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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.

The presence of circulating antigen in cattle experimentally infected with Mycobacterium bovis was demonstrated using dual-path platform (DPP) technology. The antigen capture immunoassays employed rabbit polyclonal antibody recognizing predominantly M. tuberculosis complex-specific epitopes and were able to detect soluble substances and whole cells of mycobacteria. The antigen found in serum appeared to be mostly bound to IgM, but not to IgG, within the immune complexes formed at early stages of M. bovis infection. The antigen was also detected in bile and urine, indicating possible clearance pathways. The data correlation analyses supported the idea of the role of IgM responses in antigen persistence during M. bovis infection. The antigen was detectable in serum months prior to detectable antibody seroconversion. This proof-of-concept study suggested the potential for improved immunodiagnostics for bovine tuberculosis.

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.

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.

Bovine tuberculosis (TB) in cervids remains a significant problem affecting farmed herds and wild populations. Traditional skin testing has serious limitations in certain species, whereas emerging serological assays showed promising diagnostic performance. The recently developed immunochromatographic dual-path platform (DPP) VetTB assay has two antigen bands, T1 (MPB83 protein) and T2 (CFP10/ESAT-6 fusion protein), for antibody detection. We evaluated the diagnostic accuracy of this test by using serum samples collected from groups of white-tailed deer experimentally inoculated with Mycobacterium bovis , M. avium subsp. paratuberculosis , or M. bovis BCG Pasteur. In addition, we used serum samples from farmed white-tailed deer in herds with no history of TB, as well as from free-ranging white-tailed deer culled during field surveillance studies performed in Michigan known to have bovine TB in the wild deer population. The DPP VetTB assay detected antibody responses in 58.1% of experimentally infected animals within 8 to 16 weeks postinoculation and in 71.9% of naturally infected deer, resulting in an estimated test sensitivity of 65.1% and a specificity of 97.8%. The higher seroreactivity found in deer with naturally acquired M. bovis infection was associated with an increased frequency of antibody responses to the ESAT-6 and CFP10 proteins, resulting in a greater contribution of these antigens, in addition to MPB83, to the detection of seropositive animals, compared with experimental M. bovis infection. Deer experimentally inoculated with either M. avium subsp. paratuberculosis or M. bovis BCG Pasteur did not produce cross-reactive antibodies that could be detected by the DPP VetTB assay. The present findings demonstrate the relatively high diagnostic accuracy of the DPP VetTB test for white-tailed deer, especially in the detection of naturally infected animals.

Chronic infection of cattle withLeptospira borgpeterseniiserovar Hardjo reduces animal production through reproductive failure and presents a persistent health threat to workers in the animal industry. Cattle are maintenance hosts for serovar Hardjo, and development of vaccines that establish long-term protective immunity has been problematic; induction of high titers of anti-serovar Hardjo antibody does not appear to be protective. Rather, development of an antigen-specific Th1 response appears to be critical for limiting renal colonization and urinary shedding of bacteria. In this study we compared two monovalent killed bacterial cell vaccines to assess long-term (12 months) protection against live serovar Hardjo challenge. Although neither vaccine prevented infection, renal colonization and urinary shedding of bacteria were reduced compared to those of control animals. Increased proliferation of CD4+, CD8+, and γδ T cells from vaccinated, but not control, animals was detected. In addition, NK cells from vaccinated animals and from all animals following infection, when exposed to antigenex vivo, demonstrated a gamma interferon (IFN-γ) recall response. We propose that programming NK cells to respond quickly toL. borgpeterseniiserovar Hardjo infection may be an important step toward developing protective immunity.

In the search for better tools to control bovine tuberculosis, the development of diagnostic tests with improved specificity and sensitivity has a high priority. We chose to search for novel immunodiagnostic reagents. In this study, Rv0899 (outer membrane protein A of Mycobacterium tuberculosis [OmpATb]) was evaluated as a stimulation antigen in a gamma interferon (IFN-γ) release assay to diagnose bovine tuberculosis. OmpATb induced IFN-γ responses in cattle experimentally infected with M. bovis as early and as persistently as ESAT-6 and CFP-10, the current lead diagnostic antigens. In naturally infected cattle, OmpATb stimulated IFN-γ production in 22 of 26 animals (85%). Importantly, OmpATb detected a portion of M. bovis -infected cattle which did not respond to ESAT-6 and CFP-10 (five of six cattle). The combined diagnostic sensitivity of OmpATb, ESAT-6, and CFP-10 for a preselected group consisting of naturally infected cattle with an overrepresentation of ESAT-6/CFP-10 nonresponders was 96% (25 of 26 animals). The specificity of OmpATb for uninfected cattle was 100% (27 cattle were tested; 12 of them gave false-positive results with tuberculins). In summary, our results indicate that OmpATb has the potential to enhance the sensitivity of previously described diagnostic tests based on ESAT-6 and CFP-10 and that the combined use of OmpATb, ESAT-6, CFP-10, and other proteins may achieve at least equal sensitivity to that obtained with purified protein derivative, but at a higher specificity. Further studies evaluating the diagnostic performance of OmpATb in combination with other proteins are ongoing.

Previous studies have suggested that currently available brucellosis vaccines induce poor or no protection in elk ( Cervus elaphus nelsoni ). In this study, we characterized the immunologic responses of elk after initial or booster vaccination with Brucella abortus strains RB51 (SRB51) and 19 (S19). Elk were vaccinated with saline or 10 10 CFU of SRB51 or S19 ( n = seven animals/treatment) and booster vaccinated with a similar dosage of the autologous vaccine at 65 weeks. Compared to nonvaccinates, elk vaccinated with SRB51 or S19 had greater ( P < 0.05) antibody responses to SRB51 or S19 after initial vaccination and after booster vaccination. Compared to nonvaccinated elk, greater ( P < 0.05) proliferative responses to autologous antigen after initial vaccination occurred at only a few sample times in SRB51 (6, 14, and 22 weeks) and S19 (22 weeks) treatment groups. In general, proliferative responses of vaccinates to nonautologous antigens did not differ ( P > 0.05) from the responses of nonvaccinated elk. Gamma interferon production in response to autologous or nonautologous Brucella antigens did not differ ( P > 0.05) between controls and vaccinates after booster vaccination. Flow cytometric techniques suggested that proliferation occurred more frequently in immunoglobulin M-positive cells, with differences between vaccination and control treatments in CD4 + and CD8 + subset proliferation detected only at 22 weeks after initial vaccination. After booster vaccination, one technique ([ 3 H]thymidine incorporation) suggested that proliferative responses to SRB51 antigen, but not S19 antigen, were greater ( P < 0.05) in vaccinates compared to the responses of nonvaccinates. However, in general, flow cytometric and other techniques failed to detect significant anamnestic responses to autologous or nonautologous Brucella antigens in S19 or SRB51 vaccinates after booster vaccination. Although some cellular immune responses were detected after initial or booster vaccination of elk with SRB51 or S19, our data suggest that responses tend to be transient and much less robust than previously reported in SRB51-vaccinated cattle ( Bos taurus ) or bison ( Bison bison ). These data may explain why the vaccination of elk with S19 and SRB51 induces poor protection against brucellosis.

Bovine tuberculosis persists as a costly zoonotic disease in numerous countries despite extensive eradication and control efforts. Sequential serum samples obtained from Mycobacterium bovis -infected cattle were evaluated for seroreactivity to mycobacterial antigens. Animals received M. bovis by aerosol, intratonsil, intranasal, or intratracheal inoculation. Assays included the multiantigen print immunoassay for determination of antigen recognition patterns, immunoblot analysis for sensitive kinetic studies, and the VetTB STAT-PAK test, a novel, rapid test based on lateral-flow technology. Responses to MPB83 were detected for all M. bovis -infected animals regardless of the route or strain of M. bovis used for inoculation. Other less commonly recognized antigens included ESAT-6, CFP-10, and MPB70. Responses to MPB83 were detectable as early as 4 weeks after inoculation, were boosted upon injection of purified protein derivatives for skin testing, and persisted throughout the course of each of the four challenge studies. MPB83-specific immunoglobulin M (IgM) was detected prior to MPB83-specific IgG detection; however, early IgM responses rapidly waned, suggesting a benefit of tests that detect both IgM- and IgG-specific antibodies. The VetTB STAT-PAK test detected responses in sera from 60% (15/25) of the animals by 7 weeks after challenge and detected responses in 96% (24/25) of the animals by 18 weeks. These findings demonstrate the potential for new-generation antibody-based tests for the early detection of M. bovis infection in cattle.

Mycobacterium bovis -infected white-tailed deer (WTD) in northeast Michigan are a reservoir of mycobacteria that pose a threat to both domestic animals and humans. Relatively little work has been done to characterize the immune response of WTD to M. bovis infection; however, an understanding of the immune response to infection and pathogenesis may be critical to the development of an effective vaccine. Immunological responses to infection were characterized by monitoring cytokine gene expression in M. bovis -infected and uninfected deer. Peripheral blood leukocytes (PBL) from infected WTD expressed more gamma interferon (IFN-γ), interleukin-12p40 (IL-12p40), granulocyte-monocyte colony-stimulating factor, and IL-4 mRNA than did PBL from uninfected deer; however, differences were not detected in expression of IL-10 and transforming growth factor-β mRNA. Infected animals could be divided into two groups based on pathology. Lesions were confined primarily to the lymph nodes of the head in animals with less severe pathology. Animals with more severe pathology had lesions in the lung and associated lymph nodes as well as the lymph nodes of the head. More robust IFN-γ mRNA expression correlated with pathology early in infection. These findings indicate that IFN-γ expression likely plays a role in both protection and pathogenesis.

Although tuberculin skin testing has been a hallmark of bovine tuberculosis eradication campaigns, it lacks sensitivity, can be confounded by exposure to nontuberculous mycobacteria, and cannot be repeated for 60 days due to desensitization. To overcome these difficulties, an effective whole-blood cellular immunoassay for bovine gamma interferon (IFN-γ) has been developed. The IFN-γ test is commonly used in conjunction with tuberculin skin testing as a confirmatory test following a positive response to the caudal fold test (CFT). The present study was conducted to determine the effect of different tuberculin skin-testing regimens on IFN-γ and antibody production by using calves that were experimentally infected with Mycobacterium bovis . Holstein calves were CFT tested 60 days after inoculation and the comparative cervical test (CCT) was conducted 7 (7-day CCT) or 55 (55-day CCT) days after the CFT. In both the 7-day CCT and 55-day CCT groups, IFN-γ responses increased 3 days after the CFT; this was immediately followed by a decrease to pre-skin test levels 7 days after the CFT. In both groups, the application of the CCT at 7 or 55 days after the CFT resulted in no significant increase in IFN-γ production. The administration of the CFT and the CCT to M. bovis -inoculated cattle boosted antibody responses to M. bovis PPD, rMPB83, ESAT-6, and the fusion protein Acr1-MPB83. The boosting effect was more pronounced in the 55-day CCT group. Increases in either IFN-γ or antibody production were not seen in noninoculated cattle. Measurement of both IFN-γ and antibody responses after skin testing may be useful in identifying M. bovis -infected cattle; however, the timing of collection of such samples may influence interpretation.

The only approved method of tuberculosis (TB) surveillance of reindeer within the United States is tuberculin skin testing; however, skin testing has an apparent lack of specificity, since numerous reindeer are classified as reactors, yet Mycobacterium bovis is not isolated from tissues upon necropsy. The objective of this study was to evaluate the ability of an in vitro assay (the Cervigam assay) to detect gamma interferon (IFN-γ) produced by blood leukocytes in response to mycobacterial antigens from M. bovis -infected reindeer. Thirteen male reindeer ∼9 months of age were inoculated with 10 5 CFU M. bovis in their tonsillar crypts. Stimulation of whole-blood cultures with a mitogen resulted in significant production of IFN-γ compared to that by nonstimulated samples. Responses by infected reindeer to M. bovis purified protein derivative (PPD) were as much as 3.5-fold higher than those by noninfected reindeer ( n = 4). Despite differences in responses to PPD by the two groups, reindeer within the noninfected group had responses of >0.1 change in optical density (ΔOD) (a level generally considered positive) to PPD. Mean responses by infected reindeer to a rESAT-6-CFP-10 fusion protein ( Mycobacterium tuberculosis complex specific) were as much as 20-fold higher than respective responses by noninfected reindeer at all time points. Additionally, responses by 3/4 noninfected reindeer were

White-tailed deer ( Odocoileus virginianus ) have emerged as reservoirs of bovine tuberculosis in northern America. For tuberculosis surveillance of deer, antibody-based assays are particularly attractive because deer are handled only once and immediate processing of the sample is not required. Sera collected sequentially from 25 Mycobacterium bovis -infected and 7 noninfected deer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for immunoglobulin specific to M. bovis antigens. Various routes of experimental M. bovis infection, such as intratonsillar inoculation ( n = 11), aerosol ( n = 6), and exposure to infected deer (in contact, n = 8), were studied. Upon infection, specific bands of reactivity at ∼24 to 26 kDa, ∼33 kDa, ∼42 kDa, and ∼75 kDa to M. bovis whole-cell sonicate were detected by immunoblot. Lipoarabinomannan-specific immunoglobulin was detected as early as 36 days postchallenge, and responses were detected for 94% of intratonsillarly and “in-contact”-infected deer. In MAPIA, sera were tested with 12 native and recombinant antigens coated on nitrocellulose. All in-contact-infected (8 of 8) and 10 of 11 intratonsillarly infected deer produced antibody reactive with one or more of the recombinant/native antigens. Responses were boosted by injection of tuberculin for intradermal tuberculin skin testing. Additionally, three of six deer receiving a very low dose of M. bovis via aerosol exposure produced antibody specific to one or more recombinant proteins. M. bovis was isolated from one of three nonresponding aerosol-challenged deer. Of the 12 antigens tested, the most immunodominant protein was MPB83; however, a highly sensitive serodiagnostic test will likely require use of multiple antigens.

Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium . Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-γ)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis -infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis -infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-α), IFN-γ, and nitric oxide responses by M. bovis -infected cattle to rESAT-6:CFP-10 exceeded ( P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium . Experimental infection with M. bovis , M. avium , or M. avium subsp. paratuberculosis induced significant ( P < 0.05) IFN-γ and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-γ responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis -infected calves exceeded ( P < 0.05) the corresponding responses of noninfected, M. avium -infected, and M. avium subsp. paratuberculosis -infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis , it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis .

Bovine tuberculosis in the United States has proven costly to cattle producers as well as to government regulatory agencies. While in vivo responsiveness to mycobacterial antigens is the current standard for the diagnosis of tuberculosis, in vitro assays are gaining acceptance, especially as ancillary or complementary tests. To evaluate in vitro indices of cellular sensitization, antigen-induced gamma interferon (IFN-), nitric oxide (NO), and tumor necrosis factor alpha (TNF-) responses by blood mononuclear cells from Mycobacterium bovis-infected cattle were quantified and compared. Using an aerosol model of infection, two doses of each of two strains of M. bovis (95-1315 and HC-2045T) were used to induce a range of IFN-, NO, and TNFresponses. Infection-specific increases in NO, but not in IFN- or TNF-, were detected in nonstimulated cultures at 48 h, a finding that is indicative of nonspecific activation and spontaneous release of NO. The infective dose of M. bovis organisms also influenced responses. At 34 days postinfection, IFN-, NO, and TNFresponses in antigen-stimulated cells from cattle receiving 10 5 CFU of M. bovis organisms were greater than responses of cells from cattle infected with 10 3 CFU of M. bovis organisms. The NO response, but not the IFNand TNF- responses, was influenced by infective strains of M. bovis. The TNF-, NO, and IFN- responses followed similar kinetics, with strong positive associations among the three readouts. Overall, these findings indicate that NO and TNF-, like IFN-, may prove useful as indices for the diagnosis of bovine tuberculosis. First described by Robert Koch in 1891, the tuberculin skin reaction has been the principal means of tuberculosis diagnosis for both humans and domestic animals (23). For cattle, the caudal fold skin test (CFT) is the primary approved test for tuberculosis within the United States. The CFT relies on in vivo reactivity to Mycobacterium bovis purified protein derivative (PPDb) injected intradermally into a fold of skin at the base of the tail. Cattle classified as reactors or suspect with this test are often retested by using the comparative cervical skin test, in which PPDb is injected at one site and M. avium PPD (PPDa) is injected at a separate site. The comparative cervical skin test, while technically more challenging than the CFT, provides an added ability to distinguish M. avium (including M. avium subsp. paratuberculosis) responders from M. bovis responders. An in vitro method of tuberculosis diagnosis has also been developed (41) and approved for use in the United States as a complementary test (i.e., in conjunction with the skin test) (22). The in vitro assay detects gamma interferon (IFN-) produced differentially by peripheral blood mononuclear cells (PBMC) exposed to no antigen (i.e., background response), PPDa, PPDb, or mitogen (e.g., pokeweed mitogen, PWM) (40). The assay is particularly suitable for diagnostic laboratories, as whole blood cultures are used, thus circumventing the need for cumbersome cell separation techniques. More recently, recombinant antigens specific for virulent tubercle ba

Inflammatory bowel disease (IBD) is a chronic, debilitating disorder of uncertain and perhaps multiple etiologies. It is believed to be due in part to disregulation of the immune system. Neuroimmune interactions may be involved in induction or maintenance of IBD. In the present study, we examined the potential role of a neurotransmitter, substance P, in a mouse model of IBD. We found that binding sites for substance P, and more specifically, neurokinin-1 receptors, were upregulated in intestinal tissue of mice with IBD-like syndrome. Dosing of mice with LY303870, a neurokinin-1 receptor antagonist, reduced the severity of IBD, and treatment of mice with preexisting IBD allowed partial healing of lesions. We hypothesize that blocking the binding of substance P to the neurokinin-1 receptor interrupts the inflammatory cascade that triggers and maintains intestinal lesions of IBD.