Characterization of monoclonal antibodies against avian reovirus S1133 protein sigmaA synthesized in Escherichia coli

Monoclonal antibodies (MAbs) were prepared against avian reovirus S1133 protein sigmaA (esigmaA) synthesized in Escherichia coli. MAbs were characterized and used to develop a diagnostic test. Ten MAbs were selected for competitive binding assay following coupling with horseradish peroxidase. The results indicated that these MAbs delineated two epitopes I and II of esigmaA. An immuno-dot binding assay was used to detect the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to esigmaA in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitope I was fully abolished. However, the reactivity of MAbs recognizing epitope II was not affected. MAbs 31 and 32, recognizing epitopes I and II, respectively, were selected for the cross-reactivity to heterologous reovirus strains. The results suggest that the two epitopes are highly conserved among these virus strains. A MAb capture enzyme-linked immunosorbent assay (ELISA) procedure was developed using MAbs 32 and 31 to detect reovirus protein sigmaA in samples from tendon tissues of infected bird and chicken embryo fibroblast (CEF) cell cultures. Avian reovirus sigmaA antigens in tendon specimens were detected from the inoculated birds as early as 2 days post-inoculation (PI), approximated a peak at 7 days PI, and maintained this until 16 days PI, then decreased gradually. A clear difference in absorbance values between the tendon samples of the avian reovirus- and mock-infected birds is obtained. Positive results were also obtained from avian reovirus-infected CEF and from the tendon tissues of naturally infected broilers. These results indicated that the MAb capture ELISA is a useful methods for the detection of avian reovirus from chickens suspected to have avian reovirus infections.     

Review of methodologies and a protocol for the <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of wheat

Since the first report of wheat transformation by Agrobacterium tumefaciens in 1997, various factors that influence T-DNA delivery and regeneration in tissue culture have been further investigated and modified. This paper reviews the current methodology literature describing Agrobacterium transformation of wheat and provides a complete protocol that we have developed and used to produce over one hundred transgenic lines in both spring and winter wheat varieties.     

A colorimetric quantification of softened polylactic acid and polyester filament knitted fabrics to ‘Water-spotting’

Water droplets falling onto finished fabrics can create spots which can spoil the appearance of the fabric. This study compares the propensity of softened filament polylactic acid and polyester fabrics to exhibit the adverse affects of ‘water-spotting’, and to identify suitable softeners and methods for their application to minimise and eliminate the problem. The degree of water spotting was greater on softened polylactic acid fabrics than on softened polyester fabrics. Polylactic acid and polyester fabrics with hydrophobic properties did not exhibit any water spotting. Softeners applied by an exhaustion process resulted in a finished fabric which exhibited no water spotting for either dyed polylactic acid or dyed polyester fabrics. The softening active agent was not responsible for the water spotting. The other components in the softener formulation (such as emulsifier, wetting agent), which carries the unfixed dyes with water, were found to be responsible. The water spot halo disappeared after a single machine laundering process.     

Immunoadjuvant activities of a recombinant chicken IL-12 in chickens vaccinated with Newcastle disease virus recombinant HN protein

Recombinant fowlpox virus (rFPV/HN) expressing Newcastle disease virus (NDV) HN gene and rFPV/HN/chIL-12 co-expressing chicken IL-12 (chIL-12) and HN (rHN/chIL-12) genes have been characterized. rHN/chIL-12 or rchIL-12, expressed by our previous construct rFPV/chIL-12, co-administered with rHN was assessed for adjuvant activities of chIL-12. Chickens were vaccinated with various amounts of rHN/chIL-12 mixed with mineral oil (MO), intramuscularly. Levels of hemagglutination-inhibition (HI) antibody production depended on the concentration of the injected rHN or rHN/chIL-12. The lower HI antibody titers were obtained in chicken groups rHN/chIL-12/7-rHN/chIL-12/9, receiving 60ng rHN/8ng chIL-12 with MO, 30ng rHN/4ng chIL-12 with MO or 15ng rHN/2ng chIL-12 with MO, respectively, compared to those in chicken groups rHN/7-rHN/9, receiving rHN with MO alone. However, chickens in group rHN/chIL-12/7 or rHN/chIL-12/8 and rHN with MO alone showed the same effective protection. Chicken group rHN/chIL-12/9 was even more protective than that in group rHN/9. When rchIL-12 was co-injected with 15ng rHN plus MO, chickens produced low levels of HI antibody titers; while higher levels of IFN-γ production and an effective protection rate (83%) were obtained. On the other hand, low levels of IFN-γ production and low protection response (50%) were obtained in chickens injected with rHN with MO alone. Taken together, when the concentration of rHN decreased to certain levels, rchIL-12 reduced HI antibody production. The increase in the induction of IFN-γ production might suggest the enhancement of the cell-mediated immunity which conferred the protection from the NDV challenge.     

Remotely sensed spatial dynamics of the Illex argentinus fishery, Southwest Atlantic

Illex argentinus, the Argentine short-finned squid, is an important species within the Patagonian shelf ecosystem, where it supports a major multi-national fishery. The fishing fleet operating in this region is comprised of jigging vessels which attract squid using powerful incandescent lights. These fishing lights are detectable in remotely sensed satellite imagery which makes the fishery unusually amenable to a large-scale analysis of its spatial dynamics. In this paper, long-term inter-annual variability in fleet distribution and extent is examined using imagery from the Defence Meteorological Satellite Program-Operational Linescan System (DMSP-OLS) for the period 1993–2005. The fishery was found to occupy a wide area across the shelf and slope, with regions of consistent fishing activity observed on the high seas (45–47°S) and to the north of the Falkland Islands (Malvinas). Distribution of the fishery over the 13-year study period was variable, with 28% of the fished area occupied in 1–2 years, and 7% of the area occupied in 12–13 years. Annual catch levels were positively associated with the extent of the area occupied by the fleet. Higher catches corresponded to the fishery occupying a wide latitudinal range, whereas lower catches were observed during 2004 and 2005 corresponding to a contraction of the fishery away from the south of its range. In years of very high catches, fishing took place along almost the entire latitudinal range of the species. Due to the intensity of fishing, changes in the distribution of the fleet can reflect shifts in the distribution of I. argentinus; this has potential for the long-term monitoring of this highly variable squid fishery.     

Should farmers apply fertilizer according to when their daffodils are in flower? Utilizing a “farmer‐science” approach to understanding the impact of soil temperature on spring N fertilizer application in Wales

Perennial ryegrass starts growing when soil temperatures reach 5.5°C for five consecutive days; applying N fertilizer before this risks environmental losses. To test whether daffodil flowering signified when to apply N fertilizer, farmers volunteered to take part in a citizen science study. The PROSOIL project used a “citizen science”, participatory approach to create farmer‐informed science, aiming to increase awareness of the importance of soil health. In 2014, over 300 farmers completed a “How do you manage your soil” survey. The survey included a question on the use of daffodils (Narcissus spp.) to indicate the best time to apply the first nitrogen fertilizer of the season, based on anecdotal feedback from farmers involved in the PROSOIL project. The survey recorded 7% of farmers based their first fertilizer application on when daffodils flowered. To increase farmer awareness of soil temperatures, we provided them with soil thermometers, held workshops and hosted interactive stands at agricultural events in 2014. In autumn 2014, farmers planted daffodil bulbs of the same variety, across Wales, and monitored soil temperatures. Farmers returned postcards once their daffodils were in flower, noting the soil temperature. An assessment of whether daffodil flowering date could indicate when to apply N fertilizer was made. Overall, in spring 2015, daffodils flowered when soil temperature was 6.4(±0.35)°C, suggesting daffodil flowering date is a more reliable indicator for fertilizer application, than first hypothesized. Findings show a scientific validation of local knowledge, regarding the use of daffodils to indicate the “not‐before” date for the first N fertilizer application.     

Adjuvant activity of chicken interleukin-12 co-administered with infectious bursal disease virus recombinant VP2 antigen in chickens

A recombinant fowlpox virus (rFPV/VP2) expressing infectious bursal diseases virus (IBDV) VP2 gene has been constructed. After purification and identification of rFPV/VP2, the adjuvant activity of the recombinant chicken IL-12 (rchIL-12), synthesized by our previous construct of rFPV/chIL-12, in rFPV/VP2-expressed rVP2 antigen was assessed in one-week-old specific-pathogen free chickens. The results indicated that rchIL-12 alone or rchIL-12 plus mineral oil (MO) co-administered with rVP2 antigen significantly enhanced the production of serum neutralization (SN) antibody against IBDV, compared to those with MO alone. The SN titers in groups receiving rVP2 antigen with MO alone were more inconsistent after vaccination. On the other hand, rchIL-12 significantly stimulated IFN-γ production in serum and in splenocyte cultured supernatant, suggesting that rchIL-12 alone or plus MO significantly induced a cell-mediated immune response. Finally, bursal lesion protection from very virulent IBDV (vvIBDV) challenge in chickens receiving rVP2 antigen with rchIL-12 alone or plus MO was much more effective than that with MO alone at two weeks after boosting. Taken together, rchIL-12 alone augmented in vivo the induction of a primary and also a secondary SN antibody production and a cell-mediated immunity against IBDV rVP2 antigen, which conferred the enhancement of bursal lesion protective efficacy from vvIBDV challenge. These data indicated that a potential for chIL-12 as immunoadjuvant for chicken vaccine development such as IBDV rVP2 antigen.     

Avian reovirus replication in mononuclear phagocytes in chicken footpad and spleen after footpad inoculation

Circulating monocytes and tissue macrophages were suggested to be susceptible to avian reovirus (ARV) infection. To determine if ARV infects and replicates in mononuclear phagocytes (KUL01-positive cells), we infected 3-day-old specific-pathogen-free chickens with ARV strain 2408 by inoculation of the left footpad. The left footpads and spleens were collected for analysis at 1.5 and 2.5 d after inoculation. Replication of ARV in the footpad and spleen was demonstrated by detection of the viral protein σNS using immunohistochemical testing and viral S1 RNA expression by real-time quantitative polymerase chain reaction (qPCR). Furthermore, immunofluorescent double-staining assay of cytocentrifuged cells and cryosections of the footpad and spleen for the viral protein σNS and the surface marker recognized by monoclonal antibody (MAb) KUL01 indicated that KUL01-positive cells costained with MAb H1E1, which recognizes ARV protein σNS. In addition, more ARV S1 RNA was measured by qPCR in the KUL01-positive cell samples prepared from the footpad or spleen 1.5 d after inoculation compared with non-KUL01-positive cell samples. The amounts of ARV S1 RNA in the spleen were significantly lower (P < 0.05) than the amounts in the footpad 1.5 d after inoculation. The results suggest that ARV infects mononuclear phagocytes and then replicates within these cells before migrating to the spleen, where it infects and replicates in KUL01-positive cells.     

Inhibition of the fucosterol-24(28)-epoxide cleavage enzyme of sitosterol dealkylation in Spodoptera littoralis larvae

The conversion of sitosterol into cholesterol and the effect of inhibitors on the dealkylation reaction has been studied in Spodoptera littoralis Boisd. Using a microsomal preparation from the midgut of the larvae [³H]fucosterol and [³H]fucosterol-24,28-epoxide were converted into desmosterol and cholesterol in vitro, and a series of inhibitors were tested using the latter substrate. Triadimefon [1-(4-chlorophenox)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-one] strongly inhibited the cleavage enzyme at 2 μM. In a functional group study we observed that (i) the phenoxy substituent was more strongly inhibitory than the corresponding benzyl, and (ii) when the ketone is reduced to the secondary alcohol in either phenoxy or benzyl series, inhibition decreased markedly. In the benzyl series of compounds we found that (iii) a 2,4- or 2,6-dichlorophenyl gave greater inhibition than mono-, tri-, or pentachlorophenyl derivatives, and (iv) changing the heterocyclic ring gave activity in the order 2-pyridyl=3-pyridyl=imidazole>pyrazole>triazole. In the triazole series, substitution of the hetero ring did not abolish inhibition. When either triadimefon or 22,25-diazacholesterol was incorporated into a diet (1000 ppm) containing sitosterol plus campesterol as the only sterol supplements and fed to final larval instar insects, the weight of the insects throughout the instar was reduced and the duration of the instar increased. Insects reared from the early third-instar stage on such a diet containing amounts of triadimefon had reduced percentages of cholesterol and desmosterol in the final larval instar with a proportional increase in the sitosterol level. Thus, inhibition of side chain cleavage has been obtained in vitro and in vivo, but the high concentrations of inhibitor required preclude the enzyme as a target for a novel class of insecticide.