Design and development of multiepitope chimeric antigens by bioinformatic and bacterial based recombinant expression methods, with potential application for bovine tuberculosis serodiagnosis.

Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis, is a single health concern, which causes economic losses, is a sanitary barrier and is a zoonotic concern. The golden-pattern intradermic tests have low sensitivity of about 50%. To fix this sensitivity problem, immunoassays could be a powerful tool. However, few studies produced antigens for bTB immunoassays, which needs improvements. Aim of this study was to produce multiepitope chimeric antigens (MCA) to use for bTB diagnosis. To achieve MCA design and development, extensive bibliographic search, antigenic epitope prediction, specificity, hydrophobicity, and 3D structure modeling analyses were performed, as well as cloning, expression and purification. Seven epitopes from four different target proteins (MPB-70, MPB-83, ESAT-6 and GroEL) were combined in five chimeras containing five repetitions of each epitope to enhance antibodies affinity. 3D predicted models revealed that all chimeras have a high percentage of disorder, which could enhance antibody recognition, although taking to protein instability. Each chimera was cloned into pET28a (+) expression plasmids and expressed in six Escherichia coli expression strains. Chimeras 3, 4 and 5 could be solubilized in 8 M urea and purified by ion exchange affinity chromatography. Against bTB positive and negative sera, purified chimera 5 had the best results in indirect dot blot and ELISA, as well as in lateral flow dot blot immunoassay. In conclusion, chimera 5, an MPB-83 containing MCA, could be used for further studies, aimed to develop a serologic or rapid test for bTB diagnosis.

Development and validity assessment of ELISA test with recombinant chimeric protein of SARS-CoV-2.

Serological tests developed for COVID-19 diagnostic are based on antibodies specific for SARS-CoV-2 antigens. Most of the antigens consist of a fragment or a whole amino acid sequence of the nucleocapsid or spike proteins. We evaluated a chimeric recombinant protein as an antigen in an ELISA test, using the most conserved and hydrophilic portions of the S1-subunit of the S and Nucleocapsid (N) proteins. These proteins, individually, indicated a suitable sensitivity of 93.6 and 100% and a specificity of 94.5 and 91.3%, respectively. However, our study with the chimera containing S1 and N proteins of SARS-CoV-2 suggested that the recombinant protein could better balance both the sensitivity (95.7%) and the specificity (95.5%) of the serological assay when comparing with the ELISA test using the antigens N and S1, individually. Accordingly, the chimera showed a high area under the ROC curve of 0.98 (CI 95% 0.958-1). Thus, our chimeric approach could be used to assess the natural exposure against SARS-CoV-2 virus over time, however, other tests will be necessary to better understand the behaviour of the chimera in samples from people with different vaccination doses and/or infected with different variants of the virus.

[Level of knowledge on zoonoses in dog and cat owners]. / Nível de conhecimento de tutores de cães e gatos sobre zoonoses.

OBJECTIVES: To assess knowledge about zoonoses in dog and cat owners. MATERIALS AND METHODS: After applying a questionnaire to 100 owners, data was analyzed by calculating simple means and quantifiable percentage. RESULTS: 77% of the sample owned dogs and 37% cats. All of them claimed to know that animals transmit diseases, but 74% said they were unaware of zoonoses and 80% never received information on the subject. Over 90% of respondents said they know of diseases transmitted by dogs and cats, and 94% point dogs as transmitters of rabies, with 90% being associated with bites, and 88% recognizing vaccination as prevention. Regarding leishmaniasis, 45% associated transmission by mosquito bites and 22% knew about prevention with repellent collars. Mice were associated with the transmission of leptospirosis by 57%, and 22% mentioned feces as a source of infection and 19% controlling rodents as a prevention measure. 59% of the sample reported that cats are transmitters of toxoplasmosis, that feces are the main source (26%) and that hygiene is as a prevention measure (25%). Only 9% knew about sporotrichosis, 6% recognized scratching as a form of transmission by cats and 3% indicated the treatment of diseased animals as a prevention measure. DISCUSSION: This study demonstrates the lack of awareness among the target population of the aforementioned zoonoses. CONCLUSION: The results are important to design preventive health education strategies focused on public health.