The micro‐neutralization assay is a fundamental test in virology, immunology, vaccine assessment, and epidemiology studies. Since the SARS‐CoV‐2 outbreak at the end of December 2019 in China, it has become extremely important to have well‐established and validated diagnostic and serological assays for this new emerging virus. Here, we present a micro‐neutralization assay with the use of SARS‐CoV‐2 wild type virus with two different methods of read‐out. We evaluated the performance of this assay using human serum samples taken from an Italian seroepidemiological study being performed at the University of Siena, along with the human monoclonal antibody CR3022 and some iper‐immune animal serum samples against Influenza and Adenovirus strains. The same panel of human samples have been previously tested in enzyme‐linked immunosorbent assay (ELISA) as a pre‐screening. Positive, borderline, and negative ELISA samples were evaluated in neutralization assay using two different methods of read‐out: subjective (by means of an inverted optical microscope) and objective (by means of a spectrophotometer). Our findings suggest that at least 50% of positive ELISA samples are positive in neutralization as well, and that method is able to quantify different antibody concentrations in a specific manner. Taken together, our results confirm that the colorimetric cytopathic effect‐based microneutralization assay could be used as a valid clinical test method for epidemiological and vaccine studies.

Canine brucellosis caused by Brucella canis, is an infectious disease affecting dogs and wild Canidae. Clinical diagnosis is challenging, and laboratory testing is crucial for a definitive diagnosis. Various serological methods have been described, but their accuracy is uncertain due to limited validation studies. The present study aimed to evaluate the performances of three serological tests for the diagnosis of B. canis in comparison with bacterial isolation (gold standard), in order to establish a protocol for the serological diagnosis of canine brucellosis. A panel of sera from naturally infected dogs (n = 61), from which B. canis was isolated, and uninfected dogs (n = 143), negative for B. canis isolation, were tested using microplate serum agglutination (mSAT), complement fixation performed using the Brucella ovis antigen (B. ovis-CFT), and a commercial immunofluorescence assay (IFAT). The sensitivity and specificity of the three serological methods were, respectively, the following: 96.7% (95% CI 88.8–98.7%) and 92.3 (95% CI 86.7–95.1%) for mSAT; 96.7% (95% CI 88.8–98.7%) and 96.5 (95% CI 92.1–98.2%) for B. ovis-CFT; 98.4% (95% CI 91.3–99.4%) and 99.3 (95% CI 96.2–99.8%) for IFAT. The use in of the three methods in parallel, combined with bacterial isolation and molecular methods, could improve the diagnosis of the infection in dogs.

The neutralization assays are considered the gold-standard being capable of evaluating and detecting, functional antibodies. To date, many different protocols exist for micro-neutralization (MN) assay which varies in several steps: cell number and seeding conditions, virus amount used in the infection step, virus-serum-cells incubation time and read out.

Brucella RB51 is a live modified vaccine. Its use in water buffalo has been proposed using a vaccination protocol different to that used for cattle, but knowledge of the long-term effects of RB51 vaccination in this species remains incomplete. The aim of the study was to evaluate the safety and kinetics of antibody responses in water buffaloes vaccinated according to the protocol described for the bovine species in the WOAH Manual, modified with the use of a triple dose. Water buffaloes were vaccinated with the vaccine RB51. A booster vaccination was administered at 12 months of age. When turning 23–25 months old, female animals were induced to pregnancy. RB51-specific antibodies were detected and quantified using a CFT based on the RB51 antigen. Vaccinated animals showed a positive serological reaction following each vaccine injection, but titers and the duration of the antibody differed among animals. For 36 weeks after booster vaccination, the comparison of CFT values between vaccinated and control groups remained constantly significant. Afterwards, antibody titers decreased. No relevant changes in antibody response were recorded during pregnancy or lactation. In conclusion, results indicated that the vaccination schedule applied is safe and allows for vaccinated and unvaccinated controls to be discriminated between for up to 8 months after booster vaccination.

Listeria monocytogenes is a significant concern in the food industry due to its association with outbreaks of listeriosis, particularly affecting vulnerable populations. High-throughput technologies such as RNA sequencing (RNA-seq) and proteomics offer valuable insights into the molecular responses of L. monocytogenes to stress environments. In this study, a combined transcriptomic and immunoproteomic approach was applied to explore the stress response mechanisms of the L. monocytogenes strain ST7, which was responsible for an outbreak in central Italy. The bacterium was exposed to both optimal conditions and a stress environment representative of pork product matrices (pH 5.5; 7 % NaCl) and thermal abuse prior to consumption (12 °C).Transcriptomic analysis revealed variations in gene expression related to pathogenesis, stress responses, and virulence factors under different environmental conditions. Transcriptomic analysis of Listeria involves studying the complete set of RNA transcripts produced by the bacterium under various conditions or during different stages of its lifecycle. It can provide insights into its pathogenicity and virulence mechanisms. Immunoproteomic analysis identified proteins involved in stress response pathways, including oxidoreductases and DNA repair enzymes, uniquely expressed under stress conditions. Furthermore, the study highlighted proteins linked to antibiotic resistance and cell wall biosynthesis. By delineating specific proteins crucial in the stress response pathways, these findings not only deepen our comprehension of L. monocytogenes biology but also pave the way for designing more targeted mitigation strategies to safeguard food safety and public health effectively.

The significance of Trypanosoma equiperdum as the causative agent of dourine cannot be understated, especially given its high mortality rate among equids. International movement of equids should be subject to thorough health checks and screenings to ensure that animals are not infected with Trypanosoma equiperdum. This involves the implementation of quarantine protocols, testing procedures, and the issuance of health certificates to certify the health status of the animals. Three proteins, the peptidyl-prolyl cis-trans isomerase (A0A1G4I8N3), the GrpE protein homolog (A0A1G4I464) and the transport protein particle (TRAPP) component, putative (A0A1G4I740) (UniProt accession numbers SCU68469.1, SCU66661.1 and SCU67727.1), were identified as unique to T. equiperdum by bioinformatics analysis. The proteins were expressed as recombinant proteins and tested using an indirect ELISA and immunoblotting test with a panel of horse positive and negative sera for dourine. The diagnostic sensitivity, specificity and accuracy of the i-ELISAs were 86.7%, 53.8% and 59.0% for A0A1G4I8N3; 53.3%, 58.7% and 57.9% for A0A1G4I464; and 73.3%, 65.0% and 66.3% for A0A1G4I740, respectively, while the diagnostic sensitivity, specificity and accuracy of immunoblotting were 86.7%, 92.5% and 91.6% for A0A1G4I8N3; 46.7%, 81.3% and 75.8% for A0A1G4I464; and 80.0%, 63.8% and 66.3% for A0A1G4I740. Among the three proteins evaluated in the present work, A0A1G4I8N3 provided the best results when tested by immunoblotting; diagnostic application of this protein should be further investigated using a greater number of positive and negative sera.