United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park NC 27711
Research and Development
EPA/600/S1 -87/011 Jan. 1988
4>EPA Project Summary
Cytochemical Methods for
Assessing Giardia Cysts
Viability
Donald G. Lindmark
Because of the high incidence, symp-
tomology, and waterborne dissemina-
tion of Giardia, improved methods for
determination of viability of cysts
detected after isolation from water
systems is necessary to assess poten-
tial danger to human health.
The viability of Giardia cysts has been
assessed by eosin exclusion, in vitro
excystation, and animal infectivity.
While each of these methods has its
own value, none are both rapid and
reliable.
The objective of this study was to
develop a reliable, rapid, microscopi-
cally read method for determining the
viability of Giardia cysts which is
comparable to excystation and would
provide a more practical method of
estimating cyst viability.
Though methods dependent on cyst
metabolism such as the activity of
pyruvate: ferredoxin oxidoreductase
(PFOR) and cell respiration gave prom-
ising results with trophozoites, they
proved unreliable with cysts.
Hence, exclusion of the fluorescent
dye, 3-(Dansylamido)-phenyl boronic
acid (FluoroBora I) from Giardia muris
cysts was compared with excystation
as a measure of cyst viability. The effect
of 22 different chemicals on excysta-
tion was determined and compared
with the exclusion of the fluorescent
dye FluoroBora I (FBI) under identical
conditions. These data indicate that the
dye exclusion method has potential for
use as an alternative method to excys-
tation as a measure of cyst viability.
Preliminary evidence suggests that
the method, with some modifications,
has the potential for use in the deter-
mination of the viability of Giardia
lamblia cysts.
This Project Summary was devel-
oped by EPA's Health Effects Research
Laboratory, Research Triangle Park,
NC. to announce key findings of the
research project that is fully docu-
mented in a separate report of the same
title (see Project Report ordering
information at back).
Introduction and Project Goals
Giardia lamblia is the most common
human intestinal protozoan parasite
reported in the United States and Eng-
land. The organism exists in two mor-
phologically distinct forms, the tropho-
zoite and the cyst. The infective cyst form
is transmitted via the fecal-oral route.
The high incidence, symptomology and
waterborne dissemination of Giardia has
resulted in improved methods for cyst
detection in water systems. However a
rapid, simple, reliable method for deter-
mining the viability of cysts, detected in
water systems, and exposed to water
treatment procedures is needed.
The viability of Giardia cysts has been
assessed by eosin exclusion, in vitro
excystation, and animal infectivity. While
each of these methods has its own value,
none are both rapid and reliable.
Although eosin exclusion is a rapid
method, it indicates higher cyst viability
than can be demonstrated by excystation
and shows little correlation with excys-
tation. Excystation is the most frequently
used method for determining cysts
viability. It is tedious (2-3 h), and
subjective. It cannot be used for deter-
mining the viability of individual or small
numbers of cysts. The cost, time and
large numbers of cysts required to
perform animal infectivity studies make
the method impractical for routine use.
The objective of this study was to
develop a reliable, rapid, microscopically
read method for determining the viability
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of Giardia cysts which when compared
to excystation would provide a more
practical method of estimating cyst
viability. The initial studies into develop-
ing an assay for viable cysts were
dependent on various metabolic param-
eters such as cyst respiration and specific
enzyme activities. The research was
based on some preliminary data on cyst
metabolism obtained in the author's
laboratory. Cysts of G. lamblia respire m
the presence of Qz without added exo-
genous substrate at approximately 1/10
the rate of trophozoites (8 nmoles Oz
consumed mm"1 mg protein"1 at 37°C).
Cysts after activation at low pH and
subsequent placement in nutrient
medium increase their respiration five
fold. The enzyme, pyruvate: ferredoxin
oxidoreductase (PFOR)—a major enzyme
in cell energy metabolism and an enzyme
found only in anaerobes such as Giar-
dia—is active in cyst homogenates.
These data, though preliminary, were
used in part in our attempts to develop
a metabolic dependent direct micro-
scopic measure of cyst viability.
Initially, attempts were made to assess
viability with indicators of metabolic
activity. These metabolic studies using
PFOR and cell respiration were equivocal
in that they were not effective with cysts,
Triton treated cysts or induced cysts for
measuring cyst viability. Unlike respira-
tion, PFOR activity as measured by
accumulation of nitroblue tetrazolium
(appearance of a blue color throughout
the cell) could be used for determining
G. lamblia trophozoite viability. Neither
method proved useful for determining
cyst viability possibly because of cyst
permeability.
In 1982 investigators published a
method called boronic acid-dependent
phase transfer, or "boradeption." In this
method, specific fluorescent water-
insoluble boronates are excluded by non-
viable cells in vitro. When this technique
was attempted with Giardia, the results
using FBI with Giardia lamblia trophoz-
oites, were similar to those found by
previous investigators using CHO cells,
i.e , non-viable cells excluded the dye. In
contrast, viable Giardia cysts excluded
the dye. This exclusion method using G.
muris cysts, when compared with excys-
tation, showed a high degree of asso-
ciation and a higher degree of reproduc-
ibility and precision as compared to
excystation. Preliminary evidence sug-
gests that the method, with some mod-
ifications, has the potential for use in the
determination of the viability of Giardia
lamblia cysts.
The goals of the project were the
following:
1. to develop a simple and quick
microscopically read method for
determining Giardia cyst viability,
useful to investigators with min-
imal training.
2. to correlate the new method with
the currently accepted method of
determining cyst viability,
excystation.
3. to use the new method with G.
lamblia as well as G. muris cysts.
4. to establish the reliability of the
new method on low numbers of
cysts (<50).
5. to assess the compatibility of the
new method with immunological
detection methods for Giardia cysts
in water samples.
Conclusions and
Recommendations
a. Enzymatic and metabolic methods
for determining viability of Giardia
cysts proved unfeasible at the micro-
scopic level.
b. Exclusion of the fluorescent dye 3-
(Dansylamido)-phenyl boronic acid
(FluoroBora I, FBI) from Giardia
muris cysts shows good correlation
(at cyst viability > 60%) with excys-
tation as a method for determining
cyst viability when viability of cysts
was measured after contact with
different disinfectants under identi-
cal conditions.
c. The FBI method is simple to perform,
rapid (5 mm) and can be microscop-
ically read.
d. For untreated cysts, the FBI method
shows a higher degree of precision
between investigators than excysta-
tion at viability levels above 60%.
e. The FBI method is less subjective
than excystation since (1) it relies on
the observation of fluorescence or
non-fluorescence as a measure of
viability, whereas excystation
requires differentiation between
several stages of excysting orga-
nisms and (2) the excystation proce- I
dure is long and tedious (2-3 h)
requiring control of a number of
variables (temperature, pH, reducing
conditions, etc.) not encountered in
the FBI method.
f. The FBI method can be used to
assess the efficacy of disinfectants
in a rapid manner even if these
compounds cause clumping. Clump-
ing makes the excystation method
not possible because of the difficulty
in counting.
g. The FBI method has not yet been
shown to be compatible with an
immunofluorescence method for
detecting Giardia cysts in water
samples.
h. FBI estimates of viability are consist-
ently higher than by excystation.
i. FBI can be used to estimate viability
of G. lamblia cysts if the FBI method
is compared with motility of trophoz-
oites inside the cyst during excys-
tation in contrast to excystation. This
may point to the suggestion that low
excystation rates of G. lamblia may
be due to the excystation environ- i
ment.
j. Based on the available data, the FBI
method has the potential for use in
the rapid assessment of Giardia cyst
viability in the laboratory. The FBI
method can be used for a quick
assessment of disinfectants and
chemotherapeutic agents in the
laboratory.
k. Recommendations for further work
include:
1. comparison of optimized G.
lamblia excystation method.
2. comparison of excystation with
FBI at viability 0-100 (esp <
50%).
3. if no optimal G. lamblia excys-
tation, in depth comparison of
motility within the cyst and FBI
under conditions of chemical
treatment.
4. improve compatibility of FBI with
existing detection methods (con-
ventional and immunofluores-
cence). A
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Donald G. Lindmark is with Cleveland State University, Cleveland, OH 44102.
Judith F. Sauch is the EPA Project Officer (see below).
The complete report, entitled "Cytochemical Methods for Assessing Giardia
Cysts Viability." (Order No. PB 88-124 748/AS; Cost: $14.95; subject to
change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 452GO
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