Hydrothermally Treated Chitosan Hydrogel Loaded with Copper and Zinc Particles as a Potential Micronutrient-Based Antimicrobial Feed Additive

Large-scale use of antibiotics in food animal farms as growth promoters is considered as one of the driving factors behind increasing incidence of microbial resistance. Several alternatives are under investigation to reduce the amount of total antibiotics used in order to avoid any potential transmission of drug resistant microbes to humans through food chain. Copper sulfate and zinc oxide salts are used as feed supplement as they exhibit antimicrobial properties in addition to being micronutrients. However, higher dosage of copper and zinc (often needed for growth promoting effect) to animals is not advisable because of potential environmental toxicity arising from excreta. Innovative strategies are needed to utilize the complete potential of trace minerals as growth promoting feed supplements. To this end, we describe here the development and preliminary characterization of hydrothermally treated chitosan as a delivery vehicle for copper and zinc nanoparticles that could act as a micronutrient based antimicrobial feed supplement. Material characterization studies showed that hydrothermal treatment makes a chitosan hydrogel that re-arranged to capture the copper and zinc metal particles. Systemic antimicrobial assays showed that this chitosan biopolymer matrix embedded with copper (57.6 μg/ml) and zinc (800 μg/ml) reduced the load of model gut-bacteria (target organisms of growth promoting antibiotics) such as Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Lactobacillus fermentum under in vitro conditions. Particularly, the chitosan/copper/zinc hydrogel exhibited significantly higher antimicrobial effect against L. fermentum, one of the primary targets of antibiotic growth promoters. Additionally, the chitosan matrix ameliorated the cytotoxicity levels of metal supplements when screened against a murine macrophage cell line RAW 264.7 and in TE-71, a murine thymic epithelial cell line. In this proof of concept study, we show that by using chitosan as a delivery platform, micronutrient-based metal feed additives could be used to minimize the undesirable levels of microbial population without causing significant cytotoxic effect under in vitro conditions. These findings provide the platform for further studies in target animal models to quantify the required physiological concentrations of copper and zinc when delivered via a chitosan hydrogel platform to elicit a growth promoting effect without causing any toxicity

PATHOTYPING OF NEWCASTLE DISEASE VIRUS BY MEAN DEATH TIME AND REAL-TIME PCR ASSAY: AN EMPIRICAL COMPARISON

Newcastle disease (ND) remains the most significant disease in the poultry sector and contributes to huge economic loss. Early detection and pathotyping of Newcastle disease virus associated with field infection are highly crucial. In vivo pathogenicity assaying is a sensitive and specific pathotyping tool used for the detection and identification of NDV was used until the recent past. Genome-based sequence analysis yields promising results in virulence determination. Keeping the above facts, the present study was designed to compare the efficacy of conventional and molecular assays in NDV virulence determination. In this study twelve NDV isolates (Isolate numbers 463, 464, 475, 476, 122-17C, 122-17D, 122-17E, 128-17A, 128-17D, 137, 139, 141) available in the Department of Veterinary Microbiology, Madras Veterinary College (MVC), Chennai was subjected to differentiation of virulent and avirulent strains using mean death time (MDT) in specific pathogen-free (SPF) embryonated eggs and TaqMan minor groove binding (MGB) probe real-time PCR assay. Pathotyping based on the MDT revealed two NDV isolates (isolate no. 476 and 128-17D) as velogenic strains and the remaining ten NDV isolates as lentogenic strains. Pathotyping based on TaqMan MGB probe real-time PCR assay revealed six NDV isolates (476, 128-17D, 463, 464, 475, 137) as velogenic/mesogenic strains and remaining six NDV isolates (122-17C, 122-17D, 122-17E 128-17A, 139, 141) as lentogenic strains. Using a TaqMan MGB probe in real-time PCR assay, four NDV isolates (463, 464, 475, 137) which were MDT pathotyped as lentogenic strains were re-pathotyped as velogenic/mesogenic strains, which indicates the greater sensitivity of TaqMan MGB probe real-time PCR assay in pathotyping of NDV over conventional MDT

Brucella suis urease encoded by ure1 but not ure2 is necessary for intestinal infection of BALB/c mice

BACKGROUND: In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure1 and ure2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing. RESULTS: The deduced amino acid sequence of urease-α subunits of operons-1 and -2 exhibited substantial identity with the structural ureases of alpha- and beta-proteobacteria, Gram-positive and Gram-negative bacteria, fungi, and higher plants. Four ure deficient strains were generated by deleting one or more of the genes encoding urease subunits of B. suis strain 1330 by allelic exchange: strain 1330Δure1K (generated by deleting ureD and ureA in ure1 operon), strain 1330Δure2K (ureB and ureC in ure2 operon), strain 1330Δure2C (ureA, ureB, and ureC in ure2 operon), and strain 1330Δure1KΔure2C (ureD and ureA in ure1 operon and ureA, ureB, and ureC in ure2 operon). When grown in urease test broth, strains 1330, 1330Δure2K and 1330Δure2C displayed maximal urease enzyme activity within 24 hours, whereas, strains 1330Δure1K and 1330Δure1KΔure2C exhibited zero urease activity even 96 h after inoculation. Strains 1330Δure1K and 1330Δure1KΔure2C exhibited slower growth rates in tryptic soy broth relative to the wild type strain 1330. When the BALB/c mice were infected intraperitoneally with the strains, six weeks after inoculation, the splenic recovery of the ure deficient strains did not differ from the wild type. In contrast, when the mice were inoculated by gavage, one week after inoculation, strain 1330Δure1KΔure2C was cleared from livers and spleens while the wild type strain 1330 was still present. All B. suis strains were killed when they were incubated in-vitro at pH 2.0. When the strains were incubated at pH 2.0 supplemented with 10 mM urea, strain 1330Δure1K was completely killed, strain 1330Δure2C was partially killed, but strains 1330 and 1330Δure2K were not killed. CONCLUSION: These findings suggest that the ure1 operon is necessary for optimal growth in culture, urease activity, resistance against low-pH killing, and in vivo persistence of B. suis when inoculated by gavage. The ure2 operon apparently enhances the resistance to low-pH killing in-vitro

Cataloguing functionally relevant polymorphisms in gene DNA ligase I: a computational approach

A computational approach for identifying functionally relevant SNPs in gene LIG1 has been proposed. LIG1 is a crucial gene which is involved in excision repair pathways and mutations in this gene may lead to increase sensitivity towards DNA damaging agents. A total of 792 SNPs were reported to be associated with gene LIG1 in dbSNP. Different web server namely SIFT, PolyPhen, CUPSAT, FASTSNP, MAPPER and dbSMR were used to identify potentially functional SNPs in gene LIG1. SIFT, PolyPhen and CUPSAT servers predicted eleven nsSNPs to be intolerant, thirteen nsSNP to be damaging and two nsSNPs have the potential to destabilize protein structure. The nsSNP rs11666150 was predicted to be damaging by all three servers and its mutant structure showed significant increase in overall energy. FASTSNP predicted twenty SNPs to be present in splicing modifier binding sites while rSNP module from MAPPER server predicted nine SNPs to influence the binding of transcription factors. The results from the study may provide vital clues in establishing affect of polymorphism on phenotype and in elucidating drug response

Antimicrobial Magnesium Hydroxide Nanoparticles As An Alternative To Cu Biocide For Crop Protection

In agriculture, prolonged use of copper biocides increases the risk of development of Cu resistance and its accumulation in soil, demanding an alternative. In this paper, we report antimicrobial magnesium hydroxide nanoparticles (NPs) as an alternative to Cu biocides with low cytotoxicity. To improved bioavailability, Mg hydroxide NPs were synthesized followed by coating with water-soluble capping agents, trisodium citrate (zeta potential, ζ = -22 mV) or betaine (ζ = +35 mV). Electron microscopy study confirmed the formation of ∼10-nm-sized cubical NPs with citrate and ∼100-nm-sized lamellar NPs with betaine. As-synthesized Mg hydroxide NPs inhibited bacterial growth of X. alfalfae, P. syringae, and E. coli within 4 h. Significant bacterial growth inhibition and killing were observed at 24 h post-treatment. Phytotoxicity studies on tomato plants showed no significant tissue injury. Therefore, Mg hydroxide NPs have the potential to serve as a Cu alternative

Hydrothermally Derived Water-Dispersible Mixed Valence Copper-Chitosan Nanocomposite As Exceptionally Potent Antimicrobial Agent

We report, for the first time, a one-step hydrothermal (HT) process to design and synthesize water-dispersible chitosan nanoparticles loaded with mixed valence copper. Interestingly, this HT copper-chitosan biocompatible composite exhibits exceptionally high antimicrobial properties. A comprehensive characterization of the composite indicates that the hydrothermal process results in the formation of monodispersed nanoparticles with average size of 40 ± 10 nm. FT-IR and Raman spectroscopic studies unveiled that the hydrolysis of the glycoside bonds as the origin of the depolymerization of chitosan. Furthermore, X-Ray Photoelectron Spectroscopy measurements confirmed the presence of mixed valence copper states in the composite, while UV–Vis and FT-IR studies revealed the chemical interaction of copper with the chitosan matrix. Hence, the extensive spectroscopic data provide strong evidence that the chitosan structure was rearranged to capture copper oxide nanoparticles. Finally, HT copper-chitosan composite showed a complete killing effect when tested against both Gram negative (E. coli) and Gram positive (S. aureus) bacteria at metallic copper concentration of 100 μg/ml (1.57 mM). At the same concentration, neither pure chitosan nor copper elicited such antimicrobial efficacy. Thus, we show that HT process significantly enhances the synergistic antimicrobial effect of chitosan and copper in addition to increasing the water dispersibility

Molecule modeling of human pentameric alpha(7) neuronal nicotinic acetylcholine receptor and its interaction with its agonist and competitive antagonist

The nicotinic Acetylcholine Receptor (nAChR) is the major class of neurotransmitter receptors that is involved in many neurodegenerative conditions such as schizophrenia, Alzheimer's and Parkinson's diseases. The N-terminal region or Ligand Binding Domain (LBD) of nAChR is located at pre- and post-synaptic nervous system, which mediates synaptic transmission. nAChR acts as the drug target for agonist and competitive antagonist molecules that modulate signal transmission at the nerve terminals. Based on Acetylcholine Binding Protein (AChBP) from Lymnea stagnalis as the structural template, the homology modeling approach was carried out to build three dimensional model of the N-terminal region of human alpha(7)nAChR. This theoretical model is an assembly of five alpha(7) subunits with 5 fold axis symmetry, constituting a channel, with the binding picket present at the interface region of the subunits. alpha-netlrotoxin is a potent nAChR competitive antagonist that readily blocks the channel resulting in paralysis. The molecular interaction of alpha-Bungarotoxin, a long chain alpha-neurotoxin from (Bungarus multicinctus) and human alpha(7)nAChR seas studied. Agonists such as acetylcholine, nicotine, which are used in it diverse array of biological activities, such as enhancements of cognitive performances, were also docked with the theoretical model of human alpha(7)nAChR. These docked complexes were analyzed further for identifying the crucial residues involved in interaction. These results provide the details of interaction of agonists and competitive antagonists with three dimensional model of the N-terminal region of human alpha(7)nAChR and thereby point to the design of novel lead compounds

Comparative analysis of copper and zinc based agrichemical biocide products: materials characteristics, phytotoxicity and in vitro antimicrobial efficacy

In the past few decades, copper based biocides have been extensively used in food crop protection including citrus, small fruits and in all garden vegetable production facilities. Continuous and rampant use of copper based biocides over decades has led to accumulation of this metal in the soil and the surrounding ecosystem. Toxic levels of copper and its derivatives in both the soil and in the run off pose serious environmental and public health concerns. Alternatives to copper are in great need for the agriculture industry to produce food crops with minimal environmental risks. A combination of copper and zinc metal containing biocide such as Nordox 30/30 or an improved version of zinc-only containing biocide would be a good alternative to copper-only products if the efficacy can be maintained. As of yet there is no published literature on the comparative study of the materials characteristics and phyto-compatibility properties of copper and zinc-based commercial products that would allow us to evaluate the advantages and disadvantages of both versions of pesticides. In this report, we compared copper hydroxide and zinc oxide based commercially available biocides along with suitable control materials to assess their efficacy as biocides. We present a detailed material characterization of the biocides including morphological studies involving electron microscopy, molecular structure studies involving X-ray diffraction, phytotoxicity studies in model plant (tomato) and antimicrobial studies involving surrogate plant pathogens (Xanthomonas alfalfae subsp. citrumelonis, Pseudomonas syringae pv. syringae and Clavibacter michiganensis subsp. michiganensis). Zinc based compounds were found to possess comparable to superior antimicrobial properties while exhibiting significantly lower phytotoxicity when compared to copper based products thus suggesting their potential as an alternative

Fixed-Quat: An Attractive Nonmetal Alternative To Copper Biocides Against Plant Pathogens

In this paper, we report a nonphytotoxic bactericide and fungicide formulation containing a composite of silica and quaternary ammonium compound (quat). The composite material was prepared using an acid-catalyzed sol-gel method. Positively charged quat was associated with a negatively charged silica-gel matrix, producing a stable suspension of fixed-quat gel (FQ-G). The morphology of FQ-G and the interaction of quat with silica were characterized using SEM and FTIR, respectively. Silica gel significantly reduced quat phytotoxicity when tested at 500 and 1000 μg/mL foliar-application rates. The in vitro antimicrobial efficacy of FQ-G was evaluated against Xanthomonas alfalfae, Pseudomonas syringae, and Clavibacter michiganensis, showing comparable efficacies to that of quat itself. In field conditions, its efficacy in controlling the bacterial and fungal diseases citrus canker, scab, and melanose on \u27Ray Ruby\u27 red grapefruit was evaluated. Foliar application rates at 100 and 200 μg/mL provided comparable disease control to those of several copper standards, demonstrating the potential for use as an alternative agricultural biocide