Senecio vernalis, we created propagule mixtures of four levels of genotype diversity by combining seeds across remote populations, across proximate populations, within single populations and within seed families. In a first container experiment with constant Festuca rupicola density as matrix, genotype diversity was crossed with three levels of seed density. In a second experiment, we tested for effects of establishment limitation and genotype diversity by manipulating Festuca densities. Increasing genetic diversity had no effects on abundance and biomass of S. vernalis but positively affected the proportion of large individuals to small individuals. Mixtures composed from proximate populations had a significantly higher proportion of large individuals than mixtures composed from within seed families only. High propagule pressure increased emergence and establishment of S. vernalis but had no effect on individual growth performance. Establishment was favoured in containers with Festuca, but performance of surviving seedlings was higher in open soil treatments. For S. vernalis invasion, we found a shift in driving factors from density dependence to effects of genetic diversity across life stages. While initial abundance was mostly linked to the amount of seed input, genetic diversity, in contrast, affected later stages of colonization probably via sampling effects and seemed to contribute to filtering the genotypes that finally grew up. In consequence, when disentangling the mechanistic relationships of genetic diversity, seed density and microsite limitation in colonization of invasive plants, a clear differentiation between initial emergence and subsequent survival to juvenile and adult stages is required.Genetic diversity is supposed to support the colonization success of expanding species, in particular in situations where microsite availability is constrained. Addressing the role of genetic diversity in plant invasion experimentally requires its manipulation independent of propagule pressure. To assess the relative importance of these components for the invasion of  Arabidopsis thaliana, i.e. seedling emergence, biomass, flowering duration and reproduction. However, establishing empirical relationships between invasion success and genetic diversity is not sufficient to demonstrate a mechanistic link Genetic diversity within species and populations has been proven to have profound impacts on ecosystems by affecting community structure, nutrient fluxes and productivity, thereby affecting biotic interactions, such as resistance to disturbance and invasions Taraxacum officinale) invasiveness. They found sampling effects, i.e. the presence or absence of a particular genotype identity, to be more important for explaining invasiveness of dandelions than genotypic diversity per se.Biodiversity experiments in grasslands have shown that diversity effects may comprise complementarity effects, i.e. reduced competition due to niche partitioning Establishing a mechanistic link of genetic diversity in invasions requires accounting for confounding variables, i.e. further factors that affect both genetic diversity and invasion success. Genetic diversity in invading populations is supposed to be induced by multiple introductions and, in particular, by the number of propagules released into a new range or habitat Apart from seed availability, the availability of sites suitable for seedling establishment is another major determinant of recruitment success in plant populations Senecio vernalis Waldst. & Kit. is a suitable study organism to approach these questions experimentally. Senecio vernalis, spring groundsel, is an insect pollinated therophyte, native to West Asia and expanding to northern, central and Western Europe along railways S. vernalis is characterized by the release of large amounts of achenes per individual S. vernalis in Central Europe. Further investigations have provided evidence that populations of S. vernalis in Eastern Germany differ significantly in seed traits and germination characteristics .In the present study, we aim at quantifying the role of genetic diversity during invasions in interaction with propagule pressure at the local scale and microsite availability. S. vernalis. In particular, we tested the hypotheses that i) high propagule pressure favours the establishment of S. vernalis, ii) high levels of microsite availability increase the establishment of S. vernalis. We, iii) hypothesized that, in both experiments, mixtures with higher levels of genetic diversity display higher establishment success than mixtures with lower genetic diversity. Furthermore, the effect of increased genetic diversity was expected to display interaction effects with propagule pressure and microsite availability, and to be more prominent at low levels of propagule density and microsite availability.We established two fully randomised container experiments to analyse the relationship between 1) propagule pressure and genetic diversity as well as 2) microsite availability and genetic diversity for No specific permissions were required for sampling activities, for we sampled only material at publicly accessible locations. We confirm that these locations are not privately-owned or protected in any way. The field studies did not involve endangered or protected species.seed families (SF), within populations (WP), proximate populations (PP) and remote populations (RP). As a population pool, we referred to 18 populations located in Halle , Saxony Anhalt, Germany, and in its urban periphery, with a maximum distance of 27 km and a minimum distance of 0.22 km to each other (We created seed mixtures of different levels of genetic diversity based on the assumption that genetic diversity increases with increasing geographic distance. We expected lowest levels of genetic diversity within seed families (i.e. seeds sharing a common mother plant), intermediate levels within single populations , and highest levels of genetic diversity within mixtures of seeds sampled across several populations. For the same populations as in the current study, phenotypic variation and genetic diversity was studied as a function of habitat type by Hantsch et al. (unpubl. data). The authors found overall very high phenotypic and genetic variation and only little evidence of habitat type-dependent differentiation. In particular, there was a strong differentiation among populations in germination traits. We created four levels of seed mixtures of increasing diversity in origin and increasing distance labelled ch other , Fig. S1proximate (PP) and remote population (RP). Each of the mixture replicates was composed of five populations. For the level of remote population (RP), each one initial population out of the total pool of 18 populations was selected at random. Subsequent filling of populations was done by random selection of additional four populations out of the sub-pool of those populations with a minimum distance of >13 km to the respective initial population. Accordingly, seed mixtures of proximate populations (PP) referred to eight populations of the total population pool . This restricted pool included populations located in the central part of the total study area, thus, representing the nested core area with a population distance <7 km to each other.For the two container experiments, we set up six population mixtures each for within population level (WP), we included achenes sampled across several individuals within populations. A total of 12 populations was randomly selected as replicates. For the seed family level (SF), we created a total of 18 replicates, referring to each of three populations from the groups of proximate and remote populations with each three individuals within population as replicates. The complete set of all treatments, thus, comprised a total of 42 mixtures. For details on exact composition of seed mixtures by populations and individuals see Supplementary Data (proximate population (PP). As a consequence of random assignment from a larger population pool for the mixtures of remote populations (RP), the overlap between the experiments was much smaller with each three populations being exclusively assigned to RP mixtures of the seed addition experiment  and the microsite experiment , respectively  and remote populations (RP) separately by experiment (see proximate population (PP) than in the mixtures of remote populations  and calculated mean expected germination of ment see . In a twions RP, .S. vernalis. We used containers of 20 cm diameter and a volume of 6 l filled with a substrate mixture composed by compost and sand with a ratio of 2∶1. Each container was planted with seven individuals of Festuca rupicola Heuff (Poaceae) that served as constant matrix in this experiment. Festuca rupicola individuals were raised by seeds from seed material collected in the neighbourhood of S. vernalis populations and planted as three week old seedlings a few days prior to the experiment. For seed addition of S. vernalis, we applied three levels of seed densities with 30, 60 and 90 achenes for each of the 42 seed mixtures. The intermediate level of seed number corresponded to a density of 2000 seeds m−2 following Turnbull et al. In the first experiment, we studied the role of propagule pressure, i.e. seed density, and genetic diversity for establishment of All containers were positioned randomly in one experimental plot in the Botanical Garden of the Institute of Biology/Geobotany  and regularly watered to ensure optimal water availability during the experiment. The experiment ran for 15 weeks from May to September 2009.S. vernalis early life-stage development. For this approach, we proceeded as in the first experiment but manipulated the matrix density of F. rupicola while keeping the seed density level constant at the intermediate level of 60 seeds per container. We created three levels of microsite availability by planting 5, 10 or 15 Festuca individuals per pot. In addition, we used matrix-free containers as control pots for open soil establishment. While all seed mixtures applied were anew chosen at random as described above, mixtures of genetic diversity levels were exactly repeated across all four matrix density levels. Container experiment II lasted 12 weeks from June to September 2009 and included 168 pots.A second experiment was designed for analysing effects of microsite availability and genetic diversity of propagules on establishment and growing success of S. vernalis individuals in each container. In the following we refer to the number of individuals after 1 week and after 12 weeks as “initial abundance” and “establishment”, respectively. Maximum abundance per container was assessed across all monitoring dates. In addition, individuals encountered were assigned to size classes to quantify the demographical development of individuals over time. Individuals were classified either as seedling , as small individuals , or as larger individuals . We calculated log ratios of size classes to assess the demographical development of S. vernalis during the experiment. Log size ratios were calculated as the logarithm to the base 10 of the proportion of summed individuals of size classes 2 and 3, divided by size class 1, i.e. the relative proportion of large individuals to small individuals (at the seedling stage). The more positive the log size ratio, the higher the abundance of large individuals compared to small individuals. For calculating log size ratios all values were added on by 1 to avoid division by zero.In both experiments, during the first four weeks, monitoring occurred at weekly intervals, but with increasing monitoring interval lengths thereafter. At each monitoring date, we assessed the total abundance of S. vernalis samples were dried for 48 h at 60°C for dry mass determination to yield overall productivity of mixtures.At the end of the experiments, containers of both studies were harvested. proc GLIMMIX), using Gaussian error distribution and identity link functions for both data sets and included the class variables diversity and seed density in the first container experiment and diversity and microsite availability in the second experiment as fixed factors. Calculated expected germination of a particular seed mixture applied was included as covariate into the model to account for genotype identity effects on maximum abundance calculated across all monitoring dates. In addition, we tested for effects of presence/absence for those populations in the RP mixtures that were exclusively assigned to either the Container experiment I or II. We included Pop 1, Pop 5, Pop 13 and Pop 3, Pop 4, Pop 6 in the models testing for additional covariate effects in the seed density experiment and the microsite availability experiments, respectively.Statistical analyses were done with SAS 9.2 S. vernalis. Covariation due to repeated measurements was modelled with the random statement in proc GLIMMIXWe ran an additional analysis on abundance data of all monitoring dates including ‘time’ as additional random factor to analyse cumulative establishment success of The tests of fixed effects are based on type III SS. We are aware that with applying the same model to different response variables we performed multiple tests. However, we did not apply Bonferroni correction for controlling potential type I errors, given some debate on the conservatism of this correction method Senecio vernalis individuals  displayed a more balanced and significantly higher proportion of large than of small individuals compared to seed applications from separate seed families  which displayed an intermediate log size ratio. In addition, variation in initial abundance and establishment increased with increasing diversity levels as indicated by larger Whisker ranges in There were only a few effects of genetic diversity. Log size ratio was affected by genetic diversity ; mixturelies SF; . Thus, wThere were no significant interaction effects between levels of seed density and genetic diversity in any of the variable studied.Presence/absence of exclusive populations in the RP mixtures affected the overall results only marginally, but presence of single populations accounted for additional variation depending on the variable tested .Festuca density in the recipient matrix  in the course of the experiment might hint at the possibility that positive and negative density effects have compensated each other although we cannot rule out the possibility that a wider range of seed density levels might have provided stronger effects. However, we tested only one beneficial environment, while the direction of density effects will become apparent only across a gradient in environmental adversity The initial high abundance of S. vernalis should strongly depend on the competition intensity brought about by the different Festuca matrix density treatments. However, in our experiment, initial abundance was completely unaffected by the treatments, thus displayed no evidence of microsite limitation. Moreover, at later stages, seedling abundance suffered from high mortality in the open soil, i.e. without Festuca. In contrast, these fewer individuals displayed higher individual performance and grew up to larger sizes finally resulting in increased total exotic biomass. Open soil situations, apparently, reflect more suitable sites for individual growth of S. vernalis, whereas initial facilitation for survival within the Festuca environments seems to be at the expense of competitive constraints at later stages. The sensitivity to such competitive changes during life stages, as found for S. vernalis, corresponds to theories on temporal shifts in plant interactions Acer negundo seedlings by increased survival in non-Acer stands S. vernalis abundance and performance was dependent on availability of bare ground, as provided by disturbance events. Numerous studies on invasive plant species confirm the role of disturbance on initial seedling emergence, such as the seed addition experiments with two non-native weeds of North American pastures and roadsides, Carduus nutans and C. acanthoidesS. vulgaris, congeneric to our study species, that the rate of spread was sensitive to both the size and distribution of gaps in swards S. vernalis to colonize such gap-dominated ruderal habitat types.We expected that the effect of seed addition of S. vernalis with, however, changing relative importance across subsequent early life stages. Initial abundance was mostly triggered by propagule pressure and less by microsite characteristics. Similarly, genetic diversity mattered only to a minor degree at the earliest life stages, such as for germination. However, effects of genetic diversity apparently seemed to be more decisive with regard to the probability of continued establishment, in combination with selection effects of best performing genotypes for consecutive growth. It would be promising to test for these effects on more species in replicated regions to strengthen generalization on these shifts in mechanisms. The differentiation of invasion processes into a first stage of predominant importance of propagule pressure and a second one with increasing importance of adaptation is mirrored in numerous attempts of describing the courses of invasion Click here for additional data file.Figure S2Mean expected germination of Senecio vernalis of mixtures. Values are provided for diversity levels remote populations (RP) and proximate populations (PP) separately by experiment. Experiment 1 = seed addition, Experiment 2 = microsite limitation, with n = 6 for each diversity level and experiment. Different letters indicate significant differences between levels of main factors according to the Scheffé test. Statistical details following ANOVA. Diversity F1,20 = 6.41 p = 0.020; Experiment F1,20 = 0.506, p = 0.485¸ Diversity×Experiment F1,20 = 0.991, p = 0.331.(TIF)Click here for additional data file.Table S1Location and habitat type affiliation of sampled S. vernalis populations in Eastern Germany.(DOC)Click here for additional data file.Table S2Details on exact composition of seed mixtures of S. vernalis in the four diversity levels applied by population and individual identity in the two container experiments. Ind shows the identity of seed families, “-” indicates pooled samples of a population without exact knowledge of seed family identity. Pop indicates the identity of the population referred to in the mixtures. All mixtures were exactly replicated for application to different seed density levels (Experiment 1) and Festuca rupicola density levels (Experiment 2), respectively. Potential germination capacities give the expected germination for populations and seed families determined in a previous study . Potential germination capacities of a particular seed mixture applied were calculated as the mean germination capacities across the populations involved in the respective mixture.(DOC)Click here for additional data file.Table S3Experiment 1: Propagule pressure×genetic diversity. Repeated measures GLM for number of individuals Senecio vernalis with fixed factors diversity, seed density and time. N = 126. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold p values indicate significant effects (p<0.05).(DOC)Click here for additional data file.Table S4Experiment 1: Propagule pressure×genetic diversity. GLM for maximum abundance of Senecio vernalis, including expected germination as covariate in the model. N = 126. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold numbers indicate significant effects (p<0.05).(DOC)Click here for additional data file.Table S5Experiment 1: Propagule pressure×genetic diversity and population identity effects. GLM for response variables of Senecio vernalis testing for presence/absence effects of those populations  exclusively assigned to the highest diversity level (RP = remote populations) in this experiment only. Initial abundance reflects the number of Senecio individuals after 1 week. Establishment refers to the number of Senecio individuals after 15 weeks. Abundance and total biomass were log10(x+1)-transformed prior to analysis. Log size ratio gives the proportion of number of large individuals compared to those of small individuals as logarithm to the base 10. N = 126. The results of the Scheffé post hoc tests indicate the direction of significant differences between categories of each factor. 90, 60, 30 indicates seed density levels applied. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold numbers indicate significant effects (p<0.05).(DOC)Click here for additional data file.Table S6Experiment 2: Microsite availability×genetic diversity. GLM for number of individuals, including time as additional fixed factor in the model. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold numbers indicate significant effects (p<0.05).(DOC)Click here for additional data file.Table S7Experiment 2: Microsite availability×genetic diversity. GLM for maximum abundance of Senecio vernalis, including expected germination as covariate in the model. N = 168. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold numbers indicate significant effects (p<0.05). GLM for maximum abundance of Senecio vernalis, including expected germination as covariate in the model. N = 168. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold numbers indicate significant effects (p<0.05).(DOC)Click here for additional data file.Table S8Experiment 2: Microsite availability×genetic diversity and population identity effects. GLM for response variables of Senecio vernalis testing for presence/absence effects of those populations  exclusively assigned to the highest diversity level (RP = remote populations) in this experiment only. Initial abundance reflects the number of Senecio individuals after 1 week. Establishment refers to the number of Senecio individuals after 12 weeks. Abundance and total biomass were log10(x+1)-transformed prior to analysis. Log size ratio gives the proportion of the number of large individuals compared to those of small individuals as logarithm to the base 10. N = 168. The results of the Scheffé post hoc tests indicate the direction of significant differences between categories of each factor. 15, 10, 5, 0 indicates numbers of Festuca individuals as density levels applied. Seed mixtures: RP = remote populations, PP = proximate populations, WP = within population, SF = seed family. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold numbers indicate significant effects (p<0.05).(DOC)Click here for additional data file.


Treated effluents from wastewater treatment works can comprise a large proportion of the flow of rivers in the developed world. Exposure to these effluents, or the steroidal estrogens they contain, feminizes wild male fish and can reduce their reproductive fitness. Long-term experimental exposures have resulted in skewed sex ratios, reproductive failures in breeding colonies, and population collapse. This suggests that environmental estrogens could threaten the sustainability of wild fish populations.Ne) of wild roach (Rutilus rutilus L.) living in English rivers contaminated with estrogenic effluents. Ne was estimated from DNA microsatellite genotypes using approximate Bayesian computation and sibling assignment methods. We found no significant negative correlation between Ne and the predicted estrogen exposure at 28 sample sites. Furthermore, examination of the population genetic structure of roach in the region showed that some populations have been confined to stretches of river with a high proportion of estrogenic effluent for multiple generations and have survived, apparently without reliance on immigration of fish from less polluted sites.Here we tested this hypothesis by examining population genetic structures and effective population sizes (Ne of up to 65% is still possible for the most contaminated sites because of the wide confidence intervals associated with the statistical model.These results demonstrate that roach populations living in some effluent-contaminated river stretches, where feminization is widespread, are self-sustaining. Although we found no evidence to suggest that exposure to estrogenic effluents is a significant driving factor in determining the size of roach breeding populations, a reduction in  Approximately two-thirds of the world’s freshwater is used to dilute wastewater discharges. The demand for freshwater is expected to rise by 70% by 2050  driving 2) and estrone (E1) from human excretion; 17 alpha-ethinylestradiol (EE2), a component of the female contraceptive pill [There is substantial evidence showing that experimental exposure of fish to WWTW effluents and the estrogens they contain can result in adverse health effects, including effects on reproductive development and breeding output. This has led to concerns that freshwater fish populations might also be affected with cascading consequences for freshwater ecosystems. Feminization of male fish is widespread in stretches of rivers downstream of WWTW outfalls as demonstrated in studies using wild ,4 and caive pill ; and a live pill , alterative pill , decreasive pill , alteredive pill  and chanive pill .Concern about estrogens in rivers in the United Kingdom drove a £40 M programme to evaluate the efficacy of various tertiary treatment processes in the removal of estrogens ,15. Impl2 (between 3 to 6 ng/L) in the aquatic environment could be a threat to the sustainability of fish populations. For example, a controlled exposure of an entire lake to EE2 in Canada resulted in the collapse of the fathead minnow  population within three years [Rutilus rutilus L.) to an undiluted WWTW effluent in large tanks resulted in an all-female population [2 at concentrations below 1 ng/L during the period of sexual development, have been shown to result in feminized gonads in roach [A critical question is whether chronic exposure to estrogenic effluents negatively impacts the viability of wild fish populations, but this has been difficult to address experimentally as it requires controlled experiments extending over periods of several years. Limited studies suggest that high concentrations of EEee years . Likewisee years -20 and apulation . It is npulation . Althougpulation , exposurin roach  and decrin roach . Evidencin roach  and sevein roach .Ne) – related to the breeding population of fish – may be decreased without necessarily impacting on population sizes [Ne can be several orders of magnitude smaller than census population sizes. One study found two populations of the exploited New Zealand snapper (Pagrus auratus) to have values of Ne less than 1,000 despite adult census population sizes in the millions [Morone saxatilis), a freshwater fish species, found cohorts to consist of a few, full sib families, despite an adult census size of over 300,000 [Ne influences long-term sustainability as it determines the rate at which genetic diversity is lost from a population through genetic drift [Ne, however, may act to increase the chances of losing some lethal or sub-lethal mutations through genetic purging.While these studies suggest that exposure to high concentrations of effluent could threaten the viability of fish populations, aggregates of cyprinid fish, including roach, are often found in effluent contaminated rivers. However, numbers alone may provide a misleading assessment of population sustainability as these could be sink populations maintained by substantial immigration from less contaminated locations where successful reproduction still occurs. Likewise, effective population sizes , a fish species in which feminization is widespread, in southern England. Southern England has some of the highest proportions of WWTW effluent in rivers known globally, and numerous weirs and locks which potentially confine fishes to heavily polluted stretches of river. We have used this system to evaluate whether stretches of river highly contaminated with estrogenic effluents have impaired breeding populations of roach. To do this we undertook analysis of population genetic structures of roach in the region using DNA microsatellite analysis. Microsatellite data were also used to calculate Ne and estimate levels of gene flow to determine the extent to which these populations are maintained through immigration of fish from less contaminated stretches of river.Understanding the impact of estrogenic effluents on the sustainability of fish populations is, therefore, paramount, but has been limited to date by the logistical challenges involved in undertaking long-term exposures to realistic effluent concentrations, and understanding the demographic history of wild fish populations at highly contaminated sites. In this study, we examine evidence for population impacts on wild roach (AR) ranged from 6.8 to 8.9, and expected heterozygosity (He) ranged from 0.69 to 0.75 , F = 2.1398, P = 0.00014) and observed heterozygosity (Ho)  = 1.8677, P = 0.0017) among samples were detected. Roach sampled at two relatively unpolluted sites (LamSha and LeeHUS’95) exhibited comparatively low AR and LeeHyd’95 exhibited comparatively relatively low Ho [see Additional file He were found. Additionally, there was evidence for genetic bottlenecks at two relatively unpolluted sites sampled within the rivers Arun (AruHUS) and Lee (LeeHUS), and at a polluted site in the Lee (LeeWhe)  from 32 different geographic locations in England were genotyped  indicated the majority of variation was partitioned among individuals within river locations, with river location accounting for a small 2.27%, but highly significant proportion of the genetic variation  for the study as a whole, while 262 of the 325 pairwise FST comparisons in the catchment were highly significant. There was a weak, but significant, relationship between genetic and geographic distance  within the catchment, indicating a tendency for individuals to produce offspring with fish from nearby populations rather than distant populations [see Additional file Despite Thames catchment roach appearing not to constitute a single genetic unit, distinct from roach in other regions, the study did find evidence for significant genetic structuring in roach populations within the Thames catchment. This suggests the existence of local subpopulations exchanging a limited number of effective migrants  rather than panmixia . For example, average Despite the proximity of some populations in the PCA and tree, we also found significant genetic differentiation between samples from some neighbouring stretches of the same river, sometimes occurring over small distances of separation (<10 km), for example, within the upper Lee (see below), between the Lee and the Stort, between the Blackwater and main Thames, between the Lambourn and the Kennet and within the Anglian Blackwater. In other cases, despite the separation of sampling locations by in-river impoundments such as weirs, we found no significant genetic differentiation between sites, for example, within the Stort, the main Thames, the Kennet, the Arun, the Nene and the Trent. Thus, patterns of within-river genetic structure differed between river stretches. For some other fish species analysis of genome-wide SNP data has provided greater resolution in population structure than that achievable using microsatellite data , and it Ne calculated from the microsatellite data using the approximate Bayesian computation method (Ne(ABC)), ranged from 54 to 301 for each sample, with higher precision for small Ne estimates  and predicted E2 equivalents (E2Eq), a measure of total estrogenicity of the river water due to contamination by sewage effluent , F = 0.7468, P = 0.40) or for an interaction between sample site and estrogen exposure  = 1.9954, P = 0.14) across the 28 sample sites where no recent restocking had occurred and had sufficient sample sizes for robust Ne calculation. However, the 95% confidence intervals (CI) for the model coefficient indicated Ne(ABC) could decrease by a maximum of 5.6% for each incremental increase in exposure of 1 ng/L E2Eq, or 65% at 11.6 ng/L E2Eq, equivalent to the most polluted river stretch included in this study. The inclusion of roach density as an additional covariate within the model also produced a non-significant result  = 1.3966, P = 0.26), albeit for a reduced number of sites (19). Similarly, there was no significant correlation between the other variables included in the statistical analyses  with Ne(ABC). This analysis makes the assumption that immigration of fish from remote sites is limited and this is discussed below.Estimates of Ne in roach sampled from more contaminated stretches of river compared to those sampled from less contaminated sites; all estimates above 6 ng/L E2Eq were below 100, whereas there was greater variation (54 to 301) where E2Eq was below 6 ng/L  ranged from 36 to 145 and also showed no relationship with E2Eq , and between these samples and two from its tributary, the Stort (FST ≥0.015) shown in Table A2 [Ne(ABC) estimates for these polluted sites in the upper Lee ranged from 70 to 84 (95% CI: 50 to 127) compared to only 54 (95% CI: 42 to 82) for the upstream uncontaminated location, suggesting no substantial impact of the effluent on the effective population size of these roach.The average proportion of effluent in the upper Lee downstream from Harpenden and East Hyde WWTWs ranges from 28% to 70% in different stretches. Significant genetic differentiation was detected between fish sampled from four of the five locations in this stretch of river  could not be ruled out, due to the wide 95% confidence intervals associated with the statistical model. Moreover, our analysis included relatively few samples from rivers in the highest risk category, largely because these sites are rare.In this study, analyses of population genetic structure bilities ,26. Bothbilities ), with pNe(ABC)[FST <0.002, Table As with any modelling exercise, this analysis makes assumptions that may affect the interpretation of the results. One of these assumptions is that migration between sites with different pollution profiles is limited over two to three generations, the time frame likely to have the greatest influence on Ne(ABC). This waNe(ABC). HistoloNe(ABC). This deNe(ABC) was relatively minor. Approximately 500,000 hatchery-reared roach just over one-year-old  have been introduced into the Thames catchment since 2000; broodstock for hatchery fish originate from the river Trent. The influence of restocking activities on Ne(ABC), however, is likely to be relatively minor, as the sites sampled in this study were separated by major physical barriers from sites where these introductions had occurred. Moreover, introductions prior to 2000 are unlikely to have had a large influence on Ne(ABC) as this is primarily affected by the size and variance in reproductive success of the parental generation, which would have spawned between 2004 and 2007 for most of the samples in this study. However, we cannot exclude some influence of introductions prior to 2000 as some of the summary statistics used to calculate Ne(ABC) are known to be affected by demographic processes over a longer time period [A second assumption is that no restocking of the rivers sampled had occurred or that the effect of restocking activities on e period . The effe period . Using oNe of fish populations, it suggests that roach populations can be self-sustaining despite exposure to estrogens over several generations. These findings are consistent with the fact that the prevalence of male fish with moderate to severely feminized gonads  is generally less than 10% in English rivers [Ne by reducing the reproductive dominance of large older males in estrogen-contaminated rivers. While the effects on females are less well studied, female roach exposed to an undiluted effluent for three years in large tanks were able to breed, despite the fact that this exposure caused complete gonadal feminization of males [While this study does not exclude the possibility that estrogenic effluents reduce h rivers ,25,26,46of males ; similar2/L [2 has been measured up to approximately 4 to 8 ng/L in English WWTW effluents [2 was detected in only 21 of 135 water samples from the Lee, although occasionally reaching 4 ng/L [2Eq and would be below 21 ng/L for 90% of the time. Only 1% to 3% of 10,313 individual river reaches in the UK receiving WWTW effluent were predicted to have average E2Eq >10 ng/L, and, of these, many are ditches composed almost entirely of sewage effluent [2 is approximately 10 times less potent than EE2 in inducing gonadal feminization in fish [et al. [The results of this study on wild roach populations seem to contrast with studies that have assessed population risk through long-term exposures to estrogens, where exposure to concentrations between 3 and 6 ng EE2/L -20 or to2/L  resulted2/L . The mos2/L . While Effluents ,48, for ffluents ,49, and g 4 ng/L . Consideeffluent . As E2 i in fish ,50, it i [et al.  recentlyThe population genetic structure of roach in southern England observed in this study may have been influenced by historical biogeography, migratory behaviour, human translocations and in-river barriers. Roach can be highly mobile and can migrate over 10 km, particularly in the spawning period April to June, if migration is not obstructed ; additioNe of roach populations and predicted exposure to estrogens, although because of the wide confidence intervals, a reduction in Ne of up to 65% is still possible at the most contaminated sites.Despite the widespread feminization of male roach in effluent-contaminated rivers of southern England, using nuclear DNA microsatellites we were able to identify some populations that have been confined to stretches of river with moderate to high exposure to estrogenic effluents over multiple generations. We also found no evidence of a correlation between the Southern England, particularly the region within the Thames catchment, was chosen for this study for four reasons. Firstly, it is a densely populated region with relatively low rainfall and, therefore, includes some river stretches with some of the highest concentrations of WWTW effluents in the United Kingdom . SecondlRoach was selected as the study species because it is native and widely distributed in the United Kingdom, including in rivers polluted with WWTW effluents. Additionally, widespread feminization has been reported in wild populations and with a proven association with exposure to estrogenic effluents ,19,21,57HO) and expected heterozygosity (He) using GenAlEx 6 [AR) was calculated using Fstat v2.9.3 [FST, calculated using Arlequin 3.5 [D, Dest[FST estimates was assessed based on 10,000 permutations. AMOVA was performed using Arlequin. In order to test whether fish are more likely to produce offspring with local mates, compared to mates in geographically distant locations within the Thames catchment, isolation by distance analysis was performed using the Mantel test [A2 [FST values . See Additional file To understand the extent to which roach populations are restricted to various stretches of river, several approaches were used to investigate population genetic structure. We analysed microsatellite loci variation in 1,769 fish sampled between 1995 and 2011. Each fish was genotyped at between 14 to 19 microsatellite loci. Microsatellite genotypes are provided in Additional file enAlEx 6 ; allelict v2.9.3  – see Adt v2.9.3 ,61 was uquin 3.5  and Jost [D, Dest, calcula [D, Dest. The sigtel test  in GenAltel test . Genetictel test , PCA in tel test  and a Batel test . Finallytest [A2 ,68 was uNe), which relate to the number of breeding fish and skews in breeding success, were estimated using the microsatellite genotypes. We compared Ne from sites ranging from little/no upstream WWTW effluent inputs to those where the majority of the flow can comprise WWTW effluent. Two single sample (generation) methods, that use different aspects of the microsatellite data, were used to estimate Ne for each population; the Approximate Bayesian Computation (ABC) method using ONeSAMP 1.2 [Ne(ABC); and the sibling assignment method (SA), Ne(SA)[Ne, which are calculated from the change in allele frequencies between generations, were also estimated for sites where fish had been sampled more than once using TempoFs [To test whether WWTW effluents substantially reduce the size of breeding populations, effective population sizes , Ne(SA). Tempora TempoFs  and NeEs TempoFs . For furNe(ABC). For further details see Additional file GLM were used to examine the relationship between predicted exposure to estrogenic effluents and AMOVA: analysis of molecular variance; ANOVA: analysis of variance; AR: allelic richness; CI: confidence interval; E1: Estrone; E2: Estradiol; EE2: 17 alpha-ethinylestradiol; GLM: Generalized Linear Models; He: expected heterozygosity; HWE: Hardy-Weinberg equilibrium; MCMC: Markov chain Monte Carlo; MEGA: Molecular Evolutionary Genetic Analysis; Nb: effective number of breeders; Ne: effective population size; Ne(SA): effective population size estimate calculated using the sibling assignment method; Ne(ABC): effective population size estimate calculated using the approximate Bayesian computation method; PCA: principal component analysis; SNP: single nucleotide polymorphism; WWTW: Waste Water Treatment Work (WWTW).The authors declare that they have no competing interests.PH, EN, JS, SJ, JS, JS and CT participated in the design of the research. RW modeled estrogen exposure in rivers. PH, EB and EN participated in data collection, analysis of microsatellite data and statistical analysis. PH, EN, EB, RW, JS, SJ, JS and CT wrote the paper. All authors read and approved the final manuscript.PH, the corresponding author, is a molecular ecologist and evolutionary biologist, and is a Research Fellow at the Biosciences Department at the University of Exeter.Details of microsatellite genotyping methods, population-genetic analysis and statistical analysis.Click here for fileGenetic diversity estimates for each locus in each population.Click here for fileGraph showing statistical differences in genetic diversity among roach Rutilus rutilus populations.Click here for filePairwise FST and Dest among roach population samples.Click here for fileMultidimensional scaling plots of pairwise DA distances [.stances .Click here for fileStructure analyses plots using the locprior model and analysis of optimum number of genetic units in Structure Harvester [.rvester .Click here for fileCorrelation between genetic distance and geographic distance (km) between pairs of sites for 24 population samples from the Thames catchment.Click here for fileMicrosatellite genotypes for 1,769 roach genotyped in this study.Click here for file


Liver biopsy represents the gold standard for evaluating damage and progression in patients with chronic hepatitis C (CHC); however, developing noninvasive tests that can predict liver injury represents a growing medical need. Considering that hepatocyte apoptosis plays a role in CHC pathogenesis; the aim of our study was to evaluate the presence of different apoptosis markers that correlate with liver injury in a cohort of pediatric and adult patients with CHC.Liver biopsies and concomitant serum samples from 22 pediatric and 22 adult patients with CHC were analyzed. Histological parameters were evaluated. In serum samples soluble Fas (sFas), caspase activity and caspase-generated neoepitope of the CK-18 proteolytic fragment (M30) were measured.sFas was associated with fibrosis severity in pediatric  and adult patients (advanced fibrosis p = 0.02). M30 levels were elevated in pediatric patients with severe steatosis (p = 0.01) while in adults no relation with any histological variable was observed. Caspase activity levels were higher in pediatric samples with significant fibrosis (p = 0.03) and they were associated with hepatitis severity (p = 0.04) in adult patients. The diagnostic accuracy evaluation demonstrated only a good performance for sFas to evaluate advanced fibrosis both in children (AUROC: 0.812) and adults (AUROC: 0.800) as well as for M30 to determine steatosis severity in children (AUROC: 0.833).Serum sFas could be considered a possible marker of advanced fibrosis both in pediatric and adult patient with CHC as well as M30 might be a good predictor of steatosis severity in children. Hepatitis related to Hepatitis C virus (HCV) is a progressive disease that may result in chronic active hepatitis, cirrhosis, and hepatocellular carcinoma. It is estimated that over 200 million people are infected worldwide, while 80% develop a chronic form Although liver biopsy represents the gold standard for evaluating presence, type and stage of liver fibrosis and for characterizing necroinflammation; it remains an expensive and invasive procedure with inherent risks. Thus, it cannot be performed frequently to monitor therapeutic outcomes Several less invasive diagnostic methods are currently being validated as potential tools to determine liver damage, namely serum markers and image methods, but they have not been yet incorporated in clinical practice in most countries Considering that 1) apoptosis plays a major role in the tissue development and homeostasis and in pathological processes Twenty two pediatric patients with CHC  from Hospital de Niños Ricardo Gutierrez (HNRG) and 22 adult patients  from Hospital Italiano de Buenos Aires (HIBA) were enrolled in the present study.Diagnosis was based on the presence of anti-HCV antibodies in serum at or after 18 months of age and HCV RNA in plasma at one or more separate occasions. Patients had no other causes of liver disease, autoimmune or metabolic disorders, hepatocellular carcinoma and coinfection with hepatitis B virus and/or human immunodeficiency virus. In adult cases, patients with a history of habitual alcohol consumption were excluded (>80 g/day for men and >60 g/day for women). Patients were naïve of treatment. This study has the approval of the Institutional Review Board and the Ethics Board of both HNRG and HIBA and is also in accordance with the Helsinki Declaration of 1975, as revised in 1983. A written informed consent was obtained from all the included adult patients and from parents of pediatric patients after the nature of the procedure had been fully explained.Formalin-fixed paraffin-embedded liver biopsies and serum samples at time of biopsy were used for histological and serological analysis, respectively. Histological sections were evaluated by two independent pathologists in a blind manner. Inflammatory activity and fibrosis were assessed using the modified Knodell scoring system  and METAVIR In adult cases, liver samples were not obtained from patients diagnosed as having liver cirrhosis based on clinical, biochemical and imaging findings. Although, there are no pediatric specific guidelines about the need for and timing of a liver biopsy in children, the probability of a child undergoing liver biopsy in this study reflected the current practice at our centre, which is based mostly on the national experts consensus Soluble Fas (sFas), caspase activity and caspase-generated neoepitope of the CK-18 proteolytic fragment (M30) were measured as apoptosis markers.Quantikine Human soluble Fas kit, R&D Systems Inc; and M30-Apoptosense ELISA kit, PEVIVA; respectively) according to the manufacturer's instructions. Serum concentration for each marker was determined from standard curves. Serum sFas was expressed as pg/mL and M30 as U/L.Serum sFas and M30 were determined by commercial quantitative sandwich enzyme immunoassay technique . Briefly, samples were first diluted 1∶1 in a buffer containing 50 mM Tris-HCl, 10 mM KCl, 5% glycerol, pH 7.4 and incubated with 25 µl of samples or controls diluted with an equal volume of caspase substrate for 3 h at room temperature. Then, the samples' luminescence was measured for 20 seconds in the Luminometer Junior LB 9509 (Berthold Technologies GmbH & Co. KG). Results are expressed as RLU. An activity negative control  and positive control  were included in each assay.Serum Caspase activity was determined using a chemiluminescence assay  curves by plotting the sensitivity against the reverse specificity at each value. The diagnostic value of each serum marker was assessed by the area under the ROC (AUROC). AUROC of 1.0 is characteristic of an ideal test, whereas 0.5 indicates a test of no diagnostic value. We determined the cut-off value for the diagnosis, as the maximal value at the sum of the sensitivity (Se) and specificity (Sp). The diagnostic accuracy was calculated by sensitivity, specificity and positive and negative predictive values. Area under the ROC, cut off values, positive predictive values (PPV) and negative predictive values (NPV) were determined using the MedCalc demo statistical software .Clinical, virological, and histological features of patients are described in In both groups HCV genotype 1 was predominant, 86% in pediatric cases and 77% in adults. The risk factors for HCV infection in children were 46% vertical transmission, 36% transfusion and 18% unknown. In adults, seven cases (32%) had a history of injecting drug abuse, one case (5%) described an occupational exposure to infected blood, four (18%) a transfusion as a risk factor and 10 (45%) an unknown source for transmission. The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels at time of biopsy, considering multiple biopsies from the same patient in 2 pediatric cases, were elevated in 52% and 76% serum samples of pediatric patients, respectively and in 59% and 77% serum samples of adult patients as well.Eighteen percent of pediatric biopsies showed moderate or severe hepatitis, while concerning fibrosis, bridging fibrosis (stage 2 of METAVIR) was predominant among studied biopsies (44%). In adult cases, moderate or severe hepatitis were present in 73% of biopsies and the fibrosis profile displayed was 32% stage 1, 32% stage 2 and 23% stage 3. Finally, 3 adult patients showed absence of fibrosis. The prevalence of significant fibrosis (F≥2) and advanced fibrosis (F≥3) in the pediatric cohort were 64% and 20%, respectively; meanwhile it was 54% F≥2 and 23% F≥3 in adults. Lymphoid follicles, characteristic of CHC in adults, were present in 40% of pediatric and 82% of adult specimens, whereas bile duct lesions were observed in 83% of pediatric and 95% of adult samples. Hepatocellular fat accumulation, typically a mixture of small and large droplet fat, was present in both series (64% of pediatric and 50% of adult cases). Minimal steatosis was observed in 36%, moderate in 12% and severe in 16% of pediatric biopsies; meanwhile in adults minimal, moderate and severe steatosis were present in 27%, 9% and 14%, respectively. The comparative statistical analysis of all histological parameters between pediatric and adult studied patients did not showed any significant difference except for lymphoid follicles (p = 0.01). However, it should be taken into account that adult cases with liver cirrhosis based on clinical, biochemical and imaging findings were not biopsied.Finally, aminotransferase values were not associated to any parameter of histological liver damage.Apoptosis markers were first compared between patients with CHC and healthy subjects. Then in a further analysis CHC patients apoptosis markers were related to histological parameters of liver injury, particularly fibrosis, hepatitis and steatosis severity.As it is shown in With regard to liver damage, sFas was associated with fibrosis severity in both pediatric and adult CHC patients. It was significantly increased in children with significant fibrosis (p = 0.03) and advanced fibrosis (p = 0.01), and in adults with advanced fibrosis (p = 0.02) . It is wThe M30 association profile related to the histological parameters was different between children and adults. In pediatric cases, M30 levels were elevated in patients with severe steatosis (p = 0.01)  while inThe caspase activity profile in relation to fibrosis severity was similar to that observed for sFas and M30 in both populations. Caspase activity levels were higher in those cases with significant and advanced fibrosis; however, the difference turned out to be statistically significant only in samples from children with significant fibrosis (p = 0.03). In the adult cohort only a trend of association with significant fibrosis was observed (p = 0.08). In turn, in serum samples from adult patients caspase activity was associated with hepatitis severity (p = 0.04) . No assoThe evaluation of the diagnostic performance was only assessed for those apoptosis markers which had shown to be associated with histological injury variables. When considering a less invasive test as good as a liver biopsy to evaluate liver damage, the AUROC of the marker must be equal to or greater than 0.800 The cut off value for sFas to differentiate advanced fibrosis in pediatric patients was 7416.56 pg/ml , whereas in adults it was 13806.67 pg/ml  . Serum Min vitro studies or in vivo models with whole virus demonstrated its ability to induce apoptosis. Our previous study demonstrated that apoptosis of hepatocytes is a prominent feature observed in liver biopsies of patients with CHC and that it is related to the pathogenesis of the disease 1)sFas, since it has been proposed that apoptosis triggered by Fas/FasL is a major cause of hepatocyte damage together with the observed high Fas/FasL expression levels which correlate with liver injury during CHC 2)caspase activity, since it has been reported that caspases are activated in HCV infected patient hepatocytes and are responsible for most of the morphological changes of the apoptotic cells 3)M30, since CK18 is the major intermediate filament of hepatocytes which is, in turn, a caspase substrate whose cleavage contribute to cellular collapse during apoptosis.Apoptosis has been implicated in the pathogenesis of a number of hepatic disorders, including viral hepatitis, autoimmune diseases, non-alcoholic steatohepatitis, alcohol-induced injury, cholestasis and hepatocellular cancer According to previously published data, this study showed that serum sFas levels were high in patients with CHC With respect to activation of caspases, the current results corroborate that apoptosis is an important event in CHC, as we have previously observed when investigating caspase activity and M30 expression in liver samples from CHC patients According to that previously described for adult cohorts A major clinical challenge is finding the best means for evaluating liver impairment in the increasing number of CHC infected patients Herein, based on AUROC values it was demonstrated that sFas could be a marker of advanced fibrosis both in children and adults and, in turn, M30 could be a good predictor of steatosis severity in children. However, despite the observed association between caspase activity and significant fibrosis in children as well as with hepatitis severity in adult patients, this marker would not be useful as a less invasive indicator of liver damage. Unfortunately, although there are many studies that evaluate these serum apoptosis markers in CHC patients related to liver damage, they do not assess their diagnostic value. This makes it impossible to compare the determined diagnostic accuracy of these markers for the diagnosis of liver injury severity with other reports.There are several articles that analyze AST-to-platelet ratio (APRI) and AST-to-ALT ratio (AAR) as surrogate indirect serum markers of liver fibrosis. As we previously describe when assessed in our cohorts It should not be ignored that the present study has certain limitations. First, this was in fact a retrospective study, with a quite limited number of cases, so this makes it difficult to validate the utility of serum markers. Second, due to medical management protocols from our institutions, pediatric patients without liver fibrosis (F0) and adults with cirrhosis or hepatic decompensation were not available for this study. Third, since we did not take into account biopsy length and fragmentation, the potential for sampling error and understaging of fibrosis remains possible. Anyway, the molecules here proposed turned out to be easily measurable markers, which can be interpreted in a simple manner. The study of a larger number of cases, perhaps in a multicenter study, will confirm the results obtained in this work and discuss the possibility of adding apoptosis markers to panels that included matrix deposition, clinical and biochemical parameters. Taking into account our previous results on fibrogenesis process direct markers (15) , we propIn conclusion, serum sFas could be considered a possible marker of advanced fibrosis both in pediatric and adult patient with CHC as well as M30 could be a good predictor of steatosis severity in children. Perhaps if these parameters are validated in the near future, they could be easily performed and interpreted and, therefore, could be potentially translatable to the bedside.Methods S1Technical information about immunohistochemial assays.(DOC)Click here for additional data file.Table S1AUROC for advance fibrosis.(DOC)Click here for additional data file.


Mycobacterium tuberculosis complex (MTBC). MTBC include several human-associated lineages and several variants adapted to domestic and, more rarely, wild animal species. We report an M. tuberculosis strain isolated from a wild chimpanzee in Côte d’Ivoire that was shown by comparative genomic and phylogenomic analyses to belong to a new lineage of MTBC, closer to the human-associated lineage 6  than to the other classical animal-associated MTBC strains. These results show that the general view of the genetic diversity of MTBC is limited and support the possibility that other MTBC variants exist, particularly in wild mammals in Africa. Exploring this diversity is crucial to the understanding of the biology and evolutionary history of this widespread infectious disease.Tuberculosis (TB) is caused by gram-positive bacteria known as the  Mycobacterium tuberculosis complex (MTBC)  is caused by closely related acid-fast bacteria known as the M. bovis and all other animal-associated members of MTBC but is more closely related to human-associated lineage 6 . This finding highlights critical gaps in knowledge of MTBC diversity and indicates that African wildlife, and more particularly nonhuman primates, are potential hosts of novel MTBC variants.We report microbiologically confirmed MTBC infection in a wild chimpanzee. We show that this infection was caused by a divergent MTBC strain that does belong to the clade that includes In the course of a long-term study comprising behavioral observations and disease investigations of wild chimpanzees habituated to humans in Taï National Park, Côte d’Ivoire, necropsies are performed routinely on any chimpanzee or other mammal found dead. Detailed analyses are performed to identify the causes of death of every animal , and her body condition had deteriorated over the years. Necropsy was performed and tissue samples were frozen and fixed in formalin. Frozen organ material was submerged in 70% ethanol, rinsed twice in 0.85% NaCl, shredded with a scalpel, and streaked onto Löwenstein-Jensen PACT agar . Bacteria were then cultivated on Middlebrook 7H11 agar supplemented with OADC or in Middlebrook 7H9 broth supplemented with OADC  without shaking at 37°C.gyrB using the primers MTUB-f (5′-TCGGACGCGTATGCGATATC-3′) and MTUB-r (5′-ACATACAGTTCGGACTTGCG-3′) and an annealing temperature of 65°C for the PCR. We used the DreamTaq DNA Polymerase Kit and Fermentas restriction enzymes .To investigate the possible presence of MTBC strains in other chimpanzees, samples were collected from 28 chimpanzees, many from the same community, that died in the same area within the previous 10 years. DNA was extracted from 115 tissue samples  from these 28 chimpanzees by using the QIAGEN DNeasy Blood and Tissue Kit . All tissues were tested in duplicate by using the primers MTC_IAC Fw and MTC_IAC Rv and MTC Probe as described  and 24 genomes published previously  and MIRA 3.0.0 , and all parameters were checked to have an effective sample size of >100 in the combined run. Branch robustness was assessed through their posterior probabilities .Phylogenetic analysis was performed on the basis of 13,480 high-confidence variable positions, specifying outgroup . Both cooutgroup , maximumoutgroup Figure 2,outgroup Figure 2 www.applied-maths.com/bionumerics) with the 13,480 high-confidence variable positions used for the phylogeny. The first 3 principal components accounted for 28%, 15%, and 8% of the variability and were used to generate a 3-dimensional scatter plot . We found that none of the spoligotyping patterns included in these databases (including 64 spoligotyping profiles from Côte d’Ivoire) matched the pattern of the chimpanzee strain  within the previous 10 years were tested by real-time PCR (14). However, no test results were positive. Consistent with the molecular analyses, necropsies and pathological evaluation of these animals revealed no signs suggestive of TB.M. africanum and M. tuberculosis, respectively. Direct comparison of those isolates was not possible because of the limitations of typing techniques at that time. However, close contact with humans  suggests those captive chimpanzees were infected with human strains, as reported for other captive nonhuman primates.Chimpanzees are known to be susceptible to TB, and MTBC strains have been reported previously in captive chimpanzees  was larger than the corresponding distance between any 2 strains from the same lineage. Second, our PCA analysis showed that the chimpanzee strains did not group with lineage 6. Third, spoligotyping revealed a novel pattern among 58,187 clinical isolates from 102 countries, including Côte d’Ivoire. Fourth, genome deletion analyses corroborated the distinct phylogenetic position of the chimpanzee strain compared with known MTBC lineages. Fifth, researchers and their assistants who are in proximity of the chimpanzees at Taï National Forest are regularly screened for TB, but none has ever had a positive test result.M. mungi and the Dassie Bacillus, which infect African mongooses and hyraxes, respectively . Moreover, further characterization of MTBC diversity will be crucial for understanding the origins of TB and the potential for the emergence of new strains through proximity between humans and wildlife.In conclusion, we report a microbiologically confirmed case of TB in a wild chimpanzee. Our molecular data show that the chimpanzee strain described here belongs to a novel lineage, more closely related to human-associated lineage 6 than to the other classical animal MTBC and possibly related to Mycobacterium tuberculosis complex isolates from previous studies compared with isolates from this study; genomic deletions respective to the H37Rv genome in the Chimpanzee Bacillus genome; coverage of the Chimpanzee Bacillus sequence reads in M. africanum–specific deletions; broth culture of M. tuberculosis complex isolate; maximum-likelihood phylogeny of M. tuberculosis complex isolate; observed pairwise distance between strains within lineages of M. tuberculosis; and PCA scatter plot of M. tuberculosis complex and related strains.Sequences of


These measurements represent a good approximation of the real potential of bioactive compounds in the cells of higher eucarions. The aim of the study was thus to determine in vitro and in vivo antioxidant activity of different berry juices, which reportedly contain high amounts of phenolics.Berry fruit is known for its high contents of various bioactive compounds. The latter constitute of anthocyanins, flavonols and flavanols and posses high antioxidative activity. The highly dynamic antioxidant system can be evaluated in vitro and in vivo antioxidant activity. Phenolic profiles of berries were determined with the use of a HPLC/MS system, in vitro antioxidant activity with the DPPH radical scavenging method and in vivo antioxidative activity using Saccharomyces cerevisiae. The highest diversity of individual phenols was detected for bilberry juice. The highest in vitro antioxidant capacity was determined for blackcurrant juice. A decrease in intracellular oxidation compared to control was observed in the following order: blackcurrant < chokeberry = blueberry < bilberry. The results indicate important differences in antioxidant activity of berry juices between in vitro and in vivo studies.Five different berry species were collected from several locations in central Slovenia and juice was extracted from each species separately. Juice was assessed for their In addition to the total content of phenolic compounds entering the cells, a key factor determining antioxidative activity of berry juices is also the ratio between the compounds. Where high content levels of anthocyanins and very low content levels of flavonols and hydroxycinnamic acids were measured a lower intracellular oxidation has been detected. Specifically, intracellular oxidation increased with higher consumption of hydroxycinnamic acids and lower consumption of anthocyanins in the cells. Antioxidative activity also increased when the consumption of analyzed phenols was rather low. Berry fruits are characterized by a high content and wide diversity of phenolic compounds. They differ in their structure and molecular weight and are frequently represented by phenolic acids (benzoic and cinnamic acid derivatives), tannins, stilbenes and flavonoids such as anthocyanidins, flavonols and flavanols Fruit extracts, containing phenolic compounds, are characterized by higher antioxidant activity than many isolated pure phenolic compounds or vitamins in vitro studies, antioxidant activity of specific bioactive compounds can only partly be correlated to the measured antioxidant activity in the cells. Therefore, cellular antioxidant activity assay was developed to precisely measure the antioxidant activity of selected antioxidants, dietary supplements, and foods in in vivo cell culture These phenolic compounds potentially inhibit cell damage caused by oxidation, which can lead to several pathological conditions Saccharomyces cerevisiae is an appropriate model organism for fundamental eukaryotic cellular processes such as cell antioxidative activity related to bioactive compounds Yeast in vivo studies have investigated the relationships between antioxidative activity and phenolic composition of various berry juices in a single experiment. The aim of the study was the determination of in vitro and in vivo antioxidant activity of different berry juices, which reportedly contain high amounts of phenolics. The present investigation also focused on expanding the existing initial component analysis by using high performance liquid chromatography (HPLC) equipped with a photodiode array detector (PAD) and mass spectrometer (MS) to elucidate any differences in the content and profile of selected flavonoid and phenolic acids present in different berry juices. Additionally, the uptake of these compounds was measured in the yeast cells. In literature, various methods have often been used in order to analyse the antioxidant potential of dietary substances and these assays could present the basis for further studies on antioxidant activity of specific bioactive compounds in the human body.To our knowledge, none of the No specific permits were required for the described field study. The location was not privately-owned or protected in any way so the plant material can freely be harvested by anyone.Sambucus nigra), blackcurrant (Ribes nigrum), blueberry (Vaccinium corymbosum), chokeberry (Aronia melanocarpa) and bilberry (Vaccinium myrtillus), were collected from several locations in central Slovenia . Fruit samples were harvested from July to September 2011 at full maturity. Only undamaged fruit were selected, and juice was prepared from 0.5 kg of each species by squeezing berries in a mortar. The obtained juice was then centrifuged for 10 min at 12,000 gn. The supernatant was filtered through a Chromafil AO-25/25 polyamide filter (Macherey-Nagel) and transferred to a vial prior to injection into a HPLC system, determination of antioxidant activity with the DPPH radical scavenging method and determination of intracellular oxidation.Five different berry species: elderberry (−1. The elution solvents were aqueous 1% formic acid and 5% acetonitrile (A) and 100% acetonitrile (B). Samples were eluted according to the gradient described by Marks, Mullen, & Crozier n scanning from m/z 115 to 2000. The capillary temperature was 250°C, the sheath gas and auxiliary gas were 20 and 7 units, respectively and the source voltage was 4 kV for negative ionisation and 0.1 kV for positive ionisation.Samples were analyzed using a Thermo Finnigan Surveyor HPLC system  with a PAD at 280 nm (hydroxycinnamic acids), 350 nm (flavonols) and 530 nm (anthocyanins). A Phenomenex  HPLC column C18  protected with a Phenomenex security guard column operated at 25°C was used. The injection volume was 20 µL and the flow rate maintained at 1 mL min−1.Concentrations of phenolic compounds were calculated from peak areas of the sample and the corresponding standards. The concentrations were expressed in µg mL−1 methanolic solution of DPPH was added and allowed to react in the dark at room temperature. The decrease of DPPH absorbance was measured at 520 nm at 5 min intervals by a spectrophotometer (MRX Dynex Technologies), until absorbance stabilized (30 min). Water was used as blank solution, and DPPH solution without test samples served as the control. All sample analyses were performed in triplicate. The DPPH radical scavenging activity of different berry juices was expressed as % of reacted DPPH in 30 min of reaction time.The juice of fruit samples for the determination of antioxidant activity was made according to the same protocol as for individual phenolics. The free radical scavenging activity of different berry juices was measured according to the DPPH method reported by Brand-Williams, Cuvelier and Berset S. cerevisiae ZIM 2155 was obtained from the Culture Collection of Industrial Microorganisms (ZIM) of the Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.The yeast −1; Biolife), peptone , glucose  (YEPD) medium at 28°C and 220 rpm, until their stationary phase was achieved. The cells were then centrifuged for 3 min at 4,000 ng, washed once with phosphate-buffered saline (PBS) (Merck) and suspended in PBS at a concentration of 1×108 cells mL−1. The cells were further incubated at 28°C and 220 rpm, for 96 h.The yeast cells were cultivated in a yeast extract (10 g L−1 of yeast cell suspension).The juice of 5 different fruit samples was added to the yeast cell suspensions following their 96-h incubation in PBS, at different concentrations , peptone , glucose , agar  (YEPD) medium and then after a 2-day incubation at 28°C the number of colonies was counted. The experiments were performed in biological duplicates. Data are expressed as means of CFU mL−1± SE.Cell viability was measured as CFU (colony forming units). After 2-h incubation CFU were determined by plating cell suspension on yeast extract (10 g L2DCF), which reacts with oxidants, thus revealing the presence of ROS. This was given to the cells as 2`,7`-dichlorofluorescein diacetate (H2DCFDA), which easily penetrates the plasma membrane and is hydrolysed inside the cells by non-specific esterases. The non-fluorescent H2DCF can then be oxidised to fluorescent 2`,7`-dichlorofluorescin (DCF), which is measured fluorimetrically Intracellular oxidation was estimated using 2`,7`-dichlorofluorescein , and washed three times with 50 mM potassium phosphate buffer (pH 7.8). The cell pellets were finally resuspended in 9 volumes of 50 mM potassium phosphate buffer  and incubated at 28°C for 5 min. The ROS-sensing dye H2DCFDA (Sigma) was added from a 1 mM stock solution in 96% ethanol (Merck), to a final concentration of 10 µM. After 20-min incubation at 28°C and 220 rpm, the fluorescence of yeast cell suspension was measured in a kinetic mode, using a Safire II microplate reader (Tecan). The excitation and emission wavelengths of DCF were 488 nm and 520 nm, respectively. Values of fluorescence intensity were measured at 90 min. Data are expressed as means of the relative fluorescence intensity ± SE.The cells from the 2-ml incubations were sedimented by centrifugation . The supernatants were filtered (pore size: 0.2 µm) and analysed as described in the section reporting the determination of individual phenolic compounds.For the analysis of cellular uptake the yeast cell suspension before and after exposure to berry juices was centrifuged at 14,000 O-rutinoside), cyanidin-3-O-galactoside from Sigma-Aldrich, quercetin-3-O-glucoside, quercetin-3-O-galactoside from Fluka Chemie . The chemicals for the mobile phases were HPLC-MS grade acetonitrile and formic acid from Fluka Chemie. Water for the mobile phase was twice distilled and purified with the Milli-Q system . For the determination of antioxidant capacity 1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma.The following standards were used for the quantification of phenolic compounds: chlorogenic acid (3-caffeoylquinic acid), rutin . Differences in antioxidant activity between berry juices were analyzed with one-way analysis of variance (ANOVA). Significant differences among means were determined by Duncan’s Multiple Range test with a significance level of 0.05.In all juice samples 41 different bioactive compounds were identified and classified to the following phenolic groups: hydroxycinnamic acids, flavonols and anthocyanins. The highest diversity of individual phenols was found in bilberry juice, where a total of 19 compounds were determined. The second largest diversity of phenolic compounds was measured in blueberry juice (17 compounds), followed by blackcurrant juice (15 compounds) and chokeberry juice (12 compounds). The smallest structural variability was found in elderberry juice.−1, representing from 3.2 to 40.4% total analysed phenolic compounds in different berry juices. Flavonols were the most diverse phenolic group in berry juices, but their concentration was relatively low and only represented from 1.1 to 19.2% of total analyzed phenolic compounds (40.3 to 363.6 µg mL−1). Anthocyanins were the most abundant phenolics in berry juices representing from 40.4 to 92.3% of total analyzed compounds (539.6 to 7440.9 µg mL−1)  . The hetin vitro antioxidant potential due to their high phenolic levels. In our experiment, antioxidant capacity was measured according to the DPPH method and indicated statistical differences between different berry juices. The highest antioxidant capacity was determined in blackcurrant juice, followed by elderberry, chokeberry, bilberry and blueberry juices. Additionally, statistical differences were not observed between elderberry and chokeberry juice antioxidant capacity . Results did not show statistically significant differences in the intracellular oxidation between the concentrations used  different compounds were identified from this group; the largest subgroup comprised of quercetin-glycosides, but several myricetin, kaempferol, ishoramnetin and syringetin glycosides were also determined. Even though flavonols were the most heterogeneous group identified in berry juices their content only represented from 1 to 12% total analysed phenols. The smallest flavonol diversity was determined in elderberry juice, but oppositely, their content was highest. On the other hand, the largest variability of flavonols was established for blueberry and bilberry juices, but the concentration of individual compounds was very low in blueberry juice.A high concentration of anthocyanins has previously been reported in chokeberry, bilberry, blackcurrant, and elderberry fruit in vivo antioxidant activity. This was demonstrated for bilberry and blueberry juices, where the highest levels of antioxidant activity . The highest hydroxycinnamic acids uptake was measured in blueberry and bilberry juices, and the lowest in chokeberry juice. The content of consumed flavonols ranged between 24.7 and 100%, which represents from 1.1 to 12.8 µg flavonol uptake of the cells. The anthocyanin uptake was between 64.6–100% (from 27.0 to 307.3 µg) .Saccharomyces cerevisiae. Intracellular oxidation also increased when the consumption of analyzed phenols was rather low.Furthermore, the quantification of total phenolic uptake in the cells was achieved and analysed according to individual compounds . ResultsWhich phenolic compounds, either alone or in synergism with others, contribute the most to biological effects of analyzed berries juice is still an open question. Anthocyanins are considered to be the most effective antioxidants As demonstrated in some previous reports, the differences in antioxidant capacity could be ascribed to individual molecular structures of bioactive compounds. Some possible explanations are as follows: (a) the increasing number of hydroxyl groups may increase the antioxidant activity Flavonols  possess high antioxidant activity as their structure allows a more effective antioxidant activity compared to the structure of anthocyanins. The quercetin 2,3 double bond in conjunction with a 4-oxo function in the C ring enables electron delocalization from the B ring, and showed extensive resonance, resulting in significant effectiveness for radical scavenging in vitro compared to the others, and the order of antioxidant potency was usually 3-glucoside >3-rhamnoside >3-arabinoside ≈ 3-galactoside. The differences in antioxidant potency originate from the orientation, number, and distribution of hydroxyl group of the conjugated sugars.Glycosylation of the anthocyanidins and quercetin may also modulate their antioxidant activity The contribution of individual phenolics to total antioxidant capacity was generally dependent on their structure and content levels in berry juices. The results of this study provide guidance as to the relative potential health benefits of different berries or berry juice. However, to understand the mechanisms of positive health effects after consumption of berries even better, there are still questions to be answered about bioavailability, metabolism, molecular targets, and bioactivity of phenolics in the human body.


Peste des petits ruminants (PPR) is a viral disease which primarily affects small ruminants, causing significant economic losses for the livestock industry in developing countries. It is endemic in Saharan and sub-Saharan Africa, the Middle East and the Indian sub-continent. The primary hosts for peste des petits ruminants virus (PPRV) are goats and sheep; however recent models studying the pathology, disease progression and viremia of PPRV have focused primarily on goat models. This study evaluates the tissue tropism and pathogenesis of PPR following experimental infection of sheep and goats using a quantitative time-course study. Upon infection with a virulent strain of PPRV, both sheep and goats developed clinical signs and lesions typical of PPR, although sheep displayed milder clinical disease compared to goats. Tissue tropism of PPRV was evaluated by real-time RT-PCR and immunohistochemistry. Lymph nodes, lymphoid tissue and digestive tract organs were the predominant sites of virus replication. The results presented in this study provide models for the comparative evaluation of PPRV pathogenesis and tissue tropism in both sheep and goats. These models are suitable for the establishment of experimental parameters necessary for the evaluation of vaccines, as well as further studies into PPRV-host interactions. Although originally characterized in western Africa in the early part of the 20Morbillivirus genus of single-stranded RNA viruses, with a genome size of 15 948 bp PPRV has been shown to be the largest member of the et al.Despite the ability of PPR outbreaks to cause widespread deaths in livestock, the precise viral-induced pathogenesis is still not fully understood. Although PPRV-infected animals were known to exhibit clinical signs similar to rinderpest Previous field studies in India have found that, while PPRV infection was slightly more prevalent in sheep than in goats in the target population of animals over 3 months old, outbreaks in goats tended to be more severe 2. After the third passage, transmission electron microscopy (TEM) was performed to confirm that the virus was still displaying the structural integrity typical of members of the family Paramyxoviridae. After the fourth passage, 80%–90% of cells exhibited cytopathic effect (CPE) after 17 days. Infected cells and supernatant were harvested and frozen at −70°C for subsequent challenge experiments. The virus stock was titrated using Vero cells and the TCID50 values were determined based on the method of Reed and Muench A stock of PPRV Malig (Yemen) was originally obtained from The Pirbright Institute . The viral stock was passaged four times in Vero cells (ATCC) in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Multicell Media-Gibco-BRL-USA) and 1% Penicillin/Streptomycin solution (Multicell) in a 37°C incubator with 5% COad libitum. Animal experimentation was conducted under the approval of the Canadian Science Centre for Human and Animal Health Animal Care Committee, which follows the guidelines of the Canadian Council on Animal Care. Both sheep and goats were previously screened and found negative for PPRV by real time RT-PCR and serology prior to viral infection. Each individual sheep and goat was infected with Malig PPRV . All animals were observed daily, with clinical signs recorded throughout the study. Rectal temperatures were measured daily from 1 to 13 days post infection (dpi). Oral and nasal swabs, as well as blood and sera, were collected from sheep and goats 2 days prior to infection, and 2, 4, 6, 8, 11, 13, 15, 18 and 21 dpi.Six Boer cross goats and six Rideau Arcott sheep  were housed in separate Biosafety Level 3 animal cubicles at the National Centre for Foreign Animal Disease , and were fed a complete balanced diet and water 2O2, then pretreated with proteinase K for 10 minutes. Primary antibody, a monoclonal antibody raised against PPRV strain Nigeria 75/1 , was used at a 1∶1000 dilution in 10% normal goat serum and Tris-buffered saline/0.05% Tween 20 (TBST) solution overnight at 4°C. Labelled tissue sections were then stained using a horseradish peroxidase-labelled polymer , reacted with the chromogen, diaminobenzidine (DAB). The sections were then counter-stained with Gill’s hematoxylin.One sheep and one goat were euthanized and submitted to a necropsy procedure on each of dpi 6, 8, and 11. Tissues were fixed in 10% neutral phosphate-buffered formalin. Sections were stained with haematoxylin and eosin (HE). For immunohistochemistry, paraffin tissue sections were quenched for 10 minutes in aqueous 3% H2O2, then pretreated with proteinase K for 10 minutes. The primary antibody was applied and developed as described above. The slides were then incubated for 5 minutes with Biocare denaturing solution . The second primary antibody, a mouse monoclonal antibody specific for CD68 (EMB11) (Dako), was used at a 1∶20 dilution in TBST solution overnight at 4°C. Sections were then stained using an alkaline phosphatase-labelled polymer , reacted with the chromogen, Vulcan Fast Red (VFR). The sections were then counter-stained with Gill’s hematoxylin.For double-immunostaining, paraffin tissue sections were quenched for 10 minutes in aqueous 3% Het al.2 flasks grown in a 37°C incubator with 5% CO2. Cells were evaluated daily for CPE for 20 days.Tissues from sheep  and goatet al.TTGTCCTTCTCGTCGGGCCC 3′Tam. The real-time RT-PCR reaction was performed using an ABI 7500 Sequence Detection System  and a protocol previously described by Bao et al. RNA from tissue homogenates, as well as oral/nasal swabs, was extracted using the RNeasy Mini kit  while RNA from whole blood was extracted using the QiaAmpRNA blood Mini kit  as described by the manufacturer. Quantification by real-time RT-PCR was performed using primers specific to PPRV that were designed using Genscript software (USA). The expected 130 bp fragment, covering part of the PPRV N protein gene, was previously described by Saravanan Peste des petits ruminants viral antigen was purified from virus amplified using Vero cells incubated for 17 days. The viral suspension was layered onto a PBS (pH 7.4)/20% sucrose gradient and pelleted by ultracentrifugation at 118,000×g for 2 hours. The pellet was then resuspended in PBS (pH 7.4) and stored at −80°C for use as antigen for the indirect ELISA. Ninety-six well ELISA plates  were coated with purified virus (diluted 1∶400 in carbonate buffer [pH 9.6]) and incubated overnight at 4°C. The plates were then incubated with blocking solution (5% skim milk in PBS/0.05% Tween) for 1 hour at 37°C. Serially diluted sheep and goat sera (starting at a 1∶50 dilution) were then added, incubated for 1 hour at 37°C, washed 3 times, and further incubated for 1 hour at 37°C with a 1∶1000 dilution of alkaline phosphatase-conjugated donkey secondary antibody . The plates were then washed 3 times, developed using Blu Phos™ phosphatase substrate  and absorbance measured at a wavelength of 650 nm. Endpoint titres were determined using the average optical density plus two standard deviations from 300 negative sheep sera as the cut-off value.50, in DMEM) was mixed with sheep and goat sera (in duplicate) to a volume of 200 µl, and incubated for 1 hour at 37°C. Vero cells incubated in 96-well plates were infected with the 200 µl mixture of virus and serum . The cells were then incubated for 15 to 18 days, with daily examination for CPE.Virus neutralizing antibodies were measured using VNT. Serial dilutions  of sheep and goat sera, starting from 4 to 21 dpi, were evaluated. PPRV  was used to measure sheep and goat IFN-γ based on the ability of selected bovine monoclonal antibodies to cross-react with both sheep and goat IFN-γ. Serum IFN-γ levels were measured by quantitative ELISA, as previously described by Tourais-Esteves Statistics were performed using a t-test with Excel (Microsoft) to determine differences between sheep and goats for viral RNA loads, serology and IFN-γ levels in serum.Sheep and goats were allowed to acclimate to the laboratory environment for a period of two weeks prior to experimental infection with PPRV. During that time, all experimental animals were healthy and free of disease, with normal rectal temperatures ranging from 39.5–40.3°C (sheep) and 39.1–40.1°C (goats). Following inoculation with PPRV, clinical signs, including mild depression, moderate bilateral mucopurulent nasal discharge and elevated rectal temperatures (40.5–41.1°C), were observed in goats starting at 4 dpi . In sheeThe progression of the disease was most pronounced between 6 and 8 dpi in both sheep and goats, where significant inactivity and nasal discharges were observed in nearly all animals . FurtherIn the one sheep sampled at 6 dpi, small oral ulcers (2–5 mm) were observed as well as diffuse pulmonary edema and bronchopneumonia. Mesenteric lymph nodes were enlarged and pinpoint areas of erosion were evident in the Peyer’s patches. In the goat sampled at 6 dpi, only mild enlargement of the prescapular and mesenteric lymph nodes was observed.At 8 dpi, in the single sheep and goat sampled there were enlarged mesenteric lymph nodes. The sheep had milder lesions, including 1–2 mm erosions of the oral mucosa and multifocal erosions (1.0 to 1.5 cm) throughout the ileum. The goat sampled at 8 dpi showed more severe lesions at this time point including: conjunctivitis, widespread and severe necrosis and erosion of oral mucosa , small eIn the single sheep sampled at 11 dpi, there were mild lesions including enlarged mesenteric lymph nodes and depressed and reddened Peyer’s patches . In the Following PPRV inoculation, a sheep and goat were euthanized at each of 6, 8 and 11 dpi and tissue samples were collected from multiple organs. Tissue tropism for PPRV was determined using a combination of histology, immunohistochemistry and real-time RT-PCR. In the goat sampled at 6 dpi, high levels of antigen were observed in lymphoid organs including the tonsil, spleen and parotid/bronchial lymph nodes, with some involvement of the intestine. In the goat sampled at 8 dpi, detection of virus by both IHC and real-time RT-PCR was highest in the lungs, intestines and oral mucosa. In contrast, high viral loads were detected at in the sheep sampled at 6 dpi in the lymphoid tissues, as well as the respiratory and intestinal tracts of sheep. It should also be noted that at 6 and 8 dpi considerable virus was detectable in the spleen and tonsils of both animal species. In all cases, the level of detectable virus diminished considerably at 11 dpi. When the presence of PPRV was quantified using real-time RT-PCR, the amount of viral RNA was generally consistent with the results from IHC, confirming viral replication.In both species at 6 and 8 dpi, there were prominent lesions in the palatine tonsils, which included necrosis of surface and crypt epithelium with infiltration of neutrophils, formation of syncytial cells and scattered intranuclear inclusion bodies . Lymph nSevere and widespread microscopic lesions in the pharyngeal, oral and nasal mucosa were only observed in the goat examined at 8 dpi. In the sheep at 6 and 8 dpi and the goat at 6 dpi, these lesions were smaller and only rarely observed. Lesions were characterized by multifocal erosions and formation of syncytial cells in upper cell layers, and necrosis and loss of epithelium with replacement by edema fluid and neutrophils. In the forestomachs of a few animals, multifocal areas of epithelial necrosis, with neutrophil infiltration and syncytial cell formation, were observed . PositivAt 6 dpi in the single sheep sampled and at 8 dpi in the single goat sampled, severe bronchointerstitial pneumonia was observed in the cranial and middle lung lobes . There wIn order to confirm that PPRV was replicating in sheep and goats, virus isolation was attempted on samples collected from various tissues. In sheep, virus was successfully isolated from third eyelid tissue at 6 dpi. In goats, virus was isolated from skin/lip tissue and conjunctiva at dpi 6, oral mucosa on 8 dpi and skin/lip tissue on 11 dpi. All isolations were confirmed to be PPRV by sequencing of the N gene. Virus isolation was unsuccessful in all other organs tested, including lymph nodes.In order to detect the presence of PPRV RNA at mucosal surfaces of sheep and goats, oral and nasal swabs were collected at various time points following experimental infection. Viral RNA was quantified using real-time RT-PCR. In both sheep and goats, PPRV was detectable as early as 2 dpi in oral swabs with 1/6 goats and 2/6 sheep as well as nasal swabs with 3/6 goats and 4/6 sheep showing detectable levels of viral RNA . SignifiTo measure viremia, whole blood was assessed for PPRV RNA using quantitative real-time RT-PCR. Low levels of viral RNA were detected in whole goat blood at 2 dpi in 2 goats. Significant levels of PPRV RNA were detected in goats at 4, 6 and 8 dpi, with copy numbers never exceeding 1000 copies/mL. This is in contrast to sheep, where PPRV RNA was not detected in whole blood at any time point post infection.In order to quantify PPRV-specific IgG antibodies generated in sheep and goats following viral infection, sera were collected at regular time intervals between 4 and 21 dpi. PPRV-specific antibodies were measured in serum using an indirect PPRV ELISA. Seroconversion for both sheep and goats started at 8 dpi as measured by the indirect ELISA . NeutralSerum samples obtained following PPRV inoculation were assayed and quantified for the presence of IFN-γ in response to PPRV infection. No significant IFN- γ was detected in sheep at any timepoint. Goats showed a significant increase in IFN-γ on 8 dpi compared to samples at −2 dpi .Over the past three decades, multiple studies on PPR have been performed in experimental settings Lesions and viral antigen were primarily observed in the respiratory and gastrointestinal tract, as well as within lymphoid organs, which is in agreement with previous studies While both sheep and goats had similar patterns of gross pathology, following nearly identical time courses, PPRV-induced pathology was significantly more pronounced in the single goat sampled at 8 dpi with widespread and severe oral lesions, as well as significant haemorrhaging and necrosis of cecal and colonic tissue, being observed. There were also noticeable differences in the degree of enlargement of lymph nodes between the two species, with goats being more severe. The degree to which secondary lymphatic organs, as well as mucosal and gut-associated lymphatic tissue, are affected is consistent with previous studies of morbillivirus tropism The viremia in goats elicited a more robust inflammatory response, as indicated by increased IFN-γ levels observed in goat sera compared to sheep. These results appear to be in agreement with previous work using a highly virulent Indian strain of PPRV et al.et al.In previous studies investigators administered PPRV either intranasally, subcutaneously or using both routes, as in this study. The degree of clinical progression appeared to follow a time course similar to that described in previous experiments, thereby suggesting that the route of administration did not change disease progression when compared to that of earlier studies. Recent work by Pope The histopathology, IHC, and quantitative RT-PCR results from both sheep and goats also provide some insight into the disease progression of PPR in small ruminants. In both species, significant levels of virus were detected in the lymph nodes, lymphoid tissues and digestive tract at 6 dpi. However, within two days thereafter, viral loads were lower in most lymph nodes, but the presence of virus increased in the tissues of the digestive tract in both sheep and goats. These results, when combined with the gross pathology data for both species, suggest that primary replication of PPRV may occur in the draining lymph nodes, which then seed the organs of the digestive and respiratory tract.Both sheep and goats developed clinical signs of PPR, although sheep did not have detectable viral RNA in blood, compared to goats. The reason for this difference in viral replication is not known, but demonstrates that although PPRV can infect both sheep and goats, there are differences depending on the host. A similar situation is observed with capripoxvirus where there are differences in the susceptibility of sheep and goats, although the differences are much more pronounced depending on the virus isolate involved Mannheimia haemolytica, enhanced pneumonia was observed An important discrepancy between the experimental model developed in this study (as well as most previous studies) and field conditions pertains to the lack of mortality in both sheep and goats in experimental settings, compared to the high degree of PPR-related deaths of livestock observed following field outbreaks. As mentioned in previous publications, this difference may be due to: 1) differences in the breed of sheep or goat used in the studies; 2) the overall health of the animals; 3) the virus strain/isolate used and how it was amplified or 4) the absence of other bacterial, viral or helminth pathogens, which may compromise the host immune responses. For example, when PPRV was co-administered with the bacterial pathogen The experimental PPRV-infection model developed and described in this paper in sheep and goats will now be used as a standard model, allowing for more in-depth pathogenesis studies of PPRV infection in the future as well as the evaluation of PPRV vaccines. Despite the complete recovery of experimentally infected animals, both sheep and goats developed clinical signs of disease, had detectable levels of virus replication and developed PPRV-specific antibodies.Since the 1970’s, when it was found that attenuated rinderpest virus could confer protection against PPRV, experimental vaccines based on attenuated virus, as well as recombinant viruses, have been developed. These include the attenuated Nigerian 75/1 PPRV strain, as well as the south Asian strains, Sungri 96, Arasur 87, and Coimbatoire 97