Cell mediated and humoral immune responses of white-tailed deer experimentally infected with Mycobacterium bovis

The objective of this study was to improve the understanding of immune responses of whitetailed deer (Odocoileus virginianus) infected with Mycobacterium bovis. Ten mature, female, white-tailed deer were inoculated by intratonsilar instillation of 2 x 10(3)or 2 x 10(5)colony-forming units of M. bovis. Lymphocyte proliferation and humoral response to M. bovis PPD and the M. bovis protein, MPB70 were measured. Deer were tested for exposure to M. bovis by the comparative cervical skin test. Biopsy specimens of skin test sites were examined microscopically and immunohistochemically. The comparative cervical skin test correctly identified all M. bovis -inoculated deer as exposed to M. bovis. Lymphocyte proliferative responses to MPB70 were more consistent than responses to M. bovisPPD in M. bovis -inoculated deer. Antibody responses were more prominent in deer with disseminated disease than in deer with localised disease. The cellular components of delayed-type hypersensitivity reactions at skin test sites were similar to tuberculin reactions in other species. T lymphocytes of the gamma/delta phenotype were seen in increased numbers in M. bovisPPD injection sites.     

Natural infection of red deer with bovine tuberculosis

Six bovine tuberculosis-free red deer hinds were introduced in October 1993 to a 1.8 ha enclosure, within a larger field study site known to contain tuberculous possums, and kept there for 9 months. A Mycobacterium bovis-infected possum was found in the vicinity of the deer enclosure 3 weeks after the introduction. Subsequently, a further eleven infected possums were found in the area. The deer were monitored by repeated composite antibody detection ELISA and lymphocyte transformation assays for tuberculosis, interpreted in parallel, by skin testing and by routine culturing of samples collected from potential excretion sites. Lymphocyte transformation assay evidence of M. bovis infection in four hinds was first observed 4 months after introduction. One other hind became bovine tuberculin lymphocyte transformation assay positive in the 5th month. Positive or equivocal bovine reactivity remained evident at most test episodes. A comparative cervical skin test performed in July 1994, shortly before slaughter, was positive in these five hinds. Mycobacterium bovis was recovered off swabs from the oropharyngeal tonsils of two hinds during routine sampling. Detailed necropsy of the six deer revealed a single typical tuberculous lesion in only one, but culturing of various tissue specimens ascertained that the five blood test and comparative cervical skin test-positive animals were all infected. Mycobacterium bovis was cultured from the oropharyngeal tonsils of four and medial retropharyngeal lymph nodes of two of the deer with no typical gross lesions. Six additional tuberculosis-free hinds were introduced to the enclosure in April 1994 and kept there for 12 months. Four of these animals showed a positive lymphocyte transformation assay response to M. bovis after 9 weeks, but no significant reactivity thereafter. Concurrent observational studies suggest that five of the first six deer probably became infected through close inspection and investigation of the tuberculous possums, although the possibility of deer-to-deer transmission cannot be totally excluded. The likely deer-possum contact, and thus exposure to M. bovis, was related to the curiosity and social ranking of the hinds. The second group appear to have had transient exposure to M. bovis, possibly caused by direct contact with the infected hinds introduced earlier. This group never showed any curiosity toward, or interaction with, possums during the periods of observation.     

MHC class II-restricted, CD4(+) T-cell proliferative responses of peripheral blood mononuclear cells from Mycobacterium bovis-infected white-tailed deer

White-tailed deer are significant wildlife reservoirs of Mycobacterium bovis for cattle, predators, and, potentially, humans. Infection of cattle with M. bovis stimulates an antigen-specific T-cell response, with both CD4(+) and CD8(+) cells implicated in protective immunity. Few studies, however, have examined lymphocyte subset responses to experimental M. bovis infection of white-tailed deer. In this study, a flow cytometric proliferation assay was used to determine the relative contribution of individual peripheral blood mononuclear cell subsets of M. bovis-infected white-tailed deer in the recall response to M. bovis antigen. Naive deer were challenged with M. bovis by cohabitation with infected deer. These M. bovis-challenged deer developed significant in vivo (delayed-type hypersensitivity) and in vitro (proliferative) responses to M. bovis purified protein derivative (PPD). At necropsy, typical tuberculous lesions containing M. bovis were detected within lungs and lung-associated lymph nodes of infected deer. The predominant subset of lymphocytes that proliferated in response to in vitro stimulation with PPD was the CD4(+) subset. Minimal proliferative responses were detected from CD8(+), gamma delta TCR(+), and B-cells. Addition of monoclonal antibodies specific for MHC II antigens, but not MHC I or CD1 antigens, abrogated the proliferative response. Together, these findings indicate that while CD4(+) cells from infected deer proliferate in the recall response to M. bovis antigens, this response is not sufficient to clear M. bovis and immunologic intervention may require stimulation of alternate subsets of lymphocytes.     

间接ELISA检测牛分枝杆菌抗体方法的建立及初步应用

针对现行牛结核病检疫方法结核菌素皮内变态反应(TST)的不足,本研究选用牛分枝杆菌特异性抗原MPB83、MPB70及CFP10与ESAT-6融合蛋白(CFP10-ESAT-6)分别建立了检测牛分枝杆菌特异抗体的间接酶联免疫吸附方法(ELISA)。上述3种抗原中一种以上抗原的特异性抗体为阳性时,即可判断为结核检测阳性。以TST为标准,判断ELISA方法的敏感性与特异性。结果证明,ELISA方法具有较好的敏感性(71.4%)。对ELISA检测为强阳性的奶牛进行细菌分离,并对5头结核菌分离阳性奶牛进行TST检测,结果有3头为TST阳性,2头可疑,显示出ELISA方法对严重感染牛的检测较TST具有更高的敏感性。由于TST与ELISA分别以细胞免疫与体液免疫为基础,两种检测方法结合应用可进一步提高牛结核病的检出率。     

牛结核病抗体胶体金快速检测技术的建立和应用

为了建立一种快速检测牛分支杆菌抗体的新方法用于诊断牛结核病,利用胶体金免疫层析技术原理,用原核诱导表达的牛分支杆菌抗原蛋白MPB83和MPB70分别作为胶体金标记抗原和检测线上的捕获抗原,制备牛结核抗体检测试纸条.结果表明,粒径为40 nm的胶体金制备的试纸条敏感性最高,胶体金最佳标记pH为6.0,MPB83抗原最适标记量为每毫升胶体金6.5 μg,MPB70抗原的最适包被浓度为3.0 mg/mL,抗MPB83蛋白IgG的最佳包被浓度为2.5 mg/mL,交叉试验证明试纸条不与牛的其他非相关疾病的阳性血清反应,具有较高的特异性.比较试验证明其敏感性显著高于韩国进口试纸条.在上述试验条件下生产了一批胶体金试纸条进行临床样品检测,并与细菌分离培养、结核菌素皮内变态反应(TST)和韩国试纸条比较.本试纸条与牛分支杆菌分离培养的符合率为85%,与TST的符合率为79.73%,与韩国试纸条的符合率为98.75%.快速检测牛结核抗体的免疫层析试纸条具有敏感、特异、简便、快速的特点,适用于对牛结核病进行普查和检疫,也可作为TST的辅助诊断方法,在牛结核病根除计划中具有良好的应用前景.     

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.     

牛型分枝杆菌MPB70蛋白胶乳凝集方法的建立及应用

为了研究牛结核病新型诊断试剂，提高诊断的特异性和敏感性，我们用大肠杆菌工程菌表达了牛型分枝杆菌特异性抗原MPB70并提纯蛋白建立了牛结核病乳胶凝集试验（LAT）诊断方法。MPB70是一种牛型分枝杆菌特异性分泌而卡介苗BCG缺失的蛋白质，热稳定性好，用此蛋白建立的乳胶凝集方法具有良好的敏感性、特异性以及较长的保存期，检测70份临床奶牛血清，与皮内变态反应和间接血凝方法相比较分别具有71．4％和88．6％的符合率。该方法还可鉴别诊断自然感染和疫苗免疫接种。我们建立的牛型分枝杆菌MPB70蛋白乳胶凝集试验诊断方法将分子生物学手段和经典试验方法有机结合，为临床快速检测牛结核病血清特异性抗体水平提供了行之有效的方法。     

Evaluation of relationship between plasma fibrinogen concentration and tuberculin testing in red deer

After intradermal injection of bovine purified derivative (PPD), increases in plasma fibrinogen concentration and plasma viscosity developed in red deer (Cervus elaphus) with a history of tuberculosis caused by Mycobacterium bovis. Serum haptoglobin concentrations were also found to increase under similar circumstances. The increases were reproducible and did not appear to be related to mustering, stress, or the handling associated with injection of PPD. A significant (P less than 0.05) direct relationship was found between the increase in plasma fibrinogen concentration and various markers of bovine tuberculosis infection, such as stimulation of lymphocyte transformation in response to bovine PPD and the diameter of intradermal tuberculin skin test reactions. A stronger correlation (P less than 0.01) was found with the volume of intradermal tuberculin skin test reactivity, and the strongest correlation (P less than 0.001) was with the presence of circulating antibovine PPD antibody.     

基因免疫制备牛分枝杆菌MPB64抗原单克隆抗体

为了建立针对牛分枝杆菌分泌抗原MPB64的检测手段,制备该抗原的单克隆抗体,构建真核表达载体pcDNA-mpb64,以原核表达并纯化的融合蛋白H-MPB64包被ELISA板,建立单克隆抗体检测方法。利用基因免疫方法免疫BALB/c小鼠,通过杂交瘤技术筛选出针对牛分枝杆菌分泌蛋白MPB64的单克隆抗体一株,并制备了腹水,经鉴定腹水效价达到1∶10240,采用硫酸铵沉淀法纯化腹水,纯化抗体效价为1∶8000,为今后研制分枝杆菌鉴定技术奠定基础。     

牛分枝杆菌抗原MPB70、MPB83和ESAT-6的融合表达及重组蛋白的初步应用

应用PCR方法从牛分枝杆菌Vallee株基因组中扩增获得mpb70、mpb83和esat-6三个目的基因片段。采用重叠延伸剪接技术(splicing by overlap extension,SOE)获得融合基因mpb70-mpb83后,将mpb70-mpb83和esat-6串连于同一表达载体pET32a( )中得到重组质粒pET70-83-E6。转化BL21(DE3)大肠杆菌感受态后,经IPTG诱导以可溶的形式表达融合蛋白。用Ni2 亲合层析的方法纯化该融合蛋白。Western blot分析显示:该融合蛋白能与抗牛分枝杆菌阳性血清发生特异性反应,而与牛副结核病阳性血清不反应。用该纯化蛋白初步建立了间接ELISA方法,并检测了117份临床血清样本(其中67份为PPD阳性牛血清),阳性率为39.32%(46/117)份,与PPD皮试诊断的符合率为82.05%(96/117)。     

Assessment of defined antigens for the diagnosis of bovine tuberculosis in skin test-reactor cattle

The continued use of purified protein derivative (PPD) tuberculin is considered to be the main factor which limits the specificity of diagnostic tests for bovine tuberculosis (TB). This study evaluated a whole blood interferon-gamma (IFN-gamma) assay and compared the diagnostic potential of PPD with two tuberculosis-specific antigens, ESAT-6 and MPB70. To provide estimates of sensitivity and specificity, responses were measured in 180 skin test-reacting cattle, of which 131 were confirmed as tuberculous, and in 128 cattle from TB-free herds. For the skin test reactors, there was a positive correlation between the IFN-gamma responses to PPD from Mycobacterium bovis (PPDB) and PPD from Mycobacterium avium (PPDA), indicating cross-reactivity between these complex antigens which are the basis of the skin test. In comparisons of the ESAT-6 IFN-gamma test with a PPD IFN-gamma test (using PPDB compared with PPDA), there was a decrease in sensitivity (76.3 per cent vs 89.3 per cent), but a clear increase in specificity (99.2 per cent vs 92.2 per cent). The provision of high specificity, even with lower sensitivity, offers major benefits for testing in areas with a low incidence of TB.     

Evaluation of two cocktails containing ESAT-6, CFP-10 and Rv-3615c in the intradermal test and the interferon-γ assay for diagnosis of bovine tuberculosis

The intradermal tuberculin tests and the interferon-gamma (IFN-γ) assay are the principal tests used worldwide for the ante-mortem diagnosis of bovine tuberculosis. The conventional reagent currently in use in these tests is purified protein derivative (PPD) tuberculin obtained from Mycobacterium bovis culture. The components of PPD are poorly characterized and difficult to standardize. To overcome this issue, antigens specific to the Mycobacterium tuberculosis complex are being studied. Here we have assessed the biological potency of ESAT-6, CFP-10 and Rv-3615c presented as peptide or recombinant protein cocktails in comparison with the standard bovine PPD used routinely in Spanish eradication campaigns. The study was performed in cattle (n=23) from a herd with natural M. bovis infection. Animals were simultaneously injected with PPD and the peptide and protein cocktails. The percentages of cattle reacting positively to single intradermal test were 60.9% (bovine PPD), 47.8% (peptide cocktail) and 60.9% (protein cocktail), with no significant difference between the actual skin fold thickness increases (p>0.05). The IFN-γ assay detected 60.9% of animals when stimulation was performed with bovine PPD, but decreased to 52.2% when stimulation was performed with the peptide cocktail and to 47.8% when stimulation was performed with the protein cocktail. However, no significant differences were found between IFN-γ responder frequencies (p>0.05). These results show a potential use of these defined reagents for in vivo tuberculosis diagnosis.     

11种牛分枝杆菌抗原在牛结核病诊断中的初步评价

为筛选及评价用于牛结核病诊断的抗原,本试验将CFP-10、ESAT-6、TB10.4、TB27.4、MPT51、MPT63、MPT64、MPB70、MPB83、Rv3872和Ag85B共11种牛分枝杆菌抗原分别作为包被抗原建立间接ELISA方法,比较其对牛结核病的检出率;同时利用豚鼠和牛的皮试试验评价重组蛋白作为皮试试验刺激原的潜力。此外,将重组蛋白分别刺激结核病阳性牛和阴性牛的抗凝血24 h,检测血浆中的IFN-γ水平,评价各重组蛋白作为IFN-γ释放试验刺激原的潜力。结果显示,不同重组蛋白对结核病阳性血清的反应活性不一,MPB70总检出率最高,为59.7%;其次是Ag85B、ESAT-6和MPB83,检出率均在45%以上;MPT51的检出率最低,仅为2.2%。豚鼠和牛皮试试验均显示,单个重组蛋白作为刺激原难以产生令人满意的迟发型过敏反应（delayed type hypersensitivity,DTH）,而TB10.4、TB27.4、MPT64、MPT63或Rv3872作为补充抗原,分别与CFP-10或ESAT-6混合,均可特异性地刺激结核病阳性牛产生较强的DTH反应,且与PPD-B无显著差异（P>0.05）。重组蛋白CFP-10、ESAT-6、TB10.4和MPT51均能刺激结核病牛全血释放一定的IFN-γ,其中CFP-10、CFP-10-ESAT-6串联蛋白和MPT51刺激结核病阳性牛全血释放的IFN-γ显著高于阴性牛（P<0.05）。因此,这11种牛分枝杆菌抗原并不适合单独用于牛结核病的血清学诊断、皮试试验或IFN-γ释放试验,但以CFP-10和ESAT-6为核心,TB10.4、TB27.4、MPT64、MPT63、Rv3872或MPT51作为其补充抗原,均能提高检测敏感性,有作为皮试试验和IFN-γ释放试验特异性刺激原用于牛结核病诊断的潜力。     

Blood culture and stimulation conditions for the diagnosis of tuberculosis in cervids by the Cervigam assay

Mitogen- and antigen-induced interferon-gamma (IFN-gamma) responses of peripheral blood leucocytes from cervids were evaluated by a commercial whole-blood assay. The assay was applied to Mycobacterium bovis-infected white-tailed deer and reindeer, M bovis BCG-vaccinated white-tailed deer and elk, and unvaccinated, uninfected white-tailed deer, fallow deer, elk and reindeer. The responses of the M bovis-infected white-tailed deer to pokeweed mitogen (PWM) varied with time and between individuals. The responses of the M bovis-infected reindeer to PWM and M bovis purified protein derivative (PPD) were positively associated. Samples from tuberculosis-free captive herds in various parts of the USA were also evaluated. Four per cent of fallow deer, 20 per cent of elk, 44 per cent of white-tailed deer, and 91 per cent of reindeer had responses to PWM exceeding 0.25 Delta optical density, that is, PWM stimulation minus no stimulation. The specificity of the responses to M bovis PPD and a Mycobacterium tuberculosis complex-specific antigen rESAT-6:CFP-10, excluding animals not responding to PWM, ranged from 78 per cent to 100 per cent and was dependent upon the species and the positive response cut-off value. The results show that the commercial assay is valid for the detection of TB in reindeer; however, further development of the assay will be required before it is used in surveillance programmes for white-tailed deer, fallow deer, and elk.     

Evaluation of MPB70, bovine PPD and lipoarabinomannan as antigens in ELISA for the serodiagnosis of bovine tuberculosis

The performance of the secretory protein MPB70 of Mycobacterium bovis, bovine PPD, and lipoarabinomannan (LAM) were evaluated as antigens in ELISA for detection of tuberculosis (TB) infected cattle. Sera were from 120 M. bovis infected cattle and 223 cattle from a TB free herd. ELISA results were analyzed using receiver operating characteristic (ROC) curves in relation to culture results. The areas under the three ROC curves were 71 ± 49% SE (MPB70), 71 ± 27% SE (bovine PPD), and 56 ± 4% SE (LAM).     

Mycobacterium bovis-infected white-tailed deer (Odocoileus virginianus): detection of immunoglobulin specific to crude mycobacterial antigens by ELISA.

White-tailed deer (Odocoileus virginianus) have recently emerged as a source of Mycobacterium bovis infection for cattle within North America. The objective of this study was to evaluate the antibody response of M. bovis-infected deer to crude mycobacterial antigens. Deer were experimentally inoculated with M. bovis strain 1315 either by intratonsilar instillation or by exposure to M. bovis-infected (i.e., in contact) deer. To determine the time course of the response, including the effects of antigen administration for comparative cervical skin testing, serum was collected periodically and evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (i.e., IgG heavy and light chains) reactivity to mycobacterial antigens. The reactivity to M. bovis purified protein derivative (PPDb) exceeded (P < 0.05) the reactivity to M. avium PPD (PPDa) only after in vivo administration of PPDa and PPDb for comparative cervical testing of the infected deer. The mean immunoglobulin response, as measured by ELISA, of intratonsilar-inoculated deer to a proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis strain 1315 exceeded (P < 0.05) the mean of the prechallenge responses to this antigen at approximately 1 month after inoculation and throughout the remainder of the study (i.e., approximately 11 months). This response also exceeded (P < 0.05) that of the uninfected deer. Although this is encouraging, further studies are necessary to validate the use of the proteinase K-digested M. bovis antigens in the antibody-based assays of tuberculosis.     

In vitro and in vivo evaluation of lysozyme extracts of virulent Mycobacterium bovis in guinea pigs and calves

Extracts of Mycobacterium bovis ATCC 19210 were prepared from cells following treatment with acetone 3 times, ethyl alcohol-ether 3 times (1:1, v/v), and chloroform 3 times. Cells were dried and suspended in 0.05M Tris-HCl (pH 7.5) containing lysozyme (1 mg/50 mg of dried cells). One aliquot of the lysozyme extract was filter-sterilized and 1 aliquot of the lysozyme extract was autoclaved. Delayed-type hypersensitivity responses elicited in sensitized guinea pigs, using the filter-sterilized lysozyme extract, were significantly greater than responses elicited using the autoclaved lysozyme extract (P less than 0.01). The filter-sterilized lysozyme extract and a purified protein derivative (PPD) of M bovis, at equal protein concentrations, elicited comparable delayed-type hypersensitivity responses in sensitized guinea pigs. Significant differences were not detected between the mean enzyme-linked immunosorbent assay (ELISA) values on sera collected from calves before exposure to M bovis, using each of the lysozyme extracts or the M bovis PPD (P greater than 0.05). Significant differences were detected when ELISA values obtained using each of the antigens on post-exposure serum were compared with ELISA values on serum collected from calves before exposure to M bovis (P less than 0.01). Differences were not detected in mean ELISA values on sera from cattle collected 12 months after exposure to M bovis, using each of the lysozyme extracts or M bovis PPD (P greater than 0.05); however, 8 of 8 calves were identified as positive on ELISA, using the filter-sterilized lysozyme extract, 7 of 8 calves were positive, using M bovis PPD, and 7 of 8 calves were positive, using the autoclaved lysozyme extract.     

Lymphocyte subset proliferative responses of Mycobacterium bovis-infected cattle to purified protein derivative

Despite highly successful eradication efforts in several countries, Mycobacterium bovis infection of cattle remains a significant health concern worldwide. Immune mechanisms of resistance to and/or clearance of M. bovis infection of cattle, however, are unclear. Recent studies have provided evidence supporting a role for CD4(+), CD8(+), and gammadelta TCR(+) T cells in the response of cattle to M. bovis. In the present study, we utilized a flow cytometric-based proliferation assay to determine the relative contribution of individual lymphocyte subsets in the response to M. bovis infection and/or sensitization with mycobacterial purified protein derivative (PPD). Peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle proliferated in response to in vitro stimulation with M. bovis PPD. CD4(+) T cells and gammadelta TCR(+) cells were the predominate subsets of lymphocytes responding to PPD. gammadelta TCR(+) cells also proliferated in non-stimulated cultures; however, the gammadelta TCR(+) cell proliferative response of infected cattle was significantly (p<0.05) greater in PPD-stimulated cultures as compared to non-stimulated cultures. Intradermal injection of PPD for comparative cervical testing (CCT) induced a boost in the in vitro proliferative response of CD4(+) but not gammadelta TCR(+) cells of infected cattle. Administration of PPD for CCT also boosted interferon-gamma (IFN-gamma) production by PBMC of infected cattle following in vitro stimulation with M. bovis PPD. Injection of PPD for CCT did not, however, elicit a proliferative or IFN-gamma response in cells isolated from non-infected cattle. These data indicate that CD4(+) and gammadelta TCR(+) cells of M. bovis-infected cattle proliferate in a recall response to M. bovis PPD and that the CD4(+) cell response is boosted by intradermal injection with PPD for CCT.     

Evaluation of three serological assays for the diagnosis of Mycobacterium bovis infection in brushtail possums

Three serological tests for the diagnosis of Mycobacterium bovis infection were evaluated on 29 possums (Trichosurus vulpecula) with tuberculosis and on 100 possums from a tuberculosis-free area. An indirect ELISA using M. bovis culture filtrate as the antigen had a sensitivity of 45% and a specificity of 96%, while an indirect ELISA using a M. bovis specific antigen (MPB70) had a sensitivity of 21% and a specificity of 98%. A blocking ELISA which utilised a monoclonal antibody against MPB70 had a sensitivity of 28% and a specificity of 99%. Combination of the test results of the three ELISAs resulted in an increase in sensitivity to 51% and a decrease in specificity to 93%. A previous study has shown that possums experimentally infected with M. bovis produced cellular responses to M. bovis antigens relatively early in the infection, but these responses decreased in the terminal stages of the disease. In contrast, analysis of serological responses in the current study from sequentially collected sera of possums experimentally and naturally infected with M. bovis showed that antibody was first detected late in the disease.     

Mycobacterium bovis infection in North American elk (Cervus elaphus).

A naturally occurring outbreak of Mycobacterium bovis infection in captive wild elk (wapiti) in Montana was confirmed by mycobacteriologic examination. Twenty-eight of 143 elk responded to M. bovis purified protein derivative (PPD) tuberculin injected intradermally in the cervical region (SCT). The results of comparative cervical tuberculin skin tests conducted within 9 days of SCT revealed greater responses to M. bovis PPD tuberculin than to M. avium PPD tuberculin in 23 of 28 elk responding. At necropsy, several grossly visible tuberculous lesions were observed in the parenchyma of the lung, thoracic lymph nodes, and submandibular lymph nodes. Microscopic examination of appropriately stained tissue sections revealed the presence of granulomatous lesions containing acid-fast bacilli. An enzyme-linked immunosorbent assay (ELISA) was developed using a sarkosyl extract of M. bovis (antigen) and peroxidase-labeled protein G (conjugate); reactions were detected in the sera of 8 of 9 elk responding to M. bovis PPD tuberculin. Lymphocyte blastogenic assay responses were detected using M. bovis antigens in 7 of 9 elk positive on skin tests using M. bovis PPD.