The effect of gut microbiota-derived carnosine on mucosal integrity and immunity in broiler chickens challenged with Eimeria maxima.

In the first study, an in vitro culture system was developed to investigate the effects of carnosine on macrophage proinflammatory cytokine response using an established chicken macrophage cell line (CMC), gut integrity using a chicken intestinal epithelial cell line (IEC), muscle differentiation in quail muscle cells (QMCs) and primary chicken embryonic muscle cells (PMCs), and direct anti-parasitic effect against Eimeria maxima sporozoites. Cells to be tested were seeded in 24-well plates and treated with carnosine at 4 different concentrations (0.1, 1.0, and 10.0 µg). After 18 h of incubation, cells were harvested to measure gene expression of proinflammatory cytokines in CMC, tight junction (TJ) proteins in IECs, and muscle cell growth markers in QMCs and PMCs. In vivo trials were conducted to investigate the effect of dietary carnosine on disease parameters in broiler chickens challenged with E. maxima. One hundred and twenty male broiler chickens (0-day-old) were allocated into 4 treatment groups: 1) basal diet without infection (NC), 2) basal diet with E. maxima infection (PC), 3) carnosine at 10.0 mg/kg feed with PC (HCS), and 4) carnosine at 1.0 mg/kg feed with PC (LCS). All groups except NC were orally infected with E. maxima on d 14. Jejunal samples were collected for lesion scoring and jejunum gut tissues were used for transcriptomic analysis of cytokines and TJ proteins. In vitro, carnosine treatment significantly decreased IL-1ß gene expression in CMC following LPS stimulation. In vivo feeding studies showed that dietary carnosine increased BW and ADG of chickens in E. maxima-infected groups and reduced the jejunal lesion score and fecal oocyst shedding in HCS group. Jejunal IL-1ß, IL-8, and IFN-γ expression were suppressed in the HCS group compared to PC. The expression levels of claudin-1 and occludin in IECs were also increased in HCS following carnosine treatment. In conclusion, these findings highlight the beneficial effects of dietary carnosine supplementation on intestinal immune responses and gut barrier function in broiler chickens exposed to E. maxima infection.

Differential detection of chicken heterodimeric cytokines, interleukin 12 and 23 using their subunit-specific mouse monoclonal antibodies.

Interleukin-23 (IL-23) is a recently identified member of the IL-12 family of heterodimeric cytokines that play a critical role in regulating T helper cell function. IL-12 and IL-23 share a common p40 subunit, but differ in their p35 and p19 subunits, respectively. This difference in subunit composition results in distinct signaling pathways and biological functions for IL-12 and IL-23. Here, we report the functional characterization and immunomodulatory properties of chicken IL-12 and IL-23 using the panels of newly developed mouse anti-IL-12p40, IL-12p35-α and IL-23p19 monoclonal antibodies (mAbs). Western blot and indirect ELISA analysis demonstrated that the anti-chicken IL-12p40 mAbs (chIL-12p40; #10G10F4 and #10D8G2) bound to both recombinant proteins (IL-12 and IL-23), the anti-chicken IL-12p35 mAb (chIL-12p35; #2F1) specifically recognized recombinant IL-12, and the anti-chicken IL-23p19 mAb (chIL-23p19; #15A3) exhibited specificity for recombinant IL-23, without any cross-reactivity. Two ELISAs detecting specific chicken IL-12 (#10G10F4 and #2F1) or IL-23 (#10D8G2 and #15A3) were developed using newly developed mAb combinations, #10G10F4/ #2F1 and #10D8G2/#15A3 for IL-12 and IL-23, respectively, identified through a pairing assay. The levels of IL-12 and IL-23 in Resiquimod-848 stimulated-HD11 chicken macrophage cells were monitored over time using antigen-capture sandwich ELISA developed in this study. Furthermore, the levels of chicken IL-12 and IL-23 in the circulation of Eimeria maxima (E. maxima) and Eimeria tenella (E. tenella)-infected chickens were determined. Notably, the anti-chIL-12p40 mAbs (#10G10F4 and #10D8G2) neutralized the function of both chIL-12 and chIL-23 proteins, which share the p40 subunit, while the anti-chIL-23p19 mAb (#15A3) specifically neutralized chIL-23 protein in HD11 cells in vitro. The anti-chIL-12p35 mAb (#2F1), which is specific to the p35 subunit of IL-12, showed a partial neutralizing effect on chIL-12 protein. Collectively, our study validates the specificity and significance of 2 newly developed antigen-capture immunoassays for chIL-12 and chIL-23 which will expand our understanding of the functional characteristics of IL-12 and IL-23 and their association in normal and diseased chickens. These mAbs for each subunit, anti-chIL-12p35, anti-chIL-12p40 and anti-chIL-23p19, will serve as valuable immune reagents to elucidate host immune responses against disease pathogenesis in both fundamental and applied studies of avian species.

Host-mediated beneficial effects of phytochemicals for prevention of avian coccidiosis.

Both in vitro and in vivo studies were conducted to evaluate the beneficial effects of green tea extract (GT), cinnamon oil (CO), and pomegranate extract (PO) on avian coccidiosis. In experiment (EXP) 1, an in vitro culture system was used to investigate the individual effects of GT, CO, and PO on the proinflammatory cytokine response and integrity of tight junction (TJ) in chicken intestinal epithelial cells (IEC), on the differentiation of quail muscle cells and primary chicken embryonic muscle cells, and anticoccidial and antibacterial activities against Eimeria tenella sporozoites and Clostridium perfringens bacteria, respectively. In EXP 2 and 3, in vivo trials were carried out to study the dose-dependent effect of blended phytochemicals (GT, CO, PO) on coccidiosis in broiler chickens infected with E. maxima. For EXP 2, one hundred male broiler chickens (0-day-old) were allocated into the following five treatment groups: Control group for non-infected chickens (NC), Basal diet group for E. maxima-infected chickens (PC), PC group supplemented with phytochemicals at 50 (Phy 50), 100 (Phy 100), and 200 (Phy 200) mg/kg feed diets for E. maxima-infected chickens. For EXP 3, one hundred twenty male broiler chickens (0-day-old) were allocated into the following six treatment groups: NC, PC, PC supplemented with phytochemicals at 10 (Phy 10), 20 (Phy 20), 30 (Phy 30), and 100 (Phy 100) mg/kg feed for E. maxima-infected chickens. Body weights (BW) were measured on days 0, 7, 14, 20, and 22, and jejunum samples were used to measure cytokine, TJ protein, and antioxidant enzyme responses at 8 days post-infection (dpi). Fecal samples for oocyst enumeration were collected from 6 to 8 dpi. In vitro, CO and PO reduced LPS-induced IL-1ß and IL-8 in IEC, respectively, and GT enhanced the gene expression of occludin in IEC. PO at 1.0 and 5.0 mg/mL exerted antimicrobial effect against E. tenella sporozoites and C. perfringens bacteria, respectively. In vivo, chickens fed a diet supplemented with phytochemicals showed enhanced BW, reduced oocyst shedding, and decreased proinflammatory cytokines following E. maxima challenge. In conclusion, the combination of GT, CO, and PO in the diet of broiler chickens infected with E. maxima induced enhanced host disease resistance including innate immunity and gut health, which contributed to improved growth and reduced disease responses. These findings provide scientific support for the development of a novel phytogenic feed additive formula that enhances the growth and intestinal health of broiler chickens infected with coccidiosis.

Development of a new immunodiagnostic tool for poultry coccidiosis using an antigen-capture sandwich assay based on monoclonal antibodies detecting an immunodominant antigen of Eimeria.

This study was conducted to develop an antigen-capture ELISA that detects an immunodominant antigen of Eimeria, 3-1E which is present in all Eimeria species, using a set of 3-1E-specific mouse monoclonal antibodies (mAbs). Highly sensitive 3-1E-specific antigen-capture ELISA was established using compatible mAb pairs (#318 and #320) selected from 6 mAbs (#312, #317, #318, #319, #320, and #323) with high binding activity against recombinant 3-1E protein. These anti-3-1E mAbs specifically recognized E. tenella sporozoites and a higher level of 3-1E was detected in the lysate of sporozoites than in sporocysts. Immunofluorescence assay (IFA) using 2 mAbs (#318 and #320) showed specific staining around the membrane of E. tenella sporozoites. In order to measure the changes in the 3-1E level during in coccidiosis, serum, feces, jejunal, and cecal contents were individually collected daily for 7-days postinfection (dpi) with E. maxima and E. tenella. The new ELISA was sensitive and specific for 3-1E detection in all samples collected daily from E. maxima- and E. tenella-infected chickens for a week, and the detection sensitivity ranges were 2 to 5 ng/mL and 1 to 5 ng/mL in serum, 4 to 25 ng/mL and 4 to 30 ng/mL in feces, 1 to 3 ng/mL and 1 to 10 ng/mL in cecal contents, and 3 to 65 ng/mL and 4 to 22 ng/mL in jejunal contents. Following coccidiosis, the overall 3-1E levels started to increase from 4 dpi, and the highest production was shown on 5 dpi. Among the samples collected from Eimeria-infected chickens, the highest detection level was found in the jejunal contents of E. maxima-infected chickens. Furthermore, the level of IFN-γ in serum was significantly (P < 0.05) increased from 3 dpi and peaked on 5 dpi post E. maxima infection. Post E. tenella infection, the level of IFN-γ in serum gradually (P < 0.05) increased from 2 to 5 dpi and plateaued at 7 dpi. The level of TNF-α in serum was rapidly (P < 0.05) increased from 4 dpi and those levels were kept until 7 dpi post both Eimeria infections (E. maxima and E. tenella). More importantly, the daily changes in the 3-1E levels in different samples from E. maxima- and E. tenella-infected chickens were effectively monitored with this new antigen-capture ELISA. Therefore, this new immunoassay is a sensitive diagnostic tool to monitor coccidiosis in a large field population in the commercial poultry farms before clinical symptoms develop using serum, feces, and gut samples during the entire period of infection cycle starting from 1 d after infection.

Characterization of Collagen Binding Activity of Clostridium perfringens Strains Isolated from Broiler Chickens.

Clostridium perfringens is the etiological agent for necrotic enteritis (NE) in broiler chickens, which causes a substantial economic loss of an estimated USD 6 billion annually in the global poultry industry. Collagen adhesion is involved in the NE pathogenesis in poultry. In this study, the binding capabilities of chicken C. perfringens isolates of various genetic backgrounds (netB-tpeL-, netB+tpeL-, netB+tpeL+) to collagen types I-V and gelatin were examined, and the putative adhesin protein cnaA gene was investigated at the genomic level. In total, 28 C. perfringens strains from healthy and NE-inflicted sick chickens were examined. The results on collagen adhesin-encoding gene cnaA by the quantitative-PCR results indicated that netB-tpeL- isolates had much lower copies of the detectable cnaA gene than netB+ isolates (10 netB+tpeL- isolates, 5 netB+tpeL+ isolates). Most of the virulent C. perfringens isolates demonstrated collagen-binding abilities to types I-II and IV-V, while some strains showed weak or no binding to collagen type III and gelatin. However, the netB+tpeL+ isolates showed significantly higher binding capabilities to collagen III than netB-tpeL- and netB+tpeL- isolates. The data in this study suggest that the collagen-binding capability of clinical C. perfringens isolates correlates well with their NE pathogenicity levels, especially for C. perfringens isolates carrying genes encoding crucial virulence factors and virulence-associated factors such as netB, cnaA, and tpeL. These results indicate that the presence of the cnaA gene may be correlated with C. perfringens virulence (particularly for netB+ isolates).

Effect of Dietary Organic Selenium on Growth Performance, Gut Health, and Coccidiosis Response in Broiler Chickens.

A total of 252 one-day-old Ross broilers were randomly allocated to one of six treatments in a 2 × 3 factorial arrangement with respective Eimeria challenges (non-infection and infection) and three different selenium (Se) diets. Dietary treatments were as follows: (1) Se un-supplemented control (CON), (2) inorganic Se treatment (SS; 0.3 mg/kg as sodium selenite), and (3) organic Se treatment (SY; 0.3 mg/kg as selenized yeast). Six replicate cages were allocated per treatment. Chickens in the respective Eimeria infection groups were infected with an E. acervulina, E. tenella, and E. maxima oocyst mixture (15,000 oocysts/chicken) on day 16. Growth performance was measured on days 16, 22, and 24. On day 22, intestinal samples were collected from randomly selected chickens to evaluate gut lesion scores, antioxidant enzymes, and tight junction gene expression. Blood, breast, and liver samples were collected to analyze the Se concentrations on day 24. Dietary SY supplementation improved (p < 0.05) the growth performance of the chickens regardless of the Eimeria challenge. Moreover, independent of Eimeria infection, Se supplementation elevated (p < 0.05) the heme oxygenase 1 (HMOX-1) expression in jejunal mucosa at 6 days post-infection (dpi). Duodenal junctional adhesion molecule 2 (JAM-2) expression and jejunal occludin (OCLN) were elevated (p < 0.05) with dietary SY supplementation at 6 dpi. Among Se sources, broiler chickens fed with the SY diet showed higher (p < 0.05) Se concentrations in breast muscle and serum on 8 dpi. These results confirmed the beneficial effects of dietary Se and the efficiency of organic Se compared with inorganic Se for growth improvement and muscle Se enrichment in broiler chickens regardless of coccidiosis infection.

Bacillus subtilis Expressing Chicken NK-2 Peptide Enhances the Efficacy of EF-1α Vaccination in Eimeria maxima-Challenged Broiler Chickens.

This study was conducted to investigate the synergistic effects of orally delivered B. subtilis-cNK-2 on vaccination with rEF-1α against E. maxima infection in broiler chickens. Chickens were assigned into the following five groups: control (CON, no Eimeria infection), non-immunized control (NC, PBS), component 1 (COM1, rEF-1α only), component 2 (COM2, rEF-1α plus B. subtilis empty vector), and component 3 (COM3, rEF-1α plus B. subtilis-NK-2). The first immunization was administered intramuscularly on day 4, and the second immunization was given one week later with the same concentration of components as the primary immunization. The immunization of B. subtilis spores (COM2 and COM3) was performed by oral administration given for 5 consecutive days a week later than the second immunization. On day 19, all the chickens except the CON group were orally challenged with E. maxima oocysts (1.0 × 104/chicken). The results of the in vivo vaccination showed that all the chickens immunized with rEF-1α (COM1, COM2, and COM3) produced higher (p < 0.05) serum antibodies against EF-1α on 12 days post-E. maxima infection (dpi). The COM3 group showed a significantly (p < 0.05) higher average body weight gain (BWG) on 0-6, 6-9, and 0-12 dpi compared to those of the non-immunized chickens (NC). Immunization with rEF-1α alone (COM1) reduced the gut lesion score on 6 dpi and the fecal oocyst shedding on 9 dpi, whereas co-administration with B. subtilis spores (COM2 or COM3) led to further reduction in the lesion score. E. maxima infection increased the expression levels of IFN-γ and IL-17ß in the jejunum, but these expressions were downregulated in the rEF-1α immunized (COM1) group and in the groups immunized with rEF-1α and orally treated with B. subtilis spores (COM2 or COM3) at 4 dpi. A reduced gene expression of occludin in the jejunum of the E. maxima-infected chickens on 4 dpi was upregulated following the immunization with COM2. Collectively, rEF-1α vaccination induced significant protection against E. maxima infection in the broiler chickens, and the efficacy of rEF-1α vaccination was further enhanced by co-administration with orally delivered B. subtilis spores expressing cNK-2.

Oral administration of chicken NK-lysin or recombinant chicken IL-7 improves vaccine efficacy of Eimeria tenella elongation factor-1α (EF-1α) against coccidiosis in commercial broiler chickens.

The synergistic effects of orally-delivered chicken NK-lysin peptide 2 (cNK-2) or recombinant chicken IL-7 (rchIL-7) on vaccination with recombinant Eimeria elongation factor-1α (rEF-1α) against Eimeria maxima (E. maxima) infection was investigated in broiler chickens. Chickens were divided into six groups: control (CON, no Eimeria infection), non-immunized control (NC, PBS), Vaccination 1 (VAC 1, rEF-1α plus cNK-2), Vaccination 2 (VAC 2, rchIL-7 plus cNK-2), Vaccination 3 (VAC 3, rEF-1α/rchIL-7 plus cNK-2), and Vaccination 4 (VAC 4, rEF-1α/rchIL-7 plus cNK-2). All groups, except the CON and NC, were orally treated with cNK-2 for 5 days. The first immunization, except for the VAC 4 group, was performed intramuscularly on day 4, and the second immunization was given with the same concentration of components as the primary immunization one week later. The immunization of the VAC 4 group was carried out by an oral inoculation on the same days. On day 19, all chickens except the CON group, were orally challenged with E. maxima (1.0 × 104 oocysts/chicken). The in vivo vaccination results showed that the VAC 1 and VAC 3 groups produced high (p < 0.05) levels of serum antibody titers to rEF-1α, and the VAC 3 showed enhanced (p < 0.05) levels of serum IL-7. Furthermore, the VAC 3 group showed significantly (p < 0.01) greater body weight gains at 6- and 9-days post-E. maxima infection (dpi) with reduced oocyst shedding at 6 dpi. The average jejunal lesion score of the NC group was 2.5 whereas the VAC 1 group showed a significantly (p < 0.05) lower lesion scores at 6 dpi. E. maxima infection significantly (P < 0.05) up-regulated the expression levels of cytokines (IL-6, IL-10 and IFN-γ) in the jejunum at 4 dpi, but those expressions were down-regulated in VAC 1 or VAC 3 groups. Moreover, the gene expression levels of Jam 2 and Occludin, were significantly (P < 0.05) decreased following E. maxima infection in jejunum at 4 dpi (NC), but their expressions were increased in the VAC 3 group. Collectively, these results showed that the efficacy of rEF-1α vaccination was significantly enhanced when rEF-1α vaccine co-immunized with chIL-7 or cNK-2.

Orally delivered Bacillus subtilis expressing chicken NK-2 peptide stabilizes gut microbiota and enhances intestinal health and local immunity in coccidiosis-infected broiler chickens.

We recently reported a stable Bacillus subtilis-carrying chicken NK-lysin peptide (B. subtilis-cNK-2) as an effective oral delivery system of an antimicrobial peptide to the gut with therapeutic effect against Eimeria parasites in broiler chickens. To further investigate the effects of a higher dose of an oral B. subtilis-cNK-2 treatment on coccidiosis, intestinal health, and gut microbiota composition, 100 (14-day-old) broiler chickens were allocated into 4 treatment groups in a randomized design: 1) uninfected control (CON), 2) infected control without B. subtilis (NC), 3) B. subtilis with empty vector (EV), and 4) B. subtilis with cNK-2 (NK). All chickens, except the CON group, were infected with 5,000 sporulated Eimeria acervulina (E. acervulina) oocysts on d 15. Chickens given B. subtilis (EV and NK) were orally gavaged (1 × 1012 cfu/mL) daily from d 14 to 18. Growth performances were measured on d 6, 9, and 13 postinfection (dpi). Spleen and duodenal samples were collected on 6 dpi to assess the gut microbiota, and gene expressions of gut integrity and local inflammation makers. Fecal samples were collected from 6 to 9 dpi to enumerate oocyst shedding. Blood samples were collected on 13 dpi to measure the serum 3-1E antibody levels. Chickens in the NK group showed significantly improved (P < 0.05) growth performance, gut integrity, reduced fecal oocyst shedding and mucosal immunity compared to NC. Interestingly, there was a distinct shift in the gut microbiota profile in the NK group compared to that of NC and EV chickens. Upon challenge with E. acervulina, the percentage of Firmicutes was reduced and that of Cyanobacteria increased. In NK chickens, however, the ratio between Firmicutes and Cyanobacteria was not affected and was similar to that of CON chickens. Taken together, NK treatment restored dysbiosis incurred by E. acervulina infection and showed the general protective effects of orally delivered B. subtilis-cNK-2 on coccidiosis infection. This includes reduction of fecal oocyst shedding, enhancement of local protective immunity, and maintenance of gut microbiota homeostasis in broiler chickens.

Evolution of developmental and comparative immunology in poultry: The regulators and the regulated.

Avian has a unique immune system that evolved in response to environmental pressures in all aspects of innate and adaptive immune responses, including localized and circulating lymphocytes, diversity of immunoglobulin repertoire, and various cytokines and chemokines. All of these attributes make birds an indispensable vertebrate model for studying the fundamental immunological concepts and comparative immunology. However, research on the immune system in birds lags far behind that of humans, mice, and other agricultural animal species, and limited immune tools have hindered the adequate application of birds as disease models for mammalian systems. An in-depth understanding of the avian immune system relies on the detailed studies of various regulated and regulatory mediators, such as cell surface antigens, cytokines, and chemokines. Here, we review current knowledge centered on the roles of avian cell surface antigens, cytokines, chemokines, and beyond. Moreover, we provide an update on recent progress in this rapidly developing field of study with respect to the availability of immune reagents that will facilitate the study of regulatory and regulated components of poultry immunity. The new information on avian immunity and available immune tools will benefit avian researchers and evolutionary biologists in conducting fundamental and applied research.

South Korea's Medical Tourism Competitiveness and Future Strategy: Focused on Cancer Treatment: A Review.

Background: Medical tourism is a leading industry of South Korea. Since the transition from the latest health pandemic to endemic, foreign patients have been on a rapid increase. The Korean government regards medical tourism as a major national industry and pursues medical, legal, and policy strategies with the goal of attracting 700,000 foreign patients by 2027. Methods: Examining objective data and statistics as well as academic articles, we investigated the strengths and advantages, which might persuade foreign medical tourists to visit South Korea by performing a comparative analysis with high-income or medically advanced countries among Organization for Economic Co-operation and Development (OECD) and other competing Asian countries. Results: Cancer is one of the leading causes of death in the world. Compared with other advanced countries and Asian competitors in cancer treatment, South Korea shows excellent result of major cancers treatments, accessibility to hospitals and medical professionals, and competitiveness in terms of surgery and treatment costs. Conclusion: South Korea has excellent competitiveness both in price and in cancer treatment with the outstanding results of cancer mortality rates and 5-year survival rates. The achievement of Korea's cancer treatments is due to accurate diagnosis with the advantage of PET-CT, 64-sllice CT, 3.0 TESLA MRI, etc., and the use of cutting-edge equipment, such as CyberKnife and Da Vince robotic surgery. Besides, the Korean central and local governments actively support for the promotion of Korea's medical tourism industry, such as issuance of visas for foreigners, the hospital certification system, and medical tourism convergence cluster project.

Co-administration of chicken IL-7 or NK-lysin peptide 2 enhances the efficacy of Eimeria elongation factor-1α vaccination against Eimeria maxima infection in broiler chickens.

This study was conducted to develop a recombinant Eimeria elongation factor-1α (EF-1α)-vaccination strategy against Eimeria maxima (E. maxima) infection by co-administering with chicken IL-7 (chIL-7) or chicken NK-lysin peptide 2 (cNK-2) in commercial broiler chickens. Chickens were divided into the following 5 groups: control (CON, no Eimeria infection), nonimmunized control (NC, PBS plus Montanide ISA 78 VG), Vaccination 1 (VAC1, 100 µg of recombinant EF-1α plus Montanide ISA 78 VG), Vaccination 2 (VAC2, VAC1 plus 1 µg of chIL-7), and Vaccination 3 (VAC3, VAC2 plus 5 µg of cNK-2 peptide). The first immunization except the cNK-2 injection was performed intramuscularly on day 4, and the secondary immunization was given with the same concentration of components as the primary immunization 1 wk later. All chickens except the CON group were orally inoculated with freshly prepared E. maxima (1.0 × 104 oocysts per chicken) oocysts on Day 19. The results of the in vivo vaccination trial showed that chickens of all groups immunized with recombinant EF-1α antigen (VAC1, VAC2, and VAC3) showed higher serum antibody levels to EF-1α, and co-injection with chIL-7 further increased the serum IL-7 level in the VAC2 and VAC3 groups. Chickens in the VAC2 group showed significantly (P < 0.01) higher body weight gains at 6 and 9 d post-E. maxima challenge infection (dpi) with reduced gut lesions in the jejunum at 6 dpi. The VAC3 group showed reduced fecal oocyst shedding compared to the nonimmunized and infected chickens (NC). At 4 dpi, E. maxima infection significantly (P < 0.05) up-regulated the expression levels of proinflammatory cytokines (IL-ß and IL-17F) and type Ι cytokines (IFN-γ and IL-10) in the jejunum (NC), but the expression of these cytokines was significantly (P < 0.05) down-regulated in the VAC1, VAC2, and VAC3 groups. Furthermore, E. maxima challenge infection significantly (P < 0.05) down-regulated the expressions of jejunal tight junction (TJ) proteins (Jam2 and Occludin) at 4 dpi, but their expression was up-regulated in the VAC2 and VAC3 groups. Collectively, these results show the protective effects of the EF-1α recombinant vaccine, which can be further enhanced by co-injection with chIL-7 or cNK-2 peptide against E. maxima infection.

Immunization with Pooled Antigens for Clostridium perfringens Conferred Partial Protection against Experimental Necrotic Enteritis in Broiler Chickens.

Necrotic enteritis (NE) is a multifactorial and important enteric infectious disease etiologically caused by pathogenic C. perfringens infection, accounting for the estimated loss of around USD 6 billion in the global poultry industry. The increasing incidence of NE was found to be associated with the voluntary reduction or withdrawal of antibiotic growth promoters from animal feed during recent years. Therefore, the development of effective vaccines specific to NE assumes a priority for the poultry industry. This study aimed to identify the potential C. perfringens proteins as vaccine targets for NE. Three recombinant C. perfringens proteins targeting five antigens were prepared: two chimeric proteins (alpha-toxin and NetB, fructose-1,6-bisphosphate aldolase (FBA) and a zinc metalloprotease (Zm)), and one single collagen adhesion protein (Cna). Their protection efficacies were evaluated with a potent challenge model of Eimeria maxima/C. perfringens dual infections using a netB+tpeL+ C. perfringens strain. Young chicks were immunized twice subcutaneously with adjuvanted C. perfringens proteins on Days 4 and 15. At six days after the second immunization, the chickens immunized with Cna, FBA, and Zm antigens, and alpha-toxin had much higher serum antibody titers than unvaccinated controls prior to the challenge. Following the challenge, the pooled antigen-immunized group demonstrated no mortality and the least lesion scores against virulent challenge. The results indicate that the immunization with multicomponent antigens, including C. perfringens housekeeping protein Cna, may confer partial protection.

Role of Physiology, Immunity, Microbiota, and Infectious Diseases in the Gut Health of Poultry.

"Gut health" refers to the physical state and physiological function of the gastrointestinal tract and in the livestock system; this topic is often focused on the complex interacting components of the intestinal system that influence animal growth performance and host-microbial homeostasis. Regardless, there is an increasing need to better understand the complexity of the intestinal system and the various factors that influence gut health, since the intestine is the largest immune and neuroendocrine organ that interacts with the most complex microbiome population. As we face the post-antibiotic growth promoters (AGP) era in many countries of the world, livestock need more options to deal with food security, food safety, and antibiotic resilience to maintain agricultural sustainability to feed the increasing human population. Furthermore, developing novel antibiotic alternative strategies needs a comprehensive understanding of how this complex system maintains homeostasis as we face unpredictable changes in external factors like antibiotic-resistant microbes, farming practices, climate changes, and consumers' preferences for food. In this review, we attempt to assemble and summarize all the relevant information on chicken gut health to provide deeper insights into various aspects of gut health. Due to the broad and complex nature of the concept of "gut health", we have highlighted the most pertinent factors related to the field performance of broiler chickens.

Coccidiosis: Recent Progress in Host Immunity and Alternatives to Antibiotic Strategies.

Coccidiosis is an avian intestinal disease caused by several distinct species of Eimeria parasites that damage the host's intestinal system, resulting in poor nutrition absorption, reduced growth, and often death. Increasing evidence from recent studies indicates that immune-based strategies such as the use of recombinant vaccines and various dietary immunomodulating feed additives can improve host defense against intracellular parasitism and reduce intestinal damage due to inflammatory responses induced by parasites. Therefore, a comprehensive understanding of the complex interactions between the host immune system, gut microbiota, enteroendocrine system, and parasites that contribute to the outcome of coccidiosis is necessary to develop logical strategies to control coccidiosis in the post-antibiotic era. Most important for vaccine development is the need to understand the protective role of the local intestinal immune response and the identification of various effector molecules which mediate anti-coccidial activity against intracellular parasites. This review summarizes the current understanding of the host immune response to coccidiosis in poultry and discusses various non-antibiotic strategies which are being developed for coccidiosis control. A better understanding of the basic immunobiology of pertinent host-parasite interactions in avian coccidiosis will facilitate the development of effective anti-Eimeria strategies to mitigate the negative effects of coccidiosis.

Immunological characterization of chicken tumor necrosis factor-α (TNF-α) using new sets of monoclonal antibodies specific for poultry TNF-α.

Tumor necrosis factor-α (TNF-α) is a type II transmembrane protein with either membrane-bound or soluble forms and is a prototypical member of the TNF superfamily. TNF-α is a pleiotropic cytokine associated with the regulation of systemic inflammation and host defense. Chicken TNF-α (chTNF-α) is a long-missed avian ortholog, and its immunological properties remain largely unknown compared to those of its mammalian counterparts. Here, we report the functional characterization and immunomodulatory properties of chTNF-α using a panel of newly developed anti-chTNF-α mouse monoclonal antibodies (mAbs). Using anti-chTNF-α mAbs, we determined the tissue expression of chTNF-α in lymphoid and non-lymphoid organs. A chTNF-α-specific antigen-capture sandwich ELISA was developed using compatible mAb partners by screening and validation of ten different mAbs. Employing 3G11 and 12G6 as capture and detection antibodies, respectively, the levels of native chTNF-α in the circulation of Clostridium perfringens, Eimeria, or dual C. perfringens/Eimeria-infected chickens were determined. Furthermore, intracellular expression of chTNF-α in primary immune cells or cell lines derived from chickens was validated by immunocytochemistry and flow cytometry assays using both 3G11 and 12G6 mAbs. Notably, both 3G11 and 12G6 neutralized chTNF-α-induced nitric oxide production in chicken HD11 cells in vitro. Collectively, our results enhance our understanding of the functional characteristics of chTNF-α, and these anti-chTNF-α mAbs will serve as valuable immune reagents to inform on inflammatory responses and disease pathogenesis in the fundamental and applied studies of avian species.

Clostridium perfringens-Induced Host-Pathogen Transcriptional Changes in the Small Intestine of Broiler Chickens.

Clostridium perfringens is an important opportunistic pathogen that may result in toxin-mediated diseases involving food poisoning/tissue gangrene in humans and various enterotoxaemia in animal species. It is a main etiological agent for necrotic enteritis (NE), the most financially devastating bacterial disease in broiler chickens, especially if raised under antibiotic-free conditions. Importantly, NE is responsible for losses of six billion US dollars annually in the global poultry industry. To investigate the molecular mechanisms of C. perfringens-induced pathogenesis in the gut and its microbiome mRNA levels in C. perfringens-infected and non-infected hosts, we used RNA sequencing technology to perform transcriptional analysis of both host intestine and microbiome using our NE model. The growth rate was significantly impaired in chickens infected by C. perfringens. In total, 13,473 annotated chicken genes were differentially expressed between these two groups, with ninety-six genes showing statistical significance (|absolute fold changes| > 2.0, adjusted p value < 0.05). Genes involved in energy production, MHC Class I antigen, translation, ribosomal structures, and amino acid, nucleotide and carbohydrate metabolism from infected gut tissues were significantly down-regulated. The upregulated genes were mainly engaged in innate and adaptive immunity, cellular processes, genetic information processing, and organismal systems. Additionally, the transcriptional levels of four crucial foodborne pathogens were significantly elevated in a synergic relationship with pathogenic C. perfringens infection. This study presents the profiling data that would likely be a relevant reference for NE pathogenesis and may provide new insights into the mechanism of host-pathogen interaction in C. perfringens-induced NE infection in broiler chickens.

Immunological studies on chicken interferon-kappa using an antigen-capture ELISA developed using new mouse monoclonal antibodies.

Interferon (IFN)-κ is a type I IFN that plays a central role in anti-viral defense and host immune response. The functions of type I IFNs have not been clearly defined in chickens compared to those of their mammalian counterparts. In this study, we developed an antigen-capture ELISA using newly developed mouse monoclonal antibodies (mAbs) which are specific for chicken IFN-κ (chIFN-κ) and showed that this ELISA can measure native chIFN-κ production during the activation of macrophages by polyinosinic:polycytidylic acid (poly I:C). The recombinant chicken IFN-κ expressed in Escherichia coli was used to immunize mice. Five mAbs that specifically recognized chIFN-κ were selected and characterized based on their specificity and binding activity toward chIFN-κ via indirect ELISA and western blotting. To develop a capture ELISA for chicken IFN-κ, two sets of the best capture and detection mAbs combinations were identified via pairing assays. To validate the antigen-capture assay, the production of native IFN-κ was induced in chicken HD11 macrophages using poly I:C. Furthermore, qRT-PCR was used to confirm the transcript-level expression of IFN-κ in HD11 cells at 24 and 48 h. The neutralizing effects of anti-chIFN-κ mAbs were evaluated based on their ability to block the induction of IFN-stimulated genes (ISGs) in DF-1 fibroblast cells stimulated with recombinant chIFN-κ proteins. All five mAbs blocked the mRNA expression of ISGs in a dose-dependent manner. Our results validate the specificity and utility of these newly developed mAbs for the detection of native chicken IFN-κ. This novel antigen-capture ELISA will be a valuable tool for fundamental and applied research involving IFN-κ in the normal and diseased states.