Immunized Chickens Research Articles

Application of a liposomal subunit vaccine in chickens for reduction of Campylobacter gut colonisation

Abstract Introduction Campylobacter are the most common cause of food poisoning, which manifests itself in diarrhoea of varying severity. Additionally, because of the increasing number of people with immune deficiencies, more frequent serious complications of Campylobacter infections are being observed. The main source of infection is the consumption of contaminated poultry meat, which is a consequence of the insufficiency of current hygiene and biosecurity to control Campylobacter or eliminate it from the poultry food chain. Material and Methods Two hybrid proteins, presenting selected epitopes of the Campylobacter antigens CjaD and EF-Tu, were developed based on the highly immunogenic proteins CjaA and CjaC. Four groups of chickens were vaccinated with different preparations (a mixture of both hybrid proteins encapsulated in anionic or neutral liposomes) and different doses (a single dose given on the day of hatching or two doses given on days 1 and 14 of life). The number of Campylobacter was assessed in the intestinal contents of vaccinated birds. Results No statistically significant differences in colonisation levels were observed between chickens immunised with neutral liposomes containing hybrid proteins and their non-immunised counterparts, regardless of dosage regimen. Conclusion Although immunisation of chickens did not produce the expected results, the approach used has great potential, which is worth further investigation and development.

Utilizing Flaxseed as an Antimicrobial Alternative in Chickens: Integrative Review for Salmonella enterica and Eimeria

This review provides an integrative framework for understanding flaxseed (Linum utassitissimum) as an antimicrobial alternative for poultry production. We begin by familiarizing the reader with the global legislation of antibiotics in animal husbandry; highlighting gaps and current issues for Salmonella enterica (S. enterica) and Eimeria (coccidiosis-inducing). We then discuss the natural, symbiotic characteristics of the Galliformes order (chicken-like birds) and Linum (the flaxes). The key immunological themes in this review include: (i) flaxseed’s regulation of innate and adaptive immunity in chickens, (ii) flaxseed’s ability to accelerate chicken recovery from infection with S. enterica and Eimeria, and (iii) flaxseed’s strengthening of immunity via vitamin B6 antagonism. Research indicates that whole flaxseed increases adaptive immune capacity by augmenting cecal Bacteroides and short-chain fatty acids while also attenuating the heterophil to lymphocyte ratio in chickens. Moreover, flaxseed accelerates chicken recovery from infection with Salmonella Enteritidis or Eimeria tenella; however, future work is needed to better understand (i) defatted flaxseed’s superior performance against Eimeria species and (ii) Eimeria maxima’s resilience against whole flaxseed. In the context of vitamin B6 antagonism, we propose that 15% whole flaxseed overcomes S. enterica’s insult to estrogen synthesis by sustaining the activity of phosphatidylethanolamine methyltransferase (PEMT) in liver. We also propose that 10% defatted flaxseed (as a metformin homologue) strengthens chicken immunity by safeguarding gonadal physiology and by increasing plasma thymidine bioavailability. The concepts in this review can be used as a template for conducting advanced immunological studies in poultry science.

Effect of coated calcium feeding on growth performance, carcass traits, immunity, blood biochemistry and tibial bone morphometry in commercial broiler chicken.

The bone abnormalities pose great concern in heavy, rapidly growing broilers, resulting in significant economic loss, and health issues in birds. To address these problems and prevent unnecessary mineral excretion into environment, it is crucial to optimise the inclusion level of dietary calcium in broiler's diet. The calcium utilisation by the bird is also affected by its source, particle size, bioavailability etc. Hence this study aimed to study the effect of partially replacing conventional dietary calcium source with coated calcium on growth performance, immune response, carcass traits, blood calcium and phosphorus, and mineral retention in broilers. About 416 broiler chicks were randomly distributed into four treatments, each with eight replicates of 13 birds and fed with iso-calcium, isocaloric, and isonitrogenous diet for 42 days. The T0 (control) was a basal diet while the T1, T2, and T3, were treatment diets in which conventional calcium source (limestone) was partially replaced with coated calcium by 10, 20, and 30% respectively. The birds of T3 group showed higher (P < 0.05) body weight gain, better feed conversion ratio and higher carcass yields compared to T0, T1, and T2 diet fed birds. Further the treatment groups exhibited higher (P < 0.05) cell-mediated and humoral immune responses and immune organ weights compared to control. The tibial bone weight, length and average diameter was highest (P < 0.05) in T3. Moreover, the T3 diet fed group also showed higher (P < 0.05) calcium and phosphorus content in serum and bone and lowest content of these minerals in the excreta followed by T2, T1 and control. These findings suggest that a 30% conventional calcium source with coated calcium may be used as a high-grade mineral source for broiler chicken feed.

Effect of different levels of turmeric (Curcuma longa) on productivity in broiler chickens

This research was conducted to evaluate the effect of different levels of turmeric powder on the performance, blood parameters, immunity, intestinal morphology, carcass characteristics, cecum microbial flora, fatty acid profile and sensory evaluation of breast meat of broiler chickens. A completely randomized design was used including four levels of turmeric powder (250, 500, 750 and 1000 mg/kg diet) and a control treatment (without turmeric), in 5 replications. The growth performance was measured periodically and at the end of the 42-day period, three chickens from each experimental unit were killed for carcass examination. In order to check blood parameters at the end of the course, blood samples were taken from three chickens from each experimental unit. To check the immunity of broiler chickens, the weight percentage of immune glands was also evaluated. To measure the morphology of the cecum, one chicken from each experimental unit was killed to check the desired traits. The results of the experiment showed that different levels of turmeric had a significant effect on performance, carcass characteristics, blood parameters, immunity, intestinal morphology, cecal microbial flora, fatty acid profile, and the sensory evaluation of breast meat (P&lt;0.05). The results of the experiment showed body weight, feed intake and feed conversion ratio (FCR) at the end of the period were significant in the treatments that received turmeric powder (P&lt;0.05). Survival percentage, produced body weight per square meter, and final profit was also significant (P&lt;0.05). Relative weight of abdominal fat, villus height and villus height/ crypt depth of the small intestine were significant in the treatments receiving turmeric powder (P&lt;0.05). By adding turmeric powder to the diet of broiler chickens, the microbial population of the cecum also became significant (P&lt;0.05). The concentration levels of uric acid, total cholesterol, glucose, HDL, albumin and liver enzymes alkaline phosphatase and ALT were significant in the birdsreceiving turmeric powder (P&lt;0.05). Turmeric powder in the diet of broilers also had a significant effect on chondroitinic acid, linoleic acid and total polyathenoids (P&lt;0.05). Turmeric powder was significant on the lactobacilli microbial population (P&lt;0.05). The results of this experiment showed that fat content, juicyness, crispyness, chewability, elasticity, mouthfeel and general acceptance were significantly improved (P&lt;0.05). In this research, the treatment receiving 500 mg of turmeric powder per kg had the greatest effect on the measured characteristics of broiler chickens. It can be concluded that the addition of the powder of these medicinal and aromatic plants to the diet of broiler chickens has beneficial effects on the carcass characteristics and meat quality. Keywords: fatty acid, blood plasma, abdominal cavity, triglyceride, feed conversion ratio, Curcuma longa

The effect of high levels of Glycyrrhiza glabra powder on performance, carcass characteristics, blood parameters, immunity, intestinal microbial flora, intestinal morphology and breast muscle fatty acid profile in broilers

In order to investigate the effect of licorice medicinal plant on performance, carcass characteristics, fatty acid profile of breast muscle, intestinal microbial flora, blood constitutes and immunity of broiler chickens, an experiment using a completely randomized design with 3 treatments, 4 replications, 10 chicks per repetition and a total of 120 pieces of one-day-old male broiler chickens of commercial strain Ross 308 were performed for 42 days. Experimental treatments included three different levels of licorice (0, 300 and 400 mg/kg feed). Data was analyzed using SAS statistical software and averages were compared at the 5% probability level with Duncan's multiple range test. The results showed that although variation in feed consumption, weight gain and feed conversion ratio was not significant in any of the rearing periods (P≥0.05), the highest feed consumption, weight gain and the best feed conversion ratio was observed in the final period of chickens were fed with Diet containing 400mg/kg licorice. Also, according to the data, the best European index was related to this treatment. In addition, the use of two levels of licorice had no significant effect on carcass characteristics or intestinal microbial flora; however, the highest increase in body weight and the lowest number of Lactobacillus acidophilus bacteria were observed at the higher level of licorice. However, these high levels did not lead to a decrease in abdominal fat. According to the results obtained, different levels of licorice led to a significant reduction of harmful fats such as cholesterol, triglycerides, VLDL and LDL (P&lt;0.05). Furthermore, although the level of HDL was not significant (P≥ 0.05), its highest amount was seen at high levels of licorice. In addition, the lowest amount of total protein, albumin and globulin was observed at the level of 400 mg/kg of licorice. According to these results, although the amount of glucose was lowest its level was not significant (P≥0.05), Also, the use of this same level of licorice had no significant effect on the function of the humoral immune system in response to antigen injection (SRBC Antibody against sheep red blood cell) or antibody titer against Newcastle virus and influenza (P≥0.05). Nevertheless, according to the data, the highest antibody titer against Newcastle virus and SRBC antigen was associated with this treatment. These results also showed that high levels of licorice led to an increase in the number of Bifidobacterium. Villi width and crypt depth decreased at the high dose of licorice, but the ratio of villi length to crypt depth increased. In addition, the use of different levels of licorice could not reduce the ratio of saturated to unsaturated fatty acids compared to the control group. Therefore, based on the results of this research, it is recommended to use the level of 400 mg/kg of licorice to supplement the diet of broilers of Ross 308 strain.

Adipose tissue responds to stress-induced immunosuppression affecting immune response partially by miR-145-5p/S1PR1 pathway

Stress-induced immunosuppression (SIIS) is one of the most common problems in intensive poultry production, which can cause immunized chickens to still develop diseases and bring huge losses to production. Recently, adipose tissue, as an immunomodulatory organ, has become a hot topic of attention. However, the function and mechanism of adipose tissue involved in SIIS and its influence on the immune response are still unclear. In this study, we dynamically analyzed the correlations between the T cells migration and change of sphingosine-1-phosphate receptor 1(S1PR1) gene in adipose tissue using chicken models with different immune states, and further explored the regulatory mechanisms and application. The results showed that SIIS could significantly change the expressions of lymphocytes migration related S1PR1 gene, and SIIS could inhibit the Newcastle disease virus (NDV) immune response partially by affecting the migration and proliferation of TCRα+ T cells in adipose tissue. Moreover, the miR-145-5p/S1PR1 pathway was a potential key mechanism to regulate T cells migration in adipose tissue, and circulating miR-145-5p had potential value as a molecular marker. This research can provide innovative reference for in-depth studying the immunoregulatory function and mechanism of adipose tissue.

Therapeutic efficacy of SipD/LptD-specific IgY entrapped in alginate nanoparticles against Salmonella Typhimurium infection

IntroductionSalmonella, a zoonotic pathogen causing gastroenteritis, lacks a preventive vaccine. Passive immunization with IgY antibodies derived from immunized chickens has shown potential for treating bacterial infections. This study investigated the therapeutic efficacy of entrapped IgY targeting recombinant SipD and LptD proteins from Salmonella Typhimurium. MethodsThe recombinant protein was expressed in E. coli BL21 (DE3) and purified using Ni-NTA affinity chromatography. Hens were immunized with the purified protein, and the resulting IgY was entrapped into alginate nanoparticles. The shape of spherical nanoparticles and their size in the nanometer range were determined by SEM and DLS analysis. The therapeutic efficacy of free and alginate-entrapped IgY against S. Typhimurium was evaluated in mice at 1, 50, and 100 LD50 bacterial doses. ResultsThe purified IgY concentration in each egg yolk was 6 mg/ml (35 mg/egg). Physicochemical and structural characterization revealed spherical nanoparticles with a diameter of 157.1 nm and a negatively charged surface (zeta potential of −35.6 mV). The loading efficiency of IgY into alginate nanoparticles was 95.5 %. In a challenge test with 100 LD50 of S. Typhimurium, all mice receiving alginate-entrapped IgY survived, whereas half of the mice receiving non-entrapped IgY died within 7 days. ConclusionOur results indicate that IgY antibodies entrapped in alginate nanoparticles may offer therapeutic effect against S. Typhimurium infection.

Effects of shallot (Allium ascalonicum) powder supplementation on growth, intestinal morphology, immune stimulation, and intestinal bacteria in broiler chickens

Background and Aim: Antimicrobial resistance in poultry farms is a significant global public health concern that has led farmers to explore alternative antibiotics, such as prebiotics in poultry production. This study aimed to examine the antimicrobial activity of ethanolic shallot extract (ESE) and the effects of adding shallot powder (SP) to broiler feed on broiler growth, immune response to Newcastle disease virus (NDV) vaccination, and gastrointestinal tract bacteria. Materials and Methods: We determined the antimicrobial effects of ESE against Escherichia coli O157:H7 (EOH) and Lactobacillus acidophilus TISTR 2365 (L2365) using the agar well diffusion method. We used a complete randomized design to assign 120 1-day-old Arbor Acre chicks to six groups with four replicates of five broiler chickens over 42 days. The treatment groups were as follows: T1-basal diet (B) + NDV vaccination (positive control), T2-B (negative control), T3-B + 2 g SP per kg of feed, T4-B + 2 g SP per kg of feed + NDV, T5-B + 4 g SP per kg of feed, and T6-B + 4 g SP per kg of feed + NDV. Results: The minimum inhibitory concentrations of ESE on EOH and L2365 were 62.50 and 125.00 mg/mL, respectively. The body weight gain, average daily gain, and feed conversion ratio in the 4 g SP of T5 and T6 groups were significantly improved compared with the other groups (p &lt; 0.05). The immune organ (IO) and thymus gland weights in the T4 group were significantly greater than those observed in the positive and negative control groups (p &lt; 0.05). The IO weights of the bursae of Fabricius and spleen tended to be greater in the T4 group than in the other groups. T5 group broilers had the highest ratio of villus height to crypt depth. The humoral immunity titers against NDV vaccination were improved in the SP-supplemented groups compared with the non-supplemented groups (p &gt; 0.05). SP supplementation reduced the levels of coliform (p &lt; 0.05) and E. coli in the broiler intestine by adding 4 g of SP per kg of feed. However, L2365 was more tolerant to ESE in vitro and tended to increase in line with increased SP levels. Conclusion: ESE showed strong antimicrobial activity to reduce harmful bacteria, and SP supplementation may exhibit prebiotic effects to increase broiler chicken growth, immunity, and microbial balance. Keywords: broiler chickens, food security, immune stimulation, intestinal bacteria, intestinal morphology, shallot.

Comparative analysis of changes in immune cell in the chicken spleen across different ages using flow cytometry

BackgroundConcurrent emerging and reemerging avian infectious diseases cause multiple risk factors in poultry. A body amount studies attempted to understand pathogen-associated immunity in chickens. Recent research has made progress in identifying immune functions in chicken, there are still gaps in knowledge, especially regarding immune responses during infectious diseases. A deeper understanding in chicken immune system is critical for improving disease control strategies and vaccine development.ResultsThis study proposes analytical method for chicken splenocytes, enabling the tracking changes in T cells, monocytes, and B cells across three ages. Optimized lymphocyte-activating conditions were suggested using concanavalin A and chicken interleikin-2, which facilitate immune cell activation and proliferation. Next, splenocytes from embryonic day 18, day 5, and day 30 were compared using surface markers and flow cytometry analysis. We observed an increase in T cell subsets, including activated T cells (CD4+CD44+ and CD8+CD44+), and B cells, along with a reduced monocyte population after hatching. However, morphological changes and genetic expression of functional immune molecules were limited.ConclusionsThe present findings on chicken immune system development offer valuable insights into the avian immune system, including analytical methods and the phenotypic and functional changes in immune cells. Updated immune-boosting strategies during the early stages of life are crucial for developing preventive measures against major infectious diseases in the poultry industry.

Broadly cross-reactive immune responses in chickens immunized with chimeric virus-like particles of nodavirus displaying the M2e originated from avian and human influenza A viruses

Avian influenza A viruses (IAVs) pose a persistent threat to poultry industry worldwide, despite the presence of vaccines. Additionally, reverse-zoonosis transmission potentially introduces human-originated IAVs into poultry and complicates the efforts to control the spread of influenza. Current avian influenza vaccines are primarily based upon the rapidly mutating hemagglutinin (HA) and neuraminidase (NA) glycoproteins, which limit their efficacy against diverse strains of IAVs. Hence, the highly conserved ectodomains of matrix 2 protein (M2e) of IAVs are widely studied as alternatives to the HA and NA. However, the differences in the M2e amino acid sequences between avian and human IAVs generate antibodies that do not cross-react reciprocally with IAVs from other origins. To broaden and enhance the immunogenicity of M2e, we fused two copies each of the M2e derived from avian and human IAVs at the C-terminal end of the Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (NvC). Transmission electron microscopic and dynamic light scattering analyses revealed that the chimeric protein self-assembled into virus-like particles (VLPs). Immunization of chickens with the chimeric VLPs demonstrated a robust induction of broadly reactive immune responses against both the M2e of avian and human IAVs. Additionally, the chimeric VLPs elicited the production of cytotoxic T lymphocytes (CTL), macrophages, as well as a well-balanced Th1 and Th2 population, indicating their potential in activating cell-mediated immune responses in chickens. Furthermore, the chimeric VLPs triggered the production of both Th1- and Th2-cytokines, attesting their potential in mounting a robust and balanced immune response in avian species. This study demonstrated the potential of these chimeric VLPs in stimulating and broadening cross-reactive immune responses in chickens against both avian and human IAVs.

The influence of Quercetin on behavior, performance and splenic immunity in broiler chickens

Quercetin (QRT), a potent flavonoid, holds immense mechanistic potential in enhancing various aspects of broiler chicken physiological status. This comprehensive study explores the profound еffеcts of QRT on behavior, productive performance, and immune functions, unraveling the underlying mechanisms behind its efficacy. Four hundred, one-day-old Cobb 500 chicks were placed randomly into 4 supplementation groups (100 birds /group and five replicates) and provided diets enriched with varying concentrations of QRT (0, 200, 400, and 600 ppm) for six weeks. Visual scanning revealed significant (P > 0.05) improvements in feeding and body care behaviors, accompanied by reduced instances of idleness and walking in the QRT-supplemented groups. Moreover, QRT supplementation exerted a substantial (P > 0.05) positive influence on weight gain, feed intake and the final body weight of the broilers. In-depth evaluation of immune parameters, QRT supplementation elicited significant (P > 0.05) enhancements in immune functions, including improved spleen, thymus, and bursa indices, enhanced secretion of immunoglobulin M (IgM) and immunoglobulin A (IgA), and stimulated humoral immunity against sheep red blood cells (SRBCs). Furthermore, QRT displays potent antioxidant properties, as showed by diminished splenic malondialdehyde levels and augmented activity of antioxidative stress enzymes. Remarkably, QRT supplementation elicited dose-dependent upregulation (P < 0.001) of key immune-related genes, such as intеrlеukin-4 (IL-4), interferon-γ (INF-γ), Toll-like receptor 2 (TLR2), and tumor necrosis factor-α (TNF-α) in the splenic tissue. Collectively, these mechanistic insights underscore the profound impact of QRT as a functional feed additive, fostering enhanced behavior, performance, and immune function in broiler chickens, while delivering robust antioxidant fortification.

Different dietary branched-chain amino acid ratios, crude protein levels, and protein sources can affect the growth performance and meat yield in broilers

Balanced ratios of branched-chain amino acids (BCAAs) can enhance chicken growth, immunity, and muscle synthesis. However, these ratios can be affected by changes in crude protein (CP) levels or the substitution of protein sources, leading to BCAA antagonism. This, in turn, can have a negative impact on chicken growth. In Experiment 1, a total of 960 0-d-old male Cobb 500 broilers were divided into 6 treatments with 8 replicates. Three different BCAA ratios were used in High or Low CP diets as follows: 1) Low Leu group (Low level of leucine with increased valine and isoleucine levels), 2) Med Leu group, and 3) High Leu group (High level of leucine with reduced valine and isoleucine levels) for a total of 6 diets. In Experiment 2, a total of 640 0-d-old male Cobb 500 broilers were divided into 4 treatments with 8 replicates. The four diets had either High or Low CP and one of two protein sources with the same medium levels of BCAAs: 1) the soybean meal (SBM) group, which had SBM as the main protein source (protein bound AA), and 2) the wheat middlings with non-bound AAs (WM+AA) group (non-bound AA), which had additional non-bound AAs to replace SBM. The High Leu diet had a negative effect on overall growth performance, carcass weight, breast muscle weight, and body mineral composition compared to the Low Leu and Med Leu groups, particularly in the High CP diet (P < 0.05). The SBM group showed increased growth performance, breast muscle weight, expression levels of genes promoting muscle growth, and improved bone mineral composition compared to the WM+AA group, and the High CP group intensified the negative effect of the WM+AA diet (P < 0.05). In summary, balanced BCAA ratios and SBM-based diets have positive effects on chicken growth and muscle accretion, whereas excessive leucine and non-bound AA levels in the diets may negatively affect growth performance and meat yield in chickens.

Recombinant Marek’s disease virus type 1 provides full protection against H9N2 influenza A virus in chickens

The H9N2 subtype of the avian influenza virus (AIV) poses a significant threat to the poultry industry and human health. Recombinant vaccines are the preferred method of controlling H9N2 AIV, and Marek’s disease virus (MDV) is the ideal vector for recombinant vaccines. During this study, we constructed two recombinant MDV type 1 strains that carry the hemagglutinin (HA) gene of AIV to provide dual protection against both AIV and MDV. To assess the effects of different MDV insertion sites on the protective efficacy of H9N2 AIV, the HA gene of H9N2 AIV was inserted in UL41 and US2 of the MDV type 1 vector backbone to obtain recombinant viruses rMDV-UL41/HA and rMDV-US2/HA, respectively. An indirect immunofluorescence assay showed sustained expression of HA protein in both recombinant viruses. Additionally, the insertion of the HA gene in UL41 and US2 did not affect MDV replication in cell cultures. After immunization of specific pathogen-free chickens, although both the rMDV-UL41/HA and rMDV-US2/HA groups exhibited similar levels of hemagglutination inhibition antibody titers, only the rMDV-UL41/HA group provided complete protection against the H9N2 AIV challenge, and also offered complete protection against challenge with MDV. These results demonstrated that rMDV-UL41/HA could be used as a promising bivalent vaccine strain against both H9N2 avian influenza and Marek’s disease in chickens.

Effect of the competitive exclusion culture on the growth, blood parameters, leg health, and innate immunity of broiler chickens

Abstract The primary aim of poultry production is to obtain a high yield and quality end product. To reduce the risk of disease, many direct-fed microbial products have been developed. That appears to be an excellent tool for disease prevention. We evaluated the influence of the commercial, competitive exclusion (CE) product, Broilact®, on the growth rate, hematology, serum biochemistry, and innate immunity in male ROSS-308 chickens, randomly divided into two groups (Broilact® treatment and control) raised for 42 days. The birds’ body weight was determined at 1, 7, 14, 28, 35, and 42 days, and the blood samples were collected at days 22 and 42 of life. We observed lower mortality, better gait score, and higher final body weight in the Broilact® group. At day 22, birds from the treated group presented higher white blood cells counts (WBC) and T cytotoxic lymphocyte (CD8+) counts, higher total protein (TP) (fraction globulin and albumin), and lower triglyceride (TAG) and Ca2+ plasma concentrations. No differences were found in acute phase proteins (APPs). At day 42, only the K+ and Na+ concentrations were higher, while the IL-10 was lower in treated birds’ blood serum. Our results indicate that treatment with one dose of the Broilact® product at day one of life has a beneficial influence, which improves the chickens’ performance, leg health and some serum enzymes activity, maintains electrolyte homeostasis, and influences leukocyte count with the rise of T CD8+ subpopulations.

Effects of in ovo supplementation of selenium (Se) and zinc (zn) on hatchability and production performance of broiler chickens.

The current research was conducted to assess the effect of in ovo feeding (IOF) of selenium (Se) and zinc (Zn) on hatchability, production performance, liver, intestinal morphology, antioxidant levels and expression levels of immune-related genes in broiler chickens. A total of 400 fertilized eggs were equally divided into four groups: control (non-injected), sham (in ovo injection of 0.75% NaCl), Se (@ 1.5µg/egg in ovo injection) and Zn (500µg/egg in ovo injection) groups respectively. On the seventeenth day of incubation, treatment solutions were administered into amniotic fluid of fertilized eggs. The results revealed that Se and Zn supplementation significantly (P < 0.05) enhanced hatchability, post-hatch growth, organ development, and liver antioxidant capability. Histopathological examination revealed a typical hepatocyte morphology, well-arranged cells, and a significant (P < 0.05) decrease in apoptosis in both selenium and zinc groups. Additionally, selenium and zinc produced auspicious effects on intestinal epithelium and villi surface area. Interestingly, our results revealed that IOF of Se and Zn modulated the expression of immune-related genes in comparison to the control and sham groups. Conclusively, IOF of Se and Zn augmented health and productivity by enhancing the cellular immunity in the broiler chickens, thus IOF can be utilized as an effective strategy to promote health and immunity in broiler chickens.

Improved cellular immune response induced by intranasal boost immunization with chitosan coated DNA vaccine against H9N2 influenza virus challenge

The H9N2 avian influenza virus (AIV) is spreading worldwide. Presence of H9N2 virus tends to increase the chances of infection with other pathogens which can lead to more serious economic losses. In a previous study, a regulated delayed lysis Salmonella vector was used to deliver a DNA vaccine named pYL233 encoding M1 protein, mosaic HA protein and chicken GM-CSF adjuvant. To further increase its efficiency, chitosan as a natural adjuvant was applied in this study. The purified plasmid pYL233 was coated with chitosan to form a DNA containing nanoparticles (named CS233) by ionic gel method and immunized by intranasal boost immunization in birds primed by oral administration with Salmonella strain. The CS233 DNA nanoparticle has a particle size of about 150 nm, with an encapsulation efficiency of 93.2 ± 0.12 % which protected the DNA plasmid from DNase I digestion and could be stable for a period of time at 37°. After intranasal boost immunization, the CS233 immunized chickens elicited higher antibody response, elevated CD4+ T cells and CD8+ T cells activation and increased T-lymphocyte proliferation, as well as increased productions of IL-4 and IFN-γ. After challenge, chickens immunized with CS233 resulted in the lowest levels of pulmonary virus titer and viral shedding as compared to the other challenge groups. The results showed that the combination of intranasal immunization with chitosan-coated DNA vaccine and oral immunization with regulatory delayed lytic Salmonella strain could enhance the immune response and able to provide protection against H9N2 challenge.

Chicken Immunization followed by RNA Extraction and cDNA Synthesis for Antibody Library Preparation.

Effective isolation of specific antibodies from immunological repertoires requires the generation of a diverse library against a specific antigen of interest, as well as efficient selection procedures, such as bio-panning and phage ELISA. Key to this is the generation of a good immune response in the host, followed by preparation of high-quality RNA and cDNA from which a library can be constructed by the amplification and cloning of immunoglobulin heavy and light chain genes. The first step in the construction of such an "immune library" is a successful course of immunization. Detection of a strong serum antibody titer will theoretically then result in a pool of extracted RNA that is enriched for transcripts of genes encoding the antibody of interest. Chicken antibodies have been widely used for research and diagnostic purposes, largely because of both their cross-reactivity to epitopes shared by humans, mice, primates, and other mammals, and their simple characteristics, with chickens featuring single functional copies of V H /J H and V λ /J λ gene pairs. In chickens, antibodies against an antigen of interest can be detected in the serum as soon as 5-7 d after immunization. Once the antibody titer reaches an appropriate level in the serum, the spleen, bursa of Fabricius, and bone marrow are then harvested, and antibody libraries can be prepared from extracted RNA. Here, we describe a protocol for chicken immunization with an antigen of interest, followed by RNA extraction from the relevant tissues and cDNA synthesis, which users can use for antibody library construction.

Induction of trained immunity in broiler chickens following delivery of oligodeoxynucleotide containing CpG motifs to protect against Escherichia coli septicemia

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) can promote antimicrobial immunity in chickens by enriching immune compartments and activating immune cells. Innate memory, or trained immunity, has been demonstrated in humans and mice, featuring the absence of specificity to the initial stimulus and subsequently cross-protection against pathogens. We hypothesize that CpG-ODN can induce trained immunity in chickens. We delivered single or multiple administrations of CpG-ODN to birds and mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis of peripheral blood mononuclear cells were quantified using Seahorse XFp. Next, chickens were administered with CpG-ODN twice at 1 and 4 day of age and challenged with Escherichia coli at 27 days of age. The CpG-ODN administered groups had significantly higher mitochondrial OXPHOS until 21 days of age while cellular glycolysis gradually declined by 14 days of age. The group administered with CpG-ODN twice at 1 and 4 days of age had significantly higher survival, lower clinical score and bacterial load following challenge with E. coli at 27 d of age. This study demonstrated the induction of trained immunity in broiler chickens following administration of CpG-ODN twice during the first 4 days of age to protect birds against E. coli septicemia at 27 days of age.

In vitro and in vivo evaluation of antibacterial activity and mechanism of luteolin from Humulus scandens against Escherichia coli from chicken

Resistance of Escherichia coli (E.coli) to antibiotics has steadily increased over time; hence, there is an urgent need to develop safer alternatives to antibiotics. The present study aimed to evaluate the effect of luteolin (Lut) on E. coli from chicken. The bioactive compound Lut from Humulus scandens was selected by network pharmacology and molecular docking analyses. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM) were used to observe the effects of Lut on the morphology and structure of E. coli cells. The data-independent acquisition (DIA) method was used to analyze protein expression level of E. coli before and after Lut treatment. The in vivo evaluation of the antibacterial, anti-inflammatory, and oxidative effects of Lut on E.coli was conducted using E.coli isolated strains infected the SPF chicken model. The network pharmacology analysis revealed 19 distinctive bioactive compounds such as Lut and β-sitosterol in H. scandens; furthermore, 30 core targets were selected from H. scandens. The KEGG enrichment analysis showed that the PI3K-Akt, TNF, MAPK, IL-17, JAK-STAT, and HIF-1 pathways were related from H. scandens. Based on the results of the network pharmacology analysis, Lut was subjected to screening by molecular docking analysis to determine its antibacterial effect on E. coli and the associated mechanism of action. The minimum inhibitory concentration (MIC) of Lut against E. coli standard strains was 500 µg/mL. SEM, TEM, and CLSM results indicated that Lut damaged the cell wall and cell membrane of E. coli strains and destroyed the cell structure, leading to cell death.The expression level of membrane structure, Phenylalanine metabolism and some other metabolic pathways in E.coli changed after treatment with Lut (P < 0.05). In vivo experiments in the SPF chicken model showed that Lut treatment alleviated the decline in the growth performance of chickens (P < 0.05), prevented pathological changes in the correspond ding organs and suppressed the inflammatory response induced by E. coli infection (P < 0.05), improved the immunity and antioxidant capacity of chickens (P < 0.05), and protected them against infection with E. coli strains. To summarize, Lut from H. scandens can inhibit E. coli growth by damaging the cell membrane structureand affecting the expression level of some metabolic proteins. In vivo experiments also showed that Lut can significantly reduce the damage caused by E. coli isolates on SPF chickens, improve their antioxidant capacity and immunity, and reduce inflammatory responses following E. coli infection.

Effect of orally administered B. subtilis-cNK-2 on growth performance, immunity, gut health, and gut microbiome in chickens infected with Eimeria acervulina and its potential as an alternative to antibiotics

This study investigated the best oral delivery strategy (gavage or feed) for the B. subtilis expressing the chicken anti-microbial peptide cNK-2 (B. subtilis-cNK-2) in comparison to monensin, in chickens challenged with Eimeria acervulina (E. acervulina). A total of 120 broiler chickens were randomly allocated into 5 treatment groups in a completely randomized design: 1) uninfected chickens fed with basal diet (NC), 2) E. acervulina-infected chickens fed a basal diet (PC), 3) E. acervulina-infected chickens fed a basal diet supplemented with 90 mg monensin/kg feed (MO), 4) E. acervulina-infected chickens fed a basal diet and orally gavaged with B. subtilis-cNK-2 at 1 × 1010 cfu/d (CNK-O), and 5) E. acervulina-infected chickens fed a basal diet mixed with B. subtilis-cNK-2 at 1 × 1010 cfu/kg feed (CNK-F). The challenge consisted of 5,000 sporulated E. acervulina oocysts through oral gavage on d 15. Body weights were measured on d 7, 14, 21, and 23. Duodenal tissue and digesta samples were collected at 6 d postinfection (dpi) to assess the gut integrity, oxidative stress, mucosal immunity, and the gut microbiome. Fecal samples were collected from 6 to 8 dpi to enumerate the oocyst shedding. Chickens in the CNK-O group showed improved (P < 0.05) growth performance, gut integrity, and mucosal immunity compared to PC, comparable to chickens in the MO group. Chickens in the MO, CNK-F, and CNK-O treatment groups all showed lower (P < 0.05) oocyst shedding compared to PC chickens. Moreover, distinct cytokine profile, oxidative stress measures, tight junction proteins, and shifts in the gut microbiome with associated functional changes were observed in all challenge groups. In conclusion, we showed that the oral administration of B. subtilis-cNK-2 improved growth performance, enhanced local protective immunity, and reduced fecal oocyst shedding in broiler chickens infected with E. acervulina, demonstrating potential use of B. subtilis-cNK-2 as an alternative to antibiotics to protect chickens against coccidiosis.