DE1208038B - Process for the manufacture of vaccines against Coli infections - Google Patents

Description

Process for the preparation of vaccines against Coli infections The Virulence of various strains of E. coli in mice is generally low, the LDs0 values for these organisms are usually between 10 and 108 organisms, while other pathogenic bacteria have LDs0 values of 10 to 103 organisms. At such an altitude, it is difficult to tell whether the infection or toxemia is involved causes the death of the animal, and attempts to produce vaccines or antisera, have so far not led to any great success. Albeit a poor immune response was caused in the test animals, those produced from these lines were awarded Vaccine only against infections by a homologous E. coli serum type Protection.

It has now been found that a new strain of Escherichia coli, designated as CN 5364, can provide protection against many E. coli serum types, which are pathogenic in many young warm-blooded animals.

The new strain 5364 differs from those previously described Strains in that it has a very high virulence in mice, the LD 50 values in approximately 102 (intraperitoneal), 105 (intravenous) or 10 [beta] organisms (subcutaneous) lie. In these cases, death can follow within 3 hours of the injection.

The new strain CN 5364 can be further characterized by that it results in slightly convex, creamy white glittering colonies on blood agar, and the This distinguishes the strain from its main strain RVC 383 (Royal Veterinary College), a 0.15 type of the strain according to the international classification (the surface antigen is not indicated). On the other hand, it's a typical one E. coli strain as described in Bergey's Manual of Determinative Bacteriology and by Topley and Wilson's Principles of Bacteriology and Immunity, which has the following biochemical properties. Fermented grape sugar, milk sugar, sucrose, dulcitol, mannitol and cellobiose with the production of acid and Gas. It does not ferment adonitol, inositol or inulin. He doesn't use citrate, does not liquefy gelatin, creates trace amounts of hydrogen sulfide, gives a positive methyl red reaction and a negative Voges-Proskauer reaction. Created Produces acid in milk and causes milk to curdle, produces indole, splits urea not on, reduces nitrates to nitrites and is immobile. With 5 ° / 0 horse blood agar dishes it is generally hemolytic, although hemolysis in some parts may not be present. Heamolysis, if present, is a zone of about 2 mm wide.

The new strain CN 5364 is for public use in bacterial Culture collection available from Wellcome Research Laboratories, Beckenham, Kent, England and can be grown on any common medium known to that it is suitable for the cultivation of an E. coli strain. Preferably will a Dorset egg medium used for the conservation of the trunk. In general it was found that nutrient agar and nutrient broth media are suitable and suitable for cultivation are satisfactory.

The temperature is usually around 37 "C and the pH between pH 7.0 and 7.4.

The strain CN 5364 is capable of providing protection in a wide range against E. coli serum types, which from different receptive Warm-blooded animals were separated, for example against 0.8- (OKG 7), 0.9-, 0.15-, 0.35, 0.78, 0.137 and other serum types. The strain can, for example, with formalin or inactivated by heat treatment. Inactivation or weakening of straining can also be carried out by other methods, of which are known is that they give organisms their pathogenic properties without losing their antigenic properties Effectiveness, take.

It was found that bacteria of the strain CN 5364 protection in mice 56 days after intraperitoneal injection an infestation with 1000 times the usual Give LDso dose. A passive protective material was also created by immunizing horses, for example, Calves or rabbits by subcutaneous injection of the inactivated strain and blood sampling received from the animal a few days later.

Mice were injected intraperitoneally with the antiserum thus obtained and infected 24 hours later.

The results showed that the antiserum was resistant to infestation in mice 1000 times the LD50 value and that no agglutination in the antisera occurred.

The following examples illustrate the invention.

Example 1 As a result of inexplicable variability in virulence of the Escherichia coli strain RVC 383 (serum type 015) a variant was separated off, which resulted in slightly convex, creamy white glittering colonies on blood agar that slightly are distinguishable from the flat gray, dull colonies of the RVC strain 383. The variant was also characterized by its much higher virulence versus mice.

It was listed as CN 5364 in the Wellcome Bacterial Culture Collection Research Laboratories, Beckenham, Kent.

The increased virulence towards mice is indicated by the following Experiment explained. Each strain of E. coli was inoculated into a nutrient broth and incubated overnight (18 hours) at 37 ° C. Cultures were placed in rows on the Diluted 10 times with physiological saline solution. Doses of 0.5 ml were given intraperitoneally injected into 6 week old W-Swiss mice, each weighing approximately 24 g. the Mice were observed for 7 days.

The mortality occurring within 48 hours, usually before 18 hours, was determined. Dilution of culture mortality RVC 383 CN 5364 Undiluted. . . . . . . 4/4 10 ............ 0/4 100 ............ 0/4 4/4 1000 ~ - 4/4 10000 ..... 4/4 Viable number each Milliliters thin culture 7 7th 108 12 108 E. coli strain CN 5364 was inoculated into a nutrient broth and grown for 6 hours at 37 ° C at a pH of approximately 7.2. The nutrient broth culture was used to inoculate nutrient agar dishes, which were then incubated for 20 hours. The organisms were then removed.

A vaccine was obtained from the outgrowth of E. coli strain CN 5364 the nutrient agar and by washing the cells in a saline solution containing 0.25% formaldehyde contained, manufactured. The density was set to a turbidity equivalent to the Brown tube 12, and thiomersal was added to a concentration of 0.01%.

Instead of using formaldehyde to inactivate the bacteria was heat at 60 ° C for 30 minutes or at 100 ° C for 10 minutes for preparation similar vaccine from CN 5364 was used.

Mice were formalized intraperitoneally with 0.5 ml of an inactivated strain CN 5364, which was prepared as described above and was adjusted to a turbidity equivalent to the brown tube 12, injected. 4 days later they were intraperitoneally with a homologous CN 5364 of a 0.15 strain set under attack. Protection against CN 5364 - homologous protection - was approximate 1000 times the LDs0 value for this strain.

Mice became active with an inactivated strain of CN as above 5364 and infected 4 days later with a heterologous strain 0.78. Of the Protection against 0.78 - heterologous protection - was approximately 100 times the LD50 value for 0.78.

This level of active heterologous protection was as high as the highest homologous protection, which so far could be determined for this serum type 0.78.

Example 2 A passive protective material was made by intravenous injection of horses with increased doses of 1, 2 and 3 ml of a suspension of an inactivated CN 5364 strain prepared according to the example and equivalent to a turbidity of the brown tube 5, was obtained after a period of 3 to 4 days. Blood was then drawn from the horses, antisera prepared and examined in mice. The studies showed protection up to 1000 times the LDs0 value in mice, when they got infected.

Example 3 A passive protective material was made by subcutaneous injection of calves with a single dose (10 ml) of the inactivated CN 5364 strain obtained, the strain prepared according to Example 1 and equivalent to a turbidity the brown tube 12 was set. The blood collection from calves was 3 and 7 days later carried out and the sera examined in mice. The investigations showed the presence of a highly protectable material in the event of infection or infestation took place.

Claims (2)

Claims: 1. A method for producing a vaccine for contraception or treatment of Escherichia coli infection, characterized in that it is the Inactivation of the Escherichia coli strain CN 5364, especially by exposure to it of formaldehyde or heat, and the suspension of the inactivated strain in one liquid carrier. 2. Method of making a passive vaccine for contraception or treatment of Escherichia coli infections, characterized in that it immunizing an infectious warm-blooded animal with the CN 5364 strain of Escherichia coli and the collection of the animal's serum.