Suchergebnisse

South American camelids are susceptible to tuberculosis, caused mainly by Mycobacterium bovis and M. microti. Despite the tuberculin skin test being the official test for tuberculosis, it has a very low sensitivity in these species (14-20%). Serological tests present the advantages of being rapid, easy to perform and facilitate analysis of large numbers of samples in a short period of time. Novel antigen discovery and evaluation would provide enhanced detection of specific antibodies against members of M. tuberculosis complex. Here, we describe the development and evaluation of an ELISA-type immunoassays to use in the diagnosis of tuberculosis in llamas and alpacas based on P22, a multiprotein complex obtained by affinity chromatography from bovine Purified Protein Derivative (bPPD), that showed high sensitivity and specificity in mice, cattle and goats. This work was performed in two stages. First, a preliminary panel of samples collected from tuberculosis-free (n = 396) and M. bovis-infected herds (n = 56) was assayed, obtaining high specificity (100%) and sensitivity ranging from 63 to 96%. Subsequently, the use of the serological assay was tested using samples from two herds suffering from clinical M. bovis (n = 88) and M. microti (n = 25) infection to evaluate the ability of the ELISA to detect infected animals. 11 out of 88 alpacas were positive to the ELISA in a M. bovis outbreak and 7 out of 25 in a M. microti outbreak. The P22 ELISA potentially provides a sensitive and specific platform for improved tuberculosis surveillance in camelids. ; JI-L 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). AR 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. This work was funded by the University of Surrey Innovation Voucher Scheme 2017-18 ; Sí

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). ; Peer reviewed

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 ELISAs provide a sensitive and specific test platform for improved tuberculosis surveillance in badgers. ; This work was supported by the Instituto Nacional de Investigación y Tecnología 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 (Formación de Profesorado Universitario) from the Ministerio de Educación, Cultura y Deporte of the Spanish Government (FPU2013/6000). ; Sí

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 < 0.05) for Norwegian samples. By contrast, Sp in goats was consistently low at the 100 cut-off [30.9 (CI95.4-39.5)-78% (CI95% 68.9-85)]. A higher cut-off of 150 improved Sp in Norwegian goats [97% (CI95% 91.6-99)], but still yielded a poor Sp of 56.1% (CI95% 47.3-64.6) in Spanish goats. In Norway at the 100 cut-off the Sp was 58.3 (CI95% 42.2-72.9) and 90.6% (CI95% 81-95.6) in MAP vaccinated and non-vaccinated goats, respectively, indicating interference due to MAP vaccination. Sp in sheep was between 94.4 (CI95% 91.7-96.3) and 100% (CI95% 96.3-100) depending on the cut-off and country, and no diagnostic interference due to infection with C. pseudotuberculosis was recorded. Sp in pigs was 100%, regardless the cut-off point applied, and no significant differences were observed between pigs from Norway and from Spain. 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, Fisheriesand Food and the Instituto Nacional de Investigación y Tecnología Agraria yAlimentaria of Spain (RTA2015–00043-C02–02). This is a contribution to PlanNacional de I + D + i grant CGL2017–89866 WILD-DRIVER. 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. The funding body was solely involved in funding and had no role in the design of the study, the collection, analysis, and interpretation of the data, or in writing the manuscript. ; Sí

[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. ; Peer Reviewed

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. ; Sí