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Effect of Heat on Bovine Leukosis Virus-Infected Lymphocytes
Br. uet. J. (1983). 139,291 ·
EFFECT OF HEAT ON BOVINE LEUKOSIS VIRUS-INFECTED LYMPHOCYTES
BY D. H.
ROBERTS,
M. H.
LUCAS AND
G.
WIBBERLEY
Central Veterinary Laboratory, New Haw, Weybridge, Surrey
SUMMARY
The high-temperature short-time batch procedure which simulates the commercial pasteurization process, completely inactivated bovine leukosis virus (BLV)-infected lymphocytes in milk. Heating BLV -infected lymphocytes at 50°C for 70 sec completely destroyed BLV infectivity. Destruction of BLV infectivity coincided with the loss oflymphocyte viability as indicated by the lack of response to Concanavalin A stimulation. INTRODUCTION
Bovine leukosis virus (BLV) is widely recognized as the aetiological agent of enzootic bovine leukosis. Cattle and sheep infected with BLV develop antibodies and the agar gel immunodiffusion test has been widely used in this respect (Miller & Van der Maaten, 1977, 1979). It has been established that BLV infectivity may be present in both colostrum and milk from BL V -infected cows and that neonatal calves can be infected with BLV by the oral route (Miller & Vander Maaten, 1979; Vander Maaten & Miller, 1979). BLV can be isolated from the lymphocytes of infected cattle but it is very difficult to demonstrate its presence in other tissues (Miller & Vander Maaten, 1979; Ferrer, 1979, 1980; Straub, 1982). Consequently it is assumed that transmission results from the transfer of infected cells rather than free virus. Baumgartener, Olson & Onuma (1976) have shown that BLV mixed with milk and subjected to a simulated high-temperature short-time (HTST) pasteurization procedure was inactivated. They also showed that heating BL V at 60°C for 1I 2 min destroyed the virus. It seemed desirable to add BLV -infected lymphocytes to milk and to test the effect of heat by using a simple laboratory-scale HTST milk pasteurizer. In addition the effect ofheat on viable BLV-infected lymphocytes was also studied using the criteria of mitogenic stimulation and in-vivo transmission. MATERIALS AND METHODS
Animals Four cows (three to five years old) infected with BLV were used. BLV was
292
BRITISH VETERINARY JOURNAL, 139,4
consistently isolated from the peripheral blood leukocytes of these animals. Cows (three years old) and sheep susceptible to BL V were obtained from the BL V -free stock at this laboratory; all were negative for BLV antibodies.
Separation of peripheral blood lymphocytes The mononuclear fraction of peripheral blood leukocytes was harvested using the technique described by Roberts et al. ( 1982). The lymphocytes were suspended in Eagle's MEM, counted using a haemacytometer and Giemsa staining, and sampled for viability using the trypan blue dye exclusion test. The percentage of lymphocytes estimated in these preparations was about 90%. Serology and virus isolation The demonstration of persisting antibody responses as determined by the agar gel immunodiffusion (AGID) test was used to identify animals infected with BLV. In the AGID test, leukassay-B reagents (Pitman-Moore Inc., Washington Crossing, USA) were used; the technique and interpretation of the results were in accordance with the European Communities criteria (Council Directive, 11 November 1980, 80/1102/ EEC). BLV antigen was isolated and demonstrated by indirect fluorescence using the cocultivation method (Roberts et al., 1982). Mononuclear cells prepared from peripheral blood were cocultivated with foetal bovine lung cells. All fluorescent antibody tests included the EEC reference EBL virus antiserum E 1 (Council Directive, 11 November 1980, 81/1102/EEC) and fluorescein labelled anti-bovine immunoglobulin serum prepared in rabbits (Nordic Immunological Laboratories). Lymphocyte stimulation Cultures containing 5 X 106 leukocytes (90% lymphocytes) were added to 4 ml Eagle's MEM containing 20% foetal calf serum in bijou bottles. The cultures were inoculated in triplicate with 5 p,g Concanavalin A (Sigma Chemical Co.). Cultures without stimulant served as controls. The bottles were tightly capped and incubated at 37°C for three days. Transformation and proliferation of lymphocytes stimulated by Concanavalin A (Con A) were measured by labelling DNA with tritiated thymidine (Radiochemical Centre, Amersham, England). Twenty-four hours before collection, 5 p,Ci of tritiated thymidine (methyl-T specific activity 22 Ci/mmol) were added to each culture. The cells were sedimented at 1000 g for 5 min, washed with physiological saline, and centrifuged; 5% trichloroacetic acid was added to the cell pellet. The precipitate was washed with 95% ethanol and then dissolved in 1·0 ml 2 N solution of methanolic KOH at 60°C for 30 min. A 0·4 ml amount of this sample was added to 10 ml Luma gel (Lumac Systems AG) scintillation mixture and counted in a scintillation counter. The uptake of thymidine was expressed as the mean value of triplicate determinations as counts per minute per culture. High-temperature short-time {HTST) pasteurization simulator The HTST batch procedure simulated a commercial pasteurization process (Franklin, 1965). The apparatus consisted of two parallel stainless steel plates enclosing a thin film of milk; it was kept at the test temperature in a large water bath.
EFFECT OF HEAT ON BOVINE LEUKOSIS VIRUS
293
The temperature of the water was controlled at 1°C higher than the required milk temperature treatment. Two hundred and fifty-ml quantities were poured into the unit which was allowed to remain in the water at 72·5°C for 70 sec after which it was immediately transferred to an adjacent cold water bath for cooling. Pooled milk was obtained from serologically-negative lactating cows and BLV -infected lymphocytes were added.
Experimental design l. Five hundred ml pooled milk was obtained from BLV -free cows. BLV -infected lymphocytes were prepared from a BLV-positive donor cow (no. 1) and 10 7 cells (viability 97%) added to the milk. Two hundred and fifty ml of this milk mixture were heat treated using the HTST pasteurization simulator. The susceptible sheep were allotted to two groups. Each animal was inoculated intra-peritoneally with SO ml milk containing 2 X 106 lymphocytes. One group of five received pasteurized milk and the other group of five received non-pasteurized milk. 2. BLV -infected lymphocytes from three donor cows (nos 1, 2 and 3) were prepared. The lymphocytes were suspended in Eagle's MEM containing 20% foetal calf serum . Using a water bath, 4-ml samples of the BLV -infected lymphocytes were heated in bijou bottles to 40°, 45°, 50°, 55°, 60° and 65°C for 70 sec. Heat measurements were made by suspending a thermometer in 4 ml culture medium in an open bijou bottle. After 70 sec at the required temperature the heated bijou bottles were transferred to a cold water bath. Bottles containing BLV -infected lymphocytes were kept at room temperature and used as positive controls. Each sample was then inoculated subcutaneously into two sheep. Each sheep received 6 X 105 1ymphocytes. A total of 42 sheep was inoculated. 3. BLV-infected lymphocytes from four donor cows (nos 1, 2, 3 and 4) and uninfected lymphocytes from two susceptible cows (nos 5 and 6) were prepared (viability 95 to 100% ). The lymphocytes were suspended in Eagle's MEM with 20% foetal calf serum and heated as above at 45°, 50°, 55°, 60° and 65°C. Unheated samples were kept at room temperature. The heated and unheated lymphocyte suspensions were then tested for mitogenic stimulation using Con A. The inoculated sheep were bled before inoculation and at monthly intervals for 24 weeks. Clotted blood samples were collected using vacutainers. The serum was tested for BLV antibodies using the AGID test. At 24 weeks, heparinized blood samples were collected and attempts were made to isolate BLV using the cocultivation technique (Roberts et al., 1982). The sheep were kept in isolation for 24 weeks. RESULTS
None of the sheep inoculated with pasteurized milk developed antibodies. The five sheep inoculated with non-pasteurized milk developed BLV antibodies at four weeks post inoculation . BLV was isolated from all seropositive sheep. The survival of BLV in infected lymphocytes following heat treatment at various temperatures as judged by the AGID test results in the inoculated sheep are given in Table I. BLV antibodies were detected in the sheep between four and eight weeks after inoculation and BLV was isolated from all seropositive sheep. Heating the BLV-
BRITISH VETERINARY JOURNAL, 139,4
294
infected lymphocytes at 50°C and above for 70 sec destroyed BLV. The effect of heat on BLV-infected and uninfected lymphocytes and their response to Con A is shown in Table II . Normal and BLV-infected lymphocytes heated to 50°C and above for 70 sec did not respond to Con A stimulation. The mean counts/min for temperatures above 50°C were similar to those for 50°C and consequently are not given in the Table. The stimulatory effect of Con A was significantly decreased in BL Vinfected lymphocytes compared with normal lymphocytes . Heating BLV-infected lymphocytes for 70 sec at 45°C before treating with Con A appeared to increase slightly the uptake of tritiated thymidine in BLV -infected lymphocytes. TABLE I SURVIVAL OF BLV IN INFECTED LYMPHOCYTES FROM THREE COWS (NOS I, 2 AND 3) FOLLOWING HEAT TREATMENT
Temperature treatment for 70 sec
Recipient sheep nos
20°C 40°C 45°C 50°C 55°C 60°C 65°C
7-12 13-18 19-24 25-30 31-36 37-42
ACID test
+ + +
1- 6
TABLE II EFFECT OF HEAT ON BLV-INFECTED AND UNINFECTED LYMPHOCYTES AND THEIR RESPONSE TO CONCANAVALIN A STIMULATION
Mean counts/minute Temperature treatment for 70sec
Stimulant 1
20°C 20°C 45°C 50°C
None Con A Con A Con A
Unirzfected cows
BL V-infected cows
1555 7193 14 104 633
2
3
4
231 1860 2316 76
353 5640 6867 151
372 3885 4234 66
5
1287 90 886 68 119 658
6
340 36 536 2486 57
DISCUSSION
The results demonstrate that the HTST pasteurization simu lator process destroyed BL V infectivity in infected lymphocytes when milk was heated at 50°C for 70 sec.
EFFECT OF HEAT ON BOVINE LEUKOSIS VIRUS
295
Baumgartener et al. ( 1976), using cell-free BL V, showed that heating at 56°C for I min did not destroy the virus whereas heating at 60° did. In this study, destruction ofBLV infectivity coincided with loss of lymphocyte viability as indicated by the lack of response to Con A stimulation. The difference in the results might indicate that the release of infectious virus particles from BLV-infected lymphocytes is dependent on viable cells. This study confirms the conclusion ofWeiland & Straub (1976) that the stimulatory effect of Con A was significantly decreased in BLV -infected lymphocytes in comparison with normal lymphocytes. ACKNOWLEDGEMENTS
The technical assistance of Miss S. Bushnell and Mrs acknowledged.
J.
Weedon is gratefully
REFERENCES BAUMCARTENER, L., OLSON, C . & 0NUMA, M . ( 1976) . joumal
of the American Veterinary Medical Association 169, 1189. FERRER,j. F . (1979) . Joumal of the American Veterinary Medical Associationl75, 1281. FERRER, J. F. ( 1980) . Advances in Veterinary Science and Comparative Medicine 24, I. FRANKI.IN,j. G. (1965).Joumal of Dairy Research 32, 281. MILLER,j. M. & VANDER MAATEN, M . j. {1977). Europeanjoumal of Cancer 13, 1369. MILLER,j. M . & VANDER MAATEN, M.j . (1979) . Joumal of the National Cancer l11Stitute 62,425. ROBERTS, D . H., LUCAS, M. H ., WIBBERLEY, G. & CHASEY, D. (1982). Veterinary Record 110,222. STRAUB, 0. C. ( 1982). Fourth International Symposium on Bovine Leukosis, Bologna, ed. 0 . C. Straub, p. 299. London: Martinus Nijoff. VANDER MAATEN, M. J. & MILLER, J. M. ( 1979). In Advances in Comparative Leukemia Research, ed. P. Bentvelzen, J. Hilgers and D. Yohn, p. 29. Amsterdam: Elsevier, North Holland Biomedical Press . WEILAND, F. & STRAUB, 0. C. (1976). Research in Veterinary Science 20, 340. (Accepted for publication 20 July !982)
EFFECT OF HEAT ON BOVINE LEUKOSIS VIRUS-INFECTED LYMPHOCYTES
BY D. H.
ROBERTS,
M. H.
LUCAS AND
G.
WIBBERLEY
Central Veterinary Laboratory, New Haw, Weybridge, Surrey
SUMMARY
The high-temperature short-time batch procedure which simulates the commercial pasteurization process, completely inactivated bovine leukosis virus (BLV)-infected lymphocytes in milk. Heating BLV -infected lymphocytes at 50°C for 70 sec completely destroyed BLV infectivity. Destruction of BLV infectivity coincided with the loss oflymphocyte viability as indicated by the lack of response to Concanavalin A stimulation. INTRODUCTION
Bovine leukosis virus (BLV) is widely recognized as the aetiological agent of enzootic bovine leukosis. Cattle and sheep infected with BLV develop antibodies and the agar gel immunodiffusion test has been widely used in this respect (Miller & Van der Maaten, 1977, 1979). It has been established that BLV infectivity may be present in both colostrum and milk from BL V -infected cows and that neonatal calves can be infected with BLV by the oral route (Miller & Vander Maaten, 1979; Vander Maaten & Miller, 1979). BLV can be isolated from the lymphocytes of infected cattle but it is very difficult to demonstrate its presence in other tissues (Miller & Vander Maaten, 1979; Ferrer, 1979, 1980; Straub, 1982). Consequently it is assumed that transmission results from the transfer of infected cells rather than free virus. Baumgartener, Olson & Onuma (1976) have shown that BLV mixed with milk and subjected to a simulated high-temperature short-time (HTST) pasteurization procedure was inactivated. They also showed that heating BL V at 60°C for 1I 2 min destroyed the virus. It seemed desirable to add BLV -infected lymphocytes to milk and to test the effect of heat by using a simple laboratory-scale HTST milk pasteurizer. In addition the effect ofheat on viable BLV-infected lymphocytes was also studied using the criteria of mitogenic stimulation and in-vivo transmission. MATERIALS AND METHODS
Animals Four cows (three to five years old) infected with BLV were used. BLV was
292
BRITISH VETERINARY JOURNAL, 139,4
consistently isolated from the peripheral blood leukocytes of these animals. Cows (three years old) and sheep susceptible to BL V were obtained from the BL V -free stock at this laboratory; all were negative for BLV antibodies.
Separation of peripheral blood lymphocytes The mononuclear fraction of peripheral blood leukocytes was harvested using the technique described by Roberts et al. ( 1982). The lymphocytes were suspended in Eagle's MEM, counted using a haemacytometer and Giemsa staining, and sampled for viability using the trypan blue dye exclusion test. The percentage of lymphocytes estimated in these preparations was about 90%. Serology and virus isolation The demonstration of persisting antibody responses as determined by the agar gel immunodiffusion (AGID) test was used to identify animals infected with BLV. In the AGID test, leukassay-B reagents (Pitman-Moore Inc., Washington Crossing, USA) were used; the technique and interpretation of the results were in accordance with the European Communities criteria (Council Directive, 11 November 1980, 80/1102/ EEC). BLV antigen was isolated and demonstrated by indirect fluorescence using the cocultivation method (Roberts et al., 1982). Mononuclear cells prepared from peripheral blood were cocultivated with foetal bovine lung cells. All fluorescent antibody tests included the EEC reference EBL virus antiserum E 1 (Council Directive, 11 November 1980, 81/1102/EEC) and fluorescein labelled anti-bovine immunoglobulin serum prepared in rabbits (Nordic Immunological Laboratories). Lymphocyte stimulation Cultures containing 5 X 106 leukocytes (90% lymphocytes) were added to 4 ml Eagle's MEM containing 20% foetal calf serum in bijou bottles. The cultures were inoculated in triplicate with 5 p,g Concanavalin A (Sigma Chemical Co.). Cultures without stimulant served as controls. The bottles were tightly capped and incubated at 37°C for three days. Transformation and proliferation of lymphocytes stimulated by Concanavalin A (Con A) were measured by labelling DNA with tritiated thymidine (Radiochemical Centre, Amersham, England). Twenty-four hours before collection, 5 p,Ci of tritiated thymidine (methyl-T specific activity 22 Ci/mmol) were added to each culture. The cells were sedimented at 1000 g for 5 min, washed with physiological saline, and centrifuged; 5% trichloroacetic acid was added to the cell pellet. The precipitate was washed with 95% ethanol and then dissolved in 1·0 ml 2 N solution of methanolic KOH at 60°C for 30 min. A 0·4 ml amount of this sample was added to 10 ml Luma gel (Lumac Systems AG) scintillation mixture and counted in a scintillation counter. The uptake of thymidine was expressed as the mean value of triplicate determinations as counts per minute per culture. High-temperature short-time {HTST) pasteurization simulator The HTST batch procedure simulated a commercial pasteurization process (Franklin, 1965). The apparatus consisted of two parallel stainless steel plates enclosing a thin film of milk; it was kept at the test temperature in a large water bath.
EFFECT OF HEAT ON BOVINE LEUKOSIS VIRUS
293
The temperature of the water was controlled at 1°C higher than the required milk temperature treatment. Two hundred and fifty-ml quantities were poured into the unit which was allowed to remain in the water at 72·5°C for 70 sec after which it was immediately transferred to an adjacent cold water bath for cooling. Pooled milk was obtained from serologically-negative lactating cows and BLV -infected lymphocytes were added.
Experimental design l. Five hundred ml pooled milk was obtained from BLV -free cows. BLV -infected lymphocytes were prepared from a BLV-positive donor cow (no. 1) and 10 7 cells (viability 97%) added to the milk. Two hundred and fifty ml of this milk mixture were heat treated using the HTST pasteurization simulator. The susceptible sheep were allotted to two groups. Each animal was inoculated intra-peritoneally with SO ml milk containing 2 X 106 lymphocytes. One group of five received pasteurized milk and the other group of five received non-pasteurized milk. 2. BLV -infected lymphocytes from three donor cows (nos 1, 2 and 3) were prepared. The lymphocytes were suspended in Eagle's MEM containing 20% foetal calf serum . Using a water bath, 4-ml samples of the BLV -infected lymphocytes were heated in bijou bottles to 40°, 45°, 50°, 55°, 60° and 65°C for 70 sec. Heat measurements were made by suspending a thermometer in 4 ml culture medium in an open bijou bottle. After 70 sec at the required temperature the heated bijou bottles were transferred to a cold water bath. Bottles containing BLV -infected lymphocytes were kept at room temperature and used as positive controls. Each sample was then inoculated subcutaneously into two sheep. Each sheep received 6 X 105 1ymphocytes. A total of 42 sheep was inoculated. 3. BLV-infected lymphocytes from four donor cows (nos 1, 2, 3 and 4) and uninfected lymphocytes from two susceptible cows (nos 5 and 6) were prepared (viability 95 to 100% ). The lymphocytes were suspended in Eagle's MEM with 20% foetal calf serum and heated as above at 45°, 50°, 55°, 60° and 65°C. Unheated samples were kept at room temperature. The heated and unheated lymphocyte suspensions were then tested for mitogenic stimulation using Con A. The inoculated sheep were bled before inoculation and at monthly intervals for 24 weeks. Clotted blood samples were collected using vacutainers. The serum was tested for BLV antibodies using the AGID test. At 24 weeks, heparinized blood samples were collected and attempts were made to isolate BLV using the cocultivation technique (Roberts et al., 1982). The sheep were kept in isolation for 24 weeks. RESULTS
None of the sheep inoculated with pasteurized milk developed antibodies. The five sheep inoculated with non-pasteurized milk developed BLV antibodies at four weeks post inoculation . BLV was isolated from all seropositive sheep. The survival of BLV in infected lymphocytes following heat treatment at various temperatures as judged by the AGID test results in the inoculated sheep are given in Table I. BLV antibodies were detected in the sheep between four and eight weeks after inoculation and BLV was isolated from all seropositive sheep. Heating the BLV-
BRITISH VETERINARY JOURNAL, 139,4
294
infected lymphocytes at 50°C and above for 70 sec destroyed BLV. The effect of heat on BLV-infected and uninfected lymphocytes and their response to Con A is shown in Table II . Normal and BLV-infected lymphocytes heated to 50°C and above for 70 sec did not respond to Con A stimulation. The mean counts/min for temperatures above 50°C were similar to those for 50°C and consequently are not given in the Table. The stimulatory effect of Con A was significantly decreased in BL Vinfected lymphocytes compared with normal lymphocytes . Heating BLV-infected lymphocytes for 70 sec at 45°C before treating with Con A appeared to increase slightly the uptake of tritiated thymidine in BLV -infected lymphocytes. TABLE I SURVIVAL OF BLV IN INFECTED LYMPHOCYTES FROM THREE COWS (NOS I, 2 AND 3) FOLLOWING HEAT TREATMENT
Temperature treatment for 70 sec
Recipient sheep nos
20°C 40°C 45°C 50°C 55°C 60°C 65°C
7-12 13-18 19-24 25-30 31-36 37-42
ACID test
+ + +
1- 6
TABLE II EFFECT OF HEAT ON BLV-INFECTED AND UNINFECTED LYMPHOCYTES AND THEIR RESPONSE TO CONCANAVALIN A STIMULATION
Mean counts/minute Temperature treatment for 70sec
Stimulant 1
20°C 20°C 45°C 50°C
None Con A Con A Con A
Unirzfected cows
BL V-infected cows
1555 7193 14 104 633
2
3
4
231 1860 2316 76
353 5640 6867 151
372 3885 4234 66
5
1287 90 886 68 119 658
6
340 36 536 2486 57
DISCUSSION
The results demonstrate that the HTST pasteurization simu lator process destroyed BL V infectivity in infected lymphocytes when milk was heated at 50°C for 70 sec.
EFFECT OF HEAT ON BOVINE LEUKOSIS VIRUS
295
Baumgartener et al. ( 1976), using cell-free BL V, showed that heating at 56°C for I min did not destroy the virus whereas heating at 60° did. In this study, destruction ofBLV infectivity coincided with loss of lymphocyte viability as indicated by the lack of response to Con A stimulation. The difference in the results might indicate that the release of infectious virus particles from BLV-infected lymphocytes is dependent on viable cells. This study confirms the conclusion ofWeiland & Straub (1976) that the stimulatory effect of Con A was significantly decreased in BLV -infected lymphocytes in comparison with normal lymphocytes. ACKNOWLEDGEMENTS
The technical assistance of Miss S. Bushnell and Mrs acknowledged.
J.
Weedon is gratefully
REFERENCES BAUMCARTENER, L., OLSON, C . & 0NUMA, M . ( 1976) . joumal
of the American Veterinary Medical Association 169, 1189. FERRER,j. F . (1979) . Joumal of the American Veterinary Medical Associationl75, 1281. FERRER, J. F. ( 1980) . Advances in Veterinary Science and Comparative Medicine 24, I. FRANKI.IN,j. G. (1965).Joumal of Dairy Research 32, 281. MILLER,j. M. & VANDER MAATEN, M . j. {1977). Europeanjoumal of Cancer 13, 1369. MILLER,j. M . & VANDER MAATEN, M.j . (1979) . Joumal of the National Cancer l11Stitute 62,425. ROBERTS, D . H., LUCAS, M. H ., WIBBERLEY, G. & CHASEY, D. (1982). Veterinary Record 110,222. STRAUB, 0. C. ( 1982). Fourth International Symposium on Bovine Leukosis, Bologna, ed. 0 . C. Straub, p. 299. London: Martinus Nijoff. VANDER MAATEN, M. J. & MILLER, J. M. ( 1979). In Advances in Comparative Leukemia Research, ed. P. Bentvelzen, J. Hilgers and D. Yohn, p. 29. Amsterdam: Elsevier, North Holland Biomedical Press . WEILAND, F. & STRAUB, 0. C. (1976). Research in Veterinary Science 20, 340. (Accepted for publication 20 July !982)