Background: Surveillance for circulating emerging diseases of economic importance has a major role in the rapid response to major pathogen outbreaks. Foot-and-mouth disease virus (FMDV) is one of the significant endemic viruses in Egypt. FMDV is periodically investigated for monitoring evolution and emergence of new variants. The genetic characterization of foot-and-mouth disease (FMD) virus serotype A responsible for recent outbreaks of FMD in Egypt was determined. Methods: Samples were collected from different locations and virus isolation was performed using BHK-21 cells. Viral RNA was extracted and samples were screened for FMDV using real-time RT-PCR. DNA sequence analysis was performed and computational and bioinformatics analyses were used to determine the substitution rates and phylogenetic relationship. Results: Sequence and phylogenetic analyses of full-length 1D region of FMDV samples collected from different governorates in 2020 showed close similarity to Egyptian FMDV strains from serotype A-African topotype-G-IV with genetic variation of 6.5%. Recently isolated FMDV strains showed high genetic variations from locally used vaccine strains in the major antigenic sites of VP1 region. Conclusions: Although, efforts made by the veterinary authorities to implement an effective mass vaccination plan, the recently detected FMDV strains in this study could not be subtyped using the FMDV primers routinely used for molecular serotyping. These dissimilarities raise the alarm for reconsideration of the FMDV isolates used in vaccine manufacture. Clearly close monitoring of FMD in Egypt is urgently required to define the risks of future outbreaks and to ensure appropriate control measures against FMD major outbreaks.

There is a need for an improved vaccine for tuberculosis. ESAT-6 is a cardinal vaccine antigen with unique properties and is included in several vaccine candidates in development. ESAT-6 is also the core antigen in the IFN-γ release assays (IGRA) used to diagnose latent infection, rendering IGRA tests unspecific after vaccination. This challenge has prompted the development of a companion diagnostic for ESAT-6 based vaccines, an ESAT-6 free IGRA. We screened a panel of seven potential new diagnostic antigens not recognized in BCG vaccinated individuals. Three highly recognized antigens EspC, EspF and Rv2348c were identified and combined with CFP10 in an ESAT-6 free antigen cocktail. The cocktail was prepared in a field-friendly format, lyophilized with heparin in ready-to-use vacutainer tubes. The diagnostic performance of the ESAT-6 free IGRA was determined in a cross-validation study. Compared IGRA, the ESAT-6 free IGRA induced a comparable magnitude of IFN-γ release, and the diagnostic performance was on par with Quantiferon (sensitivity 84% vs 79%; specificity 99% vs 97%). The comparable performance of the ESAT-6 free IGRA to IGRA suggests potential as companion diagnostic for ESAT-6 containing vaccines and as adjunct test for latent infection.

• FMDVs have brought great challenges to virus molecular analysis and caused several outbreaks with continuously emerging viruses in Egypt. • Samples were collected from the most recent FMDV outbreak in the Egyptian bovine in 2022. • First report on newly emerging FMDV type A of EURO-SA lineage isolated from Egypt. • Results showed that the Egyptian EURO-SA strain was related to strains detected previously in Venezuela, Brazil, and Colombia.

Background and Aim: Foot-and-mouth disease (FMD) virus causes continuous outbreaks, leading to serious economic consequences that affect animal productivity and restrict trade movement. The potential influence of the disease was due to the emergence of new strains or re-emergence of local strains with major antigenic variations due to genetic mutations. This study aims to evaluate circulating virus in samples collected from infected animals during an outbreak using antigenic characterization and identify whether there is an emergence of a new strain or mutation. Materials and Methods: Reverse-transcription polymerase chain reaction (RT-PCR) was used to screen 86 samples. Viral protein 1 (VP1) codon sequencing was performed. The virus was isolated from the samples inoculated on the baby-hamster kidney cell line and Enzyme-linked immunosorbent assay was performed for serotyping and antigen detection. Results: Based on the RT-PCR screening results, 10 positive samples were selected for sequencing. The sequences belonged to the FMD serotype A African topotype originating from the ancestor prototype Sudan/77, with which it shared 98.48% ± 1.2% similarity. The divergence with local isolates from 2020 was 9.3%. In addition, the sequences were 96.84% ± 1.01% and 95.84% ± 0.79% related to Egyptian-Damietta type 2016 and Sudanese-2018, respectively. Divergence with vaccinal strains ranged from 10% to 17%. Amino acid sequence analysis revealed that the isolates had variation in the most prominent antigenic regions (residues 35–75) and the immunogenic determinants of the G-H loop of VP1 (residues 100–146 and 161–175). Conclusion: The current isolates should be included in the locally produced vaccine to provide broader immunogenic coverage against serotype A African topotypes. Keywords: Epicenters, foot and mouth disease virus, foot-and-mouth disease virus Africa type G-IV, mutations, outbreaks, viral protein 1.

Background Toxoplasma gondii ( T. gondii ) is a globally prevalent zoonotic pathogen with major veterinary importance, causing significant livestock reproductive losses and facilitating zoonotic transmission to humans. Despite extensive efforts, there is no available efficient vaccine except for Toxovax, which is only approved for sheep and suffers from some limitations. The parasite’s complex multistage life cycle and strain diversity present major obstacles for vaccine development. Traditional vaccine strategies, including killed, attenuated, and subunit vaccines, provide only limited protection, in addition to posing potential translational drawbacks. Multi-epitope vaccine (MEV) approaches have emerged as a promising alternative that combines conserved immunodominant epitopes, enabling a robust immune response. Our study aimed to computationally design and evaluate a novel MEV candidate capable of eliciting predicted broad genotypic coverage against T. gondii. It thus, serves as a One Health candidate intended for veterinary disease control, which consequently serves the public health by reducing foodborne zoonotic transmission risk and translational potential for cross-species experimental validation. Methods Eighteen T. gondii proteins expressed in different life stages of multiple genotypes were screened for antigenicity, allergenicity, and epitope conservancy. Selected proteins were analyzed to predict MHC-I, MHC-II, and B-cell epitopes using stringent thresholds. Followed by evaluating the candidate epitopes for their cytokine induction potential and theoretical population coverage. Selected epitopes were then assembled into constructs and assessed for physicochemical properties, secondary and tertiary structure stability, immune response simulation, and molecular docking to TLR2 and TLR4. The candidate construct was subjected to complex stability analysis through molecular dynamics (MD) simulation. Codon optimization and in silico cloning were then performed for potential E. Coli expression. Results Forty-three conserved and non-allergenic epitopes were selected and constituted different constructs. Out of which, MEV-2A demonstrated high predicted antigenicity, 100 % hypothetical worldwide population coverage, Th1-skewed cytokine induction, high stability, and computational binding affinity towards TLR2 and TLR4, suggesting that MEV-2A is a promising candidate for further validation. Conclusion MEV-2A is a computationally designed and optimized multi-epitope vaccine targeting multi-stages and multi-genotypes of T. gondii, aimed to serve as a One Health candidate for livestock disease control and human zoonotic risk reduction , which warrants further experimental in vitro and in vivo validations. Clinical trial number: not applicable.