Usefulness of a quantitative real-time PCR assay using serum samples to discriminate between inactive, serologically positive and active human brucellosis

AbstractDiagnosis of brucellosis can be difficult in certain scenarios where conventional microbiological techniques have important limitations. The aim of this study was to develop a LightCycler Quantitative PCR assay in serum samples to discriminate between active and past brucellosis. In total, 110 serum samples from 46 brucellosis patients and 64 controls, including persons who had recently been treated for brucellosis, asymptomatic persons exposed to brucellosis, and patients with febrile syndromes involving a differential diagnosis with brucellosis, were studied. Brucella spp.-specific sequences of the PCR primers and probe were selected from the gene encoding an immunogenic membrane protein of 31 kDa (BCSP31). The analytical sensitivity was 1 × 101 fg of Brucella DNA. The mean threshold cycles for brucellosis patients and controls were 31.8 ± 1.7 and 35.4 ± 1.1, respectively (p <0.001). The best cut-off for bacterial DNA load was 5 × 103 copies/mL. At this cut-off, the area under the receiver operating characteristic curves was 0.963 (95% CI 0.920–1.005), with a sensitivity of 93.5% and a specificity of 98.4%. Under the assay conditions, the LightCycler Quantitative PCR in serum samples seems to be highly reproducible, rapid, sensitive and specific. It is therefore a useful method for both the initial diagnosis and the differentiation between past and active brucellosis

Rapid diagnosis of human brucellosis by SYBR Green I-based real-time PCR assay and melting curve analysis in serum samples

SUMMARYThe aim of this study was to develop a LightCycler-based real-time PCR (LC-PCR) assay and to evaluate its diagnostic use for the detection of Brucella DNA in serum samples. Following amplification of a 223-bp gene sequence encoding an immunogenetic membrane protein (BCSP31) specific for the Brucella genus, melting curve and DNA sequencing analysis was performed to verify the specificity of the PCR products. The intra- and inter-assay variation coefficients were 1.3% and 6.4%, respectively, and the detection limit was 5 fg of Brucella DNA (one genome equivalent). After optimisation of the PCR assay conditions, a standard curve was obtained with a linear range (correlation coefficient = 0.99) over seven orders of magnitude from 107 to 10 fg of Brucella DNA. The LC-PCR assay was found to be 91.9% sensitive and 95.4% specific when tested with 65 negative control samples and 62 serum samples from 60 consecutive patients with active brucellosis. The assay is reproducible, easily standardised, minimises the risk of infection in laboratory workers, and has a total processing time of < 2 h. It could therefore form a promising and practical approach for the rapid diagnosis of human brucellosis

Evaluation of the bovine ATP-binding cassette subfamily A member 13 (ABCA13) as a potential biomarker for sensitive detection of animals with focal pathological forms of subclinical paratuberculosis

Trabajo presentado al: ICP 15th International Association for Paratuberculous, Dublín, 12-16 Junio. 2022.Peer reviewe

Nuevos avances de investigación en Tuberculosis Animal, en el periodo del periodo 2018-2022, del grupo ULE

Trabajo presentado al: II Workshop Ibérico y III Nacional de Investigación en Tuberculosis Animal, Córdoba, 16-18 noviembre.2022

Tuberculosis epidemiology and spatial ecology at the cattle-wild boar inferface in norther Spain

11 páginas, 5 figuras, 3 tablas.Tuberculosis (TB) is a contagious chronic disease due to infection with Mycobacterium tuberculosis complex (MTC) bacteria. Monitoring of wildlife, especially potential reservoirs, is important for detecting changes in disease occurrence and assessing the impact of interventions. Here, we examined whether wild boar (Sus scrofa) may contribute to the re-emergence of TB in Asturias (10,604 km2), northern Spain. Although this province was declared free of TB in cattle in November 2021, MTC bacteria remain prevalent in several “hotspots,” with the European badger (Meles meles) suggested as a TB potential wild reservoir. Drawing on data from the Spanish National Bovine Tuberculosis Eradication Program and the Government of the Principality of Asturias covering the period 2014–2020, we analyzed the prevalence of TB in cattle and wild boar in this region. In hotspots (592 km2), we also investigated the ranging behavior and habitat use of five cows that belonged to farms with a history of TB and six trapped sympatric wild boar. During the observation period, TB prevalence was 0.14% among cattle overall and 0.13–0.41% in hotspots, which was much lower than the prevalence in wild boar, which was 3.15% overall and 5.23–5.96% in hotspots. Infected cattle and infected wild boar in hotspots shared the same strains of M. bovis, and GPS tracking showed spatiotemporal overlap between the species, mainly around pastures during sunrise (06:00–07:00 h) and sunset (19:00–20:00 h). Our results suggest that in addition to cattle and badgers, wild boar possibly help maintain TB in northern Spain, increasing the host richness that influences TB transmission risk in the area, which should be taken into account in monitoring and eradication efforts.Te authors would like to thank our colleagues from SERIDA, the Government of Asturias, SaBio-IREC, VISAVET, and the University of Leon for their help and support. ´ Tis 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. Tis work was partially fnanced by the Ministerio de Agricultura, Pesca y Alimentacion. Gloria Herrero- ´ Garc´ıa is funded by Junta de Castilla y Leon and FSE (grant ´ no. LE036-20).Peer reviewe