Syoji Hayasaka

An enzyme-linked immunosorbent assay to detect alkali-soluble spore surface antigens of strains of Nosema bombycis (Microspora: Nosematidae)

Abstract Spore surface antigens of strains of Nosema bombycis were extracted with alkaline solutions and used in an indirect enzyme-linked immunosorbent assay. Treatment of N. bombycis spores with 0.1 n potassium carbonate or potassium hydroxide solution at 27°C for 30 min was sufficient for the extraction of the antigens. Usually, 108 spores of N. bombycis liberated ca. 30 μg spore surface proteins. The indirect enzyme-linked immunosorbent assay detected as little as 60 ng of spore surface proteins (ca. 2000 spore-equivalent antigen). The alkali-soluble spore surface antigens of N. bombycis contained a specific antigen and were stable under storage at −20°C for more than 1 year. The serological assay separated the Nosema isolates pathogenic to the silkworm into three groups.

Cloning of a microsporidian, Nosema bombycis (Microsporida ; Nosematidae) in insect cell cultures by a limiting dilution method

Several clones of Nosema bombycis NS 001 were established using a limiting dilution of Antheraea eucalypti cell cultures inoculated with sporoplasms emerged from N. bombycis spores. The infected A. eucalypti cell cultures were diluted and transferred to 96-well plastic microplates to isolate a single infected cell in the well. Certain numbers of wells containing the single A. eucalypti cell were randomly selected, because infection of the individual cells isolated in each well was not possible to determine by phase-contrast microscopy at this time. These wells were provided with either uninfected Bombyx mori S. P. C. Bm36 or A. eucalypti cells to produce clones of N. bombycis NIS 001 from presumably a single sporoplasm parasitized in the single cell. Spores of morphological diversity were observed among the clones of N. bombycis NIS 001 isolated by this method. Generally, the characteristics of spore morphology were maintained consistently after several passages of the clones in vitro. Results of the latex adhesion test indicated that the spore surface antigens of seven clones were homologous.

A New Method for Inoculation of Poor Germinator, Nosema sp. NIS M11 (Microsporida: Nosematidae), into an Insect Cell Culture

ABSTRACT. Spores of Nosema sp. NIS M11, primed with 0.1 N KOH solution, were mixed with either physiological salt solutions or IPL‐41 medium, an insect cell culture medium, for germination. In the latter medium, only a few spores germinated, while high percentages of spore germination were obtained in physiological salt solutions, particularly in Rinaldinis solution. By using the salt solutions as inoculation media, KOH‐primed NIS M11 spores were inoculated into the Spodoptera frugiperda SF21AEII cell line. The initial infection levels were consistently higher than that obtained by using IPL‐41 medium. Among the salt solutions, Rinaldinis solution, containing KCl in place of NaCl, gave the highest percentage of initial cell infection. Increased osmolarity of salt solutions did not improve the efficiency of spore germination and infection of N. sp. NIS M11.