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.

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.

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.

A mutation scanning approach for the identification of hookworm species and analysis of population variation.

To overcome limitations in the morphological identification of different developmental stages of hookworms to species, we have established a polymerase chain reaction-linked single strand conformation polymorphism technique (PCR-SSCP) utilizing the internal transcribed spacers (ITS) of ribosomal (r)DNA. These spacers were specifically chosen because they provide reliable species markers for strongylid nematodes. ITS spacers were amplified by PCR from DNA derived from individual parasites of seven species of hookworm, then denatured and subjected to electrophoresis in a mutation detection enhancement (MDE) (non-denaturing) gel matrix. PCR SSCP analysis showed that the single-strand ITS patterns produced allowed the unequivocal identification of all species. The method also allowed the direct display of sequence variation within some species where multiple individual worms were examined. These findings demonstrate the usefulness of the SSCP approach for hookworm identification, the detection of population variation and the direct display of sequence variation in rDNA.

An index to assess the reproductive fitness of female ticks.

This paper describes an index that assesses the reproductive fitness of female ticks. The Reproductive Fitness Index (RFI) is calculated by the number of eggs that hatch into larvae as a function of the body weight of the engorged female at the time of detachment from the host. The RFI has an advantage over existing reproductive indices because it takes into account the viability of eggs laid by females. This is of ecological significance because it provides a more accurate and biologically relevant measurement in intraspecific and/or interspecific comparisons of the reproductive fitness of females.

Influence of environmental factors on oviposition and egg development in Amblyomma limbatum and Aponomma hydrosauri (Acari: Ixodidae).

This study examined the influence of temperature and light on the length of the pre-oviposition period of engorged females of two Australian ixodid ticks, Amblyomma limbatum and Aponomma hydrosauri. The hatching success and development time of eggs of both species were also compared at different temperatures and relative humidities. Darkness was found to have no effect on the duration of the pre-oviposition time or reproductive output of females of either species. In contrast, the preoviposition period of females of both species decreased with increasing temperature. Amb. limbatum females had shorter pre-oviposition periods than Ap. hydrosauri at all temperatures examined. Temperature and relative humidity had a marked effect on the hatching success of eggs. Eggs of both species had reduced hatching success at low relative humidities. Eggs failed to hatch at temperatures below 21 degrees C. Ap. hydrosauri eggs also failed to hatch at 34 degrees C while Amb. limbatum eggs failed to hatch at 36 degrees C. Within the range of temperatures suitable for egg development, the hatching times of eggs of both species decreased with increasing temperature. Amb. limbatum eggs developed faster than Ap. hydrosauri eggs at temperatures greater than 25 degrees C, but slower at cooler temperatures. These differences in the duration of their preoviposition period, and the responses of females and their eggs to different temperatures and relative humidities correlate with the different climates the two species experience throughout most of their distributional range.

A comparison of the reproductive parameters of females of two reptile tick species.

In comparisons of females of two reptile tick species Aponomma hydrosauri and Amblyomma limbatum, Ap. hydrosauri was initially larger, and after mating on the host engorged faster and remained attached for a shorter time before completing engorgement and detaching. Amb. limbatum had a longer period of engorgement, and achieved a greater engorged weight. Engorged Amb. limbatum females laid significantly more eggs than equivalent sized Ap. hydrosauri. Although the two species are ecologically similar and were collected from the same site for this study, their reproductive differences probably reflect adaptations to different conditions in their largely allopatric ranges.

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.

Sequence differences in the internal transcribed spacers of DNA among four species of hookworm (Ancylostomatoidea: Ancylostoma).

The two ribosomal DNA internal transcribed spacers (1 and 2) of the hookworms Ancylostoma caninum, A. tubaeforme, A. ceylanicum and A. duodenale were sequenced. The sequence lengths were similar among the four species, except that A. ceylanicum had slightly longer (by 5-7 bp) internal transcribed spacer 1 and 2 sequences. The predicted secondary structure of the internal transcribed spacer 2 precursor rRNA was similar for all species, despite interspecific differences in primary sequence ranging from 0.9% to 13.2%. Interspecific differences in internal transcribed spacer 1 sequence ranged from 0.9% to 7.5%. A cladistic analysis of the sequence data, using the human hookworm Necator americanus as the outgroup, provided little resolution of the phylogenetic relationships, except that A. ceylanicum occurred on a branch external to the other three species. Nonetheless, internal transcribed spacers 1 and 2 may provide useful phylogenetic information at higher taxonomic levels within the superfamily Ancylostomatoidea.

Co-evolutionary relationships between the nematode subfamily Cloacininae and its macropodid marsupial hosts.

Morphologically based phylogenies of the cloacinine genera Cyclostrongylus, Macropostrongylus, Pharyngostrongylus, Popovastrongylus, Rugopharynx, Thallostonema, Wallabinema, and Zoniolaimus were constructed and compared with the phylogeny of their respective macropodid hosts. These comparisons show some evidence of co-speciation. However, there was little consistency among trees of different nematode genera, parasite species were scattered amongst hosts and basal parasite taxa were, in some instances, parasitic in hosts belonging to derived clades. A cladistic analysis, using as characters 208 cloacinine nematode species found in 23 species of host, produced a tree largely resembling that of the host tree but with significant differences explainable by host switching among macropodids occupying similar habitat. Nematodes were moderately host-specific, but some species occurred in three or more distantly related host species indicating a degree of host switching. The results are more consistent with the hypothesis of a colonisation of macropodid hosts by cloacinine nematodes rather than a prolonged period of co-speciation although alternative interpretations of the data are also considered.

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.

Differences in a ribosomal DNA sequence of morphologically indistinguishable species within the Hypodontus macropi complex (Nematoda: Strongyloidea).

The nucleotide sequence of the second internal transcribed spacer (ITS-2) from ribosomal DNA has been determined for 3 members of the Hypodontus macropi species complex. Sequences were compared from nematodes collected from 3 species of Australian macropodid marsupial, Petrogale persephone, Macropus robustus robustus and Thylogale billardierii. The ITS-2 of each operational taxonomic unit ranged from 287 to 292 bases in length, and had a GC content of 36.6-40.1%. Differences in nucleotide sequence between nematodes from the different host species ranged from 25.0% to 28.3%. The data suggest that H. macropi from P. persephone represents a different species to those in M. r. robustus and T. billardierii. The unique feature of this study is that it represents a comparison of the ribosomal DNA sequences of nematode species which are morphologically indistinguishable but which have been demonstrated to be genetically distinct (i.e. cryptic) species based on electrophoretic data. The results also demonstrate further that morphological characters alone are often not adequate for species recognition. Differences between these 3 species of H. macropi in their recognition sites for restriction endonucleases, indicates that a PCR-RFLP approach could be used, in conjunction with allozyme electrophoresis, to establish how many species are present within the H. macropi complex.

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.

Phylogenetic relationships of Australian strongyloid nematodes inferred from ribosomal DNA sequence data.

The sequence of the second internal transcribed spacer of the ribosomal DNA was determined for the following strongyloid nematodes: Cylicocyclus insignis, Chabertia ovina, Oesophagostomum venulosum, Cloacina communis, Cloacina hydriformis, Labiostrongylus labiostrongylus, Parazoniolaimus collaris, Macropostrongylus macropostrongylus, Macropostrongylus yorkei, Rugopharynx australis, Rugopharynx rosemariae, Macropostrongyloides baylisi, Oesophagostomoides longispicularis and Paramacropostrongylus toraliformis, and compared with published sequences for species of Strongylus and for Hypodontus macropi. The resultant phylogenetic trees supported current hypotheses based on morphological evidence for the separation of the families Strongylidae and Chabertiidae, but did not support the separation of the endemic Australian genera as a distinctive clade within the Chabertiidae. The implications of this finding for the phylogenetic origins of the Australian strongyloids are discussed.