To: parasite at net.bio.net
From: max1603 at aol.com (Max1603)
Subject: Cryptosporidium
Date: 22 Mar 1995 20:28:34 -0500
Reply-to: max1603 at aol.com (Max1603)
I am looking for seminars, workshops etc. on Cryptosporidium testing in
drinking
water. I haven't found any good references either, a couple of journal
articles, but nothing relevant for what I need.
I work for a bottled water company as a Microbiologist and I need
information on
the effects of ozone, UV light, filtration etc. on the oocysts, as well as
information on filtration. I have limited experience in parasitology and
would like to obtain further training---any ideas??
C.Herold
Phoenix, AZ
--------------------------------------------------------------------------
We have organised 3 international workshops (Sydney, Seattle and
Nottingham) called "Water Microbiology for the 21st Century" which have
provided lectures and hands on training for new water microbiology
methods including Cryptosporidium testing. We plan to run another
workshop in the States later this year. I'll let you know date and venue.
We have developed a flow cytometric detection method for Crypto that
overcomes many of the difficulties associated with detecting low
numbers of oocysts in water. We are currently developing reagent kits for
use with this method (should be available later this year).
We are able to licence our technologies to laboratories and to provide
training to set them up with testing methods. However, unless you plan to
analyse a lot of samples I would not bother. The easy option is to send
samples to a commercial laboratory for analysis. Analysis is not cheap,
the going rate is over $US300. We would be more than happy to analyse
samples for you. Alternatively, contact Chuck Gerba at the University of
Arizona (a bit closer to home), fax- 602 621 6366.
We run a Cryptosporidium discussion group - crypto at rna.bio.mq.edu.au. To
subscribe send ENROL CRYPTO to listserv at rna.bio.mq.edu.au.
Here's a few references on methodologies:
1954. Lechevallier,MW; Norton,WD; Siegel,JE;
Abbaszadegan,M (1995): Evaluation of the immunofluorescence
procedure for detection of giardia cysts and cryptosporidium
oocysts in water. Applied. &. Environmental. Microbiology.
61(2, Feb), 690-697.
(Reprint available from: Lechevallier MW AMER WATER WORKS
SERV CO INC 1025 LAUREL OAK RD VOORHEES, NJ 08043 USA)
<The accurate determination of the presence of Giardia cysts
and Cryptosporidium oocysts in surface waters requires a
reliable method for the detection and enumeration of these
pathogenic organisms. Published methods have usually
reported recovery efficiencies of less than 50% for both
cysts and oocysts. Typically, the losses are greater for
Cryptosporidium oocysts than they are for Giardia cysts. The
purpose of this study was to examine procedures used for
sample collection, elution, concentration, and clarification
to determine when losses of cysts and oocysts occurred
during processing. The results showed that major losses of
cysts and oocysts occurred during centrifugation and
clarification. Depending on the centrifugation force, oocyst
losses of as high as 30% occurred for each centrifugation
step. A 1.15-specific-gravity Percoll-sucrose gradient was
needed to optimize recovery of oocysts from natural water
samples. Minor improvements in the procedure could be
accomplished by selecting a filter other than the
recommended 1-mu m-pore-size (nominal-porosity)
polypropylene filter. [References: 9]>
198. Rose,JB; Landeen,LK; Riley,KR; Gerba,CP (1989):
Evaluation of immunofluorescence techniques for detection of
Cryptosporidium oocysts and Giardia cysts from environmental
samples. Appl. Environ. Microbiol. 55(12, Dec), 3189-3196.
<Cryptosporidium and Giardia species are enteric protozoa
which cause waterborne disease. The detection of these
organisms in water relies on the detection of the oocyst and
cyst forms or stages. Monoclonal and polyclonal antibodies
were compared for their abilities to react with Giardia
cysts and Cryptosporidium oocysts after storage in water,
3.7% formaldehyde, and 2.5% potassium dichromate, upon
exposure to bleach, and in environmental samples. Three
monoclonal antibodies to Cryptosporidium parvum were
evaluated. Each test resulted in an equivalent detection of
the oocysts after storage, after exposure to bleach, and in
environmental samples. Oocyst levels declined slightly after
20 to 22 weeks of storage in water, and oocyst fluorescence
and morphology were dull and atypical. Oocyst counts
decreased after exposure to 2,500 mg of sodium hypochlorite
per liter, and fluorescence and phase-contrast counts were
similar. Sediment due to algae and clays found in
environmental samples interfered with the detection of
oocysts on membrane filters. Two monoclonal antibodies and a
polyclonal antibody directed against Giardia lamblia cysts
were evaluated. From the same seeded preparations,
significantly greater counts were obtained with the
polyclonal antibody. Of the two monoclonal antibodies, one
resulted in significantly lower cyst counts. In preliminary
studies, the differences between antibodies were not
apparent when used on the environmental wastewater samples.
After 20 to 22 weeks in water, cyst levels declined
significantly by 67%. Cysts were not detected with
monoclonal antibodies after exposure to approximately 5,000
mg of sodium hypochlorite per liter.>
108. Vesey,G; Slade,JS; Byrne,M; Shepherd,K; Fricker,CR
(1993): A new method for the concentration of
Cryptosporidium oocysts from water. J. Appl. Bacteriol.
75(1, Jul), 82-86.
<A novel method for the concentration of Cryptosporidium
oocysts from water has been developed, based upon the
precipitation of calcium carbonate. A 10 l water sample is
treated by adding solutions of calcium chloride and sodium
bicarbonate and raising the pH value to 10 with sodium
hydroxide. Crystals of calcium carbonate form and enmesh
particles in the Cryptosporidium oocyst size range. The
crystals are allowed to settle, the supernatant fluid is
discarded and the calcium carbonate precipitate dissolved in
sulphamic acid. The sample can be concentrated further by
centrifugation. Recoveries of oocysts from seeded samples of
deionized, tap and river water were in excess of 68%.>
646. Vesey,G; Hutton,P; Champion,A; Ashbolt,N; Williams,KL;
Warton,A; Veal,D (1994): Application of flow cytometric
methods for the routine detection of cryptosporidium and
giardia in water. Cytometry 16(1, 1 May), 1-6.
Macquarie Univ, Sch Biol Sci, Sydney, Nsw 2109, Australia)
<Cryptosporidium and Giardia are common causes of waterborne
disease. The currently used methods of detecting these
organisms in water rely on filtration capture,
immunofluorescence labelling, and epifluorescence
microscopy; These methods are inefficient, labour intensive,
and require a highly skilled microscopist. We describe an
alternative technique using flocculation concentration,
followed by flow cytometry with fluorescence activated cell
sorting. Environmental samples were analysed, and protozoan-
like particles were sorted and collected before confirmation
with epifluorescence microscopy. The technique was found to
be significantly more sensitive and considerably faster than
the conventional methods. (C) 1994 Wiley-Liss, Inc.>
_______________________________________________
Graham Vesey
Australian Environmental Flow Cytometry Group
School of Biological Sciences, .-.--:_:\
Macquarie University, _/ \
Sydney, : AEFCG |
Australia NSW 2109. \_ /
Tel- 612 850 8150 '-''''\__/
Fax- 612 850 8174 V
_______________________________________________
