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Further Toxocara skin sensitivity tests in animals experimentally infected with Toxocara canis
246 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 63. No. 2. 1969.
FURTHER TOXOCARA EXPERIMENTALLY Department
SKIN SENSITIVITY INFECTED WITH
R. A. WISEMAN,
A. W. WOODRUFF
of Clinical
Medicine,
Tropical
TESTS IN ANIMALS TOXOCARA CAN/S AND
L. E. PETTITT
London School of Hygiene and Tropical
Medicine
In a previous paper (WISEMAN and WOODRUFF, 1967) it was reported that positive reactions were obtained to the intradermal injection of Toxocuru antigen in both mice and monkeys after experimental infection with Toxocara cunis, although the skin reactions before infection, and the reactions with saline controls, were negative. It was concluded that the Tuxocuru skin sensitivity test was valuable for the diagnosis of infection with T. cunis and supported other evidence (WOODRUFF and THACKER, 1964; WOODRUFF, BISSERU and BOWE, 1966; WISEMAN and WOODRUFF, 1968) of its value and specificity in man. WOODRUFF et al. (1966) showed that approximately 2.1% of an apparently healthy population tested in Britain gave positive reactions to the test, and thought that this represented the level of asymptomatic or subclinical infection. The lightest infection with Toxocuru capable of provoking a positive skin reaction was not known, however, nor was it known whether a positive skin test would develop shortly after infection in the absence of other overt evidence of toxocariasis. In order to resolve this problem, 4 rhesus monkeys (C, D, E and F) were experimentally infected with different numbers of embryonated T. cunis eggs and repeatedly skin tested; the absolute eosinophil level was estimated at weekly intervals. Materials The 4 monkeys
were aged between
and methods 6 months
and 1 year, and weighed
2e-7:
kg.
All
had eosinophil counts of less than 500 per c.mm. before infection, the relative counts being :. Monkey C 2% eosinophils in 15,600 total white cells per c.mm. D 0% 9, 3, 16,700 ,, 9, ,, 9, 3, EO% F 4%
3, x
,9 14,750 ,, 11,650
J,
J,
,I
n
,,
>>
3,
3,
>,
,>
Stool examination revealed Entamoeba coli cysts and Enterobius spp. in all 4 animals; monkeys C and F were also infected with Trichuris spp. The method of preparing the antigen for the intradermal skin test has been reported previously (WOODRUFF and THACKER, 1964). In a fully positive reaction the area of induration caused by the injection of 0.1 ml. of antigen is doubled or more within 20
minutes; in a negative reaction the area of induration than the original area; in a weakly positive reaction but not fully double, the area of the original wheal. The method of collection of the T. canis eggs, of feeding to the monkeys, as well as the details of the those described in the previous report (WISEMAN and
disappears entirely or is no greater the area of induration is larger than,
their embryonation, and the method weekly intradermal testing, all follow WOODRUFF, 1967). Monkeys C, D, E and F were fed 200, l lOO, 50 and 20 embryonated eggs respectively via an intragastric tube, and were tested before infection, and then at weekly intervals, with antigen and with saline solution as a control in the contralateral arm. Blood was taken from the monkey’s femoral veins. Total white cells were estimated and eosinophil levels calculated from the differential count.
R. A. WISEMAN,
A. W.
WOODRUFF
AND
247
L. E. PETTITT
Results All 4 monkeys gave negative skin reactions to the Toxocaru antigen before infection and to the saline solution throughout the experiment. Monkey C which had been given 200 eggs, gave a fully positive reaction at the end of the 4th week following infection, and this remained fully positive until the 7th week when it became weakly positive. Thereafter it varied from weakly positive to fully positive and was weakly positive at the end of the experiment on the 19th week. Monkey D, which had been given 100 eggs, gave a weakly positive reaction at the end of the 4th week but a week later it was fully positive and remained so until the 9th week, when it became weakly positive again; afterwards it varied between weakly and fi.111~positive. Monkey E, which had been given 50 eggs, was weakly positive at the 4th week after infection, fully positive at the 5th week, but weakly positive again at the 6th week and remained so with little variation (negative at the 9th week, fully positive at the 12th and 15th weeks) until the end of the experiment. Monkey F, which had been given only 20 eggs, was negative until the 6th week when it became fully positive; it was fully positive at the 7th and 8th weeks, but thereafter either completely negative or only weakly positive. The results of the skin tests are demonstrated in Fig. 1. The white cell counts did not alter significantly during the experiment. The absolute eosinophil level of Monkey C, which was normal before infection, began to increase at the 5th week, reached a broad peak from the 9th to 12th weeks, and then slowly declined. The eosinophil counts of D and E were extremely irregular throughout RESULTS OF SKIN EXPERIMENTALLY
SENSITIVITY INFECTED
TEST IN 4 MONKEYS WITH T.CANlS
MONKEY C (200 eggs)
MONKEY (100 ws)
D
MONKEY E (50 w3d
MONKEY i (20 eggs) 01
I
NEGATIVE
23
456 WEAKLY
78 9 WEEKS AFTER POSITIVE
FIG. 1
11 13 INFECTION
15 FULLY
17 POSITIVE
19
248
FURTHER
HISTOGRAMS
Toxocara
SKIN
SENSITIVITY
OF EOSINOPHIL LEVELS EXPERIMENTALLY INFECTED
TESTS IN ANIMALS
OF RHESUS MONKEYS WITH T.CANlS
1 Monkey C (infected with 200 eggs) 2000 r g
1 Monkey F (infected with 20 eggs)
Eosinophils / cu.mm.
1000
1 2 3 4 5 6 7 8 9 1011 1213 WEEI
15
19
FIG. 2
the period of observation, but that of F showed little or no change until the 1lth week, when an irregular increase occurred which was maximal at the 15th week. The eosinophil levels of C and F are shown in Fig. 2. The mean absolute eosinophil counts of all 4 monkeys (see Fig. 3) were normal until the 5th week. From then these counts increased, again reaching a broad peak from the 9th-13th weeks, after which a slow decline occurred, although the mean count was still elevated at the 19th week. Discussion
The conversion of the skin test from negative to positive after infection with eggs of T. canis confirms the results obtained previously (WISEMAN and WOODRUFF, 1967) when 2 rhesus monkeys (1 and 2) were fed 500 and 1,000 eggs respectively. In that experiment the skin test after early conversion remained positive in both monkeys until the 36th week, and further testing (since publication of that report) has shown positive reactions still present at the 60th week. All 4 monkeys of the present series gave positive reactions to the skin test after variable periods even though very small numbers of ova had been administered, which supports the view that the skin reaction is valuable for the diagnosis of very light toxocaral infections. The results (see Fig. 1) show that the monkeys (C and D), given 200 eggs and 100 eggs respectively, produced either fully positive or weakly positive skin reactions on the 4th week after infection, and every week thereafter. However, Monkey E, given 50 eggs, and Monkey F, given 20 eggs, showed reactions which, after the initial conversion, became very variable and sometimes negative; Monkey F had more negative responses than E. It appears therefore that the permanence or otherwise of the skin reaction may be related to the size of the infecting dose, and that with very small numbers of infecting eggs the reactions may be positive only at certain stages of the disease. It is, of course, possible that repeated challenges with small doses (as may occur with children in natural circumstances) can lead to a skin reaction which remains permanently positive. The negative or only weakly positive results of the skin test after the 8th week in Monkey F, suggest that 20 eggs is probably the lowest, or nearly the lowest, dose which gives rise to a positive result. Although 20 embryonated eggs were fed to Monkey F the
R. A. WISEMAN,
A. W. WOODRUFF
AND
L. E. PETTITT
249
number of larvae which migrated through the tissues was probably considerably less, as the eggs had been kept in 1% form01 saline at room temperature for over 6 months. In other experiments where larvae are harvested from mouse livers, it has been noticed that a much smaller number of larvae are recovered if the eggs have been kept for 6 months or more than when they have been stored for the optimal time for embryonation of 6-8 weeks and then used. It appears, therefore, that positive reactions to the skin test may occur even with doses of infective eggs smaller than 20, though a result which remains positive for some time occurs only when larger numbers of eggs are ingested, as in Monkeys C and D. Although the white cell counts of the 4 monkeys did not alter appreciably during the period of observation, the absolute eosinophil levels varied. The eosinophil level in C began to rise at the 5th week after infection; it reached its maximum at the 9th to 12th weeks and then slowly declined (see Fig. 2). Monkey F, given one tenth the dosegiven to C, showed no significant change in the eosinophils until the 11th week. Previously, Monkeys 1 and 2, given 500 and 1,000 eggs respectively, had developed marked eosinophilia which reached its peak at the end of the 3rd week; these results confirm the work of OLSON and SCHULTZ (1963) who noted that with higher dosages eosinophil levels increase earlier, and the time between infection and the onset of the response diminishes. Monkey F, which gave fully positive skin reactions at the 6th, 7th and 8th weeks but not thereafter, had no other signs of hehninthic infection at the peak of positivity of the skin test. This suggests that the skin test can be positive during the course of infection even when overt clinical signs are absent, and that with low numbers of infective eggs the skin test is a sensitive indicator of infection. These factors are relevant to the 2 * 1% of positive reactors reported by WOODRUFF et al. (1966) in a symptom-free population, and confirm their supposition that subclinical infection had taken place. In the previous experiment with Monkeys 1 and 2 it was suggested that the time of conversion of the skin test might depend on the size of the infecting dose, as the more heavily infected monkey (2) developed a weakly positive reaction at the end of the 4065 E s x 3000 XI s .z sn z I: .cf? 2000
_
MEAN ABSOLUTE EOSINOPHIL COUNTS RECORDED AT 2-WEEKLY PERIODS OF 4 MONKEYS EXPERIMENTALLY INFECTED WITH T,CANIS
-
B 0 0
f zVI % 6
1000
-
2
0
I,2
3,4
5,6
7,8
9,lO WEEKS
FIG.
3
11, 12
13, 14
15, 16
19
250
FURTHER
Toxocara
SKIN
SENSITIVITY
TESTS IN ANIMALS
2nd week after infection, which was 1 week earlier than in Monkey 1. However, both gave fully positive reactions at the same time. In the present experiment Monkey C was fully positive at the end of the 4th week, while D and E were weakly positive at that stage and not fully positive until the following week. F became fully positive only at the 6th week. It appears that since the time of conversion of the skin test was delayed in the more lightly infected monkeys, it depends upon dosage. Summary
4 rhesus monkeys were infected with 200, 100, 50 and 20 embryonated eggs of Toxocara sensitivity test. Their absolute eosinophil levels were estimated weekly. Negative reactions with the antigen were obtained in all the monkeys before infection and with the saline control used throughout the experiment. Positive reactions occurred after variable periods following infection in all, although the response in those monkeys given smaller numbers was less regular, more transient and occurred later than in’ those given larger numbers. In those given small numbers, conversion of the skin test from negative to positive occurred before the eosinophil level had altered. The experiment shows that the skin tests converted after infection with T. canis in experimental animals, and suggests that a positive skin reaction is seen even when other signs of helminthic infection are absent; the conversion time depends upon dosage. T. canis respectively and tested regularly with an intradermal
REFERENCES OLSON, WISE?
L.
J. &
SCHULTZ, c. A. & WOODRUFF,
W.
(1963).
kbzn.
N. Y. Acad.
sci.,
113, 440.
R. A. W. (1967). Trans. R. Sot. trap. Med. Hyg., 61, 827. -(1968). Br. med. J., 1, 677. WOODRUFF, A. W. & THACKER, C. K. (1964). Ibid., 1, 1,001. -, BISSERU, B. 81 BOWE, J. C. (1966). Ibid., 1, 1,576.
FURTHER TOXOCARA EXPERIMENTALLY Department
SKIN SENSITIVITY INFECTED WITH
R. A. WISEMAN,
A. W. WOODRUFF
of Clinical
Medicine,
Tropical
TESTS IN ANIMALS TOXOCARA CAN/S AND
L. E. PETTITT
London School of Hygiene and Tropical
Medicine
In a previous paper (WISEMAN and WOODRUFF, 1967) it was reported that positive reactions were obtained to the intradermal injection of Toxocuru antigen in both mice and monkeys after experimental infection with Toxocara cunis, although the skin reactions before infection, and the reactions with saline controls, were negative. It was concluded that the Tuxocuru skin sensitivity test was valuable for the diagnosis of infection with T. cunis and supported other evidence (WOODRUFF and THACKER, 1964; WOODRUFF, BISSERU and BOWE, 1966; WISEMAN and WOODRUFF, 1968) of its value and specificity in man. WOODRUFF et al. (1966) showed that approximately 2.1% of an apparently healthy population tested in Britain gave positive reactions to the test, and thought that this represented the level of asymptomatic or subclinical infection. The lightest infection with Toxocuru capable of provoking a positive skin reaction was not known, however, nor was it known whether a positive skin test would develop shortly after infection in the absence of other overt evidence of toxocariasis. In order to resolve this problem, 4 rhesus monkeys (C, D, E and F) were experimentally infected with different numbers of embryonated T. cunis eggs and repeatedly skin tested; the absolute eosinophil level was estimated at weekly intervals. Materials The 4 monkeys
were aged between
and methods 6 months
and 1 year, and weighed
2e-7:
kg.
All
had eosinophil counts of less than 500 per c.mm. before infection, the relative counts being :. Monkey C 2% eosinophils in 15,600 total white cells per c.mm. D 0% 9, 3, 16,700 ,, 9, ,, 9, 3, EO% F 4%
3, x
,9 14,750 ,, 11,650
J,
J,
,I
n
,,
>>
3,
3,
>,
,>
Stool examination revealed Entamoeba coli cysts and Enterobius spp. in all 4 animals; monkeys C and F were also infected with Trichuris spp. The method of preparing the antigen for the intradermal skin test has been reported previously (WOODRUFF and THACKER, 1964). In a fully positive reaction the area of induration caused by the injection of 0.1 ml. of antigen is doubled or more within 20
minutes; in a negative reaction the area of induration than the original area; in a weakly positive reaction but not fully double, the area of the original wheal. The method of collection of the T. canis eggs, of feeding to the monkeys, as well as the details of the those described in the previous report (WISEMAN and
disappears entirely or is no greater the area of induration is larger than,
their embryonation, and the method weekly intradermal testing, all follow WOODRUFF, 1967). Monkeys C, D, E and F were fed 200, l lOO, 50 and 20 embryonated eggs respectively via an intragastric tube, and were tested before infection, and then at weekly intervals, with antigen and with saline solution as a control in the contralateral arm. Blood was taken from the monkey’s femoral veins. Total white cells were estimated and eosinophil levels calculated from the differential count.
R. A. WISEMAN,
A. W.
WOODRUFF
AND
247
L. E. PETTITT
Results All 4 monkeys gave negative skin reactions to the Toxocaru antigen before infection and to the saline solution throughout the experiment. Monkey C which had been given 200 eggs, gave a fully positive reaction at the end of the 4th week following infection, and this remained fully positive until the 7th week when it became weakly positive. Thereafter it varied from weakly positive to fully positive and was weakly positive at the end of the experiment on the 19th week. Monkey D, which had been given 100 eggs, gave a weakly positive reaction at the end of the 4th week but a week later it was fully positive and remained so until the 9th week, when it became weakly positive again; afterwards it varied between weakly and fi.111~positive. Monkey E, which had been given 50 eggs, was weakly positive at the 4th week after infection, fully positive at the 5th week, but weakly positive again at the 6th week and remained so with little variation (negative at the 9th week, fully positive at the 12th and 15th weeks) until the end of the experiment. Monkey F, which had been given only 20 eggs, was negative until the 6th week when it became fully positive; it was fully positive at the 7th and 8th weeks, but thereafter either completely negative or only weakly positive. The results of the skin tests are demonstrated in Fig. 1. The white cell counts did not alter significantly during the experiment. The absolute eosinophil level of Monkey C, which was normal before infection, began to increase at the 5th week, reached a broad peak from the 9th to 12th weeks, and then slowly declined. The eosinophil counts of D and E were extremely irregular throughout RESULTS OF SKIN EXPERIMENTALLY
SENSITIVITY INFECTED
TEST IN 4 MONKEYS WITH T.CANlS
MONKEY C (200 eggs)
MONKEY (100 ws)
D
MONKEY E (50 w3d
MONKEY i (20 eggs) 01
I
NEGATIVE
23
456 WEAKLY
78 9 WEEKS AFTER POSITIVE
FIG. 1
11 13 INFECTION
15 FULLY
17 POSITIVE
19
248
FURTHER
HISTOGRAMS
Toxocara
SKIN
SENSITIVITY
OF EOSINOPHIL LEVELS EXPERIMENTALLY INFECTED
TESTS IN ANIMALS
OF RHESUS MONKEYS WITH T.CANlS
1 Monkey C (infected with 200 eggs) 2000 r g
1 Monkey F (infected with 20 eggs)
Eosinophils / cu.mm.
1000
1 2 3 4 5 6 7 8 9 1011 1213 WEEI
15
19
FIG. 2
the period of observation, but that of F showed little or no change until the 1lth week, when an irregular increase occurred which was maximal at the 15th week. The eosinophil levels of C and F are shown in Fig. 2. The mean absolute eosinophil counts of all 4 monkeys (see Fig. 3) were normal until the 5th week. From then these counts increased, again reaching a broad peak from the 9th-13th weeks, after which a slow decline occurred, although the mean count was still elevated at the 19th week. Discussion
The conversion of the skin test from negative to positive after infection with eggs of T. canis confirms the results obtained previously (WISEMAN and WOODRUFF, 1967) when 2 rhesus monkeys (1 and 2) were fed 500 and 1,000 eggs respectively. In that experiment the skin test after early conversion remained positive in both monkeys until the 36th week, and further testing (since publication of that report) has shown positive reactions still present at the 60th week. All 4 monkeys of the present series gave positive reactions to the skin test after variable periods even though very small numbers of ova had been administered, which supports the view that the skin reaction is valuable for the diagnosis of very light toxocaral infections. The results (see Fig. 1) show that the monkeys (C and D), given 200 eggs and 100 eggs respectively, produced either fully positive or weakly positive skin reactions on the 4th week after infection, and every week thereafter. However, Monkey E, given 50 eggs, and Monkey F, given 20 eggs, showed reactions which, after the initial conversion, became very variable and sometimes negative; Monkey F had more negative responses than E. It appears therefore that the permanence or otherwise of the skin reaction may be related to the size of the infecting dose, and that with very small numbers of infecting eggs the reactions may be positive only at certain stages of the disease. It is, of course, possible that repeated challenges with small doses (as may occur with children in natural circumstances) can lead to a skin reaction which remains permanently positive. The negative or only weakly positive results of the skin test after the 8th week in Monkey F, suggest that 20 eggs is probably the lowest, or nearly the lowest, dose which gives rise to a positive result. Although 20 embryonated eggs were fed to Monkey F the
R. A. WISEMAN,
A. W. WOODRUFF
AND
L. E. PETTITT
249
number of larvae which migrated through the tissues was probably considerably less, as the eggs had been kept in 1% form01 saline at room temperature for over 6 months. In other experiments where larvae are harvested from mouse livers, it has been noticed that a much smaller number of larvae are recovered if the eggs have been kept for 6 months or more than when they have been stored for the optimal time for embryonation of 6-8 weeks and then used. It appears, therefore, that positive reactions to the skin test may occur even with doses of infective eggs smaller than 20, though a result which remains positive for some time occurs only when larger numbers of eggs are ingested, as in Monkeys C and D. Although the white cell counts of the 4 monkeys did not alter appreciably during the period of observation, the absolute eosinophil levels varied. The eosinophil level in C began to rise at the 5th week after infection; it reached its maximum at the 9th to 12th weeks and then slowly declined (see Fig. 2). Monkey F, given one tenth the dosegiven to C, showed no significant change in the eosinophils until the 11th week. Previously, Monkeys 1 and 2, given 500 and 1,000 eggs respectively, had developed marked eosinophilia which reached its peak at the end of the 3rd week; these results confirm the work of OLSON and SCHULTZ (1963) who noted that with higher dosages eosinophil levels increase earlier, and the time between infection and the onset of the response diminishes. Monkey F, which gave fully positive skin reactions at the 6th, 7th and 8th weeks but not thereafter, had no other signs of hehninthic infection at the peak of positivity of the skin test. This suggests that the skin test can be positive during the course of infection even when overt clinical signs are absent, and that with low numbers of infective eggs the skin test is a sensitive indicator of infection. These factors are relevant to the 2 * 1% of positive reactors reported by WOODRUFF et al. (1966) in a symptom-free population, and confirm their supposition that subclinical infection had taken place. In the previous experiment with Monkeys 1 and 2 it was suggested that the time of conversion of the skin test might depend on the size of the infecting dose, as the more heavily infected monkey (2) developed a weakly positive reaction at the end of the 4065 E s x 3000 XI s .z sn z I: .cf? 2000
_
MEAN ABSOLUTE EOSINOPHIL COUNTS RECORDED AT 2-WEEKLY PERIODS OF 4 MONKEYS EXPERIMENTALLY INFECTED WITH T,CANIS
-
B 0 0
f zVI % 6
1000
-
2
0
I,2
3,4
5,6
7,8
9,lO WEEKS
FIG.
3
11, 12
13, 14
15, 16
19
250
FURTHER
Toxocara
SKIN
SENSITIVITY
TESTS IN ANIMALS
2nd week after infection, which was 1 week earlier than in Monkey 1. However, both gave fully positive reactions at the same time. In the present experiment Monkey C was fully positive at the end of the 4th week, while D and E were weakly positive at that stage and not fully positive until the following week. F became fully positive only at the 6th week. It appears that since the time of conversion of the skin test was delayed in the more lightly infected monkeys, it depends upon dosage. Summary
4 rhesus monkeys were infected with 200, 100, 50 and 20 embryonated eggs of Toxocara sensitivity test. Their absolute eosinophil levels were estimated weekly. Negative reactions with the antigen were obtained in all the monkeys before infection and with the saline control used throughout the experiment. Positive reactions occurred after variable periods following infection in all, although the response in those monkeys given smaller numbers was less regular, more transient and occurred later than in’ those given larger numbers. In those given small numbers, conversion of the skin test from negative to positive occurred before the eosinophil level had altered. The experiment shows that the skin tests converted after infection with T. canis in experimental animals, and suggests that a positive skin reaction is seen even when other signs of helminthic infection are absent; the conversion time depends upon dosage. T. canis respectively and tested regularly with an intradermal
REFERENCES OLSON, WISE?
L.
J. &
SCHULTZ, c. A. & WOODRUFF,
W.
(1963).
kbzn.
N. Y. Acad.
sci.,
113, 440.
R. A. W. (1967). Trans. R. Sot. trap. Med. Hyg., 61, 827. -(1968). Br. med. J., 1, 677. WOODRUFF, A. W. & THACKER, C. K. (1964). Ibid., 1, 1,001. -, BISSERU, B. 81 BOWE, J. C. (1966). Ibid., 1, 1,576.