Experimental infection of mice with Hysterothylacium aduncum (nematoda: anisakidae) larvae from marine-farmed trout in Chile

Se probó la sobrevivencia de larvas de H. aduncum provenientes de truchas de cultivo del sur de Chile, implantadas en el abdomen de ratones y su incapacidad de producir anisakiasis en mamíferos. Mediante cirugía se introdujeron larvas L3 y L4 del nemátodo en la cavidad celómica de 6 ratones vivos y 2 ratones controles sacrificados y refrigerados. Los nemátodos murieron rápidamente en los organismos vivos (de sangre caliente), mientras que el 73% de estos helmintos sobrevivieron después de 18-22 hr de haber sido inoculados en los ratones muertos y refrigerados. Este nemátodo anisákido es menos agresivo y morfológicamente diferente al Hysterothylacium tipo MB con antecedentes zoonóticos y, por lo tanto, no podría producir anisakiasis al ser consumido por el ser humano.

Key words: anisakid, inoculation, mice.

Palabras claves: anisákido, inoculación, ratones.

INTRODUCTION

Adults, fourth-stage (L4), and third-stage (L3) larvae of Hysterothylacium aduncum (Rudolphi, 1802) were observed parasitizing the stomach, intestine and pyloric caeca of netpen-reared trout Oncorhynchus mykiss (Walbaum, 1792), coho salmon O. kisutch (Walbaum, 1792), and to a lesser degree Atlantic salmon Salmo salar Linnaeus, 1758 in the south of Chile (Carvajal & Gonzalez, 1990;Carvajal et al., 1991; González, 1998). Although this anisakid reaches maturity in fish and includes invertebrates and other fishes as intermediate hosts in it's life-cycle, an unidentified larval stage of the genus Hysterothy-lacium Warth & Magath, 1917, has been implicated in human anisakiasis (Norris & Overstreet, 1976; Overstreet & Meyer, 1981). This zoonotic disease is caused by the ingestion of raw or inadequately cooked seafood containing larval marine nematodes belonging to the family Anisakidae. These larvae penetrate the stomach or intestinal walls of the host and produce severe gastric ulcers. The following species have been related to the anisakiasis disease: Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878) , A. physeteris Baylis, 1923, and Pseudoterranova decipiens (Krabbe, 1878) (see Carvajal, 1991). Norris & Overstreet (1976) described as type MB larva, the unidentified larva of Hysterothylacium from Mississippi that adheres to the intestinal mucosa, penetrates the stomach wall and the liver, and remained in the abdominal cavity of white Swiss mice. Later, Overstreet & Meyer (1981) reported that the same type of larvae from the Gulf of Mexico caused hemorrhagic lesions in the stomach of the Rhesus monkey. Following this morphological nomenclature the larvae of H. aduncum are classified as type MA larvae.In order to evaluate the risk of anisakiasis by accidental ingestion of H. aduncum larvae from marine farmed trout in Chile, the present study reports the experimental infection of these larvae into the celomic cavity of a model mammalian host (the laboratory white mouse Mus musculus), to assess its survivorship in a warm-blooded host.

MATERIALS AND METHODS

Sixty two trout were collected during selection in marine netpen cultures in Dalcahue Channel (42° 25' 30" S; 73° 38' 30" W), southern Chile, and transported ice-chilled 200 Km to the laboratory in Puerto Montt. Trout (100 to 1.200 g) were dissected and the nematodes were placed in l µm filtered seawater at room temperature. Third and fourth-stage larvae from 5 to 11 mm long (identified according to Gonzalez & Carvajal, 1995), were selected and introduced into polyethylene tubes adapted to plastic syringes with physiological serum. After l2 hr, ap-proximately 9-15 larval nematodes were injected into the celomic cavity of 8 mice through ventral surgical incisions. The mice (20-30 g) were previously anesthetized with sodic thiopental. A dose of 1.25 mg per g of animal was injected into six mice and a dose of 2.5 mg, to the two remaining mice. The latter two, that were used as the control, died and were subsequently refrigerated to provide the temperature condition of marine fishes, their most probable definitive hosts. The animals were sacrificed with ether between 18-22 hr post-infection, dissected, and examined microscopically for larvae.

RESULTS AND DISCUSSION

All the larvae were dead in all the live mice (the burden could not be evaluated because most of the nematodes were disintegrated). In the dead-and-refrigerated hosts, 73% of the inoculated larvae (11 and 10 larvae respectively in both mice, abundance = 12.5) were recovered alive and moved very actively (Table 1).
 
 

The present work demonstrates that H. aduncum larvae obtained from salmonids reared in Chile died after their abdominal implantation in live mice. The Hysterothylacium type MB larvae that invaded albino Swiss mice, entered the stomach wall where most of them died. However, some of those that transversed the wall, survived a week within the abdominal cavity (Overstreet and Meyer, 1981). The fact that the larvae of the present work died so fast compared to the Hysterothylacium type MB larvae, suggests that there is low or no risk of anisakiasis disease outside the digestive tube for consumers of raw or inadequately cooked salmonids from Chilean marine farms. Although the experiment is not conclusive regarding the ability of the larvae to penetrate the gastric or intestinal wall of the host, it seems that Chilean H. aduncum are less aggressive than Hysterothylacium type MB. In fact, although Petter (1969a &b) related H. aduncum from Sardina pilchardus (Walbaum) to human anisakiasis in France, as do Norris and Overstreet (1976) with H. aduncum from the Gulf of México, Vermeil et al. (1975) showed that this nematode could not penetrate the stomach of the host in experimental infections carried out in rabbits. This result is in agreement with the life cycle of Hysterothylacium species described by Moller & Anders (1986) whose larvae mature in fishes, and to the particular life cycle of H. aduncum of netpen-reared salmonids in Chile described by Gonzalez (1998), where reproductive nematodes can be found in the digestive tube of trout.

In the Hysterothylacium type MB larvae that penetrated the digestive tube of white Swiss mice and Rhesus monkey (Deardorff and Overstreet, 1981), the intestinal caecum (IC) is shorter than the ventricular appendage (VA) (ratio of lengths IC:AV= 1:14-68), and the tail is bluntly rounded and without spines. These unidentified larvae are most probably an L3 according to the recent findings of Køie (1993), who showed that second-stage larvae molt to L3 within the egg. In the L3 of H. aduncum observed in the Chilean netpen-reared salmonids, the intestinal caecum is as long or longer than the ventricular appendage (ratio of lengths IC:AV=1:0.6-1.l), the bulbous tail ends in one terminal spike, and the posterior multispinous process of the L4 stage shows through the bulbous end (Carvajal et al., 1995; Gonzalez and Carvajal, 1995).

It is important to note that the zoonotical anisakids A. simplex, A. physeteris, and P. decipiens have not been reported at present in the Chilean salmonid culture systems, or in other netpen farms (Kent, 1992), despite their abundance in the native pelagic fishes (Fernández, 1985; Carvajal and Cattan, 1985; George-Nascimento & Arancibia, 1994) because of the absence of the euphausids and mysiids in the fauna related to the farm facilities located in marine inner fjords (González and Carvajal, 1995; González, 1998). These invertebrates, which are the first intermediate hosts of the zoonotical anisakids A. simnlex A. physeteris and P. deciniens (see Williams and Jones, 1976), are more common in the open ocean where no farm exists.

SUMMARY

To evaluate the potential of Hysterothylacium aduncum as an anisakiose-disease producer in mammals and therefore in human consumers, third and fourth stage larvae of the nematode from the digestive tract of Oncorhynchus mykiss reared in netpens, were introduced surgically into the celomic cavity of six live white mice and two dead-and-refrigerated mice as controls. The experiment showed that all larval nematodes died quickly in the live warm-blooded organisms, whereas 73% of the nematodes survived 18-22 hr in the dead-and-refrigerated control mice. This anisakid nematode, having a type MA larvae, is less aggressive and morphologically different from the Hysterothylacium type MB and therefore might not produce "anisakiasis".

ACKNOWLEDGEMENTS

I gratefully acknowledge Georgina Lembeye (Universidad Austral de Chile, Puerto Montt) for providing the laboratory white mice and Domingo Vera, Med. Vet. for invaluable assistance in the surgery of mice. I am also indebted to Verónica Villegas and Yenny Navarro for technical assistance as well as Juan Carvajal (Universidad de Los Lagos) and Brian Dyer (Universidad del Mar) by the critical review of the manuscript. This work was supported by the International Foundation for Science, Sweden, Grant Nº A/2076.
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Aceptado: 16.10.97.

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