Effects of heat treatment on metabolism of tobacco plants infected with Potato virus Y.

Many factors affect successful virus propagation and plant defence responses. Heat shock protein (Hsp) expression after heat shock plays an ambiguous role in viral infection. On the one hand, Hsp70 participates in plant defence response; on the other hand, Hsp70 could interact with viral proteins and facilitate virus propagation. Here, we studied metabolic adaptations of Nicotiana tabacum L. subjected to heat shock (42 °C, 2 h) before or after inoculating the plants with Potato virus Y (potyvirus). RT-qPCR and ELISA were used for potyvirus quantification. Hsp70 and Hsp90 isoforms were analysed by Western blotting. Salicylic, quinic and chlorogenic acid content was determined by LC-MS. The activity of Hatch-Slack enzymes (as markers of potyviral infection in tobacco) and glycosidases was assayed. Application of heat shock before or after inoculation showed accelerated potyviral propagation in comparison with only inoculated plants. Plants exposed to heat shock and concurrently inoculated showed higher potyviral content, higher amount of Hsp70, together with late decline of quinic acid content and low chlorogenic acid content. Spread of potyviral infection correlated with enhanced salicylic acid content and activities of enzymes of the Hatch-Slack cycle, α- and ß-galactosidase, α-mannosidase, α-glucosidase and ß-N-acetylhexosaminidase. Heat shock proteins accelerate potyviral propagation. The lower weight cytosolic and mitochondrial Hsp70 (~50-75 kDa) persist throughout the viral infection. Also, the plant defense response results in increase of salicylic and chlorogenic acids but decrease of quinic acid content.

Enzymatic outside-in cross-linking enables single-step microcapsule production for high-throughput three-dimensional cell microaggregate formation.

Cell-laden hydrogel microcapsules enable the high-throughput production of cell aggregates, which are relevant for three-dimensional tissue engineering and drug screening applications. However, current microcapsule production strategies are limited by their throughput, multistep protocols, and limited amount of compatible biomaterials. We here present a single-step process for the controlled microfluidic production of single-core microcapsules using enzymatic outside-in cross-linking of tyramine-conjugated polymers. It was hypothesized that a physically, instead of the conventionally explored biochemically, controlled enzymatic cross-linking process would improve the reproducibility, operational window, and throughput of shell formation. Droplets were flown through a silicone delay line, which allowed for highly controlled diffusion of the enzymatic cross-linking initiator. The microcapsules' cross-linking density and shell thickness is strictly depended on the droplet's retention time in the delay line, which is predictably controlled by flow rate. The here presented hydrogel cross-linking method allows for facile and cytocompatible production of cell-laden microcapsules compatible with the formation and biorthogonal isolation of long-term viable cellular spheroids for tissue engineering and drug screening applications.

[OCT-Angiography in diabetic maculopathy : Comparison between microaneurysms and the foveal avascular zone with flourescein angiography]. / OCT-Angiographie bei der diabetischen Maculopathie : Vergleich von Mikroaneurysmata und der foveolären avaskulären Zone mit der Fluoreszeinangiographie.

BACKGROUND: Optical coherence tomography angiography allows an exact visualization of retinal vascular changes. To interpret the possibilities and limitations using OCTA in clinical practice, we analysed the quality of OCTA findings concerning the evaluability. Furthermore, we correlated our OCTA findings with fluorescein angiography (FAG) in relation of the presence of microaneurysms (MA) and enlargement of the foveal avascular zone (FAZ) in diabetic maculopathy. PATIENTS AND METHODS: 30 eyes from 15 patients with diabetic maculopathy were imaged in 3 × 3mm volume scans and were depicted in OCTA mode (SSADA algorithm). By using the automatic segmentation we analysed the vascular changes in the superficial and deep capillary plexus and classified them concerning their qualitative evaluability. We analysed the number of MA in OCTA and fluorescein angiography and correlated them using an overlay technique. We analysed the presence of MA in the superficial and deep capillary plexus in OCTA as well. Furthermore we measured the enlargement of the foveal zone with both methods. RESULTS: In a large number of eyes we could not identifiy MA or measure the enlargement of the FAZ because of poor OCTA or FA quality. In the morphological analysis of our findings we identified a comparable number of MA in both methods, although the localization of the MA was different in both methods in a large number of eyes. The majority of MA was located in the deep capillary plexus. The extension of the foveal avascular zone measured in FA revealed a good correlation to the automatically measured "nonflow area" in the OCTA. CONCLUSION: OCTA allows a good visualization of characteristic vascular changes in diabetic maculopathy. In spite of technical limitations OCTA generates a three-dimensional visualisation of avascular changes. The majority of microaneurysms detected by OCTA showed a corresponding finding in FA. The central avascular zone (FAZ) in OCTA correlates well with the findings from FA. OCTA represents an important additional diagnostic tool to interpret vascular changes in DM.

Trichosporon asahii infection of a dialysis PTFE arteriovenous graft.

Trichosporon species are the causative agents of superficial skin infections, such as white piedra. Immunocompromised hosts, particularly those with underlying hematological malignancy, are at risk of developing invasive infection, which usually progresses to disseminated life-threatening disease. Peritonitis caused by Trichosporon has been described in end-stage renal disease patients treated with continuous ambulatory peritoneal dialysis. Here, we report on a Trichosporon infection of an arteriovenous graft in a patient on chronic hemodialysis. The infection was successfully treated with fluconazole and total surgical resection of the graft.

Handling of slaughter cattle in pre and post transport situations including loading and unloading on journeys up to 8 hours in Germany.

In a field study 63 commercial cattle transports to German slaughter plants including 580 bulls, cows and heifers were investigated to estimate the impact of pre and post transport aspects on animal welfare. Study 1 evaluated these aspects as part of a large model covering all possible influences at transport and slaughter on biochemical stress response and carcass and meat quality. Included were keeping system, behavioural and clinical observations at loading, unloading and lairage, transport time, loading density, mounting prevention, regrouping and lairage time. In study 2 more detailed questions about loading and unloading were answered by investigating the impact of facility design and handling criteria on cattle behaviour and heart rate. Cattle from tied housing show an increased stress response to all transport procedures. Many aspects of facility design and handling contribute to quality of loading. Cattle show calmer behaviour, turn, balk or fall less if they are not mixed and driven in small groups on driveways without impediments but with regular sideblock, not wider than the vehicle ramp. Vehicle ramps should have high and closed side gates, be inclined less than 20 degrees, and the step towards ramp should be less than 15 cm. Unloading is improved if performed level and to the back of the vehicle (behaviour, heart rate, cortisol, CK). Direct reloading should be avoided and mounting should be prevented during transport and lairage (CK, lactate, bruising). But mounting prevention devices may increase bruising if set too low (< 20 cm above withers). Longer lairage time is disadvantageous (CK, energy metabolism).

Trichinella infection in wildlife of the southwestern United States.

Several potential mammalian reservoirs of sylvatic species of Trichinella were examined from Texas, New Mexico, and Arizona. During 1998-99, tongues were collected from a black bear (Ursus americanus) in Arizona; from 9 black bears, a coyote (Canis latrans), and a mountain lion (Felis concolor) in New Mexico; and from 154 coyotes, 32 raccoons (Procyon lotor), 13 opossums (Didelphis marsupialis), 4 ocelots (Leopardus pardalis), 3 bobcats (Lynx rufus), and 5 feral hogs (Sus scrofa) in southern Texas. Larvae of Trichinella murrelli were identified by a multiple-polymerase chain reaction analysis in 1 black bear (11.1%) from New Mexico and in 7 coyotes (4.5%) of Texas, whereas Trichinella spiralis larvae were detected in the black bear of Arizona. This is the first report of Trichinella infection in wildlife of New Mexico and Texas and extends the distribution of T. murrelli into the southwestern United States near the border of Mexico.

Detection of aflatoxin-contaminated grain by three granivorous bird species.

Supplemental feeding of game species and the use of backyard feeders to attract avian wildlife are common practices throughout the United States. However, these activities may expose wildlife to aflatoxins. We tested the hypothesis that wild birds would avoid consuming aflatoxin-contaminated feed. Individual northern bobwhites (Colinus virginianus), white-winged doves (Zenaida asiatica), and green jays (Cyanocorax yncas) were presented with feeders that had four compartments, which contained milo that was contaminated with aflatoxin levels of 0, 100, 500, and 1,000 microg/kg, respectively. Feed remaining was weighed at 6, 12, 18, 24, 36, 48, 60, and 72 hr after the initiation of the trial. White-winged doves and northern bobwhites did not avoid contaminated feed. However, green jays selected against aflatoxin-tainted grain. Because white-winged doves and northern bobwhites did not avoid contaminated feed, the risk of exposure to this potentially hazardous toxin exists for these species.

Survey of aflatoxin concentrations in wild bird seed purchased in Texas.

The use of backyard feeders to attract avian wildlife is a common practice throughout the United States. However, feeding wildlife may create a problem due to aflatoxin, a harmful fungal metabolite, which can affect wildlife that are fed contaminated grain. Our study was initiated to determine if songbirds were being exposed to aflatoxin-contaminated feed throughout Texas. Bags of wild bird seed (n = 142) were purchased from grain cooperatives, grocery stores, and pet shops located in the panhandle, central, south, east, and west regions of Texas during spring and summer 1999. Aflatoxin concentrations in bird seed ranged from non-detectable to 2,780 micrograms/kg. Overall, 17% of samples had aflatoxin concentrations greater than 100 micrograms/kg, of which 83% contained corn as an ingredient. Retail establishment effects were noted in the southern and western regions of Texas, with average concentrations of aflatoxin greater from bags of bird seed purchased from grain cooperatives, followed by pet shops, then grocery stores. Regional differences in aflatoxin levels were not apparent from bags of seed purchased at pet shops: however, regional differences were noted in aflatoxin levels from seeds obtained at grocery stores and grain cooperatives. Average aflatoxin concentration from seed purchased at grocery stores was greatest in the panhandle region, followed by the remaining regions. Within grain cooperatives, the panhandle, south, and west regions of Texas exhibited higher levels of aflatoxin-contaminated bird seed than cooperatives within the east and central regions of Texas. Granivorous songbirds in Texas are exposed to aflatoxins at backyard feeders, which may be a significant morbidity and mortality factor.

Bait delivery for oral rabies vaccine to gray foxes.

Rabies is a widespread zoonotic disease that has reached epizootic proportions in gray foxes (Urocyon cinereoargenteus) in central Texas. Because each species of carnivore has different food preferences and foraging strategies, it is essential that the efficacy of a bait delivery program be examined for gray foxes prior to an oral vaccination program being attempted. Field trials were conducted to determine bait preferences of free-ranging gray foxes to selected baits and odor attractants. Baits consisted of polymer cubes made of either dog food meal or fish meal, and a wax-lard cake that was enhanced with marshmallow flavoring. Attractants added to baits exuded sulfurous, fatty, cheesy, or sweet odors and flavors. During 3,589 operable bait station nights, gray fox visitation and bait uptake rates were 9.2% and 8.3%, respectively. Gray foxes exhibited no preference in bait uptake rates between bait and odor attractant combinations. Gray foxes exhibited no difference in cumulative bait uptake rates between onroad and offroad sites; however, the uptake rate by raccoons was significantly greater for baits placed on roads than for baits randomly placed. Raccoons were the major non-target species competing for baits, being attributed with 73% of the total uptake. Visitation and bait uptake rates by raccoons significantly increased after a 7-day lethal removal of raccoons (n = 37) from the study area. Random distribution of baits is recommended; it reduced bait uptake by non-target species without adversely affecting uptake by gray foxes.

Enhanced in vitro oligonucleotide and plasmid DNA transport by VP1 virus-like particles.

PURPOSE: We developed a prokaryotic expression system to express the major capsid protein of Polyomavirus, VP1. Furthermore, we investigated the transport of single stranded (ss) and double stranded (ds) DNA, mediated through VP1 as drug delivery system into mouse fibroblasts. METHODS: To study DNA delivery we used two kinds of DNA, a ssDNA fragment (19mer) and dsDNA (plasmid pEGFPN1, 4.7 kb or a FITC-labelled dsDNA fragment, 1.8 kb). RESULTS: The uptake of VP1 capsoids loaded with FITC-labelled oligodeoxynucleotides (FODNs) was observed. VP1 pentamers loaded with condensates of dendrimer/dsDNA fragments (FITC-labelled) resulted in significantly higher fluorescence signal in the cytoplasm of NIH 3T3 cells in comparison to control experiments without VP1. Additionally, VP1 capsoids loaded with plasmid pEGFPN1 without dendrimers resulted in an approximately 10 fold higher transfection rate in comparison to blank DNA controls. CONCLUSIONS: Our results demonstrated the potential of VP1 capsoids as DNA delivery system. EGFP expression was significantly enhanced when plasmid DNA was delivered via VP1 capsoids, compared to control experiments with naked DNA.

Survival of Trichomonas gallinae in white-winged dove carcasses.

Survival of Trichomonas gallinae was examined in white-winged dove (Zenaida asiatica) carcasses to assess whether birds that have been dead up to 8 hr can be sampled reliably for this protozoan. Carcasses of 100 T. gallinae-positive white-winged doves were separated into four groups of 25 birds, representing 2, 4, 6, and 8 hr post mortem sampling intervals and placed into an environmental chamber maintained at 27 C and 75% relative humidity. Live T. gallinae were isolated in 96, 100, 100, and 92% of the carcasses at each of the respective post mortem intervals. The experiment was repeated with another 100 carcasses of T. gallinae-positive white-winged doves placed in the environmental chamber, this time maintained at 27 C and 40% relative humidity. Live T. gallinae occurred in 96, 100, 96, and 100% of the carcasses at each of the respective post mortem intervals. Across both trials, the overall ability to detect positive birds from sampling carcasses up to 8 hrs post mortem was 97%. An a posteriori experiment was conducted in which 23 and 18 carcasses from the second trial were maintained in the environmental chamber at 27 C and 40% relative humidity and resampled at 24 and 48 hr post mortem, respectively. Live trichomonads were isolated from 91 and 44% of the carcasses at 24 and 48 hr, respectively. Results suggest live T. gallinae can be obtained from dove carcasses reliably up to 8 hr and possibly up to 24 hr after host death. The ability for T. gallinae to survive within this time interval can aid wildlife personnel in monitoring this protozoan at hunter check stations or obtaining samples from recently killed birds.

Effect of climate and type of storage container on aflatoxin production in corn and its associated risks to wildlife species.

The effects of grain storage containers on aflatoxin production, and the relationship between the level of aflatoxin and the number and weight of fluorescing kernels were determined in corn (Zea maize) stored in controlled climate regimes. Two hundred and forty 100-g samples were held up to 3 mos using four types of storage containers placed in four climates. Storage containers included corn placed in metal cans, paper bags, plastic bags, and paper bags placed in plastic bags. Climates were constant during the duration of the project and included a combination of temperatures and humidities. Temperatures were 29-32 C and 14-18 C; relative humidities were 85-88% and 35-40%. In addition, corn was exposed to environmental conditions conductive for aflatoxin production and 100 g samples were randomly collected, examined under ultraviolet light for fluorescence, and then quantified for aflatoxin levels. Corn samples tested negative for aflatoxin at the beginning of the project. Main (i.e., container, climate, and month) and interactive effects were not observed. Mean levels of aflatoxin ranged from 0 to 151 microg/kg. Aflatoxin was produced regardless of type of storage container, time of storage, and climatic conditions; however, only 8% of the samples produced aflatoxin levels that exceeded 50 microg/kg. Fluorescing corn ranged from 0 to 19 kernels per sample, while aflatoxin levels ranged from 0 to 1,375 microg/kg for the same samples. No relationships were found between the number and weight of fluorescing kernels of corn and aflatoxin levels. The black light test yielded a false negative rate of 23% when in fact the aflatoxin concentrations exceeded 50 microg/kg. Therefore, quantifying fluorescing grain under UV light should not be considered a feasible alternative for aflatoxin testing of grain intended for wildlife.

Gray fox response to baits and attractants for oral rabies vaccination.

Rabies is a widespread zoonosis that recently reached epidemic proportions in gray foxes (Urocyon cinereoargenteus) in central Texas. The objectives of this study were to determine bait and attractant preferences among captive gray foxes, to determine the behavioral responses of gray foxes to selected bait-attractant combinations, and to evaluate baits as a delivery mechanism of oral rabies vaccines. Trials were conducted to determine bait preferences of captive gray foxes to selected baits and attractants. Tested baits consisted of a polymer-bound cube made of either dog food meal or fish meal, a polymer-bound cylinder made of dog food meal, and a wax-lard cake that was enhanced with marshmallow or chicken flavoring. Attractants were additives to baits that exuded sweet, sulfurous, fruity, fatty, cheesy, honey, and fishy odors and flavors. Captive gray foxes (n = 31) exhibited a preference for marshmallow wax cakes and polymer dog food baits with a lard interior and granulated sugar exterior. However, gray foxes exhibited chewing behaviors consistent with ingesting an oral vaccine only with the wax cake baits.

Responses of captive and free-ranging coyotes to simulated oral rabies vaccine baits.

The objective of this study was to develop a bait for delivering an oral rabies vaccine to free-ranging coyotes (Canis latrans) in southern Texas. Captive trials were conducted from January to April, 1994, to determine bait preferences and behavioral responses of coyotes (n = 42) to selected baits and attractants. Baits were hollow rectangular cubes made of polymer dog food or fish meal. Attractants had sweet (watermelon), fruity (raspberry), sulfurous (synthetic WU), and lard (beef lard) fragrances. Captive coyotes did not exhibit a preference for either bait bases or attractants; however, coyotes chewed dog food baits 1.6 times more than fish meal baits. Average proximity of coyotes eliciting a response to baits was 2.2 +/- 1.3 m (mean +/- SE). Captive coyotes readily accepted dog food baits containing 2 ml of liquid rhodamine B, a biological marker. Rhodamine B staining of the oropharyngeal region was evident in each captive coyote. Results from the field evaluation of baits and attractants were consistent with that of the captive trials. Of 2,070 bait station-nights conducted from February to April, 1994, coyotes comprised the greatest single species visitation and uptake rates with 31% and 28%, respectively. Bait uptake rates of free-ranging coyotes did not differ among bait-attractant combinations. Coyotes took baits 93% of the time they encountered a bait, regardless of bait type.

Efficacy of bait distributional strategies to deliver canine rabies vaccines to coyotes in southern Texas.

This study sought to develop a baiting strategy to deliver an oral rabies vaccine to free-ranging coyotes (Canis latrans) in southern Texas. To determine bait longevity, dog foodlard baits were placed (n = 50) on- and off-roads during July 1994 and January 1995. Coyote visitation and uptake rates did not differ between on-road and off-road placement of baits. To evaluate bait stations as possible visual cues, baits were placed out both with (n = 50) and without (n = 50) bait stations. A visual cue of a bait station did not affect coyote response to baits. Bait longevity was shorter during July (< or = 4 days) than January because of consumption of baits by imported fire ants (Solenopsis invicta). The effect of two different bait densities on coyote acceptance rates was determined on six 93.5 km2 study areas in southern Texas. Three study areas received a bait density of 19 baits/km2 (50 baits/mi2) and the remaining three study areas received 58 baits/km2 (150 baits/mi2). Coyote bait uptake rates, based upon the proportion of coyotes marked with either tetracycline hydrochloride or rhodamine B or both, were 83% (n = 99 coyotes) and 87% (n = 101 coyotes) for the 19 and 58 baits/km2 densities, respectively. Bait uptake rates did not differ (P > 0.54) between the two bait densities. Rodents and rabbits, which were fed baits containing tetracycline hydrochloride and a simulated oral rabies vaccine sachet containing rhodamine B, did consume the bait but not the rhodamine B sachet. These animals then were killed and fed to captive coyotes (n = 9). Canine teeth were extracted from coyotes and processed for tetracycline determination. Each coyote tested negative for tetracycline. Therefore, it was unlikely that coyote bait consumption rates were overestimated because of coyotes secondarily marking themselves by ingesting prey items that consumed baits.

Baylisascariasis in raccoons from southern coastal Texas.

Two hundred and two Baylisascaris procyonis were collected from 23 (70%) of 33 raccoons (Procyon lotor) at three localities in southern coastal Texas (USA). Abundances of B. procyonis were similar among collection localities. The presence of B. procyonis in Texas is confirmed, and this record considerably extends the potential range of baylisascariasis larval migrans in North America.

Inhibition of HIV-1 protease by short peptides derived from the terminal segments of the protease.

The active HIV-1 protease is a homodimeric enzyme. A beta-sheet consisting of N- and C-terminal segments provides the main driving force for dimerization of the inactive protomers. Several short peptides with sequences derived from the N- and C-termini of the protease were tested for inhibition of protease activity and for inhibition of HIV-1 replication in lymphocytes. Medium inhibitory activity was found with each of the peptides in the enzyme test and no inhibition of the lymphocytes was found up to 200 micrograms/ml. The enzyme tests indicate that HIV-1 protease is the target of the inhibitory action. Synergistic action could not be found with pairs of the peptides derived from the two different termini. Prolonged incubation with one of the peptides increased inhibition indicating a slow dissociation of the protease dimers. No cytotoxic effect of the inhibitors could be found below 200 micrograms/ml.