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BackgroundAnimal tuberculosis (TB) is distributed worldwide and has a wide range of wild and domestic reservoirs. Few studies concerning TB in camelids have been published in the last decade, particularly as regards Old World Camelids (OWC), but the increase in reports of TB outbreaks in these species in recent years suggests a high susceptibility to the infection.Case presentationWe studied a dromedary camel (Camelus dromedarius) herd (n = 24) in which aMycobacterium capraeinfection was detected. The TB infection was confirmed in one animal at necropsy through the detection of TB lesions, mainly in the abdominal organs, and the subsequent isolation ofM. caprae(SB0157 spoligotype). The whole herd was additionally tested using cellular and humoral based diagnostic techniques. The intradermal tuberculin test results were compared with those obtained using P22 ELISA for the detection of specific antibodies against theM. tuberculosiscomplex. The TB infected animal was a positive reactor to both the intradermal tuberculin tests and P22 ELISA, while the others were negative to all the diagnostic tests.ConclusionThe present study foundM. capraeinfection in OWC. This is the first report ofM. capraeinfection in an OWC not living in a zoo. Since the animal was born in the herd and fed with goat’s milk, this practice was suspected to be the potential source of TB infection, which was not confirmed in the other animals present in the herd. Moreover, our results highlight that the intradermal tuberculin test and the P22 ELISA could be valuable tools for the diagnosis of TB in OWC.

Recent studies show that sheep could be considered to be a maintenance host for the causative agents of animal tuberculosis (TB). The performance of diagnostic tests is not well established, and new tests need to be developed for this species. In addition, information about TB prevalence in sheep is scarce. Our objectives were to evaluate a new P22 ELISA for detection of specific antibodies against Mycobacterium tuberculosis Complex (MTC), and to assess the seropositivity in 3998 sheep from herds sampled in TB hotspot areas of northern Atlantic Spain with a low TB prevalence in cattle. Results based on 80 sheep of known infection status suggest excellent sensitivity and specificity (100% and 98%, respectively) even in a M. avium susbsp. paratuberculosis infected flock. The observed TB seroprevalence was 17.96% (698/3998; CI95% 16.31–18.67). Our results indicate that the P22 ELISA may constitute a good option for TB screening at the herd level in sheep, and that sheep are an important host and control programs should be implemented at least in hotspots or when cohabiting with other TB-infected species, i.e. cattle and goats., This study was funded by a grant from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), reference number RTA2014-00002-C02-01 (FEDER co-funded), a grant from Agencia Estatal de Investigación (AEI), reference number RTI2018-096010-B-C21 (FEDER co-funded) and the Principado de Asturias, PCTI 2018–2020 (GRUPIN: IDI2018-000237 and FEDER).

European badgers (Meles meles ) have been identified as wildlife reservoirs for Mycobacterium bovis in the UK and Ireland, and may also have a role in the epidemiology of animal tuberculosis in other European regions. Thus, detection of M. bovis‐infected badgers may be required for the purposes of surveillance and monitoring of disease levels in infected populations. Current serological assays to detect M. bovis infection in live badgers, while rapid and inexpensive, show limited diagnostic sensitivity. Here we describe and evaluate new ELISA platforms for the recognition of the P22 multiprotein complex derived from the purified protein derivative (PPD ) of M. bovis . The recognition of IgG against P22 multiprotein complex derived from PPD ‐B was tested by ELISA in the serum of badgers from the UK , Ireland and Spain. TB infection in the badgers was indicated by the presence of M. bovis in tissues by culture and histology at post‐mortem examination and TB ‐free status was established by repeated negativity in the interferon γ release assay (IGRA ). In experimentally infected badgers, humoral antibody responses against P22 developed within 45 days post‐infection. The ELISA tests showed estimated sensitivity levels of 74–82% in experimentally and naturally infected badgers with specificities ranging from 75% to 100% depending on the badger population tested. The P22 multi‐antigen based ELISA s provide a sensitive and specific test platform for improved tuberculosis surveillance in badgers., This work was supported by the Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria of Spain (INIA; RTA2015-00043-C02-02) and the TAVS-CM Programme of the Comunidad de Madrid (S2013/ABI-2747), cofinanced by the FEDER fund ‘A way to build Europe’. This work was partially supported by a FEDER co-funded grant from INIA (RTA2014-00002-C02-01). Jose Antonio Infantes Lorenzo was supported by an FPU contract-fellowship (Formacion de Profesorado Universitario) from the Ministerio de Educacion, Cultura y Deporte of the Spanish Government (FPU2013/6000).

The diagnosis of tuberculosis (TB) in goats is based mainly on the single and comparative intradermal tuberculin (SIT and CIT) tests and, exceptionally, on the interferon-gamma (IFN-γ) assay, however they are not perfect in terms of sensitivity and specificity. Nevertheless, various serological assays that provide a potential cost-effective approach for the control of TB are also available or under development, and a variety of results have been reported regarding the ability of these tests to detect infected animals, particularly in the early stages of infection. In the present study, SIT/CIT and IFN-γ tests and three different serological assays were evaluated during two consecutive herd testing events in a recently infected caprine herd (n = 447) with a high prevalence of infection in order to evaluate their performance and provide field data with which to improve the TB control programs in this species. The proportion of infected animals that tested positive among all the infected goats (T+/I+ value) in the last herd testing event ranged from 26.2% (IC95%; 19.3-34.5) to 85.7% (IC95%; 78.5-90.7) using cell-based diagnostic tests. The SIT/SCIT tests detected more infected goats than the IFN-γ test, regardless of the interpretation criteria. The T+/I+ value of serology was 83.2 (IC95%; 75.2-89), although it increased significantly (P 0.05) when using samples collected 15 days after the intradermal test (100%, IC95%; 97-100). In general, a parallel interpretation of intradermal tests with serology maximized the detection of infected goats. These results demonstrate that serological tests are valuable diagnostic tools to maximize the detection of TB infected goats, even in recent outbreaks, accelerating the eradication process.

13 páginas, 4 figuras, 1 tabla., This study was funded by a grant from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), reference number RTA2014-00002-C02 (FEDER co-funded), the Principado de Asturias, PCTI 2018–2020 (GRUPIN: IDI2018-000237 and FEDER), and Departamento de Desarrollo Económico e Infraestructuras del Gobierno Vasco. This study was also funded by Ministerio de Ciencia, Innovación y Universidades (MCIU), the Agencia Estatal de Investigación (AEI) reference project RTI2018-096010-B-C21 and Fondo Europeo de Desarrollo Regional (FEDER). DEFRA also funded APHA staff for their participation in the study and for the purchase of BCG from Statens Serum Institute (Copenhagen, Denmark).

Attaining and maintaining the Official Tuberculosis Free status continues to be a challenge when several domestic and wild hosts contribute to the maintenance of the Mycobacterium tuberculosis complex (MTC). Local tuberculosis hotspots are sometimes identified in cattle in low-prevalence regions. We have, therefore, studied one such hotspot in depth in order to produce an epidemiological diagnosis. Host population size and MTC prevalence were estimated in selected wildlife and in livestock, while on-cattle environmental DNA detection was additionally used as a proxy for risk of exposure at the farm (herd) level. Positive skin test reactors were found on16 of the 24 cattle farms studied in the period 2012–2016. Although all goats tested negative to the skin test during this period, MTC was confirmed in four sheep at slaughter, thus indicating an unknown prevalence of infection in this host species. With regard to wildlife, the prevalence of MTC infection based on culture was 8.8% in the case of wild boar (Sus scrofa), and the only road-killed badger (Meles meles) submitted for culture tested positive. Two criteria were employed to divide the cattle farms into higher or lower risk: tuberculosis testing results and environmental DNA detection. Environmental MTC DNA detection yielded significant differences regarding “use of regional pastures” and “proximity to woodland”. This study suggests that on-animal environmental DNA sampling may help when assessing contact risk as regards MTC in livestock at the herd level. This tool opens up new avenues of epidemiological research in complex multi-host settings., This work was supported by the Programa de Tecnologias Avanzadas en Vigilancia Sanitaria (TAVS) from the Comunidad de Madrid (ref. S2013/ABI-2747). JMG holds a FPI predoctoral scholarship (BES-2015- 072206), funded by MINECO. This is a contribution to MINECO grant CGL2017-89866 WildDriver and EU-FEDER. This is also a contribution to Valle de Alcudia pilot project SG-2019-02 from PDR-CLM, and to GOSTU project EU-FEADER (AEIAGRI-PNDR-MAPA, ref. 20190020007521).

We provide a temporal overview (from 2012 to 2018) of the outcomes of tuberculosis (TB) in the cattle and badger populations in a hot-spot in Asturias (Atlantic Spain). We also study the badger&rsquo, s spatial ecology from an epidemiological perspective in order to describe hazardous behavior in relation to TB transmission between cattle and badgers. Culture and single intradermal tuberculin test (SITT) were available for cattle as part of the National Program for the Eradication of TB. A field survey was also carried out in order to determine the paddocks and buildings used by each farm, and the information obtained was stored by using geographic information systems. Moreover, eighty-three badgers were submitted for necropsy and subsequent bacteriological studies. Ten badgers were also tracked, using global positioning system (GPS) collars. The prevalence of TB in cattle herds in the hot-spot increased from 2.2% in 2012 to 20% in 2016, it then declined to 0.0% in 2018. In contrast, the TB prevalence in badgers increased notably (from 5.55% in 2012&ndash, 2015 to 10.64% in 2016&ndash, 2018). Both cattle and badgers shared the same strain of Mycobacterium bovis. The collared badgers preferred paddocks used by TB-positive herds in spring and summer (when they were more active). The males occupied larger home ranges than the females (Khr95: males 149.78 &plusmn, 25.84 ha and females 73.37 &plusmn, 22.91 ha, Kcr50: males 29.83 &plusmn, 5.69 ha and females 13.59 &plusmn, 5.00 ha), and the home ranges were smaller in autumn and winter than in summer. The averages of the index of daily and maximum distances traveled by badgers were 1.88 &plusmn, (SD) 1.20 km and 1.99 &plusmn, 0.71 km, respectively. One of them presented a dispersive behavior with a maximum range of 18.3 km. The most preferred habitat was apple orchards in all seasons, with the exception of winter, in which they preferred pastures. Land uses and landscape structure, which have been linked with certain livestock-management practices, provide a scenario of great potential for badger&ndash, cattle interactions, thus enhancing the importance of the badgers&rsquo, ecology, which could potentially transmit TB back to cattle in the future.

[Background]: Serum antibody detection has potential as a complementary diagnostic tool in animal tuberculosis (TB) control, particularly in multi-host systems. The objective of the present study was to assess the specificity (Sp) of an enzyme-linked immunosorbent assay (ELISA) based on the new multiprotein complex P22 for the detection of specific antibodies against the Mycobacterium tuberculosis complex (MTC) in the four most relevant domestic animals acting as MTC hosts: cattle, goat, sheep and pig. We used sera from an officially TB-free (OTF) country, Norway, and from a non-OTF one, Spain. The samples included sera from goats that had been vaccinated against M. avium subsp. paratuberculosis (MAP) and sheep from a herd in which Corynebacterium pseudotuberculosis had been isolated., [Results]: In cattle, the Sp ranged from 92.5 (IC95% 90.7–94) to 99.4% (IC95% 98.3–99.8) depending on the cut-off used and the origin of the samples (Spain or Norway). Sp in cattle (cut-off point 100) was significantly higher (P, [Conclusions]: Due to its excellent Sp in pigs and acceptable Sp in cattle and sheep, this ELISA may constitute a suitable option for TB screening at herd level, particularly in OTF-countries., Jose Antonio Infantes-Lorenzo was supported by an FPU contract-fellowship (Formación de Profesorado Universitario) from the Ministerio de Educación, Cultura y Deporte of the Spanish Government (FPU2013/6000). Alvaro Roy is the recipient of an Industrial Doctorate contract (DI-15-08110) funded by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and the European Social Fund. Javier Bezos is recipient of a Juan de la Cierva Incorporación research contract (IJCI-2015-24805) funded by the MINECO. This work was supported by the Spanish Ministry of Agriculture, Fisheries and Food and the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria of Spain (RTA2015–00043-C02–02). This is a contribution to Plan Nacional de I + D + i grant CGL2017–89866 WILD-DRIVER.

Effective vaccines against tuberculosis (TB) are needed in order to prevent TB transmission in human and animal populations. Evaluation of TB vaccines may be facilitated by using reliable animal models that mimic host pathophysiology and natural transmission of the disease as closely as possible. In this study, we evaluated the immunogenicity and efficacy of two attenuated vaccines, BCG and MTBVAC, after each was given to 17 goats (2 months old) and then exposed for 9 months to goats infected with M. caprae. In general, MTBVAC-vaccinated goats showed higher interferon-gamma release than BCG vaccinated goats in response to bovine protein purified derivative and ESAT-6/CFP-10 antigens and the response was significantly higher than that observed in the control group until challenge. All animals showed lesions consistent with TB at the end of the study. Goats that received either vaccine showed significantly lower scores for pulmonary lymph nodes and total lesions than unvaccinated controls. Both MTBVAC and BCG vaccines proved to be immunogenic and effective in reducing severity of TB pathology caused by M. caprae. Our model system of natural TB transmission may be useful for evaluating and optimizing vaccines., This work was funded by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO, Grant Number: IPT-2012-0327-090000). AR is the recipient of an Industrial Doctorate contract (DI15-08110) funded by the MINECO and the European Social Fund. JB is the recipient of a Juan de la Cierva Incorporation Research contract (IJCI-2015-24805) funded by MINECO.

Preliminary results were presented as a Poster at XXII Simposio, Avedila, Valladolid, 16–17 November 2017., Red deer (Cervus elaphus) farming is a growing economic activity worldwide. However, the capacity of this species to act as reservoir of animal tuberculosis (TB) poses a threat to other wildlife and to livestock. Diagnostic assay accuracy in this species is therefore highly relevant for prevention and control measures. Our aim was to evaluate the diagnostic performance of the protein complex P22, obtained from Mycobacterium bovis derived purified protein derivative (bPPD), as a candidate antigen for the detection of antibodies against Mycobacterium tuberculosis complex (MTC). We assessed the performance of this new antigen in indirect enzyme-linked immunosorbent assays (ELISA) in TB-positive and TB-negative red deer, in comparison with a bPPD-based ELISA. The P22 ELISA achieved a higher specificity (Sp) and similar sensitivity (Se) in comparison with the bPPD ELISA at all the cut-off points considered. The P22 ELISA yielded optimal Sp (99.02%; 95% confidence intervals [CI95%]: 96.5–99.8) and appropriate Se (70.1%; CI95%: 63.6–76) at the selected cut-off point of 100%. These results suggest that P22 can be used as an alternative antigen in the immunodiagnosis of animal TB through the use of an ELISA-type detection of antibodies against MTC in red deer, thus contributing to the diagnosis of animal TB in this species as a measure for further disease prevention and control programs., Research funding was provided by ‘Plan Nacional’ grant AGL2014-56305 (MINECO, Spain and FEDER). J. Thomas was supported by a grant from the Indian Council of Agricultural Research-International Fellowship 2014-15 (ICAR-IF 2014-15). J.A. Infantes was supported by a FPU contract-fellowship (FPU2013/6000) (Ministry of Education, Culture and Sport, Spain). M.A. Risalde holds a ‘Juan de la Cierva program’ contract (IJCI-2014-19961) (Ministry of Economy and Competitiveness, Spain).

[Background and objectives]: Animal tuberculosis (TB) is a multi-host disease involving a wide variety of domestic and wild mammals and causing a significant economic burden and sanitary problems. Wild boar and domestic pigs (Sus scrofa) are indicators of the circulation of the Mycobacterium tuberculosis complex (MTC) and can play a role in its maintenance. The proper diagnosis of MTC contact in these species is, therefore, a key factor as regards controlling TB. The objective of the current study is to evaluate the diagnostic performance of the protein complex P22 as a candidate for use in an in-house ELISA to identify M. tuberculosis complex-specific antibodies for the diagnosis of TB in comparison to the commonly used bPPD-based ELISA (bPPD ELISA) in suids., [Methods]: We conducted a retrospective study. Sera were collected from wild boar during hunting season and from domestic pigs during routine handling, and all the animals underwent reference standard tests (detailed necropsy followed by bacteriological culture and isolation). Animal TB was confirmed to be positive in 277 animals and negative in 366 animals based on both reference standard tests. Sera from those animals were tested by P22 ELISA as well as bPPD ELISA., [Results]: Both ELISAs yielded a good diagnostic value, however, a higher sensitivity (Se) and specificity (Sp) was achieved with the P22 ELISA (Se: 84.1%; CI95%: 79.3–88.2% / Sp: 98.4%; CI95%:96.5–99.4%) when compared to the bPPD ELISA (Se: 77.3%; CI95%: 71.9–82.2% / Sp: 97.3%; CI95%: 95–98.3%). An optimum Sp of 100% (CI95%: 98.54–100%) was attained with white pigs for both the bPPD and the P22 ELISA., [Discussion]: The results suggest that serological tests for MTC-antibody detection, and particularly the P22 ELISA, are valuable tools in the diagnosis of TB in wild boar and domestic pigs when attempting to detect contact with MTC and thereby facilitate TB control and management., Research funding was provided by ‘Plan Nacional’ grant WildDriver CGL2017-89866 (MINECO, Spain and EU FEDER), the 3GdiagnoTB project (RTC-2016-4746-2, MINECO, Spain), and TAVS-CM Programme of the Comunidad de Madrid (S2013/ABI-2747). J. Thomas was supported by a grant from the Indian Council of Agricultural Research-International Fellowship 2014-15 (ICAR-IF 2014-15). MA. Risalde holds a postdoctoral contract from the Research Plan of the University of Córdoba and the Operational Program of EU-FEDER funds from Andalucía.

In recent decades, habitat change and the intensive management of wild ungulates for hunting have led to an increase in their populations in south-central Spain. This implies a higher generation of hunting waste, which can favour the transmission of infectious diseases, including tuberculosis (TB). The aim of this study was to assess the usefulness of the proper disposal of hunting waste as TB control measure in wild boar (Sus scrofa) and red deer (Cervus elaphus) during the 2008/2009 to 2016/2017 hunting seasons. Blood samples from 664 wild boar and 934 red deer were obtained in 14 game estates in two provinces in Andalusia (Area 1), where the disposal of hunting waste was implemented since the 2012/2013 hunting season. Besides, six game estates in the province of Ciudad Real, in Castilla-La Mancha (Area 2), an adjacent region where this management measure was not implemented during the studied period, were used as controls, sampling 277 wild boar and 427 red deer sera. The Mycobacterium tuberculosis complex (MTC), seroprevalence detected in wild boar from Area 1, was significantly higher before the disposal of big game hunting by-products (82.8%; 2008/2009–2012/2013) compared to the second period (61.8%; 2013/2014–2016/2017) (p, The present work was partially funded by the Spanish Ministry of Economy and Competitiveness (MINECO) research grant Ref. AGL2013-49159-C2-2-R. D. Cano-Terriza is supported by the FPU grant of the Spanish Ministry of Education, Culture and Sport.

To study complement function in mammalian leishmanioses, we developed mouse monoclonal antibodies to the human complement system components C1q, C4, factor D, factor H, factor B, properdin, C5 and C9. Antibody specificity was determined by indirect and capture ELISA and by Western blot. In flow cytometry analysis, seven antibodies recognized the cognate component on human serum-opsonized Leishmania promastigotes. Antibody reactivity was screened against promastigotes opsonized with sera of nine mammalian genera: pig, guinea pig, goat, rabbit, cat, dog, hamster, jird and rat. No antibody recognized jird epitopes on promastigotes. Anti-C4, -properdin, and -C5b reacted with the orthologous protein of all other mammals tested except cat (anti-properdin) and hamster (anti-C5b); anti-C9 only recognized the rabbit ortholog, and anti-C1q, -factor B and -factor H did not react with any of the nine orthologs. Such interspecies crossreactive antibodies can be valuable tools for analysis of mammalian complement function in infectious diseases.

In countries where bovine tuberculosis (bTB) is still prevalent the contact among different animal species in extensive systems contributes to the circulation of Mycobacterium bovis (M. bovis) and other members of the Mycobacterium tuberculosis complex (MTC). Thus, free-range pigs can develop subclinical infections and may contribute to disease spread to bovine and wildlife. Serodiagnosis has been proposed as a screening tool for detecting infected pig herds; however, the value of this method to obtain an accurate diagnosis in this species is still not clear. In this study, sensitivity (Se) and specificity (Sp) estimates of four ELISAs and a lateral flow immunochromatographic antibody assay based on different M. bovis antigens, including MPB70 and MPB83 proteins, were evaluated in naturally infected domestic free-range pigs. For this purpose, submandibular lymph nodes and blood samples from 217 pigs from both TB-infected and historically negative farms were sampled at slaughterhouse and analysed by gross examination, histopathology, bacteriological culture and qPCR. Se and Sp estimates of the 5 evaluated assays ranged from 66.1% to 78% (CI from 52.6 to 87.7%) and from 98.9% to 100% (CI from 93.8 to 100%), respectively. Results of our study suggest that all the evaluated assays could be used as a first screening tool to conduct bTB surveillance in domestic pigs at population level; however, animals from seropositive herds should later be surveyed by other methods in order to reduce false negative results., This study was financially supported by the Andalusian Government (AGR-2685-2012) and by the European Project WILDTBVAC (FP7-KBBE-613799). Cardoso-Toset F is funded by a grant of the ceiA3 Campus from the Ministry of Education, Culture and Sport and by theSantander Universities Global Division. Gómez-Laguna J is supported by a “Ramón y Cajal” contract of the Spanish Ministry of Economy and Competitiveness.

[Background]: Bovine purified protein derivative (bPPD) and avian purified protein derivative (aPPD) are widely used for bovine tuberculosis diagnosis. However, little is known about their qualitative and quantitative characteristics, which makes their standardisation difficult. In addition, bPPD can give false-positive tuberculosis results because of sequence homology between Mycobacterium bovis (M. bovis) and M. avium proteins. Thus, the objective of this study was to carry out a proteomic characterisation of bPPD, aPPD and an immunopurified subcomplex from bPPD called P22 in order to identify proteins contributing to cross-reactivity among these three products in tuberculosis diagnosis. [Methods]: Trypsin digests of bPPD, aPPD and P22 were analysed by nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry. Mice were immunised with bPPD or aPPD, and their serum was tested by indirect ELISA for reactivity against these preparations as well as against P22. [Results]: A total of 456 proteins were identified in bPPD, 1019 in aPPD and 118 in P22; 146 of these proteins were shared by bPPD and aPPD, and 43 were present in all three preparations. Candidate proteins that may cause cross-reactivity between bPPD and aPPD were identified based on protein abundance and antigenic propensity. Serum reactivity experiments indicated that P22 may provide greater specificity than bPPD with similar sensitivity for ELISA-type detection of antibodies against M. tuberculosis complex. [Conclusion]: The subpreparation from bPPD called P22 may be an alternative to bPPD for serodiagnosis of bovine tuberculosis, since it shares fewer proteins with aPPD than bPPD does, reducing risk of cross-reactivity with anti-M. avium antibodies., This work was supported by the Ministerio de Economía, Industria y Competitividad of Spain (RTC-2016-4746-2), the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria of Spain (RTA2015-00043-C02-02) and the TAVS-CM Programme of the Comunidad de Madrid (S2013/ABI-2747), cofinanced by the FEDER fund “A way to build Europe”. Jose Antonio Infantes-Lorenzo was supported by an FPU contract-fellowship (Formación de Profesorado Universitario) from the Ministerio de Educación, Cultura y Deporte of the Spanish Government (FPU2013/6000). Alvaro Roy is the recipient of an Industrial Doctorate contract (DI-15-08110) funded by the Spanish Ministerio de Economía, Industria y Competitividad and the European Social Fund.