Abstract
BackgroundAliarcobacter 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.ResultsEach 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 × 107 cells 100 mL- 1 and 1.2 × 107 cells g− 1, respectively. A. lanthieri was detected at a lower frequency (2%) with a maximum concentration in surface water of 4.2 × 105 cells 100 mL− 1; fecal samples had a prevalence and maximum density of 10% and 2.0 × 106 cells g− 1, respectively.ConclusionsThe 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.
Highlights
Aliarcobacter faecis and Aliarcobacter lanthieri are recently identified as emerging human and animal pathogens
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 assays were further validated by applying to the field isolates of each target species (A. faecis: n = 29; and A. lanthieri: n = 10)
Summary
Aliarcobacter faecis and Aliarcobacter lanthieri are recently identified as emerging human and animal pathogens. Some of the most prevalent Aliarcobacter species including A. butzleri, A. cryaerophilus, and A. skirrowii have been identified as causative agents for human (e.g., gastroenteritis, bacteremia, and sepsis) and animal (e.g., mastitis, diarrhea, abortion, and reproductive disorders) infections [5] These species have been isolated from a variety of food products, including chicken, beef, pork, and shellfish as well as various aquatic sources [6,7,8,9,10] and pose an important risk for human infection from contamination of water and food resulting from a variety of sources including livestock and poultry wastes, agricultural runoff, septic leakages, and direct or indirect inputs of wildlife fecal matter [11,12,13,14]. Non-viable or nonculturable cells of Gram-negative bacteria can potentially contaminate water by producing virulence-associated factors and toxins that can pose health risks to humans [17, 18]
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