Development and characterization of monoclonal antibodies specific for chicken interleukin-13 and their neutralizing effects in chicken primary monocytes.

Compared with mammals, the functionality of chicken cytokines is not well understood because of the unavailability of immune reagents. Mammalian interleukin (IL)-13 is an important Th2 type cytokine with well-known biological functions through its 2 receptors, IL-13 receptor (IL-13R)-α1 and IL-13Rα2. In the present study, we developed mouse monoclonal antibodies (mAb) against chIL-13 and further investigated their specificity in detecting endogenously produced chIL-13. Upon characterization of mAb using indirect ELISA and Western blot, the capture ELISA was developed for detecting chIL-13. Neutralizing effects were tested by measuring nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in primary chicken monocytes stimulated with chIL-13, lipopolysaccharide (LPS), chIL-13+LPS, or chIL-13+LPS+mAb. In addition, gene expression of chIL-13Rα1, chIL-13Rα2, and TGF-ß1 was tested in chicken monocytes treated with chIL-13 or chIL-13+mAb. Based on indirect ELISA, 5 mAb that detected recombinant chIL-13 were identified, and all of them specifically detected recombinant chIL-13 protein by Western blotting. An optimal signal was obtained with 2 mAb (#9B11 and #10A2) in a pairing assay, and these 2 mAb were used in a capture assay. A neutralization assay further revealed that chIL-13 reduced LPS-stimulated NO production and iNOS expression in monocytes and macrophage cells, and the 2 mAb (#9B11 and #10A2) abrogated these effects. In addition, chIL-13-induced expressions of chIL-13Rα2 and TGF-ß1 were neutralized by the 2 mAb. In summary, the present study showed that chIL-13 may be involved in the alternative activation of primary monocytes in chickens and that chIL-13 signaling may be regulated through chIL-13Rα2 binding and TGF-ß1 secretion. Importantly, the newly developed anti-chIL-13 mAb will serve as valuable immune reagents for future studies on the biological activity of chIL-13 and its receptors.

Beneficial effects of dietary supplementation of Bacillus strains on growth performance and gut health in chickens with mixed coccidiosis infection.

The present study investigated the effects of dietary supplementation with several Bacillus strains on growth performance, intestinal inflammatory and anti-inflammatory cytokines, anti-oxidants and tight junction (TJ) protein mRNA expression in broiler chickens challenged with mixed coccidia infection (oocysts of Eimeria tenella, Eimeria maxima and Eimeria acervulina). Ten different Bacillus strains were screened for their beneficial effects on coccidiosis challenge by measuring relative body weight gain (RBWG), lesion score, and total oocyst count. Three out of ten Bacillus strains were evaluated in depth by measuring RBWG, lesion score, total oocyst count, and the gene expression of proinflammatory (IL-6 and IL-8), anti-inflammatory (IL-10 and TGF-ß), anti-oxidant (SOD1 and HMOX1), and TJ (JAM2 and occludin) proteins. Our results showed that out of the ten different Bacillus strains, chickens fed with three strains, one Bacillus licheniformis and two Bacillus amyloliquefaciens, showed significantly higher RBWG, lower lesion scores (ceca, jejunum, and duodenum), and lower total fecal oocyst counts compared to non-Bacillus-fed control chickens. Post-coccidia challenge, the RBWG for the Bacillus-fed groups were 95-100 % as opposed to the control birds (70 %) at 6 days post infection (dpi) and 10 dpi. Similarly, the lesion scores for three organs were around 0.8-0.9 for the Bacillus-fed groups as opposed to control birds (lesion score range ∼1.4-2). The total oocyst counts were much lower in the Bacillus-fed group (10-20 folds lesser) than the control group. Furthermore, the Bacillus-fed groups showed differential gene expression at 3 dpi in different tissues, such as caecum, jejunum, and duodenum. Bacillus-fed chickens showed significant pro- and anti-inflammatory responses and higher expression of anti-oxidants and TJ proteins in the ceca, duodenum, and jejunum. Overall, our results demonstrated that dietary supplementation with Bacillus strains as direct-fed microbials (DFM) significantly improved the body weight gain after mixed coccidia challenge compared to non-Bacillus-fed and coccidia challenged control group. In conclusion, the results of this study are promising and indicate the many beneficial effects of DFM-supplementation in poultry diets to reduce the negative consequences of enteric diseases and to decrease economic losses imposed by coccidia infection in chickens.

Involvement of T Cell Immunity in Avian Coccidiosis.

Avian coccidiosis is caused by Eimeria, which is an intracellular apicomplexan parasite that invades through the intestinal tract to cause devastating disease. Upon invasion through the intestinal epithelial cells, a strong inflammatory response is induced that results in complete villous destruction, diarrhea, hemorrhage, and in severe cases, death. Since the life cycle of Eimeria parasites is complex and comprises several intra- and extracellular developmental stages, the host immune responses are diverse and complex. Interferon-γ-mediated T helper (Th)1 response was originally considered to be the predominant immune response in avian coccidiosis. However, recent studies on other avian T cell lineages such as Th17 and T regulatory cells have implicated their significant involvement in maintaining gut homeostasis in normal and disease states including coccidiosis. Therefore, there is a need to understand better their role in coccidiosis. This review focuses on research findings concerning the host immune response induced by avian coccidiosis in the context of T cell immunity, including expression of T-cell-related cytokines and surface molecules that determine the phenotype of T lymphocytes.

A three-dimensional human skin model to evaluate the inhibition of Staphylococcus aureus by antimicrobial peptide-functionalized silver carbon nanotubes.

OBJECTIVE: To investigate the toxicity and antibacterial application of antimicrobial peptide-functionalized silver-coated carbon nanotubes against Staphylococcus infection using a full thickness human three-dimensional skin model. MATERIALS AND METHODS: The three-dimensional skin formation on the scaffolds was characterized by electron microscopy and investigation of several skin cell markers by real time-reverse transcriptase polymerase chain reaction. Functionalized silver-coated carbon nanotubes were prepared using carboxylated silver-coated carbon nanotubes with antimicrobial peptides such as TP359, TP226 and TP557. Following the characterization and toxicity evaluation, the antibacterial activity of functionalized silver-coated carbon nanotubes against Staphylococcus aureus was investigated using a bacterial enumeration assay and scanning electron microscopy. For this purpose, a scar on the human three-dimensional skin grown on Alvetex scaffold using keratinocytes and fibroblasts cells was created by taking precaution not to break the scaffold beneath, followed by incubation with 5 µg/mL of functionalized silver-coated carbon nanotubes re-suspended in minimum essential medium for 2 h. Post 2-h incubation, 200 µL of minimum essential medium containing 1 × 104 colony forming units of Staphylococcus aureus were incubated for 2 h. After incubation with bacteria, the colony forming unit/gram (cfu/g) of skin tissue were counted using the plate count assay and the samples were processed for scanning electron microscopy analysis. RESULTS: MTT assay revealed no toxicity of functionalized silver-coated carbon nanotubes to the skin cells such as keratinocytes and fibroblasts at 5 µg/mL with 98% cell viability. The bacterial count increased from 104 to 108 cfu/g in the non-treated skin model, whereas skin treated with functionalized silver-coated carbon nanotubes showed only a small increase from 104 to 105 cfu/g (1000-fold viable cfu difference). Scanning electron microscopy analysis showed the presence of Staphylococcus aureus on the non-treated skin as opposed to the treated skin. CONCLUSION: Thus, our results showed that functionalized silver-coated carbon nanotubes are not only non-toxic, but also help reduce the infection due to their antibacterial activity. These findings will aid in the development of novel antibacterial skin substitutes.

Interleukin-4 (IL-4) may regulate alternative activation of macrophage-like cells in chickens: A sequential study using novel and specific neutralizing monoclonal antibodies against chicken IL-4.

In mammals, alternatively activated macrophages (AAMs) are well-recognized and are produced by stimulation with Th2 cytokines such as interleukin 4 (IL-4) and IL-13. On their mammalian counterparts, AAMs in chickens has neither been reported nor the functionality of chicken IL-4 (chIL-4) has been studied till date. Therefore, present study developed mouse monoclonal antibodies (mAbs) against chIL-4 and used these antibodies to investigate whether chIL-4 induces activation of HD11 chicken macrophage cell line. Upon characterization of mAbs using western blot, immunocytochemistry (ICC), flow cytometry and capture ELISA, activation of HD11 cells was investigated by measuring nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression, arginase activity and gene expressions of iNOS, CD80 and CD86 (associated with mammalian M1 phenotype) and chemokine (C-C motif) ligand 17 (ccl17) and mannose receptor C-type1 (MRC1L-A) (as possible chicken M2 markers) in HD11 cells treated with chIL-4, lipopolysaccharide (LPS), chIL-4+LPS, chIL-4+LPS + mAbs. The newly developed mAbs displayed wide applicability in detecting chIL-4 by capture ELISA, ICC and flow cytometry with no cross reactivities with human or mouse IL-4 and other chicken cytokines. Further, our results showed that chIL-4 inhibited NO production by LPS-stimulated HD11 cells and primary monocyte/macrophage cells with reduced iNOS expression and increased arginase activity and, induced robust expression of genes associated with M2 phenotype than M1-related genes. All these effects were neutralized by anti-chIL-4 antibodies. In summary, present study results showed a possible application of anti-chIL-4 mAbs as valuable immune reagents to explore chIL-4 functionality. In addition, our results demonstrated that chIL-4 may override LPS functionality and regulates alternative activation of HD11 cells in chicken via increased arginase activity and expression of M2 associated markers and thus may indicate the possible existence of M1/M2 paradigm in chickens.

Anti-RSV Peptide-Loaded Liposomes for the Inhibition of Respiratory Syncytial Virus.

Although respiratory syncytial virus (RSV) is one of the leading causes of acute respiratory tract infection in infants and adults, effective treatment options remain limited. To circumvent this issue, there is a novel approach, namely, the development of multifunctional liposomes for the delivery of anti RSV-peptides. While most of the peptides that are used for loading with the particulate delivery systems are the penetrating peptides, an alternative approach is the development of liposome-peptide systems, which are loaded with an RSV fusion peptide (RF-482), which has been designed to inhibit the RSV fusion and block infection. The results of this work have revealed that the liposomes themselves can serve as potential RSV inhibitors, whilst the anti-RSV-peptide with liposomes can significantly increase the RSV inhibition when compared with the anti-RSV peptide alone.

Proteomic analysis of antimicrobial effects of pegylated silver coated carbon nanotubes in Salmonella enterica serovar Typhimurium.

BACKGROUND: Synthesis of silver nano-compounds with enhanced antimicrobial effects is of great interest for the development of new antibacterial agents. Previous studies have reported the antibacterial properties of pegylated silver-coated carbon nanotubes (pSWCNT-Ag) showing less toxicity in human cell lines. However, the mechanism underlining the pSWCNT-Ag as a bactericidal agent remained unfolded. Here we assessed the pSWCNT-Ag effects against foodborne pathogenic bacteria growth and proteome profile changes. RESULTS: Measurements of bioluminescent imaging, optical density, and bacteria colony forming units revealed dose-dependent and stronger bactericidal activity of pSWCNT-Ag than their non-pegylated counterparts (SWCNT-Ag). In ovo administration of pSWCNT-Ag or phosphate-buffered saline resulted in comparable chicken embryo development and growth. The proteomic analysis, using two-dimensional electrophoresis combined with matrix assisted laser desorption/ionization time of flight/time of flight mass spectrometry, was performed on control and surviving Salmonella enterica serovar Typhimurium to pSWCNT-Ag. A total of 15 proteins (ten up-regulated and five down-regulated) differentially expressed proteins were identified. Functional analyses showed significant reduction of proteins associated with biofilm formation, nutrient and energy metabolism, quorum sensing and maintenance of cell structure and cell motility in surviving S. Typhimurium. In contrast, proteins associated with oxygen stress, DNA protection, starvation, membrane rebuilding, and alternative nutrient formation were induced as the compensatory reaction. CONCLUSIONS: This study provides further evidence of the antibacterial effects of pSWCNT-Ag nanocomposites and knowledge of their mechanism of action through various protein changes. The findings may lead to the development of more effective and safe antimicrobial agents.

Future Prospects for Scaffolding Methods and Biomaterials in Skin Tissue Engineering: A Review.

Over centuries, the field of regenerative skin tissue engineering has had several advancements to facilitate faster wound healing and thereby restoration of skin. Skin tissue regeneration is mainly based on the use of suitable scaffold matrices. There are several scaffold types, such as porous, fibrous, microsphere, hydrogel, composite and acellular, etc., with discrete advantages and disadvantages. These scaffolds are either made up of highly biocompatible natural biomaterials, such as collagen, chitosan, etc., or synthetic materials, such as polycaprolactone (PCL), and poly-ethylene-glycol (PEG), etc. Composite scaffolds, which are a combination of natural or synthetic biomaterials, are highly biocompatible with improved tensile strength for effective skin tissue regeneration. Appropriate knowledge of the properties, advantages and disadvantages of various biomaterials and scaffolds will accelerate the production of suitable scaffolds for skin tissue regeneration applications. At the same time, emphasis on some of the leading challenges in the field of skin tissue engineering, such as cell interaction with scaffolds, faster cellular proliferation/differentiation, and vascularization of engineered tissues, is inevitable. In this review, we discuss various types of scaffolding approaches and biomaterials used in the field of skin tissue engineering and more importantly their future prospects in skin tissue regeneration efforts.

Protective efficacy and immune responses by homologous prime-booster immunizations of a novel inactivated Salmonella Gallinarum vaccine candidate.

PURPOSE: Salmonella enterica serovar Gallinarum (SG) ghost vaccine candidate was recently constructed. In this study, we evaluated various prime-boost vaccination strategies using the candidate strain to optimize immunity and protection efficacy against fowl typhoid. MATERIALS AND METHODS: The chickens were divided into five groups designated as group A (non-immunized control), group B (orally primed and boosted), group C (primed orally and boosted intramuscularly), group D (primed and boosted intramuscularly), and group E (primed intramuscularly and boosted orally). The chickens were primed with the SG ghost at 7 days of age and were subsequently boosted at the fifth week of age. Post-immunization, the plasma IgG and intestinal secretory IgA (sIgA) levels, and the SG antigen-specific lymphocyte stimulation were monitored at weekly interval and the birds were subsequently challenged with a virulent SG strain at the third week post-second immunization. RESULTS: Chickens in group D showed an optimized protection with significantly increased plasma IgG, sIgA, and lymphocyte stimulation response compared to all groups. The presence of CD4(+) and CD8(+) T cells and monocyte/macrophage (M/M) in the spleen, and splenic expression of cytokines such as interferon γ (IFN-γ) and interleukin 6 (IL-6) in the immunized chickens were investigated. The prime immunization induced significantly higher splenic M/M population and mRNA levels of IFN-γ whereas the booster showed increases of splenic CD4(+) and CD8(+) T-cell population and IL-6 cytokine in mRNA levels. CONCLUSION: Our results indicate that the prime immunization with the SG ghost vaccine induced Th1 type immune response and the booster elicited both Th1- and Th2-related immune responses.

Novel cationic peptide TP359 down-regulates the expression of outer membrane biogenesis genes in Pseudomonas aeruginosa: a potential TP359 anti-microbial mechanism.

BACKGROUND: Antimicrobial peptides (AMPs) are a class of antimicrobial agents with broad-spectrum activities. Several reports indicate that cationic AMPs bind to the negatively charged bacterial membrane causing membrane depolarization and damage. However, membrane depolarization and damage may be insufficient to elicit cell death, thereby suggesting that other mechanism(s) of action could be involved in this phenomenon. In this study, we investigated the antimicrobial activity of a novel antimicrobial peptide, TP359, against two strains of Pseudomonas aeruginosa, as well as its possible mechanisms of action. RESULTS: TP359 proved to be bactericidal against P. aeruginosa as confirmed by the reduced bacteria counts, membrane damage and cytoplasmic membrane depolarization. In addition, it was non-toxic to mouse J774 macrophages and human lung A549 epithelial cells. Electron microscopy analysis showed TP359 bactericidal effects by structural changes of the bacteria from viable rod-shaped cells to those with cell membrane damages, proceeding into the efflux of cytoplasmic contents and emergence of ghost cells. Gene expression analysis on the effects of TP359 on outer membrane biogenesis genes underscored marked down-regulation, particularly of oprF, which encodes a major structural and outer membrane porin (OprF) in both strains studied, indicating that the peptide may cause deregulation of outer membrane genes and reduced structural stability which could lead to cell death. CONCLUSION: Our data shows that TP359 has potent antimicrobial activity against P aeruginosa. The correlation between membrane damage, depolarization and reduced expression of outer membrane biogenesis genes, particularly oprF may suggest the bactericidal mechanism of action of the TP359 peptide.

A novel covalent approach to bio-conjugate silver coated single walled carbon nanotubes with antimicrobial peptide.

BACKGROUND: Due to increasing antibiotic resistance, the use of silver coated single walled carbon nanotubes (SWCNTs-Ag) and antimicrobial peptides (APs) is becoming popular due to their antimicrobial properties against a wide range of pathogens. However, stability against various conditions and toxicity in human cells are some of the major drawbacks of APs and SWCNTs-Ag, respectively. Therefore, we hypothesized that APs-functionalized SWCNTs-Ag could act synergistically. Various covalent functionalization protocols described previously involve harsh treatment of carbon nanotubes for carboxylation (first step in covalent functionalization) and the non-covalently functionalized SWCNTs are not satisfactory. METHODS: The present study is the first report wherein SWCNTs-Ag were first carboxylated using Tri sodium citrate (TSC) at 37 °C and then subsequently functionalized covalently with an effective antimicrobial peptide from Therapeutic Inc., TP359 (FSWCNTs-Ag). SWCNTs-Ag were also non covalently functionalized with TP359 by simple mixing (SWCNTs-Ag-M) and both, the FSWCNTs-Ag (covalent) and SWCNTs-Ag-M (non-covalent), were characterized by Fourier transform infrared spectroscopy (FT-IR), Ultraviolet visualization (UV-VIS) and transmission electron microscopy (TEM). Further the antibacterial activity of both and TP359 were investigated against two gram positive (Staphylococcus aureus and Streptococcus pyogenes) and two gram negative (Salmonella enterica serovar Typhimurium and Escherichia coli) pathogens and the cellular toxicity of TP359 and FSWCNTs-Ag was compared with plain SWCNTs-Ag using murine macrophages and lung carcinoma cells. RESULTS: FT-IR analysis revealed that treatment with TSC successfully resulted in carboxylation of SWCNTs-Ag and the peptide was indeed attached to the SWCNTs-Ag evidenced by TEM images. More importantly, the present study results further showed that the minimum inhibitory concentration (MIC) of FSWCNTs-Ag were much lower (~7.8-3.9 µg/ml with IC50: ~4-5 µg/ml) compared to SWCNTs-Ag-M and plain SWCNTs-Ag (both 62.6 µg/ml, IC50: ~31-35 µg/ml), suggesting that the covalent conjugation of TP359 with SWCNTs-Ag was very effective on their counterparts. Additionally, FSWCNTs-Ag are non-toxic to the eukaryotic cells at their MIC concentrations (5-2.5 µg/ml) compared to SWCNTs-Ag (62.5 µg/ml). CONCLUSION: In conclusion, we demonstrated that covalent functionalization of SWCNTs-Ag and TP359 exhibited an additive antibacterial activity. This study described a novel approach to prepare SWCNT-Ag bio-conjugates without loss of antimicrobial activity and reduced toxicity, and this strategy will aid in the development of novel and biologically important nanomaterials.

Silver-coated carbon nanotubes downregulate the expression of Pseudomonas aeruginosa virulence genes: a potential mechanism for their antimicrobial effect.

The antimicrobial activity of silver-coated carbon nanotubes (AgCNTs) and their potential mode of action against mucoid and nonmucoid strains of Pseudomonas aeruginosa was investigated in vitro. The results showed that AgCNTs exhibited antimicrobial activity against both strains with minimum inhibitory concentrations of approximately 8 µg/mL, indicating a high sensitivity of P. aeruginosa to AgCNTs. AgCNTs were also bactericidal against both strains at the same minimum inhibitory concentration. Scanning and transmission electron-microscopy studies further revealed that a majority of the cells treated with AgCNTs transformed from smooth rod-shape morphology to disintegrated cells with broken/damaged membranes, resulting in leakage of cytoplasmic contents to produce ghost cells. The molecular effects of AgCNTs on P. aeruginosa genes involved in virulence and pathogenicity, stress response, and efflux pumps were evaluated for changes in their expression. Quantitative real-time PCR (qRT-PCR) showed that after exposure to AgCNTs, the expression levels of the rpoS, rsmZ, and oprD genes were significantly downregulated in both strains of P. aeruginosa compared to the untreated samples. These results suggest that the mechanism of action of AgCNTs may be attributed to their effect on cell-membrane integrity, downregulation of virulence-gene expression, and induction of general and oxidative stress in P. aeruginosa.

Immune responses to oral vaccination with Salmonella-delivered avian pathogenic Escherichia coli antigens and protective efficacy against colibacillosis.

In this study, the immune responses to and protective efficacy of a live attenuated Salmonella-delivered vaccine candidate secreting the papA, papG, iutA, and clpG antigens of Escherichia coli were evaluated against infection with avian pathogenic E. coli (APEC) in layer chickens. Primary vaccination was done at age 7 d and booster vaccination at age 5 wk. The levels of intestinal secretory immunoglobulin A specific to the 4 antigens were significantly higher in the vaccinated group than in the control group. A potent lymphocyte-proliferation response and increased levels of interferon-γ, interleukin-2, and interleukin-6 in the plasma and in culture supernatants of antigen-stimulated lymphocytes from the vaccinated group suggested significant induction of the cell-mediated immune response in this group compared with the control group. Upon challenge with a virulent APEC strain at 8 wk of age, the vaccinated group had no deaths, whereas the control group had a 15% mortality rate. In addition, the morbidity rate was significantly higher in the control group (55%) than in the vaccinated group (15%). Thus, giving primary and booster vaccination with the Salmonella-delivered APEC vaccine candidate significantly elevated both mucosal and cellular immune responses, which protected the chickens against colibacillosis.

Dans la présente étude, les réponses immunitaires et l'efficacité de protection d'un vaccin candidat administré via un Salmonella vivant atténué secrétant les antigènes papA, papG, iutA, et clpG d'Escherichia coli furent évaluées lors d'une infection chez des poules pondeuses avec un isolat d'E. coli pathogène pour les oiseaux (APEC). Une vaccination initiale fut faite à 7 jours d'âge et un rappel à l'âge de 5 semaines. Les quantités d'immunoglobulines A intestinales spécifiques aux quatre antigènes étaient significativement plus élevées dans le groupe vacciné comparativement au groupe témoin. Une réponse proliférative des lymphocytes et des niveaux augmentés d'interféron-γ, d'interleukine-2, et d'interleukine-6 dans le plasma et le surnageant de cultures de lymphocytes stimulés par des antigènes provenant des animaux du groupe vacciné suggère une induction significative de la réponse à médiation cellulaire dans ce groupe comparativement au groupe témoin. Lors d'une infection défi à 8 semaines d'âge avec une souche virulente d'APEC, aucune mortalité ne fut notée dans le groupe vacciné, alors que dans le groupe témoin on nota un taux de mortalité de 15 %. De plus, le taux de morbidité était significativement plus élevé dans le groupe témoins (55 %) comparativement au groupe vacciné (15 %). Ainsi, en donnant une primo-vaccination et un rappel avec le vaccin candidat APEC livré par Salmonella on induit des réponses significativement élevées de l'immunité humorale et de l'immunité cellulaire qui protégèrent les poulets contre la colibacillose.(Traduit par Docteur Serge Messier).

Novel pegylated silver coated carbon nanotubes kill Salmonella but they are non-toxic to eukaryotic cells.

BACKGROUND: Resistance of food borne pathogens such as Salmonella to existing antibiotics is of grave concern. Silver coated single walled carbon nanotubes (SWCNTs-Ag) have broad-spectrum antibacterial activity and may be a good treatment alternative. However, toxicity to human cells due to their physico-chemical properties is a serious public health concern. Although pegylation is commonly used to reduce metal nanoparticle toxicity, SWCNTs-Ag have not been pegylated as yet, and the effect of pegylation of SWCNTs-Ag on their anti-bacterial activity and cell cytotoxicity remains to be studied. Further, there are no molecular studies on the anti-bacterial mechanism of SWCNTs-Ag or their functionalized nanocomposites. MATERIALS AND METHODS: In this study we created novel pegylated SWCNTS-Ag (pSWCNTs-Ag), and employed 3 eukaryotic cell lines to evaluate their cytotoxicity as compared to plain SWCNTS-Ag. Simultaneously, we evaluated their antibacterial activity on Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) by the MIC and growth curve assays. In order to understand the possible mechanisms of action of both SWCNTs-Ag and pSWCNTs-Ag, we used electron microscopy (EM) and molecular studies (qRT-PCR). RESULTS: pSWCNTs-Ag inhibited Salmonella Typhimurium at 62.5 µg/mL, while remaining non-toxic to human cells. By comparison, plain SWCNTs-Ag were toxic to human cells at 62.5 µg/mL. EM analysis revealed that bacteria internalized either of these nanocomposites after the outer cell membranes were damaged, resulting in cell lysis or expulsion of cytoplasmic contents, leaving empty ghosts. The expression of genes regulating the membrane associated metabolic transporter system (artP, dppA, and livJ), amino acid biosynthesis (trp and argC) and outer membrane integrity (ompF) protiens, was significantly down regulated in Salmonella treated with both pSWCNTs-Ag and SWCNTs-Ag. Although EM analysis of bacteria treated with either SWCNTs-Ag or pSWCNTs-Ag revealed relatively similar morphological changes, the expression of genes regulating the normal physiological processes of bacteria (ybeF), quorum sensing (sdiA), outer membrane structure (safC), invasion (ychP) and virulence (safC, ychP, sseA and sseG) were exclusively down regulated several fold in pSWCNTs-Ag treated bacteria. CONCLUSIONS: Altogether, the present data shows that our novel pSWCNTs-Ag are non-toxic to human cells at their bactericidal concentration, as compared to plain SWCNTS-Ag. Therefore, pSWCNTs-Ag may be safe alternative antimicrobials to treat foodborne pathogens.

Innate immunity to recombinant QseC, a bacterial adrenergic receptor, may regulate expression of virulence genes of avian pathogenic Escherichia coli.

Certain bacterial pathogens rely on a membrane bound sensor kinase, QseC, to coordinate their virulence gene expression in a process called quorum sensing. The present study evaluated the effect of host immunity to a recombinant QseC protein, on the virulence gene expression of avian pathogenic Escherichia coli (APEC) of O78 serogroup (APECO78). For this purpose, we constructed a plasmid expressing QseC protein which is 50kDa in size and stimulated avian macrophage-like cells (AMCs) with the native form of QseC protein at different concentrations. The cell culture medium of QseC-stimulated AMCs was then used to investigate its effect on APECO78 growth rate and virulence gene expression. Growth curve analysis of APECO78 indicated that growth rate of APECO78 in Luria Bertani (LB) broth containing the culture medium of stimulated AMCs was significantly lower and was impeded at entering the exponential phase. The expression of virulence genes of APECO78 such as aufA, fliC, fimH, fyuA, iucC, iutA, msbB and vat were also significantly down-regulated. On the other hand, APECO78 grown in LB containing the cell culture medium of non-stimulated AMCs did not exhibit these changes. Additionally, stimulation with QseC effectively induced interferon gamma (IFN-γ), Toll-like receptor 4 (TLR-4) and Toll like receptor 15 (TLR-15) expression in AMCs. To summarize, our results demonstrated that recombinant QseC protein could be immunogenic and induces host immunity that regulates selective, yet major, virulence gene expression of APECO78 bacteria. Thus, present data provide evidence that QseC, a bacterial functional analog of adrenergic receptor, holds a promise as one of the vaccine candidates against APEC infections.

An experimental infection model for Escherichia coli egg peritonitis in layer chickens.

The present study describes an experimental infection model for avian pathogenic Escherichia coli (APEC)-induced egg peritonitis in layer chickens. First, a pilot study which consisted of two separate experiments was carried out to compare two routes of inoculations of APEC to induce peritonitis and to examine if the presence of egg yolk in the peritoneum would facilitate APEC-induced peritonitis. This study showed that the presence of egg yolk in the peritoneum facilitated the development of egg peritonitis when the APEC was inoculated via the intra-uterine (IU) route. Based on the results of the pilot study, 56-wk-old white leghorn hens were divided into two groups of five chickens, Group G (inoculated with E. coli APECO78 strain) and Group H (control). Both groups were inoculated with 2-3 ml of egg yolk via the intraperitoneal route (IP). Subsequently, hens in Group H were inoculated with only egg yolk whereas the hens in Group G were inoculated with 1 x 10(9) colony-forming units of APECO78 bacteria via the IU route. Parameters such as mortality, clinical signs (anorexia, depression, and egg production efficiency), gross lesion scores, bacterial loads in internal organs, and histopathology of ovary and oviduct were assessed to evaluate the success of the infection model. Group G showed 40% acute mortality, severe depression, and anorexia with markedly reduced egg production and developed peritonitis-associated lesions such as accumulation of yellowish caseous fluid in the peritoneum, salpingitis, and oophoritis. Histopathologically, ovarian and oviduct tissues from group G exhibited severe inflammatory changes such as infiltration of mononuclear cells and edema. Group G also showed significant bacterial loads in the peritoneum, ovary, and oviduct. Interestingly, deceased birds from group G had also developed mild perihepatitis and pericarditis with heavy bacterial loads in the internal organs. On the other hand, group H birds did not exhibit any of the clinical signs and remained healthy until the end of the experiment. To summarize, our results demonstrate that IP administration of egg yolk followed by IU inoculation of APECO78 induced peritonitis in laying hens. Experimental infection models are often required to understand the mechanisms of disease pathogenesis. Therefore, the present infection model will aid in the studies of pathogenesis of layer peritonitis caused by APEC and in evaluating vaccine candidates to control the disease.

Generation of a safety enhanced Salmonella Gallinarum ghost using antibiotic resistance free plasmid and its potential as an effective inactivated vaccine candidate against fowl typhoid.

A safety enhanced Salmonella Gallinarum (SG) ghost was constructed using an antibiotic resistance gene free plasmid and evaluated its potential as fowl typhoid (FT) vaccine candidate. The antibiotic resistance free pYA3342 plasmid possesses aspartate semialdehyde dehydrogenase gene which is complimentary to the deletion of the chromosomal asd gene in the bacterial host. This plasmid was incorporated with a ghost cassette containing the bacteriophage PhiX174 lysis gene E, designated as pJHL101. The plasmid pJHL101 was transformed into a two virulence genes-deleted SG. The SG ghosts with tunnel formation and loss of cytoplasmic contents were observed by scanning electron microscopy and transmission electron microscopy. The cell viability of the culture solution was decreased to 0% at 24h after the induction of gene E expression by an increase in temperature from 37°C to 42°C. The safety and protective efficacy of the SG ghost vaccine was further examined in chickens which were divided into three groups: group A (non-immunized control), group B (orally immunized), and group C (intramuscularly immunized). The birds were immunized at 7d of age. No clinical symptoms associated with FT such as anorexia, depression and greenish diarrhea were observed in the immunized chickens. Upon challenge with a virulent SG strain at 3 week post-immunization, the chickens immunized with the SG ghost via various routes were efficiently protected, as shown by significantly lower mortality and post-mortem lesions in comparison with control group. In addition, all the immunized chickens showed significantly higher antibody responses accompanied by a potent antigen-specific lymphocyte proliferative response along with significantly increased numbers of CD4⁺ and CD8⁺ T lymphocytes. Overall, our results provide a promising approach of generating SG ghosts using the antibiotic resistance free plasmid in order to prepare a non-living bacterial vaccine candidate which could be environmentally safe yet efficient to prevent FT in chickens.

Evaluation of the adjuvant effect of Salmonella-based Escherichia coli heat-labile toxin B subunits on the efficacy of a live Salmonella-delivered avian pathogenic Escherichia coli vaccine.

The present study evaluated the adjuvant effect of live attenuated salmonella organisms expressing the heat-labile toxin of Escherichia coli B subunit (LTB) on the efficacy of an avian pathogenic Escherichia coli (APEC) vaccine. The Asd(+) (aspartate semialdehyde dehydrogenase) plasmid pMMP906 containing the LTB gene was introduced into a Salmonella enterica Typhimurium strain lacking the lon, cpxR and asd genes to generate the adjuvant strain. Live recombinant Salmonella-delivered APEC vaccine candidates were used for this study. The birds were divided into three groups: group A, non-vaccinated controls; group B, immunized with vaccine candidates only; and group C, immunized with vaccine candidates and the LTB strain. The immune responses were measured and the birds were challenged at 21 days of age with a virulent APEC strain. Group C showed a significant increase in plasma IgG and intestinal IgA levels and a significantly higher lymphocyte proliferation response compared with the other groups. Upon challenge with the virulent APEC strain, group C showed effective protection whereas group B did not. We also attempted to optimize the effective dose of the adjuvant. The birds were immunized with the vaccine candidates together with 1×107 or 1×108 colony-forming units of the LTB strain and were subsequently challenged at 3 weeks of age. The 1×107 colony-forming units of the LTB strain showed a greater adjuvant effect with increased levels of serum IgG, intestinal IgA and a potent lymphocyte proliferation response, and yielded higher protection against challenge. Overall, the LTB strain increased the efficacy of the Salmonella -delivered APEC vaccine, indicating that vaccination for APEC along with the LTB strain appears to increase the efficacy for protection against colibacillosis in broiler chickens.

Construction of an attenuated Salmonella delivery system harboring genes encoding various virulence factors of avian pathogenic Escherichia coli and its potential as a candidate vaccine for chicken colibacillosis.

An attenuated Salmonella (deltalon, deltacpxR, and deltaasdA16) delivery system containing the genes encoding P-fimbriae (papa and papG), aerobactin receptor (iutA), and CS31A surface antigen (clpG) of avian pathogenic Escherichia coli (APEC) was constructed, and its potential as a vaccine candidate against APEC infection in chickens was evaluated. The birds were divided into three groups designated group A (nonvaccinated control), group B (given a single immunization), and group C (administered prime and boost immunizations). Prime and booster vaccinations with the constructions were administered to 1-day-old and 14-day-old birds, respectively. Immune responses were measured postimmunization, and the birds were challenged via an intra-air sac route with a virulent APEC strain at the second, third, and fourth weeks of age. Group B birds were partially protected against the challenge and showed increased levels of plasma immunoglobulin (Ig)G, mucosal IgA antibodies, and lymphocyte proliferation. Group C birds showed greater protection against the challenge, with significantly stronger immune responses compared with the birds in the other groups. Overall, our data suggest that the Salmonella delivery system with recombinant constructs is capable of inducing robust immune responses and induces effective protection against colibacillosis caused by APEC.

Construction of a Salmonella Gallinarum ghost as a novel inactivated vaccine candidate and its protective efficacy against fowl typhoid in chickens.

In order to develop a novel, safe and immunogenic fowl typhoid (FT) vaccine candidate, a Salmonella Gallinarum ghost with controlled expression of the bacteriophage PhiX174 lysis gene E was constructed using pMMP99 plasmid in this study. The formation of the Salmonella Gallinarum ghost with tunnel formation and loss of cytoplasmic contents was observed by scanning electron microscopy and transmission electron microscopy. No viable cells were detectable 24 h after the induction of gene E expression by an increase in temperature from 37 °C to 42 °C. The safety and protective efficacy of the Salmonella Gallinarum ghost vaccine was tested in chickens that were divided into four groups: group A (non-immunized control), group B (orally immunized), group C (subcutaneously immunized) and group D (intramuscularly immunized). The birds were immunized at day 7 of age. None of the immunized animals showed any adverse reactions such as abnormal behavior, mortality, or signs of FT such as anorexia, depression, or diarrhea. These birds were subsequently challenged with a virulent Salmonella Gallinarum strain at 3 weeks post-immunization (wpi). Significant protection against the virulent challenge was observed in all immunized groups based on mortality and post-mortem lesions compared to the non-immunized control group. In addition, immunization with the Salmonella Gallinarum ghosts induced significantly high systemic IgG response in all immunized groups. Among the groups, orally-vaccinated group B showed significantly higher levels of secreted IgA. A potent antigen-specific lymphocyte activation response along with significantly increased percentages of CD4+ and CD8+ T lymphocytes found in all immunized groups clearly indicate the induction of cellular immune responses. Overall, these findings suggest that the newly constructed Salmonella Gallinarum ghost appears to be a safe, highly immunogenic, and efficient non-living bacterial vaccine candidate that protects against FT.