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J M Pollock

Publications and source records attributed to J M Pollock.

At least 37 records · Page 2Linked to original sources
PubMed2003

Specific delayed-type hypersensitivity responses to ESAT-6 identify tuberculosis-infected cattle.

Human and bovine tuberculosis have long been detected by skin testing with purified protein derivative (PPD), a complex mix of partly denatured mycobacterial antigens with suboptimal specificity. In the present study, skin tests based on ESAT-6, a recombinantly produced antigen highly specific for tuberculosis infection, were investigated. Although ESAT-6 was strongly recognized in vitro and induced high levels of gamma interferon, initial investigations demonstrated that higher doses of ESAT-6 than of PPD were needed to induce substantial delayed-type hypersensitivity reactions. Also, the kinetics of the skin test response differed for the two reagents; PPD showed maximal response at 72 h, but the response to ESAT-6 often peaked later at 96 h. Tests based on an optimized strategy (400 micro g of ESAT-6 measured between 72 and 96 h), in cattle infected with Mycobacterium bovis (n = 22) and animals sensitized by exposure to environmental mycobacteria showed ESAT-6 to have a promising diagnostic potential (sensitivity, 82%; specificity, 100%; optimal cutoff, 3 mm), compared with PPD (sensitivity, 86%; specificity, 90%; optimal cutoff, 4 mm). Larger investigations are required to refine cutoff points for any new diagnostic test, but the present results indicate great potential for skin tests based on specific antigens for accurate in vivo diagnosis of tuberculosis.

Animals↗
PubMed2002

The WC1(+) gammadelta T-cell population in cattle: a possible role in resistance to intracellular infection.

Intracellular infections are important in veterinary medicine and detailed understanding of the associated immune responses is needed for optimal development of strategies based on diagnosis and vaccination. It is generally accepted that cell-mediated immune responses are of greatest importance in intracellular infections and recent studies from several bovine models of infection indicate that WC1(+) gammadelta T-cells have a number of possible levels of involvement, which remain incompletely defined. Investigations of experimental infection with Mycobacterium bovis in cattle have indicated that WC1(+) gammadelta T-cells are among the first cells to accumulate at initial sites of infection, an observation which has been linked with decreased numbers of these cells in the circulation within days of infection. These WC1(+) gammadelta T-cells have been shown to respond in vitro, both to protein antigens and to non-protein, phosphate containing antigens of M. bovis and to be capable of producing IFN-gamma. Studies of M. bovis infection in calves depleted of WC1(+) gammadelta T-cells by monoclonal antibody have suggested that the presence of these cells is associated with development of a Th1-biased acquired immune response. In combination, these observations allow speculation regarding a possible role for WC1(+) gammadelta T-cells as a link between the innate and acquired immune systems which is instrumental in establishing an appropriate response.

Animals↗
PubMed2002

Generation of antibodies to the signal peptide of the MPT83 lipoprotein of Mycobacterium tuberculosis.

The mpt83 gene (Rv2873) encodes the exported MPT83 lipoprotein of Mycobacterium tuberculosis. The corresponding identical mpb83 gene of Mycobacterium bovis is expressed to varying extents in different substrains of M. bovis Bacille Calmette Guerin (BCG), BCG Tokyo and BCG Moreau being high producers and BCG Danish 1331, a low producer of the MPB83 protein. Immunization with the 13-mer N-terminal part of the signal peptide of MPT83, MINVQAKPAAAASC, coupled to keyhole limpet haemocyanin (KLH) through the added C-terminal cysteine resulted in rapid antibody formation monitored by enzyme-linked immunosorbent assay (ELISA) with free immunizing peptide on the solid phase. In ELISA, with four 20-mer overlapping peptides covering the N-terminal part of the MPT83 sequence, three polyclonal rabbit antisera reacted only with the N-terminal peptide. Antigenic signal peptide could not be detected in sonicates of BCG Tokyo and BCG Moreau. After SDS-PAGE and blotting, the antibodies reacted with sonicates of recombinant Escherichia coli containing the entire mpt83 gene including the signal sequence, but not with the 22 kDa form of native MPB83 purified from BCG culture filtrate. In partition chromatography the recMPT83 partitioned in the water phase while 26 kDa MPB83 in BCG culture filtrate partitioned in the lipid phase confirming that lipidation at the N-terminal cysteine residue occurs after the splitting of the polypeptide chain by signal peptidase II.

Amino Acid Sequence↗
PubMed2002

Mycobacterium bovis infection and tuberculosis in cattle.

This review considers the possible events that can occur when cattle are exposed to Mycobacterium bovis and, where appropriate, draws on principles accepted for tuberculosis infection in humans and laboratory animal models. Consideration is given to the many complex factors which influence the outcome of challenge with tubercle bacilli. These include features inherent to the mycobacterium, the host and the environment. It is apparent that clinical disease probably occurs only in a relatively small, but undetermined, proportion of cattle that are exposed to Al. bovis. The majority of animals may clear infection or control the bacilli, possibly in a condition of latency. It is concluded that a better understanding of the dynamics of the events following M. bovis exposure and subsequent infection in cattle would be of significant benefit in developing new tools appropriate for disease control and to designing optimal approaches for their application.

Animals↗
PubMed2001

Use of ESAT-6 in the interferon-gamma test for diagnosis of bovine tuberculosis following skin testing.

The whole blood interferon-gamma (IFN-gamma) test has proven to be a practical ancillary test for re-testing cattle for bovine tuberculosis 8-28 days following tuberculin skin testing. An improvement in the specificity of the IFN-gamma test could further reduce culling of false positive animals. The primary aim of this study was to evaluate a single mycobacterial antigen, ESAT-6 in the IFN-gamma test for use in skin test-positive cattle. These skin test-positive cattle comprised 51 Mycobacterium bovis-infected animals from tuberculosis-infected herds and 85 non-infected animals from tuberculosis-free herds. The test based on ESAT-6 had a higher specificity than the test based on purified protein derivative (PPD) tuberculin, but this was offset by a small decrease in sensitivity. Use of a lower cut-off in the ESAT-6-based test improved the sensitivity, while still maintaining a very high specificity. A secondary aim in the study was to assess the ESAT-6 and PPD-based tests for detecting bovine tuberculosis in skin test-negative animals from a persistently infected herd. The PPD-based test detected the majority of the lesioned or M. bovis-culture positive animals, while the ESAT-6-based test detected a smaller proportion. The false negatives in the IFN-gamma test from both the skin test-negative and positive groups were predominantly M. bovis-culture positive animals with no visible lesions. The current study has shown that a defined specific antigen such as ESAT-6 can markedly improve the specificity of the IFN-gamma test for re-testing skin test-positive animals. An ESAT-6-based IFN-gamma test could be particularly useful to reduce the false positive rate, yet still maintain an acceptable level of sensitivity.

Animals↗
PubMed2001

Characterization of the early antibody response in bovine tuberculosis: MPB83 is an early target with diagnostic potential.

A 26-kDa antigen has been shown to be a dominant antibody target in Mycobacterium bovis-infected cattle. In this study, that antigen was used as an immunogen to raise a panel of mouse monoclonal antibodies. The majority of those bound to native protein with a molecular mass of 26 kDa and to recombinant MPB83, strongly suggesting that MPB83 is an important B-cell antigenic target in bovine tuberculosis. In order to provide assessment of the potential of measuring antibody responses to the native protein, one monoclonal antibody, 1F11, was incorporated into an enzyme-linked immunosorbant assay format to trap antigen from a crude bacterial extract. Despite some disadvantages of this format, serum samples from cattle which had been infected experimentally with M. bovis, and from tuberculin skin-test-negative and -positive field cattle were tested for the presence of antibodies. Data from the skin-test-negative cattle allowed an arbitrary cut-off value to be established and, under these conditions, test sensitivity and specificity were estimated at 37.5 and 89%, respectively. These results indicate potential for MPB83 in the development of assays for serological diagnosis of bovine tuberculosis.

Animals↗
PubMed2001

Novel monoclonal antibodies against major antigens of Mycobacterium bovis.

MPB70 and MPB83 are among the most characteristically exported proteins defining a strongly expressed phenotype of Mycobacterium bovis. These proteins are known to be homologous to osteoblast-specific factor 2. By in vitro culture of mycobacteria they appear to have a limited species distribution and to be relatively specific for M. bovis. Virtually identical genes are however, present in Mycobacterium tuberculosis. In order to facilitate further research into the immunobiology of these proteins and their potential application for differential diagnosis of tuberculosis as a result of M. bovis, we describe the reactivities of 20 monoclonal antibodies (MoAbs) to these proteins. Immunizing with bovine PPD generated 10 MoAbs. These antibodies reacted preferentially with the soluble MPB70 antigen using reducing conditions in SDS-PAGE with western blotting. Ten MoAbs were generated by immunizing mice with fractions derived from a whole cell sonic extract of M. bovis. These antibodies reacted preferentially with the surface exposed MPB83 lipoglycoprotein.

Animals↗
PubMed2001

Lymphocyte subtypes in experimentally induced early-stage bovine tuberculous lesions.

The identity of the lymphocyte subtypes constituting the lymphocytic mantle within developing early-stage lesions of bovine tuberculosis was investigated immunohistochemically in calves inoculated intranasally with 2 x 10(7) colony-forming units of a field isolate of Mycobacterium bovis. Pulmonary lesions were examined 7, 14, 21, 28 and 42 days after inoculation, and bronchial lymph node lesions at 35 days. The immunolabelling results reported were obtained with monoclonal antibodies against two T-cell epitopes (WC1+ gamma delta and CD2+) and against B-cell epitopes. Large numbers of CD2+ T-lymphocytes were observed around developing areas of necrosis throughout the study; WC1+ gamma delta cells, however, were more numerous at these sites up to and including day 21. On the other hand, aggregates of B lymphocytes did not become prominent in areas adjacent to lesions until day 42. The results suggest that these lymphocyte phenotypes play a role in the pathogenesis of early-stage lesions.

Animals↗
PubMed2001

Immune responses in bovine tuberculosis.

Knowledge of the immune responses which develop in cattle following infection with Mycobacterium bovis is essential both to the understanding of disease pathogenesis and to the logical development of immune-dependent tools, such as diagnostic tests and vaccines, which can be used to combat the disease. Studies of field cases of bovine tuberculosis (TB) and of experimental bovine models of M. bovis infection have indicated that cell-mediated immune responses (CMI) predominate within a spectrum of immunity which exists. This paper reviews aspects of recent research and indicates how knowledge of T-cell antigenic targets in bovine TB along with increasing knowledge of T-cell subpopulations and their interactions with M. bovis -infected macrophages provides opportunities for the development of better methods for disease control.

Animals↗
PubMed2001

Towards more accurate diagnosis of bovine tuberculosis using defined antigens.

Diagnostic accuracy is of paramount importance in test-and-slaughter programmes for the eradication of bovine tuberculosis (TB). Currently applied methods, such as in vivo skin testing and in vitro interferon-gamma (IFN- gamma) testing, utilize purified protein derivatives (PPDs), which are poorly-defined mixtures containing many individual antigenic components. It is known that false-positive responses to these reagents can occur in cattle which are not infected with TB, largely because of that antigenic complexity. This paper reviews recent approaches to the characterization of more precisely defined diagnostic tools which can be used to develop tests with greater specificity. For example, the low mass secreted protein ESAT-6 has been shown to be capable of differentiating TB-infected cattle from those which develop responsiveness to PPD through contact with environmental mycobacteria or vaccination with BCG. The information which has accumulated in recent years has shown that the increased specificity is associated with some decrease in test sensitivity, but the overall advantages of being able to make precise diagnostic decisions will have significant advantages in many situations.

Animals↗
PubMed2001

Pathogenesis of tuberculosis in cattle.

There has been a renewed interest in the pathogenesis of bovine tuberculosis in many countries, in an attempt to understand better its transmission, to improve diagnosis and assess the potential of vaccination. This paper, which overviews current knowledge of aspects of the pathogenesis of bovine tuberculosis, draws from studies of field cases and experimental infections and highlights deficiencies in current understanding. The pathogenesis of bovine tuberculosis has not received the same level of attention as with human tuberculosis, and in many instances, the processes involved in bovine tuberculosis have been drawn from studies of human tuberculosis or from small animal models of infection. This paper however, considers the successful emulation of naturally acquired tuberculosis using experimental cattle models and identifies the complex and integrated nature of microbiological, immunological and pathological events involved. Current understanding of the initiation of infection, immune responses, and subsequent pathology, which can vary significantly in individual animals are discussed. Whilst there are aspects of M. bovis that still remain elusive to scientific investigation, further studies on the pathogenesis of bovine tuberculosis are advocated as necessary to provide a better scientific basis on which to review control and eradication strategies, which are currently less than effective in many regions.

Animals↗
PubMed2001

In vitro responsiveness of gammadelta T cells from Mycobacterium bovis-infected cattle to mycobacterial antigens: predominant involvement of WC1(+) cells.

It is generally accepted that protective immunity against tuberculosis is generated through the cell-mediated immune (CMI) system, and a greater understanding of such responses is required if better vaccines and diagnostic tests are to be developed. gammadelta T cells form a major proportion of the peripheral blood mononuclear cells (PBMC) in the ruminant system and, considering data from other species, may have a significant role in CMI responses in bovine tuberculosis. This study compared the in vitro responses of alphabeta and gammadelta T cells from Mycobacterium bovis-infected and uninfected cattle. The results showed that, following 24 h of culture of PBMC with M. bovis-derived antigens, the majority of gammadelta T cells from infected animals became highly activated (upregulation of interleukin-2R), while a lower proportion of the alphabeta T-cell population showed activation. Similar responses were evident to a lesser degree in uninfected animals. Study of the kinetics of this response showed that gammadelta T cells remained significantly activated for at least 7 days in culture, while activation of alphabeta T cells declined during that period. Subsequent analysis revealed that the majority of activated gammadelta T cells expressed WC1, a 215-kDa surface molecule which is not expressed on human or murine gammadelta T cells. Furthermore, in comparison with what was found for CD4(+) T cells, M. bovis antigen was found to induce strong cellular proliferation but relatively little gamma interferon release by purified WC1(+) gammadelta T cells. Overall, while the role of these cells in protective immunity remains unclear, their highly activated status in response to M. bovis suggests an important role in antimycobacterial immunity, and the ability of gammadelta T cells to influence other immune cell functions remains to be elucidated, particularly in relation to CMI-based diagnostic tests.

Animals↗
PubMed2000

Specific immune-based diagnosis of tuberculosis.

Current diagnostic tests for tuberculosis based on tuberculin have poor specificity, and both BCG vaccination and exposure to non-tuberculosis mycobacteria produce a response similar to that induced by infection with Mycobacterium tuberculosis. The identification of regions of the M. tuberculosis genome that are not present in BCG and non-tuberculous mycobacteria provides a unique opportunity to develop new highly specific diagnostic reagents. We describe the current status of attempts to exploit this information and summarise recent research that has used defined antigens for an accurate and rapid test for tuberculosis infection based on the detection of T cells sensitised to M. tuberculosis either by blood tests in vitro or skin tests in vivo.

Antigens, Bacterial↗
PubMed2000

IgG isotype antibody responses to epitopes of the Mycobacterium bovis protein MPB70 in immunised and in tuberculin skin test-reactor cattle.

Serological assays may have merit in identifying animals in advanced stages of bovine tuberculosis, but most tests have had sub-optimal sensitivities and specificities. The Mycobacterium bovis protein MPB70 has been identified as a B-cell target with diagnostic potential in measurement of pre- and post-skin-test antibody responses. One observation, which has potential practical application, has been that skin testing with tuberculin boosts IgG(1) anti-MPB70 antibody responses in cattle with tuberculous lesions. However, serological cross-reactivities with bacteria, such as Nocardia asteroides, have been described for this protein. With the aim of identifying candidate reagents for improved diagnostic tests, this study investigated IgG isotype antibody responses to MPB70 at the epitope level and, because of the previous findings, focused on IgG(1) responses following skin testing. Screening of a panel of overlapping synthetic peptides using sera from cattle immunised with MPB70 and cattle infected with M. bovis showed that two regions of the protein (residues 21-70 and 101-120) contain dominant B-cell epitopes. No individual epitope appeared to be selectively recognised by one isotype of IgG antibody. Investigation of IgG(1) responses showed that recognition of the epitope within residues 51-70 was boosted strongly by tuberculin injections in skin-test positive cattle and that this memory response was generally a feature of cattle which were found to have macroscopic, tuberculous lesions.

Animals↗
PubMed2000

Assessment of defined antigens for the diagnosis of bovine tuberculosis in skin test-reactor cattle.

The continued use of purified protein derivative (PPD) tuberculin is considered to be the main factor which limits the specificity of diagnostic tests for bovine tuberculosis (TB). This study evaluated a whole blood interferon-gamma (IFN-gamma) assay and compared the diagnostic potential of PPD with two tuberculosis-specific antigens, ESAT-6 and MPB70. To provide estimates of sensitivity and specificity, responses were measured in 180 skin test-reacting cattle, of which 131 were confirmed as tuberculous, and in 128 cattle from TB-free herds. For the skin test reactors, there was a positive correlation between the IFN-gamma responses to PPD from Mycobacterium bovis (PPDB) and PPD from Mycobacterium avium (PPDA), indicating cross-reactivity between these complex antigens which are the basis of the skin test. In comparisons of the ESAT-6 IFN-gamma test with a PPD IFN-gamma test (using PPDB compared with PPDA), there was a decrease in sensitivity (76.3 per cent vs 89.3 per cent), but a clear increase in specificity (99.2 per cent vs 92.2 per cent). The provision of high specificity, even with lower sensitivity, offers major benefits for testing in areas with a low incidence of TB.

Animals↗
PubMed2000

In vitro T-cell activation of monocyte-derived macrophages by soluble messengers or cell-to-cell contact in bovine tuberculosis.

The macrophage plays a dual role in tuberculosis, promoting not only protection against mycobacteria, but also survival of the pathogen. Macrophages inhibit multiplication of mycobacteria but also act in concert with lymphocytes through presentation of antigens to T cells. Studies in animal and human infections have suggested a correlation of in vitro growth rates of mycobacteria with in vivo virulence, using uracil uptake to assess mycobacterial metabolism. This study found that blood-derived, non-activated bovine macrophages were capable of controlling Mycobacterium bovis bacillus Calmette-Gurin growth for up to 96 hr, but were permissive to intracellular growth of virulent M. bovis. The present investigation compared the in vitro modulation of these macrophage activities by cytokine-rich T-cell supernatants or cell-to-cell contact. On the one hand, treatment of cultured monocytes with mitogen-produced T-cell supernatants promoted morphological changes suggestive of an activation status, enhanced the antigen presentation capabilities of monocytes and up-regulated major histocompatibility complex class II expression. However, this activation was not associated with enhanced anti-M. bovis activity. On the other hand, incubation of infected monocytes with T-cell populations resulted in proportionally increased inhibition of M. bovis uracil uptake. This inhibition was also seen using cells from uninfected animals and indicated the necessity for cell-to-cell contact to promote antimycobacterial capability.

Animals↗
PubMed2000

Cellular interactions in bovine tuberculosis: release of active mycobacteria from infected macrophages by antigen-stimulated T cells.

The outcome of Mycobacterium bovis infections depends on the interactions of infected macrophages with T lymphocytes. Several studies in humans and in mouse models have suggested an important role for cytotoxicity in the protective immune response to mycobacterial infections, and both CD4+ and CD8+ T cells have been shown to elicit appropriate cytolytic activity. The present study investigated in vitro interactions of T cells with M. bovis-infected macrophages in bovine tuberculosis. The results showed that following interaction with antigen-stimulated peripheral blood mononuclear cells (PBMC) from infected cattle, there was an increased presence of M. bovis in the extracellular compartment of infected macrophage cultures, as measured by incorporation of [3H]uracil into mycobacterial RNA. Furthermore, out of a panel of T-cell clones from infected cattle, it was found that a higher proportion of CD8+ clones produced an increase in the number of metabolically active extracellular M. bovis organisms compared with CD4+ clones. Finally, a positive correlation between percentage of antigen-dependent release of mycobacteria and total uracil uptake by M. bovis within culture systems was detected. This could be regarded as an indication of preferential intracellular control of mycobacteria by activated macrophages.

Animals↗