Trichinella pseudospiralis from a wild pig in Texas.

In December 2001, the routine inspection of a wild boar intended for human consumption revealed the presence of Trichinella ssp. larvae. Biological, morphological and genetic analyses demonstrated the parasite to be Trichinella pseudospiralis. This is the second report of T. pseudospiralis in the United States and the first report of the parasite in a food animal species in the U.S.

Identification of strongyle eggs from anthelmintic-treated horses using a PCR-ELISA based on intergenic DNA sequences.

The efficacy of five daily fenbendazole (FBZ) treatments was tested against benzimidazole-resistant cyathostomins in naturally infected horses (n=13). Horses were treated with pyrantel embonate (PYR) to remove adult strongyles followed, 7 days later, by a 5-day course of FBZ. The PYR treatment produced an average faecal egg count reduction of 98%. All samples were negative by faecal egg count 7 days after the start of the FBZ treatment. Positive egg counts were observed from 28 days after the start of FBZ treatment and all horses displayed positive faecal egg counts by 77 days after treatment. Strongyle eggs were harvested from the faeces of the horses prior to treatment and then weekly from 42 to 70 days post-treatment. DNA was obtained from eggs in groups of ten. A PCR-ELISA, based on species-specific differences in intergenic DNA sequences, was used to identify the presence of six cyathostomin species. In pre-treatment samples, Cyathostomum catinatum was detected in nine out of the 13 horses and Cylicostephanus longibursatus, Cylicostephanus goldi and Cylicocyclus nassatus, were found in samples from eight animals. Cylicocyclus ashworthi and Cylicocyclus insigne were not detected pre-treatment. After anthelmintic treatment, C. catinatum and C. longibursatus were most frequently detected, followed by C. nassatus, C. goldi and C. ashworthi. C. insigne was detected at only one time point in a sample from a single horse.

A PCR-ELISA for the identification of cyathostomin fourth-stage larvae from clinical cases of larval cyathostominosis.

We report the use of six oligoprobes designed from intergenic spacer region sequences to identify fourth-stage larvae (L4) of the tribe Cyathostominae. Oligoprobes were designed for identification of the following species: Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicocyclus insigne, Cyathostomum catinatum, Cylicostephanus goldi, and Cylicostephanus longibursatus. A seventh probe was designed as a positive control to identify all these members of the Cyathostominae. The intergenic spacer region was amplified by PCR using conserved primers. Initially, three oligoprobes were used in Southern blot analysis. To facilitate high-throughput identification, these and a further four oligoprobes were developed for use in a PCR-ELISA. All probes were validated for their ability to detect cyathostomin PCR products in the PCR-ELISA, using DNA from morphologically identified adult parasites. Initially, 712 L4 were isolated from the diarrhoeic faeces from horses (n=17) with clinical larval cyathostominosis. PCR products from 522 of these L4 were subjected to analysis, with 413 L4 being identified as one of the aforementioned species. With reference to individual species analysis, 28.5% of the 522 L4 were identified as C. longibursatus, 25.7% as C. nassatus, 15.9% as C. ashworthi, 7.3% as C. goldi and 1.7% as C. catinatum. No L4 were identified as being C. insigne species. When L4 within faeces from individual horses were compared, no sample was found to comprise parasites of one species. The least number of species identified in a single sample was two. This study suggests that clinical larval cyathostominosis is predominantly caused by mixed-species infections.

Recommended terminology and advances in the systematics of the Cyathostominea (Nematoda: Strongyloidea) of horses.

Terminology for common names for the Tribe Cyathostominea (cyathostomins), and disease caused by the nematodes (cyathostominosis), were recommended to replace the previously used names cyathostomes and cyathostomosis, which are ambiguous, inaccurate or synonymous, by the Third Internal Workshop on the Systematics of Cyathostominea of Horses, held in Stresa, Italy, 28 August 2001. The progress by this international working group at three workshops is reviewed briefly and a list of publications is provided. Included are an annotated checklist by genus and species of 93 species level names and the recognition of 52 species, redescriptions of seven species, and the description of one new species. Upon petition by workshop participants, the International Commission on Zoological Nomenclature placed Cyathostomum tetracanthum Mehlis, 1831 on the "Official List of Specific Names in Zoology", ending more than a century of controversy over the names of cyathostomins. Some progress is described in molecular and morphological systematics and in the development of diagnostic molecular probes. A revised identification key is being prepared to the 52 species of the Tribe Cyathostominea.

Haemonchus horaki n. sp. (Nematoda: Trichostrongyloidea) from the grey rhebuck Pelea capreolus in South Africa.

In the course of a revision of species of Haemonchus Cobb, 1898 (Nematoda), commonly referred to as large stomach worms and significant pathogens of ruminants, a new species was discovered in the grey rhebuck Pelea capreolus, and the bontebok Damaliscus pygarthus, in South Africa. The new species, Haemonchus horaki, was previously reported as a long-spicule form of H. contortus (Rudolphi, 1803) Ransom, 1911. The new species, compared with H. contortus, can be distinguished by significantly longer spicules (555-615 microm vs. 383-475 microm); a synlophe with fewer ridges (26 vs. 30 in the region of the posterior part of the esophagus) that extend more posteriorly (within 1 mm of the copulatory bursa in males and postvulvar in females, vs. 2/3 to 3/4 of prebursal and prevulvar lengths); and an asymmetrical dorsal lobe with a long dorsal ray divided for more than half of its length, forming 2 branches of unequal length (vs. a dorsal ray divided for less than half of its length and forming 2 equal branches in H. contortus).

Evaluation of the specificity of five oligoprobes for identification of cyathostomin species from horses.

Here, we report evaluation of five oligoprobes designed from intergenic spacer (IGS) region sequences for identification of cyathostomin species. Oligoprobes were designed for identification of Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicostephanus longibursatus, Cylicostephanus goldi and a fifth probe designed to identify all members of this tribe. PCR amplification of IGS DNA from 16 cyathostomin species allowed sequence comparison and identification of four putative species-specific probes. Southern blotting of amplified products from 16 species showed that all probes were species-specific. The fifth probe recognised all 16 cyathostomin species but did not bind to members of the genus Strongylus. Furthermore, these probes were used to identify individual infective L3, eggs and L4 indicating that they will be invaluable to furthering the study of the epidemiology and pathogenesis of these important equine nematodes.

Synlophe patterns of the Haemonchinae of ruminants (Nematoda: Trichostrongyloidea).

The pattern of longitudinal ridges (synlophe) on the external cuticular surface of trichostrongylid nematodes has been shown to be of value for distinguishing species and determining relationships among higher taxa. In the process of studying Mecistocirrus digitatus, the large stomach worm of bovids of Asia that has been imported and established in the Americas, we observed remarkably similar synlophe patterns to those described for 3 species of Haemonchus and to those we examined in a species of Ashworthius. In all 3 genera, the synlophe is absent from the posterior part of the body. Only in Haemonchus does the synlophe extend beyond midbody. In both M. digitatus and Ashworthius sidemi, the synlophe extends posteriorly only about 1/4 of body length. In all 3 genera, the synlophe consists of about 30 ridges in the region of the esophagus with variation among species in specific areas, including additional pairs of subventral and subdorsal ridges and different lengths of sublateral ridges. This information is useful for identifying species and determining relationships among these large stomach worm parasites of cattle, sheep, goats, and farmed and wild cervids.

Phylogenetic analysis of partial mitochondrial cytochrome oxidase c subunit I and large ribosomal RNA sequences and nuclear internal transcribed spacer I sequences from species of Cyathostominae and Strongylinae (Nematoda, Order Strongylida), parasites of the horse.

Three nucleotide data sets, one nuclear (ITS-2) and two mitochondrial (COI and l-rRNA), have been investigated in order to determine relationships among species of Strongylinae and Cyathostominae, intestinal parasites of the horse. The data exhibited a strong mutational bias towards A and T and in the COI gene, silent sites appeared to saturate rapidly partly due to this substitution bias. Thus, the COI gene was found to be less phylogenetically informative than the l-rRNA and ITS-2 genes. Combined analysis of the l-rRNA and ITS-2 genes supported a monophyletic clade of the cyathostomes with Tridentoinfundibulum gobi, which had previously been classified as a nematode of' uncertain origin'. The Strongylinae grouped consistently outside the clade containing the cyathostomes and T. gobi. Molecular analysis failed to provide strong evidence for the separation of cyathostomes into classical genera, as previously defined by morphological classification.

Cerebral larva migrans in a raccoon (Procyon lotor).

During 1997, gross and histopathologic examinations were performed on an adult female raccoon (Procyon lotor) that was live-trapped in Corvallis, Oregon. Multifocal eosinophilic granulomas indicative of neural and visceral larva migrans were observed. However, within these granulomas, the presence of parasitic larva was seen only in the cerebrum. Morphologic characteristics indicated that the nematode was an ascarid larva. However, it was smaller than the larva of Baylisascaris sp. This appears to be the first documented case of cerebral larva migrans in a raccoon.

Molecular evidence for cryptic species within Cylicostephanus minutus (Nematoda: Strongylidae).

The nucleotide sequences of the first and second internal transcribed spacers of nuclear ribosomal DNA were determined for adults of Cylicostephanus minutus from different geographical origins. The lengths of first and second internal transcribed spacer sequences ranged from 370 to 372bp and 215 to 216bp, respectively. Pairwise sequence comparisons revealed that some individuals of C. minutus had identical first and second internal transcribed spacer sequences, whereas others differed by 3.0% and 7.4% in their first and second transcribed spacers, respectively. Some individuals with sequence differences originated from the same host. The levels of difference within C. minutus were higher than that between the morphologically distinct species, Cylicostephanus goldi and Cylicostephanus longibursatus (0.8% for the first internal transcribed spacer and 3.8% for the second internal transcribed spacer). The data provide support for the proposal that C. minutus represents a complex of at least two species. In order to study the population genetic structure of C. minutus, a PCR-linked single-strand conformation polymorphism technique was also established.

An annotated checklist by genus and species of 93 species level names for 51 recognized species of small strongyles (Nematoda: Strongyloidea: Cyathostominea) of horses, asses and zebras of the world.

The results of an international collaborative effort to prepare a recommended list of scientific names for the small strongyles (Nematoda: Strongyloidea: Cyathostominea) of horses, donkeys and zebras are reported. Fifty-one valid species are recognized in 13 genera, including Cyathostomum, Coronocyclus, Cylicodontophorus, Cylicocyclus, Cylicostephanus, Skrjabinodentus, Tridentoinfundibulum, Petrovinema, Poteriostomum, Parapoteriostomum, Hsiungia, Cylindropharynx and Caballonema. In addition, 42 other species level names are listed as synonyms of the 51 recognized species or as species inquirendae (10 species) or nomen nudum (one species). Numerous annotations provide information on the nomenclatural and systematics history, current status and additional studies needed.

Identification and semi-quantitation of Ostertagia ostertagi eggs by enzymatic amplification of ITS-1 sequences.

A region within the first internal transcribed spacer (ITS-1) of the ribosomal DNA repeat of Ostertagia ostertagi has been identified that is 408 base pairs (bp) in length and is comprised of a 2 x 204 bp repeat. Universal polymerase chain reaction (PCR) primers which span this region, as well as a portion of the 5.8S rDNA, generate a 1011 bp fragment using genomic DNA from O. ostertagi. However, these same primers generate only a 600 bp (approximate) fragment using DNA from Haemonchus contortus, Cooperia oncophora and Oesophagostomum radiatum, as well as other species within the genus Ostertagia. When DNA samples derived from adult parasites of the different genera were mixed and simultaneously amplified, the O. ostertagi component could be identified within the mixed DNA populations. Furthermore, a correlation was observed between relative fluorescence intensities of the 1011 bp and the 600 bp PCR fragments and the percentage of O. ostertagi DNA within a mixture of parasite DNAs. A similar high correlation was obtained between the percentage of O. ostertagi DNA and percent O. ostertagi eggs in feces containing eggs of other nematode genera. This resulted in the generation of a protocol that can determine the percentage of O. ostertagi eggs within a mixed population of gastrointestinal nematode eggs. Results indicate a detection equivalent to 0.05 eggs.

Comparisons of two polymorphic species of Ostertagia and phylogenetic relationships within the Ostertagiinae (Nematoda: Trichostrongyloidea) inferred from ribosomal DNA repeat and mitochondrial DNA sequences.

The first internal transcribed spacer DNA (ITS-1) (rDNA) and the mitochondrial (mt) DNA-derived cytochrome oxidase I gene (COX-1) were enzymatically amplified, cloned and sequenced from 6 nominal species of Ostertagiinae as well as Haemonchus contortus and Haemonchus placei. The portion of the COX-1 gene analyzed was 393 base pairs (bp) in length and contained 33 within species polymorphic base changes at 28 synonymous sites. The ITS-1 rDNA consensus sequences ranged from 392 bp (Ostertagia ostertagi/Ostertagia lyrata, Teladorsagia circumcincta) to 404 bp (H. contortus, H. placei). These data were used both in a distance analysis to assess the concept of polymorphic species within the genus Ostertagia and in parsimony analysis to assess phylogenetic relationships within a limited group of Ostertagiinae. Pairwise similarity scores of both ITS-1 and COX-1 data showed the highest number of conserved sites between the proposed dimorphic species of Ostertagia. The level of similarity was lower in the COX-1 data due to the high number of synonymous base changes. Analysis by maximum parsimony of the same data did not refute O. ostertagi/O. lyrata and Ostertagia mossil/Ostertagia dikmansi as dimorphic species and supported monophyly of these ostertagiines relative to representatives of the haemonchine outgroup. In the single most parsimonious tree from ITS-1 rDNA data, a subclade of Ostertagia spp. included forms possessing parallel synlophes and long esophageal valves that typically occur in cervid hosts.

Comparative sequence analysis of the intergenic spacer region of cyathostome species.

The ribosomal DNA intergenic spacer was amplified by the polymerase chain reaction from 16 cyathostome species using primers derived from conserved regions within the flanking 18S and 26S rRNA genes. This generated a 1.5-2.5 kb fragment which was sequenced from five species. The areas covering the 26S and 18S rRNA genes were more than 99% similar among the five species. Furthermore, in all species there existed a highly conserved region of approximately 380 bp at the 3' end of the intergenic spacer. Subsequently, two cyathostome-specific primers were designed to amplify a smaller, more variable region of the intergenic spacer. Eleven further species were amplified using these primers and analysis showed that sequence similarities varied from 40 to 97% between species. The sequence information obtained in this study is being used to develop a PCR-based assay for the differentiation of preparasitic stages of cyathostomes.

Northern bobwhites as disease indicators for the endangered Attwater's prairie chicken.

Because of limited access to the endangered Attwater's prairie chicken (Tympanuchus cupido attwateri), we used a related species, the northern bobwhite (Colinus virginianus), as a surrogate for disease evaluation. Free-living northern bobwhites (n = 62) on the Attwater Prairie Chicken National Wildlife Refuge (near Eagle Lake, Texas, USA) were examined during spring and fall 1993 for helminthic endoparasites and specific antibodies against the infectious agents responsible for nine infectious diseases. Trichostrongylus cramae, Raillietina sp., and Strongyloides avium were collected from 97, 44, and 32% of northern bobwhites examined, respectively. Dispharynx nasuta and Syngamus trachea also were found. No gross lesions due to parasites were observed. Specific antibody to Pasteurella multocida was found in 3 of 53 plasma samples. It is possible that potentially pathogenic species such as P. multocida, T. cramae, and D. nasuta could threaten sympatric Attwater's prairie chickens.

Serologic and parasitologic survey of the endangered Attwater's prairie chicken.

Because conservation biologists have postulated that infectious diseases may have potentiated the endangerment of the Attwater's prairie chicken (Tympanuchus cupido attwateri), free-living prairie chickens were surveyed from all remaining populations for helminthic endoparasites and antibody against the etiological agents of nine infectious diseases. Samples from 4 of 27 adult males were positive for anti-Pasteurella multocida antibody. All other serologic tests were negative (n = 19). We identified Dispharynx nasuta, a parasite previously associated with disease in other grouse from North America, in one of three adult Attwater's prairie chickens examined. Evidence of Trichostrongylus cramae was found for eight of nine suitable samples, which represents the first report of this parasite in prairie grouse. The mean intensity of T. cramae in Attwater's prairie chicken was 1,019.3 (Range = 3-1,906; n = 3). Further work is needed to determine whether P. multocida, T. cramae, or D. nasuta are detrimental to Attwater's prairie chicken populations. If so, conservation biologists could reduce the prevalence and incidence of these parasites and potentially gain more time to address the habitat conditions thought to be the ultimate cause of population declines.

A redescription of Cylicocyclus triramosus (Nematoda: Strongyloidea): a parasite of the zebra, Equus burchelli antiquorum.

Cylicocyclus triramosus, a rare species of Cyathostominae from the southern African Burchell's zebra (Equus burchelli antiquorum) is redescribed, and a neotype is proposed. The external leaf-crown of the species consists of 30 elements and the internal leaf-crown numerous small and clear elements. The buccal capsule is cylindrical, oval in cross section, and approximately 3 times as wide as deep. The thin walls of the buccal capsule taper anteriorly from a large ringlike, hoop-shaped thickening around the posterior margin. The dorsal gutter is nipple-shaped. The mouth collar is notched dorsally and ventrally. The dorsal ray of the copulatory bursa is slightly elongate and distinctly set off from the lateral lobes. The branches of the dorsal ray have auxiliary branches. Appendages of the genital cone consist of 2 separated, large, semilunar plates with finger-shaped processes on the margins. The vulva-to-anus distance is greater than the tail length. The species is most similar to Cylicocyclus radiatus. Cylicocyclus triramosus differs from all similar species in the presence of dorsal and ventral notches in its mouth collar. In addition, C. triramosus also has a distinctive form of male genital cone appendage. Cylicocyclus radiatus differs further from C. triramosus in lacking a dorsal gutter and having a more elongate dorsal ray and a female tail nearly equal in length to the vulva-to-anus distance.

Molecular delineation of Cylicocyclus nassatus and C. ashworthi (Nematoda:Strongylidae).

The nucleotide sequences of the first internal transcribed spacer (ITS-1), 5.8S gene and second internal transcribed spacer (ITS-2) of ribosomal DNA have been determined for Cylicocyclus nassatus, C. ashworthi and C. insignis. Pairwise comparisons revealed sequence differences between the taxa ranging from 3.8 to 6.2% for the ITS-2 and 2.2-2.7% for the ITS-1. For the ITS-1, the level of the sequence difference between C. ashworthi and C. nassatus (2.2%) was equivalent to that between C. nassatus and C. insignis (2.2%), indicating that C. ashworthi and C. nassatus represent separate species. Theoretical restriction maps were constructed from the sequence data, and a polymerase chain reaction-linked restriction fragment length polymorphism (PCR-linked RFLP) technique was established to unequivocally distinguish C. ashworthi from C. nassatus.

Systematics of gastrointestinal nematodes of domestic ruminants: advances between 1992 and 1995 and proposals for future research.

The systematics of trichostrongyloid nematodes of ruminants provides a foundation for diagnostics and responds to the need to identify eggs in feces, free-living larvae from pastures or fecal cultures and larval or adult nematodes collected from hosts. These needs are associated with diagnostic problems or research projects. Difficulties in identifying all developmental stages of trichostrongyloid nematodes of domestic ruminants still severely limit the effective diagnosis and control of these parasites. Phylogenetic hypotheses as the basis for predictive classifications have been developed only for the subfamilies of the Trichostrongylidae. This report briefly describes recent progress in the development of improved tools for identification, phylogenetic analyses and predictive classifications. It also describes future research needed on the identification and classification of trichostrongyloid nematode parasites of domestic ruminants. Nematodes included are species of the super-family Trichostrongyloidea known to be important pathogens of domestic ruminants. The information summarized is presented by nematode developmental stage and by taxonomic groups. Eggs: While eggs of some trichostrongyloid nematode parasites of ruminants can be readily identified to their genus (Nematodirus), and some to species (e.g. Nematodirus battus), most of the important pathogens (including the Ostertagiinae and Haemonchinae) cannot be identified morphologically or morphometrically even to family level. However, DNA technology has been developed for determining not only the presence of specific pathogens in eggs from fecal samples, but also for estimating the percentage of the total eggs that each pathogen comprises. This new method will make possible a rapid determination of which individual animals in a herd should be treated. Larvae: The most commonly-used method for identifying infective larvae is time-consuming (several weeks), unreliable for estimating intensities of individual species as components of mixed populations and requires highly trained specialists. Available identification keys for larvae are not well illustrated and need to be augmented. Adults: Recent advances in the identification of adult trichostrongyloids and their systematics are organized by taxonomic group. Genera included are Ostertagia, Haemonchus, Cooperia, Trichostrongylus and Nematodirus. Recently, the first phylogenetic analysis of the Trichostrongylidae family established monophyly for the family. A similar analysis of the Molineidae is needed. Ostertagia: Several studies of polymorphism summarized the phenomenon and listed 19 polymorphic species in five genera. Two studies of DNA differences within and among polymorphic species of Ostertagiinae supported earlier hypotheses that the species pairs represent polymorphic species. A phylogenetic analysis of the Ostertagiinae and generic concepts are needed. Haemonchus: A key to three species of Haemonchus provides, for the first time, morphological characteristics for the microscopical identification to species of individual adult nematodes of either sex. The Food and Drug Administration is now requiring that results of drug trials include identification of Haemonchus to species. Cooperia: Studies using random amplified polymorphic DNA methods showed a high degree of variation within and among C. oncophora/C. surnabada, but supported a polymorphic relationship for the species pair. A phylogenetic analysis of the Cooperiinae is needed. Trichostrongylus: Restriction Fragment Length Polymorphisms (RFLPs) of genomic DNA of two strains of T. colubriformis indicated a high degree of intra- and inter-strain DNA polymorphism. However, other studies demonstrated expected species level differences between T. colubriformis and T. vitrinus using Random Amplified Polymorphic DNA (RAPD) methods. Sequences of the second Internal Transcribed Spacer Region (ITS-2) ribosomal repeat showed sequence differences of 1.3-7.6% among five