Developing species-specific primers to identify Bulinus truncatus and Bulinus beccari, the intermediate hosts of Schistosoma haematobium in Saudi Arabia

This work aimed to determine the inter- and intra-specific variations in populations of Bulinus truncatus and Bulinus beccari, the intermediate hosts of Schistosoma haematobium in Saudi Arabia, and to develop species-specific primers to identify these snails as a first step in the development of multiplex PCR for simultaneously identifying the snails and diagnosing its infections in a single step. Two populations of B. truncatus were collected from Asser and Bisha (A and B), and two B. beccari populations were collected from Mahial Asser and Merba (C and D). The snails' genomic DNA was extracted and amplified using 5 different primers. The primers displayed variable intra- and inter-specific differences across the populations. The largest RAPD-PCR fragments were cloned into a vector as a preparatory step for sequencing. Similarity searches for the sequenced cloned inserts revealed no similar sequences in the GenBank database or its associated databases. Specific primers used to target the B. truncatus and B. beccari genomes were designed using the Gene Runner program and based on the DNA sequences obtained from RAPD fragment sequence analyses. Using these primers for specific PCRs resulted in expected single-band PCR products of 536 bp for B. beccari and 478 bp for B. truncatus. These results will be helpful for simultaneously identifying B. truncatus and B. beccari snails and diagnosing S. haematobium infections within the snails using single step multiplex PCR.     

Development of species-specific primers for identification of Biomphalaria arabica,the intermediate host of Schistosoma mansoni in Saudi Arabia

Schistosoma mansoni is mediated through the intermediate host Biomphalaria arabica which lives in Saudi Arabia. Molecular characterization and identification of this intermediate host are important for epidemiological studies of schistosomiasis. The present work aimed to determine the molecular variations among the populations of B. arabica found in Southern part of Saudi Arabia, and to develop species-specific primers for identification of these snails as a first step in the development of multiplex PCR for simultaneously identifying the snails and diagnosing its infections in a single step. Five populations of Saudi B. arabica snails were collected from freshwater bodies. Three populations were collected from Asser and two populations were collected from AL-Baha. Genomic DNA was extracted from snails and was amplified using five different RAPD–PCR primers. The banding patterns of amplified materials by primers P1 and P5 were identical in all populations. However, the rest primers displayed intra-specific differences among populations with variable degrees. Largest sizes of RAPD–PCR products were cloned into TA cloning vector as a preparatory step for DNA sequence analysis. After sequencing, similarity searches of obtained DNA sequences revealed that there are no similar sequences submitted to genebank data bases and its associated banks. The results obtained will be helpful in the development of simultaneous identification of B. arabica snails and diagnosis of S. mansoni infection within it in a single step by an implementation of multiplex PCR.     

Identification and differentiation of human schistosomes by polymerase chain reaction

Recent increasing number of travelers, immigrants and foreign workers from schistosomiasis endemic area has thus resulted in the importation of schistosomiasis to non-endemic countries. To avoid ova-induced pathogenicity, sensitive and specific diagnostic means at an early stage of infection are therefore crucial. In this study, we developed polymerase chain reaction (PCR) primers specific for human schistosome species. The PCR products were obtained in a species-specific manner (479 bp, Schistosoma mansoni; 365 bp, S. haematobium; 614 bp, S. japonicum; 303 bp, S. mekongi) and were detectable from 0.01 pg of total worm DNA (S. haematobium, S. japonicum, S. mekongi). The primer sets were also available for multiplex use. Although some difficulties were experienced in amplifying the parasite DNA from the infected animals, schistosome DNA could be detected from one day post infection. The PCR method described herein will therefore be beneficial to detect human schistosomiasis, after some improvements in this method.     

Infections of Larval Stages of Dicrocoelium dendriticum and Brachylaima sp. in Brown Garden Snail,Helix aspersa,in Turkey

The aim of this study was to determine the presence and prevalence of larval stages of Dicrocoelium dendriticum and Brachylaima sp. in the first intermediate host, a species of land snail, Helix aspersa, in Turkey. A total of 211 snails were collected in April-May 2014 from pastures in Mersin District. Larval stages of D. dendriticum were identified under a light microscope. Hepatopancreas from naturally infected H. aspersa snails were examined histologically. The prevalence of larval stages of D. dendriticum and Brachylaima sp. in H. aspersa snails was found to be 2.4% and 1.9%, respectively, in Mersin, Turkey. Cercariae were not matured in sporocysts at the beginning of April; however, it was observed that cercariae matured and started to leave sporocysts by early-May. Thus, it was concluded that H. aspersa acts as an intermediate host to D. dendriticumin and Brachylaima sp. in Mersin, Turkey. A digenean trematode Brachylaima sp. was seen for the first time in Turkey.     

Rapid identification of lymnaeid snails and their infection with Fasciola gigantica in Thailand

Freshwater snails of the family Lymnaeidae are the intermediate hosts of the liver fluke Fasciola worldwide. While distinct species have been identified at the molecular level in other parts of the world such data have not been published for Thailand. In this study we collected Lymnaeidae from different localities across Thailand and analyzed their 16S rDNA sequences as a molecular signature for classification. In addition to the ubiquitous Radix rubiginosa, we have confirmed the presence of Austropeplea viridis and Radix swinhoei, for the latter of which the ribosomal rDNA sequences are reported for the first time, in North-Thailand. Based on the obtained 16S rDNA data three primer pairs were designed that allowed rapid identification of these snail species by PCR. To determine their infection status, PCR primers for F.gigantica cathepsin L were used in parallel with the snail 16S rDNA species-specific primers in multiplex PCR analyses. Western blot analysis of total snail protein with a monoclonal anti-F.gigantica cathepsin L antibody confirmed positive cathepsin L PCR results. The developed diagnostic PCR will be of use in risk assessment for transmission of fascioliasis in Thailand.     

Molecular Variability in North Indian Isolates of Cylindrocladium quinquieseptatum Causing Eucalyptus Leaf and Seedling Blight

Cylindrocladium quinqueseptatum has been considered as the most destructive pathogen of Eucalyptus nurseries and plantations in north India. Genetic resistance has not been determined against this disease in Eucalyptus and genetic diversity among the fungal population in northern India is not known. Seventy three isolates from infected leaves and twigs of Eucalyptus were collected from different northern Indian state and analyzed through RAPD-PCR for screening genetic diversity. The UPGMA cluster analysis score of 284 loci permitted identification of 11 population lines and an outlier. This molecular variability prevalent among the north Indian population of the pathogen can used in identifying Cylindrocladium leaf and seedling blight resistant Eucalyptus germplasm.     

Effects of intermediate host genetic background on parasite transmission dynamics: a case study using Schistosoma mansoni

For parasites that require multiple hosts to complete their development, genetic interplay with one host may impact parasite transmission and establishment in subsequent hosts. In this study, we used microsatellite loci to address whether the genetic background of snail intermediate hosts influences life-history traits and transmission patterns of dioecious trematode parasites in their definitive hosts. We performed experimental Schistosoma mansoni infections utilizing two allopatric populations of Biomphalaria glabrata snails and assessed intensities and sex ratios of adult parasites in mouse definitive hosts. Our results suggest that the genetic background of hosts at one point in a parasite’s life cycle can influence the intensities and sex ratios of worms in subsequent hosts.     

Morphological and Molecular Characterization of Lymnaeid Snails and Their Potential Role in Transmission of Fasciola spp. in Vietnam

Freshwater snails of the family Lymnaeidae play an important role in the transmission of fascioliasis worldwide. In Vietnam, 2 common lymnaeid species, Lymnaea swinhoei and Lymnaea viridis, can be recognized on the basis of morphology, and a third species, Lymnaea sp., is known to exist. Recent studies have raised controversy about their role in transmission of Fasciola spp. because of confusion in identification of the snail hosts. The aim of this study is, therefore, to clarify the identities of lymnaeid snails in Vietnam by a combination of morphological and molecular approaches. The molecular analyses using the second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA clearly showed that lymnaeids in Vietnam include 3 species, Austropeplea viridis (morphologically identified as L. viridis), Radix auricularia (morphologically identified as L. swinhoei) and Radix rubiginosa (morphologically identified as Lymnaea sp.). R. rubiginosa is a new record for Vietnam. Among them, only A. viridis was found to be infected with Fasciola spp. These results provide a new insight into lymnaeid snails in Vietnam. Identification of lymnaeid snails in Vietnam and their role in the liver fluke transmission should be further investigated.     

Carboxylic Acids as Biomarkers of Biomphalaria alexandrina Snails Infected with Schistosoma mansoni

Biomphalaria alexandrina snails play an indispensable role in transmission of schistosomiasis. Infection rates in field populations of snails are routinely determined by cercarial shedding neglecting prepatent snail infections, because of lack of a suitable method for diagnosis. The present study aimed at separation and quantification of oxalic, malic, acetic, pyruvic, and fumaric acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to test the potentiality of these acids to be used as diagnostic and therapeutic biomarkers. The assay was done in both hemolymph and digestive gland-gonad complex (DGG) samples in a total of 300 B. alexandrina snails. All of the studied acids in both the hemolymph and tissue samples except for the fumaric acid in hemolymph appeared to be good diagnostic biomarkers as they provide not only a good discrimination between the infected snails from the control but also between the studied stages of infection from each other. The most sensitive discriminating acid was malic acid in hemolymph samples as it showed the highest F-ratio. Using the Z-score, malic acid was found to be a good potential therapeutic biomarker in the prepatency stage, oxalic acid and acetic acid in the stage of patency, and malic acid and acetic acid at 2 weeks after patency. Quantification of carboxylic acids, using HPLC strategy, was fast, easy, and accurate in prediction of infected and uninfected snails and possibly to detect the stage of infection. It seems also useful for detection of the most suitable acids to be used as drug targets.     

Conservation of protein kinase a catalytic subunit sequences in the schistosome pathogens of humans

cAMP-dependent protein kinases (PKAs) are central mediators of cAMP signaling in eukaryotic cells. Previously we identified a cDNA which encodes for a PKA catalytic subunit (PKA-C) in Schistosoma mansoni (SmPKA-C) that is required for adult schistosome viability in vitro. As such, SmPKA-C could potentially represent a novel schistosome chemotherapeutic target. Here we sought to identify PKA-C subunit orthologues in the other medically important schistosome species, Schistosoma haematobium and Schistosoma japonicum, to determine the degree to which this potential target is conserved and could therefore be exploited for the treatment of all forms of schistosomiasis. We report the identification of PKA-C subunit orthologues in S. haematobium and S. japonicum (ShPKA-C and SjPKA-C, respectively) and show that PKA-C orthologues are highly conserved in the Schistosoma, with over 99% amino acid sequence identity shared among the three human pathogens we examined. Furthermore, we show that the recently published Schistosoma mansoni and S. japonicum genomes contain sequences encoding for several putative PKA substrates with homology to those found in Homo sapiens, Caenorhabditis elegans, and Saccharomyces cerevisiae.     

A Microtus fortis protein,serum albumin,is a novel inhibitor of Schistosoma japonicum schistosomula

Schistosomiasis is an endemic parasite disease and praziquantel is the only drug currently in use to control this disease. Experimental and epidemiological evidence strongly suggests that Microtus fortis ( Mf ) is a naturally resistant vertebrate host of Schistosoma japonicum . In the present study, we found that Mf serum albumin ( Mf -albumin) and the conditioned medium of pcDNA3.1- Mf -albumin caused 46.2% and 38.7% schistosomula death rates in 96 h, respectively, which were significantly higher than that of the negative control (p < 0.05). We also found that mice injected with Mf -albumin had a 43.5% reduction in worm burden and a 48.1% reduction in liver eggs per gram (p < 0.05) in comparison to the control animals. To characterise the mechanisms involved in clearance, schistosomula were incubated with fluorescein isothiocyanate-labelled Mf -albumin and fluorescent enrichment effects were found in the gut lumen of schistosomula after 48 h of incubation. Next, digestive tract excretions from schistosomula were collected and the sensitivity of Mf -albumin to digestive tract excretions was evaluated. The results indicated that schistosomula digestive tract excretions showed indigestibility of Mf -albumin. The death of schistosomula could be partially attributed to the lack of digestion of Mf -albumin by digestive tract excretions during the development of the schistosomula stage. Therefore, these data indicate the potential of Mf -albumin as one of the major selective forces for schistosomiasis.     

Fasciola hepatica in Snails Collected from Water-Dropwort Fields using PCR

Fasciola hepatica is a trematode that causes zoonosis mainly in cattle and sheep and occasionally in humans. Fascioliasis has been reported in Korea; however, determining F. hepatica infection in snails has not been done recently. Thus, using PCR, we evaluated the prevalence of F. hepatica infection in snails at 4 large water-dropwort fields. Among 349 examined snails, F. hepatica-specific internal transcribed space 1 (ITS-1) and/or ITS-2 markers were detected in 12 snails and confirmed using sequence analysis. Morphologically, 213 of 349 collected snails were dextral shelled, which is the same aperture as the lymnaeid snail, the vectorial host for F. hepatica. Among the 12 F. hepatica-infected snails, 6 were known first intermediate hosts in Korea (Lymnaea viridis and L. ollula) and the remaining 6 (Lymnaea sp.) were potentially a new first intermediate host in Korea. It has been shown that the overall prevalence of the snails contaminated with F. hepatica in water-dropwort fields was 3.4%; however, the prevalence varied among the fields. This is the first study to estimate the prevalence of F. hepatica infection using the vectorial capacity of the snails in Korea.     

Using FAME Analysis to Compare, Differentiate, and Identify Multiple Nematode Species

We have adapted the Sherlock^® Microbial Identification system for identification of plant parasitic nematodes based on their fatty acid profiles. Fatty acid profiles of 12 separate plant parasitic nematode species have been determined using this system. Additionally, separate profiles have been developed for Rotylenchulus reniformis and Meloidogyne incognita based on their host plant, four species and three races within the Meloidogyne genus, and three life stages of Heterodera glycines. Statistically, 85% of these profiles can be delimited from one another; the specific comparisons between the cyst and vermiform stages of H. glycines, M. hapla and M. arenaria, and M. arenaria and M. javanica cannot be segregated using canonical analysis. By incorporating each of these fatty acid profiles into the Sherlock^® Analysis Software, 20 library entries were created. While there was some similarity among profiles, all entries correctly identified the proper organism to genus, species, race, life stage, and host at greater than 86% accuracy. The remaining 14% were correctly identified to genus, although species and race may not be correct due to the underlying variables of host or life stage. These results are promising and indicate that this library could be used for diagnostics labs to increase response time.     

Random Amplified Polymorphic DNA Analysis of Heterodera cruciferae and H. schachtii populations

Heterodera schachtii and H. cruciferae are sympatric in California and frequently occur in the same field upon the same host. We have investigated the use of polymerase chain reaction (PCR) amplification of nematode DNA sequences to differentiate H. schachtii and H. cruciferae and to assess genetic variability within each species. Single, random oligodeoxyribonucleotide primers were used to generate PCR-amplified fragments, termed RAPD (random amplified polymorphic DNA) markers, from genomic DNA of each species. Each of 19 different random primers yielded from 2 to 12 fragments whose size ranged from 200 to 1,500 bp. Reproducible differences in fragment patterns allowed differentiation of the two species with each primer. Similarities and differences among six different geographic populations of H. schachtii were detected. The potential application of RAPD analysis to relationships among nematode populations was assessed through cluster analysis of these six different populations, with 78 scorable markers from 10 different random primers. DNA from single cysts was successfully amplified, and genetic variability was revealed within geographic populations. The use of RAPD markers to assess genetic variability is a simple, reproducible technique that does not require radioisotopes. This powerful new technique can be used as a diagnostic tool and should have broad application in nematology.     

Clonorchiasis--a historical review of contributions of Japanese parasitologists

In the early stage of research on Clonorchis and clonorchiasis, Japanese parasitologists made a tremendous contribution on the elucidation of the life cycle of this parasite and on the epidemiology of the disease. Harujiro Kobayashi first identified cyprinoid fish as the second intermediate hosts for Clonorchis sinensis in 1912, Subsequently Parafossarulus snails were identified as the first intermediate host by Masatomo Muto in 1918. Kenso Ishisaka recorded the first human case of clonorchiasis in Japan in 1877, and two Japanese clinicians, Shigeru Matsumoto and Tsukasa Ohi, recorded the endemic nature of this disease in Korea and Taiwan respectively, in the same year, 1915.     

Comparison of complete mitochondrial genomes of pine wilt nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus (Nematoda: Aphelenchoidea) and development of a molecular tool for species identification

We determined the complete mitochondrial genome sequences for Bursaphelenchus mucronatus, one species of pinewood nematode. The genome is a circular-DNA molecule of 14,583 bp (195 bp smaller than its congener Bursaphelenchus xylophilus) and contains 12 protein-coding genes (lacking atp8), 22 tRNA genes, and 2 rRNA genes encoded in the same direction, consistent with most other nematodes. Based on sequence comparison of mtDNA genomes, we developed a PCR-based molecular assay to differentiate B. xylophilus (highly pathogenic) and B. mucronatus (relatively less virulent) using species-specific primers. The molecular identification system employs multiplex-PCR and is very effective and reliable for discriminating these Bursaphelenchus species, which are economically important, but difficult to distinguish based on morphology. The comparison of the mitochondrial genomes and molecular identification system of the two species of Bursaphelenchus spp. should provide a rich source of genetic information to support the effective control and management (quarantine) of the pine wilt disease caused by pinewood nematodes.     

Comparative Disc-Electrophoretic Protein Analyses of Selected Meloidogyne,Ditylenchus, Heterodera and Aphelenchus spp.

Disc-electrophoretic separation of soluble proteins from whole nematode homogenates yielded band profiles useful for distinguishing selected species of Meloidogyne and Ditylenchus, and the genera Heterodera, and Aphelenchus. Certain protein bands were common to all the species of Meloidogyne, whereas other bands were specific. Meloidogyne spp. and Heterodera glycines shared some protein similarities, but other genera differed distinctly. Protein profiles of Meloidogyne spp. were not significantly altered by the host on which the nematode was cultured.     

The re-discovered Maculinea rebeli (Hirschke, 1904): Host ant usage,parasitoid and initial food plant around the type locality with taxonomical aspects (Lepidoptera,Lycaenidae)

The taxonomy of the myrmecophilous Maculinea alcon group (Lepidoptera: Lycaenidae) is highly debated. The host-plant and host-ant usage of these butterflies have conventionally been important in their identification. Maculinea ‘rebeli’ has generally been considered to be the xerophilous form of Ma. alcon (Ma. alcon X hereafter) with Gentiana cruciata as initial food plant. However, the type locality and all other known sites of Ma. rebeli are found above the coniferous zone, and are well separated from the lower regions where Ma. alcon X sites are found. Furthermore, no food plant and host ant data for the nominotypic Ma. rebeli have yet been published. Our aim was therefore to identify the host ant(s) of Ma. rebeli around the type locality and compare this with the host ant usage of nearby Ma. alcon X. Nests of Myrmica spp. (Hymenoptera: Formicidae) close to the host plants were opened on one Ma. alcon X (host plant: Gentiana cruciata) and two Ma. rebeli (host plant: Gentianella rhaetica, first record, confirmed by oviposition and emerging larvae) sites just before the flying period, to find prepupal larvae and pupae. Three Myrmica species (My. lobulicornis, My. ruginodis, My. sulcinodis) were found on the two Ma. rebeli sites, which parasitized exclusively My. sulcinodis (22 individuals in 7 nests). On the Ma. alcon X site Myrmica sabuleti and My. lonae were found, with My. sabuleti the exclusive host (51 individuals in 10 nests). Ichneumon cf. eumerus parasitized both butterflies. The results highlight the differentiation of Maculinea rebeli from Ma. alcon X, from both conservation biological and ecological points of view. Thus, it should be concluded that Ma. rebeli does not simply represent an individual form of Ma. alcon but it can be considered as at least an ecological form adapted to high mountain conditions both in its initial food plant and host ant species. In addition, it should be emphasized that Ma. alcon X (= Ma. rebeli auct. nec Hirschke) cannot be synonymised with Ma. rebeli (Hirschke, 1904).