Effect of freezing on the metabolic status of L3 larvae of Anisakis simplex s. s.

The fish-borne parasite, Anisakis simplex s. s., triggers a disease called anisakiasis, that is associated with a gastrointestinal infection. The Anisakis is also associated with allergic response which may lead to anaphylactic shock. The A. simplex s. s. L3 larvae may be freeze tolerant despite when the nematodes will be cooled rapidly to -20 °C according to the sanitary authorities of the USA and the EU. The aim of this work was to study the metabolic status of A. simplex s. s. L3 larvae when frozen in terms of viability, expression of genes involved in the nematodes' survival of freezing, as well content of carbohydrates which play a cryoprotective role in thermal stress and are the main source of energy. The levels of trehalose were significantly higher after slow freezing treatment (p < .0001), than the fast freezing (p < .002). The lower temperatures induce changes, especially in trehalose synthesis gene expression, genes responsible for oxidative metabolism, and chaperone proteins, but we cannot state clearly whether these changes occur during freezing, or because they are already prevalent during cold acclimation. The induction of mentioned genes seems to be a common trait of both cold- and dehydration tolerance.

How effective is freezing at killing Anisakis simplex, Pseudoterranova krabbei, and P. decipiens larvae? An experimental evaluation of time-temperature conditions.

The consumption of raw or inadequately cooked marine fish can lead to several disorders caused by the ingestion of viable anisakid nematodes. Although anisakid larvae can be killed by subzero temperatures, making freezing an important control measure for this potential health hazard, these parasites can survive freezing under some conditions. Therefore, the aim of the present study was to experimentally evaluate the time-temperature conditions needed to kill Anisakis simplex and Pseudoterranova spp. The effectiveness of freezing was tested on two species of fish: cod, Gadus morhua from the North Atlantic, and herring, Clupea harengus membras from the southern Baltic Sea. Samples, which comprised skinless fillets of cod (n = 40) with visible parasites and whole herring (n = 240), were separately frozen at - 15, - 18, or - 20 °C for 24 h, or at - 20 °C for 48 h in the single-compressor freezer and at - 20, - 25, or - 35 °C for 24 h in the double-compressor freezer. After thawing, parasites were stained with malachite green and examined under the microscope for viability. All A. simplex and Pseudoterranova spp. larvae in cod fillets died at a temperature of - 15 °C or lower. However, freezing did not kill all the A. simplex larvae in whole herring: spontaneous movement of these parasites was observed in samples stored in the single-compressor freezer at - 15, - 18, and - 20 °C over 24 h. Our results demonstrate that the freezing procedure must consider both the capability of the freezing device and the nature of the fish product to ensure consumer safety.