Farm animal research: identifying all the customers.
Explore the source record for details and available documents.
Animal Husbandry↗
Biomedical subjects
J M Pollock
Publications and source records attributed to J M Pollock.
Testing for bovine tuberculosis--more than skin deep.
Explore the source record for details and available documents.
Animals↗
The detection of CD2+, CD4+, CD8+, and WC1+ T lymphocytes, B cells and macrophages in fixed and paraffin embedded bovine tissue using a range of antigen recovery and signal amplification techniques.
In order to develop procedures to label the main bovine leucocyte populations in paraffin embedded sections, the immunoreactivity of 25 monoclonal antibodies (mAbs) to different leucocyte antigens was assessed with formal dichromate (FD5) and 10% formalin fixation, a battery of antigen retrieval (AR) methods, and the biotin-tyramide amplification system. All the leucocyte populations investigated (CD2+, CD4+, CD8+, WC1+ T lymphocytes, B cells and macrophages) were strongly and specifically detectable under an appropriate combination of mAb, AR method and signal amplification system. CD4 and CD8 required the most stringent conditions and could only be demonstrated in FD5 fixed sections. For detection of CD2, WC1+ T lymphocytes, B cells and macrophages, all the mAbs produced immunoreactivity in FD5 or formalin fixed tissues. The need to check a range of different AR methods is stressed, as the method of choice varied for each individual mAb. The incorporation of the signal amplification system was necessary to observe a strong signal and the complete distribution of CD4, CD8 and B cells. Fixation by FD5 proved to be better than formalin for the preservation of surface antigens but it was inferior for the detection of markers which were found to show cytoplasmic immunoreactivity, such as the macrophage marker MAC387 or the B cell markers BAQ155 or IL-A59.
Animals↗
Lesions in cattle exposed to Mycobacterium bovis-inoculated calves.
Nine calves were housed for periods ranging from 24 to 117 days in close contact with cattle inoculated intranasally with Mycobacterium bovis. These "in-contact" calves were examined immunologically and bacteriologically during the period of exposure, and pathologically and immunocytochemically post mortem. Three became infected by day 14, as indicated by the detection of M. bovis in nasal mucus. In-vitro interferon-gamma production and lymphocyte proliferation were detected after stimulation of peripheral blood with M. bovis antigens in the majority of in-contact animals by day 28; this provided support for the role of immunological mechanisms in pathogenesis. Tuberculous lesions were found in the submandibular and bronchomediastinal lymph nodes and in the lungs of the in-contact calves; in distribution and appearance the lesions resembled those observed in naturally occurring disease. The distribution of M. bovis antigen and the numbers of mycobacteria within pulmonary lesions are reported. 1999 Harcourt Publishers Ltd.
Animals↗
Differentiation between Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle by using recombinant mycobacterial antigens.
Tuberculosis continues to be a worldwide problem for both humans and animals. The development of tests to differentiate between infection with Mycobacterium tuberculosis or Mycobacterium bovis and vaccination with M. bovis BCG could greatly assist in the diagnosis of early infection as well as enhance the use of tuberculosis vaccines on a wider scale. Recombinant forms of four major secreted proteins of M. bovis-MPB59, MPB64, MPB70, and ESAT-6-were tested in a whole-blood gamma interferon (IFN-gamma) assay for differentiation between cattle vaccinated with BCG and those experimentally infected with M. bovis. BCG vaccination induced minimal protection in the present study, with similar numbers of animals infected with M. bovis in BCG-vaccinated and nonvaccinated groups. Following vaccination with BCG, the animals produced moderate IFN-gamma responses to bovine purified protein derivative (PPDB) but very weak responses to the recombinant antigens. Cattle from both the BCG-vaccinated and nonvaccinated groups which were M. bovis culture positive following challenge produced IFN-gamma responses to PPDB and ESAT-6 which were significantly stronger than those observed in the corresponding M. bovis culture-negative animals. IFN-gamma responses to MPB59, MPB64, and MPB70 were significantly weaker, and these antigens could not discriminate between vaccinated animals which develop disease and the culture-negative animals. The results of the study indicate that of the four antigens tested in the IFN-gamma assay, only ESAT-6 would be suitable for differentiating BCG-vaccinated animals from those infected with bovine tuberculosis.
Animals↗
Generation of CD8(+) T-cell responses to Mycobacterium bovis and mycobacterial antigen in experimental bovine tuberculosis.
Protective immunity against tuberculosis is considered to be essentially cell mediated, and an important role for CD8(+) T lymphocytes has been suggested by several studies of murine and human infections. The present work, using an experimental model of infection with Mycobacterium bovis in cattle, showed that live M. bovis elicits the activation of CD8(+) T cells in vitro. However, a sonic extract prepared from M. bovis (MBSE) and protein purified derivative (PPDb) also induced a considerable degree of activation of the CD8(+) T cells. Analysis of proliferative responses of peripheral blood mononuclear cells, purified CD8(+) T cells, and CD8(+) T-cell clones to M. bovis and to soluble antigenic preparations (MBSE, PPDb) showed that the responses of all three types of cells were always superior for live mycobacteria but that strong responses were also obtained with complex soluble preparations. Furthermore, while cytotoxic capabilities were not investigated, the CD8(+) T cells were found to produce and release gamma interferon in response to antigen (live and soluble), which indicated one possible protective mechanism for these cells in bovine tuberculosis. Finally, it was demonstrated by metabolic inhibition with brefeldin A and cytochalasin D at the clonal level that an endogenous pathway of antigen processing is required for presentation to bovine CD8(+) cells and that presentation is also dependent on phagocytosis of the antigen.
Animals↗
Mycobacterial antigen-specific antibody responses in bovine tuberculosis: an ELISA with potential to confirm disease status.
Serological assays may help to identify animals in advanced stages of bovine tuberculosis, but most of the tests available have suboptimal sensitivities and specificities. This study was designed to determine whether the antibody responses to defined antigens (rMPB70, rMPB64 and rMPB59) of Mycobacterium bovis at the immunoglobulin subclass level could be used to develop improved serological tests. In experimentally infected cattle it was found that the predominant serum antibody response was to rMPB70, and that an IgG1 response to this antigen was boosted strongly by skin testing. Studies in naturally infected cattle suggested that this memory IgG1 anti-rMPB70 response may be able to differentiate between skin test-reactor animals with and without lesions by comparing the ratio of the antibody response before and after skin testing. The study has provided a clearer understanding of the kinetics of antibody responses to defined mycobacterial antigens at the subclass level in bovine tuberculosis and has made it possible to develop a novel ELISA system which may be useful in disease diagnosis.
Animals↗
Early lesion formation in cattle experimentally infected with Mycobacterium bovis.
Early lesion formation was examined in 13 calves inoculated intranasally with 2 x 10(7) colony-forming units of Mycobacterium bovis and killed either singly or in pairs at intervals of < or = 7 days from post-inoculation day (pid) 3 to pid 42. Immunological examinations were carried out before and after infection, and sequential necropsies were performed. M. bovis was recovered as early as pid 3, from the upper respiratory tract mucosae, retropharyngeal lymph nodes and caudal lung lobe. Gross tuberculous lesions were detected in both the upper respiratory tract mucosae and in the lungs of the calves killed from pid 14 onwards. Lesions were also present in the lymph nodes draining these areas. On histological examination, neutrophils appeared to play a key role in the earliest stages of lesion formation, and lesion mineralization was observed for the first time at pid 35. The contemporaneous development of lesions and cellular immunity, as demonstrated by in-vitro lymphocyte proliferation and interferon-gamma assay responses, provided further evidence of the role of immunopathogenic mechanisms in the development of bovine tuberculosis.
Animals↗
Recognition of a common mycobacterial T-cell epitope in MPB59 of Mycobacterium bovis.
Bovine tuberculosis, which persists as a residual level of infection in many European countries, has implications not only for the economy of farming communities but also for human health. The aim of this study was to identify a common mycobacterial antigen which was recognized in bovine tuberculosis and to characterize the response to this antigen at the epitope level. A T-cell clone, phenotype CD4+, raised from an animal experimentally infected with Mycobacterium bovis was shown to proliferate in response to a panel of sonicates derived from different mycobacterial species indicating recognition of an antigen with broad specificity. This antigen was subsequently shown to be MPB59. Recognition of MPB59 at the epitope level was determined in experimental and field cases of bovine tuberculosis using a panel of synthetic peptides (20-mers with 10-residue overlaps) incorporating the signal sequence and mature protein. The results showed that in vitro interferon-gamma was predominantly produced in response to adjacent peptides numbers 10 and 11, suggesting that the dominant epitope was contained in the overlap, correlating to residues 101-110 (YYQSGLSIVM). This epitope was recognized by 54% of tuberculous cattle of mixed breeds, which suggests that it may be genetically permissive in terms of major histocompatibility complex presentation. Sequence analysis confirmed that there were only minor differences in the amino acid composition within this region for various mycobacterial species, which could explain the common T-cell recognition described in this study. Common recognition of this epitope indicates that it would have limited potential for use as a diagnostic reagent per se but may have potential for inclusion in a subunit vaccine.
Animals↗
T-cell recognition of mycobacterial proteins MPB70 and MPB64 in cattle immunized with antigen and infected with Mycobacterium bovis.
Defined antigenic reagents and knowledge of T-cell responses are required for the design of improved diagnostic tests for bovine tuberculosis. The limited species distribution of Mycobacterium bovis antigens MPB70 and MPB64 has indicated their potential for inclusion in future tests. The strategy adopted in this study was to define bovine T-cell responses to these antigens at the epitope level, using cattle immunized with recombinant forms of the antigens, and to compare these responses with cattle which had been experimentally infected with M. bovis. Panels of synthetic peptides (20-mers with 10-residue overlaps) were used and five epitopes were identified and found to be powerful stimulators of T-cell responses in both types of animal (residues 81-100 and 174-190 for MPB70, and residues 1-20, 41-60 and 181-200 for MPB64). Further investigation in larger numbers of cattle (n = 14) of mixed breeds from tuberculosis-infected herds confirmed that each peptide produced response in several of the cattle, but no single peptide was recognized by all animals. However, the limited numbers of animals in this study suggest that peptide reagents may identify as many positive animals as the intact antigenic protein and could form components of a future diagnostic test. The use of cattle immunized with the proteins of interest has proved to be an interesting model for studying the nature of bovine T-cell responses to defined mycobacterial proteins.
Amino Acid Sequence↗
Diversity of antigen recognition by serum antibodies in experimental bovine tuberculosis.
Tuberculosis in cattle remains a major zoonotic and economic problem in many countries. The standard diagnostic assay for bovine tuberculosis, the intradermal tuberculin test, has low accuracy. Therefore, alternative immunodiagnostic methods, such as serological assays, are needed for detection of infected animals. Development of an accurate serodiagnostic test requires a detailed understanding of the humoral immune responses during bovine tuberculosis and, in particular, identification of the key antigens of Mycobacterium bovis involved in antibody production. In this study, we characterized antibody responses in cattle experimentally infected with M. bovis. Sequential serum samples were collected every 3 to 4 weeks for up to 27 months postinfection. Circulating immunoglobulin G antibody levels were measured by an enzyme-linked immunosorbent assay using 12 highly purified recombinant proteins of M. bovis. Six proteins, ESAT-6, 14-kDa protein, MPT63, MPT70, MPT51, and MPT32, were identified as major seroreactive antigens in bovine tuberculosis. A remarkable animal-to-animal variation of antigen recognition by serum antibodies was observed. Kinetic analyses of the antibody production to individual antigens during infection revealed that the heterogeneous antigen recognition profile changed markedly in a given infected animal as disease progressed.
Administration, Intranasal↗
Immunochemical characterization of the MPB70/80 and MPB83 proteins of Mycobacterium bovis.
MPB70 and MPB80 (MPB70/80) and MPB83 are closely related antigens which are highly expressed in Mycobacterium bovis. MPB70/80 are soluble secreted antigens, while MPB83 is an exported lipoprotein associated with the bacterial surface. In the present study, these antigens had different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. These differences may be explained by the fact that MPB70 and MPB83 both have two internal cysteine residues which would create ring structures by disulfide bonding. We analyzed the structures of MPB70/80 and MPB83 by using monoclonal antibodies (MAbs) raised against bovine purified protein derivative or whole M. bovis cells. MAb 1-5C reacted specifically with MPB70 and MPB80, and MAb MBS43 reacted specifically with MPB83, while the other antibodies, including several previously described MAbs, bound all three antigens. MAbs and polyclonal antibodies reacted strongly with reduced protein and less well with nonreduced protein, indicating involvement of linear epitopes. Epitopes of MAbs Bov-1, 2-6B, 1-5C, and 1-1D were mapped by using synthetic peptides of MPB70. Sequence comparison showed the peptide with the 1-5C-reactive epitope to have three residues different from those in the homologous region of MPB83. Exchanges of A for S in position 112 or Q for E in position 116 abolished the reactivity of MAb 1-5C. Polyclonal rabbit antibodies to native purified MPB70 reacted strongly with peptides 6, 7, and 8 of the N-terminal half of mature MPB70. Cattle sera of experimentally M. bovis-infected animals recognized a broader spectrum of peptides. These findings indicate that there is diagnostic potential for these proteins and that there is also a possible role for antibodies in elucidation of the host-mycobacterium relationship involving a surface-bound and exposed lipoprotein, MPB83, and its highly homologous soluble secreted MPB70/80 counterparts.
Amino Acid Sequence↗
Detection of Mycobacterium bovis infection in cattle using an immunoassay for bovine soluble interleukin-2 receptor-alpha (sIL-2R-alpha) produced by peripheral blood T-lymphocytes following incubation with tuberculin PPD.
After activation of T-lymphocytes with antigen there is an increase in the expression of interleukin-2 receptor-alpha (IL-2R-alpha) followed by the release of a soluble form of the molecule (sIL-2R-alpha) from the membrane of the stimulated cells. The present study investigates the novel use of the release of sIL-2R-alpha from activated T-lymphocytes as a marker of cell-mediated immunity (CMI) in cattle infected with Mycobacterium bovis. An enzyme immunoassay was used to detect sIL-2R-alpha produced following incubation of bovine peripheral blood mononuclear cells with mycobacterial antigens. Using this assay, 63/67 cattle naturally infected with M. bovis were identified whereas only 1/51 uninfected animals were considered to give a positive result. This assay is more convenient to use than lymphocyte proliferation assays which involve the use of radionucleosides. It should prove useful for monitoring the immunological activation of bovine T-lymphocytes in a variety of situations including the development of CMI responses in cattle to novel mycobacterial antigens or potential vaccines.
Animals↗
The potential of the ESAT-6 antigen secreted by virulent mycobacteria for specific diagnosis of tuberculosis.
Tuberculosis (TB) remains a global health problem in humans and animals, and improved diagnostic methods are needed urgently. This study examined the potential of an interferon-gamma blood test based on a recently identified low-molecular-mass secreted protein antigen, ESAT-6, for early detection of bovine TB. It was found that field cases of bovine TB and experimentally infected cattle exhibited strong in vitro interferon-y responses directed toward this antigen. Of importance, ESAT-6 reactivity was found to discriminate between cattle infected with TB and cattle sensitized by environmental mycobacteria, and the gene encoding this molecule was demonstrated to be absent from >90% of the nontuberculous mycobacterial strains isolated from healthy sensitized cattle. These results demonstrate the feasibility of using single defined antigens for the highly specific diagnosis of TB.
Animals↗
Predominant recognition of the ESAT-6 protein in the first phase of interferon with Mycobacterium bovis in cattle.
Tuberculosis continues to be a worldwide health problem for both humans and animals. The development of improved vaccines and diagnostic tests requires detailed understanding of the immune responses generated and the antigens recognized during the disease. This study examined the T-cell response which develops in cattle experimentally infected with Mycobacterium bovis. The first significant T-cell response was found 3 weeks after the onset of infection and was characterized by a pronounced gamma interferon (IFN-gamma) response from peripheral blood mononuclear cells directed to antigens in culture filtrates. Short-term culture filtrate (ST-CF) was separated into molecular mass fractions and screened for recognition by T cells from experimentally infected and field cases of bovine tuberculosis. Cattle in the early stages of experimental infection were characterized by strong IFN-gamma responses directed predominantly toward the lowest-mass (<10-kDa) fraction of ST-CF, but cattle in later stages of experimental infection (16 weeks postinfection) exhibited a broader recognition of antigens of various molecular masses. Field cases of bovine tuberculosis, in comparison, preferentially recognized low-mass antigens, characteristic of animals in the early stages of infection. The major T-cell target for this dominant IFN-gamma response was found to be the secreted antigen ESAT-6. This antigen was recognized strongly by the majority of field cases of bovine tuberculosis tested. As ESAT-6 is unique to pathogenic mycobacterial species, our study suggests that ESAT-6 is an antigen with major potential for vaccination against and specific diagnosis of bovine tuberculosis.
Animals↗
A retrospective study of red cell maternal antibodies by chemiluminescence.
Explore the source record for details and available documents.
Erythrocytes↗
Dynamic changes in circulating and antigen-responsive T-cell subpopulations post-Mycobacterium bovis infection in cattle.
Bovine tuberculosis is a threat to animal and human health in several countries. Greater understanding of the immunology of the disease is required to develop improved tests and vaccines. This study has used a model of bovine tuberculosis, established in the natural host, to investigate the dynamic changes that occur in the circulating T-cell subpopulations after infection. When the phenotypic composition of the peripheral blood lymphocytes was determined pre- and post-experimental infection, the response to disease comprised three phases. Firstly, the WC1/gamma delta T cells decreased and then increased, suggesting localization to developing lesions and clonal expansion. Secondly, the CD4:CD8 ratio increased. Thirdly, the CD4:CD8 ratio decreased to less than pre-infection measurements. The latter changes suggested sequential involvement of CD4 and then CD8 T cells. The proportion of cells expressing interleukin-2 receptor (IL-2R) also increased. Panels of T-cell clones were established at various stages post-infection and all clones that exhibited antigen responsiveness were phenotyped. T-cell clones from early infection were WC1/gamma delta and CD4 in phenotype, while CD8 clones appeared later in infection, eventually becoming dominant. Therefore, from in vivo and in vitro evidence, it was suggested that there is a dynamic progression in the T-cell subpopulations involved dominantly in responses to mycobacteria.
Animals↗
Peptide mapping of bovine T-cell epitopes for the 38 kDa tuberculosis antigen.
Mycobacterium bovis infection in cattle continues to be a problem in several regions, partly due to inadequate diagnostic tests. The aim of this study was to use an experimental model of the natural disease to identify T-cell epitopes from the mycobacterial 38 kDa antigen as potentially specific diagnostic reagents. A panel of overlapping synthetic peptides (16-mers with a five-residue overlap) were produced from the published amino acid sequence. It was found that peripheral blood lymphocytes from at least three of four experimentally infected animals, which were considered to be in either Th1- or Th1/Th2-dominated stages of anti-mycobacterial immunity, proliferated in response to five epitopes (residues 1-27, 88-107, 122-138, 243-260 and 307-328). However, in vitro production of IFN-gamma was detected only in response to epitope 122-138, indicating a role in protective immunity. The peptides were not recognized by control, uninfected animals, but all epitopes showed various degrees of recognition by animals which were field reactors to intradermal tuberculin testing. Furthermore, epitopes 1-27, 88-107 and 122-138 were recognized by four breeds of cattle and by animals from separate herds, suggesting genetic permissiveness in recognition which would be essential in the development of a diagnostic test.
Amino Acid Sequence↗