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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
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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
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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
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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
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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.
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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.
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