Anisakis spp. are marine nematodes present in fish, crustaceans, and cephalopods that can cause a foodborne zoonosis known as Anisakiasis, by ingesting raw fish infected with larvae in their third stage (L3) or infected fish processed or cooked insufficiently to inactivate the larvae. Anisakiasis manifests as gastric, intestinal forms. Additionally, allergic symptoms can occur, and even life-threatening anaphylactic shock.
One of the control measures for avoiding human infections due to the ingestion of the variety of seafood dishes containing raw or undercooked seafood is the application of treatments that inactivate the larvae present in fishery products. There is increasing information on the time-temperature conditions that can be lethal for Anisakis larvae, but little is known about the infective potential of larvae that survive heat treatments.
The objective of this work was to identify the behavioral and physiological changes of Anisakis larvae that survived thermal treatments at maximum temperatures that actually may be reached in accidentally undercooked fish in order to characterize the associated risks. To do so, Anisakis L3, thermally treated at a fixed temperature for different exposure times, were studied in terms of locomotor activity, survival in gastric juice, agar penetrative ability, and respiration capacity. Results show that, even after relatively short treatments that rendered survival ratios () close to 1 (i.e. all alive), some behavioral and physiological characteristics can be considerably impacted.
Methods
Preparation of Anisakis larvae. Five batches of Anisakis spp. larvae in the third stage were obtained from heavily infected hakes (Merluccius merluccius L.) caught at the Northeast Atlantic fishing areas and bought at a local fishmonger (Madrid, Spain) within 5-8 days’ interval from the catch to purchase. Once in the laboratory, live, non-encysted larvae were removed from the fish using forceps and cleaned with 0.145 M NaCl solution. Subsequently, sets of 50 larvae were placed in 15 mL Falcon tubes, containing 10 mL of 0.145 M NaCl, and stored at 4-5 °C with routine weekly maintenance throughout the experimental period, which typically ranged between 0 and 30 days. During the maintenance, live larvae were cleaned with 0.145 M NaCl and recounted before restoring in the tubes, while damaged, moribund, or dead larvae were discarded. Prior to each experiment, viability of the larvae was visually evaluated by verifying their typical movement.
In this study, L3 larvae were heat treated at 50 °C for different exposure times (0-320 s). Previous results revealed that these temperature-time combinations, that may actually occur in accidentally undercooked fish, induced a wide range of survival ratios, ranging from complete inactivation ( =0) to full survival ( =1), thus making it possible to characterize the associated risks.
Locomotor activity of viable larvae. The locomotor activity of Anisakis L3 that survived quasi-isothermal treatments at 50 °C for different exposure times was analyzed with an infrared locomotor tracking (ILT) system previously described by Simonetta & Golombek (2007). The system used a 96 beam sensor array to detect movement of individual larvae located in a liquid medium in 96 well microplates. To do so, it counts infrared photo-beam interruptions when larvae cross the stationary LED beam located at the center of the well in a fixed time lapse. Briefly, after the heat treatment in the thermal cycler, 170 μL of 0.145 M NaCl were added to each of the 96 wells in microplates containing one Anisakis L3 per well. Then, the microplate was covered with a transparent film and transferred to the ITL system (wMicroTracker MINI, PhylumTECH S. A., Santa Fe, Argentina) located in an incubator (Sanyo, MCO-18AIC, Electric Co., Japan) at 37 °C. The acquisition system was programmed with the detection mode “Mode 1, C. elegans” and a signal-to-noise ratio of 1.8. Light control was enabled with blue light at the Cycle/Pulse mode with 30 minutes on and 690 minutes off repetitions. After acquisition up to 22 h, the beam interruptions counted by the device were analyzed with a bin size of 30 minutes. Those larvae with more than 5 counts in any 30 min-period along the experiment were considered alive. After instrumental measurements, larvae displaying ≤5 counts were again visually checked for viability to discard false negatives. Only activity counts of alive larvae were further considered. Locomotor activity was expressed as the average beam interruptions of alive L3 detected in 30 min periods over 22 h.
Results
Locomotor activity of Anisakis L3 surviving quasi-isothermal treatments at 50 °C. The locomotor activity of surviving Anisakis L3, measured as the average of activity counts detected in the wMicroTracker MINI device during 22 h of incubation at 37 °C, showed a decreasing linear trend as the exposure time to 50 °C increased (Fig. 2). The decrease of activity counts occurred even at conditions where 100% of the larvae had been able to survive the heat treatments (Fig. 2). Thus, only 64% of the activity counts observed in untreated larvae were detected in larvae heated at 50 °C for 60 s even though, after this treatment, all larvae had been considered viable according to EFSA definition (BIOHAZ, 2010).
Thermal treatments can cause varied effects on Anisakis larvae. High temperatures can affect larval body shape and cuticle that change from a homogeneous shape with smooth cuticle to an irregular one, with breakages and swollen. Apart these structural effects, thermal treatments can also induce significant changes on behavioral and physiological characteristics of larvae. One of the features expected to change with treatment intensity until complete inactivation is the ability of the larvae to move. Some authors have evaluated changes in Anisakis mobility and motility by using viability scores assigned after visual inspection of the larval movement. However, the procedure is tedious. In other parasites, high throughput screening methods have been recently implemented; for example, for the study of anthelmintic drugs, this approach has been hardly used in Anisakis. Our results (Fig. 2) show a high sensitivity of the locomotor activity after heat treatments, even with survival ratios close to 1, suggesting early impairments of the larvae functionality.
Overall, the results show that the behavior of heat-treated Anisakis larvae can be different depending on the intensity of heating in terms of the exposure time. The changes investigated at a fixed temperature suggest that as the treatment intensity exceeds a certain exposure time, some physiological and behavioral characteristics of the larvae are modified even when the survival rate is close to 1.
Food Control, 2024. https://doi.org/10.1016/j.foodcont.2024.110564.
Aiyan Guan, Marina Usieto, Laura Otero, Susana C. Arcos, Alfonso Navas, Isabel Sánchez-Alonso, Mercedes Careche.