The Effect of Splenectomy on the Pathogenicity of Fasciola Hepatica in the Sheep

Br. vet.

J.

( 1970), 126, 15

THE EFFECT OF SPLENECTOMY ON THE PATHOGENICITY OF FASCIOLA HEPATICA IN THE SHEEP By K. B.

SINCLAIR

Animal H ealth Research Unit, University College of Wales, Aberys twy th

SU MMARY

The pathogenicity of F. hepatica was compared in groups of splenectomized and intact sheep. The recovery from the splenectomized group of a higher fluke burden with a greater proportion of larger individuals was associated with more severe clinical disease and fatalities . Liveweight loss, anaemia, eosinophilia, accelerated erythropoiesis and loss of SlCr- and s9Fe-labelled blood constituents in the faeces were all more marked in the splenectomized group, and more extensive hepatic parenchymal damage with little or no biliary hyperplasia characterized the post-mortem picture. INTRODUCTIO N

In a previous experiment (Sinclair, 1968), splenomegaly was noted in lambs infected with F. hepatica and this finding led to a consideration of the role of the spleen in the pathogenesis of the disease. It is well known that, in addition to blood cell formation and destruction, the spleen is concerned in the defence mechanism of the body, and is essential to the maintenance of the natural resistance of many animal species to some bacterial and protozoal infections. Its role, however, in resistance to infection by helminths has been inadequately investigated, and there appear to be no reports concerning fascioliasis. This paper records a study of the pathogenesis of F. hepatica in a group of splenectomized sheep together with some preliminary observations on the effects of the operation on haematology and erythrokinetics. MATERIALS AND METHODS

Plan oj the experiment Twelve castrated male Clun sheep were reared in the animal house under conditions which precluded infection with parasitic helminths. The sheep received a daily ration of cubes, and chopped hay and drinking water were available ad libitum. When they were about 2 years old and weighed 55 kg., six sheep were randomly selected for splenectomy. The operations were carried out during December 1967, and a period of 4 months was allowed for recovery. In April 1968, faecal collection bags were harnessed to the sheep which were then placed in metabolism cages prior to the intravenous injection

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of radioactively labelled autologous plasma and erythrocytes. After injection, the sheep remained in the cages for a period of 2 weeks to enable the determination of radioactivity of samples of blood, faeces and urine. Thereafter the harnesses were removed and the sheep returned to the pens where they remained for a period of 20 weeks by which time the level of radioactivity in the blood was low. In September 1968, the sheep were divided into three groups: group A comprised five of the splenectomized sheep selected at random; group B comprised five of the " normal" sheep selected at random; and the control group C comprised two sheep, one splenectomized and one "normal". The ten sheep of groups A and B were each given by mouth 3000 metacercariae of Fasciola hepatica contained in a gelatine capsule. All the sheep were observed daily, and early in J anuary 1969, they were returned to the metabolism cage prior to a second intravenous injection of radioactively labelled autologous plasma and erythrocytes. At the end of the isotope study, all the sheep were killed by the intravenous administration of barbiturate and examined immediately. Particular attention was paid to the liver, and the flukes recovered were fixed between glass and measured. Throughout the observations, samples of jugular blood were obtained into heparin at intervals of a fortnight and the sheep were weighed weekly. Samples of faeces were examined at regular intervals to confirm the absence of nematode eggs, and more frequent examinations were made after the administration of the m etacercariae in order to assess the degree of infection with F. hepatica.

Surgical technique The operation site on the left flank was clipped on the day prior to surgery and, after overnight fasting, general anaesthesia was induced and maintained with halothane. The sheep was restrained on its right side and the skin over the operation area cleansed with an alcoholic solution of cetrimide. The line of the incision followed the twelfth intercostal space commencing about 6 cm. from the vertebral extremity and extending downwards for about 12 cm. The peritoneum was opened and, with the aid of a retractor to separate the ribs, a hand inserted into the abdominal cavity and directed forwards beneath the costal arch. The spleen was located and carefully freed by blunt dissection from its attachment to the diaphragm and rumen. It was then possible to deliver the spleen through the incision and to ligate the splenic vessels. The peritoneum and muscle layers were closed with interrupted sutures of chromic catgut, and the skin with monofilament nylon. On the first 5 days after operation, the sheep received procaine penicillin intramuscularly, and the skin sutures were removed after 10 days. Healing occurred by first intention in five sheep; a localized subcutaneous infection occurred in one. Laboratory and post-mortem techniques The techniques used for the collection and examination of blood samples and for the recovery of flukes from the liver were those described by Sinclair ( I 962) except that an electronic counter* was used in determinations of the * Coulter Electronics Ltd, High Street South, Dunstable.

EFFECT OF SPLENECTOMY ON F. HEPATICA IN SHEEP

17

number of blood cells and the frequency distribution of erythrocyte volume, and packed cell volume was d etermined by microhaematocrit. Plasma iron and latent iron-binding capacity were estimated according to the methods of Landers & Zak (1958) and Ventura (1952) respectively. The number of fluke eggs p er gram offaeces was d etermined by a sedimentation technique described by Boray & Pearson (1960). The total number offluke eggs in 3 g. of wet faeces was determined and the result expressed per gram of faeces on a dry-matter basis.

Methods with radioactive materials Simultaneous labelling of erythrocytes and plasma was carried out on two occasions using solutions of sodium chromate (51Cr ) and ferric citrate (li9Fe) obtained from the Radiochemical Centre, Amersham. T en (.I.C 59Fe was added to 10 ml. autologous plasma and, after allowing 30 minutes for absorption, injected through a wide bore needle into the left jugular vein. Samples of blood were obtained into heparin from the right jugular vein, at intervals of 10 minutes for an hour, and 30 minutes for a further 3 hours after injection, for determination of the plasma clearance curve. On the occasion of the last sampling, an additional 10 ml. of blood was obtained from each sheep into acid-citrate d extrose solution and the erythrocytes labelled with 100 (.I.C 51Cr by the method of Mollison & Veall (1955). The labelled autologous erythrocytes were injected through a wide bore needle into the left jugular vein, and after allowing 15 minutes for mixing, a sample of blood was obtained from the opposite vein to provide the initial 100 p er cent level of activity due to 51Cr. The initial 59Fe "whole blood" count was calculated from the extrapolated plasma clearance curve and the determined packed cell volume. Subsequently, the radioactivity separately due to 59Fe and 51Cr was determined in samples of blood and plasma obtained daily for a period of 15 days after injection of the isotopes. The daily counts on blood were adjusted for changes in packed cell volume and decay and expressed as a percentage of the initial count. The total radioactivity due to 59Fe and to 51Cr injected into each sheep was determined from standard solutions. The activity of faeces was determined on 5 g. samples selected at random from polythene bags containing the 24-hour output of faeces from individual sheep. The daily counts of 59Fe and nCr were adjusted for decay and the 24hour output of faecal dry matter, aggregated for the lo-day period between day 2 and day I I after injection, and expressed as a percentage of the total injected activity of the respective nuclides. The activity of urine was determined on 15 ml. samples of the 24-hour output from each sheep and the similarly derived aggregate counts expressed as a percentage of the total injected activity. Blood, plasma, urine and faeces were all counted in disposable polythene tubes d esigned to fit the well-type crystal of a Packard twin-channel autogamma scintillation spectrometer. Activity was determined on aliquots of 2 ml. of blood and plasma, and 5 ml. of urine; the activity of faeces was determined by counting a sample of 5 g. equally distributed in three tubes.

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I

RESULTS

Effects of splenectomy Apart from inappetance during the first 2 or 3 days, the sheep appeared to be unaffected by the operation, and the loss of live-weight gain was small. The h aematological changes immedi ately following operation were not studi ed , but in samples of blood obtained after 2 and 4 weeks, there were no significant differ ences in erythrocyte count or volume, packed cell volume, plasma iron and iron binding capacity, total plasma protein and distribution of the various fractions. There were, however, changes in the white cell picture, and, within 2 weeks of operation, there was a highly significant increase (P < 0'01) in the leucocyte count. The leucocytosis was mainly due to an increase in circulating lymphocytes, a nd it tended to persist until the end of the observations. There were considerable differences in the results of the isotope studies on splenectomized and "normal" sheep. The initial loss of 51Cr from the blood of the splenectomized sheep was significantly less (P < 0'02 ) than in the " normal" animals although the subsequent survival curves were parallel and gave a a similar chromium half-life of about 12'5 d ays. It is well recognized that most of the 51Cr lost from the blood is excreted in the urine and, apart from a normally small amount of vascular leakage, little activity appears in the faeces, In the present experiment, there were no significant differences in the proportions of the injected dose of 51Cr which subsequently appeared in the urine and faeces of splenectomized and normal sheep , In the case of 59Fe, the splenectomized sheep showed a r etarded plasma clearance which persisted to the end of the observations, a nd a significantly accelerated (P < 0 ' 05) a nd more complete uptake of isotope into the circulating erythrocytes. There was, however, no apparent effect on marrow transit time as shown by the plateau in the utilization curve between 1 and 2 days after injection. Ther e were no significant differences in the proportions of the injected dose of 59Fe which appeared jn the urine and faeces of splenectomized and normal sheep. Up to about 2'5 per cent appeared in the faeces, mainly during the first 6 days, and less than 0'1 per cent was found in the urine. T ABL E

I

THE PROPORTION OF INTRAVENOUSLY ADMIN I STERED 51e r AND 59Fe EXCRETED PERIOD OF 10 DAYS BY NO RMAL AND SPLENECTOMIZED SHEEP INFECTED WITH

Urine

Faeces

No,

Group

INFECTED

A (Splenectomized) 3 B 5

10'93 3,62

± ±

4'19* °'92*

24'37 7' 99

± ±

1' 15** 5'07**

26' I! 23 '47

± ±

2'55 2'05

0'104 0'12

± ±

0 '03 0'03

0'27 0'31

± ±

0'1 5 0'16

1'7 0 2'04

± ±

0'59 0,8 1

25'61 26'16

± ±

1'06 1'38

0'08 0'07

± ±

0'02 0'03

C ONTROL

Normal Splenectomized

7 7

Values given are percentage mean

*P <

0'2;

DU RIN G

** P <

0' 02,

±

SE,

A

F, hepatica

EFFECT OF SPLENECTOMY ON F. HEPATICA IN SHEEP

19

For reasons of space, the haematological data obtained prior to infection have not been tabulated. The results of the isotope study, however, have been incorporated in the control curves shown in Figs. 3, 4 and 5, and in the data in Table 1.

Infections in normal and splenectomized sheep: clinical signs and pathology The changes in mean live-weight and counts of erythrocytes and eosinophils are shown in Fig. I. It is evident that splenectomy had no effect on live-weight gain or on the erythrocyte or eosinophil count in the control sheep in group C. At first, both infected groups A and B just about maintained their pre-infection live-weight, but on about day 38 a progressive loss of weight began and this coincided with the onset of anaemia. It is noteworthy, however, that there was evidence of an earlier onset of the downward trend in erythrocyte counts in group A, although individual variation precluded the demonstration of a statistical significance. In both groups, there was a peak of eosinophilia about day'.-1), but a more marked increase, independent of the continuing leucocytosis, occurred in group A. A second peak of eosinophilia occurred in group B about day 70, but therewas no evidence of this in grou~. The progressi~e loss of weight and anaemia gradually became more marked in the splenectomized group A, and it soon became clinically apparent that this group was more severely affected. Four of the sheep in group A showed considerable loss of condition, suffered periods of inappetance and elevated rectal temperature with or without abdominal pain, and two animals died of sub-acute fascioliasis ~ and 105 days after infection. Figure I shows that a more pronounced loss oflive-weighfDeganm group A on about ?ay 60 ~hen fluke eggs first appeared in the samples offaeces. It also seems likely that the decline in mean erythrocyte count became steeper at this time. Eggs were found in the faeces of sheep from both groups on day 64, and the mean numbers had increased to 1:2.8 e.p.g. in group A and 194 e.p.g. in group B on day 100. Figure 2 shows the changes in the mean frequency distribution of erythrocyte volume in groups A and B. It is evident that an increase in mean volume occurred in both groups as early as 28 days after infection, but it did not approach significance (P < 0'1) in group B until day 43, and in group A until day 86. The low level of significance was mainly due to individual variation, particularly in group A. The mean erythrocyte volume of the twelve sheep prior to infection was 37.62 cubic microns ± SE 0'98. On day 43 the mean of group B had risen to 39'94 ± 0'72 and on day 86 the mean of group A was 42'75 ± 2'9 I. It is noteworthy that a considerable increase in mean erythrocyte volume in group A occurred between days 70 and 86 and this coincided with a marked fall in the level of plasma iron and a rise in the latent iron-binding capacity which continued to the end of the observations. It is also coincided with the appearance of s4;!erocytes in the circulation of three sheep in group A, and the numbers gradually increased to 4, 7 and 10 per cent by day 100. Siderocytes were not seen in group B and plasma iron and iron-binding capacity remained within normal limits. That the spleen is involved in antibody production is well known, so that

BR ITISH VETERINARY JOURNAL,

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126, 1

changes in the plasma proteins are of particular interest. In both groups A and B, there was an increase in total plasma protein by day 14 which became significant (P < 0·05) by day 28. The rise was mainly due to an increase in the

K

)(

Group A

I!r- ---- ~

0

0

Splenec

I}

Group C

GroupB ... - - - .... Normol

+ 8

~ '"C

0-

0

4

0

.c u

:c '"'"

.!?'

4

I

'">

.. c

8

0

::;:

12

16 12

----~-

-- - --

- - ---

"'E E

.-

- ----.

-""i\,..

OD' o

"

8

6

4

x_ _ _

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7

'"Q

5

X_ _~

,E

~

,. .-A>-- ---/:r"- _--..4-- - - _ ~ .".... _- -006 -x.--------/ ;,-;",

?>- _ _ _ -v

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~

a.

0

3

'"

.~

X

'" w 0

---- o

10

20

30

--...._40

50

60

70

80

Days after infect ion

Fig. I. The changes in live-weight, erythrocyte and eosinophil counts in control (group C), a nd splenectomized (group A) and normal (group B) sheep infected with F. hepatica.

EFFECT OF SPLENECTOMY ON F. HEPATICA IN SHEEP

2I

Group A

15

10

o

:§

o c '"u

15

10

5

Ery th roc yte volume (fL 3 )

F ig.

2.

The changes in mean frequency distribution of erythrocyte volume in splenectomized (group A) and normal (group B) sheep after infection with F. hepatica.

gamma fraction which appeared to occur earlier in group A. In both groups, however, the increase was significant (P < 0'05) by day 55. Subsequently, the ga~ fraction in the splenectomized sheep of group A began to decline to less than half the value by day roo. This fall in gamma-globulin was mainly responsible for the lower total protein in group A at the end of the observations. The only other important change in plasma protein was a gradual fall in the levels of albumin in both groups commencing about day 43.

Radio-isotope studies Because of the death of two splenectomized sheep before the post-infection radio-isotope studies began, the results in this section refer to the means of three sheep in group A and five sheep in group B. The control data are the means of results obtained from the six splenectomized and six normal sheep at the preinfection radio-isotope study together with those of the two sheep in group C. The mean survival of 51Cr-Iabelled erythrocytes in the two infected groups are shown in Fig. 3. It is evident that, compared with the controls, reduced survival occurred in both groups, and that the splenectomized sheep of group A showed the greater effect. The mean chromium half-life of group A was 4'35 days, and of group B, 7'75 days. Unfortunately, individual variation was so great that a statistically significant difference between the means of group A

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126, 1

and B or between group B and the normal controls could not be found. But the difference between group A and the splenectomized controls was significant (P < 0'05). Some evidence in support of the shorter chromium half-life in group A came from the d etermination of r adioactivity in the faeces which showed that more 51Cr appeared in the faeces of group A than group B (P < 0'2). The results of the 51Cr d eterminations on faeces and urine are summarized in Table I whiCh shows that significantly (P < 0'05) greater amounts of isotope were excreted in the faeces of infected sheep. x

100 u ;;;

.~ >

=>

60

x Group A 0

~-o _

_~- - .

40

V>

-

0_

0

_

;;; 20

0..

---0____--.

. . x::. ...... .xIr'-_'Ir-__ --e __ e __ °---0 - .... _ /j,

-6-.

. . .x.. .

~

Controls

Group8 . - - - - - . Normal

........X...

C

0 SPlenect}

_ . 0_

(;-----A

. . .x.... .....

_

- - . -- _ - e _ _

-A__

... . . . x,

-ll_-_A

..........

0

-

--A

10 Days of ter inject ion

Fig. 3. The survival of 61Cr-labelled erythrocytes in the circulation of controls, splenectomized (group A) and normal (group B) sheep infected with F. hepatica.

The disappearance from the plasma of the inj ected 59Fe is shown in Fig. 4and it is evident that the isotope was taken up most rapidly in the splenectomized sheep of group A. When compared with the controls, both groups A and B showed a significant (P < 0'05) accelera tion in disappearance of 59Fe from the plasma; the difference between groups, however, was not significant. Figure 4 also shows that a r esurgence of plasma activity due to 59Fe occurred in the infected sheep with peaks at 4- days in the case of group A, and 6 days in group B; this corresponded, in both groups, to the peaks of the respective 59Fe utilization curVes shown in Fig. 5. It is clear from Fig. 5 that, compared with the controls, both infected groups showed an accelerated and more complete utilization of the nuclide, with the difference significant at the 5 per cent level. The data also indicate that the splenectomized sheep of group A showed the most complete utilization of the injected 59Fe and subsequently the most rapid decline in circulating activity, although the difference between the means of groups A and B only approached significance (P < 0'1). It is noteworthy that the latent period b etween I and 2 days after injection, which represents the marrow transit time in the controls, virtually disappeared in the infected sheep. The results of the determinations of 59Fe in urine and faeces are summarized in Table I; considerably more nuclide was excreted in the faeces of the infected sheep. Furthermore, significantly (P < 0'02) greater amounts of 59Fe were excreted in the faeces of the splenectomized sheep of group A. The output of 59Fe in the urine of all the sheep was low and there were no appar ent differences between the groups.

EFFECT OF SPLENECTOMY O N F. HEPA TICA IN SHEEP

'l~

Minutes after injection

--- --. ~

0>

10

'"

0

E V)

.2 0. 0

'c '0 C

'"u

x

X Group A

A

t:. Grou p B

o

0

sptenect}

Controls

e - - - - . -e No rmol

.t

Days after injec tion

Fig. 4. The clearance of 5"Fe from the plasm a of controls, splenectomized (group A) and normal (group B) sheep infected with F. hepatica.

The count ratio 51Crj59F e in the daily samples of blood and faecal dry matter from the individual sheep were compared, and three examples of the r esults are shown in Fig. 6. No adjustment has been made for the rate of p assage of ingesta or for individual variation in output of faeces and, apart from early differences associated with the movement of isotopes into and out of erythrocytes, it is evident that in the infected sheep, whether splenectomized or not, there was a close r elationship between the count ratios in blood and faeces. Furthermore, there was a close similarity in the shape of the curves representing the daily levels of 59Fe in the blood and faecal dry matter of individual infected sheep.

Post-mortem findings and development of the flukes \t\Then the post-mortem features of the two groups were compared, the out!standing difference was th e gener ally more extensive and severe liver lesions in the splenectomized sheep. On the other hand, although the parenchymal lesions were more severe, the anticipated white, thick-walled, projecting cord-like I

BRITISH VETERINARY JOURNAL, x Gro up A 0 IX ~

90

I

/

/,;r--~

'"

"-

0

X~

/

5

0

~

f

,/

/

C

'" u

0- -

___ _ -i>- _ _ _ _ -
X~

x

! / -"u 0---0--_0---0-

V

0..

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X_____

,,

:>

Contr ol s

0

6>(---~-----i>-

/

(To

'"c

0 SPlen eCI }

____

b---~-"""""""~ X

f

1

Group B .... - - - - .. Norma l

x"'"

x

X

I>- - - - --6

126,

_e-----· -- - - .!. - - - - - .

o

__

r /

...

~

10

8

6

4

2

14

12

Days after inj ecl io n

Fig. 5. The utiliza tio n of .9Fe in the produc tion of circula ting red blood cells in controls, splenectomi zed (group A) a nd normal (group B) sheep infected with F. hejJatica. x-

6

- - x Fae c es

1 I

'"

v>

1

\

I

u

3

I

~\

v>

C :>

\ 1

in

0

Infec l ed

I

:>

0

U

Splenec tomized In fecled

\

4

C "-

Control

I

~

If' (To

Blood

x

I I

5

Q------{)

2

x

\ \

q,

U

'b.. u.. , 'C. --o.. - -o.. _ -o

\l~ '0-0- -

2

4

14

I 2

I 4

I 6

I 8

--0-_ ..o::::!-::o:-~.::o

I 10

I 12

I 14

Days ofler injectIOn

Fig. 6. The unadjusted count ratio 5lCr!59Fe in samples of blood and dried fae ces obta ined from a control, a normal and splenectomized sheep infec ted with F. hepatica.

TABLE II

II ~

THE FINAL LIVE - WEIGHT, WEIGHT OF LIVER, SPLEEN AND GALL BLADDER AND NUMBER AND MEAN LENGTH OF FLUKES RE COVE RED

...,

()

Liver weight

Flukes Group

Noo

Total recovered

Mean length (mmo )

Live-weight (kg o)

Liver weight (g o)

Bodyweight (go/kgo )

Spice/! weight Spleen weight (g o)

Bodyweight (go/kgo )

Gall bladder weight (go )

0

'"!:I \/)

'"d

t"'

~

A

I

7* 13* 23 33

Mean B

8 16 36 47 NT

C

1[[

28

Mean * Died o

16089 17°43 17°55 16°95 18027

46 °4 59°1 47°7 44° 1 74°5

1175 99 8 12 36 1258 99 0

25"3 2 16089 25"9 1 28 °53 13°29

13 0 128 108 11 6 51

75 8

17°42

54°4

11 3 1°4

22°0

106 06

0

17 0 677 397 69 2 993

19°68 1[0 16 20 024 20 °38 [5 °84

70 °5 55°9 54°5 53° 2 4806

757 1[29 975 945 1150

10 °74 20°20 [7° 89 17°7 6 23° 66

49 32 73 47

~

586

Mean

Z

89 2 784 101 3 709 39 2

17°46

56 °5

99[02

80 12 0

175 82 173

1013 2 0[ 5 3° 2 [ 1°54 3°5 6

126

2 °32

62 06

[1 8

[ 08 5

[0 21

59[ 53 [

9° 29 7°79

65"9

56 [

8 °54

0

~

>< Z

~"tl

::... :::j

112

[ 8005

63 °6 68 02

~

...,

()

Q 0-;

Z \/)

::r::

[5"5

~ ~

"

'"d

'"

(Jl

BRITISH VETERI NAR Y JOURNAL,

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main bile ducts were absent. Instead, the walls of the bile ducts in group A were thin and transparent, and the dark-coloured contents were readily seen. The liver was c::n larged and orange-pink in colour, with bright red thread-like haemorrhagic tracts showing beneath the capsule. The ven!!:.
Group A f2]

10 r-

~

8 r-

;..:

-E -

;..:

~ ~ '/.

;..:

'/.

III


:J

CJ

-0

-

~

C


u

'
0....

I':

v

6

4

0

2

,. ,.

~

~ v:~

,.

~

~

V

~

~ V

~ ~ ~ ~ ~ ~ V

v

;..:

~ ~ ~ ~

j ~ ~ V '/.

I>-;

~ ~ '/.j V

:;

&

;..: ;..: V ;..: 5 6 7 8 9 10 II 12 131415 161718 192021222324252627282930

v:

Length of fluke (mm.) F ig. 7. The frequency distribution of the length of individual F. hepatica recovered from splenectomized (grou p A) a nd normal (group B) 'sheep.

EFFECT OF SPLENECTOMY ON F. HEPATICA IN SHEEP

27

groups. On the other hand, Table II shows that the mean number of flukes recovered from group A exceeded by 172 the mean from group B. Examination of the frequency distribution of the length of flukes recovered from each sheep revealed an almost identical mean length in group A and B. Analysis of variance, however, showed the greater variation in the mean length of flukes from the sheep of group B to be highly significant (P < 0'01 ) . The frequency distribution of the length of all the measurable flukes recovered from the two groups of sheep is shown in Fig. 7; the distribution of the length of flukes in the splenectomized sheep of group A followed an almost normal population curve while the distribution in group B indicated a lower proportion in the middle length range and a correspondingly higher proportion in the shorter length range. DISCUSSION

A review of the literature failed to reveal an account of the effects of splenectomy in the sheep. The present work has shown that the operation is straightforward and safe, with little effect on live-weight gain. Furthermore, except for a persistent leucocytosis, due mainly to an increased lymphocyte count, there were no apparent long-term haematological effects of the operation per se. It is interesting that McBride, Dacie & Shapley (1968) have recently reported leucocytosis to be a common sequel to splenectomy in man. The results of the radio-isotope studies demonstrated the importance of the spleen in regulating the amounts of 51Cr and 59Fe in the circulation. It seems likely that the regulatory function is dependent upon the normal role of the spleen in removing from the circulation effete and damaged or abnormal erythrocytes. Thus the smaller early loss of circulating 51Cr in the splenectomized sheep may be explained by the persistence of some older or damaged erythrocytes which would normally be broken down in the spleen. Similarly, the abnormal persistence in the circulation of new-formed 59Fe-labelled erythrocytes which are immature or imperfect may explain the accelerated and more complete utilization of 59Fe in the splenectomized sheep. The observations on the infected sheep clearly indicated the greater severity of the disease in the splenectomized group and, taken in conjunction with the fluke data, there can be little do~b..!J..hat resistance to F. hepatica was reduced. The precise role of the spleen in the resistancem echanism is unclear, but considerable amounts 0 antibody are produced in -the lymphoid follicles. In the present experiment, determination of plasma protein fractions provided a measure of circulating antibody and there was no lack of antibody response to infection in the splenectomized sheep. Nevertheless, the parasites d eveloped more successfully in this group. Thus there was evidence, albeit slight, that more of the administered metacercariae subsequently became adult in the liver. There was more convincing evidence, however, that a greater proportion of the fluke burden in the splenectomized sheep successfully reached m aturity and became large in size, and this was supported by the post-mortem examination of the livers. It is generally accepted that the cellular reaction in the liver plays a n important part in limiting the development of F. hepatica, and fibrosis is the

BRITISH VETERI TARY JOU R NAL, 126, 1

usu al sequel. But the hepatic changes in the splenectomized sheep here, and particularly the lack of biliary fibrosis, suggested that the usual cellular reaction was absent or delayed, thereby permitting the unres tricted development of the parasites. The considerably greater loss of r adioactivity in the faeces of the splenectomized sheep was an interesting and important finding. It could be explained by the presence of more and larger flukes , but it is also possible that the lack of hepatic fibrosis permitted a greater vascular leakage into the biliary system of the splenectomized sheep. Because the d eterminations were limited to three sheep, a firm conclusion could not be drawn, but no direc t relationship was apparent between the number of flukes r ecovered and the proportion of injected isotope excre ted in the faeces. But whatever the cause of the greater faecal loss in the splenectomized group, all the infec ted sheep showed a close relationship between the r atio 51Crj59Fe in blood and in faeces, strongly suggesting that whole blood cells pass into the biliary system. It has been suggested (Sinclair, 1965) that the anaemia of fascioliasis may be associated with the hyperfunction of the r eticUla -endothelial system. But ic is-clear from th e evidence of the present work and tha t reported by Sinclair (1968) that hypelfunction of the reticulo-endothelial system is an integral part of the resistance mechanisms, and its suppression or modification, whether by the administration of corticosteroids or splenectQ.my, leads to more severe symptoms, including anaemi a . Whilst there is some evidence of intravascular haemolysis in the more advanced stages of the disease, it must be conced ed that considerable loss of blood constituents into the biliary system occurs a nd the anaemia is probably haemorrhagic in type. The onset of the anaemi a signals an erythropoietic response demonstrated by the gradual development of macrocytosis. The delayed onset of m acrocytosis in the splenectomized sheep may be further evidence of the supposed physiological interrelationship between spleen and bone marrow. AC KNOWLEDGMENTS

I am indebted to Messrs Hirst and Isaac for technical assistance, Mr A. T . H. Batten for help with a naesthesia, and Dr A. Durrant for statistical advice. The work has been generously supported by grants from the Agricultural Research Council and the Wellcome Trust to whom I am most grateful. REFEREN CES BORAY,j. C. & PEARSON, 1. G. (1960). Aust. vet.]., 36, 331. LANDERS, j. W. & ZAK, B . (1958). Am. clin. Path., 29, 590. McBRIDE,j. A., DACIE, j. V. & SHAPLEY, R. (1968) . Br.J. Haemat., 14, 225 . MOLLISON, P. L. & V EALL, N. (1955). Br. Haemat., 1,62 . SINCLAIR, K. B. (1962). Br. vet. J., 118, 37. SINCLAIR, K. B. (1965). Br. vet. 121, 451. 1 2 4, 133. SINCLAIR, K . B. (1968). Br. vet. VENTURA, S. (1952). clin. Path., 5, 271.

J.

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J., J.,

(Accepted/or publication 8 July 1969)

EFFECT OF SPLENECTOMY ON F. HEPATICA IN SHEEP Les eflet s de la s plen e ctoDli e s u r la p athogeni cite de la Fasciola hepatica chez les Dloutons (Sinclair) ReSUDle. Une etude comparative a ete effectu ee sur la pathogenicite de la F. hepatica dans des groupes de moutons splenectomises et intacts. Le retablissement du groupe splenectomise, apres une forte concentration de douves avec une large proportion de verse de grande taille, etait associe it. des cas cliniques plus graves et une mortalite plus elevee. Perte de poids, anemie eosinophilie, erythropoiese acceleree et diminution des composants sanguins ·'Cr et ··Fe dans les excrements, etaient tous des symptomes plus accentues chez Ie groupe splenectomise; l'autopsie a revele constamment des lesions parenchymateuses hepatiques plus etendues, avec hyperplasie biliaire tres faible ou nulle. Die Wirkung der Splenektmnie auf die Vir olenz v on Fasciola hepatica behn Schaf (Sincla ir) Zu sanunenfassung. Ein Vergleich der Virulenz von F. hepatica bei Gruppen splenektomierter und normaler Schafe wird durchgefUhrt. Bei splenektomierten Schafen war der Befund eines starkeren Befalls mit h6herem Anteil groJ3er Erreger von schwereren klinischen Krankheitsbildern und einer h6heren Mortalitatsziffer begleitet. Auch andere Symptome, wie Gewichtsverlust, Anamie, Eosinophilie, eine beschleunigte Erythropoese und Verlust der markierten ·'Cr- und "Fe-Blutbestandteile im Kot waren bei Schafen der splenektomierten Gruppe starker ausgepragt. Bei der Sektion war das Bild eines ausgedehnteren Leberparenchymschadens mit geringer oder fehlender biliarer Hyperplasie charakteristisch. EI efecto de la esplenectoDlia en la patogenicidad de la Fasciola hepatica en las ovejas (Sinclair) ReSUDlen. La patogenicidad de la F. hepatica fue comparada en grupos de ovejas esplenectomizadas e intactas. La recuperacion del grupo esplenectomizado de una carga mayor de gusanos trematodos con una proporcion superior de componentes estaba asociada con enfermedad clinica mas severa y muertes. La perdida de peso activo, la anemia, la eosinofilia, la eritropoyesis acelerada y la perdida de constituyentes clasificados de la sangre de HCr y ·'Fe en las heces, se presentaron mas marcadas en el grupo esplenectomizado, y mas extenso dana parenquimatoso hepatico con poco 0 nada de hiperplasia biliar caracterizo la escena despues de la muerte.