Multi-locus sequence types of Campylobacter carried by flies and slugs acquired from local ruminant faeces.

AIMS: To assess whether flies and slugs acquire strains of Campylobacter jejuni and Campylobacter coli present in local ruminant faeces. METHODS AND RESULTS: Campylobacter was cultured from flies, slugs and ruminant faeces that were collected from a single farm in Scotland over a 19-week period. The isolates were typed using multi-locus sequence typing (MLST) and compared with isolates from cattle and sheep faeces. Campylobacter jejuni and Camp. coli were isolated from 5·8% (n=155, average of 75 flies per pool) and 13·3% (n=15, average of 8·5 slugs per pool) of pooled fly and slug samples, respectively. The most common sequence type (ST) in flies was Camp. coli ST-962 (approx. 40%) regardless of the prevalence in local cattle (2·3%) or sheep (25·0%) faeces. Two positive slug pools generated the same ST that has not been reported elsewhere. CONCLUSIONS: Despite their low carriage rate, flies are able to acquire Campylobacter STs that are locally present, although the subset carried may be biased when compared to local source. Slugs were shown to carry a previously unreported Campylobacter ST. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has demonstrated that flies carry viable Campylobacter and may contribute to the transfer of STs within and between groups of animals on farms. Further, they may therefore present a risk to human health via their contact with ready-to-eat foods or surfaces.

Campylobacter immunity and coinfection following a large outbreak in a farming community.

An outbreak of campylobacteriosis affected approximately one-half of 165 people attending an annual farmers' dance in Montrose, Scotland, in November 2005. Epidemiological investigations, including a cohort study (n = 164), identified chicken liver paté as the most likely vehicle of infection. Paté preparation involved deliberate undercooking of chicken livers by flash-frying, followed by mechanical homogenization. Typing of 32 Campylobacter strains (isolated from submitted stools) by multilocus sequence typing identified four distinct clades of Campylobacter jejuni. There was good agreement when isolates were typed by Penner serotyping, pulsed-field gel electrophoresis, and flaA short variable region sequencing but poorer agreement with phage and antibiotic susceptibility testing. At least three attendees were coinfected with two Campylobacter strains each. The outbreak was probably due to several livers contributing Campylobacter strains that survived undercooking and were dispersed throughout the paté. The study highlights improper culinary procedures as a potential human health risk and provides a striking counterexample to the "dominant outbreak strain" view of point source outbreaks of food-borne infections. It also demonstrates that previous exposure to biologically plausible sources of Campylobacter may confer protection against subsequent infection.

Experimental infection studies of UK Culicoides species midges with bluetongue virus serotypes 8 and 9.

This paper describes a rapid, standardised method for testing the susceptibility to bluetongue virus (BTV) of northern Palaearctic Culicoides species midges that can be used to assess the competence of both field-caught and laboratory-infected midges. The method has been used to show that Culicoides scoticus can replicate btv serotype 8 and BTV serotype 9 strains to more than 3 log(10) TCID50/midge, the first evidence of the potential of this species to transmit BTV.

Incursion and range expansion in the bluetongue vector Culicoides imicola in the Mediterranean basin: a phylogeographic analysis.

The bluetongue (BT) vector Culicoides imicola Kieffer (Diptera: Ceratopogonidae) has undergone widespread range expansion across most of the Mediterranean basin, concomitant with the largest BT epizootic outbreaks on record. Knowledge of the substructure of this vector expansion would be useful for identifying specific source-expansion systems. To this end we analysed the haplotype diversity of the mitochondrial cytochrome oxidase I gene in 273 C. imicola from 88 Mediterranean sites and outgroups. All the C. imicola haplotypes (n = 26) formed a single, distinct clade in comparison with haplotypes of four other species of the Imicola group from southern Africa, confirming C. imicola as a single phylospecies. Haplotype distribution showed extreme differentiation across the Mediterranean basin, with four common haplotypes each predominating in different areas. Eastern and western areas characterized by distinct BT incursions accounted for most of the molecular variance in haplotype composition. Shared common haplotypes identified one area of incursion and expansion encompassing the western half of the Mediterranean basin, with evidence of population growth, and another system encompassing Anatolian Turkey, the Aegean Islands and mainland Greece. A third area of range expansion was identified in the central Mediterranean, with a possible source in Algeria and unsampled parts of central North Africa. We conclude that the expansion of C. imicola in the Mediterranean basin consists of at least three incursions followed by expansions and that the western system experiences conditions promoting high population growth.

Widespread gene flow and high genetic variability in populations of water voles Arvicola terrestris in patchy habitats.

Theory predicts that the impact of gene flow on the genetic structure of populations in patchy habitats depends on its scale and the demographic attributes of demes (e.g. local colony sizes and timing of reproduction), but empirical evidence is scarce. We inferred the impact of gene flow on genetic structure among populations of water voles Arvicola terrestris that differed in average colony sizes, population turnover and degree of patchiness. Colonies typically consisted of few reproducing adults and several juveniles. Twelve polymorphic microsatellite DNA loci were examined. Levels of individual genetic variability in all areas were high (H(O) = 0.69-0.78). Assignments of juveniles to parents revealed frequent dispersal over long distances. The populations showed negative F(IS) values among juveniles, F(IS) values around zero among adults, high F(ST) values among colonies for juveniles, and moderate, often insignificant, F(ST) values for parents. We inferred that excess heterozygosity within colonies reflected the few individuals dispersing from a large area to form discrete breeding colonies. Thus pre-breeding dispersal followed by rapid reproduction results in a seasonal increase in differentiation due to local family groups. Genetic variation was as high in low-density populations in patchy habitats as in populations in continuous habitats used for comparison. In contrast to most theoretical predictions, we found that populations living in patchy habitats can maintain high levels of genetic variability when only a few adults contribute to breeding in each colony, when the variance of reproductive success among colonies is likely to be low, and when dispersal between colonies exceeds nearest-neighbour distances.

Sex ratio variation in gastrointestinal nematodes of Svalbard reindeer; density dependence and implications for estimates of species composition.

Estimates of the intensity and abundance of species provide essential data for ecological, evolutionary and epidemiological studies of gastrointestinal nematode communities. These estimates are typically derived from the species composition of adult males when only males have readily scorable species-specific morphological traits. Such estimation assumes that all species in the community have the same adult sex ratio. We evaluated this assumption for the trichostrongyle nematodes Ostertagia gruehneri and Marshallagia marshalli in infracommunities in Svalbard reindeer by identifying to species adult females using a polymerase chain reaction assay. The proportion of males was found to be slightly higher in O. gruehneri than in M. marshalli. Evidence for seasonal variation and density dependence in the adult sex ratio was only found for O. gruehneri. Possible demographic mechanisms for such sex ratio variation are discussed, and stochastic models that generate density-dependent sex ratios proposed. Sex ratio variation caused substantial bias in some male-based estimates of intensity of infection, while substantial and consistent bias in estimates of abundances was only evident in late winter samples. Our results suggest that estimating sex ratios can be particularly important in individual host level studies of nematode species of low abundance.

The tobacco mouse and its relatives: a "tail" of coat colors, chromosomes, hybridization and speciation.

The article reviews over 30 years' study of the chromosomal variation of the western house mice (Mus musculus domesticus) from the neighboring valleys of Poschiavo and Valtellina on the Swiss-Italian border. This is done in the context of the social and political history of this area, on the grounds that mice, as commensals, are influenced by human history. The chromosomal study of mice in this area was initiated because their unusual black coat color led a 19th century naturalist to describe the "tobacco mice" from Val Poschiavo as a separate species (Mus poschiavinus). The special coloration of the Val Poschiavo mice is matched by their chromosomes: they have 26 chromosomes instead of the usual 40. The Val Poschiavo mice are not a separate species according to the Biological Species Concept; instead they constitute a chromosome race (the "Poschiavo", POS) that is related to other races with reduced chromosome numbers that occur in N Italy (of which only those races in Val Poschiavo and Upper Valtellina have black coats). A phylogenetic analysis of mitochondrial DNA sequences suggests that the lineage of chromosome races found in N Italy was not formed during an extreme population bottleneck, although such bottlenecks have apparently occurred during the origin of individual races and certainly have influenced single populations. In one small, isolated population in Valtellina (Migiondo), two chromosome races (the POS and the "Upper Valtellina", UV, 2n = 24) became reproductively isolated from each other. In another small population (Sernio) bottlenecking led to fixation of a hybrid form with the UV karyotype and coat color, but with allozyme and microsatellite alleles characteristic of mice with the standard 40-chromosome karyotype. Two of the chromosome races in Valtellina (the UV and the "Mid Valtellina", MV, 2n = 24) also appear to be the product of hybridization. The dynamic history and patchy distribution of the house mouse chromosome races in Val Poschiavo and Valtellina in part reflects extinction-recolonization events; the formation of the UV and MV races and the introduction of the pale brown Standard race mice are believed to reflect such events. Dynamism in the chromosomal constitution of single populations is also evident from 25 years of data on the population in Migiondo. Due to change in agricultural practices, house mice in Valtellina and Val Poschiavo are becoming rarer, which is likely to have further impacts on the distribution and characteristics of the chromosome races in this area.

Molecular taxonomy and population structure of a Culicoides midge vector.

The biting midge Culicoides imicola Kieffer (Diptera: Ceratopogonidae) is the major Old World vector of the arboviruses that cause African horse sickness (AHS) and bluetongue (BT). Recently, the incidence and geographical scales of AHS and BT outbreaks in the Mediterranean Basin have increased, with serotype distribution in the BT outbreaks being geographically structured. The authors review molecular approaches for assessing the contribution of cryptic species and population subdivision in C. imicola to BT serotype structure in this region. No evidence was found for cryptic species. In contrast, evidence was found for marked matrilineal subdivision between the eastern and western parts of the Mediterranean Basin. This pattern is comparable to the geographic structure of BT serotypes, suggesting that subdivision in the insect vector potentially constrains serotype spread. The authors are presently testing this hypothesis.

Phylogenetic status and matrilineal structure of the biting midge, Culicoides imicola, in Portugal, Rhodes and Israel.

The biting midge Culicoides imicola Kieffer (Diptera: Ceratopogonidae) is the most important Old World vector of African horse sickness (AHS) and bluetongue (BT). Recent increases of BT incidence in the Mediterranean basin are attributed to its increased abundance and distribution. The phylogenetic status and genetic structure of C. imicola in this region are unknown, despite the importance of these aspects for BT epidemiology in the North American BT vector. In this study, analyses of partial mitochondrial cytochrome oxidase subunit I gene (COI) sequences were used to infer phylogenetic relationships among 50 C. imicola from Portugal, Rhodes, Israel, and South Africa and four other species of the Imicola Complex from southern Africa, and to estimate levels of matrilineal subdivision in C. imicola between Portugal and Israel. Eleven haplotypes were detected in C. imicola, and these formed one well-supported clade in maximum likelihood and Bayesian trees implying that the C. imicola samples comprise one phylogenetic species. Molecular variance was distributed mainly between Portugal and Israel, with no haplotypes shared between these countries, suggesting that female-mediated gene flow at this scale has been either limited or non-existent. Our results provide phylogenetic evidence that C. imicola in the study areas are potentially competent AHS and BT vectors. The geographical structure of the C. imicola COI haplotypes was concordant with that of BT virus serotypes in recent BT outbreaks in the Mediterranean basin, suggesting that population subdivision in its vector can impose spatial constraints on BT virus transmission.

Parentage assignment detects frequent and large-scale dispersal in water voles.

Estimating the rate and scale of dispersal is essential for predicting the dynamics of fragmented populations, yet empirical estimates are typically imprecise and often negatively biased. We maximized detection of dispersal events between small, subdivided populations of water voles (Arvicola terrestris) using a novel method that combined direct capture-mark-recapture with microsatellite genotyping to identify parents and offspring in different populations and hence infer dispersal. We validated the method using individuals known from trapping data to have dispersed between populations. Local populations were linked by high rates of juvenile dispersal but much lower levels of adult dispersal. In the spring breeding population, 19% of females and 33% of males had left their natal population of the previous year. The average interpopulation dispersal distance was 1.8 km (range 0.3-5.2 km). Overall, patterns of dispersal fitted a negative exponential function. Information from genotyping increased the estimated rate and scale of dispersal by three- and twofold, respectively, and hence represents a powerful tool to provide more realistic estimates of dispersal parameters.

Microsatellite DNA evidence for genetic drift and philopatry in Svalbard reindeer.

Mainland populations of Arctic reindeer and caribou Rangifer tarandus often undergo extensive movements, whereas populations on islands tend to be isolated and sedentary. To characterize the genetic consequences of this difference, levels of genetic diversity and subdivision of Svalbard reindeer (R. t. platyrhynchus) from two adjacent areas on Nordenskjiöldland, Spitsbergen were estimated using data from up to 14 microsatellites. The mean number of alleles per locus in Svalbard reindeer was 2.4 and mean expected heterozygosity per locus was 0.36. The latter value was significantly lower than in Canadian caribou and Norwegian reindeer but higher than in some other cervid species. Large samples of females (n = 743) and small samples of males (n = 38) from two sites approximately 45 km apart showed genetic subdivision, which could be due to local population fluctuations or limited gene flow. To our knowledge, this is the first study to report significant differentiation at microsatellite loci in Rangifer at such short geographical distances. Neither population showed genetic evidence for recent population bottlenecks when loci unbiased with respect to heterozygosity were analysed. In contrast, false signals of a recent bottleneck were detected when loci upwardly biased with respect to heterozygosity were analysed. Thus, Svalbard reindeer appeared to conform to the paradigm of island populations made genetically depauperate by genetic drift.

Efficient polymerase chain reaction detection of the second internal transcribed spacer of mucosa-derived larvae is dependent on the larval extraction method.

Methods for estimating abundance of arrested gastrointestinal larvae in large mammal hosts by digestion of the gastrointestinal mucosa are well established. The effects of digestion on the success of species identification using the polymerase chain reaction (PCR) are, however, unknown. In this study, the relationship between numerical recovery of arrested larvae and the success of PCR-typing for the second internal transcribed spacer of ribosomal genes was characterized. Fresh and prefrozen mucosa of 4 sheep yielded very similar rates of recovery and PCR detection. When sheep mucosa were digested with neutral N-acetyl cysteine, recovery increased, whereas PCR detection remained constant (60-80%) with digest duration (1-16 hr). In contrast, when sheep and Svalbard reindeer mucosa were digested with acid-pepsin, recovery increased, whereas PCR detection declined to 0 with digest duration. Thus, to optimize recovery and PCR analysis of arrested gastrointestinal nematode larvae, acid-pepsin digestion of 1-2 hr for PCR detection and 16 hr for recovery, or neutral N-acetyl cysteine digestion of 8-16 hr for both assays, should be used.

Phylogenetic analysis of the mitochondrial cytochrome oxidase subunit I gene of five species of the Culicoides imicola species complex.

The phylogenetic status of members of the Culicoides imicola Kieffer (Diptera: Ceratopogonidae) species complex of haematophagous midges is unknown, and simple means to identify the members using all life stages are unavailable. In this study, the status of three confirmed (C. imicola s.s., C. bolitinos Meiswinkel and C. loxodontis Meiswinkel) and two provisional (C. tuttifrutti Meiswinkel and C. kwagga Meiswinkel) members of the complex from South Africa was assessed using phylogenetic analysis of partial DNA and amino acid sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. The four or five individuals of each species analysed contained one or two haplotypes each. Interspecific divergence was significant and characterized by strong A <--> T transversion bias. Phylogenetic trees constructed using neighbour-joining, parsimony and maximum likelihood showed each species to be distinct. Combinations of sites for two restriction enzymes in the COI sequences were species-specific and could form the basis of a diagnostic PCR assay.

DNA evidence that Marshallagia marshalli Ransom, 1907 and M. occidentalis Ransom, 1907 (Nematoda: Ostertagiinae) from Svalbard reindeer are conspecific.

The gastro-intestinal parasitic nematodes of ruminants Marshallagia marshalli and M. occidentalis are morphs of a single species according to indirect evidence. In this study, their taxonomic status and molecular identification were assessed more directly in isolates from the abomasal nematode community of Svalbard reindeer using genetic data. DNA sequences of the first and second internal transcribed spacers of nuclear ribosomal RNA genes were obtained from individual nematodes by the polymerase chain reaction (PCR). Both taxa contained virtually identical sequences of each ITS and shared most of the polymorphisms detected. A PCR assay based on ITS-2 sequences previously developed to identify M. marshalli and Ostertagia gruehneri, the second common species in this community, gave identical results for M. marshalli and M. occidentalis. Genetic data thus confirmed that M. marshalli and M. occidentalis are conspecific.

Contrasting regulation of fecundity in two abomasal nematodes of Svalbard reindeer (Rangifer tarandus platyrhynchus).

Stability of trichostrogylid populations indicates that some form of density-dependent regulation occurs which could act through fecundity. We present evidence for intraspecific density-dependent effects in 1 of 2, dominant, abomasal nematodes species (Ostertagia gruehneri) of Svalbard reindeer (Rangifer tarandus platyrhynchus). We found evidence in O. gruehneri, for density-dependent regulation of female worm length in April, July and October 1999. However, it is only in July that female worm length explains the variation in the number of eggs in utero which is also related to egg production per female worm only in this month and not at other times of the year. The seasonal pattern in faecal egg output in this species focuses egg production in the summer months when conditions are favourable to transmission. In contrast, we found no evidence in the other common species (Marshallagia marshalli) for density-dependent regulation of female worm length during or the number of eggs in utero. Faecal egg output in M. marshalli was positively related to worm burden but not to the mean number of eggs in utero. Neither inter-specific interactions nor host body condition appeared to influence worm fecundity. The contrasting patterns of density-dependent regulation of fecundity provides further evidence for divergent life-histories in this nematode community.

Matrilineal genetic structure and female-mediated gene flow in red grouse (Lagopus lagopus scoticus): an analysis using mitochondrial DNA.

DNA sequence variation at the hypervariable 5' end of the mitochondrial control region was examined in 247 individuals to detect genetic divergence among 14 populations of red grouse (Lagopus lagopus scoticus) in northeastern Scotland. Ten haplotypes were resolved, several of which were shared among populations. Analysis of molecular variance, Nei's gamma ST, and a cladistic estimate of the amount of gene flow indicated a lack of overall population differentiation. Patterns of overall panmixia are in stark contrast to previous reports of localized subdivision among the same set of populations detected using hypervariable microsatellite markers. Because grouse cocks are territorial and show extreme natal philopatry and females are the dispersing sex, such discordance could be explained by sex-biased dispersal, with extensive female-mediated gene flow preventing mitochondrial DNA divergence. However, it is difficult to reconcile how effective dispersal of females would not homogenize both mitochondrial and nuclear structure simultaneously. We use a model that examines the spatial and temporal dynamics of diparentally and uniparentally inherited genes to show that, under realistic ecological scenarios and with specific differences in the dispersal of males and females, the local effective size of the nuclear genome can be less than that of the mitochondrial and the patterns of structuring we observe are meaningful.

Identification by polymerase chain reaction (PCR) of Marshallagia marshalli and Ostertagia gruehneri from Svalbard reindeer.

A polymerase chain reaction (PCR) assay to identify two common abomasal nematodes Marshallagia marshalli and Ostertagia gruehneri of Svalbard reindeer was developed. Species-specific PCR primers were designed from internal transcribed spacer (ITS)-2 sequences of rDNA and validated using morphologically identified adult male and female nematodes. Using the species-specific primers, a 110 bp fragment was amplified from M. marshalli and its minor morph Marshallagia occidentalis and a 149 bp fragment was amplified from Ostertagia gruehneri and its minor morph Ostertagia arctica. No PCR products were amplified from the third rare species, Teladorsagia circumcincta, or DNA from the reindeer host. The assay provides a useful tool to estimate species composition for both sexes in this nematode community.

DNA evidence that Ostertagia gruehneri and Ostertagia arctica (Nematoda: ostertagiinae) in reindeer from Norway and Svalbard are conspecific.

DNA sequences of ITS-1 and ITS-2 of rDNA were determined for 16 individual adult males each of Ostertagia gruehneri and Ostertagia arctica from Svalbard reindeer (Rangifer tarandus platyrhynchus) and Eurasian tundra reindeer (R. t. tarandus). Each ITS was virtually identical in O. gruehneri and O. arctica and the three mixed bases detected were shared by both species. Our results strongly suggest that O. gruehneri and O. arctica are dimorphic males of the same species.