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.

Advances in Skin Regeneration Using Tissue Engineering.

Tissue engineered skin substitutes for wound healing have evolved tremendously over the last couple of years. New advances have been made toward developing skin substitutes made up of artificial and natural materials. Engineered skin substitutes are developed from acellular materials or can be synthesized from autologous, allograft, xenogenic, or synthetic sources. Each of these engineered skin substitutes has their advantages and disadvantages. However, to this date, a complete functional skin substitute is not available, and research is continuing to develop a competent full thickness skin substitute product that can vascularize rapidly. There is also a need to redesign the currently available substitutes to make them user friendly, commercially affordable, and viable with longer shelf life. The present review focuses on providing an overview of advances in the field of tissue engineered skin substitute development, the availability of various types, and their application.

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.

Morphological evolution of lamellar forming polystyrene-block-poly(4-vinylpyridine) copolymers under solvent annealing.

In this work, we are reporting a very simple and efficient method to form lamellar structures of symmetric polystyrene-block-poly(4-vinylpyridine) (PS-b-P4VP) copolymer thin films with vertically (to the surface plane) orientated lamellae using a solvent annealing approach. The methodology does not require any brush chemistry to engineer a neutral surface and it is the block neutral nature of the film-solvent vapour interface that defines the orientation of the lamellae. The microphase separated structure of two different molecular weight lamellar forming PS-block-P4VP copolymers formed under solvent vapour annealing was monitored using atomic force microscopy (AFM) so as to understand the morphological changes of the films upon different solvent exposure. In particular, the morphology changes from micellar structures to well-defined microphase separated arrangements. The choice of solvent/s (single and dual solvent exposure) and the solvent annealing conditions (temperature, time etc.) has important effects on structural transitions of the films and it was found that a block neutral solvent was required to realize vertically aligned P4VP lamellae. The results of the structural variation of the phase separated nanostructured films through the exposure to ethanol are also described.

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.

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.

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.

Complete nucleotide sequence of pRS218, a large virulence plasmid, that augments pathogenic potential of meningitis-associated Escherichia coli strain RS218.

BACKGROUND: Escherichia coli is the most predominant Gram-negative bacterial pathogen associated with neonatal meningitis. Previous studies indicated that the prototypic neonatal meningitis E. coli (NMEC) strain RS218 (O18:K1:H7) harbors one large plasmid. Objectives of the present study were to analyze the complete nucleotide sequence of this large plasmid (pRS218) and its contribution to NMEC pathogenesis using in vitro and in vivo models of neonatal meningitis. RESULTS: The plasmid is 114,231 bp in size, belongs to the incompatibility group FIB/IIA (IncFIB/IIA), and contains a genetic load region that encodes several virulence and fitness traits such as enterotoxicity, iron acquisition and copper tolerance. The nucleotide sequence of pRS218 showed a 41- 46% similarity to other neonatal meningitis-causing E. coli (NMEC) plasmids and remarkable nucleotide sequence similarity (up to 100%) to large virulence plasmids of E. coli associated with acute cystitis. Some genes located on pRS218 were overly represented by NMEC strains compared to fecal E. coli isolated from healthy individuals. The plasmid-cured strain was significantly attenuated relative to the RS218 wild-type strain as determined in vitro by invasion potential to human cerebral microvascular endothelial cells and in vivo by mortalities, histopathological lesions in the brain tissue, and bacterial recovery from the cerebrospinal fluid of infected rat pups. CONCLUSIONS: The pRS218 is an IncFIB/IIA plasmid which shares a remarkable nucleotide sequence similarity to large plasmids of E. coli associated with cystitis. Both in vitro and in vivo experiments indicated that pRS218 plays an important role in NMEC pathogenesis.

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.

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.

Characterization of a novel inactivated Salmonella enterica serovar Enteritidis vaccine candidate generated using a modified cI857/λ PR/gene E expression system.

A new strategy to develop an effective vaccine is essential to control food-borne Salmonella enterica serovar Enteritidis infections. Bacterial ghosts (BGs), which are nonliving, Gram-negative bacterial cell envelopes, are generated by expulsion of the cytoplasmic contents from bacterial cells through controlled expression using the modified cI857/λ P(R)/gene E expression system. In the present study, the pJHL99 lysis plasmid carrying the mutated lambda pR37-cI857 repressor and PhiX174 lysis gene E was constructed and transformed in S. Enteritidis to produce a BG. Temperature induction of the lysis gene cassette at 42°C revealed quantitative killing of S. Enteritidis. The S. Enteritidis ghost was characterized using scanning and transmission electron microscopy to visualize the transmembrane tunnel structure and loss of cytoplasmic materials, respectively. The efficacy of the BG as a vaccine candidate was evaluated in a chicken model using 60 10-day-old chickens, which were divided into four groups (n = 15), A, B, C, and D. Group A was designated as the nonimmunized control group, whereas the birds in groups B, C, and D were immunized via the intramuscular, subcutaneous, and oral routes, respectively. The chickens from all immunized groups showed significant increases in plasma IgG and intestinal secretory IgA levels. The lymphocyte proliferation response and CD3(+) CD4(+) and CD3(+) CD8(+) T cell subpopulations were also significantly increased in all immunized groups. The data indicate that both humoral and cell-mediated immune responses are robustly stimulated. Based on an examination of the protection efficacy measured by observations of gross lesions in the organs and bacterial recovery, the candidate vaccine can provide efficient protection against virulent challenge.

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.

The Supplementation of FloraMax-B11 Did Not Affect the Bile Acid Neosynthesis and the Enterohepatic Circulation in Broiler Chickens.

Most probiotics possess bile salt hydrolase enzymes and may increase bile acid excretion and negatively affect fat digestion and absorption. Therefore, the study objective was to determine the time course effects of a commercial probiotic (P) FloraMax-B11 (FM) supplementation on bile acid neosynthesis and enterohepatic circulation in broiler chickens. Fertile Ross 708 eggs were incubated under standard commercial conditions. At hatch, chicks (n = 550) were randomly assigned to 5 treatment groups (n = 5 replicates per treatment group) with 22 birds per pen. The 5 treatment groups consisted of: control group (C, normal water from hatch to 35 days of age without supplements); P3, water supplemented with FM for the first 3 days post-hatch followed by normal water until day 35; P10, water supplemented with FM for the first 10 days post-hatch followed by normal water until day 35; P35, water supplemented with FM from hatch to day 35; and AGP, water supplemented with antibiotic growth promoter (AGP) from hatch until day 35. Ileum, liver, and plasma were collected at hatch, days 3, 10, 21, and 35 post-hatch. The relative mRNA expression of genes involved in bile acid synthesis (CYP7A1, CYP8B1, FXR, FGFR4, and FGF19) and transport (ASBT, I-BABP, OSTα, OSTß, and BSEP) as well as ileal deoxycholic acid and plasma cholic acid were determined. There was no FM and AGP interaction for any of the response criteria. No FM or AGP effects were observed (p > 0.05) for any genes, except FGF19, which expression was increased (p < 0.0001) in AGP compared to P35. No FM or AGP effects were observed (p > 0.05) for levels of deoxycholic and cholic acids. However, all the genes, deoxycholic acid, and plasma cholic acid were affected by age (p < 0.0001). In general, the data indicate that FM did not negatively impact bile acid metabolism and enterohepatic circulation, which appeared to be age dependent. However, more research should be conducted to confirm these results and investigate the effects of FM on bile acid metabolism, fat digestion, and intestinal microbiota in broiler chickens.

Temporal dynamics of the chicken mycobiome.

The microbiome is an integral part of chicken health and can affect immunity, nutrient utilization, and performance. The role of bacterial microbiota members in host health is relatively well established, but less attention has been paid to fungal members of the gastrointestinal tract (GIT) community. However, human studies indicate that fungi play a critical role in health. Here, we described fungal communities, or mycobiomes, in both the lumen and mucosa of the chicken ileum and cecum from hatch through 14 days of age. We also assessed the effects of delayed access to feed immediately post-hatch (PH) on mycobiome composition, as PH feed delay is commonly associated with poor health performance. Chicken mycobiomes in each of the populations were distinct and changed over time. All mycobiomes were dominated by Gibberella, but Aspergillus, Cladosporium, Sarocladium, Meyerozyma, and Penicillium were also abundant. Relative abundances of some taxa differed significantly over time. In the cecal and ileal lumens, Penicillium was present in extremely low quantities or absent during days one and two and then increased over time. Meyerozyma and Wickerhamomyces also increased over time in luminal sites. In contrast, several highly abundant unclassified fungi decreased after days one and two, highlighting the need for improved understanding of fungal gut biology. Mycobiomes from chicks fed during the first 2 days PH versus those not fed during the first 2 days did not significantly differ, except during days one and two. Similarities observed among mycobiomes of fed and unfed chicks at later timepoints suggest that delays in PH feeding do not have long lasting effects on mycobiome composition. Together, these results provide a foundation for future mycobiome studies, and suggest that negative health and production impacts of delayed feeding are not likely related to the development of fungal populations in the GIT.

Robust validation and performance comparison of immunogenicity assays assessing IgG and neutralizing antibodies to SARS-CoV-2.

To enable benchmarking of immunogenicity between candidate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there is a need for standardized, validated immunogenicity assays. In this article, we report the design and criteria used to validate immunogenicity assays and the outcome of the validation of serologic and functional assays for the evaluation of functional immune response and antibody titers in human serum. A quantitative cell-based microneutralization (MNT) assay, utilizing a reference standard, for detecting anti-SARS-CoV-2 spike protein-neutralizing antibodies in human serum and Meso Scale Discovery's multiplex electrochemiluminescence (MSD ECL) assay for immunoglobulin G (IgG) antibodies to SARS-CoV-2 spike, nucleocapsid, and receptor-binding domain (RBD) proteins were assessed for precision, accuracy, dilutional linearity, selectivity, and specificity using pooled human serum from coronavirus disease 2019 (COVID-19)-confirmed recovered donors. Both assays met prespecified acceptance criteria for precision, relative accuracy, dilutional linearity, selectivity, and specificity. Both assays demonstrated high specificity for the different SARS-CoV-2 antigens or virus tested, and no significant cross-reactivity with seasonal coronaviruses. An evaluation to compare the neutralizing activity in the MNT assay to the IgG measured using the MSD ECL assay showed a strong correlation between the presence of neutralizing activity and amount of antibodies against the spike and RBD proteins in sera from both convalescent and vaccinated individuals. Finally, the MNT assay was calibrated to the WHO reference standard to enable reporting of results in international units, thus facilitating comparison of immunogenicity data generated by different assays and/or laboratories. The MSD ECL assay has previously been calibrated. In conclusion, these validated assays for the evaluation of functional immune response and antibody titers following SARS-CoV-2 vaccination could provide a relatively simple standardized approach for accurately comparing immune responses to different vaccines and/or vaccination regimens.

Safety and efficacy of a virulence gene-deleted live vaccine candidate for fowl typhoid in young chickens.

The safety and efficacy of a live lon-and-cpxR-deleted Salmonella enterica serovar Gallinarum (SG) vaccine candidate (JOL916) was evaluated in young layer chickens. Vaccinated (n=25) and unvaccinated (n=25) groups were organized, respectively, at 1, 2, 3, and 4 weeks of age. One-week-old and 2-week-old chickens were orally inoculated with 2×10(7) colony-forming units of JOL916, and orally challenged with 2 x 10(6) colony-forming units of a wild-type SG strain at the third week post vaccination (w.p.v.). Doses of vaccination and challenge were increased 10-fold for 3-week-old and 4-week-old chickens. SG-antigen-specific peripheral lymphocyte proliferation response and concentrations of plasma IgG and secretary IgA in the intestine were examined at the second w.p.v. Gross lesions of the liver and spleen and recovery of the vaccine strain from the spleen were also examined at the second w.p.v. No evidence of side effects was detected by observation of general condition and body weight gain in all vaccinated groups. No, or very mild, gross lesions in the chickens were observed in the liver and/or spleen after vaccination. Significant cellular immune responses and systemic IgG responses were induced after vaccination in all age groups. Elevation of secretary IgA concentration was significant in the group, vaccinated at the age of 1 week. Depression scores after challenge were significantly lower in the vaccinated groups, as compared with the corresponding control groups. Significant reductions of death rates were observed in all vaccinated groups, as compared with the equivalent unvaccinated groups. Thus, the oral vaccination of young chickens with JOL916 was demonstrated to be safe. Moreover, it offered efficient protection against fowl typhoid.

Comparison of the safety and efficacy of a new live Salmonella Gallinarum vaccine candidate, JOL916, with the SG9R vaccine in chickens.

We evaluated a recently developed live vaccine candidate for fowl typhoid (FT)-JOL916, a lon/cpxR mutant of Salmonella Gallinarum (SG)-by comparing its safety and efficacy with that of the well-known rough mutant strain SG9R vaccine in 6-wk-old Hy-Line hens. Forty-five chickens were divided into three groups of 15 chickens each. The chickens were then intramuscularly inoculated with 2 x 10(7) colony-forming units (CFUs) of JOL916 (JOL916 group), 2 x 10(7) CFUs of SG9R (SG9R group), or phosphate-buffered saline (control group). After vaccination, no clinical symptoms were observed in any of the groups. No differences in body weight increase were detected among the three groups postvaccination. A cellular immune response was observed at 2 wk postvaccination (wpv) in the JOL916 group with the peripheral lymphocyte proliferation assay, whereas no response was detected in the SG9R group. Elevation of SG antigen-specific plasma immunoglobulin was observed 2 and 3 wpv in the JOL916 and SG9R vaccine groups, respectively. After virulent challenge on day 25 postvaccination, 0, 1, and 15 chickens in the JOL916 group, SG9R group, and control group, respectively, died by 12 days postchallenge; the death rate of the SG9R vaccine group was statistically similar to that of the JOL916 group. Postmortem examination revealed that the JOL916 vaccine offered more efficient protection than the SG9R vaccine, with significantly decreased hepatic necrotic foci scores, splenic enlargement scores, necrotic foci scores, and recovery of the challenge strain from the spleen. Vaccination with JOL916 appears to be safe and offers better protection than SG9R against FT in chickens.

Safety evaluation and immunogenicity of arabinose-based conditional lethal Salmonella Gallinarum mutant unable to survive ex vivo as a vaccine candidate for protection against fowl typhoid.

In seeking to develop a safe fowl typhoid (FT) vaccine, a novel candidate lacking cpxR, lon, and asd Salmonella Gallinarum (SG) genes was constructed with the plasmid-containing araC::P(araBAD)::asd system. A balanced-lethal host-vector system based on the essential bacterial gene for aspartate beta-semialdehyde dehydrogenase (asd) was used to construct the SG mutant strain. A plasmid (p15A ori) with an araC::P(araBAD)::asd cassette was introduced into an auxotrophic mutant to prevent ex vivo survival. The safety, immunity, and protective properties of the SG mutant were evaluated. Inoculation of the mutant at 10(6) colony-forming units (CFU) did not result in recovery in feces and internal organs, whereas inoculation at 10(8) and 10(10) CFU resulted in moderate bacterial recovery from feces and organs. Birds immunized with the mutant were challenged with a virulent SG strain at day 14 postimmunization; significantly reduced mortality and induced plasma immunoglobulin (Ig)G and mucosal IgA responses were noted. Cellular immune responses as evaluated by a peripheral lymphocyte proliferation assay were also significantly induced. The balanced-lethal host-vector system for construction of SG mutants is an effective and improved approach for safe vaccine construction against FT.

Effect of Lockdown on Diabetes Care During the COVID-19 Pandemic: Result of a Telephone-Based Survey Among Patients Attending a Diabetic Clinic in Northern India.

Background The coronavirus disease 2019 (COVID-19) pandemic has aggravated the demand for diabetes care due to restrictive measures like the lockdown affecting access to healthcare services. The current study was conducted to assess the changes in medication compliance, dietary pattern, and glucose monitoring during the lockdown period as compared to the pre-lockdown period among patients living with type 2 diabetes mellitus (T2DM) attending a diabetes clinic in northern India. Methods This cross-sectional study was conducted between May and July 2020. Information regarding the sociodemographic and clinical profiles of the patients like age, sex, income, qualification, family history of diabetes, history of smoking and alcohol, type of treatment, co-morbidities, drug adherence for T2DM, changes in the pattern of diet, physical activity, blood glucose monitoring, and drug usage during and before the lockdown was collected through telephonic interviews using a structured tool. Descriptive analysis was performed, and the chi-square and Wilcoxon sign ranks tests were used to see the association between variables. Results A total of 260 patients were enrolled in the study. A higher proportion of males reported a decrease in the consumption of cereals (13.9%), eggs (56.5%), and meat and fish (92.7%) and an increase in water intake (25.8%) while a higher proportion of females reported no change in physical activity levels (77.2%) during the lockdown against pre-COVID times. There was a significant improvement in medication adherence and glycemic control during the lockdown period as compared to the pre-lockdown times. Conclusion More time for self-care, adequate counseling about glycemic goals, and knowledge of self-monitoring of blood glucose levels helped the majority of patients in adopting a healthy lifestyle and achieve better glycemic control during the COVID-19 lockdown.