Veterinary Immunology and lmmunopathology, 9 (1985) 371--382 371 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

TRANSFER OF INFECTION-IND~ IMMUNE P~ION TO TOXOCARA CANIS 1%1 A MOUSE MODEL

JI]LIO E. CONCEPCION and OMAR O. BARRIGA

Present address: Laboratorio Veterinario Central, San Cristobal, The Dominican Republic.

Department of Veterinary Pathobiology, The Ohio State University, 1925 Coffey Road, Columbus, Ohio USA.

(Accepted 8 March 1985)

ABSTRACT

Concepcion, J.E. and Barriga, O.O., 1985. Transfer of infection-induced inrmm~e protection to Toxocara canis in a mouse model. Vet. Immunol. Immuno- pathol., 9 : 371-382.

Groups of mice were vaccinated twice with soluble extracts of em~bryonated eggs, females or males of Toxocara canis, or horse serum, and infected with 2,000 homologous embryonated eggs. Recovery of larvae on the fifth day by digestion of mesenteric lymph nodes, liver, lung, brain, and carcass revealed a slight but nonsignificant protection elicited by the parasite materials. Other groups were immunized by homologous infections. A single, 200-day-old infection increased importantly the number of larvae resulting from a challenge, possibly by inducing an immunosuppression in the host. Two infections given within ii months protected partially against the larvae of a challenge, particularly by trapping the parasites in the liver. Trans- fer of mesenteric lymph node cells from twice infected mice reduced the total number of parasites, and the liver and lung parasitism of a challenge in the recipients, whereas transfer of ser~n decreased the total number of parasites and the brain and carcass parasitism. The ccmbination of cells and ser~n acted synergistically in lungs and brain but antagonistically in liver and carcass.

INTRODUCTION

Systemic infection with Toxocara canis larvae is of universal occurrence

in dogs (Griesemer et al. 1964). Its prevalence in man is difficult to

assess because of the uncertainty of the conventioD~l diagnosis (see

Glickman et al. 1978). Nevertheless, Cantor et al. (1984) estimated that at

least 675 people were diagnosed with ocular toxocariasis in the United

States in 1981, and the Toxocara Reference Laboratory of the Hospital for

Tropical Diseases in London received 1,590 sera from the British Isles for

testing in a single year (Ree et al. 1984). In a review of the recent

literature (Barriga, in preparation), we found that I0 studies in the United

0 1 6 5 - 2 4 2 7 / 8 5 / $ 0 3 . 3 0 © 1 9 8 5 Elsevier Science Publishers B.V.

372

States and Canada reported specific anti-T, canis antibodies in 109 (6.9%)

of 1,591 apparently healthy people. Eight similar studies in Europe, Iraq

and Japan showed that 5.1% of 2,135 clinically healthy people also had

antibodies. It see~s, then, that h~man toxocariasis is a probl~n more

serious than previously believed.

A number of measures has been reconmended to prevent toxocariasis in

lower animals or in man (for exar~ple, see Jacobs et al. 1977; Schantz and

Glickman, 1979) but the figures of canine and human prevalence indicate that

they do not work.

The critical event in the epider~iological cycle of T. canis is the

persistence of larvae in the tissues of the bitch, and their subsequent

passage to her puppies in uterus and with the milk. Any method that fails

in destroying this reservoir can have only tesporary success. The advent of

fenbendazole was regarded as the first medicine able to kill the tissue

larvae consistently (Dubey, 1979) , but the drug appears to be active only on

young larvae and when administered for long periods (Abo-Shelada and

Herbert, 1984). In actual tests (Duwel, 1983), prolonged fenbendazole

treatment of the bitch still allows paranatal infection of about half tb

puppies, and does not prevent their subsequent infection with eggs from th

environment. Finally, it is dubious that dog's owners will be willing to g~

through the expense and annoyance of such a prolonged treatment without

clear and inmediate reward.

An obvious and better alternative is the creation of a vaccine able to

kill the larvae and persist effective in the presence of reinfections. It

is rather surprising, however, how little attention protective immunity to

systemic toxocariasis has commanded from researchers.

In studies in mice, Lee (1960) reported results that ir~lied that rein-

fections produced the death of pre-existing larvae, Olson (1962) found that

homologous or Ascaris infections protected partially agains a Toxocara

challenge, and Schmunis and Pacheco (1970) verified that the immunizing

infection had to last at least for 30 days to induce protection. Izzat and

Olson (1970) could not establish protection with homologous infections but

were successful with inoculation of particulated extracts of homologous

parasites in potent adjuvants. Kato (1973) reported production of protec-

tion with ccr~plete or interrupted hc~ologous infections, or with homologous

or Ascaris extracts. Kondo et al. (1976) found that homologous infections

protected the liver from the parasitism of a challenge. Sugane and Oshima

(1982, 1983) reported that muscle larvae of a primary infection were killed

by normal but not by athymic mice and that the infection of the normal mice

stimulated trapping in the liver of the larvae of a challenge.

373

Studies in dogs have been less n~nerous. Douglas and Baker (1959) found

that tissue larvae still persisted in bitches and passed to the offspring

after at least 358 days of an infection. Fernando (1968) verified that a

first infection in dogs elicited resistance against a challenge and that the

challenges suppressed the ovoposition of the primary parasites. The latter

phenomenon was confirmed by Macchioni et al. (1970). In later experiments

(Fernando et al. 1973) it was demonstrated that the resistance was directed

against the infective larvae. Oshima (1976) verified that second Toxocara

infections induced stronger ~ e reactions and resulted in a reduced

development of the parasites. Lowenstein (1981) demonstrated that a second

infection in recently whelped bitches developed higher antibody titers but

only 1.8% to 6.1% as many parasites as a primary infection.

It is evident, then, that mice and dogs are able to develop important

protective immunity to T. canis. Study of the Lmmune reactions responsible

for this protection and of the antigens that elicit it should permit the

design of artificial and effective methods of immunization against the

parasite.

Because age resistance and a putative perinatal depression of the immun-

ity in dogs ccr~plicate the investigation of anti-Toxocara protection in this

species, we decided to investigate anti-larval immunity in mice first. Here

we report some of our results.

MATERIALS AND METHODS

Animals, Parasites and Infection

Female BALB/cJ mice (The Jackson Laboratory, Bar Harbor, ME), 5-weeks-

old, were used in all experiments. T. canis specimens were obtained frcm

dogs donated by the Franklin County Animal Control. Eggs were collected by

dissecting the uteri of gravid females, suspended in 2% formalin, and

embryonated at roam temperature by bubbling air in the dark. They were then

washed 3X in 0.15M NaCI, and titered.

Infections were done by administering 2,000 embryonated eggs intragastri-

cally to each mouse. The proportion of viable eggs in the infective suspen-

sions was determined before every infection. The intensity and distribution

of the infection were determined by recovering the larvae from the mesen-

teric lyr~h nodes, liver, lungs, brain and carcass. For this, the tissues

were minced and digested separately in 0.5% pepsin with 0.75% HCl at 37°C

for 3 hours. The results of the digestion were filtered through a double

layer of gauze, centrifuged at 500 g for 5 min, and the n~ber of larvae was

estimated by examination of the entire sediment or titration of aliquots.

374

Parasite Extracts

A. Adults. The specimens were washed 3X in 0.15M NaCI, 3X in 0.15M Na2HPO 4

plus 0°I5M Na H2PO4, pH 7.2 (pH 7.2 buffer), and minced. Five grams of

minced female or male worms were suspended in 20 ml of pH 7.2 buffer,

hanogenized 5X for 60 sec at 23,000 rpm in a VirTis "45" homogenizer (The

VirTis Company, Gardiner, NY), 10X for 60 sec at 20,000 rpm in a Tekmar

homogenizer (Tekmar Company, Cincinnati, OH), and sonicated 10X for 60 sec

at 185 watts/cm 2 in a model W-10 sonicator (Heat System Ultrasonic, Inc.,

Plainview, NY). The resulting suspensions were extracted at 4°C for 24

hours and centrifuged at 20,000 g for 30 min. All procedures were done at

4°C or in ice. The resulting supernate was used as the extract.

B. Eggs. ~abryonated eggs were washed 3X with 0.15M NaCI, once with pH 7.2

buffer, suspended at about 40,000 egg/ml in I0 ml of the same buffer,

hcrnogenized 20X for 60 sec at 20,000 rpm in the Tekmar homogenizer, and

sonicated 15X for 30 sec in a W-10 sonicator. The sonicated suspension was

extracted and centrifuged as above, and the supernate was saved to be used

as the extract.

Protein content in all extracts was determined by the Folin-Lowry test

using bovine serum albumin as a standard. Analysis of protein components in

the extracts was made by sodit~n dodecyl sulfate (SDS) poly-acrylamide gel

electrophoresis (PAGE) in gels with a gradient from 4% to 30% as recommended

by Pharmacia (1980). Staining was done with Coanassie Brilliant Blue R-250,

or with silver by the modification of Victor Tsang (Center for Disease

Control, personal c~ladnication) of the technique of Morrissey (1981).

Inmunization by Vaccination with T. canis Extracts

In the definitive experiment, four groups of 12 mice were vaccinated with

egg, female, or male parasite extract, or with horse serum as a control,

respectively. Each mouse received 1,200 ~g of protein intraperitoneally as

a primary immunization and 250 ug intranuscularly a month later. After a

week, each mouse was infected with embryonated T. canis eggs, and killed 5

days later to recover the larvae and serum for hemagglutination. Production

of antibodies was determined by indirect hemagglutination (IHA) with sheep

red blood cells sensitized with female or embryonated egg extracts by the

tannic acid method (Barriga, 1975). Sert~n of a rabbit hyperimmunized with a

proportional mixture of extracts was used as a positive control.

Immunization by Previous Infections

Three groups of i0 mice each were infected with 2,000 embryonated eggs.

On day 200 of infection, one group was digested to recover the parasites and

375

the other two were infected again with the same dosis. On day 128 of this

second infection, one of the groups and a new, non-infected group of mice of

the same age were infected with 2,000 ~b~lonated eggs. Parasite burden and

antibodies were studied in all the animals 5 days after this infection. We

had, then, mice with a 200-day infection, with 334- and 133-day infections,

with 334-, 133- and 5-day infections, and with a 5-day infection.

Transfer of ~ e Resistance

Six groups of i0 mice were used as recipients of cells, serum, or cells

plus seru~ from infected or uninfected mice in the definitive experiment.

The infected mice had been infected with T. canis 130 and 30 days before

collecting the cells and sera for transfer. The infection was confirmed by

recovery of larvae. The cells were obtained from the mesenteric lyr~oh nodes

as described by Barriga (1978), but using medium 199 with Hank's salts and

L-glutamine (GIBCO Labs, Grand Island, NY) supplemented with 2.2 g of Na

bicarbonate per liter, instead of mediu~ RPMI-1640. The number of viable

cells was estimated by titration of an aliquot of the final suspension in

0.16% trypan blue in medium. The serum was collected from blood obtained by

section of the jugular vein of anesthetized animals.

Cell recipients received 2 x 106 viable cells intraperitoneally. Serum

recipients were injected intraperitoneally with 0.125 ml of ser~n/15 g of

body weight, estimated to represent 33% of their intravascular seri~n. These

mice were infected 24 hours after receiving the cells or ser~n. Recipients

of cells plus serum received 2 x 106 cells first, 0.125 ml of sert~/]5 g of

body weight 24 hours later, and were infected after 24 hours.

Statistics

The values in each group were described by mean _+ standard error of the

means; differences among groups were studied by analysis of variance and the

Duncan's rank s~n test.

RESULTS

Analysis of Antigens

The SDS-PAGE of E~bryonated egg extract (results not shown) resulted in

at least 27 protein bands that ranged from about 100,000 to less than 14,000

molecular weight. The female extract resolved into 52 bands and the male

extract in 53, in the same range. Each extract showed components exclusive

to that particular extract as well as bands cc~non to the other prepara-

tions.

376

Vaccination

The results of the vaccination with the different extracts appear in Fig.

i. The number of larvae recovered from vaccinated mice was always lower

than that retrieved from mice injected with horse serum. This was also true

for the diverse organs, except lymph nodes in which the n~aber of parasites

was always very low. The differences in number of larvae between vaccinated

and control animals, however, did not reach statistical significance. All

mice vaccinated with extracts exhibited antibodies against female extract,

at titers of 56 ± 6.4 (X z SE, range I0 to 320), regardless of the extract

with which they were vaccinated. The mice inoculated with horse serum were

all negative.

E F F E C T OF V A C C I N A T I O N W I T H P A R A S I T E E X T R A C T S ON M U R I N E T O X O C A R I A S I S

250

200

bJ i o o

• 6 0

4 0

20'

[ ] C O N T R O L

B EGGS

[ ] F E M A L E S

[ ] M A L E S

~iiiiiiii-:/~

~iiiiliL;:!/i ~iiiiii"~': J "~ ~HI I I I . j ! . :.,: I

lllllllTXi;;t

I l l l l l l ~

N ~ IIIIIlll~::i~ s:.::t ~ IIIIIIlli<.:.~:;:]

LYMPH NODES LIVER LUNG BRAIN CARCASS TOTAL

Fig. i. Distribution of Toxocara canis larvae in organs of mice vaccinated with extracts of embryonated eggs, females or males of T. canis five days after infection with 2,000 hcmologous embryonated eggs. The controls were inoculated with horse ser~n.

Immunization by Previous Infections

The 5-day infection resulted in a parasite load of 282 larvae per mouse

on average. The total parasite load and the distribution of parasites in

the organs in each case are depicted in Fig. 2. The 200-day infection did

377

not produce parasites at the time of necropsy, q~e two, 334- and 133-day

old, infections produced 999 larvae per mouse, with most of the parasites in

the brain and carcass. Three infections, at 334, 133 and 5 days before

necropsy, caused the same parasite load as two infections (994 larvae per

mouse) but most of the worms were in the brain and liver (Fig. 2) . IHA with

egg or female antigens was negative in mice with a single 5-day infection,

and gave positive reactions only at a dilution of I:i0 after 200 days of a

single infection, or after 133 days of the second infection in mice with two

infections. The sera of mice with three infections reacted with titers of

312 _+ 243 (range 20 to 1,280) to egg antigens but were negative with female

antigens.

EFFECT OF AGE AND NUMBER OF I N FECTIONS ON MURIN E TOXOCARIASIS

I 0 0 0 t r-'n 5 d INFECTION ~'q 334'~133 d INFECTIONS

"' ~ 3:34.~ 133-5 d INFECTIONS * . .~ = BOO] /..~|

"t _J " 6 0 0 o

2 0 0 z~ I

I--'-L~.!J A2' I I LIVER LUNG BRAIN CARCASS TOTAL

Fig. 2. Number of larvae recovered from mice after 1, 2 or 3 Toxocara canis infections of different duration. No larvae were found after 200 days of a single infection. Zi I means statistical significance with the first col~n of each group, Zi 2 means statistical significance with the second col~n.

Transfer of Resistance

For comparison purposes, the parasite load of mice inoculated with mater-

ials from infected mice is expressed as a percentage of the parasite load of

the control mice that received the corresponding materials from non-infected

378

animals (Fig. 3). The transfer of cells, serum, or cells plus serum from

infected mice, caused a statistically significant decrease in the total

number of larvae recovered from the recipients (Fig. 3). Transfer of cells

from immune mice produced a reduction of the number of parasites in all

organs of the recipients but the differences with the controls reached a

statistical significance of p<0.05 only in the liver (Fig. 3). The differ-

ence in the number of larvae in the lungs of control mice and mice receiving

immune cells, however, had a p=0.08 that is biologically significant, in our

opinion. This difference had been significant in pilot experiments. Transfer

of serum from infected mice caused variable changes but only the reduction

of the number of larvae in the brain and carcass of the recipients reached a

statistical significance of p<0.05 (Fig. 3). Transfer of cells plus serum

resulted in a reduced number of parasites in the liver, lung and brain of

the recipients. The changes in the number of larvae in the lymph nodes were

not significant in any case.

140-

120"

I00" >

<, u. 8 0 o

80` z

40" o.

20"

YMPH NODES

EFFECT OF TRANSFERENCE OF IMMUNE CELLS, SERUM, OR CELLS+ SERUM ON MURINE TOXOCARIASIS

IIIIIIIII

iiiiiiii i LIVER LUNG BRAIN CARCASS

[ ] CONTROL

[ ] CELLS [ ] SERUM [ ] CELLS*SERUM

TOTAL

Fig. 3. Effect of transference of cells, serum, or cells + sert~n frcln twice T. canis-infected mice on the larvae recovered 5 days after an hamologous challenge of the recipients. Control recipients received the same materials but from non-infected donors. Zi means statistical significance with the respective control.

379

DISCUSSION

Although the number of larvae recovered from vaccinated mice was not

statistically different from the control groups, the former had always fewer

parasites. Other authors (Izzat and Olson, 1970) have reported success in

stimulating inmune resistance by inoculation of particulate extracts of T.

canis in potent adjuvants. It is possible that the large number of protein

components in our preparations contained many antigens irrelevant to protec-

tion, but that competed with the resistance-inducing material. This may

explain the only moderate but consistent reduction of parasites in our

vaccinated animals. On the other hand, the protective antigens may have

remained with the particulate fraction of the parasite extract that we did

not use.

A 5-day-old infection resulted in a parasite burden equivalent to 14% of

the administered dose (Fig. 2) which agrees with our and others' (Dunsmore

et al. 1983) experience with primary infections. No parasites were

recovered, however, after 200 days of a single infection. The sera of these

latter mice gave only slight IHA reactions to T. canis larval antigens, but

uninfected control mice gave clearly negative reactions, and mice infected

at the same time and with the same inoculL~n yielded abundant larvae when

their tissues were digested only 94 days after infection. On the other

hand, mice infected twice withJm Ii months yielded abundant larvae but only

slight IHA reactions (see below) . We believe, therefore, that the mice with

a 200-day infection had been actually infected but that the T. canis larvae

survived less than 200 days in them, and antibodies had waned by that time.

Sprent (1953) also found that very few T. canis larvae survived in mice by

the 6th month of infection and all of the~ were in the brain. Dunsmore et

al. (1983) reported that larvae were found virtually only in the brain after

2 months of infection. Despite the disappearance of the larvae within this

period, the 200-day infection caused modifications of the susceptibility to

T. canis because a new infection yielded a parasite burden of 50% of the

administered dose after 133 days (Fig. 2). Our preliminary studies desDn-

strated the presence of larvae by digestion of the intestine at 24 hours of

infection, but not later. It is unlikely, then, that the much greater

recovery of parasites from the 133-day reinfection as compared with the

5-day infection was due to retention of larvae in the intestine in the

latter case. It see~ns, therefore, that a first infection prcmoted the

survival of larvae from a challenge. This may be an instance of parasite-

mediated immunosuppression as it has been often reported in the nematodes

(Barriga, 1984).

380

The second infection evidently estir~lated new and effective defense

mechanisms because a third infection did not add to the existing parasite

load (Fig. 2). From the results of a single infection, one would have

expected that the new infection contributed about 300 new parasites if

immune protection had not intervened. The nt~ber of liver parasites on the

5th day of the third infection was significantly greater than in the single

5-day infection which had already been reported by Olson (1962) and Sugane

and Oshima (1983) in mice immunized by prior infections. It seems, then,

that the anti-larval resistance was particularly effective in limiting the

migration from the liver. An important, albeit not statistically signifi-

cant, reduction of the parasites in the brain and carcass was observed in

the mice with 3 infections as cc~pared with those with 2 infections. This

suggests that the anaamestic immune response caused by the third infection

might have killed preexisting parasites, similar findings were reported by

Lee (1960) in mice and by Lowenstein (1981) in dogs. We could not detect

inportant antibody production in sert~n collected 133 days after the second

infection, but found high titers of antibodies (up to 1,280) five days after

challenging similar animals. Since mice undergoing a single 5-day infection

did not have antibodies, we interpret the above results as a declination of

the humoral response after 133 days of the second infection, followed by an

anamnestic response caused by the 5-day challenge.

The results of the transfer of cells or sert~ from infected animals

revealed that both materials carry resistance to T. canis larvae, as judged

by the reduction of the total parasite burden in about 40% in the recipients

on a challenge (Fig. 3). Intraperitoneal injection is the most practical

procedure to transfer immune materials to several recipients on the same

occasion, but it may not be the most effective. Intravenous inoculation

might have in,proved these results. The location of the protective activity

of cells and serum was different. Whereas the cells reduced the number of

larvae recovered from the liver and lungs, the serum lowered the parasite

load in brain and carcass (Fig. 3). In some cases (lung, brain) cells and

ser~n appeared to act synergistically, whereas in another case (liver), the

ser~n appeared to facilitate the infection and abolished partially the

protective activity of the cells, and in a third case (carcass) the cells

appeared to abolish the protective activity of the ser~n (Fig. 3). Frem

these results, it was evident that cells and ser~n exerted their protective

activity locally, in different body spaces. This dissociation of the cell-

mediated and antibody-mediated protection has also been reported by Armour

and Dargie (1984) in Fasciola hepatica infections of rats.

381

It is surprising that prcmotion of resistance in the liver Coy transfer

of cells) or in the lungs (by transfer of cells and ser~n) did not result in

a concurrent reduction of parasites in organs that are thought to be invaded

later in the course of the infection (such as brain and carcass, respective-

ly). It appears that the anti-larval effect in liver and lungs needs some

time to become effective so that it allows a proportion of larvae to migrate

beyond these organs. Alternatively, the parasites might reach brain and

carcass in large quantities through the lymphatic vessels, circumventing the

liver. This possibility requires further study.

Our results demonstrate that a second T. canis infection induces enough

immune resistance to kill an important proportion of the larvae of a

challenge. They suggest, therefore, that the potential for the formulation

of an effective vaccine indeed exists. We are currently studying what

manipulations are necessary to increase this protective immunity to levels

ccmpatible with practical uses.

ACKN~WIZD~

This work was partially supported by an Ohio State University Small

Research Grant.

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