Search for and detection of specific DNA fragments in high- and low-virulent strains of Nosema bombycis (Microsporida: Nosematidae)

Nosema bombycis is a highly virulent microorganism causing pebrine disease in the silkworm, Bombyx mori, and damage due to this disease has recently been successively reported in not only Japan, but also other Asian countries. Conversely, a low-virulent strain, N. bombycis Sd-NU, isolated from lawn grass cutworm (Spodoptera depravata) has been reported to exhibit no strong virulence against silkworms, unlike a highly virulent strain, N. bombycis NIS-001, derived from silkworms. There is no morphological difference between these 2 strains, and only their virulence against silkworms is different. In this study, we investigated differences in the genomic constitution between these 2 strains showing different virulence against silkworms by genome subtraction, and obtained several DNA fragments specific to the highly virulent strain. These N. bombycis NIS-001-derived DNA sequences were subjected to primer design, followed by PCR, and sequences with different sizes between N. bombycis NIS-001 and Sd-NU were amplified. The amplified sequences were not consistent between the strains on sequence analysis, clarifying that they were distinctly different. Based on these findings, Southern hybridization was performed using the DNA sequences derived from N. bombycis NIS-001 as probes, and no signals were detected in N. bombycis Sd-NU. These findings revealed that the sequences obtained by genome subtraction are present only in N. bombycis NIS-001.