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Isolation of an Adenovirus from a Pig
J.
COMPo
PATH.
81
1964. VOL. 74.
ISOLATION OF AN ADENOVIRUS FROM A PIG By
D. A. HAIG and M. C. CLARKE A.R.C., Institutefor Research on Animal Diseases, Compton
and
M. S. PEREIRA Central Public Health Laboratory, Colindale, London
INTRODUCTION Recently the list of animals from which adenoviruses have been isolated has expanded considerably and now includes, besides man, monkeys, cows, dogs, mice, chimpanzees and chickens (Pereira, Huebner, Ginsberg and van der Veen, 1963). During a survey of viral infections which is being carried out on the Compton herd of pigs a number of viruses have been isolated in tissue culture. One of these-Compton 25 R-has several of the characteristics of an adenovirus and a description of this agent is now presented. MATERIALS AND METHODS Rectal swabs were obtained from various age groups of Large White pigs maintained at Compton. The swabs were placed in 2 ml. Hartley broth containing 500 units penicillin, 500 [J.g. streptomycin and 500 units mycostatin. They were shaken and centrifuged at 2,000 r.p.m. for 10 minutes. The supernatant fluid was then inoculated into tubes containing monolayers of secondary cultures of pig-kidney cells, o· I ml. to 1'0 ml. of medium. The remainder of the fluid was stored at -60°C. Tissue culture. Secondary monolayers were prepared from kidneys obtained from week-old pigs in the Compton piggery. They were grown in a medium consisting of Earle's balanced salt solution with 0'5 per cent. lactalbumin hydrolysate,s per cent. sheep serum with 100 units penicillin, 100 [J.g. streptomycin and 25 units of mycostatin per ml. The pH was adjusted to about 7'4 by gassing with carbon dioxide. For maintenance, glucose was replaced by galactose (Cooper, Wilson and Burt, 1959). After inoculation the cultures were incubated rolling at 37°C and examined daily for 2 weeks. Cultures in which cytopathic effects were seen were frozen and thawed and subcultured into further monolayers. In some cultures flying coverslips were included. When required these were washed in saline, fixed with Bouin's solution and stained with haematoxylin and eosin. Neutralisation tests were done in cultures of pig kidney cells using lOoTCID 5o of virus with dilutions of rabbit hyperimmune sera prepared with prototype strains of adenovirus. Complement fixation tests. These were done in plastic haemagglutination trays with o· I ml. unit volumes and overnight fixation at 4°C. (Pereira, 1956). Infected and control pig kidney cultures were frozen and thawed three times, inactivated for half an hour at 56°C. and tested in doubling F
ADENOV IRUS FROM A PIG
dilutions with a high titre human serum convalescent after an adenov infection and with a rabbit hyperimmune serum prepared with adenovirus irus type 5. RESULT S
From a rectal swab obtain ed from a 12-day old pig with diarrhoea, an agent was isolate d which produc ed change s in the pig kidney culture s. On first passag e no cytopa thic change s were observ ed, but 12 days after the first subcul ture roundi ng and enlarge ment of some of the cells occurr ed with some clumpi ng. On further passag e these change s becam e appare nt on the 6th day and on subsequent passages cytopa thic effects occurr ed within 18 hours of inoculation when undilu ted inocul a were used. When re-isola tion from the origina l fluid was attemp ted it was found that cytopa thic effects appear ed on the 1 I th day of primar y culture . Coverslips prepar ed from the 7th passag e were fixed on the 4th day after inocul ation. In these, strikin g change s were observ ed in the nuclei of the cells. The chrom atin was re-arra nged and granul ar and in the early stages it appear ed to be diffusely spread . Later a densely stainin g mass accum ulated in the centre showin g regula r pattern s of granul es leaving a clear zone betwee n the mass and the nuclea r membr ane. In some cells this clear zone was vesicul ar while in others it was occupi ed by a clear pink substan ce. In additio n to these change s brightl y stainin g eosino philic bodies were seen, many with a sharply defined basoph ilic outline . Clump ing of the cells was seen, usually in groups of 3 to 5 but occasio nally up to 20. Cytopa thic effects were also produc ed in culture s of pig lung, pig testis and in a contin uous line of rhesus monke y kidney , LLC MK2 (Hull, Cherry and Tritch , 1962). No change s were found to occur in culture s of mouse embry o, chick embry o, HeLa cells, human amnio n or primar y rhesus monke y kidney . Cultur~s were tested for sensitiv ity to ether using the metho d describ ed by Andrew es and Horstm ann (1949) and to chloro form by the metho d of Feldm an and Wang (1961). No differences were found betwee n treated and untrea ted suspensions when used to infect pigkidney cell monola yers. The stabilit y of the agent at pH 4'0 was then compa red with materi al buffere d at pH 7.2. No differe nce in infectiv ity for pig-kid ney cells was observ ed after holdin g the mixtur es at 4°C. overni ght. Two families of 3 day-ol d mice were inocul ated intrace rebrall y with materi al from the 8th passage. They showed no appare nt reactio n during an observ ation period of 3 weeks. In comple ment-f ixation tests comple te fixation was demon strated with the agent and a human conval escent serum and a rabbit adenov irus type 5 antiser um up to a dilutio n of 1/128. No fixatio n was obtain ed with uninfe cted pig kidney culture s with either serum. To confirm the presen ce of the adenov irus group antigen a gel diffusion precip itation test was made as describ ed by Pereira ,
D. A. HAIG,
et
at.
Pereira and Allison (1959). The centre well contained a rabbit hyperimmune serum to adenovirus type 5. The surrounding wells contained an arc ton purified preparation of adenovirus type 5, the strain 25 R previously treated by freezing and thawing, and an uninoculated pig kidney culture similarly treated. Two lines appeared between the type 5 antigen and the type 5 serum, the inner line being continuous with a line between the type 5 serum and 25 R. No lines appeared between the serum and preparation of uninoculated pig kidney cultures. Because of the known ability of most human adenoviruses to agglutinate the erythrocytes of monkeys or rats attempts were made to demonstrate haemagglutinins. It was found that rhesus monkey cells were not agglutinated but rat cells were agglutinated to a low titre. The results of serum neutralisation tests of the strain 25 R showed no neutralisation with specific rabbit antisera to adenoviruses types I, 2, 3, 4, 5, 6, 7, 9, 10, I I, 12, 13, 14, 16, 17, 21, 25, 26, 27 and 28. DISCUSSION
The results of the complement fixation and gel diffusion tests strongly support the inclusion of the strain 25 R, isolated from a pig, among the adenoviruses. Both the cytopathic effect and the nuclear changes occurring in the pig kidney cultures ar~ typical of adenoviruses. The nuclear alterations are of the type described for the subgroup of adenoviruses types 3, 4 and 7 (Boyer, Denny and Ginsberg, 1959). Preliminary results indicate that the strain 25 R is serologically distinct from several of the commoner human adenoviruses, but full cross neutralization tests with antisera to all the human types and other animal types remain to be done. While the pig from which the agent was isolated did show diarrhoea it is not known whether this virus was associated with the disease, but work on this aspect is now being carried out. CONCLUSIONS
A virus which has the properties of an adenovirus has been isolated from a pig. It produces characteristic nuclear changes in pigkidney monolayers, and is resistant to treatment with ether, chloroform and pH 4. It was shown by complement-fixation tests and gel diffusion precipitation that this virus possesses the adenovirus group antigen. ACKNOWLEDGMENTS
We are grateful to Dr. W. S. Gordon for the interest he has taken in this work, to Mr. F. H. Summerfield for the photographs and to Miss M. E. A. Curtis for technical assistance.
ADENOVIRUS FROM A PIG
REFERENCES
Andrewes, C. H., and Horstmann, D. M. (1949). J. gen. Microbiol., 3, 290. Boyer, G. S., Denny, F. W. Jnr., and Ginsberg, H. S. (1959). J. expo Med., llO, 82 7· Cooper, P. D., Wilson, J. N., and Burt, A. M. (1959). J. gen. Microbiol., 21, 702. Feldman, H. A, and Wang, S. S. (1961). Proc. Soc. expo Biol. (N.Y.), 106, 73 6 . Hull, R. N., Cherry, W. R., and Tritch, O. J. (1962). J. expo Med., ll5, 90 3. Pereira, M. S., Pereira, H. G., and Allison, A C. (1959). Lancet, i, 551. Pereira, H. G. (1956). J. Path. Bact., 72, 105. Pereira, H. G., Huebner, R. J., Ginsberg, H. S., and van der Veen, J. (1963). Virology, 20,613. [Received for publication, September 14th, 1963]
D. A. HAIG,
et at.
2 Fig.
I.
fig .
2.
Two large nuclei of pig-kidney cells showing central granular masses with surrounding ves icular zones. X 800. An e nlarged nucle us showing chroma tin rearrangem ent and seven eosinophilic bodies. x 2250.
To face pall' 84
COMPo
PATH.
81
1964. VOL. 74.
ISOLATION OF AN ADENOVIRUS FROM A PIG By
D. A. HAIG and M. C. CLARKE A.R.C., Institutefor Research on Animal Diseases, Compton
and
M. S. PEREIRA Central Public Health Laboratory, Colindale, London
INTRODUCTION Recently the list of animals from which adenoviruses have been isolated has expanded considerably and now includes, besides man, monkeys, cows, dogs, mice, chimpanzees and chickens (Pereira, Huebner, Ginsberg and van der Veen, 1963). During a survey of viral infections which is being carried out on the Compton herd of pigs a number of viruses have been isolated in tissue culture. One of these-Compton 25 R-has several of the characteristics of an adenovirus and a description of this agent is now presented. MATERIALS AND METHODS Rectal swabs were obtained from various age groups of Large White pigs maintained at Compton. The swabs were placed in 2 ml. Hartley broth containing 500 units penicillin, 500 [J.g. streptomycin and 500 units mycostatin. They were shaken and centrifuged at 2,000 r.p.m. for 10 minutes. The supernatant fluid was then inoculated into tubes containing monolayers of secondary cultures of pig-kidney cells, o· I ml. to 1'0 ml. of medium. The remainder of the fluid was stored at -60°C. Tissue culture. Secondary monolayers were prepared from kidneys obtained from week-old pigs in the Compton piggery. They were grown in a medium consisting of Earle's balanced salt solution with 0'5 per cent. lactalbumin hydrolysate,s per cent. sheep serum with 100 units penicillin, 100 [J.g. streptomycin and 25 units of mycostatin per ml. The pH was adjusted to about 7'4 by gassing with carbon dioxide. For maintenance, glucose was replaced by galactose (Cooper, Wilson and Burt, 1959). After inoculation the cultures were incubated rolling at 37°C and examined daily for 2 weeks. Cultures in which cytopathic effects were seen were frozen and thawed and subcultured into further monolayers. In some cultures flying coverslips were included. When required these were washed in saline, fixed with Bouin's solution and stained with haematoxylin and eosin. Neutralisation tests were done in cultures of pig kidney cells using lOoTCID 5o of virus with dilutions of rabbit hyperimmune sera prepared with prototype strains of adenovirus. Complement fixation tests. These were done in plastic haemagglutination trays with o· I ml. unit volumes and overnight fixation at 4°C. (Pereira, 1956). Infected and control pig kidney cultures were frozen and thawed three times, inactivated for half an hour at 56°C. and tested in doubling F
ADENOV IRUS FROM A PIG
dilutions with a high titre human serum convalescent after an adenov infection and with a rabbit hyperimmune serum prepared with adenovirus irus type 5. RESULT S
From a rectal swab obtain ed from a 12-day old pig with diarrhoea, an agent was isolate d which produc ed change s in the pig kidney culture s. On first passag e no cytopa thic change s were observ ed, but 12 days after the first subcul ture roundi ng and enlarge ment of some of the cells occurr ed with some clumpi ng. On further passag e these change s becam e appare nt on the 6th day and on subsequent passages cytopa thic effects occurr ed within 18 hours of inoculation when undilu ted inocul a were used. When re-isola tion from the origina l fluid was attemp ted it was found that cytopa thic effects appear ed on the 1 I th day of primar y culture . Coverslips prepar ed from the 7th passag e were fixed on the 4th day after inocul ation. In these, strikin g change s were observ ed in the nuclei of the cells. The chrom atin was re-arra nged and granul ar and in the early stages it appear ed to be diffusely spread . Later a densely stainin g mass accum ulated in the centre showin g regula r pattern s of granul es leaving a clear zone betwee n the mass and the nuclea r membr ane. In some cells this clear zone was vesicul ar while in others it was occupi ed by a clear pink substan ce. In additio n to these change s brightl y stainin g eosino philic bodies were seen, many with a sharply defined basoph ilic outline . Clump ing of the cells was seen, usually in groups of 3 to 5 but occasio nally up to 20. Cytopa thic effects were also produc ed in culture s of pig lung, pig testis and in a contin uous line of rhesus monke y kidney , LLC MK2 (Hull, Cherry and Tritch , 1962). No change s were found to occur in culture s of mouse embry o, chick embry o, HeLa cells, human amnio n or primar y rhesus monke y kidney . Cultur~s were tested for sensitiv ity to ether using the metho d describ ed by Andrew es and Horstm ann (1949) and to chloro form by the metho d of Feldm an and Wang (1961). No differences were found betwee n treated and untrea ted suspensions when used to infect pigkidney cell monola yers. The stabilit y of the agent at pH 4'0 was then compa red with materi al buffere d at pH 7.2. No differe nce in infectiv ity for pig-kid ney cells was observ ed after holdin g the mixtur es at 4°C. overni ght. Two families of 3 day-ol d mice were inocul ated intrace rebrall y with materi al from the 8th passage. They showed no appare nt reactio n during an observ ation period of 3 weeks. In comple ment-f ixation tests comple te fixation was demon strated with the agent and a human conval escent serum and a rabbit adenov irus type 5 antiser um up to a dilutio n of 1/128. No fixatio n was obtain ed with uninfe cted pig kidney culture s with either serum. To confirm the presen ce of the adenov irus group antigen a gel diffusion precip itation test was made as describ ed by Pereira ,
D. A. HAIG,
et
at.
Pereira and Allison (1959). The centre well contained a rabbit hyperimmune serum to adenovirus type 5. The surrounding wells contained an arc ton purified preparation of adenovirus type 5, the strain 25 R previously treated by freezing and thawing, and an uninoculated pig kidney culture similarly treated. Two lines appeared between the type 5 antigen and the type 5 serum, the inner line being continuous with a line between the type 5 serum and 25 R. No lines appeared between the serum and preparation of uninoculated pig kidney cultures. Because of the known ability of most human adenoviruses to agglutinate the erythrocytes of monkeys or rats attempts were made to demonstrate haemagglutinins. It was found that rhesus monkey cells were not agglutinated but rat cells were agglutinated to a low titre. The results of serum neutralisation tests of the strain 25 R showed no neutralisation with specific rabbit antisera to adenoviruses types I, 2, 3, 4, 5, 6, 7, 9, 10, I I, 12, 13, 14, 16, 17, 21, 25, 26, 27 and 28. DISCUSSION
The results of the complement fixation and gel diffusion tests strongly support the inclusion of the strain 25 R, isolated from a pig, among the adenoviruses. Both the cytopathic effect and the nuclear changes occurring in the pig kidney cultures ar~ typical of adenoviruses. The nuclear alterations are of the type described for the subgroup of adenoviruses types 3, 4 and 7 (Boyer, Denny and Ginsberg, 1959). Preliminary results indicate that the strain 25 R is serologically distinct from several of the commoner human adenoviruses, but full cross neutralization tests with antisera to all the human types and other animal types remain to be done. While the pig from which the agent was isolated did show diarrhoea it is not known whether this virus was associated with the disease, but work on this aspect is now being carried out. CONCLUSIONS
A virus which has the properties of an adenovirus has been isolated from a pig. It produces characteristic nuclear changes in pigkidney monolayers, and is resistant to treatment with ether, chloroform and pH 4. It was shown by complement-fixation tests and gel diffusion precipitation that this virus possesses the adenovirus group antigen. ACKNOWLEDGMENTS
We are grateful to Dr. W. S. Gordon for the interest he has taken in this work, to Mr. F. H. Summerfield for the photographs and to Miss M. E. A. Curtis for technical assistance.
ADENOVIRUS FROM A PIG
REFERENCES
Andrewes, C. H., and Horstmann, D. M. (1949). J. gen. Microbiol., 3, 290. Boyer, G. S., Denny, F. W. Jnr., and Ginsberg, H. S. (1959). J. expo Med., llO, 82 7· Cooper, P. D., Wilson, J. N., and Burt, A. M. (1959). J. gen. Microbiol., 21, 702. Feldman, H. A, and Wang, S. S. (1961). Proc. Soc. expo Biol. (N.Y.), 106, 73 6 . Hull, R. N., Cherry, W. R., and Tritch, O. J. (1962). J. expo Med., ll5, 90 3. Pereira, M. S., Pereira, H. G., and Allison, A C. (1959). Lancet, i, 551. Pereira, H. G. (1956). J. Path. Bact., 72, 105. Pereira, H. G., Huebner, R. J., Ginsberg, H. S., and van der Veen, J. (1963). Virology, 20,613. [Received for publication, September 14th, 1963]
D. A. HAIG,
et at.
2 Fig.
I.
fig .
2.
Two large nuclei of pig-kidney cells showing central granular masses with surrounding ves icular zones. X 800. An e nlarged nucle us showing chroma tin rearrangem ent and seven eosinophilic bodies. x 2250.
To face pall' 84