Proteolitik Olmayan, Avirulan Bacillus Antracis Mutantlarının İzolasyonu veKobaylarda Mukayeseli Bağışıklık Denemesi ile Bu Mutantlardan Canlı Aşı Hazırlanması

Öz

Bu çalışmada Sterne suşu 'nun proteolitik olmayan mutantla riyle hazırlanan anthrax aşıları ve orijinal Sterne aşılannın mukayeseli bağışıklık, zararsızlık deneyleri ve aşının maliyetini etkileyen faktörler üzerinde durulmuştur . 1 - 20 cm. mesafeden 35 watt'lık ultraviole ışınlarına 45 dakika maruz bırakmakla Bac. anthracis'in Sterne suşundan (34F2 ) avirulan, proteolitik olmayan suşlar izole edildi. Proteolitik olmayan mutant suşların sütlü agarlardan besleyici agarlara pasajları yapıldı ve aerop ve % 20 co, atmosferi altında 3 er gün üretildi. Bunlardan bir çoğu proteolitik olmayan karakterini kaybetti, fakat bir mutant suş (29/la) bu şekilde yapılan 45 pasajda proteolitik olmayan karakterini muhafaza etti. 2 - Aynı metodla virulan, bir suştan da 2 proteolitik olmayan mutant suş izole edildi. Bu proteolitik olmayan mutant suşların 500 sporu kobayların deri altına injeksion edildiğinde virulan bulundular. 3 - 29 /la ve Sterne suşları, kazein digest vasatarında besleyici agarlara nazaran daha iyi sporlandı. Kazein digest vasatlarında 3 gün üretilen 29 /la mutant suşunun glyserinli süspansionu Sterne suşunun glyserinli süspansionuna nazaran % 25 daha fazla canlı spor ihtiva etmekteydi. Kazein digest vasatlarında 5 gün üretilen 29 /la suşunun süspansionları Sterne suşuna nazaran % 100- 150 daha fazla canlı spora sahipti. 4 - 29/la ve Sterne suşlarının kobaylarda yapılan mukayeseli bağışıklık testlerinde 29 /la mutant suşu, Stern e suşuna nazaran 2 - 5 misli daha etkili bulundu. S - Bu suşların 2x10 8 ·Ve 1x10 8 miktarlarındaki sporları deri altı ve adale içi kobaylara inakule edildiği zaman 29 /la mutant suşu Sterne suşundan daha emin bulundu. 6 - Ml. lerinde 8, 6, 4 ve 2 milyon spor bulunan 3 çeşit [a - % 40 glyserin, % 60 fizyolojik tuzlu su ve % 0,1 sapanin b - % 30 aluminium hydroksit, % 70 fizyolojik tuzlu su; c - Mineral yağlı Saf White Nedle Oil- B) ] aşı 29 /la mutant suşu ile hazırlandı. Keza Sterne aşıları da aynı miktar spor ihtiva edecek şekilde hazırlandı. Bu aşılardan her birinin O,S ml. i ile deri altı verilmek suretiyle 6 şar koyun aşılandı ve aşılamadan 7 ve 15 gün sonra eprüve edildi. Mineral yağlı aşı hariç her iki suştan da hazırlanan bütün aşılar çok iyi bağışıklık verdiler. 7 - Ml. inde 10 milyon spor bulunan her bir aşıdan 1- 3 ml. ile koyun ve keçiler deri altı ve adale içi inokule edildiği zaman Sterne aşısı 29 /la mutant suşundan hazırlanan aşılara nazaran daha çok reaksion verdi.

Anahtar Kelimeler

• Sterne suşları
• Sterne suşu
• Mineral yağlı Saf White Nedle

The isolation of avirulent nonpreteolytic mutant of Basillus antracis and the immunity test in guinea pigs and preparation of live vaccines from this mutant

Öz

This study has been done comparatively on the immunity and safety test on the vaccines which were prepared by a nonproteolytic mutant of Sterne's strain and the vaccines of original Sterne's and the factors of financial effect of production. 1 - Some avirulent, nonproteolytic mutants were isolated from Sterne's strain (34F2) of Bacillus anthracis by exposure by irradiation for 45 minutes at 20 cm. from the filament of a 3S W Ultraviolet lamp· These nonproteolytic mutants were madde subcultures from the milk agar to nutrient agar and were incubated aerobic and 20 % carbon dioxide atmosphere for 3 days. Many of them lost their nonproteolytic character, but a mutant strain (29/la) was stable its nonproteolytic character in about 4S subculteres by the same method. 2 - 2 nonproteolytic mutants were isolated from virulent strain by the same method. These nonproteolytic mutants were found virulent, when they were tested in guinea- pigs for virulence by subcutaneous injection of SOO spores. 3 - 29/la and Sterne's strain both sporulated better on casein digest medium than nutrient agar. The glycerinized suspensions of 29 /la mutant strain contained about 2S % mo re viable spores than the glycerinized suspensions of Sterne's strain, when they were incubated on casein digest medium for 3 days. The suspensions of 29 /la contained ab out 100- 150 % more viable spores than the suspensions of Sterne's strain, when they were incubated on casein digest medium for 5 days. 4 - Comparison of the protection of guinea- pigs induced by the vaccines of the 2 strain showed that the 29/la mutant strain was from 2- to S times more effective than the Sterne's strain. 5 - 29/la mutant strain has been found safer than Sterne's strain, when guinea- pigs were inoculated intramusculary and subcu taneously with about 2 x 10 8 and 1 x 10 8 spores of each of these strains. 6 - Three kinds of vaccnies [a - 40 %" glycerine, 60 % physiological saline, and 0,1 % saponin b - 30 % aluminium hyddroxide, 70 % physiological saline; c - mineral oil (White Needle Oil- B)] which included 8, 6, 4, and 2 million spores in ı ml. of each, were prepared by 29/la mutant strain. Sterne's vaccines were prepared also in the same amount of spores. 6 sheep of a group were vaccinated subcutaneously with O,S ml. of each of these vaccines and challenged on 7 and ıs days after vaccination. All the so vaccines which were prepared by both strains, except the vaccines of c- with mineral oil, gave very good immunity. 7 - When sheep and goats were inoculated intramusculary and subcutaneosly with 1-3 ml. of each of these vaccines which contained 10 million spores per ml., Sterne's vaccine gave more reactions than the vaccines which were prepared from 29/la mutant strain.

Kaynakça

1. Aeurbach, S. and Wright, G.G., (ı955) : Studies on immunity to anthrax.VI - İmmunizing activity of protective antigen against various strainsof Bacillus anthracis. - J . İmmunol., 75, ı29- ı33.
2. Aktan, M., (ı954) : Antraksa karşı yapılan aşılar ve üç muhtelif antraksaşısı ile tavşanlarda muafiyet kontrolu.- Ziraat Vekii.leti,. Etlik Bakteriyolojive Seroloji Enst. Neşriyatı, Güven Matbaası , Ankara , ı - ı2
3. Ananthracis by penicillin and immunogenicity of the attenuated organisms.- Bull. Azabu Vet. Coll., Japan, 8, 37 - 45.
4. Avcıl, F., ve Emre, M.N., (1964) : Türkiye'de hazırlanan Max-Sterneanthrax aşısı, -Etlik Vet. Bak. Enst. Dergisi, 2 sayı 3 - 4, 239 - 246.
5. Aygün, S., (ı934) : Türkiyede (anthrax) Dalak yanığı ve sava§ı veTürk üniversel Antraks (T.Ü.A.) aşısı üzerinde bilg~ler.- Ankara, YüksekZiraat Enstitüsü, 82 sayfa.
6. Beıton, F. and Strange, R.E., (1954) : Studies on protective 'lntigen producedin vitro from Bacillus anthracis, medium and methods of production.-British J. Exp. Path. 35, 144- 152,
7. Besredka, A., (ı921) : Vac.cinaD:m par voie cutanee charbon : cuti -infection, cuti - vaccination, cuti - immunite. -Ann. !nst. Pasteur, 35. 421-430
8. & - Boor, A.K., (ı955) : An antigen prepared in vitro effective for iınrn:nizat'bnagainst anthrax. I - :preparation and evaination of the crude protective antigen, - J. İnfect. Dis. 97, 194 - 202. Brown, E.R., Cherry, 'W. B., Moody, M:.D., and Gordon. M.A., (1955) . :the induction of motility in Bacilltis anthracis by means of BaGteriophagelysates. J. Bacteriol 69, 590-602. l Burrow, W., Moulder, J.W. and Lewert, R.M., (1963) :racis in text- book of Mikrobiology. - Saunders Comp.,London, 28, 688 - 696. Bacillus anthPhiladelphia -

9. Çizmen, F. (1946) : Antraks muafiyetinin mihanikiyeti ve prat!kte kullanılanaşıların mukayeseli denemeleri.- Y.J. Basımevi, Ankara, 78 sayfa.
10. De Armon, I.A., Jr., Klein, F., Lincoln, R.E., 1\'lahlandt, G., and Fernelius.A. L., (1961) : İınmunological studies of anthrax. I- An index to determine quantitative immunization,- J. İmmunol., 87,233- 239.
11. E!'türli, ö. ve Beşe, M., (1961) : Kombine aşıların bağışıklık etkis!ı üzerindebir araştırma.- A.Ü. Vet. Derg. 8, 188 - 196.
12. Fubra, E.S., (1966) : Nonproteolytic, avirulent Bacillus anthracis as alive vaccinE'.- J. Bacteriol. 91, 930- 933.
13. Gladstone, G.P., (1946) : İmmunity to anthrax: Protective antigen prese:ıt in cell- free culture filtrates.- Brith. J. Expt. path. 27, 394 - 410,
14. Grosso, A.M., and Perez, A.A., (19ô3) : (Experiments with the Sterne anMı.rax vaccine. I - Immunizing properties of the Sterne strain of Bacillus anthracis in subcultures in pepton agar. II- Behaviour of Sterne ac.thrax vaccine prepared with different vehicles.) -Rev. İnvest . Ganad. No : 18. pp. 319- 328, Ref. Vet. Bull. 34, (1964) : 2057.
15. Guex- Holzer, S., and Tomcsik, J., (1958) : The isolation and che m'ı::al nature ·of capsular and cell- wall haptens in a · Bacillus species. - J. of · Gen. Microblol. 14, l4 - 25:
16. Harris- Smithı, P.W., Smith, H. and Keppie, J., (1958) : Production in vitro of the toxin of Bacillus anthracis, previously recognized in vivo. - J . Gen. Mkrobiol. 19, 91- 103.
17. - Jacksoıı, F.C., Wright, G.G. and Armstrong, J., (1957) : İmmunizatioıı of catte against experimental anthrax with alum- -precipitated protective a ntigen or spore vaccine. - Amer. J. Vet. Res., 18, 771 - 777.
18. Ja~otot, H . et Virat, B., (1SHO) : Vac-cination contre L'infection Charbonneus par injection de bacteridies tuees en excipient huileux. preıniere no te? Ann. Inst. Pasteur., 98,' 297 - 300.
19. Jacotot, H. ct Virat, B., (1963) : Vaccination contre L'infection Charbonneus par injection de bacteridies Tuees en excipient huileux.- Ann. Ir..st. Pasteur., 104, 823 - 826.
20. Kelner, A., (1949a) : Effect of visible light on the recovery of streptomyces griseus conidia from ultraviolet irradiation injury.- Proc. Nat. Acad. Sci. USA. 35, 73 - 79.
21. n:eıner, A., (1949b) : Photorea ctivation of ultraviolet irradiatcd Escherchia coli, with special reference to the dose - reduction principlP. and to ultraviolet.- Induced mutatio-n.- J. Bact. 58, 511- 522.
22. - Klein, F., Hainer, B. W., Mah!amlt, s: G., u.nd Lincoln, R. E., (1963) : Immunological studies of anthrax. III. Comparison of antibody titer and immunity incl,ex after anthrax immunisation. - J. Immuncl. 91, 431- 437.
23. - Kolesove, S. G., and Mikhailov, N. A., (1956) : (I- Trial of alııminium hydroxide anthrax vaccine under field conditions. II- Immunogenic properties of alumini um hydroxide vaccine and results of large scale trials. ı - Trud Nauchno- Kontrol. !n:.ıt. Vet. preparatow, 6. 242 - 255. Ref. Vet. Bull. 28, ( 1958) : 1307 - 1308.
24. - Iiolesove, S. G., 1\'likhailov, N. A. and Borisovich, Y. E., (1S57) : (Method of preparing aluminiuro hydroxide vaccine against anthrax und the results of field trials) .- Trud. nauchno- kontrol. Inst. Vet. Preparatow. 7, 191- 200. Ref. Vet. Bull. 29, (1959) : 928. Lederberg, J ., (1948) : Problem in microbial genetics, - Heredity, 2, 145- 198. .,
25. Lee, L. A., Kidd, A. R. i\'1., aml Sterne, M., (1961) : Antibiotic treatment and its effect on anthrax·immunisation.- Vet. Record, 73, 1426.
26. Lincoln, R. E., \Valker, J. S., Klein, F., and Ha,ines, B. W., (1964) : Anthrax- Advances in Veterinary Science, Academic press., New- York, London, 9, 327 - 368. ·
27. Lin
28. Mawcchi, M., (1931) : Clin. Vet., Milano 54, 577. cited by Sterne, M., (1959) : Anthrax in infectious disease of animals, Diseases due to bacteria, by Stableforth, A. W., and Galloway, İ. A., Butterworths Scientific Publications, London, ı, 16 - 52.
29. - Newcombe, H. B., and \Vhitehead, H . A., (1951) : Photoreversal of ultraviolet indııced mutagenic and Jethal effects in Eschericl1icı coli,- J . Bact. 51. 243 - 251.
30. Nungester. W. J., (1.929) : Dissociation of Bacillus anthracis.- J . Infect. dise, 44, 73 - 125. 3,1 Özce be. İ., {1935) : Asprogime şarbon aşısı.- · Köy hocası matbaası, Ankara, 30 sayfa.
31. Ö7.cebe, İ., (1953) : Anthrax enfeksionuna karşı yeni bir aşı.- Türk Vet. Hek. Derg. 23, 916-917.
32. Pankratov. A. Y .. Tret'yakova, A. A., and Smirnov, I . I., (1960) : (Tests of imrnunity in sheep i noculated simultaneously açrainst anthrax. brucelIosis. and nox.)- Veterinariya, Moscow, No. 9, 38-40. Ref. Vet. Bull .. 31, {1961) : 357.
33. - Pa«t<ıur. L .. Chamberland et Roux. · (1881) : De L'attenuation des virus et de leur r etour a la virul ence.- Comot. Rend. Acad. Sci., 92. 429. cited bv Sterne. M .. (1959) : Anthrax in infectinus disease nf animals. dise:ıses due to bacteria, by Stableforth, A. vV., and Galloway, İ. A., Butterworths scientiflc publications. London, 1, 16 - 52.
34. - PP.r,.on <ıus. G .. Cooner. M 8 .. anrl Percival. R . C., {1956) : Studies on anthrax nr p:ned from Nonencapsulated variants of Bacillus Anthracis.Am. J. Vet. Res. 17, 153-156.