[Research Note]

The Lethal Effect of Freezing on Spirurid Nematode Larvae in Firefly Squids, Watasenia scintillans

NOBUAKI AKAO1, SETSUKO TUKIDATE1, YUKA MORI1, TAKASHI KAJIYAMA2, KAORU KONDO3 AND KOICHIRO FUJITA1

1Department of Medical Zoology, School of Medicine, Tokyo Medical & Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan

2Shimonoseki R & D Center, Nichimo co., Ltd. 3-7, Ozuki-kojima 2-chome, Shimonoseki, Yamaguchi 750-11, Japan

3Department of Parasitology, School of Medicine, Kanazawa University 13-1, Takara-machi, Kanazawa 920, Japan

Key words: Spirurina; firefly squids; freezing; creeping; larva migrans.

"Spiruroid type X" nematode larvae (Hasegawa, 1978), a causative agent of creeping eruption and ileus in humans, were recovered from the digestive organs of: sandfish, Arctoscopus japonicus (Otsuru et al., 1974); Akaska pollack, Theragra chalcogramma (Hasegawa, 1978); sagittate calamari, Ommastrephes soloanei pacificus (Okazawa et al., 1993); and firefly squids, Watasenia scintillans (Ando et al., 1992). Of these hosts, firefly squids are often eaten raw by humans. Most cases in which humans are found to have ingested Òtype XÓ larvae are reported from March to July (Ando et al., 1992). This period is consistent with the season for fishing firefly squids. In addition, some patients ate raw firefly squids just before the onset of creeping eruption. Therefore, some investigators have suggested that this type of larvae is chiefly responsible for creeping eruption and ileus (Fujihira et al., 1992; Kagei et al., 1992; Takahashi et al., 1992; Hasegawa et al., 1993; Shinozaki et al., 1993).

Recently, the consumption of raw firefly squids has rapidly increased in Japan. Finding a way to kill the spirurid nematode larvae in firefly squids would prevent a related increase in the number of cases of creeping eruption and ileus. In a previous report, we revealed that larvae in vitro died in a 14 % solution of sodium chloride within a day in vitro (Akao et al., 1994). However, this procedure is not suitable for mass preparation. We therefore examined the effect of freezing on the Òspiruroid type XÓ larvae.

In a preliminary study, 15 trays, each containing 21 firefly squids (7 rows x 3 columns), were placed side by side in a walk-in freezer (55.4 m3) for a designated period. The surface temperature of the frozen firefly squids was monitored with a digital thermometer (SK-2000MC, Iuchi, Osaka, Japan). The surface temperature of firefly squids usually required 30 minutes to fall to -32¡C; freezing times were measured and expressed from the point at which this temperature was reached. Only the squidsÕ digestive organs (the esophagus, the stomach and the intestine) were harvested. After digestion with artificial gastric juice (0.5% hydrochloric acid and 0.7% pepsin, pH 1.5), the larvae were recovered by the method of Okazawa et al. (1993). Table 1 shows the effects of freezing on the survival rate of the larvae. ÒSpiruroid type XÓ larvae were still alive when the firefly squids were kept at -32¡C for 40 minutes. However, none of the larvae survived in squids kept frozen for 60 minutes or longer. These experiments were carried out on 23 and 30 June 1994.

On the basis of the preliminary study, we conducted a further investigation on 27 February 1995, using a technique suitable for commercial preparation. Firefly squids captured in the Toyama Gulf were placed on a tray, as in the preliminary experiments, then kept in a carbon dioxide gas circulating freezer (C. P. F. Quick Freezer¨, type: G-1, D. M. L., Co. Ltd., Kanazawa, Japan). We developed this apparatus specifically for this study. The surface and core temperatures of the firefly squids were measured simultaneously during the experiment. Figure 1 shows the relationship between the surface and core temperatures of the firefly squids.

Figure 1. The relationship between the surface and core temperatures of firefly squids frozen with carbon dioxide. Carbon dioxide was supplied for 20 minutes.

Carbon dioxide was supplied for 20 minutes and the surface temperature became -32¡C within the first 8 minutes, but the core temperature was still -7.4¡C. It took 12 minutes when the surface temperature was -40¡C. The core temperature fell to -32¡C after 18 minutes. The firefly squids were then kept for 30 minutes after the surface temperature became -40¡C. This means that the core temperature was maintained below -32¡C for at least 20 minutes. As a control in this experiment, we also tried to recover the larvae from 1234 unfrozen firefly squids captured on the same day and at the same place. From the raw control, 5 larvae of Òspiruroid type XÓ were recovered, all living; from the 325 frozen squids, 11 larvae were recovered, all dead (Fig. 2).

These results suggest that keeping firefly squids frozen in a deep freezer (below -40¡C) for 40 minutes could reduce the risk of infection from Òspiruroid type XÓ larvae. During the freezing process, it is important to keep the core temperature of the squids below -32¡C rather than simply maintaining their surface temperature below -32¡C. Boiled firefly squids may be eaten safely. However, as long as the Japanese custom of eating raw fish remains unchanged, freezing squids is an alternative way to treat them to reduce the risk of infection.

Acknowledgment

We wish to thank Dr. Satoshi Shinonaga, Department of Medical Zoology, Tokyo Medical & Dental University for his assistance in preparation of the photographs. We also thank Ms Michiyo Katayama and Ms Keiko Numazaki for their technical assistance.

Reference

1) Akao, N., Okazawa, T., Nagase, H., Yoshida, M., Nakamura, H. and Kondo, K. (1994) : A parasitological survey of the spiruroid type-X larvae in firefly squids captured in the costal waters of the Hokuriku District. Hokuriku J. Public Health, 21, 56-59 (in Japanese with English Abstract).

2) Ando, K., Sato, Y., Miura, K., Chinzei, Y. and Ogawa, S. (1992) : Futher observation on the larva of the suborder Spirurina suspected as the causative agent of creeping eruption. Jpn. J. Parasitol., 41, 384-389.

3) Fujihira, K., Kawashima, A., Kurumaya, H., Akao, N., Ohyama, T. and Kondo, K. (1992) : A case reprot of creeping disease due to nematoda larva of the superfamily Spiruroidea. Rinsho Derma. (Tokyo), 34, 354-355. (in Japanese)

4) Hasegawa, H. (1978) : Larval nematodes of the superfamily Spiruroidea - a description, identification and examination of their pathogenicity. Acta Med. Biol., 26, 79-116.

5) Hasegawa, H., Sekikawa, H., Kenmotsu, M., Otsuru, M., Oka, K., Igarashi, T., Kaneko, H. and Homma, K. (1993) : A case of cutaneous larva migrans due to spirurin nematode found in Niigata Prefecture, Japan, with special references to the significance of cuticular morphology on identification. Jpn. J. Parasitol., 42, 12-17 (in Japanese with English Abstract).

6) Kagei, N., Kumazawa, H., Miyoshi, K., Kosugi, I. and Ishih, A. (1992) : A case of ileus caused by a spiruroid nematode. Int. J. Parasitol., 22, 839-841.

7) Okazawa, T., Akao, N., Oyama, T. and Kondo, K. (1993) : Prevalence and habitat of the type X larvae of the suborder Spirurina in squids. Jpn. J. Parasitol., 42, 356-360.

8) Otsuru, M., Shiraki, T. and Kenmotsu, M. (1974) : Some cases of larval or adult nematode migrans in the human tissue. Jpn. J. Parasitol., 23, 106-115 (in Japanese with English Abstract).

9) Shinozaki, M., Akao, N., Okazawa, T., Fukui, Y. and Kondo, K. (1993) : Detection of type X larva of the suborder Spirurina from a patient with a creeping eruption. Jpn. J. Parasitol., 42, 51-53.

10) Takahashi, S., Sato, T., Shimoi, K., Shinke, O. and Yoshimura, K. (1992) : A case reprot of creeping disease due to spirurid nematoda larva. Rinsho Derma. (Tokyo), 34, 341-346 (in Japanese).

Figure Legend

Figure 2. Spiruroid type X larvae recovered from raw (A) or frozen (B) firefly squids. The bar indicates 1.5 mm.