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Background   Aliarcobacter faecis  and  Aliarcobacter lanthieri  are recently identified as emerging human and animal pathogens. In this paper, we demonstrate the development and optimization of two direct DNA-based quantitative real-time PCR assays using species-specific oligonucleotide primer pairs derived from  rpoB  and  gyrA  genes for  A. faecis  and  A. lanthieri , respectively. Initially, the specificity of primers and amplicon size of each target reference strain was verified and confirmed by melt curve analysis. Standard curves were developed with a minimum quantification limit of 100 cells mL − 1  or g − 1  obtained using known quantities of spiked  A. faecis  and  A. lanthieri  reference strains in autoclaved agricultural surface water and dairy cow manure samples.    Results  Each species-specific qPCR assay was validated and applied to determine the rate of prevalence and quantify the total number of cells of each target species in natural surface waters of an agriculturally-dominant and non-agricultural reference watershed. In addition, the prevalence and densities were determined for human and various animal (e.g., dogs, cats, dairy cow, and poultry) fecal samples. Overall, the prevalence of  A. faecis  for surface water and feces was 21 and 28%, respectively. The maximum  A. faecis  concentration for water and feces was 2.3 × 10 7  cells 100 mL - 1  and 1.2 × 10 7  cells g − 1 , respectively.  A. lanthieri  was detected at a lower frequency (2%) with a maximum concentration in surface water of 4.2 × 10 5  cells 100 mL − 1 ; fecal samples had a prevalence and maximum density of 10% and 2.0 × 10 6  cells g − 1 , respectively.    Conclusions  The results indicate that the occurrence of these species in agricultural surface water is potentially due to fecal contamination of water from livestock, human, or wildlife as both species were detected in fecal samples. The new real-time qPCR assays can facilitate rapid and accurate detection in < 3 h to quantify total numbers of  A. faecis  and  A. lanthieri  cells present in various complex environmental samples.

bmc microbiology

Real-time quantitative PCR assay development and application for assessment of agricultural surface water and various fecal matter for prevalence of Aliarcobacter faecis and Aliarcobacter lanthieri