Use of PPD and phosphatide antigens in an ELISA to detect the serological response in experimental bovine tuberculosis

Five calves from tuberculosis free herds were each inoculated intranasally with 10(6) viable organisms of a field isolate of Mycobacterium bovis. Four of the calves developed acute tuberculosis. ELISAs employing protein and phosphatide extracts of M bovis as antigens were used to monitor the humoral response of the infected calves. Fourteen days after infection there was a dramatic increase in the level of antibodies demonstrated by the phosphatide antigen. This increase coincided with the first appearance of signs of the disease. The results suggest that the phosphatide antigen may be of potential value in detecting a humoral response, if present, in cattle infected with M bovis. The tests employing the protein antigen demonstrated a humoral response in only one of the infected calves and emphasises the importance of antigen selection to detect antibodies in tuberculous animals.     

Preparation of biologically active components of Mycobacterium bovis, using Triton X-100 or potassium chloride

Methods of producing antigen extracts of Mycobacterium bovis are described. Extraction of M bovis with a chaotropic salt, potassium chloride, or a nonionic detergent, Triton X-100, before autoclaving resulted in the production of soluble M bovis components. These antigens induced delayed-type hypersensitivity responses when injected intradermally in M bovis-sensitized guinea pigs. A comparison of the diameters of the hypersensitivity responses (on the basis of equal protein concentrations in the inocula) revealed that the potassium chloride extract and the Triton extract of M bovis induced similar responses compared to the response induced with a purified protein derivative of M bovis. These findings indicate alternative methods may be used for the production of biologically active extracts of M bovis.     

Immunomodulation with killed Propionibacterium acnes in guinea pigs simultaneously vaccinated with Brucella abortus strain 19

Immunomodulation with killed Propionibacterium acnes was attempted in guinea pigs simultaneously vaccinated with Brucella abortus strain 19. Two groups, each comprised of 9 guinea pigs, were injected by different routes (s.c. and or i.v.) with 1.4 mg of P. acnes and 5 X 10(8) CFU of B. abortus, S-19, while 3 other groups each received either P. acnes, B. abortus S-19, or saline (s.c.). The antibody titers to B. abortus measured at 6, 10 and 14 weeks after vaccination indicated no significant (P less than 0.01) response in the 2 groups immunopotentiated with P. acnes concurrent with B. abortus S-19 vaccination. The delayed hypersensitivity response to 3 Brucella antigens conducted 8 weeks after immunization did not show a significant difference between the B. abortus S-19 vaccinated group compared with the 2 groups immunopotentiated and vaccinated. However, the proliferative response of lymphocytes to the B. abortus soluble antigen diluted 1:100 indicated significantly enhanced blastogenesis in the (s.c.) immunopotentiated and immunized guinea pigs compared with the B. abortus S-19 vaccinated group. A slightly enhanced response was also observed in the group immunopotentiated (i.v.) and vaccinated (s.c.). The guinea pigs were challenged with B. abortus strain 2308 and necropsied 4 weeks later. The mean splenic CFU of the Brucella in the group immunopotentiated (i.v.) and vaccinated (s.c.) was significantly decreased when compared with the guinea pigs vaccinated with B. abortus S-19 alone. These findings indicated that P. acnes administered simultaneously with B. abortus S-19 vaccine was able to augment the immune response in guinea pigs. Immunomodulation as evidenced by enhanced clearance of B. abortus from the spleens of immunopotentiated animals was presumably brought about by activated macrophages or a T-cell mediated cytolytic mechanism or both.     

Observation of the Growth Trends of Mycoplasma bovis with Four Different Antigen Quantitation Methods

To explore the antigen harvest time of Mycoplasma bovis (M.bovis) and the antigen quantitation alternative method,M.bovis 08M strain was inoculated in the Thiaucourt's medium.Four growth curve plottings were made by measuring color change units (CCU),colony forming units (CFU),protein concentration and nucleic acid levels.Both the results of CCU and CFU counts showed that the growth of M.bovis was divided into four phases,the logarithmic phase appeared after being cultrured 10 h,the stationary phase appeared at 30 h with the highest number of viable cells up to 1.0×10⁸ CCU/mL and 7.7×10⁷ CFU/mL,and the decline phase started at 75 h.The protein concentration of M.bovis increased rapidly from 15 to 35 h,reached 72.06 μg/mL at 35 h,then maintained at 58.38 to 70.65 μg/mL.The nucleic acid levels of M.bovis increased rapidly from 15 h,and the cycle threshold (Ct) values were maintained between 15.32 to 17.84 after 25 h.These results indicated that there was a good correlation between the protein concentration and viable count of M.bovis at the early stationary phase,which was the best time period to harvest antigen.The protein concentration determination could be an alternative method to quantify antigen contents of M.bovis when preparing inactivated M.bovis vaccine.     

4种抗原定量方法观察牛支原体培养特征

为探索在疫苗研制过程中牛支原体抗原收获时间及抗原定量替代方法,将牛支原体08M株接种于含10%马血清的Thiaucourt's培养基,在110 h内同时监测其颜色变化单位（color change units,CCU）、菌落形成单位（colony forming units,CFU）、菌体蛋白浓度和核酸含量的变化,绘制相应曲线。活菌计数结果（CCU和CFU）显示,牛支原体生长可分为明显的4期,10 h进入对数期,30 h进入稳定期,活菌数最高可达1.0×10⁸ CCU/mL和7.7×10⁷ CFU/mL,75 h进入衰亡期;蛋白浓度从15 h开始迅速增长,至35 h蛋白浓度最高,为72.06 μg/mL,此后维持在58.38~70.65 μg/mL;核酸含量从15 h开始增长,至25 h后Ct值维持在15.32~17.84。结果表明,牛支原体蛋白含量在稳定期初期与活菌数具有良好的相关性。因此,在牛支原体灭活疫苗生产中,稳定期初期是最佳抗原收获时间,可用蛋白浓度法代替活菌计数法进行抗原定量。     

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.     

Intranasally inoculated Mycoplasma hyorhinis causes eustachitis in pigs

Specific-pathogen-free pigs were experimentally inoculated with Mycoplasma hyorhinis, Pasteurella multocida, or both bacterial isolates to evaluate the role of these bacteria in the pathogenesis of otitis media. Six pigs were inoculated intranasally with 4.4 X 10(8) colony-forming units (CFU) of M. hyorhinis. Twenty-one days later, three of these six pigs were inoculated intranasally with 5.0 X 10(8) CFU of P. multocida. Three additional pigs were also inoculated intranasally at the time with P. multocida alone. Two pigs served as uninoculated controls. Seven days later, all pigs were euthanatized. Histologically, subacute inflammation was found in 10 auditory tubes of six pigs and two tympanic cavities of two pigs inoculated with M. hyorhinis. Immunohistochemically, M. hyorhinis antigens were detected on the luminal surface of eight of 10 inflamed auditory tubes, and ultrastructural examination confirmed mycoplasmal organisms in two pigs. M. hyorhinis was isolated from the inflamed tympanic cavities of two pigs. None of the pigs inoculated only with P. multocida had otitis, and P. multocida was not isolated from the tympanic cavity. These findings indicate that M. hyorhinis can cause eustachitis but rarely otitis media in specific-pathogen-free pigs.     

2308、M28、S1330三株不同种布鲁氏菌的毒力测定

为了测定牛、羊、猪三株不同种布鲁氏菌参考强毒株的毒力,选择了牛种2308、羊种M28和猪种S1330株,分别用雌性豚鼠（Hartley）和雌性小鼠（Balb/c）对其毒力进行测定。豚鼠测毒试验中,用含不同菌数的菌液腹股沟皮下注射5只豚鼠,测定2308、M28、S1330菌株的豚鼠最小感染量（MID）,结果显示以上3种毒株对豚鼠的最小感染量分别为9 CFU、10 CFU和30CFU。小鼠测毒实验中,将2308、M28和S1330菌液按1×105CFU/0.2 mL/只腹股沟皮下注射小鼠各5只,2周后分别剖杀小鼠,取脾脏测定含菌量,平均脾含菌量分别为1676971、314765、83811CFU/g脾脏。豚鼠和小鼠测毒均显示牛种2308株毒力最强,羊种M28株次之,猪种S1330毒力最弱。本研究首次用豚鼠和小鼠同时测定了布鲁氏菌2308、M28、S1330株的毒力,补充了布鲁氏菌参考强毒株的毒力数据。     

Analysis of the antigen-specific IFN-gamma producing T-cell subsets in cattle experimentally infected with Mycobacterium bovis

Three 10 months old cattle were infected by the intratracheal route with 10(6)cfu of a field strain of Mycobacterium bovis. Blood samples were regularly collected for in vitro IFN-gamma production after antigenic stimulation. Peripheral blood cells of infected animals produced IFN-gamma in response to crude M. bovis antigens (live and heat-inactivated BCG and protein-purified derivative (PPD)) 3-4 weeks after infection. The ratio of the response to bovine PPD versus avian PPD indicated a specific sensitisation for M. bovis antigens. Three months post-infection (PI), animals were culled and M. bovis was cultured from tubercle lesions. At different time points, the frequency of specific M. bovis IFN-gamma producing CD4+, CD8+ and WC1+ T-cells in the peripheral blood was examined by flow cytometry. Two colour immunofluorescence staining of intracellular IFN-gamma and bovine cell surface molecules showed that both CD4+ and CD8+, but not WC1+, T-cells produced IFN-gamma following stimulation with PPD, live or killed BCG.In two animals analysed, the relative percentage of circulating IFN-gamma producing CD8+ cells decreased between week 5 and week 9 PI. The same evolution was not observed for IFN-gamma secreting CD4+ cells. Magnetic positive selection of T-cells from infected animals showed that CD4+ T-cells produced specific IFN-gamma only in the presence of antigen presenting cells (APCs). Positively selected CD8+ T-cells secreted IFN-gamma only in the presence of recombinant human IL-2 and APCs. In vitro depletion of the CD4+ T-cells, but not the depletion of CD8+ or WC1+ T-cells, resulted in abrogation of the specific IFN-gamma production showing the key role of this cell population for the specific IFN-gamma production.     

Diagnostic tests for Trichinella spiralis infection in pigs. A comparative study of ELISA for specific antibody and histamine release from blood cells in experimental infections.

A study on the histamine release test (HR) for the demonstration of infections with Trichinella spiralis in pigs was carried out on 18 pigs, six infected with 200 larvae, six infected with 5000 larvae and six non-infected (control group). The results obtained by HR during a 7 week infection were compared with those of the enzyme-linked immunosorbent assay (ELISA). All inoculated pigs were found to be positive on Day 40 post-inoculation (p.i.) by necropsy examination of selected muscle groups, with mean recoveries of 7.9 and 225 larvae g-1 of tissue in the low- and high-dose group, respectively. At this time, all animals of the high-dose group and five out of six animals of the low-dose group were antibody positive in ELISA with any of three coating antigens employed (a crude muscle larva extract, an excretory/secretory (ES) antigen and a purified 45 kDa antigen). HR performed on whole blood was positive in four out of six pigs of the high-dose group and one out of six pigs of the low-dose group. The earliest ELISA seroconversions took place at Day 15 p.i. with crude and ES antigens. The earliest measurable reaction in HR performed on whole blood was found on Day 19 p.i. There was considerable individual variation regarding which test was the most sensitive for the early detection of infection. Washing of the blood cells prior to antigen provocation led to a markedly improved sensitivity of HR, all animals of the high-dose and three out of six animals of the low-dose group being positive by Day 40 p.i. The time course of the development of ELISA titres and HR reactivity indicated that this effect is due to the removal of blocking antibodies.     

Evaluation of the interferon-gamma (IFN-γ) assay to diagnose Mycobacterium bovis infection in pigs

Mycobacterium bovis recognizes as hosts a wide spectrum of animal species. In particular epidemiological situations, high prevalence of infection is found also in pigs. In the present study, we evaluated the capability of the interferon-gamma (IFN-γ) assay to identify pigs infected with M. bovis. The results of the immune-diagnosis were correlated to the findings of the post mortem inspection and the bacterial culture of lymph nodes. Blood samples of 146 pigs, belonging to a local breed of Sicily reared in free or semi-free roaming conditions, were collected to assess the specificity and the sensibility of the IFN-γ assay. Thirty-one pigs, from M. bovis free herds, did not react to the IFN-γ assay, yielding a specificity of 100%. The IFN-γ assay identified 15 out of 19 animals positive to the bacterial culture and 22 out of 26 animals with tuberculous lesions, with a sensibility of 78.9-84.6%, respectively. Out of 26 reactors to the test, 15 pigs (57.7%) confirmed to be infected after the bacterial culture and 22 (84.6%) had tuberculous lesions. The IFN-γ assay was able to reveal 4 animals with no visible lesions (NVL). Together, these findings support the feasible use of the IFN-γ assay as an intra vitam tool for the surveillance and management of M. bovis infection in swine populations.     

Experimental inoculation of wild turkeys (Meleagris gallopavo) with Mycobacterium bovis

Although avian species are known to be susceptible to infection with Mycobacterium spp. organisms, much remains unknown about the susceptibility of birds to infection with M. bovis. The objective of this current study was to determine if wild turkeys (Meleagris gallopavo) can be infected with M. bovis when inoculated by the oral or intratracheal route. Six turkeys were orally inoculated and another six were inoculated via the trachea with a high dose of M. bovis, 1 x 10(5) CFU/ml. Six turkeys were sham-inoculated controls. Two turkeys from each treatment group were sacrificed on days 30, 60, and 90 postinoculation. There were no gross or microscopic lesions consistent with mycobacteriosis in the 23 inoculated turkeys over the 90-day duration of this study. Fecal cultures were also consistently negative for M. bovis when sampled before inoculation and on days 1, 30, and 60 postinoculation. Two intratracheally inoculated turkeys were positive for M. bovis in visceral tissues at 30 days postinoculation. However, this finding was only indicative of passive persistence of mycobacteria in the tissues and not of infection, as there were no attendant lesions or clinical compromise to support infection. Thus, it can be concluded that young wild turkeys are resistant to infection with M. bovis and, therefore, pose minimal threat as reservoir or spillover hosts for this organism.     

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。     

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.     

Transmission of tuberculosis from experimentally infected cattle to in-contact calves

Five of a group of six calves were inoculated with Mycobacterium bovis. Two more uninoculated calves were introduced to the group 84 days later. All the inoculated calves were subsequently shown to be excreting M bovis in nasal mucus. The uninoculated calf in the initial group of six became infected and subsequently excreted M bovis. The two uninoculated calves which were introduced later did not become infected. It was concluded that contact with nasal mucus from the infected cattle resulted in infection of the uninoculated calf and that the density of accommodation of animals excreting M bovis was an important factor in transmission of the disease.     

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.     

Vaccines designed to protect against Mycobacterium tuberculosis infection may aid the identification of novel vaccine constructs and diagnostic antigens for bovine tuberculosis

Vaccination has been identified as a promising control strategy for tuberculosis in both humans and cattle. Recent heterologous prime-boost approaches combining BCG vaccination with subunit boosts have shown considerable promise in both fields. However, the identification of further protective antigens is still a research priority. In this paper we have established the response hierarchy in Mycobacterium bovis infected or BCG vaccinated cattle of 6 Mycobacterium tuberculosis-derived proteins that were protective against M. tuberculosis in the guinea pig aerosol challenge model. Two of these proteins, Rv1806 and Rv3812, were recognised most frequently in cattle and therefore constitute potential subunit vaccine candidates that merit further evaluation in cattle. Their epitopes were mapped in infected cattle and were shown to be located primarily in the non-conserved regions of these PE/PE-PGRS protein family members. The aim of this study was to ascertain the presence of any correlation between the immunogenicity of defined antigens in different animal species. A weak association between guinea pig immunogenicity (as measured by protection) and antigenicity in M. bovis infected or BCG vaccinated cattle was found.     

Antibodies to mycobacteria in cattle not infected with Mycobacterium bovis

An indirect anti-IgG enzyme-linked immunosorbent assay (ELISA) using a whole cell sonicate of Mycobacterium bovis as the coating antigen, was used to detect anti-mycobacterial antibodies in cattle not infected with M. bovis. False positive M. bovis ELISA scores were produced in 6 cattle experimentally inoculated with Mycobacterium avium-intracellulare-scrofulaceum (MAIS) serovars 2, 8, 9, 14 and 18 and Mycobacterium flavescens, respectively. False positive ELISA results were also found in 39.5% of cattle from which other mycobacteria were cultured and in 56.4% of necropsied cattle with other pathological conditions. No M. bovis was cultured from these animals. Other groups of animals, with no pathological conditions, which had been tuberculin-tested negative, tuberculin-tested positive and never tuberculin tested showed positive ELISA results in 15.4%, 73.6% and 42.4% of the respective groups. The variation of these non-specific responses in uninfected cattle highlights the need for careful selection of negative controls in evaluating ELISAs for the diagnosis of bovine tuberculosis.     

Pulmonary lesions in guinea pigs experimentally infected with Actinobacillus pleuropneumoniae (A.p.) serovar 1

Pathological studies were carried out on the lungs of guinea pigs intratracheally inoculated with 4.6 x 10(6-8) colony forming units (CFU)/head of Actinobacillus pleuropneumoniae serovar 1. All animals in the highest dose group died within 24 hr post inoculation (hpi) and showed pulmonary lesions being hemorrhagic in nature while all animals in the lowest dose group were killed as scheduled at 11 days post inoculation (dpi) and showed only hyperplasia of peribronchial lymphoid tissues. In the middle dose group, two died within 24 hpi, two died at 9 dpi, and the remaining one was killed at 11 dpi. Two guinea pigs which died at 9 dpi showed fibrinonecrotic pleuropneumonia which is the most characteristic acute pulmonary lesion in swine, and has not yet been reproduced in laboratory animals up to the present time. This suggests that guinea pigs may be a useful laboratory animal for studying the pathogenesis of Actinobacillus pleuropneumoniae infection in swine.     

Specific antibody responses to Mycobacterium bovis in infected cattle analysed with six mycobacterial antigens in enzyme-linked immunosorbent assays

A total of 23 (15.3 per cent) of 150 cattle infected with Mycobacterium bovis and which had never been tuberculin tested showed specific antibody responses to M bovis. Their sera may be important keys to the identification of unique M bovis antigens for use in specific serodiagnostic tests. Assessment of specific and non-specific responses was done by screening sera in six indirect anti-IgG enzyme-linked immunosorbent assays using whole cell sonicates of M bovis and five members of the Mycobacterium avium-intracellulare-scrofulaceum complex as respective antigens. Sera from 16 infected cattle that had been tuberculin tested positive and nine uninfected cattle (never tuberculin tested) were also assayed for specific and non-specific responses. Three other findings emerged. First, 43 of the 150 infected animals (28.7 per cent) showed no antibody responses to any of the mycobacterial antigens used. Secondly, the cattle showing the highest antibody levels were associated with the greatest cross reactivity. Lastly, the results indicated that tuberculin injections may increase antibody responses to shared, rather than specific, M bovis antigens in infected cattle.