Sandwich ELISA system for cartilage oligomeric matrix protein in equine synovial fluid and serum

Reasons for performing study: Measurement of cartilage oligomeric matrix protein (COMP) in serum has potential for diagnosis of equine osteoarthritis (OA), but clinical use is currently limited by the lack of specificity of an inhibition ELISA as well as by baseline increases due to exercise. Improved methods for ELISA with increased antigen specificity and sensitivity are therefore required for reliable measurement. Hypothesis: Measurement of the serum level of COMP by sandwich ELISA allows identification of horses with OA. Methods: New monoclonal antibodies (mAbs) were elicited against equine cartilage COMP, their epitopes were determined and a sandwich ELISA was developed. The concentrations of COMP in synovial fluid (SF; n = 100) and sera (n = 100) from OA cases were measured by sandwich ELISA as well as by inhibition ELISA and compared with concentrations in normal joints (n = 95) and horses (n = 50). Results: Immunoblots of enzymatically cleaved COMP showed that the new mAbs recognised different epitopes located on a 20 kDa fragment between K⁶³ and K²³⁸ of the EGF‐like repeats. Inhibition ELISA with any mAb detected significantly increased levels of COMP in OA SF compared with normal SF, whereas no significant difference was detected between serum levels of COMP in OA and normal horses. Conversely, sandwich ELISA with the combination of unlabelled 2A11 × biotinylated 11F10 mAbs detected a significant increase in COMP levels in both serum and SF from OA cases compared with levels in normal animals. Conclusions and potential relevance: Measurement of serum COMP with sandwich ELISA may be useful in identifying horses with OA.     

Efficacy of open patch-grafting under cardiopulmonary bypass for pulmonic stenosis in small dogs

Objective   To assess the efficacy of an open patch-graft technique under cardiopulmonary bypass (CPB) in small dogs.
Design and methods   A retrospective analysis of 10 dogs with pulmonic stenosis. Records between 1992 and 2002 were reviewed. The effect of surgical correction was evaluated and perioperative parameters were compared between survivors and non-survivors.
Results   The postoperative pulmonary pressure gradient was reduced in all seven surviving patients. Mean ± SE was 21.5 ± 7.4 mmHg (range 3.0–54.2 mmHg) and 6/7 dogs were < 40 mmHg at 3 months postoperatively. Comparing the data between those patients that survived and those that did not, the preoperative pressure gradient (P = 0.04) and volume of the Glucose-Insulin-Kalium solution used (P = 0.001) were significantly higher in those that did not survive.
Conclusion   Open patch-grafting can be performed in small-breed dogs and decreased the pulmonary pressure gradient in survivors at 3 months postoperatively. However, this technique is more invasive than balloon valvuloplasty and should be used cautiously in severely stenosed patients.     

Nationwide seroprevalence of Neospora caninum among dairy cattle in Japan

Serum samples from 2420 clinically healthy dairy cattle, randomly selected from stored sera in 18 districts of Japan, were tested for the presence of Neospora caninum antibodies using an indirect fluorescent antibody test (titer > or =1:200). Nationwide seroprevalence is estimated at 5.7% (139/2420). Seropositive cattle were detected in all surveyed districts despite the evidence of confirmed case reports of bovine neosporosis, showing that N. caninum is widely distributed throughout Japan. Age-specific seroprevalence did not increase with cattle age, suggesting that Neospora infection is likely to be transmitted vertically rather than horizontally in Japan. Considering that N. caninum seropositive cows are thought to be more likely to abort, substantial fetal losses may be induced by N. caninum infection in Japan. Devising strategies are needed to reduce the economic impact on the Japanese dairy industry. This is the first study to investigate the nationwide seroprevalence of N. caninum in cattle in Asia.     

First application of PCR-Luminex system for molecular diagnosis of fungicide resistance and species identification of fungal pathogens

Fungicide resistance in plant pathogens is often caused by a single point mutation in a gene encoding fungicide target proteins. Such is the case for resistance to MBI-D (inhibitors of scytalone dehydratase in melanin biosynthesis) fungicides in rice blast fungus (Magnaporthe oryzae), which is caused by a mutation in the scytalone dehydratase gene that results in a replacement of valine with methionine at codon 75 of the fungicide target protein. PCR-Luminex, a novel system developed for high-throughput analysis of single nucleotide polymorphisms (SNPs) was successfully introduced to diagnose MBI-D resistance using specific oligonucleotide probes coupled with fluorescent beads. The PCR-Luminex system was further tested for its potential in identifying species causing Fusarium head blight on wheat. Four major pathogens, Fusarium graminearum (=F. asiaticum), F. culmorum, F. avenaceum, and Microdochium nivale, known to cause the disease, were tested, and the species were identified using the PCR-Luminex method. So far, this report is the first on the application of the DNA-based PCR-Luminex system in the area of crop protection and/or agricultural sciences.     

Relation between pathogenicity and genetic variation within <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

The relation between diversity of pathogenicity on clubroot-resistant (CR) cultivars of Chinese cabbage (Brassica rapa subsp. pekinensis) bred in Japan and DNA polymorphisms in 17 populations of Plasmodiophora brassicae from cruciferous plants was examined by inoculation tests and random amplified polymorphic DNA (RAPD) analysis using 18 arbitrary primers. Four pathotypes (A–D) were identified after inoculation of six CR cultivars of Chinese cabbage in the 17 populations from cruciferous crops. A relatively high level of genetic diversity was also detected among these populations in the RAPD analysis. Although the four pathotypes could not be clearly differentiated using the RAPD data, most populations of three pathotypes had a consistent location on the dendrogram. All pathotype B (virulent on five cultivars except Utage 70) and D (avirulent on all cultivars) populations, which were common in incompatible interactions with cv. Utage 70, were located in a single subcluster. All five pathotype C populations (virulent only on cv. Utage 70) except for one population grouped in another single subcluster. Because four pathotype A populations (virulent on all six cultivars, races 4 and 9) fell in different subclusters, the populations may be genetically polyphyletic. Populations from cruciferous weed Cardamine flexuosa differed remarkably from those from cruciferous crops in pathogenicity on common cultivars of Chinese cabbage and turnip and C. flexuosa, but they grouped in a single cluster with all race 9 populations from crops. Race 9 populations from crops may thus be closely related to populations from the weed rather than to races 1 and 4 from crops.     

Growth performance and physiological condition of F1 amago salmon Oncorhynchus masou ishikawae juveniles obtained from broodstock with selective breeding for growth on a low fish-meal diet

A 10-week feeding trial was conducted to evaluate the growth and physiological condition of F1 amago salmon Oncorhynchus masou ishikawae juveniles obtained from broodstock that had been selected for growth on a low fish-meal (LFM) diet. F1 juveniles from broodstock showing superior or inferior growth on a LFM diet (LFM-S, LFM-I), and those from broodstock showing intermediate growth on a fish meal-based (FM) diet (FM-M), were fed a LFM diet and a FM diet. In fish fed the LFM diet, growth of LFM-S was significantly better than FM-M at 3 weeks; however, this superiority in growth disappeared at the end of the 10-week trial. Growth of LFM-I was significantly lower than LFM-S throughout the feeding trial. Growth of fish fed the FM diet was not significantly different among groups, and higher than fish fed the LFM diet. Improvements in the blood hemoglobin concentration and morphological conditions of the distal intestine and liver were observed in LFM-S fed the LFM diet. Although the effect of selective breeding was limited in F1 amago salmon, the present results suggest that continuous selection for growth with a LFM diet has potential as a method to adapt to the limited supply of fish meal.     

Effects of tightening speed on torque coefficient in lag screw timber joints with steel side plates

To investigate the effects of tightening speed on the torque coefficient in lag screw timber joints with steel side plates, tightening tests were conducted on main timber members made from Cryptomeria japonica, Chamaecyparis obtusa and Pseudotsuga menziesii, under four tightening speed conditions (1, 4, 10, and 20 rpm). Major stick-slip behavior was observed in C. obtusa based on the relationship of tightening angle with clamp force, tightening torque, and thread torque at tightening speeds of 1 and 4 rpm. In addition, tightening speed’s effects on the torque coefficient (K) varied depending on the wood species of the main member. In P. menziesii, K was not affected by the tightening speed: the ratio of torque expended on tightening was 25% on average, and the ratio of torque expended on bearing surface friction was higher than the ratio of torque expended on thread friction.     

Monitoring of QoI fungicide resistance in Plasmopara viticola populations in Japan

BACKGROUND: The increasing occurrence of QoI fungicide resistance in Plasmopara viticola (Berk. & MA Curtis) Berl. & DeToni populations is becoming a serious problem in the control of grapevine downy mildew. In Japan, the existence of QoI‐fungicide‐resistant P. viticola was reported in 2009. RESULTS: The QoI fungicide resistance in P. viticola samples collected from vineyards in Japan in 2008 and 2009 was monitored. Resistant P. viticola were detected in the regions where QoI fungicides have been introduced in accordance with the pest management programme, whereas in Hokkaido vineyards, where QoI fungicides have not yet been introduced, QoI‐fungicide‐resistant P. viticola were not found. CONCLUSION: Japan comprises thousands of islands and is physically isolated from other countries by the sea. Monitoring the emergence, incidence and distribution of QoI fungicide resistance in P. viticola populations in Japan is necessary to improve pest management strategies for downy mildew disease in Japanese vineyards. Copyright © 2010 Society of Chemical Industry     

Goblet cell hyperplasia and muscular layer thickening in the small intestine of a cynomolgus monkey

We report here the interesting case of a 5-year-old male cynomolgus monkey with goblet cell hyperplasia and thickening of the muscular layer throughout the small intestine without exhibiting any clinical symptoms. Necropsy examination showed diffuse thickening of the intestinal wall from the jejunum to the ileum, with an appearance likened to a rubber tube. Histopathologically, marked thickening was observed in both the mucosal and muscular layers in the jejunum and ileum, and slight thickening was observed in the duodenum. Goblet cell hyperplasia with extension of the circular folds and villi was prominently observed. The mucosal surface was covered with a thick mucus layer containing desquamated mucosal epithelial cells, and both the inner and outer muscular layers were markedly thickened due to smooth muscle hypertrophy. Neither macroscopic nor histopathological examination identified any causative factors, such as infection, enteritis and intestinal stenosis, or obstruction that may have caused development of this lesion. Given these observations, this case may simply be considered of spontaneous goblet cell hyperplasia and muscular layer thickening in the small intestine of a cynomolgus monkey.