COMBINED IN ACTIVATED VACCINE,READY FOR ADMINISTRATION AND PROCESS FOR THE PREPARATION OF SUCH VACCINE,ACTIVE AGAINST EGG PRODUCTION DROP CAUSED BY ADENO LIKE VIRUSES AND AGAINST DISEASES CAUSED BY REO VIRUSES

Abstract: The vaccine is prepared by mixing a reo virus containing liquid, obtained by cultivation of a reo virus, followed by inactivation of this liquid and optionally homogenising it, with a adeno like virus (ALV) containing liquid, obtained by cultivation of an ALV, followed by inactivation of this liquid and optionally homogenising it, and adding the mixture to an oily phase containing as essential components at least one of the components selected from the group consisting of light mineral paraffenic oils, plant oils and naphtenic mineral oils, and one or more emulsifiers, such as non-ionic surface-active compounds derived from alkylene oxide and/or hexahydric alcohols and/or higher natural fatty acids such as esters or ester-ethers optionally followed by homogenising and/or addition of a preservative.

Description: Co bined inactivated vaccine, ready for administration and process for the preparation of such vaccine, active against egg production drop caused by adeno like viruses and against diseases caused by reo viruses. The invention relates to a combined inactivated vaccine, ready for administration, against infections in poultry and more particularly against egg production drops caused by adeho-like viruses (further indicated by Δ Y) and diseases caused by aviair reo viruses in poultry, to the preparation of this combined vaccine and its application. It is known that reo viruses may cause several diseases in poultry such as arthritus/tenosynovitis, myocarditis, hepatitis, hydropericardium, and diseases in the digestive tract and/or the respiratory tracts. Avian Pathology 6 (1977) pages 271 to 284, o example, gives a survey of various pathogenic microorganisms, which may cause by themselves or in combination the viral arthritis/ tenosynovitis, such as Sta hylococcus, Salmenella, Pasteurella, Ery.>-sipelotrix, Mycobacterium avium, Bacterium arthropyogenes, Escherichia venezuelensis, Streptbbacillus monoliformis, Mycoplasma gallisepticum, Mycoplasma synoviae and reo viruses, such as the Connecticut reo virus isolate S 1133* "the LZ 67 J \ virus, or the EΔM-1 virus, the reo viruses bein considered the principal disease inducers. It has been found that viral arthritis/ tenosynovitis and other diseases caused by reo virus infection plays an important role in the cause of considerable economic losses in the poultry farming in many parts of the world. Furthermore, there is proposed a method for giving protection aginst viral arthritus by vaccination during the growth period and the subsequent transfer of maternally produced antibodies to the descendants by means of vaccination with reo virus vaccines. It has been found experimentally in this connection that immunisation of breeding animals only protected the first generation descendants, but not the second generation descendants. It was known from e.g. Avian Pathology no. 3. 7. 407-41 . that a synergistic relation might exist between M.synoviae and reo virus, although not satisfactorily proved, at the experimental OMPI . WIPO induction of formerly found symptoms in chickens, such as inflammation of the synovial membrane, the tendon sheath particularly in the tarsometarsal region, and myocarditis. It also appears from prior publications, e.g. in Neth. J. - Vet. Sci. Vol. 3 > No. 1 (1070) pages 5 to 10, that reo virusses, such as the LZ 671 reo virus, are considered to be responsible predominantly for synovitis symptoms in broiler parents. Moreover, Poultry Digest of February 1978, page 102, also dicsloses that reo virus may cause growth inhibition and diarrho in broilers, and that oral infections with digestive suspensions may cause growth inhibition in chickens. Although virus material cultivated in eggs did not cause growth inhibition after oral administration, but caused "pasting", intraperitoneal injection virus cultivated in eggs caused growth inhibition. The observed disease symptoms are brought into relation with an apparently necessary combination of infectuous agents. On the other hand more recent publications discloses that the cause of rachitis-like symptoms may be primarily attributed the occurence of toxic compounds produced by moulds such as Fusarium monilifor e Sheldon. * Zbl. Vet. Med. -B, 25 (1978) pages 29 to 44, dicsloese, fo example, that rachitis symptoms observed during recent years in various countries in broilers appear to be caused primarily by toxic compounds produced by the above indicated mould, which appeared able to develop in the usual poultry feed from potentially present spores, preferably under the somewhat moist conditions of the autumn and winter seasons. Although in the years preceding those in which the published symptoms occurred, the observed rachitis symptoms coul reasonably be combatted by a citamin therapy, this therapy did n lead or lead substantially less to improvement of the disease symptoms found lateron. It has been tried, therefore, to reproduce the symptoms observed lateron by administration of feed selected especially in this connection, and to influence those symptoms by -administration of vitamins and/or minerals and to investigate those symptoms more deeply, avoiding as far ' as -possible the occurrence of diseases caused by parasites or microorganisms _--rr pathogenic for poultry, such as reo virusses, Mycoplasma gallisepticum and M.syniviae and other bacteria, by means of processes suitable for that purpose. It is concluded that, also in view of harmful properties of the metabolism products of especially Pusarium monoliforme found in recent years and the mentioned disease symptoms in broilers, there must be a causual connection, despite the fact that no accurate decisive identification of the metabolites actually responsible therefore has taken place. Prom, e.g. Feedstuf s, May 8, 1978, pages 95 and $6, it appears that the actual main cause of those rachitis symptoms has actually not yet been found. It is also known, from e.g. Avian Pathology 6, page 279 that attempts have been made in the past to oppose viral arthritis/ tenosynovitis in poultry by vaccination with reo virus vaccines, but it was definitely not obvious for an expert to use reo virus vaccines to combat, aberrations in the skeleton such as e.g. rachitis-like symptoms, osteoporose c.q. femur degeneration which become more and more serious in recent years in all parts of the world as a problem needing more and more .urgently an effective and efficient solution. A connection between the presence of adeno-like virusses (hereinafter indicated ALV) and, in part, the sudden drop in egg production and/or increasing production of eggs with soft shells and/or without shells, observed increasingly in the past years, particularly in broiler mother chickens of medium heavy lay races, is already indicated in Avian Pathology 5 (1 76) pages 261 to 272. The literature calls such symptoms sometimes "egg drop syndrome". Avian Pathology 6 (1977) J pages 405 to 413 also disclose a relation observed between the development of antibodies with regard to a he agglutinising virus, called 127 virus, and the economically very disadvantageous symptoms described hereinabove. Avian Pathology 7 (1978) pages 35 to 47 confirm the assumed relation between the presence of an adeno-like virus and the sudden drop in production described hereinabove. In addition, several charasteristic properties of the type of virus in question are described, e.g. the charasteristic agglutination of chicken' erythrocytes and the incapability of antisera of being neutral in the known adeno virus types. LuxembourgPatent Specification No. 79154 discloses a process for the preparation of a vaccine against the '"egg drop ■ syndrome", starting from an ALT characterized by a specific hemagglutination and antisera reaction. In the description and t examples this virus is called KDS-76 and -Bϋ-14 ' This vaccine is prepared by cultivating a virus in a cell culture, preferably in an avian cell culture such as chicken embryo fibroblasts and embryonal liver or kidney cells or in embryonated duck eggs. From Avian Pathology 5 ( 976) page 262, second paragraph, from Avian Pathology 6 (1977) page 40β and from Luxembourg Paten Specification 79154 > page 1, lines 7 to 11 as well, it is eviden that an egg production drop may be caused by Newcastle disease, infectious bronchitis, avian and cefalomyelitis, birdpox viruses and combinations thereof. Therefore, from a practical point of view, poultry will preferably simultaneously or a short time after each other be vaccinated with two or more vaccines against the above-mentioned more generally occurring virus diseases. The hitherto usual method for any such simultaneous administration consisted of mixing standard volumes of the separate vaccines ready for use and the administration of a vacc dose composed on the spot, the dose consisting therefore of substantially the double volume as compared to those of the separately administered vaccines, while the respective virus Moreover, amounts being diluted. an exact mixing can hardly be guaranteed, while problems due to foreign infectious matter may arise. There fore it is an object of invention to find a combined, inactivate ready for administration vaccine, effective against egg productio drops, caused by ALY and diseases caused by reo-viruses as well, which moreover meet the present requirements of the veterinary athorities in most of the relevant countries. Surprisingly it has been found that the still to a greate extent occurring aberrations in the skeleton, such as rachitis like symptoms, osteoporose c.q. femur degeneration in poultry, especially broilers, often occur with growth inhibition and diarrhoea, may be combatted by vaccinating the broiler mother animals and their descendants with inactivated vaccines derived O fro certain reo-viruses. More especially it was found that combinationsof rachitis¬ like symptoms, osteoporose c.q. femur degeneration, and egg production drops very effectively may be combatted by vaccination of broiler mother animals and their descendants with combined, ready for adminit ation, inactivated vaccines, derived from certain aviair reo-viruses and adeno-like viruses. Accordingly to another feature of the invention a process was found for the preparation of a combined inactivated ready for administration vaccine, whereby an inactivated ALV liquid is combined with an inactivated reo—virus liquid. It will be appreciated that the invention is also relating to combined inactivated virus vaccines, derived from at least an inactivated reo-virus containing liquid and from at least an inactivated ALV containing liquid, and to vaccines, derived from -more than these two mentioned virus types. According to the process of the present invention an inactivated ALV containing liquid is prepared according to methods known per se, e.g. by cultivation of the desired virus, such as a passaged virus strain 127, deposited with the Collection d'lnstitύt Pasteur, Paris, under no. 1.090, in fertilized duck eggs or in a cell culture of duck eggs, collecting the virus produced in a suspension of a titre, usual for this kind of vaccines, and in- activation of the virus liquid, suspending the virus in a suitable buffer solution, homogenising it,, mixing is with an inactivated liquid, derived from a reo-virus, e.g. prepared by the cultivation of a suitable reo-virus in a fertilized chicken egg or in an SPF chicken embryo fibroblast cell culture, collecting the cultivated virus in suspension of a titre usual for this kind of vaccine, inactivating it, suspending the virus in a suitable buffered solution and homogenising it, whereafter the liquid is added to an oily phase. For the preparation of the inactivated ALV liquid component a passaged virus strain 127, deposited with the Collection d'Institut Pasteur, Paris under no. 1.090 is preferably used, although other qua antigenicity rather similar egg production drop causing ALV virus types may be succesfully applied. The virus strain 1.090 is preferably cultivated in duck eggs. The mentioned, preferably used, passaged virus strain 127 is derived from that one, indicated in the Avian Pathology publications referred to hereinbefore. The reo-virus containing starting liquid is preferably " prepared by the cultivation of a suitable reo-virus such as the LZ 671 virus, described in detail in the past and a sample of which was deposited with the Collection d'Institut Pasteur, Paris under no. 1.092, in an impregnated chicken egg or in an SPF embry fibroblast cell culture. The ALV containing starting liquid is preferably prepared by the cultivation of a suitable ALV in duck eggs or in an SPF * duck embryo fibroblast cell culture e.g. in a monolayer rolling bottle culture or a culture of cells, attached to solid inert carriers. For this preparation the starting cells are suspended in a culture medium, consisting of at least 75-95 parts of Eagle's medium and optionally 5- 5 parts of calf serum, a 2-4% aqueous solution of sodium bicarbonate and 1-5 parts of a solution or a suspension of one or more antibiotics, such as penicellin G, streptomycin and natamycin. When the cell culture is nearly close the culture liquid is separated and the cell structure is infecte with the virus suspension, incubated at 37 C during 0.5-2 hours t attach the virus and followed by addition of maintenance medium, consisting of Eagle's medium with 2-10% of calf serum, to 10% of tryptose phosphate broth medium (30 g/l), 10-20 parts of bovin amnion fluid and the additives indicated hereinbefore. The harvest of the virus containing medium and cells is carried out 0,5-2 days after the CPE appears. The cell culture is inoculated with an ALV suspension, having an activity of ^ 30 x 0 TCID 50/rolling bottle culture. In this connection it has been found that cell suspension culture were not suitable for the process described hereinbefore. According to the suitable process for the preparation of a liquid containing a suitable reo-virus, such as the virus strai deposited under nr. I.092 with the Collection d'Institut Pasteur, Paris, the virus is cultivated in an SPF chicken embryo fibroblas cell culture, for example in a monolayer rolling bottle culture O or a culture of cells attached to solid inert carries, and preferably in a monolayer rolling bottle culture, in pre-brooded chicken eggs free of antobodies against aviair reo viruses. Preferably a cell culture is used, obtained by suspending * the starting cells in a culture medium, containing of at least 75-95 parts of Eagle's medium, with or without 5-15 parts of calf serum, an aqueous sodium bicarbonate solution of 2-4% and 1-5 parts of a solution or suspension of one or more antibiotics such as penicellin G, streptomycin and natamycin, whereafter the culture liquid is separated, when the cell culture is nearly closed. The solution or suspension of antibiotics contains e.g. 2-3 x 10 units of penicellin, 20-30 mg streptomycin and 0.4-0.8 g of natamycin per ml. After inoculation with virus suspension the culture is incubated at 37 C for 0.5-3 hours to adhere the virus, and maintenance medium is added, consisting of Eagle's maintenance medium with 4-6 parts of calf serum, 3-10 parts of tryptose phosphate broth (30 g/l), 10-20 parts of bovine amnion fluid and the additives mentioned hereinbefore, and the virus-containing medium and cells are harvested after 0.5-3 days appearance of the CPE. The cell culture is preferably inoculated with a reovirus suspension having an activity of 22 10 6 - 108 TCID 50/rolling culture bottle, preferably ^ 30 x 10 TCID 50/rolling culture bottle. The inactiyation of the starting virus liquid may be carried out by means of the usual inactivators, e.g. with β-propiolactone, combined or not combined with a stabiliser, or with fors«_ldehyde (0.02-0.5%)j more particularly by adding β-propiolactone in a concentration of 0.05-0.25% by weight (calculated on the aqueous phase weight) to the buffered virus-containing liquid, and by incubating that liquid during 1-2 hours, preferably during 0 minutes at about 37 C. Then the virus liquids are homogenised in the usual way, if necessary. If desired a preservative may be added to the aqueous phase thereafter, such as thiomersal or formaline in a buffered solution. . For the preparation of the combined inactivated vaccines, 50-200 ml of an ALV containing liquid are inactivated, optionally OMFϊ - ho ogenised and together with 150-300 ml of a reo virus containi liquid, previously being inactivated and optionally homogenised, added to 500-700 ml of an oily phase, containing as essential components at least one of the components selected from the grou consisting of light paraffinic mineral oils, plant oils and naphtenic mineral oils, and one or more suitable emulsifiers in the form of non ionic surface-active compounds derived from alkylene oxide and/or hexahydric alcohols and/or higher fatty acids of 10 to 20 carbon atoms, such as esters or ester-ethers, while optionally a preservative is added to the aqueous phase. The volume ratio of the ALV containing liquid and the reo virus containing liquid may vary from 4*3 to 1:6 and is preferab about 1 :3• It has been found that the aqueous phase must be added to the oily phase with vigorous stirring and/or homogenising in ord to obtain the desired, stable and thin-liquid final emulsion. The oily phase contains 2-20% by weight (calculated on th oily phase weight) of an emulsifier, and contains preferably 2-1 by weight of Arlacel A^ or Arlacel ^ 80 or Span® 80 and optionally 0.2-4% of Tween*^80. The components of the oily phase are preferably heated to at least 110 C in an autoclave separatel or sterily filtered as mixture. For obtaining suitable combination vaccines with the desi good properties, an ALV containing liquid having an activity of 2 3 least 10 HA units and preferably 10 HA ubits and a reo virus liquid with a titre of 10 5 ' 5 tot 10' *5 ΕIΩ- /ml and preferably 6 0 > 10 EID j - 0 /ml must be used. The vaccines prepared in this way are preferably administered to 1-30 weeks old, preferably 10-20 weeks old, broiler mother animals before their lay, It will be appreciated, that the occurring immunologic response after application of the combined vaccines, found to be of at least the same magnitude as those ones caused by the separa single virus vaccines, could in no way expected or predicted by a person skilled in the art, as this immunologic response of the combined vaccine as compared to one or both separate single vaccines might decrease until an unacceptable level on account of number undesired interactions between the virus liquid compoi -9- utually or with one of the applied auxiliaries, which could not be previously excluded. Especially it might be expected that the applied β-propiolactone should remain active for a too long time, as to one of the virus components, with reference to a less fast decomposition in the mixture. The application of the combined vaccine of the present invention involves as advantage that a smaller volume pro time has to be administered, which causes less local irritations and the administration of a smaller amount of chemicals, strange to the body, or that less separate vaccinations are necessary. Moreover the earlier mentioned problems with reference to the mixing and the occurrence of strange infectious a fer are avoided. The invention is illustrated by the following examples. Example 1 Preparation of combined ALV/reo virus vaccine a) Cultivation of ALV (passaged strain 127) In Bellco rolling culture bottles, cell cultures are made from duck eggs which were brooded for 12-13 days on DEF in Eagle's medium, containing 10% of calf serum, 0.22% by weight of JSTaHCO-- and 2% by volume of an antibiotic solution, consisting of penicillin G streptomycin and natamycin. The medium is sucked off when the cell cultures are nearly closed. Then the cell cultures are inoculated with a suspension of the passages virus strain 127, incubated at 37 C for one hour and filled up with Eagle maintenance medium containing 5% of calf serum, 4% by volume of tryptose phosphate broth (30 g/l), 10% by volume of bovine amnion fluid, 0.22% by weight of ΪTaHCO-- and 2% by volume of the above mentioned antibiotic solution. The ALV containing medium and the cells are harvested one day after the CPE appearance and when the cells are nearly coming off the glass wall. The virus suspension is frozen at -20 C or lower temperatures and is stored for vaccine production. The HA. titer and the TCI t - n of the suspension are estimated. b) Cultivation of reo virus (LZ 671 ) From chicken eggs brooded for 10 days, cell cultures are prepared in Bellco rolling culture bottles, starting from CEF in Eagle medium with 10% of new born calf serum, containing 0.22% by eigfiS?--H of HaΞCO, and 2% by volume of an antibiotic solution containing - penicellin G, streptomycin and natamycin. The medium is sucked off when the cell cultures (monolaye are nearly closed. Then the cultures are inoculated with LZ 671 virus suspension and filled up with Eagle maintenance medium containing 4% by volume of calf serum, 4% by volume of tryptose phosphate broth (30 g/l), 15% by volume of bovine amnion fluid, 0.22% by weight of ΪTaΞCO, and 3% by volume of the above mentione antiobiotic solution. The reo virus containing medium with the cells is harvest one day after appearance of CPE's. The virus suspension is froze at -20 C or lower and is stored for vaccine production. TCID o 50 this suspension is determined. c) Treatment of virus suspension The frozen virus suspensionsare defrosted and inactivated in a water bath with 0.05-0.25% of β-propiolacton at 37°C for 90 minutes. The suspensions are kept overnight at +4 C. The inactivation is controlled by testing the formation of CPE on DE or CEF followed by a haem,.absorption test. Moreover prebrooded SPF chicken eggs are incubated with t inactivated virus fluid and are subsequently controlled. The vir suspensions are homogenized with an Ultra Turrax homogenizer. If desired the suspension is diluted with PBS + 0.3% of formaline, depending on the TCID cΩ of the reo virus suspension and the BA- titer of the ALV suspension. The reo virus suspension and the ALV-suspension are mixed thereafter in the ratio of 6:4 and used for emulsion preparation d) Preparation of the emulsion The virus suspensions are mixed with the oily phase in th ratio of 6.5 parts of oil to 3 * 5 parts of virus liquid emulgated in such a way that the average drop size of the aqueous phase is about 0.05 to 0.5 μ , e.g. by means of injection of the virus suspension into the oily phase with simultaneous homogenizing wi an Ultra Turrax homogenizer or by passing the starting mixture through a colloid mill. The oily phase is composed as follows: arcol^ 2 (white paraffinic Esso oil) 93.6% by volu Arlacel®A or Arlacel 80®or Span 80® (mannide monooleate) " 6.0% by volume Tween v —' 80 (polyoxyethylene-20-sorbitan monooleate) 0.4% by volume. The components of the oily phase are separately heated to * 110 C in an autoclave or sterily filtrated as a mixture. A stable emulsion is obtained, tested by: 1) pipeting directly after emulsifying drops of emulsion onto an aqueous surface, whereby the drops do not spread out but remain intact: 2) allowing to stand at 37 C for 4 weeks, whereby a aqueous phase does not seperate. The final concentrations of the thus prepared emulsions are: 21% MDV containing buffered liquid and 14% ALV 127 containing, buffered liquid. The vaccine obtained is used in a dosage of 0.5 ml per animal intramuscularly in the breast or leg muscles or subcutaneously in the neck. Eight days after vaccination hemagglu¬ tination inhibing anti-compounds are observable. From 10 days after vaccination precipitating reo virus precipitating anti compounds are detectable. Example 2 Preperation of combined ALV/reo virus vaccine a) Cultivation of adeno-like virus (passaged strain 127) Duck eggs, which are previously brooded during ~ days, were inocultate with the passaged 127 virus strain. After 7 days incubation at 37 0, the AAF (amnion allantoic fluid) is harvested. The AAF has then a HA-titre of about 1:1024. The virus suspension is subsequently frozen at -20 C or lower and stored for vaccine production. b) Cultivation of reo-virus (strain LZ 67 ) SPF eggs brooded for ten days are inoculated with LZ 67 seed virus. After incubation at 37°C for 76 hrs -the AAF (amnion allantoic fluid) is harvested. The AAF contains than about 10 EID 5θ/ml. The virus suspension is frozen at -20 C or lower temperatures and stored for vaccine production, c) Treatment of virus suspension. The frozen virus suspensions are defrosted and inactivated QMPI Λ, ~ WIPO ~ with formaline (0.3%). The suspensions are stored overnight at +4°C. The inactivation is controlled by testing the formation of CPE's on CEF or embryonated SPF chicken eggs are inoculated with the inactivated virus fluid and are subsequently controlled. The virus suspensions are homogenised by a Ultra Turrax homogeniser. The emulsion is optionally diluted with PBS + 0.3% Formaline, depending on the TCID 50 or the EID 50. The virus suspensions of ALV and reo-virus are mixed thereafter in the ratio of 1:4 and used for emulsion preparation *p To the mixture of the virus suspensions Tween 80 is added up t 3% by weight. d) Preparation of emulsion The virus suspensions are mixed with the oily phase in a ratio of 6.5 parts of oil to 3-5 parts of the virus suspension a emulsified in such a way that a drop size of the aqueous phase i about 0.05-0.5u ; emulsifying is e.g. carried out with an Ultra Turrax homogeniser or by passing the starting mixture through a colloid mill. 93,5 6,5 The components of the oily phase are separately heated to 110 C in an autoclave or sterily filtrated as a mixture. OMP

Claims: C L A I M S. 1. Combined, inactivated, ready for administration vaccine active against at least sudden egg production drops, caused by adeno like virus, and against diseases caused by reo virus. 2. Combined, inactivated, ready for administration vaccine, active against at least sudden egg production drops, caused by adeno like virus and against aberrations of the skeleton, such as rachitis like symptoms, osteoporose c.q. femur degeneration, diarrhoea and growth inhibiton. 3- Combined, inactivated, ready for administration vaccine, according to claim 1, c h a r a c t e r i z e d i n t h a t it is derived from at least an adeno like virus containing liquid, obtained by cultivation of a passaged virus strain 127. 4. Combined, inactivated, ready for administration vaccine, according to claims 1 and 2, c h a r a c t e r i s e d i n t hat it is derived from the LZ 671 strain. 5« Combined, inactivated, ready for administration vaccine, according to claims 1 to 4 » c h a r a c t e r i s e d i n t h a t it contains an oily phase with essential components, at least one of the components selected from the group consisting of: light mineral oils, plant oils and naphtenic mineral oils, and one or more suitable emulsifiers in the form of non-ionic surface-active compounds, derived from alkylene oxiάe and/or hexahydric alcohols and/or higher natural fatty acids (C 10 to C 20 ), such as esters or ester-ethers. 6. Combined, inactivated, ready for administration vaccine, according to claims 1 to 5 . c h a r a c t e r i s e d in t h a t the volume ratio between the adeno-like virus containing liquid and the reo-virus containing liquid may vary from 4'3 to 1:6 and.is preferably about 1:3- 7- Combined, inactivated, ready for administration vaccine, according to claims 1 to 6, c h a r a c t e r i s e d i n t h a t the volume ratio between the aqueous phase, formed by both virus liquids, and the oily phase may vary from 1:1 tot 3 ' 1- and is preferably about 7:13 ' 8. Combined, inactivated, ready for administration vaccine, according to claims 1 to 7, c h a r a c t e. r i s e d i n t h a t the oily phase contains 2-20% by weight of an emulsifier (calculated on the oily phase weight), preferably 2-15% by weigh 0.2 to - 9« Process for preventing diseases caused by aviair reo-virus and sudden egg production drops caused by adeno-like virus in poultry, by vaccinating the poultry with a vaccine according to any one of the claims 1 to 8 in a manner known per 10. Process for preventing aberrations in the skeleton, su as rachitis-symptoms, osteoporose, femur degeneration, diarrhoea and growth inhibition, caused by aviair reo-virus and sudden egg production drops caused by adeno-like virus in poultry by vaccinating the poultry with a vaccine according to any one of the claims 1 to 8, in a manner known per se. 11. Process for the preparation of a combined, inactivated ready for administration vaccine against diseases caused by avia reo virus and sudden egg production drops caused by ALV, c h a r a c t e r i s e d i n t h a t a reo-virus containing liquid is inactivated, is optionally homogenised and, together w an adeno-like virus containing liquid, previously being inactiva and optionally homogenised, is added to an oily phase, containin as essential components at least one of the components selected from the group consisting of light paraffinic mineral oils (FDA quality), plant oils and naphtenic mineral oils, and one or more suitable emulsifiers in the form of non-ionic surface-active compounds, derived from alkylene oxide and/or hexahydric alcohol and/or higher fatty acids (C-, Q to C- ), such as esters and ester ethers, optionally a preservative being added to the aqueous pha 12. Process according to claim 11, c h a r a c t e r i s i n t h a t β-propiolactone is (optionally in combination with stabilizer) or formaline is used, is used as inactivator for bot virus containing liquids. 13. Process according to claim 12, c h a r a c t e r i s i n t h a t β-propiolactone is added in a concentration of 0.0 0.25% by weight or formaline in a concentration of 0.02-0.5% by weight (calculated on the aqueous phase weight). 14. Process according to claim 11 to 13 > c h a r a c t e r i s e d i n t h a t the incubation is carried out at during 1-2 hours. 15» Process according to claim 11, c h a r a c t e r i s e d i n t h a t a starting reo virus containing liquid is used, having a titre of 10 5 ' 5 -10 7 ' 5 EID 5θ/ml. 16. Process according to claim 11, c h a r a c t e r i s e d i n t h a t a starting adeno-like virus liquid is used having an 2 activity of at least 10 HA units. OMPI

Inventors: APONTOWEIL P, KOK J, KRASSELT M
Applicants: APONTOWEIL P, GIST BROCADES NV, KOK J, KRASSELT M
Application year: 1980
Main classification: A61K39
IPC codes: A61K39