Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-15
2001-08-21
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C514S002600, C530S350000, C536S023100, C536S023700, C536S024300, C536S024320, C536S024330
Reexamination Certificate
active
06277580
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
Paratuberculosis
poses a significant economic and health problem worldwide, especially in the cattle industry.
1,2
Mycobacterium avium
subspecies
paratuberculosis
3
(
M. paratuberculosis
) is the etiologic agent of
paratuberculosis
(Johne's disease), a chronic granulomatous enteritis of both domestic and wild ruminants. This organism is an intracellular pathogen that replicates within macrophage of both the gastrointestinal tract and associated lymphatic tissues.
4
The disease can be transmitted in utero, to nursing calves, or via infected fecal contamination of food. Diagnosis of subclinical
paratuberculosis
is problematic because infection progresses slowly and infected animals often do not show signs of the disease for years. Once the disease is established in a herd, there is no cure. Annual economic losses to the dairy industry are in the billions of dollars worldwide, primarily as a result of reduced milk production, decreased reproductive efficiency, and death.
In humans,
M. paratuberculosis
has been isolated from patients with Crohn's disease, a chronic enteritis with clinical symptoms similar to animals with
paratuberculosis.
5,6
M. paratuberculosis
has been implicated as a possible cause of Crohn's disease, however, the etiology of this disease remains unknown.
This invention relates to a species-specific genetic target element useful for identifying
M. paratuberculosis
and for distinguishing this organism from related bacteria by various diagnostic techniques. Probes and primer sets are disclosed for detecting target sequence in laboratory and clinical samples containing
M. paratuberculosis.
2. Description of the Prior Art
Cattle shed
M. paratuberculosis
in their feces during the subclinical and clinical stages of infection. Currently, the most sensitive test available for subclinical
paratuberculosis
requires a prolonged 8-12 week fecal culturing of the organism. Existing immunological diagnostic tests are rapid but have disadvantages resulting from poor specificity of the antigens used in the assays.
Nucleic acid diagnostic methodology is used as a rapid and sensitive way to identify specific species of mycobacteria.
7,8,9
Some mycobacterial species are genetically very closely related to
M. paratuberculosis
according to DNA-DNA hybridization analysis.
10
Genome homology ranging from 50% to nearly 100% has been reported between the ATCC 19698 reference strain of
M. paratuberculosis
and species of the
Mycobacterium avium
complex (MAC) which includes the incompletely separated
Mycobacterium avium
(subspecies avium [
M. avium
] and subspecies silvaticum [
M. silvaticum
] and
Mycobacterium intracellulare
as well as other strains not assigned to either species.
11,12,13
M. paratuberculosis
DNA is also related to DNA of other mycobacteria, such as
Mycobacterium bovis, Mycobacterium leprae,
and
M. tuberculosis.
14,15
The high percentage of genetic relatedness of
M. paratuberculosis
with other mycobacterial species requires the cloning, sequencing, and characterization of unique genetic markers (genetic elements or genes) to differentiate these closely related species. Species-specific genetic markers are useful tools for the development of new molecular diagnostic tests.
Only two species-specific genetic elements have been identified in the
M. paratuberculosis
genome.
16,17
DNA probes derived from these genetic elements have been used to detect
M. paratuberculosis
infection. One genetic element, IS900, is a 1.45 kbp insertion element found at approximately 20 copies per chromosome.
16
Similar insertion elements, which have sequences related to IS900, have been identified in closely related mycobacteria, such as
M. avium
(IS901)
18,19
and
M. silvaticum
(IS902).
20
The now commercially available IS900 DNA diagnostic kit (IDEXX Corp.), which was used in studies conducted in a
M. paratuberculosis
control program, yields an 89% specificity and a 13% sensitivity.
21
The other
M. paratuberculosis
species-specific genetic element that has been identified, F57, is a 620 bp DNA fragment not related to any known sequence, including the IS900 insertion element.
17
Southern hybridization analysis using the F57 fragment suggests that this genetic element is single-copy in the
M. paratuberculosis
genome. The F57 genetic element is currently being used as a diagnostic tool to identify
M. paratuberculosis
infection in both cattle and humans (patients with Crohn's disease).
10,17
However, the cloning and sequencing of additional
M. paratuberculosis
-specific genetic elements or genes is needed to improve or develop new rapid and sensitive nucleic acid diagnostic tests for the differentiation of
paratuberculosis
infection.
Currently, there is a need for an accurate, rapid and reliable detection of
M. paratuberculosis
infection.
SUMMARY OF THE INVENTION
We have now discovered a
M. paratuberculosis
gene, hereafter referred to as hspX, that is present as a single-copy gene in the
M. paratuberculosis
genome. This gene provides a useful target region for the construction of suitable probes and primers that are species-specific for distinguishing
M. paratuberculosis
from related mycobacteria in a test sample. Diagnostic assays for
M. paratuberculosis
could also be based on expressed protein products of the hspX gene, such as in an ELISA assay.
In accordance with this discovery, it is an object of the invention to provide a sensitive, specific, and rapid diagnostic tool for positively identifying
M. paratuberculosis
in a clinical or laboratory sample.
It is also an object of the invention to provide a target region for constructing probes and primer sets tailored to the desired specificity for detecting
M. paratuberculosis
infections.
Another object of the invention is to provide an improved method for diagnosing Johne's disease in ruminant animals.
Other objects and advantages of the invention will become readily apparent from the ensuing description.
REFERENCES:
patent: 5314801 (1994-05-01), Nycz et al.
Ellingson, J. L. E., Bolin. C. A., Stabel, J. R., Mol. Cell. Probes 12, 133-142 (1998) Identification of a gene unique toMycobacterium aviumsubspecies paratuberculosis and application to diagnois of paratuberculosis.*
Jay L. E. Ellingson et al., “Identification of a Genetic Element Unique toMycbacterium paratuberculosisand Application to Paratuberculosis Diagnosis” 97th General Meeting, American Society for Microbiology, Miami Beach, May 4-8, 1997.
Jay L. E. Ellingson et al., “Identification of a New Genetic Element Unique toMycobacterium paratuberculosisand Application to Diagnosis of Paratuberculosis”, First International Veterinary Vaccines and Diagnostics Conference, Madison, Wisconsin, Jul. 27-31, 1997.
Ellingson Jay L. E.
Stabel Judith R.
Carlson Karen Cochrane
Fado John D.
Kam Chih-Min
Ribando Curtis P.
Silverstein M. Howard
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