Immunosuppression in toxoplasmosis: Studies in mice with a clostridial vaccine and louping-ill virus vaccine

J. COMP.

PATH.

1979. VOL.

89.

375

IMhlUNOSUPPRESSION IN TOXOPLASMOSIS: IN.MICE WITH A CLOSTRIDIAL VACCINE LOUPING-ILL VIRUS VACCINE

STUDIES AND

BY

D. Animal

H. W.

REID

AND

Diseases Research Association, Moredun Edinburgh EH17 73H.

Institute, Scotland

BUXTON,

I. Pow 408 Gilmerton

Road,

INTRODUCTION

Infection with Toxoplasma go&ii has been reported to depress the immune response of mice to sheep red blood cells (SRBC) (Huldt, Gard and Olovson, 1973; Strickland, Pettit and Voller, 1973; Strickland and Sayles, 1977). In addition, Huldt et al. (1973) sh owed the same effect with inactivated polio-virus vaccine. In the U.K. T. gondii is one of the major causes of ovine abortion but its potential to diminish the immune response to antigens used in sheep vaccines has not been assessed. The aim of the present study was to examine the response of mice experimentally infected with T. gondii and subsequently inoculated with a bacterial toxoid or a viral vaccine, both of which are commonly employed in the management of sheep. MATERIALS

AND

METHODS

Random bred Swiss-white mice 4- to 5-weeks-old were used. T. gondii (Ml strain) was isolated by the inoculation of emulsified brain from a stillborn lamb intraperitoneally (i.p.) into mice and was maintained by serial passage in mice. In experiment 1 the organism was used at the 3rd brain passage and in experiment 2 at the 5th passage. The brains from donor mice were removed aseptically and homogenized in Hanks’ balanced salt solution (BSS) by passing them 10 times through an 18 gauge needle. The number of cysts in 5 standard 5 pl smears was counted and the concentration in the homogenate adjusted to 500 cysts per ml with Hanks’ BSS: penicillin (100 iu per ml) and streptomycin (745 iu per ml) were then added. Control inoculum was prepared in the same way from normal mouse brains. A commercial pulpy kidney vaccine containing purified form01 toxoids of Clostridium welchii (perfringens) type D adsorbed onto aluminium hydroxide was used. Mice were vaccinated as shown in Table 1. Mice were challenged by intravenous (i.v.) inoculation of C. zuelchii type D epsilon toxin prepared as already described (Buxton, 1978). Deaths of mice occurring within 24 h of challenge were counted and the LD50 calculated (Reed and Muench, 1938). Louping-ill virus vaccine was prepared from the supernatant fluids of louping-ill virus-infected monolayer cultures of baby hamster kidney (BHK-21) cells with formalin and concentration as previously described (Wells and Reid, 1978). The inactivated virus concentrate which contained bovine serum albumin (BSA. 0.03 g per ml) was emulsified with 2 volumes of Bayol F and Falba (Brotherston and Boyce, 1970). Control antigen was similarly prepared from supernatant fluids of uninfected cultures. Titres of serum antibody to louping-ill virus were measured with a haemagglutination inhibition (HI) test (Reid and Doherty, 1971) while indirect haemagglutination (IHA) tests were used to detect antibody to BSA (Reid, Doherty and Dawson, 1971), and 7: gondii (Jennis, 1966). 0021-9975/79/030375+05

$02.00/0

0

1979

Academic

Press

Inc.

(London)

Limited

376

et

D. BUXTON

al.

RESULTS

Experiment 1. Toxoplasmosis and C. welchii

Type D Vaccine

Four groups of mice were treated as shown in Table 1. Groups 1 and 2 were vaccinated once prior to epsilon toxin challenge while groups 3 and 4 received 2 doses of vaccine prior to challenge. After one injection of vaccine the LD50 of epsilon toxin in the toxoplasma infected mice (group 1) was 8.2 ug per 25 g bodyweight while in the control mice (group 2) it was 23.1 ug per 25 g bodyweight. Similarly the LD50 in toxoplasma infected mice in group 3 was 63.4 ug per 25 g while the LD50 in the group 4 control mice was 265.6 pg. Experiment 2. Toxoplasmosis and Louping-ill

Vaccine

Mice were divided into groups and treated as shown in Table 2; the results are recorded in Tables 3 and 4. Mice infected with T. gondii responded less to louping-ill and BSA antigens than uninfected control mice. All mice infected with T. gondii developed toxoplasma antibody and there was no difference in IHA titres to T. gondii in serum collected at days 28 and 35 (groups 1, 2 and 3). Control mice not infected with 1. gondii (groups 4, 5 and 6) were all seronegative by this test. Mice which received neither louping-ill vaccine nor adjuvant (groups 3 and 6) were negative for BSA IHA antibody and mice not treated with louping-ill vaccine (groups 2, 3, 5 and 6) were negative for louping-ill HI antibody.

TABLE EXPERIMENT

1. THE

EFFECT

(21 mice)

(2’ mice)

7

0.5 ml vaccine r-toxin challenge

i.p.

i.v

c-toxin challenge

RESPONSE

Group 3 (?3 mice)

or epsilon

8.2

toxin

in unvaccinated

0.5 ml vaccine 0.5 ml vaccme

i.p. i.p.

Control inoculum i.p. 0.5 ml vaccme i.p. 0.5 ml vaccine i.p.

i.v.

23.1

mice

TO

Group 4 ( 2.5 mice 1

100 cysts i.p.

E-toxin challenge

LD50 (pg per 25 9)

OF MICE

Treatment

Control inoculum i.p. 0.5 ml vaccine i.p. -

30

LD50

ON THE D VACCINE

TYPE

croup ?

100 cysts i.p.

23

welchii

Group1

0

21

gondii

OF ~OxO~hSma

Clostridium

Dgv

1

= 0.2 pg per 25 g body

634

weight.

i.v.

c-toxin challenge ‘654

i.v.

IMMUNOSUPPRESSION

IN

MICE

TABLE EXPERIMENT

2.~0

DEMONSTRATE MICE

No. of mice Day 0 Day 7 Day 28 Day 35

40 L,

WITH

TOXOPLASMOSIS

2 OF lbxoplasmagondii VIRUS VACCINE

THE EFFECT T O LOUPING-ILL

40 20 (0.2 ml control hoc. NIL Li Adj &alf of each group killed and serum collected Remainder killed and serum collected

2. THE

5 mg bovine

of

sample

<40

Jvo. of mice with HI titre 40 80 160 320

mice

28 28

5 14

5 4

4 1

5 1

0 0

To%%%rn! infected (Group 1) Control mice (Group 4j

Imice

35

11

3

3

2

0

1

5

3

6

3

0

2.

THE

1 0

of

samntde (16

serum

albumin.

OF

P Da&e (calculated with x2 test)

P = 0.01 >

P = 0.02

4

EFFECT OF [email protected] gondii ON THE IHA ANTIBODY MICE ~0 BOVINE SERUM ALBUMIN (BSA)

Day

20 i.p.) NIL

RESPONSE

640

infected

TABLE

OF

3

EFFECT OF Toxoplasmagondii ON THE HI ANTIBODY MICE T O LOUPING-ILL VIRUS VACCINE

Day

EXPERIMENT

RESPONSE

i.p.)

TABLE

CdZ:ZYLiJe Toxoplasma

ON THE

20

(100 c:g

Li = Louping-ill virus vaccine, 0.5 ml i.p., containing an estimated Adj = Control tissue culture fluid plus adjuvant, 0.5 ml i.p. NIL = no inoculation made.

EXPERIMENT

377

,Vo. of mice with IHA tit?-e 16 32 64 128 256

512

RESPONSE

OF

P value (calculated with x2 test)

Toxoplasma Control mice infected (n = 19)mice

2

125

i

31

;

3

ii

P = 0.07

Toxoplasma Control mice infected

35 35

14 5

1 4

1 0

1 2

; 6

0 2

P = 0.01

mice

DISCUSSION

The results indicate that mice recently infected with 1. gondii have a depressed response both to C. welchii type D vaccine, measured by epsilon toxin challenge and to lou ping-ill vaccine on the basis of serum HI titres. These findings are consistent with earlier reports which demonstrated that mice infected with T. gondii had reduced antibody and splenic plaque-forming cell responses to

378

D.

BUXTON

et

a/.

SRBC (Huldt et al., 1973; Strickland et al., 1973; Strickland and Sayles, 1977) and to inactivated polio-virus vaccine (Huldt et al., 1973). Other protozoa1 diseases also have been shown to reduce responses to certain vaccines. Children with Kala-azar produced lower antibody titres to typhoidparatyphoid A and B vaccine than uninfected children (Cassimos, Lazanakis and Thomaidis, 1966). Malarial infection of children also appeared to cause diminished antibody responses to tetanus toxoid and Salmonella &phi 0 antigen but not to S. typhi H antigen (Greenwood, Bradley-Moore, Palit and Bryceson, 1972). Furthermore, cattle infected with Ijvpanosoma congolense had depressed responses to Clostridium oedematiens vaccine (Holmes, Mamo, Thomson, Knight, Lucken, Murray, Murray, Jennings and Urquhart, 1974; Scott, Pegram, Holmes, Pay, Knight, Jennings and Urquhart, 1977), foot and mouth disease vaccine (Scott et al., 1977) and louping-ill vaccine (Whitelaw, Scott, Reid, Holmes, Jennings and Urquhart, 1979). These last authors also demonstrated that the response of cattle infected with 7. uiuax to louping-ill virus vaccine, was depressed as was the response of mice infected with ‘I: congolense and T. Drncei. In the present study a depressed antibody response to BSA incorporated in the louping-ill virus vaccine was also demonstrated. Comparable findings ha\:c also been obtained in mice infected with Plasmodium yoelii yoelii ~vherc the primary antibody response to alum-adsorbed BSA was depressed (NcBridc and Micklem, 1977). The present study gives no indication of the underlying mechanism of r. gondii induced immunosuppression although it is likely that both the B and ? lymphocytes are involved. Strickland, Ahmed and Sell (1975) have shown that B cell function is depressed for 3 weeks post inoculation in mice infected with T. gondii and that T cells are also affected for at least 3 months. Thus the efficacy of a particular vaccine in an animal infected with 7. gondii probably will depend upon both the stage of infection at vaccination and whether the response is predominantly mediated by T and/or B cells. In conclusion, it is apparent that mice recently infected with T. gondii had a reduced immune response to two sheep vaccines. Thus in addition to being a serious cause of abortion and neonatal loss it is possible that 17: gondii might also depress the response of sheep to routine vaccination schedules making them more susceptible to a variety of other disease conditions. SUMMARY

Mice experimentally infected with Toxoplasma gondii and then vaccinated with either Clostridiztm zuelchii type D vaccine or louping-ill virus vaccine gave diminished responses to both when compared with vaccinated control mice. As 1. gondii was able to depress significantly the immune response of mice to two sheep vaccines it is suggested that in addition to causing serious neonatal lossesin sheep it might also depress their response to vaccination. ACKNOWLEDGMENTS

We would like to thank Dr R. M. Barlow and Dr I. D. Aitken for helpful discussion and Mrs J. Finlayson for technical assistance.

IMMUNOSUPPRESSION

IN MICE

WITH

TOXOPLASMOSIS

379

REFERENCES

Brotherston, J. G., and Boyce, J. B. (1970). A new vaccine against louping-ill. Veterinary Record, 84, 5 14-5 15. Buxton, D. (1978). In-vitro effects of Clostridium welchii type-D epsilon toxin on guineapig, mouse, rabbit and sheep cells. Journal of Medical Microbiology, 11, 299-302. Cassimos, C., Lazanakis, S., and Thomaidis, T. (1966). Antibody response after immunisation with typhoid-paratyphoid A and B vaccine in Kala-azar. Acta Paediatrica Scandinavia, 55, 301-304. Greenwood, B. M., Bradley-Moore, A. M., Palit, A., and Bryceson, A. D. M. (1972). Immunosuppression in children with malaria. Lancet i, 169-172. Holmes, P. H., Mamo, E., Thomson, A., Knight, P. A., Lucken, R., Murray, P. K., Murray, M., Jennings, F. W., and Urquhart, G. M. (1974). Immunosuppression in bovine trypanosomiasis. Veterinary Record, 95, 86-87. Huldt, G., Gard, S., and Olovson, S. G. (1973). Effect of Toxoplasma gondii on the thymus. Nature, 244, 301-303. Jennis, F. A. (1966). Simplified haemagglutination test for toxoplasmosis using pyruvic aldehyde treated cells. Australian Journal of Experimental Biology and Medical Science, 44, 3 17-322. McBride, J. S., and Micklem, H. S. (1977). Immunosuppression in murine malaria, II. The primary response to bovine serum albumin. Immunology, 33, 253-259. Reed, L. J., and Muench, H. (1938). A simple method of estimating fifty per cent end points. The American Journal of IQgiene, 27, 493-497. Reid, H. W., and Doherty, P. C. (1971). Experimental louping-ill in sheep and lambs. I. Viraemia and antibody response. Journal of Comparative Pathology, 81, 291-298. Reid, H. W., Doherty, P. C., and Dawson, A. McL. (1971). Louping-ill encephalomyelitis in sheep. III. Immunoglobulins in cerebrospinal fluid. Journal of Comparative Pathology, 81, 537-543. Scott, J. M., Pegram, R. G., Holmes, P. H., Pay, T. W. F., Knight, P. A., Jennings, F. W., and Urquhart, G. M. (1977). Immunosuppression in bovine trypanosomiasis : field studies using foot-and-mouth disease vaccine and clostridial vaccine. Tropical Animal Health and Production, 9, 159-165. Strickland, G. T., Ahmed, A., and Sell, K. W. (1975). Blastogenic response of toxoplasma-infected mouse spleen cells to T- and B-cell mitogens. Clinical and Experimental Immunology, 22, 167-176. Strickland, G. T., Pettit, L. E., and Voller, A. (1973). Immunodepression in mice infected with Toxoplasma gondii. The American Journal qf Tropical Medicine and Hygiene, 22, 452-455. Strickland, G. T., and Sayles, P. C. (1977). D e p ressed antibody responses to a thymusdependent antigen in toxoplasmosis. Infection and Immunity, 15, 184-190. Wells, P. W., and Reid, H. W. (1978). Antibody responses to vaccination against louping-ill virus in newborn lambs. Journal of Comparative Pathology, 88, 425-43 1. F. W., and Whitelaw, D. D., Scott, J. M., Reid, H. W., Holmes, P. H., Jennings, Urquhart, G. M. (1979). Immunosuppression in bovine trypanosomiasisstudies with louping-ill vaccine. Research in Veterinary Science, 26, 102-107. [Received for publication,

October 2nd, 19781