IE84075B1 - Bacterin for the treatment of liver necrosis caused by F. Necrophorum - Google Patents

Description

PATENTS ACT, 1992 960658 BACTERIN FOR THE TREATMENT OF LIVER NECROSIS CAUSED BY F. NECROPHORUM BAYER CORPORATION BACTERIN FOR THE TREATMENT OF LIVER NECROSIS CAUSED BY F. NECROPHORUM BACKGROUND OF THE INVENTION The present invention relates to a F. necrophorum bacterin and to a process for the production thereof. —~—eFusobaetafium~necroohorum (formerly referred to as Sphaerophorus necroohorus) is an obligate anaerobic gram negative rod, which is generally recognized as playing a significant role in a variety of disease entities affecting ruminants including Chronic Footrot in sheep, Acute and Chronic Footrot in cattle, Liver Abscess in cattle, and Diphtheria in calves.

Garcia et al discloses in "Biological Characterization of Fusobacterium necrophorum Cell Fractions in Preparation for Toxin and Immunization Studies", Infection and Immunity, April 1975, pages 609-616, that preliminary trials indicated that an alum-precipitated toxoid derived from the cytoplasm of bovine liver abscesses reduced liver abscesses to a level of 10% as compared to the 35% level of the control sample.

Abe et al discloses in "Immunization of Mice Against Fusobacterium necrophorum Infection by Parenteral or Oral Administration of Vaccine", Am; J. Vet. Res., Vol. 39, No. 1, pages 115-118 (January 1978) a vaccine made with whole cell suspensions of formalin-killed F. necrophorum. This vaccine .was administered by three different routes: intraperitoneal injection of the killed cells in a saline solution, intraperitoneal injection of the killed cells with added aluminum hydroxide adjuvant and by feeding as a powder to which lyophilized bacterial cells had been added. most effective treatment (i.e., 1P injection of cells plus adjuvant) resulted in mortality rates of almost 40% after seven These bacterins have not, however, shown However, even the days post challenge. sufficient efficacy when tested under field conditions to be of commercial value.

Katitch reports in "Etude comparative sur la valeur prophylactique de 2 vaccins contre le pietin du mouton", figllg Soc. Vet. et Med. comparee, Vol. 76, No. 4 (Lyon 1974), the results of a comparison of two commercial vaccines against Foot-Rot in field studies. One of the vaccines contained only one antigen and was found to be almost completely ineffective.

The second vaccine was a multiple antigen preparation in which S. necrophorus, Staphylococcus pyogenes and W. gerfrinoens were included.

US Patent 4,061,751 discloses a treatment for foot rot and liver lesions in ruminant animals in which a 6-substituted 3-nitroimidazo[1,2-b] pyridazine is administered to the animal being treated. The preferred method of administration is oral administration.

To date, however, no effective prophylactic agent to control or prevent F. necrophorum diseases is commercially available.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel bacterin useful in the treatment of of cattle and sheep to prevent liver necrosis caused by Fusobacterium necrophorum infection.

It is a further object of the present invention to provide an inactivated F. necrophorum bacterin which provides significantly better protection against liver necrosis caused by Fusobacterium necrophorum infection than known treatments.

These and other objects which will be apparent to those skilled in the art are accomplished by inactivating a virulent isolate of Fusobacterium necrophorum with B-propriolactone. Any excess B-propriolactone may then be removed by hydrolysis.

The inactivated bacterin may then be combined with a known adjuvant to produce a vaccine suitable for parenteral administration.

DETAILED DESCRIPTION OF THE PRESENT INVENTION The present invention relates to a process for bacterin preparation in which fl-propiolactone (BPL) is used to inactivate virulent F. necroohorum isolates. Residual BPL may be hydrolyzed and an adjuvant may then be added. The bacterin prepared by this process is useful in the prevention and control of F. necroohorum in ruminant animals such as sheep, goats and cattle under normal field conditions.

The present invention also relates to a fl—propiolactone (BPL) killed bacterin prepared from virulent isolates of Fusobacterium necroohorum which is useful as an aid in protecting against diseases caused by F. necroohorum in cloven hoofed animals (i.e. cattle, sheep, goats, etc.).

Previous attempts at preparing efficacious bacterins using Fusobacterium necroohorum have been unsuccessful, probably due to the fact that critical antigens necessary for induction of immunity were not preserved by use of inactivation processes which involved heat or formaldehyde.

Applicant has found, however, through challenge studies conducted in mice that BPL inactivated, adjuvanted cultures can protect mice against experimental challenge with heterologous isolates of F. necroohorum.

The present invention can be practiced with any virulent isolate of F. necroohorum. A virulent isolate is one which is capable of producing a typical F. necroohorum lesion in cattle or sheep. Two isolates, designated 5118 and 5120 were isolated from Missouri sheep showing signs of chronic ovine Footrot and used in the development of the bacterin of the present invention. The present invention is not, however, limited to bacterins derived from these specific isolates because these isolates exhibited characteristics which are typical of all biovar (biotype) A of F. necrophorum. The characteristics relied upon to make this determination were cultural and virulence characteristics such as isolation from typical lesions, pathogenicity for mice, and growth as flat grayish colonies on blood agar. These known characteristics of F. necroohorum and techniques for determining them are disclosed in publications such as Virulence Mechanisms of Bacterial Pathogens, Roth, J (ed), Chap 21, p. 343-362, 350 "Approaches Virulence Determinants of Fusobacterium and Bacteroides spp" by David E. mery, (1988); Footrot and Foot Abscess of Ruminants, Egerton, J.R., Yong, W.K. & Rifkin, G.G., Chap. 4, pages 69-79, 70, "Foot Abscess of Cattle" by Clark, B.L. pages 69-79, 70 (CRC Press 1989).

The virulent F. necroohorum isolates used to produce the bacterins of the present invention may be grown in any of the growth media known to those in the art for periods of from hours to 24 hours, preferably from 16 to hours at temperatures of from 35 to 39°C, 36 to 38'C. Specific examples of suitable growth media include: Eugon broth (available from Baltimore Biological Laboratories (BBL)) supplemented with maltose, beef extract, yeast extract, menadione and preferably from 'l—cysteine-HCl; tryptic soy broth with dextrose (available from BBL) supplemented with beef extract and l-cysteine-HCl; and’ brain heart infusion broth (available from Difco) supplemented with yeast extract and l-cysteine-HCl. Preferred growth media are Brain Heart Infusion Broth supplemented with yeast extract and l-cysteine-HCl; and tryptic soy broth with dextrose The most preferred growth medium is Brain Heart Infusion Broth supplemented with beef extract and l-cysteine-Hcl.

.. V(available commercially from Difco Labs or BBL) supplemented with l-cysteine hydrochloride and yeast extract.

The fermented cultures are then cooled to a temperature of from 4 to 10'C, preferably from to 8’C, and most preferably from 4 to 'C. The cooled cultures are then inactivated with B-propiolactone (BPL). The 3- propiolactone is generally used 0.10 to In the inactivation in excess, typically in a quantity which is 0.15%, preferably about 0.11% v/v. procedure, the 5-propriolactone is added directly to the cooled culture and the resultant mixture is allowed to stand for a period of at least 24 hours, preferably from 48 to hours. The culture is maintained at a temperature of no 'C during the Residual fi~propiolactone more than 10'C, preferably from 4 to inactivation stage of the process. may then be removed or inactivated by any of the known procedures. One suitable procedure which may be employed is hydrolysis. More specifically, the BPL-containing culture is heated to a temperature of at least 36°C, preferably from about 36 to about 38'C and most preferably from 30 to 40‘C for a period of at least about 2 hours, preferably from» 3 to 5 hours, and most preferably about four hours.

Known preservatives may then be added to the inactivated culture. Suitable preservatives include: thimerosal added to concentrations of up 1:1000, gentamycin added up to 30 micrograms per ml and mixtures thereof. A mixture of thimerosal (preferably in a 1:10,000 final concentration) and gentamicin (30 microgram/ml) is preferred. ' The cultures may be adjuvanted in accordance with techniques known to those in the art with any of the known adjuvants. Examples of suitable adjuvants include: any of the carbopol-based, oil-based and aluminum based adjuvants which are commercially available. 10% aluminum hydroxide gel is particularly preferred.

BPL inactivated cultures of virulent £. necroohorum isolates may then be combined with an adjuvant to prepare final bacterin.

The bacterin of the present invention may be administered to any of the ruminant animals such as cattle or sheep by any of the known procedures or a combination of the known procedures for parenteral administration such as intramuscular or subcutaneous injection. In most cases, intramuscular injection is preferred. The appropriate dosage of bacterin will, of course be dependent upon the size of the animal being treated and may be readily determined by the administering veterinarian. It has been found, however, that a dose of from to 4 ml is generally suitable for adult sheep and a dose of from 2 to 6 ml is generally suitable for adult cattle.

It is also preferable to administer the bacterin in a series of doses, preferably in a series of from 2 to 3, preferably 2 injections which are given at intervals of from 2 to 6 weeks, preferably from to 5 weeks. The bacterins of the present invention which were prepared from isolates 5118 and 5120 have been found to provide very good protection of cattle and sheep from fusobacterium disease after two intramuscular injections given at intervals ranging from 2 to 6 weeks.

All percentages given in these Examples are percentages by weight, unless otherwise indicated.

EXAMPLES Example 1 The following is a description of methodology used to prepare E. necrophorum Experimental Serial X0188. This serial was used to vaccinate cattle in the South Dakota Cattle Efficacy Trial, Frozen seed cultures of E. necroohorum isolates 5118 and 5120 were inoculated onto blood agar plates prepared from brain heart infusion agar, 5% bovine blood, and 0.05% These cultures were incubated in an anaerobic glove box at 37'C for approximately 24 hours under an atmosphere of 80% N2, 10% H2, and 10% C02. A sample of the bacterial lawn from the blood agar plates was transferred using an inoculating loop into 125 ml bottles of Eugon Broth (which is commercially available from Baltimore Biological Laboratories (BBL)) supplemented with 0.5% maltose, 0.5% Beef Extract (available from BBL), 0.5% Yeast Extract (available from BBL), 50 milligram/l hemin, 500 microgram/l menadione, .05% l-cysteine hydrochloride. l-cysteine-HCl.

The culture bottles were incubated at 37'C in an anaerobic glove box for approximately 24 hours at which time the contents were used to inoculate 1500 ml flasks of Brain Heart Infusion Broth (BHI) supplemented with 1% yeast extract and 0.05% l-cysteine-HCl. Flasks were incubated at 37'C in the glove box for approximately 18 hours by which time the fermentation was complete. The flasks were then chilled to approximately 4’C in a refrigerator.

B-propiolactone (0.11% v/v) was added to each flask and each flask was then mixed to distribute the inactivating The flasks were then stored at 4'C. After 4 days, the BPL was hydrolyzed by heating the cultures to 37'C for 3 hours in a water bath. agent evenly.

To test for inactivation, samples 'from each of the inactivated cultures were streaked on BHI blood agar plates and incubated anaerobically for 24 hours. No growth was observed on the plates. An experimental bivalent bacterin was prepared by combining volumes of the S118 and 5120 inactivated cultures in a 1:1 ratio, then adjuvanting with 10% aluminum hydroxide gel (Rehydrogel, Reheis Chemical Company).

Gentamicin (30 microgram/ml) was added as a preservative.

Example 2 The following methodology was used to prepare E. necrophorum Experimental Serials 2968788 and X0988.

The only differences between Serials 2968788 and X0988 were due to use of different experimental lots of F. necrophorum 5118 and 5120 in each serial.

Frozen seed cultures of E. necrophorum isolates 5118 and 5120 were inoculated onto blood agar plates prepared from brain heart infusion broth, (Difco), 1% yeast extract (Yeast Products, Inc.), .O5% purified Agar (Difco), % fresh bovine blood and .05% 1-cysteine HCl. Following 24 hours incubation at 37'C in an anaerobic glove box under a gaseous atmosphere of % hydrogen, 10% carbon dioxide, and 80% nitrogen, loopfuls of colonial growth were transferred from blood plates to tubes containing 15 ml of BHI broth supplemented with 1% yeast extract and .05% 1-cysteine HCl. The tubes were incubated anaerobically at 37°C for approximately 8 hours. 3 ml volumes of active seed culture were then passaged into bottles containing 80 ml of Brain Heart Infusion Broth (BHI) supplemented with 1% yeast extract and 0.05% l-cysteine-HCl and incubated anaerobically at 37'C. Following 16 to 18 hours incubation, cultures were passaged again, this time into 350 ml of the Brain Heart Infusion Broth supplemented with 1% yeast (extract and 0.05% l—cysteine HCl and incubated anaerobically at 'C for approximately 8 hours.

Finally 10 liter volumes of BHI-yeast media supplemented with .05% 1-cysteine HCl were inoculated with active seed culture (3.5% to 4.5% v/v) and incubated at 37'C with constant mixing for approximately 16 hours. Cultures were chilled to 4’C in an ice bath, inactivated with B-propiolactone (0.11%), and mixed at 4'C for 4 days. Residual BPL was hydrolyzed by heating the cultures at 37'C for 4 hours. Hydrolyzed cultures were tested for inactivation in the same manner as was described in Example 1. Samples were streaked on BHI blood agar plates and incubated anaerobically for 24 hours. No growth was observed on the plates.

Inactivated cultures of 5118 and 5120 were adjuvanted individually with aluminum hydroxide gel (10% v/v) and preserved with gentamicin (30 microgram/ml). A bivalent bacterin was prepared by combining equal volumes of inactivated, adjuvanted 5118 and 5120 cultures. -10..

‘Example 3 Monovalent F. necrophorum 5118 and 5120 bacterins were prepared in the same manner using the same materials as were described in Example 1. Mouse challenge trials were conducted to determine whether the experimental monovalent bacterins could protect mice from virulent challenge with heterologous isolates of F. necrophorum. Virulent F. necrophorum isolates 5103, 5116, and 5120 were obtained from cases of ovine foot rot in 3 different Missouri sheep herds. Isolate 2382 was a bovine Liver Abscess isolate obtained from a calf slaughtered at a Each of these isolates exhibited typical colonial morphology, mouse pathogenicity and microscopic characteristics of F. necroohorum. Groups of five or ten 18 to 20 gram Swiss-Webster mice received three .2 ml subcutaneous injections of either 5118 bacterin or 5120 bacterin at weekly intervals. Equal numbers of control mice received .2 ml SC of placebo (adjuvanted PBS).

Missouri Packing Plant.

One week post final vaccination, groups of vaccinate and control mice were challenged intraperitoneally with .25 ml of 16 hour cultures of either F. necrophorum isolate 5103, 5116, 5120, or 5126.

Mice were observed and deaths recorded over a 7 day observation period. Results of the mouse challenge trials are presented In Table 1.

Results showed that monovalent bacterins.prepared from E. necroohorum isolates 5118 and 5120 could protect mice from lethal challenge. It was significant that protection was seen Thus, -propriolactone inactivated isolates S118 and 5120 bacterins appear to provide effective protective immunity under field conditions. A against a variety of heterologous isolates.

Claims (2)

Claims

1. Use of fusobacterium necrophorum isolates for the manufacture of a bacterin for treating cattle and sheep to prevent liver necrosis caused by Fusobacterium necroghorum infection in which a) a virulent strain of Fusobacterium necrophorum is isolated and b) the virulent isolate of a) is inactivated with B-propiolactone.

2. The use of Claim 1 in which the bacterin also includes an adjuvant. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS