and then by DEAE-cellulose chromatography with a gradient buffer with progressive increase in molarity. Fractions having the strongest activity to stimulate the IgE antibody response in the mouse were subjected to SDS polyacrylamide gel electrophoresis. From the results of gel filtration and polyacrylamide gel electrophoresis, the molecular weight on the partially purified allergen was estimated to be 25,000 to 27,000 daltons.

The effect of eyestalk homogenate on haemolymph vitellogenin levels in the black tiger prawn Penaeus monodon

Summary Competitive ELISA using a combination of monoclonal antibodies specific to vitellin subunits was used to monitor the fluctuation of haemolymph vitellogenin levels in Penaeus monodon during ovarian development induced by bilateral eyestalk-ablation and to monitor the effect of eyestalk homogenate injection in prawns with developing ovaries. The haemolymph vitellogenin levels were undetectable in the prawn with ovary at the resting stage but elevated sharply when the ovary began to develop, remained high during the ovary developing into the ripe stage, then fell to low levels before spawning and spent stages. The result from injection of eyestalk homogenate into prawns with developing ovaries revealed that haemolymph vitellogenin levels elevated sharply within 2 h, reached the maximal levels and remained high during 4–10 h, then declined slightly at 24 h. This response directly depended on the amount of injected eyestalk homogenate and the response was species specific. The application of eyestalk homogenate from Metapenaeus affinis demonstrated the same stimulatory effect but was less potent, whereas the eyestalk homogenate from Macrobrachium rosenbergii did not cause any changes in haemolymph vitellogenin levels. Therefore, the assay is specific and provides an indicator to monitor the activity of the putative gonad inhibiting hormone by assaying the alteration of vitellogenin levels.