Skin testing is a crucial tool for the eradication of bovine tuberculosis (bTB) and maintenance of the officially tuberculosis-free (OTF) status at the herd and regional level. According to previous estimates, the specificity of skin tests under field conditions can be very high at the animal level, close to 100%, though lower values have been also reported. Given that the cumulative effect of imperfect specificity can substantially increase the herd-level probability of reactors under OTF conditions, here we aimed to accurately estimate the performance of the single intradermal tuberculin test (SIT) in OTF herds in a high prevalence area using a Bayesian hierarchical model. The analysis of data from 3039 cattle in 42 OTF beef herds revealed a median probability of disclosing a reactor using a severe interpretation of 1.24% (95% PPI = 0.083-8.3%), with an increasing risk in older animals. Still, simulation of the outcomes of the test at the herd-level revealed a median probability of observing ≥ 1 reactors per herd of 70.8%, for a herd-level specificity of 29.2% in our population (in contrast with field data indicating that >98% of Spanish herds test negative annually). Infection could not be confirmed post-mortem in any of the reactors. Our findings indicate that, while SIT remains a reliable tool for animal-level testing in OTF herds, interpretation of results at the herd-level can be complicated, particularly when potential exposure to bTB cannot be completely ruled out. Risk-based surveillance strategies could help to minimise the impact of precautionary OTF status suspensions.

IntroductionCaprine tuberculosis (cTB) is a zoonotic disease with significant implications for animal and public health and economic impact. Therefore, accurate ante-mortem diagnosis is essential. Diagnosis in goats rely on the single and comparative intradermal tuberculin test (SITT and CITT, respectively), both of which showing limited diagnostic performance. The aim of the study was to characterize the immune and physiological responses to cTB in naturally infected goats and to identify potential in vitro biomarkers that could improve the reliability of cTB diagnosis.MethodologyThe study was conducted in a cTB infected herd and goats were classified as reactors (positive to SITT or IFN-γ release assay and considered as infected) and non-reactors. Basal production of fifteen key immune cytokines was measured in plasma samples (n = 19) and compared with levels observed after stimulation with bovine purified protein derivative (PPDb). Acute phase proteins (haptoglobin/Hp and serum amyloid-A/SAA) and oxidative stress markers (total antioxidant status/TAS and malondialdehyde/MDA) were determined in serum samples (n = 90) while complete blood count (CBC) was performed on whole-blood samples (n = 44).Results and discussionRegarding cytokine expression patterns, plasma levels of IFN-γ and IFN-γ-inducible protein 10 (IP-10) in PPDb-stimulated samples were significantly higher (p < 0.005 and p < 0.001, respectively) in reactor goats than in non-reactor. When considering the difference between PPDb and PBS-stimulated samples (Δ-cytokine), levels of Δ-IFN-γ, Δ-IP-10 and Δ-IL-1α were significantly higher (p = 0.0006, p < 0.0001 and p = 0.022, respectively) in reactor goats compared to non-reactor. In addition, significantly higher (p = 0.014) levels of serum TAS were observed in reactor vs. non-reactor goats whereas no significant differences (p > 0.05) were found for Hp, SAA and MDA. Finally, among the CBC parameters, only the percentage of mid-range cells was significantly higher (p < 0.05) in reactor vs. non-reactor goats. This study demonstrates the differential expression of immunological (IFN-γ, IP-10 and IL-1α) and physiological (TAS) biomarkers in goats reacting to TB diagnostic tests and suggest their potential as biomarkers of cTB infection. These findings could lead to the development of novel diagnostic in vitro methodologies and contribute to more effective cTB eradication strategies.

Nuclear magnetic resonance (NMR)-based metabolomics has emerged as a robust and reproducible analytical platform for investigating the molecular mechanisms underlying disease and identifying candidate biomarkers with potential clinical relevance. Although initially developed for biomedical research, its application in veterinary medicine has expanded rapidly during the last decade, driven by the growing demand for precision medicine approaches capable of improving disease diagnosis, prognosis and therapeutic monitoring. This review summarizes the current landscape of NMR metabolomics in veterinary medicine, highlighting its applications across major clinical areas, including oncology, infectious, respiratory, digestive, renal, endocrine and musculoskeletal diseases. Collectively, the available evidence demonstrates that NMR metabolomics consistently identifies disease-associated metabolic alterations, providing novel insights into pathophysiology while supporting biomarker discovery in naturally occurring animal diseases. Beyond current clinical applications, we discuss the major challenges that continue to limit routine implementation, including biological variability, standardization of analytical workflows, multicentre validation and clinical translation. Particular attention is given to recent technological advances, such as benchtop NMR instrumentation, artificial intelligence-assisted data analysis, multi-omics integration and the development of collaborative networks and standardized platforms. Together, these advances position NMR metabolomics as a promising technology to support precision veterinary medicine, facilitating its transition from biomarker discovery to routine clinical implementation.

Animal tuberculosis (TB) affects a wide range of domestic species, including goats. TB eradication programs in goats are based on cell-based techniques such as the single and comparative intradermal tuberculin test (SITT and CITT, respectively). In recent years, an ELISA technique based on the P22 protein complex (P22 ELISA), has emerged as a valuable tool for TB diagnosis. The aim of the study was to evaluate the performance of the P22 ELISA in the context of a caprine TB eradication program using serum, individual milk and bulk tank milk (BTM) samples in order to define its usefulness in classifying herds compared to SITT and CITT. Samples from 53 herds categorized based on the detection of CITT reactors (16 high-risk herds, with one or more CITT reactors, and 37 low-risk herds, with only CITT-negative goats) were analyzed. Reactors in the P22 ELISA were detected in a higher number of high-risk herds using both serum (87.5%) and individual milk (81.3%) compared to SITT (75.0%) and CITT (31.3%), while the use of BTM led to the detection of 33.3% of the herds. Individual apparent prevalence was higher using the P22 ELISA in both serum (11.0%) and milk (15.0%) compared to the SITT (6.8%) and CITT (2.5%), with also a significantly (p < 0.001) higher number of reactors in individual milk compared with the serum. Similarly, all six herds with MTBC confirmed infection showed reactors to the SITT, CITT, and individual serum and milk P22 ELISA (2 out of 5 detected using BTM), although the highest reactivity was observed using individual milk samples. In the low-risk herds, a lower number of positive herds and animals were found with the P22 ELISA using serum or individual milk (51.4%) compared to SITT (59.5%) while using CITT only 2.7% of the herds were positive and none reacted to the P22 ELISA in BTM samples. This study shows that the P22 ELISA, using serum and especially individual milk samples, could be a complementary tool for maximizing the sensitivity of intradermal testing within the framework of a caprine TB eradication program.

Goats represent a significant reservoir for tuberculosis (TB) in animals, contributing notably to public and animal health challenges, causing economic repercussions. Ante mortem diagnosis of TB is hindered by the limited sensitivity of available techniques and false-positive results from other mycobacterial infections, such as paratuberculosis (PTB). Nuclear Magnetic Resonance (NMR)-based metabolomics provides unique fingerprinting of the disease’s metabolic status, making it a promising diagnostic tool. However, conventional high-resolution NMR has limitations in veterinary practice, where high costs and large equipment size are major constraints. Benchtop NMR spectrometer is proposed as a compact, cost-effective alternative for livestock farms. The study aimed to evaluate NMR-based metabolomics as a diagnostic tool and transfer it from high-resolution to benchtop NMR spectrometers in an animal setting. Serum samples from TB-infected, PTB-infected (n = 16), and healthy control goats (HC) were analyzed by both high-resolution and benchtop NMR spectroscopy. Multivariate statistical analysis successfully differentiated groups on the basis of their metabolic profiles with both spectrometers. We identified that betaine, glucose, glycerol, and lactate are significantly capable of distinguishing between the three groups. Additionally, 3-hydroxybutyrate, creatine, glutamate, leucine, lysine, phenylalanine, threonine, and tyrosine further differentiate TB from HC. Acetate, creatine, glutamate, isoleucine, leucine, and lysine distinguish TB from PTB, while 3-hydroxybutyrate and phenylalanine serve to differentiate PTB from HC. Analyses with both high-resolution and benchtop spectrometers demonstrated high sensitivity and accuracy and reliable metabolite identification. These findings highlight NMR’s spectroscopy potential to identify biomarkers associated with TB and PTB infection, improving health management in livestock.

IntroductionBovine tuberculosis (TB) in water buffalo (Bubalus bubalis) is primarily diagnosed using intra vitam tests, as the intradermal tuberculin test (IDT) and the interferon-gamma release assay (IGRA), both of which detect cell-mediated immunity (CMI). However, a subset of infected animals fails to mount a detectable CMI response, posing a significant risk of undetected transmission. Serological tests assessing the humoral immune response could provide a valuable complementary tool for identifying infected animals that escape detection through traditional CMI-based assays.MethodsThis study analyzed 895 serum samples from water buffaloes, including 393 from TB-free herds and 502 from TB-infected herds. Animals from TB-free herds were tested using IDT and an ELISA assay, whereas those from infected herds were also tested using IGRA. We developed an ELISA assay targeting MPB70, MPB83, ESAT6, CFP10, PPDB, and P22 antigens to investigate the role of the humoral response in TB diagnosis.Results and discussionThe ELISA showed a specificity of 98.2%. However, sensitivity differed based on the antigen used: among the most reactive proteins, sensitivity was 67.5% for MPB70, 69.8% for P22, and 74.4% for PPDB. Moreover, approximately 70% of samples with discordant IDT and IGRA results, as well as those with positive IDT but inconclusive IGRA results, tested positive by serology, highlighting the potential of antibody-based detection to improve TB diagnosis in buffaloes. Our findings suggest that integrating serological testing with standard diagnostic methods could enhance the detection of infected animals, ultimately contributing to better TB control in buffalo populations.

Identifying the causes of tuberculosis (TB) chronicity in cattle herds in Spain is a complex endeavour, mainly due to the multiple factors involved in persistence and the clonal population structure of the Mycobacterium tuberculosis complex. This study assessed the genomic diversity among M. bovis isolates belonging to SB0121, the most prevalent genotype in Spain, in chronically-infected herds. A total of 70 M. bovis isolates from 22 herds, located in six Spanish provinces, in which M. bovis SB0121 was isolated in at least three different sampling events were sequenced. Forty-three isolates from wildlife and cattle herds from the same or neighbouring municipalities to the problem herds were also included to identify putative local transmission events. The within-herd analysis revealed a highly complex scenario, in which the majority (95.45 %; n = 21) of the herds were affected by highly distant strains (> 12 SNP differences), probably as a result of separate introductions. Highly similar isolates (< 6 SNPs) were retrieved in different sampling events from 11 herds, likely indicating active transmission of the outbreak strain or continued exposure to the same source of infection. The between-herd and interspecies comparison suggested the occurrence of several putative epidemiological links between cattle and wildlife species from the same or neighbouring municipalities, reflecting the complex epidemiology of the disease in some of the studied areas. The findings of this study highlight the usefulness of whole genome sequencing to study bTB breakdowns and pinpoints its potential for unravelling possible sources of persistence in cattle herds.

Caprine tuberculosis (TB) causes a zoonotic disease with significant economic and health implications. However, excluding some regions, goat herds are not subjected to official TB eradication programs. Implementing vaccination protocols for this species could provide a complementary and effective control strategy against TB. We assessed the protective efficacy and immune response associated with a heat-inactivated Mycobacterium bovis (M. bovis)-based immunostimulant (HIMB) applied intramuscularly against caprine pulmonary TB on 20 kid goats (10 immunized, 10 controls) naturally exposed to M. caprae infected goats for 10 months. TB-compatible lung lesions were assessed, alongside a local immune response analysis by immunohistochemistry of cell populations (Macrophages (MΦs), neutrophils, T, and B lymphocytes) and associated immune mediators (iNOS, TNF-α, IL-1α, IL-6, IFN-γ, TGF-β, IL-4). In the control group, 60

Tuberculosis (TB) is a notifiable zoonotic disease caused by bacteria of the Mycobacterium tuberculosis complex (MTBC) that affects a multitude of domestic and wild species. The main lesions caused by these mycobacteria are tuberculous granulomas, which determine the organism's immune response to the disease. Although TB pathogenesis in cattle has been extensively studied, information regarding its progression in other species of interest for the maintenance and transmission of TB such as goats remains limited. This study aimed to characterise the immune response developed in the lungs of goats naturally infected with mycobacteria of MTBC by assessing key cell populations and immunomodulatory molecules involved in defending against TB. Hence, twelve 6-12month-old Guadarrama kid goats, initially TB-free, were selected and exposed to M. bovis through close contact with other infected goats. Only animals that tested positive by any of the TB diagnostic methods at the end of the experiment were included in the final analysis (n = 9). Gross and microscopic lesions compatible with TB (TBL) in different organs, as well as local response to TB in lungs were evaluated. Our results revealed that after five months of exposure, 44.4 % (4/9) of the M. bovis-infected animals exhibited TBL in the lungs (TBLL+), characterized by a predominance of non-cavitary necrotic granulomas. TBLL+ animals showed significantly higher presence of neutrophils, macrophages (M Phi s) and lymphocytes along with greater expression of interferon (IFN)gamma. Conversely, the remaining animals did not present macroscopic or microscopic TBL in the lungs (TBLL-) (5/9). However, these goats displayed elevated expression of toll-like receptors (TLR)2 and TLR4 alongside heightened expression of pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) and interleukin (IL)-10. These results suggest the potential development of an effective immune response that may suppress or delay of TBL in infected animals. Further research is needed to elucidate how these molecules, which are involved in the defence against MTBC, confer protection and modulate their expression during infection for TB control.

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.

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.

The study aims to evaluate the potential use of mycobacterial ESAT6 and CFP10 antigens, Early Secretory Proteins (ESP) in the Skin Test used for bovine tuberculosis (TB) diagnosis in Water Buffalo. A pilot study was performed on 21 buffaloes from a TB outbreak and 11 buffaloes from a TB-free herd. Three concentrations of ESAT6-CFP10 (10, 20, and 30 mg) and two of ESP (50 and 100 µg) were inoculated in the Skin Test, along with PPDB, PPDA, and PBS as a negative control. Skin thickness was measured with calipers before the test and every 24 hours for 4 days. Then, to evaluate the specificity of the antigens, a field study was conducted, and 100 buffaloes from a TB-free herd were inoculated using the best antigens concentration derived from the pilot study. In the positive buffaloes, the strongest skin response was to PPDB at 24h, with some subjects becoming inconclusive at 72 and 96 h. A peak response to PPDA at 48 hours was detected, followed by a slight decrease. The response to ESP-100 µg remained high at 24 and 48 h, then decreased, remaining positive at 72 h. In the 100 TB-free buffaloes, the best specificity was observed using ESAT6-CFP10 and ESP. ESP yielded the best results, showing higher reactivity in infected animals and no reactivity in the healthy ones at 72 h. Therefore, ESP could be an excellent candidate for further extensive studies in the buffalo species to improve Skin Test performance.

Limitations in diagnostic test performance are one of the major challenges hampering the eradication of bovine tuberculosis (bTB). Non-tuberculous mycobacteria (NTM) are considered one of the main causes of non-specific reactions in the intradermal tuberculin test, the most widely used bTB diagnostic test. To determine the role of NTMs in bTB misdiagnosis in Spain, an experimental study including the NTM species most commonly found in bTB-positive animals from bTB-free farms in the country (M. avium subsp. avium (Maa), "Mycobacterium avium subsp. hominissuis" (Mah), M. bourgelatii, M. intermedium, M. kansasii and M. nonchromogenicum) was carried out on guinea pigs and cattle. First, guinea pigs were sensitized with the selected NTMs, and six weeks postsensitization four antigen mixtures (bovine-PPD, avian-PPD, P22 and ESAT6-CFP10) were inoculated intradermally and their effect was measured 24- and 48-h post-inoculation. Larger erythematous reactions were observed in guinea pigs sensitized with Mah, M. kansasii, and Maa, with significant differences in the reactions measured at the bovine-PPD inoculation site for the two first bacteria compared with other NTMs. The sensitization process was repeated in cattle, and five months post-sensitization the same antigen mixtures were inoculated in the cervical region and responses were measured at 48- and 72-h post-inoculation. A significantly higher increase in the skinfold thickness measured at the bovine-PPD inoculation site was observed in calves sensitized with Mah, Maa, M. intermedium and M. kansasii. These results demonstrate that certain NTM species may play a more significant role in bTB diagnostic interferences and show that results obtained in guinea pig and bovine models do not always coincide.

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.

Samples from the mesenteric lymph nodes (MS LNs) and ileocecal valves (ICV) of 105 goats, comprising 61 non-vaccinated and 44 vaccinated against Mycobacterium avium subspecies paratuberculosis (MAP), were collected at slaughter from a farm with a confirmed history of paratuberculosis (PTB). These goats had subclinical infections. PTB-compatible lesions in the MS LNs, ICV lamina propria (LP), and Peyer's patches (PPs) were graded separately. Furthermore, the load of acid-fast bacilli was quantified using Ziehl-Neelsen staining (ZN), MAP antigens by immunohistochemistry (IHC), and MAP DNA by PCR targeting the IS900 sequence. Gross PTB-compatible lesions were found in 39% of the goats, with 31.72% vaccinated (V) and 68.29% non-vaccinated (nV). Histopathological lesions induced MAP were observed in 58% of the animals, with 36.07% vaccinated and 63.93% non-vaccinated. The inclusion of histopathology as a diagnostic tool led to a 28% increase in diagnosed cases in MS LNs and 86.05% in ICV. Grade IV granulomas with central mineralization and necrosis were the most common lesions in MS LNs. In the ICV, mild granulomatous enteritis with multifocal foci of epithelioid macrophages was predominant, occurring more frequently in the PPs than in the LP. Furthermore, statistical differences in the presence of histopathological lesions between vaccinated and non-vaccinated goats were noted in MS LNs, ICV LPs, and ICV PPs. Non-vaccinated animals showed higher positivity rates in ZN, IHC, and PCR tests, underscoring the benefits of anti-MAP vaccination in reducing PTB lesions and bacterial load in target organs. Our findings emphasize the necessity of integrating gross and histopathological assessments with various laboratory techniques for accurate morphological and etiological diagnosis of PTB in both vaccinated and non-vaccinated goats with subclinical disease. However, further studies are required to refine sampling protocols for subclinical PTB in goats to enhance the consistency of diagnostic tools.

Mycobacterium bovis (M. bovis) is the primary agent of bovine tuberculosis (TB) in Mediterranean buffalo, which has a negative economic impact on buffalo herds. Improving TB diagnostic performance in this species represents a key step to eradicate efficiently this disease. We have recently shown the utility of the IFN-γ assay in the diagnosis of M. bovis infection in Mediterranean buffaloes (Bubalus bubalis), but other cytokines might be useful immunological biomarkers of this infection. We therefore investigated the utility of key immune cytokines as diagnostic biomarkers of M. bovis infection in this species. Thirty-six Italian Mediterranean buffaloes were used in this study: healthy animals (N = 11), infected (IFN-γ test positive, no post-mortem lesions, no M. bovis detection; N = 14), and affected (IFN-γ test positive, visible post-mortem lesions; N = 11). Heparin blood samples were stimulated with bovine purified protein derivative (PPD-B), alongside controls, and 18–24 h later plasma were collected. Levels of 14 key cytokines were measured: IFN-γ, IL-17, IL-4, IL-10, TNF, IL-1α, IL-1β, IL-6, IP-10, MIP-1α, MIP-1β, MCP-1, IL-36Ra, and VEGF-A. We observed that both infected and affected animals released higher levels of IFN-γ, IL-17, IL-10, TNF, IL-1α, IL-6, MIP-1β, in response to PPD-B compared to healthy subjects. Mycobacterium bovis infected animals released also higher levels of IL-1β and IP-10 in response to PPD-B compared to healthy subjects, whereas only tendencies were detected in affected animals. Affected animals only released MIP-1α in response to PPD-B compared to healthy subjects and in this group higher values of PPD-B specific TNF was also observed. Finally, canonical discriminant analysis (CDA) was used to generate predictive cytokine profiles by groups. Our data suggest that IL-10, TNF, IL-1α, IL-6, MIP-1β could be useful biomarkers of TB in Mediterranean Buffalo and can improve the TB diagnostic performance in this specie.

Caprine livestock are significant reservoirs of the Mycobacterium tuberculosis complex (MTBC), contributing to tuberculosis (TB) transmission among animals and humans. The P22 protein immunocomplex (P22PI), derived from bovine tuberculin, shows immunostimulating capacity and is used for TB diagnosis. This study assessed the immunogenicity and protective efficacy of P22PI in two groups of goats: 24 na & iuml;ve goats (12 immunised, 12 controls) from a TB-free herd, and 24 infected goats (12 immunised, 12 controls), referred to as pre-infected animals, from a M. bovis-infected herd. Both were exposed for 5 months to M. bovis-naturally infected goats. Reactors to single and comparative intradermal tuberculin (SIT and SCIT, respectively) tests and interferon-gamma release assay (IGRA) significantly increased (p < 0.05) in both groups 5 months' post-exposure, with no significant differences between immunised and control animals. However, immunised animals exhibited a significantly higher (p < 0.05) antibody response against P22PI. Most na & iuml;ve animals (83.3%) and all pre-infected animals developed TB-compatible lesions, with extensive necrosis in the lungs and associated lymph nodes, compared to 50% and 83.3% of control animals, respectively. These findings suggest that while P22PI stimulates an intense antibody response under the conditions of the present study, it does not confer protection against TB and may exacerbate disease severity.

The growing use of real-time PCR (qPCR) as a diagnostic method for bovine TB (bTB) requires rapid and effective DNA extraction methods, which are crucial for its success. Automated DNA extraction methods based on magnetic beads are a promising alternative to conventional silica column-based protocols (COL protocol) due to their high throughput capacity and reduced hands-on time. This study aimed to assess the performance of the MagMax CORE Nucleic Acid Purification kit and the KingFisher Flex instrument (KF protocol) as an alternative for scaling up the use of qPCR in bTB diagnosis. Performance was evaluated with two different real-time PCR (qPCR) protocols, based on the IS6110 element and the QuantiFast and VetMAX™ (QF and VM protocols) kits, on 145 frozen tissue homogenates confirmed as either bTB-positive or negative through a composite reference standard based on microbiological culture, column-based extraction, and qPCR, as well as on negative tissue samples spiked with 106 to 103 CFU/ml of M. bovis BCG. The performance of both qPCR protocols was very high on samples extracted using the KF protocol, with positive percent agreement (PPA) values of 89.04

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.