Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-07-25
2004-05-11
Johannsen, Diana B. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S023700, C536S024320, C536S024330, C536S025320, C530S350000
Reexamination Certificate
active
06733976
ABSTRACT:
FIELD OF THE INVENTION
The invention pertains to the field of detection of the presence of a pathogenic organism in the environment and in tissues of susceptible individuals. In particular, the invention pertains to the detection of the presence of the causative organism of Bacterial Kidney Disease (BKD) of salmonids.
BACKGROUND OF THE INVENTION
Bacterial Kidney Disease (BKD) is a disease of salmonids, such as salmon and trout species. BKD is a systemic illness that causes high mortality. The course of the disease is typically chronic, although acute outbreaks also occur. The disease was first reported in Scotland in 1931 and has since been reported in many countries around the world, including the United States, Canada, Chile, England, France, Germany, Iceland, Italy, Japan, Spain, Turkey, and in the Balkan peninsula.
BKD is a slowly progressing systemic illness in which the clinical signs typically are not evident until the disease is well established. External signs typically include exophthalmos and small closed blebs or open lesions. The unruptured blebs contain fluid composed of blood cells and necrotized tissue. In advanced cases, the lesions may coalesce and form large shallow ulcers. Internally, the kidneys are the organs most often affected. They become swollen and show discrete white areas that contain leucocytes, bacteria, and host cell debris. In advanced cases much kidney tissue is destroyed and hematopoietic and excretory functions are both affected. Hemorrhages occur in the body wall and testes, and peritoneal fluid is commonly present. The hind gut can be hemorrhagic and filled with white or yellow viscous fluid.
Death due to BKD most commonly is sporadic and occurs over a long period of time, although subacute outbreaks with 25 to 50% mortality occurring within a few weeks have been reported. Juvenile salmonids show the greatest susceptibility to infection with the level of susceptibility being highly variable between species. Inapparent carriers of the disease are common, as infected fish may carry the causative organism of the BKD for years without showing any clinical signs. The disease may only manifest itself clinically when such carrier fish are stressed.
The causative organism of BKD is a slow-growing gram positive bacterium,
Renibacterium salmoninarum
. This bacterium lives both extracellularly and intracellularly in the salmonid host and has been shown to survive and even to multiply within macrophages. This permits the bacterium to spread readily throughout the body. The intracellular location and the widespread distribution of
R. salmoninarum
within the body make the infection difficult to treat because antibiotics may not reach all of the locations where the organism resides. Antibiotics typically employed, such as erythromycin, are cleared rapidly by the fish and require administration at near toxic levels. Additionally, BKD disease is one of the few bacterial diseases that can be spread both horizontally and vertically.
R. salmoninarum
can be transmitted in the egg.
Because of the widespread systemic distribution and intracellular location of the BKD bacterium, the presence of latent carriers, the ability of the disease to be transmitted vertically, and the lack of an effective bacterin, BKD has proven to be very difficult to control. Therefore, because of the potential for BKD to devastate a population, control efforts are concentrated in preventing exposure of infected fish to the BKD organism. It is common practice to isolate salmon returning to hatcheries, segregate fertilized eggs until diagnostic test results are evaluated, test each female, cull eggs from females with test results that suggest the presence of the BKD organism, and segregate remaining progeny according to the test values of the females.
Presently available tests for the BKD organism present problems that have not as yet been overcome. The isolation and identification of
R. salmoninarum
in samples from clinically infected fish is relatively easy. However, the slow growth of the organism and the fact that
R. salmoninarum
is a fastidiously growing organism requiring prolonged incubation at 15° C. to produce colonies renders such tests of little utility in practice. Thus, serological methods are more usually employed to confirm the presence of the bacterium.
Immunofluorescence antibody tests (FAT) have commonly been used for demonstrating the presence of
R. salmoninarum
in infected tissues. Screening of ovarian fluid from asymptomatic brook trout populations using FAT and ELISA found that twice as many positives were detected using a FAT method than by ELISA. However, FAT reproducibility is poor for very low levels of infection, resulting in some infections being missed.
ELISA is widely used for the detection of
R. salmoninarum
, and several commercial kits are available. The most common technique used is a double antibody sandwich ELISA method. Samples of tissue (fish kidneys) collected for examination must be kept cold or frozen after collections. Tissues from clinically and sub clinically infected fish have ELISA reactions that are clearly distinct from those of uninfected fish. Unfortunately, ELISA does not work well on ovarian or seminal fluid. Since the ELISA test is performed on kidney tissue, diagnosis is not possible until after stripping eggs from the females. This requires extra manpower and space in order to strip eggs from females that will test above the culling threshold and to keep eggs segregated by female until the ELISA results are evaluated. There is about a four (4) week delay from submitting samples to a lab and receiving results. While several ELISA methods are available, it is important to note that the various evaluations as to their sensitivity and specificity have not been uniformly carried out.
Polymerase Chain Reaction (PCR) has been used for the detection and identification of
R. salmoninarum
directly within ovarian fluid or individual eggs. Present techniques rely on detection of DNA segments of the gene coding for the antigenic p57 protein. The nucleotide sequence coding for this protein is available from GenBank at the National Center for Biotechnology Information (NCBI) of the National Institutes of Health (NIH) and has been assigned Accession No. AF123888. These PCR methods are very sensitive and have a high degree of specificity. The PCR/ovarian fluid method does not detect significantly more positives than ELISA methods from the kidneys, but is more rapid as results are obtained within 1 to 2 days and, in contrast to ELISA which utilizes kidney tissues, is non-lethal as,ovarian test fluid may be obtained when stripping eggs. Other studies have found PCR to identify higher numbers of kidney tissue and ovarian fluid samples from commercially reared brood stock fish to be positive for
R. salmoninarum
than were identified by culture.
One potential problem with PCR diagnostic methods is that a PCR-positive sample may contain some proportion of dead
R. salmoninarum
with detectable levels of DNA, such as due to previous antibiotic therapy or fish that have conquered the disease. Other problems that occur when using standard PCR methods include incorrect interpretation of band presence or absence because of improper primer annealing or buffer concentration. Additionally, the number of cycles to run for correct band amplification can vary between the primers and the type of machine used. These problems are further exacerbated when attempts are made to quantify the level of bacteria present. For
R. salmoninarum
, this has proven even more difficult with the necessary use of nested PCR for detection of the pathogen, since the addition of extra primers and increased cycle numbers leads to an increase in the detection of false positive bands.
In recent years, PCR methods have been adapted to provide both for detection and for quantification of nucleic acid sequences in a sample. For example, see, Higuchi, U.S. Pat. No. 6,171,785, incorporated herein by reference. These methods employ forward and reverse primers as in standard PCR plu
Overturf Kenneth E.
Powell Madison S.
Howard Eisenberg, Esq.
Idaho Research Foundation
Johannsen Diana B.
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