Evaluation of adenosine deaminase seric activity in the diagnosis of bovine tuberculosis

Determination of seric levels of adenosine deaminase (ADA), an enzyme produced by monocytes/macrophages and lymphocytes, has been used in the diagnosis of human tuberculosis (TB). In the present study, ADA seric activity was evaluated comparatively to the comparative tuberculin test in the diagnosis of bovine tuberculosis. Two hundred fifty-six cattle were classified by origin and by the comparative tuberculin test as TB-positive animals (n = 52, from herds where the Mycobacterium bovis had previously been isolated), and TB-negative animals (n = 204, TB-free herds). The mean ADA seric value from the TB-positive group (4.45 +/- 2.33 U/L) was significantly lower (p = 0.008) than that observed in sera from the TB-negative group (6.12 +/- 4.47 U/L). When animals from a herd with clinical cases of enzootic bovine leukosis of TB-negative group were withdrawn from analysis, the mean ADA seric values of TB-negative group (5.12 +/- 3.75 U/L) was not significantly different anymore from that of the TB-positive group (p = 0.28). There was no agreement in the diagnosis of bovine TB between comparative tuberculin test and determination of ADA seric values, using two different cutoff points, being 6.12 U/L and 15.0 U/L, (kappa = -0.086 and kappa = -0.082, respectively). In conclusion, the determination of ADA seric activity was not a good auxiliary test for bovine TB, because it was not able to distinguish between TB-positive and TB-negative animals.     

An attempt to predict anergy in tuberculosis suspect cynomolgus monkeys.

Acid-fast microorganisms were identified from the tuberculous lesions of a male cynomolgus monkey (Macaca fascicularis). Twenty-two other cynomolgus monkeys housed in the same room were presumed exposed to tuberculosis (Mycobacterium spp.). In addition to standard intradermal (ID) tuberculin testing, clinicians attempted to evaluate the immune status of these monkeys in order to identify animals exhibiting false negative (anergy) ID tuberculin tests. Twenty-one of the potentially exposed monkeys were immunized with tetanus toxoid (TT). Tetanus antitoxin (TAT) titers were measured before and after immunization. The delayed cutaneous hypersensitivity (DCH) reaction to TT was evaluated using a commercially available human test panel. Some animals did not exhibit a DCH reaction to TT. At necropsy 1 of the 21 animals exhibited tuberculous lesions, and acid-fast microorganisms were identified on direct smears of lymphatic tissue of a second animal. Although reported to be of value in assessing the cellular immune status of rhesus monkeys (Macaca mulatta), the delayed cutaneous hypersensitivity response to tetanus toxoid was not helpful during this outbreak in identifying cynomolgus monkeys infected with M. tuberculosis, or in interpreting suspect ID tuberculin tests.     

Tuberculin Reactions in Bulls And Boars Sensitized with Atypical Mycobacteria from Sawdust

Due to a planned export from a combined bull and boar station, more than 70 boars and 100 bulls were examined by tuberculin tests. Distinct reactions to avian tuberculin appeared in about half of the animals. Many of them also reacted to bovine tuberculin. For diagnostic purposes, many of the reactors were slaughtered, and samples from these and from the environment were examined bacteriologically. Strains of Mycobacterium avium were isolated from only 2 out of 14 reacting boars and from none of the 23 reacting bulls. No isolation of Mycobacterium bovis was made. However, atypical mycobacterial strains, classified as Runyon Group III and IV, were isolated from 3 boars, 2 bulls, 1 pigeon and from many samples of sawdust. The isolation of identical fast-growing mycobacterial strains from the sawdust used in the pens for the reacting boars and bulls, was especially remarkable. The strains differed enzymatically and biochemically from those isolated from other sources. This indicated a possible sensitization of the animals with similar mycobacterial strains. Possible cross-reactions to avian and bovine tuberculin were investigated in tuberculin assays with guniea pigs and pigs sensitized to one of the mycobacterial strains isolated. Distinct reactions to both avian and bovine tuberculin appeared in all the animals. From these results it was concluded that the tuberculin reactions in the boars and the bulls were not due to any tuberculous infection in the herd, but to a sensitization of the animals with atypical mycobacteria.     

Paratuberculosis Vaccination Causes Only Limited Cross-Reactivity in the Skin Test for Diagnosis of Bovine Tuberculosis

Although there is a wide consensus on the efficacy of paratuberculosis vaccination to limit economic losses, its use has been restricted because of its interference in the diagnosis of tuberculosis. Data from a vaccine clinical trial in the Basque Country (Spain) has been evaluated in relationship with bovine tuberculosis intradermal test results. The trial included two herds applying a Test and Culling strategy and five applying an inactivated vaccine. The vaccine was applied to animals of all ages present in each vaccinated herd when joining the trial, and then to all the replacers within their first three months of life. Yearly testing done with the comparative intradermal test (CIT) was applied to all animals older than 6 weeks. Between 2005 and 2011, the study generated 2,033 records from Vaccinated Herds (VH) and 2,252 from Test and Cull herds (TC). Pre-vaccination positive results rate was 2.40% among the 7 herds in the single bovine intradermal tuberculin test (BSIT). Two years later it rose to 20.42% in the VH and remained below at 0.75% in the TC. Applying the CIT reduced these rates to only 0.58% in the VH and to 0.25% in the TC ons. Regarding time since each animal joined the program, the proportion of positives to BSIT was variable and, in some cases, significantly different between time points. With regard to the age of vaccination, no significant differences were found between vaccination within the first year of life and afterwards. Vaccinated animals showed seventeen times more reactions than the non-vaccinated in the BSIT, but only four times more in the CIT. In conclusion, comparative intradermal test can be a useful tool to differentiate paratuberculosis vaccine cross-reactions from specific bovine tuberculosis reactions according to the European and Spanish legislation.     

Longitudinal study of tuberculosis outcomes among immunologically naive Aché natives of Paraguay

This study documents the course of a tuberculosis epidemic in an immunologically naive group of South American Indians within fewer than 20 years after first sustained contact with outsiders. Groups of Northern Aché (ah-CHAY) of eastern Paraguay were contacted and settled on reservations between 1971-1979. Not surprisingly, the Aché are very susceptible to tuberculosis, and the epidemiological characteristics of the disease are quite different from those of populations that have had tuberculosis for centuries. Within 6 years of the first detected case of tuberculosis among the Aché, the prevalence rate of active tuberculosis cases reached 18.2%, and of infected cases among adults, 64.6%, some of the highest rates ever reported for any human group. Remarkably, males and females are equally likely to have been diagnosed with active tuberculosis, Aché children between birth and 5 years of age are least vulnerable to tuberculosis, high nutritional and socioeconomic status do not decrease the risk of disease or infection, and children immunized with BCG are less responsive to tuberculin challenge than are other children. Moreover, similar to the Yanomam?, but unlike populations of European or African descent, a high percentage of Aché with active disease test negative on tuberculin challenge tests (purified protein derivative; PPD). These differences may be due to a high prevalence of diminished cell-mediated immunity, and T-helper 2 dominance. We also hypothesize that these immunological characteristics, low genetic diversity, hostile intergroup interactions, and behavioral noncompliance to treatment protocols together contribute to the high rates of active disease observed. Existing tuberculosis control programs are poorly equipped to handle the impact of these causal complexities on the course of recent tuberculosis epidemics that have quickly spread throughout native communities of Latin America during the last decade.     

New approaches to tuberculosis surveillance in nonhuman primates

Despite significant progress in reducing the incidence of tuberculosis in nonhuman primates (NHPs) maintained in captivity, outbreaks continue to occur in established colonies, with potential serious consequences in human exposures, animal losses, disruption of research, and costs related to disease control efforts. The intradermal tuberculin skin test (TST) using mammalian old tuberculin (MOT) has been the mainstay of NHP tuberculosis surveillance and antemortem diagnosis for more than 60 years. But limitations of the TST, particularly its inability to reliably identify animals with latent TB infections, make it unsuitable for use as a single, standalone test for TB surveillance in nonhuman primates in the 21st century. Advances in technology and the availability of Mycobacterium spp. genomic sequence data have facilitated the development and evaluation of new immune-based screening assays as possible adjuncts and alternatives to the TST, including in vitro whole blood assays that measure the release of interferon gamma in response to stimulation with tuberculin or specific mycobacterial antigens, and assays that detect antibodies to highly immunogenic secreted proteins unique to M. tuberculosis, M. bovis, and other species belonging to the M. tuberculosis complex. It is becoming apparent that no single screening test will meet all the requirements for surveillance and diagnosis of tuberculosis in nonhuman primates. Instead, the use of several tests in combination can increase the overall sensitivity and specificity of screening and surveillance programs and likely represents the future of TB testing in nonhuman primates. In this article we describe the characteristics of these newer screening tests and discuss their potential contributions to NHP tuberculosis surveillance programs.     

Normal erythrocyte sedimentation rate in the elderly.

Two hundred subjects aged 60-89 were selected for a study aimed at defining a reference range for the erythrocyte sedimentation rate in the elderly. The study extended a previous survey in subjects aged 20-65. The results confirmed that the sedimentation rate increases with age and that women have higher values than men but suggested that over half of elderly patients with disease would have rates within the previously defined "normal" range. It is therefore suggested that an erythrocyte sedimentation rate exceeding 19 mm in the first hour in elderly men and 22 mm in the first hour in elderly women warrants investigation.     

Tuberculosis in patients with human immunodeficiency virus infection. Review of current concepts.

Tuberculosis is a frequent complication of human immunodeficiency virus (HIV)-induced immunosuppression. The diagnosis of extrapulmonary tuberculosis in patients with evidence of HIV infection qualifies as a criterion of the acquired immunodeficiency syndrome. Demographic characteristics of patients with tuberculosis and HIV infection vary by region and reflect the degree to which patients with Mycobacterium tuberculosis infection adopt behaviors that put them at risk for HIV infection. The clinical features of tuberculosis in patients with HIV infection are atypical. Extrapulmonary disease, tuberculin anergy, and unusual findings on chest radiographs occur most frequently when tuberculosis afflicts patients with other clinical evidence of HIV infection at the time tuberculosis is diagnosed. Treatment is effective for tuberculosis in HIV-seropositive patients, and isoniazid prophylaxis is recommended for HIV-infected patients with positive tuberculin skin tests.     

An outbreak of tuberculosis in a group of experimental baboons

A spontaneous outbreak of tuberculosis occurred in an isolated group of 21 baboons being used in an adenovirus type 12 oncogenesis study. Seventeen of the 21 animals were affected. Diagnosis was made by intradermal skin test, chest radiographs, and peripheral blood counts. Confirmation of the diagnosis was by gross and histopathology and culturing of the causative organism. Intradermal tuberculin skin tests were performed simultaneously in the eyelid (intrapalpebral) and abdomen. Fifteen of 16 baboons tested having tuberculosis showed significant reactions in the abdominal skin, whereas only 5 of the 16 had reactions in the eyelid. Infection was respiratory with extensive pathology in the pulmonary viscera. The pathology resembled that of simian tuberculosis, with miliary lesions in the spleen, liver, and in several cases, other abdominal viscera. It was characterized by caseation necrosis and an absence of calcification. Histologically, the lesions resembled the disease described in the great apes rather than the lower monkeys, with numerous Langhans giant cells. The causative organism resembled the human typeMycobacterium tuberculosis morphologically and culturally.These studies were supported in part by USPHS grant numbers GM-FR13252-02 and FR-00278-03.     

Skin reactivity to mycobacterial antigens in children living in an area of Mycobacterium xenopi endemicity

Intradermal skin tests with a 2TU dose of PPD-RT 23 prepared from M. tuberculosis and 0.1 ug/0.1 ml of PPD-RS 631 from M. xenopi were simultaneously carried out in 378 7-year-old children from two localities in North-Bohemian region's capital Ustí n. Lab., a focus of M. xenopi endemicity repeatedly confirmed since its disclosure in 1980 by positive M. xenopi isolations from humans and public water supply network. A further group 157 children serving as controls was from Prague district 4 where no presence of M. xenopi strains was ever recorded. All of these children had received routine immunization at birth with Czech BCG vaccine. The children from the two endemic localities were found to give a positive 6 mm or greater reaction to M. xenopi mycobacterin in 43.3% and 22.3%, to human tuberculin in 12.8% and 12.6%, respectively. The frequency histogram clearly separated a group of reactors with 8-18 mm indurations from a group of nonreactors showing a skin induration of 4-8 mm. The higher reactivity of this exposed child population was also reflected in a larger proportion of reactions greater to M. xenopi PPD than to human tuberculin antigen: the reactions greater by 1-5 mm accounted, respectively, for 25.1% and 20.6%, reactions greater by 6 mm or more for 23.7% and 15.9%. Among a group of children from Prague district 4, 6.4% had medium-sized and 3.8% large-sized reactions to M. xenopi antigen; the proportion of reactions greater to M. xenopi antigen than to human tuberculin accounted for only 5.1%, reactions greater to tuberculin than to sensitin were here in slight predominance. The evidenced skin sensitization to M. xenopi mycobacterin is suggested to result from the different degrees of exposure to infection by environmental mycobacteria.     

Tuberculin Activity of Mycobacterial Fractions Obtained by Chromatography

Commercial purified protein derivatives (PPD), old tuberculin (OT), the bacillary extract, and the culture filtrate of Mycobacterium tuberculosis H37Ra were submitted to Sephadex G-25 and diethylaminoethyl (DEAE)-cellulose chromatography. The ability of the fractions obtained to elicit delayed dermal hypersensitivity in M. tuberculosis H37Ra-infected guinea pigs was studied. Skin tests with Sephadex fractions in M. tuberculosis H37Ra-infected guinea pigs showed that the tuberculin activity was localized in the first fraction. All other Sephadex fractions were nonessential and nonspecifically irritating. Fractions from chromatography of Sephadex G-25 fraction 1 on DEAE-cellulose columns showed that all but the first were able to elicit delayed hypersensitivity reactions. There was a variability in the capacity to elicit the tuberculin reaction according to the fraction injected and the stage of tuberculous infection in guinea pigs. Compared to the others, the seven lots of commercial PPD were variable in composition and content. They contained both essential and nonessential materials for the tuberculin reaction. Sephadex fraction 1 would appear to be a better tuberculin as it excludes nonessential nonspecifically irritating elements and contains the complement able to elicit the tuberculin reaction. Its methodological simplicity would be economically advantageous.     

Revisiting Host Preference in the Mycobacterium tuberculosis Complex: Experimental Infection Shows M. tuberculosis H37Rv to Be Avirulent in Cattle

Experiments in the late 19th century sought to define the host specificity of the causative agents of tuberculosis in mammals. Mycobacterium tuberculosis, the human tubercle bacillus, was independently shown by Smith, Koch, and von Behring to be avirulent in cattle. This finding was erroneously used by Koch to argue the converse, namely that Mycobacterium bovis, the agent of bovine tuberculosis, was avirulent for man, a view that was subsequently discredited. However, reports in the literature of M. tuberculosis isolation from cattle with tuberculoid lesions suggests that the virulence of M. tuberculosis for cattle needs to be readdressed. We used an experimental bovine infection model to test the virulence of well-characterized strains of M. tuberculosis and M. bovis in cattle, choosing the genome-sequenced strains M. tuberculosis H37Rv and M. bovis 2122/97. Cattle were infected with approximately 10⁶ CFU of M. tuberculosis H37Rv or M. bovis 2122/97, and sacrificed 17 weeks post-infection. IFN-γ and tuberculin skin tests indicated that both M. bovis 2122 and M. tuberculosis H37Rv were equally infective and triggered strong cell-mediated immune responses, albeit with some indication of differential antigen-specific responses. Postmortem examination revealed that while M. bovis 2122/97–infected animals all showed clear pathology indicative of bovine tuberculosis, the M. tuberculosis–infected animals showed no pathology. Culturing of infected tissues revealed that M. tuberculosis was able to persist in the majority of animals, albeit at relatively low bacillary loads. In revisiting the early work on host preference across the M. tuberculosis complex, we have shown M. tuberculosis H37Rv is avirulent for cattle, and propose that the immune status of the animal, or genotype of the infecting bacillus, may have significant bearing on the virulence of a strain for cattle. This work will serve as a baseline for future studies into the genetic basis of host preference, and in particular the molecular basis of virulence in M. bovis.     

Observational Study of QuantiFERON®-TB Gold In-Tube Assay in Tuberculosis Contacts in a Low Incidence Area

Background

QuantiFERON®-TB Gold in-Tube (QFT) assay is a recently developed test to assess latent tuberculosis infection in contagious tuberculosis (TB) contact subjects.To assess the QFT assay in recently exposed contacts of active tuberculosis patients in a French area with low TB incidence but high Bacille Calmette-Guerin coverage, and evaluate progression rates to TB disease.

Methodology/Principal Findings

Between January 2007 and December 2009, 687 contacts of culture-confirmed tuberculosis cases underwent the QFT assay, with tuberculin skin test (TST) in 473, and a 34 months mean follow-up. Of 687 contacts, 148 were QFT positive, while 526 were negative and 13 indeterminate. QFT was positive in 35% of individuals with TST ≥10 mm, 47.5% with TST ≥15 mm or phlyctenular, but in 21% of cases in which two-step TST (M0 and M3) remained negative. Conversely, QFT was negative in 69% of cases with two-step TST showing conversion from negative to positive. All indeterminate QFT were associated with TST induration <10 mm in diameter. For 29 QFT-positive subjects, no chemoprophylaxis was given due to medical contraindications. Of the remaining 119 QFT-positive contacts, 97accepted chemoprophylaxis (81.5%), and 79 (81.4%) completed the treatment. Two contacts progressed to TB disease: one subject was QFT positive and had declined chemoprophylaxis, while the other one was QFT negative. QFT positive predictive value for progression to TB was 1.96% (1/51) with a 99.8% (525/526) negative predictive value.

Conclusions/Significance

Our results confirm the safety of the QFT-based strategy for assessing the TB chemoprophylaxis indication, as only one contact developed TB disease out of 526 QFT-negative subjects.     

Tuberculin Test in Children with Malnutrition

Tuberculin tests with Heaf''s multiple-puncture method, as well as intradermal tests with varying strengths of old tuberculin, were carried out on 402 children with severe malnutrition. From the radiological and bacteriological findings 51 children (12.5%) were considered to have active tuberculosis. The Heaf test was positive in only 11 of these children, but the intradermal test using 100 tuberculin units was positive in a further 18. This confirms previous findings that tuberculin sensitivity is impaired in malnourished children, and suggests that a higher dose of tuberculin is more likely to elicit a positive response.     

Increase in the Number of Tuberculosis Cases Treated following Tuberculin Skin Testing in First-Year Schoolchildren in Madagascar

Background

Tuberculosis continues to cause unacceptably high levels of disease and death worldwide. Active preventive strategies are required to improve tuberculosis control and to increase the number of cases treated in developing countries. The aim of this study was to evaluate the utility of the tuberculin skin test (TST) in first-year schoolchildren as a means of increasing the number of tuberculosis cases detected through the screening of close contacts.

Methods

All members of the households of 90 schoolchildren assigned to three groups on the basis of TST category (≤5 mm, [5–15)mm, ≥15 mm) were screened for sputum smear-positive pulmonary tuberculosis. The percentage detection of tuberculosis in close contacts was compared between TST categories.

Results

We identified 433 close contacts of the 90 schoolchildren, who were then evaluated for tuberculosis. We identified 11 cases of pulmonary tuberculosis among the close contacts (7 already on treatment and 4 previously undiagnosed): 0 in TST category ≤5 mm, 3 in TST category [5–15) mm and 8 in TST category ≥15 mm). This approach increased the detection of tuberculosis cases by a factor of 1.6 in first-year schoolchildren of the TST ≥5 mm group.

Conclusion

TST in first-year schoolchildren is a potentially effective method for improving the detection of tuberculosis in close contacts.     

Guidelines for the prevention and control of tuberculosis in non-human primates: recommendations of the European Primate Veterinary Association Working Group on Tuberculosis

Background  Effective tuberculosis (TB) control requires accurate diagnostic methods but the tuberculin skin test has serious limitations. Both false-negative and false-positive reactions are common, resulting in the spread of the infection and devastating TB outbreaks. Results of questionnaire surveys concerning TB testing practices in primate housing facilities showed great differences in testing practices. Although there was some uniformity regarding the sites of application, the amounts of tuberculin used and the time intervals for retesting, a great deal of variety was revealed considering the types of tuberculin preparations, the interpretation of tests and the susceptibility of animals.
Conclusion  Here, we summarize the most common practices as regards TB control and prevention for non-human primates, and attempt to establish a uniform guideline based upon our experience with primate husbandry and care programmes as well as recent developments in the literature. The present guideline represents a consensus recommendation intending to harmonize the existing protocols.     

Diagnosis of tuberculosis in macaques, using whole-blood in vitro interferon-gamma (PRIMAGAM) testing

During the fall of 2001, a tuberculosis outbreak caused by Mycobacterium bovis occurred in a conditioned colony of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques at Stanford University School of Medicine. During this outbreak, we evaluated the diagnostic performance of a new in vitro tuberculosis screening test (PRIMAGAM). The PRIMAGAM test measures the interferon-gamma (IFNgamma) response to purified protein derivatives (PPDs) of M. bovis and M. avium. On the basis of the results of the last test administered before necropsy, the PRIMAGAM test had good sensitivity (68%) and excellent specificity (97%), compared with the disease status, as determined by the presence or absence of gross and/or histologic lesions indicative of tuberculosis. By contrast, sensitivity and specificity of the tuberculin skin test (TST) was 84 and 87%, respectively. Both tests suffered from intermittent positive and negative reactions on repeat testing. Overall, however, there was no significant difference (P = 0.09, McNemar's chi2-test) and moderate agreement (kappa = 0.52) between these two tests. Lastly, the IFNgamma response to bovine PPD was significantly lower in infected cynomolgus macaques. Moreover, each test failed to detect tuberculosis in three cynomolgus macaques. Fortunately, they were different animals; therefore, we recommend the parallel use of the TST and PRIMAGAM test for maximal overall sensitivity in a tuberculosis screening program, especially for cynomolgus macaques.     

New approaches in the diagnosis and treatment of latent tuberculosis infection

With nearly 9 million new active disease cases and 2 million deaths occurring worldwide every year, tuberculosis continues to remain a major public health problem. Exposure to Mycobacterium tuberculosis leads to active disease in only ~10% people. An effective immune response in remaining individuals stops M. tuberculosis multiplication. However, the pathogen is completely eradicated in ~10% people while others only succeed in containment of infection as some bacilli escape killing and remain in non-replicating (dormant) state (latent tuberculosis infection) in old lesions. The dormant bacilli can resuscitate and cause active disease if a disruption of immune response occurs. Nearly one-third of world population is latently infected with M. tuberculosis and 5%-10% of infected individuals will develop active disease during their life time. However, the risk of developing active disease is greatly increased (5%-15% every year and ~50% over lifetime) by human immunodeficiency virus-coinfection. While active transmission is a significant contributor of active disease cases in high tuberculosis burden countries, most active disease cases in low tuberculosis incidence countries arise from this pool of latently infected individuals. A positive tuberculin skin test or a more recent and specific interferon-gamma release assay in a person without overt signs of active disease indicates latent tuberculosis infection. Two commercial interferon-gamma release assays, QFT-G-IT and T-SPOT.TB have been developed. The standard treatment for latent tuberculosis infection is daily therapy with isoniazid for nine months. Other options include therapy with rifampicin for 4 months or isoniazid + rifampicin for 3 months or rifampicin + pyrazinamide for 2 months or isoniazid + rifapentine for 3 months. Identification of latently infected individuals and their treatment has lowered tuberculosis incidence in rich, advanced countries. Similar approaches also hold great promise for other countries with low-intermediate rates of tuberculosis incidence.     

Statistical analysis of distribution of antibody level against Mycobacterium bovis antigens for bovine tuberculosis diagnostics

Antibody responses to purified protein derivate PPD of tuberculin and to antigens MPB63 and MPB83 of Mycobacterium bovis were determined in bovine herd (94 adult animals). Statistical approach based on approximation by multiple Gaussians with Levenberg-Marquardt algorithm for analysis of antibody level distribution against antigens examined was provided. Our results confirm that indirect ELISA with recombinant MPB83 and MPB63 as well as conventional PPD could be used for test-systems development for detection of cow tuberculosis infection at the herd level.     

Detection of Mycobacterium bovis in bovine tissue samples by the Abbott LCx Mycobacterium tuberculosis assay and comparison with culture methods.

A ligase chain reaction (LCR) DNA amplification method for the molecular diagnosis of Mycobacterium tuberculosis complex (Abbott LCx MTB) was evaluated in comparison with solid (Lowenstein Jensen), liquid (7H12, Bactec 460 system) phase culture and microscopic examination (ME) on 86 tissue samples collected from 86 intradermal tuberculin positive cattle and one pool from 4 guinea pigs experimentally infected with M. bovis. Overall, 48 samples (58.81%) were culturally positive for mycobacteria, and on the basis of biochemical characters, all the isolates were identified as M. bovis. Sensitivity was 83.92% for LCx, 53.57% for LJ, 85.71% for Bactec and 41.07% for ME. In 3 out of 25 "no visible lesion" tissue samples, M. bovis was detected only by LCx and Bactec but not by LJ and ME. The concordance in the determination of positives and negatives among the methods observed in pairs was calculated according to Cohen's K concordance coefficient and showed 81.1% of concordance of LCx vs Bactec, 68.8% LCx vs LJ, 72.2% LCx vs ME, 80.0% Bactec vs LJ, 66.7% Bactec vs ME, 85.5% LJ vs ME. Despite a certain variability in concordance rates, both Cohen's K concordance coefficients or standardized (Zk) values were statistically significant. Both LCx and Bactec appear not alternative but subsidiary to the other methods traditionally applied for direct diagnosis of bovine tuberculosis on tissue samples from cattle reacting to intradermal tuberculin test.