Comparative cellular immune responses in calves after infection with Mycobacterium avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii and M. bovis.

In the present study, calves were infected with Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. avium (M. avium), Mycobacterium kansasii (M. kansasii), or Mycobacterium bovis (M. bovis) to determine differences in cellular immunity. Comparative cellular responses were assessed upon stimulation of cells with mycobacterial whole cell sonicates respective of each infection group. Antigen-specific whole blood interferon gamma (IFN-γ) responses were observed in all infection groups compared to noninfected control calves, however, responses were more robust for M. bovis calves. Upon antigen stimulation of PBMCs, secretion of IFN-γ and IL-10 was higher for M. bovis calves compared to other infection groups. In contrast, IL-12 secretion was lower for M. bovis calves compared to MAP infected calves. Within the total PBMC population, higher numbers of CD4+, CD8+, and γδ TCR + T cells were observed for MAP and M. avium calves compared to M. bovis calves. This aligned with higher expression of CD26 on these subpopulations for MAP and M. avium calves, as well. In contrast, greater expression of CD25 was observed on CD4+ and γδ TCR + T cells and natural killer cells for M. bovis calves. Overall, similarities in cellular immune responses were observed between the closely related MAP and M. avium during infection of calves. In contrast, significant differences were noted between calves infected with MAP and M. bovis. This suggests that host immune responses to different mycobacteria may impact interpretation of diagnostic tools based upon their cellular immunity.

Evaluating the cytokine profile of the WC1+ γδ T cell subset in the ileum of cattle with the subclinical and clinical forms of MAP infection.

In the present study, we evaluated expression of IFN-γ, IL-17, TNF-α, IL-10 and TGF-ß by mucosal cells, including WC1+ γδ T cells, in ileal tissues taken from non-infected cattle and cattle naturally infected with Mycobacterium avium subsp paratuberculosis (MAP). Infected cattle were either in the subclinical or clinical stage of infection. We hypothesized that the cytokine profile of the WC1+ γδ T cell subset would be different between subclinical and clinical cattle. Our data indicate a significant increase in the numbers of WC1+ γδ T cells expressing IL-10 in clinical cattle compared to subclinical and non-infected cattle. We observed a significant increase in TGF-ß expression by non-WC1+ cells in clinically infected cattle. Expression of IFN-γ, IL-17 and TNF-α in mucosal cells, including the WC1+ γδ T cell subset, was identified in all examined groups. However, our data indicate that the stage of infection did not significantly influence expression of these proinflammatory cytokines. This study demonstrates changes in the cytokine mRNA expression profile of mucosal cells in the ileum, and specifically WC1+ γδ T cells, as cattle progress to the clinical disease. The change is characterized by an increase in expression of anti-inflammatory cytokines.

WC1+ γδ T cells from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis respond differentially to stimulation with PPD-J.

A role for γδ T cells in protection against mycobacterial infections including Johne's disease (JD) has been suggested. In neonatal calves where the risk to infection with Mycobacterium avium subsp. paratuberculosis (MAP) is high, the majority of circulating CD3+ lymphocytes are γδ TCR+. Bovine γδ T cells are divided into two major subsets based on the surface expression of workshop cluster 1 (WC1). The WC1+ subset, the predominant subset in periphery, is further divided into WC1.1+ and WC1.2+ subpopulations. The ability of γδ T cells to produce IFN-γ prior to CD4+ αß T cell activation could be crucial to the outcome of MAP infection. In the current study, cattle were naturally infected with MAP and were classified as either in the subclinical or clinical stage of infection. Compared to the control non-infected group, γδ T cell frequency in circulating lymphocytes was significantly lower in the clinical group. The observed decline in frequency was restricted to the WC1.2+ subset, and was not associated with preferential migration to infection sites (distal-ileum). γδ T cells proliferated significantly in recall responses to stimulation with purified protein derivative from MAP (PPD-J) only in subclinically infected cattle. These responses were a heterogeneous mixture of WC1.1 and WC1.2 subsets. Proliferation and IFN-γ production by the WC1.1+ γδ T cell subset was significantly higher in the subclinical group compared to the control and clinical groups. Our data indicates differences in MAP-specific ex-vivo responses of peripheral WC1+ γδ T cells of cattle with the subclinical or clinical form of JD.

Analysis of Cytokine Gene Expression using a Novel Chromogenic In-situ Hybridization Method in Pulmonary Granulomas of Cattle Infected Experimentally by Aerosolized Mycobacterium bovis.

Mycobacterium bovis is the cause of tuberculosis in most animal species including cattle and is a serious zoonotic pathogen. In man, M. bovis infection can result in disease clinically indistinguishable from that caused by Mycobacterium tuberculosis, the cause of most human tuberculosis. Regardless of host, the typical lesion induced by M. bovis or M. tuberculosis is the tuberculoid granuloma. Tuberculoid granulomas are dynamic structures reflecting the interface between host and pathogen and, therefore, pass through various morphological stages (I to IV). Using a novel in-situ hybridization assay, transcription of various cytokine and chemokine genes was examined qualitatively and quantitatively using image analysis. In experimentally infected cattle, pulmonary granulomas of all stages were examined 150 days after aerosol exposure to M. bovis. Expression of mRNA encoding tumour necrosis factor (TNF)-α, transforming growth factor-ß, interferon (IFN)-γ, interleukin (IL)-17A, IL-16, IL-10, CXCL9 and CXCL10 did not differ significantly between granulomas of different stages. However, relative expression of the various cytokines was characteristic of a Th1 response, with high TNF-α and IFN-γ expression and low IL-10 expression. Expression of IL-16 and the chemokines CXCL9 and CXCL10 was high, suggestive of granulomas actively involved in T-cell chemotaxis.

Virulence of two strains of mycobacterium bovis in cattle following aerosol infection.

Over the past two decades, highly virulent strains of Mycobacterium tuberculosis have emerged and spread rapidly in man, suggesting a selective advantage based on virulence. A similar scenario has not been described for Mycobacterium bovis infection in cattle (i.e. bovine tuberculosis). An epidemiological investigation of a recent outbreak of bovine tuberculosis in a USA dairy indicated that the causative strain of M. bovis (strain 10-7428) was particularly virulent, with rapid spread within the herd. In the present study, the virulence of this strain (10-7428) was directly compared in the target host with a well-characterized strain (95-1315) of relevance to the USA bovine tuberculosis eradication programme. Aerosol inoculation of 10(4) colony forming units of M. bovis 95-1315 (n = 8) or 10-7428 (n = 8) resulted in a similar distribution and severity of gross and microscopical lesions of tuberculosis as well as mycobacterial colonization, primarily affecting the lungs and lung-associated lymph nodes. Specific cell-mediated and antibody responses, including kinetics of the response, as well as antigen recognition profiles, were also comparable between the two treatment groups. Present findings demonstrate that M. bovis strains 95-1315 and 10-7428 have similar virulence when administered to cattle via aerosol inoculation. Other factors such as livestock management practices likely affected the severity of the outbreak in the dairy.

Persistence of Mycobacterium bovis bacillus Calmette-Guérin (BCG) Danish in white-tailed deer (Odocoileus virginianus) vaccinated with a lipid-formulated oral vaccine.

Mycobacterium bovis, the causative agent of tuberculosis in animals, has a broad host range, including humans. Historically, public health concerns prompted programs to eradicate tuberculosis from cattle in many nations. Eradication efforts decreased the prevalence of bovine tuberculosis; nevertheless, some countries encountered significant obstacles, not least of which was a wildlife reservoir of M. bovis. Efforts to decrease the size of the affected wildlife populations have neither eliminated disease nor eliminated transmission to cattle. Consequently, the use of a vaccine for wildlife is being explored. The vaccine most studied is M. bovis BCG, an attenuated live vaccine, first developed 100 years ago. The most efficient and effective means of vaccinating wildlife will be an oral vaccine. White-tailed deer in Michigan, USA, constitute a reservoir of M. bovis. White-tailed deer are a popular game species, and as such, represent a food animal to many hunters. BCG persistence in deer tissues could result in human exposure to BCG. Although non-pathogenic, BCG exposure could induce false-positive skin test results, confounding the central component of public health surveillance for TB. The objective of the present study in white-tailed deer was to evaluate persistence of lipid-encapsulated BCG and a liquid suspension of BCG after oral administration at two different dosages. Vaccine was not recovered at any time after oral consumption of a bait containing a single dose (1 × 10(8) CFU) of lipid-encapsulated BCG. However, persistence was consistent in deer consuming 10 lipid-encapsulated baits (1 × 10(9) CFU), with BCG recovered from at least one deer at 1, 3, 6, 9 and 12 months after consumption. Persistence of up to 9 months was seen in deer vaccinated with orally with a liquid suspension. Persistence of BCG was limited to lymphoid tissue and never found in samples of muscle collected at each time point. Although the risk of exposure to hunters is low, BCG persistence should be considered prior to field use in white-tailed deer.

Clinical and diagnostic developments of a gamma interferon release assay for use in bovine tuberculosis control programs.

Currently, the Bovigam assay is used as an official supplemental test within bovine tuberculosis control programs. The objectives of the present study were to evaluate two Mycobacterium bovis-specific peptide cocktails and purified protein derivatives (PPDs) from two sources, liquid and lyophilized antigen preparations. PPDs and peptide cocktails were also used for comparison of a second-generation gamma interferon (IFN-γ) release assay kit with the currently licensed first-generation kit (Bovigam; Prionics AG). Three strains of M. bovis were used for experimental challenge: M. bovis 95-1315, M. bovis Ravenel, and M. bovis 10-7428. Additionally, samples from a tuberculosis-affected herd (i.e., naturally infected) were evaluated. Robust responses to both peptide cocktails, HP (PC-HP) and ESAT-6/CFP10 (PC-EC), and the PPDs were elicited as early as 3 weeks after challenge. Only minor differences in responses to Commonwealth Serum Laboratories (CSL) and Lelystad PPDs were detected with samples from experimentally infected animals. For instance, responses to Lelystad M. avium-derived PPD (PPDa) exceeded the respective responses to the CSL PPDa in M. bovis Ravenel-infected and control animals. However, a 1:4 dilution of stimulated plasma demonstrated greater separation of PPDb from PPDa responses (i.e., PPDb minus PPDa) with the use of Lelystad PPDs, suggesting that Lelystad PPDs provide greater diagnostic sensitivity than CSL PPDs. The responses to lyophilized and liquid antigen preparations did not differ. Responses detected with first- and second-generation IFN-γ release assay kits (Bovigam) did not differ throughout the study. In conclusion, antigens may be stored in a lyophilized state without loss in potency, PC-HP and PC-EC are dependable biomarkers for aiding in the detection of bovine tuberculosis, and second-generation Bovigam kits are comparable to currently used kits.

Disparate host immunity to Mycobacterium avium subsp. paratuberculosis antigens in calves inoculated with M. avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii, and M. bovis.

The cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and a criticism of the current tests that are used for the detection of paratuberculosis. In the present study, Mycobacterium avium subsp. paratuberculosis recombinant proteins were evaluated for antigenic specificity compared to a whole-cell sonicate preparation (MPS). Measures of cell-mediated immunity to M. avium subsp. paratuberculosis antigens were compared in calves inoculated with live M. avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), Mycobacterium kansasii, or Mycobacterium bovis. Gamma interferon (IFN-γ) responses to MPS were observed in all calves that were exposed to mycobacteria compared to control calves at 4 months postinfection. Pooled recombinant M. avium subsp. paratuberculosis proteins also elicited nonspecific IFN-γ responses in inoculated calves, with the exception of calves infected with M. bovis. M. avium subsp. paratuberculosis proteins failed to elicit antigen-specific responses for the majority of immune measures; however, the expression of CD25 and CD26 was upregulated on CD4, CD8, gamma/delta (γδ) T, and B cells for the calves that were inoculated with either M. avium subsp. paratuberculosis or M. avium after antigen stimulation of the cells. Stimulation with MPS also resulted in the increased expression of CD26 on CD45RO(+) CD25(+) T cells from calves inoculated with M. avium subsp. paratuberculosis and M. avium. Although recombinant proteins failed to elicit specific responses for the calves inoculated with M. avium subsp. paratuberculosis, the differences in immune responses to M. avium subsp. paratuberculosis antigens were dependent upon mycobacterial exposure. The results demonstrated a close alignment in immune responses between calves inoculated with M. avium subsp. paratuberculosis and those inoculated with M. avium that were somewhat disparate from the responses in calves infected with M. bovis, suggesting that the biology of mycobacterial infection plays an important role in diagnosis.

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.

Adaptive immunity in the colostrum-deprived calf: response to early vaccination with Mycobacterium bovis strain bacille Calmette Guerin and ovalbumin.

Responses of the newborn calf to vaccination are frequently characterized by marginal antibody (Ab) responses. The present study evaluated effects of colostrum ingestion on the adaptive immune response of the preruminant calf to early vaccination. Colostrum-fed (CF) and colostrum-deprived (CD) calves were vaccinated at 2 d of age with Mycobacterium bovis, Pasteur strain of bacille Calmette Guerin (BCG), and ovalbumin (OVA) to track development of the adaptive immune response during the first 8 wk of life. Dams were also vaccinated with BCG prepartum. At wk 0, serum IgG(1), IgG(2), IgA, and IgM were elevated in CF calves, with IgG(1) predominating. In these calves, IgG(2), IgA, and IgM concentrations decreased with age. The CD calves, in contrast, had very low or undetectable serum immunoglobulin concentrations at wk 0 followed by an age-related increase in IgG(1), IgG(2), and IgM concentrations, suggesting endogenous production of these immunoglobulin classes. Immunoblot and ELISA analyses of Ab response to BCG vaccination indicated that colostrum ingestion was associated with measurable serum anti-mycobacterial Ab in CF calves during the first month postpartum, with substantially lower levels at 7 wk of age. Although mycobacteria-specific Ab was undetectable in CD calves at wk 0, it was present at 4 and 7 wk of age, suggesting that these calves, unlike CF calves, were capable of generating an Ab response to BCG vaccination. Antibody responses of CF and CD calves to vaccination with OVA, an antigen not present in the natural environment of dairy cattle, were of comparable magnitude and characterized by a progressive increase in Ab levels from birth (wk 0) to 7 wk of age. The disparate Ab responses of CF calves to BCG and OVA suggest that maternal antigenic experience or exposure influences Ab responses of the colostrum-fed preruminant calf to early vaccination. Ex vivo, antigen [OVA and M. bovis-derived purified protein derivative (PPDb)]-induced IFN-γ and nitric oxide responses of blood mononuclear cells (PBMC) from CF and CD calves were comparable at wk 0 and wk 7. As expected, responses were very low or nonexistent at wk 0. Responses for all calves were greater at wk 7 than at wk 0, suggesting a colostrum-independent maturation of the cell-mediated immune response capacity of the preruminant calf. The consistently greater proliferative responses of antigen-stimulated T-cell subsets at wk 7 versus wk 0 indicate the development of antigen-specific lymphocyte responses to early vaccination. Total numbers of blood leukocytes as well as numbers of lymphocytes and monocytes were unaffected by colostrum feeding; however, granulocyte numbers were higher in CD than in CF calves at wk 0. Granulocyte numbers decreased and monocyte numbers increased with age in all calves. Within the lymphocyte population, only natural killer (NK(+)) cell percentages were affected by colostrum ingestion, with higher percentages of NK(+) cells in CD calves at wk 0 and wk 7. Antigen-induced proliferation of lymphocyte subsets including IgM(+) cells was unaffected by colostrum ingestion. In conclusion, ingestion of colostrum within hours after birth inhibited the capacity of the calf to produce antigen-specific immunoglobulin (i.e., antibody) in response to vaccination, with little or no effect on cell-mediated immune responses. Although colostrum appeared to block endogenous antibody production, certain B-cell functions were retained. These findings will aid in development of new vaccination strategies for improving health of the preruminant calf.

Mediation of host immune responses after immunization of neonatal calves with a heat-killed Mycobacterium avium subsp. paratuberculosis vaccine.

A major drawback of current whole-cell vaccines for Mycobacterium avium subsp. paratuberculosis is the interference with diagnostic tests for bovine tuberculosis (TB) and paratuberculosis. The current study was designed to explore the effects of immunization with a heat-killed whole-cell vaccine (Mycopar) on diagnostic test performance and to characterize host immune responses to vaccination over a 12-month period. Neonatal dairy calves were assigned to treatment groups consisting of (i) controls, not vaccinated (n = 5), and (ii) vaccinates, vaccinated with Mycopar vaccine (n = 5). The results from this study demonstrated a rapid initiation of M. avium subsp. paratuberculosis-specific gamma interferon (IFN-γ) in vaccinated calves by 7 days, with robust responses throughout the study. Vaccinated calves also had responses to M. bovis purified protein derivative tuberculin (BoPPD) but minimal reactivity to ESAT-6/CFP-10, an M. bovis recombinant fusion protein. The levels of antigen-specific interleukin-4 (IL-4) and IL-10 were markedly decreased in vaccinated calves between days 7 and 90 of the study but thereafter were similar to the levels in controls. Vaccinated calves began to seroconvert at 4 months, with 4/5 calves having detectable M. avium subsp. paratuberculosis antibody by 6 months. The responses in test platforms for bovine TB were negligible in the vaccinate group, as only one calf had a response, which was in the suspect range of the comparative cervical skin test. Serum antibody responses to M. bovis antigens ESAT-6, CFP-10, and MPB83 were negative on the Vet TB STAT-PAK, DPP VetTB, and DPP BovidTB tests. These results suggest that the Mycopar vaccine will interfere with diagnostic tools for paratuberculosis but result in low interference with the comparative cervical skin test and emerging serologic tests for M. bovis.

Development and evaluation of an enzyme-linked immunosorbent assay for use in the detection of bovine tuberculosis in cattle.

As a consequence of continued spillover of Mycobacterium bovis into cattle from wildlife reservoirs and increased globalization of cattle trade with associated transmission risks, new approaches such as vaccination and novel testing algorithms are seriously being considered by regulatory agencies for the control of bovine tuberculosis. Serologic tests offer opportunities for identification of M. bovis-infected animals not afforded by current diagnostic techniques. The present study describes assay development and field assessment of a new commercial enzyme-linked immunosorbent assay (ELISA) that detects antibody to M. bovis antigens MPB83 and MPB70 in infected cattle. Pertinent findings include the following: specific antibody responses were detected at ∼90 to 100 days after experimental M. bovis challenge, minimal cross-reactive responses were elicited by infection/sensitization with nontuberculous Mycobacterium spp., and the apparent sensitivity and specificity of the ELISA with naturally infected cattle were 63% and 98%, respectively, with sensitivity improving as disease severity increased. The ELISA also detected infected animals missed by the routine tuberculin skin test, and antibody was detectable in bulk tank milk samples from M. bovis-infected dairy herds. A high-throughput ELISA could be adapted as a movement, border, or slaughter surveillance test, as well as a supplemental test to tuberculin skin testing.

Comparison of tuberculin activity using the interferon-gamma assay for the diagnosis of bovine tuberculosis.

In this study, interferon-gamma (IFN-gamma) responses in whole blood cultures stimulated with tuberculins from different sources were compared with regard to their diagnostic reliability in cattle experimentally and naturally infected with Mycobacterium bovis. The IFN-gamma responses to different concentrations of purified protein derivatives (PPDs) from M bovis and Mycobacterium avium were quantified. Significant differences (P<0.05) between sources and concentrations of PPDs used for stimulation were detected, indicating a need for standardisation of PPDs used in the IFN-gamma assay. Additionally, a tool named'relative potency 30' that allows rapid comparison of batches and sources of PPDs was defined.

Persistence of Mycobacterium bovis Bacillus Calmette-Guérin in white-tailed deer (Odocoileus virginianus) after oral or parenteral vaccination.

Mycobacterium bovis is the cause of tuberculosis in cattle and a serious zoonotic pathogen, most commonly contracted through consumption of unpasteurized dairy products. To control this zoonosis, many countries have developed bovine tuberculosis eradication programmes. Although relatively successful, efforts are hindered in many regions by spillover from wildlife reservoirs of M. bovis to cattle. Such is the case in the United States where spillover of M. bovis from free-ranging white-tailed deer to cattle occurs. One approach to control such inter-species transmission is vaccination of wildlife. The live, attenuated human vaccine M. bovis Bacillus Calmette-Guérin (BCG) has been shown to reduce disease severity in white-tailed deer; however, vaccine persistence within tissues has also been noted. Consumption of venison containing BCG by hunters may present a public health concern as BCG exposure, although unlikely to cause disease, could cause false positive tuberculin skin test results. To examine BCG persistence further, 42 white-tailed deer were vaccinated orally or subcutaneously (SC) with BCG Danish. Three deer from each group were killed and examined at periods ranging from 2 weeks to 11 months after vaccination. BCG was recovered from orally vaccinated deer as late as 3 months after vaccination, while BCG persisted in SC vaccinated deer for as long as 9 months. At no time was BCG isolated from meat; however, prolonged persistence was seen in lymphoid organs. Although vaccine persistence was noted, especially in SC vaccinated deer, the distribution of culture-positive tissues makes human exposure through consumption unlikely.

Bovine tuberculosis: a review of current and emerging diagnostic techniques in view of their relevance for disease control and eradication.

Existing strategies for long-term bovine tuberculosis (bTB) control/eradication campaigns are being reconsidered in many countries because of the development of new testing technologies, increased global trade, continued struggle with wildlife reservoirs of bTB, redistribution of international trading partners/agreements, and emerging financial and animal welfare constraints on herd depopulation. Changes under consideration or newly implemented include additional control measures to limit risks with imported animals, enhanced programs to mitigate wildlife reservoir risks, re-evaluation of options to manage bTB-affected herds/regions, modernization of regulatory framework(s) to re-focus control efforts, and consideration of emerging testing technologies (i.e. improved or new tests) for use in bTB control/eradication programs. Traditional slaughter surveillance and test/removal strategies will likely be augmented by incorporation of new technologies and more targeted control efforts. The present review provides an overview of current and emerging bTB testing strategies/tools and a vision for incorporation of emerging technologies into the current control/eradication programs.

Immune responses in cattle inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii.

Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare the antigen-specific immune responses to various patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis infection) and colonization without pathology (M. tuberculosis infection) to no colonization or pathology (M. kansasii infection). Delayed-type hypersensitivity and gamma interferon responses were elicited by each mycobacterial inoculation; however, the responses by the M. bovis- and M. tuberculosis-inoculated animals exceeded those of the M. kansasii-inoculated animals. Specific antibody responses were detected in all M. tuberculosis- and M. bovis-inoculated cattle 3 weeks after inoculation. From 6 to 16 weeks after M. tuberculosis inoculation, the antibody responses waned, whereas the responses persisted with M. bovis infection. With M. kansasii inoculation, initial early antibody responses waned by 10 weeks after inoculation and then increased 2 weeks after the injection of purified protein derivative for the skin test at 18 weeks after challenge. These findings indicate that antibody responses are associated with the antigen burden rather than the pathology, cellular immune responses to tuberculin correlate with infection but not necessarily with the pathology or bacterial burden, and exposure to mycobacterial antigens may elicit an antibody response in a presensitized animal.

Short communication: the preruminant calf as a model for characterizing the effects of vitamin D status in the neonate.

The objective of this study was to evaluate the feasibility of using the preruminant dairy calf as a model for evaluating effects of vitamin D status in the neonate. Because the newborn calf can be sustained during the first weeks of life solely on a fluid diet having a defined composition, has documented nutritional requirements, and is minimally affected by repeated samplings of peripheral blood, it has the potential to serve as a model for characterizing nutrient-specific effects on the growth and health of the neonate. Colostrum-fed Holstein bull calves (n = 13) entered the trial at approximately 4 d of age. All calves were fed a custom-formulated milk replacer devoid of vitamin D. Plasma 25-hydroxyvitamin D(3) concentrations in all calves were determined on a regular basis beginning at d 0. Using this information, low- and high-status groups of calves were established by subcutaneous administration of 25-hydroxyvitamin D(3). To maintain targeted plasma 25-hydroxyvitamin D(3) concentrations in low (<30 ng/mL) and high (>60 ng/mL) vitamin D-status calves, low-status calves (n = 6) received a total of 8,600 IU (2,225 IU/wk) of vitamin D during the experimental period and high-status calves (n = 7) received 54,000 IU (13,500 IU/wk). Concentrations of 25-hydroxyvitamin D(3) in low-status calves averaged 27 ng/mL, compared with 78 ng/mL in high-status calves, and were less at all sampling times from d 7 to d 28. Concentrations of 1,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3) were not correlated. Calcium, magnesium, and phosphorous concentrations were unaffected by 25-hydroxyvitamin D(3) administration; however, plasma calcium and 1,25-dihydroxyvitamin D(3) concentrations were correlated. Calcium and magnesium concentrations decreased with age but remained within normal ranges for dairy cattle. These results indicate that it is possible to predictably control vitamin D status over a 28-d period and suggest that the preruminant calf might be useful as a model for studying effects of vitamin D on growth, development, and immune function in the neonate.

Humoral immune responses of white-tailed deer (Odocoileus virginianus) to Mycobacterium bovis BCG vaccination and experimental challenge with M. bovis.

Monitoring of the kinetics of production of serum antibodies to multiple mycobacterial antigens can be useful as a diagnostic tool for the detection of Mycobacterium bovis infection as well as for the characterization of disease progression and the efficacy of intervention strategies in several species. The humoral immune responses to multiple M. bovis antigens by white-tailed deer vaccinated with BCG orally via a lipid-formulated bait (n=5), orally in liquid form (n=5), and subcutaneously (n=6) were evaluated over time after vaccination and after experimental challenge with virulent M. bovis and were compared to the responses by unvaccinated deer (n=6). Antibody responses were evaluated by using a rapid test (RT), a multiantigen print immunoassay (MAPIA), a lipoarabinomannan enzyme-linked immunosorbent assay (LAM-ELISA), and immunoblotting to whole-cell sonicate and recombinant antigen MPB83. MAPIA and RT detected minimal to no antibody responses over those at the baseline to multiple M. bovis antigens in vaccinated white-tailed deer after challenge. This was in contrast to the presence of more readily detectable antibody responses in nonvaccinated deer with more advanced disease. The LAM-ELISA results indicated an overall decrease in the level of production of detectable antibodies against lipoarabinomannan-enriched mycobacterial antigen in vaccinated animals compared to that in nonvaccinated animals after challenge. Immunoblot data were inconsistent but did suggest the occurrence of unique antibody responses by certain vaccinated groups to Ag85 and HSP70. These findings support further research toward the improvement and potential use of antibody-based assays, such as MAPIA, RT, and LAM-ELISA, as tools for the antemortem assessment of disease progression in white-tailed deer in both experimental and field vaccine trials.

Vaccination with Mycobacterium bovis BCG strains Danish and Pasteur in white-tailed deer (Odocoileus virginianus) experimentally challenged with Mycobacterium bovis.

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock and the cause for many faltering bovine tuberculosis eradication programmes. One approach in dealing with wildlife reservoirs of disease is to interrupt inter-species and intraspecies transmission through vaccination of deer or cattle. To evaluate the efficacy of BCG vaccination in white-tailed deer, 35 deer were assigned to one of three groups; one s.c. dose of 10(7) CFU of M. bovis BCG Pasteur (n = 12); 1 s.c. dose of 10(7) CFU of M. bovis BCG Danish (n = 11); or unvaccinated deer (n = 12). After vaccination, deer were inoculated intratonsilarly with virulent M. bovis. Lesion severity scores of the medial retropharyngeal lymph node, as well as all lymph nodes combined, were reduced in vaccinated deer compared to unvaccinated deer. BCG Danish vaccinated deer had no late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid-fast bacilli compared to BCG Pasteur vaccinated or unvaccinated deer where such lesions were present. Both BCG strains were isolated as late as 250 days after vaccination from deer that were vaccinated but not challenged. In white-tailed deer, BCG provides protection against challenge with virulent M. bovis. Issues related to vaccine persistence, safety and shedding remain to be further investigated.

Animal-side serologic assay for rapid detection of Mycobacterium bovis infection in multiple species of free-ranging wildlife.

Numerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n=1532), white-tailed deer (n=463), brushtail possums (n=129), and wild boar (n=177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock.