Journal of Helminihology (1977) SI, 95—104
Erythrokinetic studies on baboons (Papio anubis) with acute experimental schistosomiasis mansoni P. H. HOLMES, E. R. JAMES*, J. M. MACLEAN, G. S. NELSON* and M. G. TAYLOR* University of Glasgow, Veterinary School, Bearsden Road, Glasgow and *London School of Hygiene and Tropical Medicine, Winches Farm Field Station, Hatfield Road, St. Albans
ABSTRACT The erythrokinetics of three baboons were studied over a twelve week period after infection with 3 000 cercariae of S. mansoni and compared with three uninfected control animals. In addition to monitoring changes in haematological and parasitological parameters several radioisotopic techniques were used concurrently to measure alterations in red cell kinetics. In particular 5iCr-labelled red cells and 59Fe-ferric citrate were used to measure red cell production, circulating volume, survival time and routes of loss. All the animals remained in good condition despite heavy infection and the pathophysiological changes were in general mild and transient in nature. The principle alterations were associated with patency and consisted of a modest fall in haematocrit, some inappetence and the appearance of blood-stained mucus in the faeces. Red cell survival was reduced in the infected animals and this was correlated with an increase in faecal blood loss and haemodilution. There was no evidence of haemolytic factors or depressed erythropoiesis having any aetiological role in the anaemia. The results confirmed the complexities of schistosomal anaemia as well as the ability of primates to withstand heavy parasitic burdens for short periods with minimal pathophysiological disturbance.
Although anaemia commonly occurs in schistosome infections of man and other animals (Foy and Nelson, 1963; Warren, 1973) many aspects of its aetiology remain controversial. It is generally accepted that gastrointestinal blood loss associated with oviposition is the most important factor, while other contributing mechanisms have also been postulated. Thus, Mahmoud and Woodruff (1972) have suggested, as a result of studies in mice, that haemolytic factors from adult worms have an important role in the causation of schistosomal anaemia. Whilst Preston and Dargie (1974) have shown in sheep, that although intestinal bleeding is the major factor in causing reduced haematocrit values, haemodilution and dyshaemopoiesis are important contributing factors. However, studies of human and experimental primate schistosomiasis have not yet established the relationship between schistosomiasis and anaemia or the relative importance of the four basic mechanisms which may be responsible for anaemia, namely, blood loss, haemolysis, haemodilution and dyshaemopoiesis (Jamra et ah, 1964). In an effort to understand the pathogenesis of experimental acute schistosomiasis in primates the erythrokinetics of baboons were studied over a twelve week period after infection with 3 000 cercariae of S. mansoni using established radioisotopic techniques (Holmes, 1969—Ph.D. thesis of Glasgow University); 51Cr-labelled red cells and 59 Ferric citrate to measure red cell production, circulating volume, survival time and routes of loss. Routine haematological and parasitological measurements were performed concurrently in an attempt to relate any changes to altered red cell kinetics. 95
P. H. HOLMES et al.
MATERIALS AND METHODS Experimental Animals Young male baboons (Papio anubis) caught in the wild and weighing approximately 5 kilograms were obtained from a local animal supplier. They were maintained in quarantine for several weeks prior to the experiments and examined for evidence of previous infections, including naturally acquired schistosomiasis. The baboons were fed a commercial primate ration supplemented with fresh fruit throughout the study, and maintained individually in standard baboon cages with grid floors and draining collection trays for the separation of urine and faeces. All infection and blood sampling procedures were carried out under sedation with Vetalar* (10 mg/kg) IM. Parasitological and Haematological Techniques The Puerto Rican strain of S. mansoni used for infection was maintained in hamsters and Biomphalaria glabrata at Winches Farm. The parasitological techniques and post-mortem procedure were similar to those described previously by Taylor et al., (1973). Faecal samples were examined at regular intervals throughout the study. Heparinized blood samples obtained from the saphenous vein were used for determination of haematocrit values and plasma iron levels. The animals were killed 14 weeks after infection. Radioisotopic Measurements Two weeks before infection and at four, seven, nine and twelve weeks after infection each baboon was intravenously injected with 51Cr labelled autologous red cells (300 /xCi s l Cr per animal) and 59Fe-ferric citrate (80 /nCi per animal). Heparinized blood samples were taken at frequent intervals during the three hours following injection and samples of blood and plasma analysed for radioactivity using a multichannel automatic gamma scintillation counter**. Analysis of the red cell and plasma radioactivities provided circulating red cell volume and plasma volume by application of the dilution principle. In addition the plasma iron clearance rate was obtained from a semi-logarithmic plot of 59Fe activity against time over the first three hours, and used to calculate the plasma iron turnover rate (PITR). Between seven and twelve weeks after infection daily collections of urine and faeces were obtained, along with thrice weekly blood samples. Analysis of the radioactivities of the blood samples provided the red cell survival time and iron incorporation rate. Faecal "blood clearances" were calculated from each daily collection by dividing the total daily faecal radioactivity by the activity of the blood during the collection period. These values represent the amount of blood which has to appear in the gut to account for the faecal radioactivity. Urinary blood clearances were calculated in a similar manner. RESULTS Parasitological and Histopathological Findings Schistosome eggs were detected in the faeces of the infected baboons five weeks after infection (Fig. 1) and remained at a level of approximately 300 e.p.g. until week 12 when a fall to less than 200 e.p.g. was observed. The adult worm recovery and distribution of tissue eggs at post-mortem 14 weeks after infection are presented in Table 1. The mean adult worm burden in the three infected baboons was 717 worms, i.e. a mean recovery rate of 24 %. There was a mean of 7.3 x 103 eggs * Ketamine hydrochloride, Parke-Davis & Co. ** Nuclear Chicago, High Wycombe, England. 96
Erythrokinetics of baboons with S. mansoni
per female adult worm in the tissues and most of the eggs (mean 56 %) were detected in the large intestine with 35 % in the small intestine and 9 % in the liver. Histopathology of the liver, lungs, large and small bowel revealed active infections in all three baboons with minimal damage to the liver and extensive lesions in the large bowel and two animals showed some signs of schistosomal pneumonitis. There were a few haemorrhagic lesions in the bowel. There was no evidence of portal hypertension and the kidneys and spleen were normal. The control animals were not sacrificed at the end of the experiment.
P-m'
300-
200-
100-
15
10
FIG. 1. Faecal egg output from baboons infected with S. mansoni. Vertical axis = eggs/gram; horizontal axis = weeks after infection. TABLE 1 Worm burdens and tissue egg counts of baboons 14 weeks after infection with 3000 cercariae of S. mansoni Baboon no. 75 76 77 Mean
Individual worm burdens Total Total Final total 3 % ? % 207 (35%) 379 (65%) 586 217 (35%) 416 (65%) 633 342 (37%) 590 (63%) 932 462 (64)
255 (36)
Total tissue eggs/? worms
Individual total tissue egg counts (x 103)
717
( X 103)
1906-3 1229-2
9-21 5-66
1567-7
7-44
Distribution of tissue eggs per gram Baboon no. 75 76 77
Liver Total %
Small intestine Total %
Large intestine Total /o
1433 645
917 5884 4348
9592 3115 6754
12-0 6-7
7-7 61-3 —
80-6 32-4 97
P. H. HOLMES et al.
Haematological and Clinical Changes All the animals remained in good condition throughout the experiment with only scanty clinical signs of schistosomiasis in the infected groups. The main effects were observed between six and nine weeks after infection when there was a fall in the haematocrit of the infected animals coinciding with the appearance of bloodstained mucus in the faeces. There was a moderate degree of inappetence and a slight fall in body weight (Fig. 2). However, by 12 weeks after infection, the parasitized animals had reverted to a condition similar to that seen before infection.
Red Cell Survival The disappearance rates of 51Cr-labelled and 59Fe-labelled red cells were measured between nine and twelve weeks after infection and the results are presented in Table 2. It is
D
o-
O
-O
o
°
°
10
12
FIG. 2. Body weight and haematocrit changes in baboons infected with S. mansoni. Vertical axis upper = PCV, lower = kg; horizontal axis = weeks after infection. Key 0 - - - 0 = infected 0——% = control. 98
Erythrokinetics of baboons with S. mansoni TABLE 2 Red cell survival in baboons infected with S. mansoni
I N F E C T E D
Baboon no.
5iCr-red cell* Tidays
"Fe-red cell Tidays
75
13-8
50-7
76
15-0
56-8
77
9-6
86-0
12-8
64-5
68
16-6
244-9
74
16-3
1211
81
17-7
399-7
16-9
255-2
Mean C O N T R O L Mean 1
9-12 weeks after infection
apparent that red cell survival expressed as a half-life (T£) was markedly reduced in the infected baboons as measured by both techniques. The discrepancy between the 51Cr and 59 Fe results in both infected and control animals is due to the elution of 51Cr from labelled red cells thereby giving an abnormally shortened survival time when measured by 51Cr and the in vivo reincorporation of 59 Fe into new red cells thereby giving an overestimate of red cell survival time when measured by 59 Fe. So whilst neither method gives a "true" half-life, they nevertheless provide a valuable indication of red cell survival. Excretion of 51Cr The shortened red cell half-lives of radioisotopically labelled red cells demonstrated above indicate an increased rate of removal of red cells from the circulation of infected animals but give no evidence of how this situation arises. The decrease in red cell survival may be due to either intravascular breakdown of erythrocytes, or losses into the gut. The relative importance of these two possibilities can only be assessed by a quantitative examination of the routes of loss of 51Cr since 51Cr appears quantitatively in the faeces following loss into the gastrointestinal tract or in the urine after intravascular breakdown of labelled red cells. The results of faecal "whole blood" clearances are illustrated in Fig. 3 and show that the infected baboons were losing significantly greater amounts of blood into the gastrointestinal tract than the controls. 99
P. H. HOLMES el al.
On the other hand examination of the urinary "whole blood" clearance failed to reveal any significant differences between the infected animals (mean daily clearance 5-3 ml) and the controls (mean daily clearance 8-1 ml).
3i
2-
10
11
12
FIG. 3. Mean daily faecal blood clearance of baboons infected with 5. mansoni. Vertical axis = faecal blood clearance (ml); horizontal axis = weeks after infection. Key 0 — 0 = infected, upper 5 iCr and lower 59Fe I
= control, upper 51Cr and lower 59Fe
Ferrokinetics
The possible importance of dyshaemopoiesis as a causative factor in schistosomal anaemia was assessed by examination of the plasma iron half life. In addition, gastrointestinal loss of iron and reabsorption of haemoglobin iron were also measured. The values for plasma iron levels, plasma iron clearance and plasma iron turnover rate are shown in Fig. 4. Despite the results being somewhat variable they do indicate that there was no marked differences in the plasma iron clearance or plasma iron turnover rate between the infected and control animals. However, there was evidence of a fall in the serum iron level during the course of the infection in the parasitized baboons. The differences between the faecal losses of 51Cr and 59 Fe in the infected animals shown in Fig. 3 indicate that during the period of maximal gastrointestinal blood loss there was considerable reabsorption of haemoglobin iron (Roche, et al, 1957). 100
Erythrokinetics of baboons with 5. mansoni
160n 140120-
y
\
100-
\
o
80-
1000
0
2
4
6
8
10
12
FIG. 4. Plasma iron concentration, half-life and turnover rate in baboons infected with 5. mansoni. Vertical axis, upper = Fe concentration (tig%), middle = TJ (mins) and lower = plasma iron turnover rate ); horizontal axis = weeks after infection. Key 0 — 0 = infected # — — # = control.
Plasma, Circulating Red Cell and Blood Volumes Measurements were made of plasma, circulating red cell and blood volumes at 0, 4, 7, 9 and 12 weeks after infection and the results are presented in Fig. 5. It is apparent that several changes occurred in these volumes as the infection became patent. Initially there was little difference between the infected and control animals but by 7 weeks after infection a hypervolaemia developed in the parasitized groups due to an increase in the plasma volumes of these animals. There was however no significant change in the circulating red cell volume, thus indicating that the fall in whole body haematocrit was clearly associated with a haemodilution effect. 101
P. H. HOLMES et al.
70
60
C
rfl
I
n
50
40
30
20
10
FIG. 5. Plasma, circulating red cell, and blood volumes in baboons infected with S. mansoni. Vertical axis = ml/kg; horizontal axis = Weeks after infection. Key
= Plasma volume;
= Red cell volume;
= Total blood volume;
I = Infected, C = Control.
DISCUSSION In this experiment the erythrokinetics of baboons experimentally infected with 3000 cercariae of S1. mansoni were studied for twelve weeks post-infection. The results indicated that despite heavy parasitic burdens the clinical haematological changes associated with the infection were relatively mild and of a transient nature. The main changes were associated with oviposition and consisted of transient inappetance and a slight fall in haematocrit. The mild anaemia was found to be the result of some minor changes in the erythrokinetic status of the parasitised baboons. Namely, reduced red cell survival associated with increased faecal blood loss and haemodilution. There was no evidence of increased haemolysis or of changes in erythropoiesis. The value of baboons as experimental hosts for schistosomiasis has been established (Sadun, et ah, 1966) yet these animals have not been used to examine the pathogenesis of anaemia in schistosomiasis except for the incidental observations by Damian et al., (1976). 102
Erythrokinetics of baboons with S. mansoni
This may be partly because along with other models there is a considerable discrepancy between the worm burdens required to produce symptoms of schistosomiasis in animals and the parasitic burdens found in human cases of schistosomiasis (Cheever, 1969). This is particularly evident in the present experiment in which an average of 40 S. mansoni worm pairs per kg failed to produce marked symptoms in the baboons, yet human infections of more than 5 worm pairs per kg are rarely encountered (Cheever, 1968). In the present study changes were associated with the patency of the schistosome infection, although the mildness of these changes was surprising in view of the level of infection. Other reports of infections in baboons and other primates have generally indicated more severe effects with lower parasitic burdens, e.g. Nelson and Saoud (1968), in which 750 cercariae in rhesus monkeys produced acute schistosomiasis, although these workers found that the level of anaemia was not apparently directly related to the parasitic load, or the egg densities. Others, Bruce et al., (1963) failed to detect changes in red cell numbers after exposing rhesus monkeys to a thousand cercariae of S. mansoni, despite the presence of bloody diarrhoeas. The association of a fall in haematocrit with reduced red cell survival clearly indicates that red cell loss or haemodilution are involved in the aetiology of the anaemia. The values obtained in the control animals from 51Cr labelled red cells are in agreement with previously published findings (Huser, 1970), although the cause of the reduced red cell survival is not easily explained. The maximal blood loss in the infected animals was only 3 ml per day and though this was much greater than in comparable controls it appears unlikely to be responsible for the anaemia per se. In experiments by Huser et al., (1967) normal baboons weighing approximately 9-13 kg were found to have a mean daily faecal blood loss of 3-3 ml without any evidence of either anaemia or reduced 51Cr red cell survival and in other studies on chronic blood loss in baboons (Huser, 1970) twice weekly bleeding of 5% of the estimated blood volume at regular intervals i.e. the equivalent of 30 ml per week in the present animals, failed to bring about changes in the haematocrit levels. On the other hand losses of 50 ml per week in baboons on an iron-deficient diet were sufficient to cause marked changes in haematological and erythrokinetic indices (Huser et al., 1967). There was no evidence of haemolysis in the infected animals either as a result of "haemolytic factors" (Mahmoud and Woodruff, 1972) or haematophagia by the parasites, since the urinary level of 51Cr is very similar in both groups. The ferrokinetic aspects of the experiment reveal that although there was a slight fall in the plasma iron concentration there was no evidence of any significant changes in red cell production in the parasitised baboons. A similar failure of the erythropoietic tissues to respond to schistosomal anaemia was observed in human cases by Jamra et al., (1967) and in experimental ovine schistosomiasis by Dargie and Preston (1974). The fall in serum iron appeared to be largely due to plasma volume expansion in infected animals following patency. Although the mechanism bringing about this hypervolaemia cannot be determined from the present results the finding is consistent with previous reports in primates infected with S. mansoni (Smithers and Walker, 1961). Iron deficiency due to depletion of the iron stores has commonly been associated with longstanding chronic human schistosomiasis. This must be regarded as a potentially important contributory factor in the pathogenesis of schistosomal anaemia in primates but in the present study it is unlikely that iron deficiency as a result of blood loss could develop during the short period of the experiment when the animals were on a balanced diet. Furthermore, intestinal absorption of haemoglobin iron was clearly increased in the infected baboons. In studies on experimental chronic iron deficiency anaemias in baboons (Huser et al., 1967) induced by iron deficient diets and 103
P. H. HOLMES et al.
bleeding, excessive haemolysis was demonstrated using 51Cr labelled red cells and clearly this may contribute to the anaemia observed in cases of chronic schistosomiasis. In conclusion, the results of the present experiment both confirm the complexities of schistosomal anaemia as well as the ability of primates to withstand heavy parasitic burdens of short duration with minimal pathophysiological effects. ACKNOWLEDGEMENTS We are grateful to Tony Dobinson and Joseph Smith for their technical assistance. The studies were supported by the Edna McConnell Clark and Rockefeller Foundations.
REFERENCES BRUCE, J. I., WARREN, K. S. and SADUN, E. H. (1963) Observations on the pathophysiology of schistosomiasis mansoni in monkeys. Experimental Parasitology, 13, 194—198. CHEEVER, A. W. (1968) A quantitative postmortem study of schistosomiasis mansoni in man. American Journal of Tropical Medicine and Hygiene, 17, 38-64. CHEEVER, A. W. (1969) Quantitative comparison of the intensity of Schistosoma mansoni infections in man and experimental animals. Transactions of the Royal Society of Tropical Medicine and Hygiene, 63, 781-795. DAMIAN, R. T., GREENE, N. D., MEYER, CATHERINE, CHEEVER, A. W., HUBBARD, W. J., HA WES, MARY and CLARK, J. D. (1976) Schistosoma mansoni in baboons, III. The course and characteristics of infection, with additional observations on immunity. American Journal of Tropical Medicine and Hygiene, 25. 2, 299-306. DARGIE, J. D. and PRESTON, J. M. (1974) Pathophysiology of ovine schistosomiasis, VI. Onset and development of anaemia in sheep experimentally infected with Schistosoma mattheei—-ferrokinetic studies. Journal of Comparative Pathology, 84, 83—91. FOY, H. and NELSON, G. S. (1963) Helminths in the etiology of anaemia in the tropics with special reference to hookworms and schistosomes. Experimental Parasitology, 14, 240-262. HUSER, H. J., RIBER, E. E. and BERNARD, A. R. (1967) Experimental evidence of excess haemolysis in the course of chronic iron deficiency anaemia. Journal of Laboratory and Clinical Medicine, 69, 403-414. HUSER, H. J. (1970) Atlas of Comparative Primate Haematology. Academic Press: London. JAMRA, M., MASPES, V. and MEIRA, D. A. (1964) Types and mechanisms of anaemia in Schistosomiasis mansoni. Revista do Instituto Medicina Tropicale Sao Paulo, 6, 126-136. MAHMOUD, A. A. F. and WOODRUFF, A. W. (1972) Mechanisms involved in the anaemia of schistosomiasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 75. NELSON, G. S. and SAOUD, M. F. A. (1968) A comparison of the pathogenicity of two geographical strains of Schistosoma mansoni in rhesus monkeys. Journal of Helminthology, 62, 339-362. PRESTON, J. M. and DARGIE, J. D. (1974) Pathophysiology of ovine schistosomiasis, V. Onset and development of anaemia in sheep experimentally infected with Schistosoma mattheei—-studies with 51Cr-labelled erythrocytes. Journal of Comparative Pathology, 84, 73-81. ROCHE, M., PEREZ-GIMENEZ, M. E. and LEVY, A. (1957) Isotopic tracer method for measurement of iron loss into and re-absorbed from gastrointestinal bleeding lesions. Nature; London, 162, 12781279. SADUN, E. M., LICHTENBERG, F. von and BRUCE, J. I. (1966) Susceptibility and comparative pathology of ten species of primates exposed to infection with Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene, IS, 705-718. SMITHERS, S. R. and WALKER, R. J. (1951) Serum protein changes in monkeys infested with Schistosoma mansoniwith special reference to the metabolism of albumin. ExpzrirmntalParasitology, 11, 39-49. TAYLOR, M. G., NELSON, G. S., SMITH, MARY and ANDREWS, B. J. (1973) Studies on heterologous immunity in schistosomiasis 7. Observations on the development of acquired homologous and heterologous immunity to Schistosoma mansoni in baboons. Bulletin of the World Health Organization, 49, 47-65. WARREN, K. S. (1968) Pathophysiology and pathogenesis of hepatosplenic schistosomiasis mansoni. Bulletin of the New York Academy of Medicine, 44, 280-294. WARREN, K. S. (1973) The pathology of schistosome infections. Helminthologlcal Abstracts, 42, 591-633 Accepted 6 February, 1977. 104
Erythrokinetic studies on baboons (Papio anubis) with acute experimental schistosomiasis mansoni P. H. HOLMES, E. R. JAMES*, J. M. MACLEAN, G. S. NELSON* and M. G. TAYLOR* University of Glasgow, Veterinary School, Bearsden Road, Glasgow and *London School of Hygiene and Tropical Medicine, Winches Farm Field Station, Hatfield Road, St. Albans
ABSTRACT The erythrokinetics of three baboons were studied over a twelve week period after infection with 3 000 cercariae of S. mansoni and compared with three uninfected control animals. In addition to monitoring changes in haematological and parasitological parameters several radioisotopic techniques were used concurrently to measure alterations in red cell kinetics. In particular 5iCr-labelled red cells and 59Fe-ferric citrate were used to measure red cell production, circulating volume, survival time and routes of loss. All the animals remained in good condition despite heavy infection and the pathophysiological changes were in general mild and transient in nature. The principle alterations were associated with patency and consisted of a modest fall in haematocrit, some inappetence and the appearance of blood-stained mucus in the faeces. Red cell survival was reduced in the infected animals and this was correlated with an increase in faecal blood loss and haemodilution. There was no evidence of haemolytic factors or depressed erythropoiesis having any aetiological role in the anaemia. The results confirmed the complexities of schistosomal anaemia as well as the ability of primates to withstand heavy parasitic burdens for short periods with minimal pathophysiological disturbance.
Although anaemia commonly occurs in schistosome infections of man and other animals (Foy and Nelson, 1963; Warren, 1973) many aspects of its aetiology remain controversial. It is generally accepted that gastrointestinal blood loss associated with oviposition is the most important factor, while other contributing mechanisms have also been postulated. Thus, Mahmoud and Woodruff (1972) have suggested, as a result of studies in mice, that haemolytic factors from adult worms have an important role in the causation of schistosomal anaemia. Whilst Preston and Dargie (1974) have shown in sheep, that although intestinal bleeding is the major factor in causing reduced haematocrit values, haemodilution and dyshaemopoiesis are important contributing factors. However, studies of human and experimental primate schistosomiasis have not yet established the relationship between schistosomiasis and anaemia or the relative importance of the four basic mechanisms which may be responsible for anaemia, namely, blood loss, haemolysis, haemodilution and dyshaemopoiesis (Jamra et ah, 1964). In an effort to understand the pathogenesis of experimental acute schistosomiasis in primates the erythrokinetics of baboons were studied over a twelve week period after infection with 3 000 cercariae of S. mansoni using established radioisotopic techniques (Holmes, 1969—Ph.D. thesis of Glasgow University); 51Cr-labelled red cells and 59 Ferric citrate to measure red cell production, circulating volume, survival time and routes of loss. Routine haematological and parasitological measurements were performed concurrently in an attempt to relate any changes to altered red cell kinetics. 95
P. H. HOLMES et al.
MATERIALS AND METHODS Experimental Animals Young male baboons (Papio anubis) caught in the wild and weighing approximately 5 kilograms were obtained from a local animal supplier. They were maintained in quarantine for several weeks prior to the experiments and examined for evidence of previous infections, including naturally acquired schistosomiasis. The baboons were fed a commercial primate ration supplemented with fresh fruit throughout the study, and maintained individually in standard baboon cages with grid floors and draining collection trays for the separation of urine and faeces. All infection and blood sampling procedures were carried out under sedation with Vetalar* (10 mg/kg) IM. Parasitological and Haematological Techniques The Puerto Rican strain of S. mansoni used for infection was maintained in hamsters and Biomphalaria glabrata at Winches Farm. The parasitological techniques and post-mortem procedure were similar to those described previously by Taylor et al., (1973). Faecal samples were examined at regular intervals throughout the study. Heparinized blood samples obtained from the saphenous vein were used for determination of haematocrit values and plasma iron levels. The animals were killed 14 weeks after infection. Radioisotopic Measurements Two weeks before infection and at four, seven, nine and twelve weeks after infection each baboon was intravenously injected with 51Cr labelled autologous red cells (300 /xCi s l Cr per animal) and 59Fe-ferric citrate (80 /nCi per animal). Heparinized blood samples were taken at frequent intervals during the three hours following injection and samples of blood and plasma analysed for radioactivity using a multichannel automatic gamma scintillation counter**. Analysis of the red cell and plasma radioactivities provided circulating red cell volume and plasma volume by application of the dilution principle. In addition the plasma iron clearance rate was obtained from a semi-logarithmic plot of 59Fe activity against time over the first three hours, and used to calculate the plasma iron turnover rate (PITR). Between seven and twelve weeks after infection daily collections of urine and faeces were obtained, along with thrice weekly blood samples. Analysis of the radioactivities of the blood samples provided the red cell survival time and iron incorporation rate. Faecal "blood clearances" were calculated from each daily collection by dividing the total daily faecal radioactivity by the activity of the blood during the collection period. These values represent the amount of blood which has to appear in the gut to account for the faecal radioactivity. Urinary blood clearances were calculated in a similar manner. RESULTS Parasitological and Histopathological Findings Schistosome eggs were detected in the faeces of the infected baboons five weeks after infection (Fig. 1) and remained at a level of approximately 300 e.p.g. until week 12 when a fall to less than 200 e.p.g. was observed. The adult worm recovery and distribution of tissue eggs at post-mortem 14 weeks after infection are presented in Table 1. The mean adult worm burden in the three infected baboons was 717 worms, i.e. a mean recovery rate of 24 %. There was a mean of 7.3 x 103 eggs * Ketamine hydrochloride, Parke-Davis & Co. ** Nuclear Chicago, High Wycombe, England. 96
Erythrokinetics of baboons with S. mansoni
per female adult worm in the tissues and most of the eggs (mean 56 %) were detected in the large intestine with 35 % in the small intestine and 9 % in the liver. Histopathology of the liver, lungs, large and small bowel revealed active infections in all three baboons with minimal damage to the liver and extensive lesions in the large bowel and two animals showed some signs of schistosomal pneumonitis. There were a few haemorrhagic lesions in the bowel. There was no evidence of portal hypertension and the kidneys and spleen were normal. The control animals were not sacrificed at the end of the experiment.
P-m'
300-
200-
100-
15
10
FIG. 1. Faecal egg output from baboons infected with S. mansoni. Vertical axis = eggs/gram; horizontal axis = weeks after infection. TABLE 1 Worm burdens and tissue egg counts of baboons 14 weeks after infection with 3000 cercariae of S. mansoni Baboon no. 75 76 77 Mean
Individual worm burdens Total Total Final total 3 % ? % 207 (35%) 379 (65%) 586 217 (35%) 416 (65%) 633 342 (37%) 590 (63%) 932 462 (64)
255 (36)
Total tissue eggs/? worms
Individual total tissue egg counts (x 103)
717
( X 103)
1906-3 1229-2
9-21 5-66
1567-7
7-44
Distribution of tissue eggs per gram Baboon no. 75 76 77
Liver Total %
Small intestine Total %
Large intestine Total /o
1433 645
917 5884 4348
9592 3115 6754
12-0 6-7
7-7 61-3 —
80-6 32-4 97
P. H. HOLMES et al.
Haematological and Clinical Changes All the animals remained in good condition throughout the experiment with only scanty clinical signs of schistosomiasis in the infected groups. The main effects were observed between six and nine weeks after infection when there was a fall in the haematocrit of the infected animals coinciding with the appearance of bloodstained mucus in the faeces. There was a moderate degree of inappetence and a slight fall in body weight (Fig. 2). However, by 12 weeks after infection, the parasitized animals had reverted to a condition similar to that seen before infection.
Red Cell Survival The disappearance rates of 51Cr-labelled and 59Fe-labelled red cells were measured between nine and twelve weeks after infection and the results are presented in Table 2. It is
D
o-
O
-O
o
°
°
10
12
FIG. 2. Body weight and haematocrit changes in baboons infected with S. mansoni. Vertical axis upper = PCV, lower = kg; horizontal axis = weeks after infection. Key 0 - - - 0 = infected 0——% = control. 98
Erythrokinetics of baboons with S. mansoni TABLE 2 Red cell survival in baboons infected with S. mansoni
I N F E C T E D
Baboon no.
5iCr-red cell* Tidays
"Fe-red cell Tidays
75
13-8
50-7
76
15-0
56-8
77
9-6
86-0
12-8
64-5
68
16-6
244-9
74
16-3
1211
81
17-7
399-7
16-9
255-2
Mean C O N T R O L Mean 1
9-12 weeks after infection
apparent that red cell survival expressed as a half-life (T£) was markedly reduced in the infected baboons as measured by both techniques. The discrepancy between the 51Cr and 59 Fe results in both infected and control animals is due to the elution of 51Cr from labelled red cells thereby giving an abnormally shortened survival time when measured by 51Cr and the in vivo reincorporation of 59 Fe into new red cells thereby giving an overestimate of red cell survival time when measured by 59 Fe. So whilst neither method gives a "true" half-life, they nevertheless provide a valuable indication of red cell survival. Excretion of 51Cr The shortened red cell half-lives of radioisotopically labelled red cells demonstrated above indicate an increased rate of removal of red cells from the circulation of infected animals but give no evidence of how this situation arises. The decrease in red cell survival may be due to either intravascular breakdown of erythrocytes, or losses into the gut. The relative importance of these two possibilities can only be assessed by a quantitative examination of the routes of loss of 51Cr since 51Cr appears quantitatively in the faeces following loss into the gastrointestinal tract or in the urine after intravascular breakdown of labelled red cells. The results of faecal "whole blood" clearances are illustrated in Fig. 3 and show that the infected baboons were losing significantly greater amounts of blood into the gastrointestinal tract than the controls. 99
P. H. HOLMES el al.
On the other hand examination of the urinary "whole blood" clearance failed to reveal any significant differences between the infected animals (mean daily clearance 5-3 ml) and the controls (mean daily clearance 8-1 ml).
3i
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FIG. 3. Mean daily faecal blood clearance of baboons infected with 5. mansoni. Vertical axis = faecal blood clearance (ml); horizontal axis = weeks after infection. Key 0 — 0 = infected, upper 5 iCr and lower 59Fe I
= control, upper 51Cr and lower 59Fe
Ferrokinetics
The possible importance of dyshaemopoiesis as a causative factor in schistosomal anaemia was assessed by examination of the plasma iron half life. In addition, gastrointestinal loss of iron and reabsorption of haemoglobin iron were also measured. The values for plasma iron levels, plasma iron clearance and plasma iron turnover rate are shown in Fig. 4. Despite the results being somewhat variable they do indicate that there was no marked differences in the plasma iron clearance or plasma iron turnover rate between the infected and control animals. However, there was evidence of a fall in the serum iron level during the course of the infection in the parasitized baboons. The differences between the faecal losses of 51Cr and 59 Fe in the infected animals shown in Fig. 3 indicate that during the period of maximal gastrointestinal blood loss there was considerable reabsorption of haemoglobin iron (Roche, et al, 1957). 100
Erythrokinetics of baboons with 5. mansoni
160n 140120-
y
\
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\
o
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FIG. 4. Plasma iron concentration, half-life and turnover rate in baboons infected with 5. mansoni. Vertical axis, upper = Fe concentration (tig%), middle = TJ (mins) and lower = plasma iron turnover rate ); horizontal axis = weeks after infection. Key 0 — 0 = infected # — — # = control.
Plasma, Circulating Red Cell and Blood Volumes Measurements were made of plasma, circulating red cell and blood volumes at 0, 4, 7, 9 and 12 weeks after infection and the results are presented in Fig. 5. It is apparent that several changes occurred in these volumes as the infection became patent. Initially there was little difference between the infected and control animals but by 7 weeks after infection a hypervolaemia developed in the parasitized groups due to an increase in the plasma volumes of these animals. There was however no significant change in the circulating red cell volume, thus indicating that the fall in whole body haematocrit was clearly associated with a haemodilution effect. 101
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70
60
C
rfl
I
n
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FIG. 5. Plasma, circulating red cell, and blood volumes in baboons infected with S. mansoni. Vertical axis = ml/kg; horizontal axis = Weeks after infection. Key
= Plasma volume;
= Red cell volume;
= Total blood volume;
I = Infected, C = Control.
DISCUSSION In this experiment the erythrokinetics of baboons experimentally infected with 3000 cercariae of S1. mansoni were studied for twelve weeks post-infection. The results indicated that despite heavy parasitic burdens the clinical haematological changes associated with the infection were relatively mild and of a transient nature. The main changes were associated with oviposition and consisted of transient inappetance and a slight fall in haematocrit. The mild anaemia was found to be the result of some minor changes in the erythrokinetic status of the parasitised baboons. Namely, reduced red cell survival associated with increased faecal blood loss and haemodilution. There was no evidence of increased haemolysis or of changes in erythropoiesis. The value of baboons as experimental hosts for schistosomiasis has been established (Sadun, et ah, 1966) yet these animals have not been used to examine the pathogenesis of anaemia in schistosomiasis except for the incidental observations by Damian et al., (1976). 102
Erythrokinetics of baboons with S. mansoni
This may be partly because along with other models there is a considerable discrepancy between the worm burdens required to produce symptoms of schistosomiasis in animals and the parasitic burdens found in human cases of schistosomiasis (Cheever, 1969). This is particularly evident in the present experiment in which an average of 40 S. mansoni worm pairs per kg failed to produce marked symptoms in the baboons, yet human infections of more than 5 worm pairs per kg are rarely encountered (Cheever, 1968). In the present study changes were associated with the patency of the schistosome infection, although the mildness of these changes was surprising in view of the level of infection. Other reports of infections in baboons and other primates have generally indicated more severe effects with lower parasitic burdens, e.g. Nelson and Saoud (1968), in which 750 cercariae in rhesus monkeys produced acute schistosomiasis, although these workers found that the level of anaemia was not apparently directly related to the parasitic load, or the egg densities. Others, Bruce et al., (1963) failed to detect changes in red cell numbers after exposing rhesus monkeys to a thousand cercariae of S. mansoni, despite the presence of bloody diarrhoeas. The association of a fall in haematocrit with reduced red cell survival clearly indicates that red cell loss or haemodilution are involved in the aetiology of the anaemia. The values obtained in the control animals from 51Cr labelled red cells are in agreement with previously published findings (Huser, 1970), although the cause of the reduced red cell survival is not easily explained. The maximal blood loss in the infected animals was only 3 ml per day and though this was much greater than in comparable controls it appears unlikely to be responsible for the anaemia per se. In experiments by Huser et al., (1967) normal baboons weighing approximately 9-13 kg were found to have a mean daily faecal blood loss of 3-3 ml without any evidence of either anaemia or reduced 51Cr red cell survival and in other studies on chronic blood loss in baboons (Huser, 1970) twice weekly bleeding of 5% of the estimated blood volume at regular intervals i.e. the equivalent of 30 ml per week in the present animals, failed to bring about changes in the haematocrit levels. On the other hand losses of 50 ml per week in baboons on an iron-deficient diet were sufficient to cause marked changes in haematological and erythrokinetic indices (Huser et al., 1967). There was no evidence of haemolysis in the infected animals either as a result of "haemolytic factors" (Mahmoud and Woodruff, 1972) or haematophagia by the parasites, since the urinary level of 51Cr is very similar in both groups. The ferrokinetic aspects of the experiment reveal that although there was a slight fall in the plasma iron concentration there was no evidence of any significant changes in red cell production in the parasitised baboons. A similar failure of the erythropoietic tissues to respond to schistosomal anaemia was observed in human cases by Jamra et al., (1967) and in experimental ovine schistosomiasis by Dargie and Preston (1974). The fall in serum iron appeared to be largely due to plasma volume expansion in infected animals following patency. Although the mechanism bringing about this hypervolaemia cannot be determined from the present results the finding is consistent with previous reports in primates infected with S. mansoni (Smithers and Walker, 1961). Iron deficiency due to depletion of the iron stores has commonly been associated with longstanding chronic human schistosomiasis. This must be regarded as a potentially important contributory factor in the pathogenesis of schistosomal anaemia in primates but in the present study it is unlikely that iron deficiency as a result of blood loss could develop during the short period of the experiment when the animals were on a balanced diet. Furthermore, intestinal absorption of haemoglobin iron was clearly increased in the infected baboons. In studies on experimental chronic iron deficiency anaemias in baboons (Huser et al., 1967) induced by iron deficient diets and 103
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bleeding, excessive haemolysis was demonstrated using 51Cr labelled red cells and clearly this may contribute to the anaemia observed in cases of chronic schistosomiasis. In conclusion, the results of the present experiment both confirm the complexities of schistosomal anaemia as well as the ability of primates to withstand heavy parasitic burdens of short duration with minimal pathophysiological effects. ACKNOWLEDGEMENTS We are grateful to Tony Dobinson and Joseph Smith for their technical assistance. The studies were supported by the Edna McConnell Clark and Rockefeller Foundations.
REFERENCES BRUCE, J. I., WARREN, K. S. and SADUN, E. H. (1963) Observations on the pathophysiology of schistosomiasis mansoni in monkeys. Experimental Parasitology, 13, 194—198. CHEEVER, A. W. (1968) A quantitative postmortem study of schistosomiasis mansoni in man. American Journal of Tropical Medicine and Hygiene, 17, 38-64. CHEEVER, A. W. (1969) Quantitative comparison of the intensity of Schistosoma mansoni infections in man and experimental animals. Transactions of the Royal Society of Tropical Medicine and Hygiene, 63, 781-795. DAMIAN, R. T., GREENE, N. D., MEYER, CATHERINE, CHEEVER, A. W., HUBBARD, W. J., HA WES, MARY and CLARK, J. D. (1976) Schistosoma mansoni in baboons, III. The course and characteristics of infection, with additional observations on immunity. American Journal of Tropical Medicine and Hygiene, 25. 2, 299-306. DARGIE, J. D. and PRESTON, J. M. (1974) Pathophysiology of ovine schistosomiasis, VI. Onset and development of anaemia in sheep experimentally infected with Schistosoma mattheei—-ferrokinetic studies. Journal of Comparative Pathology, 84, 83—91. FOY, H. and NELSON, G. S. (1963) Helminths in the etiology of anaemia in the tropics with special reference to hookworms and schistosomes. Experimental Parasitology, 14, 240-262. HUSER, H. J., RIBER, E. E. and BERNARD, A. R. (1967) Experimental evidence of excess haemolysis in the course of chronic iron deficiency anaemia. Journal of Laboratory and Clinical Medicine, 69, 403-414. HUSER, H. J. (1970) Atlas of Comparative Primate Haematology. Academic Press: London. JAMRA, M., MASPES, V. and MEIRA, D. A. (1964) Types and mechanisms of anaemia in Schistosomiasis mansoni. Revista do Instituto Medicina Tropicale Sao Paulo, 6, 126-136. MAHMOUD, A. A. F. and WOODRUFF, A. W. (1972) Mechanisms involved in the anaemia of schistosomiasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 75. NELSON, G. S. and SAOUD, M. F. A. (1968) A comparison of the pathogenicity of two geographical strains of Schistosoma mansoni in rhesus monkeys. Journal of Helminthology, 62, 339-362. PRESTON, J. M. and DARGIE, J. D. (1974) Pathophysiology of ovine schistosomiasis, V. Onset and development of anaemia in sheep experimentally infected with Schistosoma mattheei—-studies with 51Cr-labelled erythrocytes. Journal of Comparative Pathology, 84, 73-81. ROCHE, M., PEREZ-GIMENEZ, M. E. and LEVY, A. (1957) Isotopic tracer method for measurement of iron loss into and re-absorbed from gastrointestinal bleeding lesions. Nature; London, 162, 12781279. SADUN, E. M., LICHTENBERG, F. von and BRUCE, J. I. (1966) Susceptibility and comparative pathology of ten species of primates exposed to infection with Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene, IS, 705-718. SMITHERS, S. R. and WALKER, R. J. (1951) Serum protein changes in monkeys infested with Schistosoma mansoniwith special reference to the metabolism of albumin. ExpzrirmntalParasitology, 11, 39-49. TAYLOR, M. G., NELSON, G. S., SMITH, MARY and ANDREWS, B. J. (1973) Studies on heterologous immunity in schistosomiasis 7. Observations on the development of acquired homologous and heterologous immunity to Schistosoma mansoni in baboons. Bulletin of the World Health Organization, 49, 47-65. WARREN, K. S. (1968) Pathophysiology and pathogenesis of hepatosplenic schistosomiasis mansoni. Bulletin of the New York Academy of Medicine, 44, 280-294. WARREN, K. S. (1973) The pathology of schistosome infections. Helminthologlcal Abstracts, 42, 591-633 Accepted 6 February, 1977. 104
