Government support under National Institutes of Health Grant Nos. AI-29882 and AI 30295, UARP Government No.: R94-SF-0555 and the University of California Academic Senate grant. The U. S. and California Governments may have certain rights in this invention.

This application is a continuation-in-part of US application Serial No. 08/827,171, filed on March 27,1997, allowed, and of US application Serial No. 08/928, 361, filed on September 12,1997, issued as the U. S. Patent 6,071,518 on June 8,2000, which is a continuation-in-part of US application Serial No. 08/700,651, filed on August 14,1996, issued as patent 6,015,882 on January 18,2000, which is a continuation-in-part of the US Application Serial No.

08/415,751 filed on April 3,1995 issued as patent 5,643,772 on July 1, 1997, which is a continuation of Ser. No.

08/071,880 filed on June 1, 1993 which is a continuation-in-part of Ser. No. 07/891,301 filed May 29, 1992.

BACKGROUND OF THE INVENTION Field of the Invention This invention concerns methods for detection of Cryptosporidium species or individual Cryptosporidium isolates and for diagnosis of prior or concurrent Cryptosporidium

infections. The method for detection of Cryptosporidium species involves detection of a Cryptosporidium surface antigens GP900, p68 or cryptopain, GP900, p68 or cryptopain antibodies, and GP900, p68 or cryptopain DNAs and RNAs using PCR primers from regions flanking different domains of GP900, p68 or cryptopain. The method for detection of Cryptosporidium isolates involves PCR amplification of portions of GP900, P68, cryptopain and flanking regions with or without restriction fragment length polymorphism analysis which yield a fingerprint for each individual isolate. The method for diagnosis of Cryptosporidium infections involves detecting a presence of GP900, p68 or cryptopain antibodies, GP900, p68 or cryptopain antigen or the DNA or RNA encoding the GP900, p68 or cryptopain antigen in biological and environmental samples.

The methods of the invention detect the Cryptosporidium antigen comprised of a protein with or without carbohydrates attached thereto, or the DNA or RNA encoding the Cryptosporidium antigen or DNA adjacent to it (flanking regions), or a mutant, variant, analog or fragment thereof.

The invention additionally concerns methods for production of recombinant Cryptosporidium antigens suitable for development of diagnostic and detection tools and kits.

BACKGROUND AND RELATED DISCLOSURES Cryptosporidium is an Apicomlexan protozoa which causes gastrointestinal disease in humans and other vertebrates. In immunocompetent host, the disease process ends when protective antibody develops. In immunocompromised hosts, the disease may become chronic resulting in wasting, diarrhea, electrolyte abnormalities, dehydration and death. There is no effective treatment for cryptosporidiosis.

The genus Cryptosporidium consists of Apicomplexan parasites that invade and develop within epithelial cells of the gastrointestinal, hepatobiliary and respiratory tracts of a wide variety of vertebrates including reptiles, birds and mammals. Cryptosporidium was recognized as a cause of animal disease for several decades before the first cases of human cryptosporidiosis were reported in 1976. However, it was not until 1982 that the magnitude of disease caused by this parasite in both AIDS patients and immunocompetent hosts began to be appreciated. Subsequently, Cryptosporidium has been found to be one of the most common causes of human diarrhea worldwide, and to be an increasingly recognized cause of diarrhea in children, animal care workers, and travelers.

(Cryptosporidium and Cryptosporidiosis in Humans, Ed. Fayer, R., CRC Press, Boca Raton (1997)).

Large water-borne outbreaks of cryptosporidiosis caused by contaminated municipal water supplies in the US or in the UK have been noted in the last decade (N. Enql. J. Med., 320: 1372 (1989), and 33: 161 (1994)). A large outbreak in Milwaukee in April 1993 involved 400 ; 000 persons and led to the subsequent deaths of more than hundred immunocompromised persons. Like a number of other waterborne outbreaks, the Milwaukee outbreak appears to have been due to contamination from farm or abattoir run-off and was specifically connected to cryptosporidiosis infected cows and calves. Nosocomial transmission in hospitals from patients to staff, patient to patient, and contaminated ice to patients and staff have also been well documented (J. Infect. Dis., 158: 647 (1985)).

Waterborne and nosocomial spread uncovered a number of biological characteristics of oocysts. First, the infectious

dose of a parasite is very low. The ID50 for human volunteers with normal immune systems is 132 oocysts (N. Enql. J. Med., 332: 855 (1995)). Second, infected hosts, for example calves, excrete large numbers of oocysts, on the order of 101°/day.

Third, the oocysts are fully sporulated and ready to infect when excreted. Fourth, the oocysts are environmentally hardy.

They remain infectious in cool, moist areas for 3-4 months and are not killed by chlorine levels achievable in drinking water. Fifth, the oocysts are quite small, 4-6 ym, and are thus difficult to filter.

The infective forms of Cryptosporidium, called sporozoites and merozoites, appear to adhere to the host cell and release the contents of anterior organelles (rhoptries, micronemes or dense granules) during the invasion process (Parasitol. Today, 8: 28 (1992)). Proteins involved in these events have in many instances been found to be the target of invasion blocking immunity in vitro and neutralization in vivo (Infect. Immun., 56: 2538 (1988)).

While the actual interaction between Cryptosporidium and the host's immune system is poorly understood, it is known that disruption of either the cellular or the humoral components can result in protracted cryptosporidiosis (Parasitol. Today, 8: 24 (1992)). Specific antibodies alone appear to be enough to neutralize the organism's infectivity.

In vitro and in vivo observations indicate that antibodies to Cryptosporidium parvum inhibit invasion and intracellular development leading to protection in challenge experiments, or amelioration of infection in established disease (Infect.

Immun., 59: 1172 (1991)).

One source of such antibodies is hyperimmune bovine colostrum (HBC) collected from cows immunized with Cryptosporidium oocysts. Calves challenged with Cryptosporidium oocysts are protected from the development of disease by the administration of HBC (Infect. Immun., 61: 4079 (1993)). Some immunocompromised AIDS patients infected with Cryptosporidium have also responded to HBC with a reduction in or disappearance of the symptoms of the disease (Gastroenterology, 98: 486 (1990)). Immunoglobulin from HBC (HBC Ig) has been found to inhibit the ability of the sporozoite to invade and/or develop intracellularly in vitro and it has been used to immunoprecipitate at least 22 different surface radioiodinated proteins of Cryptosporidium sporozoites. Western blot analysis of proteins of whole oocysts which contain sporozoite indicates that HBC predominantly recognizes two proteins of sizes 250 KD and >900 KD (Infect. Immun., 61: 4079 (1993)).

Although a connection between Cryptosporidium from water, dairy animals, pets, children in day care and hospital environments and cryptosporidiosis has been made, the relative importance of water, food, pets, sexual or casual person-to- person contact in the transmission of the parasite has not been established. The epidemiology of Cryptosporidium, particularly transmission and reservoirs of the parasite have been difficult to study because the organism cannot be propagated in vitro for the development of serological or growth characteristics as a method of systematic identification of Cryptosporidium species and individual isolates.

Therefore, availability of Cryptosporidium specific antigen, DNA and antibody markers for species identification and for differentiation of strains/isolates within species would be greatly advantageous.

While, as described above, the Cryptosporidium infection can have serious and, in some cases, fatal consequences, only limited detection and diagnostic methods, tools and kits are available.

Currently available Cryptosporidium detection methods are microbiological immunological assays and limited PCR-based detections. The microbiological detections of the organism in the stool has a very low sensitivity of detection of about 1000 organisms in one gram of stool. Sensitivity of antibody detection methods is somewhat improved but these methods still can detect the presence of the organism only when at least 500 organisms are present in one gram of stool. Attempts to use monoclonal antibodies for detection of cryptosporidiosis resulted in very expensive and not overly sensitive kits which, so far, have not generally replaced the classical microbiological detection techniques.

Thus, better and more sensitive methods for detection of Cryptosporidium are needed.

It is a primary objective of this invention to provide methods for detection of Cryptosporidium species or isolates by 1) specific detection of GP900, P68 or cryptopain antigens, 2) specific detection of GP900, P68 or cryptopain DNA fragments, and 3) detection of the presence of anti- Cryptosporidium antibodies indicating that the host has been, in the past, or is currently infected with Cryptosporidium.

All patents, patent applications and publications cited herein are hereby incorporated by reference.

SUMMARY OF THE INVENTION One aspect of this invention concerns methods and kits for detection of Cryptosporidium species or individual Cryptosporidium isolates and for diagnosis of prior or concurrent Cryptosporidium infections.

Another aspect of this invention concerns a method for detection of Cryptosporidium species involving detection of a Cryptosporidium antigen GP900, p68 or cryptopain, GP900, p68 or cryptopain antibodies, and GP900, p68 or cryptopain DNAs and RNAs using PCR primers from regions flanking different domains of GP900, p68 or cryptopain.

Still another aspect of this invention concerns a method for detection of Cryptosporidium isolates involving PCR amplification of DNA fragments which are different in different isolates, such as poly-threonine rich domains or different restriction sites which yield a fingerprint for each individual isolate differentiating it from another isolate.

Yet another aspect of this invention concerns a method for diagnosis of Cryptosporidium infections involving detection of the presence of GP900, p68 or cryptopain antibodies, GP900, p68 or cryptopain antigen or the DNA or RNA encoding the GP900, p68 or cryptopain antigen protein with or without carbohydrates attached thereto, or a mutant, variant, analog or fragment thereof in biological and environmental samples.

Still yet another aspect of this invention concerns methods for production of recombinant Cryptosporidium antigens

suitable for development of diagnostic and detection tools and kits.

Another aspect of this invention concerns antibodies, antigens, DNAs and RNAs used in methods or kits for detection of Cryptosporidium species and isolates or diagnosis of prior or concurrent Cryptosporidium infections.

Another aspect of this invention concerns polyclonal or monoclonal antibodies directed against the Cryptosporidium antigen for use in a method and kits for detection of cryptosporidiosis.

Another aspect of this invention concerns the GP900, p68 or cryptopain Cryptosporidium antigen or fragments thereof.

Still another aspect of this invention concerns a DNA and RNA encoding the Cryptosporidium antigen and fragments thereof suitable for preparation of anti-Cryptosporidium antibodies.

Still yet another aspect of the invention is the use of the GP900, p68 or cryptopain antigen, antibody, DNA or RNA for Cryptosporidium diagnosis of prior or current infection in a human or animal host or for detection of Cryptosporidium parasite in the environment.

Still another aspect of this invention concerns a DNA and RNA encoding the Cryptosporidium protein or glycoprotein comprising Cryptosporidium antigen or fragments thereof for use in production of the protein or glycoprotein for development of agents used for diagnosis or detection of Cryptosporidium infection.

Another aspect of this invention concerns the DNA sequence of 7334 bp (SEQ ID NO: 1) nucleotides encoding the GP900 protein of Iowa isolate and its upstream (5') protein coding and regulatory elements and its 3 noncoding sequence.

Another aspect of this invention concerns the DNA sequence of 5511 bp (SEQ ID NO : 2) nucleotides encoding GP900 of the Iowa isolate, and its nucleotide and size variants.

Another aspect of this invention concerns an amino acid sequence of 1832 amino acids (SEQ ID NO: 5) of GP900 of Iowa isolate, a >900 kD glycoprotein present in or on the surface of sporozoites and merozoites, and its amino acid and size variants.

Another aspect of this invention concerns the DNA sequence of 5318 bp (SEQ ID NO : 3) nucleotides encoding the GP900 protein of NINC isolate comprised'of partial ORF and 3' flanking region.

Another aspect of this invention concerns the DNA sequence of 5163 bp (SEQ ID NO : 4) nucleotides encoding GP900 of the NINC isolate comprised of the partial ORF.

Another aspect of this invention concerns an amino acid sequence of 1721 amino acids (SEQ ID NO : 6) of GP900 of NINC isolate.

Another aspect of this invention concerns the DNA sequence of 2380 nucleotides (SEQ ID NO : 25) encoding a protein portion of P68, its nucleotide and size variants and its upstream (5') protein coding and regulatory elements.

Another aspect of this invention concerns an amino acid sequence comprising 503 amino acids (SEQ ID NO: 26) of a protein portion of P68, a 50-100 kDa glycoprotein of sporozoites and merozoites, and its amino acid and size variants.

Another aspect of this invention concerns the DNA sequence of 1663 bp (SEQ ID NO : 27) of cryptopain comprised of

5'and 3'flanking sequences, its pre pro fragments (SEQ ID NO: 28) and mature enzyme (SEQ ID NO : 29).

Another aspect of this invention concerns an amino acid sequence of 401 amino acids (SEQ ID NO: 30) of cryptopain, cryptopain pre and pro fragments (SEQ ID NO: 31), and mature enzyme (SEQ ID NO: 32).

Still another aspect of this invention are PCR primers which are oligonucleotides of about 14 to about 35 bp in length synthesized using portions of the GP900, P68 or cryptopain DNA and which are used by enzymatic reactions leading to the amplification of DNA.

Yet another aspect of this invention is a DNA probe which is an oligonucleotide or a larger segment of DNA used for direct hybridization with complementary DNA or RNA sequences.

Still yet another aspect of this invention concerns a method for diagnosing Cryptosporidium infection of a subject, comprising steps: (a) contacting a body specimen, fluid or tissue obtained from the subject with an anti-Cryptosporidium monoclonal or polyclonal antibody; and (b) detecting the formation of antibody-antigen complex wherein the presence of the complex indicates positive diagnosis of the presence of a Cryptosporidium organism in the subject and wherein the absence of the complex indicates negative diagnosis of Cryptosporidium infection.

Still yet another aspect of this invention concerns a method for detecting a presence of Cryptosporidium parasite in a biological or environmental sample, said method comprising steps:

(a) contacting a biological or environmental sample with an anti-Cryptosporidium monoclonal or polyclonal antibody; and (b) detecting the formation of antibody-antigen complex wherein the presence of the complex indicates presence of the Cryptosporidium parasite in the sample and wherein the absence of the complex indicates absence of Cryptosporidium parasite in the sample.

Still yet another aspect of this invention concerns a method for detecting anti-Cryptosporidium antibody in a subject, said method comprising steps: (a) contacting a body specimen, fluid or tissue obtained from the subject with the GP900, p68 or cryptopain antigen; and (b) detecting a formation of antibody-antigen complex wherein the presence of the complex indicates positive diagnosis of the presence of a Cryptosporidium antibody in the subject and wherein the absence of the complex indicates negative diagnosis of Cryptosporidium infection.

Still another aspect of this invention is a Cryptosporidium diagnostic or detection kit comprising anti-Cryptosporidium specific monoclonal and polyclonal antibodies, Cryptosporidium GP900, P68 or cryptopain antigen according to the invention and a means for detection of an antibody-antigen complex.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the GP900 gene locus.

Figure 2 is an immunoblot of Cryptosporidium parvum oocyst/sporozoite proteins showing detection of the >900 sporozoite protein with monoclonal and polyclonal antibodies to GP900.

Figure 3 is the immunoprecipitation of 12sI surface label Cryptosporidium parvum sporozoite proteins using monoclonal and polyclonal antibodies to GP900.

Figure 4 is the MAb 7B3 indirect immunofluorescence detection of GP900 present on the surface and shed from the surface of a motile sporozoite.

Figure 5 is an immunoblot of Cryptosporidium parvum oocyst/sporozoite proteins identified by polyclonal antibodies to P68.

Figure 6 is a restriction map of P68 ORF DNA.

Figure 7 is the DNA sequence of cryptopain (SEQ ID NO: 27) comprising sequences encoding segments for the pre and pro regions (SEQ ID NO: 28) and for mature enzyme (SEQ ID NO : 29).

Figure 8 is the protein sequence (SEQ ID NO: 30) of cryptopain comprising segments for the pre and pro regions (SEQ ID NO: 31) and for mature enzyme (SEQ ID NO : 32).

Figure 9 is a genomic Southern analysis of Cryptosporidium DNA using the cryptopain probe.

Figure 10 is the genomic Southern analysis of the GP900 gene fragment of Iowa and AUCP isolate DNA hybridized with DB8 DNA probe from NINC isolate.

DEFINITIONS As used herein: "Cryptosporidium species"means any organism belonging to the genus Cryptosporidium, such as, for example, Cryptosporidium parvum or Cryptosporidium muris, but also includes other currently less well characterized organisms such as, for example, Cyclospora. Cryptosporidium species comprise Apicomlexan parasites which primarily invade cells of

the gastrointestinal tract and cause disease in a susceptible host.

"Cryptosporidium isolate"means a viable Cryptosporidium separated from the other isolates having a specific genetic characterization (fingerprint) different from other isolates which may be identified by its specific genetic characterization and distinguished from other isolates having a different fingerprint. Cryptosporidium parvum was identified and isolated from oocysts of the Iowa, NINC and AUCP-1 isolates of Cryptosporidium parvum passaged through neonatal calves, as described in Example 1, and tested for their interaction with specific anti-GP900, p68 or cryptopain antibodies. Proteins which were shown to be targets of GP900, p68 or cryptopain antibodies were prepared according to Example 2, and visualized by enzyme-linked immunosorbent assay (ELISA), chemiluminescence or with 1211 labeled Protein A followed by autoradiography as described in Example 7.

"Antigen"means a protein isolated from Cryptosporidium identified as GP900, P68 or cryptopain with or without glycoprotein attached thereto, detected in micronemes of developing merozoites and sporozoites. Antigen is present on the surface of the sporozoites and is shed from the sporozoite surface in vivo in host cells.

"GP900 antigen"means a protein with or without a carbohydrate attached thereto which defines the capacity of Cryptosporidium sporozoites and merozoites to infect host cells. When deglycosylated, the GP900 core protein has a variable molecular weight of approximately 150-250 kD. GP900 protein comprises 1832 amino acids, mutants, variants, or analogs thereof and identified as SEQ ID NO : 5 of Mr greater

than 900 kilodaltons (kDa) which may contain a covalently attached glycoprotein, said GP900 detected at the surface of sporozoites or merozoites or free in conditioned media. GP900 is the target of antibodies which are present and detectable in the tissue or fluids of the Cryptosporidium infected subject.

"P68"means an apical protein of sporozoites or merozoites represented by 503 amino acids and identified as SEQ ID NO: 26 of Mr between approximately 50 and 100 kilodaltons which is a target of antibodies which inhibit infection, invasion or adhesion of Cryptosporidium.

"Cryptopain"or"Cryptopain antigen"means a protein which is a cathepsin L-like cysteine proteinase having a function in invasion and infection of host cells by Cryptosporidium. Cryptopain is represented by a protein containing 401 amino acids and is identified as SEQ ID NO: 30 (Figure 3) comprising a protein of Mr 45 kDa. Homology to other cathepsin L-like cysteine proteinases indicates that the mature active enzyme is cleaved after amino acid 175 one residue N-terminal to a conserved prolines and comprises a 25 kDa protein of 226 amino acids (SEQ ID NO : 33). Cryptopain also includes size and sequence variant proteins which maintain the same function.

"Detection"means establishing a priority evidence for the presence or prior presence of living or dead Cryptosporidium by detecting Cryptosporidium antigens, cryptopain or Cryptosporidium DNA or RNA in the host, in a host biological sample obtained from a specimen such as stool, urine, blood, plasma, biopsy, saliva and such other samples as

appropriate or in environmental samples including water, soil, food, etc.

"Diagnosis"means establishment of the presence or prior presence of Cryptosporidium infection or disease by using the GP900, P68 or cryptopain antigen, antibody to GP900, P68 or cryptopain, GP900, P68 or cryptopain DNA or RNA as a component of a diagnostic assay according to the invention.

"Host"or"subject"means humans, or animals including birds and cattle.

"GP900 DNA"means the sequence of 5511 nucleotides identified as SEQ ID NO : 2 which encodes an amino acid portion of the protein sequence of GP900 protein (SEQ ID NO : 5) and any variant, 5'extension, mutation and fragment thereof, which corresponds to genes encoding the antigen. Variants include but are not limited to the partial sequence of GP900-NINC or Iowa.

"GP900 RNA"means the RNA sequence corresponding to GP900 DNA sequence (SEQ ID NO : 2) which encodes the amino acid sequence of GP900 protein (SEQ ID NO : 5) and any 5'extension variant, mutation and fragment thereof.

The"structure"or"structural characteristics"of GP900, P68 or cryptopain defines a protein, glycoprotein, DNA and RNA encoding the GP900, P68 or cryptopain protein and includes all structural variations, mutations and fragments exhibiting the same function.

The"functionality"or"functional characteristics"of GP900, P68 or cryptopain is defined by the interaction of antibodies to GP900, P68 or cryptopain and structural variants described, such that the antibody inhibits infection, invasion or adhesion of Cryptosporidium.

"The gene"or"genes encoding"means DNA encoding a portion or all of the GP900, P68 or cryptopain protein. One or more of these portions with or without carbohydrates attached include the targets of GP900, P68 or cryptopain antibodies.

"Sporozoites or merozoites"means any life stage which may invade host cells and any variant or mutant of said sporozoites or merozoites.

"Antibodies"means proteins which structurally interact with the target antigen and are produced when the antigen is introduced into an animal, such that they stimulate the immune system. The term also includes antibodies produced in vitro, such as antibodies produced by hybridoma cell cultures and chimeric proteins, as well as hybridoma cells and chimeric constructs introduced into the host to provide an in vivo antibody. The GP900, P68 or cryptopain protein has been identified as a target of anti-GP900, P68 or cryptopain antibodies which inhibit Cryptosporidium infection, invasion or adhesion.

"Antibodies to GP900"means proteins which structurally interact with the target antigen GP900.

"Antibodies to P68"means proteins which structurally interact with the target antigen P68.

"Antibodies to cryptopain"means proteins which structurally interact with the target antigen cryptopain.

"Antibody-antigen complex"means a detectable complex of the anti-Cryptosporidium antibody bound to the Cryptosporidium antigen.

"Monoclonal antibodies"means the monovalent antibodies produced by B cells fused to immortalized cells producing antibody specific to GP900, P68 or cryptopain.

"Polyclonal antibodies"means antibodies directed at GP900, P68 or cryptopain which are not monovalent and are the products of multiple B cells in character.

DETAILED DESCRIPTION OF THE INVENTION This invention is based on finding that cryptosporidiosis can be detected and diagnosed with a great sensitivity using methods and kits of the invention comprising specific Cryptosporidium antigens GP900, P68 and cryptopain, antibodies raised against these antigens, DNA or RNA probes or primers derived from DNA of these antigens and the mutants, variants, analogs, and fragments thereof.

The invention, therefore, primarily relates to methods for detection and diagnosis of cryptosporidiosis in human and animal subjects and the environment.

More specifically, the invention concerns detection of the presence of Cryptosporidium antigen or antibody for diagnosis of prior or current infection in humans and animals and for detection of Cryptosporidium in the environment.

Cryptosporidium antigens described herein, in particular, are suitable for diagnosis of current or prior Cryptosporidium parvum infection by virtue of detecting antibodies to Cryptosporidium.

Polyclonal or monoclonal antibodies to the Cryptosporidium antigens are suitable for detecting the presence of Cryptosporidium in biological samples of the host or in the environmental samples.

The method for detection of the Cryptosporidium antigen presence in the biological or environmental samples comprises detecting antibody-antigen complex using specific monoclonal or polyclonal anti-Cryptosporidium antibodies to detect the presence of the antigen or detecting the presence of anti- Cryptosporidium antibody with Cryptosporidium antigen.

This invention also provides DNA and RNA encoding the Cryptosporidium GP900, P68 or cryptopain molecule, or a mutant, variant, analog and fragment thereof, and methods for production of recombinant or fusion proteins for use in detection/diagnosis.

This invention additionally discloses. methods for preparation of the recombinant GP900, P68 or cryptopain antigens, mutants, variants, analogs and fragments thereof for production of monoclonal or polyclonal antibodies or probes for use in detection/diagnosis.

I. Detection of Cryptosporidium and Diagnosis of Cryptosporidiosis This invention primarily concerns methods and means for detection of Cryptosporidium in the biological and environmental samples or for diagnosis of prior or concurrent Cryptosporidium infection.

The current method provides DNA and antibody for species identification and also for differentiation of isolate within species. The antigen, DNA and antibody probes are for specific functionally important proteins and therefore may detect virulence differences as well as isolate differences.

A. Detection of Cryptosporidium in Biological and Environmental Samples

The Cryptosporidium organism which causes cryptosporidiosis cannot be grown in vitro. This fact severely limits its detection in biological samples and in the environment because regularly used microbiological methods, such as culture, cannot be used for detection of fairly virulent Cryptosporidium.

The invention described herein stems from the availability of DNA and protein sequences of the antigens GP900, P68 and cryptopain discovered previously and described in patent applications and patents by inventors, namely the U. S. Patents 6,071,518,6,015,882,5 ; 643,772, and patent applications Serial No.: 08/827, 171, filed on March 27,1997, Ser. No. 08/071,880 filed on June 1,1993, Ser.

No. 07/891,301, filed May 29,1992, hereby incorporated by reference in their entirety.

Cryptosporidium antigens GP900, P68 and cryptopain isolated from different Cryptosporidium species and isolates are differentiated by PCR amplification and/or restriction length polymorphism analysis which defines a fingerprint for each particular isolate. PCR primers from regions flanking different portions of GP900, P68 or cryptopain are used for this differentiation. For example, discovery that the poly- threonine rich domains of GP900 differentiate between isolates in fingerprinting assays enabled development of a specific method for detection of the isolates.

Diagnosis of Cryptosporidium infection and detection of organisms in environmental samples uses the principle of hybridization of complimentary strands of DNA and RNA which allows detection of the presence of the organism in a sample, by virtue of its unique nucleotide sequences. Intact RNA is

associated with viabiilty of an organism. Thus, detection of RNA in an environmental sample is be useful for determining if the environmental sample is infectious. Hybridization is the basic principle of several types of detection procedures.

Direct hybridization For direct hybridization, a portion of a diagnostic sample or an environmental sample containing the target DNA or RNA, obtained from Cryptosporidium, is prepared for hybridization and denatured so that it interacts with and binds to a DNA probe. The DNA probe is a piece of DNA, occasionally as small as 14 bp, but more typically several hundred to several thousand basepairs in length, which is denatured and allowed to interact with the prepared sample under conditions which are experimentally determined for each probe but are well known within the art. The conditions include variations in salt concentrations, temperature and washing conditions. The annealed complementary sequence of DNA containing a single sequence from the sample and a single sequence from the probe is detected by virtue of the incorporation of a label in the probe prior to the hybridization procedure. The label may be radioactive phosphate, biotin and a variety of other tags that are commercially available and are generally linked to the probe via a chemical reaction. The labeled probe is then detected in the sample via the appropriate detection procedure, i. e., for the mentioned labels, exposure to film or other methods of detection of radioactivity, reaction with a fluorescent or enzyme-linked streptaviden for detection of biotin via chemiluminescence or colorimetric assay.

Amplification of specific nucleic acids in a sample

In this application of the principle of hybridization, smaller amounts of DNA or RNA in a sample is amplified so that the signal for detection is increased and the sensitivity of the procedure is enhanced. Single stranded PCR primers, typically 14 to 35 bp in length, are synthesized using portions of the DNA of the invention. Two primers separated by a distance which may vary from less than 100 bp to more than 5 kbp, but would generally be within those limits, are selected such that they are in opposite orientations and will, when utilized in a PCR reaction, result in the detection and amplication of the DNA which lies between them. An alternate technique utilizing branched DNA may also be utilized for amplification of the signal according to methods known in the art.

Primers are typically prepared when the sequence of DNA is determined. They are single-stranded. They may be used in sequencing reaction and in PCR reactions for detection and occasionally as probes, called oligonucleotide probes, in a direct hybridization reaction. In the latter instance, however, a larger piece of double stranded DNA is typically used as a probe after it has been denatured (i. e., rendered single stranded so that it can anneal with a. DNA sequence in the sample). Herein, DNA probe is defined as a DNA segment which is an oligonucleotide or a larger segment of DNA which is used to detect complementary DNA or RNA sequences. DNA primers are defined as oligonucleotides which are used in enzymatic reactions which lead to the amplification of DNA.

List of a number of oligonucleotides presented in Tables 1,3,4 and 5 represents primers which have already been synthesized from the DNA sequence of GP900, P58 and

cryptopain. These oligonucleotides are examples of DNA which can be used as PCR primers or as hybridization probes for detection purposes. Any larger segment of DNA can be amplified by PCR from Cryptosporidium DNA and also used as a probe. In this invention all primers and probes which can be made from the DNA sequences of GP900, P68 and cryptopain and used to detect Cryptosporidium DNA in diagnostic samples or environmental samples by way of direct hybridization or amplification methods are intended to be within the scope of this invention.

The PCR and hybridization methods described herein are extremely sensitive and permits detection of even one parasite molecule per volume when the appropriate primers and conditions are used. With a small number of parasite and the specific primers, the presence of Cryptosporidium and fingerprinting of the isolate using environmental or biological sample permit using the method of the invention.

In another mode, the detection method for environmental samples comprises contacting such a sample with the antigen or antibody of the invention for purposes of detecting Cryptosporidium. The detection method in biological samples comprises contacting a tissue body fluid, biopsy or solid specimen, with the antigen or antibody of the invention for purposes of detecting the presence of Cryptosporidium.

Examples of body solid specimens are stool or tissue biopsies obtained from a subject. Examples of body fluids are blood, plasma, serum, saliva, urine, and the like.

Methods for the preparation of the tissue sample and the body fluid are standard in the art and are described, for example in Manual of Clinical Microbiology, Chapter 8,

"Collection, Handling and Processing of Specimens", 4th edition, Eds, Lennette, E. H., Balows, A., Hausler, W. J. and Shadorny, A. J., American Society for Microbiology, (1986)), and examples of environmental samples include water, soil and foods grown in the environment.

Methods for actual detection of the presence of Cryptosporidium in the biological or environmental samples include but are not limited to polymerase chain reaction (PCR) amplification of the parasite DNA and detection of such amplified DNA by hybridization with probes, immunoreaction with antibodies, direct immunofluorescence, staining, microbiological or by any other method suitable for the detection of DNA as known now in the art or will become known in the future.

B. Detection of Cryptosporidium Isolates The invention also permits detection and identification of individual Cryptosporidium isolates based on their genetic characteristics, herein called"a fingerprint". Fingerprints from each individual isolate were discovered and are further described below.

Differences between isolates of Cryptosporidium at the DNA level were previously described in J. Protozool 38: 405- 415 (1991).

The current identification of these differences and production of DNA probes which differentiate these isolates using a small amount of isolate DNA makes possible the detection of Cryptosporidium in water, food, pet, nosocomial and person-to-person nonsexual and sexual exposure. The development of isolate-specific Cryptosporidium probes also

permits determination of a latent state of Cryptosporidium infection.

Conserved PCR primers selected to amplify a polymorphic region of Cryptosporidium DNA from gastrointestinal tissue specimens of asymptomatic person will discover the presence of an organism which may later cause chronic cryptosporidiosis.

Isolate specific probes also allow the differentiation of isolates according to their specific attributes, such as virulence, infectivity or host preference, for example for man, domestic or wild animals.

Detection of isolate specific DNA The description presented above describes the detection of DNA from Cryptosporidium in general and is general enough to detect all Cryptosporidium species and isolates.

There are however, differences in sequences of different isolates of Cryptosporidium which may be exploited in order to detect and"thumbprint"different isolates. For example, the DNA of GP900 from 5 isolates has been completely or partially sequenced and contain only a limited number of variants in DNA sequence leading to the presence of different restriction sites in domains 1,3 and 5. There are also regions of variability in length within the sequence, primarily in the poly-threonine regions of domains 2 and 4. Using these two features isolate differences may be detected in the following way. PCR primers are chosen to amplify a portion of GP900 DNA, generally including one of the threonine-rich domains.

PCR fragments will be generated in all cases in which the primers were designed from the homologous isolate, in this case Iowa. In different isolates, they may be absent or vary in length. To further refine the isolate pattern, the PCR fragment is cut with restriction enzymes which cut in different locations in different isolate genomes. Comparison of the pattern of fragments as detected by electrophoretic separation will differentiate between the isolates and create a fingerprint. This example describes known differences in isolate DNA for GP900. It can be combined with PCR fragment amplification and restriction from the 5'and 3'flanking regions of GP900 (often these noncoding regions are quite variable) or from the locus of P68 and/or cryptopain to provide great sensitivity in distinguishing isolates. In

addition, primers may be synthesized in regions of known DNA polymorphisms between isolates which result in the appearance or disappearance of a restriction site. These primers may be used to further distinguish between isolates by restriction length polymorphism analysis.

The detection of Cryptosporidium isolates depends on availability of conserved PCR primers which encompass polymorphic regions but which are conserved across C. parvum isolates and allow amplification and identification of isolate specific DNA from biological or environmental samples and other sources containing minute amounts of parasite material.

For the purposes of this invention, the following isolates of Cryptosporidium parvum were obtained: isolates which are geographically distinct (8); isolates which are temporally distinct (2), namely Iowa-1 and Iowa-2 isolates or isolates surmised to have been passed from one HIV-positive person to another by direct contact in the health care setting (2), SF-1 and SF-2. The temporally distinct isolates were obtained as Iowa isolate originally isolated from a calf in Ames, Iowa. Iowa-1 isolate was obtained from the NIH after the original Iowa isolate was propagated in Arizona. The Iowa-2 isolate was collected after the original Iowa isolate was propagated sequentially through 70 animals in Washington state under conditions instituted to minimize contamination.

The geographically distinct isolates were from Peru, Brazil, Florida, Mexico, New York, AUCP-1 (Auburn, Alabama, Byron Blagburn); Iowa-1/Iowa-2 ; and SF-1/SF-2. The Peru, Brazil, Florida, Mexico and New York isolates were the same as those described in Infect. Immunol., 58 : 2071-2075 (1990) in 2-D gel experiments and in J. Protozool., in genomic Southern with

pV47-2. Aliquots of these distinct isolates were obtained from Michael Riggs and were subjected to PCR using primers which flanked the domains of GP900. This analysis showed different fragment sizes which appeared to be in domains 2 and 4 and which differentiated some but not all isolates.

Additional restriction site polymorphisms were identified when the DNA sequences were compared. Taking both observations together, better differentiation of isolates is achieved.

An average of 5 x 108 oocysts (2x109 sporozoites) of each isolate was available in a largely purified form. 109 oocysts of Iowa-2 was provided by Dr. L. Perryman. AUCP-1 and Iowa-1 isolates are available in >109 oocysts quantities. SF1 and SF2 are available in semipurified form which has been roughly quantitated at >5 x 108. Oocyts were purified and DNA isolated as previously described in J. Protozool., 38: 725-735 (1991).

Using these techniques, 50-100 mg of Cryptosporidium DNA from 5 x 108 oocysts were routinely isolated.

C. Diagnosis of Cryptosporidiosis For diagnostic detection of Cryptosporidium in a subject, the sample of the subject's tissue, body fluid or stool is obtained. The sample is then prepared to permit isolation of the parasite oocyst, for example by dissolving the stool (100 mg-3 g) in a buffer, such as TES buffer, centrifuging the suspension, separating the supernatant containing oocysts and recentrifuging to obtain oocyst pellet. After isolating the oocysts, the oocysts are then submitted to lysing conditions where the oocyst releases DNA into the lysate. Lysate is then submitted to PCR DNA amplification with specific primers prepared as 15-35 bp oligonucleotides from GP900, P68 or cryptopain gDNA, when the released DNA is amplified into

sufficient quantity. The amplified DNA is subjected to one of the, detecting methods as described above, i. e., hybridization with the specific probe, immunodetection with Cryptosporidium specific antibody, with direct immunofluorescence, etc. In the alternative, the amplified DNA is applied on a column or another stationary phase, such as microsphere, conjugated with antibody, as a mobile phase, and the antigen-antibody complex is formed and detected by fluorescence, staining, radioactivity, etc. The body fluid samples or tissue biopsies are treated similarly. Various kits and detection assays are known in the art and are commercially available. All and any may be used in practicing this invention by exchanging the primers, probes or antibodies of the kit or assay with the specific primers, probes or antibodies of the invention as described herein. The specific primers for GP900, P68 and cryptopain are listed in Tables 1, and 3-5 and the preparation of other primers as well as probes and antibodies against GP900, P68 or cryptopain are described below.

D. Kits for Detection of Cryptosporidium Kits for the diagnosis/detection of Cryptosporidium are used for determination of the presence or prior presence of the Cryptosporidium infection and environmental contamination, The kits comprise the antigen, antibody, DNA or RNA from GP900, P68 or cryptopain and a means for detecting the interaction of the materials of the invention with Cryptosporidium antigen, antibody, DNA or RNA in a host sample or Cryptosporidium antigen, DNA or RNA in an environmental sample.

The kit is utilized for the detection of endogenous antibodies/artigens/DNA/RNA produced by a subject that is

afflicted with cryptosporidiosis or for detection of Cryptosporidium antigens/DNA/RNA present in the environmental samples. Due to the extreme sensitivity of the method of the invention, even at the early stages where the parasite is commencing invasion of the subject's cells or when the parasite is extremely diluted, such as in water, some amount of the Cryptosporidium antigen DNA/RNA is detectable in stool or water samples using the kit of the invention. The kit may detect either 1) the antigen with the monoclonal or polyclonal antibodies; 2) the presence of the anti-Cryptosporidium antibody with the antigen; or 3) the presence of DNA/RNA by hybridization with DNA or RNA probe, PCR amplification with GP900, P68 or cryptopain primers or other techniques which augment a DNA/RNA presence. The reactions of antigen, antibody or DNA, RNA detected by the kit include staining, radiography, immunoprecipitation, hybridization or any other means used in the art and suitable for these purposes.

In addition to the above, the kits may also comprise additional control compounds, such as anti-antibodies, protein A/G labeled or nonlabeled, and the like, suitable for conducting the various assays referred to above.

II. Cryptosporidium Protein/Glycoprotein Antigens Cryptosporidiosis is a parasitic disease for which there are currently no effective diagnostic or detection methods available. The current invention identifies several specific Cryptosporidium antigens and provide their DNA and amino acid sequences thereby permitting detection of the minute amount of the parasite DNA and enhancing sensitivity of detection by many folds.

During the development of this invention, it has been shown that a Cryptosporidium parvum comprises several highly pathogenic proteins which differ in their genetic make-up.

These proteins were further identified as GP900, P68, cryptopain and other proteins of various sizes as described in the prior application Ser. No. 07/897, 301, incorporated by reference. Of these proteins three, namely GP900, P68 and cryptopain were further identified, cloned, tested for their pathogenicity and antibodies against these proteins were prepared.

1. GP900 Protein, Glycoprotein, Recombinant Protein and DNA/ A. Identification of Protein GP900 as Cryptosporidium Antigen Cryptosporidium antigen identified as GP900 protein is a high molecular weight glycoprotein of a Mr greater than 900 kilodaltons (kD). The GP900 protein was detected in micronemes of developing merozoites and sporozoites of the invasive stages of Cryptosporidium by immunoelectronmicroscopy and has been shown to be accessible to surface radioiodination with 125I. It is present on the surface of the sporozoites and is shed from the sporozoite surface in vivo in host cells.

When deglycosylated, the GP900 core protein has a variable molecular weight of approximately 150-250 kD.

The GP900 protein antigen has been identified as a target of anti-GP900 antibodies which bind to it and form antigen/antibody complex. The GP900 protein was found to be highly abundant and easily visualized by Coomassie blue staining of proteins on SDS-polyacrylamide gels (SDS-PAGE) and is Triton X-100 soluble and N-glycosylated.

GP900 proteins were identified and isolated from oocysts of the Iowa, AUCP-1, NINC or other isolates of Cryptosporidium parvum, as described in Example 1 and tested for their interaction with specific anti-GP900 antibodies. Proteins which were shown to be targets of GP900 antibodies were prepared according to Example 2, and visualized by enzyme-linked immunosorbent assay (ELISA), chemiluminescence or with 1251-labeled Protein A followed by autoradiography as described in Example 7.

B. GP900 Gene Cloning/Sequencinq and Genomic Southern Analysis The GP900 gene of Cryptosporidium parvum was isolated from a naturally infected neonatal calf (NINC) isolate. DNA from the calf isolate was used to prepare a gtll expression library containing overlapping clones which described a partial open reading frame (ORF) for GP900 which comprises 5163 bp nucleotides.

The complete coding sequence of the Iowa isolate GP900 gene, which shows high homology to the NINC gene, was also cloned and sequenced. The sequence was established by PCR using sequencing primers from the NINC sequence and template DNA of the Iowa isolate with subsequent cloning of overlapping sequences in pFusTrx or Bluescript for sequencing. At least two independent clones were sequenced in both directions to identify and correct PCR errors. The 5'region was sequenced from a 2.2 kb BamH1 fragment cloned into Bluescript.

C. Structure of the GP900 Gene and its Encoded Protein Sequences identified as SEQ ID NOs: 1-4 are nucleotide sequences of the GP900 gene or gene fragment of Iowa or NINC

isolates. The sequence identified as SEQ ID NOs: 5 and 6 are the corresponding proteins.

GP900 ORF encodes a multidomain protein based on predicted structural differences as seen in Figure 1. Figure 1A is a schematic drawing of the sequenced 7.6 kb locus showing the position of the open reading frame, domains, expressed fragments, Hinf 1 restriction sites (Hfl) and fragment and the hybridization probe DB8. The protein domain structure and expression clones (S34, ag4, domain 1 and domain 3) are indicated above the 7.6 kb locus. The locus is divided into a 1.5 kb 5'flanking region, a 5511 bp open reading frame and a 0.6 kb 3'flanking region. Vertical marks indicate the position of 22 Hinf 1 restriction sites within the open reading frame. Triangles mark each 1000 bp of DNA sequence.

Inverted triangles mark the proposed methionine initiation site and the termination of the open reading frame. Three Hinf 1 restriction fragments and the hybridization probe DB8 are indicated below the 7.6 kb locus. Figure 1B is deduced amino acid sequence and domains of the GP900 open reading frame. The five cysteines of domain 1 and the seven cysteines of domain 3 are underlined. The LDV motif of domain 1 is in bold print. In domain five representative 8-mer amino acid repeats and tandem repeats are underlined. A region containing the putative transmembrane segment is in bold print and underlined at amino acids 1745-1769. Figure 1C is genomic Southern analysis of the GP900 locus using DB8 as the hybridization probe. Lanes 1-4 contain digested Iowa isolate DNA, lane 5 contains AUCP-1 isolate DNA. The restriction enzymes are: lane 1, Eco R1 ; lane 2, Bgl II ; lane 3, Hin D III; lanes 4 and 5, Hinf 1.

The predicted sequence GP900 open reading frame contains 5 structurally distinct polythreonine domains (SEQ ID NOs: 7- 11) as seen in Figure 4B.

Domains 1 (SEQ ID NO : 7) and 3 (SEQ ID NO: 9) of the protein are cysteine rich domains. Domains 2 (SEQ ID NO: 8) and 4 (SEQ ID NO: 10) are mucin-like domains containing large numbers of threonines.

Domain 1 (SEQ ID NO: 7) contains 5 cysteine residues in the Iowa isolate. Domain 3 has 7 cysteines in the Iowa isolate (SEQ ID NO : 9) but only 6 cysteines in the NINC isolate (SEQ ID NO: 6). Neither domain 1 or domain 3 is highly homologous to any known sequence in GenBank or Swiss Protein Bank.

Domain 2 (SEQ ID NO: 8) and domain 4 (SEQ ID NO : 10) are composed largely of threonine residues. Both domains also contain repeats of the sequence lysine-lysine-proline or lysine-proline. Variants of domain 2 (SEQ ID NOs: 6 and 12- 20) consisting of two NINC isolates variants and eight Iowa isolates variants indicate that size and sequence variants are frequent in this domain. When the deduced protein sequence was analyzed by searches of the GenBank and Swiss Protein Bank, the greatest similarities were found between the threonine-rich regions of GP900 and other glycoproteins with either proven or putative 0-linked glycosylation including a variety of gastrointestinal mucins.

Domain 5 (SEQ ID NO: 11) is composed of a degenerate 8- mer repeat and contains a putative membrane spanning region and a cytoplasmic domain.

GP900 is both N-and O-glycosylated. GP900 has been shown to be susceptible to treatment with N-glycosi. dase F

(N-glycanase) which cleaves high mannose and complex structures.

The presence of abundant cysteines on a surface protein of Cryptosporidium which is functionally homologous to the circumsporozoite protein of malaria strongly suggests that these cysteines participate in binding phenomena and may comprise new binding motifs. Numerous apicomplexan parasite proteins, such as Plasmodium, CSP, Duffy binding protein, EBA and PFEMPI have binding domains which contain cysteine rich regions. N-or 0-linked carbohydrate moieties may also participate in binding to adjacent cells.

D. Expression of GP900 Recombinant Proteins Recombinant GP900 protein useful in the method for diagnosis and detection of Cryptosporidium was cloned and expressed using methods described in Example 10.

Briefly, the S34 insert was subcloned into the glutathione-S transferase vector, expressed as a soluble protein and purified according to supplier's instructions.

Domain 3 (amino acids 520-678) and domain 1 (amino acids 164-303) corresponding to the terminal 139 amino acids of domain 1 which appeared to be a DNA duplication of domain 3 were expressed as thioredoxin fusion proteins in the vector pTrusFux according to supplier's protocols (Invitrogen).

Sense and anti-sense PCR amplification oligonucleotides, which allowed the amplification from Iowa genomic Cryptosporidium DNA of domain 1 or domain 3 with Kpn 1 and Xba I sequences at the 5'and 3'ends respectively, were synthesized. The sense oligonucleotides were: 5'- CAGGTACCCATGAATTGGCCGGTAAGTATC-3' (SEQ ID NO: 21) for domain 1 and 5'-CAGGTACCCTCTGAAACTGAGAGTGTAATT-3'for domain 3 (SEQ

ID NO: 22). The antisense oligonucleotides were: 5'- CCTCAGATTAGTGTTTCACTCCAACACA-3'for domain 1 (SEQ ID NO : 23) and 5'-CCTCTAGATTATACGAAATCAGCTGAAGT-3'for domain 3 (SEQ ID NO: 24).

Amplified fragments were digested with Kpn I and Xba I, purified and introduced in a directional manner into the polylinker region of pTrxFus. Ligation products were transformed into G1724 E. coli and ampicillin resistant colonies were screened by hybridization of colony replicas with 12p_ labeled domain 1 and domain 3. Purified colonies were grown in 1 ml aliquots for analysis. The identity of foreign DNA was verified by sequence analysis. Growth conditions were varied with respect to time and the bacteria lysed for evaluation of soluble and insoluble proteins. Domain 1 and domain 3 were wholly soluble. Yields were maximal at 3 hours of bacterial growth. Domains 1 and 3 were purified by heat treatment and their purity and concentration determined on Coomassie stained gels. Concentration was also determined using the Bradford reagent and UV detection at 595 nm.

E. Oligonucleotide Primers Oligonucleotide primers suitable for PCR detection of GP900 antigen or a portion thereof are nucleotide sequences of about 14 to about 35 bp flanking the GP900 antigen or a portion thereof. The portion of the antigen may be any of its domains disclosed herein or a fragment thereof. The primer thus may be any 14-35 bp sequence flanking the whole GP900 antigen, any of its domain or any of its fragment.

The exemplary primers which were prepared are listed in Table 1.

Table 1 Sense primers 1. 5'-CAGGTACCCA TGAATTGGCC GGTAAGTATC-3'SEQ ID NO : 21 2.5'-CAGGTACCCT CTGAAACTGA GAGTGTAATT-3'SEQ ID NO : 22 3.5'-GGAAGGTTCA ATTGCAGG-3'SEQ ID NO: 23 4.5'-CCATTCAACC CTGTCACTGG AA-3'SEQ ID NO : 24 5.5'-GTCCATTCAA CCTGTC-3'SEQ ID NO : 25 6.5'-CAACTTATGT TGGTGTTATC GG-3'SEQ ID NO : 26 7.5'-CACTCTGGAT ATCAAACTTC A-3'SEQ ID NO : 27 8.5'-GGTTCCAGGT AAGCCACCAA T-3'SEQ ID NO : 28 9.5'-CAGGAATTTC TGCAAGTGAG TC-3'SEQ ID NO : 29 10. 5'-ATGATATTGA GTCAGGTAGA CT-3'SEQ ID NO : 30 11. 5'-CCCAATAATG AAGACACC-3'SEQ ID NO : 31 12.5'-TCAATCCACC AACTGGC-3'SEQ ID NO : 32 13.5'-CCAACAGCAC TGTCTCAGGA TC-3'SEQ ID NO : 33 14.5'-AACTTCAGGT ACTACAAAAC C-3'SEQ ID NO : 34 15.5'-CAGTCAATGG TGGAGGTG-3'SEQ ID NO : 35 16.5'-CTACTGGTAA CATTATTAAC CC-3'SEQ ID NO : 36 17.5'-AACTAATTAC CAATTCCAGG-3'SEQ ID NO : 37 18.5'-CAGATGAAGT AACAGGTTTG-3'SEQ ID NO : 38 19.5'-CAAGAGATCC AGTATCAGGA-3'SEQ ID NO : 39 20.5'-CAATTCCAGG TTCACATTC-3'SEQ ID NO : 40 21.5'-GATCACATTC TGGTACATTA T-3'SEQ ID NO: 41 22.5'-GGTCAAACCT ACCAGG-3'SEQ ID NO : 42 23.5'-CCAATTGATC CAATTAGTTA C-3'SEQ ID NO : 43 24.5'-GACTATAACA GTGGTTTATT A-3'SEQ ID NO: 44 Antisense Primers 25.5'-CCTCTAGATT AGTGTTTCAC TCCAACACA-3'SEQ ID NO : 45 26.5'-CCTCTAGATT ATACGAAATC AGCTGAAGT-3'SEQ ID NO : 46 27.5'-GATACAATGC AAGATTCGCT TCTAATACC TGC-3'SEQ ID NO : 47 28.5-CCACAAGATC TTGAACCAGA AAT-3'SEQ ID NO : 48 29.5'-ATAGTGACCA GAGGGTTG-3'SEQ ID NO : 49 30.5'-CACTAGGCTC AGTGGTCTTA GT-3'SEQ ID NO : 50 31.5'-ATGTGTGCCT TGCTGCCC-3'SEQ ID NO : 51 32.5'-AAATCCAAAC TTCTATCTGG G-3'SEQ ID NO : 52 33.5'-AGTTGGTTTG CTTGGACTTC C-3'SEQ ID NO : 53 34.5'-TTTCGTCTCT CTTGGTA-3'SEQ ID NO : 54

35.5'-ACCGATAAAC CAGACATTG TT-3'SEQ ID NO : 55 36.5'-AGTCTACCTG ACTCAATATC AT-3'SEQ ID NO : 56 37.5'-ATTGGCTTTC CTGTTGAT-3'SEQ ID NO : 57 38.5'-TGGGTTAATA ATGTTACCAG TA-3'SEQ ID NO : 58 39.5'-GGATCATATG GCAAACCAG-3'SEQ ID NO : 59 40.5'-AATGTGAACC TGGAATTGGT T-3'SEQ ID NO : 60 41.5'-CTTCATTACT TGTTGGC-3'SEQ ID NO : 61 42.5'-GTCCTGTTGT TGGATCAG-3'SEQ ID NO : 62 43.5'-CACTGAAATA TTTACCAGAG-3'SEQ ID NO : 63 It is to be understood that the primers listed in Table 1 are exemplary only and that any and all primers which meet conditions stated above are intended to be within the scope of this invention.

F. Production and Assay of GP900 Antibodies Expressed portions of the GP900 loci are targets of polyclonal and monoclonal antibodies able to detect Cryptosporidium invasion. The expression, identification and isolation of these recombinant proteins allows production of recombinant proteins and antibodies to these proteins for the purpose of detection of Cryptosporidium in hosts or the environment and for diagnosis of prior or current Cryptosporidium infection in a suitable host.

Monoclonal antibodies, which are specific for GP900, have been made according to Example 2. Three of six antibodies, namely 10C6, 7B3, and E6, made from a single fusion event in which the immunogen was an oocyst containing sporozoites, were specific to GP900, suggesting that GP900 is a highly immunogenic molecule of sporozoites. Three of eight antibodies, namely M2, M15 and M24 made from a second fusion event, in which the immunogen consisted of meronts, were also specific to GP900, suggesting that GP900 is a highly immunogenic molecule of merozoites.

Antibodies, for the purposes of this invention, are not used as therapeutic agents but only as a tool for identification of GP900 proteins or glycoproteins and as a control component of the diagnostic kit.

Polyclonal antibodies against SDS solubilized GP900 and MAb 10C6, prepared according to Example 4, which were previously shown to detect GP900, were used for detection of molecular species which are immunoprecipitable with both mono and polyclonal antibodies. A Western blot probe of oocyst/sporozoite proteins is seen in Figure 2.

Immunoprecipitation of sporozoite surface labeled proteins with mono and polyclonal antibodies as seen in Figure 3.

Figure 2 shows an immunoblot of Cryptosporidium parvum oocyst/sporozoite proteins of the AUCP-1 isolate separated by SDS-PAGE. Figure 2, lane 1 shows the MAb 10C6 culture supernatant, lane 2 shows the polyclonal anti-GP900 in 1: 5000 dilution.

As seen in Figure 2, a single molecular species, namely protein GP900, was identified at-900 kD by both monoclonal and polyclonal antibodies. Cross-immunoprecipitation studies confirmed that the same protein of approximately 900 kD size, was detected by both antibodies. At prolonged periods of detection, a less prominent ladder of bands between the 200 and 92 kD markers was observed.

Figure 3 shows immunoprecipitation of 12sI radiolabelled Cryptosporidium parvum sporozoite surface proteins of the AUCP-1 isolate separated by 5-15% SDS-PAGE. Figure 3, lane 1 shows radiolabelled Cryptosporidium parvum sporozoite surface protein control (107 sporozoites/lane). Lane 2 shows

radiolabelled Cryptosporidium parvum sporozoite surface proteins immunoprecipitated with polyclonal anti-GP900.

As seen in Figure 3, lane 2, immunoprecipitation of l25I labeled sporozoites with polyclonal anti-GP900 revealed that polyclonal anti-GP900 only detects one protein, GP900, at the surface of sporozoites. Polyclonal anti-GP900 antibody is thus an appropriate antibody for GP900.

In order to prepare reagents for specific portions of GP900 to assay their effects on sporozoite adhesion, invasion and intracellular development in vitro and infection in vivo, polyclonal antibodies were made to'purified wild type ß-galactosidase, and thioredoxin; Ag4-ß-galactosidase and S34-ß-galactosidase fusion proteins; and domain 1 thioredoxin, and domain 3 thioredoxin fusion proteins according to Examples 5 and 6.

In order to further define the antigen and S34 antibodies by removing the reactivity to p-galactosidase, affinity purified antibodies to the Ag4 and S34 portions of their fusion proteins were prepared according to Example 5. These various antibody preparations were used to probe an immunoblot of proteins from Cryptosporidium parvum oocysts/sporozoites as described in Example 7.

G. In vitro and In Vivo Assessment of Activity of Anti-GP900 and Anti-Recombinant GP900 Antibodies Since some antigens localized in the apical complex and extruded from it have been found to be adhesion molecules and the targets of inhibitory antibodies for other Apicomplexan parasites and to be suitable vaccine targets (Cell, 70: 1021 (1992); J. Immunol., 149: 548 (1992)), antibodies to fusion proteins of four expression clones were prepared from the

GP900 locus, domain l (amino acids 164-303), domain 3 (amino acids 520-678), S34 (amino acids 598-964) and Ag4 (amino acids 1030-1226) incorporated within SEQ ID NO : 5.

Immunoglobulin from unimmunized rabbits and rabbits immunized with domain 1 thioredoxin and domain 3 thioredoxin was affinity purified on protein A. Antibodies to S34 were more highly purified on a recombinant S34 affinity column and only antibodies to S34 were present in the final preparation.

Clearly, the clone S34 encodes a Cryptosporidium antigen and the antibodies specifically raised against this antigen are able to detect Cryptosporidium infection in vivo.

Thus antibodies against the recombinant S34 protein are able to detect Cryptosporidium infection in vitro and in vivo indicating the usefulness of the anti-S34 antibody for both anti-Cryptosporidium detection and diagnosis of a human or animal host.

H. GP900 Proteins, Variants and Oligonucleotide Sequences Twenty-four sequences of GP900 identified as SEQ ID NO : 1-24 are disclosed. These sequences were prepared according to methods described in patent 6,071,518.

SEQ ID NO : 1 is the 7334 bp DNA sequence of the Iowa isolate comprised of the open reading frame and 3'and 5' flanking regions.

SEQ ID NO : 2 is the 5511 bp DNA sequence of the GP900 NINC Iowa isolate and is comprised of the ORF.

SEQ ID NO: 3 is the 5318 bp partial DNA sequence GP900 NINC isolate comprised of the partial ORF and 3'flanking region.

SEQ ID NO: 4 is a 5163 bp DNA sequence of GP900 NINC isolate and is comprised of the partial ORF.

SEQ ID NO: 5 is the deduced 1832 aa sequence of GP900 of the Iowa isolate.

SEQ ID NO : 6 is the deduced 1721 partial amino acid sequence GP900 of the NINC isolate.

SEQ ID NO : 7 is an amino acid sequence of domain 1 of GP900 of the Iowa isolate having 303 amino acids.

SEQ ID NO : 8 is an amino acid sequence of domain 2 of GP900 of the Iowa isolate having 216 amino acids.

SEQ ID NO: 9 is an amino acid sequence of domain 3 of GP900 of the Iowa isolate having 159 amino acids.

SEQ ID NO: 10 is an amino acid sequence of domain 4 of GP900 of the Iowa isolate amino acids.

SEQ ID NO: 11 is an amino acid sequence of domain 5 which is 1042 amino acids.

Sequences 12-19 are size and sequence variants comprising domain 2 of Iowa isolate.

SEQ ID NO : 12 is an Iowa isolate variant sequence comprising domain 2 (95 aa domain 2 and conserved flanking amino acids), consisting of 128 amino acids.

SEQ ID NO : 13 is an Iowa isolate variant sequence comprising domain 2 (97 aa domain 2 and conserved flanking amino acids), consisting of 130 amino acids.

SEQ ID NO : 14 is an Iowa isolate variant sequence comprising domain 2 (97 aa domain 2 and conserved flanking amino acids), consisting of 130 amino acids.

SEQ ID NO : 15 is an Iowa isolate variant sequence comprising domain 2 (105 aa domain 2 and conserved flanking amino acids), consisting of 138 amino acids.

SEQ ID NO : 16 is an Iowa isolate variant sequence comprising domain 2 (91 aa domain 2 and conserved flanking amino acids), consisting of 124 amino acids.

SEQ ID NO : 17 is an Iowa isolate variant sequence comprising domain 2 (142 aa domain 2 and conserved flanking amino acids), consisting of 175 amino acids.

SEQ ID NO: 18 is an Iowa isolate variant sequence comprising domain 2 (117 aa domain 2 and conserved flanking amino acids), consisting of 150 amino acids.

SEQ ID NO: 19 is an Iowa isolate variant sequence comprising domain 2 (58 aa domain 2 and conserved flanking amino acids), consisting of 91 amino acids.

SEQ ID NO : 20 is a NINC isolate variant sequence comprising domain 2, consisting of 249 amino acids.

SEQ ID NO: 21 is a sense oligo for domain 1 (216 aa domain 3 and conserved flanking amino acids), consisting of 30 bp.

SEQ ID NO : 22 is a sense oligo for domain 3 consisting of 30 bp.

SEQ ID NO : 23 is an antisense for domain 1, consisting of 2 8 bp.

SEQ ID NO : 24 is an antisense oligo for domain 3 consisting of 29 bp.

I. Variants and Mutants Table 2 comprises an alignment of domain 2 protein variants. These consist of 8 variants (SEQ ID NOs: 12-19) of the Iowa sequence (SEQ ID NO : 5) and 1 variant (SEQ ID NO : 10) of the NINC sequence.

As seen in Table 2, domain 2 polymorphism of the GP900 contains variable numbers of threonine lysine and proline

amino acids. Those are encoded by extensive trinucleotide repeats of the DNA end Sequence (SEQ ID NO: 6). Similar trinucleotide repeat regions occur and have been characterized in the genes responsible for a number of inheritable genetic diseases of man including fragile X syndrome. Insertions and deletions in these trinucleotide repeat regions are reflected in the translated protein which is functionally different. In addition, decreased amount of protein translation has been shown to occur if the repeats are extensive. DNA insertion and deletion are thought to be related to impaired function of DNA repair enzymes and polymerases in regions of perfect repeats. Domain 2 variants reported have from 91 to 216 amino acids in length. However, even longer variants are predicted by PCR of domain 2, including a large number of variants with a domain 2 size of 1.1 kb. These data suggest that domain 2 DNA is a"hot spot"for DNA recombination/mutation.

A method of producing amplified mutants and variants is described in Example 20. Specific variants or mutants Iowa isolate compared to NINC isolate domain 2 of the SEQ ID NO : 5 are shown in Table 2.

480.19-5 PATENT<BR> TABLE 2<BR> Conservatively Modified Mutants and Variants of SEQ ID NO: 5<BR> Var 2 MGSKVYIPYT KCVGVKH.. T TTTTTTTTTT TTTTTTTTTT T........ T SEQ ID NO : 12<BR> Var 3 MGSKVYIPYT KCVGVKHTTT TTTTTTTTTT TTTTTTTTTT T........ T SEQ ID NO : 13<BR> Var 12 MGSKVYIPYT KCVGVKHTTT TTTTTTTTTT TTTTTTTTTT T........ T SEQ ID NO : 14<BR> Var 1 MGSKVYIPYT KCVGVKHTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT SEQ ID NO : 15<BR> Var 4 MGSKVYIPYT KCVGVKHTTT TTTTTTTTTT TTTTTTTTTT T......... SEQ ID NO : 16<BR> Var 11 MGSKVYIPYT KCVGVKHTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT SEQ ID NO : 17<BR> Var 9 MGSKVYIPYT KCVGVKHTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT SEQ ID NO : 18<BR> Var 10 MGSKVYIPYT KCVGVKH................................. SEQ ID NO : 19<BR> NINC MGSKVYIPYT KCVGVKHTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT SEQ ID NO : 20<BR> Var 2..................................... TTT T.........<BR> <P>Var 3..................................... TTT T.........<BR> <P>Var 12..................................... TTT T.........<BR> <P>Var 1..................................... TTT T.........<BR> <P>Var4............................................... ...<BR> <P>Var 11 TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTKKPTTT T.........<BR> <P>Var 9 TTTTTTTTTT AT......................... TTT T.........<BR> <P>Var10.............................................. ....<BR> <P>NINC TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT<BR> Var2..................................................<BR > <P>Var3............................................... ...<BR> <P>Var12.............................................. ....<BR> <P>Var1............................................... ...<BR> <P>Var4............................................... ...<BR> <P>Var11.............................................. ....<BR> <P>Var9............................................... ...<BR> <P>Var10.............................................. ....<BR> <P>NINC TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT<BR> Var 2............... TTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT<BR> Var 3............... TTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT<BR> Var 12............... KPTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT<BR> Var 1............... TTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT<BR> Var 4................ TTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT<BR> Var 11............... TTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT<BR> Var 9............... TTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT

480.19-5 PATENT<BR> Var 10...................... TTTTTTTT TTTTTTTTTT TTTTTTTTTT<BR> NINC TTTTTTTTTT TTKKPTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT<BR> Var 2 TTTKKPTTTT TTTTTTTTKK PTTTTTATTT TTTSETESVI KPDEWCWLE<BR> Var 3 TTTKKPTTTT TTTTTTTTKK PTTTTTATTT TTTSETESVI KPDEWCWLE<BR> Var 12 TTTKKPTTTT TTTTTTTTKK PTTTTTATTT TTTSETESVI KPDEWCWLE<BR> Var 1 TTTKKPTTTT TTTTTTTTKK PTTTTTATTT TTTSETESVI KPDEWCWLE<BR> Var 4 TTTKEPTTTT TTTTTTTTKK PTTTTTATTT TTTSETESVI KPDEWCWLE<BR> Var 11 TTTKKPTTTT TTTTTTTTKK PTTTTTATTT TTTSETESVI KPDEWCWLE<BR> Var 9 TTTKKPTTTT TTTTTTTTKK PTTTTTATTT TTTSETESVI KPDEWCWLE<BR> Var 10 TTT... TTTT TTTTTTTTKK PTTTTTATTT TTTSETESVI KPDEWCWLE<BR> NINC TTTTKPTTTT TTTTTTTTKK PTTTTTATTT TTTSETESVI KPDEWCWLE<BR> wherein F=phe W=trp I=ile<BR> L=leu R=arg T=thr<BR> S=ser g=gly P=pro<BR> Y=tyr E=glu H=his<BR> Z=OCH or AMB a=asp Q=gln<BR> C=cys A=ala N=asn<BR> Z=OPA V=val K=lys<BR> M=met

The NINC sequence seen in the Table 2, corresponds to amino acids 192-392 of the SEQ ID NO: 6. Iowa variant domain 2 sequences (SEQ ID NOs. 12-19) as seen in Table 2 correspond to amino acids 309-524 of SEQ ID NO: 5. Mutations or variations of the GP900 protein thus occur between isolates and within a given isolate.

2. P68 Protein, Recombinant Protein and DNA/RNA A. Identification of Protein P68 as Cryptosporidium Antigen The second antigen protein, designated P68 was identified. The P68 Cryptosporidium antigen is a smaller protein. The P68 protein was partially sequenced at the DNA level. The 3'sequence and 3'flanking regions for P68 are documented. Two P68 DNA sequences were established. SEQ ID NO: 55 comprising 1509 bp encodes a portion of P68. SEQ ID NO : 25 comprising 2380 bp encodes a portion of P68 and includes 3'flanking region. The deduced partial sequences of encoded protein P68 (SEQ ID NO: 26) were established.

A Cryptosporidium antigen designated P68 is an apical protein of sporozoites and merozoites. The protein has a size of between about 50-100 kDa. The P68 protein consists of 503 amino acids and its amino acid sequence is depicted as SEQ ID NO : 26. The P68 protein is derived from the gene S19. The DNA sequences encoding the P68 protein are depicted as SEQ ID NO: 25.

B. Cloning/Sequencing and Genomic Southern Analysis of the Gene for P68 The purification and initial characterization of the S19 clone and the description of the restriction fragment genomic expression library from which it was isolated have been described in (Infect. Immun., 60: 5132-5138 (1992)). A

recombinant eluted antibody from the clone identified a dominant 68 kDa protein on Western blot of oocyst sporozoite proteins and was localized to the anterior end of the sporozoite by indirect fluorescent antibody analysis.

Figure 5 is an immunoblot of AUCP isolate oocyst/sporozoite proteins. Lane 1 was detected with polyclonal anti-sporozoite/oocyst antibodies which had been affinity purified on the S19 fusion protein (S19-REA). As seen in Figure 5, an immunoblot with the antibody identified the protein 68 kDa of Mr less than 69 kDa marker, known as protein. Lane 2 is REA prepared on wild type ß-galactosidase as a negative control.

The S19 insert was subcloned into Bluescript and sequenced. The insert was used as a molecular probe to identify Agtll expression library clones which extended 5'and 3'from S19. A 2380 bp locus was defined. The defined portion of the locus has 1509 bp of open reading frame which remains open at the 5'end.

C. Structure of the Gene and its Encoded Protein Figure 6 shows a restriction map of P68 open reading frame (ORF) DNA.

The sequences of the P68 gene fragment are shown in SEQ ID NOs: 64 and 65. The sequence of the corresponding protein is given in SEQ ID NO: 26.

D. Oligonucleotide Primers Oligonucleotide primers suitable for PCR detection of P68 antigen or a portion thereof are nucleotide sequences of about 14 to about 35 bp flanking the P68 antigen or a portion thereof. The portion of the antigen may be any of its domain disclosed herein or a fragment thereof. The primer thus may

be any 14-30 bp sequence flanking the whole GP900 antigen, any of its domain or any of its fragment.

The exemplary primers which were prepared are listed in Table 3.

Oligonucleotide primers for detection of P68 are listed in Table 3.

TABLE 3 P68 Oliqonucleotide Primers 1.5'-TAAGGGTCAA TTATTTAACC C SEQ ID NO : 69 2.5'-TAATCCACTT CCATCACTAG SEQ ID NO : 70 3.5'-CTAATTCCGT GAGCCTTTAA C SEQ ID NO : 71 4.5'-CCTGGACTTG TTAAGCATAGAT SEQ ID NO : 72 5.5'-TAATCCACTT CCATCACTAG SEQ ID NO : 73 6.5'-CCCAGTAAGT AAGTTGTGTG SEQ ID NO : 74 7.5'-CACATTAAAG ATGCAGGCAA CC SEQ ID NO : 75 8.5'-CAGCATGTGC AACTGTTGGA SEQ ID NO : 76 9.5'-TCAGCATGTG CAACTGTTGG AT SEQ ID NO : 77 10. 5'-TCTAGAAGAC TAGCTACTGG SEQ ID NO : 78 11.5'-TTATGATGAT GAGATAATTA T SEQ ID NO : 79 12.5'-AGAAGCAAGT ATTTCATACTC SEQ ID NO : 80 13.5'-GAAAGCAGAT TCTCTGATAT SEQ ID NO : 81 14.5'-ATTAGATCTC TTCTACATCA SEQ ID NO : 82 15.5'-CCTAAGTGTT TCTTGCCCAA SEQ ID NO : 83 16.5'-CCAGTTCTGG AAGTGCTCCA SEQ ID NO : 84 17.5'-CTTGCTTCTT TATTATAATT SEQ ID NO : 85 18.5'-CATCTTCTTA ATACCTACAA CA SEQ ID NO : 86 19.5'-AATCCCCTTC AGCCTTTTG SEQ ID NO : 87 20.5'-CAATGGTTTG GTTGAACAGA SEQ ID NO : 88 21. 5'-AGTAGGATTT GATGCGCGTA SEQ ID NO : 89

Table 3 lists 21 exemplary sequences of primers suitable for. detection of P68 antigen according to the invention.

It is to be understood that the primers listed in Table 3 are exemplary only and that any and all primers which meet conditions stated above are intended to be within the scope of this invention.

E. Production of P68 Recombinant Proteins Using essentially the same methods as described for GP900 and for clones S34 and Ag4, S19 was subcloned into the pGEX expression vector to yield the expression clone GST-S19, a recombinant protein fused to glutathione S transferase.

Antibodies were raised to GST-S19 in two rabbits and to the native GST, according to methods described in Examples 4 and 5.

E. In Vitro and In Vivo Assessment of Activity of Anti-P68 Antibody Antibodies were assayed in vitro and in vivo as described in patent 6,071,518, incorporated by reference.

3. Cryptopain-Cryptosporidium Parvum Antigen Cryptopain is cathepsin L-like cysteine proteinase.

A. Cryptopain Gene Cloning, Sequencing and Genomic Southern Analysis In order to provide consistently the same antigen for production of antibodies or vaccines, and for recombinant production of fusion proteins and other agents useful for prophylactic therapeutic and diagnostic purposes, cryptopain was cloned, sequenced and genomic Southern analysis was performed to determine whether there was one or more cysteine proteinase similar to cryptopain.

Degenerative oligonucleotides were synthesized from the sequences encoding the active sites of papain like cysteine proteinases centered around the active site cysteine and histidine and around the active site arginine. The conserved cysteine and histidine residues are involved in the active site, and the cysteine residues are apparently involved in disulfide bridges. For cryptopain, the conserved cysteine is

C-24, the conserved histidine is H-164. The proposed disulfide bridges are 21-65,56-103 and 158-210.

Oligonucleotides specific for the Plasmodium vinckei cysteine proteinase were found to be suitable for and were therefore used to amplify a fragment of genomic DNA from Iowa isolate Cryptosporidium parvum oocysts.

The fragment was sequenced using methods described below and known in the art and found to encode a 459 bp portion DNA residues 869-1326 of a cysteine proteinase gene seen in Figure 7. Protein sequence of cryptopain is shown in Figure 8. The fragment (869-1326) was hybridized to an Iowa isolate genomic Southern blot which indicated that the cysteine proteinase was a single copy gene. Results are seen in Figure 9.

Figure 9 is a genomic Southern analysis of Cryptosporidium DNA using the cryptopain probe. In Figure 9, lane 1, the probe hybridizes to two Hind III fragments. These fragments are of approximate size 1.5 and 4 kb. In lane 2, the probe hybridizes with a Hae III fragment of 1.2 kb. In lane III the probe hybridizes to fragments of 1.2 and 4 kb of a Hind III/Hae III digest. In lane 4, the probe identifies fragments of 10 and 1 kb in an NsiI digest. In lane 5, the probe identifies a single band of 4 kb in an ScrII digest and in lane 6 it identifies fragments of 1.0,0.5 and 4 kb in an NsiI/ScrII digests. The presence of 1 or 2 bands greater than the size of the probe in all digests indicates that the cysteine proteinase is a single copy gene.

The 459 bp Iowa fragment was then used to identify naturally infected neonatal calf (NINC) according to Infect.

Immun., 61: 40 (1993) library clone which encoded the entire gene and 5'and 3'flanking regions. The sequence of this

clone appears in Figure 8 and is identified as (SEQ ID NO: 94). The sequence of the open reading frame was determined.

The corresponding sequences of the NINC clone and the 459 bp sequence of the Iowa cysteine proteinase isolate are identical indicating that cryptopain is highly conserved in these isolates and that its function is essential for Cryptosporidium.

SEQ ID NO : 90 is the DNA sequence of the Cryptosporidium cryptopain. The sequence (SEQ ID NO: 90) comprises 1663 base pairs and comprises 5'and 3'flanking sequences, pre, pro (SEQ ID NO: 91) and mature enzyme (SEQ ID NO : 92) sequences.

SEQ ID NO: 93 is the amino acid sequence of the cryptopain. The cryptopain contains 401 amino acids and contains pre and pro fragments (SEQ ID NO: 94), and mature enzyme (SEQ ID NO: 95).

Sequence ID NO : 96 is 459 bp residue representing a sequence of cryptopain probe.

Sequences identified as SEQ ID NOs: 97-108 are primer sequences.

Sequences SEQ ID NOs: 113-115 represent amino acid fragments of cryptopain.

Sequence SEQ ID NO : 116 represents a 1206 fragment of cryptopain DNA.

C. Structure of the Cryptopain Gene and Its Encoded Protein The function of cryptopain is highly correlated with the structure of the protein which is determined by the corresponding sequence. In addition, regulation of the function is, at least in part, dependent upon the presence of the pro sequence.

Sequence identified as SEQ ID NO : 90 (Figure 7) is a DNA sequence of cryptopain. Sequence identified as SEQ ID NO: 93 (Figure 8) is its corresponding protein. Search of the Gene Bank and Swiss Protein Bank revealed that these sequences were highly homologous to cathepsin L-like sequences of various organisms.

D. Oligonucleotide Primers Oligonucleotide primers suitable for PCR detection of cryptopain antigen or a portion thereof are about 14 to about 35 bp nucleotide sequences flanking the cryptopain antigen or a portion thereof. The portion of the antigen may be any of its domain disclosed herein or a fragment thereof. The primer thus may be any 14-35 bp sequence flanking the whole GP900 antigen, any of its domain or any of its fragment.

Oligonucleotide primers for amplification of cryptopain are listed in Table 4.

Table 4 Cryptopain Oligonucleotide Primers 1.5'-GTTGAAGTTA ATTGAAC SEQ ID NO : 97 2.5'-GGTCCGAGAA ATATATTCC SEQ ID NO : 98 3.5'-CAAAGATACA GTAACAACC SEQ ID NO : 99 4.5'-GATTCACAGT ACATATAAAA GATTCC SEQ ID NO : 100 5.5'-GAGACTCTAC TTGACTTAAA TACC SEQ ID NO 101 6.5'-ACAAACCGAG GATTACC SEQ ID NO : 102 7.5'-AACAAATTGC TGTTCACTCA AGC SEQ ID NO : 103 8. 5'-ACTCGTTTGG CTAAGTATGG ACC. SEQ ID NO : 104 9.5'-ATGCTCCTTG TGGAACCAAG SEQ ID NO : 105 10.5f-GCTAGTTTGA TGTATCC SEQ ID NO : 106 11.5'-GATTGATTAA TCACTGGATA C SEQ ID NO : 107 12.5'-GGTATATTGG TTGAGCC SEQ ID NO : 108 The sense and antisense 7B1 and 7B2 primers are listed in Table 5.

Table 5 Sense primers 1. AAAGGATCCT GC/TGGIA/TG/CITG C/TTGGGCITT SEQ ID NO : 109 2. TTTGAATTCC CAIG/CA/TA/GTTIC/T T/GIAC/TIATCCA A/GTA SEQ ID NO : 110

Antisense primers 3. CCAGGTACCA TGGACATAGG AAAC SEQ ID NO: 111 4. CCTCTAGATG CTTATATTGA TTG SEQ ID NO: 112 Primers SEQ ID NO : 109 and 110 are 7B1 sense primers.

Primers SEQ ID NO : 111 and 112 are 7B2 antisense primers for amplification of cryptopain or its fragments. These primers are degenerate primers for active site of cysteine proteinases used to identify the first cryptopain fragment in a library.

The active site cysteine shown at site 200 is embedded in a 7 amino acid all three enzymes and was one of the sites chosen to make degenerate oligonucleotides primers in Table 4.

The conserved arginine at 392 is embedded in an amino acid fragment YWL/IVRNSW (SEQ ID NO: 62). Nonetheless, the degenerate oligonucleotide 782 containing sequence for VRNFW (SEQ ID NO: 63) and the active site cysteine oligonucleotide 781 were specific enough to amplify the 459 bp fragment (SEQ ID NO : 59).

D. Production of Cryptopain Recombinant Protein Recombinant cryptopain proteins are useful as antigens for preparing antibodies which will inactivate cysteine proteinase and provide antibody probes to detect the presence of the organism in the environmental and clinical diagnostic setting. Their recombinant production is therefore important.

Recombinant proteins of the invention were produced.

Generally, the 1203 bp cryptopain open reading frame (ORF) is engineered for in frame expression as a thioredoxin fusion protein in the Invitrogen vector pTrxFus, or any other suitable vector. The vector is used to create C-terminal fusions to E. coli thioredoxin. There is a multiple cloning site which allows in frame fusion of foreign protein with thioredoxin. Between the thioredoxin and the foreign protein

there is an enterokinase cleavage site. Enterokinase treatment permits the release of thioredoxin from the protein. pTrxFus DNA is digested with for example KpNI and XbaI and the intervening fragment is removed for example, by gel purification.

Primers 7B1 and 7B2 were used to amplify the pre pro enzyme sequence from Iowa Cryptosporidium DNA. The primer 7B1 has a KpN1 site and the primer 7B2 has an XbaI site engineered into the 5'end of the oligonucleotides. These enzymes are used to digest the amplified DNA so that it could be inserted directionally and in frame into the KpnI/Xbal restriction digested pTrxFus. Then, the vector, such as pTrxFus, containing the sequence for the pre pro enzyme, is used to transform competent E. coli cells. Ampicillin resistant transformants are then analyzed for plasmid DNA by restriction with KpNI-XbaI and by sequence for the presence, orientation and reading frame of the gene. Clones containing the same gene are induced for expression of cryptopain and expression of the fusion protein, such as for example cryptopain- thioredoxin, at 57 kD, was analyzed by SDS-PAGE followed by immunoblot with antithioredoxin antibody.

Conditions of the actual preparation of recombinant cryptopain using vector pTrxFus described in application Ser.

No. 08/827,171, incorporated by reference.

Fusion protein may be purified by osmotic shock or heat treatment of cell lysates to produce highly purified fusion protein. The fusion protein is advantageously cleaved with enterokinase at a cleavage site comprising 4 asparagine and 1 lysine sequence.

Production of cryptopain may be accomplished in multiple procaryotic or eukaryotic cells, including baculovirus, insect cells, yeast and mammalian cells. Cryptopain is purified by any suitable method known in the art, such as incorporation of histidine and purification by nickel chromatography, heat treatment of fluoredoxin fusion protein with subsequent harvesting of soluble protein.

UTILITY The current invention provides a method and a means for detection of the Cryptosporidium parasite and diagnosis of infection. The following examples describe procedures used to prepare the antigens and antibodies of the invention and methods used for their detection. They are illustrative only and any modification using methods known in the art is intended to be included. The following examples are not to be considered in any way limiting.

EXAMPLE 1 Crystosporidium parvum Parasites This example illustrates the protocol used for isolation of Cryptosporidium parvum parasites.

Oocysts of the Iowa, NINC, AUCP-1 or other isolates of Cryptosporidium parvum described above were passaged through neonatal calves at the Animal Resources Services, University of California, Davis or obtained from a commercial source (Pat Mason) and the oocysts were purified and encysted. The detailed protocol is described in Infect. Immun., 61: 4079 (1993). Oocysts containing sporozoites were solubilized, resolved by SDS-PAGE and subjected to immunoblotting, according to Infect. Immun., 60: 5132 (1992).

EXAMPLE 2

Preparation of Murine Anti-Sporozoite Monoclonal Antibodies This example describes the procedure used for preparation of murine anti-sporozoite monoclonal antibodies.

Polyclonal Antibodies 10 week-old female BALB/c mice were immunized four times intraperitoneally with approximately 5x105 sonicated 105 Cryptosporidium parvum oocysts. The polyclonal antibody fraction of the ascites which was shown to react with the Cryptosporidium parvum sporozoite surface, the oocyst surface, and/or with internal antigens of the oocysts, was assessed by an IFA as described in Infect. Immun., 60: 5132 (1992).

Monoclonal Antibodies For monoclonal antibody production, mice treated as above were immunized intravenously with the supernatant from sonicated Cryptosporidium parvum oocysts three days before fusion as described in J. Immunol., 123: 1548 (1979) and J.

Parasitol., 68: 1029 (1982). Hybridoma supernatants were used as the source of antibodies.

Six sporozoite monoclonal antibodies were obtained. The 10C6, 7B3 and E6 monoclonal antibodies were determined to react with GP900. The supernatants of the corresponding hybridoma cultures were used for immunofluorescence assay (IFA) studies and Western blots.

Using the same protocol, infected MDCK cells were used to immunize mice and 3 MAbs to GP900, namely M2, M10, M24 were produced.

Polyclonal and monoclonal antibodies against antigen P68 and cryptopain are prepared in the same way and/or as described in the application Serial No. 08/827,171 or US

patents 6,071,518,6,015,882 and 5,643,772, incorporated by reference.

EXAMPLE 3 Detection of Traits of GP900 Deposited on Poly-L-lysine This example illustrates detection of GP900 traits.

In order to determine whether GP900 was shed by the Cryptosporidium sporozoite in the absence of a specific antibody, living sporozoites were allowed to glide on poly-L-lysine coated microscopic slides. Slides were fixed in formalin and GP900 detected by incubation with MAb 7B3 followed by fluorescein labeled anti-mouse second antibody.

MAb 7B3 had previously (data not shown) been shown to detect only one protein, GP900, in sporozoites.

The sporozoites were shown to be surrounded by GP900 which was shed posteriorly as the sporozoites glided on the poly-L-lysine coated slides. This reaction occurred in the absence of specific antibody which was added only for detection purposes after fixation of the sporozoites.

EXAMPLE 4 Production of Polyclonal. Anti-GP900 This example describes the procedure used for preparation of anti-GP900 polyclonal antibodies.

The Triton X-100 (1%) soluble fraction of 2 x 108 oocysts was immunoprecipitated with MAb lOC6. A >900 kD MW species was identified in gels stained with Coomassie blue in water and excised. Frozen gel containing 2 X 107 oocyst/sporozoites was pulverized and emulsified in 150 Al PI of PBS and 150 Al complete Freund's adjuvant (CFA) for intraperitoneal (IP) immunization of mice.

Subsequently, the mice were immunized (IP) three times with the same antigen dissolved in incomplete Freunds adjuvant (ICFA) at approximately 2 week intervals. The anti-GP900 antibody at a dilution of 1: 5000 recognized GP900 on Western blots.

Anti-P68 and anticryptopain polyclonal antibodies were prepared in substantially the same way and/or as described in the application Serial No. 08/827, 171 or US patents 6,071,518, 6,015,882and 5,643,772, incorporated herein by reference.

EXAMPLE 5 Production of Polyclonal Antibody Against Aq4 and S34 Fusion Proteins This example describes the procedure used for preparation of the anti-Ag4 and anti-S34 fusion protein polyclonal antibodies.

Lysogens were produced from the Ag4 and S34 gtll clones.

Cell lysates and purified protein were made using a protocol and reagents obtained from Promega. Purified fusion protein were emulsified in CFA and injected into rabbits. These injections continued at two week intervals with the substitution of ICFA. Rabbits were sacrificed at the end of 3 months and the antibody was assayed by Western analysis to verify that the antibody recognized a protein >900 kD.

The p-galactosidase and Ag4-ß-galactosidase fusion proteins were purified essentially as described by Promega except that the buffering system used was phosphate buffered saline (PBS) pH 7.4.. The purified fusion proteins were then coupled to CNBR sepharose using standard techniques. The antibodies to Ag4-ß-galactosidase were depleted by passaging serum over a CNBR sepharose column coupled to ß-galactosidase

alone. The flow through fraction was applied to a CNBR sepharose column coupled to the purified Ag4 fusion protein.

Antibodies directed against the Ag4 portion of the fusion protein were eluted in 0.1 M glycine at a pH of 2.4 and immediately neutralized in 200 Al of 2M Tris, pH 7.4. All affinity purified antibodies reacted with the fusion protein and the respective Cryptosporidium protein but not other E. coli proteins.

S34 was subcloned in GST and coupled to a column CNBR sepharose. Antibodies to S34--galactosidase were passed over this column. Antibodies directed against the S34 portion of the fusion protein were eluted in 1M Na thiocyanate and desalted and concentrated.

Anti-P68 and anti-cryptopain polyclonal antibodies were produced in substantially the same way and/or as described in the application Serial No. 08/827,171 or US patents 6,071,518, 6,015,882 and 5,643,772, incorporated herein by reference.

EXAMPLE 6 Production and Affinity Purification of Polyclonal Antibody Against GP900 Domain 1 and Domain 3 Fusion Proteins and Control Antibody This example describes production and purification of antibodies against GP900 domains 1 and 3 fusion proteins.

Purified domain 1 and domain 3 thioredoxin were prepared.

Two to three pg of fusion protein after purification were emulsified in CFA or ICFA and injected at two week intervals.

Rabbits were sacrificed at 3 months and the antibodies were assayed by immunoblot analysis to verify that they recognized GP900.

Polyclonal rabbit antisera from an unimmunized rabbit was evaluated for reactivity against Cryptosporidium antigens at a 1: 1000 dilution on immunoblot and found to be free of reactivity. One ml of polyclonal rabbit antisera, anti-domain 1 or anti-domain 3 antisera was diluted with an equal volume of 100 mM Tris (pH 8.0) and passed through a 1 ml protein A bead column 2 times. After washing with 100 mM and 10 mM Tris (pH 8.0), the column was eluted with 100 mM glycine (pH 3.0) in a stepwise fraction. Aliquots of 500 y1 were collected into 50 Al of 1.0 M Tris, pH 6. 0. Antibody concentration was determined by absorbance at 280 nm and integrity of the Ig was verified by SDS-PAGE. Positive control antibody, HBC Ig 40529 has been previously described in Infect. Immunol., 61: (10); 4079 (1993).

EXAMPLE 7 Western Analysis This example describes the Western analysis method used to identify the molecular targets of antibodies.

Oocysts (106 lane) were solubilized in denaturing sample buffer containing 5% ßME (-mercaptoethanol), resolved by SDS-PAGE and subjected to immunoblotting according to Infect.

Immunol., 60: 532 (1992). Proteins were visualized after incubation with primary antibody withal anti-rabbit or anti- mouse IgG conjugated with horseradish peroxidase or alkaline phosphatase followed by calorimetric or chemiluminescent development.

EXAMPLE 8 Southern Hybridization and Northern Blot Analysis This example describes the Southern hybridization and Northern blot methods used for analysis of GP900 used for detection of Cryptosporidium.

A. Southern Hybridization DNA was purified from 1 x 109 Cryptosporidium parvum oocysts as described in Example 1. DNA was digested with the restriction enzymes according to procedures provided by the manufacturer Promega. Digested DNAs were subjected to electrophoresis in 0.8% agarose gels in 1X TAE or 0.5X TBE.

The gel was blotted to a nylon membrane (Hybond N+, Amersham) per manufacturer's instructions. The probe was labeled with 32P-ATP and hybridized to the membrane by methods known in the art. Results are seen in Figure 6 where Lanes 1-4 show Iowa isolate DNA and Lane 5 shows AUCP isolate DNA. Lane 1, EcoRI digest; Lane 2, Bgl II digest; Lane 3, Hinf III digest; Lanes 4 and 5, Hinf I digest.

B. Northern Blot Analysis mRNA was purified from MDCK cells, or MDCK cells infected with sporozoites at a ratio of 1 oocyst/1 MDCK cell, harvested at 24 and 48 hours using guanidinium thiocyanate and oligo-dT

cellulose isolation (Ambion mRNA purification kit, Albion, Inc,, Austin, TX). Ten Hg of poly-A RNA was separated on a formamide gel, transferred and hybridized as described for Southern hybridization. The Northern blot was probed with 32p- adATP labeled domain 3 DNA and washed under stringent conditions.

EXAMPLE 9 Surface Radioiodination and Immunoprecipitation of Cryptosporidium Sporozoite Proteins This example describes the methods used for surface radio-iodination and immunoprecipitation of Cryptosporidium sporozoite proteins.

Oocysts were bleached, encysted and separated from sporozoites prior to iodination of the sporozoite surface and immunoprecipitation of surface proteins as previously described in Infect. Immun. (1993).

A membrane pellet was prepared by centrifuging 1.1 x 107 sporozoites per ml NETT (0.15 M NaCl, 5mM EDTA, 0.5 M Tris, 0.5% Triton X-100, pH 7.4) at 100,000 x g for 1 hour at 40°C.

An aliquot of membrane proteins in 2% SDS 5% p-sample buffer was prepared for total sporozoite surface protein analysis.

Aliquots of membrane proteins extracted in 2% SDS were diluted with 9 volumes NETT plus 1% high quality bovine serum albumin (BSA) obtained from Sigma; 1 volume 1% Triton X-100; proteinase inhibitors and either MAb 10C6 or anti-GP900 were added for overnight incubation. Protein A Sepharose 4B beads were added to immobilize the immunoprecipitated proteins.

Parasite proteins were solubilized in 2% SDS sample buffer containing (3-mercaptoethanol. Samples were boiled 5 minutes and separated by 5-15% gradient SDS-PAGE.

EXAMPLE 10 Cloning and Sequencing of a GP900 Locus This example illustrates the procedure used for cloning and sequencing of a GP900 locus.

The purification and initial characterization of the S34 clone and the description of the restriction fragment genomic expression library of the NINC isolate from which it was isolated have been described in (Infect. Immunol., 60 : 2343 (1992)). The Ag4 clone was isolated from the same library as an expression clone which reacted with polyclonal anti-GP900 antibody. The inserts of the S34 and Ag4 clones were subcloned into BlueScript obtained from Stratagene and sequenced in both directions using Sequenase Version 2.0 DNA Sequencing Kit (UBC) or cycle sequencing (New England Biolabs).

DBS, a 3154 bp insert, which contained the sequences of both S34 and Ag4 was identified by a double of screen of the library using these DNA inserts. PCR amplification products generated from the ends of DB8 and subsequent clones were used to screen the library to identify new clones which extend the sequence of the NINC isolate GP900 3'and 5'.

The Iowa sequence was established. The complete encoding sequence of the Iowa isolate GP900 gene, which shows high homology to the NINC gene, was also cloned and sequenced. The sequence was established by PCR using sequencing primers form the NINC sequence and template DNA of the Iowa isolate with subsequent cloning of overlapping sequences in pFusTrx or BlueScript for sequencing. At least 2 independent clones were sequenced in both directions to identify and correct PCR

errors. The 5'region was sequenced from a 2.2 kb BamH1 fragment cloned into BlueScript.

The GP900 reading frame was verified by the inframe expression of S34 and Ag 4 as ß-galactosidase fusion proteins and domains 1 and 3 as thioredoxin fusion proteins. All 4 fusion proteins elicited antibodies to GP900 when used to immunize animals (Data not shown). The FastA and blastp programs of GenBank were used to perform homology searches of the Swiss Protein database and showed homology of GP900 to mucin-like proteins.

P68 and cryptopain antigens were cloned and sequenced in substantially the same way or as described in the application Serial No. 08/827,171 or US patents 6,071,518,6,015,882 and 5,643,772.

EXAMPLE 11 Purification of Recombinant GP900 Proteins This example describes the purification procedure for GP900 proteins.

Iowa oocysts (5 x 108) were excysted at 37°C for two hours and pelleted at 4,000 x g for 10 minutes at 4°C. The supernatant was aspirated and proteinase inhibitors were added to it to a concentration of pef-abloc (AEBSF) 1 nM, leupeptin 20 AM, iodoacetamide 10 mM, PMSf 2 mM. The supernatant was concentrated by ultrafiltration to 350 Al (14.2 X) (Centricon 10, Amicon). Silver stained SDS-PAGE gel of 10 and 20 Al aliquots revealed equal amounts of 47 kD, 120 kD and >900 kD proteins. The >900 kD protein was purified by ultrafiltration and (Centricon 100) and the concentration determined by silver stain and comparison to transferrin standards.

The substantially same procedure is used for purification of recombinant P68 and cryptopain, as described in the application Serial No. 08/827,171 or US patents 6,071,518, 6,015,882 and 5,643,772, incorporated herein by reference.

EXAMPLE 12 Preparation of Mutant and Variants of GP900 This example illustrates the method used to prepare mutant or variant products.

Genomic DNA from the Iowa and several other strains was subjected to PCR amplification using primers which were situated outside of domain 2, in the distal region of domain 1 and the proximal region of domain 3. Three prominent bands of different sizes were observed when the PCR products were visualized by ethidium staining of a gel in all of these strains. As a control for TAC polymerase, DB8 DNA was also amplified by the polymerase chain reaction. Only a 700 bp amplification product from DB8 was detected indicating that the multiple bands were a product of amplification of sequences present in the genomic DNA, and were not an artifact of the PCR process.

Two of the amplification product bands were cloned into sequencing vectors and 4 clones from each of the products were sequenced to determine their relationship to the NINC domain 2 sequence (Table 1). All 8 sequences had an open reading frame indicating that they were portions of DNA which could be the blueprint for a GP900 protein. All 8 sequences appeared to have in-frame (multiples of 3) DNA deletions with respect to the NINC sequence. All 8 coded for a domain 2 which had conservation of the threonine rich regions, but all 8 differed from each other. The DNA data indicate that mutation in

domain 8 is common. The conservation of threonines and the in-frame nature of all 8 clones indicate that there are selection pressures acting at the level of the protein (presumably production of a functional protein which will allow for invasion and propagation) which determine which genotypic variants are maintained in a strain. Results show that there are mutants of GP900 which are maintained in an isolate's gene pool, presumably as variant alleles at a single locus in haploid stages of the organism.

Using a similar procedure, polymorphism of P68 and cryptopain is determined.

EXAMPLE 13 Preparation of Recombinant Domain 1 and Domain 3 This example describes the preparation of recombinant S34, Ag4, domains 1 and 3.

The oligonucleotide primers were synthesized at the Biomedical Research Center, University of California, San Francisco. A sense oligonucleotide (SEQ ID NOs: 21 and 22) are comprised of a KpnI restriction enzyme recognition site at the 5'end followed by coding sequence for the 5'end of the domain 1 sequence. The sense oligonucleotide for domain 3 is comprised of a KpnI restriction site at the 5'end followed by coding sequence for the 5'end of the domain 3 sequence. The anti-sense oligonucleotide for domain 1 is comprised of an Xbal sequence at the 5'end followed by the antisense coding sequence of the 3'end of the domain 1 sequence. The antisense Oligonucleotide for domain 3 is comprised of an Xbal sequence of the 5'end followed by the anti-sense coding sequence of the 3'end of the domain 1 sequence. When used as

a pair of PCR amplification oligonucleotides, these oligonucleotides allowed the amplification from genomic Cryptosporidium DNA of the entire domain 1 and domain 3 sequences with Kpn 1 and Xba I sequences at the 5'and 3' ends, respectively.

The oligonucleotide sequences were designed so that after Kpnl and Xba I digestion of the amplification product, the resultant fragment could be introduced in a directional manner into pTrxFus which was cut with Kpnl/XbaI. Amplified and restricted DNA was visualized on a 0.8% agarose-lXTAE gel using ethidium bromide. The amplified and endonuclease restricted band was excised from the gel and purified using a glass bead technique (Gene-Clean). pTrxFus was also digested with the enzymes KpnI and Xba I, enzymes uniquely present in the sequence in the poly linker, and the small intervening sequence was removed by gel purification as noted above. pTrxFus and domain 1 or domain 3 DNA, prepared in this manner, at 1: 1 and 1 : 5 molar ratios were ligated overnight at 14°C in the presence of ligation buffer and T4 DNA ligase at a concentration of 50-250 ng insert DNA/ 10 y1.

G1724 chemically competent cells were made as described by Invitrogen. Three to five pl of ligation mixes and control mixes were introduced into separate tubes of competent cells and the tubes were incubated on ice for 30 minutes. Tubes were incubated in a 42°C heating block for 90 seconds and placed on ice for 2 minutes. Eight hundred Al of room temperature of enriched tryptone containing broth medium was added to each tube and the tube was incubated with shaking at 30°C for 60 minutes. Twenty-five and 100 Al of each

transformation mix was plated on RMG-Ampicillin transformation plates and the plates were incubated at 30°C overnight.

Nitrocellulose membrane replicas of colonies were prepared from the transformation plates, the adherent cells lysed in alkaline solution and the DNA fixed to the membranes.

Nitrocellulose membranes were hybridized with domain 1 and DNA 3 and positive colonies were purified. DNA was extracted from the relevant bacteria and the identity of the foreign DNA verified by restriction analysis and sequence analysis.

Purified colonies were grown in 1 Al aliquots for analysis. Growth conditions were varied with respect to time (2,3,4 hours) and the bacteria lysed for evaluation of soluble and insoluble proteins.

EXAMPLE 14 Detection of Cryptosporidium in Stool Samples Using Monoclonal Anti-Cryptosporidium Antibody This example describes immunodetection of Cryptosporidium in stool samples.

After vigorous mixing, a 10-mL aliquot of stool was diluted 1: 4 with 10% buffered formalin (Fisher Scientific, Pittsburgh). The diluted sample was agitated until complete suspension was achieved. A 5-uL aliquot was removed, placed in a defined area of a glass slide, and allowed to air dry.

The slide was incubated with a fluorescein isothiocyanate- labeled monoclonal antibody M10 or 10C6 to GP900 Cryptosporidium oocysts antigen in a modified kit purchased from Meridian Diagnostics, Cincinnati. Positive and negative control reagents supplied with the kits were included in each test. Oocysts in 10 x 40 fields (2.5% of the sample area) were counted by fluorescence microscopy. If no organisms were

seen, the entire slide was examined using a X20 objective, and all oocysts were counted.

Each specimen was assayed on 3 separate days. The mean of triplicate counts in the 5-uL aliquot was standardized to the number of oocysts per milliliter of stool, taking into account the 1: 4 dilution in preservative. Total oocyst excretion per specimen was calculated by multiplying the mean concentration (oocysts/milliliter) by stool weight (grams).

Total daily oocyst excretion was the sum of oocysts in each specimen in a 24-h period.

EXAMPLE 15 Detection of Cryptosporidium in Biopsy This example describes the procedure for detection of Cryptosporidium in tissue biopsy samples.

Duodenal biopsy specimens were obtained at the time of upper gastrointestinal endoscopy. The samples were homogenized and homogenate was solubilized by subjecting the portion of homogenate to P-mercaptocthanol or sodium hydroxide. The suspension was treated similarly to the stool sample and reacted with the specific antibody 7B3 to GP900.

Additionally, the second fluorescein conjugated antibody E6, M2 or M24 is added and the formation of fluorescence for antigen-antibody complex is detected.

EXAMPLE 16 PCR Assay for Detection of Cryptosporidium DNA in Feces This example describes conditions for PCR assay for detection of DNA from Cryptosporidium.

DNA extraction of oocyst from stool was performed as follows. One-gram portions of fecal material were mixed with 40 ml of 0.35% sodium hydrochloride until suspension was formed. Twenty milliliters of the suspension was shaken for min and centrifuged at 1,600 X g for 15 min. The pellet,

resuspended in 15 ml of deionized water, was layered over a two-layer sodium chloride gradient (specific gravities, 1.1 and 1.05) interface and was diluted with 10 ml of deionized water, and half of this suspension was filtered through a 3- pm-millipore filter. The filter was washed with 3 ml Tris HC1 (pH 8). Recovery of the oocysts was completed by shaking the filters for 30 seconds in the tubes with Tris HC1. After discarding the filters, the samples were centrifuged at 16,000 X g for 3-5 min and the final pellet was recovered in 50 pi of 10 mM Tris HC1 (pH 8) were lysed for DNA extraction, as described in Example 10.

The PCR amplification of Cryptosporidium Iowa isolate domain 3 was performed as follows. For each sample analyzed, 10 pi of lysate was used as a template in 50-vil reaction mixtures containing 75 mM Tris (pH9), 20 mM (NH4) 2SO4, 0. 1% (wt/vol) Tween 20,2 mM MgCl2, 0.2 mM (each) dGTP, dATP, and dCTP, 0.6 mM dUTP, 50 pmol (each) of primers 5'-CAGGTACCCA TGAATTGGCCGGTAAGTATC-3'SEQ ID NO : 21,5'-CAGGTACCCT CTGAAACTGAGAGTGTAATT-3'SEQ ID NO: 22,5'-CCTCTAGATTA GTGTTTCACTCCAACACA-3'SEQ ID NO: 45,5'-CCTCTAGATTATACGAAATC AGCTGAAGT-3'SEQ ID NO: 46,0.5 U of uracil-N-glycosylase (UNG; Boheringer Mannheim), and 1 U of Taq DNA polymerase (Eurogentec). Reaction mixtures were incubated for 10 min at 22°C and after denaturation at 94°C for 10 min, the samples were subjected to 50 cycles of 1 min at 94°C, 90 seconds at 56°C, and 90 seconds at 72°C, followed by a 5-min extension at 72°C. PCR products were then suspended in the sample buffer containing 1.2 M urea, 3.4% saccharose, 10 mM EDTA, and 0.002% bromophenol blue, and 10 pi was analyzed on horizontal agarose gels in TAE buffer (40 mM Tris acetate, 2 mM Na2 EDTA-2H20).

Each amplification run contained a negative control

(extraction buffer) and a positive control (genomic DNA from Iowa isolated domain 3).

Example 17 Detection of Cyclosporidium in Stool This example describes the techniques used for detection of Cryptosporidium in stool samples using specific anti-GP900 antibodies.

Fresh fecal specimens were obtained for detection of Cryptosporidium antibodies. Fecal samples were diluted one in three (weight by volume) with distilled water and thoroughly mixed using a vortex mixer.

From each of the fecal samples, a sample of 10 ul was separated and smeared on marked areas of 1 cm2 on glass microscope slides. Three smears were prepared from each of the ten samples, and these were stained with either Mab 10C6, M2 or M10. Examination was performed at X400 magnification under bright-field microscopy, and fluorescence microscopy.

Total and average numbers of oocysts were recorded for every ten fields in each smear. Negative control smears were prepared from samples which were not inoculated with oocysts.

EXAMPLE 18 Preparation of Probes and Primers This example describes the procedure used for the preparation of probes and primers.

Preparation DNA Probe The probe were prepared by the modified method described in Am. J. Trop. Med. Hyg., 45: 688-699 (1991).

Purified Cyclosporidium genomic DNA of GP900, PG8 cryptopain or fragment thereof was digested to completion with the restriction enzyme Hind III, and the resultant fragments ligated into pUC18 plasmid vectors. These vectors were used to transform the DH5 a strain Escherichia coli host bacteria,

which were plated on Luria-Bertani (LB) agar supplemented with ampicillin (50 ug/ml), 20 mM isopropyl -D-thio- galactopyranoside (IPTG), and 5-bromo-4-chloro-3-indolyl -D- galactopyranoside (X-gal) (80 VgZml), and incubated overnight.

A colony hybridization procedure was performed and filters were screened with 32P-labeled, genomic Cyclosporidium DNA obtained from GP900, P68 or cryptopain Cryptosporidium antigens. Duplicate filters were screened by the same method using genomic DNA from G. lamblia, P. falciparum, Toxoplasma gondii, and Trichomonas vaginalis.

Individual colonies that hybridized only with Cyclosporidium DNA were grown overnight in 5 ml of LB medium, and the DNA was extracted by the mini-prep procedure according to method described in Molecular Cloning : A Laboratory Manual, 2nd Ed, Cold Harbor Spring Laboratory Press (1989). One clone designated GP900 as defined above, was selected and sequenced by the Sanger dideoxy chain termination method using a Sequences (United States Biochemicals, Cleveland, OH) kit.

Sequence data were analyzed with MicroGenie software (Beckman Instruments, Palo Alto, CA), and compared with GenBank and National Biomedical Research Foundation databases for homology with. DNA and amino acid sequences, respectively.

Preparation of Oligonucleotide Primers A. pair of 26-base primers and two 20-base probes homologous to the central region of the 452-base target sequence were synthesized in an Applied Biosystems (Foster City, CA) model 380 B DNA Synthesizer according to manufacturer's specifications.

Primers for detection of GP900, P68 or cryptopain were prepared as above and their sequences are listed in Tables 1 and 3-5.

EXAMPLE 19 Polymerase Chain Reaction for Detection of Cryptosporidium Isolates This example describes PCR amplification procedure used for detection of Cryptosporidium isolates.

DNA'sequence from GP900 P68, cryptopain or a fragment comprising approximately 400 bp was selected as the target for amplification by PCR. The PCR reaction mixture (final volume 103 pi) consisted of 68.5 pi of sterile, distilled, deionized water, 10 pi of 10 x PCR reaction buffer (500 mM KC1,100 mM Tris-HC1, pH 8.4,15 mM MgCl2, 0.1% gelatin), 10 pi of 2 mM nucleotide mix, 0.5 psi (2.5 units) of Taq DNA polymerase (Bethesda Research Laboratories, Gaithersburg, MD), 2 pi of 100 mM primer (1 pi each of forward and reverse primer), 2 pi of dimethylsulfoxide, and 10 pi of sample DNA.

Prior to adding the DNA samples, the mixtures were irradiated for 5 min with a UV transilluminator to destroy any contaminating DNA that might have been accidentally introduced. This mixture was centrifuged at 12,000 x g for 15 seconds and covered with 75 pi of mineral oil (Sigma, St.

Louis, MO). A positive control, consisting of linearized plasmid, purified C. parvum DNA, or amplified product, and three negative controls, consisting of reaction mixtures minus Taq DNA polymerase, primers, or template DNA, were used in each experiment. Reactions were carried out in a Perkin-Elmer (Norwalk, CT) DNA Thermal Cycler. Samples were denatured at 94°C for 1 min, annealed at 45°C for 2 min, and extended at 72°C for 3 min. This cycle was repeated 35 times, followed by a 9-min incubation at 72°C.

Following the PCR, DNA hybridization and detection was performed.

10-pl aliquots of product were electrophoresed on 6% polyacrylamide gels and 1.8% agarose gels. The gels were stained with ethidium bromide and viewed under ultraviolet light, and the resultant band patterns were photographed. The DNA bands in the agarose gels were then transferred to Hybond- N nylon membranes (Amersham, Arlington Heights, IL), using a Bio-Rad (Richmond, CA) Model 785 vacuum blotter, with a transfer time of 90 min. Transferred DNA was alkali-fixed to the membranes by soaking them on sheets of Whatman (Waltham, MA) filter paper that was saturated with 0.4 M NaOH for 20 minutes, then rinsed for 1 minute in 5 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate).

A 20-base synthetic oligonucleotide probe corresponding to the middle portion of the target segment was 3'-end-labeled with digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, IN), following the manufacturer's directions included in the Genius non-radioactive DNA labeling and detection kit.

Briefly, the following components were added in order to a 500 pi reaction tube: 4 pi of 5 x terminal transferase reaction buffer, 1.2 pi of CoCl2, 2.5 pi of digoxigenin-11-dUTP, 1 pl of probe (35 pmoles), 1 pi of dATP (0.1 nmoles), 8. 3 pi of sterile water, and 2 pi of terminal transferase. The reaction mixture was incubated at 37°C for 5 min, precipitated with 0.4 M LiCl and ethanol, vacuum-dried, and resuspended in 20 pi of TE buffer with 0.1% SDS. Molecular size markers electrophoresed with the PCR samples consisted of a Hind III digest of A DNA and a Hae III digest of cox174 RF DNA with digoxigenin-11-dUTP.

Membranes were prehybridized for 1 hr at 42°C in 5 x SSC, 5% (w/v) SDS, and 50% (v/v) formamide (hybridization solution). After prehybridization, the membranes were transferred to heat-sealable bags. Approximately 2 ml of

hybridization solution was added to the bags, followed by 10 pl of digoxigenein-11-dUTP-labeled probe and 1 ul of labeled X and OX174RF marker DNAs. The bags were heat-sealed and hybridized overnight in a circulating water bath at 42°C.

After hybridization, the membranes were washed twice (5 min/wash)'at room temperature in 50 ml of 20 x SSC, 0.1% 9w/v) SDS, followed by two washes (15 min/wash) in 0.1% (w/v) SDS at 50°C. Prior to antibody labeling, the membranes were equilibrated for 1 min at room temperature in 100 mM Tris-HCl, pH 7.5,150 mM NaCl. The membranes were then blocked in 100 mM Tris-HCl, pH 7.5,150 mM NaCl, and 2% (w/v) blocking reagent and anti-digoxigenin alkaline phosphatase conjugate at a working concentration of 150 mU/ml, and incubated at room temperature for 30 min with gentle agitation. The membranes were then washed twice (15 min/wash) in 100 mM Tris-HCl, pH 7.5,150 mM NaCl, at room temperature. After they were washed, the membranes were equilibrated for 2 min in 100 mM Tris-HCl, pH 9.5,100 mM NaCl, 50 mM MgCl2. Approximately 3 ml of chemiluminescent substrate, Lumi Phos 530@ (Boehringer Mannheim), was added to a plastic dish. The membranes were pulled through the Lumi Phos 530@ to wet them, excess liquid was allowed to drip off, and the membranes were sealed in hybridization bags. The membranes were then incubated at 37°C for 30 min to allow light emission to reach a steady state, and then exposed to Kodak (Rochester, NY) x-ray film for 5-15 min. Films were developed in a Kodak X-Omat processor.

8. The method of claim 7 wherein the detection is a direct hybridization or an amplification of specific nucleic acids in the sample.

9. The method of claim 8 wherein the amplification of the specific nucleic acid is by PCR primers.

10. The method of claim 9 wherein the PCR primers are oligonucleotides of about 14 to about 35 bp in length.

11. The method of claim 10 wherein the PCR primers are portions of the GP900, P68 or cryptopain genomic DNA.

12. The method of claim 11 wherein the primers are selected to be in opposite orientations for the detection and amplification of the DNA positioned between the primers.

13. A kit for detection of Cryptosporidium comprising: a means for contacting a biological or environmental sample with a means for detecting Cryptosporidium in said sample; a means for detection of Cryptosporidium in said sample, said means selected from an antigen, an antibody, DNA or RNA of GP900, P68 or cryptopain; and a means of detecting the presence of Cryptosporidium in the sample and by detecting a reaction between the antigen, antibody, DNA or RNA and the sample.

14. The kit of claim 13 wherein the detection means is the GP900, P68 or cryptopain antibody and the detected reaction between the Cryptosporidium and the antibody is an antigen-antibody complex.