51 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE

SCHISTOSOMA

ANAEMIA MANSONI

AND HYGIENE. Vol. 69. No. 1. 1975.

IN MICE WITH CONCOMITANT AND PLASMODIUM BERGHEI YOELll INFECTION RACHEL

LEWINSOHN*

Department of Internal Medicine, Faculty of Medicine, Federal University of Rio de Janeiro, Brazil. Lately of Hospital for Tropical Diseases, London

Schistosomiasis and malaria have a very similar geographical distribution in many areas of the tropics. Both diseases are considered to contribute heavily to the anaemia so common in tropical countries (WOODRUFF, 1970, 1972, 1973; BARRETT-CONNOR, 1972; WALTERS, 1972). Since there can be little doubt that they occur together in man with some frequency, the question arose as to how severe is the anaemia found in concomitant schistosomiasis and malaria, as opposed to infection with either. A great deal of the experimental work on malaria (reviews by ZUCKERMAN, 1964, 1969; BROWN, 1969; SCHROEDERand RISTIC, 1968; MCGREGOR,1971, 1972) has centred on the severity of the anaemia not commensurate with the parasitaemia found in most of the malarias. Many researchers consider the evidence to be strong in favour of the hypothesis that malarial anaemia is essentially due to an autoimmune haemolytic process, a view not shared, however, by MCGREGOR(1972). In 1966, Woodruff and co-workers showed that the life-span of red blood cells is shortened in patients with schistosomiasis and gross splenomegaly (WOODRUFF et al., 1966). This work was followed by the demonstration that haemolytic anaemia develops in mice experimentally infected with Schistosoma mamom (MAI-IM~IJD and WOODRUFF, 1972). By analogy with the conclusions reached in studies on two protozoa1 diseases,in which the red blood cells are not parasitized (KNIGHT, WOODRUFF and PETTITT, 1969; WOODRUFF et al., 1969,1972). it was suggested that an autoimmune process might contribute to the anaemia in schistosomiasis. ONGOM'S study (1972) on “Interaction between S. mansoni and P. berghei yoelii in mice”, was not concerned with haematological criteria. Indeed, no work seems to have been done hitherto on anaemia in concomitant schistosomiasis and malaria, in man or mouse. The questions asked in the present study were: (a) Is there any interaction between schistosomiasis and malaria in the mouse? (b) If so, what is the effect of this interaction on the anaemia observed in the concomitant infection? Materials and methods 109 male Swiss TO mice, 4-5 weeks old, were divided into 8 groups, as shown in the Table Outline of the Experiment. Baseline controls on Day 0 of the experiment consisted of 2 mice taken at random from 6 groups. They were killed, and investigations carried out as described for the other mice used in the experiment. Infection with S. mansoni

4 groups (I, II, V and VI) were exposed by the ring method to about 50 cercariae per mouse. A cercarial suspension of S. mansoni had been prepared for this purpose at Winches Farm, the Field Station of the London School of Hygiene and Tropical Medicine, where infection took place, Infection with P. berghei yoelii

3 weeks (groups I, III) and 5 weeks (groups V, VII) after exposure to cercariae, the mice were infected imraperitoneally with lo6 cells parasitized with Plasmodium berghei yoelii, strain 146-X, obtained from the NufKeld Institute of Comparative Medicine, London, by blood syringe-passaged into 3 mice. *Grateful acknowledgement is made to Professor A. W. Woodruff for providing facilities in his department; to Dr. I?. E. C. Manson-Bahr, Dr. H. A. K. Rowland, Dr. R. Knight and Dr. W. E. Ormerod for invaluable guidance and help, Dr. Voller for supplying the plasmodia, Dr. C. James for the cercariae and for carrying out the schistosomal infection of the mice, and Mr. L. E. Pettitt and Mr. H. Furze for providing facilities in their laboratories and technical assistance. Address for reprints - Rua Esteres Junior 62, Apto. 603, 20000 Rio de Janeiro, GB. ZC - 01, Brazil.

52

ANAEMIA

IN MICE

WITH

CONCOMITANT

OUTLINE

S. MANSONI

OF

P.b.y. c 2t 2t

c

P.b.y. 2t 2t

V

I5

!Al.

VI

I5

cm.

VII

I5

l

VIII

I2

--2

AND P. BERGHEI

YOELII

INFECTION

EXPFRIMENT

2 t etch week

2 t each wee6

2 t etch

*

week

t each week--

P.b.y. --2t 2t 2t

-

P.b.y. * 2t 2t

--2t

each week

I

2t each week

2t etch

week

c

etch

weex

w

I

S. m. = S. mansoni P.b.y.= P. berahei

voelii

(A)

2 mice killed

ItI

mice killed

Throughout

as baseline

experiment,

controls

all mice were bled

3 times a week.

Investigations

3 times weekly, small samples of blood were taken from all the mice. Parasitaemia was estimated by examination of thin films stained with Giemsa; reticulocyte counts were done on thin films stained with new methylene blue and counter-stained with Giemsa. At weekly intervals, 2 animals of each group were killed, the aortic arch transected and the blood

RACHEL LEWINSOHN

53

removed by heparinized Pasteur pipette, It was centrifuged, and the plasma separated and kept frozen for further use. Haemoglobin estimation was carried out by photo-electric calorimetry (cyanomethaemoglobin method). The packed cell volume (PCV) was estimated by means of the Hawksley centrifuge micro-haematocrit method. Red blood cells were counted in a Neubauer chamber. Immediately before being killed, the mice were weighed under anaesthesia. They were then bled and killed, and the spleens and livers removed and weighed separately. The livers were examined for mature worms and eggs. Indirect fluorescent antibody tests (IFATs) as described by VOLLER (1964) and KANE et al., (1971) were carried out on the sera of infected mice. Antigen slides were prepared with S. mans& cercariae obtained from Winches Farm, and with blood from mice infected with the same strain of P. b. yoelii as that used in the experiment. Factorial

design of the investgation

A factorial design was thought to be the best means of making use of experimental units. The advantages of such a design are discussed in depth by ROWLAND (1966, 1971). The information sought in the experiment concerned the effect of 3 factors on a number of observations made on mice, viz. : (A) Schistosomiasis (S. mansoni) (B) Malaria (P. berghei yoelii) (C) Duration: interval between exposure to cercariae and infection with malaria (3 weeks/5 weeks). There is, indeed, a further factor, namely the time after the start of malaria infection; this is included as the abscissa in the graphs. The effect of the factors was estimated on each of the following indices: (a) Haemoglobin level (b) Packed cell volume (c) Splenomegaly, i.e., spleen weight expressed as a percentage of body weight (d) Reticulocyte count (e) Parasitaemia. Indices (c) and (d) are not, strictly speaking, criteria for measuring anaemia, but were included because they furnish valuable ancillary information. For each index a Table as illustrated by ROWLAND (1966, 1971) was compiled, the entries being mean values. Results The large number of results obtained precluded their individual presentation in the form of Tables, conventional or factorial. The data are therefore presented in graphical form (Figs. 1 to 5). Except for the control groups, all the mice became infected, as evinced by parasitaemia andfor recovery of worms and eggs from the livers. Severe parasitaemia developed in all the mice given malaria (Figs. l-a, b, c) and reached its peak by week 3. No effect of either disease on the course of the other (“interaction”) could be deduced from the parasitaemia (Fig. l-b). Parasitaemia was higher in the mice given malaria 3 weeks after exposure to cercariae than in those given malaria 5 weeks after exposure to cercariae (Fig. l-c). Figs. 2-a and 2-b show that the effect of schistosomiasis on the anaemia produced in the mice was negligible, and its effect on the reticulocytosis and splenomegaly observed in the mice (Figs. 2-q d) was nil. Figs. 3-a and 3-b, on the other hand, demonstrate that the effect of malaria on the anaemia was considerable,though short-lived. Its course was comparable to, though not coincident with, the parasitaemia. Reticulocytosis and splenomegaly show similar readings (Figs. 3-c, d). Fig. 4 indicates that in the present study, neither disease had any effect on the other in influencing the course of the anaemia (Figs. 4-a, b), reticulocytosis or splenomegaly (Figs. 4-c, d).

54

ANAEMIA

IN MICE

WITH

CONCOMITANT

S. MANSONI

AND P. BERGHEI

YOELII

INFECTION

It can be seen from Fig. 5 that moderate splenomegaly was observed in the animals by the end of the experiment. The IFAT gave positive readings on all the sera tested, but no conclusion could be reached as to a correlation between titres and haematological data. Discussion

and conclusions

It is evident from Figs. 2, 3, 4 and 5 that malaria alone had an effect in the present investigation. Anaemia was severe for a short period in the animals with mixed infection and in those with malaria alone, but blood values returned to normal after clearance of malarial parasites from the blood, and the mice recovered without apparent ill-effect. Reticulocytosis and splenomegaly consistently corroborated the haematological findings. The fact that all the phenomena observed, i.e., anaemia, reticulocytosis and splenomegaly, were additive is brought out most strikingly in Figs. 4-a to 4-d. It may be concluded that in the present study, neither disease was affected in its course by the presence of the other, nor by the length of the interval between exposure to cercariae and infection with malaria (Fig. 5). As to the higher parasitaemia observed in the mice given malaria 3 weeks after exposure to cercariae (Fig. l-c), one of the reasons for the discrepancy may well be the great variability of values found in any kind of experimental work on rodents (HARDY, 1967).

( (1) HAEMOGLWN (01 MEAN

OF ALL

GROUPS

(b)

;i

(b)

INTERACTION

L

(concentr

1

WITH MALARIA

PACKED

CELL VOLUME

( e. g. EFFECT DF SCtUSTQ SOMIASIS ON MALARIA )

Cc) RETlCULOCYTOSlS

(c) 4D r

EFFECT OF MATION 3 WEEKS AGAINST SINCE CERCARIAL

(INTERVAL 5 WEEKS INFECTION)

Of

( d 1 SPLENOMEGALY

0

I Weeks

2 sine*

3 malaria

4 infection

FIG. 1. Parasitaemia.

5 Weeks

since

malorio

mfection

FIG. 2. Effect of schistosomiasis.

RACHEL ( o 1 HAEMOGLOSIN

(concentr

55

LEWINSOHN

)

16

(a)

[I

HAEMOGLOSM

(COI-ICE~~~)

S. mansod + p: h welit Mixed infection

(b)

PACKED

+ Cmtrol

CELL VOLUME

44 44

36

(b) I-

PACKED

CELL

(c)

RETKXLOCYTOSIS

MWME

( c ) RETICULOCYTOSIS SO

( d 1 SPLENOMEGALY

(d 1 SPLENOMEGALY =

2.6

ws 0

I Weeks

2 3 4 since malaria infection

01

5

r

0

I Weeks

FIG. 4. Interaction

FIG. 3. Effect of malaria.

(d)

0 PACKED

5

(e.g., effect of schistosomiasis on effect of malaria).

SPLENOMEGALY

CELL VOLUME

;(k*

0’

4 infection

E (bl

I

2 3 smc4 mloria

,,;,,,

I

FIG. 5. Effect of duration

I

I

(interval

i---j

I

zwx’.:::

I

~

:!opT;

:c:::

Weeks

since

molorio

infecTmn

of 3 weeks against 5 weeks between cercarial and plasmodial

infection).

56

ANAEMIA IN MICE WITH CONCOMITANT S.

MANSONI

AND

P. BERGHEI

YOELII

INFECTION

It has been shown (JORDAN, 1972; MANSON-BAHR, 1970; MANSON’S TROPICAL DISEASES, 1972) that in the early stages of schistosomiasis and malaria in the human host, the anaemia and splenomegaly are reversible. Similar observations were made in the present study on mice. Summary

1. The effect on anaemia in mice given Plasmodium berghei yoelii 3 and 5 weeks after exposure to Schistosoma mansoni cercariae, was investigated. 2. Haematological criteria (PCV and haemoglobin levels), reticulocytosis, parasitaemia and splenomegaly were used as indices. 3. Anaemia was severe in the animals given P. 6. yoelii and in those with mixed infection (P. b. yoelii -t S. mansoni). Malaria was found to dominate the picture until the clearance of the parasitaemia. The effect of the interaction between the diseases on the anaemia was nil. 4. Toward the end of the experiment, moderate splenomegaly was observed in the mice with mixed infection. REFERENCES BARRETT-CONNOR, E. (1972). Am. J. Med., 52, 242. BROWN, I. N. (1969). Advances in Immunology (F. J. Dixon, Jr. and H. G. Kunkel, eds.) Vol. 11, 267. New

York and London: Academic Press.

HARDY, J. (1967). Pathology of Laboratory

Rats and Mice. (E. Cotchin and F. J. C. Roe, eds.), p. 504. Oxford and Edinburgh : Blackwell. JORDAN, P. (1972). Schistosomiasis and disease. In Schistosomiasis, Proceedings of a Symposium on the Future of Schistosomiasis Control. (Max J. Miller, ed). New Orleans: Tulane University, La., U.S.A. KANE, G. J., MATOSSIAN, R. & BATTY, I. (1971). Ann. N.Y. Acad. Sk., 177, 134. KNIGHT, R., WOODRUFF,A. W. & PETTITT, L. E. (1967). Trans. R. Sot. trop. Med. Hyg., 61, 701. MCGREGOR, I. A. (1971). International Review of Tropical Medicine (A. W. Woodruff and D. R. Lincicome,

eds.)., 4, 1. New York and London: Academic Press.

(1972). Br. med. Bull., 26, 1, 22. MAHMOUD, A. A. F. & WOODRUFF,A. W. (1972). Trans. R. Sot. trop. Med. Hyg., 66, 75. MANSON-BAHR, P. E. C. (1970). Alimentary and Haematological Aspects of Tropical Disease. (A, W. Woodruff,

ed.), p. 400. London: Edward Arnold.

MANSON’S TROPICAL DISEASES (1972). 17th ed. (C. Wilcocks and P. E. C. Manson-Bahr, eds.). Malaria, p. 38; Schistosomiasis, p. 285. London: Bailliere-Tindall. ONGOM, V. L. (1972). Interaction between S. mansoni and Plasmodium b. yoelii in mice. M.Sc. Thesis. University of London. ROWLAND, H. A. K. (1966). Trans. R. Sot. trop. Med. Hyg., 60, 143. (1971). International Review of Tropical Medicine (A. W. Woodruff and D. R. Lincicome, eds.), 4, 175. New York and London: Academic Press. SCHROEDER,W. F. & RISTIC, M. (1968). Infectious Blood Diseases of Man and Animals (D. Weinman and M. Ristic, eds.). 1, p. 63. New York and London: Academic Press. VOLLER, A. (1964). Bull. Wld Hlth Org., 30, 343. WALTERS, J. H. (1972). In Munson’s Tropical Diseases (C. Wilcocks and P. E. C. Manson-Bahr, eds.). 17th ed., p. 21. London: Bailliere-Tindall. WOODRUFF, A. W. (1970). In Alimentary and Haematological Aspects of Tropical Disease (A. W. Woodruff, ed.), p. 207. London: Edward Arnold. (1972). Br. med. Bull, 28, 92. (1973). Trans. R. Sot. trop. Med. Hyg., 67, 313. -, KNIGHT, R. & PETTITT, L. E. (1969). “Unpublished results” (quoted in Mahmoud and Woodruff 1972). --, SHAFEI, A. Z., AUTWAD, H. K., PETTITT, L. E. & ABAZA, H. H. (1966). Trans. R. Sot. trop. Med. Hyg., 60, 343. -, TOPLEY, E., KNIGHT, R. & DOWNIE, C. G. B. (1972). Bri. J. Haemat., 22, 319. ZUCKERMAN, A. (1964). Expl. Parasit., 15, 138. (1969). Bull. Wld Hlth Org., 40, 55.

Anaemia in mice with concomitant Schistosoma mansoni and Plasmodium berghei yoelii infection.

1. The effect on anaemia in mice given Plasmodium berghei yoelii 3 and 5 weeks after exposure to Schistosoma mansoni cercariae, was investigated. 2. H...
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