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.].

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.

Assessment of Aspergillus fumigatus pathogenicity in aerosol-challenged chickens (Gallus gallus) belonging to two lineages.

Infection due to the mold Aspergillus fumigatus remains a common and life-threatening infection in many animals, especially birds. Animal models are still required to better understand the physiopathology of infection and evaluate diagnostic tools and treatment procedures. The aim of the present study was to assess the pathogenicity of A. fumigatus in two lineages of chicken (Gallus gallus): SPF White Leghorn PA12 layers and conventional JA657 broilers. Four-day-old birds were experimentally infected in an inhalation chamber in order to reproduce a "natural" contamination and to obtain a large repartition of conidia into the respiratory tract. Half of the chicks were injected subcutaneously with dexamethasone for 4 days before the infective challenge. At days 0 and 7, the effects of chicken lineage and immunosuppressive treatment on pulmonary fungal burden were analyzed using two linear mixed models. The pathogenicity of A. fumigatus varied according to the lineage: no clinical signs and no mortality were observed in layer chickens whereas more than 50% of mortality occurred in broilers. The effect of immunosuppressive treatment was also demonstrated, notably on animals weight but also on mortality.

Aspergillus fumigatus in Poultry.

Aspergillus fumigatus remains a major respiratory pathogen in birds. In poultry, infection by A. fumigatus may induce significant economic losses particularly in turkey production. A. fumigatus develops and sporulates easily in poor quality bedding or contaminated feedstuffs in indoor farm environments. Inadequate ventilation and dusty conditions increase the risk of bird exposure to aerosolized spores. Acute cases are seen in young animals following inhalation of spores, causing high morbidity and mortality. The chronic form affects older birds and looks more sporadic. The respiratory tract is the primary site of A. fumigatus development leading to severe respiratory distress and associated granulomatous airsacculitis and pneumonia. Treatments for infected poultry are nonexistent; therefore, prevention is the only way to protect poultry. Development of avian models of aspergillosis may improve our understanding of its pathogenesis, which remains poorly understood.

Relative efficiencies of two air sampling methods and three culture conditions for the assessment of airborne culturable fungi in a poultry farmhouse in France.

Fungal elements represent a significant part of the biological contaminants that could be detected in the air of animal facilities. The aim of this study was to assess the relative efficiencies of two air sampling methods and three culture conditions for the quantification of airborne culturable fungi in a poultry farmhouse in France. Air samples were collected every week throughout a 15-week period. Two devices were simultaneously used-a rotative cup air sampler (CIP 10-M, Arelco, France) and an air sampler based on filtration (AirPort MD8, Sartorius, Germany). Culture of airborne viable fungi was performed on malt extract agar (ME) and dichloran glycerol-18 (DG18) at 25 or 37°C. CIP 10-M and AirPort MD8 were shown to display comparable performances but significant differences were observed between culture conditions for Aspergillus spp. (p<0.01), Scopulariopsis spp. (p=0.02) and unidentified molds (p<0.01).

Malassezia dermatitis in dogs in Brazil: diagnosis, evaluation of clinical signs and molecular identification.

Skin carriage and quantification of Malassezia yeasts were evaluated in 180 healthy dogs (group 1) and 117 dogs with clinical signs (pruritus, erythema, lichenification/seborrhoea, excoriations and alopecia) that could be related to Malassezia dermatitis (group 2) in Brazil. The lesions in the group 2 dogs were evaluated using CADESI-03 scores. Samples were collected from five different anatomical areas. Direct examination was performed using the tape strip technique, and results were expressed as the mean number of yeasts per ×1000 microscopic field per dog. For mycological culture, a single piece of sterilized carpet was applied to the same areas sampled for cytology, and transferred onto Dixon's modified medium. Yeast populations were expressed as mean colony forming units (CFU)/plate. Malassezia isolates were characterized by polymerase chain reaction-restriction endonuclease analysis of the large subunit (LSU) of ribosomal RNA gene. The probability of culturing Malassezia from dogs with skin lesions was significantly higher (P<0.001) than from healthy dogs. There was a linear trend between CADESI-03 score and mean CFU/plate. Group 2 dogs with positive cultures had higher CADESI-03 scores than those with negative cultures (P<0.05). Almost all isolates were identified as Malassezia pachydermatis. Only one isolate (group 2) was identified as Malassezia furfur. These data suggest that dogs with skin disorders harbouring Malassezia yeasts in quantities higher than 120 mean CFU/plate should be considered as having Malassezia dermatitis. The presence of Malassezia appears to exacerbate clinical lesions in dogs.

Multiple-locus variable-number tandem repeat analysis for molecular typing of Aspergillus fumigatus.

BACKGROUND: Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) is a prominent subtyping method to resolve closely related microbial isolates to provide information for establishing genetic patterns among isolates and to investigate disease outbreaks. The usefulness of MLVA was recently demonstrated for the avian major pathogen Chlamydophila psittaci. In the present study, we developed a similar method for another pathogen of birds: the filamentous fungus Aspergillus fumigatus. RESULTS: We selected 10 VNTR markers located on 4 different chromosomes (1, 5, 6 and 8) of A. fumigatus. These markers were tested with 57 unrelated isolates from different hosts or their environment (53 isolates from avian species in France, China or Morocco, 3 isolates from humans collected at CHU Henri Mondor hospital in France and the reference strain CBS 144.89). The Simpson index for individual markers ranged from 0.5771 to 0.8530. A combined loci index calculated with all the markers yielded an index of 0.9994. In a second step, the panel of 10 markers was used in different epidemiological situations and tested on 277 isolates, including 62 isolates from birds in Guangxi province in China, 95 isolates collected in two duck farms in France and 120 environmental isolates from a turkey hatchery in France. A database was created with the results of the present study http://minisatellites.u-psud.fr/MLVAnet/. Three major clusters of isolates were defined by using the graphing algorithm termed Minimum Spanning Tree (MST). The first cluster comprised most of the avian isolates collected in the two duck farms in France, the second cluster comprised most of the avian isolates collected in poultry farms in China and the third one comprised most of the isolates collected in the turkey hatchery in France. CONCLUSIONS: MLVA displayed excellent discriminatory power. The method showed a good reproducibility. MST analysis revealed an interesting clustering with a clear separation between isolates according to their geographic origin rather than their respective hosts.

Evaluation of fungal aerosols using Temporal Temperature Gradient Electrophoresis (TTGE) and comparison with culture.

Information obtained from fungal air samples can assist in the assessment of health hazards and can be useful in proactive indoor air quality monitoring. The objective of the present study was to evaluate the PCR-TTGE technique for the analysis of fungal diversity in the air. Eleven air samples were collected in five different sites using the bioimpactor CIP 10-M (Arelco). After a 2 hours sampling period, the collection liquid was recovered for subsequent cultivation and PCR-TTGE. A set of three fungi-specific primers (Fungcont 1, Fungcont 2+GC and Fungcont 3) was designed for the partial amplification of the 18S rRNA gene. The amplification was obtained in a single reaction tube by a semi-nested PCR. For identification, the TTGE bands were extracted and sequenced. PCR-TTGE allowed the clear separation of amplicons corresponding to distinct fungal species (both Ascomycota and Basidiomycota) that may be encountered in air. The number of fungal taxa detected after culture was systematically higher than the number of taxa found using PCR-TTGE. However, few fungal species were detected by PCR-TTGE and not by cultivation, suggesting that the combination of these approaches may provide a better analysis of fungal diversity in air samples than either method alone.

Prevalence of anti-Toxoplasma gondii antibodies in serum and aqueous humor samples from cats with uveitis or systemic diseases in France.

Anti-Toxoplasma gondii antibodies were determined in serum and aqueous humor of two groups of cats in France: cats with uveitis (group 1, n=26) and cats with systemic disease (group 2, n=24) using an agglutination test. Titres above 1:64 were considered positive. IgG antibodies to T. gondii were detected in 10 serum samples from group 1 and in 10 serum samples from group 2, and in 2 aqueous humor samples from group 1 and in 1 aqueous humor samples from group 2. The distribution of ocular lesions according to the serological status of the animals indicated that lens luxation and buphthalmia were more frequent in T. gondii seropositive cats than in seronegative ones. The study reports a similar prevalence of anti-T. gondii antibodies in cats with uveitis and in cats without uveitis in France. Serological results must be analysed carefully and additional diagnostic tools is required.