Immunostimulatory Potential of Fruits and Their Extracts in Poultry.

The impact of antibiotic use for growth promotion in livestock and poultry production on the rise of antimicrobial resistance (AMR) in bacteria led to the ban of this practice in the European Union in 2006 and a restriction of antimicrobial use (AMU) in animal agriculture in Canada and the United States of America. There is a high risk of infectious diseases such as necrotic enteritis due to Clostridium perfringens, and colibacillosis due to avian pathogenic Escherichia coli in antimicrobial-free broiler chickens. Thus, efficient and cost-effective methods for reducing AMU, maintaining good poultry health and reducing public health risks (food safety) are urgently needed for poultry production. Several alternative agents, including plant-derived polyphenolic compounds, have been investigated for their potential to prevent and control diseases through increasing poultry immunity. Many studies in humans reported that plant flavonoids could modulate the immune system by decreasing production of pro-inflammatory cytokines, T-cell activation, and proliferation. Fruits, especially berries, are excellent sources of flavonoids while being rich in nutrients and other functionally important molecules (vitamins and minerals). Thus, fruit byproducts or wastes could be important resources for value-added applications in poultry production. In the context of the circular economy and waste reduction, this review summarizes observed effects of fruit wastes/extracts on the general health and the immunity of poultry.

Protective Effects of Lactobacillus plantarum 16 and Paenibacillus polymyxa 10 Against Clostridium perfringens Infection in Broilers.

This study aimed to investigate the protective effects of Lactobacillus plantarum 16 (Lac16) and Paenibacillus polymyxa 10 (BSC10) against Clostridium perfringens (Cp) infection in broilers. A total of 720 one-day-old chicks were randomly divided into four groups. The control and Cp group were only fed a basal diet, while the two treatment groups received basal diets supplemented with Lac16 (1 × 108 cfu·kg-1) and BSC10 (1 × 108 cfu·kg-1) for 21 days, respectively. On day 1 and days 14 to 20, birds except those in the control group were challenged with 1 × 108 cfu C. perfringens type A strain once a day. The results showed that both Lac16 and BSC10 could ameliorate intestinal structure damage caused by C. perfringens infection. C. perfringens infection induced apoptosis by increasing the expression of Bax and p53 and decreasing Bcl-2 expression and inflammation evidence by higher levels of IFN-γ, IL-6, IL-1ß, iNOS, and IL-10 in the ileum mucosa, and NO production in jejunal mucosa, which was reversed by Lac16 and BSC10 treatment except for IL-1ß (P < 0.05). Besides, the two probiotics restored the intestinal microbiota imbalance induced by C. perfringens infection, characterized by the reduced Firmicutes and Proteobacteria and the increased Bacteroidetes at the phyla level and decreased Bacteroides fragilis and Gallibacterium anatis at the genus level. The two probiotics also reversed metabolic pathways of the microbiota in C. perfringens-infected broilers, including B-vitamin biosynthesis, peptidoglycan biosynthesis, and pyruvate fermentation to acetate and lactate II pathway. In conclusion, Lac16 and BSC10 can effectively protect broilers against C. perfringens infection through improved composition and metabolic pathways of the intestinal microbiota, intestinal structure, inflammation, and anti-apoptosis.

Preparation and immunological characterization of an inactivated canine Clostridium perfringens type A vaccine.

Clostridium perfringens is the main cause of sudden death in dogs and currently there is no vaccine to prevent it. In this study, a canine C. perfringens type A strain was used to prepare a vaccine. C. perfringens was inactivated by formaldehyde and adjuvants were added. The safety and immunological characteristics of the inactivated C. perfringens vaccine were evaluated in mice and dogs. The results showed that the C. perfringens vaccine was safe and had immunoprotective activity. The serum antibody titre of immunized mice reached up to 6·25 × 104 . Both single immunization of 4 ml and dual immunizations of 2 ml each provided good immune protection, with five of five immunized dogs surviving. This study also studied a detoxified crude α-toxin extract vaccine. The results showed that a single immunization with 0·5 ml of the detoxified crude α-toxin extract vaccine provided immune protection, with five of five immunized dogs surviving. The inactivated C. perfringens type A vaccine can be used to prevent canine C. perfringens infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridium perfringens is the main cause of sudden death in dogs and currently there is no vaccine to prevent it. In this study, an inactivated canine C. perfringens vaccine and a detoxified crude α-toxin vaccine were prepared. The safety and protective effects of these vaccines were evaluated using mouse and dog models. The vaccines were shown to be safe and to provide immune protection effects that can be used to prevent canine C. perfringens infection.

Characterization of intestinal immune response to Clostridium perfringens infection in broiler chickens.

Necrotic enteritis toxin B (NetB)-producing Clostridium perfringens (CP) type A is the etiological agent of necrotic enteritis (NE) - an economically significant disease in broiler chickens. Understanding the immune response to CP infection in broiler chickens is becoming important to develop effective vaccines against NE. An experiment was conducted to determine the expression levels of selected cytokine genes in the intestine and cecal tonsil of CP-challenged broiler chickens. In a floor-pen housing, broiler chickens were randomly assigned to the following treatment groups: 1) bacitracin methylene disalicylate (BMD)-free control diet with no CP challenge (CX), 2) BMD-supplemented diet with no CP challenge (CM), 3) BMD-free control diet with CP challenge (PCX), or 4) BMD-supplemented diet with CP challenge (PCM). The establishment of CP infection was confirmed, with the treatment groups exposed to CP having a 1.5 to 2-fold higher CP levels (P < 0.05) compared to the non-exposed groups. On day 1 and 7 post-challenge, jejunal segments and cecal tonsils were collected from experimental chickens for quantitative real-time RT-PCR analysis to determine the expression levels of interleukin (IL)-1ß, interferon-γ (IFN-γ), IL-2, IL-13, IL-17, IL-10, and transforming growth factor (TGF)-ß genes. Levels of antibodies to CP recombinant proteins were also determined in the plasma of experimental chickens. Results indicated that on day 7 post-challenge, IL-1ß (proinflammatory cytokine), IL-13 (Th2 cytokine), and IL-17 (Th17 cytokine) were upregulated (P < 0.05) in CP-challenged PCX and PCM treatments, compared to the unchallenged (control) CX and CM treatments. A reverse trend was observed for TGF-ß (anti-inflammatory cytokine), while no change was observed in IFN-γ (Th1 cytokine). Levels of plasma antibodies (IgY) to CP recombinant proteins were higher in CP-challenged treatments (PCX and PCM; P < 0.05), compared to their corresponding controls (CX and CM). It was concluded that CP infection induced inflammatory response in the intestine of broiler chickens, and the mechanisms of inflammation are probably mediated via Th2 and Th17 cells.

Oral vaccination of broiler chickens against necrotic enteritis using a non-virulent NetB positive strain of Clostridium perfringens type A.

Necrotic enteritis (NE) is a severe disease of chickens and turkeys caused by some strains of Clostridium perfringens type A. The disease is well controlled by the use of in-feed antibiotic growth promoters (AGPs). However, due to worldwide public and regulatory pressure to reduce the use of AGPs inter alia, there is an urgent need to develop non-antibiotic based preventative measures. Vaccination would be a suitable control measure, but currently there is no commercial vaccine. NetB (necrotic enteritis toxin B-like) is a pore-forming toxin produced by C. perfringens that has been reported as an important virulence factor in the pathogenesis of NE. The present study tests a non-virulent NetB producing strain of C. perfringens (nvNetB+), with or without adjuvants, as an orally administered live vaccine. Adjuvants used were Gel 01™, Cholera toxin (CT), Escherichia coli wild type heat-labile holotoxin (LT) and mutant E. coli LT (dmLT) (R192G/L211A). Several vaccine administration regimes were tested. All vaccination regimes elicited serum and mucosal antibody responses to alpha toxin and to secreted proteins of both nvNetB+ and a very virulent NetB positive (vvNetB+) strain (p<0.0001 to p<0.05). In some vaccinated groups, there was milder intestinal pathology upon disease challenge. 55% of birds vaccinated orally at days 2, 12 with nvNetB+ adjuvanted with CT did not develop any lesions of NE by 6 days post challenge, compared to a 100% incidence of NE lesions in the unvaccinated disease challenged group.

Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on growth performance and local innate immune response of broiler chickens challenged with Clostridium perfringens.

This study evaluated the effect of yeast-derived products on growth performance, gut lesion score, intestinal population of Clostridium perfringens, and local innate immunity of broiler chickens challenged with C. perfringens. One-day-old broiler chickens were randomly assigned to eight dietary treatments providing six replicate pens of 55 birds each per treatment. Dietary treatments consisted of Control diets without and with C. perfringens challenge, and diets containing bacitracin methylene disalicylate (BMD, 55 g/tonne), nucleotides (150 g/tonne), yeast cell wall (YCW, 300 g/tonne), and a commercial product Maxi-Gen Plus (1 kg/tonne) fed to chickens challenged with C. perfringens. Diets containing 10% distillers dried grains with solubles without and with C. perfringens challenge were also used. Birds were orally challenged with C. perfringens (10(8) colony-forming units (cfu)/bird) on day 14. On day 21, intestinal samples were collected for gene expression analysis. Pathogen challenge significantly (P < 0.05) impaired feed intake, body weight gain, and feed conversion ratio (FCR) shortly after the challenge (14-21 days). Increased C. perfringens counts and intestinal lesion scores were observed for challenged birds except the BMD-containing diet. Over the entire trial (1-35 days), no difference in growth performance was observed except the BMD diet which improved FCR over the Control, challenged group. Birds receiving nucleotides showed increased expression of toll-like receptors and cytokines interleukin (IL)-4 and IL-18 compared to the Control, challenged group. Expression of macrophage mannose receptor and IL-18 was upregulated in birds receiving YCW. Increased expression of cytokines and receptors involved in innate immunity in broilers receiving nucleotides and YCW suggests the immunomodulatory properties of these products under pathogen challenge conditions.

Improvement of immunity and disease resistance in the Nile tilapia, Oreochromis niloticus, by dietary supplementation with Bacillus amyloliquefaciens.

Probiotics can be used as immunostimulants in aquaculture. The aim of this study was to evaluate the immune responses of Nile tilapia Oreochromis niloticus following feeding with Bacillus amyloliquefaciens spores at concentrations of 1 × 10(6) (G3) and 1 × 10(4) (G2) colony-forming units per gram (CFU/g) of feed compared with a basal diet with no probiotics (G1). A total of 180 fingerlings (27.7 ± 0.22 g) were divided into three groups (G1-G3 of 20 fish per group) in triplicate. Innate immunities were measured every two weeks based on serum bactericidal activity, lysozyme activity, a nitric oxide assay (mmo/l) and phagocytic activity, and the expressions of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF α) were examined after one month. Moreover, the survival of tilapia upon challenge with Yersinia ruckeri or Clostridium perfringens type D was determined at the end of feeding trial. After 15 d, the serum killing percentages and phagocytic activities were significantly higher in G3 than in G1 and G2, whereas the same parameters had significantly higher values in G3 and G2 than in G1 after 30 d. After both 15 d and 30 d, the lysozyme activities and nitric oxide assay results (mmo/l) were significantly higher in G3 than G2, and the lowest values were observed in G1. The percentage of serum killing, serum nitric oxide and serum lysozyme activity were significantly increased by the time of B. amyloliquefaciens administration independently of the probiotic dose, and the phagocytic activity percentage was significantly decreased at the end of the experiment. Dietary B. amyloliquefaciens caused significant increases in IL-1 and TNF α mRNA levels in the kidneys in the following pattern: G3 > G2 > G1. Fish that were fed B. amyloliquefaciens exhibited better relative survival percentages than the controls when challenged by Y. ruckeri or C. perfringens type D. Dietary supplementation with B. amyloliquefaciens improves immune status and disease resistance in Nile tilapia.

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.

Vaccination prepartum enhances the beneficial effects of melatonin on the immune response and reduces platelet responsiveness in sheep.

BACKGROUND: Melatonin regulates several physiological processes and its powerful action as antioxidant has been widely reported. Melatonin acts modulating the immune system, showing a protective effect on the cardiovascular system and improving vaccine administration as an adjuvant-like agent. Here, we have investigated the role of melatonin as an adjuvant of the Clostridium perfringens vaccine in prepartum sheep and whether melatonin modulates platelet physiology during peripartum. RESULTS: The experiments were carried out in peripartum sheep from a farm located in an area of Mediterranean-type ecosystem. Plasma melatonin levels were determined by ELISA and sheep platelet aggregation was monitored using an aggregometer. Here we demonstrated for the first time that plasma melatonin concentration were higher in pregnant (125 pg/mL) than in non-pregnant sheep (15 pg/mL; P < 0.05). Administration of melatonin prepartum did not significantly modify platelet function but significantly improved the immune response to vaccination against C. perfringens. CONCLUSION: Administration of melatonin as an adjuvant provides a significant improvement in the immune response to vaccine administration prepartum against C. perfringens.

Colostrum and amniotic fluid from different species exhibit similar immunomodulating effects in bacterium-stimulated dendritic cells.

The fetus and newborn are immunologically immature. Bioactive compounds in amniotic fluid (AF) and maternal milk therefore play a key role in the immunological development of the infant intestine. We hypothesized that colostrum and AF exert similar immunomodulatory effects on the developing immune system. Hence, bone marrow-derived murine dendritic cells (BMDCs) were co-incubated with Clostridium perfringens A or Escherichia coli Nissle 1917 and porcine, bovine, or human AF, colostrum/milk whey fractions. Interleukin (IL) 10, IL-12, IL-6, and tumor necrosis factor-? (TNF-?) production was measured. IL-12 production was reduced with all AFs and wheys, and IL-6 and TNF-? were also reduced by porcine AF. Porcine and bovine whey both reduced TNF-? production. Overall, the reductions were most pronounced with the porcine fluids. Only bovine fluids caused strong induction of IL-10. Overall, effects of AF and whey from same species were similar. Viability of stimulated BMDCs was not significantly affected by the fluids. Neutralization of IL-10, transforming growth factor ?, and epidermal growth factor (EGF) did not remove the IL-12-inhibiting effect, but EGF neutralization increased IL-10 production. Addition of EFG to DCs enhanced the bacterium-induced cytokine production contrary to the effect of AF and colostrums, ruling out EGF as the inhibitory component in the fluids.

Efficacy of multistrain direct-fed microbial and phytogenetic products in reducing necrotic enteritis in commercial broilers.

Our laboratory is evaluating the efficacy of direct-fed microbials (DFM) and phytogenic products to control Clostridium perfringens, a gram-positive organism associated with decreased performance and morbidity and mortality associated with necrotic enteritis, as well as some recent human food safety issues. Three experiments were conducted to evaluate a DFM (PoultryStar) and a phytogenic product (PEP125), which were administered to birds from day of hatch until termination (d 25) via the drinking water or through supplementation to a wheat-corn diet, respectively. Each experiment contained a nonchallenged negative control and a positive control wherein birds were immunocompromised with a 10x dosage of infectious bursal disease vaccine at 14 d of age and subsequently gavaged with C. perfringens (10(7) cfu/mL) daily for 3 consecutive days starting on d 17. Intestinal lesions, mortality, and log10 values of C. perfringens in the probiotic and phytogenic treatment groups were found to be lower (P<0.05) than those observed in the positive controls. These experiments suggest that the DFM and the phytogenic product could be used as potential alternatives to help control C. perfringens and necrotic enteritis.

[Cloning and expression of ScFv gene against alpha-toxin of Clostridium perfringens type A].

The VH and VL genes from a hybridoma cell line producing mouse McAb against alpha-toxin of Clostridium perfringens type A were amplified by RT-PCR. The VH and VL genes were connected thought a flexible linker (Gly4Ser)3 and the VH-linker-VL (ScFv) gene was cloned into a vector pGEM-T. The ScFv gene consists of 726 bp encoding 242 amino acid residues. Both VH and VL genes were confirmed as functionally rearranged mouse immunoglobulin variable region. According to kabat classed method, the VH and VL gene segments belong to mouse Ig heavy chain subgroup II (B) and kappa light chain subgroup III respectively. The ScFv gene was amplified inserted the expression vector pHOG21 and transformed into E coli XL1-BLUE. The ScFv protein was highly expressed in recombinant strain XL1-BLUE (pHOG-2E3) and the expression level of the ScFv was about 25% of total bacteria protein by SDS-PAGE. The neutralization assay showed that the expressed ScFv protein could neutralize the phospholipase C activities of alpha-toxin.

Human gamma delta T cells recognize alkylamines derived from microbes, edible plants, and tea: implications for innate immunity.

Approximately 4% of peripheral blood T cells in humans express a T cell receptor with markedly restricted germline gene segment usage (V gamma 2 V delta 2). Remarkably, these T cells expand 2- to 10-fold (8%-60% of all circulating T cells) during many microbial infections. We show here that these T cells recognize a family of naturally occurring primary alkylamines in a TCR-dependent manner. These antigenic alkylamines are secreted to millimolar concentrations in bacterial supernatants and are found in certain edible plants. Given the large numbers of memory V gamma 2 V delta 2 T cells in adult humans, recognition of alkylamine antigens offers the immune system a response of the magnitude of major superantigens for alpha beta T cells and may bridge the gap between innate and adaptive immunity.

Antibody response in goats vaccinated with liposome-adjuvanted Clostridium perfringens type D epsilon toxoid.

A trial was performed using 20 goats to evaluate the antibody responses to a liposome-adjuvanted Clostridium perfringens epsilon toxoid vaccine (LIPV). The antibody response was compared with those produced by epsilon toxoid vaccines prepared using aluminium hydroxide (ALV) and incomplete Freud's adjuvant (FAV). The animals were allocated to four groups at the beginning of the trial. The animals in group 1 were vaccinated with ALV, while the animals in group 2 received FAV and those in groups 3 and 4 were vaccinated with LIPV. The animals in groups 1 to 3 received three doses of the corresponding vaccine at intervals of three weeks, while those in group 4 received only 1 dose of vaccine at the beginning of the trial. A blood sample was obtained from all the goats at the beginning of the trial and then weekly for 8 weeks. The samples were analysed for epsilon toxoid antibodies by an indirect ELISA technique. No major clinical abnormalities were observed in the animals after vaccination, with the exception of those that received the FAV, which experienced transient lameness. The highest antibody response was observed in the animals vaccinated with FAV, but they presented moderate to severe inflammatory tissue reactions at the injection site. Moderately high antibody responses were obtained with the ALV, with which only minor local reactions were observed. No significant antibody responses were obtained with the LIPV, nor were local reactions observed.

Detection of Clostridium perfringens type D epsilon antitoxin in serum of goats by competitive and indirect ELISA.

Indirect and competitive ELISA techniques were developed and their ability to detect antibodies to Clostridium perfringens epsilon toxin in goat serum was compared. Different dilutions of a hyperimmune goat serum, in serum from a colostrum-deprived kid, were used as positive controls, while sera from eleven colostrum-deprived kids were used as negative controls. The epsilon toxin antibodies in the hyperimmune serum were also measured by mouse neutralisation test (MNT). The correlation coefficient between both the indirect ELISA technique and MNT was 0.99, while the same coefficient for the competitive ELISA was 0.98. Both the indirect and competitive ELISAs proved to be rapid, simple, sensitive and specific for detecting antibodies to C. perfringens epsilon toxin in serum of goats.

[The experimental validation of methods for the emergency immunoprophylaxis of gas gangrene]. / Eksperimental'noe obosnovanie metodov ékstrennoi immunoprofilaktiki gazovoi gangreny.

The effectiveness, both immunological (by an increase in the titers of antitoxins) and protective (by resistance to the inoculation of the absolute lethal dose of infective agents), of the regional (wound) revaccination with tetratoxoid (Clostridium perfringens, C. oedematiens, C. septicum, C. histolyticum) was demonstrated on the experimental model of wound infection (gas gangrene) of guinea pigs. The schedule of rapid immunization with tetratoxoid was developed, which made it possible to create good immunological preparedness (basic immunity) for subsequent revaccination in case of traumas within 6 days. The effectiveness of rapid immunization by the application of tetratoxoid on the wound was shown. This immunization ensured a considered increase in the titers of antitoxins within the first 6 days, which increased the protection of the animals from infection with each of the four causative agents of gas gangrene.

[The isolation of the ribosomal fraction of Clostridium perfringens type A and an evaluation of its protective activity]. / Poluchenie ribosomal'noi fraktsii Clostridium perfringens tipa A i otsenka ee protektivnoi aktivnosti.

A method for isolation of the ribosomal fraction (RF) from the cytoplasm of type-A C. perfringens strain BP6K was developed and its chemical and antigenic properties characterized. RF has been found to possess protective properties: two subcutaneous immunizations of mice with RF preparations adsorbed on Al(OH)3 in doses of 250 and 500 micrograms (dry weight) has ensured, on the average, the protection of 41.9% of the immunized animals from 1 DCL of type-A C. perfringens strain BP6K culture.

Colostral transfer of Clostridium perfringens type C beta antitoxin in swine.

Nine bred gilts were vaccinated with 2 doses of a Clostridium perfringens type C toxoid at a 5-week interval. Time of vaccination during gestation differed among the gilts. Clostridium perfringens beta antitoxin in colostral samples and in serum samples was titrated in mice. Blood was collected from 2 to 5 neonatal pigs from each dam (total = 32 pigs) when the pigs were 36 to 48 hours old. Antitoxin titers in colostrum were 123 to 4.5 IU/ml, indicating considerable variation in individual responses of the gilts to toxoid. Serum titers of neonatal pigs reflected colostrum titers of their dams. This colostrum-to-serum titer correlation was essentially a straight-line fit by least-squares linear regression analysis, establishing a direct proportional relationship between colostrum titers and serum titers of neonatal pigs. In the dams, a correlation was not found between colostral titers and serum titers of blood samples collected 2 weeks after collection of colostrum.