Entamoeba histolytica: genetic diversity of African strains based on the polymorphism of the serine-rich protein gene

The polymorphism of the serine-rich Entamoeba histolytica protein (SREHP) among isolates obtained from different geographic regions was analyzed by a nested PCR followed by restriction analysis. Thirteen different profiles were generated from 23 E. histolytica isolates from Cameroon, Zimbabwe and South Africa while 20 others were generated from 38 E. histolytica PCR positive stool samples from South Africa. One of the profiles was common to isolates from Cameroon, Zimbabwe and South Africa and constituted the most prevalent (26.1%) of all the profiles. However, profiles unique to each country were also observed amongst the samples. A non-significant difference was observed between isolates from diarrheic and non-diarrheic samples. Of interest, of the five HIV positive stool samples three had the same profile indicating the possibility that some E. histolytica strains might be more common/pathogenic in immuno-compromised individuals. The results obtained showed that African isolates of E. histolytica may possess extremely complex genetic structures independent of geographic location. This study indicates that certain profiles might be responsible for the presentation of intestinal amoebic symptoms. However, more extended studies need to be performed in order to confirm these observations.     

Entamoeba histolytica: the serine-rich gene polymorphism-based genetic variability of clinical isolates from Georgia

It is generally accepted that a majority of individuals infected by Entamoeba histolytica do not develop symptomatic disease. However, the parasite and the host factors contributing to the development of the disease, remain undetermined. It is also unclear why certain individuals develop extra-intestinal amebiasis without exhibiting apparent intestinal symptoms. An outbreak of amebic liver abscess in Tbilisi, Georgia in 1998-1999 suggested that the causative E. histolytica strain had an unusual propensity for extra-intestinal spread. To correlate the genetic differences with pathogenic potential of the parasite, we have examined the SREHP gene polymorphisms among Georgian E. histolytica isolates. Comparison of polymorphic patterns revealed the presence of several different genotypes of E. histolytica, thus preventing an association of a single genotype with hepatic disease, but supporting the previous finding of extensive genetic diversity among E. histolytica isolates from the same geographic origin.     

Comparative genomic hybridizations of Entamoeba strains reveal unique genetic fingerprints that correlate with virulence

Variable phenotypes have been identified for Entamoeba species. Entamoeba histolytica is invasive and causes colitis and liver abscesses but only in approximately 10% of infected individuals; 90% remain asymptomatically colonized. Entamoeba dispar, a closely related species, is avirulent. To determine the extent of genetic diversity among Entamoeba isolates and potential genotype-phenotype correlations, we have developed an E. histolytica genomic DNA microarray and used it to genotype strains of E. histolytica and E. dispar. On the basis of the identification of divergent genetic loci, all strains had unique genetic fingerprints. Comparison of divergent genetic regions allowed us to distinguish between E. histolytica and E. dispar, identify novel genetic regions usable for strain and species typing, and identify a number of genes restricted to virulent strains. Among the four E. histolytica strains, a strain with attenuated virulence was the most divergent and phylogenetically distinct strain, raising the intriguing possibility that genetic subtypes of E. histolytica may be partially responsible for the observed variability in clinical outcomes. This microarray-based genotyping assay can readily be applied to the study of E. histolytica clinical isolates to determine genetic diversity and potential genotypic-phenotypic associations.     

Entamoeba histolytica: rapid detection of indian isolates by cysteine proteinase gene-specific polymerase chain reaction

Amoebiasis, caused by Entamoeba histolytica, is still one of the major problems for developing countries like India. Early detection of the parasite is a must for its prevention and control. In this study, PCR analysis of the cysteine proteinase gene from clinical isolates of symptomatic intestinal and amoebic liver abscess (ALA) cases has been compared with the stool microscopy, serology, and ultrasonography methods. The clinical isolates negative for E. histolytica by stool microscopy demonstrated the presence of the cysteine proteinase gene by PCR amplification. Also the gene copy number was increased in ALA samples compared with intestinal cases. Hence an accurate, early, and easier detection was possible by cysteine proteinase gene amplification directly from the clinical samples.     

PCR diagnosis of Entamoeba histolytica cysts in stool samples

Amebiasis is a protozoan disease caused by Entamoeba histolytica and a potential health threat in areas where sanitation and hygiene are inappropriate. Highly sensitive PCR methods for detection of E. histolytica in clinical and environmental samples are extremely useful to control amebiasis and to promote public health. The present study compared several primer sets for small subunit (SSU) rDNA and histone genes of E. histolytica cysts. A 246 bp of the SSU rDNA gene of pure cysts contained in phosphate-buffered saline (PBS) and in stool samples was successfully amplified by nested PCR, using the 1,147-246 bp primer set, of the primary PCR products which were pre-amplified using the 1,147 bp primer as the template. The detection limit of the nested PCR using the 1,147-246 primer set was 10 cysts in both groups (PBS and stool samples). The PCR to detect histone gene showed negative results. We propose that the nested PCR technique to detect SSU rDNA can be used as a highly sensitive genetic method to detect E. histolytica cysts in stool samples.     

Genetic distinctions among clinical and environmental strains of Vibrio vulnificus

Vibrio vulnificus causes rare but frequently fatal septicemia associated with raw oyster consumption by persons with underlying hepatic or immune system dysfunction. The virulence potential of environmental reservoirs appears widely distributed, because most strains are virulent in animal models; however, several investigations recently demonstrated genetic divergence among strains from clinical versus environmental origin at independent genetic loci. The present study used PCR to screen DNA polymorphisms in strains from environmental (n = 35) or clinical (n = 33) sources, and genomic relationships were determined by repetitive extragenic palindromic DNA PCR (rep-PCR) typing. Significant (P < 0.01) association was observed for typical "clinical" or "environmental" polymorphism profiles based on strain origin. Most oyster isolates (88%), including all of those with the "environmental" profile, also formed a single rep-PCR genogroup. Clinical isolates within this group did not have the typical "clinical" profile. On the other hand, clinical isolates with the typical polymorphism profile were distributed among multiple rep-PCR genogroups, demonstrating greater genetic diversity than was evident by profiling genetic polymorphisms. Wound isolates were genetically distinct from typical blood isolates by all assays. Strains from an outbreak of wound infections in Israel (biotype 3) were closely related to several U.S. strains by rep-PCR, indicating potential reservoirs of emerging disease. Strains genetically related to blood isolates appeared to be relatively rare in oysters, as only one had the "clinical" polymorphism profile or clustered by rep-PCR. However, this study was not an extensive survey, and more sampling using rep-PCR for sensitive genetic discrimination is needed to determine the virulence potential of environmental reservoirs.     

Genetic Diversity in W- and R-type Populations of Pseudocercosporella herpotrichoides Based on DNA Restriction Fragment Length Polymorphisms

Thirty-seven isolates of Pseudocercosporella herpotrichoides belonging to W-type and 31 isolates belonging to R-type were analysed for DNA restriction fragment length polymorphisms (RFLPs). They represent diverse geographic origins and different phenotypes related to sensitivity to fungicides. Total DNA digested with EcoRI was hybridized with 22 random DNA probes from a P. herpotrichoides EcoRI-restricted DNA library. Four probes showing polymorphisms among isolates within W-type and R-type and generating a total of 44 RFLPs were retained for cluster analysis. Two main groups were distinguished corresponding to W- and R-types. The genetic diversity among isolates was greater for the W-type than for the R-type isolate, four and three distinct EcoRI-restricted mitochondrial DNA patterns were identified in W- and R-type isolates, respectively. The variability of profiles within each pathotype confirmed a higher degree of polymorphism in the W-type.     

粪便样品中大肠杆菌多态性分子研究

以粪便样品中分离到的大肠杆菌为研究对象,比较了3种不同方法在分离鉴定大肠杆菌过程中的应用。首先,通过传统方法从粪便样品中分离,筛选和确定了一批大肠杆菌疑似菌株,再用现代分子生物学方法对待鉴定的大肠杆菌疑似菌株,已知大肠杆菌MG1655以及几种其它细菌进行ARDRA(AmplifiedRibosomalDNARestrictionAnalysis)分析,最后利用ERIC-PCR技术在个体水平上分析菌株的多样性。结果表明,所有由传统方法确定的大肠杆菌疑似菌株和MG1655都属于同一ARDRA型,并与其它细菌的ARDRA条码型不同。这说明ARDRA分析得到的结果与传统分析方法的结果吻合,利用ARDRA分析可以区分大肠杆菌和其它肠道细菌。但是在本实验中ARDRA分析不能反映大肠杆菌中不同菌株之间的多样性,ERIC-PCR则可以区分它们。     

Differentiation of Entamoeba histolytica and Entamoeba dispar by PCR: a preliminary study in Izmir, Turkey

The causative agent of amoebiasis is currently attributed to two distinct species (E. histolytica and E. dispar). The aim of this study was to differentiate these species by PCR in stool samples. Isolated genomic DNA was amplified by PCR and band products of 101 bp (E. dispar) were obtained. All seven stool samples were found to be E. dispar, not E. histolytica. Our results demonstrated the significance of E. histolytica/dispar differentiation in the diagnosis of amoebiasis. This study is preliminary to our current research project entitled "Investigation of the prevalence of amoebiasis and Entamoeba species in Izmir and its hinterland".     

Genetic Variation Among Rhynchosporium secalis Populations of West Asia and North Africa as Revealed by RAPD and AFLP Analysis

In order to study genetic variation among populations of Rhynchosporium secalis, 65 isolates were sampled from the West Asian and North African regions and used for polymerase chain reaction (PCR)‐based DNA marker analyses [namely random amplified polymorphism DNAs (RAPDs) and amplified fragment length polymorphisms (AFLPs)]. The study revealed that genetic diversity among and within populations accounted for 80 and 20%, respectively, of the total genetic diversity, indicating that the local field populations of R. secalis in West Asia and North Africa originated from genetically diverse source populations. Furthermore, high genetic similarity among isolates from the same location suggests that scald populations originated from a local founder population, possibly through rain‐splash‐dispersed conidia.     

Hammondia heydorni: evidence of genetic diversity among isolates from dogs

Canine isolates of Hammondia heydorni from Argentina, Brazil, and the United States were analysed for genetic diversity. A total of 14 isolates were tested for their ability to produce amplification using three PCR assays, one targeting the common toxoplasmatiid ITS-1 region and 2 amplifying novel, H. heydorni-specific loci, HhAP7 and HhAP10. While the ITS-1 fragments could be amplified from all isolates, only six isolates were capable of amplifying the fragments from the novel loci. The PCR products were further investigated for genetic diversity using restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) techniques. Polymorphism in the digestion pattern was evident only at the HhAP10 locus, differentiating two of the Argentinean isolates from the remainder. Mobility shifts on SSCP gels revealed that the two Argentinean isolates were not only different from the other four isolates, but also differed from each other, both at the HhAP7 and HhAP10 loci. The ITS-1 fragments of all isolates were identical by RFLP. However, two distinct mobility patterns resulted when the products were electrophoresed on SSCP gels. Based on the sequence data from the ITS-1 and the two random loci, the isolates could be broadly classified into two distinct groups, within which minor polymorphisms were evident. In contrast, very little heterogeneity occurred in the sequences of corresponding ITS-1 regions of Neospora caninum and Toxoplasma gondii isolates. Thus, it is concluded that there is a considerable degree of microheterogeneity among isolates of H. heydorni. This diversity should be taken into consideration while attempting to elucidate the systematics, diagnostics, and biology of H. heydorni in relation to N. caninum.     

Plasmodium falciparum: limited genetic diversity of MSP-2 in isolates circulating in Brazilian endemic areas

The genetic polymorphism of the surface merozoite protein 2 (MSP-2) was evaluated in Plasmodium falciparum isolates from individuals with uncomplicated malaria living in a Brazilian endemic area of Peixoto de Azevedo. The frequency of MSP-2 alleles and the survival of genetically different populations clones in 104 isolates were verified by Southern blot and SSCP-PCR. Single and mixed infections were observed in similar frequencies and the rate of detection of FC27 and 3D7 allelic families was equivalent. Eight alleles were identified and among them, the sequence polymorphism was mainly attributed to variations in the repetitive region. Interestingly, in three alleles nucleotide polymorphism was identical to that detected in a previous study, conducted in 1992, in a near Brazilian endemic area. This finding demonstrated the genetic similarity between two isolate groups, besides the certain temporal stability in the allelic patterns. The implications of these data for studies on the genetic diversity are also discussed.     

不同来源鼠李糖乳杆菌的随机扩增多态DNA分析

[目的]建立鼠李糖乳杆菌(Lactobacillus rhamnosus,Lr)菌株之间的分子鉴别方法并分析不同分离株之间的遗传多样性.[方法]从56份采集自中国新疆和田和广西巴马瑶族自治县的长寿老人粪便样本中分离得到的乳酸菌中,经生理生化分析和API 50CHL试验条鉴定,获得10株Lr.对10株Lr分离株和1株Lr标准株ATCC7469进行了随机扩增多态DNA分析,从50条随机引物中筛选到5条在菌株水平上具有鉴别力的引物P14、OPG28、OPG25、P7和P4并建立和优化了Lr菌株RAPD指纹图谱扩增方法.根据RAPD结果计算菌株间的遗传相似系数并进行聚类分析.[结果]获得了清晰稳定的DNA指纹图谱,扩增产物大小在100～2000bp之间,菌株间呈现显著的DNA多态性,不同来源的Lr分离株的遗传相似系数在0.581～0.935之间,在相似系数0.80水平上可以将11株Lr菌株分为5个类群,其中分离自新疆和田的Lr菌株归在类群B和类群C,而分离自广西巴马瑶族自治县的Lr菌株归在类群D和类群E.[结论]应用RAPD方法对Lr菌株进行分子鉴别是可行的,不同来源的Lr之间存在着较大的种内遗传多态性和不同的亲缘关系.     

Genetic Distinctions among Clinical and Environmental Strains of Vibrio vulnificus

Vibrio vulnificus causes rare but frequently fatal septicemia associated with raw oyster consumption by persons with underlying hepatic or immune system dysfunction. The virulence potential of environmental reservoirs appears widely distributed, because most strains are virulent in animal models; however, several investigations recently demonstrated genetic divergence among strains from clinical versus environmental origin at independent genetic loci. The present study used PCR to screen DNA polymorphisms in strains from environmental (n = 35) or clinical (n = 33) sources, and genomic relationships were determined by repetitive extragenic palindromic DNA PCR (rep-PCR) typing. Significant (P < 0.01) association was observed for typical “clinical” or “environmental” polymorphism profiles based on strain origin. Most oyster isolates (88%), including all of those with the “environmental” profile, also formed a single rep-PCR genogroup. Clinical isolates within this group did not have the typical “clinical” profile. On the other hand, clinical isolates with the typical polymorphism profile were distributed among multiple rep-PCR genogroups, demonstrating greater genetic diversity than was evident by profiling genetic polymorphisms. Wound isolates were genetically distinct from typical blood isolates by all assays. Strains from an outbreak of wound infections in Israel (biotype 3) were closely related to several U.S. strains by rep-PCR, indicating potential reservoirs of emerging disease. Strains genetically related to blood isolates appeared to be relatively rare in oysters, as only one had the “clinical” polymorphism profile or clustered by rep-PCR. However, this study was not an extensive survey, and more sampling using rep-PCR for sensitive genetic discrimination is needed to determine the virulence potential of environmental reservoirs.     

Detection of genetic variability in various isolates of cattle tick, <Emphasis Type="Italic">Boophilus microplus</Emphasis> from Tamil Nadu,India using PCR-RAPD analysis

The genomic DNA from ten isolates of the cattle tick, Boophilus microplus collected in and around Chennai, India, was analyzed by random amplified polymorphic DNA (RAPD) using PCR. Selected five random primers were used for the study of genetic variability among different isolates of B. microplus. A high degree of genetic polymorphism with a different pattern of RAPD profiles for each tick isolate was detected with all these random primers. This variability was also confirmed by similarity coefficient values and dendrogram which were performed using mean RAPD profiles for all the primers between various isolates of ticks. The findings suggest the existence of a complex genotypic diversity of the tick B. microplus in an endemic region such as Chennai.     

Entamoeba histolytica and/or Entamoeba dispar: infection frequency in HIV+/AIDS patients in Mexico city

The objective of this work was to evaluate the frequency of Entamoeba histolytica/Entamoeba dispar intestinal infection in HIV+/AIDS subjects and their HIV- close relatives or sexual partners. Enteric parasites were investigated in stool samples by microscopic examination and E. histolytica and E. dispar were identified by PCR. We found by microscopic analysis in HIV+/AIDS group that the E. histolytica/E. dispar complex was present in 5.9% of the members, while in the HIV- group was 2.9%. With PCR we found that the E. histolytica prevalence was 25.3% in the HIV+/AIDS group and 18.5% in the HIV-group. The difference in the results obtained with the microscopic and PCR is due to the different sensibility of the procedures. Besides, we found patients who were infected with E. histolytica in both groups were asymptomatic cyst passers. Our results suggest that E. histolytica strains prevalent in the studied community appear to be of low pathogenic potential.     

Use of RAPD-PCR fingerprinting to detect genetic diversity of soil Streptomyces isolates

Random amplified polymorphic DNA (RAPD) was used for identification and assessment of genetic diversity between isolates of Streptomyces from soil. Genomic DNA from 18 Streptomyces isolates and 2 reference strains were amplified using four different 10-mer primers. Different DNA fingerprinting patterns were obtained for all the isolates. Electrophoretic and cluster analysis of the amplification products revealed incidence of polymorphism among the isolates and none of them was identical to the reference strains although there were some common amplification bands. Two highly divergent groups were determined among the isolates. The results indicate that RAPD is an efficient method for discriminating and studying genetic diversity of Streptomyces isolates.     

Entamoeba histolytica: rapid identification and differentiation of Indian isolates by riboprinting

Entamoeba histolytica infection still remains one of the major public health problem for developing countries like India. A rapid and accurate detection of this parasite is essential for prevention and control of amoebiasis. In this study, using the method of 'riboprinting' (PCR-RFLP of rRNA genes from amoeba) we have analysed 15 stool samples from symptomatic patients of amoebiasis. All 15 patients of clinical amoebiasis had E. histolytica in their stool and two of the samples also showed mixed infection of E. dispar. Apart from the known restriction enzyme sites within the amoeba SSU-rRNA genes, a new Sau3A site having a discriminatory value is identified in these E. histolytica isolates from India. Hence, it is possible to rapidly identify E. histolytica DNA and differentiating it from E. dispar using minute amounts of clinical stool samples, thus eliminating the laborious parasite culturing process. Thus, riboprinting is advantageous for clearcut identification of E. histolytica in order to decide an effective antiamoebic therapy.     

Genetic diversity of Fusarium oxysporum strains from common bean fields in Spain.

Fusarium wilt is an endemic disease in El Barco de Avila (Castilla y León, west-central Spain), where high-quality common bean cultivars have been cultured for the last century. We used intergenic spacer (IGS) region polymorphism of ribosomal DNA, electrophoretic karyotype patterns, and vegetative compatibility and pathogenicity analyses to assess the genetic diversity within Fusarium oxysporum isolates recovered from common bean plants growing in fields around El Barco de Avila. Ninety-six vegetative compatibility groups (VCGs) were found among 128 isolates analyzed; most of these VCGs contained only a single isolate. The strains belonging to pathogenic VCGs and the most abundant nonpathogenic VCGs were further examined for polymorphisms in the IGS region and electrophoretic karyotype patterns. Isolates belonging to the same VCG exhibited the same IGS haplotype and very similar electrophoretic karyotype patterns. These findings are consistent with the hypothesis that VCGs represent clonal lineages that rarely, if ever, reproduce sexually. The F. oxysporum f. sp. phaseoli strains recovered had the same IGS haplotype and similar electrophoretic karyotype patterns, different from those found for F. oxysporum f. sp. phaseoli from the Americas, and were assigned to three new VCGs (VCGs 0166, 0167, and 0168). Based on our results, we do not consider the strains belonging to F. oxysporum f. sp. phaseoli to be a monophyletic group within F. oxysporum, as there is no correlation between pathogenicity and VCG, IGS restriction fragment length polymorphism, or electrophoretic karyotype.     

Study on DNA diversity of Iranian populations of Erysiphe betae causal agent of sugar beet powdery mildew

107 samples of E. betae were collected on infected leaves from all over Iranian beet cultivation areas. Their choosing were based on geographical and host origin(sugar beet, red beet, fodder beet and wild beet). 30 isolates were single colonized and grown on sugar beet susceptible genotype 7233. 107 specimens were analyzed by restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers (ITS) and 5.8s DNA which previously amplified by the polymerase chain reaction (PCR) with 2 universal primers, ITS1 and ITS4. PCR product was affected by 9 different restriction enzymes. PCR product was a 645 bp band for all of the isolates. 3 restriction enzymes; CfoI, MspI and HaeIII could cut this fragment into smaller bands, but electrophoretic patterns were identical for all of the isolates. 30 single colonized isolates were used in RAPD experiments. In RAPD-PCR experiment genetic diversity was investigated with 30 isolates from different parts of the country. 59 random primers were used and then 21 primers that displayed good consistency and reproducibility were selected. Most of the primers revealed identical patterns between 3 to 14 bands. 5 primers that showed more polymorphism were selected to analyze 30 isolates. For these 5 primers 61 distinct bands were obtained which 62% of these bands were polymorphic. Results indicated that there is no relationship between cluster grouping and geographical origin and the isolates showed a high similarity.