Studies on the anaemia in rabbits infected with Trypanosoma brucei brucei

J.

COMP.

PATH.

1980

STUDIES

2.

VOL.

ON WITH

123

90

THE ANAEMIA TRY’PANOSOMA

HAEMATOLOGICAL

IN RABBITS INFECTED BRUCEI BRUCEI

STUDIES

ON

THE

ROLE

OF

THE

SPLEEN

BY P.

RICC:RORIE,

G.

C.

JENKINS,

J.

L.

BROWN

AND

C. E.

RAMSEY

INTRODUCTION

One of the most obvious clinical signs of trypanosomiasis is splenomegaly. Laveran and Thiroux (1907) considered the role of the spleen to be the removal of parasitic debris from the circulation following trypanolytic crises. Fiennes I 1954) expressed the opinion that the spleen is closely involved in the development of anaemia in cattle infected with Trypanosomacongolenseor 1. rivax, and that both pooling and destruction of red cells occur there. Boycott and PriceJones (1913), on the other hand, found little evidence of erythrophagocytosis above normal in the spleens of rabbits infected with 7. brucei. Anosa (1975) found that, in mice infected with T. 6. brucei, removal of the spleen increased the degree of anaemia but emphasized that this was largely due to the contribution played by the mouse spleen towards erythropoiesis (approximately half the increase in erythropoiesis which occurs in the bled mouse takes place in the spleen; Boggs, Geist and Chervenick, 1969). Similarly, in sheep infected with r. ~liz~~x,Anosa (1977) found that splenectomized animals became more anaemic than intact animals. The present study M’as undertaken to assessthe role of the spleen in rabbits infected with 7. b. bntcei (S42 and 427 stocks), particularly with respect to the developing anaemia. METHODS

Details of the breed of rabbits, the stocks of trypanosomes, their preparation and mode of injection into the host animals and the methods used for the measurement of haematological parameters are as described in the previous paper of this series (Jenkins, McCroric, Forsberg and Brown, 1980). Splenectong

Each rabbit was anacsthctized with pcntobarbitone* i.v. at a dose of 30 mg per kg. It was shaved on its left flank and a 5 cm incision made longitudinally below the left costal margin. The spleen was identified and mobilized. Its vessels were then clamped and ligated and the organ removed. After haemostasis had been achieved, the C:orrespondence to l>r P. &lcCrorie. * Ncmbutal, OO:!l--9975/80/010123

Abbott. / 15 .$02.00/O

$=!I 1980 .\crtdrmic

Press Inc.

(Lorldorl)

Limited

P. MCCRORIE;

124

et al.

peritoneum and abdominal muscleswere clohrd in layers. Interrupted xilk sutures were usedfor the skin incision. and the rabbits were bandaged to avoid cxposurc ofthr wound. Organ Counling

%r

Rabbit red cells were labelled with 20 /A Ii ;I( :r, ax dcscrihed li)r rrd c.t.11hur\.ival studies, either 30 min or 3 to 4 weeks before killing, as drscribcd in th(s trrt. .Af’tc*t death, the spleen and liver were removed, blotted and weighed. SampIps of known weight were homogenized in 5 ml of O-9 per c:cnt NaCl solution. ‘1’1~radioactivity irl the sampleswere then counted on a Packard ;\utogannna sprclrotnct~r. ‘I’hc r<~ult\ were expressedas a ratio of total spleen count to total liver count.

S$denectom-y bcfofbreInjktiolr Changes in red cell counts, haemoglobin c.onc.cntratiolls. packccl ~~11\,olutnch. mean cell volumes and reticulocyte concentrations in splencctomizcd control rabbits, splenectomized infected rabbits and non-splencctomizcd infccrcd rabbits are shown for both stocks of T. h. hrzrc~i used, S42 and J.27, in Figs. 1

to

5.

It can be seen that splenectomy not o111):delayed the onset of anaemia 1,). several days but, especially in the rabbits inlccted bvith the 427 stock, markedl) lessened its severity. Reticulocyte concentrations were sipificar~tly lower in splenectomized infected rabbits-indeed in the 427 infection thcrc. was scarcrl) any increase in their concentration relative to the non-splenectomized infected rabbits. Consequently, with respect to the S42 infection, the PC:\’ did eventuall) fall to about the same figure in both splencctomized and non-splcnectomized animals, even though the former were less anaemic in terms of red ccl1 count and Hb concentration. MCH and MC:HC: measurements remained normal. Parasitaemias were significantly highrr in the splenectomized rabbits (Figs. 6 and 7). This was particularly noticeable Lvith stock -1-27,avherc often about ten times as many parasites were clctected in the peripheral blood at any time during a parasitaemic peak as \vcre detected in non-splenectomized animals. Massive parasitaemias (100 to 200 organisms per field) were often recorded terminally in both splenectomizcd and intact animals infected with the S42 stock. Splenectomized rabbits ilrfccted Lvith this organism did IIOI survive as long as non-splenectomized animals jabout a week less, on a\:cragel. Labelling of autologous red cells with 51C;rshowed that splenectomy protrcted infected animals from a reduction in red cell life span (Figs 8. and 9; Table 1). Red cells from rabbits infected with the S42 stock survived for a slightly shorter period than those from rabbits infected with the 427 stock. Splenectomyafter Infection Nine animals were splenectomized after having been infected with

7. 6.

brucei 427, three on the 4th day after infection, two on day 7 and one each

on

days 14, 16, 22 and 31. Five of the rabbits died during the first parasitaemic peak after splenectomy, which rose to around 100 trypanosomes per field within

ANAEMIA

IN

RABBITS

INFECTED

WITH

7.

h.

brucei

125

IlOl

40. 2 ;

30. 20.

ki (L .-0 ..c

IO .

S42

OO

5

IO

15

20

25

30

35

40

45

50 I

80 70 60

[ . . . .3

50 40. 30 20

0

427

5

IO

I5

20

25

30

35

40

45

50

2 to 7 days. Three rabhits (tivo splencctomized on day 4, one on day 7) survived the first wake of parasitacmia, but died during the second (again, a massi\-c parasitaemia 11 to 16 days after splencctomy). Only one rabbit, splenertomizcd on day -1, survived the normal period for such an infection. It survi\ect 5 parasitaemias, none of them showing more than 4 trypanosomes per field. Figure 10 depicts the survival of 5iCA- labellcd autologous red cells in tkvo of the rabbits which were splenectomized mid-infection, one splenectomized on day 4, the other on day 14. In both cases, the T,,,Cr increased after splenectomy; in the animal splenectomized on day 4, the T,,C:r changed from 7.5 days to 14 days

P. MCCRORIE

126

et al.

and in the rabbit splenectomized on day 14, the T,,@r changed from 6 days to 13.5 days. The other two animals splenectomized on day 4 and the two animals splenectomized on day 7 all showed similar patterns, though to variable degrees. Animals splenectomized late in the infection did not survive long enough for meaningful red cell survival studies to be carried out.

c 0 p 5

401

c0 0 2

10.

30. 20.

O0

542

5

IO

15

20

30

25

35

40

45

e

--.I 40

45

50

5 E .-

.__._

I __

-.-

-.-I-

- -

- -

50

20 IO. 30 0 . 0

427

5

IO

15

20

25 Days

after

30

’ 35

infection

Splenectomy one day prior to infection \zas as effective in preventing severe anaemia as was splenectomy two or three months prior to infection. On the other hand, splenectomy on day 4 of infection did not prevent the rabbit becoming anaemic during the subsequent three days. Since it is not the spleen

ANAEMIA

IN

RABBITS

INFECTED

WITH

I:

b.

brucei

127

50 40 30 S42

20

0

.-G ..c

5.

IO

15

20

25

30

35

40

45

50

25

30

35

40

45

50

IO 0 0

5

IO

15

20

after

Doys

Fig.

3. Clhanges in PC:\’ during control, --~ - splenectomized rent) Controls S4’L splenectomized S42 non-splenectomized 427 splencctomized 427 note-splenectomizctl

infection infected. (4) (IO) (3 1)

(6) (14)

infection

of splenectomized and non-splenectomized - - - - non-splenectomized infected.

Initial

rabbits. PC\.

(per

35..2 f 1.9 374it I.1 38% + 0.6

35-&*0+3 39.0

+ 0.8

which is responsible for the loss of red cells between factor must be in\-oIved. The Extent of Sequestration and Destruction

day 4 and day 7, some other

in the Spleen

In non-splenectomized animals, it is clear that the spleen is involved in the initial haemolytic episode, whether the rabbits ase infected with the S42 stock or the 427 stock of T. 6. brucei. This could he explained by sequestration (pooling) of red cells within the spleen and/or destruction of red cells by splenic

P.

et a/.

MCCRORIE

S42

z2

130. 42-J

80”,““...“~‘~‘~“““..“““‘~‘~~~.. 0 5

IO

15

20 Days

25

30

after

Infection

S42 splencctonlixtl S42 non-splenectomized 427 splmectomized

( ioj (3 1) (6)

68.0 z 1-t 7 I.6 k WI 71.“+ 11.1

127

C11)

69.3:

non-.~plcnectomiz~tl

35

fl~,~~~~...~i 40 45

50

lb,-!

30 S 42

z ‘; :: 5 2

35

0

5

IO

15

25

Fig. 5. Changes in reticulocytes during infection of splcncctomized antI nol~-sr’lellectomirrcl (per cent). --- control, -. - splenectomized infected, lion-si’ienertomizcci inkrtcti.

rabbits

after

40

50

50

Days

35

45

45

20

30

40

infection

ANAEMIA

IN

RABBITS

rt

INFECTED

WITH

T. b. brucei

\i, 12( 25

00~s

after

129

30

infection

. .. : , j ; j i j / j I

t

i

25 Days

after

30

: : : . .

I t

I

35

,’.-. i I

40

Infection

Fig. 7. Changes in p:uxsit;temia during infection ol’splenrctomized and [loll-splc-llectonlized rabbit5 with 7. b. brucei 127. l - - -0 splenectomized infkrtrd rabbits, c., ----i> non-splenectomizctl infected rabbits. Data are presented Lbr t\vo indi\Gdllal rabbits. r\ similx pattern was followed in most animals.

130

Rabbit

status

(number

used in brtrc-ke/>

(t&T

Uninfected splenectomized -I Intact, infected with S42 ,i Splenectomized, infected with S42 I 4 9 Intact, infected with 427 Splenectomized, infected with 427 li

i dqa\

I :3,.5 4 0, I “.7+o.i 12.1 *IFI -1.1 +().I I:3~:3~o~I

The mean T,,Cr values k standartl errors wen computer from the data used to plot thv individrml survival

7 dq.~

calculattd rathit red

t>! ccl1

curves.

IOC 9c 00 70 60

Days

after

infection

red cell aurvival curves in splencrtonrimII and L:ig. 8. “Wr-labellcd with T. b. brucei S42 -~~- control. -- . --- splenrctomized infected.

r~on-rt,lerlectomixed

infected.

rabbits

~-

infected

non-splenectomized

ANAEMIA

macrophages. sequestration

IN

RABBITS

To ascertain and destruction,

INFECTED

WITH

7-.

the relative contributions the following experiment

b.

brucei

131

to the anaemia, was designed :

of

Four series of animals were used : Series A: 11 rabbits were infected with 7. b. brucei S42 and their red cells lahelled with 51Cr immediately prior to infection. After 3 to 4 weeks, they were killed and their liver and spleen removed for radioactive counting as described in Methods. 100 90 80 70 60 50 40

5 4

3

2

I 0

4

8

12

16 Days

Fig.

20 after

24

28

32

36

infection

9. “Cr-l:ibelled red ccl1 survivnl curves in splenectomizcd and non-splenectomk-d rabbits infected with ‘7. 6. hrucei 427. -~~ control. -- I - splenectomized infected, - ~ - non-splenectomized inkctcd. ‘I’he lines are the best fit (drawn by computer) to the values obtained for groups of rabbits rxrept the control points (0) for which thrrr was only one rabbit. ‘I’hc I-erticnl lines xe SEM.

P. MCCRORIE

132

et al.

Series B: 9 rabbits were infected with T. 6. bruceiS42 but their red cells were not labelled with 51Cr until immediately before death, 3 to 4 weeks after infection. 30 min after r-c-injection of the labelled autologous cells, by which time tht labelled red cells would have been uniformly distributed in the circulation, the animals were killed and their liver and spleen removed for counting. Series C: Red cells from 6 uninfected rabbits were labelled with jlC:r. After ii to 4 weeks, the animals were killed and tlreir liver and spleen remo\:ecl for counting. IOC 9c ea 7c 6C 50 40

30

cl n ; :

;

a

100 90 80

(b)

70 60 50 40 30

2(

IC

, \. 4

8

I2 Days

I6 after

20

,

, 24

, 28

Infection

Fig. 10. 51Cr-labellcd red cell survival curves in two rabbits infected with 7. h. bntrei Y42. EHect of splenectomy after infection. (a) splenectomy 4 day, (b1 14 days

AKAEMIA

IN

RABBITS

Series D: Red cells from uninfected min, the animals were killed counting.

INFECTED

WITH

x

h. bmcei

133

rabbits bvere labelled with 51C:r. After 30 and their liver and spleen removed for

Series A and C gave measures, in infected and uninfected animals respectively, of the amount of sequestration of red cells within the spleen plus the amount of destruction (accumulation) of red cells by the spleen over the 3 to 4 week period. Series B and D gave measures, in infected and uninfected animals respectively, only of the amount of sequestration of red cells by the spleen (relative to the liver). ‘I’he results are shown in Fig. 11 and Table 2. Each shaded area represents the ratio of 51CIr counts in the spleen to 51C:r counts in the liver in a different rabbit. The ratio in Series B rabbits was considerably greater than that- in 2.0

.”

I 5

5 L : .c : a (0

kc

,

-

05

0

L

Series

A

Series

B

Series

C

Series

Cl

P. MCCRORIE

134

et al.

Series D rabbits (by a factor of 3.7) and was about the same as that in Series C: rabbits. Thus, there was considerably more sequestration of red cells in the spleen of infected animals than in that of uninfected control rabbits. The difference between Series B and D is to some extent reflected in the relative sizes of the spleens in the two series: the average spleen size for Series B was 6.9 g and for Series D, l-6 g. Thus, the increased splenic sequestration observed in infected animals is probably due to the difference in spleen size. The sum of the effects of red cell sequestration due to the increased size of the spleen (Series B) and red cell destruction in the normal spleen over a 3 to 4 week period (Series C---Series D) was not sufficient to account for the highct level of radioactivity found in the enlarged spleen of rabbits which had been infected and 51Cr-labelled for 3 to 4 weeks (Series A). The spleen had evidently brought about the destruction of many more red cells than normal, Spleens taken after death from infected rabbits varied considerably in size but all were greater than those removed from control rabbits. DISCUSSIOS Hughes-Jones (1961) showed that, in the tlormal rabbit, the ma-jot. sitr. ol‘rctl cell destruction is the bone marrow, the liver and spleen playing minor roles. It is clear that this is not the case in our infected rabbits (Fig. 11 and Table 2:). In these animals, both red cell destruction and sequestration in the spleen wet-c markedly enhanced. There is some evidence that rabbit splcc~us contract at death (Lavcran and Thiroux, 1907) resulting in a certain amount of brood loss from the organ. Since, in our experiments, removal of the spleen was after death, some alteration in radioactivity may have resulted. This would probably have affected the measurement of red cell sequestration more than the measurement of red cc*11 destruction. Furthermore, the removal of the spleen at the end of infection would ltavc tended to minimize the changes in the radioactive counts within the organ since the majority of cells destroyed late in the infection would have bcett unlabelled. It is therefore likely that the extent of both red cell sequestration and red cell destruction was underestimated in the experiments reported here. Ne\crthclcss, it is clear that both were increased. &Ted

of

Sptenectomy

091

the Course

of the Anaemicl

Splenectomy clearly affected the course of’ anaemia in rabbits infected \vith 7. b. brucei. This was most marked in the red cell surviv~al studies; T,,,Cr values in splenectomized infected rabbits deviated only slightly from those in splenectomized control animals. Furthermore, splenectomy in mid-infection resulted in an increase in red cell life span to near normal values. Despite this effect on red cell life splan, splenectomy only partially protected against anaemia. With respect to the S42 infection, the red cell count and haemoglobin concentration did eventually fall almost to that of non-

ANAEMIA

IN

RABBITS

INFECTED

WITH

7.

b

brucei

135

splenectomized rabbits after the infection had entered its third week, presumably due to the reduced number of larger red cells. Thus, the anaemia which eventually developed in the splenectomized rabbits was not paralleled by a decrease in red cell life span. Even if the liver and bone marrow were to take on the role of the spleen in the removal of abnormal red cells, one would expect that this would be reflected in the survival curves. The developing anaemia may not therefore be haemolytic in nature but may be due to some other factor such as increased plasma volume or the anaemia of chronic disorders. The severity of this latter type of anaemia is likely to be proportional to the se\:erity of infection and may therefore be particularly important in splenectomized rabbits which have a higher parasitaemia than non-splenectomized animals. However, although the anaemia in non-splenectomized rabbits is largely due to the marked haemolytic element previously described (Jenkins it al., 1980), the other factors mentioned here with respect to splenectomized rabbits may also be contributory. Non-Specific Actiuation of the Splenic Reticuloendothelial Sjstem Laveran and Thiroux (1907) suggested that the role of the spleen in trypanosomiasis is the removal of destroyed trypanosomes. It is possible that, because of this activation of splenic macrophages, a non-specific phagocytosis occurs, resulting in an increased destruction of normal erythrocytes. Furthermore, the repeated passage of red cells through the enlarged and congested spleen may result in “conditioning” of, or damage to, otherwise normal red cells. Several Ivorkers have shown that the slow mixing and stagnation of red cells in the large extrasinusoidal compartments of infiltrated spleens has an injurious effect on the red cells as shown by increased osmotic and mechanical fragility and a decrease in K +/\aratio (Emerson, Shen, Ham, Fleming and Gastle, 19.56; hlotulsky, Casserd, Giblett, Broun and Finch, 1958a,b; Prankerd, 1960, 1963; Richmond, Donaldson, Williams, Hamilton and Hutt, 1967). Other effects of the splenic environment which would be harmful to red cells include low cholesterol and glucose levels, and low pH due to the high concentration of lactic acid (Anosa, 1977). There is some evidence to suggest that the role of the spleen in removing red cells from the circulation is of much less importance during the terminal stages of infection. Fiennes (1954) observed that in cattle, during the post critical stag< of infection, the spleen returned to a normal size and was no longer a factor in the aetiology of anaemia. Albright, Albright and Dusanic (1977) have reported that in mice infected with 7. musculi, the spleens enlarged in the first two weeks of infection and subsequently, over the following two weeks, contracted to only twice their normal weight and cell number. We, too, have observed only slightly enlarged spleens at death in some of our rabbits infected with T. 6. brucei. Role of Haemodilzction Holmes and Jennings (1976) f ound that the extent of the contribution haemodilution towards the anaemia in non-splenectomized infected rabbits

of was

136

P. MCCKORIE

et al.

dependent on the species of the infecting trypanosome. With T. 6. brucei TREU 667, they noted that, as the infection progressed, there was a marked rise in plasma volume which closely correlated with a decrease in P(:V: thcrc was no significant decrease in red cell \,olume, nor any marked rise in tht MCV values during the course of the infection. By contrast, when the infecting organism was T. con~~olense,there u-as a small increase in plasma volume and, as the infection progressed, a marked decrease in the circulating red cell volume. The latter correlated well with the decrease in PCV. Thus, haemodilution played a more important role in the actiology 01’ the, anaemia in rabbits infected with T. 6. brucei TREU 667 than it did with 7. congolense. It is possible that the non-haemolytic anacmia which eventually develops ill the splenectomized rabbits does have a haemodilution Factor in its aetiology but we have not investigated this. Increases in plasma volume arc often associated with splenomegaly in man, and splcnrctomy can often alleviatca this situation (Prankard, 1963; Richmond et nl., 1967; Pengelly, 19771. If a similar mechanism operates in trypanosome-induced splenomegaly in rabbits, it would bc surprising if haemodilution was important in the splcnectomized animal. Another possible explanation for the anacmia in infected rabbits is a defect in crythropoiesis. Our preliminary findings c~onfirm that trypanosomes have a marked effect on the marrow. In both intact (Jenkins et al., 198Oj and splcnectomized infected animals there is a marked increase in marrow cellularity, at least in part due to erythroid hyperplasia. WC are at present undertaking a more systematic and comprehensive marrokv survey and our findings will be the subject of a future publication. s u >I M A I< \ Splenectomy delayed the onset of the anacmia in rabbits infected with either Trypanosoma brucei brxei 542 or T. b. brucei 427 and, particularly in the lattcl infection, also lessened its severity. Parasitaemias were higher in the 427 infection although, terminally, massive parasitaemias were recorded in the S42 infliction. With 51Cr-labelled red cells, it was shown that there \vas an increase in both splenic sequestration and destruction of red cells in infected animals relative to controls, the degree of sequestration probably being related to the degree of splenic enlargement. Red cell T,,, \ralues were virtually unaflticted by trypanosomal infection of splenectomized rabbits, and in those splenrc-tomized in mid-infection a marked increase in T,, was observed shortly after the operation. Since the anaemia which eventually developed in the splenectomized animals was not accompanied by a reduction in T,,,, it is unlikely to be of haemolytic origin. Other possible causes are considered. ACKNOWLEDGMENTS

We are grateful to the Ministry of Overseas I>rvelopment for providing the finance for this study. We are also indebted to Dr B. Clolvin for many helpful discussions and to Mrs V. Mulligan for typing this paper.

AKAEMIA

IN

RABBITS

INFECTED

WITH

i’.

b. brucei

137

REFERENCES

:llbright, J. F., Albright, J. W., and Dusanic, D. G. (1977). Trypanosome-induced splenomegaly and suppression of mouse spleen cell responses to antigens and mitogens. Journal of fhe Retiruloendothelial Socie@, 21, 2 l-3 1. :inosa, V. 0. (1975). The effect of splenectomy on the anacmia and parasitaemia of trypanosomiasis. M.V.M. thesis, University of Glasgow, Scotland. rinosa, V. 0. (1977). Studies on the mechanism of anacmia and Ihe pathology of experimental Trlpanosoma vizJa.t infection in sheep and goats. Ph.D. thesis, University of Ibidan. Boggs, D. R., Geist, A., and C:hervcnick, I’. X. (1969). Ciontribution of th
.\lny 2nd, 19791