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Preservation of the infectious agent of tick-borne fever in the frozen state
J.
COMP. PATH.
1966.
VOL. 76.
PRESERVATION
413
OF
TICK-BORNE
THE
FEVER
INFECTIOUS IN
THE
AGENT
FROZEN
OF
STATE
BY
A.
FOGGIE
A4oredun Research Institute, Gilmcrton, Edinburgh and
W. H. R.
and
GUMSDEN
GILLIAN
J. C.
MCNEILLAGE
7ryykznosomia.k Research Unit, Royal (Dick) School of Veterinary Studies, L’niversily of Edinburgh INTRODUCTION
Sheep blood containing the tick-borne fever agent, Cytoecetes phagocytophila (Foggie, 1951), was found to remain infective for 10 days at room temperature and for 13 days at 4°C. Of numerous attempts to maintain the infection at temperatures below freezing point only one was successful. On that occasion 3 ml. of infected blood, brought rapidly down to a temperature of -79°C. and maintained there for one week, was found to be infective for test sheep. Attempts to store infected material by freeze-drying have been unsuccessful. of tick-borne fever infected blood This paper describes the storage at -79°C. by methods already found suitable for the preservation of trypanosomes (Polge and Soltys, 1957; Cunningham, Lumsden and Webber, 1963; Grainge, 1963). MATERIALS
AND
METHODS
Sheep which had not been exposed to tick infestation and guinea-pigs from the Moredun closed colony, splenectomized some weeks before use, were the test animals employed. Strains of C. phagocytophila. A sheep strain which had been passaged three times by intravenous inoculation in susceptible sheep since its isolation from the stock on Animals.
a tick infested farm and a strain which had been passaged 133 times in splenectomized guinea-pigs (Foggie and Hood, 1961) were used. Collection of infected blood. Blood from the infected donor sheep was collected from the jugular vein into 3 per cent. potassium citrate in the proportion of 4 parts blood to 1 part citrate solution. Blood from the donor guinea-pig was collected into 0.2 per cent. heparin in the proportion of 4 parts blood to 1 part heparin solution. Preservation. The method used was nearly identical to that described by Cunningham at al. (1963) in which sufficient glycerol (Analar*) or dimethyl sulphoxide (Laboratory reagent*) was added to the titrated or heparinized infected blood that it was desired to preserve to give a concentration of approximately 7.5 per cent. The resultant suspension, after mixing, was distributed with aseptic precautions by Pasteur pipette to lymph tubes, each containing about 25 ~1. After sealing, the samples were deposited in a test tube, 15 mm. in diameter and 125 mm. long, containing methanol at room temperature. After corking, the test tube was transferred to an insulating jacket with walls of Onazotef 25 mm. thick which had been deposited some hours previously at -79°C. The rate of cooling obtained by this
arrangement is such that -50°C. * Obtained
t Expanded
from
Rubber
is reached after about 1 hour (Lumsden, Robertson
Drug Houses, Poole, England. and Plastics, Croydon, England.
British
414
FREEZE-DRIED
TICK-BORNE
FEVER
AGENT
and McNeillage, in press). After 24 hours in the jacket, the lymph tubes were transferred to the permanent storage unit by manipulation under methanol at -79°C. (Grainge, 1963). Tests for infectivity. In order to determine whether the preservatives had a harmful effect on the parasite in the unfrozen state the residues of the sheep blood with glycerol and the sheep blood with dimethyl sulphoxide, after the preparation of the capillaries for storage, were retained at room temperature overnight. The following morning a & dilution in Hanks solution was made from each unfrozen sample and a test sheep was inoculated intravenously with 1 ml. of each preparation. Samples of the frozen materials were tested for infectivity after storage at -79°C. for 1 week, 11 weeks, 12 months and 18 months. The inocula were prepared by removing 2 capillaries (about 0.05 ml.) from storage allowing them to thaw rapidly on the bench and adding them to 1 ml. of Hanks solution. The test animals were inoculated within 30 minutes of the removal of the samples from the -79°C. environment. Test sheep were inoculated intravenously and test guinea-pigs intracardially, the volume of the inoculum being 1 ml. in each case. Body temperatures were recorded daily and counts of the percentage of parasitized polymorphs were made on films of blood from an ear vein stained with McNeal’s modification of Leishman’s stain. RESULTS
The results of tests on the infected sheep blood are exception all the test sheep developed typical attacks the inoculation of the samples tested. The sheep which with blood in glycerol heId at room temperature subsequently challenged with known infected blood ceptible. TABLE TESTS OF THE
INFECTWITY
OF BLOOD
shown in Table 1. With one of tick-borne fever following did not react was inoculated overnight. This sheep was and shown to be fully sus-
1
FROM
AN INFECTED
SHEEP
AFTER
STORAGE
Reactions of test sheep
Protectant
Duration of storage
-
20 hours
20 hours 7 days days 11 weeks 12
months
18 months months
Storage temperature room room - 79°C. -79°C. - 79°C. -79°C. -79°C. -79°C. - 79°C.
Maximum percentage of infected polymorpfu
Maximum body temperature “C.
92* 0
41.6 39.5 41.4 42.0 42.0 41.2 42.0 42.0 42.14 41.67
5: 80 60 80 :8” 78
Duration of
parasitaemia in abys ajh
inoculation 2 to 4to 5 to 4 to 5 to 4to 4to 4 to 5to
13 10 9 9 10 10 14 8 10
* = Reaction of donor sheep. G = Glycerol. D = Dimethyl
sulphoxide.
The results of tests on blood of infected splenectomized guinea-pigs are shown in Table 2. There was only sufficient splenectomized guinea-pig blood preserved in dimethyl sulphoxide for test inoculations after storage for 1 week and 18 months. As a splenectomized guinea-pig was not available to test the sample
A. FOOQIE TABLE TESTS ON THE
lNFECTIVITY
OF lNPECTED
415
et &. 2
SPLENECTOMIZED
GUINEA-PIG
BLOOD
STORED
AT
-79°C.
Reactions of test animals
Protectant
Duration of storage
G
7 days
D
7 days
G
11 weeks
ifi
12 18 months
D
18 months
Test animal
Maximum percenkage of infected po5vmorP~
Splenectomized guinea-pig Splenectomized guinea-pig Splenectomized guinea-pig Splenectomized guinea-pig Splenectomized Sheep guinea-pig Splenectomized guinea-pig
*60
Maximum body temperature “C.
Duration of parrm’tisemia in days afhr inoculation
39.0
3to5d
16
39.6
5 to 26
54
39.3
4 to 28
60
39.3
5 to 24
::
41.8 39.8
3to>9 5to 10
52
39.4
2to>9
* = Reaction of donor guinea-pig. d = Blood for storage was taken from the donor guinea-pig on the fourth day of reaction. The animal was found dead on the morning of the fifth day. G = Glycerol. D = Dimethyl sulphoxide.
stored for 1 year a susceptible sheep was used. All the test animals developed typical attacks of tick-borne fever following inoculation. The milder temperature reactions and more prolonged parasitaemias in the splenectomized guinea-pigs compared with the shorter, more severe reactions in the sheep are characteristic for the two species. The variations in severity and duration of the attacks in either the sheep or the guinea-pigs, as recorded in the Tables, are no greater than one would expect in groups of animals injected simultaneously with the appropriate strains of the organism. The reaction in the sheep inoculated with the stored guinea-pig blood was, however, more severe than those usually produced by this strain in sheep. DISCUSSION
The preliminary experiment showed that of the two preservatives, dimethyl sulphoxide had no deleterious effect on infectivity after overnight storage at room temperature, but glycerol suppressed the infection under these conditions. The tests on stored materials showed that C. jhzgocytophih remains viable when stored in small volumes of blood at -79°C. in the presence of either glycerol or dimethyl sulphoxide. There appeared to be no loss of infectivity after storage for 18 months and the evidence suggests that C. phagocytophila is another organism of which “stabilates” can be produced (Lumsden and Hardy, 1965). Experiments on immunity to tick-borne fever (Foggie, 195 1) and also unpublished work using laboratory animal-adapted strains have been complicated by the necessity of maintaing challenge strains of the organism by animal passage with consequent alterations of virulence. The use of stabilates in such experiments should greatly simplify their interpretation.
416
FREEZE-DRIED
TICK-BORNE
FEVER
AGENT
CONCLUSIONS
Cytoecetes phagocytophila, the infectious agent of tick-borne fever, retaiued its infectivity for test animals for at least 18 months when small volumes of infected blood from a sheep or a splenectomized guinea-pig were stored at -79°C. in the presence of either glycerol or dimethyl sulphoxide. REFERENCES
Cunningham, M. P., Lumsden, W. H. R., and Webber, W. A. F. (1963). Expl. Parusit., 14, 280. Foggie, A. (1951). J. Path. Bact., 63, 1. Foggie, A., and Hood, Catherine S. (1961). J. camp. Path., 71,413. Grainge, E. B. (1963). East Af ricun Trypunosomiusis Research Orgunisution Report, 1962-63, p. 11. Lumsden, W. H. R. and Hardy, Gillian J. C. (1965). Nature, Lond., 205, 1032. Lumsden, W. H. R., Robertson, D. H. H., and McNeillage, Gillian J. C. &it. 1. Venereal., In press. Polge, C., and Soltys, M. A. (1957). Truns. R. Sot. trap. Med. Hyg., 51, 519. [Received
for publication,
December
24th, 19651
COMP. PATH.
1966.
VOL. 76.
PRESERVATION
413
OF
TICK-BORNE
THE
FEVER
INFECTIOUS IN
THE
AGENT
FROZEN
OF
STATE
BY
A.
FOGGIE
A4oredun Research Institute, Gilmcrton, Edinburgh and
W. H. R.
and
GUMSDEN
GILLIAN
J. C.
MCNEILLAGE
7ryykznosomia.k Research Unit, Royal (Dick) School of Veterinary Studies, L’niversily of Edinburgh INTRODUCTION
Sheep blood containing the tick-borne fever agent, Cytoecetes phagocytophila (Foggie, 1951), was found to remain infective for 10 days at room temperature and for 13 days at 4°C. Of numerous attempts to maintain the infection at temperatures below freezing point only one was successful. On that occasion 3 ml. of infected blood, brought rapidly down to a temperature of -79°C. and maintained there for one week, was found to be infective for test sheep. Attempts to store infected material by freeze-drying have been unsuccessful. of tick-borne fever infected blood This paper describes the storage at -79°C. by methods already found suitable for the preservation of trypanosomes (Polge and Soltys, 1957; Cunningham, Lumsden and Webber, 1963; Grainge, 1963). MATERIALS
AND
METHODS
Sheep which had not been exposed to tick infestation and guinea-pigs from the Moredun closed colony, splenectomized some weeks before use, were the test animals employed. Strains of C. phagocytophila. A sheep strain which had been passaged three times by intravenous inoculation in susceptible sheep since its isolation from the stock on Animals.
a tick infested farm and a strain which had been passaged 133 times in splenectomized guinea-pigs (Foggie and Hood, 1961) were used. Collection of infected blood. Blood from the infected donor sheep was collected from the jugular vein into 3 per cent. potassium citrate in the proportion of 4 parts blood to 1 part citrate solution. Blood from the donor guinea-pig was collected into 0.2 per cent. heparin in the proportion of 4 parts blood to 1 part heparin solution. Preservation. The method used was nearly identical to that described by Cunningham at al. (1963) in which sufficient glycerol (Analar*) or dimethyl sulphoxide (Laboratory reagent*) was added to the titrated or heparinized infected blood that it was desired to preserve to give a concentration of approximately 7.5 per cent. The resultant suspension, after mixing, was distributed with aseptic precautions by Pasteur pipette to lymph tubes, each containing about 25 ~1. After sealing, the samples were deposited in a test tube, 15 mm. in diameter and 125 mm. long, containing methanol at room temperature. After corking, the test tube was transferred to an insulating jacket with walls of Onazotef 25 mm. thick which had been deposited some hours previously at -79°C. The rate of cooling obtained by this
arrangement is such that -50°C. * Obtained
t Expanded
from
Rubber
is reached after about 1 hour (Lumsden, Robertson
Drug Houses, Poole, England. and Plastics, Croydon, England.
British
414
FREEZE-DRIED
TICK-BORNE
FEVER
AGENT
and McNeillage, in press). After 24 hours in the jacket, the lymph tubes were transferred to the permanent storage unit by manipulation under methanol at -79°C. (Grainge, 1963). Tests for infectivity. In order to determine whether the preservatives had a harmful effect on the parasite in the unfrozen state the residues of the sheep blood with glycerol and the sheep blood with dimethyl sulphoxide, after the preparation of the capillaries for storage, were retained at room temperature overnight. The following morning a & dilution in Hanks solution was made from each unfrozen sample and a test sheep was inoculated intravenously with 1 ml. of each preparation. Samples of the frozen materials were tested for infectivity after storage at -79°C. for 1 week, 11 weeks, 12 months and 18 months. The inocula were prepared by removing 2 capillaries (about 0.05 ml.) from storage allowing them to thaw rapidly on the bench and adding them to 1 ml. of Hanks solution. The test animals were inoculated within 30 minutes of the removal of the samples from the -79°C. environment. Test sheep were inoculated intravenously and test guinea-pigs intracardially, the volume of the inoculum being 1 ml. in each case. Body temperatures were recorded daily and counts of the percentage of parasitized polymorphs were made on films of blood from an ear vein stained with McNeal’s modification of Leishman’s stain. RESULTS
The results of tests on the infected sheep blood are exception all the test sheep developed typical attacks the inoculation of the samples tested. The sheep which with blood in glycerol heId at room temperature subsequently challenged with known infected blood ceptible. TABLE TESTS OF THE
INFECTWITY
OF BLOOD
shown in Table 1. With one of tick-borne fever following did not react was inoculated overnight. This sheep was and shown to be fully sus-
1
FROM
AN INFECTED
SHEEP
AFTER
STORAGE
Reactions of test sheep
Protectant
Duration of storage
-
20 hours
20 hours 7 days days 11 weeks 12
months
18 months months
Storage temperature room room - 79°C. -79°C. - 79°C. -79°C. -79°C. -79°C. - 79°C.
Maximum percentage of infected polymorpfu
Maximum body temperature “C.
92* 0
41.6 39.5 41.4 42.0 42.0 41.2 42.0 42.0 42.14 41.67
5: 80 60 80 :8” 78
Duration of
parasitaemia in abys ajh
inoculation 2 to 4to 5 to 4 to 5 to 4to 4to 4 to 5to
13 10 9 9 10 10 14 8 10
* = Reaction of donor sheep. G = Glycerol. D = Dimethyl
sulphoxide.
The results of tests on blood of infected splenectomized guinea-pigs are shown in Table 2. There was only sufficient splenectomized guinea-pig blood preserved in dimethyl sulphoxide for test inoculations after storage for 1 week and 18 months. As a splenectomized guinea-pig was not available to test the sample
A. FOOQIE TABLE TESTS ON THE
lNFECTIVITY
OF lNPECTED
415
et &. 2
SPLENECTOMIZED
GUINEA-PIG
BLOOD
STORED
AT
-79°C.
Reactions of test animals
Protectant
Duration of storage
G
7 days
D
7 days
G
11 weeks
ifi
12 18 months
D
18 months
Test animal
Maximum percenkage of infected po5vmorP~
Splenectomized guinea-pig Splenectomized guinea-pig Splenectomized guinea-pig Splenectomized guinea-pig Splenectomized Sheep guinea-pig Splenectomized guinea-pig
*60
Maximum body temperature “C.
Duration of parrm’tisemia in days afhr inoculation
39.0
3to5d
16
39.6
5 to 26
54
39.3
4 to 28
60
39.3
5 to 24
::
41.8 39.8
3to>9 5to 10
52
39.4
2to>9
* = Reaction of donor guinea-pig. d = Blood for storage was taken from the donor guinea-pig on the fourth day of reaction. The animal was found dead on the morning of the fifth day. G = Glycerol. D = Dimethyl sulphoxide.
stored for 1 year a susceptible sheep was used. All the test animals developed typical attacks of tick-borne fever following inoculation. The milder temperature reactions and more prolonged parasitaemias in the splenectomized guinea-pigs compared with the shorter, more severe reactions in the sheep are characteristic for the two species. The variations in severity and duration of the attacks in either the sheep or the guinea-pigs, as recorded in the Tables, are no greater than one would expect in groups of animals injected simultaneously with the appropriate strains of the organism. The reaction in the sheep inoculated with the stored guinea-pig blood was, however, more severe than those usually produced by this strain in sheep. DISCUSSION
The preliminary experiment showed that of the two preservatives, dimethyl sulphoxide had no deleterious effect on infectivity after overnight storage at room temperature, but glycerol suppressed the infection under these conditions. The tests on stored materials showed that C. jhzgocytophih remains viable when stored in small volumes of blood at -79°C. in the presence of either glycerol or dimethyl sulphoxide. There appeared to be no loss of infectivity after storage for 18 months and the evidence suggests that C. phagocytophila is another organism of which “stabilates” can be produced (Lumsden and Hardy, 1965). Experiments on immunity to tick-borne fever (Foggie, 195 1) and also unpublished work using laboratory animal-adapted strains have been complicated by the necessity of maintaing challenge strains of the organism by animal passage with consequent alterations of virulence. The use of stabilates in such experiments should greatly simplify their interpretation.
416
FREEZE-DRIED
TICK-BORNE
FEVER
AGENT
CONCLUSIONS
Cytoecetes phagocytophila, the infectious agent of tick-borne fever, retaiued its infectivity for test animals for at least 18 months when small volumes of infected blood from a sheep or a splenectomized guinea-pig were stored at -79°C. in the presence of either glycerol or dimethyl sulphoxide. REFERENCES
Cunningham, M. P., Lumsden, W. H. R., and Webber, W. A. F. (1963). Expl. Parusit., 14, 280. Foggie, A. (1951). J. Path. Bact., 63, 1. Foggie, A., and Hood, Catherine S. (1961). J. camp. Path., 71,413. Grainge, E. B. (1963). East Af ricun Trypunosomiusis Research Orgunisution Report, 1962-63, p. 11. Lumsden, W. H. R. and Hardy, Gillian J. C. (1965). Nature, Lond., 205, 1032. Lumsden, W. H. R., Robertson, D. H. H., and McNeillage, Gillian J. C. &it. 1. Venereal., In press. Polge, C., and Soltys, M. A. (1957). Truns. R. Sot. trap. Med. Hyg., 51, 519. [Received
for publication,
December
24th, 19651