Anucleated live E. coli vaccine

Claim 1

A method of making an Escherichia coli (E. coli) strain which produces metabolically active, non-reproductive, anucleated live K99+E.. coli cells comprising the steps of: mixing an E. coli strain which produces, metabolically active, non-reproductive, anucleated live cells (minicells) with a K99+ enteropathogenic E. coli strain; incubating the mixed cells in a culture medium; separating minicell-producing E. coli cells which contain the K99 plasmid from other cells in the culture medium; and growing the separated minicell-producing K99+E. coli in a second culture medium.

Claim 2

A method of making an Escherichia coli (E. coli) strain which produces metabolically active, non-reproductive, anucleated live K99+E.. coli cells comprising the steps of: mixing an E. coli strain which produces, metabolically active, non-reproductive, anucleated live cells (minicells) with a K99+ enteropathogenic E. coli strain; incubating the mixed cells in a culture medium; separating minicell-producing E. coli cells which contain the K99 plasmid from other cells in the culture medium; and growing the separated minicell-producing K99+E. coli in a second culture medium.

Claim 9

A method of making a vaccine useful for preventing neonatal diarrhea in cattle comprising the steps of: tranferring the K99 plasmid from an enteropathogenic E. coli strain into a minicell-producing E. coli strain; growing the resulting minicell-producing K99+E. coli strain in broth culture medium under aerobic conditions with agitation; separating the bacterial cells from the broth culture medium by centrifugation and suspending them; and separating minicells from complete reproductive cells by sucrose gradient centrifugation.

Claim 10

A method of making a vaccine useful for preventing neonatal diarrhea in cattle comprising the steps of: tranferring the K99 plasmid from an enteropathogenic E. coli strain into a minicell-producing E. coli strain; growing the resulting minicell-producing K99+E. coli strain in broth culture medium under aerobic conditions with agitation; separating the bacterial cells from the broth culture medium by centrifugation and suspending them; and separating minicells from complete reproductive cells by sucrose gradient centrifugation.

Claim 19

A method of making a vaccine which contains metabolically active, non-reproductive, anucleated live K99+E. coli cells and which is useful for preventing neonatal diarrhea in cattle comprising the steps of: mixing an E. coli strain which produces metabolically active, non-reproductive, anucleated live cells (minicells) with a K99+ enteropathogenic E. coli strain; incubating the mixed cells at a cell density of approximately 2 to 3×10 8 cells/ml culture medium at 37° C. for about 18 hours; separating minicell-producing E. coli cells which contain the K99 plasmid from other cells in the culture medium; growing the separated minicell-producing K99+E. coli GK500 cells on agar plates containing 200 μg/ml streptomycin; selecting colonies of minicell-producing K99+E. coli GK500 and growing them in a minimal salts based synthetic medium for 15 to 18 hours at 36° to 37.5° C. under aerobic conditions with agitation; separating the bacterial cells from the synthetic medium by centrifugation and suspending them in a buffered salt solution; layering the suspension containing the bacterial cells on a first sucrose gradient, comprising a 5% sucrose solution layered over a 20% sucrose solution, and centrifuging; collecting and concentrating the top fractions of the first sucrose gradient; layering and concentrated fractions on a second sucrose gradient, comprising a linear sucrose gradient of 10-5% layered over a 10% sucrose solution, and centrifuging; and collecting, combining and concentrating the top fractions of the second sucrose gradient.

Claim 20

A method of making a vaccine which contains metabolically active, non-reproductive, anucleated live K99+E. coli cells and which is useful for preventing neonatal diarrhea in cattle comprising the steps of: mixing an E. coli strain which produces metabolically active, non-reproductive, anucleated live cells (minicells) with a K99+ enteropathogenic E. coli strain; incubating the mixed cells at a cell density of approximately 2 to 3×10 8 cells/ml culture medium at 37° C. for about 18 hours; separating minicell-producing E. coli cells which contain the K99 plasmid from other cells in the culture medium; growing the separated minicell-producing K99+E. coli GK500 cells on agar plates containing 200 μg/ml streptomycin; selecting colonies of minicell-producing K99+E. coli GK500 and growing them in a minimal salts based synthetic medium for 15 to 18 hours at 36° to 37.5° C. under aerobic conditions with agitation; separating the bacterial cells from the synthetic medium by centrifugation and suspending them in a buffered salt solution; layering the suspension containing the bacterial cells on a first sucrose gradient, comprising a 5% sucrose solution layered over a 20% sucrose solution, and centrifuging; collecting and concentrating the top fractions of the first sucrose gradient; layering and concentrated fractions on a second sucrose gradient, comprising a linear sucrose gradient of 10-5% layered over a 10% sucrose solution, and centrifuging; and collecting, combining and concentrating the top fractions of the second sucrose gradient.