Genotypes of Candida dubliniensis in clinical isolates

Amplification of specific sequences of the ITS1 and ITS2 regions and the intervening 5.8S rRNA gene has lead to the identification of four separate genotypes in Candida dubliniensis. Using primers specific for each genotype, we have studied the prevalence of these genotypes among 68 clinical isolates, mostly from Spanish patients infected by HIV. The majority of the isolates tested belonged to genotype 1 (97%), while only one isolate each from genotypes 2 (1.5%) and 3 (1.5%) were detected in the oral cavity of two patients with HIV infection.     

Genetic and phenotypic intraspecific variation in the microsporidian Encephalitozoon hellem.

Encephalitozoon hellem is a microsporidian species that causes disseminated infections in HIV-positive patients. Identical genotypes of E. hellem, as assessed by the sequence of the rDNA internal transcribed spacer, have been identified in isolates from humans and from a psittacine bird. However, by analysing the rDNA ITS of four E. hellem isolates from Switzerland (three) and Tanzania (one), two new genotypes were identified. Differences among the E. hellem isolates were also detected by Western blot analysis, but there was no absolute match between ITS genotype and antigen profile. Hence, strain variation exists in E. hellem and the ITS sequence seems a valuable marker in obtaining further insight into the epidemiology of this pathogen.     

ITS1 intra-individual variability of Ascaris isolates from Brazil

The zoonotic potential of Ascaris infecting pigs has stimulated studies of molecular epidemiology with internal transcribed spacer 1 (ITS1) as the target. The aim of this study was to determine the value of Ascaris ITS1 as a molecular marker through assessing the intra-individual genetic diversity of Ascaris isolates from two geographical areas of Brazil. DNA was extracted from single isolated eggs, ITS1 PCR was performed, and the PCR products were cloned and sequenced. Clone analysis showed high ITS1 intra-individual variability revealed by 2–4 ITS1 genotypes/haplotypes per sample (egg). Two genotypes, G1 and G6, and 13 new haplotypes were detected and characterized. The most prevalent in humans, G1 and/or the Brazilian G6, were detected in all samples. Except for genotype G1, no relationship was observed between Brazilian ITS1 genotypes/haplotypes and those previously described in China, Bangladesh, Japan, United Kingdom, Australia, and Denmark, with respect to geographic origin or host affiliation. However, an association between the two geographically separated Brazilian ITS1 isolates was observed. The ITS1 intra-individual variability revealed in this study indicated that the use of this genetic region to discriminate human and pig Ascaris genotypes should be reconsidered.     

Use of the 16S-23S rDNA internal transcribed spacer of Roseovarius crassostreae for epizootiological studies of juvenile oyster disease (JOD)

Juvenile oyster disease (JOD) in Crassostrea virginica is caused by the marine bacterium Roseovarius crassostreae. Although the 16S rRNA genes of the bacterial isolates exhibit little variation, 2 genetic signatures (GSI and GSII) may be discerned by Ava I digestion of the 16S-23S internal transcribed spacer (ITS). In this study we analyzed isolates from JOD epizootics throughout the northeastern USA (including affected adults for the first time) to better understand how oyster populations encounter and become affected by the pathogen. Isolates from a given epizootic usually had the same ITS signature; however, the involvement of both genetic signatures was occasionally detected, even within the same oyster. Sequencing was used to localize the variable Ava I site to a 100 bp region of low sequence identity, and detection of additional base changes resulted in the identification of 11 distinct genotypes. One genotype was found only in Martha's Vineyard, Massachusetts, USA and persisted in JOD survivors. Two genotypes were associated with Maine epizootics, and both were believed to be unique to that region until 2004, when one was detected in Martha's Vineyard among oysters that had survived colonization by the local genotype. Apparent competition between those 2 genotypes was also detected among a population of juveniles. Five genotypes were found only in New York, and the other 3 were isolated from both New York and from around Cape Cod, Massachusetts. Relationships between the geographic occurrence and phylogenetic relatedness of genotypes were compared with regional current patterns to identify possible mechanisms controlling their distribution.     

Molecular subtyping of clinical isolates of <Emphasis Type="Italic">Candida albicans</Emphasis> and identification of <Emphasis Type="Italic">Candida dubliniensis</Emphasis>in Malaysia

The genotypes of 221 recent isolates of Candida albicans from various clinical specimens of 213 patients admitted to the University Malaya Medical Centre, Malaysia was determined based on the amplification of a transposable intron region in the 25 S rRNA gene. The analyses of 178 C. albicansisolated from nonsterile clinical specimens showed that they could be classified into three genotypes: genotype A (138 isolates), genotype B (38 isolates) and genotype C (2 isolates). The genotyping of 43 clinical isolates from sterile specimens showed that they belonged to genotype A (29 isolates), genotype B (10 isolates), genotype C (2 isolates) and genotype D (2 isolates). The overall distribution of C. albicans genotypes in sterile and nonsterile specimens appeared similar, with genotype A being the most predominant type. This study reported the identification of C. dubliniensis (genotype D) in 2 HIV-negative patients with systemic candidiasis, which were missed by the routine mycological procedure. The study demonstrated the genetic diversity of clinical isolates of C. albicans in Malaysia.     

Characterization of Hepatitis C Virus Genotypes by Direct Sequencing of HCV 5′UTR Region of Isolates from Saudi Arabia

The current study was designed to determine the Hepatitis C Virus (HCV) genotypes in a representative sample of HCV chronically infected patients in Saudi Arabia. All HCV isolates were genotyped by sequencing of the 5′UTR region and newly identified HCV isolates were identified. Specific universal primers targeting 5′UTR region were used for both amplification and sequencing of all isolates that resulted in 244 bp fragment which represent about 80% of 5′UTR region. Most of HCV isolates in this study were genotype 4 (76.4%) where only few isolates were recognized as genotype 1 (19.6%). All results were compared to HCV reference sequences from LOS ALAMOS HCV database, considering only the complete full genomes for the main phylogenetic analysis. Sequences that showed maximum identity (98% –100%) were selected. Most isolates were identical with HCV genotype 4 references. Some isolates were similar to different subtypes of HCV genotypes 4, 1 and 6. Phylogenetic analysis showed resemblance of most isolates to similar ones from the Far East, North America and Egypt. Using sequence Weblogo, Alignment analysis of isolated HCV genotypes 4 and 1 showed 92% and 95.5% nucleotide conservation, respectively. There was no predominant nucleotide in the varied sites, in both genotypes. All isolated sequences were submitted to GenBank database.     

The occurrence and rapid discrimination of Fomes fomentarius genotypes by ITS-RFLP analysis

Sequence comparison of available Fomes fomentarius (L.) J. Kickx f. internal transcribed spacer (ITS) of ribosomal DNA sequences demonstrated genetic non-homogeneity of the species. Multiple sequence alignment indicated the presence of two genotypes with overall similarity of about 97% and a strong statistics support. Rapid and reliable method for discrimination of F. fomentarius genotypes based on restriction digestion of polymerase chain reaction (PCR)-amplified ITS sequences was developed. BseNI and SchI restriction endonucleases were found to clearly discriminate between two F. fomentarius genotypes. The method was used to study the variability in F. fomentarius isolates collected from natural forest reserves in Vihorlat Mountains (East Slovakia). In most localities both genotypes occur concurrently. The isolates belonging to the genotype A were found to be prevalent on beech (Fagus sylvatica), while genotype B tends to be found mainly on other hosts. The grouping of selected isolates was confirmed by sequence analysis. Our results indicate that F. fomentarius includes at least two sympatric cryptic species.     

IGS基因及RAPD标记分析在加特隐球酵母基因分类中的应用

根据ITS1-5.8S-ITS2区域的特异核酸序列变化,加特隐球酵母Cryptococcus gattii(≡新型隐球酵母加特变种Cryptococcu neoformans var.gattii)可分为6种基因型。本研究通过检测加特隐球酵母的IGS基因,发现其IGS序列有着更丰富的变异和信息位点。通过结合加特隐球酵母RAPD(随机扩增的多态性DNA)图谱比较研究,与IGS和ITS的序列分析结果大体一致,说明新近发现的加特隐球酵母ITS8型确实有别于以前报道过的其他加特隐球酵母ITS基因型。研究证明IGS1及IGS2基因片段分析可以作为加特隐球酵母基因分类鉴定中有效的辅助鉴别的分子生物学方法,联合多种基因分类鉴定的方法可以更有效地揭示新型隐球酵母加特变种种内不同基因亚型间的遗传进化关系。     

A Molecular Biologic Study of Enterocytozoon bieneusi in HIV-Infected Patients in Lima, Peru

ABSTRACT. A cross-sectional study was conducted to examine the genotype distribution of Enterocytozoon bieneusi in HIV-infected patients who visited two government hospitals in Lima, Peru from January 2000 through March 2003. Microsporidia were detected by microscopy in 105 (3.9%) of 2,672 patients. A total of 212 stool samples from 89 microsporidia-positive patients were genotyped by sequence analysis of the internal transcribed spacer (ITS) region of the rRNA gene. A 392-bp fragment containing the complete ITS region was amplified and sequenced. Multiple alignments and phylogenetic analysis of these ITS sequences identified 11 distinct genotypes of E. bieneusi (Peru-1 to Peru-11), 6 of which were new genotypes not reported before. The remaining 5 genotypes had nucleotide sequences identical to those previously reported in humans, cats, pigs, and wild mammals. All the 11 E. bieneusi-genotypes identified are genetically related, and members of the group have been previously found in humans, domestic animals, and some wild mammals. Thus, there is a high genetic diversity of E. bieneusi in humans in Peru, and zoonotie transmission is possible if humans are in close contact with infected animals.     

Multiple infection of TT virus (TTV) with different genotypes in Japanese hemophiliacs

To clarify the persistent TT virus (TTV) infection, we studied a possibility of multiple TTV infection by genotype analysis of isolated TTV obtained from seven Japanese hemophiliacs. The nucleotide sequences including 222 bp in the open reading frame 1 (ORF1) region of 10 TTV isolates from each patient were analyzed and classified into various TTV genotypes such as G1 to G6 by phylogenetic analysis using a N-J method. Multiple TTV genotypes were observed in all the hemophiliacs: three different TTV genotypes were found in three patients, whereas four different TTV genotypes were observed in the other three patients. The remaining patient was also infected with TTV of five different genotypes. Moreover, new TTV genotypes were found in these seven patients and tentatively designated as G7. The present findings indicate that multiple TTV infection with different TTV genotypes has occurred in Japanese hemophiliacs. They also provide valuable information to understand persistent TTV infection.     

A multiplex PCR for unequivocal differentiation of all encapsulated and non-encapsulated genotypes of Trichinella

We have developed a single PCR test for the simple and unequivocal differentiation of all currently recognised genotypes of Trichilnella. Partial DNA sequence data were generated from internal transcribed spacers ITS1 and ITS2, and from the expansion segment V region of the ribosomal DNA repeat from five species of Trichinella and two additional genotypes, designated T5 and T6. Five different PCR primer sets were identified which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern for each species and genotype including three distinct genotypes of Trichinella pseudospiralis. The banding patterns for each parasite genotype consist of no more than two well-defined DNA fragments, except isolates of T. pseudospiralis which generate multiple, closely migrating bands. The expansion segment V-derived primer set contributes at least one fragment to each genotypic pattern and, therefore, functions both as a means for differentiation as well as an internal control for the PCR. The reliability and reproducibility of each DNA banding pattern were verified using multiple geographical isolates of each Trichinella genotype. The technique was developed further to distinguish genotypes at the level of single muscle larvae using a nested, multiplex PCR, whereby the entire internal transcribed spacer region as well as the gap region of the expansion segment V of the large subunit ribosomal DNA are amplified concurrently in a first-round PCR using primer sets specific for each region, followed by the multiplex PCR for final diagnosis.     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

黑龙江省大豆菌核病菌核盘菌遗传多样性分析

了解黑龙江省不同地区侵染大豆核盘菌菌株分离物间的主要特性差异,利用PDA培养基对核盘菌进行分离和纯化,同时利用RAPD和rDNA-ITS标记方法对核盘菌进行遗传多样性分析,获得了50株纯化的核盘菌,用RAPD标记确定的遗传相似系数范围为0.54-0.98,平均相似系数为0.76,说明供试的核盘菌菌株的基因型具有一定的差异。对50个测定序列有差异的32个核盘菌ITS和5.8S rDNA片段的多序列对位分析,在ITS1区域的1-40bp种间变化较大,主要以碱基颠换和转换为进化形式。ITS2区域非常保守没有变异位点。黑龙江省核盘菌菌株在DNA水平上和ITS间隔区上具有较显著的遗传变异,显示出丰富的遗传多样性。     

Molecular evidence for the presence of a G7 genotype of Echinococcus granulosus in Slovakia

Variability in Echinococcus granulosus is very important epidemiologically since strain characteristics may influence local patterns of transmission of hydatid disease. To classify the genotype presented in pig protoscoleces of the Slovak territory, a DNA-based approach has been used. Nucleotide sequences for a 471 bp region of the mitochondrial NADH dehydrogenase 1 (ND1) gene revealed a substantial affinity of isolates examined to the G7 genotype. Only a 0.9-3.4% sequence variation was recorded for E. granulosus samples compared with the reference G7 variant. To distinguish between G7 and G9 genotypes not differing in ND1 sequences, isolates were additionally examined by PCR-RFLP analysis of the nuclear ITS1 region. The resulting two-banded pattern is characteristic for the G7 strain. The data presented thus provides the first explicit evidence of the G7 genotype in the Slovak region.     

High Levels of Endemicity of 3-Chlorobenzoate-Degrading Soil Bacteria

Soils samples were obtained from pristine ecosystems in six regions on five continents. Two of the regions were boreal forests, and the other four were Mediterranean ecosystems. Twenty-four soil samples from each of four or five sites in each of the regions were enriched by using 3-chlorobenzoate (3CBA), and 3CBA mineralizers were isolated from most samples. These isolates were analyzed for the ability to mineralize 3CBA, and genotypes were determined with repetitive extragenic palindromic PCR genomic fingerprints and restriction digests of the 16S rRNA genes (amplified ribosomal DNA restriction analysis [ARDRA]). We found that our collection of 150 stable 3CBA-mineralizing isolates included 48 genotypes and 44 ARDRA types, which formed seven distinct clusters. The majority (91%) of the genotypes were unique to the sites from which they were isolated, and each genotype was found only in the region from which it was isolated. A total of 43 of the 44 ARDRA types were found in only one region. A few genotypes were repeatedly found in one region but not in any other continental region, suggesting that they are regionally endemic. A correlation between bacterial genotype and vegetative community was found for the South African samples. These results suggest that the ability to mineralize 3CBA is distributed among very diverse genotypes and that the genotypes are not globally dispersed.     

ERIC and REP-PCR Banding Patterns and Sequence Analysis of the Internal Transcribed Spacer of rDNA of Stemphylium solani Isolates from Cotton

The genetic diversity of the Stemphylium solani isolates from cotton was assessed by Enterobacterial Repetitive Intergenic Consensus (ERIC) and Repetitive Extragenic Palindromes (REP)-PCR fingerprinting. Twenty eight monosporic isolates of S. solani from cotton were used along with five isolates from tomato and one isolate of Alternaria macrospora from cotton for comparison. The dendrogram obtained revealed clear differences between the cotton and tomato isolates as well as between the tomato isolates from different geographic regions. The genetic relationships among S. solani isolates were also analyzed by sequencing the internal transcribed spacer (ITS) region of four isolates representing the three ERIC and REP groups. The tomato isolate from the State of S?o Paulo showed a distinct ITS sequence from that of the cotton isolates and tomato isolate from the State of Goiás, giving evidence that it belongs to a different genotype of S. solani. This is the first report of the entire sequence of the ITS1-5.8S-ITS2 regions of S. solani.     

New ITS genotype of Cryptococcus gattii isolated from an AIDS patient in Brazil

Based on combinations of nine variable nucleotides at nine different base positions in the ITS1-5.8S-ITS2 region, Cryptococcus gattii strains were classified into six genotypes. A new genotype of C. gattii , designated as ITS type 8, was isolated from an AIDS patient in Brazil. The ITS type 8 strain is closely related to the ITS type 4 strain, which has been frequently isolated in Brazil and the USA, but which shows ITS-signatured nucleotide difference at each nucleotide position. The ITS type 8 strain is also differentiated from all heretofore reported ITS types of C. gattii strains in the RAPD band patterns and IGS sequence information.     

大豆疫霉根腐病菌的rDNA ITS序列分析

采用真菌核糖体基因转录间隔区(ITS)通用引物,PCR扩增了大豆疫霉根腐病菌具有差异的17个菌株的ITSI与ITS2,经过与DL2000的标准分子量DNA进行比较,得到了大约800~1000bp左右的片段,并对PCR产物进行了序列测定。以USA为外类群利用最大简约法构建了大豆疫霉根腐病菌的系统发生树,并分析了菌株之间的遗传进化关系。结果表明:不同菌株ITS1和ITS2在碱基构成上有很大差异,17个菌株大致分为4个谱系中,且来自于同一地区的菌株大都分布在同一谱系中,显示出地理上的差异。     

Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi)

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.     

Genetically unique microsporidian Encephalitozoon cuniculi strain type III isolated from squirrel monkeys

Microsporidian spores were isolated from two squirrel monkeys (Saimiri sciureus) that had been bred at an animal-breeding colony in Japan. The spores were identified as Encephalitozoon cuniculi on the basis of nucleotide sequence analysis of the small-subunit (SSU) rRNA gene. The internal transcribed spacer (ITS) gene sequence revealed that these isolates were classified into genotype III because it contained tetrarepeats of 5'-GTTT-3'. However, the sequences of the polar tube protein (PTP) gene of the monkey isolates were not identical to a reported sequence of genotype III but were quite similar to a reported sequence of genotype II. On the other hand, sequence analysis of the spore wall protein 1 (SWP-1) gene revealed that the monkey isolates did not belong to any of genotypes I, II and III. These results suggest that the present E. cuniculi isolates of squirrel monkey origin are a new subtype of E. cuniculi ITS genotype III that can cause a disseminated infection.