Waddell, Glenn H.

The significance of the transmission of animal viruses in sewage
polluted fresh and marine waters becomes an increasingly important
factor as more and more evidence is presented to support the fact
that in the case of many viral types one virus particle constitutes
an infective dose. Therefore it is very important that a method of
determining the extent of viral pollution in sewage polluted bodies
of marine and fresh water be found.
The longevity of f2 bacteriophage in both marine and fresh water
sewage polluted samples was determined, and shown to be 3 to 4 days
for marine water and 8 days for fresh water. It was also shown
that the culture medium in which the f2 phage was produced, and the
larger macromolecular polymers commonly found associated with sewage
had a favorable affect in increasing the longevity of f2 phage.
It was shown that f2 bacteriophage could be used to follow the
initial dispersion of the sewage boil arising from an ocean outfall
sewerage system. From these results it was concluded that f2
bacteriophage could be used for more detailed studies concerned
with the transmission of viruses in sewage polluted waters.

Marine bacteria are usually described as those bacteria
which grow optimally in sea water. Their complex requirements
for specific ions (sodium, potassium and magnesium)
have been used to differentiate marine from terrigenous
bacteria. These requirements, however, vary at the gene level
through mutation. Both spontaneous and induced (ultraviolet
light) mutations to loss of the ion requirement were observed.
Spontaneous frequencies were low (below 10^-6); induced frequencies,
high (10^-1 to 10^-8). Back mutations were noticed.
A stepwise mechanism was postulated for the mechanism of the
mutation involved in the appearance of ion independent
bacteria in a population of cells. Genetic analysis of the mechanism by which variations
in ion requirements occurred included determination of base
ratios and of transformation frequencies. The base ratios
of mutant and parent bacteria were similar enough and the
transformation frequencies high enough (10^-3 to 10^-5) to
show a close relationship between the two types of bacteria. This similarity indicated that, although the genome of
marine bacteria, which clearly controls the requirement
for specific ions from the values of mutation frequencies,
is altered by the action of ultraviolet light, the change
was not observable by the methods used. The change could
have been induced on the gene level to effect the synthesis
of structural or functional proteins (enzymes).

The effect of adenovirus and vaccinia infection on KB cells was studied spectrophotometrically by comparing the DNA, RNA, and protein concentrations of infected cells with normal cells. The concentration of these macromolecules in normal cells was established by assaying a large number of KB cultures at different stages of growth. Data were plotted against cell count and time. DNA, RNA, and protein concentrations were found to be linear functions of cell count within a range of 5-25 x 10^5 cells/ml. Replicate cultures of KB cells were inoculated with vaccinia or adenovirus at 10 PFU/cell and harvested at 24 hr intervals for 4 days. The cells were counted microscopically at each interval and the concentrations of DNA, RNA, and protein were determined. These results were compared to concentrations and cell counts in uninfected cells and plotted against time. Cell division was inhibited by infection with either of the viruses. At 24 hr post infection (PI) the RNA concentration in infected cells was greater than that found in normal cells. A sharp decrease in RNA content occurred after 24 hr PI until the levels in infected cells were below those in uninfected cells. DNA and protein concentrations were much lower in the infected cells than in the normal cultures. DNA and protein levels increased in the infected cells at 72 hr PI while the DNA concentration in the uninfected cells remained constant. In vaccinia-infected cells the rise in protein synthesis occurred simultaneously with a rise in hemagglutination titer. A similar increase in protein concentration paralleled a rise in complement fixation titer in adenovirus-infected cells.

The disease of equine infectious anemia
(EIA), since the first records in 1847, has been one
of the most serious and deadly virus diseases of the
Equidae. The most difficult factor concerning this
disease was adequate diagnosis. The clinical diagnosis
has proven itself undependable and until 1966 with the
Immune-adherence test the laboratory diagnosis was
inconclusive. The indirect passive hemagglutination
test was used as a possible test for the disease due
to its high sensitivity. The indirect passive hemagglutination as
the diagnostic test for EIA was based on the use of
antibody as the titrating agent instead of the virus.
The antibody was removed from the sera of infected
horses. The seperation of the antibody was
accomplished by use of a DEAE-cellulose column and an
increasing salt gradient. The various proteins were
sepRrated and measured in a spectrophotometer. The
antibody portion was collected and concentrated by
polyethylene gycol. The more specific type of antibody
(19S) was used in all tests. The indirect passive hemagglutination test
was run on a total of 92 sera of which 58 were
negative and 34 sera were positive. The test proved
reliable in all the sera tested. In support of the
IPHA the IA test was run as a comparison.