Application of multiplex quantitative polymerase chain reaction methods to detect common bacterial fish pathogens in Nile tilapia, Oreochromis niloticus, hatcheries in Costa Rica

Abstract

Edwardsiella spp., Streptococcus spp., and Francisella noatunensis subsp. orientalis are some of the most important fish pathogens affecting global tilapia, Oreochromis spp., aquaculture. In Costa Rica, the aquaculture industry is dominated by freshwater‐cultured Nile tilapia, Oreochromis niloticus, which are raised in all seven national provinces. At present, little is known regarding the diversity of pathogens present in these facilities, and definitive identification of agents associated with disease outbreaks are rare. To evaluate the prevalence of common bacterial pathogens in these systems, this study used multiplex quantitative polymerase chain reaction (qPCR) assays targeting Edwardsiella, Streptococcus, and Francisella species as a diagnostic and surveillance tool. In 2017, seven different tilapia hatcheries were visited, and 350 fingerlings were subjected to necropsy and molecular diagnostic evaluation. Fish exhibiting gross signs of disease were subjected to histological and microbiological analysis. For the first time, Edwardsiella anguillarum was recovered and molecularly confirmed from diseased tilapia in Costa Rica. In addition, F. noatunensis subsp. orientalis was identified in a region of Costa Rica where it had not been previously reported.

Pathologic changes in cultured Nile tilapia (Oreochromis niloticus) associated with an outbreak of Edwardsiella anguillarum

“…In 2018, chronic mortality (10%–30%) occurred over a four‐month period in O. niloticus fry and fingerlings at a Costa Rican aquaculture operation with a history of E. anguillarum infection (López‐Porras et al, ). The hatchery received a constant flow of untreated surface water supplied by a series of delivery canals, with fry produced from facility‐owned broodstock.…”

“…Edwardsiella ‐specific multiplex qPCR and E. anguillarum ‐specific end‐point PCR confirmed the case isolates (R18‐08; R18‐14‐1; R18‐14‐2; R18‐14‐13; R18‐14‐16; R18‐35‐2) as E. anguillarum (Griffin et al, ; Reichley et al, ) (Figure ). Fragments of the gyrB (~1,600 bp) and sodB (~400 bp) genes for the case isolates, as well as archived isolates recovered from the same farm (Eco2413; Eco2417) (López‐Porras et al, ), were amplified and sequenced as described previously (Griffin et al, ; Reichley et al, ). Isolates were 100% identical at the nucleotide level and shared 100% identity to the E. anguillarum‐ type strain ET080813 (GenBank Acc.…”

From the basics to emerging diagnostic technologies: What is on the horizon for tilapia disease diagnostics?

“…Target pathogens include TiLV, 55,56,88 ISKNV, 89 TiPV, 74 S. agalactiae 64,90 and F. orientalis. [91][92][93] Several multiplex TaqMan qPCR assays have also been developed to detect and quantify three to four pathogen species in a single PCR test, such as F. orientalis, S. iniae and S. agalactiae 94 ; A. hydrophila, A. veronii and A. schubertii 95 ; and E. ictaluri, E. tarda, E. anguillarum and E. piscicida. 96…”

“…184 was effectively used for disease surveillance on hatcheries in Costa Rica. 94 The extent to which assays can be multiplexed in this way is quite severely limited by the range of fluorochromes and the number of channels on the instrument that can detect the differing wavelength emissions. Once internal controls are accounted for, four to five pathogens per sample is the limit to which the assay can be multiplexed.…”

“…iniae and S . agalactiae 94 ; A. hydrophila , A. veronii and A. schubertii 95 ; and E. ictaluri , E. tarda , E. anguillarum and E. piscicida 96 …”