Most other bacterial pathogens are much less 
fastidious and can be readily propagated on sim¬ 
ple bacteriological media. This property is one 
reason why the facultative bacterial pathogens 
are likely candidates for microbial control. 
Unfortunately, there are several obstacles to the 
commercial production and wide-spread use of 
many biological control agents in lieu of many of 
the well-known and hazardous chemical insecti¬ 
cides. For example, the wide-scale biological con¬ 
trol of the Japanese beetle, Popilla japonica 
Newman, the European chafer, Amphimallon 
majalis Razoumowsky and other susceptible sca- 
rabaeid grubs, major pests of lawns, pastures and 
other plant life in many parts of the world, could be 
considerably facilitated by the development of an 
in vitro industrial method for spore production of 6. 
popilliae, the causative agent of milky disease in 
these insects. Spore preparations of B. popilliae 
are produced commercially by collecting living 
larvae from infested soil, injecting each grub with 
the disease organism, incubating the larvae until 
the blood becomes filled with spores and then 
grinding and mixing them with an extending mate¬ 
rial such as talc. This procedure results in an 
expensive low-yielding product incapable of 
meeting the requirements for adequate mass con¬ 
trol of these serious insect pests throughout the 
world. 
Another organism. Bacillus sphaericus. a 
potentially powerful biological control agent for 
mosquitoes, is not readily grown in cheap culture 
media, but requires rather expensive nutrient 
additives for optimum production. Under our pres¬ 
ent knowledge and technology, this organism 
would not be commercially feasible for industry to 
develop at this time and hence its widespread use 
is hindered in lieu of such organophosphate 
insecticides as malathion. The pathogenicity of 
this organism for mosquito larvae seems to reside 
in the capacity for the production of an endotoxin 
specific for its host midgut (Davidson et ai., 1975). 
Recombinant DNA incorporation of the genes 
responsible for production of this toxin and trans¬ 
formation into B. thuringiensis, the biological lepi- 
dopteran insecticide being produced at a price 
competitive with chemical insecticides, could 
yield a broader-spectrum biological control agent 
affecting several orders of economically important 
insect pests and although broader based, this new 
strain would be selective, an attribute not con¬ 
tained in many chemical insecticides. Their devel¬ 
opment and use could result in a decreased use of 
hard chemical insecticides. In fact, a recently iso¬ 
lated strain of B. thuringiensis (var. israelensis) 
has demonstrated that the parasporal crystals (6- 
endotoxin) is toxic for such pest mosquito larvae 
as Aedes aegypti and Anopheles Stephens! but is 
not toxic to larvae of Lepidoptera (de Barjac, 
1978a), Combining of these pathogenic entities 
into one strain would give commercial industry a 
very valuable and fruitful product. 
2: General Safety Considerations Pertinent for 
Approval as HV-1 Hosts 
One of the first entomopathogenic bacteria to 
be used extensively in the field was the milky dis¬ 
ease organism. Bacillus popilliae, isolated and 
described by Dutky (1940) and has been used on 
the Eastern seaboard since the early 1940's to 
control the Japanese beetle, Popillia japonica. The 
sole producers of this bacterial product is the Fair¬ 
fax Biological Laboratory at Clinton Corners, N.Y. 
and Reuter Laboratories, Inc. at Flaymarket, 
Virginia. The materials produced by Fairfax Bio¬ 
logical Laboratory is manufactured under the 
trade names of “Japanese beetle Doom" (USDA 
Reg. No. 403-9) and “Japidemic” (USDA Reg. No. 
403-14). Reuter Laboratories manufacture their 
product under the trade name of “Milky Spore” 
(ERA Reg. No. 36488-1). The results of published 
and unpublished tests demonstrated that the milky 
disease bacteria are innocuous to turf, inverte¬ 
brates and vertebrates, including man (Fleimpel, 
1971; Bailey, 1971; Fleimpel and FIrubant, 1973; 
Bulla et a!., 1975; Anonymous, 1977; Ignoffo, 
1973). The protocols for these tests were infor¬ 
mally discussed with the Environmental Protec¬ 
tion Agency and the United States Department of 
Agriculture personnel, and the tests as reported 
were conducted by qualified investigators in 
accordance with protocols found acceptable to 
and recommended by officials of these agencies. 
The following tests were conducted to obtain 
information on the safety of 8 . popilliaeXo man and 
other mammals: (1) Safety evaluation by repeated 
oral administration of 8. popilliae to rats and mon¬ 
keys; (2) Clinical examination of production per¬ 
sonnel exposed to 8. popilliae: (3) Medical and 
serological examinations of humans engaged in 
production of 8. popilliae: (4) Miorobiological 
examination of commercial preparations of 8. 
popilliae: (5) Published and unpublished studies 
demonstrating its specificity to beetle larvae and 
safety to vertebrates, including humans; (6) Acute 
dermal toxicity in guinea pigs of a spore prepara¬ 
tion of 8. popilliae: (7) Acute eye irritation potential 
study in rabbits of a spore preparation of 8. popiP 
liae: and (8) inability of 8. popilliae to grow at mam¬ 
malian or avian body temperatures. The data 
supported the relief sought by the petition, namely, 
exemption from the requirement of a tolerance. 8. 
popilliae is so specific that its use, or even its mis¬ 
use, cannot result in any threat to the safety of 
humans, or other animals. It has been shown by 
the data obtained to be completely innocuous 
wherever used and at whatever levels. The milky 
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