Identification of reliable marker genes for the detection of canine fecal contamination in sub-tropical Australia

Animal fecal contamination in aquatic environments is a major source of zoonotic diseases in humans. While concerns are focused on livestock, companion animals such as dogs can also be a source of a wide range of zoonotic pathogens. Therefore, detection of dog or canine fecal contamination in aquatic environments is important for mitigating risks. In this study, host-sensitivity and specificity of four canine fecal-associated marker genes were evaluated by analyzing 30 canine and 240 non-canine fecal samples. The application of these markers was also tested in water from an urban river under dry weather conditions. The host sensitivity values of the Bacteroides BacCan-UCD, DogBact, DF113 and DF418 were 1.00, 0.90, 0.83, and 0.90, respectively. The host specificity value of the BacCan-UCD, DogBact, DF113 and DF418 were 0.87, 0.98, 0.83, and 0.41, respectively. The mean concentrations of DF418 were highest (7.82 ± 1.13 log₁₀ gene copies (GC)/g of feces) followed by BacCan-UCD (7.61 ± 1.06 log₁₀ GC/g) and DogBact (7.15 ± 0.92 log₁₀ GC/g). The mean concentration of DF113 (5.80 ± 1.25 log₁₀ GC/g) was 1.5 to 2.5 orders of magnitude lower than the other marker genes. The DogBact marker gene was not detected in any other animal feces other than a small number of untreated sewage samples. The BacCan-UCD marker gene cross-reacted with cat, chicken, and pig fecal samples, while the DF113 marker gene cross-reacted with cat, chicken, cattle fecal and untreated sewage samples. The DF418 marker gene was detected in all sewage and animal feces and deemed not suitable for canine fecal contamination tracking in sub-tropical Australia. Canine fecal contamination was infrequently detected in environmental water samples. Based on the results obtained in this study, we recommend that at least two canine feces-associated marker genes should be used in field studies.