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The main etiologic agent of tuberculosis (TB) in livestock is Mycobacterium bovis; human TB cases caused by M. bovis are rare. Analysis of a TB outbreak caused by polyresistant M. bovis involving a human and sympatric sheep in Spain suggests local circulation of drug-resistant M. bovis strains among livestock., Generalitat de Cataluña, Ministerio de Agricultura, Pesca y Alimentación (España), Depto. de Sanidad Animal, Centro de Vigilancia Sanitaria Veterinaria (VISAVET), TRUE, pub

Author contributions J.M.G. and I.A.S. conceived and planned the study and obtained the funding. B.P.V., L.M., M.L.B., J.B., G.J.J., H.M.V., J.M.G. and I.A.S. contributed to choosing isolates and/or deciding skin test protocols and antigens. B.P.V., L.M. and M.L.B. provided isolates. G.J.J. and H.M.V. provided antigens. L.F.V., M.F., M.V.G., J.M.G. and I.A.S. performed the experiments and laboratory analysis and compiled the data. R.A.J., J.M.G. and I.A.S. analyzed and interpreted the results. R.A.J., J.M.G. and I.A.S. wrote the original draf of the manuscript. All authors reviewed the original draft and contributed to writing and editing the final version of the manuscript. Additional information Correspondence and requests for materials should be addressed to I.A.S., 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., Ministerio de Ciencia, Innovación y Universidades, INTERREG POCTEFA, European Regional Development Fund, Depto. de Sanidad Animal, Centro de Vigilancia Sanitaria Veterinaria (VISAVET), TRUE, pub

Voles are maintenance hosts of Mycobacterium microti. In line with the goal to eradicate tuberculosis (TB) in livestock, the role of this mycobacteria needs to be assessed since it might interfere with current M. bovis / M. caprae surveillance strategies. To better understand the pathogenesis of TB in voles, an experimental infection model was set up to reproduce M. microti infection in laboratory Bank voles (Myodes glareolus). Two infection routes (intragastric and intraperitoneal) and doses (10 5 and 10 6 CFU/0.1 mL) were assessed. Voles were culled at different post-infection time points. Serology, histopathology, acid-fast bacilli staining, qPCR, and mycobacterial culture from tissues were performed. In addition, qPCR from feces and oral swabs were conducted to assess bacterial shedding. The model allowed us to faithfully reproduce the disease phenotype described in free-ranging voles and characterize the pathogenesis of the infection. Most animals showed multifocal and diffuse granulomatous lesions in the liver and spleen, respectively. Less frequently, granulomas were observed in lungs, lymph nodes, muscles, and salivary gland. Mycobacterial DNA was detected in feces from a few animals but not in oral swabs. However, one contact uninfected vole seroconverted and showed incipient TB compatible lesions, suggesting horizontal transmission between voles.

The P22 ELISA was recently developed for the serodiagnosis of animal tuberculosis. Herein, the stability of the P22 antigen in different presentations and storage conditions, and the cross-reactivity with Corynebacterium pseudotuberculosis infection in small ruminants were evaluated. For the stability assay, serum samples from cows, sheep, goats, alpacas, badgers, and wild boar were used in the P22 ELISA. The cross-reactivity analysis used sera from sheep and goats with caseous lymphadenitis (CLA). Differences in the immune recognition of P22 were found when the antigen was stored at 40 °C, but without altering the negative or positive status of each sample. P22 ELISA presented 5.71 % cross-reactivity when CLA-positive sheep were evaluated, but no cross-reaction was observed among CLA-positive goat serum samples. This study showed that the P22 protein complex is stable under different formulations and temperatures, and that the assay presents a low cross-reactivity with CLA.

The main etiologic agent of tuberculosis (TB) in livestock is Mycobacterium bovis; human TB cases caused by M. bovis are rare. Analysis of a TB outbreak caused by polyresistant M. bovis involving a human and sympatric sheep in Spain suggests local circulation of drug-resistant M. bovis strains among livestock., Generalitat de Cataluña, Ministerio de Agricultura, Pesca y Alimentación (España), Depto. de Sanidad Animal, Centro de Vigilancia Sanitaria Veterinaria (VISAVET), TRUE, pub

The high-resolution WGS analyses of MTBC strains have provided useful insight for determining sources of infection for animal tuberculosis. In Spain, tuberculosis in livestock is caused by Mycobacterium bovis and Mycobacterium caprae, where wildlife reservoirs play an important role. We analyzed a set of 125 M. bovis isolates obtained from livestock and wildlife from Catalonia to investigate strain diversity and identify possible sources and/or causes of infection. Whole-genome SNP profiles were used for phylogenetic reconstruction and pairwise SNP distance analysis. Additionally, SNPs were investigated to identify virulence and antimicrobial resistance factors to investigate clade-specific associations. Putative transmission clusters (≤12 SNPs) were identified, and associated epidemiological metadata were used to determine possible explanatory factors for transmission. M. bovis distribution was heterogeneous, with 7 major clades and 21 putative transmission clusters. In order of importance, the explanatory factors associated were proximity and neighborhood, residual infection, livestock-wildlife interaction, shared pasture, and movement. Genes related to lipid transport and metabolism showed the highest number of SNPs. All isolates were pyrazinamide resistant, and five were additionally resistant to isoniazid, but no clade-specific associations could be determined. Our findings highlight the importance of high-resolution molecular surveillance to monitor bovine tuberculosis dynamics in a low-prevalence setting.

Tuberculosis (TB) in wildlife challenges epidemiological surveillance and disease control. An outbreak of TB was detected in a free-ranging wild boar population of a Natural Park in Catalonia (Spain) and the outbreak investigation was conducted in the area. During the study period (2015-2020), 278 wild boars were analysed by gross pathology, histopathology, mycobacterial culture and DVR-spoligotyping. In addition, all cattle (49) and goat (47) herds of the area were tested with tuberculin skin test. TB compatible lesions were detected in 21 wild boars, and Mycobacterium caprae was isolated in 17 of them with two different spoligotypes: SB0415 (13) and SB1908 (4). Only two goat herds showed TB positive animals that were subsequently slaughtered. M. caprae with the spoligotypes SB0416 and SB0415 were isolated from these animals. To investigate the phylogenetic relationships and the transmission chain of the outbreak, nine strains isolated from six wild boars and three goats of the study area were analysed by whole genome sequencing (WGS) followed by single nucleotide polymorphism (SNP) analysis by maximum likelihood and median-joining network inference methods. Results indicated that infected wild boars maintained M. caprae strains circulation in their own population and have likely transmitted the infection to goats, thus acting as TB reservoirs, compromising the success of livestock TB eradication campaigns and posing a risk for public health. The results also highlighted the usefulness of WGS followed by SNP analysis in providing relevant epidemiological information when detailed contact data are missing.

TFM, La tuberculosis es una importante enfermedad con grandes repercusiones en la salud pública. En el sector ganadero implica elevadas pérdidas económicas y supone un riesgo para la salud de los usuarios del sector, así como del consumidor. Así pues, para poder combatir esta enfermedad, es imprescindible encontrar métodos eficaces para tratarla y controlarla. Este trabajo aborda un aspecto poco estudiado para lograr este fin en la producción animal: el estudio de la relación de los helmintos gastrointestinales en la progresión de la infección tuberculosa. Resultados de otros estudios sugieren que los diferentes géneros y especies de simbiontes pueden jugar papeles muy distintos frente la tuberculosis. Mediante este trabajo de campo se pretende poner en evidencia esta supuesta variedad de roles, en el caso concreto del ganado caprino. Los resultados obtenidos indican que este campo merece ser estudiado con más profundidad, puesto que podría ofrecer grandes ventajas en la lucho contra esta enfermedad.

Current eradication strategies of tuberculosis (TB) in goats mainly rely on the single intradermal tuberculin test (SIT) and single intradermal cervical comparative tuberculin tests (SICCTs). TB vaccination has been proposed as a cost-effective option in high-prevalence herds or countries where economic compensation for the slaughter of positive animals is not affordable. However, TB vaccination compromises the efficiency of tuberculin-based diagnostic tests. In this study, the performance of a new diagnostic platform, based on the P22 antigenic complex, was assessed for skin test (ST), interferon-gamma release assay (IGRA), and serology under different TB scenarios. The sensitivity (Se) of diagnostic tests was assessed in TB-infected goats from the same farm (herd A, N = 77). The specificity (Sp) was assessed in two TB-negative farms (both vaccinated against paratuberculosis): one TB unvaccinated (herd B, N = 77) and another vaccinated with bacille Calmette-Guérin (BCG) (herd C, N = 68). The single (s) P22-IGRA showed the highest Se among IGRA tests (91%), and the comparative (c) P22-ST showed the highest Sp (100% in herd B and 98% in herd C). Combined interpretation of techniques enabled the best diagnostic performances. Combining the SICCT + sP22-IGRA improved Se (97%) compared to SICCT + tuberculin-based IGRA (95%), with a reduction of Sp (95 and 100%, respectively). Besides, combination of P22-ELISA with cP22-ST or SICCT elicited a similar performance in the non-vaccination context (Se: 94 and 95%; Sp: 95 and 95%, respectively), but Sp was significantly higher for the combination with cP22-ST compared to SICCT in the TB vaccination context (95 and 79%, respectively). The combination of serological tests based on P22 and MPB83 showed higher complementarity and improved 13 percentage points the Se of P22-ELISA alone. These findings suggest that either cell-mediated or antibody-based diagnostic techniques, using the P22 antigen complex, can contribute to improve t

Universitat Autònoma de Barcelon grant number D045702/B14P0024 per a Giovanna Ciaravino, In Spain, the eradication of tuberculosis (TB) in cattle has had some setbacks and therefore we need to re-evaluate the current surveillance strategies. The Spanish cattle TB surveillance system consists of three components: a) routine skin testing, b) slaughterhouse surveillance, and c) pre-movement controls. Our objectives were to assess the effectiveness of the whole surveillance system and evaluate the relative contribution of each of those components, both at national and at the province level. The effectiveness was estimated based on both the sensitivity of detection per year and the time until detection. Moreover, we evaluated the impact of different factors on that effectiveness. We used a stochastic model that simulated the spread of Mycobacterium tuberculosis-complex (MTC) infection within cattle herds that was modified to incorporate the three components of TB surveillance. Input data, at the province level, on cattle movements off-farm or the frequency of the routine screening were provided by the Spanish Ministry for Agriculture, Fisheries and Food. Under the current conditions, 96.1% of Spanish herds were detected within one year after their infection (i.e. mean sensitivity of TB surveillance), although that detection took on average 178 days. The surveillance system effectiveness was highly dependent on the routine skin testing, responsible for the detection of almost 90% of the infected herds, while slaughterhouse surveillance and pre-movement controls contributed only to the identification of a small proportion of infected herds. We observed substantial differences in the effectiveness of the surveillance components among Spanish provinces, although in general, the sensitivities were high. The most influential factor on the efficiency of TB detection in Spain was, by far, the frequency of routine controls, followed by the sensitivity of the test used. In a context of reduced funding for cattle TB eradication, the frequency of testing should be adapt

Control of animal tuberculosis (TB) through vaccination has emerged as a long-term strategy to complement test and slaughter control strategy. A pilot trial under field conditions was conducted in a goat herd with high TB prevalence to assess the efficacy of the Mycobacterium bovis BCG vaccine. Twenty-three goat kids vaccinated with BCG and other 22 unvaccinated control kids were euthanized at 18 months post-vaccination. Gross pathological and histopathological examination of target tissues was performed for detection of tuberculous lesions and assessment of vaccine efficacy. Mycobacterial culture and DNA detection were used to confirm Mycobacterium caprae infection. Vaccination significantly reduced the number of animals with TB lesions compared to unvaccinated controls (35% and 77%, respectively; P < 0.01). This reduction was even higher if only extra-pulmonary infection was considered (17% and 68%, respectively; P < 0.001). This trial demonstrates that BCG vaccination of goats can significantly reduce the TB lesion rates in high disease exposure conditions, indicating that vaccination could contribute to the control of TB in domestic goats.

Additional epidemiological information regarding tuberculosis in wild ungulates, including affected species, prevalence, associated risk factors and appropriate diagnostic methods need to be obtained in Europe. A cross-sectional study was carried out on wild artiodactyl species, including Eurasian wild boar (Sus scrofa) red deer (Cervus elaphus), roe deer (Capraelus capraelus), fallow deer (Dama dama), Spanish ibex (Capra pyrenaica hispanica) and mouflon (Ovis musimon), in Spain to assess the seroprevalence against Mycobacterium bovis or cross-reacting members of the Mycobcaterium tuberculosis complex, and to provide information on associated risk factors. Previously, two in-house indirect enzyme linked immunosorbent assays (bPPD-ELISA and MPB83-ELISA) were developed using bPPD and MPB83 antigens. Positive reference sera were selected from infected animals confirmed by culture. The M. bovis isolates belonged to spoligotypes SB0121, SB0120, SB0295, SB0265 and SB0134. Two hundred and two out of 1367 (7.5%; 95% CI: 6.1-8.9) animals presented antibodies against M. bovis by both ELISAs. Significantly higher TB seroprevalence was observed in wild boar compared to the other species analyzed. Seropositivity against M. bovis was not found in any out of 460 Spanish ibex analyzed. The logistic regression model for wild boar indicated that the seropositivity to M. bovis was associated with age, location and year of sampling, while the age was the only risk factor associated with M. bovis seroprevalence in red deer and fallow deer.

The control of bovine tuberculosis (bTB) is at a critical point in the last stage of eradication in livestock. Wildlife species recently have emerged infected with TB in Europe, particularly ungulates in the Iberian Peninsula. Epidemiological information regarding TB in wild ungulates including affected species, prevalence, associated risk factors and appropriate diagnostic methods need to be obtained in these countries. A cross-sectional study was carried out on wild artiodactyl species, including Eurasian wild boar (Sus scrofa) red deer (Cervus elaphus), roe deer (Capraelus capraelus), fallow deer (Dama dama), Spanish ibex (Capra pyrenaica hispanica) and mouflon (Ovis musimon), in Spain to assess the seroprevalence against Mycobacterium bovis or cross-reacting members of the Mycobcaterium tuberculosis complex (MTBC), and to provide information on associated risk factors. Previously, two in-house indirect enzyme linked immunosorbent assays (bPPD-ELISA and MPB83-ELISA) were developed using known TB status sera. Positive reference sera were selected from infected animals confirmed by culture. The M. bovis isolates belonged to spoligotypes SB0121, SB0120, SB0295, SB0265 and SB0134. Two hundred and two out of 1367 (7.5%; 95% CI: 6.1-8.9) animals presented antibodies against M. bovis by both bPPD-ELISA and MPB83-ELISA. Significantly higher TB seroprevalence was observed in wild boar compared to the other species analyzed. Interestingly, seropositivity against M. bovis was not found in any out of 460 Spanish ibex analyzed. The logistic regression model for wild boar indicated that the seropositivity to M. bovis was associated with age, location and year of sampling, while the only risk factor associated with M. bovis seroprevalence in red deer and fallow deer was the age. The seroprevalence observed indicates a widespread exposure to MTBC in several wild artiodactyl species in southern Spain, which may have important implications not only for conservation but also for an

The increasing interest in Rift Valley fever virus (RVFV) and its potential impact on naive animal populations deserve revisiting experimental reproduction of RVFV infection, particularly in those animal breeds for which no data about their susceptibility to RVFV infection have ever been recorded. In this study we show the susceptibility of 9-10 weeks old European sheep (Ripollesa breed) to RVFV infection, showing a mild, subacute form of disease. Four different viral isolates efficiently replicated in vivo after subcutaneous experimental inoculation, and consistent viral loads in blood and virus shedding (variable in length depending on the RVFV isolate used) were detected, showing horizontal transmission to a noninfected, sentinel lamb. RVFV infection caused transient pyrexia in adult lambs and no other clinical symptoms were observed, with the exception of corneal opacity ("blue eye") found in 3 out of 16 subcutaneously inoculated sheep. In conclusion, adult sheep from this European breed are readily infected with RVFV without apparent clinical manifestations. Copyright 2010, Mary Ann Liebert, Inc.