VERFAHRN TO INOCULATING POULTRY APPROXIMATELY PASTEURELLA MULTOCIDA

The invention concerns a procedure for inoculating poultry against Pasteurella multocida by administration of a Lebendvaccine against Pasteurella multocida, which contains a new, aviru= lenten trunk of the above 'microorganisms. “One of the usual poultry diseases is the Geflügelcholera, which is caused by Psteurella multocida. This illness is well-known also as Pasteurellosis. The illness prevails under Truthähnen and chickens forwards. The number of deaths is considerable high and the economic losses is thus considerable. The illness is sticking on and the locations remains infected over extended time intervals. Truthähne and male young chickens and/or cockerels, which are used for breeding purposes, are particularly susceptible. l0 the first weakened Vaccine of this type was developed by Pasteur (see Compt, Rend. Acad. Is. 9_! i, 673). This procedure has however the disadvantage that the bacteria returned again to their Virulenz. In Poultry Science 4/7, 1162 (1968) became i.e. from a naturally occurring trunk with low Virulenz, the Clemson university CCU) - trunk, reports. This trunk is found iu to nature, has a relatively low Virulenz, is stable and is effective to the Immunisierung of Truthähnen. With the use with young chickens it, in particular if it is orally given, is from smaller effectiveness. individual injections are effective, but represent this a serious disadvantage. Further a number of deaths rate is observed by up to 2% and even more, if the poultry accommodates other microorganisms, like e.g. Mycoplasma gallisepticum, (see oultry international, April 1975, page 12). In the last years commercial inactivated (killed) Vaccinen or bacteria was used for the Immunisierung by poultry against Geflügelcholera. The disadvantages of this procedure are that such Vaccinen lends an immunity only in relation to the special trunk of the Vaccine. There are however many trunks and occasionally arises also new trunks. The Immunisierung is not effective against such trunks. Further such Vaccinen must be given by individual injections. Aids must be consulted, which often at the injection point knot causes. By 2 or 3 injections only one immunity duration will receive from approximately 8 to 10 weeks. Many disadvantages of the conventional Vaccinen can be eliminated only by inoculating according to invention with the new Vaccine. The procedure according to invention is characterized by it that one chtvirulenten a Lebendvaccine, which the abgeschwäohten n genetically stable trunk ATCC Nr.31416 of the pathogen contains, given. The new Vaccine can be given by injections, by OS or by an aerosol. The new trunk and Vaccinen, which were made of such a trunk, have a number of important distributing, how e.g. “the weakened trunk is opposite Truthähnen and young Hfihnern avirulent completely. The weakened trunk is genetically stable and does not return not to its original Virulenz, so that with the administration at poultry, the Mycoplasma spp. is accommodated, no number of deaths is caused. The immunity stops it during a considerably longer period and is effective in relation to all trunks of Pasteurella multocida and even in relation to new trunks, which arise occasionally. The fact that the immunity is effective both in relation to homologous and heterolied trunks, is of special importance. The new trunk is the result of a controlled and reproducible mutation. The trunk differs according to a number of significant parameters of the field trunk FS-3, type X-73, which was also deposited with the ATCC and of the ATCC is available. As differences know a different size of the colonies, which are called absence a generation time extended by Hyaluronsäure of the cap and. More repetitive the stability of the new trunk depresses regarding all indicated characteristics demonstrated on intravenous] way through Truthähne. The trunk is not completely avirulent and doses as highly as i0 microorganisms per Truthahn, supplied on different administration ways, has serious side effects. The new trunk with the deposit number ATCC 31416 was developed from a virulenten field trunk, which identiziert as FS-3 of the Serotyp 1 by the Beddleston classification Nr.364080 became, which by the test of Litde et al., to. J. Res. 4, 110 (1948) one made. This trunk is called also X-73 and it is available from the ATCC under the deposit number ATCC Nr.11039. Contrary to conventional Vacclnen, which lead no side effects to a number of deaths from approximately 1 to 3%, if a hidden illness is present, have according to invention given the new Vaccinen practically. The effectiveness of the Vaccine is high (80 to 100%). The new Vaccine becomes preferably oral (preferably with dern drinking water) or given by aerosol. Oral doses are for instance I0' to I0n weakened microorganisms, preferably I0 IC, per bird. The Vacelne can be lyophilisiert and stored over extended time intervals. The lyophilisierte substance contains about 2 Dosen/mg dry material Zü and it before the use by a suitable dilution is rekonstituiert. The procedure for the production of the abgesehwächten, genetically stable new Mutante, which is avirulent and can for an effective inoculation against Geflügelcholera be used, is more near described below in the test part. The procedure contains the following substantial stages: The virulente field trunk will become on a suitable culture medium bred and after 18 h with 37 " C homogeneous colonies with a diameter from approximately 4 to 5 mm selected. The microorganisms are suspended in a buffer with a pH value of approximately 5.5 and a suitable Mutagen is added in a concentration, which leads to a survival rate from approximately 1 to 2% after 30 min with 37°C. Particularly good results were received with N-methyl-N-nitroß so-N-nitroguanidin (NTG). Other mutageue substances, like e.g. Methylmethansulfonat 28 (MMS), Äthylmethansulfonat (EMS), 5-Eromuracyl, 2-Aminopurin and Hydroxylamin, can be used ehenfa] is. After centrifugation and washing the microorganisms in a buffer with a pH value of 7,2 are suspended, diluted and presented with a concentration of approximately 100 per plate. About 95 to 90% of the colonies has a conventional Grölte and them is of the conventional type. 1 to 5% are Mutanten. Colonies with a diameter of less than about 2.5 to 3 mm after 18 h with 37 C are selected, by Relsolierung of the particulars] (, which do not go beyond this size, cleaned, on the antigens Spezifizität olonien tested, on the Virulenz tested and the avirulenten trunks are bred and on genetic stability by repetitive passage by birds tested. The trunk with the best immunogen characteristics was selected. This trunk has a generation time, which differs substantially from that of the output trunk X-73. The generation time is 27 min with 37°G and 24 min with 41°C for the output trunk and 30 min with 87°C and 39 min with 41°G with the trunk ATCC 31416. The Körpertemperatur of the birds amounts to for instance 41°C. This Mutante does not have Hyaluronsäure in its cap. The new trunk is called in the following M-3-G. The Virulenz of the Mutante is substantially decreased. During 8,80rganismen of the output trunk with i.p. - Within 2 days and isten 88 microorganisms within 24 h 5 of 5 mice kill injection 5 of 5 mice, caused M-3-G with i.p. - Injection of i0' microorganisms no death of a mouse of 10 mice. As was brought above to the Audruck, the new Vaecine does not have side effects and the immunity, which results by a Zbis malige successive oral supply with the drinking water, stops about 10 weeks or more. In the following the production of the new Vaccine, their administration and the obtained effects are more near described. Production of the Vaocine: 4S a) of trunks of field trunks, the Pasteurella multocida Serotypen, Heddleston et al., 1972” Avian Dis. 16, 703, represented, became a concentration of 10 ' Bakterien/ml with 37°C in 5 ml LB-Brühe (10 g I autotrypton, 5 g yeast excerpt, 10 g NaCI, I g glucose in I000 m! distilled water) bred. The cultures were diluted with a diluent (10 g Bactotrypton, 5 g NaC1 in I000 ml water) to a concentration of approximately 1000 bacteria/valley. Samples with 0,1 ml were applied on LB-agar (LB-Brühe, supplements agar, Difee and 200 with 10 g/l mg/l Hämin). The colonies were observed after 18 h with 37°C. All media were held at a pH value of 7,2. The trunk FS-3 (Serotyp 1), which is the trunk X-73, ATCC 11039, equivalent, resulted in colonies with an essentially homogeneous size of a diameter from 4 to 5 mm. The trunk FS-3 gave the best reproductibility of the vitality countings. The trunk FS-3 and the other trunks were examined regarding the education by colonies after the lyophilization. Erkonnte going out over 90% without loss at colony-forming units to be lyophilisiert. This trunk was selected for the Mutagenese. b) Mutagenese FS-3-Bakterlen of frozen supply cultures were thawed out, inokuliert in LB-Brühe (1 ml output culture on 10 ml LB-Brühe) and bred with 37°C in 50-ml-Erlenmayer-Kolben under Hinund le Herschütteln. The cultures were diluted after 18 h 1:60 in 5 pistons, which l0 contained ml fresh LB-Brühe, and they were bred to the Mid log phase (about 108 bacteria/valley). The cultures were harvested by centrifugation with 10000 Umdr/10 min. The sediment was washed and resuspendiert into 10 ml CIT advice buffers with a pH value of 5,5. N-methyl-n-nitrO-n-nitrosoguanidin (NTG) became the bacteria suspensions the following final concentrations: 25, 50, 100 and 200 pg/ml added. The bacteria suspensions, which contained different concentrations of NTG, were inkubiert in a Wasserbad 30 min long with 37eC (without ventilation). After these Inkubierungsperiod each culture 10 min was centrifuged long with 10000 Umdr/min. The sediment was washed twice with phosphate buffer with a pH value of 7,2. The washed sediment, which the treated bacteria contained, was again suspended in l0 ml fresh LB-Brühe and 18 h long in pistons with 37°C under ventilating and Hinund Herschütteln was inkubiert. The vitality of the bacteria was determined, by being applied immediately after Inkubierung with NTG before centrifugation on LB-agar. With the above concentrations of NTG the number of deaths amounted to 15, 40, 90 and/or 99%. The measuring of NTG was accomplished with each culture by P. multocida, which had been treated with the Mutagen. Only cultures with a number of deaths from 90 to 99% after suspending at NTG were consulted for the further selection measures. c) Isolation of the weakened Mutanten of 500 bacteria clones, which had survived the NTG treatment, were taken up by sterile Zahnstocher and cleaned genetically, by being slipped on twice on the surface by agar plates and as the individual colonies were again isolated. The cleaned clones were let grow over night with 37°C. Colonies with a diameter of less than 3 mm were selected. These were diluted in LB-Brühe 1/50, 18 h under ventilation with 37°C were bred and on the Virulenz tested, by being injected intraperitoneal with l06 bacteria at least 4 mice. The parenterally injected trunk FS-3 caused a dying of 100% with this concentration after 10 to 18 h. 11 trunks were isolated by this method, which were avirulent opposite mice. These were tested further at 5 weeks old Truthähnen. The trunks were called IT, 5T, 6T, 7T, ST, 9T, M3, M4, ITG, M3G and M4G. It was stated that 6 of the 11 trunks was weakened in Truthähnen (avirulent). The selected trunks were tested on the immunogen characteristics. It was stated that the trunk M3G was the best. This trunk has the characteristics indicated in the following table. Characteristics of Pasteurella multocida to M-3-G 1st Heddleston Serotyp I, identify by Agarpräzipitation and high-speed agglutination tests. 2. No Hyaluronsäurekapsel available. With specific anti-serum (anti) without pretreatment with Hyaluronidase agglutinierbar. 3. Generation time about 30 min with 37°C in LB-Brühe, end of 3fl min with 41°C. 4. Resistance sample against antibiotics: Streptomycin 5 pg non-sensitively Tetr acyclin 10 pg sensitively penicillin 5 p-units rather sensitively Cephalotin 3 pg very sensitively Erythromycin 5 pg resistant Chlormycetin 10 pg very sensitively Novobiocin 10 pg very sensitively Kanamycin I0 pg very sensitively Methicillin I0 pg very sensitively Nr.364080 the resistance sample was determined by disk tests (Difco) on LB-Agarplatten and by measurement of the inhibition zones according to 18 to 24 h. The parenteral trunk FS-3 is sensitive opposite Streptomycin and Erythromycin and the other Antibiotlka. 5. Avirulenz opposite mice (i.p. 1O 6 bacteria), Truthähnen and/or Truthennen (i.p. I0' bacteria). 6. No returning to the Virulenz after the run by Truthähne and/or Truthennen: 8 bacteria were injected i.v., the animals were slaughtered 20 to 30 h and the bacteria were isolated later aseptisch from the liver. Maximum bacteria number per liver: 100 to 1000. This was ten times repeated. The Endausbeutemen e at bacteria after the tenth bird bird run was reisoliert. Sensitive Truthähne and/or Truthennen were inokuliert with 101° bacteria per bird. No damage was caused to the birds. It becomes evident that the bacteria not to their. Virulenz returned. The bacteria maintained also their other characteristics, like e.g. the absence the generation time decreased of Hyaluronsäure in the cap, the formation of microorganisms with a diameter of less than 3 mm after 18 h with 37°C and (about 39 min with 41°C). 7. The bacteria survived a freezing in 8% Glycerin in the Brühe and also a lyophilization. 8. The bacteria survived at least 2 h in drinking water of 250C, which had been supplemented with 0,25% milk powder. Production of the Vaeeine the trunk M-3-G was inokuliert in 500 ml Tryptosebrühe and inkubiert over night with 37°C. The received culture became by microscopic investigation on gramgefärbte Versehmierungen and on the ability. without agglutinieren pretreatment with Hyaluronidase, examines. The trunk was applied and examined biochemical and serologisch. In such a way received culture was used as Inokulum and aseptiseh into a 40-l-Fermentator transferred, where a before-sterilized culture medium was added, the Tryptose, yeast excerpt, peptone, Dextrose and buffer salts contained. The pH value was controlled during the breeding process. The culture medium was examined periodically by phctemetrische density measurements of the taken samples for growth by M-3-G. When growth had reached its maximum, the SuspenSiCh was transferred into precooled G “fäße and the Organlsmen was concentrated by centrifugation. The concentrated product was resuspendiert in a suitable medium, like milk coal hydrates or Kaseinhydrolysat, and entered and frozen in ampuls and Glä. The frozen Vaccine was lyophilisiert and the resulting dry Vaccine became on the purity and the cultural particularly clinging as well as stability when freezing (+4°G) tested. This Vaocine was used for tests with birds. The results are below indicated. The received results show that the Vaceine of the Vaccine on basis of killed organisms, used so far, is substantially superior and that it can be substantially more simply given. Effectiveness test of the Vaccine: Sensitive Truthähne and/or Truthennen were inokuliert repetitive with the trunk -3-G. This was accomplished on different ways. With an attempt the supply took place to the experimental animals three times intramuskulär with a dosage from 7,107 to 4,10. The inokulierten Truthähne and/or Truthennen and not inokulierte control animals by injection with virulentern Pasteurella multocida was provoked, whereby the output trunk FS-3 and heterologe trunks, the sero1: were ogisch different, were used. The received results are zusämmengesteUt in table I. supplied master provoking dose homologous provoking 1.6 x 103/Vogel heterologe provoking 2.4 x 103/Vogel table I of deaths after provoking controls (not inoculated) of 16 of 19 84.2% 16 of 17 94.1% inoculated with M-3-G 0% 16.6% with a further attempt became M-3-G by Zuffihrung of the Vaccine at Truthähnen and/or. Truthennen by OS tested. A quantity of I0'° bacteria into 100 ml drinking water was supplied per bird. The supply took place three times with weekly intervals. 2 weeks after the last dose were provoked the Truthähne and/or Truthennen i.m. and on the way over the breathing system, whereby the palate column was used, in order to rub with the provoking trunk. A simulating infection was accomplished in the Peld by 108 cfu/ml. The received results are arranged in table II. The Immunisierung can be accomplished also by inhalation of an aerosol. A quantity of I0I° bacteria in 50 ml water was aerosolized within 10 min and the birds were exposed to this cloud. Table II results of the Provozlerun group of birds and 8ehandlung controls (not inoculated) zntramuskul PE ₜ EL R-3-G inoculated on the way over the drinking water hit H-3-G inoculated provoking it homo! oge¢ trunk intramuskuläre Provozlerung 72.2 provoking by the breathing tract zo/2o I100) Provozlerung IL heterologe trunk intramuskuläre Provozlerung Provozlerung by the breathing tract g/zs (o) /2o (zz, 8) 2/so /2 “6.66 8/t7 (57,1) (16,6) o/iß (o) * 13/1B = 13 dead ones of 18 provoked Testtleren the results of the performance inspections with later attempts, with which the minimum provoking dose was determined, were continued to improve.