Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

Turnover and accumulation of genetic diversity across large time-scale cycles of isolation and connection of populations.

Major climatic and geological events but also population history (secondary contacts) have generated cycles of population isolation and connection of long and short periods. Recent empirical and theoretical studies suggest that fast evolutionary processes might be triggered by such events, as commonly illustrated in ecology by the adaptive radiation of cichlid fishes (isolation and reconnection of lakes and watersheds) and in epidemiology by the fast adaptation of the influenza virus (isolation and reconnection in hosts). We test whether cyclic population isolation and connection provide the raw material (standing genetic variation) for species evolution and diversification. Our analytical results demonstrate that population isolation and connection can provide, to populations, a high excess of genetic diversity compared with what is expected at equilibrium. This excess is either cyclic (high allele turnover) or cumulates with time depending on the duration of the isolation and the connection periods and the mutation rate. We show that diversification rates of animal clades are associated with specific periods of climatic cycles in the Quaternary. We finally discuss the importance of our results for macroevolutionary patterns and for the inference of population history from genomic data.

Assisted reproductive technology and obstetric outcome in couples when the male partner has a chronic viral disease.

BACKGROUND Assisted reproductive technology (ART) with washed semen can achieve pregnancy with minimal risk of horizontal and vertical transmission of chronic viral diseases (CVD) such as human immunodeficiency virus (HIV), hepati- tis C virus (HCV) and hepatitis B virus (HBV) among serodiscordant couples. How- ever, few studies have been made of the use made by these couples of ARTs or of the obstetric results achieved. MATERIALS AND METHODS In this retrospective study, 93 men who were seropositive for HIV, HCV or HBV and who underwent assisted reproduction treatment at our centre (Hospital Universitario Virgen de las Nieves, Granada, Spain) were included. Washed semen was tested to detect viral particles. Non-infected women were tested before and after each treatment, as were the neonates at birth and after three months. RESULTS A total of 62 sperm samples were washed, and none were positive for the detec- tion of viral molecules. Semen samples from 34 HBV positive males were not washed since the female partner had immunity to hepatitis B. In total, 38 clinical pregnancies were achieved (22% per cycle and 40.9% per couple) out of 173 cycles initiated, and 28 births were achieved (16.2% per cycle and 30.1% per couple), producing 34 live births. The rate of multiple pregnancies was 21.4%. Obstetric and neonatal results were similar in the groups of couples studied. At follow-up, no seroconversion was detected in the women or neonates. CONCLUSION Sperm washing and intracytoplasmic sperm injection are shown to be a safe and effective option for reducing the risk of transmission or super infection in serodiscordant or concordant couples who wish to have a child. Pregnancies ob- tained by ART in couples when the male is CVD infected achieve good obstetric and neonatal results.

Coupled reproductive ecologies in a plant-pollinator-seed predator system

Résumé de la thèseBien que le mutualisme puisse être considéré comme une relation harmonieuse entre différentes espèces, son étude révèle plutôt une exploitation réciproque où chaque partenaire tente de maximiser ses bénéfices tout en réduisant ses coûts. Dans ce contexte, l'identification des facteurs qui favorisent ou contrarient, au cours de l'évolution, une issue mutualiste est une étape majeure pour pouvoir reconstruire les étapes clés menant à l'apparition et au maintien des interactions mutualistes. Le but de ce doctorat était l'identification des traits phénotypiques qui permettent à la plante Silene latofolia (Caryophyllacée)et à son pollinisateur - prédateur de graines, la phalène Hadena bicruris (Noctuidé), d'augmenter les bénéfices nets que chacun retire de l'interaction. Ce système d'étude est particulièrement bien approprié à l'étude de ces traits, car on peut assez facilement estimer la qualité et la quantité des descendants (fitness) des deux partenaires. En effet, la femelle papillon pond un oeuf dans la fleur qu'elle pollinise et sa larve se développe dans le fruit, consommant les graines de la plante. Ainsi, sur une même plante, il est possible d'estimer les succès respectifs de la plante et du papillon à obtenir une descendance. De plus, le conflit d'intérêt autour des graines qui sont indispensables, à la fois à la plante et au papillon, peut stimuler l'évolution de traits qui limitent la surexploitation réciproque des partenaires. Dans une première étude, j'ai montré que le papillon mâle était un pollinisateur efficace de S. latifolia et qu'ainsi, il permettait à la plante d'augmenter le nombre de graines produites (i.e.bénéfice) sans pour autant augmenter la quantité de larves sur la plante. Dans ce système, les papillons pondent un seul oeuf par fleur, déposé soit à l'intérieur de la fleur, dans le tube de corolle, soit sur le pétale. Ma seconde étude montre que les plantes répondent différemment à la présence des oeufs suivant leur position. Aussi, quand l'oeuf est placé dans la fleur, la plante a davantage tendance à ne pas développer le fruit de la fleur infesté ou bien à produire des fruits plus petits que lorsque l'oeuf est placé sur le pétale. Enfin, j'ai montré que la femelle du papillon pond plus souvent sur le pétale lorsque elle visite des fleurs dotées d'un long tube de corolle, et que les larves issues de ces oeufs ont moins de chances de réussir à pénétrer dans le fruit que les larves issues des oeufs placés à l'intérieur de la fleur. Aussi, la variation observée du site de ponte pourrait être causé par la morphologie de la fleur qui contraint le papillon à pondre sur le pétale. Vu dans leur ensemble, les résultats obtenus pendant ce doctorat suggèrent que la participation des mâles à la pollination, l'absence de développement des fruits et la profondeur du tube de corolle pourraient réduire les coûts que S. latifolia subit dans son interaction avec H. bicruris. Par ailleurs, je n'ai pas détecté de mécanismes qui permettraient au papillon de réduire les coûts que la plante pourrait lui imposer. La prochaine étape serait de déterminer l'effet des traits identifiés dans ce doctorat sur la fitness globale de la plante et du papillon pour estimer pleinement leur efficacité à réduire les coûts et à favoriser une issue mutualiste. De même, il faudrait évaluer l'effet de ces traits en populations naturelles pour identifier le rôle des facteurs environnementaux sur leur efficacité.AbstractAlthough mutualisms can be regarded as harmonious relationships between the interacting partners, they are best conceptualized as reciprocal exploitations in which each partner attempts to increase its own benefits and decrease its costs. To date, identifying the factors which promote or discourage mutualistic outcomes remains a major goal to reconstruct the ecological conditions leading to mutualisms. The aim of this PhD thesis was to identify phenotypic traits that may increase the net benefits of each partner in the interaction between the plant Silene latifolia (Caryophyllaceae) and its pollinator / seed predator, the moth Hadena bicruris (Noctuidae). This study system is particularly well suited because the fitness of both interacting species can be assessed. The female moth lays its egg in the flower it pollinated, and its offspring grows in the fruit, feeding on the seeds of the plant, which allows for the follow-up of both larva and fruit fates. Furthermore, the inherent conflict of interest over the seeds as plant progeny vs. larval resource may stimulate the evolution of traits that reduce overexploitation in both the moth and plant. In a first study, I show that male moths are efficient pollinators, hence increasing seed production without increasing oviposition. The contribution of male moths to pollination might thus improve the net benefits of the interaction for the host plant. Females of the H. bicruris moth lay a single egg per flower, and place it either inside the corolla tube or on the petal. My second study shows that plants are more likely to abort the infested flower or to produce a smaller fruit when the egg was experimentally placed inside the flower compared to plants that received an egg on the petal. Finally, female moths were found to lay their eggs more frequently on the petal when visiting a flower with a deep corolla tube, and larvae hatching from these eggs less likely to successfully attack the fruit. Variation in egg position on the flower may thus be the result of a constraint imposed by floral morphology. Overall, this PhD work suggests that the pollination by male moths, flower abortion, and deep corolla tube may efficiently reduce the costs experienced by S. latifolia in its interaction with H. bicruris. Interestingly, no apparent mechanism of costs reduction was detected for the moth. Further studies should focus on the effects of these traits (i) in the long term fitness of both the plant and the insect and (ii) their interactions with environmental factors (biotic and abiotic) that may affect their efficiency in natural populations.

Catheter entrapment in a pulmonary vein: a unique complication of pulmonary vein isolation.

Ablation strategies for the treatment of atrial fibrillation (AF) are associated with several potential complications. During electro-anatomic mapping of the left atrium (LA) before ablation, the ablation catheter was entrapped in the right inferior pulmonary vein (RIPV). After multiple unsuccessful gentle tractions, stronger maneuvers with rotation of the catheter slowly allowed its retrieval. Examination of the catheter showed a thin, translucent membrane covering its tip, suggesting complete stripping of a vein branch. Occlusion of the superior branch of the RIPV was confirmed by LA angiogram. During the following days, no pericardial effusion was noted, but the patient complained of light chest pain and mild hemoptysis, spontaneously resolving within 48 h. This case shows that catheter entrapment and mechanical disruption of a PV branch can be a rare potential complication of AF ablation. In this case, the outcome was spontaneously favorable and symptoms only included transient mild hemoptysis.

Massive nest-box supplementation boosts fecundity, survival and even immigration without altering mating and reproductive behaviour in a rapidly recovered bird population.

Habitat restoration measures may result in artificially high breeding density, for instance when nest-boxes saturate the environment, which can negatively impact species' demography. Potential risks include changes in mating and reproductive behaviour such as increased extra-pair paternity, conspecific brood parasitism, and polygyny. Under particular cicumstances, these mechanisms may disrupt reproduction, with populations dragged into an extinction vortex. With the use of nuclear microsatellite markers, we investigated the occurrence of these potentially negative effects in a recovered population of a rare secondary cavity-nesting farmland bird of Central Europe, the hoopoe (Upupa epops). High intensity farming in the study area has resulted in a total eradication of cavity trees, depriving hoopoes from breeding sites. An intensive nest-box campaign rectified this problem, resulting in a spectacular population recovery within a few years only. There was some concern, however, that the new, high artificially-induced breeding density might alter hoopoe mating and reproductive behaviour. As the species underwent a serious demographic bottleneck in the 1970-1990s, we also used the microsatellite markers to reconstitute the demo-genetic history of the population, looking in particular for signs of genetic erosion. We found i) a low occurrence of extra-pair paternity, polygyny and conspecific brood parasitism, ii) a high level of neutral genetic diversity (mean number of alleles and expected heterozygosity per locus: 13.8 and 83%, respectively) and, iii) evidence for genetic connectivity through recent immigration of individuals from well differentiated populations. The recent increase in breeding density did thus not induce so far any noticeable detrimental changes in mating and reproductive behaviour. The demographic bottleneck undergone by the population in the 1970s-1990s was furthermore not accompanied by any significant drop in neutral genetic diversity. Finally, genetic data converged with a concomitant demographic study to evidence that immigration strongly contributed to local population recovery.

PCR-based isolation of multigene families: lessons from the avian MHC class IIB.

The amount of sequence data available today highly facilitates the access to genes from many gene families. Primers amplifying the desired genes over a range of species are readily obtained by aligning conserved gene regions, and laborious gene isolation procedures can often be replaced by quicker PCR-based approaches. However, in the case of multigene families, PCR-based approaches bear the often ignored risk of incomplete isolation of family members. This problem is most prominent in gene families with highly variable and thus unpredictable number of gene copies among species, such as in the major histocompatibility complex (MHC). In this study, we (i) report new primers for the isolation of the MHC class IIB (MHCIIB) gene family in birds and (ii) share our experience with isolating MHCIIB genes from an unprecedented number of avian species from all over the avian phylogeny. We report important and usually underappreciated problems encountered during PCR-based multigene family isolation and provide a collection of measures to help significantly improving the chance of successfully isolating complete multigene families using PCR-based approaches.

Alpha 3 domain mutants of peptide/MHC class I multimers allow the selective isolation of high avidity tumor-reactive CD8 T cells.

The goal of adoptive T cell therapy in cancer is to provide effective antitumor immunity by transfer of selected populations of tumor Ag-specific T cells. Transfer of T cells with high TCR avidity is critical for in vivo efficacy. In this study, we demonstrate that fluorescent peptide/MHC class I multimeric complexes incorporating mutations in the alpha3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor can be used to selectively isolate tumor Ag-specific T cells of high functional avidity from both in vitro expanded and ex vivo T cell populations. Sorting, cloning, and expansion of alpha3 domain mutant multimer-positive CD8 T cells enabled rapid selection of high avidity tumor-reactive T cell clones. Our results are relevant for ex vivo identification and isolation of T cells with potent antitumor activity for adoptive T cell therapy.

Vascular dysfunction in children conceived by assisted reproductive technologies: underlying mechanisms and future implications.

Epidemiological studies in humans have demonstrated a relationship between pathological events during fetal development and increased cardiovascular risk later in life and have led to the so called "Fetal programming of cardiovascular disease hypothesis". The recent observation of generalised vascular dysfunction in young apparently healthy children conceived by assisted reproductive technologies (ART) provides a novel and potentially very important example of this hypothesis. This review summarises recent data in ART children demonstrating premature subclinical atherosclerosis in the systemic circulation and pulmonary vascular dysfunction predisposing to exaggerated hypoxia-induced pulmonary hypertension. These problems appear to be related to the ART procedure per se. Studies in ART mice demonstrating premature vascular aging and arterial hypertension further demonstrate the potential of ART to increase cardiovascular risk and have allowed to unravel epigenetic alterations of the eNOS gene as an underpinning mechanism. The roughly 25% shortening of the life span in ART mice challenged with a western style high-fat-diet demonstrates the potential importance of these alterations for the long-term outcome. Given the young age of the ART population, data on cardiovascular endpoints will not be available before 20 to 30 years from now. However, already now cohort studies of the ART population are needed to early detect cardiovascular alterations with the aim to prevent or at least optimally treat cardiovascular complications. Finally, a debate needs to be engaged on the future of ART and the consequences of its exponential growth for public health.

Isolation and characterisation of microsatellite loci for two species of Spinturnicid wing mites (Spinturnix myoti and Spinturnix bechsteini)

To investigate the potential for host-parasite coadaptation between bats and their wing mites, we developed microsatellite loci for two species of Spinturnix mites. For Spinturnix myoti, parasite of Myotis myotis, we were able to develop nine polymorphic loci and screened them in 100 mites from five bat colonies. For S. bechsteini, parasite of M. bechsteinii, we developed five polymorphic loci, which were also screened in 100 mites from five bat colonies. In both species, all markers were highly polymorphic (22-46 and 6-23 alleles per locus respectively). The majority of markers for both species exhibited departure from Hardy-Weinberg proportions (8 of 9 and 3 of 5, respectively). One marker pair in S. myoti showed evidence for linkage disequilibrium. As the observed departures from Hardy-Weinberg proportions are most likely a consequence of the biology of the mites, the described microsatellite loci should be useful in studying population genetics and host-parasite dynamics of Spinturnix myoti and Spinturnix bechsteini in relation to their bat hosts.

Energy requirements during reproduction and reproductive effort in shrews (Soricidae)

The energy requirements of reproducing and non-reproducing females of three species of Crocidura (C. russula, C. viaria, C. olivieri), and two species of Sorex (S. coronatus, S. minutus) were measured. Members of these two genera show different rates of metabolism and reproductive strategies (extreme altriciality and larger litter size in Sorex). During pregnancy, the daily energy intake (on either an absolute or a mass-specific basis) remained close to the non-reproductive value in all species. The absolute energy intake increased strongly after parturition and was influenced by the litter size. Peak energy intake of lactating females was extremely high, typically between 100% and 200% above the non-reproductive requirements in the Crocidura and about 300% above the non-reproductive intake in the Sorex. The mass-specific daily energy intake was reduced during lactation in the three smaller species but not in C. viaria and C. olivieri. This decrease probably involves the different thermoregulatory abilities and/or basal rate of metabolism of the pups. Average reproductive effort was about 50%, in the Crocidura species and above 150% in the Sorex species. The higher effort in the latter is partly due to a larger litter size. But in addition, extreme altriciality in the Sorex leads to an earlier increase in the energy requirements and thus is an energetically more expensive reproductive mode. The present results support the hypothesis that a higher basal rate of metabolism is associated with a higher reproductive effort in shrews.

Origin and outcome of multiple pregnancies in Bern, Switzerland, 1995-2006 and the current proposal of the Swiss parliament to revise the Swiss law of reproductive medicine: Switzerland quo vadis?

INTRODUCTION: Infertility treatments are a major source of the increase in multiple pregnancies (MPs). AIMS: The aims of the present study were (1.) to investigate the origin and maternal/neonatal outcomes of MP and (2.) to review the different measures that can be adopted to reduce these serious complications. METHODS: The study included all women with multiple births between 1 January 1995 and 31 December 2006 at the University Hospital of Bern, Switzerland. The outcomes associated with the various origins of MP (natural conception, ovarian stimulation [OS] ‒ in-vitro fertilisation [IVF-ICSI]) were analysed using a multinomial logistic regression model. An analysis of the Swiss law on reproductive medicine and its current proposed revision, as well as a literature review using Pubmed, was carried out. RESULTS: A total of 592 MP were registered, 91% (n = 537) resulted in live births. There was significantly more neonatal/maternal morbidity in MP after OS compared with natural conception and even with the IVF-ICSI group. With a policy of elective single embryo transfer (eSET), twin rates after IVF-ICSI can be reduced to <5% and triplets to <1%. CONCLUSIONS: After OS, more triplets are found and the outcome of MP is worse. MP is known to be associated with morbidity, mortality, and economic and social risks. To counteract these complications (1.) better training for physicians performing OS should be encouraged and (2.) the Swiss law on reproductive medicine needs to be changed, with the introduction of eSET policies. This would lead to a dramatic decrease in neonatal and maternal morbidity/mortality as well as significant cost reductions for the Swiss healthcare system.

Individual reproductive success and effective population size in the greater white-toothed shrew Crocidura russula.

In order to investigate the determinants of effective population size in the socially monogamous Crocidura russula, the reproductive output of 44 individuals was estimated through genetic assignment methods. The individual variance in breeding success turned out to be surprisingly high, mostly because the males were markedly less monogamous than expected from previous behavioural data. Males paired simultaneously with up to four females and polygynous males had significantly more offspring than monogamous ones. The variance in female reproductive success also exceeded that of a Poisson distribution (though to a lesser extent), partly because females paired with multiply mated males weaned significantly more offspring. Polyandry also occurred occasionally, but only sequentially (i.e. without multiple paternity of litters). Estimates of the effective to census size ratio were ca. 0.60, which excluded the mating system as a potential explanation for the high genetic variance found in this shrew's populations. Our data suggest that gene flow from the neighbourhood (up to one-third of the total recruitment) is the most likely cause of the high levels of genetic diversity observed in this shrew's subpopulations.

Three-dimensional morphological analysis of intracranial aneurysms: a fully automated method for aneurysm sac isolation and quantification

Morphological descriptors are practical and essential biomarkers for diagnosis andtreatment selection for intracranial aneurysm management according to the current guidelinesin use. Nevertheless, relatively little work has been dedicated to improve the three-dimensionalquanti cation of aneurysmal morphology, automate the analysis, and hence reduce the inherentintra- and inter-observer variability of manual analysis. In this paper we propose a methodologyfor the automated isolation and morphological quanti cation of saccular intracranial aneurysmsbased on a 3D representation of the vascular anatomy.