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.

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.

Vaccines to control tuberculosis in cattle.

The age-old cattle disease has resisted rigorous control, but the BCG vaccine may do better.

BCG vaccination reduces bovine tuberculosis transmission, improving prospects for elimination.

Bacillus Calmette-Guérin (BCG) is a routinely used vaccine for protecting children against Mycobacterium tuberculosis that comprises attenuated Mycobacterium bovis. BCG can also be used to protect livestock against M. bovis; however, its effectiveness has not been quantified for this use. We performed a natural transmission experiment to directly estimate the rate of transmission to and from vaccinated and unvaccinated calves over a 1-year exposure period. The results show a higher indirect efficacy of BCG to reduce transmission from vaccinated animals that subsequently become infected [74%; 95% credible interval (CrI): 46 to 98%] compared with direct protection against infection (58%; 95% CrI: 34 to 73%) and an estimated total efficacy of 89% (95% CrI: 74 to 96%). A mechanistic transmission model of bovine tuberculosis (bTB) spread within the Ethiopian dairy sector was developed and showed how the prospects for elimination may be enabled by routine BCG vaccination of cattle.

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.

Selection of vaccine-candidate peptides from Mycobacterium avium subsp. paratuberculosis by in silico prediction, in vitro T-cell line proliferation, and in vivo immunogenicity.

Mycobacterium avium subspecies paratuberculosis (MAP) is a global concern in modern livestock production worldwide. The available vaccines against paratuberculosis do not offer optimal protection and interfere with the diagnosis of bovine tuberculosis. The aim of this study was to identify immunogenic MAP-specific peptides that do not interfere with the diagnosis of bovine tuberculosis. Initially, 119 peptides were selected by either (1) identifying unique MAP peptides that were predicted to bind to bovine major histocompatibility complex class II (MHC-predicted peptides) or (2) selecting hydrophobic peptides unique to MAP within proteins previously shown to be immunogenic (hydrophobic peptides). Subsequent testing of peptide-specific CD4+ T-cell lines from MAP-infected, adult goats vaccinated with peptides in cationic liposome adjuvant pointed to 23 peptides as being most immunogenic. These peptides were included in a second vaccine trial where three groups of eight healthy goat kids were vaccinated with 14 MHC-predicted peptides, nine hydrophobic peptides, or no peptides in o/w emulsion adjuvant. The majority of the MHC-predicted (93%) and hydrophobic peptides (67%) induced interferon-gamma (IFN-γ) responses in at least one animal. Similarly, 86% of the MHC-predicted and 89% of the hydrophobic peptides induced antibody responses in at least one goat. The immunization of eight healthy heifers with all 119 peptides formulated in emulsion adjuvant identified more peptides as immunogenic, as peptide specific IFN-γ and antibody responses in at least one heifer was found toward 84% and 24% of the peptides, respectively. No peptide-induced reactivity was found with commercial ELISAs for detecting antibodies against Mycobacterium bovis or MAP or when performing tuberculin skin testing for bovine tuberculosis. The vaccinated animals experienced adverse reactions at the injection site; thus, it is recommend that future studies make improvements to the vaccine formulation. In conclusion, immunogenic MAP-specific peptides that appeared promising for use in a vaccine against paratuberculosis without interfering with surveillance and trade tests for bovine tuberculosis were identified by in silico analysis and ex vivo generation of CD4+ T-cell lines and validated by the immunization of goats and cattle. Future studies should test different peptide combinations in challenge trials to determine their protective effect and identify the most MHC-promiscuous vaccine candidates.

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.

Bovine Tuberculosis in Wild Boar (Sus scrofa) in Slovenia.

Mycobacteria of the Mycobacterium tuberculosis complex (MTC) are capable of infecting a wide variety of animals. Wild boar (Sus scrofa) has been recognized as an important wildlife reservoir for bovine tuberculosis. We screened wild boar in Slovenia for the presence of (1) Mycobacterium bovis in tissues and (2) antibodies to M. bovis in blood samples. In 2016 and 2017, 1284 tissue samples from 676 wild boar were subjected to cultivation. In 2018 and 2019, blood samples from 132 wild boar were examined using an ELISA kit. None of the MTC species were isolated from the tissue samples, and no antibodies to M. bovis were detected in the blood samples. Several nontuberculous mycobacteria (NTM), identified by 16S rRNA sequencing and/or matrix-assisted laser desorption ionization time-of-flight mass spectrometry, were found in the tissues of 9.8% of the wild boar: Mycobacterium nonchromogenicum, Mycobacterium peregrinum/Mycobacterium septicum, Mycobacterium avium, Mycobacterium engbaekii, Mycobacterium arupense, Mycobacterium algericum, Mycobacterium bohemicum, Mycobacterium confluentis, Mycobacterium flavescens, Mycobacterium fortuitum, Mycobacterium thermoresistibile, and Mycobacterium vaccae. Species-level identification was not possible for 21.2% of the isolates. At the time of the study, wild boar in Slovenia were not at risk from bTB; the significance of the presence of NTM in wild boar remains to be clarified and evaluated from a One Health perspective.

Contribution of herd management, biosecurity, and environmental factors to the risk of bovine tuberculosis in a historically low prevalence region.

Eradication of bovine tuberculosis (bTB) in certain historically low-prevalence regions remains elusive. A complete characterisation of the husbandry practices, biosecurity, and environment where farms are located is crucial to implement targeted in-farm risk mitigation protocols. Here, a detailed survey performed in 94 dairy cattle farms located in Navarra, a low-prevalence region of Spain between 2016 and 2020 was carried out. Data on 73 biosecurity, farm-, and environmental-level factors potentially associated with the risk of bTB occurrence were evaluated using an ordinal logistic regression model: farms were classified based on their prevalence index, a score linked to each farm to account for the severity and recurrence of bTB cases: 22.3% of the farms had a score of 1, 21.3% a score of 2, 26.6% a score of ≥ 3, and 29.8% were negative herds. A statistically significant association between a higher prevalence index and the frequency of badger sightings along with the lease of pastures to sheep during Winter was identified. Farms that detected badgers on a monthly to daily basis in the surroundings and those that leased pastures for sheep flocks during Winter were four [odds ratio, 95% CI (4.3; 1.1-17.5)] and three (3.1; 1.0-9.9) times more likely to have the highest prevalence index, respectively (predicted probabilityprevalence index≥3 = 0.7; 95% CI 0.3-0.9). Conversely, farms that used a vehicle to transport animals from holdings to pastures were less likely (0.1; <0.1-0.3) to present higher levels of prevalence index compared with farms that used none (on foot). Results suggested that the combined effect of farm- and environmental-level risk factors identified here may be hampering disease eradication in Navarra, highlighting the need to implement targeted protocols on farms and grazing plots. An increased awareness of monitoring sheep and wildlife in direct or indirect contact with cattle herds in historically low bTB prevalence areas should be raised.

Temperature impacts the bovine ex vivo immune response towards Mycoplasmopsis bovis.

Although cattle are the mammalian species with most global biomass associated with a huge impact on our planet, their immune system remains poorly understood. Notably, the bovine immune system has peculiarities such as an overrepresentation of γδ T cells that requires particular attention, specifically in an infectious context. In line of 3R principles, we developed an ex vivo platform to dissect host-pathogen interactions. The experimental design was based on two independent complementary readouts: firstly, a novel 12-14 color multiparameter flow cytometry assay measuring maturation (modulation of cell surface marker expression) and activation (intracellular cytokine detection) of monocytes, conventional and plasmacytoid dendritic cells, natural killer cells, γδ T cells, B and T cells; secondly, a multiplex immunoassay monitoring bovine chemokine and cytokine secretion levels. The experiments were conducted on fresh primary bovine blood cells exposed to Mycoplasmopsis bovis (M. bovis), a major bovine respiratory pathogen. Besides reaffirming the tight cooperation of the different primary blood cells, we also identified novel key players such as strong IFN-γ secreting NK cells, whose role was so far largely overlooked. Additionally, we compared the host-pathogen interactions at different temperatures, including commonly used 37 °C, ruminant body temperature (38-38.5 °C) and fever (≥ 39.5 °C). Strikingly, working under ruminant physiological temperature influenced the capacity of most immune cell subsets to respond to M. bovis compared to 37 °C. Under fever-like temperature conditions the immune response was impaired compared to physiological temperature. Our experimental approach, phenotypically delineating the bovine immune system provided a thorough vision of the immune response towards M. bovis and the influence of temperature towards that immune response.

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.

Difference in differences analysis evaluates the effects of the badger control policy on bovine tuberculosis in England.

Persistent tuberculosis (TB) in cattle populations in England has been associated with an exchange of infection with badgers (Meles meles). A badger control policy (BCP) commenced in 2013. Its aim was to decrease TB incidence in cattle by reducing the badger population available to provide a wildlife reservoir for bovine TB. Monitoring data from 52 BCP intervention areas 200-1600 km2 in size, starting over several years, were used to estimate the change in TB incidence rate in cattle herds, which was associated with time since the start of the BCP in each area. A difference in differences analysis addressed the non-random selection and starting sequence of the areas. The herd incidence rate of TB reduced by 56% (95% Confidence Interval 41-69%) up to the fourth year of BCP interventions, with the largest drops in the second and third years. There was insufficient evidence to judge whether the incidence rate reduced further beyond 4 years. These estimates are the most precise for the timing of declines in cattle TB associated with interventions primarily targeting badgers. They are within the range of previous estimates from England and Ireland. This analysis indicates the importance of reducing transmission from badgers to reduce the incidence of TB in cattle, noting that vaccination of badgers, fertility control and on farm biosecurity may also achieve this effect.

Live attenuated Mycobacterium bovis strains combined with the encapsulated H65 antigen as a vaccine strategy against bovine tuberculosis in a mouse model.

Mycobacterium bovis is an etiological agent of bovine tuberculosis (bTB) that also infects other mammals, including humans. The lack of an effective vaccine for the control of bTB highlights the need for developing new vaccines. In this study, we developed and evaluated an M. bovis strain deleted in the virulence genes phoP, esxA and esxB as a vaccine candidate against bTB in BALBc mice. The evaluated strains were the new live vaccine and BCG, alone or in combination with ncH65vD. The immunogen ncH65vD is a fusion protein H65, encapsulated together with vitamin D3, within the oily body of a nanocapsule composed of an antigen-loading polymeric shell. All vaccines conferred protection against the M. bovis challenge. However, no significant differences were detected among the vaccinated groups regarding bacterial loads in lungs and spleen. Mice vaccinated with the mutant strain plus ncH65vD showed negative Ziehl Neelsen staining of mycobacteria in their lungs, which suggests better control of bacteria replication according to this protection parameter. Consistently, this vaccination scheme showed the highest proportion of CD4 + T cells expressing the protection markers PD-1 and CXCR3 among the vaccinated groups. Correlation studies showed that PD-1 and CXCR3 expression levels in lung-resident CD4 T cells negatively correlated with the number of colony forming units of M. bovis in the lungs of mice. Therefore, the results suggest a link between the presence of PD-1 + and CXCR3 + cells at the site of the immune response against mycobacteria and the level of mycobacterial loads.