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The current demographic expansion of red deer populations in the northern hemisphere has led to conflicts at the levels of health, the economy, and conservation. Potential tools for deer management include the delivery of contraceptives that impair reproduction in order to control populations or the delivery of treatments/vaccines in order to control health. To successfully implement these control strategies, it is necessary to develop suitable methods with which to deliver treatments to red deer in field settings. The objectives of this study were (i) to evaluate the specificity and palatability of three bait types—alfalfa, feed, and molasses-based—for red deer (Cervus elaphus) and (ii) to describe the constraints that limit or enhance the effectiveness of the bait deployment method—on the ground or in specific red deer feeders—for the oral delivery of treatments to red deer in Mediterranean ecosystems. Our results show that alfalfa baits are the least palatable but the most deer-selective type of bait, whereas feed and molasses baits are highly palatable but low specific (mean bait consumption rates on the ground were 0.811, 0.949, and 0.955, respectively). The appropriate season for oral treatments using alfalfa baits both on the ground and in deer feeders is late summer (mean bait consumption rate on the ground for alfalfa-based baits was 0.939 in summer period). These results indicate that alfalfa baits are sufficiently palatable and specific in summer for them to be an efficient means to deliver treatments to red deer in Mediterranean ecosystems., This study was funded by the Spanish Ministry for Science, Innovation and Universities (MCIU) and the European Regional Development Fund (FEDER) through grants “AGL2014-56305” and “CGL2017-89866R.” JMG was supported by an FPI grant from MCIU (BES-2015-072206), whereas DGB was supported by the Juan de la Cierva program of MCIU. LCM acknowledges funding from grant INIA E-RTA2015-00002-C02-02 and contract PEJ2018-003155-A from MCIU.

The Eurasian wild boar (Sus scrofa) is the main wild reservoir of the Mycobacterium tuberculosis complex in Mediterranean woodlands and a key risk factor for cattle tuberculosis (TB) breakdowns. Wild boar vaccination therefore has the potential to be a valuable tool for TB control. We tested two orally delivered vaccines, heat-inactivated Mycobacterium bovis (IV) and BCG, in four sites (two per vaccine type: one Managed and one Natural or unmanaged) during four years. TB was also monitored in 15 unvaccinated sites (spatial control), as well as in all sites from one year prior to intervention (temporal control). The rationale is that by vaccinating 2–6 month old wild boar piglets we can reduce disease at the population level during the study period. This is achievable due to the fast turnover of wild boar populations. Vaccine baits were deployed using selective piglet feeders and this method proved highly successful with uptake rates of 50 to 74% in Natural sites and 89 to 92% in Managed sites. This is relevant for the potential delivery of vaccines to control other diseases, too. Local wild boar TB prevalence at the beginning of the study was already high ranging from 50 to 100%. TB prevalence increased in unvaccinated sites (6%), while a significant decline occurred in the Managed IV site (34%). Changes recorded in the remaining sites were not significant. The short-term impact of vaccination observed in the field was complemented by mathematical modelling, representative of the field system, which examined the long-term impact and showed that vaccination of piglets reduced prevalence and increased abundance at the population level. We conclude that IV could become part of integrated TB control schemes, although its application must be tailored for each specific site., This is a contribution to Plan Nacional I+D+i AGL2014-56305 from MINECO and EU FEDER, and to the EU–FP7 grant WildTBVac. E. Tanner was supported by The Maxwell Institute Graduate School in Analysis and its Applications, a Centre for Doctoral Training funded by the UK Engineering and Physical Sciences Research Council (grant EP/L016508/01), the Scottish Funding Council, Heriot-Watt University and the University of Edinburgh. I. Díez-Delgado was supported by a predoctoral grant from MINECO.

Diagnostic tests based on cell-mediated immunity are used in programmes for eradication of bovine tuberculosis (Mycobacterium bovis). Serological assays could be applied as ancillary methods to detect infected animals. Our objective was to evaluate two serological techniques: M. bovis Ab Test (IDEXX, USA) and Enferplex™ TB assay (Enfer, Ireland) in animals tested simultaneously with the single and comparative intradermal tests and the interferon-gamma assay. This work was performed at two stages. First, a preliminary panel of samples collected prior to intradermal tests from tuberculosis-free (n=60) and M. bovis-infected herds (n=78) was assayed, obtaining high specificity: 100% (M. bovis Ab Test) and 98.3% (Enferplex TB assay) but low sensitivity (detection of M. bovis infected animals): 23.9% (M. bovis Ab Test) and 32.6% (Enferplex TB assay). Subsequently, the use of serological techniques was further studied in two herds with M. bovis infection (n=77) using samples collected prior to, and 72 h and 15 days after PPD inoculation. The highest level of detection of infected animals for serology was achieved at 15 days post-intradermal tests taking advantage of the anamnestic effect: 70.4% and 85.2% in herd A, and 66.7% and 83.3% in herd B, using M. bovis Ab Test and Enferplex TB assay, respectively. Quantitative results (average values obtained with M. bovis Ab Test ELISA and degree of positivity obtained with Enferplex TB assay) were higher in animals showing lesions compatible with tuberculosis. No significant differences were observed in the number of confirmed infected animals detected with either serological technique.

En el Hospital Nacional Profesor Alejandro Posadas se estudiaron la incidencia de influenza, las características de casos y tipos y subtipos de virus circulantes de enero a agosto de 2013 inclusive, semanas epidemiológicas (SE) 1-35, y se compararon con los años 2009-2012. De fin de mayo a agosto inclusive de 2013 (SE 18 a 35) se observó un aumento del porcentaje de consulta por enfermedades respiratorias, enfermedad tipo influenza e internación por neumonía y se diagnosticaron 207 casos: 153 influenza A (FLU-A)(H1N1pdm09), 46 A(H3), ocho A(sin subtipificar). La mayor frecuencia fue en menores de 5 años, seguida por el grupo de 60 a 64.La chance de tener la enfermedad fue tres veces mayor en el grupo de 40-64 años versus 15-39 o > 64 años. La letalidad, que aumentó con la edad, fue de 7.2% y la chance de morir fue seis veces mayor en los > 64 años. El porcentaje de vacunación entre los casos fue11.6%. Ninguno de los fallecidos estaba vacunado. Luego de la pandemia de 2009 el porcentaje de consultas anuales disminuyó hasta 2012, con un aumento en el período invernal de 2013 de 52.0% con respecto a 2012. La circulación viral en 2013 fue más temprana que en los años anteriores. En 2009 y 2013 la mayor circulación fue FLU-A (H1N1pdm), en 2011 FLU-A(H3) y en 2010 y 2012 FLU-A(H3) y FLU-B.

The Eurasian wild boar (Sus scrofa) is a reservoir for tuberculosis (TB) in which vaccination is a valuable tool for control. We evaluated the protection and immune response achieved by homologous and heterologous regimes administering BCG and heat-inactivated Mycobacterium bovis (IV). Twenty-one wild boar piglets were randomly allocated in five groups: Control, homologous BCG, homologous IV, heterologous IV-BCG, heterologous BCG-IV. Significant 67% and 66% total lesion score reductions were detected in homologous IV (IVx2) and heterologous IV-BCG groups when compared with Control group (F4,16 = 6.393, p = 0.003; Bonferroni Control vs IVx2 p = 0.026, Tukey Control vs IV-BCG p = 0.021). No significant differences were found for homologous BCG (although a 48% reduction in total lesion score was recorded) and BCG-IV (3% reduction). Heterologous regimes did not improve protection over homologous regimes in the wild boar model and showed variable results from no protection to similar protection as homologous regimes. Therefore, homologous regimes remain the best option to vaccinate wild boar against TB. Moreover, vaccine sequence dramatically influenced the outcome underlining the relevance of studying the effects of prior sensitization in the outcome of vaccination., This is a contribution to Plan Nacional I + D+i AGL2011-30041 from MINECO and EU FEDER. I. Diez-Delgado was supported by a predoctoral grant from MINECO.

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.

Mycobacterium avium subspecies paratuberculosis ( M. a. paratuberculosis ) is responsible for paratuberculosis or Johne’s disease, a chronic inflammation of the gastrointestinal tract in different animal species. Some studies have also established a link between this microorganism and Crohn’s disease in humans. Although, M. a. paratuberculosis is a difficult microorganism to cultivate in the laboratory (occasionally is non-cultivable), a proper molecular characterization of M. a. paratuberculosis is necessary to better understand the epidemiology of the disease, and design strategies to eradicate it. In the present review, we compile and discuss the recent progress attained in the diagnostic and characterization of this pathogen.

Contains fulltext : 107786.pdf (Publisher’s version ) (Closed access) Mycobacterium bovis isolates from the Iberian Peninsula are dominated by strains with spoligotype patterns deleted for spacer 21. Whole-genome sequencing of three Spanish strains with spacer 21 missing in their spoligotype pattern revealed a series of SNPs and subsequent screening of a selection of these SNPs identified one in gene guaA that is specific to these strains. This group of strains from the Iberian Peninsula missing spoligotype spacer 21 represents a new clonal complex of M. bovis, defined by the SNP profile with a distinct spoligotype signature. We have named this clonal complex European 2 (Eu2) and found that it was present at low frequency in both France and Italy and absent from the British Isles. 01 juni 2012

The interferon-gamma (IFN-γ) assay is an effective tool for the diagnosis of tuberculosis (Tb) in goats. The objectives of this study were to evaluate factors that might affect assay performance: (1) the phenol concentration of the purified protein derivative (PPD, tuberculin) used; (2) dialysis of PPD; and (3) delaying antigenic stimulation of blood samples for 8, 16 and 24. h after collection. The assay was performed in duplicate with two cut-off points. Dialysis of PPD reduced test sensitivity, whereas the concentration of phenol did not significantly affect test outcome. Delaying antigenic stimulation of samples >8. h resulted in a reduction in test sensitivity, compromising the capacity of the assay to detect infected animals. Performing the assay in duplicate was unnecessary, which has implications for reducing assay costs. These findings will facilitate the effective application of the IFN-γ assay as an ancillary test in Tb eradication programmes in goats., This research was funded by The Spanish Ministry of Science and Technology and The Ministry of Environment and Rural and Marine Affairs (Project AGL2006-06206). J. Bezos is funded by The Spanish Ministry of Science and Innovation and the European Social Fund.

This study describes the attempt to trace the first Mycobacterium bovis outbreak in alpacas ( Lama pacos ) in Spain by spoligotyping and variable-number tandem-repeat (VNTR) analysis. Due to high genotype diversity, no matching source was identified, but local expansion of a clonal group was found and its significance for molecular tracing is discussed.

We have identified a clonal complex of Mycobacterium bovis isolated at high frequency from cattle in Uganda, Burundi, Tanzania, and Ethiopia. We have named this related group of M. bovis strains the African 2 (Af2) clonal complex of M. bovis. Af2 strains are defined by a specific chromosomal deletion (RDAf2) and can be identified by the absence of spacers 3 to 7 in their spoligotype patterns. Deletion analysis of M. bovis isolates from Algeria, Mali, Chad, Nigeria, Cameroon, South Africa, and Mozambique did not identify any strains of the Af2 clonal complex, suggesting that this clonal complex of M. bovis is localized in East Africa. The specific spoligotype pattern of the Af2 clonal complex was rarely identified among isolates from outside Africa, and the few isolates that were found and tested were intact at the RDAf2 locus. We conclude that the Af2 clonal complex is localized to cattle in East Africa. We found that strains of the Af2 clonal complex of M. bovis have, in general, four or more copies of the insertion sequence IS 6110 , in contrast to the majority of M. bovis strains isolated from cattle, which are thought to carry only one or a few copies.

This study aimed to maximize the sensitivity of bovine tuberculosis detection in living wild fallow deer (Dama dama) under field conditions. We evaluated the rapid test (RT; CervidTB STAT-PAK Assay, Chembio Diagnostic Systems, Inc., USA) in comparison with the comparative cervical skin test (CCT). A total of 134 fallow deer were captured between January and March 2008. At time 0, 0.1 ml of avian purified protein derivative (avian PPD; Cooper-Zeltia, Spain), 0.1 ml bovine PPD (Cooper-Zeltia, Spain), 0.1 ml negative control PBS and 0.1 ml of a positive control (the mitogen phytohaemagglutinin, PHA; containing 250 mg PHA, diluted in PBS) were injected intradermally at four shaved sites in the neck. The skin fold thickness at each injection site was measured at time 0 and 72 h (3 repeats each time). Animals with a skin test response of 2 mm or more at the bovine PPD injection site and animals with any visible reactivity in the RT were necropsied and tissues submitted for culture and for histopathology. A total of 36 fallow deer were considered reactors to bovine PPD or to the RT (apparent prevalence 27%). Regarding both bovine PPD reactivity and the skin fold increase at the PHA injection site, we found significant effects of age and sex by age interaction. Adult males had the largest responses. Mycobacterium bovis was isolated from lymphoid tissues of 21 fallow deer. Skin test sensitivity, as compared to M. bovis culture confirmed deer, was 80.1% (17/21). But, the CCT alone would have missed 4 of 21 culture confirmed animals. RT sensitivity, based on culture confirmed deer, was also 80.1% (17/21). Similarly, the RT alone would have missed another 4 of 21 culture confirmed deer. However, combining the CCT and the RT allowed for detecting all 21 culture positive fallow deer. We conclude that the combined application of the RT and the skin testing can maximize the sensitivity of bTB detection in living fallow deer, thus facilitating control programs for wildlife disease surveillance., This study was funded by Patrimonio Nacional (special thanks to M. Moreno and A. Jacoste) through contracts to IREC and TRAGSEGA (A. Rosón). This is a contribution to MCINN Plan Nacional Research grant AGL2008-03875 and FEDER, and to EU grant TB-STEP 212414. Studies on diseases shared between domestic animals and wildlife are also supported by grants and contracts from INIA and Ministerio de Medio Ambiente y Medio Rural y Marino (OAPN and SDGSPP), and Grupo Santander – Fundacion Marcelino Botin. MPM-H acknowledges a post-doctoral contract from Instituto de Salud Carlos III.

SUMMARYMycobacterium aviumsubsp.hominissuis(MAH) infection in swine may cause granulomatous lesions in lymph nodes that must undergo differential diagnosis with those caused byM. tuberculosiscomplex members. Moreover, MAH outbreaks can lead to severe economic losses due to condemnation of carcasses. A number of potential sources of infection for animals can usually be identified in contaminated farms. This report describes the application of several molecular characterization techniques in order to identify the possible environmental sources of MAH infection in an outbreak involving four breeding farms and six fattening farms. Molecular profiles obtained from MAH strains suggested a likely epidemiological link between clinical and environmental isolates cultured from sawdust and cooling systems from one breeding farm. These results highlight the potential risk posed by these environmental elements in the spread of infection and the need for implementation of adequate management practices in order to minimize this risk.

High-resolution melt (HRM) analysis can identify sequence polymorphisms by comparing the melting curves of amplicons generated by real-time PCR amplification. We describe the application of this technique to identify Mycobacterium avium subspecies paratuberculosis types I, II, and III. The HRM approach was based on type-specific nucleotide sequences in MAP1506, a member of the PPE (proline-proline-glutamic acid) gene family.

To investigate the percentage reactor animals to SCCIT in sheep and goat the present study was carried out by using bovine and avian PPDs at 7 Livestock Experiment Stations of Punjab and villages around two cities. The overall percentage of reactor animals to SCCIT at farms in sheep and goat was 0.9% and 2.4%, while it was 0.4% and 0.0% around two cities, respectively. Hundred percent of goat and 86% of sheep farms under study had positive reactor animals. The reactor animals to SCCIT around two cities were 0% in goat and 0.4% in sheep. Goats (P0.05) and sheep (P0.001) kept with large ruminants at farms had higher percentage of reactor animals. Age showed significant (P0.01) association with tuberculosis in sheep and goat. In goats of more than 6 years of age, the reactor animals to SCCIT were 5, 8 and 11 times higher than goats of 4.1-6, 3-4 and3 years of age. The reactor animals were significantly (P0.05) higher in goats of30kg (3.5%) and40kg (3.5%) body weights, while these were less in goats weighing between 30 and 40kg (1.5%). Sheep and goat breeds showed significant (P0.001; P0.05) difference in percentage of reactor animals to SCCIT. Beetal (3.9%) and Teddy breeds (2.9%) had 3.3 and 2.4 times higher percentages of reactor animals to SCCIT as compared to Pak-Angora (1.2%) breed. However, no animal of Nachhi and Dera Din Pinah breeds showed positive reaction to PPDs.

The Eurasian wild boar (Sus scrofa) is considered a reservoir for bovine tuberculosis (bTB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex in south-central Spain. The vaccination of wildlife with BCG offers an alternative to culling and to movement restriction for the control of bTB among wildlife reservoirs. In this study, we hypothesized that oral BCG immunization of wild boar would affect the expression of immunoregulatory genes and confer protection against M. bovis. Three groups were used to describe the infection, pathological findings and gene expression profiles in wild boar: BCG-vaccinated and M. bovis-challenged (vaccinated challenged group; N = 6), non-vaccinated and M. bovis-challenged (non-vaccinated challenged group; N = 4), and non-vaccinated and mock-infected (control group; N = 2) animals. M. bovis was isolated from 50% (3/6) and 75% (3/4) of vaccinated challenged and non-vaccinated challenged animals, respectively. All four wild boar from the non-vaccinated challenged group developed bTB-compatible lesions 114 days after challenge. In contrast, only 50% of vaccinated challenged wild boar developed lesions. The PBMC mRNA levels of IL4, RANTES, C3, IFN-gamma and methylmalonyl-CoA mutase (MUT) were analyzed at several days post-vaccination (dpi). When vaccinated challenged animals were compared to controls, all five genes were significantly upregulated at the time of M. bovis infection at 186 dpi but IFN-gamma levels were also upregulated at 11 and 46 dpi. The C3 and MUT mRNA levels were higher at 46 dpi, and 11 and 186 dpi, respectively, in vaccinated protected wild boar when compared to non-vaccinated challenged animals. At the end of the experiment (300 dpi), the mRNA levels of selected genes were lower in non-vaccinated challenged animals when compared to control wild boar. Exposing wild boar to a dose of 104 cfu of M. bovis by the oropharyngeal route is an adequate protocol to produce an infection model in this species. Our results suggested that oral BCG immunization of wild boar results in the upregulation of immunoregulatory genes that may be associated with protective response to M. bovis infection in this species. More studies on vaccine efficacy, delivery, and safety will be needed to confirm if oral vaccination with BCG could be used in bTB control programs for reducing M. bovis infection and clinical disease in wild boar., The study was funded by INIA-MICINN research grant FAU2006-00017. Studies on TB at IREC are also supported by Grupo Santander – Fundacion Marcelino Botin, and by TB-STEP EU FP7.

Insertion sequence IS 900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS 900 . This study, which analyzed the IS 900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.

Interferon-gamma (IFN-gamma) detection assay is being applied as an ancillary test to tuberculin tests in the diagnosis of bovine tuberculosis to detect the maximum number of infected animals. Among possible factors influencing the performance of tuberculosis-diagnostic tests, paratuberculosis, a widespread disease in Spain and other European countries, has been pointed out as a cause of false positive reactions. Still, its effect on the sensitivity of these tests in cattle has yet to be fully characterized. The impact of paratuberculosis in the apparent sensitivity of IFN-gamma assay was studied in a bullfighting cattle herd with a mixed tuberculosis-paratuberculosis infection, using culture of Mycobacterium bovis and Mycobacterium avium paratuberculosis as the gold standard to determine the infection status of every animal. A total of 218 animals were slaughtered and sampled for bacteriology after blood sampling. IFN-gamma assay showed a lower apparent sensitivity in animals with a mixed infection (50%) compared to all animals suffering tuberculosis (78.3%). This finding indicates that the presence of paratuberculosis in tuberculosis-infected herds could imply a serious impairment in the sensitivity of IFN-gamma detection test.

The role of wildlife in tuberculosis epidemiology is being widely studied since it can affect the effectiveness of eradication campaigns in cattle. The health problem is enhanced when it concerns also wildlife welfare and biodiversity conservation. This study was performed to understand the epidemiology of Mycobacterium bovis population affecting livestock and wild animals in the Doñana National Park using bacteriology and molecular characterisation techniques. Tuberculosis research was performed on 1209 cattle and wild animals (artiodactyla and carnivore) collected over 6 years in the Park. One hundred and sixty-three animals were found to be infected with M. bovis, comprising 7.96% of the cattle and 20.53% of the wild animals tested. Spoligotyping revealed nine patterns, being SB1232 and SB1230 the most prevalent (77.30% and 15.34% of infected animals, respectively). MIRU-VNTR analysis of a selected panel of 92 isolates showed eight different profiles, including several spoligotypes within the same MIRU-VNTR profile. The discriminatory capacity of both techniques in this panel was similar. The results obtained by combination of both techniques corroborate that wildlife species are infected with the M. bovis strains which are more prevalent in cattle and reveal their persistence. Genotype variation between isolates strongly suggests micro-evolutionary events in the M. bovis population in the same area. This study in the Doñana National Park exposes the risk of introduction of domestic animals into wildlife areas when there is not a warranty of disease freedom, appropriate diagnostic techniques and control measures.

This work is the first description of Mycobacterium peregrinum as an etiological agent for mycobacteriosis in farmed fishes. We report the mycobacterial infection in farmed European tench (Tinca tinca L.) which was confirmed by culture, molecular identification methods (PCRs aimed at 16S rRNA, rpobeta and hsp65 sequencing), and histopathology. Since M. peregrinum infection has been described in humans, their clinical significance in fishes should be considered of healthcare interest. With this case report, we also show that a multidisciplinary approach was needed to overcome difficulties associated to diagnosis of piscine mycobacteriosis.

Detection of infected animals is a key step in eradication programs of tuberculosis. Paratuberculosis infection has been demonstrated to compromise the specificity of the diagnostic tests. However, its effect on their sensitivity has not been clarified. In the present study, skin tests and the interferon-gamma (IFN-gamma) assay were evaluated in a goat flock (n=177) with a mixed tuberculosis-paratuberculosis infection in order to assess the possible effect of paratuberculosis on their sensitivity. Culture of mycobacteria was performed as the gold standard to determine the true infection status. All techniques showed lower sensitivities than previously described; the single intradermal tuberculin (SIT) test and the IFN-gamma assay detected 71% (62.4-78.6, 95% C.I.) of the infected animals; the single intradermal cervical comparative tuberculin (SICCT) test detected only 42.7% (34.1-51.7, 95% C.I.) of infected animals. The highest level of sensitivity was obtained when SIT test and IFN-gamma assay were combined in parallel (90.8%, 84.5-95.2, 95% C.I.). Sensitivities of the tests were also assessed by comparing animals suffering tuberculosis and animals with a mixed infection; tests were found to be more effective in the former group. Paratuberculosis seems to have a major effect in the sensitivity of the diagnostic tests under study, and therefore must be taken into account; in particular, the use of the SICCT test should be questioned when both tuberculosis and paratuberculosis are present.

Cellular immune response was evaluated in lymph nodes and lung with different granulomatous lesions from cattle naturally infected with Mycobacterium bovis. For this purpose, we assessed pro-inflammatory and anti-inflammatory cytokines by immunohistochemical assays. Immunoreaction was observed for all the cytokines analyzed. Fourteen animals displayed advanced stage IV granulomas, with intense immunoreactivity to IFN-γ and TGF-β in areas of caseous necrosis, macrophages and lymphocytes. Seven animals showed stage III granuloma, with high immunoreactivity to IFN-γ (average of 44.5% immunoreactive cells) and moderate to TNF-α and to the anti-inflammatory cytokines IL-10 and TGF-β, in relation to the proliferation of fibroblasts in granuloma periphery We found satellite stage I granulomas in 4 bovines and stage II granulomas in 2 bovines, which exhibited low immunostaining response (‐ 13%). Cytokine expression in stage III and IV granulomas was significant, with predominance of immunoreactivity to IFN-γ, thus suggesting a strong, longstanding local immune response mediated by macrophages and epithelioid cells. In addition, these two stages displayed lower reactivity to IL-10; which suggests a deficit of anti-inflammatory cytokines, suppressed immunity and persistence of the infection. High expression of TGF-β could indicate a chronic process with greater tissue damage and fibrosis. Numerous bacilli observed in necrotic areas in stage III and IV granulomas with low expression of IL-1β suggest failure in the immune response with bacterial multiplication. In this study, evidence of in situ presence of cytokines demonstrates these cytokines are involved in the development and evolution of bovine tuberculosis granulomas. Fil: Canal, Ana M.. Ministerio de la Producción; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; Argentina Fil: Pezzone, Natalia. Ministerio de la Producción; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; Argentina Fil: Cataldi, Ángel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Zumárraga, Martín José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Larzabal, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Garbaccio, Sergio. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Fernandez, Analía. Dirección General de Producción Animal; Argentina Fil: Dominguez, Lucas. Universidad Complutense de Madrid; España Fil: Aranaz, Alicia. Universidad Complutense de Madrid; España Fil: Rodriguez Bertos, Antonio. Universidad Complutense de Madrid; España

The analysis of the gyrA and gyrB genes of a panel of Mycobacterium avium subsp. paratuberculosis isolates from types I, II, and III detected type-specific single nucleotide polymorphisms. Based on these results, we developed a PCR and restriction enzyme analysis to discriminate type I and III isolates. The application of this technique would be the unique strategy to characterize these strains when there is not enough bacterial growth to perform pulsed-field gel electrophoresis and IS 900 restriction fragment length polymorphism.

Summary Spoligotyping is a typing tool used worldwide for epidemiological studies on Mycobacterium tuberculosis complex organisms; however it has received little attention regarding improvement of its discriminatory power (DP). In this study, we have evaluated a spoligotyping membrane prepared with 25 novel spacer sequences selected from a previous study [van der Zanden AG, Kremer K, Schouls LM. Improvement of differentiation and interpretability of spoligotyping for Mycobacterium tuberculosis complex isolates by introduction of new spacer oligonucleotides. J Clin Microbiol 2002; 40 :4628–39] on 308 M. bovis and 88 M. caprae Spanish isolates in comparison with the traditional spoligotyping membrane. The results obtained by combining the two membranes together revealed an improvement of 45 patterns instead of 31. The spacers used in the second membrane were able to distinguish 8 out of the 16 M. bovis types that had more than one isolate. Seven of these types were differentiated into two subtypes with the second-generation membrane, while spb-7, the most prevalent in Spain, was further differentiated into eight subtypes. This second-generation membrane also differentiates M. bovis from M. caprae . A set of 39 spacers (1, 2, 4–8, 10–15, 17–21, 23, 26–32, 37, 44–49, 51–54, 56 and 57) contain all the DP for both M. bovis and M. caprae isolates; and a set of 35 spacers (1, 2, 4–8, 10–15, 17–21, 26–32, 37, 44–48, 52–54 and 57) had all the DP for the M. bovis isolates. Our results show that the research on new spacers and the design of a new membrane may be useful for epidemiological studies of M. bovis and M. caprae isolates.

PCR-based characterization techniques have been adopted in most laboratories for Mycobacterium bovis typing. We report a molecular characterization of human multidrug-resistant M. bovis isolates and three bovine isolates that share the spoligotyping profile. The analysis of the direct repeat region showed that both groups differed in the presence of spacers not included in the current membrane. They were also distinguished by two out of the nine mycobacterial interspersed repetitive unit variable-number tandem repeat loci tested, indicating that the human infection was not acquired from the cattle from which isolates were obtained. These results highlight that a combination of techniques is required for appropriate discrimination, even for those spoligotypes that have a low frequency.

Mycobacterium caprae, a recently defined member of theMycobacterium tuberculosiscomplex, causes tuberculosis among animals and, to a limited extent, in humans in several European countries. To characterizeM. capraein comparison with otherMycobacterium tuberculosiscomplex members and to evaluate genotyping methods for this species, we analyzed 232M. capraeisolates by mycobacterial interspersed repetitive unit (MIRU) genotyping and by spoligotyping. The isolates originated from 128 distinct epidemiological settings in 10 countries, spanning a period of 25 years. We found 78 different MIRU patterns (53 unique types and 25 clusters with group sizes from 2 to 9) but only 17 spoligotypes, giving Hunter-Gaston discriminatory indices of 0.941 (MIRU typing) and 0.665 (spoligotyping). For a subset of 103M. capraeisolates derived from outbreaks or endemic foci, MIRU genotyping and IS6110restriction fragment length polymorphism were compared and shown to provide similar results. MIRU loci 4, 26, and 31 were most discriminant inM. caprae, followed by loci 10 and 16, a combination which is different than those reported to discriminateM. bovisbest.M. capraeMIRU patterns together with published data were used for phylogenetic inference analysis employing the neighbor-joining method.M. capraeisolates were grouped together, closely related to the branches of classicalM. bovis,M. pinnipedii,M. microti, and ancestralM. tuberculosis, but apart from modernM. tuberculosis.The analysis did not reflect geographic patterns indicative of origin or spread ofM. caprae.Altogether, our data confirmM. capraeas a distinct phylogenetic lineage within theMycobacterium tuberculosiscomplex.

Mycobacterium bovis infection in wildlife and feral species is a potential source of infection for livestock and a threat to protected and endangered species. The aim of this study was to identify Spanish wild animal species infected with M. bovis through bacteriological culture and spacer oligonucleotide typing (spoligotyping) of isolates for epidemiological purposes. This study included samples from red deer ( Cervus elaphus ), fallow deer ( Dama dama ), wild boar ( Sus scrofa ), Iberian lynx ( Lynx pardina ), hare ( Lepus europaeus ), and cattle ( Bos taurus ). They were collected in several geographical areas that were selected for their unique ecological value and/or known relationships between wildlife and livestock. In the areas included in this survey, M. bovis strains with the same spoligotyping pattern were found infecting several wild species and livestock, which indicates an epidemiological link. A locally predominant spoligotype was found in these areas. Better understanding of the transmission and distribution of disease in these populations will permit more precise targeting of control measures.

To better understand the evolution of the Mycobacterium tuberculosis complex, subspecies were tested for large sequence polymorphisms. Samples with greater numbers of deletions, without exception, were missing all the same regions that were deleted from samples with lesser numbers of deletions. Principal genetic groups based on single-nucleotide polymorphisms were restricted to one of the deletion-based groups, and isolates that shared genotypes based on molecular epidemiological markers were assigned almost exclusively to the same deletion type. The data provide compelling evidence that human tuberculosis did not originate from the present-day bovine form. Genomic deletions present themselves as an attractive modality to study the evolution of the M. tuberculosis complex.

To the Editor: Mycobacterium tuberculosis is the main causative agent of tuberculosis in humans. However, little attention has been paid to its transmission from humans to animals. We report M. tuberculosis infections in 3 cattle farms in Spain. The epidemiologic investigation traced humans as the source of infection, with 1 of the strains showing multidrug resistance. Recent studies have reported isolation of M. tuberculosis in cattle with prevalences of 4.7%–30.8% in African and Asian countries (1–3). In cattle, this infection occurs in countries with the highest incidence of human tuberculosis in the world. In Europe, only 14 cases of M. tuberculosis infection have been described in 3 eastern countries since implementation of eradication programs (4,5). The only reported cases of M. tuberculosis in cattle in western Europe were described in Great Britain and date back to the 1950s (6). During 2007–2009, three cases of tuberculosis caused by M. tuberculosis were detected in 3 unrelated cattle farms, 2 of them free of tuberculosis (farms 1 and 2). As part of the surveillance system of bovine tuberculosis, a pool of tissue samples from each cow (respiratory lymph nodes and lung) were homogenized with sterile distilled water, and culture was carried out by the BACTEC mycobacteria growth indicator tube 960 system (Beckton Dickinson, Madrid, Spain). Members of the M. tuberculosis complex were identified and genotyped by direct variable repeat spacer olignucleotide typing and mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) typing (7). The 3 M. tuberculosis–infected animals were

The field of molecular diagnostics in veterinary practice is rapidly evolving. An array of molecular techniques of different complexity is available to facilitate the fast and specific diagnosis of animal diseases. The choice for the adequate technique is dependent on the mission and attributions of the laboratory and requires both a knowledge of the molecular biology basis and of its limitations. The ability to quickly detect pathogens and their characteristics would allow for precise decision-making and target measures such as prophylaxis, appropriate therapy, and biosafety plans to control disease outbreaks. In practice, taking benefit of the huge amount of data that can be obtained using molecular techniques highlights the need of collaboration between veterinarians in the laboratory and practitioners.

The field of molecular diagnostics in veterinary practice is rapidly evolving. An array of molecular techniques of different complexity is available to facilitate the fast and specific diagnosis of animal diseases. The choice for the adequate technique is dependent on the mission and attributions of the laboratory and requires both a knowledge of the molecular biology basis and of its limitations. The ability to quickly detect pathogens and their characteristics would allow for precise decision-making and target measures such as prophylaxis, appropriate therapy, and biosafety plans to control disease outbreaks. In practice, taking benefit of the huge amount of data that can be obtained using molecular techniques highlights the need of collaboration between veterinarians in the laboratory and practitioners.

As from January to August 2013, epidemiological weeks 1-35 (EW), Influenza incidence, case characteristics, types and subtypes of circulating influenza virus in the Nacional Profesor Alejandro Posadas Hospital were studied, and were compared to incidences during 2009-2012. From late May to the end of August 2013 (EW18-35), an increase was observed in the proportion of patients' visits for respiratory disease, influenza-like illness and hospitalizations due to pneumonia; of 207 cases diagnosed with influenza A virus, 153 were infected by H1N1pdm09, 46 by H3, and eight without subtype. The highest proportion of cases was found in children under five years of age, followed by the group 60-64. The chances of having the illness were three times greater among the group 40-64 years old compared to 15-39 or those older than 64. Mortality, which increased with age, was 7.2%, and the odds of death were six times higher among those older than 64. Vaccination rate among the cases was 11.6%. None of the fatal cases had received the vaccine. After the 2009 pandemic, the proportions of annual patients' visits decreased until 2012; in 2013, an increase of 52.0% during the winter period compared to 2012. The viral circulation started earlier in 2013 compared to previous years. FLU-A(H1N1pdm) was the predominant circulating virus in 2009 and 2013, FLU-A(H3) in 2011, FLU-A(H3) and FLU-B in both 2010 and 2012.

*VLA Weybridge, Department of Bacterial Diseases, TB Research Group, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK. Departamento de Patologia Animal I, Facultad de Veterinaria, Universidad Complutense de Madrid, Av. Puerta de Hierro s/n, 28040 Madrid, Spain. Department of Agriculture and Rural Development, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, Northern Ireland

Isolates from the Mycobacterium tuberculosis complex cultured from caprine pathological tissue samples were biochemically and genetically characterized. The isolates were negative for nitrate reduction and niacin accumulation, they weakly hydrolysed Tween 80, were sensitive to pyrazinamide (50 micrograms ml-1) and were resistant to 1 and 2 micrograms tiophene-2-carboxylic acid hydrazide ml-1 but not to 5 or 10 micrograms tiophene-2-carboxylic acid hydrazide ml-1. Sequencing of the pncA gene revealed a polymorphism characteristic of M. tuberculosis, whereas oxyR, katG and gyrA sequences were characteristic of Mycobacterium bovis. The fingerprinting patterns obtained with IS6110, direct repeats and polymorphic G+C-rich sequence-associated RFLP and direct variable repeat-spacer oligonucelotide typing (spoligotyping) segregated these isolates from the other members of the complex. The results of this testing, together with the repeated association of this micro-organism with goats, suggest that a new member of this taxonomic complex not matching any of the classical species had been identified. This unusual mycobacterium may play a role in the epidemiology of animal and human tuberculosis in Spain. The name Mycobacterium tuberculosis subsp. caprae subsp. nov. is proposed for these isolates. The type strain of Mycobacterium tuberculosis subsp. caprae subsp. nov. is gM-1T (= CIP 105776T).

The combination of conventional investigation and DNA fingerprinting is yielding important insights into the epidemiology of Mycobacterium bovis infections. Various genetic markers used in restriction fragment length polymorphism (RFLP) have recently been exploited for fingerprinting of M. bovis isolates. The newly developed spacer oligonucleotide typing aimed to investigate the polymorphism of M. tuberculosis in the DR locus, has also been applied to the molecular typing of M. bovis isolates. This work compared the performance of the insertion sequence (IS) IS6110, IS1081 and the genetic elements polymorphic G+C-rich repeat (PGRS) and direct repeat (DR) used in RFLP analysis with spoligotyping using a group of 128 Spanish M. bovis isolates. In this study, the most sensitive technique for identifying polymorphism in M. bovis was PGRS–RFLP, closely followed by IS6110–RFLP. We propose several schemes for fingerprinting of these isolates, however, the clear geographical variations found by different authors makes the study of each local situation indispensable. An international consensus in the methods used would be desirable for efficient interlaboratory comparison of strains.

Objective To evaluate the usefulness of the gamma-interferon assay in the diagnosis of caprine tuberculosis in comparison with a single intradermal tuberculin test, and to obtain a group of animals free from this infection in a herd with a high prevalence. Design An immunological study involving four serial comparative gamma-interferon and single intradermal tuberculin tests. Animals A herd of 87 goats of Guadarrama breed. Procedure Serial testing and segregation of animals. Results We found that the number of infections detected by the gamma-interferon test was considerably greater than the number detected by the single intradermal tuberculin test. A group of 10 animals was negative to both tests in two consecutive rounds and three kids were negative in the last round of testing. Conclusions Gamma-interferon assay is appropriate for diagnosis and eradication of tuberculosis in goats. This test is able to detect early Mycobacterium bovis infection. Avian reactors with simultaneous increased reaction to bovine PPD in the gamma-interferon assay (designated as avianB reactors) should be considered test positive for M bovis. By serial testing with the gamma-interferon and the single intradermal tuberculin tests, and a policy of segregation of kids at birth, it is possible to achieve a group of animals test negative for tuberculosis from a herd of goats with high immunoreactivity to this infection.

DNA fingerprinting techniques were used to type 273 isolates of Mycobacterium bovis from Australia, Canada, the Republic of Ireland, and Iran. The results of restriction fragment length polymorphism (RFLP) analysis with DNA probes from IS 6110 , the direct repeat (DR), and the polymorphic GC-rich sequence (PGRS) were compared with those of a new PCR-based method called spacer oligonucleotide typing (spoligotyping) developed for the rapid typing of Mycobacterium tuberculosis (J. Kamerbeek et al., J. Clin. Microbiol. 35:907–914, 1997). Eighty-five percent of the isolates harbored a single copy of IS 6110 , and 81.5% of these carried IS 6110 on the characteristic 1.9-kb restriction fragment. RFLP analysis with IS 6110 identified 23 different types, RFLP analysis with the DR probe identified 35 types, RFLP analysis with the PGRS probe identified 77 types, and the spoligotyping method identified 35 types. By combining all results, 99 different strains could be identified. Isolate clusters were frequently associated within herds or were found between herds when epidemiological evidence confirmed animal movements. RFLP analysis with IS 6110 was sufficiently sensitive for the typing of isolates with more than three copies of IS 6110 , but RFLP analysis with the PGRS probe was the most sensitive typing technique for strains with only a single copy of IS 6110 . Spoligotyping may have advantages for the rapid typing of M. bovis , but it needs to be made more sensitive.

Wildlife vaccination is increasingly being considered as an option for tuberculosis control. We combined data from laboratory trials and an ongoing field trial to assess the risk of an oral Mycobacterium bovis BCG vaccine and a prototype heat-inactivated Mycobacterium bovis preparation for Eurasian wild boar (Sus scrofa). We studied adverse reactions, BCG survival, BCG excretion, and bait uptake by nontarget species. No adverse reactions were observed after administration of BCG (n = 27) or inactivated M. bovis (n = 21). BCG was not found at necropsy (175 to 300 days postvaccination [n = 27]). No BCG excretion was detected in fecal samples (n = 162) or in urine or nasal, oral, or fecal swab samples at 258 days postvaccination (n = 29). In the field, we found no evidence of loss of BCG viability in baits collected after 36 h (temperature range, 11°C to 41°C). Camera trapping showed that wild boar (39%) and birds (56%) were the most frequent visitors to bait stations (selective feeders). Wild boar activity patterns were nocturnal, while diurnal activities were recorded for all bird species. We found large proportions of chewed capsules (29%) (likely ingestion of the vaccine) and lost baits (39%) (presumably consumed), and the proportion of chewed capsules showed a positive correlation with the presence of wild boar. Both results suggest proper bait consumption (68%). These results indicate that BCG vaccination in wild boar is safe and that, while bait consumption by other species is possible, this can be minimized by using selective cages and strict timing of bait deployment., This is a contribution to Plan Nacional I+D+i AGL2011-30041 from the Ministerio de Economía y Competitividad (MINECO), Spain, and FEDER and to EU FP7 grant WildTBvac. B.B.-B. and I.D.-D. were supported by MINECO grants BES-2009-017401 and BES-2012-052490, respectively. J.A.B. was supported by MICINN grant FPU12/00980.

[Background]: Field vaccination trials with Mycobacterium bovis BCG, an attenuated mutant of M. bovis, are ongoing in Spain, where the Eurasian wild boar (Sus scrofa) is regarded as the main driver of animal tuberculosis (TB). The oral baiting strategy consists in deploying vaccine baits twice each summer, in order to gain access to a high proportion of wild boar piglets. The aim of this study was to assess the response of wild boar to re-vaccination with BCG and to subsequent challenge with an M. bovis field strain. [Results]: BCG re-vaccinated wild boar showed reductions of 75.8% in lesion score and 66.9% in culture score, as compared to unvaccinated controls. Only one of nine vaccinated wild boar had a culture-confirmed lung infection, as compared to seven of eight controls. Serum antibody levels were highly variable and did not differ significantly between BCG re-vaccinated wild boar and controls. Gamma IFN levels differed significantly between BCG re-vaccinated wild boar and controls. The mRNA levels for IL-1b, C3 and MUT were significantly higher in vaccinated wild boar when compared to controls after vaccination and decreased after mycobacterial challenge. [Conclusions]: Oral re-vaccination of wild boar with BCG yields a strong protective response against challenge with a field strain. Moreover, re-vaccination of wild boar with BCG is not counterproductive. These findings are relevant given that re-vaccination is likely to happen under real (field) conditions., Carlos Martín, Universidad de Zaragoza, kindly provided the vaccine BCG strain. Many colleagues at SaBio IREC, NEIKER and VISAVET granted valuable technical help. This is a contribution to EU FP7 grant 613779 WildTBVac and to MINECO Plan Nacional grant AGL2011-30041 and FEDER.

A total of 129 Mycobacterium bovis strains from 5 different Spanish locations were fingerprinted using the IS6110 repetitive element. We demonstrated the presence of multiple copies (from 2 to 13) of IS6110 in a large proportion (47.4%) of the M. bovis strains isolated from cattle and we showed that these strains can be successfully differentiated by means of the RFLP with IS6110. All of the M. bovis strains isolated from goats had multiple copies of IS6110 and 4 bands of 2, 1.7, 1.4 and 1.3 kb were common in all the caprine RFLP patterns. The caprine strains formed a clearly separate cluster from the bovine strains.

Current methods for detection and identification of mycobacteria from clinical specimens, with particular interest in M avium and other nontuberculous mycobacteria, are reviewed. Traditional microbiological procedures, including microscopy, comparison of the different pretreatment methods for decontamination and culture media, and DNA probes and high-performance liquid chromatography techniques are evaluated. New methods include the polymerase chain reaction, a new tool available for quick identification of mycobacteria. The protocol used for detection of the etiologic agent of six cases of bird mycobacteriosis is briefly described. Authors recommend this technique as a rapid and reliable diagnosic method.

Members of the Mycobacterium tuberculosis complex (MTBC) cause a serious disease with similar pathology, tuberculosis; in this review, bovine tuberculosis will be considered as disease caused by any member of the MTBC in bovids. Bovine tuberculosis is responsible for significant economic loss due to costly eradication programs and trade limitations and poses a threat to both endangered and protected species as well as to public health. We here give an overview on all members of the MTBC, focusing on their isolation from different animal hosts. We also review the recent advances made in elucidating the evolutionary and phylogenetic relationships of members of the MTBC. Because the nomenclature of the MTBC is controversial, its members have been considered species, subspecies or ecotypes, this review discusses the possible implications for diagnostics and the legal consequences of naming of new species.

The intradermal tuberculin tests and the interferon-gamma (IFN-γ) assay are the principal tests used worldwide for the ante-mortem diagnosis of bovine tuberculosis. The conventional reagent currently in use in these tests is purified protein derivative (PPD) tuberculin obtained from Mycobacterium bovis culture. The components of PPD are poorly characterized and difficult to standardize. To overcome this issue, antigens specific to the Mycobacterium tuberculosis complex are being studied. Here we have assessed the biological potency of ESAT-6, CFP-10 and Rv-3615c presented as peptide or recombinant protein cocktails in comparison with the standard bovine PPD used routinely in Spanish eradication campaigns. The study was performed in cattle (n= 23) from a herd with natural M. bovis infection. Animals were simultaneously injected with PPD and the peptide and protein cocktails. The percentages of cattle reacting positively to single intradermal test were 60.9% (bovine PPD), 47.8% (peptide cocktail) and 60.9% (protein cocktail), with no significant difference between the actual skin fold thickness increases (p> 0.05). The IFN-γ assay detected 60.9% of animals when stimulation was performed with bovine PPD, but decreased to 52.2% when stimulation was performed with the peptide cocktail and to 47.8% when stimulation was performed with the protein cocktail. However, no significant differences were found between IFN-γ responder frequencies (p> 0.05). These results show a potential use of these defined reagents for in vivo tuberculosis diagnosis., This study was funded by the EU Project FP7-KBBE-2007-1 “Strategies for the eradication of bovine tuberculosis (TB-STEP)”. C. Casal is recipient of a research contract assigned by Comunidad de Madrid (FINNOVA II Programme).