An Efficient Tetraplex Surveillance Tool for Salmonid Pathogens

Abstract

Fish disease surveillance methods can be complicated and time consuming, which limits their value for timely intervention strategies on aquaculture farms. Novel molecular-based assays using droplet digital Polymerase Chain Reaction (ddPCR) can produce immediate results and enable high sample throughput with the ability to multiplex several targets using different fluorescent dyes. A ddPCR tetraplex assay was developed for priority salmon diseases for farmers in New Zealand including New Zealand Rickettsia-like organism 1 (NZ-RLO1), NZ-RLO2, Tenacibaculum maritimum, and Yersinia ruckeri. The limit of detection in singleplex and tetraplex assays was reached for most targets at 10−9 ng/μl with, respectively, NZ-RLO1 = 0.931 and 0.14 copies/μl, NZ-RLO2 = 0.162 and 0.21 copies/μl, T. maritimum = 0.345 and 0.93 copies/μl, while the limit of detection for Y. ruckeri was 10−8 with 1.0 copies/μl and 0.7 copies/μl. While specificity of primers was demonstrated in previous studies, we detected cross-reactivity of T. maritimum with some strains of Tenacibaculum dicentrarchi and Y. ruckeri with Serratia liquefaciens, respectively. The tetraplex assay was applied as part of a commercial fish disease surveillance program in New Zealand for 1 year to demonstrate the applicability of tetraplex tools for the salmonid aquaculture industry.

Experimental challenge of Chinook salmon withTenacibaculum maritimumandTenacibaculum dicentrarchifulfils Koch’s postulates

“…Tenacibaculum maritimum strains in Aotearoa New Zealand belong to three O-AGC types [Cawthron Culture Collection of Microorganism (CCCM20/006, CCCM20/102 and CCCM20/133) see Table 1; 32]; Tenacibaculum dicentrarchi (CCCM21/136) used in this study were isolated from moribund Chinook salmon skin ulcers during a disease outbreak in the Marlborough Sounds, Aotearoa New Zealand. The isolates were confirmed to be T. maritimum and T. dicentrarchi by species specific PCR [31, 49, 50]. The isolates were stored at -80 °C on cryobeads (Microbeads TM ) and revived on Marine Shieh’s Medium agar [MSM, 50] and ZoBell’s Marine Agar 2216E (MA) and then incubated at 22°C for 48 h. Growth of T. maritimum on both types of media was assessed and purity checked by Gram-stain.…”

“…After incubation, bacterial colony morphology consistent with T. maritimum and T. dicentrarchi was assessed on MSSM and MA [see Figure 2 in 31]. Species identification and the O-AGC types were confirmed for the bacterial colonies using species specific PCR and mPCR [32, 49, 50, 52]. In addition, to investigate whether T. maritimum or T. dicentrarchi causes systemic infection, a swab from the anterior kidney was plated onto all four media types as described in Section 2.3.…”

“…Tissues from control fish were processed prior to samples from the treatment group and each tissue type was processed on separate days to avoid potential cross-contamination during DNA extraction. One hundred nanograms of gDNA from each tissue type or isolate were used to perform ddPCR assay using a QX200 Droplet Digital PCR System TM (Bio-Rad, Hercules, USA), as described by von Ammon et al [49]. Tenacibaculum dicentrarchi -specific primers and PCR conditions as described in Wilson et al [50] were used in droplet digital PCR to quantify T. dicentrarchi DNA in fish tissue infected with T. dicentrarchi .…”

Development of a droplet digital PCR assay for the sensitive detection of iridovirus in Andrias davidianus

“…Compared to the above PCR methods, ddPCR is less susceptible to PCR inhibitors (Guti'errez‐Aguirre et al, 2015; Li et al, 2018) and enables the absolute quantitation of nucleic acids without requiring a standard curve (Yu et al, 2022). The ddPCR method has been proven to be successful in detecting viral and bacterial infections in aquatic animals such as common carp (Wang et al, 2021), salmonid (Ammon et al, 2022; Jia et al, 2017; Orioles et al, 2022), tilapia (Zhao et al, 2023), largemouth bass (Jiang et al, 2023), shrimp (Zhang et al, 2022) and oysters (Tan et al, 2018).…”

“…The ddPCR method has been proven to be successful in detecting viral and bacterial infections in aquatic animals such as common carp (Wang et al, 2021), salmonid (Ammon et al, 2022;Jia et al, 2017;Orioles et al, 2022), tilapia (Zhao et al, 2023), largemouth bass (Jiang et al, 2023), shrimp (Zhang et al, 2022) and oysters (Tan et al, 2018).…”