The significance of fungal organisms in canine enteropathies - Establishing a qPCR and determining the prevalence of fungal organisms in canine feces

Abstract

Until now little research has been done evaluating the prevalence of fungal organisms in the gastrointestinal tract of dogs. Even less is known about the effects of fungal organisms on gastrointestinal health and disease. The aim of this study was to establish a quantitative real time PCR protocol (qPCR) to investigate the prevalence of fungal organisms in healthy dogs and dogs with signs of gastrointestinal disease, and to quantify fungal organisms in the canine feces. Fecal samples from a total of 119 dogs (49 healthy dogs and 70 dogs with signs of gastrointestinal disease) of different age, breed, and sex were obtained. DNA was extracted from 100 mg of fecal sample by a modified bead beating method using phenol/chloroform. On all samples qPCR’s were performed using universal fungal and universal bacterial primers to amplify the eukaryotic ITS2 region and parts of the bacterial 16S rRNA gene, respectively. Standard 1:10 serial dilutions containing 8,400 to 0.0084 pg of fungal DNA and 4,000 to 0.04 pg of bacterial DNA were used to calculate the starting DNA quantity for each specific sample. The obtained universal fungal data was normalized by the universal bacterial data to account for intersample variation during DNA extraction. All samples were run in duplicates on a commercially available real time PCR thermocycler. (iCylcer iQ, BioRad). The negative DNA extraction control was further investigated after PCR amplification by cloning and sequencing. The negative DNA extraction control yielded a positive signal after 33 cycles during the universal fungal qPCR assay. A cut-off value of 27 cycle times (corresponding to approximately 100-fold more starting DNA quantity than present in the negative DNA extraction control) was used to determine which samples could truly be considered positive. From all analyzed samples, 26 of the healthy dogs (53%) and 23 of the dogs with signs of gastrointestinal disease (33%) were positive for fungal DNA (p=0.0373). For all positive samples the ratio of universal fungal to universal bacterial DNA was calculated: healthy dogs had a median ratio of 0.0002779 (range 1.701x10-5 – 0.02543, p = 0.0013), and the median for the diseased animals was 0.0001879 (range 1.116x10-5 – 0.1405, p = 0.0009). This study suggests that healthy dogs have a significantly higher prevalence of fungal organisms compared to dogs with gastrointestinal signs. The relative quantities in fungal DNA between these two groups did not differ. Fungal organisms appear to be part of the normal intestinal microbiota, and the intestinal ecosystem may be disrupted in dogs with gastrointestinal disease. More research is warranted to obtain a more accurate prevalence and determine the role of fungal organisms in the gastrointestinal tract of dogs.