Comparative proteomic analysis of hemolymph from uninfected and Candidatus Liberibacter asiaticus-infected Diaphorina citri.

Hemolymph was characterized from Diaphorina citri adults infected with the phytopathogen, Candidatus Liberibacter asiaticus (CLas), and compared with that from uninfected psyllids. This study identified 5531 and 3220 peptides within infected and uninfected hemolymph using nano-LC-MS/MS. A reduced number of proteins were detected for D. citri and all known endosymbionts within infected hemolymph as compared to uninfected hemolymph. A large number of immune defense proteins were absent from D. citri hemolymph; however, a single recognition protein (PGRP), two serine protease inhibitors, three prophenoloxidase (proPO) enzymes, and a single serine protease in an uninfected D. citri were detected. The hemolymph is nearly devoid of nutrient storage proteins. This is the first proteomic analysis of D. citri hemolymph that also analyses the components contributed by all the endosymbionts. By comparing the contribution of each endosymbiont (CCR, CPA, and WB) in the presence and absence of CLas infection, this study offers initial insights regarding the hemolymph response to microbial community shifts associated with D. citri infection status. Our data also present potential protein targets for analysis and disruption of CLas transmission that may facilitate management of huanglongbing (HLB) caused by CLas in citrus.

Thymol-based submicron emulsions exhibit antifungal activity against Fusarium graminearum and inhibit Fusarium head blight in wheat.

AIMS: Fusarium graminearum is a very destructive fungal pathogen that leads to Fusarium head blight (FHB) in wheat, a disease which costs growers millions of dollars annually both in crop losses and in remediation efforts. Current countermeasures include the deployment of wheat varieties with some resistance to FHB in conjunction with timed fungicide treatments. In this article, we introduce a fungicide based on thymol, a naturally occurring plant phenolic derived from essential oils. To overcome the hydrophobicity of thymol, the thymol active was incorporated into a low-surfactant submicron emulsion with and without a carrier oil. METHODS AND RESULTS: The minimum fungicidal concentration of F. graminearum was found to be both 0·02% for thymol emulsions with and without an oil component. Time-to-kill experiments showed that thymol emulsions were able to inactivate F. graminearum in as little as 10 s at concentrations above 0·06%. Spraying the thymol emulsions (~0·1% range) on the wheat variety Bobwhite demonstrated significant reductions in FHB infection rate (number of infected spikelets). However, with 0·5% thymol, the wheat heads exhibited premature senescence. Transmission and scanning electron micrographs suggest that the mechanism of antifungal action is membrane mediated, as conidia exposed to thymol showed complete organelle disorganization and evidence of lipid emulsification. CONCLUSION: The collective experimental data suggest that thymol emulsions may be an effective naturally derived alternative to the current thymol treatments, and chemical fungicides in ameliorating FHB. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first thymol-derived nanoemulsion particles resuspended into water and not DMSO, exhibiting the same antibacterial/antifungal activity as previously described thymol and thyme oil treatments. This drastically reduces the environmental footprint thymol will leave if utilized as a fungicide treatment on field crops.

Analysis of gene transcription and relative abundance of the cys-motif gene family from Campoletis sonorensis ichnovirus (CsIV) and further characterization of the most abundant cys-motif protein, WHv1.6.

The cys-motif gene family associated with Campoletis sonorensis ichnovirus contains 10 members, WHv1.6, WHv1.0, VHv1.1, VHv1.4, AHv1.0, A'Hv0.8, FHv1.4, LHv2.8, UHv0.8, and UHv0.8a. The results of this study indicated that, within the encapsidated virion, WHv1.6 is the most abundant cys-motif gene, while the combined AHv genes are the least abundant. During parasitization of Heliothis virescens by Campoletis sonorenis, WHv1.6 transcripts were the mostly highly expressed, while the combined UHv genes had the lowest expression. Further proteomic analysis of WHv1.6 showed that it accumulates at high levels in parasitized plasma by 6 h, and is detectable in the haemocytes, fat body, malpighian tubules, nerve cord and epidermis by 2 days after parasitization. Localization experiments led us to conclude that WHv1.6 interacts with the cell membrane along with other organelles within a virus-infected cell and prevents immunocytes from spreading or adhering to a foreign surface. Similarly to VHv1.4 and VHv1.1, WHv1.6 is able to inhibit the translation of haemocyte and Malpighian tubule RNAs. Our results showed that the expression of cys-motif genes during parasitization is related to the gene copy number of each gene within the encapsidated virion and may also be dependent upon cis-regulatory element activity in different target tissues. In addition, WHv1.6 plays a major role in inhibiting the cellular encapsulation response by H. virescens.

Antibacterial effect of protamine in combination with EDTA and refrigeration.

The antimicrobial effect of protamine (clupeine) on a range of gram-positive and gram-negative foodborne pathogens and spoilage bacteria, was evaluated using an agar dilution assay and a broth dilution assay with Alamar Blue as growth indicator. Protamine was tested alone at concentrations from 0 to 10,000 microg/ml, and in combination with EDTA (0.9 mM). Assays were performed at 5 degrees C, 10 degrees C, 18 degrees C and 30 degrees C to test the effect of temperature. Minimum inhibitory concentration (MIC) values ranged from 10 microg/ml for Brochothrix thermosphacta to no inhibition at 10,000 microg/ml for bacteria such as Aeromonas hydrophila, proteolytic strains of Clostridium botulinum, Hafnia alvei and Morganella morganii. The minimum bactericidal concentrations (MBCs) were generally higher than MICs. In combination with EDTA, MICs of protamine decreased for gram-negative test strains, whereas EDTA alone inhibited gram-positive strains. The effect of assay incubation temperature was variable and not clear for most strains. Concentrations of 100-750 microg/ml protamine inhibited the five non-proteolytic C. botulinum strains, while none of the eight proteolytic strains was inhibited, indicating the possible role of proteolytic enzymes in protecting cells from protamine. Clearing zones, indicative of proteolytic activity, were observed in the opaque TSB-agarose around colonies of some but not all protamine-resistant bacteria, suggesting that this is not the only resistance mechanism. Addition of 5% (w/v) gelatin to study the effect of an increased protein concentration in the agar dilution assay showed that electrostatic interactions between protamine and the protein decreased the antimicrobial efficacy of the peptide.

Replication-defective mutants of mouse cytomegalovirus protect against wild-type virus challenge.

Five temperature-sensitive mutants (tsm9, tsm13, tsm20, tsm22, tsm30) of murine cytomegalovirus have been shown previously not to produce infectious virus in mice. In the present study, the stage at which these mutants are blocked in their replication in vitro was examined by transcriptional analysis of 4 temporally regulated marker genes (IE-1, E-1, gB and gH) using a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) coupled with an electron microscopic analysis of infected cells incubated at permissive (33 degrees C) and non-permissive (39 and/or 40 degrees C) temperatures. Replication of tsm13 appeared to be blocked at a late phase of replication after capsid formation while the block appeared to be as early as the immediate-early phase in tsm22- infected cells. In contrast, mutants tsm9, tsm20 and tsm30 were blocked at a maturation step, probably of capsid formation, as gene transcription of all 4 marker genes occurred, albeit at reduced level, at 39 and 40 degrees C but no capsids or virions were produced at 40 degrees C. Replication and transcription of mutants tsm13, tsm20 and tsm30 were also examined in infected mice. Mutant tsm13 showed no gene expression or infectious virus while mutants tsm20 and tsm30 produced no infectious virus from days 3-60 post infection, except unusually for a low titre of tsm30 (2.3 x 10(3) pfu/ml) in salivary glands 21 days post infection. Gene transcription of all 4 marker genes was observed in one or more tissues (salivary glands, spleen, kidneys, liver, thymus, heart, lungs) at one or more time points (3, 7, 10, 14, 21 days post-infection) with both mutants. Mice became infected latently with tsm20 but not tsm30, and mice previously infected with tsm20 or tsm30 were protected against a sub-lethal challenge with virulent parental virus; tsm30 also protected against a lethal challenge. This suggests that these two mutants may be good model vaccines for further studies on the mechanism of protection induced and for identification of the ts genes.

Solubility and antimicrobial efficacy of protamine on Listeria monocytogenes and Escherichia coli as influenced by pH.

The antimicrobial efficacy of protamine on Listeria monocytogenes and Escherichia coli was evaluated at concentrations from 50 to 10 000 microgram ml-1 and pH levels from 5.5 to 8.0. The minimum inhibitory concentrations decreased with increasing pH. Protamine inhibited E. coli at all pH values while L. monocytogenes was inhibited at pH 6.5 and above. The antimicrobial efficacy of protamine decreased in the presence of negatively charged gelatine B but remained almost unchanged with addition of the positively charged gelatine A. Binding studies showed that the amount of protamine adsorbed to culture media components in tryptic soy broth and bacterial cells increased with increasing pH values. The increased efficacy of protamine at alkaline pH may be explained on the basis of an increase in electrostatic affinity for the cell surface of target cells. E. coli produced a protamine-degrading enzyme, however, was still susceptible to protamine.

Separation of paralytic shellfish poisoning toxins on Chromarods-SIII by thin-layer chromatography with the Iatroscan (mark 5) and flame thermionic detection.

Thin-layer chromatography (TLC) on Chromarods-SIII with the Iatroscan (Mark-5) and a flame thermionic detector (FTID) was used to develop a rapid method for the detection of paralytic shellfish poisoning (PSP) toxins. The effect of variation in hydrogen (H2) flow, air flow, scan time and detector current on the FTID peak response for both phosphatidylcholine (PC) and PSP were studied in order to define optimum detection conditions. A combination of hydrogen and air flow-rates of 50 ml/min and 1.5-2.0 l/min respectively, along with a scan time of 40 s/rod and detector current of 3.0 A (ampere) or above were found to yield the best results for the detection of PSP compounds. Increasing the detector current level to as high as 3.3 A gave about 130 times more FTID response than did flame ionization detection (FID), for PSP components. Quantities of standards as small as 1 ng neosaxitoxin (NEO), 5 ng saxitoxin (STX), 5 ng B1-toxins (B1), 2 ng gonyautoxin (GTX) 2/3, 6 ng GTX 1/4 and 6 ng C-toxins (C1/C2) could be detected with the FTID. The method detection limits for toxic shellfish tissues using the FTID were 0.4, 2.1, 0.8 and 2.5 micrograms per g tissue for GTX 2/3, STX, NEO and C toxins, respectively. The FTID response increased with increasing detector current and with increasing the scan time. Increasing hydrogen and air flow-rates resulted in decreasing sensitivity within defined limits. Numerous solvent systems were tested, and, solvent consisting of chloroform: methanol-water-acetic acid (30:50:8:2) could separate C toxins from GTX, which eluted ahead of NEO and STX. Accordingly, TLC/FTID with the Iatroscan (Mark-5) seems to be a promising, relatively inexpensive and rapid method of screening plant and animal tissues for PSP toxins.

Fluorometric detection of paralytic shellfish poisoning toxins.

A rapid qualitative screening method was developed for the fractionation of paralytic shellfish poisoning toxins. Periodic acid, t-butyl hydroperoxide, and hydrogen peroxide were tested as oxidants for the fluorometric detection of paralytic shellfish toxins. Hydrogen peroxide was found to be the most convenient and efficient oxidant since the fluorescence can be detected after the incubation of toxins at 100 degrees C for 3-5 min. In addition to the structure of the compound, the incubation temperature and time, the amount of acid, and the peroxide concentration affect the fluorescence reaction. This method was more efficient than the previously published peroxidation methods which involved lengthy incubation periods or time-consuming pH adjustment. Also, far greater sensitivity was achieved with the new method with levels of 0.027, 0.054, 0.023, 0.003, 0.0002, and 0.0006 pmol being easily detected for saxitoxin, neosaxitoxin, gonyautoxin 1 and 4, gonyautoxin 2 and 3, C toxins, and B toxins, respectively. The method is particularly valuable for the screening of fractions separated by column chromatography.

Thermal Resistance of Paralytic Shellfish Poison in Soft-Shell Clams.

Toxic soft-shell clams ( Mya arenaria ) were collected and the meats homogenized and tested for toxicity by the A.O.A.C. mouse bioassay procedure. The homogenate was incubated at temperatures ranging from 220 to 269.5°F and toxicities measured in samples heated for various time intervals. The relationships between toxicity and the time of heating were semilogarithmic for each of the six incubation temperatures. Decimal reduction times were calculated for each heat treatment and were plotted (log scale) against heating temperature. The thermal-destruction-time (TDT) curve was linear (r2 = 0.97), indicating that the kinetics of paralytic shellfish poison destruction are similar to those of most microorganisms. The toxin levels were also analyzed by high performance liquid chromatography for 110 samples and although results compared favorably with the bioassay data, its reliability for routine assessment of toxicity was not clearly established.

Postmortem metabolism of short-finned squid muscle (Illex illecebrosus).

Postmortem biochemical changes were examined in the mantle muscle of the short-finned squid (Illex illecebrosus) in relation to the physical events associated with rigor. Unlike mammalian muscle, the major muscle phosphagen is arginine phosphate rather than creatine phosphate. Arginine phosphate levels did not change dramatically during the progress of rigor development. ATP depletion was found to be closely related to glycogen depletion as is often observed in mammalian muscle. The postmortem accumulation of octopine was related to the initial muscle glycogen content at death but a significant lag in its production was observed. The postmortem conversion of glucose to glucose-6-phosphate appeared to be the rate-limiting step in the overall conversion of glycogen to octopine. The intermediates found in the postmortem catabolism of squid muscle ATP were ADP, AMP, IMP Ino and Hx. Unlike most vertebrate fishes, AMP was found to accumulate in squid before conversion to IMP whereas accumulations of IMP and Ino were less than those normally found in vertebrate muscle.

Localization, characterization and partial purification of TMAO-ase.

1. Formaldehyde (FA) and dimethylamine (DMA) are generated in several species of fish of commercial importance. 2. The present study describes the localization, partial purification and characterization of an enzyme recovered from cod kidney tissue which is responsible for the reduction of trimethylamine oxide (TMAO) to FA and DMA. 3. The enzyme, TMAO-ase was found to be bound to purified cod kidney lysosomes and was separated into at least four distinct isozymes by isoelectric focussing.

The effects of dietary selenium and vitamin E on avian white muscle disease as measured by both chemical and physical parameters.

Commercial chicken broilers were fed a semipurified diet deficient in vitamin E and selenium from day 1 to day 13 ex ova and subsequently fed varying levels of dietary selenium and vitamin E. All birds were sacrificed on the 28th day, stored for 36 hr at 2 C to allow the onset and resolution of rigor, and frozen at -32 C until needed. Total cathepsin content of the Pectoralis major depended upon dietary vitamin E for birds receiving 0 to 12 IU/kg, whereas selenium administered at .05 to .16 ppm in the diet showed no statistically significant effect. Similarly, total protein content of P. major increased with increasing level of dietary vitamin E, but the level of dietary selenium had no effect. Muscle break strength was significantly affected by dietary selenium and vitamin E (P = .0092) interacting together. Catheptic activity and muscle protein explained 6.36% and 3.58% of the viriability in muscle break strength. Birds with more advanced avian white muscle disease showed higher break strength values. Ultrastructural deterioration of the myipathic muscle included disintegration of blood vessel walls, transverse tubules, and mitochondrial membranes as well as the obvious disruption of the myofibrillar components. Myelin figures were present in diseased, but not in normal, muscle. Accumulation of adipocytes both extracellularly and intracellularly occurred in selenium and vitamin E-deficient birds.