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Thu Mar 23 01:46:43 EST 1995

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
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??

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 
<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  

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