Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Parasitic organism or component thereof or substance...
Reexamination Certificate
2001-09-24
2004-10-26
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Parasitic organism or component thereof or substance...
C424S265100, C424S192100, C424S191100, C424S185100, C424S184100, C424S178100, C424S134100, C435S069700, C435S070100, C435S070210, C435S071100, C536S023400, C514S04400A
Reexamination Certificate
active
06808714
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to the fields of microbiology and veterinary medicine. More particularly, the invention concerns compositions and methods relating to detecting
Sarcocystis neurona.
BACKGROUND
Equine Protozoal Myeloencephalitis (EPM) is a common cause of neurologic disease in New World horses. It is caused by a parasite termed
Sarcocystis neurona
(
S. neurona
), an obligatory intracellular apicomplexan parasite whose multi-phase life cycle is completed in either one or two hosts.
S. neurona
is known to cycle naturally between opossums and both none-banded armadillos and striped skunks. Horses typically become infected by consuming infectious parasite stages found in opossum feces. Once a horse has been infected,
S. neurona
can travel to the brain and spinal cord, where merozoite stages of this parasite replicate and cause pathology.
Horses with EPM typically present with lameness, but may alternatively or additionally present with symptoms characteristic of primary brain disease. Because the parasite can inhabit any area of the central nervous system (CNS) of the horse, symptoms associated with EPM can vary widely. The degree of infection can range from subtle to severe and can involve the brain and/or the spinal cord. EPM is usually progressive.
Presently, a definitive diagnosis of EPM is made by post-mortem examination, where
S. neurona
organisms are identified in histological lesions. The organ may also be cultured from the lesion. The presence of the organism in the histologic section or when cultured from the lesion establishes the diagnosis. Heretofore, pre-mortem methods for diagnosing EPM were based on assays using whole merozoites, and not a purified protein, to probe for the presence of anti-
S. neurona
antibodies (as an indication of infection) in the horses. The use of such whole merozoites results in significant cross-reaction with non-
S. neurona
specific antibodies (e.g., those against other Sarcocystis species). This cross-reactivity obscures interpretation of results using whole merozoite-based assays.
SUMMARY
The invention relates to the discovery and characterization of a 29 kilodalton (kDa) protein found on the surface of merozoite stage
S. neurona
. This antigen, termed SnSAG-1 or SnSMA1, is an immunodominant antigen recognized on protein blots. Using purified or recombinant SnSAG-1 (i.e., rSnSAG-1) antigen, accurate assays for diagnosing EPM in horse pre-mortem have been developed. These assays involve identifying a marker indicative of the presence of the 29 kDa antigen or an antibody to this antigen in a sample to be tested. Thus, because a single purified antigen or marker is utilized in such assays, the cross-reactivity problems associated with whole-merozite based assays are obviated or much reduced.
A cDNA copy of the mRNA which encodes the SnSAG-1 antigen has been cloned from a gene library prepared from an isolate of
S. neurona
. The original clone was identified in a collection of random sequence tags prepared to characterize the cDNA library. Additional clones of the same gene sequence were obtained to identify a full length gene. The nucleotide sequence of a full-length gene clone was determined. This sequence or the clone itself can be used to gusto prepare the SnSAG-1 antigen in a recombinant or other synthetic form for use in diagnostic tests and vaccine development.
Accordingly, the invention features a composition for detecting the presence of
S. neurona
in a biological sample. In one variation, the composition includes a SnSAG-1 marker that is a purified nucleic acid including a nucleotide sequence that encodes a protein that shares at least 50% or at least 90% sequence identity with SEQ ID NO:1. In this variation, the nucleotide sequence can also encode the protein of SEQ ID NO:1. For example, the nucleotide sequence can be SEQ ID NO:3.
In a second variation of the composition, the SnSAG-1 marker is a purified polynucleotide that binds under stringent hybridization conditions to a complement of the nucleotide sequence SEQ ID NO:3, wherein the polynucleotide is at least 30 (e.g., 50, 100, or 200) nucleotides in length.
In a third variation of the composition, the SnSAG-1 marker is an isolated protein including a polypeptide that shares at least 50%, 70%, 90%, or 95% sequence identity with a fragment of the amino acid sequence of SEQ ID NO:1 that is at least 20, 50, 100, or 300 contiguous residues in length. For example, the polypeptide can include at least 20, 50, 100, or 300 contiguous amino acid residues of the sequence of SEQ ID NO:1. The polypeptide can also include the entire amino acid sequence of SEQ ID NO:1. In this composition, the protein can be a fusion protein or a recombinant protein.
In a fourth variation of the composition, the SnSAG-1 marker is a purified antibody that specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO:1, wherein the antibody is a monoclonal antibody or a monospecific polyconal antibody. The purified antibody can be labeled with a detectable label such as a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, biotin, colloidal gold, a magnetic particle, or an enzyme.
In another aspect, the invention features a method for detecting
Sarcocystis neurona
in a biological sample. This method includes the steps of: (a) providing the biological sample; and (b) analyzing the biological sample for the presence of a SnSAG-1 marker that is a nucleic acid including a nucleotide sequence that encodes a protein that shares at least 50% sequence identity with SEQ ID NO:1; a polynucleotide that binds under stringent hybridization conditions to a complement of the nucleotide sequence SEQ ID NO:3, wherein the polynucleotide can be at least 30 nucleotides in length; a protein including a polypeptide that shares at least 50% sequence identity with a fragment of the amino acid sequence of SEQ ID NO:1 that can be at least 20 contiguous residues in length; or an antibody that specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO:1. In this method, the presence of the SnSAG-1 marker in the biological sample indicates that the biological sample contains
Sarcocystis neurona.
In the variation of this method where the SnSAG-1 marker is a nucleic acid including a nucleotide sequence that encodes a protein that shares at least 50% sequence identity with SEQ ID NO:1, the nucleotide sequence can be SEQ ID NO:3.
In the variation of this method where the SnSAG-1 marker is a protein including a polypeptide that shares at least 50% sequence identity with a fragment of the amino acid sequence of SEQ ID NO:1 at least 20 contiguous residues in length, the polypeptide can include at least 20 contiguous amino acid residues of the sequence of SEQ ID NO:1. For example, the polypeptide can include the amino acid sequence of SEQ ID NO:1.
The biological sample of the method can include CNS tissue, CSF, blood, or serum. It can also be derived from a horse.
In this method of the inevtnion, the step (B) of analyzing the biological sample for the presence of a SnSAG-1 marker can include isolating RNA from the sample, generating cDNAs from the isolated RNA, and amplifying the cDNAs by PCR to generate a PCR product. The step (B) of analyzing the biological sample for the presence of a SnSAG-1 marker can also include contacting the sample with a labeled oligonucleotide probe that hybridizes under stringent hybridization conditions to the nucleotide sequence SEQ ID NO:3 or a complement of the nucleotide sequence SEQ ID NO:3; or contacting the sample to a molecule that specifically binds to an antibody that specifically binds a protein consisting of the amino acid sequence of SEQ ID NO:1. In the latter, the molecule can be immobilized on a substrate.
In one variation of the method, the biological sample includes SnSAG-1 specific antibodies that are specifically bound to the molecule immobilized on substrate. The antibodies can be detected using a secondary antibody that is labeled with a detecta
Dame John B.
Ellison Siobhan P.
Yowell Charles A.
Baskar Padma
Saliwanchik Lloyd & Saliwanchik
Smith Lynette R. F.
University of Florida
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