Characterization of the Complete Mitochondrial Genome of Ostertagia trifurcata of Small Ruminants and its Phylogenetic Associations for the Trichostrongyloidea Superfamily.

The complete mitochondrial (mt) genome of Ostertagia trifurcata, a parasitic nematode of small ruminants, has been sequenced and its phylogenetic relationship with selected members from the superfamily Trichostrongyloidea was investigated on the basis of deduced datasets of mt amino acid sequences. The entire mt genome of Ostertagia trifurcata is circular and 14,151 bp in length. It consists of a total of 36 genes comprising 12 genes coding for proteins (PCGs), 2 genes for ribosomal RNA (rRNA), 22 transfer RNA (tRNA) genes and 2 non-coding regions, since all genes are transcribed in the same direction. The phylogenetic analysis based on the concatenated datasets of predicted amino acid sequences of the 12 protein coding genes supported monophylies of the Haemonchidae, Dictyocaulidae and Molineidae families, but rejected monophylies of the Trichostrongylidae family. The complete characterization and provision of the mtDNA sequence of Ostertagia trifurcata provides novel genetic markers for molecular epidemiological investigations, systematics, diagnostics and population genetics of Ostertagia trifurcata and its correspondents.

Host specificity and species diversity of the Ostertagiinae Lopez-Neyra, 1947 in ruminants: a European perspective.

BACKGROUND: Nematodes of the subfamily Ostertagiinae appear to be rather specific to a species or family of hosts, but some are observed in a wide variety of hosts. The nematode Ostertagia leptospicularis draws special attention due to its presence or absence among the same host species in different European countries. Therefore, this paper focuses mainly on the host specificity among nematodes of the subfamily Ostertagiinae. The second aim of this study is to assess the possibility of treating O. leptospicularis as an Ostertagia species complex. METHODS: Data were gathered from post-mortem examinations of domestic and wild ruminants (n = 157), as well as bibliographical references (n = 96), which were pooled and discussed. The research area was limited to European countries, hence the studied ostertagiine species are limited to native ones; likewise, the host species. Special emphasis was placed on the mean abundance values that allowed a typical host or hosts for each nematode species to be specified. Correspondence analysis was performed to confirm the stated host specificity. RESULTS: The analysis revealed that nematodes of this subfamily tend to use ruminants from a particular subfamily as their principal host. The results indicate that Ostertagia leptospicularis, similar to Teladorsagia circumcincta, may represent a potential species complex. This nematode, as the sole member of the subfamily Ostertagiinae, occurs in almost all representatives of the Bovidae subfamily, as well as in the Cervidae. CONCLUSIONS: Despite the stated narrow host specificity, the results obtained may suggest that O. leptospicularis is not strongly connected to any host or is comparably associated with a very wide and diverse group of hosts (Cervidae, Bovidae). The Ostertagia complex may have particular cryptic species or strains typical for any individual host or group of hosts. Such a conclusion requires further investigations on a wider scale.

Phylogenetic characterization of ß-tubulins and development of pyrosequencing assays for benzimidazole resistance in cattle nematodes.

Control of helminth infections is a major task in livestock production to prevent health constraints and economic losses. However, resistance to established anthelmintic substances already impedes effective anthelmintic treatment in many regions worldwide. Thus, there is an obvious need for sensitive and reliable methods to assess the resistance status of at least the most important nematode populations. Several single nucleotide polymorphisms (SNPs) in the ß-tubulin isotype 1 gene of various nematodes correlate with resistance to benzimidazoles (BZ), a major anthelmintic class. Here we describe the full-length ß-tubulin isotype 1 and 2 and α-tubulin coding sequences of the cattle nematode Ostertagia ostertagi. Additionally, the Cooperia oncophora α-tubulin coding sequence was identified. Phylogenetic maximum-likelihood analysis revealed that both isotype 1 and 2 are orthologs to the Caenorhabditis elegans ben-1 gene which is also associated with BZ resistance upon mutation. In contrast, a Trichuris trichiura cDNA, postulated to be ß-tubulin isotype 1 involved in BZ resistance in this human parasite, turned out to be closely related to C. elegans ß-tubulins tbb-4 and mec-7 and would therefore represent the first non-ben-1-like ß-tubulin to be under selection through treatment with BZs. A pyrosequencing assay was established to detect BZ resistance associated SNPs in ß-tubulin isotype 1 codons 167, 198 and 200 of C. oncophora and O. ostertagi. PCR-fragments representing either of the two alleles were combined in defined ratios to evaluate the pyrosequencing assay. The correlation between the given and the measured allele frequencies of the respective SNPs was very high. Subsequently laboratory isolates and field populations with known resistance status were analyzed. With the exception of codon 167 in Cooperia, increases of resistance associated alleles were detected for all codons in at least one of the phenotypically resistant population. Pyrosequencing provides a fast, inexpensive and sensitive alternative to conventional resistance detection methods.

Gene expression changes in a P-glycoprotein (Tci-pgp-9) putatively associated with ivermectin resistance in Teladorsagia circumcincta.

Anthelmintic resistance in parasitic nematodes of small ruminants is widespread and, in some parts of the world, threatens the sustainability of sheep production. The genetic changes underlying resistance to anthelmintics, particularly ivermectin (IVM), remain to be determined. The majority of studies to date have investigated target site mutations; relatively little attention has been paid to the role of changes in gene expression. In this study, we investigated the expression of putative drug transporter molecules, P-glycoproteins (Pgps), in Teladorsagia circumcincta, the predominant parasitic nematode species of sheep in the UK and the major anthelmintic resistant species. Utilising a degenerate PCR approach, 11 partial Pgp sequences were identified. Constitutive differences in gene expression between an IVM-susceptible (MTci2) and a multidrug-resistant (MTci5) isolate were determined for 10 of the Pgps using the ΔΔCt TaqMan® real-time PCR method. Gene expression differences were particularly marked in one of these genes, namely Tci-pgp-9. In the MTci5 isolate, statistically significant increases in Tci-pgp-9 expression, at the mRNA level, were observed across all life-cycle stages and most notably in eggs (55-fold increase). Comparison of the partial Tci-pgp-9 nucleotide sequences from MTci2 and MTci5 also identified high levels of polymorphism. This work has shown that constitutively increased expression in Tci-pgp-9, coupled with increased sequence polymorphism, could play a role in allowing multidrug-resistant T. circumcincta to survive IVM exposure. The genetic changes underpinning these gene expression changes remain to be elucidated and need to be investigated in other isolates. These changes could form the basis of an IVM resistance marker to monitor the spread of resistance and to evaluate management practices aimed at delaying its spread.

[On the conspecificity of Ostertagia ostertagi and Ostertagia lyrata (Nematoda: Ostertagiinae)].

Partial sequences of the second internal transcribed spacer (ITS-2) rDNA were obtained for Ostertagia ostertagi and O. lyrata, which are supposedly constitute two morphologically distinct variants of the species. The 1.4% level of difference between the ITS-2 sequences of O. ostertagi and O. lyrata was reported, whereas the sequences derived from worms of the same morph were completely identical. The data obtained prevent an attribution of O. ostertagi and O. lyrata to the same species.

Benzimidazole resistance allele haplotype diversity in United Kingdom isolates of Teladorsagia circumcincta supports a hypothesis of multiple origins of resistance by recurrent mutation.

Polymorphisms in the isotype I beta-tubulin gene are important genetic determinants of benzimidazole (BZ) resistance in a number of parasitic nematode species including Teladorsagia circumcincta, a major gastrointestinal nematode of sheep. This study investigates the genetic diversity at this locus in a BZ-resistant isolate of T. circumcincta (MTci5) derived from a sheep farm in the United Kingdom (UK) that was open to animal, and therefore parasite, migration. Pyrosequencing was used to determine the frequency of single nucleotide polymorphisms (SNPs) known to be associated with BZ resistance. This was followed by a combination of single strand conformation polymorphism (SSCP) analysis and nucleotide sequencing to sample allelic diversity in a 276bp fragment immediately surrounding the isotype I beta-tubulin F200Y mutation. The genetic diversity at this locus was extremely high, with seven different haplotypes found to contain the resistant F200Y polymorphism in this single resistant isolate. Genotyping by SSCP interfaced with pyrosequencing demonstrated that the P200(Y) mutation is also present on multiple haplotypes in two other BZ-resistant T. circumcincta isolates from the UK. This contrasts with much lower levels of haplotype diversity in BZ-resistant alleles present in T. circumcincta isolates from French goat farms that are closed to parasite migration. Taken together with our knowledge of T. circumcincta population genetic structure, these results are most consistent with multiple independent origins of resistance and mixing of alleles due to the large amount of livestock movement in the UK.

Hamulonema gen. nov. for Teladorsagia hamata and Ostertagia kenyensis in the ostertagiine fauna (Nematoda: Trichostrongyloidea) from African ungulates.

Hamulonema gen. nov. is proposed for Teladorsagia hamata and Ostertagia kenyensis in the ostertagiine nematode fauna found in artiodactyl hosts from Africa. Monomorphic species representing this genus are characterized by a bilaterally symmetrical and parallel synlophe in males and females, a 2-2-1 bursal formula, an accessory bursal membrane that is strongly cuticularized and reduced, a strongly reduced dorsal lobe and ray, and robust spicules with a simple, weakly pointed, ventral process, and curved, hooklike dorsal process. Species referred to Hamulonema nov. gen. are immediately distinguished from those of Camelostrongylus, Longistrongylus, Marshallagia, Orloffia, Ostertagia, and Pseudomarshallagia in which the bursal formula is 2-1-2 in males. Hamulonema nov. gen. is distinguished from those genera having a 2-2-1 bursa, including Africanastrongylus, Cervicaprastrongylus, Hyostrongylus, Mazamastrongylus, Sarwaria, Spiculopteragia, and Teladorsagia by the structure of the synlophe, bursa, genital cone, "0" and "7" papillae, dorsal lobe, and spicules in specific instances. In the global fauna, 4 of 14 ostertagiine genera are endemic to Africa. African genera may represent morphologically divergent and discrete or historically isolated lineages reflecting a pattern of geographic and host colonization as a driver for diversification since the Miocene.

Parasites of South African wildlife. XVII. Ostertagia triquetra n. sp. (Nematoda: Trichostrongylina) from the grey rhebuck, Pelea capreolus (Forster, 1790).

Re-examination of Teladorsagia hamata (Mönnig, 1932) Durette-Desset, 1989 reported from grey rhebuck, Pelea capreolus (Forster, 1790) proved it to be a new species of Ostertagi Ransom, 1907. The new species, for which the name Ostertagia triquetra n. sp. is proposed, differs from Teladorsagia hamata in the configuration of the bursal rays (2-1-2 in the former, 2-2-1 in the latter), and in that the interno-dorsal branch of the spicules bears a process that is triangular and convex in the new species, but concave and shaped like an ice-cream scoop in Teladorsagia hamata. Ostertagia triquetra has so far been found only in grey rhebuck in the Eastern Cape Province while Teladorsagia hamata was recorded from springbok, Antidorcas marsupialis (Zimmerman, 1780) and gemsbok, Oryx gazella (Unnaeus, 1758) in the western part of the country.

Synlophe in Ostertagia cf. kasakhstanica (Nematoda: Ostertagiinae), the minor morphotype of O. bisonis from western North America.

Ostertagia cf. kasakhstanica, the putative minor morphotype of the polymorphic ostertagiine O. bisonis, is reported in Bison bison from South Dakota. Descriptions of the synlophe and details of other diagnostic characters useful in identification and differentiation of this minor morphotype from other ostertagiines in wild and domestic ruminants from western North America are presented. Laterally, the cervical synlophe is consistent with Type Ib pattern among ostertagiines and is within the range of variation previously described for O. bisonis. Among recognized minor morphotypes of ostertagiines in wild and domestic ruminants from North America, O. kasakhstanica is distinct from O. kolchida, O. dikmansi, and O. arctica based on a tapering lateral synlophe. It is further distinguished from O. lyrata by the pattern of the cervical synlophe in the lateral field, structure of the genital cone and spicules, and dimensions of the esophageal valve. Although morphologically similar to T. davtiani/T. trifucata and T. boreoarcticus f. minor, it can be readily distinguished by the 2-1-2 bursal pattern, the structure of the spicules and genital cone, and the length of the esophageal valve. Ostertagia hisonis/O. cf. kasakhstanica circulates among pronghorn, mule deer, bison, and cattle in west-central North America.

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.

Ostertagia nianqingtanggulaensis Kúng & Li, 1965 (Nematoda: Trichostrongyloidea) from sheep and goats at high altitudes in Nepal.

The trichostrongyloid nematode Ostertagia nianqingtanggulaensis Kúng & Li, 1965, previously recorded only from sheep in Tibet, is redescribed and is shown to be a common parasite of both sheep and goats at higher altitudes in the Himalayan foothills of western Nepal. Herbage examination indicated that infective larvae can overwinter on snow covered slopes. Use of tracer animals confirmed that migratory flocks are exposed to infection when grazing on alpine pastures during the summer months. In contrast, nearby non-migratory flocks kept at lower altitudes did not harbour O. nianqingtanggulaensis and tracer animals grazed with these flocks did not become infected with this species.

The ITS-2 rDNA of Teladorsagia circumcincta, T. trifurcata and T. davtiani (Nematoda: Trichostrongylidae) indicates that these taxa are one species.

There remains uncertainty about the species status of Teladorsagia circumcincta, T. davtiani and T. trifurcata. In this study, the second internal transcribed spacer (ITS-2) sequences of these taxa and 2 other members within the same subfamily (Ostertagiinae) were examined. Although some sequence variation was detected between and within single worms of each taxon of Teladorsagia, no unequivocal base differences were detected among their consensus ITS-2 sequence. In comparison, there was 9% sequence difference between O. ostertagi and O. leptospicularis and 13-15% difference between the genera Teladorsagia and Ostertagia. These findings indicate that T. circumcincta, T. davtiani and T. trifurcata represent a single species, T. circumcincta.

Epidemiology of Ostertagia in the Northwestern USA.

The two most significant environmental factors that influence the epidemiology of Ostertagia are temperature and moisture. These factors vary more in the west and northwest than in any other part of the USA because of extreme regional differences in climate, topography, and land use. Consequently, patterns of Ostertagia transmission and inhibition also vary widely from region to region and from year to year. Recent data require revisions of previously accepted concepts of northern/southern transmission and inhibition. In particular, the flaw of liberal extrapolation of information from one region to another has now been recognized. Inhibition may not occur along the coast of the Pacific Northwest. Although not completely delineated, the transition zone between northern and southern patterns of inhibition appears to be significantly wider and more variable than previously recognized; Oregon data suggest a possible indeterminate and variable band extending north of the 45th parallel and south of the 43rd parallel. Summer inhibition has now been documented in areas of Oregon and Montana. Other than the recent Oregon and Montana studies, data on this transition zone in the Rocky Mountain region and high plains are sparse to non-existent. In contrast to previously accepted doctrine, the fall rise in egg count generally exceeds the traditionally accepted spring rise in many areas. From the Pacific Northwest to the Midwest, year-round transmission patterns have been observed. The presence of geothermal ground water sources produces microclimates that favor larval survival in many areas.(ABSTRACT TRUNCATED AT 250 WORDS)

The systematics of nematodes that cause ostertagiasis in domestic and wild ruminants in North America: an update and a key to species.

Studies of medium stomach worms (Subfamily Ostertagiinae) of domestic and wild ruminants in North America have refined our understanding of the systematics, host distribution and biogeography of these nematodes. Strong support for recognition of monophyly for the Ostertagiinae has resulted from preliminary phylogenetic analyses of morphological characters among these and other trichostrongylids. Evaluation of morphological and biochemical characters in our laboratory and elsewhere has not refuted the polymorphism hypothesis where paired major and minor morphotypes are postulated to represent polymorphic species within the genera Ostertagia (Ostertagia ostertagi/Ostertagia lyrata; Ostertagia leptospicularis/Ostertagia kolchida; Ostertagia gruehneri/Ostertagia arctica; Ostertagia mossi/Ostertagia dikmansi), Teladorsagia (Teladorsagia circumcincta/Teladorsagia trifurcata (= Teladorsagia davtiani), and Marshallagia (Marshallagia marshalli/Marshallagia occidentalis). The minor morphotype of Ostertagia bisonis, recognized in Eurasia (considered to be Teladorsagia kazakhstanica), has yet to be discovered in North America. The taxonomy of these species has remained stable since an earlier overview of Ostertagia spp. and Teladorsagia spp. in North American domestic stock at the 1986 Ostertagia Workshop. Investigations since 1986 have focused on comparative morphology and the clear differentiation of Ostertagiinae, primarily from wild ruminants, that have been reported or may potentially occur in domestic bovids. Among these are species which infect wild and domestic sheep (Marshallagia marshalli/Marshallagia occidentalis), bovids (Ostertagia bisonis), and cervids (Ostertagia mossi/Ostertagia dikmansi, Ostertagia leptospicularis/Ostertagia kolchida, Ostertagia gruehneri/Ostertagia arctica, Mazamastrongylus odocoilei and Mazamastrongylus pursglovei) in North America. A key is presented for identifying 15 species of the Subfamily Ostertagiinae that are parasitic in domestic and wild ruminants of North America. Four exotic species (Camelostrongylus mentulatus, Spiculopteragia spiculoptera (= Spiculopteragia boehmi), Spiculopteragia asymmetrica and Longistrongylus curvispiculum) have been reported in North America, but only in game farms and are not included in the key. Hyostrongylus rubidus, a member of the Ostertagiinae normally parasitic in pigs and only rarely reported from domestic ruminants, is not included in the keys. Additional data from comparative morphology and molecular analyses will promote a reassessment of the generic-level systematics of the Ostertagiinae and allow an evaluation of host-parasite evolution and historical biogeography.

The identification of females within the subfamily Ostertagiinae Lopez-Neyra 1947.

As a result of the examination of many thousands of female nematodes classified as belonging to the sub-family Ostertagiinae Lopez-Neyra 1947, it became possible to compile descriptions of different female types, each of which corresponds to a polymorphic species as determined by male morphology. The female types have been designated as follows: Ostertagia ostertagi (males O. ostertagi and O. lyrata), Ostertagia circumcincta (males O. circumcincta, O. trifurcata and Teladorsagia davtiani), Ostertagia leptospicularis (males O. leptospicularis and O. kolchida) and Spiculopteragia asymmetrica (males S. asymmetrica and Apteragia quadrispiculata).

Restriction fragment length patterns of DNA from parasitic nematodes of sheep.

High molecular weight DNA obtained from sheep parasitic nematodes Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis, was digested with various restriction endonucleases. Digestion with Eco R1 produced the most informative pattern of repeat sequence bands. H contortus adult or larval DNA produced bands of 2.7, 3.0 and 1.4 kb. O circumcincta adult or larval DNA had common 2.7 and 1.4 kb bands with adult specific bands of 2.2 and 0.9 kb and a larval specific 2.08 kb band. T colubriformis adults or larval DNA produced 2.7, 1.4 and 0.79 kb bands. These preliminary results show that restriction patterns of repeat sequence DNA may be useful for the identification of various trichostrongylid species parasitic for sheep.

A concise history of the Ostertagiinae Lopez-Neyra, 1947 (Nematoda: Trichostrongyloidea) and a discussion on its composition.

After a historical introduction to the Ostertagiinae and several revisions of this group, it is proposed that the subfamily shall contain seven genera: Ostertagia, Hyostrongylus, Longistrongylus, Marshallagia, Mazamastrongylus, Spiculopteragia and Teladorsagia. The synonyms of these genera are to be found in the literature. In this proposal the genus Hyostrongylus should belong to the Ostertagiinae though it has probably an intermediate position between the Ostertagiinae and Graphidiinae. Cervicaprastrongylus and Tunisostertagia are regarded as synonyms of Hyostrongylus and Ostertagia respectively. The position of Ostertamia and Trichostrongyella is not clear but they could have their closest affinities with Teladorsagia and Mazamastrongylus respectively. The origin of the subfamily and the relationships of the genera with regard to their preferences for host groups and to their geographical distribution are discussed.

Systematics of the nematodes that cause ostertagiasis in cattle, sheep and goats in North America.

The systematics of the Ostertagiinae is unsettled with no agreement on how many genera and species are present in cattle and sheep. Ten species of Ostertagiinae are commonly parasitic in cattle and sheep. The males can be identified on the basis of differences in morphology of spicules and genital cones but the females of most species cannot be identified. The species-level systematics have been complicated by the proposal that the ten species are polymorphs of only five species. The systematics at genus level has been complicated by the use of numerous genera for the ten species, at present most commonly Ostertagia, Teladorsagia and Marshallagia. This report (1) describes the current knowledge of the systematics and (2) provides diagnoses of the genera recommended for the ten species. The morphology of the ten species was studied to determine whether characteristics could be found to identify females or to further differentiate the males. Characters studied included the system of longitudinal and surface cuticular ridges (synlophe), the internal morphology of the esophagus and the orientation of rays of the copulatory bursa. The study of species morphology resulted in additional support for the polymorphism proposal. All minor species were found to be identical to their major species in characteristics of the synlophe, esophagus and rays of the copulatory bursa. Two recent classifications of the Ostertagiinae both recommended the use of the genus Teladorsagia for the species T. circumcincta and the two minor species that occur with it, T. trifurcata and T. davtiani. Teladorsagia is characterized by a copulatory bursa in which the five lateral rays are arranged 2-2-1 and a ventral swelling of the genital cone (proconus) is absent. One pair of species, Marshallagia marshalli (major species) and Ostertagia occidentalis (minor species) should be included in the genus Marshallagia. This genus has been characterized by an elongated bursa in which the posterior pair of lateral rays are much longer than the ventral pair. The remaining two pairs of species (O. ostertagi and its minor species O. lyrata, and O. leptospicularis and its minor species O. kolchida) and O. bisonis belong in the genus Ostertagia which is characterized by a 2-1-2 arrangement of the five lateral rays of the copulatory bursa and the presence of a proconus.

[New species of Ostertagia sogdiana sp. n. (Nematoda, Trichostrongylidae) from the domestic goat in Uzbekistan]. / Novyi vid Ostertagia sogdiana sp. n. (Nematoda, Trichostrongylidae) ot domashnei kozy v Uzbekistane.

In the abomasum of domestic goat of Uzbecistan there were found ostertagiae which differ distinctly from earlier described species of the family Trichostrongylidae in size and structure of spicules and in the morphology of genital conus. The new species Ostertagia sogdiana sp. n. (fam. Trichostrongylidae) is described.