Internntiond
Journal for Parasitology. 1976. Vol. 6. pp. 247-25
I. Pergomon
Press. Printed in Great Britain.
OCULAR TOXOCARIASIS IN MICE : DISTRIBUTION OF LARVAE AND LESIONS L. J.
OLSON
The University of Texas Medical Branch, Galveston, TX 77.550, U.S.A. (Received 4 August 1975)
of larvae and lesions. InferThe distribution of TOXOCQWcanis larvae in the eye was determined for mice given a single challenge dose (C-mice) as compared to mice similarily challenged after 2 or 3 previous infections (SC-mice). Controls were mice given only the 2 or 3 previous infections and uninfected mice. Eyes were observed in si& during a 34 day post-challenge period to compare inflammatory responses in the anterior eye; histologic examination of serial sections of the eyes of these mice was done at the end of this period. Zn sim observations showed that lesions in the anterior eye converted from hemorrhagic to white cell more rapidly in the SC-mice; white cell lesions were predominant in SC-mice as early as day 5, whereas similar predominance in C-mice was not noted until days 16-21. Histology revealed that approximately 90% of these eyes were infected. Worm burdens per eye correlated more closely with total dose per mouse than with the effects of immunization. Histologic study showed that 90% of larvae observed were in the retina, but that most lesions were in the uveal tissues which harbored only 0.8 % of the total number of larvae.
Ahstract--Or_soN L. 3. 1976. Ocular Toxocariasis in mice: distribution national Journal for Parasitchgy
INDEX KEY WORDS:
6: 247-251.
Toxocar~ canis; ocular toxocariasis;
IT IS now well documented that Toxocava cunis larvae can invade the eye of man and cause serious lesions (Ferguson & Olson, 1967). This documentation began when Wilder, in 1950, reported on 46 human cases which had been diagnosed in most instances as retinoblastoma prior to enucleation of the eye. Examination of serial sections of these 46 eyes revealed the presence of nematode larvae or ‘their residual hyaline capsule’ in 24 eyes. The characteristics of the lesions in the remaining 22 eyes led Wilder to a tenative diagnosis of nematode endopht~almitis for these eyes. In 1956, Nichols identified several of the larvae in Wilder’s slides as T. canis.
murine toxocariasis.
days as either a diffuse infiltrate in the anterior chamber or as a discrete lesion on the iris. Some of these lesions were examined and shown to be primarily polymorphonuclear leukocytes, which in a few instances surrounded a larva. Based on these preliminary studies, experiments were designed to determine the distribution of T. canis larvae within the various tissues of the eye and to note any effect of previous infection on both numbers and distribution of these larvae in the eye. These experiments also permitted observations on the onset and development of hemorrhagic and white cell lesions in the anterior eye following challenge of immunized and control mice. MATERIALS
Little is known about the pathogenesis of ocular toxocariasis. Experimental work in mice has shown that these larvae, following infection via stomach tube, are capable of reaching the eye within 3 days. Viable larvae can be recovered from the eye for at least 4 months (Olson, Izzat, Petteway & Reinhart, 1970). IIence, invasion of the eye is an early event following infection of the mouse.
AND METHODS
T. cunis eggs were obtained from naturally infected dogs and given to mice by stomach intubation as previously described (Olson et al., 1970). Male mice (Swiss Yale) were purchased at IS g body weight from Texas Inbred Mice Co., Houston.
In preliminary studies of the mouse infected with a single dose of T. canis eggs, it was noted that these larvae invade the retina of this host, and also that following oral infection the first lesion observed was hemorrhage in the anterior chamber and iris. A second type of lesion developed during the next few 247
Eyes were examined in situ (without anesthetics) under a dissecting microscope (25 x) for lesions in the anterior eye; i.e. diffuse and localized hemorrhage and white cells in the anterior chamber and iris. Lesions were plotted on an eye map at each observation; hence, new and old lesions could be identified at a subsequent observation. Eyes were dissected by making an incision through the cornea and, w&h a smail spatula, the fluid, lens, and soft inner tissues were scooped out onto a glass slide and
24x -i-ABLE
1.. .!. I---/‘EK(‘ENTAGES
OF EYES
St4OWING
LESIONS
fkSON
DUKING
THE
I.J.P.
POSTf’HALLFhCE
PtKIOLf.
(NC>.
bYtS:
SC,
VOL.
6.
1076
206; c‘, 1%:
s, 92; u, 48) $ with
Day postchallenge
Hemorrhage SC
0
3
lesion White
C
Cells
SC
C
‘% showing
dominance
Hemorrhage
Iv’hite
C
SC
of Cells C
SC
0
0
0
3
0
0
0
0
6
3
2
0
6
3
0
0
12
3
0
0
a
3
0
0
35
36
31
3
12
25
16
II
37
53
45
18
4
36
31
2
9
28
26
37
13
2
26
26
6
11
30
41
37
21
5
38
24
4
13
19
19
26
13
6
11
20
6
14
17
39
31
32
6
32
25
11
16
22
22
28
21
5
11
21
9
3
20
10
22
9
5
26
11
2
24
10
8
28
13
3
0
19
IO
34
4
2
7
6
4
n
7
0
examined (25 x ) for larvae. Eyes were serially sectioned (10 pm) and stained with Harris’ hematoxylin and eosin, after fixation in buffered formalin and embedding in paraffin (Paraplast, Sherwood Medical Industries, St. Louis); infiltration of paraffin was done in a vacuum infiltrator (Lipshaw, Detroit). In counting larvae in serial sections, sections of larvae less than 500 pm apart were counted as a single larva. Three similarly designed experiments were completed in which mice were divided into four groups. Large doses of eggs were used to obtain a high percentage of infected eyes. SC-mice were given two (experiment 1) or three (experiments 2 and 3) prechallenge doses (50 eggs/g body weight) at two-three week intervals. Four weeks after the last dose, the mice were challenged (100/g). S-mice received only the prechallengc doses. C-mice received only the challenge. U-mice were uninfected controls. Eyes were examined prior to and at intervals after challenge as shown on Table 1. At the end of thz postchallenge period, mice were killed and both eyes (experi-
Analysis of these data showed that a high percentage of these eyes at the end of the post~hallenge period harbored one or more larva as determined by dissection (67-85 %) and serial sections (86-93 %) at the end of the postchallenge period (Tables 2 & 5). The number of larvae per eye ranged from 0 to 9. Observations of the eye in situ during the postchallenge period showed hemorrhage in the anterior chamber and iris as early as day two in the SC and C mice (Table 1). Discrete hemorrhage on the iris, ‘pinpoint’ to approximately 1 mm in diameter, was more common that diffuse hemorrhage in the
TABLE2--NUMRER
IN ONE OR BOTH
AND
(PERCENTAGE)
HARBORING
A LARVAE
OF MICE SHOWING IN ONE OR BOTH
Both
One
Neg.
RESULTS
LESIONS
EYES
AT THE END
Pfrcent
Lesions
Group
ments 2 and 3) of each mouse were either dissected for larvae or fixed in formalin. The results of these experiments were similar and hence have been combined.
with
THE POSTCHALLENGE
AS DETERMINED
L?li-“d?
eyes lesions
EYES DURING
OF THIS PERIOD
ma1
O”fZ
BY DISSECTION
I’frccn: with
Neg.
eyes larvae
ilRe,?n/eVe) . SC
0
s C
43 (42)
43142)
37 (54)
2 (4) 25 (36)
17(16) 44 (96) 700)
12 171)
5 (29)
I)
3(33)
6(67)
72
14(74)
63
3 (16)
0
85c3.6)
0
67c1.4)
2 (10) ~____
82 (3.0)
PERIOD
1.J.P. VOL.
Ocular toxocariasis in mice
6. 1976
internal structure and staining characteristics, these larvae, as well as those in other sites, appeared to have been viable at the time of fixation. In this connection, practially all larvae recovered by dissection, when dissection was done promptly after enucleation, were viable, i.e. motile. The distribution of larvae in the eye was similar for all three groups of mice (Table 3). Examination of serial-sections at the end of the postchallenge period showed that distribution of lesions did not parallel that for larvae (Table 4). Lesions of the retina (excluding areas of disorganization and other structural changes in which no in~ammatory cells were present) accounted for only 19.7 % of the total number of lesions; several of these lesions extended from the vitreous through the retina to the choroid. Uveitis was the dominant change with approximately one-half of all inflammatory cell reactions being located in the ciliary body, choroid and iris; choroidal inflammation frequently was located near a lesion involving the retina. The ratio of the mean number of lesions per eye (Table 4) to the mean number of larvae per eye (Table 3) was similar for all groups: SC, 0.64; S, 0.81; C, 0.68. The percentage of eyes with larvae in the absence of lesions was considerably less in the SC-mice (13%) as compared to that for S-mice (38 %) and C-mice (36%) (Table 5). Discrete lesions with a larva completely or partially in the lesion, or immediateIy adjacent to the lesion were seen infrequently; i.e. 12 lesions, or 7”/, of the total recorded (7 in vitreous-retina and retina alone; I choroid; 1 iris; and 3 anterior chamber). Discrete lesions with or without a larva were not more frequent in the SC-mice as compared to C-mice. Lesions seen on serial sections varied in cell types, possibly depending on the age of the lesion. In some lesions, polymorphonuclear leukocytes were predominant; in other lesions a granulomatous response
anterior chamber of the SC-mice in contrast to Cmice, which more frequently showed diffuse hemorrhage. The center of a discrete hemorrhage could usually be seen to lie over one of the blood vessels of the iris. Diffuse (anterior chamber) and discrete (iris) white cell lesions were first noted on day 2 in SC-mice and day 5 in C-mice. These lesions were more frequent in SC-mice, particularly during the first 2 weeks. A frequently observed pattern in the development of a given lesion on the iris was the conversion from a discrete hemorrhage to a discrete white cell lesion, which in turn cleared. It should be noted that many pinpoint hemorrhages cleared without this conversion being apparent. A similar pattern was noted for diffuse lesions in the anterior chamber. Discrete lesions tended to be more common in the periphery of the iris; i.e. outer third. Diffuse lesions sometimes involved the entire anterior chamber. No lesions were seen in U-mice during this period. S-mice were also negative during this period with the exception of one eye in each of two mice on day 34; each eye showed one small discrete white lesion on the iris. Lesions in the anterior eye were graded in terms of which type was predominant for a given eye, i.e. more frequent and or severe. Severity was based on the surface area of the lesion. Lesions, which were converting from hemorrhage to white cells, were graded on the relative surface areas of the lesion that each type occupied. In many instances no determination of predominance could be made. These data showed quite clearly that as early as day five the predominant type of lesion in the SC-mice was a white cell reaction; white cell lesions became predominant in C-mice between days 16 and 22(Table 1). Examination of serial-sections of eyes obtained at the end of the postchallenge period showed that approximately 90% of larvae were located partially or completely in the retina (Table 3). Based on
TABLE 3-PERcENT O~STRIB~I~N
OF LARVAE IN SERIAL SECTIOKSOF
(No. Group
Ant.
Post.
Cil-
c:13r-
Ch.
Ch.
iary
oid
SC
0.7
s
5.5
C
4.2
% dist.
0.4
0.4
EYES AT
THE END OF THE
POSTCHALLENGE
38; s, 16; c, 28)
SC,
Retina*
Vitr-
Optic
ems
A
B
C
D
E
2.9
4.3
56.5
32.6
0.7
0.7
h'erve
Tot31
(mean/eye)
1.4
138c3.6)
47.2
33.3
5.5
36c2.2)
5.6
2.8
52.8
30.6
2.8
72(2.5)
0.4
4.1
3.3
54.1
32.1
5.5
0.4
0.4
2.4
246
90.3
(all mice)
;: A:
partially
to choroid,
in vitreous, D:
larvae
1.4
2.8
2.0
EYES:
249
partially
B:
completely
in optic
nerve,
in retina, E:
C:
partially
in retina in ciliary
adjacent body.
PERIOD.
250
I..I.P. VOL..6. 1976
L. J. OLWY
TABLE ‘&--PERCENT DlSTRlRUTioN OF LESIONS IN EYESSERIALLY SECTIONED AT THF END OF THE POSTCIIALLENGE PERIOD.
38;S,
(No.EYEs:SC,
croup
Ant.
Post.
Cil-
Ch.
Ch.
iary
Iris
16;CJS)
Vitr-
Retinn+
oid
ecu?
A
l3
(menn/eyc)
15.9
3.4
21.6
12.5
11.4
22.7
2.2
10.2
88c2.3)
s
13.8
0
24.1
10.3
17.2
6.9
6.9
20.7
29U.8)
c
16.7
4.2
18.7
10.4
18.7
lb.7
12.5
2.1
48U.7)
X dist. 15.8
3.0
21.2
11.5
14.5
18.2
10.0
9.7
47.2
dominated; increased numbers of eosinophils were present in about one-third of these lesions. Hemorrhage was sometimes seen in the aqueous and vitreous together with white cells. A more detailed analysis of the h~stopathology of eye lesions caused by T. canis will be presented elsewhere, based on experiments in which serial-sections were obtained at intervals during the postchallenge period, rather than at the end of that period as in the current study. DISCUSSION These data indicate that 90% of those larvae which invade the eye can be found in the retina. The majority of these larvae, however, were not intimately associated with any inflammatory cells; only 20% of all lesions involved the retina. There is, of course, no way to determine from these data how long a given larva had occupied the site in which it was observed in a section. The presence of vitreal and retinal lesions in the absence of an associated larva or a residuum of a dead larva suggests that larvae, after inducing a lesion, migrate out of it. In this connection, when larvae were seen within a lesion the impression was that these larvae were viable at fixation as judged by their morphology and staining. The life span of a T. canis larva in the mouse eye is not known. Olson et al. (1970) reported recoveries from the eyes of mice infected for 4 months, which were approximately equal to that WJMBER
(PERCENTACE)OF
Group
Larvae +
EYES SHOWING LARVAE ASDETERMINEDFROM Larvae na
lesions
lesions
165
19.7
;?A: lesion involves retina and vitreous, B:
5-
Totals
SC
(all mice)
TARLE
-
Chor-
retina only
during the second week of infection; hence, a life expectancy of at least several months in this eye is probable. There was no indication that these prechallenge infections were immunogenic to the extent of preventing eye invasion following the challenge dose since the mean worm burden of SC-eyes exceeded that of S-eyes. There was a suggestion that the degree of eye invasion following challenge was reduced in the SC-mice (Tables 3 & 4). However, if one examines each of these experiments, and those in other studies in this laboratory, there is no consistent pattern of protection against eye invasion in SCmice; the differences between the SC mean and the sum of S and C means are consistently small and vary from experiment to experiment in terms of protection. Worm burdens of these eyes appear to be primarily additive, i.e. to increase with the total infective dose given the mouse. These data show that hemorrhage occurred as early as day 2, which confirms earlier findings that invasion of the eye is an early event following oral infection (Olson et al., 1970). New hemorrhagic lesions increased in frequency during the first 7 days, which is the period of increasing worm burden per eye (Olson er al., 1970). However, it was noted that new hemorrhagic lesions also developed after this period in areas of the iris, which previously were mapped as free of lesions, and in the anterior chamber. A frequent observation was to note a AND LESIONS AT THE END OF THE POSTCHALLENGE SERIALSECTIONS
Lesions only
x0 L2rvne or lesions
Percent with: Larvae
Lesions
29(X)
5(13)
l(3)
89
79
s
9061
6(3S)
1-W)
0
93
62
c
14(50)
lO(36f
3111)
l(4)
86
61
SC
3@)
PERIOD
I.J.P.voL.~.1976
Ocular toxocariasis in mice
pinpoint hemorrhage on the iris, which would be followed by one or more adjacent pinpoint hemorrhages during the time when the first lesion was enlarging and converting to a white cell lesion. These ‘satellite’ lesions suggest that a single larvae was migrating in that area of the iris causing multiple leTions. These in vivo observations of the anterior eye indicate that the eyes of mice with a history of previous infection are able to develop an accelerated inflammatory response to challenge larvae as compared to that for previously unexposed mice. By the end of the postchallenge period differences in the types and frequencies of these lesions had largely disappeared. The sharp decline in the frequency of anterior eye lesions in SC and C groups at the end of this period may reflect the migration of larvae out of the anterior eye, or the death of these larvae. In this connection, the histologic data at the close of this period showed very few larvae in those portions of the eye that were observed in vivo; i.e. anterior chamber and iris. Only 2.4% or 6 of the 246 larvae seen in sections were in the anterior-posterior chambers and iris, although 63 and 72% of SC and C eyes, respectively, showed lesions in the anterior eye during the postchallenge period. It seems reasonable to assume that a similar inflammatory response was occurring during this period in the posterior eye of these mice, although histologic data obtained at the end of this period did not show any striking differences in number, location and type of lesions among these groups of eyes. This may reflect
251
the time when these eyes were examined; i.e. at the end of the postchallenge period, at which time in vivo observations revealed no differences in type and frequency of lesions in the anterior eye of SC and C mice. In this connection, studies are underway with T. cunis in mice in which serial sections of eyes will be obtained at intervals during the postchallenge period of previously-immunized and control mice to determine if immunization results in an accelerated inflammatory response-including granuloma formation-in the retina and other posterior areas of the eye. Acknowledgemerzts-The
support of Research Grant EYO0919 from The National Institutes of Health, Bethesda, MD, U.S.A. is acknowledged. REFERENCES FERGUSONE. C. & OLSON L. J. 1967. Toxocara ocular nematodiasis. International Ophlhalmology Clinics 7: 583-603. NICHOLS R. L. 1956. The etiology of visceral larva migrans. 1. Diagnos!ic morphology of infective secondstage Toxocnra larvae. Journal of Parasitology 42: 349-362. OLSON L. J., IZZAT N. N., PETTEWAY M. B. & REINHART J. A. 1970. Ocular toxocariasis in mice. American Journal of Tropicul Medicine and Hygiene 9: 238-243.
endophthalmitis. Transactions of the American .4cademy of Ophthalmology and Otolaryngology 55: 99-109.
WILDERH. C. 1950. Nematode
Journal for Parasitology. 1976. Vol. 6. pp. 247-25
I. Pergomon
Press. Printed in Great Britain.
OCULAR TOXOCARIASIS IN MICE : DISTRIBUTION OF LARVAE AND LESIONS L. J.
OLSON
The University of Texas Medical Branch, Galveston, TX 77.550, U.S.A. (Received 4 August 1975)
of larvae and lesions. InferThe distribution of TOXOCQWcanis larvae in the eye was determined for mice given a single challenge dose (C-mice) as compared to mice similarily challenged after 2 or 3 previous infections (SC-mice). Controls were mice given only the 2 or 3 previous infections and uninfected mice. Eyes were observed in si& during a 34 day post-challenge period to compare inflammatory responses in the anterior eye; histologic examination of serial sections of the eyes of these mice was done at the end of this period. Zn sim observations showed that lesions in the anterior eye converted from hemorrhagic to white cell more rapidly in the SC-mice; white cell lesions were predominant in SC-mice as early as day 5, whereas similar predominance in C-mice was not noted until days 16-21. Histology revealed that approximately 90% of these eyes were infected. Worm burdens per eye correlated more closely with total dose per mouse than with the effects of immunization. Histologic study showed that 90% of larvae observed were in the retina, but that most lesions were in the uveal tissues which harbored only 0.8 % of the total number of larvae.
Ahstract--Or_soN L. 3. 1976. Ocular Toxocariasis in mice: distribution national Journal for Parasitchgy
INDEX KEY WORDS:
6: 247-251.
Toxocar~ canis; ocular toxocariasis;
IT IS now well documented that Toxocava cunis larvae can invade the eye of man and cause serious lesions (Ferguson & Olson, 1967). This documentation began when Wilder, in 1950, reported on 46 human cases which had been diagnosed in most instances as retinoblastoma prior to enucleation of the eye. Examination of serial sections of these 46 eyes revealed the presence of nematode larvae or ‘their residual hyaline capsule’ in 24 eyes. The characteristics of the lesions in the remaining 22 eyes led Wilder to a tenative diagnosis of nematode endopht~almitis for these eyes. In 1956, Nichols identified several of the larvae in Wilder’s slides as T. canis.
murine toxocariasis.
days as either a diffuse infiltrate in the anterior chamber or as a discrete lesion on the iris. Some of these lesions were examined and shown to be primarily polymorphonuclear leukocytes, which in a few instances surrounded a larva. Based on these preliminary studies, experiments were designed to determine the distribution of T. canis larvae within the various tissues of the eye and to note any effect of previous infection on both numbers and distribution of these larvae in the eye. These experiments also permitted observations on the onset and development of hemorrhagic and white cell lesions in the anterior eye following challenge of immunized and control mice. MATERIALS
Little is known about the pathogenesis of ocular toxocariasis. Experimental work in mice has shown that these larvae, following infection via stomach tube, are capable of reaching the eye within 3 days. Viable larvae can be recovered from the eye for at least 4 months (Olson, Izzat, Petteway & Reinhart, 1970). IIence, invasion of the eye is an early event following infection of the mouse.
AND METHODS
T. cunis eggs were obtained from naturally infected dogs and given to mice by stomach intubation as previously described (Olson et al., 1970). Male mice (Swiss Yale) were purchased at IS g body weight from Texas Inbred Mice Co., Houston.
In preliminary studies of the mouse infected with a single dose of T. canis eggs, it was noted that these larvae invade the retina of this host, and also that following oral infection the first lesion observed was hemorrhage in the anterior chamber and iris. A second type of lesion developed during the next few 247
Eyes were examined in situ (without anesthetics) under a dissecting microscope (25 x) for lesions in the anterior eye; i.e. diffuse and localized hemorrhage and white cells in the anterior chamber and iris. Lesions were plotted on an eye map at each observation; hence, new and old lesions could be identified at a subsequent observation. Eyes were dissected by making an incision through the cornea and, w&h a smail spatula, the fluid, lens, and soft inner tissues were scooped out onto a glass slide and
24x -i-ABLE
1.. .!. I---/‘EK(‘ENTAGES
OF EYES
St4OWING
LESIONS
fkSON
DUKING
THE
I.J.P.
POSTf’HALLFhCE
PtKIOLf.
(NC>.
bYtS:
SC,
VOL.
6.
1076
206; c‘, 1%:
s, 92; u, 48) $ with
Day postchallenge
Hemorrhage SC
0
3
lesion White
C
Cells
SC
C
‘% showing
dominance
Hemorrhage
Iv’hite
C
SC
of Cells C
SC
0
0
0
3
0
0
0
0
6
3
2
0
6
3
0
0
12
3
0
0
a
3
0
0
35
36
31
3
12
25
16
II
37
53
45
18
4
36
31
2
9
28
26
37
13
2
26
26
6
11
30
41
37
21
5
38
24
4
13
19
19
26
13
6
11
20
6
14
17
39
31
32
6
32
25
11
16
22
22
28
21
5
11
21
9
3
20
10
22
9
5
26
11
2
24
10
8
28
13
3
0
19
IO
34
4
2
7
6
4
n
7
0
examined (25 x ) for larvae. Eyes were serially sectioned (10 pm) and stained with Harris’ hematoxylin and eosin, after fixation in buffered formalin and embedding in paraffin (Paraplast, Sherwood Medical Industries, St. Louis); infiltration of paraffin was done in a vacuum infiltrator (Lipshaw, Detroit). In counting larvae in serial sections, sections of larvae less than 500 pm apart were counted as a single larva. Three similarly designed experiments were completed in which mice were divided into four groups. Large doses of eggs were used to obtain a high percentage of infected eyes. SC-mice were given two (experiment 1) or three (experiments 2 and 3) prechallenge doses (50 eggs/g body weight) at two-three week intervals. Four weeks after the last dose, the mice were challenged (100/g). S-mice received only the prechallengc doses. C-mice received only the challenge. U-mice were uninfected controls. Eyes were examined prior to and at intervals after challenge as shown on Table 1. At the end of thz postchallenge period, mice were killed and both eyes (experi-
Analysis of these data showed that a high percentage of these eyes at the end of the post~hallenge period harbored one or more larva as determined by dissection (67-85 %) and serial sections (86-93 %) at the end of the postchallenge period (Tables 2 & 5). The number of larvae per eye ranged from 0 to 9. Observations of the eye in situ during the postchallenge period showed hemorrhage in the anterior chamber and iris as early as day two in the SC and C mice (Table 1). Discrete hemorrhage on the iris, ‘pinpoint’ to approximately 1 mm in diameter, was more common that diffuse hemorrhage in the
TABLE2--NUMRER
IN ONE OR BOTH
AND
(PERCENTAGE)
HARBORING
A LARVAE
OF MICE SHOWING IN ONE OR BOTH
Both
One
Neg.
RESULTS
LESIONS
EYES
AT THE END
Pfrcent
Lesions
Group
ments 2 and 3) of each mouse were either dissected for larvae or fixed in formalin. The results of these experiments were similar and hence have been combined.
with
THE POSTCHALLENGE
AS DETERMINED
L?li-“d?
eyes lesions
EYES DURING
OF THIS PERIOD
ma1
O”fZ
BY DISSECTION
I’frccn: with
Neg.
eyes larvae
ilRe,?n/eVe) . SC
0
s C
43 (42)
43142)
37 (54)
2 (4) 25 (36)
17(16) 44 (96) 700)
12 171)
5 (29)
I)
3(33)
6(67)
72
14(74)
63
3 (16)
0
85c3.6)
0
67c1.4)
2 (10) ~____
82 (3.0)
PERIOD
1.J.P. VOL.
Ocular toxocariasis in mice
6. 1976
internal structure and staining characteristics, these larvae, as well as those in other sites, appeared to have been viable at the time of fixation. In this connection, practially all larvae recovered by dissection, when dissection was done promptly after enucleation, were viable, i.e. motile. The distribution of larvae in the eye was similar for all three groups of mice (Table 3). Examination of serial-sections at the end of the postchallenge period showed that distribution of lesions did not parallel that for larvae (Table 4). Lesions of the retina (excluding areas of disorganization and other structural changes in which no in~ammatory cells were present) accounted for only 19.7 % of the total number of lesions; several of these lesions extended from the vitreous through the retina to the choroid. Uveitis was the dominant change with approximately one-half of all inflammatory cell reactions being located in the ciliary body, choroid and iris; choroidal inflammation frequently was located near a lesion involving the retina. The ratio of the mean number of lesions per eye (Table 4) to the mean number of larvae per eye (Table 3) was similar for all groups: SC, 0.64; S, 0.81; C, 0.68. The percentage of eyes with larvae in the absence of lesions was considerably less in the SC-mice (13%) as compared to that for S-mice (38 %) and C-mice (36%) (Table 5). Discrete lesions with a larva completely or partially in the lesion, or immediateIy adjacent to the lesion were seen infrequently; i.e. 12 lesions, or 7”/, of the total recorded (7 in vitreous-retina and retina alone; I choroid; 1 iris; and 3 anterior chamber). Discrete lesions with or without a larva were not more frequent in the SC-mice as compared to C-mice. Lesions seen on serial sections varied in cell types, possibly depending on the age of the lesion. In some lesions, polymorphonuclear leukocytes were predominant; in other lesions a granulomatous response
anterior chamber of the SC-mice in contrast to Cmice, which more frequently showed diffuse hemorrhage. The center of a discrete hemorrhage could usually be seen to lie over one of the blood vessels of the iris. Diffuse (anterior chamber) and discrete (iris) white cell lesions were first noted on day 2 in SC-mice and day 5 in C-mice. These lesions were more frequent in SC-mice, particularly during the first 2 weeks. A frequently observed pattern in the development of a given lesion on the iris was the conversion from a discrete hemorrhage to a discrete white cell lesion, which in turn cleared. It should be noted that many pinpoint hemorrhages cleared without this conversion being apparent. A similar pattern was noted for diffuse lesions in the anterior chamber. Discrete lesions tended to be more common in the periphery of the iris; i.e. outer third. Diffuse lesions sometimes involved the entire anterior chamber. No lesions were seen in U-mice during this period. S-mice were also negative during this period with the exception of one eye in each of two mice on day 34; each eye showed one small discrete white lesion on the iris. Lesions in the anterior eye were graded in terms of which type was predominant for a given eye, i.e. more frequent and or severe. Severity was based on the surface area of the lesion. Lesions, which were converting from hemorrhage to white cells, were graded on the relative surface areas of the lesion that each type occupied. In many instances no determination of predominance could be made. These data showed quite clearly that as early as day five the predominant type of lesion in the SC-mice was a white cell reaction; white cell lesions became predominant in C-mice between days 16 and 22(Table 1). Examination of serial-sections of eyes obtained at the end of the postchallenge period showed that approximately 90% of larvae were located partially or completely in the retina (Table 3). Based on
TABLE 3-PERcENT O~STRIB~I~N
OF LARVAE IN SERIAL SECTIOKSOF
(No. Group
Ant.
Post.
Cil-
c:13r-
Ch.
Ch.
iary
oid
SC
0.7
s
5.5
C
4.2
% dist.
0.4
0.4
EYES AT
THE END OF THE
POSTCHALLENGE
38; s, 16; c, 28)
SC,
Retina*
Vitr-
Optic
ems
A
B
C
D
E
2.9
4.3
56.5
32.6
0.7
0.7
h'erve
Tot31
(mean/eye)
1.4
138c3.6)
47.2
33.3
5.5
36c2.2)
5.6
2.8
52.8
30.6
2.8
72(2.5)
0.4
4.1
3.3
54.1
32.1
5.5
0.4
0.4
2.4
246
90.3
(all mice)
;: A:
partially
to choroid,
in vitreous, D:
larvae
1.4
2.8
2.0
EYES:
249
partially
B:
completely
in optic
nerve,
in retina, E:
C:
partially
in retina in ciliary
adjacent body.
PERIOD.
250
I..I.P. VOL..6. 1976
L. J. OLWY
TABLE ‘&--PERCENT DlSTRlRUTioN OF LESIONS IN EYESSERIALLY SECTIONED AT THF END OF THE POSTCIIALLENGE PERIOD.
38;S,
(No.EYEs:SC,
croup
Ant.
Post.
Cil-
Ch.
Ch.
iary
Iris
16;CJS)
Vitr-
Retinn+
oid
ecu?
A
l3
(menn/eyc)
15.9
3.4
21.6
12.5
11.4
22.7
2.2
10.2
88c2.3)
s
13.8
0
24.1
10.3
17.2
6.9
6.9
20.7
29U.8)
c
16.7
4.2
18.7
10.4
18.7
lb.7
12.5
2.1
48U.7)
X dist. 15.8
3.0
21.2
11.5
14.5
18.2
10.0
9.7
47.2
dominated; increased numbers of eosinophils were present in about one-third of these lesions. Hemorrhage was sometimes seen in the aqueous and vitreous together with white cells. A more detailed analysis of the h~stopathology of eye lesions caused by T. canis will be presented elsewhere, based on experiments in which serial-sections were obtained at intervals during the postchallenge period, rather than at the end of that period as in the current study. DISCUSSION These data indicate that 90% of those larvae which invade the eye can be found in the retina. The majority of these larvae, however, were not intimately associated with any inflammatory cells; only 20% of all lesions involved the retina. There is, of course, no way to determine from these data how long a given larva had occupied the site in which it was observed in a section. The presence of vitreal and retinal lesions in the absence of an associated larva or a residuum of a dead larva suggests that larvae, after inducing a lesion, migrate out of it. In this connection, when larvae were seen within a lesion the impression was that these larvae were viable at fixation as judged by their morphology and staining. The life span of a T. canis larva in the mouse eye is not known. Olson et al. (1970) reported recoveries from the eyes of mice infected for 4 months, which were approximately equal to that WJMBER
(PERCENTACE)OF
Group
Larvae +
EYES SHOWING LARVAE ASDETERMINEDFROM Larvae na
lesions
lesions
165
19.7
;?A: lesion involves retina and vitreous, B:
5-
Totals
SC
(all mice)
TARLE
-
Chor-
retina only
during the second week of infection; hence, a life expectancy of at least several months in this eye is probable. There was no indication that these prechallenge infections were immunogenic to the extent of preventing eye invasion following the challenge dose since the mean worm burden of SC-eyes exceeded that of S-eyes. There was a suggestion that the degree of eye invasion following challenge was reduced in the SC-mice (Tables 3 & 4). However, if one examines each of these experiments, and those in other studies in this laboratory, there is no consistent pattern of protection against eye invasion in SCmice; the differences between the SC mean and the sum of S and C means are consistently small and vary from experiment to experiment in terms of protection. Worm burdens of these eyes appear to be primarily additive, i.e. to increase with the total infective dose given the mouse. These data show that hemorrhage occurred as early as day 2, which confirms earlier findings that invasion of the eye is an early event following oral infection (Olson et al., 1970). New hemorrhagic lesions increased in frequency during the first 7 days, which is the period of increasing worm burden per eye (Olson er al., 1970). However, it was noted that new hemorrhagic lesions also developed after this period in areas of the iris, which previously were mapped as free of lesions, and in the anterior chamber. A frequent observation was to note a AND LESIONS AT THE END OF THE POSTCHALLENGE SERIALSECTIONS
Lesions only
x0 L2rvne or lesions
Percent with: Larvae
Lesions
29(X)
5(13)
l(3)
89
79
s
9061
6(3S)
1-W)
0
93
62
c
14(50)
lO(36f
3111)
l(4)
86
61
SC
3@)
PERIOD
I.J.P.voL.~.1976
Ocular toxocariasis in mice
pinpoint hemorrhage on the iris, which would be followed by one or more adjacent pinpoint hemorrhages during the time when the first lesion was enlarging and converting to a white cell lesion. These ‘satellite’ lesions suggest that a single larvae was migrating in that area of the iris causing multiple leTions. These in vivo observations of the anterior eye indicate that the eyes of mice with a history of previous infection are able to develop an accelerated inflammatory response to challenge larvae as compared to that for previously unexposed mice. By the end of the postchallenge period differences in the types and frequencies of these lesions had largely disappeared. The sharp decline in the frequency of anterior eye lesions in SC and C groups at the end of this period may reflect the migration of larvae out of the anterior eye, or the death of these larvae. In this connection, the histologic data at the close of this period showed very few larvae in those portions of the eye that were observed in vivo; i.e. anterior chamber and iris. Only 2.4% or 6 of the 246 larvae seen in sections were in the anterior-posterior chambers and iris, although 63 and 72% of SC and C eyes, respectively, showed lesions in the anterior eye during the postchallenge period. It seems reasonable to assume that a similar inflammatory response was occurring during this period in the posterior eye of these mice, although histologic data obtained at the end of this period did not show any striking differences in number, location and type of lesions among these groups of eyes. This may reflect
251
the time when these eyes were examined; i.e. at the end of the postchallenge period, at which time in vivo observations revealed no differences in type and frequency of lesions in the anterior eye of SC and C mice. In this connection, studies are underway with T. cunis in mice in which serial sections of eyes will be obtained at intervals during the postchallenge period of previously-immunized and control mice to determine if immunization results in an accelerated inflammatory response-including granuloma formation-in the retina and other posterior areas of the eye. Acknowledgemerzts-The
support of Research Grant EYO0919 from The National Institutes of Health, Bethesda, MD, U.S.A. is acknowledged. REFERENCES FERGUSONE. C. & OLSON L. J. 1967. Toxocara ocular nematodiasis. International Ophlhalmology Clinics 7: 583-603. NICHOLS R. L. 1956. The etiology of visceral larva migrans. 1. Diagnos!ic morphology of infective secondstage Toxocnra larvae. Journal of Parasitology 42: 349-362. OLSON L. J., IZZAT N. N., PETTEWAY M. B. & REINHART J. A. 1970. Ocular toxocariasis in mice. American Journal of Tropicul Medicine and Hygiene 9: 238-243.
endophthalmitis. Transactions of the American .4cademy of Ophthalmology and Otolaryngology 55: 99-109.
WILDERH. C. 1950. Nematode
