Sequence analysis of the first internal transcribed spacer of rDNA supports the existence of the intermediate <Emphasis Type="Italic">Fasciola</Emphasis> between <Emphasis Type="Italic">F. hepatica</Emphasis> and <Emphasis Type="Italic">F. gigantica</Emphasis> in mainland China

In the present study, a polymerase chain reaction-linked single-strand conformation polymorphism (PCR-SSCP) approach combined with DNA sequencing was used to characterise samples of Fasciola spp. from different host species and geographical locations in mainland China. The first internal transcribed spacer (ITS-1) of ribosomal DNA (rDNA) was amplified by PCR from individual Fasciola and analysed by SSCP. SSCP analyses displayed three different banding profiles that allowed the identification of all Fasciola samples examined into three groups: Fasciola hepatica, F. gigantica and the “intermediate” Fasciola. Then, the ITS-1 rDNA was sequenced from representative Fasciola samples, and analysis of the complete ITS-1 sequences supported the identification of all Fasciola samples by SSCP approach. The length of the ITS-1 sequences was 422 bp for all Fasciola samples sequenced. Although there was no variation in length or composition of the ITS-1 sequences among multiple specimens within each of the taxa, F. hepatica and F. gigantica differed by 1.2% in their ITS-1 sequences, whereas the “intermediate” Fasciola was unique, in which two different ITS-1 sequences exist in the rDNA array within a single Fasciola worm. One of the sequences is identical to that of F. hepatica, and the other is identical to that of F. gigantica. This study demonstrated that PCR-SSCP analysis of the ITS-1 rDNA followed by selective sequencing provides a reliable approach for the accurate identification of Fasciola spp., and also supports the existence of the “intermediate” Fasciola between F. hepatica and F. gigantica in mainland China.     

鲁道夫对盲囊线虫rDNA ITS遗传标记的研究

本实验通过对鲁道夫对盲囊线虫(Contracaecum rudolphii)rDNA的第一及第二内转录间隔区(ITS-1及ITS-2)进行PCR扩增、PCR-SSCP分析及序列分析,以明确ITS-1及ITS-2是否可作为C.rudolphii分子分类的遗传标记.结果发现C.rudolphii rDNA ITS序列存在种间差异,并且差异显著,但是种内序列一致,没有差异.本实验证明C.rudolphii确为由两个种(C.rudolphii A和C.rudolphii B)组成的复合种,ITS-1及ITS-2可作为两个姊妹种的遗传标志.     

Prepatent periods of different Oesophagostomum spp. isolates in experimentally infected pigs

To define prepatent periods of different Oesophagostomum spp. isolates we carried out two separate experiments, one using two monospecific laboratory isolates and another using laboratory isolates as well as isolates obtained from pig herds having different management systems and with different anthelmintic treatment histories. Pigs were inoculated with 1,000–2,000 infective larvae. Fecal samples were collected daily beginning on days 15 and 16 postinoculation (p.i.). Fecal cultures were set up at different times to yield larvae that could be identified by DNA analyses. All pigs started to excrete eggs on days 18–24 p.i. The mean prepatent period was 20.2 ± 1.4 days, with no significant difference being observed between species and isolates. Prepatent periods of 17–19 days were found for the monospecific laboratory isolates of O. dentatum and O. quadrispinulatum. These findings conflict with parasitology textbooks; therefore, suggestions as to the possible reasons for the observed short prepatent periods are given. Received: 13 November 1996 / Accepted: 15 January 1997     

Genetic evidence for the existence of sibling species within Contracaecum rudolphii (Hartwich, 1964) and the validity of Contracaecum septentrionale (Kreis, 1955) (Nematoda: Anisakidae)

Specimens of Contracaecum rudolphii sensu lato (s.l.) (Nematoda: Anisakidae) from Phalacrocorax carbo sinensis from northeastern and central Italy were characterised genetically and compared with those from Phalacrocorax aristotelis from Galician coasts, Spain (identified as C. rudolphii A by multilocus enzyme electrophoresis) and with specimens of C. septentrionale from Alca torda from the Galician coasts, Spain. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified by polymerase chain reaction (PCR) from individual nematodes and the amplicons subjected to single-strand conformation polymorphism (SSCP) analysis and/or sequencing. For each ITS region, C. septentrionale specimens were distinct from those of C. rudolphii (s.l.) and C. rudolphii A based on SSCP profiles and ITS sequences. Some specimens of C. rudolphii (s.l.) had the same SSCP profiles and ITS sequences as C. rudolphii A, whereas the others had distinct SSCP profiles and ITS sequences and were suggested to represent C. rudolphii B based on host and geographical origins and genetic similarity to C. rudolphii A. While no length or nucleotide variation in the ITS-1 and ITS-2 sequences was detected within each taxon, nucleotide differences of 1.8–5.5% (ITS-1) and 5.1–12.2% (ITS-2) were detected among them. The results support the hypothesis that C. rudolphii represents a complex of at least two sibling species and provide support for the validity of C. septentrionale as a separate species. The definition of genetic markers in the ITS rDNA provides opportunities for investigating the life cycles, transmission patterns and ecology of the anisakid nematodes studied herein.Declaration: The experiments comply with the current laws of the countries in which the experiments were performed.     

Genetic characterisation of <Emphasis Type="Italic">Fasciola</Emphasis> samples from different host species and geographical localities revealed the existence of <Emphasis Type="Italic">F. hepatica</Emphasis> and <Emphasis Type="Italic">F. gigantica</Emphasis> in Niger

In the present study, 16 samples representing Fasciola (Platyhelminthes: Trematoda: Digenea) from sheep and cattle from seven geographical locations in Niger were characterized genetically by sequences of the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from individual liver flukes by polymerase chain reaction (PCR), and the amplicons were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 361/362 bp, respectively, for all liver fluke samples sequenced. Comparison of the ITS sequences of the Niger Fasciola samples examined in the present study with that of Fasciola hepatica, Fasciola gigantica, and the “intermediate Fasciola” from elsewhere revealed that the Niger Fasciola samples examined represent two species, namely F. hepatica and F. gigantica. This is the first demonstration of the existence of both F. hepatica and F. gigantica in Niger by a genetic approach, which provides foundation for further studies on F. hepatica and F. gigantica in Niger and has implications for studying the population genetic structure of the Niger Fasciola and for the diagnosis and control of the disease they cause. H. Ali and L. Ai contributed equally to this work.     

Specific PCR assays for the identification of common anisakid nematodes with zoonotic potential

Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) for six taxa of anisakids, namely, Anisakis simplex (s.s.), Anisakis typica, Anisakis pegreffii, Hysterothylacium aduncum, Hysterothylacium sp, and Contracaccum osculatum C, specific primers were designed in the ITS-1 and/or ITS-2 for each of the six anisakid taxa. These specific primers were used to develop polymerase chain reaction (PCR) tools for the identification of these anisakid taxa of sea fish by amplifying partial ITS-1 and/or ITS-2 of rDNA from anisakid nematodes. This approach allowed their specific identification, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the DNA fragments amplified. The minimum amounts of DNA detectable using the PCR assays were 0.5–1 ng. These PCR tools were then applied to ascertain the specific identity of 143 anisakid larval samples collected from fish in China, Canada, Thailand, and Indonesia, and these anisakid samples were identified to represent one of the six anisakid taxa. These PCR assays based on ITS sequences should provide useful molecular tools for the accurate identification and molecular epidemiological investigations of anisakid infections in humans and fish.     

Effect of time on migration of Oesophagostomum spp. and Hyostrongylus rubidus out of agar-gel

The agar-gel migration technique has previously been described, however, aspects regarding the effect of timing on worm migration needed further scrutiny. In the first experiment, pigs inoculated with Oesophagostomum dentatum were slaughtered simultaneously and their intestines stored at 21–23 °C until processed pairwise 2, 4, 6, 8, 12 and 18 h after slaughter. More than 95% of the worms migrated out of the agar if processed within 6 h. In the second experiment, intestines were treated immediately after slaughter and the migratory speed of adult worms or 4th-stage larvae of O. dentatum or O. quadrispinulatum, or adult Hyostrongylus rubidus were studied. For both Oesophagostomum species, more than 90% of the worms were recovered within 1 h. H. rubidus was significantly slower; however, approximately 98% of the worms had migrated out of the agar-gel by 20 h. This information is essential in planning experiments where recovery of live worms is of value. Received: 11 July 1997 / Accepted: 15 September 1997     

Molecular characterization of hard and soft ticks from Romania by sequences of the internal transcribed spacers of ribosomal DNA

In the present study, four hard tick species and one soft tick species, namely, Dermacentor marginatus, Haemaphysalis punctata, Haemaphysalis parva, Ixodes ricinus, and Dermanyssus gallinae, from south-western Romania were characterized genetically by the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA), using a hard tick, Haemaphysalis longicornis, from China for comparative purposes. The ITS rDNA was amplified by polymerase chain reaction (PCR) and sequenced from individual ticks. The lengths of the ITS-1 sequences were 238–1819 bp, and the lengths of ITS-2 were 137–1695 bp, respectively, for all ticks sequenced. While sequence variation within a hard tick species was 0–1.5%, nucleotide differences between hard tick species ranged 2–25.2%, indicating that ITS rDNA sequences provide genetic markers for the differentiation of hard ticks from Romania. Hence, a PCR-linked restriction fragment length polymorphism approach was developed for their unequivocal differentiation based on ITS-1 rDNA. This is the first characterization of ticks from Romania using a genetic approach, which provides the foundation for further studies on ticks in Romania and has implications for studying the population genetic structure of the Romanian ticks and for identification and differentiation of closely related ticks. An erratum to this article can be found at     

<Emphasis Type="Italic">Cryptosporidium</Emphasis> spp. parasitize exotic birds that are commercialized in markets,commercial aviaries,and pet shops

The purpose of this study was to genetically characterize and phylogenetically analyze the Cryptosporidium spp. isolated from exotic birds commercialized in popular markets, commercial aviaries, and pet shops located in Rio de Janeiro, Brazil. Fecal samples from individually housed birds were collected and subjected to centrifuge–flotation technique using saturated sugar solution. DNA was isolated from Cryptosporidium positive samples, and 18S subunit rDNA was amplified and processed using nested-polymerase chain reaction (PCR). To identify the protozoan species, the PCR amplicons were used for restriction fragment length polymorphism and sequencing analyses. Of the 103 analyzed fecal samples, seven (6.8%) were positive for Cryptosporidium oocysts. Sequencing and further phylogenetic analyses allowed us to identify the following species: Cryptosporidium parvum in Bengalese finch (Lonchura striata domestica) and avian genotype III in Java sparrow (Padda oryzivora) and cockatiel (Nymphicus hollandicus). The sequences of the Cryptosporidium spp. isolated from canaries (Serinus canarius) were not identifiable within the groups of known species, but they presented a higher genetic similarity with C. parvum. This is the first report in Brazil showing that C. parvum parasitizes Bengalese finches and that avian genotype III parasitizes Java sparrows.     

Characterization of <Emphasis Type="Italic">Fasciola</Emphasis> samples from different host species and geographical localities in Spain by sequences of internal transcribed spacers of rDNA

In the present study, 25 samples representing Fasciola (Platyhelminthes: Trematoda: Digenea) from nine host species and 19 geographical locations in Spain were characterized genetically by sequences of the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from individual liver flukes by polymerase chain reaction (PCR), and the amplicons were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 362 bp, respectively, for all Spanish liver fluke samples sequenced. Comparison of the ITS sequences of the Spanish Fasciola samples examined in the present study with that of Fasciola hepatica, Fasciola gigantica and the “intermediate Fasciola” revealed that all Spanish Fasciola samples examined represent the single species of F. hepatica, with only slight sequence variation in the ITS-2 (1/362, 0.3%) among the sequenced samples, but the sequence variation was not related to particular host species and/or geographical origins of the samples. The Spanish F. hepatica examined differed from Fasciola from elsewhere by two nucleotides in the ITS-2, which provided genetic marker for the differentiation of Spanish F. hepatica from Fasciola from other geographical localities. These results have implications for studying the population genetic structure of the Spanish F. hepatica and for the diagnosis and control of the disease it causes.     

Application of the PCR-sequence-specific primers for the discrimination among larval <Emphasis Type="Italic">Anisakis simplex</Emphasis> complex

The fish-borne zoonotic nematode, Anisakis simplex, is not a single specie but a complex composed of three sibling species, A. simplex sensu stricto, Anisakis pegreffii, and A. simplex C. Discrimination among these sibling species have been performed by polymerase chain reaction (PCR)–restriction fragment length polymorphism or sequencing analysis of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). In the present study, PCR-sequence-specific primers were applied for the discrimination of these parasites. Based on the sequence differences among A. simplex complex at the ITS region of rDNA, forward primer ASF1 that is expected to be specific to A. pegreffii was newly developed. The specific fragment was amplified in only A. pegreffii by PCR with ASF1, but not quite in A. simplex s. str., Anisakis physeteris, Pseudoterranova decipiens, and Contracaecum osculatum. This result suggests that PCR using the forward primer ASF1 might be applicable for the discrimination of A. simplex complex.     

Experimental hybridization between Oesophagostomum dentatum and O. quadrispinulatum in pigs

To investigate eventual hybridization between two nodular worm species of pigs, Oesophagostomum dentatum and O. quadrispinulatum, we used either mature, adult worms or 10-day-old fourth-stage larvae (L₄) as starting material, employing a nonsurgical transplantation technique. Following the transfer of adult worms the ensuing first generation of larvae gave rise to adult worms that were found by morphological examination to be purely O. dentatum. Therefore, we decided to use the immature L₄ as starting material. After the transfer of L₄ to recipient pigs, fecal cultures were established and the L₃ derived from the O. dentatum male/O. quadrispinulatum female cross gave rise to adult but infertile worms, which morphologically had the sexual characters of their parent generation, whereas other characteristics were intermediate between the two species. Attempts to reproduce the hybrid worms or the reciprocal cross were unsuccessful, indicating that hybridization between the two species is a rarely occurring phenomenon. Received: 6 April 1997 / Accepted: 15 May 1997     

Use of random amplified polymorphic DNA-polymerase chain reaction for the definition of genetic markers for species and strains of porcine Oesophagostomum

Nodular worms are common parasites of pigs, and research has recently started to focus on the biology of these nematodes. However, the methods for delineation of species at immature developmental stages and␣for␣differentiation of various lines of the same species␣remain limited. For differentiation of porcine Oesophagostomum species and strains by genomic fingerprinting, random amplified polymorphic DNA-polymerase chain reaction was performed on DNA derived from 20 larval batches of anthelmintic-susceptible and resistant strains and isolates of these nematodes and 2 ruminant Oesophagostomum spp. Polymorphic DNA markers could be amplified with 9 of the 33 primers tested. In all, 13 markers were species-specific and 6 markers could differentiate between strains or groups of strains. With a combination of the latter, artificially selected anthelmintic-resistant strains and the susceptible mother strain of O. dentatum could be delineated. When single adult worms were compared, considerable variations between strains of the same species and between individuals from the same strain could be detected. The differentiation of Oesophagostomum strains and species at all parasitic stages on the basis of genetic markers could greatly facilitate studies on the biology of these parasites. Received: 6 January 1997 / Accepted: 24 March 1997     

Genetic diversity in porcine Oesophagostomum dentatum and O. quadrispinulatum and their delineation by isoenzyme analysis

The genetic diversity in eight strains of Oesophagostomum dentatum and O. quadrispinulatum was investigated by the electrophoresis study of ten enzyme systems. The loci Idh-2, Fbp, Sdh, and Pgm were found to be diagnostic between the species examined. Both the proportion of fixed allelic differences (26.3%) and the genetic distance coefficient (D=0.54) are well above the range for differentiation of valid species. Isoenzyme patterns of susceptible and resistant lines of O. dentatum showed at polymorphic loci a reduced genetic heterogeneity in the latter group. No qualitative difference in terms of the presence/absence of alleles was observed among susceptible and resistant isolates with the enzymes studied. The detection of one possible hybrid indicates that introgression in O. dentatum and O. quadrispinuatum may occur. Received: 28 June 1997 / Accepted: 18 August 1997     

Assessing sequence variation in the internal transcribed spacers of ribosomal DNA within and among members of the Contracaecum osculatum complex (Nematoda: Ascaridoidea: Anisakidae)

The anisakid nematodes of seals from different geographical origins, previously identified as Contracaecum osculatum A, C. osculatum B, C. osculatum C, C. osculatum D, C. osculatum E and C. osculatum baicalensis by multilocus enzyme electrophoresis, were characterised using a DNA approach. The first and second internal transcribed spacers (ITS-1, ITS-2) of ribosomal DNA (rDNA) were individually amplified by polymerase chain reaction (PCR) and analysed by single-strand conformation polymorphism (SSCP) and sequencing. SSCP analyses allowed the unequivocal differentiation of all taxa except C. osculatum D from C. osculatum E. While C. osculatum D and C. osculatum E had identical ITS sequences, each of the other four taxa had distinct sequences, with interspecific differences ranging from 0.3% to 2.3%. C. osculatum C was genetically the most distinct taxon with respect to all other members of the species complex. Received: 20 November 1999 / Accepted: 23 December 1999     

Parasitic helminth fauna of the cutlass fish,<Emphasis Type="Italic"> Trichiurus lepturus</Emphasis> L., and the differentiation of four anisakid nematode third-stage larvae by nuclear ribosomal DNA sequences

The helminth fauna of the gastrointestinal tract and abdominal cavity of cutlass fish, Trichiurus lepturus L., off the Taiwanese coast of the north-western Pacific was investigated. The following helminths were found: (1) nematodes—Anisakis simplex, Hysterothylacium aduncum, Porrocaecum decipiens, Raphidascaris trichiuri; (2) digeneans—adult Lecithochirium trichiuri; and (3) cestodes—plerocercoids of Proteocephalus spp. The third-stage larvae of these four anisakid nematodes were characterized genetically using a molecular approach. The nuclear ribosomal DNA region spanning the first internal transcribed spacer (ITS-1), the 5.8S gene and the second internal transcribed spacer (ITS-2) was amplified and sequenced. Based on the sequence differences, a PCR-based restriction fragment length polymorphism method was established for the unequivocal delineation of the four species. Phylogenetic analysis showed that H. aduncum clustered with P. decipiens, whereas A. simplex was not closely related to these according to the nucleotide sequences of all rDNA.     

Molecular characterization of <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> isolates from the Philippines

Trichomonas vaginalis is a human urogenital pathogen that causes trichomoniasis, the most common nonviral parasitic sexually transmitted infection in the world. Presently, there are no reports on comparative sequence analysis as well as on the identification of phylogenetic positions of T. vaginalis isolates from the Philippines relative to known trichomonads. In this study, 5.8S rDNA and the flanking internal transcribed spacer (ITS) regions of 57 T. vaginalis isolates were sequenced. The phylogenetic positions of the isolates relative to known trichomonads were determined using the model-based (GTR+Γ+I) neighbor-joining, maximum likelihood, and Bayesian-inference analyses and the nonmodel-based maximum parsimony analysis. Construction of a phylogenetic tree showed the clustering of all the sequences in one branch together with other T. vaginalis strains obtained through basic local alignment search tool search. Sequencing of the 5.8S rDNA gene and the flanking ITS1and ITS2 regions of T. vaginalis isolates from the Philippines demonstrated low genetic polymorphism. However, comparison of the ribosomal DNA sequences may have implications on some phenotypic characteristics of T. vaginalis.     

Identification of anisakid nematodes with zoonotic potential from Europe and China by single-strand conformation polymorphism analysis of nuclear ribosomal DNA

Using genetic markers defined previously in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA), isotopic, and non-isotopic polymerase-chain-reaction-coupled single-strand conformation polymorphism (SSCP) were utilized to identify each of three anisakid species [Anisakis simplex (s.l.), Contracaecum osculatum (s.l.), and Hysterothylacium aduncum] from different host species and geographical locations in Poland and Sweden. While subtle microheterogeneity was observed within each of Anisakis simplex (s.l.) and H. aduncum, distinct SSCP profiles were displayed for each of the three species, allowing identification and differentiation of the three taxa. Subsequent sequencing of the ITS-1 and ITS-2 rDNA revealed that A. simplex (s.l.) represented Anisakis simplex s.s. and Contracaecum osculatum (s.l.) represented C. osculatum C. Application of the non-isotopic SSCP assay of ITS-2 to larval anisakid samples from different hosts and geographical locations in China revealed three distinct SSCP profiles, one of which was consistent with that of A. simplex (s.l.), and the other two had different SSCP profiles from that of C. osculatum C and H. aduncum. Sequencing of the ITS-1 and ITS-2 rDNA for representative Chinese anisakid samples examined revealed three anisakid species in China, i.e., Anisakis typica, Anisakis pegreffii, and Hysterothylacium sp. These molecular tools will be useful for identification and investigation of the ecology of anisakid nematodes in China and elsewhere.     

A single-strand conformation polymorphism (SSCP) approach for investigating genetic interactions of <Emphasis Type="Italic">Schistosoma haematobium</Emphasis> and <Emphasis Type="Italic">Schistosoma guineensis</Emphasis> in Loum,Cameroon

Single-strand conformation polymorphism (SSCP) analysis of the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA provides a molecular tool for the identification of Schistosoma haematobium, Schistosoma guineensis and the hybrids of these two species. This molecular tool was utilized to provide a detailed analysis of the interactions between S. haematobium and S. guineensis in hybrid zones of Loum, Littoral Province, Cameroon. Individual hybrid schistosomes were identified within the natural populations collected from Loum in 1990, 1999 and 2000, which would have been misidentified as S. haematobium using solely morphological and sequence criteria. This study indicates the complexities of the hybridization between S. haematobium and S. guineensis and emphasizes the importance of assessing morphological, biological and molecular data to gain insights into the interaction of these two species over time.     

Rapid identification of pathogenic yeast isolates by real-time PCR and two-dimensional melting-point analysis

There is a need in the clinical microbiological laboratory for rapid and reliable methods for the universal identification of fungal pathogens. Two different regions of the rDNA gene complex, the highly polymorphic ITS1 and ITS2, were amplified using primers targeting conserved regions of the 18S, 5.8S and 28S genes. After melting-point analysis of the amplified products, the T_m of the two PCR-products were plotted into a spot diagram where all the 14 tested, clinically relevant yeasts separated with good resolution. Real-time amplification of two separate genes, melting-point analysis and two-dimensional plotting of T_m data can be used as a broad-range method for the identification of clinical isolates of pathogenic yeast such as Candida and Cryptococcus spp.