Non-specificity of sequence characterised amplified region as an alternative molecular epidemiology marker for the identification of Salmonella enterica subspecies enterica serovar Typhi.

OBJECTIVE: Identification of Salmonella Typhi by conventional culture techniques is labour-intensive, time consuming, and lack sensitivity and specificity unlike high-throughput epidemiological markers that are highly specific but are not affordable for low-resource settings. SCAR, obtained from RAPD technique, is an affordable, reliable and reproducible method for developing genetic markers. Hence, this study investigated the use of SCAR as an alternative molecular epidemiological marker for easy identification of S. Typhi in low-resource settings. RESULTS: One hundred and twenty RAPD primers were screened through RAPD-PCR against a panel of common enterobacteriaceae for the best RAPD band pattern discrimination to develop SCAR primers that were used to develop a RAPD-SCAR PCR. Of this number, 10 were selected based on their calculated indices of discrimination. Four RAPD primers, SBSA02, SBSA03, SBSD08 and SBSD11 produced suitable bands ranging from 900 to 2500 bp. However, only SBSD11 was found to be specific for S. Typhi, and was cloned, sequenced and used to design new SCAR primers. The primers were used to amplify a panel of organisms to evaluate its specificity. However, the amplified regions were similar to other non-Typhi genomes denoting a lack of specificity of the primers as a marker for S. Typhi.

Standardization and validation of simple PCR, duplex PCR and RAPD in comparison to blood smear examination for diagnosing bovine tropical theileriosis.

Bovine Tropical Theileriosis (BTT) is an important vector-borne protozoan disease that imposing serious constraints on the health and productivity of domestic cattle. It is matter of common fact that following recovery from primary infection, cattle become persistent carriers and act as reservoirs of infection thereby, playing a critical role in disease epidemiology. The present study describes the comparative diagnostic efficiency of simplex PCR, duplex PCR and RAPD assays for detection of Theileria annulata in cattle. An optimized simple PCR and duplex PCR assay were established using TAMS F/R as primer sets encoding for 721 bp amplicon alongside a RAPD with arbitrary primer coding for 963 bp product of T. annulata. The simple PCR and duplex PCR detected pathogen with almost same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA of another pathogen without nonspecific amplifications. RAPD failed to give comparable results and suffered from limitations of sensitivity as well as specificity. The developed assays may be seen as a good tool for epidemiological studies aiming at assessing the burden of chronic infections and improving control of the associated diseases in endemic regions.

Identification and quantification of Lactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA.

Lactobacillus casei strain Shirota (LcS) has been used in the production of fermented milk products for many years and is one of the most intensively studied probiotics. To evaluate the ability of LcS to proliferate in human intestines after it has been ingested, we developed a PCR-based method to identify and quantify LcS using an LcS-specific primer set (pLcS) derived from a randomly amplified polymorphic DNA (RAPD) analysis. We confirmed the high specificity of the pLcS primer set in 167 bacterial strains (57 strains of L. casei and 110 other strains of bacteria commonly isolated from human feces). The method's ability to identify LcS matched that of an ELISA using a monoclonal antibody and a RAPD analysis in a representative sample of colonies cultured from human feces. The detection limit of quantitative PCR (qPCR) using pLcS was 10(4.6) per gram of feces. The number of LcS in feces detected with qPCR was highly and significantly correlated with the number of LcS added to fecal samples within the range of 10(4.6) to 10(9.6) per gram feces (r(2)=0.999, P<0.001). After 14 healthy subjects ingested 10(11.0) CFU of LcS daily for 7 days, 10(9.1+/-0.5) LcS g(-1) (mean+/-S.D.) was detected in the fecal samples of all subjects by qPCR, and 10(8.0+/-0.9) CFU g(-1) was detected by culture; these values were significantly different (P<0.001, paired t-test). After the subjects stopped ingesting LcS, fecal LcS counts obtained with both methods decreased daily. The values produced by the 2 methods might have differed because of an overestimation in the PCR analysis due to the presence of dead LcS cells or an underestimation in the culture system due to the use of selective culture media; however, dead LcS cells can also be beneficial as immunomodulators. We confirmed that qPCR with an LcS-specific primer set was a rapid and accurate method for determining the total amount of LcS in feces including dead or less active cells which could not be detected by culture method.

Molecular epidemiology of Pseudomonas aeruginosa in adult patients with cystic fibrosis in Northern Ireland.

Isolates (n = 51) of Pseudomonas aeruginosa obtained from the sputa of 29 adult patients attending the Regional Cystic Fibrosis Centre in Northern Ireland were compared using an enterobacterial repetitive intergenic consensus sequence (ERIC2) primer in a random amplification of polymorphic DNA (RAPD) polymerase chain reaction (PCR) method. Resulting banding patterns showed a high degree of genetic heterogeneity among all isolates from the patients examined, suggesting a non-clonal relationship between isolates from these patients, when employing this genotyping technique.

[Comparison of different primers used for the genotyping of Candida albicans clinical isolates by randomly amplified polymorphic DNA method]. / Candida albicans klinik izolatlarinin "randomly amplified polymorphic DNA" yöntemiyle genotiplendirmesinde kullanilan farkli primerlerin karsilastirilmasi.

Candida albicans causes severe infections with high mortality rates especially in immunocompromised patients. The aim of this study was to evaluate the efficiency of randomly amplified polymorphic DNA (RAPD) method and compare the discriminatory powers (DP) of different primers used for genotyping Candida albicans isolates. A total of 109 C. albicans strains recovered from throat, sputum, blood, feces, urine, vagina and wound cultures of 65 hospitalized paediatric patients with haematologic malignancy were evaluated by RAPD method using 10 different primers (OPE-03, OPE-04, OPE-12, OPE-18, OPE-19, OPE-20, OPF-10, OPF-12, P1 and P2) between June 1999-April 2003. Strains were separated into groups by analyzing band patterns derived from each primer and the DP was calculated. Reproducibility of the method was determined by evaluating randomly chosen 20 isolates with the same and different PCR devices under the same PCR conditions. C. albicans isolates generated 1-16 bands and were grouped in 41-80 genotypes depending on the primers used. DP of the RAPD method was calculated as > or = 0.90 for each primer (range between 0.90-0.99), which were accepted as reliable values. However, the strains clustered in the same group when studied with a primer could be dispersed into different groups by another primer. The reproducibility of the method was poor and the comparison of band patterns was difficult especially in isolates which generated many bands. In conclusion, for obtaining reliable results by RAPD method, using more than one primer and comparative analysis of these primers are appropriate. RAPD is an adequate method for studying small outbreaks in which a few number of isolates are evaluated, but it is laborious and unreliable for many number of isolates recovered in a long time period because of its poor reproducibility and difficulties in evaluating the strains generating many bands.

Genetic diversity in populations of Dalbulus maidis (DeLong and Wolcott) (Hemiptera: Cicadellidae) from distant localities in Brazil assessed by RAPD-PCR markers.

Populations of Dalbulus maidis (DeLong and Wolcott) from the northeastern and central-southern regions of Brazil differ morphologically, suggesting that they could be genetically isolated. Here we used the random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) technique to estimate genetic structuring of this leafhopper species among five geographically distant localities across those regions and to estimate gene flow between populations. Ten specimens were sampled per population and genotyped with RAPD markers generated from amplification with nine oligonucleotides. The percentage of polymorphic loci was 78% in relation to the total number of amplified loci, and genetic similarity either between or within populations was higher than 0.72. Cluster analysis grouped specimens from the northeastern population (Mossoró/RN) into a single group, whereas central-southern specimens were not grouped in relation to their places of origin. Overall, the genetic subdivision index (Fst) was low (or= 0.192 and Nm

Proposed minimal standards for the description of genera, species and subspecies of the Pasteurellaceae.

Principles and guidelines are presented to ensure a solid scientific standard of papers dealing with the taxonomy of taxa of Pasteurellaceae Pohl 1981. The classification of the Pasteurellaceae is in principle based on a polyphasic approach. DNA sequencing of certain genes is very important for defining the borders of a taxon. However, the characteristics that are common to all members of the taxon and which might be helpful for separating it from related taxa must also be identified. Descriptions have to be based on as many strains as possible (inclusion of at least five strains is highly desirable), representing different sources with respect to geography and ecology, to allow proper characterization both phenotypically and genotypically, to establish the extent of diversity of the cluster to be named. A genus must be monophyletic based on 16S rRNA gene sequence-based phylogenetic analysis. Only in very rare cases is it acceptable that monophyly can not be achieved by 16S rRNA gene sequence comparison. Recently, the monophyly of genera has been confirmed by sequence comparison of housekeeping genes. In principle, a new genus should be recognized by a distinct phenotype, and characters that separate the new genus from its neighbours should be given clearly. Due to the overall importance of accurate classification of species, at least two genotypic methods are needed to show coherence and for separation at the species level. The main criterion for the classification of a novel species is that it forms a monophyletic group based on 16S rRNA gene sequence-based phylogenetic analysis. However, some groups might also include closely related species. In these cases, more sensitive tools for genetic recognition of species should be applied, such as DNA-DNA hybridizations. The comparison of housekeeping gene sequences has recently been used for genotypic definition of species. In order to separate species, phenotypic characters must also be identified to recognize them, and at least two phenotypic differences from existing species should be identified if possible. We recommend the use of the subspecies category only for subgroups associated with disease or similar biological characteristics. At the subspecies level, the genotypic groups must always be nested within the boundaries of an existing species. Phenotypic cohesion must be documented at the subspecies level and separation between subspecies and related species must be fully documented, as well as association with particular disease and host. An overview of methods previously used to characterize isolates of the Pasteurellaceae has been given. Genotypic and phenotypic methods are separated in relation to tests for investigating diversity and cohesion and to separate taxa at the level of genus as well as species and subspecies.

Improvement and validation of RAPD in combination with PFGE analysis of Salmonella enterica ssp. enterica serovar Senftenberg strains isolated from feed mills.

In 1995 and 1996 a Swedish feed mill had problems due to a persistent contamination of Salmonella enterica spp. enterica serovar Senftenberg that was difficult to eliminate. Forty-eight strains isolated from the feed mill, together with unrelated strains included to evaluate the discriminatory power and reproducibility, were analysed by pulsed-field gel electrophoresis (PFGE). The source of contamination in the feed mill was identified and preventative measures were taken, that led to a resolution of the problem. A previously developed randomly amplified polymorphic DNA (RAPD) protocol was used, to evaluate a rapid and low-cost alternative to PFGE typing. The use of the alternative thermostable DNA polymerase Tth was shown to increase the reproducibility of the RAPD analysis. The reproducibility, in terms of Pearson's and Dice's similarity coefficients for duplicate runs, increased from 72.0 +/- 16.9% and 72.3 +/- 12.9% for Taq to 91.6 +/- 7.5% and 90.9 +/- 5.3% for the fingerprints obtained for the RAPD method employing Tth DNA polymerase. Simpson's index of diversity was calculated and found to be 0.580 for RAPD and 0.896 for PFGE. All of the seven RAPD types could be subdivided into one or more PFGE types, whereas none of the 22 PFGE types was divided into more than one RAPD type. RAPD provides a simple, rapid and powerful screening method that can be used to initially select isolates for further analysis by PFGE.

RAPD for the typing of coagulase-negative staphylococci implicated in catheter-related bloodstream infection.

OBJECTIVES: A rapid random amplification of polymorphic DNA (RAPD) technique was developed to distinguish between strains of coagulase-negative staphylococci (CoNS) involved in central venous catheter (CVC)-related bloodstream infection. Its performance was compared with that of pulsed-field gel electrophoresis (PFGE). METHODS: Patients at the University Hospital Birmingham NHS Foundation Trust, U.K. who underwent stem cell transplantation and were diagnosed with CVC-related bloodstream infection due to CoNS whilst on the bone marrow transplant unit were studied. Isolates of CoNS were genotyped by PFGE and RAPD, the latter employing a single primer and a simple DNA extraction method. RESULTS: Both RAPD and PFGE were highly discriminatory (Simpson's diversity index, 0.96 and 0.99, respectively). Within the 49 isolates obtained from blood cultures of 33 patients, 20 distinct strains were identified by PFGE and 25 by RAPD. Of the 25 strains identified by RAPD, nine clusters of CoNS contained isolates from multiple patients, suggesting limited nosocomial spread. However, there was no significant association between time of inpatient stay and infection due to any particular strain. CONCLUSION: The RAPD technique presented allows CoNS strains to be genotyped with high discrimination within 4h, facilitating real-time epidemiological investigations. In this study, no single strain of CoNS was associated with a significant number of CVC-related bloodstream infections.

Identification of a rice gene (Bph 1) conferring resistance to brown planthopper (Nilaparvata lugens Stal) using STS markers.

This study was carried out to identify a high-resolution marker for a gene conferring resistance to brown planthopper (BPH) biotype 1, using japonica type resistant lines. Bulked segregant analyses were conducted using 520 RAPD primers to identify RAPD fragments linked to the BPH resistance gene. Eleven RAPDs were shown to be polymorphic amplicons between resistant and susceptible progeny. One of these primers, OPE 18, which amplified a 923 bp band tightly linked to resistance, was converted into a sequence-tagged-site (STS) marker. The STS marker, BpE18-3, was easily detectable as a dominant band with tight linkage (3.9cM) to Bph1. It promises to be useful as a marker for assisted selection of resistant progeny in backcross breeding programs to introgress the resistance gene into elite japonica cultivars.

Optimization of random amplified polymorphic DNA techniques for use in genetic studies of Cuban Triatominae.

Random amplified polymorphic DNA (RAPD) technique is a simple and reliable method to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of assay. In this study, we analyzed the effect of changing the concentration of primer, magnesium chloride, template DNA and Taq DNA polymerase with the objective of determining their optimum concentration for the standardization of RAPD technique for genetic studies of Cuban Triatominae. Reproducible amplification patterns were obtained using 5 pmoL of primer, 2.5 mM of MgCl2, 25 ng of template DNA and 2 U of Taq DNA polymerase in 25 microL of the reaction. A panel of five random primers was used to evaluate the genetic variability of T. flavida. Three of these (OPA-1, OPA-2 and OPA-4) generated reproducible and distinguishable fingerprinting patterns of Triatominae. Numerical analysis of 52 RAPD amplified bands generated for all five primers was carried out with unweighted pair group method analysis (UPGMA). Jaccard's Similarity Coefficient data were used to construct a dendrogram. Two groups could be distinguished by RAPD data and these groups coincided with geographic origin, i.e. the populations captured in areas from east and west of Guanahacabibes, Pinar del Río. T. flavida present low interpopulation variability that could result in greater susceptibility to pesticides in control programs. The RAPD protocol and the selected primers are useful for molecular characterization of Cuban Triatominae.

Optimization of random amplified polymorphic DNA techniques for use in genetic studies of Cuban triatominae

La técnica de ADN polimórfico amplificado al azar (RAPD) es un método simple para detectar el polimorfismo genético del ADN. Diferentes factores afectan los perfiles de amplificación lo que se manifiesta en la presencia de bandas falsas y en la reproducibilidad del ensayo. En nuestro trabajo analizamos los cambios de la concentración de cebador, ADN molde, cloruro de magnesio y de Taq ADN polimerasa con el objetivo de determinar su concentración optima, quedando optimizada la técnica del RAPD para su utilización en estudios genéticos de Triatomíneos cubanos. Empleando una concentración de cebador de 5 pmol, 2.5 mM de MgCl2, 25 ng de ADN molde y 2 U de Taq ADN polimerasa en 25 µL de reacción, se obtuvieron patrones de amplificación reproducibles. Un total de cinco cebadores al azar fueron usados para evaluar la variabilidad genética de T. flavida. Tres de ellos (OPA-1, OPA-2 y OPA-4) produjeron patrones distinguibles y reproducibles de triatomineos. El análisis numérico según la técnica de UPGMA usando el coeficiente de similitud de Jaccard a partir de las 52 bandas de amplificación de RAPD generadas por los cinco cebadores, fue usado en la construcción del dendograma. Se obtuvieron 2 grupos bien definidos según el análisis del RAPD, mostrando concordancia con el origen geográfico, las poblaciones capturadas en áreas del occidente y el oriente de Guanahacabibes, Pinar del Río, respectivamente. T. flavida presentó una baja variabilidad genética inter-poblacional y esto puede resultar en una mayor susceptibilidad al uso de insecticidas en los programas de control. La técnica de RAPD optimizada y los cebadores seleccionados son útiles para la caracterización molecular de Triatomíneos cubanos.

Epidemiological study of invasive pulmonary aspergillosis in a haematology unit by molecular typing of environmental and patient isolates of Aspergillus fumigatus.

In order to determine the possible relationship between environmental contamination by Aspergillus fumigatus and occurrence of invasive aspergillosis, a one-year prospective study was carried out in the haematology ward of Hautepierre Hospital, Strasbourg, France. During the study period, 21 environmental isolates and 26 clinical isolates of A. fumigatus were collected. Each was genotyped using a random amplification of polymorphic DNA (RAPD) technique. Thirty-four distinct profiles were identified by RAPD analysis, indicating the great genetic diversity of A. fumigatus isolated from infected patients and from the environment. For two patients, RAPD analysis demonstrated concurrent infection by at least two different strains. In two cases, a genetic similarity was noted between isolates obtained from a patient and from the environment.

El RAPD (random amplified Polymorphism of DNA)en el entorno de los marcadores moleculares / The RAPD (random amplified Polymorphism of DNA)en the environment of those molecular markers

Los marcadores moleculares constituyen una herramienta útil en diferentes campos de la investigación, salud, producción, etc. Con éstos se pueden establecer: relaciones genéticas entre organismos y definir el estatus de especie, diagnóstico de enfermedades interviniendo directamente en la ubicación del gen en cuestión o identificando organismos que causan la enfermedad, igualmente en la producción de alimentos con ciertas características, como en la detección de alimentos transgénicos. Actualmente los investigadores están buscando moleculares que se tienen a mano, cada una de éstas tienen ventajas y desventajas. En este trabajo se pretende mostrar de manera general muchos aspectos de estos marcadores, haciendo énfasis en la técnica de RAPD.

Evaluation of randomly amplified polymorphic DNA and pulsed field gel electrophoresis techniques for molecular typing of Dermatophilus congolensis.

This study aimed to evaluate molecular typing methods useful for standardization of strains in experimental work on dermatophilosis. Fifty Dermatophilus congolensis isolates, collected from sheep, cattle, horse and a deer, were analyzed by randomly amplified polymorphic DNA (RAPD) method using twenty-one different primers, and the results were compared with those obtained by typing with a pulsed field gel electrophoresis (PFGE) method using the restriction digest enzyme Sse8387I. The typeability, reproducibility and discriminatory power of RAPD and Sse8387I-PFGE typing were calculated. Both typing methods were highly reproducible. Of the two techniques, Sse8387I-PFGE was the least discriminating (Dice Index (DI), 0.663) and could not distinguish between epidemiologically related isolates, whereas RAPD showed an excellent discriminatory power (DI, 0.7694-0.9722). Overall, the degree of correlation between RAPD and PFGE typing was significantly high (r, 0.8822). We conclude that the DNA profiles generated by either RAPD or PFGE can be used to differentiate epidemiologically unrelated isolates. The results of this study strongly suggest that at least two independent primers are used for RAPD typing in order to improve its discriminatory power, and that PFGE is used for confirmation of RAPD results.

Optimization of randomly amplified polymorphic DNA-polymerase chain reaction for molecular typing of Salmonella enterica serovar Typhi.

Optimization of the RAPD reaction for characterizing Salmonella enterica serovar Typhi strains was studied in order to ensure the reproducibility and the discriminatory power of this technique. Eight Salmonella serovar Typhi strains isolated from various regions in Brazil were examined for the fragment patterns produced using different concentrations of DNA template, primer, MgCl2 and Taq DNA polymerase. Using two different low stringency thermal cycle profiles, the RAPD fingerprints obtained were compared. A set of sixteen primers was evaluated for their ability to produce a high number of distinct fragments. We found that variations associated to all of the tested parameters modified the fingerprinting patterns. For the strains of Salmonella enterica serovar Typhi used in this experiment, we have defined a set of conditions for RAPD-PCR reaction, which result in a simple, fast and reproducible typing method.

[Evaluation of three methods for genotyping of nosocomial methicillin resistant Staphylococcus aureus isolates]. / Nozokomiyal metisiline dirençli Staphylococcus aureus izolatlarinin genotiplendirilmesinde üç ayri yöntemin incelenmesi.

Plasmid profile analysis (PPA), random amplification of polymorphic DNA (RAPD) and pulsed field gel electrophoresis (PFGE) are the three most valuable epidemiological tools for genotyping of methicillin resistant Staphylococcus aureus (MRSA) strains. Aim of this study was to evaluate these three methods in respect to their cost, reproducibility, and discriminatory power. Eighty one nosocomial MRSA isolates with unknown genetic and epidemiological relatedness from Training Hospital of Gulhane Military Medical School, were genotyped by PPA, RAPD, and PFGE methods. All isolates (100%) were typed by RAPD and PFGE, however, eight (9.9%) isolates could not be typed by PPA since they lacked plasmid DNA. Reproducibilities of all the three methods were found to be 100 percent. Discriminatory powers of PPA, RAPD and PFGE methods were calculated as 48.6%, 61.1% and 80.1%, respectively. In conclusion, out of the three methods tested, PFGE allowed the most effective discrimination of MRSA strains. However, PFGE was more time consuming and technically demanding, and required use of specialized and expensive equipment. Although PPA and RAPD were less discriminatory than PFGE, these methods were technically simple, rapid and cheaper. When PPA and RAPD were used in combination, they had equal discriminatory power to PFGE. Thus, it should be emphasized that PPA and RAPD methods could be preferred for initial screening purposes while PFGE should be used as a confirmatory test in genotyping of MRSA isolates.

Random amplified polymorphic DNA technique for identification and differentiation of old world Leishmania species.

The random amplified polymorphic DNA technique may be used to explore parasite DNA polymorphisms. We assessed its applicability to identification of Old World Leishmania species. A set of 6 random decamer primers (Al, A4, A5, A7, A10, and A15) was applied to a panel of DNA from 57 representatives of different Old World Leishmania species. The amplification profiles allowed discrimination among species belonging to different taxonomic complexes. Two criteria were used to analyze the profiles: the presence of consistent amplicons at the same electrophoretic position for isolates of the same species, and the presence of distinct amplicons for isolates of different species. Three primers--Al, A7 and A10--rendered such products.

A simple random amplified polymorphic DNA genotyping method for field isolates of Dermatophilus congolensis.

Dermatophilus congolensis is the pathogenic actinomycete that causes dermatophilosis in cattle, lumpy wool in sheep and rain scald in horses. Phenotypic variation between isolates has previously been described, but its genetic basis, extent and importance have not been investigated. Standard DNA extraction methods are not always successful for D. congolensis due to its complex life cycle, one stage of which is encapsulated. Here we describe the development of rapid and reliable DNA extraction and random amplified polymorphic DNA (RAPD) methods that can be used for genotyping D. congolensis field isolates. Our results suggest that genotypic variation between isolates correlates with host species. Several DNA extraction methods and RAPD protocols were compared. An extraction method based on incubation of the bacterium in lysozyme, sodium dodecyl sulphate (SDS) and proteinase K treatments and phenolic extraction yielded high-quality DNA, which was used to optimize RAPD-polymerase chain reaction (PCR) protocols for two random primers. An alternative rapid, non-phenolic extraction method based on proteinase K treatment and thermal shock was selected for routine RAPD typing of isolates. DNA extracted from reference strains from cattle, sheep and horse using either method gave reproducible banding patterns with different DNA batches and different thermal cyclers. The rapid DNA extraction method and RAPD-PCR were applied to 38 D. congolensis field isolates. The band patterns of the field and type isolates correlated with host species but not with geographical location.

Schistosoma japonicum strains: differentiation by RAPD and SSR-PCR.

Eight geographical isolates of Schistosoma japonicum from Taiwan and mainland China and one isolate of Schistosoma mansoni were studied by RAPD analysis using six arbitrary primers and SSR-PCR analysis using a (CA)8RY primer. The genetic distance was determined by the percentage of unshared bands. The RAPD and SSR-PCR results showed that the genetic distance between S. mansoni and S. japonicum was more than 0.900 and 0.850 respectively; the genetic distance between the eight geographical isolates of S. japonicum was 0.000 to 0.232 and 0.066 to 0.368 respectively. These results demonstrated the usefulness of RAPD and SSR-PCR for showing the differences of inter- and intra-species of Schistosoma. The results also suggest that there is genetic diversity among the different geographical strains of S. japonicum in China.