Hyperimmune egg yolk antibodies developed against Clostridium perfringens antigens protect against necrotic enteritis.

Necrotic enteritis (NE) is a widespread infectious disease caused by Clostridium perfringens that inflicts major economic losses on the global poultry industry. Due to regulations on antibiotic use in poultry production, there is an urgent need for alternative strategies to mitigate the negative effects of NE. This paper presents a passive immunization technology that utilizes hyperimmune egg yolk immunoglobulin Y (IgY) specific to the major immunodominant antigens of C. perfringens. Egg yolk IgYs were generated by immunizing hens with 4 different recombinant C. perfringens antigens, and their protective effects against NE were evaluated in commercial broilers. Six different spray-dried egg powders were produced using recombinant C. perfringens antigens: α-toxin, NE B-like toxin (NetB; EB), elongation factor-Tu (ET), pyruvate:ferredoxin oxidoreductase, a mixture of 4 antigens (EM-1), and a nonimmunized control (EC). The challenged groups were either provided with different egg powders at a 1% level or no egg powders (EN). The NE challenge model based on Eimeria maxima and C. perfringens dual infection was used. In Experiments 1 and 2, the EB and ET groups exhibited increased body weight gain (BWG; P < 0.01), decreased NE lesion scores (P < 0.001), and reduced serum NetB levels (P < 0.01) compared to the EN and EC groups. IgY against NetB significantly reduced Leghorn male hepatocellular cytotoxicity in an in vitro test (P < 0.01). In Experiment 3, the protective effect of the IgYs mixture (EM-2) against C. perfringens antigens (NetB and EFTu) and Eimeria antigens (elongation factor-1-alpha: EF1α and Eimeria profilin: 3-1E) was tested. The EM-2 group showed similar body weight, BWG, and feed intake from d 7 to 22 compared to the NC group (P < 0.05). On d 20, the EM-2 group showed comparable intestinal permeability, NE lesion scores, and jejunal NetB and collagen adhesion protein levels to the NC group (P < 0.05). In conclusion, dietary mixture containing antibodies to NetB and EFTu provides protection against experimental NE in chickens through passive immunization.

Collagen adhesin protein and necrotic enteritis B-like toxin as biomarkers for early diagnosis of necrotic enteritis in commercial broiler chickens.

Mouse monoclonal antibodies (mAbs) reactive with Clostridium perfringens collagen adhesin protein (CNA) and necrotic enteritis B-like toxin (NetB) were developed. The best capture/detection mAb pairs for CNA and NetB were selected based on their affinity and specificity to develop sandwich enzyme-linked immunosorbent assays (ELISAs) to detect CNA and NetB proteins, respectively, in jejunal digesta samples from commercial broiler farms in the United States. Prior to the analysis of samples from commercial broiler flocks, the specificity and sensitivity of the CNA and NetB ELISAs were validated using sera, jejunal digesta, and fecal samples from chickens coinfected with Eimeria maxima and CNA+/NetB+C. perfringens in an animal model of necrotic enteritis (NE). Subsequently, a total of 251 field samples were collected from 74 commercial poultry farms. Among these, 18 samples were from 6 broiler farms that used certified organics (CO), and 155 samples were from 42 farms with nonantibiotics (NA). In jejunal digesta samples, CNA levels ranged from 0.02 to 0.59 ng/mL and NetB levels ranged from 0.09 to 1.91 ng/mL. CNA and NetB levels showed a positive correlation with each other (Pearson correlation coefficient r = 0.772, P < 0.001). CNA and NetB levels in jejunal digesta were significantly decreased in CO farms compared with those from NA farms (P < 0.001). In conclusion, these new C. perfringens antigen-specific sandwich ELISAs offer a sensitive and specific means to detect C. perfringens CNA and NetB proteins as biomarkers of early NE occurrence in field samples from commercial broiler chickens.

Molecular identification and characterisation of a novel chicken leukocyte immunoglobulin-like receptor A5.

1. Leukocyte immunoglobulin-like receptor A5 (LILRA5) is a key molecule that regulates the immune system. However, the LILRA5 gene has not been characterised in avian species, including chickens. The present study aimed to identify and functionally characterise LILRA5 identified from two genetically disparate chicken lines, viz., Marek's disease (MD)-resistant (R) line 6.3 and MD-susceptible (S) line 7.2. 2. Multiple sequence alignment and phylogenetic analyses confirmed that the identity and similarity homologies of amino acids of LILRA5 in chicken lines 6.3 and 7.2 ranged between 93% and 93.7%, whereas those between chicken and mammals ranged between 20.9% and 43.7% and 21.1% to 43.9%, respectively. The newly cloned LILRA5 from chicken lines 6.3 and 7.2 revealed high conservation and a close relationship with other known mammalian LILRA5 proteins. 3. The results indicated that LILRA5 from chicken lines 6.3 and 7.2 was associated with phosphorylation of Src kinases and protein tyrosine phosphatase non-receptor type 11 (SHP2), which play a regulatory role in immune functions. Moreover, the results demonstrated that LILRA5 in these lines was associated with the activation of major histocompatibility complex (MHC) class I and ß2-microglobulin and induced the expression of the transporter associated with antigen processing. In addition, LILRA5 in both chicken lines activated and induced Janus kinase (JAK)-signal transducer and the activator of transcription (STAT), nuclear factor kappa B (NF-κB), phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) and the extracellular signal-regulated kinase (ERK)1/2 signalling pathways; toll-like receptors; and Th1-, Th2-, and Th17- cytokines. 4. The data suggested that LILRA5 has innate immune receptors essential for macrophage immune response and provide novel insights into the regulation of immunity and immunopathology.

Immune modulation, growth performance, and nutrient retention in broiler chickens fed a blend of phytogenic feed additives.

This study aimed to assess the effect of a commercial blend of phytogenic feed additives (PA), comprising 5% carvacrol, 3% cinnamaldehyde, and 2% capsicum oleoresin on the modulation of immune biomarkers of broiler chickens, their growth performance, dietary energy, and nutrient retention. Four-hundred day-old birds were assigned to one of four dietary treatments. Two control diets based on either wheat (WC) or maize (MC) were each given with and without PA at 100 g/t. Growth performance variables including feed intake (FI), weight gain (WG), and feed conversion ratio (FCR) were recorded. Dietary N-corrected apparent metabolizable energy (MEn), dry matter (DMR), nitrogen (NR), and fat retention (FR) coefficients were also determined. Gene expression of immune biomarkers (cytokines) were determined in caecal tonsil tissue from 21 d old birds. Expression of IL2, IL18, IL10, and IL17C in the caecal tonsils were upregulated (P < 0.05) in the birds fed MC-based diets compared to the WC fed birds. Feeding PA supplemented diets downregulated the expression of CD40LG (P < 0.001), IFNG, and IL6 (P < 0.05). There was a cereal type × PA interaction (P < 0.05), as expression of IFNB was downregulated in the birds fed PA supplemented MC but not WC. However, expression of IL12B was downregulated in birds fed PA supplemented WC but there was no significant (P > 0.05) change in expression levels in birds fed MC diets. Feeding MC diets gave greater FI (P < 0.001) and ME (P < 0.05), but lower FCR (P < 0.05) compared to birds fed WC diets. The WG and nutrient retention coefficients were not affected (P > 0.05) by cereal type. Supplementary PA improved FI (P < 0.05), WG (P < 0.001), FCR (P < 0.05), MEn (P < 0.05), MEn: GE ratio (P < 0.05), and FR (P < 0.05). In conclusion, dietary inclusion of PA improved overall growth performance variables, energy, and nutrient retention and intestinal cytokine expression.

Allium hookeri supplementation improves intestinal immune response against necrotic enteritis in young broiler chickens.

Three hundred birds (1 day old) were randomly assigned to 6 groups (n = 50 birds/treatment) and fed a basal diet (control) or basal diet supplemented with Allium hookeri (AH) root (1 or 3%). At day 14, half of the birds in each group were orally challenged with E. maxima 41A (1 × 104 cells/chicken), followed by C. perfringens infection (1 × 109 cfu/chicken) on day 18. Necrotic enteritis (NE)-associated infections and intestinal immune response were assessed by average body weight gain, lesion score, and oocyst shedding. The effect of dietary supplementation, AH, on transcript levels of pro-inflammatory cytokines, and tight junction proteins and mucin protein in the jejunum, were quantified by quantitative real-time (qRT)-PCR. At day 20, birds fed with diet supplementation (3% of AH) significantly weighted more than the control group. Although the NE-challenged had significantly reduced average body weight gain, there was no significance in the effect between diet × NE-challenge interactions on the average body weight gain. Among the NE-challenged groups, gut lesion score and oocyst shedding were significantly decreased in birds given AH (1 or 3%) compared to the control group. There was a correlation between diet and NE infection with regards to interleukin (IL)-17A, and inducible nitric oxide synthase (iNOS). The up-regulated transcript levels of cytokines IL-8, IL-17A, iNOS, and LITAF by NE challenged groups were significantly reduced by AH (1 or 3%) supplementation. Down-regulated expression levels of tight junction (TJ) proteins: junctional adhesion molecule 2 (JAM2), occluding, and intestinal mucin 2 (MUC2) by NE challenge, was up-regulated by the addition of AH (1 or 3%) supplementation. All TJ proteins (JAM2, ZO1, Ocluddin and MUC2) in the jejunum had a significant diet × NE-challenge interaction. These findings demonstrate that dietary supplementation of AH in chicken feed could be beneficially used to improve chicken health against NE.

Characterization and functional analyses of a novel chicken CD8α variant X1 (CD8α1).

We provide the first description of cloning and of structural and functional analysis of a novel variant in the chicken cluster of differentiation 8 alpha (CD8a) family, termed the CD8α X1 (CD8α1) gene. Multiple alignments of CD8α1 with known CD8α and CD8ß sequences of other species revealed relatively low conservation of AA residues involved in the specific and unique structural domains among CD8α genes. For example, cysteine residues that are involved in disulfide bonding to form the V domain are conserved. In contrast, the O-linked glycosylation sites (XPXX motif) are not found in the chicken CD8α1 sequence, and the A ß strand and complementarity-determining region 1 and 2 sequences are poorly conserved between chicken CD8α1 and avian CD8α. Furthermore, the alignment showed that the transmembrane regions show relatively high sequence similarity, whereas the cytoplasmic regions show relatively low similarity, indicating poor conservation. Moreover, the motif (CXCP) that is thought to be responsible for binding the p56 lymphocyte cell kinase subunit (p56) is missing in the CD8α1 sequence. The chicken CD8α1 genomic structure is similar to that of chicken CD8α, but their protein structures differ. Phylogenetic analysis showed that chicken CD8α1 grouped with known avian CD8α sequences but was somewhat distantly related to the CD8α molecules of other species. Moreover, we analyzed the signal transduction and cytokine response to CD8α1 treatment to determine the specific biological functions of chicken CD8α1 in immune cells. The results showed that chicken CD8α1 is a key regulator of the expression of genes that are associated and cooperate with transcription factors in the major histocompatibility complex class I and II promoter regions and activates Janus kinase (JAK) 1/2, signal transducer and activator of transcription (STAT), and suppressor of cytokine signaling (SOCS) 1 signaling-related genes. Immune cells that express functional CD8α1 induce proinflammatory cytokines as well as innate immune responses. Therefore, our data indicate that CD8α1 may have immunoregulatory activity by regulating the expression of proinflammatory or anti-inflammatory cytokines via its effect on immune cells.

Using genomics to identify novel antimicrobials.

There is a critical need in animal agriculture to develop novel antimicrobials and alternative strategies that will help to reduce the use of antibiotics and address the challenges of antimicrobial resistance. High-throughput gene expression analysis is providing new tools that are enabling the discovery of host-derived antimicrobial peptides. Examples of gene-encoded natural antibiotics that have gained attention include antimicrobial peptides such as human granulysin and its multi-species homolog, namely NK-lysin, which provide a protective response against a broad range of microbes and are a principal component of innate immunity in vertebrates. Both granulysin and NK-lysin are localised in cytolytic granules in natural killer and cytotoxic T lymphocytes. Host-derived NK-lysins that were first described in mammals are also found in avian species, and they have been shown to have antimicrobial activities that could potentially be used to control important poultry pathogens. Morphological alterations observed following chicken NK-lysin binding to Eimeria sporozoites and Escherichia coli membranes indicate damage and disruption of cell membranes, suggesting that NK-lysin kills pathogenic protozoans and bacteria by direct interaction. Genotype analysis revealed that chicken NK-lysin peptides derived from certain alleles were more effective at killing pathogens than those derived from others, which could potentially affect susceptibility to diseases. Although the host-derived antimicrobial peptides described in this paper may not, by themselves, be able to replace the antibiotics currently used in animal production, their use as specific treatments based on their known mechanisms of action is showing promising results.

Il est devenu impératif de développer de nouveaux agents antimicrobiens utilisables en production animale et de mettre au point des stratégies alternatives permettant de réduire l'utilisation des antibiotiques et de faire face aux enjeux de la résistance aux agents antimicrobiens. Les nouveaux outils rendus possibles par l'analyse à haut débit de l'expression génique permettent de découvrir les peptides antimicrobiens synthétisés par l'hôte. Parmi les antibiotiques naturels codés par des gènes qui suscitent actuellement le plus d'intérêt figurent les peptides antimicrobiens tels que la granulysine humaine et son homologue pluriespèces, la NK-lysine, qui déclenchent une réponse protectrice contre un large éventail d'agents microbiens et constituent l'une des principales composantes de l'immunité innée chez les vertébrés. La granulysine et la NK-lysine sont toutes deux localisées dans les granules cytolytiques des lymphocytes cytotoxiques T et des cellules NK (natural killer). Décrites dans un premier temps chez des mammifères, les NK-lysines synthétisées par l'hôte sont également présentes chez les oiseaux et il a été démontré qu'elles présentent des propriétés antimicrobiennes qui pourraient être utilisées pour contrôler d'importants agents pathogènes affectant les volailles. Les modifications morphologiques observées suite à la fixation de la NK-lysine du poulet aux sporozoïtes d'Eimeria et aux membranes d'Escherichia coli sont le signe d'un endommagement et d'une rupture des membranes cellulaires qui semblent indiquer que la NK-lysine tue les protozoaires et bactéries pathogènes par interaction directe. L'analyse génotypique a révélé que les peptides des NK-lysines de poulet encodés par certains allèles sont plus efficaces pour tuer les agents pathogènes que ceux encodés par d'autres allèles, ce qui pourrait avoir une influence sur la sensibilité aux maladies. Bien que les peptides antimicrobiens synthétisés par l'hôte décrits dans cet article ne puissent remplacer à eux seuls les antibiotiques utilisés actuellement en production animale, leur utilisation en tant que traitements spécifiques basés sur leurs propres mécanismes d'action connus, donne déjà des résultats prometteurs.

En el ámbito de la producción animal es ahora imperativo no solo dar con nuevos antimicrobianos, sino también concebir estrategias alternativas que ayuden a reducir el uso de antibióticos y a afrontar los problemas derivados de la resistencia a los antimicrobianos. El análisis de alto rendimiento de la expresión génica proporciona nuevas herramientas que están propiciando el descubrimiento de péptidos antimicrobianos sintetizados por los organismos anfitriones. Entre los ejemplos de antibióticos naturales codificados por genes que vienen suscitando interés están péptidos antimicrobianos como la granulisina humana y su homólogo multiespecífico, la NK-lisina, que inducen una respuesta protectora contra muy diversos microbios y son un componente básico de la inmunidad innata de los vertebrados. Tanto la granulisina como la NK-lisina están localizadas en el interior de los gránulos citolíticos de las células NK (natural killer) y los linfocitos T citotóxicos. Las NK-lisinas sintetizadas por el anfitrión, descritas en un principio en mamíferos, también se encuentran en especies aviares, y se ha demostrado que presentan propiedades antimicrobianas que podrían ser útiles para luchar contra importantes patógenos de las aves de corral. Las alteraciones morfológicas observadas después de que la NK-lisina de pollo se uniera a esporozoítos de Eimeria y a membranas de Escherichia coli son indicativas de deterioro y desorganización de la membrana celular, de donde se infiere que la NK-lisina mata a los patógenos protozoarios y bacterianos por interacción directa. El análisis genotípico en pollos reveló que ciertos alelos codificaban NK-lisinas que mataban a los patógenos con más eficacia que los péptidos codificados por otros alelos, lo que podría tener influencia en la sensibilidad a las enfermedades. Aunque tal vez los péptidos antimicrobianos sintetizados por el anfitrión que los autores describen en estas líneas no puedan, por sí solos, sustituir a los antibióticos actualmente utilizados en producción animal, su utilización en tratamientos específicos basados en sus mecanismos de acción conocidos está deparando resultados prometedores.

Comparing the immune responses of two genetically B-complex disparate Fayoumi chicken lines to Eimeria tenella.

The present study was conducted to compare the susceptibility of congenic Fayoumi lines to Eimeria tenella infection and to assess genetic differences in Eimeria egression. Chickens were orally inoculated with 5 × 10(4) sporulated E. tenella oocysts and challenged with 5 × 10(6) oocysts on the 10th day after the primary infection. The Fayoumi M5.1 line exhibited higher levels of body weight gain, less oocyst shedding and higher percentages of B and CD4(+)/CD8(+) T cells than the M15.2 chickens. These results demonstrate that M5.1 line is more resistant to E. tenella infection than M15.2 line. Furthermore, the percentage of sporozoite egress from peripheral blood mononuclear cells (PBMCs) was higher in the M5.1 line. The results of this study suggest that enhanced resistance of Fayoumi M5.1 to E. tenella infection may involve heightened cell-mediated and adaptive immunity, resulting in reduced intracellular development of Eimeria parasites.

Dietary sodium selenite affects host intestinal and systemic immune response and disease susceptibility to necrotic enteritis in commercial broilers.

1. This study was to evaluate the effects of supplementary dietary selenium (Se) given as sodium selenite on host immune response against necrotic enteritis (NE) in commercial broiler chickens. 2. Chicks were fed from hatching on a non-supplemented diet or diets supplemented with different levels of Se (0.25, 0.50, and 1.00 Se mg/kg). To induce NE, broiler chickens were orally infected with Eimeria maxima at 14 d of age and then with Clostridium perfringens 4 d later using our previously established NE disease model. 3. NE-associated clinical signs and host protective immunity were determined by body weight changes, intestinal lesion scores, and serum antibodies against α-toxin and necrotic enteritis B (NetB) toxin. The effects of dietary Se on the gene expression of pro-inflammatory cytokines e.g., interleukin (IL)-1ß, IL-6, IL-8LITAF (lipopolysaccharide-induced TNFα-factor), tumour necrosis factor (TNF) SF15, and inducible nitric oxide synthase (iNOS), glutathione peroxidase 7 (GPx7), and avian ß-defensins (AvBD) 6, 8, and 13 (following NE infection) were analysed in the intestine and spleen. 4. The results showed that dietary supplementation of newly hatched broiler chicks with 0.25 Se mg/kg from hatch significantly reduced NE-induced gut lesions compared with infected birds given a non-supplemented diet. The levels of serum antibody against the NetB toxin in the chicks fed with 0.25 and 0.50 mg/kg Se were significantly higher than the non-supplemented control group. The transcripts for IL-1ß, IL-6, IL-8, iNOS, LITAF, and GPx7, as well as AvBD6, 8, and 13 were increased in the intestine and spleen of Se-supplemented groups, whereas transcript for TNFSF15 was decreased in the intestine. 5. It was concluded that dietary supplementation with optimum levels of Se exerted beneficial effects on host immune response to NE and reduced negative consequence of NE-induced immunopathology.

Development and characterization of mouse monoclonal antibodies reactive with chicken IL-1ß.

Interleukin-1ß proteins from chicken, duck, goose, turkey, and pigeon share 77 to 99% amino acid sequence similarity among themselves, and only 31 to 35% sequence similarity is shared between avian and mammalian IL-1ß. There have been no antibodies that specifically detect avian IL-1ß, and the current study was conducted to develop mouse monoclonal antibodies (mAb) against chicken IL-1ß (chIL-1ß) to further define its biochemical and immunological properties. In this study, 2 mouse mAb that are specific for chIL-1ß were produced and characterized. Both mAb identified a 66.0 kDa recombinant chIL-1ß protein expressed in Escherichia coli by Western blot analysis that corresponded to the expected molecular weight of a recombinant fusion protein containing the full-length 23.0 kDa chIL-1ß protein and a 43.0 kDa maltose binding protein tag. Immunohistochemical analysis identified cells producing endogenous chIL-1ß in the cecal tonsils, bursa of Fabricius, and spleen. Purified recombinant chIL-1ß dose-dependently stimulated the proliferation and nitric oxide production by thymocytes, and both activities were inhibited by co-incubation with the 2 chIL-1ß mAb described in this paper. These mAb will be important immune reagents for basic and applied poultry research of IL-1ß in poultry.

Effects of in ovo injection with selenium on immune and antioxidant responses during experimental necrotic enteritis in broiler chickens.

This study was conducted to investigate the effects of in ovo injection of Se on modulating the immune system and antioxidant responses in broiler chickens with experimental necrotic enteritis. Broiler eggs were injected at 18 d of embryo age with either 100 µL of PBS alone or sodium selenite (Na2SeO3) in PBS, providing 0 (SS0), 10 (SS10), or 20 (SS20) µg of Se/egg. At 14 d posthatch, PBS-treated and uninfected chickens were kept as the control group, whereas the remaining chickens were orally infected with 1.0 × 10(4) sporulated oocysts of Eimeria maxima (SS0, SS10, SS20). At 18 d posthatch, E. maxima-infected chickens were orally infected with 1.0 × 10(9) cfu of Clostridium perfringens. Infected control SS0 group showed significantly decreased BW compared with the uninfected control. However, SS20 group showed significantly increased BW compared with the infected control SS0 group, whereas the BW were similar among uninfected control and infected SS10 and SS20 groups. The SS10 group showed significantly lower intestinal lesions compared with the SS0 group, and oocyst production was decreased in both SS10 and SS20 groups. Serum malondialdehyde level and catalase activity were also decreased in both SS10 and SS20 groups, whereas the superoxide dismutase level was significantly lower in the SS10 group compared with the SS0 group. The SS20 group showed significantly higher levels of transcripts for IL-1ß and IL-6 in intestine, and SS10 and SS20 groups had higher levels of transcripts for IL-8 and inducible nitric oxide synthase expression and decreased glutathione peroxidase 7 mRNA levels compared with the SS0 group. The SS10 and SS20 groups also showed increased serum antibody levels to C. perfringens α-toxin and NetB toxin compared with the SS0 group. These collective results suggest that the injection of Se into the amniotic cavity of developing eggs may be beneficial for enhancing immune and antioxidant responses in the hatched chickens exposed to the necrotic enteritis-causing pathogens.

Effect of Bacillus Subtilis-based Direct-fed Microbials on Immune Status in Broiler Chickens Raised on Fresh or Used Litter.

Type of dietary direct-fed microbials (DFMs) or poultry litter could directly influence the composition of gut microbiota. Gut microbiota plays an important role in shaping the developing immune system and maintaining the homeostasis of the mature immune system in mammal and chickens. The present study was carried out to investigate the interaction among litter, DFMs and immunity in broiler chickens exposed to a field-simulated environment. Immune status of broiler chickens was assessed by serum antibodies against Eimeria spp. and Clostridium spp. and intestinal cytokine mRNA expression. The current experimental design had a 3 ×2 factorial arrangement of treatments with three types of litter, i.e., fresh litter or used litter that was obtained from a farm with no disease outbreak (used litter) or a farm with history of a gangrenous dermatitis outbreak (GD litter), and two dietary treatments with or without DFMs. It was found that either DFM addition or type of litter significantly affected anticoccidial antibody levels of broiler chickens at d 42. In general, dietary DFMs increased the anticoccidial antibodies in the fresh-litter raised chickens, but lowered the levels in the GD-litter raised chickens. Serum antibodies against Clostridium perfringens α-toxin were significantly (p<0.05) higher in chickens raised on GD litter compared with those raised on fresh litter. Cytokine mRNA expression was significantly (p<0.05) altered by either the type of litter or DFMs. Of interest, dietary DFMs lowered interferon-γ, interleukin 1beta, and CXCLi2 cytokine mRNA expression in chickens raised on fresh litter but increased them in GD-litter raised chickens. In conclusion, dietary DFMs modulate various immune parameters of broiler chickens, but the DFM-mediated effects were dependent upon the type of litter on which chickens were raised.

Regulation of T lymphocyte subpopulations in specific pathogen free chickens following experimental fowl adenovirus Ⅷ infection

Two-day-old specific pathogen-free (SPF) chickens were divided into two groups. Group I was inoculated orally with fowl adenovirus VIII (FAV-VIII). Group II served as a negative control. Chickens were investigated at various days post-inoculation (dpi) by flow cytometric analysis for changes in T lymphocyte subpopulations in immune system and blood. In the thymus, CD3+ T lymphocytes were increased at 25 dpi, with significant increases in the FAV infected noted at 1, 12, 20dpi (p<0.05). This was accompanied by a corresponding increase of CD4+ and CD8+ T lymphocytes. In the spleen, CD3+ and CD4+ T lymphocytes were increased significantly at 30 dpi (p<0.01) whereas CD8+ and TCR γ δ+ T lymphocytes were decreased at 1 (p<0.05), 30 dpi (p<0.01). An increase of CD3+, CD4+ and CD8+ T lymphocytes was noticed in peripheral blood, and accompanied by a decrease of TCR γ δ+ T lymphocytes. These results demonstrated that infection with FAV-VIII causes significant fluctuations in T lymphocyte subpopulations in thymus, blood and spleen. It can be concluded that an infection with FAV-VIII has profound effects on the immune system, especially on cell mediated immune competency.

Development of FPV140 antigen-specific ELISA differentiating fowlpox virus isolates from all other viral pathogens of avian origin.

The FPV140 gene encodes an envelope protein of fowlpox virus (FPV). In this study, the FPV140 gene of FPV Chinese isolate HH2008 was cloned and the comparison of its sequence with other FPV isolates showed it to be highly conserved across all FPV isolates. A recombinant plasmid pET-FPV140 carrying FPV140 gene was constructed and transformed into Escherichia coli. The optimal expression condition for the FPV140 gene was developed and purified FPV140 recombinant protein was used to produce rabbit polyclonal antibody. An indirect ELISA using this anti-FPV140 polyclonal antibody was capable of distinguishing avian FPV isolates from other common avian pathogens such as mycoplasma gallisepticum, infectious laryngotracheitis virus, avian influenza virus, infectious bursal disease virus, and avian infectious bronchitis virus. This ELISA will serve as a useful diagnostic tool for the detection of FPV in clinical samples.

Clostridium perfringens alpha-toxin and NetB toxin antibodies and their possible role in protection against necrotic enteritis and gangrenous dermatitis in broiler chickens.

Necrotic enteritis (NE) and gangrenous dermatitis (GD) are important infectious diseases of poultry. Although NE and GD share a common pathogen, Clostridium perfringens, they differ in other important aspects such as clinical signs, pathologic symptoms, and age of onset. The primary virulence factors of C perfringens are its four major toxins (alpha, beta, epsilon, iota) and the newly described NE B-like (NetB) toxin. While neutralizing antibodies against some C perfingens toxins are associated with protection against infection in mammals, the serologic responses of NE- and GD-afflicted birds to these toxins have not been evaluated. Therefore, we measured serum antibody levels to C perfringens alpha-toxin and NetB toxin in commercial birds from field outbreaks of NE and GD using recombinant toxin-based enzyme-linked immunosorbent assay (ELISA). Initially, we used this ELISA system to detect antibody titers against C perfringens alpha-toxin and NetB toxin that were increased in birds experimentally coinfected with Eimeria maxima and C perfringens compared with uninfected controls. Next, we applied this ELISA to field serum samples from flock-mated birds with or without clinical signs of NE or GD. The results showed that the levels of antibodies against both toxins were significantly higher in apparently healthy chickens compared to birds with clinical signs of NE or GD, suggesting that these antitoxin antibodies may play a role in protection against NE and GD.

Immune modulation of innate immunity as alternatives-to-antibiotics strategies to mitigate the use of drugs in poultry production.

Following is invited commentary on the symposium "A Crystal Ball Look into the Future of…" delivered July 16, 2011, at the Poultry Science Association's 100th annual meeting, St. Louis, Missouri. The symposium examined various aspects that will impact the future of poultry production over the next 10 to 20 yr. Topics included genetics, nutrition, incubation, and bird health. This paper deals with various aspects of future issues affecting global feeding and nutrition of poultry.

Differential gene expression profiles of ß-defensins in the crop, intestine, and spleen using a necrotic enteritis model in 2 commercial broiler chicken lines.

Changes in the expression levels of avian ß-defensin (AvBD) mRNA were evaluated in necrotic enteritis (NE) disease model in 2 genetically disparate commercial broiler chicken lines: Ross and Cobb. The NE was initiated in the gut by a previously established co-infection model using oral Eimeria maxima infection followed by a Clostridium perfringens challenge. Among the 14 AvBD types examined, there was a tissue-specific expression of AvBD transcripts: AvBD1, AvBD7, and AvBD9 in the crop; AvBD8, AvBD10, and AvBD13; in the intestine and AvBD1 and AvBD7 in the spleen. The 2 different commercial broiler chicken lines showed differential gene expression patterns of AvBD transcripts following co-infection with E. maxima and C. perfringens, with R-line chickens generally showing higher expression levels than the C strain. Both chicken strains showed enhanced gene expression levels of proinflammatory cytokines, such as IL-1ß, IL-6, IL-17F, and TNFSF15 in spleen, and TNFSF15 in intestine, whereas IL-17F was significantly increased only in the intestine of R-line chickens following NE infection. Although the exact nature of interactions between defensins and cytokines in determining the outcome of host innate immune responses to the pathogens of NE remains to be investigated, the differences in gene expression levels of ß-defensins and proinflammatory cytokines in the intestine, crop, and spleen could explain the predisposed disease resistance and susceptibility to NE in the 2 commercial broiler chicken lines.

The long view: a selective review of 40 years of coccidiosis research.

This selective review of 40 years of coccidiosis research is one of a number on important diseases of poultry to celebrate the 40th anniversary of the birth of Avian Pathology, the journal of the World Veterinary Poultry Association, and is written for the non-specialist. The intention is to provide a flavour of the field problems and intellectual challenges, with emphasis in the areas of immunology and vaccinology that drove research in the 1970s, and to reflect on research progress since.

Regulation of T lymphocyte subpopulations in specific pathogen-free chickens following experimental fowl adenovirus-â §VIII infection.

Two-day-old specific pathogen-free (SPF) chickens were divided into two groups. Group I was inoculated orally with fowl adenovirus Ⅷ (FAV-Ⅷ). Group II served as a negative control. Chickens were investigated at various days post-inoculation (dpi) by flow cytometric analysis for changes in T lymphocyte subpopulations in immune system and blood. In the thymus, CD3(+)T lymphocytes were increased at 25 dpi, with significant increases in the FAV infected noted at 1, 12, 20dpi (p<0.05). This was accompanied by a corresponding increase of CD4(+) and CD8(+) T lymphocytes. In the spleen, CD3(+) and CD4(+) T lymphocytes were increased significantly at 30 dpi (p<0.01) whereas CD8(+) and TCR γ δ(+) T lymphocytes were decreased at 1 (p<0.05), 30 dpi (p<0.01). An increase of CD3(+), CD4(+) and CD8(+) T lymphocytes was noticed in peripheral blood, and accompanied by a decrease of TCR γ δ(+) T lymphocytes. These results demonstrated that infection with FAV-Ⅷ causes significant fluctuations in T lymphocyte subpopulations in thymus, blood and spleen. It can be concluded that an infection with FAV-Ⅷ has profound effects on the immune system, especially on cell mediated immune competency.

Effect of dietary antimicrobials on immune status in broiler chickens.

This study evaluated the effects of dietary anticoccidial drugs plus antibiotic growth promoters (AGPs) on parameters of immunity in commercial broiler chickens. Day-old chicks were raised on used litter from a farm with endemic gangrenous dermatitis to simulate natural pathogen exposure and provided with diets containing decoquinate (DECX) or monensin (COBN) as anticoccidials plus bacitracin methylene disalicylate and roxarsone as AGPs. As a negative control, the chickens were fed with a non-supplemented diet. Immune parameters examined were concanavalin A (ConA)-stimulated spleen cell proliferation, intestine intraepithelial lymphocyte (IEL) and spleen cell subpopulations, and cytokine/chemokine mRNA levels in IELs and spleen cells. ConA-induced proliferation was decreased at 14 d post-hatch in DECX-treated chickens, and increased at 25 and 43 d in COBN-treated animals, compared with untreated controls. In DECX-treated birds, increased percentages of MHC2(+) and CD4(+) IELS were detected at 14 d, but decreased percentages of these cells were seen at 43 d, compared with untreated controls, while increased TCR2(+) IELs were evident at the latter time. Dietary COBN was associated with decreased fractions of MHC2(+) and CD4(+) IELs and reduced percentages of MHC2(+), BU1(+), and TCR1(+) spleen cells compared with controls. The levels of transcripts for interleukin-4 (IL-4), IL-6, IL-17F, IL-13, CXCLi2, interferon-γ (IFN-γ), and transforming growth factorß4 were elevated in IELs, and those for IL-13, IL-17D, CXCLi2, and IFN-γ were increased in spleen cells, of DECX- and/or COBN-treated chickens compared with untreated controls. By contrast, IL-2 and IL-12 mRNAs in IELs, and IL-4, IL-12, and IL-17F transcripts in spleen cells, were decreased in DECX- and/or COBN-treated chickens compared with controls. These results suggest that DECX or COBN, in combination with bacitracin and roxarsone, modulate the development of the chicken post-hatch immune system.