Detection of an autoantibody from Pug dogs with necrotizing encephalitis (Pug dog encephalitis).

An autoantibody against canine brain tissue was detected in the cerebrospinal fluid (CSF) and serum of two Pug dogs (Nos. 1 and 2) by indirect immunofluorescence assay (IFA). Dog No. 1, a 2-year-old male, exhibited severe depression, ataxia, and generalized seizures and died 2 months after the onset of symptoms. Dog No. 2, a 9-month-old male, exhibited severe generalized seizures and died 17 months after the onset of symptoms. Histopathologic examination revealed a moderate to severe multifocal accumulation of lymphocytes, plasma cells, and a few neutrophils in both the gray and white matter of the cerebrum in dog No. 1. In dog No. 2, the cellular infiltrates were mild, but there was a severe, diffuse, and multifocal necrosis in the cerebral cortex with prominent astrocytosis. With the aid of IFA using fluorescein isothiocyanate-labeled antidog IgG goat serum and a confocal imaging system, specific reactions for glial cells were detected in the CSF of these Pug dogs but not in six canine control CSF samples. Double-labeling IFA using CSF from these Pug dogs and a rabbit antiserum against glial fibrillary acidic protein (GFAP) revealed that the autoantibody recognized GFAP-positive astrocytes and their cytoplasmic projections. By immunoblot analysis, the autoantibody from CSF of these Pug dogs recognized two common positive bands at 58 and 54 kd, which corresponded to the molecular mass of human GFAP. The role of this autoantibody for astrocytes is not yet clear. However, if the presence of the autoantibody is a specific feature of Pug dog encephalitis, it will be a useful clinical diagnostic marker and a key to the pathogenesis of this unique canine neurologic disease.     

Endoscope Application for the Mussel Watch Program of Marine Pollution Monitoring

As a trial for the complete establishment of mussel watch program, it was shown that the ciliary stroke of Mytilus edulis can be used as an indicator of its bioaccumulation of pollutants from sea water when it was measured by use of a high function OES endoscope. This is reasonable because the ciliary strokes result in the intake of phytoplankton by raising the constant water current along the cilia from an inhalant siphon transported to the mouth. The ciliary strokes showed an evident diel fluctuation with the maximal activity during the night. The contribution of mucus excretion was evident for efficient filter-feeding. The ciliary stroke and the mucus excretion were activated almost simultaneously showing similar diel fluctuation while filter-feeding. The maximal ciliary stroke was observed at 20 °C in salt solution from 30 to 40‰ salinities. The stroke follows the Arrhenius equation in the temperature range from 5 to 30 °C with a break at around 20 °C when the experiment was performed with salt solution between 10 to 33‰ salinities.     

Influence of the spawning environment on final maturation and spawning in Japanese dace, Tribolodon hakonensis

Results of the present study demonstrated that a specific combination of spawning substrate and water current was indispensable for the spawning of mature Japanese dace, Triborodon hakonensis. It became evident that the environmental factors acted as a trigger for endocrine changes leading to final maturation in this species.     

Evaluation of a stress-wave timer for the minimally destructive detection of decay in living trees in Northern-Japanese forests

The use of a stress-wave timer as a minimally destructive device for detecting decay or defect in living trees was evaluated. Measurements were conducted on five tree species (Picea jezoensis, P.glehnii, Betula platyphylla var.japonica, Abies sachalinensis, andLarix kaempferi) with or without decay. Except in sap-rottedA. sachalinensis, the apparent stress-wave velocity in most decayed trees was considerably lower than the value obtained from healthy trees. Our results showed that defect or decay in the trees was detectable more effectively by the method used in the field survey, although the device occasionally failed to detect decay that was incipient, of small extent or confined to sapwood. Other disadvantages of this method are briefly discussed in this paper.     

Survey of mutations of a histidine kinase gene BcOS1 in dicarboximide-resistant field isolates of Botrytis cinerea

Previously, we cloned a putative osmosensing histidine kinase gene (BcOS1) and revealed that a single amino acid substitution, isoleucine to serine at codon 365, conferred dicarboximide resistance in field isolates of Botrytis cinerea. This point mutation (type I) occurred within the restriction enzyme TaqI site of the wild-type BcOS1 gene. Thus, a procedure was developed for detecting the type I mutation of the BcOS1 gene using a polymerase chain reaction (PCR) in combination with restriction fragment-length polymorphism (RFLP). Diagnosis by PCR-RFLP was conducted on the 105 isolates isolated from 26 fields in Japan. All dicarboximide-sensitive isolates (49 isolates) had the wild-type BcOS1 gene, and the 43 isolates with the type I mutation were resistant to dicarboximides without exception. These data indicate that dicarboximide-resistant isolates with type I mutation are widespread throughout Japan. However, other types of dicarboximide resistance were detected among isolates from Osaka; among the 24 resistant isolates from Osaka, 12 had the BcOS1 gene without the type I mutation. BcOS1 gene sequencing of these resistant isolates classified them into two groups, type II and type III. The type II isolates have three amino acid substitutions within BcOS1p (³⁶⁸Val to Phe, ³⁶⁹Gln to His, and ⁴⁴⁷Thr to Ser). The type III isolates have two amino acid substitutions within BcOS1p (³⁶⁹Gln to Pro and ³⁷³Asn to Ser). These amino acid changes are located on the amino acid repeat domain in BcOS1p. The three types of resistant isolates were all moderately resistant to dicarboximides without significant osmotic sensitivity, and their pathogenicity on cucumber leaves was also very similar to that of the wild-type isolate.     

Control of soilborne clubroot disease of cruciferous plants by epoxydon from Phoma glomerata

Culture broth from an isolate of Phoma glomerata (no. 324, = JCM 9972) from the leaves of Viola sp., controlled the soilborne pathogen Plasmodiophora brassicae which causes clubroot disease of cruciferous plants. This effect was caused by epoxydon (5-hydroxy-3-(hydroxymethyl)-7-oxabicyclo[4.1.0]hept-3-en-2-one). Although this substance was known to have antitumour activity, phytotoxicity and antiauxin activity, no plant disease reduction had been reported previously. Epoxydon possessed neither strong antimicrobial activity nor did it induce acquired resistance. It protected crucifers from clubroot disease at 250  μ g mL⁻¹ following addition to the soil. Several antiauxins were tested for similar properties resulting in the suppression of clubroot disease and one, 2,3,5-triiodobenzoic acid, was effective at 10  μ g mL⁻¹. Clubroot reduction by epoxydon may result from antiauxin activity. This opens opportunities for a new group of agrochemicals.     

Detection of chicken anaemia virus DNA from formalin-fixed tissues by polymerase chain reaction

Chicken anaemia virus was detectable from various samples such as cell-free virus, infected cells, unfixed liver homogenates, formalin-fixed liver homogenate or formalin-fixed paraffin-embedded ( ) tissues from experimental or field infected chicks using assay. The detection limit of the first PCR assay was 1 infected cell or 10^−1·5, ₅₀ of cell-free virus (strain A2). The nested assay increased the sensitivity 10- or 100-fold. was detectable in the other 14 Japanese strains isolated from 1976 to 1994 by the assay. All the amplified products were digested with BglII, HindIII, PstI and SacI. These results suggest that the region amplified was highly conserved among the strains. The nested assay was very sensitive. However, was detectable in most field samples using the first assay. Therefore, the nested assay may not always be necessary. In contrast, the nested PCR assay was necessary to detect in tissues or formalin-fixed material. Use of the assay in detection from tissues may be most valuable in diagnosis of diseases caused by or associated with , because it allows detection of both microscopic lesions and .     

FC-6 Four isoforms of exfoliative toxin produced by Staphylococcus hyicus abolished immunofluorescence for desmoglein 1 in pig skin

Western immunoblotting studies of canine sera using Malassezia pachydermatis extracts have shown that infected dogs commonly have antibodies that recognize multiple antigens. However, reported patterns of immunoreactivity vary between different laboratories. Since culture duration influences the antigenic composition of lipid-dependent Malassezia when probed with human sera, we investigated whether the in vitro growth phase of M. pachydermatis influences immunoreactivity to canine sera. Extracts of M. pachydermatis CBS1879 grown in Sabouraud's liquid medium at 37°C for 2, 4, 6, 8 and 10 days were prepared by mechanical disruption, centrifugation, dialysis and lyophilization. Yeast growth phase was assessed by sequential colony counts and optical density measurements. Patterns of IgG immunoreactivity in high ( n  = 3) and low ( n  = 3) titre sera were compared using extracts prepared at each time point by SDS-PAGE and western immunoblotting. Protein bands of 62 and 49 kDa were recognized by all sera, and five sera recognized 98 and 68 kDa bands. Proteins of 188, 66, 58, 57, 38, 28 and 17 kDa were only recognized by high titre sera. All high titre sera recognized more bands in exponential phase (day 2) extracts when compared with decline phase (days 8–10) extracts, and two of these sera showed most bands in stationary phase (days 4–6) extracts. Bands of 62 and 57 kDa were primarily detected in exponential and early stationary phase extracts. Antigenic variation in extracts of M. pachydermatis prepared during different growth phases might explain discrepancies between previous laboratory studies of immunity to this yeast.
Funding: Government of Malaysia.