THE JOURNAL OF INFECTIOUS DISEASES. VOL. 135, xo.

4 • APRIL 1977
© 1977 by the University of Chicago. All rights reserved.

Larva-Specific Antibodies in Patients with

Visceral Larva Migrans

Raymond H. Cypess, Meryl H. Karol, John L. Zidian,
Lawrence T. Glickman, and David Gitlin
From the Department of Pediatrics, University of
Pittsburgh School of Medicine, and the Departments of
Epidemiology and Microbiology, Graduate School of
Public Health, University of Pittsburgh,
Pittsburgh, Pennsylvania

Seven of 10 patients with visceral larva migrans (VLM) had serum precipitating
antibodies specific for larval antigens of Toxocara canis as determined by double

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diffusion in agar. Such antibodies were absent in 114 normal adults and 25 patients
with collagen disease. Precipitation of ascarid components by C-reactive protein
resulted in false-positive reactions, but this precipitation was readily prevented by
appropriate absorption of sera before testing. A more sensitive assay, the enzyme-
linked immunosorbent assay, revealed high titers of antibodies to larvae of Toxocara
in all patients with VLM; the log;! titer ranged from 9 to 14. Five of eight household
relatives of these patients and four children who had had VLM two to four years
before testing had titers of 6-12. Of the 114 normal adults, 105 had titers of 0-2;
nine had titers of 3-8. Of the 25 patients with collagen disease, 23 had titers of 0, and
two had titers of 4 and 6, respectively. Additional studies of those patients indicated
that infection with Toxocara can be distinguished serologically from ascariasis,
filariasis, and trichinellosis.

Larva migrans comprises a heterogeneous group filtration, hepatomegaly, persistent eosinophilia,

of clinical disorders caused by the aberrant mi-
gration of nematode larvae through the tissues.
In visceral larva migrans (VLM), the liver and
lungs are the organs most often involved, and the
classical syndrome includes fever, pulmonary in-
Received for publication July 30, 1976, and in revised
form October 4, 1976.
This study was supported by grants no. HD 01031 and
no. AI 10490 from the National Institutes of Health, and
grant no. HSRF R63 from the Health Service Research
Foundation, Pittsburgh, Pennsylvania. Dr. Cypess is the re-
cipient of career development award no. AI 00056 from the
National Institutes of Health.
We are indebted to Drs. Eric A. Otteson and Peter
Weller of the National Institute of Allergy and Infectious
Diseases (NIAID), National Institutes of Health, for sera
from their patients with filariasis and to Dr. Franklin Neva
of NIAID for serum from his patient infected with Gnatho-
stoma. \Ve also thank Drs. Ronald Altman of the New Jer-
sey State Department of Health for sera from patients with
trichinellosis, Joseph A. Burke of the University of Kentucky
for serum from his patient with ascarid pulmonary ab-
scess, and Robert M. Weetman of Indiana University Hos-
pitals for sera from two of his patients with visceral larva
migrans.
Please address requests for reprints to Dr. Raymond H.
Cypess, Department of Epidemiology, Graduate School of
Public Health, University of Pittsburgh, Pittsburgh, Penn-
sylvania 15213.
increased serum a- or ,B-isohemagglutinins, and
hyperglobulinemia [1-3]. The patient with VLM
is usually a child aged one to five years with a
history of pica and contact with pets.
The parasites most often implicated as the
cause of VLM are dog and cat ascarids of the ge-
nus Toxocara. Dogs and cats are infected by at
least three ascarids, Toxocara canis, Toxocara
cati, and Toxascaris leonina. However, T. canis
is undoubtedly the most important of the three
in the causation of VLM because of differences in
the larval migratory patterns of the parasites,
differences in their ability to survive in aberrant
hosts, and differences between the defecation
habits of dogs and cats. At least 20% of the 30
million dogs [4, 5J in both rural and urban areas
of the United States and 6e;to-70% of the five
million dogs in the United Kingdom [6-9] are
hosts to Toxocara.
A diagnosis of VLM at present is based primar-
ily upon nonspecific criteria. None of the im-
munologic techniques available, including im-
munofluorescence [10J, indirect HA [11J, and der-
mal sensitivity to ascarid antigens (12J, distin-
guishes satisfactorily between infection with Tox-
ocara and infection with the other helminthes
that cause VLM, most notably Ascaris. Biopsies

633
634
of involved tissues in VLM frequently reveal
eosinophilic grahulomas, but larvae are uncom-
monly found, and when found are difficult to
identify. The purpose of the present study was
to determine whether patients with toxocarid
VLM produce antibodies to the parasite that can
be used as an aid in diagnosis of the disorder.
Materials and Methods
Preparation of antigens and antisera. Adult
T. canis were obtained from puppies at a local
pound, and adult Ascaris suum were obtained
from swine at a local abattoir. The live worms
were washed repeatedly with 0.15 M N aCl, and
their gravid uteri were then removed by dissec-
tion. Eggs were stripped from the uteri and
placed in I % formalin at room temperature
(about 24 C) for 21 days to induce embryonation
[13]. Free second-stage larvae were hatched from
aliquots of the embryonated eggs by the method
of Fairbairn [14]. Thus four stages of T. canis
and A. suum were obtained: unembryonated eggs
(DE), embryonated eggs containing second-
stage larvae (EE), hatched or free second-stage
larvae (HL), and adult worms (A). Each stage
was homogenized separately in 0.05 M borate buf-
fer (pH 8.6) with use of glass Ten-Broeck hom-
ogenizers and was centrifuged at 2,000 g for 30
min; the supernatant fractions were used as anti-
gens. Aliquots of each of the antigens were emul-
sified in complete Freund's adjuvant and inject-
ed into rabbits for the preparation of antisera
according to a schedule previously described
[15].
Analysis and assay of antigens. Immunochem-
ical analysis of the antigens was carried out in
1.5% agar in borate buffer (pH 8.6) by the dou-
ble diffusion method of Ouchterlony [16] and im-
munoelectrophoresis [17]. In several instances, a
precipitate formed when human sera and extracts
of Toxocara were mixed in saline; these precipi-
tates were washed three times with 0.15 M NaCl,
mixed with complete Freund's adjuvant, and in-
jected into normal rabbits to produce antisera
for the identification of the antigenic components
in these precipitates.
The enzyme-linked immunosorbent assay
(ELISA) used in this study for the detection of
antibodies in patients' sera was a modification
Cypess et al.
[18, 19] of the method of Engvall and Perlmann
[20]. The assay was performed in polystyrene
microtiter plates (Linbro Scientific, Hamden,
Conn.). The wells were coated with the appro-
priate antigen by addition of 50 pJ of 0.1 M
N aHC0 3 buffer (pH 9.6) containing 2.5 I1-g of
the antigen proteiri/ml of buffer. The plates
were incubated at 37 C for 3 hr and then rinsed
three times with 0.15 M NaCl containing 0.05%
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Tween 20. Sera to be assayed were diluted serially
with saline-Tween solution containing 0.02 M
phosphate buffer (STP), pH 7.0, and 50 11-1 of
each of the dilutions was added to the wells. The
plates were incubated at room temperature for
16 hr and then rinsed in STP.
Goat antiserum prepared against human IgG
and conjugated to horseradish peroxidase was
obtained from Miles Laboratories (Kankakee,
Ill.): the antiserum was found to be specific for
human IgG on analysis by immunoelectrophore-
sis. Antiserum (50 11-1) in dilutions of I :2,000 or
I :2,500 was added to each well. After 2 hr at
room temperature, the plates were again rinsed
with STP, and to each well was added 50 11-1 of
0.1 M phosphate buffer (pH 7.0) followed by 50
11-1 of O-dianisidine (1 mg/ml) in 0.03% H 20 2 •
The plates were read by inspection after 5 min
at room temperature. The end point was taken to
be the last dilution of serum that gave a distinct
color reaction. All samples were processed in du-
plicate. For reasons explained in Results, sera to
be assayed for specific antibodies to the larval
antigens of T. canis were absorbed with extracts
of embryonated eggs of A. suum before assay,
with use of 20 11-1 of extract/50 pJ of patient's
serum; the protein concentration of the extract
used for absorption was 4 mg/rnl. To assess non-
specific absorption of serum to the plate, we add-
ed the sera to plates containing bicarbonate buffer
instead of antigen. These titers were usually 0 and
never >1.
Sources ot serum samples. Serum was ob-
tained from the following individuals: nine chil-
dren and one adult who appeared to have VLM
(table I) and eight household relatives of these
patients; four children who had had symptoms
of VLM two to four years before serum for the
present study was collected (table I); 10 adults
with symptoms of trichinellosis who demonstrat-
ed serum titers of antibody to Trichinella spi-
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Table l. Clinical and laboratory findings in patients with suspected visceral larva migrans (VLM). c
~

Specific antibodies 5'

to embryonated eggs ;;.
of Toxocara ~
ELiSAt ~
Elevated Ova and Agar (10g2
Pets in Hepato- Leukocytes Eosino- Blood isohernag- Elevated C-reactive parasites diffusion reciprocal
Subject (sex, age) household Pica megaly (no./min 3 ) phils (%) type glutinins IgG protein in stools" plate" dilution)

Group 1
D.C. (F, 25) Yes No Yes 37,000 70 A Yes Yes No - + 13
R.C. (M, 2) Yes NK No 16,500 37 0 Yes Yes No Ascaris + 13
M.C. (F, 2) Yes Yes Yes 22,000 50 0 Yes Yes No - + 14
C.B. (M, 1Y2) Yes Yes Yes 25,500 71 A Yes Yes Yes - + 14
K.C. (M, 2) Yes Yes Not 12,800 40 0 Yes No Yes - 9
B.Z. (M, 2Y2) Yes Yes Yes 12,320 21 NK Yes Yes No Ascaris + 12
L.G. (F, 7) Yes Yes No§ 13,200 60 NK Yes Yes No + 12
A.H(M,2) Yes Yes Yes 16,000 30 0 Yes Yes No Giardia + 12
G.D (M, 4Y2) Yes NK Yes 13,800 15 0 Yes Yes No - - 9
L.L. (F, 6%) Yes Yes No§ 12,100 23 0 Yes Yes Yes - - 9
Group 2
M.B. (F, 3Y2) Yes Yes Yes 18,000 30 A Yes Yes No - + 12
M.C. (F, 1Y2) Yes Yes No 24,900 41 A Yes Yes No + 11
E.R.(M,2) No Yes Yes 49,000 69 NK NK Yes No - - 7
D.K. (F, 14) Yes No No§ 10,500 27 0 Yes NK No - 6
NOTE. Group 1 included subjects with suspected VLM at the time of study. Group 2 included subjects with suspected VLM two to four years earlier; data are from the
time of their suspected VLM. NK = not known.
*(+) = positive; (-) = negative.
tELISA = enzyme-linked immunosorbent assay.
tAsthma-like attack and pneumonia prompted investigation for VLM.
§Mentally retarded.

01
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636
ralis of > 1:5 by the bentonite flocculation test;
two children and five adults from the Cook Is-
lands with Bancroft's filariasis, at least three of
whom were also host to A scaris lumbricoides; a
child with heavy enteric infection with A . lum-
bricoides, who developed a pulmonary abscess
found to contain an adult Ascaris on biopsy; an
adult returning to the Vnited States from Thai-
land who had developed multiple recurrent eosi-
nophilic abscesses and had a positive skin test for
Gnathostoma; 25 patients with rheumatoid ar-
thritis or systemic lupus erythematosus; and 104
adult blood donors and 10 apparently normal
adults submitting blood for premarital syphilis
serology.
Results
Antigenic analysis of the four stages of T. canis
Figure 1. Double diffusion in agar: A, larva-specific
antigens in Toxocara canis embryonated eggs (TEE)
and hatched larvae (THL) revealed by rabbit anti-
serum to TEE (a-TEE) in center well; B, adult-specific
toxocara antigens (TA) revealed by rabbit antiserum
to TA (a-TA); C, TEE antigens that show some
similarity to Ascaris suum larval antigens (AEE) as
indicated by fusion of precipitin lines between TEE
and AEE; and D , antigens in Toxocara that are similar
to those in Ascaris , revealed as fused bands circling
center well. TUE = toxocara unembryonated eggs;
AUE = ascaris unembryonated eggs ; AA = adult
Ascaris; and S = normal human serum.
Cypess et al.
with the rabbit antisera prepared against these
stages revealed several antigens (figure lA) that
were present both in eggs containing larvae
(TEE) and in hatched larva (THL), but that
were not found either in the adult worm (TA) or
in unembryonated eggs (TVE) . When the rab-
bit antisera to either TEE or THL were absorbed
with extracts of T A, precipitins for antigens in TA
were removed, but antibodies to the stage-specific
Downloaded from http://jid.oxfordjournals.org/ at The University of British Colombia Library on July 30, 2015
antigens in TEE and THL remained. Similarly,
T A extracts contained antigens not detectable in
any of the other stages (figure lB). When the
rabbit antisera to T A were first absorbed with
extracts of either TEE or THL, precipitins for
these stages were removed, but antibodies to two
or more antigens in T A remained. Antigenic anal-
ysis of A. suum with the homologous antisera
yielded similar data; embryonated eggs of Ascaris
(AEE) contained antigens not present either in
ascaris adults (AA) or in ascaris unembryonat-
ed eggs (AVE), and AA extracts contained anti-
gens not found in the other stages . Each of the
rabbit antisera prepared to Toxocara con-
tained precipitins for antigens present in extracts
of A scm-is. Conversely, each of the rabbit anti-
sera to Ascaris contained precipitins against an-
tigens in Toxocara . However, the cross-reacting
antigens found in Toxocara and Ascaris were not
identical (figures IC and ID), and the antisera
could be rendered specific for the homologous
species by absorption with extracts of the other
species. Thus, by absorption of antisera to TEE
with both AEE and TA, the antisera could be
made specific for the larval stages of Toxocara:
analysis of TEE extracts with such absorbed anti-
sera revealed at least two major antigenic com -
ponents, which were termed TEE antigen A and
TEE antigen B, respectively (figure 2A).
When sera from blood donors were tested
against antigens of Toxocara by double diffusion
in agar, no visible reaction was observed. How-
ever, many of the sera from patients with either
rheumatoid arthritis or di sseminated lupus
formed precipitation lines with extracts of Toxo-
cam in the double diffusion plates (figures 2B
and 2C). Since none of these patients had a his-
tory of VLM, it was suspected that this precipi-
tation re action might be due to an interaction
between C-reactive protein in their sera and an
unknown substance present in the extracts. There-
Larva-Specific Antibodies in VI-M
Figure 2. A: Absorption of rabbit antiserum to
T oxocara canis embryonated eggs (anti-TEE) with
Ascaris suu m larval antigen s (AEE ) left antibodies for
two antigens in TEE (an t igen A and antigen B at
arrows). Band C: Precipitation lines were formed by
rheumatoid arthritis sera (RA) because of reaction of
C-reactive protein with TEE extract in center well ;
lines do not fuse (arrows) with line formed by serum
from p atient (V) with visceral larva migrans (VLM)
an d TEE extract. D: Ser a from patients with VLM
(V 1 and V 2 ) formed precip it at es with TEE that fus ed
with the antigen A b and formed between rabbit anti-
TEE and TEE (arr o ws indicate an tigen A and antigen
B). E : Reaction in B is shown af ter dissolution of
C-reactive protein precipitates in 5% trisodium citrate.
AUE = ascaris unembry on ated eggs ; AA = adult
A scaris ; and S = normal human serum.
fore, precipitat es wer e pr epa re d by addition of
aliquots of T A extract di re ctly to the sera; the
precipitates were th en wash ed and injected into
rabbits. The result ing a nt isera contained an ti bod-
ies that re acted strong ly in double diffusion plates
and on immunoelectro ph ore sis with human C·
reactive protein as well as with toxocar a anti-
637
gens. 'When C-reactive protein was removed from
the sera of the se pati ents by a bsor ptio n with ra b-
bit an tiser um specific for human C-reactive pro-
tein, the cap acity of the patients' sera to precipi-
tate ext ract s of To xocara was abolished.
Sera from seven of th e 10 patients with VLM
(table 1) formed one or more precipitin lines
with TEE extracts in do uble diffusion pla tes
and on immunoelectrophoresis: a TEE compo-
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nent precipitated by each o f the seven sera was
TEE antigen A (figure 2D) . Three of the seven
VLM sera that precipitated TEE antigen A also
precipitated components in AEE extracts. Ab-
sorption of the VLM sera with AEE extract elim-
inated the latter reaction but did not prevent the
sera fr om precipitating TEE an tigen A. Absorp-
tion with TEE extract, ho wever , eliminated pre-
cip ita tio n of both TEE and AEE, a fact indicating
tha t the pre cipitins pr esent were antibodies to
TEE that cross-reacted with AE E. One of the
sera contained sufficient C-reactive protein to pre-
cipitate components in the tox ocara extracts
other th an TEE antigen A, but the reaction with
C-reactive protein was rea dily di sti nguished from
precipitation of an tigen A in three ways. First,
addition of AEE extracts or rabbit antiserum
to human C-reactive p rotein to serum contain-
in g C-reactive protein preven ted the reaction
with C-re active protein but did not prevent pre -
cipitation of antigen A. Second. the precipitin
line formed with TEE an tigen A in double dif-
fusion plates crossed the precipitation line
formed by C-reactive protein (figure 2B). Third,
the precipitation line formed between C-reactive
protein and TEE extrac t co uld be dissolved from
the agar by soaking the pl ate s in 5% trisodium
citrate [21], wh ereas pr eci pitation of antigen A
was unaffected by this tre atment (figure 2E).
To identify th e prot eins in VLM sera capable
of precipitating antigen A, we performed elec-
trophoresis of VLM serum in agar. An extract of
TEE was then allowed to di ffuse into the agar
fro m a trough placed para llel to the elec tropho-
retic axis, and rabbit ant iseru m to human IgG
was permitted to diffuse into th e agar from a sec-
ond parallel trough. Antigen A precipitated a se-
rum protein that had the elec trophoretic mobil-
ity of IgG, and the precipitation line thus formed
fused with th e pre cipit ati on lin e for med between
the serum an d rabbit antiserum to human IgG.
638 Cypess et al.

Thus, the protein in VLM serum that precipi-
tated antigen A was IgG.
Although only seven of the 10 patients with
VLM gave positive serum reactions with TEE ex-
tracts when agar diffusion methods were used,
all 10 patients had titers of antibody to TEE of
~9 as determined by the ELISA method (table 1).
Replicate samples never differed by more than
one dilution. Serum from only two of eight house-
TEE extracts removed all antibodies to both AEE
and TEE. Thus, these sera did not contain anti-
bodies specific for AEE but did contain anti-
bodies to TEE that cross-reacted with AEE. To
avoid such cross-reactions and thus make the
ELISA method specific for TEE, we first absorbed
all sera to be assayed for antibodies to TEE with
AEE extracts.
Of the 104 sera from normal blood donors,

hold relatives of the VLM patients gave positive
precipitin reactions with TEE extracts by the
agar diffusion method, but with the ELISA meth-
od five of the eight subjects were found to have
titers of antibody to TEE of 6-9. It was clear
that ELISA was more sensitive for the detection
of antibodies to TEE than the agar diffusion
methods.
Serum from four of the VLM patients also
had titers of antibody to AEE of 4-10 as deter-
mined by the ELISA method. These antibodies
were removed completely by absorption with
AEE extract, the absorption having no signifi-
cant effect on the titers of antibody to TEE. On
the other hand, absorption of these sera with
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85 had titers of antibody to TEE of 0 with the
ELISA method; eight had a titer of 1, and 11 had
titers of 2-8. Of the 10 premarital sera studied,
nine had titers of 0, and one had a titer of 5. The
average (±SD) serum titer of antibody to TEE
in the 10 VLM patients was 11.7 ± 2.0 by ELISA
(figure 3). The four children who had had VLM
two to four years earlier had serum ti tel'S of anti-
body to TEE ranging from 6 to 12; the mean
titer was 8.5 ± 3.0. The patient with Gnathosto-
ma had no detectable serum antibodies to TEE.
Titers of antibody to TEE were 0 in all 10 patients
with trichinellosis, and only one of the seven pa-
tients with filariasis had demonstrable antibodies
to TEE; this patient, a child, had a serum titer

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(104) (10 ) (7) (10) (25) (10) (4 )

Figure 3. Mean titers (± SD) of specific antibodies to Toxocara canis embryonated eggs with use of the en-
zyrne-linked immunosorbent assay in norm al adults (blood donors and VDRL group) and in patients with filaria-
sis, trichinellosis, collagen disease (rheumatoid arthritis [RhA] and systemic lupus erythematosus [SLE] ), and
visceral larva migrans (VLM). Numbers in parentheses indicate numbers of individuals studied. The titers are
expressed as the log , of the reciprocal of the last serum dilution that gave a distinct color reaction.
Laroa-Specijic Antibodies in V LlH
of 6. Of the 25 patients with collagen disease, 23
had no detectable antibodies to TEE; two pa-
tients had titers of 4 and 6, respectively. Patients
with active or previous VLM had titers signifi-
cantly higher than those of controls or patients
with trichinellosis or filariasis (P < 0.001).
Serum from the child who had a pulmonary
abscess due to an adult Ascaris precipitated ex-
tracts of all stages of both Ascaris and Toxo-
cara in agar diffusion plates. After absorption
with rabbit antiserum to C-reactive protein, the
serum reacted with AA but not with AEE, TA,
or TEE, a result indicating the presence of anti-
bodies specific for AA and the absence of any
detectable antibodies specific for either Toxocara
or AEE.
Discussion
The prevalence of toxocarid infection in hu-
mans and the spectrum of pathology attributable
to such infection are unknown. It is obvious that
VLM represents only the most clinically overt
instances of toxocariasis, since the larvae are
known to invade critical organs, such as the eyes,
without producing symptoms of VLM [22]. Defi-
nitive diagnosis of human toxocariasis rests on
the positive identification of the larvae in the tis-
sues. In most patients with VLM, larvae are rare-
ly observed in biopsied tissue, and, when a larva
is found, it is difficult to determine its genus.
Therefore, the diagnosis of toxocariasis remains
largely presumptive, with evidence being provid-
ed by a number of serologic tests that have been
developed for the detection of antibodies to Tox-
ocara. Unfortunately, all of the tests currently in
use lack specificity [12, 23]; they fail to distin-
guish toxocariasis from ascariasis, filariasis, or
even trichinellosis. In addition, false-positive re-
actions are not uncommon because of cross-reac-
tions between the antigens used and the hetero-
phile antibodies or isohemagglutinins [2]. Toxo-
cara antigens have been used in skin testing for
toxocariasis [24], but the antigens were obtained
from adult worms, a stage not found in humans,
and the specificity of the method is uncertain
[12].
In the present study, it was observed that T.
canis contained antigens that were readily dis-
tinguishable from those of Ascaris. In addition,
639
toxocara antigens were found that were relatively
stage-specific; that is, antigens were present in
the larvae that were not present in the adult,
and vice versa. These results confirmed previous
observations [25]. Seven of the 10 patients who
were thought to have VLM on the basis of symp-
toms, clinical findings, eosinophilia, hyperglobu-
linemia, and elevated isohemagglutinins had se-
rum antibodies capable of precipitating at least
Downloaded from http://jid.oxfordjournals.org/ at The University of British Colombia Library on July 30, 2015
one of the two major antigens specific for the
larval stages of T. canis. When tested against
TEE antigen by the ELISA method, all I 0 pa~
tients with VLM had high titers of antibody in
serum.
The ELISA method proved to be more senstive
than double diffusion in agar for the detection
of serum antibodies to TEE. In both methods,
specificity for TEE antigen was attained by addi-
tion of AEE extract to the serum to be exam-
ined; this addition inhibited possible cross-reac-
tions between any antibodies to AEE that might
have been present and the TEE antigen in the
test. Absorption of the patient's serum with AEE
before testing also eliminated false-positive re-
sults in agar diffusion plates that were due to the
interaction of C-reactive protein with TEE ex-
tracts. In the absence of AEE absorption, how-
ever, confusion engendered by interaction of C-
reactive protein and TEE extracts can be avoided
by absorption of the patient's serum with anti-
serum to human C-reactive protein before test-
ing or by soaking of the developed agar plates
in 5% trisodium citrate to dissolve any C-reactive
protein-TEE precipitate [21]. For rapidity and
ease, agar diffusion can be used as a specific test
for toxocariasis, but ELISA appears to be more
sensitive. In both tests, the specific antibodies to
TEE detected were IgG proteins.
Th~ mean ELISA titer for the 104 blood do-
nors was 0.4 ± 1.26, and only eight (7.7%) of
these persons had titers ~2 SD above the mean
(>3.0). In contrast, all 14 patients with clinical
VLM had significantly elevated titers of antibody
to TEE. It was possible that the eight blood do-
nors with elevated titers had had subclinical
VLM, but unfortunately we lacked further infor-
mation on these persons. The level of antibody
in patients with trichinellosis or filariasis did not
differ significantly from that in the blood donors.
These data suggest that ELISA with TEE anti-
640 Cypess et al.

gens can serologically distinguish patients with
toxocariasis from patients infected with other
12.
parasites that cross-react with Toxocara.
The ELISA method with TEE extract as an
antigen appears to be useful in the diagnosis of
VLM due to Toxocara. The assay may also pro-
vide a means for determining the prevalence of
13.
toxocariasis in the general public and the range
of pathology that may be associated with this
minthic diseases. R. E. Krieger, New York, 1975, p.
SOS-842.
Collins, R. F., Ivey, M. H. Specificity and sensitivity of
skin test reactions to extracts of Toxocara canis and
Ascaris suum. II. Homologous 4S-hour passive cu-
taneous anaphylaxis tests with sera from infected
guinea pigs. Am. J. Trop. Med. Hyg. 24:460-464,
1970.
Tiner, J. D. The migration, distribution in the brain
and growth of Ascarid larvae in rodents. J. Infect.

particular zoonosis.
References
I. Beaver, P. C., Snyder, C. H., Carrera, G. M., Dent, J. H.,
Lafferty, J. W. Chronic eosinophilia due to visceral
larva migrans. Pediatrics 9:7-19,1952.
2. Huntley, C. C., Costas, M. C., Lyerly, A. Visceral larva
migrans syndrome: clinical characteristics and im-
munological studies in 51 patients. Pediatrics 36:
523-536, 1965.
3. Mok, C. H. Visceral larva migrans-a discussion based
on a review of the literature. Clin. Pediatr. 7:565-
573, 1968.
4. Heiner, D. C., Kevy, S. V. Visceral larva migrans. Re-
port of the syndrome in three siblings. N. Engl. J.
Med.254:629-636,1956.
5. Faulkner, L. C. Dimensions of the pet population prob-
lem. J. Am. Vet. Med. Assoc. 166:477---478, 1974.
6. Oldham, J. N. Observations on the incidence of Tox-
ocara and Toxacaris in dogs and cats from the Lon-
don area. J. Helminthol. 39:251-256, 1965.
7. Brain, L., Allan, B. Encephalitis due to infection with
Toxocara canis. Lancet 1:1355-1356, 1964.
8. Woodruff, A. W., Thacker, C. K., Shah, A. I. Infection
with animal helminths. Br. Med. J. 1:1001-1005,
1964.
9. Carding, A. H. The significance and dynamics of stray
dog populations with specific reference to United
Kingdom and Japan. J. Small Anim. Pract. 10:419-
446,1969.
10. Hogarth-Scott, R. S. Visceral larva migrans-an immu-
nofluorescent examination of rabbit and human sera
for antibodies to the ES antigens of the second
stage larvae of Toxocara canis, Toxocara cati and
Toxascaris leonina (Nematoda). Immunology 10:
217-223, 1966.
II. Arean, V. M., Crandall, C. A. Toxocariasis. In R. A.
Marcial-Rojas [ed.]. Pathology of protozoal and hel-
Downloaded from http://jid.oxfordjournals.org/ at The University of British Colombia Library on July 30, 2015
Dis. 92:105-113, 1952.
14. Fairbairn, D. The in vitro hatching of Ascaris lumbri-
coides eggs. Can. J. Zoo!. 39:153-162,1961.
15. Gitlin, D., Perricelli, A., Gitlin, J. D. The presence of
serum a-fetoprotein in sharks and its synthesis by
fetal gastrointestinal tract and liver. Compo Biochem.
Physiol. 46B:207-215, 1973.
16. Ouchterlony, o. Antigen antibody reactions in gels. IV.
Types of reactions in coordinated systems of diffu-
sion. Acta Path. Microbiol. Scand. 32:231-240, 1953.
17. Scheidegger, J. J. Vne micro-methode de l'immuno'-
electrophorese. Int. Arch. Allergy Appl. Immunol.
7:103-110,1955.
18. Voller, A., Draper, C. C., Bidwell, D., Bartlett, A. Mi-
croplate enzyme-linked immunosorbent assay for
Chagas' Disease. Lancet 1:426-428, 1975.
19. Ruitenberg, E. J., Ljungstrom, I., Steerenberg, P. A.,
Buys, J. Application of immunofluorescence and im-
munoenzyme methods in the serodiagnosis of Tri-
chinella spiralis infection. Ann. N.Y. Acad. Sci. 254:
296-303, 1975.
20. Engvall, E., Perlmann, P. Enzyme-linked immunosor-
bent assay, Elisa. III. Quantitation of specific anti-
bodies by enzyme-labeled anti-immunoglobulin in
antigen coated tubes. J. Immunol. 109:129-135, 1972.
21. Biguet, J., Tran Van Ky, P., Audrieu, S., Degaey, R.
Etude immunoelectrophoretique de la nature et de
I'ordre d'apparition des anticorps precipitants du
serum de lapins en fonction de leur mode d'immu-
nisation contre Candida albicans. Sabouraudia 4:
148-157, 1965.
22. Wilder, H. C. Nematode endophthalmitis. Trans. Am.
Acad. Ophthalmol. Otolaryngol. 55:99-109, 1950.
23. Krupp, I. M. Hemagglutination test for the detection
of antibodies specific for Ascaris and Toxocara anti-
gens in patients with suspected visceral larva mi-
grans. Am. J. Trop. Med. Hyg. 23:378-384,1974.
24. Woodruff, A. W., Bisseru, B., Bowe, J. C. Infection
with animal helminths as a factor in causing polio-
myelitis and epilepsy. Br. Med. J. 1:1576-1579, 1966.
25. Jeska, E. L. Purification and immunochemical analysis
of genus-specific cuticular antigen of Toxocara can-
is. J. Parasitol. 55:465-471, 1969.