Animals can be rendered immune to Ascaris parasites by immunization with infectious-stage larvae. The specific parasite gene products that mediate protective responses in ascariasis are unknown. We have identified a cDNA encoding Ascaris suum 14-kDa antigen (As14) and evaluated the vaccinal effect of the Escherichia coli-expressed recombinant protein (rAs14). GenBank analysis showed that As14 has low similarity at the amino acid level to a Caenorhabditis elegans gene product and to antigens of the filarial nematodes but not to other known proteins. In addition, As14 homologues were found to be expressed in human and dog roundworms. In mice that received intranasal administration of rAs14 coupled with cholera toxin B subunit (rAs14-CTB), there was a 64% reduction of recovery of larvae compared with that in the nontreated group. The vaccinated mice showed a significant increase in the total serum immunoglobulin G (IgG) levels and the mucosal IgA responses. Elevation of the rAs14-specific IgE response was also seen. Measurement of the IgG subclasses showed a higher level of IgG1 and a lower level of IgG2a antibody response in the sera of the immunized mice, suggesting that protection was associated with a type II immune response. As14 is the first protective antigen against A. suum infection to be identified. Our immunization trial results in laboratory animals suggest the possibility of developing a mucosal vaccine for parasitic diseases caused by ascarid nematodes.

Protective immunity to the pig roundworm, Ascaris suum, has been demonstrated by immunization of pigs with antigens derived from the parasite's larval stages. We identified a protective antigen commonly expressed in the human and pig Ascaris infections as a 16-kDa protein (As16), which has no similarity at the amino acid level to mammalian proteins but has some similarity to those of the filarial parasites and Caenorhabditis elegans gene product. Localization analysis revealed that the native As16 was highly expressed in the adult worm intestine, hypodermis, and cuticles. In addition, As16 was detected in the parasite excretory and secretory products. Mice intranasally vaccinated with Escherichia coli-expressed recombinant As16 (rAs16), coupled with cholera toxin B subunit, generated a significant increase in the level of rAs16-specific immunoglobulin G (IgG) and IgE in serum. Mucosal IgA levels were also increased. The recombinant protein evoked a mixed (both Th1 and Th2) type of immune response characterized by elevated levels of gamma interferon and interleukin-10 in the culture supernatants of activated spleen cells. An increased level of IgG1 and IgG2a in serum was also observed. The vaccinated mice showed a reduction by 58% in the recovery of challenged larvae compared to a nonvaccinated control. These results suggest the possibility of developing a mucosal vaccine for human and pig ascariasis.

Inorganic pyrophosphatase (PPase) is an important enzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into ortho‐phosphate (Pi). We report here the molecular cloning and characterization of a gene encoding the soluble PPase of the roundworm Ascaris suum. The predicted A. suum PPase consists of 360 amino acids with a molecular mass of 40.6 kDa and a pI of 7.1. Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a functional Family I soluble PPase containing features identical to those of prokaryotic, plant and animal/fungal soluble PPases. The Escherichia coli‐expressed recombinant enzyme has a specific activity of 937 µmol Pi·min−1·mg−1 protein corresponding to a kcat value of 638 s−1 at 55 °C. Its activity was strongly dependent on Mg2+ and was inhibited by Ca2+. Native PPases were expressed in all developmental stages of A. suum. A homolog was also detected in the most closely related human and dog roundworms A. lumbricoides and Toxocara canis, respectively. The enzyme was intensely localized in the body wall, gut epithelium, ovary and uterus of adult female worms. We observed that native PPase activity together with development and molting in vitro of A. suum L3 to L4 were efficiently inhibited in a dose‐dependent manner by imidodiphosphate and sodium fluoride, which are potent inhibitor of both soluble‐ and membrane‐bound H+‐PPases. The studies provide evidence that the PPases are novel enzymes in the roundworm Ascaris, and may have crucial role in the development and molting process.

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SUMMARYRhodococcus equi is a facultative intracellular bacterial pathogen that causes pneumonia in foals and immunosuppressed humans. There are at least three virulence levels of R. equi and these pathogenicities are associated, in mice, with the presence of virulence plasmids. This study focused on cytokine secretion, in mice, in the course of a primary infection with sublethal doses of R. equi strains of different virulence levels (virulent, intermediately virulent and avirulent). Tumour necrosis factor (TNF ) and interferon-c (IFN-c), but not interleukin-4 (IL-4) and interleukin-10 (IL-10), were induced endogenously in mice in relation to the multiplication and clearance of virulent and intermediately virulent strains of R. equi. These cytokines were not detected in mice infected with avirulent R. equi. Deaths occurred among mice treated with monoclonal antibodies (mAbs) against either TNF or IFN-c prior to sublethal dose infection with virulent and intermediately virulent strains of R. equi, but not with avirulent R. equi. These results suggested that cytokine production depended largely on the virulence levels of R. equi: TNF and IFN-c were required early during infection with virulent R. equi to limit replication and clearance of bacteria within the organs, but they were not necessary for limiting infection with avirulent R. equi. INTRODUCTIONthe submaxillary lymph nodes of pigs (mouse LD 50 =107); and avirulent R. equi, which has no virulence-associated antiRhodococcus equi is a facultative, intracellular, Gram-positive gens or plasmid (mouse LD 50 >108).10,11 Epidemiological studcoccobacillus, which causes suppurative pneumonia and ulceries have revealed that these virulent R. equi live separately in ative enteritis in foals aged 1-3 months.1 Recently, R. equi horses and pigs, and that avirulent R. equi is distributed widely has been recognized as an emerging opportunistic pathogen in our environment.12 In humans, the majority of isolates from of immunocompromised hosts, such as human immunodeficipatients with AIDS were either virulent or of intermediate ency virus (HIV )-infected patients, and the number of R. equi virulence,9 and most isolates from immunocompromised pneumonia case reports in patients with acquired immune patients without AIDS were avirulent.13 deficiency syndrome (AIDS ) has increased.2-4 Studies on R. equi infection in foal and mouse models have The discovery of virulence-associated antigens and virucontributed some understanding on the aspects of pathogenesis lence plasmids has clarified some aspects of the virulence of and immunology.1,4,14,15 However, the relative contributions R. equi.5-9 At least three virulence levels of R. equi have been of cell-mediated immunity and humoral immunity to resistance identified: virulent R. equi, which has 15 000 to 17 000 MW against R. equi infections remain unclear and paradoxical.1,4,14 antigens and a virulence plasmid of 85-90 kb, and causes Passive transfer of hyperimmune equine plasma was demonsuppurative pneumonia in foals (mouse LD 50 =106...

The protein profile of adult female Ascaris lumbricoides and Ascaris suum originating from humans and pigs, respectively, was studied using two-dimensional polyacrylamide gel electrophoresis. Six different major protein spots specific for A. lumbricoides were identified irrespective of their geographical origin and no major specific spot was encountered in A. suum. No major differences in the protein profiles between the extract by phosphate-buffered saline and urea were encountered for either Ascaris species. It is therefore possible to use 2D-PAGE as a tool for discriminating the closely related Ascaris species from humans and pigs.

An understanding of the biology of the cuticle in the larval stages of Ascariodea is of importance since the cuticle molecules not only possess a variety of functions related to survival but also have a potential role as a target for immunoprophylaxis. Thus, we made a preliminary characterization of surface proteins and antigens from 3rd-stage larvae (L3) and lung-stage larvae of Ascaris suum using two biotin-derivatives and two-dimen sional polyacrylamide gel electrophoresis (2D-PAGE). The proteins labelled with biotin comprised a total of 37 and 32 spots, with molecular weights (Air) ranging from 15 to 101 kDa and isoelectric points (pI) from 3.8 to 7.6, in L3 and lung-stage larvae, respectively. The profiles revealed that the individual spots bound to one or both biotin derivatives. In addition, stage-common and stage-specific spots were found in L3 and lung-stage larvae. 2D-PAGE/immunoblotting analysis was performed with antisera from rabbits repeatedly inoculated with A. suum L3. Nineteen spots were recognized as surface antigens, with Mr ranging from 32 to 66 kDa and pI from 4.9 to 7.6, from L3 and lung-stage larvae after alignment of the immunoblots with the profile of the surface proteins. These spots were found to include stage-common and stage-specific antigens. Identification of surface proteins by biotin labelling combined with 2D-PAGE allows a substantial shortening of sample preparation time for the target proteins, and will be a viable method for protein analysis of surface proteins and antigens of A. suum L3 and lung-stage larvae.

The protein profile and antigenic properties of lung-stage larvae of Ascaris lumbricoides and A. suum were studied using 2-dimensional electrophoresis and immunoblot analysis, respectively. The protein profiles of the 2 parasites were identical except for the presence of only 1 major protein spot specific for each. There was a complete cross-reactivity between the 2 parasites at the immunological level, and no specific antigen was recognized using specific antibody raised against the 2 parasites in rabbits.