Biology and pathogenicity of "Chlamydia"- related organisms

Abstract : This thesis investigates the pathogenicity and biology of Parachlamydia acanthamoebae and other obligate intracellular bacteria related to chlamydiae. All these Chlamydia-like organisms replicate in amoebae. Some evolved to resist to macrophages and represent possible new agents of respiratory tract infection. Using serological and molecular approaches, we showed that Parachlamydia acanthameobae likely plays a role as an etiological agent of pneumonia [1,2]. We also showed that Parachlamydia was able to enter and survive within pneumocytes and lung fibroblasts [3]. Moreover, we developed an animal model of lung infection in mice, which fulfilled the third and fourth Koch postulate [4]. Given the likely role of Parachlamydia in pneumonia, we studied its antibiotic susceptibility. We showed that Chlamydia-related organisms were resistant to quinolones, mainly due to mutations in the QRDR of gyrA [5]. To have tools to investigate the role of other Chlamydia-related bacteria in pneumonia, we developed immunofluorescence assays and assessed the rate of serological cross-reactivity between all these Chlamydia-related bacteria [6]. We also developed new diagnostic specific PCRs [2,7] and sequenced additional genes that are useful for both taxonomic and diagnostic purposes [8]. Then, we applied these serological and molecular approaches to patients with and without respiratory tract infections. This led to the identification of a possible role of Protochlamydia naegleriophila [7] and of Waddlia chondrophila in pneumonia [1]. A significant part of the thesis also investigated interactions of Parachlamydia with macrophages [9] and the host range of Chlamydia-related bacteria [10]. In conclusion, there are growing body of evidence supporting the role of Chlamydia-like organisms as agents of pneumonia. Further studies are needed to precise their pathogenic role in this setting. The diagnostic tools developed during this thesis will be useful to investigate the role of these strict intracellular bacteria in other diseases in both humans and animals [11,12]. Résumé : Le but de cette thèse est de déterminer le rôle pathogène de Parachlamydia et des bactéries apparentées aux Chlamydia ainsi que d'étudier leur biologie. Parachlamydia acanthamoebae est une bactérie intracellulaire apparentée aux Chlamydia, et qui est résistante non seulement aux amibes mais aussi aux macrophages. Par une approche sérologique et moléculaire, nous avons montré que les bactéries apparentées aux Chlamydia jouent probablement un rôle comme agent de pneumonie [1,2]. De plus, nous avons démontré que P. acanthameobae est capable d'entrer et de survivre dans les pneumocytes et fibroblastes pulmonaires [3]. Nous avons ensuite développé un modèle animal remplissant les troisième et quatrième postulats de Koch [4]. Nous avons aussi démontré que les bactéries apparentées aux Chlamydia sont résistantes aux quinolones, en raison de mutations dans la région QRDR de gyrA [5]. Afin de mieux déterminer le rôle pathogène de ces bactéries, nous avons mis au point des techniques d' immunofluorescence et déterminé la cross-réaction sérologique entre les différentes bactéries apparentées aux Chlamydia [6]. Différentes PCR diagnostiques ont aussi été développées [2,7] et des gènes supplémentaires ont été séquencés, qui seront utiles à la taxonomie ainsi qu'au développement de nouvelles méthodes diagnostiques [8]. Ces méthodes ont été appliquées à des échantillons provenant de patient avec ou sans pneumonie et ont permis l'identification du possible rôle pathogène de Protochlamydia naegleriophila [7] et de Waddlia chondrophila [1]. L'interaction de Parachlamydia avec les macrophages [9] et la permissivité de différentes cellules aux bactéries apparentées aux Chlamydia [10] ont également été étudiés dans le cadre de cette thèse. En conclusion, plusieurs nouveaux éléments viennent renforcer l'hypothèse que les bactéries apparentées aux Chlamydia sont des agents de pneumonies. Cependant, d'autres études doivent être menées pour confirmer leur rôle dans cette maladie. Les méthodes diagnostiques développées ici seront très utiles pour déterminer le rôle pathogène de ces bactéries chez les humains et animaux [11,12]

Hilbert space on probability density functions with Aitchison geometry

Compositional data analysis motivated the introduction of a complete Euclidean structure in the simplex of D parts. This was based on the early work of J. Aitchison (1986) and completed recently when Aitchinson distance in the simplex was associated with an inner product and orthonormal bases were identified (Aitchison and others, 2002; Egozcue and others, 2003). A partition of the support of a random variable generates a composition by assigning the probability of each interval to a part of the composition. One can imagine that the partition can be refined and the probability density would represent a kind of continuous composition of probabilities in a simplex of infinitely many parts. This intuitive idea would lead to a Hilbert-space of probability densitiesby generalizing the Aitchison geometry for compositions in the simplex into the set probability densities

Biology, diversity and strategies for the monitoring and control of triatomines - Chagas disease vectors

Despite the relevant achievements in the control of the main Chagas disease vectors Triatoma infestans and Rhodnius prolixus, several factors still promote the risk of infection. The disease is a real threat to the poor rural regions of several countries in Latin America. The current situation in Brazil requires renewed attention due to its high diversity of triatomine species and to the rapid and drastic environmental changes that are occurring. Using the biology, behaviour and diversity of triatomines as a basis for new strategies for monitoring and controlling the vectorial transmission are discussed here. The importance of ongoing long-term monitoring activities for house infestations by T. infestans, Triatoma brasiliensis, Panstrongylus megistus, Triatoma rubrovaria and R. prolixus is also stressed, as well as understanding the invasion by sylvatic species. Moreover, the insecticide resistance is analysed. Strong efforts to sustain and improve surveillance procedures are crucial, especially when the vectorial transmission is considered interrupted in many endemic areas.

Waddlia chondrophila: from biology to pathogenicity.

Waddlia chondrophila is an emerging pathogen causing miscarriages in humans and abortions in ruminants. The full genome of this Chlamydia-related bacterium has been recently completed, providing new insights into its biology and evolution. Moreover, new cell biology approaches and the use of novel inhibitors have allowed detailed investigations of its interaction with host cells.

Full label space reduction in MPLS networks: asymmetric merged tunneling

Traffic Engineering objective is to optimize network resource utilization. Although several works have been published about minimizing network resource utilization in MPLS networks, few of them have been focused in LSR label space reduction. This letter studies Asymmetric Merged Tunneling (AMT) as a new method for reducing the label space in MPLS network. The proposed method may be regarded as a combination of label merging (proposed in the MPLS architecture) and asymmetric tunneling (proposed recently in our previous works). Finally, simulation results are performed by comparing AMT with both ancestors. They show a great improvement in the label space reduction factor

Label Space Reduction in MPLS Networks: How Much Can A Single Stacked Label Do?

Most network operators have considered reducing LSR label spaces (number of labels used) as a way of simplifying management of underlaying virtual private networks (VPNs) and therefore reducing operational expenditure (OPEX). The IETF outlined the label merging feature in MPLS-allowing the configuration of multipoint-to-point connections (MP2P)-as a means of reducing label space in LSRs. We found two main drawbacks in this label space reduction a)it should be separately applied to a set of LSPs with the same egress LSR-which decreases the options for better reductions, and b)LSRs close to the edge of the network experience a greater label space reduction than those close to the core. The later implies that MP2P connections reduce the number of labels asymmetrically

Fine-tuning the space, time, and host distribution of mycobacteria in wildlife.

BACKGROUND We describe the diversity of two kinds of mycobacteria isolates, environmental mycobacteria and Mycobacterium bovis collected from wild boar, fallow deer, red deer and cattle in Doñana National Park (DNP, Spain), analyzing their association with temporal, spatial and environmental factors. RESULTS High diversity of environmental mycobacteria species and M. bovis typing patterns (TPs) were found. When assessing the factors underlying the presence of the most common types of both environmental mycobacteria and M. bovis TPs in DNP, we evidenced (i) host species differences in the occurrence, (ii) spatial structuration and (iii) differences in the degree of spatial association of specific types between host species. Co-infection of a single host by two M. bovis TPs occurred in all three wild ungulate species. In wild boar and red deer, isolation of one group of mycobacteria occurred more frequently in individuals not infected by the other group. While only three TPs were detected in wildlife between 1998 and 2003, up to 8 different ones were found during 2006-2007. The opposite was observed in cattle. Belonging to an M. bovis-infected social group was a significant risk factor for mycobacterial infection in red deer and wild boar, but not for fallow deer. M. bovis TPs were usually found closer to water marshland than MOTT. CONCLUSIONS The diversity of mycobacteria described herein is indicative of multiple introduction events and a complex multi-host and multi-pathogen epidemiology in DNP. Significant changes in the mycobacterial isolate community may have taken place, even in a short time period (1998 to 2007). Aspects of host social organization should be taken into account in wildlife epidemiology. Wildlife in DNP is frequently exposed to different species of non-tuberculous, environmental mycobacteria, which could interact with the immune response to pathogenic mycobacteria, although the effects are unknown. This research highlights the suitability of molecular typing for surveys at small spatial and temporal scales.

Niche dynamics in space and time.

Niche conservatism, the tendency of a species niche to remain unchanged over time, is often assumed when discussing, explaining or predicting biogeographical patterns. Unfortunately, there has been no basis for predicting niche dynamics over relevant timescales, from tens to a few hundreds of years. The recent application of species distribution models (SDMs) and phylogenetic methods to analysis of niche characteristics has provided insight to niche dynamics. Niche shifts and conservatism have both occurred within the last 100 years, with recent speciation events, and deep within clades of species. There is increasing evidence that coordinated application of these methods can help to identify species which likely fulfill one key assumption in the predictive application of SDMs: an unchanging niche. This will improve confidence in SDM-based predictions of the impacts of climate change and species invasions on species distributions and biodiversity.

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

Evaluation of RbCl and CrCl3 as markers of Triatoma brasiliensis (Hemiptera: Reduviidae) nymphs: persistence and influence of Rb and Cr on triatomine biology

In order to mark Triatoma brasiliensis, the vector of Chagas disease in Brazil, two chemical compounds, rubidium chloride (RbCl) and chromium chloride (CrCl3), were tested. First, 199 N2-N5 nymphs were fed on blood with 0.025M RbCl. Rb marker positivity ranged from 2.5% (N3)-70% (N2), with a maximum persistence of 98 days. Second, 265 N2-N5 nymphs were fed on blood containing 0.0015M CrCl3. Cr marker positivity ranged up to 93% (N5), with a maximum persistence of 119 days. Finally, we blood fed 213 T. brasiliensis to investigate whether CrCl3 altered the biology of this insect. The developmental time of T. brasiliensis was unaltered, but the survival of the Cr-marked group was lower than that of the control group. Differences in the mean fecundity of the control (mean of 156.1) and experimental (mean of 135.6) groups were not statistically significant and 100% of the egg batches of females Cr-marked as nymphs were positive. In conclusion, CrCl3 is a useful tool for marking T. brasiliensis nymphs due to its high positivity and persistence.

A comparative evaluation of endemic and non-endemic region of visceral leishmaniasis (Kala-azar) in India with ground survey and space technology

In visceral leishmaniasis, phlebotomine vectors are targets for control measures. Understanding the ecosystem of the vectors is a prerequisite for creating these control measures. This study endeavours to delineate the suitable locations of Phlebotomus argentipes with relation to environmental characteristics between endemic and non-endemic districts in India. A cross-sectional survey was conducted on 25 villages in each district. Environmental data were obtained through remote sensing images and vector density was measured using a CDC light trap. Simple linear regression analysis was used to measure the association between climatic parameters and vector density. Using factor analysis, the relationship between land cover classes and P. argentipes density among the villages in both districts was investigated. The results of the regression analysis indicated that indoor temperature and relative humidity are the best predictors for P. argentipes distribution. Factor analysis confirmed breeding preferences for P. argentipes by landscape element. Minimum Normalised Difference Vegetation Index, marshy land and orchard/settlement produced high loading in an endemic region, whereas water bodies and dense forest were preferred in non-endemic sites. Soil properties between the two districts were studied and indicated that soil pH and moisture content is higher in endemic sites compared to non-endemic sites. The present study should be utilised to make critical decisions for vector surveillance and controlling Kala-azar disease vectors.

New tools for epidemiology: a space odyssey

Geographical Information Systems (GIS) facilitate access to epidemiological data through visualization and may be consulted for the development of mathematical models and analysis by spatial statistics. Variables such as land-cover, land-use, elevations, surface temperatures, rainfall etc. emanating from earth-observing satellites, complement GIS as this information allows the analysis of disease distribution based on environmental characteristics. The strength of this approach issues from the specific environmental requirements of those causative infectious agents, which depend on intermediate hosts for their transmission. The distribution of these diseases is restricted, both by the environmental requirements of their intermediate hosts/vectors and by the ambient temperature inside these hosts, which effectively govern the speed of maturation of the parasite. This paper discusses the current capabilities with regard to satellite data collection in terms of resolution (spatial, temporal and spectral) of the sensor instruments on board drawing attention to the utility of computer-based models of the Earth for epidemiological research. Virtual globes, available from Google and other commercial firms, are superior to conventional maps as they do not only show geographical and man-made features, but also allow instant import of data-sets of specific interest, e.g. environmental parameters, demographic information etc., from the Internet.

A continuous strain-space model of viral evolution within a host

Viruses rapidly evolve, and HIV in particular is known to be one of the fastest evolving human viruses. It is now commonly accepted that viral evolution is the cause of the intriguing dynamics exhibited during HIV infections and the ultimate success of the virus in its struggle with the immune system. To study viral evolution, we use a simple mathematical model of the within-host dynamics of HIV which incorporates random mutations. In this model, we assume a continuous distribution of viral strains in a one-dimensional phenotype space where random mutations are modelled by di ffusion. Numerical simulations show that random mutations combined with competition result in evolution towards higher Darwinian fitness: a stable traveling wave of evolution, moving towards higher levels of fi tness, is formed in the phenoty space.