Characterization of the major immunogen in the excretory-secretory products of exsheathed third-stage larvae of Trichostrongylus colubriformis.

The excretory-secretory products of exsheathed third-stage larvae of Trichostrongylus colubriformis conferred some protection to guinea pigs against homologous challenge. A glycoprotein with an apparent molecular mass of approximately 94 kDa was the dominant immunogen in post-exsheathment products. Immunoblots revealed IgG antibodies to this glycoprotein in sera from multiply-infected guinea pigs and some sheep, and in sera of guinea pigs after three truncated infections which had been restricted by anthelmintic treatments to development of the third parasitic stage. IgA antibodies to this protein were also found in intestinal lymph of a naturally infected sheep. Fluorescent antibody studies indicated that this 94 kDa component was associated with cells in the central body cavity of third-stage larvae, but was absent from fourth-stage larvae or adult worms. Fractionation and protection assays in guinea pigs revealed that while the native and aggregated 94 kDa protein conferred some host protection, it was not the only protective component of the excretory-secretory products of exsheathed third-stage larvae of T. colubriformis.

A novel host-protective antigen from Trichostrongylus colubriformis.

The detergent-soluble fraction from Trichostrongylus colubriformis third-stage larvae contained a simple set of antigens, one of which (molecular weight 41,000) induced 43-51% protection in guinea pigs following immunization. Isolation and partial amino acid sequence analysis of this protective antigen showed it was parasite tropomyosin.

Cross-protection from Bacteroides nodosus vaccines and the interaction of pili and adjuvants.

The effects of vaccination of Merino sheep with the purified pili or the whole cells of Bacteroides nodosus strain 198, either in oil or alum-oil adjuvant, on the severity of foot-rot induced with the homologous strain (198) and a heterologous strain (217) were determined in a field experiment, on flood irrigated pasture. The efficacy of the whole cell vaccines was comparable to that of purified pili vaccines, against homologous challenge, when both had a similar content of pilus antigen although the purified pili vaccines induced significantly greater homologous pilus agglutinating antibody titres than the whole cell vaccines. However, against heterologous challenge, the whole cell vaccines in oil (CO) or alum-oil (CAO) provided significantly greater protection than a purified pili-in-oil (PPO) vaccine, the number of severely affected feet in sheep vaccinated with PPO being similar to that of the unvaccinated group. The group vaccinated with purified pili in alum-oil (PPAO) was intermediate between these two extremes. The superior performance of the PPAO in comparison to the PPO vaccine, against heterologous challenge, was associated with significantly higher mean ELISA titres to the outer membrane complex. Western blot analyses implicated a role in cross-protection for outer membrane proteins, in particular a protein Mr 78,000. The PPO vaccine produced fewer, smaller and less persistent vaccination reactions at the inoculation sites than did the other vaccines. Bodyweight gains in the period prior to challenge were much lower for the groups vaccinated with CO and CAO than for the controls and those vaccinated with purified pili, due presumably to the larger vaccination reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody to the 32K structural protein of infectious bursal disease virus neutralizes viral infectivity in vitro and confers protection on young chickens.

Chicken sera containing IgG antibodies specific for the 32 000 (32K) mol. wt. structural polypeptide of infectious bursal disease (IBD) virus, as assessed by Western blotting, neutralized the in vitro infectivity of tissue culture-adapted IBD virus. When injected into young chickens, the serum passively protected them from challenge with pathogenic IBD virus. Chickens immunized with the 32K structural polypeptide of IBD virus, prepared by electroelution from SDS-PAGE gels, produced antibody detectable by ELISA and the virus neutralization assay, while chickens immunized with the 37K or 41.5K viral polypeptides synthesized antibody detectable by ELISA, but only very low levels of virus-neutralizing antibody. The immunoglobulin fraction of sera obtained from chickens immunized with the 32K polypeptide, but not the 41.5K polypeptide, passively protected chickens from infection with IBD virus. It is concluded that the 32K polypeptide is a major protective immunogen of IBD virus.

Characterization by Western blotting of the immunogens of infectious bursal disease virus.

The Australian isolate of infectious bursal disease (IBD) virus (002/73) was purified from infected bursae by rate-zonal and density-equilibrium centrifugation and characterized by polyacrylamide gel electrophoresis. Two major polypeptides having approximate mol. wt. of 32 000 (32K) and 37K and three other polypeptides of approximate mol. wt. 29K, 41.5K and 91.5K were present in all preparations of virus having a buoyant density of 1.33 g/ml. Western blotting of the polypeptides of IBD virus showed that the initial antibody response of chickens infected with live virus or injected with an inactivated oil-emulsion vaccine was directed primarily towards the 32K polypeptide. Only sera obtained late in the response to live virus or following hyperimmunization contained antibodies recognizing the 29K, 37K and 41.5K polypeptides. An antibody response to the 91.5K polypeptide was not detected routinely by this technique. It was concluded that the 32K polypeptide is a major immunogen of IBD virus.

Taenia pisiformis: protective immunization of rabbits with solubilized oncospheral antigens.

Antigens were derived from hatched and activated oncospheres of Taenia pisiformis which had been separated from embryophoric debris by centrifugation on Percoll. Crude oncospheral antigen was prepared by freeze-thawing and sonication of oncospheres at 4 C, and a supernatant of crude antigen was collected following centrifugation at 100,000g. Other antigens tested were the supernatants collected after 100,000g centrifugation of crude antigen solubilized in Triton X-100, butanol, lithium diiodosalicylic acid, KCl, sodium dodecyl sulfate, or sodium deoxycholate. When groups of rabbits were immunized with the various antigens and challenged with T. pisiformis eggs, both sodium deoxycholate- and Triton X-100-solubilized antigens stimulated a level of protection similar to the crude antigen. All other antigens failed to stimulate significant protective immunity. When sodium deoxycholate-solubilized antigen was fractionated using high-performance liquid chromatography, the major host-protective components were in the fractions with molecular weight greater than 140,000. Levels of the enzyme, glutamate dehydrogenase (EC 1.4.1.2), in the serum of rabbits challenged with T. pisiformis eggs closely reflected the degree of liver damage caused by migrating larvae, and were not markedly elevated in those rabbits effectively immunized using the crude or sodium deoxycholate-solubilized antigens.

The protection given by pilus and whole cell vaccines of Bacteroides nodosus strain 198 against ovine foot-rot induced by strains of different serogroups.

A highly purified pilus vaccine prepared from cells of Bacteroides nodosus strain 198 provided a high level of protection against homologous challenge and small, not statistically significant, levels of protection against challenge with 4 other strains each from different serogroups. In a second experiment, a partially purified pilus vaccine from strain 198 induced significant immunity to 1 of 4 heterologous strains which were different from those used in the first experiment. In a third experiment a strain 198 whole cell vaccine produced significant immunity against 3 of 6 heterologous strains used in the first 2 experiments. There was no obvious relationship between the colony type, degree of piliation and level of cross-protection obtained against a particular strain. The results provide further evidence that immunogens associated with, but distinct from, the pilus are involved in cross-protection and that cross-protective antigens are common to some, but not all, strains.

Primary structure of pilin protein from Bacteroides nodosus strain 216: comparison with the corresponding protein from strain 198.

The amino acid sequence of pilin protein from Bacteroides nodosus strain 216 was determined. The protein had a calculated molecular weight of 15962 and contained the same number of amino acid residues (151) as the pilin from the previously sequenced strain 198. The sequence of the first 44 residues was common to both strains, including the unusual amino-terminal amino acid, N-methylphenylalanine. Of the remaining 107 residues, 37% of them differed between the two strains. Comparison of hydrophilicity profiles constructed from the sequence data indicated that a conserved region around residues 71-72 was probably the site of an antigenic determinant.

Analysis of the outer membrane proteins of Bacteroides nodosus, the causal organism of ovine footrot.

Examination by SDS-PAGE of lithium acetate extracts of several strains of depiliated Bacteroides nodosus revealed 6 major outer membrane proteins (including pilin). The 5 membrane proteins exhibited approximate molecular weights of 75000, 50000, 38000, 34500 and 26500 whereas pilin had a MW of 17500 for the majority of strains. All proteins were accessible to lactoperoxidase-catalysed iodination and proteins 1, 2 and 5 were shown to be glycoproteins. Several attempts to isolate individual OMC proteins in pure form by selective solubilization and gel filtration were unsuccessful, but electroelution of individual outer membrane complex proteins resolved by SDS-PAGE provided sufficient quantities of antigen for immunization of sheep and for immunochemical analysis.

Effect of pilus dose and type of Freund's adjuvant on the antibody and protective responses of vaccinated sheep to Bacteroides nodosus.

Groups of sheep were immunised twice with one or other of six vaccines consisting of purified pili from Bacteroides nodosus at three dose levels (10, 38 and 154 micrograms) and emulsified with either complete (CFA) or incomplete Freund's adjuvant (IFA). Beginning one month after vaccination the sheep were homologously challenged on irrigated pasture, with naturally transmitted foot rot for a period of 26 weeks. Statistical analyses of the number of feet per sheep with severe foot rot demonstrated that there was a significant effect of vaccinal dose but neither an adjuvant effect nor an interaction between dose and adjuvant. Similar conclusions were reached when the titres of antipilus agglutinins in the serum were analysed. By both criteria the responses to doses of 154 and 38 micrograms of pili were significantly better than to 10 micrograms, but not significantly different from each other. The IFA vaccines caused less reaction at the sites of injection than the CFA vaccines and within the former the vaccines containing 10 and 38 micrograms pilus produced less reaction than those containing 154 micrograms. Hence a vaccine containing 38 micrograms of purified pili in IFA is nearly optimal for homologous protection against severe foot rot and is acceptable in terms of the reaction at the injection site.

Electro-blot radioimmunoassay of virus-infected plant sap - a powerful new technique for detecting plant viruses.

A new technique for detecting viruses in plant sap is described. It consists of sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the infected plant sap, electrophoretic transfer of protein bands to activated paper by the Electro-Blot technique, the subsequent probing of the viral coat protein band by specific antiserum (prepared against intact virus), and detection of immune complex with 125 I-labelled protein A. The technique successfully detected tobacco mosaic virus at a sap dilution of 1 : 10,000, four strains of sugarcane mosaic virus (a potyvirus) in their perennial hosts infected for about 4 years, and five different isolated of potato leaf roll virus (a luteovirus). The latter virus occurs in extremely low concentration and is difficult to detect by the other known methods.

Activities and partial purification of extracellular proteases of Bacteroides nodosus from virulent and benign footrot.

In an attempt to differentiate virulent and benign strains of B. nodosus, the extracellular proteolytic activity of these cultures was assayed with elastin, casein and hide powder azure, and the stability to heating at 55 degrees C was determined. Broth cultures of both strains hydrolysed 125I-labelled elastin, indicating that this activity is not a unique marker of virulence. When cultures were grown in Trypticase-arginine-serine broth medium modified by omitting Na2CO3 and thioglycollic acid, the total proteolytic activity and its stability at 55 degrees C could be used to differentiate isolates causing virulent or benign footrot lesions. However, when other broth cultures were used, these parameters could no longer be used to make such a distinction. The proteases of a virulent and benign strain of B. nodosus were partially purified and characterized. Four to five closely related proteases were detected by polyacrylamide gel electrophoresis at pH 8.8 in both types of isolates. The proteases are serine-type enzymes requiring a divalent metal ion such as calcium for activity. The proteases of the benign strain were somewhat less stable to heat than the enzymes of the virulent strain. Differences in the relative mobilities of the proteases of virulent and benign strains of B. nodosus, on electrophoresis at pH 8.8, suggest that this property may be used to distinguish virulent and benign strains.

Immunization of sheep with larval antigens of Lucilia cuprina.

Sheep were immunized with antigens extracted from third-instar larvae of L. cuprina. This procedure produced substantial titres of circulating antibody as measured by solid-phase radioimmunoassay or immunodiffusion or by both techniques. However, immunization did not confer protection against subsequent implant challenge with first-instar larvae. In vitro studies indicated that pooled sera from immunized sheep (mean immunodiffusion titre = 3) significantly reduced larval survival. Antigen specificity and the modulating effects of concomitant humoral responses to larval challenge are discussed in relation to the findings.

Immunoglobulin G antibodies to the antigens of Lucilia cuprina in the sera of fly-struck sheep.

A solid-phase radioimmunoassay was used to demonstrate that sheep with myiasis caused by the larvae of the Australian sheep blowfly, L. cuprina, had serum IgG antibodies to antigens present in an extract of the ground-up larvae. Previously struck animals demonstrated a more severe myiasis than their unstruck counterparts when both groups were subjected to a standard larval challenge. The effects of immunosuppressive therapy were expressed in terms of a decrease in the total number of larvae growing to maturity and in the area of fly strike produced.

An investigation of the antigens of Ascaris lumbricoides using a radioimmunoassay and sera of naturally infected humans.

Of 5 humans from an Ascaris lumbricoides(var. suum)-endemic area who were positive for Ascaris infection (past or current) when examined by a radioallergosorbent test (RAST) for serum immunoglobulin E, only 1 contained detectable levels of Ascaris specific serum IgG antibodies. These antibodies were not detectable when the radioimmunoassay was replaced with a precipitin assay. There was a wide ringe of molecular weights and isoelectric points of antigens in Ascaris body fluid (ABF) which reacted with antibodies in this infected human. However, unlike the IgE-binding ABF antigens, the IgG-binding ABF antigens were particularly evident in one area of molecular weight and isoelectric point. This human also had IgA and IgM to Ascaris antigens whereas the others had barely detectable amounts.