Molecular detection and identification of spotted fever group rickettsiae in ticks collected from horses in Cuba.

Spotted fever group (SFG) rickettsiae are obligatory intracellular bacteria that cause disease in humans and other animals. Ixodid ticks are the principal vectors of SFG rickettsiae. The present study aimed to determine the prevalence and species identity of SFG rickettsiae in ticks and horses from urban and rural areas of western Cuba using PCR assays. Tick samples, collected from 79 horses, consisted of 14 Amblyomma mixtum adults, 111 Dermacentor nitens adults and 19 pools of D. nitens nymphs (2-5 individuals/pool). The PCR results revealed the presence of Rickettsia spp. in 64% of the A. mixtum adults, 16% of the D. nitens adults, and 11% of the pooled samples of D. nitens nymphs. In contrast, Rickettsia spp. was not detected in any of the 200 horse blood samples included in this study. DNA sequence data of the rickettsial 17 kDa antigen gene showed that Rickettsia amblyommatis was present in A. mixtum; and Rickettsia felis in D. nitens. This is the first report of R. felis in D. nitens in Cuba. The present study extends our knowledge of the potential vector spectrum and distribution of SFG rickettsiae pathogens in western Cuba.

Phylogenetic relationships of three tribes of cloacinine nematodes (Strongylida: Chabertiidae) from macropodid marsupials.

The phylogenetic relationships of 42 species of cloacinine nematodes belonging to three tribes (Coronostrongylinea, Macropostrongylinea and Zoniolaiminea) were examined based on sequence data of the first and second internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. All nematodes examined are parasites of Australian macropodid marsupials. None of the three nematode tribes was monophyletic. Paraphyly was also encountered in three genera: Papillostrongylus, Monilonema and Wallabinema. Species within the genus Thallostonema were limited to a single host genus (i.e. Thylogale), whereas species within the five principal genera (Coronostrongylus, Macropostrongylus, Popovastrongylus, Wallabinema and Zoniolaimus) were found to occur in multiple host genera. Potential modes of evolution among these nematodes are discussed.

Speciation in the genus Cloacina (Nematoda: Strongylida): species flocks and intra-host speciation.

Sequences of the first and second internal transcribed spacers (ITS1 + ITS2) of nuclear ribosomal DNA were employed to determine whether the congeneric assemblages of species of the strongyloid nematode genus Cloacina, found in the forestomachs of individual species of kangaroos and wallabies (Marsupialia: Macropodidae), considered to represent species flocks, were monophyletic. Nematode assemblages examined in the black-striped wallaby, Macropus (Notamacropus) dorsalis, the wallaroos, Macropus (Osphranter) antilopinus/robustus, rock wallabies, Petrogale spp., the quokka, Setonix brachyurus, and the swamp wallaby, Wallabia bicolor, were not monophyletic and appeared to have arisen by host colonization. However, a number of instances of within-host speciation were detected, suggesting that a variety of methods of speciation have contributed to the evolution of the complex assemblages of species present in this genus.

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.

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.

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.

Genetic variation in the mitochondrial cytochrome c oxidase subunit 1 within Progamotaenia festiva (Cestoda: Anoplocephalidae) from macropodid marsupials.

Genetic variation was examined in the anoplocephalid cestode Progamotaenia festiva, from Australian marsupials, in order to test the hypothesis that P. festiva, is a complex of sibling species and to assess the extent of host switching reported previously based on multilocus enzyme electrophoresis (MEE). Polymerase chain reaction (PCR)-based single-strand conformational polymorphism (SSCP) was used for the analysis of sequence variation in the cytochrome c oxidase subunit 1 (cox1) gene among 179 specimens of P. festiva (identified based on morphology and predilection site in the host) from 13 different host species, followed by selective DNA sequencing. Fifty-three distinct sequence types (haplotypes) representing all specimens were defined. Phylogenetic analyses of these sequence data (utilizing maximum parsimony and neighbour-joining methods) revealed 12 distinct clades. Other heterologous species, P. ewersi and P. macropodis, were used as outgroups and the remaining bile-duct inhabiting species, P. diaphana and P. effigia, were included in the analysis for comparative purposes. The latter 2 species were nested within the clades representing P. festiva. Most clades of P. festiva identified were restricted to a single host species; one clade primarily in Macropus robustus was also found in the related host species M. antilopinus in an area of host sympatry; another clade occurring primarily in M. robustus occurred also in additional kangaroo species, M. rufus and M. dorsalis. High levels of genetic divergence, the existence of distinct clades and their occurrence in sympatry provide support for the hypothesis that P. festiva represents a complex of numerous species, most of which, but not all, are host specific. Three distinct clades of cestodes were found within a single host, M. robustus, but there was no evidence of within-host speciation.

Isolation and characterization of class II myosin genes from Haemonchus contortus.

In this study, cDNAs encoding myosin from the parasitic nematode Haemonchus contortus were isolated and characterized. Several exhibited a considerable degree of sequence variation at the nucleotide and limited divergence at the amino acid levels within the various functional domains. The results suggest that the cDNAs isolated represented a single myosin heavy chain, which, by comparison with a number of other myosins, is inferred to represent a homologue of a muscle myosin (CeMHCA) of the free-living nematode Caenorhabditis elegans. The findings could have implications for investigating cytoskeletal dynamics and/or signalling pathways.

Class II myosins in nematodes-- genetic relationships, fundamental and applied implications.

Myosins are represented by a wide range of different classes of molecule, of which the most extensively studied are the class II myosins which drive muscle contraction and cell organization; the functional unit of class II myosins comprises two myosin heavy chains (MHCs). This minireview gives an update on class II MHCs of nematodes and describes a comparative analysis of MHC genes from nematodes and other organismal groups. Genetic analyses of sequence data for the four functional domains of MHCs (i.e., the SH3-like N-terminal, head, neck and tail domains) reveal a delineation between both the nematode and non-nematode myosins and between muscle and non-muscle myosins. The distinctiveness of the MHCs of nematodes suggests functional and tissue specialization. The elucidation of the functional roles of myosins and other molecules in specific signaling pathways in nematodes has the potential to lead to new intervention strategies for parasites via the specific disruption or interruption of key developmental processes, having biotechnological implications in the longer term.

Molecular and morphological evidence indicates that Pseudorhabdosynochus lantauensis (Monogenea: Diplectanidae) represents two species.

Sequences of the first internal transcribed spacer (ITS-1) and the D1-D3 domains of the large subunit (LSU) of the ribosomal DNA (rDNA) were determined for multiple specimens of 4 operational taxonomic units (OTUs) of the monogenean, Pseudorhabdosynochus lantauensis. OTUs were defined based on their collecting localities, host and/or morphological characteristics. All P. lantauensis specimens of one group (OTUs 1 and 3) differed in their sequences of the ITS-1 and partial LSU rDNA when compared with specimens of a second group (OTUs 2 and 4) by 12% and 2%, respectively. Results of the phylogenetic analyses of the LSU rDNA sequence data showed total (100%) bootstrap support for the separation of P. lantauensis into 2 distinct clades. At least 11 of the 18 nucleotide differences in the LSU sequence between the two P. lantauensis clades were derived (i.e. autapomorphic) characters when the morphologically distinct species, P. epinepheli and P. coioidesis, were used as outgroups. Furthermore, there were several autapomorphic character states for each P. lantauensis clade. This provides sufficient evidence to reject the null hypothesis that P. lantauensis represents a single species. Morphological and morphometric differences between these two clades provided additional strong support for the separation of P. lantauensis into two species. These two parasite species were found to co-exist on one of the two species of serranid fish (i.e. Epinephelus coioides) examined in the South China Sea (Guangdong Province, China).

Genetic variation in the mitochondrial cytochrome c oxidase subunit 1 within three species of Progamotaenia (Cestoda: Anoplocephalidae) from macropodid marsupials.

Sequence variation within 3 morphologically defined species of the anoplocephalid cestode genus Progamotaenia (P. ewersi, P. macropodis and P. zschokkei) was investigated using the cytochrome c oxidase subunit 1 gene. The magnitude of genetic variation detected within each morphospecies suggests that, in each instance, several cryptic species are present. Within P. ewersi, 5 genetically distict groups of cestodes were detected, 1 shared by Macropus robustus and M. parryi in Queensland, 1 in M. agilis from Queensland, 1 in Petrogale assimilis from Queensland, 1 in Macropus fuliginosus from South Australia and 1 in Wallabia bicolor from Victoria. In P. macropodis, cestodes from M. robustus from Queensland, Western Australia and the Northern Territory, M. parryi from Queensland and M. eugenii from South Australia were genetically distinct from those in Wallabia bicolor from Queensland and Victoria and from M. fuliginosus from South Australia. P. zschokkei consisted of a number of genetically distinct groups of cestodes, 1 in Lagorchestes conspicillatus and L. hirsutus from Queensland and the Northern Territory respectively, 1 in Petrogale herberti, P. assimilis and M. dorsalis from Queensland, 1 in Onychogalea fraenata from Queensland, 1 in M. agilis from Queensland and 1 in Thylogale stigmatica and T. thetis from Queensland. In general, genetic groups within each morphospecies were host specific and occurred predominantly in a particular macropodid host clade. Comparison of genetic relationships of cestodes with the phylogeny of their hosts revealed examples of colonization (P. zschokkei in M. agilis) and of host switching (P. zschokkei in M. dorsalis).

Lack of evidence for co-speciation in a parasitic nematode of grey kangaroos.

Multilocus enzyme electrophoresis was used to compare specimens of the parasitic nematode Cloacina obtusa from the stomach of the eastern grey kangaroo, Macropus giganteus and the western grey kangaroo, M. fuliginosus. Allelic variation among nematodes was detected at 17 (85%) of 20 loci, but there was only a single fixed genetic difference (at the locus for isocitrate dehydrogenase, IDH) between C. obtusa from M. fuliginosus and those from M. giganteus in areas where each host occurred in allopatry. However, this fixed difference was not apparent within the zone of host sympatry. Although electrophoretic data indicate genetic divergence among allopatric populations of C. obtusa in the two host species, the magnitude of the electrophoretic difference (5%) between these populations does not refute the hypothesis that C. obtusa represents a single species. The 'usual' situation for parasitic helminths of grey kangaroos is that pairs of parasite species occur in the two host species. This situation differs for C. obtusa, where there has been a lack of speciation following a speciation event in its macropodid marsupial hosts. This finding suggests that a speciation event in the host does not necessarily lead to a speciation event for all its parasites and further highlights our lack of understanding of which processes drive speciation in parasites.

Molecular systematics and diagnosis.

This collection of articles provides an account of six presentations delivered at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology(WAAVP) (held in New Orleans, Louisiana, USA, from 10 to 14 August 2003) in a symposium session on Molecular Systematics and Diagnosis, organised and chaired by R.B. Gasser and D.S. Zarlenga. The focus was on recent advances in molecular tools for specific and genotypic identification,diagnosis, systematics and population genetics, with special emphasis on investigations of parasitic nematodes and protists.

Structure and organization of the mitochondrial genome of the canine heartworm, Dirofilaria immitis.

This study determined the complete mitochondrial (mt) genome sequence of the canine heartworm, Dirofilaria immitis, and compared its structure, organization and other characteristics with Onchocerca volvulus and other secernentean nematodes. The D. immitis mt genome is 13814 bp in size and contains 36 of the 37 genes typical of metazoan organisms, and lacks the ATP synthetase subunit 8 gene. All of the genes are transcribed in the same direction. For the entire genome, the nucleotide contents are approximately 55% (T), approximately 19% (each for A and G) and approximately 7% (C), which is very similar to those of the protein-coding genes. In the latter genes, most (approximately 69%) third codon positions have a T, but rarely (approximately 1-9%) have an A or a C. The C content (8-12%) is higher at the first and second codon positions compared with the third position (approximately 1%). These nucleotide biases have a significant effect on the codon usage patterns and, thus, on the amino acid composition of the proteins. The mt genome organization of D. immitis is essentially the same as that of O. volvulus, but is distinctly different from other secernentean nematodes sequenced thus far. Irrespective of transpositions of transfer RNA (trn) genes and the non-coding, AT-rich region, there are 4 gene- or gene block-translocations between the mt genome of D. immitis and those of Caenorhabditis elegans, Ascaris suum and the 2 human hookworms, Ancylostoma duodenale and Necator americanus. For D. immitis, the 22 trn genes have secondary structures typical of other secernentean nematodes, and possess a TV-replacement loop instead of a TpsiC arm and loop. Like O. volvulus, the mt trnK and trnP of D. immitis use the anticodons CUU and AGG, whereas in other nematodes, UUU and UGG are employed, respectively. Also, the secondary structures of the 2 ribosomal RNA (rrn) genes are similar to the models for other nematodes. Overall, the availability of the complete D. immitis mt genome sequence provides a resource for future studies of the comparative mt genomics and of the population genetics and/or phylogeny of parasitic nematodes.

Long PCR-based amplification of the entire mitochondrial genome from single parasitic nematodes.

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.

Review of Papillostrongylus Johnston & Mawson, 1939 (Nematoda: Strongyloidea) from wallabies and kangaroos (Marsupialia: Macropodidae) using morphological and molecular techniques, with the description of P. barbatus n. sp.

The strongyloid nematode genus Papillostrongylus Johnston & Mawson, 1939, from kangaroos and wallabies, is reviewed using morphological and molecular methods. P. labiatus Johnston & Mawson, 1939 is re-described from material from the type-host, the black-striped wallaby Macropus dorsalis, from eastern Queensland, Australia, in which it is a relatively common parasite. Additional records from M. parryi and Thylogale thetis are confirmed and considered to represent examples of host-switching. A geographically disjunct population of the nematode species occurs in M. bernardus and Petrogale brachyotis in Arnhem Land, Northern Territory, but assessment of its status requires additional material. Nematodes from M. rufus, M. giganteus, M. fuliginosus and M. robustus from inland regions of Australia, formerly attributed to P. labiatus, are here assigned to a new species, P. barbatus, distinguished by the presence of an external leaf-crown, larger size, by greater spicule length in the male and by a sinuous vagina in the female. Additional hosts of P. barbatus n. sp. are Petrogale assimilis and Pet. lateralis purpureicollis. Sequence analyses of the second internal transcribed spacer of ribosomal DNA (ITS-2) also showed that P. barbatus n. sp. differed at 40 (16.7%) of the 240 alignment positions when compared with P. labiatus. Most of these interspecific sequence differences occurred in loops or bulges of the predicted precursor rRNA secondary structure, or represented partial or total compenstory base pair changes in stems.

Applications of single-strand conformation polymorphism (SSCP) to taxonomy, diagnosis, population genetics and molecular evolution of parasitic nematodes.

The analysis of genetic variation in parasitic nematodes has important implications for studying aspects of taxonomy, diagnosis, population genetics, drug resistance and molecular evolution. This article highlights some applications of PCR-based single-strand conformation polymorphism (SSCP) for the analysis of sequence variation in individual parasites (and their populations) to address some of these areas. It also describes the principles and advantages of SSCP, and provides some examples for future applications in parasitology.

Molecular approaches for studying ascaridoid nematodes with zoonotic potential, with an emphasis on Toxocara species.

Species-specific identification of ascaridoid nematodes at any developmental stage is a prerequisite for detailed investigation of the life cycles, systematics and epidemiology of this important group, and is also crucial for the diagnosis of associated infections. The morphological identification of some species and/or their larval stages can, however, present considerable difficulty. Recently, PCR-based methods, using genetic markers in the internal transcribed spacers (ITS) of ribosomal DNA, have been shown to provide reliable alternatives to more traditional methods for the specific identification of nematodes. This article provides an account of recent research on the development of PCR-based methods (utilizing ITS sequences) for the specific identification of ascaridoid nematodes of zoonotic potential, for the diagnosis of infections, and for the analysis of genetic variation within and among individual nematodes and their populations. Prospects for using these diagnostic and analytical tools to investigate epidemiological and population genetic questions relating to ascaridoid parasites are also discussed.

Single-strand conformation polymorphism analysis of genetic variation in Labiostrongylus longispicularis from kangaroos.

Single-strand conformation polymorphism (SSCP) analysis was employed to screen for sequence heterogeneity in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA of Labiostrongylus longispicularis, a parasitic strongylid nematode occuring in some species of kangaroo in different geographical regions of Australia. The results showed that most of the nematodes screened had different SSCP profiles, which were subsequently shown to correspond to polymorphisms and/or an indel in the ITS-2 sequence. These variable sites related mainly to unpaired regions of the predicted secondary structure of the precursor rRNA molecule. SSCP profiles could be used to distinguish L. longispicularis in Macropus robustus robustus (New South Wales) from L. longispicularis in Macropus robustus erubescens and Macropus rufus (South Australia). This difference corresponded to a transversional change in the ITS-2 sequence at alignment position 82. The study demonstrated clearly the effectiveness of SSCP analysis for future large-scale population genetic studies of L. longispicularis in order to test the hypothesis that L. longispicularis from different geographical regions represents multiple sibling species.