THE JOURNAL OF INFECTIOUS DISEASES. VOL. 138, NO.5. NOVEMBER 1978
© 1978 by The University of Chicago. 0022-1899/78/3805-0013$00.75
Isolation of Viruses from Cultures of Bovine Endometrial Cells P. D. Luther, P. A. Nuttall,* and R. A. Gibbons
From the Agricultural Research Council, Institute for Research on Animal Diseases,Compton, Newbury, Berkshire, United Kingdom
During investigations into the culture of endometrial cells from pregnant and nonpregnant cattle, viruses were isolated from a high proportion of' the endometrial samples. The viruses were mucosal disease virus (MDV) and bovine syncytial virus (BSV). MDV (also known as bovine viral diarrhea virus), a member of the family Togaviridae, is antigenically related to hog cholera virus [1] and morphologically similar to rubella virus [2]. BSV is a syncytium-forming "foamy" virus of the family Retroviridae [3]. Both MDV and BSV have been isolated from bovine placentas, fetal and maternal tissues, and fetal blood [4-7]. In addition, MDV has been shown to cause abortions and fetal malformations [8] and has been incriminated in bovine infertility [9]. Materials and Methods
Endometria from 19 cattle were processed to produce cell cultures; the various cell batches are listed chronologically in tables I and 2. The Received for publication January 6, 1978, and in revised form May 15, 1978. P. A. Nuttall wassupported by the Animal Health Trust. We thank E. J. Thompson, Ms. V. J. Collier, and Ms. V. R. Robinson for their assistance. The bovine syncytial virus antiserum was supplied by Dr. M. J. Van Der Maaten, National Animal Disease Laboratory, Agricultural Research Service,U.S.Department of Agriculture, Ames,Iowa. Please address requests for reprints to P. D. Luther, Institute for Research on Animal Diseases, Compton, Newbury, Berkshire, RG16 ONN, United Kingdom. • Present address: Edward Grey Institute, Zoology Department, South Parks Road, Oxford, England.
horns of the uteri were dissected longitudinally to reveal the endometrium. An area of ,..,40 mmof endometrial tissue was removed from each horn, chopped into small pieces, washed in phosphate-buffered saline, and trypsinized for I hr by the continuous flow technique [10]. The cells were suspended in Eagle's minimal essential medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 0.11 % NaRC0 3 , and penicillin, streptomycin, and mycostatin to give final concentrations of 100 units, 100 jLg, and 25 units/em", respectively. Medical flat bottles (140 em") were seeded with ,..,6 X 106 cells and incubated at 37 C until cultures were at least 75% confluent, when they were subcultured. Coverslip cultures were set up for staining with fluorescent antibody and with Giemsa. Cells were stained by the indirect fluorescent antibody technique using antiserum to the NADL (National Animal Diseases Laboratory, Ames, Iowa) strain of MDV from a gnotobiotic calf, and a rabbit antiserum to bovine antigen conjugated with fluorescein (Nordic Immunology, Tilburg, The Netherlands). Serum taken before inoculation was used as a control for fluorescence. A similar technique was used with an antiserum to BSV. Cell culture batches were screened for the presence of MDV at the secondary and subsequent passage levels. Cells were stained for the presence of BSV when syncytia were observed. Results
Success in establishing cell culture growth varied markedly from batch to batch. Three batches,
660
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
Endometrial cells from the uteri of pregnant and nonpregnant cattle were cultured. The presence of one or both of two viruses, noncytopathic mucosal disease virus and bovine syncytial virus, was demonstrated in seven of 19 endometria investigated. It was necessary to subculture the cells an average of four times to detect the viral infections. Difficulties were encountered in producing endometrial cell cultures from cows at term or near the end of term and also from older animals. The infections detected may be significant because both of the viruses isolated are capable of infecting the bovine fetus in utero and mucosal disease virus has been associated with bovine fetal diseases.
661
Viral Isolates from Bovine Endometria
Table I. Bovine endometrial cell cultures from which mucosal disease virus (MDV) and/or bovine syncytial virus (BSV) were isolated. Age of animal (years)
Calving/ servicing ratio *
1 4 5 7 8
5.5 5.5 3.5 2.25 2.5
2/4 3/3 1/6 0/2 0/9
15-day blastocyst Not pregnant Not pregnant Not pregnant 14-day blastocyst
10 16
4.5 5
2/3 2/11
16-day blastocyst Not pregnant
Cell batch no.
Stage of pregnancy
Virus demonstrated
Cell passage no. when virus was demonstrated
BSV MDV MDV MDV MDV BSV MDV MDV
4 3 4 2 4 5 5 4
"Number of calves per number of services.
enlarged vacuolated cells. Similar vacuolated cells were observed in all of the batches that were infected with MDV. When freeze-thawed extracts of the MDV-infected cell batches were inoculated into calf testis cell cultures, the calf testis cells showed fluorescence specific for MDV. However, when calf testis cell cultures were inoculated with MDV-infected or uninfected endometrial cell batches that showed vacuolation, the calf testis cells did not become vacuolated. Discussion
The response of bovine endometrial cells to subculturing in vitro appeared to reflect the state of the animal when the endometrial tissue was removed. Batches no. 2, 9, 11, and 13, which all showed poor cell growth, were from dams at full term or near the end of term. Two batches, no.
Table 2. Bovine endometrial cell cultures from which no viruses were demonstrated. Cell batch no. 2 3 6 9 11 12 13 14 15 17 18 19
Age of animal (years)
Calving/ servicing ratio"
6.5 4.5 6.5 2.5 2.5
5/5 3/3 4/6 1/1 1/1 9/17 10/13
14
13 5 days 6 4 11 3.25
4/7 1/1 8/25 1/2
"Number of calves per number of services.
Stage of pregnancy Near term Not pregnant, progesterone-treated 15-day blastocyst Term Term 15-day blastocyst Term Not pregnant Not pregnant Not pregnant 6-day blastocyst
Limit of culture growth (cell passage no.] 2 2 >17 Primary only Primary only 2 2 6 2 3 Primary .only 4
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
no. 9, 11, and 18 (table 2), showed little, if any, primary growth; six batches, no. 2, 3, 7, 12, 13, and 15 (tables 1 and 2), were capable of being subcultured only once. In contrast, batch no. 6 (table 2) has so far been subcultured 18 times. Adventitious viral infections were demonstrated in seven of the 19 batches of endometrial cells (table 1); five batches were infected with MDV alone, one batch with BSV alone, and one batch with both MDV and BSV. In one instance infection was detected in cells in the first subculture, but with most batches four passages were necessary before viral infection was demonstrated. Several of the cell batches showed a CPE that was not necessarily correlated with infection with either MDV or BSV. Cultures of batches no. 14, 15, and 19 (table 2), which did not demonstrate fluorescence for MDV or BSV, contained
Luther, Nuttall, and Gibbons
662
servations) [II]. These findings suggest that the venereal route of transmission and infection of the endometrium is a possibility. The presence of MDV and BSV in the endometria of cattle also raises the question regarding the effect of these viruses on pregnancy. In the group of animals with infected endometria, the mean conception rate was significantly lower than that in the uninfected group. However, it would be premature to state at this stage that the relationship was more than a speculative one. The number of animals involved in this preliminary investigation was small, but the data obtained reveal that infection of the bovine endometrium by MDV or BSV occurs relatively frequently in a group of culled cows from this institute's dairy herds. Extension of these studies to a larger group of animals is needed in an attempt to determine the significance of viral infections of the endometrium in relation to infertility and abortion. References 1. Darbyshire, J. H. A serological relationship between swine fever and mucosal disease of cattle. Vet. Rec. 72:331,1960. 2. Stott, E. J., Almeida, J. D., O'Reilly, K. J. Characterization of mucosal disease virus as a togavirus by electron microscopy. Microbios llA:79-83, 1974. 3. Fenner, F. The classification and nomenclature of viruses. Summary of results of meetings of the International Committee on Taxonomy of Viruses in Madrid, September 1975. Intervirology 6: 1-12,1975/6. 4. Hubbert, W. T., Bryner, J. H., Fernelius, A. L., Frank, G. H., Estes, P. C. Viral infection of the bovine fetus and its environment. Arch. Gesamte Virusforsch. 41:86-98,1973. 5. Scott, F. W., Kahrs, R. F., Parsonson, I. M. A cytopathogenic strain of bovine viral diarrhea-mucosal disease virus isolated from a bovine fetus. Cornell Vet. 62:74-84,1972. 6. Scott, F. W., Shively, J. N., Gaskin, J., Gillespie, J. H. Bovine syncytial virus isolations. Arch. Gesamte Virusforsch. 43:43-52,1973. 7. Van Der Maaten, M. J., Hubbert, W. T., Boothe, A. D., Bryner, J. H., Estes, P. C. Isolations of bovine syncytial virus from maternal and fetal blood. Am. J. Vet. Res. 34:341-343, 1973. 8. Kendrick, J. W. Bovine viral diarrhea-mucosal disease virus infection in pregnant cows. Am. J. Vet. Res. 32: 533-544, 1971. 9. Archbald, L. F., Zemjanis, R. Intrauterine infusion of the virus of bovine virus diarrhea and artificial insemination in the cow at estrus. Vet. Med. Small Anim. Clin. 72:221-225, 1977.
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
12 and 18, which also showed poor cell growth, were from older dams declared barren; batch no. 3 came from an animal which had received a course of progesterone treatment. The one batch of endometrial cells that grew well (batch no. 6) and showed no evidence of viral infection was from a dam in the very early stages of pregnancy. Thus the stage of pregnancy and the age of the dam may account for the poor cell growth in several batches of endometrial cell culture. Infection with MDV and/or BSV was demonstrated in seven of the 19 batches of endometrial cells investigated. However, because viral infection was not usually detected until the cells had been subcultured several times, infection of the batches that had poor cell growth cannot be discounted. Vacuolation of cells appeared in both MDVinfected· and uninfected batches of endometrial cell cultures. Therefore, the vacuolation was considered unlikely to be due to infection with MDV, and the type of MDV infecting the endometrial cells was considered to be noncytopathic. Because attempts to transmit a vacuolating agent to calf testis cell cultures were unsuccessful, it was concluded that the cause of vacuolation was not transmissible. The isolation of viruses from endometrial cells raises the question concerning the source of infection. In vitro infection with MDV that was possibly present in the supplementary growth factor FCS [II] can be discounted because all cell batches were cultured in the presence of heatinactivated FCS. Heating of MDV at 56 C for 30 min has been shown to inactivate the virus. Also, the materials used for the processing and growth of the endometrial cell cultures, especially trypsin and FCS, can be eliminated as sources of viral contamination. Twelve batches of calf testis cell cultures, which are sensitive to and would have demonstrated the presence of MDV and BSV, were produced and passaged an average of 12 times with materials identical to those used for the endometrial cell cultures. There was no evidence of the presence of any adventitious viruses in the calf testis cell cultures, although the methods of examination for these cultures were the same as those for the endometrial cultures. MDV and BSV have been isolated from calf testis cells (authors' unpublished ob-
663
Viral Isolates from Bovine Endometria
10. Luther, P. D. Preparation of monolayer pig kidney cell cultures by a modified continuous flow trypsinization procedure. Med. Lab. Techno!. 29: 191-194, 1972.
11.
Nuttall, P. A., Luther, P. D., Stott, E. J. Viral contamination of bovine foetal serum and cell cultures. Nature (Lond.) 266:835-837,1977.
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
© 1978 by The University of Chicago. 0022-1899/78/3805-0013$00.75
Isolation of Viruses from Cultures of Bovine Endometrial Cells P. D. Luther, P. A. Nuttall,* and R. A. Gibbons
From the Agricultural Research Council, Institute for Research on Animal Diseases,Compton, Newbury, Berkshire, United Kingdom
During investigations into the culture of endometrial cells from pregnant and nonpregnant cattle, viruses were isolated from a high proportion of' the endometrial samples. The viruses were mucosal disease virus (MDV) and bovine syncytial virus (BSV). MDV (also known as bovine viral diarrhea virus), a member of the family Togaviridae, is antigenically related to hog cholera virus [1] and morphologically similar to rubella virus [2]. BSV is a syncytium-forming "foamy" virus of the family Retroviridae [3]. Both MDV and BSV have been isolated from bovine placentas, fetal and maternal tissues, and fetal blood [4-7]. In addition, MDV has been shown to cause abortions and fetal malformations [8] and has been incriminated in bovine infertility [9]. Materials and Methods
Endometria from 19 cattle were processed to produce cell cultures; the various cell batches are listed chronologically in tables I and 2. The Received for publication January 6, 1978, and in revised form May 15, 1978. P. A. Nuttall wassupported by the Animal Health Trust. We thank E. J. Thompson, Ms. V. J. Collier, and Ms. V. R. Robinson for their assistance. The bovine syncytial virus antiserum was supplied by Dr. M. J. Van Der Maaten, National Animal Disease Laboratory, Agricultural Research Service,U.S.Department of Agriculture, Ames,Iowa. Please address requests for reprints to P. D. Luther, Institute for Research on Animal Diseases, Compton, Newbury, Berkshire, RG16 ONN, United Kingdom. • Present address: Edward Grey Institute, Zoology Department, South Parks Road, Oxford, England.
horns of the uteri were dissected longitudinally to reveal the endometrium. An area of ,..,40 mmof endometrial tissue was removed from each horn, chopped into small pieces, washed in phosphate-buffered saline, and trypsinized for I hr by the continuous flow technique [10]. The cells were suspended in Eagle's minimal essential medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 0.11 % NaRC0 3 , and penicillin, streptomycin, and mycostatin to give final concentrations of 100 units, 100 jLg, and 25 units/em", respectively. Medical flat bottles (140 em") were seeded with ,..,6 X 106 cells and incubated at 37 C until cultures were at least 75% confluent, when they were subcultured. Coverslip cultures were set up for staining with fluorescent antibody and with Giemsa. Cells were stained by the indirect fluorescent antibody technique using antiserum to the NADL (National Animal Diseases Laboratory, Ames, Iowa) strain of MDV from a gnotobiotic calf, and a rabbit antiserum to bovine antigen conjugated with fluorescein (Nordic Immunology, Tilburg, The Netherlands). Serum taken before inoculation was used as a control for fluorescence. A similar technique was used with an antiserum to BSV. Cell culture batches were screened for the presence of MDV at the secondary and subsequent passage levels. Cells were stained for the presence of BSV when syncytia were observed. Results
Success in establishing cell culture growth varied markedly from batch to batch. Three batches,
660
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
Endometrial cells from the uteri of pregnant and nonpregnant cattle were cultured. The presence of one or both of two viruses, noncytopathic mucosal disease virus and bovine syncytial virus, was demonstrated in seven of 19 endometria investigated. It was necessary to subculture the cells an average of four times to detect the viral infections. Difficulties were encountered in producing endometrial cell cultures from cows at term or near the end of term and also from older animals. The infections detected may be significant because both of the viruses isolated are capable of infecting the bovine fetus in utero and mucosal disease virus has been associated with bovine fetal diseases.
661
Viral Isolates from Bovine Endometria
Table I. Bovine endometrial cell cultures from which mucosal disease virus (MDV) and/or bovine syncytial virus (BSV) were isolated. Age of animal (years)
Calving/ servicing ratio *
1 4 5 7 8
5.5 5.5 3.5 2.25 2.5
2/4 3/3 1/6 0/2 0/9
15-day blastocyst Not pregnant Not pregnant Not pregnant 14-day blastocyst
10 16
4.5 5
2/3 2/11
16-day blastocyst Not pregnant
Cell batch no.
Stage of pregnancy
Virus demonstrated
Cell passage no. when virus was demonstrated
BSV MDV MDV MDV MDV BSV MDV MDV
4 3 4 2 4 5 5 4
"Number of calves per number of services.
enlarged vacuolated cells. Similar vacuolated cells were observed in all of the batches that were infected with MDV. When freeze-thawed extracts of the MDV-infected cell batches were inoculated into calf testis cell cultures, the calf testis cells showed fluorescence specific for MDV. However, when calf testis cell cultures were inoculated with MDV-infected or uninfected endometrial cell batches that showed vacuolation, the calf testis cells did not become vacuolated. Discussion
The response of bovine endometrial cells to subculturing in vitro appeared to reflect the state of the animal when the endometrial tissue was removed. Batches no. 2, 9, 11, and 13, which all showed poor cell growth, were from dams at full term or near the end of term. Two batches, no.
Table 2. Bovine endometrial cell cultures from which no viruses were demonstrated. Cell batch no. 2 3 6 9 11 12 13 14 15 17 18 19
Age of animal (years)
Calving/ servicing ratio"
6.5 4.5 6.5 2.5 2.5
5/5 3/3 4/6 1/1 1/1 9/17 10/13
14
13 5 days 6 4 11 3.25
4/7 1/1 8/25 1/2
"Number of calves per number of services.
Stage of pregnancy Near term Not pregnant, progesterone-treated 15-day blastocyst Term Term 15-day blastocyst Term Not pregnant Not pregnant Not pregnant 6-day blastocyst
Limit of culture growth (cell passage no.] 2 2 >17 Primary only Primary only 2 2 6 2 3 Primary .only 4
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
no. 9, 11, and 18 (table 2), showed little, if any, primary growth; six batches, no. 2, 3, 7, 12, 13, and 15 (tables 1 and 2), were capable of being subcultured only once. In contrast, batch no. 6 (table 2) has so far been subcultured 18 times. Adventitious viral infections were demonstrated in seven of the 19 batches of endometrial cells (table 1); five batches were infected with MDV alone, one batch with BSV alone, and one batch with both MDV and BSV. In one instance infection was detected in cells in the first subculture, but with most batches four passages were necessary before viral infection was demonstrated. Several of the cell batches showed a CPE that was not necessarily correlated with infection with either MDV or BSV. Cultures of batches no. 14, 15, and 19 (table 2), which did not demonstrate fluorescence for MDV or BSV, contained
Luther, Nuttall, and Gibbons
662
servations) [II]. These findings suggest that the venereal route of transmission and infection of the endometrium is a possibility. The presence of MDV and BSV in the endometria of cattle also raises the question regarding the effect of these viruses on pregnancy. In the group of animals with infected endometria, the mean conception rate was significantly lower than that in the uninfected group. However, it would be premature to state at this stage that the relationship was more than a speculative one. The number of animals involved in this preliminary investigation was small, but the data obtained reveal that infection of the bovine endometrium by MDV or BSV occurs relatively frequently in a group of culled cows from this institute's dairy herds. Extension of these studies to a larger group of animals is needed in an attempt to determine the significance of viral infections of the endometrium in relation to infertility and abortion. References 1. Darbyshire, J. H. A serological relationship between swine fever and mucosal disease of cattle. Vet. Rec. 72:331,1960. 2. Stott, E. J., Almeida, J. D., O'Reilly, K. J. Characterization of mucosal disease virus as a togavirus by electron microscopy. Microbios llA:79-83, 1974. 3. Fenner, F. The classification and nomenclature of viruses. Summary of results of meetings of the International Committee on Taxonomy of Viruses in Madrid, September 1975. Intervirology 6: 1-12,1975/6. 4. Hubbert, W. T., Bryner, J. H., Fernelius, A. L., Frank, G. H., Estes, P. C. Viral infection of the bovine fetus and its environment. Arch. Gesamte Virusforsch. 41:86-98,1973. 5. Scott, F. W., Kahrs, R. F., Parsonson, I. M. A cytopathogenic strain of bovine viral diarrhea-mucosal disease virus isolated from a bovine fetus. Cornell Vet. 62:74-84,1972. 6. Scott, F. W., Shively, J. N., Gaskin, J., Gillespie, J. H. Bovine syncytial virus isolations. Arch. Gesamte Virusforsch. 43:43-52,1973. 7. Van Der Maaten, M. J., Hubbert, W. T., Boothe, A. D., Bryner, J. H., Estes, P. C. Isolations of bovine syncytial virus from maternal and fetal blood. Am. J. Vet. Res. 34:341-343, 1973. 8. Kendrick, J. W. Bovine viral diarrhea-mucosal disease virus infection in pregnant cows. Am. J. Vet. Res. 32: 533-544, 1971. 9. Archbald, L. F., Zemjanis, R. Intrauterine infusion of the virus of bovine virus diarrhea and artificial insemination in the cow at estrus. Vet. Med. Small Anim. Clin. 72:221-225, 1977.
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
12 and 18, which also showed poor cell growth, were from older dams declared barren; batch no. 3 came from an animal which had received a course of progesterone treatment. The one batch of endometrial cells that grew well (batch no. 6) and showed no evidence of viral infection was from a dam in the very early stages of pregnancy. Thus the stage of pregnancy and the age of the dam may account for the poor cell growth in several batches of endometrial cell culture. Infection with MDV and/or BSV was demonstrated in seven of the 19 batches of endometrial cells investigated. However, because viral infection was not usually detected until the cells had been subcultured several times, infection of the batches that had poor cell growth cannot be discounted. Vacuolation of cells appeared in both MDVinfected· and uninfected batches of endometrial cell cultures. Therefore, the vacuolation was considered unlikely to be due to infection with MDV, and the type of MDV infecting the endometrial cells was considered to be noncytopathic. Because attempts to transmit a vacuolating agent to calf testis cell cultures were unsuccessful, it was concluded that the cause of vacuolation was not transmissible. The isolation of viruses from endometrial cells raises the question concerning the source of infection. In vitro infection with MDV that was possibly present in the supplementary growth factor FCS [II] can be discounted because all cell batches were cultured in the presence of heatinactivated FCS. Heating of MDV at 56 C for 30 min has been shown to inactivate the virus. Also, the materials used for the processing and growth of the endometrial cell cultures, especially trypsin and FCS, can be eliminated as sources of viral contamination. Twelve batches of calf testis cell cultures, which are sensitive to and would have demonstrated the presence of MDV and BSV, were produced and passaged an average of 12 times with materials identical to those used for the endometrial cell cultures. There was no evidence of the presence of any adventitious viruses in the calf testis cell cultures, although the methods of examination for these cultures were the same as those for the endometrial cultures. MDV and BSV have been isolated from calf testis cells (authors' unpublished ob-
663
Viral Isolates from Bovine Endometria
10. Luther, P. D. Preparation of monolayer pig kidney cell cultures by a modified continuous flow trypsinization procedure. Med. Lab. Techno!. 29: 191-194, 1972.
11.
Nuttall, P. A., Luther, P. D., Stott, E. J. Viral contamination of bovine foetal serum and cell cultures. Nature (Lond.) 266:835-837,1977.
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
