Rebecca E. A. Forder

Quantitative analyses of genes associated with mucin synthesis of broiler chickens with induced necrotic enteritis

Clostridial infection of the intestine can result in necrotic enteritis (NE), compromising production and health of poultry. Mucins play a major role in protecting the intestinal epithelium from infection. The relative roles of different mucins in gut pathology following bacterial challenge are unclear. This study was designed to quantify the expression of mucin and mucin-related genes, using intestinal samples from an NE challenge trial where birds were fed diets with or without in-feed antimicrobials. A method for quantifying mucin gene expression was established using a suite of reference genes to normalize expression data. This method was then used to quantify the expression of 11 candidate genes involved in mucin, inflammatory cytokine, or growth factor biosynthesis (IL-18, KGF, TLR4, TFF2, TNF-α, MUC2, MUC4, MUC5ac, MUC5b, MUC13, and MUC16). The only genes that were differentially expressed in the intestine among treatment groups were MUC2, MUC13, and MUC5ac. Expression of MUC2 and MUC13 was depressed by co-challenge with Eimeria spp. and Clostridium perfringens. Antimicrobial treatment prevented an NE-induced decrease in MUC2 expression but did not affect MUC13. The expression of MUC5ac was elevated in birds challenged with Eimeria spp./C. perfringens compared with unchallenged controls and antimicrobial treatment. Changes to MUC gene expression in challenged birds is most likely a consequence of severe necrosis of the jejunal mucosa.

Differences in intestinal mucin dynamics between germ-free and conventionally reared chickens after mannan-oligosaccharide supplementation

A germ-free (GF) chicken model was used to test 2 hypotheses: 1. microbial colonization of the gastrointestinal tract (GIT) influences mucin gene expression and mucin types; and 2. mannan oligosaccharide (MOS) supplementation affects GIT cells directly, without bacteria mediation, compared with bacterial-mediated effect (i.e., indirectly). Gnotobiotic isolators were used: 1) GF, 2) with a single bacteria population, and 3) conventionalized by exposure to cecal bacterial contents. Each was divided to 2 diet groups: with or without MOS (2 kg/t) for 1 wk. Results show that the absence of bacteria in the GIT caused a reduction in neutral and acidic goblet cell (GC) number and density, an increase in sulfated mucin, absence of sialylated GC, and reduced mucin 2 mRNA expression in the small intestine of GF compared with conventional birds. These results indicate a reduced development of mucin production and secretion in the absence of GIT bacteria implying a less mature small intestine mucosa, supporting our first hypothesis. Results from the single bacteria population group were not conclusive and did not support any of the hypotheses. Supplementation of MOS, regardless of microbial presence, caused a reduction in neutral GC number and density but increased neutral GC area. The MOS caused different effects on acidic mucins in conventional and GF birds, causing a reduction in sialylated GC number (conventional) and a reduction in sulfated GC density (GF), all supporting a direct effect of MOS in GF animals, in addition to an indirect effect via gut microflora.

Effects of necrotic enteritis challenge on intestinal micro-architecture and mucin profile

1. This study investigated the effect of Eimeria spp./Clostridium perfringens induced necrotic enteritis and traditional antibiotic preventatives on intestinal micro-architecture and mucin profile. 2. A total of 600 Cobb 500 broiler chickens were randomly assigned to the following three groups: (i) unchallenged, (ii) challenged, and (iii) zinc bacitracin/monensin (ZnB/monensin) (n = 25 chickens/pen, 8 pens/group). The challenged and ZnB/monensin chickens were individually inoculated with Eimeria acervulina, E. maxima and E. tenella and C. perfringens type A (EHE-NE18) at 9 and 15 d post-hatch respectively, to induce necrotic enteritis. 3. The challenge procedure significantly decreased villus height, increased villus width and increased crypt depth in the challenged compared to the unchallenged chickens. Zinc bacitracin and monensin maintained villus-crypt structure similar to that of the unchallenged chickens. 4. Mucin profile was not affected by Eimeria spp./C. perfringens challenge as demonstrated by periodic acid-Schiff and high iron diamine-alcian blue pH 2·5 staining. Zinc bacitracin and monensin decreased the number of intestinal mucin-containing goblet cells. 5. Lectin histochemistry showed a trend towards greater Arachis hypogea (PNA) reactivity in unchallenged chickens. 6. In summary, Eimeria spp./C. perfringens challenge disrupted intestinal micro-architecture; however, challenge did not appear to affect intestinal mucin profile. Traditional antibiotics, zinc bacitracin and monensin maintained micro-architecture.

Mucin Gene mRNA Levels in Broilers Challenged with Eimeria and/or Clostridium perfringens

SUMMARY The effects of Eimeria (EM) and Clostridium perfringens (CP) challenges on the mRNA levels of genes involved in mucin (Muc) synthesis (Muc2, Muc5ac, Muc13, and trefoil family factor-2 [TFF2]), inflammation (tumor necrosis factor alpha [TNF-&agr;] and interleukin-18 [IL-18]), and metabolic processes (cluster of differentiation [CD]36) in the jejunum of broilers were investigated. Two parallel experiments involving 1) EM challenge and 2) EM and CP challenges were conducted. The first experiment was a 2 × 2 study with 12 birds per treatment (N  =  48) involving fishmeal substitution (25%) in the diet (FM) and EM challenge. The treatments were: Control (FM−, EM−), Fishmeal (FM+, EM−), EM challenge (FM−, EM+), and fishmeal substitution and EM challenge (FM+, EM+). The second experiment was a 2 × 2 × 2 experiment with six birds per treatment (N  =  48) involving fishmeal (FM−, FM+), Eimeria (EM−, EM+), and C. perfringens (CP−, CP+). In both arms of the study, male broilers were given a starter diet for the whole period of 16 days, except those assigned to FM+, where 25% of the starter ration was replaced with fishmeal from days 8 to 14. EM inoculation was performed on day 9 and CP inoculation on days 14 and 15. The EM challenge birds were euthanatized for sampling on day 13; postmortem examination and sampling for the Eimeria plus C. perfringens challenge arm of the study were on day 16. In the Eimeria challenge arm of the study, fishmeal supplementation significantly suppressed the mRNA levels of TNF-&agr;, TFF2, and IL-18 pre-CP inoculation but simultaneously increased the levels of Muc13 and CD36 mRNAs. Birds challenged with Eimeria exhibited increased mRNA levels of Muc13, Muc5ac, TNF-&agr;, and IL-18. In the Eimeria and C. perfringens challenge arm, birds exposed to EM challenge exhibited significantly lower mRNA levels of Muc2 and CD36. The mRNA levels of CD36 were also significantly suppressed by CP challenge. Our results showed that the transcription of mucin synthesis genes in the jejunum of broilers is modulated by fishmeal inclusion in the diet. Furthermore, we show for the first time suppression of CD36 mRNA levels in the intestine of broilers challenged with Eimeria or C. perfringens. RESUMEN Niveles de ARN mensajero de genes de la mucina en pollos desafiados con Eimeria y/o con Clostridium perfringens. Los efectos del desafíos por Eimeria (EM) y por Clostridium perfringens (CP) en los niveles de ARN mensajero de los genes implicados en la síntesis de la mucina (Muc2, Muc5ac, Muc13 y del factor 2 de la familia trefoil [TFF2]), en la inflamación (factor de necrosis tumoral alfa [TNF-&agr;] y la interleucina 18 [IL-18]) y de los procesos metabólicos (grupo de diferenciación [CD] 36) en el yeyuno de pollos de engorde fueron investigados. Se realizaron dos experimentos paralelos que implicaron 1) desafío con Eimeria y 2) desafíos con Eimeria y C. perfringens. El primer experimento fue un estudio de 2 × 2 con 12 aves por tratamiento (N  =  48) que implica la sustitución de la harina de pescado (25%) en la dieta (FM) y el desafío con Eimeria. Los tratamientos fueron: control (FM-, EM-), Harina de pescado (FM+, EM-), desafío con Eimeria (FM-, EM+) y la sustitución de la harina de pescado con desafío con Eimeria (FM+, EM+). El segundo experimento fue un experimento 2 × 2 × 2 con seis aves por tratamiento (N  =  48) que incluyó harina de pescado (FM, FM+), Eimeria (EM- EM+) y C. perfringens (CP-, CP+). En ambas partes del estudio, a los pollos machos se les dio una dieta de iniciación para todo el período de 16 días, excepto a los asignados al grupo FM+, donde se sustituyó el 25% de la ración de iniciación con harina de pescado de los días 8 a 14. El desafío con Eimeria se realizó al día 9 y la inoculación con C. perfringens se realizó en los días 14 y 15. Se les practicó la eutanasia a las aves desafiadas con Eimeria en el día 13 con fines de muestreo; el examen post mortem y el muestreo para la parte desafiada con Eimeria y C. perfringens fue en el día 16. En la parte del estudio con el desafío por Eimeria, la suplementación de la harina de pescado suprimió significativamente los niveles de ARN mensajero de TNF-&agr;, TFF2 y de IL-18 antes de la inoculación con C. perfringens pero a la vez aumentó los niveles de ARN mensajero de Muc13 y CD36. Las aves desafiadas con Eimeria exhibieron aumento de los niveles de ARN mensajero para Muc13, Muc5ac, TNF-&agr; y de IL-18. En la parte del desafío con Eimeria y C. perfringens, las aves expuestas al desafío con Eimeria mostraron niveles significativamente más bajos de ARN mensajero para Muc2 y CD36. Los niveles de ARN mensajero de CD36 también se suprimieron significativamente por el desafío con C. perfringens. Estos resultados muestran que la transcripción de genes de la síntesis de mucina en el yeyuno de pollos de engorde es modulada por la inclusión de harina de pescado en la dieta. Además, se muestra por primera vez la supresión de los niveles de ARN mensajero de CD36 en el intestino de los pollos de engorde desafiados con Eimeria o con C. perfringens.

New biomarkers for intestinal permeability induced by lipopolysaccharide in chickens

Intestinal health is influenced by a complex set of variables involving the intestinal microbiota, mucosal immunity, digestion and absorption of nutrients, intestinal permeability (IP) and intestinal integrity. An increase in IP increases bacterial or toxin translocation, activates the immune system and affects health. IP in chickens is reviewed in three sections. First, intestinal structure and permeability are discussed briefly. Second, the use of lipopolysaccharide (LPS) as a tool to increase IP is discussed in detail. LPS, a glycolipid found in the outer coat of mostly Gram-negative bacteria, has been reported to increase IP in rats, mice and pigs. Although LPS has been used in chickens for inducing systemic inflammation, information regarding LPS effects on IP is limited. This review proposes that LPS could be used as a means to increase IP in chickens. The final section focuses on potential biomarkers to measure IP, proposing that the sugar-recovery method may be optimal for application in chickens.

Use of yeast cell wall extract as a tool to reduce the impact of necrotic enteritis in broilers

The use of a yeast cell wall extract derived from Saccharomyces cerevisiae (Actigen(®)) has been proposed as an alternative to in-feed antibiotics. This experiment was conducted to investigate the efficacy of yeast cell extract as an alternative to zinc bacitracin or salinomycin using a necrotic enteritis challenge model. A feeding study was conducted using 480-day-old male Ross 308 chicks assigned to 48 floor pens. A 2 × 4 factorial arrangement of treatments was employed. The factors were: challenge (- or +) and feed additive (control, zinc bacitracin at 100/50 mg/kg, yeast cell wall extract at 400/800/200 mg/kg, or salinomycin at 60 mg/kg in starter, grower, and finisher, respectively). Diets based on wheat, sorghum, soybean meal, meat and bone meal, and canola meal were formulated according to the Ross 308 nutrient specifications. Birds were challenged using a previously established protocol (attenuated Eimeria spp oocysts) on d 9 and 10(8) to 10(9) Clostridium perfringens (type A strain EHE-NE18) on d 14 and 15). Challenged and unchallenged birds were partitioned to avoid cross contamination. Challenged birds had lower weight gain, feed intake and livability compared to unchallenged birds on d 24 and d 35 (P < 0.05). Birds given zinc bacitracin, yeast cell wall extract, or salinomycin had improved weight gain and livability when compared to control birds given no additives. Challenge × additive interactions were observed for feed intake and weight gain on d 24 and d 35 (P < 0.01). The additives all had a greater positive impact on feed intake, weight gain, and livability in challenged than unchallenged birds. All challenged birds showed higher necrotic enteritis lesion scores in the small intestine sections when compared to unchallenged birds (P < 0.01). Birds fed yeast cell wall extract exhibited increased villus height, decreased crypt depth, and increased villus:crypt ratio when challenged. Yeast cell wall extract, zinc bacitracin, and salinomycin were effective in preventing performance decline from necrotic enteritis in the current study. This study indicates that yeast cell wall extract has promise as a tool for controlling necrotic enteritis.

Intestinal permeability induced by lipopolysaccharide and measured by lactulose, rhamnose and mannitol sugars in chickens.

Increased intestinal permeability (IP) can lead to compromised health. Limited in vivo IP research has been conducted in chickens. The objectives of the current study were to develop a model of increased IP utilizing lipopolysaccharide (LPS Escherichia coli O55:B5) and to evaluate IP changes using the lactulose, mannitol and rhamnose (LMR) sugar permeability test. In addition, fluorescein isothiocyanate dextran (FITC-d), d-lactate, zonula occludens (ZO-1) and diamine oxidase (DAO) permeability tests were employed. Male Ross chickens were reared until day 14 on the floor in an animal care facility and then transferred to individual cages in three separate experiments. In each of experiments 1 and 2, 36 chicks were randomly allocated to receive either saline (control) or LPS (n=18/group). Lactulose, mannitol and rhamnose sugar concentration in blood was measured at 0, 30, 60, 90, 120 and 180 min in experiment 1, at 60, 90 and 120 min in experiment 2 and at 90 min in experiment 3 (n=16/group). Lipopolysaccharide was injected intraperitoneally at doses of 0.5, 1 and 1 mg/kg BW in experiments 1, 2 and 3, respectively, on days 16, 18 and 20, whereas control received sterile saline. On day 21, only birds in experiments 1 and 2 were fasted for 19.5 h. Chicks were orally gavaged with the LMR sugars (0.25 gL, 0.05 gM, 0.05 gR/bird) followed by blood collection (from the brachial vein) as per time point for each experiment. Only in experiment 3, were birds given an additional oral gavage of FITC-d (2.2 mg/ml per bird) 60 min after the first gavage. Plasma d-lactate, ZO-1 and DAO concentrations were also determined by ELISA in experiment 3 (n=10). Administration of LPS did not affect IP as measured by the LMR sugar test compared with control. This was also confirmed by FITC-d and DAO levels in experiment 3 (P>0.05). The plasma levels of d-lactate were decreased (P<0.05). Plasma levels of ZO-1 were increased in the third experiment only and did not change in the first two experiments. Lipopolysaccharide at doses of 0.5 and 1 mg/kg did not increase IP in this model system. In conclusion, the LMR sugar can be detected in blood 90 min after the oral gavage. Further studies are needed for the applicability of LMR sugars tests.

A small-scale, low-cost isolation system for the incubation and rearing of low bacterial load chicks as a model to study microbial-intestinal interactions

Summary A small-scale, economical isolator system was adapted to hatch and raise chicks in a bacteria-free environment as a means to observe bacterial interactions with the intestinal mucosa during early development. The design and construction of flexible plastic isolators for incubation and brooding are described along with methodologies for preparation of eggs for entry into the isolators, incubation and hatching. Two trials were conducted, the first in August 2005 and the second in March 2006. Results from both trials showed no differences in body weights of chicks raised in isolation when compared with those raised conventionally. Growth of bacteria was detected from rectal swabs at day 2 post-hatch, with both trials, showing a light growth of Bacillus sp., coagulase-negative staphylococci and haemolytic streptococcus in trial 1, and a light growth of Bacillus cereus only in trial 2. Although not germfree, the growth of bacteria in chicks raised in isolation was decreased or absent when compared with chicks raised conventionally. Feed was negative for contamination and surface swabs of equipment were also negative until day 3 post-hatch, suggesting possible contamination within the eggs themselves. Despite the presence of bacterial species, the isolator system was successful in producing low bacterial load chicks for comparison studies with conventionally raised chicks.

Effect of restricted feed intake in broiler breeder hens on their stress levels and the growth and immunology of their offspring1

Abstract The prenatal environment has been shown to have significant effects on the lifelong health of offspring in humans and other species. Such effects have not been studied extensively in avian species but could prove important, especially in the case of severe feed restriction imposed on broiler breeder hens to prevent obesity and reduce rate of lay. Feed restriction can potentially affect not only nutrient supply to the embryo but stress hormone levels within the hen. This study investigated the impact of nutrient restriction of the breeder hen on growth rate and immune responses in the progeny with the objective to measure the impact of feed restriction of broiler breeder hens on growth and immune response of the progeny. Broiler breeder hens were feed restricted from 24 wk of age and maintained at three bodyweights; 3.4, 3.6, and 4.0 kg until 43 wk of age and behavioral and physiological measures of stress recorded. Chicks were hatched from each hen treatment and at day 7 vaccinated for infectious bronchitis virus (IBV) and at 16, 18, and 20 d old given an immune challenge of lipopolysaccharide. Growth and immune responses of these birds were then recorded. Sex ratio was affected by hen bodyweight, with a significantly increased proportion of males hatched from heavy hens. Growth rate from 35 to 42 d of age was reduced in male progeny from low bodyweight hens. Female progeny from heavy hens responded to an immune challenge by reduced live weight and increased heterophil: lymphocyte ratio, suggesting a more robust immune response in these birds than in the progeny from lower bodyweight hens. Overall, progeny from heavy hens had increased antibodies at day 35 to the vaccination of IBV compared with progeny of low bodyweight hens, also suggesting an improved immune response in these birds. Breeder hens restricted to the lowest feed level showed behaviors indicative of increased stress (object pecking) and an increased heterophil: lymphocyte ratio. Feed restriction of broiler breeder hens increased indices of stress in hens and resulted in offspring that have reduced growth rate and immune response in a sex-dependent way.

Effects of delayed feeding, sodium butyrate and glutamine on intestinal permeability in newly-hatched broiler chickens

ABSTRACT The aim of the current study was to investigate the effects of delayed feeding, and supplementation with sodium butyrate or glutamine in drinking water, on intestinal permeability (IP) in young broiler chickens. Newly-hatched male chickens (Ross 308) were allocated to four groups comprising Control, 24 h delayed fed (DF), DF supplemented with sodium butyrate (0.1%) in the drinking water and DF supplemented with glutamine (1%) in the drinking water. On days 2, 4 and 7, twelve birds per group were randomly selected, weighed and orally gavaged with fluorescein isothiocyanate dextran (FITC-d) at 2.2 mg / ml / chicken. Serum FITC-d concentration was analysed by spectrophotometry while serum diamine oxidase and d-lactic acid concentrations were analysed by microplate reader. FITC-d concentrations in the Control and DF groups were not statistically different on any day, suggesting that delayed feeding did not affect IP. Additionally, sodium butyrate increased IP compared to DF and Control on day 2 only (p < 0.05), while glutamine increased IP on all days, compared to DF and Control (p < 0.05). Diamine oxidase and d-lactic acid concentrations of all groups were not statistically different.