Revisiting the Pneumocystis host specificity paradigm and transmission ecology in wild Southeast Asian rodents.

Pneumocystis fungi are opportunistic parasites of mammalian lungs whose evolution, ecology and host specificity in natural host populations remain poorly understood and controversial. Using an extensive collection of 731 lung samples from 27 rodent species sampled in five Southeast Asian countries, and nested PCR amplification of mitochondrial and nuclear genes, we investigated the host specificity and genetic structure of Pneumocystis lineages infecting wild rodents. We also identified the rodent species playing a central role in the transmission of these parasites using network analysis and centrality measurement and we characterized the environmental conditions allowing Pneumocystis infection in Southeast Asia using generalized linear mixed models. Building upon an unprecedented Pneumocystis sampling from numerous rodent species belonging to closely related genera, our findings provide compelling evidence that the host specificity of Pneumocystis lineages infecting rodents is not restricted to a single host species or genus as often presented in the literature but it encompasses much higher taxonomic levels and more distantly related rodent host species. The phylogenetic species status at both mitochondrial and nuclear genetic markers of at least three new Pneumocystis lineages, highly divergent from Pneumocystis species currently described, is also suggested by our data. Our models show that the probability of Pneumocystis infection in rodent hosts is positively correlated to environmental variables reflecting habitat fragmentation and landscape patchiness. Synanthropic and habitat-generalist rodents belonging to the Rattus, Sundamys and Bandicota genera played a role of bridge host species for Pneumocystis spreading in these heterogeneous habitats, where they can reach high population densities. These are critical findings improving our understanding of the ecology of these enigmatic parasites and the role played by cospeciation and host switches in their evolution. Our results also confirmed the role of land-use change and habitat fragmentation in parasite amplification and spillover in rodents.

Drug-Resistant Tuberculosis, Lebanon, 2016 - 2017.

In a 12-month nationwide study on the prevalence of drug-resistant tuberculosis (TB) in Lebanon, we identified 3 multidrug-resistant cases and 3 extensively drug-resistant TB cases in refugees, migrants, and 1 Lebanon resident. Enhanced diagnostics, particularly in major destinations for refugees, asylum seekers, and migrant workers, can inform treatment decisions and may help prevent the spread of drug-resistant TB.

Correction: What Do Pneumocystis Organisms Tell Us about the Phylogeography of Their Hosts? The Case of the Woodmouse Apodemus sylvaticus in Continental Europe and Western Mediterranean Islands.

[This corrects the article DOI: 10.1371/journal.pone.0120839.].

Alphacoronaviruses Detected in French Bats Are Phylogeographically Linked to Coronaviruses of European Bats.

Bats are a reservoir for a diverse range of viruses, including coronaviruses (CoVs). To determine the presence of CoVs in French bats, fecal samples were collected between July and August of 2014 from four bat species in seven different locations around the city of Bourges in France. We present for the first time the presence of alpha-CoVs in French Pipistrellus pipistrellus bat species with an estimated prevalence of 4.2%. Based on the analysis of a fragment of the RNA-dependent RNA polymerase (RdRp) gene, phylogenetic analyses show that alpha-CoVs sequences detected in French bats are closely related to other European bat alpha-CoVs. Phylogeographic analyses of RdRp sequences show that several CoVs strains circulate in European bats: (i) old strains detected that have probably diverged a long time ago and are detected in different bat subspecies; (ii) strains detected in Myotis and Pipistrellus bat species that have more recently diverged. Our findings support previous observations describing the complexity of the detected CoVs in bats worldwide.

What do Pneumocystis organisms tell us about the phylogeography of their hosts? The case of the woodmouse Apodemus sylvaticus in continental Europe and western Mediterranean islands.

Pneumocystis fungi represent a highly diversified biological group with numerous species, which display a strong host-specificity suggesting a long co-speciation process. In the present study, the presence and genetic diversity of Pneumocystis organisms was investigated in 203 lung samples from woodmice (Apodemus sylvaticus) collected on western continental Europe and Mediterranean islands. The presence of Pneumocystis DNA was assessed by nested PCR at both large and small mitochondrial subunit (mtLSU and mtSSU) rRNA loci. Direct sequencing of nested PCR products demonstrated a very high variability among woodmouse-derived Pneumocystis organisms with a total number of 30 distinct combined mtLSU and mtSSU sequence types. However, the genetic divergence among these sequence types was very low (up to 3.87%) and the presence of several Pneumocystis species within Apodemus sylvaticus was considered unlikely. The analysis of the genetic structure of woodmouse-derived Pneumocystis revealed two distinct groups. The first one comprised Pneumocystis from woodmice collected in continental Spain, France and Balearic islands. The second one included Pneumocystis from woodmice collected in continental Italy, Corsica and Sicily. These two genetic groups were in accordance with the two lineages currently described within the host species Apodemus sylvaticus. Pneumocystis organisms are emerging as powerful tools for phylogeographic studies in mammals.

Histoplasma capsulatum and Pneumocystis spp. co-infection in wild bats from Argentina, French Guyana, and Mexico.

BACKGROUND: Histoplasma capsulatum and Pneumocystis organisms cause host infections primarily affecting the lung tissue. H. capsulatum is endemic in the United States of America and Latin American countries. In special environments, H. capsulatum is commonly associated with bat and bird droppings. Pneumocystis-host specificity has been primarily studied in laboratory animals, and its ability to be harboured by wild animals remains as an important issue for understanding the spread of this pathogen in nature. Bats infected with H. capsulatum or Pneumocystis spp. have been found, with this mammal serving as a probable reservoir and disperser; however, the co-infection of bats with both of these microorganisms has never been explored. To evaluate the impact of H. capsulatum and Pneumocystis spp. infections in this flying mammal, 21 bat lungs from Argentina (AR), 13 from French Guyana (FG), and 88 from Mexico (MX) were screened using nested-PCR of the fragments, employing the Hcp100 locus for H. capsulatum and the mtLSUrRNA and mtSSUrRNA loci for Pneumocystis organisms. RESULTS: Of the 122 bats studied, 98 revealed H. capsulatum infections in which 55 of these bats exhibited this infection alone. In addition, 51 bats revealed Pneumocystis spp. infection of which eight bats exhibited a Pneumocystis infection alone. A total of 43 bats (eight from AR, one from FG, and 34 from MX) were found co-infected with both fungi, representing a co-infection rate of 35.2% (95% CI = 26.8-43.6%). CONCLUSION: The data highlights the H. capsulatum and Pneumocystis spp.co-infection in bat population's suggesting interplay with this wild host.

Molecular detection of Histoplasma capsulatum in the lung of a free-ranging common noctule (Nyctalus noctula) from France using the Hcp100 gene.

Histoplasma capsulatum is a dimorphic fungus that is widely distributed in the tropical or subtropical areas of the world and infects several mammalian hosts, mainly bats. Infective propagules grow in bat and bird droppings. A specific molecular marker, a highly sensitive fragment of a co-activator protein-coding gene (Hcp100), was used to detect H. capsulatum in lung samples of wild and captive bats from France using a nested polymerase chain reaction. To determine whether bats in France are potential carriers of H. capsulatum, 83 bats were sampled from two regions in France. Sixty-one specimens belonging to the Pteropus rodricensis (n = 45) and Rousettus aegyptiacus (n = 16) species were collected from a zoologic park (La Palmyre, western France). Twenty-two specimens were recovered from the Natural History Museum (Bourges) including the species Plecotus austriacus (n = 1), Pipistrellus pipistrellus (n = 3), and Nyctalus noctula (n = 18). From the lung DNA samples of 83 dead bats, only one sample of an N. noctula bat from Bourges amplified the H. capsulatum Hcp100 marker. The amplified product was sequenced and revealed a high similarity to the G217B H. capsulatum reference strain sequence that was deposited in the GenBank database. This finding suggests that H. capsulatum is an environmental pathogen in France that may infect bats.

Characterizing Pneumocystis in the lungs of bats: understanding Pneumocystis evolution and the spread of Pneumocystis organisms in mammal populations.

Bats belong to a wide variety of species and occupy diversified habitats, from cities to the countryside. Their different diets (i.e., nectarivore, frugivore, insectivore, hematophage) lead Chiroptera to colonize a range of ecological niches. These flying mammals exert an undisputable impact on both ecosystems and circulation of pathogens that they harbor. Pneumocystis species are recognized as major opportunistic fungal pathogens which cause life-threatening pneumonia in severely immunocompromised or weakened mammals. Pneumocystis consists of a heterogeneous group of highly adapted host-specific fungal parasites that colonize a wide range of mammalian hosts. In the present study, 216 lungs of 19 bat species, sampled from diverse biotopes in the New and Old Worlds, were examined. Each bat species may be harboring a specific Pneumocystis species. We report 32.9% of Pneumocystis carriage in wild bats (41.9% in Microchiroptera). Ecological and behavioral factors (elevation, crowding, migration) seemed to influence the Pneumocystis carriage. This study suggests that Pneumocystis-host association may yield much information on Pneumocystis transmission, phylogeny, and biology in mammals. Moreover, the link between genetic variability of Pneumocystis isolated from populations of the same bat species and their geographic area could be exploited in terms of phylogeography.

Pneumocystis species, co-evolution and pathogenic power.

The genus Pneumocystis comprises uncultured, highly diversified microfungal organisms able to attach specifically to type-I alveolar epithelial cells and to proliferate in pulmonary alveoli provoking severe pneumonitis. The pathogenic potential of Pneumocystis species, especially of the human-associated Pneumocystis jirovecii, has stimulated a growing interest in these peculiar microfungi. However, a comprehensive understanding of basic biology and pathogenic power of Pneumocystis organisms calls for their recognition as natural, complex entities, without reducing them to their pathogenic role. For many years, the entity named "Pneumocystis carinii" was considered like an anecdotal pulmonary pathogen able to cause pneumonia in immunosuppressed hosts. Only for the last years, marked genetic divergence was documented among the Pneumocystis strains of different mammals. Cross-infection experiments showed that Pneumocystis species are stenoxenous parasites. Mainly on the basis of the Phylogenetic Concept of Species, Pneumocystis strains were considered as genuine species. Five species were described: P. carinii and Pneumocystis wakefieldiae in rats, P. jirovecii in humans, Pneumocystis murina in mice, and Pneumocystis oryctolagi in rabbits. They also present distinctive phenotypic features. Molecular techniques have revealed a high prevalence of Pneumocystis colonization in wild mammals, probably resulting from active airborne horizontal and vertical (transplacental or aerial) transmission mechanisms. Cophylogeny is the evolutionary pattern for Pneumocystis species, which dwelt in the lungs of mammals for more than 100 million years. Consistently, Pneumocystis organisms exhibit successful adaptation to colonize the lungs of both immunocompromised and healthy hosts that can act as infection reservoir. Pneumocystis pneumonia, rarely reported in wild mammals, seems to be a rather unfrequent event. A larger spectrum of Pneumocystis infections related to the heterogeneous level of immune defence found in natural populations, is, however, expected. Pneumocystis infection of immunocompetent hosts emerges therefore as a relevant issue to human as well as animal health.

Genetic differentiation in the soil-feeding termite Cubitermes sp. affinis subarquatus: occurrence of cryptic species revealed by nuclear and mitochondrial markers.

BACKGROUND: Soil-feeding termites are particularly interesting models for studying the effects of fragmentation, a natural or anthropic phenomenon described as promoting genetic differentiation. However, studying the link between fragmentation and genetics requires a method for identifying species unambiguously, especially when morphological diagnostic characters are lacking. In humivorous termites, which contribute to the fertility of tropical soils, molecular taxonomy and phylogenetic relationships are rarely studied, though mitochondrial and nuclear molecular markers are widely used in studies of pest termites. Here, we attempt to clarify the taxonomy of soil-feeding colonies collected throughout the naturally fragmented Lopé Reserve area (Gabon) and morphologically affiliated to Cubitermes sp. affinis subarquatus. The mitochondrial gene of cytochrome oxidase II (COII), the second nuclear rDNA internal transcribed spacer (ITS2) and five microsatellites were analyzed in 19 colonies. RESULTS: Bayesian Inference, Maximum Likelihood and Maximum Parsimony phylogenetic analyses, which were applied to the COII and ITS2 sequences, and Neighbor-Joining reconstructions, applied to the microsatellite data, reveal four major lineages in the Cubitermes sp. affinis subarquatus colonies. The concordant genealogical pattern of these unlinked markers strongly supports the existence of four cryptic species. Three are sympatric in the Reserve and are probably able to disperse within a mosaic of forests of variable ages and savannahs. One is limited to a very restricted gallery forest patch located in the North, outside the Reserve. CONCLUSION: Our survey highlights the value of combined mitochondrial and nuclear markers for exploring unknown groups such as soil-feeding termites, and their relevance for resolving the taxonomy of organisms with ambiguous morphological diagnostic characters.

Molecular and serological evidence of Pneumocystis circulation in a social organization of healthy macaques (Macaca fascicularis).

Simian populations represent valuable models for understanding the epidemiology of human pneumocystosis. The present study aims to describe the circulation of Pneumocystis organisms within a social organization of healthy crab-eating macaques (Macaca fascicularis) living in a natural setting in France. Animals were followed for up to 2 years. Deep nasal swab and blood samples were collected monthly from each animal under general anaesthesia. Environmental air was sampled for a 1 week period every month in the park where the macaques dwelt. Pneumocystis DNA was detected by nested-PCR of mitochondrial large subunit rRNA (mtLSU) gene in nasal swab and air samples. Anti-Pneumocystis IgG antibodies were detected in serum samples by indirect immuno-fluorescence assay. Pneumocystis DNA was detected in 168 of 500 swab samples examined (33.6 %). The number of macaques with detectable Pneumocystis DNA was highly variable from one month to another. Positive detection of Pneumocystis DNA was not related to the detection of serum anti-Pneumocystis antibody. During the second year of the study, Pneumocystis DNA was amplified more frequently from unweaned macaques than from adults or subadults. The mtLSU sequence showed marked polymorphism with eight Pneumocystis sequence types representing two distinct groups. On the whole, a constant and intensive circulation of Pneumocystis organisms within the community was observed. However, the implication of the various members of the colony was probably different and several levels of colonization by Pneumocystis may occur in immunocompetent macaques.

Phylogenetic relationships among Pneumocystis from Asian macaques inferred from mitochondrial rRNA sequences.

The presence of Pneumocystis organisms was detected by nested-PCR at mitochondrial large subunit (mtLSU) rRNA gene in 23 respiratory samples from Asian macaques representing two species: Macaca mulatta and M. fascicularis. A very high level of sequence heterogeneity was detected with 18 original sequence types. Two genetic groups of Pneumocystis could be distinguished from the samples. Within each group, the extent of genetic divergence was low (2.5+/-1.4% in group 1 and 2.3+/-1.7% in group 2). Genetic divergences were systematically higher when macaque-derived sequence types were compared with Pneumocystis mtLSU sequences from other primate species (from 5.3+/-2.7% to 19.3+/-3.0%). The two macaque-derived groups may be considered as distinct Pneumocystis species. Surprisingly, these Pneumocystis species were recovered from both M. mulatta and M. fascicularis suggesting that host-species restriction may not systematically occur in the genus Pneumocystis. Alternatively, these observations question about the species concept in macaques.

Bartonella chomelii sp. nov., isolated from French domestic cattle (Bos taurus).

Two strains of bacteria isolated from the blood of French domestic cows were found to be similar to Bartonella species on the basis of phenotypic characteristics. Genotypic analysis based on sequence comparison of the 16S rRNA and citrate synthase (gltA) genes and on DNA-DNA hybridization showed that the two isolates represent a distinct and new species of Bartonella. Moreover, the phylogenetic analysis inferred from comparison of 16S rRNA and gltA sequences demonstrated that the new Bartonella species is related to other ruminant-derived Bartonella species. The name Bartonella chomelii is proposed for the new species. The type strain of Bartonella chomelii sp. nov. is A828T (=CIP 107869T=CCUG47497T).

Phylogenetic systematics and evolution of primate-derived Pneumocystis based on mitochondrial or nuclear DNA sequence comparison.

Previous studies have demonstrated that the agent of Pneumocystis pneumonia (PcP), Pneumocystis carinii, is actually a complex of eukaryotic organisms, and cophylogeny could explain the distribution of the hosts and parasites. In the present work, we tested the hypothesis of cophylogeny between the primate-derived Pneumocystis group and their hosts. Specific strains isolated from 20 primate species, including humans, were used to produce a phylogeny of the parasites. Aligned sequences corresponding to DNA sequences of three genes (DHPS, mtSSU-rRNA, and mtLSU-rRNA) were separately analyzed and then combined in a single data set. The resulting parasite phylogeny was compared with different controversial phylogenies for the hosts. This comparison demonstrated that, depending upon which topology is accepted for the hosts, at least 61% and perhaps 77% of the homologous nodes of the respective cladograms of the hosts and parasites may be interpreted as resulting from codivergence events. This finding and the high specificity of these parasites suggests that cophylogeny may be considered the dominant pattern of evolution for Pneumocystis organisms, representing a new example of parallel evolution between primates and their specific parasites. Because the phylogeny of Pneumocystis followed very closely the differentiation of their hosts at the species level, the study of the parasites could provide valuable information on the phylogeny of their hosts. We used this information to explore controversial hypotheses of the phylogeny of the Platyrrhini by comparison with the phylogeny of their specific Pneumocystis parasites. If these organisms were closely associated as lung parasites with primates through the ages, the hypothesis of the Pneumocystis spp. being new pathogenic agents could be refuted. However, these organisms are opportunistic symbionts, becoming pathogenic whenever the immunological defences of their hosts decline. This study also provides support for the hypothesis that the different Pneumocystis species are genetically independent organisms, helping to clarify their taxonomic status.

Influence of climatic factors on Pneumocystis carriage within a socially organized group of immunocompetent macaques (Macaca fascicularis).

As monkeys-derived Pneumocystis is closely related to P. jirovecii, simian populations should be considered as valuable models for the understanding of the epidemiology of human pneumocystosis. In the present study, the impact of environmental factors on the carriage of Pneumocystis was evaluated in socially organized group of immunocompetent macaques (Macaca fascicularis). The tribe, maintained in partial release at the Primatology Center of Strasbourg in France, comprised 29 animals at the end of the study. From December 2000 to November 2002, deep nasal swab samples were collected monthly from each animal under general anaesthesia. The presence of Pneumocystis DNA was assessed by nested PCR of mtLSU rRNA gene. No case of pneumocystosis was reported during the study. Pneumocystis DNA was detected in 166 out of 481 swab samples examined (34.5%). The number of macaques with detectable Pneumocystis DNA was highly variable from one month to another. However, Pnemocystis carriage was clearly correlated to the mean precipitation rates.