Preliminary study on genetic variability of Dicrocoelium dendriticum determined by random amplified polymorphic DNA

Genetic variability of adult specimens of Dicrocoelium dendriticum has been studied using random amplified polymorphic DNA (RAPD). The worms were collected from the infected livers of different sheep from several localities in León province (NW Spain). DNA fragments were amplified by means of decamer primer oligonucleotides of arbitrary sequence. Some primers produce complex and highly variable patterns of amplified DNA in D. dendriticum. Intra- and inter-population genetic variability of adult parasites were analyzed, scoring polymorphic and monomorphic reproducible bands by means of the Jaccard similarity, and dendrograms showing genetic relationships between individuals were obtained using the FITCH method. Genetic variability seems to be high in this parasite and genetic similarity within a population (worms infecting a single animal) is similar to the average similarity between worms from different sheep. These results suggest that each sheep is infected by numerous genetically different parasites from one or more populations of infected ants.     

Isoenzymes of lactate dehydrogenase (EC 1.1.1.27) in Dicrocoelium dendriticum and Fasciola hepatica (Trematoda)

The isoenzymes of LDH (EC 1.1.1.27) were studied in Dicrocoelium dendriticum and Fasciola hepatica by horizontal electrophoresis on polyacrylamide gel. Six isoenzymes in D. dendriticum and four in F. hepatica were detected. Densitometric scans demonstrated that the enzyme pattern of LDH in D. dendriticum parasitizing hepatic tissue of Capra hircus was clearly different from the one obtained when the trematode parasitized other hosts such as Bos taurus or Ovis aries.     

The occurrence of hepatic trematodes in sheep from Emilia-Romagna

In the present investigations, 563 samples of ovine faeces from 32 flocks of Emilia-Romagna have been examined for the presence of hepatic trematodes eggs. All flocks proved positive for Dicrocoelium dendriticum and 16 for Fasciola hepatica; 511 (90,76%) out of 563 animals proved positive, with 507 (90,05%) for D. dendriticum and 50 (8,88%) for F. hepatica, 46 of these last ones were positive for both parasites.     

Detection of antibodies in wild ruminants to evaluate exposure to liver trematodes

Wild ruminants sharing pastures with domestic livestock are at risk of infection by liver trematodes. Detection of antibodies provides a very useful tool to gain more knowledge about the distribution of these parasites. Non-lethal methods are strongly encouraged for the analysis of the risk of infection among wild ruminants. A seroepidemiological survey was conducted to analyze exposure to hepatic trematodes ( Fasciola hepatica and Dicrocoelium dendriticum ) in wild ruminants from southern Spain. Blood samples were collected from 69 bovids (Mouflon + Spanish ibex) and 143 cervids (red deer + fallow deer) from Sierra de Cazorla, Segura and Las Villas Natural Park. The samples were analyzed using the excretory/secretory antigens of each trematode to determine the IgG response. All the animals were examined at necropsy for the presence of flukes, and the species, age, and gender of the animals were recorded. Fasciola hepatica were only observed in cervids (3%; 95% confidence interval [CI] = 2-8), while D. dendriticum specimens were recorded in 1% (0-8) of bovids and 4% (CI = 2-9) of the cervids. The IgG-seroprevalence against F. hepatica was significantly higher in the cervids. Statistical differences according to gender were observed. The bovids exhibited the greatest percentages of positive cases to D. dendriticum antigens, and the DdES-seroprevalence was related to age of the animals. When considering all the factors, the FhES-seroprevalence was initially distributed according to the type of ruminant (cervids), gender (male), and age (>2 yr).     

Patterns of genetic variation in native grape phylloxera on two sympatric host species

Random amplified polymorphic DNA (RAPD) markers were used to examine population genetic structure in populations of native grape phylloxera. This research asked: (i) do RAPD markers distinguish two groups corresponding to the two host plant species; and (ii) do RAPD markers distinguish groups according to spatial location, independent of host plant association? Forty‐nine phylloxera clones were collected from five pairs of adjacent individuals of two sympatric grape species in five sites along a 145 km transect in Missouri, USA. A high level of polymorphism was observed, with some evidence for structuring between host plant species and no evidence for spatial structuring. An analysis of molecular variance (amova ) found that 6.52% of the variance in RAPD banding patterns was attributable to host species and 7.96% of the variance was attributable to spatial location. A cluster analysis did not result in two groups corresponding to the two hosts, or to five groups corresponding to the geographical sites sampled. A Mantel test showed a low correlation between genetic similarity and spatial location. Two of the 93 RAPD markers were nonrandomly associated between the hosts. It is suggested that there may be a small host‐mediated effect on genetic variation but stochastic dispersal and a highly heterogeneous environment may be the primary influences on the observed polymorphism.     

Genetic variation of Casuarina equisetifolia subsp equisetifolia and C. equisetifolia subsp incana populations on the northern coast of Senegal

The genetic variation of 70 individual samples of Casuarina equisetifolia (L. Johnson) subsp equisetifolia and C. equisetifolia subsp incana growing along the northern coast of Senegal was analyzed with RAPD markers. Of the 160 primers tested, five were chosen; they generated 1396 reproducible bands and 61 polymorphic bands that were scored. This result showed a narrow genetic variation among (4.36%) and within (5.90%) C. equisetifolia subsp equisetifolia and C. equisetifolia subsp incana plantation sites. The genetic variation at each site revealed a high degree of polymorphism in Potou (5.90%) and low diversity in Retba (3.06%). In the dendrogram analyses, each sampling site was formed by two main groups. Similar results were found for the dendrograms based on the RAPD data gathered from the five different sites. These dendrograms revealed several polytomies in one of the subgroups, suggesting replication of the same specimens in different sites along the Senegalese coast. The RAPD data support the hypothesis that these populations are of the same provenance, subject to hybridization and inbreeding depression.     

Molecular variability of Spodoptera frugiperda (Lepidoptera: Noctuidae) populations associated to maize and cotton crops in Brazil

The molecular variability among 10 populations of Spodoptera frugiperda (J.E. Smith), collected from maize, Zea mays L., or cotton Gossypium hirsutum L. crops located at distinctive geographical regions in Brazil, was assessed through random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR). In total, 208 RAPD markers were evaluated, and 98% of them were polymorphic. The mean genetic similarity was 0.6621 and 0.2499 by the Simple Matching and Jaccard matrices, respectively. In general, the unweighted pair-group method with arithmetic average dendrograms separated the populations into clusters related to the geographical origin of the samples. No branch of the dendrograms underpinning a molecular association of S. frugiperda has been identified to either of the two host plants. The molecular variance analysis showed that 18 and 82% of the genetic variation was distributed among and within the groups of populations, respectively. The principal coordinate analysis reinforced the pattern of population clustering found with the unweighted pair-group method with arithmetic average method. These results suggest the occurrence of considerable gene flow between S. frugiperda populations from maize and cotton fields located in the same region in Brazil. Therefore, for an effective management of this pest, there is an urgent need for a better understanding of the gene flow of S. frugiperda populations associated to different host plants along the distribution range of this pest over time in a specific cropping system.     

利用RAPD和ISSR分子标记分析怀地黄种质遗传多样性

用RAPD与ISSR技术对怀地黄的8个品种和2个脱毒品系进行了种质遗传多样性分析。分别从80条RAPD引物和44条ISSR引物中筛选出适合怀地黄种质分析的17条RAPD引物和10条ISSR引物,用于RAPD和ISSR分析。17条RAPD引物共扩增出177条带, 多态性位点数为109; 多态性位点比率为61.58%;平均多样性指数(I)为0.3135;每个位点的有效等位基因数（Ne）是1.3641; 10条ISSR引物共扩增出110条带. 多态性位点数为79; 多态性位点比率为71.58%;平均多样性指数(I)为0.3577;每个位点的有效等位基因数（Ne）是1.4037。 基于扩增条带数据库建立了各自的Jaccard遗传相关系数矩阵,构建了相似的分子树状图,将10个供试材料分为2类:一类群含组培85.5、大田85.5、组培9302、大田9302、金状元和金白6个材料;另一类群含北京1号、大红袍、地黄9104和野生地黄4个材料。两种分子标记的分析结果呈极显著正相关(r＝0.649)。结果表明,RAPD与ISSR标记适合于怀地黄种质遗传多样性分析,ISSR标记技术是一种多态性和重复性优于RAPD技术的实用技术。     

Parasiticidal effects of peroxynitrite on ovine liver flukes in vitro

Peroxynitrite (ONOO-) is a cytotoxic anion, produced by interaction between nitric oxide and superoxide in vivo in some inflammatory cells. This study investigated its effects on Fasciola hepatica and Dicrocoelium dendriticum isolated from ovine livers and kept in bile at room temperature. Peroxynitrite was synthesized using a quenched flow reactor and assayed spectrophotometrically. It was applied at different concentrations (10(-3.5) to 10(-2.3 M)) to the flukes kept in bile. The viability of the peroxynitrite-treated flukes was compared with a control group (n=5-7 per group). Control F. hepatica and D. dendriticum lived for 226+/-11and 208+/-14min, respectively. Life times were decreased by peroxynitrite at all concentrations used (P<0.001). At the highest concentration of peroxynitrite, F. hepatica and D. dendriticum lived only for 6.1+/-0.4 and 4.1+/-4.1+/-0.2min, respectively. Correlation between peroxynitrite concentration and parasite viability was significant in the case of F. hepatica (r= -0.842; P= 0.0035). A single application of peroxynitrite can decrease the life span of ovine liver flukes. A failure in the activation of hepatic macrophages in infected animals may lead to a decreased production of free radicals and, thus, peroxynitrite. Such a failure is likely to deprive the body of a defence tool against multicellular parasites.     

Do polymorphic loci require large sample sizes to estimate genetic distances?

The coefficient of variation of estimates of three genetic distances (standard genetic distance of Nei, chord distance, FST) was examined with computer simulation to determine if large samples (per population) are necessary to precisely estimate genetic distances at loci with high levels of polymorphism. These simulations showed that loci with high mutation rates produce estimates of genetic distance with lower coefficients of variation than loci with lower mutation rates--without requiring larger sample sizes from each population. In addition, the rate at which increasing sample sizes decreases the coefficient of variation of estimates of genetic distances was shown to be approximately determined by the value of FST between the populations being sampled. When FST was greater than 0.05, sampling fewer than 20 individuals (per population) should be sufficient. When FST was less than 0.01, sampling 100 individuals (per population) or more will be useful.     

RAPD-analysis of genetic polymorphism of ducks: differences in breeds

Using randomly amplified polymorphic DNA (RAPD) obtained by polymerase chain reaction (PCR), the genetic differentiation of breeds of Peking and Musk ducks maintained at the Blagovarskii State Farm for Pedigree Poultry was studied. A comparison of genetic distances estimated by two different methods, as well as similarity dendrograms based on these estimates, was performed. The similarity dendrograms obtained using different primers and methods of construction were found to be similar. The pattern of breed clustering on these dendrograms adequately reflects their actual state known from the history and genealogy of breeds.     

Dispersal in a parasitic worm and its two hosts: consequence for local adaptation

Characterizing host and parasite population genetic structure and estimating gene flow among populations is essential for understanding coevolutionary interactions between hosts and parasites. We examined the population genetic structure of the trematode Schistosoma mansoni and its two host species (the definitive host Rattus rattus and the intermediate host Biomphalaria glabrata) using microsatellite markers. Parasites were sampled from rats. The study was conducted in five sites of the Guadeloupe Island, Lesser Antilles. Mollusks display a pattern of isolation by distance whereas such a pattern is not found neither in schistosomes nor in rats. The comparison of the distribution of genetic variability in S. mansoni and its two host species strongly suggests that migration of parasites is principally determined by that of the vertebrate host in the marshy focus of Guadeloupe. However, the comparison between genetic differentiation values in schistosomes and rats suggests that the efficacy of the schistosome rat-mediated dispersal between transmission sites is lower than expected given the prevalence, parasitic load and migration rate of rats among sites. This could notably suggest that rat migration rate could be negatively correlated to the age or the infection status of individuals. Models made about the evolution of local adaptation in function of the dispersal rates of hosts and parasites suggest that rats and mollusks should be locally adapted to their parasites.     

Dynamics of host-parasite interactions: the example of population biology of the liver fluke (Fasciola hepatica)

Knowledge of the population dynamics of parasites and their hosts is essential to build veterinary and health programs. The example chosen is that of Fasciola hepatica, a food-borne trematode responsible for severe human and animal infections on the five continents. In this paper, we review the relationships between the liver fluke and its intermediate (mollusc) and definitive (vertebrate) hosts.     

An ultrasensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3)

A capture enzyme-linked immunosorbent assay (ELISA) using a new monoclonal antibody (mAb MM3) is reported for the detection of Fasciola hepatica excretory-secretory antigens (ESAs) in feces of infected hosts. The mAb MM3 was produced by immunization of mice with a 7- to 40-kDa purified and O-deglycosylated fraction of F. hepatica ESAs, which has previously been shown to be specific for the parasite. The specificity and sensitivity of the MM3 capture ELISA were assessed using feces from sheep and cattle. Sheep feces were obtained from a fluke-free herd (with most animals harboring other nematodes and cestodes), from lambs experimentally infected with 5-40 F. hepatica metacercariae and in some cases treated with triclabendazole at 14 wk postinfection (PI), and from uninfected control lambs. Cattle feces were collected at the slaughterhouse from adult cows naturally infected with known numbers of flukes (from 1 to 154) or free of F. hepatica infection (though in most cases harboring other helminths). The MM3 capture ELISA assay had detection limits of 0.3 (sheep) and 0.6 (cattle) ng of F. hepatica ESA per milliliter of fecal supernatant. The assay detected 100% of sheep with 1 fluke, 100% of cattle with 2 flukes, and 2 of 7 cattle with 1 fluke. The false-negative animals (5/7) were probably not detected because the F. hepatica individuals in these animals were immature (5-11 mm in length). As expected, coproantigen concentration correlated positively (r = 0.889; P < 0.001) with parasite burden and negatively (r = 0.712; P < 0.01) with the time after infection at which coproantigen was first detected. Nevertheless, even in animals with low fluke burdens (1-36 parasites), the first detection of F. hepatica-specific coproantigens by the MM3 capture ELISA preceded the first detection in egg count by 1-5 wk. In all sheep that were experimentally infected and then untreated, coproantigen remained detectable until at least 18 wk PI, whereas in sheep that were experimentally infected and then flukicide treated, coproantigen became undetectable from 1 to 3 wk after treatment. None of the fecal samples from sheep or cattle negative for fascioliasis but naturally infected with other parasites including Dicroelium dendriticum showed reactivity in the MM3 capture ELISA. These results indicate that this assay is a reliable and ultrasensitive method for detecting subnanogram amounts of F. hepatica antigens in feces from sheep and cattle, facilitating early diagnosis.     

Evidence of population structuring following population genetic analyses of Fasciola hepatica from Argentina

Fasciola hepatica, the liver fluke, is a trematode parasite that causes disease of economic importance in livestock. As a zoonosis this parasite also poses a risk to human health in areas where it is endemic. Population genetic studies can reveal the mechanisms responsible for genetic structuring (non-panmixia) within parasite populations and provide valuable insights into population dynamics, which in turn enables theoretical predictions of evolutionary dynamics such as the evolution of drug resistance. Here we genotyped 320 F. hepatica collected from 14 definitive hosts from four provinces in Argentina. STRUCTURE analysis indicated three population clusters, and principal coordinate analysis confirmed this, showing population clustering across provinces. Similarly, pairwise F_ST values amongst all four provinces were significant, with standardised pairwise F_ST (F′_ST) ranging from 0.0754 to 0.6327. Therefore, population genetic structure was evident across these four provinces in Argentina. However, there was no evidence of deviation from Hardy–Weinberg equilibrium, so it appears that within these sub-populations there is largely random mating. We identified 263 unique genotypes, which gave a clonal diversity of 82%. Parasites with identical genotypes, clones, accounted for 26.6% of the parasites studied and were found in 12 of the 14 hosts studied, suggesting some clonemate transmission.     

Genetic diversity in Elymus caninus as revealed by isozyme, RAPD, and microsatellite markers.

Genetic diversity of 33 Elymus caninus accessions was investigated using isozyme, RAPD, and microsatellite markers. The three assays differed in the amount of polymorphism detected. Microsatellites detected the highest polymorphism. Six microsatellite primer pairs generated a total of 74 polymorphic bands (alleles), with an average of 15.7 bands per primer pair. Three genetic similarity matrices were estimated based on band presence or absence. Genetic diversity trees (dendrograms) were derived from each marker technique, and compared using Mantel's test. The correlation coefficients were 0.204, 0.267, and 0.164 between isozyme and RAPD distance matrices, RAPD and microsatellite distance matrices, and between isozyme and microsatellite distance matrices, respectively. The three methodologies gave differing views of the amount of variation present but all showed a high level of genetic variation in E. caninus. The following points may be drawn from this study whether based on RAPD, microsatellite, or isozyme data: (i) The Icelandic populations are consistently revealed by the three dendrograms. The congruence of the discrimination of this accession group by RAPD, microsatellite, and isozyme markers suggests that geographic isolation strongly influenced the evolution of the populations; (ii) The degree of genetic variation within accessions was notably great; and (iii) The DNA-based markers will be the more useful ones in detecting genetic diversity in closely related accessions. In addition, a dendrogram, which took into account all fragments produced by isozymes, RAPDs, and microsatellites, reflected better the relationships than did dendrograms based on only one type of marker.     

Pathogenic and molecular characterisations of pigeonpea wilt pathogen Fusarium udum

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a subpopulation of F. udum as identified by RAPD and SSR markers and pathogenicity on differential genotypes.     

The presence of Fasciola hepatica (liver-fluke) in humans and cattle from a 4,500 year old archaeological site in the Saale-Unstrut valley,Germany

During an excavation of a site of the corded ware culture in the Saale-Unstrut-Valley (ca. 3000 BC) in Germany, a soil sample from the pelvis of a human skeleton was studied under palaeoparasitological aspects. Eggs of the trematode Fasciola hepatica and of the nematode genus Capillaria were found. This is the first case of a direct association of a F. hepatica-infestation to both a prehistoric human skeleton and domesticated animal remains. Sheep and cattle bones were present at the same site and F. hepatica eggs were found in bovine samples. This strongly points toward an existing infection cycle, involving humans as a final host.