Nested–polymerase chain reaction detection of Pneumocystis carinii f. sp. canis in a suspected immunocompromised Cavalier King Charles spaniel with multiple infections

Petini, Matteo; Furlanello, Tommaso; Danesi, Patrizia; Zoia, Andrea

Polymerase chain reaction testing for CDV, CAV-2, CPiV, Angiostrongylus vasorum, Bordetella bronchiseptica, Mycoplasma spp., and Pneumocystis spp. was also performed. To investigate the presence of Pneumocystis spp., a portion of the mitochondrial small subunit rRNA gene (mtSSU rRNA gene) was amplified and sequenced by nested PCR with previously reported primers and protocol. 13 Cytological examination of the BAL fluid revealed a suppurative inflammation associated with the presence of intracellular coccobacilli bacteria. Urine culture and BAL fluid culture were positive for Escherichia coli (1,000,000 CFU) and Klebsiella pneumonia, respectively. Both microorganisms were sensitive to all antibiotic molecules tested on the antibiogram, and, surprisingly, included those used up to 4 days before presentation (i.e. amoxicillin-clavulanic acid). Polymerase chain reaction testing of BAL fluid was positive for B. bronchiseptica, Mycoplasma spp., and Pneumocystis spp. Pneumocystis identity was confirmed by sequencing of the mtSSU rRNA amplicon. The unroot phylogenetic tree constructed using mtSSU rRNA sequences showed that Pneumocystis carinii f. sp. canis was distinct from Pneumocystis spp. isolated from other animals, confirming that this was the specific species related to canine hosts (Figure 3 ). At the owner's request, at the end of all diagnostic procedures, the dog was discharged from the hospital, pending results and against medical advice. At this time, the dog had fully recovered from the anesthesia and physical examination was unchanged compared to the morning admission. On the following morning, the dog returned in acute respiratory distress. Thoracic radiographs showed a severe pneumothorax, and the animal died a short time later from cardiopulmonary arrest despite attempts to stabilize it. Post-mortem examination was declined from the owner.