18 
(1) Epidemiology studies have been performed on over 150 coli 
strains isolated from human and animal stools. These have shown 
that the majority (80%) of the strains made detectable levels of 
Shiga-like toxin. Moreover, four of four substrains of the well- 
characterized bacterium Eh_ coli K-12 were shown to make lew levels 
of the toxin. Thus, cloning of the Shiga-like toxin gene from 
clinical isolates of coli will not involve the introduction of 
a "foreign" toxin into the organism. 
(2) Production of low levels of Shiga-like toxin was observed in 2 of 
15 normal human gut flora Eh_ coli strains from asymptomatic infants. 
(3) Strains of Vibrio cholerae and Vibrio parahaemolyticus were tested 
and shewn to produce the Shiga-like toxin. Thus, the gene(s) for 
Shiga-like toxin are present in naturally occurring isolates of the 
family Vibrionaceae and not restricted to the Enterobacter iaceae . 
In volunteer studies, some of the strains of \A_ cholerae that 
produce Shiga-like toxin did not cause disease. Therefore, the 
ability to produce Shiga-like toxin is not equivalent with virulence 
in humans challenged by the oral route. 
(4) Phages frem two clinical isolates of coli have been shown to 
control high-level production of Shiga-like toxin in Eh_ coli K-12 
host strains by phage conversion. Thus, either the structural 
gene(s) for the Shiga-like toxin or regulatory genes that control 
high-level production of the toxin are present on wild-type phages 
from clinical isolates of coli . In this sense, "cloning" of 
genes that affect production of Shiga-like toxin onto phage gencmes 
has already occurred in nature. 
In addition, the U.S. Cholera Panel of the National Institute of Allergy 
and Infectious Diseases (NIAID) 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 frem candidate cholerae and ETBC 
vaccine strains. Shiga toxin is new found in many nonpathogen ic 
E. ooli, including the cannon vector host, E^ coli K-12." 
The request for reconsideration was published in the January 5, 1984, 
Federal Register (49 FR 696). During the comment period, a letter was 
received frem Dr. Werner Arber, the chairman of the Swiss Carmission for 
Experimental Genetics, which is in charge of questions related to research 
involving recombinant DNA molecules. Dr. Arber wrote that a Swiss ad hoc 
committee of experts requested by the Commission for Experimental Genetics 
had reviewed pr opposed research involving cloning of the Shiga toxin gene 
in an E. coli host-vector system. Dr. Arber wrote this committee had 
concluded that: 
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