Evidence of Airborne Excretion of Pneumocystis carinii during Infection in Immunocompetent Rats. Lung Involvement and Antibody Response

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.     

Specificity in structure-based drug design: Identification of a novel,selective inhibitor of Pneumocystis carinii dihydrofolate reductase

Specificity is an important aspect of structure-based drug design. Distinguishing between related targets in different organisms is often the key to therapeutic success. Pneumocystis carinii is a fungal opportunist which causes a crippling pneumonia in immunocompromised individuals. We report the identification of novel inhibitors of P. cariniidihydrofolate reductase (DHFR) that are selective versus inhibition of human DHFR using computational molecular docking techniques. The Fine Chemicals Directory, a database of commercially available compounds, was screened with the DOCK program suite to produce a list of potential P. carinii DHFR inhibitors. We then used a postdocking refinement directed at discerning subtle structural and chemical features that might reflect species specificity. Of 40 compounds predicted to exhibit anti-PneumocystisDHFR activity, each of novel chemical framework, 13 (33%) show IC₅₀ values better than 150 μM in an enzyme assay. These inhibitors were further assayed against human DHFR: 10 of the 13 (77%) bind preferentially to the fungal enzyme. The most potent compound identified is a 7 μM inhibitor of P. carinii DHFR with 25-fold selectivity. The ability of molecular docking methods to locate selective inhibitors reinforces our view of structure-based drug discovery as a valuable strategy, not only for identifying lead compounds, but also for addressing receptor specificity. Proteins 29:59–67, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of Pneumocystis Major Surface Glycoprotein Gene (msg) Promoter Activity in Saccharomyces cerevisiae

Major surface glycoprotein (Msg), the most abundant cell surface protein of Pneumocystis, plays an important role in the interaction of this opportunistic pathogen with host cells, and its potential for antigenic variation may facilitate evasion of host immune responses. In the present study, we have identified and characterized the promoter region of msg in 3 species of Pneumocystis: P. carinii, P. jirovecii, and P. murina. Because Pneumocystis cannot be cultured, promoter activity was measured in Saccharomyces cerevisiae, a related fungus, using a yeast vector modified to utilize the gene coding for Renilla luciferase as a reporter gene. The 5′-flanking sequences of msg from all three Pneumocystis species showed considerable promoter activity, with increases in luciferase activity up to 15- to 44-fold above baseline. Progressive deletions helped define an ∼13-bp sequence in each Pneumocystis species that appears to be critical for promoter activity. Electrophoretic mobility shift analysis using P. carinii-specific msg promoter sequences demonstrated binding of nuclear proteins of S. cerevisiae. The 144-bp 5′-flanking region of P. murina msg showed 72% identity to that of P. carinii. The 5′-flanking region of P. jirovecii msg showed 58 and 61% identity to those of P. murina and P. carinii, respectively. The msg promoter is a good candidate for inclusion in a construct designed for genetic manipulation of Pneumocystis species.     

Toxoplasma gondii: a simple high-throughput assay for drug screening in vitro

Toxoplasma gondii is the etiologic agent of toxoplasmosis. Although the combination of sulfadiazine and pyrimethamine is used as therapy for this disease, these drugs can have serious side effects and its use is limited in pregnancy. Therefore there is a need for new anti-T. gondii drugs in the clinic. Some systems for T. gondii drug screening have been described, but these have limitations and can be difficult. In order to solve these problems, we established a system to screen drugs in vitro that involved using cell viability methods to calculate drug selectivities, which are Trypan blue, [3-(4,5-dimethylthiazol-zyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliuzolium, inner salt] (MTS) method and lactate dehydrogenase (LDH) assay. These assays were simple to establish and perform. The IC₅₀ values calculated from the morphological assay were not significantly different from the EC₅₀ values calculated using the other three methods. In particular, the results of the morphological assay showed a distinct association with the MTS assay (R = 0.9841). These assays could be used for a wide range of applications in the screening of new drugs and may provide an alternative to the techniques currently used to screen for candidate anti-T. gondii compounds in vitro. In this study, we also tested many compounds and identified some that had a good anti-T. gondii effect in vitro based on the MTS assay. This simple and fast system allowed us to determine which compounds to investigate further using in vivo experiments.     

Pneumocystis carinii: Genetic Diversity and Cell Biology

As an important opportunistic pulmonary pathogen, Pneumocystis carinii has been the focus of extensive research over the decades. The use of laboratory animal models has permitted a detailed understanding of the host–parasite interaction but an understanding of the basic biology of P. carinii has lagged due in large part to the inability of the organism to grow well in culture and to the lack of a tractable genetic system. Molecular techniques have demonstrated extensive heterogeneity among P. carinii organisms isolated from different host species. Characterization of the genes and genomes of the Pneumocystis family has supported the notion that the family comprises different species rather than strains within the genus Pneumocystis and contributed to the understanding of the pathophysiology of infection. Many of the technical obstacles in the study of the organisms have been overcome in the past decade and the pace of research into the basic biology of the organism has accelerated. Biochemical pathways have been inferred from the presence of key enzyme activities or gene sequences, and attempts to dissect cellular pathways have been initiated. The Pneumocystis genome project promises to be a rich source of information with regard to the functional activity of the organism and the presence of specific biochemical pathways. These advances in our understanding of the biology of this organism should provide for future studies leading to the control of this opportunistic pathogen.     

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.     

Release of Luminal Exosomes Contributes to TLR4-Mediated Epithelial Antimicrobial Defense

Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense.     

Synthesis and SAR of alkanediamide-linked bisbenzamidines with anti-trypanosomal and anti-pneumocystis activity

A series of alkanediamide-linked bisbenzamidines was synthesized and tested in vitro against a drug-sensitive strain of Trypanosoma brucei brucei, a drug-resistant strain of Trypanosoma brucei rhodesiense and Pneumocystis carinii. Bisbenzamidines linked with longer alkanediamide chains were potent inhibitors of both strains of T. brucei. However, bisbenzamidines linked with shorter alkanediamide chains were the most potent compounds against P. carinii. N,N′-Bis[4-(aminoiminomethyl)phenyl] hexanediamide, 4 displayed potent inhibition (IC₅₀ = 2–3 nM) against T. brucei and P. carinii, and was non-cytotoxic in the A549 human lung carcinoma cell line. The inhibitory bioactivity was significantly reduced when the amidine groups in 4 were moved from the para to the meta positions or replaced with amides.     

Immune Modulation with Sulfasalazine Attenuates Immunopathogenesis but Enhances Macrophage-Mediated Fungal Clearance during Pneumocystis Pneumonia

Although T cells are critical for host defense against respiratory fungal infections, they also contribute to the immunopathogenesis of Pneumocystis pneumonia (PcP). However, the precise downstream effector mechanisms by which T cells mediate these diverse processes are undefined. In the current study the effects of immune modulation with sulfasalazine were evaluated in a mouse model of PcP-related Immune Reconstitution Inflammatory Syndrome (PcP-IRIS). Recovery of T cell-mediated immunity in Pneumocystis-infected immunodeficient mice restored host defense, but also initiated the marked pulmonary inflammation and severe pulmonary function deficits characteristic of IRIS. Sulfasalazine produced a profound attenuation of IRIS, with the unexpected consequence of accelerated fungal clearance. To determine whether macrophage phagocytosis is an effector mechanism of T cell-mediated Pneumocystis clearance and whether sulfasalazine enhances clearance by altering alveolar macrophage phagocytic activity, a novel multispectral imaging flow cytometer-based method was developed to quantify the phagocytosis of Pneumocystis in vivo. Following immune reconstitution, alveolar macrophages from PcP-IRIS mice exhibited a dramatic increase in their ability to actively phagocytose Pneumocystis. Increased phagocytosis correlated temporally with fungal clearance, and required the presence of CD4⁺ T cells. Sulfasalazine accelerated the onset of the CD4⁺ T cell-dependent alveolar macrophage phagocytic response in PcP-IRIS mice, resulting in enhanced fungal clearance. Furthermore, sulfasalazine promoted a TH2-polarized cytokine environment in the lung, and sulfasalazine-enhanced phagocytosis of Pneumocystis was associated with an alternatively activated alveolar macrophage phenotype. These results provide evidence that macrophage phagocytosis is an important in vivo effector mechanism for T cell-mediated Pneumocystis clearance, and that macrophage phenotype can be altered to enhance phagocytosis without exacerbating inflammation. Immune modulation can diminish pulmonary inflammation while preserving host defense, and has therapeutic potential for the treatment of PcP-related immunopathogenesis.     

D319 induced antifungal effects through ROS-mediated apoptosis and inhibited isocitrate lyase in Candida albicans

BackgroundCandida albicans (C. albicans) is an opportunistic pathogen that can cause superficial and life-threatening systemic infections in immunocompromised patients. However, the available clinically antifungals are limited. Therefore, the development of effective antifungal agents and therapies is urgently needed. Quinoline type of compounds were reported to possess potent anti-fungal effect. A series of quinoline derivatives were synthesized. Moreover their inhibitory activities and mechanisms on C. albicans were evaluated in this study.MethodsThe structure of D319 was identified by extensive spectroscopic analysis. The antifungal activity of D319 on C. albicans was evaluated using conventional methods, including the inhibition zone diameters with filter paper, Clinical Laboratory Standard Institute (CLSI) broth microdilution method in vitro, and in a murine model in vivo. Flow cytometry, fluorescence microscopy, western blot, knockout mutant and revertant strain techniques, and molecular modeling were applied to explore the mechanism of action of D319 in anti-Candida.ResultsD319 exhibited potent anti-Candida activity with Minimum Inhibitory Concentration value of 2.5 μg/mL in vitro. D319 significantly improved survival rate and reduced fungal burden compared to vehicle control in a murine model in vivo. The treatment of C. albicans with D319 resulted in fungal apoptosis through reactive oxygen species (ROS) accumulation in C. albicans. Furthermore, D319 inhibited the glyoxylate enzyme isocitrate lyase (ICL) of C. albicans, which was also confirmed by docking analysis.ConclusionsQuinoline compound D319 exhibited strong anti-Candida activities in vitro and in vivo models through inhibiting ICL activity and ROS accumulation in C. albicans.General significanceThis study showed that compound D319 as a novel isocitrate lyase inhibitor, would be a promising anti-Candida lead compound, which provided a potential application of this type of compounds in fighting clinical fungal infections. Furthermore, this study also supported ICL as a potential target for anti-Candida drug discovery.     

Therapeutic Potential of Caspofungin Combined with Trimethoprim-Sulfamethoxazole for Pneumocystis Pneumonia: A Pilot Study in Mice

Pneumocystis pneumonia (PcP) is a major cause of mortality and morbidity in immunocompromised patients. There are limited alternative therapeutic choices to trimethoprim-sulfamethoxazole (TMP-SMX) which is the standard first line therapy/prophylaxis for PcP. The efficacy of low doses of caspofungin and caspofungin in association with TMP-SMX standard-prophylactic dose was evaluated in an experimental model of Pneumocystis. Susceptibility of Pneumocystis spp. to low doses of caspofungin and caspofungin/TMP-SMX was evaluated in Balb/c immunosuppressed mice, infected intranasally with P. murina. Caspofungin was administered once daily at 0.1 mg/kg, 0.05 mg/kg, and 0.001 mg/kg and TMP-SMX was administered by oral gavage (12.25 mg/62.5 mg/day), for 21 days. Efficacy was calculated based on the reduction in organism burden determined through quantitative fluorescent-based real-time PCR (qPCR). Serum β-1,3-D-glucan was measured as an additional marker of infection. The present data showed that caspofungin demonstrated anti-Pneumomocystis effect. However, the doses administrated were too low to achieve Pneumocystis eradication, which suggests that echinocandin treatment should not be administrated as mono-therapy. After 21 days of treatment, P. murina was not detected in the lungs of mice with either TMP-SMX or caspofungin/TMP-SMX. The results showed that, even at the lowest concentrations tested, the efficacy of caspofungin in association with TMP-SMX was higher than the efficacy of either drug used alone. The administration of caspofungin/TMP-SMX was at least 1.4 times more effective against P. murina infection than TMP-SMX used alone. The most promising result was achieved with the combination of caspofungin 0.05 mg/kg/day with TMP-SMX 12.5 mg–62.5 mg/day, which reduced the parasite burden to undetectable levels immediately at the 14^th day of treatment, showing a highly marked anti-Pneumomocystis effect. These data suggest that the administration of low doses of caspofungin in combination with low doses of TMP-SMX may provide an improved treatment protocol for Pneumocystis infection clearance.     

Aiming for a bull’s-eye: Targeting antifungals to fungi with dectin-decorated liposomes

Globally, there are several million individuals with life-threatening invasive fungal diseases such as candidiasis, aspergillosis, cryptococcosis, Pneumocystis pneumonia (PCP), and mucormycosis. The mortality rate for these diseases generally exceeds 40%. Annual medical costs to treat these invasive fungal diseases in the United States exceed several billion dollars. In addition to AIDS patients, the risks of invasive mycoses are increasingly found in immune-impaired individuals or in immunosuppressed patients following stem cell or organ transplant or implantation of medical devices. Current antifungal drug therapies are not meeting the challenge, because (1) at safe doses, they do not provide sufficient fungal clearance to prevent reemergence of infection; (2) most become toxic with extended use; (3) drug-resistant fungal isolates are emerging; and (4) only one new class of antifungal drugs has been approved for clinical use in the last 2 decades. DectiSomes represent a novel design of drug delivery to drastically increase drug efficacy. Antifungals packaged in liposomes are targeted specifically to where the pathogen is, through binding to the fungal cell walls or exopolysaccharide matrices using the carbohydrate recognition domains of pathogen receptors. Relative to untargeted liposomal drug, DectiSomes show order of magnitude increases in the binding to and killing of Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus in vitro and similarly improved efficacy in mouse models of pulmonary aspergillosis. DectiSomes have the potential to usher in a new antifungal drug treatment paradigm.     

Clearance of Pneumocystis murina infection is not dependent on MyD88

To determine if myeloid differentiation factor 88 (MyD88), which is necessary for signaling by most TLRs and IL-1Rs, is necessary for control of Pneumocystis infection, MyD88-deficient and wild-type mice were infected with Pneumocystis by exposure to infected seeder mice and were followed for up to 106 days. MyD88-deficient mice showed clearance of Pneumocystis and development of anti-Pneumocystis antibody responses with kinetics similar to wild-type mice. Based on expression levels of select genes, MyD88-deficient mice developed immune responses similar to wild-type mice. Thus, MyD88 and the upstream pathways that rely on MyD88 signaling are not required for control of Pneumocystis infection.     

Microarray studies on effects of <Emphasis Type="Italic">Pneumocystis carinii</Emphasis> infection on global gene expression in alveolar macrophages

Background

Pneumocystis pneumonia is a common opportunistic disease in AIDS patients. The alveolar macrophage is an important effector cell in the clearance of Pneumocystis organisms by phagocytosis. However, both the number and phagocytic activity of alveolar macrophages are decreased in Pneumocystis infected hosts. To understand how Pneumocystis inactivates alveolar macrophages, Affymetrix GeneChip^® RG-U34A DNA microarrays were used to study the difference in global gene expression in alveolar macrophages from uninfected and Pneumocystis carinii-infected Sprague-Dawley rats.

Results

Analyses of genes that were affected by Pneumocystis infection showed that many functions in the cells were affected. Antigen presentation, cell-mediated immune response, humoral immune response, and inflammatory response were most severely affected, followed by cellular movement, immune cell trafficking, immunological disease, cell-to-cell signaling and interaction, cell death, organ injury and abnormality, cell signaling, infectious disease, small molecular biochemistry, antimicrobial response, and free radical scavenging. Since rats must be immunosuppressed in order to develop Pneumocystis infection, alveolar macrophages from four rats of the same sex and age that were treated with dexamethasone for the entire eight weeks of the study period were also examined. With a filter of false-discovery rate less than 0.1 and fold change greater than 1.5, 200 genes were found to be up-regulated, and 144 genes were down-regulated by dexamethasone treatment. During Pneumocystis pneumonia, 115 genes were found to be up- and 137 were down-regulated with the same filtering criteria. The top ten genes up-regulated by Pneumocystis infection were Cxcl10, Spp1, S100A9, Rsad2, S100A8, Nos2, RT1-Bb, Lcn2, RT1-Db1, and Srgn with fold changes ranging between 12.33 and 5.34; and the top ten down-regulated ones were Lgals1, Psat1, Tbc1d23, Gsta1, Car5b, Xrcc5, Pdlim1, Alcam, Cidea, and Pkib with fold changes ranging between -4.24 and -2.25.

Conclusions

In order to survive in the host, Pneumocystis organisms change the expression profile of alveolar macrophages. Results of this study revealed that Pneumocystis infection affects many cellular functions leading to reduced number and activity of alveolar macrophages during Pneumocystis pneumonia.

     

A critical role for CARD9 in pneumocystis pneumonia host defence

Caspase recruitment domains‐containing protein 9 (CARD9) is an adaptor molecule critical for key signalling pathways initiated through C‐type lectin receptors (CLRs). Previous studies demonstrated that Pneumocystis organisms are recognised through a variety of CLRs. However, the role of the downstream CARD9 adaptor signalling protein in host defence against Pneumocystis infection remains to be elucidated. Herein, we analysed the role of CARD9 in host defence against Pneumocystis both in CD4‐depleted CARD9^?/? and immunocompetent hosts. Card9 gene‐disrupted (CARD9^?/?) mice were more susceptible to Pneumocystis, as evidenced by reduced fungal clearance in infected lungs compared to wild‐type (WT) infected mice. Our data suggests that this defect was due to impaired proinflammatory responses. Furthermore, CARD9^?/? macrophages were severely compromised in their ability to differentiate and express M1 and M2 macrophage polarisation markers, to enhanced mRNA expression for Dectin‐1 and Mincle, and most importantly, to kill Pneumocystis in vitro. Remarkably, compared to WT mice, and despite markedly increased organism burdens, CARD9^?/? animals did not exhibit worsened survival during pneumocystis pneumonia (PCP), perhaps related to decreased lung injury due to altered influx of inflammatory cells and decreased levels of proinflammatory cytokines in response to the organism. Finally, although innate phase cytokines were impaired in the CARD9^?/? animals during PCP, T‐helper cell cytokines were normal in immunocompetent CARD9^?/? animals infected with Pneumocystis. Taken together, our data demonstrate that CARD9 has a critical function in innate immune responses against Pneumocystis.     

Design,synthesis and evaluation of benzoheterocycle analogues as potent antifungal agents targeting CYP51

To further enhance the anti-Aspergillus efficacy of our previously discovered antifungal lead compound 1, a series of benzoheterocycle analogues were designed, synthesized and evaluated for their in vitro antifungal activity. The most promising compounds 13s and 14a exhibited excellent antifungal activity against C. albicans, C. neoformans, A. fumigatus and fluconazole-resistant C. albicans strains, that was superior or comparable to those of the reference drugs fluconazole and voriconazole. GC–MS analyses suggested that the novel compound 13s might have a similar mechanism to fluconazole by inhibiting fungal lanosterol 14α-demethylase (CYP51). Furthermore, compounds 13s and 14a exhibited low inhibition profiles for various human cytochrome P450 isoforms as well as excellent blood plasma stability.     

In vitro attachment of Pneumocystis carinii from mouse and rat origin

Summary— The attachment of Pneumocystis carinii to lung cells could play a role in the pathophysiology of P carinii pneumonia. The trophozoite attaches to type I alveolar epithelial cells. Physical, chemical, and extracellular matrix factors, involved in the mouseor rat-derived P carinii attachment to fibroblastic cells in culture, were examined using a new model of in vitro adherence. The development of parasite filopodia penetrating deeply the host cell cytoplasm was observed using transmission electronic microscopy. Killed P carinii organisms were unable to attach to cultured cells. Also, parasites were unable to attach to killed target cells. The P carinii in vitro attachment was partially inhibited by cytochalasin B. In contrast, the parasite attachment was not affected when the target cell cytoskeleton was altered. In our work conditions, sialic acids were not involved in the attachment process. Present results showed that fibronectin (Fn) plays a role in the parasite attachment, and suggest that a specific Fn-binding receptor is present at the surface of mouse-derived P carinii organisms.     

Growth and Airborne Transmission of Cell-Sorted Life Cycle Stages of Pneumocystis carinii

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.     

Interstitial pneumonia in immunocompetent laboratory rats caused by natural infection with Pneumocystis carinii

Pneumocystis (P.) carinii is known to cause fatal pneumonia in immunocompromised rats. Cases of P. carinii interstitial pneumonia in immunocompetent rats have been shown histologically to present with perivascular lymphoid cuffs, which have previously been attributed to rat respiratory virus. This study aims to determine the prevalence and pathological characteristics of P. carinii in immunocompetent laboratory rats in experimental facilities in Japan. An epidemiological survey for this agent was performed using PCR to assess 1,981 immunocompetent rats from 594 facilities in Japan. We observed that 6 of the 1,981 rats (0.30%) from 4 out of 594 facilities (0.67%) were positive for P. carinii without infection of other known pathogens. Gross pulmonary lesions were found in 4 of the 6 affected rats. The lungs of these rats contained scattered dark red/gray foci. Histopathologically, the lungs exhibited interstitial pneumonia with lymphoid perivascular cuffs: Pneumocystis cysts were observed using Grocott’s methenamine silver stain. To our knowledge, this report is the first to reveal the prevalence of natural P. carinii infection in immunocompetent laboratory rats in Japan.