title: Pneumocystis sp. in bats evaluated by qPCR.
abstract: Molecular techniques have revealed a high prevalence of Pneumocystis colonization in wild mammals. Accurate quantification of Pneumocystis sp. is essential for the correct interpretation of many research experiments investigating this organism. The objectives of this study were to detect the presence of Pneumocystis sp. in bats by qPCR, and to distinguish colonization from infection. Probes and primers for real time PCR (qPCR) were designed based on the gene of major surface glycoprotein (MSG) of Pneumocystis sp., in order to analyze 195 lung tissue samples from bats captured (2007-2009). All samples were also analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial small subunit rRNA (mtSSU rRNA) to confirm the results. The qPCR assay was standardized using a standard curve made with the DNA extracted from bronchoalveolar lavage positive for Pneumocystis jirovecii. The average Ct was found to be between 13 and 14 (calibration curve) for the detection of infection with Pneumocystis sp. and above these values for colonization. It was considered as negative samples the ones that had Ct values equal to 50. Out of the total 195 samples, 47 (24.1%) bat lung DNA samples were positive for Pneumocystis sp. by qPCR. The most common bat species found were: Tadarida brasiliensis (23.4%), Histiotus velatus (17.0%), Desmodus rotundus (14.9%) and Molossus molossus (8.5%). The average cycle threshold of the positive samples (bats) was 25.8 and standard deviation was 1.7. The DNA samples with Ct values greater than 14 suggest that these animals might be colonized by Pneumocystis sp. Results obtained in this study demonstrated the usefulness of the qPCR procedure for identification of Pneumocystis sp. and for distinction between its colonizing or infectious status in bats.
authors: Setor de Micologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil. cavallini.sanches@ufrgs.br
authors: E M
authors: EM
authors: Cavallini Sanches
authors: L
authors: L
authors: Ferreiro
authors: C P
authors: CP
authors: Andrade
authors: S M
authors: SM
authors: Pacheco
authors: L L
authors: LL
authors: Almeida
authors: A
authors: A
authors: Spanamberg
authors: G
authors: G
authors: Wissmann
mesh_terms: D000818:Animals; D001938:Brazil; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D002353:Carrier State / Q000453:epidemiology / Q000382:microbiology / Q000662:veterinary*; D002685:Chiroptera / Q000145:classification / Q000382:microbiology*; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics; D004197:Disease Reservoirs / Q000382:microbiology*; D005656:Fungal Proteins / Q000235:genetics; D058507:Host Specificity; D008168:Lung / Q000382:microbiology; D008562:Membrane Glycoproteins / Q000235:genetics; D011010:Pneumocystis / Q000235:genetics / Q000302:isolation & purification*; D016720:Pneumocystis Infections / Q000635:transmission*; D045363:Pneumocystis carinii / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D060888:Real-Time Polymerase Chain Reaction; D021521:Ribotyping; D013045:Species Specificity
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D008562:Membrane Glycoproteins