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The population dynamics of Ascaris suum was studied by long-term exposure of pigs to infective eggs. The pigs were experimentally inoculated with 25 A. suum eggs/kg/day, and 7, 8, and 8 pigs were necropsied at weeks 4, 8, and 14 postinoculation (PI), respectively. Despite the fact that the pigs were continuously reinfected, dramatic reductions in numbers of liver lesions (white spots) and migrating lung larvae were observed as a function of time. However, even at the end of the study, a few larvae were able to complete migration, but these larvae seemed unable to mature in the small intestine. Thus, the adult worm population seemed to consist of worms from the first part of the exposure period. The noticeable decrease in number of white spots suggests that the level of exposure is not reflected in the number of white spots in the late phase of a continuous infection. The serum levels of A. suum L3-specific IgG1 and IgA were significantly elevated by week 4 PI, after which the antibody levels declined. The population dynamics and parasite regulating mechanisms are discussed for A. suum in pigs as well as for the closely related species A. lumbricoides in humans.

Experiments were conducted to investigate possible alternative routes of extraintestinal migration of Ascaris suum larvae in the pig. Pigs were infected with A. suum via injection of newly hatched larvae into cecal veins (i.v.), into cecal lymph nodes (LN), or intraperitoneally (i.p.), and control animals were inoculated orally with infective eggs (p.o.). Two pigs per inoculation route were necropsied on days 1, 4, and 13 postinoculation. The numbers of liver lesions and the percentage of larvae recovered was considerably greater in pigs inoculated i.v. or p.o. on each necropsy day. However, irrespective of inoculation route, at least a proportion of larvae passed through the livers and were able to complete migration to the small intestine by day 13. The results indicate that larval penetration of the intestinal wall is not necessary for liver-lung migration and that passage through the liver may be favorable for migrating A. suum larvae, although a delayed arrival in the small intestine cannot be ruled out for larvae following alternative routes.

The dominant proteins released by Ascaris suum during development in vitro from the L3 to L4 stage were identified as collagenous cuticular proteins by sequence analysis and susceptibility to digestion by collagenase. Under reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the collagen proteins separated into 3 groups with molecular weights estimated at 32 kDa, 54-60 kDa, and 71-91 kDa. The 32-kDa protein represents monomeric collagen; the 54-60- and 71-91-kDa components represent dimeric and trimeric forms, respectively, polymerized by nonreducible cross-links. Furthermore, the release of these forms of collagen was developmentally regulated, as exemplified by a sequential temporal progression from monomeric to dimeric to trimeric forms in association with the in vitro transition from L3 to L4. The data suggest that collagen released in vitro during development of A. suum L3 to L4 reflects the increased translation of collagen gene products and their initial assembly into higher molecular weight molecules associated with the synthesis of the L4 cuticle. A biotinylated dipeptidyl fluoromethylketone cysteine protease inhibitor (Bio-phe-ala-FMK) bound specifically to the 32-kDa collagen and, to a lesser extent, to a 30-kDa protein; binding was dependent on the presence of dithiothreitol (DTT) and was prevented by iodoacetamide. Because cysteine residues play an essential role in the initial assembly of the collagen monomers into the higher molecular weight oligomers present in the mature nematode cuticle, inhibition of molting of A. suum L3 to L4 by the cysteine protease inhibitor Z-phe-ala-FMK might be due to its binding to thiol groups of collagen monomers during a critical phase of collagen assembly. Prevention of cystine cross-links during this critical period of cuticle assembly by peptide-FMK inhibitors may represent a potential control mechanism having a novel mechanism of action.

In order to monitor the early phases of the development of Ascaris suum from domestic pigs, third-stage larvae, retrieved from the liver and the lungs, were studied by analyzing worm growth and length increase of individual transverse annuli in the cuticle. Material for study using light and scanning electron microscopy was obtained from experimental infections. The results show that the third-stage larva (not the second-stage) after emergence from the egg grows continuously, without an ecdysis in the liver. During growth, each annulus is split into a complex of 2 subannuli, each of which attains a bimodal appearance and is a prominent feature during a late phase of the third-stage larva. The results suggest that the first 2 molts occur inside the egg, a synapomorphic feature of the Ascaridoidea. The third-stage larvae of ascaridoids, with some functional similarities of the dauer-larva stage of Caenorhabditis sp., facilitate transmission of these parasitic worms to the digestive tract of the vertebrate final host (utilizing the tracheal route in A. suum), where the third and the fourth molts take place.

Studies of the morphology of Ascaris suum larvae developing in the egg during embryonation in vitro at room temperature showed that 2 molts take place within the egg. The first larval stage (L1) appeared in the egg after 17-22 days of cultivation, the first molt to the second larval stage (L2) took place from day 22 to day 27, and the second molt to the third larval stage (L3) started on day 27 and continued during the 60-day observation period. Infectivity of the eggs was studied by oral egg inoculation in mice and showed that the L3 are the infective stage for mice. Molting to the L3 stage occurs gradually over a period of 2-6 wk, and it is recommended to have an additional maturation period so the infectivity of an egg batch may reach maximum level.

Third-stage larvae (L3) of Ascaris suum develop and molt to fourth-stage larvae (L4) during in vitro cultivation; consistently greater than 80% of the larvae develop to L4 during 7 days in culture (DIC). To assess the role of proteases in this process, the effect of protease class-specific inhibitors was studied. The presence of either a serine protease inhibitor (AEBSF, 100 microM) or an aspartic protease inhibitor (pepstatin A, 100 microM) had no effect on the percentage of L4 after 7 DIC. However, the presence of either a cysteine protease inhibitor (Z-Phe-Ala-FMK, 100 microM) or an aminopeptidase inhibitor (amastatin, 100 microM) resulted in 77% and 34% reductions, respectively, in the percentage of L4 compared to untreated cultures; viability of the larvae was not affected. The effect of Z-Phe-Ala-FMK on molting was time and dose dependent. In contrast to Z-Phe-Ala-FMK, E-64, another specific inhibitor of cysteine proteases, had no effect on molting. The data support a role for an aminopeptidase and suggest a role for a cysteine protease in the development of the L3 to L4 stage of A. suum.

For studying the morphogenesis of the adult stage of Ascaris suum, worms were obtained from experimentally infected domestic pigs. Structural and morphometric features of A. suum are analyzed by light and scanning electron microscopy. It was shown that the increase in length of the worm from days 35 to 56 is based on a region-specific lengthening of individual transverse annuli in the cuticle. An increased worm size also results in an increased diameter of different cuticular sense organs. It is shown that a pair of lateral midbody papillae, the centrids, are prominent sensilla in the adult male and female worm. The centrids have an asymmetric placement, the right centrid being more anterior on the worm than the left.

Protease activity was identified in culture fluids collected during in vitro development of L3 to L4 larval stages of Ascaris suum. Fluorogenic peptide substrates with unblocked N-termini were specifically hydrolyzed indicating aminopeptidase activity; a terminal arginyl residue was preferred. Culture fluids did not hydrolyze fluorogenic peptide substrates with blocked N-termini (endopeptidase substrates). The aminopeptidase activity was inhibited by 1,10-phenanthroline (metalloprotease inhibitor) and by amastatin and bestatin (aminopeptidase inhibitors); AEBSF (serine protease inhibitor), Z-phe-ala-FMK and E-64 (cysteine protease inhibitors), and pepstatin A (aspartyl protease inhibitor) had little effect on activity. The apparent molecular weight of the aminopeptidase was estimated by sucrose density gradient centrifugation at 293 kDa. The aminopeptidase displayed an acidic isoelectric point of 4.7. The peak secretion of the aminopeptidase was temporally associated with molting and suggests a function for the protease in this complex process.