Molecular diagnosis of Pneumocystis pneumonia in dogs.
Pneumocystis pneumonia (PCP) is a life-threatening fungal disease that can occur in dogs. The aim of this study was to provide a preliminary genetic characterisation of Pneumocystis carinii f.sp.'canis' (P. canis) in dogs and thereby develop a reliable molecular protocol to definitively diagnose canine PCP. We investigated P. canis in a variety of lung specimens from dogs with confirmed or strongly suspected PCP (Group 1, n = 16), dogs with non-PCP lower respiratory tract problems (Group 2, n = 65) and dogs not suspected of having PCP or other lower respiratory diseases (Group 3, n = 11). Presence of Pneumocystis DNA was determined by nested PCR of the large and small mitochondrial subunit rRNA loci and by a real-time quantitative polymerase chain reaction (qPCR) assay developed using a new set of primers. Molecular results were correlated with the presence of Pneumocystis morphotypes detected in cytological/histological preparations. Pneumocystis DNA was amplified from 13/16 PCP-suspected dogs (Group 1) and from 4/76 dogs of control Groups 2 and 3 (combined). The latter four dogs were thought to have been colonized by P. canis. Comparison of CT values in 'infected' versus 'colonized' dogs was consistent with this notion, with a distinct difference in molecular burden between groups (CT ≤ 26 versus CT range (26 <CT < 35), respectively). Phylogenetic analyses showed that P. canis is specifically 'canine' associated, being separated from other mammalian Pneumocystis species, thereby confirming the accuracy of qPCR amplicon for Pneumocystis in dogs. Using qPCR, Pneumocystis DNA can be detected in specimens from the respiratory tract and a CT value can be interpreted to distinguish infection versus colonization.
Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), Italy.
Patrizia
P
Danesi
Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), Italy.
Silvia
S
Ravagnan
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, California, USA.
Lynelle R
LR
Johnson
Clinica Veterinaria San Marco, Padua, Italy.
Tommaso
T
Furlanello
Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), Italy.
Adelaide
A
Milani
Veterinary Pathology Diagnostic Services, Faculty of Veterinary Science, University of Sydney, Sydney, New South Wales, Australia.
Patricia
P
Martin
QML Vetnostics, Riverview Place, Metroplex on Gateway, Murrarie, QLD Australia.
Susan
S
Boyd
Brisbane Veterinary Specialist Centre, Corner Keong & Old Northern Roads, Albany Creek, QLD Australia.
Matthew
M
Best
Australian Specialised Animal Pathology (ASAP) Laboratories, Victoria, Australia.
Bradley
B
Galgut
School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia, Australia.
Peter
P
Irwin
Faculty of Veterinary Science, University of Sydney, Sydney, New South Wales, Australia.
Paul J
PJ
Canfield
Faculty of Veterinary Science, University of Sydney, Sydney, New South Wales, Australia.
Mark B
MB
Krockenberger
Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR - Pathology West, Westmead Hospital, Sydney, New South Wales, Australia.
Catriona
C
Halliday
Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Sydney Medical School-Westmead Hospital, Marie Bashir Institute for Emerging Infectious Diseases and Biosecurity, University of Sydney, Westmead Institute for Medical Research, Sydney, Australia.
Wieland
W
Meyer
Centre for Veterinary Education, B22, University of Sydney, Sydney, New South Wales, Australia.
Richard
R
Malik
D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D017931:DNA Primers; D004271:DNA, Fungal / Q000235:genetics; D004283:Dog Diseases / Q000175:diagnosis* / Q000382:microbiology* / Q000473:pathology; D004285:Dogs; D008168:Lung / Q000382:microbiology; D016533:Mycological Typing Techniques / Q000662:veterinary; D010802:Phylogeny; D045363:Pneumocystis carinii / Q000145:classification / Q000235:genetics*; D011020:Pneumonia, Pneumocystis / Q000175:diagnosis / Q000473:pathology / Q000662:veterinary*; D012313:RNA / Q000235:genetics; D000077278:RNA, Mitochondrial; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004271:DNA, Fungal; D000077278:RNA, Mitochondrial; D012313:RNA
Cavalier King Charles spaniel; Dachshund; PCR; Pneumocystis; cyst; dog; interstitial; mtLSU; mtSSU; pneumocystosis pneumonia