Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
2001-04-24
2003-12-30
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S164100, C424S176100, C424S184100, C424S185100, C424S190100, C424S197110, C424S236100, C424S234100, C424S192100, C530S350000, C530S387100, C536S023100, C536S023500, C536S023700, C435S069100, C435S069300, C435S071100, C435S252300
Reexamination Certificate
active
06669940
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is concerned with methods of cloning and expressing the leukotoxin gene from
Fusobacterium necrophorum
(
F. necrophorum
), sequencing and characterizing the leukotoxin protein expressed by this gene, truncating the gene into a series of nucleotide sequences, amplifying these sequences, expressing and recovering the polypeptides encoded by the nucleotide sequences, and utilizing the protein and the polypeptides in recombinant vaccines in order to confer effective immunity against infection caused by the production of leukotoxin by
F. necrophorum
. More particularly, it is concerned with production of an inactivated recombinant leukotoxin vaccine generated by amplifying five leukotoxin gene fragments and one upstream region through PCR, digesting the nucleotide sequences encoded by the gene fragments with restriction enzymes, expressing the polypeptide sequences coded by the nucleotide sequences through an expression vector, recovering these proteins as five truncated leukotoxin proteins (or polypeptides), purifying these proteins (or polypeptides) to apparent homogeneity, with or without inactivation of the truncated and full length proteins, and combining the inactivated recombinant leukotoxins with adjuvants.
2. Description of the Prior Art
Liver abscesses in feed lot cattle are a serious economic problem, causing condemnation of over 3 million livers and an estimated loss of $15 million annually in the United States. This estimate is based primarily on condemnation of liver and other organs, and does not include economic losses stemming from reduced feed intake, reduced feed efficiencies, decreased carcass dressing percentage and lowered weight gains. A number of studies have confirmed that cattle with abscessed livers gain less (average 4-5%) and have reduced feed efficiencies (average 7%) compared with cattle having healthy livers. The average incidence of abscessed liver in grain-fed cattle approximates 25-30%. To a lesser extent, liver abscesses in sheep and goats are also an economic problem.
F. necrophorum
is a gram-negative, rod-shaped, nonsporeforming, nonmotile, strictly anaerobic and pleomorphic organism. Morphologically, the organism varies from short rods to filamentous with pointed and rounded ends. Cell lengths range from coccoid bodies of 0.5-0.7 &mgr;m diameter to filaments over 100 &mgr;m. Surface colonies are 1-2 mm in diameter, circular, transparent to opaque, and with some strains producing &agr; or &bgr; hemolysis. The organism ferments glucose, fructose and maltose only weakly with final pH around 5.0-6.3. It ferments lactate to acetate, propionate, and butyrate. Butyrate is the major product from lactate fermentation. Indole is produced from peptone.
F. necrophorum
has been isolated from the normal flora in the oral cavity, gastrointestinal cavity, and genitourinary tract of humans and animals. The organism is also known to survive in the soil.
F. necrophorum
is a normal inhabitant of the gastrointestinal tracts of animals and humans. Virulence factors and pathogenic mechanisms that contribute to the transition of this otherwise commensal organism to a pathogen are poorly understood. A leukotoxin, endotoxin, hemolysin, hemagglutinin, and several enzymes such as deoxyribonuclease and proteases have been suggested as possible virulence factors. However, several studies implicate leukotoxin, a protein cytotoxic to ruminant polymorphonuclear cells, as the major virulence factor. The importance of leukotoxin as a virulence factor in
F. necrophorum
infections is indicated by a correlation between toxin production and ability to induce abscesses in laboratory animals, an inability of nonleukotoxin-producing strains to induce foot abscesses in cattle following intradermal inoculation, and a relationship between antileukotoxin antibody titers and protection against infection in experimental challenge studies.
F. necrophorum
is an opportunistic pathogen that is the primary etiologic agent of liver abscesses in ruminant animals. (Scanlan, et al., (1983)
Bovine rumenitis
-
liver abscess complex: a bacteriological review. Cornell Vet.
73:288-297; Nagaraja, T. G. et al., (1998)
Liver abscesses in feedlot cattle: A review. J. Anim. Sci.,
76:287-298; and Tan, et al., (1996)
Fusobacterium necrophorum infections: virulence factors pathogenic mechanism and control measures. Vet. Res. Comm.,
20:113-140). The organism has been recognized as an animal and human pathogen since the late 1800s, and is associated as a primary or secondary etiologic agent with numerous necrotic disease conditions in domestic and wild animals. In addition to liver abscesses, the organism is also the primary etiologic agent of foot rot, foot abscesses, calf diphtheria, and is frequently isolated from cases of mastitis, metritis, and necrotic lesions of the oral cavity.
Liver abscesses in cattle are part of a disease complex where the abscessation is secondary to primary foci of infection in the rumen epithelium. The pathogenesis can be summarized as follows: (1) ruminal lesions are induced by acidosis that follows rapid change in diet from high-roughage to high grain, prolonged feeding of high grain diet, or occasionally by foreign body penetration of the rumen epithelium; (2) bacteria present in the rumen invade the epithelium and form focal abscesses in the rumen wall; and (3) bacteria enter the portal circulation, and are carried to the liver where they localize in the parenchyma with subsequent abscess formation.
The ability of
F. necrophorum
to establish in the liver is attributed to the production of a toxin which is a secreted protein of high molecular weight active against leukocytes from ruminants called leukotoxin (or leucocidin). The toxin is a soluble extracellular protein that is cytotoxic to neutrophils, macrophages, hepatocytes, and ruminal cells. The leukotoxin protects against phagocytosis and is believed to aid in the establishment of
F. necrophorum
in the liver by directly impairing the normal defense mechanism and indirectly by the damage caused by cytolytic products released from neutrophils and macrophages to the hepatic cells. Therefore, the leukotoxin elaborated from
F. necrophorum
plays a critical role in
F. necrophorum
infection of the liver and is believed to be the primary virulence factor in the pathogenesis of liver abscesses (Tan et al., 1996).
Four biotypes (A, B, AB and C) of
F. necrophorum
have been described. (Langworth, (1977)
Fusobacterium necrophorum: its characteristics and role as an animal pathogen. Bacteriol. Rev.
41:373-390) Biotype A, most frequently isolated from liver abscesses, is more pathogenic than biotype B, which predominates in ruminal wall abscesses. Biotypes AB and C are rarely isolated in liver abcesses (Berg, et al., (1982)
Studies of Fusobacterium necrophorum from bovine hepatic abscesses: Biotypes, quantitation, virulence, and antibiotic susceptibility. Am. J. Vet. Res.
43:1580-1586), and biotype A has pathogenicity intermediate that of biotypes A and B while biotype C is non-pathogenic. (Shinjo, et al., (1990)
Recognition of biovar C of Fusobacterium necrophorum
(flugge)
Moore and Holdeman as Fusobacterium pseudonecrophorum sp. nov., nom. rev
. (
ex prevot
1940)
Int. J. Sys. Bacteriol.
41:395-397) Biotypes A and B, the most frequent types encountered in liver abscesses, have been assigned subspecies status: subsp. necrophorum and subsp. funduliforme, respectively (Shinjo et al., 1990). The subsp. necrophorum is more virulent, produces more leukotoxin and hemagglutinin, and is more frequently isolated from cattle liver abscesses than the subsp. funduliforme. Virulence factors and pathogenic mechanisms contributing to the formation of liver abscesses by
F. necrophorum
are poorly understood (Tan et al., 1996). However, several studies implicate leukotoxin to be a major virulence factor (Emery, et al., (1986)
Generation of immunity against Fusobacterium necrophorum in mice inoculated with extracts containing leukotoxin. Vet. Mic
Chengappa Muckatira M.
Nagaraja Tiruvoor G.
Narayanan Sanjeev K.
Stewart George C.
Baskar Padmavathi
Hovey & Williams, LLP
Kansas University Research Foundation
Smith Lynette R. F.
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