In-vitro Effects of Benzalkonium-ion Intercalated Aluminium Triphosphate on the Second-stage Larvae and Fertilized Eggs of Toxocara canis

NOBUAKI AKAO1), SETUKO TSUKIDATE1), KAORU KONDO2), AND KOICHIRO FUJITA1)

1)Department of Medical Zoology, School of Medicine, Tokyo Medical & Dental University
1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan
2)Department of Parasitology, School of Medicine, Kanazawa University
13-1, Takara-machi, Kanazawa 920, Japan

Introduction
The results of epidemiological surveys have drawn attention to the importance of sandpits contaminated with the eggs of the Toxocara species as an infectious source of toxocariasis. Many investigators reported that 13 to 92 % of sandpits in parks were contaminated by the eggs (Dada and Lindquist, 1979; Dunsmore et al., 1084; DŸwel, 1984; Horn et al., 1990; Jansen et al., 1993; Shimizu, 1993; Uga, et al., 1989; Uga, 1993; Valkounova, 1982a; 1982b). Moreover, some sandpits contained infective larvae which were the causative agent of visceral larva migrans. Although a few conventional control measures, such as steam sterilization (van Knapen, et al., 1979) or permanent fencing around the sandpit (DŸwel, 1984) have been successful, treatment either needs to be repeated or is costly.
In this paper, we report a new approach to sterilizing sand contaminated by T. canis eggs. The aim of the present study is to reduce the risk of T. canis infection from sandpits in public parks and playgrounds.
Materials and Methods

T. canis
We obtained adult female worms of T. canis from infected dogs. Fertilized eggs were collected from the uterus of the worms. The eggs were then kept in 0.5 % formaldehyde solution at 30 ¡C for 4 weeks. They were then washed free of formaldehyde, treated with sodium, and hypochlorite and hatched as second-stage larvae by the method described by Kondo et al. (1981). The larvae were collected and maintained in a culture medium, DulbeccoÕs Modification of EagleÕs Medium, (Flow Laboratories, Irvine, Scotland) at pH 7.2 at 37¡C with weekly replacement of the medium. Some fresh, fertilized eggs were used in the growth inhibition test.

Chemical
Benzalkonium-ion intercalated aluminium triphosphate (BIAT) was kindly supplied by Rasa Kogyo Co. Ltd. (Tokyo, Japan). The supplier reports the synthesis process to be as follows. BIAT was synthesized by suspending 10 g of aluminium triphosphate in 500 ml of 1 % benzalkonium chloride solution with 0.5 % n-butylamine. The suspension was stirred for 2 hours at room temperature to form the intercalation compound. The resultant product was filtered, washed with distilled water, and dried. The compound was a white slightly water-soluble powder.

Assay
For assays of larvae killed by BIAT, the larvae were incubated with serial dilution of 25 mg/ml BIAT in 24-well microplates (Corning, USA). The BIAT was dissolved in 2 % W/W dimethyl sulfoxide (Sigma Chemical Company, USA), and diluted in 0.1 M phosphate buffered saline solution (PBS, pH 7.2) to keep the dimethyl sulfoxide concentration at 2 %. The larvae were then incubated with serial diluted BIAT at 30¡C for the designated periods. After incubation, some larvae in each well was plated in 1.5 ml-polypropylene microcentrifuge tubes containing 1 ml of PBS. The larvae were then centrifuged, and supernatant fractions were aspirated and discarded. The larvae were suspended in PBS again and put on glass slide and covered with glass measuring 22 x 22 mm. The slide were subsequently incubated at 37¡C for 10 min. After this incubation, the activity of the larvae was assessed and expressed by the mobility index described previously (Kiuchi et al., 1987).
To evaluate the effect of BIAT on T. canis eggs during development, the eggs incubated with BIAT at 30¡C for 2 weeks, and then washed free from BIAT with PBS. The viability of the larvae in the eggs was assessed by gently crushing the eggshell and determining their mobility indices (Kiuchi et al., 1987). Embryonated eggs were obtained by incubating fertilized eggs for 3 weeks at 30¡C and the effects of BIAT on these embryonated eggs was examined. The embryonated eggs were incubated with BIAT at 30¡C for 30 days, and then the mobility indexes of the infective larvae were calculated as described previously (Kiuchi et al., 1987).

Results
Larvicidal effects of BIAT
In this assay, we attempted to assess the effect of BIAT on the larvae at concentrations ranging from 25 mg/ml to 0.025 mg/ml. However, we were unable to observe the larvae in the wells that contained more than 12.5 mg/ml of BIAT. Because the residual BIAT in the microcentrifuge tube affected larval mobility indices and the surpernatant was still cloudy even after washing three times with PBS at these concentrations, the data for 12.5 mg/ml or more were not shown.
Figure 1 shows the dose-dependent larvicidal effect of BIAT in vitro. At a concentration of 6.25 mg/ml, BIAT killed all larvae within 30 min.

Fig. 1 Dose dependent larvicidal effect of benzalkonium-ion intercalated aluminium triphosphate on second-stage larvae of Toxocara canis.

We noticed the larvae quavered intensely immediately after contact with BIAT, then stopped their activity. Regardless of the concentration of BIAT, all larvae died in a straight form after 6 hours of incubation (Fig. 2) . However, some larvae appeared to swell at a concentration of 3.2 mg/ml or more before they died (Fig. 3).

Effects of BIAT on T. canis eggs
We evaluated the ability of BIAT to inhibit the growth of fresh eggs of T. canis. Fertilized T. canis eggs became embryonated eggs containing larvae 2 weeks after incubation with BIAT at a concentration of 3.2 mg/ml or more at 30¡C. However, the larvae in the eggs were obviously degenerative, and were already dead when artificially hatched (Fig. 4). On the other hand, BIAT at concentrations up to 6.25 mg/ml had no effect on larvae in eggs that had already become embryonated despite continuous incubation at 30¡C for more than 30 days.

Discussion
Benzalkonium ion, the active ingredient of BIAT, is an anti-septic agent. The mode of action is assumed to be a long alkylate chain which destroys the bacterial cell membrane. However, benzalkonium ion is ordinarily used in solution, and, therefore, its persistence is limited. On the other hand, intercalated benzalkonium ion is thermostable and is not easily dissolved in water. These characteristics offer the advantage that BIAT dose not need to be applied more than once to the area requiring disinfection.
Benzalkonium ion also has a strong larvicidal effect on second-stage T. canis larvae (Prof. Tsuda, Y., Faculty of Pharmaceutical, Kanazawa University, personal communication). In the present study, we observed that the cuticle of some larvae became swollen when they came in contact with BIAT, suggesting the larvae, like bacteria, were directly impaired by the benzalkonium ion. However, almost all the larvae died in straight form without any morphological change after incubation with BIAT. This finding indicates that another mechanism might be involved in the larvicidal effect of BIAT. BIAT killed the infective larvae in eggs when fertilized eggs were continuously incubated with BIAT starting at the beginning of their development, but was ineffective for larvae which had already matured. These findings suggest that BIAT might be effective only during the early stages of the developing egg. Further study is needed to clarify this point.
BIAT has already been used as an antiseptic agent for apparel and tableware. The administation of BIAT more than 20 g/kg of body weight dose not result in a survival period of mice, and no abnormality has been reported in the mutagenicity test and skin patch test, indicating the low toxicity of BIAT. These findings are important for use in the field.
BIAT has not only a strong larvicidal effect on second-stage T. canis larvae in vitro, but also on bacteria, especially Staphylococcus aureus, Escherichia coli, Klebsiella pneuminiae and Pseudomonas aeruginosa (Isquith, et al., 1972) all of which may also be found in sandpits.
In a recent review of toxocariasis, Glickmann and Magnaval (1993) stated that there is no practical method available to purge the soil of Toxocara eggs. We believe, however, that chemical sterilization of sand is better than steam sterilization or fencing around sandpits in reducing the risk of Toxocara infection. In future studies we will attempt to ascertain the effect of BIAT on Toxocara eggs in a sandpit where fecal contamination has been established.

Acknowledgment
We wish to thank Mr. Isaku Makino (Rasa Kogyo Co. Ltd., Tokyo, Japan) for kindly supplying the BIAT.

References
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Fig. 2 All second-stage Toxocara canis larvae died within 6 hrs when they were incubated with benzalkonium-ion intercalated aluminium triphosphate (BIAT) at the concentration of 0.025 mg/ml.
Fig. 3 Some of the second-stage Toxocara canis larvae appeared to be swelling 30 minutes after incubation with benzalkonium-ion intercalated aluminium triphosphate (3.2 mg/ml, a). Larvae without BIAT showed normal appearance after the same incubation period (b).
Fig. 4 Degenerative dead larva in Toxocara canis egg after incubation with benzalkonium-ion intercalated aluminium triphosphate (3.2 mg/ml) at 30¡C for 2 weeks (a). Fertilized eggs developed into normal embryonated eggs after the same incubation period (b).