Health Canada
www.hc-sc.gc.ca
Common menu bar links
Institutional links
Isolation and Identification of Anisakid Roundworm Larvae in Fish
5.3.2 Fill funnel or vessel with normal saline (completely immersing fish tissue). Cover with plastic wrap or aluminum foil and leave apparatus overnight at room temperature. Live larvae will migrate out of the tissues and fall through the sieve and into the neck of the funnel (or into the bottom of the vessel).
5.3.3 After 16 to 18 h, drain 100 mL of sediment into a beaker. Using forceps, remove any visible worms to a culture dish containing normal saline and carefully examine the remaining suspension, using a stereoscopic microscope or magnifier, for any smaller worms.
5.4 Digestion
5.4.1 Add about 200 g of fish tissue to 750 mL of warm pepsin solution in a large beaker.
5.4.2 Place the beaker in a 37/C shaking water bath so that the level of the water is within 1 cm of the fluid level in the beaker. Shake the sample at low speed for about 15 min.
5.4.3 Adjust the sample to pH 2 with 6 N HCl, cover beaker with foil, and continue shaking for 24 h (or until the tissue is fully digested).
5.4.4 Pour the digested material through a U.S. Standard No. 18 sieve (or equivalent sized kitchen sieve) into a suitable container. Rinse the sieve with normal saline and examine the washed remains by immersing the mesh in clean normal saline. Use forceps to transfer worms to a culture dish containing normal saline.
5.4.5 Examine the material which passed through the sieve by transferring it to a clamped funnel, allowing it to settle for 1 h, and draining the sediment into a beaker as described for elution. Examine for smaller worms.
5.4.6 Alternatively, digested material can be passed through nested No. 18 and No. 140 U.S. Standard sieves which are then separated and partially immersed in normal saline. Collect and transfer to a dish of normal saline any of the larger worms caught in the No. 18 sieve. Similarly, transfer any smaller worms caught in the No. 140 sieve to normal saline.
6. Recording Results
All parasites isolated from a sample should be recorded as either live, dead (intact), or fragments. Parasites may be considered live if they demonstrate any movement during the isolation procedures. Anisakid larvae generally become much more active as they approach room temperature. In addition to numbers of parasites present per kg of product, a preliminary identification of these parasites (or a brief description) should be included if possible. The species of fish examined, the source, the total sample weight, and the weight and number of individual samples (fillets) should also be recorded, along with any other relevant information.
7. Preservation and Storage of Larvae
A variety of preservation techniques have been used for nematodes. Live or previously frozen worms should first be thoroughly fixed in glacial acetic acid or AFA, and then preserved in small glass vials containing 70% ethanol, or, preferably, an alcohol-glycerol solution. For microscopic examination, the use of a clearing agent may be necessary to make the nematode cuticle more transparent. A temporary slide mount can be prepared, after which the worms are returned to the preservative solution. Nematodes are not generally stained for microscopic examination.
8. Parasite Identification (Fig.2)
Third-stage larvae of A. simplex can generally be distinguished from P. decipiens on the basis of their size and colour. A positive identification can be made by examining the anterior gut structure (10.1, 10.8) which is easily seen in fresh or preserved larvae using a stereoscopic microscope. Third-stage larvae of Anisakis simplex are small white worms, 9-36 mm in length, with a straight anterior gut structure consisting of esophagus, ventriculus, and intestine (10.11). P. decipiens are typically reddish-brown in colour, 9-58 mm in length, and have an anteriorly projecting intestinal caecum (10.10). Contracaecum and Phocascaris spp. third- stage larvae are very difficult to distinguish morphologically and are often discussed as a single group. Third- stage larvae within the Contracaecum/Phocascaris complex can, however, be readily distinguished from those of A. simplex and P. decipiens. The former are greenish-brown in colour and 7 to 30 mm in length, with both anteriorly projecting intestinal caecum and posteriorly projecting ventricular appendix (10.8, 10.12).
