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Applied and Environmental Microbiology, September 1998, p. 3544-3545, Vol. 64, No. 9
0099-2240/98/$00.00+0
LETTERS TO THE EDITOR
Viability of Cryptosporidium parvum Oocysts:
Assessment by the Dye Permeability Assay
 |
LETTER |
The paper by Jenkins et al. (6) provides interesting
data on Cryptosporidium parvum oocyst permeability and
survival. The vital dye assay (4) relies upon oocyst
permeability and exhibits limitations, especially in assessment of
disinfectant efficacy (3). However, it is rewarding to note
that it is considered to have utility and can provide pertinent data.
Although we agree with many points addressed by Jenkins et al.
(6), we feel we should make the following three comments which, in part, reiterate conclusions from our previous work (4, 5, 7, 8). These points were perhaps overlooked by Jenkins et al.
(6).
(i) In our paper describing the vital dye assay (4),
impermeable oocysts (DAPI
PI) are not described as dead (not
viable), as suggested by Jenkins et al. (6). Rather, we
concluded (4, 8) that a further "trigger" was required
to increase oocyst permeability and thus excystation capability (see
Table 1 in reference 4). Intriguingly, in an earlier
paper by this research group (1) it appears that they did
appreciate that such oocysts (DAPI PI) were capable of becoming
viable. In this paper (1) they write, with reference to our
original paper (4), that "PI-negative, DAPI-negative
oocysts are also considered viable, but with the caveat that some
treatment, e.g., acidification, is required before excystation will
occur."
We consider oocyst permeability to be a dynamic situation (up until
death or excystation), which is reduced by incubation with saliva
(8) and storage in cow feces (7) and increased by
acidic incubation (8). Rather than simply describing oocysts as alive (viable) and dead (nonviable), our data revealed an additional oocyst state in which the oocysts were impermeable to both dyes and
became viable (able to excyst under defined conditions) after a further
trigger. Such "quiescent" oocysts could enter either stage,
becoming viable or nonviable depending upon environmental factors.
(ii) During correlation of in vitro excystation and the dye
permeability assay results, we ensured that any pretreatment was performed on both oocysts to be excysted and oocysts to be subjected to
the assay (4, 8). Jenkins et al. (6) used our
recommended pretreatment only for oocysts to be excysted and apparently
not for oocysts to be subjected to the dye assay; we are therefore not
surprised that their results differ from ours. Although we demonstrated
a strong positive correlation between in vitro excystation and DAPI+
PI oocysts, Jenkins et al. (6) did not observe this correlation. Indeed, they report a correlation between DAPI PI oocysts and excystation. If the pretreatment used for in vitro excystation by Jenkins et al. had also been used for the dye
permeability assay, we would predict that they would have observed a
correlation similar to that noted by us (4) and others
(2).
Furthermore, if both DAPI+ PI and DAPI PI oocysts are considered
to be viable, reductive arithmetic argument shows that PI alone is
being used as the indicator of viability. Here DAPI provides no
information on oocyst viability, although it may provide some
information on alteration of permeability of oocysts to this dye.
(iii) Addition of FITC-conjugated antibody to the assay to assist in
oocyst detection during survival studies is pertinent and has been used
by us (7) and others (1) as well as by Jenkins et
al. (6). However, due to the additional manipulative steps
required when DAPI and PI are used, it should be noted that, when
viability assessment is conducted simultaneously with detection, oocyst
recovery may be reduced. Simultaneous detection and viability assessment should therefore be treated cautiously for environmental monitoring in which detection of oocysts is of primary importance.
| |
REFERENCES |
| 1.
|
Anguish, L. J., and W. C. Ghiorse.
1997.
Computer-assisted laser scanning and video microscopy for analysis of Cryptosporidium parvum oocysts in soil, sediment, and feces.
Appl. Environ. Microbiol.
63:724-733[Abstract].
|
| 2.
|
Black, E. K.,
G. R. Finch,
R. Taghi-Kilani, and M. Belosevic.
1996.
Comparison of assays for Cryptosporidium parvum oocysts viability after chemical disinfection.
FEMS Microbiol. Lett.
135:187-189[Medline].
|
| 3.
|
Campbell, A. T.,
L. J. Robertson,
R. Anderson,
H. V. Smith, and J. F. W. Parker.
1997.
Viability of Cryptosporidium oocysts: assessment following disinfection with ozone, p. 97-102.
In
C. R. Fricker, J. L. Clancy, and P. A. Rochelle (ed.), International Symposium on Waterborne Cryptosporidium. Proceedings. American Water Works Association, Denver, Colo.
|
| 4.
|
Campbell, A. T.,
L. J. Robertson, and H. V. Smith.
1992.
Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes.
Appl. Environ. Microbiol.
58:3488-3493[Abstract/Free Full Text].
|
| 5.
|
Campbell, A. T.,
L. J. Robertson, and H. V. Smith.
1993.
Effects of preservatives on viability of Cryptosporidium parvum oocysts.
Appl. Environ. Microbiol.
59:4361-4362[Abstract/Free Full Text].
|
| 6.
|
Jenkins, M. B.,
L. J. Anguish,
D. D. Bowman,
M. J. Walker, and W. C. Ghiorse.
1997.
Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts.
Appl. Environ. Microbiol.
63:3844-3850[Abstract].
|
| 7.
|
Robertson, L. J.,
A. T. Campbell, and H. V. Smith.
1992.
Survival of Cryptosporidium parvum oocysts under various environmental pressures.
Appl. Environ. Microbiol.
58:3494-3500[Abstract/Free Full Text].
|
| 8.
|
Robertson, L. J.,
A. T. Campbell, and H. V. Smith.
1993.
In vitro excystation of Cryptosporidium parvum.
Parasitology
106:13-19.
|
| | | | |
L. J. Robertson
Seksjon for parasittologi
Norges veterinærhøgskole
N-0033 Oslo
Norway
|
| | | | |
A. T. Campbell
Dynal A. S.
Skøyen
0212 Oslo
Norway
|
| | | | |
H. V. Smith
SPDL
Department of Bacteriology
Stobhill NHS Trust
Glasgow G21 3UW
United Kingdom
|
| |
AUTHORS' REPLY |
In their Letter to the Editor, Robertson, Campbell, and Smith raise the
issue that we (5) may have overlooked some aspects of their
work on Cryptosporidium parvum oocyst viability (3, 4,
7, 8). First, they point out that we suggested (1, 5)
that they described impermeable oocysts (i.e., intact oocysts that do
not take up the two fluorogenic dyes DAPI and or PI [DAPI PI]) as
not viable. We were, in fact, confused by some of the terminology used
in their papers (3, 4). Our initial understanding from those
papers was that the DAPI PI oocysts were considered to be
"viable" only "after further trigger" (3) or
"viable after further stimulus" (4), the trigger or
stimulus being acid treatment. These locutions imply that the
impermeable oocysts were thought to be something other than viable
(i.e., not viable) before the "further trigger" or "further
stimulus" was applied. Furthermore, viability was defined in their
work as "the ability of an oocyst to excyst in a described
excystation protocol" (4). "Viable" (from the Old
French vie, life, derived from the Latin vita) is
generally defined as "capable of reproducing under appropriate conditions" (9). To say that something is not viable
tacitly implies that it is dead and not capable of reproducing, even if the conditions are appropriate. From these statements we concluded originally that Robertson, Campbell, and Smith thought that oocysts were not viable if they were impermeable to DAPI and PI and did not
excyst. We now recognize that that was not their interpretation. We are
grateful for their efforts to clarify their earlier work.
Our papers (1, 5) offer a further attempt to clarify the
viability issue. Our experiments (5) comparing oocyst dye permeability with in vitro excystation and mouse infectivity
demonstrate that, under the conditions of the assay, impermeable
(DAPI PI) oocysts are, in fact, excystable and infective and
therefore represent potentially infective oocysts.
In the Letter to the Editor, such phrases as "capable of becoming
viable" in reference to DAPI PI oocysts are still somewhat confusing, especially with regard to the viability status of the impermeable DAPI PI oocysts. We think that a term such as
"quiescent" or "dormant" would better describe these
impermeable, but still potentially infective, oocysts.
It is important to note that, in our work, our goal was to assess
oocyst survival in naturally contaminated calf feces, soil, and
sediment. In our experimental design, we explicitly treated the dye
permeability assay, which gives an indirect assessment of viability or
infectivity, and the in vitro excystation assay, which directly tests
excystability as two independent assays. Although our results did not
indicate a correlation between the DAPI+ PI oocysts and the
excystable oocysts in the in vitro excystation assay, they did
demonstrate a correlation between the potential for viability as
determined by the sum of DAPI PI and DAPI+ PI oocysts in the dye
permeability assay and the excystable oocysts in the in vitro
excystation assay. Therefore, our results are in concurrence with those
of Campbell et al. (3) and Black et al. (2). By
using the dye permeability assay (5), we demonstrated that a
majority of oocysts in contaminated feces as well as in fresh
sucrose-purified oocyst suspensions were impermeable to DAPI and PI
(i.e., potentially infective) and that, over time, especially at
elevated temperatures, there was an increase in the number of the more
permeable, but still potentially infective, DAPI+ PI oocysts.
Eventually, the latter were replaced by the very permeable DAPI+ PI+
(i.e., dead) oocysts. We consider this time-dependent change from
impermeable (DAPI PI) to more permeable (DAPI+ PI) to very
permeable (DAPI+ PI+) oocysts to indicate a sequence of events leading
to the ultimate inactivation of oocysts. The time- and
temperature-dependent increases in oocyst wall permeability show that
oocysts can become increasingly susceptible to detrimental environmental factors such as temperature (5) and
NH3 (6), and these factors can increase oocyst
inactivation rates, as indicated by increased permeability under
certain manure storage conditions. Therefore, for our purposes, the
inclusion of DAPI in the dye-permeability assay yields significantly
more information on the changes in oocysts that occur as they become
inactivated.
The third issue raised by Robertson, Campbell, and Smith concerns the
simultaneous use of the immunofluorescence assay and the dye
permeability assay. We found this combination of assays to be
particularly useful for identifying potentially infective and dead
oocysts in semiopaque environmental matrices such as soil, sediment,
and feces (1). It should be remembered that we were
investigating oocyst survival under various environmental conditions in
which oocyst identification was difficult because of the presence of
the opaque material in the samples. We certainly agree with Robertson
et al. that, for routine environmental monitoring purposes, such
simultaneous detection and viability assessment should be approached
with caution.
| |
REFERENCES |
| 1.
|
Anguish, L. J., and W. C. Ghiorse.
1997.
Computer-assisted laser scanning and video microscopy for analysis of Cryptosporidium parvum oocysts in soil, sediment, and feces.
Appl. Environ. Microbiol.
63:724-733.
|
| 2.
|
Black, E. K.,
G. R. Finch,
R. Taghi-Kilani, and M. Belosevic.
1996.
Comparison of assays for Cryptosporidium parvum oocysts viability after chemical disinfection.
FEMS Microbiol. Lett.
135:187-189.
|
| 3.
|
Campbell, A. T.,
L. J. Robertson, and H. V. Smith.
1992.
Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes.
Appl. Environ. Microbiol.
58:3488-3493.
|
| 4.
|
Campbell, A. T.,
L. J. Robertson, and H. V. Smith.
1993.
Effects of preservatives on viability of Cryptosporidium parvum oocysts.
Appl. Environ. Microbiol.
59:4361-4362.
|
| 5.
|
Jenkins, M. B.,
L. J. Anguish,
D. D. Bowman,
M. J. Walker, and W. C. Ghiorse.
1997.
Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts.
Appl. Environ. Microbiol.
63:3844-3850.
|
| 6.
|
Jenkins, M. B.,
D. D. Bowman, and W. C. Ghiorse.
1998.
Inactivation of Cryptosporidium parvum oocysts by ammonia.
Appl. Environ. Microbiol.
64:784-788[Abstract/Free Full Text].
|
| 7.
|
Robertson, L. J.,
A. T. Campbell, and H. V. Smith.
1992.
Survival of Cryptosporidium parvum oocysts under various environmental pressures.
Appl. Environ. Microbiol.
58:3494-3500.
|
| 8.
|
Robertson, L. J.,
A. T. Campbell, and H. V. Smith.
1993.
In vitro excystation of Cryptosporidium parvum.
Parasitology
106:13-19.
|
| 9.
|
Singleton, P., and D. Sainsbury.
1993.
Dictionary of microbiology and molecular biology, 2nd ed.
John Wiley & Sons, Chichester, United Kingdom.
|
| | | | |
Michael B. Jenkins
William C. Ghiorse
Section of Microbiology
Division of Biological Sciences
Cornell University
Ithaca, New York 14853
|
| | | | |
Lynne J. Anguish
Baker Institute
College of Veterinary Medicine
Cornell University
Ithaca, New York 14853
|
| | | | |
Dwight D. Bowman
Department of Microbiology and Immunology
College of Veterinary Medicine
Cornell University
Ithaca, New York 14853
|
| | | | |
Mark J. Walker
Environmental and Resource Sciences
University of Nevada
Reno, Nevada 89557
|
Applied and Environmental Microbiology, September 1998, p. 3544-3545, Vol. 64, No. 9
0099-2240/98/$00.00+0
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