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Vaccine Preparation by Radiation Processing Gabriela Craciun1*, Diana Martin1, Iulian Togoe2, Laurentiu Tudor2, Elena Manaila1, Daniel Ighigeanu1 and Constantin Matei1 National Institute of Lasers, Plasma and Radiation Physics, Bucharest, Romania 2 Agriculture and Veterinary Medicine University, Bucharest, Romania * [email protected] 1 A new radiation biotechnology for the acquirement of a commercial vaccine, designed for prophylaxis of ruminant infectious pododermatitis (IP), produced by gram negative bacteria Fusobacterium necrophorum (F. n.), is presented. Two different processes for preparing F. n. vaccine are used: a) the inactivation of F. n. bacteria exotoxins by microwave (MW) or/and electron beams (EB) irradiation; b) the isolation of exotoxins from F. n. cultures irradiated with MW or/and EB and the inactivation of isolated F. n. exotoxins with formalin. The EB irradiation of F. n. cultures produced simultaneously with the cells viability decrease an increasing of exotoxin quantity released in the culture supranatant as compared with classical methods. The MW irradiation is able to reduce the cells viability to zero but without an increase of exotoxin quantity in cultures supranatant. Instead of this MW irradiation, for certain conditions, is able to induce an important stimulation degree of the F. n. proliferation in cultures, from two to three log10. Two vaccine types were prepared: A1 vaccine that contains whole cell culture irradiated with MW/EB and A2 vaccine that contains cell-free culture supernatant of an MW/EB irradiated F. n. strain producing exotoxins. Also, other two vaccines are prepared: B1 and B2 that contain the same materials as A1 and A2 respectively, but without using MW/EB exposure. The vaccine efficiency is tested in ruminant farms in which IP evolves. It is expected that this new vaccine to offer a better protection, more than 60%, which is the best presently obtained result in ruminant farms. INTRODUCTION The main goal of this paper was the development of a new radiation biotechnology, based on microwaves (MW) and/or electron beams (EB) irradiation, for the acquirement of a commercial vaccine designed for prophylaxis of ruminant infectious pododermatitis (IP), produced by gram negative bacteria Fusobacterium necrophorum (F. n.). There are many studies presenting cells Keywords: Vaccine, Fusobacterium necrophorum, vaccine, microwave, electron beam International Microwave Power Institute Submission Date: 18 October 2007 Acceptance Date: 14 November 2008 Publication Date: 12 June 2009 inactivation by ionizing radiation [Smolko, 2005] but there is no previous work examining the separate and combined MW and EB treatments for bacteria inactivation. Infectious pododermatitis (IP), produced by gram negative bacteria Fusobacterium necrophorum (F. n.), are involved in many morbid conditions in animals and humans. Ruminants IP is the most dangerous because of economical loss induced. Infections induced by F. n. have different names related to their localization, they have been described in many species, but the most important frequency and economical loss affect ruminants. Detailed 43-2-65 researches have been done in order to elucidate the IP etiology and immunoprophylaxis. Nowadays, though are compulsorly rules for preventing F. n. infections (correct nutrition in accordance with age and breed, adequate humidity in shelters, maintaining of animals on clean bedding, avoiding of interdigital skin lesions, lack of exercise, etc.), in ruminat farms are frequent cases of IP induced by F. n. infections. For the Fusobacterium genus there have been described more than 18 species, but for animal pathology is of interest only F. necrophorum with 2 subspecies necrophorum and funduliforme. These bacteria induce in many animals, but especially in ruminants, primary and secondary infections called necrobacillosis among which the most important are ovines and bovines pododermatitis, foot abscess, interdigital dermatitis in ovines, calf diphteria, hepatic abscess syndrome, postparturient necrosis of the vagina and uterus in ovines and bovines, necrotic rhinitis in pigs, etc. Bacteria of the genus Fusobacterium are polymorphous bacilli, Gram negative, nonsporogenic, noncilliated, obligate anaerobe, that are isolated in pure cultures with great difficulties because of their nutritional demands and oxygen intolerance. F. n. is normally found in the digestive flora of many animal species, but frequently can be isolated from rumen, oral cavity and urogenital tract, and excreted from these it gets into natural environment. It may resist in natural environment for days and weeks. EXPERIMENTAL PROCEDURES Two different processes for preparing F. n. vaccine are used: a) the inactivation of F. n. bacteria exotoxins by EB or/and MW irradiation; b) the isolation of exotoxins from F. n. cultures irradiated with EB or/and MW and the inactivation of isolated F. n. exotoxins with formalin. Also, two vaccine types are prepared: A1 vaccine that contains whole cell culture irradiated with EB or/and MW and A2 vaccine that contains cellfree culture supernatant of an irradiated F. n. strain producing exotoxins. It is expected that 43-2-66 these new vaccines to offer a better protection, more than 60%, which is nowadays the best obtained result in ruminant farms. In order to verify this expected result, beside the A1 and A2 vaccines, other two vaccine types are prepared at the same time: B1 and B2 vaccines that contain the same materials that A1 and A2 respectively, but without using of EB or/and MW irradiation. A1 vaccine is compared with B1 vaccine and A2 vaccine with B2 vaccine. The research began with the epidemiological studies inside of a farm (“Dancu” farm for bovines, Iasi, Romania) in which IP evolves, the development of a methodology for F. n. isolation from the podal lesions and identification of the best exotoxin producing strains. By using 14 samples of podal lesions from bovines with IP, eight F. n. strains were isolated and identified by using selective isolating media. From eight analyzed F. n. stains, 407 F. n. strain that exhibited the biggest pathogenicity was selected for F. n. bacteria production and vaccine preparation. After 72 h of the F. n. strain cultures incubation in anaerobioses conditions, maximum concentration of 7.2 × 108 cells/ml was obtained. The research continued with the investigation on F. n. exotoxin cytotoxicity (hemolytic activity on horse erythrocytes, pathogenicity on laboratory animals, lecithinase activity on egg-yolk-agar) and on F. n. exotoxins antigenicity [Amoako, 1993; Berg, 1982]. Also, was investigated the effects of different EB or/and irradiation modes and doses on F. n. bacteria inactivation or proliferation. We have supposed that, for certain conditions (radiation nature, irradiation doses, F. n. cultures preparation), EB or/and MW irradiation could stimulate the F. n. proliferation in cultures and could enhance exotoxins releasing or/and their biological properties. The experimental results demonstrated that several of our suppositions are right. EXPERIMENTAL INSTALLATIONS An experimental installation for separate, successive and simultaneous MW and EB irra- Journal of Microwave Power & Electromagnetic Energy ONLINE Vol. 43, No. 2, 2009 Figure 1. General view of the MW+EB-ES used with ALIN-10 linear accelerator. diation, named MW+EB-EI, was carried out. It consist mainly of the following units: • an accelerated EB source, ALIN-10 electron linear accelerator of 6-7 MeV and adjustable absorbed dose rate up to 4 kGy/min (built in the Accelerator Laboratory of the National Institute for Laser, Plasma and Radiation Physics, Bucharest, Romania); • a mechanical and electrical proper modified MW oven (MEM-MWO) of 2.45 GHz, in which are injected both, EB and MW fields. Figure 1 shows a general view of the MW+EB-ES used with the ALIN-10 linear accelerator. Fig. 2 presents a general view of the MEM-MWO and its 2.45 GHz power supply and control unit. Magnetron power supply and control unit permits preset adjustment of the MW exposure time and MW output power. MW are generated as 10 ms pulses at 50 Hz repetition rate. The MEM-MWO is used with two different exposure geometrical configuration of its multimode cavity: EGC-1 and EGC-2. For comparative experiments with separate MW irradiation, separate EB irradiation and combined (successive and simultaneous) irradiation with MW and EB is used EGC-1 (Figure 3) that permits a radiation exposure of maximum 14 marked cylinders of PP T 309-2A type (the maximum International Microwave Power Institute Figure 2. General view of the MEM-MWO and 2.45 GHz power supply and control unit. Figure 3. EGC-1 internal configuration of the MEM-MWO multimode cavity used with 14 marked cylinders (PP T309-2A type) for separate EB and simultaneous EB+MW irradiation experiments . available volume of each T 309-2A cylinder for F. n. culture introducing in anaerobioses condtions is of 2.47 ml). The useful penetration (2.2 to 2.56 cm) of ALIN-10 accelerated electrons imposed the use of cylinders of T 309-2A types because permit a proper culture thickness for experiments with EB and EB+MW. For experi43-2-67 ments with MW only, is used EGC-2 (Figure 4) that permits a radiation exposure of maximum 16 marked cylinders of PP T 309-4A type (the maximum available volume of each T 309-4A cylinder for F. n. culture introducing in anaerobioses conditions is of 4.7 ml). RESULTS AND DISCUSSION The EB effects are related to the absorbed dose (D) expressed in Gray or J kg-1 and absorbed dose rate (D*) is expressed in Gy s-1 or J kg-1 s-1. The MW effects are related to SAR (Specific Absorption Rate) which is equivalent to D* and SA (Specific Absorption) which is equivalent to D. The MW absorbed energy depends strongly on the volume and the geometrical configuration of the exposed samples in an oven multimode cavity. Only a small amount of offered MW energy is absorbed by small sample volumes [Persch, 1995]. Different sample volumes absorb different MW power levels from the same offered MW power in the exposure applicator as shown in Figure 5. In this case SAR and SA depend strongly on sample volume and geometry, and could be given by W/sample and J/sample, respectively, pointing out each time the sample nature, mass and geometry as well as applicator type and exposure geometry. In view of this argument we determined prior our experiments the dependence of the absorbed MW power amount versus volume and exposure geometry of the samples used in experiments. Some results regarding separate MW irradiation and separate EB irradiation are given in Figures 6, 7 and 8. Figure 6 demonstrates that, with respect to the control sample (2×107 CFU/ml), the number of F.n. colony forming units per ml (CFU/ml), at 24 h incubation after MW irradiation, decreases with the increasing of sample absorbed MW energy from 3.5 × 106 CFU/ml for 57 J/sample to zero for 223 J/sample. Figure 7 shows that, at the same finale temperature, 37ºC and 40ºC, the CFU/ml number for MW irradiated samples is smaller than the CFU/ml 43-2-68 Figure 4. EGC-2 internal configuration of the MEM-MWO multimode cavity used with 16 marked cylinders (PP T309-4A type) for separate MW exposure experiments. Figure 5. MW mean power absorbed by different sample volumes and geometrical exposure configurations versus magnetron mean current. number for CH (classical heating) samples, by a factor of 2.3×103 and 102, respectively. At higher temperature of 48-50ºC, the CH effects and MW effects are comparable. Figure 8 demonstrated that starting from 40 kGy all population cells lost drastically them viability. The EB irradiation of F. n. cultures produced simultaneously with the cells viability decrease an increasing of exotoxin quantity released in the Journal of Microwave Power & Electromagnetic Energy ONLINE Vol. 43, No. 2, 2009 Figure 6. The effects of different MW irradiation modes on the CFU/ml number. Figure 7. Comparison of the CH (classical heating) and MW effects on the CFU/ml number of F. n. Figure 8. The effects of different EB irradiation doses on the CFU/ml number of F. n. Table 1. Antibody titer values versus vaccine types and inoculation stages. Vaccine Antibody titer (IU/ml) A1 128-256 A2 128-512 B1 64-128 B2 128-1024 256-512 512-2048 128-512 512-2048 International Microwave Power Institute for 0.5 ml for 1 ml after 14 days 43-2-69 culture supranatant as compared with classical methods that is important for vaccine preparation productivity. The MW irradiation is able to reduce the cells viability to zero but without an increase of exotoxin quantity in cultures supranatant. Instead of this MW irradiation, for certain conditions (certain culture composition, low initial temperature, SAR level in the range of 6-7 W/sample, 9-10 complete rotations inside MEM-MWO multimode cavity), is able to induce an important stimulation degree of the F. n. proliferation in cultures, from two to three log10, during of short times (several tens of seconds). This effect was verified three times with the same result. This is a very important feature for vaccine preparation productivity. Additional use of EB to MW reduces the F. n. proliferation rate by one to two log10, but a proper EB+MW procedure seems to increases both, exotoxin quantity and proliferation rate. This irradiation procedure is now under study. The measured hemolytic titer and lecithinase activity titer of the F. n. exotoxins isolated from irradiated cultures was from 512 to 1024 IU/ml and from 128 to 512 IU/ml, respectively. F. n. exotoxin pathogenicity test on white mice (Swiss type) is high: after inoculating with 0.5 ml supernatant of F. n. culture five mice from five died. The immune capacity of the A1, A2, B1 and B2 vaccines was tested on the great white rabbits, of 2.5 kg each, by the evaluation of the specific antibody titer values after underskin inoculation with different vaccine types (A1, A2, B1 and B2) in two stages: the first inoculation used 0.5 ml vaccine and the second inoculation, after 14 days, 1 ml vaccine. The results are presented in Table 1. Nowadays, the vaccines efficacy is tested on the 30 bovines divided in two lots: Lot A tested with A1 and A2 vaccines and Lot B tested with B1 and B2 vaccines. The more details and results will be reported in the future papers after the patent procedure finishing. 43-2-70 CONCLUSIONS This work opened up promising new prospects for vaccine preparation. In certain conditions (radiation nature, irradiation doses, F. n. cultures preparation), the EB or/and MW irradiation may stimulate the F. n. proliferation in cultures and may enhance exotoxins releasing or/and their biological properties. The use of EB or/and MW irradiation could be applied to improve vaccine preparation productivity and efficacy against many diseases. REFERENCES Amoako, K.K., Goto, Y. and Shinjo, T. (1993). “Comparison of extracellular enzymes of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. Funduliforme” J. Clin. Microbiology, 31, pp.2244 – 2247. Berg, J.N and Scanlan C.M (1982). “Study of Fusobacterium necrophorum from bovine hepatic abscesses: biotypes, quantitation, virulence and antibiotic susceptibility” Am. J. Vet. Res., 43, pp.1580 – 1586. Persch, C. and Schubert, H. (1995). “Characterization of household microwave ovens by their efficiency and quality parameter.” Proc. 5th International Conference on Microwave and High Frequency Heating, England UK, pp.S31.1-S31.4 Smolko, E. E. and Lombardo, J. H. (2005). “Virus inactivation studies using ion beams, electron and gamma irradadiation” Nucl. Instr. And Meth. In Phys. Res., B236, pp.249-253. Journal of Microwave Power & Electromagnetic Energy ONLINE Vol. 43, No. 2, 2009