Alteration in immune response of mice with dual infection of Toxocara canis and Japanese encephalitis virus

Alteration in immune response of mice with dual infection of Toxocara canis and Japanese encephalitis virus

ASHOK K. GUPTA* AND KHORSHED M. PAVRI

National Institute of Virology, 20-A, Dr Ambedkar Road, Post Box No. 11, Pune 41 I 001, India

Abstract Concurrent dual infections of mice with Toxocara canis and Japanese encephalitis virus (JEV)

showed a distinct synergistic effect of the parasite on sublethal JEV infection. Dose-dependent effects were evident in the mice infected dually and with T. canis alone; leucocytosis with suppression of eosinophilia in the peripheral blood and reduction in migration inhibition factor produced by spleen cells in response to T. canis and JEV antigens were recorded, particularly in the former group. These changes were accompanied by reductions in both antibody response to sheep erythrocytes and T cell numbers in the spleen. No appreciable changes were recorded in the mice infected with JEV alone. Thus, the T cell immuno-suppression induced by T. canis might have triggered the viral encephalitis caused by JEV in the dual infections.

Introduction Japanese encephalitis (JE), caused by a mosquito-

borne flavivirus (family Flaviviridae), has a wide distribution in south-east Asia and the western Pacific region (WHO, 1979; RODRIGUES? 1984). In India JE is increasingly being recognized m different parts of the country (RODRIGUES, 1984). Some characteristic features of JE in India are (i) a scattered, almost exclusively rural distribution; (ii) it predominantly affects children; (iii) high clinical to sub-clinical ratio (between 1:30 and 1:300 and higher) and (iv) occurr- ence of the disease mainly among the lowest socio- economic groups.

These features led us to a search for the role of parasites as special risk factors in JE. A substantial proportion of JE patients suffer from concurrent infections with intestinal parasites (LIU et aE., 1957). High levels (two-fold increases or higher) of total serum IgE were found in laboratory-proved JE cases during the acute stage of infection (PAVRI et al., 1980). These levels declined during convalescence.

Earlier studies in China (LIU et al., 1957) and in India (SHANKAR et al., 1983) have provided evidence for association between cerebral cysticercosis and JE. A synergistic effect of Toxocaru canis (MOCHIZUKI et al., 1954; PAVRI et al., 1975) or Trichinella spiralis (LUBINIECKI et al., 1974) infection on Japanese encephalitis virus (JEV) infection in adult mice has been reported. LUBINIECKI et al. (1974) recorded such an effect on JEV infection due to the immunosuppression caused by T. spiralis in dual infections. Here, we present data showing significant immunosuppression, particularly in T cell-mediated immune mechanisms, in mice dually infected with Toxocara canis and JEV.

Materials and Methods Infectious agents, animals and experimental design

In all experiments, T. canis second stage larvae,\pa.in- tained in vitro, were used (GUPTA, 1984). The excr+ory- secretory (ES) antigen was prepared from the pooled spent

*For reprint requests.

medium collected from the cultures. Swiss male mice, 3 to 4 weeks old, were infected with the larvae by feeding.

An In&an strain of JEV, P 20778, was used in all the experiments. The virus was inoculated into mice sub- cutaneously (SC); young adult mice infected SC do not show overt illness. JEV was given 3 d after the larval feeding, so that the viraemic phase would coincide with active larval migration. Normal uninfected controls, mice infected with the parasite only, and mice infected with the virus only, were included in all the experiments. 2 experiments were con- ducted, one using a low dose and the other a high dose of 7’. canis and JEV.

Total and differential leucocyte counts Pre-infection and post-infection (PI) values for the total

and differential leucocyte counts (TLC and DLC) were determined in all mice using blood taken from the tail vein according to standard methods (DACIE & LEWIS, 1975).

Larval detection Larval migration was studied by examining a small

portion of the liver, lungs and brain microscopically (MOCHIZUKI~~ al., 1954; PAVRI et al., 1975). No attempt was made to quantitate the larvae present in tissues; all organs which showed the presence of larvae were scored as positive.

Virus isolation It has been established that, although, mice dually

infected with T. canis and JEV have levels of viraemia similar to those of mice infected with the virus alone! the rate of virus isolation from the brain of mice dually mfected was significantly higher (PAW et al., 1975). Accordingly, we attempted isolation of JEV from brain tissue of mice infected with virus, using the method described previously (PAW er al., 1975). A previously described method was also used to confirm that each isolate contained JEV antigens (PAVRI & SHAIKH, 1966).

Migration inhibition facrm (MZF) assays Assays were carried out as described by BLOOM &

BENNETT (1971). Single cell suspensions obtained from spleens were incubated with T. canis (20 pg) and JEV (50 haemagglutination units) antigens in minimum essential medium containing 10% foetal calf serum (FCS) or goat serum (GS). Medium containing GS was used for most studies, since it gave results similar to those obtained using medium containing FCS. JEV antigen was prepared by sucrose-acetone treatment (CLARKE & CASALS, 1958). A 10% suspension of normal mouse brain in normal saline was used

836 IMMUNE RESPONSE OF MICE TO

as control. The percentage migration was calculated by comparing the area of cell migration in the presence of antigen with that in its absence, and expressed as a migration index.

Antibodies to T. canis and JEV antigens Antibodies to T. cunis ES antigen were detected by the

agar gel diffusion (AGD) test as described by GU~TA (1984). Human blood group A- and B-like substances have been demonstrated in ES of T. canis larvae (SMITH et al., 1983). Therefore, ES antigen was also checked against human blood group (ABO) sera in the AGD test and no reaction was recorded. The antibodies to JEV were detected using a neutralization test (HAMMON St SATHER, 1969). Suckling mice 2 d old were challenged intraperitoneally (II’) with the virus-serum mixtures.

Antibodies to sheep red blood cells (SRBC) The effect of dual infection on the immune response to

SRBC was studied in the mice infected with the low doses of T. canis and JEV. Primary and secondary antibody responses were measured in the mice inoculated IP with one (PI da2 2/5)* and 2 (PI days 2/5 and 22/25) doses of SRBC (10 cells/mouse). The mice were bled 5 d after the inoculation of SRBC and the levels of antibody present in the sera were determined using the haemagglutination test. The sera of dually and singly infected mice which did not receive SRBC were included as controls. The experiment was repeated to confirm the results obtained initially. The effect of ES products of the larvae on the primary immune response to SRBC was also determined by inoculating the fluid IP into mice, one day before giving SRBC. Serum samples were obtamed 5 d later and assayed for presence of antibodies.

Enumeration of B- and T-cells B- and T-cells were counted in the mice infected with low

doses of T. canis and JEV. Single cell suspensions of spleens were prepared and fractionated on lymphocyte separation fluid. B- and T-cell enumeration was carried out according to the procedure described by BHATIA et al. (1983).

Results Fate of infected animals and recovery of infectious agents

The dosages and number of mice used in the 2 experiments are given in the Table. When low doses of-the parasite and the virus were used for infection, sickness or death was observed only in the dually

*2 d after giving JEV and 5 d after giving T. canis.

Toxocara AND JEV INFECTIONS

infected mice; of 38 dually infected mice, 2 became sick and 3 died (13.2%). When higher infective doses were used, 6 of the 38 (15.8%) mice infected with T. canis died, compared to 18 of the 45 (40%) dually infected mice. This difference was statistically significant (P<O.O5). The mice receiving JEV alone showed no ill effect even when given the higher dose.

Infection with JEV did not affect the larval migration pattern in mice infected with T. canis; in the mice receiving the higher dose migration started earlier and persisted longer than in mice which received the low dose. With the higher dose the larvae were detected in the brain on PI day 2, compared to PI day 3 in those receiving the low dose. Similarly, larvae were detected in the liver and lungs for 11 d in the former, compared to 4 d in those receiving the low dose.

JEV was isolated from dually infected mice only. In the frrst experiment JEV was isolated from brain tissue of 5 out of 13 mice killed up to PI day 17. No JEV was detected in another 20 mice sampled from PI days 24 to 180. When the higher challenge doses were used, 16 of 22 mice examined up to PI day 17 yielded the virus. Again, no JEV could be isolated from mice examined after this time up to PI day 180.

Total leucocyte count and differential leucocyte count The results of TLC and DLC are presented in Figs

1 and 2. The mice infected with low and high doses of JEV alone exhibited a slight leucopaenia on PI day 3 (6467 + 969 cells/mm3 with low dose and 3320 rtr 843 cells/mm3 with high dose) against the respective pre-infection values in the same mice (7533 + 1122 cells/mm3 and 4200 + 807 cells/mm3), after which the counts returned to the normal range. Such changes in TLC were accompanied with a transient lympho- paenia on PI day 3, after which lymphocyte numbers were in the normal range. In contrast, the mice infected dually or with the parasite alone showed leucocytosis accompanied with marked differences in the numbers of lymphocytes, blast cells and eosin- ophils. The leucocytosis and the number of blast cells were of high magnitude in dually infected mice, particularly those receiving the high dose. The levels of lymphocytes and the blast cells declined with the

Table-Experimental design, and sickness and death of infected mice

Group of mice

Route of inocula-

tion.

T. canis larvae JEV Dual

Oral

SC As above

infection Uninfected controls

Experiment I Number of

Experiment II Number of

mice sick mice sick or died/ or died

Infective number Infective number dose per of mice dose per of mice mouse tested. mouse tested

800-1000 0/37t 1400-1600 6/38$(15.8%)

105’5LD5d0.03 ml o/33 lO”“~,D~~~;~3 ml 0129 As above 5/38+(13.2%) 18/45$(40%)

0132 0129

T. canis low dose vs high dose P<O.O5. Dual infection low dose vs high dose FcO.05.

A. K. GUPTA AND K. M. PAVRI 837

TIME AFTER INFECTION ( DAYS 1

Fig. 1. Total leucocyte counts. Normal control mice q ; mice with T. canis atone@; JEV alone q n; dual infection n . Each value represents the mean of 4-6 observations on different PI days; each bar represents one standard deviation. #PI day 3 for JEViPI day 6 for T. cams.

advent of eosinophilia in the peripheral blood. Eosi- nophilia occurred earlier, and was higher, in the mice receiving the lower dose of T. canis alone. Dually infected mice receiving the higher dose showed a low and bnly transient eosinophilia around PI day 31/34.

Migration inhibition factor Production of MIF (migration index of ~80%)

against T. canis ES antigen was lower in the dually infected mice, compared to those infected with the parasite alone (Fig. 3A). Production of MIF against JEV antigen was much lower in the dually infected mice, compared to that recorded in the mice infected with the virus alone (Fig. 3B). With the higher dose, particularly with dual infections, there was a marked reduction in the production of MIF. In both experiments, an inverse correlation was recorded between MIF production to JEV and virus isolation from the brain.

In the first experiment with the lower dose, a nositive correlation was recorded between MIF nro- huction to T. canis and the appearance of eosinophilia in the dually infected mice (r = O-77) and in those infected with the parasite alone (r = 0.85). With the higher dose also, a positive though lower correlation between MIF production to T. canis antigen and eosinophilia was recorded in the mice infected dually (r = 0.66) or with T. canis alone (r = 0.67).

Antibodies to T. canis and JEV antigens There was no difference in antibody production to

T. canis ES antigen between mice infected dually and

T,,,,E AFTER INFECTION ( DAYS 1

Fig. 2. Differential leucocyte counts. Mice infected with JEV, low dose x---x; JEV, high dose x--x; T. canis, low dose e---o; T. cnnis, high dose O-O; dual infection, low dose A---A; dual infection, high dose A-A. Each value represents the mean of 4-6 observations, vertical bar represents one standard deviation. Range for normal controls represents the mean of 4-6 observations on different PI days. #PI day 3 for JEViPI day 6 for T. canis. * Normal and pre-infection values.

those with the parasite alone. Antibodies at 1:2 dilution were detected on PI day 20 and at 1:8 dilution on PI day 27; they seemed to persist up to PI day 183. There was no apparent difference between antibody titres in the mice receiving the low and high doses.

The appearance of antibodies to JEV was delayed by one week in the dually infected mice. With the lower dose, low levels of antibodies were recorded on PI day 17 in the mice infected with the virus alone. In the dually infected mice, antibodies appeared on PI day 24. With the higher dose, the antibodies were detected on PI day 10 in the mice infected with the virus alone and on PI day 10117 in those infected with both T. canis and JEV. There was, however, no marked difference between the antibody levels. No antibodies were detected in the sera collected on PI day 180 in the mice infected dually or with the virus alone. In both experiments, no inverse correlation, as seen with MIF, was recorded between antibody production and virus isolation from the brain.

Antibodies to SRBC The results of these tests are depicted in Fig. 4.

Sera of normal and infected mice which did not receive SRBC were all negative for antibodies. The haemagglutinin titres were significantly lower in the mice with dual infections, particularly at the early stage (Fig. 4A). A slight decrease in antibody titre was also recorded in the mice infected with T. canis alone. No decrease in the haemagglutinin titres was recorded in those infected with the virus alone.

838 IMMUNE RESPONSE OF MICE TO Toxocara AND JEV INFECTIONS

i I 3v ’ , /’ J(

TIME AFTER INFECTION ( DAYS I

Fig. 3. Migration indices. (A) T. canis and (B) JEV antigens in mice infected with JEV, low dose x---x; JEV, high dose x-x; T. canis, low dose a- -a; T. Curtis, high dose O-O; dual infection, low dose A- -A; dual infection, high dose A-A. Each value represents the mean of 4-6 observations; vertical bars represent 1 standard deviation. Range in normal controls (*) and against normal mouse brain (**) represent means of 4-6 observations on different PI days. The spleen cells taken from mice with dual infections or JEV alone showed higher migration indices (loo-104%) against normal mouse brain, compared to normal controls and those infected with T. canis alone (%-l&7%). #PI day 3 for JEViPI day 6 for T. canis.

The effect of ES on immune response to SRBC is shown in Fig. 4B. Mice injected with ES fluid showed a similar decrease in antibody titre to SRBC to that seen in mice infected with T. canis larvae. No such effect was observed in the mice injected with saline extract of T. canis larvae only. Dose-dependent effects of ES products on antibody titres were recorded when different doses of ES fluid were injected. Control mice were injected similarly with culture medium only.

B and T cells The proportions of B and T cells in the normal and

infected mice are shown in Fin. 5. Normal B cell level in the control (uninfected) n&e was 24-80 + 2.03%. In the mice infected with JEV alone, no change was recorded in B cell levels on PI days 7 or 28 (P>O.OS). In mice infected dually, and those with T. canis alone, a significant increase m B cells was recorded on PI days 7/10 and 28/3 1, compared to the values in normal mice (P<O*OOl). This rise in B cell numbers was significantly higher in the dually infected mice on PI day 7/10, compared to that of the mice infected with the parasite alone (P<O901).

The nomal T cell level in the control mice was 44.50 f 1.50%. In the mice infected with JEV alone,

F 1:x-

<1:2- 0 I

1:16.

8 I:&

F I=

& 1:4,

B t= a 1:2.

c1:2.

CNS c 2-ME

PRIMARY

(81

?N S c 2-ME

SECONDARY

0.25 0.5 I.0

PRIMARY Fig. 4. Haemagglutinating antibody titres to sheep red blood cells (SBBC) (A) in mice not receiving excretory - secretory (ES) antigens, and (B) in mice receiving ES antigens. Normal control mice (SBBC not injected) 0; normal controls (SBBC injected) l7J; T. canis alone q ; JEV alone q ; dual infection n ; intraperitoneal injection of saline extract of T. canis larvae&j’; intraperitoneal injection of ES fluid of T. canis larvae a. Sera were’colleced on 5th day of tirst (primary) or second (secondary) SBBC injections. Each value represents the titre of 4-6 pooled sera. NS, normal saline; 2-ME, 2-mercaptoethanol.

::. :::... : 8: I

5 :: :: 00 :- - 88:

DO0 8 oDoo”

**. 8

:: :’

i:. : :

o- ?/IO # 28/31 7110 28131 I I I I

6 - CELL T-CELL

TIME AFTER INFECTION t DAYS 1

Fig. 5. Percentages of B- and T-cells. Normal control mice 0; JEV alone x; T. canis alone 0; dual infection A. At least 2 normal control mice were included with each experimental group. #PI day 7 for JEV/PI day 10 for 2’. canis.

A. K. GUPTA AND K. M. PAVRI 839

T cells showed a decrease on PI day 7 (P<O.O5), compared to the normal controls. On PI day 28, the T cell level was normal. In mice infected dually or with T. canis alone, T cell level was lower on both PI days 7110 and 28/31, compared to the normal controls (P<O*OOl). The decrease in T cells in the dually infected mice was significantly greater, particularly on PI day 7/10, compared to the mice infected with the uarasite alone or with IEV alone (P<O~OOl).

Discussion MOCHIZLJKI et al. (1954) and PAVRI et al. (1975)

recorded a synergistic effect of T. canis on sublethal JEV infection in mice. The present study confirms their findings and shows a dose-dependent effect. A statistically significant difference (P<O*OS) was noted between the dually infected mice receiving the higher dose of parasite and virus, compared to the mice given the lower dose. An association between sickness and death and isolation of the virus from the brain in the dually infected mice suggested that the virus might ultimately be responsible for the acute illness and death of the mice.

role of CM1 in defence against JEV infection, particularly during early stages. The T cell immunosuppression induced by T. canis larvae in the experimental model thus seems to be an important predisposing factor triggering the viral encephalitis due to JEV. Our preliminary studies in nude athymic (nu/nu) mice (BL6) showed that the virus alone produces sickness and death when injected SC (105’5LD&0*03 ml). The virus was isolated from the brain and-the lungs of the sick or moribund mice. In contrast, no sickness or death was recorded in Swiss and BL6 (nu/+) mice and no virus was isolated from the brain and the lungs (Lad, Gupta & Pavri, unpublished observation).

The present study indicated that some haematological and immunological features were also dose- dependent. The dose-response effect of the parasite on eosinophilia was interesting. CALDWELL et al. (1980) and LLOYD et al. (1983) recorded that T. cunis infections in humans and animals induce T cell imunosuppression with a corresponding suppression of eosinophilia. KAYES & OAKS (1980) also reported a significantly reduced eosinophil response in T. cunis infected mice depleted of their functional T lvmpho- cytes. We have also recorded a correlation between the levels of eosinoohiha and MIF oroduction to T. canis antigen. The significantly reduSed MIF production in dually infected mice and the lowered eosinophilia in our experiments provide evidence of a marked suppression in T cell-mediated immunity, particularly in the dually infected mice.

We had earlier noted changes in B and T cells numbers in the peripheral blood, similar to those seen in the present study using the spleen (Gupta & Pavri, unpublished observation). The decrease in T cells was correlated with a marked reduction in MIF as well as the immune response to SRBC, a T cell-dependent antigen. This effect on the immune response to SRBC wasevidently due to the metabolites produced by the narasite in the ES fluid and not due to the adsorotion of antibodies on the surface of the parasite. A similar effect of ES or extracts of adult worms on the immune response to SRBC was recorded in T. spirulis infection in mice by BARRIGA (1975).

Studies in experimental animals infected with JEV have established the role of immune response, parti- cularlv cell-mediated immunitv (CMI). in orotection from JEV infection (HUANG, -1982; MAT&JR et al., 1983). Moreover, antibody production to JEV has been found to be thymus-dependent (MORI et al., 1970). Significant MIF production to JEV in experimental infections has been recorded as early as PI day 3 (HUANG, 1982; MATHUR et al., 1983) and an inverse correlation has been recorded with viral encephalitis (HUANG, 1982). We have shown an inverse correlation between MIF and virus isolation from the brain in both the experiments. We did not see such correlation for antibodies. This confirms the

Acknowledgements We are grateful to Shri V. L. Ayachit and Mrs V. J. Lad

for invaluable technical assistance. The help of Mrs N. Athalye with statistical analysis is gratefully acknowledged.

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Accepted for publication 19 January I987

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Alteration in immune response of mice with dual infection of Toxocara canis and Japanese encephalitis virus