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.
Some compounds articulated around a piperazine or an ethylenediamine linker have been evaluated in vitro to determine their activity in the presence of a 3T6 fibroblast cell line and an axenic culture of Pneumocystis carinii, respectively. The most efficient antifungal derivatives, namely N,N-bis(benzamidine-4-yl)ethane-1,2-diamine (compound 6, a diamidine) and N-(benzamidine-4-yl)-N-phenylethane-1,2-diamine (compound 7, a monoamidine), exhibited no cytotoxicity and were evaluated in vivo in a rat model. Only the diamidine 6 emerged as a promising hit for further studies.
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.
Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms) can be transmitted by aerial route from host to host.
To better understand the role of immunocompetent hosts in the diffusion of Pneumocystis in the environment, airborne shedding of Pneumocystis carinii in the surrounding air of experimentally infected Sprague Dawley rats was quantified by means of a real-time PCR assay, in parallel with the kinetics of P. carinii loads in lungs and specific serum antibody titres. Pneumocystis-free Sprague Dawley rats were intratracheally inoculated at day 0 (d0) and then followed for 60 days. P. carinii DNA was detected in lungs until d29 in two separate experiments and thereafter remained undetectable. A transient air excretion of Pneumocystis DNA was observed between d14 and d22 in the first experiment and between d9 and d19 in the second experiment; it was related to the peak of infection in lungs. IgM and IgG anti-P. carinii antibody increase preceded clearance of P. carinii in the lungs and cessation of airborne excretion. In rats receiving a second challenge 3 months after the first inoculation, Pneumocystis was only detected at a low level in the lungs of 2 of 3 rats at d2 post challenge and was never detected in air samples. Anti-Pneumocystis antibody determinations showed a typical secondary IgG antibody response. This study provides the first direct evidence that immunocompetent hosts can excrete Pneumocystis following a primary acquired infection. Lung infection was apparently controlled by the immune response since fungal burdens decreased to become undetectable as specific antibodies reached high titres in serum. This immune response was apparently protective against reinfection 3 months later.
Our understanding of the correlation of Mycobacterium bovis Bacille Calmette-Guerin (BCG)-mediated immune responses and protection against Mycobacterium tuberculosis (Mtb) infection is still limited. We have recently characterized a Wistar rat model of experimental tuberculosis (TB). In the present study, we evaluated the efficacy of BCG vaccination in this model. Upon Mtb challenge, BCG vaccinated rats controlled growth of the bacilli earlier than unvaccinated rats. Histopathology analysis of infected lungs demonstrated a reduced number of granulomatous lesions and lower parenchymal inflammation in vaccinated animals. Vaccine-mediated protection correlated with the rapid accumulation of antigen specific CD4(+) and CD8(+) T cells in the infected lungs. Immunohistochemistry further revealed higher number of CD8(+) cells in the pulmonary granulomas of vaccinated animals. Evaluation of pulmonary immune responses in vaccinated and Mtb infected rats by real time PCR at day 15 post-challenge showed reduced expression of genes responsible for negative regulation of Th1 immune responses. Thus, early protection observed in BCG vaccinated rats correlated with a similarly timed shift of immunity towards the Th1 type response. Our data support the importance of (i) the Th1-Th2 balance in the control of mycobacterial infection and (ii) the value of the Wistar rats in understanding the biology of TB.
Despite the availability of many animal models for tuberculosis (TB) research, there still exists a need for better understanding of the quiescent stage of disease observed in many humans. Here, we explored the use of the Wistar rat model for the study of protective immunity and control of Mycobacterium tuberculosis (Mtb) infection.
To better understand the diffusion of Pneumocystis in the environment, airborne shedding of Pneumocystis carinii in the surrounding air of experimentally infected rats was quantified by means of a real-time polymerase chain reaction assay, in parallel with the kinetics of P. carinii loads in their lungs. P. carinii DNA was detected in the air 1 week after infection and increased until 4-5 weeks after infection before stabilizing. A significant correlation was shown between lung burdens and the corresponding airborne levels, suggesting the possibility of estimating the fungal lung involvement through quantification of Pneumocystis in the exhaled air.
Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP) is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i) mating/fusion leading to a diploid status or (ii) asexual mitotic division or (iii) both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the complexity of its modes of proliferation.
At the end of the 20th century the unique taxonomically enigmatic entity called Pneumocystis carinii was identified as a heterogeneous group of microscopic Fungi, constituted of multiple stenoxenic biological entities largely spread across ecosystems, closely adapted to, and coevolving in parallel with, mammal species. The discoveries and reasoning that led to the current conceptions about the taxonomy of Pneumocystis at the species level are examined here. The present review also focuses on the biological, morphological and phylogenetical features of Pneumocystis jirovecii, Pneumocystis oryctolagi, Pneumocystis murina, P. carinii and Pneumocystis wakefieldiae, the five Pneumocystis species described until now, mainly on the basis of the phylogenetic species concept. Interestingly, Pneumocystis organisms exhibit a successful adaptation enabling them to dwell and replicate in the lungs of both immunocompromised and healthy mammals, which can act as infection reservoirs. The role of healthy carriers in aerial disease transmission is nowadays recognized as a major contribution to Pneumocystis circulation, and Pneumocystis infection of nonimmunosuppressed hosts has emerged as a public health issue. More studies need to be undertaken both on the clinical consequences of the presence of Pneumocystis in healthy carriers and on the intricate Pneumocystis life cycle to better define its epidemiology, to adapt existing therapies to each clinical context and to discover new drug targets.
Three compounds were isolated from Acnistus arborescens, a tree commonly used in South and Central America in traditional medicine against several infectious diseases, some of which are caused by fungi. Bioassay-guided fractionation of a MeOH extract of leaves, based on its anti-Pneumocystis carinii activity, led to the isolation of compounds 1-3. Mono- and bidimensional NMR analyses enabled identification of two new withanolides, (20R,22R)-5beta,6beta-epoxy-4beta,12beta,20-trihydroxy-1-oxowith-2-en-24-enolide (1) and (20R,22R)-16beta-acetoxy-3beta,4beta;5beta,6beta-diepoxy-12beta,20-dihydroxy-1-oxowith-24-enolide (2), and withanolide D (3). Antifungal activity on 13 fungi responsible for human infections (five dermatophytes, one nondermatophyte mold, six yeasts, and Pneumocystis carinii) was examined. Cytotoxicity of these compounds was also evaluated in vitro.
The separation of Pneumocystis carinii life-cycle stages while preserving infectivity is a hitherto unresolved challenge. We describe an original, reproducible, and efficient method for separating trophic from cystic forms of P. carinii using a high-speed cell sorter. The large amounts of highly purified (99.6+/-0.3%) infectious trophic and cystic forms can now be used to elucidate the poorly understood P. carinii life cycle.
Screening of the antifungal activities of ten Guadeloupean plants was undertaken to find new extracts and formulations against superficial mycoses such as onychomycosis, athletes foot, Pityriasis versicolor, as well as the deep fungal infection Pneumocystis pneumonia. For the first time, the CMI of these plant extracts [cyclohexane, ethanol and ethanol/water (1:1, v/v)] was determined against five dermatophytes, five Candida species, Scytalidium dimidiatum, a Malassezia sp. strain and Pneumocystis carinii. Cytotoxicity tests of the most active extracts were also performed on an HaCat keratinocyte cell line. Results suggest that the extracts of Bursera simaruba, Cedrela odorata, Enterolobium cyclocarpum and Pluchea carolinensis have interesting activities and could be good candidates for developing antifungal formulations.
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.
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