J. gen. Virol.
(I977), 38, I83-I86
183
Printed in Great Britain
The Growth of Virulent African Swine Fever Virus in Pig Monocytes and Macrophages (Accepted lO August 1977) SUMMARY The replication of virulent Afiican swine fever virus (ASFV) in cultures of monocytes and macrophages derived from pig bone marrow (PBM) and pig leukocyte (PL) cells was investigated by light microscopy, immunofluorescence, haemadsorption and infective virus release. Monocytes showed a high rate of infection and complete destruction within 2 to 3 days, whereas macrophages had only a very low level of infection and survived to form persistently infected cultures. These observations may explain the decrease in sensitivity of PBM and PL cells for ASFV assay after extended periods of incubation and suggest that the macrophage may be one of the cell types concerned with virus persistence in the pig.
Virulent African swine fever virus (ASFV) is usually cultured and assayed in pig bone marrow (PBM) or pig leukocyte (PL) cultures. The cell type in which the virus grows and shows haemadsorption is adherent to glass (Malmquist & Hay, 196o) and is presumably of the monocyte-macrophage series. At the time of the development of these tissue culture systems for virus assay it was implied that the sensitivity of both PBM and PL cultures decreased if they were not inoculated within the first week (Hess & DeTray, 196o; Malmquist & Hay, I96O; Sidorov, 1971). This observation led us to look more closely at the relationship between ASFV and the cells of the monocyte-macrophage series. The virulent Kirawira isolate (Greig & Plowright, 197o) of ASFV was used in all experiments as a suspension of spleen taken from acutely infected pigs or as a clarified suspension after 2 to 3 passages in PBM cells. PBM cultures were prepared from the cells washed out of fragments of red bone marrow derived from the long bones of 2 kg pigs (Plowright, Parker & Staple, 1968). To prepare PL cultures, buffy coat cells were placed on FicollHypaque gradients (B6yum, 1968), centrifuged at 200 g for 20 min and the interface cells removed, washed three times and suspended in growth medium. The medium used for growth and maintenance of cells in all the experiments was RPMI 1640 containing 5 % heatinactivated foetal calf serum. PBM cultures were set up in plastic flasks (Falcon Plastics no. 3o18) at a concentration of 5 x lO7 cells/flask and non-adherent cells were removed at 48 h. PL cultures were similarly established at 3 x lO7 cells/flask and non-adherent cells removed after 4 h. Cell morphology was examined by preparing slides in a cytospin centrifuge (Shandon Elliott, Runcorn, Cheshire, U.K.) and staining cells with Wright's stain. We adopted the morphological criteria and terminology of van Furth et aI. (I972) for defining the cell types present. Cells from the PL population adhered to the surface of the plastic flask in 1 to 2 h. Cytospin preparations of these adherent cells showed that 80 to 95 % of them were monocytes, the remaining cells being mainly neutrophils. By 24 h the monocytes had increased 95 to IOO % and these cells ranged in diameter from 9 to 15 #m (mean I I/~m) and had a round to oval nucleus containing a fairly open chromatin pattern which was surrounded by faintly basophilic cytoplasm (Fig. I b). During cultivation for lO to 14 days, most of the adherent cells became detached from the plastic but the few
I84
Short communications
Fig. i. Cytospin preparations of pig leucocytes stained with Wright's stain. (a) Macrophages after 14 days in culture; (b) monocytes after 5 h in culture. remaining cells divided to form small islands and eventually complete monolayers. The number of islands of cells varied from 5 to zoo but usually about IOO islands developed in each flask. Cytospin preparations showed that these cells were morphologically homogeneous, ranging in size from 2o to 70 # m (mean 38 #m), contained m a n y vacuoles, displayed large pseudopodia (Fig. I a) and were assumed to be fully differentiated cells of the mononuclear phagocytic series. They stained rapidly with neutral red (Cohn & Wiener, 1963) and ingested carbonyl iron particles. All these characteristics are properties of macrophages. Virus growth curves in PBM cultures were performed within 3 days (PBM monocyte preparations) or cultures were maintained for at least IO days with daily media changes before virus inoculation (PBM macrophage preparations). Similarly, PL cultures were used within 6 to 24 h (PL monocyte preparations) or maintained for lo to I4 days with repeated changes of medium (PL macrophage preparations) and then inoculated with virus. PBM or PL cultures were inoculated with virus at a multiplicity of infection of o.I. After an adsorption period of I h at 37 °C, cultures were washed five times in phosphate buffered saline (PBS), fresh medium added and the cultures incubated at 37 °C. Samples of supernatant fluid were removed at intervals for virus assay. I f cells in culture were still viable 6 days after virus inoculation, the remaining medium was removed, the cell sheet washed twice with PBS and fresh medium added. After this, the medium was removed every 2 to 3 days for virus assay and the cell sheets washed twice in PBS before fresh medium was added. Virus assays were performed by making Io-fold dilutions of samples which were inoculated on to PBM cells cultured in tubes. The presence of Virus was determined by haemadsorption 6 days later and titres were expressed as log10 5o % haemadsorbing doses (HADs0) per ml. Cells for immunofluorescent staining were grown on coverslips. At various times after
Short communications
I85
T a b l e i. Virulent ASFV growth in PL monoeyte and PL macrophage preparations Monocytes Days postinfection
Virus titre*
o o'75 !
2"25 5"75 6"75
2
7"0
3
6"75
5 Io
6-o --§
2 0
-
40 80
---
-
Macrophages
Fluorescent cells (~) t
Haemadsorbing cells ( ~ ) "~
NT;~ NT 59 (z8-67) 95 (9o-98) 98 (96-1oo) NT --
NT NT 5 (2-8) 66 (31-74) 73 (4o-79) NT --
-
-
---
-
-
---
Virus titre 2.o 3'o 3'5 3"75 4"0 4"25 3"5 3"75
Fluorescent cells (~) t
Haemadsorbing cells (~) ~
NT NT 0.6 (o'4-o'9) 0'4 (o'3-o'5) 0'4 (o'3-o'5) NT NT 0.6
2 × lO-4 (1-5 × Io -4) NT NT 2 × lO-4
(0'2-0"9)
(1- 3 × IO-4)
NT NT
NT NT
4'25 3'75
NT NT 2 × lO-4 (I-4 × IO-4) NT
* Log~0 HADs0/ml, mean of. Ia experiments. ~- Mean and range of 4 experiments. NT: not tested. § - - , Complete cytopathic effect; cultures discarded. infection, cells were fixed for 5 min in acetone a n d stained with fluorescein-conjugated a n t i - A S F V globulin for 3o rain at 37 °C. A f t e r three 2o m i n washes in PBS, coverslips were m o u n t e d in p h o s p h a t e buffered glycerol a n d viewed with an ultraviolet microscope. T h e i n o c u l a t i o n o f virulent A S F V o n to cultures o f m o n o c y t e s p r e p a r e d f r o m either P B M o r P L cells caused c o m p l e t e cell d e s t r u c t i o n within 2 to 3 days. T h e time c o u r s e o f infective virus release in P L m o n o c y t e cultures a n d the n u m b e r o f cells shown to be infected b y immunofluorescence a n d h a e m a d s o r p t i o n are presented in T a b l e I. Results f r o m P B M m o n o c y t e cultures were similar. I n c o n t r a s t to this, the i n o c u l a t i o n o f virus o n to m a c r o p h a g e p r e p a r a t i o n s caused relatively little change, W h e n c o m p a r e d with u n i n o c u l a t e d cultures, however, small areas o f 6 to I o r o u n d e d u p cells were o b s e r v e d 48 h posti n o c u l a t i o n a n d these foci never b e c a m e extensive. A s s a y o f m e d i u m f r o m P L a n d P B M m a c r o p h a g e cultures showed t h a t virus r e p l i c a t i o n c o n t i n u e d for at least 3 m o n t h s . D a t a f r o m P L m a c r o p h a g e cultures are shown in T a b l e I . D u r i n g this period, cell cultures r e m a i n e d a p p a r e n t l y healthy and, a p a r t f r o m some small foci o f r o u n d e d u p cells, were m o r p h o l o g i c a l l y indistinguishable f r o m u n i n o c u l a t e d cultures. T h e m o n i t o r i n g o f m a c r o p h a g e s b y i m m u n o f l u o r e s c e n c e showed t h a t between ~ a n d Io cells p e r Iooo were infected b y 48 h a n d this p r o p o r t i o n r e m a i n e d c o n s t a n t over the p e r i o d tested (Table I). H a e m a d s o r p t i o n b y persistently infected cells was consistently m u c h less sensitive a n d o n l y I lr~ i o 6 cells was shown to c o n t a i n virus (Table I). Infective centre assays could n o t be carried o u t since virulent isolates o f A S F V have a very low efficiency o f plaquing. Thus, as far as we could determine, persistently infected m a c r o p h a g e cultures c o n t a i n e d a m a x i m u m o f 1 % infected cells. It was also n o t possible to d e t e r m i n e w h e t h e r the i m m u n o f l u o r e s c e n t cells were associated with the very small c y t o p a t h i c foci, because the rour, d e d u p cells w e r e r e a d i l y d e t a c h e d f r o m the m o n o l a y e r s d u r i n g the i m m u n o f l u o r e s c e n t s~aining p r o c e d u r e . H o w e v e r , since no foci were o b s e r v e d i n c o n t r o l cultures, those o b s e r v e d in i n o c u l a t e d cultures most p r o b a b l y occurred as the result o f virus infection.
186
Short communications
In common with other workers, we have shown that the early adherent cells from both blood and bone marrow preparations are rapidly destroyed by ASFV (Hess & DeTray, 196o; Malmquist & Hay, I96O; Moulton & Coggins, I968) and immunofluorescence and haemadsorption show that a high proportion of them are infected. Monocytes maintained in culture gave rise to homogeneous populations of morphologically fully differentiated macrophages. In contrast to the monocyte, macrophage populations were not destroyed when infected with virulent ASFV. This suggests that the reduced sensitivity of I-week-old PL and PBM cultures used for the routine assay of ASFV (Hess & DeTray, 196o; Malmquist & Hay, 196o; Sidorov, I 9 7 0 is due to the differentiation of susceptible monocytes into less susceptible macrophages. As well as differing markedly from monocytes in their susceptibility to infection with virulent ASFV, macrophage cultures also became persistently infected. Since only some monocytes developed into macrophages, it is possible that a population of macrophages was selected which only became persistently infected as the result of changes produced during cultivation. Persistent infection of macrophages in vitro would therefore have no correlation with events occurring in vivo. However, two observations (data not shown) suggest that cells of the monocyte-macrophage series, capable of becoming persistently infected, are present in the peripheral blood of normal pigs. When monocytes were infected after only 6 h in culture, thus ruling out the possibility of a major population change, a few cells survived and developed into persistently infected macrophages. Secondly, monocytes cultured from pigs infected with acute ASF developed into persistently infected macrophages. These observations imply that the selection of a resistant cell type during prolonged cultivation did not occur and also suggest that the macrophage may well be one of the cell types which are involved with the persistence of ASFV in vivo. We wish to thank B. L. Ellis and S. M. Williams for excellent technical assistance. R. C. WARDLEY
The Animal Virus Research Institute Pirbright, Woking, Surrey
P . J. WILKINSON REFERENCES
B6YtSM,A. 0968). Isolation of mononuclear cells and granulocytes from human blood. Scandinavian Journal of Clinical and Laboratory Investigation 2x (Suppl. 97), 77-89. CO~rN,Z. A. & WEINER,E. (I963). The particulate hydrolases of macrophages. II. Biochemical and morphological response to particle ingestion. Journal of Experimental Medicine 118, IOO9-IO2O. GREIG,A. & PLOWRmHT,W. 0970). The excretion of two virulent strains of African swine fever virus by domestic pigs. Journal of Hygiene 68, 673~682. HESS, W. R. & DETRAY, D. E. (1960). T h e u s e o f leukocyte cultures for d i a g n o s i n g A f r i c a n swine fever (ASF). Bulletin of Epizootic Diseases of Africa 8, 317-32o. MALMQUIST, W. A. & HAY, D. (1960). H e m a d s o r p t i o n a n d cytopathic effect p r o d u c e d by A f r i c a n swine fever virus in swine b o n e m a r r o w a n d buffy coat cultures. American Journal of Veterinary Research 2I, lO4-Io8. MOULTON, S. & COGGINS, L. (1968). Synthesis a n d cytopathogenesis o f A f r i c a n swine fever virus in porcine cell cultures. American Journal of Veterinary Research 29, 219-232. PLOWRIGIIT, W., PARKER, J. & STAPLE, R. F. (1968). T h e g r o w t h of a virulent strain o f A f r i c a n swine fever virus in domestic pigs. Journal of Hygiene 66, I I 7 - I 3 4 . SlDOROV, M. A. (1971). Leucocyte cultures f r o m cells o f peritoneal fluid a n d their sensitivity to A f r i c a n swine fever virus. Doklady Vsesoyuznoi Akademii Sel' skokhoz. 12, 29-31. VAN FURTH, R., COHN, Z. A., HIRSCH, S. G., HUMPHREY, J. H., SPECTOR, W. G. & LANGEVOORT, H. L. (1972). T h e m o n o n u c l e a r p h a g o c y t e system: a n e w classification o f m a c r o p h a g e s , m o n o c y t e s a n d their precursor cells. Bulletin of the Worm Health Organization 46, 845-852.
(Received I I June 1977)
(I977), 38, I83-I86
183
Printed in Great Britain
The Growth of Virulent African Swine Fever Virus in Pig Monocytes and Macrophages (Accepted lO August 1977) SUMMARY The replication of virulent Afiican swine fever virus (ASFV) in cultures of monocytes and macrophages derived from pig bone marrow (PBM) and pig leukocyte (PL) cells was investigated by light microscopy, immunofluorescence, haemadsorption and infective virus release. Monocytes showed a high rate of infection and complete destruction within 2 to 3 days, whereas macrophages had only a very low level of infection and survived to form persistently infected cultures. These observations may explain the decrease in sensitivity of PBM and PL cells for ASFV assay after extended periods of incubation and suggest that the macrophage may be one of the cell types concerned with virus persistence in the pig.
Virulent African swine fever virus (ASFV) is usually cultured and assayed in pig bone marrow (PBM) or pig leukocyte (PL) cultures. The cell type in which the virus grows and shows haemadsorption is adherent to glass (Malmquist & Hay, 196o) and is presumably of the monocyte-macrophage series. At the time of the development of these tissue culture systems for virus assay it was implied that the sensitivity of both PBM and PL cultures decreased if they were not inoculated within the first week (Hess & DeTray, 196o; Malmquist & Hay, I96O; Sidorov, 1971). This observation led us to look more closely at the relationship between ASFV and the cells of the monocyte-macrophage series. The virulent Kirawira isolate (Greig & Plowright, 197o) of ASFV was used in all experiments as a suspension of spleen taken from acutely infected pigs or as a clarified suspension after 2 to 3 passages in PBM cells. PBM cultures were prepared from the cells washed out of fragments of red bone marrow derived from the long bones of 2 kg pigs (Plowright, Parker & Staple, 1968). To prepare PL cultures, buffy coat cells were placed on FicollHypaque gradients (B6yum, 1968), centrifuged at 200 g for 20 min and the interface cells removed, washed three times and suspended in growth medium. The medium used for growth and maintenance of cells in all the experiments was RPMI 1640 containing 5 % heatinactivated foetal calf serum. PBM cultures were set up in plastic flasks (Falcon Plastics no. 3o18) at a concentration of 5 x lO7 cells/flask and non-adherent cells were removed at 48 h. PL cultures were similarly established at 3 x lO7 cells/flask and non-adherent cells removed after 4 h. Cell morphology was examined by preparing slides in a cytospin centrifuge (Shandon Elliott, Runcorn, Cheshire, U.K.) and staining cells with Wright's stain. We adopted the morphological criteria and terminology of van Furth et aI. (I972) for defining the cell types present. Cells from the PL population adhered to the surface of the plastic flask in 1 to 2 h. Cytospin preparations of these adherent cells showed that 80 to 95 % of them were monocytes, the remaining cells being mainly neutrophils. By 24 h the monocytes had increased 95 to IOO % and these cells ranged in diameter from 9 to 15 #m (mean I I/~m) and had a round to oval nucleus containing a fairly open chromatin pattern which was surrounded by faintly basophilic cytoplasm (Fig. I b). During cultivation for lO to 14 days, most of the adherent cells became detached from the plastic but the few
I84
Short communications
Fig. i. Cytospin preparations of pig leucocytes stained with Wright's stain. (a) Macrophages after 14 days in culture; (b) monocytes after 5 h in culture. remaining cells divided to form small islands and eventually complete monolayers. The number of islands of cells varied from 5 to zoo but usually about IOO islands developed in each flask. Cytospin preparations showed that these cells were morphologically homogeneous, ranging in size from 2o to 70 # m (mean 38 #m), contained m a n y vacuoles, displayed large pseudopodia (Fig. I a) and were assumed to be fully differentiated cells of the mononuclear phagocytic series. They stained rapidly with neutral red (Cohn & Wiener, 1963) and ingested carbonyl iron particles. All these characteristics are properties of macrophages. Virus growth curves in PBM cultures were performed within 3 days (PBM monocyte preparations) or cultures were maintained for at least IO days with daily media changes before virus inoculation (PBM macrophage preparations). Similarly, PL cultures were used within 6 to 24 h (PL monocyte preparations) or maintained for lo to I4 days with repeated changes of medium (PL macrophage preparations) and then inoculated with virus. PBM or PL cultures were inoculated with virus at a multiplicity of infection of o.I. After an adsorption period of I h at 37 °C, cultures were washed five times in phosphate buffered saline (PBS), fresh medium added and the cultures incubated at 37 °C. Samples of supernatant fluid were removed at intervals for virus assay. I f cells in culture were still viable 6 days after virus inoculation, the remaining medium was removed, the cell sheet washed twice with PBS and fresh medium added. After this, the medium was removed every 2 to 3 days for virus assay and the cell sheets washed twice in PBS before fresh medium was added. Virus assays were performed by making Io-fold dilutions of samples which were inoculated on to PBM cells cultured in tubes. The presence of Virus was determined by haemadsorption 6 days later and titres were expressed as log10 5o % haemadsorbing doses (HADs0) per ml. Cells for immunofluorescent staining were grown on coverslips. At various times after
Short communications
I85
T a b l e i. Virulent ASFV growth in PL monoeyte and PL macrophage preparations Monocytes Days postinfection
Virus titre*
o o'75 !
2"25 5"75 6"75
2
7"0
3
6"75
5 Io
6-o --§
2 0
-
40 80
---
-
Macrophages
Fluorescent cells (~) t
Haemadsorbing cells ( ~ ) "~
NT;~ NT 59 (z8-67) 95 (9o-98) 98 (96-1oo) NT --
NT NT 5 (2-8) 66 (31-74) 73 (4o-79) NT --
-
-
---
-
-
---
Virus titre 2.o 3'o 3'5 3"75 4"0 4"25 3"5 3"75
Fluorescent cells (~) t
Haemadsorbing cells (~) ~
NT NT 0.6 (o'4-o'9) 0'4 (o'3-o'5) 0'4 (o'3-o'5) NT NT 0.6
2 × lO-4 (1-5 × Io -4) NT NT 2 × lO-4
(0'2-0"9)
(1- 3 × IO-4)
NT NT
NT NT
4'25 3'75
NT NT 2 × lO-4 (I-4 × IO-4) NT
* Log~0 HADs0/ml, mean of. Ia experiments. ~- Mean and range of 4 experiments. NT: not tested. § - - , Complete cytopathic effect; cultures discarded. infection, cells were fixed for 5 min in acetone a n d stained with fluorescein-conjugated a n t i - A S F V globulin for 3o rain at 37 °C. A f t e r three 2o m i n washes in PBS, coverslips were m o u n t e d in p h o s p h a t e buffered glycerol a n d viewed with an ultraviolet microscope. T h e i n o c u l a t i o n o f virulent A S F V o n to cultures o f m o n o c y t e s p r e p a r e d f r o m either P B M o r P L cells caused c o m p l e t e cell d e s t r u c t i o n within 2 to 3 days. T h e time c o u r s e o f infective virus release in P L m o n o c y t e cultures a n d the n u m b e r o f cells shown to be infected b y immunofluorescence a n d h a e m a d s o r p t i o n are presented in T a b l e I. Results f r o m P B M m o n o c y t e cultures were similar. I n c o n t r a s t to this, the i n o c u l a t i o n o f virus o n to m a c r o p h a g e p r e p a r a t i o n s caused relatively little change, W h e n c o m p a r e d with u n i n o c u l a t e d cultures, however, small areas o f 6 to I o r o u n d e d u p cells were o b s e r v e d 48 h posti n o c u l a t i o n a n d these foci never b e c a m e extensive. A s s a y o f m e d i u m f r o m P L a n d P B M m a c r o p h a g e cultures showed t h a t virus r e p l i c a t i o n c o n t i n u e d for at least 3 m o n t h s . D a t a f r o m P L m a c r o p h a g e cultures are shown in T a b l e I . D u r i n g this period, cell cultures r e m a i n e d a p p a r e n t l y healthy and, a p a r t f r o m some small foci o f r o u n d e d u p cells, were m o r p h o l o g i c a l l y indistinguishable f r o m u n i n o c u l a t e d cultures. T h e m o n i t o r i n g o f m a c r o p h a g e s b y i m m u n o f l u o r e s c e n c e showed t h a t between ~ a n d Io cells p e r Iooo were infected b y 48 h a n d this p r o p o r t i o n r e m a i n e d c o n s t a n t over the p e r i o d tested (Table I). H a e m a d s o r p t i o n b y persistently infected cells was consistently m u c h less sensitive a n d o n l y I lr~ i o 6 cells was shown to c o n t a i n virus (Table I). Infective centre assays could n o t be carried o u t since virulent isolates o f A S F V have a very low efficiency o f plaquing. Thus, as far as we could determine, persistently infected m a c r o p h a g e cultures c o n t a i n e d a m a x i m u m o f 1 % infected cells. It was also n o t possible to d e t e r m i n e w h e t h e r the i m m u n o f l u o r e s c e n t cells were associated with the very small c y t o p a t h i c foci, because the rour, d e d u p cells w e r e r e a d i l y d e t a c h e d f r o m the m o n o l a y e r s d u r i n g the i m m u n o f l u o r e s c e n t s~aining p r o c e d u r e . H o w e v e r , since no foci were o b s e r v e d i n c o n t r o l cultures, those o b s e r v e d in i n o c u l a t e d cultures most p r o b a b l y occurred as the result o f virus infection.
186
Short communications
In common with other workers, we have shown that the early adherent cells from both blood and bone marrow preparations are rapidly destroyed by ASFV (Hess & DeTray, 196o; Malmquist & Hay, I96O; Moulton & Coggins, I968) and immunofluorescence and haemadsorption show that a high proportion of them are infected. Monocytes maintained in culture gave rise to homogeneous populations of morphologically fully differentiated macrophages. In contrast to the monocyte, macrophage populations were not destroyed when infected with virulent ASFV. This suggests that the reduced sensitivity of I-week-old PL and PBM cultures used for the routine assay of ASFV (Hess & DeTray, 196o; Malmquist & Hay, 196o; Sidorov, I 9 7 0 is due to the differentiation of susceptible monocytes into less susceptible macrophages. As well as differing markedly from monocytes in their susceptibility to infection with virulent ASFV, macrophage cultures also became persistently infected. Since only some monocytes developed into macrophages, it is possible that a population of macrophages was selected which only became persistently infected as the result of changes produced during cultivation. Persistent infection of macrophages in vitro would therefore have no correlation with events occurring in vivo. However, two observations (data not shown) suggest that cells of the monocyte-macrophage series, capable of becoming persistently infected, are present in the peripheral blood of normal pigs. When monocytes were infected after only 6 h in culture, thus ruling out the possibility of a major population change, a few cells survived and developed into persistently infected macrophages. Secondly, monocytes cultured from pigs infected with acute ASF developed into persistently infected macrophages. These observations imply that the selection of a resistant cell type during prolonged cultivation did not occur and also suggest that the macrophage may well be one of the cell types which are involved with the persistence of ASFV in vivo. We wish to thank B. L. Ellis and S. M. Williams for excellent technical assistance. R. C. WARDLEY
The Animal Virus Research Institute Pirbright, Woking, Surrey
P . J. WILKINSON REFERENCES
B6YtSM,A. 0968). Isolation of mononuclear cells and granulocytes from human blood. Scandinavian Journal of Clinical and Laboratory Investigation 2x (Suppl. 97), 77-89. CO~rN,Z. A. & WEINER,E. (I963). The particulate hydrolases of macrophages. II. Biochemical and morphological response to particle ingestion. Journal of Experimental Medicine 118, IOO9-IO2O. GREIG,A. & PLOWRmHT,W. 0970). The excretion of two virulent strains of African swine fever virus by domestic pigs. Journal of Hygiene 68, 673~682. HESS, W. R. & DETRAY, D. E. (1960). T h e u s e o f leukocyte cultures for d i a g n o s i n g A f r i c a n swine fever (ASF). Bulletin of Epizootic Diseases of Africa 8, 317-32o. MALMQUIST, W. A. & HAY, D. (1960). H e m a d s o r p t i o n a n d cytopathic effect p r o d u c e d by A f r i c a n swine fever virus in swine b o n e m a r r o w a n d buffy coat cultures. American Journal of Veterinary Research 2I, lO4-Io8. MOULTON, S. & COGGINS, L. (1968). Synthesis a n d cytopathogenesis o f A f r i c a n swine fever virus in porcine cell cultures. American Journal of Veterinary Research 29, 219-232. PLOWRIGIIT, W., PARKER, J. & STAPLE, R. F. (1968). T h e g r o w t h of a virulent strain o f A f r i c a n swine fever virus in domestic pigs. Journal of Hygiene 66, I I 7 - I 3 4 . SlDOROV, M. A. (1971). Leucocyte cultures f r o m cells o f peritoneal fluid a n d their sensitivity to A f r i c a n swine fever virus. Doklady Vsesoyuznoi Akademii Sel' skokhoz. 12, 29-31. VAN FURTH, R., COHN, Z. A., HIRSCH, S. G., HUMPHREY, J. H., SPECTOR, W. G. & LANGEVOORT, H. L. (1972). T h e m o n o n u c l e a r p h a g o c y t e system: a n e w classification o f m a c r o p h a g e s , m o n o c y t e s a n d their precursor cells. Bulletin of the Worm Health Organization 46, 845-852.
(Received I I June 1977)
