Bee chitosan nanoparticles loaded with apitoxin as a novel approach to eradication of common human bacterial, fungal pathogens and treating cancer.

Antimicrobial resistance is one of the largest medical challenges because of the rising frequency of opportunistic human microbial infections across the globe. This study aimed to extract chitosan from the exoskeletons of dead bees and load it with bee venom (commercially available as Apitoxin [Api]). Then, the ionotropic gelation method would be used to form nanoparticles that could be a novel drug-delivery system that might eradicate eight common human pathogens (i.e., two fungal and six bacteria strains). It might also be used to treat the human colon cancer cell line (Caco2 ATCC ATP-37) and human liver cancer cell line (HepG2ATCC HB-8065) cancer cell lines. The x-ray diffraction (XRD), Fourier transform infrared (FTIR), and dynamic light scattering (DLS) properties, ζ-potentials, and surface appearances of the nanoparticles were evaluated by transmission electron microscopy (TEM). FTIR and XRD validated that the Api was successfully encapsulated in the chitosan nanoparticles (ChB NPs). According to the TEM, the ChB NPs and the ChB NPs loaded with Apitoxin (Api@ChB NPs) had a spherical shape and uniform size distribution, with non-aggregation, for an average size of approximately 182 and 274 ± 3.8 nm, respectively, and their Zeta potential values were 37.8 ± 1.2 mV and - 10.9 mV, respectively. The Api@ChB NPs had the greatest inhibitory effect against all tested strains compared with the ChB NPs and Api alone. The minimum inhibitory concentrations (MICs) of the Api, ChB NPs, and Api@ChB NPs were evaluated against the offer mentioned colony forming units (CFU/mL), and their lowest MIC values were 30, 25, and 12.5 µg mL-1, respectively, against Enterococcus faecalis. Identifiable morphological features of apoptosis were observed by 3 T3 Phototox software after Api@ChB NPs had been used to treat the normal Vero ATCC CCL-81, Caco2 ATCC ATP-37, and HepG2 ATCC HB-8065 cancer cell lines for 24 h. The morphological changes were clear in a concentration-dependent manner, and the ability of the cells was 250 to 500 µg mL-1. These results revealed that Api@ChB NPs may be a promising natural nanotreatment for common human pathogens.

In ovo nano-silver and nutrient supplementation improves immunity and resistance against Newcastle disease virus challenge in broiler chickens.

Silver nanoparticles (AgNPs) interact with the microbes and host immune system to protect against diseases. Fertile broiler eggs (n = 900) were allotted to six groups: un-injected control, sham (sterile water), AgNPs (50 µg), AgNPs+Amino acids (Methionine-10 mg + Arginine-25 mg), AgNPs+Vitamins (Vit B1-72µg + Vit B6-140µg), and AgNPs+Trace Elements (Zn-80 µg and Se-0.3 µg) and incubated for 18 days. On 18th embryonic day, 0.6 ml test solution was injected at the broad end of egg using 25 mm needle and transferred to hatcher. Post-hatch, half of the chicks from each group were vaccinated with Newcastle disease (ND) vaccine, and the other half were kept as unvaccinated unit and reared for 42 d with standard management practices. Hatchability, 1st and 42nd d body weight, feed intake, and feed conversion ratio were similar between treatment groups in both vaccinated and unvaccinated units. The relative weight of bursa Fabricius and thymus was similar, but spleen weight was higher (P ≤ 0.05) in AgNPs, AgNPs+Vits, and AgNPs+TEs chicks than control group. Cellular immune response (against mitogen phytohemagglutinin-P) was higher (P ≤ 0.05) in AgNPs+TEs chicks, whereas HA titer against sheep red blood cells antigen, serum IgG, IgM, and HI titer against ND vaccine was apparently higher in AgNPs+Vits group chicks than control. No clinical symptoms were observed in the vaccinated groups except for a few control birds 6 days postchallenge (PC). Three days PC, unvaccinated birds show depression, off feed, greenish diarrhea, and nasal discharge and the control group started dying. The highest cumulative infection (CI) was observed in sham (79.17%) and un-injected control (75%), but lowest in AgNPs+AAs birds (58.33%) on 3rd dpi. The CI reached 100% on 5th dpi in control groups and AgNPs, and 91.67% and 93.75% in AgNPs+TEs and AgNPs+AAs group, respectively. The AgNPs+TEs and AgNPs+AAs group birds lived for more than 90 h compared to 75 h in control groups and also had higher IL-6 and IL-2 gene expressions at 24 h PC. It was concluded that 50 µg/egg AgNPs with vitamins (B1 and B6) and trace elements (Zn and Se) improved performance, but AgNPs with trace elements and amino acids enhanced immune response and resistance against vND virus challenge in broilers.

Bioactive-loaded nanodelivery systems for the feed and drugs of livestock; purposes, techniques and applications.

Advances in animal husbandry and better performance of livestock results in growing demands for feed and its nutrients, bioactive compounds (bioactives), such as vitamins, minerals, proteins, and phenolics, along with drugs/vaccines. To protect the feed bioactives in unintended circumstances, they can be encapsulated to achieve desired efficacy in animal feeding and nanoencapsulation gives more potential for better protection, absorption and targeted delivery of bioactives. This study reviews structures, properties, and methods of nanoencapsulation for animal feedings and relevant drugs. Essential oil (EOs) and plant extracts are mostly encapsulated bioactives and phytochemicals for poultry diets and chitosan is found as most effective nanocarrier to load EOs and plant extracts. Nanoparticles (NPs) and nanocapsules are frequently studied nanocarriers, which are mostly processed by using the ionotropic/ionic gelation. Nanofibers, nanohydrogels and nanoemulsions are not found yet for their application in feed bioactives. These nanocarriers can have an improved protection, stability, and controlled release of feed bioactives which benefits to additional nutrition for the growth of livestock regardless of the low stability and water solubility of bioactives. For ruminants' feeds, nano-minerals, vitamins, phytochemicals, essential fatty acids, and drugs are encapsulated by NPs to facilitate the delivery to target organs through direct penetration, to improve their bioavailability, to generate more efficient absorption in cells and tissues, and protect them from rapid degradation. Furthermore, safety and regulatory issues, as well as advantages and disadvantages of nanoencapsulation application in animal feeds are also discussed. The review shows an accurate design of NPs can largely mask safety issues with straightforward approaches and awareness of safety concerns is fundamental for better designing of nanoencapsulation systems and commercialization. This review gives an insight of understanding and potential of nanoencapsulation in ruminants and poultry feedings to obtain a better bioavailability of the nutrients and bioactives with improved safety and awareness for better designing of nanoencapsulating systems.

In ovo Inoculation of Bacillus subtilis and Raffinose Affects Growth Performance, Cecal Microbiota, Volatile Fatty Acid, Ileal Morphology and Gene Expression, and Sustainability of Broiler Chickens (Gallus gallus).

Banning antibiotic growth promoters has negatively impacted poultry production and sustainability, which led to exploring efficient alternatives such as probiotics, probiotics, and synbiotics. Effect of in ovo injection of Bacillus subtilis, raffinose, and their synbiotics on growth performance, cecal microbial population and volatile fatty acid concentration, ileal histomorphology, and ileal gene expression was investigated in broilers (Gallus gallus) raised for 21 days. On 300 h of incubation, a total of 1,500 embryonated eggs were equally allotted into 10 groups. The first was non-injected (NC) and the remaining in ovo injected with sterile distilled water (PC), B. subtilis 4 × 105 and 4 × 106 CFU (BS1 and BS2), Raffinose 2 and 3 mg (R1 and R2), B. subtilis 4 × 105 CFU + raffinose 2 mg (BS1R1), B. subtilis 4 × 105 CFU + raffinose 3 mg (BS1R2), B. subtilis 4 × 106 CFU + raffinose 2 mg (BS2R1), and B. subtilis 4 × 106 CFU + raffinose 3 mg (BS2R2). At hatch, 60 chicks from each group were randomly chosen, divided into groups of 6 replicates (10 birds/replicate), and fed with a corn-soybean-based diet. In ovo inoculation of B. subtilis and raffinose alone or combinations significantly improved body weight, feed intake, and feed conversion ratio of 21-day-old broilers compared to NC. Cecal concentrations of butyric, pentanoic, propionic, and isobutyric acids were significantly elevated in R1, R2, BS2R1, and BS2R2, whereas isovaleric and acetic acids were significantly increased in R1 and BS2R1 compared to NC. Cecal microbial population was significantly altered in treated groups. Ileal villus height was increased (p < 0.001) in BS1, R2, and BS2R2 compared to NC. The mRNA expression of mucin-2 was upregulated (p < 0.05) in synbiotic groups except for BS1R1. Vascular endothelial growth factor (VEGF) expression was increased (p < 0.05) in BS2, R1, BS1R1, and BS1R2 compared to NC. SGLT-1 expression was upregulated (p < 0.05) in all treated birds except those of R1 group compared to NC. The mRNA expressions of interleukin (IL)-2 and toll-like receptor (TLR)-4 were downregulated (p < 0.05) in BS2 and R1 for IL-2 and BS1R1 and BS2R2 for TLR-4. It was concluded that in ovo B. subtilis, raffinose, and synbiotics positively affected growth performance, cecal microbiota, gut health, immune responses, and thus the sustainability of production in 21-day-old broilers.

Antioxidant and antimicrobial activities of Spirulina platensis extracts and biogenic selenium nanoparticles against selected pathogenic bacteria and fungi.

This study investigated the antimicrobial and antioxidant activity of three Spirulina extracts (methanol, acetone, and hexane) and the biological selenium nanoparticles (SeNPs) fabricated by Bacillus subtilis AL43. The results showed that Spirulina extracts exhibited antimicrobial activity against tested pathogens. Besides, Spirulina extracts significantly scavenged ABTS and DPPH radicals in a dose-dependent manner. The methanolic extract had higher total phenolic content, antimicrobial activity, and antioxidant activity than other extracts. The selenium nanoparticles were synthesized by Bacillus subtilis AL43 under aerobic conditions and were characterized as spherical, crystalline with a size of 65.23 nm and a net negative charge of -22.7. We evidenced that SeNPs possess considerable antimicrobial activity against three gram-positive, three gram-negative bacteria, and three strains from both Candida sp. and Aspergillus sp. Moreover, SeNPs were able to scavenge ABTS and DPPH radicals in a dose-dependent manner. An association was found between the total phenolic content of Spirulina and SeNPs and their biological activities. Our results indicate that Spirulina and SeNPs with significant antimicrobial and antioxidant activities seem to be successful candidates for safe and reliable medical applications.

Spirulina platensis and biosynthesized selenium nanoparticles improve performance, antioxidant status, humoral immunity and dietary and ileal microbial populations of heat-stressed broilers.

This study was conducted to assess the impact of dietary incorporation of Spirulina platensis and selenium nanoparticles (SeNPs) individually or in combinations on growth performance, antioxidant status, humoral immune response, and microbial populations in diet and ileum of heat-stressed broilers. Ross-308 one-day chicks (n = 450) were fed one of 9 experimental diets with five replicate cages in 2 phases for 35 d. The experimental diets were a control basal diet without supplementation or with 0.1 mg SeNPs, 0.2 mg SeNPs, 5 g Spirulina, 10 g Spirulina, 0.1 mg SeNPs + 5 g Spirulina, 0.1 mg SeNPs + 10 g Spirulina, 0.2 mg SeNPs + 5 g Spirulina and 0.2 mg SeNPs + 10 g Spirulina per kg diet. Dietary supplementation with Spirulina and SeNPs significantly (P < 0.05) increased body weight gain and European production efficiency factor. Serum GPx and SOD were significantly (P < 0.05) increased with dietary Spirulina and SeNPs supplementation, while, TBARS was decreased (P < 0.05). Circulating immunoglobulin IgM, IgA and IgG were increased in treated birds compared to the control ones, while the antibody titers to IBD, AIV, and NDV were not significantly altered. The results showed that SeNPs and Spirulina exhibited dose-dependent antimicrobial activities against ileal counts of total bacterial, total molds and yeast, coliform, E. coli, Salmonella spp. and Enterococcus spp. However, ileal populations of Lactic acid bacteria were increased with dietary Spirulina and SeNPs in a dose-dependent manner. The microbial load in broilers' diets was reduced by dietary incorporation of S. platensis and SeNPs. These results indicate that Spirulina and SeNPs can be potentially used as growth promoters and antioxidant, immunostimulant, and antimicrobial agents in heat-stressed broilers.

Synergistic effect of Spirulina platensis and selenium nanoparticles on growth performance, serum metabolites, immune responses, and antioxidant capacity of heat-stressed broiler chickens.

This study examined the effects of dietary Spirulina platensis (SP) at levels of 0, 5, and 10 g.kg-1 and selenium nanoparticles (SeNPs) at 0, 0.1, and 0.2 mg.kg-1, individually and in combination, on heat-stressed broiler chickens for 5 weeks. Four hundred fifty one-day-old Ross-308 chicks were allocated to 9 dietary groups with 5 replicates (10 chicks each). The control diet was consisted of corn-soybean-based basal diet. The obtained results displayed a significant increase in final body weight (p = 0.005) and weight gain during the periods from 22 to 35 days (p = 0.002) and 1 to 35 days (p = 0.005) in birds fed supplemented diets compared to those fed control diet, with the highest being in birds fed with both 10 g SP and 0.1 mg SeNPs. Feed conversion ratio was also improved in birds fed supplemented compared to control group. Dietary supplements significantly improved carcass dressing (p < 0.001), carcass yield (p = 0.001) percentages, and blood lipid profile. Blood triiodothyronine was higher (p = 0.005) with all treated diets except that contain 5 g SP compared to the control, with the highest being in birds fed diet contains 5 g SP + 0.2 mg SeNPs. Immunoglobulin subclasses IgG, IgM, and IgA were higher in birds fed supplemented diets compared to the control group. Antibody titers to Newcastle disease, avian influenza, and infectious bursal disease were numerically increased with dietary supplementation compared to the control group. Dietary treatments increased (p < 0.001) glutathione peroxidase and superoxide dismutase (SOD) levels, except diet contains 5 g SP for SOD level and decreased (p < 0.001) malondialdehyde level. It is concluded that dietary inclusion of SP and SeNPs, particularly their combination at levels 5 g SP plus 0.2 mg SeNPs kg-1 and 10 g SP plus 0.1 mg SeNPs kg-1, improved growth performance, carcass yield, immunity, and antioxidant capacity of heat-stressed broilers.

Managing Gut Microbiota through In Ovo Nutrition Influences Early-Life Programming in Broiler Chickens.

The chicken gut is the habitat to trillions of microorganisms that affect physiological functions and immune status through metabolic activities and host interaction. Gut microbiota research previously focused on inflammation; however, it is now clear that these microbial communities play an essential role in maintaining normal homeostatic conditions by regulating the immune system. In addition, the microbiota helps reduce and prevent pathogen colonization of the gut via the mechanism of competitive exclusion and the synthesis of bactericidal molecules. Under commercial conditions, newly hatched chicks have access to feed after 36-72 h of hatching due to the hatch window and routine hatchery practices. This delay adversely affects the potential inoculation of the healthy microbiota and impairs the development and maturation of muscle, the immune system, and the gastrointestinal tract (GIT). Modulating the gut microbiota has been proposed as a potential strategy for improving host health and productivity and avoiding undesirable effects on gut health and the immune system. Using early-life programming via in ovo stimulation with probiotics and prebiotics, it may be possible to avoid selected metabolic disorders, poor immunity, and pathogen resistance, which the broiler industry now faces due to commercial hatching and selection pressures imposed by an increasingly demanding market.

Nutritional manipulation to combat heat stress in poultry - A comprehensive review.

Global warming and climate change adversely affect livestock and poultry production sectors under tropical and subtropical conditions. Heat stress is amongst the most significant stressors influencing poultry productivity in hot climate regions, causing substantial economic losses in poultry industry. These economic losses are speculated to increase in the coming years with the rise of global temperature. Moreover, modern poultry strains are more susceptible to high ambient temperature. Heat stress has negative effects on physiological response, growth performance and laying performance, which appeared in the form of reducing feed consumption, body weight gain, egg production, feed efficiency, meat quality, egg quality and immune response. Numerous practical procedures were used to ameliorate the negative impacts of increased temperature; among them the dietary manipulation, which gains a great concern in different regions around the world. These nutritional manipulations are feed additives (natural antioxidants, minerals, electrolytes, phytobiotics, probiotics, fat, and protein), feed restriction, feed form, drinking cold water and others. However, in the large scale of poultry industry, only a few of these strategies are commonly used. The current review article deliberates the different practical applications of useful nutritional manipulations to mitigate the heat load in poultry. The documented information will be useful to poultry producers to improve the general health status and productivity of heat-stressed birds via enhancing stress tolerance, oxidative status and immune response, and thereby provide recommendations to minimize production losses due to heat stress in particular under the growing global warming crisis.

Impact of multi-strain probiotic, citric acid, garlic powder or their combinations on performance, ileal histomorphometry, microbial enumeration and humoral immunity of broiler chickens.

Heat stress, one of the critical obstacles to poultry sector in subtropical and tropical countries, reduces performance, immune response, and animal welfare. This study examined the effect of dietary inclusion of probiotic (PRO), citric acid (CIT), garlic powder (GAR) or their combinations on growth, blood constituents, ileal microflora and morphology and humoral immunity of broiler chickens subjected to cyclic heat stress. Four hundred ninety one-day-old Ross-308 broiler chicks were randomly allocated to 7 groups with 7 replicates of 10 birds each as follows: control (C) group received the basal diet without supplements, PRO, CIT and GAR groups supplemented with 0.5 g kg-1 multi-strain probiotic mixture (MPM), citric acid and garlic powder, respectively. PRO-CIT and PRO-GAR groups treated with 0.5 g kg-1 MPM, and 0.5 g kg-1 citric acid and garlic powder, while CIT-GAR group fed diet with 0.5 g kg-1 of citric acid and garlic powder. Results revealed that dietary supplements and their combinations improved (P < 0.001) growth performance and decreased abdominal fat of heat-stressed birds. Dietary supplements decreased (P < 0.01) serum concentrations of cholesterol, triglycerides and LDL, while HDL was elevated (P < 0.05). Feed additives reduced (P < 0.01) ileal enumeration of Escherichia coli and total coliform while Lactobacillus count was increased (P < 0.05) only in MPM-enriched groups. Supplementation of these natural products improved (P < 0.01) ileal architecture while humoral immune response was not significantly influenced except antibody titre against Newcastle disease virus which was increased (P < 0.05) in MPM-supplemented groups. Conclusively, addition of the dietary supplements and their combinations, particularly, probiotic and citric acid combination can improve productive performance, and intestinal flora and histomorphometry of broilers exposed to cyclic heat stress.

Approaches to prevent and control Campylobacter spp. colonization in broiler chickens: a review.

Campylobacter, Gram-negative bacteria, is the most common cause of acute bacterial enteritis in human beings, both in developing and developed countries. It is believed that poultry, in particular broiler chickens, is the main host of human infection with Campylobacter. Handling and consumption of contaminated chicken meat are the usual modes of transmission. Prevention and reduction of Campylobacter colonization in poultry farms will cut off the road of infection transmission to humans throughout the food chain. With the incidence of antibiotic resistance and with growing concern about superbugs, the search for natural and safe alternatives will considerably increase in the coming years. In this review, we will discuss the prevalence and risk factors of Campylobacter colonization in broiler chickens and sources of infection. This review also provides extensive and recent approaches to prevent and control Campylobacter colonization in broiler chickens, including biosecurity measures, natural feed/drinking water additives with antimicrobial properties, bacteriocins, bacteriophages, antimicrobial peptides, and vaccination strategies to prevent and control the incidence of human campylobacteriosis.

Modulation of Heat-Shock Proteins Mediates Chicken Cell Survival against Thermal Stress.

Heat stress is one of the most challenging environmental stresses affecting domestic animal production, particularly commercial poultry, subsequently causing severe yearly economic losses. Heat stress, a major source of oxidative stress, stimulates mitochondrial oxidative stress and cell dysfunction, leading to cell damage and apoptosis. Cell survival under stress conditions needs urgent response mechanisms and the consequent effective reinitiation of cell functions following stress mitigation. Exposure of cells to heat-stress conditions induces molecules that are ready for mediating cell death and survival signals, and for supporting the cell's tolerance and/or recovery from damage. Heat-shock proteins (HSPs) confer cell protection against heat stress via different mechanisms, including developing thermotolerance, modulating apoptotic and antiapoptotic signaling pathways, and regulating cellular redox conditions. These functions mainly depend on the capacity of HSPs to work as molecular chaperones and to inhibit the aggregation of non-native and misfolded proteins. This review sheds light on the key factors in heat-shock responses for protection against cell damage induced by heat stress in chicken.

The role of polyphenols in poultry nutrition.

In the last two decades, poultry and animal industries became increasingly interested in using plant-based feed supplements, herbs and their derivatives to retain or enhance their health and productivity. These health benefits for the host mainly attributed to the secondary plant metabolites, namely polyphenols. Polyphenols are renowned for their antioxidant, immunomodulatory, anti-mutagenic and anti-inflammatory properties. However, despite these advantages of polyphenols, they have been characterized by poor absorption in the gut and low concentration in target cells that compromise their role as effective antioxidants. The low bioavailability of polyphenols necessitates the need for further investigations to harness their full potential in poultry farms. This review is existing evidence about the bioavailability of polyphenols and their antioxidant, immunomodulatory, antimicrobial, detoxification properties and their impacts on poultry performance.

The Toxicological Aspects of the Heat-Borne Toxicant 5-Hydroxymethylfurfural in Animals: A Review.

The incidence of adverse reactions in food is very low, however, some food products contain toxins formed naturally due to their handling, processing and storage conditions. 5-(Hydroxymethyl)-2-furfural (HMF) can be formed by hydrogenation of sugar substances in some of manufactured foodstuffs and honey under elevated temperatures and reduced pH conditions following Maillard reactions. In previous studies, it was found that HMF was responsible for harmful (mutagenic, genotoxic, cytotoxic and enzyme inhibitory) effects on human health. HMF occurs in a wide variety of food products like dried fruit, juice, caramel products, coffee, bakery, malt and vinegar. The formation of HMF is not only an indicator of food storage conditions and quality, but HMF could also be used as an indicator of the potential occurrence of contamination during heat-processing of some food products such as coffee, milk, honey and processed fruits. This review focuses on HMF formation and summarizes the adverse effects of HMF on human health.