ABSTRACT
Due to the indistinguishable morphology between Entamoeba histolytica (pathogenic) and Entamoeba dispar (non pathogenic), PCR-based assays were conducted. Based on microscopy, suspected Entamoeba cells were detected in 30 out of 455 fecal samples obtained from individuals residing at Thai/Myanmar border region. The target genes for PCR amplification included genes encoding small subunit rRNA (SSU-rRNA), chitinase and serine rich Entamoeba protein. PCR primers derived from SSU-rRNA gene amplified both E. histolytica and E. dispar genes producing an amplicon of 1,080 bp, and detected 3 out of 30 samples. PCR primers derived from chitinase gene of E. histolytica generating amplicons of 500 and 1,260 bp, samples were positive in 12 out of 30 samples. Due the large difference of gene encoding serine rich protein between E. histolytica and E. dispar, two specific sets of primers were designed. SREH-primer set, specific for E. histolytica, generated amplicons of 550 and 700 bp and detected 22 out of 30 samples. SED-primer set, specific to E. dispar, produced an amplicon of 550 bp, and together with a nested primer pair generating an amplicon of 477 bp, detected 16 out of 30 samples. Thus, detection of single and mixed infections of the two Entamoeba species could be effectively achieved directly from DNA extracted from feces without the need to culture the parasites.
Subject(s)
Dysentery, Amebic/diagnosis , Entamoeba histolytica/isolation & purification , Feces/parasitology , Polymerase Chain Reaction/methods , Animals , Dysentery, Amebic/epidemiology , Entamoeba histolytica/genetics , Humans , Myanmar/epidemiology , Prevalence , RNA, Ribosomal/genetics , Thailand/epidemiologyABSTRACT
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.
Subject(s)
Acanthamoeba/isolation & purification , Contact Lenses/parasitology , Nucleic Acid Amplification Techniques/methods , Acanthamoeba/genetics , Animals , DNA Primers/chemistry , DNA, Protozoan/analysis , DNA, Ribosomal/analysis , Humans , RNA, Protozoan/genetics , RNA, Ribosomal, 18S/genetics , Sensitivity and Specificity , Time FactorsABSTRACT
The VP19 gene encoding a structural envelope protein of white spot syndrome virus was cloned into an expression vector and introduced into E. coli. The objective was to produce a recombinant VP19 structural protein. After induction, the recombinant VP19 protein (rVP19) was produced, purified by SDS-PAGE and used for immunization of Swiss mice for polyclonal antibody production. The mouse anti rVP19 antiserum had specific immunoreactivity to the viral antigen in WSSV infected Penaeus monodon as verified by immunohistochemistry and Western blot. The production of monoclonal antibodies against this rVP19 may be useful in order to combine with anti-VP28 monoclonal antibodies for enhancing the sensitivity of various WSSV serological assays.
Subject(s)
Antibodies, Viral/immunology , DNA, Viral/immunology , Viral Envelope Proteins/immunology , Virology/methods , White spot syndrome virus 1/immunology , Animals , Antibodies, Monoclonal/metabolism , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Ethidium/metabolism , Genes, Viral , Immunohistochemistry , Mice , Penaeidae/virology , Plasmids/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolismABSTRACT
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.