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

A lateral flow assay for the rapid diagnosis of Mycobacterium bovis infection in wild boar

The native Eurasian wild boar (Sus scrofa ) is a reservoir of Mycobacterium bovis , the causative agent of animal tuberculosis (TB), a chronic disease in livestock, companion animals and wild mammals. Cases of M. bovis infection in wild boar or feral pig have been reported worldwide, making early detection a priority in the eradication of the disease. Point‐of‐care diagnostic tests, such as low cost lateral flow assays, provide high specificity and sensitivity and can be performed on site , an essential requirement for a rapid screening of wildlife. A lateral flow assay, LFA, (INgezim TB CROM Ab) for the detection of M. bovis ‐specific antibodies in wild boar serum and blood has been developed based on MPB83, one of the major immunogenic antigens of the bacterium. A total of 140 samples of wild boar serum, well‐characterized by Mycobacterium tuberculosis complex culture and TB compatible post‐mortem lesions, have been analysed with LFA, and results were compared with one in‐house and two commercial Enzyme‐linked Immunosorbent Assays (ELISA), INgezim TB Porcine and INgezim Tuberculosis DR. In experimental samples, the achieved values of sensitivity of the different techniques ranged from 84.3% to 92.1% and the specificity was 100% in all of them. In field animals, specificity ranged from 96% to 100%, whereas sensitivity ranged from 48% to 64% in juvenile wild boar, increasing to 93.3%–100% in adult wild boar. In particular, the total sensitivity and specificity values obtained with the new LFA were 83% and 97%, respectively, indicating that INgezim TB CROM Ab could be used as a first approach for the surveillance of TB in wild boar, with a special applicability for animal‐side testing.Part of this research was funded by the EU, Seventh Research Framework Program FP7‐KBBE‐2013‐7 under grant number nº 613799 (WildTBVac). This is a contribution to the WildDriver grant CGL2017‐89866 from MINECO and EU‐FEDER. We are very grateful to Dr. Mercedes Dominguez and her laboratory (Instituto de Salud Carlos III, Unidad de Inmunología Microbiana, Majadahonda, Spain) for their generous supply of P22 protein complex.Peer reviewe

Monitoring Anti-NS1 Antibodies in West Nile Virus-Infected and Vaccinated Horses

West Nile virus (WNV) is a zoonotic arboviral pathogen affecting humans, birds, and horses. Vaccines are available for veterinary use, which efficiently prevent the infection in horses. Most common diagnostic tools rely on the identification of the agent (RT-PCR, virus isolation), or on the detection of antibodies (IgM and IgG) recognizing structural proteins of the virus or neutralizing virus infection in cell cultures (virus-neutralization tests). The recent emergence of WNV in different parts of the world has resulted in an increase in the vaccination of horses in many countries. Methods for differentiation between infected and vaccinated animals (“DIVA” assays) would be useful for surveillance and control purposes but are still not available. A usual approach in this regard is the use of antibodies to nonstructural proteins as markers of nonvaccinated, infected animals, and the nonstructural NS1 protein of WNV has been proposed as a candidate for such a marker. The aim of this study was to test the hypothesis that NS1 can be a useful antigen in DIVA assays for differentiating WNV vaccinated and infected horses in field conditions. For that, we examined serum samples from either vaccinated and infected horses both from experimental infections/vaccinations (under controlled conditions) and from the field, exposed to natural infection or vaccinated in response to a risk of infection. The overall conclusion of the study is that NS1 antigen can effectively differentiate WNV infected from vaccinated horses in experimental (controlled) conditions, but this differentiation might be difficult depending on the conditions prevailing in the field

Development and evaluation of a new epitope-blocking ELISA for universal detection of antibodies to West Nile virus

West Nile virus (WNV) is an emerging zoonotic pathogen with a wide range of hosts, including birds, horses and humans. The development and evaluation of the performance of a new enzyme-linked immunosorbent assay (ELISA) are described for rapid detection of WNV-specific antibodies in sam- ples originating from an extensive range of vertebrates susceptible to WNV infection. The assay uses a monoclonal antibody (MAb) which binds whole virus particles and neutralizes infection in vitro by recognizing a neutralizing epitope within the envelope (E) glycoprotein of the virus. This MAb, labelled with horseradish peroxidase, was used to compete with WNV-specific serum antibodies for virus-binding in vitro. The epitope-blocking ELISA was optimized in a manner that enabled its validation with a number of experimental and field sera, from a wide range of wild bird species, and susceptible mammals. The new ELISA exhibited high specificity (79.5–96.5%) and sensitivity (100%), using the virus-neutralization test as reference standard. It also required a much lower volume of sample (10 pl per analysis) com- pared to other ELISAs available commercially. This new method may be helpful for diagnosis and disease surveillance, particularly when testing samples from small birds, which are available in limited amountsPeer reviewe

Absence of protection from West Nile virus disease and adverse effects in red legged partridges after non-structural NS1 protein administration

The red-legged partridge (Alectoris rufa) is a competent host for West Nile virus (WNV) replication and highly susceptible to WNV disease. With the aim to assess in this species whether the inoculation of non-structural protein NS1 from WNV elicits a protective immune response against WNV infection, groups of partridges were inoculated with recombinant NS1 (NS1 group) or an unrelated recombinant protein (mock group), and challenged with infectious WNV. A third group received no inoculation prior to challenge (challenge group). The NS1 group failed to elicit detectable antibodies to NS1 while in the mock group a specific antibody response was observed. Moreover, no protection against WNV disease was observed in the NS1 group, but rather, it showed significantly higher viral RNA load and delayed neutralizing antibody response, and suffered a more severe clinical disease, which resulted in higher mortality. This adverse effect has not been observed before and warrants further investigations.This work was financially supported by the European Commission (HEALTH.2010.2.3-3-3 261391 EuroWestNile project).Peer Reviewe

Evaluation of five serologic assays for bovine tuberculosis surveillance in domestic free-range pigs from southern Spain

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.Peer Reviewe

Learning about Student Performance from Moodle logs in a Higher Education Context

Ponencia presentada en: 2022 XII International Conference on Virtual Campus (JICV). IEEE, 2022. p. 1-4.Learning Management Systems (LMS) are educational tools used to plan, implement and assess the learning-teaching procedure. LMS are continuously acquiring data from students in their learning progress when they interact with the platform. In this work, we use statistically-based approaches to learn about student performance solely using data from Moodle LMS, which is widely used along the educational institutions around the world. Using logged data from several subjects and degrees, our results indicate that the number of interactions with the LMS is a good indicator of student performance and can potentially be used as a measure to track students’ learning progress