Duration of immunity against Mycobacterium bovis following neonatal vaccination with bacillus Calmette-Guérin Danish: significant protection against infection at 12, but not 24, months.

Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against bovine tuberculosis, caused by infection with virulent M. bovis. In two independent experiments, we assessed the duration of the protective immunity induced in calves by neonatal vaccination with BCG Danish. Protection from disease was assessed at 12 and 24 months postvaccination in cattle challenged via the endotracheal route with M. bovis. We also assessed antigen-specific immune responses to assess their utility as correlates of protection. At 12 months postvaccination, significant reductions in lung and lymph node pathologies were observed compared to nonvaccinated M. bovis-challenged control cattle. At 24 months post-BCG vaccination, there was a reduction in lung and lymph node pathology scores and in bacterial burden. However, when comparing vaccinated and control groups, this did not reach statistical significance. Vaccination induced long-lived antigen (purified protein derivative [PPD])-specific gamma interferon (IFN-γ) release in whole-blood cultures, which remained above baseline levels for more than 20 months (approximately 90 weeks). The number of antigen-specific IFN-γ-secreting central memory T cells present at the time of M. bovis challenge was significantly higher in vaccinated than in control animals at 12 months postvaccination, but not at 24 months. Vaccination of neonatal calves with BCG Danish induced protective immune responses against bovine TB which were maintained for at least 12 months postvaccination. These studies provide data on the immunity induced by BCG vaccination in calves; the results could inform vaccination strategies for the control of bovine TB in United Kingdom cattle herds.

Identification of surrogates and correlates of protection in protective immunity against Mycobacterium bovis infection induced in neonatal calves by vaccination with M. bovis BCG Pasteur and M. bovis BCG Danish.

Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against infection with virulent M. bovis, the causative agent of bovine tuberculosis (bTB). We compared two strains of BCG, Pasteur and Danish, in order to confirm that the current European human vaccine strain (BCG Danish) induced protective immunity in calves, and we assessed immune responses to determine correlates of protection that could assist future vaccine evaluation in cattle. Both vaccine strains induced antigen (purified protein derivate [PPD])-specific gamma interferon (IFN-γ) in whole-blood cultures. These responses were not significantly different for BCG Pasteur and BCG Danish and peaked at week 2 to 4 postvaccination. Vaccination with either BCG Danish or BCG Pasteur induced significant protection against bTB, with reductions in both lesion score and bacteriological burden evident in both groups of vaccinated calves compared with nonvaccinated control calves. Measurement of IFN-γ-expressing T lymphocytes postvaccination and postchallenge revealed both correlates and surrogates of protective efficacy. The frequency of central memory T lymphocytes present at 12 weeks postvaccination (at the time of M. bovis challenge) correlated significantly with protection. Conversely, the number of IFN-γ-expressing effector T cells present after M. bovis challenge was correlated with disease. These results demonstrate that vaccination of neonatal calves with either BCG Pasteur or BCG Danish induces protective immune responses against TB. In addition, we show that measurement of antigen-specific T lymphocyte populations may provide a reliable means for identifying protective vaccine candidates.

The effect of tuberculin testing on the development of cell-mediated immune responses during Mycobacterium bovis infection.

Protection against tuberculosis (TB) is associated with Th1-type cell-mediated immunity (CMI). Whilst the intradermal injection of partially purified derivatives of tuberculin (PPD) represents the classic test assessing the delayed type hypersensitivity (DTH) response used in both humans and cattle for diagnosing TB, it has been suggested that the test may modulate host CMI responses. To investigate the kinetics of the development of the DTH response and its subsequent effect on CMI responses, groups of 6-month old calves were inoculated intranasally with 8 x 10(4) cfu of Mycobacterium bovis, subjected to the comparative intradermal tuberculin test (TT) using bovine and avian PPD (PPD-B, PPD-A) at various time intervals post-infection, and immune responses compared. These included DTH, lymphocyte proliferation, IgG production, and synthesis of the cytokines: IFNgamma, IL-10, IL-4, IL-6, and IL-13. All animals were subjected to post-mortem examination. The kinetics of the development of the DTH response assessed in the TT was such that infected cattle could be identified as early as 3 weeks post-infection, which correlated with the detection of an antigen-specific IFNgamma response. Transient increases in plasma-derived IFNgamma as a result of TT during an established TB infection were more pronounced when blood was stimulated with PPD-A compared with PPD-B stimulation. This has the potential to mask diagnosis of infection as a result of the stronger avian-bias if the IFNgamma test is used the week following TT. Disease pathology was not affected by TT. A transient failure to a second TT was observed in 1 of 30 animals and the time (post-infection) at which the TT is administered may be of significance. In serum, IgG responses to PPD-B, which were undetectable prior to TT, were elevated after TT and were most pronounced in cattle that were TT at 6 weeks post-infection. Other cytokines were also affected by the TT; IL-4 mRNA levels increased and IL-6 mRNA levels decreased, whilst PPD-B specific IL-10 protein synthesis was enhanced. These observations may offer the potential for further diagnostic assays that could complement the TT and IFNgamma test.

Exposure to Mycobacterium avium induces low-level protection from Mycobacterium bovis infection but compromises diagnosis of disease in cattle.

We assessed the effect of exposure to Mycobacterium avium on the development of immune responses and the pathogenesis of disease observed following Mycobacterium bovis challenge. A degree of protection against M. bovis was observed in calves which were pre-exposed to M. avium as assessed by the extent of lesions and bacterial load compared to the M. bovis alone group. The immune response following M. bovis challenge in cattle previously inoculated with M. avium was biased towards antigens (PPD) present in M. avium, whereas the response following M. bovis alone was biased towards antigens present in M. bovis, indicating an imprinting of memory to avian antigens on T lymphocytes. A consequence of the memory to M. avium antigens was failure to diagnose M. bovis infection by the skin test or the IFN(gamma) assay in some of the animals which had lesions of tuberculosis at necropsy. The use of M. bovis specific antigens ESAT-6 and CFP-10 increased IFN(gamma) test specificity in animals previously exposed to M. avium but the responses to these antigens were lower than those observed in animals exposed to M. bovis alone. The implication is that responses to M. avium, although providing some immunity, may mask diagnosis of M. bovis infection, even when specific antigens are employed, potentially contributing to disease transmission in the field.

Vaccination of neonatal calves with Mycobacterium bovis BCG induces protection against intranasal challenge with virulent M. bovis.

Vaccination of neonates with Mycobacterium bovis bacillus Calmette-Guerin (BCG) may be a strategy that overcomes reduced vaccine efficacy associated with exposure to environmental mycobacteria in humans and cattle. Preliminary comparisons indicated that 2-week-old calves produced an immune response to vaccination at least as intense as that observed in adults. Subsequently, five gnotobiotic hysterotomy derived calves aged 1 day were inoculated with BCG and 3 months later were challenged intranasally with virulent M. bovis. The number of tissues with lesions and the pathological extent of these lesions was reduced significantly in vaccinates. Furthermore, lesions were evident in the lung or associated chest lymph nodes of four of five controls but none of five vaccinates. BCG vaccination reduced significantly the level of bacterial colonization. However, lesions in the head associated lymph nodes were observed in three of five BCG-vaccinated cattle. Levels of interferon gamma (IFN-gamma) detected by enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot (ELISPOT) in individual vaccinated animals at challenge did not correlate with subsequent resistance and in general immune responses post-challenge were lower in vaccinated calves. Low IL-10 responses were evident but IL-4 was not detected. Responses to ESAT-6 and/or CFP-10 were evident in four of four control calves that had lesions. Two of the BCG vaccinates with lesions did not produce a response to ESAT-6 and CFP-10, indicating that these antigens did not distinguish vaccinated immune animals from vaccinated animals with lesions. Overall, vaccination of neonatal calves with BCG induced significant protection against disease and has potential as a strategy for the reduction of the incidence of bovine tuberculosis.

Interaction of antigen presenting cells with mycobacteria.

The interaction of mycobacteria with antigen presenting cells is a key feature in the pathogenesis of tuberculosis and the outcome of this interaction is pivotal in determining whether immunity or disease ensues. Human and mouse macrophages and dendritic cells (DC) have been shown to become infected with mycobacteria and to produce a response to infection that reflects their suggested role in immunity. Thus, macrophages elicit anti-microbial mechanisms for elimination of mycobacteria and DC up-regulate expression of molecules that aid their stimulation of T lymphocytes. We have examined the effects of infection with the avirulent strain Mycobacterium bovis BCG and with virulent M. bovis on bovine antigen presenting cells. Differences in the intracellular survival of bacteria within DC and macrophages were observed with higher numbers of bacteria maintained within DC following infection compared to macrophages. BCG was killed more effectively than M. bovis. Alterations in the expression of cell surface molecules involved in antigen presentation and the stimulation of T cells, including MHC II and CD40, were observed following infection of bovine antigen presenting cells. In addition infected DC secreted IL-12, TNFalpha and IL-10 whereas macrophages produced TNFalpha, IL-10 and little IL-12. Generally responses were more marked when virulent M. bovis was used compared to BCG. These studies indicate that infection of bovine antigen presenting cells by mycobacterial species results in the induction of both innate and adaptive immune responses that are critical for the outcome of infection.

Development of an ELISA for bovine IL-10.

The objective of the study was to develop an assay for bovine IL-10 that could be applied to analyses of immune responses and advance understanding of a variety of diseases of cattle. Recombinant bovine IL-10 (rbo IL-10) was transiently expressed in Cos-7 cells and shown to inhibit the synthesis of IFN gamma by bovine cells stimulated with antigen in vitro. Mice were immunised with a plasmid containing a cDNA insert encoding rbo IL-10 and inoculated with rbo IL-10. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-10 in an ELISA. Some of these mAb neutralised the ability of rbo IL-10 to inhibit IFN gamma synthesis by antigen-stimulated bovine cells. A pair of mAb was identified that together could be used to detect both recombinant and natural bovine IL-10 present in supernatant of PBMC stimulated with ConA. A luminescent detection method was applied to the ELISA making it more sensitive. Using this method native IL-10 was detected in supernatants of PBMC, diluted blood and undiluted blood from cattle immunised with Mycobacterium bovis BCG or ovalbumin and incubated in vitro with antigen indicating the applicability of the assay to a number of in vitro culture systems.

The enzymes of lecithin biosynthesis in human neonatal lungs. IV. Phosphorylcholine cytidyltransferase.

Phosphorylcholine cytidyltransferase, the enzyme which catalyzes the transfer of phosphorylcholine to cytidine 5'-triphosphate to form CDP-choline, was studied for the first time in human neonatal lung. The assay of product synthesis was linear for 10-20 min and up to 12 mg protein. The pH optimum was 6-6.5. The Km of CTP was 2.0 times 10- minus 3 M, and the Km of phosphorylcholine was 0.25 times 10- minus 3 M. The true Vmax was 10 nmol CDP-choline/mg protein/10 min. The enzyme was stable under frozen conditions. Oxygen had no apparent affect on enzyme activity.