In vivo delivery of bovine viral diahorrea virus, E2 protein using hollow mesoporous silica nanoparticles

Our work focuses on the application of mesoporous silica nanoparticles as a combined delivery vehicle and adjuvant for vaccine applications. Here we present results using the viral protein, E2, from bovine viral diarrhoea virus (BVDV). BVDV infection occurs in the target species of cattle and sheep herds worldwide and is therefore of economic importance. E2 is a major immunogenic determinant of BVDV and is an ideal candidate for the development of a subunit based nanovaccine using mesoporous silica nanoparticles. Hollow type mesoporous silica nanoparticles with surface amino functionalisation (termed HMSA) were characterised and assessed for adsorption and desorption of E2. A codon-optimised version of the E2 protein (termed Opti-E2) was produced in Escherichia coli. HMSA (120 nm) had an adsorption capacity of 80 [small mu ]g Opti-E2 per mg HMSA and once bound E2 did not dissociate from the HMSA. Immunisation studies in mice with a 20 [small mu ]g dose of E2 adsorbed to 250 [small mu ]g HMSA was compared to immunisation with Opti-E2 (50 [small mu ]g) together with the traditional adjuvant Quillaja saponaria Molina tree saponins (QuilA, 10 [small mu ]g). The humoral responses with the Opti-E2/HMSA nanovaccine although slightly lower than those obtained for the Opti-E2 + QuilA group demonstrated that HMSA particles are an effective adjuvant that stimulated E2-specific antibody responses. Importantly the cell-mediated immune responses were consistently high in all mice immunised with Opti-E2/HMSA nanovaccine formulation. Therefore we have shown the Opti-E2/HMSA nanoformulation acts as an excellent adjuvant that gives both T-helper 1 and T-helper 2 mediated responses in a small animal model. This study has provided proof-of-concept towards the development of an E2 subunit nanoparticle based vaccine.

Organic contaminants - occurrence and biological effects in the Baltic Sea

The Baltic Sea was studied with respect to selected organic contaminants and their ecotoxicology. The research consisted of analyses of total hydrocarbons, polycyclic aromatic hydrocarbons, bile metabolites, hepatic ethoxyresorufin-O-deethylase (EROD) activity, polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs). The contaminants were measured from various matrices, such as seawater, sediment and biota. The methods of analysis were evaluated and refined to comparability of the results. Polyaromatic hydrocarbons, originating from petroleum, are known to be among the most harmful substances to the marine environment. In Baltic subsurface water, seasonal dependence of the total hydrocarbon concentrations (THCs) was seen. Although concentrations of parent polycyclic aromatic hydrocarbons (PAHs) in sediment surface varied between 64 and 5161 ug kg-1 (dw), concentrations above 860 ug kg-1 (dw) were found in all the studied sub-basins of the Baltic Sea. Concentrations commonly considered to substantially increase the risk of liver disease and reproductive impairment in fish, as well as potential effects on growth (above 1000 ug kg-1 dw), were found in all the studied sub-basins of the Baltic Sea except Kattegat. Thus, considerable pollution in sediments was indicated. In bivalves, the sums of 12 PAHs varied on a wet weight basis between 44 and 298 ug kg-1 (ww). The predominant PAHs were high molecular weight and the PAH profiles of M. balthica differed from those found in sediment from the same area. The PAHs were both pyrolytic and petrogenic in origin, and a contribution from diesel engines was found, which indicates pollution of the Baltic Sea, most likely caused by the steadily increasing shipping in the area. The HPLC methods developed for hepatic EROD activity and bile metabolite measurements proved to be fast and suitable for the study of biological effects. A mixed function oxygenase enzyme system in Baltic Sea perch collected from the Gulf of Finland was induced slightly: EROD activity in perch varied from 0.30 14 pmol min-1 mg-1 protein. This range can be considered to be comparable to background values. Recent PAH exposure was also indicated by enhanced levels (213 and 1149 ug kg-1) of the bile metabolite 1-hydroxypyrene. No correlation was indicated between hepatic EROD activity and concentration of 1-hydroxypyrene in bile. PCBs and OCPs were observed in Baltic Sea sediment, bivalves and herring. Sums of seven CBs in surface sediment (0 5 cm) ranged from 0.04 to 6.2 ug kg-1 (dw) and sums of three DDTs from 0.13 to 5.0 ug kg-1 (dw). The highest levels of contaminants were found in the most eastern area of the Gulf of Finland where the highest total carbon and nitrogen content was found and where the lowest percentage proportion of p,p -DDT was found. The highest concentrations of CBs and the lowest concentration of DDTs were found in M. balthica from the Gulf of Finland. The highest levels of DDTs were found in M. balthica from the Hanö Bight, which is the outer part of the Bornholm Basin close to the Swedish mainland. In bivalves, the sums of seven CBs were 72 108 ug kg-1 (lw) and the sums of three DDTs were 66 139 ug kg-1 (lw). Results from temporal trend monitoring showed, that during the period 1985 2002, the concentrations of seven CBs in two-year-old female Baltic herring were clearly decreased, from 9 16 to 2 6 ug kg-1 (ww) in the northern Baltic Sea. At the same time, concentrations of three DDTs declined from 8 15 to 1 5 ug kg-1 (ww). The total concentration of the fat-soluble CBs and DDTs in Baltic herring muscle was shown to be age-dependent; the average concentrations in ten-year-old Baltic herring were three to five-fold higher than in two-year-old herring. In Baltic herring and bivalves, as well as in surface sediments, CB 138 and CB153 were predominant among CBs, whereas among DDTs p,p'-DDD predominated in sediment and p,p'-DDE in bivalves and Baltic herring muscle. Baltic Sea sediments are potential sources of contaminants that may become available for bioaccumulation. Based on ecotoxicological assessment criteria, cause for concern regarding CBs in sediments was indicated for the Gulf of Finland and the northern Baltic Proper, and for the northern Baltic Sea regarding CBs in Baltic herring more than two years old. Statistical classification of selected organic contaminants indicated high-level contamination for p,p'-DDT, p,p'-DDD, p,p'-DDE, total DDTs, HCB, CB118 and CB153 in muscle of Baltic herring in age groups two to ten years; in contrast, concentrations of a-HCH and g-HCH were found to be moderate. The concentrations of DDTs and CBs in bivalves is sufficient to cause biological effects, and demonstrates that long-term biological effects are still possible in the case of DDTs in the Hanö Bight.

Analysis of Crude Protein and Allergen Abundance in Peanuts (Arachis hypogaea cv. Walter) from Three Growing Regions in Australia

The effects of plant growth conditions on concentrations of proteins, including allergens, in peanut (Arachis hypogaea L.) kernels are largely unknown. Peanuts (cv. Walter) were grown at five sites (Taabinga, Redvale, Childers, Bundaberg, and Kairi) covering three commercial growing regions in Queensland, Australia. Differences in temperature, rainfall, and solar radiation during the growing season were evaluated. Kernel yield varied from 2.3 t/ha (Kairi) to 3.9 t/ha (Childers), probably due to differences in solar radiation. Crude protein appeared to vary only between Kairi and Childers, whereas Ara h 1 and 2 concentrations were similar in all locations. 2D-DIGE revealed significant differences in spot volumes for only two minor protein spots from peanuts grown in the five locations. Western blotting using peanut-allergic serum revealed no qualitative differences in recognition of antigens. It was concluded that peanuts grown in different growing regions in Queensland, Australia, had similar protein compositions and therefore were unlikely to show differences in allergenicity.

Purification, crystallization and partial characterization of the antitumour and insecticidal protein subunit from the σ-endotoxin of Bacillus thuringiensis var. thuringiensisstar, open

The antitumour protein from the α-endotoxin of Bacillus thuringiensis var. thuringiensis has been purified, crystallized and partially characterized. The same protein also shows the insecticidal activity. According to amino acid analysis it is an acidic protein with a molecular weight of approx. 13 000.

Silica vesicles as nanocarriers and adjuvants for generating both antibody and T-cell mediated immune resposes to Bovine Viral Diarrhoea Virus E2 protein

Bovine Viral Diarrhoea Virus (BVDV) is widely distributed in cattle industries and causes significant economic losses worldwide annually. A limiting factor in the development of subunit vaccines for BVDV is the need to elicit both antibody and T-cell-mediated immunity as well as addressing the toxicity of adjuvants. In this study, we have prepared novel silica vesicles (SV) as the new generation antigen carriers and adjuvants. With small particle size of 50 nm, thin wall (similar to 6 nm), large cavity (similar to 40 nm) and large entrance size (5.9 nm for SV-100 and 16 nm for SV-140), the SV showed high loading capacity (similar to 250 mu g/mg) and controlled release of codon-optimised E2 (oE2) protein, a major immunogenic determinant of BVDV. The in vivo functionality of the system was validated in mice immunisation trials comparing oE2 plus Quil A (50 mu g of oE2 plus 10 mu g of Quil A, a conventional adjuvant) to the oE2/SV-140 (50 mu g of oE2 adsorbed to 250 mu g of SV-140) or oE2/SV-140 together with 10 mu g of Quil A. Compared to the oE2 plus Quil A, which generated BVDV specific antibody responses at a titre of 10(4), the oE2/SV-140 group induced a 10 times higher antibody response. In addition, the cell-mediated response, which is essential to recognise and eliminate the invading pathogens, was also found to be higher [1954-2628 spot forming units (SFU)/million cells] in mice immunised with oE2/SV-140 in comparison to oE2 plus Quil A (512-1369 SFU/million cells). Our study has demonstrated that SV can be used as the next-generation nanocarriers and adjuvants for enhanced veterinary vaccine delivery. (C) 2014 Elsevier Ltd. All rights reserved.

Digestion of forages in the rumen is increased by the amount but not the type of protein supplement

Three polyester bag experiments were conducted with fistulated Bos indicus steers to determine the effect of the amount and type of nitrogen (N) supplement on the digestion rate of forages different in quality. In Experiment 1, test substrates were incubated in polyester bags in the rumen of steers fed ryegrass, pangola grass, speargrass and Mitchell grass hays in a 4 by 4 Latin-square design. In Experiment 2, test substrates were incubated in polyester bags in the rumen of steers fed speargrass hay supplemented with urea and ammonium sulfate (US), branched-chain amino acids with US (USAA), casein, cottonseed meal, yeast and Chlorella algae in a 7 by 3 incomplete Latin-square design. In Experiment 3, test substrates were incubated in polyester bags in the rumen of steers fed Mitchell grass hay supplemented with increasing amounts of US or Spirulina algae (Spirulina platensis). The test substrates used in all experiments were speargrass, Mitchell grass, pangola grass or ryegrass hays. Digestion rate of the ryegrass substrate was higher than that of the speargrass substrate (P < 0.05) in Experiment 1. Supplementation with various N sources increased the degradation rate and effective degradability of all incubated substrates above that apparent in Control steers (P < 0.05; Experiment 2). Supplementation of US and Spirulina increased degradation rate and effective degradability of ryegrass, pangola grass and Mitchell grass substrates above that apparent in Control steers (P < 0.05; Experiment 3). However, there was no further response on digestion rate of the substrates in increasing supplementation levels either for US or Spirulina. In conclusion, rate of digestion was affected by forage physical and anatomical properties. Supplementation with various N sources increased rate of digestion when the Control forage ration was very low in N but once a minimum level of N supplementation was reached, irrespective of form of N or other potential growth factors, there was no further increase in rate of digestion.

Immediate, cumulative and residual effects of short- and long-term low plane of nutrition on milk protein and casein composition

Immediate and residual effects of two lengths of low plane of nutrition (PON) on the synthesis of milk protein and protein fractions were studied at the Mutdapilly Research Station, in south-east Queensland. Thirty-six multiparous Holstein-Friesian cows, between 46 and 102 days in milk (DIM) initially, were used in a completely randomised design experiment with three treatments. All cows were fed on a basal diet of ryegrass pasture (7.0 kg DM/cow.day), barley-sorghum concentrate mix (2.7 kg DM/cow.day) and a canola meal-mineral mix (1.3 kg DM/cow.day). To increase PON, 5.0 kg DM/cow.day supplemental maize and forage sorghum silage was added to the basal diet. The three treatments were (C) high PON (basal diet + supplemental silage); (L9) low PON (basal diet only) for a period of 9 weeks; and (L3) low PON (basal diet only) for a period of 3 weeks. The experiment comprised three periods (1) covariate – high PON, all groups (5 weeks), (2) period of low PON for either 3 weeks (L3) or 9 weeks (L9), and (3) period of high PON (all groups) to assess ability of cows to recover any production lost as a result of treatments (5 weeks). The low PON treatment periods for L3 and L9 were end-aligned so that all treatment groups began Period 3 together. Although there was a significant effect of L9 on yields of milk, protein, fat and lactose, and concentrations of true protein, whey protein and urea, these were not significantly different from L3. There were no residual effects of L3 or L9 on protein concentration or nitrogen distribution after 5 weeks of realimentation. There was no significant effect of low PON for 3 or 9 weeks on casein concentration or composition.

Effect of varying the proportion of molasses in the diet on intake, digestion and microbial protein production by steers

The present experiment was conducted to determine the efficiency of microbial protein production in the rumen and intake by cattle fed high-molasses diets. Intake and microbial crude protein (MCP) production were measured along with the concentration of rumen ammonia-nitrogen (N) and volatile fatty acids (VFA), pH and the rate of digestion of roughage in the rumen. Eight Brahman crossbred steers weighing 211 ± 19.3 (± s.d.) kg were used in a double 4 × 4 Latin square design. Steers were allocated to one of four total mixed rations: control (pangola hay only), 25M (25% molasses/urea mix + 75% hay), 50M (50% molasses/urea + 50% hay), and 75M (75% molasses/urea + 25% hay). The production and efficiency of production of MCP (EMCP) of the diet increased quadratically as the level of molasses in the diet increased. The EMCP from the molasses/urea mix was estimated as 166 g MCP/kg digestible organic matter (DOM), a relatively high value. Intake of dry matter (DM) and DOM increased quadratically, reaching a peak when molasses was ~50% (as fed) of the ration. Digestibility of DM increased quadratically and that of neutral detergent fibre decreased linearly with increasing level of molasses in the diet. Molasses inclusion in the diet had no effect on rumen pH, ammonia and VFA concentration in the rumen fluid, plasma urea-N, urine pH or ruminal fractional outflow rate of ytterbium-labelled particles and Cr-EDTA. It was concluded that a diet with a high level of molasses (>50%) and supplemented with adequate N had high EMCP, and that low MCP production was not a factor limiting intake or performance of cattle consuming high-molasses diets.

Pregnancy suppression by active immunization against gestation-specific riboflavin carrier protein

A riboflavin carrier protein isolated from chickens cross-reacts with a gestation-specific rodent carrier for riboflavin. Active immunization of female rats of proved fertility with the purified chicken carrier protein completely yet reversibly suppressed early pregnancy without impairing implantation per se. Concurrently there were no discernible adverse effects on maternal health in terms of weight gain, vitamin status, and fertility.

Effect of varying the proportion of molasses in the diet on intake, digestion and microbial protein production by steers

The present experiment was conducted to determine the efficiency of microbial protein production in the rumen and intake by cattle fed high-molasses diets. Intake and microbial crude protein (MCP) production were measured along with the concentration of rumen ammonia-nitrogen (N) and volatile fatty acids (VFA), pH and the rate of digestion of roughage in the rumen. Eight Brahman crossbred steers weighing 211 ± 19.3 (± s.d.) kg were used in a double 4 x 4 Latin square design. Steers were allocated to one of four total mixed rations: control (pangola hay only), 25M (25% molasses/urea mix + 75% hay), 50M (50% molasses/urea + 50% hay), and 75M (75% molasses/urea + 25% hay). The production and efficiency of production of MCP (EMCP) of the diet increased quadratically as the level of molasses in the diet increased. The EMCP from the molasses/urea mix was estimated as 166 g MCP/kg digestible organic matter (DOM), a relatively high value. Intake of dry matter (DM) and DOM increased quadratically, reaching a peak when molasses was ∼50% (as fed) of the ration. Digestibility of DM increased quadratically and that of neutral detergent fibre decreased linearly with increasing level of molasses in the diet. Molasses inclusion in the diet had no effect on rumen pH, ammonia and VFA concentration in the rumen fluid, plasma urea-N, urine pH or ruminal fractional outflow rate of ytterbium-labelled particles and Cr-EDTA. It was concluded that a diet with a high level of molasses (>50%) and supplemented with adequate N had high EMCP, and that low MCP production was not a factor limiting intake or performance of cattle consuming high-molasses diets.

Spirulina (Spirulina platensis) algae supplementation increases microbial protein production and feed intake and decreases retention time of digesta in the rumen of cattle

Cattle consuming pastures low in protein have low liveweight gain due to low rumen degradable protein (RDP) supply and thus low microbial crude protein (MCP) production and efficiency of MCP production [EMCP, g MCP/kg digestible organic matter (DOM)]. Nitrogen supplements can increase MCP production and EMCP of cattle grazing low protein pastures. The objective of this study was to compare the effects of supplementation with a non-protein-N source (NPN), in this case urea and ammonium sulfate (US), with a single-cell algal protein source (Spirulina platensis), on intake, microbial protein supply and digestibility in cattle. Nine cannulated Bos indicus steers [initial liveweight 250.1 ± 10.86 (s.d.) kg] were fed Mitchell grass hay (Astrebla spp; 6.1 g N, 746 g NDF/kg DM) ad libitum and were supplied with increasing amounts of US (0, 6, 13, 19 and 33 g US DM/kg hay DM) or Spirulina 0, 0.5, 1.4, 2.5 and 6.1 g Spirulina DM/kg W.day in an incomplete Latin square design. The response of MCP production and EMCP to increasing amounts of the two supplements was different, with a greater response to Spirulina evident. The MCP production was predicted to peak at 140 and 568 g MCP/day (0.64 and 2.02 g MCP/kg W.day) for the US and Spirulina supplements, respectively. The highest measured EMCP were 92 and 166 g MCP/kg DOM for the US and Spirulina treatments at 170 and 290 g RDP/kg DOM, respectively, or a Spirulina intake of 5.7 g DM/kg W.day. Increasing RDP intake from US and Spirulina resulted in an increase in Mitchell grass hay intake and rumen NH3-N concentration and reduced the retention time of liquid and particulate markers and digesta DM, NDF and lignin in the rumen with greater changes due to Spirulina. Total DM intake peaked at a Spirulina supplement level of 4.6 g Spirulina DM/kg W.day with a 2.3-fold higher DOM intake than Control steers. Rumen NH3-N concentrations reached 128 and 264 mg NH3-N/L for the US and Spirulina treatments with a significant increase in the concentration of branched-chain fatty acids for the Spirulina treatment. The minimum retention time of liquid (Cr-EDTA; 23 and 13 h) and particulate (Yb; 34 and 22 h) markers in the rumen were significantly lower for Spirulina compared with US and lower than unsupplemented animals at 24 and 34 h for Cr-EDTA and Yb, respectively. Spirulina could be provided safely at much higher N intakes than NPN supplements. The results suggest that, at an equivalent RDP supply, Spirulina provided greater increases than US in MCP production, EMCP and feed intake of Bos indicus cattle consuming low protein forage and could also be fed safely at higher levels of N intake.

Importance of non-conserved distal carboxyl terminal amino acids in two peptidases belonging to the M1 family: Thermoplasma acidophilum Tricorn interacting factor F2 and Escherichia coli Peptidase N

Enzymes belonging to the M1 family play important cellular roles and the key amino acids (aa) in the catalytic domain are conserved. However, C-terminal domain aa are highly variable and demonstrate distinct differences in organization. To address a functional role for the C-terminal domain, progressive deletions were generated in Tricorn interacting factor F2 from Thermoplasma acidophilum (F2) and Peptidase N from Escherichia coli (PepN). Catalytic activity was partially reduced in PepN lacking 4 C-terminal residues (PepNΔC4) whereas it was greatly reduced in F2 lacking 10 C-terminal residues (F2ΔC10) or PepN lacking eleven C-terminal residues (PepNΔC11). Notably, expression of PepNΔC4, but not PepNΔC11, in E. coliΔpepN increased its ability to resist nutritional and high temperature stress, demonstrating physiological significance. Purified C-terminal deleted proteins demonstrated greater sensitivity to trypsin and bound stronger to 8-amino 1-napthalene sulphonic acid (ANS), revealing greater numbers of surface exposed hydrophobic aa. Also, F2 or PepN containing large aa deletions in the C-termini, but not smaller deletions, were present in high amounts in the insoluble fraction of cell extracts probably due to reduced protein solubility. Modeling studies, using the crystal structure of E. coli PepN, demonstrated increase in hydrophobic surface area and change in accessibility of several aa from buried to exposed upon deletion of C-terminal aa. Together, these studies revealed that non-conserved distal C-terminal aa repress the surface exposure of apolar aa, enhance protein solubility, and catalytic activity in two soluble and distinct members of the M1 family.

Protein A: nature's universal anti-antibody

Staphylococcal protein A specifically interacts with immunogobulins. This fact is being used in various disciplines of biology and some of the unique properties of protein A and their applications are summarized in this review.