Genotypic variation among Douglas-fir tussock moth nucleopolyhedrovirus (OpNPV) isolates in the western United States.

Periodic outbreaks of the Douglas-fir tussock moth (Orgyia pseudotsugata) in forests of western North America generally end with a sudden collapse due primarily to an epizootic caused by a nucleopolyhedrovirus (NPV) that occurs naturally within O. pseudotsugata populations. We genotypically characterized NPV populations from Washington State, Oregon, Idaho, New Mexico and California for the first time. Of 159 infected tussock moth samples, restriction fragment length polymorphism (RFLP) analysis showed that 125 (78.6%) contained single nucleopolyhedrovirus (OpSNPV), 28 (17.6%) contained multiple nucleopolyhedrovirus (OpMNPV), and six (3.8%) contained both OpSNPV and OpMNPV. In comparison, our previous studies in the southern interior of British Columbia showed that all 298 samples examined were infected with OpMNPV, and none were infected with OpSNPV. More than half of the Washington OpSNPV samples shared the same genotype, but most OpSNPV genotypes were rare or unique: across the five states, 31 of the 43 different OpSNPV genotypes were each only found in a single sample. In contrast, only four different OpMNPV genotypes were found, and 29 of the 34 OpMNPV samples shared the same genotype, designated genotype AA. This strain of OpMNPV has been developed, registered and used in both Canada and the United States to control outbreaks of the Douglas-fir tussock moth. It is also the most common genotype in southern British Columbia. The estimated degree of genetic divergence ranged from 0% to 4.19% for the various OpSNPV genotypes and from 0% to 3.16% for the OpMNPV genotypes (based on number of shared bands). This is the first description of the genotypic diversity in a population of OpSNPV, and the first genotypic characterization of NPVs infecting O. pseudotsugata in the USA.

In vitro culture of Lambdina fiscellaria lugubrosa nucleopolyhedrovirus in heterologous cell lines.

Infections of two heterologous insect cell lines derived from Malacosoma disstria (Md108) and Choristoneura fumiferana (Cf70) by the Lambdina fiscellaria lugubrosa nucleopolyhedrovirus (LafiNPV-W) were characterized. Cytopathic effects characteristic of LafiNPV-W infection, including rounding of cells, nuclear hypertrophy, and occlusion body (OB) production, were observed in both cell lines. Budded virus titers were slightly higher in Md108 cells than Cf70 cells (5.8 x 10(7) versus 3.1 x 10(7) TCID(50) units mL(-1)). Viral replication kinetics and cytopathic effects induced by LafiNPV-W infection were very similar in both cell lines. Actin rearrangements and redistribution of heterochromatin and euchromatin were observed within 24 h post-inoculation (hpi), and large quantities of nucleocapsids and virions were observed by electron microscopy at 48 hpi in both cell lines. Cf70 cultures produced OBs with numerous embedded virions, while OBs in Md108 cultures contained few virions or were empty with nucleocapsids packed in the nucleoplasm between OBs. In bioassays against second instar L. fiscellaria lugubrosa, OBs derived from LafiNPV-W-infected Md108 cells induced significantly lower levels of mortality than OBs derived from LafiNPV-W-infected Cf70 cells or from infected L. fiscellaria fiscellaria larvae.

Development of a dipstick immunoassay to detect nucleopolyhedroviruses in Douglas-fir tussock moth larvae.

In this paper, we describe the development of a novel field detection system for the identification of Orgyia pseudotsugata nucleopolyhedrovirus (OpNPV) and OpNPV infections in Douglas-fir tussock moth (O. pseudotsugata) (DFTM) larvae, utilizing antibodies in a dipstick immunoassay. The dipstick method is sensitive to a minimum of 10ng of extracted virus protein, or approximately 1070 virus occlusion bodies, and is sufficiently sensitive to detect OpNPV infections in DFTM prior to mortality. Additionally, the method can be used to unambiguously detect virus in infected larvae without purification of the test sample. This research provides a novel tool for on-site assessment of the incidence of OpNPV in field populations of DFTM, and has the potential to improve the biological control of the DFTM by facilitating on-site pest management decisions.

Recovery of nontarget Lepidoptera on Vancouver Island, Canada: one and four years after a gypsy moth eradication program.

The gypsy moth (Lymantria dispar) is a destructive defoliator that is not established in British Columbia, Canada, because of successful eradication programs involving the microbial insecticide Bacillus thuringiensis var. kurstaki (Btk). In 1999, three aerial applications of Btk were made over two areas, totaling 12,805 ha, on southern Vancouver Island, Canada. The impacts of these Btk applications on nontarget Lepidoptera were studied from 1999 to 2004 on Garry oak (Quercus garryana) and common snowberry (Symphoricarpos albus). In 1999, lepidopteran larvae were collected from S. albus foliage at 24 urban parks and from Q. garryana foliage at 28 oak-dominated habitats. The initial impacts (i.e., 1999 data) were published previously, and the present paper is a continuation of the same study. We tested two hypotheses: Reductions of nontarget Lepidoptera would be more severe at 12 to 13 months postspray than at one to two months postspray, and recovery would be significant, though not necessarily complete, at four years postspray. The total number of nontarget Lepidoptera on S. albus and Q. garryana was significantly reduced in the treatment sites in each year of the study: the reduction was greatest in 2000. Relative to the reference sites, each of 11 species that were initially reduced by the Btk applications showed an increase in the treatment sites between 2000 and 2003, by which time only four species remained significantly reduced in the treatment sites. The uncommon species were significantly reduced in 1999 and 2000 but not in 2003, indicating that some recovery had occurred. Limitations and economic implications of the present study are discussed.

Development and evaluation of methods to detect nucleopolyhedroviruses in larvae of the Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough).

Various molecular methods are used to detect pathogenic microorganisms and viruses within their hosts, but these methods are rarely validated by direct comparison. Southern hybridization, enzyme-linked immunosorbent assay (ELISA), and a novel DNA extraction/PCR assay were used to detect Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) in Douglas-fir tussock moth larvae. PCR was more sensitive than Southern hybridization and ELISA at detecting semipurified virus. ELISA, however, was the most accurate method for detecting virus within larvae, given that Southern hybridization and PCR produced false-negative results (31% and 2.5%, respectively). ELISA may be preferable in some applications because virus infections can be quantified (r(2) = 0.995). These results may be applicable to both applied and academic research that seeks to accurately identify the incidence of viruses and microorganisms that regulate insect populations.

Genotypic variation and presence of rare genotypes among Douglas-fir tussock moth multicapsid nucleopolyhedrovirus (OpMNPV) isolates in British Columbia.

The Douglas-fir tussock moth (Orgyia pseudotsugata) multicapsid nucleopolyhedrovirus (OpMNPV) is periodically applied to suppress Douglas-fir tussock moth populations in British Columbia and in the western United States. The strain of OpMNPV in the product currently used for suppression is not genetically distinct from naturally occurring OpMNPV. To separate the mortality caused by the applied virus from that caused by the naturally occurring virus, a rare and genetically distinct strain of OpMNPV must be applied. To learn more about the genotypic diversity of OpMNPV populations in BC and to identify rare strains in this region, viral DNA was extracted from larvae reared from 208 field-collected egg masses found in five geographic regions of British Columbia and subjected to REN analysis. Nine, 12, and 9 different genotypes were detected using PstI, SalI, and HindIII, respectively. When the PstI, SalI, and HindIII profiles for each pure (single strain) isolate were grouped and considered as a combined PstI-SalI-HindIII genotype, 23 different genotypes were identified among 185 isolates. Nine rare OpMNPV genotypes were selected as ideal candidates for use as a potential 'marker strain' to accurately determine the efficacy of the treatment.

Characterization and lime treatment of olive mill wastewater.

Seventeen olive mill wastewater (OMW) samples were characterized. For characterization of the samples, amount of total, fixed, volatile, suspended, volatile-suspended solids, COD, oil-grease, polyphenol, volatile phenol, nitrogen and reducing sugar were determined. Effects of lime treatment on the waste samples were investigated. Reduction of contents of the samples treated with the lime was described. The effect of the addition of lime on an artificial phenolic mixture was also examined.

High sensitivity to autoxidation in neonatal calf erythrocytes: possible mechanism of accelerated cell aging.

The suspension viscosity, formation of methaemoglobin and production of malondialdehyde (MDA) associated with the non-enzymatic oxidation of polyunsaturated fatty acids during auto-oxidation conditions in vitro have been compared in erythrocytes from young calves (2, 4 and 6 weeks of age) and mature cattle. The autoxidation conditions were designed to simulate the oxidative stress to which neonatal erythrocytes are exposed in vivo. Characterisation of lipid peroxidation was also undertaken by a combination of lipid fluorescent measurements and quantification of the superoxide dismutase (SOD) activities of the erythrocytes. The results demonstrated that high SOD activities in the erythrocytes of the neonatal calf was insufficient to afford protection against the increased autoxidation of haemoglobin and subsequent accumulation of lipid peroxidation products. High levels of methaemoglobin formation and lipid peroxidation were able to provide an explanation for an observed reduction in rheological adaptability (increased suspension viscosity) and an accelerated aging of the neonatal cells under in vivo conditions.

Reduced lipid peroxidation capacity and desaturation as biochemical markers of aging.

The lipid peroxidation capacity, the lipid composition and the fatty acid composition of the major lipid fractions have been compared in the liver tissue of mice at different ages. Compared with mice at 2 months of age, there was a notable reduction in peroxidation capacity in the livers of mice at 12 and 26 months of age. These observations were associated with changes in the proportions of the major C18, C20 and C22 polyunsaturated fatty acids expressed in all the major lipid fractions and were accompanied by alterations in lipid composition. From the relationship between these features it is suggested that, under in vivo conditions, regulation of the aging processes may be intimately related to the metabolism of the polyunsaturated fatty acids and associated aspects of lipid metabolism.

Vitality diagnostics and preventive medicine for the well-being of people at advanced age.

This paper emphasises the importance of vitality diagnostics in relation to healthy ageing and prevention of age-associated diseases. Ageing, reducing the reserve capacity, decreases the adaptability of various systems and increases the risk of functional disorders. The early recognition and treatment of functional disorders in vitality diagnostics laboratories provides an opportunity to prevent or delay the onset of degenerative diseases related to advanced age.

Increased proportion of docosahexanoic acid and high lipid peroxidation capacity in erythrocytes of stroke patients.

BACKGROUND AND PURPOSE: Intracellular accumulation of lipid peroxides that derive from the autoxidation of membrane polyunsaturated fatty acids reduces the deformability of erythrocytes contributing to the hemorheological disturbances observed in acute cerebral ischemia. The present study deals with the biochemical background of increased lipid peroxidation capacity in the erythrocytes of stroke patients. METHODS: A complete clinical and laboratory assessment was made of 24 men and 18 women (aged 50 to 78 years; 64.5 +/- 13.9 years, mean +/- SD) who had an ischemic hemispheric lesion of the brain. Lipid peroxide content, lipid peroxidation capacity, superoxide dismutase activity, and fatty acid composition of erythrocytes were compared in stroke patients and 22 healthy subjects matched for age. The lipid peroxide content of the erythrocytes was estimated before and after the autoxidative test; the results were expressed as nanomoles of malondialdehyde per gram of hemoglobin. The increase of the lipid peroxide content in the erythrocytes during the autoxidative test measures the lipid peroxidation capacity. RESULTS: In comparison with healthy subjects (1.45 +/- 0.30 nmol MDA/g Hb per 24 hours), the lipid peroxidation capacity was found to be significantly higher (4.18 +/- 0.41 nmol MDA/g Hb per 24 hours) (P < .01) in the erythrocytes of stroke patients. The stroke patients could be divided into two groups on the basis of lipid peroxidation capacity of their erythrocytes. Twenty patients had erythrocytes with high lipid peroxidation (< 4 nmol MDA/g Hb per 24 hours), and 22 patients had very high lipid peroxidation capacity (> 4 nmol MDA/g Hb per 24 hours). There was no significant difference in superoxide dismutase activity in the erythrocytes of patients compared with healthy subjects. Before the autoxidative test was conducted, the fatty acid composition in the erythrocytes of stroke patients with very high lipid peroxidation capacity was measured and found to be generally normal; only the proportion of docosahexanoic acid (22:6 n-3) was markedly (P < .01) increased. CONCLUSIONS: The results suggest that the erythrocytes of ischemic stroke patients with very high lipid peroxidation capacity displaying an abnormal fatty acid composition are much more vulnerable to lipid peroxidation. The increased proportion of docosahexanoic acid and the high lipid peroxidation capacity of erythrocytes play a pathogenetic role and explain the hemorheological disturbances observed in the microcirculation of stroke patients.

[Effect of paracetamol on the acid hemolysis of erythrocytes, with special reference to the intracellular glutathione level]. / Paracetamol hatása a vörösvérsejtek savi haemolysisére, különös tekintettel az intracellularis glutathion szintre.

The authors give account of their study concerning the effect of paracetamol on the acid haemolysis, and glutathione content of human erythrocytes. In vitro, paracetamol decreased intracellular glutathione content in a dose dependent manner. This may lead to an increased sensitivity of erythrocytes to the haemolytic effect of hydrochloric acid, what may explain the dose dependent decrease of time of haemolysis, presented in form of dynamic haemolysis curve. In vivo four hours following the oral intake of 500 mg paracetamol, although the intracellular glutathione content did not decrease significantly, shortening of the time of acid haemolysis could be demonstrated in more than half of the persons studied. No haemolysis was caused by the given dose of the drug.

The effect of silibinin (Legalon) on the the free radical scavenger mechanisms of human erythrocytes in vitro.

The effect of Legalon was investigated parallel with that of Adriblastina (doxorubicin) and paracetamol on some parameters characterizing the free radical scavenger mechanisms of human erythrocytes in vitro and on the time of acid hemolysis performed in aggregometer. Observations suggest that Adriblastina enhances the lipid peroxidation of the membrane of red blood cells, while paracetamol causes significant depletion of intracellular glutathione level, thus decreasing the free radical eliminating capacity of the glutathione peroxidase system. Legalon on the other hand, is able to increase the activity of both superoxide dismutase and glutathione peroxidase, which may explain the protective effect of the drug against free radicals and also the stabilizing effect on the red blood cell membrane, shown by the increase of the time of full haemolysis.

[Auto-oxidation test for the determination of lipid peroxidation capacity of liver tissue homogenates of young and old mice]. / Autoxidationstest zur Bestimmung der Lipidperoxidationskapazität von Lebergewebshomogenaten junger und alter Mäuse.

The activity of superoxide dismutase (SOD) and the lipid-peroxidation capacity, i.e., the autoxidation of lipids during incubation at 37 degrees C for 3 h under aerobic conditions was measured in liver tissue homogenates of young (1.5-month-old) and old (24-month-old) inbred female Balb/c mice. The lipid peroxidation capacity was found to be significantly higher in young mice than in old ones. The pretreatment of young mice with allopurinol, a specific inhibitor of xanthine oxidase, remarkably decreased the lipid peroxidation capacity without any modification of SOD activity. SOD levels were equal in young and old mice. Our results suggest that the lipid peroxidation capacity of liver tissue depends, not only on the level of polyunsaturated fatty acids, but, in general, also on the state of a finely regulated counterbalance of pro- and antioxidant factors. The autoxidation test seems to be a suitable in vitro method for measuring the lipid peroxidation capacity of liver tissue.

Lipid peroxidation and superoxide dismutase activity in muscle and erythrocytes in adult muscular dystrophies and neurogenic atrophies.

Lipid peroxidation (LP) and superoxide dismutase (SOD) activity were determined in erythrocytes and skeletal muscle obtained from patients with limb-girdle and facioscapulohumeral muscular dystrophies, neurogenic atrophies and from age-matched control subjects. Neither lipid peroxidation nor SOD activity in erythrocytes of patients differed from control values. SOD activity and LP in muscle specimens were also normal in types of neurogenic atrophy. Lipid peroxidation in the muscle from patients with adult types of muscular dystrophy had a tendency to be increased. The values were widely scattered, the highest being obtained in the older patients with long duration of disease.

Effect of 2-mercaptoethanol on some metabolic indices of ageing of CBA/Ca inbred mice.

It has been shown in several studies that 2-mercaptoethanol (2-ME) improves the life span and aspects of life performance of laboratory animals. To obtain further details on the beneficial effects of 2-ME, a long-term study has been performed on male CBA/Ca inbred mice treated with this antioxidant. Four month-old mice were each given 4 micrograms of 2-ME in physiological saline via intraperitoneal (i.p.) injection 3 x per week. Measurements were made of the following: cold tolerance (heat performance), apparent total body protein turnover (T1/2), changes in the major lipid and fatty acid compositions of the liver, superoxide dismutase activity and formation of malondialdehyde and observations on a range of pathological changes. It was found that the basal rectal temperatures of the treated mice were higher and in the oldest group, heat performance capacity was better than those of the controls. After about 1 year of age the apparent biological half-life time of total body protein (T1/2) was observed to be shorter in the treated mice. Significant increases were observed to occur in the proportions of the polyunsaturated fatty acids in the lipids of the liver in the mice injected with 2-ME. Although no differences were observed in the superoxide dismutase activities, malondialdehyde concentrations in the livers of the experimental mice were significantly increased. Autopsy data showed that Dunn-sarcomas associated with amyloidoses occurred more frequently in the untreated mice.

The effect of oxidative stress on inbred mice of different ages.

The effect of oxidative stress applied for 2 months was compared in adult (14-month-old) and aged (27-month-old) female BALB/c mice. The stress was administered in two ways. One group of animals received a high dose of H2O2 (0.5% H2O2 in drinking water) the other one was given a low dose of H2O2 (0.4 ml of 0.5% H2O2 by esophageal cannula every alternate day). After a 2-month treatment the activity of superoxide dismutase (SOD), catalase and lipid peroxidation capacity were measured in liver homogenates of mice. The activity of catalase and capacity of lipid peroxidation gradually decreased as a function of age while the SOD activity did not reduce even in older mice. The aged-mice could tolerate a high dose of H2O2 mainly by increasing SOD activity. As a result of intake of low dose of H2O2 in old animals SOD activity was decreased and lipid peroxidation capacity was getting higher suggesting that in this life period the liver tissue is very sensitive to chronic, oxidative stress effects of low dose.

Superoxide anion, superoxide dismutase and lipid peroxidation in the murine macrophage cell line C4M phi.

A remarkable increase in the production of superoxide radicals and SOD activity was measured in suspension of the murine macrophage cell line C4M phi treated with Lentinan (4-10 X 10(3) micrograms/5 X 10(6) cells). In activated macrophages the decrease of lipid peroxidation could be interpreted as a consequence of enhanced SOD activity.

Superoxide dismutase, catalase and lipid peroxidation in liver of young mice of different ages.

The lipid peroxidation and activities of main enzymes involved in the peroxide metabolism were measured at resting conditions and after completion of a 2-month treatment with H2O2. At the start of the experiments 1- and 3-month-old female BALB/c mice received either a high dose of H2O2 (0.5% H2O2 in drinking water; intake 12.5 x 10(-3) g/day/mouse) or a low dose of H2O2 (0.5 ml of 0.5% H2O2 by esophageal canula on every alternate day; intake 1.0 x 10(-3) g/day/mouse). The following conclusions can be drawn: a relative higher increase in activity of catalase could be induced at the age of 3 months than at the age of 5 months by a high dose of H2O2. The activity of superoxide dismutase (SOD) was not changed by a high dose of H2O2. A moderate increase in the activity of catalase and a remarkable decrease in SOD-activity resulted from a low-dose H2O2 treatment at the age of 5 months. The level of lipid peroxidation was increased by the low-dose and not influenced by the high-dose H2O2 treatment in 5-month-old-mice.