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.

Organic or Inorganic Zinc for Laying Hens? A Systematic Review and Meta-analysis of the Effects of Zinc Sources on Laying Performance, Egg Quality, and Zinc Excretion.

The higher availability of zinc (Zn) from organic than inorganic sources is already established, but more assertive and cost-friendly protocols on the total replacement of inorganic with organic Zn sources for laying hens still need to be developed. Because some discrepancy in the effects of this replacement in laying hen diets is noticeable in the literature, the objective of this meta-analysis was to properly quantify the effect size of total replacing inorganic Zn with organic Zn in the diet of laying hens on their laying performance, egg quality, and Zn excretion. A total of 2340 results were retrieved from Pubmed, Scielo, Scopus, WOS, and Science Direct databases. Of these, 18 primary studies met all the eligibility criteria and were included in this meta-analysis. Overall, the replacement of inorganic Zn with organic Zn, regardless of other factors, improved (p < 0.01) egg production by 1.46%, eggshell thickness by 0.01 mm, and eggshell resistance by 0.11 kgf/cm2. Positive results of the same nutritional strategy on egg weight and Zn excretion were only observed at specific conditions, especially when organic Zn was supplemented alone in the feed, not combined with other organic minerals. Therefore, there is evidence in the literature that the total replacement of inorganic Zn with organic Zn improves egg production, eggshell thickness, and eggshell resistance. Factors such as hen age and genetics, organic Zn source, concentration of Zn in the feed, and the strategy of its supplementation have to be more carefully considered in protocols designed to address egg weight and Zn excretion by the hen.

Roles of viable but non-culturable state in the survival of Campylobacter jejuni.

Despite being considered fragile and fastidious, Campylobacter jejuni is the most prevalent cause of foodborne bacterial gastroenteritis, and chicken meat is considered the main vehicle of transmission to humans. This agent can survive adverse conditions in the form of biofilms, but extreme stress (nutritional, oxidative and thermal) promotes the acquisition of a state called viable but not culturable (VBNC). The emergence of this pathogen worldwide and the recent international requirements in its control instigated us to qualitatively and quantitatively estimate the time required for the acquisition of the VBNC form in 27 strains of C. jejuni, characterize morphological aspects, determine its adaptive and invasive potential and perform comparative metabolomic evaluation. Extreme stress promoted the complete acquisition of the VBNC form in a mean time of 26 days. Starting from an average initial count of 7.8 log CFU/mL, the first four days determined the greatest average reduction of the culturable form of 3.2 log CFU/mL. The scanning and transmission image analyses showed a transition from the typical viable form (VT) to the VBNC form, with initial acquisition of the straight rod shape, followed by loss of the flagella and subdivision into two to 11 imperfect cocci arranged in a chain and rich in cellular content, until their individual release. RT-PCR identified the presence of ciaB and p19 transcripts in the 27 cultivable C. jejuni strains, a character maintained in the VBNC form only for p19 and in 59.3% (16/27) of the VBNC strains for the ciaB gene. The average inoculation of 1.8 log CFU/mL of C. jejuni VBNC into primary chicken embryo hepatocyte cells promoted the occurrence of apoptosis processes significantly after 24 hours of contact by one of the strains tested. In C. jejuni VBNC, we detected higher expression of metabolites linked to protective and adaptation mechanisms and of volatile organic precursor compounds indicative of metabolism interruption. The oscillations in the time of acquisition of the VBNC form together with the presence of transcripts for ciaB and p19, the identification of cell lysis and metabolites that ensure the maintenance of the pathogen alert to the fact that C. jejuni VBNC remains virulent and adapted to stress, which makes evident the potential danger of this latent form, which is not detectable by official methodologies.

Selection of Brucella abortus mimetic epitopes for fast diagnostic purposes in cattle.

Bovine brucellosis is a disease that significantly impacts animal production and human health. Although many sensitive diagnostic tests are used, there is still no ideal fast serological test for all epidemiological situations. In this context, we developed peptides that mimic regions of antigenic proteins of Brucella abortus and can be used in serological diagnosis. RESULTS: From phage display technology, we randomly selected nine clones of phage displaying peptide binders to B. abortus. These clones were sequenced and translated. After molecular docking analysis, two peptides (Ba4 and Ba9) were selected, chemically synthesized, and verified for their potential diagnostic value. By enzyme-linked immunoassay (ELISA), Ba9 showed a sensitivity of up to 97.5% to detect antibodies circulating in animals with brucellosis. We incorporated the peptide Ba9 onto a bioelectrode (graphite modified with poly-3-hydroxyphenylacetic acid). Then, direct serum detection was demonstrated by differential pulse voltammetry, micrographs, and topographic analyses in addition to the average roughness coefficient (Ra) and the value of the mean squared deviation of the roughness (Rms). CONCLUSION: This work shows that the mimetic epitope of B. abortus can be useful for developing new platforms for diagnosing brucellosis. In addition, we propose a fast test based on an electrochemical sensor using graphite modified with poly-3-hydroxyphenylacetic acid.

Lipid nanoparticles based on natural matrices with activity against multidrug resistant bacterial species.

Lately, the bacterial multidrug resistance has been a reason to public health concerning around world. The development of new pharmacology therapies against infections caused by multidrug-resistant bacteria is urgent. In this work, we developed 10 NLC formulations composed of essential oils (EO), vegetable butter and surfactant. The formulations were evaluated for long-term and thermal cycling stability studies in terms of (particle size, polydispersion index and Zeta potential). In vitro antimicrobial assays were performed using disk diffusion test and by the determination of the minimum inhibitory concentration (MIC) performed with fresh and a year-old NLC. The most promising system and its excipients were structurally characterized through experimental methodologies (FTIR-ATR, DSC and FE-SEM). Finally, this same formulation was studied through nanotoxicity assays on the chicken embryo model, analyzing different parameters, as viability and weight changes of embryos and annexes. All the developed formulations presented long-term physicochemical and thermal stability. The formulation based on cinnamon EO presented in vitro activity against strains of Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa isolated from humans and in vivo biocompatibility. Considering these promising results, such system is able to be further tested on in vivo efficacy assays.

Time series evaluation of condemnation at poultry slaughterhouses enable to export in Southeastern Brazil (2009-2019): a tool for optimizing resources in the poultry production chain.

BACKGROUND: Even with the technological advances in management, health and genetics applied to poultry farming worldwide, there is still a high rate of carcasses condemnation at slaughterhouses, which result in losses for the poultry production chain. Thus, this work aimed to evaluate the condemnation occurrence index (COI) and adjusted seasonal index (ASI) of poultry (turkey, griller, and heavy chicken) between 2009 and 2019, in a slaughterhouse enable to export in southeastern Brazil. Data were obtained from official spreadsheets from the Brazilian Federal Inspection Service (FIS) and used to calculate the COI, correlation analysis between the main causes of condemnation, and ASI assessments throughout the year. RESULTS: Seven percent (55,594,318) of the poultry carcasses were condemned (partial or total), and the most frequent causes, contamination, and contusion/traumatic injury, amounted to 63.5% of the total condemnation. There was a trend of increasing condemnation throughout the time series evaluated, with COI varying between 45,282-149,809 condemnations per 1,000,000 poultry slaughtered. Considering the ASI, it was identified that for ascitic syndrome, July has a higher index value (1.63) than the months between January-June (P < 0.05). CONCLUSIONS: The main causes of condemnation were contamination and contusion/traumatic injury, both technological causes. ASI showed that in July there is a greater carcasses condemnation due to ascitic syndrome than in the months between January and June. The variations observed in the ASIs can provide subsidies for preventive measures and optimization of human and financial resources, generating positive impacts on food safety, productivity, and profitability of the sector.

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.

Alternative use of phage display: phage M13 can remain viable in the intestines of poultry without causing damage.

Phage display (PD) is a tool for developing new molecules to control pathogens. Peptides selected by PD are commonly synthesised and tested, but the use of phage M13 displaying the selected peptides as a direct biding in the intestinal tract has not yet been tested. This study evaluated whether phage M13 can remain viable in the chicken gastrointestinal tract and whether it causes injury or humoral immune response. We inoculated phage M13 or E. coli ER2738 (ECR) infected with M13 into birds at different ages. We found the virus in faeces at 5 or 13 days after inoculation, just when it infected the ECR. The presence of phage M13 or ECR did not result in gut injuries and had no impacts on weight gain and bird health. Furthermore, the levels of IgY were similar in all treatments, which indicates that the virus can be used in chicken until 42 days without being recognised by the immune system. This work provides a scientific basis for the use of PD as a tool in numerous applications to control different pathogens.

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.

Hybrid Nanobeads for Oral Indomethacin Delivery.

The oral administration of the anti-inflammatory indomethacin (INDO) causes severe gastrointestinal side effects, which are intensified in chronic inflammatory conditions when a continuous treatment is mandatory. The development of hybrid delivery systems associates the benefits of different (nano) carriers in a single system, designed to improve the efficacy and/or minimize the toxicity of drugs. This work describes the preparation of hybrid nanobeads composed of nanostructured lipid carriers (NLC) loading INDO (2%; w/v) and chitosan, coated by xanthan. NLC formulations were monitored in a long-term stability study (25 °C). After one year, they showed suitable physicochemical properties (size < 250 nm, polydispersity < 0.2, zeta potential of −30 mV and spherical morphology) and an INDO encapsulation efficiency of 99%. The hybrid (lipid-biopolymers) nanobeads exhibited excellent compatibility between the biomaterials, as revealed by structural and thermodynamic properties, monodisperse size distribution, desirable in vitro water uptake and prolonged in vitro INDO release (26 h). The in vivo safety of hybrid nanobeads was confirmed by the chicken embryo (CE) toxicity test, considering the embryos viability, weights of CE and annexes and changes in the biochemical markers. The results point out a safe gastro-resistant pharmaceutical form for further efficacy assays.

A New Approach to Inhibiting Triple-Negative Breast Cancer: In Vitro, Ex Vivo and In Vivo Antiangiogenic Effect of BthTx-II, a PLA2-Asp-49 from Bothrops jararacussu Venom.

Phospholipases A2 (PLA2) represent a superfamily of enzymes widely distributed in living organisms, with a broad spectrum of pharmacological activities and therapeutic potential. Anti-angiogenic strategies have become one of the main tools in fighting cancer. In this sense, the present work reports the inhibition of tumor angiogenesis induced by Asp-49 BthTX-II using in vitro, ex vivo and in vivo approaches. We demonstrate that BthTx-II inhibited cell adhesion, proliferation, and migration of human umbilical vein endothelial cells (HUVEC), as well as caused a reduction in the levels of endothelial growth factor (VEGF) during in vitro angiogenesis assays. BthTx-II was also able to inhibit the sprouting angiogenic process, by the ex vivo germination assay of the aortic ring; in addition, this toxin inhibited the migration and proliferation of HUVEC in co-culture with triple-negative breast cancer cells (e.g., MDA-MB-231 cells). Finally, in vivo tumor suppression and anti-angiogenic activities were analyzed using MDA-MB-231 cells with Matrigel injected into the chorioallantoic membrane of chicken embryo (CAM) for 7 days treatment with BthTx-II, showing a considerable reduction in vessel caliber, on the size and weight of tumors. Together, these results suggest an important antiangiogenic and antitumor role for BthTx-II, as a potential prototype for the development of new tools and antitumor drugs in cancer therapy.

Increased selenium concentration in the synthesis of CdSe magic-sized quantum dots affects how the brain responds to oxidative stress.

CdSe magic-sized quantum dots (MSQDs) have been widely used as fluorescent probes in biological systems due to their excellent optical properties with a broader fluorescence spectrum and stable luminescence in biological media. However, they can be cytotoxic and alter the redox balance depending on the amounts of Cd2+ adsorbed on their surface. Thus, the present study aimed to evaluate whether increases in selenium concentration in the synthesis of CdSe-MSQDs decrease the oxidative stress caused by Cd2+ -based quantum dots. CdSe-MSQDs synthesized with different concentrations of selenium were investigated against oxidative stress in the brain of chicken embryos by examining total antioxidant capacity, lipid peroxidation, thiol, and glutathione contents, as well as the activities of glutathione peroxidase, superoxide dismutase (SOD), catalase (CAT), and glutathione reductase. In addition, the vascularization of the chorioallantoic membrane (CAM) analysis was performed. Higher selenium concentrations alter the surface defect levels (decrease free Cd2+ ) and controlled the oxidative effects of CdSe-MSQDs by reducing the lipid peroxidation, restoring the glutathione defense system and the antioxidant enzymes SOD and CAT, and maintaining the vascular density of the CAM. The current findings reinforce the study of the effects of the presence of Cd2+ ions on the surface of quantum dots, changing toxicity, and aiming interesting strategies of nanomaterials in biological systems.

Influence of age and sex on the blood biochemical constituent values of broiler breeders during the egg-laying stage.

This study aimed to evaluate the influence of age and sex on the blood biochemical constituents of broiler breeders during the egg production stage. The analysis was performed in an industrial broiler breeder farm, and blood samples were collected from males and females at five different ages. Biochemical elements analysed in each serum sample were total proteins, albumin, globulins, uric acid, total cholesterol and triglycerides, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), creatine kinase (CK), alkaline phosphatase (PAL), calcium and phosphorus, beside the glycaemic status. At most ages, females had higher values of total proteins, albumin, globulins, triglycerides, cholesterol, glucose, calcium, phosphorus, Ca/P (calcium and phosphorus) ratio and gamma-glutamyltransferase (GGT). The values of uric acid, aspartate aminotransferase (AST), creatine kinase (CK) and alkaline phosphatase (PAL) were higher in males. The lowest protein values were found at 28 and 60 weeks old. The mean albumin values were significantly higher at 44 and 52 weeks old in males and females. A trend of increasing globulin values with increasing age up to 52 weeks old was observed. Although calcium and phosphorus did not vary according to age, the Ca/P ratio was lowest at 36 weeks old. Comparing the means of both sexes, the AST and GGT values were significantly higher in 60-week-old birds. The highest serum levels of CK occurred at 28 and 52 weeks old. The physiological levels of serum biomarkers presented in this work are important for evaluating productivity performance, welfare and disease indication in breeding flocks.

Comparative genomics of a novel clade shed light on the evolution of the genus Erysipelothrix and characterise an emerging species.

Erysipelothrix sp. isolates obtained from a deadly outbreak in farmed turkeys were sequenced and compared to representatives of the genus. Phylogenetic trees-supported by digital DNA:DNA hybridization and Average Nucleotide Identity-revealed a novel monophyletic clade comprising isolates from pigs, turkeys, and fish, including isolates previously described as E. sp. Strain 2. Genes coding for the SpaC protein, typically found in E. sp. Strain 2, were detected in all isolates of the clade. Therefore, we confirm E. sp. Strain 2 represents a unique species that may be isolated from a broad host range, and the name "Erysipelothrix takahashiae" is suggested. Core genome analysis showed that the pathogenic species of this genus, E. rhusiopathiae and the clade E. sp. Strain 2, are enriched in core functionalities related to nutrient uptake and transport, but not necessarily homologous pathways. For instance, whereas the aerobic DctA transporter may uptake C4-dicarboxylates in both species, the anaerobic DcuC transporter is exclusive of the E. sp. Strain 2. Remarkably, the pan-genome analysis uncovered that genes related to transport and metabolism, recombination and repair, translation and transcription in the fish isolate, within the novel clade, have undergone a genomic reduction through pseudogenization. This reflects distinct selective pressures shaping the genome of species and strains within the genus Erysipelothrix while adapting to their respective niches.

First Report of Genetic Variability of Erysipelothrix sp. Strain 2 in Turkeys Associated to Vero Cells Morphometric Alteration.

Erysipelas is a disease caused by the Erysipelothrix genus, whose main species is the E. rhusiopathiae, the causative agent of animal erysipelas and human erysipeloid. We isolated Erysipelothrix sp. strain 2 (ES2) from turkey's organs during an outbreak in Brazilian commercial and breeder flocks with sepsis and high mortality levels. We studied 18 flocks, accounting for 182 samples, being eight flocks (84 samples) as ES2 positive with individuals demonstrating clinical symptoms and high mortality. We obtained the genetic variability of 19 samples with PFGE and found two clones, both from the same flock but different samples, and two clusters. Interestingly, we found 15 strains with high genetic variability among and within flocks. We have found a positive association between the proximity of ES2 positive turkey flocks and commercial swine sites through epidemiological analysis. We infected Vero cells with two different isolates and three distinct concentrations of ES2. After performing the morphometry, we recorded enlargement of the nucleus and nucleolus. Moreover, we performed fluorescence assays that resulted in apoptotic and necrotic cells. We demonstrated that ES2 could multiply in the extracellular medium and invade and survive inside Vero cells. For the first time, our finds show that ES2 may have similar behavior as E. rhusiopathiae as a facultative intracellular microorganism, which may represent a hazard for humans.

Hybrid Pectin-Liposome Formulation against Multi-Resistant Bacterial Strains.

This work describes the development of a gastroresistant antimicrobial formulation composed of two carriers, pectin and liposomes, intended to improve the efficiency of norfloxacin (NOR) against multi-resistant bacterial strains. The formulations showed physicochemical stability for 180 days (4 °C) in terms of size, polydispersity, and zeta potential of the vesicles, prolonging the in vitro release of NOR for 11 h. The hybrid nanocarriers improved the in vitro antimicrobial activity against different multidrug-resistant bacterial strains, such as Salmonella sp., Pseudomonasaeruginosa, E. coli and Campylobacterjejuni, in comparison to commercial NOR and liposomal suspensions. The in vivo toxicity assay in chicken embryos revealed that the hybrid systems were not toxic in any of the different parameters analyzed, a result also corroborated by the analyses of biochemical biomarkers of the chicken-embryos liver function.

Nanocarriers From Natural Lipids With In Vitro Activity Against Campylobacter jejuni.

Campylobacter jejuni (CJ) is the most prevalent zoonotic pathogen of chicken meat and related products, which may lead to gastroenteritis and autoimmune diseases in humans. Although controlling this bacterium is important, CJ strains resistance against traditional antibiotic therapy has been increased. Vegetable oils and fats are natural biomaterials explored since the Ancient times, due to their therapeutic properties. Nanotechnology has promoted the miniaturization of materials, improving bioavailability and efficacy, while reducing the toxicity of loaded active molecules. In this work, a screening of 28 vegetable oils was firstly performed, in order to select anti-CJ candidates by the disc diffusion test. Thus, the selected liquid lipids were used as active molecules in nanostructured lipid carriers (NLC) formulations. The three resultant systems were characterized in terms of particle size (~200 nm), polydispersity index (~0.15), and zeta potential (~-35mV), and its physicochemical stability was confirmed for a year, at 25°C. The structural properties of NLC were assessed by infrared (FTIR-ATR) and differential scanning calorimetry (DSC) analyses. The spherical nanoparticle morphology and narrow size distribution was observed by transmission electron microscopy (TEM) and field emission scanning electron (FE-SEM) analyses, respectively. Then, the in vitro antimicrobial activity test determined the minimum inhibitory concentration (MIC) of each formulation against CJ strains, in both free (1-3 mg/ml-1) and sessile (0.78 mg/ml-1) forms. Finally, the in vitro biocompatibility of NLC was demonstrated through cell viability using VERO cell line, in which F6 was found twice less cytotoxic than pure olibanum oil. Considering the abovementioned achieved, F6 formulation is able to be evaluated in the in vivo anti-CJ efficacy assays.