Molecular diagnosis of Pneumocystis pneumonia in dogs

Abstract

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.

Confirmed case of Pneumocystis pneumonia in a Maltese Terrier × Papillon dog being treated with toceranib phosphate

“…A PCR panel including Toxoplasma and Neospora , as well as distemper, Bartonella , Borrelia and Cryptococcus , was negative. A recently validated real‐time PCR assay was positive and confirmed the presence of P. canis within the bronchoalveolar lavage fluid . Immunoglobulin quantification through radial immunodiffusion showed no significant abnormalities (Table ).…”

“…In dogs, clinical disease resulting from P. carinii f.sp . canis has been recorded secondary to hereditary immunosuppressive diseases, as well as acquired immunodeficiency secondary to distemper . Cases of P. canis secondary to acquired non‐infectious immunosuppression have not been reported in dogs, which is in stark contrast to the human literature, where the incidence of pneumocystis pneumonia (PCP) is as high as 15–20% in patients receiving multidrug chemotherapy protocols, even allowing for the tendency towards more aggressive chemotherapy protocols in human medicine.…”

Molecular detection ofPneumocystisin the lungs of cats

“…28,38 In the dog and horse, clinical PCP is generally seen in young animals and related to inherited immunodeficiency states, for example, combined immunodeficiency in Arabian horses 37 and PCP in Cavalier King Charles Spaniels and miniature dachshunds, and their hybrids. 21,36,39,40 Rare spontaneous cases of Pneumocystis pneumonia (PCP) in pigs (adult and piglets) are mostly linked with immunosuppression from adverse environmental conditions (stressful farming conditions, overcrowding, high ambient ammonia concentrations, perhaps complicated by suboptimal nutrition) and/or genetic predispositions. [41][42][43] In pigs, Pneumocystis is generally eliminated from the lungs or constrained to small quiescent foci unless such immunosuppressive conditions are present.…”

“…A 320-340 bp fragment of the mtSSU rRNA gene was amplified by nested PCR with two primer pairs (Paz 112-10F and Paz 112-10R as external primers; Paz 112-13 and Paz112-14 as internal primers) and PCR conditions as reported previously. 21…”

CT findings in Pneumocystis carinii pneumonia in five dogs

“…Special stains such as Grocott‐Gomori methenamine silver, or PCR and in situ hybridisation are often required to detect and characterise the organism (Lobetti , Danesi et al . , Weissenbacher‐Lang et al . ).…”