Search

Borrelia burgdorferi has been implicated as the causative agent of borreliosis in dogs, which is characteristically a limb/joint disorder, but can be associated with multiple-organ dysfunction. Attempts to reproduce this disease by inoculating dogs with B burgdorferi have not been successful. In the study of this report, B burgdorferi from Ixodes dammini ticks was used to induce signs of limb/joint dysfunction, fever, anorexia, depression, and systemic infection in dogs. A pure culture of this bacterium from the blood of an infected dog has been used to fulfill Koch's postulates for B burgdorferi as the causative agent of limb/joint dysfunction associated with borreliosis in dogs.

The immunogenicity and efficacy of a commercial Borrelia burgdorferi bacterin was evaluated for stimulation of the host immune response and protection against clinical disease associated with experimentally induced borreliosis in dogs. A total of 30 vaccinated and 24 control dogs were used in 3 separate studies. The vaccine was given IM as two 1-ml doses separated by a 3-week interval. Two weeks or 5 months following the last vaccination, the dogs were challenge inoculated with 7 daily doses of a virulent preparation of a B burgdorferi field isolate through intraperitoneal, subcutaneous, and intradermal routes with or without glucocorticoid administration at the same time. The development of B burgdorferi spirochetemia and clinical disease in the dogs after challenge exposure was studied. Serum samples were obtained from the dogs at various times during the study for serum neutralizing antibody determination and protein immunoblot antibody assay against various geographic isolates of B burgdorferi. Challenge exposure induced limb/joint disorder, fever, anorexia, signs of depression, and B burgdorferi spirochetemia in the nonvaccinated control dogs. The vaccine was found to elicit cross-reactive serum neutralizing and protein immunoblot antibody responses in dogs to various isolates of B burgdorferi and to protect the vaccinated dogs against experimentally induced borreliosis.

Specifically purified antibody to either domain I, domain III, or to subregions of domains I or III of serum albumin was added to refolding mixtures containing reduced serum albumin but no other refolding catalyst. It was found that the refolding of reduced albumin was greatly enhanced by the presence of specific antibody in the refolding mixture, that this enhancement was restricted to that domain for which the added antibody was directed, and that antibody-mediated enhancement of refolding in the NH2-terminal portion of each domain was delayed as compared to that seen in the COOH-terminal portion of each domain. Thus, an apparent COOH-terminal to NH2-terminal pathway of refolding within each domain was observed, which is consistent with the proposed evolutionary pathway of the albumin molecule and also consistent with the proposed presence of a nucleation center in the COOH-terminal double disulfide loop of each domain.

Four antigenic regions of native bovine pancreatic ribonuclease have been located by using antibodies that react specifically with segments 1--13, 31--79, and 80--124. These antibodies were purified by affinity chromatography on columns to which these peptide segments were bound. Analysis of precipitin curves indicates that there are at least three antigenic determinants to which antibody molecules can bind simultaneously in the presence of excess antibodies. Analysis of binding data, however, for each purified specific antibody preparation, carried out by the method of Berzofsky et al. [Berzofsky, J. A., Curd, J. G., & Schechter, A. N. (1976) Biochemistry, 15, 2113], leads to an estimate of four for the number of antigenic determinants in ribonuclease; this estimate had also been made earlier by Stelos et al. [Stelos, P., Fothergill, J. E., & Singer, S. J. (1960) J. Am. Chem. Soc. 82, 6034]. We find that one determinant is associated with each of segments 1--13 and 80--124 and two with segment 31--79. No antigenic activity could be detected for segment 14--29 either in native ribonuclease or in the free fragment. These conclusions are based on (1) the use of specific peptides to isolate purified antibodies by affinity chromatography, (2) immunoprecipitation of an antigenic peptide from the peptic digest of ribonuclease, (3) competitive inhibition studies with various peptide and protein fragments [cyanogen bromide fragments 1--13, 31--79, and 80--124, the tryptic peptides 40--61 and 105--224, S-peptide, S-protein, and des(121--124)-RNase], and (4) comparison and evaluation of the published effects on antigenicity of chemical and enzymatic modifications and changes in sequence among homologous ribonucleases. These approaches provide evidence that the four antigenic determinants are localized around the alpha-helical portion of segment 1--10, somewhere in segment 40--61, at the beta bend in segment 63--75, and either at the beta bend or beta sheet in segment 87--104 of native ribonuclease.

An immunological method is used to follow the folding of different portions of the reduced bovine pancreatic ribonuclease molecule during air oxidation. Antibodies that react specifically with segments 1-13, 31-79, and 80-124 of native ribonuclease, as they are folded, were purified by affinity chromatography, using antiserum to native ribonuclease and columns to which the ribonuclease fragments were attached. The kinetics of reaction between these prufied antibodies and refolded portions that are produced when reduced rebonuclease is oxidized by air demonstrate the presence of intermediate states of folding, and are consistent with folding of the anti-genic determinants in the order 80-124, 1-13, and 31-79. The relative stabilities of each of these segments to thermal denaturation in the native protein provide additional evidence that the native conformation of region 80-124 is a very stable one in the intact molecule. On the basis of these two types of evidence, it appears that segment 80-124 contains a nucleation site for the folding of the protein molecule.