PCR y MALDI-TOF MS: dos alternativas a la identificación fenotípica de Trueperella pyogenes

Premio extraordinario de Trabajo Fin de Máster curso 2012-2013. Medicina, Sanidad y Mejora Animal.En nuestro estudio hemos realizado la identificación fenotípica, genética y proteómica de un total de 70 cepas de origen porcino aisladas a partir de lesiones de carácter supurativo, a las que se les había realizado una caracterización previa en base a sus características morfológicas, hemolíticas y serológicas como la reacción de aglutinación con el antisuero frente al grupo G de Lancefield. La caracterización fenotípica se llevó a cabo mediante el sistema comercial API Coryne 2.0 (software de lectura Apiweb V 3.0) que identificó 67 cepas como Trueperella pyogenes con perfiles bioquímicos variables, dos cepas como Arcanobacterium haemolyticum y una cepa como Microbacterium spp. Para la identificación genética se emplearon técnicas de PCR convencional: la amplificación del gen plo sirvió para la caracterización de los aislamientos de T. pyogenes, mientras que la amplificación del gen aln se utilizó en el caso de A. haemolyticum. Las 67 cepas identificadas mediante API Coryne como T. pyogenes fueron plo+, mientras que en el resto no pudo determinarse la presencia del gen. El gen aln, no fue amplificado en ninguna de los aislamientos estudiados, estando este resultado en discordancia con el obtenido mediante el sistema API Coryne. Por último, el estudio proteómico se realizó con el sistema MALDI-TOF MS (Matrix-assisted laser desorption ionization-time of flight mass spectrometry) que se reveló como una técnica válida para la identificación rutinaria de las cepas de T. pyogenes a un nivel similar a las técnicas de PCR.In our study we have performed the phenotypic, genetic and proteomic identification of a total of 70 bacterial porcine strains isolated from suppurative lesions, which were previously characterized based on their morphological, hemolytic and serologic characteristics as the agglutination reaction with the antiserum against Lancefield G group. The phenotypic characterization was carried out by using the commercial system API Coryne 2.0 (apiweb reading software V 3.0), identifying 67 strains as Trueperella pyogenes with variable biochemical profiles, two strains as Arcanobacterium haemolyticum and one strain as Microbacterium spp. For the genetic identification, conventional PCR techniques were used: the plo gene amplification was used for the characterization of T. pyogenes isolates, while the aln gene amplification was used in the case of A. haemolyticum. The 67 strains identified by API Coryne as T. pyogenes were plo+, while the presence of this gene was not found in the rest of the strains. The Aln gene was not amplified in any of the studied isolates, being this result in disagreement with that obtained with the API Coryne system. Finally, the proteomic study was performed by using MALDI-TOF MS (Matrixassisted laser desorption ionization-time of flight mass spectrometry), which was demonstrated as a valid method for the routine identification of T. pyogenes strains, with similar results to those obtained by means of PCR techniques

Caracterización imunoproteómica de derivados proteicos de "Mycobacterium bovis, Mycobacterium avium (subespecies avium y paratuberculosis) y Corinebacterium pseudotuberculosis"

La tuberculosis animal es una zoonosis bacteriana producida por bacterias del complejo Mycobacterium tuberculosis, especialmente M. bovis y M. caprae que representa un importante problema sanitario en el hombre y los animales (domésticos y salvajes). Por esta razón, en la Unión Europea, los bovinos están sometidos a un programa de erradicación que se basa en el “diagnóstico y sacrificio” de los animales reaccionantes a las pruebas de diagnóstico oficiales, cuyo reactivo básico es la tuberculina bovina o PPDb. Esta es un derivado proteico purificado de M. bovis de composición y titulación compleja. Por todo ello nos planteamos el estudio de este reactivo, para a través del conocimiento de la composición de las tuberculinas poder mejorar el diagnóstico. En primer lugar, llevamos a cabo una caracterización inmunoproteómica utilizando anticuerpos monoclonales (AcM). Obtuvimos 4 AcMo tipo que nos han permitido identificar cinco proteínas inmunodominantes presentes en la PPDb: MPB70, MPB83, HspX y ESAT-6, exclusivas del MTBC, y meromycolate acyl carrier protein presente también en la PPDa (derivado proteico purificado de M. avium subsp. avium). Las cuatro primeras forman parte de un complejo multiproteico, que se obtiene siempre por inmunopurificación, a partir de la PPDb, con el AcM SIM 377-18 el cual reconoce un epítopo común en las proteínas MPB70 y MPB83. A este complejo multiproteico lo denominamos P22, dado que en el análisis por western-blot muestra una proteína mayoritaria con movilidad electroforética a la altura de 22 kDa

Effect of the Inoculation Site of Bovine and Avian Purified Protein Derivatives (PPDs) on the Performance of the Intradermal Tuberculin Test in Goats From Tuberculosis-Free and Infected Herds

The single and comparative intradermal tuberculin (SIT and CIT) tests are used for the ante-mortem diagnosis of caprine tuberculosis (TB). The tuberculin injection site has been associated with a different performance of the test in cattle. In contrast to that required in cattle in Europe (cervical injection), it can be carried out in the scapular region in goats. Nevertheless, there are no previous data concerning the effect of the injection site on the performance of the test in goats. The aim of the present study was to evaluate the effect of two different inoculation sites (cervical and scapular) on the performance of the SIT/CIT tests. This was done by intradermally inoculating 309 goats from two infected herds and one TB-free herd with both avian and bovine PPDs in the mid-cervical and scapular regions. None of the animals from the TB-free herd had positive reactions, and the number of reactors was not significantly higher, regardless of the inoculation site, in the high and low prevalence herds. However, significantly higher increases in skin fold thickness were observed on the cervical site when compared to the scapular site after the avian and bovine PPD inoculations in the TB-free herd (p < 0.001) and after the bovine PPD injection in the high prevalence herd (p = 0.003). The presence of clinical signs was also more evident on the cervical site when using avian and bovine PPDs in the high prevalence herd (p < 0.01). In contrast, increases in higher skin fold thickness were observed on the scapular site when compared to the cervical site after the bovine and avian PPD inoculations were employed in the low prevalence herd (p < 0.01). These results suggest that the cervical injection of PPDs may improve the sensitivity of the intradermal tuberculin test in high TB prevalence caprine herds, mainly owing to the increased presence of local clinical signs and a better performance of the CIT test. Moreover, specificity was not affected when using standard interpretations, although further analyses in a great number of herds are required in order to confirm these findings.This study was funded by the Herramientas para alcanzar la erradicación de la tuberculosis caprina (GoaTBfree) project (PID2019-105155RB-C31) and the Spanish Government's Ministerio de Agricultura, Pesca y Alimentación. JO was supported by an FPU (Formación de Profesorado Universitario) contract-fellowship provided by the Spanish Ministerio de Ciencia, Innovación y Universidades (FPU18/05197).S

Evaluation of P22 antigenic complex for the immuno-diagnosis of Tuberculosis in BCG vaccinated and unvaccinated goats

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

Evaluation of four ELISA assays to diagnose Mycobacterium tuberculosis complex infection in pigs

Resumen del trabajo presentado al 8th European Symposium of Porcine Health Management and 24th International Pig Veterinary Society Congress, celebrados en Dublin (Irlanda) del 7 al 10 de junio de 2016.[Introduction]: In countries in which bovine tuberculosis (bTB) is still prevalent or is re-emerging the contact among different animal species in extensive systems may contribute to the circulation of Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex (MTC) and the spread of this disease. Thus, free-range pigs may be infected by MTC, developing subclinical infections, which are not detected until meat inspection procedures at slaughterhouse. Serodiagnosis has been recently proposed as a reliable screening tool for detecting infected herds. In this study four ELISA assays using different M. bovis peptides/proteins (MPB70+MPB83, INGENASA; treated bovine purified protein derivative, t-bPPD; bPPD1; and bPPD2 VACUNEK) as coating antigens were evaluated to diagnose MTC infection in pigs. [Materials and Methods]: Submandibular lymph nodes (SLN) and blood samples from 129 free-range pigs raised on Southern Spain farms with a history of condemnation due to tuberculosis-like lesions were sampled at slaughterhouse. SLN were tested by gross examination, histopathology, bacteriological culture and qPCR. Ninety-seven out of these animals were classified as bTB positive cases (compatible lesions and MTC detection by means of culture and qPCR) or bTB negative cases (absence of compatible lesions and negative MTC detection) and used as reference method. When necessary different cut-off values were evaluated. [Results]: All assays had a very good concordance between them (k ≥ 0.82). The MPB70+MPB83 based ELISA had the best sensitivity (Se) (78%, CI95 67.4%>88.5%) and a good concordance with the reference method (k=0.69). The t-bPPD and the bPPD1 in-house assays presented a slightly reduced Se (71.2%, CI95 59.6%>82.7%; and 66.1%, CI95 54%>78.2%; respectively) and a moderate concordance with the reference method (k=0.57 and 0.52, respectively). When the bPPD2 based ELISA was evaluated, similar Se to the previous ones was obtained using a cut-off of 0.35 (Se: 66.1%, CI95 54%>78.2%; k=0.52). Conclusion` +: These results suggest that despite the fact that MPB70+MPB83 ELISA presented the best results all four evaluated ELISA assays could be used as a screening tool to conduct TB surveillance in pigs at a population level. In addition, a cut-off of 0.35 is recommended for bPPD2 ELISA in order to obtain better diagnostic values.This study was financially supported by the Council of Economy, Science, Innovation and Employment of the Andalusian Government (AGR-2685-2012) and by the European Project WILDTBVAC (FP7-KBBE-613799).Peer Reviewe

Valutazione della gestione dei sottoprodotti no destinati al consumo umano e provenienti dalla attività cinegetica, come misura di controllo sulla tubercolosi bovina, dovuta al numero elevato e al ruolo che hanno gli ungulati silvestri

Trabajo presentado al 35º Encuentro GEEFSM (Groupe d’Etudes sur l’Eco-pathologie de la Faune Sauvage de Montagne), celebrado en Cofrentes, Muela de Cortes (España) del 1 al 4 de junio de 2017.[ES]: La tuberculosis (TB) es una enfermedad de declaración obligatoria sometida a programas de erradicación en diferentes países europeos. Pese a los esfuerzos realizados, en determinadas regiones del centro-sur de España se ha producido un estancamiento o incluso un aumento en la prevalencia de TB en el ganado bovino en los últimos años. El incremento en las densidades de ungulados silvestres, así como el papel de estas especies como reservorios naturales de la TB, son factores potencialmente implicados en la persistencia de esta enfermedad en el ganado bovino en estas regiones. En respuesta a esta situación, el gobierno regional de Andalucía redactó la Orden del 2 de Mayo de 2012, que desarrolla las normas de control de subproductos animales no destinados al consumo humano en la práctica cinegética de caza mayor. El objetivo del presente estudio fue evaluar la eficacia de dicha Orden como medida de control de la TB en ungulados silvestres en España. Durante las temporadas cinegéticas 2009/2010 a 2016/2017 se obtuvieron muestras de sangre de un total de 1181 jabalíes (Sus scrofa) y 1514 ciervos (Cervus elaphus), procedentes de 69 cotos de caza mayor en Andalucía. Así mismo, en Castilla-La Mancha (CLM, zona control), donde no tiene efecto la Orden del 2 de Mayo de 2012, se obtuvieron muestras de sangre de 330 jabalíes y 467 ciervos en 13 cotos geográficamente próximos a las áreas de muestreo de Andalucía, durante el mismo periodo. El 26,9% de los ungulados silvestres procedentes de Andalucía, así como el 61,9% de CLM, se muestrearon durante las temporadas cinegéticas 2009/2010 a 2012/2013, mientras que el 73,1% y el 38,8% de las muestras recogidas en Andalucía y CLM, respectivamente, se obtuvieron durante el periodo 2013/2014 a 2016/2017. La detección de anticuerpos frente a TB se realizó mediante ensayo inmunoenzimático “inhouse” (MPB83/P22-ELISA). Así mismo, se tomaron 112 muestras (91 jabalíes y 21 ciervos) de lesiones compatibles con TB en Andalucía. Estas muestras se sometieron a cultivo y espoligotipado. Un animal se consideró infectado por TB si mostró resultado positivo a ELISA o cultivo. En Andalucía, antes de la Orden (periodo 2009/2010 a 2012/2013), el 77,3% (177/229) de los jabalíes y el 10,7% (53/496) de ciervos fueron positivos a TB; mientras que el 47,7% (454/952) de los jabalíes y el 9,2% (94/1018) de los ciervos, presentaron seropositividad tras la implantación de la misma. En jabalí, la seroprevalencia fue significativamente mayor (P<0,001) antes de la implantación de la Orden. En ciervo, no se observaron diferencias estadísticamente significativas entre periodos (P=0,210). En CLM, el 42,7% (96/225) de los jabalíes y el 10,8% (29/268) de los ciervos muestreados antes de la Orden, presentaron anticuerpos frente a TB; mientras que el 44,8% (47/105) de los jabalíes y el 11,6% (23/199) de los ciervos analizados tras su entrada en vigor, fueron seropositivos. En ninguna de las especies se detectaron diferencias estadísticamente significativas entre animales muestreados antes y después de la Orden. La disminución significativa obtenida en la seroprevalencia en jabalí en Andalucía tras la implantación de la Orden, podría estar asociada al comportamiento carroñero de esta especie. Nuestros resultados indican que la gestión correcta de subproductos animales procedentes de la actividad cinegética de caza mayor, tiene una consecuencia directa en la disminución de la prevalencia de TB en las poblaciones de ungulados silvestres. Al igual que en Andalucía, la implementación de este tipo de medidas en otras regiones puede ser una herramienta complementaria para el control de la TB en fauna silvestre, e indirectamente, en especies domésticas simpátricas.Peer reviewe

P22 protein complex in the serodiagnosis of animal tuberculosis: Antigenic stability and cross-reactivity with Corynebacterium pseudotuberculosis infection

The P22 ELISA was recently developed for the serodiagnosis of animal tuberculosis. Herein, the stability of the P22 antigen in different presentations and storage conditions, and the cross-reactivity with Corynebacterium pseudotuberculosis infection in small ruminants were evaluated. For the stability assay, serum samples from cows, sheep, goats, alpacas, badgers, and wild boar were used in the P22 ELISA. The cross-reactivity analysis used sera from sheep and goats with caseous lymphadenitis (CLA). Differences in the immune recognition of P22 were found when the antigen was stored at 40 °C, but without altering the negative or positive status of each sample. P22 ELISA presented 5.71 % cross-reactivity when CLA-positive sheep were evaluated, but no cross-reaction was observed among CLA-positive goat serum samples. This study showed that the P22 protein complex is stable under different formulations and temperatures, and that the assay presents a low cross-reactivity with CLA.This work is a result of the I+D+i research project RTI2018–096010-B-C21, funded by the Spanish MCIN/AEI/10.13039/501100011033/ Ministry of Science, Innovation and the European Regional Development Funds (FEDER Una manera de hacer Europa), and of PCTI 2021–2023 (GRUPIN: IDI2021–000102) funded by Principado de Asturias and FEDER. TDB is a Ph.D. fellow the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES, 88887.511077/2020–00). RDP is a Technological Development fellow from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - Proc. 313350/2019–1).Peer reviewe

Serological technique for detecting tuberculosis prevalence in sheep in Atlantic Spain

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).Peer reviewe

A new test to detect antibodies against Mycobacterium tuberculosis complex in red deer serum

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).Peer reviewe