AU3129800A - Methods for detection of cryptosporidium oocysts - Google Patents

Description

AUSTRALIA

Patents Act 1990 Macquarie Research Ltd, Australian Water Technologies Pty Ltd

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Methods for detection of Cryptosporidium oocysts The following statement is a full description of this invention including the best method of performing it known to us:- 2 Technical Field The present invention relates to antibodies reactive to recently excysted oocysts of Crvptosporidium and methods of detecting viable Cryptosporidium oocysts using the antibodies.

Background Art The protozoan parasite Cryptosporidium is amongst the most common pathogens responsible for diarrhoeal disease in humans (Current 1986).

Infection occurs when Cryptosporidium oocysts shed in the faeces of infected individuals are ingested by new hosts. Recently, several large outbreaks of cryptosporidiosis have occurred in which drinking water has been identified as the source of infection (Smith and Rose 1990, Badenoch 1990). Surveys have shown that many surface water supplies are contaminated with Cryptosporidium oocysts (LeChevallier et al 1991, Rose 1988).

S:i The detection of Cryptosporidium oocysts in water relies on the 15 concentration of particulate matter including oocysts from large volumes of water prior to staining with fluorescently labelled antibodies. Until recently, detection and identification of fluorescently labelled oocysts required examination of the sample using epifluorescence microscopy. The tedious and labour intensive nature of this detection method, in particular the amount of fluorescent microscopy required, limited the monitoring work which could be performed. The development of flow cytometric detection methods has alleviated many of these problems and enabled the routine monitoring of water for the presence of Cryptosporidium oocysts (Vesey et al 1994). A major limitation of this methodology, however, is the lack of oocyst viability measurements.

A further problem with the currently employed method for flow cytometric analysis of water samples for Cryptosporidium is the requirement of a flow cytometer that can sort particles for subsequent microscopical examination. Sorting flow cytometers are expensive and sophisticated instruments that require a highly skilled operator.

The flow cytometric method involves staining of samples with a fluorescently labelled monoclonal antibody specific to the surface of Cryptosporidium oocysts and then analysis with a sorter flow cytometer.

Particles with the fluorescence and light scatter characteristics of Cryptosporidium oocysts are sorted onto a microscope slide and examined manually using epifluorescence microscopy to confirm their identity as oocysts. This confirmation step is necessary because the cytometer is unable to distinguish oocysts from all other particles present in water samples. The particles that the cytometer can mistake as oocysts are autofluorescent particles such as algae or particles that bind the oocyst-specific antibody.

Analysis flow cytometers are available which are simple to operate and relatively inexpensive. These cytometers, however, are unable to perform sorting. To enable the detection of Cryptosporidium oocysts using one of these analysis cytometers the discrimination achieved by the cytometer must be improved so that non-oocyst particles are not mistaken as oocysts. The present inventors have shown previously that it is possible to detect a single specific microorganism in turbid water samples with an analysis cytometer (Vesey et al 1994B) if the microorganism is labelled with two different antibodies.

Korich et al (1993) reported on the use of a monoclonal antibody for 15 assessing Cryptosporidium oocyst viability. The antibody recognises an epitope on the inside of the oocyst and the antibody will only bind to an oocyst after being ruptured. The method described the use of the antibody to determine if oocysts have a ruptured wall. An oocyst with a ruptured wall, however, will not be viable and excysted oocysts are not the same as ruptured oocysts. The antibody therefore is not particularly suitable to detect viable oocysts.

The presence of non-viable Cryptosporidium oocysts in drinking water is of little significance to public health. If oocysts are viable, however, the risk to public health is enormous. There is therefore a real need to develop an effective method for determining the viability of Cryptosporidium oocysts in water. Furthermore, a method for assessing oocyst viability that is applicable to flow cytometry would enable the technique to be applied to the routine monitoring of water.

Disclosure of the Invention In a first aspect, the present invention consists in a method of detecting the presence of viable Cryptosporidium oocysts in a sample containing Cryptosporidium oocysts, the method comprising the steps of: a) treating the sample so as to cause any viable oocysts of Cryptosporidium in the sample to excyst; b) exposing the treated sample to an antibody that binds specifically to recently excysted Cryptosporidium oocysts such that the antibody binds to excysted Cryptosporidium oocysts in the sample; and c) detecting the presence of oocyst-bound antibody in the sample.

It will be appreciated that any treatment of the sample that causes oocysts to undergo excystation would be suitable. It is, however, presently preferred that the oocysts are caused to excyst by incubating the sample at about 37°C under acidic conditions, followed by incubating the sample under neutral to alkaline conditions at about 37 0 C. More preferably, the oocysts are caused to excyst by incubating the sample at 370C at pH 2 to 4 for 10 to minutes, followed by incubating the sample at 37°C at pH 7 to 9 for 10 to minutes. The sample can be washed between the steps to facilitate the removal of the buffers and replacement with fresh buffers at the required pH.

:Recently excysted Crvptosporidium oocysts are defined as oocysts that have excysted and contain antigens that are initially present upon being excysted but are not present on mature Cryptosporidium sporozoites. The antigens present and characteristic of recently excysted Cryptosporidium oocysts are usually present within less than about two hours, preferably less than about one hour, from treatment to cause the oocysts to excyst. The oocysts can be analysed within this period or preserved to maintain the antigens for subsequent analysis. Usually, the oocysts are exposed to an antibody within one hour or less from being excysted to ensure optimal binding. It has been found that the antigen or antigens present on recently excysted oocysts to which antibodies can be made do not remain intact over prolonged periods without some form of preservation of the excysted oocysts.

After otocysts have been caused to excyst, usually within less than about two hours, preferably one hour or less, the sample can be assayed for the presence of recently excysted oocysts or preserved for later testing. It will be appreciated that the period from being excysted to testing can be increased if the treated sample or oocysts are preserved in some manner. For example, freezing of the sample has been found to preserve the antigenicity of the excysted oocyst past the period after treatment.

The present inventors have found that short-lived antigens are present on recently excysted Cryptosporidium oocysts and that specific antibodies can be raised against these antigens. These antibodies can be used to detect viable Cryptosporidium oocysts in samples. It will be appreciated that by following the teaching of the present invention useful antibodies can be produced against recently excysted Cryptosporidium oocysts.

In a preferred embodiment of the first aspect of the present invention, the sample is analysed by flow cytometry or microscopy to detect the oocystbound antibody. The binding of the antibody to the recently excysted oocysts can be measured indirectly by further treating the sample with a fluorescently-labelled ligand that binds specifically to the antibody and measuring the binding of the labelled ligand to the oocyte-bound antibody.

Alternatively, the antibody can be fluorescently labelled prior to use and the binding of the antibody to the recently excysted oocysts can be detected by measuring directly the fluorescence of the bound antibody.

In a further preferred embodiment of the first aspect of the present invention, the antibody is a monoclonal antibody, more preferably the monoclonal antibody is Cry4, Cry5 or Cry6, and most preferably Cry4.

S 15 In another preferred form of the method of the present invention, the treated oocysts are exposed to a first antibody that binds specifically to recently excysted oocysts and a second antibody that binds specifically to the surface of Cryptosporidium oocysts. Preferably the first and second antibodies are labelled with different fluorescent markers such that antibody binding can be detected by measuring the respective fluorescence of each fluorescent marker with a simple analysis-only flow cytometer.

The second antibody preferably binds to both viable and non-viable oocysts and the detection of the binding of one or more of the antibodies to the oocysts can be used to indicate the presence Cryptosporidium oocysts in the sample. In a preferred form, the first antibody is Cry4, Cry5 or Cry6, preferably Cry4, and the second antibody is Cry26. When the two antibodies are used as described above, it is possible to differentiate between viable and non-viable oocysts in a sample with the one test.

The method of the present invention is particularly suitable for the detection of viable Cryptosporidium parvum oocysts.

In a second aspect, the present invention consists in an antibody that binds specifically to recently excysted oocysts of Cryptosporidium.

Preferably, the antibody is a monoclonal antibody and more preferably the monoclonal antibody is Cry4, Cry5 or Cry6.

In a third aspect, the present invention consists in an hybridoma cell producing a monoclonal antibody that binds specifically to recently excysted oocysts of Cryptosporidium. More preferably the hybridoma cell produces the monoclonal antibody Cry4, Cry5 or Cry6.

In a fourth aspect, the present invention consists in a ligand or ligands of recently excysted oocysts of Cryptosporidium that is specifically bound by the monoclonal antibody Cry4, Cry5 or Cry6.

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will be described with reference to the following examples and drawings.

Brief Description of the Drawings Figure 1 shows flow cytometric analysis of pure excysted oocysts labelled with monoclonal antibodies; Figure 2 shows flow cytometric analysis of pure non-excysted oocysts reacted with monoclonal antibodies; Figure 3 shows flow cytometric analysis of pure excysted oocysts S: 15 stained with Cry4; and Figure 4 shows flow cytometric analysis of environmental samples seeded with excysted oocysts and treated with two antibodies Cry26 and Cry4; Modes for Carrying Out the Invention MATERIALS AND METHODS Cryptosporidium oocysts. Cryptosporidium parvum oocysts cultured in lambs and purified by density gradient centrifugation were purchased from the Moredun Animal Research Institute, Edinburgh.

Monoclonal antibodies. Two female balbC mice were injected with oocyst preparations as presented in Table 1. Mice were sacrificed, spleen cells dissected and fused with NS1 mouse myeloma cells and the resulting hybridomas cloned. Clones were screened for anti-oocyst antibody production by screening against fresh and excysted oocyst preparations with flow cytometry.

Excysted oocysts were prepared by excysting oocysts and then washing in saline solution.

Excysted SDS treated oocysts were prepared by excysting oocysts, treating with 1% sodium deoxycholate at 21 0 C for 10 minutes and then washing in saline solution.

Table 1. Description of inoculation program.

S. Mouse 1 Mouse 2 Days Antigen Route Days Antigen Route 0 1.5 x 10" sonicated I.P. 0 1 x 10 sonicated I.P.

oocysts in FCA ___oocysts in FCA 33 0.3 x 106 sonicated I.P. 53 9.1 x 10" excysted I.P.

oocysts in FIA oocysts in FIA 82 1 x 106 heat killed (80 0 C I.P. 83 3.1 x 10" excysted I.P.

for 10 minutes) oocysts SDS treated oocysts in FIA 103 1 x 106 sonicated oocysts I.P. 225 3 x 106 gamma I.P.

in FIA irradiated oocysts in

FIA

121 1 x 103 sonicated oocysts I.V. 259 5 x 106 gamma I.V.

in saline irradiated oocysts in saline solution 121 4 x 106 sonicated oocysts I.P 281 2.5 x 106 gamma I.P.

in saline irradiated oocysts in saline solution 282 2.5 x 106 gamma I.V.

irradiated oocysts in saline solution 286 2.5 x 10" gamma I.V.

irradiated oocysts in saline solution FCA Freund's complete adjuvant FIA Freund's incomplete adjuvant I.P. intraperitoneal I.V. intravenous Sonicated oocysts were prepared by sonicating a heat killed (80 0 C for minutes) oocysts suspension until no intact oocysts were visible using light microscopy.

The fusion of mouse 2 resulted in a clone (Cry26) that produced antibody specific to oocyst outer walls (similar to commercially available antibodies to Cryptosporidium oocysts). The fusion of mouse 1 spleen cells resulted in three clones (Cry4, Cry5 and Cry6) that produced an antibody that reacted with an oocyst internal antigen. A number of other clones produced antibodies that also recognised oocyst internal antigens. Figures 1 and 2 represents flow cytometric analysis of oocysts and excysted oocysts reacted against a selection of the antibodies. All antibodies except for Cry26 produced more highly fluorescent particles when reacted with excysted oocysts than with non-excysted oocysts. This demonstrates that the 8 antigens, recognised by these antibodies, are only accessible in open oocysts and are therefore internal.

Oocysts 105 (existed or fresh) were aliquotted into each well of a 96 well plate, and 100 ul hybridoma super supernatant added plus 10 pl1 of FITCcoupled sheep anti-mouse antibody (1/40 dilution) Silenus. Samples were incubated at 370C for 30 minutes, then mixed with 200 pl of phosphate buffered saline and analysed by flow cytometry (Vesey, et al, 1994B). The flow cytometer was calibrated with dilutions of a commercially available anti-Cryptosporidium antibody so that positive and negative controls were defined.

Analysis of all 41 clones by ELISA, indirect immuno-fluorescence and Western blot produced the results presented in Table 2. All antibodies were positive by ELISA, with results ranging from weakly positive to strongly positive A range of antigen sites were identified by 15 immunofluorescence including sporozoites, oocyst walls and the interior of oocysts. A series of different antigen-binding patterns were identified by Western blot analysis.

Western blots of Cryptosporidium parvum antigens were probed with hybridoma supernatants containing monoclonal antibodies. Solubilised intact oocyst proteins were separated by SDS-polyaccrylamide gel electrophoresis under reducing conditions. Each lane consisted of 1 x 106 oocysts and detection of bound antibodies was by HRP-conjugated antimouse antibodies and 4-Chloro-l-napthol. A series of different antigenbinding patterns were identified by Western blot analysis. Each group of antibodies that reacted to a particular antigen on a given site on the oocyst (for example interior, surface, wall, sporozoite) had a characteristic protein binding pattern.

Antibodies Cry26 and Cry4 were purified using EZ-Sep (Amrad Phamacia Biotech, Boronia, Australia) according to the manufacturer's instructions. Purified Cry26 antibody was conjugated with CY3 (Biological Detection Systems, PA, USA) according to the manufacturer's instructions.

0O Table 2. Characteristics of monoclonal antibodies generated against Crvptosporidium parvum oocysts Mabs jsotype ELISA IFAT Cryl M ND** Cry2 M ND Cry3 M ND Cry4 M oocyst interior M oocyst interior Cry6 M oocyst interior Cry7 M sporozoite surface Cry8 ND sporozoite surface Cry9 M sporozoite surface Cryl 0 M ND Cryll1 M ND M ND M ND M ND Cryl 5 ND ND Cryl 6 M sporozoite surface Cry17 M ND Cryl 8 M ND Cryl 9 M sporozoite surface C ry2O M s porozoite surface Cry2l M ND Cry22 M ND Cry23 M ND Cry24 M +4 sporozoite surface Cry25 M oocyst interior Cry26 M oocyst wall C ry27 M sporozoite surface Cry28 ND sporozoite Cry29 M sporozoite surface ND sporozoite surface Cry3l M oocyst wall Cry32 ND oocyst interior o y33 M sporozoite surface Cry34 M ++sporozoite ND sporozoite Cr-y36 M sporozoite surface Cry37 ND oocyst Cry38 M sporozoite surface Cry39 M ND M ND Cry4l sporozoite surface I I *IFAT, Indirect Fluorescence Antibody Test ND, Not Detected Oocyst preparation. Oocysts (1 x 108) were surfaced sterilised by suspending in 1 ml of 70% ethanol for 5 min and then washing by centrifuging at 13000g for 2 min, discarding the supernatant and resuspending in phosphate buffered saline (PBS), pH 7.4. Excystation was then performed by suspending in 1 ml of acidified PBS, pH 2.75, incubating for 30 min at 37°C, then washing by centrifuging at 13000g for 2 min and resuspending in PBS, pH 7.4 with 0.1% sodium deoxycholate and 0.22% sodium hydrogen carbonate. After a further 30 min incubation at 37 0

C

the sample was centrifuged at 13000g for 5 min and fixed by resuspension in 1 ml of PBS, pH 7.4 with 1% formalin (excysted oocyst suspension).

Staining pure oocysts. An aliquot (10 til -106 oocysts) of excysted oocyst suspension was mixed with 200 p1 of Cry4 (approximately 0.005 mg/ml) and incubated at 37 0 C for 10 min prior to the addition of 5 il of a goat anti-mouse FITC conjugated antibody (Silenus Laboratory, Melbourne, 15 Australia; Product DDAF). After a further 10 min incubation at 37°C the sample was analysed by flow cytometry.

Staining seeded environmental samples. An aliquot (10 tl) of excysted oocyst suspension that had been stained with Cry4 as above was mixed with 500 p1 (equivalent to 5 litres unconcentrated sample) of a river water sample that had been concentrated by flocculation (Vesey et al 1994A) and fixed in 4% formalin.

Filtered (0.22 um) bovine serum albumin fraction V was then added to a final concentration of 2% before the addition of 20 i1l of CY3 conjugated Cry26 antibody (approximately 0.055 mg/ml). The sample was 25 incubated at 370 for 10 min and then analysed by flow cytometry.

Flow cytometry analysis. A coulter Elite flow cytometer (Coulter Corporation, Miami, USA) and Becton Dickinson Facscan flow cytometer (Becton Dickinson, San Francisco, USA) were used to analyse samples as described previously (Vesey et al 1994B). Sorted samples were further examined using epifluorescence microscopy.

RESULTS

Flow cytometric analysis of pure excysted oocysts labelled with Cry4 and FITC resulted in four distinct populations 2, 3 and 4) (Figure 3).

Analysis of the sample prior to excystation resulted in only two unlabelled populations labelled (population 1 and 4) (Figure Population 3 reacts strongly with the antibody Cry4, population 2 reacts weakly whereas populations 1 and 4 do not react with Cry4. Examination of the samples using microscopy revealed that the brightly fluorescing population (population 3) is completely excysted empty oocysts and the weakly fluorescing population (population 2) is partially excysted oocysts. The two non-fluorescent populations 1 and 4 were observed to be full oocysts and empty oocysts. The higher light scatter of population 4 indicates that this population represents the full oocysts. Therefore population 1 must represent the empty oocysts.

The lack of populations 2 and 3 in the sample that was not excysted indicates that the antigen recognised by Cry4 is only present in recently excysted oocysts.

Fig. 3 gives a pattern that is characteristic of antibodies that react specifically with recently excysted Cryptosporidium oocysts. It will be 20 appreciated that the flow cytometric analysis as described can be used to determine whether any particular antibody reacts to recently excysted Cryptosporidium oocysts as defined herein.

Examination of the samples with microscopy revealed that the preexcystation sample contained 26% empty oocysts, the post-excystation sample contained 53% empty oocysts. This indicates that 27% of the oocysts in the original sample were viable and capable of complete excystation. The percentage of oocysts in the excysted sample that appeared in population 3 was 29%. This confirms that population 3 represents freshly excysted oocysts. The partially excysted oocysts represented by population 2 are viable oocysts that are potentially infections. This type of population has not previously been recognised by standard methods presently in use. The present results therefore demonstrate that by treating oocysts to an excystation procedure prior to staining with Cry4 it is possible to determine oocyst viability.

It was often found that if oocysts were excysted without surface sterilising with ethanol then no reaction with Cry4 could be detected, even though the number of empty oocysts increased after excystation. High numbers of motile bacteria were observed in these samples, whereas no motile bacteria were observed in samples that had been treated with ethanol.

Results of the analysis of environmental water samples that had been seeded with oocysts stained with both the Cry26 and Cry4 antibodies are presented in Figure 4. The first graph represents side scatter versus red fluorescence (ie, the fluorescence due to binding of CY3 labelled Cry26 and the second graph represents side scatter versus green fluorescence (ie, the fluorescence due to binding of FITC labelled Cry4). A box was drawn on the first graph around an area containing the stained oocysts. This box was then used to gate graph 2 (ie, the only particles that appear on graph 2 are those that appeared in the box). Two distinct populations are present on graph 2, highly fluorescent viable oocysts and non-fluorescent, non-viable oocysts and debris. The viable oocysts are completely separated from all other particles, 15 thus allowing enumeration.

:Crvyptosporidium oocysts are surrounded by an extremely robust oocyst wall that can protect the organism in the environment. When a viable oocyst is exposed to a temperature of 37 0 C in acidic solution, followed by an alkaline solution, the sporozoites rapidly break out of the oocysts wall and oo' 20 swim away leaving behind an empty oocyst (Current 1986).

The monoclonal antibody Cry4 recognises an internal antigen in empty Clyptosporidium oocysts. The antigen is not accessible in oocysts that have not excysted nor is it present in oocysts that have excysted prior to the excystation treatment. Furthermore, the antigen recognised by Cry4 is removed if oocysts are excysted in the presence of bacteria. This would indicate that the antigen is destroyed by bacterial enzymes.

Previously, oocyst viability has been determined on pure samples of oocysts by performing excystation and then manually counting the number of full and empty oocysts. This method is tedious and labour intensive. The development of the antibody Cry4 or other antibodies that react specifically with recently excysted oocysts will enable immunofluorescence assays employing flow cytometry or other automated technologies to replace this manual methodology.

Methods for determining the viability of small numbers of Clyptosporidium oocysts based on the uptake or exclusion of the fluorogenic vital dyes propridium iodide (PI) and 4'6-diamidino-2-phenylindole (DAPI) have been reported (Campbell et al 1992). The authors report that dead oocysts take up PI and fluoresce red, whereas live oocysts exclude PI but are permeable to DAPI resulting in the sporozoites within the oocysts fluorescing blue. The method has proven useful for viability studies on pure oocysts (Robertson et al 1992).The application of the method to the routine monitoring of Cryptosporidium oocysts in water, however, has been limited due to problems with incorporating the technique into flow cytometric detection methods. The present inventors have experienced problems with the method when analysing turbid environmental samples. In particular, the demand for DAPI by some of the particulate matter present in these samples is higher than the demand for DAPI by the oocysts. This results in the particulate matter staining bright blue and the oocysts not staining.

Increasing the concentration of DAPI to ten times that recommended by Campbell et al (1992) results in fluorescent oocysts but the fluorescence of the background becomes unacceptably high. Furthermore, the stains PI and DAPI are not specific to oocysts, they may stain any particle that contains DNA. Therefore, these stains cannot be used as specific labels to enable detection with an analysis-only flow cytometer.

The use of the Cry4 antibody to stain samples after they have been 20 excysted enable the routine detection of viable Cryptosporidium oocysts in environmental samples. The present inventors have found that the antigen recognised by Cry4 rapidly degrades in samples containing bacterial activity.

Therefore, oocysts which have excysted in the environment prior to sample collection will no longer contain the antigen recognised by Cry4.

The recent development of a flow cytometric detection method has solved many of the problems associated with the routine monitoring of Cryptosporidium oocysts in water (Vesey et al 1993, 1994A). Water utilities are now able to process significant numbers of samples whilst achieving high sensitivities. Unfortunately, the application of the method has been limited by the high cost and sophistication of the flow cytometer. At present, a flow cytometer which can sort is required for the detection of oocysts in water. It has been shown, however, that a simple and less expensive analysis-only cytometer is capable of detecting a single specific microorganism in turbid water samples when the organism is labelled with two specific probes (Vesey et al 1994B), such as antibodies. Until now it has not been possible to label Cryptosporidium oocysts with more than one antibody because all 14 commercially available antibodies recognise the same epitope on the surface of the oocyst wall (Moore et al 1995). By performing excystation and then staining with an antibody which reacts specifically to recently excysted oocysts and a surface antibody according to on method of the present invention it is now possible to dual label oocysts and detect them using a simple analysis-only flow cytometer.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

o* *go• *o *o References Badenoch, J. 1990. Cryptosporidium in water supplies. HMSO Publications, London.

Campbell, Robertson and Smith H.V. 1992 Viability of Cryptosporidium parvum oocysts; correlation of in vitro excystation with exclusion of fluorogenic vital dyes. Appl. Environ. Microbiol., 58: 3488-3493.

Current, 1986. Cryptosporidium: its biology and potential for environmental transmission. Crit. Revs. Environ. Conf. 17: 21-51.

Korich, Yozwiak, Marshal, Sinclair, and Sterling, R.S. 1993. Development of a test to assess Cryptosporidium parvum oocyst viability: correlation with infectivity potential. Report to the American Water Works Association Research Foundation.

LeChevallier, Norton, and Lee, R.G. 1991. Evaluation of a method to detect Giardia and Cryptosporidium in water, p.483-498. In Hall, J.R. and Glysson, Monitoring Water in the 1990's; Meeting New Challenges. American Society for Testing and Materials.

Robertson, Campbell, Smith, H.V. 1992. Survival of Cryptosporidium parvum oocysts under various environmental pressures.

Appl. Environ. Microbiol. 58: 3494-3500.

Smith, and Rose, J.B. 1990 Waterborne cryptosporidiosis.

Parasitol Today 6: 8-12.

Vesey, Slade, Byrne, Shepherd, Dennis, and Fricker, C.R. (1993A). A new method for the concentration of Cryptosporidium oocysts from water. J. Appl. Bacteriol. 75, 87-90.

•Vesey, Slade, Byrne, M, Shepherd, Dennis, and Fricker, C.R. (1993B). Routine monitoring of Cryptosporidium oocysts in water using flow cytometry. J. Appl. Bacteriol. 75: 82-86.

Vesey, Hutton, Champion, Ashbolt, Williams, K,L., Warton, and Veal, D.A. (1994A). Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water.

Cytometry 16: 1-6.

Vesey, Narai, Ashbolt, Williams, and Veal, D. (1994B).

Detection of specific microorganisms in environmental samples using flow cytometry. In "Methods in Cell Biology Flow Cytometry Second Edition", Vol 42: 489-522. Eds Academic Press Inc., New York.

Claims (22)

1. A method of detecting the presence of viable Cryptosporidium oocysts in a sample containing Cryptosporidium oocysts, the method comprising the steps of: a) treating the sample so as to cause any viable oocysts of Cryptosporidium in the sample to excyst; b) exposing the treated sample to an antibody that binds specifically to recently excysted Cryptosporidium oocysts such that the antibody binds to excysted Cryptosporidium oocysts in the sample; and c) detecting the presence of oocyst-bound antibody in the sample.

2. The method according to claim 1 such that the Cryptosporidium is Cryptosporidium parvum.

3. The method according to claim 1 or 2 wherein the oocysts are caused to excyst by incubating the sample at about 37 0 C under acidic conditions, followed by incubating the sample under neutral to alkaline conditions at about 370C.

4. The method of claim 3 wherein the oocysts are caused to excyst by incubating the sample at 37 0 C at pH 2 to 4 for 10 to 60 minutes, followed by incubating the sample at 370C at pH 7 to 9 for 10 to 60 minutes. 20

5. The method of claim 4 wherein the oocysts are caused to excyst by incubating the sample at 37 0 C at pH 2.75 for 30 minutes, followed by incubating the sample at 370C at pH 7.4 for 30 minutes.

6. The method according to any one of claims 1 to 5 wherein the recently excysted Cryptosporidium oocysts are excysted oocysts in the sample up to about one hour after the treatment of step

7. The method according to any one of claims 1 to 6 wherein the antibody is fluorescently labelled prior to use and the binding of the antibody to the recently excysted oocysts is detected by measuring directly the fluorescence of the oocyte-bound antibody.

8. The method according to any one of claims 1 to 7 wherein the binding of the antibody to the recently excysted oocysts is measured indirectly by further treating the sample with a fluorescently-labelled ligand that binds specifically to the antibody and measuring the binding of the labelled ligand to the oocyte-bound antibody.

9. The method according to any one of claims 1 to 8 wherein the sample is analysed by flow cytometry or microscopy to detect the oocyst-bound antibody.

The method according to any one of claims 1 to 9 wherein the antibody is a monoclonal antibody.

11. The method according to claim 10 wherein the monoclonal antibody is selected from the group consisting of Cry4, Cry5 and Cry6.

12. The method according to claim 11 wherein the monoclonal antibody is Cry4.

13. The method according to any one of claims 1 to 12 wherein the excysted oocysts are exposed to a first antibody that binds specifically to recently excysted oocysts and a second antibody that binds specifically to the surface of Cryptosporidium oocysts.

14. The method according to claim 13 wherein the first and second 15 antibodies are labelled with different fluorescent labels such that antibody Sbinding is detected by measuring the respective fluorescence of each fluorescent label with a simple analysis-only flow cytometer.

The method according to claim 14 wherein the second antibody binds to both viable and non-viable oocysts and the detection of the binding of one 20 or more of the antibodies to the oocysts is used to indicate the presence Cryptosporidium oocysts in the sample.

16. The method according to claim 15 wherein the first antibody is Cry4 and the second antibody is Cry26.

17. An isolated antibody that binds specifically to recently excysted oocysts of Cryptosporidium.

18. The antibody according to claim 17 being a monoclonal antibody selected from the group consisting of Cry4, Cry5 and Cry6.

19. The antibody according to claim 18 being the monoclonal antibody Cry4.

20. An hybridoma cell producing a monoclonal antibody that binds specifically to recently excysted oocysts of Cryptosporidium.

21. The hybridoma cell which produces the monoclonal antibody selected from the group consisting of Cry4, Cry5 and Cry6.

22. An isolated ligand or ligands of recently excysted oocysts of Cryptosporidium specifically bound by the monoclonal antibody Gry4, or Cry6. Dated this third day of May 2000 Macquarie Research Ltd, Australian Water Technologies Pty Ltd Patent Attorneys for the Applicant: B *FRICE &CO