Bovis Purified Protein Derivative Research Articles

Vaccination of white-tailed deer (Odocoileus virginianus) with Mycobacterium bovis bacille Calmette-Guérin (BCG) results in positive tuberculin skin test results in a dose-dependent fashion

Mycobacterium bovis is the cause of tuberculosis in most mammalian species, most notably cattle and other members of the family Bovidae; however, many species of the family Cervidae are also susceptible. In North America, tuberculosis has been identified in both captive and free-ranging cervids. Captive cervids are tested for tuberculosis following many of the same guidelines applied to cattle, including intradermal tuberculin testing using M. bovis purified protein derivative (PPD). Both captive and free-ranging deer and elk have been implicated as the source of infection for many cattle herds. Vaccination with the human vaccine M. bovis BCG has been considered as one possible tool to aid in eradication of tuberculosis from cattle and both captive and free-ranging cervids. Studies in cattle have demonstrated that BCG vaccination can induce false positive intradermal tuberculin test reactions in some cattle. Similar findings have been reported for red deer. We orally vaccinated white-tailed deer with BCG and showed that vaccination can induce false positive skin test reactions in some deer and that the rate of false positive reactions is greater with a higher vaccine dose.

Biomarkers of cell-mediated immunity to bovine tuberculosis

Whole blood based assays, particularly interferon gamma (IFN-γ) release assays (IGRAs), are used for the diagnosis of both bovine and human tuberculosis (TB). The aim of the current study was to evaluate a panel of cytokines and chemokines for potential use as diagnostic readouts indicative of Mycobacterium bovis (M. bovis) infection in cattle. A gene expression assay was used to determine the kinetics of the response to M. bovis purified protein derivative and a fusion protein consisting of ESAT-6, CFP10, and Rv3615c upon aerosol infection with ∼104 cfu of M. bovis. The panel of biomarkers included: IFN-γ, CXCL9, CXCL10, CCL2, CCL3, TNF-α, IL-1α, IL-1β, IL-1Ra, IL-22, IL-21 and IL-13. Protein levels of IFN-γ, CXCL9, and CXCL10 were determined by ELISA. Findings suggest that CXCL9, CXCL10, IL-21, IL-13, and several acute phase cytokines may be worth pursuing as diagnostic biomarkers of M. bovis infection in cattle.

Seroprevalence ofMycobacterium bovisinfection in warthogs (Phacochoerus africanus) in bovine tuberculosis-endemic regions of South Africa

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M.bovis), has been reported in many species including suids. Wild boar are important maintenance hosts of the infection with other suids, that is domestic and feral pigs, being important spillover hosts in the Eurasian ecosystem and in South Africa, warthogs (Phacochoerus africanus) may play a similar role in M.bovis-endemic areas. However, novel diagnostic tests for warthogs are required to investigate the epidemiology of bTB in this species. Recent studies have demonstrated that serological assays are capable of discriminating between M.bovis-infected and uninfected warthogs (Roos etal., ). In this study, an indirect ELISA utilizing M. bovis purified protein derivative (PPD) as a test antigen was used to measure the prevalence and investigate risk factors associated with infection in warthogs from uMhkuze Nature Reserve and the southern region of the Greater Kruger National Park (GKNP). There was a high overall seroprevalence of 38%, with adult warthogs having a higher risk of infection (46%). Seroprevalence also varied by geographic location with warthogs from Marloth Park in the GKNP having the greatest percentage of positive animals (63%). This study indicates that warthogs in M.bovis-endemic areas are at high risk of becoming infected with mycobacteria. Warthogs might present an under-recognized disease threat in multi-species systems. They might also serve as convenient sentinels for M.bovis in endemic areas. These findings highlight the importance of epidemiological studies in wildlife to understand the role each species plays in disease ecology.

Divergent macrophage responses to Mycobacterium bovis among naturally exposed uninfected and infected cattle.

Mycobacterium bovis, the causative agent of bovine tuberculosis (TB), is a successful pathogen that remains an important global threat to livestock. Cattle naturally exposed to M. bovis normally become reactive to the M. bovis-purified protein derivative (tuberculin) skin test; however, some individuals remain negative, suggesting that they may be resistant to infection. To better understand host innate resistance to infection, 26 cattle from herds with a long history of high TB prevalence were included in this study. We investigated the bactericidal activity, the production of reactive oxygen and nitrogen species and the TB-related gene expression profile after in vitro M. bovis challenge of monocyte-derived macrophages from cattle with TB (n=17) and from non-infected, exposed cattle (in-contacts, n=9). The disease status was established based on the tuberculin skin test and blood interferon-gamma test responses, the presence of visible lesions at inspection on abattoirs and the histopathology and culture of M. bovis. Although macrophages from TB-infected cattle enabled M. bovis replication, macrophages from healthy, exposed cattle had twofold lower bacterial loads, overproduced nitric oxide and had lower interleukin (IL)-10 gene expression (P⩽0.05). Higher mRNA expression levels of inducible nitric oxide synthase, C-C motif chemokine ligand 2 and IL-12 were observed in macrophages from all in-contact cattle than in macrophages from their TB-infected counterparts, which expressed more tumour necrosis factor-α; however, the differences were not statistically significant owing to individual variation. These results confirm that macrophage bactericidal responses have a crucial role in innate resistance to M. bovis infection in cattle.

Antigen-Specific IP-10 Release Is a Sensitive Biomarker of Mycobacterium bovis Infection in Cattle

The most widely used ante-mortem diagnostic tests for tuberculosis in cattle are the tuberculin skin test and the interferon-gamma (IFN-γ) release assay, both of which measure cell-mediated immune responses to Mycobacterium bovis infection. However, limitations in the performance of these tests results in a failure to identify all infected animals. In attempting to increase the range of diagnostic tests for tuberculosis, measurement of the cytokine IP-10 in antigen-stimulated blood has previously been shown to improve the detection of M. tuberculosis and M. bovis infection, in humans and African buffaloes (Syncerus caffer), respectively. In the present study, 60 cattle were identified by the single intradermal comparative tuberculin test as tuberculosis reactors (n = 24) or non-reactors (n = 36) and the release of IFN-γ and IP-10 in antigen-stimulated whole blood from these animals was measured using bovine specific ELISAs. There was a strong correlation between IP-10 and IFN-γ production in these samples. Moreover, measurement of the differential release of IP-10 in response to stimulation with M. bovis purified protein derivative (PPD) and M. avium PPD distinguished between reactor and non-reactor cattle with a sensitivity of 100% (95% CI, 86%–100%) and a specificity of 97% (95% CI, 85%–100%). These results suggest that IP-10 might prove valuable as a diagnostic biomarker of M. bovis infection in cattle.

Characterization of effector and memory T cell subsets in the immune response to bovine tuberculosis in cattle.

Cultured IFN-γ ELISPOT assays are primarily a measure of central memory T cell (Tcm) responses with humans; however, this important subset of lymphocytes is poorly characterized in cattle. Vaccine-elicited cultured IFN-γ ELISPOT responses correlate with protection against bovine tuberculosis in cattle. However, whether this assay measures cattle Tcm responses or not is uncertain. The objective of the present study was to characterize the relative contribution of Tcm (CCR7+, CD62Lhi, CD45RO+), T effector memory (Tem, defined as: CCR7-, CD62Llow/int, CD45RO+), and T effector cells (CCR7-, CD62L-/low, CD45RO-), in the immune response to Mycobacterium bovis. Peripheral blood mononuclear cells (PBMC) from infected cattle were stimulated with a cocktail of M. bovis purified protein derivative, rTb10.4 and rAg85A for 13 days with periodic addition of fresh media and rIL-2. On day 13, cultured PBMC were re-stimulated with medium alone, rESAT-6:CFP10 or PPDb with fresh autologous adherent cells for antigen presentation. Cultured cells (13 days) or fresh PBMCs (ex vivo response) from the same calves were analyzed for IFN-γ production, proliferation, and CD4, CD45RO, CD62L, CD44, and CCR7 expression via flow cytometry after overnight stimulation. In response to mycobacterial antigens, ~75% of CD4+ IFN-γ+ cells in long-term cultures expressed a Tcm phenotype while less than 10% of the ex vivo response consisted of Tcm cells. Upon re-exposure to antigen, long-term cultured cells were highly proliferative, a distinctive characteristic of Tcm, and the predominant phenotype within the long-term cultures switched from Tcm to Tem. These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem). The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection.

Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle.

Several serological tests designed to detect antibodies to immunodominant Mycobacterium bovis antigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when used after the injection of purified protein derivative (PPD) for skin testing, which significantly boosts M. bovis-specific antibody responses. The present findings demonstrate the onset and duration of boosted antibody responses after the injection of M. bovis PPD for the caudal fold test (CFT) and Mycobacterium avium and M. bovis PPDs for the comparative cervical test (CCT), administered in series in cattle experimentally infected with M. bovis. While skin tests boosted the responses to certain antigens (i.e., MPB83 and MPB70), they did not affect the responses to other antigens (e.g., ESAT-6, CFP10, MPB59, and MPB64). Administration of the CCT 105 days after the CFT resulted in an even greater secondary boost in antibody responses to MPB83 and MPB70 and to a proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis. Both IgM and IgG contributed to the initial boost in the MPB83/MPB70-specific antibody response after the CFT. The secondary boost after the CCT was primarily due to increased IgG levels. Also, the avidity of antibodies to MPB83 and MPB70 increased after the CCT in M. bovis-infected cattle. The avidity of antibodies to the WCS-PK antigens increased in the interval between the CFT and the CCT but did not increase further after the CCT. Together, these findings demonstrate that the administration of PPDs for skin tests results in additive enhancement (i.e., when the CFT and CCT are performed in series), both qualitative and quantitative, of MPB83/MPB70-specific antibody responses.

Bovine central memory T cells are highly proliferative in response to bovine tuberculosis infection. (VET2P.1033)

Abstract Long-term (i.e., 14 days) cultured IFN-γ ELISPOT assays measure central memory T cell (Tcm) responses in both humans and cattle. With bovine tuberculosis, vaccine-elicited long-term IFN-γ ELISPOT responses correlate with protection. In other species, Tcm’s posses low activation threshold and are highly proliferative. It has been shown that Tcm’s accounts for 75% of long-term cultured cells. The objective of this study was to access the proliferative capability of Tcm cells compared to 6 day cultured cells (n = 6) in response to aerosol Mycobacterium bovis infection. Long-term cultured PBMCs from infected cattle were stimulated with M. bovis purified protein derivative (PPDb), rESAT-6:CFP10 (E:C), rTB10.4 and rAg85A for 13 days. Next, cells were stained with CellTrace dye and re-stimulated with E:C in the presence of fresh autologous adherent cells for an additional six days (20 days). In response to E:C, long-term cultured were mainly CD4+ cells and were more proliferative (~ 48%) than 6d cultured cells (27%; p=0.001). Long-term cultured cells were: ~30% Tcm (CCR7+, CD45RO+), ~48% Tem (CCR7-, CD45RO+), ~8% effector (CCR7-, CD45RO-) and <1% of naïve cells (CCR7+, CD45RO-). Six day cultured cells were: ~16% Tcm (p=0.01), ~47% Tem, ~42% effector (p=0.001) and < 1% naïve cells. These findings demonstrate that bovine Tcm cells present high proliferative capability as compared to short-term cultured cells.

Differences in immune cell function between tuberculosis positive and negative Asian elephants

Tuberculosis is an important health concern for Asian elephant (Elephas maximus) populations worldwide, however, mechanisms underlying susceptibility to Mycobacterium tuberculosis are unknown. Proliferative responses assessed via brominated uridine incorporation and cytokine expression measured by real-time RT-PCR were evaluated in peripheral blood mononuclear cell (PBMC) cultures from 8 tuberculosis negative and 8 positive Asian elephants. Cultures were stimulated with Mycobacterium bovis purified protein derivative (PPD-B), M.tuberculosis culture filtrate protein (CFP)-10, and Mycobacterium avium PPD (PPD-A). Following stimulation with PPD-B, proliferation was higher (α=0.005) in positive samples; no significant differences were detected following CFP-10 or PPD-A stimulation. Tumor necrosis factor (TNF)-α, interleukin (IL)-12, and interferon (IFN)-γ expression was greater in samples from positive elephants following stimulation with PPD-B (α=0.025) and CFP-10 (α=0.025 TNF-α and IL-12; α=0.005 IFN-γ). Stimulation with PPD-A also produced enhanced IL-12 expression in positive samples (α=0.025). Findings suggested that differences in immune cell function exist between tuberculosis positive and negative elephants. Proliferative responses and expression of TNF-α, IL-12, and IFN-γ in response to stimulation with PPD-B and CFP-10 differ between tuberculosis positive and negative elephants, suggesting these parameters may be important to tuberculosis immunopathogenesis in this species.

MycobacteriumBovisOrnithine Carbamoyltransferase, MB1684, Induces Proinflammatory Cytokine Gene Expression by Activating NF-κB in Macrophages

Mycobacterium bovis is the etiological factor of bovine tuberculosis (BTB), posing a significant problem to domestic cattle. The bacterium is also zoonotic, affecting human health worldwide. Macrophage evasion of the bacterium involves mycobacterial molecules such as MB1684 (ornithine carbamoyltransferase). In this study, we confirmed a concentration-dependent decrease in proliferation of Ana-1 macrophages when treated with rMB1684 when compared with mycobacterium bovis purified protein derivative of tuberculosis (MbPPD) or phosphate buffer solution incubation groups. We examined the activation of nuclear factor-kappa B (NF-κB) upon exposure to MB1684, and its role in MB1684-induced upregulation of interferon (IFN)-γ and proinflammatory cytokines (interleukin [IL]-1β, IL-6, and tumor necrosis factor-α) in Ana-1 macrophages. The levels of proinflammatory cytokines and IFN-γ were significantly high in MB1684-treated Ana-1 macrophages. The treatment led to an increase in NF-κB activation and a high expression of the just mentioned proinflammatory cytokines. NF-κB inhibition significantly abrogated MB1684-induced upregulation of proinflammatory cytokine mRNA expression, which suggests that MB1684-induced activation of NF-κB in turn stimulates gene expression of IFN-γ and proinflammatory cytokines in Ana-1 macrophages. The experiment was repeated in bone marrow macrophages, a more in-vivo-like model system, and similar results validated our conclusion. Further, we identified the possibility of the application of MB1684 antigen for the detection of BTB in cattle serum.

The role of gamma delta T cells in immunity to Mycobacterium bovis infection in cattle

Accumulating evidence suggests that γδ T cells play a critical role in the early response to Mycobacterium bovis and may be key in bridging innate and adaptive immunity following infection. In vitro, γδ T cells proliferate and produce robust amounts of IFNγ in response to complex, protein and non-protein mycobacterial antigens including M. bovis purified protein derivative (PPD), heat shock proteins and cell wall components such as mycolylarabinogalactan peptidoglycan (mAGP). Vaccination with Bacille Calumette-Guerin (BCG), as well as infection with virulent M. bovis, induces an increase in the frequency and activation of WC1+ γδ T cells circulating in the blood. Gamma delta T cells are rapidly recruited to the lungs and draining lymph nodes following BCG vaccination, and accumulate in developing lesions early following M. bovis infection. In Severe Combined Immuno-deficient (SCID)-bo mice, depletion of γδ T cells prior to M. bovis infection results in impaired granuloma formation, suggesting a role for γδ T cells in immune cell recruitment and lesion development. In vivo depletion of WC1+ γδ T cells from calves prior to M. bovis infection results in significantly reduced levels of M. bovis specific IgG2 and IFNγ, and increased IL-4 production compared to non-depleted control animals, suggesting that γδ T cells may also play a role in shaping the character of the adaptive M. bovis specific immune response. Whereas it is clear that γδ T cells are responding during M. bovis infection, much remains to be understood about their function in vivo and their ability to shape the innate and adaptive immune responses. This review focuses on recent advances in our understanding of γδ T cell biology with a particular emphasis on the immune response of γδ T cells in cattle during M. bovis infection.

Proteomic analysis of purified protein derivative of Mycobacterium tuberculosis

BackgroundPurified protein derivative (PPD) has been used for more than half a century as an antigen for the diagnosis of tuberculosis infection based on delayed type hypersensitivity. Although designated as “purified,” in reality, the composition of PPD is highly complex and remains ill-defined. In this report, high resolution mass spectrometry was applied to understand the complexity of its constituent components. A comparative proteomic analysis of various PPD preparations and their functional characterization is likely to help in short-listing the relevant antigens required to prepare a less complex and more potent reagent for diagnostic purposes.ResultsProteomic analysis of Connaught Tuberculin 68 (PPD-CT68), a tuberculin preparation generated from M. tuberculosis, was carried out in this study. PPD-CT68 is the protein component of a commercially available tuberculin preparation, Tubersol, which is used for tuberculin skin testing. Using a high resolution LTQ-Orbitrap Velos mass spectrometer, we identified 265 different proteins. The identified proteins were compared with those identified from PPD M. bovis, PPD M. avium and PPD-S2 from previous mass spectrometry-based studies. In all, 142 proteins were found to be shared between PPD-CT68 and PPD-S2 preparations. Out of the 354 proteins from M. tuberculosis–derived PPDs (i.e. proteins in either PPD-CT68 or PPD-S2), 37 proteins were found to be shared with M. avium PPD and 80 were shared with M. bovis PPD. Alignment of PPD-CT68 proteins with proteins encoded by 24 lung infecting bacteria revealed a number of similar proteins (206 bacterial proteins shared epitopes with 47 PPD-CT68 proteins), which could potentially be involved in causing cross-reactivity. The data have been deposited to the ProteomeXchange with identifier PXD000377.ConclusionsProteomic and bioinformatics analysis of different PPD preparations revealed commonly and differentially represented proteins. This information could help in delineating the relevant antigens represented in various PPDs, which could further lead to development of a lesser complex and better defined skin test antigen with a higher specificity and sensitivity.

Evaluation of Gamma Interferon (IFN-γ)-Induced Protein 10 Responses for Detection of Cattle Infected with Mycobacterium bovis: Comparisons to IFN-γ Responses

Gamma interferon (IFN-γ)-induced protein 10 (IP-10) has recently shown promise as a diagnostic biomarker of Mycobacterium tuberculosis infection of humans. The aim of the current study was to compare IP-10 and IFN-γ responses upon Mycobacterium bovis infection in cattle by using archived samples from two aerosol inoculation studies. In the first study (10(4) CFU M. bovis by aerosol, n = 7), M. bovis purified protein derivative (PPDb)-specific IP-10 and IFN-γ gene expression was detected as early as 29 days after challenge. PPDb-specific IP-10 and IFN-γ mRNA responses followed a similar pattern of expression over the course of this study and were highly correlated (r = 0.87). In the second study (10(5) CFU M. bovis by aerosol, n = 5), IP-10 and IFN-γ (protein) responses to mycobacterial antigens were compared following challenge. IFN-γ responses to mycobacterial antigens were detected at 29 days after challenge and were sustained during the remainder of the study. IFN-γ responses to mycobacterial antigens exceeded corresponding responses in nonstimulated cultures. IP-10 responses to mycobacterial antigens exceeded preinfection responses at 7, 29, and 63 days after challenge. In contrast to IFN-γ responses, IP-10 responses to mycobacterial antigens generally did not exceed the respective responses in nonstimulated cultures. IP-10 responses to medium alone and to mycobacterial antigens followed a similar pattern of response. Correlations between IP-10 and IFN-γ (protein) responses were modest (r ≈ 0.50 to 0.65). Taken together, these findings do not support the use of IP-10 protein as a biomarker for bovine tuberculosis using the current testing protocol and reagents; however, mRNA-based assays may be considered for further analysis.

Mycobacterium bovis BCG and purified protein derivative-induced reduction in the CD80 expression and the antigen up-take function of dendritic cells from patients with cervical cancer

ObjectiveClinical diagnostic studies have reported that co-existence of malignancy and tuberculosis in regions with high incidence of tuberculosis may be possible. However, simultaneous occurrence of cervical cancer (CaCx) and tuberculosis has been reported for it rarity. Our analysis of prevalence of mycobacterial infection among the study group of patients with CaCx had revealed co-infection of mycobacterium species including Mycobacterium tuberculosis. Since M. tuberculosis potentially affects the dendritic cells-mediated protective immune response, the main aim of this study was to analyse the effect of the mycobacterial infection on the immunological functions of dendritic cells (DC) in patients with CaCx. Study designInitially, the prevalence of mycobacterial infection was analysed in the blood samples of patients with CaCx using ELISA and PCR techniques. Later to determine the role of mycobacterial infection on immunosuppression of DC in the progression of CaCx, dendritic cell-mediated immune suppression induced by Mycobacterium bovis BCG (M. bovis BCG) and purified protein derivative (PPD) was studied in patients with CaCx. Expression of co-stimulatory molecules CD80 and CD86, and the antigen processing function of the macrophage-derived immature dendritic cells (imDC) were analysed. ResultsWhen compared to untreated imDC, the PPD and M. bovis BCG-treated imDC displayed 15% and 46.7% reduced expression of CD80, respectively. However, the M. bovis BCG and PPD did not have any significant reduction in the CD86 expression. Moreover, the dextran up-take was reduced by 24.4% and 35.8% with PPD and M. bovis BCG treatment, respectively. In addition, the M. bovis BCG-treated imDC derived from patients with CaCx showed a marginal reduction of lymphocyte proliferation and the PPD-treated imDC did not show a significant stimulation of lymphocyte proliferation. ConclusionThese results indicate that the PPD and M. bovis BCG inhibit the maturation and antigen up-take property of macrophage-derived imDC in patients with cervical cancer, suggesting that the M. tuberculosis infection may favour the progression of cervical cancer through immunosuppressed imDC.

Serologic Tests for Detecting Antibodies against Mycobacterium Bovis and Mycobacterium Avium Subspecies Paratuberculosis in Eurasian Wild Boar (Sus Scrofa Scrofa)

New tools to detect exposure of free-range Eurasian wild boar (Sus scrofa scrofa) to pathogenic mycobacteria would be valuable for improved disease surveillance and wildlife management. Two hundred sera from wild boar of known Mycobacterium bovis infection status were used to evaluate test suitability for the detection of antibodies against M. bovis and Mycobacterium avium subsp. paratuberculosis (or cross-reacting members of the M. avium complex). Two traditional enzyme-linked immunosorbent assays were evaluated using M. bovis purified protein derivative (bPPD) and paratuberculosis protoplasmatic antigen 3 (PPA3) as antigens, respectively, and a new point-of-care test format for bovine tuberculosis (bTB) that uses the innovative dual-path platform (DPP TB) test. The effect of individual factors (sex, age, lesions) on the diagnostic performance of the serologic tests was also determined. Although the DPP had a sensitivity of 89.6% and a specificity of 90.4%, for bPPD, the sensitivity was 79.2% and the specificity 100%. Both tests had a kappa agreement of 0.80. Sixty-five of 68 (95.6%) wild boar sera with antibodies against the PPA3 antigen corresponded to known M. bovis-infected wild boar. Significant differences were not observed in the bPPD and DPP readings among lesion categories or between age classes. A slight sex-related difference in sensitivity toward males in the DPP was found, but it was not detected in the bPPD enzyme-linked immunosorbent assay. The results support the use of antibody-based diagnostic tests for both large-scale and individual bTB testing of Eurasian wild boar and suggest that wild boar cannot be used as sentinels for infections caused by M. avium complex members.

Genetic correlations between measures of Mycobacterium bovis infection and economically important traits in Irish Holstein-Friesian dairy cows

Mycobacterium bovis is the primary agent of tuberculosis (TB) in cattle. The failure of Ireland and some other countries to reach TB-free status indicates a need to investigate complementary control strategies. One such approach would be genetic selection for increased resistance to TB. Previous research has shown that considerable genetic variation exists for susceptibility to the measures of M. bovis infection, confirmed M. bovis infection, and M. bovis-purified protein derivative (PPD) responsiveness. The objective of this study was to estimate the genetic and phenotypic correlations between economically important traits and these measures of M. bovis infection. A total of 20,148 and 17,178 cows with confirmed M. bovis infection and M. bovis-PPD responsiveness records, respectively, were available for inclusion in the analysis. First- to third-parity milk, fat, and protein yields, somatic cell count, calving interval, and survival, as well as first-parity body condition score records, were available on cows that calved between 1985 and 2007. Bivariate linear-linear and threshold-linear sire mixed models were used to estimate (co)variance components. The genetic correlations between economically important traits and the measures of M. bovis infection estimated from the linear-linear and threshold-linear sire models were similar. The genetic correlations between susceptibility to confirmed M. bovis infection and economically important traits investigated in this study were all close to zero. Mycobacterium bovis-PPD responsiveness was positively genetically correlated with fat production (0.39) and body condition score (0.36), and negatively correlated with somatic cell score (−0.34) and survival (−0.62). Hence, selection for increased survival may indirectly reduce susceptibility to M. bovis infection, whereas selection for reduced somatic cell count and increased fat production and body condition score may increase susceptibility to M. bovis infection.

Antibody response to a sterile filtered PPD tuberculin in M. bovis infected and M. bovis sensitized cattle

BackgroundBovine tuberculosis, caused by Mycobacterium bovis, afflicts approximately 50 million cattle worldwide and is detected by the tuberculin skin test (TST). While it has long been recognized that purified protein derivative (PPD) tuberculin is composed of a mixture of M. bovis derived protein components, little is known about the quality, relative quantity and identity of the proteins that make up PPD tuberculin. We manufactured a sterile filtered PPD tuberculin (SF-PPD) from a nine-week-old M. bovis culture supernatant in order to characterise the culture filtrate proteins (CFP) which make up M. bovis PPD tuberculin and to compare the antibody response of M. bovis infected versus M. bovis sensitized cattle.ResultsSF-PPD resolved into approximately 200 discrete spots using two-dimensional polyacrylamide gel electrophoresis (2-DE) while fewer than 65 spots could be discerned from 2-DE gels of tuberculin derived from autoclaved culture supernatant. Two dimensional Western blot analyses indicated that sera from M. bovis sensitized cattle recognized additional SF-PPD antigens as compared to M. bovis infected cattle at seven weeks post infection/sensitization. However, application of a comparative tuberculin skin test resulted in an antibody boosting response to the same set of M. bovis CFPs in both the M. bovis infected and M. bovis sensitized cattle.ConclusionsWe concluded that it is the heat sterilization of the M. bovis CFPs that causes severe structural changes to the M. bovis proteins. This work suggests that M. bovis infected cattle and cattle artificially sensitized to M. bovis with an injection of heat killed cells exhibit similar antibody responses to M. bovis antigens.

Genetics of tuberculosis in Irish Holstein-Friesian dairy herds

Information is lacking on genetic parameters for tuberculosis (TB) susceptibility in dairy cattle. Mycobacterium bovis is the principal agent of tuberculosis in cattle. The objective of this study was to quantify the genetic variation present among Irish Holstein-Friesian dairy herds in their susceptibility to M. bovis infection. A total of 15,182 cow and 8,104 heifer single intradermal comparative tuberculin test (SICTT, a test for M. bovis exposure and presumed infection) records from November 1, 2002, to October 31, 2005, were available for inclusion in the analysis. Data on observed carcass TB lesions from abattoirs were also available for inclusion in the analysis. The only animals retained were those present in a herd during episodes in which at least 2 animals showed evidence of infection; this ensured a high likelihood of exposure to M. bovis. Linear animal models, and sire and animal threshold models were used to estimate the variance components for susceptibility to M. bovis-purified protein derivative (PPD) responsiveness and confirmed M. bovis infection. The heritability estimates from the threshold sire models were biased upward because the relatedness between dam-daughter pairs was ignored. The threshold animal model produced heritability estimates of 0.14 in cows and 0.12 in heifers for susceptibility to M. bovis-PPD responsiveness, and 0.18 in cows for confirmed M. bovis infection susceptibility. Therefore, exploitable genetic variation exists among Irish dairy cows for susceptibility to M. bovis infection. Sire rankings from the linear and threshold animal models were similar, indicating that either model could be used for the analysis of susceptibility to M. bovis-PPD responsiveness. A favorable genetic correlation close to unity was observed between susceptibility to confirmed M. bovis infection and M. bovis-PPD responsiveness, indicating that direct selection for resistance to M. bovis-PPD responsiveness will indirectly reduce susceptibility to confirmed M. bovis infection. Data from the national TB eradication program could be used routinely to estimate breeding values for susceptibility to M. bovis infection.

Evaluation of granulysin and perforin as candidate biomarkers for protection following vaccination with Mycobacterium bovis BCG or M. bovis delta-RD1 (81.10)

Abstract Development of improved vaccines against tuberculosis (TB) is directly correlated to the investigation of new and better correlates of protection after vaccination. We examined the kinetics of granulysin, perforin, IFN-gamma and Fas-L responses to M. bovis purified protein derivative (PPD) stimulation by peripheral blood mononuclear cells from BCG- and non-vaccinated cattle. Gene expression profiles following PPD stimulation showed significant increases in transcripts for granulysin and IFN-gamma in both CD4+ and CD8+ T cells in BCG-vaccinated as compared to non-vaccinated animals. Perforin & IFN-gamma examined by flow cytometry, showed a difference of 1-2% more PPD-specific cells in BCG-vaccinated than non-vaccinated animals. In a second trial, M. bovis BCG vaccinated animals had lower disease and pathological scores as compared to M. bovis Delta-RD1(a new vaccinate candidate) and the control group. Gene expression of granulysin and perforin were significantly increased in both vaccine groups (M. bovis BCG and M. bovis Delta-RD1 vaccinated) as compared to control after vaccination and challenge. IFN-gamma expression was increased only after vaccination and secretion was higher in the control, as compared to both vaccine groups demonstrating no correlation with protection upon vaccination.

Evaluation of Granulysin and Perforin as Candidate Biomarkers for Protection Following Vaccination withMycobacterium bovisBCG orM. bovisΔRD1

The development of improved vaccines against tuberculosis (TB) is directly linked to the investigation of new and better correlates of protection after vaccination against TB. Cloning and characterization of bovine homologues of the antimicrobial protein granulysin (Bo-lysin) and perforin by our group could be used as potential biomarkers for TB vaccination efficacy. In the present study, we examined the kinetics of granulysin, perforin, IFNgamma and Fas-L responses to Mycobacterium bovis purified protein derivative (PPD) stimulation by peripheral blood mononuclear cells from M. bovisDeltaRD1-, BCG- and non-vaccinated cattle. Gene expression profiles following PPD stimulation showed significant increases in transcripts for granulysin and IFNgamma in both CD4(+) and CD8(+) T cells in BCG-vaccinated as compared with non-vaccinated animals. Perforin and IFNgamma examined by flow cytometry, showed a difference of 1-2% more PPD-specific cells in BCG-vaccinated than non-vaccinated animals. In the vaccine trial, granulysin and perforin were significantly increased in both vaccine groups as compared with control after vaccination and challenge. IFNgamma expression was increased only after vaccination and secretion was higher in the control, non-protected group as compared with both vaccine groups demonstrating no correlation with protection upon vaccination. In summary, results shown here provide evidence that granulysin and perforin are prospective candidates as biomarkers of protection after vaccination against TB.