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Cystic Echinococcosis is a parasitic disease caused by the cestode Echinococcus granulosus widely distributed in Africa. Monitoring of this parasite requires access to cyst samples on intermediate hosts observed at the slaughterhouse. In order to facilitate sampling in the field and analysis, the French National Reference Laboratory for Echinococcus spp. has developed a tissue derived from DNA sampling with FTA ® card technology. The DNA samples were taken by applying the FTA ® paper on the germinal layer after opening the cysts. The sampling technique was validated using frozen cysts (n = 76) stored in the laboratory and from field samples (n = 134) taken at the slaughterhouse by veterinarian technicians during meat inspection in Morocco, Mali and Mauritania. DNA was extracted after several weeks of storage at room temperature. PCR assays were performed using primers for generic cestode (cox1) and amplified fragments were sequenced. All samples taken in the lab and 80% of field samples were capable of molecular characterization. Cyst-derived DNA from FTA ® samples can be useful for easy sampling, storage and rapid, safe and cheap shipment. The use of the FTA methodology will facilitate studies in the field to investigate the presence and genetic characterization of E. granulosus sensu lato in African countries., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Pacific bluefin tuna (PBT), Thunnus orientalis, due to its high average price on the market is an economically valuable fish species. Infections by blood flukes from the genus Cardicola (Trematoda: Aporocotylidae) represent a growing concern for the cage culture of bluefin tuna in Japan, Australia and Southern Europe. The accumulation of numerous Cardicola eggs in the fish gills causes severe pathology that has been linked to mortality in PBT juveniles up to one year old. The only effective treatment used to mitigate the infection is the oral administration of the antihelminthic drug praziquantel (PZQ) to the affected fish. However, with the need to minimise therapeutic drug use in aquaculture it is hoped that immunoprophylaxis can provide a future alternative to protect the PBT juveniles against Cardicola infection. Currently, little is known of the host immune response to these parasites and of their infection dynamics. In this study, using real-time qPCR we aimed to quantitatively detect C. orientalis and C. opisthorchis DNA within the gills and heart of cultured PBT juveniles and to investigate the host immune response at the transcriptional level in the gills. The research focused mainly during early stages of infection soon after young PBT were transferred to culture cages (from 14 to 77 days post-transfer). An increase (up to 11-fold) of immune-related genes, namely IgM, MHC-I, TCR-β and IL-1β was observed in the PBT gills infected with Cardicola spp. (28-77 days post-transfer). Furthermore, IgM (19-fold increase) and MHC-I (11.5-fold increase) transcription was strongly up-regulated in gill samples of PBT infected with C. orientalis relative to uninfected fish but not in fish infected with C. opisthorchis. Cardicola-specific DNA was first detected in the host 14 days post-transfer (DPT) to sea-cages which was 55 days earlier than the first detection of parasite eggs and adults by microscopy. Oral administration of PZQ did not have an immediate effect on parasite DNA presence in the host and the DNA presence started to reduce after 24 days only in the host heart. The results provide evidence of an immune response in early age sea-cage cultured juveniles of PBT naturally infected with C. orientalis and C. opisthorchis. This response, whilst not protective against primary infection, provides evidence that immunisation at an early age may have potential as a health strategy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Human infection with Capillaria philippinensis is accidental; however, it may end fatally if not diagnosed and treated in the proper time. The first case was detected in the Philippines in 1963, but later reported in other countries around the world, including Egypt. In this report, molecular diagnosis using a specific nested PCR for detection of C. philippinensis in faeces is described based on the amplification of small ribosomal subunit. The test showed sensitivity and specificity, as it detected all the positive cases and gave no cross-reaction with human DNA and DNA of other tested parasites. This method can be very useful not only for improvement of diagnosis, but also to understand the different environmental routes of transmission by detection of C. philippinensis DNA-stages in the possible fish intermediate hosts and reservoir animal host, helping to improve strategies for surveillance and prevention of human disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

An experimental study in hamsters was performed to evaluate the capability for detecting Schistosoma mansoni DNA in serum and fecal samples during the pre and post-egg-laying periods of infection using TaqMan® Real-Time PCR system (qPCR), was compared with the circumoval precipitin test (COPT) and the Kato-Katz technique, especially among individuals with low parasitic burden. Twenty-four hamsters were infected with cercariae. Three hamsters were sacrificed per week under anesthesia, from 7 days post infection (DPI) up to 56 DPI. A serum sample and a pool of feces were collected from each hamster. The presence of S. mansoni eggs in fecal samples was evaluated by Kato-Katz method and in the hamsters gutby histopathology. Detection of S. mansoni DNA was performed using qPCR and S. mansoni antibody using COPT. The first detection of eggs in feces by Kato-Katz method and S. mansoni DNA in feces by qPCR occurred 49 DPI. Nevertheless, S. mansoni DNA was detected in serum samples from 14 up to 56 DPI. COPT was positive at 35 DPI. The results not only confirm the reliability of S. mansoni DNA detection by qPCR, but also demonstrate that serum is a trustworthy source of DNA in the pre patent infection period., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Caenorhabditis elegans is an important system for the study of cell cycle regulation in the context of animal development. One of the most powerful features of C. elegans is the invariant cell lineage in which somatic cells initiate cell division at specific times within the developmental program. The cell lineage is fully known and provides the foundation for the analysis of cell cycle progression at single-cell resolution in a multicellular organism. In this chapter, we describe the different types of cell cycles observed in C. elegans, and provide methods and reagents for the analysis of cell cycle progression as well as specific cell cycle phases. We also provide strategies for the analysis and proper interpretation of cell cycle and checkpoint mutants., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Programmed cell death or apoptosis is a common cell fate during the development of multicellular organisms, including C. elegans. C. elegans has been proven to be an excellent model organism for studies of programmed cell death. Its transparency and the knowledge of its cell lineage, including its invariant life vs. death fate of all cells, allow programmed cell death to be studied in vivo at single cell resolution in a way that no other systems can currently match. This advantage, in combination with sophisticated genetic manipulations, has facilitated the identification and characterization of many genes important for different aspects of programmed cell death. In addition to the ability to observe life and death of cells directly under Nomarski optics or by using fluorescent transgene products in living animals, several methods have been developed to study other aspects of apoptosis. For example, fragmentation of chromosomal DNA and the identity of phagocytic cells that engulf cell corpses can be analyzed using specific staining methods. Proteases and nucleases involved in cell killing and DNA degradation, respectively, can be studied using specific biochemical assays. These cell death assays have been instrumental in elucidating the functions of cell death-related genes and the mechanisms by which they affect cell death. In this chapter, we will review and describe these methods in detail., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Strongyloides venezuelensis is a parasitic nematode that has been used as a model to study human and animal strongyloidiasis. In this study, we compared the sensitivity between traditional methodologies and PCR assay to characterize the dynamics of S. venezuelensis infection and its migration route in Lewis rats subcutaneously infected with 4000 L3. The dynamics of the infection was determined by counting the number of eggs and by detecting parasite deoxyribonucleic acid in faeces samples. Both techniques similarly detected the infection at day 6 after larvae inoculation. However, PCR performed with the genus primer showed higher sensitivity during the recovery phase. Histological analysis and PCR assay were then used to follow parasite tissue migration. S. venezuelensis migration route included the muscular fibers below the skin, the pulmonary alveoli and the small intestine vilosities. The sensitivity of these two techniques to detect parasite's presence in these tissues was statistically similar., (Copyright © 2010 Elsevier Inc. All rights reserved.)

A specific PCR assay for the detection of Schistosoma japonicum DNA in rabbit fecal and serum samples was developed by amplifying a 230-bp fragment from the sequence information of the clone G55A of the highly repetitive retrotransposon SjR2. The minimum amount of DNA detectable using the PCR assay was 0.8pg, and the expected PCR product was amplified when DNA equivalent of 1.1 egg from feces was used as template. In the meantime, serum anti-worm IgG was examined by ELISA. ELISA gave positive results at 4-6 weeks post-infection depending on the cercarial doses. The parasite eggs were detected in feces at 7 weeks post-infection. In contrast, S. japonicum DNA was detected in sera at first week post-infection, and it became negative at 10 weeks post-treatment, whereas the anti-worm IgG was still at high levels at 23 weeks post-treatment. These data demonstrated that the PCR assay established provides a potential tool for the early diagnosis and therapy evaluation for S. japonicum infection in humans.

The four currently recognized mermithid (Nematoda) species parasitizing black flies (Diptera: Simuliidae) from Northeast America were distinguished using discriminatory PCR primers aimed at COI and 18S rDNA. Isomermis wisconsinensis, Gastromermis viridis and Mesomermis camdenensis were easily differentiated using either genomic target, even for juvenile mermithids damaged beyond morphological recognition. However, specimens from Mesomermis flumenalis being identical in external morphology and producing a unique-sized PCR product were classified by sequence data into four clearly distinguished molecular variants. This quartet was made of two winter and two summer 'physiological variants', including one which also belonged to, but diverged early from the rest of the Mesomermis genus. Combining the multiplex PCR and sequencing approaches allowed for the characterization of a multiple parasitism which simultaneously implicated I. wisconsinensis and two M. flumenalis variants. With another instance where parasites were identified by morphology only, this is the first report of black fly parasitism by multiple mermithid species. A phylogenetic tree built by combining our sequences to previous GenBank entries likely indicates a monophyletic origin for the mermithid family, but also suggests that differentiation between parasite genera sometimes occurred before the evolutionary emergence of the actual host group.

The etiological agents of human trichinellosis are distributed worldwide in domestic and wild animals. In Spain, two morphologically indistinguishable Trichinella species have been described--Trichinella spiralis and Trichinella britovi--that are perpetuated in both domestic and sylvatic cycles. The present work reports a double natural infection involving these species in a wild boar killed by hunters in the Province of Cáceres, Spain. After artificial digestion of the boar's muscles, nine larvae/g were collected. These were characterized by multiplex-PCR and Western-blotting using the Trichinella-specific monoclonal antibodies US5 and US9, and both T. spiralis and T. britovi were detected. The mechanism by which this wild boar came to acquire a mixed infection remains unclear.

RNA interference (RNAi) mediated by short interfering RNA (siRNA) is a powerful reverse genetics tool and holds enormous therapeutic potential for various diseases, including parasite infections. siRNAs bind their complementary mRNA and lead to degradation of their specific mRNA targets. RNAi has been widely used for functional analysis of specific genes in various cells and organisms. In this paper, we tested the potential of silencing the expression of the Mago nashi gene in Schistosoma japonicum by siRNAs derived from shRNA expressed by mammalian Pol III promoter H1. Schistosomula, transformed from cercariae by mechanical shearing of the tails, were electroporated with Mago nashi shRNA expression vector. Aliquots of parasites were harvested at days 1, 3, and 5 after electroporation, respectively. Levels of Mago nashi mRNA and protein were determined by RT-PCR and Western blotting analysis. The results showed that shRNA expressed from mammalian Pol III promoter H1 specifically reduced the levels of Mago nashi mRNA and proteins in S. japonicum. Changes in testicular lobes were apparent when parasites were introduced into mammalian hosts. Thus, vector-mediated gene silencing is applicable to S. japonicum, which provides a means for the functional analysis of genes in this organism.

Until now, only a few systematic surveys of entomopathogenic nematodes (EPN) have been conducted in Middle Eastern countries. Many of the recovered EPN species in this region have shown to own distinctive qualities that enable their survival in unique environments, such as high temperatures and low moisture levels tolerance. These new species and strains, with unique environmental tolerances, are more suitable for their consideration in pest management programs in xerophytic regions. With this background in mind, we recently conducted a survey of EPN in Jordan. This study records for the first time the diversity and distribution of these nematodes and their bacterial symbionts in this country. Jordan's three geographic regions: (1) the highlands, (2) Jordan valley and (3) the desert region were sampled. Within each region, natural habitats and agricultural regions characteristic to each region were considered for sampling purposes. Four EPN species including three Steinernema and one Heterorhabditis were recovered. Nematodes were identified using a combination of molecular markers and classic morphological diagnostic tools. Bacterial symbionts were identified by analysis of 16S rRNA sequences. Abiotic characteristics such as soil type, soil pH, and elevation were also recorded. We herein report the diversity of EPN species in Jordan and discuss their potential in Biocontrol and IPM programs for this country.

Isolation and identification of native nematode-bacterial associations in the field are necessary for successful control of endemic pests in a particular location. No study has yet been undertaken to recover and identify EPN in metropolitan France. In the present paper, we provide results of a survey of EPN and their symbiotic bacteria conducted in Hérault and Gard regions in Southern France. Molecular characterization of isolated nematodes depicted three different Steinernema species and one Heterorhabditis species, H. bacteriophora. Steinernema species recovered were identified as: S. feltiae and S. affine and an undescribed species. Xenorhabdus symbionts were identified as X. bovienii for both S. feltiae and S. affine. Phylogenetic analysis placed the new undescribed Steinernema sp. as closely related to S. arenarium but divergent enough to postulate that it belongs to a new species within the "glaseri-group". The Xenorhabdus symbiont from this Steinernema sp. was identified as X. kozodoii. All Heterorhabditis isolates recovered were diagnosed as H. bacteriophora and their bacterial symbionts were identified as Photorhabdus luminescens. Molecular characterization of these nematodes enabled the distinction of two different H. bacteriophora strains. Bacterial symbiontic strains of these two H. bacteriophora strains were identified as P. luminescens ssp. kayaii and P. luminescens ssp. laumondii.

During a random survey of entomopathogenic nematodes in the provinces of Sichuan and Gansu (eastern Tibet) in 2004, soil samples from several sites were collected and tested for the incidence of entomopathogenic nematodes. A new species was collected in this survey and it is described herein as Steinernema cholashanense n. sp. Steinernema cholashanense n. sp. is characterized by morphology and morphometry of the IJ and male. For the IJ, the new species can be recognized by the average body length 843 microm, esophagus length 125 microm, H%=39% and E%=81%. The lateral field pattern is 2, 5, 7, 4, 2. The male of the first generation is characterized by spicule shape and length and especially with prominent velum and the presence of a mucron on both generations. The average body length of the IJ of S. cholashanense n. sp. (843 microm) is shorter than that of S. oregonense (980 microm), S. kraussei (951 microm) and S. litorale (909 microm), similar to that of S. feltiae (849 microm), but longer than that of S. weiseri (740 microm), S. jollietti (711 microm) and S. hebeiense (658 microm). Esophagus length of the new species (125 microm) is closer to that of S. jollieti (123 microm) but longer than that of S. weiseri (113 microm) and shorter than that of S. oregonense (132 microm), S. kraussei (134 microm) and S. feltiae (136 microm). E% of the new species (81) is similar to that of S. kraussei (80), but smaller than that of S. jollieti (88), S. weiseri (95), S. oregonense (100) and S. feltiae (119). Spicule head length of the new species is almost the same as its width, this character is similar to that of S. kraussei but it is different from this species by its prominent velum. The new species can be recognized further by characteristics of sequences of ITS and D2D3 regions and cross hybridization with closely related species, S. feltiae, S. kraussei and S. oregonense.

In the present study, we have extended earlier taxonomic, biochemical and experimental investigations to characterize two species of Taenia from carnivores in Kenya by use of the sequences of a variable domain (D1) of nuclear ribosomal DNA and the cytochrome c oxidase subunit 1 and NADH dehydrogenase 1 genes of mitochondrial DNA. Emphasis was placed on the characterization of Taenia madoquae from the silver-backed jackal (Canis mesomelas) and Taenia regis from the lion (Panthera leo), given the previous absence of any DNA sequence data for them, and on assessing their genetic relationships with socioeconomically important taeniids. The study showed that T. regis was genetically most closely related to T. hydatigena, and T. madoquae to T. serialis, T. multiceps or T. saginata. The present findings provide a stimulus for future work on the systematic relationships and epidemiology of lesser-known taeniid cestodes in Africa and other continents, employing mitochondrial sequence data sets.

A single step novel multiplex polymerase chain reaction (PCR) has been developed for simultaneous detection of human filarial parasites, Brugia malayi and Wuchereria bancrofti, from blood samples and mosquitoes. The primers used were novel and have been tested with the parasite DNA amplifying 188bp (BM) and 129bp (WB) DNA fragments, specific to B. malayi and W. bancrofti, respectively, in a single reaction. The specificity of the PCR product was confirmed by DNA sequencing and slot blot hybridization assay. The test was found highly sensitive for both B. malayi and W. bancrofti by detecting the parasitaemia up to the level of one microfilaria per reaction. The assay was further evaluated on 98 blood samples and 144 mosquito samples collected from filarial endemic areas. The PCR was found to be more efficient in comparison to microscopy by detecting 8% and 5% more filarial parasites in field-collected blood and mosquito samples, respectively. This novel PCR that offers scope for simultaneous detection of both the parasites may be used as a diagnostic tool for the detection of filariasis in population and can be adopted for rapid surveillance and monitoring of mosquitoes for use in the effective control of filariasis.

The rumen flukes of 37 cows (Bos indicus) from Chiang Mai and Lamphun provinces were investigated, and the overall prevalence of infection was 78.38% (29/37). Three species were found: Paramphistomum epiclitum, Orthocoelium streptocoelium, and Fischoederius elongatus with prevalences of infection of 75.68%, 48.65%, and 40.54%, respectively. Genomic DNA was amplified by polymerase chain reaction based on the high annealing temperature-random amplification of polymorphic DNA (HAT-RAPD) technique. Five random 10-mer oligonucleotide primers (OPA2, OPA4, OPB18, OPC9, and OPH11) produced distinct banding patterns in three species. No genetic variations in these three species were identified using 10 arbitary primers.

Steinernema sichuanense n. sp. is characterized by male, female and IJ. For male, the spicules are robust with prominent rostrum; gubernaculum has blunt anterior end; cuneus is arrow-shaped, pointed posteriorly. Second-generation male has a prominent mucron. For female, tail usually has one to four papillae-like projections on tail tip; post anal swelling is absent. For IJ, body length is about 710 microm; lateral field has six ridges; the formula of lateral field is 2, 5, 6, 4, 2 with two prominent submarginal ridges; tail usually has a dorsal depression. In Steinernema affine/intermedium group, the IJ of S. sichuanense n. sp. differs from S. affine by its absence of the internal tail spine; differs from Steinernema beddingi by its six ridges in lateral field compared to 4 for S. beddingi. For male mucron is absent in both generations of S. affine, S. intermedium and S. beddingi, whereas it is present in the second-generation of S. sichuanense sp. n. Morphology and morphometrics of spicules and gubernacula of the four species in S. affine/intermedium group are quite different based on SEM photographs. For female, the postanal swelling is absent in the first-generation of S. sichuanense n. sp. whereas S. affine and S. intermedium have slight swelling and S. beddingi has conspicuous swelling. The new species is further recognized by characterization of sequences of ITS and D2/D3 regions of the ribosomal DNA. The symbiotic bacterium associated to S. sichuanense belongs to the species Xenorhabdus bovienii.

In the present study, we utilised the method of AFLP to screen for genetic variation within and among individuals of the blood-feeding human hookworm Necator americanus (Nematoda) from Africa, Asia and South America. A total of 45 adult worms (i.e. 20 from Ghana, 16 from Colombia and 9 from Nepal) were subjected to analysis using the restriction enzyme/primer combination HindIII+AG/BglII+AC. Cluster analysis divided N. americanus into multiple, genetically distinct groups, consistent with previous findings using ribosomal and mitochondrial DNA data sets. The results demonstrated the usefulness of AFLP fingerprinting for establishing genetic variation within N. americanus and reinforce its applicability to other parasitic helminths of human and/or veterinary health importance.

In order to investigate the feasibility of a microarray-based method for diagnostics of plant-parasitic nematodes, we have developed a DNA oligonucleotide microarray to detect the nematode species Meloidogyne chitwoodi, which is listed as a quarantine organism in Europe. Oligonucleotide capture probes were designed from nematode SCAR and satellite DNA sequences and spotted onto epoxy-coated glass slides. PCR products were generated using specific primers, labeled with Cyanine 3 or Cyanine 5 fluorescent dyes, and hybridized overnight to the microarray. This methodology allowed the specific detection of M. chitwoodi DNA in pure and mixed samples (i.e. when M. chitwoodi DNA was mixed with DNA from a congeneric nematode species). Simultaneous hybridization of the microarray with two amplified targets labeled with different dyes proved to be efficient, without any competition between the targets. These results illustrate a significant step forward in the development of the DNA chip technology for nematode detection, and constitute to our knowledge the first report of production and use of oligonucleotide microarrays for the detection of plant-parasitic nematodes, using the quarantine species M. chitwoodi as a test organism.

The detection of Echinococcus granulosus in dogs is important for epidemiological surveillance and evaluation of cystic hydatic disease control programs. We report the efficacy of two PCR-based methods to detect patent and pre-patent infection in dogs experimentally infected with E. granulosus. The detection is based on amplification of a fragment of a mitochondrial gene (Mit-PCR) and a DNA repetitive element (Rep-PCR) of E. granulosus. We tested the ability of both methods to detect several genotypes of the parasite. Both PCR methods could detect E. granulosus in pre-patent and patent periods, even when microscopical observation of eggs resulted negative in fecal samples. The Mit-PCR produced the same amplification pattern for all the parasite genotypes tested while the amplification patterns with the Rep-PCR differed among groups of strains. Fecal samples collected from dogs of an endemic area were diagnosed with more sensitivity than arecoline hydrobromide purgation. These molecular methods could be applied in the confirmation of coproantigen-positive fecal samples and to verify the success of control programs.

We tested the hypothesis that the cestode Schistocephalus solidus is capable of premature gamete exchange as a plerocercoid in the last intermediate stickleback host. The existence of such a reproductive mode is suggested by the highly advanced gonadal development in the plerocercoid and the large fitness gain of outcrossing. In addition, eggs from selfing cestodes have a higher hatching rate when the cestode originated from a doubly infected stickleback than when it came from a singly infected fish. We hatched eggs from 10 singly breeding cestodes that originated from doubly infected sticklebacks with the prediction that some should be outcrossed and share alleles with both the breeding cestode and the second cestode in the coinfection if the hypothesis is correct. However, all of the 430 tested larvae matched only the alleles of the breeding cestode. It is therefore very unlikely that S. solidus engaged in gamete exchange in its fish host. We suggest an alternative hypothesis to explain the higher hatching rate of eggs produced by cestodes from doubly infected fish as compared to those from single infections.

Fascioliasis is of well-known veterinary importance and an increasing human health problem, with reported cases in the five continents. The causative agents, Fasciola hepatica and Fasciola gigantica, present geographical distributions, which overlap in many regions of Africa and Asia, and in which the differentiation of both species is usually difficult because of the many variations in their morphological characteristics. Moreover, in humans, liver fluke classification cannot be achieved by clinical, pathological, coprological or immunological methods. The differential diagnosis between F. hepatica and F. gigantica infection is very important because of their different transmission and epidemiological characteristics. A simple and rapid PCR-restriction fragment length polymorphism (RFLP) assay, using the common restriction enzymes Ava II and Dra II, is described to distinguish between both fasciolid species. It is based on a 618-bp-long sequence of the 28S rRNA gene recently obtained from liver fluke populations of South America, Europe and Africa. This sequence showed a few nucleotide differences between both fasciolids and no intraspecific variations within each species. This assay provides unambiguous results and may be useful for both individual subject diagnosis and epidemiological surveys of humans and animals in endemic regions of sympatry.

Mitochondrial genome sequences provide useful markers for investigating population genetic structures because of their maternal inheritance and high evolutionary rates. There is, however, a paucity of information on mitochondrial genomes for many parasitic organisms, including nematodes, which appears to relate mainly to technical limitations and (for modestly funded laboratories) the cost associated with full mitochondrial genome sequencing. In this article, we describe a simple, relatively inexpensive long-PCR approach for the amplification (using two sets of primers) of the entire mitochondrial genome from individual parasitic nematodes for subsequent sequencing, which overcomes these limitations. We employed two species of human hookworm (Ancylostoma duodenale and Necator americanus; order Strongylida) to establish the long-PCR conditions, and then extended its use to a number of other species of parasitic nematode of the class Secernentea (orders Strongylida, Ascaridida and Rhabditida). The long-PCR method for the amplification of the entire mitochondrial genome from single nematodes, coupled with direct sequencing of amplicons, provides a useful tool for the comparative analysis of genome organisation and evolution of a range of nematode groups. It also creates a platform for molecular ecological and population genetic studies., (Copyright 2002 Elsevier Science Ltd.)

The metacercariae of the lung flukes, Paragonimus westermani and P. miyazakii, are of known medical importance as the pathogens causing human paragonimiasis. They are both found in the same freshwater crab species in Japan and are morphologically quite similar. The aim of the present study was to establish molecular methods for accurate discrimination between individual metacercariae of the two species. In the first step, we amplified and sequenced the second internal transcribed spacer (ITS2) region of ribosomal DNA. Searches of nucleotide databases revealed that the ITS2 sequences generated from the metacercarial DNA were identical to those previously reported for the adults of the respective species. Utilizing a nucleotide difference between the two species, we have established two PCR-based techniques; PCR-restriction fragment length polymorphism and direct PCR-amplification using species-specific primers. Both techniques showed that the individual metacercariae of the two species could be unequivocally discriminated from one another. The present results suggest that the ITS2 region is useful for species discrimination irrespective of the life cycle stages of the lung flukes. Established techniques can thus be used for epidemiological investigations of the prevalence of human lung fluke metacercariae.

Eleven species belonging to superfamily Ascaridoidea, which infect marine and freshwater fish, mammals, and fish-eating birds, were analyzed using a PCR-RFLP method. The following species were investigated: Anisakis pegreffi, A. physeteris, and A. simplex (parasites of fish and mammals), Contracaecum osculatum, C. radiatum, and C. rudolphi (parasites of mammals and fish-eating birds), Hysterothylacium aduncum (a parasite of fish), Porrocaecum angusticolle, P. crassum, P. depressum, and P. ensicaudatum (parasites of fish-eating birds). PCR-amplified rDNA regions encompassing ITS1, 5.8S rDNA, and ITS2 produced on templates of genomic DNA isolated from all investigated species were digested with TaqI, AluI, BsuRI, and RsaI endonucleases. Restriction patterns showed that endonuclease TaqI is the most useful enzyme for identification of all investigated species. No variations in restriction patterns within each species were detected. Therefore, we propose that the PCR-RFLP assay described in this report may be used for identification of marine and freshwater parasites from superfamily Ascaridoidea.

Many lakes around the world are contaminated with bird schistosome cercariae, which penetrate into human skin, causing an itching dermatitis called "swimmers' itch." Bathers could be forewarned from exposure to the larvae and ecological examinations could be performed, when a sensitive method to detect the parasites in aquatic systems, where lots of organisms hinder microscopic examinations, would be available. For this purpose we cloned, sequenced, and analyzed a 396 bp tandem repeated DNA sequence from Trichobilharzia ocellata (ToSau3A), and employed it for developing molecular detection assays. It hybridized with less than 100 pg DNA from different Trichobilharzia species (T. ocellata, Trichobilharzia franki, and Trichobilharzia regenti), but not with 10 ng DNA from other related or sympatric trematodes. A PCR assay, amplifying this sequence with the same specificity, detected 100 fg T. ocellata DNA, 1 cercaria in 0.5 g plankton, and 2 cercariae in 0.5 g host snail tissues.

The sensitive, rapid and species-specific diagnosis of Brugia infections in humans or animal models is important in determining the level of parasitemia and the efficacy of chemotherapy or vaccinations. The HhaI family of highly repeated DNA sequences from Brugia have been useful in polymerase chain reaction (PCR)-based diagnosis of brugian filarial infections in blood samples and in mosquitoes. A PCR assay was developed using a biotinylated primer, a non-biotinylated primer and a species-specific chemiluminescent probe [tris[2,2'bipyridine] ruthenium (II) chelate, TBR] to detect PCR amplified Hhal family repeats. Individual blood samples from jirds infected with Brugia malayi or B. pahangi and with different levels of microfilaremia were tested in this assay. Known concentrations of Brugia DNA and DNA from the blood of uninfected control jirds were used as positive and negative controls, respectively. The PCR products generated by this method were analyzed using a semi-automated quantitative (Q)-PCR system. The levels of parasite DNA can be calculated from the luminosity units generated. Significant amounts of parasite DNA were detected in blood samples from infected jirds, and these values were correlated with the levels of microfilaremia. In contrast, reductions in circulating microfilaria following treatment with ivermectin correlated with low levels of measurable DNA. Using this system, we were also able to detect HhaI repeat DNA in the spleens of B. pahangi- infected jirds at 56 days post-infection when circulating microfilariae were not readily detectable. The results indicate that the species-specific Hhal Q-PCR detection and quantification method is rapid and sensitive, is useful in the detection of Brugia DNA in blood and other tissues and is suited for use in clinical settings because it does not require radioactive isotopes and gel-based protocols.

Filariasis is still a public health problem in tropical countries. The most common causative agents of human filariasis are Wuchereria bancrofti and Brugia malayi. Traditional methods used to detect filarial parasites in human, animal and vector populations are tedious, time consuming, and confer little guarantee of sensitivity and species specificity. We have developed a rapid and specific method to detect filarial parasite DNAs in blood and mosquito samples using the polymerase chain reaction (PCR) technique. The primers used are MF/F and MF/R which amplify a 1.5 kb glutathione peroxidase gene of filarial worms. Using the restriction fragment length polymorphism (RFLP) technique, these PCR products will be further digested with restriction enzymes either Hpa I, Pst I, Alu I or Hinf I to differentiate the genus of filaria. This PCR-RFLP technique can be apply to use in diagnosis and to differentiate between species of filaria in humans the reservoir host and the mosquito vector in endemic areas, (Copyright 2000 Academic Press.)

The nematode Caenorhabditis elegans has been shown to be an excellent model organism with which to study the mechanisms of programmed cell death because of its powerful genetics and the ability to study cell death with single-cell resolution. In this chapter, we describe methods that are commonly used to examine various aspects of programmed cell death in C. elegans. These methods, in combination with genetic analyses, have helped identify and characterize many components of the C. elegans cell death pathway, illuminating the mechanisms by which these components affect programmed cell death.

A polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis of the expansion segment 5 (domain IV) of the large subunit of ribosomal DNA was employed to characterize seven isolates of Trichinella from China (A-G), including six of pig origin from regions in Dengxian (A), Tianjin (B), Harbin (D), Baoshan (E), Xinye (F) and Xian (G), and one of canine origin from Changchun (C). Isolates A, D, E, F and G were classified as Trichinella spiralis based on SSCP patterns, while the patterns for isolates B and C were consistent with those of Trichinella nativa or Trichinella T6. The results were supported by random amplified polymorphic DNA (RAPD) analysis using five decamer primers and were in accordance with ecological information for the isolates. Single strand conformation polymorphism results also allowed the direct display of mutational sequence variation in the expansion segment among the five isolates of T. spiralis from China, indicating its usefulness for studying population variation within that species., (Copyright 1998 Academic Press Limited)

Phenoloxidase (PO) is the key enzyme for melanin synthesis and plays an important role in the defense and recognition of pathogens in insects and other arthropods. We now report the upregulated transcription of the gene encoding the precursor of PO, prophenoloxidase, in Onchocerca-infected Simulium damnosum s.l., the main vector of human and bovine onchocerciasis in subsaharan Africa. Using homology-based generic primers in a polymerase chain reaction-based targeted differential display, the gene itself was identified and partially sequenced.

Setaria species are filarial parasites which inhabit the peritoneal cavity of cattle and other ungulates. The parasite is generally considered to be nonpathogenic in its natural hosts, but the transmission of the infective larvae through mosquito vectors to its abnormal hosts (goats, sheep, or horses) can result in a serious and often fatal neuropathological disorder commonly referred to as cerebrospinal nematodiasis. We have previously described the cloning and characterization of a repetitive DNA sequence of Setaria digitata that could be used as a diagnostic probe to detect the parasite in host and vector populations. Here we report the development of a rapid nonradioactive hybridization assay using an oligonucleotide probe based on the above cloned repetitive sequence.

Oesophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodular worm) is of major human health significance in northern Togo and Ghana where Necator americanus (human hookworm) also exists at high prevalence. However, very little is known about the transmission patterns of O. bifurcum, partly due to the difficulty in differentiating O. bifurcum from N. americanus at some life-cycle stages using morphological features. To overcome this limitation, a molecular approach utilizing genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA was developed. The ITS-2 sequence of each species was determined, and specific oligonucleotide primers were designed to the regions of greatest sequence difference between the species. Utilizing these primers, rapid PCR assays were developed for the specific amplification of DNA of O. bifurcum or N. americanus, which have the potential to confirm the identity of eggs from faeces and larvae from the intestine or environment. The application of species-specific PCR has important implications for studying the epidemiology and population biology of O. bifurcum.

Fourteen species of parasitic nematodes (order Strongylida) were characterized using a polymerase chain reaction-linked single strand conformation polymorphism technique (PCR-SSCP). The rDNA region spanning the second internal transcribed spacer (ITS-2) was amplified from parasite DNA by PCR. The PCR products were then denatured and subjected to electrophoresis on a non-denaturing gel matrix. PCR-SSCP of the single stranded ITS-2 molecules generated characteristic and reproducible patterns for each species, and allowed the rapid delineation of all of the 14 species in one step. The method also allowed the display of variation in patterns within some species between different geographical isolates. These findings demonstrate the usefulness of PCR-SSCP of ITS-2 for the rapid identification of nematode species and indicate its potential for resolving variation in the ITS-2 sequence within a species.

At some stages of development, it is impossible to identify the porcine nodular worms Oesophagostomum dentatum and O. quadrispinulatum to the species level using morphological parameters. A molecular approach utilizing genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA was developed to overcome this limitation. The ITS-2 sequence of each species was determined, and specific oligonucleotide primers were designed to regions of greatest sequence difference between the species. Utilizing these primers, rapid PCR procedures were developed for the specific amplification of DNA of O. dentatum or O. quadrispinulatum, which are now used routinely to monitor the purity of larval cultures and to confirm the identity of larvae derived from the intestine or faeces. The application of specific PCR has major implications for studying the population biology of nodular worms in the pig model.

A simple method to design a polymerase chain reaction (PCR) primer for parasites of interest was developed using Dirofilaria immitis as a test sample. The method involved the cloning and sequencing of randomly amplified DNA of the parasite, and designing a primer based on the resulting DNA sequence. Using the primer, DNA fragments of the expected length were amplified by a regular PCR with genomic DNA of Dirofilaria immitis.

Seven species of closely-related nematode parasite (Trichostrongylus axei, T. colubriformis, T. probolurus, T. retortaeformis, T. rugatus, T. vitrinus and T. tenuis) were characterized using a polymerase chain reaction-linked restriction fragment length polymorphism technique (PCR-RFLP). The rDNA region spanning the first and second internal transcribed spacers as well as the 5.8S rDNA gene (ITS+) was amplified from isolates of each of the seven species, digested separately with six restriction endonucleases (Dra I, Hinf I, Rsa I, Vsp I, Nla III and Tsp 509 I) and the fragments separated by agarose gel electrophoresis. PCR-RFLP of ITS+ produced characteristic patterns for each Trichostrongylus species examined. No variation in RFLP patterns was observed among different isolates for species where multiple isolates were examined. The present study demonstrates that the ITS+ provides genetic markers for the species identification of closely-related parasitic nematodes, and indicates the usefulness of these markers for diagnostic purposes, and epidemiologic and molecular-systematic studies on parasites and other eukaryotic organisms.

Digestion of genomic DNA of Trichinella spiralis with EcoRI reveals the presence of several families of repetitive DNA. Two small repeats of 516 and 604 bp, respectively, have been cloned and sequenced. The two repeats are related to each other and to a 1.1-kb repetitive DNA. Similar sequences are not detected in other species by hybridisation. There are about 270 copies of each small repeat per haploid genome and these are organised in large arrays of > or = 50 kb. The basic repetitive unit appears to contain one copy of each repeat. This repetitive DNA is transcribed in muscle-stage larvae although it is not protein coding.

We have determined the levels of superoxide dismutase (SOD) in different stages of the lymphatic filarial nematode parasite of man, Brugia malayi. Adult male worm extracts showed the highest levels of enzyme activity at 34.5 U mg-1, and there was no significant difference in the overall levels of SOD in extracts of adult female worms and microfilariae (27.1 and 26.7 U mg-1, respectively). SOD activity was detected in the culture medium of parasites maintained in vitro, with particularly high levels of specific activity in media in which males and females were maintained (357 and 339 U mg-1, respectively), indicative of active secretion. In all cases, this was accounted for predominantly by CuZn SOD, assessed by potassium cyanide inhibition. Northern blots with cDNA probes specific for cytoplasmic and extracellular CuZn SODs indicated that levels of mRNA for the cytoplasmic form were similar between adults and microfilariae, whereas expression of the extracellular form was 10x higher in adult worms. Western blots with an antibody to recombinant CuZn SOD demonstrated that higher levels of the extracellular protein were present in adult male worms, whereas the cytoplasmic form was present in roughly equivalent amounts in males, females, and microfilaria. Iodination and immunoprecipitation experiments indicated that the extracellular enzyme was accessible to surface labeling of both male and female adult worms, but not microfilaria. Immuno-electron microscopy showed that CuZn SOD was localised predominantly in the hypodermis of adult parasites, with an asymmetric distribution in the intercordal regions suggestive of compartmentalisation into several distinct syncytia. No labeling was evident in the cuticle, and thus the accessibility of the extracellular enzyme to extrinsic iodination in adult worms remains unclear. No binding of antibody was demonstrable in the glandular region of the oesophagus or the uterus of females, presumed to be major sites of synthesis for secreted proteins. Dense labeling was observed in the seminal fluid surrounding spermatazoa in the vas deferens of male parasites. These data also suggest that, as observed in mammals, nematode spermatazoa are particularly susceptible to oxidative damage and are protected during storage by secreted anti-oxidant enzymes.