Detection of antibody to Theileria parva schizonts and cell surface membrane antigens of infected lymphoblastoid cells by immunoperoxidase techniques.

Peroxidase-labeled antibody procedures were described for detecting bovine antibodies reactive with intracellular Theileria parva schizonts and cell surface membrane antigens of infected lymphoblastoid cells. Indirect tests were performed where the reacting bovine antibodies were localized with affinity purified rabbit-anti-bovine IgG coupled to horseradish peroxidase. A 4- to 8-fold increase in sensitivity for detecting bovine antibodies was obtained with unlabeled rabbit-anti-bovine IgG which in turn was detected with peroxidase labeled goat-anti-rabbit IgG. The T. parva infected cells used as antigen were attached to poly-l-lysine treated glass slides and all reaction steps were performed on the slides. The intracellular schizonts and cell surface staining reactions were dependent upon the status of the cells; acetone-fixed cells were required for schizont reactions and viable unfixed cells for cell surface membrane reactions. Sera from cattle stimulated in various ways with T. parva were examined by the techniques. Cattle infected by stabilate inoculation or inoculated with infected autologous lymphoblastoid cells developed relatively high levels of antibody to schizonts, but no detectable antibody to cell surface membrane antigens. This would indicate that parasite antigens do not occur on the surface of infected lymphoblasts. Cattle inoculated with infected allogeneic lymphoblasts developed low-levels of antibody to schizonts and readily demonstrable antibody to cell surface antigens. The immunoperoxidase procedures have certain advantages over immunofluorescence in that light microscopy is used; therefore, the reactions do not fade which permits a more detailed examination and provides a relatively permanent record, the preparations can be counterstained, and the reagents may be used for immunoelectron-microscopy. The procedures could provide suitable alternatives to immunofluorescence methods for East Coast fever investigations and other systems having intracellular and/or cell surface membrane antigens.

A histocompatibility barrier to immunization against East Coast fever using Theileria parva-infected lymphoblastoid cell lines.

Histocompatibility may be a barrier to the infection of cattle when Theileria parva parva-infected tissues or in vitro cultured macroschizont-infected lymphoblastoid cell lines are used for immunization. By inoculating 10(3) and 10(5) infected cells into autologous recipients infection was achieved and immunity engendered. Cell lines inoculated into BoLA matched recipients did not produce patent infections but some recipients developed antibodies to the parasite and 3/5 were immune to challenge. No evidence of infection or immunity was found in BoLA half matched or mismatched cattle. This result suggests that there is an histocompatibility barrier to infection using T. p. parva-infected lymphoblastoid cells.

Surface membrane components of Theileria spp piroplasms and associated complement fixation activity.

Theileria parva (parva) piroplasms were examined for complement fixation (CF) activity with sera from cattle immune to T parva (parva) (Muguga). The piroplasms were found to be highly antigenic, and the antigens responsible for this CF reactivity were completely insoluble in aqueous media. The antigens were traced through a variety of fractionation procedures and the fractions examined by electrophoresis in the presence of sodium docedyl sulphate. Antigenically active preparations were found always to contain two polypeptides having molecular weights of 21,500 and 34,000. When intact piroplasms were surface labelled with iodine 125 these same two polypeptides were the most heavily labelled, which suggested that the antigens responsible for the CF reactivity were plasma-membrane bound. Electrophoretic analysis of fractions containing plasma membrane components from piroplasms of three species of Theileria (T parva [parva], T mutans and T taurotragi) enabled interspecific differentiation to be made. Three stocks of T parva (parva) could not be differentiated by this means; such different stocks, from various locations within East Africa, appeared to be identical chemically.

Effect of trypsin and chymotrypsin on the polypeptides of large and small plaque variants of foot-and-mouth disease virus: relationship to specific antigenicity and infectivity.

Large and small plaque variants of A12 foot-and-mouth disease virus were shown to have specific antigenic determinants. Large plaque virus antigenic specificity was destroyed by trypsin treatment, but the small plaque antigen was resistant despite cleavage of the trypsin-sensitive polypeptide. The cleavage of polypeptide VP3 by trypsin resulted in the formation of a new antigen not present on untreated virus. The effects of chymotrypsin and trypsin on the polypeptides of the plaque variants have been examined and related to changes in antigenicity, infectivity, and exposure of the polypeptides at the surface of the capsid. The results are discussed in relation to the orientation of the trypsin-sensitive polypeptide in the virus capsid.

Effect of trypsin treatment on the antigenic characteristics of plaque variants of type-O 1 and type Asia-1 foot-and-mouth disease viruses.

Antigenic differences were demonstrated between the large and small plaque variants of both types O1 and Asia-1 foot-and-mouth disease viruses. Treatment of the large and small plaque variants of the viruses with trypsin essentially abolished the observed antigenic differences. Thus, these plaque variants have antigenically different trypsin-sensitive determinants that may influence their immunogenicity and infection capabilities.

Complement-fixation analysis of four subtypes of foot-and-mouth disease virus type A.

Complement-fixation patterns were established for four subtypes of foot-and-mouth disease virus by block assays against homologous and heterologous antiserum. Inhibition of fixation by excess antigen was observed in most homologous systems but rarely in the heterologous systems. The heterologous antibody titers were, in all instances, considerably lower than those for the homologous systems. Although relatively high dilutions of antiserum may be desirable for subtyping, higher concentrations of antibody should be used for determining serological types.

Screening of sera for antibodies to foot-and-mouth disease viral antigens by radial immunodiffusion.

Radial immunodiffusion procedures have been developed for testing bovine sera for the presence of antibody activity against three foot-and-mouth disease virus types and a virus infection-associated antigen. Reactions with virus antigens were obtained as early as 4 days after infection, and the virus type causing the response was identified. The procedure had the advantages of great sensitivity for the detection of antibody and the ease with which antibodies to a variety of antigens could be detected.

Immunochemical studies of foot-and-mouth disease. V. Antigenic variants of virus demonstrated by immunodiffusion analyses with 19S but not 7S antibodies.

Three antigenic variants of foot-and-mouth disease virus, type A, strain 119, were demonstrated in Ouchterlony analyses utilizing serum collected from guinea pigs 7 days postinfection (DPI). Such antisera contain antibodies of the 19S class. Guinea pig antisera that contained antibodies of the 7S class were unable to distinguish between the antigenic variants. Similarly, 19S antibody was able to demonstrate antigenic differences in trypsin- and chymotrypsin-treated viruses that were not detected by 7S antibody-containing antisera. One of the antigenic variants of virus is apparently the wild type and is tentatively considered to have two antigenic determinant groupings termed the a- and b-sites (140S-ab). The 140S-ab variant was the sole or predominant antigenic type produced in guinea pigs and in large plaque-forming- and tissue culture-low passage sources of the virus. Another antigenic variant appears to possess only the b-site (140S-b) and was the major constituent in tissue culture-high passage virus preparations. The third variant, a small plaque former, was also devoid of the a-site and contains an antigenic determinant that is related to, but not identical with, the b-site. This variant appears to be a minor constituent of tissue culture-high passage virus. 7-DPI serum could be absorbed with a suitable concentration of tissue culture-high passage virus to remove antibody reactive with the b-determinant site. This absorbed serum still precipitated 140S-ab virus by virtue of still containing antibody reactive with the a-determinant site; however, the neutralizing activity was eliminated. This suggests that the b-site is critical with respect to neutralization while the a-site is noncritical.

Immunochemical studies of foot-and-mouth disease. III. Acridine orange staining of agar gel precipitin reactions.

Agar gel diffusion precipitin reactions obtained with foot-and-mouth disease virus antigens were examined by an acridine orange staining procedure. After treatment with 0.01% acridine orange, precipitin bands produced by the virus particles (140 S antigen) fluoresced flame red when examined under ultraviolet light and with 0.001% to 0.0001% acridine orange, a yellow green. Treatment with RNase prior to staining inhibited the reaction, whereas, treatment with DNase did not. This was in accord with the known RNA nature of foot-and-mouth disease virus. Precipitin bands formed by the 75 S, 12 S, and infection-associated (VIA) antigenic components did not take up acridine orange, an indication that they do not contain detectable amounts of nucleic acid.