- Email: [email protected]
Precipitin reactions in monkeys (Macaca sinica) experimentally infected with Toxocara canis and in children with visceral larva migrans syndrome
J. COMP.
YATH.
1Y7U.
VOL.
407
NJ.
PRECIPITIN REACTIONS IN MONKEYS (MACACA EXPERIMENTALLY INFECTED WITH TOXOCARA AND IN CHILDREN WITH VISCERAL LARVA MIGRANS SYNDROME
SZNZCA) CANZS
BY
S. T. FERNANDO Department
of
Veterinary
and B. VASUDEVAN of Ceylon,
Science, University
Peradeniya,
Ceylon
and
M. H. M. HAMZA, and
I. K. T. PANDITHA-GUNAWARDENE H. T. SAMARASINGHE
Lady Ridgeway
Hospital,
Colombo, C&on
INTRODUCTION
canis is one of the most common causes of larva migrans in children which is often character&d by persistent eosinophilia, fever of varying duration, cough, pica, loss of weight and sometimes hepatomegaly (Beaver, 1956 ; Smith and Beaver, 1953). Puppies in Ceylon show a high prevalence of Toxocaru canis infection (90 per cent. or more), and counts of Toxocaru eggs in the faeces reveal 4,000 to 12,000 eggs per gramme in puppies 4 to 22 weeks old (Fernando, 196813). In consequence the soil may be contaminated by large numbers of T. canis eggs. In the last nineteen months, several cases of persistent eosinophilia (50 per cent. or over) with cough, fever of varying duration, muscle and joint pains were encountered in children. Preliminary examination of the sera by gel-diffusion revealed precipitins to Ascaris Zumbricoides or to T. canis or to both species. Differential diagnosis of T. canis infection in children by gel-diffusion is difficult because of the occurrence of marked serological cross-reactions between T. cunis and A. lumbricoides. It was decided, therefore, to study the nature of the precipitin reaction in monkeys experimentally infected with T. cunis and compare it with that of the children showing precipitins specific to Toxocuru-antigens. Toxocara
MATERIALS
AND METHODS
Antigens. Saline extracts of the embryonated eggs and adult worms of Toxocara canis from puppies and of Ascaris lumbricoides from children collected after piperazine citrate medication were used as the main antigens, and for comparison, saline extracts of Ascaridia galli and Ancylostoma caninum. The methods of culture of the worm eggs to the infective stage and the preparation of the different antigens were the same as were those described earlier (Fernando, 1968a). The nitrogen content of the antigens determined by the method of Lanni, Dillon and Beard (1950) was adjusted to 0.25 mg./ml. in 1 /lO,OOO thiomersal. The antigens from the infective eggs and
408
PRECIPITIN
REACTIONS
ASSOCIATED
WITH
~oxocara
canis
adult worms of T. canis and of A. lumbricoides are referred to in most of the text as TE, TC, AE and AL respectively and those from the adult worms of A. galli and A. canium as AG and AC. Sera. Bloods from children with larva migrans syndromes were allowed to clot overnight and the sera collected after centrifugation were stored at -2OOC. Experimental hosts. Monkeys (Macaca sinica) weighing 4 to 5 kg., of unknown age, trapped in the nearby jungles were used. They were kept in the laboratory for a minimum period of 4 weeks before they were infected. Faecal examinations did not reveal any parasitic ova. For the production of antibodies to T. canis infection, six monkeys (1 to 6) were infected orally with two dosesof 2,000 ova with an interval of one week between the dosesfollowed by a dose of 100,000 ova four weeks after the first dose. Two monkeys (7,8) were the uninfected controls. The animals were bled from the external saphenousveins weekly, commencing at least two weeks before the initial infection. The serum samplescollected from the blood were stored at -2OOC. Serology. Gel-diffusion and immuno-electrophoresis were the serological tests used. Since marked variations occur in the antibody responseof an animal in the course of an infection, serum sampleswere screened for precipitins by the agar diffusion test, and the sera which gave the maximal reaction in intensity and in the number of bands were selected for detailed study. In general the serum samples collected during the first 4 weeks after the challenge showed the maximal reaction. Gel-difusion precipitin test. The test (Ouchterlony, 1949) was carried out as described earlier (Fernando, 1968a) in 1.3 per cent. Noble agar (Difco) containing 1/lO,OOO thiomersal. Agar prepared in Tris-buffer pH 9.0 (Zweig and Whitaker, 1967) was used in preference to veronai buffer (Mayer, Osler, Bier and Heidelberger, 1946) since the former gave a clearer precipitin reaction. Immuno-electrophoresis. Immuno-electrophoresis was carried out in the EEL equipment* in Verona1acetate buffer, pH 8.6, ionic strength 0.1, and in Tris-buffer, Noble agar (1.3 per cent.) with l/10,000 thiomersal (Kabat and Mayer, 1961). The gels were poured on glassplates (17 x 5 cm.) to a thickness of 4 mm. The different antigen preparations were subjected to electrophoresis at a potential of 15 V./cm. width of the gel for 3 hours. In somecasesafter electrophoresis,blocks of agar were removed from about the levels of the different precipitin arcs. The precipitin reactions of the sera were compared with the whole antigen and with the electrophoretically separated antigen components contained in the agar blocks. In addition, when different precipitin lines appeared after immuno-electrophoresis, wells were dug a little away from and below the levels of the extremities of the precipitin lines and the corresponding whole antigens were added. By this method it was also possible to compare the precipitin reactions of the sera with the whole and electrophoretically fractioned antigens, and to ascertain the specificities of the different antigen fractions which separate out on electrophoresis. Diffusion took place at room temperatures (26Oto 28OC.) for 3 to 7 days in moist chambers. To ascertain the extent of the separation of the protein fractions in the antigens, one agar slide, after each run, was fixed in 2 per cent. acetic acid in 96 per cent. ethanol to precipitate the protein fractions. Agar gels with the immuno-precipitins or the precipitated antigen fractions were washed, mounted, dried and stained with amido black lOB, azocarmine 10B or ponseau red for proteins, and with oil red 0 for lipid components (Grabar and Burtin, 1964). Absorptive studies. In order to confirm the specificity of the precipitins, where possiblesomeserum sampleswere absorbedwith equal volumes of the corresponding antigens at room temperatures (26’ to 28OC.) for one hour, followed by about 18 hours at 0 to 4OC. before being used in the immuno-diffusion tests.The control serum sampleswere incubated with an equal volume of normal saline to allow for any possible variations in the precipitin reaction due to the dilution of the samplesby the absorbing antigen. * Evans
Electroselenium
Ltd.,
England.
S. T.
FERNANDO
et
al.
409
RESULTS
Precipitin Reaction of the Sera of Monkeys with T. canis in Diflusion Test
Infected
Pattern of the reaction. The serum samples were examined for precipitins with the saline extracts of embryonated eggs and adult ~01711s of T. canis and A. Zumbricoides (TE, TC, AE and AL) and saline extracts of adult worms of A. galli and A. caninum (AG and AC). The sera of four monkeys (1, 3, 4, 6) showed precipitins to both TE and TC, but those of two (2, 5) very strong precipitins with TE only. In general, the precipitins which appeared about one week after the larger dose of 100,000 ova lasted 7 to 9 weeks. Precipitins were not observed in the sera of the two controls (7, 8). Nature of the reaction. The sera of two monkeys (2, 5) which reacted only with TE showed a minimum of four bands virtually overlapping one another (Fig. 6). The sera which reacted with TE and TC, however, showed a minimum of 2 to 3 bands with TE and 2 bands with TC. One of the bands with TE, that closest to the serum wells, showed partial identity with the one of the two bands with TC which was situated closer to the the antigen (TC) well. A representative reaction of the sera of these four monkeys (1, 3, 4, 6) seen in Fig. 1 shows that a minimum of 5 antibodies was present in the sera. It was also apparent that TE and TC show marked qualitative or quantitative differences in their antigen components.
Fig.
1.
Gel-diffusion precipitin reaction of the serum of a monkey (3) showing precipitins with both Toxocuru adult-worm and egg antigens; peripheral wells contain the sera and the numbers denote the weeks aRer infection with the larger dose; 2,000 ova on days 0 and 7; 100,000 ova on day 28.
of A. lumbricoides, A. galli and A. caninum Antigens with the sera of Toxocara-infected monkeys. The reactions of the sera of the Toxocara-infected monkeys with the homologous antigens (TE, TC) from T. canis and with the heterologous antigens (AE, AL) from A. Zumbricoides, AG from A. galli and AC from A. caninum were compared. A representative pattern of the sera examined is shown in Fig. 2. The sera of the 4 monkeys (1, 3, 4, 6) which reacted with both TE and TC showed at least one common band with TC, AL and AG, in addition to a minimum of two bands specific to TE and one Cross-reactivity
410
PRECIPITIN
REACTIONS
ASSOCIATED
wrm
lhxmzra
canis
specific to TC indicating that at least one antibody component is ascaroid specific, in that it is specific to a common antigen component shared by T. canis, A. lumbricoides and A. galli. This group specific antigen was not present in detectable amounts in TE although it was present in TC. The sera of the monkeys (2, 5) which reacted only with TE, but not with TC, did not react with the heterologous antigens (AL, AG). Ancylostoma antigen (AC), however, did not react with the sera.
Fig.
2.
Gel-diffusion test on he sera of two monkeys (2, 6) to show serological cross-reaction among 7: canis, A. lumbrico’ ld! sand A. galli; S 32, serum of monkey 2 which reacted only with Toxocara egg antigen; S 36 serum of monkey 6 which reacted with both Toxocara egg and adult worm antigens; sera collected 2 weeks after the larger dose; an antigen component in Toxocara adult worm, antigen (TC) is shared by A. lumbricoides and A. galli adult worm extracts; 2,000 ova on days Ojand 7; 100,000 ova on day 28.
Immtlno-electrophoresis. The relative position of the precipitin arcs the sera of the monkeys (1, 3, 4, 6) with TE, TC and AL subjected to phoresis are shown in the Figs. 3 and 4. The specific antigen components only in TE in the gel-diffusion tests (Fig. 1) migrated to the distal part
I
Fig.
3.
0
TE
k -
-r
s40
TC
against electropresent of the
+ I
The immuno-electrophoretic precipitin pattern shown by a serum of a monkey (3) which reacted with both adult worm and egg antigens of T. canti with the electro heretically separated adult worm and egg antigens of 1. canis; S 40, serum of the monkey P3) collected 3 weeks after infection with the larger dose; 2,000 ova on days 0 and 7; 100,000 ova on day 28.
cathode while the ascaroid group specific antigen component shared by TC, AL and AG separated into two fractions. The position of the two precipitin arcs corresponding to these fractions of the ascaroid group specific antigen component
S. T.
FERNANDO
et
411
al.
indicated that one fraction had migrated to the proximal part of the anode while the other had shown little migration towards either pole. Since these two precipitin arcs were united or continuous at the adjoining extremities it is probable that (Grabar and they have identical specificities, but have different mobilities Burtin, 1964). Blocks of agar removed from about the levels of these two ascaroid specific precipitin arcs after an initial electrophoresis of TC, AL and AG were transplanted into agar gels and resubjected to immuno-electrophoresis.
--
L Fig.
4.
0
TE
s40
I f
The immuno-electrophoretic precipitin pattern developed with the serum of a monkey (3) which reacted with both adult worm and egg antigens of T. canis against the electrophoretically separated egg antigen of T. canis and adult-worm antigen of A. lumbricoides; S40, serum of the monkey (3) collected 3 weeks after infection with the larger dose; 2,000 ova on days 0 and 7; 100,000 ova on day 28.
The positions of the precipitin arcs showed that the two fractions had migrated to the same position as in the original electrophoresis indicating that this phenomenon is due to the difference in the mobilities of these fractions and not to the retention of a part of this antigen component by the agar. The Toxocara specific antigen component shared by TE and TC in the geldiffusion test (Fig. 1) formed an arc at the middle of the cathode area on either side of the serum reservoir. The Toxocara-specific antigen present in TE only (Fig. 1) was also associated with the antigen component which formed the arc at the middle of the anode area against TE indicating that it has a minimum of two antigen determinants. The sera of the monkeys (2, 5) which reacted only with TE (Fig. 6) showed a wide band consisting of a minimum of 3 to 4 almost overlapping lines of precipitin, about the distal part of the cathode area (Fig. 7). of the Sera of Children with Larva Migrans Syndrome Samples from fifty-three children with larva migrans syndromes were examined for precipitins against antigens prepared from adult worm and embryonated egg extracts of T. canis and A. lumbricoides: twenty-three showed precipitins to the antigens of either one or both species. The sera could be classed into four main groups (Table 1). It appears from the results that the reaction of group 1 sera is specific to A. lumbricoides antigens, whereas the group 2 sera show precipitating antibody to an antigen component common to A. lumbricoides and T. canis. The reaction of the sera of group 3, shown in Fig. 8, is mainly referable to an infection with T. canis. Although four children from this group showed a patent infection with A. lumbricoides, specific precipitins were not observed in their sera. The precipitins common Precipitin
Reactions
412
PRECIPITIN
REACTIONS
ASSOCIATED TABLE
FAECAL
Group
No. of sera
AND
SERUM
WITH
iroXOC@YZ
1
EXAMINATIONS OF CHILDREN MIGRANS SYNDROME
Preczpitin
CaniS
reaction
WITH
LARVA
Parasitological
findings
tested 1
7
2
6
3
10
4
30
A single band of precipitin only \vith AE and AL cnpAn band with TE, TC, AE Two overlapping bands with TE only and a single faint band with TE, TC, AE and AL Negative for precipitins
TE, egg extracts of T. canis. TC, adult worm AL, adult worm extracts of A. lumbricoides.
extracts
Two children showed ova in the faeces Three children showed ova in the faeces Four children showed ova in the faeces
A. lumbricoides
Eleven children showed ova in the faeces
.4. bmbricoides
of 7. canis. Al%, egg extracts
A. lumbricoides ‘4. lumbricoides
of A. lumbricoides.
to an antigen present in T. canis and A. lumbricoides were also identical with those seen in group 2. The antibody to the antigen component common to T. canis and A. lumbricoides antigens (TE, TC, AE, AL) was absorbed with TC, AE and AL, but the specific precipitins to TE were not. Absorption with TE, however, removed both the precipitins to TE and those to TC, AE and AL. Comparison
of Human and Monkey
Sera
Only six sera from children (Table 1, group 3) were available. The two TE specific precipitin bands in the sera of the children showed identity with one of the TE specific precipitin bands in the sera of the monkeys infected with T. canis (Fig. 5). In immuno-electrophoresis assays the specific precipitin arcs to TE in the children and monkey sera respectively developed at about the same levels in the distal part of the cathode area (Fig. 9).
Fig.
5.
Geldiffusion test on the serum of a child (Table I, Group 3) with specific precipitins to T. canis egg antigens showing a reaction of identity with a Toxocara- egg-antigen specific precipitin band of the serum of a monkey (3); CS 7 serum of the child; S 46, serum of the monkey collected 4 weeks after the larger dose; both sera were absorbed with A. lumbricoides adult worm, antigen. Monkey infected with 2,000 ova on days 0 and 7 and 100,000 ova on day 28.
S.
T.
FERNANDO
et
al.
413
DISCUSSION
It is evident from this study that a minimum of 5 to 7 antibody components developed in the sera of the monkeys during the peak period of antibody response. Although they all received the same doses of infective ova the nature of the precipitin reaction varied : for instance, two of the six revealed a minimum of 4 antibody components specific to 4 antigen components present in the egg extracts of T. canis. The sera of these two monkeys did not show any reaction with the A. lumbricoides and A. galli extracts indicating specificity for T. canis. The sera of four, however, showed a minimum of five antibody components, four of which seemed to be specific to T. canis and one appeared to be ascaroid specific since it was specific to an antigen component common to T. canis, A. lumbricoides and A. galli. The egg and adult worm extracts of T. canis showed a striking difference in their antigens. The egg extracts appeared to contain at least 3 antigens specific for T. canis, one of which was shared by the adult worm extract. In addition to the specific antigen common to the adult worm and egg extracts, the adult worm extract also revealed a specific component not present in the Toxocara egg extracts in detectable amounts. The ascaroid specific antigen, however, was present in the adult worm extract, but the egg extract did not possess this antigen in detectable amounts. The Toxocara-specific antigens in the embryonated egg extract showed a cathodal migration. The ascaroid specific antigens, however, separated into two fractions, one of which showed little migration towards either pole and the other migrated to the anode. The pattern of the precipitin arcs shown by the sera against these fractions suggested that they have identical specificities with different mobilities. A marked serological cross-reaction between A. lumbricoides and T. canis antigens in the indirect haemagglutination and in the in vitro circumlarval precipitation tests has been reported previously (Sadun, Norman and Allain, 1957 ; Jung and Pacheco, 1960; Olson, 1960). Studies on antigenic analysis of T. canis while confirming the serological relationship among T. canis and the closely related nematodes Ascaris suum and A. Zumbricoides have also shown the presence of two genus specific antigens in T. canis (Jeska, 1967). Jeska, however, used antibodies produced in rabbits by inoculation with the whole worm suspensions. In the present study, however, the antibodies which were stimulated in monkeys (Macaca sinica) by oral infection with T. canis ova were used to evaluate the specificity of the precipitin reaction and to ascertain the extent of serological crossreactivity among T. canis, A. lumbricoides and A. galli. The specific precipitins to Toxocara-egg antigens in the sera of ten of the fifty-three children examined suggest the possibility of occurrence of this infection in the ten with larva migrans syndrome. SUMMARY
Six monkeys (Macaca sinica) were experimentally infected with Toxocara canis ova and their humoral antibody response was studied by immuno-diffusion precipitin tests using as antigens saline extracts of embryonated eggs and adult worms of T. canis and Ascaris lumbricoides respectively. In addition, saline
414
PRECIPITIN
REACTIONS
ASSOCIATED
WITH
?-OXOCcZrcZ
Ca?Zi.Y
extracts of adult worms of Ascaridia galli and Ancylostoma caninum were used to elucidate the comparative serological cross-reactivity among T. canis, A. lumbricoides, A. galli and A. caninum. The monkey sera revealed a minimum of 4 to 5 species specific antibodies and at least one group antibody specific to a common antigen component shared by T. canis and the related ascaroids, A. lumbricoides and A. galli. The specific antigens were present mainly in Toxocara embryonated egg extracts while the ascaroid group specific component was present in the Toxocara adult worm extracts. Both types of antigens differed in their electrophoretic mobilities. Fifty-three serum samples from children with larva migrans syndromes were examined for precipitins to adult and embryonated egg extracts of Ascaris Zumbricoides and of Toxocara canis. A specific precipitin reaction consisting of a minimum of two overlapping bands against Toxocara egg antigen was observed in the sera of 10 children. These sera also showed an ascaroid group specific precipitin reaction consisting of a minimum of one band with the antigens of Toxocara and Ascaris. The sera of seven children showed a reaction consisting of at least one with the A. lumbricoides antigens only. Six sera showed an ascamid group specific reaction consisting of one common band with the adult worm and egg extracts of A. lumbricoides and T. canis antigens. ACKNOWLEDGMENTS
The writers thank Professor P. Seneviratna for the facilities provided and for his constructive criticism of the manuscript. The technical assistance of Mr. W. G. Senaratne and Mr. S. G. Wijeratnebanda are gratefully acknowledged. This investigation was supported by a grant from the University of Ceylon, Peradeniya. REFERENCES
Beaver, P. C. (1956). Expt. Parasit., 5, 587. Fernando, S. T. (1968a). Parasitology, 58, 91; (1968b). Ibid., 523. Analysis, pp. 34. Elsevier Grabar, P., and Burtin, P. (1964). Zmmuno-electrophoretic Publishing Company; Amsterdam, London, New York. Jeska, E. (1967). J. Immunol.,
98, 1290.
Jung, R. E., and Pacheco, G. (1960). Amer. 1. trap. Med. Hyg., 6, 185. pp. 638. Kabat, E. A., and Mayer, M. M. (1961). E x p erimental Immunochemistry, Charles C. Thomas; Springfield, Illinois, U.S.A. Lanni, F., Dillon, M. L., and Beard, J. W, (1950). Proc. Sot. exp. Biol. Med., 74, 4. Maya h5L5M., Osler, A. G., Bier, 0. G., and Heidelberger, M. (1946). J. exp. Med., Olson, ‘L. J.1 Richards, B., and Ewert, A. (1960). Tex. Rep. Biol. Med., 18, 254. Olson, L. J. (1960). Ibzd., 473. Ouchterlony, 0. (1949). Acta path. microbial. Stand., 26, 507. Sadun, E. H., Norman, L., and Allain (1957). Amer. J. trop. Med., 9, 562. Smith, M. H. D., and Beaver, P. C. (1953). Paediutrics, 12, 491. Zweig, G., and Whitaker, J. R. (1967). Paper Ch romatography and Electrophoresis in Stabili&zg Media, I, pp. 148. Academic Press; New York and London. [Receizled for publication,
October 27th, 19691
Fig. 6.
Gel-diffusion test on two sera of the monkeys (2. .5) showing precipitins only with ~oxocara egg antigens; S 32 and S 35, the sera of monkeys 2 and 5 respectively, collected two weeks after infection with the larger dose; 2,000 ova on days 0 and 7; 100,000 ova on day 28. Fig. 7. The immuno-electroohoretic orecinitin uattern develooedI with the serum of a monkey (5) which reacted only with the egg antigen of 7. canis in the gel-diffusion test, against the electrophoretically separated 7. canic rgq and adult worm antigens; S 41, serum of the mo&ey collected 3 weei; after the larger dose; 2,000 ova on days 0 and 7; 100,000 ova on day 28. Fig. 8. Gel-diffusion test on the serum (CSI) of a child (Table I, Group 3) showing specific precipitins with Toxocara egg antigen; the serum also sho\vs a precipitin band to an antigen component common to r. cani, and .l. I
I
I
lumhricoide~.
Fig.
9. The immuno-electrophoretic precipitin pattern developed \vith the serum of a child (Table I, Group 3) and of a monkey i.5) with the electrophoretirally separated Toxocara egg antigen; CS 7, serum of a child showing specific precipitins to Tooxocarc2 egg antigen ; S 41 serum of the monkey (5 3 weeks after the larger dose; both sera were absorbed with .A. hhricoide\ adult worm antigen. Monkey infected with 2,000 ova on days 0 and 7 and 100,000 ova on day 28.
S.
T.
FERNANDO
et
a/.
YATH.
1Y7U.
VOL.
407
NJ.
PRECIPITIN REACTIONS IN MONKEYS (MACACA EXPERIMENTALLY INFECTED WITH TOXOCARA AND IN CHILDREN WITH VISCERAL LARVA MIGRANS SYNDROME
SZNZCA) CANZS
BY
S. T. FERNANDO Department
of
Veterinary
and B. VASUDEVAN of Ceylon,
Science, University
Peradeniya,
Ceylon
and
M. H. M. HAMZA, and
I. K. T. PANDITHA-GUNAWARDENE H. T. SAMARASINGHE
Lady Ridgeway
Hospital,
Colombo, C&on
INTRODUCTION
canis is one of the most common causes of larva migrans in children which is often character&d by persistent eosinophilia, fever of varying duration, cough, pica, loss of weight and sometimes hepatomegaly (Beaver, 1956 ; Smith and Beaver, 1953). Puppies in Ceylon show a high prevalence of Toxocaru canis infection (90 per cent. or more), and counts of Toxocaru eggs in the faeces reveal 4,000 to 12,000 eggs per gramme in puppies 4 to 22 weeks old (Fernando, 196813). In consequence the soil may be contaminated by large numbers of T. canis eggs. In the last nineteen months, several cases of persistent eosinophilia (50 per cent. or over) with cough, fever of varying duration, muscle and joint pains were encountered in children. Preliminary examination of the sera by gel-diffusion revealed precipitins to Ascaris Zumbricoides or to T. canis or to both species. Differential diagnosis of T. canis infection in children by gel-diffusion is difficult because of the occurrence of marked serological cross-reactions between T. cunis and A. lumbricoides. It was decided, therefore, to study the nature of the precipitin reaction in monkeys experimentally infected with T. cunis and compare it with that of the children showing precipitins specific to Toxocuru-antigens. Toxocara
MATERIALS
AND METHODS
Antigens. Saline extracts of the embryonated eggs and adult worms of Toxocara canis from puppies and of Ascaris lumbricoides from children collected after piperazine citrate medication were used as the main antigens, and for comparison, saline extracts of Ascaridia galli and Ancylostoma caninum. The methods of culture of the worm eggs to the infective stage and the preparation of the different antigens were the same as were those described earlier (Fernando, 1968a). The nitrogen content of the antigens determined by the method of Lanni, Dillon and Beard (1950) was adjusted to 0.25 mg./ml. in 1 /lO,OOO thiomersal. The antigens from the infective eggs and
408
PRECIPITIN
REACTIONS
ASSOCIATED
WITH
~oxocara
canis
adult worms of T. canis and of A. lumbricoides are referred to in most of the text as TE, TC, AE and AL respectively and those from the adult worms of A. galli and A. canium as AG and AC. Sera. Bloods from children with larva migrans syndromes were allowed to clot overnight and the sera collected after centrifugation were stored at -2OOC. Experimental hosts. Monkeys (Macaca sinica) weighing 4 to 5 kg., of unknown age, trapped in the nearby jungles were used. They were kept in the laboratory for a minimum period of 4 weeks before they were infected. Faecal examinations did not reveal any parasitic ova. For the production of antibodies to T. canis infection, six monkeys (1 to 6) were infected orally with two dosesof 2,000 ova with an interval of one week between the dosesfollowed by a dose of 100,000 ova four weeks after the first dose. Two monkeys (7,8) were the uninfected controls. The animals were bled from the external saphenousveins weekly, commencing at least two weeks before the initial infection. The serum samplescollected from the blood were stored at -2OOC. Serology. Gel-diffusion and immuno-electrophoresis were the serological tests used. Since marked variations occur in the antibody responseof an animal in the course of an infection, serum sampleswere screened for precipitins by the agar diffusion test, and the sera which gave the maximal reaction in intensity and in the number of bands were selected for detailed study. In general the serum samples collected during the first 4 weeks after the challenge showed the maximal reaction. Gel-difusion precipitin test. The test (Ouchterlony, 1949) was carried out as described earlier (Fernando, 1968a) in 1.3 per cent. Noble agar (Difco) containing 1/lO,OOO thiomersal. Agar prepared in Tris-buffer pH 9.0 (Zweig and Whitaker, 1967) was used in preference to veronai buffer (Mayer, Osler, Bier and Heidelberger, 1946) since the former gave a clearer precipitin reaction. Immuno-electrophoresis. Immuno-electrophoresis was carried out in the EEL equipment* in Verona1acetate buffer, pH 8.6, ionic strength 0.1, and in Tris-buffer, Noble agar (1.3 per cent.) with l/10,000 thiomersal (Kabat and Mayer, 1961). The gels were poured on glassplates (17 x 5 cm.) to a thickness of 4 mm. The different antigen preparations were subjected to electrophoresis at a potential of 15 V./cm. width of the gel for 3 hours. In somecasesafter electrophoresis,blocks of agar were removed from about the levels of the different precipitin arcs. The precipitin reactions of the sera were compared with the whole antigen and with the electrophoretically separated antigen components contained in the agar blocks. In addition, when different precipitin lines appeared after immuno-electrophoresis, wells were dug a little away from and below the levels of the extremities of the precipitin lines and the corresponding whole antigens were added. By this method it was also possible to compare the precipitin reactions of the sera with the whole and electrophoretically fractioned antigens, and to ascertain the specificities of the different antigen fractions which separate out on electrophoresis. Diffusion took place at room temperatures (26Oto 28OC.) for 3 to 7 days in moist chambers. To ascertain the extent of the separation of the protein fractions in the antigens, one agar slide, after each run, was fixed in 2 per cent. acetic acid in 96 per cent. ethanol to precipitate the protein fractions. Agar gels with the immuno-precipitins or the precipitated antigen fractions were washed, mounted, dried and stained with amido black lOB, azocarmine 10B or ponseau red for proteins, and with oil red 0 for lipid components (Grabar and Burtin, 1964). Absorptive studies. In order to confirm the specificity of the precipitins, where possiblesomeserum sampleswere absorbedwith equal volumes of the corresponding antigens at room temperatures (26’ to 28OC.) for one hour, followed by about 18 hours at 0 to 4OC. before being used in the immuno-diffusion tests.The control serum sampleswere incubated with an equal volume of normal saline to allow for any possible variations in the precipitin reaction due to the dilution of the samplesby the absorbing antigen. * Evans
Electroselenium
Ltd.,
England.
S. T.
FERNANDO
et
al.
409
RESULTS
Precipitin Reaction of the Sera of Monkeys with T. canis in Diflusion Test
Infected
Pattern of the reaction. The serum samples were examined for precipitins with the saline extracts of embryonated eggs and adult ~01711s of T. canis and A. Zumbricoides (TE, TC, AE and AL) and saline extracts of adult worms of A. galli and A. caninum (AG and AC). The sera of four monkeys (1, 3, 4, 6) showed precipitins to both TE and TC, but those of two (2, 5) very strong precipitins with TE only. In general, the precipitins which appeared about one week after the larger dose of 100,000 ova lasted 7 to 9 weeks. Precipitins were not observed in the sera of the two controls (7, 8). Nature of the reaction. The sera of two monkeys (2, 5) which reacted only with TE showed a minimum of four bands virtually overlapping one another (Fig. 6). The sera which reacted with TE and TC, however, showed a minimum of 2 to 3 bands with TE and 2 bands with TC. One of the bands with TE, that closest to the serum wells, showed partial identity with the one of the two bands with TC which was situated closer to the the antigen (TC) well. A representative reaction of the sera of these four monkeys (1, 3, 4, 6) seen in Fig. 1 shows that a minimum of 5 antibodies was present in the sera. It was also apparent that TE and TC show marked qualitative or quantitative differences in their antigen components.
Fig.
1.
Gel-diffusion precipitin reaction of the serum of a monkey (3) showing precipitins with both Toxocuru adult-worm and egg antigens; peripheral wells contain the sera and the numbers denote the weeks aRer infection with the larger dose; 2,000 ova on days 0 and 7; 100,000 ova on day 28.
of A. lumbricoides, A. galli and A. caninum Antigens with the sera of Toxocara-infected monkeys. The reactions of the sera of the Toxocara-infected monkeys with the homologous antigens (TE, TC) from T. canis and with the heterologous antigens (AE, AL) from A. Zumbricoides, AG from A. galli and AC from A. caninum were compared. A representative pattern of the sera examined is shown in Fig. 2. The sera of the 4 monkeys (1, 3, 4, 6) which reacted with both TE and TC showed at least one common band with TC, AL and AG, in addition to a minimum of two bands specific to TE and one Cross-reactivity
410
PRECIPITIN
REACTIONS
ASSOCIATED
wrm
lhxmzra
canis
specific to TC indicating that at least one antibody component is ascaroid specific, in that it is specific to a common antigen component shared by T. canis, A. lumbricoides and A. galli. This group specific antigen was not present in detectable amounts in TE although it was present in TC. The sera of the monkeys (2, 5) which reacted only with TE, but not with TC, did not react with the heterologous antigens (AL, AG). Ancylostoma antigen (AC), however, did not react with the sera.
Fig.
2.
Gel-diffusion test on he sera of two monkeys (2, 6) to show serological cross-reaction among 7: canis, A. lumbrico’ ld! sand A. galli; S 32, serum of monkey 2 which reacted only with Toxocara egg antigen; S 36 serum of monkey 6 which reacted with both Toxocara egg and adult worm antigens; sera collected 2 weeks after the larger dose; an antigen component in Toxocara adult worm, antigen (TC) is shared by A. lumbricoides and A. galli adult worm extracts; 2,000 ova on days Ojand 7; 100,000 ova on day 28.
Immtlno-electrophoresis. The relative position of the precipitin arcs the sera of the monkeys (1, 3, 4, 6) with TE, TC and AL subjected to phoresis are shown in the Figs. 3 and 4. The specific antigen components only in TE in the gel-diffusion tests (Fig. 1) migrated to the distal part
I
Fig.
3.
0
TE
k -
-r
s40
TC
against electropresent of the
+ I
The immuno-electrophoretic precipitin pattern shown by a serum of a monkey (3) which reacted with both adult worm and egg antigens of T. canti with the electro heretically separated adult worm and egg antigens of 1. canis; S 40, serum of the monkey P3) collected 3 weeks after infection with the larger dose; 2,000 ova on days 0 and 7; 100,000 ova on day 28.
cathode while the ascaroid group specific antigen component shared by TC, AL and AG separated into two fractions. The position of the two precipitin arcs corresponding to these fractions of the ascaroid group specific antigen component
S. T.
FERNANDO
et
411
al.
indicated that one fraction had migrated to the proximal part of the anode while the other had shown little migration towards either pole. Since these two precipitin arcs were united or continuous at the adjoining extremities it is probable that (Grabar and they have identical specificities, but have different mobilities Burtin, 1964). Blocks of agar removed from about the levels of these two ascaroid specific precipitin arcs after an initial electrophoresis of TC, AL and AG were transplanted into agar gels and resubjected to immuno-electrophoresis.
--
L Fig.
4.
0
TE
s40
I f
The immuno-electrophoretic precipitin pattern developed with the serum of a monkey (3) which reacted with both adult worm and egg antigens of T. canis against the electrophoretically separated egg antigen of T. canis and adult-worm antigen of A. lumbricoides; S40, serum of the monkey (3) collected 3 weeks after infection with the larger dose; 2,000 ova on days 0 and 7; 100,000 ova on day 28.
The positions of the precipitin arcs showed that the two fractions had migrated to the same position as in the original electrophoresis indicating that this phenomenon is due to the difference in the mobilities of these fractions and not to the retention of a part of this antigen component by the agar. The Toxocara specific antigen component shared by TE and TC in the geldiffusion test (Fig. 1) formed an arc at the middle of the cathode area on either side of the serum reservoir. The Toxocara-specific antigen present in TE only (Fig. 1) was also associated with the antigen component which formed the arc at the middle of the anode area against TE indicating that it has a minimum of two antigen determinants. The sera of the monkeys (2, 5) which reacted only with TE (Fig. 6) showed a wide band consisting of a minimum of 3 to 4 almost overlapping lines of precipitin, about the distal part of the cathode area (Fig. 7). of the Sera of Children with Larva Migrans Syndrome Samples from fifty-three children with larva migrans syndromes were examined for precipitins against antigens prepared from adult worm and embryonated egg extracts of T. canis and A. lumbricoides: twenty-three showed precipitins to the antigens of either one or both species. The sera could be classed into four main groups (Table 1). It appears from the results that the reaction of group 1 sera is specific to A. lumbricoides antigens, whereas the group 2 sera show precipitating antibody to an antigen component common to A. lumbricoides and T. canis. The reaction of the sera of group 3, shown in Fig. 8, is mainly referable to an infection with T. canis. Although four children from this group showed a patent infection with A. lumbricoides, specific precipitins were not observed in their sera. The precipitins common Precipitin
Reactions
412
PRECIPITIN
REACTIONS
ASSOCIATED TABLE
FAECAL
Group
No. of sera
AND
SERUM
WITH
iroXOC@YZ
1
EXAMINATIONS OF CHILDREN MIGRANS SYNDROME
Preczpitin
CaniS
reaction
WITH
LARVA
Parasitological
findings
tested 1
7
2
6
3
10
4
30
A single band of precipitin only \vith AE and AL cnpAn band with TE, TC, AE Two overlapping bands with TE only and a single faint band with TE, TC, AE and AL Negative for precipitins
TE, egg extracts of T. canis. TC, adult worm AL, adult worm extracts of A. lumbricoides.
extracts
Two children showed ova in the faeces Three children showed ova in the faeces Four children showed ova in the faeces
A. lumbricoides
Eleven children showed ova in the faeces
.4. bmbricoides
of 7. canis. Al%, egg extracts
A. lumbricoides ‘4. lumbricoides
of A. lumbricoides.
to an antigen present in T. canis and A. lumbricoides were also identical with those seen in group 2. The antibody to the antigen component common to T. canis and A. lumbricoides antigens (TE, TC, AE, AL) was absorbed with TC, AE and AL, but the specific precipitins to TE were not. Absorption with TE, however, removed both the precipitins to TE and those to TC, AE and AL. Comparison
of Human and Monkey
Sera
Only six sera from children (Table 1, group 3) were available. The two TE specific precipitin bands in the sera of the children showed identity with one of the TE specific precipitin bands in the sera of the monkeys infected with T. canis (Fig. 5). In immuno-electrophoresis assays the specific precipitin arcs to TE in the children and monkey sera respectively developed at about the same levels in the distal part of the cathode area (Fig. 9).
Fig.
5.
Geldiffusion test on the serum of a child (Table I, Group 3) with specific precipitins to T. canis egg antigens showing a reaction of identity with a Toxocara- egg-antigen specific precipitin band of the serum of a monkey (3); CS 7 serum of the child; S 46, serum of the monkey collected 4 weeks after the larger dose; both sera were absorbed with A. lumbricoides adult worm, antigen. Monkey infected with 2,000 ova on days 0 and 7 and 100,000 ova on day 28.
S.
T.
FERNANDO
et
al.
413
DISCUSSION
It is evident from this study that a minimum of 5 to 7 antibody components developed in the sera of the monkeys during the peak period of antibody response. Although they all received the same doses of infective ova the nature of the precipitin reaction varied : for instance, two of the six revealed a minimum of 4 antibody components specific to 4 antigen components present in the egg extracts of T. canis. The sera of these two monkeys did not show any reaction with the A. lumbricoides and A. galli extracts indicating specificity for T. canis. The sera of four, however, showed a minimum of five antibody components, four of which seemed to be specific to T. canis and one appeared to be ascaroid specific since it was specific to an antigen component common to T. canis, A. lumbricoides and A. galli. The egg and adult worm extracts of T. canis showed a striking difference in their antigens. The egg extracts appeared to contain at least 3 antigens specific for T. canis, one of which was shared by the adult worm extract. In addition to the specific antigen common to the adult worm and egg extracts, the adult worm extract also revealed a specific component not present in the Toxocara egg extracts in detectable amounts. The ascaroid specific antigen, however, was present in the adult worm extract, but the egg extract did not possess this antigen in detectable amounts. The Toxocara-specific antigens in the embryonated egg extract showed a cathodal migration. The ascaroid specific antigens, however, separated into two fractions, one of which showed little migration towards either pole and the other migrated to the anode. The pattern of the precipitin arcs shown by the sera against these fractions suggested that they have identical specificities with different mobilities. A marked serological cross-reaction between A. lumbricoides and T. canis antigens in the indirect haemagglutination and in the in vitro circumlarval precipitation tests has been reported previously (Sadun, Norman and Allain, 1957 ; Jung and Pacheco, 1960; Olson, 1960). Studies on antigenic analysis of T. canis while confirming the serological relationship among T. canis and the closely related nematodes Ascaris suum and A. Zumbricoides have also shown the presence of two genus specific antigens in T. canis (Jeska, 1967). Jeska, however, used antibodies produced in rabbits by inoculation with the whole worm suspensions. In the present study, however, the antibodies which were stimulated in monkeys (Macaca sinica) by oral infection with T. canis ova were used to evaluate the specificity of the precipitin reaction and to ascertain the extent of serological crossreactivity among T. canis, A. lumbricoides and A. galli. The specific precipitins to Toxocara-egg antigens in the sera of ten of the fifty-three children examined suggest the possibility of occurrence of this infection in the ten with larva migrans syndrome. SUMMARY
Six monkeys (Macaca sinica) were experimentally infected with Toxocara canis ova and their humoral antibody response was studied by immuno-diffusion precipitin tests using as antigens saline extracts of embryonated eggs and adult worms of T. canis and Ascaris lumbricoides respectively. In addition, saline
414
PRECIPITIN
REACTIONS
ASSOCIATED
WITH
?-OXOCcZrcZ
Ca?Zi.Y
extracts of adult worms of Ascaridia galli and Ancylostoma caninum were used to elucidate the comparative serological cross-reactivity among T. canis, A. lumbricoides, A. galli and A. caninum. The monkey sera revealed a minimum of 4 to 5 species specific antibodies and at least one group antibody specific to a common antigen component shared by T. canis and the related ascaroids, A. lumbricoides and A. galli. The specific antigens were present mainly in Toxocara embryonated egg extracts while the ascaroid group specific component was present in the Toxocara adult worm extracts. Both types of antigens differed in their electrophoretic mobilities. Fifty-three serum samples from children with larva migrans syndromes were examined for precipitins to adult and embryonated egg extracts of Ascaris Zumbricoides and of Toxocara canis. A specific precipitin reaction consisting of a minimum of two overlapping bands against Toxocara egg antigen was observed in the sera of 10 children. These sera also showed an ascaroid group specific precipitin reaction consisting of a minimum of one band with the antigens of Toxocara and Ascaris. The sera of seven children showed a reaction consisting of at least one with the A. lumbricoides antigens only. Six sera showed an ascamid group specific reaction consisting of one common band with the adult worm and egg extracts of A. lumbricoides and T. canis antigens. ACKNOWLEDGMENTS
The writers thank Professor P. Seneviratna for the facilities provided and for his constructive criticism of the manuscript. The technical assistance of Mr. W. G. Senaratne and Mr. S. G. Wijeratnebanda are gratefully acknowledged. This investigation was supported by a grant from the University of Ceylon, Peradeniya. REFERENCES
Beaver, P. C. (1956). Expt. Parasit., 5, 587. Fernando, S. T. (1968a). Parasitology, 58, 91; (1968b). Ibid., 523. Analysis, pp. 34. Elsevier Grabar, P., and Burtin, P. (1964). Zmmuno-electrophoretic Publishing Company; Amsterdam, London, New York. Jeska, E. (1967). J. Immunol.,
98, 1290.
Jung, R. E., and Pacheco, G. (1960). Amer. 1. trap. Med. Hyg., 6, 185. pp. 638. Kabat, E. A., and Mayer, M. M. (1961). E x p erimental Immunochemistry, Charles C. Thomas; Springfield, Illinois, U.S.A. Lanni, F., Dillon, M. L., and Beard, J. W, (1950). Proc. Sot. exp. Biol. Med., 74, 4. Maya h5L5M., Osler, A. G., Bier, 0. G., and Heidelberger, M. (1946). J. exp. Med., Olson, ‘L. J.1 Richards, B., and Ewert, A. (1960). Tex. Rep. Biol. Med., 18, 254. Olson, L. J. (1960). Ibzd., 473. Ouchterlony, 0. (1949). Acta path. microbial. Stand., 26, 507. Sadun, E. H., Norman, L., and Allain (1957). Amer. J. trop. Med., 9, 562. Smith, M. H. D., and Beaver, P. C. (1953). Paediutrics, 12, 491. Zweig, G., and Whitaker, J. R. (1967). Paper Ch romatography and Electrophoresis in Stabili&zg Media, I, pp. 148. Academic Press; New York and London. [Receizled for publication,
October 27th, 19691
Fig. 6.
Gel-diffusion test on two sera of the monkeys (2. .5) showing precipitins only with ~oxocara egg antigens; S 32 and S 35, the sera of monkeys 2 and 5 respectively, collected two weeks after infection with the larger dose; 2,000 ova on days 0 and 7; 100,000 ova on day 28. Fig. 7. The immuno-electroohoretic orecinitin uattern develooedI with the serum of a monkey (5) which reacted only with the egg antigen of 7. canis in the gel-diffusion test, against the electrophoretically separated 7. canic rgq and adult worm antigens; S 41, serum of the mo&ey collected 3 weei; after the larger dose; 2,000 ova on days 0 and 7; 100,000 ova on day 28. Fig. 8. Gel-diffusion test on the serum (CSI) of a child (Table I, Group 3) showing specific precipitins with Toxocara egg antigen; the serum also sho\vs a precipitin band to an antigen component common to r. cani, and .l. I
I
I
lumhricoide~.
Fig.
9. The immuno-electrophoretic precipitin pattern developed \vith the serum of a child (Table I, Group 3) and of a monkey i.5) with the electrophoretirally separated Toxocara egg antigen; CS 7, serum of a child showing specific precipitins to Tooxocarc2 egg antigen ; S 41 serum of the monkey (5 3 weeks after the larger dose; both sera were absorbed with .A. hhricoide\ adult worm antigen. Monkey infected with 2,000 ova on days 0 and 7 and 100,000 ova on day 28.
S.
T.
FERNANDO
et
a/.