Accuracy of molecular diagnostic assays for detection of Mycobacterium bovis: A systematic review and meta-analysis.

Bovine tuberculosis (bovine TB) is a chronic wasting disease of cattle caused primarily by Mycobacterium bovis. Controlling bovine TB requires highly sensitive, specific, quick, and reliable diagnostic methods. This systematic review and meta-analysis evaluated molecular diagnostic tests for M. bovis detection to inform the selection of the most viable assay. On a per-test basis, loop-mediated isothermal amplification (LAMP) showed the highest overall sensitivity of 99.0% [95% CI: 86.2%-99.9%] and specificity of 99.8% [95% CI: 96.2%-100.00%]. Quantitative real-time polymerase chain reaction (qPCR) outperformed conventional PCR and nested PCR (nPCR) with a diagnostic specificity of 96.6% [95% CI: 88.9%-99.0%], while the diagnostic sensitivity of 70.8% [95% CI: 58.6-80.5%] was comparable to that of nPCR at 71.4% [95% CI: 60.7-80.2%]. Test sensitivity was higher with the input of milk samples (90.9% [95% CI: 56.0%-98.7%]), while specificity improved with tests based on major M. bovis antigens (97.8% [95% CI: 92.3%-99.4%]), the IS6110 insertion sequence (95.4% [95% CI: 87.6%-98.4%]), and the RD4 gene (90.7% [95% CI: 52.2%-98.9%]). The design of the currently available molecular diagnostic assays, while mostly based on nonspecific gene targets, prevents them from being accurate enough to diagnose M. bovis infections in cattle, despite their promise. Future assay development should focus on the RD4 region since it is the only target identified by genome sequence data as being distinctive for detecting M. bovis. The availability of a sufficiently accurate diagnostic test combined with the routine screening of milk samples can decrease the risk of zoonotic transmissions of M. bovis.

Diagnostic accuracy of the Enferplex Bovine TB antibody test using individual milk samples from cattle.

Bovine tuberculosis is usually diagnosed using tuberculin skin tests or at post-mortem. Recently, we have developed a serological test for bovine tuberculosis in cattle which shows a high degree of accuracy using serum samples. Here, we have assessed the performance of the test using individual bovine milk samples. The diagnostic specificity estimate using the high sensitivity setting of the test was 99.7% (95% CI: 99.2-99.9). This estimate was not altered significantly by tuberculin boosting. The relative sensitivity estimates of the test using the high sensitivity setting in milk samples from comparative skin test positive animals was 90.8% (95% CI: 87.1-93.6) with boosting. In animals with lesions, the relative sensitivity was 96.0% (95% CI: 89.6-98.7). Analysis of paired serum and milk samples from skin test positive animals showed correlation coefficients ranging from 0.756-0.955 for individual antigens used in the test. Kappa analysis indicated almost perfect agreement between serum and milk results, while McNemar marginal homogeneity analysis showed no statistically significant differences between the two media. The positive and negative likelihood ratio were 347.8 (95% CI: 112.3-1077.5) and 0.092 (95% CI: 0.07-0.13) respectively for boosted samples from skin test positive animals. The results show that the test has high sensitivity and specificity in individual milk samples and thus milk samples could be used for the diagnosis of bovine tuberculosis.

Bacteriological culture and direct PCR for detecting the Mycobacterium tuberculosis complex in the Italian eradication campaign: a decade of experience at the National Reference Laboratory.

AIMS: Our study evaluates the capacity of direct real-time PCR for detecting Mycobacterium tuberculosis complex (MTBC), with a focus on diagnostic performances and the feasibility of implementing this protocol in an eradication campaign. Specifically, we compare the effectiveness of the direct PCR method to various culture systems used by the Italian National Reference Laboratory over the last decade to detect MTBC. METHODS AND RESULTS: Bovine tissue samples were routinely tested and analyzed for bovine tuberculosis (bTB) confirmation using microbiological culture (solid and liquid media), histopathological analysis, and a direct PCR assay targeting IS6110, an insertion sequence specific to the MTBC that is widely used for tuberculosis diagnosis. The direct real-time PCR demonstrated a high concordance (K = 0.871) with microbiological culture, as well as good sensitivity (91.84%) and specificity (95.24%). In contrast, histopathology demonstrated lower concordance (K = 0.746) and performance levels (sensitivity 91.41%, specificity 82.88%). Liquid media promoted faster and more efficient growth of MTBC than solid media. M. bovis and M. caprae had the comparable ability to respond to the direct real-time PCR test and grow on the microbiological medium. CONCLUSIONS: This study confirms that direct real-time PCR can detect MTBC with high diagnostic accuracy within a few days. This study found no significant differences in performance between culture media and direct PCR for M. bovis and M. caprae.

Assessing the impact of various tuberculin PPD brands on bovine tuberculosis diagnosis.

Although several brands of tuberculin purified protein derivatives (PPDs) are available for diagnosing bovine tuberculosis (bTB), comparative studies to determine their diagnostic accuracy are infrequent. In Ecuador we compared two different PPD brands for bTB diagnosis using skin testing and measuring skin thickness increase. Additionally, we evaluated four PPD brands, including those used for skin testing, in the Bovine Tuberculosis Interferon Gamma Test (IFN-γ test) measuring IFN-γ induction in whole blood. The study included 17 naturally tuberculosis-infected PPD and IFN-γ test positive bovines. Both the field and laboratory results showed significant differences in classifying the 17 bovines as bTB positive or negative. We hypothesize that several factors, such as the genetic background of the cows, sensitization to environmental mycobacteria, M. bovis strains involved in the bTB infection, and the manufacturing procedures of the PPDs, could have influenced the immune reaction toward the different tuberculin PPD brands. Our study emphasizes the necessity for comparative studies aimed at determining the diagnostic accuracy of PPD brands for bTB diagnosis as well as the development of standardized methods for PPD production and potency determination.

Design and development of multiepitope chimeric antigens by bioinformatic and bacterial based recombinant expression methods, with potential application for bovine tuberculosis serodiagnosis.

Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis, is a single health concern, which causes economic losses, is a sanitary barrier and is a zoonotic concern. The golden-pattern intradermic tests have low sensitivity of about 50%. To fix this sensitivity problem, immunoassays could be a powerful tool. However, few studies produced antigens for bTB immunoassays, which needs improvements. Aim of this study was to produce multiepitope chimeric antigens (MCA) to use for bTB diagnosis. To achieve MCA design and development, extensive bibliographic search, antigenic epitope prediction, specificity, hydrophobicity, and 3D structure modeling analyses were performed, as well as cloning, expression and purification. Seven epitopes from four different target proteins (MPB-70, MPB-83, ESAT-6 and GroEL) were combined in five chimeras containing five repetitions of each epitope to enhance antibodies affinity. 3D predicted models revealed that all chimeras have a high percentage of disorder, which could enhance antibody recognition, although taking to protein instability. Each chimera was cloned into pET28a (+) expression plasmids and expressed in six Escherichia coli expression strains. Chimeras 3, 4 and 5 could be solubilized in 8 M urea and purified by ion exchange affinity chromatography. Against bTB positive and negative sera, purified chimera 5 had the best results in indirect dot blot and ELISA, as well as in lateral flow dot blot immunoassay. In conclusion, chimera 5, an MPB-83 containing MCA, could be used for further studies, aimed to develop a serologic or rapid test for bTB diagnosis.

Comparative analysis of tuberculin and defined antigen skin tests for detection of bovine tuberculosis in buffaloes (Bubalus bubalis) in Haryana state, India.

BACKGROUND: Bovine tuberculosis (bTB) is a chronic disease that results from infection with any member of the Mycobacterium tuberculosis complex. Infected animals are typically diagnosed with tuberculin-based intradermal skin tests according to World Organization of Animal Health which are presently in use. However, tuberculin is not suitable for use in BCG-vaccinated animals due to a high rate of false-positive reactions. Peptide-based defined skin test (DST) antigens have been identified using antigens (ESAT-6, CFP-10 and Rv3615c) which are absent from BCG, but their performance in buffaloes remains unknown. To assess the comparative performance of DST with the tuberculin-based single intradermal test (SIT) and the single intradermal comparative cervical test (SICCT), we screened 543 female buffaloes from 49 organized dairy farms in two districts of Haryana state in India. RESULTS: We found that 37 (7%), 4 (1%) and 18 (3%) buffaloes were reactors with the SIT, SICCT and DST tests, respectively. Of the 37 SIT reactors, four were positive with SICCT and 12 were positive with the DST. The results show that none of the animals tested positive with all three tests, and 6 DST positive animals were SIT negative. Together, a total of 43 animals were reactors with SIT, DST, or both, and the two assays showed moderate agreement (Cohen's Kappa 0.41; 95% Confidence Interval (CI): 0.23, 0.59). In contrast, only slight agreement (Cohen's Kappa 0.18; 95% CI: 0.02, 0.34) was observed between SIT and SICCT. Using a Bayesian latent class model, we estimated test specificities of 96.5% (95% CI, 92-99%), 99.7% (95% CI: 98-100%) and 99.0% (95% CI: 97-100%) for SIT, SICCT and DST, respectively, but considerably lower sensitivities of 58% (95% CI: 35-87%), 9% (95% CI: 3-21%), and 34% (95% CI: 18-55%) albeit with broad and overlapping credible intervals. CONCLUSION: Taken together, our investigation suggests that DST has a test specificity comparable with SICCT, and sensitivity intermediate between SIT and SICCT for the identification of buffaloes suspected of tuberculosis. Our study highlights an urgent need for future well-powered trials with detailed necropsy, with immunological and microbiological profiling of reactor and non-reactor animals to better define the underlying factors for the large observed discrepancies in assay performance, particularly between SIT and SICCT.

The impact of changing the cut-off threshold of the interferon-gamma (IFN-γ) assay for diagnosing bovine tuberculosis in Ireland.

In Ireland, the interferon-gamma (IFN-γ) assay is routinely used as an ancillary test interpreted in parallel with the single intradermal comparative tuberculin test (SICTT) to maximize the detection of bovine tuberculosis (bTB) infected animals. Up until 2018, a positive test result was recorded in the IFN-γ ELISA assay following whole blood stimulation with purified protein derivative (PPD)-bovine (B), PPD-avian (A) and nil sample (N), using the interpretation criteria, B-N > 50 optical density units (OD), B > 100 and B-A > 0. Following a review of available data, the threshold of the B-A component changed to B-A > 80. As predicting the impact of changing the cut-off thresholds for the IFN-γ test de novo is challenging, the aims of this study were to follow animals that initially tested negative using the new IFN-γ assay interpretation criteria and investigate their future risk of disclosure with bTB, with a focus on animals that otherwise would have been removed when using the older interpretation criteria (0 < B-A ≤ 80). Enrolled animals (n = 28,669 cattle from 527 herds) were followed up for two years (2019-2021), or to point of bTB detection or death. At the end of follow-up, 1151 (4.0%) of enrolled animals were bTB cases. The majority of these cases were diagnosed using SICTT (80.5%). The cumulative number of positive animals that would have been removed if the old cut-off (0 < B-A ≤ 80) was used amounted to 1680 cattle (5.9% of the enrolled cohort). Of these, 127 (7.5%) were diagnosed with bTB during follow-up. In contrast, 1024 of the 1151 cattle which subsequently tested positive during the study period following a negative IFN-γ test would not have been identified with the old or new IFN-γ cut-off criteria. Survival analysis showed that animals that would have been removed under the old interpretation criteria were at increased risk of a positive diagnosis with bTB during follow-up compared to other test negative animals. A newly developed risk prediction model (using a Cox proportional hazard model) showed that age, animal number of SICTT tests, number of inconclusive SICTT tests, B-A (IFN-γ assay), B-N (IFN-γ assay), animals from store herds and the percentage of the rest of the herd that were positive during the breakdown were statistically significantly associated with bTB detection. However, inclusion of the IFN-γ OD variables did not show added value in terms of prediction performance of the model.

Temporal dynamics of the early immune response following Mycobacterium bovis infection of cattle.

Bovine tuberculosis is an infectious disease of global significance that remains endemic in many countries. Mycobacterium bovis infection in cattle is characterized by a cell-mediated immune response (CMI) that precedes humoral responses, however the timing and trajectories of CMI and antibody responses determined by newer generation assays remain undefined. Here we used defined-antigen interferon-gamma release assays (IGRA) and an eleven-antigen multiplex ELISA (Enferplex TB test) alongside traditional tuberculin-based IGRA and IDEXX M. bovis antibody tests to assess immune trajectories following experimental M. bovis infection of cattle. The results show CMI responses developed as early as two-weeks post-infection, with all infected cattle testing positive three weeks post-infection. Interestingly, 6 of 8 infected animals were serologically positive with the Enferplex TB assay as early as 4 weeks post-infection. As expected, application of the tuberculin skin test enhanced subsequent serological reactivity. Infrequent M. bovis faecal shedding was observed but was uncorrelated with observed immune trajectories. Together, the results show that early antibody responses to M. bovis infection are detectable in some individuals and highlight an urgent need to identify biomarkers that better predict infection outcomes, particularly for application in low-and-middle income countries where test-and-slaughter based control methods are largely unfeasible.

Investigation of the association between the Enferplex bovine tuberculosis antibody test and the future risk of bovine tuberculosis in irish cattle in infected herds: a pilot field study.

The Single Intradermal Comparative Tuberculin Test (SICTT) and the interferon-gamma (IFN-γ) assay are the approved diagnostic tests for bovine tuberculosis (bTB) in Ireland. The aim of this pilot study was to explore if there was any added diagnostic benefit from applying the Enferplex bTB test (an antibody test) in severe bTB herd breakdowns after the removal of cattle that had tested positive to the SICTT and the IFN-γ test. In addition to the normal bTB testing and management protocols, the animals in these herds that tested negative to SICTT and the IFN-γ test were followed forward for a period of two years. All animals were tested by Enferplex at enrolment. The time to subsequent bTB detection (diagnosed with SICTT/IFN-γ tests or detection of visible lesions at routine slaughter) for animals that tested positive or negative to the Enferplex bTB test at the start of the study was compared using Kaplan-Meier survival curves and Cox based survival models. Of the 484 enrolled animals (from 11 herds), 171 (35.3%) and 151 (31.1%) initially tested positive in the Enferplex assay under the high sensitivity and high specificity interpretation settings respectively. The results of the survival analysis showed that there was no difference in the survival time to a positive diagnosis with bTB during the follow-up period between animals initially classified as positive and negative by the Enferplex test. Further research is warranted to explore the potential benefit of using the Enferplex test in other scenarios.

Enferplex bovine TB antibody test and bovine TB diagnosis: letter to the editor.

Bovine tuberculosis is usually diagnosed using tuberculin skin and interferon gamma tests. However, it is clear these tests miss infected animals due to poor sensitivity. The Enferplex Bovine TB antibody test has been validated by the World Organisation for Animal Health as fit for purpose in diagnosing bovine TB. A recent paper by Madden and colleagues (Veterinary Research Communications published online 17 August 2023) presented data on the future risk of Enferplex test antibody positive animals developing bovine TB. We argue in this communication that this does not make sense. Also, the study design did not include measuring antibodies at the point of censure of the animals and hence the survival analysis performed was meaningless. Most significantly, the study misses the point that skin and interferon gamma tests fail to detect a significant proportion of infected animals identified by the Enferplex test.

Using machine learning to improve bovine tuberculosis control in herd level outbreaks.

Bovine tuberculosis (bTB), a chronic disease of cattle, is caused by the Mycobacterium bovis infection. Despite having a serious social and economic impact in the United Kingdom and Ireland, there is no antemortem gold standard diagnostic test. Tuberculin skin tests (CICT) are commonly used as a control measure with the interferon gamma (IFN-γ) assay being applied in certain circumstances. This paper utilizes data gathered describing tuberculin regression in reactors (test positive cattle) following the CICT at 72 ± 4 h post injection in herds with large bTB outbreaks. The work then applies machine learning techniques (Decision Trees, Bagging Trees and Random Forests, alongside several balancing approaches) to predict which cattle were likely to be truly infected with tuberculosis, enabling identification of atypical breakdowns that require extra investigation and providing a mechanism for quality assurance of the existing CICT bTB surveillance scheme. The analysis showed that Random Forests (RF) trained using SMOTE balancing had the joint best performance and accuracy (0.90). The importance of the two components of the interferon gamma assay within the RF model also indicated that varying the assay threshold for large outbreaks would be beneficial. Furthermore, the combined use of the RF and IFN- γ models could lead to the improved detection of infection within breakdown herds, reducing the scale and duration of outbreaks. An additional use of these models would be for quality assuring the current bTB surveillance based on CICT and post mortem inspection. Quality control is well recognized as an essential component of a disease surveillance/eradication programme.Clinical Relevance- Bovine tuberculosis remains a disease that is hard to control on a national level. The use of the machine learning model could lead to significant improved detection of infection within breakdown herds, reducing the scale and duration of outbreaks. Advanced modelling, such as this, has the potential to strengthen the efficacy of disease surveillance and the eradication strategy and can meaningfully contribute to animal disease national control plans.

Histopathological and molecular methods as complementary diagnostic in case of lymphadenopathies suggestive of bovine tuberculosis.

Bovine tuberculosis (TB) is a chronically evolving zoonotic infectious disease caused by Mycobacterium bovis. Anatomopathological examination during post mortem inspection in bovines is the main resource engaged in sanitary slaughter; however, it is very troublesome since many granulomatous inflammatory processes have similar morphological characteristics. Thus, this study aims to use complementary diagnosis methods (histopathological and polymerase chain reaction - PCR assays) to confirm the macroscopic assessment of lymphadenopathies indicative of tuberculosis in bovines slaughtered in a refrigerated slaughterhouse in Tailândia city, PA, Brazil. Fifty­one samples were collected from lesions indicative of tuberculosis in pre­scapular and pre­pectoral lymph nodes (or different lymphadenitis) in condemned carcasses. Histological processing employed routine techniques carried out at the Laboratory of Animal Pathology of the Federal Rural University of the Amazon, while the PCR assay was performed at the Bacteriology Laboratory of the Evandro Chagas Institute. Results showed that 1.96% of the histopathology samples corresponded to inflammatory processes typical of TB and that, in PCR, 4.25% of the samples had the amplification profile of the M. bovis species. These results indicate the importance of adding complementary methods to assist the sanitary inspection line and make inspection more efficient in its decisions.

Enhancing bovine tuberculosis screening at Dhaka city in Bangladesh: Integrating gamma interferon blood test as ancillary testing with tuberculin skin test.

Tuberculin skin test (TST) is the standard method for screening of bovine tuberculosis (bTB). However, gamma interferon blood test has been introduced in the bTB control program as an ancillary testing with TST in many countries of the world. The objective of this study was to recommend this screening test as an ancillary testing with TST for field application in Bangladesh. In this study 577 cattle of different age, sex and breeds from twenty nine (29) cattle herds were examined to determine skin response against bTB through single intradermal comparative tuberculin test (SICTT) that comprised of positive (n = 81), inconclusive (n = 44) and negative (n = 452) animals. Of which 74 animals that included positive (n = 63), inconclusive (n = 8) and negative (n = 3) animals were taken under this study. Blood samples were collected in heparinized tube and stimulated overnight with bovine and avian purified protein derivatives (PPDs) for the secretion of gamma interferon, and measured via sandwich ELISA. Cohen's kappa statistics was performed for the evaluation of agreement between the two tests. The agreement obtained between two tests was fair (Kappa agreement, K = 24.0%, 95% CI = 16.9-30.5%, P = 0.037). Of positive (n = 63), inconclusive (n = 8) and negative (n = 3) status of animals at SICTT, 82.54% (n = 52), 62.50% (n = 5), and 33.33% (n = 1) were found to be bTB positive respectively through this ancillary test. This test notably corroborates to TST result. A considerable number of inconclusive TB status animals were found to be positive through this gamma interferon assay. Therefore, this test could be used as an ancillary test with TST to maximize the proportion of bTB estimation in the infected cattle herd for early detection of zoonotic tuberculosis in Bangladesh before transmission at the animal-human interface.

Zoonotic tuberculosis in a high bovine tuberculosis burden area of Ethiopia.

Background: Tuberculosis (TB) is a major cause of ill health and one of the leading causes of death worldwide, caused by species of the Mycobacterium tuberculosis complex (MTBC), with Mycobacterium tuberculosis being the dominant pathogen in humans and Mycobacterium bovis in cattle. Zoonotic transmission of TB (zTB) to humans is frequent particularly where TB prevalence is high in cattle. In this study, we explored the prevalence of zTB in central Ethiopia, an area highly affected by bovine TB (bTB) in cattle. Method: A convenient sample of 385 patients with pulmonary tuberculosis (PTB, N = 287) and tuberculous lymphadenitis (TBLN, N = 98) were included in this cross-sectional study in central Ethiopia. Sputum and fine needle aspirate (FNA) samples were obtained from patients with PTB and TBLN, respectively, and cultures were performed using BACTEC™ MGIT™ 960. All culture positive samples were subjected to quantitative PCR (qPCR) assays, targeting IS1081, RD9 and RD4 genomic regions for detection of MTBC, M. tuberculosis and M. bovis, respectively. Results: Two hundred and fifty-five out of 385 sampled patients were culture positive and all were isolates identified as MTBC by being positive for the IS1081 assay. Among them, 249 (97.6%) samples had also a positive RD9 result (intact RD9 locus) and were consequently classified as M. tuberculosis. The remaining six (2.4%) isolates were RD4 deficient and thereby classified as M. bovis. Five out of these six M. bovis strains originated from PTB patients whereas one was isolated from a TBLN patient. Occupational risk and the widespread consumption of raw animal products were identified as potential sources of M. bovis infection in humans, and the isolation of M. bovis from PTB patients suggests the possibility of human-to-human transmission, particularly in patients with no known contact history with animals. Conclusion: The detected proportion of culture positive cases of 2.4% being M. bovis from this region was higher zTB rate than previously reported for the general population of Ethiopia. Patients with M. bovis infection are more likely to get less efficient TB treatment because M. bovis is inherently resistant to pyrazinamide. MTBC species identification should be performed where M. bovis is common in cattle, especially in patients who have a history of recurrence or treatment failure.

Evaluation of the diagnostic accuracy of the serological test for paratuberculosis in cattle according to tuberculosis status.

BACKGROUND: Enzyme-linked immunosorbent assays (ELISAs) are the most widely used diagnostic tools in bovine paratuberculosis (bPTB) control. However, their diagnostic accuracy may be compromised by bovine tuberculosis (bTB) infection, as both diseases share diagnostic targets. METHODS: The bPTB and bTB infection status of 228 animals was determined using microbiological tissue culture as a reference test. The diagnostic performance (sensitivity, specificity, likelihood ratios and predictive values) of the bPTB-ELISA on blood serum samples, taking into account the bPTB animal-level prevalence of the area and the bTB status of the animals, was evaluated. RESULTS: A sensitivity of 40.7% (95% confidence interval [CI]: 27.5%-53.9%) and a specificity of 94.7% (95% CI: 91.4%-98.0%) were obtained for bPTB-ELISA in all animals. A bPTB-ELISA-positive animal would have a post-test probability of 70% or more of being infected in areas with a bPTB prevalence of 23% or more. A negative bPTB-ELISA result, in areas with a bPTB prevalence of 41% or less, would rule out the disease with more than 70% certainty. In bTB-positive animals, sensitivity increased (94.4% [95% CI: 81.4%-100%] vs. 25.1% [95% CI: 11.8%-38.4%]) and specificity decreased (82.6% [95% CI: 71.8%-93.4%] vs. 99.4% [95% CI: 98.0%-99.9%]). The bPTB-ELISA is a good tool to rule out bPTB co-infection in bTB-positive animals, while in bTB-negative animals, it allows confirmation of disease with more than 70% probability if disease prevalence is 6% or more. LIMITATIONS: The observed differences could be enhanced by the effect of frequent application of the intradermal tuberculin test, which was unknown in the animals studied. CONCLUSIONS: These results provide useful guidance for the application and interpretation of ELISA as a tool for bPTB disease control.

LAP-MALDI MS Profiling and Identification of Potential Biomarkers for the Detection of Bovine Tuberculosis.

Detecting bovine tuberculosis (bTB) primarily relies on the tuberculin skin test, requiring two separate animal handling events with a period of incubation time (normally 3 days) between them. Here, we present the use of liquid atmospheric pressure (LAP)-MALDI for the identification of bTB infection, employing a three-class prediction model that was obtained by supervised linear discriminant analysis (LDA) and tested with bovine mastitis samples as disease-positive controls. Noninvasive collection of nasal swabs was used to collect samples, which were subsequently subjected to a short (<4 h) sample preparation method. Cross-validation of the three-class LDA model from the processed nasal swabs provided a sensitivity of 75.0% and specificity of 90.1%, with an overall classification accuracy of 85.7%. These values are comparable to those for the skin test, showing that LAP-MALDI MS has the potential to provide an alternative single-visit diagnostic platform that can detect bTB within the same day of sampling.

Performance of two commercial serological assays for bovine tuberculosis using plasma samples.

In the bovine tuberculosis diagnosis, the use of plasma samples (already available for IFNÉ£ assays) in serological tests might facilitate the work in the field. Here, the performance of two commercial serological tests (ELISA IDEXX M. bovis Ab test and Enferplex Bovine TB antibody test) were evaluated using plasma samples from cattle in Belgium. Specificity values estimated from 567 plasma samples collected from bTB-free cattle were 98.4% when using the ELISA IDEXX M. bovis Ab test, and were 96.5% and 93.3% when using the high specificity and high sensitivity settings of the Enferplex Bovine TB antibody test, respectively. Sensitivity values were calculated relative to SICCT-positive (N = 117) and IFNÉ£-positive (N = 132) animals originating from M. bovis-infected herds. Overall, the multiplexed Enferplex Bovine TB antibody test had better sensitivity (mean: 32.5% and 43.4% for the high specificity and sensitivity settings, respectively) compared to the ELISA IDEXX M. bovis Ab test (mean: 12%). Data obtained from plasma samples in the current study were compared to a previous study using both serological tests with sera. In conclusion, both serological tests showed comparable performance with both matrix; although overall specificity values with the Enferplex Bovine TB antibody test were lower when using plasma samples than sera.

Rapid detection of Mycobacterium bovis in bovine cytological smears and tissue sections by peptide nucleic acid fluorescence in-situ hybridization.

BACKGROUND: Bovine tuberculosis is the leading cause of death in cattle and other species worldwide. Quick and precise identification of mycobacteria is critical to control the occurrence of tuberculosis in cattle. METHODS: We developed a fluorescent peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) approach to detect Mycobacterium bovis and Mycobacterium avium in cytological smears and tissue sections of bovines suspected of having tuberculosis. PNA-FISH was conducted on smears of lung and lymph node tissues. Standard bovine mycobacterial cultures were used to standardize the probes using 50 % formamide for M. bovis and 30 % formamide for M. avium. M. bovis probe (MTBCcy3), which was standardized at hybridization conditions of (55 °C and 40 % formamide) concentrations, was positive in all cytological smears. RESULTS: Four out of twenty five samples tested positive in tissue sections observed as a bright red fluorescence with a cy3 filter (MTBC probe). No results were observed with (MAVTAMRA) probe for M. avium which was standardized at hybridization conditions of (55 °C and 30 % formamide). No fluorescence was observed in the control tissue sections. Additionally, the results were juxtaposed with those of other commonly used detection methods such as immunohistochemistry and Polymerase Chain Reaction (PCR) by targeting the esxA gene. None of the samples tested positive for M. avium infection. CONCLUSION: PNA-FISH can be used to obtain cytological impression smears and tissue sections. When compared to PCR it consumes less time in the diagnosis of bovine tuberculosis in post mortem cases.

Evaluation of the performance of the IFN-γ release assay in bovine tuberculosis free herds from five European countries.

The diagnostic methods for granting and maintenance of the official tuberculosis-free (OTF) status and for intra-Community movement of cattle are the tuberculin skin tests (single or comparative) and the interferon-γ (IFN-γ) release assay (IGRA). However, until now, IGRAs have been primarily applied in infected farms in parallel to the skin test to maximize the number of infected animals detected. Therefore, an evaluation of the performance of IGRAs in OTF herds to assess whether if their specificity is equal to or higher than that of the skin tests is needed. For this, a panel of 4365 plasma samples coming from 84 OTF herds in six European regions (five countries) was assembled and analysed using two IGRA kits, the ID Screen® Ruminant IFN-g (IDvet) and the Bovigam™ TB Kit (Bovigam). Results were evaluated using different cut-offs, and the impact of herd and animal-level factors on the probability of positivity was assessed using hierarchical Bayesian multivariable logistic regression models. The percentage of reactors ranged from 1.7 to 21.0% (IDvet: S/P ≥ 35%), and 2.1-26.3% (Bovigam: ODbovis-ODPBS ≥ 0.1 and ODbovis-ODavium ≥ 0.1) depending on the region, with Bovigam disclosing more reactors in all regions. The results suggest that specificity of IGRAs can be influenced by the production type, age and region of origin of the animals. Changes in the cut-offs could lead to specificity values above 98-99% in certain OTF populations, but no single cut-off yielding a sufficiently high specificity (equal or higher than that of skin tests) in all populations was identified. Therefore, an exploratory analysis of the baseline IFN-γ reactivity in OTF populations could help to assess the usefulness of this technique when applied for the purpose of maintaining OTF status.