Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-09-18
2001-03-20
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S184100, C424S190100, C424S264100, C424S001210, C435S007100, C435S007200, C435S243000, C435S245000, C435S252100, C435S340000, C435S351000, C436S501000, C436S512000, C530S388400
Reexamination Certificate
active
06204003
ABSTRACT:
BACKGROUND OF THE INVENTION
Feline infectious anemia (FIA) is a serious, and sometimes fatal, feline disease with worldwide distribution. The frequency of the disease has been difficult to estimate, due to difficulties detecting the causative agent,
Haemobartonella felis
. One study estimated that the causative agent was present in 7.5% of the ill cats studied and 3.6% of cats which appeared healthy, while another study reported infection rates as high as 23.2% in animals which had been referred for treatment. (Grindem, C. B. et al., “Risk Factors for
Haemobartonella felis
Infection in Cats,”
JAVAM
196:96 (1990); Nash, A. S. and Bobade, P. A. , “
Haemobartonella felis
Infection in Cats from the Glasgow Area,”
The Veterinary Record,
Oct. 11, 1986, page 373). Cats having low packed cell volume (PCV), a lack of vaccinations generally and against feline enteritis and respiratory tract virus infections specifically, catbite abscesses, a history of roaming outdoors and Feline Leukemia Virus (FeLV) positive status are at a greater risk for FIA. (Grindem et al., supra) Some studies have found a correlation between gender and risk of FIA, although other studies have failed to detect this association. (Hayes, H. M., “Feline Infectious Anaemia. Risk by Age, Sex, and Breed; Prior Disease; Seasonal Occurrence; Mortality,”
J. Small Anim. Pract.
14: 797 (1973)). Although the disease is curable by treatment with antibiotics, there is no induction of protective immunity. Infection may occur through a variety of routes, including ingestion of infected material, open wounds resulting from fighting, or transplacental transmission.
In addition to severe anemia, clinical manifestations of
Haemobartonella felis
infection include depression, weakness, anorexia, weight loss, paleness of mucous membranes, and, occasionally, splenomegaly. (Harvey, J. W. “Hemobartonellosis” in Clinical Microbiology and Infectious Diseases of the Dog and Cat, C. E. Greene Ed., W. B. Saunders Co. (1984)). Without intervention, approximately one third of cats with uncomplicated acute haemobartonellosis perish from severe anemia. (Holzworth, J. “Anemia in the Cat,”
J. Am. Vet. Med. Assoc.,
126:471-488, (1956); Splitter, E. J. et al.,“Feline Infectious Anemia,”
Vet. Med.,
51:17-22, (1956)). However, some cats experience both an immune response to the bacteria and a regenerative bone marrow response, which allows erythrocyte production to exceed erythrocyte destruction. These animals eventually recover from the disease, with the recovery time lasting one month or more. (Harvey, J. W. and Gaskin, J. M., “Experimental Feline Haemobartonellosis,”
J. Am. Anim. Hosp. Assoc.,
13:28-38, (1977)).
Unfortunately, cats that recover from acute infections with
Haemobartonella felis
remain infected with the pathogen for considerable time periods and may even remain infected throughout their lifetimes. (Splitter, E. J. et al. 1956, supra; Harvey, J. W. and Gaskin, J. M., “Feline Haemobartonellosis: Attempts to Induce Relapses of Clinical Disease in Chronically Infected Cats,”
J. Am. Anim. Hosp. Assoc.,
14:453-456, (1978)). In addition, carrier cats may not exhibit any symptoms of infection. The lack of observable symptoms increases the likelihood that the disease will escape treatment and that the carrier will transmit the organism to other animals.
Haemobartonella felis
feeds and multiplies on the surface of red blood cells within the circulation, leading to progressive erythrocyte damage and shortened erythrocyte lifespans (Maede, Y., “Studies on Feline Haemobartonellosis. IV. Lifespan of Erythrocytes of Cats Infected with
Haemobartonella felis,”
Jpn. J Vet. Sci., 37:269-272, (1975)). The anemia appears to result from the induction of autoantibodies which result in lysis of the host's own red blood cells. It has been hypothesized that bacterial attachment to the cell surface exposes hidden antigens on the surface or alters antigens normally expressed on the surface. The antibodies produced against the erythrocytes result in a false positive Coombs test.
An alternative hypothesis suggests that cell death is due to antibodies against
Haemobartonella felis
. It has been proposed that antibodies against
Haemobartonella felis
cause erythrocyte death by binding to bacteria on the surface of the cell and inducing complement fixation, thereby triggering lysis of the erythrocyte.
Following infection, the levels of bacteria found in the blood may rise and decline rapidly. (Maede, Y. and Hata, R., “Studies on Feline Haemobartonellosis. II. The Mechanism of Anemia Produced by Infection with
Haemobartonella felis
,” Jpn.
J Vet. Sci.,
37:49-54, (1975); Harvey and Gaskin, (1977), supra). This rapid and extensive fluctuation may be a consequence of splenic sequestration of infected erythrocytes and release of noninfected erythrocytes. (Maede, 1975, supra.).
Prior to the present invention, the most commonly employed method for detecting
Haemobartonella felis
infection relied on blood film staining. However, this approach suffers from numerous drawbacks. Because the bacteremia associated with
Haemobartonella felis
infection is episodic, it is often difficult to detect
Haemobartonella felis
using such traditional cytological approaches. In addition, the pleomorphic nature of
Haemobartonella felis
may cause the organism to be confused with stain precipitation or cellular debris. (C. B. Grindem et al. supra; C. M. R. Turner et al. Letters The Veterinary Record, Nov. 22, 1986 page 534; H. C. Carney and J. J. England, “Feline Hemobartonellosis,”
Feline Infectious Diseases
23: 79 (1993)).
Thus, there is a need for improved tests for
Haemobartonella felis
infection. In particular, given the low levels of
Haemobartonella felis
present during some stages of the disease, there is a need for sensitive diagnostics capable of detecting the low levels of circulating organisms present during some phases of the infection or in carrier cats harboring low residual levels of the organism. In addition, there is a need for selective tests capable of readily distinguishing
Haemobartonella felis
from other morphologically or genetically similar organisms. Finally, there is a need for tests which may be rapidly performed. The diagnostics described herein provide all of the preceding advantages.
SUMMARY OF THE INVENTION
The present invention relates to methods for detecting the presence of
Haemobartonella felis
in a mammal and isolated or purified nucleic acids containing at least 15 consecutive bases of the
Haemobartonella felis
16S and/or 23S ribosomal RNA genes. In addition to being specific for
Haemobartonella felis
, the present assay provides high sensitivity due to the fact that the 16S and 23S ribosomal RNA genes or the transcripts thereof are present in multiple copies in the genome.
As used herein, the term “isolated” requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
As used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition. In other words, “purified” means that the nucleic acid of the
Haemobartonella felis
16S and/or 23S ribosomal RNA genes are enriched in concentration relative to other cellular components or nucleic acids. This enrichment is at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude.
One aspect of the present invention is a method for detecting the presence of
Haemobartonella felis
in a mammal comprising obtaining a nucleic acid sample from a body fluid of said mammal, contacting said nucleic acid sample with at least one nucleic acid probe from the
Haemobartonella felis
16S or 23S ribosomal RNA genes, said probe being specific for
Haemobartonella felis
; and determining whether said
Cutting John A.
Steele J. Kevin
Telford David L.
Knobbe Martens Olson & Bear LLP
Stucker Jeffrey
Synbiotics Corporation
Winkler Ulrike
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