Molecular Analysis of Echinostome Metacercariae from Their Second Intermediate Host Found in a Localised Geographic Region Reveals Genetic Heterogeneity and Possible Cryptic Speciation

Echinostome metacercariae are the infective stage for humans and animals. The identification of echinostomes has been based until recently on morphology but molecular techniques using sequences of ribosomal RNA and mitochondrial DNA have indicated major clades within the group. In this study we have used the ITS2 region of ribosomal RNA and the ND1 region of mitochondrial DNA to identify metacercariae from snails collected from eight well-separated sites from an area of 4000 km² in Lamphun Province, Thailand. The derived sequences have been compared to those collected from elsewhere and have been deposited in the nucleotide databases. There were two aims of this study; firstly, to determine the species of echinostome present in an endemic area, and secondly, to assess the intra-specific genetic diversity, as this may be informative with regard to the potential for the development of anthelmintic resistance and with regard to the spread of infection by the definitive hosts. Our results indicate that the most prevalent species are most closely related to E. revolutum, E. trivolvis, E. robustum, E. malayanum and Euparyphium albuferensis. Some sites harbour several species and within a site there could be considerable intra-species genetic diversity. There is no significant geographical structuring within this area. Although the molecular techniques used in this study allowed the assignment of the samples to clades within defined species, however, within these groupings there were significant differences indicating that cryptic speciation may have occurred. The degree of genetic diversity present would suggest the use of targeted regimes designed to minimise the selection of anthelmintic resistance. The apparent lack of geographic structuring is consistent with the transmission of the parasites by the avian hosts.     

Haplorchis taichui, Witenberg, 1930: Development of a HAT-RAPD marker for the detection of minute intestinal fluke infection

Specific primers to determine the presence of an intestinal fluke, Haplorchis taichui, were investigated using the high annealing temperature random amplified polymorphic DNA (HAT-RAPD) PCR, and 18 arbitrary primers (Operon Technologies), to generate different polymorphic DNA profiles. Thirteen kinds of parasites were used to compare fingerprints. A 256 bp HAT-RAPD marker, generated from the OPP-11 primer, was found to be H. taichui-specific, and this marker was cloned, transformed, and sequenced. From the sequence data, a pair of primers were designed with Genetyx-MAC ver.11 and indicated as: Hap-t F 5′-GGC CAA CGC AAT CGT CAT CC-3′ and Hap-t R 5′-GCG TCG GGT TTC AGA CAT GG-3′. These specific primers were tested for efficacy and specificity by amplifying them with all 13 parasites DNAs in PCR reaction. A 256 bp amplicon was generated, which was shown to have a positive result, only for H. taichui DNA. It revealed no cross-reaction with any of the other tested parasite species. The minimum DNA template, needed for detection by PCR, was 0.1 picogram (pg). The successful development of H. taichui-specific primers is expected to be beneficial for epidemiological studies and for prevention and control of these parasitic infections.     

Morphological and molecular profiling of Spirogyra from northeastern and northern Thailand using inter simple sequence repeat (ISSR) markers

Green algae, Spirogyra (Chlorophyta), are found in a wide range of habitats including small stagnant water bodies, rivers, and streams. Species identification of Spirogyra based on morphological characteristics has proven to be a difficult process. An accurate identification method is required to evaluate genetic variations. This study is aimed at investigating the molecular profiling of 19 samples of Spirogyra from northern and northeastern Thailand. The morphological characteristics of each sample were recorded, viz. cell dimensions (width and length), along with the number and arrangement of chloroplast spirals/pyrenoids. With regard to a correlation of the biological and ecological parameters, conductivity was clearly significantly related to the number of pyrenoids. While DO is negatively related to the number of chloroplast spirals. Molecular studies with 10 ISSR primers were amplified to examine the DNA fingerprints. Morphological characters were determined to be significantly different by revealing 5 traits (P < 0.05) for all specimens. In addition, the DNA markers of all specimens were investigated using 10 ISSR primers. The results show that the PCR technique amplified 108 fragments. An analysis of the DNA fragments grouped all samples by ISRR-PCR, which were then separated into two groups according to their distribution.     

Opisthorchis viverrini and Haplorchis taichui: Development of a multiplex PCR assay for their detection and differentiation using specific primers derived from HAT-RAPD

Specific primers for the detection of Opisthorchis viverrini and Haplorchis taichui were investigated by using the HAT-RAPD PCR method. Fourteen arbitrary primers (Operon Technologies) were performed for the generation of polymorphic DNA profiles. The results showed that a 319 bp fragment generated from the OPA-04 primer was expected to be O. viverrini-specific while a 256 bp fragment generated from the OPP-11 primer was considered to be H. taichui-specific. Based on each sequence data, two pairs of specific primers were designed and sequences of each primer were as follows; H. taichui; Hapt_F5′-GGCCAACGCAATCGTCATCC-3′and Hapt_R1 5′-CTCTCGACCTCCTCTAGAAT-3′ which yielded a 170 bp PCR product. For O. viverrini, OpV-1F: 5′-AATCGGGCTGCATATTGACCGAT-3′ and OpV-1R: 5′-CGGTGTTGCTTATTTTGCAGACAA-3′ which generated a 319 bp PCR product. These specific primers were tested for efficacy and specific detection for all parasites DNA samples. The results showed that 170 and 319 bp specific PCR products were generated as equivalent to positive result in H. taichui and O. viverrini, respectively by having no cross-reaction with any parasites tested. PCR conditions are recommended at 68 °C annealing temperature and with 0.5 mM magnesium chloride (Mg Cl₂). Additionally, specific primers developed in this study were effective to determine the presence of both parasites in fish and snail intermediate hosts, which the DNA of O. viverrini was artificially spiked since it is rarely found in northern Thailand.The H. taichui and O. viverrini-specific primers successfully developed in this study can be use for epidemiological monitoring, preventing management and control programs.     

Prevalence of Haplorchis taichui in field-collected snails: a molecular approach

The prevalence of the cercarial stage of an intestinal trematode, Haplorchis taichui, in thiarid snails (Gastropoda: Thiaridae) was investigated using light microscope and species-specific PCR procedures. A total of 988 snails were collected from Mae Taeng district, Chiang Mai province, northern Thailand, which comprised of 3 species; Melanoides tuberculata, Tarebia granifera, and Thiara scabra. The overall prevalence of pleurolophocercous cercariae was 21.7% as determined by the morphology. For genetic detection of H. taichui infection in snails, 2 primers Hapt_F (5'-GGCCAACGCAATCGTCATCC-3') and Hapt_R (5'-GCGTCGGGTTTCAGACATGG-3'), were used. The genomic DNA of H. taichui, which was used as a positive control, gave an amplification of the 256 bp fragment. The overall prevalence of H. taichui from specific PCR was 9.7%. The proportion of H. taichui among the pleurolophocercous cercariae in this study was 44.9%.     

Echinostoma revolutum: Freshwater Snails as the Second Intermediate Hosts in Chiang Mai,Thailand

The occurrence of 37-collar spined echinostome metacercariae in freshwater snails was investigated in 6 districts of Chiang Mai Province, Thailand, from October 2011 to April 2012. A total of 2,914 snails that belong to 12 species were examined, and 7 snail species (Clea helena, Eyriesia eyriesi, Bithynia funiculata, Bithynia siamensis siamensis, Filopaludina doliaris, Filopaludina sumatrensis polygramma, and Filopaludina martensi martensi) were found infected with echinostome metacercariae. The prevalence of metacercariae was the highest in Filopaludina spp. (38.5-58.7%) followed by B. funiculata (44.0%), E. eyriesi (12.5%), B. siamensis siamensis (8.2%), and C. helena (5.1%). Metacercariae were experimentally fed to hamsters and domestic chicks, and adult flukes were recovered from both hosts at days 15 and 20 post-infection. The adult flukes were identified based on morphological features, morphometrics, host-parasite relationships, and geographical distribution. They were compatible to Echinostoma revolutum or Echinostoma jurini, with only minor differences. As the adults were recovered from both hamsters and chicks, our specimens were more compatible to E. revolutum rather than E. jurini (reported only from mammals). This is the first report for metacercariae of E. revolutum in the snail host, C. helena, and also confirmed that Filopaludina spp., E. eryresi, and Bithynia spp. act as the second intermediate hosts of E. revolutum under natural conditions, which are indigenously distributed in Chiang Mai province.     

Co-infection with Opisthorchis viverrini and Haplorchis taichui detected by human fecal examination in Chomtong district, Chiang Mai Province, Thailand

Diseases caused by the liver fluke, Opisthorchis viverrini and the minute intestinal fluke, Haplorchis taichui, are clinically important, especially in the Northeast and North regions of Thailand. It is often difficult to distinguish between these trematode species using morphological methods due to the similarity of their eggs and larval stages both in mixed and co-infections. A sensitive, accurate, and specific detection method of these flukes is required for an effective epidemiological control program. This study aimed to determine the prevalence of O. viverrini and H. taichui infections in human feces by using formalin-ether sedimentation and high annealing temperature random amplified polymorphic DNA (HAT-RAPD) PCR methods. Fecal specimens of people living along the Mae Ping River, Chomtong district were examined seasonally for trematode eggs using a compound microscope. Positive cases were analyzed in HAT-RAPD, DNA profiles were compared with adult stages to determine the actual species infected, and specific DNA markers of each fluke were also screened. Our results showed that out of 316 specimens, 62 were positive for fluke eggs which were pre-identified as O. viverrini and H. taichui. In addition, co-infection among these two fluke species was observed from only two specimens. The prevalence of H. taichui infections peaked in the hot-dry (19.62%), gradually decreased in the rainy (18.18%), and cool-dry seasons (14.54%), respectively. O. viverrini was found only in the hot-dry season (6.54%). For molecular studies, 5 arbitrary primers (Operon Technologies, USA) were individually performed in HAT-RAPD-PCR for the generation of polymorphic DNA profiles. The DNA profiles in all 62 positives cases were the same as those of the adult stage which confirmed our identifications. This study demonstrates the mixed infection of O. viverrini and H. taichui and confirms the extended distribution of O. viverrini in Northern Thailand.     

Effect of niclosamide on the tegumental surface of Haplorchis taichui using scanning electron microscopy

The effect of niclosamide on the tegument of adult Haplorchis taichui (Trematoda: Heterophyidae) exposed in vitro was observed by scanning electron microscope. Adult worms were incubated in Tyrode's solution containing 0.01, 0.1, 1.0, and 10 microg ml(-1) of niclosamide for 30 min, 1, 6, 12 and 24 h. Control groups were incubated in Tyrode's solution without niclosamide and worms remained active until 24 h. In 0.01 microg ml(-1) of niclosamide, worms showed slightly active movements up to 1 h after incubation, while in 0.1 microg ml(-1) solution a few worms showed only slightly active movements after 30 min. Tegumental changes were determined by scanning electron microscopy. Swelling and blebbing of the tegument were observed on both ventral and dorsal sides. After longer periods, extensive swelling and blebbing of the tegument became more severe and there was a loss of the apical plasma membrane in some regions. Empty spine sockets occurred, and small perforations penetrated the basal lamina, followed by some lesions. Destruction of both surfaces was more pronounced on the posterior compared with the anterior regions.     

Experimental Life History and Biological Characteristics of Fasciola gigantica (Digenea: Fasciolidae)

This study was conducted to investigate the life history, morphology, and maturation of larval stages and adult worms of Fasciola gigantica in experimental mice. Lymnaea auricularia rubiginosa was used as the intermediate host, and Oryza sativa was used for encystment of the metacercariae, while Mus musculus was used as the definitive host for maturation study. Fresh eggs from the gall bladder of water buffaloes fully developed into embryonated ones and hatched out at days 11-12 after incubation at about 29ºC. Free-swimming miracidia rapidly penetrated into the snail host, and gradually developed into the next larval stages; sporocyst, redia, and daughter redia with cercariae. Fully-developed cercariae were separated from the redia and shed from the snails on day 39 post-infection (PI). Free-swimming cercariae were immediately allowed to adhere to rice plants, and capsules were constructed to protect metacercariae on rice plants. Juvenile worms were detected in intestines of mice at days 3 and 6 PI, but they were found in the bile duct from day 9 PI. Juvenile and adult flukes were recovered from 16 mice experimentally infected with metacercariae, with the average recovery rate of 35.8%. Sexually mature adult flukes were recovered from day 42 PI. It could be confirmed that experimentally encysted metacercariae could infect and develop to maturity in the experimental host. The present study reports for the first time the complete life history of F. gigantica by an experimental study in Thailand. The obtained information can be used as a guide for prevention, elimination, and treatment of F. gigantica at environment and in other hosts.