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Effect of radiation on certain animal viruses in liquid swine manure
Int. J..4ppl. Radiat. lsot. Vol. 34. No. 5. pp. 793--795. 1983 Printed in Great Britain
0020-708X 83,050793-03503.00/0 Pergamon Press Ltd
Effect of Radiation on Certain Animal Viruses in Liquid Swine Manure JOZSEF
SIMON,
ENDRE
MOCSARI,
and VILMOS
MARIA DI GLERIA
FELKAI
Phylaxia Veterinary Biologicals and Feedstuffs Co.. Szallas u. 5, H-1107 Budapest. Hungary Central Veterinary Institute. Tabornomk u. 2, H-1149 Budapest, Hungary The virucidal effect of 6°Co "/-radiation was studied in cell culture medium and in liquid swine manure involving the most important porcine viruses that can be spread by liquid manure. The radiation doses, 20 and 30 kGy, were determined in preliminary experiments. At a radiation dose of 30 kGy, the activity of extracellular and cell-associated test viruses, except swine vesicular disease virus (SVDV). was completely destroyed both in cell culture medium and in liquid swine manure. The infectivity of SVDV decreased significantly (P < 0.01). by 7.0 logto TCIDs0, both in cell culture medium and in liquid manure and this value corresponded to the international effectiveness demand for a disinfectant. The results showed that the safe disinfection virus in liquid swine manure by ionizing radiation requires a radiation dose of 30 kGy.
Introduction SOLUTIONS for the economical and safe utilization of liquid swine manure, a new by-product of the largescale swine farms with an industrial type of management, have been searched for in many countries for many years. It is known that besides well-utilizable and valuable nutritive materials for plants and animals, liquid swine manure also contains polluting materials and different kinds of microorganisms which can survive in it for a long time. Thus, the protection of the environment and of the health of livestocks and people requires the proper disinfection of liquid manure prior to its utilization. For this purpose, several physical, chemical and biological methods are available. Recently, physical disinfecting methods---implying S°Co 7-radiation----came into the limelight. In Hungary, the sterilization of sewage, sewage sludge and liquid manure by ionizing radiation has been studied for several years. The results of these studies were previously reported. ~1-5~ The present study was undertaken to clarify the virucidal effect of 6°Co 7-radiation in liquid swine manure. The experiments involved the most important viruses that can be spread by swine manure. M a t e r i a l s and M e t h o d s In the preliminary experiment, the 5-DVIII strain of porcine enterovirus (serogroup 1, Teschen group, PEV-I)`t6~ was used. In the main experiment, the following viruses were employed: the England 72 (UKG-72) strain of swine vesicular disease virus
(SVDV), 17~ the Cr strain of type C foot-and-mouth disease virus (FMDV-C, Phylaxia, Budapest), a virulent strain of Aujeszky's disease virus (ADV) isolated at the Central Veterinary Institute, the FS 216/64 strain of transmissible gastroentcritis virus (TGEV), ts~ as well as the Oregon C24V strain of bovine viral diarrhoea virus (BVDV). c9~The letter strain served as a model for the classcical swine fever virus. The propagation and the determination of the activity of PEV-1, SVDV and ADV was carried out on monolayers of a porcine embryonic kidney cell line (IB-RS-2),~t°j FMDV-C, TGEV and BVDV were cultivated on baby hamster kidney (BHK-21, Clone 13),ttl~ on secondary swine thyroid and on bovine testicle cell monolayers, respectively. 5 mL aliquots of each virus-infected cell culture medium were dispersed into sterile glass ampules. The ampules were heat sealed. Liquid swine manure was previously sterilized by 6°Co "/-radiation and adjusted to pH 7.0. Its dry matter content was 5Yo. Each virus was diluted to 1 : 10 in liquid swine manure and 40 mL aliquots were dispersed into sterile bottles of 50 mL. The irradiation was carried out by a s°Co 7-source. The activity of the source was 6.23 pBq. The eight rod-shaped 6°Co "/-sources were arranged in two parallel rows. The samples were placed in the geometrical centre of the sources. The dose rate was 6 kGy/h. The optimal radiation doses were determined in a preliminary experiment. For this purpose, the PEV-I was used because this virus is extremely stable over a wide range of physico-chemical treatments. In this experiment, 10, 20 and 30 kGy radiation doses were applied. According to the results, 20 and 30 kGy 793
794
Jo:sef Simon et al. Control 20kGy
8
6
I0 kGy 151
[]
30kGy
Im
r
4
o_
a I 0.5
L
~//ii ii l Cell associated
( x t racelluIar virus
virus
doses were selected for the radiation of all the other test viruses. Virus activity was assayed before and after radiation. Serial tenfold dilutions of both the virusinfected cell culture media and liquid swine manures were made in Hanks' balanced salt solution containing 2 ~ foetal calf serum. 0.1 mL of each dilution was inoculated in quadruplicate onto cell monolayers in culture tubes. The titres (log~o TCIDso/0.1 mL, TCID : tissue culture infective dose) were calculated according to the method of K~.RSER.(121
FIG. 1
Control l-I
20kGy ~l
30kGy gll
8
F[•l|l.
s 4
~-_o
3
_o
Results and Discussion
2
I
o.s
V i r u s in liquid manure
V i r u s in cell culture medium
rf~nure.
F;o. 2
Control [ ] -J
E
8 7
0
J
E_o
Results of the preliminary experiment involving the PEV-1 are shown in Fig. I. Irradiation with 10 kGy reduced the infective titre of both the extracellular and the cell-associated virus by 5.0 logto TCIDs0. The cell-associated virus lost its activity after a radiation with 20 kGy. The activity of the extracellular virus, however, was only destroyed after irradiation with 30 kGy. The results of the radiation of the other test viruses are shown in Figs 2-6. Following irradiation with 20 kGy, TGEV (Fig. 2), ADV (Fig. 3) and BVDV (Fig. 4) lost their activity both in cell culture medium and in liquid swine
2 0 kG y P'A 8-
.J
- 3 0 kGy E
E
m
7--
6
20kGy [ ]
30kGy Im
Irxtracellular virus
Ceil asseciated virus
5
5 4
Control [ ]
O
i
3 2
!
~
2
I G5
iI
F-
O. I.
!
Virus in cell cu Iture medium
F~'=#]II I ] Virus in liquid mQflum
Fzc. 5
FIG. 3
Control
Control I-I .J E "% 0
o
8
.J
@
.% 0
7
E
- 3 0 kGy Ill
6 S
o_ old
4
o
9
20kGy [ ]
7
0
n
20kay
~:~
30
k a y Im
n
6 5 o_ o _o
3
4 3 2
2 I--
Virus in liquid manure
I 0.5
IV i r u s In cell culture medium
FtG. 4
~-/i ii ii Virus in liquid manure
i 0.5 [xf r~llul(~r virus
Cell associated virus
F[O. 6
Virus in liquid manure
795
l,'ir,lcidal effect of °°Co 7-radiation
After application of a radiation dose of 20 kGy. the cell-associated FMDV-C was completely inactivated in cell culture medium. The same dose reduced the infective titre of the extracellular virus by 5.5 logto TCID~o. while that of the cell-associated virus was only diminished by 4.5 logto TCIDso (Fig. 6). Applying the same radiation dose. the activity of SVDV was reduced by 2.0 logto TCID~o in liquid swine manure. After an irradiation with a dose of 30 kGy, the infective titre of the extracellular and the cell-associated virus in cell culture medium and that of SVDV diluted in liquid swine manure was equally decreased by 7.0 logto TCIDso. However, SVDV survived both in the cell culture medium and in liquid manure, its infective titres were 1.5 and 1.0 loglo TCIDso, respectively.
Conclusions The activity of extracellular and cell-associated test viruses, except SVDV, was completely destroyed by applying a radiation dose of 30 kGy both in cell culture medium and in liquid swine manure. The infectivity of SVDV, however, also decreased significantly (P < 0.01), by 7.0 logto TCIDso, both in cell culture medium and in liquid swine manure. As a result of the radiation with 20 kGy, ADV, BVDV and TGEV completely lost their activity. On the other hand, FMDV-C. SVDV and PEV-I survived this radiation dose both in cell culture medium and in liquid swine manure. Although the titre of these viruses decreased by at least 4.0 logto TCIDso
:,.R I. 34 5---FJ
in cell culture medium, even if the virus was cellassociated, and this value corresponds to the international effectiveness-demand for a disinfectant, the titre decrease of SVDV and FMDV-C was only 2.0 and 3.5 logto TCIDso in liquid swine manure, respectively. Accordingly, it can be concluded that the safe disinfection of the virus infected liquid swine manure by 6°Co y-radiation requires a radiation dose of 30 kGy.
References 1. SIMONJ. and TAMASIG. IAEA SM 194/'306.(1976). 2. SIMONJ., GACS F. and URBANA. IAEA Proc. Munich (1975). 3. SIMO.~J., SOLYOM F. FELKAIV. and OROSZLANVP. Proc. Conf. of ESNA, Munich (1976). 4. SIMONJ. and D[ GLERtAM. Proc. Conf. ofESNA. Brno (1978). 5. SIMON J.. SZEMEREDI GY. and DI GLERIA M. Abstr. 6th Int. Congr. of Radiation Research, Tokyo (1979). 6. SZENT-IvANY]T. Acta Microbiol. Acad. Sci. Hung. 17, 267 (1970). 7. DAWEP. S., FORMANA. J. and SMALEC. J. Nature 241, 540 (1973). 8. CARTWRIGHT S. F.. HARRIS H. M.. BLANDFORD T. B.. FINCHAM I. and GrrTER M. J. Comp. Pathol. 75, 397 (1965). 9. GILL~PIEJ. H., BAKERJ. A. and McENTEE K. Cornell Vet. 50, 74 (1960). I0. CASTROM. P. Ar O. Inst. Biol. Sao Paulo 31, 63 (1964). I 1. MACPttERSONI. A. and STOKERG. P. Virology 16, 147 (1962). 12. KXRBER G. Arch. Exp. Pathol. Pharmakol. 162, 480 (1931).
0020-708X 83,050793-03503.00/0 Pergamon Press Ltd
Effect of Radiation on Certain Animal Viruses in Liquid Swine Manure JOZSEF
SIMON,
ENDRE
MOCSARI,
and VILMOS
MARIA DI GLERIA
FELKAI
Phylaxia Veterinary Biologicals and Feedstuffs Co.. Szallas u. 5, H-1107 Budapest. Hungary Central Veterinary Institute. Tabornomk u. 2, H-1149 Budapest, Hungary The virucidal effect of 6°Co "/-radiation was studied in cell culture medium and in liquid swine manure involving the most important porcine viruses that can be spread by liquid manure. The radiation doses, 20 and 30 kGy, were determined in preliminary experiments. At a radiation dose of 30 kGy, the activity of extracellular and cell-associated test viruses, except swine vesicular disease virus (SVDV). was completely destroyed both in cell culture medium and in liquid swine manure. The infectivity of SVDV decreased significantly (P < 0.01). by 7.0 logto TCIDs0, both in cell culture medium and in liquid manure and this value corresponded to the international effectiveness demand for a disinfectant. The results showed that the safe disinfection virus in liquid swine manure by ionizing radiation requires a radiation dose of 30 kGy.
Introduction SOLUTIONS for the economical and safe utilization of liquid swine manure, a new by-product of the largescale swine farms with an industrial type of management, have been searched for in many countries for many years. It is known that besides well-utilizable and valuable nutritive materials for plants and animals, liquid swine manure also contains polluting materials and different kinds of microorganisms which can survive in it for a long time. Thus, the protection of the environment and of the health of livestocks and people requires the proper disinfection of liquid manure prior to its utilization. For this purpose, several physical, chemical and biological methods are available. Recently, physical disinfecting methods---implying S°Co 7-radiation----came into the limelight. In Hungary, the sterilization of sewage, sewage sludge and liquid manure by ionizing radiation has been studied for several years. The results of these studies were previously reported. ~1-5~ The present study was undertaken to clarify the virucidal effect of 6°Co 7-radiation in liquid swine manure. The experiments involved the most important viruses that can be spread by swine manure. M a t e r i a l s and M e t h o d s In the preliminary experiment, the 5-DVIII strain of porcine enterovirus (serogroup 1, Teschen group, PEV-I)`t6~ was used. In the main experiment, the following viruses were employed: the England 72 (UKG-72) strain of swine vesicular disease virus
(SVDV), 17~ the Cr strain of type C foot-and-mouth disease virus (FMDV-C, Phylaxia, Budapest), a virulent strain of Aujeszky's disease virus (ADV) isolated at the Central Veterinary Institute, the FS 216/64 strain of transmissible gastroentcritis virus (TGEV), ts~ as well as the Oregon C24V strain of bovine viral diarrhoea virus (BVDV). c9~The letter strain served as a model for the classcical swine fever virus. The propagation and the determination of the activity of PEV-1, SVDV and ADV was carried out on monolayers of a porcine embryonic kidney cell line (IB-RS-2),~t°j FMDV-C, TGEV and BVDV were cultivated on baby hamster kidney (BHK-21, Clone 13),ttl~ on secondary swine thyroid and on bovine testicle cell monolayers, respectively. 5 mL aliquots of each virus-infected cell culture medium were dispersed into sterile glass ampules. The ampules were heat sealed. Liquid swine manure was previously sterilized by 6°Co "/-radiation and adjusted to pH 7.0. Its dry matter content was 5Yo. Each virus was diluted to 1 : 10 in liquid swine manure and 40 mL aliquots were dispersed into sterile bottles of 50 mL. The irradiation was carried out by a s°Co 7-source. The activity of the source was 6.23 pBq. The eight rod-shaped 6°Co "/-sources were arranged in two parallel rows. The samples were placed in the geometrical centre of the sources. The dose rate was 6 kGy/h. The optimal radiation doses were determined in a preliminary experiment. For this purpose, the PEV-I was used because this virus is extremely stable over a wide range of physico-chemical treatments. In this experiment, 10, 20 and 30 kGy radiation doses were applied. According to the results, 20 and 30 kGy 793
794
Jo:sef Simon et al. Control 20kGy
8
6
I0 kGy 151
[]
30kGy
Im
r
4
o_
a I 0.5
L
~//ii ii l Cell associated
( x t racelluIar virus
virus
doses were selected for the radiation of all the other test viruses. Virus activity was assayed before and after radiation. Serial tenfold dilutions of both the virusinfected cell culture media and liquid swine manures were made in Hanks' balanced salt solution containing 2 ~ foetal calf serum. 0.1 mL of each dilution was inoculated in quadruplicate onto cell monolayers in culture tubes. The titres (log~o TCIDso/0.1 mL, TCID : tissue culture infective dose) were calculated according to the method of K~.RSER.(121
FIG. 1
Control l-I
20kGy ~l
30kGy gll
8
F[•l|l.
s 4
~-_o
3
_o
Results and Discussion
2
I
o.s
V i r u s in liquid manure
V i r u s in cell culture medium
rf~nure.
F;o. 2
Control [ ] -J
E
8 7
0
J
E_o
Results of the preliminary experiment involving the PEV-1 are shown in Fig. I. Irradiation with 10 kGy reduced the infective titre of both the extracellular and the cell-associated virus by 5.0 logto TCIDs0. The cell-associated virus lost its activity after a radiation with 20 kGy. The activity of the extracellular virus, however, was only destroyed after irradiation with 30 kGy. The results of the radiation of the other test viruses are shown in Figs 2-6. Following irradiation with 20 kGy, TGEV (Fig. 2), ADV (Fig. 3) and BVDV (Fig. 4) lost their activity both in cell culture medium and in liquid swine
2 0 kG y P'A 8-
.J
- 3 0 kGy E
E
m
7--
6
20kGy [ ]
30kGy Im
Irxtracellular virus
Ceil asseciated virus
5
5 4
Control [ ]
O
i
3 2
!
~
2
I G5
iI
F-
O. I.
!
Virus in cell cu Iture medium
F~'=#]II I ] Virus in liquid mQflum
Fzc. 5
FIG. 3
Control
Control I-I .J E "% 0
o
8
.J
@
.% 0
7
E
- 3 0 kGy Ill
6 S
o_ old
4
o
9
20kGy [ ]
7
0
n
20kay
~:~
30
k a y Im
n
6 5 o_ o _o
3
4 3 2
2 I--
Virus in liquid manure
I 0.5
IV i r u s In cell culture medium
FtG. 4
~-/i ii ii Virus in liquid manure
i 0.5 [xf r~llul(~r virus
Cell associated virus
F[O. 6
Virus in liquid manure
795
l,'ir,lcidal effect of °°Co 7-radiation
After application of a radiation dose of 20 kGy. the cell-associated FMDV-C was completely inactivated in cell culture medium. The same dose reduced the infective titre of the extracellular virus by 5.5 logto TCID~o. while that of the cell-associated virus was only diminished by 4.5 logto TCIDso (Fig. 6). Applying the same radiation dose. the activity of SVDV was reduced by 2.0 logto TCID~o in liquid swine manure. After an irradiation with a dose of 30 kGy, the infective titre of the extracellular and the cell-associated virus in cell culture medium and that of SVDV diluted in liquid swine manure was equally decreased by 7.0 logto TCIDso. However, SVDV survived both in the cell culture medium and in liquid manure, its infective titres were 1.5 and 1.0 loglo TCIDso, respectively.
Conclusions The activity of extracellular and cell-associated test viruses, except SVDV, was completely destroyed by applying a radiation dose of 30 kGy both in cell culture medium and in liquid swine manure. The infectivity of SVDV, however, also decreased significantly (P < 0.01), by 7.0 logto TCIDso, both in cell culture medium and in liquid swine manure. As a result of the radiation with 20 kGy, ADV, BVDV and TGEV completely lost their activity. On the other hand, FMDV-C. SVDV and PEV-I survived this radiation dose both in cell culture medium and in liquid swine manure. Although the titre of these viruses decreased by at least 4.0 logto TCIDso
:,.R I. 34 5---FJ
in cell culture medium, even if the virus was cellassociated, and this value corresponds to the international effectiveness-demand for a disinfectant, the titre decrease of SVDV and FMDV-C was only 2.0 and 3.5 logto TCIDso in liquid swine manure, respectively. Accordingly, it can be concluded that the safe disinfection of the virus infected liquid swine manure by 6°Co y-radiation requires a radiation dose of 30 kGy.
References 1. SIMONJ. and TAMASIG. IAEA SM 194/'306.(1976). 2. SIMONJ., GACS F. and URBANA. IAEA Proc. Munich (1975). 3. SIMO.~J., SOLYOM F. FELKAIV. and OROSZLANVP. Proc. Conf. of ESNA, Munich (1976). 4. SIMONJ. and D[ GLERtAM. Proc. Conf. ofESNA. Brno (1978). 5. SIMON J.. SZEMEREDI GY. and DI GLERIA M. Abstr. 6th Int. Congr. of Radiation Research, Tokyo (1979). 6. SZENT-IvANY]T. Acta Microbiol. Acad. Sci. Hung. 17, 267 (1970). 7. DAWEP. S., FORMANA. J. and SMALEC. J. Nature 241, 540 (1973). 8. CARTWRIGHT S. F.. HARRIS H. M.. BLANDFORD T. B.. FINCHAM I. and GrrTER M. J. Comp. Pathol. 75, 397 (1965). 9. GILL~PIEJ. H., BAKERJ. A. and McENTEE K. Cornell Vet. 50, 74 (1960). I0. CASTROM. P. Ar O. Inst. Biol. Sao Paulo 31, 63 (1964). I 1. MACPttERSONI. A. and STOKERG. P. Virology 16, 147 (1962). 12. KXRBER G. Arch. Exp. Pathol. Pharmakol. 162, 480 (1931).