Dynamic changes of virus load in supernatant of primary CEK cell culture infected with different generations of avian infectious bronchitis virus strains Sczy3 as revealed by real-time reverse transcription-polymerase chain reaction.

Infectious bronchitis virus (IBV) can multiply effectively in chick embryo kidney (CEK) cells after adapting to the chick embryo. To investigate the dynamic changes in IBV load in the supernatant of primary CEK cells, we developed an SYBR Green I-based real-time polymerase chain reaction assay to quantify nucleic copy numbers of the IBV-Sczy3 strain. The 20, 54, and 87th generations of CEK-adapted IBV-Sczy3 strains were used to infect CEK cells, and then nucleic copy numbers in the samples of supernatant collected at 12, 24, 36, 48, 60, and 72 h were detected. The results showed that the rapid growth period of the virus load of all the 3 generations was approximately 12-36 h post-infection; the peak of the virus load appeared at 36 h post-infection and then decreased gradually in the order of 20th > 54th > 87th for the 3 generations of CEK-adapted strains; the dynamic change curve of the IBV load in the supernatant of primary CEK cells showed a single peak. The results of this study provide a useful reference for CEK-adapted IBV field strains and the production of CEK-attenuated IBV vaccine.

First Report of Cladobotryum protrusum causing Cobweb Disease on the Edible Mushroom Coprinus comatus.

Coprinus comatus is one of the most commercially important mushrooms in China. Its fruiting body possesses rich nutritional and medicinal value. In November 2013, unusual symptoms were observed on C. comatus on a mushroom farm in Wuhan, Hubei, China. At first, fruiting bodies were covered by white and cobweb-like mycelia. Later, the cap and stipe turned brown or dark before rotting and cracking. The pathogen was isolated from infected tissue of C. comatus. Colonies of the pathogen on potato dextrose agar (PDA) medium first appeared yellowish, followed by an obvious ochraceous or pinkish color. Aerial mycelia grew along the plate wall, cottony, 1 to 4 mm high. Conidiophores were borne on the tops of hyphae, had two to four branches, and were cylindrical, long clavate, or fusiform. Conidia were borne on the tops of the branches of conidiophores, had one to two separates, and were long and clavate. The spores ranged from 15.3 to 22.1 µm long and were 5.1 to 8.3 µm wide, which was consistent with the characteristics of Cladobotryum protrusum (1). The species was identified by ribosomal internal transcribed spacer sequencing. The ribosomal ITS1-5.8S-ITS2 region was amplified from the isolated strain using primers ITS1 and ITS4. A BLAST search in GenBank revealed the highest similarity (99%) to C. protrusum (GenBank Accession Nos. FN859408.1 and FN859413.1). The pathogen was grown on PDA at 25°C for 3 days, and the inoculation suspension was prepared by flooding the agar surface with sterilized double-distilled water for spore suspension (1 × 105 conidia/ml). In one treatment, the suspension was sprayed on casing soil (106 conidia/m2) and mixed thoroughly with it, then cased with treated soil for 2 to 3 cm thickness on the surface of compost in cultivation pots (35 × 25× 12 cm), with sterile distilled water as a control (2). Eight biological replicates were included in this treatment. In the second treatment, mycelia plugs (0.3 × 0.3 cm) without spore production were added to 20 fruiting bodies. Mushrooms treated with blank agar plugs (0.3 × 0.3 cm) were used as a control. The plugs were covered with sterilized cotton balls to avoid loss of moisture. Tested cultivation pots were maintained at 18°C and 85 to 95% relative humidity. In the samples where casing soil was sprayed with conidia suspension, white mildew developed on the pileus, and a young fruiting body grew out from the casing soil. Eventually, the surface of the mushroom was overwhelmed by the mycelia of the pathogen and the pileus turned brown or black. For the other group inoculated with mycelia plugs, only the stipe and pileus inoculated with mycelia turned brown or dark; it rotted and cracked 2 to 3 days later. The symptoms were similar to those observed on the C. comatus cultivation farm. Pathogens re-isolated from pathogenic fruiting bodies were confirmed to be C. protrusum based on morphological characteristics and ITS sequence. To our knowledge, this is the first report of the occurrence of C. protrusum on the edible mushroom C. comatus (3). Based on the pathogenicity test results, C. protrusum has the ability to severely infect the fruiting body of C. comatus. References: (1) K. Põldmaa. Stud. Mycol. 68:1, 2011. (2) F. J. Gea et al. Plant Dis. 96:1067, 2012. (3) W. H. Dong et al. Plant Dis. 97:1507, 2013.

Pharmacokinetics of cytotoxin from Chinese cobra (Naja naja atra) venom.

The pharmacokinetics of cytotoxin from Chinese cobra (Naja naja atra) venom was studied in rabbits after i.v. and i.m. injection of 0.2 and 0.5 mg.kg-1, respectively. The plasma levels of the cytotoxin were analysed by a biotin-avidin enzyme-linked immunosorbent assay. The plasma concentration-time course after i.v. administration fitted a two-compartment open model. The half-life (mean +/- S.D.) of the alpha-phase was 5.8 +/- 0.6 min and that of the beta-phase 3.5 +/- 0.2 hr. Apparent volume of distribution was 1.7 +/- 0.3 litres.kg-1, and clearance was 5.6 +/- 1.4 ml.min-1. A rapid absorption was observed after i.m. injection with peak plasma level of 260 +/- 90 ng.ml-1 reached within 13.6 +/- 2.4 min. The absorption rate constant was 0.16 +/- 0.03 ml-1. The area under the plasma concentration-time curve was 33 +/- 15 micrograms.min.ml-1.

[Biotin-avidin-ELISA for cytotoxin purified from cobra venom].

A technique combining the biotin-avidin amplifying system with sandwich enzyme-linked immunosorbent assay (B-A-ELISA) was developed for the microassay of the cytotoxin from Naja naja atra venom. The horse-peroxidase was used as the marker and polystyrene microhemagglutination plates as solid carrier. The sensitivity was 0.5 ng.ml-1. The assay range was 1.25-320 ngml-1 with coefficient of variation of 4.9% (intragroup) and 8.1% (intergroups), respectively.

[Distribution of 131I-cytotoxin 14 from Naja naja atra venom in rats].

Cytotoxin 14 (CT14) from Naja naja atra venom was labelled with 131I by chloramine-T method and its tissue distribution was studied in rats. The highest concentration of the cytotoxin was found in kidney, 5979 dpm per mg weight, 14 times more than that of the control animals, at 0.5 h after i.v. injection and high concentrations were found in liver, spleen, pancreas, and adrenal. CT14 was also found in brain at 2 h after injection, 50 dpm per mg weight, 3 times more than that of the control.

[Pharmacokinetics of cytotoxin 14 from Naja naja atra venom].

The pharmacokinetics of cytotoxin 14 (CT 14) from Naja naja atra venom was studied with biotin-avidin enzyme-linked immunosorbent assay. The plasma concentration-time curve of CT 14 after bolus i.v. 0.2 mg.kg-1 into rabbits was found to fit a two-compartment open model. T1/2 alpha was 5.8 +/- 0.6 min and T1/2 beta was 3.5 +/- 0.2 h. A rapid absorption was seen after im injection (0.5 mg.kg-1) to rabbits. The peak level was 260 +/- 90 ng.ml-1 and Tpeak was 13.6 +/- 2.4 min. The absolute bioavailability was 34 +/- 9%. It was shown that CT 14 was absorbed by gut. The peak level was 75 +/- 15 ng.ml-1 after a gastric gavage of 50 mg.kg-1 to rats.