RESUMEN
Infectivity of and immune responses to 28 Finnish Borrelia burgdorferi sensu lato isolates was studied in 3-4-week-old outbred NMRI and inbred BALB/c/Hy laboratory mice; rabbits were also inoculated. Twenty-one isolates were found to detectably infect mice. A variation among isolates in degree of infectivity was observed. Higher infection rate and higher average ELISA readings were recorded for intradermal than intraperitoneal inoculations. The results suggest differences between Borrelia genospecies in organotropism. The ear was frequently infected by representatives of all genospecies; among high infectivity experiments, this rate was highest, 100%, in infections by Borrelia afzelii. Further differences between genospecies specific organ distributions: B. burgdorferi sensu stricto and Borrelia garinii isolates seemed to infect the bladder relatively more frequently than B. afzelii did; B. afzelii isolates infected heart relatively more frequently than others did. Genospecies specific differences were demonstrated between antigens in reactivity, i.e. in their 'sensitivity' as reagents of ELISA and IFA methods to measure isolate specific immune responses. Antigens from two B. afzelii isolates differed clearly in sensitivity.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Borrelia burgdorferi/inmunología , Enfermedad de Lyme/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Borrelia burgdorferi/clasificación , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidad , Ensayo de Inmunoadsorción Enzimática , Genotipo , Enfermedad de Lyme/microbiología , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , ConejosRESUMEN
Twenty-three experimental cattle, mainly calves, were each inoculated 1-3 times with one of ten Finnish Borrelia burgdorferi sensu lato strains. All three genospecies were represented. Borreliae were administered mainly by both intravenous (about 10(6) to 10(9) spirochaetes) and intradermal (10(4)) routes, and on six occasions subcutaneously (10(3)) only. For infectivity control and comparison purposes mice and rabbits were inoculated simultaneously. Immune responses in cattle were monitored both with whole-cell sonicate enzyme-linked immunosorbent assay (IgG-ELISA) and indirect immunofluorescent assay (IgM-IgG-IFA). Five Finnish strains and the American strain B31 were used as antigens. No clinical signs of borreliosis were observed. Of the strains, 7/10 were interpreted by the immune responses to have caused relatively short-term subclinical infections of varying intensity. Borreliae could not be isolated from blood or other organ specimens of cattle. A rough estimate of the mean infectious dose in the conditions of experiments is 10(6) to 10(7) organisms. In conclusion, the overall result appears to argue a low susceptibility of cattle to clinical borreliosis, at least when infected by Finnish strains of the agent. Significant antigen-specific differences were observed both by ELISA and IFA in detection and quantification of immune responses. As a rule, the homologous antigen was found to be the most sensitive. Genospecies differences were mostly distinct. Antigens of two Borrelia garinii isolates proved practically equal in sensitivity, whereas major differences were displayed between two Borrelia afzelii antigens. In an IFA study, an American (B31) and a Finnish B. burgdorferi sensu stricto strain proved equally sensitive as antigens. In two relatively strong primary immune responses the antigen-specific measurement differences were such that diagnostically in a cross-sectional study only the homologous antigen or an antigen of the same genospecies would have been sufficiently sensitive to show a positive result.
Asunto(s)
Grupo Borrelia Burgdorferi/clasificación , Enfermedades de los Bovinos , Enfermedad de Lyme/veterinaria , Animales , Anticuerpos Antibacterianos/sangre , Grupo Borrelia Burgdorferi/genética , Grupo Borrelia Burgdorferi/patogenicidad , Bovinos , Ensayo de Inmunoadsorción Enzimática , Femenino , Finlandia , Técnica del Anticuerpo Fluorescente Indirecta , Genotipo , Inmunoglobulina G/sangre , Enfermedad de Lyme/diagnóstico , Enfermedad de Lyme/inmunología , Masculino , Ratones , América del Norte , Conejos , Sensibilidad y EspecificidadRESUMEN
The binding of 80 Streptococcus dysgalactiae mastitis isolates from 51 farms to plasma and connective tissue proteins fibronectin (29 kDa N-terminal fragment), vitronectin, collagen type I, fibrinogen, alpha 2-macroglobulin, IgG, and albumin was studied. All isolates boiund the bovine 29 kDa fibronectin fragment and the binding of bovine fibrinogen, caprine albumin, bovine alpha 2-macroglobulin-trypsin complexes and caprine IgG was also very frequent (92.5, 92.5, 72.5% and 87.5%, respectively). Binding to human vitronectin was observed in 55% of the isolates, whereas only 20% of the isolates bound human type I collagen. None of the isolates bound native alpha 2-macroglobulin. Nearly all isolates (91%) bound more than 3 ligands. The bacterial binding sites for these proteins (termed here receptors) occurred in different combinations of which the combination fibronectin-, albumin-, fibrinogen-, vitronectin-, alpha 2-macroglobulin- and IgG-receptor was the most common. More than one isolate was obtained from 10 farms. The isolates from 5 farms showed close similarity of binding profiles within the farm, indicating that they were of similar origin and suggesting that the binding characteristics were relatively stable. Wider variation among the isolates obtained from other 5 farms was detected. The different isolates of the same farm origin varied mostly in the binding of albumin, IgG and fibrinogen. Interestingly, a difference in the number of receptors between isolates from two different sampling areas was observed. The binding profiles offer a new phenotypic method for epidemiological studies and may also when combined with genetical studies provide more insight both into the role of the bacterial plasma and connective tissue protein receptors in the infection process and the regulation of receptor expression.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Mastitis Bovina/microbiología , Streptococcus/clasificación , Animales , Bovinos , Femenino , Humanos , Fenotipo , Unión ProteicaRESUMEN
Binding of human alpha 2-macroglobulin (alpha 2M) to group G streptococci and to their immunoglobulin G (IgG)-binding proteins (protein G) was investigated. Native alpha 2M bound specifically to strain G-148 with an apparent dissociation constant of (2.2 +/- 1.5) x 10(-9) M. Proteinase-complexed alpha 2M did not compete for the binding sites, and 125I-labelled proteinase-complexed alpha 2M did not bind to the bacteria. Binding of native alpha 2M to the cells was not affected by IgG or protein G consisting of only IgG-binding domains. 125I-labelled recombinant protein G did not bind to native or proteinase-complexed alpha 2M. However, a lysate of G-148 cells inhibited binding of alpha 2M to the bacteria, and immobilized wild-type protein G bound alpha 2M directly from fresh human plasma. In 13 group G streptococcal isolates, IgG-binding proteins were immunologically identified as protein G. In 11 isolates, these molecules reacted also with alpha 2M and human serum albumin (HSA). Western blots (immunoblots) of two wild-type protein G variants revealed identical bands reactive with goat IgG, HSA, and native alpha 2M. Digestion of wild-type protein G with clostripain destroyed in both variants the binding sites for alpha 2M but not for albumin and IgG. N-terminal fragments of protein G (lacking the IgG-binding region) bound both alpha 2M and HSA, whereas a similar HSA-binding peptide lacking the first 80 amino acids did not react with alpha 2M. Our findings are consistent with a specific binding site for native alpha 2M in the N-terminal region of protein G and suggest that binding of alpha 2M via IgG-binding proteins may be a general feature of human group G streptococci.
Asunto(s)
Proteínas Bacterianas/metabolismo , Streptococcus/metabolismo , alfa-Macroglobulinas/metabolismo , Humanos , Técnicas In Vitro , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Streptococcus/inmunologíaRESUMEN
Thirty-seven Borrelia burgdorferi strains, isolated in 1992 from Ixodes ricinus in Finland, were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting and indirect immunofluorescence assay (IFA) with five to nine monoclonal antibodies (MAbs). By SDS-PAGE results and reactivities to MAbs H3TS, J 8.3, I 17.3, and D6, the 37 isolates were assigned to the species B. burgdorferi sensu stricto (n = 7), Borrelia afzelii (n = 17), or Borrelia garinii (n = 13). Twenty more isolates examined only by IFA and with part of the MAbs were distributed as follows: 9 B. burgdorferi sensu stricto and 11 other species. Among 16 of 37 isolates displaying a SDS-PAGE patterns considered typical of that of B. garinii, 3 were negative by the test with MAb D6; the rest were positive. The three MAb D6-negative isolates reacted with MAb J 8.3 but not with MAb I 17.3. It is suggested that these isolates of a previously undescribed type represent atypical B. afzelii strains deficient in the expression of OspB proteins. The misleading species designation by the SDS-PAGE result is described. The IFA results were generally consistent with those obtained by immunoblotting. The exception was for 3 of 29 isolates that were positive with MAb H5332 by immunoblotting but that were IFA negative. In the present material of 57 strains, all 16 B. burgdorferi sensu stricto isolates originated from the Aland Islands. B. afzelii and B. garinii were isolated from all three regions where ticks were collected. The distributive difference seems to offer a basis for comparative clinico-epidemiological studies of Lyme borreliosis.
Asunto(s)
Antígenos Bacterianos , Grupo Borrelia Burgdorferi/aislamiento & purificación , Lipoproteínas , Animales , Anticuerpos Monoclonales , Antígenos de Superficie/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Técnicas de Tipificación Bacteriana , Vacunas Bacterianas , Grupo Borrelia Burgdorferi/clasificación , Grupo Borrelia Burgdorferi/inmunología , Electroforesis en Gel de Poliacrilamida , Finlandia/epidemiología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Immunoblotting , Enfermedad de Lyme/epidemiología , Enfermedad de Lyme/microbiología , Dodecil Sulfato de Sodio , Especificidad de la Especie , Garrapatas/microbiologíaRESUMEN
The application of stabilised streptococcal cells for the purification of both immunoglobulin 1 (IgG1) and IgG2 subclasses from goat sera was evaluated. Guanidinium chloride extracted, lyophilised cells of the Lancefield Group C Streptococcus dysgalactiae Sc1 strain showed strong binding to goat IgG, reaching a capacity of approximately 1.4 mg IgG per 100 mg cells (dry weight). The IgG preparation obtained was of high quality. In immunoelectrophoretic analysis the preparation appeared to consist of pure IgG, whereas the high pressure liquid chromatography (HPLC) gel filtration and immunoblot analyses showed a very slight contamination (less than 1.3% of the total probe) with alpha 2-macroglobulin. The presence of both IgG subclasses in the preparation was verified by HPLC ion exchange chromatography. The adsorption procedure proved to be efficient and easy to perform without advanced technical equipment and the cells were reusable. As these streptococcal cells bind both IgG subclasses, this method presents an economical way for the small scale purification of goat IgG. Additionally the streptococcal cells may conveniently substitute Protein A bearing Staphylococcus aureus cells in various immunological assays.
Asunto(s)
Cabras/inmunología , Inmunoglobulina G/aislamiento & purificación , Isotipos de Inmunoglobulinas/aislamiento & purificación , Receptores Fc , Streptococcus , Adsorción , Animales , Cromatografía Líquida de Alta Presión/veterinaria , Cromatografía por Intercambio Iónico/veterinaria , Electroforesis en Gel de Poliacrilamida/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Inmunoelectroforesis/veterinaria , Receptores Fc/inmunología , Streptococcus/citología , Streptococcus/inmunologíaRESUMEN
Whey samples were screened for the presence of the proteinase inhibitor alpha 2-macroglobulin (alpha 2M). From an enzymic test, alpha 2M levels in normal whey varied in the range 0.49-0.84% of the serum level, whereas in mastitis whey the activity was markedly increased, reaching values between 0.91 and 138.5% (median 7.2%) of standard serum level. In mastitis milk samples but not in normal milk alpha 2M was also detected by double immunodiffusion and Western blotting. The proteinase inhibitor was purified from a mastitis milk sample with high alpha 2M activity (138.5% of serum level). In SDS-PAGE, native-PAGE and in double immunodiffusion analysis the inhibitor appeared indistinguishable from plasma-derived alpha 2M. The alpha 2M preparation from mastitis whey migrated essentially as native alpha 2M, representing the 'slow' form of the molecule. Treatment with trypsin transformed the alpha 2M preparation to the electrophoretic 'fast' form, but treatment with methylamine had only a minor effect. The receptor recognition sites were not exposed on the isolated alpha 2M molecule but could be readily exposed by treatment of the proteinase inhibitor with trypsin, which further proved that the isolated alpha 2M was in the entire native, functionally active state.