Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-05-20
2002-12-17
Swartz, Rodney P (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S139100, C424S163100, C424S164100, C424S184100, C424S185100, C424S190100, C424S234100, C424S258100, C435S004000, C435S007100, C435S007200, C435S007350, C435S252800, C530S300000, C530S350000
Reexamination Certificate
active
06495334
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of cloning and expressing a truncated form of a fimbrial gene and the use of the truncated fimbrial gene product in an immunodiagnostic assay and for immunoprophylaxis.
BACKGROUND OF THE INVENTION
Foodborne infections cause an estimated 6.5 million cases of human illness and 9000 deaths annually in the United States alone. Bacterial infections by Salmonella are the most commonly reported cause of foodborne outbreaks.
Salmonella enteritidis
(SE) is the dominant Salmonella serotype isolated from cases of food poisoning. Many of these outbreaks are thought to be due to infected poultry products, particularly eggs and egg products.
The best way to prevent infection in human populations is to diagnose and treat the infected animal prior to human consumption. Because the greatest threat of food poisoning from Salmonella is from poultry products, there is a need for a method to detect birds that are infected with SE.
Some current diagnostic methods rely on conventional bacteriologic cultures. However, these procedures are relatively slow, often taking up to 3 to 4 days to provide even a presumptive diagnosis. Additionally, the great susceptibility of SE to physical and chemical factors such as desiccation, radiation, low temperature, heating, or chemical preservatives, causes traditional bacteriologic culture methods to generally have a low sensitivity. Consequently, many birds or animals that are infected with SE are often not detected when conventional bacterial cultures are used.
Other diagnostic methods rely on the detection of serum antibodies specific to SE. Although several serological methods such as micro-agglutination, serum plate agglutination, latex particle agglutination microantiglobulin, ELISA have previously been employed, these assays lack either the sensitivity or specificity necessary to detect SE infected birds, or the tests are too difficult to perform in a routine laboratory or field setting. Consequently, widespread application of these tests for the detection of SE infections has been impractical.
A useful antigenic determinant that is found on many species of Enterobacteriaceae are fimbriae, proteinaceous filamentous surface structures composed of protein subunits called fimbrin. Upon infection, birds make antibodies to this SE fimbrial antigen. Therefore, the SE fimbrial antigen is useful in a diagnostic assay for the presence of SE in poultry.
SE is known to have at least four distinct fimbria, designated Sef14, Sef17, Sef18 and Sef21. These proteins are encoded by SefA, AgfA, SefD and FinA genes, respectively.
Although the gene encoding Sef14 has been identified and its DNA nucleotide sequence determined (Trucotte and Woodward,
Journal of General Microbiology,
139:1477-1485 (1993)), an effective diagnostic method using this surface antigen has not been developed, partially due to the difficulty of efficiently producing the fimbriae proteins in purified form and in large quantities. Additionally, expression of Sef14 fimbriae by cultured
Salmonella enteritidis
is highly dependent on the growth medium composition. In a study by Thorns et al.,
International Journal of Food Microbiology,
21:47-53 (1994), only peptone water pH 7.2 supported the expression of Sef14 by all
Salmonella enteritidis
strains examined. [Consequently, previous diagnostic assays using Sef14 have used antibodies against Sef14 and not the antigen itself.]
Thorns et al.,
Journal of Clinical Microbiology,
34(4):792-797 (1996),have disclosed an ELISA for the detection of
Salmonella enteritidis
based on antibodies to an uncharacterized fraction of cell lysates having SEF14 activity. This assay suffers from the problems previously mentioned. Thorns isolates SEF14 from harvested
Salmonella enteritidis
486/86 (phage type 4) rendering the process highly dependent on the growth medium and susceptible to the difficulties in efficiently producing the fimbriae proteins in purified form and in large quantities.
Hence, there is a need for a sensitive, specific and routine antigen and method to reliably detect SE infection in birds, preferably a method that is easily adaptable to large-scale screening of poultry flocks.
The use of recombinant techniques as presented herein afford a process for efficiently producing a truncated or modified form of SEF14. The truncated or modified form of SEF14 is easily isolated to a high degree of purity in large quantities suitable for use in diagnostic assays for large-scale screening.
SUMMARY OF THE INVENTION
The present invention provides a sensitive, specific, routine antigen and assay to reliably detect SE-infected animals. Specifically, the present invention provides a truncated form of the Sef14 antigen that can be easily produced in purified form and in large quantities and used in the method of the invention. The novel Sef14 antigen, when coupled to a substrate such as latex beads, provides a diagnostic assay for SE, particularly useful in large-scale screening of poultry flocks.
REFERENCES:
patent: 5510241 (1996-04-01), Thorns
patent: WO 92/06197 (1992-04-01), None
patent: WO 92/06198 (1992-04-01), None
patent: WO 93/20231 (1993-10-01), None
Feutrier, J. et al., “Purification and Characterization of Fimbriae fromSalmonella enteritidis”, J. Bacteriol., vol. 168, No. 1, pp. 221-227 (Oct. 1986).
Clouthier, S. et al., “Characterization of Three Fimbrial Genes,, sefABC, ofSalmonella enteritidis”, Journal of Bacteriology, vol. 175, No. 9, pp. 2523-2533 (May 1993).
Ogunniyi, A. et al., “ASalmonella enteritidis11RX Pilin Induces Strong T-Lymphocyte Responses”,Infection and Immunity, vol. 62, No. 12, pp. 5376-5383 (Dec. 1994).
Peralta, R. C. et al., “Passive immunisation against experimental salmonellosis in mice by orally administered hen egg-yolk antibodies specific for 14-kDa fimbriae ofSalmonella enteritidis”, The Pathological Society of Great Britain and Ireland, pp. 29-35 (1994).
Rajashekara, G. et al., “Application of Recombinant Fimbrial Protein for the Specific Detection ofSalmonella enteritidisInfection in Poultry”,Diagn Microbiol Infect Dis, vol. 32, pp. 147-157 (1998).
Rajashecara, G. et al., “A rapid strip immunoblot assay for the specific detection ofSalmonella enteritidisinfection in chickens”, International Journal of Food Microbiology, 8 pages (1999).
Thorns, C. et al., “Characterisation of monoclonal antibodies against a fimbrial structure ofSalmonella enteritidisand certain other serogroup D salmonellae and their application as serotyping reagents”,Research in Veterinary Science, vol. 53, pp. 300-308 (1992).
Thorns, C. et al., “The use of latex particle agglutination to specifically detectSalmonella enteritidis”, International Journal of Food Microbiology, vol. 21, pp. 47-53 (1994).
Thorns, C. et al., “Development and Application of Enzyme-Linked Immunosorbent Assay for Specific Detection ofSalmonella entertidisInfections in Chickens Based on Antibodies to SEF14 Fimbrial Antigen”,Journal of Clinical Microbiology, vol. 34, No. 4, pp. 792-797 (Apr. 1996).
Turcotte, C. et al., “Cloning, DNA nucleotide sequence and distribution of the gene encoding the SEF14 fimbrial antigen ofSalmonella enteritidis”, Journal of General Microbiology, vol. 139, pp. 1477-1485 (1993).
Kapur Vivek
Nagaraja Kakambi V.
Rajashekara Gireesh
Regents of the University of Minnesota
Swartz Rodney P
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