PCR-RFLP patterns of four isolates of Trichinella for rDNA ITS1 region

We have studied the genetic differences among four isolates of Trichinella including a new strain of Trichinella spiralis (ISS 623) recently found from a human case who took a badger in Korea. Because they have a different host origin and came from geographically separated regions, we supposed the genetic pattern of the isolates might be different as had been previously reported. It was analysed by PCR-RFLP analysis of the rDNA repeat that can readily distinguish a species or strain from others. Isolated genomic DNA of each isolate of Trichinella larvae was amplified with ITS1 specific primers and digested with restriction endonucleases. The PCR product of ITS1 was confirmed using Southern blot analysis to be a 910 bp fragment. The restriction fragments of each isolate had variable patterns when it was digested with Rsa 1 only. According to the RFLP patterns, the estimated genetic divergence between each isolate was different. In conclusion, four isolates of Trichinella including a new strain of T. spiralis obtained from a Korean patient may have genetic differences in the ITS1 region and the Shanghai isolate was genetically more similar to the Japanese unknown isolate than others in the ITS1 region.     

Genetic heterogeneity of Pneumocystis carinii from rats of several regions and strains

Pneumocystis carinii is a major opportunistic pathogen which has been found in the lungs of a wide variety of mammalian host species, and the fact suggests the possibility of intraspecific variation. Until now, P. carinii from different mammalian species are differentiated as subspecies, and the rats are known to be infected by two subspecies. The present study investigated genetic heterogeneity of P. carinii isolates from two strains of rats in Korea and China by molecular karyotyping, RFLP and sequencing analysis. Karyotypes of P. carinii were grouped into three, two from two strains of rats in Korea and one from rats in China. However RFLP of PCR product of ribosomal and MSG gene of the P. carinii isolates showed same pattern. The sequence homology rates of alpha-tubulin DNA of the P. carinii isolates were 96% in Seoul Wistar rats, 93% in Seoul Sprague-Dawley rats, and 85% in Chinese Sprague-Dawley rats. The present finding confirmed that P. carinii from rats in Korea are grouped into two karyotype strains which are different from that of P. carinii from rats in China. The Chinese isolate shows a little different sequences of alpha-tubulin DNA.     

Two new genotypes of Plasmodium vivax circumsporozoite protein found in the Republic of Korea

The gene encoding Plasmodium vivax circumsporozoite protein (PvCSP) exhibits polymorphism in many geographical isolates. The present study was designed to investigate polymorphism in PvCSP gene of P. vivax isolates in Korea. Thirty isolates, obtained from indigenous cases in Yonchon-gun, Kyonggi-do in 1997, were subjected for sequencing and RFLP analysis of the repeat and post-repeat regions of PvCSP gene and two genotypes (SK-A and SK-B) were identified. The genotype of 19 isolates was SK-A and that of 11 isolates was SK-B. Although the number of 12-base repeats present in SK-A was three while two were found in a Chinese strain CH-5, the repeat sequence of SK-A was identical to that of CH-5 except for one base substitution. Compared with known data there was no identical isolates with SK-B, but the sequence of SK-B was similar to that of a North Korean (NK) isolate. These results indicate that two genotypes of PvCSP coexist in the present epidemic area of Korea and the present parasite may originate from East Asia. RFLP would be useful to classify genotypes of P. vivax population instead of gene sequencing.     

Genetic analyses of Acanthamoeba isolates from contact lens storage cases of students in Seoul, Korea.

We conducted both the small subunit ribosomal DNA (SSU rDNA) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and mitochondrial (mt) DNA RFLP analyses for a genetic characterization of Acanthamoeba isolates from contact lens storage cases of students in Seoul, Korea. Twenty-three strains of Acanthamoeba from the American Type Culture Collection and twelve clinical isolates from Korean patients were used as reference strains. Thirty-nine isolates from contact lens storage cases were classified into seven types (KA/LS1, KA/LS2, KA/LS4, KA/LS5, KA/LS7, KA/LS18, KA/LS31). Four types (KA/LS1, KA/LS2, KA/LS5, KA/LS18) including 33 isolates were regarded as A. castellanii complex by riboprints. KA/LS1 type was the most predominant (51.3%) in the present survey area, followed by KA/LS2 (20.9%), and KA/LS5 (7.7%) types. Amoebae of KA/LS1 type had the same mtDNA RFLP and riboprint patterns as KA/E2 and KA/E12 strains, clinical isolates from Korean keratitis patients. Amoebae of KA/LS2 type had the identical mtDNA RFLP patterns with A. castellanii Ma strain, a corneal isolate from an American patient as amoebae of KA/LS5 type, with KA/E3 and KA/E8 strains from other Korean keratitis patients. Amoebae of KA/LS18 type had identical patterns with JAC/E1, an ocular isolate from a Japanese patient. Three types, which remain unidentified at species level, were not corresponded with any clinical isolate in their mtDNA RFLP and riboprint patterns. Out of 39 isolates analyzed in this study, mtDNA RFLP and riboprint patterns of 33 isolates (84.6%) were identical to already known clinical isolates, and therefore, they may be regarded as potentially keratopathogenic. These results suggest that contact lens wearers in Seoul should pay more attention to hygienic maintenance of contact lens storage cases for the prevention of Acanthamoeba keratitis.     

The detection of non-RoTat 1.2 Trypanosoma evansi

The majority of Trypanosoma evansi can be detected using diagnostic tests based on the variant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypanozoon antigen type (RoTat) 1.2. Exceptions are a number of T. evansi isolated in Kenya. To characterize T. evansi that are undetected by RoTat 1.2, we cloned and sequenced the VSG cDNA from T. evansi JN 2118Hu, an isolate devoid of the RoTat 1.2 VSG gene. A 273 bp DNA segment of the VSG gene was targeted in PCR amplification for the detection of non-RoTat 1.2 T. evansi. Genomic DNA samples from different trypanosomes were tested including 32 T. evansi, 10 Trypanosoma brucei, three Trypanosoma congolense, and one Trypanosoma vivax. Comparison was by PCR amplification of a 488 bp fragment of RoTat1.2 VSG gene. Results showed that the expected 273 bp amplification product was present in all five non-RoTat 1.2 T. evansi tested and was absent in all 27 RoTat 1.2-positive T. evansi tested. It was also absent in all other trypanosomes tested. The PCR test developed in this study is specific for non-RoTat 1.2 T. evansi.     

Molecular identification of a Trichinella isolate from a naturally infected pig in Tibet, China

The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial large-subunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.     

Morphologic and genetic identification of Taenia tapeworms in Tanzania and DNA genotyping of Taenia solium

Species identification of Taenia tapeworms was performed using morphologic observations and multiplex PCR and DNA sequencing of the mitochondrial cox1 gene. In 2008 and 2009, a total of 1,057 fecal samples were collected from residents of Kongwa district of Dodoma region, Tanzania, and examined microscopically for helminth eggs and proglottids. Of these, 4 Taenia egg positive cases were identified, and the eggs were subjected to DNA analysis. Several proglottids of Taenia solium were recovered from 1 of the 4 cases. This established that the species were T. solium (n = 1) and T. saginata (n = 3). One further T. solium specimen was found among 128 fecal samples collected from Mbulu district in Arusha, and this had an intact strobila with the scolex. Phylegenetic analysis of the mtDNA cox1 gene sequences of these 5 isolates showed that T. saginata was basal to the T. solium clade. The mitochondrial cox1 gene sequences of 3 of these Tanzanian isolates showed 99% similarity to T. saginata, and the other 2 isolates showed 100% similarity to T. solium. The present study has shown that Taenia tapeworms are endemic in Kongwa district of Tanzania, as well as in a previously identified Mbulu district. Both T. solium isolates were found to have an "African/Latin American" genotype (cox1).     

Sequence Variation in Superoxide Dismutase Gene of Toxoplasma gondii among Various Isolates from Different Hosts and Geographical Regions

Toxoplasma gondii, an obligate intracellular protozoan parasite of the phylum Apicomplexa, can infect all warm-blooded vertebrates, including humans, livestock, and marine mammals. The aim of this study was to investigate whether superoxide dismutase (SOD) of T. gondii can be used as a new marker for genetic study or a potential vaccine candidate. The partial genome region of the SOD gene was amplified and sequenced from 10 different T. gondii isolates from different parts of the world, and all the sequences were examined by PCR-RFLP, sequence analysis, and phylogenetic reconstruction. The results showed that partial SOD gene sequences ranged from 1,702 bp to 1,712 bp and A + T contents varied from 50.1% to 51.1% among all examined isolates. Sequence alignment analysis identified total 43 variable nucleotide positions, and these results showed that 97.5% sequence similarity of SOD gene among all examined isolates. Phylogenetic analysis revealed that these SOD sequences were not an effective molecular marker for differential identification of T. gondii strains. The research demonstrated existence of low sequence variation in the SOD gene among T. gondii strains of different genotypes from different hosts and geographical regions.     

Comparisons of Isolates of Heterodera avenae using 2-D PAGE Protein Patterns and Ribosomal DNA

Six geographic isolates of Heterodera avenae, including two isolates each from Sweden, Australia, and the United States, were compared on the basis of 2-D PAGE protein patterns and the complete DNA sequence for the two internal transcribed ribosomal DNA spacers (rDNA ITS1 and ITS2) and the 5.8S rRNA gene. The protein pattern data and rDNA ITS sequence data both indicated that the Swedish Gotland strain of H. avenae differed markedly from the rest of the isolates. Protein patterns for the Australia isolates differed more from a Swedish strict H. avenae isolate and isolates from Oregon and Idaho, than the two U.S. isolates and the Swedish strict H. avenae isolate differed from each other. Except for the Gotland strain isolate, the rDNA ITS sequences were highly conserved among all of the H. avenae isolates, just as we earlier found them to be conserved among species of the schachtii group of Heterodera.     

Genetic relatedness among Trypanosoma evansi stocks by random amplification of polymorphic DNA and evaluation of a synapomorphic DNA fragment for species-specific diagnosis.

In this study we employed randomly amplified polymorphic DNA patterns to assess the genetic relatedness among 14 Brazilian Trypanosoma evansi stocks from domestic and wild hosts, which are known to differ in biological characteristics. These akinetoplastic stocks were compared with one another, to three Old World (Ethiopia, China and Philippines) dyskinetoplastic stocks of T. evansi, and also with Trypanosoma equiperdum, Trypanosoma brucei brucei, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Randomly amplified polymorphic DNA analysis showed limited heterogeneity in T. evansi stocks from different hosts and geographical regions of the world, or in other species of the subgenus Trypanozoon. However, minor variations generated random amplification of polymorphic DNA analysis disclosed a pattern consisting of a unique synapomorphic DNA fragment (termed Te664) for the T. evansi cluster that was not detected in any other trypanosome species investigated. Pulsed field gel electrophoresis analysis demonstrated that the Te664 fragment is a repetitive sequence, dispersed in intermediate and minichromosomes of T. evansi. Based on this sequence, we developed a conventional PCR assay for the detection of T. evansi using crude preparations of blood collected either on glass slides or on filter paper as template DNA. Our results showed that this assay may be useful as a diagnostic tool for field-epidemiological studies of T. evansi.     

Trypanosoma evansi: cloning and expression in Spodoptera frugiperda [correction of fugiperda] insect cells of the diagnostic antigen RoTat1.2

A complementary DNA encoding the variant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypanozoon antigenic type (RoTat)1.2, currently used for experimental serological diagnosis of T. evansi infection in livestock, was cloned as a recombinant plasmid and sequenced. A recombinant baculovirus containing the coding region of RoTat1.2 VSG was constructed to express the protein in Spodoptera frugiperda [corrected] insect cells. From this, sufficient quantities of the recombinant protein are being produced for empirical and wide-scale objective assessment of the diagnostic potential of this antigen. The gene encoding the RoTat1.2 VSG was shown by PCR to be present in the genomes of many different cloned isolates of T. evansi, but not T. brucei, from geographically separate regions of Africa, Asia, and South America. With the recombinant RoTat1.2 at hand, it is now possible to investigate the extent to which epitopes on this VSG are conserved among different T. evansi isolates.     

Double-stranded RNA virus in Korean isolate IH-2 of Trichomonas vaginalis

In this study, we describe Korean isolates of Trichomonas vaginalis infected with double-stranded (ds) RNA virus (TVV). One T. vaginalis isolate infected with TVV IH-2 evidenced weak pathogenicity in the mouse assay coupled with the persistent presence of a dsRNA, thereby indicating a hypovirulence effect of dsRNA in T. vaginalis. Cloning and sequence analysis results revealed that the genomic dsRNA of TVV IH-2 was 4,647 bp in length and evidenced a sequence identity of 80% with the previously-described TVV 1-1 and 1-5, but only a 42% identity with TVV 2-1 and 3 isolates. It harbored 2 overlapping open reading frames of the putative capsid protein and dsRNA-dependent RNA polymerase (RdRp). As previously observed in the TVV isolates 1-1 and 1-5, a conserved ribosomal slippage heptamer (CCUUUUU) and its surrounding sequence context within the consensus 14-nt overlap implied the gene expression of a capsid protein-RdRp fusion protein, occurring as the result of a potential ribosomal frameshift event. The phylogenetic analysis of RdRp showed that the Korean TVV IH-2 isolate formed a compact group with TVV 1-1 and 1-5 isolates, which was divergent from TVV 2-1, 3 and other viral isolates classified as members of the Giardiavirus genus.     

Molecular phylogenetics and diagnosis of soil and clinical isolates of Halicephalobus gingivalis (Nematoda: Cephalobina: Panagrolaimoidea), an opportunistic pathogen of horses

Phylogenetic relationships among six isolates of Halicephalobus gingivalis (Stefanski, 1954), a species with pathogenic potential in horses and humans, were evaluated using DNA sequences from the nuclear large-subunit ribosomal RNA (LSU rDNA) gene. Sequences from nematodes obtained from in vitro cultures (soil or clinical sources), or isolated from infected horse tissues, were compared. Gene sequences from a fatal equine clinical case from southern California and a free-living isolate recovered from southern California soil showed no fixed differences. Sequences from isolates representing two fatal equine cases from North America, one from Ontario, Canada and another from Tennessee also showed no fixed differences. In contrast, two equine cases from Tennessee had 18 fixed differences for this LSU region, the greatest observed among isolates from horses. Phylogenetic analysis of six Halicephalobus sequences and four outgroup taxa by maximum parsimony yielded one tree with five well-supported clades. This phylogeny did not group isolates of Halicephalobus strictly by region of geographic isolation or source of sample, and depicted one clinical and one soil isolate as sister taxa. These results confirm that free-living environmental isolates are potential sources of infection for horses. The phylogeny also reveals that diverse isolates can cause infections in horses within a relatively limited geographic region, and conversely that genetically similar sister taxa can be recovered from geographically distant localities. PCR primers that selectively amplify Halicephalobus DNA were designed and tested based on comparison of closely related nematodes as inferred from phylogenetic analysis.     

Extreme DNA sequence variation in isolates of Aspergillus fumigatus

DNA sequence analysis of the alkaline protease gene was used to investigate two Aspergillus fumigatus strains isolated from ostriches (QLD1 and NSW3) and one environmental isolate (FRR 1266) that have shown genetic variation in previous analyses. The results showed that the QLD1 sequence was virtually identical to the published sequences for three human isolates but NSW3 differed in >6% and FRR 1266 in >10% of the nucleotides that were analysed. An RFLP assay was designed to determine the distribution of these (and other) genetic variants among environmental and clinical isolates of A. fumigatus.     

Characterization of Heterorhabditis Isolates by PCR Amplification of Segments of mtDNA and rDNA Genes

Restriction digests of amplified DNA from the mitochondrial genome and the nuclear ribosomal internally transcribed spacer region have been evaluated as genetic markers for species groups in Heterorhabditis. Six RFLP profiles have been identified. These profiles supported groupings determined by cross-breeding studies and were in agreement with less definitive groupings based on other biochemical and molecular methods. Digestion patterns of both amplification products provided strong evidence for the recognition of species groups, which include Irish, NW European, tropical, and a H. bacteriophora complex. The H. bacteriophora complex could be further resolved into three genotypes represented by H. zealandica, the H. bacteriophora, Brecon (Australian) type isolate for H. bacteriophora, and a grouping composed of isolates NC1, V16, HI82, and HP88. All cultures obtained of the H. megidis isolate were identical to the NW European group. These results could be used to aid monitoring of field release of Heterorhabditis as well as allowing a rapid initial assessment of taxonomic grouping.     

Randomly Amplified Polymorphic DNA Differs with Burrowing Nematode Collection Site,but not with Host Range

The genetic variability of 12 burrowing nematode (Radopholus sp.) isolates from Central America, the Caribbean, and Florida, and one isolate from Ivory Coast were compared with RAPD analysis. A high degree of genetic similarity (>0.82) was determined for isolates from the Western Hemisphere. Genome similarity was greatest among isolates collected within a country. Among isolates collected in Central America and the Caribbean, burrowing nematodes from Belize and Guatemala were genetically more distant. However, the genome of the isolate from Ivory Coast was most dissimilar (>0.30). These results suggest that African and American burrowing-nematode isolates may have had different origins or that they have been geographically isolated for a sufficient amount of time to have accumulated genetic changes detectable by RAPD analysis. No relationship was found between the genomic similarity and extent of reproduction or damage to banana or citrus roots. Morphometric analysis involving eight of the isolates indicated that they were morphologically identical and values for morphometric parameters were well within the range previously published for banana and citrusparasitic burrowing nematodes.     

Reproductive Fitness and Random Amplified Polymorphic DNA Variation among Isolates of Pratylenchus vulnus

The reproductive fitness of seven isolates of Pratylenchus vulnus from different geographical areas and hosts was assessed in monoxenic cultures (carrot), and greenhouse cultures (plum, sour orange, and quince). The genetic makeup of the different isolates was compared by Random Amplified Polymorphic DNA (RAPD-PCR). The apple (PvAP-S) and apricot (PvAT-F) isolates reproduced less in monoxenic cultures than the rose (PvRO-S) and walnut (PvWA-A and PvWA-U) isolates. On plum, the rose isolate (PvRO-S) reproduced better than the apple (PvAP-S) and walnut isolate from the United States (PvWA-U). On sour orange, the apple (PvAP-S), unknown origin (PvU-UK), and walnut isolate from Argentina (PvWA-A) multiplied well, whereas the walnut isolate from the United States (PvWA-U), apricot (PvAT-F), and rose (PvRO-S) did not. On quince, the apple (PvAP-S) and walnut (PvWA-U) isolates showed a higher reproduction than the one from unknown origin (PvU-UK). RAPD-PCR patterns among the seven P. vulnus isolates were similar, although high intraspecific varibility was detected. Very few bands of P. neglectus were shared by any population of P. vulnus. A high degree of similarity was found among the patterns corresponding to the rose (PvRO-S), apple (PvAP-S), walnut from the United States (PvWA-U), and unknown origin (PvUK-U) isolates. The apricot isolate (PvAT-F) was the most dissimilar among the seven isolates. No correlation could be established between the genetic variation of P. vulnus detected by RAPD-PCR and reproductive fitness. Results demonstrate high genetic varibility between geographically separated populations of P. vulnus.     

Molecular and Biochemical Diversity Among Isolates of Radopholus spp. from Different Areas of the World

Eleven isolates of Radopholus similis from various banana-growing areas around the world and one isolate of R. bridgei from turmeric in Indonesia were compared using DNA and isoenzyme analysis. The polymerase chain reaction (PCR) was used to amplify a fragment of ribosomal DNA (rDNA), comprising the two internal transcribed spacers (ITS) and the 5.8S gene. Restriction fragment length polymorphisms (RFLPs) in this rDNA fragment were used to compare the 10 isolates. The analysis of this rDNA region revealed little variation among the isolates tested. However, data also were obtained by random amplified polymorphic DNA (RAPD) analysis of total DNA, and a hierarchical cluster analysis of these data arranged the R. similis isolates into two clusters. The first cluster consisted of isolates from Nigeria, Cameroon, Queensland, and Costa Rica; the second was comprised of isolates from Guinea, Guadeloupe, the Ivory Coast, Uganda, and Sri Lanka. The isolate of R. bridgei from turmeric in Indonesia appeared to be more divergent. This grouping was consistent with that obtained when phosphate glucose isomerase (PGI) isoenzyme patterns were used to compare the R. similis isolates. The results from both RAPD analysis and PGI isoenzyme studies indicate that two gene pools might exist within the R. similis isolates studied. No correlation could be detected between the genomic diversity as determined by RAPD analysis and either geographic distribution of the isolates or differences in their pathogenicity. The results support the hypothesis that R. similis isolates have been spread with banana-planting material.