Catfish [channel (Ictalurus punctatus) and hybrid catfish (♀ I. punctatus × ♂ I. furcatus)] are some of the most cultured finfish species domestically, and catfish aquaculture is one of the most crucial industries in the southeastern United States. In recent decades, bacterial infections caused by Flavobacterium covae, Edwardsiella ictaluri, and virulent Aeromonas hydrophila have been the largest contributing factors to losses of fingerling and market-sized catfish all throughout western Alabama and other southeastern regions. To address the growing need for a rapid diagnostic test, a series of loop-mediated isothermal amplification (LAMP) assays have been developed, field tested, and validated across multiple sample types, collection environments, and test settings.
The first iteration of the LAMP assay was formulated using single-target primer and probe mixes (monoplex) that could successfully confirm the presence of virulent (vAh) and non-virulent strains of Aeromonas hydrophila. These initial tests indicated that positive results can be obtained from genomic DNA, viable bacterial colonies suspended in sterile ddH2O or TE buffer, swabs from infected livers, spleens, and kidneys, or external composite swabs from the mouth, gills, naris, eyes, operculum, and vent. These successful laboratory tests led to the development of two quadruplex LAMP assays designed specifically for catfish pathogens. One contained genus-level primers and probes for Aeromonas, Flavobacterium, and Edwardsiella strains with an Ictalurus internal control probe. The other assay was able to detect vAh, A. veronii, and A. sobria, all within a single 20 μL.reaction. Under laboratory conditions both quadruplex assays could obtain positive results on all bacterial targets, regardless of input sample type used. Once all LAMP assays were successfully validated in the lab, both quadruplex LAMP assays were taken to a local catfish operation and tested pond-side using a portable qPCR machine (Maverick, MAx16).
Six moribund fish across three different ponds were collected and swabbed both internally and externally. Each swab was placed in 2 mL of 30 mM NaOH + 0.01% tannic acid solution and vigorously mixed. A 10-fold serial dilution of each swab sample was prepared, and 4 µL of dilute swab sample was added to the 20 µL reaction mixture and subjected to a constant temperature. The LAMP assays were able to accurately indicate the presence of vAh, A. veronii, A. sobria, and Edwardsiella under 35 minutes. While the LAMP assays were running, each catfish was necropsied and the spleen, posterior kidney, and liver were plated on Tryptic Soy Agar (TSA) plates to cultivate any pathogenic bacterial species that result in positive LAMP results. After re-isolation and sequencing of six bacterial isolates of interest, two of the assembled genomes produced a 100% 16S match to hyper-virulent A. hydrophila and A. veronii. The sequencing results of another 14 isolates will be provided later. These encouraging results speak to the robustness and accuracy of these LAMP assays and may prove to be a more widely used and viable time-saving diagnostic test in the not-so-distant future.