DK154804B - Process for producing a modified, live Pasteurella haemolytica vaccine - Google Patents

Description

1 DK 154804 B

The present invention relates to a method of producing a modified live Pasteurella haemolytica vaccine capable of inducing immunity in cattle without serious side effects.

Pasteurella haemolytica is known to infect cattle and cause respiratory illnesses and has been implicated in the etiology of transport fever syndrome. [Jensen et al., "Diseases of Feedlot

Cattle ”, 3rd edition, Lea & Febiger, Philadelphia (1979), pages 59-65]. Presently, pasteurellosis in livestock is combated with varying degrees of success in administering vaccines, antimicrobials or a combination thereof.

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Known vaccines for pasteurellosis contain killed whole cells (bacteria), live attenuated bacteria or cell fractions, with or without an adjuvant. They lacked efficacy in non-toxic doses.

Commercial bacteria, which usually contain one or more strains of formaldehyde Pasteurella, are undesirable due to several factors. At least two doses of one. such vaccine given at several day intervals is necessary for effective protection (Collins, supra). They are often unsuccessful and have been observed to cause transient endotolic shock [Larsonm.fi., J.Am.Vet.Med. Assn. 155: 495 (1969)].

A number of combination vaccines 25 containing killed Pasteurella are also known and used. For example, killed Pasteu rella has been combined for use in cattle with bovine infections rhinotracheitis virus [Matsuoka et al., J.Am.Vet.Med.Assn. 160 (3): 333 (1972)], with bovine parainfluenza-3 virus [Sampson et al., Vet.Méd.Small Anim. Clin. 67 (12): 1354 (1972), U.S. Patent 50 No. 3,501,770 and U.S. Patent 3,526,696], in quadrivalent form with bovine infectious rhinotracheitis virus, bovine viral diarrhea-mucosal disease virus and bovine parainfluenza-3 virus (U.S. Patent No. 3,634,587) and with Salmonella typhimurium (U.S. Patent No. 4,167,560). Bird vaccines, especially for 55 use of poultry cholera, containing, killed Pasteurella alone

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or in combination with other bacteria or viruses are also known.

Bird vaccines and bovine vaccines containing live Pasteurella have also been developed.

Recently, a cattle vaccine for transport fever, administered by intradermal injection and comprising a live culture of a field strain of Pasteurella haemolytica in a brain / heart-infusion soup, is disclosed in U.S. Patent No. 4,171,354.

jLQ Ribosomal vaccines made from various organisms, including Pasteurella haemolytica, are described in Belgian Patent No. 857,014. A potassium thiocyanate extract of Pasteurella haemolytica serotype 1 was found to be immunogenic and to produce cross-immunity in mice against Pasteurella multocida 15 type Å [Mukkur, Infect. Immune. 18 (3): 583 (1977)].

Until the present invention, it has not been believed that chemical modification of Pasteurella bacteria and the manufacture of a safe and highly effective vaccine for the protection of animals, 2Q especially economically important animals for human consumption, against destruction of transport fever and other associated diseases with Pasteurella, has been achieved. The vaccine produced by the novel modified Pasteurella organisms has also been shown to be cross-protective against various mark isolates of Pasteurella species.

One aspect of the method according to the invention consists in safe and effective vaccines for the protection of cattle against diseases in them. upper respiratory organs that are associated with 2g see Pasteurella infection, including what is commonly called shipping fever. Modified live Pasteurella haemolytica monovalent vaccine has been prepared for administration by the subcutaneous, intranasal or, preferably, intramuscular route. . For administration to bovine animals, this vaccine preferably contains from ca.

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DK 154804 B

7 11 1.0 x 10 to approx. 1.0 x 10 CFU (colony forming units) per dose of the modified live Pasteurella haemolytic organisms together with a suitable carrier and / or stabilizer. The vaccine is administered in one or two doses, preferably two, of 2.0 to 5.0 ml each, depending on the nature and size of the animal being vaccinated, as well as the organism metal, i.e. the number of organisms per dosage.

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A bivalent vaccine consisting of vaccinal amounts of the modified live Pasteurella haemolytica and the one in Ans. No. 1187/81 modified live Pasteurella multocida may also be used.

ΤΠ administration to cattle contains such a vaccine from approx. 1.0 x 10 to approx. 1.0 x 10H CFU per dose of each of the modified live Pasteurella strains together with a suitable carrier and / or stabilizer. This vaccine may be administered by the subcutaneous, intranasal or, preferably, intramuscular route in one or two doses, preferably two, of 2.0 ml to 5.0 ml each.

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Another aspect of the method of the present invention consists of the novel modified Pasteurella haemolytica organism. It was deposited in The American Type Culture Collection in Rockville, Maryland, on March 5, 1980 and has been filed with Deposit No. 31612. The organisms are freely available upon application upon issue of the present application or other equivalent application in another country as a patent. for a period of at least 30 years or at least 5 years after the last inquiry.

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DK 154804 B

The Pasteurella bacteria used to prepare the vaccine prepared by the method of the invention were isolated from lung tissue from infected animals and identified by standard identification methods. The isolate was found to be 5 virulent in vaccination of mice and hamsters. Propagation of the bacteria was carried out in a liquid medium consisting of tryptose soup supplemented with thiamine (Difco Laboratories, Detroit, Michigan). Tryptosis agar with 5% sheep blood was used as the medium to determine the colony characteristics of each of the Pa-10 steurella parent bacteria. Parents The Pasteurella strain is chemically modified with acridinium salts such as 3,6-bis-dimethylaminoacridinium chloride (acri di n-orange), 2.8 (3.6) diamino-10-methylacridinium chloride and 2.8 (3.6) diaminoacridinium chloride ( acriflavine HCl) at concentrations from ca. 0.1 pg per ml to 15 approx. 150 pg per day. ml in a medium consisting of tryptosis supplemented with thiamine soup. The organisms are passed 10 to 30 times. After modification, the morphological properties of the bacteria change from smooth, shiny and mucous colonies of 1.5 - 2.0 mm in diameter after incubation at 37 ° C for 18 hours to rough, 20 matte and dot-shaped colonies of 0.5 - 1.0 mm after incubation. There are no other differences between the bacteria before and after the modification. All other properties are as described in Bergey's Manual of Determinative Bacteriology, 8th edition 1974. The chemically modified bacteria can be further cultured and passed into any suitable growth medium, e.g. in tryptose bui1-ion supplemented with thiamine, or in the medium described in the present application.

The vaccine prepared by the method of the invention 30 is prepared by standard methods well known in the art, e.g. by combining the bacteria with a suitable carrier and / or stabilizer.

The invention thus relates to a method of producing a modified live Pasteurella haemolytica vaccine capable of inducing immunity in cattle without serious side effects, which is a method of chemically modifying the virulent Pasteurella haemolytica strain

DK 154804 B

5 ATCC No. 31611 at 10-30 passes of it in the presence of an acridinium salt, after which the obtained modified bacterium Pasteurella haemolytica ATCC No. 31612 is grown in a suitable growth medium and combined with a carrier for the vaccine.

DETAILED DESCRIPTION OF THE INVENTION.

The process of the invention is conveniently carried out in that the growth medium has the composition: 10

Ingredients grams / liter of water

Low molecular weight obtained by hydrolysis of proteins ... 10.0 - 40.0 15 Hydrochloric acid hydrolyzate of casein. 5.0 - 20.0

Bankreatic hydrolyzate of casein, containing all amino acids originally found in casein ............ 5.0 - 20.0 20 Uncreatic hydrolyzate of casein, containing amino acids, peptides and major protein fractions ... .. 5.0 - 20.0 Yeast Extract ................. 5.0 - 20.0 N. Sodium Chloride ............. 0.5 - 3.0 25

The above ingredients are combined and sterilized by autoclaving. A solution of 20.0 - 80.0 grams per liter of sucrose is sterilized separately by autoclaving and added to the other ingredients when cold. The pH is adjusted to 7.4 - 7.6 with 10N sodium hydroxide solution.

From 1 to 4 parts of the growth medium containing the modified organism is combined with 1 part stabilizer and lyophilized.

An example of a suitable stabilizer is the following:

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Solution 1

Ingredients' · Gram / liter of water

Potassium hydroxide (anhydrous) 0.2 - 0.8 L-glutamic acid 0.5-2.0

Potassium phosphate, dibasic (anhydrous) 1.0 - 4.0 Potassium phosphate, monobasic (") 0.3 - 1.5

Sucrose 50.0 - 200.0

The ingredients are combined and sterilized by autoclaving.

10 Solution 2

Ingredients Gram / liter of water

Gelatin (Knox) 100.0 - 300.0 Autoclaved for 4 hours to hydrolyze.

2 parts of solution 2 are added to 3 parts of solution 1 to prepare the stabilizer solution.

N

20! §2i® £ i22i_f2H§ £ i22_22_} £ §i2iskjnodification_of_Pasteurella haem2lytica_vaccine_stammeiu

The Pasteurella haemolytica parent strain used to produce the modified living organism 25 of the invention (ATCC No. 31611) was isolated from aseptically removed lung tissue submitted to the University of Nebraska, Department of Veterinary Sciences death of transport fever and frozen at -50 ° C.

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Samples of the frozen tissue were thawed and smeared on the surfaces of sheep blood agar plates. After incubation at 37 ° C for 24 hours, a pure culture of colonies resembling Pasteurella species was observed. Several colonies were selected 35 and seeded on experimental media for identification. The results of the tests showed that the organism was Pasteurella

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Other colonies of the organism were seeded on a medium of tryptose bouillin supplemented with thiamine. After incubation at 37 ° C for 24 hours, a further passage was made in the same medium. A volume of stabilizer solution, as described above, was added to the growth of the organism. The mixture of growth and stabilizer was dispensed into 2.0 ml aliquots and subjected to lyophilization. The lyophilized parental organism was stored at 4 ° C. Colonies 5 o of the parental organism after incubation at 37 C for 24 hours were circular, shiny and alimony. The colonies were approx. 2.0 mm in diameter.

A lyophilized sample of the parental organism was rehydrated, seeded on a medium of tryptose broth supplemented with 1.5 µg of acriflavine HCl / ml and subjected to three µ passages in this broth. By the following passages, the concentration of acriflavine HCl was increased. At the 6th passage, the concentration of acriflavine HCl was 15.0 pg per day. ml.

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By the 10th passage, the colonies of the organism were spot-shaped, matte and rough with a diameter of ca. 0.5 mm.

Alongside the changes in the size and properties 2q of the colonies of the acriflavin-treated organisms, a marked reduction in the virulence of the modified organisms was observed. The LD5Q values on mice for the parent strain and the chemically modified strains were 2.9 x 10 3 and 7.3 x 10 6 CFU, respectively. On hamsters 25, the LD ^ q values for the parental organism and for the chemically modified strain were 1.1 x 10 6 and - 6.2 x 10 8 CFU, respectively.

After 12 passes in medium supplemented with acriflavin HCl, 3Q the modified organism was passed 15 times in a medium not supplemented with acriflavin HCl. The material from the 15th passage yielded LDsg values on mice and hamsters which were almost identical to those produced by material prior to passage in acriflavin-free medium, but after modification in acriflavin 25 HCl supplemented with medium, showing that the modification is stabi1.

Claims (5)

8 DK 154804 B Preparation and Use of the Modifier For the preparation of a vaccine, additional amounts of the modified Pasteurella haemolytica (12th passage) are grown in a suitable medium such as that described above combined with a stabilizer and lyophilized. An example of a stabilizer that can be used is described above. The use and effect of the vaccine on cattle is described in an article in the American Journal of Veterinary Research, 10 Vol. 44, No 10, (1983) pages 1848-1852. v ' A process for the preparation of a modified live Pasteurella haemolytica vaccine capable of inducing immunity in cattle without serious: side effects characterized by chemical modification of the 'virulent Pasteurella haemolytica strain ATCC No. 31611 by 20 to 30 passages of the in the presence of an acridinium salt, after which the obtained modified bacterium Pasteurella haemolytica ATCC No. 31612 is grown in a suitable growth medium and combined with a carrier for the vaccine. A method according to claim 1 for producing a further equal amount of the modified Pasteurella haemolytica bacteria, characterized by growing the bacteria for a sufficient time to enable growth of a larger amount of the bacteria. 30 Process according to claim 2, characterized in that the growth medium is tryptose broth supplemented with thiamine. Process according to claim 2, characterized in that the growth medium has the composition: DK 154804 B Ingredients Gram / liter of water Low molecular weight obtained by hydrolysis of proteins ... 10.0 - 40.0 Hydrochloric acid hydrolyzate of casein. 5.0 '- 20.0 5 Pancreatic hydrolyzate of casein, containing all amino acids originally found in casein ............ 5.0 - 20.0 Pancreatic hydrolyzate of casein, jLo containing amino acids, peptides and major protein fractions ... ... 5.0 - 20.0 Yeast extract ....... 5.0 - 20.0 v Sodium chloride .............. 0.5 - 3.0] _5 Method according to claim 2, 3 or 4, characterized in that the growth medium contains a stabilizer. 20 25 30 35