Detection of hemorrhagic septicemia virus of salmonid fishes by use of an enzyme-linked immunosorbent assay containing high sodium chloride concentration and two noncompetitive monoclonal antibodies against early viral nucleoproteins.

Inclusion of high-ionic strength buffers helped us to develop a sandwich ELISA to detect hemorrhagic septicemia virus (HSV) in cell culture and infected trout tissue extracts. For maximal sensitivity of 0.1 to 0.2 ng/well/100 microliters or about 10 to 50 TCID50/well/100 microliters, trout extracts were diluted 1:1 and assayed for the earliest synthesized nucleoprotein N. Simultaneous binding of the N protein from HSV in the sample to the wells coated with monoclonal antibody (2D5 against the N protein) and to the peroxidase-labeled monoclonal antibody (2C9 against the N protein) proceeded during a 2-hour incubation at 20 to 22 C (room temperature). The response was linear between 6 to 60 ng/well of purified virus. Monoclonal antibodies used were noncompetitive with each other and reacted with F1, F2, 23.75, and 5 Spanish isolates of HSV, but not with infectious hematopoietic necrosis or infectious pancreatic necrosis viruses. Tissue specimens with low content of HSV virus may now be assayed directly without use of cell culture, rapidly, and with high precision, during the acute phase of the disease in salmonid fishes.     

A Quantitative Polymerase Chain Reaction Assay for the Detection and Quantification of Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus 3)

Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R² = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR.

Received April 20, 2015; accepted November 10, 2015     

单克隆抗体夹心ELISA试验检测鸡新城疫病毒抗原的研究

从10株抗鸡新城疫病毒(NDV)特异性单克隆抗体(MAbs) 中筛选出2株MAbs—M22和F23,分别用作包被抗体和酶标抗体而建立的MAbs—夹心ELISA试验,对提纯NDV的最小检出量为25ng/ml病毒蛋白.NDV鸡阳性血清对本试验的特异性阻断作用和与其他禽类病毒交叉反应的阴性结果,证明本试验对NDV的特异性。从临床疑为鸡新城疫的724只病鸡的口腔棉试样品中共检出阳性502只,其中200个样品与病毒分离试验结果完全一致,两者均查出阳性鸡176只。     

大中型输配水渠道工程系统优化设计

提出了大中型输配水渠道工程系统优化设计的定量定性混合系统动态规划模型。模型以计算分析期内总支出费用最小为目标函数,各渠段纵坡和定性规划方案为系统变量,渠道不冲不淤流速、水头损失、投资等为约束条件。克服了目前优化方法中不能综合考虑各渠段断面设计参数、配套建筑物设计参数和位置、渠道断面形状、衬砌方式优化的缺陷,为大中型输配水渠道工作系统优化提出了一个新的方法。     

One-step purification of the major rainbow trout immunoglobulin

Salmonid immunoglobulin purification has been hampered by time-consuming, labour-intensive, conventional chromatographic procedures. In this report we describe the use of immunoaffinity chromatography for the purification of immunoglobulins from trout serum in a single step. An anti-heavy chain monoclonal antibody (1G7) which reacts with more than 85% of total trout immunoglobulins was used to purify the trout immunoglobulin. The purity achieved was higher than 95%, and the immunoglobulins recovered were fully immunogenic. This method served equally well in isolating immunoglobulins from the sera of other salmonids (coho salmon and chinook salmon). The procedure should be useful in the standardization of salmonid immunoglobulin reagents.     

Quantitative trait locus mapping for meat quality traits in an Iberian x Landrace F2 pig population

An experimental F2 cross between Iberian and Landrace pig strains was performed to map quantitative trait loci (QTL) for diverse productive traits. Here we report results for meat quality traits from 369 F2 animals with records for pH 24 h postmortem (pH 24 h), muscle color Minolta measurements L* (lightness), a* (redness), and b* (yellowness), H* (hue angle), C* (chroma), intramuscular fat (IMF) and haematin pigment content measured in the longissimus thoracis. Pigs were genotyped for 92 markers covering the 18 porcine autosomes (SSC). Results of the genome scan show evidence for QTL for IMF (SSC6; F = 27.16), pH 24 h (SSC3; F = 7.73), haematin pigments (SSC4 and SSC7; F = 8.68 and 9.47 respectively) and Minolta color measurements L* (SSC4 and SSC7; F =16.42 and 7.17 respectively), and a* (SSC4 and SSC8; F = 8.05 and 7.36 respectively). No QTL were observed for the color measurements b*, H*, and C*. Alternative models fitting epistasis between QTL were also tested, but detected epistatic interactions were not significant at a genome-wise level. In this work we identify genomic regions related with meat quality traits. Improvement by traditional selection methods is complicated, and finer mapping would be required for their application in introgression programs.     

Antibody response to a fragment of the protein G of VHS rhabdovirus in immunised trout

A fragment (called frg#11, amino acids, aa 56-110) of the protein G (pG) of viral haemorrhagic septicaemia virus (VHSV) was designed after previous results showed it to be recognised by approximately 40% of the trout immunised to VHSV [Dis. Aquat. Organ. 34 (1999) 167]. frg#11 was then cloned, expressed, purified and used to study the production of antibodies to its epitopes in trout immunised to VHSV. Anti-frg#11 trout antibodies could be detected in serum from individual trout surviving VHSV exposure, immunised by injection with purified VHSV or DNA-immunised with its pG gene whereas it was not detected in non-infected and non-immunised trout. The trout serum antibodies which reacted more strongly by ELISA using solid-phase frg#11 (continuous or linear epitopes on the sequence of the pG) had the lowest VHSV-neutralising activity (epitopes which are pG conformation-dependent). Because antibodies recognising continuous as well as conformation-dependent epitopes of the pG seem to be involved in protective trout immunological responses to VHSV, the estimation of anti-frg#11 antibodies could help to the dissection of the complex trout antibody response to VHSV infections. In addition, these preliminary results suggest that the determination of anti-frg#11 antibodies might also be used to complement in vitro viral neutralising assays which seem to be restricted to pG conformation-dependent epitopes.     

Microphytic crusts,shrub patches and water harvesting in the Negev Desert: the Shikim system

Human-made contour banks are a central component of theShikim water harvesting system in Israels Negev Desert.Efficient water capture depends on the presence of a stable microphytic crustwhich directs surplus surface runoff into the banks where it is stored. We usedsimulated rainfall to examine the impact of soil surface disturbance on runoffand sediment transport, and the effect of this on the efficiency of resourcecapture within the Shikim system. Two disturbance regimes:1) removal of the microphytic crust only, and 2) removal of the crust and shrubpatches by cultivation, were compared with an undisturbed control. In theundisturbed state, 32% of rainfall was redistributed as runoff. This runoffpenetrated approximately 27% deeper under the shrub patches compared with themicrophytic crust. When the microphytic crust was destroyed by simulatedtrampling, the runoff coefficient declined to 13%, and there was no significantdifference in water penetration between shrub and crust patches. Completedestruction of the shrub hummocks and crust by cultivation resulted in adeclinein the runoff coefficient to 6%. The result of sustained disturbance in thesepatchy Negev shrublands is a breakdown in spatial heterogeneity, a loss ofecosystem function, a reduction in ecosystem goods and services such as plantdiversity and production, and ultimately a reduction in pastoral productivity.These results reinforce the view that microphytic crusts are critical for theefficient operation of the Shikim water harvesting system.Given that practices such as cultivation and trampling which disturbmicrophyticcrusts result in enhanced infiltration, crusts should be left intact tomaximisethe water harvesting efficiency in these desert landscapes.This revised version was published online in May 2005 with corrections to the Cover Date.     

Morphological and physiological responses of beech (Fagus sylvatica) seedlings to grass-induced below ground competition

We examined morphological and physiological responses of beech (Fagus sylvatica L.) seedlings to grass-induced below ground competition in full-light conditions. Two-year-old beech seedlings were grown during two growing seasons in 160-l containers in bare soil or with a mixture of five grass species widely represented in semi-natural meadows of central France. At the end of the second growing season, beech seedlings in the presence of grass showed significant reductions in diameter and height growth, annual shoot elongation, and stem, root and leaf biomass, but an increase in root to shoot biomass ratio. Grasses greatly reduced soil water availability, which was positively correlated with daily seedling diameter increment. Beech seedlings seemed to respond to water deficit by anticipating stomatal closure. There was evidence of competition for nitrogen (N) by grasses, but its effect on seedling development could not be separated from that of competition for water. By labeling the plants with 15N, we showed that beech seedlings absorbed little N when grasses were present, whereas grasses took up more than 97% of the total N absorbed in the container. We conclude that, even if beech seedlings display morphological and physiological adaptation to below ground competition, their development in full-light conditions may be strongly restricted by competition from grass species.     

Isotopic evidence and quantification assessment of in situ RDX biodegradation in the deep unsaturated zone

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is an explosive compound whose extensive use has resulted in significant contamination of soils and groundwater worldwide. We studied its in situ biodegradation along the unsaturated zone beneath an explosives wastewater lagoon using compound-specific isotope analysis (CSIA) of RDX in the unsaturated zone, together with biodegradation slurry experiments under anaerobic conditions. We found the highest degradation potential of RDX and its nitroso derivatives in the upper part of the soil profile while in the lower parts, RDX-degradation potential was lower and the nitroso derivatives tended to persist. This was also observed in the field, as reflected by the isotopic composition of RDX along the profile. We also found a correlation between biodegradation potential and clay content: biodegradation was further enhanced in layers characterized by high-clay content or in those influenced by the high-clay layers. In addition, in the presence of high organic matter content, further enhancement of biodegradation was observed. We obtained different isotopic enrichment factors (?) for RDX biodegradation in different sections of the unsaturated profile and suggest that different degradation pathways exist simultaneously in situ, in variable proportions. Using the range of enrichment factors, we were able to assess the biodegradation extent of RDX at different sampling points along the profile, which ranged between 30 and 99.4%. The novel application of CSIA together with slurry experiments provides better insight into degradation processes that are otherwise difficult to detect and assess.