Validation of a quantitative PCR assay for detection and quantification of ‘Candidatus Xenohaliotis californiensis’

Abstract

Withering syndrome (WS), a serious disease affecting abalone Haliotis spp., is caused by infection from an intracellular Rickettsia-like organism (WS-RLO). Diagnosis of the disease currently relies on a combination of histological examination and molecular methods (in situ hybridization, standard PCR, and sequence analysis). However, these techniques only provide a semi-quantitative assessment of bacterial load. We created a real-time quantitative PCR (qPCR) assay to specifically identify and enumerate bacterial loads of WS-RLO in abalone tissue, fecal, and seawater samples based on 16S rDNA gene copy numbers. The qPCR assay designed to detect DNA of the WS-RLO was validated according to standards set by the World Organisation for Animal Health. Standard curves derived from purified plasmid dilutions were linear across 7 logs of concentration, and efficiencies ranged from 90.2 to 97.4%. The limit of detection was 3 gene copies per reaction. Diagnostic sensitivity was 100% and specificity was 99.8%. The qPCR assay was robust, as evidenced by its high level of repeatability and reproducibility. This study has shown for the first time that WS-RLO DNA can be detected and quantified in abalone tissue, fecal, and seawater samples. The ability to detect and quantify RLO gene copies in a variety of materials will enable us to better understand transmission dynamics in both farmed and natural environments.

Quantifying chelonid herpesvirus 5 in symptomatic and asymptomatic rehabilitating green sea turtles

“…Repeatability of the ChHV5 UL30 assay was determined by a single person using qPCR to test 3 ChHV5 DN A-positive samples each of FP, nontumored skin, blood, plasma, urine, and cloacal swab samples spanning the qPCR assay linear operating range on the Stratagene platform (Friedman et al 2014). Samples were tested in replicates of 8, and an intra-assay coefficient of variation (CV) of 0 to 20% was considered acceptable (Pfaffl 2004).…”

Differing responses of red abalone (Haliotis rufescens) and white abalone (H. sorenseni) to infection with phage-associated Candidatus Xenohaliotis californiensis

“…Prior to challenge, feces from all tanks were tested for 16S rRNA Ca. Xc genes by quantitative PCR (qPCR) following a validated protocol (Friedman et al, 2014b). Although fecal qPCR is treated only as a proxy for live pathogen, it has been shown to be the most sensitive assay for Ca.…”

“…Although fecal qPCR is treated only as a proxy for live pathogen, it has been shown to be the most sensitive assay for Ca. Xc detection (Friedman et al, 2014b). To support the absence of infection, five animals from both the red and white groups were sacrificed for histologic examination.…”

“…While Friedman et al (2014a) and Friedman et al (2014b) demonstrated significant improvement in black abalone survival when challenged with phage-containing Ca. Xc, the mechanism by which the phage reduces pathogenicity is unclear.…”

Fishing diseased abalone to promote yield and conservation

“…In marine systems, this strategy faces added challenges, such as difficulties and costs associated with diagnosing cryptic infections. Recently, presumptive diagnoses of many marine diseases have been streamlined with rapid, inexpensive and non-invasive methods (table 1), including observing external parasites [14,16,23,26], morphological and behavioural changes in hosts [20,27,30], and non-lethal immunological and molecular assays [29,31]. For instance, polymerase chain reaction (PCR) methods can detect genes specific to the WS bacterium in abalone faeces [29].…”