Seung I. Jang

Effects of direct-fed microbials on growth performance, gut morphometry, and immune characteristics in broiler chickens

This study was conducted to compare growth performance, gut morphometry, and parameters of local and systemic immunity in broiler chickens fed for 22 consecutive days with a diet supplemented with Bacillus spp. as direct-fed microbials (DFM), a commercial product incorporating 3 DFM, or a nonsupplemented diet. Direct-fed microbials did not significantly modify BW gain and most failed to affect serum antibody levels in response to immunization with a recombinant Eimeria protein. However, altered intestinal morphometric measurements were readily apparent in DFM-fed chickens as revealed by increased villus height and crypt depth compared with non-DFM-fed controls. In addition, serum levels of alpha-1-acid glycoprotein as an inflammatory marker were reduced in DFM-fed birds, whereas splenic lymphocyte proliferation, intestine intraepithelial lymphocyte subpopulations, and cytokine mRNA levels in intraepithelial lymphocytes were increased, decreased, or unchanged compared with controls depending on the DFM used. These results provide a rational scientific basis for future studies to investigate DFM as immunomodulating agents to enhance host protective immunity against enteric pathogens in broiler chickens.

Effects of dietary supplementation with phytonutrients on vaccine-stimulated immunity against infection with Eimeria tenella

Two phytonutrient mixtures, VAC (carvacrol, cinnamaldehyde, and Capsicum oleoresin), and MC (Capsicum oleoresin and turmeric oleoresin), were evaluated for their effects on chicken immune responses following immunization with an Eimeria profilin protein. Chickens were fed with a non-supplemented diet, or with VAC- or MC-supplemented diets, immunized with profilin, and orally challenged with virulent oocysts of Eimeria tenella. Immunity against infection was evaluated by body weight, fecal oocyst shedding, profilin antibody levels, lymphocyte recall responses, cytokine expression, and lymphocyte subpopulations. Following immunization and infection, chickens fed the VAC- or MC-supplemented diets showed increased body weights, greater profilin antibody levels, and/or greater lymphocyte proliferation compared with non-supplemented controls. Prior to Eimeria infection, immunized chickens on the MC-supplemented diet showed reduced IFN-γ and IL-6 levels, but increased expression of TNFSF15, compared with non-supplemented controls. Post-infection levels of IFN-γ and IL-6 were increased, while IL-17F transcripts were decreased, with MC-supplementation. For VAC-supplemented diets, decreased IL-17F and TNFSF15 levels were observed only in infected chickens. Finally, immunized chickens fed the MC-supplemented diet exhibited increased MHC class II(+), CD4(+), CD8(+), TCR1+, or TCR2(+) T cells compared with nonsupplemented controls. Animals on the VAC-containing diet only displayed an increase in K1(+) macrophages. In conclusion, dietary supplementation with VAC or MC alters immune parameters following recombinant protein vaccination against avian coccidiosis.

Protective effect of hyperimmune egg yolk IgY antibodies against Eimeria tenella and Eimeria maxima infections.

Avian coccidiosis is caused by several distinct species of Eimeria protozoa and is the major parasitic disease of poultry of economic importance. As an alternative strategy to control avian coccidiosis without using prophylactic medications, we have investigated the efficacy of inducing passive immunity against coccidiosis by orally feeding hyperimmune IgY antibodies. In this study, a commercially available egg yolk powder, Supracox (SC), a purified IgY fraction of egg yolk prepared from hens hyperimmunized with three major species of Eimeria oocysts, were continuously fed to young chicks from hatch. Upon orally infecting these broiler chicks with Eimeria tenella and Eimeria maxima oocysts at 1 week of age, they showed significantly higher body weight gains (P<0.05) compared to the untreated controls. Furthermore, SC-fed chicks showed significantly less intestinal lesions and reduced fecal oocyst output compared to the untreated controls following oral infections with E. tenella and E. maxima. These results provide clear evidence that passive immunization of chicks with hyperimmune egg yolk IgY antibodies provide significant protection against E. tenella or E. maxima infections.

Vaccination with Clostridium perfringens recombinant proteins in combination with Montanide™ ISA 71 VG adjuvant increases protection against experimental necrotic enteritis in commercial broiler chickens

This study was performed to compare four Clostridium perfringens recombinant proteins as vaccine candidates using the Montanide™ ISA 71 VG adjuvant in an experimental model of necrotic enteritis. Broiler chickens were immunized subcutaneously with purified clostridial recombinant NetB toxin, pyruvate: ferredoxin oxidoreductase (PFO), α-toxin, or elongation factor-Tu (EF-Tu), or with vehicle control, in conjunction with ISA 71 VG, and intestinal lesion scores, body weight gains, NetB toxin and PFO antibody levels, and proinflammatory cytokine and chemokine levels were measured as outcomes of protection following oral co-infection with C. perfringens and Eimeria maxima. Birds immunized with all recombinant proteins plus ISA 71 VG showed significantly reduced gut lesions compared with the ISA 71 VG-only group. Birds immunized with NetB toxin or PFO plus ISA 71 VG exhibited significantly increased body weight gains compared with the ISA 71 VG alone group. Greater NetB toxin antibody titers were observed in the NetB/ISA 71 VG group, and greater PFO antibody titers were evident in the PFO/ISA 71 VG group, each compared with the other three vaccine/adjuvant groups. Finally, decreased levels of gene transcripts encoding interleukin-8, tumor necrosis factor superfamily 15, and LPS-induced TNF-α factor were observed in the intestinal lymphocytes of chickens immunized with NetB toxin, PFO, α-toxin, and/or EF-Tu in the presence of ISA 71 VG compared with ISA 71 VG alone. All parameters evaluated were equal in co-infected chickens given ISA 71 VG alone compared with infected/adjuvant-free birds, indicating that the adjuvant itself did not have a disease protective effect. These results suggest that vaccination with clostridial recombinant proteins, particularly NetB toxin or PFO, in combination with ISA 71 VG enhances protective immunity against experimental necrotic enteritis in broiler chickens.

Dietary supplementation of young broiler chickens with Capsicum and turmeric oleoresins increases resistance to necrotic enteritis

The Clostridium-related poultry disease, necrotic enteritis (NE), causes substantial economic losses on a global scale. In the present study, a mixture of two plant-derived phytonutrients, Capsicum oleoresin and turmeric oleoresin (XT), was evaluated for its effects on local and systemic immune responses using a co-infection model of experimental NE in commercial broilers. Chickens were fed from hatch with a diet supplemented with XT, or with a non-supplemented control diet, and either uninfected or orally challenged with virulent Eimeria maxima oocysts at 14 d and Clostridium perfringens at 18 d of age. Parameters of protective immunity were as follows: (1) body weight; (2) gut lesions; (3) serum levels of C. perfringens α-toxin and NE B-like (NetB) toxin; (4) serum levels of antibodies to α-toxin and NetB toxin; (5) levels of gene transcripts encoding pro-inflammatory cytokines and chemokines in the intestine and spleen. Infected chickens fed the XT-supplemented diet had increased body weight and reduced gut lesion scores compared with infected birds given the non-supplemented diet. The XT-fed group also displayed decreased serum α-toxin levels and reduced intestinal IL-8, lipopolysaccharide-induced TNF-α factor (LITAF), IL-17A and IL-17F mRNA levels, while cytokine/chemokine levels in splenocytes increased in the XT-fed group, compared with the animals fed the control diet. In conclusion, the present study documents the molecular and cellular immune changes following dietary supplementation with extracts of Capsicum and turmeric that may be relevant to protective immunity against avian NE.

Induction of passive immunity in broiler chickens against Eimeria acervulina by hyperimmune egg yolk immunoglobulin Y

The protective effect of hyperimmune IgY fraction of egg yolk prepared from hens hyperimmunized with multiple species of Eimeria oocysts on experimental coccidiosis was evaluated in young broilers. Chickens were continuously fed from hatch with a standard diet containing hyperimmune IgY egg yolk powder or a nonsupplemented control diet and orally challenged at d 7 posthatch with 5.0 x 10(3) sporulated Eimeria acervulina oocysts. Body weight gain between d 0 and 10 and fecal oocyst shedding between d 5 and 10 postinfection were determined as parameters of protective immunity. Chickens given 10 or 20% hyperimmune IgY egg yolk powder showed significantly increased BW gain and reduced fecal oocyst shedding compared with control birds fed the nonsupplemented diet. In another trial, lower IgY concentrations (0.01, 0.02, and 0.05%) were used to treat birds with 1.0 x 10(4) oocysts of E. acervulina. Total oocyst shedding was significantly (P < 0.05) reduced in chickens fed the 0.02 and 0.05% hyperimmune IgY supplemented-diets compared with animals fed the nonsupplemented diet. Similarly, chickens fed 0.5% of hyperimmune IgY egg yolk powder diet and challenged with 1.0 x 10(4) oocysts exhibited reduced oocyst shedding compared with the control birds given 0.5% of IgY from nonimmunized hen eggs, although BW gain was not affected. We conclude that passive immunization of chickens with anti-coccidia IgY antibodies provide protective immunity against coccidiosis challenge infection.

Effect of Bacillus-based direct-fed microbials on Eimeria maxima infection in broiler chickens

The effect of dietary Bacillus-based direct-fed microbials (DFMs; eight single strains designated as Bs2084, LSSAO1, 3AP4, Bs18, 15AP4, 22CP1, Bs27, and Bs278, and one multiple-strain DFM product [AVICORR]) on growth performance, intestinal lesions, and innate and acquired immunities were evaluated in broiler chickens following Eimeria maxima (EM) infection. EM-induced reduction of body weight gain and intestinal lesions were significantly decreased by addition of 15AP4 or Bs27 into broiler diets compared with EM-infected control birds. Serum nitric oxide levels were increased in infected chickens fed with Bs27, but lowered in those given Bs2084, LSSAO1, 3AP4 or 15AP4 compared with the infected controls. Recombinant coccidial antigen (3-1E)-stimulated spleen cell proliferation was increased in chickens given Bs27, 15AP4, LSSAO1, 3AP4, or Bs18, compared with the infected controls. Finally, all experimental diets increased concanavalin A-induced splenocyte mitogenesis in infected broilers compared with the nonsupplemented and infected controls. In summary, dietary Bacillus subtilis-based DFMs reduced the clinical signs of experimental avian coccidiosis and increased various parameters of immunity in broiler chickens in a strain-dependent manner.

Dietary Curcuma longa enhances resistance against Eimeria maxima and Eimeria tenella infections in chickens

The effects of dietary supplementation with an organic extract of Curcuma longa on systemic and local immune responses to experimental Eimeria maxima and Eimeria tenella infections were evaluated in commercial broiler chickens. Dietary supplementation with C. longa enhanced coccidiosis resistance as demonstrated by increased BW gains, reduced fecal oocyst shedding, and decreased gut lesions compared with infected birds fed a nonsupplemented control diet. The chickens fed C. longa-supplemented diet showed enhanced systemic humoral immunity, as assessed by greater levels of serum antibodies to an Eimeria microneme protein, MIC2, and enhanced cellular immunity, as measured by concanavalin A-induced spleen cell proliferation, compared with controls. At the intestinal level, genome-wide gene expression profiling by microarray hybridization identified 601 differentially expressed transcripts (287 upregulated, 314 downregulated) in gut lymphocytes of C. longa-fed chickens compared with nonsupplemented controls. Based on the known functions of the corresponding mammalian genes, the C. longa-induced intestinal transcriptome was mostly associated with genes mediating anti-inflammatory effects. Taken together, these results suggest that dietary C. longa could be used to attenuate Eimeria-induced, inflammation-mediated gut damage in commercial poultry production.

Bacillus subtilis-based direct-fed microbials augment macrophage function in broiler chickens

The present study was conducted to evaluate the function of Bacillus subtilis-based direct-fed microbials (DFMs) on macrophage functions, i.e., nitric oxide (NO) production and phagocytosis in broiler chickens. DFMs used in this study were eight single strains designated as Bs2084, LSSAO1, 3AP4, Bs18, 15AP4, 22CP1, Bs27, and Bs278, and one multiple strain DFM product (Avicorr™) containing equal amount of Bs2084, LSSAO1 and 15AP4. NO concentrations were monitored in plasma and in the supernatants from the peripheral blood-derived monocytic cells (PBMC)-derived macrophages stimulated by either chicken recombinant interferon gamma (IFNγ) or lipopolysaccharide (LPS) from Escherichia coli or Salmonella typhi. In addition, phagocytosis of fluorescent beads and green fluorescent protein (GFP)-labeled Salmonella by PBMC-derived macrophage was assayed. Plasma NO levels were significantly higher in groups given 3AP4 or Bs27 diets compared with the control group at days 7 and 14. NO production by PBMC-derived macrophages stimulated with IFNγ or LPS was apparent, although the effect was strain-dependent. Phagocytosis of fluorescent beads or GFP-labeled Salmonella by macrophages was augmented in groups on DFM-supplemented diets compared with those fed the control diet. This study describes the immunomodulatory effects of Bacillus-based DFMs on innate immunity in broiler chickens.

High-throughput gene expression analysis of intestinal intraepithelial lymphocytes after oral feeding of carvacrol, cinnamaldehyde, or Capsicum oleoresin

Among dietary phytonutrients, carvacrol, cinnamaldehyde, and Capsicum oleoresin are well known for their antiinflammatory and antibiotic effects in human and veterinary medicine. To further define the molecular and genetic mechanisms responsible for these properties, broiler chickens were fed a standard diet supplemented with either of the 3 phytochemicals and intestinal intraepithelial lymphocytes were examined for changes in gene expression by microarray analysis. When compared with chickens fed a nonsupplemented standard diet, carvacrol-fed chickens showed altered expression of 74 genes (26 upregulated, 48 downregulated) and cinnamaldehyde led to changes in the levels of mRNAs corresponding to 62 genes (31 upregulated, 31 downregulated). Most changes in gene expression were seen in the Capsicum-fed broilers with 98 upregulated and 156 downregulated genes compared with untreated controls. Results from the microarray analysis were confirmed by quantitative real-time PCR with a subset of selected genes. Among the genes that showed >2.0-fold altered mRNA levels, most were associated with metabolic pathways. In particular, with the genes altered by Capsicum oleoresin, the highest scored molecular network included genes associated with lipid metabolism, small molecule biochemistry, and cancer. In conclusion, this study provides a foundation to further investigate specific chicken genes that are expressed in response to a diet containing carvacrol, cinnamaldehyde, or Capsicum oleoresin.