24 
In response to Dr. King Holmes' stated concerns. Dr. Levine said the Working 
Group on Toxins, in devising its guidelines for Appendix F, had considered 
toxicity to primates to be of paramount importance ard more relevant than 
data generated with 40 guinea pigs or 40 mice. He emphasized that the 
primate data of Braham, Dack, and Riggs show that 20,000 monkey parenteral 
lethal doses will not cause adverse effect when administered by means of 
an intestinal pouch. 
Dr. Levine said he did not believe coli would present a problem by 
colonizing the skin or peritoneal areas; if coli is going to present a 
problem, it will present a problem in the gut as the numbers of E. coli in 
the gut are orders of magnitude greater than in other areas of the body. 
Dr. Levine said that EL_ coli strains vhich cause hemorrhagic colitis, such 
as 933 Eh_ coli 0157H7, are smooth coli strains capable of colonizing 
the human gut. These strains also have other virulence factors. Neverthe- 
less, these strains are not widespread pathogens. He argued that if strains 
such as 0157H7 which possess so many virulence characteristics are not 
widespread pathogens, it is inconceivable that a rough Eb_ coli strain, 
such as Eb_ coli K-12 vhich does not colonize or possess virulence factors, 
would become a widespread pathogen. 
Dr. Levine said the infinitesimal risks perceived to be associated with 
cloning the Shiga toxin gene in coli K-12 must be weighed against the 
actual benefits. He said research with the Shiga toxin gene is very 
important to the development of a cholera vaccine. He explained that 
live attenuated cholera vaccines vhich lack cholera toxin are a major step 
forward in controlling cholera by immunoprophylaxis. These vaccines, 
however, still cause a mild diarrhea in perhaps a third of the recipients. 
Thus, this vaccine is not sufficiently attenuated for public health use. 
Dr. Levine said the mild diarrhea may be explained in two ways: (1) the 
diarrhea is a response of the intestine to colonization by the live 
bacterial strain, or (2) other diarrhea-causing toxins may be produced by 
the live attenuated strain. Dr. O'Brien and her covorkers have shewn 
that some cholera vaccine strains do produce Shiga toxin. Shiga toxin 
thus may play a role in causing the mild diarrhea associated with the live 
attenuated cholera vaccine strains. This possibility must be tested by 
cloning the Shiga toxin gene and deleting it from the vaccine strains. 
Delaying this research will adversely affect public health. 
Dr. Holmes asked Dr. Levine to explain why if non-invasive cholerae 
vaccine strains may cause Shiga toxin induced diarrhea, would there not be 
similar concerns about an Eb_ coli strain producing Shiga toxin? Dr. Levine 
replied that to be a concern the bacterium must possess accessory virulence 
properties. These virulence properties need not include invasiveness; the 
organisms must, however, possess characteristics that maintain the bacteria 
in a special proximity to the intestinal cells. Dr. Levine said the 
V. cholerae vaccine strains colonize the small bowel in contrast to 
e 7 coli K-12 strains vhich will not colonize the small bcwel . 
[477] 
