B. Laarveld

Immunomodulatory Activities of Oat β-Glucan In Vitro and In Vivo

Previous studies have shown that β‐glucans extracted from yeast or fungi potentiate immune responses. In the present study, the immunomodulatory activities of β‐(1→3, 1→4)‐glucan, derived from oats, were investigated. The ability of oat β‐glucan (OβG) to stimulate IL‐1 and TNF‐α release from murine peritoneal macrophages and the murine macrophage cell line P338D1, was assessed. In vitro stimulation of macrophages with OβG resulted in the production of IL‐1 in a dose and time‐dependent manner, whereas only small amounts of TNF‐α could be detected in the culture supernatants. OβG also induced the production of IL‐2, IFN‐γ and IL‐4 secretion in a dose‐dependent manner in cultured spleen cells. The intraperitoneal administration of OβG in mice resulted in the accumulation of leucocytes, predominantly macrophages, in the peritoneal cavity. Furthermore, OβG was tested for its ability to enhance non‐specific resistance to a bacterial challenge in mice. Survival of mice challenged with Staphylococcus aureus was enhanced by a single intraperitoneal administration of 500 μg of OβG 3 days prior to bacterial challenge. In conclusion, these studies demonstrated that OβG possesses immunomodulatory activities capable of stimulating immune functions both in vitro and in vivo.

Effects of Commensal Bacteria on Intestinal Morphology and Expression of Proinflammatory Cytokines in the Gnotobiotic Pig

A germ-free neonatal pig model was established to determine the effects of bacterial colonization by different species on small intestinal morphology and proinflammatory cytokine gene expression. Two experimental groups of 16 pigs were aseptically delivered by cesarian section and allocated into 4 gnotobiotic isolators. Pigs were either maintained germ-free (GF), or were orally inoculated with either a single strain of nonpathogenic Escherichia coli (EC) or Lactobacillus fermentum (LF) or conventionalized with adult porcine feces (CV). After 13 days tissue samples were collected at 5 regions corresponding to 5%, 25%, 50%, 75%, and 95% of the small intestine (SI) length. In Experiment 2, the GF isolator became contaminated with Staphylococcus epidermidis (SE). In general, intestinal responses to bacterial colonization were similar among GF, LF, and SE pigs, and intestinal responses in EC pigs were more similar to CV pigs. Responses to bacterial colonization were most pronounced in the distal SI regions (50%–95%), suggesting that nonmicrobiai factors may be more important in the proximal SI. Relative to CV pigs, the distal intestines of GF, LF, and SE pigs were characterized by long villi, shallow crypts, Increased relative intestinal mass, and decreased lamina propria cellularity, whereas SI morphology was intermediate in EC pigs. Relative expression of proinflammatory cytokines interleukin-1β (IL-1β) and IL-6 generally increased distally in the SI and was highest in EC and CV pigs. We observed regional variation in SI morphology and proinflammatory cytokine expression, which differed with bacterial species. This study demonstrates that bacterial species differentially affect intestinal morphology and expression of proinflammatory cytokines and suggests that neonatal bacterial colonization patterns may have long-term effects on intestinal health and development

Isolation and evaluation of immunological adjuvant activities of saponins from Polygala senega L.

We have identified saponins in the root of Polygala senega L., a plant indigenous to the Canadian prairies, which display immunopotentiation activity to protein and viral antigens. By two-step extraction and hemolytic activity-guided fractionation by silica flush chromatography six saponin fractions were generated and their HPLC profiles determined. Two dominant fractions, designated as PS-1 and PS-2, were tested for adjuvant activity in mice immunized with ovalbumin, and hens immunized with rotavirus. The resulting adjuvant activity was compared with that of Quil A saponin. The P. senega saponins increased specific antibody levels to the antigens, in both mice and hens. In mice, there was a preferential increase of the IgG2a subclass, and upon in vitro secondary antigen stimulation, high IL-2 and IFN-gamma levels were observed in spleen cell cultures from P. senega saponins-immunized animals. The saponins were tested for their toxicity by lethality in mice and were found to be less toxic at the same dose than their counterpart Quil A. The results of this study indicated the potential of P. senega saponins as vaccine adjuvants to increase specific immune responses.

β-Glucan, extracted from oat, enhances disease resistance against bacterial and parasitic infections

The effect of beta-glucan, extracted from oats, on the enhancement of resistance to infections caused by Staphylococcus aureus and Eimeria vermiformis was studied in mice. In vitro study using macrophages isolated from the peritoneal cavity showed that beta-glucan treatment significantly enhanced phagocytic activity. In vivo study further demonstrated that beta-glucan treatment induced a significant (P<0.05) protection against the challenge with 5 x 10(8) of S. aureus in mice. Fecal oocyst shedding in the C57BL/6 mice infected with E. vermiformis was diminished by beta-glucan treatment by 39.6% in intraperitoneal and 28.5% in intragastric group compared to non-treated control. Patency period was shorter and antigen (sporozoites and merozoites) specific antibodies were significantly (P<0.05-0.01) higher in beta-glucan-treated group compared to non-treated control group. There were an increasing number of splenic IFN-gamma-secreting cells in glucan-treated group via intraperitoneal route, which might be responsible for the enhancement of the disease resistance. Glucan treatment was able to effectively change the lymphocytes population (Thy 1.2(+), CD4(+) and CD8(+) cells) in the mesenteric lymph nodes and Peyers patches in mice infected with E. vermiformis. In conclusion, the oral or parenteral oat beta-glucan treatment enhanced the resistance to S. aureus or E. vermiformis infection in the mice.

Sub-clinical necrotic enteritis in broiler chickens: Novel etiological consideration based on ultra-structural and molecular changes in the intestinal tissue

The present study revealed several previously not recognized etiological details in the development of necrotic enteritis (NE) in broilers. We provide evidence that the pathological process leading to mucosal epithelium necrosis follows morphologically distinct phases commencing at the basal domain of the mucosal epithelium and then progressively invading the entire lamina propria. Initially mucosal epithelium appears normal, but as the pathological changes progress throughout the lamina propria, the adjacent enterocytes begin to show features of necrotic cell death and the necrotic process of the epithelium progresses from being focal to locally extensive. Ultra-structural examination showed that primary changes occur at the level of basal and lateral domains of the enterocytes, whereas the apical domain of enterocytes remains intact even in the face of advanced necrotic changes. This indicates that the mucosal necrosis does not result from direct damage to the mucosal epithelium. Rather, the necrotic death of enterocytes is a consequential effect of the destruction of lamina propria, the extra-cellular matrix, and intercellular junctions. The nature of these morphological changes indicates that initiation of the pathological process leading to NE involves proteolytic factors affecting the extra-cellular matrix and cellular junctions. Further studies revealed that, indeed, the elevated activity of collagenolytic enzymes in the mucosal milieu and in intestinal tissue represents an integral component of the pathological process leading to NE. In the first instance we discovered that Clostridium perfringens strains isolated from field cases of NE secrete several potent collagenolytic enzymes. In the second instance we observed that, in comparison to controls, broilers challenged with C. perfringens isolated from field cases of NE show high levels of several collagenolytic enzymes in the intestinal tissue. A major component of the overall collagenolytic activity detected in the intestinal tissue was identified by zymography as matrix metalloproteinases (MMPs). Dominant activity was associated with MMP-2. We confirmed using immuno-histochemistry that this enzyme is expressed at high levels in mucosal tissue showing signs of NE. The high levels of collagenolytic activities, in particular associated with MMP-2, demonstrated in our studies are consistent with the nature of morphological changes observed primarily in extra-cellular matrix (ECM) at the basal domain of enterocytes, as well lateral domains of enterocytes. The lack of changes at the level of apical domain of mucosal epithelium indicates that the lipolytic aspect of alpha toxin in NE is not an essential factor in primary lesions development. Taken together, our findings indicate that the early lesions leading to NE are associated with virulence factors that induce proteolytic activity, rather than lipolytic activity.

Immunomodulatory Effects of Oat β-Glucan Administered Intragastrically or Parenterally on Mice Infected with Eimeria vermiformis

The immunostimulatory effect of intragastrically or parenterally administered β‐(1→3; 1→4) glucan, extracted from oats (OβG), on disease resistance to Eimeria vermiformis was studied in C57BL/6 mice. Multiple administrations of OβG by intragastric or subcutaneous routes reduced fecal oocyst shedding compared to the non‐treated control group. The administration of OβG by subcutaneous route resulted in higher levels of total serum immunoglobulins and antigen (sporozoite and merozoite)‐specific immunoglobulins as compared with the non‐treated group. To evaluate the effect of a single subcutaneous dose, groups of mice were treated with OβG 2 days before E. vermiformis infection, at the time of infection and at 2 or 6 days after infection. From day 11 post‐infection the oocyst discharge was significantly diminished (P< 0.05‐0.01) in the OβG‐treated groups, except in those treated 6 days after infection, as compared to the non‐treated control group. The proliferative responses to E. vermiformis sporozoite antigen of lymphocytes isolated from the spleen were significantly increased (P < 0.05) when OβG was administered 2 days before or at the time of E. vermiformis infection. Lymphocyte proliferative responses to merozoite antigen were not influenced by treatment. In conclusion, OβG appeared to up‐regulate immune mechanisms and provide enhanced resistance against eimerian coccidiosis in mice.

β-(1→3, 1→4) Oat glucan enhances resistance to Eimeria vermiformis infection in immunosuppressed mice

The effect of intragastrically or parenterally administered beta-glucan, extracted from oats, on the enhancement of disease resistance to Eimeria vermiformis was studied in C57BL/6 mice. Groups of mice were immunosuppressed with dexamethasone (DXM), infected with oocysts of E. vermiformis and treated with oat beta-glucan by the intragastric (i.g.) or subcutaneous (s.c.) routes. Faecal oocyst shedding was reduced in the beta-glucan-treated groups compared to the non-treated group. Immunosuppressed mice which received no beta-glucan treatment showed more severe clinical signs of the disease and a 50% mortality, while minimal clinical signs and no mortality were recorded in the beta-glucan-treated groups. Total IgG, IgG1, IgG2a, IgM and IgA immunoglobulins in the serum of beta-glucan-treated groups were overall higher than those in the non-treated group. Specific IgG anti-sporozoite and merozoite immunoglobulins in serum were significantly higher in the beta-glucan-treated groups than in the non-treated animals. No significant differences were found in the levels of intestinal IgA anti-sporozoite and anti-merozoite immunoglobulins. IFN-gamma- and IL-4-secreting cells, in response to sporozoite antigen, were detected in the spleen and mesenteric lymph nodes of the beta-glucan-treated groups only. In conclusion, the i.g. and s.c. oat beta-glucan treatment increased the resistance to E. vermiformis infection in immunosuppressed mice.

Dietary amino acids affect intestinal Clostridium perfringens populations in broiler chickens

An experiment was performed to examine the effect of protein source and dietary amino acid profile on intestinal levels of C. perfringens in broiler chickens. Broiler chickens (age = 14 d; n = 192) were fed diets containing 400 g kg-1 crude protein with fish meal, meat/bone meal, feather meal, corn gluten meal, soy protein concentrate, pea protein concentrate, or potato protein concentrate as the primary protein source along with a control diet containing 230 g kg-1 crude protein. The birds were orally inoculated daily, with 1 mL (~1.0 × 108 CFU mL-1) of an overnight culture of C. perfringens between 14 and 21 d of age, killed at 28 d of age and C. perfringens numbers in ileum and cecum were enumerated. Birds fed fish meal, meat/bone meal, feather meal and potato protein concentrate had significantly higher intestinal C. perfringens counts than the birds fed corn gluten meal, soy or pea protein concentrates or the control diet (P < 0.05). The glycine content of the diets and ileal contents was significant...

Adjuvant action of Chenopodium quinoa saponins on the induction of antibody responses to intragastric and intranasal administered antigens in mice

Saponins extracted from the seed of Chenopodium quinoa (quinoa) were studied for their ability to act as mucosal adjuvants upon their intragastric or intranasal administration together with model antigens in mice. Quinoa saponins, co-administered intragastrically or intranasally with cholera toxin or ovalbumin, potentiated specific IgG and IgA antibody responses to the antigens in serum, intestinal and lung secretions. The potentiating effect of the saponins appeared, to some extent, mediated by increased permeability of the mucosa, allowing increased uptake of the antigen. The intragastric administration of 99mTc-radio-labeled human serum albumin together with quinoa saponins revealed an increased presence of the radiolabeled protein in blood, liver, spleen and lungs of mice. This study indicates the potential of quinoa saponins as adjuvants for mucosally administered vaccines.

The ontogeny of serum insulin-like growth factor-I concentration in foals: Effects of dam parity, diet, and age at weaning

The effects of dam parity, age at weaning, and preweaning diet were examined in the ontogeny of serum insulin-like growth factor-I (IGF-I) concentrations in foals. Foals born to 13 primiparous and 19 multiparous draft-cross mares were weighed and bled near birth. About one-half of the foals in each group were weaned early (about 13 wk old); the remaining foals were weaned late (about 16 wk of age). Pooled values for serum IGF-I concentrations between birth and 17 wk of age were higher (P < 0.065) for foals born to multiparous (386 ng/ml) than to primiparous mares (237.5 ng/ml). Colts (378 ng/ml) had higher (P < 0.05) serum IGF-I concentrations than fillies (254.5 ng/ml), regardless of dam parity. Colts (173.5 kg) also tended (P = 0.12) to be heavier than fillies (159.2 kg). Weaning, whether at 13 or 16 wk of age, reduced (P < 0.05) growth rates and serum IGF-I concentrations. Serum IGF-I values recovered to preweaning values within 1-3 wk postweaning concurrent to an improved weight gain. Fifteen 1-d-old foals in a second study were fed milk replacer for 7 wk and were compared with five foals that nursed their mares for 8 wk. During the first 2 wk, replacer-fed foals (0.46 kg/d) did not gain as rapidly (P < 0.03) as mare-nursed foals (1.73 kg/d). The associated serum IGF-I values for replacer foals (139.4 ng/ml) were lower (P < 0.0001) than values for mare-nursed foals (317.4 ng/ml). Despite similarity in gains for both groups there-after, serum IGF-I concentrations of replacer-fed foals were only 36 and 60% of values obtained for mare-nursed foals at 8 (weaning) and 18 wk of age, respectively. The intrinsic differences between mare-nursed and milk-replacer foals in serum IGF-I concentrations persisted to 1 yr of age despite similarities in dietary management and body weight of the foals. At 1 yr of age, the serum IGF-I concentration of mare-nursed foals (1,203 ng/ml) was 48% higher than that of replacer-fed foals (815 ng/ml). These data indicate that dam parity, sex of foal, and preweaning nutrition affect the ontogeny of serum IGF-I concentration in the foal. The chronic, persistent difference in serum IGF-I values created by the early nutritional management of growing animals has implications in the interpretation of longitudinal serum IGF-I studies in all species.