PCR protocol for detection of Vibrio ordalii by amplification of the vohB (hemolysin) gene

Abstract

Vibrio ordalii is the causative agent of atypical vibriosis and has the potential to cause severe losses in salmonid aquaculture. To prevent and control outbreaks, a rapid, reproducible, sensitive, and effective diagnostic method is needed. We evaluated a new conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) protocol using a primer set (VohB_Fw-VohB_Rv) designed to amplify a 112 bp fragment flanking the vohB gene (coding for hemolysin production), against 24 V. ordalii strains isolated from different fish species, the V. ordalii type strain, and 42 representative related and unrelated bacterial species. The primer set was species-specific, recognizing all V. ordalii strains evaluated, with no cross-reaction with the other bacterial species. A sensitivity of 103 copies of the vohB gene was obtained with a standard curve. When the VohB_Fw-VohB_Rv qPCR protocol was applied to Atlantic salmon seeded tissues (kidney, liver, spleen, and muscle), the detection limit ranged from 5.27 × 102 to 4.13 × 103 V. ordalii CFU ml-1, i.e. 62 to 145 copies of the vohB gene, using the previously calculated standard curve. The conventional PCR also detected V. ordalii, but the total reaction time was 1 h longer. When the qPCR protocol was applied to naturally infected cage-cultured Atlantic salmon samples, 5 of 8 fish tested positive for V. ordalii, but only one of them was diagnosed as positive by direct cultivation on agar. We conclude that the PCR protocol evaluated is fast, specific, and sensitive enough to detect V. ordalii in infected tissues and is an important tool for secure diagnosis of atypical vibriosis, and is therefore helpful for the control of the disease through the prompt detection within fish populations.

Evidence for the facultative intracellular behaviour of the fish pathogen Vibrio ordalii

“…Both have been used in previous studies (Ruiz et al, , ). The identity of each strain was confirmed as V. ordalii by standard phenotypical procedures (Silva‐Rubio, Acevedo, et al, ) and using the PCR‐based analysis described by Avendaño‐Herrera et al (). The strains were routinely cultivated under aerobic conditions at 18 ºC for 48–72 hr on trypticase soy agar/broth (TSA/TSB, Oxoid) supplemented with 1% (w/v) NaCl (TSA‐1 and TSB‐1, respectively).…”

Comparative pathogenesis of piscirickettsiosis in Atlantic salmon (Salmo salar L.) post‐smolt experimentally challenged with LF‐89‐like and EM‐90‐like Piscirickettsia salmonis isolates

“…Non‐vaccinated post‐smolt Atlantic salmon, Salmo salar L., strain Marine Harvest (Marine Harvest Chile), with an average size of 118.4 g were used for the cohabitation challenge 30 days after being transferred to water with 15 g/L salinity. Prior to this, 30 fish were randomly tested and screened to ensure that they were enzootic pathogen‐free using PCR for piscine reovirus (PRv) (Palacios et al., ), infectious pancreatic necrosis virus (IPNv) (Skjesol et al., ), infectious salmon anaemia virus (ISAv) (Snow et al., ), Flavobacterium psychrophilum (Del Cerro, Mendoza, & Guijarro, ), Vibrio ordalii (Avendaño‐Herrera et al., ), Renibacterium salmoninarum (Suzuki & Sakai, ) and P. salmonis (Karatas et al., ). All fish also were examined by histology to discard microscopic lesions before the challenge.…”

Cell-surface properties of Vibrio ordalii strains isolated from Atlantic salmon Salmo salar in Chilean farms

“…The Atlantic salmon isolates were obtained from internal organs (kidney or spleen) of diseased cage-cultured Atlantic salmon. The identity of each isolate was initially confirmed as V. ordalii by standard phenotypical procedures (Silva-Rubio et al 2008, Poblete-Morales et al 2013) and using the PCR-based analysis described by Avendaño-Herrera et al (2014). In addition, profiles of our isolates were compared with those of the V. ordalii type strain ATCC 33509…”

“…These fish were analysed with plating and PCR techniques to confirm the absence of V. ordalii as well as other bacterial fish pathogens according the protocols described by Avendaño-Herrera et al (2014) and Tapia-Cammas et al (2011), respectively. The pool of mucus samples was centrifuged for 5 min at 2655 × g to remove particulate material, and the supernatant (3 ml) was dissolved in sterile seawater (1:1), mixed thoroughly, filter-sterilized onto 0.22 µm and stored at −20°C until use.…”

“…All fish were maintained in tanks at 18 ± 1°C with aeration during the course of the experiments (21 d), with 50% of the tank water refreshed daily. Dead fish were removed from each tank daily and examined to confirm whether the bacterium inoculated was the cause of mortality by the following methods: (1) kidney, liver, spleen and external lesions were directly streaked onto TSA-1 plates and incubated at 18°C for 1 wk and (2) pure or mixed cultures as well as fish tissues (kidney, liver and spleen) were also tested using the specific PCR (Avendaño-Herrera et al 2014). In addition, the identity of each isolate was confirmed as V. ordalii by standard phenotyping procedures (SilvaRubio et al 2008, Poblete-Morales et al 2013).…”