Mycobacterium avium subsp. paratuberculosis in muscle, lymphatic and organ tissues from cows with advanced Johne's disease.

Blood, liver, kidney, lymph nodes and muscle tissue were obtained from the carcasses of five cows with advanced Johne's disease. Samples from the raw tissues, from cooked muscle tissues and from cooked hamburger patties that contained chopped mesenteric lymph nodes were collected aseptically. Each sample was divided into two portions, one of which was decontaminated. Both portions were homogenized. Homogenates were spread on selective agar for the recovery of Mycobacterium avium subsp. paratuberculosis (Map) and inoculated into a Map growth medium with the organism being detected in the cultures by PCR procedures and Ziehl-Neelsen staining. Map were recovered at numbers > 10(3) cfu/g from 7 of 15 liver and mesenteric and ileocaecal lymph node samples; and at lesser numbers from 5 of 15 kidney and superficial inguinal and prescapular lymph node samples. The numbers recovered from decontaminated and not decontaminated portions of each sample were generally similar. Map was recovered from 1 and detected in 6 of 50 not decontaminated portions of samples of raw, chilled or frozen meat; and detected in 1 of 15 not decontaminated samples of meat cooked to 61 degrees C, and in 1 of 40 samples of meat cooked to >/=70 degrees C. Map was detected in 2 of 4 samples of mesenteric lymph nodes cooked to 61 degrees C, but not in samples cooked to > or =70 degrees C. The findings indicate that Map may be present in meat from infected animals at low numbers, but that any such organisms are likely to be inactivated when meat is cooked to a well done condition.

Comparison of serological tests and faecal culture for the detection of Mycobacterium avium subsp. paratuberculosis infection in cattle and analysis of the antigens involved.

Three hundred and forty-one sera from cattle in Western Australia and 106 sera from Mycobacterium paratuberculosis faecal culture positive cattle were used to evaluate the performance of two absorbed enzyme-linked immunosorbent assays (ELISA) (one locally produced, the other a commercial test) and a complement fixation test (CFT) for the detection of Johne's disease in cattle. The diagnostic sensitivity (47.2%) of the local ELISA was significantly higher than that of the commercial ELISA (31.1%), and significantly higher than that for the complement fixation test (17.9%) and immunoblot (20.8%). Diagnostic specificity for the two ELISAs was 99.7% and 97.9% and similar for CFT and immunoblot (97.1% and 97.7%, respectively). The diagnostic sensitivity rose for both ELISAs and the CFT as the number of M. paratuberculosis isolated from the faeces increased. The ELISA antigen was characterised by polyacrylamide gel electrophoresis and electrophoretic immunoblotting and was found to consist mostly of a carbohydrate-type macromolecule of 32-42 kDa. This macromolecule was identified as lipoarabinomannan (LAM) by using a LAM-specific monoclonal antibody in immunoblots and purified LAM in absorption experiments. By applying more complex antigen preparations in immunoblots, serum antibodies against proteins of 47, 37, 30, 24 and 21 kDa, and against the 32-42 kDa carbohydrate component were frequently found in infected cattle, and of these the 47 kDa protein and the 32-42 kDa antigen were immuno-dominant. Pre-absorption of the sera with M. phlei sonicate indicated that the protein antigens contributed markedly to non-specific serological cross-reactions, while the 32-42 kDa non-protein macromolecule appeared to be specific.

Purification, characterization and immunochemical properties of a novel 60-kDa protein of Vibrio anguillarum strains.

Vibrio anguillarum strains expressed increased amounts of a novel 60-kDa protein when cells were grown at physiologically elevated temperatures. The relative amounts of the 60-kDa protein were unaltered by changes in osmolarity or ionic concentration of the growth medium in cells grown at optimal growth temperatures. The N-terminal amino acid sequence analysis of the V. anguillarum 60-kDa protein showed extensive (94-89%) sequence identity with the 60-kDa heat shock protein of Yersinia enterocolitica and with Serratia rubidaea GroEL protein. Monoclonal antibodies against the Y. enterocolitica chaperonin reacted with the 60-kDa protein from V. anguillarum strains, and with a temperature-induced protein of similar molecular mass in other Gram-negative pathogens of fish.

Structural characterization of the lipopolysaccharide O-antigen and capsular polysaccharide of Vibrio ordalii serotype O:2.

Structures of the capsular and O-chain polysaccharides of Vibrio ordalii serotype O:2, the causative agent of vibriosis in salmonid fish, were determined by high-field NMR techniques, mass spectrometric methods and partial hydrolysis. Both polymers were shown to be composed of linear tetrasaccharide repeating units, having the structure: carbohydrate sequence [see text].

Influence of culture conditions on expression of the 40-kilodalton porin protein of Vibrio anguillarum serotype O2.

Vibrio anguillarum serotype O2 strains express a 40-kDa outer membrane porin protein. Immunoblot analysis revealed that antigenic determinants of the V. anguillarum O2 40-kDa porin were conserved within bacterial species of the genus Vibrio. The relative amounts of the V. anguillarum O2 40-kDa porin were enhanced by growth of V. anguillarum O2 in CM9 medium containing 5 to 10% sucrose or 0.1 to 0.5 M NaCl. In contrast, the levels of the porin were significantly reduced when cells were grown at 37 degrees C, and a novel 60-kDa protein was also observed. However, the osmolarity or ionic concentration of the growth medium did not influence expression of the 60-kDa protein. Growth in medium containing greater than 0.6 mM EDTA reduced production of the V. anguillarum O2 40-kDa porin and enhanced levels of a novel 19-kDa protein. Thus, expression of the V. anguillarum O2 40-kDa porin was osmoregulated and possibly coregulated by temperature. The N-terminal amino acid sequence of the V. anguillarum O2 40-kDa protein and the effect of environmental factors on the cellular levels of the porin suggested that the V. anguillarum O2 40-kDa porin was functionally similar to the OmpC porin of Escherichia coli. However, pore conductance assays revealed that the V. anguillarum O2 40-kDa porin was a general diffusion porin with a pore size in the range of that of the OmpF porin of E. coli.

Analysis of culture filtrate and cell wall-associated antigens of Mycobacterium paratuberculosis with monoclonal antibodies.

Proteins secreted by Mycobacterium species have been suggested as major immune targets in the early phase of infection. In this study, we sought to identify specific antigens in culture filtrates and in soluble cell extracts of Mycobacterium paratuberculosis. The release of antigens into the culture medium during growth of the bacilli and the distribution of specific epitopes within the Mycobacterium species were investigated by immunoblot analysis with monoclonal antibodies (MAbs) raised against M. paratuberculosis antigens. MAb B6A interacted with a cellular antigen with an apparent molecular mass of 34.5 kDa in lysates of M. paratuberculosis. MAb B6A did not interact with lysates from any other mycobacterial species, suggesting recognition of an M. paratuberculosis species-specific epitope. MAb FL1-A1 reacted with an antigen of 44.3 kDa in M. paratuberculosis and a 9-kDa antigen in Mycobacterium kansasii. MAb PII-B1 reacted with concanavalin A (ConA)-binding cellular and filtrate molecules of M. paratuberculosis and with lysates of Mycobacterium kansasii and Mycobacterium avium 18. The affinity-purified glycosylated antigens migrated as a diffuse band of between 35 and 45.6 kDa and reacted strongly with ovine and bovine paratuberculosis serum and polyclonal serum against M. tuberculosis lipoarabinomannan antigens. These glycoconjugates were the earliest antigens detected in culture filtrates of M. paratuberculosis. Deglycosylation of the ConA-binding molecules with alpha-mannosidase enzyme abolished the reaction with MAb PII-B1 and with bovine but not ovine paratuberculosis serum, suggesting selective immunogenicity in the different animal species.

Structural studies of the lipopolysaccharide O-antigen and capsular polysaccharide of Vibrio anguillarum serotype O:2.

Vibriosis caused by Vibrio anguillarum affects salmonid and marine fish species worldwide and is considered to be one of the most serious threats to the success of commercial fish farming. In the course of this study, it was found that V. anguillarum serotype O:2 strains produce an acidic capsular polysaccharide having the identical structure to that of the O-chain polysaccharide. One-dimensional and two-dimensional nuclear magnetic resonance techniques, together with partial hydrolysis and various specific modifications, were used to determine the structure of these polysaccharides. It is proposed that both O-chain and capsular polysaccharide of V. anguillarum serotype O:2 are composed of linear tetrasaccharide repeating units having the following structure, in which Glc2NAc3NAN represents 2-acetamido-3-amino-2,3-dideoxy-D-glucuronamide, Man2NAc3AmA is 3-acetamidino-2-acetamido-2,3-dideoxy-D-mannuronic acid. Am represents an acetamidino group, Gal(NAc)2A is 2,3-diacetamido-2,3-dideoxy-L-galacturonic acid, Bac(NAc)2 is 2,4-diacetamido-2,4,6-trideoxy-D-glucose (N,N'-diacetylbacillosamine) and Fo is formyl.

Cloning and expression of rfb genes from Vibrio anguillarum serotype O2 in Escherichia coli: evidence for cross-reactive epitopes.

Vibrio ordalii and Vibrio anguillarum O2 express lipopolysaccharide (LPS) O antigens containing both specific and cross-reactive epitopes. The localization of these epitopes on the O antigen is not known. We have cloned and expressed the rfb gene cluster for O-antigen synthesis from V. anguillarum O2 (rfbVaO2) in Escherichia coli. E. coli DH5 alpha containing the recombinant plasmid pAM86 expressed O antigens which reacted with polyclonal antisera to V. ordalii and to V. anguillarum O2 LPS and with monoclonal antibody (MAb) 7B4, which is specific for V. anguillarum O2 O antigens. The recombinant strains were also protected from bactericidal killing by normal fish serum. Surprisingly, the LPS expressed from the cloned rfbVaO2 genes also reacted with MAb A16, which is specific for V. ordalii O antigens. Western immunoblot analysis revealed that MAb 7B4 reacted with recombinant LPS bearing shorter O-antigen repeat units, while MAb A16 reacted with the longer O antigens. Similar results were obtained when pAM86 was transformed into E. coli CLM4, which has a deletion spanning the sbcB-rfb region, indicating that the changes in antigenic profiles of O antigens from the recombinant strains were not due to genes within the E. coli rfb cluster. These data suggest that the epitope recognized by the MAb A16 is expressed by V. anguillarum O2 strains but it is apparently not accessible to the antibody in the native O polysaccharide. Cloning of the rfbVaO2 gene cluster resulted in expression of a novel O antigen. The modification(s) which leads to the alterations in antigenic profile of these recombinant LPS remains to be determined.

Characterization of concanavalin A-binding glycoproteins from procyclic culture forms of Trypanosoma congolense, T. simiae and T. brucei brucei.

Concanavalin A-binding glycoproteins were obtained from procyclic culture forms (PCFs) of Trypanosoma congolense, T. simiae, and T. b. brucei strains. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that glycoproteins of 38.5, 30.5, and 27 kDa were conserved between the different species and strains of the procyclic parasites. There were few similarities in the profiles of the high-molecular-weight glycoconjugates between the parasites. Monoclonal antibody analysis revealed that the 38.5- and 27-kDa glycoproteins were intracellular molecules and that they contained cross-reactive antigenic determinants. Surface biotinylation of PCF T. congolense K45/1 identified surface-accessible glycoproteins of 81.5, 59, and 38-42 kDa. By use of lectin blots and enzymatic deglycosylation studies, we demonstrated that the 81.5-, 59-, 38.5-, and 27-kDa glycoproteins contained N-linked oligosaccharide chains with both high-mannose-type and complex-type oligosaccharides, and the 81.5- and 59-kDa surface glycoproteins contained sialic acid residues. The glycoproteins identified in this study provide a starting point for further structure and function studies.

Monoclonal antibodies against Vibrio anguillarum O2 and Vibrio ordalii identify antigenic differences in lipopolysaccharide O-antigens.

Monoclonal antibodies (mAbs) that recognize distinct species-specific antigenic epitopes in O-antigens from Vibrio anguillarum O2, O2a and certain O2b strains (mAb 7B4) and from Vibrio ordalii strains (mAbs A16 and 7D11) were generated. Western immunoblot analysis using these mAbs revealed that vibrio strains grown in the presence of fresh rainbow trout blood expressed lipopolysaccharide (LPS) with longer (high molecular mass) O-antigens and extracellular capsular layers when compared to strains grown without rainbow trout blood. We also generated mAbs that react with O-antigens from V. anguillarum serotype O1 (mAbs 7B8, 7B5 and 1C3) and serotype O3 (mAbs 13A1 and 14C5) strains. These mAbs provide rapid and accurate diagnostic reagents for serological differentiation of V. ordalii from serotype O2 strains of V. anguillarum, and for serotyping of these pathogenic vibrios.

Protein changes in bovine lymphoblastoid cells induced by infection with the intracellular parasite Theileria parva.

Protein and glycoprotein changes induced in bovine lymphoblasts by infection with Theileria parva were analyzed by high-resolution two-dimensional gel electrophoresis. Uninfected and infected cloned bovine T and B lymphoblasts were biosynthetically labeled with [35S]methionine and their two-dimensional autoradiographic patterns were compared with each other and with the pattern obtained using purified labeled schizonts. Ten proteins were found in infected cells which were not present in uninfected cells, and seven of these were detected in preparations of purified schizonts. Four glycoproteins were detected on the surface of infected cells labeled with [3H]borohydride while a major glycoprotein present on uninfected cells disappeared or was reduced in infected cells. Other minor changes in protein and glycoprotein patterns were also observed.

Surface carbohydrates of procyclic forms of African trypanosomes studied using fluorescence activated cell sorter analysis and agglutination with lectins.

Living culture form procyclics of Trypanosoma brucei brucei, T.b. rhodesiense, T.b. gambiense, T. congolense and T. simiae were tested for binding of eight different lectins. The binding of fluorescein isothiocyanate (FITC)-conjugated lectins was measured using a fluorescence activated cell sorter (FACS) and by agglutination with unlabelled lectins. Five of the lectins failed to bind to any of the procyclic organisms in both tests. All parasites bound concanavalin A (Con A) and all T.b. brucei, T.b. rhodesiense and T. congolense procyclics bound Ricinus communis agglutinin 120 (RCA) and wheat germ agglutinin (WGA). Trypanosoma b. gambiense procyclics failed to bind RCA and thus could be easily discriminated from other subspecies of T. brucei. Similarly, T. simiae did not bind WGA, unlike T. congolense, the other species of the genus Nannomonas. All positive reactions were inhibited by 0.2 M concentrations of the relevant sugars. The results indicate that all species and subspecies of the procyclic culture forms tested have surface-exposed structures resembling alpha-D-mannose moieties and that T.b. brucei, T.b. rhodesiense and T. congolense have surface-exposed molecules resembling D-galactose and N-acetyl D-glucosamine (or sialic acid) moieties. Molecules resembling D-galactose and N-acetyl D-glucosamine residues are absent or inaccessible in T.b. gambiense and T. simiae respectively. A group of T. congolense clones of parasite stocks isolated at Kilifi on the Kenyan coast showed quantitatively different binding of RCA when compared to the other T. congolense clones tested indicating that these organisms differ in surface carbohydrate structure.

Identification and isolation of a variant surface glycoprotein from Trypanosoma vivax.

The protozoan Trypanosoma vivax is one of the most important agents of African trypanosomiasis, a disease that hinders the productive use of livestock in one-third of the African continent. Trypanosoma vivax is also present in the Caribbean and in South America, posing a threat to the livestock industries of the tropical and subtropical world. Much less is known of the biology of this trypanosome than of the better studied T. brucei and T. congolense. One of the variant surface glycoproteins (VSGs) of a West African stock of T. vivax was identified, purified, and partially characterized by the use of a combination of highly resolving techniques to maximize information from the relatively small amount of parasite material available. The molecular weight of the isolated protein (46,000) is smaller than that of VSGs from other species. As with T. brucei VSGs the protein from T. vivax is complexed with sugars and incorporates 3H when living trypanosomes are incubated with [3H]myristic acid, but the T. vivax molecule is more hydrophobic than the T. brucei molecule. The small size of the T. vivax VSG may have a bearing on the functional and evolutionary relationships of variant antigens in trypanosomes.

Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa.

A rapid colony immunoblot screening procedure was used to demonstrate the surface localization of porin protein F on bacterial colonies of Pseudomonas aeruginosa. By this method, we demonstrated that protein F was accessible to four different specific monoclonal antibodies in a wide variety of both mucoid and nonmucoid P. aeruginosa strains. Controls were performed to demonstrate that, using this procedure, only surface-exposed epitopes bound monoclonal antibodies and that nonspecific binding of monoclonal antibodies either to cells lacking protein F or to mucoid exopolysaccharide did not occur. Monoclonal antibodies MA4-4, MA2-10, and MA4-10, specific for protein F, also interacted with colonies of Pseudomonas putida and Pseudomonas syringae, whereas the protein F specific monoclonal antibody MA5-8 interacted only with P. aeruginosa strains. Using the above-named monoclonal antibodies, we investigated the antigenic structure of protein F. Monoclonal antibodies MA4-4, MA2-10, and MA4-10 bound to 29-31 kilodalton proteolytic fragments produced after papain or trypsin digestion of purified protein F or of protein F in outer membranes or intact cells. Antibody MA5-8 did not interact with proteolytically digested protein F but did interact with two of the six fragments produced after partial cyanogen bromide cleavage of protein F. Antibodies MA4-4, MA2-10, and MA4-10 did not interact with protein F after reduction of its internal disulphide bonds with 2-mercaptoethanol; in contrast, the reactivity of MA5-8 was unaffected. This data suggests that there are at least two distinct highly conserved surface epitopes on porin protein F.

Immunotherapeutic potential of monoclonal antibodies against Pseudomonas aeruginosa protein F.

To unambiguously demonstrate the immunotherapeutic potential of outer membrane porin protein F from Pseudomonas aeruginosa, a series of monoclonal antibodies have been isolated and demonstrated to be specific for protein F by Western blotting procedures. The antibodies recognize a surface-exposed antigenic site that is conserved on all Pseudomonas aeruginosa strains tested to date. One of these monoclonal antibodies named MA4-4 resulted in passive protection against subsequent infections by Pseudomonas aeruginosa in two different mouse infection models. In vitro studies using human polymorphonuclear leukocytes suggested that this antibody opsonized Pseudomonas aeruginosa for phagocytosis. The data suggest that immunotherapy based on porin protein F has definite potential for success.

Monoclonal antibodies against bacterial outer membrane antigens.

Monoclonal antibodies have proved to be highly specific tools for defining the antigenic epitopes of Pseudomonas aeruginosa outer membrane macromolecules. In this article we have highlighted the use of monoclonal antibodies in the study of lipopolysaccharide heterogeneity and in particular have demonstrated that single monoclonal antibodies can recognize epitopes on lipid A which are conserved in all Gram negative bacteria tested. Monoclonal antibodies against P. aeruginosa outer membrane proteins have been used to demonstrate the strong conservation of specific antigenic sites in all P. aeruginosa strains tested. In the case of one monoclonal antibody, specific for outer membrane lipoprotein H2, the antigenic site recognized by the antibody was also found to be conserved in all group 1 Pseudomonads. The implications of these monoclonal antibodies to bacterial taxonomy is discussed. Monoclonal antibodies against two separate conserved surface epitopes on outer membrane protein F were isolated and differentiated according to their reactions with 2 mercaptoethanol-reduced protein F and with proteolytic and cyanogen bromide peptide fragments of protein F. One of these protein F-specific monoclonal antibodies has been demonstrated to have immunotherapeutic potential.

Monoclonal antibodies specific for Escherichia coli J5 lipopolysaccharide: cross-reaction with other gram-negative bacterial species.

Four monoclonal antibodies against Escherichia coli J5 were studied. Each of these monoclonal antibodies reacted with purified lipopolysaccharides from E. coli J5, the deep rough mutant Salmonella minnesota Re595, Agrobacterium tumefaciens, and Pseudomonas aeruginosa PAO1 as well as with the purified lipid A of P. aeruginosa. Enzyme-linked immunosorbent assays using the outer membranes from a variety of gram-negative bacteria demonstrated that these lipid A-specific monoclonal antibodies interacted with between 84 and 97% of the gram-negative bacterial species tested. One of the monoclonal antibodies, 5E4, was shown to interact with 34 of the 35 outer membrane or lipopolysaccharide antigens tested. Immunoenzymatic staining of Western electrophoretic blots of separated P. aeruginosa outer membrane components was used to demonstrate that antibody 5E4 interacted with a similar fast-migrating band, corresponding to rough lipopolysaccharide, from all 17 serotype strains and all 14 clinical isolates of P. aeruginosa. Similarly, iodinated goat anti-mouse immunoglobulin was used to detect the binding of monoclonal antibody 8A1 to a fast-migrating band on Western electrophoretic blots of purified lipopolysaccharides from Klebsiella pneumoniae and both smooth and rough strains of E. coli, Salmonella typhimurium, and S. minnesota. These results suggest considerable conservation of single antigenic sites in the lipid A of gram-negative bacteria.

Antibody to Pseudomonas aeruginosa mucoid exopolysaccharide and to sodium alginate in cystic fibrosis serum.

Antibodies in cystic fibrosis (CF) sera to Pseudomonas aeruginosa mucoid exopolysaccharide and to sodium alginate (a polysaccharide from seaweed chemically similar to mucoid exopolysaccharide) were measured in sera of CF patients to determine if the exopolysaccharide is immunogenic. An enzyme-linked immunosorbent assay was used to test sera from 26 CF patients (18 colonized with pseudomonas and eight non-colonized) and 26 healthy controls. CF patients colonized with pseudomonas had more antibody to mucoid exopolysaccharide (P = 0.0008) and to sodium alginate (P = 0.0008) than did non-colonized CF patients. Virtually none was found in healthy controls. Duration of colonization was correlated with the level of antibody to sodium alginate (P = 0.003) but not with antibody to mucoid exopolysaccharide. Mucoid exopolysaccharide is immunogenic in patients with CF.

Surface localization of Pseudomonas aeruginosa outer membrane porin protein F by using monoclonal antibodies.

Hybridomas secreting highly specific monoclonal antibodies against porin protein F of Pseudomonas aeruginosa were isolated. These antibodies interacted with protein F in outer membranes isolated from strains representing the 17 serotypes of P. aeruginosa and from another 15 clinical isolates from patients with cystic fibrosis. The cell surface localization of antigenic sites on protein F was shown by indirect immunofluorescent techniques with these monoclonal antibodies. No fluorescence was observed on a protein F-deficient strain H283 of P. aeruginosa. Another monoclonal antibody specific for outer membrane lipoprotein H2 of P. aeruginosa showed no fluorescence on intact, wild-type bacterial cells, but was able to interact with a rough, LPS-deficient mutant.