Raveendra R. Kulkarni

Immunization of Broiler Chickens against Clostridium perfringens-Induced Necrotic Enteritis

ABSTRACT Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens. Currently, no vaccine against NE is available and immunity to NE is not well characterized. Our previous studies showed that immunity to NE followed oral infection by virulent rather than avirulent C. perfringens strains and identified immunogenic secreted proteins apparently uniquely produced by virulent C. perfringens isolates. These proteins were alpha-toxin, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase (PFOR), fructose 1,6-biphosphate aldolase, and a hypothetical protein (HP). The current study investigated the role of each of these proteins in conferring protection to broiler chickens against oral infection challenges of different severities with virulent C. perfringens. The genes encoding these proteins were cloned and purified as histidine-tagged recombinant proteins from Escherichia coli and were used to immunize broiler chickens intramuscularly. Serum and intestinal antibody responses were assessed by enzyme-linked immunosorbent assay. All proteins significantly protected broiler chickens against a relatively mild challenge. In addition, immunization with alpha-toxin, HP, and PFOR also offered significant protection against a more severe challenge. When the birds were primed with alpha-toxoid and boosted with active toxin, birds immunized with alpha-toxin were provided with the greatest protection against a severe challenge. The serum and intestinal washings from protected birds had high antigen-specific antibody titers. Thus, we conclude that there are certain secreted proteins, in addition to alpha-toxin, that are involved in immunity to NE in broiler chickens.

Insights into the role of Toll-like receptors in modulation of T cell responses.

The innate immune receptors, such as Toll-like receptors (TLRs), are intimately involved in the early sensing of invading microorganisms or their structural components. Engagement of TLRs with their ligands results in activation of several downstream intracellular pathways leading to activation of innate and adaptive immune system cells. It was initially thought that TLRs are primarily expressed by antigen-presenting cells (APCs), such as macrophages and dendritic cells, and that interactions between microbial ligands and TLRs in these cells will indirectly result in activation of cells of the adaptive immune system, especially T cells. However, it has now become evident that TLRs are also expressed by various T cell subsets, such as conventional αβT cells, regulatory T cells, and γδT cells as well as natural killer T cells. Importantly, it appears that at least in some of these T cell subsets, TLRs are functionally active, because stimulation of these cells with TLR agonists in the absence of APCs results in exertion of effector or regulatory functions of T cells. The present review attempts to summarize the recent findings related to TLR expression in different T cell subsets and the direct role of TLRs in the induction and regulation of T cell responses, including those responses that occur at mucosal surfaces. In addition, the potential use of TLR agonists for steering T cell responses as a prophylactic or therapeutic strategy in the context of infectious, allergic or autoimmune diseases is explored.

Oral immunization of broiler chickens against necrotic enteritis with an attenuated Salmonella vaccine vector expressing Clostridium perfringens antigens.

Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens but currently no effective vaccine is available. Our previous study showed that certain C. perfringens secreted proteins when administered intramuscularly protected chickens against experimental infection. In the current study, genes encoding three C. perfringens proteins: fructose-biphosphate-aldolase (FBA), pyruvate:ferredoxin-oxidoreductase (PFOR) and hypothetical protein (HP), were cloned into an avirulent Salmonella enterica sv. typhimurium vaccine vector. Broiler chickens immunized orally with recombinant Salmonella expressing FBA or HP proteins were significantly protected against NE challenge. Immunized birds developed serum and mucosal antibodies to both clostridial and Salmonella antigens. This study showed the oral immunizing ability of two C. perfringens antigens against NE in broiler chickens through an attenuated Salmonella vaccine vector.

Synthetic double-stranded RNA oligonucleotides are immunostimulatory for chicken spleen cells

Abstract Toll-like receptors (TLRs) are an integral part of the innate immune system that recognize microbe-derived molecular patterns and initiate innate and adaptive defenses against invading pathogens. TLR3 and TLR7 are involved in sensing virus-associated single-stranded and double-stranded RNA (dsRNA) molecules in cellular endosomes to activate the type I interferon pathway in mammals. Although certain synthetic dsRNA molecules have been identified to show immunostimulation in mammalian cells, very little is known about the ability of these sequences to stimulate avian cells. The current study investigated immunostimulatory properties of four synthetic oligonucleotide sequences using chicken splenocytes. Expression of TLR3 and 7, type I interferons and several other cytokines as well as TLR signaling pathway-related genes at different time points post-stimulation was quantified by real-time PCR. A dose-dependent increase in expression of TLR3 was observed in splenocytes treated with poly-UGUGU (poly-UG) and β-galactosidase dsRNA molecules. TLR3 and TLR7 gene expression was significantly up-regulated upon stimulation with all four dsRNA molecules. Furthermore, in a time course study, a significant increase was noted in the expression of TLR3, TLR7, interferon (IFN)-α, IFN-β, interleukin (IL)-1β, IL-6 as well as 2′,5′-OAS in splenocytes treated with poly-UG. In conclusion, the present study demonstrated the immunostimulatory properties of dsRNA oligonucleotides, especially those that contain a poly-UG motif, in chickens.

A live oral recombinant Salmonella enterica serovar typhimurium vaccine expressing Clostridium perfringens antigens confers protection against necrotic enteritis in broiler chickens.

ABSTRACT Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens, and there is currently no effective vaccine for NE. We previously showed that in broiler chickens protection against NE can be achieved through intramuscular immunization with alpha toxin (AT) and hypothetical protein (HP), and we subsequently identified B-cell epitopes in HP. In the present study, we identified B-cell epitopes in AT recognized by chickens immune to NE. The gene fragments encoding immunodominant epitopes of AT as well as those of HP were codon optimized for Salmonella and cloned into pYA3493, and the resultant plasmid constructs were introduced into an attenuated Salmonella enterica serovar Typhimurium χ9352 vaccine vehicle. The expression of these Clostridium perfringens proteins, alpha toxoid (ATd) and truncated HP (HPt), was confirmed by immunoblotting. The protection of broiler chickens against experimentally induced NE was assessed at both the moderate and the severe levels of challenge. Birds immunized orally with Salmonella expressing ATd were significantly protected against moderate NE, and there was a nonsignificant trend for protection against severe challenge, whereas HPt-immunized birds were significantly protected against both severities of challenge. Immunized birds developed serum IgY and mucosal IgA and IgY antibody responses against Clostridium and Salmonella antigens. In conclusion, this study identified, for the first time, the B-cell epitopes in AT from an NE isolate recognized by chickens and showed the partial protective ability of codon-optimized ATd and HPt against NE in broiler chickens when they were delivered orally by using a Salmonella vaccine vehicle.

Clostridium perfringens antigens recognized by broiler chickens immune to necrotic enteritis.

ABSTRACT Little is known about immunity to necrotic enteritis (NE) in chickens. A recent study of broiler chickens showed that protection against NE was associated with infection-immunization with virulent but not with avirulent Clostridium perfringens.In the current study, six secreted antigenic proteins unique to virulent C. perfringens that reacted to serum antibodies from immune birds were identified by mass spectrophotometry; three of these proteins are part of the VirR-VirS regulon.

Immunization of Broiler Chickens Against Clostridium perfringens–Induced Necrotic Enteritis Using Purified Recombinant Immunogenic Proteins

Abstract This study identified and assessed secreted proteins of Clostridium perfringens additional to those previously described for their ability to protect broiler chickens against necrotic enteritis (NE). Secreted proteins of virulent and avirulent C. perfringens were electrophoretically separated and reacted with serum of chickens immune to NE. Three immunoreactive protein bands unique to the virulent C. perfringens were identified by mass spectrometry as the toxin C. perfringens large cytotoxin (TpeL), endo-beta-N-acetylglucosaminidase (Naglu), and phosphoglyceromutase (Pgm). The genes encoding Naglu and Pgm proteins were cloned, and their gene products were purified as histidine-tagged recombinant proteins from Escherichia coli and used in immunizing chickens. Immunized and nonimmunized control broiler chickens were then challenged with two different strains (CP1, CP4) of C. perfringens and assessed for the development of NE. Of the two immunogens, Pgm immunization showed significant protection of broiler chickens against experimental NE, although protection reduced as challenge severity increased. However, birds immunized with Naglu were protected from challenge only with strain CP4. Birds immunized with these proteins had antigen-specific antibodies when tested in an enzyme-linked immunosorbent assay. In conclusion, this study demonstrated the partial efficacy of additional secreted proteins in immunity of broiler chickens to NE. The study also showed that there may be differences in the protective ability of immunogens depending on the infecting C. perfringens strain.

Costimulatory activation of murine invariant natural killer T cells by toll-like receptor agonists.

Invariant NKT (iNKT) cells are glycolipid-reactive lymphocytes with anti-microbial properties. Toll-like receptor (TLR)-primed antigen-presenting cells are known to activate iNKT cells, however, the expression and function of TLRs in iNKT cells remain largely unknown. Here, we show that TCR-activation of murine iNKT cells by α-GalactosylCeramide (α-GalCer) or anti-CD3 antibodies can result in increased expression of TLR genes. TLR3, 5 and 9-mediated costimulation of TCR-preactivated iNKT cells resulted in enhancement of iNKT cell activation, as determined by their cytokine production. Expression of TLR3 and 9 at protein level was also confirmed in TCR-activated iNKT cells. Furthermore, TCR-preactivation followed by TLR9-costimulation of iNKT cells increased their ability to induce maturation of dendritic cells. Thus, our findings show that iNKT cells can up-regulate their TLR expression upon TCR activation and a subsequent TLR-signaling in these cells can lead to their enhanced activation, suggesting a new possible mode of iNKT cell activation.

CpG oligonucleotide-mediated co-stimulation of mouse invariant natural killer T cells negatively regulates their activation status

Invariant natural killer T (iNKT) cells play important roles in antimicrobial defense and immune-regulation. We have previously shown that iNKT cells express certain toll-like receptors (TLR), and that TLR co-stimulation of iNKT cells in the presence of suboptimal concentrations of T cell receptor (TCR) agonists enhances cellular activation. In the present study, we investigated the regulatory effects of CpG oligonucleotides in mouse primary hepatic and splenic iNKT cells and in DN32.D3 iNKT cells. We show that CpG treatment of iNKT cells in the presence of higher concentrations of TCR agonists (α-GalCer or anti-CD3 mAb) results in the up-regulation of TLR9 in iNKT cells with a concurrent reduction in their cellular activation, as assessed by their production of IL-2, IL-4 and IFN-γ compared with controls. CpG-mediated down-regulation of iNKT cell activation has been found to depend, at least in part, on signaling by MyD88, a critical adapter moiety downstream of TLR9 signaling. Mechanistically, iNKT cells treated with CpG in the presence of TCR agonists show inhibition of MAPK signaling as determined by the levels of ERK1/2 and p38 MAPKs. Furthermore, CpG treatment leads to an increased induction of phosphatases, DUSP1 and SHP-1, that seem to impede MAPK and TCR signaling, resulting in the negative regulation of iNKT cell activation. Our findings therefore suggest a novel regulatory role for CpG in iNKT cells in the mediation of a negative feedback mechanism to control overactive iNKT cell responses and hence to avoid undesirable excessive immunopathology.