W. I. Muir

Immunity, vaccination and the avian intestinal tract

Defence of the intestinal mucosal surface from enteric pathogens is initially mediated by secretory IgA (SIgA). As oral immunization of non-replicating antigen induces minimal SIgA antibody titers, novel immunization strategies which selectively induce mucosal immune responses in mammals are now being assessed in chickens. The strategies reviewed include the route of antigen delivery, the incorporation of antigenic components in delivery vehicles, the inclusion of immunomodulators in the vaccine formula or in the diet, and manipulation of intestinal microflora. The differences in anatomical organization and immunological mechanisms between birds and mammals must be considered when manipulating avian intestinal immunity with the latest immunotechnologies developed for mammals. Our knowledge of the function and functioning of the avian mucosal system is discussed. Progress in our understanding of this system, the location of precursor IgA B cells and antigen sampling by these sites is not as advanced as knowledge of the mammalian system, highlighting the need for ongoing research into the avian application of novel vaccination strategies.

Dietary supplementation with vitamin E modulates avian intestinal immunity

The effect of dietary vitamin E on immunoglobulin A (IgA) antibody production, which acts as the first line of defence at the intestinal mucosa, has not been evaluated in chickens. In the present study the impact of the inclusion of supplementary levels of vitamin E to the diet, on total and antigen-specific IgA antibody titres, T-cell subsets and Ia+ cells, was assessed. From hatching, chickens received a maize-based diet which was supplemented with either 25, 250, 2500 or 5000 mg dl-alpha-tocopherol acetate/kg. Primary immunisation with tetanus toxoid (T. toxoid) emulsified in a vegetable oil-in-water adjuvant was administered by the intraperitoneal route at 21 d of age. At 35 d of age all birds received an oral booster vaccination of T. toxoid. Significantly higher total IgA antibody titres were present in the day 42 intestinal scrapings of birds receiving the 5000 mg/kg vitamin E-supplemented diet (VESD) (P=0.05) and a notable increase was observed in birds receiving the 250 mg/kg VESD (P=0.06). At days 21 and 42 total serum IgA antibody titres of birds receiving the 250 mg/kg VESD was significantly higher (P<0.05) than the control birds. Following immunisation with T. toxoid, birds receiving the 250 and the 5000 mg/kg VESD had elevated anti-T. toxoid IgA antibody titres in final day intestinal scrapings, which, for the latter group was statistically significant (P=0.02). Both of these groups also demonstrated increased titres of anti-T. toxoid IgA in the serum at day 42. Birds receiving the 250 mg/kg VESD exhibited a notable increase in the percentage of T-helper cells and Ia+ cells in peripheral blood on day 26. The results illustrate the potential for some levels of dietary vitamin E supplementation to act as an immunomodulator of total and antigen-specific IgA antibody.

Induction of specific IgA responses in rats after oral vaccination with biodegradable microspheres containing a recombinant protein

Diseases which affect mucosal surfaces cause considerable mortality and morbidity. New vaccine technologies are now available which justify a reappraisal of oral delivery not only for infectious disease control but also to control mucosal physiological processes such as fertility. Biodegradable microspheres have been investigated for their use as an oral delivery vehicle in rats using a recombinant antigen derived from fox sperm. Unencapsulated antigen administered in saline by the oral route produced a negligible response although an improved response was obtained if administered directly into the duodenum. This response was considerably enhanced if Peyers patch (PP) priming was performed by direct injection of antigen in Freunds complete adjuvant (FCA) prior to intraduodenal (ID) delivery. In contrast, microencapsulated antigen given orally produced a substantial response, which was predominantly IgA specific, and almost equal in magnitude to that obtained by PP priming and ID boosting with native antigen. Direct ID delivery produced a similar response but when PP were primed with microencapsulated antigen in FCA the response to ID boosting was greater than with any of the other protocols investigated. These data demonstrate the efficacy of biodegradable microspheres in producing an IgA antibody response following oral vaccination.

The effect of nutritional status and muscle fiber type on myogenic satellite cell fate and apoptosis

Satellite cells (SC) are multipotential stem cells that can be induced by nutrition to alter their cellular developmental fate, which may vary depending on their fiber type origin. The objective of the current study was to determine the effect of restricting protein synthesis on inducing adipogenic transdifferentiation and apoptosis of SC originating from fibers of the fast glycolytic pectoralis major (p. major) and fast oxidative and glycolytic biceps femoris (b. femoris) muscles of the chicken. The availability of the essential sulfur amino acids Met and Cys was restricted to regulate protein synthesis during SC proliferation and differentiation. The SC were cultured and treated with 1 of 6 Met/Cys concentrations: 60/192, 30/96 (control), 7.5/24, 3/9.6, 1/3.2, or 0/0 mg/L. Reductions in Met/Cys concentrations from the control level resulted in increased lipid staining and expression of the adipogenic marker genes peroxisome proliferator-activated receptor gamma and stearoyl-CoA desaturase during differentiation in the p. major SC. Although b. femoris SC had increased lipid staining at lower Met/Cys concentrations, there was no increase in expression of either adipogenic gene. For both muscle types, SC Met/Cys, concentration above the control increased the expression of peroxisome proliferator-activated receptor gamma and stearoyl-CoA desaturase during differentiation. As Met/Cys concentration was decreased during proliferation, a dose-dependent decline in all apoptotic cells occurred except for early apoptotic cells in the p. major, which had no treatment effect (P < 0.05). During differentiation, decreasing Met/Cys concentration caused an increase in early apoptotic cells in both fiber types and no effect on late apoptotic cells except for an increase in the p. major 7.5/24 mg/L of Met/Cys treatment. In general, the viability of the SC was unaffected by the Met/Cys concentration except during proliferation in the p. major 0/0 mg/L of Met/Cys treatment, which increased SC viability. These data demonstrate the effect of nutrition on SC transdifferentiation to an adipogenic lineage and apoptosis, and the effect of fiber type on this response in an in vitro context.

The effect of nutritional status on myogenic gene expression of satellite cells derived from different muscle types

Satellite cells (SC) are a multipotential stem cell population responsible for facilitating posthatch muscle fiber hypertrophy. The proliferation and differentiation of SC is sensitive to nutritional regimen, and the SC response to nutrition varies depending upon their muscle type of origin. The objective of the current study was to determine the effect of altering protein synthesis on the expression of several key genes regulating SC activity and the effect of muscle type. Satellite cells isolated from the fast glycolytic pectoralis major and the fast oxidative and glycolytic biceps femoris were studied. These genes included the myogenic regulatory factors myogenic determination factor 1 (MyoD) and myogenin, the cell-membrane associated proteoglycans syndecan-4 and glypican-1, the extracellular matrix proteoglycan decorin, and the transcription factor paired box 7. Protein synthesis potential varied by the concentration of the sulfur amino acids Met and Cys during SC proliferation and differentiation. The SC were cultured and treated with 1 of 6 Met/Cys concentrations: 60/192, 30/96 (control), 7.5/24, 3.0/9.6, 1.0/3.2, or 0/0 mg/L. A consistent pattern of gene expression emerged following Met/Cys manipulation as increasing reductions in mRNA expression for all genes were observed as Met/Cys concentration decreased, whereas increased Met/Cys concentration caused either no change or had a small negative effect on mRNA expression. Reduced paired box 7 expression would limit myogenic specification of SC, whereas decreased myogenic regulatory factor expression would affect subsequent myogenic development of the SC. Decreased levels of decorin affect SC response to growth factors like myostatin and transforming growth factor β, and extracellular matrix organization. These data highlight the importance of nutrition on the expression of genes critical for satellite cell activation, proliferation and differentiation, and growth factor signal transduction.

The effect of nutritional status on myogenic satellite cell proliferation and differentiation

Early posthatch satellite cell (SC) mitotic activity is a critical component of muscle development and growth. Satellite cells are stem cells that can be induced by nutrition to follow other cellular developmental pathways. The objective of the current study was to determine the effect of restricting protein synthesis on the proliferation and differentiation of SC, using variable concentrations of Met and Cys to modulate protein synthesis. Broiler pectoralis major SC were cultured and treated with 1 of 6 different Met/Cys concentrations: 60/192, 30/96 (control), 7.5/24, 3/9.6, 1/3.2, or 0/0 mg/L. The effect of Met/Cys concentration on SC proliferation and differentiation was measured, and myonuclear accretion was measured by counting the number of nuclei per myotube during differentiation. The 30/96 mg/L Met/Cys treatment resulted in the highest rate of proliferation compared with all other treatments by 72 h of proliferation (P < 0.05). Differentiation was measured with Met/Cys treatments only during proliferation and the cultures receiving normal differentiation medium (R/N), normal proliferation medium and differentiation medium with variable Met/Cys (N/R), or both proliferation and differentiation receiving variable Met/Cys treatments (R/R). Differentiation responded in a dose-dependent manner to Met/Cys concentration under all 3 of these treatment regimens, with a degree of recovery in the R/N regimen cells following reinstatement of the control medium. Reductions in both proliferation and differentiation were more pronounced as Met/Cys concentrations were further reduced, whereas increased differentiation was observed under the increased Met/Cys concentration treatment when applied during differentiation in the N/R and R/R regimens. The number of nuclei per myotube was significantly decreased in the severely Met/Cys restricted treatments (P < 0.05). These data demonstrate the sensitivity of pectoralis major SC to nutritional availability and the importance of optimal nutrition during both proliferation and differentiation for maximizing SC activity, which will affect subsequent muscle mass accretion.

Investigation of the site of precursors for IgA-producing cells in the chicken intestine

Bursectomized chicks received lymphocyte single cell suspensions harvested from the bursa of Fabricius (BF), ileal lymphoid aggregate (ILA), caecal tonsils (CT), spleen and peripheral blood. Four days after cell transfer, repopulation of the duodenal and CT lamina propria in age‐matched recipient bursectomized chickens with IgA‐secreting plasma cells was determined. The results indicate the highest level of reconstitution with cells derived from BF, but substantial numbers of IgA‐secreting plasma cells were also observed in a number of birds that received lymphocytes originating from the ILA and CT.

Intraperitoneal immunization promotes local intestinal immunity in chickens

The limited success in stimulating protective immunity in the intestine by traditional vaccination approaches has led to the search for novel strategies to improve intestinal immunity. In mammalian species we have demonstrated that whereas oral immunization produces poor intestinal responses, immunization by the intraperitoneal route using appropriate formulations is an effective means of priming the intestinal lymphoid tissue for an enhanced mucosal response to orally administered antigen. In the experiments reported here we extend these findings to chickens and compare the intestinal IgA response to a non-replicating antigen administered by the oral route alone or in oil emulsion formulations administered subcutaneously or intraperitoneally with or without oral boosting. Despite repeated daily dosing, oral immunization of unprimed animals produced only a small intestinal response. However, priming by the intraperitoneal route with antigen in either Freunds adjuvant or a biodegradable oil-in-water emulsion (Auspharm adjuvant) resulted in a markedly improved response, especially when additional oral boosting was given. The data also demonstrate that Quil A, a saponin derivative, further boosts the response when co-administered with the oil emulsion intraperitoneally. This study illustrates the efficacy of systemic immunization by the intraperitoneal route in priming for an intestinal IgA response in chickens.

Interferon-γ is expressed in both gut and spleen during Eimeria tenella infection.

The effect of primary infection and subsequent challenge with Eimeria tenella on interferon-γ (IFN-γ ) production in the spleen and caeca of Light Sussex chickens was assessed. The ability of splenocytes to proliferate and produce IFN-γ in response to mitogen stimulation ex vivo was determined. Differences in the kinetics of IFN-γ production suggested that the spleens of infected birds contain a subpopulation of T cells, primed to produce IFN-γ , which migrate from the spleen in response to a secondary infection. IFN-γ mRNA expression was detected by hybridization of an anti-sense chicken IFN-γ riboprobe to splenic sections from infected birds and caecal sections from challenged birds. Hybridization was to T-cell areas in the spleen, and to cells in the lamina propria and intraepithelial compartments of the caecum. This is the first direct demonstration of IFN-γ expression in chickens at the site of E. tenella infection, and also the first indication that IFN-γ may be involved in the immune response to challenge.

Influence of chick hatch time and access to feed on broiler muscle development

The effect of hatch time and the timing of access to feed on growth rate and breast muscle development was assessed in Ross 308 broiler chickens. Chicks were removed from the incubator upon hatching, and classified as early (EH), midterm (MH), or late (LH) hatchers, based on the duration of their incubation. Feed and water were available either immediately at hatch, or 24 h after the conclusion of the hatch period. Hatchling body weight was uniform regardless of hatch time. Subsequently, bodyweight was increased in EH compared to LH birds following immediate access to feed, until 7 d in female, and 14 d in male birds. Relative breast weight was increased until 28 d in birds with immediate access to feed, and also EH and MH birds regardless of access to feed. Pectoralis major muscle morphology and expression of the myogenic regulatory factors myogenic determination factor 1 (MYOD1) and myogenin, and the proteoglycans syndecan-4, glypican-1, and decorin were measured. Myogenin and glypican-1 stimulate satellite cell (SC) differentiation. Glypican-1 expression was unaffected by treatment. A late increase in myogenin expression was observed in MH birds with delayed access to feed, and all LH birds. Syndecan-4 and MYOD1, expressed in proliferating SC, and decorin, which stimulates satellite cell proliferation and differentiation, were variably upregulated in the first wk posthatch in the same birds. These data suggest SC were activated and proliferating, but had reduced differentiation in later hatching and feed deprived birds. Conversely, EH birds with immediate access to feed had maximal myofiber width at 7 d, while fiber width was increased in birds with immediate access to feed compared to those with delayed access to feed through 40 d of age. These results demonstrate that delaying chick access to feed for 24 h upon removal from the incubator will impair muscle growth. Additionally, hatch time influences muscle development, with accelerated muscle growth in EH and MH, compared to LH birds, irrespective of access to feed.