Optimization of a Plaque Neutralization Test (PNT) to Identify the Exposure History of Pacific Herring to Viral Hemorrhagic Septicemia Virus (VHSV)

Abstract

Methods for a plaque neutralization test (PNT) were optimized for the detection and quantification of viral hemorrhagic septicemia virus (VHSV) neutralizing activity in the plasma of Pacific Herring Clupea pallasii. The PNT was complement dependent, as neutralizing activity was attenuated by heat inactivation; further, neutralizing activity was mostly restored by the addition of exogenous complement from specific-pathogen-free Pacific Herring. Optimal methods included the overnight incubation of VHSV aliquots in serial dilutions (starting at 1:16) of whole test plasma containing endogenous complement. The resulting viral titers were then enumerated using a viral plaque assay in 96-well microplates. Serum neutralizing activity was virus-specific as plasma from viral hemorrhagic septicemia (VHS) survivors demonstrated only negligible reactivity to infectious hematopoietic necrosis virus, a closely related rhabdovirus. Among Pacific Herring that survived VHSV exposure, neutralizing activity was detected in the plasma as early as 37 d postexposure and peaked at approximately 64 d postexposure. The onset of neutralizing activity was slightly delayed in fish reared at 7.4°C relative to those in warmer temperatures (9.9°C and 13.1°C); however, neutralizing activity persisted for at least 345 d postexposure in all temperature treatments. It is anticipated that this novel ability to assess VHSV neutralizing activity in Pacific Herring will enable retrospective comparisons between prior VHS infections and year-class recruitment failures. Additionally, the optimized PNT could be employed as a forecasting tool capable of identifying the potential for future VHS epizootics in wild Pacific Herring populations.

Received November 7, 2016; accepted January 14, 2017 Published online April 4, 2017     

Association mapping in a simulated barley population

The analysis of association mapping populations is frequently complicated by substructure within the population. If this is unaccounted for, it generally results in many false positive marker-trait associations. In this study, we simulate a large barley population, modelling inbreeding and selection, and compare five models for analysing marker-trait associations. One of these includes no population substructure, one includes information about geographical origin and type of barley to represent structure within the population and three use different approaches that have been proposed for estimating marker-based kinship. Kinship methods reduced the number of false positives substantially compared to the other models but none of these approaches had a clear advantage over the others. One solution is to fit more than one model and to consider as candidate associations those markers that are significant by all models. None of the approaches were very successful at detecting true associations at the lowest level of heritability considered (25%), suggesting it is important to consider power in association mapping studies to avoid missing some true associations and overestimating others.     

Stability of resistance to Phytophthora infestans in potato: an international evaluation

Ten institutions in nine countries joined together to test the stability of resistance of 14 potato genotypes to the oomycete pathogen Phytophthora infestans in three separate trials. Seven of the genotypes were tested in one trial involving seven locations, and all 14 were tested in two subsequent trials, each involving eight locations. Stability of resistance was tested with nonparametric tests and with an additive main effects and multiplicative interaction (AMMI) model. Overall, resistance to P. infestans was robust; resistant genotypes were consistently resistant in all locations and trials. The nonparametric analysis indicated that specific genotypes were basically stable across sites for resistance. In trial 3, the Z statistic for overall stability was significant at 0·05%, indicating a significant level of interaction across the trial, but there were no significant interactions for specific genotypes in this trial. The genotype by environment (G × E) effect of the AMMI model was highly significant in both trials, but the mean square of G × E was less than 10% of the genotype effect in each trial. The first two principal components (PCA1 and PCA2) of the AMMI analyses together explained 75 and 80% of the interaction effects in trials 2 and 3, respectively. Based on both nonparametric and AMMI analyses, Ecuador and Argentina were locations of relatively high interaction effects for both trials 2 and 3, although in Ecuador this interaction was not associated with any particular potato genotype. Other locations also had high interaction effects, but these occurred in only one trial. The genotypes Chata Blanca and, to a lesser extent, Torridon were relatively unstable in trials 2 and 3, but in the case of Torridon, resistant, this did not represent a significant loss of resistance.     

Factors affecting the development and control of black dot on potato tubers

Field trials were carried out over a 4 year period (2004–2007) to determine the effect of agronomic factors, specifically cultivar resistance, irrigation, crop duration and chemical control (in‐furrow application of azoxystrobin), on black dot development on potato tubers grown in fields where soilborne inoculum of Colletotrichum coccodes was present. In 2004, 2005 and 2006, two field trials were performed each year and in 2007, 19 mini‐field trials were carried out across Scotland and England. Cultivar resistance was clearly demonstrated to be an effective method of reducing black dot disease severity on tubers (described here as the percentage of unmarketable tubers, i.e. those with symptoms covering a surface area of >10%). In the four field trials carried out in 2004 and 2005, in irrigated and fungicide‐untreated plots, 43·8% of tubers of cv. Maris Piper were unmarketable, compared with 17·0% of tubers of cv. Sante. Assessments of disease development on underground plant parts (stems, stolons and roots) revealed that cultivar resistance acted only at the tuber level, as disease symptoms on other parts were often high irrespective of published disease resistance ratings. Irrigation increased the severity of disease on tubers in two trials (England 2004 and 2006), but its effect was less significant when rainfall was high. Delaying harvest by 2 weeks increased disease severity in all six trials, whilst application of azoxystrobin consistently reduced black dot severity. There were significant interactions between factors. The results clearly show how black dot disease severity can be reduced through an integrated approach to disease management.     

Evaluation of action thresholds for chronic rice insect pests in the Philippines. I. Less frequently occurring pests and overall assessment

Action thresholds as decision tools for insecticide application were developed and tested against the major insect pests of rice at four sites in the Philippines over a 13-year period. Action threshold treatments were compared to the farmers' practice, prophylactic insecticide usage, and an untreated check. Yield loss data using the insecticide check method partitioned yield losses over three crop growth stages in the same test fields. Chronic pests that exceeded action thresholds in 79% of fields were whorl maggot Hydrellia philippina Ferino (Diptera: Ephydridae), defoliators Naranga aenescens Moore and Rivula atimeta (Swinhoe) (Lepidoptera: Noctuidae), leaffolders Cnaphalocrocis medinalis (Guenée) and Marasmia patnalis Bradley (Lepidoptera: Pyralidae), and stemborers Scirpophaga incertulas (Walker) and S. innotata (Walker) (Lepidoptera: Pyralidae). Minor chronic pests reached threshold levels in only one site each: rice bug Leptocorisa oratorius (F.) (Koronadal), whitebacked planthopper Sogatella furcifera (Horvath) (Zaragoza) and green leafhopper Nephotettix virescens (Distant) (Guimba); brown planthopper Nilaparvata lugens (Stål) did not exceed a threshold in any field. Stemborers were the most important pest group in terms of yield loss. Despite the insecticide check method underestimating losses, a mean crop loss of 0.62 t/ha was measured which showed ample scope for corrective action. But loss was evenly distributed across crop growth stages (0.15?–?0.24 t/ha) reducing the impact of insecticides. Action threshold treatments overall outyielded the untreated check, more so in the two sites with highest pest density. The benefit of thresholds was to reduce insecticide usage, as a cost saving. However all the practices showed poor economic returns including the farmers' practice. Farmers' practice employed low insecticide dosages and timing was not consistent with pest damage, but yields were often similar to threshold treatments. Farmers appear to use insecticide more for risk aversion than for profit. The best threshold characters when evaluated against resulting pest density and yield loss criteria showed accuracies >?90% correct decisions. Future work is needed to improve the insecticide response rather than monitoring tools. Thresholds need to be incorporated into improved crop management, which was often found suboptimal by farmers, to take advantage of the high levels of tolerance in modern high tillering cultivars. Crop husbandry practices which improve yield potential such as selection of longer maturing varieties and nitrogen fertilizer may be a more effective pest management strategy than insecticides.     

New analogue of gymnodimine from a Gymnodinium species

A spiroimine, gymnodimine B (1), was isolated from cells recovered by filtration from cultures of a marine dinoflagellate, Gymnodinium sp. Its structure was identified by one- and two-dimensional NMR spectroscopy and mass spectrometry. Gymnodimine B is similar in structure to gymnodimine (2) but contains an exocyclic methylene at C-17 and an allylic hydroxyl group at C-18.     

Environmental conditions and isozyme polymorphism in Chenopodium album L.

Electrophoretic variations within four enzyme systems (α-esterase, acid phosphatase, malate dehydrogenase and leucine amino peptidase), were studied for various garden and field populations of Chenopodium album. A correlation was found between the degree of polymorphism for the electrophoretic traits and environmental conditions. The amount of phenotypic polymorphism is very high in gardens, intermediate in an untreated and, low in herbicide-treated fields and nil for a triazine resistant population in a maize (Zea mats L.) monoculture. Furthermore, the tetraploid versus hexaploid ratio is higher within garden populations. Higher variability in garden populations is due, in part, to a high proportion of polymorphic phenotypes, and in part to a low effect of selection pressure in such types of habitat. Although there is a difference between phenotypes of populations from acid soils and those from more basic ones, we cannot be sure that the variation of polymorphism is due only to selection.     

Heliotropium europaeum poisoning of sheep with low liver copper concentrations and the preventive efficacy of cobalt and antimethanogen

In a field experiment in the Mallee district of Victoria, Merlno xBorder Leicester ewes and wethers grazed Heliotropium europaeum (heliotrope) over periods of 3 to 4 months in 4 successive years. By the end of the second year 12% (14 of 120) of the sheep had died; after 4 years the loss attributable to heliotrope was between 18% and 35%. Mortality was not affected by intraruminal treatment with cobalt or antimethanogen. At the end of the experiment the highest concentration of copper in the liver was 1.95 mmol/kg wet weight (approximately 413 micrograms/g dry weight). The relatively low mortality from primary heliotrope poisoning and the low concentration of copper in the liver of sheep grazing the plant are discussed in relation to the contrasting situation that prevails in the Riverina area of New South Wales. The importance of local environmental factors in the management of heliotrope grazing by sheep is emphasised, particularly in relation to the number of seasons in which the plant may be a major component of the diet.