Unlocking the power of Libidibia ferrea extracts: antimicrobial, antioxidant, and protective properties for potential use in poultry production.

Plant extracts are increasingly recognized as potential prophylactic agents in poultry production due to their diverse bioactive properties. This study investigated the phytochemical and biological properties of Libidibia ferrea (L. ferrea), a plant species native to the Caatinga region of northeastern Brazil. The aim of this study was to identify secondary metabolites and to demonstrate the antimicrobial, antioxidant and protective effects of the plant extract. Three extracts were produced: EHMV, a hydroalcoholic extract from the maceration of pods, and EEMC and EEMV ethanolic extracts from the maceration of peels and pods, respectively, from L. ferrea. High-performance liquid chromatography (HPLC-MS/MS) and atomic absorption spectroscopy (AAS) were used to characterize the metabolites and metals. The antimicrobial activity against Salmonella Galinarum (SG), Salmonella pullorum (SP), Salmonella Heidelberg (SH) and Avian pathogenic Escherichia coli (APEC) was evaluated alone and in combination with probiotic bacteria (Bacillus velenzensis) using agar diffusion and the bactericidal minimum concentration (CBM). The antioxidant potential of the extracts was evaluated in 5 in vitro assays and 6 assays in 3t3 cells. The toxicity of EHMV was tested, and its ability to combat SP infection was demonstrated using a chicken embryo model. The results showed that EHMV exhibited significant antimicrobial activity. The combination of EHMV with BV had synergistic effects, increased antimicrobial activity and induced bacterial sporulation. Composition analysis revealed the presence of 8 compounds, including tannins and phenolic compounds. In vitro antioxidant tests demonstrated that total antioxidant capacity(TAC) activity was increased, and the extract had strong reducing power and notable metal chelating effects. Analysis of 3T3 cells confirmed the protective effect of EHMV against oxidative stress. Toxicity assessments in chicken embryos confirmed the safety of EHMV and its protective effect against SP-induced mortality. EHMV from L. ferrea is rich in proteins and contains essential metabolites that contribute to its antimicrobial and antioxidant properties. When associated with probiotic bacteria such as B. velezensis, this extract increases the inhibition of SH, SG, SP, and APE. The nontoxic nature of EHMV and its protective effects on chicken embryos make it a potential supplement for poultry.

Chicken embryos are a valuable model for the selection of Bacillus subtilis for probiotic purposes.

Bacillus subtilis (BS) has been used as an excellent probiotic; however, some BS strains seem to be opportunist pathogens or do not present inhibitory effects in the pathogenic bacteria, so the characterization of BS strains for use in animals is mandatory. This study aimed to select nonpathogenic strains of BS, which can inhibit Salmonella spp., avian pathogenic Escherichia coli (APEC), and Campylobacter jejuni (CJ) using a chicken embryo as a model. We tested nine (9) strains of BS isolated from several sources (named A to I) in in vitro by tests of mucin degradation activity, haemolytic activity, apoptosis, and necrosis in fibroblasts from chickens. After the in vitro test, we tested the remaining seven (7) strains (strains A to G) in a chicken embryo (CE) as an in vivo model and target animal. We inoculated 3 log CFU/CE of each strain via allantoic fluid at the 10th day postincubation (DPI). Each treatment group consisted of eight CEs. At the 17th DPI we checked CE mortality, gross lesions, CE weight, and whether BS strains were still viable. To perform the cytokine, total protein, albumin, and reactive C protein analysis, we collected the CE blood from the allantoic vessel and intestine fragments in the duodenum portion for histomorphometric analysis. After the results in CEs, we tested the inhibition capacity of the selected BS strains for diverse strains of Salmonella  Heidelberg (SH), S. Typhimurium (ST), S. Enteritidis (SE), S. Minnesota (SM), S. Infantis (SI), Salmonella var. monophasic (SVM), APEC and C. jejuni. After the in vitro trial (mucin degradation activity, haemolytic activity, apoptosis, and necrosis), we removed two (2) strains (H and I) that showed ß-haemolysis, mucin degradation, and/or high apoptosis and necrosis effects. Although all strains of BS were viable in CEs at the 17th DPI, we removed four (4) strains (A, B, D, F) once they led to the highest mortality in CEs or a high albumin/protein ratio. C. jejuni inoculated with strain G had greater weight than the commercial strain, which could be further used for egg inoculation with benefits to the CE. From the tests in CEs, we selected the strains C, E, and G for their ability to inhibit pathogenic strains of relevant foodborne pathogens. We found that the inhibition effect was strain dependent. In general, strains E and/or G presented better or similar results than commercial control strains in the inhibition of SH, ST, SI, APEC, and two (2) strains of CJ. In this study, we selected BS strains C, E and G due to their in vitro and in vivo safety and beneficial effects. In addition, we emphasize the value of CE as an in vivo experimental model for assessing BS's safety and possible benefits for poultry and other animals.

Physiological Changes in Chicken Embryos Inoculated with Drugs and Viruses Highlight the Need for More Standardization of this Animal Model.

Several studies have been developed using the Gallus gallus embryo as an experimental model to study the toxicity of drugs and infections. Studies that seek to standardize the evaluated parameters are needed to better understand and identify the viability of CEs as an experimental model. Therefore, we sought to verify whether macroscopic, histopathological, blood count, metabolites and/or enzymes changes and oxidative stress in CE of different ages are specific to the model. To achieve this goal, in ovo assays were performed by injecting a virus (Gammacoronavirus) and two drugs (filgrastim and dexamethasone) that cause known changes in adult animals. Although congestion and inflammatory infiltrate were visible in the case of viral infections, the white blood cell count and inflammation biomarkers did not change. Filgrastim (FG) testing did not increase granulocytes as we expected. On the other hand, CE weight and red blood cell count were lower with dexamethasone (DX), whereas white blood cell count and biomarkers varied depended on the stage of CE development. Our work reinforces the importance of standardization and correct use of the model so that the results of infection, toxicity and pharmacokinetics are reproducible.