- Email: [email protected]
Immunological studies on Nippostrongylus brasiliensis infection in the rat: Experiments with irradiated larvae
EXPERIMENTAL
PARASITOLOGY
17,
Immunological
51-56
(1965)
Studies on Nippostrongylus Infection in the Rat: Experiments with Irradiated Larvae Zd.
Prochazkal
of Glasgow
University
(Submitted
and Veterinary
for publication,
W.
Mulligan
School,
Glasgow,
4 June
brasiliensis
Scotland
1964)
1965. Immunological studies on Nippostrongylus PROCHAZKA, ZD., AND MULLIGAN, W. brasiliensis infection in the rat: experiments with irradiated larvae. Experimental Parasitology 17, 51-56. The development in rats of Ni$#ostrongylus brmiliensis larvae treated with X-ray doses of 0, 50, 100, 180 Kr was studied. The intestinal burdens at Day 10 from 2000 larvae so treated were, respectively, 855, 800, 103, 0; the lung burdens at the same time were, respectively, 17, 19, 32 and 94. Larvae treated with X-ray doses of 50 and 100 Kr conferred as good an immunity as normal larvae. Larvae treated with 180 Kr had little or no immunogenic action. Differences in the degree of attenuation produced by the same dose of X-rays on different preparations of N. brasiliensis larvae may be associated with differences in inherent infectivity of the larvae.
Helminth larvae suitably treated with ionizing radiation do not develop normally in the host animal. This effect was first demonstrated experimentally by Tyzzer and Honeij ( 1916) using Trichinella spiralis, and most of the early irradiation work on helminths was carried out with this parasite. The use of irradiated larvae as immunizing agents has widened the scopeof this field of study, and the effect of larval irradiation has now been investigated in a number of different hostparasite systems (see review by Urquhart et al., 1962). In the caseof Nippostrongylus brasiliensis, X-ray treatment of the infective larvae leads not only to a reduction in the total number of worms which develop from a standard doseof larvae, but also to differences in their distribution between lungs and small intestine (Jennings et al., 1963). It seemedlikely therefore l Present Veterinary Czechoslovakia.
address: Research
Radiobiology Institute,
Department, Brno-Medlanky,
51
that by using suitably irradiated preparations of N. brasiliensisone could study the relative immunological importance of the lung and intestinal phasesof this infection. The present paper dealswith someexperiments carried out along these lines. MATERIALS
AND
METHODS
The culture of infective larvae, irradiation, and infection procedures were all as described previously (Jennings et a2., 1963). Throughout the experiments the standard culture procedures were maintained, and all larvae used were harvested 7 days after setting up the culture. On each occasion on which larvae were required a large single pool was prepared. It was not possible to use the same pool for stages of an experiment that were separatedin time, e.g., different but identically prepared pools were used for the immunizing and challenge phasesof the first experiment. Likewise different pools were needed for different experiments.
52
PROCHAZKA
AND
viously (Jennings et al., 1963). The results are summarized in Table II. These figures provided information on the degree of attenuation produced by the different X-ray doses. The results in Table II show that very little attenuation results from an X-ray dose of .50Kr, i.e., the total numbers of worms recovered and their distribution were essentially the same in Groups D1 and Ar. With a dose of 100 Kr (Group B,) very significant attenuation occurred as manifested by a reduced gut burden compared to Group D1 and an increased lung/gut ratio. This situation had gone to the extreme in Group Cl where a further increase in the lung burden occurred and no worms were recovered from the small
RESULTS
Immunogenic Effect of Nippostrongylus brasiliensis, Larvae Treated with Different Doses of x-rays A plan of the experiment is given in Table I. Four groups (D, A, B, and C) of twenty rats each were inoculated (Day 0) with larvae treated with X-ray doses of 0, 50, 100, and 180 Kr, respectively. Each rat received 2000 larvae. Faeces from each group were examined for eggs from Day 6 onwards and on Day 10 seven rats from each group (i.e., Subgroups Di, Ar, B1, and C,) were killed and the numbers of worms in lungs and small intestines were determined as described prePlan
of Experiment
Sub groups
Group D
D2
c2
7 9
C3 1
4 10
X
Development
Group No. Dl Al Bl Cl
in Rats
X-Ray dose to larvae Wr) 0 50
Eggs/gm (7)
+++
+-t+ 0
+++ +
+
100
0
0
0
180
0
0
0
9
-
4
-
-
9
9
-
-
-
10
10
with
Different
4
Doses of X-rays
Mean numbers -C SD of worms recovered at autopsy on Day 10
faeces on Day (8)
9
7
Treated
4
-
-
II Larvae
9
-
-
TABLE brasiliensis
4 9
7
2000 irradiated larvae ( 180 Kr )
9
-
2000 irradiated larvae (100 Kr)
-
-
-
2000 irradiated larvae (SO Kr)
Kill and examine (Day 30)
9
7
of Nippostrongylus
(6)
-
-
4
B3 1 Cl
Challenge (Day 20)
7
7 9
B2
Larvae
-
4
I
Bl
C
Kill and examine (Day 10)
treatment 0)
2000 normal larvae
7 9
‘42 ‘43
brasiliensis
4
D.3 I Al
B
Immunizing (Day
7 9
Dl
A
TABLE I Irradiated Nippostrongylus
with
No. of rats
MULLIGAN
(9)
(10)
Small intestine
+++ ++
+++ ++
855 & 162 800 k 300
+
+
0
103 _t
0
0
21
Lungs 17-c 8 19k 7 32 2 15 94 c 19
N. brasiliensis
INFECTION
intestine. During the period Day 6 to Day 10, faecal egg output was assessed semiquantitatively (Table II). The egg output of group A was substantially less than that of the control Group D showing that although a dose of 50 Kr did not significantly reduce the number of worms which developed, their reproductive capacity was impaired. Only occasional eggs were found in the faeces from Group B, and none at all in the case of Group C. Of the remaining 13 rats in each group, nine (in Subgroups DB, AZ, BP, and C,) were challenged with 2000 larvae each along with a group of susceptible controls (Group X) on Day 20, faecal egg counts were carried out from Day 25 onwards and all rats were killed on Day 30. The numbers of worms recovered from lungs and small intestines of these animals are shown in Table III. The remaining four rats in each group (Subgroups Da, Aa, Ba, and Ca) were not subjected to challenge on Day 20 but were killed on Day 30 to give a measure of the “background” worm burden that might still be present as a result of the primary infections given on Day 0. In the nine rats of each group which were challenged with 2000 larvae the immunity, as assessed by the number of worms which developed from the challenge, was similar in TABLE Challenge
Larval Challenge (Day 20)
Group
of Rats
(27)
(28)
0 0 6 44 34 -
0 0 0.2 33 31 -
2000 ea.
0 19
D3 A3 B3
25 -
I None
c3
a Where worms in D,, A,, etc.
were
found
in groups
D,,
THE
53
RAT
Groups A, B, and D (see Table III), i.e. an experience with 2000 larvae treated either with 50 Kr or 100 Kr gave rise to an immunity comparable to that induced by a primary dose of 2000 normal larvae. However, in Group C where the radiation dose was 180 Kr no significant immunity developed. This is shown clearly in Table III by the worm burdens and faecal egg counts in this group. It is interesting that in this group although a substantial number of worms arose from the immunizing dose of irradiated larvae all of these were present in the lungs at Day 10. The results obtained with the four rats of each group killed on Day 30 without challenge showed that at this time only very few worms were left from the immunizing infection given on Day 0. Where worms were found the numbers were subtracted as a blank from the results in the appropriate groups. Immunogenic Eflect of Different Doses of Irradiated Nippostrongylus brasiliensis Larvae The relationship in helminth systems between the size of the immunizing dose and the degree of immunity produced has received little investigation. It seemed useful to study this relationship with irradiated larvae because of their potential role as immunizing agents. An X-ray dose of 1OOKr was chosen III Nippostrangylus
brasiliensis
A,,
Larvae
NeP numbers f SD of worms recovered at autopsy (Day 30)
X 103 on Day
0 0
c2
X
Irradiated
(26)
I
B2
with
Eggs/gm
D2 -42
Immunized
IN
(29) 0 0 0.4 60
(30)
51
57
0 0 0.2 50
-
Small
intestine
84 -c 34% 47 k 503 f 627 2 3
138 26 75 217 357
Lungs
4k2 623 lo&
6
Sk5 6-+4 0
-
-
-
0
0
-
-
-
4 0
0 0
etc. the numbers
were
subtracted
from
the totals
recovered
PROCHAZKA
54
Plan
of Experiment
to Study
AND
MULLIGAN
TABLE IV the Immunogenic Eflect
of
Different
Primary dose of larvae (Day 0)
Doses
Kill and examine (Day IO)
Group No.
Sub Rroups
No. of rats
K
Kl
7 14
50
-
4
irradiateda
-
K2 K3
L
L2 L3
M
M2 M3
N
Nl N2 N3
P Q a Irradiated
larvae
used were
Number
Group
Kl
irradiated”
7 15
2000 normal -
-
with
an X-ray
250a 5cW 2OOoa 2000 normal
Ml
Nl P with
100
15 -
15
4 9 4
-
7
13 -
13 4 -
15
15
dose of 100 Kr.
TABLE recovered from Nippostrongylus
SW
Ll
-
7 -
worms Irradiated
-
9
-
2000
Dose of Iarvae (Day 0)
No.
a Treated
of
14
-
500
7 13 4
treated
-
14
-
7
irradiated=
Kill and examine (Day 30)
-
-
250 irradiated”
Larvae
4 7
7 9 4
Ml
Challenge (Day 20)
7
7 15 4
Ll
Irradiated
of
V Rats Given brasiliensis
Different Larvae
Doses
of
Mean number worms recovered on Day Small
intestine 0
0 0.3 11 f
445 t
5.4
110
c SD of at autopsy 10
Lungs 0.6 k 1.5 f 0.7 k 7.3 k 2.0?
0.6 1.5 0.7 6.0 1.5
Kr.
because the previous experiment showed that it produced a suitable degree of attenuation with no detectable loss of immunizing power. A plan of the experiment is shown in Table IV. Groups of rats (K, L, M, and N) each divided into three sub-groups as indicated, were infected with doses of 50, 250, 500, and 2000 irradiated larvae per rat. Group P consisted of 7 rats and they were given 2000 normal larvae of the batch used for irradiation. On Day 10 the rats in the Subgroups K1, L1, M1, and N1 were killed and examined for worms to show how many had developed from the various doses of irradiated larvae.
The rats in group P were killed and examined at the same time, these figures giving a measurement of the infectivity of the larvae used for irradiation. The results of this stage of the experiment are summarized in Table V. The rats in Subgroups KZ, L2, M2 and N2 were challenged with 2000 larvae each on Day 20 along with 15 control rats (Group Q). All were killed on Day 30 along with the rats of Subgroups KS, La, MS, and Na. The results are summarized in Table VI. It is clear from the figures shown in Tables V and VI that the results of this experiment differ from those of the previous experiment in
N. brasiliensis
Challenge
of Rats
Immunized
Larval Challenge (Day 20)
Group No.
MS! 32
1 r
Q
J
TABLE Doses
Diflerent
Eggs/gm
2000 ea.
a The rats of Group in Group N3.
with
INFECTION
x
IN
THE
VI of Irradiated
103 on Day
(28)
(29)
29 29 18 12 70
70 64 36 6 86
60 70 33 16.4 60
48 64 46 1.3 110
were
brasiliensis
all negative
one respect, i.e., the numbers of worms developing from the doses of irradiated larvae were very much smaller. In the previous experiment the administration of 2000 larvae treated with 100 Kr gave rise to an intestinal worm burden on Day 10 of 103 worms. In the present experiment the same dose of larvae gave rise to only 11 worms. It would appear that the larvae in the present experiment were more severely attenuated. Note that the larvae used for irradiation in the two experiments differed significantly in their degree of infectivity (compare Group D1 in Table II with Group P in Table V). Apart from this anomaly the results of the two experiments are in agreement. Only in Group N where a significant number of worms was found in the intestine at Day 10 was any measurable immunity observed. DISCUSSION
Elucidation of the mechanism of helminth immunity is made difficult by the complexity of the parasites. It is often hard to find out the relative immunological importance of different stages of development and/or different phases in the migratory path for those parasites which migrate in the host. An analysis of the situation is greatly helped by agents which can interfere with development or migration at selected points. The impairment of development in the host which results from X-irradiation of infective larvae may in some cases be put to use for this purpose.
Larvae
Neta numbers f SD of worms recovered at autopsy (Day 30)
(27)
and N,
55
Nippostrongylus
(26)
IS,, La, Ma,
RAT
on Day
Small 671 799 574 100 932 30 except
intestine 5~ & ” 2 f
151 198 287 174 126
for an average
Lungs 0.9 1.6 2.0 2.6 2.0
k ‘-c k k j,
1.2 1.8 1.6 2.7 3.1
of 1 worm
The experiments reported here show that by use of a suitable dose of X-rays one can obtain a preparation of N. brasiliensis larvae which gives rise to no worms in the small intestine at Day 10, although substantial numbers are present in the lungs at this time. Furthermore such a preparation of larvae seems to have little immunogenic power. This would indicate that the intestinal phase is of great immunological importance. It should be pointed out that in the case of this preparation of larvae (Group C) we cannot guarantee that worms do not become transiently established in the intestine at some time after Day 10. We know that of the approximately 90 worms present in the lungs at Day 10 all have disappeared by Day 30, but they may in fact have moved to the gut prior to being eliminated from the host. It would be useful to study the fate of the relatively larger number of parasites that are still present in the lungs at Day 10 when irradiated as opposed to normal larvae are administered to rats. The attenuation phase of the second experiment reported here is not in line with that of the first. In seeking for an explanation of this discrepancy it may be worth noting that the larvae used for irradiation differed significantly in their inherent infectivity in these two experiments. This raises the question of the relationship between infectivity and radiosensitivity. It may well be that the same dose of radiation will not produce the same degree
56
PROCHAZKA
of damage in larval preparations of different degrees of infectivity. This is a problem that should be investigated. ACKNOWLEDGMENTS
This work was supported by a grant from the Agricultural Research Council, and carried out during the tenure of a Fellowship from the International Atomic Energy Agency by one of the authors (Zd. Prochazka). REFERENCES JENNINGS,
G. M.
F.
W.,
MULLIGAN,
1963.
Variables
AND
URQUHART,
in the X-ray
W.,
“inactiva-
AND
MULLIGAN
tion” of Experimental
Nippostrongylus Parasitology
brasiliensis 13, 367-373.
larvae.
E. L., AND HONEIJ, J. A. 1916. The effect of radiation on the development of Irichinella spir&, with respect to its application to the treatment of other parasitic diseases. Journal of Parasitology 3, 43-56.
TYZZER,
URQUHART,
G. M.,
JARRETT,
W. 1962. “Helminth vances in Veterinary and E. L. Jungherr, Academic Press, New
W. F. H., AND MULLIGAN, Immunity,” Science” (C. eds.) Vol. York.
In “AdA. Brandly 7, 87-129.
PARASITOLOGY
17,
Immunological
51-56
(1965)
Studies on Nippostrongylus Infection in the Rat: Experiments with Irradiated Larvae Zd.
Prochazkal
of Glasgow
University
(Submitted
and Veterinary
for publication,
W.
Mulligan
School,
Glasgow,
4 June
brasiliensis
Scotland
1964)
1965. Immunological studies on Nippostrongylus PROCHAZKA, ZD., AND MULLIGAN, W. brasiliensis infection in the rat: experiments with irradiated larvae. Experimental Parasitology 17, 51-56. The development in rats of Ni$#ostrongylus brmiliensis larvae treated with X-ray doses of 0, 50, 100, 180 Kr was studied. The intestinal burdens at Day 10 from 2000 larvae so treated were, respectively, 855, 800, 103, 0; the lung burdens at the same time were, respectively, 17, 19, 32 and 94. Larvae treated with X-ray doses of 50 and 100 Kr conferred as good an immunity as normal larvae. Larvae treated with 180 Kr had little or no immunogenic action. Differences in the degree of attenuation produced by the same dose of X-rays on different preparations of N. brasiliensis larvae may be associated with differences in inherent infectivity of the larvae.
Helminth larvae suitably treated with ionizing radiation do not develop normally in the host animal. This effect was first demonstrated experimentally by Tyzzer and Honeij ( 1916) using Trichinella spiralis, and most of the early irradiation work on helminths was carried out with this parasite. The use of irradiated larvae as immunizing agents has widened the scopeof this field of study, and the effect of larval irradiation has now been investigated in a number of different hostparasite systems (see review by Urquhart et al., 1962). In the caseof Nippostrongylus brasiliensis, X-ray treatment of the infective larvae leads not only to a reduction in the total number of worms which develop from a standard doseof larvae, but also to differences in their distribution between lungs and small intestine (Jennings et al., 1963). It seemedlikely therefore l Present Veterinary Czechoslovakia.
address: Research
Radiobiology Institute,
Department, Brno-Medlanky,
51
that by using suitably irradiated preparations of N. brasiliensisone could study the relative immunological importance of the lung and intestinal phasesof this infection. The present paper dealswith someexperiments carried out along these lines. MATERIALS
AND
METHODS
The culture of infective larvae, irradiation, and infection procedures were all as described previously (Jennings et a2., 1963). Throughout the experiments the standard culture procedures were maintained, and all larvae used were harvested 7 days after setting up the culture. On each occasion on which larvae were required a large single pool was prepared. It was not possible to use the same pool for stages of an experiment that were separatedin time, e.g., different but identically prepared pools were used for the immunizing and challenge phasesof the first experiment. Likewise different pools were needed for different experiments.
52
PROCHAZKA
AND
viously (Jennings et al., 1963). The results are summarized in Table II. These figures provided information on the degree of attenuation produced by the different X-ray doses. The results in Table II show that very little attenuation results from an X-ray dose of .50Kr, i.e., the total numbers of worms recovered and their distribution were essentially the same in Groups D1 and Ar. With a dose of 100 Kr (Group B,) very significant attenuation occurred as manifested by a reduced gut burden compared to Group D1 and an increased lung/gut ratio. This situation had gone to the extreme in Group Cl where a further increase in the lung burden occurred and no worms were recovered from the small
RESULTS
Immunogenic Effect of Nippostrongylus brasiliensis, Larvae Treated with Different Doses of x-rays A plan of the experiment is given in Table I. Four groups (D, A, B, and C) of twenty rats each were inoculated (Day 0) with larvae treated with X-ray doses of 0, 50, 100, and 180 Kr, respectively. Each rat received 2000 larvae. Faeces from each group were examined for eggs from Day 6 onwards and on Day 10 seven rats from each group (i.e., Subgroups Di, Ar, B1, and C,) were killed and the numbers of worms in lungs and small intestines were determined as described prePlan
of Experiment
Sub groups
Group D
D2
c2
7 9
C3 1
4 10
X
Development
Group No. Dl Al Bl Cl
in Rats
X-Ray dose to larvae Wr) 0 50
Eggs/gm (7)
+++
+-t+ 0
+++ +
+
100
0
0
0
180
0
0
0
9
-
4
-
-
9
9
-
-
-
10
10
with
Different
4
Doses of X-rays
Mean numbers -C SD of worms recovered at autopsy on Day 10
faeces on Day (8)
9
7
Treated
4
-
-
II Larvae
9
-
-
TABLE brasiliensis
4 9
7
2000 irradiated larvae ( 180 Kr )
9
-
2000 irradiated larvae (100 Kr)
-
-
-
2000 irradiated larvae (SO Kr)
Kill and examine (Day 30)
9
7
of Nippostrongylus
(6)
-
-
4
B3 1 Cl
Challenge (Day 20)
7
7 9
B2
Larvae
-
4
I
Bl
C
Kill and examine (Day 10)
treatment 0)
2000 normal larvae
7 9
‘42 ‘43
brasiliensis
4
D.3 I Al
B
Immunizing (Day
7 9
Dl
A
TABLE I Irradiated Nippostrongylus
with
No. of rats
MULLIGAN
(9)
(10)
Small intestine
+++ ++
+++ ++
855 & 162 800 k 300
+
+
0
103 _t
0
0
21
Lungs 17-c 8 19k 7 32 2 15 94 c 19
N. brasiliensis
INFECTION
intestine. During the period Day 6 to Day 10, faecal egg output was assessed semiquantitatively (Table II). The egg output of group A was substantially less than that of the control Group D showing that although a dose of 50 Kr did not significantly reduce the number of worms which developed, their reproductive capacity was impaired. Only occasional eggs were found in the faeces from Group B, and none at all in the case of Group C. Of the remaining 13 rats in each group, nine (in Subgroups DB, AZ, BP, and C,) were challenged with 2000 larvae each along with a group of susceptible controls (Group X) on Day 20, faecal egg counts were carried out from Day 25 onwards and all rats were killed on Day 30. The numbers of worms recovered from lungs and small intestines of these animals are shown in Table III. The remaining four rats in each group (Subgroups Da, Aa, Ba, and Ca) were not subjected to challenge on Day 20 but were killed on Day 30 to give a measure of the “background” worm burden that might still be present as a result of the primary infections given on Day 0. In the nine rats of each group which were challenged with 2000 larvae the immunity, as assessed by the number of worms which developed from the challenge, was similar in TABLE Challenge
Larval Challenge (Day 20)
Group
of Rats
(27)
(28)
0 0 6 44 34 -
0 0 0.2 33 31 -
2000 ea.
0 19
D3 A3 B3
25 -
I None
c3
a Where worms in D,, A,, etc.
were
found
in groups
D,,
THE
53
RAT
Groups A, B, and D (see Table III), i.e. an experience with 2000 larvae treated either with 50 Kr or 100 Kr gave rise to an immunity comparable to that induced by a primary dose of 2000 normal larvae. However, in Group C where the radiation dose was 180 Kr no significant immunity developed. This is shown clearly in Table III by the worm burdens and faecal egg counts in this group. It is interesting that in this group although a substantial number of worms arose from the immunizing dose of irradiated larvae all of these were present in the lungs at Day 10. The results obtained with the four rats of each group killed on Day 30 without challenge showed that at this time only very few worms were left from the immunizing infection given on Day 0. Where worms were found the numbers were subtracted as a blank from the results in the appropriate groups. Immunogenic Eflect of Different Doses of Irradiated Nippostrongylus brasiliensis Larvae The relationship in helminth systems between the size of the immunizing dose and the degree of immunity produced has received little investigation. It seemed useful to study this relationship with irradiated larvae because of their potential role as immunizing agents. An X-ray dose of 1OOKr was chosen III Nippostrangylus
brasiliensis
A,,
Larvae
NeP numbers f SD of worms recovered at autopsy (Day 30)
X 103 on Day
0 0
c2
X
Irradiated
(26)
I
B2
with
Eggs/gm
D2 -42
Immunized
IN
(29) 0 0 0.4 60
(30)
51
57
0 0 0.2 50
-
Small
intestine
84 -c 34% 47 k 503 f 627 2 3
138 26 75 217 357
Lungs
4k2 623 lo&
6
Sk5 6-+4 0
-
-
-
0
0
-
-
-
4 0
0 0
etc. the numbers
were
subtracted
from
the totals
recovered
PROCHAZKA
54
Plan
of Experiment
to Study
AND
MULLIGAN
TABLE IV the Immunogenic Eflect
of
Different
Primary dose of larvae (Day 0)
Doses
Kill and examine (Day IO)
Group No.
Sub Rroups
No. of rats
K
Kl
7 14
50
-
4
irradiateda
-
K2 K3
L
L2 L3
M
M2 M3
N
Nl N2 N3
P Q a Irradiated
larvae
used were
Number
Group
Kl
irradiated”
7 15
2000 normal -
-
with
an X-ray
250a 5cW 2OOoa 2000 normal
Ml
Nl P with
100
15 -
15
4 9 4
-
7
13 -
13 4 -
15
15
dose of 100 Kr.
TABLE recovered from Nippostrongylus
SW
Ll
-
7 -
worms Irradiated
-
9
-
2000
Dose of Iarvae (Day 0)
No.
a Treated
of
14
-
500
7 13 4
treated
-
14
-
7
irradiated=
Kill and examine (Day 30)
-
-
250 irradiated”
Larvae
4 7
7 9 4
Ml
Challenge (Day 20)
7
7 15 4
Ll
Irradiated
of
V Rats Given brasiliensis
Different Larvae
Doses
of
Mean number worms recovered on Day Small
intestine 0
0 0.3 11 f
445 t
5.4
110
c SD of at autopsy 10
Lungs 0.6 k 1.5 f 0.7 k 7.3 k 2.0?
0.6 1.5 0.7 6.0 1.5
Kr.
because the previous experiment showed that it produced a suitable degree of attenuation with no detectable loss of immunizing power. A plan of the experiment is shown in Table IV. Groups of rats (K, L, M, and N) each divided into three sub-groups as indicated, were infected with doses of 50, 250, 500, and 2000 irradiated larvae per rat. Group P consisted of 7 rats and they were given 2000 normal larvae of the batch used for irradiation. On Day 10 the rats in the Subgroups K1, L1, M1, and N1 were killed and examined for worms to show how many had developed from the various doses of irradiated larvae.
The rats in group P were killed and examined at the same time, these figures giving a measurement of the infectivity of the larvae used for irradiation. The results of this stage of the experiment are summarized in Table V. The rats in Subgroups KZ, L2, M2 and N2 were challenged with 2000 larvae each on Day 20 along with 15 control rats (Group Q). All were killed on Day 30 along with the rats of Subgroups KS, La, MS, and Na. The results are summarized in Table VI. It is clear from the figures shown in Tables V and VI that the results of this experiment differ from those of the previous experiment in
N. brasiliensis
Challenge
of Rats
Immunized
Larval Challenge (Day 20)
Group No.
MS! 32
1 r
Q
J
TABLE Doses
Diflerent
Eggs/gm
2000 ea.
a The rats of Group in Group N3.
with
INFECTION
x
IN
THE
VI of Irradiated
103 on Day
(28)
(29)
29 29 18 12 70
70 64 36 6 86
60 70 33 16.4 60
48 64 46 1.3 110
were
brasiliensis
all negative
one respect, i.e., the numbers of worms developing from the doses of irradiated larvae were very much smaller. In the previous experiment the administration of 2000 larvae treated with 100 Kr gave rise to an intestinal worm burden on Day 10 of 103 worms. In the present experiment the same dose of larvae gave rise to only 11 worms. It would appear that the larvae in the present experiment were more severely attenuated. Note that the larvae used for irradiation in the two experiments differed significantly in their degree of infectivity (compare Group D1 in Table II with Group P in Table V). Apart from this anomaly the results of the two experiments are in agreement. Only in Group N where a significant number of worms was found in the intestine at Day 10 was any measurable immunity observed. DISCUSSION
Elucidation of the mechanism of helminth immunity is made difficult by the complexity of the parasites. It is often hard to find out the relative immunological importance of different stages of development and/or different phases in the migratory path for those parasites which migrate in the host. An analysis of the situation is greatly helped by agents which can interfere with development or migration at selected points. The impairment of development in the host which results from X-irradiation of infective larvae may in some cases be put to use for this purpose.
Larvae
Neta numbers f SD of worms recovered at autopsy (Day 30)
(27)
and N,
55
Nippostrongylus
(26)
IS,, La, Ma,
RAT
on Day
Small 671 799 574 100 932 30 except
intestine 5~ & ” 2 f
151 198 287 174 126
for an average
Lungs 0.9 1.6 2.0 2.6 2.0
k ‘-c k k j,
1.2 1.8 1.6 2.7 3.1
of 1 worm
The experiments reported here show that by use of a suitable dose of X-rays one can obtain a preparation of N. brasiliensis larvae which gives rise to no worms in the small intestine at Day 10, although substantial numbers are present in the lungs at this time. Furthermore such a preparation of larvae seems to have little immunogenic power. This would indicate that the intestinal phase is of great immunological importance. It should be pointed out that in the case of this preparation of larvae (Group C) we cannot guarantee that worms do not become transiently established in the intestine at some time after Day 10. We know that of the approximately 90 worms present in the lungs at Day 10 all have disappeared by Day 30, but they may in fact have moved to the gut prior to being eliminated from the host. It would be useful to study the fate of the relatively larger number of parasites that are still present in the lungs at Day 10 when irradiated as opposed to normal larvae are administered to rats. The attenuation phase of the second experiment reported here is not in line with that of the first. In seeking for an explanation of this discrepancy it may be worth noting that the larvae used for irradiation differed significantly in their inherent infectivity in these two experiments. This raises the question of the relationship between infectivity and radiosensitivity. It may well be that the same dose of radiation will not produce the same degree
56
PROCHAZKA
of damage in larval preparations of different degrees of infectivity. This is a problem that should be investigated. ACKNOWLEDGMENTS
This work was supported by a grant from the Agricultural Research Council, and carried out during the tenure of a Fellowship from the International Atomic Energy Agency by one of the authors (Zd. Prochazka). REFERENCES JENNINGS,
G. M.
F.
W.,
MULLIGAN,
1963.
Variables
AND
URQUHART,
in the X-ray
W.,
“inactiva-
AND
MULLIGAN
tion” of Experimental
Nippostrongylus Parasitology
brasiliensis 13, 367-373.
larvae.
E. L., AND HONEIJ, J. A. 1916. The effect of radiation on the development of Irichinella spir&, with respect to its application to the treatment of other parasitic diseases. Journal of Parasitology 3, 43-56.
TYZZER,
URQUHART,
G. M.,
JARRETT,
W. 1962. “Helminth vances in Veterinary and E. L. Jungherr, Academic Press, New
W. F. H., AND MULLIGAN, Immunity,” Science” (C. eds.) Vol. York.
In “AdA. Brandly 7, 87-129.