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Pathogenesis of Trypanosoma brucel infection in sheep
J. COMP.
PATH.
1975. VOL.
PATHOGENESIS
II.
33
85.
OF INFECTION
TRY-PANOSOMA
IN
CEREBRO-SPINAL
BRUCE1
SHEEP
FLUID
CHANGES
BY
B. 0. Department
IKEDE
and G. J, Losos
of Veterinary Pathology,
University
of Ibadan, Nigeria
INTRODUCTION
In late stages of African trypanosomiasis in man, the trypanosomes often invade the cerebrospinal fluid (CSF) and provoke cellular and chemical alterations that are of diagnostic significance (Apted, 1970; Baker, 1970). Following experimental infections of monkeys and chimpanzees with Tq$xznosoma rhodesiense,T. gambienseor T. brucei, the parasites invade the CSF and produce changes similar to those in man (Wolbach and Binger, 1912; Peruzzi, 1928; Corson, 1938; Stewart, 1950; Baker and Taylor, 1971). Corson (1931, 1934) found trypanosomes and increased cell count in the CSF of sheep, goats and antelopes experimentally infected with T. rhodesiense.Parasites have also been demonstrated in the CSF of horses (Laveran and Mesnil, 1970; Nietz and McCully, 1971), sheep and cattle (Ikede and Losos, 1972a,b) infected with T. brucei, but the accompanying pathological changes have not been adequately described. As part of studies on the pathogenesis of 1: brucei infection in sheep, the CSF of normal and infected sheep was examined and the results are presented in this communication. MATERIALS
AND
METHODS
T. brucei strain 8118 (Ikede and Losos, 1972a) was used. The feeding and management of the experimental animals are described elsewhere (Ikede and Losos, 1974). Twenty-two sheep were infected and there were 11 controls. Parasitaemia was monitored throughout the experiment by daily examination of wet mounts of venous blood. Collection and examination of CSF. CSF was collected from each animal only when moribund or immediately after euthanasia. The suboccipital puncture technique, as described by Cornelius and Kaneko ( 1963) was used to collect up to 5 ml. of CSF. In a few cases, this was unsuccessful and the lumbar site was used. The general appearance of the CSF was noted; the presence of trypanosomes in the fluid, as well as in blood, was determined by examining wet mount preparations at 100 and 400 magnifications. When parasites were detected in the CSF by this method, they were counted in a haemocytometer using a diluting fluid containing toluidine blue and formalin. Part of the CSF was also centrifuged at 1500 rev./min. (about 400 g.) for 7 min. and the deposit was examined for trypanosomes in wet preparations and in thick smears stained with Giemsa. The remaining CSF was injected i.p. into a young albino rat which was subsequently examined weekly for 4 weeks for trypanosomes in the tail blood. Leucocytes in the CSF were counted in a haemocytometer as described by Benjamin C
34
B.
0.
IKEDE
AND
G. J.
LOSOS
(1962). If the total count was above 70 cells per mm. 3, differential counts were done on the stained thick smears of the centrifuged deposit. Total protein concentrations were determined by the sulphosalicylic acid method using proteinometer standards, * and also by the Pandy’s test (Benjamin, 1962). RESULTS
Samples CSF samples were obtained from the infected sheep between the 62nd day after inoculation, when the first animal died, and at the 150th day, when the experiment was terminated. Samples were also obtained from 8 of the control sheep killed between the 105th and 150th day. Appearance The CSF obtained from the 8 uninfected sheep and from 15 out of the 22 infected sheep was clear and colourless. In 5 infected animals it was slight to moderately cloudy. In 2 cases, one of which was noticed after centrifugation, there was contamination with fresh blood and these were excluded from all further tests. Presence of Typanosomes Trypanosomes were found in the CSF of 18 of the remaining 20 infected cases. Eight were detected by direct examination of the fluid without centrifugation, 7 only after centrifugation and 3 only by rat inoculation. In the first group, the trypanosomes were motile and counts varied from 1 to 150 parasites per mm.3 of CSF. In stained smears, trypomastigote forms of T. brucei were observed. Some of the parasites were adherent to the leucocytes whereas others were phagocytized by macrophages. At the time the CSF was collected parasitaemia was undetectable in 9 animals, scanty in 7 and heavy in the remaining 4. Leucocyte Count in CSF In uninfected sheep, the total cell count varied from 1 to 18 per mm.3 Leucocytes in smears of centrifuged deposit were generally too few to be differentially counted, but the predominant cells were small and large lymphocytes and a few macrophages and endothelial cells. Total leucocyte counts were done on samples from 17 infected sheep and the values in 4 of these were similar to those of the control group (1 to 22 per mm.3). In the remaining infected animals, the cell count was increased slightly in 4 (30 to 66 per mm.3), moderately in 7 (70 to 410 per mm.3) and markedly in 2 (7 10 to 2094 per mm.3). Mean differential counts in 9 animals with total counts above 70 per mm.3 were as follows: small lymphocytes 73.3 per cent., large mononuclear cells 26.2 per cent., neutrophils 1.5 per cent. and endothelial cells 1-O per cent. Morular cells of Mott were occasionally present. Total Proteins The concentration of total proteins in the CSF was determined in 8 control and 16 of the infected sheep. Values for control animals were between 10 and 25 * Gallenkamp
and
Co.
Ltd,
London.
T.
bW%‘i
INFECTION
IN
SHEEP:
II.
CEREBRO-SPINAL
FLUID
35
mg. per 100 ml. and the Pandy’s test was negative. In the infected group 8 animals had values similar to those of the control group. In 4 others the values were between 26 and 50 mg. per 100 ml. while the remaining 4 had values above 50 mg. It was only in this last group that the Pandy’s test was positive. DISCUSSION
Trypanosomes were detectable in the CSF of 18 of the 20 infected sheep by using three different methods: direct examination of a drop of the fluid, examination of a drop of the centrifuged deposit and rat inoculation. Many more cases were detected after centrifugation than by direct examination; a few additional cases were detected by rat inoculation. These findings are similar to those reported in human sleeping sickness (Baker, 1970). Owing to technical difficulties, CSF examination is generally not recommended for routine diagnosis of trypanosomiasis in animals. However, under experimental conditions, it would be useful for the study of drug action on trypanosomes that have invaded the central nervous system. It is noteworthy that parasitaemia was scanty or undetectable in all but 4 infected sheep at the time of necropsy. The probability of accidental introduction of trypanosomes into the CSF from blood contamination during withdrawal is thus negligible. Moreover, such contamination should be evident after centrifugation (Chandler, 1957). Invasion of the CSF by the parasites was accompanied by a moderate to marked increase in the cell content in 9 animals and an increase in the protein content in 4 compared with the controls. The predominant cells in the fluid were lymphocytes, macrophages and plasma cells. Morular cells were rarely observed. The relative amount of the protein components was not studied, but in man a rise in gamma globulins and especially the IgM fraction is characteristic (Apted, 1970). The alterations observed in this study are consistent with the neurological signs reported elsewhere in these animals (Ikede and Losos, 1974). Peruzzi (1928) stated that in monkeys and cattle infected with the brucei group of trypanosomes, the CSF was invaded by parasites originating from the choroid plexus where they multiplied greatly in the interstitial tissue. Recently, Ormerod and Venkatesan (197 1) concluded from their studies in rats that the capillaries of the choroid plexus were probably the most important site of multiplication of r. brucei when parasitaemia was low or undetectable. It seems possible that the trypanosomes in the CSF of these sheep originated from a similar site but this was not investigated. SUMMARY
Eighteen of 20 sheep experimentally infected with I: brucei for 2 to 5 months had trypanosomes in the CSF in the terminal stages. The total cell count was significantly raised in 9 animals (70 to 2095 cells per mm.3), the predominant cells being lymphocytes and macrophages with occasional plasma and morular cells. Total protein concentration was increased in 4 animals (51 to 100 mg. per 100 ml. and positive Pandy’s test). Although routine CSF examination for trypanosomes may not be practicable in farm animals, it is recommended for chemotherapeutic studies on parasites invading the central nervous system.
36
B.
0.
IKEDE
AND
G. J.
LOSOS
ACKNOWLEDGMENTS
This study was supported by Grant Foundation. We also wish to thank Mr
66024 Allocation 74, from Isaac Obiwale for secretarial
the Rockefeller assistance.
REFERENCES
Apted,
F. I. C. (1970). Clinical Manifestations and Diagnosis of Sleeping Sickness. In The African Tryjanosomiases, pp. 661-683, H, W. Mulligan and W. H. Potts, Eds. Allen and Unwin, London. Baker, J. R. (1970). Techniques for the Detection of Trypanosome Infections. In The African Trypanosomiases, pp. 67-68, H. W. Mulligan and W. H. Potts, Eds. Allen and Unwin, London. Baker, J. R., and Taylor, A. E. R. (1971). Experimental infections of the chimpanzee (Pan troglodytes) with Trypanosoma brucei brucei and Typanosoma brucei rhodesiense. Annals of Tropical Medicine and Parasitology, 65, 471-485. Benjamin, M. M. (1962). Outline of Veterinary Clinical Pathology, 2nd edit., pp. 144 148. Iowa State University Press, Ames. Chandler, R. L. (1957). Observations on the cerebrospinal fluid in some cases of trypanosomiasis in cattle and sheep. British Veterinary Journal, 113,482-483. Cornelius, C. E., and Kaneko, J. J. (1963). Clinical Biochemistry of Domestic Animals, 1st edit., pp. 369-397. Academic Press, New York. Corson, J. F. (1931). Trypanosoma rhodesiense in the cerebrospinal fluid of sheep and goats. Annals of Tropical Medicine and Parasitology, 25, 145. Corson, J. F. (1934). The cerebrospinal fluid of some small antelopes infected with T. rhodesiense. Annals of Tropical Medicine and ParasitoloD, 28, 243-244. Corson, J. F. (1938). The cerebrospinal fluid of monkeys (Cercopithecus sp.) infected with a strain of Trypanosoma rhodesiense. Annals of Tropical Medicine and Parasitology, 32, 197-199. Ikede, B. O., and Losos, G. J. (1972a). Pathology of the disease in sheep produced experimentally by Trypanosoma brucei. Veterinary Pathology, 9, 278-289. Ikede, B. O., and Losos, G. J. (1972b) Pathological changes in cattle infected with Trypanosoma brucei. Veterinary Pathology, 9, 272-277. Ikede, B. O., and Losos, G. J. (1974). Pathogenesis of Trypanosoma brucei infection in sheep. I. The Clinical Signs. Journal qf Comparative Pathology, 84, 23-31. Laveran, A., and Mesnil, F. (1907). Trypanosomes and Trypanosomiasis, translated by D. Nabarro. Baillihe, Tindall and Cox, London. Nietz, W. O., and McCully, R. M. (1971). Clinicopathological study on experimental Trypanosoma brucei infections in horses. Part I. Development of clinically recognizable nervous symptoms in nagana-infected horses treated with subcurative doses of antrypol and berenil. Onderstepoort Journal of Veterinary Research, 38, 127-140. Ormerod, W. E., and Venkatesan, S. (1971). An amastigote phase of the sleeping sickness trypanosome. Transactions of the Royal Society of Tropical Medicine and Hygiene, 65, 736-741. Peruzzi, M. (1928). Pathologico-anatomical and Serological Observations on the Trypanosomiases. Final Report of the League of Nations International Committee on Human Sleeping Sickness, Section 5, pp. 245-328. Geneva. Stewart, G. R. (1950). Experimental neuro-trypanosomiasis in the monkey. Transactions of the Royal Society of Tropical Medicine and Hygiene, 43, 364. Wolbach, S. B., and Binger, C. A. L. (1912). A contribution to the parasitology of trypanosomes. Journal of Medical Research, 27, 83-107. [Receivedfor publication,
February 27th, 19741
PATH.
1975. VOL.
PATHOGENESIS
II.
33
85.
OF INFECTION
TRY-PANOSOMA
IN
CEREBRO-SPINAL
BRUCE1
SHEEP
FLUID
CHANGES
BY
B. 0. Department
IKEDE
and G. J, Losos
of Veterinary Pathology,
University
of Ibadan, Nigeria
INTRODUCTION
In late stages of African trypanosomiasis in man, the trypanosomes often invade the cerebrospinal fluid (CSF) and provoke cellular and chemical alterations that are of diagnostic significance (Apted, 1970; Baker, 1970). Following experimental infections of monkeys and chimpanzees with Tq$xznosoma rhodesiense,T. gambienseor T. brucei, the parasites invade the CSF and produce changes similar to those in man (Wolbach and Binger, 1912; Peruzzi, 1928; Corson, 1938; Stewart, 1950; Baker and Taylor, 1971). Corson (1931, 1934) found trypanosomes and increased cell count in the CSF of sheep, goats and antelopes experimentally infected with T. rhodesiense.Parasites have also been demonstrated in the CSF of horses (Laveran and Mesnil, 1970; Nietz and McCully, 1971), sheep and cattle (Ikede and Losos, 1972a,b) infected with T. brucei, but the accompanying pathological changes have not been adequately described. As part of studies on the pathogenesis of 1: brucei infection in sheep, the CSF of normal and infected sheep was examined and the results are presented in this communication. MATERIALS
AND
METHODS
T. brucei strain 8118 (Ikede and Losos, 1972a) was used. The feeding and management of the experimental animals are described elsewhere (Ikede and Losos, 1974). Twenty-two sheep were infected and there were 11 controls. Parasitaemia was monitored throughout the experiment by daily examination of wet mounts of venous blood. Collection and examination of CSF. CSF was collected from each animal only when moribund or immediately after euthanasia. The suboccipital puncture technique, as described by Cornelius and Kaneko ( 1963) was used to collect up to 5 ml. of CSF. In a few cases, this was unsuccessful and the lumbar site was used. The general appearance of the CSF was noted; the presence of trypanosomes in the fluid, as well as in blood, was determined by examining wet mount preparations at 100 and 400 magnifications. When parasites were detected in the CSF by this method, they were counted in a haemocytometer using a diluting fluid containing toluidine blue and formalin. Part of the CSF was also centrifuged at 1500 rev./min. (about 400 g.) for 7 min. and the deposit was examined for trypanosomes in wet preparations and in thick smears stained with Giemsa. The remaining CSF was injected i.p. into a young albino rat which was subsequently examined weekly for 4 weeks for trypanosomes in the tail blood. Leucocytes in the CSF were counted in a haemocytometer as described by Benjamin C
34
B.
0.
IKEDE
AND
G. J.
LOSOS
(1962). If the total count was above 70 cells per mm. 3, differential counts were done on the stained thick smears of the centrifuged deposit. Total protein concentrations were determined by the sulphosalicylic acid method using proteinometer standards, * and also by the Pandy’s test (Benjamin, 1962). RESULTS
Samples CSF samples were obtained from the infected sheep between the 62nd day after inoculation, when the first animal died, and at the 150th day, when the experiment was terminated. Samples were also obtained from 8 of the control sheep killed between the 105th and 150th day. Appearance The CSF obtained from the 8 uninfected sheep and from 15 out of the 22 infected sheep was clear and colourless. In 5 infected animals it was slight to moderately cloudy. In 2 cases, one of which was noticed after centrifugation, there was contamination with fresh blood and these were excluded from all further tests. Presence of Typanosomes Trypanosomes were found in the CSF of 18 of the remaining 20 infected cases. Eight were detected by direct examination of the fluid without centrifugation, 7 only after centrifugation and 3 only by rat inoculation. In the first group, the trypanosomes were motile and counts varied from 1 to 150 parasites per mm.3 of CSF. In stained smears, trypomastigote forms of T. brucei were observed. Some of the parasites were adherent to the leucocytes whereas others were phagocytized by macrophages. At the time the CSF was collected parasitaemia was undetectable in 9 animals, scanty in 7 and heavy in the remaining 4. Leucocyte Count in CSF In uninfected sheep, the total cell count varied from 1 to 18 per mm.3 Leucocytes in smears of centrifuged deposit were generally too few to be differentially counted, but the predominant cells were small and large lymphocytes and a few macrophages and endothelial cells. Total leucocyte counts were done on samples from 17 infected sheep and the values in 4 of these were similar to those of the control group (1 to 22 per mm.3). In the remaining infected animals, the cell count was increased slightly in 4 (30 to 66 per mm.3), moderately in 7 (70 to 410 per mm.3) and markedly in 2 (7 10 to 2094 per mm.3). Mean differential counts in 9 animals with total counts above 70 per mm.3 were as follows: small lymphocytes 73.3 per cent., large mononuclear cells 26.2 per cent., neutrophils 1.5 per cent. and endothelial cells 1-O per cent. Morular cells of Mott were occasionally present. Total Proteins The concentration of total proteins in the CSF was determined in 8 control and 16 of the infected sheep. Values for control animals were between 10 and 25 * Gallenkamp
and
Co.
Ltd,
London.
T.
bW%‘i
INFECTION
IN
SHEEP:
II.
CEREBRO-SPINAL
FLUID
35
mg. per 100 ml. and the Pandy’s test was negative. In the infected group 8 animals had values similar to those of the control group. In 4 others the values were between 26 and 50 mg. per 100 ml. while the remaining 4 had values above 50 mg. It was only in this last group that the Pandy’s test was positive. DISCUSSION
Trypanosomes were detectable in the CSF of 18 of the 20 infected sheep by using three different methods: direct examination of a drop of the fluid, examination of a drop of the centrifuged deposit and rat inoculation. Many more cases were detected after centrifugation than by direct examination; a few additional cases were detected by rat inoculation. These findings are similar to those reported in human sleeping sickness (Baker, 1970). Owing to technical difficulties, CSF examination is generally not recommended for routine diagnosis of trypanosomiasis in animals. However, under experimental conditions, it would be useful for the study of drug action on trypanosomes that have invaded the central nervous system. It is noteworthy that parasitaemia was scanty or undetectable in all but 4 infected sheep at the time of necropsy. The probability of accidental introduction of trypanosomes into the CSF from blood contamination during withdrawal is thus negligible. Moreover, such contamination should be evident after centrifugation (Chandler, 1957). Invasion of the CSF by the parasites was accompanied by a moderate to marked increase in the cell content in 9 animals and an increase in the protein content in 4 compared with the controls. The predominant cells in the fluid were lymphocytes, macrophages and plasma cells. Morular cells were rarely observed. The relative amount of the protein components was not studied, but in man a rise in gamma globulins and especially the IgM fraction is characteristic (Apted, 1970). The alterations observed in this study are consistent with the neurological signs reported elsewhere in these animals (Ikede and Losos, 1974). Peruzzi (1928) stated that in monkeys and cattle infected with the brucei group of trypanosomes, the CSF was invaded by parasites originating from the choroid plexus where they multiplied greatly in the interstitial tissue. Recently, Ormerod and Venkatesan (197 1) concluded from their studies in rats that the capillaries of the choroid plexus were probably the most important site of multiplication of r. brucei when parasitaemia was low or undetectable. It seems possible that the trypanosomes in the CSF of these sheep originated from a similar site but this was not investigated. SUMMARY
Eighteen of 20 sheep experimentally infected with I: brucei for 2 to 5 months had trypanosomes in the CSF in the terminal stages. The total cell count was significantly raised in 9 animals (70 to 2095 cells per mm.3), the predominant cells being lymphocytes and macrophages with occasional plasma and morular cells. Total protein concentration was increased in 4 animals (51 to 100 mg. per 100 ml. and positive Pandy’s test). Although routine CSF examination for trypanosomes may not be practicable in farm animals, it is recommended for chemotherapeutic studies on parasites invading the central nervous system.
36
B.
0.
IKEDE
AND
G. J.
LOSOS
ACKNOWLEDGMENTS
This study was supported by Grant Foundation. We also wish to thank Mr
66024 Allocation 74, from Isaac Obiwale for secretarial
the Rockefeller assistance.
REFERENCES
Apted,
F. I. C. (1970). Clinical Manifestations and Diagnosis of Sleeping Sickness. In The African Tryjanosomiases, pp. 661-683, H, W. Mulligan and W. H. Potts, Eds. Allen and Unwin, London. Baker, J. R. (1970). Techniques for the Detection of Trypanosome Infections. In The African Trypanosomiases, pp. 67-68, H. W. Mulligan and W. H. Potts, Eds. Allen and Unwin, London. Baker, J. R., and Taylor, A. E. R. (1971). Experimental infections of the chimpanzee (Pan troglodytes) with Trypanosoma brucei brucei and Typanosoma brucei rhodesiense. Annals of Tropical Medicine and Parasitology, 65, 471-485. Benjamin, M. M. (1962). Outline of Veterinary Clinical Pathology, 2nd edit., pp. 144 148. Iowa State University Press, Ames. Chandler, R. L. (1957). Observations on the cerebrospinal fluid in some cases of trypanosomiasis in cattle and sheep. British Veterinary Journal, 113,482-483. Cornelius, C. E., and Kaneko, J. J. (1963). Clinical Biochemistry of Domestic Animals, 1st edit., pp. 369-397. Academic Press, New York. Corson, J. F. (1931). Trypanosoma rhodesiense in the cerebrospinal fluid of sheep and goats. Annals of Tropical Medicine and Parasitology, 25, 145. Corson, J. F. (1934). The cerebrospinal fluid of some small antelopes infected with T. rhodesiense. Annals of Tropical Medicine and ParasitoloD, 28, 243-244. Corson, J. F. (1938). The cerebrospinal fluid of monkeys (Cercopithecus sp.) infected with a strain of Trypanosoma rhodesiense. Annals of Tropical Medicine and Parasitology, 32, 197-199. Ikede, B. O., and Losos, G. J. (1972a). Pathology of the disease in sheep produced experimentally by Trypanosoma brucei. Veterinary Pathology, 9, 278-289. Ikede, B. O., and Losos, G. J. (1972b) Pathological changes in cattle infected with Trypanosoma brucei. Veterinary Pathology, 9, 272-277. Ikede, B. O., and Losos, G. J. (1974). Pathogenesis of Trypanosoma brucei infection in sheep. I. The Clinical Signs. Journal qf Comparative Pathology, 84, 23-31. Laveran, A., and Mesnil, F. (1907). Trypanosomes and Trypanosomiasis, translated by D. Nabarro. Baillihe, Tindall and Cox, London. Nietz, W. O., and McCully, R. M. (1971). Clinicopathological study on experimental Trypanosoma brucei infections in horses. Part I. Development of clinically recognizable nervous symptoms in nagana-infected horses treated with subcurative doses of antrypol and berenil. Onderstepoort Journal of Veterinary Research, 38, 127-140. Ormerod, W. E., and Venkatesan, S. (1971). An amastigote phase of the sleeping sickness trypanosome. Transactions of the Royal Society of Tropical Medicine and Hygiene, 65, 736-741. Peruzzi, M. (1928). Pathologico-anatomical and Serological Observations on the Trypanosomiases. Final Report of the League of Nations International Committee on Human Sleeping Sickness, Section 5, pp. 245-328. Geneva. Stewart, G. R. (1950). Experimental neuro-trypanosomiasis in the monkey. Transactions of the Royal Society of Tropical Medicine and Hygiene, 43, 364. Wolbach, S. B., and Binger, C. A. L. (1912). A contribution to the parasitology of trypanosomes. Journal of Medical Research, 27, 83-107. [Receivedfor publication,
February 27th, 19741