Molecular characterization of Prototheca in 11 symptomatic dogs

Abstract

Protothecosis is an uncommon disease caused by algae of the genus Prototheca. In dogs, the infection is usually first localized to the colon but has the propensity to later disseminate hematogenously to many other organs, with marked tropism for the eyes and central nervous system. Diagnosis is established by culture and/or evidence of Prototheca organisms in cytologic or histologic preparations. Species characterization, however, requires molecular investigations. Our laboratory set up a real-time PCR targeting portion D1/D2 of the 28S rRNA for identification of Prototheca species from both positive cultures (of rectal swabs and urine) and formalin-fixed, paraffin-embedded tissue. Prototheca bovis, P. ciferrii, and P. wickerhamii were characterized in 11 dogs with systemic or cutaneous protothecosis. Prototheca identifications were phylogenetically consistent with the new taxonomy proposed for this genus based on the mitochondrial cytochrome b gene. As a pilot study, we screened feces and rectal scrapes from 200 asymptomatic dogs, using 2 cohorts of stray and owned animals, to determine the prevalence of intestinal carriage of Prototheca spp. The Prototheca-negative results from both cohorts of healthy dogs suggest that predisposing factors related to the host probably contribute more to the acquisition of clinical disease than exposure to contaminated environments.

Keywords: 28S rRNA; FFPE; canine protothecosis; cytb mtDNA; cytology; real-time PCR.

Figure 1.

Phylogenetic tree based on the Prototheca 28S rRNA sequences. Prototheca sequences produced from our study are shown in red (from culture Prototheca isolates), and those from FFPE tissue are presented in red boldface. Chlamydomonas reinhardtii sequence was used as an outgroup. The tree was constructed using the maximum likelihood method. Bootstrap values shown at the main nodes represent the probabilities based on 1,000 replicates. (T) = type strains (color version of the figure is available online).

Figure 2.

Phylogenetic tree based on the Prototheca cytb mitochondrial DNA sequences. Prototheca sequences produced from our study are shown in red. Chlamydomonas reinhardtii sequence was used as an outgroup. The tree was constructed using the maximum likelihood method. Bootstrap values shown at the main nodes represent the probabilities based on 1,000 replicates. (T) = type strains (color version of the figure is available online).