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Trypanosoma theileri as a contaminant of tissue origin in cultures of fetal bovine kidney cells in vitro
LETTERS
394
TO
THE
EDITORS
States Department of Health, Education, and Welfare Public Health Service National Institutes of Healfh
F. It. ARIN~NTI R. J. HUEBNER
Ihited
National
Institute
of Allergy
and Infectious Diseases Laborator,y of Infectious Diseases Bethesda 14, Maryland EZeceived .2pril 23, 1959 Trypanosoma
theileri
as a Contaminant
of Fetal
Bovine
Kidney
of Tissue Origin Cells
in Cultures
in Vitro
Microorganisms representing essentially t,he entire microbiological spect,rum have been, on occasions, recovered as contaminants from cultures of various tissues in vitro. The larger forms (molds, yeasts, and bacteria) are undoubtedly of extraneous origin. The pleuropneumonia-like organisms are doubtlessly also of extraneous origin, but may be carried in serial culture as inapparent contaminants unless the appropriate cult.ural methods are used t,o detect their presence (1). In the case of viruses, however. it appears that, these are of tissue origin in many instances (2). Since no report,s could be found indicating that protozoa, either as extraneous or tissue contaminants, have ever been isolated in tissue culture, it was ronsidered of interest. t.o report the recovery of a trypanosome from a primary culture of fetal bovine kidney cells. In view of this finding, these agent,s must, be regarded tissues of certain mammalian as potential contaminants of in vitro cult.ivated species. We routinely obtain fetal t,issues of catt.le and sheep from a local slaughtjer plant for the preparation of Cssue cultures. The gravid uterus is removed by personnel on the killing floor, while the individual we dispat,ch to obtain t.he tissues thoroughly hoses down the uterus with hot water before opening it and removing the fetus. The abdominal cavity of the fetus is then opened carefully and the required organs are removed with sterile instruments and taken to the laboratory where the cultures are prepared. We have followed essentially this procedure for the past several years and have experienced lit,tle or no trouble from coincident,al contamination. Recently, a primary monolayer culture of fetal bovine kidney cells in Roux according to the method of Madin flasks prepared, with slight modifications, et al. (S), was observed to be cloudy at the time the first fluid change was scheduled to be made (the sixth day after preparation of the culture), and the cells were found to he almost entirely sloughed off the glass. Low-power examination of a wet mount of the culture revealed the presence of large, motile bodies somewhat resembling vibrios. However, a stained preparation indicated that the forms were mature t,rypanosomes. This finding was confirmed by more critical morphologic studies. It was found that the trypanosome could multiply, in the absence of cells, in
LETTERS
TO THE
EDITORS
395
tissue culture medium containing 10% by volume of lamb serum and the usual concentration of penicillin and streptomycin, although multiplication appeared to be more rapid when cells were present. It thrived also in fetal bovine kidne) cultures of the infectious bovine rhinotracheitis virus in which qtolysis eventually became complete. The presence of the trypanosome appearecl to have no effect on the virus. Guinea pigs and mice, which were inoculated with the trgpanosome, remained clinically normal, and trypanosomes were not observed in either the peritoneal fluid or in the organs of these animals several weeks after inoculation. Unfortunately, the original culture contained a mold and, in attempting to rid it of this contaminant bp differential centrifugation and by treat ment wit,h mycostatin, the trypanosome was lost before fmther st,udies could be carried out. Although positive identification of the trypanosome report,ed herein was not made, the fact that it was obtained from cattle and shown to be innocuous to guinea pigs and mice indicated that it was undoubtedly T~ypanosorna theileri-a non-pathogenic-protozoan (4) which is of wide distribution in catt,le. There is ample evidence that intrauterine t,ransmission of trypanosomes occurs in man (5) and in both domest,ic and experimental animals (6-9). In view of this, and the fact t,hat t,here was little or no opportunit,y for the fet,al organs to have become cont,aminated with mat,ernal blood, it was concluded that the trypanosome reported herein must have been present in the kidney at the time it was removed from the fetus. Regardless of the identity of the trppanosome and the manner in which it gained access to the fetal tissues, the unusual feature is the fact that, despite the washing and trypsinization procedures to which tissues are subjected in the preparat,ion of in vitro cultures, these organisms can apparently persist, in sufficient niimbers t,o become established as culture contaminants. 11 is doubtful whether trypanosomal rontaminat,ion of tissue cultures will be of more than passing academic interest in this country, other t,han possibly in connection wit.h bovine tissues. In tropical and subtropical areas of the world, however, where protozoan infections of t,he domestic species of animals are quit,e common, it is possible that contamination with this type of agent might prove to be troublesome. REFERENCES 1. ROBINSON, L. B., WICHELHAUSEN, R. H., and ROIZMAN, B., Scierlce 134, 11471148 (1956). 2. HULL, R. N., MINNER, J. R., and SMITH, J. W., iln2. J. Hyg. 63, 204-215 (1956). 3. MADIN, S. H., ANDRIESE, I’. C., and DARBY, N. B., .47rz. J. Vet. Research 18, 932-941 (1957). 4. WENYON, C. M., Protozoology: A Man/La1 for Medical Men, Veterinarians and Zoologists, Vol. 1, p. 778; Vol. 2, p. 779. William Wood, New York, 1926. 6. MUHLENS, P., Arch. Schifls-u. l’ropen Hyg. 33, 181 (1929). 6. PIGNEUR, G., Ann. sot. belge med. trop. 12, 545 (1932). 7. DIKMANS, G., MANTHEI, C. A., and FRANK, A. H., Cornell Vef. 47, 344-353 (1957).
396
LETTERS
TO
THE
EDITORS
F. C., and MANSJOER, M., Hemera Zoa 61, 97 (1954). 8. KRANEVELD, 9. ROBIN, C., and JOSPIN,M., Bd. sot. puthol. Ezotique 30,372 (1937). School of Veterinary Medicine University of California Davis, California Received May 5, 2959 Plaque
Production
BEVERLY
L). LG~HOL~~ JOHANNES STORZ D.G.MCJXERCHER
by the Polyoma
Virus’
Eddy ef al. (1) have shown that the polyoma virus destroys mouse embryo cells cultivated in vitro. We have found that this characterist,ic of t,he virus could be utilized for its assay by production of plaques. Recently, Sachs et al. (2) have reported a similar development,. The cells used were monolayers of either primary or secondary mouse embryo cultures. The embryos were collected shortly before birth a.nd trypsinized, following removal of the heads. Trypsinization was according to the technique commonly employed for monkey kidney cells (8). The resulting cells were suspended in modified Eagle’s medium.2 Of t,his suspension, 5 ml, containing 3 X 106 cells, cultures were usually well was seeded in each 64.mm pet.ri dish. Primary developed, having usable confluent monolayers after 2-4 days’ incubation at, 37”. Because these cultures sometimes contained tissue lumps which interfered with plaque production, they were usually trypsinized and three secondary culture plates were made from two primary plates. These formed confluent, sheets of cells and were usable after l-3 days. Optimal conditions for assaying the virus proved to be as follows. The monolayers are washed t,wice with 5 ml of tris-buffered saline (pH 7.4). Then 0.1 ml of the virus suspension is added dropwise onto the center of the cell layer and t,he plat,e is incubated for 30 minutes at 37”. Subsequently, it, is overlaid with 10 ml medium containing O.9o/0 agar in modified Eagle’s medium with the addition of 2% horse serum. The plates are then incubated at 37” in a well-humidified incubator having a CO2 atmosphere appropriate for maintaining the pH of t,he culture medium between 7 and 7.4. The plaques start to appear after about 10 days of incubation. To make them recognizable, on the tenth day a volume of 3 ml of a solution of equal parts of a 1OP (w/v) solution of neut,ral red in water and twofold concentrated Eagle’s is added. The neutral red solution is placed over the agar-containing medium and left, there overnight. When the liquid is removed, the plaques stand out, as pale areas over the red background. The number of visible plaques continues to increase for 4-5 days and then becomes stable. The size of the plaques is about 1 mm in diameter at 10 days, and increases to about 3-4 mm. That the plaques are produced by the polyoma virus was proved by showing that plaque formation is very strongly inhibit,ed by antipolyoma mouse serum ’ Aided by grants from the American Cancer Society States Public Health Service. 2 Fourfold concentration of amino acids and vitamins.
and from
the Iinitetl
394
TO
THE
EDITORS
States Department of Health, Education, and Welfare Public Health Service National Institutes of Healfh
F. It. ARIN~NTI R. J. HUEBNER
Ihited
National
Institute
of Allergy
and Infectious Diseases Laborator,y of Infectious Diseases Bethesda 14, Maryland EZeceived .2pril 23, 1959 Trypanosoma
theileri
as a Contaminant
of Fetal
Bovine
Kidney
of Tissue Origin Cells
in Cultures
in Vitro
Microorganisms representing essentially t,he entire microbiological spect,rum have been, on occasions, recovered as contaminants from cultures of various tissues in vitro. The larger forms (molds, yeasts, and bacteria) are undoubtedly of extraneous origin. The pleuropneumonia-like organisms are doubtlessly also of extraneous origin, but may be carried in serial culture as inapparent contaminants unless the appropriate cult.ural methods are used t,o detect their presence (1). In the case of viruses, however. it appears that, these are of tissue origin in many instances (2). Since no report,s could be found indicating that protozoa, either as extraneous or tissue contaminants, have ever been isolated in tissue culture, it was ronsidered of interest. t.o report the recovery of a trypanosome from a primary culture of fetal bovine kidney cells. In view of this finding, these agent,s must, be regarded tissues of certain mammalian as potential contaminants of in vitro cult.ivated species. We routinely obtain fetal t,issues of catt.le and sheep from a local slaughtjer plant for the preparation of Cssue cultures. The gravid uterus is removed by personnel on the killing floor, while the individual we dispat,ch to obtain t.he tissues thoroughly hoses down the uterus with hot water before opening it and removing the fetus. The abdominal cavity of the fetus is then opened carefully and the required organs are removed with sterile instruments and taken to the laboratory where the cultures are prepared. We have followed essentially this procedure for the past several years and have experienced lit,tle or no trouble from coincident,al contamination. Recently, a primary monolayer culture of fetal bovine kidney cells in Roux according to the method of Madin flasks prepared, with slight modifications, et al. (S), was observed to be cloudy at the time the first fluid change was scheduled to be made (the sixth day after preparation of the culture), and the cells were found to he almost entirely sloughed off the glass. Low-power examination of a wet mount of the culture revealed the presence of large, motile bodies somewhat resembling vibrios. However, a stained preparation indicated that the forms were mature t,rypanosomes. This finding was confirmed by more critical morphologic studies. It was found that the trypanosome could multiply, in the absence of cells, in
LETTERS
TO THE
EDITORS
395
tissue culture medium containing 10% by volume of lamb serum and the usual concentration of penicillin and streptomycin, although multiplication appeared to be more rapid when cells were present. It thrived also in fetal bovine kidne) cultures of the infectious bovine rhinotracheitis virus in which qtolysis eventually became complete. The presence of the trypanosome appearecl to have no effect on the virus. Guinea pigs and mice, which were inoculated with the trgpanosome, remained clinically normal, and trypanosomes were not observed in either the peritoneal fluid or in the organs of these animals several weeks after inoculation. Unfortunately, the original culture contained a mold and, in attempting to rid it of this contaminant bp differential centrifugation and by treat ment wit,h mycostatin, the trypanosome was lost before fmther st,udies could be carried out. Although positive identification of the trypanosome report,ed herein was not made, the fact that it was obtained from cattle and shown to be innocuous to guinea pigs and mice indicated that it was undoubtedly T~ypanosorna theileri-a non-pathogenic-protozoan (4) which is of wide distribution in catt,le. There is ample evidence that intrauterine t,ransmission of trypanosomes occurs in man (5) and in both domest,ic and experimental animals (6-9). In view of this, and the fact t,hat t,here was little or no opportunit,y for the fet,al organs to have become cont,aminated with mat,ernal blood, it was concluded that the trypanosome reported herein must have been present in the kidney at the time it was removed from the fetus. Regardless of the identity of the trppanosome and the manner in which it gained access to the fetal tissues, the unusual feature is the fact that, despite the washing and trypsinization procedures to which tissues are subjected in the preparat,ion of in vitro cultures, these organisms can apparently persist, in sufficient niimbers t,o become established as culture contaminants. 11 is doubtful whether trypanosomal rontaminat,ion of tissue cultures will be of more than passing academic interest in this country, other t,han possibly in connection wit.h bovine tissues. In tropical and subtropical areas of the world, however, where protozoan infections of t,he domestic species of animals are quit,e common, it is possible that contamination with this type of agent might prove to be troublesome. REFERENCES 1. ROBINSON, L. B., WICHELHAUSEN, R. H., and ROIZMAN, B., Scierlce 134, 11471148 (1956). 2. HULL, R. N., MINNER, J. R., and SMITH, J. W., iln2. J. Hyg. 63, 204-215 (1956). 3. MADIN, S. H., ANDRIESE, I’. C., and DARBY, N. B., .47rz. J. Vet. Research 18, 932-941 (1957). 4. WENYON, C. M., Protozoology: A Man/La1 for Medical Men, Veterinarians and Zoologists, Vol. 1, p. 778; Vol. 2, p. 779. William Wood, New York, 1926. 6. MUHLENS, P., Arch. Schifls-u. l’ropen Hyg. 33, 181 (1929). 6. PIGNEUR, G., Ann. sot. belge med. trop. 12, 545 (1932). 7. DIKMANS, G., MANTHEI, C. A., and FRANK, A. H., Cornell Vef. 47, 344-353 (1957).
396
LETTERS
TO
THE
EDITORS
F. C., and MANSJOER, M., Hemera Zoa 61, 97 (1954). 8. KRANEVELD, 9. ROBIN, C., and JOSPIN,M., Bd. sot. puthol. Ezotique 30,372 (1937). School of Veterinary Medicine University of California Davis, California Received May 5, 2959 Plaque
Production
BEVERLY
L). LG~HOL~~ JOHANNES STORZ D.G.MCJXERCHER
by the Polyoma
Virus’
Eddy ef al. (1) have shown that the polyoma virus destroys mouse embryo cells cultivated in vitro. We have found that this characterist,ic of t,he virus could be utilized for its assay by production of plaques. Recently, Sachs et al. (2) have reported a similar development,. The cells used were monolayers of either primary or secondary mouse embryo cultures. The embryos were collected shortly before birth a.nd trypsinized, following removal of the heads. Trypsinization was according to the technique commonly employed for monkey kidney cells (8). The resulting cells were suspended in modified Eagle’s medium.2 Of t,his suspension, 5 ml, containing 3 X 106 cells, cultures were usually well was seeded in each 64.mm pet.ri dish. Primary developed, having usable confluent monolayers after 2-4 days’ incubation at, 37”. Because these cultures sometimes contained tissue lumps which interfered with plaque production, they were usually trypsinized and three secondary culture plates were made from two primary plates. These formed confluent, sheets of cells and were usable after l-3 days. Optimal conditions for assaying the virus proved to be as follows. The monolayers are washed t,wice with 5 ml of tris-buffered saline (pH 7.4). Then 0.1 ml of the virus suspension is added dropwise onto the center of the cell layer and t,he plat,e is incubated for 30 minutes at 37”. Subsequently, it, is overlaid with 10 ml medium containing O.9o/0 agar in modified Eagle’s medium with the addition of 2% horse serum. The plates are then incubated at 37” in a well-humidified incubator having a CO2 atmosphere appropriate for maintaining the pH of t,he culture medium between 7 and 7.4. The plaques start to appear after about 10 days of incubation. To make them recognizable, on the tenth day a volume of 3 ml of a solution of equal parts of a 1OP (w/v) solution of neut,ral red in water and twofold concentrated Eagle’s is added. The neutral red solution is placed over the agar-containing medium and left, there overnight. When the liquid is removed, the plaques stand out, as pale areas over the red background. The number of visible plaques continues to increase for 4-5 days and then becomes stable. The size of the plaques is about 1 mm in diameter at 10 days, and increases to about 3-4 mm. That the plaques are produced by the polyoma virus was proved by showing that plaque formation is very strongly inhibit,ed by antipolyoma mouse serum ’ Aided by grants from the American Cancer Society States Public Health Service. 2 Fourfold concentration of amino acids and vitamins.
and from
the Iinitetl