Understanding how pathogens or vaccine antigens are targeted to dendritic cell (DC) subsets is important for disease pathogenesis studies and vaccine design. We characterised the sub-populations of migrating bovine DC with functional and phenotypic diversity present in pseudoafferent lymph draining the skin. These skin draining DC exist as a series of maturation dependent subsets with differential capacities for antigen uptake and cytokine expression, and include both Langerhans’ cells (LC) and dermal derived cells. Furthermore, Mycobacterium bovis Bacille Calmette Guerin, a vaccine which is administered by the intradermal route, was only taken up by a small number of the migrating DC, which were SIRPα+ and expressed the mannose receptor and CD1b. This was evident following in vitro infection and also in vivo following inoculation of green fluorescent BCG over the lymphatic cannulation site. Only the SIRPα+ DC were able to present antigen to T cells isolated from BCG vaccinated calves. Furthermore, presentation of BCG antigens by DC to T lymphocytes was ineffective compared to mycobacterial proteins. However, mycobacterial antigen 85 was delivered more effectively to DC via an adenoviral vector and the magnitude of the subsequent antigen-specific T cell response was significantly increased. This study further extends our understanding of the biology of migrating DC, identifies potential explanations for the modest success of BCG vaccination and demonstrates that targeted delivery of antigens via adenoviruses to DC can improve antigen presentation.

Bovine tuberculosis (bTB) is increasing in incidence in the UK. Effective control strategies could involve vaccination; BCG, either alone or in prime-boost strategies, remains the most effective vaccine against bovine tuberculosis. However, BCG vaccination of cattle would require development of diagnostic tests able to accurately discriminate Mycobacterium bovis-infected from BCG-vaccinated animals. Herein, we demonstrate that the detection of secreted IFN-gamma following short term culture (4h) of whole blood with purified protein derived from M. bovis (PPD-B) allows such discrimination. This reflects, in part, the differential kinetics of IFN-gamma secretion in infected compared to vaccinated cattle. This is the first study to demonstrate that accurate, rapid distinction of BCG-vaccinated from M. bovis-infected cattle can be achieved in a short time period without the need for production of M. bovis-specific antigens, complex antigen mixtures or extensive laboratory procedures. We were also able to detect PPD-specific IFN-gamma release during short term culture of blood from a number of humans with active TB indicating that this test may have wider application and is potentially useful for the rapid diagnosis of disease in humans.

A shift in the balance of T(Helper) (T(H))1/T(H)2 cytokine production by maternal peripheral blood leukocytes is regarded as a common important feature of successful mammalian pregnancy. Although the phenomenon has been studied extensively in animals with invasive hemochorial placentae, the paradigm has not been studied in detail in species with less-invasive placentae, such as sheep that have a synepitheliochorial placenta. Sixteen sheep were immunised with the nominal antigen chicken egg albumin (Ova) and antigen-specific humoral and cellular responses were established in all sheep. The 16 sheep were synchronised, 11 were mated and successfully conceived, the remaining 5 served as non-pregnant controls. Peripheral blood mononuclear cells (PBMC) were isolated approximately every 2 weeks and restimulated in vitro with either Ova or the T cell mitogen concanavalin A (ConA), and cell proliferation and cytokine production measured. There were no detectable differences in antigen-driven PBMC proliferation, interferon-gamma (IFN-gamma), interleukin (IL)-4 or IL-10 production between pregnant and non-pregnant sheep. Also, there were no appreciable differences in ConA-induced IFN-gamma, IL-4 or IL-10 between the groups. These data suggest that a shift in T(H)1/T(H)2 cytokine production does not occur in pregnant sheep and indicate that further comparative reproductive immunology studies on species with non-invasive placentation will be informative of materno-fetal interactions and immune regulation during pregnancy.

Concurrent infection of cattle with bovine viral diarrhoea virus (BVDV) and Mycobacterium bovis is considered to be a possible risk factor for onward transmission of bovine tuberculosis (BTB) in infected cattle and is known to compromise diagnostic tests. A comparison is made here of M. bovis shedding (i.e. release) characteristics from 12 calves, six experimentally co-infected with BVDV and six infected with M. bovis alone, using simple models of bacterial replication. These statistical and mathematical models account for the intermittent or episodic nature of shedding, the dynamics of within-host bacterial proliferation and the sampling distribution from a given shedding episode. We show that while there are distinct differences among the shedding patterns of calves given the same infecting dose, there is no statistically significant difference between the two groups of calves. Such differences as there are, can be explained solely in terms of the shedding frequency, but with all calves potentially excreting the same amount of bacteria in a given shedding episode post-infection. The model can be thought of as a process of the bacteria becoming established in a number of discrete foci of colonization, rather than as a more generalized infection of the respiratory tract. In this case, the variability in the shedding patterns of the infected calves can be explained solely by differences in the number of foci established and shedding being from individual foci over time. Should maximum exposure on a particular occasion be a critical consideration for cattle-to-cattle transmission of BTB, cattle that shed only intermittently may still make an important contribution to the spread and persistence of the disease.

Abstract Tuberculosis (TB) remains one of the leading causes of death due to infectious disease. Current aims to improve vaccines against TB have focused on enhancing immunity conferred by the attenuated M. bovis BCG (BCG) vaccine. The design of a vaccine to specifically boost the protective features of BCG requires a greater understanding of the immune response induced by BCG vaccination. In this work we show that vaccination with BCG induces a population of CD8+CD45RO+ T cells that proliferate, produce IFN-gamma, up-regulate expression of perforin and granulysin, and lyse BCG-infected macrophages, following antigen specific stimulation. To determine whether a similar response could be detected in vivo, vaccinated animals were re-inoculated intradermally with BCG or PBS eight weeks after primary BCG vaccination. Draining lymph nodes were removed 3 days after the second inoculation for analysis by immunofluorescence and confocal microscopy for the presence of activated CD8+ T cells. Clusters of CD8+CD3+perforin+ cells were readily detectable in lymph nodes from animals re-inoculated with BCG but absent in the animals given PBS in the second inoculum. These results suggest that the memory CD8+ T cells induced by BCG vaccination have the capacity to mount secondary responses to mycobacteria in vivo. Support for these studies was provided by the BBSRC, DEFRA and the Jenner Institute.

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.

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB) and of a proportion of human TB. Protection against TB requires Th1 responses and worsening of disease is associated with Th2 responses. To help clarify the nature of the response to mycobacteria, the responses from M. bovis-BCG vaccinated cattle boosted with live mycobacteria (BCG), bacterial soluble antigens (PPD) or PBS were evaluated. The results indicated that macrophages may be the major cell population ingesting and presenting mycobacteria in BCG boosted animals, while B-cells seem able to ingest and present PPD to T-cells in PPD boosted animals.

Dendritic cells (DC) are important cells at the interface between innate and adaptive immunity. DC have a key role in antigen processing and presentation to T cells. Effector functions of DC related to innate immunity have not been explored extensively. We show that bovine monocyte-derived DC (mDC) express inducible nitric oxide synthase (iNOS) mRNA and protein and produce NO upon triggering with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes (HKLM). An immunocytochemical analysis revealed that a sizeable subset (20-60%) copiously expresses iNOS (iNOShi) upon IFN-gamma/HKLM triggering, whereas the other subset expressed low levels of iNOS (iNOSlo). Monocyte-derived macrophages (mMphi) are more homogeneous with regard to iNOS expression. The number of cells within the iNOSlo mDC subset is considerably larger than the number of dead cells or cells unresponsive to IFN-gamma/HKLM. The large majority of cells translocated p65 to the nucleus upon triggering by IFN-gamma/HKLM. A contamination of mDC with iNOS-expressing mMphi was excluded as follows. (i) Cell surface marker analysis suggested that mDC were relatively homogeneous, and no evidence for a contaminating subset expressing macrophage markers (e.g. high levels of CD14) was obtained. (ii) iNOS expression was stronger in iNOShi mDC than in mMphi. The use of maturation-promoting stimuli revealed only subtle phenotypic differences between immature and mature DC in cattle. Nevertheless, these stimuli promoted development of considerably fewer iNOShi mDC upon triggering with IFN-gamma/HKLM. Immunocytochemical results showed that although a significant proportion of cells expressed iNOS only or TNF only upon triggering with IFN-gamma/HKLM, a significant number of cells expressed both iNOS and TNF, suggesting that TNF and iNOS producing (TIP) DC are present within bovine mDC populations obtained in vitro.

Humoral and cellular immune responses to Fasciola gigantica experimental infection in buffaloes were studied. The results showed that 33.4 ± 9.1% of the infection dose was recovered as adult flukes from infected animals at necropsy. Significant differences of weight gain between infected and non-infected buffaloes was observed at 4 MPI (months post-infection). Anti F. gigantica excretory–secretory products (FgESP)-IgG levels increased significantly from 3 WPI (weeks post-infection) and displayed a peak at 13 WPI. Western blot indicated that in FgESP six major bands of 11.5, 19.0, 23.4, 29.8, 47.5 and 53.2 kDa were recognized by F. gigantica-infected buffaloes sera after 0 WPI. Eosinophil numbers increased significantly from 3 WPI in F. gigantica-infected buffaloes and displayed a peak at 8 WPI. Peripheral blood mononuclear cells (PBMC) proliferation induced by FgESP increased from 2 WPI with a peak at 5 WPI. IFNγ secretion by FgESP-stimulated PBMC appeared early from 1 WPI with three peaks at 2, 5 and 8 WPI, respectively. IL-10 production was observed from 2 WPI with two peaks at 4 and 9 WPI, respectively. Our results suggested that buffaloes were highly susceptible to F. gigantica infection, and this susceptibility could be associated with the late and weak cellular immune response in the early phase of infection and the Th0-like response throughout the infection.

SummaryCytokine expression in lymph nodes from cattle inoculated intranasally with Mycobacterium bovis was compared to that of non-infected animals using real-time polymerase chain reaction. The effect of M. bovis infection, 4 months post-challenge, was to suppress the expression of anti-inflammatory cytokines interleukin (IL)-4 and IL-10 as well as the pro-inflammatory cytokines tumour necrosis factor (TNF) and IL-6. Expression of interferon (IFN)-γ and IL-12 was maintained. Animals vaccinated with bacille Calmette–Guérin responded differently to challenge with M. bovis. In particular, no decrease in expression of IL-4 or IL-6 was observed following challenge of vaccinated animals and decreased IFN-γ was detected. Also, vaccinated animals had higher levels of IL-4 and IL-10 transcripts compared to unvaccinated animals following challenge. These changes in cytokine expression levels led to a significant shift in the IFN-γ/IL-4 or IFN-γ/IL-10 ratio within the lymph node following challenge. Challenged animals generally showed a strong Th1 bias that was not seen in animals vaccinated prior to challenge. An inverse correlation between the level of pathology and bacterial load within the lymph node and the expression of IL-4, IL-10 and TNF was also observed. These results suggest that in the lymph nodes of cattle with established tuberculosis and a persisting bacterial infection, maintenance of the pro-inflammatory response in combination with a suppressed anti-inflammatory response may control the infection but contribute to host-induced tissue damage. Vaccination, which reduces the bacterial load and consequently the IFN-γ response, may result in less suppression of anti-inflammatory cytokines.

ABSTRACTMycobacterium bovisis the causative agent of bovine tuberculosis, a disease that is increasing in incidence in United Kingdom cattle herds. In addition to increasing economic losses, the rise in bovine tuberculosis poses a human health risk. There is an urgent requirement for effective strategies for disease eradication; this will likely involve vaccination in conjunction with current test and slaughter policies. A policy involving vaccination would require an accurate diagnosis ofM. bovis-infected animals and the potential to distinguish these animals from vaccinates. Currently used diagnostic tests, the skin test and gamma interferon (IFN-γ) blood test, have a sensitivity of up to 95%. A further complication is thatM. bovisBCG-vaccinated animals are also scored positive by these tests. We tested the hypothesis that the quantification of IFN-γ-producing lymphocytes by flow cytometric analysis of intracellular IFN-γ expression would provide a more accurate discrimination ofM. bovis-infected animals from BCG vaccinates. Significant numbers of IFN-γ-expressing CD4+T cells were detected following culture of heparinized blood fromM. bovis-infected animals, but not from BCG vaccinates, with purified protein derived fromM. bovis(PPD-B) or live mycobacteria. Only 1 of 17 BCG-vaccinated animals had a significant number of CD4+T lymphocytes expressing IFN-γ, compared with 21/22M. bovis-infected animals. This assay could allow an accurate diagnosis ofM. bovisand allow the discrimination of BCG-vaccinated cattle from infected cattle.

In vitro infection of bovine cells of many origins with the cytopathogenic bovine viral diarrhea virus (cpBVDV) results in the induction of alpha/beta interferon (IFN-alpha/beta), whereas noncytopathogenic BVDV (ncpBVDV) isolates have been shown not to induce IFN-alpha/beta in vitro. Similarly, cpBVDV induces IFN-alpha/beta in the early bovine fetus, but ncpBVDV does not. However, acute infection of naive cattle with ncpBVDV results in IFN-alpha/beta production. In this study, we identified and characterized a minor population of cells, present in lymph nodes that produce IFN-alpha in response to ncpBVDV. These cells expressed the myeloid markers CD14, CD11b, and CD172a but did not express CD4 and CD45RB. We also established that these cells produced IFN-alpha in the absence of detectable productive infection.

In the present study we compared the ELISPOT and antibody in lymphocyte supernatants (ALS) assays as surrogate measures of mucosal immunity. In separate studies, 20 inpatient volunteers received oral doses of 6 x 10(8) or 4 x 10(9)cfu of ETEC strain E24377A (LT+, ST+, CS1+, CS3+) and 20 subjects received 1 (n = 9) or 2 (n = 11) oral doses of the attenuated ETEC vaccine, PTL-003 expressing CFA/II (CS1+ and CS3+) (2 x 10(9)cfu/dose). Peripheral blood mononuclear cells (PBMCs) from all subjects were assayed for anti-colonization factor or toxin-specific IgA antibody responses using the ALS and ELISPOT procedures. ALS responses were measured using a standard ELISA, as well as by time-resolved fluorescence (TRF). Following challenge with E24377A, significant anti-CS3, CS1 and LT ALS responses were detected in the lymphocyte supernatants of 75-95% of the subjects. A similar proportion (75%) of subjects mounted an ALS response to CFA/II antigen after vaccination with the PTL-003 vaccine. Inter-assay comparisons between ALS and ELISPOT methods also revealed a high degree of correlation in both immunization groups. ALS sensitivity versus the ELISPOT assay for LT, CS3 and CS1-specific responses following challenge were 95%, 94% and 78%, respectively and 83% for the ALS response to CFA/II antigen after vaccination with PTL-003. Correlation coefficients for the LT and CS3 antigens were 0.94 (p<0.001) and 0.82 (p<0.001), respectively after challenge and 0.78 (p<0.001) after vaccination. The association between ALS and ELISPOT for the CS1 antigen was however, significant only when ALS supernatants were tested by TRF (r = 0.91, p<0.001). These results demonstrate the value and flexibility of the ALS assay as an alternative to ELISPOT for the measurement of mucosal immune responses to ETEC antigens, particularly when the complexities of ELISPOT may make it impractical to perform.

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.

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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.