A monoclonal antibody‐based ELISA for the analysis of the insecticide flucythrinate in environmental and crop samples

A competitive enzyme‐linked immunosorbent assay (ELISA) has been developed for the detection of the insecticide flucythrinate in environmental and food samples. Two types of haptens, the acid moiety that is the hydrolyzed product of flucythrinate, and the carboxylated propyl derivative of the alcohol moiety, were used to prepare monoclonal antibodies (MAbs). Five MAbs, which raised against the former hapten, were reactive with flucythrinate. Among them, MAb F1A27‐4 showed the highest activity toward flucythrinate, and did not cross‐react with other pyrethroids such as cycloprothrin, fenvalerate, fluvalinate, etofenprox and silafluofen. The assay conditions of indirect competitive ELISA with MAb F1A27‐4 were studied to optimize the detection of flucythrinate in environmental and food samples. Incubation at 4 °C in the assay buffer, pH 8, with 300 mM sodium chloride improved the sensitivity. The addition of rabbit serum albumin or rabbit antiserum and the presence of 50 ml litre^?1 of methanol reduced matrix effects of the samples. Under optimized conditions, the ELISA detected flucythrinate spiked in water, soil, and extracts of apple and tea samples down to 10 mg litre^?1, 0.2 mg litre^?1, 0.3 mg litre^?1 and 0.3 mg litre^?1, respectively. The mean recovery and CV ranged from 91% to 120% and from 5% to 12%, respectively. The ELISA results in apple samples correlated well with those from LC–MS analysis (r² = 0.99, n = 12). © 2001 Society of Chemical Industry     

IgE sensitivity and cross-reactivity to crude and purified mite allergens (Der f 1, Der f 2, Der p 1, Der p 2) in atopic dogs sensitive to Dermatophagoides mite allergens

The present study investigates IgE-reactivity to crude and purified mite allergens by intradermal skin test (IDST), Immunodot method, and ELISA in atopic dogs sensitive to mite allergens, as well as the allergenic cross-reactivity between Dermatophgoides (D) farinae (DF) and D. pteronyssinus (DP) in dogs by IgE-ELISA inhibition. IDST and Immunodot method for crude mite allergens were performed for atopic dogs and 16 atopic dogs showed sensitivity to mite allergens. Of the 16 dogs, all dogs had anti-DF IgE and 11 had anti-DP IgE. We measured specific IgE to purified major allergens (Der f 1, Der f 2, Der p 1, Der p 2). Of the 16 atopic dogs, six had anti-Der f 1 IgE and seven had anti-Der f 2 IgE. Similarly, of the 16 dogs, six had anti-Der p 1 IgE and seven had anti-Der p 2 IgE. However, eight dogs had no specific IgE to these mite allergens. These dogs may be sensitive to other major mite allergens except Der 1 and Der 2. In the dogs that had both anti-DF and DP IgE, IgE binding to DF was greatly inhibited by DP, and reciprocal inhibition was observed. Based on these data, it appears that there is a strong cross-reactivity between DF and DP in dogs. Similarly, a cross-reactivity between DF and DP in purified allergens was also observed. IDST and Immunodot method are useful methods for the diagnosis of atopic diseases in dogs, and ELISA is a useful method for further investigation of IgE-reactivity for the allergens.     

Protective effect of a lignan-containing flaxseed extract against CCl(4)-induced hepatic injury

Carbon tetrachloride (CCl(4)) -induced hepatotoxicity is a commonly used model for investigating lipid peroxidation-related tissue injury. In the present study, the effect of flaxseed extract was observed on histological sections, glutathione-content and DNA strand breaks. Lignan-containing flaxseed extract (1.6 g/kg body weight/day) was daily administered with intragastric injection to rats for three days, on the fourth day, CCl(4) (2 g/kg) was intraperitoneally injected. Liver tissue was sampled at 24 hr after administering CCl(4). Liver-necrosis was observed in CCl(4)-injected rats without pretreatment of flaxseed extract. Pretreatment of flaxseed extract reduced extent of the necrosis found 24 hr after the intraperitoneal administration of CCl(4). Pretreatment of flaxseed extract protect against CCl(4)-induced decrease of reduced glutathione-content measured from reactions with 5,5'-dithiobis-(2-nitrobenzoic acid) and also protect against the elevation of DNA strand breaks in the liver cells measured by comet assay. Flaxseed-extract appears to protect liver cells against CCl(4)-induced necrosis.     

Determination of nitrotyrosine by HPLC-ECD and its application

3-nitrotyrosine, a product of tyrosine nitration, is a useful indicator of oxidative damage. We modified the previously reported HPLC-electrochemical detection (ECD) method: specifically, a through-type porous carbon electrode was used as a reducing electrode instead of the mercury-gold amalgam electrode, because the response of the latter changes over time. A combination of reverse-phase HPLC and electrochemical detector passed through -800 mV reduction potential and subsequently under +250 mV oxidation potential allows measurement of 3-nitrotyrosine. The detection limit of this assay was less than 10 fmol. In mice to which lipopolysaccharide (LPS) was administered intraperitoneally, plasma 3-nitrotyrosine levels were elevated, corresponding to LPS dosage. These findings suggest that the improved HPLC-ECD method can be used as a specific and sensitive assay of biological 3-nitrotyrosine and can be applied clinically.     

Black root rot of cucurbits caused by Phomopsis sclerotioides in Japan and phylogenetic grouping of the pathogen

Although the causal agent of black root rot of Cucurbitaceae in Japan has been proposed as Phomopsis sclerotioides, the species identification of the pathogen has remained inconclusive because of a lack of spore formation. We confirmed that a Japanese isolate of Phomopsis sp. obtained from a diseased pumpkin root produced pycnidia containing α spores in sterilized bean pods. In phylogenetic analyses of rDNA-ITS regions, nine Japanese Phomopsis sp. isolates from melon, watermelon grafted onto bottle gourd, and pumpkin diagnosed with black root rot, formed a single clade with P. sclerotioides standard isolates. We identified the causal agent of the black root rot of melon, pumpkin, bottle gourd, and watermelon in Japan as P. sclerotioides and propose the Japanese name “Phomopsis-negusare-byo” for the disease. Patterns of random amplified polymorphic DNA (RAPD) of these Japanese isolates were also similar to those of P. sclerotioides, thus supporting the species identification. However, mycelial incompatibilities were found for many combinations among these P. sclerotioides isolates, suggesting some genotypic variations of this fungus in Japan at a level that the RAPD analyses cannot discriminate. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB201430 to AB201444     

Butt rot ofChamaecyparis obtusa (Sieb. et Zucc.) Endlicher trees caused byPerenniporia subacida in Shikoku district, Japan: Pathogen, distribution of damaged trees in the stand, and soil investigation

The rot ofChamaecyparis obtusa (hinoki) trees was studied in the northern part of Kagawa Prefecture, Japan. Among 158 hinoki examined, butt rot was found in 28.6, 58.3, and 100% of trees in the 29, 30, and 34-year-old stands, respectively. All trees with butt rot, and 11 trees without it had rotted roots. Thirteen hinoki were peeled byCervus nippon (sika deer) and all of them were infected with butt rot. The maximum height of rot in deer-damaged trees was 2.6 m from the ground level, but was 1.9 m in undamaged trees. About eighty-two percent of rotted trees showed rotted areas of less than 40 cm² on the cross section of stems at the ground level. White mycelia and black flecks sometimes appeared in the rotted wood. Basidiocarps of polyporaceous fungus were often found on felled logs and rotted stumps of hinoki and identified asPerenniporia subacida. Basidiomycetous fungus was isolated frequently from rotted wood of roots and stems, and determined to beP. subacida by comparative study on cultural characteristics. An inoculation experiment and wood-decay test proved that the fungus was the cause of the rot of hinoki. Few absorbing roots of living trees were found in the clayey subsurface soil of the high dry bulk density and the less soil aeration. Some absorbing roots had root rot and the rot spread from the base of the absorbing root to the central part of the woody root. This is the first report on the rot of hinoki caused byP. subacida in Japan.     

Transfer of blood urea nitrogen to cecal microbial nitrogen is increased by fructo‐oligosaccharide feeding in guinea pigs

The present study was conducted to determine the mechanism by which nitrogen (N) availability is improved by fructo‐oligosaccharide (FOS) in guinea pigs. Adult male guinea pigs were fed a commercial pellet diet (50 g/day) with either 5% glucose or 5% FOS for 7 days in individual metabolism cages. After 7 days of feeding the diet, ¹⁵N‐urea was administered intravenously 1 h before slaughter under anesthesia. The amount and concentration of total, protein, bacterial, ammonia and urea N and the ¹⁵N atom % excess were measured in blood, liver, gut contents and urine. The ¹⁵N atom % excess of total and protein N, and the amount of total, protein and bacteria N and ¹⁵N in the cecum were significantly increased by the consumption of FOS. Furthermore, the concentration and amount of short‐chain fatty acids were significantly increased by the consumption of FOS. In contrast, the amount of urinary ¹⁵N was significantly decreased by the consumption of FOS. These results suggest that consumption of FOS increases transfer of blood urea N into the large intestine for bacterial N synthesis, which is subsequently re‐absorbed by cecotrophy, and contributes to the increase of N utilization in guinea pigs.     

Reliability of estrous detection in Holstein heifers using a radiotelemetric pedometer located on the neck or legs under different rearing conditions

To evaluate the efficiency and accuracy of estrous detection using a new pedometry system that can measure the hourly activity of cattle, pedometers were attached to the neck and the hind legs of 15 Holstein heifers. Heifers were reared in pasture for grazing, an open paddock, or in a tie-stall barn (an additional pedometer was attached to a front leg of each of these heifers). The most recent 24 h-total number of steps was compared for each 1 h-interval with the mean value of the preceding days during the reference period (RP). The neck pedometer detected all 10 instances of estrous activity (100%) for the grazing heifers at 1.3 times the thresholds value for a 5-day RP but with only 32% accuracy. The hind leg pedometer, however, obtained 100% efficiency and 83% accuracy at 1.4 times the threshold value for a 7-day RP. The efficiencies and accuracies in detecting 12 instances of estrous activity under the paddock condition were 92 and 65% (neck, 1.3-fold, 7-day RP) and 92 and 100% (hind leg, 1.6- or 1.7-fold, 7-day RP), respectively. Under the tie stall condition, the neck pedometers detected 92% of 23 instances of estrous activity with 34% accuracy (1.2-fold, 3-day RP), and the efficiencies and accuracies of the leg pedometers were 78 and 78% (hind leg, 1.4-fold, 4- or 6-day RP) and 87 and 83% (front leg, 1.4-fold, 7-day RP), respectively. Prediction of ovulation time was more precisely with the leg pedometers than with those under the tie stall conditions. Our preliminary results indicate that this new pedometer system has practical value for estrous detection in heifers under different rearing conditions, which affect the criteria required for detection. Furthermore, they also indicate that a leg pedometer can reliably detect estrus and that a neck pedometer may only be capable of detecting estrus under paddock rearing conditions.     

Morphological identity of a taxonomically unassigned cytochrome c oxidase subunit I sequence from stomach contents of juvenile chum salmon determined using polymerase chain reaction

In a previous report, we analyzed the stomach contents of juvenile chum salmon Oncorhynchus keta by morphological observation and also by molecular identification using the mitochondrial cytochrome c oxidase subunit I (COI) region. However, one of the most frequently detected COI sequences could not be assigned to any specific taxon, even at the phylum level. In the present study, we conducted in situ hybridization (ISH) on the stomach contents of juvenile chum salmon using the COI sequence and polymerase chain reaction amplification of a 18S ribosomal RNA gene from the tissue sections where ISH signals were detected. As a result, the organism that was enigmatic at the phylum level was found to be an appendicularian. Moreover, Oikopleura longicauda collected from the bay where the juvenile chum salmon samples were obtained was shown to have the same COI sequences as this taxonomic “orphan” COI sequence from the stomach contents. The present results suggest that the COI sequences previously deposited in public databases for “Oikopleura” are actually derived from taxonomic groups other than appendicularians, and that this may have hampered our understanding of prey richness in the stomach or gut of certain marine animals based on DNA barcoding.