Enzyme-linked immunosorbent assay as complement of intradermal skin test for the detection of mycobacterium bovis infection in cattle.

Diagnostic tests based on cell-mediated immunity are used in programs for the control and eradication of bovine tuberculosis (bTB), which is mainly caused by Mycobacterium bovis. Additional serological assays could be performed as an ancillary method to detect an infected animal that fails to produce an immune response against the intradermal reaction (IDR), the official bTB test. In this study, we evaluated the effectiveness of an enzyme-linked immunosorbent assay (ELISA) that uses bovine PPD as a capture antigen as a complement to the IDR in herds with confirmed cases of bTB. The study was conducted in two stages. First, a panel of 200 serum samples was analyzed by ELISA. The sensitivity and specificity obtained were 60% and 99%, respectively. The subsequent stage consisted of evaluating 7,494 bovines from 14 selected dairy farms. The number of animals yielding a IDR negative/ELISA positive result were 200. A necropsy analysis of 33 of these IDR negative/ELISA positive animals revealed that 30 (91%) presented granulomatous lesions and positive M. bovis isolation. This finding confirmed bTB in most cases. Altogether, the results obtained in the present study suggest that the combined use of IDR and ELISA is an effective strategy to improve the control of bTB in endemic herds.

Identification of bovine tuberculosis biomarkers to detect tuberculin skin test and IFNγ release assay false negative cattle.

Bovine tuberculosis (bTB) is an important animal and zoonotic disease, which causes severe economic losses. The main focus of this study was to assess the predictive power of previously identified biomarkers of bTB in infected animals that were negative to the tuberculin skin test (TST). We studied 16 animals with bTB, in which the disease was confirmed by necropsy, and 16 healthy animals. The level of expression of ten biomarkers (CXCL9, THBS1, MMP9, IL-22, CXCL10, IFNγ, IL-17, FYVE, CD14, IL-1R) was evaluated by RT-qPCR upon stimulation or not of peripheral blood mononuclear cells with PPDb (purified protein derivative of bovine tuberculin). In this assay, CXCL9, THBS1, MMP9, IL-22 and IFNγ changed their expression level depending on the bTB status. In addition, we evaluated different biomarker candidates simultaneously to infer the animal condition. By performing an analysis with classification trees, we found that the sturdiest combination was IL-22, IFNγ and IL-1R. On the other hand, CXCL10, IFNγ and IL-22's expression distinguished between bTB positive animals that were negative to TST (TST false negative animals) and the bTB negative groups. Thus, these biomarkers are promising candidates to be tested as an ancillary diagnostic assay. In addition, the expression of CXCL10 and IL-22 exhibited also significant differences between the bTB positive animals that were undetectable by IFNγ release assay (IGRA) and TST tests (TST and IGRA false negative animals) and the bTB negative groups. Therefore, CXCL10 and IL-22 constitute candidate biomarkers that could complement the two most widely used diagnostic tests.

Mycobacterium bovis ESAT-6, CFP-10 and EspC antigens show high conservation among field isolates.

ESAT-6, CFP-10 and EspC are virulence factors that have been extensively assayed for bovine and human tuberculosis diagnosis due their potent T-cell inducing activities. While polymorphisms of ESAT-6 and CFP-10 were analyzed, with the description of CFP-10 variants in M. tuberculosis, this fact has not been explored in M. bovis field isolates. The coding sequences of esxA (ESAT-6), esxB (CFP-10) and mb3645c (EspC) from 58 M. bovis strains exhibiting genomic variability (spoligotyping) were analyzed. Two genes -esxA and esxB - remained invariant while mb3645c exhibited one synonymous polymorphism (G to A mutation, position 66bp) in one isolate, compared to M. bovis AF2122/97 reference strain. All isolates exhibited a synonymous nucleotide polymorphism simultaneously (G to A mutation, position 255bp), compared to M. tuberculosis H37Rv reference strain. This study confirms the high conservation for ESAT-6, CFP-10 and EspC in local M. bovis field isolates and reinforce the use of these three antigens in the diagnosis of bovine tuberculosis. Further studies should be performed to globally confirm these findings.

HBV-HDV en donantes de sangre: prevalencia y análisis molecular / Hepatitis B virus and Hepatitis D virus in blood donors: prevalence and molecular analysis in an Argentinean population

The aim of this report was to determine retrospectively the prevalence of hepatitis viruses infection by both HBV and HDV, and to identify the genotype in a population of blood donors. From 42,055 sample of donors, the authors study the hepatitis B virus and hepatitis D virus prevalence and molecular analysis in an Argentinean population. The results obtained are detailed in the article.