Molecular identification of trematode parasites infecting the freshwater snail Bithynia siamensis goniomphalos in Thailand.

Digenetic trematodes are important parasites of humans and animals. They have complex life cycles and typically infect a gastropod as the first intermediate host. Bithynia siamensis goniomphalos, the first intermediate host of the liver fluke, Opisthorchis viverrini, harbours a wide variety of other trematode species. Morphological details of cercariae of 20 trematode taxa from B. s. goniomphalos, collected mainly in Thailand from 2009 to 2014, were provided in an earlier paper. Correct identification to the species or genus level based on morphology of these cercariae is generally not possible. Therefore, we used molecular data to improve identification and to investigate the diversity of the species of trematodes infecting B. s. goniomphalos. We were successful in extracting, amplifying and sequencing portions of the 28S ribosomal RNA (rRNA) gene for 19 of these 20 types of cercaria, and the internal transcribed spacer 2 region for 18 types. BLAST searches in GenBank and phylogenetic trees inferred from the 28S rRNA sequences identified members of at least nine superfamilies and 12 families. Only a few cercariae could be assigned confidently to genus or species on the basis of the sequence data. Matching sequence data from named adult trematodes will be required for definitive identification. There is clearly a great diversity of trematode species utilizing B. s. goniomphalos in Thailand.

Trematode diversity in the freshwater snail Bithynia siamensis goniomphalos sensu lato from Thailand and Lao PDR.

In order to obtain a comprehensive understanding of trematode diversity in Bithynia siamensis goniomphalos sensu lato, the first intermediate host of the liver fluke Opisthorchis viverrini s.l., the prevalence of larval trematode species was investigated in different localities in Thailand and Lao People's Democratic Republic (Lao PDR). In Thailand, snail samples were collected from 29 localities in the nine provinces: Buri Ram, Surin, Chaiya Phum, Maha Sarakham, Khon Kaen, Kalasin, Mukdahan, Sakon Nakhon and Nakhon Phanom. In Lao PDR, snail samples were collected from 21 localities in Vientiane Province and six localities in Savannakhet Province. Snails were identified by standard morphological criteria and then examined for trematode infection using the cercarial shedding method. Twenty different types of cercariae were detected and identified, based on morphological criteria. Virgulate type 1 emerged as the most common cercaria, with an average prevalence of 10.90% (range 0.26-54.22%) in Thailand and 6.58% (range 1.15-89.77%) in Lao PDR. Opisthorchis viverrini s.l. cercariae were the fourth most common in Thailand, with an average prevalence of 1.59% (0.15-6.93), while in Lao PDR their prevalence was 0.96% (0.08-8.37). The high diversity of trematode cercariae observed in this study indicates that B. s. goniomphalos s.l. is highly susceptible to infection with a variety of trematode species. However, the role of non-opisthorchiid trematodes as fish-borne parasites in human health is not fully known and further molecular identification is required.

Genetic differentiation of Echinostoma revolutum and Hypodereaum conoideum from domestic ducks in Thailand by multilocus enzyme electrophoresis.

Echinostomes are common intestinal parasites causing zoonotic disease, which are endemic worldwide. Of the four species of medically important echinostomes reported in Thailand, two species, Echinostoma revolutum and Hypodereaum conoideum, have been detected in poultry. These two parasites are morphologically similar and are sometimes difficult to distinguish. In the present study, multilocus enzyme electrophoresis was used to differentiate E. revolutum from H. conoideum collected from domestic ducks in Thailand. The parasites were compared using 22 enzymes with 30 presumptive enzyme loci. The two species of echinostome could be distinguished at 17 of the 30 enzyme loci. Several loci were polymorphic within each species, suggesting that these can be used to examine their population genetics.

Unique biological rhythm in the reproductive behaviour of female ticks of reptiles.

We report the discovery of a biological rhythm in the reproductive behaviour of the tick Bothriocroton hydrosauri that was absent in Amblyomma limbatum, a species that occurs on the same species of reptile host. Female B. hydrosauri mated in autumn or winter delayed oviposition until the following spring, while there was no diapause in conspecific females mated in spring or early summer. Initiation of ovipositional diapause in ticks is usually related to photoperiodic stimuli, but this was not the case for B. hydrosauri. The sinusoidal pattern in pre-oviposition times of B. hydrosauri females mated in different months in the laboratory suggests an internal seasonal time-keeping mechanism. We hypothesize that hormones imbibed by females during their bloodmeal may provide environmental cues associated with the induction of diapause. Irrespective of the mechanism underlying the rhythm, diapause by B. hydrosauri females mated during autumn or winter is of adaptive advantage because it synchronizes oviposition with favourable environmental conditions for egg hatching and increases the chance of larvae finding a host. The lack of a similar biological rhythm in A. limbatum may be a reflection of the different environmental conditions this species experiences throughout most of its range as compared with B. hydrosauri.

Impact of temporal changes and host factors on the genetic structure of a population of Opisthorchis viverrini sensu lato in Khon Kaen Province (Thailand).

The population genetics of 317 individual Opisthorchis viverrini from Khon Kaen Province Thailand, from 4 different years and 4 cyprinid fish species was examined using multilocus enzyme electrophoresis of enolase (Enol), phosphoglucomutase (Pgm) and triose phosphate isomerase (Tpi). Allele and genotype frequencies for Enol and Pgm were consistent irrespective of year or host species. No heterozygote deficiency was detected for Enol. Significant heterozygote deficiencies were detected in 3 of 4 years for Pgm. For Tpi, allele frequencies of the most common allele and genotype frequency varied between years and among individuals from different host species. Heterozygote deficiencies for Tpi were detected in 2 years. No significant heterozygous deficiencies were detected among O. virerrini from different fish species in 2005, except at Pgm and Tpi from Puntioplites protozsron. There was no statistical significance in pairwise FST values between O. viverrini from Cyclocheilichthys armatus in different years or different host species in 2005. Significant departures from Hardy-Weinberg expectations and a high rate of gene flow in a population of O. viverrini are discussed in terms of self- and cross-fertilisation, natural selection, non-random mating, the Wahlund effect, presence of null alleles, intensity of infection, biology and ecology of their intermediate cyprinid hosts.

Mitochondrial DNA sequence variation among geographical isolates of Opisthorchis viverrini in Thailand and Lao PDR, and phylogenetic relationships with other trematodes.

The present study compared the genetic variation among 14 different geographical isolates of Opisthorchis viverrini sensu lato from Thailand and Lao PDR using sequence data for 2 mitochondrial DNA genes, the subunit 1 of NADH dehydrogenase gene (nad1) and cytochrome c oxidase gene (cox1). Four different nad1 haplotypes were detected among isolates, all of which were identical at the amino acid sequence level. Nucleotide sequence variation among 14 isolates ranged from 0 to 0.3% for nad1. Two different cox1 haplotypes were detected among isolates. These two haplotypes differed at 2 nucleotide positions, one of which resulted in a change in the amino acid sequence. Nucleotide sequence variation among isolates for cox1 ranged from 0 to 0.5%. Comparison of cox1 sequences of O. viverrini to those of other trematodes revealed nucleotide differences of 13-31%. A phylogenetic analysis of the cox1 sequence data revealed strong statistical support for a clade containing O. viverrini and 2 other species of opisthorchid trematodes; O. felineus and Clonorchis sinsensis.

Distributions of the paralysis ticks Ixodes cornuatus and Ixodes holocyclus in south-eastern Australia.

OBJECTIVE: To describe the actual and potential geographic distributions of Ixodes cornuatus and I holocyclus in south-eastern Australia. PROCEDURE: Examination of ticks from museum collections and trapped animals were made. (Bioclimatic analysis BIOCLIM) was used to predict potential distributions. RESULTS: I holocyclus was collected from rodents (Rattus fuscipes, R lutreolus, R rattus), wombats (Vombatus ursinus), cats and dogs in Gippsland and I cornuatus was collected from rodents (R fuscipes), wombats, cats and dogs in central Victoria. All life-cycle stages of both species were collected during the warmer months of the year. The known distribution of the two species was established from specimens in museum collections and suggested that a boundary between the two may exist in eastern Gippsland. BIOCLIM suggested that the area immediately to the east of Melbourne was climatically suitable for I holocyclus, although no endemic foci of infection are currently known from this region. The potential distribution of I cornuatus included east Gippsland and the Otway Ranges, areas in which the tick is not currently known to occur. CONCLUSIONS: I holocyclus and I cornuatus have more restricted distributions than current collections suggest and therefore may have the possibility to extend their geographical ranges in the future.

Untangling the Complexity of Liver Fluke Infection and Cholangiocarcinoma in NE Thailand Through Transdisciplinary Learning.

This study demonstrates how a transdisciplinary learning approach provided new insights for explaining persistent Opisthorchis viverrini infection in northern Thailand, as well as elucidating problems of focusing solely on the parasite as a means of addressing high prevalence of cholangiocarcinoma. Researchers from diverse backgrounds collaborated to design an investigative homestay program for 72 Singaporean and Thai university students in five northeast Thai villages. The students explored how liver fluke infection and potential cholangiocarcinoma development are influenced by local landscape dynamics, aquatic ecology, livelihoods, food culture and health education. Qualitative fieldwork was guided daily by the researchers in a collaborative, co-learning process that led to viewing this health issue as a complex system, influenced by interlinked multidimensional factors. Our transdisciplinary experience has led us to believe that an incomplete understanding of these linkages may reduce the efficacy of interventions. Further, viewing liver fluke infection and cholangiocarcinoma as the same issue is inadvisable. Although O. viverrini infection is an established risk factor for the development of cholangiocarcinoma, multiple factors are known to influence the likelihood of acquiring either. Understanding the importance of the current livelihood transition, landscape modification and the resulting mismatch between local cultures and new socio-ecological settings on cholangiocarcinoma initiation and liver fluke transmission is of critical importance as it may help readjust our view of the respective role of O. viverrini and other socioeconomic risk factors in cholangiocarcinoma etiology and refine intervention strategies. As demonstrated in this study, transdisciplinary approaches have the potential to yield more nuanced perspectives to complex diseases than research that focuses on specific aspects of their epidemiology. They may therefore be valuable when designing effective solutions to context-sensitive diseases such as liver fluke infection and cholangiocarcinoma.

Molecular epidemiology: assumptions and limitations of commonly applied methods.

An understanding of the epidemiology of a disease (i.e. its aetiology, transmission patterns) is crucial for the development and implementation of effective management practices. This requires sound epidemiological data. It is therefore important that scientists understand the assumptions and limitations of the methods used to gather such data. The aim of this paper is to discuss some of the assumptions and limitations of PCR-based methods used in studies of epidemiology. Since its development, PCR has had a major impact in the biological sciences. The ability to selectively amplify a specific region of the genome from a small amount of DNA makes this technique particularly useful as a diagnostic tool. A variety of PCR-based methods are available which can be used to identify strains and species of parasites. Some of these methods, such as random amplification of polymorphic DNA, have intrinsic properties which can limit their application. Other methods, such as PCR-restriction fragment length polymorphism, require the availability of a sound taxonomic or genetic framework for the development of any diagnostic system for a particular organism. The problems encountered developing diagnostic probes in the absence of such a framework will be discussed using Giardia intestinalis as an example.

Paradigms and expectations: the nature of research and diagnostics.

Researchers are judged by their best work. No matter what disasters happen at the researcher's bench, there is time to repeat experiments, discard ambiguous data and work steadily toward a clearer understanding of the research problem. The work practices of researchers are matters of personal choice, because the process of peer review prior to publication considers only the work that is reported and not all work done by the researcher. On the other hand, the diagnostician's credibility is directly linked to every experiment performed, because a customer is waiting for the result. This paper contrasts aspects of the work of researchers and diagnosticians and concludes that researchers could benefit from understanding the framework within which diagnosticians operate.

Multilocus enzyme electrophoresis: a valuable technique for providing answers to problems in parasite systematics.

The aim of this review is to highlight the effectiveness of the technique of multilocus enzyme electrophoresis in answering questions relating to the systematics of parasites and to highlight errors in the way the technique has been used and the results interpreted. We have approached this topic by answering specific questions that we have been asked by colleagues and students not necessarily familiar with the technique, the method of data analysis and its application. Although the technique has been applied to provide answers for taxonomic and population genetics studies, it remains under-utilised, perhaps because of recent advances in newer molecular technology. Rather than not acknowledge or dismiss the value of more traditional technology, we suggest that researchers examine problems in the systematics of parasites by the comparison of data derived from morphological, biochemical and molecular techniques.

Multiparasite communities in animals and humans: frequency, structure and pathogenic significance.

Individual humans and animals are subject to infection by a variety of parasites (broadly defined to include viruses, bacteria and other non-protozoan microparasites) at any one time. Multiple parasite infections occur frequently in populations of wild animals as well as in humans from developing countries. In some species and regions, hosts with multiple infections are more common than hosts with either no infection or a single infection. Studies, predominantly on animals, show that a wide variety of environmental and host-dependent factors can influence the structure and dynamics of the communities of parasites that make up these multiple infections. In addition, synergistic and competitive interactions can occur between parasite species, which can influence the likelihood of their successful transmission to other hosts and increase or decrease their overall pathogenic impact. This review summarises aspects of our current knowledge on the frequency of multiparasite infections, the factors which influence them, and their pathogenic significance.

Sibling species within Macropostrongyloides baylisi (Nematoda: Strongyloidea) from macropodid marsupials.

Macropostrongyloides baylisi from four different species or subspecies of host were analysed electrophoretically at 27 enzyme loci. The results revealed the existence of two species, one in Macropus giganteus and the other in M. robustus robustus, M.r. erubescens and M.r. parryi, that had fixed genetic differences at 33% of loci. Populations of nematodes from two subspecies of M. robustus, M.r. robustus from Queensland and M.r. erubescens from South Australia, had fixed genetic differences at two (7.4%) of 27 loci and were considered to belong to the same species. No fixed genetic differences were detected between nematodes from M. parryi and M.r. robustus. A discriminant function analysis of morphological data assigned 96% of specimens to groups defined on the basis of the host species or subspecies from which they were obtained. This separation of Ma. baylisi into host-specific groups did not, however, totally correlate with the electrophoretic data. The species of M. baylisi in M. giganteus was genetically more distinct from the sibling species in M. robustus/M. parryi than to a related but morphologically dissimilar nematode, Ma. yamagutii from M. fuliginosus. This suggests an evolutionary parallel between host and parasite at the genetic level which is not reflected by morphological differences.

Detection by allozyme electrophoresis of cryptic species of Hypodontus macropi (Nematoda: Strongyloidea) from macropodid marsupials.

Allozyme electrophoresis of 98 Hypodontus macropi from eight different species of hosts using 24 enzymes revealed a complex of at least six sibling species, with 15-50% fixed genetic differences between taxa. Except for the taxon parasitizing Macropus rufus/M. robustus, pairs of parasite taxa were, in each case, sympatric at each locality examined, thus supporting the conclusion that they represent valid species. The existence of a series of host-specific nematode taxa explains many of the inconsistencies noted previously in the host distribution of H. macropi. Comparison of parasite allozyme phenograms with host phylogeny suggests that four of the speciation events could be attributable to cospeciation and two to host switching. A clear case of host switching between M. rufus/M. robustus and M. fuliginosus was found.

An electrophoretic analysis of patterns of speciation in Cloacina clarkae, C. communis, C. petrogale and C. similis (Nematoda:Strongyloidea) from macropodid marsupials.

An electrophoretic study was conducted on Cloacina clarkae, C. communis, C. petrogale and C. similis based on 19 enzyme loci. C. communis was widely distributed in Macropus robustus, showing some genetic variation among populations but occasionally switching to other macropodid hosts (M. agilis, M. antilopinus). C. similis occurred in members of the Petrogale penicillata complex, Macropus dorsalis and Thylogale billardierii, but showed no evidence of genetic differentiation in spite of its occurrence in different host species and in geographically distinct regions of Australia. C. clarkae from Macropus eugenii was genetically indistinguishable from C. similis and was considered synonymous with it. C. petrogale occurred in a similarly diverse range of hosts and geographical regions to C. similis, but was represented electrophoretically as 4 distinct genetic species, 1 in Petrogale assimilis, a second in P. lateralis purpureicollis, a third in Macropus parryi in Queensland and a fourth in M. eugenii in South Australia. Although the host and geographical ranges of C. similis and C. petrogale are analogous, the genetic uniformity of the former and diversity of the latter illustrate the incomplete understanding we have of the immediate causes of speciation in nematodes.

Giardia intestinalis: detection of major genotypes by restriction analysis of gene amplification products.

The polymerase chain reaction (PCR) has been used to amplify a 0.52 kb segment of Giardia intestinalis DNA, using primers specific for nucleotide sequences conserved within two genes (tsp11 and tsa417) that encode homologous, cysteine-rich trophozoite surface proteins. Using products amplified from axenic isolates belonging to genetic groups I and II (defined on the basis of allozyme electrophoresis data), restriction endonuclease analysis revealed both tsp11-like and tsa417-like fragments within all samples. The study also identified among the amplification products of group II organisms an additional fragment, containing a novel PstI site, that is not detected in the reaction products of group I isolates. The recovery of three distinct PCR products from each group II isolate was verified by cloning the fragments into the plasmid vector pGEM-7. Fragments containing the new PstI site possess the ClaI site common to both tsp11 and tsa417-like fragments, but they lack the HindIII site which characterizes tsp11-like fragments and also lack the PstI and KpnI sites which characterize tsa417-like fragments. Spot-blot analyses using cloned fragments of all three types as probes showed strong homologous hybridization but weak heterologous hybridization, indicating that each type differs substantially in nucleotide sequence from the others. Because the samples of Giardia DNA used in the PCR were purified from cultures that had been established from single trophozoites, the data indicate that individual trophozoites belonging to genetic group II possess three homologous genes defined by these related fragments. The presence of a PstI site in the amplified segment of the newly-discovered third gene of group II organisms provides a simple diagnostic means of differentiating group I and II isolates.

Apparent lack of genetic variation within Pelecitus roemeri (Nematoda: Filarioidea) from three Australian species of macropodid marsupial.

An electrophoretic study of Pelecitus roemeri from Macropus robustus, M. giganteus and Wallabia bicolor revealed no genetic differences at 23 enzyme loci. The genetic data support the existing morphological evidence that P. roemeri from these three hosts represents a single species. The data show no genetic variation between nematodes from the same or different host species collected in northern and southern Australia. This result is discussed briefly in relation to Price's model of parasite speciation.

Interspecific differences in the movements of female ticks on reptiles.

Movements of females of two reptile ticks, Amblyomma limbatum and Aponomma hydrosauri were studied to determine whether the behaviour of the species on hosts was similar or different. Experiments were conducted to determine if the movement of females on hosts prior to mating was influenced by the delayed attachment of conspecific males. A field experiment was also undertaken to determine whether data obtained during laboratory experiments reflected the behaviour of females on hosts in the field. Both in laboratory and field experiments, a significantly greater proportion of Ap. hydrosauri females changed their site of attachment prior to mating. Individual Ap. hydrosauri females made significantly more positional changes than Amb. limbatum females. These interspecific differences occurred irrespective of the absence of conspecific males or presence of non-conspecific adults on hosts. These data may have important ecological implications in relation to the success of colonizing females at their parapatric boundary.

Influence of temperature and relative humidity on the moulting success of Amblyomma limbatum and Aponomma hydrosauri (Acari: Ixodidae) larvae and nymphs.

This study compared the duration of the moulting periods of engorged larvae and nymphs of the ixodid ticks, Amblyomma limbatum and Aponomma hydrosauri, at different temperature/relative humidity regimes, and examined the relationships between the engorged weight of ticks and their weights after moulting. The results showed that for each species, there was a significant relationship between the weights of unfed nymphs and engorged larvae, and the weights of unfed adults and engorged nymphs. The weight of engorged nymphs was also a good indicator of their sex, with female ticks having heavier weights as engorged nymphs. Temperature and relative humidity had a marked effect on the moulting success of engorged ticks of both species. Aponomma hydrosauri larvae and nymphs were able to moult at lower temperatures than Amb. limbatum but most ticks, except Ap. hydrosauri larvae, failed to moult at 13 degrees C. Additionally, there was a marked decrease in the pre-moult times of ticks at higher temperatures, with larvae taking less time to moult than nymphs. At temperatures greater than 21 degrees C, Amb. limbatum took less time to moult than Ap. hydrosauri but this interspecific difference was less marked for nymphs. The interspecific differences in the responses of engorged larvae and nymphs to different temperatures and relative humidities correlated with interspecific differences in off-host behaviour and with the different climates the two species experience throughout most of their distributional range.

Comparison of the levels of intra-specific genetic variation within Giardia muris and Giardia intestinalis.

The extent of intra-specific genetic variation between isolates of Giardia muris was assessed by allozyme electrophoresis. Additionally, the levels of allozymic variation detected within G. muris were compared with those observed between members of the two major assemblages of the morphologically distinct species Giardia intestinalis. Four isolates of G. muris were analysed. Three (Ad-120, -150, -151) were isolated from mice in Australia, while the fourth (R-T) was isolated from a golden hamster in North America. The 11 isolates of G. intestinalis (Ad-1, -12, -2, -62, representing genetic Groups I and II of Assemblage A and BAH-12, BRIS/87/HEPU/694, Ad-19, -22, -28, -45, -52, representing genetic Groups III and IV of Assemblage B) were from humans in Australia. Intra-specific genetic variation was detected between G. muris isolates at four of the 23 enzyme loci examined. Similar levels of variation were found within the genetic groups that comprise Assemblages A and B of G. intestinalis. These levels of intra-specific variation are similar to those observed within other morphologically-distinct species of protozoan parasites. We suggest that the magnitude of the genetic differences detected within G. muris provides an indication of the range of genetic variation within other species of Giardia and that this can be used as a model to delineate morphologically similar but genetically distinct (cryptic) species within this genus.