Olga Mínguez

Evaluation of the sensitivity and specificity of bovine tuberculosis diagnostic tests in naturally infected cattle herds using a Bayesian approach.

Test-and-slaughter strategies have been the basis of bovine tuberculosis (BT) eradication programs worldwide; however, eradication efforts have not succeeded in certain regions, and imperfect sensitivity and specificity of applied diagnostic techniques have been deemed as one of the possible causes for such failure. Evaluation of tuberculosis diagnostic tools has been impaired by the lack of an adequate gold standard to define positive and negative individuals. Here, a Bayesian approach was formulated to estimate for the first time sensitivity (Se) and specificity (Sp) of the tests [single intradermal tuberculin (SIT) test, and interferon-gamma (IFN-γ) assay] currently used in Spain. Field data from the first implementation of IFN-γ assay (used in parallel with SIT test 2-6months after a first disclosure SIT test) in infected beef, dairy and bullfighting cattle herds from the region of Castilla and Leon were used for the analysis. Model results suggested that in the described situation: (i) Se of SIT test was highly variable (40.1-92.2% for severe interpretation, median=66-69%), and its Sp was high (>99%) regardless interpretation criteria; (ii) IFN-γ assay showed a high Se (median=89-90% and 83.5% for 0.05 and 0.1 cut-off points respectively) and an acceptable Sp (85.7% and 90.3% for 0.05 and 0.1 thresholds) and (iii) parallel application of both tests maximized the combined Se (95.6% using severe SIT and 0.05 cut-off point in the IFN-γ assay). These results support the potential use of the IFN-γ assay as an ancillary technique for routine BT diagnosis.

Effect of paratuberculosis on the diagnosis of bovine tuberculosis in a cattle herd with a mixed infection using interferon-gamma detection assay

Interferon-gamma (IFN-gamma) detection assay is being applied as an ancillary test to tuberculin tests in the diagnosis of bovine tuberculosis to detect the maximum number of infected animals. Among possible factors influencing the performance of tuberculosis-diagnostic tests, paratuberculosis, a widespread disease in Spain and other European countries, has been pointed out as a cause of false positive reactions. Still, its effect on the sensitivity of these tests in cattle has yet to be fully characterized. The impact of paratuberculosis in the apparent sensitivity of IFN-gamma assay was studied in a bullfighting cattle herd with a mixed tuberculosis-paratuberculosis infection, using culture of Mycobacterium bovis and Mycobacterium avium paratuberculosis as the gold standard to determine the infection status of every animal. A total of 218 animals were slaughtered and sampled for bacteriology after blood sampling. IFN-gamma assay showed a lower apparent sensitivity in animals with a mixed infection (50%) compared to all animals suffering tuberculosis (78.3%). This finding indicates that the presence of paratuberculosis in tuberculosis-infected herds could imply a serious impairment in the sensitivity of IFN-gamma detection test.

Humans as source of Mycobacterium tuberculosis infection in cattle, Spain.

To the Editor: Mycobacterium tuberculosis is the main causative agent of tuberculosis in humans. However, little attention has been paid to its transmission from humans to animals. We report M. tuberculosis infections in 3 cattle farms in Spain. The epidemiologic investigation traced humans as the source of infection, with 1 of the strains showing multidrug resistance. Recent studies have reported isolation of M. tuberculosis in cattle with prevalences of 4.7%–30.8% in African and Asian countries (1–3). In cattle, this infection occurs in countries with the highest incidence of human tuberculosis in the world. In Europe, only 14 cases of M. tuberculosis infection have been described in 3 eastern countries since implementation of eradication programs (4,5). The only reported cases of M. tuberculosis in cattle in western Europe were described in Great Britain and date back to the 1950s (6). During 2007–2009, three cases of tuberculosis caused by M. tuberculosis were detected in 3 unrelated cattle farms, 2 of them free of tuberculosis (farms 1 and 2). As part of the surveillance system of bovine tuberculosis, a pool of tissue samples from each cow (respiratory lymph nodes and lung) were homogenized with sterile distilled water, and culture was carried out by the BACTEC mycobacteria growth indicator tube 960 system (Beckton Dickinson, Madrid, Spain). Members of the M. tuberculosis complex were identified and genotyped by direct variable repeat spacer olignucleotide typing and mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) typing (7). The 3 M. tuberculosis–infected animals were <9 months of age (Table). As described (6), the possibility of infection in young animals could be more probable than infection in older cows. Table Relevant information about Mycobacterium tuberculosis infection in 3 cattle farms in Spain* M. tuberculosis–infected animals from farms 1 and 3 were detected by the intradermal tuberculin test (Table). The animal without immunologic response (farm 2) was detected because an M. bovis infection was confirmed in the herd, and all animals were slaughtered. Confirmation of infection by culture without immunologic response is rare, although the high sensitivity of the mycobacteria growth indicator tube system could detect a low bacterial load in the initial stages of infection. Recent implementation of liquid systems in animal health laboratories has enabled detection of M. tuberculosis when it is compared with results using only conventional methods. Moreover, no tuberculosis-compatible lesions were observed in the 3 animals, similar to previous studies (6). On the basis of these facts, M. tuberculosis transmission was not detected among cattle in the following intradermal tuberculin tests. Co-infection with other mycobacteria (M. avium subsp. hominissuis) was found in the same animal from farm 1 (Table). This co-infection suggested the immunocompromised status of the animal and hence a high susceptibility to M. tuberculosis infection. Moreover, M. bovis was isolated from 52% (16/31) of all animals from farm 2 that showed a positive reaction to the intradermal tuberculin skin test, making remarkable the absence of co-infection with M. bovis in the M. tuberculosis–infected animal. Therefore, the lack of M. tuberculosis transmission within this herd contrasts with the M. bovis dissemination. The veterinary services reported these findings to the National Public Health System, and an epidemiologic investigation was conducted on the cattle farms to determine the source of infection. In all cases, staff of the farms had active tuberculosis (Table). Three different strains were characterized: SIT2537 (octal code 777617777720771), 253533233433236252211423 (farm 1); SIT1564, 3′52334232455457251213423 (farm 2); and SIT58, 254343243232325262213423 (farm 3) (Table). The MIRU-VNTR pattern and spoligotype are shared by Spanish human and cattle isolates from farm 1; SIT2537 is an uncommon profile that has been detected in Brazil and Spain (according to the SITVIT2 database). The human strain showed multidrug resistance to isoniazid, rifampin, and ethionamide. In cattle and human isolates, genes associated with isoniazid and rifampin resistance were studied (8) and rpoB analysis confirmed rifampin resistance (Ser531Leu). In farm 2, the origin of the farm worker was eastern Europe and the cattle isolate showed an SIT1564 profile, which is found only in 6 human isolates in the SpolDB4 database, all from Poland, Bulgaria, and Russia. On farm 3, human and cattle isolates from Spain shared identical spoligotype and MIRU-VNTR patterns. The profile SIT58 is frequent in Spain (9) and other countries with historical links to Spain, mainly the south American countries (79/114 according to SpolDB4). A well-designed program for eradicating bovine tuberculosis helps to detect M. tuberculosis infection by immune response or by bacteriologic culture. The use of liquid systems and results of epidemiologic studies (Spanish Database of Animal Mycobacteriosis, mycoDB.es) (S. Rodriguez, unpub. data) are recommended for prompt confirmation of the M. tuberculosis complex infection and for enhancing the sensitivity of culture. In addition, the Spanish Ministry of Environment, Rural and Marine Affairs has reinforced the need to improve cooperation between human and animal health systems to minimize the risk for M. tuberculosis complex transmission from animals to humans or vice versa and to control infection in all susceptible animal species (10).

Evaluation of specificity of tuberculosis diagnostic assays in caprine flocks under different epidemiological situations.

The aim of this study was to evaluate the specificity of the most widely used tuberculosis (TB) diagnostic tests, single intradermal tuberculin (SIT) and single comparative intradermal tuberculin (SCIT) tests and interferon-gamma (IFN-γ) assay in 937 animals from eight TB-free caprine flocks under different epidemiological situations. Maximum specificity was found using SCIT test (99.4-100% depending on the interpretation criteria) while SIT test and IFN-γ assay showed a slightly lower overall specificity (97.6-99.2% and 96.4-98.4% respectively). Specificity of the SIT test in a Corynebacterium pseudotuberculosis infected flock was significantly (P<0.05) lower if a severe interpretation criterion was applied. Similarly, specificity values of SIT test and particularly IFN-γ assay in a paratuberculosis (PTB)-vaccinated flock were lower than those observed in non-vaccinated flocks. Higher proportion of false positive reactors to TB tests (SIT and IFN-γ assay) were observed among animals positive in the PTB-ELISA in PTB vaccinated flock. These results demonstrate that TB diagnostic tests show an adequate specificity when performed in goats from TB-free flocks in most situations. However, certain factors such as C. pseudotuberculosis infection and paratuberculosis vaccination can have a negative impact in the most sensitive tests.

Evaluation of single and comparative intradermal tuberculin tests for tuberculosis eradication in caprine flocks in Castilla y León (Spain).

Goats can act as reservoirs for tuberculosis (TB) infection. The main etiological agents of TB in goats are Mycobacterium caprae and Mycobacterium bovis and they infect also a wide range of domestic and wild animals and humans. Control programmes based mainly on the application of single and comparative intradermal tuberculin (SIT and SCIT respectively) tests are being implemented in certain regions of Spain with a high density of caprine flocks as Castilla y León, including goats with epidemiological relationship with cattle. The aim of this study was to evaluate the performance of the intradermal tests in naturally TB-infected caprine flocks from this region. The study was performed using data from 17,450 goats in 54 different flocks that were classified as TB-infected in the control programmes executed in 2010 and 2011. Data from 1237 goats from 7 dairy flocks depopulated after the first intradermal testing were used to estimate the sensitivity (Se) using bacteriology as the gold-standard. Overall Se of the SIT test using the severe interpretation was 43.9% (CI 95%, 40.4-47.4) and decreased to 38.8% (CI 95%, 35.5-42.3) using the standard interpretation. Overall Se of the SCIT test ranged between 21.3% (CI 95%, 17.6-25.4) and 7% (CI 95%, 4.9-9.8) depending of the interpretation criteria. A significant weak positive correlation was found between age and skin fold thickness (Spearmans test p<0.05). Results from this study yielded, in general, low Se values probably due the systematic detection and slaughter of reactors as a consequence of the eradication programme in previous years or the presence of factors that may interfere in the diagnosis. Therefore, these results suggest the necessity of including ancillary diagnostic tools and/or strict interpretation criteria to maximize detection of positive animals in infected settings.

Comparison of depopulation and S19-RB51 vaccination strategies for control of bovine brucellosis in high prevalence areas

RB51 vaccination can minimise the diagnostic problems associated with S19 vaccination of adult cattle, but its use for bovine brucellosis (BB) control remains controversial. Here, the evolution of BB prevalence in five high prevalence areas in Spain subjected to different control measures is described: herd depopulation of infected herds (I–III) or mass vaccination with RB51 and S19-RB51 vaccination of replacement heifers (IV–V). Annual data from the eradication campaigns were analysed at the special incidence area (SIA) level and the time to obtain herd prevalence levels of <1 per cent (‘controlled status’) was obtained at the local veterinary unit (LVU) level and compared using Coxs proportional hazard model. A higher annual rate of decrease in herd prevalence was observed in the SIAs subjected to vaccination (46.9%, 95% CI 43.5% to 50.0%) compared with those managed using stamping out (14.9%, 95% CI 9.6% to 19.9%). No significant differences in the time to achieve controlled status were observed between the stamping-out and vaccination strategies used at the LVU level, with median times of 60 (stamping-out LVUs) and 63 (vaccination LVUs) months. These results suggest that RB51 mass vaccination, in combination with the S19-RB51 vaccination of replacement heifers and strict implementation of other eradication measures, may provide results at least comparable with those resulting from a herd depopulation based strategy.

The use of serological tests in combination with the intradermal tuberculin test maximizes the detection of tuberculosis infected goats

The diagnosis of tuberculosis (TB) in goats is based mainly on the single and comparative intradermal tuberculin (SIT and CIT) tests and, exceptionally, on the interferon-gamma (IFN-γ) assay, however they are not perfect in terms of sensitivity and specificity. Nevertheless, various serological assays that provide a potential cost-effective approach for the control of TB are also available or under development, and a variety of results have been reported regarding the ability of these tests to detect infected animals, particularly in the early stages of infection. In the present study, SIT/CIT and IFN-γ tests and three different serological assays were evaluated during two consecutive herd testing events in a recently infected caprine herd (n = 447) with a high prevalence of infection in order to evaluate their performance and provide field data with which to improve the TB control programs in this species. The proportion of infected animals that tested positive among all the infected goats (T+/I+ value) in the last herd testing event ranged from 26.2% (IC95%; 19.3-34.5) to 85.7% (IC95%; 78.5-90.7) using cell-based diagnostic tests. The SIT/SCIT tests detected more infected goats than the IFN-γ test, regardless of the interpretation criteria. The T+/I+ value of serology was 83.2 (IC95%; 75.2-89), although it increased significantly (P < 0.05) when using samples collected 15 days after the intradermal test (100%, IC95%; 97-100). In general, a parallel interpretation of intradermal tests with serology maximized the detection of infected goats. These results demonstrate that serological tests are valuable diagnostic tools to maximize the detection of TB infected goats, even in recent outbreaks, accelerating the eradication process.

Evaluation of the Performance of the IDvet IFN-Gamma Test for Diagnosis of Bovine Tuberculosis in Spain

In Spain, the national bovine tuberculosis (bTB) eradication program is based on yearly skin testing of every ≥6 weeks old animal using the single or comparative tuberculin test and parallel use of the interferon-gamma (IFN-γ) assay as an ancillary diagnostic test in infected herds. There are several versions of the latter. Recently, a new commercial IDvet IFN-γ assay has been authorized for use in the program, but there is limited scientific evidence about its performance in different epidemiological settings. Therefore, two studies to evaluate the performance of the IDvet assay were conducted. In study 1, a concordance analysis between the new IDvet and the Bovigam IFN-γ assay in use in Spain for over 10 years was conducted. In study 2, results from the IDvet assay when applied in tandem with a single intradermal tuberculin (SIT) test were used to evaluate the concordance between both tests and to estimate their sensitivity (Se) and specificity (Sp) using a Bayesian latent-class model. Field data from cattle herds located in Madrid and Castilla y Leon (Spain) were collected. For study 1, herd selection was based on a high expected prevalence of reactors to the IFN-γ assay, while herds were selected at random to estimate Se and Sp of the new IDvet assay in study 2. Agreement between the results obtained with both kits for IFN-γ assay was poor (Kappa = 0.20), and a receiver operating characteristic (ROC) analysis indicated a low Se of the new IDvet relative to the Bovigam in a heavily bTB infected population. The Bayesian latent-class analysis estimated the Se of the IDvet assay to be 36.7% [95% probability posterior interval (PPI) 14.7–78.8%] with estimated Sp close to 100% when the cut-off recommended by the manufacturer (35) was applied. At the alternative cut-off values of 16 and 4, the estimated Se of the IDvet assay increased to 49.0% (PPI: 24.8–94.1%) and 56.0% (PPI: 30.8–96.3%), respectively, while maintaining a high specificity. The results suggest that the new IDvet assay may have lower sensitivity than the Bovigam for diagnosis of bTB in cattle herds in Spain, and that adjusting its cut-off might be considered.