Parasitology (1975), 70, 263-271
263
Infections with Eimeria maxima and Eimeria acervulina in the fowl: effect of previous infection with the heterologous organism on oocyst production M. ELAINE ROSE Houghton Poultry Research Station, HougMon, Huntingdon, Cambs. PEll 2DA {Received 16 September 1974) SUMMARY
Judged by oocyst production, infections with Eimeria acervulina in birds immunized with E. maxima were consistently higher than in birds which had not been immunized. Oocyst production of E. maxima in birds previously infected with E. acervulina was greater, in three out of four experiments, than in control chickens, but some of the differences were slight. The findings are discussed but no satisfactory explanation can be offered. In general there was little or no difference between the oocyst production of the individual species when present as single or mixed infections. INTRODUCTION
The work described here was originally undertaken to determine whether the form of non-specific cellular immunity described by Mackaness & Blanden (1967), and considered to be particularly active in infections with intracellular organisms, was involved in immunity to the Eimeria. This form of cellular immunity is evoked by a specific immunological reaction but is non-specific in its effects, which can be directed against micro-organisms other than the inducing one; the enhanced resistance is considered to be due to the 'activation' of macrophages. Evidence for the participation of macrophages in immunity to Eimeria infections (Huff & Clark, 1970; Patton, 1970; Rose, 1974) indicated that non-specific cellular responses in Eimeria infections should be examined. Consequently, although immunity to the Eimeria has in the main been shown to be speciesspecific, the effect of immunization with one species of Eimeria on subsequent infection at different time intervals by another was investigated, infections being measured by oocyst production. Two species of Eimeria which parasitize the intestine of the fowl were used — E. acervulina and E. maxima. These species appear to be immunologically distinct (no cross-protection when tested in the usual way), and also differ in the evocation of host response; a single inoculum of very small numbers (50—100) of oocysts of E. maxima results in rapid and complete immunity whereas more than one inoculum of several thousands of oocysts of E. acervulina are needed to induce complete immunity.
264
M. ELAINE ROSE
METHODS
Animals H.P.R.S. Light Sussex chickens, reared and maintained coccidia-free (Long, 1974) before experimental infection, were used in all experiments. Infective materials These were the Houghton strains of E. maxima and E. acervulina. Methods for counting oocysts for dosing and in faeces have been described (Horton-Smith & Long, 1959; Long & Rowell, 1958). Design of experiments (a) Immunized with E. maxima and subsequently infected with E. acervulina Experiments 1 and 2. Groups of 2-week-old birds were given approximately 500 oocysts of E. maxima on day 0, and the test inocula consisting of approximately 500 oocysts of E. acervulina were given on days 7, 14 or 21, as there is evidence that the temporal relationship between stimulation and challenge is important in non-specific cellular resistance. Control chicks, not initially infected with E. maxima, were given the oocysts of E. acervulina to provide a basis for comparison. Birds from test and control groups were dosed alternately from the same oocyst suspension. In two further experiments (3 and 4), the test inocula of oocysts of E. acervulina were given 14 days after the immunizing inoculum of E. maxima, with or without a ' boosting' inoculum of oocysts of E. maxima. This was done because non-specific resistance may be specifically recalled by re-exposure to the original organism (Nelson, 1972) and, in some cases, is effective only when homologous and heterologous organisms are given simultaneously. Oocyst production of both species was measured in test and control groups. (b) Immunized with E. acervulina and subsequently infected with E. maxima Four experiments (5-8) of the same design as Exps. 3 and 4 above, but with the reverse procedure, were carried out. The inocula of E. acervulina, however, consisted of approximately 10000 oocysts in order to stimulate a reasonably good immunity. In all experiments total oocyst production was measured throughout patency.
RESULTS
(a) Oocyst production of E. acervulina in birds immunized with E. maxima The results of experiments 1 and 2, in which the test inocula of E. acervulina oocysts were given 7, 14 or 21 days after the initial inoculum of E. maxima, are given in Table 1. This shows that for all three time intervals, oocyst production of E. acervulina in groups previously infected with E. maxima was consistently higher than in control groups not previously infected. In both experiments the
Infections with Eimeria spp. in the fowl
265
Table 1. Effect of previous infection with Eimeria maxima on subsequent infection with Eimeria acervulina: oocysts of Eimeria acervulina given 1, 2 or 3 weeks after the inoculum of Eimeria maxima: chicks 2 weeks old on day 0 Pre-infection with E. maxima on day 0 i
Exp. no.
Group no.
It
1 2 3 4 5 6 7 1 2 3 4 5 6 7
2§
Test infection with E. acervulina
^
Oocysts given* + + + + + + + +
Nos. oocysts produced! Day given 7 2-2 7 0 14 5-3 14 0 21 2-1 21 0 1-4 7 7-7 7 0 14 9-9 14 0 21 9-1 21 0 7-8
* 476 oocysts of E. maxima inoculated. 1 Twelve birds per group.
Nos. of oocysts given
Nos. oocysts produced!
476 476 580 580 520 520
251 11-2 51-3 20-2 64-4 13-3
524 524 475 475 486 486
490 191 93-7 26-6 91-4 24-6
Increase in nos. of oocysts of E. acervulina in groups previously infected with E. maxima 124 154 384
157 252 272
f Millions per bird. § Ten birds per group.
effect was least with the shortest interval (7 days) between the two inocula but, even in these groups, oocyst production was more than double that in the 'unstimulated' birds. The daily pattern of oocyst production was unaffected. In Exps. 3 and 4 the time interval between the two inocula was constant at 14 days and 'boosting' doses of E. maxima were given to some groups to determine whether this specific restimulation would affect the infections with E. acervulina. Results are given in Table 2. Immunization by the dose of E. maxima given on day 0 is shown by the lack of oocyst production to the second inoculum given on day 14 (group 1) and this immune state was not affected by the simultaneous administration of oocysts of E. acervulina (group 5). The viability of the 'boosting' inoculum of E. maxima was evident from the production of oocysts in groups 2 and 6 (previously uninfected with E. maxima). In Exps. 3 and 4, as in 1 and 2, more oocysts of E. acervulina were produced in groups of birds previously infected with E. maxima (groups 3 and 5) than in ' unstimulated' controls (groups 4 and 6); the daily pattern of oocyst production was unaltered. In the four experiments in which oocyst production by E. acervulina in groups of birds 'stimulated' with E. maxima infection was compared with control
266
M. ELAINE ROSE
Table 2. Effect of previous infection with Eimeria maxima on oocyst production of Eimeria acervulina with and without re-stimulation Inoculations on day 14 A
i
Infection
E. maxima oocysts
"OTltVl
A
Exp. Group E. maxima no.
no.*
on day 0"j"
3
1
+
2
-
3
+
4
5
+
6 4
1
+
2
3
+
4
-
5
+
6
Nos. given
Nos. produced |
476 476
0 11
. 495 495
A
Nos. given
0 7 0 6
Nos. produced:):
UJLLt/(-> t e l l
YV1LX1
E. maxima (%)
m
480 480
t
. 476 476 495 495
E. acervulina oocysts
Increase in nos of oocysts of E. acervulina in groups previously
480 480 .
.
478 478
0 5
478 478
34 7 29 9
16 5 11 5
386 222
220 120
* Ten birds per group. f Exp. 3: 478 oocysts given, oocyst production, millions/bird = 2-6 and 2-1 (two groups each of 15 birds). Exp. 4: 468 oocysts given, oocyst production, millions/bird = 6-4 and 5-4 (two groups each of 15 birds). X Millions/bird.
unstimulated groups, at least twice as many oocysts were found in the faeces of the stimulated groups irrespective of whether a 'boosting' inoculum of oocysts of E. maxima was given simultaneously with the test inoculum of E. acervulina oocysts or not (two experiments). (b) Oocyst production of E. maxima in birds immunized with E. acervulina These experiments (5—8) were identical in design to experiments 3 and 4 above, but the procedures were reversed, the initial ('stimulating') inoculum consisting of oocysts of E. acervulina (approximately 10000) and the test inoculum E. maxima (approximately 500). The results, given in Table 3, show more variation than was obtained in the experiments above. In three out of four experiments oocyst production of E. maxima in groups previously given E. acervulina was higher than in the controls (compare groups 4 with 3, and 6 with 5), but the differences between groups 3 and 4 in Exp. 5 and between 5 and 6 in Exp. 8 were small. In Exp. 7 there were no differences. The reason for this is not obvious; the initial inoculum with E. acervulina produced a reasonable infection, shown by the oocyst production figures and by the development of immunity to the second inoculum of E. acervulina (compare group 1 with 2, and 5 with 6). The administration of a 'boosting' inoculum with E. acervulina simultaneously with the test inoculum of E. maxima did not have a consistent effect; in one experiment (7) there was no effect, in
Infections with Eimeria spp. in the fowl
267
Table 3. Effect of previous infection with Eimeria acervulina on oocyst production of Eimeria maxima with and without restimulation with Eimeria acervulina
Inoculations on day 14 A
r
Infection wi+Vi 7/7 W1LI1 JJJ.
Exp. Group acervulina no. no. on day Of + 1 5 2
3 4 5
+ +
6 6
1
+
2
3
+
7
1
+
2
8
3 4 5 6 1
+ + +
2
3 4 5 6
+ +
A
"l (
Nos. given 9000 9000
Nos. producedj
.
9000 9000 10000 10000
10000 10000 10000 10000
. 0-2 290 2 212 . 3 171 3 239
. .
10000 10000 10000 10000
10000 10000
Nos. given
0-1 337
263
Infections with Eimeria maxima and Eimeria acervulina in the fowl: effect of previous infection with the heterologous organism on oocyst production M. ELAINE ROSE Houghton Poultry Research Station, HougMon, Huntingdon, Cambs. PEll 2DA {Received 16 September 1974) SUMMARY
Judged by oocyst production, infections with Eimeria acervulina in birds immunized with E. maxima were consistently higher than in birds which had not been immunized. Oocyst production of E. maxima in birds previously infected with E. acervulina was greater, in three out of four experiments, than in control chickens, but some of the differences were slight. The findings are discussed but no satisfactory explanation can be offered. In general there was little or no difference between the oocyst production of the individual species when present as single or mixed infections. INTRODUCTION
The work described here was originally undertaken to determine whether the form of non-specific cellular immunity described by Mackaness & Blanden (1967), and considered to be particularly active in infections with intracellular organisms, was involved in immunity to the Eimeria. This form of cellular immunity is evoked by a specific immunological reaction but is non-specific in its effects, which can be directed against micro-organisms other than the inducing one; the enhanced resistance is considered to be due to the 'activation' of macrophages. Evidence for the participation of macrophages in immunity to Eimeria infections (Huff & Clark, 1970; Patton, 1970; Rose, 1974) indicated that non-specific cellular responses in Eimeria infections should be examined. Consequently, although immunity to the Eimeria has in the main been shown to be speciesspecific, the effect of immunization with one species of Eimeria on subsequent infection at different time intervals by another was investigated, infections being measured by oocyst production. Two species of Eimeria which parasitize the intestine of the fowl were used — E. acervulina and E. maxima. These species appear to be immunologically distinct (no cross-protection when tested in the usual way), and also differ in the evocation of host response; a single inoculum of very small numbers (50—100) of oocysts of E. maxima results in rapid and complete immunity whereas more than one inoculum of several thousands of oocysts of E. acervulina are needed to induce complete immunity.
264
M. ELAINE ROSE
METHODS
Animals H.P.R.S. Light Sussex chickens, reared and maintained coccidia-free (Long, 1974) before experimental infection, were used in all experiments. Infective materials These were the Houghton strains of E. maxima and E. acervulina. Methods for counting oocysts for dosing and in faeces have been described (Horton-Smith & Long, 1959; Long & Rowell, 1958). Design of experiments (a) Immunized with E. maxima and subsequently infected with E. acervulina Experiments 1 and 2. Groups of 2-week-old birds were given approximately 500 oocysts of E. maxima on day 0, and the test inocula consisting of approximately 500 oocysts of E. acervulina were given on days 7, 14 or 21, as there is evidence that the temporal relationship between stimulation and challenge is important in non-specific cellular resistance. Control chicks, not initially infected with E. maxima, were given the oocysts of E. acervulina to provide a basis for comparison. Birds from test and control groups were dosed alternately from the same oocyst suspension. In two further experiments (3 and 4), the test inocula of oocysts of E. acervulina were given 14 days after the immunizing inoculum of E. maxima, with or without a ' boosting' inoculum of oocysts of E. maxima. This was done because non-specific resistance may be specifically recalled by re-exposure to the original organism (Nelson, 1972) and, in some cases, is effective only when homologous and heterologous organisms are given simultaneously. Oocyst production of both species was measured in test and control groups. (b) Immunized with E. acervulina and subsequently infected with E. maxima Four experiments (5-8) of the same design as Exps. 3 and 4 above, but with the reverse procedure, were carried out. The inocula of E. acervulina, however, consisted of approximately 10000 oocysts in order to stimulate a reasonably good immunity. In all experiments total oocyst production was measured throughout patency.
RESULTS
(a) Oocyst production of E. acervulina in birds immunized with E. maxima The results of experiments 1 and 2, in which the test inocula of E. acervulina oocysts were given 7, 14 or 21 days after the initial inoculum of E. maxima, are given in Table 1. This shows that for all three time intervals, oocyst production of E. acervulina in groups previously infected with E. maxima was consistently higher than in control groups not previously infected. In both experiments the
Infections with Eimeria spp. in the fowl
265
Table 1. Effect of previous infection with Eimeria maxima on subsequent infection with Eimeria acervulina: oocysts of Eimeria acervulina given 1, 2 or 3 weeks after the inoculum of Eimeria maxima: chicks 2 weeks old on day 0 Pre-infection with E. maxima on day 0 i
Exp. no.
Group no.
It
1 2 3 4 5 6 7 1 2 3 4 5 6 7
2§
Test infection with E. acervulina
^
Oocysts given* + + + + + + + +
Nos. oocysts produced! Day given 7 2-2 7 0 14 5-3 14 0 21 2-1 21 0 1-4 7 7-7 7 0 14 9-9 14 0 21 9-1 21 0 7-8
* 476 oocysts of E. maxima inoculated. 1 Twelve birds per group.
Nos. of oocysts given
Nos. oocysts produced!
476 476 580 580 520 520
251 11-2 51-3 20-2 64-4 13-3
524 524 475 475 486 486
490 191 93-7 26-6 91-4 24-6
Increase in nos. of oocysts of E. acervulina in groups previously infected with E. maxima 124 154 384
157 252 272
f Millions per bird. § Ten birds per group.
effect was least with the shortest interval (7 days) between the two inocula but, even in these groups, oocyst production was more than double that in the 'unstimulated' birds. The daily pattern of oocyst production was unaffected. In Exps. 3 and 4 the time interval between the two inocula was constant at 14 days and 'boosting' doses of E. maxima were given to some groups to determine whether this specific restimulation would affect the infections with E. acervulina. Results are given in Table 2. Immunization by the dose of E. maxima given on day 0 is shown by the lack of oocyst production to the second inoculum given on day 14 (group 1) and this immune state was not affected by the simultaneous administration of oocysts of E. acervulina (group 5). The viability of the 'boosting' inoculum of E. maxima was evident from the production of oocysts in groups 2 and 6 (previously uninfected with E. maxima). In Exps. 3 and 4, as in 1 and 2, more oocysts of E. acervulina were produced in groups of birds previously infected with E. maxima (groups 3 and 5) than in ' unstimulated' controls (groups 4 and 6); the daily pattern of oocyst production was unaltered. In the four experiments in which oocyst production by E. acervulina in groups of birds 'stimulated' with E. maxima infection was compared with control
266
M. ELAINE ROSE
Table 2. Effect of previous infection with Eimeria maxima on oocyst production of Eimeria acervulina with and without re-stimulation Inoculations on day 14 A
i
Infection
E. maxima oocysts
"OTltVl
A
Exp. Group E. maxima no.
no.*
on day 0"j"
3
1
+
2
-
3
+
4
5
+
6 4
1
+
2
3
+
4
-
5
+
6
Nos. given
Nos. produced |
476 476
0 11
. 495 495
A
Nos. given
0 7 0 6
Nos. produced:):
UJLLt/(-> t e l l
YV1LX1
E. maxima (%)
m
480 480
t
. 476 476 495 495
E. acervulina oocysts
Increase in nos of oocysts of E. acervulina in groups previously
480 480 .
.
478 478
0 5
478 478
34 7 29 9
16 5 11 5
386 222
220 120
* Ten birds per group. f Exp. 3: 478 oocysts given, oocyst production, millions/bird = 2-6 and 2-1 (two groups each of 15 birds). Exp. 4: 468 oocysts given, oocyst production, millions/bird = 6-4 and 5-4 (two groups each of 15 birds). X Millions/bird.
unstimulated groups, at least twice as many oocysts were found in the faeces of the stimulated groups irrespective of whether a 'boosting' inoculum of oocysts of E. maxima was given simultaneously with the test inoculum of E. acervulina oocysts or not (two experiments). (b) Oocyst production of E. maxima in birds immunized with E. acervulina These experiments (5—8) were identical in design to experiments 3 and 4 above, but the procedures were reversed, the initial ('stimulating') inoculum consisting of oocysts of E. acervulina (approximately 10000) and the test inoculum E. maxima (approximately 500). The results, given in Table 3, show more variation than was obtained in the experiments above. In three out of four experiments oocyst production of E. maxima in groups previously given E. acervulina was higher than in the controls (compare groups 4 with 3, and 6 with 5), but the differences between groups 3 and 4 in Exp. 5 and between 5 and 6 in Exp. 8 were small. In Exp. 7 there were no differences. The reason for this is not obvious; the initial inoculum with E. acervulina produced a reasonable infection, shown by the oocyst production figures and by the development of immunity to the second inoculum of E. acervulina (compare group 1 with 2, and 5 with 6). The administration of a 'boosting' inoculum with E. acervulina simultaneously with the test inoculum of E. maxima did not have a consistent effect; in one experiment (7) there was no effect, in
Infections with Eimeria spp. in the fowl
267
Table 3. Effect of previous infection with Eimeria acervulina on oocyst production of Eimeria maxima with and without restimulation with Eimeria acervulina
Inoculations on day 14 A
r
Infection wi+Vi 7/7 W1LI1 JJJ.
Exp. Group acervulina no. no. on day Of + 1 5 2
3 4 5
+ +
6 6
1
+
2
3
+
7
1
+
2
8
3 4 5 6 1
+ + +
2
3 4 5 6
+ +
A
"l (
Nos. given 9000 9000
Nos. producedj
.
9000 9000 10000 10000
10000 10000 10000 10000
. 0-2 290 2 212 . 3 171 3 239
. .
10000 10000 10000 10000
10000 10000
Nos. given
0-1 337
