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.

Spoligotype-specific risk of finding lesions in tissues from cattle infected by Mycobacterium bovis.

BACKGROUND: Although the pathogenic effect of members of the Mycobacterium tuberculosis complex in susceptible hosts is well known, differences in clinical signs and pathological findings observed in infected animals have been reported, likely due to a combination of host and pathogen-related factors. Here, we investigated whether Mycobacterium bovis strains belonging to different spoligotypes were associated with a higher risk of occurrence of visible/more severe lesions in target organs (lungs and/or lymph nodes) from infected animals. A large collection of 8889 samples belonging to cattle were classified depending on the presence/absence of tuberculosis-like lesions and its degree of severity. All samples were subjected to culture irrespective of the presence of lesions, and isolates retrieved were identified and subjected to spoligotyping. The association between the presence/severity of the lesions and the isolation of strains from a given spoligotype was assessed using non-parametric tests and Bayesian mixed multivariable logistic regression models that accounted for origin (region and herd) effects. RESULTS: Results suggested a difference in severity in lesioned samples depending on the strain's spoligotype. An association between specific spoligotypes and presence of lesions was observed, with a higher risk of finding lesions in animals infected with strains with spoligotypes SB0120, SB0295 and SB1142 compared with SB0121, and in those coming from certain regions in Spain. CONCLUSIONS: Our results suggest that strains belonging to certain spoligotypes may be associated with a higher probability in the occurrence of gross/macroscopic lesions in infected cattle, although these observational findings should be confirmed in further studies that allow accounting for the effect of other possible confounders not considered here, and ultimately through experimental studies.

Evaluation of the immunogenicity and efficacy of BCG and MTBVAC vaccines using a natural transmission model of tuberculosis.

Effective vaccines against tuberculosis (TB) are needed in order to prevent TB transmission in human and animal populations. Evaluation of TB vaccines may be facilitated by using reliable animal models that mimic host pathophysiology and natural transmission of the disease as closely as possible. In this study, we evaluated the immunogenicity and efficacy of two attenuated vaccines, BCG and MTBVAC, after each was given to 17 goats (2 months old) and then exposed for 9 months to goats infected with M. caprae. In general, MTBVAC-vaccinated goats showed higher interferon-gamma release than BCG vaccinated goats in response to bovine protein purified derivative and ESAT-6/CFP-10 antigens and the response was significantly higher than that observed in the control group until challenge. All animals showed lesions consistent with TB at the end of the study. Goats that received either vaccine showed significantly lower scores for pulmonary lymph nodes and total lesions than unvaccinated controls. Both MTBVAC and BCG vaccines proved to be immunogenic and effective in reducing severity of TB pathology caused by M. caprae. Our model system of natural TB transmission may be useful for evaluating and optimizing vaccines.

Proteomic characterisation of bovine and avian purified protein derivatives and identification of specific antigens for serodiagnosis of bovine tuberculosis.

BACKGROUND: Bovine purified protein derivative (bPPD) and avian purified protein derivative (aPPD) are widely used for bovine tuberculosis diagnosis. However, little is known about their qualitative and quantitative characteristics, which makes their standardisation difficult. In addition, bPPD can give false-positive tuberculosis results because of sequence homology between Mycobacterium bovis (M. bovis) and M. avium proteins. Thus, the objective of this study was to carry out a proteomic characterisation of bPPD, aPPD and an immunopurified subcomplex from bPPD called P22 in order to identify proteins contributing to cross-reactivity among these three products in tuberculosis diagnosis. METHODS: Trypsin digests of bPPD, aPPD and P22 were analysed by nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry. Mice were immunised with bPPD or aPPD, and their serum was tested by indirect ELISA for reactivity against these preparations as well as against P22. RESULTS: A total of 456 proteins were identified in bPPD, 1019 in aPPD and 118 in P22; 146 of these proteins were shared by bPPD and aPPD, and 43 were present in all three preparations. Candidate proteins that may cause cross-reactivity between bPPD and aPPD were identified based on protein abundance and antigenic propensity. Serum reactivity experiments indicated that P22 may provide greater specificity than bPPD with similar sensitivity for ELISA-type detection of antibodies against M. tuberculosis complex. CONCLUSION: The subpreparation from bPPD called P22 may be an alternative to bPPD for serodiagnosis of bovine tuberculosis, since it shares fewer proteins with aPPD than bPPD does, reducing risk of cross-reactivity with anti-M. avium antibodies.

Genetic diversity assessment of Tunisian Mycobacterium bovis population isolated from cattle.

BACKGROUND: The genetic diversity of M. bovis in Tunisia is still underestimated despite the implementation of an eradication program. The lack of data about spatial distribution of the M. bovis population hinders the control of bovine tuberculosis (bTB) progress. This study represents the largest molecular analysis of M. bovis isolates in Tunisia. It is aimed to upgrade the understanding of bTB epidemiology and the geographical distribution of the infection. Tuberculosis research was performed in cattle (n = 149) with TB-compatible lesions collected over 5 months from a slaughterhouse located in Sfax, Tunisia. RESULTS: Ninety-four animals were found to be infected by M. bovis and two others by M. caprae. Spoligotyping revealed twenty-five patterns, SB0120, SB0134, and SB0121 being the most prevalent profiles (36.4%, 11.4%, and 7.2%, respectively). Three new spoligotypes were detected: SB2345, SB2344 and SB2343. MIRU-VNTR analysis classified the isolates in seventy-three profiles and showed a large genotypic variety observed within the main spoligotype which was split into several MIRU-VNTR types: 29 in SB0120 (h = 0.983), 10 in SB0134 (h = 0.981) and 7 in SB0121 (h = 1). Genotyping revealed a common pattern in different geographic regions. It also showed that Sfax, located in southern-Tunisia, represents a high-risk area with an elevated genetic diversity. CONCLUSIONS: Spatial analysis may provide insights into disease transmission, which affects the effectiveness of eradication campaigns in cattle.

Risk factors associated with negative in-vivo diagnostic results in bovine tuberculosis-infected cattle in Spain.

BACKGROUND: Despite great effort and investment incurred over decades to control bovine tuberculosis (bTB), it is still one of the most important zoonotic diseases in many areas of the world. Test-and-slaughter strategies, the basis of most bTB eradication programs carried out worldwide, have demonstrated its usefulness in the control of the disease. However, in certain countries, eradication has not been achieved due in part to limitations of currently available diagnostic tests. In this study, results of in-vivo and post-mortem diagnostic tests performed on 3,614 animals from 152 bTB-infected cattle herds (beef, dairy, and bullfighting) detected in 2007-2010 in the region of Castilla y León, Spain, were analyzed to identify factors associated with positive bacteriological results in cattle that were non-reactors to the single intradermal tuberculin test, to the interferon-gamma (IFN-γ) assay, or to both tests applied in parallel (Test negative/Culture + animals, T-/C+). The association of individual factors (age, productive type, and number of herd-tests performed since the disclosure of the outbreak) with the bacteriology outcome (positive/negative) was analyzed using a mixed multivariate logistic regression model. RESULTS: The proportion of non-reactors with a positive post-mortem result ranged from 24.3% in the case of the SIT test to 12.9% (IFN-γ with 0.05 threshold) and 11.9% (95% CI 9.9-11.4%) using both tests in parallel. Older (>4.5 years) and bullfighting cattle were associated with increased odds of confirmed bTB infection by bacteriology, whereas dairy cattle showed a significantly lower risk. Ancillary use of IFN-γ assay reduced the proportion of T-/C + animals in high risk groups. CONCLUSIONS: These results demonstrate the likelihood of positive bacteriological results in non-reactor cattle is influenced by individual epidemiological factors of tested animals. Increased surveillance on non-reactors with an increased probability of being false negative could be helpful to avoid bTB persistence, particularly in chronically infected herds. These findings may aid in the development of effective strategies for eradication of bTB in Spain.

Evaluation of the Effect of a Recent Comparative Intradermal Tuberculin Test on the Humoral Diagnosis of Paratuberculosis Using Serum and Milk Samples from Goats.

Paratuberculosis (PTB) and tuberculosis (TB) are two mycobacterial diseases with a severe economic and health impact on domestic ruminants. The ante mortem diagnosis of PTB is hampered, among other factors, by the limited sensitivity of all the available diagnostic techniques. Since TB-infected goats subjected to the comparative intradermal tuberculin test (CITT) may experience a booster effect on their antibody titer and a potential enhancement to the sensitivity of humoral techniques for tuberculosis, in the present study we aimed to evaluate this diagnostic strategy on the humoral diagnosis of PTB in serum and milk samples collected from a caprine herd that was TB free and PTB infected. The results from 120 goats indicated a significant increase (p < 0.001) in the quantitative response detected using an ELISA technique, conducted using serum and milk samples taken 15 and 30 days after performing a CITT (day 0 of the study); although, it did not translate into a significant increase in the number of reactors during any of the testing events (0, 3,15, 30 and 60 days post-CITT). Additionally, the number of ELISA-positive animals was higher for the serum versus the milk samples at both 15 and 30 days post-CITT. The increase in the quantitative ELISA result suggested a diagnostic strategy that maximizes ELISA sensitivity, mainly using serum samples, in PTB-infected herds; although, it may depend on individual differences and the interpretation criteria.

Effect of a recent intradermal test on the specificity of P22 ELISA for the diagnosis of caprine tuberculosis.

Caprine tuberculosis (TB) is a zoonotic disease caused by members of the Mycobacterium tuberculosis complex. TB eradication programs in goats are based on the single and comparative intradermal tuberculin tests (SITT and CITT, respectively). Antibody-based diagnostic techniques have emerged as potential diagnostic tools for TB. P22 ELISA has been previously evaluated using samples collected after the intradermal tuberculin tests to maximize the sensitivity, a phenomenon known as booster effect. However, there is no information available on whether the use of this diagnostic strategy could lead to a decrease of its specificity (Sp). The aim of the present study was to elucidate the interference effect of a recent CITT on the Sp of the P22 ELISA in serum and milk samples collected at different times after the CITT from a TB-free herd (n = 113). The number of reactors to P22 ELISA was significantly higher (p < 0.01) on serum samples collected 15 days post-CITT compared to day 0, showing a decrease in Sp from 99.1% (95% CI; 95.2-99.8%) to 88.5% (95% CI; 81.3-93.2%). The number of reactors and the quantitative values of P22 ELISA were significantly higher (p < 0.01) in serum samples compared to milk. No significant (p > 0.05) changes in the Sp of the P22 ELISA were observed throughout the different time samplings using milk No significant (p > 0.05) changes were observed on days 30 and 60 post-CITT. In conclusion, the booster effect strategy may significantly decrease the Sp of P22 ELISA in TB-free herds when serum samples are used but not when milk is tested.

Non-tuberculous mycobacteria: occurrence in skin test cattle reactors from official tuberculosis-free herds.

Non-tuberculous mycobacteria (NTM) are considered a relevant cause of non-specific reactions to the most widely applied bovine tuberculosis (bTB) test, the intradermal tuberculin test. In order to establish which NTM species might act as a potential source of such diagnostic interference, a collection of 373 isolates obtained from skin test positive cows from 359 officially tuberculosis-free (OTF) herds, culled in the framework of the bTB eradication campaign in Spain, were identified at the species level through PCR and Sanger sequencing of the 16S rDNA, hsp65 and rpoB genes. Of the 308 isolates for which a reliable identification was achieved, 32 different mycobacterial species were identified, with certain species being most represented: among M. avium complex members (n = 142, 46.1%), M. avium subsp. hominissuis (98; 69.0%) was the most abundant followed by M. avium subsp. avium (33, 23.2%), and M. intracellulare (7, 4.9%). Among non-MAC members (n = 166, 53.9%), M. nonchromogenicum (85; 27.6%) and M. bourgelatii (11; 5.6%) were the predominant species. In addition, mixed results were obtained in 53 isolates presenting up to 30 different genotypes, which could be indicative of new mycobacterial species. Our results represent a first step toward characterizing the diversity of NTM species that could interfere with official diagnostic tests for bTB eradication in Spain.

Splitting of a prevalent Mycobacterium bovis spoligotype by variable-number tandem-repeat typing reveals high heterogeneity in an evolving clonal group.

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

Wild boar tuberculosis in Iberian Atlantic Spain: a different picture from Mediterranean habitats.

BACKGROUND: Infections with Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex (MTC) are shared between livestock, wildlife and sporadically human beings. Wildlife reservoirs exist worldwide and can interfere with bovine tuberculosis (TB) eradication efforts. The Eurasian wild boar (Sus scrofa) is a MTC maintenance host in Mediterranean Iberia (Spain and Portugal). However, few systematic studies in wild boar have been carried out in Atlantic regions. We describe the prevalence, distribution, pathology and epidemiology of MTC and other mycobacteria from wild boar in Atlantic Spain. A total of 2,067 wild boar were sampled between 2008 and 2012. RESULTS: The results provide insight into the current status of wild boar as MTC and Mycobacterium avium complex (MAC) hosts in temperate regions of continental Europe. The main findings were a low TB prevalence (2.6%), a low proportion of MTC infected wild boar displaying generalized TB lesions (16.7%), and a higher proportion of MAC infections (4.5%). Molecular typing revealed epidemiological links between wild boar and domestic - cattle, sheep and goat - and other wildlife - Eurasian badger (Meles meles) and red fox (Vulpes vulpes) - hosts. CONCLUSIONS: This study shows that the likelihood of MTC excretion by wild boar in Atlantic habitats is much lower than in Mediterranean areas. However, wild boar provide a good indicator of MTC circulation and, given the current re-emergence of animal TB, similar large-scale surveys would be advisable in other Atlantic regions of continental Europe.

Disseminated avian mycobacteriosis in a free-living grey heron (Ardea cinerea).

Wild birds share with humans the capacity for moving fast over large distances. During migratory movements, birds carry pathogens that can be transmitted between species. One of these concerning pathogens is Mycobacterium spp. A necropsy was performed in a grey heron (Ardea cinerea) that had been medically treated for a polyarthritic process. Grossly, firm white-yellowish nodules of various size, resembling granulomas, were observed in right carpal joint, both patellar joints, neck musculature, palate, pharynx, larynx, nasal sinuses, pericardial sac, air sacs, proventriculus and intestinal serosa, pancreas, kidneys, adrenal glands, and oviduct. Microscopically, these lesions were composed of multinucleated giant cells, large macrophages, and lymphocytes, with a central zone of necrosis and in some cases with peripheral fibrosis. Acid-fast bacilli were detected within these lesions. Lesions were cultured and Mycobacterium avium subsp. avium was identified. To the best of our knowledge this is the first description of mycobacteriosis in grey herons. Although the grey heron is currently considered a least-concern species, avian mycobacteriosis remains a threat on the conservation management of endangered avian species, and constitutes a public health concern as well.

Bovine tuberculosis in Spain, is it really the final countdown?

Bovine tuberculosis (bTB) is a severe zoonotic disease that has major impacts on both health and the economy, and which has been subjected to specific eradication programmes in many countries for decades. This manuscript highlights the relevance of this disease in the context of the European Union (EU) and summarizes the epidemiological situation and the main tools (e.g. antemortem diagnostic tests, slaughterhouse surveillance, laboratories, comprehensive databases, etc.) used to control and eradicate bTB in the various EU countries with a focus on the situation in Spain. A comprehensive description of the specific bTB epidemiological situation in Spain is provided, together with an assessment of the evolution of different epidemiological indicators throughout the last decades. Moreover, the main features of the Spanish bTB eradication programme and its control tools are described, along with the studies carried out in Spain that have allowed the updating of and improvement to the programme over the years with the aim of eradication, which has been established for 2030.

Once bitten twice shy: Risk factors associated with bovine tuberculosis recurrence in Castilla y Leon, Spain.

Persistence of bovine tuberculosis (bTB) in cattle herd remains a major challenge in disease elimination due to the ineffectual removal of all infected animals in a bTB breakdown. Characterization of herds with a higher probability of experiencing further bTB breakdowns can help to implement specific risk-based policies for disease control and eradication. Here, our aim was to identify herd- and breakdown-level risk factors in bTB infected herds in Castilla y Leon, Spain, associated with a decreased time to recurrence and an increased risk of recurrence using a mixed effects Cox proportional hazards model and a multivariable logistic regression model, respectively. Results revealed that location (province), herd size and number of incoming animals/contacts were good predictors of a decreased time to bTB recurrence and an increased risk of becoming a recurrent herd. Additionally, the duration of the previous outbreak and the number of IFN-γ herd-tests applied in it were associated with increased odds of (an early) recurrence. Risk factors identified here can be used for early identification of herds in which bTB eradication may be more challenging and that should thus be subjected to increased control efforts. The characterization of high-risk herds may help to minimize the risk of reinfection and emphasize early detection and removal of bTB positive animals in the herd.

Differences in skin test reactions to official and defined antigens in guinea pigs exposed to non-tuberculous and tuberculous bacteria.

The single and comparative intradermal tuberculin tests (SITT and CITT) are official in vivo tests for bovine tuberculosis (TB) diagnosis using bovine and avian purified protein derivatives (PPD-B and PPD-A). Infection with bacteria other than Mycobacterium tuberculosis complex (MTC) can result in nonspecific reactions to these tests. We evaluated the performance of the skin test with PPDs and new defined antigens in the guinea pig model. A standard dose (SD) of Rhodococcus equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis, M. avium subsp. avium, M. avium subsp. hominissuis, M. scrofulaceum, M. persicum, M. microti, M. caprae and M. bovis, and a higher dose (HD) of M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis were tested using PPD-B, PPD-A, P22, ESAT-6-CFP-10-Rv3615c peptide cocktail long (PCL) and fusion protein (FP). The SD of R. equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare and M. avium subsp. paratuberculosis did not cause any reactions. The HD of M. nonchromogenicum, M. monacense, M. intracellulare, and M. avium subsp. paratuberculosis and the SD of M. avium subsp. hominissuis, M. scrofulaceum and M. persicum, caused nonspecific reactions (SIT). A CITT interpretation would have considered M. avium complex and M. scrofulaceum groups negative, but not all individuals from M. nonchromogenicum HD, M. monacense HD and M. persicum SD groups. Only animals exposed to M. bovis and M. caprae reacted to PCL and FP. These results support the advantage of complementing or replacing PPD-B to improve specificity without losing sensitivity.

Eradication of bovine tuberculosis at a herd-level in Madrid, Spain: study of within-herd transmission dynamics over a 12 year period.

BACKGROUND: Eradication of bovine tuberculosis (bTB) through the application of test-and-cull programs is a declared goal of developed countries in which the disease is still endemic. Here, longitudinal data from more than 1,700 cattle herds tested during a 12 year-period in the eradication program in the region of Madrid, Spain, were analyzed to quantify the within-herd transmission coefficient (ß) depending on the herd-type (beef/dairy/bullfighting). In addition, the probability to recover the officially bTB free (OTF) status in infected herds depending on the type of herd and the diagnostic strategy implemented was assessed using Cox proportional hazard models. RESULTS: Overall, dairy herds showed higher ß (median 4.7) than beef or bullfighting herds (2.3 and 2.2 respectively). Introduction of interferon-gamma (IFN-γ) as an ancillary test produced an apparent increase in the ß coefficient regardless of production type, likely due to an increase in diagnostic sensitivity. Time to recover OTF status was also significantly lower in dairy herds, and length of bTB episodes was significantly reduced when the IFN-γ was implemented to manage the outbreak. CONCLUSIONS: Our results suggest that bTB spreads more rapidly in dairy herds compared to other herd types, a likely cause being management and demographic-related factors. However, outbreaks in dairy herds can be controlled more rapidly than in typically extensive herd types. Finally, IFN-γ proved its usefulness to rapidly eradicate bTB at a herd-level.

MALDI-TOF Mass Spectrometry as a Rapid Screening Alternative for Non-tuberculous Mycobacterial Species Identification in the Veterinary Laboratory.

Non-tuberculous mycobacteria (NTM) are difficult to identify by biochemical and genetic methods due to their microbiological properties and complex taxonomy. The development of more efficient and rapid methods for species identification in the veterinary microbiological laboratory is, therefore, of great importance. Although MALDI-TOF Mass Spectrometry (MS) has become a promising tool for the identification of NTM species in human clinical practise, information regarding its performance on veterinary isolates is scarce. This study assesses the capacity of MALDI-TOF MS to identify NTM isolates (n = 75) obtained from different animal species. MALDI-TOF MS identified 76.0% (n = 57) and 4% (n = 3) of the isolates with high and low confidence, respectively, in agreement with the identification achieved by Sanger sequencing of housekeeping genes (16S rRNA, hsp65, and rpoB). Thirteen isolates (17.3%) were identified by Sanger sequencing to the complex level, indicating that these may belong to uncharacterised species. MALDI-TOF MS approximated low confidence identifications toward closely related mycobacterial groups, such as the M. avium or M. terrae complexes. Two isolates were misidentified due to a high similarity between species or due to the lack of spectra in the database. Our results suggest that MALDI-TOF MS can be used as an effective alternative for rapid screening of mycobacterial isolates in the veterinary laboratory and potentially for the detection of new NTM species. In turn, Sanger sequencing could be implemented as an additional method to improve identifications in species for which MALDI-TOF MS identification is limited or for further characterisation of NTM species.

Extracellular matrix proteins (fibronectin, collagen III, and collagen I) immunoexpression in goat tuberculous granulomas (Mycobacterium caprae).

The lesion resulting from the interaction between Mycobacterium and the host immune response is the tuberculous granuloma. Tuberculous granulomas, except in incipient stages, are partially or totally encapsulated by connective tissue. The aim of this study was to assess the immunoexpression of the extracellular matrix proteins fibronectin, collagen III, and collagen I in granulomas caused by Mycobacterium caprae in goats (Capra aegagrus hircus) to understand capsule development at different granuloma stages. For this purpose, a retrospective study of 56 samples of tuberculous granulomas in lung (n = 30) and mediastinal lymph node (n = 26) from 17 goats naturally infected with M. caprae in stages I (n = 15), II (n = 14) and III (n = 27) was carried out. Fibronectin immunoreaction was extracellular, fibrillar-reticular in the center of stage I, II and III granulomas and peripheral in stages II and III granulomas. Collagen III immunoexpression was extracellular and fibrillar in the center of stages I, II and III tuberculous granulomas in lung and mediastinal lymph node, and progressive expression was observed in the periphery of stages II and III granulomas. Finally, collagen I immunoexpression was extracellular and fibrillar, showing a progressive loss of central expression and an increase in peripheral expression in stage III granulomas compared to stage I granulomas. Immunoexpression of these extracellular matrix proteins could help understand fibrogenesis and dating in tuberculous granuloma in both animal models and humans.

Use of Whole-Genome Sequencing to Unravel the Genetic Diversity of a Prevalent Mycobacterium bovis Spoligotype in a Multi-Host Scenario in Spain.

Despite the efforts invested in the eradication of bovine tuberculosis in Spain, herd prevalence has remained constant in the country during the last 15 years (~1.5-1.9%) due to a combination of epidemiological factors impairing disease control, including between-species transmission. Here, our aim was to investigate the molecular diversity of Mycobacterium bovis isolates belonging to the highly prevalent SB0339 spoligotype in the cattle-wildlife interface in different regions of Spain using whole-genome sequencing (WGS). Genomic data of 136 M. bovis isolates recovered from different animal species (cattle, wild boar, fallow deer, and red deer) and locations between 2005 and 2018 were analyzed to investigate between- and within-species transmission, as well as within-herds. All sequenced isolates differed by 49-88 single nucleotide polymorphisms from their most recent common ancestor. Genetic heterogeneity was geographic rather than host species-specific, as isolates recovered from both cattle and wildlife from a given region were more closely related compared to isolates from the same species but geographically distant. In fact, a strong association between the geographic and the genetic distances separating pairs of M. bovis isolates was found, with a significantly stronger effect when cattle isolates were compared with wildlife or cattle-wildlife isolates in Spain. The same results were obtained in Madrid, the region with the largest number of sequenced isolates, but no differences depending on the host were observed. Within-herd genetic diversity was limited despite the considerable time elapsed between isolations. The detection of closely related strains in different hosts demonstrates the complex between-host transmission dynamics present in endemic areas in Spain. In conclusion, WGS results a valuable tool to track bTB infection at a high resolution and may contribute to achieve its eradication in Spain.

Benchtop nuclear magnetic resonance-based metabolomic approach for the diagnosis of bovine tuberculosis.

Even though enormous efforts and control strategies have been implemented, bovine tuberculosis (TB) remains a significant source of health and socioeconomic concern. The standard method used in TB eradication programs for in vivo detection is the tuberculin skin test. However, the specificity of the tuberculin skin test is affected by infection with non-tuberculous mycobacteria or by vaccination. Thus, some animals are not correctly diagnosed. This study aimed first to identify a plasma metabolic TB profile by high-field (HF) nuclear magnetic resonance (NMR) spectroscopy and second measure this characteristic TB metabolic profile using low-field benchtop (LF) NMR as an affordable molecular technology for TB diagnosis. Plasma samples from cattle diagnosed with TB (derivation set, n = 11), diagnosed with paratuberculosis (PTB, n = 10), PTB-vaccinated healthy control (n = 10) and healthy PTB-unvaccinated control (n = 10) were analyzed by NMR. Unsupervised Principal Component Analysis (PCA) was used to identify metabolic differences between groups. We identified 14 metabolites significantly different between TB and control animals. The second group of TB animals was used to validate the results (validation set, n = 14). Predictive models based on metabolic fingerprint acquired by both HF and LF NMR spectroscopy successfully identified TB versus control subjects (Area under the curve of Receiver Operating Characteristic over 0.92, in both models; Confidence Interval 0.77-1). In summary, plasma fingerprinting using HF and LF-NMR differentiated TB subjects from uninfected animals, and PTB and PTB-vaccinated subjects who may provide a TB-false positive, highlighting the use of LF-NMR-based metabolomics as a complementary or alternative diagnostic tool to the current diagnostic methods.