Amyloodinium ocellatum is a cosmopolitan, ectoparasitic dinoflagellate that infects a wide variety of marine and brackish-water fishes. The life cycle of the parasite is direct and consists of three stages: the trophont which attaches to and feeds on the host, the tomont which is free-living and reproductive, and the dinospore which hatches from the tomont and infects new hosts. Each tomont can produce up to 256 dinospores. Because of its tolerance to a wide range of temperatures and salinities, its lack of host specificity, and its high reproductive capacity it is considered one of the most troublesome parasites in warm water marine aquaculture.
Early diagnosis of infections is critical to the effective management of outbreaks considering the prolificity of the parasite. The current gold standard for diagnosis is microscopic examination of gill tissue, but light infections may be missed, leading to false-negative outcomes and/or other organisms may be misdiagnosed as Amyloodinium, leading to false-positive ones. Moreover, microscopy cannot detect the parasite in the water prior to establishment of the infection. Both a conventional PCR and a LAMP assay are available. The LAMP is technically easier, quicker, highly specific, and more sensitive than PCR, but sensitivity was not assessed for detection of the parasite in either tissue or water beyond the benchtop scale. The objective of this study is to compare the sensitivity of light microscopy to that of the LAMP assay for detection of Amyloodinium ocellatum in gill tissue and water in simulated field conditions.
Forty-two 10-gallon aquariums were filled with 20 L of 25 psu artificial seawater and inoculated with either 0, 1, 10, 100, 1000, 5000, or 10,000 laboratory-grown dinospores (six tanks per dose), resulting in concentrations of 0, 0.05, 0.5, 5, 50, 250, and 500 dinospores/L, respectively. Ten 1-L aliquots from each of 21 tanks (3 tanks per dose) were removed and filtered on a 0.22 µm 47 mm nitrocellulose filter membrane and stored at -80 ?. The other 21 tanks (3 per dosage) received four naïve spotted seatrout (Cynoscion nebulosus) reared from captive broodstock spawns in recirculating systems. After 7 days or upon fish death, 4 gill arches per fish (2 from each side of the fish) were examined via microscopy then preserved in 95% ethanol for DNA extraction and analysis with the LAMP assay.
The lowest dinospore concentration that could be detected by the LAMP assay in water samples was 0.5 dinospores/L, while positive detections occurred only at 5 dinospores/L or higher when using LAMP or microscopy on gill samples. Thus, LAMP of water samples is a superior diagnostic tool. Simulations of replicated assays indicated that a 95% probability of detection was achieved with 10 replicated assays of water samples when the dinospore concentration is as low as 1 dinospore/L.