Complete Genome Sequence of Streptococcus pneumoniae Serotype 19F Strain EF3030.

We report the complete genome sequence of Streptococcus pneumoniae EF3030, a serotype 19F isolate that colonizes the nasopharynx of mice while being mostly noninvasive. Such attributes make this strain highly attractive in pneumococcal carriage studies. The availability of its complete genomic sequence is likely to advance studies in the field.

High-resolution profiles of the Streptococcus mitis CSP signaling pathway reveal core and strain-specific regulated genes.

BACKGROUND: In streptococci of the mitis group, competence for natural transformation is a transient physiological state triggered by competence stimulating peptides (CSPs). Although low transformation yields and the absence of a widespread functional competence system have been reported for Streptococcus mitis, recent studies revealed that, at least for some strains, high efficiencies can be achieved following optimization protocols. To gain a deeper insight into competence in this species, we used RNA-seq, to map the global CSP response of two transformable strains: the type strain NCTC12261T and SK321. RESULTS: All known genes induced by ComE in Streptococcus pneumoniae, including sigX, were upregulated in the two strains. Likewise, all sets of streptococcal SigX core genes involved in extracellular DNA uptake, recombination, and fratricide were upregulated. No significant differences in the set of induced genes were observed when the type strain was grown in rich or semi-defined media. Five upregulated operons unique to S. mitis with a SigX-box in the promoter region were identified, including two specific to SK321, and one specific to NCTC12261T. Two of the strain-specific operons coded for different bacteriocins. Deletion of the unique S. mitis sigX regulated genes had no effect on transformation. CONCLUSIONS: Overall, comparison of the global transcriptome in response to CSP shows the conservation of the ComE and SigX-core regulons in competent S. mitis isolates, as well as species and strain-specific genes. Although some S. mitis exhibit truncations in key competence genes, this study shows that in transformable strains, competence seems to depend on the same core genes previously identified in S. pneumoniae.

Comprehensive Transcriptome Profiles of Streptococcus mutans UA159 Map Core Streptococcal Competence Genes.

In Streptococcus mutans, an oral colonizer associated with dental caries, development of competence for natural genetic transformation is triggered by either of two types of peptide pheromones, competence-stimulating peptides (CSPs) (18 amino acids [aa]) or SigX-inducing peptides (XIPs) (7 aa). Competence induced by CSP is a late response to the pheromone that requires the response regulator ComE and the XIP-encoding gene comS. XIP binds to ComR to allow expression of the alternative sigma factor SigX and the effector genes it controls. While these regulatory links are established, the precise set of effectors controlled by each regulator is poorly defined. To improve the definition of all three regulons, we used a high-resolution tiling array to map global changes in gene expression in the early and late phases of the CSP response. The early phase of the CSP response was limited to increased gene expression at four loci associated with bacteriocin production and immunity. In the late phase, upregulated regions expanded to a total of 29 loci, including comS and genes required for DNA uptake and recombination. The results indicate that the entire late response to CSP depends on the expression of comS and that the immediate transcriptional response to CSP, mediated by ComE, is restricted to just four bacteriocin-related loci. Comparison of the new data with published transcriptome data permitted the identification of all of the operons in each regulon: 4 for ComE, 2 for ComR, and 21 for SigX. Finally, a core set of 27 panstreptococcal competence genes was identified within the SigX regulon by comparison of transcriptome data from diverse streptococcal species. IMPORTANCES. mutans has the hard surfaces of the oral cavity as its natural habitat, where it depends on its ability to form biofilms in order to survive. The comprehensive identification of S. mutans regulons activated in response to peptide pheromones provides an important basis for understanding how S. mutans can transition from individual to social behavior. Our study placed 27 of the 29 transcripts activated during competence within three major regulons and revealed a core set of 27 panstreptococcal competence-activated genes within the SigX regulon.

Genome editing by natural genetic transformation in Streptococcus mutans.

Classical mutagenesis strategies using selective markers linked to designed mutations are powerful and widely applicable tools for targeted mutagenesis via natural genetic transformation in bacteria and archaea. However, the markers that confer power are also potentially problematic as they can be cumbersome, risk phenotypic effects of the inserted genes, and accumulate as unwanted genes during successive mutagenesis cycles. Alternative mutagenesis strategies use temporary plasmid or cassette insertions and can in principle achieve equally flexible mutation designs, but design of suitable counter-selected markers can be complex. All these drawbacks are eased by use of direct genome editing. Here we describe a strategy for directly editing the genome of S. mutans, which is applied to the widely studied reference strain UA159 (ATCC 700610) and has the advantage of extreme simplicity, requiring construction of only one synthetic donor amplicon and a single transformation step, followed by a simple PCR screen among a few dozen clones to identify the desired mutant. The donor amplicon carries the mutant sequence and extensive flanking segments of homology, which ensure efficient and precise integration by the recombination machinery specific to competent cells. The recipients are highly competent cells, in a state achieved by treatment with a synthetic competence pheromone.

Sequencing of the ß-tubulin genes in the ascarid nematodes Parascaris equorum and Ascaridia galli.

Benzimidazoles (BZ) are used to control infections of the equine roundworm Parascaris equorum and the poultry roundworm Ascaridia galli. There are still no reports of anthelmintic resistance (AR) to BZ in these two nematodes, although AR to BZ is widespread in several other veterinary parasites. Several single nucleotide polymorphisms (SNP) in the ß-tubulin genes have been associated with BZ-resistance. In the present study we have sequenced ß-tubulin genes: isotype 1 and isotype 2 of P. equorum and isotype 1 of A. galli. Phylogenetic analysis of all currently known isotypes showed that the Nematoda has more diversity among the ß-tubulin genes than the Vertebrata. In addition, this diversity is arranged in a more complex pattern of isotypes. Phylogenetically, the A. galli sequence and one of the P. equorum sequences clustered with the known Ascaridoidea isotype 1 sequences, while the other P. equorum sequence did not cluster with any other ß-tubulin sequences. We therefore conclude that this is a previously unreported isotype 2. The ß-tubulin gene sequences were used to develop a PCR for genotyping SNP in codons 167, 198 and 200. No SNP was observed despite sequencing 95 and 100 individual adult worms of P. equorum and A. galli, respectively. Given the diversity of isotype patterns among nematodes, it is likely that associations of genetic data with BZ-resistance cannot be generalised from one taxonomic group to another.

Population genetics of Parascaris equorum based on DNA fingerprinting.

The large roundworm of horses, Parascaris equorum is considered ubiquitous in breeding operations, and is regarded as a most important helminth pathogen of foals. Over the past decade, this parasite has been reported increasingly resistant to anthelmintic drugs worldwide. This paper reports analysis of the population genetic structure of P. equorum. Adult parasites (n=194) collected from Sweden, Norway, Iceland, Germany, Brazil and the USA were investigated by amplified restriction fragment length polymorphism (AFLP) analysis. The genetic variation was low (Hj=0.12-0.4), for the global population of worms. This was accompanied by a weak degree of population structure (Fst=0.2), low gene flow (Nm=1.0) and low mutation rate (4 Nµ=0.07). Thus, the low genetic diversity is probably a result of a low mutation rate in DNA, although the gene flow (due to global movement of horses) is large enough to allow the spread of novel mutations. Surprisingly, isolates from Icelandic horses were not found to be different from other isolates, in spite of the fact that these have been isolated for thousands of years. The study indicates that the global P. equorum population is essentially homogenous, and continents do not appear to be strong barriers for the population structure of this species. Consequently, the potential spread of rare anthelmintic resistance genes may be rapid in a homogenous population.

Simultaneous repair of two large cranial defects using rapid prototyping and custom computer-designed titanium plates: a case report.

Custom titanium cranioplasty plates, manufactured by a variety of techniques, have been used to repair a range of cranial defects. The authors present a case where two relatively large, adjacent cranial defects were repaired by custom computer-designed titanium plates. The two plates were designed and fabricated simultaneously using a unique methodology. A 28-year-old woman underwent a corpus callosotomy for medically intractable epilepsy. The surgery was complicated by unexpected haemorrhage which necessitated a second craniotomy. Subsequent deep infection required the removal of bilateral bone flaps, presenting a challenge in the reconstruction of extensive, bilateral but asymmetrical cranial defects. The patient underwent a head computed tomography scan, from which a rapid-prototype model of the skull was produced. The surfaces for the missing cranial segments were generated virtually using a combination of software products and two titanium plates that followed these virtual contours were manufactured to cover the defects. The cranioplasty procedure to implant both titanium cranial plates was performed efficiently with no intra-operative complications. Intra-operatively, an excellent fit was achieved. The careful planning of the plates enhanced the relative ease with which the cranial defects were repaired with an excellent cosmetic outcome.

Prospects for elucidating the phylogeny of the Apicomplexa.

The Apicomplexa is traditionally considered to contain four clearly defined groups, but the phylogenetic relationships among these groups, and their subsequent taxonomic arrangement, are currently unclear. Here, I consider five of the important criteria that need to be met before radical taxonomic changes are made, in relation to phylogenetic analyses. Unfortunately, at least four of these criteria indicate that the prospects for elucidating the phylogeny and taxonomy of the Apicomplexa are not good in the short term. A lot more pertinent data need to be collected, both in terms of taxa sampled and genes sequenced, before any concordance among phylogenies is likely to emerge; and someone (e.g. a consortium) needs to coordinate the active collection and analysis of these data if any worthwhile progress is to be made.

Characterization of bovine lymphocytes stimulated in vitro by Dictyocaulus viviparus homogenate.

Adult Dictyocaulus viviparus homogenate induced proliferation of lymphocytes from naïve cattle. We characterized the responding cells by carboxyfluorescein diacetate succinimidyl ester (CFSE) loading, for detection of proliferation, and antibody labelling for cell surface molecules. Lymphocytes expressing CD4, CD8 and gamma/delta TCR, rather than Ig expressing cells, proliferated after in vitro stimulation with D. viviparus homogenate. Of gamma/delta TCR expressing cells, both CD8, WC1.1 and WC1.2 co-expressing cells proliferated. Moreover, gamma/delta T cells expressing MHC class II proliferated to a higher extent than those negative for MHC class II. Of CD4 and CD8 expressing lymphocytes, both those co-expressing CD45R and CD45R0 proliferated. Among CD4 expressing lymphocytes, those that were CD45R0 positive had a larger proportion of proliferated cells than did CD45R positive cells. Compared to stimulation with Con A, the proportion of dividing cells after D. viviparus stimulation was smaller although the cells had divided more times. Furthermore, we also compared in vitro responses of peripheral blood mononuclear cells collected before and after two subsequent infections with D. viviparus, but no clear acquired responses could be detected. Overall, this suggests that most T lymphocytes are stimulated by the D. viviparus homogenate rather than any particular lymphocyte subpopulation.

Genetic differences in internal transcribed spacer 1 between Dermanyssus gallinae from wild birds and domestic chickens.

We investigated the presence of the poultry red mite or the chicken mite, Dermanyssus gallinae De Geer, Acari: Dermanyssidae, in wild bird populations in four different geographical regions of Sweden. The mites identified as D. gallinae were compared genetically with D. gallinae from egg-producing poultry farms in the same regions. The small subunit (SSU) gene, the 5.8S ribosomal RNA (rRNA) gene and the two internal transcribed spacers (ITS) of the rRNA genes were used in the genetic analysis. All D. gallinae mites had identical SSU rRNA, 5.8S rRNA and ITS2 sequences independent of their origin. By contrast, we identified significant differences in the ITS1 sequences. Based on the differences in the ITS1 sequences, the mites could be divided into two genotypes, of wild and domesticated origin, with no variation within the groups. These results imply that wild bird populations are of low importance, if any, as natural reservoirs of D. gallinae in these four geographical regions of Sweden.

Emergency percutaneous coronary intervention (PCI) for the care of patients with ST-elevation myocardial infarction (STEMI).

There is general consensus that emergency percutaneous coronary intervention (PCI) is the preferred treatment for patients with ST-elevation myocardial infarction (STEMI), so long as it can be delivered in a timely fashion, by an experienced' operator and cardiac catheterization laboratory (CCL) team. STEMI is both a functional and structural issue. Although it has been recognized since the work of pioneering cardiologists and surgeons in Spokane, Washington, that approximately 88% of patients presenting within 6 hours of onset of STEMI have an occluded coronary artery, it is the pathophysiology of myocardial necrosis, and the varied consequences of necrosis that characterize STEMI. Accordingly, experience' of both primary operator and cardiac catheterization laboratory (CCL) crew, in performing an emergency PCI for STEMI, are as much a function of experience with the treatment of complex MI patients, as experience with coronary intervention. Rapidly achieving normal coronary artery flow, at both the macro and micro vascular levels, is the recognized key to aborting the otherwise progressive wavefront' of myocardial necrosis. The time urgency of decisions (Time is muscle') make emergency PCI for patients with on-going necrosis, more like emergency room (ER) care, than like most in-hospital or outpatient care. In general, most patients with acute coronary syndromes (ACS) are currently thought to have plaque rupture and/or erosion with subsequent thrombosis and embolization. Consequences of thrombo-embolism, such as slow flow' or no-reflow' are in addition to, the structural (anatomic) considerations of PCI in stable patients (such as ostial location; bifurcation involvement; heavy calcification; tortuosity of lesion or access to it; length of disease; caliber of infarct-artery; etc.). Good quality studies have provided strong support for the specific added value of glycoprotein IIb/IIIa inhibitors (especially abciximab), dual antiplatelet therapy (the addition of the thienopyridine, clopidogrel, to aspirin use), and bare-metal stents (BMS), for a broad range of STEMI patients. The added value of drug-eluting stents (DES) to bare-metal stents (BMS), primarily in terms of reducing restenosis and repeat revascularization, is supported by several randomized trials, and a number of registries, despite its being off-label' from a regulatory standpoint. The recognition of late stent thrombosis (LST) has raised additional issues, in choosing between these two options for specific STEMI patients. The added value of a number of other mechanical approaches to coronary thrombus, such as thrombus removal devices, and/or distal protection, are more controversial, and perhaps, patient specific. Whether intravascular ultrasound guidance (IVUS) for stent use should be used for the majority, or even a specific minority, of STEMI patients, is also controversial; late-stent thrombosis provides a counter-point. The advantages of developing a network approach to STEMI care, so as to optimize the number of patients receiving timely reperfusion, have been demonstrated in Prague, Denmark, and Minneapolis, among many places. The benefits of both bivalirudin (anti-thrombin drug with efficacy against clot-bound thrombin, which does not appear to stimulate platelets) and abciximab (glycoprotein IIb/IIIa inhibitor which is antibody to platelet receptors), as PCI adjuncts generally, and for STEMI patients, in particular, are supported by multiple trials. The specific choice of administering the bolus dose of either, or both, drugs via intra-coronary (IC) injection follows the precedents' of IC thrombolytics, and IC small-vessel vasodilators for no-reflow', but it has not been tested by prospective, randomized trials. Although rapid reperfusion is the first objective, one cannot ignore the other components of the oxygen delivery chain, and the importance of each of these components to on-going delivery of oxygen to all vital organs. A balance must be struck between doing those control' things which serve to stabilize other vital components of the oxygen-delivery chain, without digressing too long from the job of re-establishing brisk coronary flow. The clinical and angiographic heterogeneity of STEMI patients and the array of available therapeutic approaches make it impossible to obtain specific randomized trial direction for many of the clinical decisions in an individual emergency PCI for STEMI. There are a range of reasonable/ appropriate therapeutic choices for a given emergent PCI performed by multiple experienced and competent operators. The treatment of STEMI, and high-risk non-STEMI, patients, by means of emergent PCI, is among the most challenging and rewarding arenas in contemporary medicine.

Biological control of sheep parasites using Duddingtonia flagrans: trials on commercial farms in Sweden.

Trials were conducted on 3 commercial sheep farms in Sweden to assess the effect of administering spores of the nematode trapping fungus, Duddingtonia flagrans, together with supplementary feed to lactating ewes for the first 6 weeks from turn-out on pastures in spring. Also control groups of ewes, receiving only feed supplement, were established on all 3 farms. Groups were monitored by intensive parasitological investigation. The ewes and their lambs were moved in late June to saved pastures for summer grazing, the lambs receiving an anthelmintic treatment at this time. After approximately 6 weeks on summer pasture the lambs were weaned, treated a second time with anthelmintic, and returned to their original lambing pastures for finishing. Decisions as to when lambs were to be marketed were entirely at the discretion of the farmer co-operators. No difference in lamb performance was found between the two treatments on all three farms. This was attributed to the high levels of nutrition initially of the ewes limiting their post-partum rise in nematode faecal egg counts in spring, which in turn resulted in low levels of nematode infection on pastures throughout the autumn period. Additionally, pastures were of good quality for the lambs during the finishing period, so they grew at optimal rates as far as the farmers were concerned.

Population genetics of the bovine/cattle lungworm (Dictyocaulus viviparus) based on mtDNA and AFLP marker techniques.

Mitochondrial DNA (mtDNA) sequence data and amplified fragment length polymorphism (AFLP) patterns were compared for the lungworm Dictyocaulus viviparus, a nematode parasite of cattle. Eight individual D. viviparus samples from each of 8 herds in Sweden and 1 laboratory isolate were analysed, with the aim of describing the diversity and genetic structure in populations using different genetic markers on exactly the same DNA samples. There was qualitative agreement between the whole-genome AFLP data and the mtDNA sequence data, both indicating relatively strong genetic differentiation among the Swedish farms. However, the AFLP data detected much more genetic variation than did the mtDNA data, even after allowing for the different inheritance patterns of the markers, and indicated that there was much less differentiation among the populations. The mtDNA data therefore seemed to be more informative about the most recent history of the parasite populations, as the general patterns were less obscured by detailed inter-relationships among individual worms. The 4 mtDNA genes sequenced (1542 bp) showed consistent patterns, although there was more genetic variation in the protein-coding genes than in the structural RNA genes. Furthermore, there appeared to be at least 3 distinct genetic groups of D. viviparus infecting Swedish cattle, 1 of which was predominant and showed considerable differentiation between farms, but not necessarily within farms. Second, the 2 smaller genetic groups occurred on farms where the predominant group also occurred, suggesting that these farms have had multiple introductions of D. viviparus.

Characterization of an atypical antigen from Sarcoptes scabiei containing an MADF domain.

We have cloned a cDNA encoding a novel antigen from a Sarcoptes scabiei (Acari) cDNA library by immunoscreening with sera from S. scabiei-infected dogs. The antigen is encoded by a 2,157 bp mRNA with a predicted open reading frame of 719 amino acids (molecular weight 79 kDa). Our sequence analysis identified the presence of a MADF domain in the N-terminus, and downstream of this domain there was a region of low sequence complexity. This latter region contained several blocks of triplets and quadruplets of polar amino acids (Asn, Gln and Ser), and these 3 amino acids represented 39.7% of all amino acids. The antigen was named Atypical Sarcoptes Antigen 1 (ASA1) since the MADF domain normally is found in proteins involved in transcriptional regulation. In addition, 15 out of 62 S. scabiei-infected dogs reacted with a purified recombinant version of ASA1 in Western blot analysis. With immunohistochemistry we could show that ASA1 is expressed throughout the parasite, and that IgG specific for ASA1 binds to the inside wall of the mite's burrow. To our knowledge, this is the first description of an antigen containing an MADF domain.

How old are the extant lineages of Toxoplasma gondii?

Most known isolates of Toxoplasma gondii belong to one of only three lineages, which are presumed to be clonal. Three models have been proposed for the evolutionary relationship of these lineages to the other extant lineages: Model (a) proposing that all lineages are derived from a most recent common ancestor (MRCA) in the distant past, Model (b) that all lineages are derived from a MRCA in the very recent past, and Model (c) that the clonal lineages share a recent MRCA but are related to the other lineages only in the distant past. Here, I test these models using DNA intron and coding-sequence data for loci at 14 genes, using three different methods to calculate the time of the MRCA. All of the calculations agree that the MRCA of the clonal lineages was > 70% of the age of the MRCA of all lineages, thus favouring Model (a). The MRCA may have existed approximately 150,000 years ago, with the clonal lineages expanding in prevalence approximately 10,000 years ago.

Multivessel percutaneous coronary intervention: a new paradigm for a new century.

Percutaneous coronary intervention (PCI) was introduced in 1977, to treat single, discrete lesions in patients with stable symptoms, and favorable anatomy. Increased operator experience and numerous technical advances, notably including bare-metal stents (BMS) and then drug-eluting stents (DES), and a number of adjunctive pharmacologic modalities, have allowed for the gradual application of PCI to far more diverse clinical and anatomic subsets. The proper role of PCI, compared with both medical therapy and coronary artery bypass graft (CABG) surgery, is evolving. This manuscript is synthesized from literature review of randomized clinical trials, and some cohort investigations, and 21 years of PCI experience, which has been focused on high-risk patients. Current American College of Cardiology/ American Heart Association/ Society for Cardiac Angiography and Interven-tions (ACC/AHA/ SCA&I) Guidelines, and most research, are based upon a perspective (conventional paradigm') that categorizes patients primarily based upon number of major' coronary (left anterior descending artery, circumflex and right) arteries with a >70% stenosis, and whether left ventricular function is normal or abnormal (left ventricular ejection fraction < or >0.50). This paradigm developed when: a) CABG was the only revascularization option, and b) medical therapy was quite limited. All of the trials demonstrating survival benefit with aspirin, clopidogrel, statins, angiotensin-converting-enzyme inhibition (ACE-I) or angiotensin receptor blockers (ARB), and beta-blockers; coupled with the PCI versus thrombolytics trials in ST-elevation myocardial infarction (STEMI), and non-STEMI strategy trials, both of which include revascularization almost exclusively by PCI, make this conventional paradigm, outdated and counterproductive. Attempts to compare CABG and PCI have de-emphasized their very different advantages and disadvantages. The new paradigm makes major division of patients based upon whether patients have stable or unstable: a) ischemia and b) hemodynamics, and c) whether they are having acute myocardial infarction (MI). The first issue to be settled is whether the patient is likely to benefit from revascularization. If little or no benefit can be envisioned, the patient should be managed medically. Unstable ischemia and unstable hemodynamics, and acute MI, all favor emergent or urgent revascularization. Clinical features, which generally favor PCI for revascularization, include hemodynamic instability, STEMI and non-STEMI; and severe comorbidity, particularly cerebral, pulmonary, or hepatic comorbidity. Anatomic features, which generally favor CABG, include unprotected left main stenosis, especially involving the bifurcation, one or more graftable chronic total occlusions (CTO), and bifurcation disease with large important side-branches. Old age and severely reduced left ventricular function are associated with higher risks, with either CABG or PCI. Small caliber and diffusely diseased vessels may imply lower expectation of success, by either CABG or PCI.

Testing the hypothesis of recent population expansions in nematode parasites of human-associated hosts.

It has been predicted that parasites of human-associated organisms (eg humans, domestic pets, farm animals, agricultural and silvicultural plants) are more likely to show rapid recent population expansions than are parasites of other hosts. Here, we directly test the generality of this demographic prediction for species of parasitic nematodes that currently have mitochondrial sequence data available in the literature or the public-access genetic databases. Of the 23 host/parasite combinations analysed, there are seven human-associated parasite species with expanding populations and three without, and there are three non-human-associated parasite species with expanding populations and 10 without. This statistically significant pattern confirms the prediction. However, it is likely that the situation is more complicated than the simple hypothesis test suggests, and those species that do not fit the predicted general pattern provide interesting insights into other evolutionary processes that influence the historical population genetics of host-parasite relationships. These processes include the effects of postglacial migrations, evolutionary relationships and possibly life-history characteristics. Furthermore, the analysis highlights the limitations of this form of bioinformatic data-mining, in comparison to controlled experimental hypothesis tests.

Genetic diversity assessed by amplified fragment length polymorphism analysis of the parasitic nematode Dictyocaulus viviparus the lungworm of cattle.

We have examined the population genetic structure in a collection of nine isolates of the parasitic lungworm Dictyocaulus viviparus. Eight of the isolates were sampled from cattle in geographically separated farms throughout south-central Sweden, and one isolate was a laboratory strain that has been maintained in experimentally infected calves for almost four decades. A total of 72 worms were examined, with eight individual worms from the same individual host representing each isolate. The genetic variation as revealed by amplified fragment length polymorphism analysis using four selective primer combinations was high. Depending on the primer combination a total of 66-79 restriction fragments were amplified, with 26-44 peaks of similar complexity from each of the isolates. The heterozygosity within populations was relatively small, as were the population mutation and immigration rates, which seemed to be in neutral equilibrium. The genetic diversity was therefore reasonably well structured in the field; and the laboratory isolate was quite distinct from the field samples. There was no relationship between the patterns of genetic diversity and the geographical proximity of the farms. The estimates of heterozygosity were much larger and more consistent than those previously estimated for this nematode species using mitochondrial sequencing, and the genetic structuring was thus much less pronounced and the gene flow greater. We attribute these differences in estimation to the broader sampling of loci available using amplified fragment length polymorphism markers, which may therefore constitute a superior technique for the study of patterns of lungworm diversity. Furthermore, the data estimating gene flow for D. viviparus was less than previously reported for closely related species in North America. This might be related to different rates of movements of infected hosts. It seems likely that lungworm infections are rather persistent on different farms, and the sudden outbreaks of disease that can be observed with host movements are most likely to be related to the introduction of susceptible stock.