SUMMARYAcid proteases hydrolysing haemoglobin, first described by Timms & Bueding (1959), were studied to investigate the interspecific and intraspecific distribution in adult parasitic helminths. Acid protease activity was detected in a variety of adult helminths, irrespective of their habitats, namely, parasitic nematodes, Angiostrongylus cantonensis, Dirofilaria immitis, Trichuris muris and Ascaris suum, a trematode, Paramphistomum sp. and cestodes, Diphyllobothrium erinacei and Hymenolepis nana. Much higher specific activity was observed in the blood nematodes, A. cantonensis and D. immitis than in the gastro-intestinal helminths examined. The intraspecific distribution of the acid protease activity within organs was studied in those nematodes from which the organs could be anatomically separated. The highest specific activity was observed in the intestine of A. cantonensis, D. immitis and A. suum. The activity was also found in other organs of these nematodes, namely, the reproductive organs and the body wall, but to a much lesser extent. The pH optima for the hydrolysis of haemoglobin were found to be in the pH range of 3·1 to 4·6. Each of the enzyme solutions from the 7 species of helminths hydrolysed haemoglobin faster than the other proteins examined but some activity on myoglobin, casein, albumin and other proteins was demonstrated. Pepstatin, a potent inhibitor of carboxyl proteases, inhibited the hydrolysis of haemoglobin by the acid proteases of the 7 species of the parasites. Acid protease activity from A. cantonensis and A. suum was not inhibited in the presence of thiol-, serine- and metallo-protease inhibitors at a concentration of 10−6 M. This fact, together with the inhibitory effect of pepstatin, suggests that the enzyme activity is likely to be due to carboxyl protease(s). Acid proteases such as cathepsin D and pepsin were simultaneously used for comparison of some enzymological characteristics. The substrate specificity of the enzyme solutions from the helminths seemed to resemble that of cathepsin D rather than that of pepsin. The effects of pH and pepstatin on the acid protease activity of the helminths were similar to those on pepsin and cathepsin D.

Evidence has been presented showing two kinds of acidic protease activities in adult Schistosoma mansoni, Dirofilaria immitis, Angiostrongylus cantonensis and Ascaris suum. A haemoglobinolytic activity without adding any SH-containing compounds was maximal at pH 3.5, 2.5, 30 and 3.5 in S. mansoni, D. immitis, Angiostrongylus cantonensis and Ascaris suum respectively. The inhibitor studies demonstrated that this activity is ascribable to carboxyl protease(s). In the presence of dithiothreitol, activity on Azocoll (azo-dye coupled hide powder) was maximal at pH 4.6, 4.6, 3.5 and 5.6 in S. mansoni, D. immitis, Angiostrongylus cantonensis and Ascaris suum respectively. The effects of inhibitors demonstrated that this activity belongs to the thiol protease class. The intraspecific distribution of the protease activities was studied in some of the nematodes from which the organs could be anatomically separated. The distribution patterns of the haemoglobinolytic and azocollytic activities were quite different in An. cantonensis and much the same in As. suum. Based on the present results, acidic haemoglobinolytic activities reported in adult S. mansoni by other authors are thought to be due to carboxyl and thiol protease(s) respectively.

Flubendazole and mebendazole given orally at 10 mg/kg/day 5-7 days post-infection (total 30 mg/kg) were found to eliminate 93-100% of Angiostrongylus cantonensis larvae in mice and rats. No significant difference was observed between the effects of the 2 drugs. The effectiveness of the drugs decreased with the increase in days post-infection on which medication was administered. It was found possible to treat A. cantonensis adults in rats by administering flubendazole or mebendazole at 10 mg/kg/day for 10 consecutive days. The drugs exhibited better anthelmintic efficacy in a divided dosing regimen than in a single dosing regimen.

Flubendazole or thiabendazole were administered orally to mice harbouring larvae of Angiostrongylus cantonensis, Trichinella spiralis, Diphyllobothrium erinacei or Hymenolepis nana 5-10, 28-33, 21-26 and 1-6 days post-infection respectively. All the mice infected with A. cantonensis were completely cured after treatment with flubendazole at 5 mg/kg/day for 6 days. No noticeable damage was found in the cerebral hemispheres of the mice treated with flubendazole and examined under a dissecting microscope. On the other hand, larvae were found in or on the cerebral hemispheres showing obvious haemorrhage in the control mice. The mice treated with flubendazole gained weight while the control mice lost weight. No larvicidal effect of thiabendazole on A. cantonensis was found at a dose of 10 mg/kg/day for 6 days. The mean reduction of larval T. spiralis in mice treated with flubendazole at 5, 50 and 100 mg/kg/day for 6 days was 64, 100 and 99% respectively. In comparison, thiabendazole showed no efficacy against T. spiralis larvae at 50 or 100 mg/kg/day. Mice harbouring plerocercoids of D. erinacei or H. nana larvae were not cured with either flubendazole or thiabendazole at 50 or 100 mg/kg/day for 6 days.

SummaryAcid phosphatase activity was demonstrated in the intact blood nematodes, Dirofilaria immitis and Angiostrongylus cantonensis. Biochemical studies on acid phosphatase, using intact females and whole worm, body-wall and visceral homogenates were undertaken to clarify the characteristics and the distribution of the enzyme. In D. immitis, high acid phosphatase activity was observed at pH 3·8–5·8 and in A. cantonensis, at pH 4·5–6·0. Molybdate, fluoride, copper and zinc ions and L(+)-tartrate were effective inhibitors of the enzymes of both parasites while cobalt and magnesium ions and D(−)-tartrate had no noticeable effect on the activity. When the effect of substrate concentration on the phosphatase activity was studied, kinetic curves of Michaelis–Menten type were obtained with the 2 species of intact worms as well as their homogenates. The reproductive organs and body wall of D. immitis showed high total acid phosphatase activity. In A. cantonensis, the majority of the enzyme was localized in the body wall. The activity of intact A. cantonensis expressed as μg Pi/h/mg dry weight decreased with increase in mean worm weight. The characteristics of the acid phosphatase of the 2 nematodes are compared with those of other parasitic helminths and of acid phosphatase of mammalian origin. The localization of the phosphatase responsible for the hydrolysis of the external substrate has been discussed for D. immitis and A. cantonensis based on results of the kinetics and distribution of the enzyme.