8 
containment for the proposed studies of cloned genes for Shiga-like toxin 
in laboratory strains of EL coll lacking ability to colonize or invade the 
intestinal mucosa in man. 
The U.S. Cholera Panel of the National Institute of Allergy and Infectious 
Diseases (NIAID) in a meeting on October 16, 1983, reviewed and critiqued 
the Microbiology and Infectious Diseases Program dealing with cholera and 
E. coli (ETEC) vaccine development. It was the consensus of the panel 
that it be recommended that NIH reconsider the ban: 
"...on shiga toxin cloning experiments in containment facilities other 
than P4. This strict requirement will prevent most laboratories from 
deleting the Shiga gene from candidate cholera and ETEC vaccine 
strains . Shiga toxin is new found in many nonpathogenic EL_ coli , 
including the common vector host, EL_ coli K-12." 
At the February 6, 1984, RAC meeting, Dr. Levine discussed the proposal. 
He said the genus Shigella comprises bacteria that cause diarrheal disease 
and dysentery. These organisms were first identified at the end of the 
19th century when they were causing epidemic disease. Kioshi Shiga identi- 
fied the organisms through their agglutination by serum from convalescent 
patients. The genus was named after Er. Shiga. 
In 1903, Conradi demonstrated that a cell-free lysate of Shigella when 
inoculated into rabbits resulted in rear limb paralysis. This led to the 
hypothesis that Shigella produced a neurotoxin. Over the ensuing 50 years, 
many other Shigella serotypes were recognized. In about 1960, Vicari and 
Olitsky demonstrated that partially purified toxin was cytotoxic to cells 
in tissue culture. In 1968, Keusch showed that these organisms cause 
secretion of fluid in isolated rabbit ileal loops. 
Shiga's bacillus was thus shewn to be producing a neurotoxin, a cy to toxin, 
and an enterotoxin. Over the next several years, evidence began to accumulate 
demonstrating that a single toxin was responsible for all three biological 
activities. Towards the late 1970s, several groups purified the toxin, and 
it was shewn that many Shigella serotypes produce the toxin. Dr. O'Brien 
contributed to this research . 
Dr. Levine said he would like to digress and recount to the RAC the genesis 
of Appendix F, Con toinment Conditions for Cloning of Genes Coding for the 
Biosynthesis of Molecules Toxic for Vertebrates , br. Levine said he was a 
member of the Working Group on Toxins that met in the spring of 1981 to 
develop guidelines for the cloning of toxin genes. In the course of the 
discussions, two positions developed regarding classification. One group 
felt that guidelines for the cloning of toxin genes should be based solely 
on the pharmacological effect of the toxin. The other group felt that 
other additional factors had to be considered. Nonetheless, both groups 
agreed that genes for toxins such as botulinum toxin and tetanus toxin 
should be cloned only under the most stringent containment conditions. 
These toxins are extraordinarily powerful and a few molecules, so few that 
[253] 
