Immunization of mice against infection with Taenia taeniaeformis using various antigens prepared from eggs,oncospheres, developing larvae and strobilocerci

Rajasekariah G. R., Rickard M. D. and Mitchell G. F. 1980. Immunization of mice against infection with Taenia taeniaeformis using various antigens prepared from eggs, oncospheres, developing larvae and strobilocerci. International Journal for Parasitology10: 315–324. Antigens were collected during in vitro incubation of oncospheres, 3-week-old larvae and strobilocerci of T. taeniaeformis. Supernatants of these in vitro products centrifuged at 500 g contained antigens which stimulated a significant degree of protective immunity when injected into mice. However, centrifugation of the strobilocercus preparation at 4500 g yielded supernatants which failed to induce immunity. Suspensions of eggs and oncospheres disrupted by sonication stimulated a high level of immunity as also did 4500 g supernatants of the sonicated preparations. Centrifugation of sonicated oncospheres at 100,000 g yielded a supernatant which stimulated significantly less immunity than the 4500 g supernatant, although the protective capacity was not totally abolished. The pellet from 100,000 g centrifugation of sonicated oncospheres induced almost absolute immunity. These results are consistent with the suggestion that the ‘functional’ antigens in the preparations tested may initially be membrane-associated or particulate in nature and that sonication causes partial solubilization. Supernatants prepared from homogenised strobilocerci and centrifuged at 4500 g also stimulated protective immunity and presumably contain soluble antigens. No evidence is available to suggest whether or not the strobilocercus antigens which stimulated protective immunity are identical to those found in oncospheral preparations. Immunity was stimulated by subcutaneous, intraperitoneal and intramuscular injections of antigen and both Freund's complete adjuvant and Bordetella pertusiss vaccine were effective as adjuvants. Using sonicated oncospheres, a high level of immunity was stimulated without the use of adjuvant.     

Immunization against Taenia taeniaeformis in mice: studies on the characterization of antigens from oncospheres

Mature eggs of Taenia taeniaeformis hatched readily in the presence of sodium hypochlorite and no loss in infectivity of oncospheres for mice was observed after hatching. Crude and sodium deoxycholate-solubilized antigens (termed TtO-DOC) prepared from such oncospheres stimulated high levels of protection against T. taeniaeformis infection in immunized mice similar to those described previously for oncospheres prepared by other methods. Mice immunized with TtO-DOC antigens that had been exposed to potassium metaperiodate remained significantly protected against infection. Exposure of TtO-DOC antigens to pronase and thermolysin, or to trypsin, significantly reduced the ability of these antigens to protect mice against infection. These data suggest that the antigens which immunize mice against infection include protein components. ¹²⁵I-labelled TtO-DOC antigens were immunoprecipitated with sera from mice infected with T. taeniaeformis and the immunoprecipitates analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoprecipitation with sera from C3H/He mice infected for 28 days revealed a single major labelled protein antigen having a relative molecular mass (M_r) of 31,000. Sera from 5-month infected C3H/He mice immunoprecipitated at least thirteen labelled antigens, including one at M_r 31,000. Attempts to use SDS-PAGE separated proteins to immunize mice showed that oncosphere antigens exposed to the reducing conditions prior to SDS-PAGE lost their ability to protect mice against infection. It was concluded that SDS-PAGE was an unsatisfactory technique for the isolation of a host protective fraction of TtO-DOC antigens. TtO-DOC proteins were resolved by PAGE performed in the presence of sodium deoxycholate (DOC-PAGE) and mice were vaccinated with cut-outs from the gel. A fraction of the DOC-polyacrylamide gel was found to be effective in immunizing mice against infection. Thus, although the characteristics of the protein antigens in this DOC-PAGE fraction have yet to be determined, an important fractionation technique has been identified. It was shown that partial removal of DOC from oncosphere antigen preparations solubilized in 1% DOC was required for the antigen to stimulate protective immunity. These findings will facilitate further antigen characterization studies towards the development of a defined-antigen vaccine in murine cysticercosis. This is particularly so as attempts to raise anti-oncospheral monoclonal antibodies capable of passively transferring protection to mice by using crude antigen preparations to immunize donor mice have not been successful.     

Resistance of larvae of common species of White Sea invertebrates to extreme changes in salinity

This study examines the effect of sharp changes in salinity on pelagic larvae of ten common species of invertebrates from the brackish White Sea (Mollusca, Polychaeta, Echinodermata, Cnidaria, Ascidia). For five species, the low salinity resistance limit was in the range of 8–12‰: for the gastropod Littorina littorea, it was below 8‰; for Dyaphana sp. and the bivalves Hiatella arctica and Heteranomia ovata, it was more than 12‰; and for the ascidian Styela rustica, it was 16‰. About 50% of larvae of four investigated species were able to withstand high salinity and survived at 36–40 and even 50‰ (Littorina). Larvae of littoral-sublittoral species proved to be more euryhaline than larvae of sublittoral species.     

Schistosoma japonicum: Immunological response to circulating polysaccharide antigens in rabbits with a light infection

To study the detectability of circulating polysaccharide antigens and the immunological response to such antigens in rabbits with a light Schistosoma japonicum infection, sera of five rabbits infected with 50 cercariae were studied up to 29 weeks post infection (p.i.). While one rabbit developed no worm burden, the other rabbits developed low worm burdens (4 to 16 worms). In the sera of these rabbits, the only polysaccharide antigen demonstrable with immunoelectrophoresis (IEF), was the circulating anodic antigen (CAA). With the enzyme-linked immunosorbent assay (ELISA), CAA was detectable from 5 to 6 weeks p.i. in the sera of the two rabbits with the highest number of worm couples. The lowest CAA level which was detectable in unconcentrated sera from which serum proteins had been removed was 125 ng CAA/ml, corresponding with a worm burden of 4.5 worm/kg body wt. During the entire infection, CAA-specific immune complexes were only demonstrable in very low concentrations. Antibodies against polysaccharide antigens were assessed with immunofluorescent antibody (IFA) on Rossman's fixed sections of adult worms, with the ELISA, and with IEF. Specific IgA, IgG, and IgM antibodies were detectable from 2 to 3 weeks p.i. with IFA and ELISA. These early antibodies were shown to be directed against gut-associated antigens, while antibodies against parenchyma-associated antigens were found later in the infection. With IEF, antibodies against two trichloroacetic acid (TCA)-soluble antigens were detectable, including the major, S. japonicum-specific antigen 2.     

A comparison of in vitro and in vivo estimates of the viability of Taenia pisiformis eggs aged under controlled conditions, and their ability to immunise against a challenge infection

Storing the eggs of T. pisiformis for periods of 0 to 9 days at a temperature of 36–38°C and relative humidity of 87–93 % revealed at least 4 stages in the ageing process. Firstly, a stage in which eggs hatched and activated in vitro and were also fully infective for rabbits. Secondly, a stage in which eggs hatched and activated in vitro but underwent only partial development in rabbits. Thirdly, a stage in which eggs hatched and activated in vitro, and presumably therefore in vivo, but no evidence of infection could be found when they were fed to rabbits. Fourthly, a stage in which eggs either did not hatch, or if they did hatch, the oncosphere was rapidly digested. In vitro techniques for assessing viability of T. pisiformis eggs were not a reliable guide to their infectivity for rabbits. ‘Senescent’ eggs, that is eggs which produced evidence of early infection in rabbits but did not complete their development to cysticerci, failed to produce immunity to challenge infection with T. pisiformis eggs.     

Gnathostoma binucleatum: excretion-secretion antigen analysis obtained from advanced third-stage larvae in in vitro culture

The paper describes an introductory characterization of antigenic stimulation of excretion-secretion products (ESP) of Gnathostoma binucleatum advanced third-stage larvae cultured in vitro and proteinases present in this products. Excretory and secretory proteins were obtained after 10 larvae were maintained in 5% CO(2) RPMI medium. The supernatant was collected each week for two months. The proteins were dialyzed, concentrated, and separated in 10% SDS-PAGE gels under reducing conditions and transferred to nitrocellulose paper for immunoblot analyses. G. binucleatum immunized mice serum was used to determine protein antigenicity. Four proteins of 40, 80, 120, and 208 kDa persisted for two months and three proteins, 80, 120, and 208 kDa were recognized for antibodies of mice. In SDS-PAGE gelatin substrate gels ESP resolved as two proteins with molecular weight of 80 and 208 kDa that were sensitive to a metalloproteinase inhibitor, and thus it may be inferred that they might be used for diagnosis of gnathostomiasis.     

Toxoplasma gondii: DNA vaccination with bradyzoite antigens induces protective immunity in mice against oral infection with parasite cysts

Infection of the host by Toxoplasma gondii leads to an acute systemic dissemination of tachyzoites, followed by a chronic phase, in which bradyzoites, enclosed in cysts, persist in the brain, the heart, and other tissues. Among putative vaccine candidates, the bradyzoite antigens BAG1 and MAG1 look promising since they are preferentially expressed during the chronic stage of the parasite. This work focused on studying the immunogenicity of bradyzoite antigens in a mouse model of chronic toxoplasmosis. A mixture of plasmids directing the cytoplasmic expression of MAG1 and BAG1 in mammalian cells was used to immunize mice. We show here that immunized mice developed, preferentially, specific anti-MAG1 and anti-BAG1 IgG2a subclass antibodies, indicating a shift towards a Th1-like response after DNA immunization. We then demonstrated that DNA immunization followed by challenge infection elicited effective protection in mice, suggesting that bradyzoite antigens should be considered in the design of vaccines against toxoplasmosis.     

Resistance to RHD virus in wild Australian rabbits: Comparison of susceptible and resistant individuals using a genomewide approach

Deciphering the genes involved in disease resistance is essential if we are to understand host–pathogen coevolutionary processes. The rabbit haemorrhagic disease virus (RHDV) was imported into Australia in 1995 as a biocontrol agent to manage one of the most successful and devastating invasive species, the European rabbit (Oryctolagus cuniculus). During the first outbreaks of the disease, RHDV caused mortality rates of up to 97%. Recently, however, increased genetic resistance to RHDV has been reported. Here, we have aimed to identify genomic differences between rabbits that survived a natural infection with RHDV and those that died in the field using a genomewide next‐generation sequencing (NGS) approach. We detected 72 SNPs corresponding to 133 genes associated with survival of a RHD infection. Most of the identified genes have known functions in virus infections and replication, immune responses or apoptosis, or have previously been found to be regulated during RHD. Some of the genes identified in experimental studies, however, did not seem to play a role under natural selection regimes, highlighting the importance of field studies to complement the genomic background of wildlife diseases. Our study provides a set of candidate markers as a tool for the future scanning of wild rabbits for their resistance to RHDV. This is important both for wild rabbit populations in southern Europe where RHD is regarded as a serious problem decimating the prey of endangered predator species and for assessing the success of currently planned RHDV variant biocontrol releases in Australia.     

Trypanosoma brucei: loss of variable antigens during transformation from bloodstream to procyclic forms in vitro.

The loss of variable antigen from Trypansoma brucei during transformation from the bloodstream to the procyclic form in vitro has been monitored by agglutination and immunofluorescence reactions using antisera against both forms. Maximum agglutination of transforming trypanosomes with anti-culture form sera was obtained in 36–48 hr coinciding with loss of the surface coat as seen by electron microscopy. Agglutination with antisera against homologous bloodstream forms, however, reached a constant minimum but still positive level after 7–9 days: absorption of such antisera with culture or heterologous bloodstream forms reduced this period of persistent agglutinability to 72–84 hr, suggesting that the sera contained antibodies to “common” (surface membrane) antigens which became exposed when the surface coat was lost during transformation. The indirect immunofluorescence reaction provided a direct correlation of loss of antigen with loss of coat. The majority of trypanosomes lost detectable variable antigen by 36–48 hr, but a few flagellates, morphologically resembling bloodstream forms, retained the coat and capacity for labeling up to 84 hr; the numbers of such persistent bloodstream forms were shown to be sufficient to give a positive agglutination reaction for the population as a whole up to this time. Variable antigen appeared to be lost by dilution over the entire trypanosome surface rather than in patches or caps and the relevance of this observation to the process of antigenic variation is discussed.     

Homogeneous antibodies directed against human cell surface antigens: I. The mouse spleen fragment culture response to T and B cell lines derived from the same individual

The use of the mouse spleen fragment culture system is extended to the production of antibodies to human lymphoblastoid cell lines. These antibodies were tested for reactivity against the immunizing cell line, and against a second cell line which had been derived from the same human blood sample. Many of the antibodies were found to discriminate between the 2 isogenic lines. These results demonstrate the potential of the mouse spleen fragment culture system to provide homogeneous reagents which detect distinguishing markers on closely related human cells.