DE1076892B - Process for the production of a vaccine for the simultaneous immunization of dogs or foxes against distemper and contagious hepatitis - Google Patents

Description

Process for the preparation of a vaccine for simultaneous immunization of dogs or foxes against distemper and hepatitis contagiosa canis The invention relates to a method for producing a combined vaccine for immunization of dogs or foxes against distemper and hepatitis contagiosa canis (H.c.c.). the Distemper and the H.c.c. are two dangerous and widespread viral diseases the dogs. The foxes and fur animals of the marten family also suffer from distemper (Mustellids). The H.c.c. virus is not only for dogs but also for foxes (fox encephalitis) pathogenic.

One can successfully fight dogs and other susceptible animals Immunize distemper. The immunization methods currently practiced are based on the Administration of a vaccine that is a live, egg-adapted, non-pathogenic virus contains. In addition, there is also the simultaneous administration of a pathogenic virus in connection with immune serum and the inoculation of a vaccine with inactivated Virus applied. Canine distemper became effective by using the above vaccines fights. However, these vaccinations do not provide protection against the H.c.c.

From the Swiss patent specification 200 180 and the USA patent specification 2 173 440 it is known that mixed vaccines can be produced by using vaccines, which contain various killed bacteria or yeast-like fungi with each other mixed.

In the German Auslegeschrift 1 027 367 a mixed vaccine for the simultaneous immunization of dogs against infectious hepatitis and distemper which contains live H.c.c. virus and live distemper virus.

The use of two live viruses according to Auslegeschrift 1 027 367 in a vaccine cannot be done safely. The living, modified, Distemper virus adapted to the hen's egg is a real modification; H., the transmission of the egg-adapted distemper virus from dog to dog and the associated Passages do not lead to any re-modification and consequent recovery of pathogenicity. The living, through passages on canine kidneys or other canine tissues However, the H.c.c. virus that has been cultivated and thereby become non-pathogenic is not a genuine one Modification. If applied to healthy, susceptible dogs, it can cause a Undergo reverse modification and become pathogenic again.

As is well known, the H.c.c. virus is excreted in the urine and on this Pathways picked up by other dogs, leading to increased H.c.c. leads.

There has now been a method of making a vaccine for simultaneous immunization of dogs or foxes against distemper and hepatitis contagiosa canis (H.c.c.) found, which is characterized in that one Vaccine for simultaneous gene immunization of dogs or foxes against distemper and H.c.c. can be produced by containing the distemper virus which has been worked up into a suspension Parts of the embryonated hen's egg with a formaldehyde-inactivated H.c.c. virus, that from dog livers and / or dog spleen by extraction or from tissue culture is obtained and its virus-damaging formaldehyde content by adding a 1.5 to 2 times the stoichiometric bisulfite solution was set, mixed and freeze-dried. Here it is advantageous if the H.c.c. antigen by an aluminum hydroxide solution is adsorbed.

It is preferable to proceed in such a way that a mixing ratio of a congestion fraction, which consists of about 500/0 of a modified, congestion virus-containing Chorioallantoic material, 400/0 beef bouillon of pe 7.6 and 100/0 500 / o glucose solution consists of approximately the same amount of H.c.c., which consists of about 750/0 of a inactivated extract containing H.c.c. virus and 250/0 of a 20% aluminum hydroxide solution exists, complies.

The distemper H.c.c. Vaccine (SH-Vaccine) obtained according to the method establishes a connection between a living, egg-adapted, non-pathogenic distemper virus and an inactivated H.c.c. virus. The manufacture of such a mixed vaccine brings various advantages. Compared to making two separate According to the procedure, vaccine preparations are made after mixing the distemper and H.c.c. vaccine share of the material, labor and energy expenditure by about half lowered. In particular, there is a saving in filling and packaging materials and a substantial reduction in the cost of lyophilization. In addition, the administration of only one vaccine instead of two is a simplification and cheaper vaccination procedures.

According to the invention, the distemper and Hcc components of the 5 H vaccines must be brought into a special form so that the two components cannot be adversely affected. The living distemper virus in particular is very sensitive and is easily damaged and destroyed by chemical, thermal and other influences. For example, the formaldehyde contained in the Hcc vaccine component would destroy the modified distemper virus within an hour if it were mixed with the congestion component. We have succeeded in eliminating the virus-damaging properties caused by formaldehyde (CH2O) by adding bisulfite (NaH SO,). One proceeds z. B. in such a way that one first quantitatively determines the free and bound formaldehyde. The free formaldehyde in the Hcc virus content is then reduced by adding 150 to 200% of the stoichiometric amount of bisulfite, e.g. B. Sodium bisulfite, chemically bonded according to the equation The Hcc vaccine component is now combined with the distemper vaccine component while cooling with ice, and the vaccine mixture is filled and lyophilized.

The SH vaccine obtained according to the method was tested for their immunizing properties against distemper as well as against H.c.c. checked.

Of the sub-edges with a dose of 2 ml each of the combined vaccine vaccinated dogs all withstood artificial infection with active distemper and H.c.C. virus. In contrast, all of the 7 unvaccinated congestion control dogs fell ill in the fever typical for virus distemper.

A part of these congestion control dogs was at the height of the so-called second rise in fever killed.

This rise in fever is due to the colonization and multiplication of the Virus marked in the organs. When examining the organs in the complement fixation reaction (KBR) a positive result for distemper was obtained. Nine not vaccinated H.c.c. control dogs became more or less severely ill with H.c.c., four of them perished after a few days. In the KBR, an infection of the organs with the H.c.c. virus be detected.

In addition to testing in animal experiments, the SH vaccine according to the invention checked for their canine virus content on pre-incubated hen's eggs. There were also twelve further test dogs were vaccinated subcutaneously with 2 ml each of the SH vaccines described above.

Blood samples were taken immediately before vaccination and 3 weeks after taken and the serum tested in the virus neutralization test on the pre-incubated hen's egg. All twelve dogs had none before vaccination, but a considerable 3 weeks later Blood serum levels of antibodies that neutralize the congestion virus are able to neutralize an infectious dose of virus.

The main advantage of the vaccine according to the invention is that with excellent immunity infection of non-vaccinated animals is excluded is.

Example a) Production of the stagnation part of the SH Vaccine Bei der Production of the live, egg-adapted, non-pathogenic distemper virus containing distemper will proceed as follows.

Chicken eggs that have been pre-incubated for 8 days are x-rayed and the location of the embryo is marked. The eggshell is drilled at the designated point, so that an opening is created through which the chorioallantoic skin with 0.2 ml 40% suspension is infected. This suspension contains modified, dated Distemper virus obtained from eggs in distilled water, the 104 / o of a 1/15 molar phosphate buffer solution of the PEE 7.6 was added. The inoculation opening is made with paraffin-soaked Japanese Tissue paper closed. The eggs treated in this way are post-incubated at 370 ° C. for 5 days and then opened. The membranes overgrown with distemper virus are harvested, collected, finely ground in the homogenizer with ice cooling and with a stabilizer solution worked up to a suspension as follows. For example, it becomes 500 ml des a stabilizer solution is added to the crushed chorioallantoic material composed of 400 ml of beef broth of p 7.6 and 100 ml of a 500 / above glucose solution. The proportion of congestion in the SH vaccine therefore consists of 500/0 virus-containing chorioallantoic material, 400/0 beef broth with a pH of 7.6 and 10% of a 500% glucose solution. b) Manufacturing of the H.c.c. part of the SH vaccine In the manufacture of the H.c.c. part of the SH vaccine the procedure is as follows.

350 g of a dog's liver and spleen containing H.c.c. finely chopped in the homogenizer while cooling with ice, and the material is taken under Add 700 ml of distilled water. worked up to a suspension. The suspension will collected in a bottle with 0.50 / 0, that is 5.25 ml 350 / above formaldehyde solution, added and shaken vigorously for 1 hour in the shaker. The suspension will subsequently frozen and thawed three times and 1 week at refrigerator temperature (about 40 C) kept.

The suspension treated in this way is then heated to 3000 for 30 minutes Centrifuged rpm and the supernatant aspirated. To 750 ml of the extract obtained 250 ml of a 2% own aluminum hydroxide solution are added. One thus obtains 1000ml of the inactivated H.c.c. virus-containing vaccine component, which is made up of 75% H.c.c. virus-containing extract and 250/0 of a 20 / above aluminum hydroxide solution. A sample of the H.c.c. vaccine content is used to determine the free and bound Formaldehyde content.

The free formaldehyde is increased by adding 1, S-fold stoichiometric Amount of bisulfite set. For example, is the free formaldehyde content 100 mg0 / o, then to 11 H.c.c. vaccine portion are 26.9 ml of a 200% bisulfite solution added. c) Production of the H.c.c. part of the SH vaccine by a tissue culture H.c.c. virus obtained from a fresh dog's kidney is as sterile as possible Conditions the renal cortex won. This is collected in a Petri dish and up to 4 to 5 mm large chunks. These bits of tissue are treated with a phosphate buffer solution (PH 7.5) according to R. Dulbecco and M. Vogt (J. of Exp. Med., 99, p. 167, 1954). This is followed by trypsination the pieces of kidney cortex with a 0.250% trypsin solution in a water bath at 370 C.

The trypsinization turns the pieces of organs into individual cells cleaved. The suspension of these detached cells is collected and the Trypsinization interrupted by hypothermia in an ice water bath. By centrifugation initially at 1000 and after washing twice with phosphate buffer solution (pH 7.5) at 600 rpm, the trypsin solution and that contained in the suspension Blood cells removed. 1 ml of washed kidney cells are now z. B. in a Mixture of 95 ml of culture medium 199 according to J. F. Morgan, A. J.

Morton and R.C. Parker (Proc. Soc. Exp. Biol.

Med., V. 73, p. 1, 1950) and 5 ml of calf serum bloated. To 100ml this cell suspension is an antibiotic mixture consisting of 20,000 I. E. penicillin, 20,000 y streptomycin and 20,000 y neomycin, added and with a 2.80 / above Sodium bicarbonate solution containing an addition of 0.0020 / 0 phenol red, a pB value set of 6.8. With this cell suspension, sterile culture vessels, e.g. Fernbach flasks, Erlenmeyer flasks or rolled rim tubes, with about 8% of their capacity loaded. About 4 to 6 days after the approach, the epithelial lawn must grow out Kidney cells are fed by replacing the nutrient solution and, if necessary, in the following days the p-value was adjusted with a sodium bicarbonate solution will. As soon as the cell lawn is fully developed, it is coated with a suspension, the one bred from dog liver and through some passages to the tissue culture contains adapted H.c.c. virus, inoculated. The H.c.c. virus is released after about 6 to 7 days after a full cytopathogenic effect has developed and The dead epithelial cells are detached by pouring off the cell suspension harvested. The H.c.c. virus content or the antigenicity of the suspension is determined by Inoculation of culture tubes in different dilutions (virus titration) or checked by examination in the complement fixation reaction (KBR). The one from the Cell suspension containing H.c.c. virus obtained from tissue culture is 0.1 to 0.20 / 0 350% formaldehyde solution added and for inactivation for 1 week at refrigerator temperature (approx. 40 C) preserve. Then 250 ml of a 20 / oigen are added to 750 ml of the suspension Aluminum hydroxide solution added.

The free formaldehyde is determined and before mixing with the The proportion of stagnation is bound by the addition of 1, 5 times the stoichiometric amount of bisulfite. d) Mixture of the distemper and H.c.c. part of the SH vaccine 1000 ml of the distemper part are mixed with 1000 ml of the bisulfite-treated H.c.c. portion while cooling with ice mixed and filled into vials of 2 ml each.

The distemper-H.c.c.-Vaccine mixture obtained in this way is added while standing Frozen at a temperature of - 400 C and lyophilized in the freeze dryer. This converts the vaccine into a durable and storable form.

PATENT CLAIMS: 1. Process for the manufacture of a vaccine for simultaneous immunization of dogs or foxes against distemper and hepatitis contagiosa canis (H.c.c.), characterized in that one from dog livers and / or - H.c.c. virus-containing spleens obtained by extraction or from tissue culture Material inactivated in a manner known per se by means of formaldehyde, then the free formaldehyde content by adding a solution that is 1.5 to 2 times stoichiometric Contains an amount of acid salts of sulphurous acid, especially sodium bisulphite, sets and the product obtained in this way with an embryonated chicken egg in Mixes the suspension containing the congestion virus obtained in a known manner and the Freeze-dried mixture.

Claims (1)

2. The method according to claim 1, characterized in that one is a Mixing ratio of a stowage part, which consists of about 50 ovo of a modified, chorioallantoic material containing congestion virus, 404 / o beef broth of pH 7.6 and 10% 500 / o glucose solution, with an almost equally large H.c.c. proportion, that of about 750/0 of an inactivated H.c.c. virus-containing extract and 25 0 / o a 20% aluminum hydroxide solution. Documents considered: German Auslegeschrift No. i 027367; Swiss Patent No. 200 180; U.S. Patent No. 2,173 440