GROWTH OF PAENIBACILLUS-LARVAE, THE CAUSATIVE AGENT OF AMERICAN FOULBROOD IN HONEY-BEES AND BACILLUS-POPILLIAE, THE CAUSATIVE AGENT OF MILKY DISEASE IN BEETLE LARVAE IN LEPIDOPTERAN CELL-CULTURES

TNM-FH Lepidopteran insect cell culture medium containing 10% fetal bo vine serum (FBS), while allowing limited vegetative growth of Paenibac illus larvae (wild-type strain), the causative agent of American foulb rood, contained no viable vegetative cells upon subculture, nor were a ny heat resistant spores produced in this medium alone. However, TNM-F H medium containing embryonic or midgut cells from Trichoplusia ni, he mocytes from Estigmene acres, ovarian and embryonic cells from Spoctop tera frugiperda, embryonic cells from Plutella xylostella, Spodoptera exigua and Pseudaletia unipuncta or ovarian cells from Lymantria dispa r, supported both heavy vegetative cell growth and moderate production of heat resistant spores. EX-CELL 405 serum-free insect cell culture medium alone appeared to contain the appropriate nutrients required fo r both vegetative growth and sporulation of P. larvae. However, in the presence of embryonic cells from T. ni, limited vegetative growth occ urred and the P. larvae cells appeared to die off. This was confirmed by the fact that no colony growth occurred upon subculture, nor were a ny heat resistant spores detected. This was true also in the presence of fat body cells from T. ni, except that a limited number of spores ( 4,000/ml) were detected in the form of colony-forming units (CFU) on p lates following heating to 80 degrees C for 20 minutes. In a parallel study with a wild-type strain of Bacillus popilliae, vegetative cells grew only in TNM-FH medium in the presence of mid-gut BTI-Tn-MG and ov arian (Tn-368) cells of T. ni. No heat resistant spores, however, were detected in any of the cultures. When BTI-Tn-MG and Tn-368 cells were further challenged with four variant cultures of B. popilliae, vegeta tive growth and limited sporulation were achieved. The BTI-Tn-MG cell line in TNM-FH medium produced as many as 12,000 spores/ml after 21 da ys in culture.