INTRODUCTION
Recently we have demonstrated that a chemical medium can be used as a substitute for infected-rat feces for harvesting the third-stage filariform larvae (L
3) of
Strongyloides venezuelensis from eggs in a polyvinyl culture bag (
Baek et al., 1998a). The egg hatch rate and larval metamorphosis to the L
3 stage are greatly influenced by incubation temperatures, concentration of culture medium and numbers of eggs inoculated in the culture bags. We have also showed that larval development rate to the L
3 stage is significantly different according to the in vitro culture methods. In a subsequent experiment, the viability of
S. venezuelensis in different stages maintained in vitro has been assessed (
Baek et al., 1998b). Eggs, L
3 and adults of
S. venezuelensis obtained from experimentally infected rats can survive for an extended period in various culture media at different temperatures. The development of intestinal nematode from the free-living stage (e.g., L
3 stage) to the parasitic adult stage depends on several factors related to host (e.g., species, strain, age, sex and resistance) and parasite species (e.g., strain, infective dose and culture age of L
3) (
Solomon and Haley, 1966;
Carter and Wilson, 1989;
Sato and Toma, 1990;
Khan et al., 1993). Taira et al. (
1994) have demonstrated that the inoculation routes of
S. venezuelensis L
3 significantly influence the worm burdens. Additionally, studies on morphological development and infectivity of free-living stage (e.g., L
3) and parasitic adult nematodes are considered useful in analysing the mode of action of anthelmintics and resistance thereto (
Martin, 1997). However, there has been, so far, no report on the comparative analysis of morphological development and infectivity of L
3 stage and parasitic adults of
Strongyloides species according to the methods of in vitro cultures. Therefore this study was attempted to report on the effects of in vitro culture methods including currently established nutrient broth culture (
Baek et al., 1998a) on morphological development of L
3 and their infectivity to rats; recovery rate of migrating stage larvae (MSL
3) and adults from the lungs and the small intestines, respectively, morphological development of the corresponding adults, worm burdens in the small intestines, fecundity, egg outputs and persistence of infection.
DISCUSSION
Strongyloides venezuelensis, a natural parasite of the rat (
Brumpt, 1934), initiate a parasitic life cycle by the L
3, which penetrate host's skin, migrate to the lungs and then travel to the small intestine where they eventually develop to the adult worms. It has been considered as a suitable model for the study of host-parasite interaction of strongyloidiasis since the parasite can be easily passaged in the laboratory and large numbers of eggs and larvae are obtained from rats exposed to 10,000-30,000 L
3 (
Taira et al., 1994). In addition, this nematode does not cause sudden death even in massive infection in rats (
Taira et al., 1995) as compared with another closely related species,
Strongyloides ratti, which can cause sudden death in rats and Mongolian gerbils. The morphology of L
3 obtained from different conventional in vitro cultures as well as those of the in vivo derived adult worms have been reported previously (
Little, 1966;
Hasegawa et al., 1988;
Taira et al., 1994). After primary infection with
S. venezuelensis, the subsequent development to the parasitic adult stage in a single and/or concurrent infections with
S. ratti, pulmonary and intestinal worm burdens, kinetics of egg outputs, persistence of infection and immunological responses have been reported in rats (
Werthiem, 1970a,
1970b;
Carter and Wilson, 1989), mice (
Sato and Toma, 1990), Mongolian gerbils (
Tsuji et al., 1993) and Syrian golden hamsters (
Shi et al., 1994). In the present study, we have demonstrated that in vitro culture methods have significant effects on morphological development to the L
3 stage, however, they do not influence the infectivity of the larvae to rats.
A significantly greater body length but not body width of the L
3 were observed in FPC than those in NBC and FC, which also reflected on subsequent morphological development of the corresponding adult worms with an increased body length but not body width. The reason for such differences in morphological dimensions of the L
3 and of corresponding adults according to the culture methods is not clear. It may be explained that an increased body length of the L
3 harvested from FPC is due to the use of infected-rat feces directly in the culture device without isolating and repeatedly washing the eggs in SS as was done in NBC and FC (
Baek et al., 1998a). Thus the larval microenvironment was not physically interfered in FPC and the larvae gain optimum morphological development. However, in the previous report, we have shown that the saturated salt solution used for the isolation of eggs does not affect the egg hatch rate, and yields of the L
3 are higher in NBC as compared to FPC (
Baek et al., 1998a). The reason for higher morphological development of adult worms corresponding to FPC is difficult to explain. Morphological dimensions with respect to body length of the L
3 recorded in NBC and FC except for FPC were observed smaller than those described for
S. venezuelensis L
3 by several authors (
Little, 1966;
Wertheim, 1970a;
Hasegawa et al., 1988;
Taira et al., 1994). These findings suggest that growth and development of the L
3 are influenced not only by composition and/or methods of in vitro cultures but also by geographical and/or strain variations (
Hasegawa et al., 1988). Irrespective of the culture methods, morphological dimensions with respect to body length of adults recovered from Fischer (F344) rats on day 7 PI were greater than those reported by Little (
1966) and Taira et al. (
1994), but smaller than those reported by Wertheim (
1970a) and Hasegawa et al. (
1988). This variation may be due to the differences in rat strain, infective dose, parasite strain and stage of infection at which the worms were recovered.
Infectivity of the L
3 was not different according to the culture methods although recovery rate of the MSL
3 and adult worms was slightly higher in those corresponding to FPC than those corresponding to NBC and FC. However, infectivity has been known to be significantly influenced by the inoculation routes; higher infection rate was resulted from subcutaneous and percutaneous inoculation than from oral administration of L
3 (
Taira et al., 1994). The overall recovery rate of MSL
3 and adults in the present study are much higher than that observed in rats (
Taira et al., 1994), but much lower than those in mice (
Sato and Toma, 1990;
Khan et al., 1993;
Korenaga et al., 1995) and Mongolian gerbils (
Horii et al., 1993). In case of
Nippostrongylus brasiliensis infection, the development rate of adults was significantly higher with mouse strain than with rat strain of the parasite (
Solomon and Haley, 1966). Taken together, it is suggested that infectivity of the L
3 of intestinal nematodes to rats are significantly affected by the host species, routes of inoculation and parasite strains but not by in vitro culture methods.
A significantly higher fecundity of the parasitic females as well as higher EPG counts were observed in rats infected with the L
3 harvested from NBC than those from FC (P<0.05). The reason for such difference is not clear in this study. The fecundity of the parasitic females recovered from Fischer rats infected with the L
3 from NBC except FPC and FC are almost similar to that reported in mice by Sato and Toma (
1990). The time course of egg discharge, worm burdens and persistence of infection observed in Fischer rats were almost similar irrespective of the culture methods which further indicates that culture methods do not influence the infectivity of the L
3.
In our previous studies, we have determined suitable media and optimum temperature for in vitro culture of L
3 and maintenance of
S. venezuelensis in different stages, all of which are considered important for understanding the biology of the parasite (
Baek et al., 1998a,
1998b). The present results showed that larval morphological development is significantly influenced by the culture methods but their infectivity to the host is not. Our previous findings may contribute to the understanding and manipulating the life cycle for experimental studies with
Strongyloides species, and the present study provides a useful measure in analysing the mode of action of anthelmintics and resistance thereto.