Early detection of bovine leukemia virus by using an enzyme-linked assay for polymerase chain reaction-amplified proviral DNA in experimentally infected cattle.

Bovine leukemia virus is the causative agent of bovine leukosis and has been described in many countries throughout the world. We describe here a sensitive and readily applicable assay for the detection of bovine leukemia proviral DNA. Detection relies on initial amplification of proviral DNA by using polymerase chain reaction (PCR) followed by an enzyme-linked assay (PCR-ELA). Amplification is carried out by using one biotinylated primer and a second primer containing the GCN4 protein binding site. DNA is detected by a colorimetric assay after it is coupled to GCN4-coated plates and subsequently incubated with horseradish-streptavidin peroxidase and the appropriate substrate to produce a chromogenic reaction. It was possible to detect proviral DNA for all of eight bovine leukemia virus-infected calves by 2 weeks postinfection. Use of the more conventional agar gel immunodiffusion assay failed to reveal the presence of the virus in any of the animals up to 4 weeks postinfection. The PCR-ELA detected as little as 0.1 to 0.2 ng of amplified DNA per well, which compares very favorably with ethidium bromide staining of gels, by which 1 to 2 ng per lane was detected. This method lends itself to mass screening, is carried out in a similar way to an enzyme-linked immunosorbent assay, and does not require gel electrophoresis or the use of radioactive gene probes.

Molecular cloning of Taenia taeniaeformis oncosphere antigen genes.

Infection of mice with the cestode Taenia taeniaeformis exhibits several important features common to other cestode infections, including the ability to vaccinate with crude antigen mixtures. Partial purification of the protective oncosphere antigens has been reported with a cutout from deoxycholate (DOC) acrylamide gels; this cutout was called fraction II (FII), and comprises approximately 10% of total DOC-soluble oncosphere antigen. Western blots of DOC gels probed with anti-FII antisera revealed a series of 3-5 discrete bands within the FII region. Further fractionation of the FII antigens on DOC gels was impractical due to limitations in supply of oncospheres, so a cDNA library was constructed from 150 ng of oncosphere mRNA and screened with alpha-FII antisera. Two distinct clone families were identified, oncA and oncB. Antibodies affinity-purified on either of two representative members, oncA1 and oncB1, recognised all the FII bands. Individual FII bands excised from a DOC gel resolved into an overlapping series of molecules when re-run on SDS-PAGE, indicating that each FII band consisted of several polypeptides of differing molecular weight. Immunoprecipitates resolved on SDS-PAGE revealed that alpha-FII recognised 3 major oncosphere antigens, of 62, 34 and 25 kDa; antisera against oncB precipitated both the 34- and 25-kDa antigens, whereas alpha-oncA antisera precipitated the 62-kDa antigen. We conclude that oncA and oncB encode the major antigens in the FII complex. The 62-kDa antigen encoded by oncA1 was the only common antigen precipitated by anti-FII and two other antisera raised against different protective extracts, suggesting that it may be a protective component in all three. Southern blot results indicate that oncA and oncB are distinct genes present at low copy number in the genome. Evidence is also presented suggesting that some cestode mRNAs, including oncA, may use variant polyadenylation signals.

Vaccination against ovine cysticercosis using a defined recombinant antigen.

Cysticercosis caused by larval tapeworms is a major public health problem and a cause of substantial economic losses in the farm-animal industries. Taenia ovis in sheep is a particularly important example. Immunity to reinfection with the larvae has a central role in regulating natural transmission of the parasites, and vaccination with antigens from the early larval oncosphere stage can induce complete protection against infection. As it is impractical to obtain enough oncospheres for a commercial vaccine against these tapeworms, an alternative approach is to use recombinant DNA methods to generate a cheap and plentiful supply of antigens. We report here the expression in Escherichia coli of complementary DNA encoding T. ovis antigens as fusion proteins with the Schistosoma japonicum glutathione S-transferase. Vaccination of sheep with these fusion proteins gave significant, although not complete, immunity against challenge infection with T. ovis eggs. Commercial development of a vaccine is being pursued.