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Moraxella bovis cytotoxin, cytotoxin gene, antibodies and vaccines for prevention and treatment of moraxella bovis infections

a technology of moraxella bovis and cytotoxin, which is applied in the field of moraxella bovis cytotoxin and a gene encoding moraxella bovis cytotoxin, can solve the problems of insufficient control measures that could substantially reduce this expense, the potential for adulteration of the nation's food supply, and the success of antimicrobial treatment of ibk

Inactive Publication Date: 2007-11-22
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent is about a vaccine for preventing infectious bovine keratoconjunctivitis, which is caused by a bacteria called Moraxella bovis. The vaccine contains a purified or recombinant version of the bacteria's cytotoxin, which is a protein that causes damage to cells. The patent also includes DNA sequences and amino acid sequences of the cytotoxin. The technical effect of the patent is to provide a tool for preventing a common and potentially damaging disease in cattle.

Problems solved by technology

Effective control measures that could substantially reduce this expense are not available as the commercially available vaccines have low efficacy.
Control of IBK using antimicrobial treatments has been only partially successful.
Antimicrobials have been used to eliminate the carrier state in experimental and field trials, but these treatments have drawbacks that include emergence of resistant bacteria in cattle and the general environment, potential for adulteration of the nation's food supply, high cost and marginal economic benefit.
Topical and oral administration of antibiotics has proven somewhat effective for treatment of pinkeye, but the high cost and losses associated with the ongoing disease often outweigh the benefits of such treatment.
Topical therapy alone does not eliminate M. bovis from non-ocular sites such as nasal choanae and vagina where it is known to reside.
So far, vaccines prepared from killed or live bacteria, isolated pili, or ribosomes have had limited benefit for immunization of cattle in the field (Proc. Am. Assoc. Bov. Pract., 20:26-32 (1987)).
These studies suggest that because of the pili heterogeneity pilus based vaccines will not be 100% effective at preventing IBK.

Method used

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  • Moraxella bovis cytotoxin, cytotoxin gene, antibodies and vaccines for prevention and treatment of moraxella bovis infections
  • Moraxella bovis cytotoxin, cytotoxin gene, antibodies and vaccines for prevention and treatment of moraxella bovis infections
  • Moraxella bovis cytotoxin, cytotoxin gene, antibodies and vaccines for prevention and treatment of moraxella bovis infections

Examples

Experimental program
Comparison scheme
Effect test

example 1

Isolation of Moraxella bovis Bacteria

[0193] This example describes isolation of a cytolytic strain of Moraxella bovis T+ and a noncytolytic subculture T−.

[0194] A hemolytic, pathogenic strain of Moraxella bovis (T+) was isolated from a beef cow with infectious bovine keratoconjunctivitis. Cytolytic EPP and GLN 63 strains were furnished by Dr. G. W. Pugh (Ames, Iowa), and additional isolates were obtained from field specimens that were submitted to the laboratory for confirmation of bacterial identification. These additional isolates were recovered from clinically affected cattle located in regions of the United States that included southern Georgia (Tifton 1, Tifton 2), northern Georgia (GA 3), northern California (LS-2, LS-4, LS-6), southern California (LS-3, LS-8), Washington (LS-5), and North Carolina (LS-7). Isolate were identified and propagated as described in Acta Path. Microbiol. Scand., 80B, 629-640 (1972) and in Identification of Non-Enteric Gram-Negative Bacteria, US De...

example 2

Purification and Concentration of the Cytotoxin

[0195] This example describes the procedure used for purification and concentration of the cytotoxin when produced in M. bovis shaker cultures.

[0196] The cytotoxin was produced in broth shaker cultures that were inoculated with lawn cultures of M. bovis. The inoculum was harvested from the surface of 10, 20 hour sheep blood agar plate lawns of M. bovis with sterile cotton tipped applicators. The bacteria on the swab were suspended into 10 ml of heart infusion broth (Difco Laboratories, Detroit, Mich.) spiked with 1.5 mM CaCl2.

[0197] Flasks were inoculated with the suspension, and were incubated at 35.5±0.5° C. on a rotary shaker set at 200 oscillations per minute. The cultures were removed from the incubator when the optical densities (420 nM) reached 1.85. For purification, the cultures (living whole cells) were centrifuged for one hour at 13,000×g (4° C.) and the supernatants (centrifuged supernatants) were filtered through a steri...

example 3

Cytotoxin Preparation for Neutralization Studies

[0202] This example describes the procedure used for preparation of cytotoxin for neutralization studies.

[0203]Moraxella bovis lawns were grown on trypticase soy agar plates supplemented with 5% sheep blood. After 24 hours of incubation, the bacterial cells were harvested by flooding the plate with 10 ml of Tris buffered saline solution (TBS CaCl2 50 mM Tris, 150 mM NaCl, 1.5 mM CaCl2, pH 8.0), and suspending the bacterial growth with a sterile inoculating loop. The suspensions were collected from the surface of the agar, and then were centrifuged at 27,000×G for one hour. After centrifugation, the supernatants were harvested, filtered through 0.22 μM polyethersulfone membrane (Gelman Biosciences, Ann Arbor, Mich.), and tested for cytolytic activity.

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Abstract

Moraxella bovis cytotoxin, cytotoxin gene, antibodies and vaccines for prevention and treatment of Moraxella bovis infections Moraxella bovis cytotoxin and a gene encoding Moraxella bovis cytotoxin. Identification, isolation, cloning and identification of nucleotide sequence of the Moraxella bovis genes mbxA, mbxB, mbxC and mbxD, partial purification of the native cytotoxin, preparation of partially purified native and a recombinant Moraxella bovis cytotoxin, identification of an amino acid sequence of the cytotoxin, preparation of antibodies against the Moraxella bovis cytotoxin, preparation of vaccines against Moraxella bovis. Method for prevention and treatment of infectious bovine keratoconjunctivitis caused by Moraxella bovis.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional application under 35 U.S.C. § 121 of U.S. patent application Ser. No. 09 / 884,696, filed on Jun. 19, 2001, the disclosure of which is incorporated herein by reference in its entirety for all purposes.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The current invention concerns Moraxella bovis cytotoxin and a gene encoding Moraxella bovis cytotoxin. In particular, the invention concerns identification, isolation, cloning and identification of nucleotide sequence of the Moraxella bovis gene mbxA and adjacent genes mbxB, mbxC and mbxD, partial purification of the native cytotoxin, preparation of a purified native and recombinant Moraxella bovis cytotoxin, an amino acid sequence of the cytotoxins, preparation of antibodies against the Moraxella bovis cytotoxin, preparation of vaccines against Moraxella bovis and a method for prevention and treatment of infectious bovine keratoconjunctivit...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395A61K39/00C07H21/00C12N1/20C12N15/00C07K14/21C07K16/12
CPCA61K39/00A61K2039/53C07K16/1214C07K14/212A61K2039/55577
Inventor GEORGE, LISLE W.ANGELOS, JOHN A.HESS, JOHN F.
Owner RGT UNIV OF CALIFORNIA
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