Pharyngostrongylus thylogale n. sp. (Nematoda: Strongylida) from the stomachs of macropodid marsupials defined by morphological and molecular criteria.

Pharyngostrongylus thylogale n. sp. (Nematoda: Strongylida) is described from the stomach of the red-legged pademelon, Thylogale stigmatica (Gould) (Marsupialia: Macropodidae) from north-eastern Queensland and Papua New Guinea, having formerly been confused with P. iota Johnston & Mawson, 1939. Pharyngostrongylus thylogale n. sp. differs from all congeners in having 12 labial crown elements rather than eight or 16. Pharyngostrongylus iota was found in T. stigmatica, but only in southern Queensland and northern New South Wales, in the subspecies T. s. wilcoxi, compared with P. thylogale n. sp. which was found in T. s. stigmatica in northern Queensland and T. s. oriomo in Papua New Guinea. Differences in the sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA of P. thylogale n. sp. and ten congeners support the erection of the new species, and the validity of the morphospecies examined. However, results of the phylogenetic analyses of the molecular data also provide evidence for the existence of cryptic species within P. kappa Mawson, 1965. No obvious co-evolutionary relationships were observed between parasite species and their macropodid marsupial hosts.

Detection of cryptic species of Rugopharynx (Nematoda: Strongylida) from the stomachs of Australian macropodid marsupials.

Sequences of the internal transcribed spacers of nuclear ribosomal DNA (ITS-1 and ITS-2) were determined for species of the genus Rugopharynx and Rugonema labiatum, nematodes from the stomachs of macropodid marsupials. Phylogenetic analyses of the aligned sequence data were conducted. The relationships provided molecular support for all species currently recognised, some of which are based on minor morphological differences and on multilocus enzyme electrophoretic data, but also indicated that additional, cryptic species exist within the genus. In addition, the genus Rugonema is placed as a synonym of Rugopharynx, its sole species becoming Rugopharynx labiatum n. comb. The molecular data provided some insights into the evolution of complex buccal capsule morphologies within the genus, but there was no evidence of co-evolution between the macropodid hosts and their parasites.

Phylogenetic relationships of species of the oesophageal parasitic nematode genera Cyclostrongylus and Spirostrongylus (Strongyloidea: Chabertiidae: Cloacininae) with their wallaby hosts (Marsupialia: Macropodidae).

A phylogeny for seven species of Cyclostrongylus and the monotypic genus Spirostrongylus (Nematoda: Chabertiidae), all highly host specific parasites of the oesophagi of wallabies (Marsupialia: Macropodidae), was constructed using sequence data for the first and second internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. There was no evidence for co-speciation, or for the sympatric or synxenic speciation of Cyclostrongylus alatus and Cyclostrongylus perplexus, both of which are parasites of Macropus rufogriseus. Rather, host switching, correlating with geographical distributions, appeared to provide some explanation of the pattern of speciation observed.

The phylogenetic relationships of endemic Australasian trichostrongylin families (Nematoda: Strongylida) parasitic in marsupials and monotremes.

The phylogenetic relationships of the endemic (or largely endemic) Australasian trichostrongylin nematode families Herpetostrongylidae, Mackerrastrongylidae and Nicollinidae as well as endemic trichostrongylin nematodes currently placed in the families Trichostrongylidae and Molineidae were examined using the complete large subunit (28S) ribosomal RNA gene. The Herpetostrongylinae proved to be monophyletic. However, representatives of the Nicollinidae nested with the Herpetostrongylinae. The Mackerrastrongylidae was also a monophyletic group and included Peramelistrongylus, currently classified within the Trichostrongylidae. The Globocephaloidinae, currently considered to be a subfamily of the Herpetostrongylidae, was excluded from the family in the current analysis. Ollulanus and Libyostrongylus, included for the first time in a molecular phylogenetic analysis, were placed within the Trichostrongylidae. This study provided strong support for the Herpetostrongylidae (including within it the Nicollinidae, but excluding the Globocephaloidinae) and the Mackerrastrongylidae as monophyletic assemblages. Additional studies are required to resolve the relationships of the remaining endemic Australasian trichostrongylin genera.

Genetic variation within the Hypodontus macropi (Nematoda: Strongyloidea) complex from macropodid marsupial hosts in Australia.

Genetic variation was investigated in the strongylid nematode Hypodontus macropi from macropodid marsupials using the second internal transcribed spacer of ribosomal DNA. A total of 547 specimens from ten species of hosts, representing all of the known hosts of the parasite, from across the Australian continent was examined. Phylogenetic analyses revealed distinct genetic clades in each of Macropus agilis, M. dorsalis, M. rufogriseus, M. bicolor, Petrogale persephone, Thylogale billardierii and T. stigmatica. A further clade contained all specimens from M. robustus and M. rufus, together with two examples of host switching by nematodes into M. fuliginosus. The latter clade was subdivided into three subclades, one comprising specimens occurring in M. robustus erubescens, M. rufus and M. fuliginosus, the second in M. r. woodwardi and the third in M. r. robustus suggesting a relationship between the subclades and the subspecies of M. robustus. The extent of the genetic differences and the fact that several of them occur in broad sympatry suggests that H. macropi as currently defined morphologically may represent as many as ten cryptic species. Limited evidence was found for co-speciation between hosts and parasites; rather most relationships were better explained by host switching.

Molecular evidence for a cryptic species within the parasitic nematode Macroponema comani (Strongyloidea: Cloacininae).

Nematodes resembling Macroponema comani, a common parasite of eastern grey kangaroos, Macropus giganteus, in eastern Australia were collected from an unexpected host species, the northern wallaroo, Macropus robustus woodwardi, in the Northern Territory, representing a highly disjunct occurrence. Although these specimens showed no morphological differences when compared with Ma. comani from M. giganteus, sequencing of the first and second internal transcribed spacers ITS-1 and ITS-2 of the nuclear ribosomal DNA revealed seven base pair differences in each spacer region between the two taxa. These differences included a number of autapomorphies. Sequences from both taxa differed significantly from those of Ma. beveridgei, a common parasite of the common wallaroo, Macropus robustus robustus and the euro, M. robustus erubescens. Based on these findings, the specimens in M. r. woodwardi are considered to represent a crypic species.

Phylogenetic relationships of species within the tribe Labiostrongylinea (Nematoda: Cloacinidae) from Australian marsupials based on ribosomal DNA spacer sequence data.

Parasitic nematodes of the tribe Labiostrongylinea (Family Cloacinidae) occur in the stomachs of a wide variety of potoroid and macropodid marsupials in Australia, Papua Indonesia and Papua New Guinea. The aim of the present study was to infer the evolutionary relationships of the five genera of labiostrongyline nematodes that occur in Australian potoroids and macropodids using sequence data of the nuclear first and second internal transcribed spacers of ribosomal DNA. The phylogenetic analyses resulted in the separation of the Labiostrongylinea into two major groups reflecting coevolution between hosts and parasites. Two nematode species belonging to the genus Potorostrongylus formed a sister group to the remaining species of the Labiostrongylinea. This genus occurs exclusively in potoroid marsupials, which are considered to be basal to the macropodid marsupials. The second major group included species of Labiostrongylus, Labiosimplex, Labiomultiplex and Parazoniolaimus, all of which occur in macropodids. These species formed two distinct clades, one predominating in the host genera Thylogale and Onychogalea, and the second in the genus Macropus, which includes the more recent macropodids. However, there is also evidence of colonisation by both nematode clades of relatively unrelated hosts. In addition, genetic differences among individuals of Lm. eugenii from geographically isolated populations of M. eugenii, and among Ls. longispicularis from different subspecies of M. robustus suggest the existence of sibling species that may have arisen by allopatric speciation. The broad coevolutionary relationship between the labiostrongyline nematodes and their marsupial hosts therefore represents a mixture of potential cospeciation and colonisation events.

Detection of prohibited animal products in livestock feeds by single-strand conformation polymorphism analysis.

Single-strand conformation polymorphism (SSCP) analysis of amplicons produced from a mitochondrial DNA region between the tRNA(Lys) and ATPase8 genes was applied for the detection of animal product within livestock feeds. Identification of prohibited animal (cattle, elk, sheep, deer, and goat) and nonprohibited animal (pig and horse) products from North America was possible based on the differential display of the single-stranded DNA fragments for the different animal species on SSCP gels. This method allowed specific detection and identification of mixed genomic DNA from different animal species. Trace amounts of cattle-derived materials were also detected in pig meat and bone meal and in grain-based feeds fortified with 10, 5, 1, or 0% porcine meat and bone meal. This study demonstrates the applicability of SSCP analyses to successfully identify the origin of animal species derived materials potentially present in animal feeds.

Detection and identification of Tetratrichomonas in a preputial wash from a bull by PCR and SSCP.

Motile Tritrichomonas foetus-like trichomonads were found during microscopic examination of a wet mount sample of a preputial wash collected from a bull. Staining of the organisms with a modified Wright-Giemsa stain revealed that several had four anterior flagella of unequal length instead of the three anterior flagella of equal length characteristic of T. foetus. Limited propagation of these organisms was achieved in InPouch medium but no growth occurred in modified Diamond's medium. The 5.8S rRNA gene and the flanking internal transcribed spacers were amplified from the trichomonad gDNA of two preputial wash samples and a fecal sample taken from the affected bull. Amplicons were subjected to single-strand conformation polymorphism (SSCP) analyses. The SSCP banding patterns of the amplicons from gDNA of the preputial wash samples were different from those of a T. foetus control sample. These unknown trichomonads were not detected in the fecal sample. The gDNA extracted from preputial washes was also subjected to PCR using primers developed to amplify the 16S rDNA of the non-T. foetus trichomonads, Tetratrichomonas and Pentatrichomonas spp. Amplicons were produced from the gDNA of the two preputial washes but not from the T. foetus gDNA control sample. The 16S rDNA sequences obtained from the trichomonads in the two preputial washes samples were 100% similar to that of a Tetratrichomonas species previously isolated from an Angus bull from the United States.

Analysis of genetic variation in Globocephaloides populations from macropodid marsupials using a mutation scanning-based approach.

Three species of Globocephaloides, parasitic nematodes occurring in macropodid marsupials in different areas of Australia, were characterized by the sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA. Samples were subjected to PCR-coupled SSCP analysis and targeted sequencing, in order to assess genetic variation within and among individuals from different host species. Both SSCP and sequence data supported the current classification of morphospecies. Contrary to a previous hypothesis that cryptic species exist within the Globocephaloides trifidospicularis "complex", no or minor (0.2%) variation was detected among individuals from different hosts or geographical origins. Within G. macropodis populations, there was a consistent difference in both the ITS-1 (5.2%) and ITS-2 (7.1%) sequences between individuals derived from Macropus agilis and those from Macropus dorsalis. Although the results suggested that G. macropodis from each host species represented sibling species, future morphological study of individuals representing each G. macropodis genotype is warranted to provide further support for this hypothesis. (Nucleotide sequence data have been deposited in the GenBank database under accession nos. GQ131400-GQ131409.).

Genetic divergence between island and continental populations of the parasitic nematode Labiosimplex australis in Australia.

Sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal (r) DNA were characterised for Labiosimplex australis, a parasitic nematode of macropodid marsupials from continental Australia and from island populations which have been isolated from mainland Australia for relatively short periods of time (<40,000 years). The results showed that the geographically isolated populations of L. australis on Kangaroo Island and Tasmania were genetically different from each other and from populations on the mainland. There were two unequivocal nucleotide differences sites within the ITS-1 rDNA sequence between the two island populations; however, the ITS-1 sequences of individuals from mainland populations contained one or both of these nucleotides. In contrast, L. australis from each island population had unique nucleotides in ITS-2 sequence that were not detected in any individual from the mainland. Although these results are consistent with the hypothesis that L. australis represents a single species, the genetic divergence in the ITS-2 sequences amongst individuals from different isolated populations suggests that the island populations of L. australis may be in the initial stages of allopatric speciation.

Rapid discrimination of Salmonella isolates by single-strand conformation polymorphism analysis.

A molecular typing technique was developed for the differentiation of Salmonella isolates based on single-strand conformation polymorphism (SSCP) analysis of amplicons generated by PCR. Amplicons from parts of the fimA (both the 5' and 3' ends), mdh, invA, and atpD genes were generated separately from a panel of Salmonella strains representing Salmonella bongori, and four subspecies and 17 serovars of Salmonella enterica. These amplicons were subjected to SSCP analysis for differentiation of the salmonellae on the basis of different conformational forms arising due to nucleotide sequence variations in the target genes. Several distinct SSCP banding patterns (a maximum of 14 each for atpD and fimA 3' end) were observed with this panel of Salmonella strains for amplicons generated from each target gene. The best discrimination of Salmonella subspecies and serovar was achieved from the SSCP analysis of a combination of at least three gene targets: atpD, invA, and either mdh or fimA 3' end. This demonstrates the applicability of SSCP analysis as an important additional method to classical typing approaches for the differentiation of foodborne Salmonella isolates. SSCP is simple to perform and should be readily transferable to food microbiology laboratories with basic PCR capability.

Nucleotide alterations in the D3 domain of the large subunit of ribosomal DNA among 21 species of equine strongyle.

The expansion segments or divergent (D) domains in the large subunit (LSU) of the ribosomal DNA have been suggested as genetic markers for taxonomic and/or phylogenetic studies of parasites. In the present study, we assessed the degree of sequence variation in the D3 domain and flanking core regions of the LSU for 21 species of equine strongyles (Strongylida: Strongylidae) and determined which positions in the secondary structure of the LSU were associated with the nucleotide alterations. No intraspecific sequence variation was detected in 17 species, for which multiple individual worms were available. Mutations in sequence among species were detected at 19 nucleotide positions, most of which were located in the D3 domain. Fifteen alterations were transitions, three were transversions and one represented a site of multiple mutations. In relation to the secondary structure element of D3, 26% of these mutations were located in unpaired regions (i.e., end of loops, or in bulges of helices) and thus did not appear to alter the pairing arrangement in the helices of the secondary structure. Many of the other mutations represented partial or complete compensatory base pair changes. The magnitude of interspecific nucleotide variation in the D3 domain (0-4%) was considerably less than that recorded for some other nematode groups (enoplids and thelastomatoids), indicating that this region alone is of limited value for taxonomic and phylogenetic studies for strongyles of equids but is interesting in relation to the evolution of ribosomal DNA.

The evolutionary origins of nematodes within the order Strongylida are related to predilection sites within hosts.

The evolutionary relationships of the different groups of nematodes within the order Strongylida based on morphological data have been speculative and the subject of conjecture. In this paper, we present a multigene phylogenetic analysis, using sequence data of the 18S and 28S ribosomal RNA genes from representatives of all four suborders and seven superfamilies of the Strongylida, to test existing hypotheses proposed for the relationships of the suborders based on morphological data sets. The results obtained demonstrated that the Strongylida is a monophyletic assemblage, with only the Metastrongylina (but not the other suborders) forming a distinct monophyletic clade. We show that, in contrast to all previous hypotheses, one major lineage comprises taxa which occur exclusively in the pulmonary, circulatory or nervous systems of marsupial and eutherian mammals, whereas a second lineage comprises species occurring in the gastrointestinal tracts or perirenal tissues of vertebrates, or in the lungs of birds. The findings suggest that the predilection site of adult nematodes and host type reflect the evolutionary origin of the different taxonomic groups within the Strongylida.

Single-strand conformation polymorphism (SSCP) analysis as a new diagnostic tool to distinguish dorsal-spined larvae of the Elaphostrongylinae (Nematoda: Protostrongylidae) from cervids.

Single-strand conformation polymorphism (SSCP) was used to genetically differentiate morphologically indistinguishable first-stage larvae (L(1)) of the six species of elaphostrongyline nematodes. A partial fragment (317-336bp) of the first internal transcribed spacer (pITS-1) plus 5' flanking region (76bp of the 18S gene) of the nuclear ribosomal DNA (rDNA) was amplified from individual L(1) of known identity and subjected to SSCP. The results showed that the four species of elaphostrongylines found in North American cervids, Parelaphostrongylus tenuis, P. andersoni, P. odocoilei and Elaphostrongylus rangiferi, could be distinguished from one another based on their distinct (i.e. species-specific) SSCP profiles. In addition, E. alces, a species that occurs in moose in Fennoscandinavia, also had a distinct SSCP profile with respect to the other species of elaphostrongylines. However, the SSCP profiles of E. cervi could not be distinguished from those of E. rangiferi because of a lack of interspecific sequence differences in this region of the ITS-1. The distinct SSCP profiles for the other species were consistent with the interspecific differences in ITS-1 sequences, which ranged from 2 (between P. tenuis and P. andersoni) to 59bp (between genera). The pITS-1 SSCP approach was also used to identify unknown elaphostrongyline L(1) from different hosts and localities in North America. The ability to distinguish between L(1) of the four elaphostrongyline species that occur in North American cervids has important diagnostic and epidemiological implications.

Bighorn sheep, a new host record for Parelaphostrongylus odocoilei (Nematoda: Protostrongylidae).

Larval nematodes with a dorsal spine on the tail were recovered from fecal samples of California bighorn sheep (Ovis canadensis californiana) in northeastern Washington State, USA. The identity of these dorsal-spined larvae (DSL) was established by single-strand conformation polymorphism (SSCP) analyses of a partial fragment of the first internal transcribed spacer of the ribosomal DNA. The SSCP profiles of individual DSL from bighorn sheep were compared with those of DSL of five protostrongylid species (Parelaphostrongylus andersoni, P odocoilei, P. tenuis, Elaphostrongylus rangiferi, and Muellerius capillaris) but were identical to only those of P. odocoilei. This study represents the first confirmed identification of P. odocoilei in bighorn sheep.

A method for extracting genomic DNA from individual elaphostrongyline (Nematoda: Protostrongylidae) larvae and differentiation of Elaphostrongylus spp. from Parelaphostrongylus spp. by PCR assay.

This article reports a rapid and effective method for the extraction and purification of genomic DNA (gDNA) from individual first-stage larvae (L1) of elaphostrongyline nematodes that had been stored frozen or fixed in 95% ethanol for 1 to 5 years. The method was highly effective for L1s of all 6 species of elaphostrongylines, based on polymerase chain reaction (PCR) amplification of a partial fragment of the first internal transcribed spacer (ITS-1) of the ribosomal DNA. Differences were detected in the sizes of partial ITS-1 amplicons between the 2 elaphostrongyline genera, Elaphostrongylus and Parelaphostrongylus. The reliability of the ITS-1 PCR assay was tested by using L1s of unknown identity from Newfoundland and Labrador, Canada. The ability to consistently isolate gDNA from individual L1s, together with a simple PCR-based method to distinguish between Parelaphostrongylus and Elaphostrongylus, have important implications for diagnostic testing and for conducting epizootiological studies on these parasites of veterinary importance.

First complete large subunit ribosomal RNA sequence and secondary structure for a parasitic nematode: phylogenetic and diagnostic implications.

The complete sequence and secondary structure of the large subunit of nuclear ribosomal RNA(LSUrRNA) were determined for the parasitic nematode Labiostrongylus bipapillosus (order Strongylida). Its LSU rRNA sequence was shorter (by 18 bp) than that of the free-living nematode, Caenorhabditis elegans (order Rhabditida), the only other species within the Nematoda for which a complete LSU rRNA sequence has been determined. Interspecific differences in sequence were greater in the 12 D domains compared with the core segments, with the secondary structure being maintained by partial or complete compensatory base pair changes. The magnitude of interspecific sequence difference in each D domain (except for D6 and D12) was similar, suggesting that several domains contain informative genetic markers for phylogenetic studies of the phylum Nematoda at different taxonomic levels. The LSU rRNA may also provide species-specific markers for the identification of some bursate nematodes of veterinary and medical importance.

Redescription of Zoniolaimus mawsonae Beveridge, 1983 (Nematoda: Strongyloidea) and the description of Z. latebrosus n. sp. from the red kangaroo Macropus rufus (Marsupialia: Macropodidae) based on morphological and molecular data.

This study shows that the description of Z. mawsonae given by Beveridge (1983) represented a composite of two species. Hence, Z. mawsonae Beveridge, 1983 is re-described and a new species, Z. latebrosus, is described. Males of the two species differ in the lengths of their spicules (0.94-1.23 mm in Z. mawsonae compared with 1.53-1.95 mm in Z. latebrosus) and in several characteristics of the bursa and genital cone. Females of the two species can be identified based on the shape of the posterior end of the body, that of Z. mawsonae being markedly swollen. Molecular data, obtained from single strand conformation polymorphism (SSCP) analysis and DNA sequencing of the second internal transcribed spacer of ribosomal DNA, provided additional support for the separation of the two species.