NO119491B - - Google Patents

Description

Method for producing a vaccine against infectious gastoenteritis virus (intestinal catarrh).

The invention relates to a method for the production of

vaccine against infectious gastroenteritis hereinafter referred to as S.G.E.

Infectious gastroenteritis is a highly contagious and widespread pig disease that causes significant economic losses. S.G.E. can affect pigs of all breeds and all ages, but causes widespread mortality only in very young pigs. S.G.E. was first described by Doyle and Hutchings in 19^6 (J.A.V.M.A., Vol 108, pages 257-259), and since that time the disease has been reported in many American states and in other countries.

It has been practice in the area to feed infected intestinal tract

from infected pig to sow three or more weeks before the pigs give birth.

In this way, passive immunity is transferred to the piglets. This

however, the practice is unhealthy as it serves to further spread the disease.

It has previously been generally known cultivation of a

virus in several successive passages in tissue culture fluid until the virus is no longer pathogenic. However, the present invention lies in the finding that by shortening the passage interval time there is an improvement in the survival of none of the S.G.E. viruses. Cultivation of S.G.E. virus in several consecutive tissue cultures is previously known, but no non-virulent antigen is developed which would immunize pregnant sows who would then in turn pass on passive immunity to their children. No S.G.E. vaccine has previously been developed that includes protection for sows and piglets.

While the inactivated or reduced live vaccine is preferably produced on canine or porcine kidney tissue culture, it can

..also grown in other tissue cultures such as monkey etc.

The purpose of the invention is to provide new, inactivated or diluted vaccines containing viruses produced in tissue cultures, which vaccines are intended for use in the immunization of sows for the protection of these and their offspring during lactation due to all other pig types that are immunologically adult against.S.G.E.

The invention therefore relates to a method for the production of a vaccine in that virulent infectious gastroenteritis

virus is inoculated into a first tissue culture, in which the virus grows adults during the intended incubation, the virus particles are removed from said culture and introduced into another tissue culture and this incubation, inoculation and repeated incubations of the virus from one culture to the other continue until the virus is no longer pathogenic , the method being characterized by allowing the virus to grow in the various tissue cultures for a period of 6-24 hours, preferably 8-12 hours, the non-pathogenic virus from the last tissue culture being added to a stabilizing medium and incubated at approx. 25°C, preferably for 5 days.

Dilution of S.G.E. virus (the term "dilution" is used here in the sense of virulence reduction) occurs by growing or propagating the virus in tissue cultures at intervals of 6-24 hours until the virus no longer causes symptoms of S.G.E. in piglets. The virus is transmitted in series at intervals of 6-24 hours until

the virus is non-pathogenic and until a vaccine prepared from the virus will stimulate immunogenesis in pigs without causing symptoms of

S.G.E. and without spreading foreign viruses.

Kidneys are preferably used to produce the tissue cultures, although it will be understood that other tissue types can also be used.

After initiation of growth for S.G.E. virus and propagation of the virus, a vaccine is prepared by harvesting the virus-containing culture, preferably with the addition of a stabilizer and then incubating the vaccine to inactivate the S.G.E. virus. The vaccine can be stored as a box or it can be frozen, e.g. freeze-dried.

The inactivated S.G.E. vaccine is used to immunize sows and their suckling piglets against S.G.E. by injecting the vaccine into the pig before it gives birth to stimulate the production of antibodies that protect the sow, and which are also transferred to the milk and thereby protect the suckling piglets. Purka can be injected intramuscularly or subcutaneously.

Unless otherwise stated, all quantities by weight and percentages are given by volume.

Example 1.

The original starting virus was prepared from piglets infected with S.G.E. The mucous membrane was removed from the intestinal tract of infected pigs. This mucosa was then diluted with a buffered saline solution, horse serum added and homogenized. The buffered salt solution consisted of 16 g KH 2 PO 1 , 34 g Na 2 PO 3 and 8.5 parts NaCl in 1000 ml aqueous solution. One part by volume of horse serum was added to 9 parts by volume of saline solution. A part of the mucosa (mucous skin) was added to 4 parts by volume of the finished saline solution. This buffered salt solution is completely different from "Hank's Balanced salt solution" and other salt solutions commonly used as nutrient fluids for tissue cultures.

The homogenate was centrifuged and filtered through a Seitz filter to render the homogenate bacteriologically sterile. All the above steps were carried out at between 0 and 4°C.

The filtrate prepared in example 1 was then added to different tissue culture systems, one-layer systems or suspension systems which had been prepared according to usual methods. The medium usually used consisted of 9 to 9.5 parts by volume of Hank<*>'s Balanced Salt Solution (Hanks, J. Cell Comp. Physical, vol. 31, pages 235-260, 1948), and 0.5 to 1 part by volume of inactivated horse serum . Other media that can be used for the initiation and transport of infected cultures include a) "Earle's BSS" plus 5-10% horse serum and b) Medium 199 described by Morgan et al (proe.Soc.Exp. Biol. and Med., vol. 73, pages 1-8, 1959) added 5-10% horse serum. If desired, lactalbumin hydrolyzate can be added in a final concentration of 0.5% to the above-mentioned media. In the cultivation and dilution of viruses according to the present invention, any non-toxic nutrient in the form of liquid tissue culture can be used. In the following examples, the medium used is 9 parts Hank's Balanced Salt Solution and 1 part inactive horse serum (calculated as parts by volume).

The pH of the medium was adjusted to 7.2 to 7.6 by incubating the cultures at 35 to 38°C. Serial transfer of S.G.E. virus in tissue cultures was performed at intervals of 24 hours or less, most often between 6 and 14 hours, by inoculating freshly prepared tissue cultures with pooled dilutions from previous transfer. Harvested and pooled fluids were also inoculated orally in piglets at every fifth transfer to check for the presence of the virus and/or if the virus had survived.

S.G.E. virus virulent for piglets was found

in strengths (titer) of at least 10 J after 5 to 15 transfers. Isolation and cultivation of S.G.E. virus in tissue cultures has been carried out with material from various pigs infected with S.G.E. In each case, the presence of virus was demonstrated by oral inoculation of piglets, which developed clinical symptoms of S.G.E. and died. Moreover, the presence of virus was demonstrated by electron microscopic examination of tissue culture fluids. Virus particles observed in these tissue culture fluids were identical to virus particles isolated from intestinal mucosa extracts from infected piglets.

Electron microscopic identification of this S.G.E. virus was performed at 5, 10, 15 and 40 tissue culture passages. At higher transmission stages (20-40), the virulence of the virus became less and less for piglets until the virus became avirulent.

It has been found that the tissue culture techniques of the invention yield viruses in high yield from tissues originating from pigs, sheep, cattle, dogs, martens and cats. Other animal tissue can also be used to produce propagation of S.G.E. virus. Production of vaccine from tissue types other than pig tissue, especially tissue from animals of the canine genus, e.g. dogs and foxes, eliminate unwanted contamination in the form of viruses in pigs. Canine virus that can be found in the cultured dog tissue or dog tissue cells used in the present method,

are not pathogenic to pigs, and therefore have no effect on

pigs receiving a vaccine based on canine tissue cultures.

The virus produced according to the invention can be diluted according to potency or stabilizers or other non-toxic compounds can be added. For use as a vaccine, the virus can be dried, e.g. by freeze-drying, or can be prepared in liquid form.

Administration of the vaccine of the invention can in practice

only used on pigs, which can produce protective antibodies. Since piglets are immunologically immature and at the same time most susceptible to S.G.E., they cannot be successfully vaccinated. However, the piglets can be given immunity by injecting the S.G.E. vaccine into the sow before it comes down with the litter. Piglets that nurse these vaccinated sows are immune to S.G.E.

Example 2.

The filtrate was prepared as indicated in Example 1, and S.G.E. virus was cultured according to the procedure described above, using 9 parts of Hank's Balanced Salt Solution and 1 part of inactivated horse serum. Virus-containing fluids from the 10th to 12th tissue culture transfer were diluted 1 to 20, and inoculated into regular Povitsky flasks containing a single layer of canine kidney cells. The flasks were incubated at 37°C for 10 hours, during which no cytopathic effect could be observed. The single layers were released into the liquid by freezing and thawing. The identity of the fluid's viral content was determined by oral inoculation of 1 ml doses of every 10th serial dilution into piglets kept in individual separate cages. Infection titres for the virus of at least 10 were found.

When it is desired to prepare an inactivated vaccine, the virus-containing culture is combined with a stabilizing medium and incubated at 25°C for preferably 5 days.

After incubation, the vaccine can be freeze-dried or stored

at low temperature, e.g. -41°C or -35°C The inactivation of the virus was confirmed by the fact that oral administration of 10 ml of liquid did not induce S.G.E. in piglets.

Example 3.

The effectiveness of the vaccines prepared according to Example 2 was tested by intramuscular inoculation in sows during the last 6 weeks before farrowing. All sows were of special pathogen-free litters and were taken from pig farms that had not had S.G.E. for over 20 years. 48 hours after birth, 2 or 3 pigs were born

(control) from each litter taken from the sow and isolated in separate rooms. These control piglets, which did not receive any milk from the sow for 2 days and were therefore exposed to S.G.E. as well as all piglets that stayed with the sow, were exposed to oral infection at the age of 4 days with 1000 infection doses S.G.E. Piglets originating from a non-vaccinated sow (control) were also exposed to infection, and the results appear in table 1.

Sows vaccinated with 2 or 4 ml of inactivated S.G.E. vaccine before litter descent transferred a passive immunity to S.G.E. to their piglets. Control piglets that were taken from the sow and isolated and exposed to infection two days later with 1000 infection doses of S.G.E., usually developed watery diarrhea and vomiting after 24 to 48 hours, and died after 4-5 days after infection. Experimentally infected immune piglets nursing a vaccinated sow showed no symptoms of S.G.E. as long as they got milk from the sow. In some cases, sows that were not experimentally infected were exposed to a mild fever, diarrhea and agalactia (lack of milk production) for one or two days. Because of this, the piglets could not get milk from the sow and therefore had diarrhoea. When the sow again became able to

give milk, the piglets got rid of diarrhoea. Other experiments have also confirmed the observation that a constant supply of sow's milk is necessary for the piglets to protect them against S.G.E.

Preferably, the vaccination should consist of a minimum of 2 ml

dose given within 6 weeks before sows give birth.

Non-vaccinated sows, on the other hand, did not confer immunity to S.G.E. to the piglets. These piglets developed watery diarrhea and vomiting after 24-48 hours and died within 6-7 days of infection. The vaccine was also an effective immunization agent for swine. Non-vaccinated sows had more severe symptoms of S.G.E. compared to vaccinated sows.

Example 4.

Liquids containing S.G.E. virus propagated or cultured as described in Example 2 during 40 transfers on dog kidney, was pooled with stabilization medium in a ratio of 50% liquid to 50% stabilization medium, and freeze-dried. The avirulence of the vaccine was tested by several oral inoculations corresponding to 5 or 10 ml of undiluted tissue culture fluid in piglets. One could not observe any S.G.E. symptoms during a post-inoculation observation period of 10 days to 2 weeks. Presence of S.G.E. virus was confirmed by electron microscopic examination of the fluids.

Example 5.

The efficacy of the diluted vaccine prepared as in Example 4 was examined by intramuscular inoculation of sows 17 or 34 days before litter descent. The results are listed in Table 2.

Diluted S.G.E. vaccine provided effective immunization of sows (active) and their piglets (passive). Control piglets that were taken from the sow and infected 48 hours later with 1000 infection doses of S.G.E. got typical symptoms of S.G.E. and died. None of the immune pigs that diet vaccinated sows died, even ora 1 had watery diarrhea for a day. The unvaccinated sow was susceptible to S.G.E., and did not transfer immunity to S.G.E. to the piglets.

Claims (1)

Method for producing a vaccine by inoculating virulent, infectious gastroenteritis virus into a first tissue culture in which the virus is allowed to grow during continued incubation, the virus particles are removed from said culture and introduced into another tissue culture and this incubation, inoculation and repeated incubations of the virus from one culture to the next continues until the virus is no longer pathogenic, characterized by allowing the virus to grow in different tissue cultures for a period of 6-24 hours, preferably 8-12 hours, whereby the non-pathogenic virus from the last tissue culture is placed in a stabilizing medium and incubated at approx. 25°C, preferably for 5 days.